University of Southern California Dissertations and Theses

Molecular and behavioral mechanisms of circatidal biological rhythms in intertidal mollusks

(USC Thesis Other)

Molecular and behavioral mechanisms of circatidal biological rhythms in intertidal mollusks

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content MOLECULAR AND BEHAVIORAL MECHANISMS OF CIRCATIDAL BIOLOGICAL
RHYTHMS IN INTERTIDAL MOLLUSKS

By
Jacqueline Lin

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOLOGY)

Department of Biological Sciences
Marine Biology and Biological Oceanography
August 2018

ii
Dedication

This work is dedicated to my family and friends, for always believing in me, especially when I could not
believe in myself.

iii
Acknowledgements

This work would not have been possible without the support and guidance of Andrew Gracey, my
advisor and committee chair. Thank you for seeing potential in me and taking me on as a student. Thank
you for your advice, insight, and reassurances throughout the past several years. I am grateful to the rest
of my committee as well: Suzanne Edmands, Michael Habib, and David Bottjer. Thank you for your
support and for your enthusiasm for my project. To all of my committee members, thank you for all of
the stimulating discussions at our meetings and for providing me with interesting new lenses with which
to look at my data.
I would also like to thank my lab mates, Kwasi Connor and Megan Hall. To Kwasi, thank you
for paving the way and setting up the foundation for my project. Thank you also for your help and
friendship, even after you graduated. To Megan, my lab twin, you were so integral to my graduate school
experience. Thank you for your constant support and for allowing me to bask in the warmth of your
friendship. Thank you for always being there for me throughout the roller coaster of PhD student life. I
am also deeply indebted to Leslie Pratt, my stalwart undergraduate. Thank you for the many hours you
spent assisting me with the limpet components of my project, and thank you for giving me the
opportunity to mentor you.
On the administrative side, I would like to thank Linda Bazilian, Douglas Burleson, Adolfo Dela
Rosa, and Donald Bingham. Thank you for keeping me on track and for patiently answering my endless
questions about graduation requirements, financial support, and the ins-and-outs of AHF.
Finally, I would like to thank my family, without whom I would have given up a long time ago.
To Mum, Dad, Tracy, Francis, and Matthew, thank you for your unwavering support and encouragement.
Thank you for being my first and foremost champions, always gently prodding me towards success.

iv
TABLE OF CONTENTS

Dedication ii

Acknowledgements iii

Table of Contents iv

Introduction 1

References 3

Chapter 1 6

Entrainment and persistence of rhythmic expression of
candidate molluscan circatidal genes in California ribbed
mussel Mytilus californianus and Pacific oyster Crassostrea gigas

References 26

Chapter 2 48

Behavioral characterization of the circatidal oscillator in
Boreal limpet Lottia paradigitalis

References 68

Chapter 3 88

Multi-tissue transcriptomic analysis of locomotor rhythms and
circatidal gene expression in free-running Boreal limpet Lottia
paradigitalis

References 102

Conclusion 132

References 133

1
INTRODUCTION

The marine intertidal zone is a dynamic environment. Because it lies at the interface between
land and sea, denizens of the intertidal zone must be prepared to face highly contrasting environmental
conditions with every changing of the tide. For many intertidal organisms, high tide and submergence
bring cool temperatures, ample oxygen concentrations, water flow, and opportunity to feed, especially for
sessile filter feeders and other animals dependent on planktonic food sources. Conversely, low tide and
emergence present intertidal organisms with a host of potential stresses, including thermal stress, hypoxia,
desiccation risk, limited feeding opportunity, and predation risk (Tessmar-Raible et al. 2011). Since the
ebb and flood of the tide results in such contrasting conditions and is a predictable, regularly-occurring
event, it would be to the advantage of intertidal organisms to have an endogenous time-keeping
mechanism that allows them to track and anticipate the changing of the tide, and therefore make the
appropriate behavioral and physiological adjustments necessary to survive and thrive – the circatidal
clock.
Many intertidal organisms have been shown to exhibit biological rhythms that reflect the two
major environmental oscillations that they experience: circadian rhythms (~24hr) that track the day-night
cycle and circatidal rhythms (~12.4hr) that track the changing of the tide (Wilcockson & Zhang 2008, Hut
& Beersma 2011). The circadian clock is an extensively studied system with much of the molecular
mechanism of its central oscillator already elucidated across several different taxa (Doherty & Kay 2010).
And rightly so, because nearly all organisms on Earth are subject to the effects of the day-night cycle.
The circadian clock allows organisms to optimize the regulation of behavioral and physiological
processes throughout the day and night in order to ensure maximum growth and fitness (Yerushalmi &
Green 2009, Nagel & Kay 2012). This is true across all of the taxa in which the circadian clock
mechanism has been elucidated, including cyanobacteria (Ouyang et al. 1998), plants (Dodd et al. 2005),
mammals (Takahashi et al. 2008), and insects (Numata et al. 2015). Indeed, any irregularity in an
organism’s circadian rhythm can lead to disease phenotypes and loss of fitness. For cyanobacteria,

2
rhythmic strains are able to outcompete arrhythmic strains (Ouyang et al. 1998), while in mammals
deviations for the normal circadian rhythms can result in sleep disorders, cardiovascular disease, cancer,
metabolic disorders, and immune-related disease (Hastings et al. 2003, Green et al. 2008, Arjona et al.
2012).
Just as the circadian clock is necessary for the survival of all organisms subject to the day-night
cycle, we hypothesize that the circatidal clock confers a similar fitness advantage for those organisms
living in the harsh and varying intertidal zone. However, the majority of research on circatidal rhythms
has been focused on only behavior or the measurement of a single metabolite or single physiological
output (Naylor 1988, Tessmar-Raible et al. 2011, De La Iglesia & Johnson 2013). It is only within the
past few years that researchers have started to attempt to elucidate the molecular mechanism of the
circatidal clock (Takekata et al. 2012, Zhang et al. 2013, Mat et al. 2014).
This dissertation aims to understand the molecular and behavioral mechanisms of circatidal
biological rhythms through time-course experiments with the sessile bivalves California mussel Mytilus
californianus and Pacific oyster Crassostrea gigas, as well as the locomotory Boreal limpet Lottia
paradigitalis. The sessile nature of M. californianus and C. gigas renders them even more vulnerable
than mobile organisms to the fluctuating conditions of the intertidal zone, making an anticipatory
physiological mechanism, such as that of the circatidal clock, especially necessary for their survival. The
movement of locomotory animals, such as limpets, provides a phenotypic framework in which to
corroborate gene expression patterns detected in sessile animals.
In Chapter 1, we sought to identify a set of candidate genes likely to be involved in the central
circatidal oscillator in M. californianus and C. gigas. Our previous work has shown that M. californianus
exhibits circatidal gene expression rhythms while living in simulated as well as field intertidal and
subtidal conditions (Connor & Gracey 2011). However, it was not known whether these rhythmic gene
expression patterns were the output of an endogenous molecular mechanism such as the circatidal clock
or merely a response to the different environmental changes brought by the ebb and flood of the tide.
Persistent circatidal gene expression patterns in free-running animals maintained under constant

3
laboratory conditions with no environmental cues would provide evidence for an endogenous molecular
oscillator, as well as produce a source of potential genes to further investigate.
In Chapter 2, we investigated the behavioral mechanism of the circatidal clock in L. paradigitalis,
focusing on the limpet’s locomotor activity. Although rhythmic movement behavior has been shown to
be endogenous in other limpet species (Della Santina & Naylor 1994, Gray & Hodgson 1999, Gray &
Williams 2010), that had not been confirmed in the genus Lottia, nor had the likely zeitgeber (“time-
giver,” i.e. environmental cue that sets the clock) been identified. In this chapter, we examined the free-
running locomotor activity of limpets entrained to field conditions and simulated laboratory conditions.
We also probed the plasticity of the circatidal oscillator through entrainment of limpets to non-circatidal
tidal cycles. Finally, we sought to identify the likely zeitgeber that sets the limpets in motion.
In Chapter 3, we combined gene expression and locomotor activity into one investigation in L.
paradigitalis. Although limpet movement has been extensively studied for some time now (e.g. Beckett,
1968; Davies et al., 2006; Hartnoll and Wright, 1977), the underlying molecular mechanism is unknown.
Because limpets tend to move with a circatidal rhythm, we investigated the circatidal gene expression
profiles in limpet head and foot, and identified genes associated with bouts of movement.
Overall, this dissertation aims to provide support for the hypothesis of a circatidal clock in
intertidal organisms, as well as establish a solid starting point from which to conduct further
investigations into the behavioral and molecular mechanisms of the circatidal clock.

References
Arjona A, Silver AC, Walker WE, Fikrig E (2012) Immunity’s fourth dimension: approaching the
circadian-immune connection. Trends Immunol 33:607–12
Beckett TW (1968) Limpet movements. An investigation into some aspects of limpet movements,
especially homing behaviour. Tane 14:43–63
Connor KM, Gracey AY (2011) Circadian cycles are the dominant transcriptional rhythm in the intertidal
mussel Mytilus californianus. Proc Natl Acad Sci U S A 108:16110–5

4
Davies MS, Edwards M, Williams GA (2006) Movement patterns of the limpet Cellana grata (Gould)
observed over a continuous period through a changing tidal regime. Mar Biol 149:775–787
Dodd A, Salathia N, Hall A, Kévei E, Tóth R (2005) Plant circadian clocks increase photosynthesis,
growth, survival, and competitive advantage. Science (80- ) 309:630–633
Doherty CJ, Kay SA (2010) Circadian Control of Global Gene Expression Patterns. Annu Rev Genet
44:419–444
Gray D, Hodgson A (1999) Endogenous rhythms of locomotor activity in the high-shore limpet, Helcion
pectunculus (Patellogastropoda). Anim Behav 57:387–391
Gray DR, Williams GA (2010) Knowing when to stop: Rhythms of locomotor activity in the high-shore
limpet, Cellana grata Gould. J Exp Mar Bio Ecol 391:125–130
Green CB, Takahashi JS, Bass J (2008) The meter of metabolism. Cell 134:728–42
Hartnoll RG, Wright JR (1977) Foraging movements and homing in the limpet Patella vulgata L. Anim
Behav 25:806–810
Hastings MH, Reddy AB, Maywood ES (2003) A clockwork web: circadian timing in brain and
periphery, in health and disease. Nat Rev Neurosci 4:649–61
Hut RA, Beersma DGM (2011) Evolution of time-keeping mechanisms: early emergence and adaptation
to photoperiod. Philos Trans R Soc Lond B Biol Sci 366:2141–54
La Iglesia HO De, Johnson CH (2013) Biological clocks: Riding the tides. Curr Biol 23:R921–R923
Mat AM, Massabuau J-C, Ciret P, Tran D (2014) Looking for the clock mechanism responsible for
circatidal behavior in the oyster Crassostrea gigas. Mar Biol 161:89–9
Nagel DH, Kay SA (2012) Complexity in the wiring and regulation of plant circadian networks. Curr Biol
22:R648-57
Naylor E (1988) Clock-Controlled Behaviour in Intertidal Animals. In: Chelazzi G, Vannini M (eds)
Behavioral Adaptation to Intertidal Life. Springer US, Boston, MA, p 1–14
Numata H, Miyazaki Y, Ikeno T (2015) Common features in diverse insect clocks. Zool Lett 1:10
Ouyang Y, Andersson CR, Kondo T, Golden SS, Johnson CH (1998) Resonating circadian clocks

5
enhance fitness in cyanobacteria. Proc Natl Acad Sci U S A 95:8660–4
Santina P Della, Naylor E (1994) Endogenous rhythms in the homing behaviour of the limpet Patella
vulgata Linnaeus. J Molluscan Stud 59:87–91
Takahashi JS, Hong H-K, Ko CH, McDearmon EL (2008) The genetics of mammalian circadian order
and disorder: implications for physiology and disease. Nat Rev Genet 9:764–775
Takekata H, Matsuura Y, Goto SG, Satoh A, Numata H (2012) RNAi of the circadian clock gene period
disrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket. Biol Lett 8:488–
91
Tessmar-Raible K, Raible F, Arboleda E (2011) Another place, another timer: Marine species and the
rhythms of life. Bioessays 33:165–72
Wilcockson D, Zhang L (2008) Circatidal clocks. Curr Biol 18:753–755
Yerushalmi S, Green RM (2009) Evidence for the adaptive significance of circadian rhythms. Ecol Lett
12:970–81
Zhang L, Hastings MH, Green EW, Tauber E, Sladek M, Webster SG, Kyriacou CP, Wilcockson DC
(2013) Dissociation of Circadian and Circatidal Timekeeping in the Marine Crustacean Eurydice
pulchra. Curr Biol 40:1–11

6
CHAPTER 1
Entrainment and persistence of rhythmic expression of candidate molluscan circatidal genes in
California ribbed mussel Mytilus californianus and Pacific oyster Crassostrea gigas

Abstract
Since the ebb and flow of the tide is a regular temporal occurrence associated with highly
contrasting environmental conditions, it follows that intertidal organisms should have a dedicated,
endogenous time-keeping mechanism that aids in anticipating these environmental fluctuations and
making the appropriate physiological changes. As a first step in elucidating the molecular mechanism of
the circatidal clock, we sought to identify a set of candidate genes likely to be involved in the central
oscillator in California ribbed mussel Mytilus californianus and Pacific oyster Crassostrea gigas. Our
previous work has shown that M. californianus exhibit circatidal gene expression rhythms while exposed
to simulated intertidal conditions, as well as intertidal and subtidal conditions in the field. If any of those
rhythmic genes are under the control of the circatidal clock, then they should continue to show circatidal
rhythmicity even in the absence of environmental cues. In this study, M. californianus and C. gigas were
entrained to field intertidal and subtidal conditions, then transferred to free-running constant conditions.
Gill samples were taken every 2 hours for 48 hours. RNA was isolated, and microarray and RNAseq
analysis were performed. Results show that M. californianus and C. gigas continue to show circatidal
gene expression, even under constant conditions in the absence of environmental cues. Moreover, a core
set of candidate genes was identified that showed circatidal rhythmicity across all of the treatments. This
set of candidate genes will allow us to continue to investigate the molecular mechanism of the circatidal
clock.

Introduction
Importance of biological clocks
Biological clocks are important to many organisms on Earth, since they are subject to the day-

7
night cycles of Earth rotating on its axis. The circadian clock allows organisms to optimize the regulation
of physiological processes throughout the day to ensure maximum growth and fitness (Yerushalmi &
Green 2009, Nagel & Kay 2012). This is true in a many taxa of organisms, including cyanobacteria
(Ouyang et al. 1998, Lambert et al. 2016), plants (Dodd et al. 2005), and mammals (Takahashi et al.
2008). Indeed, aberrations in circadian rhythms can lead to disease phenotypes and loss of fitness. In
mammals, these include sleep disorders, cardiovascular disease, cancer, metabolic disorders, and
immune-related disease (Hastings et al. 2003, Green et al. 2008, Arjona et al. 2012), while with
cyanobacteria, rhythmic strains are able to outcompete arrhythmic strains (Ouyang et al. 1998, Woelfle et
al. 2004).
The mammalian circadian clock is composed of interconnected transcription-translation positive
and negative feedback loops that control the expression of the core circadian genes. This cycle lasts for
approximately 24 hours, and sets the pace for the circadian clock (Langmesser et al. 2008). These core
genes show the most persistent circadian oscillations (Hughes et al. 2009), so we would expect the genes
involved in the central circatidal oscillator to also show the most persistent oscillations, even in free-
running animals. Previous research in our lab has identified a group of genes that oscillate with a
circatidal rhythm in animals maintained under various tidal conditions. Having identified this set of tidal
genes, we wanted to investigate if circatidal rhythms would persist in animals kept in free-running
conditions, thus providing strong evidence for these genes being involved in the central circatidal
oscillator. Identifying the candidate genes is the necessary first step in investigating the molecular
mechanism of the circatidal clock.

Rhythmic gene expression in Mytilus californianus
Prior research by our lab has started to characterize the biological rhythms in M. californianus
and C. gigas, specifically the rhythms of gene expression. In a previous experiment, M. californianus
were acclimated to a simulated intertidal regime with alternating 6-hour high and low tides, combined
with a 12-hour light-dark cycle (Connor & Gracey 2011). Gill samples were taken every 2 hours for 96

8
hours, and relative transcript abundance was later measured by microarray hybridization. At the 80-hour
time point, a warm low tide was simulated by a heat shock event with an increase of 7°C higher than the
normal low tide temperature. 2756 transcripts were found to cycle with a period between 10 and 28
hours. Of these transcripts, 236 oscillated with a circatidal rhythm, while 2365, approximately 10-fold
more transcripts, oscillated with a circadian rhythm. While circatidal transcriptional rhythms are present
in M. californianus, circadian rhythms are the dominant rhythms (Connor & Gracey 2011). However,
following the moderate heat shock event, periodicity was abolished in 24% of the transcriptome, with
37% and 17% of the circatidal and circadian genes, respectively, losing periodicity. Considering the
importance of biological rhythms and the potential for disease pathologies and loss of fitness in the face
of loss of rhythmicity, this could pose a serious problem for organisms in the context of increasing
temperatures and climate change.
In order to corroborate the results from the simulated tidal environment, a similar experiment was
with M. californianus acclimatized to field intertidal and subtidal environments such that the intertidal
animals spent approximately half of each tidal cycle emerged and approximately half submerged, while
the subtidal animals were always submerged (Connor & Gracey 2011). Gill samples were taken every 2
hours for 50 hours, and relative transcript abundance was later measured by microarray hybridization.
Many of the genes identified as tidal in the simulation study, such as cAMP responsive element binding-
like 2 (CREBL2), also exhibited tidal rhythmicity under field conditions. Between the simulated and field
studies, 44 genes were identified as tidal genes among intertidal, as well as subtidal, animals in both
experiments. This raises the possibility that a circatidal oscillator exists within subtidal organisms as
well.

Circadian clock organization
If the circatidal clock is its own separate oscillator, then what are the core genes and how do they
interact? The extensive body of circadian clock research is a likely source for the answer to this question.
The structure of the central oscillator of the circadian clock is very similar in all of the organisms that it

9
has been described in (Doherty & Kay 2010). It consists of interconnected transcription-translation
positive and negative feedback loops that control the expression of the core circadian genes.
In the mammalian circadian clock, transcription factors BMAL1 and CLOCK heterodimerize and
induce the expression of many target genes, including per and cry. The BMAl1/CLOCK heterodimer
binds the E-box regions of the promoters of per1, per2, and per3, as well as cry1 and cry2, forming the
positive loop of the oscillator (Hirayama et al. 2007, Yan et al. 2008). The PER and CRY proteins form a
complex that moves into the nucleus and inhibits the BMAL1/CLOCK heterodimer, thus inhibiting its
own expression (Kume et al. 1999). This is the negative loop of the oscillator. When PER/CRY levels
decrease, BMAL1/CLOCK is able to start the cycle over again. This cycle lasts for approximately 24
hours, and sets the pace for the circadian clock (Langmesser et al. 2008). The Drosophila and
Arabidopsis central circadian oscillators follow the same pattern, but with different genes (Figure 1).
Given that clock mechanisms in different species are all constructed in a similar fashion, we
hypothesize that the central oscillator of the circatidal clock is likely to also be composed of
interconnected transcription-translation positive and negative feedback loops, and that our candidate
circatidal genes may belong to the same protein families as the core circadian clock genes are involved in
the central oscillator.

Proposed mechanisms of the circatidal clock
There are three main hypotheses explaining the mechanism behind the generation of circatidal
biological rhythms. The first is the circalunidian clock hypothesis, which postulates that 12.4 hour
rhythms are the result of two coupled circalunidian (~24.8 hour) oscillators running in perfect antiphase to
each other. This hypothesis regards the circalunidian and circadian clocks as the same, because the free-
running periods of the two clocks are very similar (Palmer 2000). It was originally proposed as an
explanation for the significant difference in the observed rates of phase shift in the two daily activity
peaks in pliant-pendulum crab Helice crassa in free-running conditions. If each of the two daily peaks
were controlled by separate circalunidian clocks and these two circalunidian clocks became uncoupled as

10
a result of free-run, then that would allow for the two activity peaks to shift at different rates.
Furthermore, this hypothesis also provided an explanation for the splitting of one peak but not the other,
which Palmer (2000) also observed in H. crassa. This hypothesis is supported by studies on other
species, including clam Chione stutchburyi (Williams et al. 1993), crab Macrophthalmus hirtipes
(Williams 1998), and fish Lipophrys pholis (Northcott et al. 2009).
The second hypothesis is that of a co-opted circadian clock – that is, in intertidal organisms, the
mechanism of the circadian clock has been converted to run on a 12.4 hour rhythm (Wilcockson & Zhang
2008). The implication with this hypothesis is that circatidal rhythms are the dominant rhythms within
intertidal organisms, because the circadian machinery is entrained to the tidal cycle and no longer
generates 24 hour rhythms. When released from tidal conditions, then, it has the potential to free-run with
either a 12.4 or a 24 hour rhythm. This hypothesis is supported by studies done with animals such as
Washington clam Saxidomus pupuratus (Kim et al. 2003) and oyster C. gigas (Mat et al. 2014), where it
was observed that animals in constant, free-running conditions exhibited a combination of circadian and
circatidal activity rhythms, including: circatidal rhythms only, circadian rhythms only, and an interval of
circatidal rhythms followed by an interval of circadian rhythms.
The third hypothesis is that of a dedicated circatidal clock, which has its own machinery that
generates a 12.4 hour rhythm (Reid & Naylor 1989). However, interaction between the circatidal and
circadian clocks can result in modulation of the 12.4 peaks.
The shared basis of the circalunidian and co-opted circadian clock hypotheses is that the circatidal
clock and the circadian clock are one and the same. However, many recent studies have provided strong
evidence that the circatidal clock and the circadian clock are separate from one another. Prior research in
our lab has found that a much greater proportion of rhythmic transcripts in M. californianus subjected to
simulated tidal conditions or field tidal conditions have a circadian rhythmicity rather than a circatidal
rhythmicity (Connor & Gracey 2011). In the simulated tidal environment, 2,365 transcripts showed a
circadian rhythm, compared to 236 transcripts that showed a circatidal rhythm, a ten-fold difference. In
field intertidal animals, 501 transcripts were circadian, versus 109 transcripts that were circatidal.

11
Similarly, in field subtidal animals, 1,224 transcripts were circadian, versus 41 that were circatidal. Since
circadian rhythms are the dominant, but not sole, transcriptional rhythms in animals kept in tidal
conditions, this suggests that there are two time-keeping mechanisms in M. californianus. If a co-opted
circadian clock must entrain to tidal cues in order to run with circatidal rhythmicity, then animals
maintained in tidal conditions with continued exposure to tidal cues should show circadian rhythmicity in
most, if not all, of their rhythmic transcripts. Since this is not the case, it rules out the possibility of a co-
opted circadian clock mechanism, at least in M. californianus.

Evidence for dedicated circatidal clock mechanism
Experiments involving circadian clock disruption provide compelling evidence for separate
circadian and circatidal clock mechanisms. Although technically a terrestrial organism, the mangrove
cricket Apteronemobius asahinai forages on the mangrove forest floor at low tide and rests at high tide,
thus showing a circatidal activity pattern. A. asahinai also show a circadian modification of circatidal
rhythms, manifested as a suppression of activity during the day. Takekata et al. (2012) showed that RNA
interference (RNAi) via injection of double stranded per RNA (dsper) disrupted circadian activity but not
circatidal activity. As described above, per is a core circadian gene, and its expression is necessary for
regular circadian oscillations and activity. If the circadian clock is responsible for producing circatidal
rhythms, then RNAi of one of its core genes should result in cessation of circatidal rhythms. However,
circatidal activity rhythms continued with no difference before and after dsper injection.
Takekata et al. (2014) further confirmed the results of their RNAi experiments when they showed
that circatidal rhythms persist even in A. asahinai from which the optic lobe had been removed. In
crickets, the master circadian clock is localized in brain, specifically in the optic lobes. The optic lobes
were surgically removed either unilaterally or bilaterally from A. asahinai and activity rhythms were
recorded. There was a significant difference in the percentage of individuals exhibiting circadian
rhythmicity between the intact and optic lobe- removed groups, with no individuals from which the optic
lobes had been bilaterally removed showing circadian rhythmicity. The majority of individuals

12
maintained circatidal rhythmicity, and free-running circatidal rhythms were not statistically different
between individuals with optic lobes removed and individuals retaining their optic lobes. The combined
results from these A. asahinai studies certainly provide important evidence for a dedicated circatidal clock
separate from the circadian clock; however, A. asahinai are terrestrial organisms, and thus similar results
found in an intertidal organism would be more convincing still.
Zhang et al. (2013) found that circadian and circatidal phenotypes in intertidal crustacean
Eurydice pulchra could be separated through both environmental and molecular manipulation. The
light/dark cycle is a classic zeitgeber of the circadian clock. When E. pulchra were subjected to constant
bright light, their circadian chromatophore cycle was severely disrupted, while their tidal swimming
behavior remained unchanged. Furthermore, the E. pulchra homolog of tim (Eptim) lost its circadian
rhythm of gene expression. Tim is a core central oscillator gene in the Drosophila circadian clock
mechanism (Doherty & Kay 2010). RNAi against the E. pulchra homolog of per (Epper) resulted in
dampened chromatophore cycling as well as Eptim expression. However, circatidal swimming rhythm
and levels remained the same. Since the circalunidian and co- opted circadian clock hypotheses both
posit the circadian clock as the mechanism responsible for producing 12.4 hour rhythms, disruption of the
circadian clock should disrupt the 12.4 hour rhythms. These studies have shown that this is not the case,
thus providing strong support for the dedicated circatidal clock hypothesis. We hypothesize that intertidal
mollusks possess a separate, dedicated circatidal clock, which may interact with the circadian clock.

Methods
Collection and experimental set-up
California ribbed mussel Mytilus californianus were collected at low tide from Zuma Beach,
Malibu, CA, then immediately transported to our laboratory aquarium, located in a temperature-controlled
environmental chamber at USC set at 15°C, to be held until the start of entrainment. Prior to the time
course, mussels were placed into two purse cages that were suspended from the dock at the USC Wrigley
Marine Science Center, Catalina Island, CA, such that one cage was exposed to an intertidal tidal regime

13
and the other was exposed to a subtidal regime, and allowed to entrain for one month. The same was
performed simultaneously with one cage of Pacific oyster Crassostrea gigas, exposed to a subtidal regime
only.
After entrainment, mussels were transferred to free-running conditions of constant submergence,
constant flow, constant temperature (15°C), and constant low light. Animals were not fed during the
experiment. Oyster gill tissue was dissected out to make ex vivo tissue cultures, also kept in constant
conditions, in 0.2µm-filtered seawater with Gibco Antibiotic-Antimycotic (final concentrations: 100U/ml
penicillin, 100U/ml streptomycin, and 0.25µg/ml Fungizone). Four individuals from each treatment were
sampled every 2 hours for 48 hours, for a total of 24 time points. At each time point, gill tissue was
excised and flash frozen on dry ice, then stored at -80°C for later processing.
RNA was isolated from tissue samples using TRIzol (Invitrogen, Carlsbad, CA), according to
manufacturer instructions. Isolated RNA was further purified using glass-fiber spin columns (Qiagen,
Hilden, Germany), according to manufacturer instructions. For each time point of each treatment, RNA
was pooled with equal amounts of RNA from each individual.

Microarray analysis
RNA pools from both mussel treatments were polyA+ selected and amplified, then split into two
equal volumes. Each half was labelled with either Cy3 or Cy5 mono-reactive dyes (GE Healthcare Life
Sciences, Marlborough, MA). Labelled RNA was hybridized to an in-house manufactured cDNA
microarray containing 10,410 putatively non-redundant cDNA. Each labelled RNA sample was
hybridized to two or four arrays with dye reversal in an interwoven loop of hybridizations.
Hybridized arrays were imaged using an Agilent scanner (Agilent Technologies, Santa Clara,
CA) and spot intensities were quantified using the Agilent Feature Extraction software (version 9.5.1).
Spot median pixel intensities without background correction were collected, spatial intensity trends
removed, and individual channels normalized using joint lowess transformation. Relative expression of

14
each gene in each hybridization loop was estimated using MAANOVA version 0.98-7 for R (Wu et al.
2003). Gene expression data were centered by dividing the relative expression of each gene by the mean
expression of that gene across samples from the same treatment. Rhythmic genes were identified using
JTK_CYCLE, a nonparametric statistical algorithm for R that identifies rhythmic components in large
genomic datasets. It applies the Jonckheere-Terpstra-Kendall algorithm to alternative hypothesized group
orderings based on user-defined periods and phases, as well as cosine curves, in order to optimize period
and phase to minimize the p-value of Kendall’s tau correlation between an experimental time series and
the group orderings (Hughes et al. 2010, R Core Team 2014).

RNA-seq analysis
RNA pools from the oyster ex vivo and mussel subtidal treatments were used to create Illumina
sequencing libraries using an optimized in-house protocol. Briefly, RNA was polyA+ selected, reverse
transcribed, size fragmented, then PCR-enriched. Completed, barcoded libraries were submitted to the
USC Epigenome Facility for sequencing on the Illumina HiSeq 2000 platform. Reads were mapped to
the Crassostrea gigas genome (Zhang et al. 2012) and our own Mytilus californianus transcript database
using Bowtie (Langmead et al., 2009). Read counts were calculated using RSEM (Li & Dewey 2011).
Gene expression data were centered by dividing the read counts of each gene by the median of that gene
across samples from the same treatment. Rhythmic genes were identified using the JTK_CYCLE
algorithm for R (Hughes et al. 2010, R Core Team 2014).

Metadata analysis
Gene expression data were analyzed using a guided approach. Transcripts we have previously
found to exhibit tidal gene expression rhythms (FDR-adjusted p < 0.05, Connor & Gracey, 2011) were
probed in this dataset, and rhythmicity was assessed using JTK_CYCLE for R (Hughes et al. 2010, R
Core Team 2014).

15
Nearest neighbor cluster analysis was performed as well to identify co-expression modules,
groups of genes that consistently peak in expression together throughout the time course. These analyses
were seeded with the fos and C/EBPE expression profiles, and co-expression modules were identified as
comprised of the genes that had a Pearson correlation > 0.6 (Gracey et al. 2008). Overall candidate tidal
genes were identified as those that exhibited significant tidal rhythmicity (FDR-adjusted p < 0.05) in
previous experiments (Connor & Gracey 2011) and also exhibited tidal rhythmicity in all three of the
treatments of this experiment.
Venn diagrams and overlap lists were generated using Venny (version 2.0) (Oliveros 2015). The
significance of the overlap lists was assessed using the hypergeometric distribution in R (R Core Team
2014).

Functional annotation
Transcripts were assigned Gene Ontology (GO) terms using DAVID (Ashburner et al. 2000,
Huang et al. 2009a, b, Carbon et al. 2017). GO term enrichment was assessed using REVIGO with
medium (0.7) allowed similarity (Supek et al. 2011).

Results
Time course gene expression profiles of mussels and oysters subjected to three different
entrainment regimes were obtained and analyzed. In each treatment, circatidal gene expression rhythms
persisted during free-running.

Free-running mussels entrained to a field intertidal regime
During free-running, mussels entrained to a field intertidal regime continued to show circatidal
rhythms of gene expression, with 165 transcripts persisting in oscillations (Figure 2). Interestingly, when
examining the expression profiles of previous genes of interest from mussels in a simulated intertidal
environment (Connor & Gracey 2011) within this dataset, many of the same genes continued to show

16
circatidal rhythmicity during free-running, peaking at or around anticipated low tide (Figure 3).
Moreover, when cluster analysis was performed to find co-expression modules of genes that exhibit a
similar expression profile to two genes of particular interest (fos and C/EBPE), there was significant
overlap between the clusters found for mussels in a simulated intertidal environment and free-running
mussels entrained in a field intertidal environment (Figure 4).

Free-running mussels entrained to a field subtidal regime
Mussels entrained to a field subtidal regime continued to exhibit circatidal gene expression during
free-running, with 116 transcripts having a period of 10-14 hours (Figure 5). When comparing the
expression profiles of genes of interest from mussels in a simulated intertidal regime, all of the same
genes also exhibit circatidal rhythmicity in free-running mussels entrained to a field subtidal regime, with
transcripts peaking at or around anticipated low tide (Figure 6). When cluster analysis was performed to
find co-expression modules of genes that exhibit a similar expression profile to fos and C/EBPE, there
was significant overlap between the clusters found for mussels in a simulated intertidal environment and
free-running mussels entrained to a field subtidal environment (Figure 7).

Free-running ex vivo tissue culture of oysters entrained to a field subtidal regime
Free-running ex vivo tissue culture of oysters entrained to a field subtidal regime exhibited
circatidal gene expression rhythms (Figure 8). When comparing expression profiles of previously
identified genes of interest between whole mussels in simulated intertidal conditions and free-running ex
vivo tissue culture of oysters entrained in field subtidal conditions, it can be seen that some of the same
genes continue to exhibit circatidal rhythmicity, although they were not necessarily in the same phase as
each other (Figure 9). Hypergeometric analysis revealed significant overlap (p = 1.29e-14) between the
Fos and C/EBPE co-expression modules of both the mussels in a simulated intertidal environment and the
free-running ex vivo tissue culture of oysters entrained in a field subtidal environment (Figure 10).

17
Functional analysis of genes of interest
In order to determine their molecular function, Gene Ontology (GO) terms were assigned to the
genes in the fos and C/EBPE co-expression modules. The highest frequency GO terms for each treatment
are listed in Table 1. Many of the same GO terms, including DNA binding, RNA binding, and ATP
binding, appear in all four lists. Interestingly and additionally, when only the overlap lists of each of the
Venn diagrams were compared, the most represented GO terms included sequence-specific DNA binding,
transcription factor activity, protein dimerization, and protein binding (GO:0043565, GO:0003700,
GO:0046983, and GO:0005515, respectively).

Discussion
Evidence for an endogenous molecular oscillator
Using time course transcriptomic methods, we investigated whether or not intertidal bivalve
mollusks, specifically mussels and oysters, continue to exhibit circatidal gene expression rhythms in the
absence of environmental cues (free-running in constant conditions). We found that they did, indicating
that they contain an endogenous molecular mechanism that anticipates the changing of the tides, rather
than merely adjusting their gene expression in response to the changing of the tides (Figures 2, 5, 8). A
similar ability to maintain persistent gene expression rhythms in constant conditions is one of the
hallmarks of the circadian clock (Roenneberg et al. 2003).
Circatidal gene expression continued in free-running for at least three tidal cycles, peaking at or
around times of anticipated low tide (Figures 3, 6, 9). Presumably, the circatidal oscillator requires
resetting within three tidal cycles, which is consistent with the mechanism of the circadian clock, which
also requires resetting by the light/dark cycle after several days of free-running (Sharma &
Chandrashekaran 2005). In the case of the mussels entrained to the field intertidal regime, some of the
genes appear to continue peaking near anticipated low tide throughout the remainder of the time course
(Figure 3). This suggests that an intertidal regime, with emergence from the water, provides a stronger

18
entraining signal than does a subtidal one, which makes sense, given the more extreme differences in
environmental conditions between high and low tides experienced in an intertidal regime.

Circatidal genes in common between the three different treatments
A common theme in circadian research is that the genes with the most persistent circadian
expression rhythms are the ones most likely to be involved in or with the central oscillator. The common
circatidal genes identified in this study are listed in Table 2. Examining all of the Venn diagram overlap
lists revealed that five genes exhibit circatidal expression rhythms across all three treatments, as well as in
Connor and Gracey (2011): C/EBP, fos, jun, immediate early response gene 5 (IER5), and 3-
hydroxybutyrate dehydrogenase type 2 (BDH2). Notably, four of these five genes are all classified as
immediate early genes, which, as suggested by their name, comprise the first level of response to cellular
stimuli; the importance of immediate early genes will be discussed below.
Across the three mussel treatments, additional persistent tidal genes include carbonic anhydrase,
Y-box factor homolog, complement C1q-like protein 3 (C1QL3), protein quiver (QVR, aka SLEEPLESS
(SSS)), feline leukemia virus subgroup C receptor-related protein 2 (FLVCR2), Mdm2-binding protein
(MTBP), and protein BTG1. These proteins are involved in either homeostasis or cell cycle regulation,
which make sense, given the need for intertidal animals to adjust their internal physiologies in order to
survive in the contrasting conditions of low and high tide. Between the two treatments involving
entrainment to a field subtidal regime, circatidal expression of von Willebrand factor D and EGF domain-
containing protein (VWDE) persisted during free-running. Although not much is currently known about
VWDE as a whole, von Willebrand factor D (VWD) acts as a carrier protein in hemestasis, and the EGF
domain is predicted to be calcium-binding, suggesting a role in signal transduction (Wagner 1990).
Interestingly, epidermal growth factor (EGF) activates extracellular regulated kinase (ERK), which
regulates Fos expression (Nakakuki et al. 2010).

19
Immediate early genes
Four of the five genes that persist in circatidal expression rhythms across all of the treatments are
immediate early genes: IER5, fos, jun, and C/EBP. This is significant because immediate early genes are
the first response to stimuli such as immune challenges and stress. They are able to respond so quickly
because the required transcription factors and their corresponding protein kinases already exist in the cell
and merely require activation (Herschman 1991, Bahrami & Drabløs 2016). Because de novo synthesis
of other proteins (eg. transcription factors) is not required, immediate early gene expression is fast and
unaffected by translational inhibitors. Their expression is transient as well as rapid with expression
peaking within 30 to 60 minutes after stress stimulus (Cullinan et al. 1995). They are also generally
shorter and contain fewer exons than other genes, allowing for faster translation (Bahrami & Drabløs
2016). Other hallmarks of immediate early genes that contribute to their rapid expression include low
nucleosome occupancy (ie. more readily available DNA for transcription) and constitutive RNA
polymerase II recruitment to inactive genes (Fowler et al. 2011).
Immediate early genes tend to code for transcription factors, regulating the expression of target
genes (secondary response genes) in response to stimulus, which is reflected in their enrichment in the
GO terms “transcription factor” and “DNA binding” (Tullai et al. 2007). This is consistent with the most
common GO terms that we found across the three Venn diagram overlap lists.
Immediate early genes can be divided into two groups based on how quickly their induction takes
place after stimulus: the fast immediate early genes, which activate right away, and the slow or delayed
immediate early genes, which exhibit a slower induction profile, but still do so in the absence of de novo
protein synthesis (Bahrami & Drabløs 2016). Immediate early response gene 5 (IER5) falls into the latter
category (Williams et al. 1999). It is involved in the response to ionizing radiation and thermal stress,
both of which are major stressors faced by intertidal organisms at low tide. IER5 has been shown to be
induced by ionizing radiation, and may be involved with apoptosis caused by radiation, possibly through
disruption of cell cycle checkpoints (Ding et al. 2009). Furthermore, it participates in non-homologous
end joining-mediated DNA double strand break repair (Yu et al. 2017).

20
IER5 can also be induced by thermal stress. There are two heat shock elements (HSEs) in its
promoter, which are bound by heat-activated heat shock factor 1 (HSF1), the master regulator and
transcriptional activator of chaperone genes in response to thermal, heavy metal, and toxic stresses. IER5
overexpression promotes efficient recovery from heat stress by activating heat shock protein (HSP)
transcription and increasing protein chaperone activity, resulting in increased refolding of heat-denatured
proteins during recovery (Ishikawa & Sakurai 2015, Asano et al. 2016). IER5 can also recruit protein
phosphatase 2 (PP2A) to HSF1 in order for PP2A to dephosphorylate HSF1 at Ser121, Ser307, Ser314,
Thr323, and Thr367, which results in further HSF1 activation beyond the level of heat shock activation
(Asano et al. 2016). Because of the interaction between IER5 and HSF1, it appears that heat is an
important stress that that the circatidal oscillator accounts for.

bZIP transcription factors
Many basic leucine zipper (bZIP) transcription factors are under direct control of the central
circadian oscillator and have been implicated in accomplishing output functions of the circadian clock
(Harmer et al. 2001, Gachon 2007, Hirayama & Sassone-Corsi 2009). bZIP transcription factors are so
named because they contain a highly conserved basic region that binds DNA and a leucine-zipper motif
(Landschulz et al. 1988). bZIP transcription factors dimerize through a leucine-zipper motif to form
homodimers and heterodimers, and these dimers determine which DNA sites can be bound (Eferl &
Wagner 2003, Hess et al. 2004, Reinke et al. 2013). bZIP transcription factors are known to diversify
binding specificity by through cross-family dimerization. Fos-Jun proteins have been reported to interact
with over 50 different proteins, including C/EBP (Chinenov & Kerppola 2001).
Several of the candidate circatidal genes we have identified code for bZIP transcription factors.
These include fos, jun, cAMP responsive element binding protein-like 2 (CRBL2), and CCAAT/enhancer
binding protein epsilon (C/EBPE). If these genes are closely associated with the circatidal central
oscillator, then it is important to characterize their activity in order to begin to understand the
transcriptional regulation and regulatory output of the circatidal clock.

