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Comparative physiological studies of marine invertebrate larvae from Antarctic and temperate environments

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Comparative physiological studies of marine invertebrate larvae from Antarctic and temperate environments

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Content COMPARATIVE PHYSIOLOGICAL STUDIES OF MARINE
INVERTEBRATE LARVAE FROM ANTARCTIC AND TEMPERATE
ENVIRONMENTS

by

Allison Jeanette Green

____________________________________________________________________

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)

December 2006

Copyright 2006 Allison Jeanette Green
ii
Acknowledgements
When I was in the fourth grade, there was a teacher at my school with the
prefix ‘Dr.’ to her name. I remember being surprised to find out that she was not a
medical doctor, but instead a doctor of philosophy. I did not know up to that point
that you could be called ‘Doctor’ without being a medical doctor. Since I knew I did
not want to be a medical doctor, I decided then and there that I would someday get
my PhD., so I could be called ‘Doctor’. I loved school and the prospect of getting
the highest scholastic degree possible was very appealing to me.
This seemingly ridiculous goal for a fourth-grader remained a goal of mine as
I went through school. It was when I was the ripe old age of 13 that I decided I
would get a PhD. in Marine Biology. I always saw the ocean as outer space on
Earth. It was a frontier that needed to be explored, and I wanted to be one of the
people who did it. So, not only was Marine Biology in general an interest, but also
the biology of the deep-sea. I kept this resolve throughout school and here I am
today, receiving my Ph.D. in Marine Biology. To work towards a goal such as this
for most of your life is not something you can do by yourself. I can attribute my
success in reaching my goals to many people I have been fortunate to have known
and be surrounded by. I know that this small acknowledgement will never truly
convey the gratitude and admiration I have for these people, but it’s a start.
Considering my quest for this degree started when I was a young girl, I have
to say thank you to my family first and foremost. My parents, sister and
grandparents have believed in me from the start, and were especially supportive
iii
when I started the graduate program at USC. I think that the ultimate goal of most
children is to make their parents proud. This is definitely true for myself, and that
was a motivation that never died throughout my time at USC. The amount of
support, love, patience and understanding that is necessary to put up with a graduate
student and her crazy schedules, stress levels and lack of financial resources is too
much to measure, and I received all of those things ad libitum from my family. I
couldn’t have gotten through without their continued support and inspiration, and I
know that the work ethic they all taught me through my life was pivotal in making
this happen.
I have also been fortunate enough to have lab mates that have turned into a
second family to me. We always have called ourselves a classic ‘dysfunctional
family’, because we argue, often don’t communicate properly and sometimes really
dislike one another. However, the family part is also there, meaning we truly care
for one another and will help one another out whenever needed. I am so thankful for
all of these people: Doug, Dave, Eli, Mike, Amanda-they have made my life in
general better, and definitely got me through graduate school. I counted on them for
their expertise, their advice and their patience. I have to give special thanks to Doug
Pace, because I consider him one of my closest friends. In the summer of 1999, he
took three new graduate students up to a field marine lab and not only taught us how
to run an oyster season and do research science, but also managed three young, crazy
kids. Doug has taught me so much of what I have learned in graduate school, and he
iv
has also helped me in some of my darkest times to heal. I literally owe him my life
and I will miss him dearly as we both move on in our lives.
Last but not least, I would like to thank my committee members: Dr. Suzanne
Edmands, Dr. David Caron, Dr. Dennis Hedgecock, Dr. David Bottjer and in
particular my Ph.D. advisor, Dr. Donal Manahan. My committee is made up of some
of the most well respected scientists in their fields, and their expertise and experience
were crucial to my research and this document. In addition, in my last year of
graduate school, I found myself in a situation that made the completion of my degree
much more difficult. My committee members worked with me through this and
were extremely understanding, accommodating and patient-above and beyond what I
ever could have expected from them. I thank them sincerely for this.
I want to thank my advisor, Dr. Donal Manahan in particular for his guidance
and support during my graduate career. As I mentioned before, my dream was to be
able to explore frontiers of the ocean, and because of him, I was able to make that
dream come true. He entrusted me to be a part of field teams that participated in
multi-million dollar research in two of the most ‘extreme’ places on Earth-the deep-
sea hydrothermal vents, and McMurdo Sound, Antarctica. I was able to dive in the
Alvin submersible to see the hydrothermal vents first-hand and go to the bottom of
the world - places that very few people can even dream of going. The significance of
these events in my life is not lost on me, and I am deeply grateful for the
opportunities that Donal afforded me during my tenure in his lab. In addition to this,
v
Donal offered valuable advice and guidance for my work that has made it into a
thesis that I am extremely proud of.
This, of course, is not an exhaustive list of the people who made my goals
attainable and possible. In addition to individuals, I also am grateful to the National
Science foundation, the United States Department of Agriculture, the staff of the
Bodega Marine Laboratory, the Wrigley Institute for Environmental Studies and its
staff, the crew of the R/V Atlantis, the Alvin Group and the support staff of
McMurdo Station for their financial and technical support that has made this work
possible. My tenure at USC will never be forgotten and will always be a very special
time of my life. It was filled with laughter, tears, exhilaration, defeat, hope, despair
and most of all, friends. Thank you to all that have made an almost lifelong dream
come true.

vi
Table of Contents
Acknowledgements ii
List of Tables viii
List of Figures ix
Abstract xi
Introduction 1
Introduction References 15
Chapter 1: Feeding state, aerobic enzyme activity and respiration in
echinoderm larvae
Chapter 1 Abstract 21
Introduction 22
Materials and Methods 24
Results 27
Discussion 40
Chapter 1 References 53

Chapter 2: Metabolic efficiency in fast-growing larvae
Chapter 2 Abstract 57
Introduction 58
Materials and Methods 62
Results 67
Discussion 74
Chapter 2 References 83

Chapter 3: High growth efficiencies in Antarctic larvae
Chapter 3 Abstract 86
Introduction 86
Materials and Methods 91
Results 103
Discussion 117
Chapter 3 References 130

Chapter 4: O:N atomic ratios in Antarctic and temperate planktotrophic
echinoderm larvae
Chapter 4 Abstract 136
Introduction 138
Materials and Methods 141
vii
Results 145
Discussion 164
Chapter 4 References 173

References 176

viii
List of Tables
Table 1: Total Protein-specific Citrate Synthase Activity 39
Table 2: Ingestion Rates in Temperate and Antarctic larvae 104

Table 3: Biochemical Composition of Embryos and Larvae
of Antarctic and Temperate Echinoderms 146

Table 4: O:N Atomic Ratios in Antarctic and Temperate
Echinoderm Species 160

ix
List of Figures
Figure 1: Asterina miniata. Total protein content of embryos and larvae 29
from all feeding treatments

Figure 2: Asterina miniata. Respiration rates in embryos and larvae from
all feeding treatments 30

Figure 3: Strongylocentrotus purpuratus and Lytechinus pictus.
Respiration rates in embryos and larvae from all feeding
treatments 32

Figure 4: Asterina miniata. Respiration rates versus total protein
content 34

Figure 5: Lytechinus pictus. Respiration rates versus total protein content 36

Figure 6: Asterina miniata. Total citrate synthase activity in embryos
and larvae from all feeding treatments 37

Figure 7: Asterina miniata. Respiration rates versus total citrate
synthase activity 41

Figure 8: Respiration rates versus total citrate synthase activity 42

Figure 9: Effect of food on metabolic rates in echinoderm larvae 51

Figure 10: Maternal effects on respiration rates in larvae produced
from the factoral crosses of lines 35 and 51 69

Figure 11: Respiration rates in inbred and hybrid larvae through
the first six days of development 70

Figure 12: Total protein content of inbred and hybrid larvae through
the first six days of development plotted on a log-linear scale 73

Figure 13: Total citrate synthase activity plotted with respiration
rates 76

Figure 14: The cumulative cost of development in inbred and hybrid
larvae over the first six days of development 78

Figure 15: Algal concentration over experimental incubation time 98
x
Figure 16: Algal ingestion rates and gross protein growth efficiencies in
Strongylocentrotus purpuratus 105

Figure 17: Algal ingestion rates and gross protein growth efficiencies in
Sterechinus neumayeri 108

Figure 18: Algal ingestion rates and gross protein growth efficiencies in
Odontaster validus 109

Figure 19: Algal ingestion rates and gross protein growth efficiencies in
Odontaster meridionalis 111

Figure 20: Gross protein growth efficiencies in Lytechinus pictus and
Asterina miniata 114

Figure 21: Average gross protein growth efficiencies in all species 116

Figure 22: Biochemical composition of pre-feeding embryos and
feeding larvae of all species. 148

Figure 23: Respiration and ammonia-N excretion rates in larvae
of Lytechinus pictus 153

Figure 24: Respiration and ammonia-N excretion rates in larvae
of Asterina miniata 155

Figure 25: Respiration and ammonia-N excretion rates in larvae
of Sterechinus neumayeri 159

Figure 26: Respiration and ammonia-N excretion rates in larvae
of Odontaster validus 163

xi
Abstract
Life histories of many marine invertebrates include a pelagic, larval stage.
The larval stages of these marine invertebrates are considered the most vulnerable as
they are subjected to highly variable environmental conditions (i.e. temperature,
salinity). Larvae that are planktotrophic during their pelagic life history stage are
also subjected to fluctuating nutrient levels as they disperse. All of these factors
make the study of larval physiology and biochemistry important in understanding
how larvae cope with a highly variable environment to successfully metamorphose.
In this thesis, the larvae of a number of marine invertebrate species are
studied with respect to respiration and biochemical aspects of metabolism. The
central question answered is how changes in one or more environmental parameters
affect the overall metabolism of a larva. The studies encompass a variety of
comparative approaches to this question, including multi-species comparisons and
comparisons of larval families of the same species with different genotypes. The
effects of different endogenous nutrient conditions on growth, metabolism and an
enzyme involved in aerobic respiration are studied in three echinoderm species in
Chapter One. These results show that relationships between whole-animal
metabolism and the amount of a mitochondrial enzyme exist in the larval species
studied. Chapter Two looks at larvae of the Pacific oyster produced from factoral
crosses of inbred adult oysters. Hybrid larvae grow faster than inbred larvae, despite
similar metabolic rates between each group. This indicates that hybrid oyster larvae
are metabolically more efficient than inbred larvae. Chapters Three and Four
xii
explore the effects of temperature on larval physiology by comparing the
metabolism, gross protein growth efficiencies and nitrogen excretion between larvae
of temperate and Antarctic echinoderms. Antarctic echinoderms have high gross
protein growth efficiencies relative to temperate larvae. The high retention of
protein in Antarctic echinoderm larvae is also indicated by the amount of nitrogen
excreted.
This body of work contributes to the body of knowledge in the field of larval
biology and physiology. The studies presented here employ robust comparative
approaches that are unique in this research field.

1
Introduction

In modern invertebrates, an organism’s mode of development occurs on the
continuum that exists between maximal indirect development and direct
development (Peterson et al., 1997). Maximal indirect development is defined as a
pattern of development where the embryo stages (i.e. cleavage stages, blastula and
gastrula) are followed by an intermediate stage. The structures formed in this
intermediate (i.e. larval) stage are used for either locomotion or feeding and are lost
during the formation of the juvenile (Strathmann, 1993; Peterson et al., 1997).
Direct development is defined as the pattern of development where the embryo
stages are followed by the formation of a juvenile that has features of the adult body
plan (Peterson et al., 1997). The maximal indirect developmental mode is present in
many groups of invertebrates, including echinoderms and mollusks (Peterson et al.,
1997). In sea urchins, maximal indirect development is considered to be primitive to
other modes of development and in molluscs, while many groups have a larval stage,
most of the cells in the larva are later used for the formation of the juvenile,
diverging from the maximal indirect developmental mode (Peterson et al., 1997).
Nearly every metazoan phylum contains members that display varying degrees of
indirect development, and only three highly derived groups consist of organisms that
all utilize direct development (Peterson et al., 1997).
The larval stages of marine invertebrate organisms that do have some degree
of an indirect mode of development are extremely diverse in their form and function
(McEdward, 1995). A functional difference that is seen within and among groups is
2
how a particular species attains nutrients during the larval stage. The most common
feeding modes are lecithotrophy (larvae are non-feeding and all larval nutritional
needs are obtained from the endogenous nutrients in the egg) and planktotrophy
(larval stages with specialized feeding structures and guts that feed on particulate
nutrients, mainly phytoplankton) (McEdward, 1995). Thorson (1950) observed that
over 70% of marine invertebrate species with a larval stage were planktotrophic.
Although planktotrophy is predominant as the nutritional mode in marine
invertebrate larvae, it is considered to be ancestral to lecithotrophy, and has been lost
more than it has been gained through evolutionary time (Strathmann, 1978;
Strathmann, 1993). In addition, once planktotrophy has been lost in a species, it is
most likely to be irreversible.
Thorson (1950) deemed pelagic larval stages of marine species to be
extremely vulnerable to mortality due to changing environmental conditions (e.g.
temperature, salinity) and predation. Species with planktotrophic larvae are also
affected by variable amounts of suitable particulate nutrients (Pechenik, 1987).
Marine invertebrate species with pelagic larval life history stages vulnerable to high
levels of mortality may experience fluctuations in the benthic adult populations on
yearly time scales (Thorson, 1950). These fluctuations may be in part due to varying
larval supply and recruitment, as these factors have been shown to influence the
population dynamics of many marine species (Roughgarden et al., 1988; Minchinton
and Scheibling, 1991; Ekert, 2003). The larval stages of marine invertebrates are
distinct from juvenile and adult stages in their morphology, physiology and ecology
3
in that they are part of the plankton, and may disperse great distances before finding
a suitable habitat to settle on and metamorphose (Strathmann, 1993; McEdward,
1995; Peterson et al., 1997). Understanding how all of these aspects of a of larva’s
biology are affected by varying environmental conditions is therefore extremely
important in understanding the overall recruitment and population dynamics of
marine invertebrate species.
Effects of exogenous nutrient levels on larval growth and development
As previously stated, the larval stages of marine invertebrates are subject to
extremely high rates of mortality, sometimes up to 99% of a given cohort (Morgan,
1995). In planktotrophic larvae, nutrient quality and quantity been cited as a major
factor in determining their survivorship or limiting their growth and development
(Paulay et al., 1985; Pechenik, 1987; Rumrill, 1990; Basch, 1996). Species that have
maximal indirect development as part of their life history produce larvae that are
known as obligate planktotrophs, which means that they are completely dependent
on receiving exogenous nutrients in order to develop and/or grow and eventually
metamorphose [e.g. the sea urchin, Lytechinus spp. cannot develop past the 4-arm
pluteus stage without feeding on exogenous nutrients (Herrera et al., 1996)].
Variability, or ‘patchiness’ of phytoplankton concentrations in the ocean is well
documented (Mackas et al., 1985; Davis et al., 1992; Martin, 2003). This patchiness
of food availability, in addition to water currents that could possibly transport larvae
to unfavorable nutritional habitats indicate that planktotrophic larvae can be exposed
to the extremes of phytoplankton concentration (Allison, 1994). Studies have shown
4
that many species of planktotrophic larvae are indeed food limited at some point in
their development (Paulay et al., 1985; Olson and Olson, 1989; Fenaux et al., 1994;
Harms et al., 1994; Hansen, 1999; Reitzel et al., 2004).
It has been shown that marine invertebrate larvae possess the ability to cope
with food limitation using various behavioral, morphological and physiological and
biochemical mechanisms (reviewed by Pechenik, 1987; Rumrill, 1990; Morgan,
1995). There have been many studies that have looked at the effects of varying
nutrient levels on the morphology of marine invertebrate larvae. Many species of
planktotrophic larvae are phenotypically plastic in their development, meaning that
larvae from the same cohort are capable of displaying different phenotypes of their
feeding structures depending on the level of exogenous nutrients they are exposed to.
This type of phenotypic variation of larval development in which all variations are
caused solely by external stimuli is known as polyphenism (Greene, 1999). Marine
invertebrate larvae that are exposed to low nutrient concentrations generally display
enlarged feeding structures that increase the probability of capturing particles
(echinoid larvae: Strathmann et al., 1992; Hart and Strathmann, 1994; Shilling, 1995;
mollusk larvae: Strathmann et al., 1993; Klinzing and Pechenik, 2000). In contrast,
larvae exposed to high nutrient concentrations develop with reduced feeding
structure sizes and increased growth rates of the juvenile rudiment (Fenaux et al.,
1994; Hart and Strathmann, 1994). Due to the phenotypic plasticity that occurs in
larvae, morphology has been cited as a possible measure of nutritional condition or
feeding history in field-caught larvae (Paulay et al., 1985; Strathmann et al., 1993;
5
Fenaux et al., 1994; Reitzel et al., 2004). However, it is also noted that
morphological measurements should be supplemented with other indicators due to
the confounding effects of variables other than nutrition on morphology (e.g.
temperature, food quality and genetic variation) (Klinzing and Pechenik, 2000).
The feeding history of a planktotrophic larva is also influential on their
physiology and biochemistry during growth and development. Metabolic rates are
greatly affected by the amount of exogenous nutrients planktotrophic larvae receive.
Metabolic rates are often lower in larvae that do not receive any exogenous nutrients
(‘starved’) or are fed at low food concentrations than in larvae that are fed at high
food concentrations (Lucas et al., 1979; Dawirs, 1983; Dawirs, 1984; Sprung, 1984;
MacDonald, 1988; Beiras and Camacho, 1994; Marsh et al., 1999; Meyer et al.,
2002; Moran and Manahan, 2004; Pace and Manahan, 2006). Rates of protein
synthesis are also lower in unfed echinoderm larvae, resulting in protein synthesis
accounting for a smaller amount of the larvae’s total energy budget (Pace and
Manahan, 2006). These differences in metabolic rates between larvae fed at high
food concentrations and larvae that are either unfed or fed at low food concentrations
are most likely due to differences in size. Growth rate increases as a function of food
concentration (MacDonald, 1988; Beiras and Camacho, 1994), and metabolic rates
have been shown to scale isometrically with size in numerous species of marine
invertebrate larvae (Beiras and Camacho, 1994; Hoegh-Guldberg and Manahan,
1995; Shilling, 1995). A combination of morphological and physiological measures
6
has been cited as a possible index of feeding history in field-caught larvae (Shilling,
1995).
The effects of varying nutrient levels on biochemical aspects of
planktotrophic larval development have also been studied. Through measurements
of total lipid and lipid classes, lipids are indicated as the primary source of energy to
unfed larvae of many marine invertebrate species (Lucas et al., 1979; Gallager et al.,
1986; Meyer et al., 2003; Moran and Manahan, 2004; Sewell, 2005). Larvae are also
able to accumulate storage lipids when given food, which contributes to their overall
growth and provides additional reserves to sustain them if they are exposed to low
food concentrations (Gallager et al., 1986; Meyer et al., 2003; Moran and Manahan,
2004; Reitzel et al., 2004). The total protein content of planktotrophic larvae is also
greatly affected by the level of nutrients they are exposed to, as protein is a major
constituent of biomass in planktotrophic larvae (Lucas et al., 1979; Shilling and
Manahan, 1994; Marsh et al., 1999; Moran and Manahan, 2004; Pace et al., 2006).
As in total biomass and metabolic rates, increases in food concentration lead to
increases in total protein content and protein growth rates (Marsh et al., 1999; Pace
and Manahan, 2006). While many species of marine invertebrate larvae use lipids as
their primary energy substrate in times of starvation or low food, they are able to
utilize protein as an energy substrate once lipid stores are depleted (Lucas et al,
1979). Previous studies have also examined the effects of exogenous nutrient levels
on levels of certain enzymes in marine invertebrate larvae. Leong and Manahan
(1999) measured the total amount of Na
+
-K
+
-ATPase activity and also the
7
physiologically active amounts of this enzyme in fed and unfed larvae of an
Antarctic sea urchin. Enzymes involved in aerobic and anaerobic metabolism (e.g.
citrate synthase and pyruvate kinase, respectively) have also been studied in marine
fish and invertebrate larvae exposed to different nutrient levels (Clarke et al., 1992;
Marsh et al., 1999; Moran and Manahan, 2004).
The effects of food availability on larval survival, growth, morphology,
physiology, biochemistry and metamorphic success have been studied extensively.
Many species of planktotrophic larvae have been shown to be food limited at some
point during their development, which highlights the importance of understanding
how larvae are affected by food limitation. Given the complex nature of the
environments larvae encounter, finding a single index of feeding history may not be
possible. However, a combination of these measures may offer an accurate picture
of the nutritional environment a planktotrophic larva has been exposed to.
Effects of temperature on larval growth and development
Environmental temperature is another factor that strongly influences the
growth and development of a larva (Pechenik, 1987; Hoegh-Guldberg and Pearse,
1995). The effects of temperature on larval growth and development will be
discussed here in the context of comparing marine invertebrate species living in
either temperate or polar environments. As previously stated, there are numerous
developmental modes that are used by marine organisms, and correlates have been
used in order to predict the developmental mode of marine invertebrates including
habitat, fecundity, egg size, dispersal potential and the size at metamorphosis
8
(Thorson, 1950; McEdward, 1995). A particularly controversial correlate, known as
‘Thorson’s Rule’, involves the prediction of non-pelagic, non-feeding larval stages to
be predominant in organisms living at high latitudes or at low temperatures (Clarke,
1992). This correlate was based on Thorson’s work in the early 20
th
century
studying species found in Greenland (Thorson, 1936), and other data from the
researchers studying the Antarctic and deep sea environments (Thorson, 1950). The
species reviewed were determined to be mostly brooders, with others displaying
demersal, lecithotrophic development. The theory behind this discovery was that at
high latitudes and in the deep-sea, where temperatures are low and suitable food for
planktotrophic larvae is not abundant, a pelagic, feeding larvae is selected against
(Clarke, 1992).
However, work throughout the second half of the 20
th
century has shown that
organisms in the Southern Ocean display a much larger breadth of developmental
modes (Clarke, 1992). Picken (1979) showed that out of 11 species of prosobranch
gastropods, while 10 of these species were brooders, there was one species (Nacella
concinna) that had a pelagic larval stage. Hain and Arnaud (1992) studied the
developmental mode of Antarctic mollusks in the high Weddell Sea by direct
observation. Again, although most species were brooders, two species were found
that had a pelagic, feeding larval stage. Pearse et al. (1991) reviewed the
developmental modes of a number of species in McMurdo Sound, Antarctica, and
found 8 species that had planktotrophic pelagic larvae, and 4 species that had pelagic
lecithotrophic larvae. Two of these species, Sterechinus neumayeri and Odontaster
9
validus, are two of the most abundant invertebrate species in McMurdo Sound,
indicating that the larvae are able to receive enough nutrients during their
development to settle and grow successfully. While there is evidence that supports
some of the trends outlined by Thorson, it is apparent that they are not the ‘rule’
amongst the benthic organisms in the Antarctic.
The rates of development in marine invertebrate larvae are also affected by
temperature. Larvae of polar species develop more slowly than temperate species
[e.g. 2-10 times slower in Antarctic echinoderm species (Stanwell-Smith and Peck,
1998)]. These rates can be increased with slight increases in temperature, but
Antarctic species are not tolerant of a wide range of temperatures that temperate or
tropical species may be (Peck, 2002). This is most likely due to the relatively
stenothermal nature of the waters of the Antarctic. Waters in the open Southern
Ocean fluctuate a small amount from -2º to +1ºC on a yearly basis (Stanwell-Smith
and Peck, 1998), and the water temperature of more sheltered areas, such as
McMurdo Sound (open waters only in the summer), has an annual variation of only
± 0.2ºC (Clarke, 1991a).
The effects of temperature on physiology and biochemistry can be seen in
both adults and larvae of marine invertebrate species. All physiological processes
that require a change in free energy will be affected by changes in temperature
(Clarke, 1991b). Therefore, physiological rates such as metabolic rate are expected
and have been found to be lower in ectotherms living in cold waters (Peck, 2002).
This is also true in the larvae of polar marine invertebrates (Shilling and Manahan,
10
1994). It is thought that these reduced metabolic respiratory costs may be an
advantageous to polar organisms in that they can potentially shunt energy that is
ingested into fitness related processes such as growth and thus have higher growth
efficiencies than warmer-water organisms (Clarke, 1991b). In contrast, other
physiological and cellular processes, such as microtubule function in polar fishes,
show temperature compensation, which is when a rate or process is elevated to a
level similar to that of warmer-water organisms (Williams et al., 1985). Marsh et al.,
2001, showed that protein synthesis is temperature compensated in the embryos and
larvae of the Antarctic sea urchin, Sterechinus neumayeri. Despite low metabolic
rates, measured rates of protein synthesis in these larvae were as high as rates
measured in temperate sea urchin embryos and larvae. This results in an extremely
low energetic cost of protein synthesis in these larvae, which may again be
advantageous to their growth and survival in the cold, oligotrophic waters of
McMurdo Sound.
The growth, development and physiology of marine invertebrate larvae
species are greatly affected by the temperature at which they live. While the effects
of exposure to different temperatures over evolutionary time scales has been the
focus of this discussion, it is important to note that ectothermic organisms, including
marine invertebrate larvae, are also affected by temperature on much shorter-time
scales (days, week, or years). There have been numerous studies comparing the
growth, development and physiology of marine invertebrate larvae in which
temperatures have been manipulated to reflect a natural range of temperatures an
11
organism may be exposed to during their larval period. Temperature has been shown
to play an important role in determining the overall success of marine invertebrate
larvae on both short and long time scales.
Effect of genetic variation on larval growth and development
As previously discussed, phenotypic variation in the morphology of the
feeding structures of planktotrophic larvae of the same cohort that have been
exposed to different levels of food is most likely caused solely by the external
environment (polyphenism). Phenotypic variation can also be a result of both the
genetic make-up of a larva and its external environment (developmental reaction
norm), or can be solely the result of their genotype (genetic polymorphism) (Greene,
1999). It is extremely difficult to ascertain the level at which endogenous and
exogenous environments each contribute to the phenotypic variation observed in any
organism, including marine invertebrate larvae. Studies on juvenile and adult marine
invertebrates (in particular, marine bivalves) have been able to distinguish groups of
animals based on their genotype and place them in identical environmental
conditions. Any variations that are subsequently measured between the genetic
groups are therefore based solely on the genotypes of those groups, and not the
external environment (Koehn and Shumway, 1982; Hawkins et al., 1989; Hawkins
and Day, 1996; Bayne and Hawkins, 1997; Bayne, 1999; Bayne et al., 1999a, b).
Using this approach, these studies were able to ascertain that the phenotypic
variation seen between groups or individuals (e.g. growth, metabolic rates) was
based solely on their genotype.
12
The genetic basis of phenotypic variation has been studied to a lesser extent
in marine invertebrate larvae. Hedgecock et al. (1995) introduced an experimental
approach where adult oysters of known genotype are mated using controlled crosses
to produce larvae of different genotypes. These crosses result in larvae and juveniles
with variable growth rates (fast- and slow-growing). Genotype-based differences in
growth rates could be seen in larvae as early as six days of age (Hedgecock et al.,
1996).
The most obvious and well-documented phenotypic variation in individuals
of differing genotype is variation in growth. The vast amount of variation in growth
has led to the study of the physiological mechanism(s) that may determine an
individual’s growth rate. Studies on adult bivalves with genotype-dependent
differences in growth have attempted to explain the observed differences in terms of
Winberg’s (1956) scope for growth equation. This equation states that G = C – (F +
U + R), where (G) is growth, (C) is energy consumed, (F) and (U) are energy loss
due to feces and excreta, respectively, and (R) is energy loss due to respiration.
Bayne (1999) postulated that faster growth (G) could be attributed to one of three
models. One model indicates an increase in the amount of energy consumed (C),
without increasing the amount of energy lost through respiration (energy acquisition
model). A second model indicates a preferential allocation of energy to growth
(energy allocation model). The third model shows a situation where fast-growing
animals are metabolically efficient in that their growth-specific energy loss due to
respiration is lower than slower-growing animals (metabolic efficiency model). This
13
third model, metabolic efficiency, has been cited in numerous studies of adult
bivalves as the basis of faster growth (Koehn and Shumway; 1982, Scott and Koehn;
1990, Bayne et al., 1999a). Faster growth in later-stage veliger larvae of the Pacific
oyster, Crassostrea gigas has also been attributed to metabolic efficiency
(Hedgecock et al., 1996; Pace et al. 2006). The genetic basis of phenotypic variation
has not been extensively studied in marine invertebrate larvae, but has been shown to
be an important factor in determining traits associated with larval fitness, such as
growth and metabolism.
Summary
Larval biology has been a well-established field of study for numerous
decades, with much work being dedicated to the study of marine invertebrate larvae.
This field has remained active because of the importance of larval survival and
metamorphic success to the population dynamics of adult benthic marine
invertebrates. Understanding how larvae survive in the water column despite
environmental fluctuations and predation can hopefully lead to a better
understanding of how and why adult populations may vary from year to year.
My research has utilized both interspecific and conspecific (as described by
Hedgecock et al., 1995) approaches in this work to determine the effects of varying
environments on larval physiology. It should be noted that while interspecific
comparative approaches have been used to explain an organism’s physiology and
biology for many years, they have been met with criticism over the past two decades.
Garland and Adolph (1994) caution researchers against making inferences about
14
adaptations based on the comparison of only two species of organisms. When
comparing species, one must take into consideration the evolutionary processes that
occur when species differentiate. Comparing the physiology of two species and
assuming any differences are due to adaptation or genetic differences could possibly
lead to false conclusions from the experimental results. They cite numerous ways to
combat this problem, such as comparing multiple species (i.e. at least three species),
or the inclusion of phylogenetic data into the final comparative analysis. The studies
in Chapters 1,3 and 4 compare the physiology of multiple species (4-6) within and
between different environments to overcome possible confounding factors inherent
in interspecific comparisons. Chapter 2 discusses a study that avoids interspecific
comparisons completely and explores physiological and biochemical differences
amongst larvae of the same species with different genotypes. My thesis adds to the
field of larval biology through studies of various physiological and biochemical
aspects of marine invertebrate larvae when exposed to various nutritional,
temperature and genetic environments.
15
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21
Chapter 1: Feeding state, aerobic enzyme activity and respiration in
echinoderm larvae
Chapter 1 Abstract
There have been numerous studies citing the morphological and behavioral
mechanisms that planktotrophic marine invertebrate larvae have to cope with
stressful environmental conditions such as food limitation (poor nutrient quantity
and/or quality). There is less known about how larvae may respond physiologically,
depending on their feeding history. In this study, larvae of the temperate
echinoderms Asterina miniata, Lytechinus pictus, and Strongylocentrotus
purpuratus, were reared under a range of algal rations (i.e. 0 to 40 algal cells μl
-1
).
Total protein growth, metabolic rate and total activity of a key regulatory enzyme of
aerobic metabolism, citrate synthase (CS), were measured in all treatments
throughout larval development to address questions of how physiological responses
scale with one another and with nutrient level. In higher food treatments of 20, 30
and 40 algal cells μl
-1
, larvae of A. miniata and L. pictus had a higher rate of protein
growth, increasing metabolic rates, and a linear relationship between protein content
and respiration rate and CS activity and respiration rate (ANOVA, P< 0.05 for all
treatments). The protein growth rates and metabolic rates in larvae of A. miniata, S.
purpuratus and L. pictus reared under low-food environments (0, 2, 4 and 8 algal
cells μl
-1
) remained low throughout development, and in some cases, decreased in
the unfed treatments (i.e. 0 algal cells μl
-1
). In all species, neither total protein nor
CS activity showed significant relationships to metabolic rate in the low feeding
22
treatments (ANOVA, P >0.05 for both treatments). The physiological state of these
larvae reflected the amount of exogenous nutrients they had received during their
development, with a possible threshold of food limitation occurring at or around 4
algal cells μl
-1
. The total protein content and activity of CS is a good indicator of
metabolic rate in larvae that are well fed through development, and in conjunction
with morphological and developmental measures, may be a useful index of
physiological state in larvae.

