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Physiological rates during development of marine invertebrates in temperate and polar oceans
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Physiological rates during development of marine invertebrates in temperate and polar oceans
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Content
PHYSIOLOGICAL RATES DURING DEVELOPMENT OF MARINE
INVERTEBRATES IN TEMPERATE AND POLAR OCEANS
by
David Ward Ginsburg
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
(BIOLOGICAL SCIENCES)
May 2007
Copyright 2007 David Ward Ginsburg
ii
Acknowledgements
The research presented in this dissertation could not have been completed without
the intellectual mentorship and financial assistance that I received from the Marine
Environmental Biology program at USC. I would like to thank my graduate advisor,
Dr. Donal Manahan, for his advice and training during my tenure in his laboratory.
At a time when my options for applying to Ph.D. programs seemed few and far
between – Donal took a chance on me, and for that I will always be grateful. Many
thanks to my good friends and colleagues from ‘Team Larvae in the Manahan Lab’
who helped make my dissertation research both above and below the water possible:
D. Pace, A. Green, R. Robbins, E. Meyer, P. Yu, M. Moore, P. Von Dippe, E.
Yamiguchi, K. Ruffino, E. Rogers, K Watkins, and M. Ginsburg. Special thanks to
my dissertation committee for their assistance with the design and analysis of various
components of my research, and their time and patience in seeing me through the
degree progress: Dr. Dave Caron, Dr. Frank Corsetti, Dr. Dennis Hedgecock, and Dr.
Rob Maxson. Lastly, I am indebted to my wife, Virginia, who, through thick and thin
has stood by my side and provided me with emotional and moral support.
iii
Table of Contents
Acknowledgements ii
List of Tables iv
List of Figures v
Abstract vii
Introduction
Introduction references
1
10
Chapter 1:
Nutritional status of marine invertebrate larvae in the
ocean: a study of the sea urchin Lytechinus pictus.
Chapter references
17
46
Chapter 2:
Developmental physiology of Antarctic sea stars with
different life-history modes.
Chapter references
55
82
Chapter 3:
Energetic cost of protein synthesis during early
development in the temperate sea urchin
Strongylocentrotus purpuratus.
Chapter references
88
114
Bibliography 119
Appendix I:
Rates of protein synthesis and respiration in embryos
and larvae of the Antarctic sea star Porania
antarctica.
Appendix references
135
148
Appendix II:
Direct measurements of the energetic cost of
metabolism in the Antarctic sea star Odontaster
meridionalis using inhibitors of protein synthesis.
Appendix references
150
161
iv
List of Tables
Table 1
Summary of rates of protein synthesis for larvae of L.
pictus reared in the laboratory and in the field. 37
Table 2
Amino acid composition of embryos and larvae of the
Antarctic sea stars A. hodgsoni and O. meridionalis. 62
Table 3
Fractional rates of protein synthesis during embryonic
and larval development of A. hodgsoni and O.
meridionalis. 72
Table 4
Amino acid composition of protein in embryos and
larvae of the sea urchin S. purpuratus. 94
Table 5
Rates of alanine transport for embryos and larvae of
S. purpuratus in the presence and absence of emetine. 103
Table 6
Amino composition and mole-percent of amino acids
in proteins of embryos and larvae of the Antarctic sea
star P. antarctica. 139
Table 7
Alanine transport rates and alanine pool content for
embryos and larvae of O. meridionalis with and
without protein synthesis inhibitors. 155
Table 8
Energetic cost of protein synthesis during early
development in the temperate sea urchin
Strongylocentrotus purpuratus. 159
v
List of Figures
Figure 1 Design of field chamber culture system. 23
Figure 2 Testing field chamber performance in the laboratory. 29
Figure 3
The use of physiological rate measurements for
determining the nutritional condition of larvae of L.
pictus. 31
Figure 4 Physiological growth capacities of larvae of L. pictus. 34
Figure 5
Temporal changes in rates of protein synthesis in
larvae of L. pictus reared in the laboratory and field. 38
Figure 6
Alanine transport, free amino acid pool, and total
protein in embryos and larvae of A. hodgsoni. 64
Figure 7
Alanine transport, free amino acid pool, and total
protein in embryos and larvae of O. meridionalis. 67
Figure 8
Rates of protein synthesis and respiration in embryos
and larvae of A. hodgsoni. 70
Figure 9
Rates of protein synthesis and respiration in embryos
and larvae of O. meridionalis. 73
Figure 10
Temperature compensation in embryos of Antarctic
and temperate asteroids. 78
Figure 11
Changes in rates of
14
C-alanine incorporated into
embryonic protein of S. purpuratus with increasing
concentrations of emetine. 95
Figure 12
Free alanine pool and total protein content during
early development of S. purpuratus. 97
Figure 13
Metabolic and protein synthesis rates during early
development of S. purpuratus. 99
vi
Figure 14
Protein synthesis and metabolic rates during early
development of S. purpuratus in the presence and
absence of emetine. 104
Figure 15
Metabolic energy expenditure during early
development of S. purpuratus. 111
Figure 16
Alanine transport, free amino acid pool, and protein
content of embryos and larvae of P. antarctica. 140
Figure 17
Rates of protein synthesis during early stages of
development of P. antarctica. 143
Figure 18
Rates of oxygen consumption during early stages of
development of P. antarctica. 145
Figure 19
Change in rates of respiration and protein synthesis in
embryos and larvae of O. meridionalis with and
without protein synthesis inhibitors. 156
vii
Abstract
The goal of this dissertation was to investigate the nutritional and physiological state
of benthic marine invertebrates from both temperate and polar habitats. In Chapter 1,
larvae of the temperate sea urchin Lytechinus pictus reared in the field were
compared to those reared on cultured phytoplankton diets in the laboratory. A
biochemical index of physiological state was established using an in situ culturing
system and measurements of protein synthesis to define the nutritional status of
larvae growing in the ocean. Protein synthesis rates for larvae reared in the field were
~50% less than larvae fed at near-maximal physiological capacities in the laboratory.
Research presented in Chapter 2 focused on the physiological and metabolic
requirements of early development in the Antarctic asteroids Acodontaster hodgsoni
and Odontaster meridionalis. Despite differences in egg size and developmental
mode, A. hodgsoni and O. meridionalis maintained high rates of protein synthesis
while sustaining a low metabolic rate. Additionally, fractional rates of protein
synthesis in embryos of O. meridionalis were two-fold greater than its temperate
counterpart (standardized to -1 °C) indicating that the Antarctic species is cold-
adapted. The goal of Chapter 3 was to determine how metabolic energy is partitioned
during early development of the temperate sea urchin Strongylocentrotus purpuratus.
The energetic cost of protein synthesis was 8.6 J (mg protein)
-1
. Combined, protein
synthesis ATP utilization by the sodium pump accounted for ~90% of total
respiration during development. The information provided in this dissertation will
viii
substantially increase understanding of the physiology of organisms developing and
growing in different oceanic environments.
1
Introduction
The ecology and life-history strategies of benthic marine invertebrates have received
considerable attention for more than 100 years (reviewed by Young 1990, Grosberg
& Levitan 1992). In echinoderms, specifically the sea urchins and sea stars
(Echinoidea and Asteroidea, respectively), the relationship between egg size and
developmental life-history mode is well known (reviewed by Emlet et al. 1987,
McEdward & Miner 2001). In general, species that produce small eggs (<250 µm
diameter) are maternally endowed with a low protein and lipid content and have a
feeding (planktotrophic) larval mode, whereas species with larger eggs (>250 µm),
are provisioned with greater amounts of endogenous energy stores (typically lipid)
and have a nonfeeding (lecithotrophic) larval mode (Jaeckle 1995, Villinski et al.
2002). Numerous studies on species with feeding and nonfeeding nutritional modes
have compared phenotypic changes during embryonic and larval development
(McEdward & Miner 2001). However, the metabolic demands (e.g., energetic costs)
that underlie these different life-history traits are poorly understood.
The amount of energy utilized during early development can have important
consequences on larval performance such as survival, feeding ability, and time in the
plankton (Crisp 1974, Hoegh-Guldberg & Emlet 1997, Bertram & Strathmann 1998,
Levitan 2000). Although numerous studies have focused on the supply of larvae and
its importance in structuring benthic marine communities (Mileikovsky 1971, Lewin
2
1986, Roughgarden et al. 1988, Morgan 1995, Caley et al. 1996, Hughes et al. 2000),
fundamental questions regarding the nutritional and physiological condition of
planktonic organisms are relatively undefined (e.g., Huntley & Boyd 1984). For
example, food availability often correlates with larval recruitment events (e.g.,
Lamare & Barker 1999), yet the causative mechanisms of such processes can only be
inferred (Cushing 1990).
Key questions to be answered in the field of larval biology include: Are natural
populations of larvae surviving under near-starvation conditions or are they well fed?
To what extent are the rates of macromolecular synthesis in embryos and larvae
affected by changing developmental and cellular needs? Do embryos and larvae
living in “extreme environments” have unique physiological capabilities? The
research presented in this dissertation addresses the physiological questions outlined
above by (1) establishing a biochemical index with which to measure the nutritional
condition of larvae growing under natural conditions; (2) measuring the
physiological energetics of embryos and larvae living in the extreme cold; and (3)
quantifying the costs of embryonic and larval development in a temperate
environment.
The major theme of this dissertation is to investigate the nutritional and
physiological state of embryos and larvae of benthic marine invertebrates during
specific stages of development. Research presented in this volume was performed
3
with a variety of echinoderm species in both temperate and polar habitats. These
organisms, specifically the sea urchins, represent excellent research “model
organisms” (Davidson 1976) to investigate physiological rate processes during early
developmental stages. The species of echinoderms selected for these experiments
were used because of their seasonal availability, high fecundity, different life-history
strategies, and their experimental tractability for studies of protein metabolism. This
research was based upon well-established methods for in vivo measurements of
protein synthesis (Berg & Mertes 1970, Fry & Gross 1970, Goustin & Wilt 1981)
and oxygen consumption (Gerdes 1983, Gnaiger 1983, Marsh & Manahan 1999)
during the embryonic and larval stages of development. Protein was the primary
macromolecule of interest for this research because it is a major (in some cases
>50%) biochemical constituent of the eggs, embryos, and larvae of echinoderms
(Turner & Lawrence 1979, Shilling & Manahan 1990, 1994).
Physiological condition in the field
In Chapter 1, the goal was to establish a biochemical index and an in situ culturing
system (i.e., experimental field enclosures) to assess the physiological growth
capacity of larvae of the sea urchin Lytechinus pictus in the ocean. This work offers a
direct comparison of larvae of L. pictus reared on unknown concentrations of natural
foods in the field to those reared on cultured phytoplankton diets in the laboratory.
More importantly, this research provides a direct index from which to address the
4
topic of food availability in the field rather than extrapolating from studies based in
the laboratory.
Experimental field enclosures have long been used as a means to study organisms in
their natural environment. For instance, studies dating back to the 19
th
Century have
employed such enclosures for investigating the behavior of a range of different
organisms from bacteria to marine fishes (see Sakshaug & Jensen 1978, Øiestad
1990, and references therein). As pointed out by Grice & Reeve (1982), field-based
enclosure systems can provide a link between the variable conditions of the natural
world and the controlled environment of the laboratory. Although the term ‘field
enclosure’ is used to describe the particular field culture system used in this study,
such culture techniques are referred to by a variety of different names in the
literature (e.g., cage or diffusion cultures: Sakshaug & Jensen 1978, Furnas 1982;
meso- or macrocosms: Grice & Reeves 1982, Lalli 1990; in situ chambers: Olson &
Olson 1989).
Results from this first chapter provide a better understanding of the physiological
growth capacity of larvae in the plankton and are important for models of dispersal
and recruitment. Moreover, these data provide a starting point for incorporating the
physiological characteristics of planktonic organisms into the design of ecologically
sustainable marine reserves (e.g., Grantham et al 2003, Gaylord et al. 2005, Cowen
5
2006). Experimental observations on the nutritional condition of marine organisms
also can be applied towards the effective management of marine fisheries.
Developmental energetics in the cold
Research presented in Chapter 2 focuses on the physiological and metabolic
requirements of early development in two species of Antarctic sea stars: one with a
lecithotrophic mode of development (Acodontaster hodgsoni) and the other with a
planktotrophic larval mode (Odontaster meridionalis). Interestingly, few studies
have examined the metabolic costs associated with these different life-history types
in cold-water species. While previous physiological investigations have offered a
general description of the energetics of nonfeeding development (e.g., Shilling &
Manahan 1994, Hoegh-Guldberg & Emlet, 1997, Bryan 2004), the metabolic costs
for a given stage of embryonic or larval development are not fully understood.
The most comprehensive information to date on the energetic cost of development in
the cold is based on a single species, the Antarctic sea urchin Sterechinus neumayeri.
Embryos and larvae of this species are reported to maintain one of the lowest
metabolic costs of protein synthesis known for all animals (Marsh et al. 2001).
However, whether this unique efficiency is limited to S. neumayeri or is widespread
among other Antarctic species remains unknown. As such, this question provided the
impetus for examining the rates and costs of protein synthesis during early stages of
6
development in the sea stars A. hodgsoni and O. meridionalis. These findings present
a framework for determining the fundamental energy requirements that an embryo
will require to reach larval metamorphosis, and eventually the juvenile stage.
Overall, very little is known regarding the physiological development of benthic
marine invertebrates living in cold-water environments (Pearse & Lockhart 2004,
Pörtner 2006). Nowhere, perhaps, is this more apparent than in research presented on
O. meridionalis and on a third species of Antarctic asteroid Porania antarctica in
this dissertation. For instance, only three studies are known to have investigated the
development of embryos and larvae from these particular species of asteroids (Bosch
1989, Hoegh-Guldberg & Pearse 1995, Stanwell-Smith & Peck 1998). The paucity
of physiological data on the early developmental stages of cold-water species comes
as somewhat of a surprise given that approximately 90% (by volume) of the living
biosphere exists at temperatures less than 5 °C (see Broad 1998). Data presented in
this dissertation will not only improve our understanding of the overall physiological
characteristics of polar marine organisms, but will provide insight to how organisms
are able to persist in extreme environments.
Allocation of metabolic energy during early development
The primary goal of Chapter 3 was to determine how metabolic energy is partitioned
during the transition from embryonic to larval stages of development in the
7
temperate sea urchin Strongylocentrotus purpuratus. The energetic costs of specific
stages of development were established using measured rates of protein synthesis
and oxygen consumption combined with published values for sodium pump activity
(see Leong & Manahan 1997). Both protein synthesis and the sodium pump are
energetically expensive processes (Hochachka 1988, Houlihan et al. 1990) and,
together, can account for ~60-75% of the total energetic budget during early
development (Pace & Manahan 2006).
The degree to which such changes in metabolism affect the physiological state (e.g.,
growth) of embryos and larvae was investigated in Chapter 3. Specifically, the
energetic cost of metabolism during embryonic and larval development was
calculated using an inhibitor of protein synthesis (e.g., Aoyagi et al. 1988). The
inhibitor chosen for these experiments was emetine, which binds to ribosomes and
blocks the process of peptide elongation during the synthesis of proteins (Grollman
1968). Emetine is as an effective inhibitor of protein synthesis in embryos and larvae
of marine invertebrates (Fenteany & Morse 1993, Pace & Manahan 2006). The
advantage of this approach is that it can be used to measure the energetic cost of
protein synthesis for specific stages of development, which is not possible with other
methods used to calculate costs (e.g., Pannevis & Houlihan 1992, Marsh et al. 2001).
Patterns of metabolic partitioning can be used to determine the metabolic
expenditures required for development. As previously mentioned, both protein
8
synthesis and the sodium pump can account for a large portion of the metabolic
energy budget during early development. Presumably, the remaining fraction of
available energy (i.e., ≤30%, Pace & Manahan 2006) must be allocated towards
additional physiological functions such as the integumental transport of organic
solutes (reviewed by Gomme 2001) and ciliary swimming (Qian et al. 1990, Wendt
2000, Strathmann et al. 2006). Bennett & Marshall (2005) suggest that as much as
25% of the energy reserves in the larval ascidian Diplosoma listerianum are used for
swimming. Based on this estimate, less than 5% of the metabolic budget outlined
above would be available for all other biological processes. Overall, research
presented in this chapter highlight the importance cellular energy metabolism and its
affects on the survival and growth of larval forms in the plankton.
Concluding remarks
Using a novel combination of laboratory and field experiments, the research
presented here provides a mechanistic analysis of the metabolic processes that
regulate the growth and development of these organisms. These data offer a better
understanding of how changing environmental conditions, ranging from the
availability of particulate foods to extreme shifts in temperature, can affect the
development of larvae and the rate at which they grow in the ocean. Additionally,
little is known about the physiological bases of nonfeeding development compared to
that of the metabolism and developmental energetics of species with feeding
9
developmental modes. This is especially true for the larval forms of Antarctic
echinoderms. Experimental studies of species with nonfeeding life histories have
primarily described the composition of biochemical energy reserves, rather than
quantify the actual physiological costs of early stages of development as presented in
each of the chapters outlined above.
The research presented in this dissertation helps to define the nutritional and
physiological status of larval forms in nature, making it an important contribution to
the field of larval ecology and physiology. These data provide novel information on:
(1) the nutritional status of larvae growing under natural conditions in the field; (2)
rates of protein synthesis and metabolism in nonfeeding and feeding larval forms
living in the cold biosphere, and (3) the metabolic energy expenditure for early
stages of development in a temperate species of sea urchin. Physiological data on
embryonic and larval condition can be used to determine important parameters for
these organisms such as total metabolic output, the energy that is gained through
feeding, and potential time in the water column. Such information can provide
insight into how embryos and larvae are able to adapt to different environments, and,
ultimately, improve our understanding of the ecological structure of benthic marine
communities.