21
Fos and jun are both oncogenes that, along with activating transcription factors (ATF) and Maf
family proteins, comprise the activating protein 1 (AP-1) complex (Curran & Jr 1988, Shaulian 2010).
These proteins are able to homodimerize and heterodimerize in order to form the AP-1 complex, with the
exception that Fos is unable to homodimerize (Shaulian 2010). The specific combination of proteins in
the dimers determines the promoter or enhancer DNA sequences AP-1 can bind, and therefore control
which genes are regulated by AP-1 at any given time. For example, Jun-Fos heterodimers preferentially
bind the TPA-responsive element (TRE), while Jun-ATF heterodimers are more likely to bind the cAMP-
responsive element (CRE) (Chinenov & Kerppola 2001). Conversely, cAMP-responsive element binding
protein (CREB) is able to bind AP-1 sites in addition to CRE, although with lower affinity (Hurst & Jones
1987, Yamamoto et al. 1988). Members of the AP-1 complex are very unstable proteins with rapid
nuclear turnover (Vesely et al. 2009).
Other factors affecting AP-1 activity include transcription, post-translational modifications, and
interactions with other proteins (Eferl & Wagner 2003). Notably among post-translational modifications,
the cJun N-terminal kinase (JNK) phosphorylates and regulates Jun proteins, and its pathway is widely
recognized as being involved in responses to environmental stress, including UV radiation and osmotic
shock (Kyriakis & Avruch 2001, Li et al. 2002, Dhanasekaran & Reddy 2008). Furthermore, AP-1 itself
is highly sensitive to changes in redox state, and hypoxia can induce Jun transcription, lending further
support to AP-1 proteins as important players in the circatidal clock. AP-1 has been shown to cooperate
with hypoxia inducible factor (HIF-1) in response to low oxygen, although no physical interaction has
been observed and AP-1 is also able to function independently of HIF-1 in hypoxic cells (Laderoute
2005). The involvement of AP-1 in so many different stress responses underscores its likelihood of being
involved with the circatidal oscillator.
There may also be a circadian connection to AP-1. In the rat pineal gland, it has been shown that
some AP-1 proteins experience nocturnal regulation, with increased expression of cFos, junB, and junD
after the onset of darkness. This is interesting because the pineal gland is where melatonin production
occurs, suggesting a connection between AP-1 proteins and the sleep/wake cycle (Carter 1997). If AP-1

22
is involved in both the circadian and circatidal mechanisms, then it could represent the agent of cross-talk
between the two different clocks.
C/EBP transcription factors regulate many processes, including energy metabolism, immunity,
cell cycle, cellular proliferation, and response to environmental stimuli (Poli 1998, Tsukada et al. 2011).
There are six isoforms of C/EBP, denoted by Greek letters a through z in chronological order of
discovery, with the ratio of isoforms controlled by the mammalian target of rapamycin (mTOR) signaling
pathway (Calkhoven et al. 2000, Tsukada et al. 2011). This ratio is important because C/EBPZ (also
known as CHOP) generally represses the transactivation activity of the other isoforms (Chih et al. 2004).
Like other bZIP transcription factors, C/EBPs dimerize in order to bind DNA and regulate
downstream gene expression (Landschulz et al. 1989). They can dimerize with other C/EBP family
members, as well as CREB/ATF and Fos/Jun proteins (Newman & Keating 2003, Chih et al. 2004). In
addition to the basic DNA-binding region and leucine zipper dimerization motif found in all bZIP
proteins, all C/EBP transcription factors also contain a C-terminal extension of the leucine zipper.
Although the N-terminus is fairly divergent between the isoforms, the transactivation domains within the
N-terminus are highly conserved (Ramji & Foka 2002). The bZIP module does not vary among isoforms,
so the tail region allows for diversification of protein-protein interactions; as such, C/EBP homodimers
are able to bind a broader range of DNA sequences than CREB/ATF family proteins or AP-1 (Listman et
al. 2005, Tsukada et al. 2011). However, when C/EBPZ and ATF3 heterodimerize, the resulting
heterodimer is unable to bind ATF/CRE sites; C/EBPZ acts as a negative regulator of stress-induced
ATF, although it is itself stress-induced (Chen et al. 1996). Factors that induce C/EBPZ expression
include oxidative stress, UV light, DNA damage, and cellular stress (Ramji & Foka 2002), all of which
are stresses intertidal organisms can be exposed to during low tide.
C/EBP expression is regulated by several of the other candidate circatidal genes. This level of
interconnectedness is promising, as the central circadian oscillator is also composed of a set of genes and
proteins that regulate each other (Figure 1). The proximal promoter region of C/EBPA contains NF-kB

23
binding sites (Legraverend et al. 1993). CREB controls C/EBPB transcription by binding a CRE-like
sequence close to the TATA box (Niehof et al. 1997). The C/EBPZ promoter contains an AP-1 binding
site, and it has been shown that subjecting cells to oxidative stress results in Fos-Jun heterodimers binding
this site (Guyton et al. 1996). The C/EBPZ promoter also contains a composite C/EBP-ATF binding site,
which is bound by CREB-ATF complexes, as well as C/EBPB (Fawcett et al. 1999).
C/EBPB has also been found to interact with the p50 subunit of NF-kB (which was identified as a
candidate circatidal gene in Connor and Gracey (2011)) in solution, but it has not been definitely shown
that this interaction also occurs when the complex is bound to DNA. However, it has been shown that
both proteins are necessary for inflammation immune responses (LeClair et al. 1992). Importantly,
related to the role of C/EBPA in metabolism, C/EBPA has been shown to oscillate with a circadian
rhythm in mouse liver cells, with C/EBPA under transcriptional control of CLOCK (Kawasaki et al.
2013). Additionally, C/EBPB appears to be in control of circadian autophagy rhythms in mouse liver
cells as well, maintaining nutrient homeostasis (Ma et al. 2011).
Although it did not significantly persist in circatidal rhythmicity across all of the treatments in
this experiment, CRBL2 is still an important candidate tidal gene, because it did qualitatively persist
across all of the treatments (Figures 3, 6, 9), as well as exhibit robust circatidal expression rhythms in
Connor and Gracey (2011). CREB protein family members contain a centrally-located kinase-inducible
domain (KID), which contains phosphorylation sites for cAMP-dependent protein kinase (PKA) and
casein kinases 1 and 2 (CK1 and CK2, respectively). The activation domains, Q1 and Q2, are located on
either side of the KID, and the bZIP module is located at the C-terminus (Brindle et al. 1993). The KID is
highly conserved across species, suggesting that CREB activation mechanisms elucidated in mammals
will likely apply in other species as well. CREB-induced transcription requires phosphorylation of CREB
at Ser133, but activity can be modulated by phosphorylation at additional residues or through protein-
protein interactions (Shaywitz & Greenberg 1999).
As suggested by its name, CREB can become activated when there is an accumulation of cAMP,
an important intracellular second messenger. The cAMP stimulates PKA to phosphorylate CREB.

24
Phosphorylation of CREB recruits its transcriptional coactivator, CREB-binding protein (CBP), which in
turn recruits and stabilizes the RNA polymerase II complex on the DNA strand (Mayr & Montminy
2001). The magnitude of CREB activation is proportional to the intensity and duration of the cAMP
stimulus (Hagiwara et al. 1993). In addition to cAMP and calcium, other stress-related stimuli that can
result in CREB phosphorylation include survival signals, hypoxia, and UV light (Deak et al. 1998, Du &
Montminy 1998, Beitner-Johnson et al. 2001). Other processes CREB is involved in include cellular
proliferation and differentiation, hormonal control of metabolism, and memory (Shaywitz & Greenberg
1999).
Like other bZIP transcription factors, CREB dimerizes in order to regulate target genes, but rather
than dimerizing first and then binding the DNA, CREB proteins bind to DNA as monomers, then
dimerize over the CRE (Brindle et al. 1993). CREB is able to heterodimerize with ATF-1, but compared
to the CREB homodimer, the CREB/ATF-1 heterodimer has a shorter half-life when bound to the CRE
(Kobayashi & Kawakami 1995). Besides CREB, other genes that have a CRE-binding motif in or near
their promoter regions include aryl hydrocarbon receptor, C/EBPB, Per1, Jun, Fos, and Cyclin A (Mayr &
Montminy 2001). Because it has a CRE in its promoter region, Fos expression can be induced by cAMP
or Ca
2+
signaling, mediated by CREB (Sassone-Corsi et al. 1988).
Interestingly, CREB has been shown to exhibit circadian oscillations in the SCN (Obrietan et al.
1999). It has also been shown that CREB-binding protein is involved in the transcriptional regulation of
the CLOCK/CYCLE heterodimer in Drosophila (Lim et al. 2007). Importantly, part of the light signal
transduction cascade of the circadian clock includes the phosphorylation of CREB, which induces the
protein’s transactivational activity. Cell membrane depolarization and increased intracellular Ca
2+

concentration stimulates the calmodulin-dependent kinases that phosphorylate CREB (Sheng et al. 1991).
Once CREB is phosphorylated, it is able to bind CRE sites, leading to the downstream result of a light-
induced phase shift. Hamsters that were briefly exposed to light at night began to accumulate
phosphorylated CREB in their suprachiasmatic nucleus (SCN) within five minutes of exposure (Ginty et
al. 1993). This represents a key initial step in coupling extracellular light stimulus to the setting of the

25
circadian clock (Ding et al. 1997). If CREB is involved with the circatidal oscillator, then this pathway
could represent a point of interaction between the two clock mechanisms.

3-hydroxybutyrate dehydrogenase type 2
3-hydroxybutyrate dehydrogenase type 2 (BDH2) is the one circatidal gene out of the five genes
found across all of our treatments that is not an immediate early gene. Rather, it is a member of the short-
chain dehydrogenase/reductase (SDR) superfamily. SDR proteins contain a conserved N-terminus that
binds an NAD(H) and NADP(H) cofactor, and a variable C-terminus that binds the substrate. The active
site at the central fold of an SDR protein consists of a parallel seven-stranded b-sheet flanked by a-
helices, and it is this region that differs between the classical SDR proteins and the extended SDR
proteins. Classical SDR proteins have a much smaller a-helical substrate binding region, whereas
extended SDR proteins have a much larger substrate binding region containing a two-stranded b-sheet
and three a-helices (Bray et al. 2009).
BDH2 is a non-membrane bound, classical SDR that prefers an NAD(H) cofactor (Bray et al. 2009). It
has been shown to play a role in iron homeostasis by catalyzing the rate-limiting step in the production of
mammalian siderophores, small iron-binding molecules that facilitate iron trafficking; this role is
supported by the presence of an iron-responsive element in the 3’ untranslated region of the BDH2 gene
(Liu et al. 2012). Binding and transporting of free intracellular iron reduces the levels of reactive oxygen
species and provides mitochondria with necessary iron (Devireddy et al. 2010), which is important
because of the high potential for oxidative stress during low tide. BDH2 is also important in fat
metabolism and ketone body utilization in the TCA cycle (Guo et al. 2006).

Future studies
Data from these experiments will provide an important groundwork for the study of the
mechanisms of the circatidal clock. What is currently known about the candidate circatidal genes we

26
have identified – in particular their tendency to be involved in stress responses, as well as their roles as
transcriptional regulators – provide compelling evidence for their involvement in the central circatidal
oscillator. As a follow-up to this study, we have begun to develop custom antibodies against some of the
candidate genes, in order to probe the activity of the proteins expressed by these genes. Due to the
interconnectedness of the genes and proteins involved in central oscillators (similar to that in the circadian
system), identification and characterization of a few of the key players of the circatidal clock should lead
to the identification and characterization of additional components through experiments involving
techniques such as time course co-immunoprecipitations and RNA interference (RNAi), ultimately
leading to the construction of likely models of the molluscan circatidal oscillator.

References
Arjona A, Silver AC, Walker WE, Fikrig E (2012) Immunity’s fourth dimension: approaching the
circadian-immune connection. Trends Immunol 33:607–12
Asano Y, Kawase T, Okabe A, Tsutsumi S, Ichikawa H, Tatebe S, Kitabayashi I, Tashiro F, Namiki H,
Kondo T, Semba K, Aburatani H, Taya Y, Nakagama H, Ohki R (2016) IER5 generates a novel
hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis. Sci Rep 6:1–19
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS,
Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE,
Ringwald M, Rubin GM, Sherlock G (2000) Gene ontology: tool for the unification of biology. The
Gene Ontology Consortium. Nat Genet 25:25–9
Bahrami S, Drabløs F (2016) Gene regulation in the immediate-early response process. Adv Biol Regul
62:37–49
Beitner-Johnson D, Rust RT, Hsieh TC, Millhorn DE (2001) Hypoxia activates Akt and induces
phosphorylation of GSK-3 in PC12 cells. Cell Signal 13:23–27
Bray JE, Marsden BD, Oppermann U (2009) The human short-chain dehydrogenase/reductase (SDR)
superfamily: A bioinformatics summary. Chem Biol Interact 178:99–109

27
Brindle P, Linke S, Montminy M (1993) Protein-kinase-A-dependent activator in transcription factor
CREB reveals new role for CREM repressors. Nature 364:821–824
Calkhoven CF, Müller C, Leutz A (2000) Translational control of C/EBPalpha and C/EBPbeta isoform
expression. Genes Dev 14:1920–32
Carbon S, Dietze H, Lewis SE, Mungall CJ, Munoz-Torres MC, Basu S, Chisholm RL, Dodson RJ, Fey
P, Thomas PD, Mi H, Muruganujan A, Huang X, Poudel S, Hu JC, Aleksander SA, McIntosh BK,
Renfro DP, Siegele DA, Antonazzo G, Attrill H, Brown NH, Marygold SJ, Mc-Quilton P, Ponting
L, Millburn GH, Rey AJ, Stefancsik R, Tweedie S, Falls K, Schroeder AJ, Courtot M, Osumi-
Sutherland D, Parkinson H, Roncaglia P, Lovering RC, Foulger RE, Huntley RP, Denny P,
Campbell NH, Kramarz B, Patel S, Buxton JL, Umrao Z, Deng AT, Alrohaif H, Mitchell K,
Ratnaraj F, Omer W, Rodríguez-López M, C. Chibucos M, Giglio M, Nadendla S, Duesbury MJ,
Koch M, Meldal BHM, Melidoni A, Porras P, Orchard S, Shrivastava A, Chang HY, Finn RD,
Fraser M, Mitchell AL, Nuka G, Potter S, Rawlings ND, Richardson L, Sangrador-Vegas A, Young
SY, Blake JA, Christie KR, Dolan ME, Drabkin HJ, Hill DP, Ni L, Sitnikov D, Harris MA, Hayles
J, Oliver SG, Rutherford K, Wood V, Bahler J, Lock A, Pons J De, Dwinell M, Shimoyama M,
Laulederkind S, Hayman GT, Tutaj M, Wang SJ, D’Eustachio P, Matthews L, Balhoff JP,
Balakrishnan R, Binkley G, Cherry JM, Costanzo MC, Engel SR, Miyasato SR, Nash RS, Simison
M, Skrzypek MS, Weng S, Wong ED, Feuermann M, Gaudet P, Berardini TZ, Li D, Muller B,
Reiser L, Huala E, Argasinska J, Arighi C, Auchincloss A, Axelsen K, Argoud-Puy G, Bateman A,
Bely B, Blatter MC, Bonilla C, Bougueleret L, Boutet E, Breuza L, Bridge A, Britto R, Hye- A-Bye
H, Casals C, Cibrian-Uhalte E, Coudert E, Cusin I, Duek-Roggli P, Estreicher A, Famiglietti L,
Gane P, Garmiri P, Georghiou G, Gos A, Gruaz-Gumowski N, Hatton-Ellis E, Hinz U, Holmes A,
Hulo C, Jungo F, Keller G, Laiho K, Lemercier P, Lieberherr D, Mac- Dougall A, Magrane M,
Martin MJ, Masson P, Natale DA, O’Donovan C, Pedruzzi I, Pichler K, Poggioli D, Poux S, Rivoire
C, Roechert B, Sawford T, Schneider M, Speretta E, Shypitsyna A, Stutz A, Sundaram S, Tognolli
M, Wu C, Xenarios I, Yeh LS, Chan J, Gao S, Howe K, Kishore R, Lee R, Li Y, Lomax J, Muller

28
HM, Raciti D, Auken K Van, Berriman M, Stein, Paul Kersey L, W. Sternberg P, Howe D,
Westerfield M (2017) Expansion of the gene ontology knowledgebase and resources: The gene
ontology consortium. Nucleic Acids Res 45:D331–D338
Carter DA (1997) Rhythms of cellular immediate-early gene expression: More than just an early
response. Exp Physiol 82:237–244
Chen BP, Wolfgang CD, Hai T (1996) Analysis of ATF3, a transcription factor induced by physiological
stresses and modulated by gadd153/Chop10. Mol Cell Biol 16:1157–1168
Chih DY, Park DJ, Gross M, Idos G, Vuong PT, Hirama T, Chumakov AM, Said J, Koeffler HP (2004)
Protein partners of C/EBPε. Exp Hematol 32:1173–1181
Chinenov Y, Kerppola TK (2001) Close encounters of many kinds: Fos-Jun interactions that mediate
transcription regulatory specificity. Oncogene 20:2438–2452
Connor KM, Gracey AY (2011) Circadian cycles are the dominant transcriptional rhythm in the intertidal
mussel Mytilus californianus. Proc Natl Acad Sci U S A 108:16110–5
Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ (1995) Pattern and time course of immediate
early gene expression in rat brain following acute stress. Neuroscience 64:477–505
Curran T, Jr BF (1988) Fos and Jun: the AP-1 connection. Cell 55:395–397
Deak M, Clifton AD, Lucocq JM, Alessi DR (1998) Mitogen- and stress-activated protein kinase-1
(MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB.
EMBO J 17:4426–4441
Devireddy LR, Hart DO, Goetz DH, Green MR (2010) A mammalian siderophore synthesized by an
enzyme with a bacterial homolog involved in enterobactin production. Cell 141:1006–1017
Dhanasekaran DN, Reddy EP (2008) JNK signaling in apoptosis. Oncogene 27:6245–6251
Ding JM, Faiman LE, Hurst WJ, Kuriashkina LR, Gillette MU (1997) Resetting the biological clock:
mediation of nocturnal CREB phosphorylation via light, glutamate, and nitric oxide. J Neurosci
17:667–675
Ding K-K, Shang Z-F, Hao C, Xu Q-Z, Shen J-J, Yang C-J, Xie Y-H, Qiao C, Wang Y, Xu L-L, Zhou P-

29
K (2009) Induced expression of the IER5 gene by γ-ray irradiation and its involvement in cell cycle
checkpoint control and survival. Radiat Environ Biophys 48:205–213
Dodd A, Salathia N, Hall A, Kévei E, Tóth R (2005) Plant circadian clocks increase photosynthesis,
growth, survival, and competitive advantage. Science (80- ) 309:630–633
Doherty CJ, Kay SA (2010) Circadian Control of Global Gene Expression Patterns. Annu Rev Genet
44:419–444
Du K, Montminy M (1998) CREB Is a Regulatory Target for the Protein Kinase Akt/PKB. J Biol Chem
273:32377–32379
Eferl R, Wagner EF (2003) AP-1: a double-edged sword in tumorigenesis. Nat Rev Cancer 3:859–868
Fawcett TW, Martindale JL, Guyton KZ, Hai T, Holbrook NJ (1999) Complexes containing activating
transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the
CCAAT/enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression
during the stress response. Biochem J 339 ( Pt 1:135–41
Fowler T, Sen R, Roy AL (2011) Regulation of primary response genes. Mol Cell 44:348–360
Gachon F (2007) Physiological function of PARbZip circadian clock-controlled transcription factors. Ann
Med 39:562–71
Ginty D, Kornhauser J, Thompson M, Bading H, Mayo K, Takahashi J, Greenberg M (1993) Regulation
of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science
(80- ) 260:238–241
Gracey AY, Chaney ML, Boomhower JP, Tyburczy WR, Connor K, Somero GN (2008) Rhythms of
Gene Expression in a Fluctuating Intertidal Environment. Curr Biol 18:1501–1507
Green CB, Takahashi JS, Bass J (2008) The meter of metabolism. Cell 134:728–42
Guo K, Lukacik P, Papagrigoriou E, Meier M, Lee WH, Adamski J, Oppermann U (2006)
Characterization of Human DHRS6, an Orphan Short Chain Dehydrogenase/Reductase Enzyme. J
Biol Chem 281:10291–10297
Guyton KZ, Xu Q, Holbrook NJ (1996) Induction of the mammalian stress response gene GADD153 by

30
oxidative stress: role of AP-1 element. Biochem J 314 ( Pt 2:547–54
Hagiwara M, Brindle P, Harootunian A, Armstrong R, Rivier J, Vale W, Tsien R, Montminy MR (1993)
Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is
rate limited by nuclear entry of protein kinase A. Mol Cell Biol 13:4852–4859
Harmer S, Panda S, Kay S (2001) Molecular bases of circadian rhythms. Annu Rev Cell Dev Biol
17:215–253
Hastings MH, Reddy AB, Maywood ES (2003) A clockwork web: circadian timing in brain and
periphery, in health and disease. Nat Rev Neurosci 4:649–61
Herschman HR (1991) Primary Response Genes Induced by Growth Factors and Tumor Promoters. Annu
Rev Biochem 60:281–319
Hess J, Angel P, Schorpp-Kistner M (2004) AP-1 subunits: quarrel and harmony among siblings. J Cell
Sci 117:5965–73
Hirayama J, Sahar S, Grimaldi B, Tamaru T, Takamatsu K, Nakahata Y, Sassone-Corsi P (2007)
CLOCK-mediated acetylation of BMAL1 controls circadian function. Nature 450:1086–90
Hirayama J, Sassone-Corsi P (2009) Transcription Control and the Circadian Clock. In: Encyclopedia of
Neuroscience.p 1071–1080
Huang DW, Sherman BT, Lempicki RA (2009a) Systematic and integrative analysis of large gene lists
using DAVID bioinformatics resources. Nat Protoc 4:44–57
Huang DW, Sherman BT, Lempicki RA (2009b) Bioinformatics enrichment tools: Paths toward the
comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13
Hughes ME, DiTacchio L, Hayes KR, Vollmers C, Pulivarthy S, Baggs JE, Panda S, Hogenesch JB
(2009) Harmonics of circadian gene transcription in mammals. PLoS Genet 5:e1000442
Hughes ME, Hogenesch JB, Kornacker K (2010) JTK_CYCLE: an efficient nonparametric algorithm for
detecting rhythmic components in genome-scale data sets. J Biol Rhythms 25:372–80
Hurst HC, Jones NC (1987) Identification of factors that interact with the E1A-inducible adenovirus E3
promoter. Genes Dev 1:1132–1146

31
Ishikawa Y, Sakurai H (2015) Heat-induced expression of the immediate-early gene IER5 and its
involvement in the proliferation of heat-shocked cells. FEBS J 282:332–340
Kawasaki H, Doi R, Ito K, Shimoda M, Ishida N (2013) The Circadian Binding of CLOCK Protein to the
Promoter of C/ebpα Gene in Mouse Cells. PLoS One 8:3–8
Kim W, Huh H, Je J, Han K (2003) Evidence of two-clock control of endogenous rhythm in the
Washington clam, Saxidomus purpuratus. Mar Biol 142:305–309
Kobayashi M, Kawakami K (1995) ATF-1CREB heterodimer is involved in constitutive expression of the
housekeeping Na,K-ATPase alpha 1 subunit gene. Nucleic Acids Res 23:2848–55
Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin X, Maywood ES, Hastings MH, Reppert
SM (1999) mCRY1 and mCRY2 are essential components of the negative limb of the circadian
clock feedback loop. Cell 98:193–205
Kyriakis JM, Avruch J (2001) Mammalian Mitogen-Activated Protein Kinase Signal Transduction
Pathways Activated by Stress and Inflammation. Physiol Rev 81:807–869
Laderoute KR (2005) The interaction between HIF-1 and AP-1 transcription factors in response to low
oxygen. Semin Cell Dev Biol 16:502–513
Lambert G, Chew J, Rust MJ (2016) Costs of Clock-Environment Misalignment in Individual
Cyanobacterial Cells. Biophys J 111:883–891
Landschulz WH, Johnson PF, Mcknight SL (1988) The Leucine Zipper: A Hypothetical Structure
Common to a New Class of DNA Binding Proteins. Science (80- ) 240:1759–1764
Landschulz W, Johnson P, McKnight S (1989) The DNA binding domain of the rat liver nuclear protein
C/EBP is bipartite. Science (80- ) 243:1681–1688
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short
DNA sequences to the human genome. Genome Biol 10:R25
Langmesser S, Tallone T, Bordon A, Rusconi S, Albrecht U (2008) Interaction of circadian clock proteins
PER2 and CRY with BMAL1 and CLOCK. BMC Mol Biol 9:41
LeClair KP, Blanar MA, Sharp PA (1992) The p50 subunit of NF-kappa B associates with the NF-IL6

32
transcription factor. Proc Natl Acad Sci U S A 89:8145–9
Legraverend C, Antonson P, Flodby P, Xanthopoulos G (1993) High level activity of the mouse
CCAAT/enhancer binding protein (C/EBPalpha) gene promoter involves autoregulation and several
ubiquitous transcription factors. Nucleic Acids Res 21:1735–1742
Li T, Dai W, Lu L (2002) Ultraviolet-induced junD activation and apoptosis in myeloblastic leukemia
ML-1 cells. J Biol Chem 277:32668–76
Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a
reference genome. BMC Bioinformatics 12:323
Lim C, Lee J, Choi C, Kim J, Doh E, Choe J (2007) Functional role of CREB-binding protein in the
circadian clock system of Drosophila melanogaster. Mol Cell Biol 27:4876–4890
Listman JA, Wara-Aswapati N, Race JAE, Blystone LW, Walker-Kopp N, Yang Z, Auron PE (2005)
Conserved ETS domain arginines mediate DNA binding, nuclear localization, and a novel mode of
bZIP interaction. J Biol Chem 280:41421–41428
Liu Z, Lanford R, Mueller S, Gerhard GS, Luscieti S, Sanchez M, Devireddy L (2012) Siderophore-
mediated iron trafficking in humans is regulated by iron. J Mol Med 90:1209–1221
Ma D, Panda S, Lin JD (2011) Temporal orchestration of circadian autophagy rhythm by C/EBPβ.
EMBO J 30:4642–4651
Mat AM, Massabuau J-C, Ciret P, Tran D (2014) Looking for the clock mechanism responsible for
circatidal behavior in the oyster Crassostrea gigas. Mar Biol 161:89–9
Mayr B, Montminy M (2001) Transcriptional regulation by the phosphorylation-dependent factor CREB.
Nat Rev Mol Cell Biol 2:599–609
Nagel DH, Kay SA (2012) Complexity in the wiring and regulation of plant circadian networks. Curr Biol
22:R648-57
Nakakuki T, Birtwistle MR, Saeki Y, Yumoto N, Ide K, Nagashima T, Brusch L, Ogunnaike BA, Okada-
Hatakeyama M, Kholodenko BN (2010) Ligand-specific c-fos expression emerges from the
spatiotemporal control of ErbB network dynamics. Cell 141:884–896

33
Newman JRS, Keating AE (2003) Comprehensive identification of human bZIP interactions with coiled-
coil arrays. Science (80- ) 300:2097–2101
Niehof M, Manns MP, Trautwein C (1997) CREB controls LAP/C/EBP beta transcription. Mol Cell Biol
17:3600–3613
Northcott SJ, Gibson RN, Morgan E (2009) The persistence and modulation of endogenous circatidal
rhythmicity in Lipophrys pholis (Teleostei). J Mar Biol Assoc United Kingdom 70:815
Obrietan K, Impey S, Smith D, Athos J, Storm DR (1999) Circadian Regulation of cAMP Response
Element-mediated Gene Expression in the Suprachiasmatic Nuclei. J Biol Chem 274:17748–17756
Oliveros J (2015) Venny. An interactive tool for comparing lists with Venn’s diagrams.
:http://bioinfogp.cnb.csic.es/tools/venny/index.htm
Ouyang Y, Andersson CR, Kondo T, Golden SS, Johnson CH (1998) Resonating circadian clocks
enhance fitness in cyanobacteria. Proc Natl Acad Sci U S A 95:8660–4
Palmer JD (2000) The clocks controlling the tide-associated rhythms of intertidal animals. Bioessays
22:32–7
Poli V (1998) The role of C/EBP isoforms in the control of inflammatory and native immunity functions.
J Biol Chem 273:29279–29282
R Core Team (2014) R: A Language and Environment for Statistical Computing.
Ramji DP, Foka P (2002) CCAAT/enhancer-binding proteins: structure, function and regulation.
Biochem J 365:561–75
Reid D, Naylor E (1989) Are there separate circatidal and circadian clocks in the shore crab Carcinus
maenas. Mar Ecol Prog Ser 52:1–6
Reinke AW, Baek J, Ashenberg O, Keating AE (2013) Networks of bZIP protein-protein interactions
diversified over a billion years of evolution. Science 340:730–4
Roenneberg T, Daan S, Merrow M (2003) The Art of Entrainment. J Biol Rhythms 18:183–194
Sassone-Corsi P, Visvader J, Ferland L, Mellon PL, Verma IM (1988) Induction of proto-oncogene fos
transcription through the adenylate cyclase pathway: characterization of a cAMP-responsive

34
element. Genes Dev 2:1529–1538
Sharma VK, Chandrashekaran MK (2005) Zeitgebers (time cues) for biological clocks. Curr Sci 89:1136–
1146
Shaulian E (2010) AP-1 - The Jun proteins: Oncogenes or tumor suppressors in disguise? Cell Signal
22:894–899
Shaywitz AJ, Greenberg ME (1999) CREB: A Stimulus-Induced Transcription Factor Activated by A
Diverse Array of Extracellular Signals. Annu Rev Biochem 68:821–861
Sheng M, Thompson MA, Greenberg ME (1991) CREB: a Ca(2+)-regulated transcription factor
phosphorylated by calmodulin-dependent kinases. Science 252:1427–30
Supek F, Bošnjak M, Škunca N, Šmuc T (2011) Revigo summarizes and visualizes long lists of gene
ontology terms. PLoS One 6
Takahashi JS, Hong H-K, Ko CH, McDearmon EL (2008) The genetics of mammalian circadian order
and disorder: implications for physiology and disease. Nat Rev Genet 9:764–775
Takekata H, Matsuura Y, Goto SG, Satoh A, Numata H (2012) RNAi of the circadian clock gene period
disrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket. Biol Lett 8:488–
91
Takekata H, Numata H, Shiga S (2014) The Circatidal Rhythm Persists without the Optic Lobe in the
Mangrove Cricket Apteronemobius asahinai. J Biol Rhythms 29:28–37
Tsukada J, Yoshida Y, Kominato Y, Auron PE (2011) The CCAAT/enhancer (C/EBP) family of basic-
leucine zipper (bZIP) transcription factors is a multifaceted highly-regulated system for gene
regulation. Cytokine 54:6–19
Tullai JW, Schaffer ME, Mullenbrock S, Sholder G, Kasif S, Cooper GM (2007) Immediate-early and
delayed primary response genes are distinct in function and genomic architecture. J Biol Chem
282:23981–23995
Vesely PW, Staber PB, Hoefler G, Kenner L (2009) Translational regulation mechanisms of AP-1
proteins. Mutat Res - Rev Mutat Res 682:7–12

35
Wagner DD (1990) Cell Biology of von Willebrand Factor. Annu Rev Cell Biol 6:217–242
Wilcockson D, Zhang L (2008) Circatidal clocks. Curr Biol 18:753–755
Williams BG (1998) The lack of circadian timing in two intertidal invertebrates and its significance in the
circatidal/circalunidian debate. Chronobiol Int 15:205–218
Williams M, Lyu MS, Yang YL, Lin Edward P, Dunbrack R, Birren B, Cunningham J, Hunter K (1999)
Ier5, a novel member of the slow-kinetics immediate-early genes. Genomics 55:327–334
Williams BG, Palmer JD, Hutchinson DN (1993) Comparative studies of tidal rhythms XIII. Is a clam
clock similar to those of other intertidal animals? Mar Behav Physiol 24:1–14
Woelfle M, Ouyang Y, Phanvijhitsiri K, Johnson C (2004) The Adaptive Value of Circadian Clocks: An
Experimental Assessment in Cyanobacteria. Curr Biol 14:1481–1486
Wu H, Kerr MK, Cui X, Churchill GA (2003) MAANOVA: A Software Package for the Analysis of
Spotted cDNA Microarray Experiments. In: Parmigiani G, Garrett ES, Irizarry RA, Zeger SL (eds)
The Analysis of Gene Expression Data: Methods and Software. Springer New York, New York,
NY, p 313–341
Yamamoto KK, Gonzalez GA, Biggs WH, Montminy MR (1988) Phosphorylation-induced binding and
transcriptional efficacy of nuclear factor CREB. Nature 334:494–498
Yan J, Wang H, Liu Y, Shao C (2008) Analysis of gene regulatory networks in the mammalian circadian
rhythm. PLoS Comput Biol 4:e1000193
Yerushalmi S, Green RM (2009) Evidence for the adaptive significance of circadian rhythms. Ecol Lett
12:970–81
Yu XP, Wu YM, Liu Y, Tian M, Wang JD, Ding KK, Ma T, Zhou PK (2017) IER5 is involved in DNA
double-strand breaks repair in association with PAPR1 in hela cells. Int J Med Sci 14:1292–1300
Zhang G, Fang X, Guo X, Li L, Luo R, Xu F, Yang P, Zhang L, Wang X, Qi H, Xiong Z, Que H, Xie Y,
Holland PWH, Paps J, Zhu Y, Wu F, Chen Y, Wang J, Peng C, Meng J, Yang L, Liu J, Wen B,
Zhang N, Huang Z, Zhu Q, Feng Y, Mount A, Hedgecock D, Xu Z, Liu Y, Domazet-Lošo T, Du Y,
Sun X, Zhang S, Liu B, Cheng P, Jiang X, Li J, Fan D, Wang W, Fu W, Wang T, Wang B, Zhang J,

36
Peng Z, Li Y, Li N, Wang J, Chen M, He Y, Tan F, Song X, Zheng Q, Huang R, Yang H, Du X,
Chen L, Yang M, Gaffney PM, Wang S, Luo L, She Z, Ming Y, Huang W, Zhang S, Huang B,
Zhang Y, Qu T, Ni P, Miao G, Wang J, Wang Q, Steinberg CEW, Wang H, Li N, Qian L, Zhang G,
Li Y, Yang H, Liu X, Wang J, Yin Y, Wang J (2012) The oyster genome reveals stress adaptation
and complexity of shell formation. Nature 490:49–54
Zhang L, Hastings MH, Green EW, Tauber E, Sladek M, Webster SG, Kyriacou CP, Wilcockson DC
(2013) Dissociation of Circadian and Circatidal Timekeeping in the Marine Crustacean Eurydice
pulchra. Curr Biol 40:1–11

37

Figure 1. The central oscillators of Mus musculus, Drosophila melanogaster, and
Arabidopsis thaliana all consist of similar transcription-translation positive and
negative feedback loops. From Doherty and Kay (2010)

38

Figure 2. Heat map showing circatidal gene expression in mussels entrained to a field
intertidal regime. 165 genes exhibited circatidal expression rhythms (period = 10 -
14hr, p < 0.05) during free-running. The corresponding anticipated tide and light/dark
cycle are included for reference.

row min row max
MI1
MI2
MI3
MI4
MI5
MI6
MI7
MI8
MI9
MI10
MI11
MI12
MI13
MI14
MI15
MI16
MI17
MI18
MI19
MI20
MI21
MI22
MI23
MI24
Clone.ID
Clone.ID UID name Acc.. geneID ADJ.P PER LAG AMP
row min row max
MI1
MI2
MI3
MI4
MI5
MI6
MI7
MI8
MI9
MI10
MI11
MI12
MI13
MI14
MI15
MI16
MI17
MI18
MI19
MI20
MI21
MI22
MI23
MI24
Clone.ID
Clone.ID UID name Acc.. geneID ADJ.P PER LAG AMP
Time
-1
0
1
2
3
4
5
Light/dark
Tide

39

Figure 3. Comparison of expression patterns in tidal genes of interest, as exhibited in mussels a)
experiencing a simulated intertidal regime and b) free-running after entrainment to a field intertidal
regime. In both treatments, gene expression peaks at or around low tide.
-1
0
1
2
3
4
5
Tide
Light/dark
-1.5
-1
-0.5
0
0.5
1
1.5
Fos-related antigen 2 CCAAT/enhancer-binding protein epsilon
CCAAT/enhancer-binding protein epsilon Transmembrane protein 47
Protein quiver Carbonic anhydrase 2
CCAAT/enhancer-binding protein epsilon Probable isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial
Y-box factor homolog Angiopoietin-2
Complement C1q-like protein 3
a) b)
Time
Relative expression (log
2
)

40

Figure 4. Venn diagram of genes with expression profiles highly correlated (Pearson > 0.6) with fos
and C/EBPE in mussels experiencing a simulated intertidal regime and mussels free-running after
entrainment to a field intertidal regime. The overlap was found to be significant (p = 0) using the
hypergeometric distribution.
Simulated intertidal
Free-running after
field intertidal
23 22 631

41

row min row max
X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
X12
X13
X14
X15
X16
X17
X18
X19
X20
X21
X22
X23
X24
Probeset
Probeset Name BH.Q ADJ.P PER LAG AMP
Time
row min row max
X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
X12
X13
X14
X15
X16
X17
X18
X19
X20
X21
X22
X23
X24
Probeset
Probeset Name BH.Q ADJ.P PER LAG AMP
row min row max
-1
0
1
2
3
4
5
Light/dark
Tide
Figure 5. Heat map showing circatidal gene expression in mussels entrained to a field
subtidal regime. 116 genes exhibited circatidal expression rhythms (period = 10 -
14hr, p < 0.05) during free-running. The corresponding anticipated tide and light/dark
cycle are included for reference.

42

-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
3
Fos-related antigen 2 Fos-related antigen 2
CCAAT/enhancer-binding protein epsilon CCAAT/enhancer-binding protein epsilon
Transmembrane protein 47 Protein quiver
Carbonic anhydrase 2 CCAAT/enhancer-binding protein epsilon
Probable isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial Y-box factor homolog
Angiopoietin-2 Complement C1q-like protein 3
-1
0
1
2
3
4
5
Tide
a) b)
Light/dark
Time
Relative expression (log
2
)
Figure 6. Comparison of expression patterns in tidal genes of interest, as exhibited in mussels a)
experiencing a simulated intertidal regime and b) free-running after entrainment to a field subtidal
regime. In both treatments, gene expression peaks at or around low tide.

43

Figure 7. Venn diagram of genes with expression profiles highly correlated
(Pearson > 0.6) with fos and C/EBPE in mussels experiencing a simulated
intertidal regime and mussels free-running after entrainment to a field subtidal
regime. The overlap was found to be significant (p = 0) using the
hypergeometric distribution.
Simulated intertidal
Free-running after
field subtidal
16 29 448

44

Figure 8. Heat map showing circatidal gene expression in ex vivo tissue culture samples from
oysters entrained to a field subtidal regime. 248 genes exhibited circatidal expression rhythms
(period = 10 - 14hr, p < 0.05) during free-running. The corresponding anticipated tide and
light/dark cycle are included for reference.
row min row max
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
T21
T22
T23
T24
UID
UID ipr GO kegg KEGG_map swissprot trembl ADJ.P PER LAG AMP
row min row max
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
T21
T22
T23
T24
UID
UID ipr GO kegg KEGG_map swissprot trembl ADJ.P PER LAG AMP
Time
-1
0
1
2
3
4
5
Tide
Light/dark

45

Figure 9. Expression of a, b) carbonic anhydrase, c) structural maintenance of chromosomes protein,
d, e) C/EBP family members, f) DNA-binding protein inhibitor ID-2, g) angiopoietin, h) heat shock
protein 70 B2, i) cAMP responsive element binding-like protein 2, j, k) Fos, l) Jun, m) transcription
factor AP-1, and n) protein quiver in whole mussels in a simulated intertidal environment (left side of
each graph) and in free-running ex vivo tissue cultures of oysters entrained to a field subtidal
environment (right side of each graph).

-1
-0.5
0
0.5
1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
-1
-0.5
0
0.5
1
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
-4
-3
-2
-1
0
1
2
3
-1.5
-1
-0.5
0
0.5
1
1.5
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
-2
-1.5
-1
-0.5
0
0.5
1
1.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
-4
-3
-2
-1
0
1
2
3
4
-5
-4
-3
-2
-1
0
1
2
3
-2
-1.5
-1
-0.5
0
0.5
1
1.5
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
-1
0
1
2
3
4
5
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)
l)
m)
n)
-1
0
1
2
3
4
5
Tide
Light/dark
Relative expression (log
2
)
Time

46

Figure 10. Venn diagram of genes with expression profiles highly correlated (Pearson > 0.6) with
fos and C/EBPE in mussels experiencing a simulated intertidal regime and free-running ex vivo
tissue cultures from oysters entrained to a field subtidal regime. The overlap was found to be
significant (p = 2.13e-11) using the hypergeometric distribution.
10 158
35
Simulated intertidal,
mussel
Ex vivo oyster, field
subtidal

47

GO ID GO term GO ID GO term GO ID GO term GO ID GO term
GO:0016787 hydrolase activity GO:0000166 nucleotide binding GO:0016787 hydrolase activity GO:0036094 small molecule binding
GO:0003676 nucleic acid binding GO:0046872 metal ion binding GO:0003676 nucleic acid binding GO:0003676 nucleic acid binding
GO:0016740 transferase activity GO:0005524 ATP binding GO:0016740 transferase activity GO:0016740 transferase activity
GO:0000166 nucleotide binding GO:0016491 oxidoreductase activity GO:0000166 nucleotide binding GO:0000166 nucleotide binding
GO:0046872 metal ion binding GO:0003677 DNA binding GO:0046872 metal ion binding GO:0046872 metal ion binding
GO:0005524 ATP binding GO:0005215 transporter activity GO:0005524 ATP binding GO:0005524 ATP binding
GO:0016491 oxidoreductase activity GO:0016301 kinase activity GO:0016491 oxidoreductase activity GO:0016491 oxidoreductase activity
GO:0003677 DNA binding GO:0048037 cofactor binding GO:0003677 DNA binding GO:0003677 DNA binding
GO:0003723 RNA binding GO:0003723 RNA binding GO:0003723 RNA binding GO:0016301 kinase activity
GO:0008270 zinc ion binding GO:0016887 ATPase activity GO:0008270 zinc ion binding GO:0003723 RNA binding
Simulated intertidal mussel Free-running intertidal mussel Free-running subtidal mussel Free-running ex vivo subtidal oyster
Table 1. The top ten highest frequency molecular function GO terms assigned to the Fos and
C/EBPE co-expression modules for each treatment
All treatments and Connor and Gracey (2011) Both mussel treatments and Connor and Gracey (2011)
CCAAT-enchancer-binding protein (C/EBP) Carbonic anhydrase
Fos Y-box factor homolog
Jun (Transcription factor AP-1) Complement C1q-like protein 3 (C1QL3)
Immediate early response gene 5 (IER5 ) Protein quiver (QVR)
3-hydroxybutyrate dehydrogenase type 2 (BDH2 ) Feline leukemia virus subgroup C receptor related protein 2 (FLVCR2 )
Mdm2-binding protein (MTBP)
Protein BTG1
Table 2. Summary lists of circatidal genes identified across all treatments of the present
study as well as in Connor and Gracey (2011), and of circatidal genes identified across both
mussel treatments of the present study as well as in Connor and Gracey (2011).

48
CHAPTER 2
Behavioral characterization of the circatidal oscillator in Boreal limpet Lottia paradigitalis

Abstract
Living in an environment with conditions that vary with the ebb and flow of the tide, many taxa
of intertidal animals exhibit circatidal behavior rhythms. For many of these animals, this pattern persists
even when they are released into free-running (constant environmental conditions in the laboratory),
suggesting the presence of an endogenous oscillator that controls the rhythms, rather than the rhythms
occurring as a response to the changing of the tides. A few studies have shown that circatidal locomotor
rhythms are endogenous in different species of limpets. However, not much else is known about the
limpet circatidal oscillator beyond that observation. In this study, we investigated the circatidal oscillator
in the Boreal limpet Lottia paradigitalis. We first confirmed that circatidal locomotor rhythms persist in
free-running in this species, after entrainment to either field or laboratory tidal cycles. We then sought to
determine the plasticity of the circatidal oscillator by analyzing their locomotor rhythms after entrainment
to tidal cycles of periods that differed from 12.4 hours. We found that limpets that were entrained to non-
circatidal tidal cycles still exhibited circatidal or near-circatidal locomotor rhythms when released into
free-running, suggesting that the circatidal oscillator in this species is quite rigid. We also sought to
identify the zeitgeber (“time-giver,” ie. environmental cue) that sets their oscillator with the correct period
by analyzing their locomotor rhythms after entrainment to cycles of individual zeitgebers: emersion, light,
flow, and temperature. Out of the four zeitgebers tested, flow appeared to be the most effective, with
limpets adhering most closely to the flow rhythms during both entrainment and free-running. These
results give further insight into the mechanism of the limpet circatidal clock. When combined with
corresponding gene expression data (Chapter 3), these results will enable us to begin to build a model of
the limpet circatidal clock.

49
Introduction
Rhythmic movement behaviors in intertidal organisms
Free-running circatidal movement rhythms were first described in the flatworm Convoluta
roscoffensis (Gamble & Keeble 1903). Since it is in a symbiotic relationship with the phytoplankton
Tetraselmis convolutae, it emerges from the sand during low tide in order to expose the T. convolutae to
the sun. It burrows back into the sand right before the onset of high tide. When removed to constant
laboratory conditions, C. roscoffensis continued this pattern for 5 days. Since then, similar patterns of
circatidal movement rhythms observed in the field have been found to persist in free-running laboratory
conditions, in many different taxa, including crustaceans, annelids, mollusks, echinoderms, and
vertebrates (Tessmar-Raible et al. 2011).
There are several examples of endogenous circatidal rhythmic behavior in mollusks, roughly
divided between observations of shell-gaping in bivalves and locomotor activity in gastropods. Beentjes
and Williams (1986) observed shell gaping and siphon extension in New Zealand cockle Chione
stutchburyi kept in constant laboratory conditions. They found activity peaks during times of high water,
lasting up to 20 tidal cycles. Ameyaw-Akumfi and Naylor (1987) found similar results in mussel Mytilus
edulis, with shell-gaping activity under constant conditions occurring with a circatidal rhythmicity.
However, this only persisted for four tidal cycles. In experiments in four species of mid- and upper-
littoral neritids, Zann (1973) found that they all exhibited endogenous circatidal locomotor rhythms when
released into free-running conditions, with activity rhythms persisting for 3-6 days. Petpiroon and
Morgan (1983) and Ng and Williams (2006) found similar results in the periwinkle Littorina nigrolineata
and chiton Acanthopleura japonica, respectively.

