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The Effect of Temperature on Larval Survival and Development of the Giant California Sea Cucumber Parastichopus californicus in a hatchery Setting

Item

Title (dcterms:title)

Eng The Effect of Temperature on Larval Survival and Development of the Giant California Sea Cucumber Parastichopus californicus in a hatchery Setting

Date (dcterms:date)

2016

Creator (dcterms:creator)

Eng Baird, Kendra

Subject (dcterms:subject)

Eng Environmental Studies

extracted text (extracttext:extracted_text)

The Effect of Temperature on Larval Survival and Development of The Giant California
Sea Cucumber Parastichopus californicus in a Hatchery Setting

by
Kendra Baird

A Thesis
Submitted in partial fulfillment
of the requirements for the degree
Master of Environmental Studies
The Evergreen State College
December 2016

© 2016 by Kendra Baird All rights reserved.

This Thesis for the Master of Environmental Studies Degree
by
Kendra Baird

has been approved for
The Evergreen State College
by

________________________
Erin Martin, Ph.D.
Member of the Faculty

________________________
Date

ABSTRACT
THE EFFECT OF TEMPERATURE ON LARVAL SURVIVAL AND
DEVELOPMENT OF PARASTICHOPUS CALIFORNICUS IN A HATCHERY
SETTING
Kendra Baird
The current global demand for sea cucumber products cannot be met through
commercial harvesting alone, which has led to a recent increased interest in developing
aquaculture techniques for sea cucumbers. Currently, there is no commercial-scale
aquaculture of the Giant California sea cucumber Parastichopus californicus, and
development is in the early research stage. The goal of this experiment was to determine
the optimal temperature range P. californicus larvae should be reared at in hatcheries.
The experiment was conducted at Puget Sound Restoration Fund’s Kenneth K. Chew
Center for Shellfish Research and Restoration hatchery located at NOAA’s Manchester
Research Station in Manchester, WA. Larvae were reared in five temperature treatments
(12°, 15°, 18°, 21°, and 24°C) for 24 days, and survival and developmental stages were
measured and scored, respectively. Larvae developed quicker with an increase in
temperature, but survival was lower with the temperature increase. Survival was best at
12°C, with a mean survival of 14%, but took the longest to develop. The 24°C treatment
had negligible survival, but almost all larvae counted were at the final pentactula phase
on day 24. Of the temperatures tested for rearing P. californicus in a hatchery setting,
12°C is recommended for obtaining the highest survival for maximum production.
Understanding proper larval conditions for maximizing larval growth and survival will
further the advancement of aquaculture of P. californicus.

Table of Contents
LIST OF FIGURES.............................................................................................................v
LIST OF TABLES.............................................................................................................vii
ACKOWLEDGEMENTS................................................................................................viii
INTRODUCTION...............................................................................................................1
LITERATURE REVIEW....................................................................................................3
Natural History........................................................................................................4
Ecology........................................................................................................4
Spawning.....................................................................................................6
Eggs.............................................................................................................7
Larval Development....................................................................................8
Sea Cucumber Market............................................................................................10
Sea Cucumber Fisheries.........................................................................................11
Washington State Sea Cucumber Fishery..............................................................13
Aquaculture of Sea Cucumbers.............................................................................16
Spawning...................................................................................................17
Larval Culture............................................................................................18
Juvenile Grow Out.....................................................................................18
Integrated Multi-Trophic Aquaculture.......................................................19
The Effects of Temperature on Sea Cucumber Larvae..........................................20
METHODS........................................................................................................................21
Broodstock Collection...........................................................................................21
Spawning................................................................................................................23
Experimental System.............................................................................................34
Inoculation.............................................................................................................36
Larval Maintenance...............................................................................................36
Sampling................................................................................................................37
Statistical Analysis ................................................................................................37
RESULTS..........................................................................................................................38
Survival..................................................................................................................38
Development..........................................................................................................40
DISCUSSSION..................................................................................................................44
Spawning………………………………................................................................44
Survival..................................................................................................................45
Development..........................................................................................................46
LITERATURE CITED......................................................................................................49



iv

List of Figures
Figure 1. Photograph of Parastichopus californicus..........................................................5
Figure 2. Illustration of a male and female releasing gametes during spawning................7
Figure 3. Photograph of an oocyte......................................................................................8
Figure 4. Photographs of early auricularia developing into late auricularia.......................9
Figure 5. Photographs of the late auricularia metamorphosing into doliolaria...................9
Figure 6. Photographs of the development of pentactula larvae.......................................10
Figure 7. Graph of Washington State ‘s historical Sea Cucumber landings.....................13
Figure 8. Map of the harvest districts in Washington State managed by WDFW............18
Figure 9. Map of Clam Bay, WA......................................................................................21
Figure 10. Photograph of broodstock holding tank...........................................................22
Figure 11. Photograph of spawning broodstock...............................................................24
Figure 12. Photograph of spawning broodstock with the addition of algae.....................25
Figure 13. Photograph of using ultrasound on the broodstock.........................................26
Figure 14. Photograph of a sea cucumber being weighed................................................27
Figure 15. Photograph of fluorescent light being shone on the broodstock.....................30
Figure 16. Photograph of desiccation...............................................................................31
Figure 17. Photograph of eggs being syphoned................................................................32
Figure 18. Photograph of a male spawning.......................................................................33
Figure 19. Photograph of the 24° C system......................................................................35
Figure 20. Photograph of 12°, 15°, 18°, and 21°C systems..............................................35
Figure 21. Photograph of all of the systems......................................................................36
Figure 22. Graph of the mean survival of larvae throughout the experiment...................39
Figure 23. Graph of the mean percentage of larvae at each developmental stage on day
11........................................................................................................................................41
Figure 24. Graph of the mean percentage of larvae at each developmental stage on day
17........................................................................................................................................42



v

Figure 25. Graph of the mean percentage of larvae at each developmental stage on day
24........................................................................................................................................42



vi

List of Tables
Table 1. Developmental schedule for P. californicus larvae at 11±0.5°C..........................8
Table 2. Weights of the broodstock collected...................................................................27
Table 3. Spawning attempts..............................................................................................34
Table 4. Mean survival of larvae.......................................................................................38
Table 5. ANOVA table of survival on day 5 ....................................................................39
Table 6. ANOVA table of survival on day 11...................................................................39
Table 7. ANOVA table of survival on day 17 ..................................................................40
Table 8. ANOVA table of survival on day 24...................................................................40
Table 9. Mean percentage of larvae at each developmental stage....................................43
Table 10. ANOVA table of the percentage of auricularia larvae on day 11.....................43
Table 11. ANOVA table of the percentage of auricularia larvae on day 17.....................43
Table 12. ANOVA table of the percentage of pentactula larvae on day 17......................44
Table 13. ANOVA table of the percentage of pentactula larvae on day 24......................44



vii

Acknowledgements
This project would not have been possible without the support from many
different people and organizations. I first want to thank my reader Dr. Erin Martin who
assisted with experimental design, thesis writing, preparation for presentations, and for
being a fantastic and supportive mentor throughout my studies at Evergreen State
College. I would also like to thank Puget Sound Restoration Fund for partnering with me
on the project and providing the space, materials, and support for this experiment.
Specifically, I would like to thank Hatchery Manager Ryan Crim who assisted with
experimental design, sampling, and provided mentorship. I would also like to thank
PSRF staff Alice Helker, Stuart Ryan, Laura Spencer, and all the other volunteers who
assisted with sampling and larval care. I would really like to thank Dr. Henry Carson
from WDFW for sharing WDFW’s information with me for use for this study. This
project would not have been possible without financial contributions from The Evergreen
State College Student Foundation, Greg Lineberry, Ashley Andrews, Kelsey Jones, Misty
Speers, and Brandt Detering. Finally, I would like to thank all of my friends, family, and
colleagues who provided support throughout my studies.



viii

INTRODUCTION
There has been an increase in demand for sea cucumber products in recent years,
primarily from Asia. The current global demand cannot be met through commercial
harvest alone, due to limited fishing opportunities over concerns for the sustainability of
the fishery. The sustainability of sea cucumber fisheries is a major concern because sea
cucumbers are especially vulnerable to overfishing because they can be easily and
effectively targeted in shallow waters, display a slow growth rate, late age of maturity,
and a low recruitment rate, which results in a slow population replenishment (Bruckner,
2005; Bruckner et al., 2003; Uthicke and Benzie, 2000; Uthicke et al., 2004). Many
countries such as Japan and New Zealand have been able to recover populations by
limiting fishing opportunities. However, the sustainability of the local sea cucumber
Parastichopus californicus fishery in Washington State is a major concern because recent
surveys have found that in areas of Puget Sound 90% of the historic populations have
been harvested out. This has resulted in a decrease in fishing opportunities through
emergency closures and decreased quotas set and enforced by Washington Department of
Fish and Wildlife (WDFW).
P. californicus is the largest of the 12 species of sea cucumbers found in Puget
Sound, with an average length of 30 cm, but can reach up to 50 cm (Clark, 1922). Recent
studies indicate that the feeding behavior of sea cucumbers plays an important role in
their local ecosystems (Uthicke 2001a). They process large volumes of benthic sediments
from which they assimilate bacterial, fungal, and detrital organic matter. Their feeding
results in the horizontal redistribution and bioturbation of sediments, and the recycling of
nutrients (Crozier, 1918; Haukson, 1979; Lawrence, 1982; Moriarty, 1982; Slater and
Carton, 2009; Uthicke, 1999, 2001b). As sea cucumbers feed, they turn over the top
layers of sediment and transform sediments into finer particles, inhibiting the buildup of
detritus material that contributes to anoxic conditions (CITES, 2002; Michio et. al, 2003).
These findings suggest that the loss of P. californicus have a large impact on the nutrient
cycling in Puget Sound.
P. californicus currently supports a valuable commercial fishery in both Canada
and the United States (Bruckner, 2006; Department of Fisheries and Oceans Canada
statistics). Currently, the United States sea cucumber fishery is managed by individual
states within state waters, and by NOAA Fisheries in coordination with Regional Fishery
Management Council in waters 200 miles off the coast (Bruckner, 2006). The
Washington State fishery is a co-operative management agreement between WDFW and
treaty tribes, with quotas being split about half and half. Washington management
measures for sea cucumbers include seasonal closure from April-June during spawning
season, spatial closures, license of collectors, and annual quotas set for each management
zone (Bruckner, 2005).



