BRIEF RESEARCH REPORT
published: 22 June 2021
doi: 10.3389/fmars.2021.684989

Predation of Juvenile Japanese Sea
Cucumber Apostichopus japonicus
by Kelp Crab Pugettia ferox
Nobuharu Inaba 1* , Takuma Matsumoto 2 , Hiroshi Kawai 1 , Yuji Anaguchi 3 andKohei Matsuno 2,41

Civil Engineering Research Institute for Cold Region, Public Works Research Institute, Sapporo, Japan, 2 Faculty/Graduate
School of Fisheries Sciences, Hokkaido University, Hakodate, Japan, 3 Ocean Construction Co., Ltd., Kurashiki, Japan,
4
Arctic Research Center, Hokkaido University, Sapporo, Japan

Edited by:
Libin Zhang,Institute of Oceanology (CAS), China
Reviewed by:
Qinzeng Xu,
First Institute of Oceanography,
Ministry of Natural Resources, China
Reiji Masuda,
Kyoto University, Japan*Correspondence:
Nobuharu Inaba
inaba-n@ceri.go.jp
Specialty section:
This article was submitted toMarine Fisheries, Aquacultureand Living Resources,
a section of the journal
Frontiers in Marine Science
Received: 24 March 2021
Accepted: 24 May 2021
Published: 22 June 2021
Citation:
Inaba N, Matsumoto T, Kawai H,
Anaguchi Y and Matsuno K (2021)
Predation of Juvenile Japanese Sea
Cucumber Apostichopus japonicus
by Kelp Crab Pugettia ferox.
Front. Mar. Sci. 8:684989.
doi: 10.3389/fmars.2021.684989

The predation of commercially important Japanese sea cucumber Apostichopus
japonicus by the kelp crab Pugettia ferox, widely distributed in coastal northeast Asia,
was examined in field sampling and laboratory experiments. The ossicles of A. japonicus
were detected from the stomach contents of a natural population of P. ferox at the rateof 32.4% of 68 individuals collected within artificial intermediate sea cucumber reefs
for releasing hatchery-produced juveniles in December 2018. In the following laboratory
experiments, a high mortality rate (7.7 ± 2.4 individuals day−1 ) of juvenile A. japonicus(15.35 ± 2.47 mm) was observed despite the different sizes and sex of P. ferox tested. It
was also confirmed that a maximum of five sea cucumbers was killed and cut into small
pieces within the first 2 h. Smaller and younger P. ferox individuals (adolescent) between
carapace widths of 14.2–17.8 mm actively decorated themselves using pieces ofchopped sea cucumber after feeding. Attached pieces of sea cucumber were observed
to be fully eaten within a week, suggesting a possible strategy by P. ferox of short-term
food storage as well as mimicry. This study demonstrates considerable evidence that
predation mortality by sufficiently mobile P. ferox on commercially important A. japonicus
can be significant, causing high mortality at the early life stage in the natural environment,
especially in areas releasing hatchery-produced juveniles. It is also worth noting that theutilization of freshly chopped sea cucumbers as decoration material and food storage isa unique and novel ecological trait of P. ferox.
Keywords: sea cucumber, Apostichopus japonicus, Pugettia ferox, predation, decorator crab

INTRODUCTION
The Japanese sea cucumber Apostichopus japonicus (Selenka, 1867), an Echinoderm of
the class Holothuroidea, is widely distributed along the coast of southern far-east Russia,
South Korea, northern China, Japan, and United States (Choo, 2008). A. japonicus isone of the common temperate sea cucumber species that has been harvested, traded,
and traditionally consumed as seafood and a tonic in south-east Asia for centuries(Akamine, 2015). Japan has exported sea cucumbers, including A. japonicus, to China asa major commodity at least for 350 years (Akamine, 2004). In particular, A. japonicus
harvested from Hokkaido, the northern island of Japan, is regarded as the highest
quality due to characteristics of a greater number, large lined-up warts, and thicker flesh

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Crab Predation on Apostichopus japonicus(PCL), and wet weight were measured as body size indexes(Figure 2). After the measurement, the stomach of each P. ferox
was dissected using a scalpel. The stomach contents were placed
into a microtube and dissolved in a sodium hypochlorite solution(NaClO, 5%). Then, microscopic observation using Axiovert 135(Zeiss, Germany) was performed to check whether the ossicles of
A. japonicus exist in the samples.

