Kobe University Repository : Kernel

タイトルTit le

Rapid degradat ion of longer DNA fragments enables the improvedest imat ion of distribut ion and biomass using environmental DNA

著者Author(s)

Jo, Toshiaki / Murakami, Hiroaki / Masuda, Reiji / Sakata, Masayuki K. /Yamamoto, Satoshi / Minamoto, Toshifumi

掲載誌・巻号・ページCitat ion Molecular Ecology Resources,17(6):e25-e33

刊行日Issue date 2017-11

資源タイプResource Type Journal Art icle / 学術雑誌論文

版区分Resource Version author

権利Rights

© 2017 John Wiley & Sons Ltd. This is the peer reviewed version of thefollowing art icle: [Molecular Ecology Resources, 17(6):e25-e33, 2017],which has been published in final form atht tp://dx.doi.org/10.1111/1755-0998.12685. This art icle may be used fornon-commercial purposes in accordance with Wiley Terms andCondit ions for Self-Archiving.

DOI 10.1111/1755-0998.12685

JaLCDOI

URL http://www.lib.kobe-u.ac.jp/handle_kernel/90004748

PDF issue: 2020-06-13

1

Title: 1

Rapid degradation of longer DNA fragments enables the improved estimation of distribution 2

and biomass using environmental DNA 3

4

Authors: 5

Toshiaki Jo1, Hiroaki Murakami2, Reiji Masuda2, Masayuki Sakata1, Satoshi Yamamoto1, and 6

Toshifumi Minamoto1 7

8

Affiliations: 9

1Graduate School of Human Development and Environment, Kobe University: 3-11, 10

Tsurukabuto, Nada-ku, Kobe City, Hyogo 657-8501, Japan 11

2Maizuru Fisheries Research Station, Kyoto University: Nagahama, Maizuru, Kyoto 625-12

0086, Japan 13

14

Corresponding author: 15

Toshifumi Minamoto 16

Graduate School of Human Development and Environment, Kobe University: 3-11, 17

Tsurukabuto, Nada-ku, Kobe City, Hyogo 657-8501, Japan 18

2

Tel / FAX: +81-78-803-7743 19

Email: minamoto@people.kobe-u.ac.jp 20

21

Running head: Longer eDNA improves fish biomass estimation 22

23

Keywords: 24

decay rate; DNA fragment length; echo intensity; environmental DNA (eDNA); Japanese 25

Jack Mackerel (Trachurus japonicus); quantitative real-time PCR 26

27

28

3

Abstract 29

The advent of environmental DNA (eDNA) analysis methods has enabled rapid and wide-30

range ecological monitoring in aquatic ecosystems, but there is a dearth of information on 31

eDNA degradation. The results of previous studies suggest that the decay rate of eDNA varies 32

depending on the length of DNA fragments. To examine this hypothesis, we compared 33

temporal change in copy number of long eDNA fragments (719 bp) with that of short eDNA 34

fragments (127 bp). First, we isolated rearing water from a target fish species, Japanese Jack 35

Mackerel (Trachurus japonicus), and then quantified the copy number of the long and short 36

eDNA fragments in 1 L water samples after isolating the water from the fish. Long DNA 37

fragments showed a higher decay rate than short fragments. Next, we measured the eDNA 38

copy numbers of long and short DNA fragments by using field samples, and compared them 39

with fish biomass as measured by echo intensity. Although a previous study suggested that 40

short eDNA fragments could be overestimated because of non-target eDNA from a nearby 41

fish market and carcasses, the eDNA concentrations of long fragments were correlated with 42

echo intensity. This suggests that the concentration of longer eDNA fragments reflects fish 43

biomass more accurately than the previous study by removing the effects of the fish market 44

and carcasses. The length-related differences in eDNA have a substantial potential to improve 45

estimation of species biomass. 46

47

4

Introduction 48

Global biodiversity loss is currently one of the most critical ecological challenges, particularly 49

in the ocean (Dulvy et al. 2003; Worm et al. 2006), but it is generally difficult to obtain 50

accurate information about species distribution and population size. For example, traditional 51

survey methods such as visual surveys, capturing, and tracking with biotelemetry, require 52

substantial efforts and costs (Henderson et al. 1966; Brill et al. 1993). Moreover, the accuracy 53

of species identification depends on the observer’s ability. 54

Environmental DNA (eDNA) analysis is a new monitoring method that can 55

overcome such problems (Ficetola et al. 2008; Takahara et al. 2012; Minamoto et al. 2012). 56

