DNA and eDNA-based tracking of the North African sharptooth catfish Clarias gariepinus

Journal Pre-proof DNA and eDNA-based tracking of the North African sharptooth catfish Clarias gariepinus Aya Ibrahim Elberri, Asmaa Galal-Khallaf, Sarah Emad Gibreel, Said Fathallah ElSakhawy, Islam El-Garawani, Sobhy El-Sayed Hassab ElNabi, Khaled MohammedGeba PII:

S0890-8508(19)30384-6

DOI:

https://doi.org/10.1016/j.mcp.2020.101535

Reference:

YMCPR 101535

To appear in:

Molecular and Cellular Probes

Received Date: 6 October 2019 Revised Date:

15 February 2020

Accepted Date: 16 February 2020

Please cite this article as: Elberri AI, Galal-Khallaf A, Gibreel SE, El-Sakhawy SF, El-Garawani I, ElSayed Hassab ElNabi S, Mohammed-Geba K, DNA and eDNA-based tracking of the North African sharptooth catfish Clarias gariepinus, Molecular and Cellular Probes (2020), doi: https://doi.org/10.1016/ j.mcp.2020.101535. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Statement Aya Ibrahim Elberri: Collection of water samples, DNA extraction, Primers testing. Asmaa Galal-Khallaf: Designing of the qPCR test, software data analyses, participation in manuscript revision & discussion preparation, and manuscript writing, response to editors and reviewers´ comments, Supervision of Elberri PhD Thesis. Sarah Emad Gibreel: Participation in the population genetic analyses. Said Fathallah El-Sakhawy: Collection of fish samples along the River Nile, participation in manuscript revision. Islam El-Garawani: Supervision of Elberri PhD Thesis. Sobhy El-Sayed Hassab ElNabi: Supervision of Elberri PhD Thesis. Khaled Mohammed-Geba: Collection of samples, primers design, analysis of populations´ genetic data, software data analyses, results analysis and discussion, response to editors and reviewers´ comments, Supervision of Elberri PhD Thesis.

DNA and eDNA-based tracking of the North African sharptooth catfish

1

Clarias gariepinus.

2 3 1‡

1‡

1

Aya Ibrahim Elberri , Asmaa Galal-Khallaf , Sarah Emad Gibreel , Said

4

Fathallah El-Sakhawy2, Islam El-Garawani1, Sobhy El-Sayed Hassab ElNabi1,

5

Khaled Mohammed-Geba(1,*, ‡).

6 7

1. Genetic Engineering and Molecular Biology Division, Department of

8

Zoology, Faculty of Science, Menoufia University, 32511 Shebin El-

9

Kom, Menoufia, Egypt.

10

2. Physiology Department, Faculty of Veterinary Medicine, Sadat City University, Sadat City, Menoufia, Egypt.

11 12 13

(*) Corresponding author:

14

Dr. Khaled Mohammed-Geba, Molecular Biology and Biotechnology Lab,

15

Department of Zoology, Faculty of Science, Menoufia University, 32511

16

Shebin El-Kom, Menoufia, Egypt.

17

E-mail1: [email protected],

18

E-mail2: [email protected]

19 20

(‡)

Authors equally contributing to the article

21 22

1

Abstract

23 24

The African sharptooth catfish, Clarias gariepinus, contributes much to

25

the River Nile ecosystem by its high omnivorosity, sturdiness, growth rates,

26

and fecundity. It was globally appreciated as a key fluvial aquaculture species.

27

Yet, it is also one of the top world freshwater aliens. Monitoring the genetic

28

diversity of different economically and ecologically important species as well

29

as development of markers that aid their tracing and abundance are

30

fundamental. This is chiefly due to the growing international threats of

31

environmental pollution, reduction, and loss of biodiversity. Herein, the genetic

32

diversity of C. gariepinus along the River Nile in Egypt was assessed through

33

sequencing of the mitochondrial cytochrome oxidase subunit I (COI). Also, a

34

qPCR assay based on C. gariepinus 16srDNA was developed to assess the

35

species abundance through environmental water DNA samples (eDNA). The

36

results showed low genetic diversity of that species in Egypt. Moreover, its

37

populations exhibited high rates of fixation. Testing its eDNA-based marker

38

resulted in an unambiguous quantitative trend in situ, in agreement with

39

reports of local fishermen. These eDNA signals were strong at least 1 Km

40

upstream to the initial sampling areas, even where no C. gariepinus fishing

41

activities are carried out. This possibly indicated a degree of homogenous

42

species-abundance in each of the studied areas. Finally, the results identified

43

a need for better conservation strategies for C. gariepinus, since its low

44

diversity in the Egyptian River Nile may represent a threat against its

45

persistence under the continuously changing environmental conditions.

46

Moreover, using non-invasive sampling methods, e.g. based on aquatic eDNA

47

quantification, can aid much the detection of areas of abundance of C.

48

gariepinus, especially for both the economic importance it contributes and the

49

invasive power it possesses.

50

Keywords: Clarias gariepinus, eDNA tracking, genetic diversity, population

51

genetics, qPCR.

