SC/66a SP-WP3

Preliminary report of efficiency and practicability ofbiopsy sampling, faecal sampling and prey species
identification from genetic analyses in 2014, and workplan for non-lethal research in JARPN IITOSHIHIRO MOGOE1, TSUTOMU TAMURA1, HIDEYOSHI YOSHIDA2, TOSHIYA KISHIRO2, GENTAYASUNAGA1, TAKEHARU BANDO1, KENJI KONISHI1, KAZUYOSHI NAKAI1, NAOHISA KANDA1,3,
TOURU KITAMURA3, KOICHIRO NAKANO4, HIROSHI KATSUMATA4, YOSHIHIRO HANDA4, AND
HIDEHIRO KATO5
1

Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan
National Research Institute of Far Seas Fisheries, 2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648, Japan
3
JAPAN NUS Co., Ltd., 7-5-25 Nishi-Shinjuku, Shinjuku-Ku, Tokyo 160-0023, Japan
4
FASMAC Co., Ltd., 5-1-3 Midorigaoka, Atsugi-Shi, Kanagawa 243-0041, Japan
5
Tokyo University of Marine Science and Technology, 5-7 Konan 4, Minato-ku, Tokyo 108-8477, Japan
2

Contact e-mail: mogoe@cetacean.jp

ABSTRACTThis paper is the preliminary report of efficiency and practicability of biopsy sampling, faecal sampling and preyspecies identification from genetic analyses in 2014. It also includes a work-plan for non-lethal research in
JARPN II. Biopsy sampling of sei and Bryde’s whales has been conducted in many surveys and has been shown
to be reasonably efficient. However, the efficiency (the number of obtained samples per targeted individuals) ofbiopsy sampling was lower than that of lethal sampling. Furthermore, it seems to take too much effort to conduct
biopsy sampling for common minke whales as an alternative to lethal sampling in JARPN II. We also haveconcluded that sampling of faeces from swimming whales is inefficient because of the rare encounter excretionand that sampling of faeces from sea water seems to be highly biased depending on the prey species consumed
by whales. The preliminary study of DNA analyses of the content of large intestine of whales using nextgeneration sequencing (NGS) technologies clearly indicated that the genetic prey ID only from the contents of
the large intestine is insufficient to understand feeding habits of the whales, because of their low identification
rate of prey species in the large intestine contents. Furthermore, the prey species compositions identified in thelarge intestine were quite different from those in the stomach. We sampled blubber and prey samples in the 2014
survey, however the analyses of stable isotope and fatty acid could not be conducted because of logistical reason.
We are going to analyse these samples and evaluate how these results contribute to the elucidation of the role ofwhales compared to conventional stomach contents analyses.

KEYWORDS: SCIENTIFIC PERMITS; COMMON MINKE WHALE; BRYDE’S WHALE; SEI WHALE
INTRODUCTION
Some non-lethal techniques have proven practical in Japan’s whale research and therefore biopsy sampling has
been conducted in Japanese Special Permit surveys through JARPA, JARPA II, JARPN and JARPN II especiallyin the dedicated sighting surveys. However, we did not evaluate the efficiency of non-lethal techniques such as
biopsy sampling, and faeces observation and sampling.
Following the March 31, 2014 Judgment of the International Court of Justice (ICJ) in the case whaling in the
Antarctic (Australia v. Japan: New Zealand intervening), the Government of Japan voluntarily reviewed the stateof JARPN II. In the process of the review it was decided to evaluate the potential use of the non-lethal
techniques.
Here we report the preliminary results of the evaluation of non-lethal sampling from a technical point of view
addressing the following points:(i) whether a tissue and other samples can be obtained by a non-lethal method (e.g. biopsy sampling andfaeces collection);1