Environmental DNA, which is the DNA shed by organisms into the environment (Ficetola et 57

al. 2008; Lodge et al. 2012; Thomsen et al. 2012a), is thought to derive from skin, urine, 58

feces, and mucus (Martellini et al. 2005; Ficetola et al. 2008; Merkes et al. 2014; Barnes et al. 59

2016). The presence of a target species can be estimated by detecting eDNA from water 60

samples without locating or capturing individuals (Lodge et al. 2012). These advantages of 61

eDNA analysis have enabled quick and wide-range assessments of species presence/absence, 62

biodiversity, and abundance in freshwater (Thomsen et al. 2012a; Fukumoto et al. 2015; 63

Yamanaka et al. 2016; Dougherty et al. 2016) and marine environments (Foote et al. 2012; 64

Thomsen et al. 2012b; Port et al. 2016; Yamamoto et al. 2016). 65

5

However, some technical challenges still remain unexplored in eDNA 66

methodologies. For example, it is difficult to know when the detected eDNA was released 67

from an individual: how many hours have passed since the eDNA was shed? Environmental 68

DNA has been shown to persist in aquatic environments or terrestrial soils for hours to 69

months (Dejean et al. 2011; Goldberg et al. 2013; Barnes et al. 2014; Merkes et al. 2014). 70

Thus, the species that released the detected eDNA might already be absent at the time of 71

eDNA detection. In addition, applications of eDNA analysis to migratory fish species require 72

knowledge of time scale information because precise timing and location information is 73

required to monitor these species. 74

Previous studies might suggest the answer to this problem. It has been shown that the 75

detected copy number decreases exponentially or biphasically after removal of the target 76

species (Dejean et al. 2011; Barnes et al. 2014; Maruyama et al. 2014; Eichmiller et al. 2016; 77

Minamoto et al. 2017), that there is a negative correlation between the length of DNA 78

fragments and the detected copy number (Deagle et al. 2006), and that the difference in 79

detection using eDNA metabarcoding might be a result of longer persistence of the shorter 80

12S rRNA fragment (~100 bp) than the longer cytochrome b (CytB) fragment (460 bp) in lake 81

water (Hänfling et al. 2016). According to these findings, we can hypothesize that the decay 82

rate of eDNA varies depending on the length of DNA fragments: a longer DNA fragment 83

6

decays more rapidly than a shorter one. In this study, to test this hypothesis, we compared 84

temporal change in the copy number of a long eDNA fragment (719 bp) with that of a short 85

eDNA fragment (127 bp), using Japanese Jack Mackerel (Trachurus japonicus) as a model 86

species. We first developed primers and probes that targeted a longer DNA fragment than 87

previous studies of eDNA did. Then, we isolated rearing water from the target fish and 88

monitored the copy number of the long and short eDNA fragments in water samples for 48 h. 89

In addition to the tank experiment, we quantified longer eDNA fragments in field samples 90

obtained in a previous survey (Yamamoto et al. 2016), and compared the result with the 91

distribution of biomass estimated from echo sounder data. 92

93

Materials and methods 94

Primers and probe development 95

In this study, we used two primer/probe sets that specifically amplified the Japanese Jack 96

Mackerel DNA, targeting two different DNA fragments of the same gene Cytb. One set of 97

primers and probe, which targeted a short DNA fragment (hereafter “Primer S”), was taken 98

from Yamamoto et al. 2016. Primer S was designed to specifically amplify a 127-bp fragment 99

of the mitochondrial CytB gene: forward primer, 5′-CAG ATA TCG CAA CCG CCT TT-3′; 100

reverse primer, 5′-CCG ATG TGA AGG TAA ATG CAA A-3′; probe, 5′-FAM-TAT GCA 101

7

CGC CAA CGG CGC CT-TAMRA-3′ (Yamamoto et al. 2016). Another set of primers and 102

probe, which targeted a long DNA fragment (hereafter “Primer L”), was designed to 103

specifically amplify a 719-bp fragment of the mitochondrial CytB gene with Primer Express 104

