Aquaculture 468 (2017) 156161

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Aquaculture

journal homepage: www.elsevier.com/locate/aquaculture

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,
a
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7th Nanhai Road, Qingdao 266071, China
b
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
Keywords:
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

Sea cucumbers are organisms inhabiting in almost all marine habi-
tats, from tropical to polar areas. In China and Southeast Asia, sea cu-
cumbers are also a highly valued traditional medicine and food
product and support signicant wild capture sheries in many parts of
the world (Ferdouse, 2004). With a burgeoning sea cucumber sheries
business, acquiring precise in situ data on the stock amount, growth
rate, and migration scope of the sea cucumber is becoming more impor-
tant for the people to manage the aquaculture farm well. Mark-recap-
ture techniques are conventional method which is feasible for the
investigation, yet because of the lack of hard tissue, it is very difcult
for this animal to retain any physical marks on its body.
Many physical, chemical and molecular methods have been applied to
tag holothurians. Fluorescent dyeing of ossicles or genetic ngerprint
markers have been proven to be the most effective and safe methods of
marking sea cucumbers (Purcell, Blockmans, 2009; Uthicke, Benzie,
2002; Uthicke et al., 2004); however, visible external tags (such as T-
bar, scratch mark) are cheaper and more convenient and easy to detect
in eld studies. Previous studies on physical or external tagging on
Corresponding authors.
E-mail addresses: gaofeicas@126.com (F. Gao), hshyang@qdio.ac.cn (H. Yang).
on the tagging method, the species and the individual size. Conand
(1990) found that most sea cucumber species reject T-bar tags inserted
into their body wall. Juvenile Holothuria scabra can reject all T-bar tags
within 1 month after tagging, but can retain 60% of coded-wire tags for
1 year (Purcell et al., 2006). Another study on Cucumaria frondosa gave
the opposite result: all single-anchor T-bar tags remained embedded in
the sea cucumbers for up to 100 days (Kirshenbaum et al., 2006). Scratch
marks are visible up to a month with no indication of any considerable
behavioral change, and it has been applied successfully on some holothu-
rian species for behavioral studies (Reichenbach, 1999; Mercier et al.,
2000; Shiell, 2006; Navarro et al., 2013; Siegenthaler et al., 2015). Gener-
ally, many of the techniques have yielded important success, yet also have
a variety of shortcomings as summarized by Gianasi et al. (2015). The
major problem for the external tagging methods (such as T-bars) is the
rejection by the tegument or severe necrosis around the tag. More studies
on the exact recovery time after tagging and the short-term side effects
are needed. New feasible tagging method is also needed to be invented.
The sea cucumber Apostichopus japonicus is an important economic
species for aquaculture and stock enhancement in many Asian countries
(Yang et al., 2015). Unlike other holothurian species, A. japonicus has
much soft body tissue and lacks a coriaceous epidermis. Our previous
studies have tried integument embedding and papillae puncture