SC/66a/WP**(ii) whether enough number of samples can be obtained by the non-lethal method for statistical analysis;(iii) whether the sample obtained by the non-lethal method can produce scientific information compatible tothat produced by lethal sampling; and(iv) whether the cost for obtaining the sample/producing scientific information is reasonable.
Points (i) and (ii) above are technical, (iii) is analytical, and (iv) is a logistical evaluation.
Experiments related to the above points were carried out in 2014 and will be continued for an additional 2 or 3
years.
The main objectives of JARPN II are to elucidate the feeding ecology of whales and the construction of
ecosystem models based on interspecific interactions among whales and their prey species in the western North
Pacific. Prey information such as prey species and amount of food taken has been collected for common minke(Balaenoptera acutorostrata), sei (B. borealis) and Bryde’s (B. edeni) whales. Therefore the experiments willaddress whether feeding information derived from non-lethal methods is comparable to that from lethal methods.
As potential alternatives to stomach contents analyses from lethal sampling, stable isotope and fatty acidanalyses of biopsy samples and genetic analyses of faecal samples have been reported in many marine mammal
studies, however we did not conduct these analyses because of a logistical reason.
The ICJ judgement also pointed out that the other similar research program (JARPA II) has not conducted
practicability experiments for non-lethal sampling. To respond to this, we first tested the practicability of biopsy
sampling and sampling of excreted faeces from swimming whales targeted in JARPN II. Evaluation of whether
the samples obtained from biopsy and faecal sampling methods can achieve the objectives of JARPN II which
require quantitative information on feeding of baleen whales was made.
This paper reports the progress of non-lethal sampling and preliminary results of the evaluation of non-lethal
methods in the 2014 JARPN II with reference to points (i) and (ii) above. Evaluation related to the other points
and an overall evaluation will be reported based on the data from JARPN II research for 3 years from 2014.

MATERIALS AND METHODS
The feasibility studies were conducted in three components of JARPN II in 2014, i.e., Offshore component andCoastal components off Sanriku and off Kushiro. Detailed research methods and results are reported in the cruise
reports submitted to this meeting (Mogoe et al., 2015; Tamura et al., 2015; Yoshida et al., 2015). Different
survey platforms (research vessels) were used for the Offshore and Coastal components. Larger vessels wereused in the offshore survey. Equipment used for biopsy sampling was also different for the above noted
components as described below.

Sampling methodology (Biopsy, excretion and contents from large intestine)i) Offshore component
The offshore component of JARPN II was conducted from 16 May to 29 July 2014 using two sighting andsampling vessels (Yushin Maru (YS1: 724GT) and Yushin Maru No.2 (YS2: 747GT))(Fig. 1). Biopsy sampling
and observation of excretions and sampling faeces were conducted. Contents of stomachs and large intestines
were collected from sampled whales on the board of a research-base vessel, the Nisshin Maru (NM: 8,145GT) in
order to study whether faeces can produce comparable information to that from stomach contents.
A dedicated sighting survey, which is independent from the sampling survey, was carried out from 11 May to 29
June 2014, using a research vessel (Yushin Maru No.3 (YS3: 742GT)). In this sighting survey, only observationof excretions from swimming whales was conducted.
i)-1 Biopsy sampling
Using two sighting and sampling vessels (YS1 and YS2), biopsy sampling was conducted for common minke, sei
and Bryde’s whales using a compound crossbow (Fig.2).

i)-2 Observation of excretion and faecal sampling
Observation of excretion from the whales and sampling of excreted faeces were conducted for common minke,
sei and Bryde’s whales. The observed time is defined as duration of approaching to the whale within 0.2 n.mile
to end of chasing or observation. If an observer found faeces near surface of the sea water, the faeces were2

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sampled by circle net with 100µm mesh size (Fig. 4). The sampled faeces were stored at -20°C until analyses. As
an experiment for faecal sampling practicability, contents of large intestines obtained from four sei whales andfour Bryde’s whales were released to the sea, then attempts to collect these were made after one and threeminutes using the circle net in order to compare the contents of large intestines and faeces released to the sea.

i)-3 Sampling of large intestine contents of whales sampled
For testing the potential of prey species identification from excreted faeces sampled using genetic analysis, 50gof large intestine contents were collected from 90 sei and 25 Bryde’s whales and stored at -20°C for later
examination.