52 53

2

Introduction

54

Catfishes are known in all continents of the world except Australia, with

55

a fossil record extending back to the late Cretaceous (Nelson, 2006). In the

56

modern era, the order Siluriformes encompasses 478 genera and 3093

57

species. Most of them are freshwater inhabitants, together with the marine

58

families Ariidae and Plotosidae. Despite catfishes being of low market value,

59

they contribute much to the international aquaculture products trade (FAO

60

2014). The sharptooth catfish Clarias gariepinus occupies a very wide

61

geographical range in Africa, its native homelands from the North to the South

62

(Ellender and Weyl 2014). It was recorded as an invasive species in many

63

countries, such like India, Thailand, and Brazil, having negative impacts on

64

the aquatic, amphibian, and avian biota (Cambray, 2003; Kiruba-Sankar et al.

65

2018). Massive introgression between C. gariepinus and the Thailand walking

66

catfish, C. microcephalus was identified in Thailand, representing a serious

67

concern for the balance of the normal populations of C. macrocephalus there

68

(Na-Nakorn et al. 2014).

69

Environmental DNA is the DNA fraction that is present and collectable

70

from different ecological niches such as soil, freshwater, seawater, snow or air

71

rather than invasively sampling from the individual organism itself (Ficetola et

72

al. 2008; Sigsgaard et al. 2015). eDNA-based measurements have become

73

widely applied in aquatic biodiversity monitoring, as they reduce the need for

74

destructive or invasive sampling methods. Aquatic organisms´ eDNA is

75

continuously liberating from their bodies through shedding of dermal, mucous,

76

intestinal, and gamete cells, as well as scales, urine, and feces (Geerts et al.

77

2018; Harper et al. 2019). This intracellular DNA (i.e. tissue or cells)

78

3

transforms over time to subcellular eDNA (i.e. mitochondria, ribosomes or free

79

floating nucleotide strands) as cells degrade (Moushomi et al. 2019). eDNA

80

analyses overcome the need for the tools and efforts required for animal

81

dissection and tissue sampling, as well as the necessity of target organisms´

82

abundance, and whether there are concerns about their conservation

83

statuses. Hence, it also saves time and money consumed for sampling and

84

sample preparation, and even the extensive taxonomic knowledge needed for

85

targeted species´ identification (Jo et al. 2017; Geerts et al. 2018; Harper et

86

al. 2019). eDNA-based species abundance estimation has given very similar

87

results

techniques

88

(Lacoursière‐Roussel et al. 2016; O'Donnell et al. 2017). Moreover, molecular

89

methods show good success in tracking the historical patterns of distribution

90

of species, population dynamics, ecosystem health and trophic interactions

91

(Chain et al. 2016; Yamamoto et al. 2017). This, indeed, contributes much to

92

the enhancement of aquatic conservation and fisheries management

93

strategies (Goldberg et al. 2016; Evans et al. 2017).

94

to

abundance

estimation

using

regular

fisheries

Clarias gariepinus (Clariidae: Siluriformes), is a typical African sturdy,

95

air-breathing catfish (Picker 2013). It is believed to be the most widely

96

distributed fish species in Africa. It is considered the second most

97

economically important freshwater fish in Africa, after tilapia (Ikpeme et al.

98

2015). In Egypt, this species is the third most important one for the

99

aquaculture sector, providing about 17,895 tons to the annual Egyptian

100

aquaculture production (FAO 2018). The C. gariepinus sturdiness, rapid

101

growth rates in comparison to other species in the genus Clarias, and relative

102

ease in aquaculture lead to its propagation to countries far from its African

103

4

main homelands, including Jordan, Lebanon, Syria, India, Indonesia, Vietnam,

104

and Brazil (Dunham et al. 2000; Mehanna et al. 2018, FAO 2018). However,

105

some aquaculture practices can make it vulnerable to stock declining due to

106

reduction of genetic resources. For example, there is a tendency for

107

continuous use of aquaculture-bred strains for restocking, which lead in some

108

cases to 30% or more reduction in growth due to inbreeding (Megbowon et al.

109

2013).

110

The current work was carried out to assess the genetic diversity of the

111

sharptooth catfish C. gariepinus in Egypt. Also, it aimed to provide a novel,

112

universal, economic, non-invasive, eDNA-based tool for exploring C.

113

gariepinus abundance/biomass in different tributaries in the Egyptian River

114

Nile, as a case study that can be replicated later in any other area in the world

115

where this species exists as native, invasive, or introduced for aquaculture.

116 117

5

2. Materials and methods

118

2.1. C. gariepinus Egyptian populations´ analysis

119

2.1.1. Collection of samples

120

A total of 90 caudal fin clips of C. gariepinus were collected from the

121

local fish markets that receive fresh landings from the River Nile in the cities

122

of Shebin El-Kom (coordinates: 30.581603, 31.018031), Assiut (27.262462,

123

31.123200) and Aswan (24.093597, 32.895600), as 30 samples from each

124

location (Fig. 1). Samples of Assiut and Aswan were collected from the main

125

stem of the Nile. Samples of the Shebin El-Kom city were collected from Bahr

126

Shebin irrigation canal, a tributary from the River Nile in the North of Egypt.

127

Preserved in 100 % Ethyl Alcohol, the samples were immediately transferred

128

to the Lab of Molecular Biology and Biotechnology at the Faculty of Science in

129

Menoufia University for genetic analyses.