(Brown and Eddy, 2015). The declining wild population ofsea cucumber by overfishing, habitat loss, and human-induced
pollution have been described by Purcell et al. (2013), andA. japonicus was also added to the ICUN Red List of threatened
species as “Endangered” in the same year (Mercier and Hamel,2013). Although Japan has taken the initiative in conservation
measures such as regulating the total annual catch, harvest
size and adopting a closed season to promote reproduction,
the wild stocks of A. japonicus have declined at least 30%
in the past 30 years (Choo, 2008). The release of hatcheryproduced juveniles is promoted to enhance the wild populationof A. japonicus (Battaglene, 1999; Purcell, 2004). To rebuild thewild stocks around the coast of Hokkaido, the vigorous release ofjuveniles has been carried out (Hokkaido Government, 2020).
Predation is one of the determining factors that influence thebenthic invertebrate community structure in aquatic ecosystems(Bell et al., 2005). Numerous reports have documented that early
juveniles of invertebrates such as bivalves, decapod crustaceans,
and echinoderms, etc., are vulnerable to predation, and asignificant number of juveniles are assumed to be killed by
predators (Gosselin and Qian, 1997). Aquatic animals, including
marine mammals, sea birds, fish, asteroids, crustaceans, andgastropods, have also been documented to prey on holothurians(Francour, 1997). However, few studies have documented
the significance of predators on A. japonicus (Yu et al.,2015). The kelp crab Pugettia ferox, recently reclassified asa new species, is a much larger crab species than formerly
assigned Pugettia quadridens, which distributes more northward(Ohtsuchi and Kawamura, 2019). P. quadridens is known as anotorious predatory crab negatively affecting fishery resources
such as juveniles of Ezo abalone and sea urchins in Japan(Shibui, 1971; Kawai and Agatsuma, 1996; Hoshikawa, 2003).
On our study site, a remarkable increase of the P. ferox
population within artificial intermediate sea cucumber reefs
for releasing hatchery-produced juvenile A. japonicus has been
observed (Figures 1A–C), and the ossicles of A. japonicus
were detected continuously from the stomach contents of
P. ferox (Figure 1D) as well as active predation (Figure 1E
and Supplementary Movie 1). To our best knowledge, there
is no literature on quantitative predation on A. japonicus by
P. ferox. This report provides novel findings of predation
on A. japonicus juveniles by P. ferox through microscopic
observations of stomach contents in the natural population andpredation experiments.

Laboratory Predation Experiment
Pugettia ferox (n = 9, four Males, five Females) collected in thesame reefs mentioned above at the end of June 2019 were broughtback to the laboratory alive. P. ferox were kept individually inclear aquaria (31 cm length × 18 cm width) filled to a depth of
5 cm of seawater at 13 ± 1◦ C which is the local water temperatureof releasing A. japonicus juveniles, under a 12 h light/12 h darkcycle (12:12 LD) without feeding for a week before the predation
experiments. An aquaria with no P. ferox was also prepared ascontrol (C). CW, PCL, cheliped propodus length (CPL), andwet weight of P. ferox were measured as indexes of body size
(Figure 2 and Table 1). Body length, width, and wet weightof A. japonicus juveniles used as prey were also measured inadvance. All A. japonicus were immersed in L-menthol for 20–
30 min (Hatanaka and Tanimura, 1994), and the body length
and width were measured, then standard body length (SBL),
which is a widely used method for accurate measurement ofsea cucumbers, was estimated (Yamana et al., 2011). A digitalvernier caliper was used for all the measurements to thenearest 0.1 mm. A. japonicus juveniles used in this study werepurchased from Hokkaido Aquaculture Promotion Corporation.
Ten A. japonicus individuals were randomly selected and placed
into each aquaria for predation experiments. The average SBL
and wet weight of A. japonicus used were 15.35 ± 2.47 mm(Mean ± SD) varied from 10.82 to 19.99 mm, and 0.1 ± 0.04 gvaried from 0.03 to 0.23 g, respectively. A. japonicus werecounted after 12 h (the end of a light cycle) and 24 h (theend of a dark cycle). All the P. ferox decorated with a pieceof A. japonicus were observed continuously for another week
in the same aquaria after removing the remaining A. japonicus
and organic residuals. The densities of A. japonicus juveniles
per unit area adopted in the experiments were based on thedensities observed in the field. The depth of seawater in theaquaria applied was to eliminate the inaccessibility due tovertical attachment by A. japonicus during the experiments. The
experiments mentioned above were repeated three times andthen preserved in 70% ethanol to examine the ontogenetic stages(Ohtsuchi and Kawamura, 2019).

