THE ROLE OF ENERGY RESERVES ON MORTALITY AND STRESS-TOLERANCE
THRESHOLDS DURING THE EARLY BENTHIC PHASE IN INTERTIDAL
INVERTEBRATES

by

SHANNON ROCHELLE MENDT
B.Sc. Simon Fraser University, 2013

A thesis submitted in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
in Environmental Science
in the Department of Biological Sciences
Thompsons Rivers University

Thesis examining committee:
Dr. Louis Gosselin (PhD), Thesis Supervisor, Department of Biological Sciences,
Thompson Rivers University
Dr. Robert Higgins (PhD), Supervisory Committee Member, Department of Biological
Sciences, Thompson Rivers University
Dr. Mark Rakobowchuk (PhD), Supervisory Committee Member, Department of Biological
Sciences, Thompson Rivers University
Dr. Jan Pechenik (PhD), External Examiner, Department of Biology, Tufts University

© Shannon Rochelle Mendt, 2021

ii

Thesis Supervisor: Professor Dr. Louis Gosselin

ABSTRACT
For many intertidal marine invertebrate species, mortality rates during the early
benthic phase are often very high and highly variable, which can lead to fluctuations in the
abundance and distribution of adult invertebrate populations and the structure of the intertidal
community. One of the major factors hypothesized to contribute to this mortality is
insufficient energy reserves at the onset of the early benthic phase. Yet the role of energy
reserves in regulating early survivorship has not been directly tested, and so remains
speculative. This thesis explores both the direct and indirect impacts of energy reserves on
mortality during the early benthic phase.
Recently settled or hatched individuals of six invertebrate species were collected from
natural populations, maintained without food, and their survivorship was monitored.
Contrary to expectations, starved individuals of all six species had high survivorship through
the critical first 10 d of the early benthic phase, with half of the species experiencing <2%
mortality, and the remaining three species experiencing only 6 – 12% mortality, and no
difference in short-term survivorship was detected among starved individuals of three
different size classes (a proxy for energy reserves) of Nucella. ostrina hatchlings. This study
is the first to reveal that depleted energy reserves are not a primary direct cause of high
mortality at the start of the early benthic phase, as had previously been hypothesized.
Indirect impacts of initial energy reserves were then examined by determining the
extent to which energy reserves affect acute tolerance thresholds of early benthic phase
individuals to two of the most challenging intertidal environmental stressors: desiccation and
high emersion temperature. Levels of energy reserve were controlled by maintaining early
benthic phase individuals of two invertebrate species without food. These were then exposed
to an experimental range of emersion temperatures and desiccation periods. Duration of
starvation, an indirect measure of energy levels, had no effect on tolerance to emersion
temperature but did significantly impact tolerance to desiccation. This suggests that
desiccation is likely a more important selective pressure on intertidal invertebrates and could
favor the evolution of greater energy reserves at the onset of the early benthic phase.

iii

Interestingly, acute tolerance thresholds to both stressors were significantly reduced for
smaller individuals relative to those with greater body mass, suggesting that body size – and
particularly the surface area: volume ratio may be a better indicator of vulnerability to abiotic
stressors than energy content. Together, these findings reveal that depleted energy reserves at
the onset of the early benthic phase can affect rates of early benthic phase mortality through
indirect effects, by making individuals more vulnerable to certain stressors encountered in
early benthic life. These findings are important because they reveal that although insufficient
energy reserves are not a major source of early mortality, they can still impact recruitment
and community structure in the intertidal zone.

KEYWORDS: Early benthic phase, mortality factors, recruitment, tolerance thresholds,
starvation, energy reserves, emersion temperature, desiccation

iv

TABLE OF CONTENTS
ABSTRACT ............................................................................................................................. ii
TABLE OF CONTENTS ...................................................................................................... iv
ACKNOWLEDGEMENTS .................................................................................................. vi
LIST OF FIGURES .............................................................................................................. vii
LIST OF TABLES .................................................................................................................. x
CHAPTER 1: General Introduction ..................................................................................... 1
LITERATURE CITED ......................................................................................................... 5
CHAPTER 2: Role of depleted initial energy reserves in early benthic phase mortality
of six marine invertebrate species ......................................................................................... 9
INTRODUCTION................................................................................................................. 9
METHODS.......................................................................................................................... 13
Study species .................................................................................................................. 13
Study site and collection of animals ............................................................................. 14
Procedures for rearing EBP individuals without food ............................................... 15
Relationship between organic matter content in EBP individuals and duration of
starvation ........................................................................................................................ 16
Ability of EBP individuals to survive exclusively on their initial energy reserves .. 17
Ability to recover after delayed feeding in EBP invertebrates .................................. 19
Data analysis .................................................................................................................. 20
RESULTS............................................................................................................................ 20
Relationship between organic matter content in EBP individuals and duration of
starvation ........................................................................................................................ 20
Ability of EBP individuals to survive exclusively on their initial energy reserves .. 21
DISCUSSION ..................................................................................................................... 27
Relationship between organic matter content in EBP individuals and duration of
starvation ........................................................................................................................ 27
Ability of EBP individuals to survive exclusively on their initial energy reserves .. 28
Ability to recover after delayed feeding for EBP invertebrates ................................ 31
Ecological implications .................................................................................................. 33
LITERATURE CITED ....................................................................................................... 34
CHAPTER 3: The role of initial energy reserves on stress tolerance thresholds during
the early benthic phase for intertidal invertebrates .......................................................... 42

v

INTRODUCTION............................................................................................................... 42
METHODS.......................................................................................................................... 44
Study site and species collection ................................................................................... 44
Establishing treatment levels of energy reserves ........................................................ 45
Emersion temperature tolerance .................................................................................. 47
Desiccation tolerance ..................................................................................................... 49
Data analysis .................................................................................................................. 51
RESULTS............................................................................................................................ 51
Emersion temperature tolerance .................................................................................. 51
Desiccation tolerance ..................................................................................................... 55
DISCUSSION ..................................................................................................................... 59
Effect of estimated energy reserves on tolerance to elevated emersion temperature
......................................................................................................................................... 59
Effect of estimated energy reserves on tolerance to desiccation ............................... 60
Does the relationship between energy reserves and stress tolerance contribute to
EBP mortality? .............................................................................................................. 62
LITERATURE CITED ....................................................................................................... 64
CHAPTER 4: General Conclusion ...................................................................................... 68
SUMMARY OF RESULTS ................................................................................................ 68
RELEVANCE OF FINDINGS FOR POLICY AND MANAGEMENT ........................... 69
Impacts of climate change on intertidal communities ..................................................... 70
Marine invertebrate aquaculture and hatchery operations ............................................ 70
Marine invertebrate fisheries management ..................................................................... 71
DIRECTIONS FOR FUTURE STUDY ............................................................................. 72
LITERATURE CITED ....................................................................................................... 73
APPENDIX A. ....................................................................................................................... 76
APPENDIX B. ....................................................................................................................... 80

vi

ACKNOWLEDGEMENTS
I would first like to thank my supervisor, Dr. Louis Gosselin, for his enthusiastic and
patient guidance throughout the duration of this research project, and for the hours spent
searching for snail eggs with our noses buried in Fucus. Thank you as well to the members of
my committee, Dr. Rob Higgins and Dr. Mark Rakobowchuk, for their support and advice.
This project would not have been possible without the directors and staff at the
Bamfield Marine Sciences Centre providing the facilities and equipment necessary, and
keeping me housed, fed, and caffeinated during my time at the station. I extent my gratitude
to the Huu-ay-aht First Nation for granting us access to their ancestral territory. And a big
thank you as well to the entire community of Bamfield who made my many months living in
such a remote location an absolute joy.
Thank you to Anna Skurikhina, Bree Iwabuchi, and Renée Gaudet and everyone else
who I roped into helping me with field and laboratory work, and to Hil Hamilton for
answering my often-frantic methodology and statistic questions. And finally, thank you to all
my friends and family, and especially to my partner, Reid Yochim, who’s relentless and
unyielding support and encouragement (for all my hairbrained schemes) keeps me sane and
motivated.
This research was funded by NSERC Discovery Grants (RGPIN-2014-04779 and
RGPIN-2020-04935) to LA Gosselin. Additional financial support was provided by a BC
Graduate Scholarship, and a TRU ENVS Fellowship Award and Ken Lepin Graduate Student
Award (Thompson Rivers University). Permits required for this research included: DFO
animal collection permits (XR 151 2018, XMCFR 17 2019), Bamfield Marine Sciences
Centre AUPs (RS-18-08, RS-19-04), and Huu-ay-aht First Nations heritage investigation
permits (HFN 2018-004, HFN 2019-007).

vii

LIST OF FIGURES
Figure 2.1. Two hypothetical frequency distributions (bell curves) of energy reserves among
individuals at the onset of the EBP. The dashed vertical line represents a minimum energy
threshold required for EBP survival. ...................................................................................... 12
Figure 2.2. Map of field sites in Barkley Sound near Bamfield, British Columbia: Ross Islets
(R), Wizard Islet (W), Prasiola Point (P), Dixon Island (D), and Grappler Inlet (G). Map
modified from Gosselin and Chia (1995). .............................................................................. 14
Figure 2.3. Relationship between OM:ash weight ratio and the duration of starvation,
starting at the onset of the EBP, for Nucella ostrina hatchlings. OM is used as an indicator of
total energy content and ash weight is used as an index of initial total body mass. Each value
represents a combined sample of 52 - 60 individuals. Each batch represents a single
collection of hatchlings from the field. ................................................................................... 21
Figure 2.4. Percent survivorship of starved B. glandula as a function of the duration of
starvation, starting at the onset of the EBP. The point where the curve intersects the dashed
line represents the starvation LD50. ....................................................................................... 22
Figure 2.5. Survivorship of individuals starved for the first 10 d since the onset of EBP for
six benthic intertidal species. Each point represents the average % survivorship and
associated standard errors calculated from GLMs for each species (Table 2.1). ................... 23
Figure 2.6. Effect of initial body size of Nucella ostrina hatchlings on (A) the survivorship at
5 d and (B) the starvation LD20. Small = 0.80 – 1.10 mm shell length; Medium = 1.11 – 1.40
mm shell length; Large = 1.41 – 1.80 mm shell length. Each mean value and associated
standard error are calculated from generalized linear models (GLM) for 6 – 7 batches of each
size class. Different letters above the bars indicate values that were significantly different
based on Tukey HSD tests (p < 0.05). .................................................................................... 25
Figure 2.7. Percent of individuals able to recover, as a function of duration of starvation in
(A) C. dalli and (B) N. ostrina. Equations of the regression lines: (A) y = 84.8758-1.2184x,
and (B) y = 83.8095-1.6190x. The point where the model intersects the dashed line indicates
the duration of starvation resulting in only 50% of individuals being able to recover when
fed. .......................................................................................................................................... 26

viii

Figure 2.8. Effect of duration of starvation on (A) shell diameter prior to feeding, and (B)
subsequent growth of EBP B. glandula. Values represent average shell diameter ± SE at the
end of the period of starvation (A) and average growth ± SE during the 30 d feeding period
(B). Different letters above error bars on the bar graph indicate values that were significantly
different. Equation of the regression line in (B): y = 1.76033 – 0.023276x. .......................... 27
Figure 3.1. Acute temperature tolerance thresholds, measured as average temperature at
death, as a function of duration of starvation in EBP. (A) B. glandula and (B) N. ostrina. The
30 d N. ostrina group was removed from this analysis due to inconsistent results for this
batch of hatchlings and mass mortality among the remaining hatchlings of the same batch
that were held in the same cage. In both species, starvation was initiated at the onset of the
EBP. Error bars represent SE. Bars identified with different letter are significantly different
based on Dunn’s multiple comparison test. ............................................................................ 53
Figure 3.2. Effect of initial body size on acute temperature tolerance, as measured by
average temperature at death, for EBP N. ostrina. Error bars represent SE. .......................... 54
Figure 3.3. Effect of exposure to desiccation conditions on the survivorship of newly
metamorphosed (not starved) EBP B. glandula. Each point represents the proportion of
individuals from a group of 10 that survived the desiccation treatment for a given duration
(h). The point where the curve intersects the dashed line represents the desiccation LD 50; in
this case, LD50 = 13.98 h......................................................................................................... 55
Figure 3.4. Acute desiccation tolerance thresholds, measured as LD 50, as a function of
duration of starvation in EBP (A) B. glandula and (B) N. ostrina. Error bars represent SE.
Bars identified with different letters are significantly different based on GLM analysis. ...... 57
Figure 3.5. Effect of exposure to desiccation conditions on the survival of newly hatched N.
ostrina of two size classes (Small = 0.074 – 0.192 mg; Large = 0.402 – 0.833 mg). Each
point represents the proportion of individuals from a group of 10 that survived desiccation
treatment for a given duration (h). The points where the curves intersect the dashed line
represent the desiccation LD50 for each size class. ................................................................. 58
Figure A1. Relationship between % survivorship and the duration of starvation, starting at
the onset of the EBP, for five species of benthic invertebrates. Results from the generalized
linear models (GLMs) are shown in Table 2.1 in Chapter 2. ................................................. 79
Figure B1. Relationship between the proportion of individuals surviving and the duration of
the desiccation treatment to which they were exposed for groups of B. glandula starved for

ix

(A) 0 d, (B) 10 d, (C) 20 d, and (D) 30 d. Results from the generalized linear models (GLMs)
are shown in Table 3.3 in Chapter 3. ...................................................................................... 80
Figure B2. Relationship between the proportion of individuals surviving and the duration of
the desiccation treatment to which they were exposed for groups of N. ostrina starved for (A)
0 d, (B) 10 d, (C) 20 d, (D) 30 d, (E) 40 d, and (F) 50 d. Results from the generalized linear
models (GLMs) are shown in Table 3.3 in Chapter 3............................................................. 81

x

LIST OF TABLES

Table 2.1. Shell lengths (SL) and corresponding body masses (wet weight, WW) for N.
ostrina hatchlings. The conversion equation is: WW = 2.98 * log(SL) - 3.84, from Hamilton
& Gosselin (2020). .................................................................................................................. 18
Table 2.2. Starvation LD50 and associated standard error (SE) from generalized linear model
(GLM) analysis of survival as a function of duration of starvation for each of the 6 species.
The z statistic represents the strength of the relationship between the % survivorship and the
duration of starvation; df = degrees of freedom...................................................................... 23
Table 2.3. Results of the GLM analysis of percent survivorship as a function of duration of
starvation for each size class from each batch of N. ostrina hatchlings. Z statistics indicate
the fit of the relationship between duration of starvation and % survivorship; df = degrees of
freedom. The binomial link function was used in all GLMs. ................................................. 24
Table 3.1. Shell lengths (SL) and corresponding body masses (wet weight, WW) for N.
ostrina hatchlings. The conversion equation is: WW = 2.98 * log(SL) - 3.84, from Hamilton
& Gosselin (2020). .................................................................................................................. 46
Table 3.2. Experimental range of emersion temperatures and desiccation periods used for
each species. Ranges were set to include treatments that result in 0 – 100% mortality. ........ 48
Table 3.3. Desiccation LD50 and associated standard error (SE) for groups of B. glandula
and N. ostrina starved for different durations of time since the onset of EBP. LD50 values
were calculated from generalized linear models (GLM) of the relationship between the
proportion of individuals surviving and the duration of exposure to the desiccation treatment.
The z statistic represents the strength of this relationship; df = degrees of freedom; p values
represent the probability that survivorship varied as a function of duration of exposure to the
desiccation treatment. ............................................................................................................. 56
Table A1. Number of batches and number of individuals in each batch for all six benthic
invertebrate species in the experiment assessing the survivorship of EBP individuals unable
to replenish energy reserves. ................................................................................................... 76
Table A2. GLM equations for the relationship between % survivorship and duration of
starvation, starting at the onset of the EBP, for all six species. .............................................. 77

xi

Table A3. Starvation LD20 and percent survivorship at 5 d starvation, and their associated
standard errors (SE), calculated from generalized linear models (GLM) of survival as a
function of duration of starvation for each size class of each batch of Nucella ostrina
hatchlings. n represents sample size for each size class of each batch. .................................. 77
Table A4. Recovery LD50 and associated standard error (SE) from linear model analysis of
the proportion of individuals able to recover from different durations of starvation. The z
statistic represents the strength of the relationship between the % survivorship and the
duration of starvation; df = degrees of freedom...................................................................... 78

1

CHAPTER 1: General Introduction
Recruitment rates for many species of intertidal invertebrates are highly variable, which
leads to significant spatial and temporal fluctuations in adult populations. Recruitment rates
are determined by both the supply of larvae colonizing the habitat, and their success in
reaching adulthood, and thus recruiting to the adult population (Keough and Downes 1982;
Hadfield 1986). Larval (i.e. pre-settlement) mortality is high for many intertidal species
(Jablonski and Lutz 1983; Gaines and Roughgarden 1985), and can impact recruitment when
larval supply is low. However, larval supply is generally not limiting, and so its impact on
population dynamics is often small (Hunt and Scheibling 1997; Fraschetti et al. 2003).
Conversely, post-settlement mortality, or mortality during the early benthic phase, has been
shown to heavily affect adult population abundance and distribution for many intertidal
species (Connell 1985; Hughes 1990; Stoner 1990; Miller and Waldbusser 2016).
The early benthic phase (EBP) is the period immediately following settlement or hatching
into the benthic environment, and it can encompass both pre- and post-metamorphic life
stages (Sandee et al. 2016). The beginning of the EBP marks a major transition from the
larval stage, that is either pelagic or contained within a protective egg capsule, to independent
benthic life. This transition to the benthic intertidal zone is also associated with many biotic
and abiotic challenges. For this reason, the EBP is characterized by very high mortality, with
the most critical period occurring in the first 24 – 48 h (Guillou and Tartu 1994; Gosselin and
Qian 1997; Hunt and Scheibling 1997). Many new cohorts experience 30 – 100% mortality
in the first few days or weeks following settling or hatching into the benthic environment
(Gosselin and Qian 1997; Hunt and Scheibling 1997). Mortality rates during this time frame
are also highly variable, not only among species, but also among and within cohorts of the
same species (Jarrett 2000; Phillips 2017). For example, mortality rates of newly settled
barnacles range from 22% to 87% in the first 48 hours after settlement (Hunt and Scheibling
1997). Some of this variability may be attributed to different magnitudes of mortality factors,
such as predation, competition, and abiotic stress, being experienced at different times, or in
different locations (Gosselin and Qian 1997; Dahlhoff et al. 2001; Helmuth and Hofmann
2001). However, the specific causes of variability in mortality rates among and within
cohorts are not fully understood. These findings suggest that individuals are most vulnerable

2

at the very beginning of independent benthic life, and that understanding the rates and
variability in mortality during the EBP may be critical for determining fluctuations in adult
populations.
The beginning of independent benthic life is associated with many challenges that
contribute to the high rates of EBP mortality. The most significant of these challenges are the
abiotic factors linked to periods of emersion during low tide including elevated temperatures
and desiccation stress (Gosselin and Chia 1995; Helmuth and Hofmann 2001; Miller et al.
2009; Shanks 2009). Benthic invertebrates possess many morphological, physiological, and
behavioural adaptations to combat these abiotic stressors, but their natural tolerances can be
exceeded by the environmental conditions they experience, resulting in extensive mortality
(Foster 1971; Gosselin and Chia 1995; Miller et al. 2009; Jenewein and Gosselin 2013a;
Jenewein and Gosselin 2013b). This is especially true for EBP individuals, as their small
body size and lack, or underdevelopment, of protective structures makes them more
vulnerable (Foster 1971; Gosselin 1997, Hamilton and Gosselin 2020).
Initial energy reserves held by the individual at the onset of EBP have been proposed as
another major factor in determining success and survival through the critical first days
(Gosselin and Qian 1997; Hunt and Scheibling 1997; Jarrett and Pechenik 1997), as
individuals with greater energy reserves tend to grow faster and have lower mortality than
their lower energy counterparts (Goulden et al. 1987; Phillips 2002; Emlet and Sadro 2006).
It has been suggested that many benthic invertebrates may begin the EBP with relatively low
energy reserves, as many species transition through energy intensive stages, such as
metamorphosis, before beginning independent benthic life (Lucas et al. 1979; Wendt 2000;
Thiyagarajan et al. 2003; Bryan 2004; Bennett and Marshall 2005). Many species also have a
non-feeding phase directly before or following metamorphosis, during which they are
entirely dependent upon their stored energy to complete metamorphosis and support
metabolism throughout the first hours or days of early benthic life (Lucas et al. 1979;
Pechenik et al. 1993; Gosselin and Chia 1994). Additionally, there is evidence that levels of
energy reserves, as measured by organic content or size at hatching, at the onset of the EBP
may vary considerably among individuals, and that energy stores are variable both among
and within cohorts (Jarrett and Pechenik 1997; Jarrett 2003; Lloyd and Gosselin 2011).
Beginning the EBP with low energy reserves greatly impacts the size and growth rates of