130

2.1.2. DNA extraction, polymerase chain reaction (PCR) amplification of

131

cytochrome oxidase subunit I (COI) gene

132

DNA extraction was carried out using the method described in

133

Mohammed-Geba et al. (2016a). Briefly, 200 µL of TNES-urea buffer with 2.4

134

U mL-1 Proteinase K solution (ThermoFischer Scientific) were added to the

135

individually lysing fin clips, with incubation at 65 ºC. Then, 54 µL of 5 M NaCl

136

were added, the tubes were repeatedly inverted for mixing, then centrifuged at

137

4,000 g for 10 min. The aqueous supernatant was removed to a new, sterile

138

tube, and 200 µL of freeze-cold isopropyl alcohol were added with shacking to

139

precipitate the DNA. After 11,000 g centrifugation for 10 min, the supernatant

140

was replaced by 400 µL of 70 % ethanol for washing the DNA pellet. The

141

tubes were centrifuged for 5 min at 11,000 g, the ethanol was completely

142

6

removed. Finally, 30 µL of Tris EDTA buffer (10 mM TRIS.HCL pH8, 2 mM

143

EDTA pH8) were added for DNA pellet resuspension. DNA quality was

144

checked by running in a 1 % agarose gel electrophoresis stained with 0.5 µg

145

mL-1 ethidium bromide (ThermoFisher Scientific). Intra-specific hypervariable

146

5´ region of the barcode of life, COI gene, was PCR-amplified in all samples,

147

using the universal primers described by Ward et al. (2005). The amplification

148

reaction for each sample was set up as 50 ng of template DNA, 1X MyTaq™

149

Red Mix (Bioline), 0.4 µM of each primer, and 200 ng mL-1 BSA, to a total

150

volume of 25 µL. PCRs were carried out in the thermal cycler TC512 (Techne,

151

UK). The amplified products were sent to Macrogen Inc. (South Korea) for

152

conventional Sanger chain termination sequencing method.

153

2.1.3. GenBank comparisons

154

C. gariepinus COI gene sequences were reviewed and manually

155

trimmed for removing non-informative nucleotide peaks. Edited sequences

156

were compared to archived reference sequences in GenBank database and

157

BOLD

(https://blast.ncbi.nlm.nih.gov/Blast.cgi,

158

http://www.boldsystems.org/index.php/IDS_OpenIdEngine). These sequences

159

were aligned using CLUSTALW (Thompson et al. 1994) integrated in Mega 6

160

software (Tamura et al. 2013). This alignment was later uploaded to DNAsp

161

6.0 Software (Rozas et al. 2017) in Fasta format to determine different

162

haplotypes, if present. Egyptian C. gariepinus COI sequences were then

163

submitted to the GenBank/EMBL/DDBJ International Databases for assigning

164

accession numbers.

165 166

2.1.4. Populations´ genetic analyses

7

167

Good-quality COI sequences obtained from C. gariepinus in the current

168

study, together with GenBank COI sequences from Nigeria (17), Thailand

169

(10), Turkey (21) and India (15) were retrieved. All the sequences used were

170

linked to the BOLD database, and all were checked for absence of premature

171

stop codons that mark nuclear copies of mitochondrial genes (NuMTs). No

172

regions with number of COI sequences below 10 were included in the

173

analyses. The sequences were aligned using ClustalW integrated to Mega 6

174

software, then trimmed to remove the initial and final non-informative

175

background peaks. Also, the nucleotide areas that were not common among

176

our sequences and those retrieved from the GenBank database in the

177

sequences´ extremities were removed. The obtained final 608-bp common

178

COI fragment after trimming was used for subsequent population analyses.

179

The resulting file was uploaded to DNAsp 6.0 Software to determine the

180

haplotypes existing in common and separately within the selected COI

181

fragment, haplotype (Hd) and nucleotide (π) diversity indices in each site, as

182

well as the index of raggedness, the r of Harpending (1994) and the R2

183

parameter of Ramos-Onsins and Rozas (2002) for identifying recent

184

populations´ expansions. Later on, the haplotypes determined through DNAsp

185

6.0 Software were uploaded to the program PopArt 1.7. (Leigh and Bryant

186

2015) to draw a median-joining haplotype network and further demonstrate

187

the interrelationships among different haplotypes. The same software was

188

applied to produce a map for world haplotypes´ distribution in the main

189

countries where economic and/or ecological significance for this species were

190

mostly recorded.

191

8

Pairwise genetic differences between samples were assessed using

192

the F-statistics, based on haplotypes frequencies, and the Fst value, based on

193

haplotypes frequencies and sequence divergences between them, all being

194

estimated using the software ARLEQUIN 3.5.1.3 (Excoffier and Lischer 2010).

195

Moreover, neutrality analysis was performed by determining the D test

196

statistic of Tajima (1989), whose negative values arise from selection or rapid

197

population growth (Tajima, 1989; Borrell et al. 2012).

198

2.2. Development of C. gariepinus-specific eDNA Assay

199

2.2.1. Design and testing of species-specific eDNA primers

200

Sequences of the 16srDNA mitochondrial gene were exported to the

201

Primer3Plus online algorithm (Untergasser et al. 2007) to design species-

202

specific pair of primer. The primers that exhibited the highest identity with C.

203

gariepinus from all areas in the world, as compared to GenBank database, an

204

annealing temperature of about 50 ºC, the least self-complementation, and

205

the least nucleotides repetition were chosen and synthesized in Vivantis

206

(Malysia). Moreover, a second forward primer was designed for a shorter

207

amplicon size (150bp), more suitable for quantitative PCR (qPCR) analysis.