MATERIALS AND METHODS

Statistical Analysis

Field Sampling and Observation of
Stomach Content

The differences in body size indexes of CW, PCL, and wet weight
between the ossicle-detected and not-detected groups from thefield sampling and mortality rate between day and night, maleand female, and ontogenetic stages (adolescent and full-grown)in the predation experiments were assessed by using a Student’s
t-test after confirming the population of two tested groups arenormally distributed with equal variance. The occurrence rate ofossicle-detected and not-detected was compared between maleand female using a chi-square test. Relationships between the

A total of 68 P. ferox were collected in December 2018 from
artificial intermediate sea cucumber reefs, deployed at a depthof 4–5 m within a fishing port (41.2744N, 140.1452E) locatedin southwestern Hokkaido, Japan. Samples were preserved in
70% ethanol immediately after sampling in the field for latermeasurements and stomach content observations. Maximum
carapace width (CW) and post-pseudorostral carapace length

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FIGURE 1 | (A) An artificial intermediate sea cucumber reef for releasing hatchery-produced juvenile Apostichopus japonicus. (B) Attached juvenile A. japonicus (on scallop shells as a substrate within the reef. (C) Observed Pugettia ferox ( ) within the same reef. (D) Ossicles of A. japonicus detected from the stomach
contents of P. ferox. (E) Active predation of juvenile A. japonicus by P. ferox. (F) Results of the laboratory predation experiments. An aquaria with no P. ferox wasused as a control (C). (G) Picture of A. japonicus killed by a female P. ferox within the first 2 h. (H) Decoration of P. ferox using pieces of chopped sea cucumber.

mortality rate and all four body size indexes (CW, PCL, CPL, andwet weight) were examined using correlation analyses (Pearson’s
correlation coefficient) to determine if two numeric variables

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are significantly linearly related. The statistical analyses were
performed using StatView statistical software (version 5.0) andR version 3.6.2.

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groups (p > 0.05), the average of wet weight between the ossicledetected and not-detected groups in male crabs (p < 0.05) weresignificantly different (Figure 3). Moreover, the ossicles weredetected more frequently in females (1:1.44) despite the sex ratioof male and female P. ferox examined stomach contents was
1:0.94. No statistical significance was seen in the occurrence rateof ossicle-detected and not-detected groups between male andfemale crabs (p > 0.05).

Mortality Rate and Predatory Behavior
The high mortality rate [7.7 ± 2.4 individuals day−1(Mean ± SD)] of juvenile A. japonicus was confirmed despite thevarious sizes and sexes of P. ferox beside no mortality in control(Figure 1F). Even though there were positive relationships
between the mortality rate and all four body size indexes (CW,
PCL, CPL, and wet weight) in the male crabs, no significant
linear correlation was detected from either male or female crabs(p > 0.05). All crabs actively searched for food right after thestart of experiments, and a maximum of five sea cucumbers was
confirmed to be killed by a female P. ferox and cut into small
pieces within the first 2 h (Figure 1G). Although tested P. ferox
were confirmed to forage for food during the daytime, thenumber of A. japonicus killed during the night was significantly
higher (p < 0.001). No statistical significance was confirmed in
mortality rate between male and female and ontogenetic stages(p > 0.05). During experiments, all of the smaller and younger
individuals (adolescent) between CWs of 14.2–17.8 mm actively
decorated themselves using chopped sea cucumber pieces during
and/or after feeding (Figure 1H). However, attached pieces ofsea cucumber were observed to be fully eaten within a week inall experiments.

DISCUSSIONFIGURE 2 | Maximum carapace width (CW), post-pseudorostral carapace
length (PCL), and cheliped propodus length (CPL) of P. ferox measured asbody size indexes.