3.0 (Thermo Fisher Scientific, Waltham, MA, USA) with default settings, using sequences of 105

the Japanese Jack Mackerel CytB gene, which was used in the previous study (Yamamoto et 106

al. 2016), from the National Center for Biotechnology Information. 107

Then we checked the specificity of both primers as follows. Each 20 µL TaqMan 108

reaction contained 2 µL DNA extract (one individual of Japanese Jack Mackerel or Amberfish 109

[Decapterus maruadsi], the species most closely related to the target species in the surveyed 110

area, was used as a template), a final concentration of 900 nM forward and reverse primers 111

and 125 nM TaqMan probe in1 × Taqman Gene Expression PCR Master Mix (Thermo 112

Fisher Scientific). Using both primer sets, quantitative PCR (qPCR) was performed with the 113

following conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 114

60°C. For each DNA sample, qPCR was performed in duplicate. In addition, a 2-µL pure 115

water sample was analyzed simultaneously, in duplicate, as a negative control (PCR negative 116

control). Quantitative PCR was performed using a StepOnePlus Real-Time PCR system 117

(Thermo Fisher Scientific). Additionally, qPCR products were verified on 2% agarose gels 118

stained with Midori Green (NIPPON Genetics Co, Ltd., Japan). 119

8

120

Tank experiment 121

Experimental setup and water sampling 122

We conducted tank experiments to verify that the decay rate of eDNA varies depending on the 123

length of the DNA fragments. The experiment was conducted at the Maizuru Fisheries 124

Research Station of Kyoto University on August 9–11, 2015. Three black polycarbonate 200-125

L tanks were prepared and three Japanese Jack Mackerels were kept in each tank for 1 week 126

prior to the experiments. Total length (TL) and weight of each Japanese Jack Mackerel used 127

for this experiment was measured after the experiment (Table 1). Filtered seawater, which 128

was pumped up from 6 m depth at the station, was used as inlet water into each tank (900 129

mL/min). In each tank, the temperature was kept constant using a chiller, and aeration was 130

performed using a pump. Fish were fed a small amount of krill every morning until the day 131

before water sampling. We cleaned the bottom of each tank an hour after feeding to eliminate 132

the effect of the feces, and on the sampling day the fish were starved. For sampling, 100 L of 133

each rearing water was transferred to other tanks from which we sampled. Soon after isolating 134

rearing water, we collected 1 L of sampling tank water. The time when we started the first 135

water sampling was defined as time 0, and the water was sampled at 0.5, 1, 1.5, 2, 4, 6, 8, 10, 136

12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, and 48 h after time 0 (hereafter, those time points 137

9

are referred as time 0.5–48). There were 22 total sampling time points. At each sampling 138

time, we also filtered 1 L of artificial seawater as a filtration negative control. Moreover, 1 L 139

of inlet water was sampled from each tank at time 24 to evaluate the background Japanese 140

Jack Mackerel eDNA concentration in the inlet water, because the seawater was collected 141

from the sea, where Japanese Jack Mackerel potentially occur. 142

At each sampling time, we immediately filtered the 1-L sample through a 47-mm 143

diameter glass microfiber filter GF/F (nominal pore size 0.7 µm; GE Healthcare Life Science, 144

Little Chalfont, U.K.). Filtering devices (i.e., filter funnels [Magnetic Filter Funnel, 500 mL 145

capacity; Pall Corporation, Westborough, MA, USA], 1-L beakers, tweezers, and sampling 146

bottles used for water sampling) were bleached after every use, using 0.1% sodium 147

hypochlorite solution for at least 5 minutes. The filters were placed in a freezer immediately 148

after filtration until eDNA extraction. 149

150

DNA extraction 151

Total eDNA was extracted from each filter using a DNeasy Blood and Tissue Kit (Qiagen, 152

Hilden, Germany). Briefly, a sample filter was placed in the suspended part of a Salivette tube 153

(Sarstedt, Nümbrecht, Germany). Then, 420 µL solution, composed of 20 µL Proteinase K, 154