ii) Coastal Component off Sanriku
The coastal component off Sanriku of JARPN II was conducted from 26 April to 11 June 2014. Four small typewhaling catcher boats were used as the sighting and sampling vessels (Taisho Maru No.28 (47.3GT), Koei Maru
No.8 (32.0GT), Katsu Maru No.7 (32.0GT) and Sumitomo Maru No.51 (30.0GT))(Fig. 1). Biopsy and faecal
sampling experiments were conducted by these vessels. Contents from large intestine were collected fromanimals sampled in order to study whether faeces can produce comparable information to that from stomach
contents.

ii)-1 Biopsy sampling
As a general rule, biopsy sampling was conducted using a compound crossbow after the catch of one common
minke whale by each research vessel.

ii)-2 Observation of excretion and faecal sampling
Observation of excretion was conducted for common minke whales targeted for lethal and biopsy sampling. The
observed time is defined as duration from the start of chasing for the sampling to hit time of the harpoon or
biopsy sampling dart to the animal or to time of abandonment for chasing. In addition, observation of excretion
was conducted after the first catch of common minke whales for each research vessel per day as a general rule. If
an excretion was observed, sampling of faeces was attempted using the circle net with 100µm mesh. To test thefeasibility of sampling faeces for common minke whales at the sea, contents from large intestines collected fromthree common minke whales were released to the sea water by use of a 100L plastic tub then attempts were madeto collect these using the circle net after 15, 30 and 60 seconds.

ii)-3 Sampling of large intestine contents of whales sampled
For testing the potential of prey species identification from excreted faeces sampled using genetic analysis, 100gof large intestine contents from three common minke whales were also collected and stored -20°C for later
examination.

iii) Coastal component off Kushiro
The coastal component off Kushiro was conducted from September 4 - 24 with additional sighting survey on
September 2 and 3. The four sighting and sampling vessels that conducted the coastal component off Sanriku
were used. Biopsy and faecal sampling experiments were conducted by these vessels. Contents from large
intestine were collected from animals sampled.
iii)-1 Biopsy sampling
Biopsy sampling was attempted on four days: afternoon of September 2, 3, and 15, and morning of September 19.
During that period, biopsy sampling was conducted without lethal sampling, using two compound crossbows, an
air gun (LKARTS-Norway), and a Larsen gun (Fig.2).

iii)-2 Observation of excretion and faecal sampling3

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Observation of excretion was conducted in the same manner as in the Coastal component off Sanriku, forcommon minke whales targeted for lethal and biopsy sampling. To test the feasibility of sampling of faeces ofcommon minke whales at the sea, contents of large intestines collected from six animals sampled were released
to the sea from the deck of vessels, then attempts were made to collect these using the circle net (100µm mesh)after 1 minute.

iii)-3 Sampling of large intestine contents of whales sampled
The experiments were carried out in same manner as for the Coastal component off Sanriku. 100g of large
intestine contents were collected from six common minke whales and divided into two halves. One half of eachof the samples from the 6 minke whales was stored at -20°C for later genetic analysis. The other half was used
for the feasibility test of faeces sampling, as described above.

Analytical methodologyi)-1 Tissue and prey samples for stable isotope and fatty acids analyses
Blubber samples from all whales sampled in both Offshore and Coastal components and main prey species fromstomach contents were stored at -20°C. These samples are going to be used for stable isotope and fatty acids
analyses. The number of analyses will be decided based on the budget. The results of the analyses will bereported to the 2016 IWC/SC.

i)-2 The DNA analyses of the contents of large intestine by next-generation sequencing (NGS) technologies
50g of the content of the large intestines were collected from the sampled whales and the samples were stored at-20°C until start of DNA extraction. Some of the samples (from ten sei whales, six Bryde’s whales, and ninecommon minke whales) were analysed. Total genomic DNA was extracted from each sample. A set of universal
primers for amplifying a part of the COI region of mtDNA was used for PCR amplification (Leray et al. 2013;
the barcode of life data systems). Amplified PCR products were analysed using an Illumina MiSeq (NGS) to
identify the prey species. These results were then compared to those observed from stomach content on an
individual base.

RESULTS and DISCUSSION
1. Effort for non-lethal research activity
1-1. Offshore component
For the sighting/sampling vessels, out of 282.9 hours search effort during the survey, total effort that was spentin the sighting survey for non-lethal experiments was 89.6 hours. These efforts were 31.7% of total research time.
For sighting vessels, total effort that was spent for the sighting survey of non-lethal experiment was 262.4 hours(Table 1).