In general, holothurians, including A. japonicus, are known tohave few predators (Francour, 1997), owing to wide-ranging
anti-predator behaviors, including detachment, body shapechange, shedding body parts, and toxicity, in exchange for
their slow-moving behavioral characteristics (Mosher, 1956;
Bakus, 1968; Kropp, 1982; Bingham and Braithwaite, 1986;
Morton, 1991; Aminin, 2016; Kamyab et al., 2020). Indeed, upon
attack by P. ferox, often observed antipredator behaviors by
A. japonicus were the contractions of their body and detachmentsfrom the attached surface in this study. The contraction of
A. japonicus is caused by external stimuli using catch connective
tissue, which controls the body wall’s stiffness for resisting
predators (Motokawa, 1984). Some studies in Holothuria scabra
and Stichopus horrens demonstrated successful escapes frompredators by detachment (Kropp, 1982; Morton, 1991). On theother hand, Dance et al. (2003) reported heavy predations byfish after releasing juveniles of tropical sea cucumber Holothuria
scabra to the wild, indicating the importance of evaluating impact
by predators. Carnivorous fish, crabs, gastropods, and sea stars
are suggested to be candidates as predators for A. japonicus
juveniles (Francour, 1997; Yu et al., 2015), and sea star Asterina
pectinifera is considered as the principal predator of A. japonicus

RESULTS
Measurement of P. ferox andA. japonicus Ossicle in Stomach Content
Averages of CW, PCL, and wet weight of the collected P. ferox
were 13.40 ± 5.40 mm (Mean ± SD) varied from 4.08 to
25.26 mm, 18.45 ± 6.34 mm varied from 6.42 to 34.71 mm, and1.73 ± 2.05 g varied from 0.03 to 9.42 g, respectively. The ossiclesof A. japonicus were detected from the stomach contents at a rateof 32.4%, and averages of CW, PCL, and wet weight of P. ferox
containing ossicles were 12.53 ± 3.00 mm varied from 6.80 to
20.33 mm, 18.10 ± 3.17 mm varied from 11.53 to 24.92 mm, and1.12 ± 0.85 g varied from 0.18 to 4.11 g, respectively. Although
there were no statistically significant differences in body size
indexes of CW, PCL in both male and female crabs, and wet
weight in females between the ossicle-detected and not-detected

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TABLE 1 | Measured carapace width at the widest part (CW), post-pseudorostral carapace length (PCL), an average of the right and left cheliped propodus length (CPL),
and wet weight of Pugettia ferox (n = 9, four Males, five Females) used in the laboratory predation experiments.
Sample ID

Sex

Ontogenetic stage

Wet weight (g)Carapace width(mm)

Post-pseudorostral
carapace length(mm)

Cheliped propodus
length (mm)8.3 ± 0.7

1

F (Female)

Adolescent

1.34

14.2

17.1

2

F

Adolescent

1.48

14.2

17.5

8.3 ± 0.15

3

M (Male)Adolescent

2.34

17.0

21.1

12.5 ± 1.35

4M

Adolescent

2.78

17.8

21.0

12.9 ± 0.05

5M

Full-grown

5.19

22.4

23.3

17.8 ± 0.1

6M

Full-grown

14.77

27.9

31.2

28.5 ± 0.2

7F

Full-grown

3.14

16.0

22.1

9.5 ± 0.3

8F

Full-grown

4.15

20.9

22.9

11.0 ± 0.4

9F

Full-grown

2.66

15.8

20.2

8.2 ± 0.05

FIGURE 3 | Box plot of wet weight between the ossicle-detected and not-detected groups in male and female P. ferox collected within artificial intermediate sea
cucumber reefs for releasing hatchery-produced juveniles in December 2018. An open circle ( ) represents the average wet weight. The top and the bottom line of
the box show the first and third quartiles, and the middle line is the median. The upper (lower) whisker displays the maximum (minimum) value. Asterisk (∗ ) indicatesstatistical significance (p < 0.05).

