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Aquaculture 468 (2017) 156161

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A new tagging method and its early stress effect on the sea cucumber
Apostichopus japonicus
Qiang Xu a,b, Fei Gao b,, Libin Zhang a, Tao Zhang a, Hongsheng Yang a,
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7th Nanhai Road, Qingdao 266071, China
State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, 58th Renmin Road, Haikou 570228, China

a r t i c l e i n f o a b s t r a c t

Article history: The sea cucumber Apostichopus japonicus is a widely exploited species that is highly valued as a traditional med-
Received 1 April 2016 icine and as a food product in China, Singapore, Japan and many other countries of Southeast Asia. With the fast
Received in revised form 5 September 2016 development of sea cucumber sheries and aquaculture, in-situ investigation is necessary to assess the stock
Accepted 1 October 2016
amount, growth and migration of cultured sea cucumbers. Unfortunately, this work is limited for the lacking of
Available online 04 October 2016
effective tagging method for the species. In this study, a calcareous-ring-threading method (CRT method) was
tried to tag the sea cucumber A. japonicus. The early stress response of the sea cucumber to tagging operation
Calcareous-ring-threading method and invasive tag was monitored so as to evaluate the reliability of the new method. This method had a tag reten-
Stress response tion rate of 84%, which was higher than the T-bar method (65%), during a 1-month experiment. The results
Catecholamine showed that the side effects on large individuals (wet weight above 40 g) disappeared after 2 days. The individ-
Defecation amount uals with a wet weight below 40 g had less tolerance on the new tagging method and couldn't recover after
Invasive tagging method 7 days. It is feasible to select large A. japonicus individuals with a wet weight above 60 g for this new tagging
method. Based on the result of present study, the CRT method is considered to be applicable for tagging A.
japonicus individuals with wet weights above 60 g.
2016 Elsevier B.V. All rights reserved.

1. Introduction holothurians yielded varying results, which were primarily dependent

on the tagging method, the species and the individual size. Conand
Sea cucumbers are organisms inhabiting in almost all marine habi- (1990) found that most sea cucumber species reject T-bar tags inserted
tats, from tropical to polar areas. In China and Southeast Asia, sea cu- into their body wall. Juvenile Holothuria scabra can reject all T-bar tags
cumbers are also a highly valued traditional medicine and food within 1 month after tagging, but can retain 60% of coded-wire tags for
product and support signicant wild capture sheries in many parts of 1 year (Purcell et al., 2006). Another study on Cucumaria frondosa gave
the world (Ferdouse, 2004). With a burgeoning sea cucumber sheries the opposite result: all single-anchor T-bar tags remained embedded in
business, acquiring precise in situ data on the stock amount, growth the sea cucumbers for up to 100 days (Kirshenbaum et al., 2006). Scratch
rate, and migration scope of the sea cucumber is becoming more impor- marks are visible up to a month with no indication of any considerable
tant for the people to manage the aquaculture farm well. Mark-recap- behavioral change, and it has been applied successfully on some holothu-
ture techniques are conventional method which is feasible for the rian species for behavioral studies (Reichenbach, 1999; Mercier et al.,
investigation, yet because of the lack of hard tissue, it is very difcult 2000; Shiell, 2006; Navarro et al., 2013; Siegenthaler et al., 2015). Gener-
for this animal to retain any physical marks on its body. ally, many of the techniques have yielded important success, yet also have
Many physical, chemical and molecular methods have been applied to a variety of shortcomings as summarized by Gianasi et al. (2015). The
tag holothurians. Fluorescent dyeing of ossicles or genetic ngerprint major problem for the external tagging methods (such as T-bars) is the
markers have been proven to be the most effective and safe methods of rejection by the tegument or severe necrosis around the tag. More studies
marking sea cucumbers (Purcell, Blockmans, 2009; Uthicke, Benzie, on the exact recovery time after tagging and the short-term side effects
2002; Uthicke et al., 2004); however, visible external tags (such as T- are needed. New feasible tagging method is also needed to be invented.
bar, scratch mark) are cheaper and more convenient and easy to detect The sea cucumber Apostichopus japonicus is an important economic
in eld studies. Previous studies on physical or external tagging on species for aquaculture and stock enhancement in many Asian countries
(Yang et al., 2015). Unlike other holothurian species, A. japonicus has
Corresponding authors. much soft body tissue and lacks a coriaceous epidermis. Our previous
E-mail addresses: (F. Gao), (H. Yang). studies have tried integument embedding and papillae puncture
0044-8486/ 2016 Elsevier B.V. All rights reserved.
Q. Xu et al. / Aquaculture 468 (2017) 156161 157