Limpets and the intertidal environment
Limpets are gastropod mollusks commonly found in rocky intertidal zones. As herbivores in the
intertidal zone, limpets exhibit circatidal (~12.4 hour) rhythmic foraging behavior, returning to their home
scars at the end of each foraging period (Galbraith 1965, Little 1989). Several genera of limpets,

50
including Lottia, home, returning to a designated home scar or home area just before low tide after
moving and foraging at the onset of and during high tide (Cook, et al., 1969; Galbraith, 1965). However,
even within a conspecific population, not all individuals will reliably home (Beckett 1968).
Homing may be based on mnemotaxis, involving the memorization of landmarks within the
substrate, and trail-following, but neither hypothesis has been fully supported experimentally. There is no
direct evidence for the mnemotaxis hypothesis, while evidence for the trail-following hypothesis is
incomplete. Specific evidence against trail-following include: outgoing and return trails do not always
overlap, limpets physically removed from their home sites are still able to return, and experimental trail
removal does not deter limpets from homing to the correct location (Galbraith 1965, Beckett 1968, Cook
et al. 1969, Thomas 1973, Chelazzi et al. 1988). Trail polarity may be based on chemical or physical cues
in the mucus, but this has not been concretely shown. Cook (1969) suggests that limpets can perhaps
follow old, previously laid trails home, but this idea still concedes that return to the home site is not
dependent on the outgoing trail.
Limpets have two main modes of sensing the environment: the eyes and the mantle tentacles. A
limpet’s eyes are located on the head, at the base of the tentacles. The eyes consist of shallow
indentations filled with light-sensing cells that contain black pigment granules. Given the primitive
nature of their eyes, limpets’ vision is quite poor, and they instead rely more heavily on sensory
information from their mantle tentacles. The entire margin of the mantle is lined with tentacles that are
able to extend and withdraw individually, giving limpets 360-degree perception. These tentacles are
responsible for sensing tactile and chemical stimulation, but can also sense light to a small degree (Heller
2015).
A major stressor that limpets need to avoid during low tide is heat. High temperatures at low tide
are magnified by the fact that limpet bodies are always warmer than the surrounding air and rock
(Vermeij 1972, Wolcott 1973). When they are submerged, their body temperature equilibrates with the
cooler temperatures of the surrounding water. As such, throughout the course of one day, limpets can
experience a large range of temperatures, from 5 to 34°C (Grainger 1968).

51
Limpet feeding schedules seem to be aimed at reducing risk desiccation, with foraging
expeditions occurring solely during submergence. Expeditions are oriented shoreward during the flood
and seaward during the ebb. Experiments with Patella depressa indicate that even a sublethal level of
desiccation leads to osmotic imbalance, reduced muscle action, and reduced adhesion, which all have the
very real possibility of resulting in death of the animal (Branch 1981).
In addition to the normal challenges associated with the changing tide, reasons for specific
rhythmic timing of foraging and homing include defense against predators (Wells 1980, Garrity &
Levings 1983) and avoidance of osmotic stress (Little et al. 2009). Shorebirds do not dive for prey, and
instead forage when at least part of the intertidal zone is exposed (Evans 1988). As osmoconformers,
limpets’ sole defense against salinity stress is to clamp down on the rock, shutting out the external
environment (Hoyaux et al. 1976).
Limpets’ main defense against both wave action and low tide desiccation is to clamp down on
their substrate. For this reason, by secreting chemical compounds and by physically scraping away the
rock using their radulas, individuals of some limpet species form a personalized home scar that they alone
fit into. They achieve their strong grip on the substrate using their powerful shell muscle, which extends
from the shell into the foot. This is aided by the sticky mucus the foot secretes. Strong adhesion to the
substrate is important because if a limpet is dislodged and flipped over, it is unable to right itself on its
own (Heller 2015).

Rhythmic locomotor behavior in limpets
A few studies have shown that limpet rhythmic behaviors are endogenous and not merely
immediate responses to the ebb and flow of the tide, with limpets of several different genera exhibiting
persistent tidal locomotor rhythms after removal to constant laboratory conditions. Della Santina and
Naylor (1994) found that rhythms of homing behavior were endogenous in the common European limpet
Patella vulgata. Limpets were immediately transferred to constant laboratory conditions of constant
darkness, 14°C, and emersion with continuous fine water spray after collection. The limpets exhibited

52
both circatidal and circadian rhythmicity that persisted in free-running for four days. Gray and Hodgson
(1999) performed similar experiments with the high-shore prickly limpet Helcion pectunculus. Limpets
were transferred to constant laboratory conditions of constant darkness, 25°C, and emersion in moist air
following collection. Limpets continued to exhibit circatidal movement with circadian modulation
through three days of free-running. Gray and Williams (2010) observed locomotor rhythms in Cellana
grata, a tropical, high-shore limpet. After collection, limpets were moved to constant laboratory
conditions of 25.5°C, constant darkness or white light, and constant immersion, emersion, or seawater
spray. In all white light treatment combinations, circatidal movement persisted for at least two days. In
all constant darkness treatments, circatidal movement persisted for at least four days. Interestingly and
notably, limpets maintained in constant white light and constant seawater spray exhibited persistent
circatidal locomotor rhythms for 30 days. However, the underlying molecular mechanism remains
unknown, and even potential zeitgebers have not been extensively studied. The adaptive nature and
circatidal rhythmicity of this behavior suggest that the circatidal clock is involved in its timing.
The majority of previous biological rhythms research in our lab has been conducted in mussels
and oysters. However, as sessile bivalves, mussels and oysters do not have a visible rhythmic phenotype.
As such, we are investigating the possibility of using the rhythmic movement behavior of limpets of
genus Lottia as a phenotypic reporter assay. If foraging rhythms are endogenous and under control of the
circatidal clock, then Lottia limpets could serve as a convenient assay with which we can easily test and
verify molecular data, which would be especially advantageous given that the L. gigantea genome has
been published (Simakov et al. 2013).
It is to our advantage that the circadian clock is so well-studied, as this allows us to apply
principles and strategies from circadian research to our investigation of the as yet not elucidated
molecular mechanism of the circatidal clock. One of the advantages of studying the circadian clock lies
in the development of consistent reporter assays, based on forward genetic approaches, which are easily
used to identify and verify new findings. We aim to eventually use limpets in a similar manner as wheel-
running mice are used (Siepka & Takahashi 2005). Briefly, mice are screened for wheel-running rhythms

53
that deviate from the LD 12:12 (light/dark) cycle to which they have been entrained, and the genes
responsible for this mutant phenotype are identified. In addition, there exist several transgenes as well as
strains of transgenic organisms employing a luciferase and circadian clock gene fusion product for the
majority of the main circadian model organisms (Welsh et al., 2005): Synechoccocus (Kondo et al. 1993),
Arabidopsis thaliana (Millar et al., 1992), Drosophila melanogaster (Brandes et al. 1996), and Mus
musculus (Wilsbacher et al. 2002). In this approach, luciferase fluorescence as a result of transgene
expression becomes the observable phenotype.
However, before any of these techniques can be developed for limpets and the circatidal clock,
we must first establish the baseline of what a wild-type rhythmic gene expression profile looks like in
limpets and connect that to normal movement behaviors. Additional questions addressed in this study
include the plasticity of the circatidal clock and identification of possible zeitgebers of the circatidal clock
in limpets.

Methods
Collection
Boreal limpets Lottia paradigitalis sized ~2cm were collected during low tide from the mid- to
upper intertidal zone at Will Rogers State Beach, Santa Monica, CA, then immediately transported to our
laboratory tidal simulation aquarium, located in a temperature-controlled environmental chamber at USC
set at 15°C. Will Rogers State Beach is located on Santa Monica Bay, which experiences a mixed semi-
diurnal tidal regime, with a tidal range of ~2.7m.

Free-running period of field-entrained limpets
Limpets (n = 50) sized ~2cm were collected during low tide at Will Rogers State Beach, Santa
Monica, CA, then immediately transported to the laboratory and released into free-running conditions, in
a seawater microcosm in a temperature-controlled (15°C) environmental chamber. Limpets were kept
submerged and in constant darkness, with constant low flow.

54
Tidal simulation aquarium conditions and overall experimental set-up
Limpets sized ~2cm were collected during low tide at Will Rogers State Beach, Santa Monica,
CA, then immediately transported to our laboratory tidal simulation aquarium, located in a temperature-
controlled environmental chamber set at 15°C. Limpets were positioned such that they were submerged
during high tide and emerged during low tide, and entrained to the tidal cycle of interest. Water flow was
modulated with Turbelle stream water pumps (Tunze, Penzberg, Germany) such that the water was slack
at the times flanking the changing of the tides, with step-wise increase and decrease in flow; maximum
flow occurred at the middle of high tide. Light (AquaSun FR20T12/VHO, Zoo Med Laboratories, San
Luis Obispo, CA) was set to a LD 12:12 cycle. All settings of the tidal simulation aquarium were
programmed and controlled using an Apex Aquacontroller (Neptune Systems, Morgan Hill, CA).

Entrainment of limpets to laboratory simulated 12.5-hour tidal cycle
After collection, limpets (dark treatment n = 51, light treatment n = 52) were entrained to a tidal
cycle with a period of 12.5 hours in the tidal simulation aquarium. After at least one week of entrainment,
limpets were released into free-running conditions, with constant submergence and low flow, and either
constant darkness or constant light.

Plasticity of free-running period
After collection, limpets were entrained to tidal cycles of various periods: 9, 15, or 24 hours (n =
16, 74, and 64, respectively) in the tidal simulation aquarium. After two weeks of entrainment, limpets
were released into free-running conditions of constant submergence, flow, and darkness.

Zeitgebers of limpet rhythmic movement
After collection, limpets (n = 20) were moved to a separate experimental seawater microcosm and
entrained to various cycles of one of four different zeitgebers: emersion, light, flow, and temperature. For

55
each treatment, limpets were kept in constant darkness, low flow, and submergence with only the
zeitgeber of interest oscillating.

Light/dark
Limpets were placed in the tidal simulation aquarium and entrained for at least one week to a
light/dark cycle with a period of either 12 or 24 hours, LD 6:6 and LD 12:12, respectively. Entrainment
took place under constant temperature, constant submergence, and constant low flow. Following
entrainment, the light cue was removed and limpets were released into free-running under constant dark.

Emersion
Limpets were placed in the tidal simulation aquarium and entrained for at least one week to an
emersion/submersion cycle with a period of 12 hours (emersion:submersion 6:6). Entrainment took place
under constant temperature and constant darkness, and there was no modulation of flow intensity flanking
the ebb and flow of the tide. Following entrainment, the emersion cue was removed and limpets were
released into free-running under constant submergence.

Flow
Limpets were placed in a seawater microcosm and entrained for at least one week to a flow cycle
with a period of either 6 or 12 hours, flow high:low 3:3 and high:low 6:6, respectively. Flow alternated
between low flow and high flow intensities, matching the period of the treatment. For the 6-hour period
treatment, limpets experienced alternating cycles of three hours with low flow, then three hours of high
flow intensity. For the 12hr period treatment, limpets experienced oscillating cycles of six hours with low
flow, then six hours of high flow intensity. Entrainment took place under constant temperature, constant
darkness, and constant submergence. Following entrainment, the flow cue was removed and limpets were
released into free-running under constant low flow.

56
Temperature
Limpets were placed in a seawater microcosm and entrained for at least one week to a
temperature cycle with a period of 12 hours, temperature low:high 6:6. Water temperature was
modulated between 15 and 25°C such that limpets were exposed to a high temperature episode (23-25°C)
lasting six hours, occurring every six hours. This was done using an Autotune temperature controller
(OMEGA, Norwalk, CT) and Theo aquarium heater (150W, Hydor, Italy). Temperature was recorded
using a Thermochron temperature logger (iButtonLink, Whitewater, WI). Entrainment took place under
constant darkness, constant submergence, and constant low flow. Following entrainment, the temperature
cue was removed and limpets were released into free-running under constant temperature at 15°C.

Movement video collection and analysis
In all experiments, limpet movement was recorded using a USB webcam (Toogoo, China)
connected to a dedicated laptop (Lenovo ThinkPad). Time lapse videos were made of limpet movement
throughout the experiment. Each video was scored as described in Gray and Williams (2010), with slight
modifications. Each video frame was scored for presence and absence of average movement, wherein
movement was documented if over 20% of the group was moving. The number of active limpets was
also recorded for each frame. If a limpet moved out of frame, it was not counted until it returned, so
activity was calculated as the proportion of moving limpets divided by all visible limpets. The same
person blindly (ie. without prior knowledge of experimental conditions) scored all of the videos in order
to maintain consistency. Reanalysis revealed that the two scoring methods did not produce significantly
different results (unpub).
Lomb-Scargle periodogram analysis was performed using the ActogramJ plug-in (Schmid et al.,
2011) for ImageJ (Abràmoff et al. 2004) to determine significant (p<0.05) periods in movement. This
periodogram is derived from Fourier spectral analysis, and is calculated as follows:
!" ω =
1
2(
)

*
+
− * cos0(2
+
−3)
+
)
cos
)
0(2
+ +
− 3)
+
*
+
− * sin0(2
+
−3)
+
)
sin
)
0
+
(2
+
– 3)

57
given a time series of N data points *
+
= *(2
+
) collected at times t
j
where j = 1,2,…N, with a mean of *,
and where t is defined as:
3 =
9
):
tan
=9
>?@) : A
B B
CD>) :
B
A
B

PN gives the normalized power as a function of angular frequency w (Ruf 1999).

Results
Free-running limpet movement immediately after various modes of entrainment was tested.
Results are summarized in Table 1.

Field-entrained
Free-running movement was tested in field-entrained limpets in order to determine their naturally
occurring rhythmicity. Limpets entrained in the field and released into constant conditions immediately
after collection exhibited tidal free-running movement overall, with a period of 12.85 hours (Fig. 1a,b).
However, closer inspection of the actograms revealed that tidal free-running movement lasted for 5 tidal
cycles before the limpets switched to a circadian period for the remainder of the experiment (Fig. 1a,c,d).
Importantly, movement occurred only during times of subjective high tide (Fig. 2).

Lab-entrained 12.5-hour tidal cycle
Limpets were entrained to simulated 12.5-hour tidal cycles in the laboratory in order to determine
the viability of laboratory-based rhythmicity experiments. Limpets entrained to a simulated tidal cycle
and released into constant conditions with constant light exhibited a circatidal free-running period of
11.88 hours (Fig. 3b,d), which was slightly shorter than their entraining period of 12.42 hours (Fig. 3a,c).
In contrast, while entrained limpets released into constant conditions with constant darkness also
exhibited a circatidal free-running period of 12.83 hours (Fig. 4b,d), this was slightly longer than the
entraining period of 12.45 hours (Fig.4a,c).

58
Lab entrained tidal cycles of various periods
In order to determine the plasticity of the circatidal oscillator, limpets were entrained to tidal
cycles with various periods. Limpets entrained to 9, 15, and 24 hour tidal cycles exhibited free-running
movement with circatidal or near circatidal rhythmicity in all three treatments (Fig. 5-7). Limpets
entrained to the 9-hour tidal cycle showed the most robust return to circatidal movement with a free-
running period of 12.56 hours (Fig. 5d), while limpets entrained to the 15 and 24 hour tidal cycles moved
with a majority circatidal period of 12.25 hours, with minor non-circatidal peaks, (Fig. 6d) and a near
circatidal period of 15.97 hours (Fig. 7d), respectively.

Lab-entrained with only one variable
In order to determine the zeitgeber of the circatidal oscillator, limpets were entrained with a
single rhythmic variable, while all other environmental variables were held constant.

Light/dark
Limpets entrained to a 12-hour light/dark cycle (ie. LD 6:6) exhibited circatidal rhythmicity
during entrainment with a period of 12.38 hours, moving during the light phases (Fig. 8a,c), but upon
release into constant conditions switched to a circadian movement rhythm with a period of 24.9 hours
(Fig. 8b,d). Interestingly, limpets entrained to a 24-hour light/dark cycle (LD 12:12) maintained a
circatidal movement rhythm with a period of 11.88 hours during entrainment (Fig. 9a,c). When released
into free-running, these limpets exhibited neither a circatidal nor circadian movement rhythm overall with
a period of 16.23 hours; however, secondary spectral peaks in the free-running periodogram demonstrate
underlying circatidal (period = 11.86 hours) and circadian (period = 23.32 hours) movement rhythms
(Fig. 9b,d).

59
Emersion
Limpets entrained to a 12-hour emersion cycle (emersion:submersion 6:6) exhibited a circadian
free-running period of 24.6 hours overall, with minor circatidal peaks (Fig. 10d). However, closer
examination of individual days during the free-running episode revealed a circatidal movement period of
12.17 hours during the first 24 hours of free-running before the limpets switched to a circadian movement
period of 24.08 hours for the remainder of the experiment (Fig. 10e,f).

Flow
During entrainment, limpets entrained to 6 and 12 hour flow cycles (flow high:low 3:3 and
high:low 6:6, respectively) exhibited rhythmic movement matching their respective flow cycles (Fig.
11a,c; Fig.12a,c). When released into free-running, limpets entrained to 6 and 12 hour flow cycles
exhibited movement matching their entraining flow cycles for the first four cycles of free-running (24
hours and 48 hours, respectively) (Fig. 11b,e; Fig. 12b,e). Subsequent movement was negligible.

Temperature
Limpets entrained to a 12-hour temperature cycle (temperature low:high 6:6) did not exhibit
circatidal rhythmicity, even during entrainment (Fig. 13a,c), although minor spectral peaks of nearly half
the power of the major peak did indicate a circatidal component. Overall free-running movement was
equally lacking in circatidal rhythmicity (Fig. 13b,d); however, limpets did exhibit circatidal movement
during the first 48 hours of free-running with a period of 13.02 hours (Fig. 13e). Comparing limpet
movement with the temperature cycle, there was only a vague tendency for movement to occur during the
phases in which temperature decreased or was anticipated to decrease, but this was not a consistent
pattern and did not occur at every temperature dip (Fig. 14).

60
Discussion
Persistence of circatidal locomotor rhythms after natural and laboratory tidal entrainment
Limpets of genus Lottia appear to possess an internal oscillator that directs the rhythm of their
movement. Whether they were entrained to tidal conditions in the field or simulated tidal conditions in
the laboratory, they continued to exhibit circatidal movement patterns upon release into free-running (Fig.
1-4). Due to the lack of environmental cues during free-running, this provides strong evidence for an
endogenous mechanism that was set by the tidal regime, which is consistent with previous findings in
other limpet species, including Patella vulgata (Della Santina and Naylor 1994), Helcion pectunculus
(Gray & Hodgson 1999), and Cellana grata (Gray & Williams 2010).
Other animals exhibiting endogenous circatidal movement rhythms include the chiton
Acanthopleura japonica (Ng & Williams 2006), shore crab Carcinus maenas (Reid & Naylor 1990),
marine isopods Exosphaeroma truncatitelson (Holloway 2014) and Eurydice pulchra (Zhang et al. 2013),
mud snail Hydrobia ulvae (Vieira et al. 2010), and mangrove cricket Apteronemobius asahinai (Satoh et
al. 2009), indicating that this phenomenon is not specific to just Lottia, and even goes beyond marine
gastropods. Although these organisms are of disparate taxa, they all have in common the fact that they
live in the intertidal zone.

Persistence of circatidal locomotor rhythms after entrainment to cycles with non-tidal periods
Interestingly, limpets entrained to 9, 15, and 24-hour tidal cycles reverted to circatidal or near
circatidal movement when released into free-running, despite closely following their respective tidal cycle
during entrainment (Fig. 5-7), suggesting that the circatidal clock is not very plastic, at least in limpets
from a semidiurnal coast. Limpets entrained to the 9 and 15-hour tidal cycles switched back much more
readily than limpets entrained to the 24-hour tidal cycle, which makes sense, because 9 and 15 hours are
much closer to 12.4 hours; moreover, if, as has been previously suggested, there is a connection between
the circatidal and circadian clocks, then the 24-hour tidal cycle may have stimulated the shared circadian
clock components.

61
In comparative studies between different species of Carcinus crabs, C. maenas from a non-tidal
harbor along a tidally-influenced coastline exhibited circadian, then circatidal locomotor activity when
released into constant conditions in the laboratory (Naylor 1960). Since these crabs are usually subject to
a daily rhythm only, this switch to circatidal movement suggests a genetic basis for their tidal timing
mechanism. Meanwhile, C. aestuarii (formerly C. mediterraneus), a species native to the Mediterranean
Sea, which is commonly accepted to have negligible tides, exhibits circadian rhythmicity when moved to
constant laboratory conditions (Naylor 1988). This makes sense because the lack of tidal cues makes the
circadian cues the only zeitgebers that C. aestuarii experience.
Barnwell (1968) performed some interesting transplantation experiments in fiddler crabs of genus
Uca. Populations of U. mordax live on the Caribbean coast of Costa Rica, where the tidal regime
alternates between diurnal and semidiurnal patterns. In environmental conditions, these crabs exhibit a
generally circadian activity rhythm, with no obvious circatidal pattern. When individuals from this
population were transplanted to the Pacific coast of Costa Rica and entrained for 5 days to the
consistently semidiurnal tidal regime there, they exhibited circatidal locomotor activity when transferred
to constant laboratory conditions. Similar experiments were performed on U. minax originating from a
Mississippi bayou that experiences diurnal tides (Barnwell 1968). Crabs kept under constant temperature
and LD 12:12 in the laboratory exhibited only a weak circadian rhythmicity and no circatidal rhythmicity.
However, after the crabs were transplanted to Woods Hole and entrained to the semidiurnal tidal regime
for 26 days, all of the experimental crabs demonstrated tidal locomotor activity. These results suggest
that the circatidal machinery defaults to a circatidal rhythm when given the proper cues.

Identification of potential zeitgebers
The main zeitgebers of the circatidal clock have not been definitively determined. Palmer (1973)
suggested five possible zeitgebers: inundation (submersion), mechanical agitation, hydrostatic pressure,
temperature, and light.

62
Emersion/submersion
Limpets entrained to alternating bouts of 6 hours of submersion and 6 hours of emersion
exhibited an overall circadian locomotor rhythm when released into free-running (Fig. 10b,d). However,
closer inspection of the actograms revealed that their movement was circatidal for the first 24 hours of
free-running (Fig. 10b,e). Additionally, their movement was circatidal and followed the submersion cycle
during entrainment (Fig. 10a,c). Combined, this suggests emersion provides a weak entrainment cue for
the circatidal clock in limpets, but that it is not strong enough on its own to set the clock, which is
consistent with findings in other animals. In another limpet, Helcion pectunculus, Gray and Hodgson
(1999) found that a 24-hour pulse of emersion resulted in circatidal movement persisting for two tidal
cycles, according to the timing of the pulse. However, a pulse of submersion only dampened limpet
movement, with no significant rhythmicity. Williams and Naylor (1969) subjected Carcinus crabs to
12.4-hour immersion cycles with the crabs submerged for 6.2 hours and emerged for 6.2 hours while the
air and water temperatures were maintained at a constant 19°C. When the crabs were released into free-
running at the same temperature, they did not exhibit significant rhythmicity in their movement. Jones
and Naylor (1970) found similar results in the speckled sea louse Eurydice pulchra.

Pressure
Although we did not directly test hydrostatic pressure as a zeitgeber, the emersion treatment is a
close approximation, because of the water column that sits on top of the limpets during submersion.
Pressure cycles have been found to induce a tidal rhythm in submerged Carcinus crabs. Entrainment for
three days to square wave pressure cycles between atmospheric and above-atmospheric pressures resulted
in persistent tidal movement rhythms for six tidal cycles in constant conditions (Palmer 1973). The
cumacean Dimorphostylis asiatica has also been shown to entrain to pressure cycles with an amplitude of
0.3 atm (Akiyama 2004). However, experiments in other animals do not support pressure as a main
zeitgeber. Eurydice entrained to pressure cycles lasting 30 minutes every 12 hours exhibited a tidal
rhythm when released into free-running conditions, but the patterns did not match the entraining pattern

63
(Jones & Naylor 1970). Experiments comparing activity rhythms of Synchelidium entrained in either a
sealed bottle anchored in place (exposure to varying pressure) or a sealed bottle attached to a float
(constant pressure) showed no difference (Enright 1963). Animals kept under constant pressure during
the entraining period exhibited the same circatidal activity rhythms as animals exposed to a varying
pressure cycle when released into free-running conditions.

Light
Limpets entrained to LD 6:6 exhibited circatidal movement rhythms during entrainment (Fig.
8a,c), but switched to circadian and non-circatidal rhythms when released into free-running (Fig. 8b,d).
Strangely, limpets entrained to a 24-hour light/dark only cycle exhibited an overall rhythm that was
neither circatidal nor circadian (Fig. 9b,d), although further examination of secondary spectral peaks in
the periodogram does reveal circatidal and circadian components in their movement (Fig. 9d). Light is
already well established as a major zeitgeber of the circadian clock. If the circatidal clock does share the
circadian clock machinery, then light should at least contribute to the setting of the circatidal clock.
However, if the circatidal clock is a separate system, then it would not be productive for it to be set by
something that occurs with a daily rhythm. Under natural LD and laboratory LD 12:12 cycles, many
different taxa have exhibited persistent tidal activity rhythms (Palmer 1973). It is important to note that
for many of these taxa, the experiments did not record free-running rhythms, so their results are consistent
with our entrainment – but not free-running – results. In other experiments, after entrainment to an LD
6:6 light cycle for 2.5 days, Excirolana did not show any evidence of entrainment (Enright 1965). Gibson
(1967) found similar results in similar experiments with the combtooth blenny Blennius pholis.

Flow as mechanical agitation
Limpets entrained to a 6-hour flow-only cycle (flow high:low 3:3) adhered to their entrainment
flow (Fig. 11a,c), and this rhythm persisted for the first four tidal cycles of free-running (Fig. 11b,e).
Limpets entrained to the 12-hour flow-only cycle (flow high:low 6:6) also adhered to their entrainment

64
flow, both during entrainment and during free-running (Fig. 13). Combined, these results strongly
suggest that flow or mechanical agitation could be a major zeitgeber of the circatidal clock. Out of all of
the potential zeitgebers tested in this study, flow appears to be the most promising candidate.
Indeed, in experiments with Excirolana, mechanical agitation in the form of periodic stirring (6
hours on, 6 hours off) of the water the animals were staying in resulted in multiple days of free-running
circatidal swimming (Enright 1963). This occurred after two and a half days of agitation cycles. Further
experiments by Enright (1963) using cyclic chemical regimes, feeding, and oxygen tensions did not result
in entrainment of the Excirolana. Jones and Naylor (1970) achieved similar results in Eurydice with just
30 minutes of stirring at 12-hour intervals. Even a single, one-hour pulse of agitation was enough to
induce an almost circatidal rhythm. Klapow (1972) elaborated on these findings in Excirolana by
alternating the agitation duration between 30 minutes and 120 minutes, in order to simulate a mixed
semidiurnal tide. He found that not only did the animals’ circatidal swimming rhythms persist in free-
running, but the amplitudes of the activity peaks mirrored the agitation cycles, with greater amplitudes
exhibited during anticipated 120-minute agitation. Mole crabs Emerita talpoida can also entrain to
mechanical agitation cycles (Forward et al. 2007). Crabs were placed in beakers on an orbital shaker,
which shook in pattern of 15 seconds on then 15 seconds off for a 4-hour duration coinciding with
subjective high tide. Crabs were entrained for four tidal cycles, then released into free-running.
Circatidal locomotor rhythmicity persisted for three tidal cycles, coinciding with subjective high tide,
under constant conditions. Similar results were found in the same experiment repeated with mechanical
agitation occurring at subjective low tide, mid-flood, and mid-ebb during entrainment. However, the
littoral prawn Palaemon does not seem to entrain to mechanical agitation, at least in the form of
alternating 6-hour cycles of running and still water (Rodriguez and Naylor 1972). Even after 10 days of
entrainment, the prawns did not exhibit circatidal movement activity when released into free-running.

65
Temperature
Limpets entrained to alternating cycles of 6 hours at 15°C followed by 6 hours at 25°C exhibited
neither circatidal nor circadian rhythmicity during entrainment and free-running (Fig. 13a-d). They did
exhibit circatidal rhythmicity during the first 24 hours of free-running (Fig. 13e), but temperature does not
seem like a promising zeitgeber, since limpet movement did not match the entraining temperature pattern.
Furthermore, when comparing movement and temperature peaks, there appears to be no discernible
pattern or relationship (Fig. 14).
Experiments with Carcinus show that temperature can contribute to entrainment of circatidal
rhythms, but only under specific conditions. Williams and Naylor (1969) subjected crabs kept in
otherwise constant moist air conditions to temperature cycles with a 4°C or 11°C differential. After
entraining to 10 temperature cycles, the crabs exposed to the 11°C differential exhibited circatidal
locomotor rhythms that persisted for 3 days in free-running conditions, while the crabs exposed to the 4°C
differential did not exhibit persistent locomotor rhythms. However, when the 4°C temperature
differential was combined with an immersion cycle, this combination was able to induce persistent
locomotor rhythms. Moreover, while Enright (1963) did not specifically test entraining temperature
cycles in Excirolana, he did suggest that temperature would be an unreliable zeitgeber, because of
possible temperature changes independent of the tidal cycle, such as irregular water mixing in the surf and
irregular cool-down periods due to varying cloud cover.

Summary of zeitgebers
Out of the four potential zeitgebers tested, flow appears to be the most effective at setting the
circatidal clock in limpets, as shown by the limpets’ close adherence to the various flow cycles, both
during entrainment and during free-running. Importantly, even when the limpets were entrained to a flow
cycle that was neither circatidal nor circadian in period, they still adhered to the entraining rhythm when
released into free-running. Emersion provided a weak cue; even though limpets were entrained to a

66
circatidal emersion rhythm, their movement rhythms only persisted for two tidal cycles during free-
running. Light and temperature do not seem to be effective zeitgebers of the circatidal clock in limpets.

Variability in persistence of rhythms
In this study, all of the results are presented as group averages and proportions because of the
inherent inter-individual variability of wild animals, consistent with previous studies on circatidal activity
rhythms. However, although not all individuals exhibited perfect rhythms during every anticipated tidal
cycle, these overall results still provide evidence for an endogenous circatidal oscillator. In experiments
on the swimming activity of the beach amphipod Synchelidium, freshly collected amphipods kept in
constant laboratory conditions demonstrated robust circatidal rhythmicity for three days before the
oscillations dampened out (Enright 1963). In similar experiments on the swimming activity of the sand-
beach isopod Excirolana chiltoni, Klapow (1972) and Enright (1972) found that while there was a
discernible endogenous circatidal pattern, there was also a great deal of inter-individual variability in
period. Under constant laboratory conditions, the New Zealand cockle Austrovenus stutchburyi exhibits
circatidal gaping behavior corresponding with anticipated high tide (Williams & Pilditch 1997). This
pattern persists for several days before dampening out. Williams and Pilditch (1997) found that tidal
pulses of algae reestablished the circatidal gaping pattern; however, the pattern only persisted for two
tidal cycles after an algae pulse. In free-running anomuran mole crab Emerita asiatica, Chandrashekaran
(1965) found that swimming and oxygen consumption rhythms were circatidal, matching the tidal cycles
of the collection site for three days. From the fourth day on, the crabs exhibited decreased levels of
activity as well as lack of rhythmicity. In the absence of an ideal, well-established genetic model system,
as well as considering the complexity of the intertidal environment, it makes sense that not all individuals
will exhibit a consistent, perfect 12.4-hour rhythm.

67
Ecological relevance
Possession of a circatidal clock would be an advantageous adaptation to life in the intertidal zone.
An endogenous circatidal oscillator makes sense because the mid- and upper- intertidal zones are not
completely inundated as soon as tide comes in; wave action represents noise on top of the larger tidal
oscillation (Enright 1970). Having an internal oscillator that follows the greater tidal pattern decreases
the chances of a misguided response to a passing wave rather than actual full submersion. Moreover,
Enright (1970) suggests that an internal oscillator can maintain consistent timing in the face of abnormal
exogenous entraining factors such as heavy storm conditions.
Intertidal arthropods and vertebrates generally exhibit rhythmic zonal migration, moving with the
tide in order to stay in their preferred surroundings (ie. water or air), whereas rocky intertidal chitons and
gastropods, due to the high energy expenditure for movement, stay in the same zone, alternating between
emergence and submergence (Chelazzi et al. 1988).
Another example of a situation in which an internal timer makes sense is in the case of burrowing
animals or other animals that are not able to keenly sense the surrounding environment during their
inactive phases. In this case, not all individuals of a population necessarily emerge from their burrows
during every anticipated active phase, but because they are able to time the phases internally, when they
do eventually emerge, they do so during the correct time with other population members (Naylor 1988).

Future studies
Other suggested circatidal zeitgebers that we did not specifically address in this study include
salinity and hydrostatic pressure. Since we the intertidal zone is such a complex environment, it is likely
that there is not one single zeitgeber that sets the circatidal clock, so it would be interesting to test
different combinations of two or more of the potential zeitgebers together to find the optimal minimum
combination required to reliably set the clock. Also important will be the connection between gene
expression and circatidal movement in limpets (discussed in Chapter 3), in order to begin to elucidate the
molecular mechanism of the molluscan circatidal clock.

68
References
Abràmoff MD, Magalhães PJ, Ram SJ (2004) Image processing with imageJ. Biophotonics Int 11:36–41
Akiyama T (2004) Entrainment of the circatidal swimming activity rhythm in the cumacean
Dimorphostylis asiatica (Crustacea) to 12.5-hour hydrostatic pressure cycles. Zoolog Sci 21:29–38
Ameyaw-Akumfi C, Naylor E (1987) Temporal patterns of shell-gape in Mytilus edulis. Mar Biol
95:237–242
Barnwell FH (1968) The Role of Rhythmic Systems in the Adaptation of Fiddler Crabs to the Intertidal
Zone. Am Zool 8:569–583
Beckett TW (1968) Limpet movements. An investigation into some aspects of limpet movements,
especially homing behaviour. Tane 14:43–63
Beentjes MP, Williams BG (1986) Endogenous circatidal rhythmicity in the New Zealand cockle Chione
stutchburyi (Bivalvia, Veneridae). Mar Behav Physiol 12:171–180
Branch GM (1981) The Biology of Limpets: physical factors, energy flow, and ecological interactions.
Oceanogr Mar Biol 19:235–380
Brandes C, Plautz JD, Stanewsky R, Jamison CF, Straume M, Wood K V, Kay SA, Hall JC (1996) Novel
features of drosophila period Transcription revealed by real-time luciferase reporting. Neuron
16:687–92
Chandrashekaran MK (1965) Persistent tidal and diurnal rhythms of locomotory activity and oxygen
consumption in Emerita asiatica (M.-EDW.). Z Vgl Physiol 50:137–150
Chelazzi G, Focardi S, Deneubourg J-L (1988) Analysis of Movement Patterns and Orientation
Mechanisms in Intertidal Chitons and Gastropods. In: Chelazzi G, Vannini M (eds) Behavioral
Adaptation to Intertidal Life. Springer US, Boston, MA, p 173–184
Cook SB (1969) Experiments on homing in the limpet Siphonaria normalis. Anim Behav 17:679–682
Cook A, Bamford OS, Freeman JDB, Teideman DJ (1969) A study of the homing habit of the limpet.
Anim Behav 17:330–339
Enright JT (1963) The tidal rhythm of activity of a sand-beach amphipod. Z Vgl Physiol 46:276–313

69
Enright JT (1965) Entrainment of a Tidal Rhythm. Science (80- ) 147:864–867
Enright JT (1970) Ecological aspects of endogenous rhythmicity. Ann Rev Ecol Syst 1:221–238
Enright JT (1972) A virtuoso isopod. J Comp Physiol 77:141–162
Evans PR (1988) Predation of Intertidal Fauna by Shorebirds in Relation to Time of the Day, Tide and
Year. In: Chelazzi G, Vannini M (eds) Behavioral Adaptation to Intertidal Life. Springer US,
Boston, MA, p 65–78
Forward RB, Thaler AD, Singer R (2007) Entrainment of the activity rhythm of the mole crab Emerita
talpoida. J Exp Mar Bio Ecol 341:10–15
Galbraith R (1965) Homing Behavior in the Limpets Acmaea digitalis and Lottia gigantea. Am Midl Nat
74:245–246
Gamble F, Keeble F (1903) The Bionomics of Convoluta Roscoffensis, with Special Reference to Its
Green Cells. Proc R Soc London 72:93–98
Garrity S, Levings S (1983) Homing to scars as a defense against predators in the pulmonate limpet
Siphonaria gigas (Gastropoda). Mar Biol 72:319–324
Gibson RN (1967) Experiments on the tidal rhythm of Blennius pholis. J Mar Biol Assoc United
Kingdom 47:97
Grainger JNR (1968) The Relation between Heat Production, Oxygen Consumption and Temperature in
Some Poikilotherms. In: Locker A (ed) Quantitative Biology of Metabolism: Models of Metabolism,
Metabolic Parameters, Damage to Metabolism, Metabolic Control. Springer Berlin Heidelberg,
Berlin, Heidelberg, p 86–90
Gray D, Hodgson A (1999) Endogenous rhythms of locomotor activity in the high-shore limpet, Helcion
pectunculus (Patellogastropoda). Anim Behav 57:387–391
Gray DR, Williams GA (2010) Knowing when to stop: Rhythms of locomotor activity in the high-shore
limpet, Cellana grata Gould. J Exp Mar Bio Ecol 391:125–130
Heller J (2015) Patellogastropoda: Limpets. In: Sea Snails. Springer International Publishing, Cham, p
37–53

70
Holloway SC (2014) A Study of the Endogenous Activity Rhythms of the marine isopod Exosphaeroma
truncatitelson. Biol Rhythm Res:1
Hoyaux J, Gilles R, Jeuniaux C (1976) Osmoregulation in molluscs of the intertidal zone. Comp Biochem
Physiol -- Part A Physiol 53:361–365
Jones DA, Naylor E (1970) The swimming rhythm of the sand beach isopod Eurydice pulchra. J Exp Mar
Bio Ecol 4:188–199
Klapow LA (1972) Natural and artificial rephasing of a tidal rhythm. J Comp Physiol 79:233–258
Kondo T, Strayer C A, Kulkarni RD, Taylor W, Ishiura M, Golden SS, Johnson CH (1993) Circadian
rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria. Proc
Natl Acad Sci U S A 90:5672–6
Little C (1989) Factors Governing Patterns of Foraging Activity in Littoral Marine Herbivorous Molluscs.
J Molluscan Stud 55:273–284
Little C, Morritt D, Paterson DM, Stirling P, Williams GA (2009) Preliminary observations on factors
affecting foraging activity in the limpet Patella vulgata. J Mar Biol Assoc United Kingdom 70:181
Millar AJ, Short SR, Chua NH, Kay SA (1992) A novel circadian phenotype based on firefly luciferase
expression in transgenic plants. Plant Cell 4:1075–1087
Naylor E (1960) Locomotory rhythms in Carcinus maenas (L.) from non-tidal conditions. J Exp Biol
37:481–488
Naylor E (1988) Clock-Controlled Behaviour in Intertidal Animals. In: Chelazzi G, Vannini M (eds)
Behavioral Adaptation to Intertidal Life. Springer US, Boston, MA, p 1–14
Ng JSS, Williams GA (2006) Intraspecific variation in foraging behaviour: Influence of shore height on
temporal organization of activity in the chiton Acanthopleura japonica. Mar Ecol Prog Ser 321:183–
192
Palmer JD (1973) Tidal rhythms: The clock control of the rhythmic physiology of marine organisms. Biol
Rev 48:377–418
Petpiroon S, Morgan E (1983) Observations on the tidal activity rhythm of the periwinkle Littorina

71
nigrolineata (Gray). Mar Behav Physiol 9:171–192
Reid DG, Naylor E (1990) Entrainment of Bimodal Circatidal Rhythms in the Shore Crab Carcinus
maenas. J Biol Rhythms 5:333–347
Rodriguez G, Naylor E (1972) Behavioural Rhythms in Littoral Prawns. J Mar Biol Assoc United
Kingdom 52:81
Ruf T (1999) The Lomb-Scargle Periodogram in Biological Rhythm Research: Analysis of Incomplete
and Unequally Spaced Time-Series. Biol Rhythm Res 30:178–201
Santina P Della, Naylor E (1994) Endogenous rhythms in the homing behaviour of the limpet Patella
vulgata Linnaeus. J Molluscan Stud 59:87–91
Satoh A, Yoshioka E, Numata H (2009) Entrainment of the circatidal activity rhythm of the mangrove
cricket, Apteronemobius asahinai, to periodic inundations. Anim Behav 78:189–194
Schmid B, Helfrich-Förster C, Yoshii T (2011) A new ImageJ plug-in “ActogramJ” for chronobiological
analyses. J Biol Rhythms 26:464–7
Siepka SM, Takahashi JS (2005) Methods to record circadian rhythm wheel running activity in mice.
Methods Enzymol 393:230–9
Simakov O, Marletaz F, Cho S-J, Edsinger-Gonzales E, Havlak P, Hellsten U, Kuo D-H, Larsson T, LVJ,
Arendt D, Savage R, Osoegawa K, Jong P de, Grimwood J, Chapman JA, Shapiro H, Aerts A,
Otillar RP, Terry AY, Boore JL, Grigoriev IV, Lindberg DR, Seaver EC, Weisblat DA, Putnam NH,
Rokhsar DS (2013) Insights into bilaterian evolution from three spiralian genomes. Nature 493:526–
31
Tessmar-Raible K, Raible F, Arboleda E (2011) Another place, another timer: Marine species and the
rhythms of life. Bioessays 33:165–72
Thomas RF (1973) Homing Behavior and Movement Rhythms in the Pulmonate Limpet, Siphonaria
pectinata Linnaeus. Proc Malacol Soc London 40:303–311
Vermeij GJ (1972) Intraspecific shore-level size gradients in intertidal molluscs. Ecology 53:693–700
Vieira S, Coelho H, Nolasco R, Serôdio J, Barnes R, Queiroga H (2010) The circatidal rhythm of the

72
estuarine gastropod Hydrobia ulvae (Gastropoda: Hydrobiidae). Biol J Linn Soc 100:439–450
Wells R (1980) Activity pattern as a mechanism of predator avoidance in two species of Acmaeid limpet.
J Exp Mar Bio Ecol 48:151–168
Welsh DK, Imaizumi T, Kay SA (2005) Real-time reporting of circadian-regulated gene expression by
luciferase imaging in plants and mammalian cells. Methods Enzymol 393:269–88
Williams BG, Naylor E (1969) Synchronization of the locomotor rhythm of Carcinus. J Exp Biol:715–
725
Williams BG, Pilditch CA (1997) The Entrainment of Persistent Tidal Rhythmicity in a Filter-Feeding
Bivalve Using Cycles of Food Availability. J Biol Rhythms 12:173–181
Wilsbacher LD, Yamazaki S, Herzog ED, Song E-J, Radcliffe LA, Abe M, Block G, Spitznagel E,
Menaker M, Takahashi JS (2002) Photic and circadian expression of luciferase in mPeriod1-luc
transgenic mice invivo. Proc Natl Acad Sci U S A 99:489–94
Wolcott TG (1973) Physiological Ecology and Intertidal Zonation in Limpets (Acmaea): A Critical Look
at “Limiting Factors.” Biol Bull 145:389–422
Zann LP (1973) Relationships between intertidal zonation and circatidal rhythmicity in littoral
gastropods. Mar Biol 18:243–250
Zhang L, Hastings MH, Green EW, Tauber E, Sladek M, Webster SG, Kyriacou CP, Wilcockson DC
(2013) Dissociation of Circadian and Circatidal Timekeeping in the Marine Crustacean Eurydice
pulchra. Curr Biol 40:1–11

73

Table 1. Summary of entrainment experiments observing movement in Boreal limpet Lottia
paradigitalis. * indicates that the number of tidal cycles noted was the number for the entire duration of
free-running recorded.