1

Managing the fishery is difficult because sea cucumbers are difficult to size, sex,
age, and tag. Enforcing fishery management techniques such as minimum and maximum
sizes and the harvest of only males would be difficult due to their plasticity, seasonal
weight variance, and lack of external evidence of dimorphism (Carson et al., 2016). Past
attempts to tag sea cucumbers have been unsuccessful, which makes it difficult to obtain
growth, mortality, and longevity measurements (Carson et al., 2016). Currently, the
Washington Department of Fish and Wildlife assesses populations through abundance,
harvest weight, and catch-per-unit effort (CPUE) measurements, obtained by video and
dive surveys, and harvest logbooks (Carson et al., 2016).
The development of aquaculture for P. californicus would relieve pressures on
wild stocks, provide additional income and products for shellfish growers, provide more
information on the biology of the species, and allow for restoration projects in areas
where the wild populations have significantly declined. While the demand and market for
sea cucumbers exists, there is still little information regarding broodstock conditioning,
larval and juvenile production, and grow out, impairing the development of hatchery
methods (Zamora and Jeffs, 2013). Research on P. californicus has primarily focused on
stock assessment, fisheries management, and the use as a deposit feeder in Integrated
Multi-Trophic Aquaculture (IMTA) to reduce organic accumulation.
Commercial aquaculture has only been developed for a few species of tropical sea
cucumbers globally (Duy, 2012; Gamboa et al., 2012; Huiling et al., 2004; Renbo and
Yuan, 2004; Xiyin et al., 2004). Currently, there is no commercial level production of P.
californicus, but the Alutiiq Pride Shellfish Hatchery in Alaska has started to develop
spawning and larval grow-out techniques. They have made advancements in overcoming
transport stress of adults during collection, holding and conditioning of broodstock,
spawning, larval culture, settlement, and nursery culturing (Development of red sea
cucumber (Parastichopus californicus) poly-aquaculture for nutrient uptake and seafood
export, unpublished). The non-profit organization Puget Sound Restoration Fund (PSRF)
has started to develop aquaculture techniques for sea cucumbers, with a focus on
continuing to advance the develop of methods for broodstock maintenance, spawning
methods, larval culture, and nursery culturing for grow out.
One of the most difficult early life stages to grow in hatcheries is the larval
phase. Early life stages are the most sensitive to environmental conditions, and therefore,
it is extremely important to know the optimal ranges of physical stressors such as salinity
and temperature, as well as, nutritional requirements and the ideal diets. Larvae
experience the most damage from temperature extremes (Strathmann and McEuen,
1987). Most species can tolerate a relatively wide ambient temperature range within their
natural habitat, although optimal performance is typically restricted to a specific thermal
range (Meng et al., 2009; Pawson, 1966). The range varies between species and life
stages, but typically broadens with development (Costlow et al., 1960; Crisp and Ritz,


2

1967). Studies have shown that echinoderm larvae display a high sensitivity to
temperature, which results in increased mortality, delayed development, reduced growth,
and reduced metabolic rate (Bressen et al., 1995; Przeslawski 2005; Roller and Stickle,
1989, 1993). This is hypothesized to be due to multiple factors including, but not limited
to; oxidative stress, lysosomal destablilisation, increased lipid peroxidation, a change in
immune ability, and the decrease in concentrations of soluble proteins, soluble sugars and
ions (Chang et al., 2006; Deschaseaux et al., 2010, 2011; Liu et al., 2013; Wang et al.,
2012).
Studies have examined the effect of temperature on the survival and growth of
other tropical species of sea cucumbers. Apostichopus japonicas and Holothuria spinifera
larvae rapidly develop at higher temperatures and slower with delayed metamorphosis at
lower temperatures (Asha and Muthiah, 2005; Li Li et al., 2011). Hamel and Mercier
(1996) discovered lower temperatures increased the duration between developmental
stages, but did not increase mortality at each developmental stage for the sea cucumber
Cucumania Grandosa. Liu et al. (2010) observed that A. japonicas larvae grew faster
with increasing temperatures within a suitable range, but extreme temperatures restrained
development. High temperatures resulted in weak-digestion, decreased-absorbing of
nutrients, increased oxygen consumption rate and ammonia excretion rate (Villarreal and
Ocampo, 1993).
In order to grow P. californicus at a large-scale production level in hatcheries, it is
imperative to know the effects of temperature on survival, growth, and development in
order to determine the ideal temperature range that larvae should be reared in. The
objective of this study is to determine the optimal thermal range that P. californicus
larvae should be reared at in hatcheries. The application of this research will further the
advancement of the development of aquaculture techniques for P. californicus and
provide important information to Puget Sound Restoration Fund (PSRF) in order to grow
P. californicus in their hatchery for future restoration efforts throughout Puget Sound.
LITERATURE REVIEW
For my thesis I researched the effect of temperature on P. californicus larval
survival and development in order to determine the thermal range that larvae should be
reared in for optimal growth and survival in hatcheries. There has been an increased
interest in developing aquaculture techniques for P. californicus in recent years due to the
increased demand for sea cucumber products. Wild populations in Washington State have
dramatically declined due to over harvesting, and as a result, fishing opportunities have
been limited, creating a further demand to develop the methods and techniques for
farming. Techniques have been developed for many tropical species globally, but there is
little information pertaining to farming P. californicus. My thesis was completed in
partnership with Puget Sound Restoration Fund (PSRF). PSRF will use the findings from


3

this study to develop aquaculture techniques for rearing larvae and juveniles at their
Kenneth K. Chew Center for Shellfish Research and Restoration hatchery in Manchester,
WA, in order to out plant juveniles throughout Puget Sound for restoration work at areas
where populations have experienced significant declines.
The literature review will provide information on the natural history,
reproduction, and larval development of P. californicus to understand the biology and
ecology of the species. Next, the history of the global and local fisheries will be
examined to understand the changes that have occurred over time to the wild populations
and the relationship to the market and market potential in Washington State. The
aquaculture techniques that have been established for related tropical species and the
recent advancements in aquaculture of P. californicus will be briefly discussed in order to
understand the current state of development and discuss where future research efforts
should be focused. Finally, the effect of temperature on echinoderm and sea cucumber
larvae will be discussed in order to understand the importance of this physical variable
for the production success of hatcheries.
Natural History
Ecology
At least 12 species of sea cucumbers are native to Puget Sound (Strathmann and
McEuen, 1987). P. californicus is the largest species with an average length of 30 cm, but
can reach up to 50 cm (Clark, 1922). They are typically reddish-brown in color but
sometimes appear pinto or albino (Figure 1) (Cameron, 1985; Strathmann and McEuen,
1987). They are found off the west coast of Northern America and range from Baja, CA
to British Columbia (Cameron, 1985). This particular species is found in Rocky low
intertidal and subtidal zones up to 249 meters deep, and prefer areas away from strong
wave action such as quiet bays (Cameron, 1985; Ricketts and Calvin, 1968). They can
migrate randomly, covering 3.9-100 meters in a day (Cameron and Fankboner, 1989;
Muse, 1998). They are epibenthic and prefer sand and shell substrate, kelp beds, and
rocks (Fankboner and Cameron,1985; Feder et al., 1974; Woodbey et al., 2000).
Population densities may reach 0.5 individuals per m2 in the wild (Sloan, 1985).



4

Figure 1. Photograph of Parastichopus californicus. Photo Credit: Kendra Baird.

Like other sea cucumbers, P. californicus eviscerate their gut, respiratory tree,
circulatory system, and gonads when they are subjected to predation or physical stress as
a defense mechanism (Garcia-Arraras and Greenberg, 2001). It takes only a few weeks to
regenerate the lost organs, but it can take 1-3 months to regenerate sufficiently to be fully
functional (Cameron, 1985; Swan, 1961). P. californicus atrophy their internal organs on
a seasonal basis in the fall, as they enter a state of winter dormancy, and regenerate
organs over a period of several weeks in the spring (Fankboner 2002; Fankboner and
Cameron, 1985; Swan, 1961).
Recent studies are indicating that the feeding behavior of sea cucumbers plays an
important role in their local ecosystems (Uthicke 2001a). P. californicus are deposit
feeders and process large volumes of benthic sediments from which they assimilate
bacterial, fungal, and detrital organic matter. Their feeding results in the horizontal
redistribution and bioturbation of sediments, and the recycling of nutrients (Crozier,
1918; Haukson, 1979; Lawrence, 1982; Moriarty, 1982; Slater and Carton, 2009;
Uthicke, 1999, 2001b). Methews et al. (1990) suggests that sea cucumbers turn over the
top few millimeters of sediment for over 90% of the sea floor. Studies further suggest that
sea cucumbers may affect the physical nature of the sea floor. Eradication of sea
cucumbers in localized areas resulted in the hardening of the sea floor, which
subsequently eliminated habitat for benthic and infaunal organisms (CITES, 2002).
As sea cucumbers feed they turn over the top layers of sediment and transform
sediments into finer particles, inhibiting the buildup of detritus material that contributes
to anoxic conditions (CITES, 2002; Michio et. al, 2003). Studies in tropical coral reef
ecosystems have shown that sea cucumbers play an important role in the nutrient cycling
of nitrogen and phosphorus. The sediments are typically high in nitrogen and phosphorus,
but studies found that the presence of sea cucumbers resulted in the translocation of



5

nitrogen and phosphorus into the water column, where they become available to other
organisms to utilize (Uthicke, 2001a,b).
Natural predation is not limiting on sea cucumber populations, but known
predators include the sea stars Solaster dawsonii and pycnopodia helianthoides, fish such
as kelp bass Paralabrax clathratus and California sheephead Semicossyphus pulcher, and
crabs (Fankboner and Cameron, 1985; Quast 1968; Sewell, 1990; Washington
Department of Fisheries, 1976; Woods, 1993). Sea cucumbers also serve as a host for
many parasitic and symbiotic organisms. The scale worm Arctonoe pulchra is commonly
found living on the ventral side within the tube feet, the flatworm Anoplodium hymanae
lives in the body cavity, the flatworm Wahlia pulchella lives in the upper intestine, the
gastropod Enteroxenos parastichopoli attaches to the intestine, and the snail vitriolina
Columbiana attaches to the outside skin. (Cameron, 1985; Fankboner and Cameron,
1985; Kozloff and Shinn, 1987; Shinn, 1983, 1984).
Spawning
P. californicus reach sexual maturity at four years of age (Cameron and
Fankboner, 1989). Sexes are separate with no external evidence of sexual dimorphism,
and occur at a 1:1 ratio in the wild (Cameron, 1985). Individuals spawn repeatedly during
the season (Washington Department of Fisheries, 1976). Adults may migrate to shallower
water for spawning, with spawning frequently observed 5-12 meters below the surface by
divers (Cameron 1985; Courtney, 1927). Spawning events may correlate with bright
sunshine days and high phytoplankton blooms (Cameron, 1985; Cameron and Fankboner,
1986; Muse, 1998).
The spawning season in the wild has been observed to extend from late April to
August, with the peak of the season occurring May to mid July in the San Juan Islands
(Cameron, 1985; Courtney,1927; Bovard and Osterud, 1918; Johnson and Johnson, 1950;
MacGinitie and MacGinitie 1949; McEuen, 1986; Mortensen, 1921; Smiley 1984, 1986a,
1986). Spawning has been observed in August in Newport Bay, CA and June through
August in British Columbia (Cameron and Fankboner, 1986).
During spawning adults assume a distinctive posture as they release the gametes
into the water column. One third to one half of the anterior end is raised vertically from
the substrate with the “head” curved forward to the substrate (Figure 2). The gonophore
opens at the point of maximum elevation above the substratum on the anterior dorsal
surface of the animal (Cameron, 1985). Gametes of each sex are easily distinguishable
from one another in situ. Sperm is cream colored and occurs in a thick cloud. Oocytes are
orange and disperse immediately in water, which can make it difficult to notice when a
female has begun to spawn (Cameron, 1985). Females have a significantly larger gonad
index (the calculation of the gonad mass as a proportion of the total body mass) at



6

maturity than males, which indicates that females have a much greater reproductive effort
than males (Cameron, 1985).

Figure 2. Illustration of a male and female P. californicus releasing gametes during spawning.
Credit: Cameron, 1985.