3

individuals (Miller 1993; Pechenik et al. 1996; Thiyagarajan et al. 2005; Emlet and Sadro
2006), which can in turn have implications for survivorship (Moran 1999; Phillips 2002;
Thiyagarajan et al. 2002; Phillips 2017). It is possible that some portion of individuals may
begin the EBP with such critically low energy stores that they are near a minimum threshold
necessary to survive and would either inevitably die of energy depletion very soon after
metamorphosis, or need to quickly feed to replenish energy stores in order to survive. These
direct effects of low energy on mortality remain largely untested but have the potential to
play a vital role in determining which individuals survive through the first few days of the
EBP.
The amount of energy held by the individual at the onset of the EBP has a variety of other
effects on the individual’s performance. Individuals with low energy reserves, as estimated
by low organic content, small larval size, or poor feeding conditions, often grow more slowly
and have a longer time to maturity than individuals with greater energy stores (Moran and
Emlet 2001; Marshall et al. 2003; Thiyagarajan et al. 2003; Torres et al. 2016). Stored energy
may also play an essential role in stress tolerance, as energy demands can increase during
exposure to environmental stress (Sokolova et al. 2012), such that limited energy reserves
may inhibit the physiological mechanisms utilized by intertidal invertebrates to tolerate
adverse conditions in the intertidal zone. These physiological mechanisms include a reduced
metabolic rate, the production of protective proteins such as heat shock proteins, and the
synthesis of new proteins to replace those that have been denatured (Sokolova and Pörtner
2001; Somero 2002; Berger and Emlet 2007). Each of these responses have an associated
energetic cost, and some portion of individuals may possess energy stores low enough that
their physiological tolerances to these environmental stressors are impacted. In this way, low
energy reserves could indirectly lead to increased likelihood of mortality. However, the
influence of initial energy reserves on tolerance to environmental stressors in EBP
invertebrates has been mostly speculative.
The overall goal of this thesis was to examine the extent to which initial energy reserves
at the onset of the EBP impact the rates of EBP mortality in intertidal invertebrates. I
examined both the direct effect of energy content in EBP individuals on their survival as well
as the indirect effects of energy content, by determining the relationship between initial
energy reserves and vulnerability to physiological stress. The specific objectives of this

4

project were (1) to determine the proportion of individuals that begin the EBP with energy
reserves that are close to or below a minimum energy threshold, (2) examine the effect of
delayed feeding on the performance of EBP invertebrates, and (3) examine how initial energy
content affects acute tolerance thresholds to two major environmental stressors: (a) high
emersion temperature and (b) desiccation. Exploring these direct and indirect impacts of
initial energy content on EBP mortality will aid our understanding of the factors that
contribute to the high rates of EBP mortality among intertidal invertebrates, and thus the
mechanisms that drive the variable population dynamics of many intertidal species.
Chapter 2 of this thesis focuses on the direct effects of initial energy content on mortality
and performance during the first days and weeks of the EBP. To assess what proportion of
individuals begin the EBP close to or below a minimum energy threshold, individuals of six
invertebrate species were maintained in the laboratory without food for up to 100 d and their
survivorship throughout this period was monitored. To examine the effect of a delay of
feeding on performance, groups of three intertidal species were exposed to different
durations of starvation (0 – 50 d) before providing individuals with food. Each individual
was measured at the end of the starvation period and again after a 30 d feeding period, and
the impact of different durations of feeding delay on survivorship and growth was
determined.
Chapter 3 examines the indirect effects of energy on EBP mortality by determining the
relationship between initial energy content and physiological tolerance to the two most
important environmental stressors in the intertidal zone: desiccation and elevated
temperature. EBP individuals of two species were collected and groups with different
quantities of energy reserve were established. Since direct measurement of energy content in
live individuals was not possible, two proxy methods were used to quantify energy reserves:
duration of starvation prior to experimentation, and initial body mass. The various energy
treatment groups were then exposed to a range of temperatures and durations in a low
humidity environment in a series of controlled laboratory experiments. Acute tolerance
thresholds to each stressor were then determined and compared between the different energy
treatment groups.
Chapter 4 provides a summary of the major findings of this thesis, implications of the
findings for policy, and possible directions for future study.

5

LITERATURE CITED
Bennett CE, Marshall DJ. 2005. The relative energetic costs of the larval period, larval
swimming and metamorphosis for the ascidian Diplosoma listerianum. Mar. Freshw.
Behav. Physiol. 38:21–29.
Berger MS, Emlet RB. 2007. Heat-Shock Response of the Upper Intertidal Barnacle Balanus
glandula: Thermal Stress and Acclimation. Biol. Bull. 212:232–241.
Bryan PJ. 2004. Energetic cost of development through metamorphosis for the seastar
Mediaster aequalis (Stimpson). Mar. Biol. 145:293–302.
Connell J. 1985. The consequences of variation in intertidal settlement vs. post-settlement
mortality in rocky intertidal communities. Exp. Mar. Biol. Ecol. 93:11–45.
Dahlhoff EP, Buckley BA, Menge BA. 2001. Physiology of the Rocky Intertidal Predator
Nucella ostrina along an Environmental Stress Gradient. Ecology 82:2816–2829.
Emlet RB, Sadro SS. 2006. Linking stages of life history: How larval quality translates into
juvenile performance for an intertidal barnacle (Balanus glandula). Integr. Comp. Biol.
46:334–346.
Foster BA. 1971. Desiccation as a factor in the intertidal zonation of barnacles. Marine
Biology 29:12–29.
Fraschetti S, Giangrande A, Terlizzi A, Boero F. 2003. Pre- and post-settlement events in
benthic community dynamics. Oceanol. Acta 25:285–295.
Gaines S, Roughgarden J. 1985. Larval settlement rate: A leading determinant of structure in
an ecological community of the marine intertidal zone. Proc. Natl. Acad. Sci. 82:3707–
3711.
Gosselin LA. 1997. An ecological transition during juvenile life in a marine snail. Mar. Ecol.
Prog. Ser. 157:185–194.
Gosselin LA, Chia FS. 1994. Feeding habits of newly hatched juveniles of an intertidal
predatory gastropod, Nucella emarginata (Deshayes). J. Exp. Mar. Bio. Ecol. 176:1–13.
Gosselin LA, Chia FS. 1995. Characterizing temperate rocky shores from the perspective of
an early juvenile snail: the main threats to survival of newly hatched Nucella
emarginata. Mar. Biol. 122:625–635.
Gosselin LA, Qian P. 1997. Juvenile mortality in benthic marine invertebrates. Mar. Ecol.

6

Prog. Ser. 146:265–282.
Goulden CE, Henry L, Berrigan D. 1987. Egg size, postembryonic yolk, and survival ability.
Oecologia 72:28–31.
Guillou J, Tartu C. 1994. Post-larval and juvenile mortality in a population of edible cockle
Cerastoderma edule (L.) from northern Brittany. Netherlands J. Sea Res. 33:103–111.
Hamilton HJ, Gosselin LA (2020) Ontogenetic shifts and interspecies variation in tolerance
to desiccation and heat at the early benthic phase of six intertidal invertebrates. Mar.
Ecol. Prog. Ser. 634:15-28.
Hadfield M. 1986. Settlement and recruitment of marine invertebrates: a perspective and
some proposals. Bull. Mar. Sci. 39:418–425.
Helmuth BST, Hofmann GE. 2001. Microhabitats, thermal heterogeniety, and patterns of
physiological stress in the rocky intertidal zone. Biol. Bull. 201:374–384.
Hughes TP. 1990. Recruitment Limitation, Mortality, and Population Regulation in Open
Systems: A Case Study. Ecology 71:12–20.
Hunt HL, Scheibling RE. 1997. Role of early post-settlement mortality in recruitment of
benthic marine invertebrates. Mar. Ecol. Prog. Ser. 155:269–301.
Jablonski D, Lutz R. 1983. Larval Ecology of Marine Benthic Invertebrates: Paleobiological
Implications. Biol. Rev. 58:21–89.
Jarrett JN. 2000. Temporal variation in early mortality of an intertidal barnacle. Mar. Ecol.
Prog. Ser. 204:305–308.
Jarrett JN. 2003. Seasonal variation in larval condition and post-settlement performance of
the barnacle Semibalanous balanoides. Ecology 84:384–390.
Jarrett JN, Pechenik JA. 1997. Temporal variation in cyprid quality and juvenile growth
capacity for an intertidal barnacle. Ecology 78:1262–1265.
Jenewein BT, Gosselin LA. 2013a. Ontogenetic shift in stress tolerance thresholds of Mytilus
trossulus: Effects of desiccation and heat on juvenile mortality. Mar. Ecol. Prog. Ser.
481:147–159.
Jenewein BT, Gosselin LA. 2013b. Early benthic phase mortality of the barnacle Balanus
glandula is influenced by Fucus spp. cover but not by weather-related abiotic conditions.
J. Exp. Mar. Bio. Ecol. 449:28–35.

7

Keough MJ, Downes BJ. 1982. Recruitment of marine invertebrates: the role of active larval
choices and early mortality. Oecologia 54:348–352.
Lloyd MJ, Gosselin LA. 2011. Role of maternal provisioning in controlling interpopulation
variation in hatching size in the marine snail Nucella ostrina. Biol. Bull. 213:316–324.
Lucas MI, Walker G, Holland DL, Crisp DJ. 1979. An energy budget for the free-swimming
and metamorphosing larvae of Balanus balanoides (Crustacea: Cirripedia). Mar. Biol.
55:221–229.
Marshall DJ, Bolton TF, Keough MJ. 2003. Offspring size affects the post metamorphic
performance of a colonial marine invertebrate. Ecology 84:3131–3137.
Miller CA, Waldbusser GG. 2016. A post-larval stage-based model of hard clam Mercenaria
mercenaria development in response to multiple stressors: Temperature and
acidification severity. Mar. Ecol. Prog. Ser. 558:35–49.
Miller LP, Harley CDG, Denny MW. 2009. The role of temperature and desiccation stress in
limiting the local-scale distribution of the owl limpet, Lottia gigantea. Funct. Ecol.
23:756–767.
Miller SE. 1993. Larval period and its influence on post-larval life history: comparison of
lecithotrophy and facultative planktotrophy in the aeolid nudibranch Phestilla sibogae.
Mar. Biol. 117:635–645.
Moran AL. 1999. Size and Performance of Juvenile Marine Invertebrates: Potential Contrasts
Between Intertidal and Subtidal Benthic Habitats. Am. Zool. 39:304–312.
Moran AL, Emlet RB. 2001. Offspring Size and Performance in Variable Environments :
Field Studies on a Marine Snail. Ecol. Soc. Am. 82:1597–1612.
Pechenik JA, Hammer K, Weise C. 1996. The effect of starvation on acquisition of
competence and post- metamorphic performance in the marine prosobranch gastropod
Crepidula fornicata (L.). J. Exp. Mar. Bio. Ecol. 199:137–152.
Pechenik JA, Rittschof D, Schmidt AR. 1993. Influence of delayed metamorphosis on
survival and growth of juvenile barnacles Balanus amphitrite. Mar. Biol. 115:287–294.
Phillips NE. 2002. Effects of Nutrition-Mediated Larval Condition on Juvenile Performance
in a Marine Mussel. Ecology 83:2562–2574.
Phillips NE. 2017. Variability in early post-settlement mortality in intertidal mussels and the
role of size at settlement. Mar. Biol. Res. 13:726–732.

8

Sandee SD, Van Hamme JD, Gosselin LA. 2016. Testing microbial pathogens as a cause of
early juvenile mortality in wild populations of benthic invertebrates. Mar. Ecol. Prog.
Ser. 562:53–63.
Shanks AL. 2009. Barnacle settlement versus recruitment as indicators of larval delivery. I.
Effects of post-settlement mortality and recruit density. Mar. Ecol. Prog. Ser. 385:205–
216.
Sokolova IM, Frederich M, Bagwe R, Lannig G, Sukhotin AA. 2012. Energy homeostasis as
an integrative tool for assessing limits of environmental stress tolerance in aquatic
invertebrates. Mar. Environ. Res. 79:1–15.
Sokolova IM, Pörtner HO. 2001. Physiological adaptations to high intertidal life involve
improved water conservation abilities and metabolic rate depression in Littorina
saxatilis. Mar. Ecol. Prog. Ser. 224:171–186.
Somero GN. 2002. Thermal physiology and vertical zonation of intertidal animals: Optima,
limits, and costs of living. Integr. Comp. Biol. 42:780–789.
Stoner DS. 1990. Recruitment of a tropical colonial ascidian relative importance of pre
settlement vs post settlement processes. Ecology 71:1682–1690.
Thiyagarajan V, Harder T, Qian PY. 2002. Relationship between cyprid energy reserves and
metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica). J.
Exp. Mar. Bio. Ecol. 280:79–93.
Thiyagarajan V, Harder T, Qiu J-W, Qian P-Y. 2003. Energy content at metamorphosis and
growth rate of the early juvenile barnacle Balanus amphitrite. Mar. Biol. 143:543–554.
Thiyagarajan V, Hung OS, Chiu JMY, Wu RSS, Qian PY. 2005. Growth and survival of
juvenile barnacle Balanus amphitrite: interactive effects of cyprid energy reserve and
habitat. Mar. Ecol. Prog. Ser. 299:229–237.
Torres G, Giménez L, Pettersen AK, Bue M, Burrows MT, Jenkins SR. 2016. Persistent and
context-dependent effects of the larval feeding environment on post-metamorphic
performance through the adult stage. Mar. Ecol. Prog. Ser. 545:147–160.
Wendt DE. 2000. Energetics of larval swimming and metamorphosis in four species of
Bugula (Bryozoa). Biol. Bull. 198:346–356.

9

CHAPTER 2: Role of depleted initial energy reserves in early benthic phase mortality
of six marine invertebrate species 1

INTRODUCTION
Recruitment rates of benthic marine invertebrates are highly variable and can heavily
impact the abundance and distribution of adult populations (Connell 1985; Stoner 1990; Hunt
and Scheibling 1998; Miller and Waldbusser 2016). This is largely due to high rates of
mortality during the first few days of independent benthic life (Thorson 1966; Keough and
Downes 1982; Hadfield 1986; Osman et al. 1989; Guillou and Tartu 1994; Gosselin and Qian
1997; Hunt and Scheibling 1997; Ellien et al. 2000), a period identified as the early benthic
phase (EBP) that encompasses early juvenile life and may also include pre-metamorphic
larval life (Sandee et al. 2016). In addition, EBP mortality rates are highly variable not only
among species, but also among and within cohorts of the same species (Gosselin and Qian
1996; Jarrett and Pechenik 1997; Jarrett 2000; Phillips 2017). The mechanisms responsible
for the high rates and extensive variability in EBP mortality are not yet fully understood and
remain an area of active research.
A factor that has been proposed as a major cause of mortality during early benthic life is
depleted energy reserves, when individuals begin the EBP with minimal energy stores
(Gosselin and Qian 1997; Hunt and Scheibling 1997; Jarrett and Pechenik 1997). Energy
reserves at the start of independent benthic life are known to affect other relevant aspects of
EBP performance, including the subsequent size and growth rate of individuals throughout
juvenile life (Miller 1993; Pechenik et al. 1996; Miller and Emlet 1999; Pechenik et al. 2002;
Thiyagarajan et al. 2003; Thiyagarajan et al. 2005; Emlet and Sadro 2006). Previous studies
have also revealed a correlation between initial body size and mortality, with smaller
individuals experiencing higher rates of mortality than larger EBP individuals (Moran 1999;
Phillips 2002; Thiyagarajan et al. 2002; Phillips 2017). Those studies have suggested the
relationship between body size and mortality may be due to increased vulnerability to
predation, competition, or abiotic stressors, but it might also be due to insufficient initial

1

A version of this chapter has been published:
Mendt SR, Gosselin LA (2021) Role of depleted initial energy reserves in early benthic phase mortality of six
marine invertebrate species. Ecology and Evolution 11: 8882–8896. https://doi.org/10.1002/ece3.7723

10

energy reserves directly causing EBP mortality. While these indirect lines of evidence are
consistent with the hypothesis that depleted initial energy reserves directly cause EBP
mortality, the hypothesis remains untested.
For the depleted initial energy hypothesis to be correct, there must be variation in initial
energy reserves, with some individuals having relatively low energy levels. Accordingly,
variation in organic content, a proxy for energy reserves, at the onset of the EBP among
individuals does occur in at least some species, with some individuals beginning the EBP
with relatively low organic content (Jarrett and Pechenik 1997; Moran and Emlet 2001). This
variation is largely due to differences in maternal provisioning or in food availability to the
larvae, as well as competent larvae of many species transitioning through energetically
demanding periods of development prior to the onset of the EBP (Lucas et al. 1979;
Thiyagarajan et al. 2003). The most energetically demanding of these developmental periods
is metamorphosis, which involves extensive rearrangement of tissues and loss or replacement
of numerous body structures (Wendt 2000; Thiyagarajan et al. 2002, 2003; Bryan 2004;
Bennett and Marshall 2005). Additionally, development in some species includes a
substantial non-feeding phase immediately before, during, or following the onset of the EBP
(Anderson 1994; Gosselin and Chia 1994). During these non-feeding periods, and until
metamorphosis is completed and feeding begins, individuals are solely reliant upon their
internal energy stores for resting and active metabolism (Lucas et al. 1979; Pechenik et al.
1993; Gosselin and Chia 1994). Accordingly, it has been suggested that some portion of
individuals may begin the EBP with such critically low energy stores that they are near a
minimum threshold necessary to survive (Jarrett and Pechenik 1997); those individuals
would either inevitably die of energy depletion very soon after metamorphosis, or need to
quickly feed to replenish energy stores to survive. We illustrate this hypothesis in Figure 2.1,
representing two hypothetical frequency distributions of energy reserves among EBP
individuals as well as a theoretical minimum threshold of energy reserves represented by the
vertical line. Curve A represents the null hypothesis, whereby all individuals have sufficient
energy to survive, in which case the depletion of energy reserves would not be a direct cause
of EBP mortality. Conversely, curve B represents the current hypothesis, that a proportion of
individuals begin the EBP below the minimum energy reserve threshold, and thus inevitably
die during or shortly after metamorphosis. In addition, if the hypothesis represented by curve

11

B is correct, then a substantial proportion of EBP individuals would have energy reserves
only slightly larger than the minimum threshold; these individuals would need to feed
immediately to survive or would otherwise drop below the threshold and die. Thus, a goal of
the present study was to test these two hypotheses to establish the role of energy reserves as a
direct cause of EBP mortality.
While EBP mortality rates are often very high, they are also highly variable among and
within populations (Gosselin and Qian 1997; Hunt and Scheibling 1997; Jarrett 2000;
Phillips 2017). The hypothesis that variation in energy reserves (bell curves in Figure 2.1) is
a cause of the variability in EBP mortality is partly supported by evidence (Jarrett and
Pechenik 1997; Phillips 2006) of natural variation among individuals in the amount of
organic content or lipid stores present at the onset of the EBP. Internal energy reserves can
vary depending on the source of energy, and in some cases upon the quantity of food
available prior to the beginning of the EBP. For species with planktotrophic larvae, EBP
energy reserves originate primarily from particulate food consumed during their larval phase.
For these species, energy reserves can vary among individuals depending on the quantity and
quality of food they encounter (Jarrett 2003; Moran and Manahan 2004), whether they
transition through a non-feeding phase, and whether they encounter conditions that delay
settlement (Pechenik et al. 1993; Phillips 2002; Thiyagarajan 2003, 2007). For direct
developing species, EBP energy reserves originate entirely from maternal provisioning, and
thus may vary with the condition of the maternal parent (Moran and McAlister 2009; van der
Sman et al. 2009) or with the genetic programming of the population or species (Rivest 1983;
Allen and Marshall 2013). In some cases, unfertilized nurse eggs are provided to the embryos
to consume as they develop; for these species, energy levels can further vary among
individuals of the same cohort depending on the quantity of nurse eggs consumed before
hatching (Jablonski and Lutz 1983; Lloyd and Gosselin 2011; Marko et al. 2014). All these
differences, both interspecific and intraspecific, result in individuals beginning the EBP with
vastly different amounts of internal energy reserves, and this might play a substantial role in
determining which individuals survive through the first few days of independent benthic life.
Additionally, any of these factors has the potential to shift the frequency distribution of
energy reserves to the left or right, as depicted by the bell curves A and B in Figure 2.1, or

12

even alter the shape of these curves and thus also affect the proportion of the individuals
falling below the minimum threshold for survival.

Proportion of individuals

Minimum
energy
threshold

B

A

Energy reserves at onset of EBP
Figure 2.1. Two hypothetical frequency distributions (bell curves) of energy reserves among
individuals at the onset of the EBP. The dashed vertical line represents a minimum energy
threshold required for EBP survival.