208

In order to test primer efficiency in 16srDNA amplification, 15 mg-liver

209

samples were obtained from C. gariepinus, Bagrus bajad, Chrysichthys

210

ruppelli, and Malapterurus electricus specimens coming from local fish

211

markets to the Faculty of Science, Menoufia University for purposes of

212

practical teaching, after anaesthetizing the fishes with lethal doses of clove oil.

213

The catfish species tested were chosen in base of their co-existence with C.

214

gariepinus in the Northern River Nile waters. Total DNA was extracted using

215

QIAamp DNA Mini kit (QIAGEN, Cat. No. 51304) following the manufacturer

216

9

instructions. The DNA was subjected to PCR for amplification of the 16srDNA

217

gene using the newly designed C. gariepinus 16srDNA-specific primers, in the

218

same PCR conditions mentioned in section 2.1.1. herein. In order to optimize

219

the best conditions for primer annealing, a gradient of annealing temperatures

220

between 50-60 ºC was applied over replicates of the same PCR reaction, in a

221

PCR program consisting of an initial denaturation step at 95°C for 3 min,

222

followed by 35 cycles of 95°C for 30s, 55°C ±5°Cfor 30s, and 72°C for 1 min.

223

A final extension of 72°C for5 min was appended. PCR products were

224

electrophoresed

in

UV-

225

transillumination

(TransillumintorTi1,

resulting

226

positive amplicons, if any, were sent for sequencing to Macrogen Inc. (Seol,

227

South Korea).

228

2.2.3. Field trial

229

a

1%agarose

gel,

and

visualized

Biometra,Germany).

using

The

One-liter water samples in triplicates were collected from just above the

230

riverine sediments of each of the following four main canals in Menoufia

231

governorate, Egypt: Bahr Shebin (Coordinates: 30.581603, 31.018031 and

232

30.5667129, 31.013454), El-Atfy (30.557611, 31.066975 and 30.5520328,

233

31.0654790), Bahr Seif (30.542083, 30.983160 and 30.534444, 30.988021)

234

and Al-Nenaeia (30.694269, 30.824215 and 30.683351, 30.825784) (Fig.2).

235

All of them are irrigation canals, except El-Atfy (also known as Al-Atf), that is

236

an agricultural drainage canal. Sampling was carried out for two areas per

237

canal: one that was reported by fishermen to exhibit catfish abundance, and

238

another random one that is 1 km upstream to the first point. Different sampling

239

boats were used for sampling as fishing areas in each canal are managed by

240

different fishermen. As a blank, 1 L samples of dechlorinated tap waters were

241

10

obtained. This water belongs to Shebin El-Kom city potable water treatment

242

station, whose inlet takes directly from Bahr Shebin Canal and the water is

243

subjected to different chemical and biological water treatments for water

244

purification. The blank was processed in the same way as the water samples

245

for eDNA extraction and qPCR running. To avoid any possible cross-

246

contamination among different sampling points/triplicates, single-use, 1 L

247

plastic jars were applied for obtaining each of the sampling triplicates from

248

every sampling point and canal assayed. The samples were directly

249

transferred on ice to the Laboratory of Biotechnology and Molecular Biology

250

Lab at the Faculty of Science in Menoufia University, where they were

251

immediately processed for eDNA extraction.

252

2.2.4. eDNA extraction and quantitative PCR (qPCR)

253

50 mL of each sample were concentrated by centrifugation, as the best

254

way to harvest free cells and free DNA in comparison to filtration (Boström et

255

al. 2004). The supernatant was removed completely, then the sediment was

256

processed using QIAmp DNA extraction kit (QIAGEN) following the

257

manufacturer’s instructions. DNA quantity and quality were assessed through

258

spectrophotometric measurement at A260/A280 and running a 1 % agarose

259

gel electrophoresis. As a way to produce a standard curve from serial DNA

260

concentrations and their Ct values, without using a single sample from our

261

DNA, but applying the same kind of sample in the same time, a random DNA

262

pool was produced from different samples, and its DNA concentration was

263

adjusted to 100 ng µL-1. Three half-serial dilutions were prepared from this

264

initial concentration. Triplicates from each concentration were amplified

265

through qPCR, using 1 µL of DNA, 1 µL of C. gariepinus 16srDNA-specific

266

11

primers, 10 µL of TOPreal™ qPCR 2X PreMIX (Cat. No. RT500S,

267

enzynomics, South Korea), 200 ng BSA, and completed to 20 µL using

268

deionized waters. The reactions were run in the PikoReal™ Real-Time PCR

269

System (Model Number: TCR0024) available at the Central Laboratories of

270

National Institute of Oceanography. The qPCR program consisted of an initial

271

denaturation step at 95 ºC for 3 min, followed by 35 cycles of 95 °C for 30s

272

and 58°C for 1 min. and finishing with a melting curve from 60 °C till 95 °C for

273

20 min to check for the absence of primer–dimer artifacts and non-specific

274

amplifications. Non-template controls (NTCs) were used as negative controls.

275

The relation between cycles of amplification (Ct) and the ng of DNA in each of

276

the serial dilutions was plotted to calculate the regression coefficient (r2) and

277

the straight line equation. Based on the results of this assay, the

278

concentrations of all samples were adjusted, and run in the qPCR through the

279

same conditions.

