A NEW IDENTIFICATION METHOD FOR CLARIAS GARIEPINUS, CLARIAS MACROCEPHALUS AND THEIR HYBRIDS
USING PCR-RFLP
DOI:10.30825/5.EJPAU.164.2018.21.4

Remigiusz Panicz¹, Jacek Sadowski², Paulina Hofsoe-Oppermann², Mirosław Półgęsek^2
1 Department of Meat Technology, Faculty of Food Technology and Fisheries,
West Pomeranian University of Technology, Szczecin, Poland
² Division of Aquaculture, Faculty of Food Technology and Fisheries, West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

A high tolerance to a wide spectrum of environmental factors and excellent taste of its meat explain why the African sharptooth catfish (Clarias gariepinus) is widely used for creating interspecific hybrids with the broadhead catfish (Clarias macrocephalus), a species which is characterized by a high growth rate. However, when the spawning stock on a fish farm is being supplemented with new individuals, there is a serious concern about introducing hybrids instead of purebred individuals. Therefore, it is necessary to develop a quick and reliable method of hybrid identification. In the presented study species diversity was assessed based on PCR and sequence analysis of the internal transcribed spacer 1 (ITS1) and cytochrome oxidase subunit 1 (COI) sequences. Additionally, three restriction enzymes (Fok I, Eco47I and HhaI) were selected and analysed with PCR-RFLP of COI sequences. ITS1 sequence comparisons revealed 0.64% diversity between analysed species and were submitted into GenBank database (KJ136021 and KJ136022). Analysis of COI sequences revealed over 14% variation between two species and based on obtained species-characteristic restriction patterns, a PCR-RFLP test has been developed.

Key words: catfish hybrids, Claridae, cytochrome oxidase subunit I, internal transcribed spacer 1, species delineation.

INTRODUCTION

Fish species from the Clariidae family are an important element of aquaculture in Asia, Europe and Africa. Different databases describe from 104 up to 119 species which belong to the family Claridae and originate from various water bodies in Africa and Southeast Asia [7, 8]. In the African aquaculture, the most common species is sharptooth catfish, Clarias gariepinus (Burchell 1822), whereas in Asian walking catfish, Clarias batrachus (Linnaeus 1758), broadhead catfish, Clarias macrocephalus (Günther 1864) and Hong Kong catfish, Clarias fuscus (Lacepède 1803). For a long time, European aquaculture had a great interest in two catfish species i.e. C. gariepinus and sampa, Heterobranchus longifilis [2, 24]. However, in the recent years, interspecific catfish hybrids have drawn much more attention, especially C. macrocephalus × C. gariepinus [16] and H. longifilis × C. gariepinus due to high growth and late maturation [20, 22]. Global trade in fish and growing aquaculture production pose a serious challenge to species identification additionally complicated by hybrid production. The standard method for identification of fish species relies on taxon-specific morphological features which, undoubtedly disappear or became mixed in hybrids. In such cases, molecular methods emerge as an alternative approach to confirm species authenticity. Among numerous available molecular markers, cytochrome oxidase subunit I (COI) has been the most widely used in species identification and serve as a universal DNA barcode. COI barcodes have been routinely applied for fish classification since the establishment of Fish Barcode of Life Initiative (FISH-BOL), a global effort to coordinate an assembly of a standardised reference sequence library for all fish species [4, 26]. The second region analysed in our study was the internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA (rDNA). This region allows for an error-free identification of potential interspecific hybrids and is especially useful for breeding programs where several species and their hybrids are crossed simultaneously [28]. Genetic control of fish groups used in farming is necessary since species identification based on biometric features is not free from errors. In the case of C. macrocephalus it is especially important as it may create fertile hybrids with C. gariepinus. If such hybrids escape from a fish farm, they may genetically pollute and negatively affect populations in diverse natural water bodies, since both catfish species are highly voracious [21, 25]. Furthermore, whenever the spawning stock on a fish farm is supplemented with fish living at large, there is a serious danger that hybrids will be introduced instead of purebred individuals. Fast identification of fish species and their hybrids plays a significant role in stock monitoring. Therefore, the aim of our study was to develop a time-saving and efficient method for the identification of selected species and their hybrids from the Clariidae family.