1-2. Coastal component of Sanriku
Out of 571.1 hours searching effort during the survey, total effort that was spent in the sighting survey for nonlethal experiments was 60.8 hours. These efforts were expended 10.6% of the total research time (Table 1).

1-3. Coastal component of Kushiro
Out of 309.5 hours of searching effort during the survey, total effort that was spent in the searching for nonlethal experiment was 58.6 hours. These efforts were 18.9% of total research time (Table 1).

2. Biopsy sampling2-1. Offshore4

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Thirty-four hours were taken for the biopsy sampling. For sei whales, 33 schools (42 animals) were encounteredand targeted for biopsy sampling (Table 2). Of the 63 darts launched for 42 individuals, 21 darts hit and 16
biopsy samples were collected. The average sampling time was 80 minutes per a sample, and the sampling
efficiency (the number of obtained samples per targeted individuals) was 0.38. In the case of lethal sampling, theaverage sampling time was calculated as 27 minutes, and sampling efficiency was 0.87.
For Bryde’s whales, 38 schools (39 animals) were encountered and targeted for biopsy sampling (Table 2). Of
the 67 darts launched for 39 individuals, 31 darts hit and 25 biopsy samples were collected. The average
sampling time was 32 minute per sample and sampling efficiency is 0.64. In case of lethal sampling, the average
sampling time was calculated as 13 minutes, and sampling efficiency was 0.93.
There was no chance to conduct biopsy sampling for common minke whales due to few sightings.

2-2. Coastal component off Sanriku
No common minke whales were encountered during the biopsy experiment.

2-3. Coastal component of Kushiro
During the biopsy searching, 11 schools (12 animals) were encountered and 9 animals were targeted for biopsy
sampling (Table 2). Of the 14 darts launched for 8 animals chasing for 7.63 hours, five biopsy samples werecollected from five individuals. The average sampling time was 92 minutes per sample. The sampling efficiency
was 0.56. In case of lethal sampling, it was calculated as 60 minutes for sampling a whale; 32 minutes shorter
than for biopsy sampling. If all the 51 samples were collected only with biopsy sampling, time for collecting thesamples increased from 51.0 hours to 78.2 hours (around 1.5 times). Biopsy sampling is likely to be affected inrelation to larger wind scale, especially for common minke whales. Biopsy dart is smaller and lighter than
harpoon for lethal sampling, and smaller body size with no visible blow in this species make it more difficult forchasing the species than for larger baleen whale species, e.g., sei and Bryde’s whales. The average time ofbiopsy sampling in the common minke whale was summarized by Beaufort wind scale (Table 2). Twenty-threeminutes was calculated as the time for sampling at the wind scale ‘1’ and 194 minutes at the scale ‘2’.There wasno sampling chance at the scale ‘0’ (calm condition). In case of lethal sampling, it was calculated as 28, 45, 66
and 75 minutes for the scales ‘0’, ‘1’, ‘2’ and ‘3-4’, respectively. From the comparison of the average samplingtime for the biopsy and lethal samplings at the same wind scale, it is noted that efficiency of biopsy samplingchanged significantly related to the wind scale. The comparison of efficiency between biopsy and lethal
sampling methods should be conducted taking the wind scale into account.

3. Observation of excretion and faecal sampling
3-1. Offshore
For sei whales, 679 individuals were observed (Table 3). During the observation effort of 81 hours, excretion
was recognized for 21 individuals. Faeces samples were obtained from three individuals using circle net (Fig. 3).
The average sampling time was over 25 hours per a sample.
For Bryde’s whales, 158 individuals were observed (Table 3). During the observation effort of 38 hours,
excretion was recognized for three individuals. However all faeces sampling efforts failed. Because, all faeces
dispersed or sink before sampling.
For minke whales, four individuals were observed (Table 3). During the observation effort of 16 minutes, faeces
were not recognized.
The re-sampling experiment of contents from the large intestine of sei whales (Fig. 4) showed that the contents
derived from whales feeding on copepods tend to float while the faeces from whales feeding on fish tend to sink.
This represents serious biases in the analysis of feeding habits by faeces samples.