For individuals starting the EBP with only slightly more internal energy than the
minimum threshold, rapidly accessing a new food source to replenish their energy reserves
could be vital to their survival. A delay in feeding at this point might drop them below the
threshold, and thus push them beyond their “point of no return” (commonly referred to as
PNR in other publications), a pivotal point beyond which animals are unable to recover even
if provided with food (Blaxter and Hempel 1963; Moran and Manahan 2004). For individuals
that do not feed before this point, both their survivorship and ability to grow are likely to be
impaired (Takami et al. 2000; Roberts et al. 2001; Chen et al. 2005). The existence and
timing of a point of no return has been studied for the larval phase of several species of

13

intertidal invertebrates (Moran and Manahan 2004; Yan et al. 2009; Gebauer et al. 2010;
Espinoza et al. 2017) but has not been well established for EBP individuals of most species.
Determining the starvation point at which an individual’s energy reserves are so heavily
depleted that they are unable to recover may be useful for understanding how energy reserves
impact EBP survivorship and growth.
The main purpose of this study was to determine the role of energy content in regulating
survivorship during the critical first days of independent benthic life, thus helping to better
understand the mechanisms controlling EBP mortality, and thus recruitment, of benthic
invertebrates. The first specific objective of the study was (1) to establish whether energy
reserves of EBP individuals could be experimentally controlled by starvation; this was
addressed by determining the relationship between total organic content of EBP individuals
and the duration of starvation they had experienced. This was followed by two additional
specific objectives: (2) to test the hypothesis (Figure 2.1, curve B) that a large proportion of
individuals begin the EBP with critically low energy stores such that they either die or
require rapid access to food to survive the first days of independent benthic life, and (3) to
examine the effect of delayed feeding on the performance (growth and survival) of EBP
invertebrates over a period of several weeks.

METHODS
Study species
This study examined the role of initial energy reserves on survivorship in six species of
benthic intertidal invertebrates. All animals used in this study were from wild populations
and had developed through the embryonic and planktonic larval phases in the field. Species
were selected based on sufficient availability of early benthic phase (EBP) specimens in the
field. All six species are abundant on the west coast of Vancouver Island, Canada. The
barnacles Balanus glandula Darwin 1854 and Chthamalus dalli Pilsbry 1916, as well as the
mussel Mytilus trossulus Gould 1850, colonize exposed hard substrata in the mid to high
intertidal zone. Nucella ostrina Gould 1852 and Nucella lamellosa Gmelin 1791 are
predatory snails that inhabit the mid-intertidal zone. Finally, Petrolisthes cinctipes Randall
1840 and P. eriomerus Stimpson 1871 are small (~2 cm) porcellanid crabs that inhabit
cryptic habitats, primarily the undersides of large rocks (Jensen 1989), in the low to mid

14

intertidal zone; EBP individuals of these two species could not be readily distinguished, so
these were grouped together and designated as Petrolisthes spp.
These species have different sources and quantities of energy reserves at the beginning
of the EBP. Nucella ostrina and N. lamellosa are lecithotrophic, and thus depend entirely on
maternal provisioning for EBP energy reserves. The four other species, however, have
planktotrophic larvae, and a large portion of their EBP energy reserves come from the food
they consume during the larval stage. Additionally, B. glandula and C. dalli have a nonfeeding cyprid larval stage prior to metamorphosis, and thus use up some energy stores prior
to the EBP, whereas M. trossulus and Petrolisthes spp. are able to feed up to and throughout
metamorphosis. By including species with different developmental histories, this study
explored how EBP survivorship is impacted by energy reserves across species with different
modes of energy acquisition prior to entering the EBP.

Study site and collection of animals
Research was conducted at the Bamfield Marine Sciences Centre (BMSC) on the west
coast of Vancouver Island from May to September of 2019. Animals were collected from
five rocky intertidal shore sites located in Barkley Sound (Figure 2.2). Sites were selected
based on the availability of EBP animals of each species. Collected animals were brought to
and maintained at BMSC, where all experiments were conducted.

Figure 2.2. Map of field sites in Barkley Sound near Bamfield, British Columbia: Ross Islets
(R), Wizard Islet (W), Prasiola Point (P), Dixon Island (D), and Grappler Inlet (G). Map
modified from Gosselin and Chia (1995).

15

Juvenile barnacles (B. glandula and C. dalli) were collected as described by Sandee et
al. (2016) and Hamilton & Gosselin (2020). Small rocks (5 – 10 cm diameter) were gathered
from the intertidal zone and brought to the laboratory where all small settlers were dislodged
under a dissecting microscope, and a perimeter was marked on the rock around the cleared
area with nail polish. Marked rocks were then returned to the field and left for 48-72 h (4-6
tidal cycles) to allow for new settlement, after which rocks were recovered and examined for
newly settled barnacles. At that age, barnacles would still be reliant upon energy reserves
obtained during the larval phase, as juvenile barnacles do not begin to feed until 2 – 5 d postmetamorphosis (Rainbow and Walker 1977). Ripe egg capsules of both Nucella species (N.
ostrina and N. lamellosa) were collected from intertidal substrata using fine-tipped forceps as
in Gosselin & Chia (1995). Egg capsules were returned to the laboratory and held in small
cages in aerated seawater for 72 h, after which all newly hatched juvenile snails were
removed and retained for experiments. EBP M. trossulus were extracted from tufts of the
filamentous algae Cladophora columbiana. Extracted mussels were measured and only
recently settled juveniles measuring 250 – 600 μm shell length (Phillips 2017) were retained
for experiments. Petrolisthes spp. megalopae were collected from the underside of large
rocks in the intertidal zone. Megalopae were carefully removed with a pair of soft insect
forceps and transported to BMSC in small containers. All animals were brought to the
laboratory within 3 h of collection.

Procedures for rearing EBP individuals without food
To experimentally control the energy reserves of EBP individuals, most experiments in
this study involved rearing EBP individuals without food. In all experiments described
below, the following procedures were used to rear EBP individuals of each species while
ensuring they did not have access to food. All animals were held in seawater filtered to 1.0
μm to ensure no food particles were available for feeding. M. trossulus, however, can capture
particles < 1.0 μm (Strohmeier et al. 2012), so filtered water used for this species was also
autoclaved and then vacuum-filtered to 0.45 μm. Rocks containing newly-settled barnacles
were kept in 1 L containers, each with an air stone for aeration. All other animals were kept
in small cages (1.5 – 600 mL) with mesh-covered cut-out windows to allow for water flow
while preventing escapement. Water was maintained at 15 – 17°C, which approximates

16

average sea surface temperatures during the summer months (Iwabuchi and Gosselin 2019).
Filtered water was replaced every second day and holding containers and cages were rinsed
and scrubbed to prevent the growth of algae.

Relationship between organic matter content in EBP individuals and duration of
starvation
To determine if starvation could be used as a method for predictably depleting energy
reserves in EBP individuals, we tested the hypothesis that organic content decreases as a
function of the duration of starvation. This experiment was carried out with N. ostrina
hatchlings, and total organic matter (OM) was measured by ash-free dry weight (AFDW).
AFDW is an effective, albeit simple, indicator of energy content (Moran and McAlister
2009). It does not, however, reflect the energetic value of any single type of organic matter,
such as triacyglycerols (Podolsky 2002), which are the primary source of metabolizable
energy for marine invertebrates (Sewell 2005; Whitehill and Moran 2012). Thus, it is
assumed that the first OM to be depleted by starved EBP individuals will be these energetic
lipids, followed by other forms of OM, resulting in a progressive decrease in AFDW.
This experiment was carried out twice, using two different batches of hatchlings. Batch 1
was collected from Ross Islets on July 17, 2019 and Batch 2 was collected from Prasiola
Point on July 26, 2019. Only intermediate-sized N. ostrina hatchlings (1.11 – 1.40 mm shell
length) were used. Hatchlings from each batch were kept in 50 mL cages, with 100 – 200
individuals in each cage and maintained without food following the procedure stated above.
Every 10 d, beginning immediately after hatching, samples of 52 – 60 unfed N. ostrina
hatchlings from each batch were removed from their respective holding cages. These were
briefly rinsed in distilled water to remove adhering salts and then stored at -80°C. AFDW of
each sample was obtained using a procedure adapted from Pechenik et al. (1993). For each
sample of 52-60 frozen snails, the sample (all snails together) was placed in a pre-weighed
foil pan and dried at 65°C for 48 h. After drying, the pan was weighed on a Fisher Scientific
accuSeries balance with an instrument precision of ±0.01 mg to quantify total dry weight and
then placed in a muffle furnace at 500°C for 5 h. The pan was then placed in a sealed
desiccator containing silica gel desiccant for 20 min to cool, and then reweighed to determine
ash weight (i.e. the weight of the sample without the OM). AFDW, a measure of OM content

17

of each sample, was calculated by subtracting the ash weight from the initial dry weight. Ash
weight consisted almost entirely of the shell, and shell weight was not expected to decline
with starvation, so ash weight was used as an indicator of initial snail body mass prior to
starvation. The OM estimate (AFDW) was therefore divided by the ash weight to obtain the
OM:ash weight ratio; this ratio standardized results for all snails, accounting for any minor
differences in initial body size among individuals.

Ability of EBP individuals to survive exclusively on their initial energy reserves
Survivorship of EBP individuals unable to replenish energy reserves
This experiment determined, for each of the six species, the proportion of individuals
that begin the EBP with energy reserves close to the minimum threshold required for
survival. EBP individuals were collected on several dates throughout the spring and summer,
and each batch of individuals was tested separately as a replicate trial. For each species, 2-8
batches of individuals were tested, each batch consisting of 9-200 individuals (Appendix A).
The number of batches collected for each species, as well as the number of individuals in
each batch, were dependent on the availability of EBP individuals in the field. All animals
were held in 50 mL cages without food, as described above, for up to 100 d starting
immediately after the onset of the EBP. Every 10 d, survival was assessed by examining all
individuals for movement under a dissecting microscope. If an animal did not show any signs
of movement within 2 min, their tissues were gently contacted with a probe and they were
observed for an additional 2 min. If no movement was observed at this point, the individual
was recorded as dead and removed from the experiment. The proportion of individuals
surviving each 10 d period was recorded.
Influence of initial body mass on survivorship of EBP individuals unable to replenish energy
reserves
There is a strong correlation between body mass and quantity of organic matter among
EBP invertebrates (Moran and Emlet 2001). Specifically, larger individuals have larger
stores of lipids (Phillips 2002; Emlet and Sadro 2006), which are the primary metabolizable
energy source for many intertidal species (Thiyagarajan et al. 2002; Sewell 2005; Whitehill
and Moran 2012). Thus, body mass was used as a proxy for energy reserves to determine
their influence on survivorship. To determine how the ability to survive periods of starvation

18

is influenced by body mass at the start of the EBP, the survivorship of three size classes of
newly hatched N. ostrina were examined. Most N. ostrina egg capsules contain 7-20
fertilized embryos that will hatch into juvenile snails, as well as 500 – 600 unfertilized nurse
eggs that the embryos consume as they develop inside the capsule (Lloyd and Gosselin 2011;
Marko et al. 2014). N. ostrina hatchlings consume different numbers of nurse eggs while in
the capsule, and therefore hatchlings range considerably in size upon emergence, with larger
hatchlings having consumed more nurse eggs and thus having greater energy reserves than
smaller hatchlings. Newly hatched N. ostrina were sorted into three shell length (SL)
categories: small (0.81 – 1.10 mm SL), medium (1.11 – 1.40 mm SL), and large (1.41 – 1.80
mm SL). These SL measurements were then converted to body mass (wet weight, WW)
using a regression equation for N. ostrina from Hamilton & Gosselin (2020) (Table 2.1).
Eight batches of hatchlings were collected throughout the spring and summer; each batch
was sorted into the three size classes, and each size class of a given batch contained 11-217
hatchlings (Appendix A). In some batches, certain size classes were not included in this
experiment because too few individuals of the size class were available. Hatchlings were held
in 50 mL cages without food for up to 70 d, beginning at the time of hatching (i.e. the onset
of EBP). Every 10 d, survival was assessed by examining all individuals under a dissecting
microscope, as described above, and the number of survivors of each size class was recorded.

Table 2.1. Shell lengths (SL) and corresponding body masses (wet weight, WW) for N.
ostrina hatchlings. The conversion equation is: WW = 2.98 * log(SL) - 3.84, from Hamilton
& Gosselin (2020).
Size class

Shell length (SL)

Body mass (WW)

in mm

in mg

Small

0.81 – 1.10

0.074 – 0.192

Medium

1.11 – 1.40

0.197 – 0.394

Large

1.41 – 1.80

0.402 – 0.833

19

Ability to recover after delayed feeding in EBP invertebrates
The ability of EBP individuals to recover from periods of starvation was examined in
three species: B. glandula, C. dalli, and N. ostrina. EBP individuals of each species were
reared without food, beginning at the onset of EBP, for various durations (0, 10, 20, 30, 40 or
50 d) before being fed for 30 d. Growth during the 30 d feeding period was determined by
measuring the size (rostro-carinal diameter for barnacles, SL for N. ostrina) of each
individual at the end of the starvation period (i.e. just before feeding began), and then again
at the end of the 30 d feeding period. The number of individuals that died during the 30 d
feeding period was also recorded for each group. All individuals of the same species were
within a narrow size range at the beginning of the experiment. The experimental design for
each of the three species was as follows: 6 duration of starvation treatments per species X 10
– 15 individuals per starvation treatment.
During the feeding period, B. glandula and C. dalli were held in 1 L aerated containers
and fed a mixture of diatoms (Chaetoceros muelleri and C. gracilis) and flagellates
(Tertaselmis spp.) that had been reared in the laboratory. Algae were provided at a total
concentration of 1x106 cells/mL, determined by counting algal cells with a hemocytometer.
Water and algae were changed every second day. To feed N. ostrina hatchlings, each
individual was placed in a small (1.5 mL) cage with 10 juvenile mussels (M. trossulus), a
preferred prey species for EBP N. ostrina (Gosselin and Chia 1994). N. ostrina feed by
drilling a small hole through the shell of their prey with their radula, making it possible to
determine whether the hatchling had fed.
To assess the effect of delayed feeding on the performance of EBP invertebrates,
individuals were categorized as either recovering (i.e. alive and showing growth) or not
recovering (i.e. dead, or alive but with no growth) following the 30 d feeding period. The
percent of individuals that were recovering in each starvation treatment was then calculated
for each species. Additionally, the growth (i.e. change in size) of each EBP individual during
the feeding period was calculated to assess the effects of delayed feeding on the ability to
resume growth when food becomes available.

20

Data analysis
In all experiments, survivorship data followed a binomial distribution. All survivorship
data were analyzed using generalized linear models (GLM) with a binomial link function to
determine the starvation LD50 for each species, and the % survivorship after 5 d starvation
and the starvation LD20 for each size class of N. ostrina. The fit of each GLM model was
tested by comparing the residual deviance to the residual degrees of freedom, which
indicated goodness of fit for each model (i.e. residual deviance / residual df < 1). One-way
ANOVA and the Tukey HSD multiple comparisons test were used to compare the %
survivorship after 5 d starvation and the starvation LD 20 among the three size classes of N.
ostrina hatchlings. The Shapiro-Wilk normality test and the Bartlett test of homogeneity of
variances were used to assess the assumptions of ANOVA. All statistical analyses were
conducted using R statistical software (version 3.5.0) (R Core Team 2018).

RESULTS
Relationship between organic matter content in EBP individuals and duration of
starvation
The relationship between organic matter (OM):ash weight ratio and duration of
starvation starting at the onset of the early benthic phase (EBP)(Figure 2.3) was effectively
described by an exponential decay model: 𝑦 = 𝑦0 + 𝑎 ∗ 𝑒

, where y0 is the intercept, a is

the lower limit reached for y, x is the duration of starvation, and b is the decay coefficient.
The OM:ash weight ratio of Nucella ostrina was significantly related to the duration of
starvation in both batches of N. ostrina hatchlings (Batch 1: F = 583.95, df = 6, R2 = 0.996, p
< 0.001; Batch 2: F = 175.45, df = 5, R2 = 0.989, p = 0.006). Based on this analysis,
approximately half the OM content of an individual was utilized by day 10 of starvation
(Batch 1: 51.05%, Batch 2: 49.35%).

21

OM:ash weight ratio

0.5
Batch 1
Batch 2

0.4
0.3
0.2
0.1
0.0
0

10

20

30

40

50

Duration of starvation,
since onset of EBP (d)
Figure 2.3. Relationship between OM:ash weight ratio and the duration of starvation,
starting at the onset of the EBP, for Nucella ostrina hatchlings. OM is used as an indicator of
total energy content and ash weight is used as an index of initial total body mass. Each value
represents a combined sample of 52 - 60 individuals. Each batch represents a single
collection of hatchlings from the field.

Ability of EBP individuals to survive exclusively on their initial energy reserves
Survivorship of EBP invertebrates unable to replenish energy reserves
Survivorship was impacted by the duration of starvation experienced by the individuals
(e.g. Balanus glandula, Figure 2.4). The relationship, assessed using generalized linear
model (GLM) with a binomial link function, was significant in four of the six species (Table
2.2). For Mytilus trossulus and Petrolisthes spp. the relationship was not quite significant,
likely due to the modest sample sizes, as only two batches of individuals were collected for
each of these species, whereas 5 – 9 replicate batches were examined in each of the other
four species. The trends of decreasing survivorship as a function of duration of starvation for
M. trossulus and Petrolisthes spp. were nevertheless similar to those of the other four species
(Appendix A).

22

Survivorship (%)

100
80
60
40
20
0
0

20

40

60

80

100

Duration of starvation,
since onset of EBP (d)
Figure 2.4. Percent survivorship of starved B. glandula as a function of the duration of
starvation, starting at the onset of the EBP. The point where the curve intersects the dashed
line represents the starvation LD50.

The duration of starvation that is lethal to 50% of individuals (starvation LD 50), as
determined from the above GLM analyses, varied substantially among species (Table 2.2).
Starvation LD50 ranged from 27.9 ± 4.75 d (average ± SE) for Nucella lamellosa to 78.4 ±
7.59 d for B. glandula. When survivorship of all six species during the first 10 d of starvation
was combined in a single regression analysis, the percent survivorship declined significantly,
but moderately (Linear regression: F = 7.589, n = 6, R 2 = 0.26, p = 0.012) (Figure 2.5). As a
result, survivorship remained high throughout these first 10 d. After 5 d of starvation,
average survivorship was >90% in all species; after 10 d starvation average survivorship was
still >90% in four of the six species (B. glandula, C. dalli, N. ostrina, and Petrolisthes spp),
and >85% for the remaining two (N. lamellosa and M. trossulus).

23

Table 2.2. Starvation LD50 and associated standard error (SE) from generalized linear model
(GLM) analysis of survival as a function of duration of starvation for each of the 6 species.
The z statistic represents the strength of the relationship between the % survivorship and the
duration of starvation; df = degrees of freedom.
Starvation
Species

LD50 (d)

SE

z statistic

df

p

B. glandula

78.4

7.59

-3.40

62

< 0.001

C. dalli

69.3

7.20

-3.23

42

0.001

N. ostrina

55.9

9.66

-2.56

51

< 0.001

N. lamellosa

27.9

4.75

-2.99

30

0.003

M. trossulus

58.3

14.28

-1.76

16

0.079

Petrolisthes spp.

50.8

5.70

-1.59

12

0.113

100

Survivorship (%)

90
80
B. glandula
C. dalli
N. ostrina
N. lamellosa
M. trossulus
Petrolisthes spp.

70
60
50
0

5

10

Duration of starvation,
since onset of EBP (d)
Figure 2.5. Survivorship of individuals starved for the first 10 d since the onset of EBP for
six benthic intertidal species. Each point represents the average % survivorship and
associated standard errors calculated from GLMs for each species (Table 2.1).