200 µL Buffer AL, and 200 µL pure water, was put on the filter and the tube was incubated at 155

10

56°C for 30 min. After incubation, the liquid held in the filter was collected by centrifugation. 156

To increase the yield of eDNA, the filter was re-washed with 200 µL TE buffer for 1 minute 157

and the liquid was again gathered by centrifugation. We added 500 µL ethanol to the collected 158

liquid, and transferred the mixture to a spin column. Subsequently, we followed the 159

manufacturer’s instructions and total eDNA was eluted in 100 µL AE buffer. The eDNA 160

samples were placed in a freezer until quantitative PCR. 161

162

Quantification of eDNA using qPCR 163

To evaluate the amount of eDNA derived from Japanese Jack Mackerel at each time point, 164

quantification of the copy number of CytB genes was performed using real-time TaqMan 165

PCR with the StepOnePlus Real-Time PCR system. To quantify the number of Japanese Jack 166

Mackerel CytB genes in each 2-µL eDNA solution sample, we simultaneously performed 167

qPCR using a dilution series of standards containing 3 × 101 – 3 × 104 copies of a linearized 168

plasmid that contained synthesized artificial DNA fragments of the full CytB gene sequence 169

of Japanese Jack Mackerel. In addition, a 2-µL pure water sample was analyzed 170

simultaneously as a negative control in the PCR (PCR negative control). Each 13.3-µL 171

TaqMan reaction contained 2 µL DNA extract, a final concentration of 900 nM forward and 172

reverse primers, and 125 nM TaqMan probe in 1 × Taqman Gene Expression PCR Master 173

11

Mix. Quantitative PCR with Primer S was performed with the following conditions: 2 min at 174

50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C. The qPCR with Primer L 175

was performed with the following conditions: 2 min at 50°C, 10 min at 95°C, 55 cycles of 15 176

s at 95°C, 30 s at 60°C, and 1 min at 72°C. All qPCRs for eDNA extract, standards, and PCR 177

negative control were performed in triplicate. The DNA concentration of each water sample 178

was calculated by averaging the triplicate. We treated all positive replicates as successfully 179

quantified (no “limit of quantification” was set). Each replicate with non-detection (PCR-180

negative) was regarded as containing 0 copies (Ellison et al. 2006). The performance of the 181

qPCR assays is shown in Table S1. 182

We used a linear mixed model to evaluate the differences in the decay rate of eDNA 183

depending on the amplification target length of each primer set with R version 3.2.4 (R Core 184

Team 2016) using the function lmer of the R package lme4 (Bates et al. 2015). In this model, 185

we considered log-transformed eDNA concentrations in each tank as the dependent variable, 186

and each time point (hour) and primer set (Primer S or L) were included as explanatory 187

variables. Tank replicates were included as random effects. The slopes of the two regression 188

lines, one based on each primer set, should be different if a significant interaction effect of the 189

explanatory variables is observed. Note that, as the temperature of each tank before time 2 190

was higher than it was after time 4 (Fig. S1), we also ran the models using only the data after 191

12

time 4, because it has been shown that eDNA degrades rapidly in warmer environments 192

(Strickler et al. 2015; Roussel et al. 2016). The significance threshold was set at 0.05. 193

194

Application to field samples 195

Quantification of Japanese Jack Mackerel’s eDNA was performed using qPCR with Primer L. 196

The eDNA samples used here were those used in Yamamoto et al. 2016, and thus eDNA 197

concentrations with Primer S were cited from Yamamoto et al. 2016. Seawater sampling was 198

conducted on June 18, 2014 in west Maizuru Bay, Japan. Seawater samples (1 L) for eDNA 199

analyses were collected both from the sea surface using buckets and from ~1.5 m above the 200

bottom of the sea using Van Dorn water samplers at 47 sites. Quantitative PCR reaction 201

conditions for Primer L were the same as above. Quantitative PCR for seawater samples, 202

standards, and PCR negative control were performed in duplicate. The DNA concentration in 203

each water sample was calculated by averaging the duplicates. We treated all positive 204

replicates as successfully quantified. Each replicate with non-detection was regarded as 205

containing 0 copies (Ellison et al. 2006). The performances of the qPCR assays are shown in 206