methods to secure the markers, but the markers were rejected within The needle and thread used in the method should be as ne as possible
3 days and the necrosis of the wound was serious (Sun et al., 2013). to minimize potential injury to the body. A metal embroidering needle
The key problem which limits the retention of tag is the strong (2.3 cm long, = 0.26 mm) were selected rst as the threading tool.
ejecting ability of sea cucumber's body. An enclosed calcareous ring is Three kinds of threads were pretested on small sea cucumbers (ave.
a common skeletal structure in the body of many sea cucumber species. 5 g in wet weight) including nylon shing line, polypropylene line and
It is an internal ring of usually 10 calcareous plates circling the pharynx surgical suture. Severe necrosis around the lines occurred after 3 days
or the anterior part of the gut. Our hypothesis is that threading the tag except nylon shing line (Sun et al., 2013). Based on the result, a smooth
on calcareous ring could prolong its retention time. In this study, a nylon shing line (20 cm long, = 0.128 mm) is selected.
new calcareous-ring-threading method (CRT method) was designed The sea cucumbers were tagged according to the following procedure
and applied to tag the sea cucumber A. japonicus. A size category that (Fig. 1). Two embroidering needles were threaded with a shing line.
is feasible for the CRT method was screened. The tag retention rate The rst needle pierced the calcareous ring inside of the mouth and
was calculated during a 1-month experiment. A short-term stress effect exited the dorsal body wall, leading the rst end of the line through
on different sizes of sea cucumber was assessed based on the defecation the calcareous ring. The second needle pierced the dorsal body wall
amount and endocrine indices, aiming to evaluate the side effects and next to the calcareous ring, leading the second end of the line out of
reliability of the tagging method. the upper lip of the sea cucumber (Fig. 1A, B). The two free ends of the
line were joined with a short, colored heat-shrink tube, and then, a
2. Materials and methods glass Radio Frequency Identication (RFID) tag (8 mm long, =
0.14 mm) was plugged in (Fig. 1C, D). The line and tag were bound to-
2.1. Tagging method gether tightly after the tube was briey heated using a heat gun (temper-
ature below 240 C) (Fig. 1E). The excess line was cut off with scissors
The key feature of the CRT method is threading through the calcare- (Fig. 1F). The entire process only took 3 min or less. The tag can be recog-
ous ring of the sea cucumber, and xing an external tag on the thread. nized by the tube color or the RFID electronic code after radio scanning.

Fig. 1. Demonstration of the tagging procedure for the calcareous-ring-threading (CRT) method. Two embroidering needles pierced the inside and outside of the calcareous ring to the
dorsal body wall (A), leading the nylon line tied on the calcareous ring (B). The two ends of the line were tubed with a short heat-shrink tube with a glass RFID (Radio Frequency
Identication) tag that was plugged in (C, D). The tube was heated by a hot-wind gun and shrunk tightly (E). Excessive line was cut off and operation completed (F).
158 Q. Xu et al. / Aquaculture 468 (2017) 156161