Introduction
When looking at the recruitment patterns and rates of successful settlement in
marine invertebrate larvae, the end result of larval development is often the focus of
the study. This does not explain how the larva actually survived to get to
metamorphosis. In a cohort of planktotrophic, marine invertebrate larvae, high
levels of mortality are common (Rumrill, 1990; Morgan, 1995). Planktotrophic
marine invertebrate larvae are subject to heavy predation, changing environmental
conditions and varied nutrient availability (Thorson, 1950), which can consequently
lead to changes in their developmental times and trajectories, growth and time spent
in the plankton (Pechenik, 1987). In order to understand the effects such a variable
environment may have on embryos and larvae and ultimately to understand
recruitment and ecosystem dynamics, it is necessary to study the physiology of the
organism during its developmental stages.
23
There has been a lot of debate about whether planktotrophic marine
invertebrate larvae are food limited (defined as not having an adequate amount or
quantity of exogenous nutrients to achieve growth and development) during their
planktonic period. Many studies have shown that the larvae of certain species do
appear to be subject to some period of food limitation during their development
(Paulay et al., 1985; Olson and Olson, 1989; Fenaux et al., 1994; Reitzel et al.,
2004). In previous laboratory studies on the effects of food limitation on
invertebrate larvae, it has been found that both growth rates (as measured by either
morphology or total protein content) and the rates of development are reduced when
larvae do not receive an adequate supply of particulate nutrients (Paulay et al., 1985;
Strathmann et al., 1992; Strathmann et al., 1993; Allison, 1994; Feneaux et al., 1994;
Hart and Strathmann, 1994; Basch, 1996; Pechenik et al., 1996; Leong and Manahan,
1999; Marsh et al., 1999; Klinzing and Pechenik, 2002; Pace and Manahan, 2006).
While there has been a lot of work exploring the effects of varying exogenous
nutrient levels on the growth and development of planktotrophic larvae, much less is
known about how the actual physiological state of these larvae may change
depending on the amount of exogenous nutrients available to them during their
development. It has been shown that metabolic rates as measured by oxygen
consumption are often lower in larvae of echinoderm (Marsh et al., 1999), crustacean
(Dawirs, 1983) and mollusk (Sprung, 1984) species when they are starved (receiving
no particulate nutrients). There has also been work that has followed lipid content
24
and composition through development in larvae reared under low food conditions
(Gallager et al., 1986; Reitzel et al., 2004; Meyer and Oettl, 2005; Sewell, 2005).
In this study, the larvae of three temperate echinoderms, Asterina miniata,
Strongylocentrotus purpuratus and Lytechinus pictus were reared with levels of algal
nutrients that ranged from 0 (unfed) to 40 algal cells μl
-1
. The larvae were fed a
large range of algal concentrations to determine whether or not there is a critical
threshold of nutrient concentration that will elicit a physiological response (e.g.
changes in metabolic rate). In addition to metabolic rate, total protein content and
the total activity of citrate synthase, a key regulatory enzyme in aerobic metabolism
(Hochachka et al., 1970) were measured throughout development as potential indices
of the physiological state and feeding history of these larvae. Finally, questions of
metabolic scaling are addressed by looking at the relationships that may or may not
exist between these physiological and biochemical aspects and with nutrient level.
This study shows that the larvae of all three echinoderm species do not show a
measurable response in either aerobic metabolism or protein growth unless they are
fed at or above a nutrient concentration of 4 algal cells μl
-1
. The commonalities seen
among echinoderm species show that there may, in fact, be a critical threshold of
food limitation during larval development.
Materials and Methods
Larval culturing
Adult A. miniata, L. pictus, and S. purpuratus were induced to spawn by
injection of 1-methyladenine (A. miniata) and 0.5 M KCl (L. pictus and S.
25
purpuratus). Eggs from several females were pooled for fertilization with the sperm
from one male. Embryos and larvae from all species were cultured under similar
conditions. Animals were reared at 15 ± 1°C, at a density of 10 individuals ml
-1
in
either 200-l (A. miniata) or 20-l culture vessels (S. purpuratus, L. pictus) and mixed
using motor-driven paddles (30-40 rpm). Water was changed and algae replenished
every 3-4 days by gently siphoning the cultures onto a mesh sieve and re-suspending
them into fresh 0.2 μm pore-size filtered seawater.
Feeding treatments
Once a feeding stage was reached in all three species, larvae were pooled and
split evenly into their respective feeding treatments. Treatments that received algal
nutrients were fed a 1:1 mixture of the unicellular algae species Rhodomonas lens
and Dunaliella tertiolecta. As natural food conditions are most likely fluctuating
between high and low concentrations, we fed the larvae in this study a wide range of
algal concentrations. The larvae of A. miniata were split into four feeding
treatments, which were either unfed (received no algal nutrients) or fed 4, 8, 20, or
40 algal cells μl
-1
. Larvae of L. pictus were split into two feeding treatments: 0
(unfed) or 30 algal cells μl
-1
. Larvae of S. purpuratus were split into two, low
feeding rations at 2 and 4 algal cells μl
-1
.
Respiration Rate Measurements
Metabolic rate was determined in embryos and larvae by measuring oxygen
consumption throughout development as in Marsh and Manahan, 1999. In brief,
animals were placed into small biological oxygen demand vials (400-600μl) filled
26
with oxygen saturated 0.2 μm pore- size filtered seawater. Each experiment
consisted of 7-10 replicates, with the numbers of animals ranging from 100-900
embryos and 25-500 larvae, depending on their size and stage of development.
These ranges were tested to ensure that there were no density-dependent effects on
oxygen consumption rates. Animals were incubated for 4 hours at 15 ± 0.5°C to
achieve a measurable reduction in oxygen in the vials. End-point measurements
were then taken by injecting 300-500μl of sample onto a Clark-type electrode,
maintained at 15 ± 0.5°C, using a re-circulating water bath. The change in PO
2
was
monitored using a Strathkelvin Universal Interface and software.
Total Protein Content
The total protein content of embryos and larvae was measured with the
Bradford assay (Bradford, 1976), as modified for larvae by Jaeckle and Manahan
(1989). Frozen embryos and larvae (stored at -80°C) were homogenized in a known
volume of de-ionized water. A standard curve was created using Bovine Serum
Albumin (Bio-Rad Laboratories). Homogenates and standards were then extracted
in trichloroacetic acid (TCA) for 20 minutes at 4°C. The TCA-insoluble fraction
was dissolved in 1.0 N NaOH at 56°C for 30 minutes, and then acidified with 1.67 M
HCl. Concentrated Bradford dye reagent (Bio-Rad Laboratories) was used as a
colorimetric reagent and added to the acidified solution. The absorbance of all
samples was determined at 595 nm after the dye developed for 20 minutes.
27
Enzymatic Activity
Total citrate synthase (CS: E.C. 4.1.3.7) activity was measured in vitro in all
three species with the method described by Srere (1969) as modified for larvae by
Marsh et al. (1999). Citrate synthase activity was followed spectrophotometrically at
an absorbance of 412 nm by measuring the formation of mercaptide ions from
DTNB (Ellman’s reagent) reacting with the free -SH groups formed from the release
of Coenzyme A during the reaction. Measurements were made using a reaction
medium of 50 mM histidine buffer (pH 8.0, 23ºC), 0.4 mM Acetyl Co-A, 0.5 mM
oxaloacetate, and 0.25 mM DTNB. Background activity was measured using only
the tissue homogenate (enzyme), Acetyl Co-A, and DTNB. After establishing a
steady background activity (10 minutes), oxaloacetate was added to drive the
reaction. Increasing absorbance was measured for 15 minutes, or until a liner trace
was seen. Background activity was subtracted from the reaction rate after
oxaloacetate was added. The assay was run with varying amounts of tissue
homogenate of each species prior to final analysis to ensure that there was no
substrate limitation on enzyme activity.

Results
Total protein content
The total protein content in larvae of the sea star, Asterina miniata was
dependent on the amount of exogenous nutrients the larvae received during their
development. The total protein content of larvae of A. miniata that received 8 cells
28
μl
-1
did not change significantly through 26 days of age (ANOVA: df, 1,9, F = 4.6,
P>0.05). Larvae that received 4 cells μl
-1
also did not have a significant increase in
protein content through 45 days of age, but did significantly increase by 50 days of
age (ANOVA: df, 1, 22, F = 9.15, P<0.01). The total protein content of larvae that
received 0 algal cells μl
-1
(unfed) decreased significantly through 44 days of age at a
rate of -0.006 ng protein larva
-1
day
-1
(ANOVA: df, 1,20, F = 4.6, P<0.05) (Figure 1).
The total amount of protein increased 17-fold in larvae that received 40 algal cells μl
-
1
and almost five-fold in larvae that received 20 algal cells μl
-1
(Figure 1). The total
protein content in larvae of the echinoid Strongylocentrotus purpuratus that were fed
2 or 4 algal cells μl
-1
did not significantly during the experimental time period (18
days) (2 algal cells μl
-1
: ANOVA, df, 1,9, F = 2.98, P>0.1; 4 algal cells μl
-1
:
ANOVA, df, 1,11, F = 0.22, P>0.2). The total protein content in larvae of
Lytechinus pictus that received no exogenous nutrients also did not change
significantly over time (ANOVA: df, 1,7, F = 0.55, P>0.2). The total amount of
protein in larvae of L. pictus that were fed 30 algal cells μl
-1
increased 7-fold during
their larval development through 16 days of age.
Respiration rates
The respiration rates in larvae of Asterina miniata were affected by the
amount of exogenous nutrients the larvae received during their development
similarly to total protein content (Figure 2). Individual respiration rates increased at
a rate of 3.2 ± 0.75 pmol O
2
larva hr
-1
day
-1
in larvae fed 8 algal cells μl
-1
and 1.2 ±
0.5 pmol O
2
larva hr
-1
in larvae fed 4 algal cells μl
-1
. The respiration rates in larvae
29
Figure 1. Asterina miniata. Total protein content in embryos and larvae from all
feeding treatments. Each data point represents the mean of three replicates ± SE
mean. 40 algal cells μl
-1
(●) regression: y = 0.17x – 0.84, R
2
= 0.97, P<0.001. 20
algal cells μl
-1
(۟∆) regression: y = 0.03x + 0.08, R
2
= 0.94, P<0.001. 8 algal cells μl
-1

(۟□) regression: y = 0.01x + 0.22, R
2
= 0.37, P>0.05. 4 algal cells μl
-1
(۟■) regression:
0.004x + 0.15, R
2
= 0.30, P< 0.05. 0 algal cells μl
-1
(۟▼) regression: y = -0.006x +
0.31, R
2
= 0.20, P<0.05.

Age (Days)
0 10 20 30 40 50 60
Total Protein Content (μg ind
-1
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
40 cells μl
-1
20 cells μl
-1
0 cells μl
-1
4 cells μl
-1
8 cells μl
-1

30
Figure 2. Asterina miniata. Respiration rates in embryos and larvae from all
feeding treatments. Symbols are the same as in Figure 1. Each data point represents
the slope of the regression between total oxygen consumed (pmol hour
-1
) and
number of individuals ± SE slope. Symbols are the same as Figure 1. 40 algal cells
μl
-1
regression: y = 11.8x – 10.6, R
2
= 0.93, P<0.001. 20 algal cells μl
-1
regression:
y = 7.72x + 38.3, R
2
= 0.75, P<0.01. 8 algal cells μl
-1
regression: y = 3.25x + 8.28,
R
2
= 0.59, P<0.01. 4 algal cells μl
-1
regression: y = 1.22x + 37.9, R
2
= 0.20, P<0.05.
0 algal cells μl
-1
regression: y = -0.91 + 46.3, R
2
= 0.39, P<0.01.

Age (Days)
0 5 10 15 20 25 30 35 40 45 50
Respiration rate (pmol O
2
ind
-1
hr
-1
)
0
75
150
225
300
375
450
525
40 cells μl
-1
20 cells μl
-1
8 cells μl
-1
4 cells μl
-1
0 cells μl
-1

31
that were given no exogenous nutrients throughout the experimental time period
decreased significantly at a daily rate of –0.91 ± 0.25 pmol O
2
larva hr
-1
(ANOVA:
df, 1,22, F = 13.48, P<0.01). Respiration rates increased in larvae fed 40 algal cells
μl
-1
at a daily rate of 11.8 ± 1.3 pmol O
2
larva hr
-1
and a daily rate of 7.7 ± 1.8 pmol
O
2
larva hr
-1
in larvae fed 20 algal cells μl
-1
.
The respiration rates in larvae of S. purpuratus that were fed 2 algal cells μl
-1

did not change significantly throughout the time period studied (ANOVA: df, 1,4, F
= 0.50, P>0.2), and the respiration rates of larvae that were fed 4 algal cells μl
-1

increased at a rate of 0.89 ± 0.24 pmol O
2
larva hr
-1
day
-1
(ANOVA: df, 1,4, F = 14.1,
P<0.05; Figure 3A). The respiration rates did not change significantly over time in
larvae of L. pictus that received no algal nutrients (ANOVA: df, 1,3, F = 0.10, P>0.2)
and increased at a rate of 11.2 ± 0.92 pmol O
2
larva hr
-1
day
-1
in larvae that were fed
30 algal cells μl
-1
(ANOVA: df, 1,4, F = 148.6, P<0.01; Figure 3B).
Total protein content in relation to respiration rates
The relationship between protein content and respiration rates in the embryos
and larvae of all three species studied are shown in Figures 4 and 5. When the total
protein content and respiration rates larvae of Asterina miniata in the lower feeding
treatments (0, 4 and 8 algal cells μl
-1
) are pooled, there was a significant linear
relationship between the two (ANOVA: df, 1, 62, F = 19.5, P< 0.01; Figure 4A).
When the larvae of A. miniata that were fed at higher concentrations of exogenous
nutrients (20 and 40 algal cells μl
-1
) are pooled, there was also a linear relationship
between increases in total protein content and respiration rates (ANOVA: df, 1, 23,
32
Figure 3. Respiration rates in echinoid embryos and larvae from different feeding
treatments. Each data point represents the slope of the regression between total
oxygen consumed (pmol hour
-1
) and number of individuals ± SE slope. A)
Strongylocentrotus purpuratus. 4 algal cells μl
-1
(●) regression: y = 0.89x – 1.08, R
2

= 0.82, P<0.05. 2 algal cells μl
-1
(∆) regression: y = -0.08x + 6.17, R
2
= 0.14, P>0.2.
B) Lytechinus pictus. 30 algal cells μl
-1
(●) regression: y = 11.2x – 42.2, R
2
= 0.92,
P<0.01. 0 algal cells μl
-1
(∆) regression: y = -0.13x + 8.01, R
2
= 0.05, P>0.2.
Age (Days)
0 2 4 6 8 10 12 14 16 18 20
Respiration Rate (pmol O
2
ind
-1
hr
-1
)
2
4
6
8
10
12
14
16
18
20
4 cells μl
-1
2 cells μl
-1
A.
B.
Age (Days)
0 2 4 6 8 10 12 14 16 18
Respiration Rate (pmol O
2
ind
-1
hr
-1
)
0
20
40
60
80
100
120
140
160
180
0 cells μl
-1
30 cells μl
-1

33

Figure 4. Asterina miniata. Respiration rates vs. total protein content. Symbols are
the same as in Figure 1. Each data point has bi-directional error bars that represent
protein content and respiration rates as above. A) Embryos, 0, 4, 8 algal cells μl
-1

regression: y = 109.8x + 21.4, R
2
= 0.24, P<0.001. B) Embryos, 20 and 40 algal
cells μl
-1
regression: y = 76.8 + 37.4, R
2
= 0.67, P<0.0001. C) Embryos, all feeding
treatments regression: y = .009x – 0.15, R
2
= 0.66, P<0.0001.

34
Figure 4.
Total Protein Content (μg individual
-1
)
0 1 2 3 4 5 6
Respiration Rate (pmol O
2
individual
-1
hour
-1
)
0
100
200
300
400
500
600
Total Protein Content (μg individual
-1
)
0.0 0.2 0.4 0.6 0.8 1.0
Respiration Rate (pmol O
2
individual
-1
hour
-1
)
0
50
100
150
200
A.
B.
Total Protein Content (μg individual-1)
0 1 2 3 4 5 6
Respiration Rate (pmol O
2
individual
-1
hour
-1
)
0
100
200
300
400
500
600
C.

35
F = 44.7, P< 0.01; Figure 4B). Figure 4C shows the relationship between total
protein content and respiration rates in larvae from all feeding treatments (ANOVA:
df, 1, 82, F = 154.8, P< 0.001
There was no significant relationship in either treatment between total protein
content and respiration rate in the larvae of Strongylocentrotus purpuratus that were
fed 2 or 4 algal cells μl
-1
(2 algal cells μl
-1
: ANOVA, df, 1, 3, F = 1.37, P> 0.2; 4
algal cells μl
-1
: ANOVA, df, 1, 3, F = 0.04, P> 0.2). There was no significant
relationship between the two variables in larvae of Lytechinus pictus that were not
fed any exogenous nutrients (ANOVA, df, 1, 7, F = 3.19, P> 0.1), but there was a
significant relationship between increases in total protein content and respiration
rates in larvae that were fed 30 algal cells μl
-1
(ANOVA, df, 1, 8, F = 30.9, P< 0.001)
(Figure 5).
Total Citrate Synthase Activity
The total amount of citrate synthase (CS) activity (a measure of
mitochondrial density) was also dependent on feeding regime in the larvae of
Asterina miniata (Figure 6). The total CS activity did not change significantly in
larvae fed 8 algal cells μl
-1
through the first 40 days of development (ANOVA, df, 1,
11, F = 0.07, P> 0.2). Total CS activity also did not increase significantly in larvae
fed 4 algal cells μl
-1
through the first 40 days of development, but did begin to
increase significantly by 45 days of age (ANOVA, df, 1, 19, F = 4.89, P< 0.05).
Activity decreased significantly at a rate of -0.07 ± 0.03 pmol citrate larva
-1
min
-1

day
-1
in larvae that were given no exogenous nutrients (ANOVA, df, 1, 20, F = 6.78,
36
Figure 5. Lytechinus pictus. Respiration rates vs. total protein content. Symbols
are the same as in Figure 3. Each data point has bi-directional error bars that
represent protein content and respiration rates as above. Embryos, 30 algal cells μl
-1

regression: y = 0.49 + 4.13, R
2
= 0.73, P<0.01. Embryos, 0 algal cells μl
-1

regression: y = 0.34 – 2.79, R
2
= 0.32, P>0.1.
Total Protein Content (ng ind
-1
)
20 40 60 80 100 120
Respiration Rate (pmol O
2
ind
-1
hr
-1
)
0
20
40
60
80
100
120
140
160
180

37
Figure 6. Asterina miniata. Total citrate synthase activity in embryos and larvae
from all feeding treatments. Symbols are the same as Figure 1. Each data point
represents the mean of three replicates ± SE mean. 40 algal cells μl
-1
regression: y =
3.02x – 5.69, R
2
= 0.83, P<0.01. 20 algal cells μl
-1
regression: y = 3.45x – 14.5, R
2
=
0.88, P<0.01. 8 algal cells μl
-1
regression: y = 0.02x + 8.64, R
2
= 0.007, P<0.1. 4
algal cells μl
-1
regression: y = 0.37 + 2.43, R
2
= 0.21, P<0.05. 0 algal cells μl
-1

regression: y = -0.07 + 6.10, R
2
= 0.26, P<0.05.