http://dx.doi.org/10.1016/j.aquaculture.2016.10.002
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
3 days and the necrosis of the wound was serious (Sun et al., 2013).
The key problem which limits the retention of tag is the strong
ejecting ability of sea cucumber's body. An enclosed calcareous ring is
a common skeletal structure in the body of many sea cucumber species.
It is an internal ring of usually 10 calcareous plates circling the pharynx
or the anterior part of the gut. Our hypothesis is that threading the tag
on calcareous ring could prolong its retention time. In this study, a
new calcareous-ring-threading method (CRT method) was designed
and applied to tag the sea cucumber A. japonicus. A size category that
is feasible for the CRT method was screened. The tag retention rate
was calculated during a 1-month experiment. A short-term stress effect
on different sizes of sea cucumber was assessed based on the defecation
amount and endocrine indices, aiming to evaluate the side effects and
reliability of the tagging method.
2. Materials and methods
2.1. Tagging method
The key feature of the CRT method is threading through the calcare-
ous ring of the sea cucumber, and xing an external tag on the thread.
The needle and thread used in the method should be as ne as possible
to minimize potential injury to the body. A metal embroidering needle
(2.3 cm long, = 0.26 mm) were selected rst as the threading tool.
Three kinds of threads were pretested on small sea cucumbers (ave.
5 g in wet weight) including nylon shing line, polypropylene line and
surgical suture. Severe necrosis around the lines occurred after 3 days
except nylon shing line (Sun et al., 2013). Based on the result, a smooth
nylon shing line (20 cm long, = 0.128 mm) is selected.
The sea cucumbers were tagged according to the following procedure
(Fig. 1). Two embroidering needles were threaded with a shing line.
The rst needle pierced the calcareous ring inside of the mouth and
exited the dorsal body wall, leading the rst end of the line through
the calcareous ring. The second needle pierced the dorsal body wall
next to the calcareous ring, leading the second end of the line out of
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
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-
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
(Fig. 1F). The entire process only took 3 min or less. The tag can be recog-
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
Sea cucumbers (n = 150) were collected from the rocky subtidal
zone (depth b 8 m) at Laoshan Bay, Qingdao, China, in March 2013.
The sea cucumbers were starved for 2 days to empty their guts, after
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
tanks in the aquaculture research lab and provided with sand-ltered
seawater. The water temperature was 10 0.5 C. Aeration was provid-
ed continuously, and approximately two-thirds of the water volume was
exchanged every day to ensure the water quality. Individuals were fed
with articial feed (3:1 dry weight ratio of sea mud and Sargassum sp.
powder, screened by 60 m mesh net) during this period, and the feed
amount is 1015% of total wet weight of sea cucumbers in the tank.
2.3. Experiment 1: effect on the catecholamine level
The tagging operation and tag retained on the body may cause stress
to the sea cucumber, especially during the rst few days, and the reaction
level may vary depending on the size of tested individual. We hypothesis
that larger sea cucumbers have better tolerance to tags than smaller ones.
Catecholamine is considered to be sensitive and can rapidly (within 1 h)
reacts to stresses (Tan et al., 2016). Epinephrine (E) and dopamine (DA)
were analyzed to quantify the intensity of the stress responses of the sea
cucumbers. For each size category, four replicate individuals were sam-
pled initially and at 1 h, 6 h, 12 h, 24 h, 48 h, 72 h and 7 days after tagging.
It is very difcult to withdraw coelomic uid from the body cavity within
seconds because of frequent blocking of the syringe needle by inner body
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
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-
ferred into freezing tubes and stored at 80 C for further analysis.
The catecholamines (epinephrine, E; dopamine, DA) in the coelomic
uid were analyzed using commercial kits (Enzyme Immunoassay for
the quantitative determination of E and DA in the serum, plasma and
tissue homogenates, Cusabio, Wuhan, China). The detection sensitivi-
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-
sured for the 5 standards (linear, y-axis) against the corresponding con-
centrations (logarithmic, x-axis). The concentration of E and DA in the
samples was then taken from the curve.
2.4. Experiment 2: effect on the defecation amount
A. japonicus has a continuous feeding and defecating pattern. The
defecation amount is selected as another indicator to assess the stress
of the retained tag to the sea cucumber. The size categories of sea cu-
cumbers for this experiment were the same as those in Exp. 1. After
adapting for 2 weeks in four 500 L tanks to the culture conditions,
each individual was reared separately in a 30 L tank and four replica-
tions were set for each size. The amount of daily articial feed was gen-
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
at the second day. The water temperature was 10 0.5 C. Aeration
was provided continuously, and approximately two-thirds of the
water volume was exchanged every day. After water was drained out,
sea cucumber feces was carefully siphoned out to keep its strip shape
Table 1
Size categories of the tagged sea cucumbers.
Indices Size category
S M1 M2 B
Weight range b40 g 4060 g 6190 g N90 g
Ave. SD (g) 34.23 6.87 52.76 9.18 79.60 12.27 120.68 30.73
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
for the whole 24 h was sampled every day during the week before tag-
ging and at 1, 2, 3, 4, and 7 days after tagging. Feces samples were oven-
dried at 60 C for 48 h and weighed.
2.5. Experiment 3: retention of the CRT tags
The retention rate of the CRT method was compared with that of the
T-bar method, which has been widely tested on other sea cucumber
species. For the T-bar method, 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 (Fig. 2). The size of the sea cucumber for
the experiment was determined to be 6090 g due to the relatively
smaller stress response and better recovery concluded in Experiments
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 experiment period. Aeration was provided continuously, and ap-
proximately half of the water volume was exchanged every day. Appro-
priate amount of articial feed (1015% of sea cucumber's wet weight)
was fed each day. After acclimation for 2 weeks, 45 sea cucumbers were
tagged by the CRT method with a colored heat-shrink tube and 90 were