2.2. Experimental animals and collected on a mesh net (50 m). The residual feed in the tank
was cleaned out and fresh water was then added. The feces excreted
Sea cucumbers (n = 150) were collected from the rocky subtidal for the whole 24 h was sampled every day during the week before tag-
zone (depth b 8 m) at Laoshan Bay, Qingdao, China, in March 2013. ging and at 1, 2, 3, 4, and 7 days after tagging. Feces samples were oven-
The sea cucumbers were starved for 2 days to empty their guts, after dried at 60 C for 48 h and weighed.
which they were wet weighed and divided into four size categories
(Table 1). Then, they were held separately for 15 days in four 500-L 2.5. Experiment 3: retention of the CRT tags
tanks in the aquaculture research lab and provided with sand-ltered
seawater. The water temperature was 10 0.5 C. Aeration was provid- The retention rate of the CRT method was compared with that of the
ed continuously, and approximately two-thirds of the water volume was T-bar method, which has been widely tested on other sea cucumber
exchanged every day to ensure the water quality. Individuals were fed species. For the T-bar method, a numbered T-bar (2.3 cm long, stem
with articial feed (3:1 dry weight ratio of sea mud and Sargassum sp. = 0.6 mm) is xed on the dorsal body wall of the sea cucumber by
powder, screened by 60 m mesh net) during this period, and the feed punching with a tagging gun (Fig. 2). The size of the sea cucumber for
amount is 1015% of total wet weight of sea cucumbers in the tank. the experiment was determined to be 6090 g due to the relatively
smaller stress response and better recovery concluded in Experiments
2.3. Experiment 1: effect on the catecholamine level 1 and 2. The experiment was carried out in an indoor concrete tank
with a 5-m3 water column. The water temperature was 12 1 C during
The tagging operation and tag retained on the body may cause stress the experiment period. Aeration was provided continuously, and ap-
to the sea cucumber, especially during the rst few days, and the reaction proximately half of the water volume was exchanged every day. Appro-
level may vary depending on the size of tested individual. We hypothesis priate amount of articial feed (1015% of sea cucumber's wet weight)
that larger sea cucumbers have better tolerance to tags than smaller ones. was fed each day. After acclimation for 2 weeks, 45 sea cucumbers were
Catecholamine is considered to be sensitive and can rapidly (within 1 h) tagged by the CRT method with a colored heat-shrink tube and 90 were
reacts to stresses (Tan et al., 2016). Epinephrine (E) and dopamine (DA) tagged with numbered T-bars (2.3 cm long, stem = 0.6 mm) on the
were analyzed to quantify the intensity of the stress responses of the sea dorsal body wall with a tagging gun.
cucumbers. For each size category, four replicate individuals were sam- All tagged sea cucumbers were reared in the same tank. The moni-
pled initially and at 1 h, 6 h, 12 h, 24 h, 48 h, 72 h and 7 days after tagging. toring lasted for 30 days. The daily retention rates of the two tagging
It is very difcult to withdraw coelomic uid from the body cavity within methods were calculated by counting the number of each of the two
seconds because of frequent blocking of the syringe needle by inner body types of tags that had fallen off each day.
tissues, and it may also disturb the hormone level, so the coelomic uid
(23 mL ind1) of the A. japonicus was collected after quick dissection 2.6. Statistics
and stored into Eppendorf tubes. The coelomic uid was centrifuged at
1500 rpm for 10 min at 4 C to remove the cellular debris and then trans- 2.6.1. Experiment 1
ferred into freezing tubes and stored at 80 C for further analysis. The duration of the tag remained was dened as the stress factor.
The catecholamines (epinephrine, E; dopamine, DA) in the coelomic The hormone concentrations at each duration time were compared
uid were analyzed using commercial kits (Enzyme Immunoassay for with the initial value before tagging for signicance test using one-
the quantitative determination of E and DA in the serum, plasma and way ANOVA. Least Signicant Difference (LSD) was selected as the
tissue homogenates, Cusabio, Wuhan, China). The detection sensitivi- Post Hoc testing method.
ties were 11.9 pg/mL and 31.25 pg/mL for E and DA, respectively. The
calibration curve was obtained by plotting the extinction values mea- 2.6.2. Experiment 2
sured for the 5 standards (linear, y-axis) against the corresponding con- t-test is used to detect difference of defecation amount among days
centrations (logarithmic, x-axis). The concentration of E and DA in the before tagging, then all the data were averaged as the initial value. The
samples was then taken from the curve. defecation amounts at each duration time were compared with the ini-
tial value for signicance test using one-way ANOVA. Least Signicant
2.4. Experiment 2: effect on the defecation amount Difference (LSD) is selected as the Post Hoc testing method.