10
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17
Chapter 1
Nutritional status of marine invertebrate larvae in the ocean:
a study of the sea urchin Lytechinus pictus.
Abstract
Planktotrophic larvae of the temperate sea urchin Lytechinus pictus reared on
unknown concentrations of natural foods in the field were compared to those reared
on cultured phytoplankton diets in the laboratory. A biochemical index of
physiological state was established using an in situ culturing system and
measurements of protein synthesis to define the nutritional status of larvae growing
in the ocean. Based on control studies in the laboratory, the size, growth, and
survival of larvae reared in laboratory and field chambers were not significantly
different. Thus, direct comparisons between cohorts of larvae reared simultaneously
in the laboratory and the field were possible. The physiological growth capacity of
larvae reared in the laboratory spanned a 10-fold range in rates of protein synthesis
(from 0.21 ± 0.01 to 2.2 ± 0.15 ng larva
-1
h
-1
) between unfed and fed treatments.
When removed from the laboratory and placed in the field, protein synthesis rates for
ocean-fed larvae were ~50% less than larvae fed at near-maximal capacities in the
laboratory (0.63 ± 0.06 and 1.2 ± 0.08 ng larva
-1
h
-1
, respectively). Based on these
18
results, the predicted longevity of sea urchin larvae in the plankton was estimated to
be approximately 2 times longer than is suggested by laboratory-based studies.
Introduction
The topic of food availability as it relates to the larvae of benthic marine
invertebrates has been studied for more than half a century (reviewed by Olson &
Olson 1989, Fenaux et al. 1994, Phillips 2004, Pechenik 2006). Many species with
planktotrophic larval stages of development can remain in the water column for
weeks-to-months until competent to settle and metamorphose (Crisp 1976,
Strathmann 1985). During this time, the survival and recruitment success of larvae
can be affected by a combination of biological (Thorson 1950, Pechenik 1987,
Johnson & Shanks 2003) and physical (Jackson & Strathmann 1981, Pineda 1991)
processes. Numerous studies have focused on the dynamics of larval supply and its
importance in structuring benthic communities (Roughgarden et al. 1988, Eckman
1996). However, our current knowledge regarding the extent to which planktonic
organisms growing in the ocean are limited by the availability of food, and whether
they are ever capable of reaching their maximal growth rates, is equivocal (e.g.,
tintinnids: Stoecker et al. 1983; copepods: Huntley & Boyd 1984; echinoderm
larvae: Fenaux et al. 1994; fish larvae: Cushing 1990; marine phytoplankton: Crosbie
& Furnas 2001).
19
Complex assemblages of particulate foods are the primary source of energy for most
invertebrate larvae (Strathmann 1987). Heterotrophic bacteria (Douillet 1993),
phagotrophic protists (Baldwin & Newell 1991), detritus (Qian & Chia 1990), and
dissolved organic matter (Manahan 1990), also supplement larval nutrition. Given
the wide variety, and possibly infinite combinations of food items in the ocean,
studies of larval growth rates have relied on experiments performed in the laboratory
with either cultured mono- or mixed-phytoplankton diets (e.g., Boidron-Métairon
1995). Controlled laboratory diets do not represent natural food conditions (Harms et
al. 1994, Vargas et al. 2006) and may be deficient in any one of a number of
different combinations of essential elements and biochemicals required by animals
from their food (Sterner & Schulz 1998).
In general, the temporal and spatial distribution of phytoplankton foods is
hypothesized to limit larval growth (Thorson 1950, Crisp 1974). Despite numerous
attempts to address this issue, conflicting data make it difficult to evaluate whether a
natural diet is in fact food-limiting. The majority of research on this topic is based on
laboratory studies in which larvae are fed a natural seawater diet supplemented with
cultured phytoplankton. Such experiments, however, do not directly assess the
nutritional status of larvae in their natural environment. While some studies have
indicated larval growth and development is limited by the availability of
phytoplankton foods (e.g., bivalves: Fotel et al. 1999; echinoderms: Paulay et al.
1985, Fenaux et al. 1994, Reitzel et al. 2004; crustaceans: Harms et al. 1994, Desai
20
& Anil 2002), in others no effect was observed (e.g., echinoderms: Olson et al. 1985;
polychaetes: Hansen 1999).
Given the dynamic nature of the ocean, isolating a specific patch of water for the
purpose of collecting replicate biological and chemical samples is difficult (cf. Coale
et al. 1998). Experimental field enclosures, however, can provide a link between the
variable conditions of the natural world and the controlled environment of the
laboratory. Such enclosures have been used for more than four decades to examine
the growth and survival of marine organisms, and have made it possible to conduct
experiments under ambient conditions of temperature, light, and food (see reviews:
Strickland 1967, Sakshaug & Jensen 1978, Olson & Olson 1989, Furnas 1990). The
application of field enclosures for ecological studies of fishes, phytoplankton, and
pollution are described in detail elsewhere (see Grice & Reeve 1982, Lalli 1990, and
references therein). Few studies have used such methodology to investigate the
physiological condition of invertebrate larvae reared in the ocean (e.g., Olson 1985,
Preston 1992, Davis et al. 1996, Hansen 1999, Desai & Anil 2002).
The present study provides a direct comparison of planktotrophic larvae reared on
unknown concentrations of natural foods in the field to those reared on cultured
phytoplankton diets in the laboratory. The developmental stages of the sea urchin
Lytechinus pictus were chosen for this study because of their experimental
tractability for investigating rate processes (Davidson 1976). The findings presented
21
here are based upon in vivo measurements of protein synthesis, which were used to
establish a biochemical index of larval nutritional state. Protein was chosen as the
primary macromolecule of interest for this research because it is a major biochemical
component of the eggs, embryos, and larvae of echinoderms (Turner & Lawrence
1979, Shilling & Manahan 1994). Protein synthesis rates, combined with in situ
culturing techniques, were used to: (1) measure growth, (2) define physiological
state, and (3) make predictions about the potential lifespan of invertebrate larvae in
the ocean.
Materials and Methods
Laboratory cultures. Adults of the sea urchin Lytechinus pictus were induced to
spawn with intracoelomic injections of 0.5 M KCl. Embryos and larvae of L. pictus
were maintained as separate cohorts. For all cultures, 95 to 100% fertilization
success was achieved. Embryos were placed into 20-l containers (herein referred to
as ‘laboratory chambers’) at a concentration of 5-10 ml
-1
. Animals were kept in
suspension using motorized, rotary paddles and maintained in sterile-filtered (0.2 µm
pore size) seawater. Culture water was replaced every 3 to 4 days with newly filtered
seawater. Temperature was monitored in the laboratory and field with underwater
temperature loggers (Onset Computer Corp.). Larval cultures used solely for
laboratory experiments were maintained at 15 ± 0.02
o
C, whereas cultures used for
both laboratory and field experiments were maintained at ambient seawater
22
temperature (18 ± 0.07
o
C). For each experiment, developmental synchronicity
among cultures was monitored by measuring the larval length (n = 50) from either
the left or right postoral arm-tip to the dorsal apex of the larval body.
Field cultures. Field experiments were performed in Big Fisherman’s Cove, located
off Santa Catalina Island, California, USA. A floating platform (Fig. 1), moored in 8
m of water, was built to support the following: 2 flow-through, 20-l plexiglass
containers (herein referred to as ‘field chambers’; 20 cm diameter × 33 cm length);
DC gearmotors (Dayton Electric); 12 V marine battery, and a solar panel charger
(ICP Solar, Inc.). Larvae of Lytechinus pictus were removed from the laboratory and
placed inside field chambers at a concentration of 1.5 ml
-1
. The top and bottom of
each chamber was sealed with a watertight, detachable lid covered with mesh screen
(53 µm pore size). This design prevented larvae from escaping while still allowing
flow and access to natural foods. Chambers were suspended 3-4 m underwater and
mixed (vertical displacement 15 cm) with electric motors (1-2 rpm) mounted on the
platform’s surface. Particulate material trapped on the exterior surfaces of the mesh
screens was removed daily.
Performance validation of field chambers. The flushing rate of field chambers was
tested in the laboratory. Individual 20-l field chambers were placed inside 200-l
culture vessels filled with seawater (i.e., 10× dilution). Field chambers were filled
with 10-l deionized water. Every 1-2 min, water samples were removed from the
Figure 1
Design of field chamber culture system.
a
b
c
d
a
b
c
d
(a) 20-l field chamber; (b) DC gearmotor for vertical mixing; (c) battery box
containing 12 V marine battery; (d) and solar panel charger. Diagram not to scale.
23
24
inside of each field chamber. The salinity (‰) of each sample was analyzed with a
refractometer. Six separate assays were conducted in which field chambers were
equipped with mesh screens of different sizes (53 or 90 µm pore size), and were
either mixed (1-2 rpm) or left immobile to examine the effects of flow. Additionally,
the survival, size, and growth of larvae of Lytechinus pictus reared in both laboratory
and field chambers were examined. Field chambers were placed inside a 200-l
culture vessel as described above. Larvae (6-day-old) were placed inside replicate (n
= 2) laboratory and field chambers and fed equal amounts of the algae Rhodomonas
sp. and Dunaliella tertiolecta at a concentration of 10,000 cells ml
-1
. Every 12 h (4
time points), known numbers of larvae were removed from laboratory and field
chambers for measurements of larval length and protein content. Survivorship was
calculated by recording the mean number of larvae present at the beginning and end
of the experiment.
The effects of sample handling (e.g., time between sample collection and analysis)
and the use of physiological rate measurements for determining larval nutritional
status over short time periods (<2h) also was investigated. Larvae of L. pictus (6-
day-old) were placed inside laboratory chambers and fed Rhodomonas sp. at a
concentration of 5,000 cells ml
-1
. After feeding for 24 hours, algal food was removed
by siphoning the suspension of both algae and larvae from laboratory chambers onto
a mesh screen (80 µm pore size). Larvae were concentrated onto the screen, washed,
and resuspended in filtered seawater inside a clean, laboratory chamber. Known
25
numbers of larvae were removed periodically (i.e., 0, 1, and 2 hours after algal food
was removed) from laboratory chambers for measurements of protein synthesis and
oxygen consumption.
Laboratory and field experiments. The nutritional condition of larvae (4-6-day-
old) of Lytechinus pictus was examined in the laboratory and the field. Individual
cohorts were maintained under experimental conditions for 48 hours. Larvae reared
in the laboratory were divided into ‘fed’ and ‘unfed’ treatments. Fed larvae received
equal amounts of the algae Rhodomonas sp. and Dunaliella tertiolecta at different
concentrations ranging from high (40,000-15,000 cells ml
-1
), intermediate (10,000
cells ml
-1
), to low (5,000 cells ml
-1
) rations. Unfed larvae received no particulate
algal food. Larvae reared in the field were exposed to natural conditions (herein
referred to as ‘ocean-fed’). At the end of each experiment, known numbers of larvae
were removed for measurements of protein synthesis rates.
Additionally, temporal changes in the nutritional condition of larvae of L. pictus
were examined. Larvae were maintained under experimental conditions as described
above. Measurements of protein synthesis were performed daily over a 4-day period.
Larvae were removed from unfed treatments and placed in the field every 24 hours.
Surface seawater samples (650 ml) were collected daily for measurements of
Chlorophyll a (Chl a) following the method described by Parsons et al. (1984). In the
laboratory, samples were filtered through a Whatman GF/F glass-fiber filter and
26
extracted overnight in 7-ml acetone (90%). Chlorophyll measurements were
corrected for phaeopigments (i.e., Chl b, Chl c) by acidification with HCl (37%).
Sample absorbances were measured at 665 and 750 nm with a spectrophotometer.
Protein synthesis. Rates of protein synthesis were measured in vivo in larvae of
Lytechinus pictus following the methods described by Pace & Manahan (2006).
Known numbers of larvae were placed into 20-ml glass scintillation vials at a
concentration of 750 ml
-1
. Animals were exposed to a 10 µM solution of
14
C-alanine
(PerkinElmer, Wellesley, MA; specific activity 6.2 MBq µmol
-1
) in 12-ml filtered
seawater (15
o
C). Every 2-5 min (5 to 6 time points), larvae were removed (375-750)
and collected onto a Nuclepore filter membrane (8 µm pore size). Samples were
washed with filtered seawater to remove excess radioactivity and stored at -80
o
C for
later analysis.
Rates of protein synthesis were calculated using the amount of
14
C-alanine
incorporated into larval protein and the intracellular specific activity of alanine in the
free amino acid pool. Radioactive alanine incorporated into protein was precipitated
with cold, 5% trichloroacetic acid (TCA). The free amino acid pools of larvae were
extracted with ethanol (70%) and the specific activity of alanine quantified with
reverse-phase high performance liquid chromatography (HPLC) following the
protocol described by Welborn & Manahan (1995). The amount of radioactivity in
each sample was measured with a liquid scintillation counter after the addition of 5-
27
ml of scintillation cocktail (Scintisafe, Fisher). The mole-percent of alanine (7.8%)
incorporated into protein was converted to units of protein mass based on the amino
acid composition (average molecular weight of protein 129.3 g mol
-1
) of embryonic
and larval protein in L. pictus (Pace and Manahan 2006). Protein content was
analyzed using a modified Bradford assay (Jaeckle & Manahan 1989).
Respiration. Rates of oxygen consumption in larvae of Lytechinus pictus were
measured continuously using a polarographic oxygen sensor. This method is useful
for observing short-term changes (e.g., <1h) in respiration (cf. end-point
determination; see Marsh & Manahan 1999). Larvae (50-125) were suspended in
filtered seawater and placed inside a 100-µl microrespiration chamber (Strathkelvin
Instruments, Glasgow, UK) maintained at 15
o
C. Respiration measurements were
monitored for 20-30 min with a Clark-type microelectrode following an equilibration
period of 10-15 min. After each assay, larvae were removed from the
microrespiration chamber and enumerated.
Statistics. All error values represent ± 1 SE. Statistical comparisons were performed
using the “R” statistical software package (R Development Core Team, 2005). Prior
to statistical analysis, data were tested for homogeneity of variances using an F test.
Flushing rates expressed as percent values were arcsine square-root transformed
before comparison by one-way ANOVA (Sokal & Rohlf 1995) to test when the
28
“field chambers” were fully flushing with seawater. Statistical significance was
accepted when p < 0.05.
Results
Laboratory and field chamber controls
Flushing rates are expressed as a percent of the initial salinity of seawater used in
each experiment (Fig. 2a). The rate of flushing between an individual field chamber
and that of a 200-l culture vessel reached equilibrium within ~6 min as there was no
significant change in salinity detected after this time point (one-way ANOVA; F
1, 20
= 0.93; p > 0.05). Larval survivorships (Fig. 2b) in laboratory and field chambers
(>91%, respectively) were not statistically different from each other (one-way
ANOVA; F
1, 14
= 1.1; p = 0.31). Comparison of the regressions of larval length
against age (Fig. 2c) demonstrated that there was no significant difference in the size
of larvae reared in laboratory and field chambers (two-way ANOVA; F
1, 6
= 0.62; p
= 0.46). Similarly, there were no significant change in the rates of protein growth
(Fig. 2d) during the same time period (two-way ANOVA, F
1, 6
= 0.04; p = 0.85). The
use of physiological rate measurements for determining larval nutritional status over
short time periods is shown in Fig. 3. There were no significant differences in either
rates of protein synthesis (one-way ANOVA; F
2, 39
= 1.35; p = 0.27) or oxygen
consumption (F
1, 4
= 3.27; p = 0.21). Similar results were obtained when just the
29
Figure 2
Testing field chamber performance in the laboratory.
(a) Field chamber flushing rates. White symbols: chambers equipped with 90 µm
mesh and mixed; grey symbols: chambers equipped with 90 µm mesh and kept
stationary; black symbols: chambers equipped with 53 µm mesh and mixed. Changes
in (b) survival; (c) size; and (d) protein content in larvae of L. pictus reared in
laboratory and field chambers over developmental time. White symbols: field
chambers; grey symbols: laboratory chambers. Error values are ± SE.
Figure 2 (continued)
30
Time (minutes)
0 2 4 6 8 101214
% Seawater
40
50
60
70
80
90
100
% Larval survival
0
20
40
60
80
100
20-l Field
Chambers
20-l Lab
Chambers
Age (days-post-fertilization)
6.0 6.5 7.0 7.5 8.0
Larval length (µm)
0
50
510
525
540
555
Age (days post-fertilization)
6.0 6.5 7.0 7.5 8.0
Protein content (ng larva
-1
)
20
30
40
50
60
a
c
b
d
31
Figure 3
The use of physiological rate measurements for determining the
nutritional condition of larvae of L. pictus.
(a) Change in rates of protein synthesis and (b) respiration in larvae of L. pictus
during short-term food deprivation. Black symbols: larvae provided with algal food;
white symbols: larvae with algal food removed. Protein synthesis values are means ±
SE (n = 5-8); respiration values are individual slopes.
Figure 3 (continued)
32
Protein synthesis (ng larva
-1
h
-1
)
0.0
0.5
1.0
1.5
2.0
Time without algal food (hours)
0.0 0.5 1.0 1.5 2.0
Respiration (pmol O
2
larva
-1
h
-1
)
a
b
0
10
20
30
40
33
TCA-insoluble protein fraction was quantified. Over a 2 h time period, the rates of
14
C alanine incorporated into protein ranged from 0.33 ± 0.03 to 0.49 ± 0.13 Bq
larva
-1
h
-1
and were not significantly different from each other (one-way ANOVA,
F
2, 6
= 0.26; p = 0.78).