Age (Days)
0 5 10 15 20 25 30 35 40 45 50
Citrate Synthase Activity (pmol citrate ind
-1
min
-1
)
0
20
40
60
80
100
120
140
160
40 cells μl
-1
20 cells μl
-1
8 cells μl
-1
4 cells μl
-1
0 cells μl
-1

38
P< 0.05. Total CS activity increased in larvae of Asterina miniata fed 40 algal cells
μl
-1
at a rate of 3.02 ± 0.55 pmol citrate larva
-1
min
-1
day
-1
and in larvae fed 20 algal
cells μl
-1
, CS activity increased at a rate of 3.45 ± 0.53 pmol citrate larva
-1
min
-1

day
-1
. The total citrate synthase activity in both feeding treatments of larvae of the
echinoid S. purpuratus decreased significantly throughout the experimental time
period (18 days) (2 algal cells μl
-1
: ANOVA, df, 1, 12, F = 6.39, P< 0.05; 4 algal
cells μl
-1
: ANOVA, df, 1, 13, F = 4.54, P = 0.05). Total CS activity decreased
significantly in the unfed treatment of larvae of L. pictus, but increased significantly
at a rate of 1.84 pmol citrate larva
-1
min
-1
day
-1
in the larvae that were fed 30 algal
cells μl
-1
(0 algal cells μl
-1
: ANOVA, df, 1, 10, F = 22.5, P< 0.01; 30 algal cells μl
-1
:
ANOVA, df, 1, 14, F = 237.9, P< 0.001).
Citrate synthase activity per unit protein was calculated for all species and
feeding treatments (Table 1). The means of protein-specific CS activity were similar
between the embryos of A. miniata and L. pictus (Student’s t-test: df, 1,4, t = -1.4,
P>0.2). There was no significant difference between the means of protein-specific
CS activity in the larvae given high food rations (20, 30 and 40 algal cells μl
-1
) of A.
miniata and L. pictus (Student’s t-test: df, 1,4, t = 1.85, P>0.1). When the means of
protein-specific CS activity of larvae given low food rations (0,2,4 or 8 algal cells μl
-
1
) are compared between the echinoids and asteroid species, there is no significant
difference between the two (Student’s t-test: df, 1,21, t = -0.67, P>0.2). The overall
mean of protein-specific CS activity in the asteroid species (0.04 ± 0.004 pmol CS
(ng protein)
-1
min
-1
did not differ significantly from the mean protein-specific CS
39
Table 1. Total Protein-specific Citrate Synthase Activity (pmol min
-1
ng
protein
-1
)

Feeding
Treatment

Asterina miniata

Lytechinus pictus

Strongylocentrotus
purpuratus
Mean Range Mean Range Mean Range

Embryos

0.05
0.03-
0.07

0.07
0.04-
0.08
__ __

Larvae: 0-8 cells
μl
-1

0.04
0.01-
0.12

0.03
0.03-
0.09

0.02
0.01-0.03

Larvae: 20-40
cells μl
-1

0.04
0.01-
0.09

0.09
0.03-
0.17
__

40
Respiration rate as a function of total citrate synthase activity
Respiration rates were plotted against CS activity for all treatments and
species. There was no significant relationship between the two variables in larvae of
Asterina miniata from lower feeding treatments (0, 4 and 8 algal cells μl
-1
)
(ANOVA: df, 1,40, F = 2.39, P>0.1; Figure 7A). There was also a significant, linear
relationship between total CS activity and respiration rate in the larvae of A. miniata
that were fed 20 and 40 algal cells μl
-1
(ANOVA: df, 1,15, F = 74.9, P<0.05; Figure
7B).
There was no significant relationship between metabolic rate and total CS
activity in the larvae of Strongylocentrotus purpuratus and Lytechinus pictus that
were fed 0, 2 or 4 algal cells μl
-1
(ANOVA: df, 1,17, F = 1.23, P > 0.2; Figure 8A).
The resulting relationship between respiration rates and CS activity in the larvae of
the treatment L. pictus fed 30 algal cells μl
-1
was similar to that seen in A. miniata, as
the higher food ration resulted in a highly significant linear relationship between
respiration rates and CS activity (ANOVA: df, 1,7, F = 80.77, P < 0.001; Figure 8B).
Discussion
Respiration rates, protein content and aerobic enzyme activities were
measured in the larval stages of three temperate echinoderm species to investigate
the effects of feeding history on the physiological state of larvae and possible
relationships that may exist between these physiological and biochemical
parameters. The respiration rates in larvae of all of the species studied were greatly
influenced by the nutritional environment in which they were cultured. In the larvae
41
Figure 7. Asterina miniata. Respiration rate vs. total citrate synthase activity. Each
data point has bi-directional error bars that represent citrate synthase activity and
respiration rates as above. A) Embryos, 0, 4, 8 algal cells μl
-1
(□) regression: y =
0.90x + 33.2, R
2
= 0.06, P>0.1. B) Embryos, 20 and 40 algal cells μl
-1
(●)
regression: y = 2.82x + 54.5, R
2
= 0.83, P<0.001.
Total Citrate Synthase Activity (pmol citrate ind
-1
min
-1
)
0 20 40 60 80 100 120 140 160
Respiration Rate (pmol O
2
ind
-1
hr
-1
)
0
100
200
300
400
500
600
A.
Total Citrate Synthase Activity (pmol citrate ind
-1
min
-1
)
0 5 10 15 20 25 30 35
Respiration Rate (pmol O
2
ind
-1
hr
-1
)
0
20
40
60
80
100
B.

42
Figure 8. Respiration rate vs. total citrate synthase activity. Each data point has bi-
directional error bars that represent citrate synthase activity and respiration rates as
above. A) Strongylocentrotus purpuratus and Lytechinus pictus. Embryos, 0, 2,4
algal cells μl
-1
(□) regression: y = 0.96x + 6.05, R
2
= 0.07, P>0.2. B) Lytechinus
pictus. Embryos, 30 algal cells μl
-1
(●) regression: y = 6.7x – 2.3, R
2
= 0.97,
P<0.001.

0 2 4 6 8 10 12 14 16 18 20 22 24
Total Citrate Synthase Activity (pmol citrate ind
-1
min
1
)
0
20
40
60
80
100
120
140
160
180
Respiration Rate (pmol O
2
ind
-1
hr
-1
)
0 1 2 3 4 5
Total Citrate Synthase Activity (pmol citrate ind
-1
min
1
)
0
2
4
6
8
10
12
14
16
18
20
22
A.
B.
Respiration Rate (pmol O
2
ind
-1
hr
-1
)

43
of A. miniata and L. pictus fed high levels of algal nutrients (i.e. 20, 30, 40 algal cells
μl
-1
), respiration rates and total protein content increased with age. This is a well-
documented phenomenon in planktotrophic larvae of marine invertebrates when they
are given exogenous nutrients during their development (Marsh et al., 1999; Moran
and Manahan, 2004; Pace and Manahan, 2006; Pace et al., 2006). Between 60 and
70% of the increases in respiration rate can be attributed to the changes in size (i.e.
total protein content), in these treatments, as shown in Figures 4 and 5. The
respiration rates of larvae of A. miniata and S. purpuratus that were fed either 4 or 8
algal cells μl
-1
increased significantly through the duration of the culture. The
respiration rates of larvae of all three species that were unfed or fed at 2 algal cells
μl
-1
either did not change significantly, or decreased over the experimental time
period. The total protein content did not increase significantly in any of the species
fed at lower food rations (0, 2, 4 and 8 algal cells μl
-1
) during the first 40 days of
development, and in fact decreased in the unfed treatments of A. miniata. This is
also a common phenomenon in unfed marine invertebrate larvae, indicating a period
of starvation (Garcia-Esquivel, 2002; Moran and Manahan, 2004). There was an
increase in total protein in the larvae fed 4 algal cells μl
-1
between 45 and 50 days of
age, indicating that a food ration of 4 algal cells μl
-1
or more can sustain growth
given an adequate amount of time. The linear relationship between total protein
content and respiration rates in the lower feeding treatments of A. miniata was still
significant, but not nearly as strong as in the higher feeding treatments (R
2
= 0.24
and 0.66, respectively). In these larvae, even small changes in respiration rates can
44
be attributed to small changes in size. The lower feeding treatments of the two
echinoid species did not show a significant relationship between total protein content
and respiration rates. This is not surprising as there were no significant increases in
either total protein content or respiration rates during the experimental time period in
these larvae.
The total amount of citrate synthase activity in all species studies was
affected by nutrient level in similar fashion to protein content and respiration rates.
These results agree with studies showing that nutrition, age, and size all play a role in
determining the total CS activity in larval stages (Segner & Verreth, 1995; Marsh et
al., 1999; Meyer et al., 2002; Garcia-Esquivel et al., 2002; Moran and Manahan,
2004). A physiological relationship that has been shown to exist in marine
organisms is the strong correlation between metabolic rate (i.e. rate of oxygen
consumption) and the activities of enzymes involved with aerobic metabolism
(Childress and Somero, 1979; Sullivan and Somero, 1980; Torres and Somero, 1988;
Thuesen and Childress, 1993; Seibel, et al., 2000). One such enzyme is citrate
synthase (CS), an enzyme involved in the Kreb’s cycle, which has been correlated to
metabolic rate in a number of marine fishes (Childress and Somero, 1975; Sullivan
and Somero, 1980; Torres and Somero, 1988), and invertebrates (Thuesen and
Childress, 1993; Seibel, et al., 2000). CS activity has also been found to closely
follow metabolic rate in the early life stages of shrimp (Lemos, et al., 2003), oyster
larvae (Moran and Manahan, 2004) and Antarctic echinoderm larvae (Marsh et al.,
1999). Citrate synthase is a mitochondrial enzyme that facilitates the formation of
45
citrate from Acetyl Co-A and Oxaloacetate. This reaction is the driving force of the
Kreb’s cycle, and citrate synthase is considered to be an integral part of the
regulation of aerobic respiration (Hochachka et al., 1970; Hochachka et al., 1975).
The total amount of CS activity is commonly used as a measure of mitochondrial
density and can be used to calculate the maximum aerobic capacity of a tissue or
organism (Hochachka and Somero, 1984).
In the higher feeding rations, the total citrate synthase activity in the larvae of
A. miniata and L. pictus followed respiration rates very closely (Figures 7B and 8B),
resulting in a strong linear relationship between the two. This indicates that in
echinoderm larvae fed high rations of algal nutrients, total citrate synthase activity is
a good indicator of metabolic rate and of mitochondrial density. In larvae of all three
species studied that were fed lower amounts of algal nutrients, there was no
significant relationship between total CS activity and metabolic rate (Figures 7A and
8A). The respiration rates and total citrate synthase activities changed very little if at
all relative to higher feeding treatments, which explains the lack of relationship in
these larvae. In the highest feeding treatments, if this relationship is examined in
only the embryological stages, there is no significant relationship, further
demonstrating this point. In order to see a significant relationship between
respiration rate and CS activity, there has to be high growth, which translates into
higher respiration rates and total CS activity. This agrees with studies where larval
respiration rate and CS activity are well correlated, because in these studies, the
larvae are also fed throughout their development (Clarke et al., 1992; Marsh et al.,
46
1999; Lemos et al., 2003; Moran & Manahan, 2004). This has also been an issue
when calculating the scaling exponent of body mass to respiration rates in very small
invertebrates (Banse, 1982). The amount of error associated with measurements
made on animals of such a narrow body mass range confounds the identification of
the actual physiological relationships that may exist. While it is possible to measure
extremely accurate scaling exponents in larvae (Hoegh-Guldberg and Manahan,
1995), measurements of respiration rates and citrate synthase activity may be
confounded by error in the lower feeding treatments of echinoderm larvae. Although
the methods used are extremely sensitive, the relationships that may exist are may
not be seen at the lower end of the larvae's metabolic scope. In the higher feeding
treatments, both variables increase to a level sufficient to override any error that may
be inherent in the measurements.
Interestingly, when the total amount of citrate synthase activity was
normalized to protein content (pmol citrate larva
-1
min
-1
ng protein
-1
), there were no
differences between larvae regardless of species, developmental stage or feeding
treatment. This has been documented in the larvae the Pacific oyster, Crassostrea
gigas, where larvae that were fed and unfed had no difference in their protein-
specific CS activity at anytime during the experiment (Garcia-Esquivel et al., 2002).
Larvae of the red drum, Sciaenops ocellatus, do not show differences in protein-
specific CS activity between fed and unfed larvae, but do show a difference in
weight-specific CS activity between the two groups (Clarke, et al., 1992). Berges, et
47
al. (1990) showed that in adults of the brine shrimp, Artemia franciscana, the index
of growth chosen to normalize physiological rates could change the resulting values.
In addition to there being no differences between fed and unfed larvae in
protein-specific CS activity, there was also no consistent increase or decrease in
protein-specific CS activity through time within or among species. Later
developmental stages of fed larvae of the Antarctic sea urchin, Sterechinus
neumayeri, have consistent protein-specific CS activity, which suggested the
increase in cell number associated with developmental stage in these larvae was
equivalent to the increase in the total number of mitochondria (Marsh, et al., 1999).
Unfed post larvae of C. gigas did not have a decrease in protein-specific CS activity
during the first four days of starvation, despite a decrease in their total protein
content (Garcia-Esquivel et al., 2002). This indicated that an organism may be able
to selectively conserve proteins that are crucial to its functional integrity.
The stage of development that is reached and the rate at which development occurs
in echinoderm larvae is dependent on temperature, salinity, and nutritional
environment to which they are exposed (Paulay, et al., 1985; Pechenik, 1987;
Rumrill, 1990; Hoegh-Guldberg & Pearse, 1995). Therefore, it is difficult to
ascertain in wild-caught larvae what environment they have encountered during their
larval period, in particular the feeding histories of planktotrophic larvae. Previous
studies have looked to morphological differences between planktotrophic larvae that
have been exposed to both high and low levels of particulate nutrients as possible
indices of their condition and nutritional history. The larvae of many species have
48
been shown to have a developmental plasticity where they are able to increase the
total surface area of their feeding apparatus to increase particulate intake when they
are food limited. In contrast, when these same larvae are exposed to high nutrient
levels, they are able to preferentially allocate their resources into development and
growth, and therefore have significantly smaller feeding structures (Strathmann et
al., 1992; Strathmann et al., 1993; Fenaux et al., 1994; Hart and Strathmann, 1994;
Klinzing and Pechenik, 2000). While the morphology of a larva’s feeding structures
may be indicative of its feeding history, it cannot be used alone to determine the
actual condition of the larva (Klinzing and Pechenik, 2000). In the present study, the
actual physiological state was examined in three species of temperate echinoderm
larvae that were fed a large range of concentrations of algal nutrients. The results of
this study show that there are indeed physiological and biochemical indicators of
feeding history in these larvae, including total protein content, metabolic rates, and
the activities of an enzyme involved in aerobic metabolism. Within the range of
feeding treatments used, there was a distinct threshold of nutrient levels that allowed
the larvae to grow and develop quickly (algal concentrations > 4 algal cells μl
-1
). At
nutrient levels below this threshold, protein mass could be maintained and growth
could occur given enough time at that ration. However, this still indicates food
limitation in these larvae, as it is not optimal for larvae to extend their time in the
plankton due to predation pressures and increased chance of becoming entrained in
unfavorable water currents (Pechenik, 1987). In the unfed treatments, larvae were
able to sustain protein mass and metabolic rates throughout most of the experimental
49
time period, but started to show decreases in both of these variables after a certain
period of not receiving exogenous nutrients. This period of time was much longer in
the asteroid larvae than either of the echinoid species (50 days of age compared to
14-16 days of age, respectively), indicating that these larvae are more tolerant of
periods of starvation. It has been shown that certain echinoderm species are more
tolerant to starvation than others (Olson and Olson, 1989), and the results of this
study agree with calculations of larval life span based on metabolic rates and
endogenous nutrients within eggs of these species (Shilling and Manahan, 1994).
The physiological state of a larva may be a useful indicator of the feeding
history they have experienced in the field. When metabolic rates measured for all
three species on larvae that were 14-16 days of age are plotted against feeding
treatment, the increases in metabolic rates in all three species follow the same
trajectory as the amount of exogenous nutrients available to them increases (Figure
9). When a power curve is fitted through this data, the increases in metabolic rate
follow a trajectory much like that seen in nutrient uptake kinetics. In the lower
concentrations of algal nutrients, metabolic rate is greatly affected by even the
smallest increase or decrease in nutrient concentrations. However, as larvae are
exposed to increasing concentrations, the effects on metabolic rate are much less.
This is similar to nutrient uptake kinetics in that as the concentration of substrate
increases, the transporter becomes saturated and can no longer increase it’s activity
regardless of further increases in substrate concentration. Planktotrophic larvae are
limited by the size and ability of their feeding structures in the amount of exogenous
50
Figure 9. Effect of food on metabolic rates in echinoderm larvae. Each data point
represents the respiration rate from larvae that were between 14 and 16 days old.
Power regression: y = 14.6 x
0.65
, df 1, 8, F = 81.2, P<0.0001. Symbols:
Strongylocentrotus purpuratus (□); Lytechinus pictus (∆); Asterina miniata (●).

51
Figure 9.

Feeding Treatment (cells μl
-1
)
0 10 20 30 40 50
0
20
40
60
80
100
120
140
160
180

52
nutrients they can take in. These larvae may also be limited by their ability to
assimilate the nutrients they do take in if the nutrients are moving through their guts
too quickly. These factors in turn limit the growth rates of these larvae, and hence
their metabolic rate. While there appears to be a lower threshold of nutrients that a
larva needs to grow, there also appears to be an upper threshold of growth rates that
can be achieved. This study offers another perspective on the debate of whether
planktotrophic larvae experience food limitation in the plankton. The measures of
physiological state used show that when larvae experience varying particulate
nutrient levels are able to maintain their protein mass and metabolic rates at an
adequate level for survival in the face of low nutrient levels. The combination of
measuring morphological and developmental status with physiological state may be
useful in ascertaining the feeding history and status of planktotrophic larvae in the
field.

53
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57
Chapter 2: Metabolic efficiency in fast-growing larvae

Chapter 2 Abstract

Variation in growth and other fitness-related traits occurs both within and
among cohorts of marine invertebrate larvae. Faster development and growth in
larvae may be the result of the exogenous environment they are in, their genetic
make-up, or a combination of both. In this study, we studied the mechanisms of
faster development and growth in the larvae of the Pacific oyster, Crassostrea gigas.
We were able to produce larvae with variable growth rates (inbred and hybrid larvae)
through crosses of inbred adult oysters, and cultured them under the same exogenous
environmental conditions (i.e. food and temperature). Overall metabolism was
measured in C. gigas trochophore and early veliger stage larvae to increase our
understanding of larval energetics and explore possible mechanisms of faster growth
in these larvae. An enzyme involved with intermediary metabolism, citrate synthase
(CS), was also studied in these larvae to explore any relationships that may exist
between CS and metabolic rate. No significant changes in metabolic rate or CS
activity occurred in pre-feeding larvae, but both began to increase after feeding
began at two days post-fertilization. Differential growth in both shell length and
total protein content occurred in all inbred and hybrid genotypes by six days post-
fertilization. Metabolic rate did not change significantly through time in inbred and
hybrid larvae at any stage of development (ANOVA, P>0.05). However, the initial
“set-point” of metabolism (y-intercepts, ANOVA, P<0.05) was different in larvae
produced from females from different lines (i.e. egg effects). When the metabolic
58
rates at individual developmental stages were analyzed, significant differences
between certain crosses did occur, but consistent patterns did not exist between
inbred and hybrid larvae. The larvae of C. gigas do not show marked increases in
metabolic rate until the onset of feeding occurs, in contrast to the rapid growth seen
upon commencement of feeding. The total amount of citrate synthase activity over
time was also not significantly different between inbred and hybrid larvae (ANOVA,
P>0.05). Increases in CS activity closely followed metabolic rates from Day 1-6 of
age, indicating a strong relationship between the two in oyster larvae. The increase
in total CS activity occurred without an increase in protein-specific activity. This
shows that there is an actual increase in the maximum aerobic capacity of the larvae
as they grow and develop. Despite the differential growth that occurred between
inbred and hybrid larvae, metabolic rate and CS activity did not differ. These results
indicate that in pre-feeding and early larval stages, oyster larvae that develop and
grow at a faster rate are more metabolically efficient than their more slowly growing
counterparts.

Introduction

Within species, there can be a large amount of variation in growth rates.
When culturing species that are economically important, such as the Pacific oyster,
Crassostrea gigas, it is beneficial to understand the mechanism(s) behind this
variation. This knowledge can be useful when trying to separate fast-growing
animals from slower-growing animals. It could also lead to the discovery of
59
indicators of fast growth rates that can be seen early in development (i.e. larval
stages).
A phenomenon that has occurred in previous studies in natural populations of
bivalve molluscs is the positive correlation between faster growth rates and other
fitness-related traits and increasing levels of allozyme heterozygosity (Mitton and
Grant, 1984, Zouros et al., 1987). This phenomenon was first noticed in certain plant
species, such as maize, and was described with the term ‘heterosis’ (Shull 1948).
This term includes not only the visible effects of increased heterozygosity (hybrid
vigor), but also the mechanisms that cause hybrid vigor. His definition itself
recognizes that heterosis cannot have a single cause or mechanism, as growth is a
culmination of many individual processes. It is still unclear what the physiological
or biochemical mechanisms of faster growth rates are in these animals, or in fast-
growing organisms in general.
There have been several models proposed to explain the physiological
components of variation in growth rates that utilize the balanced energy equation of
Winberg (1956). This equation states that: G = C – (F + U + R), where (G) is
growth, (C) is energy consumed, (F) and (U) are energy loss due to feces and
excreta, respectively, and (R) is energy loss due to respiration. According to Bayne
et al. (1999b.), there are three possible ways this equation can be manipulated within
an animal to produce an increase in growth (G). One model indicates an increase in
the amount of energy consumed (C), without increasing the amount of energy lost
through respiration (energy acquisition model). A second model postulates a
60
preferential allocation of energy to growth. The third model shows a situation where
fast-growing animals are metabolically efficient in that their growth-specific energy
loss due to respiration is lower than slower-growing animals. This model has been
supported in studies of adult and larval bivalves, from both natural and cultured
populations (Koehn and Shumway; 1982, Scott and Koehn; 1990, Bayne et al.,
1999b; Pace et al. 2006). In each of these studies, metabolic efficiency was greater
in animals with higher levels of genetic heterozygosity.
When inbred adults of the Pacific oyster, Crassostrea gigas, are crossed
within and among families, the larvae produced (inbred and hybrid) show variable
growth rates as early as six days post- fertilization (Hedgecock et al., 1996). These
variations not only occur between inbred and hybrid larvae, but can also be seen
among inbred and hybrid crosses (Pace et al., 2006). The fact that fast and slow-
growing oyster larvae within the same species can consistently be produced allows
the possible physiological mechanisms of fast growth to be studied. In a
comprehensive study of the physiology of inbred and hybrid veliger larvae by Pace,
et al. (2006), there were no genotype-dependent differences in respiration rates,
despite consistently faster growth rates in hybrid larvae. In addition, while the
feeding rates of hybrid larvae scaled with increasing size in a similar fashion to
inbred larvae, they had a higher initial ‘set-point’ of feeding rates. This meant that at
any given size, hybrid larvae were able to feed at a faster rate than inbred larvae.
The ability of these hybrid larvae to grow and feed faster while expending the same
amount of energy points to a possible combination of metabolic efficiency and
61
energy acquisition models as a mechanism of faster growth in oyster larvae. While
Pace et al., 2006 focused on later stages of larval development (mid-to-late veliger),
where feeding may comprise a major proportion of a larva’s metabolism, it is unclear
whether this metabolic efficiency would occur in pre-feeding, trochophore stages
and/or early veliger stages from two to six days of age when the ability to feed is just
beginning. As the mechanism of faster growth in later stages of oyster larvae (Pace
et al., 2006) and adult oysters (Koehn and Shumway, 1982) is metabolic efficiency,
the early stages of faster-growing larvae are also likely to be metabolically efficient.
In this study, inbred and hybrid larvae with variable growth rates were
produced from crosses of adult inbred oysters to test the hypothesis that metabolic
efficiency enables hybrid larvae to develop and grow at faster rates than inbred
larvae in early developmental stages. As in Pace et al., 2006, this approach was used
to produce larvae with different growth rates that were cultured under the same
environmental conditions (i.e. food and temperature). Respiration rates were
measured for both inbred and hybrid larvae through the first six days of development
in both pre-feeding and feeding larval stages. The total activity of citrate synthase,
an enzyme involved in aerobic metabolism, was also measured to quantify the
mitochondrial densities in both inbred and hybrid larvae. By concentrating on pre-
feeding and very early feeding larval stages of C. gigas, we were able to test whether
the mechanisms of faster growth and development remains the same or varies
depending on developmental stage.