24

Influence of initial body mass on survivorship of EBP individuals unable to replenish energy
reserves
Survivorship was significantly affected by the duration of starvation for each batch of all
three size classes of N. ostrina hatchlings (Table 2.3). The shape of this relationship,
however, differed between size classes. The impact of starvation on survivorship of N.
ostrina in each size class during the critical first days of the EBP was examined using two
different approaches: (1) determining the proportion of individuals that survived 5 d of
starvation after hatching, and (2) determining the number of days of starvation required to
cause 20% mortality of hatchlings (starvation LD20). The starvation LD20 was used instead of
the LD50 because some of the Medium and Large size class batches did not reach 50%
mortality during the time frame of the experiment.
Table 2.3. Results of the GLM analysis of percent survivorship as a function of duration of
starvation for each size class from each batch of N. ostrina hatchlings. Z statistics indicate
the fit of the relationship between duration of starvation and % survivorship; df = degrees of
freedom. The binomial link function was used in all GLMs.
Batch
1
2
3
4
5

6

7
8

Size class
Small
Medium
Small
Medium
Large
Medium
Large
Large
Small
Medium
Large
Small
Medium
Large
Small
Medium
Large
Small
Large

n
13
29
106
90
217
37
56
59
11
25
15
57
152
76
34
115
85
68
34

Estimate
-0.056
-0.034
-0.092
-0.078
-0.036
-0.088
-0.045
-0.050
-0.108
-0.033
-0.036
-0.088
-0.058
-0.051
-0.102
-0.042
-0.080
-0.077
-0.105

SE
0.010
0.015
0.006
0.007
0.004
0.012
0.006
0.007
0.028
0.012
0.018
0.009
0.006
0.009
0.014
0.008
0.016
0.014
0.034

z value
-5.37
-2.34
-15.78
-11.20
-8.22
-7.14
-7.16
-6.87
-3.87
-2.67
-2.03
-9.05
-10.17
-5.99
-7.57
-5.33
-5.12
-5.38
-3.05

df
8
5
7
7
7
4
7
6
5
5
5
5
5
5
5
5
5
4
4

p
<0.001
0.020
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.008
0.042
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.002

25

The three size classes survived equally well without food in the short-term, but their
fate eventually differed as time without food became extended. Survivorship after 5 d of
starvation did not differ significantly among the three size classes (one-way ANOVA: F =
1.207, df = 16, p = 0.325), with survivorship ranging from 93 - 96 % (Figure 2.6A). The
starvation LD20, however, did differ significantly among size classes (one-way ANOVA: F =
5.718, df = 16, p = 0.013) (Figure 2.6B). The LD20 for the Small size class was significantly
shorter than for the Large size class (Tukey HSD: p = 0.010), whereas the LD 20 for the
Medium size class was not significantly different from either the Small (p = 0.132) or the
Large (p = 0.453) size classes.
B)
100

50

90

40

Starvation LD20 (d)

Survivorship (%) at 5 d starvation

A)

80
70
60
50

b
ab

30

a
20
10
0

Small

Medium

Size Class

Large

Small

Medium

Large

Size Class

Figure 2.6. Effect of initial body size of Nucella ostrina hatchlings on (A) the survivorship at
5 d and (B) the starvation LD20. Small = 0.80 – 1.10 mm shell length; Medium = 1.11 – 1.40
mm shell length; Large = 1.41 – 1.80 mm shell length. Each mean value and associated
standard error are calculated from generalized linear models (GLM) for 6 – 7 batches of each
size class. Different letters above the bars indicate values that were significantly different
based on Tukey HSD tests (p < 0.05).

Ability to recover after delayed feeding for EBP invertebrates
The duration of starvation experienced before being offered food had a significant or
nearly significant effect on the percent of individuals able to recover (i.e. survive and grow
during the 30 d feeding period) for C. dalli (Linear regression: F = 17.56, n = 6, R2 = 0.81, p
= 0.014) and N. ostrina (F = 7.08, n = 6, R2 = 0.64, p = 0.056) (Figure 2.7). The duration of
starvation resulting in only 50% of individuals being able to recover (i.e. the recovery LD 50)

26

was >20 d in both species, but was somewhat longer in C. dalli than in N. ostrina (C. dalli:
28.6 ± 4.2 d (SE); N. ostrina: 20.9 ± 6.6 d).

Figure 2.7. Percent of individuals able to recover, as a function of duration of starvation in
(A) C. dalli and (B) N. ostrina. Equations of the regression lines: (A) y = 84.8758-1.2184x,
and (B) y = 83.8095-1.6190x. The point where the model intersects the dashed line indicates
the duration of starvation resulting in only 50% of individuals being able to recover when
fed.

In B. glandula, the ability of individuals to recover was not significantly affected by the
duration of starvation they experience (Linear regression: F = 0.512, n = 6, R 2 = 0.11, p =
0.512). Once B. glandula individuals were provided with food after any period of starvation,
very few deaths were observed (3 deaths, each from a different starvation group, out of a
total of 88 individuals), and all surviving individuals exhibited some growth during the 30 d
feeding period. Thus, to analyze the effect of starvation on recovery of B. glandula, the
average growth throughout the 30 d feeding period was calculated for each treatment (i.e.
each duration of starvation) in order to assess the effect of starvation on their ability to grow
once food becomes available. Additionally, each individual was measured at the end of the
starvation period just prior to feeding, and an average shell diameter was calculated for each
treatment group to assess if any growth occurred during the starvation period. The three
individuals that died during the 30 d feeding period were excluded from this analysis.
Shell diameter at the end of the starvation period differed among treatment groups
(Figure 2.8A) (one-way ANOVA: F = 7.90, df = 74, p < 0.001). This indicates that even in

27

the absence of particulate food, EBP B. glandula deposited some new calcium carbonate into
their shells, thus increasing their shell diameter. However, the increase in shell diameter was
moderate (up to 0.15 mm, or 22%, increase) and significant increases did not occur beyond
20 d of starvation. In contrast, growth during the 30 d feeding period was significantly and
strongly affected by the duration of starvation experienced (Figure 2.8B) (Linear regression:
F = 65.93, n = 79, R2 = 0.46, p < 0.001). Individuals starved for longer periods of time grew
at a substantially slower rate than those starved for a shorter period once particulate food was
introduced.

A)

B)

1.0

2.0

Shell diameter prior
to feeding (mm)

abc

ab

bc

Growth during feeding
period (mm / 30 d)

ab

0.8

c

a
0.6
0.4
0.2
0.0
0

10

20

30

40

Duration of starvation (d)

50

1.5

1.0

0.5

0.0

0

10

20

30

40

50

Duration of starvation prior to feeding (d)

Figure 2.8. Effect of duration of starvation on (A) shell diameter prior to feeding, and (B)
subsequent growth of EBP B. glandula. Values represent average shell diameter ± SE at the
end of the period of starvation (A) and average growth ± SE during the 30 d feeding period
(B). Different letters above error bars on the bar graph indicate values that were significantly
different. Equation of the regression line in (B): y = 1.76033 – 0.023276x.

DISCUSSION
Relationship between organic matter content in EBP individuals and duration of
starvation
Energy content, as estimated by organic matter (OM), in early benthic phase (EBP)
Nucella ostrina declined rapidly, and in a predictable way, as a function of duration of
starvation. The OM:ash weight ratio, a proportional estimate of energy content (relative to
initial body size), significantly decreased as the period of starvation increased. The depletion
of energy content was particularly rapid in the first weeks of starvation; the OM:ash weight

28

ratio dropped by half after only 10 d of starvation, and decreased by an additional 19% by
day 20. In both batches of hatchlings, the OM:ash weight ratio levelled off by day 40 at
approximately 0.14, in other words when the mass of remaining organic matter in the body
was 14% of the mass of the inorganic material, the latter mostly consisting of the shell. This
continuous reduction in OM content is consistent with hatchlings progressively using up their
available energy reserves when not able to feed, and confirms the assumption that ash-free
dry weight (AFDW) is an effective measure of energetic lipids. The minimum OM:ash
weight ratio of 0.14 is likely determined by the amount of organic matter that is difficult to
use as an energy source by the hatchlings. Almost all organic compounds that can readily be
metabolized to provide usable energy, mainly energetic lipids such as triacylglycerols
(Thiyagarajan et al. 2002; Sewell 2005; Lee et al. 2006; Whitehill and Moran 2012; Gosselin
et al. 2019), would thus be utilized within 40-50 d of the onset of the EBP if starved, leaving
compounds such as structural lipids and proteins as the bulk of the remaining organic matter.
Since OM predictably decreases as a function of the duration of starvation, this finding
confirmed that duration of starvation is a useful proxy for metabolizable energy content at the
onset of the EBP.

Ability of EBP individuals to survive exclusively on their initial energy reserves
Survivorship of EBP invertebrates unable to replenish energy reserves
Short-term starvation of individuals from the onset of the EBP did not have the same
implications for survivorship as longer-term starvation. Unsurprisingly, for all six species in
this study, starvation eventually caused high mortality. However, a large proportion of
individuals of all species were capable of surviving prolonged periods of starvation; in 5 of
the 6 species, more than 50% of individuals were still alive after 50 d of starvation, and some
individuals survived starvation for more than 100 d. In addition, survivorship during
starvation decreased gradually within each species, suggesting considerable variation in
either the amount of initial energy reserves or in metabolic rate among individuals of a given
species at the onset of early benthic life. Together, these results indicate that a large
proportion of EBP individuals can survive for remarkably extended periods of time solely on
the internal energy reserves obtained either by larval feeding in planktotrophic species or
from maternal provisioning in direct-developing species.

29

The most significant finding of this study was that all six species of invertebrates were
highly tolerant of starvation in the short-term, i.e. the first few days of the EBP. Survivorship
was minimally impacted by starvation during the first 10 d of the EBP, with three species
experiencing < 2% mortality, and the remaining three species experiencing only 6 – 12%
mortality through this time period. This finding is not consistent with the starting hypothesis
that a substantial proportion of individuals would die in the first few days of starvation due to
depleted energy stores (Figure 2.1, curve B). Rather, these low rates of mortality suggest that
for each of the examined species, very few individuals, if any, enter the EBP with such
critically low energy reserves that they are below or only slightly above the minimum
threshold needed to survive.
This finding of very low mortality of starved individuals during the first days of early
benthic life is surprising. Many species of intertidal invertebrates have extremely high
mortality rates in the field, especially during the first 10 d of the EBP, often ranging from 30100% (Gosselin and Qian 1997; Hunt and Scheibling 1997). More specifically, high
mortality during the first 3-10 d of early benthic life have been reported for wild populations
of a variety of intertidal taxa, some of which were included in the present study: barnacles
(Gosselin and Qian 1996; Shanks 2009; Gosselin and Jones 2010; Jenewein and Gosselin
2013a), gastropods (Spight 1975; Moran and Emlet 2001), mussels (Bownes and McQuaid
2009; Von Der Meden et al. 2012; Phillips 2017), and crabs (Spitzer et al. 2003). Several
studies have hypothesized that the high rate of mortality during the first few days of the EBP
might be due in part to a substantial portion of individuals having insufficient internal energy
reserves at the beginning of the EBP (Gosselin and Qian 1997; Jarrett and Pechenik 1997;
Phillips 2002; Thiyagarajan et al. 2002; Thiyagarajan et al. 2007; Phillips 2017). The present
study, however, demonstrated that under controlled conditions, where individuals were
isolated from other mortality factors (e.g. predation, competition, environmental stressors),
survivorship remained high during the first weeks of the EBP even when animals were
starved and thus relied solely on existing energy reserves present at the onset of the EBP.
Interestingly, the impact of starvation on early survivorship was similar regardless of mode
of larval development or source of EBP energy reserves. The four species with
planktotrophic larvae (B. glandula, C. dalli, M. trossulus, and Petrolisthes spp.) as well as
the two direct developing, lecithotrophic species (N. ostrina and N. lamellosa) all had

30

remarkably high survivorship throughout the first 10 d of EBP when starved. This pattern
was also unaffected by whether the larvae continue to feed through to competence and
settlement or experience some period of pre-EBP starvation as a result of having a nonfeeding phase. Together, these findings fail to reject the theoretical null hypothesis depicted
by curve A in Figure 2.1, revealing that depleted energy reserves are not a significant cause
of the high mortality rates observed during the first weeks of the EBP.
Influence of initial body mass on survivorship of EBP individuals unable to replenish energy
reserves
Differences in starvation tolerance among the three size classes of newly hatched N.
ostrina depended on the temporal scale at which these were examined. When considering
longer-term impacts, the time required to reach 20% mortality (i.e. starvation LD 20) differed
among size classes, with small individuals reaching this point an average of 23 d earlier than
their larger counterparts. This is consistent with the idea that smaller individuals have a
smaller reserve of metabolizable energy and reach a minimum energy threshold more quickly
during periods of starvation than larger individuals. However, the impact of size in
determining starvation tolerance was subtle, as a difference was only observed between the
largest and smallest size classes; the intermediate size class did not differ from either of the
other size classes in their ability to tolerate starvation. The advantage bestowed by the larger
energy reserve of large hatchlings was also moderate; even small individuals were able to
survive for extended periods of time without replenishing energy stores.
The modest long-term energetic advantage of a larger body size was not apparent on a
shorter time scale: there was no significant effect of hatchling size on survivorship during the
first 5 d of the EBP. This indicates that even the smallest size class of N. ostrina hatchlings
have sufficient energy upon hatching to survive through the first 5 d without replenishing
their energy stores, consistent with the null hypothesis that nearly all individuals enter the
EBP with sufficient energy reserves (Figure 2.1, curve A). This is not entirely surprising for
N. ostrina, as hatchlings of this species do not begin feeding until 3 – 10 d after hatching
(Gosselin and Chia 1994). These findings nevertheless further demonstrate that depletion of
energy stores is not a significant cause of EBP mortality.
Offspring size is often a good indicator of offspring fitness; indeed, this is a central tenet
of life history theory (Sinervo 1990, Stearns 1992, Roff 1992). For benthic invertebrates,

31

larger larval size often translates into larger EBP size, which in turn leads to greater
performance (survival and growth) during the EBP (Jarrett and Pechenik 1997; Phillips 2002;
Thiyagarajan et al. 2003; Marshall and Keough 2004; Emlet and Sadro 2006). While many of
these studies have focused on the larval stage, very few studies have examined the
relationship between offspring size and offspring performance after the onset of EBP. The
present study found no significant difference in short-term survivorship among starved
individuals of different initial body sizes, and only modest differences in long-term
survivorship. These findings are not dissimilar to those of Moran and Emlet (2001), who
found increased survivorship for larger hatchlings in longer outplant experiments (36 – 54 d),
but no significant difference in shorter duration outplants (9 d). This indicates that the shortterm response to starvation and energy depletion is independent of the size of recently
hatched individuals.
In populations of many benthic species, larger offspring do tend to outperform their
smaller counterparts; larger offspring grow more quickly (Moran and Emlet 2001; Marshall
et al. 2003; Thiyagarajan et al. 2003; Torres et al. 2016), reach maturity earlier (Moran and
Emlet 2001; Marshall et al. 2003), and often have lower rates of mortality (Phillips 2002;
Thiyagarajan et al. 2003; Emlet and Sadro 2006) than smaller offspring. The results of the
present study, however, indicate that the larger energy reserve of a larger body size does not
lead to greater intrinsic performance during the first days of the EBP. Rather, having a larger
body size may provide other benefits that allow for greater success. A larger body size may
allow individuals to handle and consume prey more efficiently and thus grow at a faster rate
(Palmer 1990; Moran and Emlet 2001). Being larger, or being able to grow quickly to reach
larger body sizes, also decreases risk of predation (Paine 1976; Rumrill 1990; Bashevkin and
Pechenik 2015) and vulnerability to desiccation (Gosselin 1997) by reaching a size refuge
(i.e. growing large enough to considerably reduce vulnerability to stressors in their
environment).

Ability to recover after delayed feeding for EBP invertebrates
Although EBP individuals could survive extended periods of starvation, the ability of
Chthamalus dalli, Nucella ostrina, and Balanus glandula to recover once access to food was
regained was negatively affected by the duration of starvation to which they were exposed at

32

the onset of the EBP. The effect, however, was expressed differently among species. In C.
dalli and N. ostrina, the proportion of individuals able to survive and begin to grow once
food was introduced decreased with increasing duration of starvation. Conversely, in B.
glandula the proportion of individuals able to recover was not impacted by the duration of
starvation, but the rate of growth throughout the 30 d feeding period decreased significantly
as the duration of starvation increased.
In C. dalli and N. ostrina, recovery was impacted by starvation, and in both species the
duration of starvation that resulted in only 50% of individuals able to recover was
considerably shorter than the starvation LD50, found in the previous experiment. Thus,
although individuals are able to survive on internal energy stores for extended periods of
time, they may still be destined to die if unable to replenish those energy stores much sooner
than the LD50. One possible explanation for the difference between the starvation LD 50 and
the starvation period resulting in 50% recovery is that extended periods of starvation may
cause EBP individuals to lose the capacity to feed by inducing a reabsorption or atrophy of
digestive structures (Takami et al. 2000; Espinoza et al. 2017). From that point on,
individuals would then continue to deplete all remaining available internal energy reserves
but be incapable of efficiently feeding and thus replenishing those energy stores if food were
to become available again. We did note that C. dalli exhibited behaviours associated with
feeding, such as extending and beating their cirri, throughout the 30 d feeding period even in
individuals that did not grow; cirral beating in those individuals most likely served primarily
for gas exchange. Similarly, each N. ostrina, including those that did not grow, attacked at
least one juvenile mussel during the 30 d feeding period, as evidenced by drill marks
observed on the valves of dead mussels. This suggests that although EBP individuals are still
capable of exhibiting behaviours associated with feeding, extended periods of starvation may
have reduced their ability to digest or absorb nutrients, even if they are able to access food
items. Another possible explanation for the inability of individuals to recover from prolonged
periods of starvation is that they were unable to extract sufficient energy from consumed
food to both replenish the energy they had spent during the starvation period and also begin
to grow. In this case more food, or a longer feeding period, may be required before these
animals can fully recover.

33

The recovery of starved B. glandula after regaining access to food differed relative to the
other species in this study. Regardless of the duration of starvation experienced in this study,
virtually all B. glandula individuals showed some ability to recover. The rate of growth
throughout the 30 d feeding period, however, was significantly impacted by the duration of
starvation. The 0 d group, which did not experience any starvation, increased in size five
times more during the 30 d feeding period than the group starved for 50 d. This considerable
reduction in growth rate following starvation may have further implications for survivorship,
as slower growing individuals will take longer to reach a size refuge, and thus remain
susceptible to factors such as competition (Connell 1961; Pechenik 1990), predation (Osman
and Whitlach 1995; Gosselin and Rehak 2007), and desiccation (Hamilton and Gosselin
2020) for a longer period of time.
B. glandula also differed from the other species in terms of growth of shell material
while being starved (i.e. prior to feeding). During the first 20 d of starvation, some growth
was apparent in all groups of EBP B. glandula. While growth throughout the starvation
period was modest, it indicates that EBP B. glandula are capable of depositing new shell
material and thus increasing their shell diameter for the first few weeks of the EBP even
without feeding, and thus without replenishing their energy stores. This suggests that
increasing shell size during the EBP is not entirely reliant on obtaining food, nor is it
exclusively stimulated by the growth of body tissues. However, the increase in shell diameter
in newly settled B. glandula that have been without food for any duration was greatly
reduced when compared to individuals that had fed.
Finally, in all three species studied in this experiment, an extended period of starvation
following the onset of the EBP eventually had a negative impact on the ability to recover,
either by directly causing mortality or by impacting growth. Although depletion of energy
was not found to be a direct, major source of early mortality among EBP individuals, these
findings suggest that EBP individuals with reduced energy reserves would still be at a
marked disadvantage for longer-term survival in the intertidal zone.

Ecological implications
Cohorts of benthic invertebrates in the natural environment suffer extremely high
mortality rates during the first days and weeks of the EBP. This study revealed that

34

insufficient energy reserves are not likely a direct cause of this EBP mortality, as had
previously been hypothesized (Gosselin and Qian 1997; Hunt and Scheibling 1997; Jarrett
and Pechenik 1997; Phillips 2017). For those individuals that survive through the larval
phase and make it to the start of the EBP, energy reserves obtained through larval feeding or
maternal provisioning thus appear to be sufficient to sustain the metabolism of the individual
through the critical first days of early benthic life. This further indicates that low food
availability during the first 5-10 days of the EBP would not be a direct cause of mortality
during that time. EBP survivorship in wild populations might nevertheless be impacted by
initial energy reserves through indirect effects. Low energy reserves may cause EBP
individuals to have low tolerance thresholds to environmental stressors, such as extreme
temperatures (Miller et al. 2009; Jenewein and Gosselin 2013b; Hamilton and Gosselin
2020), desiccation (Foster 1971; Gosselin and Chia 1995; Miller et al. 2009; Jenewein and
Gosselin 2013a; Jenewein and Gosselin 2013b), and low salinity (Qiu and Qian 1999;
Thiyagarajan et al. 2007). If so, this could explain previously reported associations between
low energy reserves and high EBP mortality rates in some species (Phillips 2002;
Thiyagarajan et al. 2003; Emlet and Sadro 2006). The role of energy levels on stress
tolerance thresholds, and the possible indirect effects of initial energy reserves on EBP
mortality, are not well understood and require further investigation.

LITERATURE CITED
Anderson, DT. 1994. Barnacles: structure, function, development, and evolution. London,
New York, USA: Chapman & Hall.
Allen RM, Marshall DJ. 2013. Phenotypic links among life-history stages are complex and
context-dependent in a marine invertebrate: Interactions among offspring size, larval
nutrition and postmetamorphic density. Functional Ecology 27:1358–1366.
Bashevkin SM, Pechenik JA. 2015. The interactive influence of temperature and salinity on
larval and juvenile growth in the gastropod Crepidula fornicata (L.). J. Exp. Mar. Bio.
and Ecol. 470:78–91.
Bennett CE, Marshall DJ. 2005. The relative energetic costs of the larval period, larval
swimming and metamorphosis for the ascidian Diplosoma listerianum. Mar. Freshw.
Behav. Physiol. 38:21–29.