Eggs
Large females can have fecundities up to 8.92x106 (Strathmann and McEuen,
1987). Oocytes are 185-210 µm in diameter and surrounded by a jelly coat about 30 µm
thick (Cameron, 1985; Strathmann and McEuen, 1987) (Figure 3). The oocytes possess a
distinct germinal vesicle and nucleolus (Cameron, 1985). Oocytes are negatively buoyant
and will sink into the water column or to the bottom of tanks; therefore must be syphoned
out of spawning tanks during spawning events in hatcheries (Cameron, 1985). Oocytes
collected from excised ovaries are enclosed within an ovarian capsule. Once they are
exposed to seawater the oocytes exit the capsule, becoming distorted during the process
(Cameron, 1985). Strip spawning is not effective with P. californicus because only less
than one percent of oocytes undergo germinal vesicle breakdown within the ovarian
tubules on their own, indicating they need external triggers to begin meiosis (Cameron,
1985).



7

Figure 3. Photograph of an oocyte collected in the lab taken with an Iphone and a
compound scope. Credit: Kendra Baird.

Larval Phases
Most benthic marine invertebrates have a pelagic planktonic larval phase
characterized by high dispersal and high mortality (Chen, 2003; Miller, 1995; Rumrill,
1990; Smiley, 1986b). This period may extend from three weeks to several months for P.
californicus, and larvae can develop asynchronously (Cameron, 1985; Miller, 1995;
Strathmann and McEuen, 1987). The development schedule for P. californicus at 11°C is
reported by Strathmann and McEuen (1987) and is included in Table 1. The development
rate of larvae is strongly influenced by temperature and food supply, therefore, it is
expected to vary in different conditions (Miller, 2001). The advancement of aquaculture
and hatchery methods for this species will be dependent upon determining these factors,
along with other physical and biological parameters such as salinity and stocking density.
Planktrophic larvae feed primarily algal cells through the capture of particles, and
through absorption of dissolved organic molecules. Food is processed in the gut and fecal
matter is expelled through a ventral anus. Because the development of P. californicus
requires feeding on external matter, eggs are smaller and contain little lipid reserves
compared to other species (Miller, 2001).
2
cell

32
cell

64
cell

Blastula

Gastrula

Early
larvae

Early
doliolaria

Settlement

Armored

2.75h

10.75h

12 h

19h

27 h
(290 um)

3.254.25d
(1120 um)

52 d
(410-550
um)

60-61d
(245-365
um)

61-65 d
(299 um)

Table 1. Developmental schedule for P. californicus larvae at 11±0.5°C reported by Strathmann
and McEuen (1987). Measurements in parenthesis indicate the size.



8

The first larval phase of P. californicus is auricularia (Figure 4, Table 1). This
phase typically lasts 35-52 days. Individuals range from 425-1,120 µm in length during
this phase (Strathmann and McEuen, 1987). Early auricularia develop a looped band of
cilia used for swimming and feeding. Further development produces arms and lobes over
which the single, continuous band is lopped. The width of the band varies and there are 37 cilia across the band, one cilium per cell. Encircling the mouth is an aboral band of cilia
that is continuous with a ciliary band at the top of the esophagus (Strathmann, 1971,
1974, 1975). Calcareous ossicles are present in the posterolateral lobes. The larva is
typically transparent with occasional tinting of the ciliary band (Strathmann and McEuen,
1987).

Figure 4. Photographs of early auricularia developing into late auricularia. Photos were taken
with an Iphone and a compound scope. Photo Credit: Kendra Baird.

As auricularia begin to metamorphose into simplified doliolaria, the single ciliary
band rearranges into five transverse rings of cilia (Strathmann and McEuen, 1987).
Further reduction results in the compact barrel-shaped doliolaria (Figure 5). The
metamorphosis to doliolaria results in a 90% decrease in body volume, and individuals
range from 410-550 µm in length (Strathmann and McEuen, 1987). During
metamorphosis the mouth withdrawals to form the atrium and the larvae do not feed
during this stage. This is a very brief larval phase that only lasts 24-48 hours (Cameron,
1985; Strathmann and McEuen, 1987).

Figure 5. Photographs of the late auricularia metamorphosing (left and middle) into doliolaria
(right). Photos were taken with an Iphone and a compound scope. Photo Credit: Kendra Baird.

The final larval stage is the pentactula phase, which is marked by the emergence
of a single tube foot at the posterior end (Figure 6). The period to settlement followed by


9

completion of metamorphosis may be as long as 131 days (McEuen, 1986). Individuals
range from 245-1,120 µm in length during this phase (Strathmann and McEuen, 1987).
Ciliated bands persist for a short while, and larvae at this stage may be found swimming
or crawling (Cameron, 1985). Calcareous ossicles begin to form and cover the larval
surface at metamorphosis (Miller, 2001). 3-5 days after metamorphosis, button ossicles
form and the organisms take on a spiny appearance. During or just after settlement, the
pentactulae grow a single ventroposterior podium (Strathmann and McEuen, 1987). They
use the knobbed “sticky” tip of the podium to attach to substratum at settlement
(Strathmann and McEuen, 1987). McEugene (1986) suggests that settling larvae probably
attach to undersides of rock in calm coves, bays, and fjords, since that is where juveniles
1-11cm long have been observed. In southern British Columbia, juveniles are regularly
observed in dense mats of filamentous red algae, on polychaete tubes, and in crevices on
rock walls (Cameron, 1985). Settled Juveniles will continue to grow into adults and
sexually mature at four years old.

Figure 6. Photographs of pentactula larvae developing over time. Photos were taken with an
Iphone and a compound scope. Photo Credit: Kendra Baird.

Sea Cucumber Market
Sea cucumbers are a delicacy in many Asian countries and are also used for
nutritional supplements, arthritis treatments for humans and pets, and traditional
medicinal treatments (Bruckner, 2005; Chen, 2003; Feindel, 2002; Fredalina et al., 1999).
Modern medical research has discovered many biochemical products that can be
extracted from sea cucumbers, many of which have medicinal potential for antibacterial,
antiviral, anti-oxidant, anticoagulant, antimicrobial, and cancer fighting compounds
(Avilov et al., 1998; David and MacDonald, 2002; Findlay et al., 1983; Haug et al., 2002;
Kelly, 2005; Li et al., 2008; Mamelona et al., 2007; Roginsky et al., 2004; Silchenko et
al., 2007; Tipper et al., 2003; Trotter et al., 1995, 1997; Yayli and Findlay, 1999; Zhong
et al., 2007).
Most sea cucumbers harvested in the United States are exported to Asia, to the
countries of China, Hong Kong, Taiwan, and Korea (Bruckner, 2005). There are also
small commercial markets of P. californicus in North America in cities such as New



10

York, San Francisco, and Vancouver (Bruckner, 2005). The global demand for sea
cucumber products has driven the price to increase in recent years. In the early 1980’s the
value was USD $0.07 per kg and in 2005 had risen to USD $0.82 per kg (Bruckner,
2005). Processed sea cucumbers can be sold for up to USD $9.00 per kg (Bruckner,
2005). The total ex-vessel revenue (the value established by determining the average
price for an individual species, harvested by a specific gear, in a specific area) for
Washington and California fisheries has varied from USD $1,000,073 in 1999 to a
maximum of USD $4,848,999 in 1993, with the majority of the revenue associated with
the Washington State fishery (Bruckner, 2005).
Sea cucumbers can be sold raw or preserved. Traditional methods of preserving
include drying, smoking, canning, or freezing (Bruckner, 2005). They are prepared by
being gutted and then boiled or roasted. The end with the tentacles is removed and the
body wall is slit lengthwise to remove the viscera and scrape the muscles off the body
wall (Washington Department of Fisheries, 1976). The body wall and muscles are
typically boiled, dried, and salted before export (Bruckner 2005). The longitudinal
muscles are one of the most common parts consumed in the United States, because they
can be prepared like clams. Intestine and gonads can be processed in high-priced
delicacies, and were sold for up to USD $23.20 per pound and USD $45.30 per pound in
1974, respectively (Washington Department of Fisheries, 1976).
Sea Cucumber Fisheries
In the United States commercial fisheries of sea cucumbers began in Washington
State in 1971, followed by California in 1978, Alaska in 1981, Maine in 1988, and
Oregon in 1993 (Bruckner, 2005). P. californicus currently supports a valuable
commercial fishery in Canada and the United States. In 2003, landings were 807.4 metric
tons in Alaska, 132.6 metric tons in California, 0.312 metric tons in Washington, and
520.7 metric tons in British Columbia (Bruckner, 2005). In 2007, the landings in Canada
were 623 metric tons worth CAN $2.65 million (Hannah et al., 2012). In 2011, the
landings in the United States were 1,101 metric tons worth USD $3.4 million
(Department of Fisheries and Oceans Canada statistics).
The sustainability of sea cucumber fisheries is a major concern in the United
States. Sea cucumber populations are especially vulnerable to overfishing because they
can be easily and effectively targeted in shallow waters by harvesters, and display slow
growth, late age maturity, and low rate of recruitment, which results in slow population
replenishment (Bruckner, 2005, Bruckner et al., 2003; Uthicke, 2004; Uthicke and
Benzie, 2000). As broadcast spawners, they are prone to the Allee effect, meaning that a
low population size or density below the critical population threshold will result in a
population crash to extinction. If the populations are not dense enough, and individuals
are too spread out from one another, gametes cannot reach each other during spawning,


11

which results in a population collapse and inhibits recovery (Allee, 1938; Bruckner,
2005; Courchamp et al., 1999; Uthicke et al., 2009; Uthicke and Benzie, 2000).
The loss of sea cucumbers not only affects the marine ecosystem, but the decrease
in fishing opportunities can have a major impact on the economies of coastal
communities and a loss of cultural value. The fishery provides income, food, and is
culturally important to many Native American tribes (Kinch et al., 2008). In 1988, the sea
cucumber fishery became the most highly valued fishery outside of tuna fishing season
and represented 80% of the value of all non-fish marine products in the Maldives
(Joseph, 2005). The First Nations of Canada and the native communities in Alaska still
harvest sea cucumbers for subsistence and social and ceremonial use (Mathews et al.,
1990; Wein et al., 1996). In 1990, yearly harvests ranged from 150 to 700 sea cucumbers
per household (Mathews et al., 1990).
In the United States sea cucumber fisheries are managed by individual states in
state waters and by NOAA Fisheries in coordination with Regional Fishery Management
Council in waters 200 miles off the coast (Bruckner, 2006b). Stock assessments and
management of sea cucumbers are hindered by large gaps of knowledge about biological
information such as the amount of time spent in larval phases, recruitment, and minimum
density needed to successful reproduction, which is necessary for sustainable
management (Toral-Granda et al., 2008). Managing the fisheries is difficult because sea
cucumbers are difficult to size, sex, age, and tag. Enforcing fishery management
techniques such as minimum and maximum sizes and the harvest of only males would be
difficult to enforce due to their plasticity and lack of external evidence of dimorphism
(Carson et al., 2016). Sea cucumbers can expand and contract in length and diameter,
expel or retain water, and their weights vary seasonally due to atrophy (Carson et al.,
2016). Past attempts to tag sea cucumbers have been unsuccessful, which makes it
difficult to obtain migration, growth, mortality, and longevity measurements (Carson et
al., 2016). WDFW’s population assessments are determined by abundance, harvest
weight, and catch-per-unit effort (CPUE) measurements, recorded in dive surveys and
harvest logbooks (Carson et al., 2016).
Some countries have successfully managed sea cucumber fisheries despite the
management hurdles. Japan was able to recover populations by limiting fishing through
implementing fishery laws, rights systems, permits, and fishery co-operatives and
restocking depleted areas (Akamine, 2004). New Zealand established a conservative
harvesting limit, with a total allowable commercial catch of 35 metric tons (Ministry of
Fisheries, 2011), which is relatively small when compared to 1,000, 6,000, and 400
metric ton harvest limits for Japan, Korea, and British Columbia, respectively (Hamel
and Mercier, 2008).