Physiological growth capacity of larvae
Protein synthesis rates for unfed larvae increased more than 10-fold (0.21 ± 0.01 to
2.21 ± 0.15 ng larva
-1
h
-1
) when fed a high-ration diet at 15,000 algal cells ml
-1
, and
remained relatively unchanged (mean 1.97 ± 0.16 ng larva
-1
h
-1
) as the amount of
food was increased from 30,000 to 45,000 algal cells ml
-1
(Fig. 4a). The relationship
between measured rates of protein synthesis and concentrations of algal food was
statistically significant (one-way ANOVA, F
1, 4
= 18.8; p < 0.05). A similar trend
was observed for the total protein content of larvae (Fig. 4b). After feeding was
initiated, protein content increased 36% in larvae fed an intermediate (10,000 algal
cells ml
-1
) diet, and leveled off (mean 77.5 ± 2.35 ng larva
-1
) at higher feeding
rations (i.e., 15,000-45,000 algal cells ml
-1
). Changes in protein content with algal
food concentration were significantly different (one-way ANOVA, F
1, 4
= 13.9; p <
0.05).
34
Figure 4
Physiological growth capacities of larvae of L. pictus.
Changes in (a) rates of protein synthesis and (b) protein content in larvae of L. pictus
fed equal amounts of the algae Rhodomonas sp. and Dunaliella tertiolecta at
different concentrations. Values are means ± SE (n = 3-8). The observed data were
described using a logistic model with the greatest adjusted r
2
value. The equation for
each model is as follows: protein synthesis (ng larva
-1
h
-1
) = 2.1(1 – e
-0.1*Age
)
(adjusted r
2
= 0.78); protein content (ng larva
-1
) = 24.3(1 – e
-0.13*Age
) + 55.1 (adjusted
r
2
= 0.84).
Figure 4 (continued)
35
Amount of food (algal cells × 10
3
ml
-1
)
0 5 10 15 20 25 30 35 40 45
Protein synthesis (ng larva
-1
h
-1
)
0
1
2
3
0 5 10 15 20 25 30 35 40 45
Total protein (ng larva
-1
)
50
60
70
80
90
Amount of food (algal cells × 10
3
ml
-1
)
a
b
36
Larval feeding status in the ocean
Rates of protein synthesis for unfed and fed larvae in the laboratory provided a
physiological range with which to compare rates of synthesis for ocean-fed larvae
(Table 1). For all cohorts studied, the mean rate of protein synthesis for unfed larvae
was 0.22 ± 0.01 ng larva
-1
h
-1
. On average, rates of protein synthesis doubled (0.48 ±
0.03 ng larva
-1
h
-1
) in larvae fed a low-ration of 5,000 algal cells ml
-1
, and increased
more than 5-fold (1.2 ± 0.08 ng larva
-1
h
-1
) in larvae fed an intermediate (10,000
algal cells ml
-1
) diet. The mean rate of protein synthesis in ocean-fed larvae was 0.63
± 0.06 ng larva
-1
h
-1
, which was nearly 3 times greater than that of unfed larvae.
Fig. 5 shows the temporal change in rates of protein synthesis for larvae reared in the
laboratory and field. Rates of protein synthesis in unfed larvae remained constant
throughout the period of development studied (one-way ANOVA of linear
regression: protein synthesis (ng larva
-1
h
-1
) = 0.23 – 0.003*(Age); p = 0.52, n = 22).
When removed from the laboratory and placed in the field (i.e., days 5, 6, 7, and 8),
protein synthesis rates for ocean-fed larvae were between 18 to 67% greater than
unfed larvae. The overall rates of protein synthesis for ocean-fed larvae, however,
significantly decreased throughout this experiment (one-way ANOVA of linear
regression: protein synthesis (ng larva
-1
h
-1
) = 0.62 – 0.04*(Age); p < 0.05, n = 8).
Based on the linear regressions described above, rates of protein synthesis for larvae
reared in the field as compared to the laboratory changed by as much as 17% daily.
37
Table 1
Summary of rates of protein synthesis for larvae of L. pictus reared in the laboratory and in the field.
Protein synthesis rate (ng protein larva
-1
h
-1
)
Treatment Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5 Cohort 6 Cohort 7 Mean
Lab-fed@10
0.86±0.14
NS
0.89±0.22
0.92±0.11
NS
1.29±0.13
1.38±0.13
NS
1.45±0.11
1.62±0.22
NS
1.58±0.34
1.04±0.19
1.17±0.29
1.01±0.04
1.20±0.08
Lab-fed@5
0.46±0.09
NS
0.40±0.06
0.46±0.07
0.50±0.06
0.56±0.02
0.48±0.03
Ocean-fed
0.37±0.09
NS
0.37±0.05
0.99±0.11
NS
0.76±0.05
0.70±0.07
NS
0.59±0.04
0.64±0.10
NS
0.72±0.02
0.56±0.07
ND
ND
0.63±0.06
Lab-unfed
0.22±0.07
NS
0.24±0.04
0.20±0.02
NS
0.18±0.03
0.23±0.16
NS
0.21±0.08
0.22±0.05
NS
0.23±0.04
0.22±0.02
0.22±0.04
0.21±0.01
0.22±0.01
All values are means ± SE (n = 5-8)
Lab-fed@10; larvae fed near maximal growth capacity (10,000 algal cells ml
-1
)
Lab-fed@5; larvae provided low-food ration (5,000 algal cells ml
-1
)
NS
indicates protein synthesis rates are not statistically different (ANOVA, p > 0.05)
ND indicates values not determined
Figure 5
Temporal changes in rates of protein synthesis in larvae of L. pictus
reared in the laboratory and field.
Age (days post-fertilization)
56789
Protein synthesis (ng larva
-1
h
-1
)
0.15
0.20
0.25
0.30
0.35
0.40
Rates of protein synthesis for unfed larvae (white symbols) reared in the laboratory,
and for ocean-fed larvae (black symbols) placed in the field every 24 hours. Protein
synthesis values are means ± SE (n = 5-8).
38
39
Surface seawater concentrations of Chl a varied daily. For example, Chl a decreased
more than 2-fold from 0.68 to 0.30 µg l
-1
between days 6 and 7, and then increased to
0.57 µg l
-1
on day 9. Average Chl a concentration for all samples analyzed (days 6 to
9) was 0.49 ± 0.06 µg l
-1
.
Discussion
The availability of food and its effects on the size and distributions of marine
populations is a longstanding debate in marine ecology (Hjort 1914, Thorson 1950,
Steele 1974, Huntley & Boyd 1984, Carr et al. 2003). Specifically, it has been
problematic to use laboratory-based studies to extrapolate the growth potential of
planktonic organisms in the field (Stoecker et al. 1983, Olson 1985, Fenaux et al.
1994). In the water column, particulate food is distributed in three-dimensional space
(Siegel 1998), temporally patchy (Mackas et al. 1985), and extremely dilute
(Conover 1968), making it difficult to assess the biomass available to zooplankton
species (Sterner & Schulz 1998). As a result, incomplete knowledge about the
natural diet of marine zooplankton, as well as the lack of accurate quantitative
techniques, has constrained efforts to determine the nutritional and physiological
condition of organisms in nature.
40
Physiological growth capacity of larvae
The degree to which natural food levels in the ocean are sufficient to support the
high metabolic needs of larvae for optimal growth is not fully understood (Thorson
1950, Crisp 1974, Phillips 2004). As pointed out by Fenaux et al. (1994), natural
concentrations of particulate food in seawater must be sufficient because when used
in laboratory studies, larval forms do not starve to death. Olson and Olson (1989)
reviewed the topic of larval starvation and its importance to the recruitment success
of 26 species of marine invertebrates. In general, they concluded that larval
starvation plays a significant role in the growth and survival of crustaceans and
molluscs, yet is rarely important for echinoderms. In this study, the growth potential
for larvae of Lytechinus pictus was determined for animals reared in the laboratory
and field. As demonstrated in Table 1 (cohorts 1-5), the physiological growth
potential of ocean-fed larvae was 2 to 6 times greater than that defined under unfed
laboratory conditions.
The nutritional condition of larvae has been defined using a variety of experimental
methods ranging from morphological indices, chlorophyll concentrations, and
histological assays (e.g., morphometrics: McEdward 1984; Chl a abundance:
Checkley 1980; lipid-specific stain: Gallager & Mann 1986) to biochemical indices
such as total protein and lipid content (Moran & Manahan 2004), digestive enzyme
activities (Harms & Meyer-Harms 1994), and RNA: DNA ratios (Buckley 1984).
41
While providing estimates of the general condition of the organism at the time it was
examined, such methods are poorly suited for providing quantitative estimates of
maximal growth rates in the ocean. Measurements from this study on the nutritional
and physiological status of larvae will help predict larval lifespan in the plankton.
Physiological state of field-reared larvae
Results from this study demonstrate that the rearing of planktotrophic larvae of the
temperate sea urchin Lytechinus pictus in their natural environment can provide a
direct, quantitative estimate of their nutritional condition. An in situ culturing system
was established that allows for direct comparisons between the laboratory and the
field. Specifically, the natural feeding state of larvae reared in waters off southern
California was measured. The methodology for rearing echinoderm larvae in situ
was based on controlled experiments conducted in the laboratory. Carefully designed
control studies are necessary to minimize the potential for experimental artifacts that
can affect the outcome of such studies, such as unnatural concentrations of food and
the irregular flow of water through field chambers (see reviews by Olson & Olson
1989, Boidron-Métairon 1995). As demonstrated in Fig. 2a, the complete flushing of
field chambers occurred rapidly (~6 min) suggesting that larvae, when placed in the
field, would not encounter abnormally low concentrations of food. Moreover, no
significant differences among the survival, size, and growth of larvae of L. pictus
42
reared in laboratory and field chambers were observed (Fig. 2b-d). Thus comparisons
between the laboratory and natural environment were possible.
A biochemical index of the physiological growth capacities of larvae of L. pictus was
established in this study. Larvae were fed a standard algal diet consisting of
Rhodomonas sp. and Dunaliella tertiolecta over a range of concentrations and the
resulting change in protein growth and synthesis was measured. Previous studies of
the nutritional condition of echinoderm larvae using these same species of algae have
demonstrated that the optimal level of food required for maximal feeding and
development can range from ~5,000-15,000 algal cells ml
-1
(e.g., Strathmann 1971,
Lucas 1982, Basch 1996). Feeding conditions in the laboratory were experimentally
manipulated to produce a 10-fold difference (0.2-2.2 ng larva
-1
h
-1
) in the rates of
protein synthesis between unfed and fed larvae (Fig. 4a). The rates of protein
synthesis for ocean-fed larvae were compared to those reared under defined food
rations in the laboratory (Table 1). Among the different cohorts of L. pictus
examined simultaneously in the laboratory and field, the average rate of protein
synthesis for ocean-fed varied by as much as 23% (estimated using 95% confidence
interval). Rates of protein synthesis in ocean-fed larvae were 4-fold greater than
unfed larvae (Table 1; 0.63 and 0.22 ng larva
-1
h
-1
, respectively), but were, on
average, 2 times less than larvae fed at near-maximal capacities (1.2 ng larva
-1
h
-1
; cf.
Fig. 4a).
43
Longevity of larvae in the ocean
The dispersal of pelagic larvae in the plankton and its importance in structuring
benthic marine communities has been the focus of many studies (Roughgarden et al.
1988, Grantham et al. 2003, Edwards & Richardson 2004). During the planktonic
period, numerous environmental factors affect the survival and recruitment success
of larvae. However, as pointed out by Eckert (2003) and Shanks & Eckert (2005),
little is known regarding the planktonic duration and actual dispersal distance for the
majority of marine invertebrate species. As a result, both larval longevity and
dispersal have often been inferred from a variety of measures ranging from genetic
methods and developmental life-history modes (reviewed by Palumbi 1994) to
otolith microchemistry (Swearer et al. 1999) and elemental fingerprinting (Zacherl et
al. 2003, Becker et al. 2005).
In this study, the predicted longevity of larvae in the plankton as a function of the
amount of natural food available was determined using rates of protein synthesis in
larvae of Lytechinus pictus fed different concentrations of food in the laboratory. The
amount of time required for sea urchin larvae to reach competency for settlement,
depending on environmental conditions, can range from 21 to 74 days (Cameron &
Hinegardener 1974, Strathmann 1987, Grantham et al. 2003). Thus, it can be inferred
that given the best of conditions, the average amount of time larvae spend in the
plankton is ~48 days. Based on biochemical and physiological indices to define
44
nutritional status (Fig. 4a, Table 1), ocean-fed larvae may spend longer periods in the
water column as food availability decreases. Larvae grown under natural conditions
(Table 1; mean protein synthesis rate 0.63 ng larva
-1
h
-1
), would be predicted to
spend ~70% longer in the plankton than larvae fed a high ration (>15,000 algal cells
ml
-1
) diet. On average, larvae could spend >2 times longer (e.g., >100 days) in the
plankton due to reduced growth rates than is suggested from laboratory-based
studies. These results support the view that some members of a given larval cohort
can withstand prolonged starvation (Moran & Manahan 2004). Hence, the top-
down48 control of meroplanktonic communities by predation may play a larger role
than previously expected (Verity & Smetacek 1996, Pitta et al. 1998).
Results from this study provide a framework with which to define the condition (e.g.,
nutritional status, lifespan, etc.) of larvae in the ocean, and offer a temporal
“snapshot” of the availability of food in the field. For example, rates of protein
synthesis for unfed larvae in the laboratory remained constant, while those for ocean-
fed larvae increased between 18 and 67% daily (Fig. 5). Estimates of phytoplankton
abundance, as measured by surface seawater concentrations of Chl a, ranged
between 0.3-0.7 µg l
-1
and are similar to those recorded in the surface waters in the
southern California Bight (Eppley 1992, Mantyla et al. 1995). While neither estimate
of the immediate food availability described above is mutually exclusive, these
results highlight the need to better understand how the timing and availability of
45
particulate algal foods affect the nutritional condition of larvae (Mackas et al. 1985,
Phillips 2002).
In conclusion, these findings provide a link between the physiological capacities of
planktotrophic larvae reared on unknown natural foods in the field as compared to
those reared on controlled phytoplankton diets in the laboratory. Such studies are
crucial for determining how food availability affects the growth and survival of
larvae in the plankton, which is an important component of dispersal and recruitment
success.
46
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55
Chapter 2
Developmental physiology of Antarctic sea stars
with different life-history modes.
Abstract
Rates of protein synthesis and respiration were measured in Antarctic sea stars with
different modes of development. Acodontaster hodgsoni hatches from a large (~550
µm diameter) egg and develops as a pelagic lecithotrophic larva. Odontaster
meridionalis hatches from a smaller (~220 µm diameter) egg and has a pelagic
planktotrophic larval mode. Fractional rates of protein synthesis in embryos and
larvae were 0.25% and 0.50% h
-1
for A. hodgsoni and O. meridionalis, respectively.
The fractional rate for embryos of O. meridionalis at -1 °C was 2-fold greater than
that of embryos of a temperate species of sea star (Asterina miniata), when the rate
of the latter species was calculated at -1 °C (measured at 15 ºC) with a Q
10
value of
2.0). In A. hodgsoni, rates of oxygen consumption increased 3-fold from 50.2-153.5
pmol O
2
embryo
-1
h
-1
during embryogenesis and ranged between 112.5-155.8 pmol
O
2
larva
-1
h
-1
during
nonfeeding larval stages of development. In O. meridionalis,
oxygen consumption increased 7-fold from 50.2-153.5 pmol O
2
embryo
-1
h
-1
during
embryonic development and between 16.1-35.8 pmol O
2
larva
-1
h
-1
in unfed larvae.
56
Likewise, in fed larvae, respiration rates increased more than 3-fold (23.8-81.5 pmol
O
2
larva
-1
h
-1
). These findings support the hypothesis that embryos of Antarctic
echinoderms maintain high rates of protein synthesis while sustaining low rates of
respiration, and have physiological adaptations that enable them to development at
low environmental temperatures.
Introduction
Antarctic echinoderms live in one of the coldest and most seasonally food-limited
habitats in the world (Clarke 1988). Although the developmental mode and life-
history strategies of these organisms are relatively well known (Clark 1963, Bosch &
Pearse 1990, Pearse et al. 1991), the mechanisms that set physiological rates for a
given stage of development are not fully understood. Moreover, despite the
predominance of nonfeeding (lecithotrophic) modes of development among
Antarctic echinoderm species (Pearse 1994, Pearse & Lockhart 2004), previous
measurements of physiological rate processes in Antarctic organisms have focused
primarily on species with feeding (planktotrophic) larvae (Leong & Manahan 1999,
Marsh et al. 1999, 2001). Few studies have examined the energetic costs required for
different modes of development (reviewed by Jaeckle 1995, Hoegh-Guldberg &
Emlet 1997, Moreno & Hoegh-Guldberg 1999), and only one (Shilling & Manahan
1994) has investigated this topic with invertebrate larval forms in polar waters.
57
The present study focuses on the physiological and metabolic requirements for
marine invertebrates to develop in the cold. The synthesis of proteins is an
energetically demanding process and accounts for a large proportion (up to 50%) of
metabolism in animals (Houlihan et al. 1988, 1990, Hawkins 1991, Fraser et al.
