Mitochondrial Cytochrome b based Identification of Some Commercially Important Fishes Found in the Arabian Sea Coast of Pakistan

Fish industry is serving as a backbone of the economy of many countries. In order to gain high and easy profit, commercial frauds of mislabeling has been reported earlier in many different cases. Chances of mislabeling a relatively less expensive fish with an expensive one particularly if they are morphologically similar gets very high as it is not easy to differentiate them. This problem increases when it is the case of processed fish as morphological characters are not available for the identification. DNA based methods are likely to solve this dilema. To our knowledge no report has been made on the DNA based methods of marine fish identification though there are some reports of DNA based fresh water fish identification. We have used the cytochrome b gene of mitochondria for performing DNA sequencing and PCR-RFLP of 22 fish species belonging to different families of commercial importance and have shown that these fish can be successfully identified by these techniques.

Fish industry is serving as a backbone of the economy of many countries.In order to gain high and easy profit, commercial frauds of mislabeling has been reported earlier in many different cases.Chances of mislabeling a relatively less expensive fish with an expensive one particularly if they are morphologically similar gets very high as it is not easy to differentiate them.This problem increases when it is the case of processed fish as morphological characters are not available for the identification.DNA based methods are likely to solve this dilema.To our knowledge no report has been made on the DNA based methods of marine fish identification though there are some reports of DNA based fresh water fish identification.We have used the cytochrome b gene of mitochondria for performing DNA sequencing and PCR-RFLP of 22 fish species belonging to different families of commercial importance and have shown that these fish can be successfully identified by these techniques.
F ish and fishery products are one of the major sources of foreign exchange earning of Pakistan.Hundreds of highly valuable fish species are found in the Arabian Sea Coast of Pakistan that are exported worldwide as per demand (Hand Book of Fisheries, 2012;Psomadakis et al., 2015).Various factors have increased the potential of fraud, such as increase in fish consumption, rise in international trade and high demand of certain species, thus fish are mislabeled and relatively cheaper fish are sold by labelling as an expensive one (Civera, 2003;Rasmussen and Morrissey, 2008).Although mislabeling is not always deliberate as sometimes different species with similar morphology in a catch cannot be distinguished, however deliberate mislabeling is more frequent (Blanco-Fernandez et al., 2021).The fraud in seafood can range from involving millions of dollars in multinational imports to individual retailers and restaurants (Hellberg and Morrissey, 2011).Strict laws have been established by various regulatory organizations that require proper information like origin, processing method and identification of the fish species (Moretti et al., 2003).Although some protein based methods are in use worldwide for their identification of species, including Iso-Electric Focusing (IEF), Capillary Electrophoresis (CE), High Performance Liquid Chromatography (HPLC) and Enzyme-Linked Immuno-Sorbent Assay (Kvasnička, 2005;Asensio et al., 2000), but these techniques works only with the relatively fresh samples as their results are not reliable with processed samples and protein moieties tends to lose their biological activities and occurrence of structural conformational changes after the catch and process (Rasmussen and Morrisey, 2008).Forensic science is now recognizing the importance of DNA methodologies for the identification and differentiation of animals and it is now also used in the legal proceedings (Withler et al., 2004).This is the reason why various DNA based methods are developed to identify tissue source or even organisms that also includes commercially important fish.DNA methods are more reliable and sensitive when compared to the existing protein-based methods (Teletchea, 2009) as DNA is found to be the same regardless of the type of the tissue and age of the organism in contrast to the proteins that can vary (Civera, 2003).Due to their specific sensitivity and stability, DNA based results are more reliable even with the processed samples (Teletchea, 2009).
This study deals with molecular identification of some marine fishes found in Arabian Sea coast of Pakistan using mt cytochrome b as marker gene.