Treatment Entrainment period (hr) Free-running period (hr) # of tidal cycles persisted
Field entrainment ~12.4 12.85 5
Simulated tide, free-running in constant light 12.5 11.88 5*
Simulated tide, free-running in constant darkness 12.5 12.83 4*
Simulated tide 9 12.56 12*
Simulated tide 15 12.25 10*
Simulated tide 24 15.97 9*
Only light/dark oscillations 12 24.9 4
Only light/dark oscillations 24 16.23 9*
Only emersion/immersion oscillations 12 12.17 2
Only flow oscillations 6 6.07 4
Only flow oscillations 12 12.63 4
Only temperature oscillations 12 13.02 4

74

Figure 1. Limpets entrained in the field then moved to constant laboratory conditions exhibit circatidal
and circadian locomotor rhythms. a) Actogram of limpet movement over the course of 7 days in constant
laboratory conditions. Lomb-Scargle periodograms showing significant movement periods (p<0.05) b)
over the entire time course, c) during the first 5 tidal cycles, d) during the remainder of the experiment.

26.08
0 6 12 18 24
Days
1
5
Time of day
a)
d)
c)
b)
12.85
12.4
26.28
500
500
1000
500
1000
0
0
0
Period (hr)
PN PN PN

75

Figure 2. Limpet movement under constant laboratory conditions (black) in relation to the corresponding
anticipated tide (blue) occurring at the collection site during the course of the experiment.

0
2
4
6
8
10
12
14
5/11/15 11:02 5/12/15 11:02 5/13/15 11:02 5/14/15 11:02 5/15/15 11:02 5/16/15 11:02 5/17/15 11:02 5/18/15 11:02
Time
Activity Anticipated tide

76

Figure 3. Limpets entrained to a simulated laboratory 12.5-hour tidal cycle then released into constant
laboratory conditions with constant light exhibit circatidal locomotor rhythms. Actograms showing limpet
movement during a) entrainment and b) free-running. Lomb-Scargle periodograms showing significant
movement periods (p<0.05) during c) entrainment and d) free-running.
0 6 12 18 24
Time of day
a)
d)
c)
b)
Days Days
12.42
11.88
Period (hr)
500
1000
0
PN
400
800
0
PN

77

Figure 4. Limpets entrained to a simulated laboratory 12.5-hour tidal cycle then released into constant
laboratory conditions with constant dark exhibit circatidal locomotor rhythms. Actograms showing limpet
movement during a) entrainment and b) free-running. Lomb-Scargle periodograms showing significant
movement periods (p<0.05) during c) entrainment and d) free-running.

0 6 12 18 24
Time of day
a)
d)
c)
b)
Days Days
12.45
12.83
500
1000
0
PN
500
1000
0
PN
Period (hr)

78

Figure 5. Limpets entrained to a simulated laboratory 9-hour tidal cycle then released into constant
laboratory conditions exhibit circatidal locomotor rhythms. Actograms showing limpet movement during
a) entrainment and b) free-running. Lomb-Scargle periodograms showing significant movement periods
(p<0.05) during c) entrainment and d) free-running over the entire time course, e) the first 24 hours of
free-running, f) the remainder of the experiment.

0 6 12 18 24
Time of day
Days Days
a)
d)
c)
b)
e)
f)
9.02
12.68
9.95
12.77
Period (hr)
1000
2000
0
PN
1000
2000
0
PN
1000
2000
0
PN
300
0
200
100
PN

79

Figure 6. Limpets entrained to a simulated laboratory 15-hour tidal cycle then released into constant
laboratory conditions exhibit circatidal locomotor rhythms. Actograms showing limpet movement during
a) entrainment and b) free-running. Lomb-Scargle periodograms showing significant movement periods
(p<0.05) during c) entrainment and d) free-running.

0 6 12 18 24
Time of day
Days Days
a)
d)
c)
b)
Period (hr)
1000
2000
0
PN
500
0
PN
15.07
12.28

80

Figure 7. Limpets entrained to a simulated laboratory 24-hour tidal cycle then released into constant
laboratory conditions exhibit near circatidal locomotor rhythms. Actograms showing limpet movement
during a) entrainment and b) free-running. Lomb-Scargle periodograms showing significant movement
periods (p<0.05) during c) entrainment and d) free-running.

0 6 12 18 24
Time of day
a)
d)
c)
b)
Days Days
Period (hr)
1000
2000
0
PN 1000
2000
0
PN
24.05
15.97

81

Figure 8. Limpets entrained to a 12-hour light/dark only cycle (LD 6:6) then released into constant
laboratory conditions exhibit circadian locomotor rhythms. Actograms showing limpet movement during
a) entrainment and b) free-running. Lomb-Scargle periodograms showing significant movement periods
(p<0.05) during c) entrainment and d) free-running.

0 6 12 18 24
Time of day
Days Days
a)
c)
b) d)
15.97 24.9
Light/dark
12.38
500
1000
0
PN
200
400
0
PN
Period (hr)

82

Figure 9. Limpets entrained to a 24-hour light/dark only cycle (LD 12:12) then released into constant
laboratory conditions exhibit circatidal locomotor rhythms during entrainment and neither circatidal nor
circadian rhythms during free-running. Actograms showing limpet movement during a) entrainment and
b) free-running. Lomb-Scargle periodograms showing significant movement periods (p<0.05) during c)
entrainment and d) free-running.

0 6 12 18 24
Time of day
11.87
23.32
Days Days
a)
d)
c)
b)
Light/dark
11.88
16.23
400
800
0
PN
400
800
0
PN
Period (hr)

83

Figure 10. Limpets entrained to a 12-hour emersion only cycle (emersion:submersion 6:6) then released
into constant laboratory conditions exhibit circadian and circatidal locomotor rhythms. Actograms
showing limpet movement during a) entrainment and b) free-running. Lomb-Scargle periodograms
showing significant movement periods (p<0.05) during c) entrainment, d) free-running over the entire
time course, e) the first 24 hours of the time course, f) the remainder of the time course.

0 6 12 18 24
Time of day
14.7
Days Days
a)
d)
c)
b)
e)
f)
24.08
12.45
24.6
12.17
1000
2000
0
PN
400
600
0
200
PN
400
0
200
PN
0
200
PN
Period (hr)

84

Figure 11. Limpets entrained to a 6-hour flow only cycle (flow high:low 3:3) then released into constant
laboratory conditions. Actograms showing limpet movement during a) entrainment and b) free-running.
Lomb-Scargle periodograms showing significant movement periods (p<0.05) during c) entrainment, d)
free-running over the entire time course, e) the first 24 hours of the time course, f) the remainder of the
time course.

0 6 12 18 24
Time of day
Days Days
a)
d)
c)
b)
f)
e)
6.02
17.02
6.07
15.08
Period (hr)
1000
3000
0
2000
PN
100
300
0
200
PN
100
0
200
PN
100
300
0
200
PN

85

Figure 12. Limpets entrained to a 12-hour flow only cycle (flow high:low 6:6) then released into constant
laboratory conditions. Actograms showing limpet movement during a) entrainment and b) free-running.
Lomb-Scargle periodograms showing significant movement periods (p<0.05) during c) entrainment, d)
free-running over the entire time course, e) the first 48 hours of the time course.

0 6 12 18 24
Time of day
a)
d)
c)
b)
e)
Days Days
Period (hr)
200
600
0
400
PN 200
600
0
400
PN
1000
3000
0
2000
PN
12.12
12.62
12.63

86

Figure 13. Limpets entrained to a 12-hour temperature only cycle (temperature low:high 6:6) then
released into constant laboratory conditions. Actograms showing limpet movement during a) entrainment
and b) free-running. Lomb-Scargle periodograms showing significant movement periods (p<0.05) during
c) entrainment, d) free-running over the entire time course, e) the first 48 hours of the time course.

0 6 12 18 24
Time of day
Days Days
a)
d)
c)
b)
e)
11.63
18.37
46.85
13.02
200
600
0
400
PN
200
600
0
400
PN
200
0
400
PN
Period (hr)

87

Figure 14. Limpet movement (black) during 12-hour temperature only cycle (temperature low:high 6:6,
red) and during free-running in relation to the anticipated temperature cycle (grey). The blue arrow marks
time of release into free-running conditions.

18
19
20
21
22
23
24
25
2/5/17 5:16 2/7/17 5:16 2/9/17 5:16 2/11/17 5:16 2/13/17 5:16 2/15/17 5:16
Series1 Series2 Series3
Temperautre Activity
Time

88
CHAPTER 3
Multi-tissue transcriptomic analysis of locomotor rhythms and circatidal gene expression in free-
running Boreal limpet Lottia paradigitalis

Abstract
The marine intertidal zone is a regularly-changing environment, oscillating between very
different conditions during high tide versus low tide. As such, limpets exhibit circatidal locomotor
rhythms, foraging during high tide and staying sheltered in place during low tide. Some studies have
shown that this rhythmic movement behavior is endogenous, rather than a response to the ebb and flow of
the tide. Many other intertidal animals exhibit similar endogenous circatidal behavior rhythms. Coupled
with the highly contrasting environmental conditions experienced during high versus low tide, these
endogenous behavior rhythms suggest that intertidal organisms have an endogenous time-keeping
mechanism that allows them to anticipate the changing of the tides and to make the appropriate behavioral
and physiological adjustments – the circatidal clock. Although there has been some work done
investigating circatidal gene expression rhythms in various intertidal animals, the molecular mechanisms
of endogenous limpet movement and the circatidal clock remain unknown. In this study, we investigated
those two questions in two different limpet tissues: head and foot. We identified some genes of interest
that persist with circatidal expression in free-running limpets. We also found that genes associated with
limpet movement are enriched for feeding, locomotion, and muscle activity. These results provide a
foundation for further investigations of the molecular mechanism of the circatidal clock in limpets.

Introduction
Limpets and the intertidal zone
Limpets are herbivorous gastropod mollusks that live in the intertidal zone (Branch, 1985). They
exhibit circatidal (~12.4hr) foraging behavior, moving during high tide and returning to their home scars
or home areas before low tide (Galbraith 1965, Little et al. 1988, 2009). This seems to be a strategy for

89
reducing desiccation risk, which is important, because experiments with Patella depressa show that even
a sublethal level of desiccation leads to osmotic imbalance, reduced muscle tension, and reduced
adhesion, all of which can result in death (Branch 1981). Heat stress is another hazard of low tide,
especially because limpet bodies are always warmer than the already high temperatures of the
surrounding air and rock (Vermeij 1972, Wolcott 1973).
Other reasons for the specific timing of foraging expeditions include defense against predators
(Wells 1980, Garrity & Levings 1983) and avoidance of osmotic stress (Little et al. 1990). Shorebirds do
not dive for prey, and only forage when at least part of the intertidal zone is exposed (Evans 1988). Since
limpets cannot adhere to the substrate as well and are thus more easily picked off while they are in
motion, it is a matter of survival for them not to be moving when predators are foraging (Branch 1981).
And as osmoconformers, limpets’ sole defense against osmotic stress is to clamp down on the substrate
and shut out the external environment (Hoyaux et al. 1976). Indeed, clamping down is also their main
defense against wave action and desiccation. This is why some limpet species physically and chemically
bore into the rock in order to create a perfectly-fitting home scar. Their strong shell muscle grips the
rock, aided by a sticky mucus secreted by the foot (Davies & Hawkins 1998). Strong adhesion is
important because if a limpet is dislodged and flipped over, it is unable to right itself on its own (Heller
2015).
Because of the highly contrasting environmental conditions between high versus low tide,
especially the different potential stresses of low tide, and because of the extensive documentation of
endogenous circatidal movement rhythms in various intertidal animals, it has been hypothesized the
intertidal organisms contain a molecular mechanism (similar to the circadian clock) that allows them to
track and anticipate the changing of the tides, allowing them to regulate their physiologies accordingly.
This is the hypothesized circatidal clock, of which the molecular mechanism is currently unknown.

90
Endogenous locomotor rhythms in limpets
Some studies have shown that rhythmic locomotor activity in limpets is endogenous and not just
a reaction to the changing of the tides. Della Santina and Naylor (1994) found that rhythmic homing
behavior in the common European limpet Patella vulgata was endogenous. They transferred limpets
from the field to constant laboratory conditions of constant darkness, 14°C, and emersion with continuous
fine water spray, and found that free-running limpets persisted in circatidal and circadian movement
rhythms for four days.
Gray and Hodgson (1999) conducted similar experiments with the high shore prickly limpet
Helcion pectunculus. Limpets were transferred from the field to constant laboratory conditions of
constant darkness, 25°C, and emersion in moist air. They persisted in circatidal movement with circadian
modulation for three days of free-running.
Gray and Williams (2010) conducted similar but more complex experiments with the high shore
tropical limpet Cellana grata. Limpets were transferred from the field to constant laboratory conditions
of constant darkness or white light, 25.5°C, and constant immersion, emersion, or seawater spray. In all
white light treatments, free-running limpets exhibited circatidal movement for at least two days. In all
darkness treatments, free-running limpets exhibited circatidal movement for at least four days.
Interestingly, free-running limpets kept in constant white light and constant seawater spray exhibited
persistent circatidal movement for 30 days. However, the molecular mechanisms underlying endogenous
rhythmic limpet movement is not known; nor is it known if these locomotor rhythms are under control of
the circatidal clock.

Rhythmic gene expression in intertidal mollusks
To date, we are aware of only three other studies that have examined the transcriptomes
underlying biological rhythms in mollusks: California ribbed mussel Mytilus californianus (Connor &
Gracey 2011), Pacific oyster Crassostrea gigas (Payton et al. 2017), and Red Sea limpet Cellana rota

91
(Schnytzer et al. 2018). Connor and Gracey (2011) subjected M. californianus to a simulated intertidal
regime with alternating 6-hour high and low tides, with a 12:12 LD cycle. Gill samples were taken every
two hours for 96 hours, and gene expression was measured using microarray analysis. 236 transcripts
were found to oscillate with a circatidal rhythm (period=10-14hr), compared with 2365 transcripts that
oscillated with a circadian rhythm (period=24-28hr). This was followed up with a similar experiment
with M. californianus acclimatized to field intertidal and subtidal conditions, with intertidal animals
emerged for half of each tidal cycle and submerged for half of each tidal cycle, while subtidal animals
were always submerged. Gill samples were taken every two hours for 50 hours, and gene expression was
measured using microarray analysis. Many of the circatidal genes identified in the simulation experiment
also exhibited circatidal rhythmicity under field conditions, with 44 transcripts exhibiting circatidal
rhythmicity across all treatments.
Payton et al. (2017) entrained C. gigas to a 9:15 LD cycle with constant submergence and fed
either harmful or non-harmful algae. Gill samples were taken every four hours for 52 hours, and gene
expression was measured using RNA-seq. Oysters fed non-harmful algae exhibited circadian expression
in 1300 transcripts, while oysters fed harmful algae exhibited circadian expression in only 630 transcripts.
However, oysters fed non-harmful algae exhibited circatidal expression in 1576 transcripts, compared
with 2207 circatidal transcripts in oysters fed harmful algae. Interestingly, some of the circatidal
transcripts were of genes involved in the circadian clock network.
Schnytzer et al. (2018) collected C. rota limpets directly from the field for RNA-seq analysis.
Head samples collected every four hours for two days were analyzed. 367 transcripts were found to be
circatidal, while 221 transcripts were found to be circadian. Interestingly, some of the circatidal genes
they found are somehow related to the circadian clock, while the core clock genes cryptochrome (cry),
clock, and Bmal1 exhibited circadian expression rhythms that were not significant. However, although
they also examined limpet locomotor activity, they did not produce corresponding gene expression data.
These studies provide a basic foundation for investigating the molecular mechanisms of the
circatidal clock insofar as they provide a list of potential genes to further investigate. However, none of

92
them examine the transcriptomes of free-running animals, so it is difficult to distinguish if those gene
expression rhythms are part of an endogenous clockwork or if they are only a response to the oscillations
between the extremely different environmental conditions associated with high and low tides. It is
necessary to identify persistent circatidal genes in free-running animals in order to conclude that the
circatidal gene expression is endogenous and subsequently determine key components of the circatidal
clock. In this study, we aim to investigate the gene expression profiles associated with free-running
limpet movement, as well as identify candidate circatidal genes in Boreal limpet Lottia paradigitalis
released into constant laboratory conditions after entrainment to a simulated intertidal environment.

Methods
Collection and experimental set-up
Boreal limpets Lottia paradigitalis sized ~2cm were collected from the mid- to upper intertidal
zone at Will Rogers State Beach (Santa Monica, CA) at low tide and immediately transported to our
laboratory tidal simulation aquarium located at USC in a temperature-controlled environmental chamber,
which is maintained at 15°C. Will Rogers State Beach is located in Santa Monica Bay, which
experiences a mixed semi-diurnal tidal regime, with a tidal range of ~2.7m.
Limpets were housed in custom-built cages that allow for clear viewing through the top. 4
limpets were kept in each cage. Two cages with six limpets each were designated as the viewing cages.
The movement of these limpets (n=12) was video recorded throughout entrainment and free-running.
Limpets were positioned such that they were submerged during high tide and emerged during low
tide, and entrained to a 12.4-hour tidal cycle (alternating submergence and emergence in 6.2 hour
intervals) with 12:12 LD cycle. Water flow was modulated using Turbelle stream water pumps (Tunze,
Penzberg, Germany) such that the water was slack during the times flanking the changing of the tides,
with step-wise increase and decrease in flow; maximum flow occurred at the middle of high tide. Light
(AquaSun FR20T12/VHO, Zoo Med Laboratories, San Luis Obispo, CA) was set to a LD 12:12 cycle.
All of the tidal simulation aquarium settings were programmed and controlled using an Apex

93
Aquacontroller (Neptune Systems, Morgan Hill, CA). After an entrainment period of at least 2 weeks,
limpets were released into free-running conditions of constant temperature, darkness, flow, and
submergence.
Starting from initial release into free-running conditions (t0), one cage of 4 limpets was sampled
every two hours, for a total of 48 hours. At each time point, the head, foot, and hepatopancreas were
excised out of each limpet, then submerged and stored in RNAlater (Invitrogen, Carlsbad, CA).
Harvested tissues were stored in RNAlater at -20°C until further processing.
Head and foot tissue were removed from RNAlater and RNA was isolated using the phenol-
chloroform extraction method with TRIzol reagent, according to the manufacturer instructions
(Invitrogen, Carlsbad, CA). Isolated RNA was further purified using glass-fiber spin columns (Qiagen,
Hilden, Germany), according to manufacturer instructions. For each tissue type, equal amounts of RNA
from each individual were pooled for every time point.

RNA-seq analysis
Pooled RNA from each time point was used to create Illumina sequencing libraries using an
optimized in-house protocol. Briefly, RNA was subjected to polyA+ selection using oligodT magnetic
beads (SeraMag, Pittsburgh, PA), reverse transcribed (MMLV-RT HP, Epicentre, Madison, WI), size-
fragmented, and then PCR-enriched (KAPA Real-time Library Amplification Kit, Wilmington, MA).
Libraries were submitted to the University of Oregon Genomics and Cell Characterization Core
Facility for sequencing on the Illumina HiSeq 4000 platform with paired-end 100bp reads.
A de novo transcriptome assembly was created using IDBA (Peng et al. 2010) and a putative non-
redundant set of 19,457 transcripts were selected for mapping RNA-seq reads. The reads were mapped to
the assembly and transcript abundance, calculated as transcripts per million (TPM) per sample, was
estimated using Salmon (Patro et al. 2017). Hierarchical clustering analysis and visual inspection of the
data was used to determine that time point five was an outlier in both tissues sampled, so time point five
was removed from gene expression analysis (Gould 2018). Gene expression data were centered by

94
dividing the relative expression of each gene by the median expression of that gene across the 24 samples
collected from the same tissue.

Data analysis
Rhythmic genes were identified using JTK_CYCLE, a nonparametric statistical algorithm for R
that identifies rhythmic components in large genomic datasets. It applies the Jonckheere-Terpstra-
Kendall algorithm to alternative hypothesized group orderings based on user-defined periods and phases,
as well as cosine curves, in order to optimize period and phase to minimize the p-value of Kendall’s tau
correlation between an experimental time series and the group orderings (Hughes et al. 2010, R Core
Team 2014). Transcripts were assigned Gene Ontology (GO) terms using DAVID (Ashburner et al.
2000, Huang et al. 2009, Carbon et al. 2017). GO term enrichment was assessed using REVIGO with
medium (0.7) allowed similarity (Supek et al. 2011). Enrichment was reported as uniqueness values,
which correspond to overrepresentation of GO terms in relation to average background representation.

Movement video analysis
Limpet movement was recorded using a USB webcam (Toogoo, China) connected to a dedicated
laptop. Time lapse video was made of limpet movement throughout the experiment. Video of
entrainment and free-running was scored as described in Gray and Williams (2010), with slight
modifications. Each video frame was scored for presence and absence of average movement, wherein
movement was documented if over 20% of the group was moving. The number of active limpets was also
recorded for each frame. If a limpet moved out of frame, it was not counted until it returned, so activity
was calculated as the proportion of moving limpets divided by all visible limpets. The same person
blindly (ie. without prior knowledge of experimental conditions) scored all of the videos in order to
maintain consistency. Reanalysis revealed that the two scoring methods did not produce significantly
different results (unpub).
Lomb-Scargle periodogram analysis was performed using the ActogramJ plug-in (Schmid et al.,

95
2011) for ImageJ (Abràmoff et al. 2004) to determine significant (p<0.05) periods in movement. This
periodogram is derived from Fourier spectral analysis, and is calculated as follows:
!" ω =
1
2(
)

*
+
− * cos0(2
+
−3)
+
)
cos
)
0(2
+ +
− 3)
+
*
+
− * sin0(2
+
−3)
+
)
sin
)
0
+
(2
+
– 3)

given a time series of N data points *
+
= *(2
+
) collected at times t
j
where j = 1,2,…N, with a mean of *,
and where t is defined as:
3 =
9
):
tan
=9
>?@) : A
B B
CD>) :
B
A
B

PN gives the normalized power as a function of angular frequency w (Ruf 1999).

Results
Free-running limpet movement
Limpets entrained to a simulated intertidal regime exhibited circatidal movement during
entrainment and when released into free-running constant conditions, with movement periods of 12.45
and 12.02 hours, respectively (Figure 1).

Gene expression in head tissue samples from free-running limpets
Tissue samples were also taken during free-running in order to examine the gene expression
profiles in head and foot. Limpet head samples exhibited significant rhythmic gene expression, with 386
transcripts displaying significant expression with a period between 10 and 28 hours (p<0.05, Figure 2a).
When the phase of the circatidal (period=10-14hr) genes was analyzed, it was revealed that the greatest
number of transcripts peaked during anticipated high tide, approaching anticipated low tide (Figure 2b).
When the same analysis was done with circadian (period=22-28) genes, it was revealed that the greatest
number transcripts peaked at the anticipated transition from dark to light (Figure 2c).
Overall, in limpet head tissue samples, 85 transcripts cycled with a circatidal period, while 164
transcripts cycled with a circadian period (Figure 3). Many of the circatidal transcripts peaked during

96
anticipated high tide, which also coincided with bouts of limpet movement for the first three tidal cycles
of free-running. In order to assess the function of these circatidal genes, GO terms were assigned (Table
1). The majority of the assigned GO terms that were consistent with movement and foraging: metabolic
process (GO:0008152), rhythmic process (GO:0048511), locomotory behavior (GO:0007626), hydrogen
peroxide metabolic process (GO: 0042743), and response to activity (GO:0014823). These are
exemplified by the expression profiles of three genes in particular: myoglobin, hepatocyte growth factor
receptor (HGFR), and acidic mammalian chitinase (AMCase), all of which are related to foraging (Figure
4). Myoglobin and HGFR expression peaks almost match perfectly with bouts of limpet movement,
while AMCase peaks during non-movement (Figure 4).

Gene expression in foot tissue samples from free-running limpets
Free-running limpets exhibited significant gene expression rhythms in foot tissue samples, with
691 transcripts displaying gene expression rhythms with a period between 10 and 28 hours (p<0.05,
Figure 5a). Examining the phase of the rhythmic transcripts revealed that the greatest number of
circatidal (period=10-14hr) transcripts peaked towards the end of anticipated low tide (Figure 5b).
Meanwhile, the greatest number of circadian (period=22-28hr) peaked towards the end of the anticipated
dark phase (Figure 5c).
Overall, in limpet foot tissue samples, 174 transcripts exhibited circatidal gene expression
rhythms, while 416 transcripts exhibited circadian gene expression rhythms (Figure 6). The majority of
circatidal transcripts peaked at the just before anticipated high tide and the onset of movement. In order
to determine the function of these circatidal genes, GO terms were assigned (Table 2). Among the
assigned GO terms, notable terms include sensory perception of sound (GO:0007605), locomotory
behavior (GO:0007620), response to iron ion (GO:0010039), skeletal muscle satellite cell proliferation
(GO:0014841), amyloid precursor protein catabolic process (GO:0042987), and hematopoietic stem cell
homeostasis (GO:0061484).

97
Based on amplitude of oscillations and GO terms, interesting genes with circatidal rhythmicity
include stress-related genes peroxiredoxin-5 and multidrug and toxin extrusion protein 1 (aka multi-
antimicrobial extrusion protein 1, MATE1), neurological genes endothelin-converting enzyme 2 (ECE2)
and 4-aminobutyrate aminotransferase (ABAT), and work and muscle-related genes isocitrate
dehydrogenase (IDH), dynein heavy chain, and sarcoplasmic calcium-binding protein (SCP). These
genes have fairly similar expression profiles, peaking at or around anticipated high tide and also during
bouts of limpet movement (Figure 7).

Discussion
Using a combination of movement and transcriptomic analyses, we investigated free-running
circatidal gene expression in two different tissues (head and foot) in limpets. Consistent with results
found in Chapter 2, as well as previous studies on limpet movement (Della Santina & Naylor 1994, Gray
& Hodgson 1999, Gray & Williams 2010), the limpets in this study continued to exhibit circatidal (period
= 12.02hr) locomotor rhythms even in constant free-running conditions (Figure 1), providing additional
evidence for an endogenous mechanism that controls limpet movement.
Among genes exhibiting a circatidal expression period in head tissue samples from free-running
limpet, the greatest number had peaks during high tide (Figure 2b), the time during which limpets are
most likely to move. Indeed, GO term enrichment of circatidal transcripts in limpet head was dominated
by GO terms consistent with movement and foraging: metabolic process (GO:0008152), rhythmic process
(GO:0048511), locomotory behavior (GO:0007626), hydrogen peroxide metabolic process (GO:
0042743), and response to activity (GO:0014823) (Table 1). Locomotory behavior and response to
activity are obviously consistent with movement. The enrichment for genes associate with metabolic
processes makes sense as well because it is well-documented that one of the main reasons limpets move is
in order to forage and feed (Hartnoll & Wright 1977, Little et al. 1988, Della Santina & Chelazzi 1991,
Evans & Williams 1991). The presence of genes related to hydrogen peroxide metabolic processes is
consistent with the work that has to be done during movement, as it relates to respiration and supports the

98
increased demand for ATP during limpet movement (Santini & Chelazzi 1996, Santini et al. 2004). The
hydrogen peroxide metabolic process GO term refers to the metabolism of hydrogen peroxide (a reactive
oxygen species) resulting from cellular respiration, preventing it from causing DNA damage and cell
dysfunction or even death (Boveris & Chance 1973, Milev & Reddy 2015). Importantly, rhythmic
process appears as a GO term assigned to these circatidal genes, underscoring that these genes have been
associated with rhythmic processes in other contexts as well.
Several interesting circatidal genes in limpet head were consistent with the GO term annotations,
including myoglobin, hepatocyte growth factor receptor (HGFR, aka c-Met, which is encoded by the
MET gene), and acidic mammalian chitinase (AMCase), all of which are related to foraging. Myoglobin
and HGFR expression peaks almost match perfectly with bouts of limpet movement, while AMCase
peaks during non-movement (Figure 4). Myoglobin is vital to foraging, because the muscles attached to
the radula (the rasping tongue that limpets use to feed) are the most powerful muscles in the limpet body
and are therefore the only muscles supplied with myoglobin (iron-based oxygen-binding protein) rather
than the hemocyanin (copper-based oxygen-binding protein) that circulates through the rest of the body
(Heller 2015). HGFR, or c-Met, is a receptor tyrosine kinase, and its ligand is hepatocyte growth factor
(HGF), also known as scatter factor. This ligand name is appropriate, because one of the main
responsibilities of c-Met is cell-scattering, which is a vital process in wound repair. Foraging is the main
time during which a limpet might become injured. C-Met is also important in long-range migration of
skeletal muscle progenitor cells, as well as proliferation and survival of hepatocytes (Organ & Tsao 2011)
As implied by its name, AMCase breaks down chitin, which is often found in arthropod exoskeletons and
fungi cell walls, but can also be found in the cell walls of some algae (Pearlmutter & Lembi 1978, Boot et
al. 2001). We suggest that AMCase aids limpets in their digestion of the algae they feed on, and that this
takes place during bouts of non-movement at low tide, when the limpets have ample time to digest the
long-chain polysaccharide. The other plant-based digestive enzyme that exhibited significant rhythmic
expression, xylose isomerase, had a period of 18 hours, so it could not be counted as circatidal, but two of
its expression peaks correspond with AMCase expression peaks (not shown).

99
Genes exhibiting circatidal expression rhythms in limpet foot peaked more often during
anticipated low tide, towards the end of low tide (Figure 5b). Therefore, it makes sense that the GO terms
assigned to these genes are not as obviously movement-related. However, closer inspection of some of
the notable terms -- sensory perception of sound (GO:0007605), response to iron ion (GO:0010039),
skeletal muscle satellite cell proliferation (GO:0014841), amyloid precursor protein catabolic process
(GO:0042987), and hematopoietic stem cell homeostasis (GO:0061484) – suggests that they are
consistent with preparing to move and forage. Examining Table 2, the assignment of the GO term
locomotory behavior (GO:0007626) stands out as a potential inconsistency (Table 2). However, it may
be partially explained by the observation that this species of limpets tends to start wiggling around in its
home area prior to actually setting out to forage (personal observation).
Sensory perception of sound may be related to the sound of the swash, waves breaking onto the
shore; although there are no studies on the effect of swash sounds on limpets, it has been shown that
movement in the clam Donax variabilis can be entrained to the sound of the swash (Ellers 1995a, b). The
skeletal muscle satellite cell proliferation term makes sense because the foot muscle is the main effector
of movement (Fisher 1904, Branch 1981). Not much is known about amyloid proteins apart from their
role in causing Alzheimer’s disease, which is characterized by a gradual decline in cognitive function
(Murphy & Iii 2010). Since learning and memory have been suggested to be involved in limpet
movement (Heller 2015), it may be that catabolizing amyloid proteins is part of the memory and learning
process. Enrichment in genes responding to iron ion is interesting because the radula is impregnated with
iron and requires daily iron inputs in order to form new teeth and maintain optimal radula condition
(Shaw et al. 2008). Hematopoietic stem cells are produced in the bone marrow and important for immune
function because they are the precursors of leukocytes (Gunsilius et al. 2001). Limpets are more likely to
be exposed to pathogens when they are moving and foraging, so this could represent a preparation of
immune defenses. This is especially interesting because the genes these GO terms have been assigned to
tend to peak during low tide, the times during which our previously identified candidate tidal genes from

100
Chapter 1 generally peak, and one of our candidate tidal genes is complement C1q-like protein, a member
of the complement system, which is a part of the innate immune system.
Based on oscillation amplitude and GO terms, a number of interesting genes were identified
among the circatidal genes in foot tissue samples from free-running limpets entrained to a simulated
intertidal cycle. These genes have fairly similar expression profiles, peaking at or around anticipated high
tide and also during bouts of limpet movement (Figure 7). Peroxiredoxin-5 and multidrug and toxin
extrusion protein 1 (aka multi-antimicrobial extrusion protein 1, MATE1) are stress-related genes.
Peroxiredoxin-5 is an antioxidant that can localize to many subcellular compartments to protect against
oxidative stress (Knoops et al. 2011). Importantly, due to their antioxidant properties and ability to
maintain redox homeostasis, the whole family of peroxiredoxins has been suggested to be inextricably
linked to the evolution of circadian rhythms (Edgar et al. 2012). Endothelin-converting enzyme 2 (ECE2)
and 4-aminobutyrate aminotransferase (ABAT) are both related to neurological function. ECE2 is a
metalloprotease that may have a role in processing peptides important in learning and memory (Devi &
Miller 2013), while ABAT is involved in GABA metabolism (Park et al. 1993). Muscle and work-related
genes of interest include isocitrate dehydrogenase (IDH), dynein heavy chain, and sarcoplasmic calcium-
binding protein (SCP). IDH is especially interesting, because it was previously identified as a candidate
circatidal gene across all three of the mussel treatments in Chapter 1.

Variability in tissue-specific gene expression
For this study, we chose to analyze head and foot tissues in limpet. The head was chosen because
it contains the cerebral ganglia, the closest anatomical structure to a brain that limpets possess (Fisher
1904). In circadian research, the brain, and more specifically, the suprachiasmatic nucleus (SCN), is the
central pacemaker of the circadian clock, controlling the circadian rhythms in the rest of the body
(Takahashi 1995). The foot was chosen because it is the main effector of limpet movement (Heller 2015),
in addition to being the most plentiful and easily accessible tissue in limpets. While there are interesting
circatidal genes in each of the separate tissues, when they were compared against each other, only three

101
genes were significantly circatidal in expression rhythms in both tissues: protocadherin-9, GTPase IMAP
family member 4 (GIMAP4), and a contig that we were not able to annotate. Although the functions of
specific protocadherin family members are not thoroughly understood, the group overall plays a role in
cell-cell interactions, particularly cell-cell adhesion in neural tissues (Hayashi & Takeichi 2015).
GIMAP4 is a lymphocyte cell-survival regulator, promoting apoptosis in mature T cells (Nitta &
Takahama 2007).
Based on our results from Chapter 1, we would have expected more overlap, and for the
overlapping genes to be transcriptional regulators. However, a main difference between Chapter 1 and
this study is that gill tissue (the most abundant and easily accessible tissue in bivalves) was sampled
across all treatments in Chapter 1, while in this study, we sampled two different tissues, head and foot. It
has been shown throughout circadian research that different tissue types sampled from the same animal
will not necessarily exhibit the same numbers of cycling transcripts, nor will those transcripts necessarily
match across different tissue types or even cycle with the same phase (Yan et al. 2008). This may be due
to tissue-specific sets of clock-controlled genes with different phase distributions (Korenčič et al. 2014).
In a recent study analyzing gene expression in 64 different tissues in baboons, the number of rhythmic
transcripts in any given tissue could range over ten-fold from fewer than 200 to over 3000 (Mure et al.
2018). Moreover, there was only limited overlap between different tissues of the genes that exhibited
rhythmic expression, and no single transcript was significantly rhythmic across all of the tissues sampled,
even the known core circadian clock genes. Mure et al. (2018) suggest that this may be due to differing
composition of transcriptional activators, repressors, and modulators in different tissues, resulting in
tissue-specific input and output mechanisms of even the core circadian genes. Therefore, it may be due to
tissue-specific mechanisms that we did not find many circatidal transcripts in common between limpet
head and foot.

102
Future studies
The results of these experiments provide support for an endogenous circatidal oscillator in
limpets, although whether or not it is the same mechanism as in mussels and oysters remains to be seen
through further experimentation. Given the list of genes identified in this study, and due to the
interconnectedness of genes and proteins involved in central oscillators like the circadian clock,
identification of a few important genes close to the circatidal central oscillator will lead to the discovery
of even more circatidal clock components, through experiments involving more transcriptome screens,
co-immunoprecipitations, and RNA interference (RNAi). This will ultimately lead to the construction of
network models of the limpet circatidal clock, adding an important piece to our understanding of these
animals.

References

Abràmoff MD, Magalhães PJ, Ram SJ (2004) Image processing with imageJ. Biophotonics Int 11:36–41
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS,
Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE,
Ringwald M, Rubin GM, Sherlock G (2000) Gene ontology: tool for the unification of biology. The
Gene Ontology Consortium. Nat Genet 25:25–9
Boot RG, Blommaart EFC, Swart E, Ghauharali-van der Vlugt K, Bijl N, Moe C, Place A, Aerts JMFG
(2001) Identification of a Novel Acidic Mammalian Chitinase Distinct from Chitotriosidase. J Biol
Chem 276:6770–6778
Boveris A, Chance B (1973) The mitochondrial generation of hydrogen peroxide. General properties and
effect of hyperbaric oxygen. Biochem J 134:707–716
Branch GM (1981) The Biology of Limpets: physical factors, energy flow, and ecological interactions.
Oceanogr Mar Biol 19:235–380
Branch GM (1985) Limpets: Evolution and Adaptation. In: The Mollusca: Evolution. Academic Press,
Inc., p 187–220

103
Carbon S, Dietze H, Lewis SE, Mungall CJ, Munoz-Torres MC, Basu S, Chisholm RL, Dodson RJ, Fey
P, Thomas PD, Mi H, Muruganujan A, Huang X, Poudel S, Hu JC, Aleksander SA, McIntosh BK,
Renfro DP, Siegele DA, Antonazzo G, Attrill H, Brown NH, Marygold SJ, Mc-Quilton P, Ponting
L, Millburn GH, Rey AJ, Stefancsik R, Tweedie S, Falls K, Schroeder AJ, Courtot M, Osumi-
Sutherland D, Parkinson H, Roncaglia P, Lovering RC, Foulger RE, Huntley RP, Denny P,
Campbell NH, Kramarz B, Patel S, Buxton JL, Umrao Z, Deng AT, Alrohaif H, Mitchell K,
Ratnaraj F, Omer W, Rodríguez-López M, C. Chibucos M, Giglio M, Nadendla S, Duesbury MJ,
Koch M, Meldal BHM, Melidoni A, Porras P, Orchard S, Shrivastava A, Chang HY, Finn RD,
Fraser M, Mitchell AL, Nuka G, Potter S, Rawlings ND, Richardson L, Sangrador-Vegas A, Young
SY, Blake JA, Christie KR, Dolan ME, Drabkin HJ, Hill DP, Ni L, Sitnikov D, Harris MA, Hayles
J, Oliver SG, Rutherford K, Wood V, Bahler J, Lock A, Pons J De, Dwinell M, Shimoyama M,
Laulederkind S, Hayman GT, Tutaj M, Wang SJ, D’Eustachio P, Matthews L, Balhoff JP,
Balakrishnan R, Binkley G, Cherry JM, Costanzo MC, Engel SR, Miyasato SR, Nash RS, Simison
M, Skrzypek MS, Weng S, Wong ED, Feuermann M, Gaudet P, Berardini TZ, Li D, Muller B,
Reiser L, Huala E, Argasinska J, Arighi C, Auchincloss A, Axelsen K, Argoud-Puy G, Bateman A,
Bely B, Blatter MC, Bonilla C, Bougueleret L, Boutet E, Breuza L, Bridge A, Britto R, Hye- A-Bye
H, Casals C, Cibrian-Uhalte E, Coudert E, Cusin I, Duek-Roggli P, Estreicher A, Famiglietti L,
Gane P, Garmiri P, Georghiou G, Gos A, Gruaz-Gumowski N, Hatton-Ellis E, Hinz U, Holmes A,
Hulo C, Jungo F, Keller G, Laiho K, Lemercier P, Lieberherr D, Mac- Dougall A, Magrane M,
Martin MJ, Masson P, Natale DA, O’Donovan C, Pedruzzi I, Pichler K, Poggioli D, Poux S, Rivoire
C, Roechert B, Sawford T, Schneider M, Speretta E, Shypitsyna A, Stutz A, Sundaram S, Tognolli
M, Wu C, Xenarios I, Yeh LS, Chan J, Gao S, Howe K, Kishore R, Lee R, Li Y, Lomax J, Muller
HM, Raciti D, Auken K Van, Berriman M, Stein, Paul Kersey L, W. Sternberg P, Howe D,
Westerfield M (2017) Expansion of the gene ontology knowledgebase and resources: The gene
ontology consortium. Nucleic Acids Res 45:D331–D338
Connor KM, Gracey AY (2011) Circadian cycles are the dominant transcriptional rhythm in the intertidal

104
mussel Mytilus californianus. Proc Natl Acad Sci U S A 108:16110–5
Davies MS, Hawkins SJ (1998) Mucus from Marine Molluscs. Elsevier Masson SAS
Devi LA, Miller LK (2013) Endothelin-Converting Enzyme-2. Handb Proteolytic Enzym 1:632–635
Edgar RS, Green EW, Zhao Y, Ooijen G van, Olmedo M, Qin X, Xu Y, Pan M, Valekunja UK, Feeney
KA, Maywood ES, Hastings MH, Baliga NS, Merrow M, Millar AJ, Johnson CH, Kyriacou CP,
O’Neill JS, Reddy AB (2012) Peroxiredoxins are conserved markers of circadian rhythms. Nature
485:459–64
Ellers O (1995a) Discrimination Among Wave-Generated Sounds by a Swash-Riding Clam. Biol Bull
189:128–137
Ellers O (1995b) Behavioral Control of Swash-Riding in the Clam Donax variabilis. Biol Bull 189:120–
127
Evans PR (1988) Predation of Intertidal Fauna by Shorebirds in Relation to Time of the Day, Tide and
Year. In: Chelazzi G, Vannini M (eds) Behavioral Adaptation to Intertidal Life. Springer US,
Boston, MA, p 65–78
Evans MR, Williams GA (1991) Time Partitioning of Foraging in the Limpet Patella vulgata. J Anim
Ecol 60:563–575
Fisher WK (1904) The anatomy of Lottia gigantea Gray. Zool Jahrbuchern Abteilung fur Anat und Ontog
der Tiere 20:1–66
Galbraith R (1965) Homing Behavior in the Limpets Acmaea digitalis and Lottia gigantea. Am Midl Nat
74:245–246
Garrity S, Levings S (1983) Homing to scars as a defense against predators in the pulmonate limpet
Siphonaria gigas (Gastropoda). Mar Biol 72:319–324
Gould J (2018) Morpheus.
Gracey AY, Chaney ML, Boomhower JP, Tyburczy WR, Connor K, Somero GN (2008) Rhythms of
Gene Expression in a Fluctuating Intertidal Environment. Curr Biol 18:1501–1507
Gray D, Hodgson A (1999) Endogenous rhythms of locomotor activity in the high-shore limpet, Helcion

105
pectunculus (Patellogastropoda). Anim Behav 57:387–391
Gray DR, Williams GA (2010) Knowing when to stop: Rhythms of locomotor activity in the high-shore
limpet, Cellana grata Gould. J Exp Mar Bio Ecol 391:125–130
Gunsilius E, Gastl G, Petzer A. (2001) Hematopoietic stem cells. Biomed Pharmacother 55:186–194
Hartnoll RG, Wright JR (1977) Foraging movements and homing in the limpet Patella vulgata L. Anim
Behav 25:806–810
Hayashi S, Takeichi M (2015) Emerging roles of protocadherins: from self-avoidance to enhancement of
motility. J Cell Sci 128:1455–1464
Heller J (2015) Patellogastropoda: Limpets. In: Sea Snails. Springer International Publishing, Cham, p
37–53
Hoyaux J, Gilles R, Jeuniaux C (1976) Osmoregulation in molluscs of the intertidal zone. Comp Biochem
Physiol -- Part A Physiol 53:361–365
Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists
using DAVID bioinformatics resources. Nat Protoc 4:44–57
Hughes ME, Hogenesch JB, Kornacker K (2010) JTK_CYCLE: an efficient nonparametric algorithm for
detecting rhythmic components in genome-scale data sets. J Biol Rhythms 25:372–80
Knoops B, Goemaere J, Eecken V Van der, Declercq J-P (2011) Peroxiredoxin 5: structure, mechanism,
and function of the mammalian atypical 2-Cys peroxiredoxin. Antioxid Redox Signal 15:817–829
Korenčič A, Košir R, Bordyugov G, Lehmann R, Rozman D, Herzel H (2014) Timing of circadian genes
in mammalian tissues. Sci Rep 4:1–9
Little C, Morritt D, Paterson DM, Stirling P, Williams GA (1990) Preliminary observations on factors
affecting foraging activity in the limpet Patella vulgata. J Mar Biol Assoc United Kingdom 70:181–
196
Little C, Partridge JC, Teagle L (2009) Foraging Activity of Limpets in Normal and Abnormal Tidal
Regimes. J Mar Biol Assoc United Kingdom 71:537
Little C, Williams GA, Morritt D, Perrins JM, Stirling P (1988) Foraging behaviour of Patella vulgata L.