Table S1. Three of the detected DNA samples were commercially sequenced, and all were 207

confirmed as target sequences. 208

13

We calculated correlation coefficients for echo intensity and DNA concentrations of 209

each primer set with R version 3.2.4. Here, echo intensity data were also cited from 210

Yamamoto et al. 2016, who obtained echo intensity, using a calibrated quantitative echo 211

sounder, as a biomass index of Japanese Jack Mackerel. An acoustic survey was also 212

conducted on June 18, 2014 in west Maizuru Bay, Japan. The echo sounder surveys started 213

from the mouth of the bay and moved southwest to the end of the bay (the location of 214

Maizuru Bay is shown in Fig. 2). It can be assumed that signals detected via echo sounder in 215

June in Maizuru Bay predominantly indicated Japanese Jack Mackerel (see Yamamoto et al. 216

2016 for detail). Five levels of horizontal range (buffer area) and four levels of vertical range 217

were set to define the water columns reflecting the spatial pattern of eDNA concentration 218

inside the bay. Horizontal ranges were within a 10, 30, 50, 150, and 250 m radius from each 219

sampling station, and vertical ranges were within 2, 5, and 10 m from both the surface and 220

bottom at each sampling station, as well as the entire vertical range of the sea. Because neither 221

surface nor bottom distribution of eDNA satisfied the normality and homoscedasticity 222

assumptions, which was verified by performing Shapiro-Wilk and Bartlett tests (P < 0.05), 223

Spearman’s rank correlation coefficients were used for the comparison of eDNA data and 224

Japanese Jack Mackerel distribution. The significance threshold was the same as above. In 225

this analysis, the sites where no eDNA was detected with either primer set were eliminated. 226

14

227

Results 228

Primers and Probe development 229

Primer L was designed as below: forward primer, 5′-AAT CCT CAC AGG TCT TTT CCT 230

AGC TA-3′; reverse primer, 5′-ATT GAT CGG AGA ATG GCG TAT G-3′; probe, 5′-FAM-231

TAC CAT TCG TCA TTG CAG CCT TCT TTG TTC-TAMRA-3′, producing a 719-bp 232

amplicon. As a result of qPCR and agarose gel electrophoresis, Japanese Jack Mackerel DNA 233

was amplified by both S and L primer sets, while amplification of Amberfish DNA was not 234

observed. We checked primer specificity using NCBI Primer Blast, and only CytB gene 235

sequences of Japanese Jack Mackerel were hit as complete match sequences to our designed 236

primers. 237

238

Degradation curves for long and short amplicons 239

Depending on the length of the DNA fragments, slopes of the two regression lines based on 240

all eDNA concentrations at each time point differed significantly (P < 0.05; Fig.1). Although 241

one of filtration negative controls (at time 8) and one of the inlet water samples showed 242

eDNA amplification, these copy numbers were much fewer than those of experimental tanks. 243

In addition, all of the PCR negative controls showed no eDNA amplification. Thus, the 244

15

effects of Japanese Jack Mackerel eDNA included in the inlet water and cross-contamination 245

among samples during filtration and qPCR could be neglected. 246

In another model, which used only the data after time 4, the slopes of the two 247

regression lines also differed significantly (P < 0.001). The decay curves of primers S and L 248

were estimated as CS (t) = 507.3e-0.044t and CL (t) = 158.74e-0.09t, respectively, where Ci(t) is eDNA 249

concentration at time t as measured by the Primer i (S or L) (Fig. 1). 250

251

Comparison of eDNA and echo intensity in the field survey 252

The qPCR data from seawater samples with each primer set and echo intensity data were 253

compared. The distribution of Japanese Jack Mackerel eDNA concentrations in west Maizuru 254

Bay is shown in Fig. 2. The copy number of eDNA differed significantly between the surface 255

and the bottom with both primer sets (Wilcoxon signed rank test; P < 0.05). With Primer L, 256

Japanese Jack Mackerel eDNA was detected at 15/47 sites (surface) and 8/47 sites (bottom), 257

while it was detected with Primer S at 46/47 sites (surface) and 40/47 sites (bottom). For 258