12

After British Columbia experienced the boom-and-bust pattern they reduced
quotas, added license restrictions, and implemented adaptive management and
subsequently, catches are beginning to recover (Hand et al., 2008). Alaska sets their
harvest limit based on the lower 90% bound of a biomass estimate. Areas are fished on a
3-year rotation schedule and separate areas are left closed as controls (Clark et al., 2009).
In California, both P. californicus and Parastichoous parvimensis are harvested. A
special permit was required from 1992 to 1993 for sea cucumber harvest. Separate
permits for each gear type and a limit on the total number of permit were implemented in
1997. There are no restrictions on catch, but trawling is prohibited in some conservation
areas (Rogers-Bennett and Ono, 2001; Schroeter et al., 2001). Surveys have reported that
harvested sites in California showed densities that were 50-80 percent lower than in nonfished areas (Bruckner, 2006). In Oregon, the annual fishery lasts for three weeks in
October (DFO, 2002). The commercial fishery is a small limited–entry dive fishery that
is managed by individual quotas (DFO, 2002). All landings are monitored by an
independent industry-funded firm (Bruckner, 2006).
Washington State Sea Cucumber Fishery
Commercial exploitation of P. californicus in Washington State has reduced
populations from historic levels, with a decline of up to 90% in some areas. The fishery
was established in 1971 and occurred without restrictions until 1987. The early 1980’s
commercial harvest and value was low (125-181 metric tons per year and USD $0.060.13 per kg). The annual harvest began to increase in 1988 from 952 tons and peaked in
1992 at 1880 metric tons (Bruckner, 2005). Following signs of overfishing, the state
implemented a rotational harvest from 1987 to 1992 (Bradbury, 1994). And in 1994
seasonal harvest and specific harvest districts were finally adopted (Bruckner, 2005).

Figure 7. Graph of Sea Cucumber landings with inflation adjusted value over time in Washington
State. Credit: WDFW.



13

The fishery in Washington State is a co-operative management agreement
between WDFW and treaty tribes, with quotas being split about half and half.
Washington management measures include seasonal closure from April-June during
spawning season, spatial closures, license of collectors, and annual quotas set for each
management zone. Seven areas have been closed, two for human health reasons. There is
a prohibition on trawling and shrimp areas. Regulations for the trawl fishery include 1) a
ban on trawling in waters less than about 20 m deep; 2) temporal closures during softshell Dungeness crab (reproductive) periods; 3) specific fishing locations; and 4)
restrictions on gear type and size, including maximum beam width for beam trawl gear
and minimum mesh size for otter trawl gear. Fish receiving tickets are submitted to
WDFW after each fishing trip. This data is used to determine when the annual tribal and
commercial harvest quota is reached. Fishermen also submit monthly harvest logs that
include the date, vessel name or boat registration number, location fished, pounds landed,
average depth of harvest, number of divers, and total diver hours spent fishing. There are
46 licensed commercial divers, with a license reduction program initiated in 2002, with a
goal of reducing the total number of licenses to 25 (Bruckner, 2005). Recreational
licenses also allow individuals to harvest up to 10 animals a day for personal use. There
are also a low number of Scientific Collection Permits granted, which allow individuals
and organizations to collect a small number of individuals for research purposes.
Each management zone designated by WDFW has individual quotas set using
models and estimates from catch-effort data and video surveys and dive surveys. WDFW
uses the 5% biomass rule to set the harvest limits, as suggested by Uthicke (2004), for the
sustainable fishery of P. californicus. Only 5% of the population should be harvested to
minimize the effect on the population and ensure the sustainability of the fishery. Surveys
have determined that District 1 (Figure 8) has a current biomass estimate of 6,000,000
pounds. The 2013 quota was 11.4% of the biomass. WDFW is lowing the harvest rate by
1% a year, until the ideal 5% is reached. The current 2016 quota is at 8%. Populations in
District 2 (Figure 8) have stabilized and may be rebounding in the in the western half of
the district. Current biomass estimate is 3,886,516 pounds, which indicates that District 2
can support continued harvest. However, the eastern area of the district was found to have
a much lower biomass than the west, and as a result, the district has now been split into
two separate management zones with a 128,000-pound quota for the west and a 42,000pound quota for the east to meet the 5% harvest rate for each section of District 2.
WDFW also designated no-take zones in the Strait of Juan de Fuca to act as a reservoir
and to allocate a reference site where there is an absence of harvest. Surveys have
determined that the Central Puget Sound District (District 3) (Figure 8) has been
subjected to a high level of historic harvest and has greatly reduced the biomass in
comparison to the virgin state. The biomass estimate for District 3 is 183,000 pounds, 7%
of the historic biomass. Harvest has been closed in District 3 until evidence of recovery is
obtained. South Puget Sound (District 5) (Figure 8) biomass is estimated to be 437,135


14

pounds. The quota is now set at 22,000 for the 5% harvest rate. WDFW has focused
recovery efforts on continuing to monitor activities, develop the ability to tag to estimate
age, growth, mortality, and behavior, as well as research population genetic information.
These efforts will help develop and enforce regulations for sustainable management of P.
californicus in Washington State (WDFW, unpublish data).

Figure 8. Map of the harvest districts in Washington State managed by WDFW. Credit: WDFW.


15

Aquaculture
The current demand for sea cucumber products cannot be met through
commercial harvesting alone, which has led to an increased interest in developing
aquaculture techniques for sea cucumbers globally (Ren et al., 2016). There has been
progress made and aquaculture techniques have been established for tropical species in
countries such as New Zealand and Australia, for the temperate species Japanese sea
cucumber Apostichopus japonicus; and the tropical sandfish Holothuria scabra (Duy,
2012; Gamboa et al., 2012; Huiling et al., 2004; Renbo and Yuan, 2004; Xiyin et al.,
2004). Hatchery and grow-out technology is still being developed for many other species
of interest including; Autralostichopus mollis, Isostichopus fuscus, Athyonidium chilensis,
Cucumaria frondosa, Stichopus horrens, Holothuria fuscogilva, Actynopiga spp., and
Parastichopus californicus (Guisado et al., 2012; Jimmy et al., 2012; Nelson et al., 2012;
Mercier et al., 2012; Paltzat et al., 2008). However, large-scale commercial aquaculture
has not yet been established for P. californicus, and it remains in the early developmental
stage (Zamora and Jeffs, 2013). There is still little information regarding broodstock
conditioning, larval and juvenile production, and grow out. Current research is primarily
focused on stock assessment and fisheries management, and the use as a deposit feeder in
Integrated Multi-trophic Aquaculture (IMTA) to reduce organic accumulation. (Zamora
and Jeffs, 2013).
The Alutiiq Pride Shellfish Hatchery in Alaska was one of the first hatcheries to
start developing aquaculture techniques for P. californicus. They began developing
spawning and larval grow-out techniques in 2010. They have since made advancements
in overcoming transport stress of adults during collection, holding and conditioning
broodstock, spawning, larval culture, settlement, and nursery culturing (Development of
red sea cucumber (Parastichopus californicus) poly-aquaculture for nutrient uptake and
seafood export, unpublished). Puget Sound Restoration Fund (PSRF) received Salton
Kennedy Grant from NOAA in partnership with Pacific Shellfish Institute, University of
Washington, Alutiiq Pride Shellfish Hatchery, and Washington Department of Fish and
Wildlife to research P. californicus. PSRF will develop aquaculture techniques for sea
cucumbers, with a focus on continuing to develop methods for broodstock maintenance,
spawning methods, larval culture, and nursery culturing. The purpose of their research is
to develop aquaculture techniques for future restoration work. They hope to grow out
juveniles at the Kenneth K. Chew Center for Shellfish Research and Restoration hatchery
to out plant to areas of Puget Sound that have experienced significant population
declines. The findings from this study will contribute to the advancement of PSRF’s
work. Determining the optimal temperature range to rear larvae at for maximum survival
and development will improve production success.



16

Spawning
The first step of developing hatchery techniques for P. californicus is to
determine reliable methods for inducing spawning in order for hatcheries to have access
to large number of gametes and subsequent larvae. Many species of sea cucumbers, if
collected early in the spawning season, will release gametes within 1-2 weeks in the
laboratory (McEuen 1986, Strathmann and McEuen 1987). At the height of the spawning
season, Psolus Chitonoides frequently spawns in early morning hours in response to
increase in light intensity, Cucumaria miniata discharges gametes in late morning and
afternoon, and Eupentacta quinquesemite spawns during evening and early night hours.
Stopping seawater flow and warming the water triggered spawning in Cucumaria lubrica
and Cucumaria pseudocurat. Jordan (1972) found that decreasing seawater temperature
by 2°C, then warming it again with the addition of dense concentrations of Dunaliella
sp., induces Calceolaria frondosa to spawn.
Alutiiq Pride Shellfish Hatchery has had success with inducing spawning in P.
californicus by raising the seawater temperature at least 2-4°C. Males began to release
sperm first, and females began about an hour after the first male began. Females release
oocytes for 20-30 minutes (Ren et al., 2016). During spawning, slight disturbances
typically do not completely stop the release of gametes, and individuals resume spawning
shortly after being handled. Isolating individuals into separate buckets is possible to
collect gametes in order to manipulate crosses if the sea cucumbers are carefully handled
and rinsed.
If methods for inducing spawning are not successful, then it may be possible to
induce meiosis through chemical and artificial means. Studies have found that meiosis
can be induced in oocytes of P. californicus by using radial nerve factors (RNF) extracted
from holothuroids or pycnopodia and prepared in accordance to the methods and Smiley
(1984), Strathmann and Sato (1969), and Maruyama (1986). Unlike other echinoderms,
1-methyladenine does not induce maturation in P. californicus, but purine does show
potential. Smiley (1984) found that low concentrations of dithiothretol (10mM for 10
minutes or less) can be successful in inducing maturation in oocytes, but only a low
percentage develop normally into larvae. Until methods for inducing meiosis in oocytes
has resulted in a high percentage of normally developed larvae, artificial fertilization of
ova will not be a practical option for hatcheries.
When obtaining gametes via dissection is recommended to slit the body wall
lengthwise, away from the mid-dorsal axis to avoid severing the gonoduct. When very
ripe, ovarian tubes spontaneously release oocytes through the gonoduct into the dish
(Strathmann and McEuen, 1987). Active sperm can be obtained through dissection and
using forceps to open the testis tubule. Sperm can be also activated by adding aqueous
NH4Cl to final concentration of 7-10 mM (Smiley, 1986b). The optimal time for