3-2. Coastal component off Sanriku
On the 49 common minke whales, observation for excretion was conducted for 44.8 hours (Table 3). Throughout
the experiment, excretion was not observed and therefore there was no opportunity to collect the faecal samples.
In the feasibility test for faeces sampling using contents of the large intestine it was observed that contents fromwhales feeding on sand lace tend to sink (Fig. 5).

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3-3. Coastal component off Kushiro
On the 89 common minke whales, observation of excretion was conducted for 60.6 hours (Table 3). One case of
excretion was observed, but a faecal sample could not be collected. It dispersed before sampling. Contents oflarge intestines from six common minke whales were released to the sea from the deck of sampling vessels (Fig.
6). A part of them could be sampled using net.

4. Preliminary consideration of efficiency of biopsy and faecal samplingThis study shows the technical aspects of biopsy and faecal sampling, especially their sampling efficiency.
Biopsy sampling from larger whales (sei and Bryde’s whales) was shown to be comparatively reasonable
regarding sampling efficiency. However, sampling efficiency was lower than that of lethal sampling. We also
have reached the conclusion that the sampling of faeces from swimming whales is inefficient because of the rareencounter with excretion and that sampling of faeces from the sea water seems to be highly biased depending on
the prey species of the whales. However, faecal sampling experiments will be continued in the 2015 and 2016
surveys to more completely evaluate the potential contribution of non-lethal methods to the research objectivesof JARPN II as response to the ICJ judgement and, taking account of recommendations by the Review Panel of
JARPA II.

5. DNA analyses of large intestine contents of whales using next-generation sequencing (NGS) technologies
Ten, 6 and 9 large intestine contents samples from sei, Bryde’s and common minke whales were provided fornext-generation sequencing (NGS) analysis respectively. The preliminary results were shown in Table 4 andAppendix 1.
Success rate of genetic species identification was 40.0% in the sei whale samples, 50.0% in the Bryde’s whale
samples, 66.7% in the common minke whale samples collected from Sanriku, and 50.0% in the common minke
whale samples collected from Kushiro (Table 4). The comparison with the results from the analysis of thestomach contents showed 0% match in the sei whale samples, 33.3% match in the Bryde’s whale samples, 50.0%
match in the common minke whale Sanriku samples, and 33.3% match in the common minke whale Kushiro
samples (Table 4). The comparison of results between stomach contents and large intestine is shown inAppendix 1.
Preliminary findings from data in Table 4 and Appendix 1 are as follows:
1. The prey species compositions identified in the large intestine were quite different from those in thestomach contents.
2. Although this method provides some prey information even from a whale without stomach contents, insome cases, no prey species was identified in the large intestine of the individuals notwithstanding the fullstomach contents. In addition, the prey identification rate differed among the whale species.
3. Prey of the prey species were also detected (e.g., Copepoda: Acartia clausii in the individual 14NPCSM019 is known as a major prey of sand lance and not as a prey of common minke whales). This is criticalpoint in feeding ecology study of whales.
The next step to be conducted especially for points 1 and 2 above is to examine whether these results were due toeither biological, technical or both reasons. One of the technical reasons which we encountered in this study wasthat almost all of the PCR products predominantly contained the fragments of the whale sequences, causing low
prey species identification rate in the samples. Development of a method which avoids amplifying the whale
sequences (e.g., Shehzad et al., 2012) is now under way, so a better resolution will be obtained in near future.
Likewise, a solution should be found to separate amplification of prey of the prey species. This preliminary study
clearly indicates that the genetic prey ID only in the large intestine (and faeces) is insufficient to understandfeeding habits of the whales.

6. Work plan of Stable isotope and fatty acid analyses
We sampled blubber and prey samples in the 2014 survey, but the analyses of stable isotope and fatty acid could
not be conducted because of a budget shortfall, however the samples will be analysed to evaluate how theseanalyses results contribute the elucidation of the role of whales in the ecosystem compared to the conventional
stomach contents analyses.