2002). Protein synthesis plays an important role in establishing the physiological
state for developmental stages of Antarctic and temperate species of echinoderms
(Marsh et al. 2001, Pace & Manahan 2006, Chapter 3, this dissertation). Recent work
has demonstrated that during early development of the Antarctic sea urchin
Sterechinus neumayeri rates of protein synthesis are high, and together with the
physiologically-active fraction of the sodium pump, account for nearly 90% of total
metabolic energy expenditure (Pace & Manahan submitted). Furthermore, these
authors provide evidence that such rates can be maintained with both a low energetic
cost of protein synthesis and a low rate of metabolism. However, whether these
biochemical rates are unique to S. neumayeri or widespread among other Antarctic
echinoderms is uncertain, since little is known regarding the physiology and
developmental energetics of organisms from high-latitude environments.
Echinoderms (e.g., sea urchins) represent excellent research “model organisms”
(Davidson 1976) to investigate physiological rate processes of embryonic and larval
forms. The species of asteroids (Acodontaster hodgsoni and Odontaster
meridionalis) selected for these experiments were chosen because of their seasonal
availability, high fecundity, and their experimental tractability for studies of protein
58
metabolism (Bosch & Pearse 1990, Pearse et al. 1991, Shilling & Manahan 1994).
Both A. hodgsoni and O. meridionalis are common in benthic habitats off McMurdo
Sound, Antarctica (Dayton et al. 1974, Bosch & Pearse 1990) and have circumpolar
distributions (Clark 1963). Females of A. hodgsoni display lecithotrophic
development and produce large (~550 µm diameter) eggs, whereas females of O.
meridionalis produce smaller (~200 µm diameter) eggs and have a planktotrophic
developmental mode (McClintock et al. 1988, Bosch & Pearse 1990).
The findings presented in this study are based upon in vivo measurements of
macromolecular synthesis and respiration during early development of A. hodgsoni
and O. meridionalis. For each species of sea star investigated the rates of (1) protein
synthesis, (2) oxygen consumption, and (3) the utilization of energy reserves were
determined during embryonic and larval development. These data provide insight
into the biochemical and energetic adaptations exhibited by benthic marine
invertebrates with different life-history strategies.
Materials and Methods
Animal culturing. During the 2004 austral autumn season, adult Acodontaster
hodgsoni and Odontaster meridionalis were collected by SCUBA divers from two
different locations (Intake Jetty and Arrival Heights) in McMurdo Sound, Antarctica.
Gametes were obtained using standard methods to spawn sea stars (injections of 1
59
mM 1-methyladenine). Fertilization success was >95% for all cultures. Embryos
from each species of asteroid were placed into 20-l culture containers at a
concentration of 5-10 ml
-1
, and maintained as separate cohorts. Animals were kept in
suspension by gently mixing cultures with motorized, rotary paddles (5-10 rpm). All
cultures were maintained in sterile-filtered (0.2 µm pore-size), ambient temperature
(-1.5 °C) seawater. Culture water was replaced with newly filtered seawater every 3
to 4 days.
Once competent to feed, larvae (26-day-old) of O. meridionalis were divided into
either ‘fed’ or ‘unfed’ treatments. Fed larvae received the alga Dunaliella tertiolecta
at a concentration of 15,000 cells ml
-1
. Unfed larvae received no particulate algal
food. Prior to initiating feeding experiments, suspensions of D. tertiolecta were
maintained at -1 °C to determine the viability of this species at low temperatures.
Algal suspensions were observed over a 3-day period, which represented the
approximate interval between days when algal food rations for larvae were
replenished. Preliminary experiments indicated that algal cells were actively motile
and decreased <10% from their initial concentration. Additionally, when transferred
to warmer seawater (i.e., from -1 to 4 °C), algal cells were revived after 48 hours thus
confirming that fed larvae received viable algal food in this study. Developmental
synchronicity between fed and unfed cultures of O. meridionalis was monitored after
each water change by recording the size of individual larvae (n = 50). The body
60
lengths of larvae were measured as the distance between either the left or right
postoral arm-tip to the dorsal apex of the larval body.
Protein metabolism and growth. Rates of alanine transport and protein synthesis
were measured in vivo in embryos and larvae of Acodontaster hodgsoni and
Odontaster meridionalis following the methods described by Vavra & Manahan
(1999). Known numbers of animals were placed into a 20-ml glass scintillation vial
and exposed to a 10 to 15 µM solution of
14
C-alanine (PerkinElmer, Wellesley, MA;
specific activity 6.2 MBq µmol
-1
) in filtered seawater (i.e., A. hodgsoni: 250 animals
in 4 ml; O. meridionalis: 12,000 animals in 12 ml). Time course experiments were
maintained for ~1.5 h at -1
o
C. Embryos or larvae (A. hodgsoni: n = 200; O.
meridionalis: n = 375 to 750, depending on developmental stage and treatment) were
removed every 5-10 min, collected onto an 8-µm (pore size) Nuclepore filter
membrane, and washed two times with 15-ml cold seawater to remove
unincorporated isotope. All samples were flash-frozen in liquid nitrogen and stored
at -80 °C.
The amount of
14
C-alanine incorporated into whole-animal protein and the specific
activity of alanine in the free amino acid pools of either embryos or larvae was
analyzed for each sample. Radioactive alanine incorporated into protein was
precipitated with cold, trichloroacetic acid (5%). Animals were placed in ethanol
(70%) and extracted overnight. The specific activity of alanine was quantified with
61
reverse-phase high performance liquid chromatography (HPLC) following the
methods of Welborn & Manahan (1995). Scintillation cocktail (Scintisafe, Fisher)
was added to each sample and the amount of radioactivity determined by liquid
scintillation counting. Rates of protein synthesis were calculated using the mole-
percent of alanine and average molecular weight of amino acids in hydrolyzed
protein of embryos and larvae of A. hodgsoni and O. meridionalis (Table 2).
Fractional rates of protein synthesis (i.e., the percentage of proteins newly
synthesized h
-1
) were calculated for each species of sea star as the ratio between
protein synthesis rate and total protein content for a specific developmental stage or
treatment. Total protein content was analyzed using a modified Bradford assay
(Jaeckle & Manahan 1989).
Respiration. Rates of oxygen consumption for embryos and larvae of Acodontaster
hodgsoni and Odontaster meridionalis were measured using the end-point micro-
biological oxygen demand (µBOD) method (Marsh & Manahan 1999). Individual
animals were placed inside µBOD vials (n = 8-10) and suspended in oxygen-
saturated, filtered seawater. The number of embryos or larvae used for each assay
depended upon the species of asteroid and developmental stage being investigated
(i.e., A. hodgsoni: 5 to 50 individuals per vial; O. meridionalis: 25 to 250 individuals
per vial). Vials with no animals present served as controls to correct for background
measurements of respiration. After a 6 to 9 h incubation period at -1
o
C, oxygen
tension was measured by injecting 500-µl of seawater from each vial onto a
62
Table 2
Amino acid composition of embryos and larvae of the Antarctic
sea stars A. hodgsoni and O. meridionalis.
Percent composition in protein
Amino acid A. hodgsoni O. meridionalis
Cysteine
†
0.4 ± 0.1
Methionine 1.1 ± 0.1 1.3 ± 0.2
Histidine 1.5 ± 0.2 1.9 ± 0.1
Tyrosine 2.8 ± 0.0 2.3 ± 0.1
Phenylalanine 4.0 ± 0.2 3.9 ± 0.0
Arginine 5.0 ± 0.4 4.9 ± 0.1
Isoleucine 5.4 ± 0.3 5.0 ± 0.1
Proline 7.0 ± 1.1 5.3 ± 0.1
Threonine 6.1 ± 0.2 6.0 ± 0.1
Lysine 5.6 ± 0.2 7.2 ± 0.1
Valine 5.3 ± 1.4 6.7 ± 0.1
Serine 7.0 ± 0.2 6.6 ± 0.1
Leucine 7.9 ± 0.2 8.0 ± 0.1
Alanine 8.3 ± 0.2 7.6 ± 0.2
Glycine 11.1 ± 1.0 9.8 ± 0.4
Aspartate, Asparagine
†
11.4 ± 0.1 10.9 ± 0.1
Glutamate, Glutamine
†
10.5 ± 0.3 12.2 ± 0.2
§
MW
p
(g mol
-1
) 124.2 ± 1.25 126.5 ± 0.41
Values are means ± SE, n = 3-7 developmental time points (A. hodgsoni: days 9, 17,
and 31 post-fertilization; O. meridionalis: 8, 23, 39, 55, 62, 83, and 118 days post-
fertilization)
†
During acid hydrolysis of proteins, asparagine and glutamine deaminate to form
aspartic acid and glutamic acid, respectively; cysteine and tryptophan are often lost
§
MW
p
, average mole-percent corrected molecular weight of amino acids in the
protein pool of embryos and larvae of A. hodgsoni and O. meridionalis. MW
p
calculated by, (1) multiplying the mole-percent value for each amino acid by its
corresponding molecular weight, and (2) summing all mole-percent corrected
weights of the amino acids
63
polarographic oxygen sensor housed inside a microrespiration chamber (Model RC-
100; Strathkelvin Instruments, Glasgow, UK). At the end of each assay, animals
were removed from their respective vials and enumerated.
Statistical analysis. Error values reported throughout this study represent ± 1 SE.
Prior to analysis of variance (ANOVA), data were first tested for heteroscadiscity
and for data expressed as percents, and arcsine root transformation was used to
achieve homogeneity (Sokal & Rohlf 1995). All significance levels were set at p <
0.05 using the “R” statistical software package (R Development Core Team, 2005).
Results
Alanine transport, free alanine pool, and total protein content
Rates of alanine transport in embryos and larvae of Acodontaster hodgsoni (Fig. 6a)
significantly increased between 3 and 39 days post-fertilization (one-way ANOVA,
F
1, 26
= 321.9, p < 0.001). In contrast, the free amino acid pool content of embryos
and larvae (Fig. 6b) showed a marked decrease during the same time period (one-
way ANOVA, F
1, 16
= 108.2, p < 0.001). The total protein content measured in eggs,
embryos, and larvae of A. hodgsoni ranged from 5.1 ± 0.11 to 3.2 ± 0.08 µg per
individual
(Fig. 6c). Protein content decreased steadily (days 0-39) at a rate of 25.3 ±
4.7 ng individual
-1
day
-1
(one-way ANOVA, F
1, 29
= 28.8, p < 0.001).
64
Figure 6
Alanine transport, free alanine pool, and total protein in embryos and
larvae of A. hodgsoni.
Change in (a) alanine transport rates; (b) free alanine pool; and (c) total protein
content during embryological and larval development. Transport values are slopes ±
SE (n = 3-4); free alanine pool and protein values are means ± SE (n = 3-6). Where
not shown, errors fell into the graphical representation of the data point. Dark grey:
eggs; grey symbols: embryos; black symbols: larvae.
Figure 6 (continued)
65
Age (days post-fertilization)
0 7 14 21 28 35 42
Alanine transport
(pmol individual
-1
h
-1
)
0
10
20
30
40
50
Age (days post-fertilization)
0 7 14 21 28 35 42
Free alanine pool content
(pmol individual
-1
)
0
300
600
900
1200
Age (days post-fertilization)
0 7 14 21 28 35 42
Total protein
(µg individual
-1
)
a
b
c
2
3
4
5
6
7
66
For Odontaster meridionalis, rates of alanine transport more than doubled between
days 4 and 7 (from 0.96 ± 0.12 to 2.4 ± 0.16 pmol embryo
-1
h
-1
), and remained
relatively unchanged until the larval stage at day 26 (Fig. 7a). Alanine transport rates
for unfed and fed larvae increased significantly during the period of development
studied (to 135 days) (one-way ANOVA, p < 0.05, respectively). The free alanine
pool content for embryos and larvae of O. meridionalis is shown in Fig. 7b. Between
days 4 and 26, free alanine pool content decreased 60% in embryos (from 37.8 ± 1.1
to 15.2 ± 0.74 pmol embryo
-1
). After day 30, the concentration of alanine in unfed
larvae (mean 20.5 ± 0.85 pmol larva
-1
) remained constant (one-way ANOVA, F
1, 9
=
3.1, p = 0.11). The free alanine pool content of fed larvae, however, increased
significantly throughout the remainder of the study to day 135 (one-way ANOVA,
F
1, 9
= 11.5, p < 0.01). During early development of O. meridionalis, total protein
content ranged from 262 ± 6.8 to 306 ± 4.7 ng individual
-1
from the egg (day 0) to
the larval stage at day 28 (Fig. 7c). Protein content of fed larvae increased steadily
(days 32-135) at a rate of 2.8 ± 0.25 ng individual
-1
d
-1
(one-way ANOVA, F
1, 15
=
124.9, p < 0.001). The amount of protein in unfed larvae did not change over >100
days tested (one-way ANOVA, F
1, 14
= 0.09, p = 0.77).
Protein synthesis and respiration rates
Rates of protein synthesis in embryos of Acodontaster hodgsoni increased between 5
and 23 days post-fertilization, and then slightly declined during larval stages of
67
Figure 7
Alanine transport, free alanine pool, and total protein in embryos and
larvae of O. meridionalis.
Change in (a) alanine transport rates; (b) free alanine pool; and (c) total protein
content in embryos and larvae. Transport values are slopes ± SE (n = 3-4); free
alanine pool and protein values are means ± SE (n = 3-6). Where not shown, errors
fell into the graphical representation of the data point. Dark grey symbols: eggs; grey
symbols: embryos; white symbols: unfed larvae; black symbols: fed larvae.
Figure 7 (continued)
68
Age (days post-fertilization)
0 30 60 90 120 150
Alanine transport
(pmol individual
-1
h
-1
)
0
2
4
6
8
Age (days post-fertilization)
0 30 60 90 120 150
Free alanine pool content
(pmol individual
-1
)
10
20
30
40
50
60
Age (days post-fertilization)
0 30 60 90 120 150
Total protein
(ng individual
-1
)
150
300
450
600
750
a
b
c
69
development (to 39 days) (Fig. 8a). Overall, there was a significant change in rates of
protein synthesis with developmental age (one-way ANOVA, F
1, 18
= 5.3, p < 0.05).
Fractional rates of protein synthesis for embryos and larvae were similar (paired-
sample Student’s t-test, p > 0.05) (Table 3). Rates of oxygen consumption for A.
hodgsoni are shown in Fig. 8b. Respiration rates did not significantly change
throughout the period of development studied (days 9-39) (one-way ANOVA; F
1, 9
=
2.5, p > 0.05).
Rates of protein synthesis during embryonic and larval development of Odontaster
meridionalis are shown in Fig. 9a. For embryos and unfed larvae, protein synthesis
rates did not significantly change during the period of development examined (days
4-135) (one-way ANOVA, F
1, 16
= 0.68, p = 0.42). However, once larvae were fed
(day 30), rates of protein synthesis increased significantly (one-way ANOVA, F
1, 9
=
34.2, p < 0.001). Fractional rates of protein synthesis in embryos and fed larvae were
similar (paired-sample Student’s t-test, p > 0.05), but were more than 1.5 times lower
in unfed larvae (paired-sample Student’s t-test, p < 0.05) (Table 3). Rates of oxygen
consumption for embryos and larvae of O. meridionalis are shown in Fig. 9b.
Respiration rates increased in embryos (days 4 to 26), and then leveled off during
larval stages of development (to day 135) (one-way ANOVA, F
1, 16
= 14.9, p < 0.01).
Respiration rates for fed larvae (days 30-135) increased significantly from 24 ± 3.2
to 82 ± 9.9 pmol O
2
larva
-1
h
-1
(one-way ANOVA, F
1, 9
= 33.7, p < 0.001).
70
Figure 8
Rates of protein synthesis and respiration in embryos and larvae of A.
hodgsoni.
Change in rates of (a) protein synthesis and (b) respiration during early development.
Protein synthesis (n = 5-8) and respiration (n = 6-10) values are slopes ± SE. Grey
symbols: embryos; black symbols: larvae. Where not shown, errors fell into the
graphical representation of the data point. Grey symbols: embryos; black symbols:
larvae. The observed data were described using a logistic model with the greatest
adjusted r
2
value. The equation for each model is as follows: protein synthesis (ng
individual
-1
h
-1
) = 17.8*(1 – e
-0.15*Age
) – 6.9 (adjusted r
2
= 0.45); respiration (pmol O
2
individual
-1
h
-1
) = 620.6*(1 – e
-0.21*Age
) – 481.3 (adjusted r
2
= 0.56).
Figure 8 (continued)
71
Age (days post-fertilization)
0 7 14 21 28 35 42
Protein synthesis
(ng individual
-1
h
-1
)
0
4
8
12
16
Age (days post-fertilization)
0 7 14 21 28 35 42
Respiration
(pmol O
2
individual
-1
h
-1
)
a
b
30
60
90
120
150
180
72
Table 3
Fractional rates of protein synthesis during
embryonic and larval development of A.
hodgsoni and O. meridionalis.
Sea star species
(stage/treatment)
Fractional protein
synthesis rate
(% h
-1
)
A. hodgsoni
Embryos 0.21 ± 0.03 (13)
Larvae 0.29 ± 0.02 (7)
O. meridionalis
Embryos 0.64 ± 0.09 (7)
Unfed larvae 0.35 ± 0.04 (11)
Fed larvae 0.52 ± 0.05 (11)
Values are means ± SE
Sample sizes for each stage/treatment in parentheses
73
Figure 9
Rates of protein synthesis and respiration in embryos and larvae of O.
meridionalis.
Change in rates of (a) protein synthesis and (b) respiration during early development.