Primer S, eDNA concentrations of surface samples were significantly higher than those of 259

bottom samples (P < 0.05), while for Primer L, eDNA concentrations between the surface and 260

the bottom showed a marginally significant difference (P = 0.05097). The average 261

16

concentrations with Primer L were 25.4 copies/L (surface) and 4.7 copies/L (bottom), while 262

those with Primer S were 479.1 copies/L (surface) and 317.9 copies/L (bottom). 263

Spearman’s rank correlation coefficients between eDNA concentration and echo 264

intensity are shown in Table 2. On the surface, eDNA concentrations with Primer L showed a 265

significantly positive correlation with echo intensity of 150 or 250 m in radius horizontally 266

and the entire water column vertically (i.e., from surface to bottom). These correlation 267

coefficients were 0.61 (P = 0.02) and 0.59 (P = 0.02) for the radius of 150 m and 250 m, 268

respectively. On the other hand, eDNA concentrations with Primer S showed no significant 269

correlation with any echo intensity data sets. 270

For bottom collected samples, those eDNA concentrations found with Primer L had 271

no significant correlations with any echo intensity data sets, while those with Primer S had a 272

significant negative correlation with echo intensity of 50 m in radius horizontally and 2 m 273

vertically, and the correlation coefficient was -0.35 (P = 0.03). However, there was no 274

correlation between them when excluding the two outlier sites (see the discussion). 275

276

Discussion 277

In this study, we were able to successfully show that decay rate of eDNA varied depending on 278

the length of the DNA fragment. Previously, some studies have indicated that, though longer 279

17

DNA fragments are present at lower concentrations in the field, they may represent more 280

recent biological information (Hänfling et al. 2016; Bista et al. 2017). However, our study is 281

the first to directly measure the degradation rates of shorter and longer eDNA fragments. Our 282

results might expand the application of eDNA techniques such as monitoring in time series 283

and estimating population abundance and biomass. 284

In the tank experiment, we used a linear mixed model to evaluate the differences in 285

the decay rate of eDNA depending on the length of DNA fragments, except the datasets 286

before time 2 because the temperature of each tank before time 2 was higher than at later 287

times (Fig. S1), so we considered that eDNA data before time 2 should be divided from those 288

of later. Actually, eDNA decay in this experiment showed a period of rapid decay (i.e., the 289

initial 2 h) followed by a period of slower decay, which is considered to correspond with the 290

change in temperature. The effect of temperature on eDNA degradation has been shown 291

previously (Strickler et al. 2015; Lacoursière-Roussel et al. 2016), and eDNA decay rate is 292

correlated with water temperature. On the other hand, Eichmiller et al. (2016) showed that 293

common carp eDNA exhibited biphasic exponential decay, characterized by rapid decay for 3294

−8 days followed by slow decay, in spite of a constant temperature during the experiment. 295

Further study would be needed to clarify the underlying mechanisms. 296

18

Under the assumption that eDNA decay starts after it is shed from individuals, eDNA 297

concentration at time 0 should theoretically be the same regardless of the length of the DNA 298

fragment, but eDNA concentration at time 0 estimated with Primer S was about 10 times as 299

much as that estimated with Primer L. This difference in eDNA concentration at time 0 300

suggests that eDNA had already degraded before it was released into the environment. For 301

instance, if feces are the origin of eDNA, the DNA must have already degraded when the 302

feces were released from the body. The two exponential decay curves based on Primer S and 303

L intersect with each other at t = -25.3 h, indicating that eDNA started to degrade the day 304

before sampling. For example, gut cell DNA included in feces should already be decayed 305

before release from the body. Similarly, other hypothetical sources of eDNA, such as mucus 306

and epithelia (Martellini et al. 2005; Merkes et al. 2014; Barnes et al. 2016), might be 307

decayed before shedding. Our findings suggest that the time point at which DNA molecules 308

start to degrade is not always equal to the point when eDNA is released into the environment 309

from the individuals. 310

Based on previous studies, we hypothesized that longer DNA fragments show lower 311

detection probabilities because longer DNA fragments could be more damaged by 312

environmental factors. In this study, the fragment sizes we used were 127 bp and 719 bp, and 313

other fragment sizes were not tested. The length-dependent change of DNA decay rate could 314