62
Materials and Methods
Experimental Design
Gametes used for larval culture were obtained from inbred adult Crassostrea
gigas that were grown and conditioned in Dabob Bay, Washington. The inbred lines
originated from either self-fertilized hermaphrodites, or from full-sib mating of
inbred oysters (Hedgecock et al., 1995, McGoldrick and Hedgecock, 1997; Pace et
al., 2006). Larvae were produced as the result of factorial mating between the inbred
lines designated as line 35 and line 51, which resulted in inbred (♂35 ×♀35 and ♂15
× ♀51) and hybrid larvae (♂51 × ♀35 and ♂35 × ♀51). Over 16 million larvae in a
total of eight larval families were produced and tested in this study. Further details
of experimental design are given in Pace et al., 2006.
The results presented here are from two, separate cultures (designated as
Culture 1 and Culture 2). The larvae from Culture 1 were intensively sampled for
the first two days of development, while the larvae from Culture 2 were sampled at
longer time intervals from fertilization to 6 days of development.
Fertilization and Culturing
Sex was determined for adult oysters by inspecting small samples taken from
their gonads. Eggs and sperm were inspected for quality, and one female and one
male from each inbred line were chosen for fertilization based on the amount of
gametes and their quality. After washing and counting eggs, they were divided into
separate containers to be fertilized by the appropriate sperm, producing inbred and
hybrid embryos. Prior to fertilization, 200-L culture vessels were filled with 0.2 μm
63
pore-size filtered seawater (FSW) and maintained at 23ºC ± 2ºC. Larval crosses
were randomly assigned to vessels, and were stocked at a final density of 5 animals
ml
-1
. Cultures were aerated and the water was changed after two days post
fertilization, and every 4 days thereafter. The cultures were fed starting at two days
post fertilization with the unicellular algae, Isochrysis galbana, at a concentration of
30,000 algal cells ml
-1
with additional algae being added every two days.
Growth (change in population mean shell length with time)
Shell lengths were measured daily from Days 2 through 6 of development to
determine growth rates in all inbred and hybrid larval crosses. For each
measurement, 50 larvae were sampled from each cross and shell length was
measured using an ocular micrometer. The mean of these measurements was plotted
against time and the slope of the resulting linear regression equals the growth rate of
that cross (μm
–1
larva
-1
day
-1
). Errors presented represent the standard error of the
slope.
Sampling
Larvae from all four crosses were removed for respiration rate measurements
and for biochemical (e.g. total protein content, citrate synthase activity) analysis
throughout the duration of the culture. During very early development (0-42 hours),
sampling occurred every six hours (Culture 1). In the crosses that were studied
throughout later trochophore and early veliger stages (Culture 2) sampling occurred
every 12 to 24 hours. To sample, cultures were gently drained onto a 20μm mesh
sieve, rinsed with FSW, and enumerated for sampling. Larvae were then placed
64
immediately into experimental vials for respiration rate measurements or frozen at -
80ºC for later biochemical analysis. Samples for shell length measurements were
taken every day from two to six days of age for all cultures, and at sampling points
for longer duration cultures.
Total Protein Content Analysis
Total protein content in all species was measured on larval samples taken in
conjunction with the measurement of clearance and respiration rates. To measure
total protein content, a modified Bradford assay was used (Bradford, 1976 as
modified by Jaeckle and Manahan, 1989). Briefly, larvae were homogenized with a
known volume of deionized water. Homogenate was transferred to tubes and
deionized water was added, if needed, to tubes to bring all samples up to an equal
volume. A standard curve was constructed using known concentrations of Bovine
Serum Albumin (Bio-Rad Laboratories). Coomassie Brilliant Blue G-250 (Bio-Rad
Laboratories) was added to all of the tubes as a colorimetric reagent. The absorbance
of the standards and samples were read at 595nm at least 20 minutes, and no more
than 40 minutes after adding the dye.
Metabolic Rate Measurements
Respiration rate was used as a measure of overall metabolism in C. gigas
larvae. Respiration rates were measured using the micro-respiration technique
described in Moran and Manahan, 2004.. At each time point, once the larvae had
been sampled and enumerated, they were placed into small biological oxygen
demand vials (500-700μl) filled with 0.2 μm Nuclepore® filtered seawater.
65
Trochophore and early veliger oyster larvae were placed into the vials volumetrically
using mean of 4 counts taken on 50-100 μl of concentrated sample (5-7000 larvae
ml
-1
) ± a coefficient of variance (CV) of < 10% so that a range of known larval
densities (50 - 1000) could be achieved. Once loaded into the vials, the larvae were
incubated at a constant temperature of 23 ± 0.5ºC for 2-3 hours, during which a
measurable (<10%) reduction in oxygen tension (PO
2
) occurred. At the end of the
incubation time, the vials were opened and a sample (250-500μl) was removed with
a gas-tight syringe and injected onto a Clark-type electrode kept at a constant
temperature of 23ºC to measure PO
2
. A universal interface (Strathkelvin
Instruments) was used to convert PO
2
readings into μmoles/L oxygen. The slope of
the regression between total oxygen consumed and the number of individuals per
vial was used as the individual larval respiration rate (see Moran and Manahan,
2004). Errors presented represent the standard error of the slope.
Total Citrate Synthase Activity
Total citrate synthase (CS: E.C. 4.1.3.7) activity was measured in vitro for
larval samples using the method described by Srere (1969), as modified by Marsh
and Manahan, 1999 and Moran and Manahan, 2004. Citrate synthase activity was
measured spectrophotometrically at an absorbance of 412 nm by measuring the
formation of mercaptide ions from DTNB (Ellman’s reagent) reacting with the free -
SH groups formed from the release of coenzyme A (CoASH) during the reaction.
Measurements were made using a reaction medium of 50 mM histidine buffer (pH
8.0, 23ºC), 0.4 mM Acetyl Co-A, 0.5 mM oxaloacetate, and 0.25 mM DTNB.
66
Background activity was measured using only the tissue homogenate (enzyme),
Acetyl Co-A, and DTNB. Background activity was measured for 5-7 minutes, and
then oxaloacetate was added to drive the reaction. Increasing absorbance was
measured for about 15 minutes, or until a liner trace was seen. Background activity
was subtracted from the reaction rate after oxaloacetate was added for a total CS
activity in pmol citrate produced individual
-1
min
-1
. Measurements using varying
amounts of enzyme (i.e. tissue homogenate) were made prior to final analysis to
ascertain optimal sensitivity and to ensure that there was no substrate limitation on
enzyme activity.
Statistical analysis of genotype effects
In both Cultures 1 and 2, differences in growth rates were determined by
comparison of the linear regressions of shell length against age (in days) using
ANOVA (shell length = Age + Family + Age*Family). Comparisons of metabolic
rate, citrate synthase activity, protein content and protein-specific citrate synthase
activity over developmental time were made similarly between inbred and hybrid
larvae using ANOVA of the combined regressions. Data that increased
exponentially were log-transformed and plotted on a linear-log scale for ANOVA
regression comparisons. Where possible, the two inbred and two hybrid crosses
were pooled for analysis. All statistical analyses were conducted using the statistical
program “R” (R Development Core Team, 2005).

67
Results
Growth
Heterosis for growth occurred between inbred and hybrid crosses of both
Culture 1 and 2 within the first six days of development. Growth rates in hybrid
larvae of Culture 1 shown in Figure 10 (♂35 × ♀51 and ♂51 × ♀35) from days two-
six of development were 11.6 ± 0.89 and 9.7 ± 0.61 μm larva
-1
day
-1
(Figure 10
inset). Inbred crosses (♂35 × ♀35 and ♂51× ♀51) had growth rates 6.7 ± 0.43 and
5.6 ± 1.0 μm larva
-1
day
-1
(Figure 10 inset). There was a significant effect of the
interaction term (Age*Family) on size when the growth rates of the pooled hybrid
larvae were compared with the pooled inbred larvae (two-way ANOVA, df = 4, 10;
F = 10.8; P<0.05). In larvae from Culture 2, shown in Figure 11, there was no
significant effect of the interaction term (Age*Family) on size over the first six days
of development between pooled inbred and hybrid larvae (two-way ANOVA, df = 4,
20; F = 0.93; P>0.05). While the growth rates themselves were not significantly
different, the slope of the hybrid larval growth rate had a significantly higher
intercept (two-way ANOVA, df = 1, 20; F = 23.0; P<0.01) than the inbred larvae.
This means that at any given point during the first six days of development, the
hybrid larvae were significantly larger in size than the inbred larvae. For example,
on Day 6 of development, the pooled inbred larvae had an average shell length of
106.2μm larva
-1
and the pooled hybrid larvae had an average shell length of
122.8μm larva
-1
(t-test, P<0.01; Figure 11 inset).
68
Physiological and Biochemical Analyses-Culture 1, the first 48 hours of development
Metabolic Rates
The respiration rates of the combined inbred crosses (♂35 × ♀35 and ♂51 ×
♀51) increased over larval development at a rate of 0.33 ± 0.16 pmol O
2
larva
-1

hour
-1
(one-way ANOVA: df = 1, 32, F = 4.37, P<0.05). The respiration rates in
larvae of the combined hybrid crosses (♂35 × ♀51 and ♂51× ♀35) increased at a
rate of 0.52 ± 0.17 pmol O
2
larva
-1
hour of development
-1
(one-way ANOVA: df = 1,
25, F = 9.70, P<0.01). When these regressions of respiration rate over time were
compared between inbred and hybrid larvae, there were no significant differences P>
0.20), the y-intercepts of the regressions were significantly different from one
another (ANOVA of combined regressions: df = 1, 56, F = 14.3, P< 0.001) (Figure
10). This result indicates a maternal genotype effect on metabolism in these larvae.
The larvae produced from the inbred Line 35 female had a higher initial ‘set-point’
of metabolism. The mean of the individual respiration rate measurements in larvae
produced from the Line 35 female over the first 48 hours of development was
therefore significantly higher than in larvae produced from the Line 51 female (23.4
± 2.35 pmol larva
-1
hour
-1
(N = 29) vs. 13.4 ± 1.7 pmol larva
-1
hour
-1
(N = 28);
ANOVA, P<0.05).
Citrate Synthase Activity
Total CS activity did not change significantly in any of the pooled inbred
crosses studied during this time period (one-way ANOVA: df = 1, 28, F = 3.08,
69
Figure 10. Comparison of maternal effects on respiration rates in larvae produced
from the factoral cross of lines 35 and 51. Each data point is an individual
respiration rate measurement. ○ = ♂35x ♀35, ▲ = ♂35x ♀51, ● = ♂51x ♀35,
= ♂51x ♀51. Dashed line represents the regression of respiration rates over time
in larvae from the Line 35 female. The solid line represents the regression of
respiration over time in larvae from the Line 51 female. Inset: Differential growth
between inbred and hybrid crosses, at six days post fertilization using inbred adult
oyster lines 35 and 51. Each bar represents the growth rate (one-way ANOVA ± SE
slope). Shaded bar indicates hybrid crosses (♂51x ♀35 and ♂35x ♀51), cross-
hatched bar indicates inbred crosses (♂35x ♀35 and ♂51x ♀51).

Age (hours)
0 10 20 30 40 50
Respiration Rate (pmol O
2
larva
-1
hour
-1
)
0
10
20
30
40
50
60
35x35 51x35 35x51 51x51
Growth Rate (μm day
-1
)
0
2
4
6
8
10
12
14

70
Figure 11. Respiration rates in inbred and hybrid larvae through the first six days of
development. Each data point is an individual respiration rate measurement.
Symbols are the same as in Figure 1. The dotted, solid, dash-dotted and dashed lines
line represent the regressions between respiration rates over time in the ♂35x ♀35,
♂35x ♀51, ♂51x ♀35 and ♂51x ♀51 crosses, respectively. Inset: Day 6 shell
lengths of inbred and hybrid crosses between lines 35 and 51. Each bar represents
the mean of 50 measurements ± SE mean.

Age (hours)
0 20 40 60 80 100 120 140 160
Respiration Rate (pmol O
2
larva
-1
hour
-1
)
0
10
20
30
40
50
Shell Length (μm)
85
90
95
100
105
110
115
35x35 35x51 51x35 51x51

71
P>0.10). There were also no significant changes in CS activity over time in the
larvae of the hybrid crosses (one-way ANOVA: df = 1, 27, F = 0.09, P> 0.20).
There were no significant differences between the regressions of total CS activity
over time amongst the individual crosses (ANOVA of combined regressions, four
variables: df = 1, 53, F = 2.72, P> 0.10). The average total CS activity of all larvae
was 0.90 ± 05 pmol of citrate produced larva
-1
min
-1
(N = 57). Unlike metabolic
rates, there were no significant differences in either the slopes or y-intercepts of CS
activity over time based on maternal contribution (ANOVA of combined
regressions: slopes, df = 1, 53, F = 3.31, P> 0.05; y-intercepts, df = 1, 54, F = 0.32,
P> 0.20).
Physiological and Biochemical Analyses-the first six days of development
Metabolic rates
There was an overall increase in respiration rates over the first six days of
development in all larvae of all crosses due to the addition of exogenous nutrients at
48 hours of age (Figure 11). The increase in respiration rates through the first six
days of growth in the larvae in all crosses was mostly due to growth in the larvae
(Figure 11B, one-way ANOVA of respiration rate vs. total protein content: ♂35 ×
♀35: df = 1, 12, F = 13.7, P< 0.01; ♂35 × ♀51: df = 1, 10, F = 9.96, P< 0.02; ♂51 ×
♀35: df = 1, 12, F = 34.6, P< 0.0001; ♂51 × ♀51: df = 1, 11, F = 28.4, P< 0.001).
The slopes or intercepts of the regressions of pooled inbred and hybrid respiration
rates over time were not significantly different (ANOVA of combined regressions:
slopes, df = 1, 76, F = 1.55, P> 0.10; y-intercepts, df = 1, 77, F = 2.19, P> 0.10),
72
despite the fact that at Day 6, hybrid larvae were significantly larger than inbred
larvae (Student t-test of pooled inbred and hybrid Day 6 shell lengths: df = 1, 164, P-
stat = 9.29, P< 0.05) (Figure 11 inset).
Total protein content was measured in conjunction with metabolic rate in
larvae from all the crosses studied throughout the first six days of development. As
with metabolic rates, the addition of exogenous nutrients resulted in an increase of
total protein content over time in all crosses (Figure 12). The total protein content of
hybrid larvae increased at a significantly faster rate than inbred larvae (ANOVA of
combined regressions: df = 1, 54, F = 20.7, P< 0.0001), which agrees with the
growth results obtained from shell length measurements (Figure 11 inset).
Respiration rates were normalized for total protein content at each time point for all
crosses. The protein-specific respiration rates (pmol O
2
ind
-1
hr
-1
ng protein
-1
)
remained relatively constant throughout the first six days of development in both
inbred (♂35 × ♀35 and ♂51 × ♀51) and hybrid (♂35 × ♀51 and ♂51 × ♀35) larvae
(one-way ANOVA: inbreds, df = 1, 9, F = 0.36, P> 0.20; hybrids, df = 1, 9, F = 0.04,
P> 0.20). Protein-specific respiration did not differ significantly amongst inbred and
hybrid larvae (ANOVA of combined regressions: df = 1, 16, F = 0.11, P> 0.20).
Citrate synthase activity
Total CS activity was measured in conjunction with metabolic rate in
larvae from all the crosses studied throughout the first six days of development.
Total CS activity increased as a function of time in all crosses studied. There were
no significant differences in the slopes of log-transformed CS activity over time in
73
Figure 12. Total protein content of inbred and hybrid larvae through the first six
days of development plotted on a log-linear scale. Each data point represents an
individual replicate measurement. Symbols and lines are the same as in Figure 2.

Age (Hours)
0 20 40 60 80 100 120 140 160
Total Protein Content (ng larva
-1
)
1
10
100

74
three of the four crosses (♂35 × ♀35, ♂35 × ♀51, ♂51 × ♀35, ANOVA of
combined regressions, three variables: df = 1, 34, F = 0.19, P> 0.20). The larvae in
the inbred cross, ♂51 × ♀51 had a significantly higher increase in CS activity over
time than the other three crosses [y (log CS activity) = 0.006 (age) – 0.46, R
2
= 0.93,
P<0.05 and y = 0.004x – 0.30, R
2
= 0.81, P<0.05, respectively]. The increase in CS
activity followed the increase in metabolic rates very closely in both inbred and
hybrid larvae (Figure 13), indicating that total CS activity is a good indicator of
metabolic rate in these larvae. When CS activity was normalized to protein content,
it again followed a similar pattern as protein-specific respiration rates. Protein-
specific CS activity remained relatively constant in both inbred and hybrid larvae
throughout the ♀51), df = 1, 9, F = 0.004, P> 0.20; Hybrids (♂35 × ♀51 and ♂51 ×
♀35), df = 1, 9, F = 1.03, P> 0.20) and there were no significant differences between
inbred and hybrid larvae when protein-specific CS activity was regressed against
time (ANOVA of combined regressions: df = 1, 16, F = 0.11, P> 0.20).
Discussion
Controlled-genetic crosses of the Pacific oyster Crassostrea gigas were used
in this study to produce larvae that had differential growth (i.e. fast- or slow-
growing). Overall metabolism, the activity of a key regulatory enzyme of aerobic
metabolism and growth were measured in these larvae in order to test the hypothesis
that metabolic efficiency (i.e. the energetic cost of somatic growth is lower in fast-
growing larvae) is the predominant mechanism responsible for faster growth in the
early larval stages of hybrid oysters. In all of the crosses studied, hybrid
75
Figure 13. Crassostrea gigas: Total citrate synthase activity (О) plotted with
respiration rates (▲) in A) hybrid and B) inbred larvae through the first six days of
development. The y-axis on the left is respiration rate in pmol O
2
larvar
-1
hour
-1
, and
the y-axis on the right offset is total CS activity in pmol larva
-1
minute
-1
. Each
respiration rate data point represents the mean regression of total oxygen consumed
against the number of larvae ± the SE slope. Each CS data point represents the mean
of three replicate measurements ± SE mean.

76
Figure 13.

0 20 40 60 80 100 120 140 160
Respiration Rate (pmol O
2
larva
-1
hour
-1
)
0
5
10
15
20
25
30
35
Citrate Synthase Activity (pmol larva
-1
min
-1
)
0
1
2
3
4
5
6
7
Age (Hours)
0 20 40 60 80 100 120 140 160
Respiration Rate (pmol O
2
larva
-1
hour
-1
)
0
5
10
15
20
25
30
35
Citrate Synthase Activity (pmol larva
-1
min
-1
)
-1
0
1
2
3
4
5
6
7
A.
B.

77
offspring developed faster (observed trochophore ‘streaming’ at earlier time point
than inbred larvae), the larvae were consistently larger and often had faster growth
rates than inbred larvae. Respiration rates were positively correlated with increasing
size in both inbred and hybrid larvae, which is consistent with previous studies on C.
gigas larvae (Gerdes, 1983). There was no consistent pattern of differences in
respiration rate between inbred and hybrid larvae when they were compared over
time and at stage-specific time points. In fact, when respiration rates are converted
into energy units using a mean oxyenthalpic equivalent value of 484kJ mol O
2
-1

(Gnaiger, 1983), the cumulative cost of development in inbred and hybrid larvae
through the first six days of development were 643.7 and 591.9μJ, respectively
(Figure 14). These values of cumulative cost of development are within the same
range as previously measured values in Pacific oyster larvae (6-day-old larvae
cumulative cost: ca. 400 μJ in Moran and Manahan, 2004; 6-day-old larvae
cumulative cost: ca. 600 μJ in this study).
Rodhouse & Gaffney (1984) showed that adults of the oyster Crassostrea
virginica showed differences in growth based on differing levels of heterozygosity,
but showed no differences in metabolic rate between fast- and slow-growing
animals. In a study by Bayne et al. (1999a), metabolic rates were measured in inbred
and hybrid adults of C. gigas and also showed no consistent pattern of differences in
metabolic rate among the different crosses, despite faster growth on average in
hybrid oysters.

78
Figure 14. The cumulative cost of development in inbred and hybrid larvae over the
first six days of development. □ = Inbred larvae (♂35x ♀35 and ♂51x ♀51), ● =
Hybrid larvae (♂51x ♀35 and ♂35x ♀51). Dashed line represents the cumulative
cost of development in inbred larvae. The solid line represents the cumulative cost
of development in hybrid larvae. The cumulative cost of development in inbred
larvae follows the exponential equation y = 28.4(1.0)
x
(R
2
= 0.99, P<0.01). The
cumulative energetic cost of development in hybrid larvae follows the exponential
equation y = 32.4 (1.0)
x
(R
2
= 0.99, P<0.01).

Age (Hours)
0 20 40 60 80 100 120 140 160
Cumulative Ehergetic Cost (μJ)
0
100
200
300
400
500
600
700

79
Total citrate synthase (CS) activity is a measure of the total mitochondrial
density in tissues and organisms (Hochachka and Somero, 1984) and was also
measured in inbred and hybrid larvae. Total CS activity over time did not differ
greatly between inbred and hybrid larvae, and CS activity increased significantly
faster over time in only one hybrid cross out of a total of eight inbred and hybrid
larval families. These results agree with the metabolic rate measurements and
indicate that the aerobic potential of these larvae is not genotype-dependent. In
addition, total CS activity increased concomitantly with metabolic rates in feeding
inbred and hybrid larvae. This indicates not only that total CS activity is a good
index of metabolic rate in oyster larvae (Moran and Manahan, 2004), but also that
the increases in total respiration rates were closely related to increases in total
mitochondrial density in both inbred and hybrid larvae (Marsh and Manahan, 1999).
Protein-specific CS activity did not increase significantly over time in either inbred
or hybrid crosses. This result also agrees with similar measurements previously
made on oyster larvae, and indicates that the increase in total CS activity
corresponded with an increase in total protein content (Moran and Manahan, 2004).
The early stages of fast-growing, hybrid larvae have a lower energy-
expenditure (represented by respiration rate) per unit of protein growth than slow-
growing, inbred larvae (13.0 μJ energy ng protein
-1
in hybrid larvae cf.25.1 μJ
energy ng protein
-1
in inbred larvae). Therefore, during the first six days of
development, hybrid oyster larvae appear to fit the metabolic efficiency model when
explaining their faster growth. This model has been cited as the primary mechanism
80
behind faster growth in adults of several marine invertebrate species from both
natural and experimentally crossed inbred and hybrid populations (Diehl, 1986;
Hawkins and Day, 1996; Cruz, 1997; Bayne et al., 1999a, 1999b; Bayne, 1999;
Bayne, 2000). Later staged (veliger), fast-growing larvae of C. gigas have also been
shown to require 2 times less energy for an equivalent increase in shell length as
slow-growing inbred larvae (Pace et al., 2006). Previous studies on feeding rates and
assimilation efficiencies in the larvae of C. gigas have shown that hybrid veliger
larvae have higher feeding rates than inbred larvae at any given size despite having
similar metabolic rates (Pace, et al., 2006). This finding would indicate that fast-
growing hybrid larvae are able to acquire a greater amount of energy through algal
nutrients than inbred larvae, as well as being more metabolically efficient.
There are several different aspects of metabolism that have been proposed as
possible mechanisms governing metabolic efficiency in faster-growing organisms.
Protein metabolism (i.e. synthesis, degradation) contributes largely to the total
amount of energy expended in an organism, including invertebrate larvae (Pace and
Manahan, 2006), and has been highlighted in adults as the process that can lead to a
higher metabolic efficiency overall (Hawkins and Day, 1996; Bayne and Hawkins,
1997). Comparing inbred and hybrid veliger larvae, Pace et al. (2006) were able to
calculate that 50% of the difference in the amount of energy required for a fast-
growing, hybrid larva to grow the same amount as a slow-growing, inbred larvae
could be accounted for by differential feeding abilities, while the other 50% could be
accounted for by differential protein metabolism. The fact that there were no
81
differences in either overall metabolic rates or maximum aerobic potential in the
early feeding stages of fast- and slow-growing larvae in the present study potentially
indicates that differences in feeding and protein metabolism may also account for
differential growth in these stages of C. gigas development. As differences in
feeding rates cannot account for the faster development observed in pre-feeding
trochophore stages of hybrid C. gigas, it presents the possibility that differences in
protein metabolism may account for a larger (if not the entire) percentage of the
differences in development.
Previous studies on the underlying mechanisms of heterosis for growth in
bivalve molluscs have concentrated on juvenile or adult stages (Diehl, 1986;
Hawkins, 1996; Hawkins and Day, 1996; Bayne and Hawkins, 1997; Cruz, 1997;
Bayne, 1999a, 1999b; Bayne, 2000), or on mid-to-late veliger larval stages (Pace et
al., 2006). The current study confirmed the occurrence of differential development
and growth as early as six days of development, and also compared the metabolism
between fast-growing, hybrid larvae and slow-growing, inbred larvae to determine
which energetic model may explain faster development and growth in trochophore
and very early veliger developmental stages (1-6 days in age). The fact that hybrid
larvae did not have significantly different metabolic rates than inbred larvae, yet on
average demonstrated faster developmental and growth rates is consistent with the
metabolic efficiency model described earlier. However, previous studies of hybrid
oyster larvae have indicated both the metabolic efficiency and energy allocation
model are possible explanations of faster growth (Pace et al., 2006). Considering the
82
cumulative nature of metabolism, it is not surprising that fast-growing oyster larvae
utilize numerous energetic mechanisms. This study has shown that in trochophore
and early-staged veliger larvae of the Pacific oyster, Crassostrea gigas, faster
development and growth can be attributed at least partially to metabolic efficiency.

83
Chapter 2 References

Bayne B.L. 1999. Physiological components of growth differences between
individual oysters (Crassostrea gigas) and a comparison with Saccostrea
commercialis. Phsiol. Biochem. Zool. 72(6): 705-713.

Bayne B.L., Hedgecock D., McGoldrick D., Rees R. 1999 a. Feeding behaviour and
metabolic efficiency contribute to growth heterosis in Pacific oysters
[Crassostrea gigas (Thunberg)]. J. Exp. Mar. Biol. Ecol. 223:115-130.

Bayne B.L., Svensson S., Nell J.A. 1999b. The physiological basis for faster growth
in the Sydney rock oyster, Saccostrea commercialis. Biol. Bull. 197: 377-
387.

Bayne B.L. 2000. Relations between variable rates of growth, metabolic costs and
growth efficiencies in individual Sydney rock oysters (Saccostrea
commercialis). J. Exp. Mar. Biol. Ecol. 251(2): 185-203.

Bayne B.L., Hawkins A.J.S. 1997. Protein metabolism, the costs of growth and
genomic heterozygosity: Experiments with the mussel Mytilus
galloprovincialis (Lamark). Physiol. Zool. 70(4): 391-402.

Bradford M. M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem. 72: 248-254.

Cruz P., Ibarra A.M. 1997. Larval growth and survival of two catarina scallop
(Argopecten circularis, Sowerby, 1835) populations and their reciprocal
crosses. J. Exp. Mar. Biol. Ecol. 212: 95-110.

Diehl W.J., Gaffney P.M., Koehn R.K. 1986. Physiological and genetic aspects of
growth in the mussel Mytilus edulis. 1. Oxygen consumption, growth and
weight loss. Physiol. Zool. 59(2): 201-211.

Gerdes D. 1983. The Pacific oyster Crassostrea gigas: Part II. Oxygen
consumption of larvae and adults. Aquaculture. 31: 221-231.

Gnaiger E. 1983. Calculation of energetic and biochemical equivalents of
respiratory oxygen consumption. Pp. 337-345 in Polarographic Oxygen
Sensors: Aquatic and Physiological Applications, E. Gnaiger, H. Forstner,
eds. Springer-Verlag, Berlin.

84
Hawkins A.J.S., Day A.J. 1996. The metabolic basis of genetic differences in
growth efficiency among marine animals. J. Exp. Mar. Biol. Ecol. 203: 93-
115.

Hedgecock D., McGoldrick D.J., Bayne B.L. 1995. Hybrid vigor in Pacific oysters:
an experimental approach using crosses among inbred lines. Aquaculture.
137: 285-298.

Hedgecock D., McGoldrick D.J., Manahan D.T., Vavra J. Appelmans N., Bayne
B.L. 1996. Quantitative and molecular genetic analyses of heterosis in
bivalve molluscs. J. Exp. Mar. Biol. Ecol. 203: 49-59.

Hochachka P.W., Somero G.N. 1984. Biochemical Adaptation. Princeton
University press, Princeton.

Koehn R.K, Shumway S.E. 1982. A genetic/physiological explanation for
differential growth rate among individuals of the American oyster,
Crassostrea virginica (Gmelin). Mar. Biol. Lett. 3: 35-42.

Launey S., Hedgecock D. 2001. High genetic load in the Pacific oyster Crassostrea
gigas. Genetics. 159: 255-265.

Mitton J.B., Grant M.C. 1984. Associations among protein heterozygosity, growth
rate and developmental homeostasis. Annu. Rev. Ecol. Syst. 15: 479-499.

Moran A.L., Manahan D.T. 2004. Physiological recovery from prolonged
‘starvation’ in larvae of the Pacific oyster Crassostrea gigas. J. Exp. Mar.
Biol. Ecol. 306: 17-36.

Pace D.P., Manahan D.T. 2006. Fixed metabolic costs for highly variable rates of
protein synthesis in sea urchin embryos and larvae. J. Exp. Biol. 209: 158-
170.

Pace D.A, Marsh, A.G., Leong P.K., Green A.J., Hedgecock D., Manahan D.T.
2006. Physiological bases of genetically determined variation in growth of
marine invertebrate larvae: A study of growth heterosis in the bivalve
Crassostrea gigas. J. Exp Mar. Biol. Ecol. 335(2): 188-209.