Protein synthesis (n = 5-8) and respiration (n = 6-10) values are slopes ± SE. Grey
symbols: embryos; black symbols: larvae. Where not shown, errors fell into the
graphical representation of the data point. Grey symbols: embryos; white symbols:
unfed larvae; black symbols: fed larvae.
Figure 9 (continued)
74
Age (days post-fertilization)
0 30 60 90 120 150
Protein synthesis
(ng individual
-1
h
-1
)
0
2
4
6
Age (days post-fertilization)
0 30 60 90 120 150
Respiration
(pmol O
2
individual
-1
h
-1
)
0
20
40
60
80
100
a
b
75
Discussion
Antarctic marine invertebrates exhibit high rates of protein synthesis and low
metabolic rates (Marsh et al. 2001, Peck 2002, Clarke & Fraser 2004). Specific
examples of this phenomenon have been measured in both adults (limpet: Fraser et
al. 2002) and early developmental stages (sea urchin: Marsh et al. 2001; sea star:
Shilling & Manahan 1994, Peck & Prothero-Thomas 2002). The research presented
here supports the hypothesis that Antarctic organisms possess energetically efficient
metabolic processes, which enable them to survive under extreme environmental
conditions (Clarke 1983, Peck 2002, Clarke & Fraser 2004, Pearse & Lockhart
2004). Additionally, these findings provide insight to the developmental physiology
and energetics of Antarctic echinoderms with different life-history strategies.
Changes in protein synthesis
Protein synthesis plays a major role in setting metabolic rates in echinoderms (Marsh
et al. 2001, Pace & Manahan 2006). Measurements of in vivo rates of protein
synthesis can provide information on the developmental energetics of embryos and
larvae, as well as provide an index for establishing physiological state during later
stages of development (cf. Chapter 1, this dissertation). Recent work has shown that
embryos and larvae of the Antarctic sea urchin Sterechinus neumayeri have
potentially novel physiological adaptations that enhance their survival and growth
76
when temperatures are low and food is only sporadically available (Leong &
Manahan 1999, Marsh et al. 1999, 2001). To date, these physiological capabilities
are known for only early developmental stages of S. neumayeri and the sea star
Odontaster validus (Shilling & Manahan 1994) thus the question of whether they
extend to other Antarctic species remains open.
In this study, rates of protein synthesis were measured in the embryos and larvae of
the asteroids Acodontaster hodgsoni and O. meridionalis. In A. hodgsoni, a species
with a nonfeeding mode of development, protein synthesis increased nearly 5-fold in
embryos (2.7 to 12.7 ng protein embryo
-1
h
-1
) and then stabilized during larval stages
of development (Fig. 8a). In contrast, a different trend was observed for rates of
protein synthesis in O. meridionalis, which has a feeding mode of development.
Despite a more than 3-fold increase (0.8 to 2.7 ng protein embryo
-1
h
-1
) in rates of
protein synthesis during the onset of gastrulation (~day 7), the synthesis of proteins
in embryos and unfed larvae of O. meridionalis remained relatively constant during
development (Fig. 9a). For fed larvae, however, rates of protein synthesis increased
nearly 6-fold (0.8-4.5 ng larva
-1
h
-1
) when provided with algal food. Differences
between the mean fractional rates of protein synthesis for both species at 0.25% (A.
hodgsoni) and 0.50% (O. meridionalis) were different by 50% (Table 3). This
difference was statistically significant (two-way ANOVA, F
1, 45
= 4.3, p = 0.045;
Table 3). This range of fractional rates of protein synthesis measured at -1 °C is
consistent with other rates reported for other species of Antarctic sea stars (O.
77
validus at 0.1-1.1% h
-1
: Pace 2002; Porania antarctica at 0.1-6.0% h
-1
, Appendix I,
this dissertation).
Fractional rates of protein synthesis measured during embryonic development in this
study were compared to those reported for a temperate species of asteroid (Asterina
miniata: 1.0% h
-1
, Pace 2002) with a similar egg size and life-history mode (Fig. 10).
This comparison was conducted to assess whether whole-organism protein
metabolism in Antarctic asteroids is compensated to low temperatures (i.e., rates at
low temperatures are higher than predicted based on comparable species measured at
higher temperatures) (see Clarke 1991 for discussion of these comparative issues).
When the fractional rate of protein synthesis reported for the temperate species is
calculated at -1 °C (as used in this study for Antarctic species), using a Q
10
of 2.0
(cf. Torres & Somero 1988), the rate measured for O. meridionalis is elevated by an
approximate factor of two over its temperate counterpart (0.64 and 0.33% h
-1
,
respectively). This analysis did not include A. hodgsoni, as there is no comparable
physiological data set for a temperate species of asteroid with a nonfeeding mode of
development. These findings demonstrate that fractional rates of protein synthesis in
embryos of O. meridionalis are indeed cold-adapted, as described by previous
investigations of polar organisms (Scholander et al. 1953, Wohlschlag 1964, Torres
& Somero 1988, Marsh et al. 2001).
Figure 10
Metabolic cold adaptation in embryos of Antarctic and temperate
asteroids.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Fractional protein synthesis (% h
-1
)
Rate
@
15 °C
Rate
@
-1.0 °C
Asterina
miniata
Odontaster
meridionalis
Rate
@
-1.0 °C
Fractional rates of protein synthesis measured during embryonic development of the
Antarctic asteroid O. meridionalis, and the temperate asteroid A. miniata (from Pace
2002). All values are means ± SE. The temperatures at which measurements were
made are indicated above each bar. Hatched bars represent fractional rates at -1.0 °C.
Q
10
extrapolation for A. miniata estimated using value of 2.0 (cf. Torres & Somero
1988).
78
79
Changes in metabolic rate
Metabolic rate, measured as the amount of oxygen consumed, represents the total
energy expenditure of an organism and provides the fuel for a number of basic
cellular processes such as the synthesis of proteins (Robertson et al. 2001, Pace &
Manahan 2006), tissue growth (Rombough 1994, Marsh et al. 1999), and the
maintenance of ion gradients (Leong & Manahan 1997, 1999, Maxime 2002).
Measurements of respiration are valuable predictors of metabolic energy
consumption during early life-history stages of development. For example, Shilling
& Manahan (1994) demonstrated that the low metabolic rates of embryos and larvae
of a variety of Antarctic echinoderms potentially allow for survival in the water
column for up to 5 years without food. Likewise, Strathmann et al. (2006) suggest
that low rates of respiration in tadpole larvae of the Antarctic ascidian Cnemidocarpa
verrucosa extend developmental rates in cold waters by nearly 4-fold as compared to
temperate species.
In this study, rates of oxygen consumption increased 3-fold (50.2 to 153.5 pmol O
2
embryo
-1
h
-1
) during embryogenesis in Acodontaster hodgsoni and then leveled off in
the nonfeeding larval stages of development (Fig. 8b). A similar trend was observed
for embryos and larvae of Odontaster meridionalis, which has a feeding mode of
development (Fig. 9b). Oxygen consumption steadily increased in embryos (from 2.5
to 17.6 pmol O
2
embryo
-1
h
-1
) and remained relatively constant for unfed larvae.
80
Once feeding was initiated (day 30), however, larval respiration rates increased more
than 3-fold over the period of development investigated. Overall, rates of oxygen
consumption observed for embryos and larvae in this study were as much as 3-times
lower than those reported for species of asteroids from temperate regions (cf. Hoegh-
Guldberg & Emlet 1997, Bryan 2004).
Physiological development in the cold
Few studies have examined the metabolic costs required for different modes of
development (Hoegh-Guldberg & Emlet 1997, Moreno & Hoegh-Guldberg 1999).
Species with planktotrophic life-history stages are provisioned with protein-rich
energy stores to fuel early stages of development until they are capable of feeding on
their own in the plankton (Turner & Lawrence 1979, Jaeckle 1995). Alternatively,
embryos and larvae with lecithotrophic life-history stages are provisioned with large
lipid reserves and are not required to feed in the water column (George et al. 1997,
Bertram & Strathmann 1998). Which factors might select for these different
developmental modes, however, is a longstanding debate in larval ecology (Thorson
1950, Strathmann 1978, 1985, Emlet et al. 1987, Pearse 1994).
Early ideas about the relationship between life-history patterns and latitudinal
gradients suggested that animals living in cold water should exhibit physiological
rates similar to those from warmer environments (reviewed by Pearse & Lockhart
81
2004). Simply stated, this view suggested that as environmental temperatures
decline, physiological rates should increase (e.g., Krogh 1934). As a result, marine
invertebrates with pelagic dispersing larvae would be more likely to adopt a
nonfeeding life-history strategy to accommodate periods when food is either limited
or completely absent (e.g., Orton 1920, Thorson 1950, Mileikovsky 1971). Recent
investigations have refuted these views though and suggest that factors other than sea
temperatures (e.g., biochemical adaptations; reviewed by Pörtner 2006) are
responsible for shaping the evolutionary life-histories of Antarctic species.
Results presented here support the hypothesis that Antarctic marine organisms have
unique physiological adaptations that enable them to survive and grow under
extreme environmental conditions. Despite concerns regarding physiological
comparisons between just two-species (see examples from mammalian literature,
Garland & Adolph 1994), designing experiments with many species of echinoderms
that are congeners found in Antarctic and temperate regions is not possible due to the
unique and endemic nature of the Antarctic species. Nonetheless, the comparisons
presented here provide the first description of protein synthesis rates from Antarctic
asteroids with different life-history strategies. Measurements of rate of protein
synthesis during early development for species living in different environments
provide physiological insights into rates and costs of development for stages that are
similar in size and form (e.g., embryos).
82
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implications for the use of egg size in life-history models. In: Stancyk SE
(ed) Reproductive ecology of marine invertebrates. University of South
Carolina Press, South Carolina, p 25-40
Vavra J, Manahan DT (1999) Protein metabolism in lecithotrophic larvae
(Gastropoda: Haliotis refescens). Biol Bull 196:177-186
Welborn JR, Manahan DT (1995) Taurine metabolism in larvae of marine molluscs
(Bivalvia, Gastropoda). J Exp Biol 198:1791-1799
Wohlschlag DE (1964) Respiratory metabolism and ecological characteristics of
some fishes in McMurdo Sound, Antarctica. In: Lee, MD (ed) Scale effects in
animal locomotion. Academic Press, New York, p 203-232
88
Chapter 3
Energetic cost of protein synthesis during early development in the
temperate sea urchin Strongylocentrotus purpuratus.
Abstract
During early stages of development, high mass-specific metabolic rates support
energetically-demanding processes such as the synthesis of proteins and the
maintenance of cellular ion gradients. In this study, the relationship was quantified
between these major biochemical processes and their role in ‘setting’ metabolic rates
during embryonic and larval development. Oxygen consumption and protein
synthesis rates were measured in the sea urchin Strongylocentrotus purpuratus. At
the onset of gastrulation, there was a ~3-fold increase in mass-specific metabolic
rate, while rates of protein synthesis did not change in proportion. The energetic cost
of protein synthesis during early development was 8.6 J (mg protein)
-1
. Combining
data for protein synthesis, respiration and previously published rates of ATP
utilization by the sodium pump, a series of energy budgets were calculated for
different stages of development. Protein synthesis and the sodium pump accounted
for ~90% of total respiration throughout development. These findings suggest that
other important physiological processes, such as locomotion and feeding, require
small amounts of energy.
89
Introduction
Species of marine invertebrates with a planktotrophic life-history mode are
provisioned with energy stores to fuel the embryonic stages of development until
they are capable of feeding in the plankton (reviewed by Levin & Bridges 1995). In
echinoderms, the endogenous nutrients (e.g., protein, lipid) provided from the egg to
the embryo are required for successful development to the larval stage (Turner &
Lawrence 1979, Cognetti 1982, Emlet et al. 1987, Villinski et al. 2002). Thus, given
a finite amount of maternally endowed reserves, the total amount of energy utilized
by embryos can have profound consequences on larval performance and survival
(e.g., Lucas et al. 1979, Pechenik 1987, Bertram & Strathmann 1998, Wendt 2000,
Moran & Manahan 2003).
During early development, metabolic energy is allocated toward basic cellular
processes such as the synthesis of proteins (Hawkins 1991, Vavra & Manahan 1999),
production of new tissues (Rombough 1994, Marsh et al. 1999), and the maintenance
of ion gradients (Mitsunaga-Nakatsubo et al. 1992, Leong & Manahan 1997). Energy
also is potentially allocated towards physiological processes such as the production
of secondary metabolites (Lindquist & Hay 1996, McClintock & Baker 1997) and
swimming (Qian et al. 1990, Wendt 2000, Strathmann et al. 2006). As previously
mentioned, the amount of metabolically available energy within an embryo can
effect the growth and survival potential of later stages of development (Boulekbache
90
1981, Shilling & Bosch 1994). For example, both protein synthesis and the sodium
pump account for ~75% of the total energetic budget in embryos and larvae of the
sea urchin Lytechinus pictus (Pace & Manahan 2006) thus leaving only a small
proportion of energy available for other biological processes. Whether such internal
partitioning of metabolic energy is common among other temperate echinoderms is
uncertain since the physiological costs of development in marine invertebrates are
not fully understood.
The present study was undertaken to investigate how organismal metabolic budgets
are affected by changing ontogenetic and cellular requirements, as well as to define
the energetic cost of metabolism during the transition from embryonic (prefeeding)
to larval (feeding) stages of development in the temperate sea urchin
Strongylocentrotus purpuratus. The early developmental stages of S. purpuratus
were chosen for this study because of their experimental tractability for
investigations of physiological rate processes (see Leong & Manahan 1997, Pace &
Manahan 2006). For embryos and larvae of S. purpuratus, the rates of (1) oxygen
consumption; (2) protein synthesis; and (3) the energetic cost of protein synthesis
during early development were determined.
91
Materials and Methods
Animal cultures. Gravid adults of the sea urchin Strongylocentrotus purpuratus
were induced to spawn with intracoelomic injections of 0.5 M KCl. Embryos and
larvae were maintained as separate cohorts (n = 3). After verifying the fertilization
success (>95%) of each cohort, embryos were placed into 20-l containers at a
concentration of 5-10 ml
-1
. Each chamber was mixed with a rotary paddle and
maintained in UV-irradiated, filtered (0.2 µm pore size) seawater maintained at 15
o
C. Known numbers of animals were periodically removed at different stages of
embryonic and larval development from culture containers for parallel measurements
of protein synthesis and respiration.
Respiration. Rates of oxygen consumption for embryos and larvae of
Strongylocentrotus purpuratus were measured using the micro-biological oxygen
demand (µBOD) method (see Marsh & Manahan 1999 for details). Individual
animals (25-500, depending on developmental stage) were placed inside µBOD vials
(n = 8-10) containing oxygen-saturated, filtered seawater and incubated at 15
o
C for
5 to 6 h. Control vials with no animals present were set up in parallel to correct for
background respiration. At the end of each incubation period, oxygen tension was
measured by injecting 500-µl of seawater from each vial onto a polarographic
oxygen sensor housed inside a microrespiration chamber (Model RC-100;
Strathkelvin Instruments, Glasgow, UK). Embryos or larvae were then removed from
92
their respective vials and enumerated. Respiration rates were converted to energy
units using an average oxyenthalpic value of 484 kJ (mol O
2
)
-1
(Gnaiger 1983).
Amino acid transport and protein synthesis. Time course experiments were
conducted to determine the rates of amino acid transport and protein synthesis in
embryos and larvae of Strongylocentrotus purpuratus (see Manahan 1983, Pace &
Manahan 2006 for details). Known numbers of animals (750 ml
-1
) were placed into
20-ml glass scintillation vials and exposed to a 10 to 15 µM solution of
14
C-alanine
(PerkinElmer, Wellesley, MA; specific activity 6.2 MBq µmol
-1
) in 12-ml filtered
seawater. Vials were incubated at 15
o
C for ~30 min, during which time aliquots
(500-1000 µl) were removed, collected onto a Nuclepore filter membrane (8 µm pore
size), and washed 2 times with sterile seawater (15-ml) to remove unincorporated
radioactivity.
Rates of protein synthesis were calculated using the specific activity of
14
C-alanine
in the intracellular free amino acid pool and the incorporation of radioactivity into
the trichloroacetic acid (TCA)-insoluble protein fraction of each sample. The free
amino acid pools of embryos and larvae were extracted with ethanol (70%) and the
specific activity of alanine fraction determined with reverse-phase high performance
liquid chromatography (HPLC) (Welborn & Manahan 1995). All samples containing
radioactive material were mixed with scintillation cocktail (EcoLume, MP
Biomedicals) and counted with a liquid scintillation counter. The mole-percent of
93
alanine (7.8%) incorporated into protein was converted to units of protein mass
based on the amino acid composition of protein (average molecular weight 125.7 g
mol
-1
) in embryonic and larval (12- and 72-h-old, respectively) stages of S.
purpuratus (Table 4). Fractional rates of protein turnover were calculated by
expressing protein synthesis rates as a percentage of total protein content for a given
stage of development. Protein content of embryos and larvae was measured using a
modified Bradford assay (Jaeckle & Manahan 1989).
Cost of development. The energetic cost of protein synthesis in embryos (18- and
36-h-old) and larvae (72-h-old) of Strongylocentrotus purpuratus was determined by
quantifying the difference in protein synthesis and metabolic rates in the presence
and absence of the protein synthesis inhibitor emetine (Sigma Chemical Co.). In
eukaryotes, emetine binds to ribosomes and blocks the process of peptide elongation
during protein synthesis (Grollman 1968). Preliminary experiments with embryos
(18-h-old) examined the relationship between the rates of
14
C-alanine incorporated
into protein with increasing concentrations of emetine. Specifically, the lowest
effective doses of emetine (50 µM) inhibited >90% of radioactivity incorporated into
protein (Fig. 11). The concentrations of emetine used in this study are within the
range known to inhibit protein synthesis in other species of echinoderms (e.g., Pace
& Manahan 2006).