17

insemination is immediately following germinal vesicle breakdown. High levels of
polyspermy are noted to occur when sperm is added to ripe oocytes after the formation of
the first polar body (Smiley, 1986b). It is recommended to collect fertilized eggs with a
syphon, and collect onto a wet 45-60µm screen. It also recommended to rinse the
fertilized eggs with seawater to remove excess sperm.
Larval Culture
Larval culture of P. californicus is currently hindered by poor survival to
metamorphosis. The rate of development for larvae is variable and influenced by
maturation rate, time of fertilization, water temperature, stocking density, quantity and
quality of food, and individual variation in developmental rate (Strathmann and McEuen,
1987). Cultures of embryos and larvae should be stirred occasionally to prevent
coalescence in dense aggregations in the tanks or bowls, although, they can be reared
successfully in static systems with frequent water changes every 1-5 days (McEugen,
1986; Strathmann and McEugen 1987). The recent study by Ren et al. (2016) determined
that P. californicus larvae performed well on mono-species microalgal diets containing
Chaetoceros calcitrans, Chaetoceros muelleri, Dunaliella tertiolecta, Pavlova lutheri,
and Tahitian sp., but performed best with bi-species microalgal diets containing C.
calcitrans. It is recommended to have a stocking density between 0.3-1.0 individual per
ml (Agudo, 2006; Palzat, 2008). Studies have found that survival to settlement in sea
cucumbers is dependent upon stocking densities, however, more studies are needed to
understand the relationship between stocking densities and survival during different
planktonic phases (Battaglene et al., 1999; Duy, 2012; Purcell, 2012).
Juvenile Care
Methods for growing and feeding newly settled sea cucumbers have been heavily
adopted from methodologies for culturing herbivorous gastropods, such as, abalone
(Battaglene et al., 1999). Once sea cucumber larvae began to settle and metamorphose
into the early juvenile stage they are normally transferred to nursery raceways, ponds, or
off shore sea pens (Chen, 2004; Purcell et al., 2012). On shore nursery systems are the
preferred method for rearing juvenile p. californicus because environmental conditions,
feeding, and predation can be controlled. Lavitra et al. (2010) found that the growth rates
of juveniles in nursery tanks were highest at lower stocking densities, and were relatively
unaffected by sediment quality. Pond cultures are primarily used in tropical regions and
China. They can be very cost effective if pre-existing ponds, such as ones used in shrimp
production, can be utilized. However, creating new ponds can be expensive and labor
intensive (Renbo and Yuan, 2004). Not all species perform well in ponds (Mercier et al.
2012, Purcell, 2012).



18

Ocean-based aquaculture or “sea ranching” is a method to culture sea cucumbers
held in pens, suspended from a raft in cages or placed directly on the sea floor (Chen,
2004; Purcell and Simutoga,, 2008; Renbo and Yuan, 2004). This method has also been
used in China and other tropical regions (Chen, 2004; Renbo and Yuan, 2004; Mills et
al., 2012). The pens prevent the sea cucumbers from migrating, theft, and helps to
designate ownership (Purcell et al., 2012). Research has also evaluated the potential to
release juvenile sea cucumbers without any confinement (Bowman, 2012; Fleming, 2012;
Juinio-Menez et al., 2012; Purcell, 2012; Purcell and Simutoga, 2008,). Studies have
demonstrated that A. mollis and H. scabra will not move great distances and can be
harvested years later, if the habitat conditions and size of the site are adequate (Mercier et
al., 2000; Purcell and Kirby, 2006; Slater and Carton, 2010). However, survival of the
released juveniles is a major concern because the majority is not surviving market size in
the wild (Juinio-Menez et al., 2012; Purcell and Simutoga, 2008).
Developing cost-effective methods and technology will be important for scaling
up the aquaculture of P. californicus to commercial levels of production (Purcell et al.,
2012). The development of broodstock management, larval rearing, nursery culture, and
techniques for transporting juveniles are key to successful commercial aquaculture
development. If more reliable methods for obtaining high quality gametes throughout the
year in large numbers can be developed, then there is more potential for the success of
aquaculture for P. californicus.
Integrated Multi-Trophic Aquaculture
Recent studies indicate that P. californicus may have the potential as an organicextractive species, to be co-cultured under salmon Salmo salar and sablefish Anoplopoma
fimbria, or under suspended Pacific oysters Crassostrea gigas (Ahlgren 1998, Hannah et
al., 2012, Paltzat et al. 2008). Results of these studies yielded high growth and survival.
Integrated multi-trophic aquaculture (IMTA) is the culture of two or more compatible
species, which occupy different trophic levels, in one system (Bardach, 1986; Zamora
and Jeffs, 2012). Successfully integrated IMTA systems closely mimic natural ecosystem
functions (Folke and Kautsky, 1992). Species at lower trophic level consume the different
types of waste produced by the species at the higher trophic levels. Examples include
macroalgae can absorb dissolved nutrients, filter-feeding shellfish can consume fine
particulates, and deposit feeders can consume heavier particulates (Chopin et al., 2001).
Studies suggest that sea cucumbers have the potential to reduce fish farm waste,
mitigating the negative environmental impacts of fish pens, while providing an additional
remunerative product to the companies.
Commercial-scale IMTA systems have already been established in other areas of
the globe. In Canada blue mussels Mytilus edulis and kelp Saccharina litissima and
Alaria esculenta are grown adjacent to Atlantic salmon S. salar (Neori et al., 2007; Reid


19

et al., 2009; Ridler et al., 2007). In China, S. japonicus grew well when co-cultured with
scallops Chlamys farreri and Argopecten irradians and Pacific oysters C. gigas in both
closed and open systems (Zhou et al., 2006). Shrimp are also commonly raised in coculture with sea cucumbers (Martinez-Porchas et al., 2010; Yaqing et al., 2004). In New
Zealand, A. mollis had high survival and growth rates when cultured below the greenlipped mussels Perna canaliculus (Slater and Carton, 2007).
The Effect of Temperature on Sea Cucumber Larvae
One of the most important variables for hatcheries to control during larval culture
is seawater temperature. Echinoderms display a high sensitivity to thermal stress
(Przeslawski, 2015). Sea cucumbers are ectothermic and they depend on surrounding
seawater temperature in regulate their internal temperature, which in turn, controls most
of the biochemical and physiological processes (An et al., 2007). Most species can
tolerate a relatively wide ambient temperature range within their natural habitat, although
optimal performance is typically restricted to a specific thermal range (Meng et al., 2009;
Pawson, 1966). The range varies between species life stages, but typically broadens with
development (Costlow et al., 1960; Crisp and Ritz, 1967).
Early life stages are the most sensitive to environmental conditions, and
experience the most damage from temperature extremes (Strathmann and McEuen,
1987). The developmental rates of marine invertebrates generally increase with
temperature until a lethal maximum is approached (Strathmann and McEuen, 1987).
Studies have shown that echinoderm larvae show increased mortality, delayed
development, reduced growth, and reduced metabolic rate in response to temperature
stress (Bressen et al., 1995; Przeslawski 2005; Roller and Stickle, 1989, 1993). This is
hypothesized to be due to multiple factors including but not limited to: oxidative stress,
lysosomal destablilisation, increased lipid peroxidation, a change in immune ability, and
the decrease in concentrations of soluble proteins, soluble sugars and ions (Chang et al.,
2006; Deschaseaux et al., 2010; Deschaseaux et al., 2011; Liu et al., 2013; Wang et al.,
2012). A study by McEdward (1985) suggests that larvae cannot increase their feeding
capacity at high temperatures to meet the energy demands of the increased metabolism
for the accelerated development that occurs at higher temperatures thus, leading to
deformation and mortality.
Studies have examined the effect of temperature on the survival and growth of
other species of sea cucumbers. A. japonicas and H. spinifera larvae rapidly developed at
higher temperatures and slower with delayed metamorphosis at lower temperatures (Asha
and Muthiah, 2005; Li Li et al., 2011). The extended larval phase exposes organisms to
the hazards present in planktonic life (Davis & Calabrese, 1969; Garcia de Severyn,
2000, Stickney 1964). Hamel and Mercier (1996) discovered for the sea cucumber
Cucumania Grandosa, lower temperatures increased the duration between developmental


20

stages, but did not increase mortality at each developmental stage. Liu et al. (2010)
observed that larvae grew faster with increasing temperatures in suitable range, but
extreme temperatures restrained the development of larvae in A. japonicas. High
temperatures resulted in weak-digestion, decreased-absorbing, increasing of oxygen
consumption rate and ammonia excretion rate (Villarreal, 1993). Li Li et al. (2011) found
that thermal stress affected mid-auricularia A. japonicas larvae more than early and late
auricularia, and the metamorphic period between the auricularia and the doliolaria stage
had the highest tolerance to temperature and salinity changes. The low survival of midauricularia larvae is due to their poor ability to adjust to physiologically in response to
changes in environmental conditions (Kashenko, 2002). In order to grow P. californicus
at a large-scale in hatcheries it is imperative to know the effects of temperature on
survival, growth, and development in order to determine the ideal temperature range that
larvae should be reared at.
METHODS
This study was conducted in collaboration with Puget Sound Restoration Fund
(PSRF) at the Kenneth K. Chew Center for Shellfish Research and Restoration hatchery
located at National Oceanic and Atmospheric Administration’s (NOAA) Manchester
Research Station in Manchester, WA.
Broodstock

Figure 9. Map of Washington with Clam Bay indicated by the red marker.

Divers collected 74 large adult P. californicus from Clam Bay (47.5712079°,
-122.5481897°) on March 12, 2016 (Figure 9). Four additional adults were also collected


21

on May 14, 2016. Divers targeted larger individuals in the wild in order to collect adults
that were most likely to be sexually mature and ready to spawn. The sea cucumbers were
placed in 5 gallon buckets and transferred to the hatchery within 1 hour of harvest. The
sea cucumbers were then housed in outdoor 4200 L tanks on a flow through system with
ambient seawater filtered to 5 µm. The tanks were lightly aerated with oxygen distributed
through a manifold built from PVC pipe and placed on the bottom on the tank. Cylinder
blocks and macroalgae were also placed inside the tanks. The sea cucumbers were
observed to accumulate mostly in the cylinder blocks and on the tank walls. Half of the
tank lid was left open each day in order to allow the broodstock to be exposed to sunlight
in case photoperiod affects gonad development and reproductive conditioning, and to
allow natural algae to grow as a secondary food source (Figure 10).

Figure 10. Photograph of outside broodstock holding tank. Photo Credit: Kendra Baird.



22

The waste from the sea cucumbers was syphoned out once a week, and algae was
only scrubbed off the tank once it became noticeably thick. The broodstock were fed
Otohime B1 and C2 fish diet but displayed a preference for the algae that grew naturally
on the tank walls. Evisceration was rare, but organs were removed from the tank as soon
as they were observed in order to limit stress on other individuals. Once an individual
eviscerates they are not able to spawn for the remainder of the season and must wait a
year to regenerate their gonads.
Spawning
Agudo et al (2016) was referenced for methods to induce spawning. The dates for
spawning were chosen within a few days of a new moon because divers had previously
observed P. californicus spawning in the wild around a new moon. When we attempted
to spawn we brought in 29-30 individuals from the outside holding tanks and placed them
inside a clean 660 L tote (Figure 11). We used a combination of many different methods
to stimulate spawning. For thermal stimulation, the seawater was heated to 16-19°C
before adding the broodstock to the spawning tote. For food stimulation, we added
combinations of Pavlova pinguis, Chaetoceros muelleri, and Chaetoceros sp. to the water
until it was visibly cloudy (Figure 12).