280 281

2.2.6. Statistical analysis

282

C. gariepinus 16s rDNA abundance, as measured using qPCR in the

283

eDNA samples, were statistically analyzed. For different sampling points from

284

the same canal, Student t-test was applied. For differences among the four

285

assayed canals, One-Way Analysis of Variance (ANOVA) was applied, with

286

the Least Significant Difference (LSD) as a post-hoc test. Statgraphics

287

Centurion IX program was used for both analyses, t-test and ANOVA.

288

Differences were considered significant when P < 0.01.

289 290

12

Results

291

2.1. C. gariepinus genetic population richness

292

COI gene sequences of the sampled catfish exhibited 99 % - 100 %

293

sequence identities with GenBank references of C. gariepinus. Our samples

294

were found to belong to 4 haplotypes, deposited in the GenBank database

295

with the acc. Nos. MK335910.1-MK335913.1. Table 1 shows the details of

296

haplotypes found in the current study, including numbers of sequences for

297

each haplotype, and their geographical distribution. The analyzed 150 C.

298

gariepinus COI sequences (63 from GenBank, 87 Egyptian in vivo ones)

299

lacked NuMTs, as checked using in silico translation to primary amino acids

300

sequences using Mega 6 software. All COI sequences belonged to 14

301

haplotypes, four of which (i.e. Hap 1, 4, 6, and 14) were found in Egypt. Most

302

of the Egyptian samples belonged to Hap 14, that was unique to Egypt. Hap 1

303

also included samples from all the Egyptian points. However, this haplotype

304

was also shared with Thailand and Nigeria. Hap 4 was purely African, being

305

found in the southern-most sampling site in Egypt, i.e. Aswan, and out of

306

Egypt it was reported in Nigeria. Hap 6, a pure Northern one, included

307

samples from the Egyptian North, i.e. of Shebin El-Kom city, along with all the

308

Turkish sequences. Haplotypes´ network assumed a double star-shape, with

309

most haplotypes spanning around the two central ones, Hap 1 and Hap 14

310

(Fig. 3). All Egyptian and African sequences were directly related to Hap 1, at

311

most

Turkish

312

representatives were directly related to the world-wide distributed Hap 1. This

313

last haplotype was the closest to the purely Asian group of haplotypes, i.e.

314

through

a

single

mutation

13

separating

them

from

it.

Haplotypes 5 and 7-12. This group was centered Hap 5, that included solely

315

Thai and Indian samples.

316

Egyptian, Nigerian, Thai, and Indian populations showed signs of

317

population expansion, as identified by negativity of Tajima´s D statistic, low R2

318

values, and the haplotypes´ distribution (Tables 1,2). For pairwise Fst, the

319

values in general exhibited very high fixation among different populations of

320

this species in different countries (Table 3). Fixation of the Egyptian

321

population followed an ascending pattern with Nigeria, Turkey, Indonesia,

322

India, Thailand, and Brazil. The fixation was almost complete between the

323

Egyptian population and the last three ones.

324

2.2. eDNA analyses

325

2.2.1. Primer design

326

The primers that fitted the selection criteria mentioned above were termed

Cgar16srDNAFw1

(5´-CTTAGTTATAGCTGGTTGCCTA-3´),

327

and

328

Cgar16srDNARv1 (5´-CAGGGCAGGCAAGACCTCCT-3´). The PCR product

329

of these primers was a 650 bp band, whose sequence exhibited 99.8%

330

identity with C. gariepinus16srDNA (acc. No. JF280894.1), 94 % with C.

331

gabonensis (acc. No. JX899749.1), and 92.1 % with C. batrachus (acc. No.

332

KM259918.1). Much lower identities (< 80%) were found with other siluriform

333

species. No 16srDNA amplicons were obtained for the other catfish species

334

that co-exist in the Northern River Nile, i.e. B. bajad, C. ruppelli, and M.

335

electricus, at all tested annealing temperatures.

336

2.2.3. Field trial

337

For the qPCR, we have used the primer CgarqPCR16SFw-2019 (5´-

338

ACTCACAACCCAAATCGTTAAT-3´) as a forward primer, with the above-

339

14

mentioned primer Cgar16srDNARv1 as a reverse one. The expected PCR

340

amplicon was 150 bp, as identified through in silico analysis and agarose gel

341

electrophoretic

eDNA

342

concentrations resulted in an efficiency of 1 and an r2 of 0.998. Based on the

343

standard curve results, 50 ng concentration was found to be in the middle of

344

the linear range. Therefore, DNA concentrations in all our eDNA samples

345

were adjusted to 50 ng. The quantity of C. gariepinus eDNA was found to be

346

the highest significantly in El-Atfy canal, followed by Al- Nenaeia, then 2-3

347

folds lesser in both Bahr Seif and Bahr Shebin. These two latter canals did not

348

vary significantly from one other in the quantities of C. gariepinus eDNA.

349

Finally, there were no significant differences between the ¨abundant¨ and

350

¨random¨ sampling points in each canal from where the eDNA samples were

351

obtained (Fig. 4).

352

checking.

The

standard

curve

for

different

353

15

Discussion

354

The current work analysed, at the molecular level, the genetic population

355

diversity of the North African sharptooth catfish C. gariepinus in Egypt. It was

356

possible to identify also some genetic relationships among the Egyptian population

357

of C. gariepinus and different populations of the same species in other areas in

358

Africa and Asia. The results obtained herein exhibited significant signs for C.