MATERIALS AND METHODS

Material for the study consisted of fin clips collected from C. gariepinus (n=8) and C. macrocephalus (n=7) individuals. Samples preserved in 75% EtOH were transported to the laboratory where DNA was extracted using peqGOLD Tissue DNA Kits (PeqLab). Quality and quantity of DNA isolates were assessed by separation in 1.5% agarose gel and spectrophotometric measurements using a NanoDrop 2000 (Thermo Scientific). Species-specific DNA barcodes were analysed based on amplification of partial fragment of the internal transcribed spacer 1 (ITS1) and the first cytochrome oxidase subunit 1 (COI) according to method described by Kijewska et al. [15], and Wyatt et al. [29], and Ivanova et al. [12], respectively with needed modifications. Briefly, polymerase chain reaction (PCR) were performed using a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems) and prepared using the GoTaq® G2 Flexi DNA Polymerase kit (Promega) comprising 1× PCR buffer, 200 μM of dNTPs (Thermo Fisher Scientific), 2.5 mM of MgCl₂ (25 mM), 0.5 U of Taq-polymerase, 0.2 μM of each primer (Genomed), 1 μL of DNA template, topped up with milli-Q water to 25 μl. Samples were initially denatured at 94°C for 5 min, followed by 35 cycles of 45 s denaturation at 94°C, 45 s annealing at 61°C (ITS1), 30 s annealing at 54°C (COI), and 60 s extension at 72°C, and followed by a final extension at 72°C for 7 min. All PCR products (n = 15) were assessed by 2% gel electrophoresis, visualized using Gel Doc™ XR gel documentation system (BioRad) and sequenced on both strands by means of direct Sanger sequencing by Genomed (Warsaw, Poland). Raw sequences were assembled using BioEdit v. 7.0.5 and submitted to BLAST to retrieve the records from the nucleotide GenBank database [9]. In order to develop the PCR-RFLP test, three restriction enzymes (FokI, Eco47I and HhaI) were selected, based on the virtual digestion of obtained sequences in WebCutter 2.0 software. Digestion of PCR products was performed according to the assay kit instructions and resulting fragments were separated on high-resolution 3% agarose gel (Micropor Omega, Prona).

RESULTS

PCR resulted in single bands of anticipated size, i.e. 624 bp for ITS1 and 652 bp for COI (Fig. 1). Analyses performed using BioEdit v. 7.0.5 software revealed that ITS1 sequences (C. gariepinus, n=8 and C. macrocephalus n=7) were identical for each species, whereas interspecies comparisons revealed 0.64% of difference. Next, ITS1 sequences were also compared against GenBank nucleotide repository using BLASTn and as a new haplotype identifies and submitted into GenBank database under accession numbers KJ136021 (C. gariepinus) and KJ136022 (C. macrocephalus). Comparison of the COI of sequences revealed 14% variability between of catfish species. Analysis using BLASTn showed that COI sequences of C. gariepinus were identical with those in GenBank found under the accession numbers GU701825.1, GU701826.1, and GU701827.1. Whereas COI sequences obtained for C. macrocephalus represented a new haplotype and were submitted into GenBank under accession number KJ136023. Digestion of COI amplicons by FokI, Eco47I and HhaI allowed for the unequivocal identification of C. gariepinus and C. microcephalus samples (Fig. 2, Tab. 1).

Fig. 1. Sample PCR amplification of ITS1 and COI sequences yielded DNA fragments of single bands. Ma – DNA ladder (100 – 3000 bp); Mb – DNA ladder (100 – 1000 bp); lanes 1, 2, 5 and 6 - samples of C. gariepinus; lanes 3, 4, 7 and 8 – samples of C. macrocephalus.

Fig. 2. Agarose gel electrophoresis of COI restriction bands after digestion with FokI, Eco47Iand HhaI. M – DNA ladder (100 – 1000 bp); lanes 1, 2 and 3 – C. macrocephalus samples; lanes 4, 5 and 6 - C. gariepinus samples; nrs – no recognition site (undigested DNA); size of the bands in bp; number in the circle depict very weak 69 bp bands.

Table 1. The size of COI fragments resulted from digestion with three restriction enzymes, FokI, Eco47Iand HhaI.