106
in an Irish sea-lough. J Exp Mar Bio Ecol 120:1–21
Milev NB, Reddy AB (2015) Circadian redox oscillations and metabolism. Trends Endocrinol Metab:1–8
Mure LS, Le HD, Benegiamo G, Chang MW, Rios L, Jillani N, Ngotho M, Kariuki T, Dkhissi-Benyahya
O, Cooper HM, Panda S (2018) Diurnal transcriptome atlas of a primate across major neural and
peripheral tissues. Science (80- ) 359
Murphy MP, Iii HL (2010) Alzheimer’s Disease and the β-Amyloid Peptide. J Alzheimer’s Dis 19:1–17
Nitta T, Takahama Y (2007) The lymphocyte guard-IANs: regulation of lymphocyte survival by
IAN/GIMAP family proteins. Trends Immunol 28:58–65
Oliveros J (2015) Venny. An interactive tool for comparing lists with Venn’s
diagrams. :http://bioinfogp.cnb.csic.es/tools/venny/index.htm
Organ SL, Tsao MS (2011) An overview of the c-MET signaling pathway. Ther Adv Med Oncol 3:S7–
S19
Park J, Osei YD, Churchich JE (1993) Isolation and characterization of recombinant mitochondrial 4-
aminobutyrate aminotransferase. J Biol Chem 268:7636–7639
Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017) Salmon provides fast and bias-aware
quantification of transcript expression. Nat Methods 14:417–419
Payton L, Perrigault M, Hoede C, Massabuau JC, Sow M, Huvet A, Boullot F, Fabioux C, Hegaret H,
Tran D (2017) Remodeling of the cycling transcriptome of the oyster Crassostrea gigas by the
harmful algae Alexandrium minutum. Sci Rep 7:1–14
Pearlmutter N, Lembi C (1978) Localization of chitin in algal and fungal cell walls by light and electron
microscopy. J Histochem Cytochem 26:782–791
Peng Y, Leung HCM, Yiu SM, Chin FYL (2010) IDBA - A practical iterative De Bruijn graph De Novo
assembler. Lect Notes Comput Sci (including Subser Lect Notes Artif Intell Lect Notes
Bioinformatics) 6044 LNBI:426–440
R Core Team (2014) R: A Language and Environment for Statistical Computing.
Ruf T (1999) The Lomb-Scargle Periodogram in Biological Rhythm Research: Analysis of Incomplete

107
and Unequally Spaced Time-Series. Biol Rhythm Res 30:178–201
Santina P Della, Chelazzi G (1991) Temporal organization of foraging in two Mediterranean limpets,
Patella rustica L. and P. coerulea L. J Exp Mar Bio Ecol 153:75–85
Santina P Della, Naylor E (1994) Endogenous rhythms in the homing behaviour of the limpet Patella
vulgata Linnaeus. J Molluscan Stud 59:87–91
Santini G, Burrows MT, Chelazzi G (2004) Bioeconomics of foraging route selection by limpets. Mar
Ecol Prog Ser 280:189–198
Santini G, Chelazzi G (1996) Energy maximization vs. time minimization in the foraging of the limpet
Patella vulgata. J Anim Ecol 65:599–605
Schmid B, Helfrich-Förster C, Yoshii T (2011) A new ImageJ plug-in “ActogramJ” for chronobiological
analyses. J Biol Rhythms 26:464–7
Schnytzer Y, Simon-Blecher N, Li J, Ben-Asher HW, Salmon-Divon M, Achituv Y, Hughes ME, Levy O
(2018) Tidal and diel orchestration of behaviour and gene expression in an intertidal mollusc. Sci
Rep 8:4917
Shaw JA, Macey DJ, Brooker LR (2008) Radula synthesis by three species of iron mineralizing molluscs:
Production rate and elemental demand. J Mar Biol Assoc United Kingdom 88:597–601
Supek F, Bošnjak M, Škunca N, Šmuc T (2011) Revigo summarizes and visualizes long lists of gene
ontology terms. PLoS One 6
Takahashi J (1995) Molecular Neurobiology and Genetics of Circadian Rhythms in Mammals. Annu Rev
Neurosci 18:531–553
Vermeij GJ (1972) Intraspecific shore-level size gradients in intertidal molluscs. Ecology 53:693–700
Wells R (1980) Activity pattern as a mechanism of predator avoidance in two species of Acmaeid limpet.
J Exp Mar Bio Ecol 48:151–168
Wolcott TG (1973) Physiological Ecology and Intertidal Zonation in Limpets (Acmaea): A Critical Look
at “Limiting Factors.” Biol Bull 145:389–422
Yan J, Wang H, Liu Y, Shao C (2008) Analysis of gene regulatory networks in the mammalian circadian

108
rhythm. PLoS Comput Biol 4:e1000193

109

Figure 1. Limpets entrained to a simulated intertidal cycle then released into constant laboratory
conditions. Actograms showing limpet movement during a) entrainment and b) free-running.
Lomb-Scargle periodograms showing significant movement periods (p<0.05) during c)
entrainment and d) free-running.
0 6 12 18 24
Time of day
Days Days
a)
b)
c)
d)
12.45
12.02
Period (hr)
Days
e)

110

Figure 2. Head tissue
samples from free-running
limpets entrained to a
simulated intertidal cycle
exhibit circatidal and
circadian gene expression
rhythms. a) Histogram
showing the period length
of 386 statistically
significant (p<0.05)
transcripts. b) Histogram
showing the phase of 85
transcripts that had a
period of 10-14hr. A phase
of zero means that peak
expression coincided with
the start of the free-
running time course. The
anticipated tidal cycle is
included for reference,
where black indicates
anticipated submergence
and white indicates
anticipated emergence. c)
Histogram showing the
phase of 164 transcripts
that had a period of 22-
28hr. A phase of zero
means that peak
expression coincided with
the start of the free-
running time course. The
anticipated light/dark cycle
is included for reference,
where white indicates light
and black indicates dark.
0
10
20
30
40
50
60
10 12 14 16 18 20 22 24 26 28
Frequency
Period (hours)
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7 8 9 10 11
Frequency
Phase (hours)
Tide
Light/dark
a)
b)
c)
0
5
10
15
20
25
30
35
40
45
50
0 2 4 6 8 10 12 14 16 18 20 22 24
Frequency
Phase (hours)

111

row min row max
FMD_TPM_T1
FMD_TPM_T2
FMD_TPM_T3
FMD_TPM_T4
FMD_TPM_T5
FMD_TPM_T6
FMD_TPM_T7
FMD_TPM_T8
FMD_TPM_T9
FMD_TPM_T10
FMD_TPM_T11
FMD_TPM_T12
FMD_TPM_T13
FMD_TPM_T14
FMD_TPM_T15
FMD_TPM_T16
FMD_TPM_T17
FMD_TPM_T18
FMD_TPM_T19
FMD_TPM_T20
FMD_TPM_T21
FMD_TPM_T22
FMD_TPM_T23
FMD_TPM_T24
FMD_TPM_T25
id
id Name Uniprot.acc Uniprot ADJ.P PER LAG AMP
row min row max
FMD_TPM_T1
FMD_TPM_T2
FMD_TPM_T3
FMD_TPM_T4
FMD_TPM_T5
FMD_TPM_T6
FMD_TPM_T7
FMD_TPM_T8
FMD_TPM_T9
FMD_TPM_T10
FMD_TPM_T11
FMD_TPM_T12
FMD_TPM_T13
FMD_TPM_T14
FMD_TPM_T15
FMD_TPM_T16
FMD_TPM_T17
FMD_TPM_T18
FMD_TPM_T19
FMD_TPM_T20
FMD_TPM_T21
FMD_TPM_T22
FMD_TPM_T23
FMD_TPM_T24
FMD_TPM_T25
id
id Name Uniprot.acc Uniprot BH.Q ADJ.P PER LAG AMP
row min row max
FMD_TPM_T1
FMD_TPM_T2
FMD_TPM_T3
FMD_TPM_T4
FMD_TPM_T5
FMD_TPM_T6
FMD_TPM_T7
FMD_TPM_T8
FMD_TPM_T9
FMD_TPM_T10
FMD_TPM_T11
FMD_TPM_T12
FMD_TPM_T13
FMD_TPM_T14
FMD_TPM_T15
FMD_TPM_T16
FMD_TPM_T17
FMD_TPM_T18
FMD_TPM_T19
FMD_TPM_T20
FMD_TPM_T21
FMD_TPM_T22
FMD_TPM_T23
FMD_TPM_T24
FMD_TPM_T25
id
id Name Uniprot.acc Uniprot BH.Q ADJ.P PER LAG AMP
Circatidal
Circadian
Time
Light/dark
Tide
Movement
Figure 3. Heat maps showing significant (p<0.05) circatidal (85, period=10-14hr) and circadian (164,
period=22-28hr) transcripts in head tissue samples from free-running limpets entrained to a simulated
intertidal cycle. Corresponding anticipated tidal cycle and anticipated light/dark cycle are included for
reference. Limpet movement actogram is included for comparison. Grey entries in the heat map denote
the time point that was removed.

112

representative GO ID GO term Uniqueness
locomotory behavior GO:0008152 metabolic process 0.998
GO:0048511 rhythmic process 0.992
response to activity GO:0007626 locomotory behavior 0.985
response to activity GO:0098609 cell-cell adhesion 0.985
response to activity GO:0042743 hydrogen peroxide metabolic process 0.944
response to activity GO:0043473 pigmentation 0.938
response to activity GO:0016192 vesicle-mediated transport 0.928
response to activity GO:0014823 response to activity 0.911
response to activity GO:2000677 regulation of transcription regulatory region DNA binding 0.883
response to activity GO:0060048 cardiac muscle contraction 0.851
Table 1. Top ten most-represented GO categories of genes with circatidal expression rhythms
in head tissue samples from free-running limpets entrained to a simulated intertidal regime.
Uniqueness corresponds to overrepresentation of that GO category in relation to average
background representation.

113

-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
-1.5
-1
-0.5
0
0.5
1
1.5
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
Relative expression (log
2
)
Time
a)
b)
c)
Tide
Light/dark
Movement
Figure 4. Circatidal gene expression of foraging-related genes a) myoglobin, b) hepatocyte
growth factor receptor, and c) acidic mammalian chitinase in head tissue samples from free-
running limpets entrained to a simulated intertidal cycle. Anticipated tidal cycle and
anticipated light/dark cycle are included for reference. Corresponding limpet movement
actogram is included for comparison.

114

Figure 5. Foot tissue
samples from free-
running limpets
entrained to a
simulated intertidal
cycle exhibit
circatidal and
circadian gene
expression rhythms.
a) Histogram showing
the period length of
691 statistically
significant (p<0.05)
transcripts. b)
Histogram showing
the phase of 174
transcripts that had a
period of 10-14hr. A
phase of zero means
that peak expression
coincided with the
start of the free-
running time course.
The anticipated tidal
cycle is included for
reference, where black
indicates anticipated
submergence and
white indicates
anticipated
emergence. c)
Histogram showing
the phase of 416
transcripts that had a
period of 22-28hr. A
phase of zero means
that peak expression
coincided with the
start of the free-
running time course.
The anticipated
light/dark cycle is
included for reference,
where white indicates
light and black
indicates dark.
0
20
40
60
80
100
120
140
160
180
10 12 14 16 18 20 22 24 26 28
Frequency
Period (hours)
0
10
20
30
40
50
60
0 1 2 3 4 5 6 7 8 9 10 11
Frequency
Phase (hours)
Tide
Light/dark
a)
b)
c)
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14 16 18 20 22 24
Frequency
Phase (hours)

115
row min row max
Foot_FMD_T1
Foot_FMD_T2
Foot_FMD_T3
Foot_FMD_T4
Foot_FMD_T5
Foot_FMD_T6
Foot_FMD_T7
Foot_FMD_T8
Foot_FMD_T9
Foot_FMD_T10
Foot_FMD_T11
Foot_FMD_T12
Foot_FMD_T13
Foot_FMD_T14
Foot_FMD_T15
Foot_FMD_T16
Foot_FMD_T17
Foot_FMD_T18
Foot_FMD_T19
Foot_FMD_T20
Foot_FMD_T21
Foot_FMD_T22
Foot_FMD_T23
Foot_FMD_T24
Foot_FMD_T25
id
id Name Uniprot.acc Uniprot BH.Q ADJ.P PER LAG AMP
row min row max
Foot_FMD_T1
Foot_FMD_T2
Foot_FMD_T3
Foot_FMD_T4
Foot_FMD_T5
Foot_FMD_T6
Foot_FMD_T7
Foot_FMD_T8
Foot_FMD_T9
Foot_FMD_T10
Foot_FMD_T11
Foot_FMD_T12
Foot_FMD_T13
Foot_FMD_T14
Foot_FMD_T15
Foot_FMD_T16
Foot_FMD_T17
Foot_FMD_T18
Foot_FMD_T19
Foot_FMD_T20
Foot_FMD_T21
Foot_FMD_T22
Foot_FMD_T23
Foot_FMD_T24
Foot_FMD_T25
id
id Name Uniprot.acc Uniprot BH.Q ADJ.P PER LAG AMP
row min row max
Foot_FMD_T1
Foot_FMD_T2
Foot_FMD_T3
Foot_FMD_T4
Foot_FMD_T5
Foot_FMD_T6
Foot_FMD_T7
Foot_FMD_T8
Foot_FMD_T9
Foot_FMD_T10
Foot_FMD_T11
Foot_FMD_T12
Foot_FMD_T13
Foot_FMD_T14
Foot_FMD_T15
Foot_FMD_T16
Foot_FMD_T17
Foot_FMD_T18
Foot_FMD_T19
Foot_FMD_T20
Foot_FMD_T21
Foot_FMD_T22
Foot_FMD_T23
Foot_FMD_T24
Foot_FMD_T25
id
id Name Uniprot.acc Uniprot BH.Q ADJ.P PER LAG AMP
Light/dark
Tide
Movement
Time
a)
b)
Figure 6. Heat maps showing significant (p<0.05) a) circatidal (174, period=10-14hr) and
b) circadian (416, period=22-28hr) transcripts in foot tissue samples from free-running
limpets entrained to a simulated intertidal cycle. Corresponding anticipated tidal cycle and
anticipated light/dark cycle are included for reference. Limpet movement actogram is
included for comparison. Grey entries in the heat map denote the time point that was
removed.
row min row max
Foot_FMD_T1
Foot_FMD_T2
Foot_FMD_T3
Foot_FMD_T4
Foot_FMD_T5
Foot_FMD_T6
Foot_FMD_T7
Foot_FMD_T8
Foot_FMD_T9
Foot_FMD_T10
Foot_FMD_T11
Foot_FMD_T12
Foot_FMD_T13
Foot_FMD_T14
Foot_FMD_T15
Foot_FMD_T16
Foot_FMD_T17
Foot_FMD_T18
Foot_FMD_T19
Foot_FMD_T20
Foot_FMD_T21
Foot_FMD_T22
Foot_FMD_T23
Foot_FMD_T24
Foot_FMD_T25
id
id Name Uniprot.acc Uniprot BH.Q ADJ.P PER LAG AMP

116

Table 2. Top ten most-represented GO categories of genes with circatidal expression
rhythms in foot tissue samples from free-running limpets entrained to a simulated
intertidal regime. Uniqueness corresponds to overrepresentation of that GO category
in relation to average background representation.
GO ID GO term Uniqueness
sensory perception of sound GO:0007626 locomotory behavior 0.984
sensory perception of sound GO:0007620 copulation 0.959
sensory perception of sound GO:0042987 amyloid precursor protein catabolic process 0.95
sensory perception of sound GO:1902774 late endosome to lysosome transport 0.923
sensory perception of sound GO:0014841 skeletal muscle satellite cell proliferation 0.922
sensory perception of sound GO:0010039 response to iron ion 0.898
sensory perception of sound GO:0060021 palate development 0.882
sensory perception of sound GO:0036292 DNA rewinding 0.867
sensory perception of sound GO:0007605 sensory perception of sound 0.862
sensory perception of sound GO:0061484 hematopoietic stem cell homeostasis 0.857

117

-1.5
-1
-0.5
0
0.5
1
-2
-1.5
-1
-0.5
0
0.5
1
1.5
-3
-2
-1
0
1
-1.5
-1
-0.5
0
0.5
1
1.5
-0.5
0
0.5
1
-3
-2
-1
0
1
2
3
-1.5
-1
-0.5
0
0.5
1
-1.2
-0.8
-0.4
0
0.4
0.8
Time
Relative expression
(log
2
)
Light/dark
Tide
Movement
a)
b)
c)
d)
f)
g)
e)
h)
Figure 7. Circatidal gene expression of stress-related genes a) peroxiredoxin-5 and b) multidrug and toxin
extrusion protein 1, neurological genes c) endothelin-converting enzyme 2 and d) 4-aminobutyrate
aminotransferase, and work and muscle-related genes e) isocitrate dehydrogenase, f) dynein heavy chain, g)
sarcoplasmic calcium-binding protein, and h) myotrophin in foot tissue samples from free-running limpets
entrained to a simluated intertidal cycle. Anticipated tidal cycle and anticipated light/dark cycle are included
for reference. Corresponding limpet movement actogram is included for comparison.

118

Supplemental table 1. List of significant rhythmic genes in head tissue samples from free-running
limpets entrained to a simulated intertidal cycle. Genes are ordered by period, then lag, then amplitude.
Contig Name Uniprot accession Uniprot ID ADJ.P PER LAG AMP
Above3000_transcript-100_15594 Unclassifiable EST No hits found N/A 0.01151434 10 0 0.85488397
top500-3000_transcript-100_5411 Unclassifiable EST No hits found N/A 0.01843601 10 1 2.22571547
Above3000_transcript-100_9454 Zinc finger protein 233 A6NK53 ZN233_HUMAN 0.00903557 10 1 1.06066548
idba_c812 Dual oxidase maturation factor 1 Q1HG43 DOXA1_HUMAN 0.04002766 10 4 59.4828096
Above3000_transcript-100_23606 Unclassifiable EST No hits found N/A 0.0289876 10 4 3.862452
Above3000_transcript-100_131 Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16 O60231 DHX16_HUMAN 0.04002766 10 4 2.09957221
mira_extended_contigs_c1049 Integrin-linked kinase-associated serine/threonine phosphatase 2C Q0IIF0 ILKAP_BOVIN 0.03219362 10 4 1.64649019
Above3000_transcript-100_8253 NEDD8-conjugating enzyme ubc12 O74549 UBC12_SCHPO 0.00478726 10 5 8.07886238
Above3000_transcript-100_14476 Unclassifiable EST No hits found N/A 0.04002766 10 5 2.66422809
idba_c888 Lysine-specific demethylase 8 Q497B8 KDM8_RAT 0.04955868 10 5 1.44906933
idba_c7240 Probable ATP-dependent RNA helicase DHX35 Q9H5Z1 DHX35_HUMAN 0.04955868 10 5 0.54760152
Above3000_transcript-100_6833 H/ACA ribonucleoprotein complex subunit 2-like protein Q6PBV6 NHP2_DANRE 0.01013747 10 6 4.16392415
idba_c918 Exosome complex component RRP45 Q3SWZ4 EXOS9_BOVIN 0.04955868 10 6 3.68331746
idba_c10186 Mothers against decapentaplegic homolog 5 Q56I99 SMAD5_CHICK 0.04955868 10 6 2.7028835
Above3000_transcript-100_12196 Myoglobin P51537 MYG_HALMK 0.01151434 10 6 2.30490966
Above3000_transcript-100_11015 Unclassifiable EST No hits found N/A 0.01289121 10 6 1.78990028
idba_c7157 SRR1-like protein Q9UH36 SRR1L_HUMAN 0.03219362 10 6 1.21290203
mira_extended_contigs_c1252 Succinyl-CoA:3-ketoacid coenzyme A transferase 1, mitochondrial P55809 SCOT1_HUMAN 0.03219362 10 7 5.65825821
Above3000_transcript-100_11115 WD repeat-containing protein 47 Q8CGF6 WDR47_MOUSE 0.03219362 10 7 2.5681192
Above3000_transcript-100_8947 Histone-lysine N-methyltransferase, H3 lysine-79 specific Q8TEK3 DOT1L_HUMAN 0.00903557 10 7 2.3089932
idba_c8496 DENN domain-containing protein 4C A6H8H2 DEN4C_MOUSE 0.04002766 10 7 1.7654114
idba_c4363 Syndetin Q5ZKV9 VPS50_CHICK 0.04955868 10 8 2.02472584
Above3000_transcript-100_23459 Mitogen-activated protein kinase-binding protein 1 Q6DFF9 MABP1_XENLA 0.02316879 10 8 1.67500727
idba_c7752 Unclassifiable EST No hits found N/A 0.04479317 10 8 1.56630693
top500-3000_transcript-100_7044 Unclassifiable EST No hits found N/A 0.03219362 10 8 1.24383795
Above3000_transcript-100_5412 Unclassifiable EST No hits found N/A 0.00705605 10 8 0.77408005
mira_extended_contigs_c111 Exportin-4 Q9C0E2 XPO4_HUMAN 0.03219362 10 8 0.66077007
mira_extended_contigs_c976 Unclassifiable EST No hits found N/A 0.04955868 10 9 1.26727713
Above3000_transcript-100_26936 Protein lin-28 homolog Q9VRN5 LIN28_DROME 0.04479317 10 9 0.9428659
Above3000_transcript-100_15897 Monocarboxylate transporter 12 Q6P2X9 MOT12_XENTR 0.020556 10 9 0.64798912
Above3000_transcript-100_2422 Syntaxin-7 Q5R602 STX7_PONAB 0.04002766 12 0 5.25245263
mira_extended_contigs_c48 Protocadherin-9 Q9HC56 PCDH9_HUMAN 0.02578158 12 0 1.2081105
idba_c4010 Drebrin-like protein A6H7G2 DBNL_BOVIN 0.03219362 12 1 5.54169272
idba_c4151 UTP--glucose-1-phosphate uridylyltransferase Q07130 UGPA_BOVIN 0.04002766 12 1 4.75853483
Above3000_transcript-100_6776 Synaptosomal-associated protein 25 P36975 SNP25_DROME 0.03219362 12 1 2.68934612
idba_c8015 Unclassifiable EST No hits found N/A 0.01631603 12 1 1.21121859
idba_c8650 Protein ERGIC-53 Q62902 LMAN1_RAT 0.02578158 12 2 9.3019296
idba_c7061 Rho-related GTP-binding protein RhoG P84096 RHOG_MOUSE 0.04002766 12 2 6.43679957
mira_extended_contigs_c1356 Serine/threonine-protein kinase PLK2 Q9R012 PLK2_RAT 0.01631603 12 2 1.63422789
idba_c319 Unclassifiable EST No hits found N/A 0.00793367 12 3 7.54046032
Above3000_transcript-100_26454 Protein archease Q2EGP9 ARCH_ICTPU 0.020556 12 3 6.43968651
idba_c4378 Unclassifiable EST No hits found N/A 0.01631603 12 3 1.77937977
idba_c9066 Transmembrane protein 205 Q5REM8 TM205_PONAB 0.020556 12 3 1.70488218
Above3000_transcript-100_10551 Unclassifiable EST No hits found N/A 0.03219362 12 3 1.52680052
Above3000_transcript-100_11808 Zinc finger CCHC domain-containing protein 2 Q69ZB8 ZCHC2_MOUSE 0.01151434 12 3 1.47656341
Above3000_transcript-100_5864 Unclassifiable EST No hits found N/A 0.04955868 12 3 1.47213498
idba_c9149 Unclassifiable EST No hits found N/A 0.01013747 12 4 7.60292136
idba_c7949 Protrudin Q6P7B7 ZFY27_RAT 0.00163627 12 4 2.24861936
mira_extended_contigs_c1709 Unclassifiable EST No hits found N/A 0.01460362 12 4 1.27442279
Above3000_transcript-100_2189 Patatin-like phospholipase domain-containing protein 4 P41247 PLPL4_HUMAN 0.00478726 12 4 1.13132878
Above3000_transcript-100_21013 Unclassifiable EST No hits found N/A 0.04479317 12 4 1.12690476
top500-3000_transcript-100_8634 Unclassifiable EST No hits found N/A 0.020556 12 4 1.00683874
Above3000_transcript-100_27291 Unclassifiable EST No hits found N/A 0.00793367 12 5 2.39038614
idba_c1480 60S acidic ribosomal protein P0 Q9U3U0 RLA0_CERCA 0.04002766 12 7 106.622752
mira_extended_contigs_c1487 60S ribosomal protein L4-B P02385 RL4B_XENLA 0.01013747 12 7 104.583141
Above3000_transcript-100_23958 Zinc finger SWIM domain-containing protein 7 Q9CWQ2 ZSWM7_MOUSE 0.01631603 12 7 0.92555628
top500-3000_transcript-100_10525 Acidic mammalian chitinase Q9BZP6 CHIA_HUMAN 0.01289121 12 8 20.4386597
idba_c6488 Zinc finger protein ubi-d4 A Q9W638 REQUA_XENLA 0.04955868 12 8 2.71429832
mira_extended_contigs_c607 Unclassifiable EST No hits found N/A 0.04002766 12 8 0.79283694
idba_c196 Unclassifiable EST No hits found N/A 0.01631603 12 9 4.86855464
mira_extended_contigs_c1001 Adhesion G-protein coupled receptor D1 A6QLU6 AGRD1_BOVIN 0.02578158 12 9 3.01894397
idba_c9880 Unclassifiable EST No hits found N/A 0.02578158 12 10 3.69079387
idba_c7536 GTPase IMAP family member 4 Q9NUV9 GIMA4_HUMAN 0.04479317 12 11 37.9176359
top500-3000_transcript-100_9897 Unclassifiable EST No hits found N/A 0.00282946 12 11 10.8969695
mira_extended_contigs_c2657 Hepatocyte growth factor receptor Q108U6 MET_LOXAF 0.00478726 14 0 5.70852926
idba_c3409 Anaphase-promoting complex subunit 1 Q9H1A4 APC1_HUMAN 0.00163627 14 0 1.64618772
Above3000_transcript-100_14633 Unclassifiable EST No hits found N/A 0.00548284 14 1 1.170115
Above3000_transcript-100_24632 Unclassifiable EST No hits found N/A 0.04002766 14 1 0.98151725
idba_c7372 DNA polymerase subunit gamma-2, mitochondrial Q0VC30 DPOG2_BOVIN 0.04002766 14 2 3.43696039
top50_transcript-100_94 Unclassifiable EST No hits found N/A 0.02578158 14 3 129.0672
idba_c2902 Protein arginine N-methyltransferase 9 A0JMU5 ANM9_XENLA 0.01013747 14 4 1.46008217
idba_c5545 Uncharacterized protein MJ1187 Q58588 Y1187_METJA 0.01013747 14 5 0.84168458
idba_c2783 Carnitine O-acetyltransferase P52826 CACP_COLLI 0.020556 14 7 3.04699913
Above3000_transcript-100_23087 Hydramacin-1 B3RFR8 HYDMA_HYDVU 0.04955868 14 8 1995.90053
idba_c1606 WD repeat-containing protein 19 Q3UGF1 WDR19_MOUSE 0.01289121 14 9 2.58525911
Above3000_transcript-100_16673 Nuclease EXOG, mitochondrial Q0IH72 EXOG_XENLA 0.04002766 14 10 4.96110838
idba_c530 Adenylate kinase B4J672 KAD2_DROGR 0.04955868 14 10 3.02545182
Above3000_transcript-100_15282 Polyphosphoinositide phosphatase Q91WF7 FIG4_MOUSE 0.03219362 14 11 4.19105194
Above3000_transcript-100_18284 Unclassifiable EST No hits found N/A 0.02578158 14 11 1.92445474

119

Above3000_transcript-100_18284 Unclassifiable EST No hits found N/A 0.02578158 14 11 1.92445474
mira_extended_contigs_c601 Unclassifiable EST No hits found N/A 0.01631603 14 11 1.52576655
idba_c8522 Unclassifiable EST No hits found N/A 0.01013747 14 12 3.48645822
mira_extended_contigs_c1088 Sterol regulatory element-binding protein cleavage-activating protein Q12770 SCAP_HUMAN 0.02578158 14 12 1.93461693
idba_c10298 Unclassifiable EST No hits found N/A 0.00617842 14 13 2.60474627
idba_c3488 Unclassifiable EST No hits found N/A 0.01151434 14 13 1.09606572
Above3000_transcript-100_21115 Unclassifiable EST No hits found N/A 0.01843601 14 13 0.61994915
mira_extended_contigs_c2675 BTB/POZ domain-containing protein 17 Q6GLJ1 BTBDH_XENLA 0.00617842 16 2 40.5875767
Above3000_transcript-100_25706 Unclassifiable EST No hits found N/A 0.00548284 16 4 3.2260985
Above3000_transcript-100_770 Gamma-butyrobetaine dioxygenase P80193 BODG_PSESK 0.04002766 16 5 0.84233194
idba_c2723 GTP-binding protein GEM Q5R541 GEM_PONAB 0.01289121 16 5 0.83346924
mira_extended_contigs_c2305 Unclassifiable EST No hits found N/A 0.01289121 16 7 1.29634381
Above3000_transcript-100_1970 NAD-dependent protein deacetylase sirtuin-3, mitochondrial Q9NTG7 SIR3_HUMAN 0.04955868 16 7 0.56332828
top500-3000_transcript-100_8563 NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, Q0MQH5 NDUS6_PONPY 0.01631603 16 8 16.4736406
Above3000_transcript-100_15847 U6 snRNA-associated Sm-like protein LSm4 Q9Y4Z0 LSM4_HUMAN 0.02578158 16 8 3.08157347
Above3000_transcript-100_20563 39S ribosomal protein L11, mitochondrial Q9VFJ2 RM11_DROME 0.04955868 16 9 4.85292952
Above3000_transcript-100_20408 Dehydrogenase/reductase SDR family member 7B Q0VFE7 DRS7B_XENTR 0.04002766 16 9 2.03945612
idba_c8583 Unclassifiable EST No hits found N/A 0.01631603 16 9 1.27607848
mira_extended_contigs_c173 Complement C3 (Fragment) Q00685 CO3_LETCA 0.020556 16 10 3.11860324
idba_c7376 Unclassifiable EST No hits found N/A 0.01289121 16 10 1.73019218
idba_c9620 WD repeat-containing protein 81 Q5ND34 WDR81_MOUSE 0.00215771 16 10 1.17854442
Above3000_transcript-100_4807 Transmembrane protein 179B Q7T392 T179B_DANRE 0.04955868 16 10 0.95816434
Above3000_transcript-100_16024 Unclassifiable EST No hits found N/A 0.02316879 16 10 0.62606881
idba_c8639 Cytochrome P450 3A24 Q29496 CP3AO_SHEEP 0.01631603 16 11 8.30678712
mira_extended_contigs_c1211 Ectonucleoside triphosphate diphosphohydrolase 1 O18956 ENTP1_BOVIN 0.04002766 16 11 2.29572823
mira_extended_contigs_c952 Long-chain fatty acid transport protein 1 Q6PCB7 S27A1_HUMAN 0.00369019 16 11 2.23618206
mira_extended_contigs_c2228 Golgi SNAP receptor complex member 2 O35166 GOSR2_MOUSE 0.04002766 16 11 1.54246452
idba_c4989 Microsomal triglyceride transfer protein large subunit Q865F1 MTP_PIG 0.04002766 16 11 1.47505109
idba_c8170 Endothelial differentiation-related factor 1 homolog Q5ZMC0 EDF1_CHICK 0.03219362 16 12 16.2184856
idba_c4606 Cytochrome P450 3A24 Q29496 CP3AO_SHEEP 0.00478726 16 12 6.72954142
Above3000_transcript-100_25980 MAM and LDL-receptor class A domain-containing protein 2 (Fragment) B3EWZ6 MLRP2_ACRMI 0.04955868 16 12 4.42752189
idba_c9299 Peroxisomal trans-2-enoyl-CoA reductase Q99MZ7 PECR_MOUSE 0.020556 16 12 2.74601171
mira_extended_contigs_c684 Solute carrier organic anion transporter family member 4C1 Q8BGD4 SO4C1_MOUSE 0.04002766 16 12 1.64460415
Above3000_transcript-100_20296 Tudor domain-containing protein 1 A9CPT4 TDRD1_ORYLA 0.00249359 16 12 1.00823669
mira_extended_contigs_c1776 Teneurin-m O61307 TENM_DROME 0.03219362 16 12 0.61440225
idba_c7908 Unclassifiable EST No hits found N/A 0.02578158 16 13 5.49776017
idba_c1622 Neurogenic locus notch homolog protein 1 P21783 NOTC1_XENLA 0.00793367 16 13 2.06196934
top500-3000_transcript-100_2936 Ubiquitin carboxyl-terminal hydrolase 25 Q9UHP3 UBP25_HUMAN 0.04955868 16 13 1.91420594
mira_extended_contigs_c1145 Phospholipase D1 O08684 PLD1_CRIGR 0.03219362 16 14 2.52581017
idba_c7296 Extracellular tyrosine-protein kinase PKDCC Q5RJI4 PKDCC_MOUSE 0.04955868 16 14 0.50810059
idba_c8586 H(+)/Cl(-) exchange transporter 7 P51799 CLCN7_RAT 0.00478726 16 15 1.59149619
idba_c2517 Unclassifiable EST No hits found N/A 0.020556 18 1 2.1712739
Above3000_transcript-100_17253 Unclassifiable EST No hits found N/A 0.00793367 18 2 3.11942419
idba_c5745 Low molecular weight phosphotyrosine protein phosphatase P11064 PPAC_BOVIN 0.020556 18 4 7.37084007
idba_c9655 Methylosome protein 50 Q99J09 MEP50_MOUSE 0.04002766 18 4 1.03835944
top500-3000_transcript-100_1493 Cytochrome b-c1 complex subunit Rieske, mitochondrial Q69BJ9 UCRI_AOTAZ 0.04955868 18 5 6.71495823
idba_c9835 Unclassifiable EST No hits found N/A 0.04002766 18 5 4.03042804
Above3000_transcript-100_10531 Glutaredoxin-2, mitochondrial Q32L67 GLRX2_BOVIN 0.03219362 18 5 3.22864956
Above3000_transcript-100_25377 Methyltransferase-like protein 5 Q8K1A0 METL5_MOUSE 0.01013747 18 5 2.81997932
idba_c2632 WAP four-disulfide core domain protein 1 Q8JG33 WFDC1_CHICK 0.04002766 18 5 2.47835836
mira_extended_contigs_c1176 Potassium voltage-gated channel subfamily H member 7 Q9NS40 KCNH7_HUMAN 0.04955868 18 5 1.05230217
top500-3000_transcript-100_3491 NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, P0CB97 NDUS8_PONAB 0.04002766 18 6 41.9954437
Above3000_transcript-100_20295 Marginal zone B- and B1-cell-specific protein Q9D8I1 MZB1_MOUSE 0.01289121 18 6 10.5622725
top500-3000_transcript-100_723 Activator of 90 kDa heat shock protein ATPase homolog 1 O95433 AHSA1_HUMAN 0.01631603 18 6 9.80302931
idba_c6973 NFU1 iron-sulfur cluster scaffold homolog, mitochondrial B4R3T1 NFU1_DROSI 0.00010263 18 6 4.10162026
Above3000_transcript-100_10659 Unclassifiable EST No hits found N/A 0.04955868 18 6 3.80952718
Above3000_transcript-100_22186 Histone H3 P0CO04 H3_CRYNJ 0.020556 18 6 3.31934944
idba_c9500 G patch domain-containing protein 4 Q4V842 GPTC4_XENLA 0.01289121 18 6 2.4620256
mira_extended_contigs_c1744 Uncharacterized membrane protein C776.05 O94673 YG75_SCHPO 0.00369019 18 6 2.18289944
idba_c7155 Probable low-specificity L-threonine aldolase 2 Q9FPH3 THA2_ARATH 0.04002766 18 6 1.84655208
mira_extended_contigs_c1004 Unclassifiable EST No hits found N/A 0.04955868 18 6 1.38936512
idba_c3848 Ribosomal protein S6 kinase delta-1 Q96S38 KS6C1_HUMAN 0.04002766 18 6 1.23379385
idba_c7626 Caspase-3 Q5IS54 CASP3_PANTR 0.01289121 18 6 1.11696991
Above3000_transcript-100_7388 Trafficking protein particle complex subunit 5 Q2NL13 TPPC5_BOVIN 0.02578158 18 7 5.74771111
idba_c9886 Histone H2AX Q7ZUY3 H2AX_DANRE 0.04955868 18 7 3.64681554
idba_c8838 Xylose isomerase Q3YVV0 XYLA_SHISS 0.04955868 18 7 3.25230299
mira_extended_contigs_c1537 Activating signal cointegrator 1 complex subunit 2 Q91WR3 ASCC2_MOUSE 0.020556 18 7 2.34037566
Above3000_transcript-100_23647 Condensin-2 complex subunit H2 Q28GV1 CNDH2_XENTR 0.03219362 18 7 2.23085684
Above3000_transcript-100_13258 TIMELESS-interacting protein Q0IHI4 TIPIN_XENLA 0.03219362 18 7 1.79479399
Above3000_transcript-100_26126 Unclassifiable EST No hits found N/A 0.01460362 18 7 1.65139061
mira_extended_contigs_c1676 Unclassifiable EST No hits found N/A 0.01460362 18 7 1.55012868
Above3000_transcript-100_14251 Zinc finger protein 85 Q03923 ZNF85_HUMAN 0.03219362 18 7 1.10785672
mira_extended_contigs_c661 Cell division cycle protein 20 homolog Q5H7C0 CDC20_PIG 0.03219362 18 7 0.66134566
Above3000_transcript-100_23269 Unclassifiable EST No hits found N/A 0.00617842 18 8 7.42083111
mira_extended_contigs_c1726 Negative elongation factor B Q9Y113 NELFB_DROME 0.04955868 18 8 3.86665363
Above3000_transcript-100_9377 Retinoblastoma-binding protein 5 Q8BX09 RBBP5_MOUSE 0.03219362 18 8 3.63878705
idba_c7078 Ribosome biogenesis protein TSR3 homolog Q5HZH2 TSR3_MOUSE 0.04955868 18 8 2.39873955
idba_c6806 Uncharacterized protein C7orf26 homolog Q8BGA7 CG026_MOUSE 0.04955868 18 8 1.84565901
mira_extended_contigs_c2157 Unclassifiable EST No hits found N/A 0.04002766 18 8 1.80629579
Above3000_transcript-100_37 Organellar oligopeptidase A, chloroplastic/mitochondrial Q94AM1 OOPDA_ARATH 0.04002766 18 8 1.35403878
mira_extended_contigs_c711 Cyclic nucleotide-gated cation channel beta-1 Q14028 CNGB1_HUMAN 0.03219362 18 8 0.95185341