359

gariepinus populations´ expansion in Nigeria, Thailand, and India. In both these last

360

countries, C. gariepinus was introduced for aquaculture but many escapes occurred

361

and introgression with native species, besides reduction of some others, were

362

recorded (Barua et al. 2000; Krishnakumar et al. 2011; Radhakrishnan et al. 2011).

363

The Egyptian population of C. gariepinus, however, did not show significant

364

expansion. Moreover, its high fecundity, rapid growth, and omnivorous feeding

365

should have positively contributed to higher levels of genetic diversity, rather than

366

the low diversity found in the current study. In addition, the high homogeneity of the

367

hydro-geological conditions of the Northern River should have been accompanied by

368

higher level of species genetic diversity. This homogeneity comes from several

369

factors. For examples, the sediment provided to the Nile comes only from the Blue

370

Nile (50–61%) and Atbara (30–42%) (Padoan et al. 2011). Moreover, natural barriers

371

in the Northern River Nile, i.e. to the North of Aswan´s high dam, are absent. Yet, the

372

limited genetic diversity we found for C. gariepinus can be attributed to some

373

behavioral, populational, and ecological factors. Mark-recapture studies have pointed

374

to that C. gariepinus population is dominated by old and large males, i.e. length and

375

ages-skewed populations, which suppresses the recruitment of younger individuals

376

(Booth et al. 2010). Also, it is believed that the North African C. gariepinus population

377

belongs to one of three major phylogeographic groups of this species in Africa; that

378

16

are i) the East, South and Central African; ii) the North African; and iii) the Levant

379

(Giddelo et al. 2002). Hence, the possibility of having low intra-specific variability

380

within C. gariepinus population in the area of study covered by the current work may

381

be reasonable. The current study pointed to high genetic fixation among the

382

Egyptian and Nigerian populations. Also, the Egyptian population of C. gariepinus

383

showed low genetic diversity. However, other African populations showed clear

384

genetic diversities and populations´ structuring. as in Kenya and Nigeria (Ikpeme et

385

al. 2015; Barasa et al. 2016, 2017). These contrasting situations among Egypt and

386

other countries in Africa may further support the wide-African populations´ division as

387

mentioned by Giddelo et al. (2002). High fixation values were also obtained upon

388

comparing the African populations of C. gariepinus with the Asian ones. These

389

fixation values were even higher than comparing among African populations

390

themselves. This can be directly attributed to that the Asian stocks, mainly the Indian

391

ones, are belonging to different genetic lineages (Lal et al. 2003). Also, that higher

392

intra-continental than inter-continental genetic fixation can be a reason of

393

introgressions with native Clarias species due to escapes from aquaculture or

394

accidental introductions. Introgressions are very well known in South Eastern Asian

395

countries. Most Thai population is actually from hybrids of C. gariepinus x C.

396

microcephalus, which are responsible for 50,000 tons of Thailand fish production

397

annually (Yi et al. 2003). Therefore, high fixation between these populations and the

398

Egyptian pure ones can be reasonable.

399

Another aspect that could be achieved in the current study was the

400

development of C. gariepinus eDNA-based qPCR-assay, which can contribute much

401

to the identification of natural abundance of this species. In Egypt, there are still

402

many deficiencies in the recording the fluvial landings. Furthermore, the studies

403

17

about conservation status of different Northern Nile species are scarce. Hence,

404

development of tools for biodiversity monitoring are both ecologically and

405

economically significant. Many species in the River Nile have disappeared or

406

severely declined due to the excessive pollution, coming chiefly with industrial

407

wastewater, oil pollution, municipal wastewater, and agricultural drainage (El-

408

Sheekh, 2009). Many of the existing species are showing signs of genotoxicity

409

and/or low levels of genetic diversity, such like the Nile pufferfish Tetraodon lineatus

410

(Mohammed-Geba et al. 2016b), the snail Bulinus truncates (Zein-Eddine et al.

411

2017) and the Nile tilapia Oreochromis niloticus (Hassanien et al. 2004). Pollution

412

directly impacted the hereditary material, reproduction, growth and development of

413

Nile species including C. gariepinus itself, as already found in response to heavy

414

metals, industrial and agricultural effluents, and other pollutants (El-Assal et al. 2014;

415

Mahrous et al. 2006; Osman et al. 2012,2017). This continued species declining,

416

being for environmental changes or as a result of increased pollution, enforces a

417

continuous, efficient monitoring of species, i.e. for purposes of conservation and

418

sustainable fisheries management. Development of an eDNA-based tool specific for

419

a species like C. gariepinus can contribute much to the identification of areas that

420

can serve, form one side, as abundant fisheries for it, especially in the countries

421

where C. gariepinus is an essential economic resource, as in Egypt, South Africa,

422

Kenya, Netherlands, and Thailand (FAO 2005a-e). From the other side, such tools

423

can be applied for controlling C. gariepinus invasion and identification of hotspots of

424

its spread, e.g. through escapes, in the countries where this species was recorded

425

as a strongly destructive alien, such like in India, Bangladesh, and China, and Brazil-

426

among many others (Barua et al. 2000; Krishnakumar et al. 2011; Radhakrishnan et

427

al. 2011). Regarding the application of field eDNA abundance in reporting the density

428

18

of commercial species, it is accepted that the amount of eDNA at a site is positively

429

correlated with the target species density. This was concluded after extensive works

430

in the field and laboratory eDNA in different aquatic native and invasive animals. For

431

examples, Takahara et al. (2012) identified a direct linear correlation between

432

common carp´s biomass in Iba-Naiio Lagoon in China and the fish´s eDNA

433

quantities. Pilliod et al. (2013) upon assaying 13 streams in Idaho found a positive

434

correlation between Rocky Mountain tailed frogs (Ascaphus montanus) and Idaho

435

giant salamanders (Dicamptodon aterrimus) and their eNDA levels, with the eDNA

436

concentrations did not vary significantly with sample location in the stream, time of

437

day, or distance downstream from animals. Likewise, Lacoursière‐Roussel et al.