19

be clarified using other fragment sizes, such as ~300 bp and ~500 bp; further studies are 315

needed to clarify this. 316

In the field survey, targeting short DNA fragments, the copy number of Japanese 317

Jack Mackerel eDNA at the surface was significantly higher than that at the bottom 318

(Wilcoxon signed rank test; P < 0.05), while there was a marginally significant difference 319

between the copy numbers at the surface and at the bottom when targeting long DNA 320

fragments (P = 0.05097). Thus, eDNA of Japanese Jack Mackerel is distributed more at the 321

sea surface than at the bottom. It has been reported that when Japanese Jack Mackerel larvae 322

were collected in the East China Sea, over 95% were collected in the upper 30 m layer (Sassa 323

et al. 2006). The differences of the eDNA distribution between the surface and the bottom in 324

our study may be correlated with this distribution. 325

We compared the echo intensity and eDNA concentrations measured with two 326

primer sets (S and L) to clarify whether the eDNA decay rate varies depending on the length 327

of DNA fragments in the field, as it was thought that these decay rates should be the same in 328

the field and in the tank experiment (Fig. 2). On the sea surface, eDNA concentrations with 329

Primer L showed a significantly positive correlation with echo intensity of 150 or 250 m in 330

radius horizontally and the entire water column vertically, while those with Primer S showed 331

no significant correlation with any echo intensity data sets (Table 2). This result suggests that 332

20

detection of longer eDNA can improve the accuracy of estimations of fish distribution or 333

biomass. Yamamoto et al. (2016) considered a wholesale fish market in Maizuru Bay as an 334

additional source of Japanese Jack Mackerel eDNA, and they were able to evaluate a partial 335

correlation between eDNA concentrations and echo intensity by including the inverse of the 336

distance of each sampling station from the fish market as an explanatory variable in their 337

statistical models. On the other hand, we were able to evaluate a correlation without 338

considering any effects of the fish market. Primer S targets shorter DNA fragments that would 339

include “old” or “non-fresh” eDNA. Therefore, it should be more affected by eDNA 340

contamination from the fish market. Whereas Primer L, which targets longer DNA fragments, 341

can detect relatively “fresh” eDNA compared to that detected by Primer S. Environmental 342

DNA from the fish market should be more degraded and therefore we could observe the 343

relationships between eDNA concentration with Primer L and echo intensity, excluding the 344

effect of the fish market. On the sea bottom, eDNA concentrations with Primer L showed no 345

significant correlation with any echo intensity data sets, while those with Primer S showed a 346

negative correlation with echo intensity of 50 m in radius horizontally and 5 m vertically 347

(Table 2). However, a significant correlation was not observed for Primer S when excluding 348

two outlier sites (St. 2 and 27). At these sites, there were much higher eDNA concentrations 349

than at other sites, which was referred to as “exogenous DNA” in Yamamoto et al. 2016. In 350

21

particular, St. 2 is close to the fish market, which was considered a major source of Japanese 351

Jack Mackerel eDNA (Yamamoto et al. 2016). Also at St. 27, for instance, eDNA might be 352

released from dead individuals that may accumulate there due to the specific features of the 353

site such as seafloor dips or rocks. It has already been reported that high concentrations of 354

eDNA from silver carp carcasses can be detected for at least 28 days (Merkes et al. 2014), so 355

the release of eDNA from carcasses might be possible. Contrastingly, eDNA concentrations 356

with Primer L at these sites were very low or zero, suggesting that this is “non-fresh” eDNA; 357

eDNA from carcasses or from the fish market has already been degraded when released. 358

Previous studies have focused on the influences of environmental factors on eDNA 359

persistence (Dejean et al. 2011; Thomsen et al. 2012a; Burnes et al. 2014, Strickler et al. 360