35

Blaxter JHS, Hempel G. 1963. The influence of egg size on herring larvae (Clupea harengus
L.). ICES J. Mar. Sci. 28:211–240.
Bownes SJ, McQuaid CD. 2009. Mechanisms of habitat segregation between an invasive and
an indigenous mussel: Settlement, post-settlement mortality and recruitment. Mar. Biol.
156:991–1006.
Bryan PJ. 2004. Energetic cost of development through metamorphosis for the seastar
Mediaster aequalis (Stimpson). Mar. Biol. 145:293–302.
Chen Y, Ke CH, Zhou SQ, Li FX. 2005. Effects of food availability on feeding and growth
of cultivated juvenile Babylonia formosae habei (Altena & Gittenberger 1981). Aquac.
Res. 36:94–99.
Connell J. 1985. The consequences of variation in intertidal settlement vs. post-settlement
mortality in rocky intertidal communities. Exp. Mar. Biol. Ecol. 93:11–45.
Connell JH. 1961. The influence of interspecific competition and other factors on the
distribution of the barnacle Chthamalus stellatus. Ecology 42:710–723.
Ellien C, Thiebaut É, Barnay AS, Dauvin JC, Gentil F, Salomon JC. 2000. The influence of
variability in larval dispersal on the dynamics of a marine metapopulation in the eastern
Channel. Oceanol. Acta 23:423–442.
Emlet RB, Sadro SS. 2006. Linking stages of life history: How larval quality translates into
juvenile performance for an intertidal barnacle (Balanus glandula). Integr. Comp. Biol.
46:334–346.
Espinoza V, Viana MT, Rosas C, Uriarte I, Far A. 2017. Effect of starvation on the
performance of baby octopus (Robsonella fontaniana) paralarvae. Aquac. Res. 48:5650–
5658.
Foster BA. 1971. Desiccation as a factor in the intertidal zonation of barnacles. 29:12–29.
Gebauer P, Paschke K, Anger K. 2010. Seasonal variation in the nutritional vulnerability of
first-stage larval porcelain crab, Petrolisthes laevigatus (Anomura: Porcellanidae) in
southern Chile. J. Exp. Mar. Bio. Ecol. 386:103–112.
Gosselin LA. 1997. An ecological transition during juvenile life in a marine snail. Mar. Ecol.
Prog. Ser. 157:185–194.
Gosselin LA, Chia FS. 1994. Feeding habits of newly hatched juveniles of an intertidal
predatory gastropod, Nucella emarginata (Deshayes). J. Exp. Mar. Bio. Ecol. 176:1–13.

36

Gosselin LA, Chia FS. 1995. Characterizing temperate rocky shores from the perspective of
an early juvenile snail: the main threats to survival of newly hatched Nucella
emarginata. Mar. Biol. 122:625–635.
Gosselin LA, Gallego R, Peters-Didier J, Sewell MA. 2019. Field evidence of
interpopulation variation in oocyte size of a marine invertebrate under contrasting
temperature and food availability. Mar. Ecol. Prog. Ser. 619:69–84.
Gosselin LA, Jones LA. 2010. Effects of solar radiation on barnacle settlement, early postsettlement mortality and community development in the intertidal zone. Mar. Ecol. Prog.
Ser. 407:149–158.
Gosselin LA, Qian P. 1997. Juvenile mortality in benthic marine invertebrates. Mar. Ecol.
Prog. Ser. 146:265–282.
Gosselin LA, Qian PY. 1996. Early post-settlement mortality of an intertidal barnacle: A
critical period for survival. Mar. Ecol. Prog. Ser. 135:69–75.
Gosselin LA, Rehak R. 2007. Initial juvenile size and environmental severity: Influence of
predation and wave exposure on hatching size in Nucella ostrina. Mar. Ecol. Prog. Ser.
339:143–155.
Guillou J, Tartu C. 1994. Post-larval and juvenile mortality in a population of edible cockle
Cerastoderma edule (L.) from northern Brittany. Netherlands J. Sea Res. 33:103–111.
Hamilton HJ, Gosselin LA (2020) Ontogenetic shifts and interspecies variation in tolerance
to desiccation and heat at the early benthic phase of six intertidal invertebrates. Mar.
Ecol. Prog. Ser. 634:15-28.
Hadfield M. 1986. Settlement and recruitment of marine invertebrates: a perspective and
some proposals. Bull. Mar. Sci. 39:418–425.
Hunt HL, Scheibling RE. 1997. Role of early post-settlement mortality in recruitment of
benthic marine invertebrates. Mar. Ecol. Prog. Ser. 155:269–301.
Hunt HL, Scheibling RE. 1998. Spatial and temporal variability of patterns of colonization
by mussels (Mytilus trossulus, M. edulis) on a wave-exposed rocky shore. Mar. Ecol.
Prog. Ser. 167:155–169.
Iwabuchi BL, Gosselin LA. 2019. Long-term trends and regional variability in extreme
temperature and salinity conditions experienced by coastal marine organisms on
Vancouver Island, Canada. Bull. Mar. Sci. 95:337–354.

37

Jablonski D, Lutz R. 1983. Larval Ecology of Marine Benthic Invertebrates: Paleobiological
Implications. Biol. Rev. 58:21–89.
Jarrett JN. 2000. Temporal variation in early mortality of an intertidal barnacle. Mar. Ecol.
Prog. Ser. 204:305–308.
Jarrett JN. 2003. Seasonal variation in larval condition and post-settlement performance of
the barnacle Semibalanous balanoides. Ecology 84:384–390.
Jarrett JN, Pechenik JA. 1997. Temporal variation in cyprid quality and juvenile growth
capacity for an intertidal barnacle. Ecology 78:1262–1265.
Jenewein BT, Gosselin LA. 2013a. Early benthic phase mortality of the barnacle Balanus
glandula is influenced by Fucus spp. cover but not by weather-related abiotic
conditions. J. Exp. Mar. Bio. Ecol. 449:28–35.
Jenewein BT, Gosselin LA. 2013b. Ontogenetic shift in stress tolerance thresholds of Mytilus
trossulus: Effects of desiccation and heat on juvenile mortality. Mar. Ecol. Prog. Ser.
481:147–159.
Jensen G. 1989. Gregarious settlement by megalopae of the porcelain crabs Petrolisthes
cinctipes (Randall) and P. eriomerus Stimpson. J. Exp. Mar. Bio. Ecol. 131:223–231.
Keough MJ, Downes BJ. 1982. Recruitment of marine invertebrates : the role of active larval
choices and early mortality. Oecologia 54:348–352.
Lee RF, Hagen W, Kattner G. 2006. Lipid storage in marine zooplankton. Mar. Ecol. Prog.
Ser. 307:273–306.
Lloyd MJ, Gosselin LA. 2011. Role of maternal provisioning in controlling interpopulation
variation in hatching size in the marine snail Nucella ostrina. Biol. Bull. 213:316–324.
Lucas MI, Walker G, Holland DL, Crisp DJ. 1979. An energy budget for the free-swimming
and metamorphosing larvae of Balanus balanoides (Crustacea: Cirripedia). Mar. Biol.
55:221–229.
Marko PB, Moran AL, Kolotuchina NK, Zaslavskaya NI. 2014. Phylogenetics of the
gastropod genus Nucella (Neogastropoda: Muricidae): Species identities, timing of
diversification and correlated patterns of life-history evolution. J. Molluscan Stud.
80:341–353.
Marshall DJ, Bolton TF, Keough MJ. 2003. Offspring size affects the post metamorphic
performance of a colonial marine invertebrate . Ecology 84:3131–3137.

38

Marshall DJ, Keough MJ. 2004. Variable effects of larval size on post-metamorphic
performance in the field. Mar. Ecol. Prog. Ser. 279:73–80.
Von Der Meden CEO, Porri F, McQuaid CD. 2012. New estimates of early post-settlement
mortality for intertidal mussels show no relationship with meso-scale coastline
topographic features. Mar. Ecol. Prog. Ser. 463:193–204.
Miller BA, Emlet RB. 1999. Development of newly metamorphosed juvenile sea urchins
(Strongylocentrotus franciscanus and S. purpuratus): Morphology, the effects of
temperature and larval food ration, and a method for determining age. J. Exp. Mar. Bio.
Ecol. 235:67–90.
Miller CA, Waldbusser GG. 2016. A post-larval stage-based model of hard clam Mercenaria
mercenaria development in response to multiple stressors: Temperature and
acidification severity. Mar. Ecol. Prog. Ser. 558:35–49.
Miller LP, Harley CDG, Denny MW. 2009. The role of temperature and desiccation stress in
limiting the local-scale distribution of the owl limpet, Lottia gigantea. Funct. Ecol.
23:756–767.
Miller SE. 1993. Larval period and its influence on post-larval life history: comparison of
lecithotrophy and facultative planktotrophy in the aeolid nudibranch Phestilla sibogae.
Mar. Biol. 117:635–645.
Moran AL. 1999. Size and Performance of Juvenile Marine Invertebrates : Potential
Contrasts Between Intertidal and Subtidal Benthic Habitats. Am. Zool. 39:304–312.
Moran AL, Emlet RB. 2001. Offspring Size and Performance in Variable Environments:
Field Studies on a Marine Snail. Ecol. Soc. Am. 82:1597–1612.
Moran AL, Manahan DT. 2004. Physiological recovery from prolonged 'starvation' in larvae
of the Pacific oyster Crassostrea gigas. J. Exp. Mar. Bio. Ecol. 306:17–36.
Moran AL, McAlister JSMC. 2009. Egg Size as a Life History Character of Marine
Invertebrates: Is It All It’s Cracked Up to Be? Biol. Bull. 216:226–242.
Osman R, Whitlatch R, Zajac R. 1989. Effects of resident species on recruitment into a
community larval settlement versus post-settlement mortality in the oyster Crassostrea
virginica. Mar. Ecol. Prog. Ser. 54:61–73.
Osman RW, Whitlach RB. 1995. Predation on early ontogenetic life stages and its effect on
recruitment into a marine epifaunal community. Mar. Ecol. Prog. Ser. 117:111–126.

39

Paine RT. 1976. Size-Limited Predation: An Observational and Experimental Approach with
the Mytilus-Pisaster Interaction. Ecology 57:858–873.
Palmer AR. 1990. Predator size, prey size, and the scaling of vulnerability: hatchling
gastropods vs. barnacles. Ecology 71:759–775.
Pechenik JA. 1990. Delayed metamorphosis by larvae of benthic marine invertebrates: Does
it occur? Is there a price to pay? Ophelia 32:63–94.
Pechenik JA, Hammer K, Weise C. 1996. The effect of starvation on acquisition of
competence and post- metamorphic performance in the marine prosobranch gastropod
Crepidula fornicata (L.). J. Exp. Mar. Bio. Ecol. 199:137–152.
Pechenik JA, Jarrett JN, Rooney J. 2002. Relationships between larval nutritional experience,
larval growth rates, juvenile growth rates, and juvenile feeding rates in the prosobranch
gastropod Crepidula fornicata. J. Exp. Mar. Bio. Ecol. 280:63–78.
Pechenik JA, Rittschof D, Schmidt AR. 1993. Influence of delayed metamorphosis on
survival and growth of juvenile barnacles Balanus amphitrite. Mar. Biol. 115:287–294.
Phillips NE. 2002. Effects of Nutrition-Mediated Larval Condition on Juvenile Performance
in a Marine Mussel. Ecology 83:2562–2574.
Phillips NE. 2006. Natural variability in size and condition at settlement of 3 species of
marine invertebrates. Integr. Comp. Biol. 46:598–604.
Phillips NE. 2017. Variability in early post-settlement mortality in intertidal mussels and the
role of size at settlement. Mar. Biol. Res. 13:726–732.
Podolsky RD. 2002. Fertilization ecology of egg coats: Physical versus chemical
contributions to fertilization success of free-spawned eggs. J. Exp. Biol. 205:1657–1668.
Qiu JW, Qian PY. 1999. Tolerance of the barnacle Balanus amphitrite amphitrite to salinity
and temperature stress: effects of previous experience. Mar. Ecol. Prog. Ser. 188:123–
132.
Rainbow PS, Walker G. 1977. The functional morphology and development of the
alimentary tract of larval and juvenile barnacles (Cirripedia: Thoracica). Mar. Biol.
42:337–349.
Rivest BR. 1983. Development and the influence of nurse egg allotment on hatching size in
Searlesia dira (Reeve, 1846) (Prosobranchia : Buccinidae). J. Exp. Mar. Bio. Ecol.
69:217–241.

40

Roberts RD, Lapworth C, Barker RJ. 2001. Effect of starvation on the growth and survival of
post-larval abalone (Haliotis iris). Aquaculture 200:323–338.
Rumrill SS. 1990. Natural mortality of marine invertebrate larvae. Ophelia 32:163–198.
Sandee SD, Van Hamme JD, Gosselin LA. 2016. Testing microbial pathogens as a cause of
early juvenile mortality in wild populations of benthic invertebrates. Mar. Ecol. Prog.
Ser. 562:53–63.
Sewell MA. 2005. Utilization of lipids during early development of the sea urchin Evechinus
chloroticus. Mar. Ecol. Prog. Ser. 304:133–142.
Shanks AL. 2009. Barnacle settlement versus recruitment as indicators of larval delivery. I.
Effects of post-settlement mortality and recruit density. Mar. Ecol. Prog. Ser. 385:205–
216.
Sinervo B. 1990. The evolution of maternal investment in lizards: an experimental and
comparative analysis of egg size and its effect on offspring performance. Evolution.
44:279–294.
van der Sman J, Phillips NE, Pfister CA. 2009. Relative effects of maternal and juvenile food
availability for a marine snail. Ecology 90:3119–3125.
Spitzer PM, Heck KL, Valentine JF. 2003. Then and now: A comparison of patterns in blue
crab post-larval abundance and post-settlement mortality during the early and late 1990s
in the Mobile Bay System. Bull. Mar. Sci. 72:435–452.
Stoner DS. 1990. Recruitment of a tropical colonial ascidian relative importance of pre
settlement vs post settlement processes. Ecology 71:1682–1690.
Strohmeier T, Alunno-Bruscia M, Duinker A, Cranford PJ. 2012. Variability in particle
retention efficiency by the mussle Mytilus edulis. 412:96–102.
Takami H, Kawamura T, Yamashita Y. 2000. Starvation tolerance of newly metamorphosed
abalone Haliotis discus hannai. Fish. Sci. 66:1180–1182.
Thiyagarajan V, Harder Tilmann, Qian PY. 2003. Effects of TAG/DNA ratio and age of
cyprids on post-metamorphic growth and survival in the barnacle Balanus amphitrite. J.
Mar. Biol. Assoc. United Kingdom 83:83–88.
Thiyagarajan V, Harder T, Qian PY. 2002. Relationship between cyprid energy reserves and
metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica). J.
Exp. Mar. Bio. Ecol. 280:79–93.

41

Thiyagarajan V, Harder T., Qiu JW, Qian PY. 2003. Energy content at metamorphosis and
growth rate of the early juvenile barnacle Balanus amphitrite. Mar. Biol. 143:543–554.
Thiyagarajan V, Hung OS, Chiu JMY, Wu RSS, Qian PY. 2005. Growth and survival of
juvenile barnacle Balanus amphitrite: interactive effects of cyprid energy reserve and
habitat. 299:229–237.
Thiyagarajan V, Pechenik JA, Gosselin LA, Qian PY. 2007. Juvenile growth in barnacles:
Combined effect of delayed metamorphosis and sub-lethal exposure of cyprids to lowsalinity stress. Mar. Ecol. Prog. Ser. 344:173–184.
Thorson G. 1966. Some factors influencing recruitment and establishment of marine benthic
communities. Netherlands J. Sea Res. 3:267–293.
Torres G, Giménez L, Pettersen AK, Bue M, Burrows MT, Jenkins SR. 2016. Persistent and
context-dependent effects of the larval feeding environment on post-metamorphic
performance through the adult stage. Mar. Ecol. Prog. Ser. 545:147–160.
Wendt DE. 2000. Energetics of larval swimming and metamorphosis in four species of
Bugula (Bryozoa). Biol. Bull. 198:346–356.
Whitehill EAG, Moran AL. 2012. Comparative larval energetics of an ophiuroid and an
echinoid echinoderm. Invertebr. Biol. 131:345–354.
Yan X, Zhang Y, Huo Z, Yang F, Zhang G. 2009. Effects of starvation on larval growth,
survival, and metamorphosis of Manila clam Ruditapes philippinarum. Acta Ecol. Sin.
29:327–334.

42

CHAPTER 3: The role of initial energy reserves on stress tolerance thresholds during
the early benthic phase for intertidal invertebrates

INTRODUCTION
Intertidal invertebrates are extremely vulnerable during their early benthic phase
(EBP), the period immediately following settlement or hatching into the benthic
environment, and which can encompass both pre- and post-metamorphic life stages. Most
new cohorts of intertidal invertebrates suffer mortality rates of 30 - 100% in the first few
days of the EBP (Gosselin and Qian 1997; Hunt and Scheibling 1997). This high level of
mortality can drastically impact recruitment rates, and thus influence overall population
abundance and distribution (Connell 1985; Stoner 1990; Hunt and Scheibling 1997). Though
generally high, EBP mortality is also highly variable not only among species, but also among
cohorts of a single species, and even among individuals within a cohort (Jarrett 2000; Phillips
2002). Many factors can cause mortality during the EBP, however the mechanisms
responsible for the high degree of variation in mortality are still not fully understood.
Among the most important factors contributing to EBP mortality are those associated
with aerial exposure at low tide and with the unique challenges associated with these periods
of emersion. Two of the most significant of these environmental stressors are desiccation and
elevated emersion temperature, both of which have been shown to cause significant mortality
in many species of intertidal invertebrates (Foster 1971; Gosselin and Chia 1995; Helmuth
and Hofmann 2001; Miller et al. 2009; Jenewein and Gosselin 2013a). Indeed, these two
stressors define the upper limit of the vertical range for many intertidal species (Connell
1961; Foster 1971). Loss of moisture from body tissues (i.e. desiccation) and elevated
temperatures both have the potential to disrupt a range of physiological processes including
protein stability, organ function, and gas exchange (Buckley et al. 2001; Sokolova and
Pörtner 2001; Miller et al. 2009). EBP individuals are particularly vulnerable to these
stressors owing to their very small body size, and thus large surface-to-volume ratio and
small reservoir of water in their tissues, which greatly reduces their ability to tolerate adverse
environmental conditions (Lowell 1984; Gosselin 1997).
Individuals fed reduced rations throughout the larval phase often have lower success
rates through the EBP than those reared on full rations (Phillips 2002; Thiyagarajan et al.

43

2003; Emlet and Sadro 2006). This suggests that low initial energy reserves might lead to
higher EBP mortality, and thus energy reserves at the onset of the EBP have previously been
proposed as a factor that may play a major role in determining whether an individual will
survive the critical first days of early benthic life (Gosselin and Qian 1997; Hunt and
Scheibling 1997; Jarrett and Pechenik 1997). This hypothesis is supported by evidence that
individuals within a cohort differ considerably in size and amount of organic content held at
the start of the EBP, both of which are proxies for energy reserves (Marshall et al. 2003,
Emlet and Sadro 2006, Phillips 2006), with some individuals possessing relatively low
energy reserves at the onset of the EBP (Lucas et al. 1979; Jarrett and Pechenik 1997;
Thiyagarajan et al. 2003). Additionally, limiting larval diet can negatively impact growth rate
during juvenile life (Woodcock and Benkendorff 2008; Pechenik and Tyrell 2015; Gilman
and Rognstad 2018), leaving individuals more vulnerable to mortality factors for a longer
period of time. In wild populations, differences in energy reserves among EBP individuals
could be due to different rates of feeding in the larval stage, or delayed metamorphosis
(Thiyagarajan et al. 2003). Many species also transition through energy intensive stages or
non-feeding periods prior to entering the EBP, during which initial energy reserves can
become depleted (Lucas et al. 1979; Wendt 2000; Bryan 2004; Bennett and Marshall 2005).
Recent research, however, has revealed that the vast majority of individuals entering the EBP
likely have adequate energy reserves to survive the critical first days to weeks of early
benthic life, and that depleted energy reserves are not an important direct cause of EBP
mortality (Chapter 2). Initial energy reserves might nevertheless have indirect effects on EBP
mortality by altering tolerance thresholds to environmental stressors; these indirect effects of
initial energy reserves are therefore the focus of the present study.
The physiological mechanisms and adaptations utilized by EBP individuals to tolerate
adverse conditions are thought to be energy dependent, as energy demand increases during
exposure to environmental stress (Sokolova et al. 2012). These mechanisms can include
increasing the production of protective proteins such as heat shock proteins, synthesizing
new proteins to replace those that have been damaged, restructuring cellular membranes, and
shifting gene expression to accommodate these adaptive mechanisms (Sokolova and Pörtner
2001; Hofmann et al. 2002; Somero 2002; Berger and Emlet 2007). Many of these
mechanisms have an associated energetic cost, and thus individuals with limited energy

44

reserves may be unable to adequately respond to the physiological stressors they encounter.
In this way, low energy reserves could indirectly lead to increased EBP mortality by reducing
the ability of the individual to respond to environmental stressors such as desiccation and
high emersion temperature.
Consequently, the purpose of this study was to assess the indirect impacts of depleted
energy reserves on EBP mortality by testing the hypothesis that depleted initial energy
content affects vulnerability to physiological stress during the EBP. Two species of intertidal
invertebrates, the barnacle Balanus glandula and the snail Nucella ostrina, were used as
model organisms. The specific goals of this study were to examine the extent to which initial
energy content affects acute tolerance thresholds of EBP individuals to the two most
challenging environmental stressors in the intertidal zone: (1) desiccation and (2) high
emersion temperature. Since direct measurement of energy content in live individuals was
not possible, two proxy methods were used to estimate energy reserves: duration of
starvation prior to experimentation, and initial body mass.

