Technical Reports of the Institute of Cetacean Research (2018)

pp. 50–55

Technical Report (not peer reviewed)

Field and analytical protocol for the evaluation of novel non-lethaltechniques in the Japanese whale research programs
Toshihiro Mogoe*
Institute of Cetacean Research, 4–5 Toyomi-cho, Chuo-ku, Tokyo 104–0055, Japan*Contact e-mail: mogoe@cetacean.jp

ABSTRACTThis paper describes the ICR ﬁeld and analytical protocol used to evaluate novel non-lethal techniques, which
potentially can be used to respond to scientiﬁc questions related to NEWREP-A and NEWREP-NP. The protocolincludes four primary questions, which should be responded to in order to evaluate the techniques. This paper
also illustrates the use of this protocol to evaluate the use of faeces for studies of feeding ecology of whales(a non-lethal technique).

INTRODUCTION
The International Whaling Commission Scientiﬁc Committee (IWC SC) recommended that a ﬁeld and analytical
protocol should be developed to assist the evaluation of
the utility of novel non-lethal techniques (IWC, 2016).
Such techniques could potentially be used to respondto scientiﬁc questions related to the Japanese whale
research programs (see Goto and Inoue, this issue). In
response to this recommendation, scientists from theInstitute of Cetacean Research (ICR) developed a protocol
consisting of several questions that should be responded
to in order to evaluate novel non-lethal techniques (seeMogoe et al., 2015).
The objective of this paper is to describe this ﬁeld andanalytical protocol. The paper also illustrates the use ofthis protocol to evaluate the use of faeces for studies offeeding ecology of whales, a non-lethal technique.

DESCRIPTION OF THE FIELD AND ANALYTICAL
PROTOCOL
The questions
The protocol consists of four questions, which are listed
and explained in this section.
The ﬁrst question (Q1) is whether a tissue or otherkinds of samples can be obtained by a non-lethal technique (for example whether or not faeces can be collected from the sea surface during a research period).
The second question (Q2) is whether a suﬃcient number of samples for statistical analysis can be obtained by
the non-lethal technique.
The third question (Q3) is whether the sample ob50

tained by the non-lethal technique can produce scientiﬁc information comparable to that produced by a lethal
sampling technique.
The fourth question (Q4) is whether the cost for obtaining the sample and for producing scientiﬁc information from the sample is reasonable.
Q1 and Q2 above are technical in nature. Q3 is analytical while Q4 is of a logistic nature.
Formulating the questions to evaluate a non-lethaltechnique
The ﬂow chart of Figure 1 shows a systematic applicationof the protocol to evaluate a novel non-lethal technique.
The fundamental question to be responded to throughthis chart is whether a novel non-lethal technique canreplace the lethal-sampling.
The formulation of the four questions forms a basis
to objectively discuss the feasibility and practicability of
non-lethal techniques, particularly from a perspectiveof whether particular research objectives are achievablethrough non-lethal techniques.
Criteria for questions
The criteria for Q1 is simple. If at least one tissue or
sample can be taken during the research period by the
non-lethal technique, the answer is ʻyesʼ otherwise theanswer is ʻno.ʼ
For Q2 the sampling eﬃciency of a non-lethal technique needs to be compared with that of a lethal technique. For example in the case of faeces sampling (seebelow), the number of samples collected by some unit ofeﬀort is compared with the number of whales (stomachs)

Field and analytical protocol for the evaluation of novel non-lethal techniques in the Japanese whale research programs

Figure 1.

Systematic application of the four questions to evaluate the feasibility of novel non-lethal techniques.

sampled by the same unit.
In Q3 the key issue is whether the analysis of the tissue or samples obtained by the non-lethal technique
can respond to speciﬁc scientiﬁc questions by producing
scientiﬁc information of similar or better quality than that
produced by the lethal-technique. For example in thecase of faeces samples (see below), the key question will
be whether the analysis of faeces samples can produce
similar or better scientiﬁc information than the lethal
technique (stomach content analysis) to respond questions of prey consumption (qualitative and quantitative)
and prey preference, which are important input data forecosystem models.
Criteria for Q4 require further consideration but thesimplest approach for an evaluation could be dividing
the overall cost for research by the number of samples
obtained.

APPLICATION OF THE PROTOCOL TO EVALUATE
THE USE OF FAECES FOR FEEDING ECOLOGYSTUDIES
Currently reliable information on prey consumption andprey preferences of whales is obtained through the analyses of stomach contents. The fundamental question iswhether the analysis of faeces samples can provide suchreliable information.

Field and laboratory data were obtained, which are explained below. Data collected were the basis of responses
to the four questions in Figure 1.
Field data
The ﬁeld experiments on the evaluation of the faeces
sampling for studies of feeding ecology of North Paciﬁc common minke, Brydeʼs and sei whales were carriedout during the 2014 and 2015 JARPNII surveys. Yushin
Maru-type vessels were used for the experiments inthe oﬀshore component of JARPNII (Bando et al., 2016)(sighting and sampling vessels: SSVs, and sighting vessels:SVs). Smaller vessels were used in the coastal component(Kishiro et al., 2016).
Table 1 shows a summary of the eﬀort (time) spent on
both whale observation and sampling (lethal) and faeces
observation and sampling (non-lethal).
Observation and sampling of faeces
Observation of excretion and sampling of faeces in identiﬁed individuals of common minke, sei and Brydeʼs whales
were made from the platforms of the survey vessels. The
eﬀort was deﬁned as the observation time from conﬁrmation of the whale species (0.2 n.miles) to the end ofobservation/sampling. If an observer found faeces near
the sea surface, the faeces were sampled by circle net51

Technical Reports of the Institute of Cetacean Research (2018)
Table 1Eﬀort (hours) spent in whale observation and sampling (lethal)and faeces observation and sampling (non-lethal) surveys. See
text for details.
Oﬀshore Oﬀshore Coastal Coastal
SSVs
SVs
Sanriku Kushiro2014 JARPNII
Whale observationand sampling eﬀort 193.3
Faeces observationand sampling eﬀort 89.6
Total eﬀort
282.9
Rate (%) of
non-lethal eﬀort
31.7
2015 JARPNII
Whale observationand sampling eﬀort
Faeces observationand sampling eﬀort
Total eﬀort
Rate (%) of
non-lethal eﬀort

Figure 2.

—

510.3

250.9

262.4
262.460.8
571.1

58.6
309.5

100.0

10.6

18.9

61.5

—596.4

521.7

99.5
161.0

547.5
547.5

54.6
650.9

65.4
587.1

61.8

100.0

8.4

11.1

Sampling of sei whale faeces in the 2015 JARPNII
oﬀshore survey.

with 100 µm mesh size (Figure 2). The sampled faeces
were stored using polyethylene bottles at −20°C.
Observation and sampling of whales
Whales were sighted and sampled along the predetermined track-lines. The eﬀort was deﬁned as the timefrom conﬁrmation of whale species to the time of theshooting.
Analytical proceduresThe idea was to compare the results on prey species inthe stomach contents (direct observations) and in faeces
samples (DNA analyses). As this comparison was not pos52

sible for a same individual, the contents in intestine wasused as a proxy for faeces. Therefore prey species in thestomach (direct observation) and intestine (DNA analyses) was compared for a same individual. In addition,
prey species in the faeces samples collected were also
investigated by DNA analyses.
In the case of genetic analysis, total genomic DNA wasextracted using the standard phenol/chloroform extractions protocol of the GENTRA PUREGENE DNA extraction
kit (QIAGEN) following the companyʼs manual. Extracted
DNAs were stored in TE buﬀer. After PCR ampliﬁcation,
the products were analyzed using an Illumina MiSeq(next-generation DNA sequencers) to identify the preyspecies.
Analysis of stomach content followed standard procedure (Tamura et al., 2016).

RESULTS
Response to question 1
A total of 1,808 experiments were made (1,179 for sei,
393 for Brydeʼs and 236 for common minke whales), for377.2 h (Table 2). Excretion was observed in 38 individuals (30 sei, 6 Brydeʼs, and 2 common minke whales). Of
these, faeces was obtained successfully from ﬁve seiwhales. For Brydeʼs and common minke whales, faeces
could not be obtained due to sinking or spreading of faeces before sampling.
Therefore at the present, the answer to Q1 is ʻyesʼ
for sei whales and ʻnoʼ for common minke and Brydeʼswhales.
Response to question 2
Table 2 shows that the observation eﬀort and number ofexcretions observed in the research period. The numberof faeces observed is extremely low, 38 cases in 1,808
individual experiments (2.1%) for all surveys and species
combined. These percentages were 2.5%, 1.5%, 0.0%
and 0.0% for sei, Brydesʼs, common minke (oﬀshore) andcommon minke (coastal), respectively.
These percentages contrast with those of the eﬃciencyof whale (stomach) sampling. For all surveys and species
combined there were 297 targeted individuals of which
151 were sampled (50.8%). The percentages by species
were 93.6%, 89.3% and 50.8% for sei, Brydeʼs and minke(coastal) whales, respectively.
Sampling eﬃciency of faecal sampling is substantially lower than the whale (stomach) sampling eﬃciency.
Therefore the response to Q2 is ʻnoʼ for sei, Brydeʼs andcommon minke whales.

Field and analytical protocol for the evaluation of novel non-lethal techniques in the Japanese whale research programs
Table 2
The results of faecal sampling in the 2014 and 2015 surveys.
Species2014 JARPNII
Sei
Brydeʼs
C. minke (Oﬀshore)
C. minke (Sanriku)
C. minke (Kushiro)
2015 JARPNII
Sei
Brydeʼs
C. minke (Oﬀshore)
C. minke (Sanriku)
C. minke (Kushiro)
Total
Sei
Brydeʼs
C. minke (Oﬀshore)
C. minke (Coastal)

Vesseltype

Number ofexperiments(school)

Number ofexperiments(individuals)

Observationeﬀort (hours)

Observation ofexcretion(number)

Faecalsampling(number)

SSVs
SVs
SSVs
SVs
SSVs
SVs
SSVs
SSVs

192
1349430224989

34633311642224989

75.1
5.9
25.4
12.7
0.1
0.2
44.8
60.6

11101
2000130000000

SSVs
SVs
SSVs
SVs
SSVs
SVs
SSVs
SSVs

193
133
113
70203359259241
147
8820335951.6
7.7
27.4
2.4
0.90.0
31.0
31.4

632
1000120000000652
307
6230

1,179393
6230

140.3
67.9
1.2
167.8

30
602

5000

Response to question 3Comparison between direct observation of stomach contents and DNA analysis of intestine
Table 3 shows a comparison of the prey species foundin the stomach (direct observation) and in the intestine(DNA analysis) of sei, Brydeʼs and common minke whales.
As indicated earlier, intestine was used as a proxy forfaeces.
There is no good correspondence between the preys
identiﬁed in the stomach and the intestine of the same
individuals. In several cases the species could not beidentiﬁed by the DNA analysis notwithstanding the fullstomach of the whales.
A prey of prey species was also observed. For example
Acartia clausii was identiﬁed by the DNA analysis in theindividual 14NPCS-M019. This species is known as amajor prey of sand lance, which is a known prey speciesof the common minke whale.
The results of prey identiﬁcation by the DNA analysis
was somewhat diﬀerent between the upper/middle partsof the small intestine and the large intestine.

DNA analysis of faeces
Table 4 shows the results of the DNA analysis for prey
identiﬁcation from faeces in three sei whales. The DNA
analysis could not detect the prey species from the faecesof other three sei whales, which suggest low eﬃciencyof this technique to identify prey species from faeces
samples.
Therefore the response to Q3 is ʻnoʼ for sei, Brydeʼs andcommon minke whales.
Respond to question 4This question is not addressed in the present study. However whale ﬁeld surveys are extremely expensive. Funding for some of the surveys involving sampling of whales
are provided in part by the sale of by-products.

CONCLUDING REMARKS
Discussions on the feasibility of novel non-lethal techniques in whale research have so far been controversial
and inconclusive. This is due a lack of an objective protocol guiding these discussions. The ICR has developed53

Technical Reports of the Institute of Cetacean Research (2018)
Table 3Results of detected prey species in enteral content using next generation DNA sequencers.
Species
SeiBrydeʼsC.minkeID Number

Prey species observedby stomach contents

Prey species estimatedPrey species estimatedby NGS—Middle part ofby NGS—Large intestine
small intestine

14NPSE001

Mackerels (90%) andMackerels and Japanese Mackerels and Japanese No identiﬁed
Japanese anchovy (10%) anchovyanchovy

14NPSE006

Copepods (99%) andkrill (1%)

Krill

No identiﬁed

Krill

14NPSE018

Mackerels

Mackerels and Paciﬁcsaury

Paciﬁc saury

No identiﬁed

14NPSE044

Japanese sardine (50%), Japanese sardine andJapanese anchovy (40%) Japanese anchovyand Mackerels (10%)

No identiﬁed

No identiﬁed

14NPSE048

Copepods (80%) andPaciﬁc saury (20%)

Paciﬁc saury

Paciﬁc saury

Krill

14NPSE052

Copepods

Paciﬁc saury

Paciﬁc saury

Paciﬁc saury

14NPSE067

Copepods

No identiﬁed

No identiﬁed

No identiﬁed

14NPSE070

Mackerels

Mackerels and Paciﬁcsaury

Mackerels

No identiﬁed

14NPB005

Japanese anchovy

Japanese anchovy

Japanese anchovy

No identiﬁed

14NPB006

Mackerels

Japanese anchovy

No identiﬁed

Light ﬁsh(Maurolicus muelleri)

14NPB009

Japanese anchovy
Japanese anchovy(90%), Japanese sardine(8%) and Mackerels (2%)

Japanese anchovy

Japanese anchovy,
Japanese sardine andkrill

14NPB010

Japanese anchovy

Japanese anchovy

No identiﬁed

No identiﬁed

14NPB016

Japanese anchovy

Japanese anchovy

Japanese anchovy

Krill

14NPB019

Japanese anchovy (99%) Japanese anchovy
and mackerels (1%)

Japanese anchovy

No identiﬁed

14NPCS-M013

Sand lance——

Sand lance

14NPCS-M019

Sand lance——

Copepoda(Acartia clausii)

14NPCS-M021

Sand lance——

No identiﬁed

14NPCK-M013

Japanese sardine——

No identiﬁed

14NPCK-M015

Japanese sardine——

No identiﬁed

14NPCK-M016

Japanese sardine——Japanese sardine

14NPCK-M017

Walleye pollock andJapanese sardine——

No identiﬁed

14NPCK-M019

Japanese sardine——

Japanese anchovy

14NPCK-M027

Walleye pollock andJapanese common squid——

Japanese common squidand krill

NGS: next-generation sequencing.

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Prey species estimatedby NGS—Upper part ofsmall intestineField and analytical protocol for the evaluation of novel non-lethal techniques in the Japanese whale research programs
Table 4Prey species in faeces of sei whales identiﬁed by the NGS.
Species

ID NumberResults

Sei
Sei
Sei

140527SEI
140528SEI
150529SEI

Copepoda (Oithona similis)
Copepoda (Oithona similis)
Euphausiacea, Calanoida

a protocol with four questions that should be responded
to in order to evaluate the feasibility of novel non-lethaltechniques. An objective evaluation of the available datain the context of the four questions will make possible
more useful discussions and conclusions on the feasibility of a given novel non-lethal technique. In this study
the protocol was used to evaluate the feasibility of theanalysis of whale faeces in studies on feeding ecology
speciﬁcally to respond to questions on prey consumption
and prey preferences. Results of the evaluation following
the protocol suggest that at this stage of knowledge, suchtechnique is not feasible and therefore cannot replace
the analysis of stomach content to respond the samequestions.

ACKNOWLEDGEMENTS
The author would like to thank all captains, crews andresearchers, who were involved in the JARPNII surveys.

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