A. japonicus has a continuous feeding and defecating pattern. The 2.6.3. Experiment 3
defecation amount is selected as another indicator to assess the stress A Chi-square Test was conducted to test whether there was signi-
of the retained tag to the sea cucumber. The size categories of sea cu- cant difference (95% condence intervals) in the daily retention rates
cumbers for this experiment were the same as those in Exp. 1. After between the two tagging methods.
adapting for 2 weeks in four 500 L tanks to the culture conditions, The differences were considered signicant at a probability level of
each individual was reared separately in a 30 L tank and four replica- P b 0.05. All of the statistical analysis was performed using the software
tions were set for each size. The amount of daily articial feed was gen- SPSS 19.0.
erally 1015% of sea cucumber's wet weight, and the amount was
adjusted to be a little excessive so that the residual feed was no N 2 g 3. Results
at the second day. The water temperature was 10 0.5 C. Aeration
was provided continuously, and approximately two-thirds of the 3.1. Stress effect on the catecholamine level
water volume was exchanged every day. After water was drained out,
sea cucumber feces was carefully siphoned out to keep its strip shape The small-sized sea cucumbers (group S) had higher E and DA con-
centration base lines than the other groups, so the dynamic change of
Table 1 endocrine was demonstrated in separate gures. The hormone concen-
Size categories of the tagged sea cucumbers. tration increased from 163.33 25.15 to 475.24 512.43 ng mL1 for
Indices Size category E and 152.92 15.63 to 488.60 413.14 ng mL1 for DA after 6 h, yet
both changes were statistically insignicant (Fig. 3A, P=0.402; Fig. 4A,
S M1 M2 B
P=0.113). After 7 d, signicantly high level of E and DA concentrations
Weight range b40 g 4060 g 6190 g N90 g (2727.20 1988.95 and 1080.66 460.11 ng mL1, respectively) were
Ave. SD (g) 34.23 6.87 52.76 9.18 79.60 12.27 120.68 30.73
recorded (Fig. 3A, 4A; P = 0.001).
Q. Xu et al. / Aquaculture 468 (2017) 156161 159

Fig. 2. Demonstration of the T-bar tagging operation. A numbered T-bar (2.3 cm long, stem = 0.6 mm) is xed on the dorsal body wall of the sea cucumber by punching with a tagging
gun (A & B).

For sea cucumbers larger than 40 g, the E concentration of group M1 3.2. Stress effect on defecation
signicantly increased from 104.81 14.14 to 192.83 130.25 ng
mL1 after 24 h (P = 0.016); the DA increased from 104.30 18.44 The impact of tagging on defecation amount is shown in Fig. 5. There
to 181.14 115.38 ng mL1 (P = 0.019) (Fig. 3B, 4B), and both of E was no signicant difference among daily defecation amount within all
and DA recovered to average level at 48 h. Group M2 and B showed a groups before tagging by t-test. The defecation amounts averaged as
stable endocrine level throughout the entire experimental period 2.25 0.48, 4.38 1.55, 5.49 0.53 and 5.86 1.31 gdw1 for
(Figs. 3B and 4B). group S, M1, M2 and B respectively.
After tagging, all groups showed a signicant increase of defecation
amount at the 2nd day, which were 4.45 1.67 (P = 0.041), 11.36
2.49 (P = 0.002), 13.67 2.89 (P b 0.001) and 13.70 3.24

Fig. 3. Dynamic change of the epinephrine (E) concentration in the coelomic uid of the Fig. 4. Dynamic change of the dopamine (DA) concentration in the coelomic uid of the
sea cucumber Apostichopus japonicus with different sizes after the calcareous-ring- sea cucumber Apostichopus japonicus with different sizes after the calcareous-ring-
threading tag operation. Signicant increase was found in group S (at 7th day) and M1 threading tag operation. Signicant increase was found in group S (at 7th day) and M1
(24 h). * indicates signicant difference (P b 0.05) was detected. (24 h). * indicates signicant difference (P b 0.05) was detected.
160 Q. Xu et al. / Aquaculture 468 (2017) 156161

sized individuals of A. japonicus have a strong expelling ability for exog-

enous materials embedded in their body wall. The newly developed CRT
method had a better retention rate than T-bar method from the 26th
day. There were still 16% of the sea cucumbers lost their tags in the 1st
month. The possible cause is the prociency of the tagging operator:
the rst tagging needle did not puncture through the calcareous ring
properly; training is necessary for the technician to avoid the wrong