investigation of relatively unknown ecological information of
P. ferox may be crucial for the areas putting efforts into recovering
wild stocks of A. japonicus, and/or establishing efficient releasemethods of hatchery-produced A. japonicus juvenile.
In general, body size, ontogenetic stages, and sex are essential
determinants influencing the feeding behaviors of brachyuran
crabs (Kolts et al., 2013; Williner and Collins, 2013). For instance,larger males could consume larger prey, but female and smaller
male crabs may be more efficient feeders, depending on theirhandling ability and strength (Kolts et al., 2013; Tina et al., 2015).

juvenile, ingesting 1.8 individuals with an average length of
15.9 mm on average per day in laboratory experiments (Hatanaka
et al., 1994). In the present study, the calculated mortality
rate of 7.7 ± 2.4 A. japonicus individuals (15.35 ± 2.47 mm)
per day by P. ferox was over four times higher than Asterina
pectinifera, suggesting the importance of specifying potential
predators as well as evaluating their impact. Considering spatial
distribution and habitat overlap of the two species (Liu, 2015;
Ohtsuchi and Kawamura, 2019), the presented phenomenon may
be widespread in the natural environment. Thus, the future

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In the present study, the ossicles were detected more frequently
in females with a broader size range, and no ossicle was foundin male crabs over 15 mm in CW (ca. 1.56 g in wet weight).
Together with the high mortality rate of female crabs confirmedin laboratory study, these results indicate female P. ferox pose agreater risk on juvenile A. japonicus than males. Regarding thesize of sea cucumber, Purcell and Simutoga (2008) documented
that size at release significantly affected the survival of juvenile
sandfish Holothuria scabra by predation. The predation by
sea star Asterina pectinifera was substantially reduced when
A. japonicus juveniles with an average length of 30.1 mmcompared with an average length of 15.9 mm (Hatanaka et al.,1994). Therefore, sex and ontogeny-related feeding behaviors andthe predator-prey body size relationships may need to be carefully
investigated as a future task for a more accurate evaluation of the
impact on A. japonicus by P. ferox.
Pugettia ferox is referred to as decorator crab, using
their specialized setae to hook extraneous materials from theenvironment for use as camouflage (Ohtsuchi and Kawamura,2019). The functions of decoration camouflage are to reduce
the probability of detection, recognition, and consumption by
predators (Hultgren and Stachowicz, 2011). P. ferox utilize
various combinations of pieces of red and brown algae, branchedcolonies of bryozoans, or hydrozoans for their decorationmaterials (Ohtsuchi and Kawamura, 2019). In the present study,
all of the smaller and younger (adolescent) P. ferox actively usedchopped sea cucumber pieces to decorate themselves during
and/or after feeding (Figure 1H and Supplementary Movie 2).
Our observation was consistent with the decoration behavior ofother brachyuran crabs that juvenile decorated more frequently
than adult (Hultgren and Stachowicz, 2009; De Carvalho et al.,2016), explained with their susceptibility to predation and habitatchanges (Todd et al., 2009). There are also crabs known todecorate with noxious organisms, such as algae, sponges, orother invertebrates, to deter predators (Stachowicz and Hay,
1999). A. japonicus produce saponins, which act as a feeding
deterrent and/or warning effect to potential predators (Van
Dyck et al., 2011). Assuming that P. ferox exhibit the same
decorating behavior using A. japonicus in nature, the risk offeeding pressure by predators may be significantly reduced.
However, decorated pieces of sea cucumbers were fully eaten
within a week when no food was given thereafter. In this regard,
our observation was rather similar to the results presented byWoods and Mclay (1994) that the attached materials take a roleas short-term food storage and/or play multiple roles (Hay, 1992).
It is intriguing to deepen our knowledge on these novel andunique ecological traits of decoration in reaction to the presenceor absence of predators and food resources in order to understandadaptation and natural selection of P. ferox, as well as offering animportant perspective on A. japonicus fishery.

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AUTHOR CONTRIBUTIONSNI and HK conceived and designed the research. NI, TM, and YA
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ACKNOWLEDGMENTS
We express sincere gratitude to the members of Fisheries
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especially Sawako Shirai, a technical assistant. We would also
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Conflict of Interest: YA was employed by the company Ocean Construction
Co., Ltd.
The remaining authors declare that the research was conducted in the absence ofany commercial or financial relationships that could be construed as a potential
conflict of interest.
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June 2021 | Volume 8 | Article 684989