438

(2016) could elucidate biomass-dependent eDNA concentrations for the Lake Trout

439

Salvelinus namaycush in twelve lakes in Québec, Canada. These eDNA

440

concentrations were only correlated to fish biomass in the sampled lakes, with no

441

effect of water temperature, dissolved oxygen, pH or turbidity. Cai et al. (2017)

442

generated a sensitive qPCR assay that enabled the detection of even very minute

443

amounts of Procambarus clarkii destructive to the UNISCO Natural Heritage site of

444

Honghe-Hani landscape in China. Schmelzle and Kinziger (2016) provided a

445

valuable eDNA-based assay for exploring the sites of presence and abundance of

446

the endangered tidewater goby (Eucyclogobius newberryi). This test succeeded in

447

both localization of the species in points where it was not found before using

448

conventional monitoring techniques i.e. seining, and in identification of species

449

abundance in different sites. Finally, not only invasive, endangered, or economic

450

species in waters could be localized and quantified through their eDNA traces, but

451

also the nearly-extinct aquatic species. This approach was effectively used to

452

quantify and localize the weather loach Misgurnus fossilis from different points in the

453

19

Danish freshwaters, including a historical site where the last record for this species

454

was in 1995 (Sigsgaard et al. 2015).

455

Yet, and in spite of the advantages it provides for detection of aquatic species,

456

some serious considerations should be taken into account before applying eDNA

457

quantification for species abundance detection. In some cases, it showed poor

458

correspondence with species relative abundance, or even the species are very

459

challenging for detection using their eDNA, especially for their hard exoskeletons,

460

such like the cases of the invasive rusty crayfish Orconectes rusticus and the

461

invasive spiny water flea (Bythotrephes longimanus), respectively (Dougherty et al.

462

2016; Walsh et al. 2019). eDNA detection frequency of a given species increases

463

with increasing its relative abundance in water (Dougherty et al. 2016). Such

464

detection can be affected by water clarity, being the clearer the water the better the

465

species detection probability (Dougherty et al. 2016). Reduction in water clarity may

466

come from different contaminants (for instance dissolved organic Carbon, resulting in

467

humic acid inhibition) that are themselves potent PCR inhibitors (Jane et al. 2015).

468

Inhibitors from biochemical processes of phytoplankton and plant species present in

469

the areas of sampling must also be considered and eliminated through application of

470

powerful filtering, as well as a robust DNA-extraction procedure (McKee et al. 2015).

471

Some species show seasonality in their abundance, and this further reduces their

472

detection probability (Young et al. 2011). Other species move actively in the water

473

body, and their eDNA circulates widely around by the wave action and currents.

474

Hence, the increase in eDNA content is related to the species general or season-

475

specific activity, as in cases of reproduction migrations (Dougherty et al. 2016,

476

Larson and Olden 2016). Taking large volumes of waters for eDNA sampling may

477

reduce random variation inherent in smaller sample volumes (Dougherty et al. 2016).

478

20

Therefore, it is strongly recommended to precede the eDNA sampling process with a

479

good knowledge about spatial and temporal patterns of distribution of the target

480

species. Besides, the ecological conditions related to the sampling location and/or

481

the specific habitats of that species should be considered.

482

In conclusion, clear signs of reduction in genetic diversity of C. gariepinus was

483

detected in the River Nile in Egypt. Yet, such reduction can be secondary to different

484

factors related to a wide African populations´ structuring, some dominance factors

485

within the same population, and/or pollution. The high genetic fixation in the Asian

486

populations of C. gariepinus may be a result of the introgression of C. gariepinus

487

with native species of the genus Clarias, or to the multiple introductions and genetic

488

stocks there. The newly designed set of primers can be applied in future works for

489

assessment of genetic diversity of this species in different locations, applying more

490

advanced tool, like next generation sequencing (NGS) technologies. Finally, the C.

491

gariepinus eDNA-based qPCR assay, designed in the current study, could detect

492

significant variations in its eDNA concentrations in several canals related to the

493

Northern River Nile in Egypt. Such assay can be suggested for identifying the

494

regions of abundance and reduction of C. gariepinus where this species exists, being

495

either a key economic resource, or an alien with highly competitive nature.

496 497

Acknowledgement

498

The authors would like to appreciate their deep thanks to the anonymous

499

reviewers and the Editor-in-Chief of Molecular and Cellular Probes for their valuable

500

comments, dedication and sincere guidance that helped so much to enhance the

501

manuscript to the best possible way. Also, many thanks to Dr. Mustafa G. Khallaf,

502

Cleveland Dental Institute (USA) and Dr. Hazem Galhom, Department of English

503

21

Language, Faculty of Arts, Menoufia University (Egypt) for their thorough English

504

language review. Also, we deeply appreciate the precious aids provided by Assoc.