23

Figure 11. Photograph of broodstock inside the spawning tote during a spawning attempt. Photo
Credit: Kendra Baird.



24

Figure 12. Photograph of broodstock in the spawning tank with algae in the water to stimulate
spawning. Photo Credit: Kendra Baird.

Our attempts to spawn on 05/19/2016 and 05/20/2016 using thermal stimulation
and algae only resulted in 3 and 5 males spawning, respectively. We used different
individuals on the 20th than previously used on 19th. On 06/01/2016 Fish Biologist Rick
Goetz from NOAA assisted with an ultrasound on two individuals (Figure 13). We
attempted to sex the individuals and assess gonad development. However, we were
unable to identify the organs through ultrasound. A male was dissected and sperm was
extracted. The testes were observed to be very full, which led us to believe that we were
at the peak of the spawning season. However, we attempted another spawn using thermal
and food stimulation in addition to adding the extracted sperm to the water, but still only
4 males spawned. Eviscerated eggs were collected from the out side holding tanks and we
attempted to strip spawn using the eviscerated eggs and the extracted sperm from the
previous dissection. The sperm was diluted and combined with the eggs and the fertilized
eggs were carefully rinsed with seawater to wash off excess sperm. After two hours the



25

fertilized eggs were counted and examined. Only 0.4% had been fertilized and there was
a 0% survival.

Figure 13. Photograph of NOAA biologist Rick Goetz assisted with an ultrasound to determine
gonad development. Photo Credit: Kendra Baird.

Concerned that thermal shock and the addition of algae were not drastic enough to
induce spawning, additional spawning methods were implemented in hopes of getting
females to spawn. For the spawn on 06/17/2016, individuals were weighed and only
those over 782 grams were used for spawning, as the literature suggested these
individuals should be at least 4 years old and therefore sexually mature (Figure 14, Table
2). A fluorescent light was also shone on them for 24 hours prior to spawning in addition
to thermal and food stimulation (Figure 15). Only one male spawned during this attempt.
On 06/20/2016 the spawning totes were drained and the broodstock was left out of the
water and dried for 30 minutes before being lightly sprayed with a chemical sprayer, and



26

the tank refilled with 16°C seawater and algae added (Figure 16). No sea cucumbers
spawned during this attempt.

Figure 14. Photograph of a sea cucumber being weighed on a scale. Photo Credit: Kendra Baird.

Number
1
2
3
4
5
6
7
8
9



Weight (grams)
169.4
199.1
285.2
330.5
338.4
346.3
347.7
363.2
375.3

Date Collected
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
27

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48



383.0
415.2
418.9
419.7
421.8
422.8
436.4
439.3
453.1
476.5
484.0
526.0
528.8
533.3
543.5
546.7
570.3
578.3
587.6
593.1
611.6
617.7
621.6
622.4
628.3
658.0
658.8
659.1
661.8
666.1
673.3
673.5
683.1
688.7
700.0
723.5
745.3
758.6
769.2

3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
6/14/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
28

49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78

771.6
783.1
787.6
791.4
820.6
829.5
834.0
843.7
867.0
871.4
898.8
954.3
971.1
973.8
985.2
1,033.4
1,088.8
1,105.7
1,106.8
1,122.8
1,152.2
1,202.3
1,220.1
1,304.6
1,305.8
1,325.5
1,390.0
1,816.6
2,028.9
2,110.1

3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
6/14/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
3/12/16
6/14/16
3/12/16
6/14/16

Table 2. Table of the weights of the broodstock collected. Bold indicates the 29 individuals that
were used for spawning on 6/17/16.



29

Figure 15. Photograph of fluorescent light being shone on the broodstock in the spawning tote for
24 hours prior to spawning attempt. Photo Credit: Kendra Baird.



30

Figure 16. Photograph of the broodstock being dried for 30 minutes during a spawning attempt to
induce spawning. Photo Credit: Kendra Baird.

On 07/15/2016 the heated water line set at 16°C was turned on to one of the
outside holding tanks and the broodstock began to spawn. A single female began
releasing oocytes. Males in the holding tank next to it also began spawning, even though
they were on separate systems and on ambient water. Individuals were collected from the
outside tanks and brought into the hatchery and placed inside the spawning tank filled
with 16° C seawater. 5 males and 1 female spawned. The fertilized eggs were carefully
syphoned out of the tank and collected onto a 48 µm submerged screen (Figure 17). The
fertilized eggs were carefully rinsed with seawater to remove excess sperm to prevent
polyspermy. The eggs were carefully collected into a tri-pour beaker, homogenously
mixed, and sub sampled to determine the density and total count. PSRF conducted two
more spawns on August 4, 2016 and September 7, 2016, and were able to get successful
spawns with only a temperature stimulation with the seawater temperature increased to



31

20°C. Males were noted to spawn first, and females begun to release eggs within an hour
after the first male began to spawn. When sea cucumbers spawned they maintained a
unique posture, with their anterior end raised in the water column and the head curled
forward parallel to the surface of the tank (Figure 18). Sperm was thick and milky white
and easily distinguished in situ. Eggs were slightly orange, appeared granular, and
dissipated quickly making it difficult to notice the release. Females and males
occasionally swayed their bodies when releasing gametes. Table 3 contains a summary of
all the spawning attempts conducted during this study.

Figure 17. Photograph of eggs being syphoned out of the tank after being released by a female.
Photo Credit: Kendra Baird.



32

Figure 18. Photograph of a male spawning and displaying the typical posture seen while releasing
gametes. Photo Credit: Kendra Baird.



33

Time
0931-1600

Stimulatio
n
Thermal

05/20/16

0916-1245

Algae
Thermal

06/01/16

1010-1345

06/17/16

0900-1552

Date
05/19/16

Algae
Thermal
Algae
Gonad
Extraction
Weight

Treatment
19°C
Pavlova pinguis &
Chaetoceros muelleri
20°C
Chaetoceros muelleri &
Chaetoceros sp.
20°C
Chaetoceros muelleri
Extracted sperm was added to
the water
Individuals > 782 grams
Exposed to light for 24 hours
prior to spawning
20°C
Chaetoceros muelleri
For 30 minutes

♂
3

♀ Successful?
0 No

5

0 No

4

0 No

1

0 No

Light
Thermal
Algae
06/20/16 0945-1407 Desiccation
0
0 No
Water
Pressure
Sprayed lightly
Thermal
16°C
Algae
Chaetoceros muelleri
07/15/16 1640-1830 Thermal
16°C
5
1 Yes*
08/04/16 1430-1630 Thermal
20°C
5+ 3 Yes
09/07/15 1330-1600 Thermal
20°C
3
1 Yes
Table 3. Table of spawning attempts. The date, time of day, stimulation used, number of males
and females that spawned, and if the spawn was successful is noted. * indicates the spawn used to
inoculate the experiment.

Experiment: The Effect of Temperature on Larval Survival and Development
System
This experiment was comprised of five different water baths held at five different
temperature treatments (12°, 15°, 18°, 21°, and 24°C). The range was chosen to replicate
the temperature range that a hatchery would be able to chill and heat to. Teco TK500
chillers were set with a temperature error range of +/- 0.01°C. Each system included two
connected 39" x 18.5" x 11.5" totes filled with freshwater to maintain a constant
temperature, and an external 500 gph Danner Mag Drive pump was connected with a
hose and PVC pipe to circulate water through the system (Figure 19). There were six
replicates for each temperature treatment, each consisting of 4L containers. In addition,
there were three 2 gallon buckets, which held clean saltwater for the future water changes
heated or chilled to the same temperature as the replicates, per system. The replicate
containers were filled with 3L of UV sterilized and 1µm filtered seawater and to maintain
a constant temperature, the level of the outside freshwater bath was filled to the 2.5 L on
the out side of buckets to remain negatively buoyant. Each replicate was lightly aerated



34

with oxygen from an air stone. The totes, hoses, and pipes were insulted, and both totes
contained a Styrofoam lid to insulate temperature and ensure all systems on the top and
bottom of the tables had the same amount of light. One table held the 24°C water bath
system, and the other table held the 12°, 15°, 18°, and 21°C systems (Figure 19, 20, 21).

Figure 19. Photograph of the 24°C system without the lids. The first tote (left) connected directly
to the inflow from the Teco TK500 chiller and contained the six experimental replicates. The
second tote (right) contained three 2 gallon buckets filled with clean water for future water
changes. The freshwater flowed out from the right tote the external pump (pictured in the bottom
center), which pumped directly back to the chiller. Photo Credit: Kendra Baird.

Figure 20. A photograph of the table that contained four of the systems. The 12°C system is on
the bottom shelf and the 15°C system is on the top shelf. Photo Credit: Kendra Baird.



35

Figure 21. Photograph of the two tables that contained all of the systems. The bottom left corner
is the 12°C system, the upper left corner is the 15°C system, the middle upper system is the 21°C
system, the bottom middle is the 18°C system (not pictured, behind the larval sink), and the 24°C
water bath is on the top of the second table on the right. Photo Credit: Kendra Baird.

Inoculation
Fertilized eggs from the July 15, 2016 spawn were used. The spawn was a cross
between one female and five males. Each replicate for each treatment was inoculated
with 3,000 fertilized eggs for a stocking density of one individual per milliliter on
7/15/16. All five water baths were held at 16°C until the embryos hatched into the fist
larval stage auricularia on July 18, 2016 (day three post fertilization) to ensure that
temperature did not affect hatching rate or survival to hatchment. The water was not
changed and the larvae were not fed until the experiment started on July 18, 2016.
Larval Maintenance
Once the experiment began on 7/18/16 The larvae were fed either C. muelleri or
Chaetoceros calcitrans, which ever was available, and Pavlova sp. at a density of 30,000
cells per ml every other day after water changes. During water changes the larval sink
was plugged and filled about an inch with water from the 2 gallon buckets held in the
same water bath as the replicates, in order to keep the temperature that the larvae were
exposed to consistent throughout the experiment and to limit stress on the organisms.
Once the water level was too high in the sink after a couple of water changes, the sink
was drained slightly.
During water changes, the larvae were carefully poured from the containers onto
the submerged 48-60 µm screen. The smallest available mesh size was used to relieve the
pressure on the gelatinous larvae, when held on the screen. The bucket was back rinsed to
ensure all larvae were emptied from the bucket and were not stuck on the side of the dry


36

container. Water changes were a time intensive and careful process. The larvae were
never pulled directly out of the water, to make sure they were not squished against the
screen. As the screen was slowly lifted, the larvae were carefully rinsed with a squirt
bottle to the water line until all the larvae were accumulated to one section of the screen.
If samples were being taken, the larvae from the screen were poured into a 250 ml cup for
subsampling before being returned to the cleaned container. Each container was scrubbed
with the industrial chemical Vortex to kill bacteria and pathogens and then rinsed with
freshwater, before being refilled with 3 L of clean seawater. Each 2-gallon bucket was
also treated with the cleaning agent Vortex and rinsed with freshwater before being
refilled and returned to the water bath. The screens were sprayed with freshwater between
each replicate and treated with Vortex between each temperature treatment.
Sampling
Samples were taken on days 0, 5, 11, 17, and 24. Samples were taken once a week
and the experiment continued until competent pentactula larvae were observed. The
experiment was concluded on day 24 to ensure that larvae were not settling on the
containers of the higher temperatures, as they wouldn’t be countable. Larvae were
concentrated into a 100 ml volume, homogenized with a home made larval plunger, and a
6 ml subsample was collected from each replicate, for a minimum of 100 individuals to
be counted. If samples were not counted the same day as collected, they were fixed in
Lugol’s iodine solution for counting and scoring the following day. Larvae were
observed under a Zeiss Srerri DV4 dissecting scope.
Survival was determined by comparing the number of survivors (i.e. present
larvae) to the initial number of larvae, and accounting for sampling loss. Development
was determined by scoring the developmental stage (e.g. auricularia, dolioloria,
pentactula) of each larva. Larvae were considered doliolaria when they shrunk into the
barrel shape and rings of cilia were observed. Pentactula was marked by the emergence
of a tube foot.
Statistical Analysis
Mean survival and percentage of larvae at each developmental stage were
calculated for each replicate container and used in subsequent statistical analyses. Oneway analysis of variance (ANOVA) was performed to examine the effects of temperature
on survival and development using the software JMP Pro 12 for Mac. Differences among
treatment means were determined by Tukey’s tests, the differences being considered
significant when P<0.05.