	Length of restriction fragments (bp)
	Fok I (GGATG(N)9NNNN)	Eco47I (GGWCC)	HhaI (GCGC)
C. gariepinus	–	583, 69	–
C. macrocephalus	336, 316	–	361, 291

DISCUSSION

Results obtained in our study showed that PCR-RFLP test may be easily applied to distinguish C. gariepinus and C. macrocephalus species. Furthermore, a new, previously unpublished COI barcode for C. macrocephalus was obtained in the course of the studies and submitted to the GeneBank database (KJ136023). The proposed method allows also fish products (e.g. fillets) which might be erroneously labelled on the market. In the case of significant differences in growth rates of respective catfish species, a correct identification may help fish breeder to correct estimates of production plan for catfish. Attempts to distinguish individuals based on morphological differences are time-consuming and often require scrupulous measurements [14], whereas test developed in this study is fast and easy to conduct, and the obtained results might be directly applied in practice [6]. Additionally, an important advantage of the test is the fact that during one analysis two alleles of a single gene are identified (identification of homo- and heterozygotes). Therefore, PCR-RFLP tests are often useful in activities focused on fish breeding or on population genetic where variation among examined individuals must be clearly determined [10, 18]. Amplification, followed by digestion with one of the three restriction enzymes FokI, Eco47I or HhaI, allow for an unequivocal identification of parent species selected for hybrid production (Fig. 2). Results apart from being useful for distinguishing C. gariepinus from C. macrocephalus the above results may also be helpful in identification of their hybrids, i.e. strictly speaking, for the identification of the species of females used in breeding programs. Additionally, C. gariepinus (female) x C. microcephalus (male) hybrids have spines in pectoral fins which are unique for the latter species. Thus, identification of those hybrids is possible owing to the fact that mitochondrial COI marker is maternally inherited and mentioned phenotypic feature [19, 23].

In the present study, a fragment of internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA was also analysed. Due to rapid evolution and homogenizing forces of concerted evolution and molecular drive, this region is commonly used in population studies as it allows for unequivocal identification of species [5]. In the present studies, it was determined that the level of genetic variation between the examined catfish species amounted to 0.64% and was sufficient to distinguish both species. Sequence variants can be used in the course of further studies on catfish species. It is worth mentioning that in the future identification of both the original species and their hybrids is especially important from the point of view of protecting the biodiversity of organisms in the environment. Hybrid breeding should be strictly supervised since an unintentional release of hybrids may result in introducing new diseases into the natural environment [11, 17], interbreeding between the hybrids and wild fish [27] or competition for food [13] Other studies confirm that introduction of hybrids to the natural environment might pose a serious problem for an autochthonic fauna [3, 1].

CONCLUSIONS

Aquaculture production is one of the main solutions to food shortages which are expected in the nearest future and directly related to the growing human population. Production of interspecific hybrids in most cases result in sterile fish which in general grow faster and combine advantageous traits inherited from parental lines. However, monitoring of such breeding programs requires fast and reliable methods for unequivocal identification of hybrids, a uniform phenotype with a combination of characteristics from the parents. PCR-RFLP test and ITS1 markers developed in our study provide a solution to identify C. gariepinus x C. microcephalus hybrids. Moreover, results provide a unique opportunity also to confirm the authenticity of catfish products (e.g. fillets) sold on the market. In the author’s opinion, our method might be easily adjusted and applied to the needs of the aquaculture sector, but also utilized in the identification of catfish which escaped from aquaculture facilities. 

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Accepted for print: 22.10.2018

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Remigiusz Panicz
Department of Meat Technology, Faculty of Food Technology and Fisheries,
West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4
71-550 Szczecin
Poland

email: rpanicz@zut.edu.pl

Jacek Sadowski
Division of Aquaculture, Faculty of Food Technology and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4
71-550 Szczecin
Poland

email: jsadowski@zut.edu.pl

Paulina Hofsoe-Oppermann
Division of Aquaculture, Faculty of Food Technology and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4
71-550 Szczecin
Poland

email: phofsoe@zut.edu.pl

Mirosław Półgęsek
Division of Aquaculture, Faculty of Food Technology and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4
71-550 Szczecin
Poland
email: mpolgesek@zut.edu.pl

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