120

mira_extended_contigs_c711 Cyclic nucleotide-gated cation channel beta-1 Q14028 CNGB1_HUMAN 0.03219362 18 8 0.95185341
idba_c2511 Ubiquitin-protein ligase E3B Q08CZ0 UBE3B_XENTR 0.03219362 18 8 0.88826471
Above3000_transcript-100_21130 F-box only protein 22 Q78JE5 FBX22_MOUSE 0.020556 18 8 0.70866171
idba_c381 Enoyl-[acyl-carrier-protein] reductase, mitochondrial Q6GQN8 MECR_DANRE 0.01289121 18 9 1.12410745
idba_c5213 Probable Xaa-Pro aminopeptidase 3 B7ZMP1 XPP3_MOUSE 0.00793367 18 9 0.96353198
top500-3000_transcript-100_6272 Protocadherin-23 Q6V1P9 PCD23_HUMAN 0.04479317 18 9 0.86214277
Above3000_transcript-100_3499 Beta-lactamase domain-containing protein 2 Q09621 LACT2_CAEEL 0.01289121 18 12 1.87151578
idba_c6086 Protein Wnt-11b P49893 WN11B_XENLA 0.04955868 18 12 1.2236992
Above3000_transcript-100_1117 Adhesion G protein-coupled receptor A2 E7FBY6 AGRA2_DANRE 0.01013747 18 13 0.64092654
top500-3000_transcript-100_4580 AT-rich interactive domain-containing protein 2 Q68CP9 ARID2_HUMAN 0.04002766 18 14 3.92653143
idba_c1945 Short-chain dehydrogenase TIC 32, chloroplastic Q6RVV4 TIC32_PEA 0.03219362 18 14 1.73656233
idba_c6371 Mitogen-activated protein kinase kinase kinase 4 Q9Y6R4 M3K4_HUMAN 0.04955868 18 14 0.95414938
Above3000_transcript-100_6288 NmrA-like family domain-containing protein 1 Q0VCN1 NMRL1_BOVIN 0.04955868 18 17 1.10878162
idba_c2605 Protein RER1 Q498C8 RER1_RAT 0.00793367 20 2 4.63500522
Above3000_transcript-100_5598 Complexin Q95PA1 CPLX_DORPE 0.01289121 20 2 3.25001939
mira_extended_contigs_c929 Cubilin O60494 CUBN_HUMAN 0.00793367 20 2 2.37105137
Above3000_transcript-100_25218 Unclassifiable EST No hits found N/A 0.04955868 20 2 0.86478894
idba_c6431 Unclassifiable EST No hits found N/A 0.04955868 20 2 0.70068873
top500-3000_transcript-100_941 Enterin neuropeptides Q95P23 ENPP_APLCA 0.01631603 20 3 13.4844888
top500-3000_transcript-100_1039 Neurensin-1 P97799 NRSN1_MOUSE 0.00163627 20 3 11.552619
Above3000_transcript-100_5230 Unclassifiable EST No hits found N/A 0.02578158 20 3 7.28874038
Above3000_transcript-100_3747 Unclassifiable EST No hits found N/A 0.02578158 20 3 5.25005024
idba_c8667 BTB/POZ domain-containing protein 2 Q9BX70 BTBD2_HUMAN 0.00793367 20 3 4.0673719
Above3000_transcript-100_11490 Unclassifiable EST No hits found N/A 0.01289121 20 3 3.33039357
idba_c5833 Protein unc-93 homolog A A2VE54 UN93A_BOVIN 0.01013747 20 3 2.23018403
top500-3000_transcript-100_7216 Protein PRQFV-amide Q86MA7 PRQFV_APLCA 0.04955868 20 4 8.31774957
idba_c4199 Protein RNA-directed DNA methylation 3 F4JW79 RDM3_ARATH 0.01289121 20 4 7.98386047
idba_c8394 Peptidyl-glycine alpha-amidating monooxygenase B P12890 AMDB_XENLA 0.00369019 20 4 7.83406997
top500-3000_transcript-100_2054 Casein kinase II subunit beta P28021 CSK2B_XENLA 0.04002766 20 4 7.48978092
top500-3000_transcript-100_777 Solute carrier family 25 member 46 Q5ZIG3 S2546_CHICK 0.00617842 20 4 6.98586109
idba_c6523 Unclassifiable EST No hits found N/A 0.020556 20 4 6.42494828
Above3000_transcript-100_5261 Ras-like protein family member 11B Q6IMA7 RSLBB_RAT 0.03219362 20 4 3.8186455
idba_c1751 ATP-dependent DNA helicase Q4 O94761 RECQ4_HUMAN 0.02578158 20 4 2.99622639
Above3000_transcript-100_7873 Con-Ins Im1 A0A0B5A7M7 INS1_CONIM 0.04955868 20 4 1.80240352
Above3000_transcript-100_4711 Serine/threonine/tyrosine-interacting protein A Q4V7N3 STYXA_XENLA 0.04955868 20 4 1.36749502
mira_extended_contigs_c1142 Tubulin alpha-1 chain P06603 TBA1_DROME 0.02578158 20 5 25.9217759
idba_c5641 CDGSH iron-sulfur domain-containing protein 3, mitochondrial B1AR13 CISD3_MOUSE 0.00014344 20 5 14.7460029
idba_c7795 Unclassifiable EST No hits found N/A 0.00369019 20 5 8.39028617
idba_c4147 Histone-lysine N-methyltransferase EHMT1 Q5DW34 EHMT1_MOUSE 0.00215771 20 5 4.21817232
idba_c796 N6-adenosine-methyltransferase subunit METTL3 Q8C3P7 MTA70_MOUSE 0.04955868 20 5 3.49435908
idba_c1891 tRNA (guanine-N(7)-)-methyltransferase non-catalytic subunit wdr4 A4IGH4 WDR4_DANRE 0.02578158 20 5 3.12225757
Above3000_transcript-100_28383 Ribonuclease P protein subunit p21 Q8R040 RPP21_MOUSE 0.00617842 20 5 2.34013171
idba_c6757 Vitelline envelope sperm lysin receptor Q8WR62 VERL_HALRU 0.01631603 20 5 2.2754721
idba_c5882 Dihydrolipoyllysine-residue succinyltransferase component of Q90512 ODO2_TAKRU 0.00369019 20 6 5.33083843
idba_c3534 Post-GPI attachment to proteins factor 2 Q3TQR0 PGAP2_MOUSE 0.03219362 20 6 3.39487128
mira_extended_contigs_c2115 Selenocysteine-specific elongation factor Q9JHW4 SELB_MOUSE 0.04002766 20 6 2.42746099
mira_extended_contigs_c339 GATOR complex protein MI Q802U2 MIO_DANRE 0.03219362 20 6 2.28076143
Above3000_transcript-100_634 Mitochondrial chaperone BCS1 Q7ZTL7 BCS1_XENLA 0.04955868 20 6 1.57501884
idba_c2977 GTP-binding protein 10 Q6DHF7 GTPBA_DANRE 0.04955868 20 6 1.19773989
idba_c3256 Glutamyl-tRNA(Gln) amidotransferase subunit A, mitochondrial F1QAJ4 GATA_DANRE 0.01631603 20 6 0.69710811
top500-3000_transcript-100_3746 Unclassifiable EST No hits found N/A 0.04002766 20 6 0.43108553
Above3000_transcript-100_11445 Transcription elongation factor SPT4 Q6DGQ0 SPT4H_DANRE 0.020556 20 7 7.93141913
idba_c803 Cytochrome c oxidase assembly protein COX15 homolog Q08DG6 COX15_BOVIN 0.00478726 20 7 4.79807589
idba_c10636 Unclassifiable EST No hits found N/A 0.04955868 20 8 12.682322
idba_c1245 28S ribosomal protein S5, mitochondrial Q99N87 RT05_MOUSE 0.00282946 20 8 7.1635724
Above3000_transcript-100_1997 Citrate lyase subunit beta-like protein, mitochondrial Q8R4N0 CLYBL_MOUSE 0.00051009 20 9 1.64159877
idba_c5994 Endophilin-B1 Q32LM0 SHLB1_BOVIN 0.04002766 20 10 1.29413552
mira_extended_contigs_c1639 Filamin-A Q9VEN1 FLNA_DROME 0.00617842 20 12 3.38071553
idba_c1143 Protein grainyhead P13002 ELF1_DROME 0.03219362 20 12 1.58054382
idba_c5217 Vacuolar protein sorting-associated protein 54 Q5SPW0 VPS54_MOUSE 0.01013747 20 12 0.48004419
Above3000_transcript-100_20906 Unclassifiable EST No hits found N/A 0.04002766 20 14 4.74552637
Above3000_transcript-100_19551 Protein FAM167A Q5RFZ7 F167A_DANRE 0.04955868 20 15 2.35739307
Above3000_transcript-100_17292 Unclassifiable EST No hits found N/A 0.01460362 20 17 0.98729113
idba_c9063 Unclassifiable EST No hits found N/A 0.01013747 20 18 1.57695348
Above3000_transcript-100_27524 3-oxo-5-beta-steroid 4-dehydrogenase Q8VCX1 AK1D1_MOUSE 0.020556 20 19 2.74433304
idba_c7331 DOMON domain-containing protein FRRS1L B1AXV0 FRS1L_MOUSE 0.01013747 22 0 6.0996431
idba_c7327 Unclassifiable EST No hits found N/A 0.00019903 22 0 5.76749808
idba_c3305 Unclassifiable EST No hits found N/A 0.02578158 22 0 5.01169285
idba_c3379 1-acyl-sn-glycerol-3-phosphate acyltransferase epsilon Q9NUQ2 PLCE_HUMAN 0.04955868 22 0 2.29322649
Above3000_transcript-100_191 Transcription factor COE3 O08791 COE3_MOUSE 0.00051009 22 0 1.66091888
idba_c2099 Delta and Notch-like epidermal growth factor-related receptor Q8JZM4 DNER_MOUSE 0.01631603 22 0 1.27639916
Above3000_transcript-100_6369 Cytoplasmic polyadenylation element-binding protein 2 Q812E0 CPEB2_MOUSE 0.00903557 22 0 0.8239484
idba_c7670 Buccalin P20481 BUCC_APLCA 0.00369019 22 1 18.6232594
idba_c1623 Complement C1q-like protein 4 Q4ZJM9 C1QL4_MOUSE 0.00215771 22 1 13.9986142
top500-3000_transcript-100_8974 Unclassifiable EST No hits found N/A 0.00793367 22 1 11.0984008
idba_c8445 Neuropeptide prohormone-4 A0A0F7YYX3 CPROH_CONVC 0.00478726 22 1 10.1774353
Above3000_transcript-100_378 Protein lev-9 Q22328 LEV9_CAEEL 0.00037524 22 1 6.69505459
idba_c4046 Tetraspanin-7 Q7YQK9 TSN7_PONPY 0.00215771 22 1 6.49219485
idba_c4384 Unclassifiable EST No hits found N/A 0.04002766 22 1 5.34295046
idba_c9662 Unclassifiable EST No hits found N/A 0.00092475 22 1 5.09173592
idba_c9973 Polypeptide N-acetylgalactosaminyltransferase 3 P34678 GALT3_CAEEL 0.01631603 22 1 1.09766095

121

idba_c9973 Polypeptide N-acetylgalactosaminyltransferase 3 P34678 GALT3_CAEEL 0.01631603 22 1 1.09766095
idba_c3673 Unclassifiable EST No hits found N/A 0.01289121 22 2 12.1306509
idba_c7099 Diphthine methyl ester synthase Q9CWQ0 DPH5_MOUSE 0.01289121 22 2 5.82111728
idba_c1772 Suppressor of lurcher protein 1 Q93212 SOL1_CAEEL 0.020556 22 2 3.17124522
idba_c5826 Gigaxonin Q9H2C0 GAN_HUMAN 0.00282946 22 2 2.16648608
idba_c4051 Piwi-like protein 2 A2CEI6 PIWL2_DANRE 0.04002766 22 2 1.59482702
mira_extended_contigs_c2065 Coiled-coil domain-containing protein 141 Q6ZP82 CC141_HUMAN 0.00092475 22 2 1.15456961
Above3000_transcript-100_27179 Unclassifiable EST No hits found N/A 0.03611064 22 2 0.43111381
mira_extended_contigs_c637 THO complex subunit 6 homolog Q5XJS5 THOC6_DANRE 0.03219362 22 3 6.78862108
idba_c5806 Coiled-coil domain-containing protein 84 Q4VA36 CCD84_MOUSE 0.0289876 22 3 0.97403959
top500-3000_transcript-100_6760 Unclassifiable EST No hits found N/A 0.03611064 22 4 2.21556
idba_c8850 Probable ubiquitin carboxyl-terminal hydrolase MINDY-4 Q3UQI9 MINY4_MOUSE 0.020556 22 4 1.12483718
idba_c7547 Titin A2ASS6 TITIN_MOUSE 0.00037524 22 5 1.97508359
idba_c9064 Glutathione S-transferase kappa 1 Q9Y2Q3 GSTK1_HUMAN 0.03219362 22 6 5.00689937
Above3000_transcript-100_4552 Actin-binding Rho-activating protein B5SNZ6 ABRA_PIG 0.04955868 22 7 3.47948579
Above3000_transcript-100_26662 Unclassifiable EST No hits found N/A 0.01460362 22 7 2.69904709
idba_c8959 von Willebrand factor D and EGF domain-containing protein Q8N2E2 VWDE_HUMAN 0.04955868 22 8 1.05991276
Above3000_transcript-100_1507 C-myc promoter-binding protein Q7Z401 MYCPP_HUMAN 0.04955868 22 8 0.94500225
mira_extended_contigs_c1201 Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein Q5ZK62 ACAP2_CHICK 0.01631603 22 10 1.5510458
mira_extended_contigs_c1553 60S ribosomal protein L23a P62752 RL23A_RAT 0.04955868 22 11 134.952306
idba_c376 Protein hedgehog P56674 HH_DROHY 0.04955868 22 12 8.76365093
Above3000_transcript-100_11052 Kinesin-like protein KIF17 Q9P2E2 KIF17_HUMAN 0.04955868 22 12 1.09663494
idba_c9167 Unclassifiable EST No hits found N/A 0.00423873 22 13 1.44012054
Above3000_transcript-100_2135 Tight junction-associated protein 1 Q9DCD5 TJAP1_MOUSE 0.020556 22 14 1.1402804
mira_extended_contigs_c996 Unclassifiable EST No hits found N/A 0.04955868 22 15 4.3599398
idba_c7823 Unclassifiable EST No hits found N/A 0.020556 22 15 3.20753818
Above3000_transcript-100_17051 Unclassifiable EST No hits found N/A 0.00793367 22 16 2.03270148
mira_extended_contigs_c1877 N-sulphoglucosamine sulphohydrolase P51688 SPHM_HUMAN 0.04955868 22 18 0.92686284
mira_extended_contigs_c1903 Protein amnionless Q99JB7 AMNLS_MOUSE 0.04955868 22 19 5.30956936
Above3000_transcript-100_24886 Epoxide hydrolase 4 Q8IUS5 EPHX4_HUMAN 0.04002766 22 20 5.08218574
Above3000_transcript-100_4609 Unclassifiable EST No hits found N/A 0.01289121 22 20 3.77325755
idba_c4258 Serine/threonine-protein kinase DCLK1 O15075 DCLK1_HUMAN 0.01631603 22 20 1.91136973
idba_c9777 Unclassifiable EST No hits found N/A 0.04002766 22 21 14.778705
Above3000_transcript-100_5783 Unclassifiable EST No hits found N/A 0.03611064 22 21 1.99117875
Above3000_transcript-100_13227 Sodium-dependent noradrenaline transporter O55192 SC6A2_MOUSE 0.00617842 22 21 1.91432261
mira_extended_contigs_c2509 Unclassifiable EST No hits found N/A 0.020556 22 21 0.95317853
idba_c8603 Unclassifiable EST No hits found N/A 0.04955868 24 0 4.73173761
top500-3000_transcript-100_654 Mitochondrial ornithine transporter 1 Q9Y619 ORNT1_HUMAN 0.00215771 24 2 4.97733877
idba_c2713 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex assembly factor 3 Q6DFN1 NDUF3_XENTR 0.020556 24 4 2.14349557
idba_c8806 Amyloid protein-binding protein 2 Q92624 APBP2_HUMAN 0.00793367 24 4 0.91358532
mira_extended_contigs_c749 Titin A2ASS6 TITIN_MOUSE 0.01289121 24 5 1.02325829
idba_c5378 Proteasome subunit alpha type-6 P60901 PSA6_RAT 0.02578158 24 8 8.07393809
top500-3000_transcript-100_3658 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 5 Q63362 NDUA5_RAT 0.00051009 24 8 6.33755201
idba_c10235 60S ribosomal protein L32 Q962T1 RL32_SPOFR 0.00617842 24 9 76.2871313
mira_extended_contigs_c1523 60S ribosomal protein L44 Q6BLK0 RL44_DEBHA 0.02578158 24 10 115.220829
Above3000_transcript-100_16859 Unclassifiable EST No hits found N/A 0.020556 24 10 7.2110297
mira_extended_contigs_c2393 Unclassifiable EST No hits found N/A 0.04955868 24 10 0.78007242
idba_c9137 60S ribosomal protein L35 Q90YT4 RL35_ICTPU 0.020556 24 11 73.3419324
idba_c8421 Caspase-3 Q8MJC3 CASP3_RABIT 0.04002766 24 14 2.25672351
Above3000_transcript-100_22614 Unclassifiable EST No hits found N/A 0.0289876 24 15 1.53441464
Above3000_transcript-100_9685 Unclassifiable EST No hits found N/A 0.04002766 24 16 1.53429072
Above3000_transcript-100_15150 N-fatty-acyl-amino acid synthase/hydrolase PM20D1.2 Q08BB2 P2012_DANRE 0.01013747 24 17 1.24354857
idba_c10014 ATP-sensitive inward rectifier potassium channel 12 F1NHE9 KCJ12_CHICK 0.00215771 24 19 3.45810218
idba_c9239 Protein amalgam P15364 AMAL_DROME 0.04002766 24 19 1.52066177
idba_c4097 E3 ubiquitin-protein ligase RNF19A Q9NV58 RN19A_HUMAN 0.020556 24 20 5.64595915
Above3000_transcript-100_23922 Unclassifiable EST No hits found N/A 0.04955868 24 20 1.36914859
idba_c5169 Protein sprouty homolog 2 Q08E39 SPY2_BOVIN 0.03219362 24 21 9.35367744
idba_c8381 Unclassifiable EST No hits found N/A 0.02578158 24 21 6.14892456
top500-3000_transcript-100_313 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase Q91309 F26_LITCT 0.01013747 24 21 4.38003755
idba_c7181 Neuronal acetylcholine receptor subunit alpha-4 Q5IS77 ACHA4_PANTR 0.03219362 24 21 2.54837961
Above3000_transcript-100_4798 Synaptotagmin-7 Q62747 SYT7_RAT 0.00282946 24 21 2.1868999
Above3000_transcript-100_27182 Tripartite motif-containing protein 2 F7H9X2 TRIM2_CALJA 0.020556 24 21 0.86320166
idba_c832 Neuroendocrine convertase 2 P16519 NEC2_HUMAN 0.01289121 24 22 24.5186015
Above3000_transcript-100_18813 Unclassifiable EST No hits found N/A 0.02578158 24 22 20.7036195
idba_c248 Secernin-3 Q17QS0 SCRN3_BOVIN 0.04002766 24 22 8.24633143
Above3000_transcript-100_9897 Unclassifiable EST No hits found N/A 0.04955868 24 22 7.26374239
idba_c1902 Glutamate receptor ionotropic, NMDA 1 Q05586 NMDZ1_HUMAN 0.00068896 24 22 5.12421581
idba_c4019 Potential protein lysine methyltransferase SET5 Q4PBP5 SET5_USTMA 0.00478726 24 22 4.91773286
Above3000_transcript-100_4531 Microtubule cross-linking factor 1 Q9Y4B5 MTCL1_HUMAN 0.04002766 24 22 2.94030415
idba_c8474 Protein rolling stone O44252 ROST_DROME 0.00027422 24 22 2.82697349
idba_c3538 Glutamate receptor P26591 GLRK_LYMST 0.00215771 24 22 2.81525019
idba_c8258 Unclassifiable EST No hits found N/A 0.02578158 24 22 2.24743814
Above3000_transcript-100_1160 Acetylcholine receptor subunit alpha-like P91766 ACH1_MANSE 0.04002766 24 22 1.07341921
idba_c5012 Major facilitator superfamily domain-containing protein 6-A Q1LUQ4 MFD6A_DANRE 0.03219362 24 23 2.56669862
idba_c6705 Voltage-dependent calcium channel gamma-5 subunit Q9UF02 CCG5_HUMAN 0.04955868 24 23 1.40331298
Above3000_transcript-100_22597 Neuronal acetylcholine receptor subunit beta-4 P30926 ACHB4_HUMAN 0.020556 26 0 3.32144213
idba_c1930 BPTI/Kunitz domain-containing protein (Fragment) P86733 KCP_HALAI 0.03219362 26 2 19.2648697
top50-500_transcript-100_2183 Unclassifiable EST No hits found N/A 0.04002766 26 4 499.013689
idba_c449 Myophilin Q24799 MYPH_ECHGR 0.00478726 26 6 29.790374
idba_c4316 High mobility group protein DSP1 Q24537 HMG2_DROME 0.020556 26 6 24.5473669
idba_c4907 Protein-lysine methyltransferase METTL21D Q9H867 MT21D_HUMAN 0.04955868 26 6 2.07380842

122

idba_c4907 Protein-lysine methyltransferase METTL21D Q9H867 MT21D_HUMAN 0.04955868 26 6 2.07380842
idba_c3639 1-acyl-sn-glycerol-3-phosphate acyltransferase delta Q9NRZ5 PLCD_HUMAN 0.00617842 26 7 1.33412329
Above3000_transcript-100_26188 Unclassifiable EST No hits found N/A 0.01843601 26 8 2.24971891
Above3000_transcript-100_2986 Unclassifiable EST No hits found N/A 0.0289876 26 8 0.7045018
Above3000_transcript-100_20779 Unclassifiable EST No hits found N/A 0.00282946 26 9 8.40828346
Above3000_transcript-100_17233 RING finger protein nhl-1 Q03601 NHL1_CAEEL 0.04955868 26 9 3.76240523
mira_extended_contigs_c1546 Receptor-type tyrosine-protein phosphatase U B3DK56 PTPRU_DANRE 0.02578158 26 13 4.69091916
top50-500_transcript-100_1578 Unclassifiable EST No hits found N/A 0.020556 26 14 0.20924633
idba_c7737 Unclassifiable EST No hits found N/A 0.03219362 26 15 4.18689168
idba_c6262 Synaptosomal-associated protein 29 Q9Z2P6 SNP29_RAT 0.03219362 26 18 4.91733458
mira_extended_contigs_c2369 E3 ubiquitin-protein ligase CHFR Q5RF77 CHFR_PONAB 0.01289121 26 18 2.66988176
Above3000_transcript-100_768 Metalloprotease mig-17 Q20930 MIG17_CAEEL 0.04002766 26 18 1.64136631
Above3000_transcript-100_11918 Metabotropic glutamate receptor 2 Q14416 GRM2_HUMAN 0.00044267 26 18 1.56182263
mira_extended_contigs_c2338 Alpha-aminoadipic semialdehyde dehydrogenase Q2KJC9 AL7A1_BOVIN 0.04002766 26 19 7.98339289
mira_extended_contigs_c568 Pseudouridine-metabolizing bifunctional protein C1861.05 Q9USY1 YOW5_SCHPO 0.01631603 26 19 3.70807627
idba_c3515 Organic cation transporter protein Q9VCA2 ORCT_DROME 0.04002766 26 19 3.40276453
mira_extended_contigs_c2301 Ionotropic receptor 25a E9NA96 IR25A_DROME 0.02578158 26 19 2.07760594
idba_c1666 UPF0692 protein C19orf54 Q5BKX5 CS054_HUMAN 0.03219362 26 20 2.72865241
mira_extended_contigs_c2452 Unclassifiable EST No hits found N/A 0.02578158 26 20 1.05674121
idba_c1825 LIM domain-binding protein 3 O75112 LDB3_HUMAN 0.04002766 26 21 12.0250088
Above3000_transcript-100_2573 Unclassifiable EST No hits found N/A 0.03219362 26 21 2.84328662
Above3000_transcript-100_15930 Unclassifiable EST No hits found N/A 0.01289121 26 23 4.71986423
idba_c4871 Arrestin domain-containing protein 3 Q7TPQ9 ARRD3_MOUSE 0.04955868 26 23 2.1523489
idba_c461 Ornithine aminotransferase, mitochondrial Q9VW26 OAT_DROME 0.00793367 26 24 8.19157821
idba_c10203 Unclassifiable EST No hits found N/A 0.03219362 26 24 4.01266481
idba_c1214 E3 ubiquitin-protein ligase UBR2 Q8IWV8 UBR2_HUMAN 0.00793367 26 24 3.17003837
idba_c7252 Patched domain-containing protein 3 Q0EEE2 PTHD3_MOUSE 0.02578158 26 24 1.45052562
Above3000_transcript-100_4095 Lys-63-specific deubiquitinase BRCC36 B5X8M4 BRCC3_SALSA 0.03219362 28 2 1.70452827
idba_c6035 Unclassifiable EST No hits found N/A 0.04955868 28 2 1.1739408
idba_c2084 Chymotrypsinogen A P00766 CTRA_BOVIN 0.04955868 28 3 9.35121919
idba_c87 Unclassifiable EST No hits found N/A 0.01289121 28 4 65.6600774
idba_c8156 Ankyrin repeat domain-containing protein 39 Q0P5B9 ANR39_BOVIN 0.04002766 28 5 1.57064326
Above3000_transcript-100_27972 Protein lin-37 homolog Q1RMQ5 LIN37_BOVIN 0.01289121 28 5 0.74340752
idba_c6301 Gamma-secretase subunit pen-2 Q86BE9 PEN2_DROME 0.04002766 28 8 5.15404264
idba_c5323 Probable elongation factor 1-beta O96827 EF1B_DROME 0.01631603 28 9 67.3029516
idba_c2075 JmjC domain-containing protein 8 Q96S16 JMJD8_HUMAN 0.03219362 28 9 5.59761284
idba_c10648 Aminoacylase-1A Q6AYS7 ACY1A_RAT 0.04002766 28 10 9.89992344
idba_c7794 Unclassifiable EST No hits found N/A 0.02578158 28 10 9.26266891
top500-3000_transcript-100_8363 Unclassifiable EST No hits found N/A 0.03219362 28 11 8.89452862
Above3000_transcript-100_14195 Unclassifiable EST No hits found N/A 0.04955868 28 16 2.10014745
Above3000_transcript-100_2588 Store-operated calcium entry regulator STIMATE Q7TSW6 STIMA_RAT 0.04002766 28 17 1.25159314
Above3000_transcript-100_12622 Unclassifiable EST No hits found N/A 0.00793367 28 17 0.87977094
idba_c972 MAP kinase-interacting serine/threonine-protein kinase 2 Q66I46 MKNK2_XENTR 0.00369019 28 18 61.0365148
idba_c5816 Casein kinase I isoform alpha P67963 KC1A_XENLA 0.03219362 28 18 8.08868003
mira_extended_contigs_c276 Prolyl endopeptidase P23687 PPCE_PIG 0.04955868 28 18 3.82660663
Above3000_transcript-100_13513 ADP-ribosylation factor-related protein 1 Q32LJ2 ARFRP_BOVIN 0.04955868 28 18 2.70081627
top500-3000_transcript-100_3702 Coatomer subunit beta' P35606 COPB2_HUMAN 0.01013747 28 19 13.5992912
idba_c7371 Sodium-coupled neutral amino acid transporter 9 Q3B8Q3 S38A9_RAT 0.01631603 28 19 1.75461477
idba_c6760 Putative G-protein coupled receptor F59B2.13 P34488 YMJC_CAEEL 0.03219362 28 19 0.91428907
mira_extended_contigs_c2548 Major facilitator superfamily domain-containing protein 1 Q9H3U5 MFSD1_HUMAN 0.02578158 28 20 4.46160408
top500-3000_transcript-100_8947 Unclassifiable EST No hits found N/A 0.00369019 28 20 3.77149544
Above3000_transcript-100_480 Kelch-like protein 24 Q56A24 KLH24_RAT 0.02578158 28 20 3.05910586
top500-3000_transcript-100_9257 Unclassifiable EST No hits found N/A 0.03219362 28 20 2.43138172
idba_c7796 Unclassifiable EST No hits found N/A 0.03219362 28 20 1.96546906
Above3000_transcript-100_349 Excitatory amino acid transporter 1 O57321 EAA1_AMBTI 0.01631603 28 20 1.94768815
Above3000_transcript-100_2925 Rho guanine nucleotide exchange factor 17 Q96PE2 ARHGH_HUMAN 0.04002766 28 20 1.81079016
idba_c1042 Putative alkyl/aryl-sulfatase YjcS P32717 YJCS_ECOLI 0.04002766 28 20 0.53464697
idba_c4454 Mitochondrial import inner membrane translocase subunit Tim17-A Q99595 TI17A_HUMAN 0.00282946 28 21 18.5191648
idba_c502 F-box/WD repeat-containing protein 5 Q4KLI9 FBXW5_RAT 0.020556 28 21 7.41819219
idba_c3965 Kelch-like protein 20 Q6DFF6 KLH20_XENLA 0.020556 28 21 6.16579011
Above3000_transcript-100_2529 DmX-like protein 2 Q8TDJ6 DMXL2_HUMAN 0.01631603 28 21 5.25028199
idba_c6879 Solute carrier family 22 member 21 Q9WTN6 S22AL_MOUSE 0.02578158 28 21 4.08123437
Above3000_transcript-100_389 MFS-type transporter SLC18B1 Q6NT16 S18B1_HUMAN 0.02578158 28 22 4.01088998
Above3000_transcript-100_27045 Unclassifiable EST No hits found N/A 0.04955868 28 22 1.92847818
idba_c10429 Unclassifiable EST No hits found N/A 0.04002766 28 23 7.22816148
mira_extended_contigs_c167 Aldehyde dehydrogenase family 16 member A1 A6QR56 A16A1_BOVIN 0.01289121 28 23 4.15275752
Above3000_transcript-100_23970 Progestin and adipoQ receptor family member 3 Q6TCG8 PAQR3_MOUSE 0.00123373 28 24 5.21170932
idba_c1173 Unclassifiable EST No hits found N/A 0.03219362 28 27 4.96669611

123

Supplemental table 2. List of significant rhythmic genes in foot tissue samples from free-running
limpets entrained to a simulated intertidal cycle. Genes are ordered by period, then lag, then amplitude.
Contig Name Uniprot accession Uniprot ID ADJ.P PER LAG AMP
Above3000_transcript-100_1162 DNA annealing helicase and endonuclease ZRANB3 E1BB03 ZRAB3_BOVIN 0.00068896 10 0 1.37801288
idba_c6079 Putative ammonium transporter 3 Q21565 AMT3_CAEEL 0.020556 10 0 0.609601
mira_extended_contigs_c2360 Unclassifiable EST No hits found N/A 0.03219362 10 2 3.03138745
Above3000_transcript-100_16779 Unclassifiable EST No hits found N/A 0.01013747 10 2 2.29436564
idba_c7534 Leucine-rich repeat-containing protein 4B P0CC10 LRC4B_RAT 0.020556 10 2 0.77202237
idba_c9262 Sodium- and chloride-dependent glycine transporter 1 A7Y2W8 SC6A9_XENLA 0.020556 10 3 46.8960257
top500-3000_transcript-100_8673 Unclassifiable EST No hits found N/A 0.04955868 10 3 19.3528214
Above3000_transcript-100_2124 RCC1 and BTB domain-containing protein 1 Q8NDN9 RCBT1_HUMAN 0.00617842 10 3 5.66433438
idba_c7872 Kelch-like protein 9 Q6ZPT1 KLHL9_MOUSE 0.020556 10 3 5.62578168
idba_c1622 Neurogenic locus notch homolog protein 1 P21783 NOTC1_XENLA 0.00068896 10 3 3.45858319
idba_c4031 Unclassifiable EST No hits found N/A 0.04955868 10 3 2.54114095
Above3000_transcript-100_833 Synaptic vesicle 2-related protein Q2XWK0 SVOP_XENLA 0.03611064 10 3 1.05259138
Above3000_transcript-100_488 Kelch-like protein 24 Q56A24 KLH24_RAT 0.04002766 10 3 0.38485135
idba_c2621 Unclassifiable EST No hits found N/A 0.03219362 10 4 49.7039198
idba_c10431 Endothelin-converting enzyme 2 Q80Z60 ECE2_MOUSE 0.00163627 10 4 23.0910931
Above3000_transcript-100_12136 Peroxiredoxin-5, mitochondrial P30044 PRDX5_HUMAN 0.04955868 10 4 8.20817153
idba_c5340 Uncharacterized protein in xynA 3'region (Fragment) P40983 YOR6_CALSR 0.00478726 10 4 5.38716691
idba_c9327 Probable proline iminopeptidase O83041 PIP_LEPBY 0.01631603 10 4 4.7360644
idba_c8321 Transmembrane protein 127 Q504G0 TM127_DANRE 0.04002766 10 4 4.65700844
mira_extended_contigs_c2709 Unclassifiable EST No hits found N/A 0.00793367 10 4 3.32778063
idba_c1188 Unclassifiable EST No hits found N/A 0.03219362 10 4 2.04032197
mira_extended_contigs_c307 Acyl-CoA synthetase short-chain family member 3, mitochondrial Q5REB8 ACSS3_PONAB 0.03219362 10 4 1.9248753
idba_c2202 Glutamyl-tRNA(Gln) amidotransferase subunit B, mitochondrial B0W3H3 GATB_CULQU 0.00793367 10 4 1.70624725
mira_extended_contigs_c2358 Probable 2-oxoglutarate dehydrogenase E1 component DHKTD1, Q5R7H0 DHTK1_PONAB 0.01289121 10 4 1.4906808
Above3000_transcript-100_3598 Glutaredoxin domain-containing cysteine-rich protein CG31559 Q9VNL4 GRCR1_DROME 0.020556 10 4 1.27788585
idba_c6041 Unclassifiable EST No hits found N/A 0.020556 10 4 0.60624825
mira_extended_contigs_c524 Dynein heavy chain 8, axonemal Q96JB1 DYH8_HUMAN 0.04002766 10 5 2.24063418
Above3000_transcript-100_4134 Uncharacterized protein C15orf41 homolog Q6NRW5 CO041_XENLA 0.04002766 10 6 1.78983434
mira_extended_contigs_c1877 N-sulphoglucosamine sulphohydrolase P51688 SPHM_HUMAN 0.04955868 10 6 0.42001188
mira_extended_contigs_c2702 Unclassifiable EST No hits found N/A 0.01631603 12 0 5.29149324
Above3000_transcript-100_25715 UPF0193 protein EVG1 homolog Q9D9S1 EVG1_MOUSE 0.020556 12 1 5.43383171
Above3000_transcript-100_26703 Unclassifiable EST No hits found N/A 0.04479317 12 2 6.36731625
mira_extended_contigs_c300 Ataxin-10 Q9ER24 ATX10_RAT 0.04955868 12 2 1.5382256
idba_c319 Unclassifiable EST No hits found N/A 0.03219362 12 3 13.5243983
mira_extended_contigs_c1988 PIH1 domain-containing protein 1 Q7ZWY2 PIHD1_XENLA 0.02578158 12 3 3.90702271
idba_c661 Filamin-A P21333 FLNA_HUMAN 0.02578158 12 3 0.48937906
top50-500_transcript-100_470 Unclassifiable EST No hits found N/A 0.04002766 12 4 32.8411672
Above3000_transcript-100_17533 Ubiquitin-conjugating enzyme E2 G2 Q17QG5 UB2G2_BOVIN 0.04955868 12 4 5.50104521
Above3000_transcript-100_28173 Unclassifiable EST No hits found N/A 0.03219362 12 4 2.11994361
idba_c3240 Unclassifiable EST No hits found N/A 0.01289121 12 5 2.98719027
Above3000_transcript-100_13998 Unclassifiable EST No hits found N/A 0.04955868 12 5 2.54761204
idba_c4488 Growth/differentiation factor 8 Q98TB4 GDF8_OREMO 0.04955868 12 5 1.52191441
top50_transcript-100_31 4-aminobutyrate aminotransferase, mitochondrial P61922 GABT_MOUSE 0.04002766 12 6 110.445251
Above3000_transcript-100_9505 Unclassifiable EST No hits found N/A 0.00369019 12 6 2.72248768
mira_extended_contigs_c1349 Acetolactate synthase, mitochondrial Q5KPJ5 ILVB_CRYNJ 0.03219362 12 6 0.47717015
Above3000_transcript-100_17096 Unclassifiable EST No hits found N/A 0.04955868 12 7 2.53440116
Above3000_transcript-100_10410 Uncharacterized shell protein 26 (Fragment) B3A0P4 USP26_LOTGI 0.01013747 12 7 0.64542232
Above3000_transcript-100_14277 GTPase IMAP family member 4 Q9NUV9 GIMA4_HUMAN 0.00215771 12 8 5.82673595
Above3000_transcript-100_12558 Connector enhancer of kinase suppressor of ras 3 Q5SGD7 CNKR3_RAT 0.03611064 12 8 3.91371158
Above3000_transcript-100_3517 Creatine transporter Q91502 SC6A8_TORMA 0.020556 12 8 2.94753361
Above3000_transcript-100_755 Kelch domain-containing protein 2 Q3KRE6 KLDC2_RAT 0.01631603 12 8 2.76616602
mira_extended_contigs_c2450 DNA helicase MCM8 Q9UJA3 MCM8_HUMAN 0.03611064 12 8 0.81225215
idba_c4055 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase P46978 STT3A_MOUSE 0.01289121 12 9 12.7485911
idba_c6728 Arylesterase B5BLW5 ARE_SULSF 0.02578158 12 9 8.09201333
Above3000_transcript-100_1163 Vacuolar protein sorting-associated protein 26B Q8C0E2 VP26B_MOUSE 0.02578158 12 9 4.28350757
Above3000_transcript-100_615 Unclassifiable EST No hits found N/A 0.01013747 12 9 2.52255324
Above3000_transcript-100_4931 CDK2-associated and cullin domain-containing protein 1 Q8R0X2 CACL1_MOUSE 0.01289121 12 9 2.24755623
Above3000_transcript-100_11773 SNARE-associated protein Snapin P60192 SNAPN_RAT 0.03219362 12 9 1.79456064
Above3000_transcript-100_8944 BarH-like 1 homeobox protein Q9BZE3 BARH1_HUMAN 0.01013747 12 9 1.78213165
idba_c1180 General transcription factor 3C polypeptide 5 Q8R2T8 TF3C5_MOUSE 0.04002766 12 9 1.77140519
Above3000_transcript-100_16056 Meiotic recombination protein REC8 homolog O95072 REC8_HUMAN 0.01631603 12 9 1.72087959
Above3000_transcript-100_18146 Neuronal acetylcholine receptor subunit alpha-9 Q9PTS8 ACHA9_CHICK 0.04002766 12 9 1.54604125
Above3000_transcript-100_7132 TBC1 domain family member 5 Q92609 TBCD5_HUMAN 0.04955868 12 9 0.95108797
mira_extended_contigs_c423 Protocadherin-9 Q9HC56 PCDH9_HUMAN 0.00369019 12 9 0.26406655
idba_c9316 Transformer-2 protein homolog beta P62997 TRA2B_RAT 0.00369019 12 10 13.8974622
idba_c6344 Ubiquitin carboxyl-terminal hydrolase O01391 UCHL_APLCA 0.04955868 12 10 6.53887096
idba_c3647 Arginine kinase O15990 KARG_LIOJA 0.04002766 12 10 6.53023684
idba_c5870 Syntaxin-17 P56962 STX17_HUMAN 0.01013747 12 10 5.24089073
mira_extended_contigs_c898 Epidermal growth factor-like protein 7 Q9UHF1 EGFL7_HUMAN 0.01631603 12 10 4.66684041
Above3000_transcript-100_13390 Unclassifiable EST No hits found N/A 0.03219362 12 10 4.53824173
idba_c4465 Zinc finger CCCH domain-containing protein 15 Q803J8 ZC3HF_DANRE 0.03219362 12 10 3.20410518
Above3000_transcript-100_1193 Isocitrate dehydrogenase [NAD] subunit gamma, mitochondrial Q58CP0 IDH3G_BOVIN 0.03219362 12 10 3.18179384
Above3000_transcript-100_3767 Unclassifiable EST No hits found N/A 0.04955868 12 10 2.5907696
idba_c3113 X-ray repair cross-complementing protein 6 P23475 XRCC6_MOUSE 0.04955868 12 10 2.33737983
Above3000_transcript-100_6291 Zinc finger protein 84 P51523 ZNF84_HUMAN 0.02578158 12 10 1.61038372
Above3000_transcript-100_713 Protein smg8 Q0VA04 SMG8_XENTR 0.04002766 12 10 1.35618979
Above3000_transcript-100_3013 RING finger protein 17 Q99MV7 RNF17_MOUSE 0.01151434 12 10 0.94959933
mira_extended_contigs_c979 HEAT repeat-containing protein 6 Q7ZY56 HEAT6_XENLA 0.00027422 12 10 0.73207949
Above3000_transcript-100_7873 Con-Ins Im1 A0A0B5A7M7 INS1_CONIM 0.01289121 12 10 0.61291362

124

Above3000_transcript-100_7873 Con-Ins Im1 A0A0B5A7M7 INS1_CONIM 0.01289121 12 10 0.61291362
idba_c9639 Unclassifiable EST No hits found N/A 0.04002766 12 11 2.85694439
Above3000_transcript-100_10530 Unclassifiable EST No hits found N/A 0.00051009 12 11 2.44788806
Above3000_transcript-100_321 Beta-catenin-like protein 1 Q4V8K2 CTBL1_RAT 0.02578158 12 11 2.01057682
mira_extended_contigs_c1304 Multidrug and toxin extrusion protein 1 Q96FL8 S47A1_HUMAN 0.04955868 12 11 1.50982094
idba_c476 Protein NRDE2 homolog Q80XC6 NRDE2_MOUSE 0.03219362 12 11 1.13422332
idba_c104 Target of EGR1 protein 1 Q17QN2 TOE1_BOVIN 0.00617842 12 11 1.02429862
top50-500_transcript-100_2379 Unclassifiable EST No hits found N/A 0.04955868 14 0 2.37602551
idba_c3068 Glutathione S-transferase U16 Q9XIF8 GSTUG_ARATH 0.03219362 14 0 2.12238507
mira_extended_contigs_c1640 Laccase-9 Q9LFD1 LAC9_ARATH 0.04002766 14 0 0.52578198
top500-3000_transcript-100_10159 Tubulin alpha-2/alpha-4 chain P41383 TBA2_PATVU 0.00478726 14 1 170.793934
Above3000_transcript-100_17219 Unclassifiable EST No hits found N/A 0.00793367 14 1 1.005462
top50_transcript-100_366 Unclassifiable EST No hits found N/A 0.01289121 14 2 18.3123732
mira_extended_contigs_c317 NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial P25708 NDUV1_BOVIN 0.04002766 14 2 18.11882
Above3000_transcript-100_16261 Rieske domain-containing protein Q8K2P6 RFESD_MOUSE 0.04002766 14 2 4.01569229
idba_c1442 4-aminobutyrate aminotransferase, mitochondrial P80147 GABT_PIG 0.02578158 14 5 84.3540065
idba_c6959 Sarcoplasmic calcium-binding proteins I, III, and IV P04569 SCP1_BRALA 0.020556 14 6 13.7223909
idba_c3382 Unclassifiable EST No hits found N/A 0.04002766 14 6 3.28811689
Above3000_transcript-100_2855 TGF-beta-activated kinase 1 and MAP3K7-binding protein 1 Q8CF89 TAB1_MOUSE 0.020556 14 6 3.1329165
top500-3000_transcript-100_5864 Unclassifiable EST No hits found N/A 0.03611064 14 6 0.76548198
top500-3000_transcript-100_6402 3'(2'),5'-bisphosphate nucleotidase 1 O95861 BPNT1_HUMAN 0.04002766 14 7 21.283483
top500-3000_transcript-100_9200 Unclassifiable EST No hits found N/A 0.00282946 14 7 15.256678
mira_extended_contigs_c1353 Phosphatidylinositol glycan anchor biosynthesis class U protein Q8CHJ1 PIGU_RAT 0.02578158 14 7 10.8550246
Above3000_transcript-100_11792 Helicase with zinc finger domain 2 E9QAM5 HELZ2_MOUSE 0.04955868 14 7 8.67661174
idba_c5308 Sorting nexin-2 Q2TBW7 SNX2_BOVIN 0.01289121 14 7 7.27085588
top500-3000_transcript-100_5609 Oxysterol-binding protein-related protein 8 B9EJ86 OSBL8_MOUSE 0.00793367 14 7 7.20478064
Above3000_transcript-100_23716 Unclassifiable EST No hits found N/A 0.01013747 14 7 6.75381127
top500-3000_transcript-100_415 Peroxisomal carnitine O-octanoyltransferase O19094 OCTC_BOVIN 0.00215771 14 7 5.86554161
Above3000_transcript-100_22716 Unclassifiable EST No hits found N/A 0.01289121 14 7 5.76621203
idba_c504 Renin receptor Q5R563 RENR_PONAB 0.020556 14 7 5.25421881
Above3000_transcript-100_28196 Multiple epidermal growth factor-like domains protein 10 Q96KG7 MEG10_HUMAN 0.01631603 14 7 4.93286918
Above3000_transcript-100_4040 Failed axon connections homolog D3ZAT9 FAXC_RAT 0.00617842 14 7 4.93099058
Above3000_transcript-100_18425 Unclassifiable EST No hits found N/A 0.04002766 14 7 4.58433943
Above3000_transcript-100_6579 Calcium-regulated heat stable protein 1 Q9CR86 CHSP1_MOUSE 0.04955868 14 7 4.18153393
idba_c1604 Translocating chain-associated membrane protein 1 Q9GKZ4 TRAM1_BOVIN 0.00123373 14 7 3.68176394
idba_c5432 Protein FAM92A Q1LU86 FA92A_DANRE 0.01013747 14 7 3.60618377
Above3000_transcript-100_23117 Tubulin polyglutamylase TTLL11 A4Q9F4 TTL11_MOUSE 0.01631603 14 7 3.44648866
idba_c7031 UBX domain-containing protein 6 Q2KIJ6 UBXN6_BOVIN 0.04955868 14 7 3.02978179
top500-3000_transcript-100_3370 E3 ubiquitin-protein ligase Ubr3 Q9W3M3 UBR3_DROME 0.03611064 14 7 2.87051147
idba_c10476 Stathmin Q6DUB7 STMN1_PIG 0.04955868 14 7 2.85694121
mira_extended_contigs_c2114 Transmembrane protein 237 Q96Q45 TM237_HUMAN 0.04955868 14 7 2.68510224
idba_c1782 Leucine-rich repeat-containing protein 43 Q95JT3 LRC43_MACFA 0.04002766 14 7 2.52147402
mira_extended_contigs_c2644 Transcription factor E2F4 Q8R0K9 E2F4_MOUSE 0.04955868 14 7 2.50489289
idba_c5364 Charged multivesicular body protein 1a Q6PHF0 CHM1A_DANRE 0.01013747 14 7 2.16681029
mira_extended_contigs_c2681 1-phosphatidylinositol 3-phosphate 5-kinase Q9Y2I7 FYV1_HUMAN 0.03219362 14 7 2.09972353
Above3000_transcript-100_11318 3-hydroxybutyryl-CoA dehydrogenase P77851 HBD_THETC 0.01289121 14 7 2.04323878
idba_c5064 Zinc finger protein 830 Q63ZM9 ZN830_XENLA 0.04955868 14 7 1.96507909
Above3000_transcript-100_8255 Deoxynucleotidyltransferase terminal-interacting protein 1 Q99LB0 TDIF1_MOUSE 0.020556 14 7 1.92028617
Above3000_transcript-100_3716 Mitotic spindle assembly checkpoint protein MAD1 Q80YF0 MD1L1_CRIGR 0.01013747 14 7 1.9192119
idba_c5151 Protein FAM50 homolog Q299F9 FAM50_DROPS 0.04955868 14 7 1.80887849
mira_extended_contigs_c2653 Centromere protein J Q569L8 CENPJ_MOUSE 0.020556 14 7 1.64091164
Above3000_transcript-100_3626 Structural maintenance of chromosomes flexible hinge Q6P5D8 SMHD1_MOUSE 0.03611064 14 7 1.41369278
idba_c6269 RING finger protein 17 Q4R3G4 RNF17_MACFA 0.04955868 14 7 1.37704449
top500-3000_transcript-100_8607 Unclassifiable EST No hits found N/A 0.02578158 14 7 1.34985995
Above3000_transcript-100_3202 Unclassifiable EST No hits found N/A 0.01289121 14 7 1.29569539
idba_c9119 Mediator of RNA polymerase II transcription subunit 12-like protein Q86YW9 MD12L_HUMAN 0.03611064 14 7 1.21894143
mira_extended_contigs_c1064 Crossover junction endonuclease MUS81 Q640B4 MUS81_XENTR 0.04002766 14 7 1.03527928
mira_extended_contigs_c296 Unclassifiable EST No hits found N/A 0.00478726 14 7 1.01051216
Above3000_transcript-100_121 Unclassifiable EST No hits found N/A 0.04955868 14 7 0.93237491
Above3000_transcript-100_1415 Cell growth regulator with RING finger domain protein 1 Q99675 CGRF1_HUMAN 0.02578158 14 7 0.33671806
idba_c340 Unclassifiable EST No hits found N/A 0.04002766 14 8 19.7497775
idba_c152 Receptor-type tyrosine-protein phosphatase T Q99M80 PTPRT_MOUSE 0.02578158 14 8 19.6194986
idba_c7710 Myotrophin Q7T2B9 MTPN_DANRE 0.04955868 14 8 19.1142266
idba_c1828 Unclassifiable EST No hits found N/A 0.020556 14 8 16.1514962
idba_c663 Baculoviral IAP repeat-containing protein 2 Q13490 BIRC2_HUMAN 0.04955868 14 8 15.8154142
idba_c9170 Unclassifiable EST No hits found N/A 0.04955868 14 8 13.5768688
Above3000_transcript-100_26391 Unclassifiable EST No hits found N/A 0.02578158 14 8 13.2181606
Above3000_transcript-100_9598 Unclassifiable EST No hits found N/A 0.00478726 14 8 11.9764272
idba_c10504 Multiple epidermal growth factor-like domains protein 10 A0JM12 MEG10_XENTR 0.01631603 14 8 10.7458399
idba_c6899 Unclassifiable EST No hits found N/A 0.01013747 14 8 10.4582525
Above3000_transcript-100_15287 Transcription and mRNA export factor ENY2 B5FZ63 ENY2_TAEGU 0.020556 14 8 8.40325858
idba_c7068 Methionine-R-sulfoxide reductase B3, mitochondrial Q8BU85 MSRB3_MOUSE 0.04955868 14 8 6.58975066
Above3000_transcript-100_12769 Transcription initiation factor TFIID subunit 13 Q5R9W6 TAF13_PONAB 0.01013747 14 8 6.49484473
Above3000_transcript-100_22232 Unclassifiable EST No hits found N/A 0.00369019 14 8 6.05563348
idba_c9352 Splicing factor U2AF 65 kDa subunit P26369 U2AF2_MOUSE 0.04002766 14 8 5.51494605
mira_extended_contigs_c2288 Nicastrin Q92542 NICA_HUMAN 0.03219362 14 8 4.85673853
idba_c1212 Copine-8 Q86YQ8 CPNE8_HUMAN 0.02578158 14 8 4.68191487
Above3000_transcript-100_101 PH and SEC7 domain-containing protein 3 Q2PFD7 PSD3_MOUSE 0.00068896 14 8 3.93222576
Above3000_transcript-100_20703 Unclassifiable EST No hits found N/A 0.02578158 14 8 3.28942574
idba_c1426 Bardet-Biedl syndrome 7 protein homolog Q8K2G4 BBS7_MOUSE 0.020556 14 8 2.97478727
idba_c6479 Unclassifiable EST No hits found N/A 0.04955868 14 8 2.7984656