O n l i n e F i r s t A r t i c l e
Materials and methods A total of sixty six fish samples belonging to twenty two different species shown in Table I were collected in triplicate, from the Arabian Sea coast and the Karachi Fish Harbor, Pakistan.After taxonomic confirmation, on the basis of morphology as per FAO Field Identification Guide for Species Identification (Psomadakis et al., 2015) in collaboration with Biological Section of the Marine Fisheries Department, Karachi, Pakistan, all the samples were shifted to the laboratory and kept in the freezer at -20 ⁰ C till further use.
The genomic DNA was extracted from 300mg muscles of fish samples (in replicates) by Phenol-Chloroform Isoamyl-Alcohol method (Taggart et al., 1992).The extracted DNA quality and purity was checked by visualizing on 1% agarose gel containing 0.5µg/ml ethidium bromide after electrophoresis.
A set of primers was used for the amplification of the ~464bp region of mt.cytochrome b gene L14735 5'-AAAAACCACCGTTGTTATTCAACTA -3' and H15149 5' -GCNCCTCARAATGAYATTTGTCCTCA -3' (Russell et al., 2000).Briefly PCR amplification was done at the annealing temperature of 56 ⁰ C after optimizing on Gradient Thermal Cycler (D-Lab, China).The amplification reaction was carried out by mixing 30 µl of 2X prepared PCR master mix (Bioron, Germany), 1 µm of each primer, 50 ng extracted DNA template in a final reaction mixture volume of 60 µl raised with molecular grade water (Bioron, Germany).The conditions set for the PCR amplifications were initial denaturation at 95 ⁰ C for 10 min, followed by 45 cycles of 95 ⁰ C for 1 min denaturation, annealing at 56 ⁰ C for 1 min and extension at 72 ⁰ C for 1 min with the final extension of 10 min at 72 ⁰ C. Amplified PCR products were visualized by electrophoresis on 2% agarose gel having 0.5 µg/ml ethidium bromide.For each species, the one best amplified product in terms of purity and concentration, was selected for further analysis.The selected amplified DNA products were commercially sequenced (Macrogen, Korea) and used for virtual digestion by Restriction Mapper Version III, for getting the exact DNA fragment sizes (DNA sequence not shown).
For RFLP analysis PCR amplicons of about 464bp were digested with the two restriction endonucleases NlaIII and HaeIII (Thermo Scientific) separately.15 µl PCR product, 1 µl (10 U/µl) restriction enzyme, 2 µl of 10x Tango buffer supplied by the manufacturers, and 2 µl molecular grade water, in the final volume of 20 µl constituted the reaction mixture.The reaction mixture was incubated at 37 ⁰ C for 3 h.The digested products were visualized by electrophoresis on 4% agarose gel containing 0.5 µg/ml ethidium bromide alongside 50bp DNA ladder (Bioron Life Sciences).

Results and discussion
The aim of the current research was to use the DNA PCR-RFLP to identify and differentiate various commercially important marine fish species of Pakistan.
Studies have shown the usefulness of this technique in the identification of various species (Pfeiffer et al., 2004;Yahia and Kamata, 2018).Though generation or deletion of additional restriction sites due to intraspecific variations and incomplete digestion are the possible drawbacks of PCR-RFLP (Lockley and Bardsley, 2000), this technique is still considered to be one of the most powerful technique for the identification and differentiation of species.We also used the same technique for the identification of the twenty two fish species belonging to different genera caught from the coast of Pakistan that are of commercial importance as are exported to various countries.The genomic DNA successfully extracted from all the fish samples was used for the amplification of the mt cytochrome b gene fragment by using specific primers.All the fish DNA samples generated good quality DNA fragments of approximately 430 bp with no apparent contamination when ran on the agarose gels.Other studies also used approximately the same size cytochrome b gene fragments for the identification of various species (Russell et al., 2000).I.