37

RESULTS
Survival
Out of all treatments, the coolest temperature generally had the highest number of
individuals surviving over the duration of the experiment. Further, for all treatments,
survivorship declined with time. Each replicate for each treatment was initially inoculated
with 3,000 individuals. On days 11, 17, and 24 the larvae reared at 12°C had the highest
survival of the treatments, with the mean number of individuals surviving being 1178,
897, and 435 individuals, respectively, declining with time (Table 4). The survival of the
larvae reared in 24°C was the highest of all treatments on day five (2175 individuals) but
then drastically decreased to 164 individuals on day 11 (which was the lowest value of all
treatments). Survival was negligible on days 17 and 24 (Table 4). With the exception of
day 5, higher survival over all time periods correlated with the colder the temperature
treatment, with the highest survival at 12°, 15°, 18°, 21° and 24°C respectively (Table 4,
Figure 22).
The effect of temperature on survival was statistically significant. The one-way
ANOVA on differences of survival of larvae reared in different temperatures indicated a
high level of significance on all of the sampling days; with p=0.0024 on day five and
p<0.0001 on days 11, 17, and 24 (Tables 5, 6, 7 and 8). Day 5 (F(4,25)=5.5661,
p=0.0024) pair-wise comparison test indicated a statistically significant difference
between the 18° and 24°C, and 21° and 24°C treatments (Table 5). Day 11
(F(4,25)=8.9448, p=0.0001) pair-wise comparison test indicated a statistically significant
difference between the 12° and 21°C, 12° and 24°C, 15° and 21°C, and 15° and 24°C
(Table 6), with the cooler temperature always having the highest number of surviving
individuals.
Day 17 (F(4,25)=22.3659, p=< .0001) pair-wise comparison test indicated a
statistically significant difference between the 12°C and all the other treatments, and
between 15° and 21°C, and 15° and 24°C (Table 7), with the cooler temperature always
having the highest number of surviving individuals. Day 24 (F(4,25)=15.0585 p<.0001)
pair-wise comparison test also indicated a statistically significant difference between the
12°C and all other treatments as well, and between the 15° and 24°C treatments (Table
8), also with the cooler temperature always having the highest number of surviving
individuals
Temperature
(°C)
12
15
18
21
24
5
1800 ± 84.8
1675 ± 189.4
1433.3 ± 153.9 1436.1 ± 157.6
2175 ± 110.0
11
1177.8 ± 108.2 966.7± 209.9
644.4 ± 73.7
352.8 ± 158.7
163.9 ± 108.9
17
897.2 ± 93.2
483.3 ± 106.2 261.1 ± 55.6
127.8 ± 68.7
Nil
24
434.5 ± 57.0
188.7 ± 48.4
131.7 ± 18.7
104.2 ± 51.8
Nil
Table 4. Mean (±S.E., n=6) survival of the number of individuals of P. californicus larvae
observed at different temperatures.
Days



38

Figure 22. Graph of the mean survival of P. californicus larvae at different temperatures.

Treatments
Between groups
Within Groups
Total
HSD
18
21

sum of squares
2261537
2539398.1
4800935.2

df
4
25
29

Mean square
565384
101576

F ratio
5.5661

P value
< 0.0024

24
0.0038
0.004

Table 5. ANOVA table on survival of P. californicus larvae on day 5 at different temperatures
with results of Tukey post-hoc HSD test.

Treatments
Between groups
Within Groups
Total
HSD
12
15

sum of squares
4217314.8
2946759.3
7164074.1

df
4
25
29

21
0.0028
0.0351

24
0.0002
0.0036

Mean square
1054329
117870

F ratio
8.9448

P value
< 0.0001

Table 6. ANOVA table on survival of P. californicus larvae on day 11 at different temperatures
with results of Tukey post-hoc HSD test.



39

Treatments
Between groups
Within Groups
Total
HSD
12
15

sum of squares
2981629.6
833194.4
3814824.1

df
4
25
29

Mean square
745407
33328

F ratio
22.3659

15

18
<.000
1

21

24

< .0001
0.0187

< .0001
0.0009

0.0049

P value
< 0.0001

Table 7. ANOVA table on survival of P. californicus larvae on day 17 at different temperatures
with results of Tukey post-hoc HSD test.

Treatments
Between groups
Within Groups
Total
HSD
12
15

sum of squares
623834.2
258921
882755.2

df
4
25
29

Mean square
155959
10357

F ratio
15.0585

15
0.0026

18
0.0002

21
< .0001

24
< .0001
0.0305

P value
< .0001

Table 8. ANOVA table on survival of P. californicus larvae on day 24 at different temperatures
with results of Tukey post-hoc HSD test.

Development
The effect of temperature on development was statistically significant. The 12°C
treatment had the highest mean percentage of the first larval stage of auricularia and the
lowest mean percentage of the third larval pentactula stage throughout the experiment
(Table 9). The larvae were first observed to metamorphose to dolioloria on day 11 in the
18°, 21°, and 24°C treatments (Figure 23, Table 9). The highest occurrence of dolioloria
was in the 18°C treatment, with a mean percentage of 29.4% (Table 9). The larvae were
observed to metamorphose into the final pentactula stage on day 17 in all the treatments,
with the highest occurrence in the 18°C treatment at a mean percentage of 59% (Figure
23, Table 9). On day 24 the highest mean percentage of larvae at the pentactula stage
correlated with the highest temperatures occurring at 98%, 85%, 80%, 74%, and 46% in
24°, 21°, 18°, 15°, and 12°C, respectively (Figure 25, Table 9).
The one-way ANOVA on differences of percentage of larvae at each
developmental stage in different temperatures indicated a high level of significance
between auricularia on day 11 with p=0.0176, between auricularia and pentactula on day
17 with p <.0001 for both, and between dolioloria on day 24 with p=0.0021 (Tables 10,
11, 12, and 13). Day 11 auricularia (ANOVA; F(4,25)=3.6621, p=0.0176) pair-wise
comparison test indicated a significant different between the 12° and 21°C treatments,
and between the15° and 21°C treatments (Table 10). Day 17 auricularia (ANOVA;


40

F(4,25)=15.3509, p<.0001) pair-wise comparison test indicated a statistically significant
difference between 12°C and all other temperature treatments, and between 15° and 24°C
(Table 11). Day 17 pentactula (ANOVA; F(4,25)=14.157, p<.0001) pair-wise
comparison test indicated a statistically significant difference between 15° and 12°C,
between 15° and 24°C, between 18° and 12°C, between 18° and 21°C, and between 18°
and 24°C treatments (Table 12). Day 24 auricularia (ANOVA; F(4,25)=2.6162,
p=0.0593) pair-wise comparison test indicated a statistically significant difference
between the 12° and 24°C treatments (Table 13).
100%
90%
Percent of Larvae

80%
70%
60%
50%

Pentacula

40%

Doliolaria

30%

Auricularia

20%
10%
0%
12

15

18

21

24

Temperature (C°)

Figure 23. Stacked bar graph of the mean percentage of larvae at each developmental stage reared
in different temperatures on day 11.



41

100%
80%
70%
60%



Percent of Larvae

90%

50%

Pentacula

40%

Doliolaria

30%

Auricularia

20%
10%
0%
12

15

18

21

24

Temperature (C)

Figure 24. Stacked bar graph of the mean percentage of larvae at each developmental stage reared
in different temperatures on day 17. *There were not enough live larvae on day 17 to get a count.
100%
90%
Percent of Larvae

80%
70%
60%
50%

Pentacula

40%

Doliolaria

30%

Auricularia

20%
10%
0%
12

15

18

21

24

Temperature (C)

Figure 25. Stacked bar graph of the mean percentage of larvae at each developmental stage reared
in different temperatures on day 24.



42

Days

Developmental
Stage

Temperature (°C)
12
15
18
21
24
5
Auricularia
100±0
100±0
100±0
100±0
100±0
Doliolaria
0±0
0±0
0±0
0±0
0±0
Pentacula
0±0
0±0
0±0
0±0
0±0
11
Auricularia
100±0
100±0
86±4.4
70.6±16.7 98.1±20.7
Doliolaria
0±0
0±0
14±04.4
29.4±9.8
1.9±1.6
Pentacula
0±0
0±0
0±0
0±0
0±0
17
Auricularia
93.6±1.3
48.8±11.9 22.8±2.3
36.7±16.2 0±0
Doliolaria
5.6±1.6
15±4.5
18.1±3.9
38.9±16.2 0±0
Pentacula
0.84±0.6
36.1±1.6
59±4
24.3±10.3 0±0
24
Auricularia
29.9±3.6
18.2±5.6
9±1.3
14.9±13
1.9±1.3
Doliolaria
24.1±7
7.8±3.6
11.1±4.9
0.1±.1
0±0
Pentacula
46±6.5
74±9.1
80±5.2
85±13
98±20.7
Table 9. Mean (±S.E., n=6) percentage of P. californicus larvae at each developmental stage at
different temperatures.

Treatments
Between groups
Within Groups
Total
HSD
12
15

sum of squares
12804.48
21853.192
34657.672

df
4
25
29

Mean square
3201.12
874.13

F ratio
3.6621

P value
0.0176

21
0.0348
0.0348

Table 10. ANOVA table of the percentage of P. californicus auricularia larvae on day 11 at
different temperatures with results of Tukey post-hoc HSD test.

Treatments
Between groups
Within Groups
Total
HSD
12
15

sum of squares
30374.181
12366.627
42740.807

df
4
25
29

15
0.0145

18
<.0001

Mean
square
7593.55
494.67

21
0.0001

F ratio
15.3509

P
value
<.0001

24
<.0001
0.0067

Table 11. ANOVA table of the percentage of P. californicus auricularia larvae on day 17 at
different temperatures with results of Tukey post-hoc HSD test.



43

Treatments
Between groups
Within Groups
Total
HSD
15
18

sum of squares
15218.427
6718.594
21937.021

df
4
25
29

12
0.0081
<.0001

21

Mean square
3804.61
268.74

F ratio
14.157

P value
<.0001

24
0.0065
0.0011 <.0001

Table 12. ANOVA table of the percentage of P. californicus pentactula larvae on day 17 at
different temperatures with results of Tukey post-hoc HSD test.