METHODS
Study site and species collection
This research was conducted at the Bamfield Marine Sciences Centre from May to
September, in 2018 and 2019. Specimens of two species of intertidal invertebrates were
collected from rocky intertidal shore sites in Barkley Sound, on the west coast of Vancouver
Island, Canada: Balanus glandula were collected from Wizard Islet (N 48°51.19, W
125°09.56) and Dixon Island (N 48°51.06, W 125°07.20), and Nucella ostrina were collected
from Dixon Island, Prasiola Point (N 48°49.05, W 125°10.12), and Ross Islets (N 48°52.23,
W 125°09.67). Sites were selected based on availability of early benthic phase (EBP)
individuals of these two species.
The two species examined in this study differ in their source and quantity of initial
energy reserves at the beginning of the EBP. N. ostrina are lecithotrophic, and thus depend
entirely on maternal provisioning, primarily via ingestion of nurse eggs in the egg capsule.
Uneven division of nurse eggs among capsule mates results in some individuals having
greater energy reserves and larger body size at hatching (Moran and Emlet 2001; Lloyd and
Gosselin 2011; Marko et al. 2014). B. glandula have planktotrophic larvae so the majority of

45

their EBP energy reserves are obtained from the food they consume during the larval stage.
They also transition through a non-feeding cyprid stage and undergo an energy intensive
metamorphosis, such that some individuals may have more depleted energy reserves at the
onset of the EBP (Wendt 2000; Thiyagarajan et al. 2003).
EBP B. glandula were collected as described by Sandee et al. (2016). Small (5 – 10
cm diameter) rocks were collected from the intertidal zone, cleared of all small barnacles,
and a perimeter was marked around the cleared area with nail polish before being returned to
the intertidal zone. Rocks were then recovered 48 – 72 h later and examined for newly settled
barnacles; rocks containing new settlers were retained for experiments. EBP N. ostrina were
collected by removing ripe egg capsules from intertidal substrata using fine-tipped forceps,
as in Gosselin & Chia (1995). Egg capsules were returned to the laboratory and held in small
cages in aerated seawater for 72 h; all newly hatched individuals were then collected and
retained for experiments.

Establishing treatment levels of energy reserves
To assess how the vulnerability of EBP invertebrates to physiological stressors is
affected by the individual’s energy reserves, the first step was to obtain groups of individuals
with different levels of energy content. The measurement of energy content, for example by
quantifying organic content, in such small animals is a destructive process, such that
individuals in which energy content had been quantified could not then be used for
experiments. Consequently, two proxy methods were used to obtain relative estimates of
energy reserves in live EBP individuals: duration of starvation and body mass. Energy
reserves were estimated by duration of starvation by rearing groups of individuals of a
similar body size without food for different periods of time. Organic content, an effective
measure of energy content (Moran and McAlister 2009), predictably decreases as a function
of the duration of starvation (N. ostrina: Chapter 2; Moran and Emlet 2001). All newly
metamorphosed B. glandula obtained for these experiments were 0.60 – 0.68 mm shell
diameter, a very narrow range of body sizes.. N. ostrina, however, varied considerably in size
at hatching (Lloyd and Gosselin 2011; Marko et al. 2014). All newly-hatched snails were
thus measured and sorted into three size classes based on shell length (SL): small (0.8 – 1.10
mm SL), medium (1.11 - 1.40 mm SL), and large (1.41 – 1.80 mm SL). Only medium size

46

class N. ostrina (1.11 – 1.40 mm SL). Only medium size class N. ostrina (1.11 – 1.40 mm
SL) were used when testing the influence of duration of starvation on tolerance thresholds.
All EBP individuals were held in water that was filtered to 1.0 μm, aerated, and maintained at
15-17 °C. The water was replaced every second day and containers and cages were rinsed
and scrubbed to prevent the growth of biofilm, thus ensuring no food particles were
available. A sample of individuals were removed from the starvation holding cages every 10
d, beginning on day 0 and continuing to day 60, and used in tolerance experiments.
Accordingly, groups of individuals representing seven distinct energy level treatments were
obtained: 0, 10, 20, 30, 40, 50, and 60 d durations of starvation.
The second approach used to establish distinct energy reserve treatments involved
sorting individuals by initial body size, and using body mass as a proxy for energy reserves.
Body mass and quantity of organic matter are strongly correlated among EBP invertebrates
(Moran and Emlet 2001) with larger individuals having greater stores of lipids, which are the
primary source of metabolizable energy for intertidal species (Phillips 2002; Thiyagarajan et
al. 2002; Emlet and Sadro 2006; Whitehill and Moran 2012). This method was only feasible
for N. ostrina, as there was insufficient variation in body size (0.60 – 0.68 mm shell
diameter) of newly metamorphosed B. glandula at our field sites. As previously mentioned,
N. ostrina, had considerable variation in size at hatching and individuals in two shell length
(SL) categories were used in stress experiments: small (0.8 – 1.10 mm SL) and large (1.41 –
1.80 mm SL). These SL measurements were then converted to wet weight (WW) body mass
(Table 3.1) using a regression equation for N. ostrina from Hamilton & Gosselin (2020). All
individuals were held without food in the laboratory for a 10 d acclimation period before
tolerance experiments were conducted.
Table 3.1. Shell lengths (SL) and corresponding body masses (wet weight, WW) for N.
ostrina hatchlings. The conversion equation is: WW = 2.98 * log(SL) - 3.84, from Hamilton
& Gosselin (2020).
Size class

Shell length (SL)

Body mass (WW)

in mm

in mg

Small

0.8 – 1.10

0.074 – 0.192

Large

1.41 – 1.80

0.402 – 0.833

47

Emersion temperature tolerance
To determine if emersion temperature tolerance varies as a function of energy
reserve, individuals in each energy level treatment (i.e. duration of starvation or size class)
were exposed to five temperature treatments (Table 3.2). The range of temperatures to which
each species was exposed was determined in preliminary trials and included temperatures
that resulted in 0 - 100% mortality; the low end of the temperature range resulting in no
mortality served as a control for the experiment. Immediately before each experiment, all
individuals were examined under a dissecting microscope to ensure they were alive, and also
that there were no cracks in their shells, as this might have affected their tolerance. Rocks
containing newly-settled B. glandula were then blot dried and transferred to sealed 591 ml
plastic Ziplock® containers. N. ostrina hatchlings were first placed in small cages made of
plastic microcentrifuge tubes with the lower half removed and covered with 600 μm mesh to
prevent their escape. These small cages were then placed within separate, sealed 591 ml
containers, one small cage per container. A sheet of paper towel saturated with seawater was
also placed in each container to maintain a high humidity level (>90% RH), as well as a data
logger (Hygrochron iButton® model DS1923) that recorded temperature and relative
humidity every 15 min. All replicate containers were then placed in an incubator set at the
lowest temperature in the experimental range for 6 h. Following the 6 h temperature
treatment, all rocks and small cages were removed from containers and submerged in 15 17°C seawater for a 6 h recovery period, and then examined again under a dissecting
microscope to assess survivorship. The 6 h treatment and recovery intervals were chosen to
approximate a natural tidal cycle, so that animals were exposed to emersion conditions for a
duration similar to that experienced in the intertidal zone. Dead individuals were recorded
and removed, then rocks containing the surviving B. glandula were blot dried and surviving
N. ostrina were placed back into small cages. All rocks and small cages were transferred
back into their respective containers and placed into the incubator set to the next
experimental temperature (i.e. increased by 2 °C). This process was repeated until the final
experimental temperature was reached. Temperature within the incubators was stable
throughout all experiments and stayed within ±0.5 degrees of the intended temperature for
each treatment. It took up to 30 min for the internal temperature of each container to reach
equilibrium with the incubator, but once the target temperature was reached it remained

48

stable for the remainder of the 6 h treatment. Relative humidity remained above 90% for the
duration of the experiment in all treatments. The temperature at which each individual died
was recorded and an average temperature at death was calculated for all the individuals in
each replicate container.

Table 3.2. Experimental range of emersion temperatures and desiccation periods used for
each species. Ranges were set to include treatments that result in 0 – 100% mortality.
Species

Experimental emersion

Experimental desiccation

temperature treatments (°C)

duration treatment (h)

B. glandula

36, 38, 40, 42, 44

4, 6, 8, 10, 12, 14, 16, 18, 20

N. ostrina

26, 28, 30, 32, 34

0.25, 0.5, 1.0, 1.5, 2.0, 3.0

For the experiments using duration of starvation as a proxy for energy reserves, a
single collection of at least 200 individuals of each species was used, and animals were
maintained as stated above. All individuals in this experiment were collected from Dixon
Island; B. glandula and N. ostrina were collected on 21 May 2019 and 14 June 2019,
respectively. Using only individuals from a single collection for each species ensured that
they had all experienced similar environmental conditions prior to collection. At the start of
the experiment (day 0), and then again after each 10 d interval, 30 individuals were removed
from the starvation holding tanks and used in the emersion temperature experiments as
described above. The experimental design for each of the two species was: x 7 durations of
starvation x 3 replicate groups of individuals per duration of starvation x 10 individuals per
replicate group.
The experiment using body mass as a proxy for energy reserves was designed slightly
differently. N. ostrina were collected from the field on three separate dates, twice from Dixon
Island and once from Prasiola Point, and each of these three batches of individuals was used
in a different trial. Comparisons of tolerance thresholds between size classes were only
carried out between groups from the same trial, thus accounting for temporal and spatial
differences among groups. Following the 10 d acclimation period, 30 - 50 individuals of each
size class from each trial were used in the emersion temperature experiments as described

49

above. The experimental design for this experiment was: 3 trials x 2 size classes per trial x 3
– 5 replicate groups of individuals per size class x 10 individuals per replicate group.

Desiccation tolerance
The effect of energy reserves on tolerance to desiccation stress was determined by
exposing the various energy treatment groups to a low humidity environment for set periods
of time. The durations of exposure to desiccation used in this experiment were determined in
preliminary trials and set to include treatment periods that result in 0 – 100% mortality
(Table 3.2). Desiccation conditions were quantified using vapour pressure deficit (VPD) as in
Jenewein and Gosselin (2013). VPD accounts for both temperature and relative humidity
(RH) and is an effective measure of desiccation potential. Given that B. glandula and N.
ostrina occupy different microhabitats and levels in the intertidal zone, they normally
experience different degrees of desiccation stress. Additionally, EBP B. glandula are known
to have a higher desiccation tolerance threshold than EBP N. ostrina (Hamilton and Gosselin
2020). Thus, the two species were exposed to different VPD levels in the present experiment;
B. glandula were exposed to a VPD of 2.0 kPa, whereas N. ostrina were exposed to the less
stressful VPD of 1.6 kPa. Both VPD measurements are common in the intertidal zone during
low tide throughout the summer months (Jenewein and Gosselin 2013b). To ensure this
experiment tested desiccation tolerance and not temperature stress, desiccation trials were
performed in a temperature-controlled incubator set well below the temperature tolerance
threshold for each species, as determined in the emersion temperature experiment described
above: B. glandula trials were carried out at 22°C, and N. ostrina trials were carried out at
19°C. The RH needed to obtain the above VPD measurements (approximately 20 – 28% RH
for B. glandula and 25 - 31% RH for N. ostrina) was then achieved by placing a large pan of
silica desiccant in the bottom of the incubator as well as two small fans to create a continuous
air flow. A data logger (Lascar Electronics, model EL-USB-2) was also placed in the
incubator to record temperature and RH every 10 min, allowing the calculation of VPD. For
all desiccation tolerance experiments, VPD remained stable and close to the target set for
each species, although there were slight fluctuations among trials. Additionally, it took up to
10 min for the internal RH of the incubator to stabilize after the door had been closed, thus
the first RH reading was omitted from the average VPD calculation. Throughout experiments

50

involving B. glandula and N. ostrina, VPD ranged from 1.91 – 2.10 kPa and 1.56 – 1.69 kPa,
respectively.
Before each experiment, all individuals were examined under a dissecting microscope
to ensure they were alive, and that there were no cracks in their shells, as this may have
impacted their ability to tolerate desiccation conditions. Rocks containing B. glandula were
blot dried and then placed in a small desiccator with silica desiccant crystals for 10 min to
remove any remaining water on the rock surface. N. ostrina were placed in small dry cages
using a pair of flexible insect forceps. The rocks or small cages were then transferred into
open 591 mL containers and placed into the prepared incubator for a set period of time.
Following the desiccation treatment, animals were removed from the incubator and
immediately submerged in seawater for 12 h to recover, after which they were checked for
survival and the number of live and dead individuals in each treatment was recorded.
Contrary to the emersion temperature experiments, individuals that survived a desiccation
treatment were not used again in further treatments; a different set of individuals were used
for each desiccation duration treatment.
For the experiment using duration of starvation as a proxy for energy reserves, more
individuals were required than could be obtained from a single field collection. Accordingly,
the experiment involved 2 - 4 trials per desiccation period; each trial used EBP individuals
from one field site collected on a same day, but the collection sites and days differed among
trials. Data from all trials were combined for analysis, and trial was included as a fixed
factor. At 10 d intervals, beginning on day 0 and ending on day 30 for B. glandula and day
50 for N. ostrina, groups of 7-12 individuals were removed from holding tanks and assigned
to one of the desiccation duration treatments. The experimental design for each of the two
species was as follows: x 4 – 6 duration of starvation treatments per species x 6 – 9
desiccation duration treatments per starvation treatment x 2 – 4 replicate groups of
individuals per desiccation treatment x 7 – 12 individuals per replicate group.
For experiments using body mass as a proxy for energy reserves, three replicate trials
were conducted, each using animals from a separate collection (i.e. collected from different
locations on different days). Immediately prior to a desiccation trial, six groups of 5 – 10
individuals of each size class were removed from holding tanks and assigned to one of the six
desiccation duration treatments. Thus, the experimental design was: 2 size classes x 6

51

desiccation duration treatments per size class x 3 replicate groups of individuals per
desiccation treatment x 5 – 10 individuals per replicate group.

Data analysis
Data from the emersion temperature experiments were not normally distributed, and so
did not meet the requirements for ANOVA. Thus, the Kruskal-Wallis non-parametric
analysis of variance was used to compare temperature at death among the various energy
reserve treatments (i.e. durations of starvation). When the Kruskal-Wallis test revealed a
significant difference, a Dunn’s multiple comparisons test was then used to compare each
starvation treatment to the 0 d control to assess how tolerance was impacted by starvation. To
compare emersion temperature tolerance between size classes, a randomized block ANOVA
was used, with trial as the blocking factor. Although the data did not meet the requirements
for ANOVA, the p-value was deemed sufficiently low as to be unaffected by using a
parametric test (Blanca et al. 2017). A significance value of 0.05 was used for both the
Kruskal-Wallis and ANOVA statistical tests.
Survivorship data from all desiccation experiments followed a binomial distribution and
were thus analysed using generalized linear models (GLM) with a binomial link function.
Each energy reserve treatment (i.e. duration of starvation or size class) was analyzed
separately, and their desiccation LD50 was determined from the resulting curve. To identify
differences in acute desiccation tolerance among the various energy reserve treatments, all
survivorship data for a given species were fit to a single GLM. In all cases, trial was included
as a fixed factor to account for potential differences between batches collected from different
sites or at different times and a significance value of 0.05 was used. All statistical analyses
were conducted using R statistical software (version 3.5.0) (R Core Team 2018).

RESULTS
Emersion temperature tolerance
Effect of starvation duration on emersion temperature tolerance
Although there was significant variation in average temperature at death among the
duration of starvation treatments in both species (Kruskal-Wallis test; Balanus glandula: χ2 =
17.85, df = 6, p = 0.007; Nucella ostrina: χ 2 = 15.55, df = 5, p = 0.008), results for the non-

52

starved control treatment were not significantly different from any of the starved treatments
(Figure 3.1). Duration of starvation for up to 60 d had no apparent effect on acute emersion
temperature tolerance.
Results from the 30 d starvation treatment for N. ostrina were removed from the
analysis due to uncharacteristically low temperature tolerance; 100% of individuals in that
group died at 30°C, a temperature that was only lethal to a few individuals in any other
starvation treatment. In addition, very high mortality also occurred among other individuals,
remaining in the same holding cage, that were not used in the temperature tolerance
experiment. It was apparent that the individuals in this holding cage had become unhealthy
and their ability to tolerate emersion conditions was impacted. The 40, 50, and 60 d
starvation treatments involved hatchlings from the same batch of animals, collected at the
same time from the same location, as those in the 0, 10, 20 and 30 d starvation treatment, but
those in the 40, 50 and 60 d treatments were not held in the same holding cage as the ones
used in the 30 d treatment. In addition, individuals remaining in the holding cage for the 40,
50 and 60 treatments did not experience high mortality.

53

A) B. glandula
42

Temperature at death (°C)

a
41

a
ab

ab

ab

ab
b

40

39

38
0

10

20

30

40

50

60

B) N. ostrina
Temperature at death (°C)

33
32

a
ab

31

a

ab

ab
b

30
29
28
0

10

20

30

40

50

60

Duration of starvation,
since onset of EBP (d)

Figure 3.1. Acute temperature tolerance thresholds, measured as average temperature at
death, as a function of duration of starvation in EBP. (A) B. glandula and (B) N. ostrina. The
30 d N. ostrina group was removed from this analysis due to inconsistent results for this
batch of hatchlings and mass mortality among the remaining hatchlings of the same batch
that were held in the same cage. In both species, starvation was initiated at the onset of the
EBP. Error bars represent SE. Bars identified with different letters were significantly
different based on Dunn’s multiple comparison test.

54

Effect of body mass on emersion temperature tolerance
Acute temperature tolerance in early benthic phase (EBP) N. ostrina, as measured by
temperature at death (Figure 3.2), differed significantly between the two size classes
(Randomized block ANOVA using trial as the blocking factor: F = 71.02, 2,250, p < 0.001). In
each trial, the large size class had significantly higher tolerance thresholds than the small size
class; the differences between the size classes were 1.47°C, 0.89°C, and 2.12°C in trials 1, 2,
and 3 respectively. This indicates that a greater initial body mass provides better tolerance to
increased emersion temperatures. Additionally, within each size class (large or small),
temperature at death varied significantly among trials (p < 0.001) (i.e. among groups
collected from different sites or on different dates). This suggests that temperature tolerance
thresholds can vary among similarly-sized hatchlings depending on where or when they
completed encapsulated development.

Temperature at death (°C)

33

32

31

30

29

Small

Large
Size Class

Figure 3.2. Effect of initial body size on acute temperature tolerance, as measured by
average temperature at death, for EBP N. ostrina. Error bars represent SE. These means are
significantly different based on a randomized block ANOVA (p <0.001).

55

Desiccation tolerance
Effect of starvation duration on desiccation tolerance
Within each starvation treatment, survivorship was significantly impacted by the
duration of the desiccation treatment in both species (Figure 3.3 and Appendix B), as
determined by separate generalized linear model (GLM) analyses of each treatment (Table
3.3). The duration of desiccation resulting in 50% mortality (i.e. the desiccation LD 50)
decreased with increasing duration of starvation (Table 3.3).

Survivorship (proportion)

1.0
0.8
0.6
0.4
0.2
0.0
0

5

10

15

20

Duration of desiccation treatment (h)
Figure 3.3. Effect of exposure to desiccation conditions on the survivorship of newly
metamorphosed (not starved) EBP B. glandula. Each point represents the proportion of
individuals from a group of 10 that survived the desiccation treatment for a given duration
(h). The point where the curve intersects the dashed line represents the desiccation LD 50; in
this case, LD50 = 13.98 h.

56

Table 3.3. Desiccation LD50 and associated standard error (SE) for groups of B. glandula
and N. ostrina starved for different durations of time since the onset of EBP. LD50 values
were calculated from generalized linear models (GLM) of the relationship between the
proportion of individuals surviving and the duration of exposure to the desiccation treatment.
The z statistic represents the strength of this relationship; df = degrees of freedom; p values
represent the probability that survivorship varied as a function of duration of exposure to the
desiccation treatment.