O n l i n e F i r s t A r t i c l e
Although different studies have used various restriction enzymes (Xu et al., 2016) for the identification of fish species through RFLP, restriction enzymes NlaIII and HaeIII were used in this study and were found to be sufficient to differentiate the studied fish samples.The amplified ~ 430 bp partial mtDNA cytochrome b gene fragments digested with the restriction enzymes NlaIII and HaeIII generated specific DNA banding patterns for the twenty two species studied shown in Figure 1A and  B. The sizes of the bands were also confirmed by the virtual digestion of the DNA sequences of the cytochrome b gene DNA fragments obtained by Sanger sequencing commercially (Macrogen, Korea) by Restriction Mapper Version III as shown in the Table I and was found to match the banding pattern obtained by performing the PCR RFLP of the cytochrome b gene fragment.For many of the fish species used in this study, one of the two restriction enzymes was found to be enough to give a characteristic banding pattern.In the cases where one of the two enzymes did not cut the cytochrome b gene fragment at all or the banding pattern was also found to be same or similar to other specie(s), the other enzyme gave a distinctive DNA banding pattern.The enzymes NlaIII and HaeIII mostly gave two to four fragments when used for digesting the cytochrome b gene DNA fragments of the fish species used in this study.
The cytochrome b gene DNA fragment sizes of Escualosa thoracata, Thryssa vitrioritis and Eleutheronema tetradactylum obtained after the digestion with the restriction enzyme NlaIII gave were found to be almost same as Escualosa thoracata gave three restriction fragments of 272, 83 and 80 bp, Thryssa vitrioritis gave four bands of 272, 80,75 and 8 bp and Eleutheronema tetradactylum also generated three bands of 272, 88 and 75 bp.However, the use of the

O n l i n e F i r s t A r t i c l e
S. putnamae, Ilisha striatula and Netuma aff.bilineata species did not show any restriction site for the enzyme HaeIII in the amplified cytochrome b gene fragment but the use of NlaIII generated quite distinct fragment pattern.Of all the fish species studied Acanthopagrus butcheri was the only species that did not contain the restriction sites for the enzymes NlaIII and HaeIII in the amplified cytochrome b gene fragment.Thus, the two restriction enzymes NlaIII and HaeIII were found to be enough to distinguish and identify the twenty-two marine fish species used in this study as was also observed by Cocolin et al. (2000) with their fish samples.This study also highlighted the importance of the use of molecular biology techniques instead of the conventional morphological based identification of the fish species.At least five fish species were found to be identified incorrectly through the morphological basis when their DNA sequence were matched with the database on NCBI.The DNA sequence obtained with the twenty two fish species showed BLAST matching from around 91% to 100% with the sequence available in the NCBI database.

Conclusions
Our results have shown that the use of PCR RFLP is a powerful and sensitive technique to identify and distinguish all the twenty-two fish species used in this study.The use of the two restriction enzymes NlaIII and HaeIII is enough for all the twenty-two species used in this study to be identified and distinguished.The data has been shared with the Marine Fisheries Department, Pakistan and is the step towards the establishment of the database for the marine fish species found in Pakistan.

Fig. 1 .
Fig. 1. (A and B) Restriction fragment length polymorphism pattern of the amplified fragment of cytochrome b gene of the fish species with the use of enzymes NlaIII and HaeIII after running on 4% agarose gel containing 0.5 µg/ ml Ethidium Bromide alongside 50bp (L 50) DNA ladder (Bioron Life Sciences).U/C -Uncut PCR amplified DNA fragment.The symbols used for the fish species are shown in TableI.

Table I . Fish species identified morphologically and on the basis of the amplified fragment of the cytochrome b gene DNA sequence.
restriction enzyme HaeIII gave distinctive DNA fragment sizes to easily differentiate them i.e.Escualosa thoracata gave 289, 135 and 11 bp, Thryssa vitrioritis gave 221, 154 and 60 bp and Eleutheronema tetradactylum gave 153, 135,117 and 30 bp.Similarly, Hilsa kelee and Anodontostoma chacunda both species gave three bands with NlaIII of 183, 167 and 87 bp and 180, 179 and 73 respectively, but with the restriction enzyme HaeIII generated 178, 142, 111 and 6 bp and 400 and 32 bp, respectively that are quite distinguishable.Similar was the case with Katsuwonus pelamis compared with Sphyraena putnamae and Thunnus albacares compared with Euthynnus affinis, where the other gave distinctive fragment sizes.Sardinella longiceps, Nematalosa nasus, Matching Accession numbers in the NCBI data base with the percentage match is also given.The band sizes from the virtual digestion with the Restriction mapper version III with the enzymes NlaIII and HaeIII is also given.Five fish species identified differently with the DNA sequence compared to the morphological identification are shown in bold.