94
Table 4
Amino acid composition of protein in embryos
and larvae of the sea urchin S. purpuratus.
Amino acid Percent composition in protein
Cysteine
†
0.7 ± 0.16
Methionine 0.7 ± 0.18
Histidine 1.9 ± 0.07
Tyrosine 1.9 ± 0.04
Phenylalanine 3.7 ± 0.07
Arginine 4.9 ± 0.00
Isoleucine 4.9 ± 0.13
Proline 5.2 ± 0.09
Threonine 6.1 ± 0.01
Lysine 6.7 ± 0.30
Valine 7.1 ± 0.53
Serine 7.5 ± 0.26
Leucine 7.6 ± 0.21
Alanine 7.8 ± 0.25
Glycine 9.8 ± 0.99
Aspartate, Asparagine
†
11.7 ± 0.73
Glutamate, Glutamine
†
11.9 ± 0.16
§
MW
p
(g mol
-1
) 125.7 ± 0.67
Values are means ± SE, n = 2 developmental time points
(12 and 72 hours post-fertilization).
†
During acid hydrolysis of proteins, asparagine and
glutamine deaminate to form aspartic acid and glutamic
acid, respectively; tryptophan is often lost.
§
MW
p
, average mole-percent corrected molecular weight of
amino acids in the protein pool of embryos and larvae of S.
purpuratus. MW
p
calculated by multiplying the mole-
percent value for each amino acid by its corresponding
molecular weight, and summing all mole-percent corrected
weights of the amino acids.
Figure 11
Change in rates of
14
C-alanine incorporated into embryonic protein of S.
purpuratus with increasing concentrations emetine.
Emetine concentration (µM)
0 100 200 300 400
Radioactivity in protein (Bq embryo
-1
h
-1
)
0
3
6
9
12
15
Change in rates of
14
C-alanine incorporated into protein in embryos (18-h-old) of S.
purpuratus with increasing concentrations of emetine. Values are means ± SE (n = 1-
2). Dose response data fitted to an exponential decay equation (see Results for
details).
95
96
Statistical analysis. All data are presented as either means or slopes of linear
regressions with the appropriate standard error (SE). Statistical analyses were
undertaken with the “R” statistical software package (R Development Core Team,
2005). Data were tested for heteroscedasticity between treatments, as judged by an
equality of variance test (F test). Statistical significance was accepted when p < 0.05.
Results
Free alanine pool and total protein content
The amount of alanine in the free amino acid pool of embryos and larvae of
Strongylocentrotus purpuratus (Cohort 2) did not significantly change between 6 and
72 hours post-fertilization (ANOVA of linear regression: free alanine pool (pmol
individual
-1
) = 0.001*(Age) + 1.61, p > 0.05; n = 8) (Fig. 12a). The protein content
of embryos and larvae from all cohorts did not change (mean 15.3 ± 0.12)
throughout embryogenesis (ANOVA of linear regression: total protein (ng
individual
-1
) = 0.01*(Age) + 14.5, p > 0.05; n = 18) (Fig. 12b).
Metabolic and protein synthesis rates
Metabolic rates were compared amongst embryos and larvae from two separate
cohorts of S. purpuratus (Fig. 13a). For Cohort 1, there was a significant increase in
97
Figure 12
Free alanine pool and total protein content during early development of
S. purpuratus.
Change in (a) free alanine pool and (b) protein content during development of
embryos and larvae of S. purpuratus. Values are means ± SE (n = 3-6). Where not
shown, errors fell within the graphical representation of the data point. Black
symbols: Cohort 1; grey symbols: Cohort 2; white symbols: Cohort 3.
Figure 12 (continued)
98
Age (days post-fertilization)
0 122436486072
Total protein (ng individual
-1
)
12
14
16
18
Age (days post-fertilization)
0 122436 486072
Free amino acid pool content
(pmol ala individual
-1
)
0.5
1.0
1.5
2.0
2.5
3.0
a
b
99
Figure 13
Metabolic and protein synthesis rates during early development of S.
purpuratus.
Change in (a) metabolic and (b) protein synthesis rates during embryonic and larval
development of S. purpuratus. Metabolic rate values are slopes ± SE (n = 8-10);
protein synthesis values are means ± SE (n = 5-6). Metabolic rate data were
described using a ‘step-function’ sigmoidal model with the greatest adjusted r
2
value
(see text). Where not shown, errors fell within the graphical representation of the
data point. Black symbols: Cohort 1; grey symbols: Cohort 2; white symbols: Cohort
3.
Figure 13 (continued)
100
Age (hours post-fertilization)
0 122436486072
Respiration (µJ individual
-1
h
-1
)
0
2
4
6
8
Age (hours post-fertilization)
0 122436 486072
Protein synthesis (ng individual
-1
h
-1
)
b
a
0.0
0.2
0.4
0.6
0.8
1.0
101
metabolic rate (from 0.68-1.9 µJ embryo
-1
h
-1
) between 4 and 28 hours post-
fertilization (two-way ANOVA, F
3, 22
= 7.03, p < 0.003). When pooled with data for
blastulae from another spawn (Cohort 3), no significant increase in metabolic rate
during early embryogenesis was measurable (one-way ANOVA, F
1, 3
= 1.72, p >
0.05). Among later stages of development, metabolic rates for both Cohorts 1 and 3
remained unchanged after gastrulation (36-72 h-old) at a mean rate of 5.7 ± 0.12 µJ
individual
-1
h
-1
(one-way ANOVA, F
1, 4
= 0.03, p > 0.05). The important finding here
is that there was a ~3-fold increase in metabolic rate after the onset of gastrulation at
~36h (one-way ANOVA, F
1, 9
= 20.2, p < 0.002). This change in metabolic rate can
be described with a ‘step-function’ sigmoidal model with the greatest adjusted r
2
value, where metabolic rate (µJ individual
-1
h
-1
) = 1.8 + 3.8*(Age)
59.6
*[(30.1)
59.6
+
(Age)
59.6
]
-1
.
Rates of protein synthesis were measured in three separate cohorts of S. purpuratus
for developmental stages spanning 4 to 72 hr post-fertilization (Fig. 13b). For
developmental stages in Cohorts 1 and 2, protein synthesis rates did not change
significantly with age (two-way ANOVA to compare regressions, F
1, 12
= 1.83, p >
0.05). The intercepts of the lines for each of these cohorts were significantly different
(two-way ANOVA, F
1, 12
= 54.7, p < 0.001), showing differences in absolute rates of
protein synthesis starting with gametes from different adults. For these data, protein
synthesis rates for each cohort are given as separate linear regressions (Fig. 3b). The
average rate of protein synthesis for embryos and larvae were 0.26 ± 0.04 and 0.51 ±
102
0.03 ng individual
-1
h
-1
for Cohorts 1 and 2, respectively. The average rate of protein
synthesis from a separate spawn (Cohort 3) of embryos and larvae was 0.38 ± 0.06
ng individual
-1
h
-1
. Specifically, this value fell within the range of rates for Cohort 1
and 2. Fractional rates of protein turnover for embryos and larvae from all cohorts of
S. purpuratus ranged from 2.6-3.5% h
-1
(mean 2.9 ± 0.10% h
-1
) for different stages
of development (4 to 72-h-old).
Cost of protein synthesis measurements
Metabolic costs of embryos and larvae of Strongylocentrotus purpuratus were
determined with the protein synthesis inhibitor, emetine. For each developmental
stage (i.e., blastula, gastrula, and pluteus) examined in this study, emetine did not
significantly affect rates of
14
C-alanine transport (Table 5), which is an important
step for verifying that non-specific cellular processes were not altered in the presence
of this drug. The change in rates of protein synthesis and respiration for specific
stages of development is shown in Fig. 14. The maximum height of each histogram
bar represents either the metabolic or protein synthesis rate when emetine was
absent, whereas the solid component represents the rate when emetine was present.
Based on the difference between each of these respective rates, the energetic cost
was determined by calculating the proportion of metabolic rate attributed to the rate
of protein synthesis. For each time point analyzed, the energetic cost was as follows:
103
Table 5
Rates of alanine transport for embryos and larvae of S. purpuratus in
the presence and absence of emetine.
Stage
Age
Treatment
Alanine transport rate
(pmol individual
-1
h
-1
)
ANOVA
(p-value)
Blastula 18-h-old
Control
Emetine (50 µM)
1.61 ± 0.06
1.62 ± 0.05
0.87
ns
Blastula 18-h-old
Control
Emetine (100 µM)
1.61 ± 0.06
1.60 ± 0.12
0.95
ns
Gastrula 36-h-old
Control
Emetine (100 µM)
2.83 ± 0.35
3.19 ± 0.38
0.89
ns
Pluteus 72-h-old
Control
Emetine (100 µM)
3.96 ± 0.35
4.02 ± 0.04
0.53
ns
Values are slopes ± SE of the increase in
14
C-alanine with time (n = 3 to 4 time course
measurements).
Numbers in parentheses indicate concentration of emetine for each experiment
ns
indicates alanine transport rate for a given developmental stage is not statistically
significantly different (ANOVA, p > 0.05)
104
Figure 14
Protein synthesis and metabolic rates during early development of S.
purpuratus in the presence and absence of emetine
Change in metabolic rate (MR) and protein synthesis (PS) in embryos and larvae
when emetine was either absent (total height of each bar) or present (height of solid
component). Cost of protein synthesis using emetine: (a) blastula, 8.2 and 8.9 J (mg
protein)
-1
(50 and 100 µM, respectively); (b) gastrula, 8.9 J (mg protein)
-1
(100 µM);
(c) pluteus, 8.3 J (mg protein)
-1
(100 µM). All values are means ± SE; n = 6,
metabolic rate; n = 6, protein synthesis.
Figure 14 (continued)
105
Metabolic rate (µJ embryo
-1
h
-1
)
0
1
2
3
4
Protein synthesis (ng embryo
-1
h
-1
)
0.0
0.1
0.2
0.3
0.4
Protein synthesis (ng embryo
-1
h
-1
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Metabolic rate (µJ embryo
-1
h
-1
)
0
1
2
3
4
5
6
7
Protein synthesis (ng larva
-1
h
-1
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Metabolic rate (µJ larva
-1
h
-1
)
0
1
2
3
4
5
6
a. Blastula (18-h-old)
b. Gastrula (36-h-old)
c. Pluteus (72-h-old)
PS MR
PS MR
PS MR
PS MR
50 µM 100 µM
100 µM
100 µM
106
blastula, 8.2 and 8.9 J (mg protein)
-1
(50 and 100 µM emetine, respectively);
gastrula, 8.9 J (mg protein)
-1
(100 µM emetine); pluteus, 8.3 J (mg protein)
-1
(100
µM emetine).
Discussion
Rates of protein synthesis and respiration are important physiological parameters for
assessing the energetic expenditure required for embryonic and larval development.
However, the relationship between these physiological processes and their role in
establishing metabolic rates during early development are not fully understood. For
example, changes in metabolic rate may reflect periods of increased energetic
demand that are required not only for specific stages of development, but are
necessary to support physiologically expensive processes such as the biosynthesis of
macromolecules and the maintenance of intracellular ion concentrations (Hochachka
1988, Leong & Manahan 1997, 1999, Marsh et al 1999, 2001, Pace & Manahan
2006). The present study was undertaken to examine how metabolic energy is
partitioned during early stages of development of the temperate sea urchin
Strongylocentrotus purpuratus, and provides a framework with which to compare the
developmental physiology of other species of echinoderms.
107
Metabolic activity during development
The total metabolic expenditure for embryonic and larval stages of development of
Strongylocentrotus purpuratus measured in this study ranged from 0.7-6.0 µJ
individual
-1
h
-1
(Fig. 13a) and is similar to previous studies (measured as moles O
2
consumed) with this species (Shilling & Manahan 1990, 1994, Hoegh-Guldberg &
Manahan 1995, Leong & Manahan 1997, Marsh & Manahan 1999). The increase in
metabolic rate observed between 28 and 36 hours post-fertilization is explained by
the onset of the gastrulation phase of development. Increased metabolic activity is
required to support the reorganization of the blastula embryo to an advanced
(triploblastic) stage of development. Such bursts of metabolic activity are reported
for other temperate (Leong & Manahan 1997, Mitsunaga-Nakatsubo et al. 1992) and
polar (Marsh et al. 2001) species of echinoderms.
Although stage-specific rates of protein synthesis were variable among different
cohorts of embryos and larvae, no significant changes were observed during the
period of development examined in this study (Fig. 13b). Pace & Manahan (2006)
report a similar finding for the temperate sea urchin Lytechinus pictus, in which
protein synthesis remained constant during embryogenesis. Measured rates of protein
synthesis for all cohorts of S. purpuratus presented in this study (mean 0.38 ± 0.03
ng individual
-1
h
-1
) agree with those reported previously for this species (cf. 0.3 to
0.9 ng embryo
-1
h
-1
, reviewed by Davidson 1976, Goustin & Wilt 1980).
108
Fractional rates of protein turnover (1.8 ± 0.17% h
-1
) during early stages of
development were nearly 2-times the literature value (cf. 1.1% h
-1
, Goustin & Wilt
1980). Previous reports on rates of turnover in temperate species of sea urchins are
based on measurements of protein content obtained with the Lowry assay, which can
overestimate the total amount of protein by more than 50% (Chu & Casey 1986,
Berges et al. 1992). If the percentage of total body protein turned over in 18-h-old
blastula-stage embryos (mean protein synthesis rate 0.35 ng embryo
-1
h
-1
; Fig. 13b)
measured in this study is recalculated using the same protein content value (cf. 40 ng
embryo
-1
) as Goustin & Wilt (1980), the new rate (0.9% h
-1
) would be nearly
identical to the value reported previously. This recalculated value is consistent with
turnover rates (estimated with the Lowry protein assay) for other species of
temperate sea urchins (e.g., Arbacia punctulata: 1.9% h
-1
, Fry & Gross 1970;
Lytechinus pictus: 0.8% h
-1
, Berg & Mertes 1970).
Metabolic cost of protein synthesis
The average cost of protein synthesis for embryos and larvae of Strongylocentrotus
purpuratus was 8.6 J (mg protein)
-1
(Fig. 14). This cost is based on measurements of
protein synthesis using the inhibitory drug, emetine. The advantage of this approach
is that it can be used to measure the cost of protein synthesis for specific stages of
development, which is not possible with other methods for calculating metabolic
costs. For instance, an energetic cost estimate can be calculated from the slope of the
109
relationship between respiration and protein synthesis (cf. correlative method,
Pannevis & Houlihan 1992, Marsh et al. 2001).
Costs of protein synthesis for S. purpuratus presented in this study are consistent
with the metabolic costs reported for embryos and larvae of the temperate sea urchin
Lytechinus pictus (8.4 J (mg protein)
-1
, Pace & Manahan 2006). Results from
experiments presented here suggest that the energetic cost of protein synthesis in S.
purpuratus is fixed and does not change in response to the developmental stage or
size of the animal (cf. variable cost in fish, Smith & Houlihan 1995). The cost of
protein synthesis reported in this study is in a range similar to a variety of organisms
(mussels: 11.4 J mg
-1
, Hawkins et al. 1989; cod fish: 8.7 J mg
-1
, Lyndon et al. 1989;
chickens: 5.4 J mg
-1
, Aoyagi et al. 1988).
Metabolic partitioning during development
Protein synthesis and the sodium pump play important metabolic roles during the
development of sea urchins (Leong & Manahan 1997, 1999, Marsh et al. 2001, Pace
& Manahan 2006). Based on the percentage of total metabolism used by these
physiological processes, an energetic budget can be established for specific stages of
development of S. purpuratus (see Fig. 14, metabolic rate data for blastulae,
gastrulae, and pluteus-stage larvae). Leong and Manahan (1997) measured the
physiologically-active portion of the sodium pump during development of S.
110
purpuratus and found that this enzyme accounted for 14 to 37% of metabolism
(depending on stage of development). Based on the cost of protein synthesis
measured (from Figure 14) of 8.6 J (mg protein)
-1
, the percentage of metabolism
accounted for by protein synthesis was 82% for blastulae, 52% for gastrulae, and
57% for larvae (Fig. 15). This calculation is based on the average rate of protein
synthesis (Fig. 13b) for all cohorts examined: [average rate of protein synthesis (ng
individual
-1
h
-1
) = 0.45 – 0.002*(Age), n = 19)]. When combined, both protein
synthesis and the sodium pump accounted for up to 96% of the total metabolic
expenditure of embryonic and larval stages of S. purpuratus. These results are
consistent with the allocation of energy for major metabolic processes that have been
established for temperate and polar species of sea urchins (at 15 ºC Lytechinus pictus
= 59-77%, Pace & Manahan 2006; at -1.5 ºC Sterechinus neumayeri = 68-81%,
Marsh et al. 2001).