4.2. Side effect of tagging

It is essential to assess the security of any tagging method and the

duration of the necessary accommodation rearing after tagging. Tan et
al. (2016) reported the quick response of hormones in A. japonicus to
damp-transporting handling stress (packing 200 sea cucumbers into a
Fig. 5. Defecation amount of the sea cucumber Apostichopus japonicus with different sizes polyethylene bag for 1 h and transport). Noradrenaline (NA) and dopa-
before and after the calcareous-ring-threading tag operation. Signicant increase was mine (DA) concentrations signicantly increased to sixfold and three-
found in all four groups at the 2nd day, and values recovered to normal levels thereafter,
except for another signicant increase occurred in group S at the 7th day. * and **
fold higher levels, respectively, in 0.51 h after the beginning of damp
indicate signicant difference at two levels (P b 0.05 and P b 0.001) were detected. transportation. After the end of the transport, NA and DA concentrations
declined and returned to basal values at 2 h (Tan, et al., 2016). Wang
(2008) also reported that E and DA concentration changed signicantly
gdw1(P = 0.006) for four groups, respectively. Then, all groups recov- at 1 h after the beginning of acute temperature and salinity stress. In
ered to the initial level thereafter except group S, which reached to a present study, the whole tagging operation time is limited within
maximum defecation amount of 5.48 1.82 gdw1 (P = 0.008) at 3 min, yet the earliest increase of catecholamine concentration was ob-
the 7th day. served at 6 h after tagging in group S (although statistically insignicant,
Figs. 3A and 4A), so the impact of tagging operation itself to catechol-
3.3. Retention rate of the CRT tags amine is very small and can be ignored. Instead, the epinephrine and do-
pamine values revealed the stress response of the sea cucumber to the
During the 1-month tag retention experiment, no mortality or tissue retained tag in its body. The small sea cucumbers in group S showed a
necrosis was found and all of the sea cucumbers ate normally. The re- signicant stress response to the tags at day 7th. In other groups, only
tention rate of the T-bar method decreased gradually from 96.7% (day group M1 at 24 h duration showed a signicant increase of hormone,
10) to 65% at the end of the experiment, whereas the CRT method but it recovered to average level right after that. The bumping value
retained a high retention rate of over 90% until day 23 and 84% until seems to be caused by other unexpected factors, not the retained tag.
day 30 (Fig. 6). From day 26, CRT method showed signicantly higher The result indicates that large sea cucumbers have better tolerance to
retention rate than T-bar method by Chi-square test (X2 = 5.36, P = external environmental stresses than small ones. Our previous tagging
0.02). Whenever either of the two types of tags fell off, the remaining study with juvenile A. japonicus showed that all sea cucumbers (approx-
holes on the body were observed to heal within 2 days. imately 10 g) died within 10 days after calcareous ring tagging, mostly
because of tissue necrosis around the thread (Sun et al., 2013). There-
4. Discussion fore, it is feasible to select large A. japonicus individuals with a wet
weight above 60 g for CRT tagging.
4.1. Retention of the CRT tags Under normal conditions, the defecation amount can reect the
feeding activity of the deposit-feeding sea cucumbers. In the present
In the present study, the retention rate of the T-bar tag on A. study, sea cucumbers of all size groups had a stable daily defecation
japonicus decreased to 65% within 1 month, indicating that even large- amount during the 7 days before tagging (Fig. 5). However, 2 days
after tagging, sea cucumbers of all four size groups exhibited a signi-
cant increase in the defecation amount. Shiell (2006) found that stress
caused by handling and invasive tagging on the body wall could result
in a higher moving activity of Holothuria whitmaei during the rst
three days, and thereafter, activity recovered to the normal level. In
present study, the abnormal increase of defecation amount at the 2nd
day is probably a kind of manifestation of higher moving activity,
which caused by the integrated impact of tagging handle and retained
tags. The defecation amount of sea cucumbers larger than 40 g recov-
ered to the normal level from 3rd day, which coincides well with the re-
port on H. whitmaei. And it also indicates that the integrated impact of
handling and invasive tagging on the digestive physiology of large sea
cucumbers (N40 g) can disappear after 2 days.
Based on the result of present study, the CRT method is considered to
be applicable for tagging A. japonicus individuals with wet weights
above 60 g for short-term group recognition and studies such as migra-
tion behavior in the eld.