2015). In our study, for instance, eDNA degradation might have been slowed at lower 361

temperatures (Strickler et al. 2015; Roussel et al. 2016), UV radiation might damage DNA 362

nucleic acids (Sage et al. 1996; Ravanat et al. 2001; Pilliod et al. 2014), and water chemistry 363

might also influence eDNA persistence (Barnes et al. 2014; Eichmiller et al. 2016). However, 364

it remains unknown how these environmental factors can influence eDNA persistence in the 365

field, especially in marine environments. Answering these questions would be important 366

when applying eDNA analysis to field surveys. 367

22

We were able to successfully show that the decay rate of eDNA varied depending on 368

the length of the DNA fragment, and our findings showed the possibility of obtaining time-369

scale information from eDNA. With primer sets that target longer DNA fragments than in 370

previous eDNA studies, we can selectively detect newly released eDNA. Such longer eDNA 371

fragments indicate fresher biological information in the field. Thus, by selecting the detected 372

fragment length, we can extract time-scale information from eDNA. For instance, detection of 373

longer eDNA fragments enables us to obtain more accurate distribution information (Hänfling 374

et al. 2016; Bista et al. 2017), which would contribute to revealing the route of migratory 375

organisms. Various fish species are known to migrate on different scales (Tesch 1978; Heard 376

1991; Arai et al. 1999; Yamanaka and Minamoto 2016). The time scale information obtained 377

using the results of our study may enable us to understand the details of fish migration. On the 378

other hand, the primer/probe sets we designed in our study targeted a CytB gene of Japanese 379

Jack Mackerel that might be too long to be sufficiently informative. The primer/probe sets 380

that target a shorter fragment size than Primer L and longer than Primer S (e.g., 300–500 bp), 381

would be more informative and also detectable for a reasonable period of time. Detection of 382

longer eDNA fragments might be able to dramatically improve the study of ecological 383

monitoring. 384

385

23

Acknowledgements 386

We thank Dr. Atushi Ushimaru and Dr. Yasuoki Takami (Kobe University) for helpful 387

comments and suggestions to the experimental design and interpretation of results. This work 388

was supported by JST CREST Grant Number JPMJCR13A2, Japan. 389

390

Data accessibility: 391

392

The raw data of qPCR experiments and echo intensity data are included in support 393

information. 394

395 Author contributions: 396 397 T.J., H.M., R.M., and T.M. designed the experiments. T.J., H.M., M.S., and S.Y. performed 398 tank experiments. T.J. analyzed the data. T.J., H.M., R.M., S.Y., and T.M. wrote the paper. 399 400 Support Information: 401 402 Additional Supporting Information may be found in the online version of this article: 403 404

Fig. S1: The shift of water temperature in the tank experiment. Each line (solid red, dotted 405

blue, and dashed green) shows the shift in water temperature in each tank. 406

407

Table S1. R2 values, slopes, and Y intercepts of the calibration curves and the polymerase 408

chain reaction (PCR) efficiencies for each qPCR experiment performed in this study. These 409

values are represented as mean ± 1 SD. 410

411

24

Table S2. Raw values of eDNA concentrations (copies per 2μL) in tank samples with Primer 412

S. 413

414

Table S3. Raw values of eDNA concentrations (copies per 2μL) in tank samples with Primer 415

L. 416

417

Table S4. Raw values of eDNA concentrations (copies per 2μL or 1L) in field samples with 418

Primer L for surface (left tables) and bottom (right tables). 419

420

Table S5. Detailed information on echo intensity data for the sea surface, cited from 421

Yamamoto et al. (2016). 422

423

Table S6. Detailed information on echo intensity data for the sea bottom, cited from 424

Yamamoto et al. (2016). 425

426

25

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Figures 564 565 Fig. 1. Decay curves for Japanese Jack Mackerel eDNA in the tank experiments. Dots show 566 eDNA concentrations (average of triplicate) at each time point (blue: Primer S, red: Primer L) 567 and solid lines show regression curves excluding the initial 2 hours of data. Error bars show 568 standard deviation (SD). 569

570

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12

34

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30

Fig. 2. The distribution of Japanese Jack Mackerel eDNA concentrations and echo intensity in 572 west Maizuru Bay (surface and bottom). The level of the estimated eDNA concentrations is 573 indicated by colors between red (relatively high concentration) and blue (low concentration or 574 zero), as well as the echo intensity by echo sounder as indicated by colors between dark 575 yellow (relatively high intensity) and white (low intensity or zero). Gray areas indicate land 576 masses. Spatial approximation was performed using a regularized spline with a tension 577 parameter of 40. 578 579

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