Rodhouse P.G., Gaffney P.M. 1984. Effect of heterozygosity on metabolism during
starvation in the American oyster Crassostrea virginica. Mar. Biol. 80(2):
179-187.

85
Scott T.M. Koehn R.K. 1990. The effect of environmental stress on the relationship
of heterozygosity to growth rate in the coot clam Milinia lateralis (Say). J.
Exp. Mar. Biol. Ecol. 135(2): 109-116.

Shull G.H. 1948. What is ‘heterosis’? Genetics. 33: 439-446.

Srere P.A. 1969. Citrate Synthase. Pp. 3-11 in Methods in Enzymology, vol 13.
J.M. Lowenstein, ed. Academic Press, New York.

Winberg G.G. 1956. Rate of energy and food requirements of fishes. Fish Res. Bd.
Can. Trans. Ser. 194: 1-202.

Zouros E., Romero-Dorey M., Mallet A.L. 1987. Heterozygosity and growth in
marine bivalves: further data and possible explanations. Evolution. 42:
1332-1341.
86
Chapter 3: High growth efficiencies in Antarctic larvae
Chapter 3 Abstract
Antarctic sea urchin larvae synthesize protein for 1/25
th
of the energy
required in any other animal. This unique, efficient metabolism might be the basis
for high growth efficiency in cold ocean environments. We studied growth efficiency
in larvae of the Antarctic sea urchin Sterechinus neumayeri, and the Antarctic sea
stars Odontaster validus and O. meridionalis. The following series of measurements
were made over a total period of larval development and growth of 2 months at -
1.5ºC: clearance rates of algae by larvae; rates of protein growth; and metabolic
rates. The same set of measurements was made on the temperate sea urchins
Strongylocentrotus purpuratus and Lytechinus pictus and the temperate sea star,
Asterina miniata for comparison. Gross protein growth efficiency for larvae was
calculated for each culture – i.e., ratio of protein growth to amount of protein
obtained from algae. Resulting gross protein growth efficiencies were between 60-
90% for all Antarctic species and between 18-30% for temperate species. The
protein growth efficiencies seen in the Antarctic species are exceptionally high for
invertebrate larval forms and suggest that these larvae, living for most of their life
span in food-limited environments, can maximize growth rates by having unique
macromolecular synthetic processes.
Introduction
Larval stages of benthic marine invertebrates are characterized by structures
that are not necessary to form the juvenile (and therefore adult) organism
87
(McEdward and Janies, 1993). In addition to being morphologically distinct from
later life history stages, larvae are ecologically distinct from juvenile and adult
stages; planktonic organisms that must feed, swim and disperse to a suitable benthic
environment for metamorphosis and settlement (McEdward, 1995). While many
studies focus on adult metabolism and energetic budgets, much less work has been
done to describe the energetics of larval development. The characteristics of marine
invertebrate larvae would lead one to believe that the energetics and overall
metabolism of larvae would also differ from juvenile and adult stages. Considering
this, coupled with the high vulnerability of these stages to mortality (Thorson, 1950),
studying the physiology of larvae becomes an integral part of understanding an
organisms’ overall ecology and life history.
The waters of the Antarctic have a high diversity of organisms, including
many species of invertebrates. The developmental modes of these organisms have
been well studied, and a variety of life history strategies have been documented
(Pearse et al., 1991; Clarke, 1992). During the first half of the 20
th
century, it was
believed that at higher latitudes and lower sea temperatures, the pre-dominant mode
of development among invertebrates would be comprised of a non-feeding, non-
pelagic larval stage (Thorson, 1950). This prediction was coined ‘Thorson’s Rule’,
as it was based on surveys conducted by Thorson in Greenland on prosobranch
gastropods (Thorson, 1936) and other data from researchers studying Antarctic and
deep sea environments (Thorson, 1950). The theory behind this rule was that at high
latitudes and in the deep sea, where temperatures were low and suitable food for
88
planktotrophic larvae is not abundant, pelagic, feeding larvae would be selected
against (Clarke, 1992).
This rule has since been revisited with the addition of data from Antarctic
organisms (Pearse et al., 1991; Clarke, 1992), and while Thorson’s rule holds true for
certain groups of organisms (e.g. Arctic prosobranch gastropods), there are many
exceptions among Antarctic invertebrates. Picken (1979) showed that out of 11
species of prosobranch gastropods, 10 were brooders, and one (Nacella concinna)
had a pelagic larval stage. Hain and Arnaud (1992) studied the developmental mode
of Antarctic molluscs in the high Weddell Sea by direct observation. Again,
although most species were brooders, two species were found that had a pelagic,
feeding larval stage. Pearse et al. (1991) reviewed the developmental modes of a
number of species in McMurdo Sound, Antarctica, and found 8 species that had
planktotrophic pelagic larvae, and 4 species that had pelagic lecithotrophic larvae.
Two of the most abundant invertebrate species in McMurdo Sound, Antarctica are
the echinoderms, Odontaster validus (asteroid) and Sterechinus neumayeri
(echinoid) indicating that the larvae are able to receive enough nutrients during their
development to settle and grow successfully. While there is evidence that supports
some of the trends outlined by Thorson, it is apparent that many different
developmental modes are utilized amongst the benthic organisms in the Antarctic.
The waters of McMurdo Sound, and the Southern Ocean in general have a
degree of seasonality in primary production that is much greater than more temperate
waters (Clarke, 1988; Rivkin, 1991). There is a single phytoplankton bloom that
89
follows the retreating ice edge during austral summer that persists for about 60 days
(Smith and Nelson, 1985). This bloom accounts for almost 40% of total annual
primary production in the Southern Ocean (Smith and Sakshaug, 1990).
Planktotrophic larvae depend on particulate algae in order to fully develop to
metamorphosis, which would lead to the prediction that Antarctic species such as O.
validus and S. neumayeri would spawn at a time that coordinated with the
phytoplankton bloom. This is not the case, as the primary spawning seasons for O.
validus and S. neumayeri are during austral winter (June through September), and
early summer (October through December), respectively (Pearse et al., 1991). This
raises the question of how these larvae are able to survive in the plankton for up to
several months with little or no particulate food available to them. It has been shown
that echinoderm larvae are able to feed on bacteria (Rivkin et al., 1986) and take up
dissolved organic material (DOM) through their integument (Manahan, 1990). In
addition, Antarctic echinoderm larvae have lower metabolic rates when compared to
temperate echinoderm larvae (e.g. Mean metabolic rates: Temperate asteroid
Asterina miniata, 45.75 pmol O
2
μg
-1
hr
-1
cf. Antarctic asteroid Odontaster validus,
2.22 pmol O
2
μg
-1
hr
-1
from Shilling and Manahan, 1994). This is common among
species of marine ectotherms that live at lower temperatures (Peck, 2002). These
low metabolic rates allow the larvae to continue to survive and develop using the
endogenous nutrients provided in the eggs for several months to a year (Shilling and
Manahan, 1994; Marsh et al., 1999).
90
The eggs of many echinoderm species contain a large amount of protein,
usually constituting around 50% of the egg’s total biomass (McClintock and Pearse,
1986; Shilling and Manahan, 1994). Protein is an important building block in all
organisms, as it constitutes the machinery for numerous physiological processes and
is the primary constituent of biomass. In addition to the large amount of protein
provided in echinoderm eggs, this importance is also demonstrated by the amount of
energy organisms devote to the process of protein synthesis. In the temperate sea
urchin, Lytechinus pictus, the amount of total metabolism allocated to protein
synthesis can range from around 16% in unfed larvae (larvae that receive no
particulate nutrients) to as much as 75% in rapidly growing larvae fed with high
amounts of exogenous nutrients (Pace and Manahan, 2006). The rates and metabolic
cost of protein synthesis were examined in embryos and larvae of the Antarctic
echinoderm, S. neumayeri by Marsh et al. (2001), revealing protein synthesis rates
that were comparable to temperate echinoderm larvae. Coupled with the low
metabolic rates measured in these stages, the resulting metabolic cost of protein
synthesis in both embryos and larvae was only 0.45 J mg protein synthesized
-1
, 1/25
th

the value reported for most organisms. The combination of low metabolic rates and
low cost of protein synthesis provide a possible explanation of how these
planktotrophic larvae are able to survive and develop under the extreme conditions
of Antarctic waters (Marsh et al., 2001).
This unique, efficient metabolism could possibly be the basis for high protein
growth efficiency in cold ocean environments. Carter et al. (1998) found that in
91
juveniles of the flounder, Pleuronectes flesus, the gross protein growth efficiencies
(Protein growth rate/Amount of protein ingested) were positively correlated with the
amount of synthesized protein retained. In this study, we tested the hypothesis that
Antarctic echinoderm species with planktotrophic larvae would have higher gross
protein growth efficiencies than temperate echinoderm species. To do this, we
studied protein growth efficiency in larvae of the Antarctic sea urchin Sterechinus
neumayeri, and the Antarctic sea stars Odontaster validus and O. meridionalis
through measurements of ingestion rates and protein growth during development.
For comparison, we made similar measurements with the temperate sea urchins
Strongylocentrotus purpuratus and Lytechinus pictus, and the temperate sea star
Asterina miniata. High protein growth efficiencies in Antarctic invertebrate larval
forms would suggest that these larvae, living for most of their life span in food-
limited environments, are able to maximize growth rates by having unique
macromolecular synthesis processes.

Materials and Methods
Larval culturing
Temperate species

Marinus, Inc (Long Beach, CA) collected adults of the echinoid species
Strongylocentrotus purpuratus and Lytechinus pictus and asteroid species Asterina
miniata off the Southern California coast during their respective reproductive
seasons. Adult sea urchins were induced to spawn by intracoelomic injection of 0.5
92
M KCl; sea stars were similarly injected with a 1 mM solution of the hormone, 1-
methyladenine (1MA) in order to induce spawning. In all species, eggs from several
females were inspected for quality and then pooled for fertilization and the sperm
from one male was used for fertilization.
Once a fertilization success of >90-95% was achieved, embryos were placed
into 20-l culturing vessels with 0.2 μm-filtered seawater (FSW) at a final density of
10 embryos ml
-1
. Cultures were stirred constantly using motor-driven paddles in
order to keep embryos and larvae suspended and evenly distributed. The water of
each culture was changed every 3 days by gently siphoning the animals down onto
mesh sieves of sizes ranging from 20-80μm, depending on the stage of development.
The animals were enumerated and then placed into fresh 0.2μm pore-size filtered
seawater.
Polar species
Adult Odontaster validus and Odontaster meridionalis were collected from
various sites along McMurdo Sound, Antarctica between mid-August and October.
Adult Sterechinus neumayeri were also collected periodically from various sites in
McMurdo Sound between October and December. In both asteroid and echinoid
species, similar protocols were followed regarding adult spawning and subsequent
treatment of gametes and embryos as in the temperate species outlined above.
Fertilized embryos were placed into 200-l culture containers filled with 0.2μm pore-
size filtered seawater (FSW) at a final density of 5 embryos ml
-1
. Culture
temperature was maintained at -1.5°C by immersing the culture vessels in large
93
aquarium tanks with constant ambient seawater flow from McMurdo Sound.
Animals were kept in suspension using paddles driven by slowly rotating motors.
The cultures were inspected twice daily to ensure no settling had occurred. Culture
water was changed every four days by siphoning the larvae gently onto an 80μm
sieve. After enumeration, the larvae were placed into fresh 0.2μm pore-size FSW.
Feeding treatments
In all species studied, protein growth was achieved by feeding larval stages at
algal rations of 15-30 algal cells μl
-1
. Once a feeding stage was reached (3-5 days
post-fertilization), larvae of S. purpuratus L. pictus and A. miniata received the alga
Rhodomonas sp. at a concentration of 30 cells μl
-1
. Once a feeding stage was
reached in the Antarctic species (20-25 days post-fertilization), larval growth was
measured between cultures that were fed Dunaliella tertiolecta at either a
concentration of 15 or 30 algal cells μl
-1
. There were no differences in growth rate
between the 15 and 30 cells μl
-1

of D. tertiolecta (ANOVA, P>0.05), so the lower
concentration was used in subsequent cultures to maintain cleaner culturing
conditions for the larvae. In this study, while both algal species were comprised of
similar amounts of lipid (average Dunaliella tertiolecta: 13.9 ± 1.9 pg lipid cell
-1
;
Rhodomonas spp.: 16.8 ± 4.7 pg lipid cell
-1
; Student’s t-test, P>0.05, N = 5) the algae
Rhodomonas sp. typically had a higher average protein content than Dunaliella
tertiolecta (41.3 ± 0.04 vs. 23.2 ±1.8 pg protein per cell; Student t-test, P<0.05, N =
19), and was the preferred nutrient choice for echinoderm culturing. Cells of
Rhodomonas spp. were observed microscopically after being placed in
94
–1.5ºC seawater. The observed cells ceased movement and began to clump together.
Protein growth did not occur in larvae fed Rhodomonas spp., perhaps due to the
clumping of the algal cells. Cultures of D. tertiolecta were tested for viability at -
1.5°C, and the cells continued to move, did not clump together and decreased from
their initial concentration by only 1.5% over three days. Once these algae were
returned to their original growing temperatures (23°C) after three days at -1.5°C, in
less than three days the culture began to grow again at a similar rate to cultures never
subjected to cold temperatures (growth rates of 42.4 ± 9.1 cells μl
-1
day
-1
cf. 44.5 ±
3.8 cells μl
-1
day
-1
) (D. Ginsburg, unpublished data, 2006).
Ingestion rate measurements
General protocol
Ingestion rates were calculated from the clearance rates of algal cells by
larvae in all species studied. The concentration of Chlorophyll a (Chla) was
measured in samples taken before and after larvae were incubated in 25ml
scintillation vials with FSW and a concentration of algal cells equivalent to that of
their respective culture conditions. Clearance rate (μl larva
-1
hour
-1
) was then
calculated using the equation:
ln C
1
– ln C
2

CR = *V
n * t

where C
1
is the initial algal concentration (cells μl
-1
), C
2
is the final algal
concentration(cells μl
-1
), V is the volume of the experimental chamber (μl), n is the
95
number of animals used in the assay, and t is the time that the animals were
incubated (hours).
Ingestion Rate Experiments with Temperate Species: Strongylocentrotus. purpuratus,
Lytechinus pictus, Asterina miniata
Larvae of all three species were allowed to clear their guts prior to a feeding
experiment by removing animals from their culture vessels, rinsing them with FSW
and resuspended them in FSW for 16 ± 1 hr with no added particulate food. To test
for the optimal length of time the larvae should be removed from particulate food
prior to an experiment was run, the larvae of Lytechinus pictus were placed into
experimental vials with food to measure ingestion rates after 2, 4, 6, 12, 16 and 24
hours of being without particulate food. Ingestion rates dropped almost 5-fold in the
first 6 hours after being removed from food (65.2 ± 4.5 to 13.7 ± 5.5 algal cells larva
-
1
hr
-1
). The ingestion rates then began to rise and reached a maximum ingestion rate
of 74.7 ± 2.6 algal cells larva
-1
hr
-1
at 16 hours after being removed from particulate
food. Ingestion rates did not change significantly in larvae removed from food
between 16 and 24 hours (74.7 ± 2.6 to 65.6 ± 6.2 algal cells larva
-1
hr
-1
; slope:
ANOVA, P>0.05, N = 8).
Once the larvae had cleared their guts they were enumerated and placed into
25 ml scintillation vials at a final concentration of 50-75 larvae ml
-1
. Dunaliella
tertiolecta or Rhodomonas spp. were added to the vials at a concentration equivalent
to the respective culture conditions (i.e. 15 or 30 algal cells μl
-1
). The vials were
filled to an equal volume and allowed to incubate in the dark at 15°C for no more
96
than four hours. Algal depletion rates were measured with the larvae of S.
purpuratus and L. pictus to ascertain the period of incubation time during which the
larvae depleted the algal cells in a linear fashion (Figure 15A and B). A 4 hr
incubation time fell within this range, so it was used as the standard incubation time
of subsequent ingestion rate measurements. Controls that contained larvae but no
algal cells, and with algal cells but no larvae present were incubated concurrently.
Initial algal controls were also taken in order to assess whether algal growth occurred
during the incubation period. Samples of larvae and of the algae were taken and
frozen immediately at -80°C for later protein content analysis.
Upon completion of the incubation period, larvae were removed from the
experimental chamber and the seawater was filtered onto a GF/F filter at 100-200
mmHg vacuum to collect the algal cells. Chlorophyll a was extracted from the filters
according to EPA method 445. Briefly, GF/F filters were placed into 15ml of 90%
acetone and homogenized. The Chla was allowed to extract in the dark at 4°C for
12-18 hours. The extracts were then read on a Turner-10-AU Field Fluorometer.
The amount of Chla present in a sample was calculated as the difference between the
fluorescence before and after acidification of a sample with 500 μl of 1.2 M HCl.
The total amount of Chla (μg in a sample was divided by the amount of Chla per cell
of D. tertiolecta or Rhodomonas sp to determine the algal concentration of that
sample (cells μl
-1
). Ingestion rates were calculated as outlined above as the number
of cells cleared larva
-1
hr
-1
. The mean of 3-5 experimental replicates was used for
the final calculations of gross protein growth efficiency (protein growth rate/protein
97
Figure 15. Algal concentration over experimental incubation time. A)
Strongylocentrotus purpuratus. Each data point represents a single experimental
replicate. Measurements of algal concentration using both Chl-a analysis (●) and
hemacytometer counts (□) are shown on the same plot. Inset: data plotted on a log-
linear scale to show linear decrease in algal concentration over time. B) Lytechinus
pictus. Chl-a analysis of algal concentration over time. Inset: same as above. C)
Sterechinus neumayeri. Chl-a analysis of algal concentration over time. Inset: same
as above. D) Sterechinus neumayeri. Pictures of algae auto-fluorescence in guts
over 24 hours after larvae were removed from food.

98
Figure 15.
Time of Incubation (hours)
0 2 4 6
Algal Cell Concentration (# cell ml
-1
)
0
5
10
15
20
25
30
35
Time of Incubation (hours)
Duration of all ingestion
rate experiments
A.
0 1 2 3 4 5 6
Algal Concentration (cells ml
-1
)
0
10
20
30
40
50
60
B.
0 2 4 6 8 10
Algal Concentration (cells ml
-1
)
0
10
20
30
40
50
C.
D.
Time of Incubation (hours)
Time of Incubation (hours)
1.0
3.0
7.0
11.0
55.0
55.0
11.0
7.0
0.0
0 2 4 6
Algal Cell Concentration (# cell ml
-1
)
Time of Incubation (hours)
55.0
11.0
7.0
0
0 2 4 6
Time of Incubation (hours)
Algal Concentration (cells ml
-1
)
Algal Concentration (cells ml
-1
)
0
0.0
2 4 6 8

99
ingested). Ingestion rates calculated from the total amount of Chl-a were cross
calibrated in four separate experiments with actual hemacytometer counts of algal
cells in conjunction with Chl-a analysis (e.g. Figure 15A). In all trials, ingestion
rates calculated by Chl-a analysis (N = 10) were not significantly different
from that calculated from direct hemacytometer counts (N = 15) (all trials: t-test,
P>0.05).
Ingestion Rate Experiments in Antarctic Species: Sterechinus neumayeri, Odontaster
validus, Odontaster meridionalis
Ingestion rates were measured similarly in larvae S. neumayeri, O. validus
and O. meridionalis as in the temperate species with some modifications. Larvae of
S. neumayeri were removed from culture vessels and placed in FSW with no
particulate food for 24-hours prior to ingestion rate measurements. This time period
was decided upon based on microscopic observations of gut fluorescence (Figure
15D). The presence of algae in the guts of larvae was observed at regular intervals
on an epifluorescent microscope (excitation at 425 nm) from 0-24 hours after
removal from food. The fluorescence was reduced to negligible amounts by 24
hours, indicating that the larvae’s guts were empty. The depletion of gut
fluorescence in larvae of O. validus was also observed, and it was found that these
larvae required 48 hours to sufficiently clear their guts. This 48-hour time period
was also used for larvae of O. meridionalis.
Algal depletion rates were measured in larvae of S. neumayeri (Figure 15C).
Algal depletion was linear for the first 6 hr of incubation, so in subsequent ingestion
100
rate measurements, a 6-hr incubation time was used. In addition to cross-calibration
of Chl-a analysis with hemacytometer counts, larvae of S. neumayeri were observed
feeding under a microscope. Larvae were placed on a glass slide on a cooling stage
maintained at -1.5°C. Algal cells were added to the slide at a similar concentration
as in the clearance rate assays. An individual larva was observed for 30- minute time
intervals and the number of cells ingested counted. This was repeated for nine
separate larvae. Larvae were mobile on the slide throughout the observations, and
algal cells remained separate and mobile. There was no significant difference
between the average ingestion rate measured by direct observation (17.8 ± 3.7 algal
cells larva
-1
hr
-1
) and with Chl-a analysis (14.4 ± 1.6 algal cells larva
-1
hr
-1
) (student
t-test, P>0.05)
Biochemical analyses
Total protein content analysis
Total protein content in all species was measured on larval samples taken in
conjunction with the measurement of clearance and respiration rates. To measure
total protein content, a modified Bradford assay was used (Bradford, 1976 as
modified by Jaeckle and Manahan, 1989). Briefly, larvae were homogenized with a
known volume of deionized water. Homogenate was transferred to tubes and
deionized water was added, if needed, to bring all samples up to an equal volume. A
standard curve was constructed using known concentrations of Bovine Serum
Albumin (Bio-Rad Laboratories). Coomassie Brilliant Blue G-250 (Bio-Rad
Laboratories) was added to all of the tubes as a colorimetric reagent. The absorbance
101
of the standards and samples were read at 595nm at least 20 minutes, and no more
than 40 minutes after adding the dye.
Total Lipid Content of Algae
Total lipid content and lipid classes were measured according to Moran and
Manahan, 2004. Algal samples of 500,000 to 1 million cells were homogenized in a
known volume of deionized water and transferred to glass scintillation vials for lipid
extraction. Samples were extracted in 1:1:0.5 water/methanol/chloroform with an
added stearyl alcohol internal standard. Using a 1μl capillary tube, three replicate
samples of extracted lipid samples were placed onto thin-layer Chromarods (Iatron
laboratories). Developing the Chromarods in a 60:6:0.1 hexane/diethyl ether/formic
acid mixture separated lipid classes. After developing, the Chromarods were dried
for 10 minutes at 100ºC and then analyzed using an Iatroscan MK-5 flame ionization
detector. Resulting chromatograms were compared to standard lipid class
chromatograms (L-a-phosphatidylcholine (phospholipid), cholesterol, tripalmitin
(triacylglycerol), squalene, palmitic acid (free fatty acid), lauric acid palmityl ester
(wax ester) and stearyl alcohol (fatty alcohol internal standard). Total lipid content
and the amounts of specific lipid classes per individual algal cell were calculated as
the mean of two to three replicates ± SE mean.
Statistical analysis of protein growth rates
Protein growth rates for all species studied were calculated using the slope of
the regression between total protein content (ng larva
-1
) and age (days). To
determine whether the increase in total protein was linear over time, an ANOVA test
102
for non-linearity was performed on the data. This tests the non-linear components of
a regression (sums of squares). If the critical value for the non-linear components of
the regression is below 0.05, the regression is considered to be non-linear (see
Manahan and Richardson, 1983 for application of this test). If the increase in total
protein content over the entire experimental time period (e.g. Egg-Day 13 in the
larvae of L. pictus) was statistically non-linear, growth rate was calculated from the
linear portion of the curve.
Calculations
Gross Protein Growth Efficiency
Gross protein growth efficiency in all species was calculated in the following
manner. The ingestion rate (algal cells ingested larva
-1
hr
-1
) was multiplied by the
average amount of protein in each cell of either D. tertiolecta or Rhodomonas spp.
(ng cell
-1
) resulting in the amount of protein being ingested per individual larva hr
-1

and expressed as a rate per day. The average protein growth rate of each culture (ng
larva
-1
day
-1
) was then divided by the total protein ingested (ng per individual larva
day
-1
), which resulted in percent gross growth efficiency. For example, in a 6-day-
old pluteus larva of S. purpuratus, an ingestion rate of 31 ± 2.8 cells larva
-1
hr
-1
is
multiplied by the average protein content of a Rhodomonas spp. algal cell (measured
as 30 ± 3.0 pg cell
-1
), resulting in the amount of protein (ng) ingested by a larva hr
-1

of 0.93 ng protein larva
-1
hr
-1
, equivalent to 26.78 ng protein larva
-1
day
-1
. The
protein growth rate of the larvae is equal to the slope of the line between log-
transformed total protein values and age in days (9.2 ± 0.35 ng protein larva
-1
day
-1
).
103
The protein growth rate per larva is divided by the amount of protein ingested per
larva per day and multiplied by 100 resulting in a percent gross protein growth
efficiency (34%). The error associated with gross protein efficiency was calculated
using the “Delta Method” of error propagation (Taylor 1982; Vardeman and Jobe
2001):
Results

Ingestion rates
The ingestion rates for all species studied are listed in Table 2. Ingestion
rates of larvae of Strongylocentrotus purpuratus at different points during their
development are shown in Figure 16A. The ingestion rates or protein growth rates at
any time point in two, replicate cultures were not significantly different among
replicates (protein growth: ANOVA, P>0.05; ingestion rates: t-test, P>0.05), so the
results from the replicates were pooled. The average clearance rates of the larvae fed
with Rhodomonas spp. at a concentration of 30 cells μl
-1
was dependent on
developmental age, and ranged from 1.03 ± 0.1 (6-day-old larva) to 2.9 ± 0.3 (13-
day-old larva) μl larva
-1
hr
-1
, resulting in average ingestion rates that ranged from
30.8 ± 2.9 to 86.1 ± 10.9 algal cells hr
-1
larva
-1
, respectively. Two cultures of larvae
of Lytechinus pictus had significantly different protein growth rates (ANOVA,
P<0.05) and were not pooled for further analysis. The clearance rates of larvae of L.
pictus did not increase significantly with age (ANOVA, P>0.05), and the overall
average clearance rate was 2.0 ± 0.44μl hour
-1
larva
-1
and the overall average