5. Conclusion
Fig. 6. Retention rate of calcareous-ring-threading tags on the sea cucumber Apostichopus
japonicus compared with T-bar tag during one month. The dashed line shows that
signicant difference was found between values of the two methods from day 26 by This study indicates that the side effect of tagging on A. japonicus in-
Chi-square test. dividuals with wet weights above 60 g could disappear after 2 days. The
Q. Xu et al. / Aquaculture 468 (2017) 156161 161

selection of proper line and threading method, together with limiting Mercier, A., Battaglene, S.C., Hamel, J.F., 2000. Periodic movement, recruitment and size-
related distribution of the sea cucumber Holothuria scabra in Solomon Islands.
the operation time, can minimize possible damage to the animal. It is Hydrobiologia 440 (13), 81100.
feasible to select large A. japonicus individuals larger than 60 g (wet Navarro, P.G., Garcia-Sanz, S., Barrio, J.M., Tuya, F., 2013. Feeding and movement patterns
weight) for the CRT operation, which has a higher retention rate of the sea cucumber Holothuria sanctori. Mar. Biol. 160 (11), 29572966.
Purcell, S.W., Blockmans, B.F., 2009. Effective uorochrome marking of juvenile sea cu-
(84%) than the T-bar method (65%) during a 1-month experiment. cumbers for sea ranching and restocking. Aquaculture 296 (34), 263270.
Further eld studies should be carried out to conrm whether it is Purcell, S.W., Blockmans, B.F., Nash, W.J., 2006. Efcacy of chemical markers and physical
also feasible for other long-term in situ studies. tags for large-scale release of an exploited holothurian. J. Exp. Mar. Biol. Ecol. 334 (2),
Reichenbach, N., 1999. Ecology and shery biology of Holothuria fuscogilva
Acknowledgements (Echinodermata: Holothuroidea) in the Maldives, Indian Ocean. Bull. Mar. Sci. 64
(1), 103113.
Shiell, G.R., 2006. Effect of invasive tagging on the activity of Holothuria whitmaei
We thank Dr. Tianlong Qiu for his help in experimental management
[Echinodermata: Holothuroidea]: a suitable mark-recapture method for short-term
and sampling. Many thanks are also dedicated to Mrs. Song for her assis- eld studies of holothurian behaviour. Mar. Freshw. Behav. Physiol. 39 (2), 153162.
tance in rearing the tagged sea cucumbers and keeping a daily record of Siegenthaler, A., Canovas, F., Gonzalez-Wanguemert, M., 2015. Spatial distribution pat-
the dropped tags. This study was sponsored by the NSFC-Shandong Joint terns and movements of Holothuria arguinensis in the Ria Formosa (Portugal). J. Sea
Res. 102, 3340.
Fund for Marine Science Research Centers (grant No. U1406403), the Sun, L., Qiu, T., Xu, Q., Liu, H., Lin, C., Yang, H., 2013. Evaluation of different methods for
Non-Prot Marine Sector of the State Oceanic Administration (grant external tagging of Apostichopus japonicus. Mar. Sci. (Beijing) 37 (7), 16.
No. 201205023, 201305043) and the Science and Technology Innova- Tan, J., Sun, X., Gao, F., Sun, H., Chen, A., Gai, C., Yan, J., 2016. Immune responses of the sea
cucumber Apostichopus japonicus to stress in two different transport systems. Aquac.
tion Plan of Aoshan (grant No. 2015ASKJ02-05). Res. 47, 21142122.
Uthicke, S., Benzie, J.A.H., 2002. A genetic ngerprint recapture technique for measuring
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