6SC/66a/WP**REFERENCES
Deagle, B.E., Tollit, D.J., Jarman, S.N., Hindell, M.A., Trites, A.W., and Gales, N.J. 2005. Molecular scatology
as a tool to study diet: analysis of prey DNA in scats from captive Steller sea lions. Molecular Ecology
14:1831-42.
International Whaling Commission. 2015. Report of the Expert Workshop to Review the Japanese JARPA II
Special Permit Research Programme, 24-28 February 2014, Tokyo, Japan. J. Cetacean Res. Manage.
(Suppl.) 16:369-409.
Mogoe, T., Bando, T., Ito, N., Nakamura, G., Hiruda, H., Oikawa, H., Isoda, T., Kumagai, S., Sato, H.,
Sakamoto, N., Miyagawa, N., Takahashi, M., Fukumoto, S., Ota, M., Furuyama, Y., Hirose, A., Kato,
Kouichiro, Hayashi, R., Yoshii, K. and Kato, HIdehiro. 2015. Cruise Report of the Second Phase of theJapanese Whale Research Program under Special Permit in the Western North Pacific (JARPN III) (part II) –Coastal component off Sanriku in spring 2014. Paper SC/66a/SP** presented to the IWC ScientificCommittee, May 2015 (unpublished). **pp.
Leray, M., Yang, J.Y., Meyer, C.P., Mills, S. C., Agudelo, N., Ranwez, V., Boehm, J. T. and Machida, R.J. 2013.
A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding
metazoan diversity: application for characterizing coral reef fish gut contents. Frontiers in Zoology.10:34.
Shehzad, W, Riaz, T, Nawaz, M. A, Miquel, C, Poillot, C, Shah, S. A., Pompanon, F, Coissac, E, and Taberlet, P.
2012. Carnivore diet analysis based on next-generation sequencing: application to the leopard cat(Prionailurus bengalensis) in Pakistan. Molecular Ecology 21:1951–65.
Tamura, T., Kanda, N., Nakai, K., Sakamoto, N., Okitsu, Y., Kato, K., Yoshii, K., Mori, M., Tsunekawa, M.,
Kawabe, S., Yamaguchi, F., Honnma, H. and Ogawa, T. 2015. Cruise Report of the Second Phase of theJapanese Whale Research Program under Special Permit in the Western North Pacific (JARPN II) in 2014
(part I) – Offshore component –. Paper SC/66a/SP** presented to the IWC Scientific Committee, May 2015
(unpublished). 15pp.
Yoshida, H., Ito, N., Kishiro, T., Miyashita, T., Nakai, K., Nakamura, G., Maeda, H., Ishida, K., Takahashi, M.,
Ota, M., Furuyama, Y., Kato, K., Hayashi, R., Hiruda, H., Kumagai, S., Sakamoto, N., Kimura, Y., Teshima,I., and Kato, H. 2015. Cruise Report of the Second Phase of the Japanese Whale Research Program underSpecial Permit in the Western North Pacific (JARPN II) in 2014 (Part III) - Coastal component off Kushiro -.
Paper SC/66a/SP** presented to the IWC Scientific Committee, May 2015 (unpublished). 13pp.

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Table 1. The research effort time (hours) of each survey.
Offshore SSVs
(hours)
193.3
89.6
31.7
282.9

Item/Area
Lethal effort
Non-lethal effort
Rate(%)of non lethal/total
Total

Offshore SV(hours)
262.4
100.0
262.4

Sanriku(hours)
510.3
60.8
10.6
571.1

Kushiro(hours)
250.9
58.6
18.9
309.5

Table 2. The results of biopsy samplingWhalespecies

Ship

Targeted

NumberNumberNumberindividuals

of

of

of

EffortSampling

Averageeffeciency

sampling timeshootshits

samples(minute)(sample per trial)(minute per sample)

(A)

(B)

(C)

(D)

(E)

(D)/(B)(E)/(D)Sei whale

SSVs

42

63

21

16

1275

0.25

80

Bryde's whale
Common minke
whale (Sanriku)
Common minke
whale (Kushiro)

SSVs

39

67

31

25

789

0.37

32

SSVs

0

0

0

0

-

-

-

SSVs

9

14

5

5

458

0.36

92
(23)a
(194)ba：efficiency of sampling at Beaufort wind scale‘1’；b：efficiency at scale ‘2’.