104
Table 2. Ingestion rates in temperate and Antarctic echinoderm larvae
Species Age (days) Ingestion
Rate (algal cells larva
-1
hr
-1
)
± SE mean
Sterechinus neumayeri 26
31
33
44
48
48
52
57
29.2 ± 3.9
22.1 ± 2.6
24.6 ± 5.2
26.5 ± 3.7
10.7 ± 2.5
23.5 ± 3.6
12.9 ± 2.9
17.7
Odontaster validus 34
42
47
60
61
72
95
13.8 ± 1.2
7.1 ± 1.7
12.4 ± 0.75
14.7 ± 0.15
25.5 ± 0.99
11.2 ± 2.4
15.1 ± 0.90
Odontaster meridionalis 49
62
72
9.6 ± 2.4
13.9 ± 4.4
7.1 ± 0.53
Strongylocentrotus
purpuratus
6
8
13
30.8 ± 2.9
46.7 ± 2.0
86.1 ± 10.1
Asterina miniata 10
11
14
16
21
23
23
26
74.6 ± 6.8
190.7 ± 15.7
235.7 ± 17.6
149.3 ± 13.6
307.6 ± 47.1
295.0 ± 37.8
394.6 ± 74.6
419.4 ± 40.6
Lytechinus pictus 4
5
6
7
8
10
11
13
93.1 ± 2.3
50.3 ± 5.1
121.3 ± 1.4
27.1 ± 1.3
10.3 ± 4.0
34.1 ± 2.6
54.1 ± 2.6
87.8

105
Figure 16. S. purpuratus. A) Algal ingestion rates through development. Each bar
represents the mean of 3-5 experimental replicates ± SE mean. B) Gross protein
growth efficiencies through development. Each bar represents the gross protein
growth efficiency (%) ± SE of larvae at the age shown. The dotted line represents
the average gross protein growth efficiency of all three measurements.
6 8 13
Gross Growth Efficiency (%)
0
10
20
30
40
50
60
70
B.
Age (Days)
A.
Age (Days)
Ingestion Rates (algal cells larva
-1
hr
-1
)
0
20
40
60
80
100
120
6 8 13

106
larvae of the temperate asteroid, Asterina miniata. Clearance rates increased with
age in the larvae of this species, ranging from 4.5 ± 0.8 (10-day old larva) to 14.0 ±
1.2 (26-day old larva)μl larva
-1
hr
-1
the three cultures. The ingestion rates ranged
from 74.6 ± 6.8 to 419.4 ± 40.6 algal cells hr
-1
larva
-1
, respectively.
Ingestion rates were also measured in three Antarctic species. The larvae of
Sterechinus neumayeri had average clearance rates ranging from 0.4 ± 0.1 (44-day-
old larva fed 30 cells Dunaliella tertiolecta μl
-1
) to 1.0 ± 0.2 μl larva
-1
hour
-1
(26-
day-old larva fed 15 cells μl
-1
) which translated into average ingestion rates ranging
from 12.9 ± 2.9 to 29.2 ± 5.3 algal cells larva
-1
hr
-1
with an overall average of 20.9 ±
2.3 algal cells larva
-1
hr
-1
(Figure 17A). The larvae of Odontaster validus (fed 30
cells D. tertiolecta μl
-1
) had clearance rates that ranged from 0.5 ± 0.1 to 1.6 ± 0.1 μl
larva
-1
hr
-1
, with ingestion rates of 7.1 ± 1.7 to 25.6 ± 1.0 algal cells larva
-1
hr
-1

(overall average of 6.9 ± 1.1 algal cells larva
-1
hr
-1
) (Figure 18A). The larvae of
Odontaster meriodionalis had similar clearance rates to O. validus with values
ranging from 0.5 ± 0.04 to 0.6 ± 0.2 μl larva
-1
hr
-1
, translating into ingestion rates
that ranged from 6.2 ± 0.4 to 11.0 ± 2.6 algal cells larva
-1
hr
-1
(overall average of 8.1
± 1.3 algal cells larva
-1
hr
-1
) (Figure 19A).
Protein content
In the temperate species, protein content increased non-linearly with age
(ANOVA test for non-linearity, Total N = 88, P<0.05 in all three species). In order
to calculate a protein growth rate that could be used in protein growth efficiency

107
Figure 17. Sterechinus neumayeri. Algal ingestion rates through development.
Each bar represents the mean of 3-5 experimental replicates ± SE mean. White bars
represent measurements from cultures fed 15 algal cells μl
-1
. Gray bars represent
measurements from cultures fed 30 algal cells μl
-1
. B) Gross protein growth
efficiencies through time. Each bar represents the gross protein growth efficiency
(%) ± SE of larvae at the age shown. The dotted line represents the average gross
protein growth efficiency of all measurements excluding measurements from Days
48 and 52.

108
Figure 17.
Gross Protein Growth Efficiency (%)
0
20
40
60
80
100
Age (days)
A.
B.
26 31 33 44 48 48 52 57
26 31 33 44 48 48 52 57
Age (days)
163% 134%
Ingestion Rate (algal cells larva
-1
hr
-1
)
0
5
10
15
20
25
30
35

109
Figure 18. Odontaster validus. A) Algal ingestion rates through development.
Each bar represents the mean of 3-5 experimental replicates ± SE mean. B) Gross
protein growth efficiencies through development. Each bar represents the gross
protein growth efficiency (%) ± SE of larvae at the age shown. The dotted line
represents the average gross protein growth efficiency of all measurements excluding
Days 42 and 72.
34 42 47 60 61 72 95
Ingestion Rate (algal cells hr
-1
ind
-1
)
0
5
10
15
20
25
30
Age (Days)
34 42 47 60 61 72 95
Gross protein Growth Efficiency (%)
0
20
40
60
80
100
174% 110%
A.
B.

110
measurements, the log-transformed total protein content was plotted against age in
days. Larvae of the purple sea urchin, S. purpuratus, had similar growth rates
between replicates (ANOVA, P>0.05), so an overall protein growth rate was
calculated as 9.2 ± 0.4 ng protein day
-1
larva
-1
. The average protein and lipid
contents of the Rhodomonas spp. used in these cultures were 30 ± 0.3 pg protein
algal cell
-1
and 16.8 ± 4.7 pg lipid algal cell
-1
. The larvae of the white sea urchin, L.
pictus, had significantly different growth rates between replicates: 18.4 ± 2.3 and 9.6
± 1.6 ng protein day
-1
larva
-1
in larvae from Replicate A and B, respectively
(ANOVA, P<0.05). The average protein and lipid contents of the Rhodomonas spp.
used in these cultures were 41.3 ± 0.04 pg protein algal cell
-1
and 21.4 ±4.4 pg lipid
algal cell
-1
. Larvae of the bat star, Asterina miniata, had similar protein growth rates
in all three replicates, and were combined for an overall protein growth rate of 43.7 ±
3.2 pg protein day
-1
larva
-1
. The average protein and lipid contents of the
Rhodomonas sp. used in these cultures were 48.0 ± 4.1 pg protein algal cell
-1
and
12.1 ± 1.0 pg lipid algal cell
-1
. Total protein content was also measured in all
Antarctic species studied. Protein growth over the period of development studied
(60-100 days) was linear in nature (ANOVA test for non-linearity, Total N = 34,
P>0.05), and therefore total protein content values did to have to be log-transformed.
The overall protein growth rate for the echinoid S. neumayeri was 8.2 ± 1.8 ng
protein day
-1
larva
-1
. The average protein and lipid contents of the Dunaliella
tertiolecta used in these cultures were 20.0 ± 0.10 pg protein algal cell
-1
and 11.8 ±

111
Figure 19. Odontaster meridionalis. A) Algal ingestion rates through development.
Each bar represents the mean of 3-5 experimental replicates ± SE mean. B) Gross
protein growth efficiencies through development. Each bar represents the gross
protein growth efficiency (%) ± SE of larvae at the age shown. The dotted line
represents the average gross protein growth efficiency of all measurements.
49 62 72
Ingestion Rate (algal cells hr
-1
larva
-1
)
0
5
10
15
20
Age (days)
49 62 72
Gross Protein Growth Efficiency (%)
0
20
40
60
80
100
A.
B.

112
4.2 pg lipid algal cell
-1
. The asteroid O. validus had an overall protein growth rate of
8.7 ± 2.0 ng protein day
-1
larva
-1
. The average protein and lipid contents of the D.
tertiolecta used in these cultures were 26.0 ± 1.1 pg protein algal cell
-1
and 12.1 ± 1.2
pg lipid algal cell
-1
. The other asteroid species studied, O. meridionalis, had a much
lower protein growth rate of 2.08 ± 0.22 ng protein day
-1
larva
-1
(data from D.
Ginsburg, 2006). The average protein and lipid contents of the D. tertiolecta used in
these cultures were 20.0 ± 2.0 pg protein algal cell
-1
and 17.8 ±6.1 pg lipid algal
cell
-1
.
Gross protein growth efficiency
Gross protein growth efficiencies were calculated for all species at each time
point using the respective ingestion rates, protein growth rates, and average protein
content of the algal cultures used for each species’ culture. In conjunction with
increases in ingestion rates through development, gross protein growth efficiencies
decreased over time for S. purpuratus, (e.g. 34% for 6-day-old larvae and 16% for
13-day-old larvae), with an average gross protein growth efficiency of 27.9 ± 7.7%
(Figure 16B). Larvae of L. pictus had an average gross protein growth efficiency of
23.4 ± 3.6%, while the larvae of A. miniata had an average gross protein growth
efficiency of 16.7 ± 2.3% (Figure 20A and 20B).
All three Antarctic species demonstrated higher gross protein growth
efficiencies than the temperate species studied. The average gross protein growth
efficiency in the larvae of S. neumayeri was 93.2±13.1% (Figure 18B). The two
extremely high protein growth efficiencies (48 days-163% and 52 days-134%)
113
Figure 20. Gross protein growth efficiencies. A) Lytechinus pictus. Gross protein
growth efficiencies through growth. Each bar represents the gross protein growth
efficiency (%) ± SE of larvae at the age shown. Gray bars represent Replicate
Culture A, white bars represent Replicate Culture B. The dotted line represents the
average gross protein growth efficiency of all measurements. B) Asterina miniata.
Gross protein growth efficiencies through development. Each bar represents the
gross protein growth efficiency (%) ± SE of larvae at the age shown. Gray bars
represent Replicate Culture A, white bars represent Replicate Culture B and Black
bars represent Replicate Culture C. The dotted line represents the average gross
protein growth efficiency of all three measurements.

114
Figure 20.
Age (Days)
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Gross Protein Growth Efficiency (%)
0
10
20
30
40
50
B.
Age (Days)
4 5 6 7 10 11 13
Gross Protein Growth Efficiency (%)
0
10
20
30
40
50
A.

115
shown in the results are driven by the relatively low ingestion rates measured at
those time points (48 days: 10.7 ± 2.5 and 52 days: 12.9 ± 2.9 algal cells larva
-1
hr
-1
).
The average gross protein growth efficiency in larvae of S. neumayeri excluding
those values was 75.0 ± 5.5%. Larvae of O. validus had similarly high gross protein
growth efficiencies, with an average of 88.4 ± 13.1% over all time points measured
(Figure 19B). Again, the gross protein growth efficiency values exceeding 100% (42
days-174%, 72 days-110%) can be explained by relatively low ingestion rate rates
measured at those time points (42 days: 7.1 ± 1.7 and 72 days: 11.2 ± 2.4 algal cells
larva
-1
hr
-1
). The average gross protein growth efficiency in larvae of O. validus
excluding those values was 80.7 ± 8.7%. The larvae of O. meridionalis had lower
gross protein growth efficiencies than the other two Antarctic species studied with an
average of 58.9 ± 11.2% gross protein growth efficiency.
While gross protein growth efficiencies did not differ among temperate or
among polar species, they did differ significantly between temperate and polar
species (Figure 21; Antarctic: N = 18, ANOVA, P>0.05; Temperate: N = 19,
ANOVA, P>0.05). There was however, a significant difference between the gross
protein growth efficiencies between all temperate and all polar species (Figure 21;
Student’s t-test, N = 33, P<0.05). The average gross protein growth efficiency of
Antarctic larvae was ca. 80%. Conversely, the temperate species had a relatively
low average gross protein growth efficiency of ca. 25%. This is an approximately 3-
fold difference in gross protein growth efficiencies between larvae from the two
habitats.
116
Figure 21. Average gross protein growth efficiencies of all six species studied.
Each bar represents the mean gross protein growth efficiencies of that species ± SE
mean. The solid line represents the average gross protein growth efficiency of all
temperate species combined and the dotted line represents the average gross protein
growth efficiency of all polar species combined.
Gross Protein Growth Efficiency (%)
0
20
40
60
80
100
L. pictus A. miniata S.purpuratus S.neumayeri O. validus O. merdionalis

117
Discussion
In this study, gross protein growth efficiency was calculated in the larvae of
three Antarctic echinoderm species through measurements of the total amount of
protein the larvae accrued as biomass (i.e. total protein growth) and the amount of
protein consumed by feeding larvae (via measuring the ingestion rates of algae).
These same measurements were also made in the larvae of three temperate,
echinoderm species. The degree of efficiency with which larvae were able to retain
the protein they ingested was compared between the two groups, and the Antarctic
larvae were found to have a much higher efficiency in protein retention than
temperate larvae.
There are a variety of methods to measure the clearance and ingestion rates in
larvae, including observations of individual larvae (Strathmann, 1971; Hart, 1991;
Ayukai, 1994) or using the natural fluorescence inherent in phyto-pigments to
measure the presence of algae in the gut and algal depletion (Gerdes, 1983; Sprung,
1984; Ishii, 1990; Pace et al., 2006). In this study, we used the change in the amount
of Chl-a present in an experimental chamber before and after incubation with
feeding larvae (measured through the difference in fluorescence). This method has
also been successfully employed in grazing rate experiments of the Antarctic krill,
Euphausia superba (Haberman et al., 2003). End-point measurements could be
made with this method, which allowed us to conduct experiments with high
replication within each experiment. Starving the larvae before each feeding
experiment maximized clearance rates- a protocol commonly used in zooplankton
118
feeding studies (Rivkin et al., 1986; Ayukai, 1994; Okaji et al., 1997; Haberman et
al., 2003; Graeve et al., 2005). Cross-calibration was conducted several times in
different species between actual hemacytometer counts of algal depletion and Chl-a
measurements (e.g. Figure 15A). These measurements showed no significant
difference in measured ingestion rates between the two methods (4 experiments:
Total N = 43, Student’s t-test on all experiments, P>0.05), which validated the use of
Chl-a as a method of determining ingestion rates in these echinoderm larvae.
Furthermore, actual microscopic observations of feeding were conducted on larvae
and resulted in ingestion rates consistent with the Chl-a measurements (17.8 ± 3.7
cells larva
-1
hr
-1
vs. 14.4 ± 1.6 cells larva
-1
hr
-1
, respectively; Student’s t-test,
P>0.05). Additionally, clearance and the resulting ingestion rates obtained in this
study were in agreement with previously published values for both Antarctic and
temperate echinoderm larvae [Pearse et al., 1991: Antarctic asteroid bipinnaria
larvae-ca. 11 algal cells larva
-1
hr
-1
; Strathmann, 1971: Asteroid bipinnaria larvae-ca.
400 algal cells larva
-1
hr
-1
, four-armed plutei larvae-ca. 30 algal cells larva
-1
hr
-1
;
Hart, 1991: six-armed plutei larvae-ca. 120 algal cells larva
-1
hr
-1
; Ayukai, 1994:
(with Q
10
correction, late bipinnaria larvae- ca. 444 algal cells larva
-1

hr
-1
)].
An issue that occurs with the culture of any planktotrophic, marine
invertebrate larva is which algal species will result in larval growth and the potential
development to metamorphosis. The algal species that are used to culture larvae
successfully in the laboratory may not be the species that they would eat in their
119
natural environment. This is especially true in studies of Antarctic marine
invertebrate larvae. In this study, the Antarctic echinoderm larvae were fed either
Dunaliella tertiolecta, or Rhodomonas spp., both of which are not endemic to
McMurdo Sound, or polar waters in general. These species were used in accordance
with the protocols of previous studies on these organisms, in which both species
have been used successfully in the culture of Antarctic echinoderm larvae (Bosch et
al., 1987). Due to this and the unavailability of pure cultures of Antarctic algal
species, D. tertiolecta and Rhodomonas spp. were used. While these species have
repeatedly been shown to be and adequate food supply for Antarctic larvae (Bosch et
al., 1987; Shilling and Manahan, 1994; Marsh et al., 1999), we do not know how it
compares in quality to the Antarctic algae that these larvae will actually encounter
during their planktonic period. This raises the issue of how differing food quality (in
terms of lipid and protein content) may have affected the gross protein growth
efficiencies measured in the Antarctic echinoderm larvae in this study. Both protein
and lipid content were measured in the two species of algae used in this study.
Individual algal cells of Dunaliella tertiolecta were comprised of an average of about
60% protein and 40% lipid. The individual cells of Rhodomonas spp. were
comprised of an average of about 70% protein and 30% lipid. The larvae of both
temperate and Antarctic echinoderms maintained an average protein content of 60%
and lipid content of 40% regardless of the species of algae they were fed (i.e.
Dunaliella tertiolecta or Rhodomonas spp.). The average protein to lipid ratios were
also consistent between the temperate and Antarctic asteroids, maintaining a
120
biochemical composition of about 70% protein and 30% lipid. This ratio was
maintained regardless of the algae they were fed.
The fact that the biochemical composition of the larvae differs from that of
their algal food is not unusual. In a study on the larvae of the sand dollar,
Dendraster excentricus, Shiopu et al. (2006) found that there were inconsistencies
between the fatty acid composition of the algae that was fed to the larvae and of the
larvae themselves. The larvae were able to take components of the algae they ingest
convert it into the fatty acids they need. In fact, it was found that algae that did not
have the essential fatty acids (EFA) that the larvae needed were the best food sources
because the larvae were able to convert the components of that algae into the EFAs.
The inconsistencies between the biochemical composition of the algae that were
given to the larvae and the larvae themselves in this study agrees with these results.
What this possibly means is that while some foods may be better than others for
obtaining a faster growth or development rate, the biochemistry of the larva makes it
possible for the for it to get what it needs from the food it ingests. These larvae are
geared towards getting as much protein as possible from the food they ingest,
possibly through a number of different biochemical pathways. Therefore, the
regardless of the food they are given, they will demonstrate high protein growth
efficiencies.
Ingestion rates measured for the larvae of the Antarctic sea urchin,
Sterechinus neumayeri, averaged around 20 cells Dunaliella tertiolecta ingested
larva
-1
hr
-1
, and did not change significantly throughout the duration of the culture
121
(Figure 18A, ANOVA, P>0.05). Antarctic echinoderm larvae develop at a much
slower rate than their temperate counterparts, with larval stages that may last up to
several months to a year compared to a 1-2 months in temperate species (Bosch et
al., 1987; Clarke, 1992; Shilling and Manahan, 1994; Stanwell-Smith and Peck,
1998). Our experimental time period for the larval cultures of Antarctic species used
here ranged from 60-90 days, (with measurable feeding occurring in all species at
around 20-25 days of age). The total protein content in larvae of the Antarctic
species during this time increased between 43 (O. meridionalis)-125% (S.
neumayeri). Total protein content increases of 325 (S. purpuratus)-850 % (A.
miniata) occurred within the duration of the cultures in larvae of the temperate
species. The relatively small change in size in terms of protein content in the
Antarctic larvae may explain the lack of increases in ingestion rates throughout the
experimental time period. The ingestion rates measured for larvae of the two
asteroids, Odontaster validus and Odontaster meridionalis were on average lower
than in the larvae of S. neumayeri, (16 and 8 cells D. tertiolecta ingested larva
-1
hr
-1
,
respectively; Figure 19A & 20A). The protein-specific ingestion rates, however,
were not significantly different between the two groups (Asteroids: 0.02 ± 0.003
algal cells larva
-1
hr
-1
ng protein
-1
; Echinoid: 0.09 ± 0.04 algal cells larva
-1
hr
-1
ng
protein
-1
; Student’s t-test, N = 33, P>0.05). The values obtained for larvae of O.
validus agree with previously published values of about 17 cells D. tertiolecta larva
-1

hr
-1
(Pearse et al., 1991). The ingestion rates of larvae living in McMurdo Sound
122
(–1.5 °C) are lower than those measured in temperate species (15ºC). Some
physiological processes, such as metabolism, suspension feeding or growth, are
much lower in organisms that live in cold waters (Clarke, 1983; Peck, 1989; Shilling
and Manahan, 1994; Ahn and Shim, 1998; Marsh and Manahan, 1999; Peck, 2002;
Heilmayer and Brey, 2003; Heilmayer et al., 2004)
This study compared the gross protein growth efficiencies in planktotrophic,
echinoderm larvae from both Antarctic and temperate waters. A previous study by
Marsh et al. (2001) conducted with embryos and non-growing larvae of the Antarctic
sea urchin Sterechinus neumayeri revealed that they are able to synthesize protein at
similar rates as temperate urchin larvae, but have low metabolic rates, resulting in a
very low metabolic cost of protein synthesis (0.45 J mg protein synthesized
-1
). It is
thought that this unique physiology allows these larvae to survive for several months
in the food-limited environment of the Southern Ocean. The low cost of protein
synthesis allows larvae to synthesize proteins at a rate higher than necessary for
development and maintenance of cellular processes, without depleting their
endogenous resources before exogenous nutrients become available to them.
This study led us to hypothesize that once exogenous nutrients (i.e.
monocellular algae) were present in the water column during the brief phytoplankton
bloom that occurs during austral summer in McMurdo Sound (Rivkin, 1991), these
planktotrophic, echinoderm larvae would be able to turn ingested protein into protein
biomass very efficiently. This finding would be important ecologically, because it
might help reconcile the apparent mismatch of larval production with peak
123
phytoplankton production in the Southern Ocean. Cushing (1975, 1990) put forth an
explanation for variation in recruitment called the ‘match/mismatch hypothesis’.
According to this hypothesis, maximum recruitment would occur if adults spawned
their gametes to produce feeding larval stages during a time that coincided with peak
phytoplankton blooms. In the Antarctic echinoderms studied here, the spawning
season is mismatched from peak phytoplankton production, which according to the
hypothesis would result in poor recruitment of these species. While there may be
variation in recruitment from year to year, the species in this study are three of the
most abundant benthic invertebrate species in McMurdo Sound (Pearse et al., 1991).
Reproductive seasons for many Antarctic benthic invertebrates occur during
austral winter and early in austral summer (Pearse et al., 1991; Stanwell-Smith et al.,
1999; Pearse and Bosch, 2002). There are many possible reasons as to why this is a
common reproductive season amongst these organisms despite the mismatch from
the phytoplankton bloom. Embryos of the sea star, Odontaster meridionalis, suffer
increased mortality than embryos of O. validus and S. neumayeri when exposed to
even slightly higher temperatures than experienced during winter (-1.8 °C), which
may be why the adults need to spawn during the winter months (Stanwell-Smith et
al., 1998). Predation may be less of a threat to embryos and larvae during the winter
months, allowing larvae a higher chance of survival in the water column (Stanwell-
Smith et al., 1998). The changes in photoperiod that occur between austral winter
and summer may be a cue that allows adult asteroids to be more synchronous when
spawning mature gametes (Pearse & Bosch, 2002). While these are all feasible
124
explanations for timing of the reproductive seasons in Antarctic benthic
invertebrates, it is unknown whether they are the actual reasons for the mismatch
between spawning seasons and phytoplankton production in animals (Stanwell-
Smith et al., 1998).
The explanation for this mismatch may not actually lie within the adult
populations, but instead within the larvae themselves. An increase in the gross
protein growth efficiency would allow larvae to take greater advantage of a short
period of phytoplankton availability. It is feasible that larvae with high protein
growth efficiencies would be able to accrue enough protein and biomass to
metamorphose successfully during the brief period of time that algae were present in
the water column. In this study, the larvae of the three Antarctic echinoderm species
studies accumulated protein at average gross efficiencies of 60% (O. meridionalis),
81% (O. validus) and 75% (S. neumayeri). Gross protein growth efficiency was
measured using the same methods in the larvae of three temperate echinoderm
species, resulting in average gross protein growth efficiencies of 30% (S. purpuratus
and L. pictus, respectively) and 18% (A. miniata) (Figure 7). Comparing the means
of these ranges results in an almost three-fold difference between gross protein
growth efficiency between animals from the two environments.
The high growth efficiencies reported here are theoretically possible. Callow
(1977) calculated the minimum costs expended during the processes necessary for
growth and maintenance and determined that the best possible net growth efficiency
that can be achieved by any growing heterotroph is between 70-80%. Assimilation
125
efficiencies can reach >80% and sometimes close to 100%. High net growth and
assimilation efficiencies can result in a gross growth efficiency of 60-80% (Porter,
1982; Verity, 1985). The larvae of the Antarctic krill, Euphasia superba have been
shown to have carbon assimilation efficiencies of 72-90%, which is thought to allow
them to grow rapidly during short periods of high phytoplankton concentrations
(Meyer et al., 2003). Values of gross growth efficiencies (based on carbon content)
reported for protozoan and metazoan zooplankters (dinoflagellates, ciliates,
copepods, etc.) living in temperate waters range from 24-32% (Straille, 1997). .
Growth efficiencies measured in Antarctic protozoan species (based on either carbon
or volume) are similar to temperate species with a range from about 40-50% (J.
Rose, per. comm.). Using data from a previous study on the developmental
energetics of the larvae of Sterechinus neumayeri (Marsh et al., 1999) and data
measured on larvae from the same species in this study, we were able to calculate the
approximate carbon growth efficiencies in the larvae of Sterechinus neumayeri.
According to Figure 3 of Marsh et al., 1999, the total elemental carbon and nitrogen
increased at a rate of ca. 13.8 ng C+N larva
-1
day
-1
. As the ratio of C:N in feeding
larvae was found to be 3.8 (Marsh et al., 1999, Figure 2), the total organic carbon
increased at a rate of 10.9 ng C larva
-1
day
-1
. The larvae in this study were fed a 1:1
ratio of the algal species Dunaliella tertiolecta and Rhodomonas lens. The
approximate biochemical composition of a phytoplankton cell is 45% protein, 29%
carbohydrate and 4% lipid (Rios et al., 1998). Using the protein and lipid contents
measured in this study, we were able to calculate the approximate amount of
126
carbohydrates in each cell of these algal species (i.e. the amount of carbohydrate in
an algal cell is equal to 64% of the total protein content). The percentage of protein,
carbohydrate and lipid that is made up of carbon is 53%, 44% and 78%, respectively
(Gnaiger and Bitterlich, 1984). Using these percentages and the total amounts of
protein, carbohydrate and lipid, we were able to calculate the total organic carbon
content in an individual cell of D. tertiolecta (29 pg cell
-1
) and R. lens (46 pg cell
-1
).
Since the larvae in marsh et al., 1999 were fed equal amounts of both algal species,
we can assume that the mean carbon content of the algae consumed was 38 pg C
cell
-1
. Using an average algal ingestion rate by the larvae of S. neumayeri of 22.7
algal cells larva
-1
hour
-1
(this study), the total amount of carbon ingested by a larva
per day is ca. 21 ng C. Therefore, the carbon growth efficiency of the larvae of S.
neumayeri is 53% (10.9 ng C larva
-1
day
-1
/21 ng C ingested larva
-1
day
-1
). It makes
sense that this number is not as high as the gross protein growth efficiencies, because
it includes the efficiency of lipid and carbohydrate turnover. According to Marsh et
al., 1999, the elemental carbon: nitrogen ratios in Sterechinus neumayeri decreased
from 5.0 in eggs to 3.8 in both fed and unfed larvae, indicating that lipid reserves
were being used as the primary energy substrate. This would explain the lower
carbon growth efficiencies calculated here. Regardless, the estimated carbon growth
efficiency is higher than the gross protein growth efficiencies calculated in the larvae
of temperate echinoderm species. These results in would also indicate that protein
ingested by larvae was not used as a main energy substrate, but instead retained as
protein biomass. The combination of high gross protein growth efficiencies
127
measured and lower carbon growth efficiencies calculated in the larvae of these
Antarctic echinoderms agree with this data, in that they indicate the use of substrates
other than protein as fuel for their development.
Studies on juvenile and adult fish species have shown that in growing
organisms, increased protein growth efficiencies are correlated with increased
retention of protein (Carter et al., 1993; Carter et al., 1998; Dolby et al., 2004).
These studies also measured rates of protein synthesis and degradation in
conjunction with gross protein growth efficiency (protein growth/protein
consumption), and found that there was a negative relationship between rates of
protein synthesis and degradation (protein turnover) and protein growth efficiencies.
In these juvenile and adult fish, lower rates of protein turnover were correlated
significantly with higher protein growth efficiencies. The present study focuses on
one objective of a larger study that is looking at the rates and metabolic costs of
protein synthesis in growing, larval stages of these Antarctic echinoderms, not only
on a whole-organism level, but also through in vitro cell-free systems (other
objectives studied by D.A. Pace, M. Moore, D. Ginsburg). These studies can
possibly elucidate any relationships that may occur between rates of protein
synthesis and the extremely high protein retention efficiencies of growing Antarctic
echinoderm larvae.
The results of this study may offer an additional explanation of how Antarctic
planktotrophic larvae are able to survive up to several months with little or no
particulate nutrients. These larvae also have the ability to take up dissolved organic
128
material (DOM) from the surrounding waters, which may contribute to fulfilling the
metabolic and structural needs of early larval development (Manahan, 1990). In
addition, Antarctic echinoderm larvae have low metabolic rates (relative to temperate
echinoderm larvae), which allows their endogenous protein and lipid stores to be
used at a low rate (Shilling and Manahan, 1994). Embryos and early, non-growing
larval stages of the Antarctic sea urchin, S. neumayeri, are able to synthesize proteins
very efficiently (Marsh et al., 2001). The timing of reproduction in adults may be
advantageous to larvae by making them poised to take the greatest advantage of
available nutrients. Antarctic larvae may be able to survive and develop to
metamorphosis successfully, despite a lack of particulate nutrients during a large
portion of their larval lives, by having the ability to convert any protein that is
ingested almost completely into protein biomass through having extremely high
protein growth efficiencies. The ability of these larvae to retain protein efficiently
could have positive effects on their subsequent metamorphosis and juvenile survival.
It has been shown that exposure to a low food environment during larval stages, can
lead to a delay in the development of the juvenile rudiment (Hart and Strathmann,
1994; Fenaux et al., 1994), a decreased chance of metamorphic success (Eckert,
1995), or a smaller size at metamorphosis (Hart and Strathmann, 1994; Eckert,
1995). While there may be variation in recruitment success from year to year in
these Antarctic echinoderm species, their ubiquitous abundance in the Southern
Ocean attests to the fact that an adequate proportion of the larvae are able to
consistently and successfully metamorphose and grow as juveniles, despite a long
129
period without particulate nutrients. The combination of using their endogenous
nutrients and DOM efficiently and having a high protein growth efficiency once
algal nutrients are available may offer an explanation of how these larvae are able to
survive for long periods of time without particulate nutrients and retain a sufficient
amount of ingested protein to grow and develop during the brief phytoplankton
bloom.