Patterns of metabolic partitioning are used to determine the major processes
responsible for energetic expenditures during development. As demonstrated in this
study, protein synthesis and the sodium pump accounted for ~90% of metabolic rate
through early development of S. purpuratus (Fig. 15). There is a dramatic 3-fold
increase in respiration rate around gastrulation (Fig. 13a). This increase in respiration
cannot be explained by an increase in organic mass, as in these prefeeding stages of
development mass would not be expected to increase and, in fact, total protein
content remained constant (Fig. 12b). What biochemical process might account for
111
Figure 15
Metabolic energy expenditure during early development of S. purpuratus
Total energy metabolism during early developmental stages (18- to 72-h-old) of S.
purpuratus was calculated from metabolic rate data (Fig. 4, see text). Percentage of
total metabolism attributed to protein synthesis (grey area) was calculated for each
developmental stage (age) given above each pie chart from the linear equation:
protein synthesis (ng individual
-1
h
-1
) = 0.45 – 0.002*Age (see text). These values
were converted into energetic units using an energetic cost of 8.6 J (mg protein)
-1
calculated in this study. Values for sodium pump activity (‘ATPase’ = white area)
were taken from Leong & Manahan (1997). “Unaccounted” (solid area) = fraction of
metabolism not accounted for by protein synthesis or sodium pump.
Figure 15 (continued)
112
Blastula (18-h-old)
MR = 3.0 µJ individual
-1
h
-1
82% Protein synthesis
14% ATPase
4% Undefined
Gastrula (36-h-old)
MR = 5.8 µJ individual
-1
h
-1
52% Protein synthesis
32% ATPase
16% Undefined
Pluteus (72-h-old)
MR = 5.1 µJ individual
-1
h
-1
57% Protein synthesis
37% ATPase
6% Undefined
113
the observed rapid increase in respiration? Protein synthesis cannot responsible, as
rates of synthesis did not change in proportion to respiration for this stage of
development (Fig. 13b, cf. Fig. 13a). Hence, the proportion of metabolic rate
accounted for by protein synthesis is constant for early developmental stages (<28h,
Fig. 13a). With the 3-fold increase in respiration rate for developmental stages at 36-
h-old and later, the proportion of metabolic rate accounted for by protein synthesis
decreased in these stages. For instance, protein synthesis accounted for 82% of
respiration in 18-h-old blastulae and decreased to 52% in gastrulae (Fig. 15).
Concomitantly, the sodium pump accounted for an increased percent of respiration in
these developmental stages at 14% and 32% in blastulae and gastrulae, respectively.
The degree to which changes in metabolism affect the physiological potential (e.g.,
development, growth, feeding, locomotion) of embryos and larvae is poorly
understood. A major conclusion from the current study is that all other metabolic
processes, other than protein synthesis and the sodium pump, could only account for
between 4 to 16% of respiration throughout embryogenesis to the larval stage
(average for the “undefined” fraction of metabolism for 3 developmental stages
studied was 9%). This narrowing of metabolic options, in terms of energy use for all
other biological processes at 9%, suggests that some important physiological
processes – such as locomotion and feeding – require very small amounts of energy.
114
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135
Appendix I
Rates of protein synthesis and respiration in embryos and larvae
of the Antarctic sea star Porania antarctica.
Introduction
The developmental mode and life-history strategies of Antarctic echinoderms are
relatively well known (Clark 1963, Bosch & Pearse 1990, Pearse et al. 1991).
However, the metabolic demands that underlie the development of embryos and
larvae in this extreme environment are not fully understood. Previous investigations
of the energetics and physiological rates of early development in Antarctic
echinoderms have focused primarily on just two species: the sea star Odontaster
validus (e.g., McClintock et al. 1988, Peck & Prothero-Thomas 2002) and the sea
urchin Sterechinus neumayeri (e.g. Leong & Manahan 1999, Marsh et al. 2001). As a
result, the degree to which these findings extend to embryos and larvae of other
echinoderms, and polar species in general, is equivocal.
The purpose of this study was to examine the developmental physiology of
embryonic and larval development in the sea star Porania antarctica of which very
little is known. Field collections of P. antarctica in the shallow-water habitats of
136
McMurdo Sound, Antarctica are rare (see Bosch 1989), and only 6 individuals (out
of 39 SCUBA dives) were collected for the experiments presented here. Females
from this species of asteroid produce large eggs (~550 µm) and larvae have a feeding
(planktotrophic) mode of development (Bosch 1989). In this study, measurements of
(1) protein synthesis; (2) oxygen consumption; and (3) protein content were
quantified in embryos and larvae of P. antarctica.
Materials and Methods
Spawning and culturing methods. Adults of the sea star Porania antarctica were
collected by SCUBA during November 2002 at New Harbor, located at the mouth of
the Taylor Dry Valley on the Antarctic continent. Animals were transferred to the
Crary Laboratory aquarium facility at McMurdo Station, placed in running seawater
tanks and maintained at constant temperature (-1° C). Gametes were obtained by
intracoelomic injections of 1 mM 1-methyladenine. Eggs were pooled from 3
different females and fertilized with sperm from 1 male. After verifying that
fertilization success was >95%, embryos were placed into 20-l culture containers at a
concentration of 5 ml
-1
. Cultures were maintained in filtered (0.2 µm pore-size)
seawater, which was replaced every 3-4 days. Six developmental stages, from 1-day-
old embryos to 20-day-old larvae, were used for studies presented here.
137
Physiological rate measurements. The methods used to quantify rates of oxygen
consumption, alanine transport and protein synthesis are described in detail in
Chapters 2 and 3 of this dissertation. Measurements of respiration rates were
determined using the end-point micro-biological oxygen demand (µBOD) method
(see Marsh & Manahan 1999). Alanine transport and protein synthesis rates were
measured following the method outlined by Vavra & Manahan (1999). However,
modifications were made to enable the use of this protocol with the embryos and
larvae of Porania antarctica, which are relatively large and positively buoyant
(Bosch 1989). Known numbers of individuals (150-200) were removed from cultures
and placed in 1 ml of filtered seawater (20 ml glass vial) in which
14
C-alanine was
added to a final concentration 15 µM (PerkinElmer, Wellesley, MA; specific activity
6.1 MBq µmol
-1
). Vials were incubated at -1° C for 1.5 to 2 h, during which time
animals were removed (4-10 individuals per 10-20 µl aliquot; 5-7 time points) and
placed through three successive washes with 20-ml filtered seawater (60 ml total
volume). Each sample was then collected onto a 8 µm (pore size) Nuclepore filter
membrane. Preliminary experiments demonstrated that the concentration of
radioactivity remaining in the final wash vial was 10
9
times less than at the start of
the experiment (8.3×10
-9
and 92.5 Bq ml
-1
, respectively), thus confirming that
unincorporated isotope was sufficiently removed from embryos and larvae.
Rates of protein synthesis were calculated using the mole-percent of alanine (8.3%)
and average molecular weight (124.3 g mol
-1
) of amino acids in hydrolyzed protein
138
of embryos and larvae of P. antarctica (Table 6). Fractional rates of protein turnover
were calculated as the percentage of protein newly synthesized hour
-1
(i.e., the ratio
between protein synthesis rate and protein content for a specific developmental
stage). Protein content was analyzed using a modified Bradford assay (Jaeckle &
Manahan 1989).
Statistics. All statistical analyses were performed with the “R” statistical software
package (R Development Core Team, 2005). Error values represent ± 1 SE.
Results and Discussion
Alanine transport, free amino acid pool, and protein content
Rates of alanine transport in embryos and larvae of Porania antarctica (Fig 16a)
increased significantly between 1 and 20 days post-fertilization (ANOVA, p = 0.04).
These data were described using a logistic growth function: transport rate (pmol
alanine individual
-1
h
-1
) = 38.8(1 – e
-0.07*Age
) (adjusted r
2
= 0.72). In contrast, the
concentration of alanine in the free amino acid pools of embryos and larvae (Fig.
16b) showed a marked decrease during the same time period (ANOVA of linear
regression: free amino acid pool (pmol alanine individual
-1
) = 761.3 – 21.7*(Age), p
< 0.01; n = 5).
139
Table 6
Amino acid composition and mole-percent of
amino acids in proteins of embryos and larvae of
the Antarctic sea star P. antarctica.
Amino acid Percent composition in protein
Methionine 1.4 ± 0.3
Histidine 1.6 ± 0.1
Tyrosine 2.9 ± 0.0
Phenylalanine 3.4 ± 0.7
Arginine 5.1 ± 0.2
Isoleucine 5.1 ± 0.2
Proline 2.5 ± 1.3
Threonine 6.0 ± 0.1
Lysine 5.8 ± 0.1
Valine 5.9 ± 0.7
Serine 7.9 ± 0.6
Leucine 8.0 ± 0.1
Alanine 8.3 ± 0.7
Glycine 12.2 ± 0.8
Aspartate, Asparagine
†
11.9 ± 0.8
Glutamate, Glutamine
†
12.0 ± 0.5
§
MW
p
(g mol
-1
) 124.3 ± 1.00
Values are means ± SE, n = 3 developmental time points
(9, 17, and 22 days post-fertilization)
†
During acid hydrolysis of proteins, asparagine and
glutamine deaminate to form aspartic acid and glutamic
acid, respectively; cysteine and tryptophan are often lost
§
MW
p
, average mole-percent corrected molecular weight of
amino acids in the protein pool on embryos and larvae of
P. antarctica. MW
p
calculated by, 1) multiplying the mole-
percent value for each amino acid by its corresponding
molecular weight, and 2) summing all mole-percent
corrected weights of the amino acids
140
Figure 16
Alanine transport, free amino acid pool, and protein content of embryos
and larvae of P. antarctica.
Change in (a) alanine transport rates; (b) free amino acid pool content; and (c) total
protein during early stages of development of Porania antarctica. Transport values
are slopes ± SE (n = 5-7); free amino acid pool and protein values are means ± SE (n
= 3-7). Where not shown, errors fell into the graphical representation of the data
point. White symbols: eggs; grey symbols: embryos; black symbols: larvae.
Figure 16 (continued)
141
Age (days post-fertilization)
0 4 8 121620
Alanine transport rate
(pmol individual
-1
h
-1
)
0
10
20
30
40
Age (days post-fertilization)
0 4 8 121620
Total protein
(µg individual
-1
)
0.5
1.0
1.5
2.0
2.5
3.0
Age (days post-fertilization)
0 4 8 121620
Free amino acid pool content
(pmol alanine individual
-1
)
200
400
600
800
1000
a
b
c
142
Protein content of eggs, embryos, and larvae of P. antarctica (Fig. 16c) decreased
significantly throughout the period of development examined (ANOVA of linear
regression: protein content (µg individual
-1
) = 1.75 – 0.04*(Age), p = 0.03; n = 6).
These data are consistent with protein content values reported for the Antarctic sea
star Acodontaster hodgsoni (cf. 0.027 µg individual
-1
d
-1
; Shilling & Manahan 1994,
Chapter 2, this dissertation), which, despite a lecithotrophic life-history mode, has a
comparable egg size (~550 µm; Bosch & Pearse 1990) to that of P. antarctica.
Protein synthesis and respiration
Rates of protein synthesis for embryos of Porania antarctica increased 35-fold
between 1 and 14 days post-fertilization (1.81 ± 0.5 and 63.5 ± 7.88 ng protein
embryo
-1
h
-1
, respectively) and for larvae (day 20) were 54.9 ± 3.54 ng protein larva
-1
h
-1
(Fig. 17a). Fractional rates of protein turnover ranged from 0.11 to 6.0 % h
-1
for
different stages of development (days 1-20). Overall, rates of protein synthesis for
embryos and larvae increased significantly with age (ANOVA of linear regression:
protein synthesis (ng protein individual
-1
h
-1
) = 3.52*(Age) – 4.71, p = 0.03; n = 5).
Similarly, respiration rates for embryos increased 6-fold from 85.1 ± 15.8 to 571.4 ±
47.2 pmols O
2
h
-1
embryo
-1
between 5 and 14 days post-fertilization, and for larvae
(day 20) were 719.7 ± 90.6 pmols O
2
h
-1
larva
-1
(Fig. 18a). The change in respiration
rate with age was statistically significant (ANOVA of linear regression: respiration
(pmol O
2
h
-1
individual
-1
) = 44.6*(Age) – 137.6, p = 0.02; n = 4).
143
Figure 17
Rates of protein synthesis during early stages of development of P.
antarctica.
(a) Change in rates of protein synthesis during development and growth of embryos
and larvae of Porania antarctica. Protein synthesis values are slopes ± SE (n = 5-7)
Where not shown, errors fell into the graphical representation of the data point. Each
symbol corresponds to a series of time course measurements of protein synthesis
with respect to time at a specific stage of development: (b) 1-d-old; (c) 5-d-old; (d)
10-d-old; (e) 14-d-old; (f) 20-d-old. Grey symbols: embryos; black symbols: larvae.
Figure 17 (continued)
144
Age (days post-fertilization)
0 4 8 121620
Protein synthesis
(ng embryo
-1
h
-1
)
0
20
40
60
80
Time (hours)
0.0 0.5 1.0 1.5
Protein synthesis
(ng embryo
-1
)
4
6
8
10
Time (hours)
0.0 0.5 1.0 1.5 2.0
Protein synthesis
(ng embryo
-1
)
6
8
10
12
Time (hours)
0.0 0.5 1.0 1.5
Protein synthesis
(ng embryo
-1
)
0
25
50
75
Time (hours)
0.0 0.5 1.0 1.5
Protein synthesis
(ng embryo
-1
)
25
50
75
100
125
Time (hours)
0.0 0.5 1.0 1.5 2.0
Protein synthesis
(ng larva
-1
)
b 1-d-old
c 5-d-old
d 10-d-old
e 14-d-old
f 20-d-old
a
0
25
50
75
100
125
145
Figure 18
Rates of oxygen consumption during early stages of development of P.
antarctica.
(a) Change in rates of respiration during development and growth of embryos and
larvae of Porania antarctica. Respiration values are slopes ± SE (n = 5-6) Where not
shown, errors fell into the graphical representation of the data point. Each symbol
corresponds to a series of time course measurements of oxygen consumption with
respect to number of individuals at a specific stage of development: (b) 5-d-old; (c)
10-d-old; (d) 14-d-old; (e) 20-d-old. Grey symbols: embryos; black symbols: larvae.
Figure 18 (continued)
Age (days post-fertilization)
0 5 10 15 20
Respiration
(pmol O
2
h
-1
individual
-1
)
0
150
300
450
600
750
900
Number of embryos
46 8 1012
Respiration
(pmol O
2
h
-1
)
0
200
400
600
800
Number of embryos
010 20 30 40
Respiration
(pmol O
2
h
-1
)
0
3000
6000
9000
12000
Number of embryos
0 5 10 15 20
Respiration
(pmol O
2
h
-1
)
0
3000
6000
9000
12000
Number of larvae
510 15 20 25
Respiration
(pmol O
2
h
-1
)
0
5000
10000
15000
20000
b 5-d-old c 10-d-old
d 14-d-old
e 20-d-old
a
146
147
Physiological rate measurements presented in this study were unexplainably higher
than those reported for embryos and larvae from other species of Antarctic
echinoderms investigated in this dissertation (i.e., Chapter 2). However, upon further
inspection, there appear to be no inconsistencies with how data were collected or
analyzed. For example, individual time point measurements of protein synthesis and
respiration for embryos and larvae of P. antarctica (Fig. 17b-f and Fig. 18b-e,
respectively) were consistently linear with respect to time or number of individuals.
Conclusions
Few studies have examined rates of protein metabolism and oxygen consumption
during early development in polar marine invertebrates. In general, the availability
and fecundity of animals in McMurdo Sound has constrained efforts to measure and
compare the energetic metabolism of phylogenetically related groups of organisms.
Although the findings presented in this study are preliminary, they provide, to the
best of my knowledge, the first description of the rates of macromolecular synthesis
during the early stages of development for this species of Antarctic sea star. These
results suggest that embryos and larvae of Porania antarctica are capable of
maintaining high rates of protein synthesis and turnover as reported for other species
of Antarctic echinoderms (sea urchins: Marsh et al. 2001; sea stars: Chapter 2, this
dissertation).
148
Appendix I References
Bosch I (1989) Contrasting modes of reproduction in two Antarctic asteroids of the
genus Porania, with a description of unusual feeding and non-feeding larval
types. Biol Bull 177:77-82
Bosch I, Pearse JS (1990) Developmental types of shallow-water asteroids in
McMurdo Sound, Antarctica. Mar Biol 104:41-46
Clark HES (1963) The Fauna of the Ross Sea. Part 3. Asteroidea. NZ Dept Sci Ind
Res Bull 151:1-84
Jaeckle WB, Manahan DT (1989) Growth and energy imbalance during the
development of a lecithotrophic molluscan larva (Haliotis refescens). Biol
Bull 177:237-246
Leong PKK, Manahan DT (1999) Na
+
/K
+
ATPase activity during early development
and growth of an Antarctic sea urchin. J Exp Biol 202:2051-2058
Marsh AG, Manahan DT (1999) A method for accurate measurements of the
respiration rates of marine invertebrate embryos and larvae. Mar Ecol Prog
Ser 184:1-10
Marsh AG, Maxson RE, Manahan DT (2001) High macromolecular synthesis with
low metabolic cost in Antarctic sea urchin embryos. Science 291:1950-1952
McClintock JB, Pearse JS, Bosch I (1988) Population structure and energetics of the
shallow-water Antarctic sea star Odontaster validus in contrasting habitats.
Mar Biol 99:235-246
Peck LS, Prothero-Thomas E (2002) Temperature effects on the metabolism of
larvae of the Antarctic starfish Odontaster validus, using a novel
microrespirometry method. Mar Biol 141:271-276
149
Pearse JS, McClintock JB, Bosch I (1991) Reproduction of Antarctic marine
invertebrates: tempos, modes and timing. Am Zool 31:65-80
R Development Core Team (2005) R: A language and for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria, http://www.R-
project.org
Shilling FM, Manahan DT (1994) Energy metabolism and amino acid transport
during early development of Antarctic and temperate echinoderms. Biol Bull
187:398-407
Vavra J, Manahan DT (1999) Protein metabolism in lecithotrophic larvae
(Gastropoda: Haliotis refescens). Biol Bull 196:177-186
150
Appendix II
Direct measurements of the energetic cost of metabolism in the
Antarctic sea star Odontaster meridionalis
using inhibitors of protein synthesis.