505

Prof. Eman Mamdouh Abbas and Dr. Ibrahim Sahraby (National Institute of

506

Oceanography and Fisheries, Alexandria Branch, Egypt for their aids and providing

507

the qPCR machine, the working station for that assay and the device´s

508

consumables, and to Prof. Hassan Abdelrahman, ex-head of

Physiology

509

Department, Faculty of Veterinary Medicine, Sadat City University, for his helps

510

during samples´ collection.

511 512

22

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746 747

32

Figures

748

749

Fig. 1. Map for areas of C. garipinus populations´ collection, i.e. Shebin El-Kom

750

(coordinates: 30.581603, 31.018031), Assiut (27.262462, 31.123200) and Aswan

751

(24.093597, 32.895600). Photo credits GoogleMapsTM (details below the map)

752 753

33

754

755

Fig. 2. Map for areas of water samples´ collected for eDNA assessment. Photo credits

756

GoogleMapsTM (details below the map). Yellow points: usual fishing areas,

757

White points: randomly selected areas. A:Bahr Shebin (Coordinates:

758

30.581603, 31.018031 and 30.5667129, 31.013454), B: El-Atfy/Al-Atf Canal

759

(30.557611, 31.066975 and 30.5520328, 31.0654790), C: Bahr Seif (30.542083,

760

30.983160 and 30.534444, 30.988021), D: Al- Nenaeia canal (30.694269,

761

30.824215

762

and

30.683351,

34

30.825784).

763

A

B

Fig.3. Median-joining haplotype network for COI barcode region of C. gariepinus (A) and the distribution of world haplotypes (B) in the 5 main countries where this species is present as native or invasive, viz. Egypt, Nigeria, India, Thailand and Turkey. The branch length is proportional to the number of substitutions. Circles represent haplotypes, and their diameters are proportional to the haplotype frequencies.

35

764 765 766 767 768 769 770 771 772 773 774 775 776 777 778

779

780 781 782

Fig. 4. ANOVA-analysed, qPCR-assessed abundance data for C. gariepinus eDNA in

783

El-Atfy, Al-Nenaeia, Bahr Seif, and Bahr Shebin canals, besides qPCR results

784

for the field blank waters and negative PCR controls. Brackets with asterisk (*)

785

above show significant variations (LSD as post-hoc test, P<0.001) among

786

sampling canals. ns: non-significant differences. Dark Grey: Abundant

787

sampling

788

points;

Light

Grey:

36

Random

sampling

points.

Table 1: Haplotypes (Hap) of C. gariepinus COI gene analysed in the current study and in different

789

countries where this species presents key economic value for aquaculture or key environmental

790

concern as an alien. .

791

Haplotype

Hap 1

No. of sequences in each haplotype

49

Countries

Accession numbers

Egypt,

MK335910.1, HM882809- HM882811, HM882813,

Nigeria,

HM882814, HM882817, HM882823- HM882828,

Thailand,

HM882830, HM882831, JF292311

Hap 2

1

Nigeria

HM882812

Hap 3

1

Nigeria

HM882820

Hap 4

3

Egypt,

MK335911.1 , HM882821 Nigeria

JF292310, JF292312, JF292313, JF292315, JF292316Thailand, Hap 5

JF292320, JQ699199, JQ699201, JQ699203, JX024324-

17 India

JX024327, MF189951. Egypt, Hap 6

MK335912.1, JQ623925, KC500413- KC500432

22 Turkey

Hap 7

1

India

JQ699200

Hap 8

1

India

JQ699202

Hap 9

1

India

JX024320

Hap 10

1

India

JX024321

Hap 11

1

India

JX024322

Hap 12

1

India

JX024323

Hap 13

1

India

JX260853

Hap 14

50

Egypt

MK335913.1

37

Table 2: Genetic diversity parameters. n: total number of sequences, S: no. of segregated sites, nh: number of haplotypes, Ph: number of site-specific

792

haplotypes, Hd: haplotype diversity index, r: raggedness index; R2: Ramos Onsins and Rozas ststistic; and π: nucleotide diversity index.* :

793

P<0.05, N/A not available data for absence of polymorphic sits.

794 795

Egypt Nigeria Thailand Turkey India

n 87 17 10 21 15

S 3 4 8 0 22

nh 4 4 2 1 8

Ph 1 0 0 0 7

Hd 0.522 0.331 0.2 0 0.733

38

π 0.00104 0.00106 0.00296 0 0.00566

r 0.1962 0.3609 0.72 0 0.1068

D (Tajima) -0.10078 -1.57683* -1.87333* 0 -2.26743*

R2 0.0968 0.1311 0.3 N/A 0.1285

Table 3: Fst pairwise values. All values were found significant (ARLEQUIN 3.5.1.3, 100 permutations for significance, P<0.01).

Egypt Nigeria Thailand Turkey India

796 797

Egypt

Nigeria

Thailand

Turkey

India

0.00 0.37 0.91 0.74 0.89

0.00 0.87 0.78 0.8

0.00 0.921 0

0.00 0.83

0.00

798 799 800 801 802

39

Hughlights • • • •

Clarias gariepinus is one of the top aquaculture species in the world. C. gariepinus was recorded as an invasive species in many rivers. In its natural habitats it shows very low genetic diversity. Mitochondrial DNA markers could be designed for its effective environmental tracking.