130
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136
Chapter 4: O:N atomic ratios in Antarctic and temperate planktotrophic
echinoderm larvae
Chapter 4 Abstract
The adults of two abundant Antarctic echinoderms, Sterechinus neumayeri
and Odontaster validus produce embryos and feeding larvae several months prior to
the main phytoplankton bloom that occurs in McMurdo Sound, Antarctica. There
are many factors that allow these larvae are able to survive for long periods of time
without exogenous nutrients, including low metabolic rates, and the ability to take up
dissolved organic material (DOM) from the surrounding waters. Once particulate
nutrients (i.e. monocellular algae) become available, it is unknown what energy
substrate (namely protein or lipid) is the primary fuel for their growth and
development. The energy substrate an organism is catabolizing can be determined
by calculating the O:N atomic ratio from the measurement of oxygen consumption
and nitrogen excretion rates. In this study, the biochemical composition (total
protein and lipid content) of pre-feeding eggs and embryos and the feeding larval
stages was measured in the Antarctic echinoderms S. neumayeri and O. validus and
the temperate echinoderms Lytechinus pictus and Asterina miniata. In addition, the
O:N atomic ratios were calculated from measured oxygen consumption and nitrogen
excretion rates in all four species. The eggs and pre-feeding embryos of both the
Antarctic and temperate echinoids (S. neumayeri and L. pictus) embryos and larvae
were comprised of approximately 50% protein and 50% lipid. The eggs and pre-
feeding embryos of the Antarctic and temperate asteroid species (O. validus and A.
137
miniata) were made up of around 65% protein and 35% lipid. The larvae of both of
the temperate species had increased amounts of protein and decreased amounts of
lipids. The storage lipids (triacylglycerols) were depleted during embryogenesis, and
were never built up again during their larval stages. This pattern was also seen in the
Antarctic echinoid, S. neumayeri, but the larvae of the Antarctic asteroid, O. validus
were able to build up lipid stores in the form of triacylglycerol during their larval
period. While the total protein content in these larvae increased over time, the
percent composition of protein decreased. The O:N ratios reflected these changes in
biochemical composition in all species studied. Both temperate species had O:N
ratios that indicated the use of protein as the primary energy substrate during larval
development. The early staged embryos of S. neumayeri and O. validus had high
O:N ratios, indicating lipid catabolism. As the embryos reached a larval stage,
however, the O:N ratios decreased, indicating a switch to protein catabolism as the
lipids in the eggs were depleted. The O:N ratios in the larvae of S. neumayeri
fluctuated between values that indicated switched between protein and lipid
catabolism and a combination of both protein and lipid as energy substrates. The
larvae of O. validus had O:N ratios that remained high, indicating the primary use of
lipid. The different patterns of O:N ratios in the embryos and larvae of the Antarctic
species compared to the temperate species highlight the differences in feeding
environments that these animals see during their developmental time period.
Antarctic larvae are able to use the energy sources in the food they ingest differently
138
than temperate larvae, which may allow them to take greater advantage of particulate
food once it becomes available to them.

Introduction
The waters of the Southern Ocean characterized by low temperatures (-1.8ºC
to 2.0ºC) and highly seasonal patterns of primary production (Clarke, 1988). There
is a phytoplankton bloom that generally occurs between November and January
(Clarke, 1988). It follows the retreating ice edge, and thus is able to expand into
more sheltered areas such as McMurdo Sound (Smith and Nelson, 1985). The
marine organisms that inhabit Antarctic waters generally have slower growth
(Clarke, 1983) and developmental (Stanwell-Smith and Peck, 1998) rates than
temperate or tropical species. While the low temperature of the water may account
for the slower developmental rates, particularly in non-feeding larval forms, the slow
growth rates can mostly be attributed to the amount of food available to Antarctic
marine organisms (Clarke, 1988). Many Antarctic species have developed over
wintering strategies to cope with the time period where algal and particulate nutrients
are in low supply. For instance, adults of the Antarctic krill, Euphasia superba, have
been known to use lipid reserves during winter months, reduce their metabolism, use
alternative food sources (benthopelagic feeding) and may actually use protein to a
degree that there is biomass shrinkage in the winter months (Meyer et al., 2002).
Larvae of this species are required to use different over wintering strategies as they
have very small lipid reserves and are unable to exploit benthopelagic habitats for
139
alternative food sources (Meyer and Oettl, 2005). Adults of the Antarctic sea urchin,
Sterechinus neumayeri, reduce their metabolic rates and use lipid and carbohydrate
reserves in addition to protein reserves during the winter season (Brockington and
Peck, 2001). Adults of the Antarctic limpet, on the other hand, utilize proteins only
in periods of nutritional stress, preferentially using lipids otherwise (Fraser et al.,
2002). These examples point out the many different feeding ecologies and energetic
strategies used by Antarctic organisms to cope with long periods of low nutrient
availability.
There are many species of Antarctic marine invertebrates that have complex
life histories including pelagic, feeding larvae (Pearse et al., 1991). Amongst these
species are the echinoid, Sterechinus neumayeri and the asteroid, Odontaster validus,
which are two of the most abundant species found in McMurdo Sound, Antarctica
and are ubiquitously found in the Southern Ocean (Pearse et al., 1991). The adults of
the O. validus release their gametes into the water column during the austral winter
(June through September), while the adults of S. neumayeri spawn primarily in the
beginning of austral summer (October through December) (Pearse et al., 1991).
These spawning seasons produce larvae that reach a feeding stage (~20 days of age)
well before the primary phytoplankton bloom occurs. This apparent ‘mismatch’ of
these species to an abundant food supply raises the question of how the larvae are
able to survive long periods with little or no particulate algal nutrients.
Previous studies have shown that these larvae are able to utilize the maternal
reserves provided to them during this period of low nutrient availability. Low
140
metabolic rates in these larvae allow them to maintain sufficient levels of these
nutrient reserves (predominantly protein and lipid) to fuel development until
particulate food is available to them (Shilling and Manahan, 1994). In addition, it
has been shown that planktotrophic larvae are able to utilize alternative food sources
during periods of low nutrient availability, much like the Antarctic krill cited above.
Antarctic echinoderm larvae are able to take up dissolved organic material (DOM)
through their integument (Manahan, 1990), and have also been shown to have the
ability to ingest bacteria as an alternative food source (Rivkin et al., 1991).
It is not currently known how these larvae are utilizing their endogenous
reserves prior to being exposed to particulate algal nutrients, and what they use as
their primary energetic substrate once they are able to feed on algae in the water
column. As cited in the examples given previously, Antarctic organisms are able to
preferentially utilize either protein or lipid, depending on the nutrient environment
they are in, and also depending on that particular organisms’ physiological needs. A
common way to determine what substrate an organism is using as it’s primary energy
source is by calculating its atomic O:N ratio through measurements of oxygen
consumption and ammonia-N excretion rates. This has been found to be a useful
tool in the study of zooplankton feeding ecology. Mayzaud and Conover (1988)
calculated the O:N ratios for different zooplankters and showed that O:N ratios
between 3 and 16 indicate pure protein metabolism, and O:N ratios between 50 and
60 indicate the equal use of lipid and protein as metabolic substrates. O:N ratios
above 60 indicate pure lipid metabolism.
141
In this study, rates of oxygen consumption and ammonia-N excretion were
measured in the pre-feeding embryos and well-fed larvae of two Antarctic
echinoderm species, Odontaster validus and Sterechinus neumayeri and used to
calculate the O:N ratios through growth and development. These results were
compared to similar measurements performed on two temperate echinoderm species,
Lytechinus pictus and Asterina miniata.
Materials and Methods
Larval culturing
Temperate species-Asterina miniata and Lytechinus pictus

Marinus, Inc. (Long Beach, CA) collected adults of the echinoid species
Lytechinus pictus and asteroid species Asterina miniata off the Southern California
coast during their respective reproductive seasons. Adult sea urchins were induced
to spawn by intracoelomic injection of 0.5 M KCl; sea stars were similarly injected
with a 1 mM solution of the hormone, 1-methyladenine (1MA) in order to induce
spawning. Eggs from several females were inspected for quality and then pooled for
fertilization and the sperm from one male was used for fertilization.
Once a fertilization success of >90-95% was achieved, embryos were placed
into 20-l culturing vessels with 0.2 μm pore-size filtered seawater at a final density
of 10 embryos ml
-1
. Cultures were stirred constantly to keep embryos and larvae
suspended and evenly distributed. Once larvae reached a feeding, larval stage
(approximately 3-4 days of age), larvae of both species were fed the monocellular
algae, Rhodomonas salinas, at a concentration of 30 cells μl
-1
. The water of each
culture was changed every 3 days by gently siphoning the animals down onto mesh
142
sieves of sizes ranging from 20-80μm, depending on the stage of development. The
animals were enumerated and then placed into fresh 0.2μm pore-size filtered
seawater.
Polar species-Odontaster validus and Sterechinus neumayeri
Adult Odontaster validus and were collected from various sites along
McMurdo Sound, Antarctica between mid-August and October. Adult Sterechinus
neumayeri were also collected periodically from various sites in McMurdo Sound
between October and December. In both the asteroid and echinoid species, similar
protocols were followed regarding adult spawning and subsequent treatment of
gametes and embryos as in the temperate species outlined above. Fertilized
embryos were placed into 200-l culture containers filled with 0.2-μm pore-size
filtered seawater at a final density of 5 embryos ml
-1
. Culture temperature was
maintained at -1.5°C by immersing the culture vessels in large aquarium tanks with
constant ambient seawater flow from McMurdo Sound. Animals were kept in
suspension and the cultures were inspected twice daily to ensure no settling had
occurred. In both species, once a feeding, larval stage was reached (approximately
20 days of age), cultures were fed the monocellular algae, Dunaliella tertiolecta, at a
concentration of 15 cells μl
-1
. Culture water was changed every four days by
siphoning the larvae gently onto an 80μm sieve. After enumeration, larvae were
placed into fresh 0.2-μm pore-size filtered seawater.
143
Biochemical Analysis
A known amount of embryos and larvae were sampled at each developmental
time point and stored at -80°C for later biochemical analysis (total protein content,
total lipid and lipid classes). To measure total protein content, a modified Bradford
assay was used (Bradford, 1976 as modified by Jaeckle and Manahan, 1989).
Briefly, a known number of larvae were homogenized with a known volume of
deionized water. A standard curve was constructed using known concentrations of
Bovine Serum Albumin (Bio-Rad Laboratories). Coomassie Brilliant Blue G-250
(Bio-Rad Laboratories) was added to all of the tubes as a colorimetric reagent. The
absorbance of the standards and samples were read at 595 nm at least 20 minutes,
and no more than 40 minutes after adding the dye. Total protein content per
individual embryo or larvae was calculated as the mean of three replicates ± SE
mean.
Total lipid content and lipid classes were measured according to Moran and
Manahan, 2004. Samples were homogenized in a known volume of deionized water
and transferred to glass scintillation vials for lipid extraction. Samples were
extracted in 1:1:0.5 water/methanol/chloroform with an added stearyl alcohol
internal standard. Using a 1μl capillary tube, three replicate samples of extracted
lipid samples were placed onto thin-layer Chromarods (Iatron laboratories).
Developing the Chromarods in a 60:6:0.1 hexane/diethyl ether/formic acid mixture
separated lipid classes. After developing, the Chromarods were dried for 10 minutes
at 100ºC and then analyzed using an Iatroscan MK-5 flame ionization detector.
144
Resulting chromatograms were compared to standard lipid class chromatograms (L-
a-phosphatidylcholine (phospholipid), cholesterol, tripalmitin (triacylglycerol),
squalene, palmitic acid (free fatty acid), lauric acid palmityl ester (wax ester) and
stearyl alcohol (fatty alcohol internal standard). Total lipid content and the amounts
of specific lipid classes per individual embryo or larvae were calculated as the mean
of two to three replicates ± SE mean.
Ammonia Excretion
In order to determine whether embryos and larvae primarily utilize protein or
lipid to fuel development, ammonia excretion rates were measured. Using the
method of Solorzano as modified by Strickland and Parsons (1982), ammonia release
was measured in both non-feeding embryos and feeding larval stages. At each
developmental time point, animals were placed in a 20 ml glass scintillation vial at a
density of 3-4000 animals ml
-1
in ammonium-free 0.2-μm pore-size filtered seawater
(FSW). After stocking the experimental vial, an initial 1 ml sample was taken and
the remainder the animals were incubated at -1.5°C. Subsequent samples were taken
each hour from hours 2-7 of incubation. All samples and a standard curve were
processed immediately and absorbance readings were taken at 640 nm. Ammonia
excretion rates were attained from the slope of the resulting regression.
To account for any background chemical or biological ammonia production
or loss, a separate 20 ml scintillation vial was stocked with water that the animals
were in when sampled from the main culture (‘supernatant’) at the same ratio of
animals to FSW in the experimental chambers. During analysis, if there was a
145
significant increase or decrease in NH
3
in the supernatant during the experiment
(ANOVA, P<0.05), the slope of that regression was subtracted from the
experimental NH
3
excretion rate.
Rates of Oxygen Consumption
Metabolic rates were measured via oxygen consumption in conjunction with
NH
3
excretion measurements using the method as described in Marsh and Manahan,
1999. In brief, animals were placed into airtight micro-biological oxygen demand
vials (500-700μl) with oxygen-saturated 0.2-μm pore-size filtered seawater. The
number of animals varied with species and developmental stage, but was kept at a
density that would ensure no greater than a 20% drop in oxygen during the
experimental time period. The vials were incubated at -1.5°C for 5-7 hours. An end-
point sample was injected from each individual vials onto a Clark-type electrode and
the animals were enumerated. The experimental measurements were compared
against measurements of control vials with no animals and used to calculate the total
amount of oxygen consumed per vial. This number was plotted against the number
of animals in each vial and the slope of the final regression was the oxygen
consumption rate in pmol O
2
hour
-1
larva
-1
.
Results
Biochemical composition
Temperate species-Lytechinus pictus and Asterina miniata
The values of total protein and lipid and breakdown of lipid classes for all
species are seen in Table 3. The average percent biochemical compositions (total
146
Cholesterol
(ng ind
-1
)
9.7 ± 1.3
0
58.5
18.8

173.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.26 ± 0.20

40.1
27.5
15.9 ± 10.8
39.3
Triacylglycerol
(ng ind
-1
)
9.7 ± 1.3
0
58.5
18.8

173.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
66.4 ± 4.66

405.3
269.5 ± 167.2
258.5 ± 131.9
294.7 ± 178.4
Phospholipids
(ng ind
-1
)
29.8 ± 5.6
50.8
189.6
514.8

234.1
177.3 ± 70.6
240 ± 108.8
111.9 ± 6.8
388.4
248
103.9 ± 27.2
465.5 ± 37.5
81.5 ± 31.7
50.6 ± 10.9

506.2
435.6 ± 192.6
443.9 ± 191.2
294.7 ± 131.9
Total Lipid
(ng ind
-1
)
39.5 ± 6.9
59.5
253.4
580.7

452.6
186.1 ± 70.2
240 ± 108.8
111.9 ± 6.8
388.4
256.4
103.9 ± 27.2
465.5 ± 37.5
81.5 ± 31.7
119.3 ± 11.9

951.6
732.7 ± 329.1
718.2 ± 332.4
628.7 ± 329.4
Total Protein
(ng ind
-1
)
119.6 ± 1.6
296.6 ± 1.3
135.0 ± 4.8
187.6 ± 11.3

194.1 ± 5.7
245.3 ± 9.8
253.5 ± 8.6
285.3 ± 32.3
281.6 ± 8.7
215.6 ± 4.1
443.9 ± 89.1
611.2 ± 47.4
325.0 ± 11.9
215.9 ± 0.93

240.4 ± 11.4
245.3 ± 0.12
293.6 ± 4.2
256.3 ± 9.9
Age (Days)
0
15
21
28

36
39
46
49
51
56
72
78
0
18

34
40
42
56
Culture
Replicate
Non-feeding
Embryo

1
2
3
2
3
1
3
2
Non-feeding
Embryo

1
1
1
1
Table 3. Biochemical composition of embryos and larvae of Antarctic and temperate echinoderms
Species
Sterechinus
neumayeri
Odontaster
validus

147
Cholesterol
(ng ind
-1
)
2.4
0.0
4.7 ± 0.61
20.1

0.0
0.0
0.0
23.7
0.0
0.0
18.2 ± 9.8
11.1
6.4
0.0
0.0
0.0
0.0
4.2 ± 0.79
2.0 ± 0.11
5.9 ± 0.65
15.2 ± 2.0
Triacylglycerol
(ng ind
-1
)
40.7
0.0
0.0
0.0

0.0
0.0
0.0
41.2
0.0
0.0
0.0
0.0
9.0 ± 3.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Phospholipids
(ng ind
-1
)
107.8
64.2 ± 7.8
147.4 ± 4.2
385.5 ± 22.1

196.7 ± 36.8
50.0 ± 5.4
228.6 ± 70.2
148.9 ± 37.9
266.4 ± 18.8
387.9 ± 33.2
326.6 ± 30.0
440.5 ± 107.5
34.2 ± 4.7
61.7
114.3 ± 5.8
295.0
18.0 ± 6.0
30.9 ± 5.8
17.0 ± 1.8
113.9 ± 81.1
112.2 ± 14.6
Total Lipid
(ng ind
-1
)
150.9
64.2 ± 7.8
152.1 ± 3.6
405.6 ± 22.1

196.7 ± 36.8
50.0 ± 5.4
228.6 ± 70.2
213.8 ± 37.9
266.4 ± 18.8
387.9 ± 33.2
344.8 ± 39.1
451.6 ± 107.5
45.4 ± 9.5
61.7
114.3 ± 5.8
295.0
18.0 ± 6.0
35.1 ± 6.6
19.0 ± 1.7
119.8 ± 59.9
127.3 ± 16.5
Total Protein
(ng ind
-1
)
293.4 ± 46.4
430.4 ± 101.8
272.9 ± 55.6
381.9 ± 28.7
459.0 ± 7.2
436.8 ± 18.8
645.8 ± 18.0
490.8 ± 68.8
679.3 ± 112.6
800.7 ± 22.2
862.5 ± 13.3
943.9 ± 51.4
1348.7 ± 44.8
36.3 ± 2.5
51.3 ± 1.5
46.7 ± 8.3
48.4 ± 3.7
75.5 ± 0.9
146.0 ± 5.3
96.0 ± 6.2
142.7 ± 2.9
208.4 ± 6.0
Age (Days)
0
9
9
9
14
16
16
20
20
20
23
23
26
0
5
6
7
8
10
11
13
14
Culture
Replicate
Eggs
1
2
3
2
1
3
1
2
3
2
3
1
Eggs
1
1
2
1
2
1
2
1
Table 3 (continued). Biochemical composition of embryos and larvae of Antarctic and temperate
echinoderms
Species
Asterina
miniata
Lytechinus
pictus

148

Figure 22. Biochemical composition of the pre-feeding embryos (PF) and feeding
(F) larvae of Lytechinus pictus (Lp), Asterina miniata (Am), Sterechinus neumayeri
(Sn) and Odontaster validus (Ov). Each bar is divided into the average %
composition of protein and lipid classes. = Total protein (ng individual
-1
), =
Cholesterol, = Triacylglycerol, = Polar Lipid.