Introduction
Few studies have examined the energetic costs of early development in polar marine
invertebrates. The most comprehensive information to date is based on a single
species, the Antarctic sea urchin Sterechinus neumayeri. Embryos and larvae of S.
neumayeri are reported to maintain one of the lowest metabolic costs of protein
synthesis known for all animals (Marsh et al. 2001). However, whether this unique
efficiency is limited to S. neumayeri or is widespread among other Antarctic
echinoderm species remains unknown.
The goal of this study was to address this question by establishing the energetic costs
of metabolism during embryonic and larval development of the Antarctic sea star
Odontaster meridionalis using inhibitors of protein synthesis. An obligate predator
of sponges, O. meridionalis is relatively abundant in the shallow benthic habitats of
151
McMurdo Sound, Antarctica (Dayton et al. 1974, Bosch & Pearse 1990). Other than
a single study on the effects of temperature on the developmental rates and viability
of embryos (Stanwell-Smith & Peck 1998), almost nothing is known regarding the
developmental physiology of this particular species of asteroid. The research
presented here is a preliminary look at the energetic costs of protein synthesis for
embryos and larvae of O. meridionalis, and provides insight to the physiological and
metabolic requirements for marine invertebrates to develop and grow in the cold.
Materials and Methods
Spawning and animal culturing. Research was conducted at McMurdo Station,
Antarctica during September 2004. Adults of the sea star Odontaster meridionalis
were hand-collected by SCUBA divers from Arrival Heights, which is located
approximately 2 miles north of McMurdo Station. Animals were induced to spawn
following intracoelomic injections of 1 mM 1-methyladenine and maintained in
sterile seawater held at -1.0 °C as described in Chapter 2 (this dissertation). Fertilized
eggs were placed into 20-l culture containers at a concentration of 10 ml
-1
and mixed
with rotary paddles using standard laboratory methods (Shilling & Manahan 1994).
Culture seawater was replaced every 3-4 days. Known numbers of animals were
removed from culture containers at 3 separate developmental time points (16-day-old
gastrulae, 21-day-old gastrulae, and 48-day-old larvae) for parallel measurements of
protein synthesis and respiration.
152
Measurements of metabolic activity. A detailed description of the methods used to
quantify rates of oxygen consumption, amino acid transport, and protein synthesis
for embryos and larvae of Odontaster meridionalis is provided in Chapter 2 of this
dissertation. Respiration rates were aliquoted into micro-biological oxygen (µBOD)
demand vials and measured using the end-point determination method following the
protocol outlined by Marsh & Manahan (1999).
14
C-labeled alanine (PerkinElmer,
Wellesley, MA, USA; 6.1-6.3 MBq µmol
-1
) was used to determine rates of alanine
transport from seawater and absolute rates of protein synthesis as described by Vavra
& Manahan (1999). Protein synthesis rates were calculated using the mole-percent of
alanine (7.6%) and average molecular weight (126.5 g mol
-1
) of amino acids in
hydrolyzed whole-animal protein from embryos and larvae of O. meridionalis (see
Chapter 2, Table 2; this dissertation). All physiological rate measurements were
maintained at -1.0 °C.
Cost of protein synthesis. The energetic cost of protein synthesis for embryos and
larvae of Odontaster meridionalis was measured directly using inhibitors of protein
synthesis (see Pace & Manahan 2006, Chapter 3, this dissertation). Rates of
respiration and protein synthesis were measured in the presence and absence of the
antibiotic drugs emetine and anisomycin (Sigma Chemical Co.), which are known to
inhibit protein synthesis in other species of marine invertebrates (e.g., Pace &
Manahan 2006 and references therein). Little is known, however, regarding the
developmental physiology of O. meridionalis. Thus the optimal concentrations of
153
emetine and anisomycin required for the inhibition of protein synthesis in this
species, let alone the efficacy of these particular inhibitors, was not known at the
onset of this study.
The concentrations of inhibitory drugs used in the experiments presented here were
assumed to be similar to those used for blocking protein synthesis in temperate
echinoderms (e.g., Chapter 3, this dissertation). Given the preliminary nature of
measuring the energetic cost of protein synthesis in O. meridionalis, emetine and
anisomycin were each used across a range of different concentrations, and at
different stages of embryonic and larval development. Specifically, gastrula-stage
embryos (16 and 21-day-old) were exposed to different concentrations (5-50 µM) of
emetine, whereas unfed bipinnaria-stage larvae (48-day-old) were exposed to
different concentrations (0.2-20 µM) of anisomycin.
Statistics. Statistical tests were performed with the “R” statistical software package
(R Development Core Team, 2005). All error values represent ± 1 SEM.
Results and Discussion
Rates of alanine transport from seawater for embryos and larvae of the sea star
Odontaster meridionalis ranged from 1.92 ± 0.18 to 3.11 ± 0.05 pmol individual
-1
h
-1
154
when inhibitors of protein synthesis were absent (Table 7). For each stage of
development (i.e., gastrula embryo to bipinnaria larva) examined, rates of alanine
transport did not significantly change in the presence of either emetine or anisomycin
(linear regressions compared by two-way ANOVA, p > 0.05; n = 4 for each
developmental stage). In the absence of inhibitors, the amount of alanine in the free
amino acid pools of embryos and larvae ranged between 19.2 ± 1.62 to 26.4 ± 6.16
pmol individual
-1
(Table 7). No significant change in the free amino acid pool
content of alanine was measurable when either emetine or anisomycin were present
(linear regressions compared by two-way ANOVA, p > 0.05; n = 4 for each
developmental stage). These results are consistent with those reported for other
species of echinoderms (e.g., Pace & Manahan 2006, Chapter 3, this dissertation)
and were an important first step for demonstrating that the concentrations of emetine
and anisomycin used in this study did not adversely affect the transport and
subsequent incorporation of alanine into the intracellular free amino acid pools of
embryos and larvae.
The energetic cost of protein synthesis during specific stages of development of
Odontaster meridionalis was determined by measuring the difference between rates
of respiration and protein synthesis in the presence and absence of the inhibitory
drugs emetine and anisomycin (Fig. 19). The maximum height of each histogram bar
represents rates of respiration and protein synthesis when inhibitors were absent,
whereas the solid component represents the rates when inhibitors were present.
155
Table 7
Alanine transport rates and alanine pool content for embryos and
larvae of O. meridionalis with and without protein synthesis
inhibitors.
Stage/age
Treatment
Alanine
transport rate
(pmol individual
-1
h
-1
)
Free amino
acid pool content
(pmol Ala individual
-1
)
Gastrula
(16-day-old)
Control
Emetine (5 µM)
2.27 ± 0.11
ns
1.88 ± 0.44
23.2 ± 3.01
ns
23.4 ± 3.78
Gastrula
(16-day-old)
Control
Emetine (10 µM)
2.27 ± 0.11
ns
2.19 ± 0.09
23.2 ± 3.01
ns
23.4 ± 2.50
Gastrula
(16-day-old)
Control
Emetine (25 µM)
2.27 ± 0.11
ns
2.33 ± 0.19
23.2 ± 3.01
ns
25.6 ± 3.13
Gastrula
(21-day-old)
Control
Emetine (5 µM)
1.92 ± 0.18
ns
1.85 ± 0.28
26.4 ± 6.16
ns
25.1 ± 5.43
Gastrula
(21-day-old)
Control
Emetine (10 µM)
1.92 ± 0.18
ns
1.70 ± 0.10
26.4 ± 6.16
ns
26.8 ± 8.22
Gastrula
(21-day-old)
Control
Emetine (50 µM)
1.92 ± 0.18
ns
1.47 ± 0.16
26.4 ± 6.16
ns
28.4 ± 4.34
Unfed larva
(48-day-old)
Control
Anisomycin (0.2 µM)
3.11 ± 0.05
ns
2.94 ± 0.73
19.2 ± 1.62
ns
19.5 ± 0.50
Unfed larva
(48-day-old)
Control
Anisomycin (2.0 µM)
3.11 ± 0.05
ns
3.39 ± 0.56
19.2 ± 1.62
ns
21.2 ± 0.52
Unfed larva
(48-day-old)
Control
Anisomycin (20 µM)
3.11 ± 0.05
ns
2.92 ± 0.12
19.2 ± 1.62
ns
18.2 ± 1.18
Transport rate values are slopes ± SE of the increase in
14
C-alanine with time (n = 4).
Numbers in parentheses indicate concentration of inhibitor used for each experiment.
‘ns’ indicates transport rates are not statistically significant for a given stage of embryonic or
larval development (p > 0.05).
156
Figure 19
Change in rates of respiration and protein synthesis in embryos and
larvae of O. meridionalis with and without protein synthesis inhibitors.
Change in metabolic rate (MR) and protein synthesis (PS) in embryos and larvae of
O. meridionalis when emetine or anisomycin were either absent (total height of each
bar) or present (solid component). Respiration values were converted into energetic
units using the average oxyenthalpic equivalent of 484 kJ (mol O
2
)
-1
for protein and
lipid (Gnaiger 1983). Metabolic rate values are slopes ± SE (n = 8); Protein synthesis
values are means ± SE (n = 4).
Protein synthesis (ng individual
-1
h
-1
)
0
2
4
6
8
10
157
Metabolic rate (µJ individual
-1
h
-1
)
0
5
10
15
20
510 25
PS
Emetine (µM)
PS PS
510 50
PS
Gastrula
(16-day-old)
Gastrula
(21-day-old)
PS PS
0.2 2 20
PS
Anisomycin (µM)
PS PS
Unfed larva
(48-day-old)
Emetine (µM)
MR MR MR MR MR MR MR MR MR
Figure 19 (continued)
158
Respiration rates were converted to energy units using a mean oxyenthalpic value of
484 kJ (mol O
2
)
-1
for protein and lipid (Gnaiger 1983). Costs of protein synthesis for
gastrula-stage embryos ranged from 0.7 to 6.4 J (mg protein)
-1
, while unfed
bipinnaria-stage larvae ranged from 7.0 to 87.0 J (mg protein)
-1
(Table 8).
The different concentrations of emetine and anisomycin used in this study were
within a range known to inhibit protein synthesis in the Antarctic sea urchin
Sterechinus neumayeri (Pace & Manahan submitted). One drawback to using
inhibitors for studies of metabolism is that non-specific cellular processes may be
altered. While neither emetine nor anisomycin had a negative effect on rates of
alanine transport (Table 7), these inhibitors may have differentially affected
respiration relative to protein synthesis (i.e., respiration rate is altered, but protein
synthesis remains the same). Based on results presented here, this may have been the
case, as one outlier demonstrated an unrealistically high cost of protein synthesis (cf.
87 J (mg protein)
-1
, Table 8) at the lowest concentration (0.2 µM) of anisomycin
tested for unfed larvae. The average cost of protein synthesis, when analyzed without
the outlier present, was 4.2 J (mg protein)
-1
. Although this cost is approximately 10
times greater than previously reported for the sea urchin S. neumayeri (i.e., 0.41-0.45
J mg
-1
protein synthesized: Marsh et al. 2001), it is in a range that is consistent with
costs of protein synthesis measured in temperate species of echinoderms (e.g.,
Lytechinus pictus: 8.4 J (mg protein)
-1
, Pace & Manahan 2006; Strongylocentrotus
purpuratus: 8.6 J (mg protein)
-1
, Chapter 3, this dissertation).
159
Table 8
Energetic costs of protein synthesis during embryonic
and larval development of O. meridionalis.
Stage/age
Treatment
Energetic cost
J (mg protein)
-1
Gastrula
(16-day-old)
Emetine (5 µM) 5.2
Gastrula
(16-day-old)
Emetine (10 µM) 0.9
Gastrula
(16-day-old)
Emetine (25 µM) 0.7
Gastrula
(21-day-old)
Emetine (5 µM) 6.4
Gastrula
(21-day-old)
Emetine (10 µM) 2.4
Gastrula
(21-day-old)
Emetine (50 µM) 3.0
Unfed larva
(48-day-old)
Anisomycin (0.2 µM) 87
Unfed larva
(48-day-old)
Anisomycin (2.0 µM) 7.0
Unfed larva
(48-day-old)
Anisomycin (20 µM) 7.7
Numbers in parentheses indicate concentration of inhibitor used
for each experiment.
160
Furthermore, the complete inhibition of protein synthesis was not observed with the
concentrations of emetine and anisomycin used in this study (Fig. 19). Rates of
protein synthesis were never observed to reach a minimum, stable level as the
concentration of inhibitor was increased (cf. Chapter 3, Fig. 14a; this dissertation),
which is a fundamental requirement to accurately determine the energetic cost of
protein synthesis using inhibitory drugs. As demonstrated in Table 8, costs of protein
synthesis calculated for embryos and larvae of O. meridionalis varied by as much as
10-fold with the outlier (see above) removed. Neither emetine nor anisomycin were
tested at higher doses since the water solubility of these inhibitors is significantly
affected as their concentration is increased.
In conclusion, these data are an important step forward in analyzing the metabolism
of Antarctic marine invertebrates, and is the first study that I am aware of to directly
measure the energetic cost of metabolism in an Antarctic sea star using inhibitors of
protein synthesis. Future studies might explore the efficacy of other protein synthesis
inhibitors such as cycloheximide and pactamycon, but it is possible that such
antibiotic drugs are ineffective in this particular species of sea star, and for that
matter, asteroids in general.
161
Appendix II References
Bosch I, Pearse JS (1990) Developmental types of shallow-water asteroids in
McMurdo Sound, Antarctica. Mar Biol 104, 41-46
Dayton PK, Robilliard GA, Paine RT, Dayton LB (1974) Biological accommodation
in the benthic community at McMurdo Sound, Antarctica. Ecol Monogr
44:105-128
Gniager E (1983) Calculation of energetic and biochemical equivalents of respiratory
oxygen consumption. In: Gnaiger E, Forstner H (eds) Polarographic oxygen
sensors: Aquatic and physiological applications. Springer-Verlag, New York,
NY
Marsh AG, Manahan DT (1999) A method for accurate measurements of the
respiration rates of marine invertebrate embryos and larvae. Mar Ecol Prog
Ser 184:1-10
Marsh AG, Maxson RE, Manahan DT (2001) High macromolecular synthesis with
low metabolic cost in Antarctic sea urchin embryos. Science 291:1950-1952
Pace DA, Manahan DT (2006) Fixed metabolic costs for highly variable rates of
protein synthesis in sea urchin embryos and larvae. J Exp Biol 209:158-170
Pace DA, Manahan DT (submitted) Cost of protein synthesis and energy allocation
during development of Antarctic sea urchin embryos and larvae. Biol Bull
R Development Core Team (2005) R: A language and for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria, http://www.R-
project.org
Shilling FM, Manahan DT (1994) Energy metabolism and amino acid transport
during early development of Antarctic and temperate echinoderms. Biol Bull
187:398-407
162
Stanwell-Smith D, Peck LS (1998) Temperature and embryonic development in
relation to spawning and field occurrence of larvae of three Antarctic
echinoderms. Biol Bull 194:44-52
Vavra J, Manahan DT (1999) Protein metabolism in lecithotrophic larvae
(Gastropoda: Haliotis refescens). Biol Bull 196:177-186
Abstract (if available)
Abstract
The goal of this dissertation was to investigate the nutritional and physiological state of benthic marine invertebrates from both temperate and polar habitats. In Chapter 1, larvae of the temperate sea urchin Lytechinus pictus reared in the field were compared to those reared on cultured phytoplankton diets in the laboratory. A biochemical index of physiological state was established using an in situ culturing system and measurements of protein synthesis to define the nutritional status of larvae growing in the ocean. Protein synthesis rates for larvae reared in the field were ~50% less than larvae fed at near-maximal physiological capacities in the laboratory. Research presented in Chapter 2 focused on the physiological and metabolic requirements of early development in the Antarctic asteroids Acodontaster hodgsoni and Odontaster meridionalis. Despite differences in egg size and developmental mode, A. hodgsoni and O. meridionalis maintained high rates of protein synthesis while sustaining a low metabolic rate. Additionally, fractional rates of protein synthesis in embryos of O. meridionalis were two-fold greater than its temperate counterpart (standardized to -1 °C) indicating that the Antarctic species is cold-adapted. The goal of Chapter 3 was to determine how metabolic energy is partitioned during early development of the temperate sea urchin Strongylocentrotus purpuratus. The energetic cost of protein synthesis was 8.6 J (mg protein)-1. Combined, protein synthesis ATP utilization by the sodium pump accounted for ~90% of total respiration during development. The information provided in this dissertation will substantially increase understanding of the physiology of organisms developing and growing in different oceanic environments.
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Asset Metadata
Creator
Ginsburg, David Ward
(author)
Core Title
Physiological rates during development of marine invertebrates in temperate and polar oceans
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Biology
Degree Conferral Date
2007-05
Defense Date
12/15/2006
Publisher
University of Southern California
(original),
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(digital)
Tag
Antarctic,development,marine invertebrate,OAI-PMH Harvest,physiological rate,temperate
Language
English
Advisor
Manahan, Donal T. (
committee chair
), Caron, David A. (
committee member
), Corsetti, Frank A. (
committee member
), Hedgecock, Dennis (
committee member
), Maxson, Robert E. (
committee member
)
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