Treatments
Between groups
Within Groups
Total
HSD
12

sum of squares
2356.0536
2586.2621
4942.3157

df
4
25
29

21
0.0033

24
0.0032

Mean square F ratio
589.014
5.6937
103.45

P value
0.0021

Table 13. ANOVA table of the percentage of P. californicus pentactula larvae on day 24 at
different temperatures with results of Tukey post-hoc HSD test.

DISCUSSION
Larval development of sea cucumbers has been extensively described in previous
studies (Dautov 1997; Hamel and Mercier, 1996; Mashanov and Dolmatov, 2000;
McEuen and Chia, 1991; Morgan, 2008a; Smiley, 1986; Strathmann, 1971). The effect of
egg source, fertilization, food availability, diets, and environmental conditions on larval
survival, growth, and development have been examined for select species globally (Asha
and Muthiah 2006; Kashenko 1998, 2002; Martinez and Richmond 1998; Morgan, 2001,
2008, 2009b, 2009c; Ren et al 2016). However, despite all of the available information,
there is still little information directly pertaining to sea cucumber aquaculture techniques.
Furthermore, most studies have focused tropical sea cucumber species and there is little
information regarding ideal environmental parameters for rearing P. californicus in
hatcheries. Previous studies found that the developmental rates of marine invertebrates
generally increase with temperature until a lethal maximum is approached (Strathmann
and McEuen, 1987). Echinoderm larvae show increased mortality, delayed development,
reduced growth, and reduced metabolic rate in response to temperature stress (Bressen et
al., 1995; Przeslawski 2005; Roller and Stickle, 1989, 1993). The optimal thermal range
for rearing larvae in a hatchery has been determined for a few other tropical species
globally, however, there has not been any studies conducted to determine the optimal
thermal range to rear P. californicus larvae in hatcheries. It will be imperative to develop


44

reliable spawning techniques in order for hatcheries to have access to large number of
gametes. Developing reliable spawning methods and determining the optimal thermal
range for rearing larvae at in a hatchery will further advance the development of
aquaculture techniques for P. californicus.
Spawning
The first step of developing hatchery techniques for P. californicus is to
determine reliable methods for inducing spawning in order for hatcheries to have access
to large number of gametes and subsequently larvae. Methods for inducing spawning in
sea cucumbers have been developed for many species globally, however, spawning
methods for P. californicus are still in the early development stage and further studies
need to be conducted in order to develop more reliable methods.
Spawning became a bottleneck for this experiment. Per our communication with
Alutiiq Pride Shellfish Hatchery, Lummi Tribe, and after reviewing published studies
conducted by Cameron and Fankboner (1986) in the San Juan Islands and Alaska, we
expected our broodstock to spawn much earlier in the season from April to August, with
the peak of the spawning occurring May to mid-July. When we attempted to spawn our
broodstock collected from Clam Bay, WA, at the hatchery from mid-May to early
September we did not have a successful spawn until July 15, 2016, and from that point
forward we had success until our last spawn attempt on September 7, 2016, suggesting
our broodstock had a later spawning window than observed in previous studies in the San
Juan Islands and Alaska. Our broodstock was collected from a different geographical
location much further south than the ones observed in previous studies. It is not
surprising that our population has a different spawning season window, due to the
difference in physical parameters such as location, seawater temperature, salinity, food
availability, and seawater chemistry.
The amount of stimulation and spawning methods used did not appear to effect
spawning success, as all successful spawns only required thermal shock, indicating that
time of season is more important than method and degree of stimulation used for
inducing spawning. Alutiiq Pride Shellfish Hatchery only used thermal stimulation for
successfully inducing spawning. When we did not have a successful spawn from midMay to early July, we used other methods in hopes of triggering females to release
oocytes. Along with thermal shock, we also tried the addition of algae, gonad extraction,
light stimulation, desiccation, water pressure, and using larger individuals without
success. The successful spawns on July 15, August 4, and September 7 only required
thermal stimulation.
Experiment
Temperature is an important environmental parameter that hatcheries must control
during larval rearing because it strongly affects the survival and metamorphosis rate for



45

each stage of sea cucumber larvae (Ito and Kitamura, 1998; McGurk, 1984). Studies have
indicated that thermal stress results in increased mortality, delayed development, and
reduction in growth (Bressen et al., 1995; Przeslawski 2005; Roller and Stickle, 1989,
1993). The study by McEdward (1985) suggests that larvae cannot increase their feeding
capacity at high temperatures to meet the energy demands of the increased metabolism
for the accelerated development that occurs at higher temperatures thus, leading to
deformation and mortality.
Survival
In this study the larvae reared at the highest temperature (24°C) had the highest
survival of all treatments initially on day 5 (73%), but survival dramatically declined by
day 11 (5%), which resulted in decreased survival directly correlating with increased
temperature for the remainder of the experiment. The initially high survival in the 24°C
treatment could be explained by pathogens initially dying off. Larvae are extremely
sensitive to pathogens present in culture tanks. Higher temperatures result in the
increased growth of potentially pathogenic ciliates and bacteria in cultures, which leads
to the proliferation of invaders and fouling organisms that bring their own microflora and
disease problems (Cook et al., 2005; Gruffydd and Beaumont, 1972; His et al, 1989;
Olafsen, 2001). Typically, pathogens flourish in heated conditions and have a higher
transmission rate between shellfish larvae in heated conditions (Dorfmeier et al. 2011). If
the temperature range exceeds the thermal threshold of the pathogen, then larvae
wouldn’t have to fight off infection. The 24°C treatment could have killed off the
pathogens initially, resulting in the highest survival of the temperature treatments.
However, the larvae were not able to keep up with the metabolic demands of the heated
conditions and quickly declined in survival by day 11. It is important to note that data
was not collected on pathogens, and this theory is speculative, but is consistent with the
literature and findings from other shellfish researchers.
Larvae reared at 12°C had the highest survival of all the treatments after day 5
with a 14% survival at the end of the experiment on day 24, which was significantly
higher than the other treatments, leading us to conclude that hatcheries should rear larvae
at a maximum temperature of 12°C for optimal production. This temperature corresponds
to the average water temperature of 12.7-13.33°C for July-September, the time of
spawning in the Seattle, WA area, as reported by NOAA in the Water Temperature of All
Coastal Regions database. This indicates that hatcheries using ambient water pumped in
from the sound may be able to rear larvae during the spawning season at 12°C without
having to heat or cool the water.
Our findings agree with previous studies, which found that increased thermal
stress results in lower sea cucumber larval survival. Too high or too low temperature
outside of the threshold range results in low survival and delayed metamorphosis (Asha
and Muthia, 2005; Hamel and Mercier, 2005; Kashenko, 1998; Sui, 1990). All previous



46

temperature studies on larval survival, growth, and development have focused on tropical
species and mostly on the early auricularia stage. Li Li et al (2011) found that 21-24°C
was optimal for the early development of the tropical species A. japonicas. Asha and
Muthia (2005) had similar results with the tropical species H. spinifera and determined
the ideal temperature range that larvae should be reared at is 28-31°C, which is on the
higher end of the temperature range of their natural environment (25.5-31.2°C).
Development:
The assessment of larval development during rearing is critical for determining
the competence of sea cucumbers to successfully complete the larval cycle and to reach
settlement (Morgan 2001, 2008b, 2009b, 2009c). My findings agreed with previous larval
studies, which found that, thermal stress affects the metamorphosis and development of
sea cucumber larvae (Sui, 1990). Sea cucumber larvae develop rapidly at high
temperatures but display slower growth and delayed metamorphosis at lower
temperatures as seen in other cultured species (Asha and Muthiah, 2005; Ito et al, 1998;
Li Li et al, 2011; Liu et al, 2010; Pan et al., 1997; Sui, 1990). The extension of the larval
period requires hatcheries to use more time and resources to care for the delicate life
phase.
In this experiment, decreased temperatures resulted in delayed metamorphosis.
The lowest temperature treatment (12°C) had the highest mean percentage of auricularia
larvae and the lowest mean percentage of pentactula throughout the experiment. The
larvae were first observed to metamorphose into the second larval phase of dolioloria on
day 11 in the treatments of 18°, 21°, and 24°C. The final larval phase pentactula was first
observed on day 17, with the highest occurrence in the 18°C treatment at a mean
percentage of 59%. On day 24 there was a statistically significant difference between the
coldest treatment (12°C) and the highest treatment (24°C), with the lowest percentage of
pentactula occurring in the 12°C. treatment.
Similarly, Hamel and Mercier (1996), Ito and Kitamura (1998), and Li Li et al.
(2011) also reported rapid development of C. frondosa, I. japonicas, and H. spinifera
larvae, respectively, at higher temperatures than the prevailing natural environmental
conditions for the species. Li Li et al (2011) found higher survival, growth, and
metamorphosis occurred at 21° and 24°C for A. japonicas larvae, and no metamorphosis
to doliolaria were observed in the lowest temperature treatment of 18°C. Asha and
Muthiah (2015) determined that water temperatures of 28°C would be optimal for the
normal growth and development of H. spinifera auricularia larvae.
Larval culture of P. californicus is currently hindered by poor survival to
metamorphosis, and this was evident in this study. The overall survival rates of this study
were significantly lower than previous studies conducted on sea cucumber larvae.
Previous studies focused on tropical species and only focused on the early auricularia and
doliolaria stages. Ren et al (2016) evaluated the effect of microalgal diets on body length,



47

survival, and metamorphosis of P. californicus auricularia larvae. At day 16 the highest
survival was 62.4% and the lowest was 38.7% found in the unfed treatment, which was
higher than the highest survival of 30% on day 17 of this experiment. Low survival could
be due to the gap in knowledge of other parameters such as salinity, stocking density,
photoperiod, and the limited genetic variation, as the experiment was inoculated with the
cross of five males and a single female.
In conclusion, P. californicus only need thermal stimulation to induce spawning
when females are ripe. Our broodstock collected from Clam Bay, WA appears to have a
spawning season ranging from mid-July to mid-September. The findings from this
experiment agree with previous studies that found that temperature stress results in
increased mortality and delayed development. Our findings indicate that hatcheries
should rear P. californicus larvae at a maximum of 12°C for optimal survival and
production. However, more studies should be conducted to determine the lower thermal
threshold, which may be lower than 12°C. Rearing larvae at 12°C will require hatcheries
to use more resources to care for the larvae during the extended larval phase. 12°C is
within the ambient temperature range of Seattle area and should be feasible for many
small hatcheries to maintain while rearing larvae in July-September.
This experiment suggests that the development of aquaculture of P. californicus is
possible, but further studies need to be conducted to better understand broodstock
conditioning, spawning, larval culturing, and juvenile grow out. Further studies need to
focus on determining the optimal conditions for larval rearing such as salinity, stocking
density, types of culture tanks, DO, diets, and other physical characteristics to increase
the survival rates of larvae in order for aquaculture to be a reasonable option for
hatcheries. The development of aquaculture of P. californicus would relieve pressure on
wild stocks in Washington State and allow PSRF and other restoration work to be carried
out in Puget Sound. Once hatcheries are able to produce large numbers of juveniles, then
they can out plant them to the populations that have been greatly reduced by overfishing.
I would expect broodstock collected in other areas to have varying spawning seasons, as
seen with our broodstock. I would also expect that larvae should have similar optimal
thermal ranges, but future studies should determine if 12°C is also optimal for larvae
spawned from broodstock collected in other areas.



48

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