tagged with numbered T-bars (2.3 cm long, stem = 0.6 mm) on the
dorsal body wall with a tagging gun.
All tagged sea cucumbers were reared in the same tank. The moni-
toring lasted for 30 days. The daily retention rates of the two tagging
methods were calculated by counting the number of each of the two
types of tags that had fallen off each day.
2.6. Statistics
2.6.1. Experiment 1
The duration of the tag remained was dened as the stress factor.
The hormone concentrations at each duration time were compared
with the initial value before tagging for signicance test using one-
way ANOVA. Least Signicant Difference (LSD) was selected as the
Post Hoc testing method.
2.6.2. Experiment 2
t-test is used to detect difference of defecation amount among days
before tagging, then all the data were averaged as the initial value. The
defecation amounts at each duration time were compared with the ini-
tial value for signicance test using one-way ANOVA. Least Signicant
Difference (LSD) is selected as the Post Hoc testing method.
2.6.3. Experiment 3
A Chi-square Test was conducted to test whether there was signi-
cant difference (95% condence intervals) in the daily retention rates
between the two tagging methods.
The differences were considered signicant at a probability level of
P b 0.05. All of the statistical analysis was performed using the software
SPSS 19.0.
3. Results
3.1. Stress effect on the catecholamine level
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
endocrine was demonstrated in separate gures. The hormone concen-
tration increased from 163.33 25.15 to 475.24 512.43 ng mL1 for
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,
P=0.113). After 7 d, signicantly high level of E and DA concentrations
(2727.20 1988.95 and 1080.66 460.11 ng mL1, respectively) were
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
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
to 181.14 115.38 ng mL1 (P = 0.019) (Fig. 3B, 4B), and both of E
and DA recovered to average level at 48 h. Group M2 and B showed a
stable endocrine level throughout the entire experimental period
(Figs. 3B and 4B).
3.2. Stress effect on defecation
The impact of tagging on defecation amount is shown in Fig. 5. There
was no signicant difference among daily defecation amount within all
groups before tagging by t-test. The defecation amounts averaged as
2.25 0.48, 4.38 1.55, 5.49 0.53 and 5.86 1.31 gdw1 for
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
Fig. 5. Defecation amount of the sea cucumber Apostichopus japonicus with different sizes
before and after the calcareous-ring-threading tag operation. Signicant increase was
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 **
indicate signicant difference at two levels (P b 0.05 and P b 0.001) were detected.
gdw1(P = 0.006) for four groups, respectively. Then, all groups recov-
ered to the initial level thereafter except group S, which reached to a
maximum defecation amount of 5.48 1.82 gdw1 (P = 0.008) at
the 7th day.
3.3. Retention rate of the CRT tags
During the 1-month tag retention experiment, no mortality or tissue
necrosis was found and all of the sea cucumbers ate normally. The re-
tention rate of the T-bar method decreased gradually from 96.7% (day
10) to 65% at the end of the experiment, whereas the CRT method
retained a high retention rate of over 90% until day 23 and 84% until
day 30 (Fig. 6). From day 26, CRT method showed signicantly higher
retention rate than T-bar method by Chi-square test (X2 = 5.36, P =
0.02). Whenever either of the two types of tags fell off, the remaining
holes on the body were observed to heal within 2 days.
4. Discussion
4.1. Retention of the CRT tags
In the present study, the retention rate of the T-bar tag on A.
japonicus decreased to 65% within 1 month, indicating that even large-
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
Chi-square test.
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
operation.
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
polyethylene bag for 1 h and transport). Noradrenaline (NA) and dopa-
mine (DA) concentrations signicantly increased to sixfold and three-
fold higher levels, respectively, in 0.51 h after the beginning of damp
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
at 1 h after the beginning of acute temperature and salinity stress. In
present study, the whole tagging operation time is limited within
3 min, yet the earliest increase of catecholamine concentration was ob-
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-
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
retained tag in its body. The small sea cucumbers in group S showed a
signicant stress response to the tags at day 7th. In other groups, only
group M1 at 24 h duration showed a signicant increase of hormone,
but it recovered to average level right after that. The bumping value
seems to be caused by other unexpected factors, not the retained tag.
The result indicates that large sea cucumbers have better tolerance to
external environmental stresses than small ones. Our previous tagging
study with juvenile A. japonicus showed that all sea cucumbers (approx-
imately 10 g) died within 10 days after calcareous ring tagging, mostly
because of tissue necrosis around the thread (Sun et al., 2013). There-
fore, it is feasible to select large A. japonicus individuals with a wet
weight above 60 g for CRT tagging.
Under normal conditions, the defecation amount can reect the
feeding activity of the deposit-feeding sea cucumbers. In the present
study, sea cucumbers of all size groups had a stable daily defecation
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
This study indicates that the side effect of tagging on A. japonicus in-
dividuals with wet weights above 60 g could disappear after 2 days. The
 Q. Xu et al. / Aquaculture 468 (2017) 156161
selection of proper line and threading method, together with limiting
the operation time, can minimize possible damage to the animal. It is
feasible to select large A. japonicus individuals larger than 60 g (wet
weight) for the CRT operation, which has a higher retention rate
(84%) than the T-bar method (65%) during a 1-month experiment.
Further eld studies should be carried out to conrm whether it is
also feasible for other long-term in situ studies.
Acknowledgements
We thank Dr. Tianlong Qiu for his help in experimental management
and sampling. Many thanks are also dedicated to Mrs. Song for her assis-
tance in rearing the tagged sea cucumbers and keeping a daily record of
the dropped tags. This study was sponsored by the NSFC-Shandong Joint
Fund for Marine Science Research Centers (grant No. U1406403), the
Non-Prot Marine Sector of the State Oceanic Administration (grant
No. 201205023, 201305043) and the Science and Technology Innova-
tion Plan of Aoshan (grant No. 2015ASKJ02-05).
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