Table 3. The results of faecal sampling
Area

Bryde's whale
Common minke whale

CoastalObservation
effort(hours)
75.1
5.9
25.4
12.7
0.1
0.2

SSVs

49

44.8

0

0

-

SSVs

89

60.6

1

0

-

Ship
type

Sei whale
Offshore

SSVs
SV
SSVs
SV
SSVs
SV

Number ofexperiments(inds.)
346
333
116
42
2
2

Whale
speciesCommon minke whale(Sanriku)
Common minke whale(Kushiro)

Observation
Faecalof excretion sampling(number)(number)
11
3
10
0
1
0
2
0
0
0
0
0

Efficiency
of sampling(hours per sample)
25.0-

Table 4. The preliminary results of DNA analyses of large intestine of whales using next-generation
sequencing (NGS) analysis
WhalespeciesNumberofanalyses(inds.)

Numberofprey detected(inds.)

Detection
rate(%)

Matchingof stomach
contents(% of yes)

Sei whale(Offshore)

10

4

40.0

0.0

Bryde's whale(Offshore)

6

3

50.0

33.3

Minke whale(Sanriku)

3

2

66.7

50.0

Minke whale(Kushiro)

6

3

50.0

33.3

8

SC/66a/WP**a) Yushin Maru (724GT)b) Sumitomo Maru No.51 (30GT)

Fig. 1. Research vessels used as the sighting and sampling vessel at the offshore component (a) and coastal
component (b) of the JARPN II.

Fig. 2. The equipment for biopsy sampling. Compound crossbow (left); air gun (LKARTS-Norway; right upper);Larsen gun (right lower).

Fig. 3. The faecal sampling experiment using circle net at the sea (offshore component).

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Fig. 4. The experiment for faecal sampling using contents from large intestine of whales sampled (offshore
component). Contents from large intestine (left); contents immediately after throwing into the sea(middle); contents at 1 minute after throwing into the sea and sampling using ring net (right).

Fig. 5. The experiment for faecal samples using contents from large intestine of whales sampled (coastal
component off Sanriku).

Fig. 6. The experiment for faecal samples using contents from large intestine of whales samples, conducted at
the deck of a sighting and sampling vessel (coastal component off Kushiro).

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Appendix 1. The comparison of results between stomach contents and large intestine.

SpeciesSei

ID numberPrey species estimating by
next-generation sequencing(NGS)

14NPSE001

Mackerels (90%) andNo detected
Japanese anchovy (10%)

14NPSE006

Copepods (99%) andkrill (1%)

Krill

14NPSE018

Mackerels

No detected

14NPSE044

Japanese sardine (50%),
Japanese anchovy (40%) No detected
and Mackerels (10%)

14NPSE048

Copepods (80%) and
Pacific saury (20%)

Krill

14NPSE052

Copepods

Pacific saury

14NPSE067

Copepods

No detected

14NPSE070

MackerelNo detected

Japanese anchovy

No detected

14NPB006

Mackerels

Lightfish(Maurolicus muelleri )14NPB009

Japanese anchovy (90%),
Japanese anchovy, JapaneseJapanese sardine (8%)
sardine and krill
and mackerels (2%)

14NPB010

Japanese anchovy

No detected

14NPB016

Japanese anchovy

Krill

14NPB019

Japanese anchovy (99%)
No detected
and mackerels (1%)

14NPCS-M013

Sand lance

Sand lance

14NPCS-M019

Sand lance

Copepoda (Acartia clausii )14NPCS-M021

Sand lance

No detected

14NPCK-M013

Japanese sardine

No detected

14NPCK-M015

Japanese sardine

No detected

14NPCK-M016

Japanese sardine

Japanese sardine

14NPCK-M017

Walleys pollock andJapanese sardine

No detected

14NPCK-M019

Japanese sardine

Japanese anchovy

14NPCK-M027

Walleys pollock andJapanese common sqiod

Japanese common sqiod andkrill

Bryde's 14NPB005

Minke

Prey species estimatingby stomach contents

11