Lp PF Lp F Am PF Am F Sn PF Sn F Ov PF Ov F
% Composition
0
20
40
60
80
100

149
protein and lipid classes) in eggs, embryos (pre-feeding) and larvae (feeding) for all
species are shown in Figure 22. The eggs of the temperate echinoid Lytechinus
pictus were comprised of 44.4% protein and 55.6% lipid (36.3 ± 2.5 ng protein egg
-1
,
45.4 ± 9.5 ng lipid egg
-1
; a 1:1.3 protein: lipid ratio). The total lipid was made up of
mostly polar lipids, which constituted 75.3% of the total lipid (41.9% of the egg).
The remainder of the total lipid was made up of 19.8% triacylglycerols (TAG)
(11.0% of the egg) and 4.8% cholesterol (2.7% of the egg). Similar calculations
were made for feeding stages of the larvae of L. pictus (5-14 days in age) using the
mean percentages of total protein, total lipid and lipid classes at each developmental
time point. These calculations revealed that the feeding stages of the larvae of L.
pictus were comprised of an average of 56.2 ± 9.1% protein and 43.8 ± 9.1% lipid
[e.g. 5-day old larva: 51.3 ± 1.5 ng protein larva
-1
, 61.7 ng lipid larva
-1
(100% polar
lipid); a 1:1.2 protein: lipid ratio]. The total lipid in feeding larvae was made up of
95.1 ± 2.0% polar lipids (42.2 ± 9.3% of larva) and 4.9 ± 2.0% cholesterol (1.4 ±
0.59% of larva). Triacylglycerols were depleted and no longer present in feeding
larvae.
The eggs of the temperate asteroid, Asterina miniata were comprised of
66.0% protein and 34% lipid (293.4 ± 46.4 ng protein egg
-1
, 182.2 ± 81.7 ng lipid
egg
-1
, a 1.6:1 protein: lipid ratio). The total lipid was made up of 71.4% polar lipids
(24.3% of egg), 27% TAG (9.2% of egg) and 1.6% cholesterol (0.54% of egg).
Feeding larvae of A. miniata (9-26 days of age) were comprised of 72.5 ± 5.8%
protein and 27.5 ± 5.8% lipid [e.g. 16-day old larva: 436.8 ± 42.0 ng protein larva
-1
,
150
196.7 ± 36.8 ng lipid larva
-1
(100% polar lipid); a 2.2:1 protein: lipid ratio]. The
total lipid was comprised almost completely of polar lipids (95.8 ± 4.5%; 26.3 ±
5.7% of larva), and small amounts of triacylglycerol (1.75% of total lipid; 0.42% of
larva) and cholesterol (2.44 ± 1.77% of total lipid; 0.76 ± 0.52% of larva).
Polar species-Sterechinus neumayeri and Odontaster validus
The percent biochemical compositions of the pre-feeding embryo and feeding
larval stages in the polar species are also shown in Figure 22. The eggs and pre-
feeding embryo stages of the Antarctic echinoid Sterechinus neumayeri (0-28 days of
age) consisted of an average of 56.3 ± 13.8% protein and 43.6 ± 13.8% total lipid
[e.g. 21-day old embryo: 135.0 ± 4.8 ng protein embryo
-1
, 253.4 ng lipid embryo
-1

(74% polar lipid, 23% triacylglycerol and 2.1% cholesterol); a 1:1.9 protein: lipid
ratio]. The total lipid was comprised of 81.1 ± 3.5% polar lipid (35.7 ± 12.2% of
embryo), 12.7 ± 6.5% triacylglycerol (5.5 ± 2.9% of embryo), and 6.2 ± 3.3%
cholesterol (2.4 ± 1.32% of embryo). Feeding larval stages of S. neumayeri (36-78
days of age) consisted of 54.4 ± 9.4% protein 45.6 ± 9.4% lipid [e.g. 56-day old
larva: 215.6 ± 4.1 ng protein larva
-1
, 256.4 ng lipid larva
-1
(96.7% polar lipid, 3.3%
cholesterol); a 1:1.2 protein: lipid ratio]. Total lipid was comprised of 92.9 ± 9.7%
polar lipid (40.8 ± 7.4% of the larva), 3.4% triacylglycerol (4.7% of the larva) and
1.3% cholesterol (2.3% of larva).
The eggs and pre-feeding stages of the Antarctic asteroid, Odontaster validus
(0-18 days of age) were comprised of 72.2 ± 7.8% protein and 27.8 ± 7.8% lipid [e.g.
18-day old embryo: 215.9 ± 0.93 ng protein embryo
-1
, 443.3 ± 1.6 ng lipid embryo
-1

151
(42.4% polar lipid, 55.6% triacylglycerol and 1.9% cholesterol); a 1:2.6 protein: lipid
ratio]. The total lipid was comprised of 71.2 ± 28.8% polar lipid (17.6 ± 2.5% of
embryo), 27.8% triacylglycerol (9.9% of larva) and 0.95% cholesterol (0.34% of
larva). The average biochemical composition of feeding, larval stages of O. validus
(34-56 days of age) were much different than that seen in the embryo stages. Larvae
were comprised of 25.9 ± 2.2% protein and 74.1 ± 2.2% lipid [e.g. 56-day old larva:
256.3 ± 9.9 ng protein larva
-1
, 628.7± 329.4 ng lipid larva
-1
(46.9% polar lipid,
46.9% triacylglycerol and 6.3% cholesterol); a 1:1.6 protein: lipid ratio]. The total
lipid was comprised of 55.6 ± 3.1% polar lipids (41.2 ± 2.5% of larva), 40.1 ± 2.8%
triacylglycerol (30.2 ± 2.2% of larva) and 4.1 ± 0.86% cholesterol (3.1± 0.61% of
larva). Unlike the other species studied, the larvae of O. validus were able to store
lipids in the form of triacylglycerol.
Rates of Oxygen Consumption and Nitrogen Excretion
Temperate species
An example of the data set used to calculate the rate if ammonia excretion in
the larvae of the temperate echinoid, Lytechinus pictus, is shown in Figure 23A. The
rates of oxygen consumption and ammonia-N excretion of larvae of the temperate
echinoid, Lytechinus pictus, are plotted together in Figure 23B. Ammonia-N
excretion rates increased in conjunction with oxygen consumption rates over the
culture’s duration. Respiration rates increased non-linearly following the equation: y
= 4.4e
0.20x
. Ammonia-N excretion rates also increased non-linearly following the
equation: y = 0.70e
0.16x
.
152
Figure 23. Lytechinus pictus. A) Typical data set for nitrogen excretion experiment.
Each data point represents the amount of nitrogen in one experimental vial (see
Methods). The slope of the regression represents the amount of ammonia-N excreted
per larva per hour. B) Respiration rates and ammonia-N excretion rates of temperate
echinoderm larvae. Each data point represents the slope of the regression between
total oxygen consumed and the number of larvae ± SE slope. □ = Respiration Rate.
Each data point represents the slope between total ammonia excreted larva
-1
and time
(hours) ± SE slope. ● = ammonia-N excretion rate. The increase in respiration rates
(dashed line) follows the equation y = 4.4e
0.20x
, R
2
= 0.90. The increase in
ammonia-N excretion rates (solid line) follows the equation y = 0.70e
0.16x
, R
2
=
0.96.

153
Figure 23.
Incubation Time (Hours)
0 1 2 3 4 5 6 7
Nitrogen Excretion (pmol N larva
-1
)
0
2
4
6
8
10
12
14
16
18
A.
y = 1.9x + 4.6
R
2
= 0.99
Age (Days)
0 2 4 6 8 10 12 14 16 18
Respiration Rate (pmol O
2
larva
-1
hour
-1
)
0
20
40
60
80
100
120
140
160
180
NH
3
Excretion Rate (pmol larva
-1
hour
-1
)
0
2
4
6
8
10
12
14
B.

154
Figure 24. Asterina miniata. A) Typical data set for nitrogen excretion experiment.
Each data point represents the amount of nitrogen in one experimental vial (see
Methods). The slope of the regression represents the amount of ammonia-N excreted
per larva per hour. B) Respiration rates and ammonia-N excretion rates of temperate
echinoderm larvae. Each data point represents the slope of the regression between
total oxygen consumed and the number of larvae ± SE slope. □ = Respiration Rate.
Each data point represents the slope between total ammonia excreted larva
-1
and time
(hours) ± SE slope. ● = ammonia-N excretion rate. The increase in respiration rates
(dashed line) follows the equation y = y = 44.3e
0.04x
, R
2
= 0.62. There was no
significant change in ammonia-N excretion rate over time (ANOVA: df, 1,6, F =
0.36, P>0.2).

155
Figure 24.
Incubation Time (Hours)
0 1 2 3 4
Nitrogen Excretion (pmol larva
-1
)
0
5
10
15
20
25
30
A.
y = 5.0x + 8.3
R
2
= 0.99
Age (Days)
0 5 10 15 20 25 30
Respiration Rate (pmol O
2
larva
-1
hour
-1
)
0
20
40
60
80
100
120
140
160
180
NH
3
Excretion Rate (pmol larva
-1
hour
-1
)
0
5
10
15
20
25
B.

156
An example of the data set used to calculate the rate if ammonia excretion in the
larvae of the temperate asteroid, Asterina miniata, is shown in Figure 24A.The
rates of oxygen consumption and ammonia-N excretion of larvae of the temperate
asteroid, Asterina miniata, are shown in Figure 24B. Ammonia-N excretion rates did
not change significantly throughout the experimental time period (ANOVA: df, 1,6,
F = 0.36, P>0.2). Respiration rates increased non-linearly following the equation:
44.3e
0.04x
.
Polar species
An example of the data set used to calculate the rate if ammonia excretion in
the larvae of the Antarctic echinoid, Sterechinus neumayeri, is shown in Figure
25A.The rates of oxygen consumption and ammonia-N excretion in the embryos and
larvae of Sterechinus neumayeri are shown in Figure 25B. The rates of ammonia-N
excretion did not change significantly throughout the experimental time period
(ANOVA: df, 1,21, F = 1.02, P>0.1). The rates of oxygen consumption increased
non-linearly following the equation: 10.8e
0.02x
.
An example of the data set used to calculate the rate if ammonia excretion in
the larvae of the Antarctic asteroid, Odontaster validus, is shown in Figure 26A.The
rates of oxygen consumption and ammonia-N excretion in the embryos and larvae of
Odontaster validus are shown in Figure 26B. The rates of ammonia-N excretion
increased until 40 days of age, but were lower in 44-day old larvae. Respiration rates
increased non-linearly following the equation: 4.4e
0.05x
.
157
O:N Atomic Ratio
The O:N atomic ratios for all species studied are shown in Table 4. O:N was
calculated using the equation (Oxygen Consumption Rate)*2/ (Nitrogen Excretion
158

Figure 25. Sterechinus neumayeri. A) Typical data set for nitrogen excretion
experiment. Each data point represents the amount of nitrogen in one experimental
vial (see Methods). The slope of the regression represents the amount of ammonia-N
excreted per larva per hour. B) Respiration rates and ammonia-N excretion rates of
Antarctic echinoderm larvae. □ = Respiration Rate. Each data point represents the
slope of the regression between total oxygen consumed and the number of larvae ±
SE slope. ● = ammonia-N excretion rate. Each data point represents the slope
between total ammonia excreted larva
-1
and time (hours) ± SE slope. The increase in
respiration rates (dashed line) follows the equation y = 10.8e
0.02x
, R
2
= 0.32. There
was no significant increase in ammonia-N excretion rates (solid line) through the
experimental time period (ANOVA: df, 1,21, F = 1.02, P>0.1).

159
Figure 25.

Incubation Time (Hours)
0 1 2 3 4 5 6 7
Nitrogen Excretion (pmol N larva
-1
)
0
2
4
6
8
10
12
y = 0.90x + 4.9
R
2
= 0.92
A.
Age (Days)
0 20 40 60 80
Respiration Rate (pmol O
2
larva
-1
hour
-1
)
0
50
100
150
200
NH
3
Excretion Rate (pmol larva
-1
hour
-1
)
0
1
2
3
4
5
6
B.

160
Table 4. O:N Atomic Ratios in Antarctic and Temperate
Echinoderm Species
Species
Culture
Replicate
Age (Days) O:N atomic ratio
Lytechinus
pictus
1
2
1
2
1
6
7
8
10
17
15.7 *
22.4 *
43.7 *
10.0 *
26.0 *
Asterina
miniata
2
1
2
3
1
2
1
9
11
14
16
21
23
26
12.5 *
29.2 *
20.7 *
12.4 *
38.5 *
24.7 *
20.6 *
Sterechinus
neumayeri
Non-feeding
embryo

3
2
3
1
2
3
1
2
3
1
15
21
28

36
39
43
46
49
51
56
63
66
70
74.0 ‡
18.7 *
24.5 *

78.9 ‡
65.3 ‡
22.1 *
41.1 *
34.7 *
54.2 †
219.4 ‡
57.1 †
70.5 ‡
58.4†
Odontaster
validus
Non-feeding
embryo

1
1
17
19
24

39
55
259.9‡
1275.3 ‡
40.0*

11.8 *
361.6 ‡

161
Rate). O:N ratios < 50 indicated protein as the primary energy substrate; O:N ratios
between 50-60 indicated an equal use of protein and lipid as energy substrates, and
an O:N ratio > 60 indicated lipid as the primary energy substrate (after Mayzaud and
Conover, 1988).
Temperate species
The O:N atomic ratios for the larvae of both Lytechinus pictus and Asterina
miniata were all below 50 throughout the experimental time period, indicating that
they used protein as their primary source of energy throughout their development.
This corresponds with the lack of triacylglycerol in these larvae, which are storage
lipids that are used as energy reserves. These O:N ratios measured here indicate that
the larvae of these temperate echinoderms primarily use their protein stores
throughout their development as the primary energy substrate. It is possible that any
lipid that is ingested may be used immediately for energy as well, as there is no
accumulation of storage lipids throughout development.
Polar species
The non-feeding embryos of the Antarctic sea urchin, Sterechinus neumayeri,
had an O:N ratio of 74.0 at 15 days of age, indicating the predominant use of lipid as
their energy source. However, as their embryological development continued
through 28 days of age, the O:N ratio decreased dramatically to 18.7 (21-day old
embryo) and 24.5 (28-day old embryo), indicating a shift to protein use for energy.
Once a feeding, larvae stage was reached and the larvae received algal nutrients, the
O:N ratio increased again to above 50 in larvae 36 and 39 days of age. For the
162
Figure 26. A) Odontaster validus. Typical data set for nitrogen excretion
experiment. Each data point represents the amount of nitrogen in one experimental
vial (see Methods). The slope of the regression represents the amount of ammonia-N
excreted per larva per hour. B) Respiration rates and ammonia-N excretion rates of
Antarctic echinoderm larvae. □ = Respiration Rate. Each data point represents the
slope of the regression between total oxygen consumed and the number of larvae ±
SE slope. ● = ammonia-N excretion rate. Each data point represents the slope
between total ammonia excreted larva
-1
and time (hours) ± SE slope. The increase in
respiration rates (dashed line) follows the equation: y = 4.4e
0.05x
, R
2
= 0.96.

163
Figure 26.
Incubation Time (Hours)
0 1 2 3 4 5
Nitrogen Excretion (pmol larva
-1
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
y = 0.3x + 2.1
R
2
= 0.92
A.
Age (Days)
0 20 40 60
Respiration Rate (pmol O
2
larva
-1
hour
-1
)
0
20
40
60
80
Nitrogen Excretion Rate (pmol larva
-1
hour
-1
)
0
2
4
6
8
B.

164
remainder of their development, however, the O:N ratios shifted between values that
indicated protein use (43-49 days of age), lipid use (56 and 66 days of age) and a
combination of both lipid and protein (51 and 70 days of age). This is in contrast to
the strict use of protein as an energy substrate seen in the temperate echinoid species.
The O:N ratios in embryos of the Antarctic asteroid, Odontaster validus,
followed a similar pattern to S. neumayeri. From the ages of 17-19 days post
fertilization, the O:N ratios were well above 50, indicating lipid as the primary
energy source. As they reached a feeding, larval stage (24 days post fertilization) the
embryos began to use protein as their primary energy substrate as indicated by the
O:N ratio (40.0). The larvae were still primarily using protein by 39 days post
fertilization (O:N of 11.8), but by 55 days of age they had switched to using lipid as
their primary energy substrate (O:N of 361.6). These results suggest that these
Antarctic echinoderms preferentially use lipid as their primary source of energy, but
are able to utilize protein if needed. As larvae, they are able to switch between
energy sources or use both when necessary.
Discussion
Polar marine environments present many challenges to the organisms living
there, namely cold temperature and highly oligotrophic conditions. The
planktotrophic larvae of marine invertebrates are required to survive for months
without particulate nutrients before the main phytoplankton bloom occurs. It is
currently unknown what metabolic substrates these larvae use to fuel their
development prior to feeding and how they utilize particulate nutrients once they are
165
available. In this study, the substrates used to fuel the development of an Antarctic
echinoid and asteroid (Sterechinus neumayeri and Odontaster validus, respectively)
were determined and compared to similar measurements in a temperate echinoid and
asteroid species (Lytechinus pictus and Asterina miniata, respectively). The
biochemical composition (total protein and lipid classes) of pre-feeding embryos and
feeding larvae was followed throughout development, and ammonia-N excretion and
respiration rates were measured to calculate the O:N atomic ratios during
development.
The biochemical composition of protein and lipids differed between the
temperate species. The eggs of L. pictus were comprised of about half protein and
half lipid, while the eggs of A. miniata were comprised of about 60% protein and
40% lipid. In both species, however, there was an increase in total protein content
and concomitant decrease in total lipids once the larval stages were able to feed on
particulate nutrients. The lipid class composition also changed in both temperate
species once a feeding larval stage was reached. The eggs of these species contained
mostly polar lipids (over 70% of total lipid), but also contained triacylglycerol and
sterol lipids (ranging from 11-27% and 1.6- 2.7%, respectively). Polar lipids and
sterols are primarily lipid classes that are found in cellular membranes, while
triacylglycerol lipids are storage lipids that are used as an energy source in
organisms. Once a feeding, larval stage was reached in both L. pictus and A.
miniata, polar lipids comprised over 95% of the total lipid content, with the
remainder being made up of sterols, indicative of increasing cell number.
166
Triacylglycerols were completely depleted by the time a larval stage was reached,
and did not reappear at any point during their development. This absence of storage
lipids in the larvae of temperate echinoderms has also been seen in the larvae of the
purple sea urchin, Strongylocentrotus purpuratus (Meyer et al., in submission).
These findings indicate that the maternally provisioned stores of triacylglycerols
were used to fuel embryonic development, and any storage lipid that was in the algae
ingested by larvae was quickly turned over and used as an energy source.
The pre-feeding embryos and feeding larvae of the Antarctic sea urchin,
Sterechinus neumayeri, had similar biochemical compositions as L. pictus. The eggs
and embryos of S. neumayeri were comprised of about half protein and half lipid.
Over 80% of the total lipid was made up of polar lipids, and also contained
triacylglycerol and sterol lipids (13% and 5%, respectively). The feeding larvae of S.
neumayeri were also similar to the larvae of L. pictus in that the total protein content
increased and the total lipid content decreased. In addition, triacylglycerol lipids
were completely depleted by the time a larval stage was reached and did not reappear
at any point during larval development. This result agrees with a previous study that
measured the C:N content in the eggs and embryos of S. neumayeri and showed that
the embryos had a lower C:N content than the eggs, indicating that lipids were used
to fuel early development in this organism (Marsh et al., 1999).
The pre-feeding embryos of the Antarctic sea star, Odontaster validus, had
similar protein to lipid ratios as A. miniata. Out of the combined protein and lipid
contents, about 70% was comprised of protein and 30% lipid. The total lipid content
167
was made up of over 70% polar lipid, with the remainder being made up of
triacylglycerol and sterol lipid classes (28% and 1%, respectively). The feeding,
larval stages of O. validus showed a much different pattern of biochemical
composition than the other three species studied. The protein to lipid ratio switched
from 2.6:1 in eggs and pre-feeding embryos to 1:2.9 in feeding larvae. Although the
percent protein decreased in feeding larvae, there was an overall increase in total
protein content, indicating both protein and lipid growth during larval development.
The total lipid was comprised of only 55% polar lipids and 4% sterols and unlike the
larvae of other species the larvae of O. validus contained 40% triacylglycerol, an
increase from the levels in eggs and embryos. The fact that there was a decrease in
the percent protein composition in these larvae, and they were able to accumulate
storage lipids from exogenous nutrients indicates that they may have been using both
protein and lipid as energy sources during their larval stages.
The rates of oxygen consumption and ammonia-N excretion were measured
and used to calculate the O:N atomic ratios during development in both the
temperate and Antarctic echinoderms. O:N atomic ratios are helpful in determining
the primary energy substrate(s) (e.g. protein, lipid) being used by an organism,
including zooplankton (Mayzaud and Conover, 1988). Very low O:N ratios (3-16)
indicate the sole use of protein as the primary energy substrate, while high O:N ratios
(> 60) indicate that only lipid catabolism is occurring. Ratios that fall in between
these extremes indicate that an organism is using both lipid and protein as energy
substrates to different degrees. The O:N atomic ratios in larvae of the temperate
168
echinoderm species studied remained below 60 throughout the experimental time
period (Table 3), indicating that protein was used as the primary energy substrate in
these animals. There were time points in both species when the O:N ratio went
above 16, which indicated that although protein was still the primary energy source,
lipid was also being catabolized for energy (Brockington and Peck, 2001). The
increased O:N ratios, combined with the fact that the larvae of L. pictus or A. miniata
depleted their storage lipids (triacylglycerol) during embryogenesis and did not
rebuild any lipid stores during their larval stages indicates high turnover rates of lipid
that was ingested through the algal nutrients (Meyer et al., 2002). These results for
the larvae of temperate echinoderm species agree with previous studies on marine
invertebrates from similar environments. Lemos et al. (2003) calculated an O:N
ratio of 3.7 in the veliger larvae of the tropical mussel, Perna perna. In another
study of the tropical shrimp, Farfantepenaeus paulensis, ontogenetic changes in
energy usage occurred. The O:N ratios of the embryo and early larval stages, was
low (<30), but once lipid reserves had been built up in the later larval stages of F.
paulensis, the O:N ratios increased above 60, which indicating a switch from protein
to lipid as primary energy sources (Lemos and Phan, 2001).
The larvae of the polar echinoderm species had different patterns of energy
substrate usage than the temperate species. At 15 days of age, pre-feeding embryos
of Sterechinus neumayeri had an O:N atomic ratio of 74.0, which indicates the use of
lipid as the sole energy substrate. As embryogenesis continued, the O:N ratio
decreased to around 20, which indicated a the addition of protein as an source of
169
energy. These changes in O:N ratios are reflected in the changes in biochemical
composition. The O:N ratios decreased as the lipid stores were depleted in the
embryos. This also agrees with a previous study where C:N ratios indicated the
primary use of lipids in early development of S. neumayeri (Marsh et al., 1999). The
larvae of Antarctic krill, Euphasia superba, are also able to switch from a primarily
lipid metabolism to protein metabolism during periods of starvation (Meyer and
Oettl, 2005). Larval stages of krill have small lipid reserves compared to adult krill,
and during their over wintering period are required to use protein stores once these
reserves are depleted. The O:N ratios in the larvae of S. neumayeri fluctuated
between values indicating mainly lipid catabolism (40% of experimental time
period), protein catabolism (30% of experimental time period) and a combination of
lipid and protein catabolism (30% of experimental time period). The fact that these
larvae to switch between protein and lipid use suggests that they are able to utilize
the particulate nutrients they encounter to the fullest degree. In addition, the
preferential use of lipid as an energy substrate allows the larvae to accrue protein
biomass with higher efficiency, which is imperative to successful development and
growth to metamorphosis.
The embryos of the Antarctic asteroid, Odontaster validus, showed a similar
pattern of substrate use as S. neumayeri. Extremely high O:N ratios up to 19 days of
age indicate the sole use of lipids as an energy substrate. By the 24 days of age, a
feeding larval stage had been reached, but they had not yet been given algal
nutrients. At this point, the O:N ratio decreased significantly to 40, which indicated
170
that protein was being used as the primary energy substrate. After a lag after feeding
began, the O:N ratios again began to increase to very high levels, indicating that
lipids were being used as the sole energy source for their development. These results
also correlate well with the build up of triacylglycerols (storage lipids) in these
larvae. The larvae of O. validus are able to utilize lipids as energy that allows a
greater portion of the protein they ingest to be used towards biomass growth and
development. The larvae of Antarctic copepods (Clarke, 1988) and krill (Meyer et
al., 2002; Meyer and Oettl, 2005) have been shown to preferentially use lipids as
their energy source.
In a previous study (Green and Manahan, in prep), the gross protein growth
efficiency in the larvae of three Antarctic echinoderms (Sterechinus neumayeri,
Odontaster validus, and Odontaster meridionalis) was measured and compared to
the larvae of three temperate echinoderms (Lytechinus pictus, Strongylocentrotus
purpuratus and Asterina miniata). The resulting gross protein growth efficiencies of
the Antarctic echinoderm larvae were quite high (an average of around 80%),
indicating that the majority of the protein that they ingested was converted into
protein biomass. In contrast, the larvae of the temperate echinoderms had gross
protein growth efficiencies that averaged around 30%. The results of the present
study agree with the gross protein growth efficiencies measured in the larvae of these
species. The O:N ratios in the temperate larvae studied remained in the range
indicative of protein usage, which would result in a lower gross protein growth
efficiency. The O:N ratios in the Antarctic larvae were on average higher than the
171
temperate larvae, indicating lipid catabolism. The primary use of lipids as an energy
source would allow the ingested protein to be used towards biomass growth,
resulting in higher growth efficiencies.
There are differing views of what influences O:N atomic ratios in marine
invertebrates. There is evidence that point to food quality as the main influence of
energy usage in these animals (Ikeda and Dixon, 1984; Meyer and Oettl, 2005). If
the source of food is high in protein, protein catabolism will dominate, and vice
versa. The temperate echinoderm larvae in this study were fed the alga,
Rhodomonas spp. Out of total lipid and protein content, protein comprises almost
80% of these algal cells, with lipid making up only around 20% (average protein
content of Rhodomonas spp. cell: 41.3 ± 0.04 pg cell
-1
; average lipid content: 16.8 ±
4.7 pg cell
-1
). This large disparity could account for the primary use of protein as an
energy substrate and the lack of lipid storage in these larvae. The Antarctic
echinoderm larvae in this study were fed the algal species Dunaliella tertiolecta,
which is comprised of 62% protein and 38% lipid (average protein content of
Dunaliella tertiolecta cell: 23.2 ± 1.8 pg cell
-1
; average lipid content: 13.9 ± 1.9 pg
cell
-1
). The higher percentage of lipid in these algal cells could account for the
greater usage of lipids and also for the combined use of lipid and protein in these
larvae. However, the fact that the Antarctic larvae often had O:N ratios high enough
to indicate pure lipid catabolism despite the algae being predominantly made up of
protein, indicates that they preferentially use lipid rather than protein. Again, this
172
allows these larvae to accrue protein biomass with greater efficiency, which is
extremely important to animals developing in a food-limited environment.
Mayzaud and Conover (1988) argue that O:N ratios reflect the food
catabolized only if the diet is completely balanced with all of the nutrients and amino
acids the animal needs. Otherwise, O:N ratios are regulated by the fate of each
biochemical fraction of the food being catabolized, not the elemental composition of
the food itself. Therefore, animals that have different metabolic pathways may show
differential use of protein and lipid as energy sources. The patterns of O:N ratios
and the gross protein growth efficiencies of Antarctic and temperate echinoderm
larvae (Green and Manahan, in prep) could be a result of differing metabolisms of
animals from varying environments.
The present study highlights the unique physiology of Antarctic echinoderm
larvae through their energy usage during their development. Regardless of food
quality, Antarctic larvae are able to utilize lipids to a greater degree than temperate
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long periods with no particulate nutrients that they encounter during their larval life
span.
173
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Abstract (if available)

Abstract Life histories of many marine invertebrates include a pelagic, larval stage. The larval stages of these marine invertebrates are considered the most vulnerable as they are subjected to highly variable environmental conditions (i.e. temperature, salinity). Larvae that are planktotrophic during their pelagic life history stage are also subjected to fluctuating nutrient levels as they disperse. All of these factors make the study of larval physiology and biochemistry important in understanding how larvae cope with a highly variable environment to successfully metamorphose.

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Asset Metadata

Creator Green, Allison Jeanette (author)

Core Title Comparative physiological studies of marine invertebrate larvae from Antarctic and temperate environments

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Biology

Publication Date 11/20/2006

Defense Date 10/03/2006

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

Tag Antarctic,invertebrate,larvae,marine,metabolism,OAI-PMH Harvest,temperate

Place Name Antarctica (continents)

Language English

Advisor Manahan, Donal T. (committee chair), Bottjer, David J. (committee member), Caron, David A. (committee member), Edmands, Suzanne (committee member), Hedgecock, Dennis (committee member)

Creator Email allisong@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m178

Unique identifier UC175997

Identifier etd-Green-20061120 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-37679 (legacy record id),usctheses-m178 (legacy record id)

Legacy Identifier etd-Green-20061120.pdf

Dmrecord 37679

Document Type Dissertation

Rights Green, Allison Jeanette

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu