Seattle-Kobe
International Academic
Collaboration Initiative

SKIACIThe 1st Workshop

Development ofEnvironmental DNA Research

March 1, 2021, 4:00 pm - 7:00 pm PST(March 2, 2021, 9:00 - 12:00 JST)
This workshop is held online via Zoom.
Organized by the Kobe University Science Shop
Supported by the Assistance Grant for Activities to Promote International Exchange, Kobe University
Cooperated by Prof. Toshifumi Minamoto, Kobe University Graduate School of Human Development andEnvironment, and Prof. Ryan Kelly, University of Washington College of the Environment

Inquiry (e-mail): ss-stf@rdix.h.kobe-u.ac.jp1Seattle-Kobe
International Academic
Collaboration Initiative (SKIACI)The 1st Workshop:
Development of Environmental DNA Research
March 1, 2021, 4:00 pm - 7:00 pm PST (March 2, 2021, 9:00 - 12:00 JST)
Opening

4:00 pm - 4:15 pm (9:00 - 9:15 JST)

Message from Prof. Hideyuki Yamamoto (Executive Director,
KULOS), Introduction of participantsPart 1 4:15 - 5:15 pm
Talk 1

4:15 pm - 4:30 pm (9:15 - 9:30 JST)

Estimation of the spawning period of Japanese seacucumber,
Apostichopus japonicus
Daiki Takeshita (Kobe U)

Talk 2

4:30 pm - 4:45 pm (9:30 - 9:45 JST)

Fish community dynamics over the past two decades
revealed through DNA metabarcoding
Zack Gold (UW)

Talk 3

4:45 pm - 5:00 pm (9:45 - 10:00 JST)

Performance of benzalkonium chloride (BAC) as thepreservative of environmental DNA in seawater targeting
longer and nuclear DNA fragments and fish community
Toshiaki Jo (Kobe U)

Discussion 1

5:00 pm - 5:15 pm (10:00 - 10:15 JST)Part 2 5:30 - 6:50 pm
Talk 45:30 pm - 5:45 pm (10:30 - 10:45 JST)

Reconstructing past biological information in Lake Biwa
using sedimentary environmental DNA approach
Masayuki K. Sakata (Kobe U)

Talk 5

5:45 pm - 6:00 pm (10:45 - 11:00 JST)

Tracking a marine invasion front using molecular surveys
Abigail Keller (UW)

Talk 66:00 pm - 6:15 pm (11:00 - 11:15 JST)

Assessing nutritional status of carp by environmental
nucleic acid analysis
Mingyang Jiang (Kobe U)

2

Discussion 2

6:15 pm - 6:30 pm (11:15 - 10:30 JST)

Communication

6:30 pm - 6:50 pm (11:30 - 10:50 JST)

Closing

6:50 pm - 7:00 pm (11:50 - 12:00 JST)

Seattle-Kobe International Academic Collaboration Initiative
The 1st Workshop“Development of Environmental DNA Research”
March 2, 2021

(Ebina)  I would like to start the workshop.  I am Kuniyoshi Ebina of Kobe University’s Science Shop.  The Science Shop
usually works to connect ordinary people and experts, but today we are going to connect experts in Seattle and Kobe.
At the start, we would like to have a message from the Executive Director of the Kobe University Liaison Office in Seattle(KULOS), Prof. Yamamoto.

Opening Message

Hideyuki Yamamoto (Executive Director, Kobe University Liaison Office in Seattle)
Hello, everyone.   My name is Hideyuki Yamamoto, Director of the Office of the Americas and Executive Director of
KULOS.  It is my pleasure to give the opening speech for the Seattle-Kobe International Academic Collaboration Initiative(SKIACI) 1st Workshop “Development of Environmental DNA Research,” in conjunction with the Research Oriented On-site
Training (ROOT) Program in Kobe University.  I am very glad that it can be held online, despite the pandemic situation of
COVID-19.
I would like to express my gratitude to our guest speakers from the University of Washington, as well as the speakers
from Kobe University.  I am also glad to hear that Kevan Yamahara of the Monterey Bay Aquarium Research Institute (MBARI)
will join us.  I would like to extend my gratitude to all the people involved in the workshop, especially the organizer, Prof. Ebina,
for putting everything together and for inviting me as a guest speaker.
Before starting today’s workshop, I would like to take advantage of this opportunity to briefly introduce Kobe University’s
Office of the Americas and the recently established KULOS.  Kobe University has partnership agreements with 31 North
American universities and eight Latin American universities for academic collaboration and student exchange.  The Office of
the Americas serves as a hub for developing the international network of academic exchange between Kobe University andits counterparts in the Americas.  One of our recent major initiatives is the establishment of KULOS in 2020.  It is our firstliaison office in the mainland U.S., located in the Hyogo Business and Cultural Center in Downtown Seattle.
Like Hyogo Prefecture, the State of Washington has developed an international port city by actively incorporating thecultures of numerous foreign countries into its own.  It is a pleasure to have our liaison office in Seattle, which is a sister city of
Kobe.  I wish that KULOS will contribute to the globalization of research and education in Kobe University by providing venues
for international symposia and workshops for faculty members, scholars, and students in various academic fields, as well as
global educational activities for students of various levels.  KULOS is highly expected to increase international partnerships
between Kobe University and its counterparts in the West Coast, especially the University of Washington.
I hope today’s workshop will be successful and meaningful for everyone involved in it, and beneficial in enhancing
academic interest and developing academic collaboration between Kobe University and the University of Washington.
(Ebina) Thank you, Prof. Yamamoto.   Now, I would like to outline today’s meeting.   We would like to start with selfintroductions.  Let us start from the Japanese side with Prof. Minamoto.

3

Self-introductions(Minamoto)I am Toshifumi Minamoto from Kobe

and surveys using targeted eDNA quantification.

University.   I am the PI of the eDNA laboratory in Kobe
University. It is a great opportunity for us to have this(Allan)  I am Eily Allan.  I am a postdoc with Ryan at the
University of Washington.  I am also very excited to hear all

workshop between our lab and Ryan’s lab.

of your new and exciting work.

(Jo) I am Toshiaki Jo from Minamoto Lab.  I am a PhD

student, but I might get my PhD this month.  My researchinterest is the characteristics and dynamics of eDNA.

(Kelly) Kevan Yamahara is not in our lab, but he is a friend
and I invited him to join us because he is doing related
work.

(Sakata) I am Masayuki Sakata from Minamoto Lab. I
am a PhD student, but I will graduate soon.  I am interested(Yamahara)   My name is Kevan Yamahara.   I work at
MBARI.  Most of my research is focused on the design,in eDNA in sediment.

research, and development of autonomous sampling(Yasashimoto)I am Tetsu Yasashimoto.

My study

subject is eDNA metabarcoding for Odonata.

instruments focused mostly on applications for eDNA
monitoring.  I have known Ryan Kelly for a long time and it
is great to be here.

(Takeshita) I am Daiki Takeshita. I am a Master’s student.
(Wu) I am Luhan Wu.  I am a second-year doctoral student
studying the spawning period of fish by using eDNA.

(Gallego)  I am Ramon Gallego.  I am a postdoc at NOAA

Fisheries in Seattle.  I was previously a postdoc in Ryan’s lab.  
I do mostly eDNA metabarcoding.  I am interested in boththe technical aspects of metabarcoding, decontamination(Jiang)  My name is Mingyang Jiang.  I was born in China.  
I am a second-year Master’s student in Kobe University,and quantification, and also the correlation between eDNA
surveys and environmental evaluations.

doing research on eRNA.

(Kihara)(D’Agnese)   I am Erin D’Agnese.   I am a postdoc in
I am Natsumi Kihara.   I am a fourth-year

Ryan’s lab.  I am very excited to be here and learn what

undergraduate student.

everybody is doing.

(Higashisaka)  I am Hayato Higashisaka.  I am a junior(Gold) I am Zack Gold.  I am a postdoc in Ryan’s lab.  I

in Kobe University.

am at NOAA’s Northwest Fisheries Science Center.  I workon a variety of eDNA projects, but broadly on how to use(Ebina)  Next, let us move to Prof. Kelly’s lab.

eDNA metabarcoding to reconstruct marine ecosystems.

(Kelly)   I am Ryan Kelly, a professor at the University(Ebina)  There are some other guests, but we would likeof Washington.   I am excited to be here.   Thank you forto start the first talk.  The first speaker is Daiki Takeshita.  

inviting us. It is an honor to meet all the people making all“Estimation of the spawning period of Japanese seaof the amazing papers.  It is too much work for us to keep

cucumber, Apostichopus japonicus.”

up with.

(Keller)  I am Abby Keller.  I am a Master’s student working
with Ryan.  Today, I will be presenting about using eDNA tomanage invasive species.

(Ramon-Laca)  I am Ana Ramon-Laca.  I am a technician
at the National Oceanic and Atmospheric Administration(NOAA), specialized in running basic DNA diagnostics.  I
am involved in various eDNA projects, mainly on fisheries
4

Talk 1

Estimation of the spawning period of Japanese sea
cucumber, Apostichopus japonicus
Daiki Takeshita (Kobe University)

Eastern hellbender

Introduction(Pugh et al. 2018, Fig. 1)

・Estimating spawning period or sites via eDNA concentration(Spear et al. 2015, Erickson et al. 2016, Buxton et al. 2017)

・Date: June 9–14, 2020

・Water volume: 100L

Few studies in marine environment

・Water running (exchange rate：4 times/day)(Bayer et al. 2019, Wang et al. 2021)

・Possibility to capture ejaculation from

Settings of the tank experiments

Spear et al. 2015, Fig. 2-B

・Habituation → injection

nu-eDNA/mt-eDNA (Bylemans et al. 2017)→ observation → dissection

Practicality: unknown

・Water temperature:

ObjectMacquarie perch

12℃ → 15℃ (after injection)(Piggott 2016, Fig. 1)

Verification of capturing reproduction by eDNA
conc. and the ratio in marine environment

Bylemans et al. 2017, Fig. 4

IDkubi-1kubi-2kubi-3kubi-4arti-1arti-2arti-3arti-4NC

Injection

No. ofinds.

Cubifrin: 6
inducesspawning 5
artificial
1
seawater0

Estimating the spawning period or sites via eDNA
concentration has been one of the purposes of eDNA

Using individuals of this species, tank experiments

analysis, however there have been few studies in a marine

were conducted from June 9 to 14, 2020.   The water

environment.  According to a previous study, the possibility

volume was 100 L and the water condition was runningto capture ejaculation from nuclear eDNA concentration

with an exchange rate of four times per day.  The outline ofto mitochondrial eDNA concentration was suggested,

this experiment was habituation, injection, observation, andhowever the practicality of the method has been unknown.  

then dissection.  The water temperature was raised fromThe object of this study was the verification of capturing

12°C to 15°C after injection.   Nine tanks were prepared

reproduction by eDNA concentration and the ratio in afor this experiment, with one tank without individuals as

marine environment.

a negative control.   Four of the tanks were for injecting
cubifrin which induces spawning, and the others were forinjecting artificial seawater.

Target species
・Japanese sea cucumber (Apostichopus japonicus): benthic echinoderm,aquatic resource which may be depleted (Purcell et al. 2010)

Sampling

・Inhabits in western Pacific Ocean (Kanno et al. 2005)
・Spawning season: April–June (Minami et al. 2019)

Water

Tissues & feces

・250mL×1 from each tank・sperms, eggs, and body tissuesof breeding individuals
・feces at the bottom of tanks

Filtration (GF/F, pore size: 0.7μm)
AITS 2012 (modified)

The target species was the Japanese sea cucumber,a species of benthic echinoderm which is an aquatic
resource of Japan which may be depleted.  This species

DNA extraction

qPCR (nuclear DNA: 18S gene 126bp & mitochondrial DNA: COI gene 131bp)

inhabits the western Pacific Ocean.  The spawning season
is April to June.

For water samples, the volume was 250 mL from each

The lifecycle is as follows.  First, they hatch from eggs,

tank.  They were filtered, and then DNA was extracted andthen they spend two weeks as floating larvae, and thenqPCR was performed.  Sperm, eggs, and body tissues ofthey move to benthic life and spend two to three years until

breeding individuals were collected, as well as feces at thesexual maturity.   They aestivate in summer, recover frombottom of tanks with breeding individuals.

aestivation in autumn, and become active in winter andspring.
5

Results of observation and dissection
ID

Injection

No. ofinds.

kubi-1
6
kubi-2 Cubifrin:
induceskubi-3 spawning5
kubi-4arti-1arti-2arti-3arti-4

artificial
1
seawater

Repro- SexualTypeduction maturity
♂×1
♀×1
♀×1

♂×1
♀×1
♀×1
♂×1

♂×1

♂×1

immature
ejaculated
oviposited
ovipositedimmature

From the results of the statistical analysis, the eDNA
concentrations of “ejaculated” and “oviposited” weresignificantly higher than “immature.”   This could be due
to gametes or increased activity during reproduction,observed as head shaking during oviposition.

On theother hand, injection and water temperature did not have asignificant effect.

mature (♂)
ejaculated
immature

The ratio of (e)DNA concentration

These are the results of observation and dissection.
For “kubi-2” and “arti-3,” one of the individuals ejaculated.  
Sexual maturity was confirmed from dissection, so thesetanks were considered as “ejaculated.”   For “kubi-3”
and “kubi-4,” one of the individuals oviposited.   Sexual
maturity was confirmed from dissection, so these tanks
were considered as “oviposited.”  For “arti-2,” remarkableejaculation was not observed, however sexual maturity

・Eggs with small nu-DNA/mt-DNA caused the values below the PI?

was confirmed, so this tank was regarded as “mature.”  

The decrease in ratio can be used to capture oviposition.

The others were regarded as “immature.”

These are the results of the ratio of nu-eDNA
concentration over mt-eDNA concentration.   The pink

eDNA concentration in tanks

band indicates a 95% prediction interval of the ratio
converted into a common logarithm of tanks without
reproduction.  After oviposition, the values lower than thisprediction interval were recorded.  This could be causedby eggs because they have a low ratio value.  This study
newly suggested that a decrease in the ratio can be used
to capture oviposition.

A series of field surveys
・Site: Maizuru Fishery Research Station of Kyoto University, Japan・Period: April 2015–March 2016

These are the results of eDNA concentration.   Thehorizontal axis indicates time after injection, with time 0・Sample: 1L×1 for each point (P1, P2, P5)
・Frequency: once a week

indicating immediately after injection.   The vertical axis
indicates eDNA concentration.

Comparison of eDNA concentration
・“ejaculated” and “oviposited”＞“immature”
due to gametes or increased
activity during reproduction?
Shaking head during oviposition

Next is about a series of field surveys.  The study site
was the Maizuru Fishery Station of Kyoto University.  Thestudy period was April 2015 to March 2016.  The volume
of samples was 1 L, with one replication for each point (P1,
P2, and P5).  The sampling frequency was once a week.

・Injection and water temperature

did not affect significantly.

These are the results of eDNA concentration.   Thevertical axis indicates eDNA concentration, added one

6

To

eDNA concentration in the fieldsummarize,

tankexperiments

showedthepossibility of using a decrease in the ratio as an index ofoviposition.  Field surveys showed capturing reproduction

・Winter&spring: highof the species via the ratio may be difficult, but it may besummer: low

put into practical use via eDNA concentration.  Of course,

autumn: increased
consistent with the ecology

eDNA analysis is expected to be used in reproduction, but

・remarkably high (May 12&19)

as the lunar periodicity of reproduction.

in the spawning season
eDNA analysis can be used

it is also expected to be used for observing ecology such

(Ebina) Thank you very much, Daiki.  Any questions or

for monitoring this species

comments?
copy.  In winter and spring, eDNA concentration was high.  
Individuals become active in these seasons. In summer,
eDNA was rarely detected or low.  Individuals aestivate in(Kelly)The use of two different loci to make theinferences about the species is really interesting. I think

this season.   In autumn, eDNA concentration increased.  

the idea of using them in combination is very powerful.  

Individuals recover from aestivation in this season. The

Did you measure inhibition?  Did you see different inhibition

fluctuation of eDNA concentration was consistent with

between the two different loci, 18S and COI?

the ecology of the Japanese sea cucumber. In addition,
eDNA concentration was remarkably high in the spawningseason.   From this, eDNA analysis can be used for themonitoring of this species.

(Takeshita) I used Environmental Master Mix.  It is strong

for inhibition, so in this study, I assumed there was no
inhibition of detecting eDNA.

(Allan) What filter pore size did you use?

nu-eDNA/mt-eDNA in the field(Takeshita) 0.7 μm.
(Allan) For the tissues and feces, did you just use a bloodand tissue kit and a standardized mass of DNA?

(Takeshita) I used the QIAGEN blood and tissue kit.  The・Even in the samples with high concentration, the ratio wassimilar to the others.

kit was used to extract eDNA for water samples, and theSperms (nu-DNA/mt-DNA: high) and eggs (low) offset the ratio?

same kit was used for tissues and feces.

High eDNA concentration came from floating larvae?

However,fornucleareDNA

concentrationtomitochondrial eDNA concentration, the ratio was similar(Allan) I like the plot where you compared the water
samples to the tissues and feces.   I wanted to see theto others, even in samples with high concentration.  This

pore size, thinking about what you might be capturing in

could be because sperm and eggs can offset the ratio or

the water.  It is probably a combination of those things.

because the high eDNA concentration might have comefrom floating larvae.

(Takeshita) In this study, the filter pore size was 0.7 μm.  
For this filter, eDNA can be captured in or out of organelles,
so maybe a little eDNA out of the organelles may not be
detected in some cases, but I did not care about the poresize of the filters in this study.

(Ebina) Thank you, Daiki.   The next speaker is Zack

Gold from the University of Washington.  “Fish community
dynamics over the past two decades revealed throughDNA metabarcoding.”
7

Talk 2Fish community dynamics over the past two decades
revealed through DNA metabarcoding
Zack Gold (University of Washington)

I am going to be talking about marine heatwave-driven

and they get every hydrographic variable that you could

shifts in larval fish community dynamics over the past two

want.  It is currently run by NOAA and state agencies in thedecades revealed through DNA metabarcoding.

State of California.

I want to acknowledge all of the coauthors andWith this data of 70 years, we now know the answer.  

funders.  There are a lot of people who have helped move

It was actually both the environment and overfishing which

this project along, so I would like to thank them for their

led to the collapse of sardines.   This was highlighted by

support.

Francisco Chavez’s work at MBARI in 2003, but also
by some work at the University of Washington from Tim

In the 1940s and 1950s, in both Japan and California,

Essington, a professor in the School of Aquatic and Fishery

there was a dramatic crash in sardine fisheries.  In California

Sciences (SAFS).  We now know what caused the shift,in particular, this led to a really huge economic crisis.

but it is too late to go back and prevent the collapse of thesardine fishery.

Cannery

Row

inMontereywas

economicallydevastated by the loss of the fishery.   It was the largest

That brings up the limitations of the CalCOFI survey.  

fishery in the United States at the time.   At both the

It is really time and labor-intensive. Oftentimes, this leads

federal and state governments, they had no idea how thisto a four to five-year delay between when they capture ahappened because a fishery had never been overfishednet sample and when they actually identify all the species

before.

of fish and invertebrates in the sample.  It also requires thehighest level of taxonomic expertise.  It is very difficult to

At the time, scientists pointed fingers at each other,

identify fish eggs and larvae.  A lot of the eggs do not haveclaiming either the environment or overfishing as the cause.  

any morphological characteristics, so you cannot identify

No one knew the answer, so the California Cooperative

them at the species level.

Oceanic Fisheries Investigations (CalCOFI) program wasdeveloped.

That leaves us largely blind to real-time ecosystem
dynamics, and so if we have a marine heatwave like we
did in the Northeast Pacific from 2014 to 2016, we will
not know what happened to those ecosystems until 2019

CalCOFI Sampling History

when we finally get the data turned around.  By then, we
have already been fishing for five years.

 Grid Surveys Started 1950
 Quarterly cruises

What that means is we need a new monitoring Hydrographic and biologicaltool that is rapid, reliable, standardized, accurate, anddata

cost-effective.   That is the promise of eDNA and DNA

 Led by NOAA SWFSC,

metabarcoding technologies.

CDFW, and SIO

What we did here is to compare DNA metabarcoding

8

The idea was to get independent information fromwith microscopy in a non-destructive way.   There is afisheries on fish recruitment and ichthyoplankton data

picture of a CalCOFI net tow sample preserved in ethanol.  

using a grid survey. It is actually one of the longest marine

We do not touch or disturb the sample.  We filter the ethanol

ecosystem surveys taken continuously to date.

They

and do DNA metabarcoding side-by-side with microscopy

combine hydrographic and biological data. They do net

work so that we can have a comparison of what we aretows that capture both zooplankton and ichthyoplankton,

find under a microscope with what we get with eDNA.

STUDY DESIGN

Amplification Efficiency Predicts Detection

 4 Stations
 23 years (1996,1998-2019) 3 Technical PCR

Replicates each 2 barcodes - MiFish

Sebastes

SD O
OffshoreffshoreeThis is the study design.  We picked four stations thatare representative of the main habitats that we have: theCalCOFI Data

We are finding that species with high amplification efficiency
have the highest detection or true positive rate.

California Current, the countercurrent, and more variable
sites.   We have 23 years of data, dating back to 1996.  
We have three technical PCR replicates for two MiFish

Amplification Efficiency Predicts Abundance

universal primers, as well as the MiSebastes, a rockfishspecific primer that I helped develop with a PhD student at

Spearman’s ρ isa metric ofcorrelation

the University of Washington.

Ranges -1 to 1
With 0 being nocorrelation

Before jumping into the results, I think it is importantto understand PCR bias.   Ryan, Ramon, and Andrew
Shelton (2019) have a great theoretical framework for

Kelly et al. 2019

understanding how the propagation of small biases ofcertain species amplify slightly better than other species

At the same time, the modeling efforts also looked

for the same barcode results in driving the ratios thatat how well we can track biomass for a species.   For a

we see of different species, basically leading to somespecies with high amplification efficiency, again, we expect

species amplifying much better than others for the samethat to correlate quite well.

metabarcoding assay.

Amplification Efficiency Predicts Detection

Kelly et al. 2019

Amplification Efficiency Predicts Abundance

We actually showed similar results with the CalCOFI
data.   The species with high amplification efficiency had

What they were able to demonstrate with modelingthe strongest correlation in biomass.   This is exciting

efforts is that for species with high amplification efficiency,

because it ties the theory to what we expect from empirical

we expect there to be a strong likelihood of detection.  We

data.  This demonstrates that understanding amplification

can detect a species that has good amplification efficiency,

efficiency is important for interpreting eDNA metabarcoding

but our ability to detect a species with low amplification

data for ecological results.

efficiency in a given assay is going to be quite low.
Why are the real fits not as good as the theory?  There
We compared that to the data we generated from ourare a couple of reasons. One is these are paired bongo

CalCOFI study and we actually found very similar results.  

nets, so the ethanol and formaldehyde are coming from9

different sides of the nets, and it is possible that they are

set up mock community experiments ahead of time to be

not the exact same ichthyoplankton communities and thatable to know how well each species amplifies for a given

there is microspatial structuring.  We are also comparing

locus.

two imperfect methods.   Microscopy cannot identify
very many species, and many eggs are hard to identify.

How do we use this towards accurate reconstruction

Also, extracellular DNA does not really matter.  With DNA

of marine ecosystems, going back to the bigger question

metabarcoding, it does not matter what the source is.  

of how marine ecosystems are changing in response to

It can amplify it.   As long as there is mitochondrial DNA

marine heatwaves?

floating around in the ethanol, the DNA metabarcoding will
pick it up.  However, DNA metabarcoding is not a perfectmethod either.

What we were able to show is that eDNA is able toidentify two main groups of species.  We have warm andcold-associated species assemblage groups. The vast

What we are able to show is that the amplification

majority of the species are subtropical.  A lot of them are

efficiency is the key threshold.  We can use this information

myctophids.   We are seeing these species showing up

then to consider if these are species we can trust.  If wein marine heatwave events, whereas a lot of the fisherytrust the true positive rate and the correlation with biomass,

targets like hake, sardine, anchovy, and some of the main

we can use these species to focus the ecological analyses

rockfish taxa are recruiting much better in colder years.

because we know that our method is working well forthese species.

Looking at the whole ecosystem, we can see the1998 El Nino, which was one of the warmest years on

Amplification Efficiency Predicts Abundance

record, with the marine heatwaves which were a little bitcooler, but still three of the top seven hottest years everrecorded in the California Current.  Those are all clustering
together and we are getting a lot of tropical and subtropical
species showing up in our coastal waters associated withthese marine heatwave events.

CalCOFI Data

However, it is not an easy and straightforward story.  
We also got unexpectedly high anchovy recruitment in

These are a couple of examples.   One is Pacific

the California Current during some of the warmest years

sardine.  We show that the eDNA index tracks quite wellever recorded. That helped break the idea that has beenwith the log of abundance.   We see the same thing forlongstanding in the literature that sardines are warm-

anchovy.   These are two of the forage fish species thatassociated and anchovies are cold-associated taxa.  

are the targets of the CalCOFI work.  This is really exciting

eDNA metabarcoding was able to pick that up and track

because it is showing that we can actually get estimates ofthis interesting phenomenon that happened. Basically,

relative abundance from eDNA data, which is going to be

climate change and marine heatwaves that are becoming

important for ecological analyses.

more frequent are going to impact our fisheries and our
ecosystems in unexpected and non-linear ways, so we

How do you use amplification efficiency?   We wereable to show that amplification efficiencies track wellneed a tool that gives us the biggest and fullest picture ofmarine ecosystems, and eDNA can provide that.

with biomass and detection rates, but you can calculate

10

these amplification efficiencies from mock community

With eDNA, we can get a much broader view of

estimates.   This is done in a paper by McLaren, Willis,

biodiversity.   We are getting about three times as manyand Callahan(2019).   They did very good analyses

species as we are identifying via microscopy.   A lot of

demonstrating that from different extraction methods, asthem are important fishery targets.   eDNA can provide

well as PCR biases, you can then correct data to use this.  

quantitative estimates which are going to be important for

It is demonstrating that amplification efficiency is important

a lot of ecological analyses. In addition, it is much cheaper

for the interpretation of metabarcoding results.  Also, they

and automatable, so it is scalable to something like the sizeare easy to do because you just need to get tissues andof the CalCOFI where they are collecting upwards of 1,000

samples in net tows per year.  Also, we can get faster results
which are going to be needed for dynamic management.  
Realistically, we can expect results in a couple of months
instead of four to five years, with enough investment.  Oneinteresting advantage is that we can access archived
samples from 23 years ago without damaging them, so
future studies can still look at the eggs or larvae and use
the ethanol preservative to get ecosystems reconstructed.

with metabarcoding.

(Yamanaka) How do you obtain basic information on theamplification efficiency for each species by using a mockcommunity?   I believe the results will be affected by thebiomass ratio between species in the mock samples.  How doyou treat the issue related to these variations?

(Gold) I recommend the Mclaren, Willis, Callahan (2019)
paper.   It has really good examples of three different mock(Ebina) Thank you very much, Zack. Do you have any
questions or comments?

(Jo)  In real-world eDNA metabarcoding experiments, how
can we know the amplification efficiency?

communities and how they take information and calculate theamplification efficiency.   Even if you make all the species ofinterest even and add the same concentrations of extracted
tissue sample to the mock community, you can at least knowthat they are all even starting concentrations.   You would
expect with perfect metabarcoding, the same number of(Gold) It is something that you cannot directly calculate from areads from every single species put in a mock community.  We

water sample collected from seawater, but it is something thatknow that never happens. So you can start even, look at the

you can calculate by doing a mock community experiment.  

relative abundance of each different species, and calculate theFor example, if you took all the fish species that show up in

amplification efficiencies.  The paper takes three microbiome

Maizuru Bay and you put them in a mock community, you

examples with multiple different mock communities, and thencould see what the relative amplification efficiencies are,demonstrates how to do those calculations.

especially if you put them all at known starting concentrations.  
That number is static. It is inherent to the method you areusing, the marker you are using, and the species that are in thesample.  So you could do a little mock community experimentand go back to samples that you have already collected oranalyses that you have already done, and you can correct thedata once you know what the amplification efficiencies are.
Something that Ryan and I have been working on is thatyou can estimate the amplification efficiency.   Basically, youfind ones that have high amplification efficiency.   It is fairly(Kelly) The evidence we have so far is that the compositionof the community does not change amplification efficiency
very much.  It seems there are more primers than DNA, somaybe there is no real competition.

(Minamoto) I could not catch the methodology. Did you

extract DNA from the museum samples of individuals orfrom ethanol?

(Gold) Basically, it is the cod end of a plankton tow that got

easy because they are usually obvious and show up in many

stored in ethanol, so there are hundreds of species in eachsamples and have high read counts.   Where it becomesjar.  Also, they have been untouched for 23 years.  Theydifficult is if you have a rare species.   It might have reallyjust put them in the museum.  We took a large pipette andhigh amplification efficiency, but if they only show up in one

pipetted the ethanol off the top of the sample, and then put

sample, you cannot distinguish them from the backgroundit into a filter and did the same as we would with seawater

noise of other species. Therefore, I think a mock community

eDNA.   I think on average it was 125 mL of ethanol thatis ultimately the best way to get at that, but you can also figure

went into the sample, and then we got the filter and did aout which species are doing well if they are showing up in all of

QIAGEN DNeasy extraction kit.   We then amplified them

your samples with high reads.  Those species clearly amplify

for the MiFish universal primer, as well as the MiSebastes

quite well for your metabarcoding locus of choice.

rockfish primer, and used the CALeDNA protocols.  They

(Kelly) The equation we have been using is amplicons~ [biomass] * (1+a)^NPCR-cycles, so if you have someare very similar to the ones that Kevan uses at MBARI.

(Ebina) Thank you very much, Zack.  We would like toinformation about biomass and some information about

move to the next speaker, Toshiaki Jo.   “Performance ofthe number of amplicons, then you can estimate thebenzalkonium chloride as the preservative of environmental

amplification efficiency, “a.”DNA in seawater targeting longer and nuclear DNA

Reading the Cusick paper, there are many options to thinkfragments and fish community.”

about how to improve these kinds of quantitative questions
11

Talk 3

Performance of benzalkonium chloride as the preservative ofenvironmental DNA in seawater targeting longer andnuclear DNA fragments and fish community
Toshiaki Jo (Kobe University)

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Because of its high cost-performance, BAC has


As I introduced before, I am a PhD student inbeen used in many eDNA studies, particularly in Japan,

Minamoto Lab at Kobe University. My primary research

however the performance of eDNA preservation has beeninterest is the characteristics and dynamics of eDNA fromlimitedly evaluated by species-specific assays targeting

macro-organisms such as fish and vertebrates.   This is

short fragments of mitochondrial DNA in freshwater andsometimes referred to as the ecology of eDNA.  It includes

brackish ecosystems.   Therefore, I set two questions.  

source, state, transport, and fate.

Q1: Is BAC effective to preserve different fragment sizesof mitochondrial and nuclear DNA in marine ecosystems?  
Q2: Is BAC effective to preserve the community information,$"'!

such as richness and composition, inferred by eDNA

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based monitoring of invasive and endangered species
in freshwater ecosystems where eDNA in water samples
must be preserved after sampling as soon as possible12 )/-3&.&4", ($ &4"#"2$/%*.("33"9'/2#;**3)!02*-&23*9"&4",
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    because it degrades rapidly.   Among the strategies forI will briefly explain the design of the experiment.  In the

eDNA preservation, benzalkonium chloride (BAC) is known

tank experiment, rearing seawater of the stock tank withas an inexpensive and simple preservative of eDNA in

Japanese jack mackerel was transported into two samplingwater samples.   This preservative was used for the firsttanks.  BAC was then added to one of the sampling tanks,

time in Yamanaka et al. (2017), which showed that theand rearing water was collected and filtered for 60 hours.  In

addition of BAC substantially suppresses the degradation

the field sampling, seawater samples were collected fromof mitochondrial eDNA from freshwater fish in water

Maizuru Bay in Kyoto Prefecture.  BAC was added to half

samples.  In the field, we only need to add 1 mL of BAC

of them, and then water samples were filtered after water

solution to 1 L of water sample and mix it.

collection, after six hours, and after a day.

To answer Q1, four types of mackerel eDNA wereplots represent the decay rate constants in the initial andquantified by real-time PCR.   I targeted 164 base pairsecondary phases in biphasic degradation. In the tankfragments of cytochrome b gene and nuclear internal

experiment, the addition of BAC suppressed the initialtranscribed spacer 1 region, and more than 600 baserapid degradation, but not the following slower degradation

pair fragments of both genes. Monophasic or biphasic

for all types of mackerel eDNA.

exponential models were then fitted to each eDNA decay
curve to estimate decay rate constants. In the monophasic

In addition, the yields of target eDNA from the fieldmodel, the eDNA decay rate is consistent during timesamples hardly decreased throughout a day by thepassage. On the other hand, in the biphasic model, rapid

addition of BAC.

eDNA degradation occurs which is followed by a slower
degradation, divided by a breakpoint.  To answer Q2, eDNA
metabarcoding was performed targeting the fish community
in Maizuru Bay.  MiFish-U primers were used, and iSeq raw
reads were preprocessed and analyzed.
 0$%%$"1(3$1-./$0$/3$#(%%$/$,1%/ &+$,10(7$0-%+(1-"'-,#/( * ,# ,2"*$ /(,+ /(,$$"-0601$+0 ,)$5.$/(+$,1

40

60

80

100

0

20

40

60

80

Time point [hour]Time point [hour]

Nuclear rRNA ITS1 Short (164 bp)

Nuclear rRNA ITS1 Long (603 bp)

40

60

80

100

3
2

5

10

)$ $ ""###'"$$

BAC addition
Species name / Time point [hour]

Yes
0

Yes
6

Yes
24

No
0

No
6

No
24

Acanthogobius flavimanus
Acanthopagrus schlegelii
Aluterus monoceros
Chaenogobius gulosus
Decapterus maruadsi
Dentex tumifrons
Dictyosoma burgeri
Engraulis japonicus
Epinephelus awoara
Equulites rivulatus
Hyporhamphus sajori
Konosirus punctatus
Lagocephalus spadiceus
Lateolabrax japonicus
Leucopsarion petersii
Mugilogobius abei
Nuchequula nuchalis
Omobranchus elegans
Oplegnathus fasciatus
Ostorhinchus semilineatus
Pagrus major
Parablennius yatabei
Paralichthys olivaceus
Pseudolabrus sieboldi
Scomber japonicus
Scomberomorus niphonius
Seriola quinqueradiata
Siganus fuscescens
Sphyraena pinguis
Takifugu spp.
Thamnaconus modestus
Total

0
1
1
0
0
1
0
1
0
0
1
0
0
1
0
1
1
0
0
0
0
1
1
1
1
1
1
0
0
1
0
16

0
1
1
0
0
0
0
1
0
1
1
1
0
1
0
0
0
1
0
0
0
1
0
1
1
1
1
1
1
1
1
18

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
0
1
0
1
1
1
1
1
1
1
1
29

0
0
0
0
0
0
0
1
0
0
1
1
0
1
0
0
0
0
0
0
0
1
0
1
0
1
1
0
0
1
0
9

0
1
0
0
1
0
1
0
1
0
1
0
0
1
0
0
0
1
1
0
1
0
0
0
0
0
1
1
0
1
0
13

1
1
1
0
1
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
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1
1
1
1
1
1
0
15

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These are the results for Q2.   The list represents all

-1

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0

20

40

60

80

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100

5

10

15

20

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Time point [hour]Time point [hour]-,-.' 0("1  $5.)1

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Nuclear rRNA ITS1 Short (164 bp)

0

log10(eDNA conc.) [copies/2 µL template DNA]

5
4
3
2
1

20

0

100

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Mitochondrial cytochrome b Short (164 bp)

Mitochondrial cytochrome b Long (682 bp)

0

log10(eDNA conc.) [copies/2 µL template DNA]0

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Mitochondrial cytochrome b Short (164 bp)#11'$01 /1-%4 1$/"-**$"1(-,1(+$ < -#$*0$*$"1(-,! 0$#-,

DI=CAGMFKEH@$LNBJI>?
1 $"-,"$,1/ 1(-, 11(+$1"-.($09 ) #$" 6/ 1$"-,01 ,1'-2/ ) #$" 6/ 1$"-,01 ,1 1(,(1( *.' 0$'-2/ ) #$" 6/ 1$"-,01 ,1 10$"-,# /6.' 0$'-2/ 18 !/$ ).-(,1'-2/marine and brackish fish species detected from seawater
samples.  In total, 33 fishes were detected.  31 species
were detected from the samples with the addition of BAC,
and 24 species were detected from those without theThese are the results for Q1.  The decay rate curves

addition of BAC.  I then compared the number of detectedof each type of mackerel eDNA in the tank experiment andfish species between BAC treatments and compared thefield sampling are shown.  The x-axis shows the sampling

similarities of community compositions between BAC

time points and the y-axis shows the log-transformed

treatments and sampling time points.

eDNA concentrations.   The circular plots in each box
represent the datasets from the treatment with BAC, and       
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Biphasic degradation models were supported in the tank



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experiment, while monophasic models were supported(stress = 0.016)

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A7-9:0312'.,>4GB8/=;()*   ! $  A7-  5+,E?,DCF6/<@()*The left figure shows the summary of species richness
points.   Each Venn diagram represents a comparisonof the number of fish species detected from seawater

$!,&' #$) /  ) %$(*&&'((! *)$%)! %'"")-&(%#!'"  ZRDUSC\[ANWJ`T9IMO>:CPCaF6ANWBDE?K@^_8A6<; "(#&" $ /- "(%)') '"-'()'%*%*)--  ) %$ ZRBGH X]DMLJQ8=E?K@VY8A7A<;

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(  ) %$(*&&'(()') %$%+' %*()-&(%    "  ""$"" "$" 
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in the field sampling.  The addition of BAC increased theyields of all types of target eDNA at the start of experiment.

&% ! "
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NMDS2

the triangular plots represent the treatment without BAC.  



samples between BAC treatments.   Each numeralwith a two-way arrow represents the Jaccard-based
dissimilarity indices where a lower value indicates that the

The decay rate constants derived from the modelcompositions are more similar between treatments.   Infittings in the tank experiment are shown.  Black and white

addition, the right figure represents the NMDS product13

based on the Jaccard-based index for the fish community
among each BAC treatment and time point.   At all time(Jo) We might have confirmed biphasic degradation if thesampling time points were prolonged or increased.

points, the addition of BAC increased the number of fish
species from seawater samples.   Moreover, the additionof BAC decreased the dissimilarities of the fish community
among time points, implying that time series changes of(Kelly) It seems fantastic. Do you recommend everyone
to use BAC or is there any situation in which you would not
use BAC?

community compositions were mitigated by the addition of(Jo) The addition of BAC increased initial concentrations,

BAC.

but depending on water chemistries, maybe the addition
6..#3:+4%644+0/< *'13'4'/5456&:+/&+%#5'&#*+)*7'34#5+-+5:0( +/13'4'37+/)7#3+0645:1'40(#26'064'+/7#3+064'/7+30/.'/5#-%0/&+5+0/4 4'(('%5+7'5013'4'37'&+(('3'/5 4'(('%5+7'5013'4'37'5*'%0..6/+5:(3#).'/54+;'40(.+50%*0/&3+#-#/&+/(03.#5+0/+/('33'&$:'.'5#$#3%0&+/)

/6%-'#3+/.#3+/''%04:45'.4 +/%3'#4'&5*'41'%+'43+%*/'44#/& 46113'44'&'&')3#&#5+0/#/&.+5+)#5'5*'5+.'4'3+'4%*#/)'40(+/%3'#4'&+54+/+5+#-%0/%'/53#5+0/4

of BAC completes not only eDNA but also some PCR
inhibition substances.  For example, with very turbid water,
the addition of BAC would maybe rather cause PCR
inhibition.

%0..6/+5:%0.104+5+0/4

<:4+.1-:#&&+/)+/508#5'34#.1-'4$05*5*'26#/5+5#5+7'%01:/6.$'3 #/&26#-+5#5+7'41'%+'43+%*/'44#/&%0.104+5+0/+/(03.#5+0/0/#26'064'%#/'(('%5+7'-:$'13'4'37'&(Yamahara)&63+/)53#/41035#5+0/505*'-#$03#503:8*+%*806-&#--0850.03'13'%+4'-:'45+.#5'
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<gT?YW[mRCG[=Qb_dSSoPINSSi@pq^}IK

Do you think you can modify the BAC

protocol to be added to filters instead of water samples?

<nVSSj^uafdeRvJ[LGOSl `c>tl t<xr|w^lRoOE[P~BY\[<^pJ[HPRXZR{MFhzCXUyqSXZsQkD

RQ[L]A(Jo) Maybe, but I have not tried it.

%,/08-'&)'.'/54"'5*#/,3#,#5#0$'!/+7(034611035+/).0-'%6-#3'91'3+.'/5#/&$+0+/(03.#5+%43')#3&+/)'.'5#$#3%0&+/) *+4456&:8#44611035'&$:3#/5+/+&(03'4'#3%*'--083#/56.$'3   In summary, the present study indicated a highversatility of BAC in preserving various types of aqueous(Yamanaka)   I can answer the question.   I have tried

to preserve an eDNA sample in a Sterivex cartridge filter.  

eDNA in various environmental conditions.  In Q1, I foundI added the BAC to water at 0.01% final concentration,that the addition of BAC suppressed eDNA degradation

filtered the water with Sterivex, capped both ends with luer

and increased its initial concentrations for different

lock caps, and transferred the sample at room temperaturefragment sizes of mitochondrial and nuclear genes.   Into the lab during a few days of travel back to the university.  

Q2, I also found that the addition of BAC increased the

I did not confirm the efficiency, but it worked.

species richness in water samples and mitigated the timeseries changes of community compositions. By simply
adding BAC into water samples, both the quantitative andqualitative information on aqueous eDNA can effectively
be preserved during transportation to the laboratory, which(Ebina) Thank you very much, Toshiaki.  The next speaker
is Masayuki Sakata.   “Reconstructing past biologicalwould allow to more precisely estimate species abundance

information in Lake Biwa using sedimentary environmental

and biodiversity via eDNA analysis.

DNA approach.”(Ebina) Thank you very much, Toshiaki. Do you have any
questions or comments?

(Allan) I think the results you found between the biphasic

and the monophasic with the field are interesting.  Do you
have a hypothesis for why you did not see biphasic in thefield sampling?

(Jo)  The difference can be explained by the lower initialconcentration of eDNA or fewer sampling time points in thefield sampling relative to the tank experiment.

(Allan) So you are already at the second phase or you are

just not getting the resolution between time 0 and six hours?
14(Yamahara)  That is good news.

Talk 4

Reconstructing past biological information in Lake Biwa using
sedimentary environmental DNA approach
Masayuki Sakata (Kobe University)

My name is Sakata.  I am a PhD student in Minamoto

However, since eDNA in sediment is preserved forLab.  I am mainly in charge of eDNA metabarcoding.  I ama long time, it can be used to overcome this challenge.

interested in eDNA in sediment.   This is some research

Therefore, this research was conducted with the following

related to that.

two purposes.   First, an attempt was made to detect
past fish eDNA in sediment cores.  Second, the temporalvariation of eDNA concentrations was tested to see if it

could be a tracer of biomass variation.

        
  

! ! 

                    !# !#!"!$  

45.0 N 

 

35.4 N35.0 N

Lake Biwa

30.0 NThe loss of biodiversity is a serious environmental
130.0 E

140.0 E

35.2 N

problem on the planet. It is reported to be particularly 
 

40.0 N  ! !!   #!
 !#!! ! 

150.0 E

serious in freshwater environments.  Biodiversity monitoring    

35.0 Nis important to conserve biodiversity. In monitoring, it is136.0 E

important to understand where, what, and how much

136.2 E

species inhabit.   However, even if a diversity survey

The sediment cores used in this study were

was conducted in an environment, it would not be able

collected off the coast of Lake Biwa in 2019.  Lake Biwa

to understand the original biodiversity that has alreadyis the largest lake in Japan.  The sediment cores weredecreased.  It is important to know what kind of species

collected with a gravity corer.  The diameter is about 10

originally inhabited an area in order to achieve the goal of

cm and the length is about 30 cm.

ecosystem reconstruction.
Sediment cores contain the remains and fossils oforganisms and have been used to obtain information on
past organisms.  However, in the case of fishes, despite

&$%! &"%"$")"$

their importance in freshwater ecosystems, they do not 

leave direct traces such as remains in sediment, so pastreconstruction is a challenge.


                       
 




              

  

  

 


 
 

#%%#$&  

*&$&"!

 





!+%%

&&%&!+%%+#/$$%%"! "&&!)%#$"$ "#%,  &!&+$

-& $!& "$&.

  &)%'%%!!&"$"" %%!&)%("$    "!+This figure shows the workflow of eDNA analysis in the
study.  First, the sediment cores were sliced at a thicknessof 1 cm.   Next, three 10-gram samples were collectedfrom each sediment slice and eDNA was extracted.  Theextracted eDNA was analyzed by species-specific real-time
15

PCR for two target fish species, both of which are native toLake Biwa, to examine whether past fish eDNA could be

"$#"
 &"#")#!  #!#
$# "#%#!

detected in the sediment cores.  Next, type II regression
model fitting was performed to examine the relationship 

between eDNA variation and biomass variation.   CPUE 

""##!!""

data was used as an indicator of biomass, but it was only
available for Plecoglossus altivelis, so this analysis was
performed only for one species.

         
       ' *!!""  (  

Here, the relationship between eDNA concentration
and CPUE is shown.  In this graph, the vertical axis shows  

eDNA concentration and the horizontal axis shows CPUE.  
The analysis was conducted using data from 1975 to

2019 when CPUE data was available.  Statistical analysis
showed that there was no significant relationship between
the two factors, but a positive trend was observed.

 " 
Through dating, it was estimated that the sediment   ! #! !         

cores contained sediment of about the past 100 years.  
This graph shows the results for Gymnogobius isaza.
The vertical axis shows the eDNA concentration and thehorizontal axis shows the estimated date of sediment.  
The results of the analysis showed that the eDNA of thisspecies was detected in sediment slices corresponding to!%  %  %#!
!!!#! ! !"!  !

about 35 years ago at the oldest.

            

%" %
!  ! !

   

!!! $    !%!" 
 !"!   !

In this study, fish eDNA was detected in past
sediments.   Two species of eDNA could be detected in
sediment layers corresponding to 35 years ago and 100   years ago, at the oldest, respectively. Fish information



was a gap in past reconstructions, especially in freshwater
ecosystems, however sedimentary eDNA analysis may
have the potential to overcome this gap.
On the other hand, there are several issues. One ofthem is the difference in temporal detection limits amongfish species, even though both target species must have
inhabited Lake Biwa during the period when the sediments

This graph shows the results for Plecoglossus altivelis.

This can be attributed to several

The vertical axis shows the eDNA concentration and thefactors.   For example, differences in initial concentration

horizontal axis shows the estimated date of sediment.  

due to different biomass and release rates, or spatially

The results of the analysis showed that the eDNA of thisheterogeneous distribution of eDNA in sediments.   The

species was detected in sediment slices corresponding toresolution of such issues is a future challenge.

about 100 years ago at the oldest.
16

contained them.

sediment examples and test for detection or no detection.

  
 %  " " !$ #
 " 

maybe from 400 or 500 years ago, so no detection would
probably mean degradation.

(D’Agnese) I saw that your timeline went back 100 



(Minamoto) We have records of species in Lake Biwa

years. Is that as long as the core sample that you took or
was it only a portion of it?
 

(Sakata) In this study, the max age was 100 years ago.  
Maybe we will collect longer cores and examine older

In conclusion, two purposes were set for this study.  

environments.

The first purpose of detecting past fish eDNA in sedimentcores was achieved.   On the other hand, the second
purpose of tracking biomass variation by eDNA was notcompletely achieved, but a positive trend was suggested.  

(Ebina) Thank you, Masayuki.  The next speaker is Abigail

Keller.   “Tracking a marine invasion front using molecularsurveys.”

Therefore, one future issue to achieve this purpose. I amvery much looking forward to the future development of
past reconstruction using sedimentary eDNA.

(Ebina) Thank you, Masayuki.  Do you have any questions
or comments?

(Kelly) This is quite exciting.   I did not realize that you
could get DNA from so long ago in sediment.  I was justreading your paper about the comparison of sedimentand water DNA, and so this is the logical extension of thatwork.  Of course, there is some degradation over time.  Is
that a term in the model for CPUE?  How are you thinking
about degradation over time so that you are not comparing
directly 1920 and today?

(Sakata)

I understand that problem, but this is a

proof of concept.   The comparison of CPUE and eDNA
concentration is a challenging issue, but a positive trend
was suggested.  In the future, maybe we will calculate thedegradation rate in sediment cores to compare CPUE andeDNA concentration.

(Allan) When you see no DNA in earlier years and thenyou start to see DNA 30 or so years ago, how would you
separate out whether it is an introduction of a species or a
detection limit because of decay?  Are there other tools or
historical data that you could use?

(Sakata) That is a very difficult problem.  For example,
after the introduction of an alien species, we will collect17

Talk 5Tracking a marine invasion front using
molecular surveys
Abigail Keller (University of Washington)

I am Abby Keller.   I am a Master’s student at the
University of Washington working with Ryan Kelly.

   

     

This study focuses on the European green crab.   ItIn 2012, they made their way into the Salish Sea
which is a shared water body between British Columbia in
Canada and Washington in the United States.

is one of the most harmful aquatic invasive species in theworld.  It is also one of the most widespread.  It is foundon all continents except Antarctica.  They are successful
invaders because they are robust and can survive awide range of temperatures and conditions.   They also
reproduce in large abundances. The picture in the topleft is a female green crab with about 400 eggs.   They
can negatively impact ecosystems because they dig up
eelgrass habitats, outcompete native shore crabs, and arealso aggressive and voracious predators. This predation
has been designated as the singular main cause of thecollapse of a soft-shell clam fishery on the East Coast of
the United States.  As waters are warming, they are able toexpand into new ranges and survive in new locations.

Managers are really concerned about habitat
alteration and fishery resources in the Salish Sea,so they were interested in developing an early
detection eDNA monitoring tool for the green crab.  

      

We used and refined an existing qPCR assay thatwas developed by scientists in British Columbia,
and then to test it in the field, we collected water
samples at 27 sites all around the Salish Sea andalso along the coast of Washington.  Green crabs
like marshy, soft-bottom habitats, so 20 of thesesites were highly suitable for green crabs andseven of them were unsuitable.   Across these    

sites, we have a range of known presence andabundances of green crabs.

In the 1990s, they were first discovered on the West
Coast of the U.S. and have since then been making their
way north.
18

What we are trying to understand is how thedetection or non-detection of green crabs relates   
   state, which is binary.  It is either truly occupied or truly notoccupied by a species.  We were finding that this binary   
# !'%'%$

latent state was insufficient for our data because each site ')                crabs. The probability of detecting green crabs depends &(             
seemed to have a different underlying abundance of green
on abundance, so the estimates of occupancy dependon abundance as well.  We wanted to find a way to make      

better use of the quantitative information that we had.  We   

to find a way to incorporate both the trapping data and thealso had information from trapping efforts, so we wanted

to the presence and absence or density of green

eDNA qPCR data so that they can mutually inform eachcrabs in the environment.

other.

&" *) $+#0)
"$%$(,"- (    
 

 ' $"-%$%, $%&' $" +%  

     ! %  
 $  "       

A few government groups conduct regular monitoring

In order to use all available information to estimate

for green crabs. They put out a baited trap, leave it there

green grab density and presence, we developed this joint

overnight, come back the next day, and then remove any

model that uses both trapping data and qPCR information.  

green crabs caught in the trap.  We have some estimates

The symbols and colors are the parameters estimated byof green crab densities from these trapping efforts at many

the model.   We used trapping data that were collectedof our sites.  These trapping efforts are conducted by bothwithin a three-week window of when our water samples

the U.S. government and the Washington Department offor eDNA were detected so that they both would capture

Fish and Wildlife.  In addition, Washington Sea Grant has a

the same environmental status, but with different forms ofcommunity science group called the Crab Team that helps

sampling.

expand monitoring efforts.

The number of crabs per trap at site “i” is anindependent draw from a negative binomial distribution,!  $  #
   !#                 

 '$!%,&** +$*#%&*)' $"%          and the mean of that distribution is the true green crab
density.   We then incorporated our qPCR false positive
detections.  The number of detections at site “i” is being
drawn from a binomial distribution with a probability
detection that is broken up into two parts.  The first part,
p11, is our true positive probability of detection. This is ourlink between the qPCR data and the trapping data.  As thetrue green crab density increases, so does the true positive
probability of detection.  False positives are an issue with

Occupancy modeling is a technique that was

eDNA surveys, so p10 is our false positive probability. Thisoriginally developed for wildlife studies, but recently theyis the probability of a molecular detection, given that thehave been applied to understand detection histories fromtrue green crab density is actually zero.

single-species eDNA data.   These models estimate theAlso, this model is formulated under a Bayesian

probability of occupancy of a species at a site, as wellframework.  In this type of statistics, you can incorporate

as the probabilities of true positive and false positive

extra information into the distributions that inform thedetections. These models are contingent upon the latentestimated parameters, so we included an informative prior19

distribution on p10 using information from our negative
controls.  This model is useful because it incorporates two

" .!   $ *#%$ '"""$#types of data that arise in different ways from the same   ""#$(0!  $$ "$(*/ " $# 



shared underlying crab density.



We wanted to directly compare how the addition of
eDNA information influences crab density estimates.  Thisis the output of two separate models.   One model uses

only trapping data and the other is our joint model thatuses both qPCR and trapping data.  For each paired box  

As green crab density increases, the probability ofdetecting them with molecular methods also increases.  
This is fundamental to the model.

This probability ofdetection then reaches a saturation point at higher densities.
Therefore, if you do enough qPCR application, you can get
a decent estimate of the probability of molecular detection
and then relate it back to the underlying green crab density.

plot, the left is the estimated density from the joint modeland the right is the estimated density from the trap-onlymodel.  Across all of our sites, there were differing trapping
efforts.  Some of our sites had 60 traps set over the threeweek period and some only had three traps set, so I used
two different color schemes.  The lighter colors represent
the sites that had a larger trapping effort and the darker
colors represent the sites that had a lower trapping effort.
The sites where the estimates of green crab density!
  


  

diverge between the two models are those where there
was a lower trapping effort.  For example, with DEL and TIT,
by adding qPCR data to the model, it narrows the credibility
intervals so that we can more confidently say that green
crabs are likely not there.   On the other hand, with STA
and RAA, by adding qPCR information, we can estimate
non-zero estimates of green crab density and say that they
are likely at low abundances that cannot be detected using
such a low number of traps set.

Using this joint model of both qPCR and trapping data,

we have been able to improve estimates of green crab
density and track the invasion front. The map on the left
is trapping data over the three-week time period that weused that overlapped with our eDNA collection.   This is
the number of crabs caught per trap. There has been ahistorically high abundance of green crabs at the coast, as
well as at Drayton Harbor at the border of Canada and the     "
 "$ ##"#

United States.
The map on the right is the output of our model. Thisis the estimated green crab density that is informed byboth eDNA and trapping data.  We have some non-zeroestimates of green crab density in places that they weretrapped.  In particular, the two sites in the bottom-right aremuch farther south than green crabs have ever been seen
before.
20

I think the biggest conclusion is that eDNA sampling isuseful to supplement low trapping efforts to see if they are
there at low abundances or not.  What I found interestingis that the two sampling methods of trapping and qPCR
eDNA arise from the same underlying crab density inconsistent ways.

    !# " (Keller) Yes, I considered a positive detection as any
amplification below 38 cycles in any of the replicates.

(Kelly) The model treats each replicate separately, so it
would be counted as one detection out of 15 attempts.

(Takeshita) You did qPCR with 15 replications.  Why did
you choose 15?  I feel that it is a lot of replications.

(Keller) I took five water samples at each site, and thenThese are the limitations.   At least in the southernsites where we are detecting green crab eDNA where
we have never seen them before, maybe we are actuallydetecting larval DNA because there is not a great way of
distinguishing life stages.   Maybe we are detecting nonviable organisms, maybe it is a crab molt, or maybe it isfor each water sample, I did three replicates.  In retrospect,
I think you would want so many replicates, especially forgreen crabs, because they have a relatively low shedding
rate, so they are harder to detect than other taxa like fish
which are maybe shedding mucus in higher abundances.  
Therefore, by having the high replication, we were able toeDNA that has been resuspended from the sediment.  

better estimate the probability of detection.

However, I think from a management perspective, these(Takeshita) I have another question about qPCR

uncertainties might not matter as much, because this isreally most useful to be able to figure out where to increase
and direct future management and removal trapping efforts
for monitoring the invasion front.

detection.   How many copies are the limit of detection

and the limit of quantification?   The detection may be
dependent on the limit of detection.

(Keller) The limit of detection is about 16 copies.  I am

not sure about quantification.  We were finding that most

(Ebina)  Thank you very much, Abby.  Any questions orof our positive qPCR had high cycle numbers, so beyondcomments?

the limit of quantification, we were doing qPCR, but not(Jo) How did the crab invade the U.S.?  Did it move by

were probably not using qPCR to its full capability.

itself or was it moved by some anthropogenic activity?

(Keller) Most likely anthropogenic activity. I think overlonger distances, it is probably ballast water in ships.  Over

shorter distances, you can have a recreational boat wherea crab is accidently stuck in someone’s shoe, but I think forthe most part, probably ballast water.  A lot of the sites thathave historically high abundances are harbors where you
would expect ballast water exchange to be happening.

(Jo)  Are the crabs used for recreation or for eating?
(Keller) Certain areas have been trying to turn them intoa culinary delicacy to try to manage them, but they areusually not eaten.

(Jo)  You performed real-time PCR to detect the invasive
crabs.  What is the threshold of detection or non-detectionin the 15 PCR replicates?

(Keller) The threshold of detection is about 38 cycle
thresholds.

quantitatively.  If we got a hit, it was one.  In that sense, we(Takeshita) 16 copies per 1 L?
(Keller) I think it was 16 copies per 1 µL.
(Minamoto) You incorporated the false positive rate into
your model.  How did you estimate the false positive rate?

(Keller)  We have negative controls at different stages ofthe process like the qPCR stage and the extraction stage.  
Given that, we estimated that the probability of a false
positive detection was probably less than 0.01, and so wegenerously set it at 0.01.

(Kelly) The model is a Bayesian framework, so we canuse the negative controls to inform our prior distribution
on the false positive rate, and then let the model find theposterior distribution for that parameter.

(Ebina) Thank you, Abigail.  Let us move to the last talkby Mingyang Jiang.  “Assessing nutritional status of carpby environmental nucleic acid analysis.”(Jo) Do you mean you considered the target eDNA wasdetected if one of the 15 replicates was eDNA positive?

21

Talk 6

Assessing nutritional status of carpby environmental nucleic acid analysis
Mingyang Jiang (Kobe University)

My name is Mingyang. I am a second-year Master’s
student in Kobe University.  I am doing research on eRNA.

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organisms. This research focused on the nutritional statusof the organism.

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.0& -(1,5(2'$Compared with traditional methods, eDNA has

8 .5$4$02'$0$(1-./0$4(.31123#7.%$4 +3 2(-&2'$-320(2(.- +12 231.% -
.0& -(1,31(-&$ -#$
8 !)$"2(4$ 4$0(%75'$2'$0$ $" -!$31$#2.$4 +3 2$-320(2(.- +
12 231some benefits.   For example, we do not have to spend
a lot of time and money, and it is not invasive.   eDNA

The RNA and DNA ratio in tissues is used as andetects species by analyzing DNA released by organisms

index of growth, nutritional status, and condition of

into the environment.   It does not require morphologicalmacroorganisms.   Compared with poorly-nourishedtaxonomic expertise because it identifies species based

individuals, well-nourished individuals have a higher

on their DNA sequences.  eDNA analysis has been widely

RNA to DNA ratio.   However, there is no previous study

used in studies, but it is difficult to identify the status andevaluating the nutritional status of an organism using eRNAdevelopment stage of an organism with eDNA.  In order toand eDNA.  This study verifies if the eRNA to eDNA ratio

solve such problems, eRNA analysis is being considered.

can be used to evaluate nutritional status.

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8 '(10$1$ 0"'%."31$#.-2'$-320(2(.- +12 231.%2'$.0& -(1,In recent years, research on the detection of eukaryotic
RNA from environmental water has increased.   RNAIn this experiment, I used common carp as thedegrades rapidly after cell death, so it can be consideredexperimental organism.   The experiment was conducteda “living” signal of an organism.  RNA expression patterns

for three weeks.  One carp was placed in each of 12 waterchange due to physiological conditions and environmental

tanks.  Six of the carp as the feeding group were fed dailystress.  If RNA can be detected from environmental water,

with an automatic feeder.  The other six were not fed as theit may be possible to extract more detailed biologicalnon-feeding group during the entire experiment.

information from water such as the health status of living
22/.+""!%*#$,#",

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used to test whether the passage of days and the; "#4&23#-0,&2&&%*.(7#4&26/,5-&feeding or non-feeding had a significant effect on eDNA

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concentration.  The random variable was the water tank.  

"#4&23#-0,*.(  &#$)'/2&#.%&*,42#4*/. =- The response variable was the eDNA concentration.  The

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explanatory variables were the passage of days, feeding; &842#$4*/.&#39 ,//%!*335&*4

or non-feeding, and the interaction between days and; &842#$4*/.5$,&/ 0*. ,53+*4
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feeding or non-feeding.  It was found that the feeding or; &#,4*-&5$,&#2-#2+&2!

non-feeding did not affect the eDNA concentration.

I took 0.5 L of water samples twice a week each for

eDNA and eRNA.  I took carp muscle tissue as a tissue
DNA, as well as RNA to synthesize complementary DNA.  
After that, real-time PCR was performed on the nuclear 

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marker, ITS1.

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sample on the last day of the experiment, then extracted$*#"-%*"#+* "*.,.%+*

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These are the results of changes in eRNA concentration.  
A generalized linear mixed model was used to test whether'&&%*.( ./.'&&%*.('&&%*.( ./.'&&%*.() ! %! %!# '% %* ) !#%("# %%#'$ !$  %#  %' % 
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the passage of days and the feeding or non-feeding had
a significant effect on eRNA concentration.  It was foundthat, over time, eRNA concentration decreased in the nonfeeding condition compared to the feeding condition.

#!&" % !  #!&"!(! # $&%$%"  

These are the results of carp condition factor before
and after the experiment.   Before the experiment, there

group.

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after the experiment, there was a significant difference
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group and the non-feeding group. On the other hand,

was no significant difference in CFK between the feeding 

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9%"72'&&%*.(/1./.'&&%*.(0   These are the results of changes in the eRNA toeDNA ratio.  A generalized linear mixed model was used
to test whether the passage of days and the feeding or
non-feeding had a significant effect on the eRNA to eDNA
ratio.  It was found that, over time, the eRNA to eDNA ratio
decreased in the non-feeding condition compared to the8'$ %$$#(-& .0 -.-%$$#(-& #(# -.2 %%$"2 2'$ $ ".-"$-20 2(.-/  

feeding condition.

These are the results of changes in eDNA
23

ratio.   It is possible to monitor and improve the growth   /'!

(Ebina)'&&%*.(./.'&&%*.(

Do you have any(D’Agnese)  How did you take the muscle tissue?  Did

you euthanize the fish and excise muscle?

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Thank you, Mingyang.

questions or comments?

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8process of protecting rare species and aquaculture.

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8and mortality of fish using the eRNA to eDNA ratio in the
%*"*0%,+*)"*.(1.",+*.$"(-.!2
%*.%--/"-+*.$"(-.!2

These are the results of the RNA and DNA ratio on thelast day.  It was found that the ratio of RNA to DNA of ITS1
in tissues in the feeding group was higher than in the non-

(Jiang) Yes.
(D’Agnese) Did you do a post-mortem necropsy withpathology to see if there were any underlying health issues

feeding group.   Similarly, the ratio of RNA to DNA in theor were these fish previously known to be healthy?  How did

environmental water was higher in the feeding group thanyou verify that the lack of RNA expression was nutritionalin the non-feeding group.

stress and not potentially other underlying confounding
health issues?

 

(Jiang) Sorry, I do not understand what you are talking 7123#713&&$1212' 22'$-320(2(.- +12 231.%%(1'" -!$$12(, 2$#!72'$

about.

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2.2'$2.2 +-3"+$(" "(#0 2(.&$-$0 ++731$# 1 -(-#$6.%-320(2(.- +12 231My study suggested that the nutritional status of fish

(D’Agnese) Were the fish verified as healthy to start with?
(Jiang) Yes.
(D’Agnese) So you did not necessarily do a necropsy
with any pathology post-mortem?

can be estimated by the ratio of eRNA to eDNA.   eRNA
concentration decreased with the non-feeding condition.  
Non-feeding may cause the deteriorated nutritional status
and decreased RNA expression.  In this experiment, ITS1
was used to compare the nucleic acid ratio in tissues.  Itis necessary to examine whether the nucleic acid ratio of
ITS1 corresponds to the total nucleic acid ratio generally
used as an index of nutritional status.

(Jiang) Yes.
(D’Agnese) If they were healthy, that is fine.
(Kelly) This is very exciting because I think it is a wayof telling live animals from dead animals. You compared
good condition animals and poor condition animals, but
live and dead would be even bigger.  It would be an evenbigger difference and that is very exciting for people who 

want to know if they found a live fish or a dead fish.  I think
this is a way to do that.

  

          (Gold) If you were taking a sample in the field and not            

      from an aquarium, what kind of sampling do you think you
would need to take to be able to distinguish the status ofa fish, whether it is fed or not fed, to a fish that was maybe
there from a couple of days ago?  You would expect the

In conclusion, my study suggested that the nutritional
status of fish can be estimated from the eRNA to eDNA
24

eRNA ratio to be lower because DNA lasts longer thanRNA.

(Jiang) I think it is difficult to distinguish between goodcondition fish and bad condition fish because of the RNA
degradation, but it is a problem that we are going to solve.

(Minamoto)I think our method can be used inaquaculture or some limited conditions.

(Yamanaka)  I want to know the validity.  You used the

RNA and DNA concentrations in tissue samples of carp as
an indicator of the condition of fish, but I am not sure it is
the best indicator for your study.  In your experiment, you
used starvation as the trigger to change the health statusof fish, but is there any other better indicator to measure
the condition of fish in this case?  Maybe using a different
tissue would give you a better relationship.  What do you
think about that?

(Jiang) You mean if I use other tissues I can get better
results?

(Yamanaka) Yes.   I am not sure, but maybe different

types of stress will affect different tissues.   It could be a
future experiment theme.  Please think about that.

(Ebina) Thank you very much, Mingyang.   Now, we
would like to move to the overall discussion.

25

Discussion(Ebina)   First, I would like to ask anybody who did not

you would with other organic molecules in cores or older

make a self-introduction yet to do so now.

samples.

(Yamanaka)(Kelly) You could use eels, Hiroki.   In your new paper,

I am Hiroki Yamanaka, an associate

professor at Ryukoku University.

I think you had examples of places eels have been
introduced, with known abundances over time.

(Doi) I am Hideyuki Doi from the University of Hyogo. I am
working on eDNA things also.

(Gallego) You could try mRNA, cDNA, and metabarcodingin that order to see if you also see a change in the mRNA

(Saito) I am Tatsuya Saito. I am studying the degradation

with time.

of eDNA at the University of Hyogo.

(Ebina)  Okay, now we would like to move to the overall
discussion.  Do you have any comments or questions?

(Kelly) I do not have anything particularly intelligent to say,but it is so nice to see people’s faces when we read yourpapers and to try to make friends across an ocean. Thisis real treat for all of us and I would just like to say thank(Yamanaka)

I want to discuss the future usage ofyou very much.  I think it is exciting to see all of the different

sedimentary eDNA.  Does anyone have any idea of how toapproaches and technological advances come together.

calibrate the eDNA concentration in each layer of the core?

I think it is a particularly exciting time to see the science,
imagination, and creativity that are just now coming out(Minamoto) We can sometimes estimate the degradationof the eDNA in sediment from the concentration of theof the lab, being used by government agencies to makedecisions in the real world.

profile or organic materials.

(Kelly) You could maybe do a one or two-year study.  If(Ramon-Laca)   Jo, can you explain what BAC does?  
How does it preserve the sample?

there is an exponential decay, most of the decay happens
quickly in the time that you do the study, and so you could
bury some fish extracts in the sediment at different levels(Jo)  BAC is a kind of cationic surfactant which adsorbs
the cell surface of microbes and inactivates them.

for a time and then calibrate the degradation with a known
sample over the first year or two.  If the exponential decay(Ramon-Laca) So it prevents degradation by microbes?

is consistent, you could carry it out over time. You cannot
go back in time to 100 years ago, but you might be able to(Jo) Yes, it is considered that BAC can indirectly preserve

constrain the possible values of 100 years ago from more

eDNA by suppressing enzymatic degradation.  A detailed

recent samples.

explanation can be seen in Yamanaka et al. (2017) andsome citations in it.

(Gold) Another way you could calibrate it is if there wasa well-studied system.  For example, a river that we have

(Yamahara)  Back to Hiroki’s question, I was wondering if

been keeping track of species abundance for a really long

there was a way to use the ratio of eRNA to eDNA to looktime and we know a lot about the abundance.   It couldat degradation over time. In the individual slices, if youeven be better if you picked a system where there wasare looking for one given species, you may presume thata complete loss of a species or an introduction of a new

there should be both eRNA and eDNA in each slice.  That

species.  There could be a natural experiment somewhere.  

is a chronological record that you already have based on

A dam removal could be a useful experiment to calibrate

the slices and depth, but if you also look at the eRNA tothe degradation rates.

eDNA ratio, there are going to be some degradation anddifferences between each individual slice.  There could be

26(D’Agnese) I have an off-the-wall idea.  You could maybe

a way to get an approximation of eDNA age from looking

partner it with some DNA and use stable isotope metrics

at that ratio as it changes chronologically in the sedimentof measuring the half-life of DNA molecules in samples likecore, because one would expect the eDNA to stick aroundmuch longer than the eRNA.

Minamoto and Prof. Doi.  It seems like a big development.  
Where will you be taking the quantification next?

(Kelly)  The size fractionation of the DNA is also a possibility

for looking at age.  They are maybe independent ways ofassessing age.   If you have a few independent lines ofevidence, you can constrain the possible age.

(Doi) We just tested in mesocosms.  It is a kind of mockcommunity.   Actually, this technique is very difficult to
directly apply to field samples because it needs many
DNA copies in the PCR templates, but Dr. Hoshino, a(Yamanaka)   Thank you for your ideas.   I think wecoauthor of this paper, and I are now thinking about a

can use some of your ideas to develop new techniquesmore developed qSeq technique that needs much lessto calibrate the data.   I am not an expert, but probably

DNA copies in the PCR templates.   I am not sure, but

multiple factors like pressure and oxygen concentration will

we should probably provide a more sensitive technique for

affect the degradation in each slice of the core, so it will be

qSeq in the near future.

hard work, but I think we have to do it.

(Gold)   I have a question for everyone.   I was reallyfascinated by reading one of Hideyuki Doi’s papers about(Kelly)I heard from Hiroki about the potential for a

Japanese eDNA meeting at some point in the future.   Isthere any update? Is there a possibility of that meeting?

the number of water bottle replicates and technical PCR
replicates needed to saturate species diversity in an
aquarium.  Going forward, what would you recommend in(Yamanaka)   Thank you for asking.   We actually
postponed the meeting to next year, 2022.  We are still notterms of taking samples from coastal marine ecosystems?

sure if we can make it an in-person meeting or not, but the

How many water bottle replicates and how many PCR

place has already been decided.  It will be held in Kyoto if it

replicates do you expect we should be taking to saturate

is in person.  It could be a good attraction for empowering

diversity?

researchers, so I want to make it an in-person meeting, but
we are still not sure.  We are looking at the situation related(Doi)   We actually tested how many filters and PCR

replicates affect the detection of the community using

to COVID-19.  I will let you know when we decide how todeal with it.

MiFish metabarcoding and we found PRC replicates to be
more important than filter replicates.  That means that weshould increase PCR replicates more than filter replicates,(Kelly) In the United States, different people inmanagement agencies and the government are thinking

but I am not sure how we should take water bottle

about what to do with this new technology and this new

replicates for different habitats.  I would like to mention thatway of understanding the world, and how it relates to ourwe should keep many PCR replicates – for example, eight

old way of understanding the world.  In Japan, have theor 16.  We can then maybe take more species richness

government science and management agencies adopted– probably 85%.  We usually use eight replicates for the

eDNA as a tool?  How is that going?

metabarcoding, but for field volume or field replicates, it isa difficult question.  We usually use 1 L or 10 L in marine
habitats and only one site per coastal regional, but I think it(Minamoto) I think it is the very early stage of usage of

eDNA in Japan, especially for the Japanese government.  

depends on the aim of the study or survey.

Yamanaka-san, please help me.

(Minamoto)   We have some data about the sampling(Yamanaka) Hideyuki and Toshifumi are board members

habitat.   In our experience, in lakes or rivers, we do not

Japanese government.   They are talking about how tohave to take so many field replicates, but in the case ofgo forward with the new techniques of eDNA and eRNA.  

dam reservoirs, we have to take many field replicates to

The Japanese government has big interest in the newdetect enough species, so it highly depends on the habitat.

technology, but they are still watching how it will be because

design and the number of replicates. It depends on theof a working group hosted by some ministries of thethere are many black boxes.  This is my personal opinion,(Kelly)  I was interested about the qSeq paper from Prof.

but I think most of the governmental officers want to use27

it as a quantification method of fishery resources.  There

are still trying to figure that out, but we are going in thatare unknown factors affecting the eDNA concentrations,direction.

so they are now looking at the progress of the research touse it as a quantification tool for the fishery species, but weare still talking frequently with government side to promote

MinION?  I have heard that it is far lower than the benchtop

the usage of this new technique.

machine, so how do you deal with it?

(Kelly) It is the same here. The government is interested(Yamahara)

to count fish.  They need quantification, but there are many

you are looking for very specific taxonomic groups, they

other uses as well such as finding endangered species

have been developing some algorithms to look at all theor invasive species, or even being able to understand

individual sequences and aligning them so that you canwater pollution and water quality in a practical way.  I thinkget some confidence in your error for your base calls

right now it is with the academic scientists and engineers

individually.  That is just an issue with MinION and I think itlike Kevan to try and make this a tool that works and towill get better.  In my personal opinion, I do not like using

demonstrate that, and then as it becomes easy to use, theit, but it is the only format that will allow us to do in situ

government will become happier with using it.

sequencing at the moment.

(Yamanaka) Kevan, I am very interested in the progress(Ebina) Thank you very much, everybody.   Prof. Itoh,in this as a technology, but there are people who only wantof your research work on the robot behind you.  What kindof development are you working on right now?  I rememberthat your robot is working on species-specific detection.  
How about metabarcoding?  Are you trying to develop arobot for that?

(Yamahara)

Yes.   We have two instruments, thesecond-generation ESP and the third-generation ESP.  On
the second-generation ESP, we are not developing any
new detection technologies.   What we have now is ourprobe arrays and qPCR, and I think that those will be themain detection technologies that we will focus with on that.  
On the third-generation ESP, we are always investigating
new technologies to do detection, whether it is specificlike qPCR or digital PCR.  We are also working on a thingcalled surface plasmon resonance.
We are also working with Oxford Nanopore MinION
for onboard sequencing.  The difficulty that we are facingwith that is data because it is gigabytes of data.  How doyou send that over a telecommunications network?  When
you are out in the middle of the ocean, you are sending
everything by satellite.   It is very expensive and you can
only transfer kilobytes of data, so we are working on data
reduction.  Right now, we have a prototype system that weare working on that goes from sample selection all the way
through to MinION sequencing, but the data reduction isa bottleneck that we are having issues with, as well as thesequencing part, and so there are some trade-offs.  We
28(Yamanaka)   How is the sequencing quality of the

There still are problems with their basecalling, but it is getting better and better.   I think whenwould you please make the closing remarks?

Closing(Itoh)  I am Masayuki Itoh, the Director of Kobe University Science Shop, the host of today’s workshop.  My background isastrophysics, but I still enjoyed the talks today.  I am very happy to see this event happen because I have been observing
Prof. Ebina working so hard to prepare.  One of the objectives of the Science Shop is to promote dialogue and collaboration
between citizens and scientists.  From that point of view, eDNA seems to have very high potential.  I hope to see anothermeeting to discuss such aspects of this wonderful technique.  To conclude, I would like to express my gratitude to Prof. Kelly,Prof. Minamoto, Prof. Ebina, and all the other participants today.

Participants in SKIACI 2021
Toshifumi Minamoto (Kobe Univ)
Daiki Takeshita (Minamoto Lab, Kobe U)
Toshiaki Jo (Minamoto Lab, Kobe U)
Masayuki K. Sakata (Minamoto Lab, Kobe U)
Mingyang Jiang (Minamoto Lab, Kobe U)
Takehiro Matsumoto (Minamoto Lab, Kobe U)
Qianqian Wu (Minamoto Lab, Kobe U)
Natsumi Kihara (Minamoto Lab, Kobe U)
Hayato Higashisaka (Minamoto Lab, Kobe U)
Tetsu Yasashimoto (Minamoto Lab, Kobe U)
Luhan Wu (Minamoto Lab, Kobe U)
Ryan Kelly (Univ of Washington)
Zack Gold (Kelly Lab, NOAA)
Abigail Keller (Kelly Lab, UW)
Ana Ramón-Laca (Kelly Lab, UW)
Elizabeth Allan (Kelly Lab, UW)Disha Patel (Kelly Lab, UW)
Joe Duprey (Kelly Lab, UW)Erin Rose D’Agnese (Kelly Lab, UW)
Joe Duprey (Kelly Lab, UW)Disha Patel (Kelly Lab, UW)
Ramon Gallego (NOAA)
Kevan Yamahara (Monterey Bay Aquarium Research Institute)
Hiroki Yamanaka (Ryukoku Univ)
Hideyuki Doi (Univ Hyogo)
Tatsuya Saito (Univ Hyogo)
Junko Kobayashi (Hyogo Business & Cultural Center, Seattle)
Hideaki Kawachi (Hyogo International Association)
Hideyuki Yamamoto (KULOS, Kobe Univ)
Masayuki Itoh (Kobe Univ SciShop)
Kuniyoshi Ebina (Kobe Univ SciShop)

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