Species

B. glandula

N. ostrina

Duration of

Desiccation

starvation (d)

LD50 (h)

SE

z statistic

df

p

0

13.98

0.45

-7.60

22

< 0.001

10

11.94

0.37

-7.97

23

< 0.001

20

8.14

0.43

-8.56

27

< 0.001

30

5.87

0.53

-4.37

13

< 0.001

0

1.22

0.07

-7.02

18

< 0.001

10

1.23

0.05

-7.43

24

< 0.001

20

1.19

0.05

-7.09

23

< 0.001

30

0.95

0.05

-6.45

23

< 0.001

40

0.98

0.07

-5.10

11

< 0.001

50

0.91

0.08

-3.27

5

0.001

In both species, acute desiccation tolerance thresholds differed significantly among
the starvation treatments, as determined by GLM analysis of the combined survivorship data
from all starvation treatments of a given species. For B. glandula, desiccation tolerance
differed significantly among each starvation treatment, with the 0 d control group having the
highest tolerance threshold (pairwise comparisons among starvation treatments: z values < 3.36, df = 92, p < 0.001) and tolerance decreasing gradually for each sequential starvation
treatment period (Figure 3.4A). For N. ostrina, however, the trend towards lower desiccation
tolerance was not gradual. There was no significant difference in desiccation tolerance
among 0, 10, and 20 d starvation treatments (pairwise comparisons: z values > 0.001, df =
90, p > 0.71). Desiccation tolerance was, however, significantly lower for longer starvation
treatments (30, 40, and 50 d) when compared to the shorter starvation treatments (pairwise
comparisons: z values < -1.76, df = 90, p < 0.005) (Figure 3.4B).

57

A) B. glandula
16

a

Desiccation LD50 (h)

14

b

12
10

c

8

d

6
4
2
0
0

10

20

30

B) N. ostrina
1.4

Desiccation LD50 (h)

a

a

a

1.2

b

1.0

b

b

0.8

0.6
0

10

20

30

40

50

Duration of starvation,
since onset of EBP (d)
Figure 3.4. Acute desiccation tolerance thresholds, measured as LD 50, as a function of
duration of starvation in EBP (A) B. glandula and (B) N. ostrina. Error bars represent SE.
Bars identified with different letters are significantly different based on GLM analysis.

58

Effect of body mass on desiccation tolerance
Within each N. ostrina size class, survivorship decreased significantly as a function of
the duration of the desiccation treatment (Figure 3.5), as determined by GLM analysis (Small
size class: z value = -5.46, df = 17, p < 0.001; Large size class: z value = -6.26, df = 17, p <
0.001). In addition, when comparing size classes, the desiccation LD 50 was significantly
shorter for the small size class than for the large size class, as determined by GLM analysis
of combined survivorship data from both size classes (z value = -5.46, df = 23, p < 0.001).
Small individuals had a desiccation LD50 of 0.71 ± 0.05 h, whereas the desiccation LD50 of
the large size class was 132% longer, at 1.65 ± 0.10 h.

Survivorship (proportion)

1.0

Small
Large

0.8
0.6
0.4
0.2
0.0
0.0

0.5

1.0

1.5

2.0

2.5

3.0

Duration of desiccation treatment (h)
Figure 3.5. Effect of exposure to desiccation conditions on the survival of newly hatched N.
ostrina of two size classes (Small = 0.074 – 0.192 mg; Large = 0.402 – 0.833 mg). Each
point represents the proportion of individuals from a group of 10 that survived desiccation
treatment for a given duration (h). The points where the curves intersect the dashed line
represent the desiccation LD50 for each size class.

59

DISCUSSION
Effect of estimated energy reserves on tolerance to elevated emersion temperature
In early benthic phase (EBP) invertebrates, organic matter, a proxy for energy
content, can decrease by half after only 10 d of starvation and approach a minimum after 40 –
50 d of starvation (Chapter 2), at which point nearly all metabolizable organic compounds
have likely been utilized. Yet, the ability of EBP individuals to tolerate elevated emersion
temperature was not impacted by the duration of the starvation period they had experienced.
In both Balanus glandula and Nucella ostrina, EBP individuals starved for 50 or 60 d had
comparable acute temperature tolerance thresholds to individuals that had not been starved.
This suggests that the mechanisms employed by EBP individuals to tolerate acute elevated
emersion temperatures are not energetically intensive. The production of heat-shock proteins
(HSPs) is one common strategy among invertebrate species to cope with the physiological
challenges of increased environmental temperatures (Sokolova et al. 2012). Since the
production and activation of these specialized proteins is energetically expensive (Krebs and
Loeschcke 1994; Somero 2002), individuals with more depleted energy reserves were
expected to produce fewer HSPs and thus be more vulnerable to elevated temperatures.
However, our results indicate that individuals with highly depleted energy reserves, as
estimated by duration of starvation, were just as tolerant of acute exposure to high
temperatures as individuals with a full complement of energy reserves. This suggests that
temperature tolerance may be determined by other physiological and biochemical
mechanisms that require minimal energy expenditure, such as metabolic rate depression
(Storey 1998) and reduced ATP turnover rates (Sokolova & Pörtner 2001). Alternatively,
benthic invertebrates may already possess protective mechanisms, such as a store of HSPs,
upon entering the EBP, protecting them from the physiological stress of elevated temperature
during emersion without requiring the use of stored energy. The quantity of stored HSPs, as
well as the capacity of EBP invertebrates to utilize other suggested tolerance methods, should
be the focus of future research.
Emersion temperature tolerance in N. ostrina hatchlings was significantly impacted
by their initial body mass. For N. ostrina hatchlings, a greater initial body mass is indicative
of having consumed more nurse eggs during embryonic development (Rivest 1983; Gallardo
et al. 2004; Lloyd and Gosselin 2011), such that larger individuals have a larger reserve of

60

stored energy (particularly lipids) upon hatching than their smaller counterparts (Moran and
Emlet 2001). However, given that no relationship was detected in the previous experiment
between temperature tolerance and duration of starvation, the effect of body mass on
temperature tolerance was likely due to body size itself rather than to a difference in initial
energy reserve. Body size can be an important determinant of tolerance thresholds, as found
in this and other studies (Lowell 1984; Gosselin 1997, Hamilton and Gosselin 2020), largely
owing to the lower surface area:volume (SA:V) ratio and increased water reservoir in the
tissues of larger individuals. These traits would enhance temperature tolerance in large
hatchlings by decreasing the rate of heat exchange with the external environment, and
perhaps also the ability to carry out evaporative cooling, thus decreasing the rate of warming
of the body. It is also possible that the capacity for other physiological or biochemical
mechanisms, such as those discussed in the previous paragraph, may differ between snails of
different body size. This remains speculative, however, and may be explored in future
studies.
An interesting finding of the body size experiment was that tolerance thresholds also
differed significantly among replicate groups of hatchlings of the same body size. These
replicate groups, however, were collected from different field sites or on different dates,
indicating some natural variation in temperature tolerance within and among cohorts of the
same species. The mechanisms controlling this variation remain unclear but may involve
geographical (i.e. interpopulation) and seasonal differences in tolerance thresholds.

Effect of estimated energy reserves on tolerance to desiccation
Unlike emersion temperature, vulnerability to desiccation was impacted by estimated
initial energy reserves; in both species, EBP individuals starved for a longer duration of time
had lower desiccation tolerance thresholds than those that had not been starved. This suggests
the mechanisms or strategies employed by EBP intertidal invertebrates to withstand the
challenge of desiccation are more dependent on energy-mediated processes than those used
for acute temperature tolerance. Such physiological mechanisms of desiccation tolerance
may include production of protective proteins, such as certain heat-shock proteins (i.e.
HSP70) (Hayward et al. 2004), LEA proteins, and Trehalose (Hibshman et al. 2020), as well
as the synthesis of new proteins to replace those that become damaged. Many of these

61

responses have a significant energetic cost, such that individuals with reduced energy
reserves may be unable to properly utilize these mechanisms and thus be less tolerant of
desiccation stress (Sokolova 2013). The specific energy-dependent mechanisms of
desiccation tolerance utilized by intertidal invertebrates require further research.
The effect of duration of starvation on desiccation tolerance thresholds appears to be
different in the two species. In B. glandula, desiccation tolerance decreased gradually with
increasing duration of starvation such that individuals in each starvation treatment had a
significantly lower tolerance of desiccation than individuals starved for shorter durations. B.
glandula transition through a non-feeding cyprid stage prior to completing an energetically
expensive metamorphosis shortly after settlement (Anderson 1994, Lucas et al. 1979;
Thiyagarajan et al. 2003), such that some individuals likely begin the EBP with very limited
energy reserves. Any further depletion of energy, such as a delay in feeding, could also
further restrict the physiological mechanisms of desiccation tolerance described above. For
N. ostrina, desiccation tolerance thresholds were impacted differently; rather than decreasing
gradually, desiccation tolerance was unchanged for groups starved for up to 20 d but was
significantly reduced in groups starved for longer durations. Since N. ostrina hatchlings
begin the EBP with relatively large energy reserves obtained through maternal provisioning
(Spight 1976; Marko et al. 2014), it is likely they have enough stored energy to maintain
physiological mechanisms of desiccation tolerance for a more extended period. Prior research
has revealed that metabolizable energy reserves in EBP N. ostrina, as estimated by organic
matter content, decline rapidly throughout the first 20 – 30 d when starved, decreasing by 58
– 69% during this period (Chapter 2). After that point, metabolizable energy reserves quickly
approach a minimum, and are thus likely sufficiently reduced to begin impacting any energy
dependent mechanisms of desiccation tolerance.
As was the case for emersion temperature tolerance, desiccation tolerance thresholds
were also affected by initial body mass; N. ostrina hatchlings in the large size class had
significantly higher tolerance thresholds than those in the small size class. The higher organic
content, and thus larger reserves of energy, present in larger individuals likely contributed to
the increased desiccation tolerance in the larger size class, in addition to the physical
characteristics of larger individuals, such as having a smaller SA:V ratio and a larger
reservoir of water in their tissues (Lowell 1984; Gosselin 1997). Together, these factors have

62

a large impact on the ability of EBP individuals of different sizes to tolerate desiccation
stress and likely determine tolerance thresholds.

Does the relationship between energy reserves and stress tolerance contribute to EBP
mortality?
The present study investigated the indirect effects of initial energy reserves on EBP
mortality, and more specifically on the vulnerability of EBP individuals to two of the most
significant abiotic stressors in the intertidal zone, desiccation and elevated emersion
temperature (Gosselin and Chia 1995; Miller et al. 2009). One approach used to obtain
groups of individuals with distinct levels of initial energy reserve was to sort them according
to initial body size, and vulnerability to both environmental stressors did vary as a function of
body size, with larger individuals being more tolerant of desiccation and elevated
temperatures than smaller individuals. This outcome, however, was likely primarily due to
the physical characteristics of a larger body size, such as a smaller SA:V ratio, rather than
difference in energy stores between the size classes, especially in the case of temperature
tolerance.
The most interesting finding of this study is that depleted energy reserves, as
estimated by duration of starvation, can indirectly lead to EBP mortality by reducing
tolerance to desiccation. Desiccation stress is likely a more important selective pressure on
EBP individuals than emersion temperature, regardless of energy reserves. Acute temperature
tolerance thresholds of EBP individuals of these two species are higher than most
temperatures experienced in the field during the summer. All temperature treatments in this
study were within the range of low-tide summer temperatures recorded in the intertidal zone
in Barkley Sound (Jenewein and Gosselin 2013). However, the temperatures necessary to
cause significant mortality, even in the most vulnerable experimental groups, were at the
high end of the natural temperature range (> 30°C) in the intertidal zone, and those high
temperatures usually persist only for short periods of time (< 2.5 h) before the surfaces are
immersed by the incoming tide (Jenewein and Gosselin 2013b). Desiccation tolerance
thresholds, however, were lower than durations of aerial exposure occurring in most midand upper-intertidal habitats. The duration of desiccation required to cause significant
mortality among individuals in all starvation treatments of both species was shorter than

63

durations regularly experienced in mid- and upper-intertidal habitats in the study area, such
that EBP individuals would be vulnerable to desiccation stress during many daytime summer
low tides. This indicates EBP individuals are substantially more vulnerable to desiccation
conditions in their natural habitats than to the temperature conditions in those same habitats.
In addition, individuals with low energy reserves would be most vulnerable to desiccation
stress, and thus less likely to survive the EBP, whereas those entering the EBP with greater
stores of energy are more likely to survive desiccation during the critical first few days of the
EBP. In this way, desiccation may be acting as a selective pressure favoring the evolution of
greater energy reserves at the onset of the EBP, especially in species that inhabit areas of the
intertidal with the greatest level of desiccation stress, such as the upper-intertidal zone.
The findings of this study suggest energy reserves at the start of the EBP may be an
important indirect cause of EBP mortality by reducing desiccation tolerance thresholds. An
additional consequence of this indirect effect is that variation in initial energy reserves would
likely be a contributing factor in the extensive variation in intraspecific EBP mortality rates.
In all the experiments of this study, however, tolerance thresholds to both stressors also
varied among individuals and replicate groups within the same starvation treatment (i.e. with
the same energy reserves). Replicate batches of individuals were obtained from different
locations or at different times of the spring and summer, suggesting differences in tolerance
thresholds may be linked to the location or time when each batch of animals was collected.
This could have resulted from differences among populations, or temporal differences in
environmental conditions experienced by individuals from different batches (Jarrett and
Pechenik 1997; Jarrett 2003). Additionally, in the experiment examining the relationship
between duration of starvation and emersion temperature tolerance, significant differences in
tolerance thresholds also occurred among starvation treatments, but those differences were
random with regards to the duration of starvation each group had experienced (i.e. the
amount of remaining energy reserve). Together, these findings suggest tolerance thresholds
are also influenced by factors other than initial energy reserves. Such factors could include
latent effects resulting from the environmental conditions experienced during the embryonic
and larval stages, altering their ability to tolerate similar stressors during the subsequent EBP
(Pechenik 2006; Nasrolahi et al. 2016), or transgenerational effects resulting from the
conditions experienced by the parents (Agrawal et al. 1999; Marshall 2008; Donelan and

64

Trussell 2015). Additionally, parasites or bacterial infections might negatively impact
tolerance thresholds. A better understanding of the mechanisms controlling EBP mortality
requires further research on how these additional factors influence acute tolerance thresholds
in BP intertidal invertebrates.

LITERATURE CITED
Agrawal AA, Laforsch C, Tollrian R. 1999. Transgenerational induction of defences in
animals and plants. Nature 401:60–63.
Berger MS, Emlet RB. 2007. Heat-Shock Response of the Upper Intertidal Barnacle Balanus
glandula: Thermal Stress and Acclimation. Biol. Bull. 212:232–241.
Buckley BA, Owen M, Hofmann G. 2001. Adjusting the thermostat: the threshold induction
temperature for the heatshock response in intertidal mussels (genus Mytilus) changes
as a function of thermal history. J. Exp. Biol. 204:3571–3579.
Connell J. 1985. The consequences of variation in intertidal settlement vs. post-settlement
mortality in rocky intertidal communities. Exp. Mar. Biol. Ecol. 93:11–45.
Connell JH. 1961. The influence of interspecific competition and other factors on the
distribution of the barnacle Chthamalus stellatus. Ecology 42:710–723.
Donelan SC, Trussell GC. 2015. Parental effects enhance risk tolerance and performance in
offspring. Ecology 96:2049–2055.
Emlet RB, Sadro SS. 2006. Linking stages of life history: How larval quality translates into
juvenile performance for an intertidal barnacle (Balanus glandula). Integr. Comp.
Biol. 46:334–346.
Foster BA. 1971. Desiccation as a factor in the intertidal zonation of barnacles. Mar. Biol.
29:12–29.
Gallardo CS, Diaz MA, Varela C. 2004. Growth and survival of early juvenile trumulco snail
Chorus giganteus (Gastropoda: Muricidae ) fed two prey species. New Zeal. J. Mar.
Freshw. Res. 38:767–773.
Gilman SE, Rognstad RL. 2018. Influence of food supply and shore height on the survival
and growth of the barnacle Balanus glandula (Darwin). J. Exp. Mar. Bio. Ecol.
498:32–38.

65

Gosselin LA. 1997. An ecological transition during juvenile life in a marine snail. Mar. Ecol.
Prog. Ser. 157:185–194.
Gosselin LA, Chia FS. 1995. Characterizing temperate rocky shores from the perspective of
an early juvenile snail: the main threats to survival of newly hatched Nucella
emarginata. Mar. Biol. 122:625–635.
Gosselin LA, Qian P. 1997. Juvenile mortality in benthic marine invertebrates. Mar. Ecol.
Prog. Ser. 146:265–282.
Hamilton HJ, Gosselin LA (2020) Ontogenetic shifts and interspecies variation in tolerance
to desiccation and heat at the early benthic phase of six intertidal invertebrates. Mar.
Ecol. Prog. Ser. 634:15-28.
Helmuth B. 1998. Intertidal mussle microclimates: Predicting the body temperature of a
sessile invertebrate. Ecol. Monogr. 68:51–74.
Helmuth BST, Hofmann GE. 2001. Microhabitats, thermal heterogeniety, and patterns of
physiological stress in the rocky intertidal zone. Biol. Bull. 201:374–384.
Hofmann GE, Buckley BA, Place SP, Zippay ML. 2002. Molecular chaperones in
ectothermic marine animals: Biochemical function and gene expression. Integr.
Comp. Biol. 42:808–814.
Hunt HL, Scheibling RE. 1997. Role of early post-settlement mortality in recruitment of
benthic marine invertebrates. Mar. Ecol. Prog. Ser. 155:269–301.
Jarrett JN. 2000. Temporal variation in early mortality of an intertidal barnacle. Mar. Ecol.
Prog. Ser. 204:305–308.
Jarrett JN. 2003. Seasonal variation in larval condition and post-settlement performance of
the barnacle Semibalanous balanoides. Ecology 84:384–390.
Jarrett JN, Pechenik JA. 1997. Temporal variation in cyprid quality and juvenile growth
capacity for an intertidal barnacle. Ecology 78:1262–1265.
Jenewein BT, Gosselin LA. 2013a. Early benthic phase mortality of the barnacle Balanus
glandula is influenced by Fucus spp. cover but not by weather-related abiotic
conditions. J. Exp. Mar. Bio. Ecol. 449:28–35.
Jenewein BT, Gosselin LA. 2013b. Ontogenetic shift in stress tolerance thresholds of Mytilus
trossulus: Effects of desiccation and heat on juvenile mortality. Mar. Ecol. Prog. Ser.
481:147–159.

66

Krebs RA, Loeschcke V. 1994. Costs and Benefits of Activation of the Heat-Shock Response
in Drosophila melanogaster. Funct. Ecol. 8:730.
Lloyd MJ, Gosselin LA. 2011. Role of maternal provisioning in controlling interpopulation
variation in hatching size in the marine snail Nucella ostrina. Biol. Bull. 213:316–
324.
Lowell RB. 1984. Desiccation of intertidal limpets: Effects of shell size, fit to substratum,
and shape. J. Exp. Mar. Bio. Ecol. 77:197–207.
Lucas MI, Walker G, Holland DL, Crisp DJ. 1979. An energy budget for the free-swimming
and metamorphosing larvae of Balanus balanoides (Crustacea: Cirripedia). Mar. Biol.
55:221–229.
Marko PB, Moran AL, Kolotuchina NK, Zaslavskaya NI. 2014. Phylogenetics of the
gastropod genus Nucella (Neogastropoda: Muricidae): Species identities, timing of
diversification and correlated patterns of life-history evolution. J. Molluscan Stud.
80:341–353.
Marshall DJ. 2008. Transgenerational plasticity in the sea: Context-dependent maternal
effects across the life history. Ecology 89:418–427.
Miller LP, Harley CDG, Denny MW. 2009. The role of temperature and desiccation stress in
limiting the local-scale distribution of the owl limpet, Lottia gigantea. Funct. Ecol.
23:756–767.
Moran AL, Emlet RB. 2001. Offspring Size and Performance in Variable Environments :
Field Studies on a Marine Snail. Ecol. Soc. Am. 82:1597–1612.
Nasrolahi A, Havenhand J, Wrange AL, Pansch C. 2016. Population and life-stage specific
sensitivities to temperature and salinity stress in barnacles. Sci. Rep. 6:1–10.
Pechenik JA. 2006. Larval experience and latent effects - Metamorphosis is not a new
beginning. Integr. Comp. Biol. 46:323–333.
Pechenik JA, Tyrell AS. 2015. Larval diet alters larval growth rates and post-metamorphic
performance in the marine gastropod Crepidula fornicata. Mar. Biol. 162:1597–1610.
Phillips NE. 2002. Effects of Nutrition-Mediated Larval Condition on Juvenile Performance
in a Marine Mussel. Ecology 83:2562–2574.
Rivest BR. 1983. Development and the influence of nurse egg allotment on hatching size in
Searlesia dira (Reeve, 1846) (Prosobranchia : Buccinidae). J. Exp. Mar. Bio. Ecol.