125

idba_c6479 Unclassifiable EST No hits found N/A 0.04955868 14 8 2.7984656
mira_extended_contigs_c1780 WASH complex subunit 4 Q2M389 WASC4_HUMAN 0.02578158 14 8 2.58263575
mira_extended_contigs_c1692 Conserved oligomeric Golgi complex subunit 6 Q9Y2V7 COG6_HUMAN 0.04955868 14 8 2.53360213
idba_c5024 Unclassifiable EST No hits found N/A 0.04002766 14 8 2.23869901
idba_c1635 GRIP and coiled-coil domain-containing protein 2 Q8CHG3 GCC2_MOUSE 0.00282946 14 8 2.14172921
mira_extended_contigs_c2219 Putative phospholipase B-like 2 Q3TCN2 PLBL2_MOUSE 0.04002766 14 8 2.06021978
mira_extended_contigs_c1679 Type I inositol 3,4-bisphosphate 4-phosphatase Q9EPW0 INP4A_MOUSE 0.04955868 14 8 2.02432933
top500-3000_transcript-100_8137 Unclassifiable EST No hits found N/A 0.00705605 14 8 1.66887064
idba_c3272 Tetratricopeptide repeat protein 13 Q8NBP0 TTC13_HUMAN 0.00282946 14 8 1.57662344
Above3000_transcript-100_4749 Polycomb complex protein BMI-1 P25916 BMI1_MOUSE 0.04955868 14 8 1.39274828
Above3000_transcript-100_4128 La-related protein 7 Q4G0J3 LARP7_HUMAN 0.04002766 14 8 1.25709762
idba_c10226 Serine/arginine-rich splicing factor 4 Q08170 SRSF4_HUMAN 0.00369019 14 9 15.652859
top500-3000_transcript-100_344 Septin-11 Q9NVA2 SEP11_HUMAN 0.03219362 14 9 5.37524491
idba_rep_c10663 Zinc finger protein 84 P51523 ZNF84_HUMAN 0.01289121 14 9 3.7910229
idba_c2851 DnaJ homolog subfamily C member 28 Q8VCE1 DJC28_MOUSE 0.03219362 14 9 2.37569423
Above3000_transcript-100_27044 Protein angel homolog 2 Q5VTE6 ANGE2_HUMAN 0.03219362 14 13 2.47799066
mira_extended_contigs_c2675 BTB/POZ domain-containing protein 17 Q6GLJ1 BTBDH_XENLA 0.01013747 16 2 26.1590254
mira_extended_contigs_c2052 Eukaryotic translation initiation factor 2D P0CL18 EIF2D_RABIT 0.01631603 16 2 1.52720322
Above3000_transcript-100_25078 Unclassifiable EST No hits found N/A 0.04955868 16 2 1.02297421
idba_c8094 Unclassifiable EST No hits found N/A 0.03219362 16 3 9.11909654
idba_c8538 High mobility group protein B1 Q9YH06 HMGB1_CHICK 0.04002766 16 4 9.32290027
Above3000_transcript-100_25548 Unclassifiable EST No hits found N/A 0.03219362 16 4 8.53342863
idba_c7433 Down syndrome critical region protein 3 homolog O35075 DSCR3_MOUSE 0.04002766 16 4 3.61576737
idba_c6272 Protein SFI1 homolog A9CB34 SFI1_PAPAN 0.00369019 16 4 2.32444278
mira_extended_contigs_c873 Transmembrane and ubiquitin-like domain-containing protein 1 Q6P135 TMUB1_DANRE 0.04002766 16 4 1.78032181
Above3000_transcript-100_1266 Leucine-rich repeat serine/threonine-protein kinase 2 Q5S006 LRRK2_MOUSE 0.01843601 16 4 0.6684512
idba_c375 Cytochrome P450 3A41 Q9JMA7 CP341_MOUSE 0.04002766 16 5 10.5713256
Above3000_transcript-100_10096 Condensin complex subunit 2 Q8C156 CND2_MOUSE 0.020556 16 5 2.77058986
mira_extended_contigs_c722 Leucine-rich repeats and immunoglobulin-like domains protein 1 P70193 LRIG1_MOUSE 0.04955868 16 5 2.5166344
Above3000_transcript-100_253 Transcription factor 25 Q9BQ70 TCF25_HUMAN 0.04955868 16 5 1.66288004
Above3000_transcript-100_3274 E3 ubiquitin-protein ligase TRIM33 Q56R14 TRI33_XENLA 0.00282946 16 6 3.9898638
mira_extended_contigs_c1082 Zinc finger protein 479 Q96JC4 ZN479_HUMAN 0.00617842 16 6 1.43369966
idba_c4565 Dystonin Q91ZU6 DYST_MOUSE 0.04002766 16 6 0.60584715
Above3000_transcript-100_13602 G1/S-specific cyclin-E O15995 CCNE_HEMPU 0.01631603 16 7 2.97172956
idba_c5884 Ubiquitin carboxyl-terminal hydrolase 3 Q91W36 UBP3_MOUSE 0.03219362 16 7 2.09770368
Above3000_transcript-100_17377 E3 ubiquitin-protein ligase RNF38 Q8BI21 RNF38_MOUSE 0.03219362 16 7 1.77513996
idba_c4978 Actin P90689 ACT_BRUMA 0.03219362 16 8 1.15106908
Above3000_transcript-100_24971 Unclassifiable EST No hits found N/A 0.04955868 16 9 2.18498877
idba_c3602 Unclassifiable EST No hits found N/A 0.0289876 16 9 2.0330469
top500-3000_transcript-100_9522 Unclassifiable EST No hits found N/A 0.04479317 16 9 1.681612
Above3000_transcript-100_4724 Ubiquitin carboxyl-terminal hydrolase 34 Q70CQ2 UBP34_HUMAN 0.01843601 16 9 0.8028653
idba_c9636 Unclassifiable EST No hits found N/A 0.02578158 16 11 38.2092977
top500-3000_transcript-100_3810 Oligosaccharyltransferase complex subunit ostc-B Q5M9B7 OSTCB_XENLA 0.03219362 16 11 7.44234377
idba_c10175 GTP cyclohydrolase 1 feedback regulatory protein P99025 GFRP_MOUSE 0.00051009 16 11 4.19650391
mira_extended_contigs_c126 N-acetylserotonin O-methyltransferase-like protein O95671 ASML_HUMAN 0.00793367 16 11 0.66551971
idba_c5348 Myosin heavy chain, striated muscle P24733 MYS_ARGIR 0.04002766 18 0 0.4273711
Above3000_transcript-100_19753 E3 ubiquitin-protein ligase MIB2 Q5ZIJ9 MIB2_CHICK 0.04955868 18 1 15.7839138
idba_c3235 Protein phosphatase 1 regulatory subunit 7 Q32PL1 PP1R7_DANRE 0.04002766 18 1 2.80034367
idba_c4428 Eukaryotic translation initiation factor eIF1 P42678 ETIF1_ANOGA 0.020556 18 2 18.1533049
top500-3000_transcript-100_2054 Casein kinase II subunit beta P28021 CSK2B_XENLA 0.03219362 18 2 8.51920765
top500-3000_transcript-100_8025 Sphingosine-1-phosphate lyase 1 Q8CHN6 SGPL1_RAT 0.04955868 18 2 3.65204955
idba_c9706 Pancreatic secretory granule membrane major glycoprotein GP2 P25291 GP2_CANLF 0.04002766 18 3 239.920477
idba_c2055 Dynein heavy chain 6, axonemal Q9C0G6 DYH6_HUMAN 0.00793367 18 3 9.84475851
top500-3000_transcript-100_919 Enolase O02654 ENO_DORPE 0.02578158 18 4 15.373263
idba_c1048 Lachesin Q24372 LACH_DROME 0.04955868 18 4 8.9711797
idba_c2386 Voltage-gated hydrogen channel 1 Q5M7E9 HVCN1_XENLA 0.020556 18 4 4.28845414
idba_c4739 Splicing factor 3B subunit 3 Q921M3 SF3B3_MOUSE 0.01013747 18 4 2.8733977
Above3000_transcript-100_3227 Unclassifiable EST No hits found N/A 0.01289121 18 4 2.1136348
idba_c3126 Zinc phosphodiesterase ELAC protein 2 Q8CGS5 RNZ2_RAT 0.01289121 18 4 1.91848535
Above3000_transcript-100_14528 Osmotic avoidance abnormal protein 3 P46873 OSM3_CAEEL 0.02316879 18 4 1.66485463
idba_c3256 Glutamyl-tRNA(Gln) amidotransferase subunit A, mitochondrial F1QAJ4 GATA_DANRE 0.04002766 18 4 1.64058036
Above3000_transcript-100_4568 Protein jagunal Q7K1V5 JAGN_DROME 0.04002766 18 5 1.84680876
Above3000_transcript-100_25789 Probable RNA-directed DNA polymerase from transposon BS Q95SX7 RTBS_DROME 0.02578158 18 5 1.71444686
mira_extended_contigs_c255 Proton myo-inositol cotransporter Q3UHK1 MYCT_MOUSE 0.01460362 18 5 0.41725381
Above3000_transcript-100_628 Sonic hedgehog protein A Q92008 SHH_DANRE 0.00705605 18 5 0.17833445
mira_extended_contigs_c460 Unclassifiable EST No hits found N/A 0.03219362 18 6 0.21543422
idba_c548 Acyl-coenzyme A thioesterase 9, mitochondrial Q9Y305 ACOT9_HUMAN 0.01289121 18 7 2.24199519
Above3000_transcript-100_5994 Aspartate aminotransferase Q4J8X2 AAT_SULAC 0.020556 18 7 1.61458924
Above3000_transcript-100_23160 Unclassifiable EST No hits found N/A 0.00051009 18 7 1.31665687
Above3000_transcript-100_24371 GPI ethanolamine phosphate transferase 3 Q8TEQ8 PIGO_HUMAN 0.01013747 18 7 1.20906421
idba_c3973 Gamma-glutamyl hydrolase A7YWG4 GGH_BOVIN 0.02578158 18 8 16.7241
idba_c3570 Cytochrome P450 4F6 P51871 CP4F6_RAT 0.020556 18 8 0.98134754
Above3000_transcript-100_1921 Uncharacterized transporter slc-17.2 Q03567 SL172_CAEEL 0.03219362 18 8 0.84255733
Above3000_transcript-100_5356 Unclassifiable EST No hits found N/A 0.01631603 18 8 0.75540147
Above3000_transcript-100_2778 Box C/D snoRNA protein 1 Q3UFB2 BCD1_MOUSE 0.04955868 18 9 2.12575249
idba_c2466 Inositol-3-phosphate synthase 1-A Q7ZXY0 INO1A_XENLA 0.03219362 18 9 1.38995414
idba_c6126 Unclassifiable EST No hits found N/A 0.00369019 20 0 19.5412965
idba_c8180 Medium-chain specific acyl-CoA dehydrogenase, mitochondrial P41367 ACADM_PIG 0.01631603 20 0 7.53882496
Above3000_transcript-100_14634 Aquaporin-10 G5CTG7 AQP10_MILTA 0.00163627 20 0 3.58720078
top500-3000_transcript-100_1866 Unclassifiable EST No hits found N/A 0.01289121 20 0 2.53618413
idba_c2058 Ufm1-specific protease 2 Q5ZIF3 UFSP2_CHICK 0.00369019 20 0 2.0992777

126

idba_c2058 Ufm1-specific protease 2 Q5ZIF3 UFSP2_CHICK 0.00369019 20 0 2.0992777
Above3000_transcript-100_1270 Kelch domain-containing protein 10 Q5U3Y0 KLD10_RAT 0.04955868 20 0 1.60030886
idba_c6389 Leucine-zipper-like transcriptional regulator 1 Q8N653 LZTR1_HUMAN 0.00014344 20 0 1.24943647
top500-3000_transcript-100_1020 Tetraspanin-33 Q3SYV5 TSN33_BOVIN 0.020556 20 1 14.965679
mira_extended_contigs_c1433 Probable V-type proton ATPase subunit H 2 Q619W9 VATH2_CAEBR 0.00617842 20 1 10.1966376
idba_c4622 Electron transfer flavoprotein subunit alpha, mitochondrial P13803 ETFA_RAT 0.02578158 20 1 9.85087268
mira_extended_contigs_c193 Lysosomal aspartic protease Q03168 ASPP_AEDAE 0.04955868 20 1 7.1698029
Above3000_transcript-100_3965 Unclassifiable EST No hits found N/A 0.02578158 20 1 6.73633973
top500-3000_transcript-100_11569 E3 ubiquitin-protein ligase UBR3 Q5U430 UBR3_MOUSE 0.020556 20 1 6.09635806
idba_c3108 Zinc finger DHHC domain-containing protein 4 Q8I0G4 ZDHC4_CAEEL 0.04955868 20 1 5.48532217
mira_extended_contigs_c2670 eIF-2-alpha kinase GCN2 Q9QZ05 E2AK4_MOUSE 0.02578158 20 1 2.45896277
idba_c3574 Epoxide hydrolase 4 Q8IUS5 EPHX4_HUMAN 0.01631603 20 2 5.52172473
Above3000_transcript-100_3303 Protein AMN1 homolog Q32L08 AMN1_BOVIN 0.00215771 20 2 4.52897226
Above3000_transcript-100_10160 Histidine ammonia-lyase P42357 HUTH_HUMAN 0.04955868 20 2 2.56720898
Above3000_transcript-100_3374 Dihydropteridine reductase P11348 DHPR_RAT 0.01289121 20 2 2.51958816
idba_c5393 Nucleoside diphosphate-linked moiety X motif 19 Q5PQ50 NUD19_XENLA 0.01289121 20 2 2.15029015
Above3000_transcript-100_12032 BTB/POZ domain-containing protein KCTD7 Q5ZJP7 KCTD7_CHICK 0.00478726 20 2 1.54725924
Above3000_transcript-100_9310 Uncharacterized protein YMR196W Q04336 YM54_YEAST 0.020556 20 2 1.32394184
mira_extended_contigs_c1664 Unclassifiable EST No hits found N/A 0.03611064 20 2 0.41502572
idba_c5037 Unclassifiable EST No hits found N/A 0.04479317 20 2 0.37615959
top500-3000_transcript-100_2304 Selenoprotein W Q568W0 SELW_DANRE 0.020556 20 3 53.1155868
Above3000_transcript-100_23478 Unclassifiable EST No hits found N/A 0.04002766 20 3 1.9946807
idba_c693 Glutathione reductase, mitochondrial A2TIL1 GSHR_CALJA 0.00793367 20 4 4.30326484
idba_c9288 Protein SCO1 homolog, mitochondrial A1A4J8 SCO1_BOVIN 0.02578158 20 4 3.20567549
idba_c3158 Hydroxysteroid dehydrogenase-like protein 2 Q66KC4 HSDL2_XENTR 0.01631603 20 5 4.76087253
Above3000_transcript-100_5713 Unclassifiable EST No hits found N/A 0.01631603 20 5 4.45926568
Above3000_transcript-100_6453 AUGMIN subunit 2 O48767 AUG2_ARATH 0.04955868 20 5 1.82749308
idba_c4006 Unclassifiable EST No hits found N/A 0.00903557 20 9 0.34366167
mira_extended_contigs_c1553 60S ribosomal protein L23a P62752 RL23A_RAT 0.01631603 20 11 178.111316
idba_c8373 Unclassifiable EST No hits found N/A 0.01289121 20 11 75.9765562
top500-3000_transcript-100_2650 Ras-like GTP-binding protein rhoA Q22038 RHO1_CAEEL 0.02578158 20 13 81.1811991
Above3000_transcript-100_26315 Unclassifiable EST No hits found N/A 0.03219362 20 14 2.50186913
idba_c671 Coiled-coil domain-containing protein 112 A0AUP1 CC112_MOUSE 0.01289121 20 15 1.59484399
top500-3000_transcript-100_5583 Cyclin-L1 Q9UK58 CCNL1_HUMAN 0.00793367 20 16 9.29194685
mira_extended_contigs_c231 Unclassifiable EST No hits found N/A 0.03219362 20 16 2.40630294
idba_c4340 Unclassifiable EST No hits found N/A 0.04955868 20 19 16.3012268
idba_c567 Sucrase-isomaltase, intestinal P14410 SUIS_HUMAN 0.01289121 20 19 2.20177212
top500-3000_transcript-100_142 Lysosomal protective protein Q3MI05 PPGB_BOVIN 0.00068896 22 0 26.3410209
Above3000_transcript-100_23850 Multiple epidermal growth factor-like domains protein 6 O75095 MEGF6_HUMAN 0.00793367 22 0 16.8510659
Above3000_transcript-100_1782 Unclassifiable EST No hits found N/A 0.00478726 22 0 4.61564729
idba_c808 ATP synthase subunit beta, mitochondrial P56480 ATPB_MOUSE 0.00617842 22 1 58.7796237
idba_c899 Glutathione S-transferase Q8MU52 GST_PLAFA 0.00027422 22 1 25.1303268
idba_c4707 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, P0CB82 NDUA9_PONPY 0.01631603 22 1 9.46305732
idba_c8297 Unclassifiable EST No hits found N/A 0.01289121 22 1 4.44964973
idba_c5694 WD repeat-containing protein 54 Q9H977 WDR54_HUMAN 0.04002766 22 1 3.61606347
Above3000_transcript-100_26904 Unclassifiable EST No hits found N/A 0.00793367 22 1 3.53646375
idba_c1515 Activating signal cointegrator 1 complex subunit 1 Q9D8Z1 ASCC1_MOUSE 0.03219362 22 1 2.99407184
mira_extended_contigs_c1048 AP-5 complex subunit zeta-1 Q3U829 AP5Z1_MOUSE 0.020556 22 2 2.35563715
idba_c9064 Glutathione S-transferase kappa 1 Q9Y2Q3 GSTK1_HUMAN 0.01289121 22 3 5.10138157
Above3000_transcript-100_2604 Unclassifiable EST No hits found N/A 0.04955868 22 3 1.72864132
Above3000_transcript-100_17898 Chymotrypsin-like serine proteinase P35003 CTRL_HALRU 0.03219362 22 3 1.4012028
Above3000_transcript-100_18020 Unclassifiable EST No hits found N/A 0.03219362 22 3 1.38954331
idba_c9802 28S ribosomal protein S12, mitochondrial O35680 RT12_MOUSE 0.04955868 22 7 8.53507018
idba_c1171 Probable protein disulfide-isomerase A6 P38661 PDIA6_MEDSA 0.01289121 22 7 3.43130425
idba_c5498 Density-regulated protein homolog Q9VX98 DENR_DROME 0.01013747 22 8 8.83971723
idba_c10070 60S ribosomal protein L24 Q962T5 RL24_SPOFR 0.01631603 22 9 822.208731
mira_extended_contigs_c1523 60S ribosomal protein L44 Q6BLK0 RL44_DEBHA 0.03219362 22 9 205.660747
top50-500_transcript-100_1309 60S ribosomal protein L36 Q90YT2 RL36_ICTPU 0.00051009 22 9 195.656671
idba_c7844 60S ribosomal protein L28 P41105 RL28_MOUSE 0.02578158 22 9 156.711967
idba_c8204 60S ribosomal protein L35a P04646 RL35A_RAT 0.04002766 22 9 102.880908
idba_c10297 Unclassifiable EST No hits found N/A 0.00793367 22 9 14.2481097
mira_extended_contigs_c2188 Transmembrane protein 189 Q99LQ7 TM189_MOUSE 0.01013747 22 9 13.7596446
mira_extended_contigs_c1461 Tubulin alpha-8 chain Q6AY56 TBA8_RAT 0.04002766 22 10 64.5698609
top500-3000_transcript-100_8314 Unclassifiable EST No hits found N/A 0.03219362 22 11 9.47059295
idba_c8117 Unclassifiable EST No hits found N/A 0.01631603 22 11 8.73912988
idba_c3952 AP-3 complex subunit delta-1 O54774 AP3D1_MOUSE 0.03219362 22 11 3.33517944
idba_c1674 Unclassifiable EST No hits found N/A 0.01631603 22 11 0.60444053
top500-3000_transcript-100_8614 Unclassifiable EST No hits found N/A 0.04955868 22 12 5.48230565
idba_c1078 Regulator of nonsense transcripts 2 Q9HAU5 RENT2_HUMAN 0.01013747 22 12 3.58440559
Above3000_transcript-100_10119 Unclassifiable EST No hits found N/A 0.02578158 22 13 3.37549443
idba_c7888 Unclassifiable EST No hits found N/A 0.00903557 22 13 0.74929277
Above3000_transcript-100_14427 Unclassifiable EST No hits found N/A 0.04955868 22 15 3.31567744
idba_c9500 G patch domain-containing protein 4 Q4V842 GPTC4_XENLA 0.04002766 22 15 1.31997249
idba_c10212 Unclassifiable EST No hits found N/A 0.04955868 22 16 0.53915266
Above3000_transcript-100_15279 Tubulin polyglutamylase TTLL4 Q14679 TTLL4_HUMAN 0.020556 22 17 5.10153925
mira_extended_contigs_c2214 Sodium- and chloride-dependent taurine transporter Q9MZ34 SC6A6_BOVIN 0.04955868 22 18 18.5574376
Above3000_transcript-100_8171 BDNF/NT-3 growth factors receptor Q91987 NTRK2_CHICK 0.00163627 22 18 8.1049053
idba_c1645 E3 ubiquitin-protein ligase ZSWIM2 Q8NEG5 ZSWM2_HUMAN 0.03219362 22 18 5.34539846
idba_c2847 Multivesicular body subunit 12B Q9H7P6 MB12B_HUMAN 0.04002766 22 18 3.70613544
Above3000_transcript-100_8055 Thrombospondin type-1 domain-containing protein 7B Q6P4U0 THS7B_MOUSE 0.00215771 22 18 3.49748449
idba_c5947 Unclassifiable EST No hits found N/A 0.020556 22 18 1.68782924

127

idba_c5947 Unclassifiable EST No hits found N/A 0.020556 22 18 1.68782924
mira_extended_contigs_c233 Ectopic P granules protein 5 homolog A5WUT8 EPG5_DANRE 0.02578158 22 18 1.61307815
mira_extended_contigs_c1177 Xaa-Pro dipeptidase P12955 PEPD_HUMAN 0.04955868 22 19 15.4129981
idba_c10014 ATP-sensitive inward rectifier potassium channel 12 F1NHE9 KCJ12_CHICK 0.00019903 22 19 11.3339636
idba_c7694 Netrin-1 O95631 NET1_HUMAN 0.01631603 22 19 9.43412322
Above3000_transcript-100_26861 Protein phosphatase 1D O15297 PPM1D_HUMAN 0.02578158 22 19 7.68073811
idba_c7722 Otoferlin Q5SPC5 OTOF_DANRE 0.00617842 22 19 2.42128777
idba_c254 Sodium/potassium-transporting ATPase subunit beta P25169 AT1B1_ARTSF 0.020556 22 20 127.82047
Above3000_transcript-100_21488 Unclassifiable EST No hits found N/A 0.020556 22 20 36.4083483
idba_c4715 Superoxide dismutase [Mn], mitochondrial P07895 SODM_RAT 0.00051009 22 20 19.1343456
idba_c1926 Retinoid-inducible serine carboxypeptidase Q920A5 RISC_MOUSE 0.020556 22 20 7.21016279
idba_c1728 Unclassifiable EST No hits found N/A 0.01631603 22 20 4.92098166
idba_c821 Multidrug resistance-associated protein 1 P33527 MRP1_HUMAN 0.04002766 22 20 4.32782018
Above3000_transcript-100_2194 Pyridoxine-5'-phosphate oxidase Q5E9K3 PNPO_BOVIN 0.04955868 22 20 2.95098499
Above3000_transcript-100_12840 Xanthine dehydrogenase P10351 XDH_DROME 0.01843601 22 20 2.21194951
Above3000_transcript-100_5039 Unclassifiable EST No hits found N/A 0.04955868 22 20 1.41357858
Above3000_transcript-100_10632 E3 ubiquitin-protein ligase PDZRN3 P68907 PZRN3_RAT 0.01013747 22 21 9.31246549
Above3000_transcript-100_24035 Mitotic checkpoint serine/threonine-protein kinase BUB1 O43683 BUB1_HUMAN 0.020556 22 21 6.19451208
idba_c3381 Non-specific lipid-transfer protein O62742 NLTP_RABIT 0.00123373 22 21 5.94353231
Above3000_transcript-100_4250 Probable acyl-CoA dehydrogenase 6 P34275 IVD_CAEEL 0.00369019 22 21 4.09121518
Above3000_transcript-100_16561 Zinc finger protein 431 E9QAG8 ZN431_MOUSE 0.00123373 22 21 1.24566193
idba_c885 Chitotriosidase-1 Q13231 CHIT1_HUMAN 0.01289121 22 21 0.50798127
mira_extended_contigs_c1524 Guanylate cyclase soluble subunit beta-2 P22717 GCYB2_RAT 0.02578158 24 3 0.28807813
top500-3000_transcript-100_7161 Unclassifiable EST No hits found N/A 0.00369019 24 4 5.72094358
mira_extended_contigs_c2704 Unclassifiable EST No hits found N/A 0.01843601 24 4 0.74815292
idba_c6067 Pseudouridine-5'-phosphatase Q08623 HDHD1_HUMAN 0.00617842 24 5 4.17515371
top500-3000_transcript-100_9625 Unclassifiable EST No hits found N/A 0.020556 24 5 0.66008984
top500-3000_transcript-100_8004 Dynein light chain roadblock-type 2 Q9DAJ5 DLRB2_MOUSE 0.03219362 24 6 72.4080561
top50-500_transcript-100_954 40S ribosomal protein S11 P41115 RS11_XENLA 0.00369019 24 7 149.681831
idba_c10235 60S ribosomal protein L32 Q962T1 RL32_SPOFR 0.00478726 24 7 47.2468526
idba_c8675 FAS-associated death domain protein Q13158 FADD_HUMAN 0.00215771 24 7 12.2495155
idba_c9197 60S ribosomal protein L26 P61255 RL26_MOUSE 0.00793367 24 8 38.1278054
idba_c67 40S ribosomal protein S24 Q962Q6 RS24_SPOFR 0.020556 24 10 302.894571
idba_c2894 Unclassifiable EST No hits found N/A 0.00793367 24 10 1.97302255
top500-3000_transcript-100_6294 Unclassifiable EST No hits found N/A 0.00369019 24 10 1.67528976
idba_c2853 Protein MAK16 homolog A Q66L33 MK16A_XENLA 0.04955868 24 11 1.54141111
idba_c5829 Probable peptidyl-tRNA hydrolase Q8BW00 PTH_MOUSE 0.020556 24 14 3.09020654
idba_c2345 Unclassifiable EST No hits found N/A 0.02578158 24 14 2.67249664
Above3000_transcript-100_26635 A disintegrin and metalloproteinase with thrombospondin motifs 18 Q8TE60 ATS18_HUMAN 0.01843601 24 14 1.13431241
mira_extended_contigs_c1562 Helicase with zinc finger domain 2 Q9BYK8 HELZ2_HUMAN 0.04002766 24 15 12.7698802
Above3000_transcript-100_27415 Protein-S-isoprenylcysteine O-methyltransferase O60725 ICMT_HUMAN 0.00478726 24 15 7.42806411
idba_c5157 Ankyrin repeat domain-containing protein 60 Q9BZ19 ANR60_HUMAN 0.00617842 24 15 6.7461328
Above3000_transcript-100_25557 Unclassifiable EST No hits found N/A 0.020556 24 15 6.03649776
Above3000_transcript-100_608 Kinesin-like protein KIF12 Q9D2Z8 KIF12_MOUSE 0.02578158 24 15 2.47710324
idba_c5092 Unclassifiable EST No hits found N/A 0.03219362 24 15 1.79611274
top500-3000_transcript-100_5662 Unclassifiable EST No hits found N/A 0.04955868 24 15 1.38449987
Above3000_transcript-100_27256 Unclassifiable EST No hits found N/A 0.00617842 24 16 9.54945799
top500-3000_transcript-100_10048 Unclassifiable EST No hits found N/A 0.02578158 24 16 6.60596514
mira_extended_contigs_c1359 LON peptidase N-terminal domain and RING finger protein 1 Q17RB8 LONF1_HUMAN 0.02578158 24 16 4.99292412
Above3000_transcript-100_3256 Unclassifiable EST No hits found N/A 0.00478726 24 16 1.95250567
mira_extended_contigs_c1747 Unclassifiable EST No hits found N/A 0.04002766 24 16 1.58531855
idba_c6241 Unclassifiable EST No hits found N/A 0.04479317 24 16 0.5416194
top500-3000_transcript-100_868 Max dimerization protein 1 P50538 MAD1_MOUSE 0.01013747 24 17 52.1205534
mira_extended_contigs_c1491 Sodium-dependent phosphate transport protein 2B Q27960 NPT2B_BOVIN 0.00478726 24 17 16.718873
idba_c3957 Serine incorporator 1 Q3MHV9 SERC1_BOVIN 0.00793367 24 17 13.0554603
idba_c197 UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 P81436 OGT1_RABIT 0.00617842 24 17 8.86892746
idba_c6262 Synaptosomal-associated protein 29 Q9Z2P6 SNP29_RAT 0.03219362 24 17 6.29549559
Above3000_transcript-100_26966 Unclassifiable EST No hits found N/A 0.00478726 24 17 4.53277544
mira_extended_contigs_c2311 Ubiquitin-like modifier-activating enzyme 5 X1WER2 UBA5_DANRE 0.00215771 24 17 4.40518845
idba_c5995 Unclassifiable EST No hits found N/A 0.00282946 24 17 3.31089245
idba_c9829 E3 ubiquitin-protein ligase MARCH8 Q0VD59 MARH8_BOVIN 0.04955868 24 17 2.87430633
idba_c4777 E3 ubiquitin-protein ligase KCMF1 Q9P0J7 KCMF1_HUMAN 0.03219362 24 17 2.33213027
idba_c2901 Palmitoyltransferase ZDHHC6 Q9CPV7 ZDHC6_MOUSE 0.03219362 24 17 2.23353784
mira_extended_contigs_c2631 Chloride intracellular channel exc-4 Q8WQA4 EXC4_CAEEL 0.00793367 24 18 27.7491252
top500-3000_transcript-100_573 Ectoine dioxygenase Q7W977 ECTD_BORPA 0.020556 24 18 18.1523475
idba_c3341 Vesicle-associated membrane protein 4 O70480 VAMP4_MOUSE 0.00163627 24 18 10.4016007
idba_c3515 Organic cation transporter protein Q9VCA2 ORCT_DROME 0.04955868 24 18 8.9023379
mira_extended_contigs_c2079 Dual specificity mitogen-activated protein kinase kinase 1 (Fragment) Q91447 MP2K1_SERCA 0.00478726 24 18 8.62606527
idba_c4911 Unclassifiable EST No hits found N/A 0.01013747 24 18 7.95053081
Above3000_transcript-100_5017 Scavenger receptor class B member 1 Q8SQC1 SCRB1_PIG 0.02578158 24 18 5.85146842
Above3000_transcript-100_604 Protein farnesyltransferase subunit beta P49355 FNTB_BOVIN 0.020556 24 18 1.50773073
Above3000_transcript-100_20436 ADP-ribosylation factor P91924 ARF_DUGJA 0.00617842 24 19 11.4474326
idba_c739 H(+)/Cl(-) exchange transporter 3 O18894 CLCN3_RABIT 0.020556 24 19 10.4221699
idba_c680 Alpha-N-acetylgalactosaminidase Q90744 NAGAB_CHICK 0.01631603 24 19 9.21769904
idba_c727 Phosphoglucomutase-2 Q96G03 PGM2_HUMAN 0.00123373 24 19 8.29491974
idba_c1499 Transcriptional adapter 2-beta Q5RBN9 TAD2B_PONAB 0.01013747 24 19 5.06071161
Above3000_transcript-100_16327 Unclassifiable EST No hits found N/A 0.01460362 24 19 3.86111026
idba_c7459 Xylan 1,4-beta-xylosidase D5EY15 XYL3A_PRER2 0.01013747 24 20 10.4613153
Above3000_transcript-100_2529 DmX-like protein 2 Q8TDJ6 DMXL2_HUMAN 0.03219362 24 20 8.09288732
idba_c8491 Transmembrane emp24 domain-containing protein 2 Q15363 TMED2_HUMAN 0.00617842 24 20 4.5571137
Above3000_transcript-100_2131 Unclassifiable EST No hits found N/A 0.04002766 24 20 4.15367074

128

Above3000_transcript-100_2131 Unclassifiable EST No hits found N/A 0.04002766 24 20 4.15367074
mira_extended_contigs_c2540 Leucine-rich repeat-containing protein 74A Q0VAA2 LR74A_HUMAN 0.020556 24 20 3.34260831
idba_c9740 Amiloride-sensitive sodium channel subunit gamma-2 O13263 SCNNH_XENLA 0.020556 24 20 2.41075453
mira_extended_contigs_c1948 WD repeat-containing protein 76 Q4KLQ5 WDR76_XENLA 0.04955868 24 22 1.69718426
idba_c7893 Uncharacterized oxidoreductase YrbE O05389 YRBE_BACSU 0.01631603 24 22 0.95392806
top500-3000_transcript-100_10704 Zinc finger protein 330 homolog Q9VAU9 ZN330_DROME 0.020556 24 23 5.29547
Above3000_transcript-100_15411 N-acylphosphatidylethanolamine synthase Q9ZV87 NAPES_ARATH 0.00019903 24 23 2.81598487
Above3000_transcript-100_9610 Myoglobin P51537 MYG_HALMK 0.01289121 26 0 3.37799476
idba_c3752 Unclassifiable EST No hits found N/A 0.04955868 26 1 10.1861048
Above3000_transcript-100_13942 A-kinase anchor protein 14 O35817 AKA14_RAT 0.04955868 26 1 9.7997455
idba_c7696 Proteasome subunit beta type-1-B Q9IB83 PSB1B_CARAU 0.00282946 26 1 7.23776541
idba_c5206 Regulator of microtubule dynamics protein 1 Q96DB5 RMD1_HUMAN 0.01289121 26 1 1.79456418
Above3000_transcript-100_5786 EEF1A lysine methyltransferase 1 Q6GN98 EFMT1_XENLA 0.01013747 26 1 1.79044988
idba_c6428 Unclassifiable EST No hits found N/A 0.00051009 26 2 39.6851338
idba_c10395 Fatty acid-binding protein 12 A6NFH5 FBP12_HUMAN 0.00478726 26 2 38.6012556
idba_c7937 39S ribosomal protein L27, mitochondrial Q32PC3 RM27_BOVIN 0.01013747 26 2 8.0341271
Above3000_transcript-100_20421 Unclassifiable EST No hits found N/A 0.01013747 26 2 3.95324592
idba_c8581 RNA-binding protein with serine-rich domain 1-B Q3KPW1 RNP1B_XENLA 0.00123373 26 3 12.2627667
idba_c6394 Splicing factor U2AF 26 kDa subunit Q7TP17 U2AF4_RAT 0.00478726 26 3 10.3277733
idba_c128 COMM domain-containing protein 10 Q9Y6G5 COMDA_HUMAN 0.020556 26 3 5.78366819
Above3000_transcript-100_7987 Protein canopy 4 Q2L6K8 CNPY4_DANRE 0.00478726 26 3 5.13583994
idba_c1739 Microspherule protein 1 Q96EZ8 MCRS1_HUMAN 0.020556 26 3 2.97971191
Above3000_transcript-100_3799 Unclassifiable EST No hits found N/A 0.03219362 26 3 1.73797301
Above3000_transcript-100_14652 U1 small nuclear ribonucleoprotein C C3Z1P5 RU1C_BRAFL 0.03219362 26 5 9.84803984
Above3000_transcript-100_13436 Unclassifiable EST No hits found N/A 0.00369019 26 5 4.48606891
idba_c7943 Biogenesis of lysosome-related organelles complex 1 subunit 2 Q66KB9 BL1S2_XENTR 0.04955868 26 5 3.87420906
idba_c10529 Unclassifiable EST No hits found N/A 0.04955868 26 5 1.33596035
top500-3000_transcript-100_3877 Unclassifiable EST No hits found N/A 0.04002766 26 6 12.1060354
idba_c8840 Protein IMPACT-B A9UMG5 IMPTB_XENTR 0.04002766 26 6 4.06124022
idba_c9003 Ribosome biogenesis protein NSA2 homolog Q9QYU7 NSA2_RAT 0.00793367 26 7 12.7346933
idba_c5218 Coiled-coil domain-containing protein 166 P0CW27 CC166_HUMAN 0.03219362 26 7 5.1808091
Above3000_transcript-100_21552 Mitotic-spindle organizing protein 2 Q6DC17 MZT2_DANRE 0.00037524 26 8 12.1157329
top500-3000_transcript-100_6273 Unclassifiable EST No hits found N/A 0.01013747 26 8 3.24182526
Above3000_transcript-100_9925 Unclassifiable EST No hits found N/A 0.00215771 26 10 5.76736956
Above3000_transcript-100_2640 ELKS/Rab6-interacting/CAST family member 1 Q8IUD2 RB6I2_HUMAN 0.020556 26 11 2.1306287
mira_extended_contigs_c2062 Coiled-coil domain-containing protein 13 Q8IYE1 CCD13_HUMAN 0.04955868 26 11 1.92644949
idba_c7095 Coiled-coil domain-containing protein 66 Q6NS45 CCD66_MOUSE 0.02578158 26 13 1.28836588
idba_c785 Midnolin homolog Q8SXD4 MIDN_DROME 0.04002766 26 14 15.4133918
idba_c2255 Purine nucleoside phosphorylase P55859 PNPH_BOVIN 0.04002766 26 14 7.858276
Above3000_transcript-100_8234 Tubulin polyglutamylase TTLL7 A4Q9F0 TTLL7_MOUSE 0.03219362 26 14 3.95802278
Above3000_transcript-100_24115 Insulin receptor substrate 1-B P84770 IRS1B_XENLA 0.00080685 26 14 2.88837193
mira_extended_contigs_c1497 Unclassifiable EST No hits found N/A 0.01631603 26 14 2.10630511
idba_c10207 Nicotinamidase P21369 PNCA_ECOLI 0.00215771 26 14 2.09692816
Above3000_transcript-100_11312 Zinc finger protein 311 Q5JNZ3 ZN311_HUMAN 0.02578158 26 14 1.64045167
mira_extended_contigs_c1233 Equilibrative nucleoside transporter 3 Q80WK7 S29A3_RAT 0.00369019 26 14 1.60559325
mira_extended_contigs_c2128 Protein bicaudal D homolog 2 Q921C5 BICD2_MOUSE 0.04955868 26 14 1.4028493
Above3000_transcript-100_8238 GAS2-like protein 1 Q99501 GA2L1_HUMAN 0.01843601 26 14 1.26421748
idba_c2915 Protein CREBRF homolog Q9VC61 CRERF_DROME 0.02578158 26 15 27.9751913
mira_extended_contigs_c2643 Extracellular sulfatase Sulf-1 Q8VI60 SULF1_RAT 0.020556 26 15 18.8645804
idba_c9472 Baculoviral IAP repeat-containing protein 7-B A9ULZ2 BIR7B_XENLA 0.00369019 26 15 17.6169743
idba_c7566 Glycine-rich domain-containing protein 1 Q9ZQ47 GRDP1_ARATH 0.01631603 26 15 12.2064034
idba_c9171 Cytokine-inducible SH2-containing protein Q2HJ53 CISH_BOVIN 0.04955868 26 15 11.2442678
top500-3000_transcript-100_7044 Unclassifiable EST No hits found N/A 0.02578158 26 15 10.5311874
Above3000_transcript-100_13370 cAMP-responsive element modulator P79145 CREM_CANLF 0.01013747 26 15 7.28360466
top500-3000_transcript-100_216 Spermine oxidase Q9NWM0 SMOX_HUMAN 0.02578158 26 15 6.85033382
mira_extended_contigs_c319 Serine/threonine-protein kinase N2 A7MBL8 PKN2_DANRE 0.00282946 26 15 6.23043063
idba_c1325 Ubiquitin-conjugating enzyme E2 A Q9Z255 UBE2A_MOUSE 0.01013747 26 15 6.18554562
Above3000_transcript-100_937 Tubulin-specific chaperone cofactor E-like protein Q8C5W3 TBCEL_MOUSE 0.00617842 26 15 4.40146712
mira_extended_contigs_c892 Unclassifiable EST No hits found N/A 0.00793367 26 15 4.32761618
Above3000_transcript-100_15272 E3 ubiquitin-protein ligase RBBP6 P97868 RBBP6_MOUSE 0.00163627 26 15 3.0222207
mira_extended_contigs_c60 Reducing polyketide synthase swnK D4AU31 SWNK_ARTBC 0.01013747 26 15 2.86235994
idba_c8474 Protein rolling stone O44252 ROST_DROME 0.00478726 26 15 2.81212761
idba_c9705 Neuromedin-U receptor 1 O55040 NMUR1_MOUSE 7.29E-05 26 15 2.30990113
mira_extended_contigs_c706 Sodium-dependent glucose transporter 1 A4QN56 MFS4B_DANRE 0.00617842 26 15 1.92614155
idba_c5808 Transcription cofactor vestigial-like protein 4 Q80V24 VGLL4_MOUSE 0.00793367 26 15 1.45283715
idba_c3395 GTP-binding protein RAD O88667 RAD_MOUSE 0.04955868 26 15 1.1462459
idba_c7997 Testin Q2YDE9 TES_BOVIN 0.00282946 26 15 0.91946951
idba_c9613 Putative molluscan insulin-related peptide(s) receptor Q25410 MIPR_LYMST 0.04955868 26 15 0.74195937
idba_c972 MAP kinase-interacting serine/threonine-protein kinase 2 Q66I46 MKNK2_XENTR 0.00793367 26 16 105.688074
idba_c112 Nuclear receptor subfamily 1 group D member 1 Q63503 NR1D1_RAT 0.02578158 26 16 98.4249055
top500-3000_transcript-100_5778 cAMP-responsive element-binding protein-like 2 A4IGK3 CRBL2_XENTR 0.03219362 26 16 28.9822532
idba_c6628 Unclassifiable EST No hits found N/A 0.03219362 26 16 23.6182854
idba_c4595 cAMP-specific 3',5'-cyclic phosphodiesterase 4D Q01063 PDE4D_MOUSE 0.00019903 26 16 19.4009149
mira_extended_contigs_c2283 Ras and EF-hand domain-containing protein Q08CX1 RASEF_XENTR 0.04002766 26 16 15.1887389
idba_c4292 Alanine--glyoxylate aminotransferase 2, mitochondrial Q9BYV1 AGT2_HUMAN 0.00617842 26 16 14.893782
idba_c5816 Casein kinase I isoform alpha P67963 KC1A_XENLA 0.00282946 26 16 14.4777405
idba_c7309 Neutrophil collagenase O88766 MMP8_RAT 0.00092475 26 16 13.6285127
idba_c5169 Protein sprouty homolog 2 Q08E39 SPY2_BOVIN 0.01631603 26 16 9.98924199
Above3000_transcript-100_6934 Tripartite motif-containing protein 45 Q5BIM1 TRI45_BOVIN 0.020556 26 16 8.25115797
idba_c5300 Disintegrin and metalloproteinase domain-containing protein 10 O35598 ADA10_MOUSE 0.04955868 26 16 7.57110226
idba_c760 Calcium-independent phospholipase A2-gamma Q9NP80 PLPL8_HUMAN 0.00163627 26 16 7.41523118