67

69:217–241.
Sokolova IM, Frederich M, Bagwe R, Lannig G, Sukhotin AA. 2012. Energy homeostasis as
an integrative tool for assessing limits of environmental stress tolerance in aquatic
invertebrates. Mar. Environ. Res. 79:1–15.
Sokolova IM, Pörtner HO. 2001. Physiological adaptations to high intertidal life involve
improved water conservation abilities and metabolic rate depression in Littorina
saxatilis. Mar. Ecol. Prog. Ser. 224:171–186.
Somero GN. 2002. Thermal physiology and vertical zonation of intertidal animals: Optima,
limits, and costs of living. Integr. Comp. Biol. 42:780–789.
Spight TM. 1976. Ecology of hatching size for marine snails. Oecologia 24:283–294.
Stoner DS. 1990. Recruitment of a tropical colonial ascidian relative importance of pre
settlement vs post settlement processes. Ecology 71:1682–1690.
Thiyagarajan V, Harder T, Qian PY. 2002. Relationship between cyprid energy reserves and
metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica). J.
Exp. Mar. Bio. Ecol. 280:79–93.
Thiyagarajan V, Harder T, Qiu J-W, Qian P-Y. 2003. Energy content at metamorphosis and
growth rate of the early juvenile barnacle Balanus amphitrite. Mar. Biol. 143:543–
554.
Whitehill EAG, Moran AL. 2012. Comparative larval energetics of an ophiuroid and an
echinoid echinoderm. Invertebr. Biol. 131:345–354.
Woodcock SH, Benkendorff K. 2008. The impact of diet on the growth and proximate
composition of juvenile whelks, Dicathais orbita (Gastropoda: Mollusca).
Aquaculture 276:162–170.

68

CHAPTER 4: General Conclusion
SUMMARY OF RESULTS
To further our understanding of the role of initial energy reserves on the high rates of
early benthic phase (EBP) mortality among intertidal invertebrates, I (1) determined the
proportion of individuals that begin the EBP with energy reserves that are close to or below a
minimum threshold necessary for survival; (2) determined the effect of delayed feeding from
the onset of the EBP on the performance of individuals; and (3) examined to what extent
initial energy reserves affect acute tolerance thresholds to two major abiotic stressors
encountered throughout the EBP: (a) desiccation and (b) high emersion temperature. The
most important finding of this research is that, contrary to previous hypotheses, depleted
energy reserves have minimal direct impact on EBP mortality, as all six species of
invertebrates examined were highly tolerant of starvation throughout the critical first few
days.
The first study of this thesis examined the direct impacts of estimated initial energy
content on performance during the EBP. Past research has indicated that the period of highest
mortality for many species of benthic invertebrates is the first 24 to 48 hours after the onset
of the EBP, after which time mortality levels off (Gosselin and Qian 1997; Hunt and
Scheibling 1997), and it has been hypothesized that depleted or insufficient energy reserves
at the onset of EBP may be partially responsible for this early mortality. In the current study,
all species were highly tolerant of starvation throughout the first 10 d after the onset of the
EBP, with three species experiencing < 2% mortality, and the remaining three experiencing
only 6 – 12% mortality during the first 10 d period. Furthermore, five of the six species that
were examined only reached 50% mortality after >50 d of starvation with the exception of N.
lamellosa which experienced 50% mortality at 27.9 d. This suggests EBP individuals can
survive with only the energy stores acquired during their larval stages or provided by
maternal inputs for an extended period, and leads us to conclude that the vast majority of
individuals do not begin the EBP with energy reserves below or only slightly above the
minimum threshold to survive.
This study also examined the effect of different durations of delay in feeding on the
recovery and performance of EBP invertebrates of three species, and found that delayed

69

feeding from the onset of the EBP negatively impacted performance (i.e. survivorship and
growth) for all three species examined. For two of the species (C. dalli and N. ostrina), the
duration of delay in feeding that resulted in only 50% of individuals being able to recover
(i.e. survive and grow) was considerably shorter than the starvation LD 50 from the previous
experiment. This suggests that even if individuals can survive on their initial energy reserves
for extended periods of time, they may nevertheless fail to recover if they are unable to feed
and replenish their energy stores much sooner. For the final species, B. glandula, recovery
was possible for all individuals, regardless of the delay in feeding. However, the rate of
growth throughout the subsequent feeding period was greatly impacted, with non-starved
individuals growing five times more than individuals who had experienced a 50 d delay in
feeding. Such a drastic reduction in growth rate could impact survivorship by making
individuals more vulnerable to other mortality factors for a longer period of time.
The second study in this thesis examined potential indirect effects of initial energy
reserves on EBP mortality by determining the role of energy reserves in tolerance to two
major environmental stressors, desiccation and high emersion temperature. For both species
that were examined, the acute tolerance threshold to desiccation, but not to emersion
temperature, was significantly reduced among individuals with lower energy reserves (i.e.
starved for a longer duration from the onset of EBP). This suggests that having low energy
reserves makes individuals more vulnerable to some environmental conditions experienced
during the EBP, and thus may lead to increased EBP mortality. Additionally, initial body size
had a significant effect on tolerance to both emersion temperature and desiccation, as larger
individuals had higher tolerance thresholds to both stressors, indicating the body size at the
onset of the EBP may play a larger role in determining vulnerability to environmental
stressors than the amount of stored energy an individual possesses.

RELEVANCE OF FINDINGS FOR POLICY AND MANAGEMENT
The findings of this research have substantive implications for the following areas of
policy and management: (1) climate change impacts on communities of coastal marine
organisms, (2) marine invertebrate aquaculture operations, in particular hatcheries, and (3)
marine invertebrate fisheries management.

70

Impacts of climate change on intertidal communities
The findings of this study highlight the vulnerability of EBP invertebrates to stressors
such as desiccation and elevated emersion temperature, both of which are associated with the
ongoing impacts of climate change. Average air temperature in coastal British Columbia is
increasing at a faster rate than the global average (1.4 °C for coastal BC versus 0.8 °C global
average over the past century), and extreme weather events, including periods of extreme
heat, are becoming more common (Lemmen 2016). The warmest summer sea surface
temperature has also increased over the past century at a rate of 0.81-1.13 °C (Iwabuchi and
Gosselin 2019). With these long-term climatic changes, important abiotic stressors such as
desiccation and emersion temperature are increasing in magnitude and are increasingly likely
to reach or exceed lethal levels for many intertidal invertebrate species. An example of the
acute effects of climate change on intertidal life was recently observed in British Columbia,
encompassing the study area of this research. An extreme heat wave during the summer of
2021 lead to rock surface temperatures as high as 50 °C being recorded along the rocky
intertidal zone of British Columbia, causing mass mortality of intertidal animals near
Vancouver B.C. (Migdal 2021). This assessment, however, only examined adult individuals,
that are significantly more tolerant to abiotic stressors than individuals in the EBP. It is thus
likely that temperatures of this magnitude will also cause drastic mortality events for EBP
individuals of many species. If these types of extreme weather events coincide with peak
spawning or hatching periods, this could severely impact recruitment of those species
affected, which could in turn lead to substantial impacts on population dynamics and
community structure. As such, it is important that the protection of rocky intertidal
ecosystems and the species that inhabit them are taken into consideration when developing
regulations for climate change management and adaptation and future CO 2 emissions.
Furthermore, since EBP individuals are substantially more vulnerable to these abiotic
stressors (Lowell 1984; Gosselin 1997), it is key that tolerance thresholds of the most
vulnerable life stage be considered when making decisions about climate change mitigation
strategies.
Marine invertebrate aquaculture and hatchery operations
Marine invertebrate aquaculture is a thriving industry in coastal BC, with 25 different
invertebrate species being farmed on the Pacific coast (DFO 2021), generating an estimated

71

$41 million annually (Pinfold 2013). For most invertebrate aquaculture industries, the most
unpredictable and complex period is the hatchery and nursery phase, i.e. the rearing of larvae
and early juveniles into healthy and adequately sized “seed” that can then be put into the
“growout” phase of the farming operation. Survivorship is significantly lower through these
early phases, and growth rates can be difficult to standardize (Roberts et al. 2001). While
adequate feeding throughout the larval stage is undoubtedly critical for development and
survivorship, the results of this study indicate that low food availability during the first 5-10
days following metamorphosis into the EBP would not directly cause significant mortality.
However, this study also demonstrates that even a brief delay in feeding after the onset of the
EBP can significantly impact growth for some species of marine invertebrates. Thus, to
optimize growth and success it is critical that invertebrate hatcheries ensure adequate food is
available to their stock immediately following the onset of the EBP. The variability among
collection batches in the present study also demonstrates some evidence of the latent effects
of conditions experienced during the larval or embryonic phase impacting tolerance to certain
stressors, such as desiccation stress, later in life. Thus, if hatchery stock is exposed to
inadequate feeding or stressful conditions (i.e. temperature, salinity, acidity, etc.) it could
have impacts on survivorship during the growout phase of the farming operation. These types
of latent impacts are too often obscured in the aquaculture industry, as the hatchery/nursery
facilities are often separate from the farming facilities making it difficult to trace mortality
events in the farm back to conditions experienced in the hatchery. Nevertheless, it is
important to consider these effects as they have potential to greatly reduce the amount of
product produced by farms.
Marine invertebrate fisheries management
Many species of marine invertebrate species such as crabs, shrimp, and prawns are
actively harvested, either recreationally or as part of a commercial fishery. However, these
invertebrate fisheries are often not as meticulously managed as finfish fisheries, and
overharvesting, or unregulated harvesting, can lead to reduced populations of these species
over time (Anderson et al. 2011). Many factors, such as sex, size, and location, are
considered when fishery managers are establishing allowable catches. However, the results
of this study highlight the importance of also considering rates of EBP survivorship on a
local scale when making these management decisions. Invertebrates are considerably more

72

vulnerable to abiotic changes in the environment during this early life stage (Hamilton and
Gosselin 2020), and local disturbances could result in variable recruitment rates of EBP
individuals to the adult population, which could greatly impact population size and
distribution in future years. Monitoring these changes in recruitment could thus allow
fisheries managers to set harvest quotas at an appropriate level to maintain a sustainable
fishery. Failure to account for these small-scale differences in population recruitment could
potentially lead to drastic changes in population abundance over time, which could in turn
lead to shifting baseline syndrome, or even extirpation, for these important invertebrate
species as has been observed in many finfish fisheries (Pauly 1995).

DIRECTIONS FOR FUTURE STUDY
The results of this study indicate that initial energy reserves at the onset of the EBP
have minimal direct impact on EBP mortality rates but do influence mortality through
indirect effects, such as reducing early growth and increasing susceptibility to desiccation
stress. Yet there are many factors that influence survivorship throughout the critical first few
days to weeks of the EBP that were not addressed in the current study. To further our
understanding of the overall effects of initial energy reserves on EBP success, future research
should examine the role of energy reserves on these other mortality factors. Focus should be
placed on other abiotic stressors that are likely to increase in magnitude with future effects of
climate change such as elevated sea surface temperature, reduced salinity, reduced dissolved
oxygen, and increased ocean acidity (Talloni-Álvarez et al. 2019). Of further interest are the
effects of low initial energy reserves on biotic mortality factors such as predation,
competition, and food acquisition. Furthering our understanding of the relationships between
initial energy reserves and these additional factors will allow us to better understand and
predict changes in populations and how they will be impacted by future changes to their
environment.
The current study demonstrates that tolerance to some stressors, such as desiccation,
are likely mediated by energy dependent mechanisms. However, the physiological
mechanisms of desiccation tolerance utilized by intertidal invertebrates are not well
understood. Future research in this field should focus on determining the specific
physiological and biochemical mechanisms responsible for the desiccation tolerance of these

73

species. One method of desiccation tolerance suggested in this thesis is that benthic
invertebrates may enter the EBP already possessing a large store of protective proteins, such
as heat-shock proteins. This would allow them to tolerate adverse conditions experienced in
the intertidal zone without depleting their energy reserves. The presence of these stores of
protective proteins are, however, unknown. Future research should aim to determine the
tolerance potential of pre-EBP (i.e. larval) individuals of intertidal species.
The findings of this study also demonstrate that tolerance thresholds are influenced by
factors other than initial energy reserves, as replicate groups of the same starvation treatment
(i.e. same amount of remaining energy reserves) but obtained at different collection sites or
times exhibited some significant differences in acute tolerance thresholds. Previous studies
have shown that for many species of intertidal invertebrates, environmental conditions
experienced in the larval or embryonic stages can have significant latent effects on some
aspects of later juvenile fitness (Emlet and Sadro 2006; Pechenik 2006; Pechenik and Tyrell
2015). However, little is known about how such latent effects could impact vulnerability
during the EBP to the various environmental stressors to which they are exposed. Future
research should aim to understand how environmental conditions experienced during the
larval or embryonic stage might alter vulnerability to similar stressors following the onset of
the EBP. Such latent effects could considerably impact population dynamics and fluctuations
in population over time and space in the intertidal zone.

LITERATURE CITED
Anderson SC, Flemming JM, Watson R, Lotze HK. 2011. Rapid global expansion of
invertebrate fisheries: Trends, drivers, and ecosystem effects. PLoS One 6:1–9.
Department of Fisheries and Oceans. c2021. Shellfish and Invertebrates [Internet]. Canada.
[cited 2021 Aug 9]. Available from: https://www.pac.dfo-mpo.gc.ca/fmgp/commercial/shellfish-mollusques/index-eng.html
Emlet RB, Sadro SS. 2006. Linking stages of life history: How larval quality translates into
juvenile performance for an intertidal barnacle (Balanus glandula). Integr. Comp.
Biol. 46:334–346.
Gosselin LA. 1997. An ecological transition during juvenile life in a marine snail. Mar. Ecol.
Prog. Ser. 157:185–194.

74

Gosselin LA, Qian P. 1997. Juvenile mortality in benthic marine invertebrates. Mar. Ecol.
Prog. Ser. 146:265–282.
Hamilton HJ, Gosselin LA (2020) Ontogenetic shifts and interspecies variation in tolerance
to desiccation and heat at the early benthic phase of six intertidal invertebrates. Mar.
Ecol. Prog. Ser. 634:15-28
Hunt HL, Scheibling RE. 1997. Role of early post-settlement mortality in recruitment of
benthic marine invertebrates. Mar. Ecol. Prog. Ser. 155:269–301.
Iwabuchi BL, Gosselin LA. 2019. Long-term trends and regional variability in extreme
temperature and salinity conditions experienced by coastal marine organisms on
Vancouver Island, Canada. Bull. Mar. Sci. 95:337–354.
Lemmen DS, Warren FJ, James TS, Mercer Clarke, C.S.L editors (2016): Canada's Marine
Coasts in a Changing Climate; Government of Canada, Ottawa ON
Lowell RB. 1984. Desiccation of intertidal limpets: Effects of shell size, fit to substratum,
and shape. J. Exp. Mar. Bio. Ecol. 77:197–207.
Migdal, Alex. 2021 July 6. More than a billion seashore animals may have cooked to death in
B.C. heat wave, says UBC researcher. CBC News. [Internet] [cited 2021 July 28].
Available from: https://www.cbc.ca/news/canada/british-columbia/intertidal-animalsubc-research-1.6090774
Pauly D. 1995. Anecdotes and the shifting baseline syndrome of fisheries. Trends Ecol. Evol.
10:430.
Pechenik JA. 2006. Larval experience and latent effects - Metamorphosis is not a new
beginning. Integr. Comp. Biol. 46:323–333.
Pechenik JA, Tyrell AS. 2015. Larval diet alters larval growth rates and post-metamorphic
performance in the marine gastropod Crepidula fornicata. Mar. Biol. 162:1597–1610.
Pinfold, G. 2013. Socio-Economic impact of aquaculture in Canada for Fisheriea and
OCeans Canada aquaculture management directorate.
Roberts RD, Lapworth C, Barker RJ. 2001. Effect of starvation on the growth and survival of
post-larval abalone (Haliotis iris). Aquaculture 200:323–338.
Talloni-Álvarez NE, Sumaila RU, Le Billon P, Cheung WWL. 2019. Climate change impact
on Canada’s Pacific marine ecosystem: The current state of knowledge. Mar. Policy
104:163–176.

75

76

APPENDIX A.
Table A1. Number of batches and number of individuals in each batch for all six benthic
invertebrate species in the experiment assessing the survivorship of EBP individuals unable
to replenish energy reserves.
Species

B. glandula

C. dalli

N. ostrina

N. lamellosa

M. trossulus
Petrolisthes spp.

Batch #
1
2
3
4
5
6
7
8
1
2
3
4
5
1
2
3
4
5
6
7
1
2
3
4
5
1
2
1
2

# individuals
16
41
34
9
21
88
25
15
20
34
17
27
171
29
90
37
131
21
68
47
196
198
170
200
166
34
39
72
40

Total # of individuals

249

269

449

930

73
112

77

Table A2. GLM equations for the relationship between % survivorship and duration of
starvation, starting at the onset of the EBP, for all six species.
Species
B. glandula
C. dalli
N. ostrina
N. lamellosa
M. trossulus
Petrolisthes spp.

Equation
y=1/(1+exp(-5.2253+0.0666x))
y=1/(1+exp(-4.7558+0.0686x))
y=1/(1+exp(-3.3032+0.0591x))
y=1/(1+exp(-3.1759+0.1139x))
y=1/(1+exp(-2.4995+0.0419x))
y=1/(1+exp(-8.7306+0.1720x))

Table A3. Starvation LD20 and percent survivorship at 5 d starvation, and their associated
standard errors (SE), calculated from generalized linear models (GLM) of survival as a
function of duration of starvation for each size class of each batch of Nucella ostrina
hatchlings. n represents sample size for each size class of each batch.
Batch
1
2
3
4
5

6

7
8

Size class

n

Starvation
LD20 (d)

SE

Survivorship (%)
at 5 d starvation

SE

Small
Medium
Small
Medium
Large
Medium
Large
Large
Small
Medium
Large
Small
Medium
Large
Small
Medium
Large
Small
Large

13
29
106
90
217
37
56
59
11
25
15
57
152
76
34
115
85
68
34

20.4
44.7
21.6
44.3
69.0
20.7
38.3
31.9
26.3
22.7
32.3
12.9
26.1
32.1
10.8
44.8
49.1
30.2
39.1

5.8
8.7
1.4
1.6
3.4
3.4
3.0
2.8
3.9
6.7
7.9
2.2
1.6
2.5
2.5
3.5
2.8
2.1
3.5

90.5
93.9
94.8
98.9
97.6
94.0
94.6
93.9
97.6
87.8
91.5
88.9
93.1
94.1
87.9
95.6
99.3
96.5
99.3

3.9
2.6
1.0
0.4
0.5
2.6
1.5
1.6
2.1
3.9
4.3
2.5
1.2
1.5
3.4
1.1
0.4
1.3
0.7

78

Table A4. Recovery LD50 and associated standard error (SE) from linear model analysis of
the proportion of individuals able to recover from different durations of starvation. The z
statistic represents the strength of the relationship between the % survivorship and the
duration of starvation; df = degrees of freedom.
Species
C. dalli
N. ostrina

Recovery
LD50 (d)
28.6
20.9

SE

F value

n

R2

p

4.2
6.6

17.56
7.08

6
6

0.81
0.64

0.014
0.056

79

Figure A1. Relationship between % survivorship and the duration of starvation, starting at
the onset of the EBP, for five species of benthic invertebrates. Results from the generalized
linear models (GLMs) are shown in Table 2.1 in Chapter 2.

80

APPENDIX B.
B) 10 d

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

Survivorship (proportion)

A) 0 d

0.0

0.0
0

5

10

15

0

20

D) 30 d

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

Survivorship (proportion)

C) 20 d

5

10

15

20

5

10

15

20

0.0

0.0
0

5

10

15

Duration of desiccation treatment (h)

20

0

Duration of desiccation treatment (h)

Figure B1. Relationship between the proportion of individuals surviving and the duration of
the desiccation treatment to which they were exposed for groups of B. glandula starved for
(A) 0 d, (B) 10 d, (C) 20 d, and (D) 30 d. Results from the generalized linear models (GLMs)
are shown in Table 3.3 in Chapter 3.

81
B) 10 d

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

Survivorship (proportion)

A) 0 d

0.2

0.2

0.0
0

1

2

3

4

0.0
0

D) 30 d

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

0.0

Survivorship (proportion)

C) 20 d

0

1

2

3

0

4

F) 50 d

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

Survivorship (proportion)

E) 40 d

0.0
0

1

2

3

Duration of desiccation treatment (h)

4

1

2

3

4

1

2

3

4

1

2

3

4

0.0
0

Duration of desiccation (h)

Figure B2. Relationship between the proportion of individuals surviving and the duration of
the desiccation treatment to which they were exposed for groups of N. ostrina starved for (A)
0 d, (B) 10 d, (C) 20 d, (D) 30 d, (E) 40 d, and (F) 50 d. Results from the generalized linear
models (GLMs) are shown in Table 3.3 in Chapter 3.