129

idba_c760 Calcium-independent phospholipase A2-gamma Q9NP80 PLPL8_HUMAN 0.00163627 26 16 7.41523118
idba_c4057 Patatin-like phospholipase domain-containing protein 2 Q96AD5 PLPL2_HUMAN 0.01013747 26 16 6.57791952
idba_c2357 WD repeat-containing protein 86 Q86TI4 WDR86_HUMAN 0.03219362 26 16 6.52659771
idba_c5541 Liprin-beta-1 Q8C8U0 LIPB1_MOUSE 0.01289121 26 16 6.10589393
mira_extended_contigs_c1970 Protein PTHB1 Q6AX60 PTHB1_XENLA 0.04955868 26 16 5.92659198
mira_extended_contigs_c962 Glutamate receptor ionotropic, kainate 2 P42260 GRIK2_RAT 0.01289121 26 16 5.70175942
idba_c6777 Ubiquitin-conjugating enzyme E2 H P62257 UBE2H_MOUSE 0.04955868 26 16 5.29447475
Above3000_transcript-100_22362 Protein FAM135B Q9DAI6 F135B_MOUSE 0.03219362 26 16 3.82948916
Above3000_transcript-100_389 MFS-type transporter SLC18B1 Q6NT16 S18B1_HUMAN 0.01289121 26 16 3.80432623
mira_extended_contigs_c2497 Neuronal acetylcholine receptor subunit alpha-2 Q15822 ACHA2_HUMAN 0.03219362 26 16 3.70942242
idba_c1939 Glycine-rich domain-containing protein 2 Q9SZJ2 GRDP2_ARATH 0.00163627 26 16 3.40628645
idba_c8899 N-terminal kinase-like protein Q28FH2 SCYL1_XENTR 0.020556 26 16 2.78456264
Above3000_transcript-100_2548 Sorting nexin-13 Q6PHS6 SNX13_MOUSE 0.04955868 26 16 2.46293335
Above3000_transcript-100_14924 ATP-dependent DNA helicase Q1 P46063 RECQ1_HUMAN 0.01460362 26 16 2.13692672
Above3000_transcript-100_1290 Integrator complex subunit 6-A Q2TAF4 INT6A_XENLA 0.03219362 26 16 1.81280718
idba_c9284 Unclassifiable EST No hits found N/A 0.03219362 26 16 1.64649955
Above3000_transcript-100_3298 Coactosin P34121 COAA_DICDI 0.04955868 26 16 1.4776018
Above3000_transcript-100_1971 Ras-related protein Rab-33B O35963 RB33B_MOUSE 0.04002766 26 16 1.23291545
mira_extended_contigs_c896 Unclassifiable EST No hits found N/A 0.020556 26 16 1.05495028
Above3000_transcript-100_7830 Protein CLEC16A Q80U30 CL16A_MOUSE 0.01289121 26 16 1.03863804
mira_extended_contigs_c251 Neuron navigator 3 Q80TN7 NAV3_MOUSE 0.04955868 26 16 0.84332065
idba_c2992 Unclassifiable EST No hits found N/A 0.03219362 26 17 5.55041329
idba_c5177 Octopamine receptor Oamb Q7JQF1 OAMB_DROME 0.00068896 26 17 2.81494525
idba_c2278 Innexin unc-7 Q03412 UNC7_CAEEL 0.01631603 26 17 2.63951895
idba_c9595 Sex peptide receptor Q8SWR3 SPR_DROME 0.04955868 26 17 2.48725553
idba_c975 Methylmalonate-semialdehyde dehydrogenase [acylating], mitochondrial Q07536 MMSA_BOVIN 0.00617842 26 18 17.2958061
idba_c248 Secernin-3 Q17QS0 SCRN3_BOVIN 0.04955868 26 18 15.0880368
Above3000_transcript-100_19993 Neutrophil collagenase P22894 MMP8_HUMAN 0.04955868 26 18 14.7859525
top500-3000_transcript-100_8221 Ras GTPase-activating protein 3 Q9QYJ2 RASA3_RAT 0.03219362 26 18 13.233811
idba_c10272 Sulfate anion transporter 1 P45380 S26A1_RAT 0.00369019 26 18 7.16702503
idba_c502 F-box/WD repeat-containing protein 5 Q4KLI9 FBXW5_RAT 0.04002766 26 18 6.98316383
mira_extended_contigs_c625 Sialin Q9NRA2 S17A5_HUMAN 0.020556 26 18 6.91187863
mira_extended_contigs_c449 Solute carrier family 13 member 2 Q13183 S13A2_HUMAN 0.04002766 26 18 4.17587248
idba_c3204 Prickle-like protein 2 Q7Z3G6 PRIC2_HUMAN 0.01289121 26 18 1.81250259
Above3000_transcript-100_4194 Endoplasmic reticulum aminopeptidase 1 Q9NZ08 ERAP1_HUMAN 0.04002766 26 18 1.63917693
idba_c8695 Mitochondrial coenzyme A transporter SLC25A42 Q0P483 S2542_DANRE 0.03219362 26 18 1.50190718
top500-3000_transcript-100_4991 Charged multivesicular body protein 1b Q5ZKX1 CHM1B_CHICK 0.01013747 26 19 23.4717732
idba_c4323 H(+)/Cl(-) exchange transporter 3 Q9R279 CLCN3_CAVPO 0.01631603 26 19 9.13659195
mira_extended_contigs_c765 Pleckstrin homology domain-containing family A member 8 F1MS15 PKHA8_BOVIN 0.01631603 26 19 3.97844403
idba_c2534 Unclassifiable EST No hits found N/A 0.01013747 26 19 2.5954365
top500-3000_transcript-100_241 DnaJ homolog subfamily C member 3 Q27968 DNJC3_BOVIN 0.01631603 26 19 2.06227587
Above3000_transcript-100_16947 Unclassifiable EST No hits found N/A 0.04002766 26 20 1.78406364
mira_extended_contigs_c2431 NHL repeat-containing protein 2 Q5ZI67 NHLC2_CHICK 0.03219362 26 20 0.71439741
idba_c8496 DENN domain-containing protein 4C A6H8H2 DEN4C_MOUSE 0.020556 26 22 2.82662436
mira_extended_contigs_c1552 Unclassifiable EST No hits found N/A 0.01013747 26 23 1.28334189
top50-500_transcript-100_1747 Unclassifiable EST No hits found N/A 0.04002766 28 0 44.3708635
top500-3000_transcript-100_1909 NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, P42026 NDUS7_BOVIN 0.01631603 28 0 23.3169331
idba_c8086 Vacuolar ATPase assembly integral membrane protein vma21 Q28GR4 VMA21_XENTR 0.02578158 28 0 6.89365189
top50-500_transcript-100_443 Unclassifiable EST No hits found N/A 0.04955868 28 0 5.11923319
Above3000_transcript-100_3048 Ankyrin repeat domain-containing protein 40 Q6AI12 ANR40_HUMAN 0.020556 28 0 2.31753753
mira_extended_contigs_c16 Unclassifiable EST No hits found N/A 0.04955868 28 0 0.79425557
Above3000_transcript-100_11231 Unclassifiable EST No hits found N/A 0.04955868 28 1 19.6257027
top500-3000_transcript-100_4594 Reactive oxygen species modulator 1 Q6NYD1 ROMO1_DANRE 0.03219362 28 1 10.0919468
top50-500_transcript-100_574 Inter-alpha-trypsin inhibitor heavy chain H3 Q9GLY5 ITIH3_RABIT 0.03219362 28 1 6.02914243
idba_c4543 40S ribosomal protein S30 P62864 RS30_RAT 0.04955868 28 2 189.973661
Above3000_transcript-100_11893 Unclassifiable EST No hits found N/A 0.04002766 28 2 29.2959869
idba_c2716 Exosome complex component RRP41 Q7YRA3 EXOS4_BOVIN 0.01631603 28 2 6.48711428
top500-3000_transcript-100_3658 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 5 Q63362 NDUA5_RAT 0.01289121 28 3 3.11520135
Above3000_transcript-100_1608 LINE-1 retrotransposable element ORF2 protein O00370 LORF2_HUMAN 0.04955868 28 3 1.04009963
top500-3000_transcript-100_6864 Unclassifiable EST No hits found N/A 0.00793367 28 6 9.48747018
Above3000_transcript-100_22550 NudC domain-containing protein 2 Q8WVJ2 NUDC2_HUMAN 0.04955868 28 6 1.22947484
top500-3000_transcript-100_5634 Glutathione S-transferase alpha-4 P14942 GSTA4_RAT 0.00617842 28 7 5.57831378
idba_c3429 Surfeit locus protein 6 homolog Q9I8B0 SURF6_XENLA 0.03219362 28 7 5.21993297
Above3000_transcript-100_13438 Unclassifiable EST No hits found N/A 0.04955868 28 7 4.2522835
Above3000_transcript-100_13952 Unclassifiable EST No hits found N/A 0.01631603 28 7 3.24924193
mira_extended_contigs_c318 Cactin Q8WUQ7 CATIN_HUMAN 0.020556 28 7 1.94171664
Above3000_transcript-100_7803 PRKR-interacting protein 1 homolog Q6GNG8 PKRI1_XENLA 0.01631603 28 9 3.11275193
idba_c8145 Unclassifiable EST No hits found N/A 0.00793367 28 9 1.0466135
idba_c9247 Unclassifiable EST No hits found N/A 0.00617842 28 9 1.04134767
top500-3000_transcript-100_1274 Unclassifiable EST No hits found N/A 0.04002766 28 11 3.25530749
mira_extended_contigs_c818 Probable imidazolonepropionase Q9DBA8 HUTI_MOUSE 0.01631603 28 11 0.74910468
Above3000_transcript-100_25861 Unclassifiable EST No hits found N/A 0.00215771 28 12 11.4981969
idba_c1808 Unclassifiable EST No hits found N/A 0.00793367 28 12 5.96569445
idba_c1462 GRB10-interacting GYF protein 1 Q99MR1 GGYF1_MOUSE 0.00369019 28 12 4.49145671
idba_c3324 Solute carrier family 35 member E1 homolog Q9VR50 S35E1_DROME 0.04002766 28 12 0.63241085
top500-3000_transcript-100_10570 Troponin I Q7M3Y3 TNNI_CHLNI 0.04002766 28 13 31.0449582
Above3000_transcript-100_27361 TNF receptor-associated factor 1 Q13077 TRAF1_HUMAN 0.00068896 28 13 7.29397474
Above3000_transcript-100_25735 NudC domain-containing protein 3 Q8R1N4 NUDC3_MOUSE 0.04955868 28 13 5.34901531
Above3000_transcript-100_6495 Unclassifiable EST No hits found N/A 0.04955868 28 13 4.5056335
mira_extended_contigs_c508 Retinal-specific ATP-binding cassette transporter O35600 ABCA4_MOUSE 0.03219362 28 13 4.11744814
top500-3000_transcript-100_6191 Unclassifiable EST No hits found N/A 0.04955868 28 13 4.04897757

130

top500-3000_transcript-100_6191 Unclassifiable EST No hits found N/A 0.04955868 28 13 4.04897757
Above3000_transcript-100_4626 Upstream activation factor subunit spp27 O74503 UAF30_SCHPO 0.04002766 28 13 3.02516262
idba_c2009 Unclassifiable EST No hits found N/A 0.00617842 28 13 2.86419754
idba_c557 RUN domain-containing protein 3B Q4R7B9 RUN3B_MACFA 0.04955868 28 13 1.65999681
idba_c9521 Mushroom body large-type Kenyon cell-specific protein 1 Q95YM8 MBLK1_APIME 0.020556 28 13 1.51546913
idba_c8962 Unclassifiable EST No hits found N/A 0.03219362 28 13 0.85060491
idba_c8390 Unclassifiable EST No hits found N/A 0.00617842 28 14 21.5262142
idba_c4374 Chordin-like protein 2 Q8VEA6 CRDL2_MOUSE 0.00014344 28 14 18.5734748
idba_c8155 Sodium- and chloride-dependent glycine transporter 1 P48067 SC6A9_HUMAN 0.00051009 28 14 10.9044316
Above3000_transcript-100_1400 Tetraspanin-5 Q68VK5 TSN5_RAT 0.00068896 28 14 8.8740699
mira_extended_contigs_c659 Vesicle-associated membrane protein 7 Q5ZL74 VAMP7_CHICK 0.00163627 28 14 8.29579585
idba_c8931 Bcl-2-like protein 1 Q07816 B2CL1_CHICK 0.01289121 28 14 7.66742824
idba_c7775 Dual specificity protein phosphatase 3 Q5RD73 DUS3_PONAB 0.03219362 28 14 7.43794239
idba_c3338 WD repeat and SOCS box-containing protein 1 Q7T2F6 WSB1_DANRE 0.01289121 28 14 5.12426284
idba_c6131 Hemicentin-1 Q96RW7 HMCN1_HUMAN 0.020556 28 14 4.88756733
idba_c7600 Unclassifiable EST No hits found N/A 0.04002766 28 14 4.63432552
idba_c3172 WD repeat domain phosphoinositide-interacting protein 2 Q5ZHN3 WIPI2_CHICK 0.01289121 28 14 3.41334798
Above3000_transcript-100_11667 Growth hormone-regulated TBC protein 1-A Q6PBU5 GRT1A_DANRE 0.03611064 28 14 2.99755929
idba_c10189 Ceramide glucosyltransferase Q5BL38 CEGT_XENTR 0.00793367 28 14 2.98327502
top500-3000_transcript-100_8956 Unclassifiable EST No hits found N/A 0.00548284 28 14 2.78632387
idba_c5086 Polycomb protein SCMH1 Q8K214 SCMH1_MOUSE 0.02578158 28 14 2.57289146
idba_c26 Neurotrypsin Q5G270 NETR_GORGO 0.00123373 28 14 2.31348863
idba_c4857 Bifunctional arginine demethylase and lysyl-hydroxylase JMJD6 Q6PFM0 JMJD6_DANRE 0.02578158 28 14 2.23639278
Above3000_transcript-100_10711 Unclassifiable EST No hits found N/A 0.01289121 28 14 2.15814505
mira_extended_contigs_c2665 Acyl-CoA-binding domain-containing protein 5 Q8RWD9 ACBP5_ARATH 0.04002766 28 14 2.08758569
Above3000_transcript-100_4672 Corepressor interacting with RBPJ 1 Q5ZI03 CIR1_CHICK 0.04955868 28 14 1.99805466
idba_c4482 Ubiquitin thioesterase Zranb1 Q7M760 ZRAN1_MOUSE 0.01631603 28 14 1.99083722
Above3000_transcript-100_831 CUGBP Elav-like family member 3-B Q7ZWM3 CEL3B_XENLA 0.04002766 28 14 1.79438104
mira_extended_contigs_c1232 Vesicular glutamate transporter 2 Q9JI12 VGLU2_RAT 0.01013747 28 14 1.52738459
mira_extended_contigs_c2154 Kelch-like protein 12 Q5U374 KLH12_DANRE 0.04955868 28 14 1.38996475
idba_c827 Beta-4C adrenergic receptor P43141 ADB4C_MELGA 0.01631603 28 14 1.33692113
idba_c5017 WD repeat-containing and planar cell polarity effector protein fritz Q32NR9 FRITZ_XENLA 0.00163627 28 14 1.23516281
Above3000_transcript-100_17084 Unclassifiable EST No hits found N/A 0.00163627 28 14 1.23422802
idba_c3110 Octopamine receptor beta-2R G3M4F8 OCTB2_CHISP 0.00478726 28 14 1.04646147
Above3000_transcript-100_14744 Unclassifiable EST No hits found N/A 0.00793367 28 14 0.98782959
Above3000_transcript-100_992 Choline/ethanolamine kinase Q9Y259 CHKB_HUMAN 0.00478726 28 14 0.98079175
mira_extended_contigs_c793 H(+)/Cl(-) exchange transporter 7 Q4PKH3 CLCN7_BOVIN 0.04002766 28 14 0.56588412
idba_c431 NAD-dependent alcohol dehydrogenase Q4J781 ADH_SULAC 0.00617842 28 15 102.619053
idba_c7573 Dendritic arbor reduction protein 1 Q9VZN4 DAR1_DROME 0.020556 28 15 59.3576
idba_c8416 Glutamate dehydrogenase, mitochondrial P82264 DHE3_CHAAC 0.00793367 28 15 44.6850396
mira_extended_contigs_c1453 Phosphatidate phosphatase LPIN3 Q9BQK8 LPIN3_HUMAN 0.01289121 28 15 30.1395844
idba_c1121 Methenyltetrahydrofolate synthase domain-containing protein Q52L34 MTHSD_XENLA 0.02578158 28 15 19.9438697
top500-3000_transcript-100_8742 cAMP-responsive element-binding protein-like 2 Q5BJU6 CRBL2_RAT 0.04002766 28 15 16.1436586
idba_c1196 Collagen alpha-1(XXI) chain Q96P44 COLA1_HUMAN 0.01289121 28 15 13.8775617
mira_extended_contigs_c1870 Gelsolin-like protein 1 Q7JQD3 GELS1_LUMTE 0.00793367 28 15 12.6609039
idba_c10548 La-related protein 6 Q8BN59 LARP6_MOUSE 0.01289121 28 15 11.2623245
idba_c4671 Protein HEXIM Q4V7W3 HEXIM_XENLA 0.00617842 28 15 9.23981946
top50-500_transcript-100_2271 Phosphatidate phosphatase LPIN3 Q99PI4 LPIN3_MOUSE 0.00282946 28 15 8.75682593
idba_c6243 Lysoplasmalogenase-like protein TMEM86A Q9D8N3 TM86A_MOUSE 0.04955868 28 15 7.64224675
idba_c416 Endothelin-converting enzyme 1 P42892 ECE1_HUMAN 0.020556 28 15 6.94289057
idba_c776 Ran-binding protein 9 P69566 RANB9_MOUSE 0.04955868 28 15 6.33949443
idba_c2800 Interleukin-17 receptor D Q7T2L7 I17RD_CHICK 0.00123373 28 15 4.54094075
mira_extended_contigs_c1106 Matrix metalloproteinase-17 Q9R0S3 MMP17_MOUSE 0.03219362 28 15 4.48978441
Above3000_transcript-100_23644 Glutamate receptor 1 P34299 GLR1_CAEEL 0.02578158 28 15 4.25752741
idba_c7086 Ubiquitin-conjugating enzyme E2 R2 Q29503 UB2R2_RABIT 0.04002766 28 15 4.03779327
top500-3000_transcript-100_8962 Fatty-acid amide hydrolase 2-A Q6DH69 FAH2A_DANRE 0.01289121 28 15 4.02949961
idba_c8866 Probable phosphomannomutase Q9XUE6 PMM_CAEEL 0.01289121 28 15 3.76843013
idba_c4204 BRD4-interacting chromatin-remodeling complex-associated protein-like Q8CHH5 BICRL_MOUSE 0.02578158 28 15 3.71586311
idba_c401 Peptidyl-prolyl cis-trans isomerase FKBP14 Q5R941 FKB14_PONAB 0.01631603 28 15 3.58070106
mira_extended_contigs_c2459 Glutamate receptor 2 P19491 GRIA2_RAT 0.020556 28 15 2.84383003
idba_c8692 Glutathione hydrolase 1 proenzyme P07314 GGT1_RAT 0.00282946 28 15 2.84119889
Above3000_transcript-100_1069 Homeobox protein Meis1 O00470 MEIS1_HUMAN 0.04955868 28 15 2.62747586
mira_extended_contigs_c2693 Zinc finger SWIM domain-containing protein 5 Q9P217 ZSWM5_HUMAN 0.04002766 28 15 2.62145202
idba_c9278 ADP-ribosylation factor 1 Q94650 ARF1_PLAFA 0.03219362 28 15 2.51759642
mira_extended_contigs_c482 Acetyl-CoA carboxylase P11029 ACAC_CHICK 0.03219362 28 15 2.51178542
idba_c1821 Protein O-mannosyl-transferase 2 F1Q8R9 POMT2_DANRE 0.00793367 28 15 2.16934209
mira_extended_contigs_c664 Acid-sensing ion channel 1 Q1XA76 ASIC1_CHICK 0.03219362 28 15 1.94479467
idba_c8433 Prostaglandin E2 receptor EP4 subtype Q95KZ0 PE2R4_PANTR 0.03219362 28 15 1.94478972
idba_c5256 Pre-mRNA cleavage complex 2 protein Pcf11 O94913 PCF11_HUMAN 0.03219362 28 15 1.77130814
idba_c3737 Protein unc-13 homolog D B2RUP2 UN13D_MOUSE 0.00215771 28 15 1.52504689
Above3000_transcript-100_1061 Serine/threonine-protein kinase TBK1 Q6DFJ6 TBK1_XENLA 0.04479317 28 15 1.5102026
Above3000_transcript-100_682 Inositol-pentakisphosphate 2-kinase Q4JL91 IPPK_DANRE 0.00793367 28 15 1.14845879
idba_c7870 Unclassifiable EST No hits found N/A 0.00617842 28 15 1.10057706
mira_extended_contigs_c2110 E3 ubiquitin-protein ligase UBR5 Q80TP3 UBR5_MOUSE 0.01631603 28 15 1.06827005
Above3000_transcript-100_19821 Zinc transporter 5 Q5ZLF4 ZNT5_CHICK 0.03219362 28 15 0.96783968
idba_c1373 Importin-5 O00410 IPO5_HUMAN 0.01631603 28 15 0.89394914
Above3000_transcript-100_861 Unclassifiable EST No hits found N/A 0.04479317 28 15 0.38472478
Above3000_transcript-100_7610 Protocadherin gamma-A10 Q9Y5H3 PCDGA_HUMAN 0.02316879 28 15 0.20179979
idba_c9258 Unclassifiable EST No hits found N/A 0.00051009 28 16 71.8470899
mira_extended_contigs_c1597 Unclassifiable EST No hits found N/A 0.00163627 28 16 67.1686544
mira_extended_contigs_c1831 Protein transport protein Sec61 subunit alpha Q25147 SC61A_HALRO 0.00068896 28 16 39.9655812

131

mira_extended_contigs_c1831 Protein transport protein Sec61 subunit alpha Q25147 SC61A_HALRO 0.00068896 28 16 39.9655812
mira_extended_contigs_c2201 Unclassifiable EST No hits found N/A 0.03219362 28 16 36.0376957
idba_c8972 Innexin unc-9 O01393 UNC9_CAEEL 0.00282946 28 16 24.5754262
idba_c456 La-related protein 6 Q8BN59 LARP6_MOUSE 0.00163627 28 16 24.4472645
mira_extended_contigs_c157 Fatty acid hydroxylase domain-containing protein 2 Q9GKT2 FXDC2_MACFA 0.03219362 28 16 13.7116844
mira_extended_contigs_c529 Pikachurin B4F785 EGFLA_RAT 0.04955868 28 16 12.9370871
mira_extended_contigs_c375 Protein disulfide-isomerase A6 homolog Q9V438 PDIA6_DROME 0.00369019 28 16 12.1178261
top500-3000_transcript-100_8331 Serine/threonine-protein kinase 3 Q7ZUQ3 STK3_DANRE 0.020556 28 16 10.0088094
idba_c1770 Protein DD3-3 Q58A42 DD3_DICDI 0.03219362 28 16 9.41189319
idba_c9546 Mesencephalic astrocyte-derived neurotrophic factor P55145 MANF_HUMAN 0.03219362 28 16 7.29850234
idba_c3457 Macrophage mannose receptor 1 P22897 MRC1_HUMAN 0.00478726 28 16 6.69096751
Above3000_transcript-100_13969 High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A Q9I7S6 PDE9A_DROME 0.020556 28 16 5.43571509
Above3000_transcript-100_480 Kelch-like protein 24 Q56A24 KLH24_RAT 0.01289121 28 16 5.1689831
idba_c8172 ATP-dependent Clp protease ATP-binding subunit clpX-like, Q9JHS4 CLPX_MOUSE 0.04955868 28 16 4.68340439
mira_extended_contigs_c1810 G patch domain-containing protein 1 Q24K12 GPTC1_BOVIN 0.04002766 28 16 4.11488205
Above3000_transcript-100_22068 Unclassifiable EST No hits found N/A 0.0289876 28 16 3.95805708
mira_extended_contigs_c1079 N-acetylgalactosaminyltransferase 7 Q8MV48 GALT7_DROME 0.00478726 28 16 3.65041754
idba_c1498 ADP-ribosylation factor GTPase-activating protein 1 Q8N6T3 ARFG1_HUMAN 0.00793367 28 16 3.51644909
mira_extended_contigs_c1841 HEAT repeat-containing protein 5B Q8C547 HTR5B_MOUSE 0.01013747 28 16 3.24975741
Above3000_transcript-100_4400 Ras-related protein Rab-30 Q923S9 RAB30_MOUSE 0.04002766 28 16 3.05249866
mira_extended_contigs_c1182 Protein transport protein Sec24C P53992 SC24C_HUMAN 0.00617842 28 16 2.90666637
Above3000_transcript-100_6787 Kv channel-interacting protein 4 Q99MG9 KCIP4_RAT 0.00617842 28 16 2.85596611
mira_extended_contigs_c2011 Cationic amino acid transporter 4 Q8BLQ7 CTR4_MOUSE 0.02578158 28 16 2.67697192
idba_c9921 Serine/threonine-protein kinase unc-51 Q23023 UNC51_CAEEL 0.04002766 28 16 2.57486358
Above3000_transcript-100_6597 Cyclic nucleotide-gated channel rod photoreceptor subunit alpha Q90980 CNG3_CHICK 0.04002766 28 16 2.493815
Above3000_transcript-100_15955 Kinesin-like protein KIF24 Q5T7B8 KIF24_HUMAN 0.0289876 28 16 2.45565316
idba_c2467 DNA polymerase zeta catalytic subunit Q61493 REV3L_MOUSE 0.04479317 28 16 2.29718275
idba_c5534 M-phase phosphoprotein 8 Q3TYA6 MPP8_MOUSE 0.03219362 28 16 2.11500659
mira_extended_contigs_c570 Acid-sensing ion channel 4 Q708S4 ASI4A_DANRE 0.00123373 28 16 2.10244625
idba_c1533 Syndetin Q8CI71 VPS50_MOUSE 0.01289121 28 16 1.95831031
Above3000_transcript-100_10015 Muscarinic acetylcholine receptor gar-2 Q09388 ACM2_CAEEL 0.00705605 28 16 1.2490419
Above3000_transcript-100_12898 Unclassifiable EST No hits found N/A 0.03219362 28 16 0.59266791
Above3000_transcript-100_16340 Sodium/calcium exchanger 3 P57103 NAC3_HUMAN 0.03611064 28 17 4.33857104
mira_extended_contigs_c736 TBC1 domain family member 25 A1A5B6 TBC25_MOUSE 0.04002766 28 17 2.91762865
Above3000_transcript-100_2118 Beta-1,4-N-acetylgalactosaminyltransferase bre-4 A8Y1P7 BRE4_CAEBR 0.01013747 28 17 2.8995359
idba_c1079 Unclassifiable EST No hits found N/A 0.04002766 28 18 8.87414343
idba_c7276 Protein dimmed B6VQA1 DIMM_DROME 0.00068896 28 18 6.39973781
idba_c9984 Glucose transporter type 1 Q8IRI6 GTR1_DROME 0.020556 28 18 5.25758641
Above3000_transcript-100_7186 WD repeat-containing protein 81 E7FEV0 WDR81_DANRE 0.04955868 28 18 0.89731002
idba_c3812 SPRY domain-containing SOCS box protein 3 Q28DT9 SPSB3_XENTR 0.04955868 28 19 3.64379125
Above3000_transcript-100_11782 Major facilitator superfamily domain-containing protein 9 Q8NBP5 MFSD9_HUMAN 0.04002766 28 19 2.75223213
idba_c4847 Plasminogen P00747 PLMN_HUMAN 0.00369019 28 19 1.67134799
idba_c6760 Putative G-protein coupled receptor F59B2.13 P34488 YMJC_CAEEL 0.03219362 28 20 2.79522988
idba_c2765 Ubiquitin-like protein 7 Q91W67 UBL7_MOUSE 0.00282946 28 26 2.30745984
mira_extended_contigs_c1418 GPI mannosyltransferase 2 Q5KR61 PIGV_RAT 0.04955868 28 26 1.53021089
idba_c9031 60S ribosomal protein L22 Q28IL6 RL22_XENTR 0.020556 28 27 108.904102
top500-3000_transcript-100_3193 Unclassifiable EST No hits found N/A 0.02578158 28 27 62.3104313
idba_c449 Myophilin Q24799 MYPH_ECHGR 0.01289121 28 27 33.1881607
Above3000_transcript-100_13921 Cytochrome c oxidase assembly factor 6 homolog Q2M2S5 COA6_BOVIN 0.01289121 28 27 6.25392602
idba_c5450 Toll-like receptor 6 Q704V6 TLR6_BOVIN 0.02578158 28 27 4.443925
idba_c8302 Unclassifiable EST No hits found N/A 0.03611064 28 27 1.94592887
Above3000_transcript-100_506 Sphingomyelin phosphodiesterase 3 Q9NY59 NSMA2_HUMAN 0.03219362 28 27 1.77680943
Above3000_transcript-100_22069 Bile salt-activated lipase P19835 CEL_HUMAN 0.04002766 28 27 1.58941836

132
CONCLUSION

Taken together, the results from this dissertation provide support for the existence of a circatidal
clock in intertidal organisms. Results from Chapters 2 and 3 show that circatidal locomotor activity in
limpets persists in free-running in the absence of environmental cues, while results from Chapters 1 and 3
show that circatidal gene expression patterns persist in free-running mussels, oysters, and limpets. That
both of these types of biological rhythms can persist in constant conditions suggests that there is an
endogenous molecular mechanism controlling them.
In Chapter 1, we identified a set of candidate bivalve circatidal genes that persisted in circatidal
expression rhythms in whole mussels and even ex vivo oyster tissue cultures. We found that these
candidate genes peaked at or around times of anticipated low tide. These candidate genes are all known
to be involved in different stress responses, which makes sense, given the host of potential stresses that
intertidal organisms face during low tide. Notably, a large proportion of the candidate genes are classified
as immediate early genes, which act as the first line responders to stress stimuli (Herschman 1991,
Bahrami & Drabløs 2016). Since immediate early genes tend to code for transcription factors (Tullai et
al. 2007), this lends further support for these candidate genes being involved in the central circatidal
oscillator; many of the core circadian genes are transcription factors and transcriptional regulators as well
(Hirayama & Sassone-Corsi 2005).
In Chapter 2, we investigated the circatidal clock through the lens of limpet movement. We
found that limpet movement rhythms were quite persistent, returning to their circatidal ~12.4hr period
when entrained to tidal cycles of both longer and shorter periods. We also identified water flow as a
likely zeitgeber of the circatidal clock in limpets. This makes sense, because wave action is the most
overt harbinger of the transition between high and low tides (Enright 1970). Identification of a zeitgeber
is important to inform future studies on the entrainment pathway. Just as identifying light as the main
zeitgeber of the circadian clock has led to deeper understanding of its mechanism (Golombek &

133
Rosenstein 2010), so too can identification of water flow as a potential zeitgeber lead to greater
understanding of the circatidal clock mechanism.
In Chapter 3, we investigated the gene expression patterns associated with free-running limpet
movement and identified candidate circatidal genes in limpet. We found that genes associated with
movement were enriched for feeding, locomotion, and muscle activity, which is consistent with
movement and the fact that the main motivation for limpet movement is in order to forage and feed
(Hartnoll & Wright 1977, Little et al. 1988, Evans & Williams 1991). Although these candidate circatidal
genes are not all the same as those identified in mussels and oysters in Chapter 1, they still provide
evidence for an endogenous circatidal oscillator in limpets; whether that mechanism is the same as the
one in bivalves remains to be seen.
The results from this dissertation provide a starting point from which future investigation into the
circatidal clock can begin. If the circatidal clock is structured similarly to the circadian clock, with highly
interconnected transcription/translation feedback loops involving extensive protein dimerization (Harmer
et al. 2001), then starting with the potential zeitgeber and the candidate genes we have identified can lead
to discovering network relationships with other genes and proteins, ultimately resulting in elucidation of
the molecular mechanism of the circatidal clock.

References
Bahrami S, Drabløs F (2016) Gene regulation in the immediate-early response process. Adv Biol Regul
62:37–49
Enright JT (1970) Ecological aspects of endogenous rhythmicity. Ann Rev Ecol Syst 1:221–238
Evans MR, Williams GA (1991) Time Partitioning of Foraging in the Limpet Patella vulgata. J Anim
Ecol 60:563–575
Golombek DA, Rosenstein RE (2010) Physiology of circadian entrainment. Physiol Rev 90:1063–102
Harmer S, Panda S, Kay S (2001) Molecular bases of circadian rhythms. Annu Rev Cell Dev Biol
17:215–253

134
Hartnoll RG, Wright JR (1977) Foraging movements and homing in the limpet Patella vulgata L. Anim
Behav 25:806–810
Herschman HR (1991) Primary Response Genes Induced by Growth Factors and Tumor Promoters. Annu
Rev Biochem 60:281–319
Hirayama J, Sassone-Corsi P (2005) Structural and functional features of transcription factors controlling
the circadian clock. Curr Opin Genet Dev 15:548–56
Little C, Williams GA, Morritt D, Perrins JM, Stirling P (1988) Foraging behaviour of Patella vulgata L.
in an Irish sea-lough. J Exp Mar Bio Ecol 120:1–21
Tullai JW, Schaffer ME, Mullenbrock S, Sholder G, Kasif S, Cooper GM (2007) Immediate-early and
delayed primary response genes are distinct in function and genomic architecture. J Biol Chem
282:23981–23995

Abstract (if available)

Abstract The marine intertidal zone is a regularly-changing environment, oscillating between very different conditions during high tide versus low tide. Since the ebb and flow of the tide is a regular temporal occurrence associated with highly contrasting environmental conditions, it follows that intertidal organisms should have a dedicated, endogenous time-keeping mechanism that aids in anticipating these environmental fluctuations and making the appropriate physiological changes. As a first step in elucidating the molecular mechanism of the circatidal clock, we sought to identify a set of candidate genes likely to be involved in the central oscillator in California ribbed mussel Mytilus californianus and Pacific oyster Crassostrea gigas. Our previous work has shown that M. californianus exhibit circatidal gene expression rhythms while exposed to simulated intertidal conditions, as well as intertidal and subtidal conditions in the field. If any of those rhythmic genes are under the control of the circatidal clock, then they should continue to show circatidal rhythmicity even in the absence of environmental cues. In this study, M. californianus and C. gigas were entrained to field intertidal and subtidal conditions, then transferred to free-running constant conditions. We performed time course microarray and RNAseq analysis, which revealed that M. californianus and C. gigas continued to show circatidal gene expression, even under constant conditions in the absence of environmental cues. Moreover, a core set of candidate genes was identified that showed circatidal rhythmicity across all of the treatments. Next, we investigated circatidal locomotor rhythms in the Boreal limpet Lottia paradigitalis. Living in an environment with conditions that vary with the ebb and flow of the tide, many taxa of intertidal animals exhibit circatidal behavior rhythms. For many of these animals, this pattern persists even when they are released into free-running (constant environmental conditions in the laboratory), suggesting the presence of an endogenous oscillator that controls the rhythms, rather than the rhythms occurring as a response to the changing of the tides. Limpets exhibit circatidal locomotor rhythms, foraging during high tide and staying sheltered in place during low tide. A few studies have shown that these rhythms are endogenous in different species of limpets. However, not much else is known about the limpet circatidal oscillator beyond that observation. We first confirmed that circatidal locomotor rhythms persist in free-running in this species, after entrainment to either field or laboratory tidal cycles. We then sought to determine the plasticity of the circatidal oscillator by analyzing their locomotor rhythms after entrainment to tidal cycles of periods that differed from 12.4 hours. We found that limpets that were entrained to non-circatidal tidal cycles still exhibited circatidal or near-circatidal locomotor rhythms when released into free-running, suggesting that the circatidal oscillator in this species is quite rigid. We also sought to identify the zeitgeber (“time-giver,” i.e. environmental cue) that sets their oscillator with the correct period by analyzing their locomotor rhythms after entrainment to cycles of individual zeitgebers: emersion, light, flow, and temperature. Out of the four zeitgebers tested, flow appeared to be the most effective, with limpets adhering most closely to the flow rhythms during both entrainment and free-running. Finally, we investigated the molecular mechanisms of endogenous limpet movement, using time course RNAseq analysis of two different limpet tissues: head and foot. We identified some genes of interest that persist with circatidal expression in free-running limpets. Combining the gene expression results with the corresponding movement results, we also found that genes associated with limpet movement are enriched for feeding, locomotion, and muscle activity. Taken together, the results from this dissertation provide support for the existence of a circatidal clock in intertidal organisms. These results provide a starting point from which future investigation into the circatidal clock can begin. If the circatidal clock is structured similarly to the circadian clock, with highly interconnected transcription/translation feedback loops involving extensive protein dimerization, then starting with the potential zeitgeber and the candidate genes we have identified can lead to discovering network relationships with other genes and proteins, ultimately resulting in elucidation of the mechanism of the circatidal clock.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Simulated and field environmental effects on the transcriptome and metabolome of mussel Mytilus californianus

PDF

The evolution of pollution tolerance in the marine copepod Tigriopus

PDF

Transgenerational inheritance of thermal tolerance in two coral species in the Florida Keys

PDF

Transcriptional and morphological impacts of copper on Mytilus californianus larval development in current and future ocean conditions

PDF

Genetic architecture underlying variation in different traits in the Pacific oyster Crassostrea gigas

PDF

Phylogeography, reproductive isolation, and the evolution of sex determination mechanisms in the copepod Tigriopus californicus

PDF

Using molecular techniques to explore the diversity, ecology and physiology of important protistan species, with an emphasis on the Prymnesiophyceae

PDF

Comparative behavior and distribution of biologically relevant trace metals - iron, manganese, and copper in four representative oxygen deficient regimes of the world's oceans

PDF

The molecular adaptation of Trichodesmium to long-term CO₂-selection under multiple nutrient limitation regimes

Asset Metadata

Creator Lin, Jacqueline (author)

Core Title Molecular and behavioral mechanisms of circatidal biological rhythms in intertidal mollusks

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Biology (Marine Biology and Biological Oceanography)

Publication Date 08/09/2020

Defense Date 04/24/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biological rhythms,circatidal rhythms,intertidal zone,limpet,mussel,OAI-PMH Harvest,oyster

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Gracey, Andrew (committee chair), Bottjer, David (committee member), Edmands, Suzanne (committee member), Habib, Michael (committee member)

Creator Email j.lin42@gmail.com,linjacqu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-68145

Unique identifier UC11672478

Identifier etd-LinJacquel-6716.pdf (filename),usctheses-c89-68145 (legacy record id)

Legacy Identifier etd-LinJacquel-6716.pdf

Dmrecord 68145

Document Type Dissertation

Format application/pdf (imt)

Rights Lin, Jacqueline

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA