Identification of sea snake meat adulteration in meat products using PCR-RFLP of mitochondrial DNA

Sunutch Suntrrchun,Lwn Chnhome,Montri Sumonth

a Queen Saovabha Memorial Institute,Thai Red Cross Society,Bangkok 10330,Thailand

b Ranong Marine Fisheries Station,Ranong 85000,Thailand

ABSTRACT PCR-RFLP based technique for identification of sea snakes in Thai waters was achieved by developing species-specific markers.To distinguish between sea snake species,the PCR products of cytochrome b(Cyt b),12S and 16S rRNA were sequenced and cut with different restriction endonuclease,Alu I and Hinf I.Each enzyme generated different-sized fragments which specific to Cyt b of eight sea snake species.However,the identical pattern was found among Hydrophis group.This result could be resolved by using these enzymes 12S rRNA digestion.This technique was successfully applied to blood,shed skin,raw meat,cooked meat,sea snake-fish binary admixture,and sea snake-pork binary admixture.Hence,it could be applied for identification when sea snake meat adulteration in meat products and sold as meatballs to reduce production costs.Hopefully,this technique would improve sea snake species identification when morphological examination is no longer possible because the animals are already processed.This is very important to track when sea snake species are being hunted and also used to assess the conservation and management of the sea snakes in Thai waters,especially the Gulf of Thailand.

Keywords:Sea snakes PCR-RFLP Adulteration

1.Introduction

Sea snakes are venomous elapid snakes,which are distributed throughout tropical and subtropical waters of the Gulf of Thailand,Andaman Sea,Indian and Pacific Oceans.Sea snakes are marine reptiles that are distinguished from the land snakes by their laterally compressed fin-like tail.Sea snakes belong to the family Elapidae and to the two subfamilies Hydrophiinae and Laticaudinae[1,2].Hydrophiinae are the viviparous or true sea snakes,which have laterally flattened tails and large and symmetrical shields on the head.They have upwards valvular nostrils on top of the snout and can close them tightly to exclude water.Viviparous sea snake,include more than 62 species that occupy diverse marine habitats and typically,spend their entire lives at sea,except the species that have secondarily moved into freshwater rivers and lakes.Only six species of viviparous sea snakes are abundantly found in the gulf of Thailand and the Andaman Sea Coast in Southern Thailand[3].These snakes includeEnhydrina schistosa(Beaked sea snake),Hydrophis caerulescens(Dwarf sea snake),Hydrophis cyanocinctus(Blue-banded sea snake),Hydrophis obscurus(Russell’s sea snake),Lapemis curtus(Short sea snake),andPelamis platura(Yellowbellied sea snake) [4].Laticaudinae are oviparous amphibious sea snakes or sea kraits.Although normally feed exclusively in the water,they spend substantial amounts of time on land where they digest their prey,slough their skins,mate and lay eggs.Laticaudais comprised by eight species and this is the only genus laying eggs on land.Two species of Laticaudinae sea kraits,Laticauda colubrina(Yellow-lipped sea krait),andLaticauda laticaudata(Brown-lipped sea krait),are broadly distributed across the waters of Continental Southeastern Asia to the Southwestern Pacific[5,6].Bites have become unusual with the advent of modern fishing methods,but still are a potentially serious hazard for the marine environment as their venom contains potent neurotoxins and myotoxins,more lethal than the venoms of many terrestrial snakes[7,8].

Southeast Asia is a principal region for sea snakes harvesting and trading.Sea snake harvest in the Gulf of Thailand via south-ern Vietnam is one of the largest harvests of venomous snakes and marine reptiles in the world [3,6].Although there have been several studies on the conservation aspects of terrestrial snakes in this region,the exploitation of sea snakes(marine elapids)in Southeast Asia,has received less attention from limited reports.Globally,9%of sea snakes are threatened,6%are near threatened,and 34%are data deficient,as defined by the International Union for the Conservation of Nature (IUCN) [8].Sea snakes are usually harvested by squid fishing vessels and trawlers that originated from ports in Malaysia and Thailand.Cao et al.[3]and Tu et al.[8]reported data the measured average biomass of sea snakes were annually harvested from the Gulf of Thailand was 80 tons,roughly equal to 225,500 snakes.Nearly every part of the snake,including skin,blood,and meat,is used for food,medicines,leather and other purposes[9].Sea snakes are exploited for food either raw or cooked.Sea snake meat is believed sometimes to be mixed with fish or pork and sold as fish ball or pork ball to reduce production costs.This illegal substitution raised significant public attention and caused serious social effects,surveillance of this meat adulteration has become a crucial challenge for government agencies and laboratories[10].

Table1 Classification of sea snakes used in sequence analyses.

Techniques for species identification have been investigated and developed as the key approaches for identifying meat adulteration during the past few years [2,5].Species identification based on protein analysis had been widely used and regarded as a reliable method.However,it is difficult to identify the species origin in processed products since the proteins are denatured and morphological features are no longer detectable after processing.This is especially important for threatened species due to illegal trading[11].Molecular tools such as DNA Barcoding based on mitochondrial Cyt b,12S and 16S rRNA for species identification,have recently started to be applied towards a wide variety of animals[12-14].

The aim of this study was to develop PCR-RFLP technique as species-specific DNA marker for identification of blood,shed skin,raw meat,cooked meat and binary admixtures of sea snakes in Thai waters,especially the Gulf of Thailand.These potential applications could protect sea snakes from overharvesting for illegal trading,protecting consumers from species fraud and adulteration in meat products,and concerning biological conservation of sea snakes.

2.Material and methods

2.1.Blood and shed skin samples

Forty-three blood samples and shed skins were obtained from viviparous sea snakes belonging to the speciesHydrophis cyanocinctus(HC),Hydrophis caerulescens(HCA),Hydrophis obscurus(HO),Pelamis platura(PEP),Enhydrina schistose(ES),andLapemis curtus(LCU).In addition,four sea kraits of the speciesLaticauda colubrina(LC)andLaticauda laticaudata(LLA)were also included(Table1).

2.2.Meat samples

Meat samples,HC,PEP,ES,LCU,and LC sea snakes,were obtained after a postmortem examination from a Veterinary Clinic of Snake Farm,Queen Saovabha Memorial Institute,The Thai Red Cross Society.Samples were collected aseptically in sterile collection bottles.They were cut into small pieces and then cooked by boiling(90°C,30 min),autoclaving (121°C,20 min),patties (72°C,30 min),and pan frying with vegetable oil.Binary admixtures of fish:sea snake and pork:sea snake were prepared in 50:50,60:40,70:30,80:20 and 90:10 proportions.

2.3.DNA extraction

Shed snake skins and processed meat products were cut into small pieces and minced.Meanwhile,binary admixtures were blended vigorously to make a homogeneous admixture.DNA extraction was performed using Genomic DNA extraction mini kit (Tissue) (RBC Bioscience,Taipei,Taiwan).DNA from the blood was extracted using a Genomic DNA extraction kit(Blood/Bacteria/Cultured cells)(RBC Bioscience,Taipei,Taiwan).

2.4.Oligonucleotide primers and amplification

Oligonucleotide primers of Cyt b,12S and 16S ribosomal RNA was designed based on a NCBI GenBank database (Table2).DNA amplification using PCR was carried out with 50 μl reaction buffer containing 10xbuffer,100 mM of each dNTP,25 mM MgCl2,50 pmol/μl of sense and antisense primers,Taq DNA polymerase and 10 μl DNA template.The amplification was preceded on a thermocycle (MWG Biotech,USA) at 94°C,3 min,followed by 40 cycles of 94°C/56°C/72°C one minute each with a final extension of 72°C for 7 min.The final products were electrophoresed on a 1.5%agarose gel containing ethidium bromide in 1xTAE buffer along with appropriate molecular size markers.The gel fragment containing the amplified product was excised and extracted using Gel/PCR DNA fragments extraction kit(RBC Bioscience,USA).

Table2 Oligonucleotide primers of Cyt b(tRNA),12S and 16S ribosomal RNA were designed based on NCBI GenBank database:mitochondrion,complete genome of Laticauda laticaudata (FJ 587153.1), Laticauda semifasciata (AB701344.1), Naja naja(NC 010225.1),Naja atra(NC 011389.1),Ophiophagus hannah(NC011394.1),Bungarus fasciatus (NC011393.1), Bungarus multicinctus (NC011392.1),and Daboia russellii (NC011391.1).DNA sequencing was carried out using the same primers used in the PCRs by 1st BASE sequencing(Malaysia-http://www.base-asia.com).

Fig.1.PCR amplification of Cyt b gene of different species from 8 sea snakes frequently found in Thai waters.Lane 1:MW marker 100 bp,lane 2:HC,lane 3:HCA,lane 4:HO,lane 5:LC,lane 6:ES,lane 7:PEP,lane 8:LCU,and lane 9:LLA.

2.5.Nucleotide sequencing

DNA sequencing was carried out using the same primers used in the PCR by 1st BASE sequencing (Malaysia-http://www.base-asia.com).

2.6.Restriction fragment length polymorphism(RFLP)analysis

Cyt b,12S,and 16S ribosomal RNA gene sequences obtained in this study were subjected to electronic RFLP using the Webcutter 2.0 software.Based on these preliminary results,two enzymes(Alu I and Hinf I)were selected to obtain different restriction digestion patterns among the venomous sea snakes(See Tables 4 and 5).Fifteen microliters of PCR product(approximately 1 μg)were separately digested with 1 U of Alu I and Hinf I (Vivantis,USA),in a final volume of 20 μl for 4 h at 37°C.The final products were electrophoresed on a 2% agarose gel containing ethidium bromide in 1xTAE buffer along with appropriate molecular size markers.

3.Results

PCR amplification products of Cyt b gene for HC,HCA,HO,LC,ES,PEP,LCU,and LLA sea snakes were shown in Fig.1.The size of the amplified product was 1144 bp in all species.Forty seven numbers represented by the eight species of sea snakes were tested and sequences have been deposited in the NCBI database with the accession numbers from KX239640 to KX239647.Cyt b gene from HC and HCA in Thai waters shared the highest number of homologies among Asian and Australia HC and HCA lineages.DNA sequencing of Cyt b gene from other sea snakes,including HO,LC,ES,PEP,LCU,and LLA in Thai waters were also closely related to the sequences of sea snakes mostly found in Southeast Asia (data not shown).A specific primer for Cyt b was subsequently tested in different varieties of sea snakes and used for PCR-RFLP studies.The uniform amplification was observed in heat processed meats at 72°C and 90°C for 30 min and meats subjected to deep frying.The intensity of signal reduced with heat processed meats compared with fresh meat products.However,no band obtained after 121°C for 30 min autoclaving,this could be due to complete degradation of DNA(Fig.2).

The mitochondrial Cyt b sequence of eight venomous snakes was assembled and subjected to the electronic RFLP.Following the electronic RFLP,two enzymes Alu I and Hinf I were then selected to obtain different restriction digestion patterns among the venomous sea snakes(Tables 3 and 4).These two enzymes generated reproducible species-specific restriction patterns and allowed to discriminate eight snake species on agarose gel.Expected restriction fragments of Cyt b gene were obtained except LLA (Fig.3A).In addition,Fig.3B showed aberrant or unexpected PCR-RFLP pattern of LLA which might come from incomplete digestion of the enzyme or variation in DNA sequence.However,Cyt b gene,from HC and HCA,generated identical patterns among sea snakes when both Hinf I and Alu I have been used which came from the highest number of homologies of Cyt b gene.These results could be resolved by using additional gene such as 12S rRNA with these Hinf I and Alu I restriction enzymes (Fig.4).Partial sequences of 12S and 16S rRNA from eight species venomous sea snakes in Thailand were also deposited in GenBank under the accession number from KX239648 to KX239663.Different cooking methods also did not affect the PCR-RFLP pattern and the results were similar in fresh and processed meat products(data not shown).Specificity did not vary with cooking methods.

Fig.2.PCR amplification of Cyt b gene of different processed meat products from sea snakes using PEP as an example.Lane 1:MW marker 100 bp,lane 2:Fresh meat products,lane 3:Pan frying,lane 4:Patties (72°C,30 min),lane 5:Boiling(90°C,30 min),and lane 6:Autoclaving(121°C,20 min).Binary admixtures between fish and PEP were also shown.Lane 7:Combination;fish and PEP at 50:50,lane 8:Combination;60:40,lane 9:Combination;70:30,lane 10:Combination;80:20,and lane 11:Combination;90:10.

Table3 Summary of predicted size of DNA segments(Cyt b gene; 1144 bp) based on electronic restriction fragment length polymorphism analysis with the Webcutter 2.0 program.

Table4 Summary of predicted size of DNA segments (12S rRNA gene; 528 bp) based on electronic restriction fragment length polymorphism analysis with the Webcutter 2.0 program.

Similar studies were conducted on binary admixtures(fish:sea snake and pork:sea snake).For this purpose,various combinations of binary admixtures were prepared.Only DNA samples from 50:50 and 60:40 proportions of binary admixtures successfully amplified with Cyt b primer set.However,the intensity of the signal reduced with decreased proportions of sea snake in binary admixtures(Fig.2).It seems likely that during PCR,the amplification may be proportional to the respective quantity of DNA present.The obtained results could correspond to the proportions of the mixtures.These results can be very important to track when sea snakespecies are being hunted and also used to assess the conservation and management of the sea snakes in Thai waters,especially the Gulf of Thailand.

Fig.3.PCR-RFLP patterns of sea snakes for Cyt b gene obtained by digestion with enzyme Hinf I.Fig.3A;lane 1:100 bp molecular weight marker,lane 2:HC,lane 3:HCA,lane 4:HO,lane 5:LC,lane 6:ES,lane 7:PEP,and lane 8:LCU.Additional gel in Fig.3B;lane 9:100 bp molecular weight marker and lane 10:LLA.

Fig.4.PCR-RFLP patterns of HC and HCA for 12S rRNA gene obtained by digestion with enzyme Hinf I and Alu I.Lane 1 and 7:100 bp molecular weight marker;lane 2 and 5:HC;land 3 and 6:HCA.

4.Discussion

Sea snakes in the Gulf of Thailand were captured by squid fishing vessels.The boats were equipped with electric lamps used to attract squids at night.Sea snakes were harvested opportunistically at night due to their attraction to the board lights.There is an increasing trend to commercialize sea snakes,rather than discard bycatch in most artisanal fisheries of Southeast Asia and the South China Sea.In 1995,there were 20-30 squid fishing vessels harvesting sea snakes.In 2013,there were approximately 900 squid fishing vessels in Vietnam.Every year 80 tonnes of sea snakes are harvested in the Gulf of Thailand.Identification of snakes obtained during sea snake trading sessions indicated thatLapemis curtusandHydrophis cyanocinctusconstituted more than 85%of the total snake biomass harvested.The approximate proportion by weight for these 2 species in the catch varied from 50:50%to 80:20%[3,6].

Sea snake trading is a dangerous business for both snakes and fishermen,due to the snakes are in danger of being overharvested and the fishermen could get bitten.Most of the sea snakes end up in Vietnam and China for snake products.Nearly all parts of the snake were used for food,drink or medicine.Snake meat is a delicacy.It can be fried or eaten in soups.The snake’s organs,including the heart were considered potent elixirs and the gall bladder was a benefit to pregnant women.The blood has been often mixed with alcohol to enhance health benefits.Sea snakes were also submerged in rice wine and served as a drink[3].It is believed that sea snakes were the source of Rheumatin and people also made “sea snake glue” which is sold to cure point pain,back pain,anorexia,and insomnia and to strengthen tendons and bones.A restaurant in the Ryukyu Islands of Japan has the meat and skin of black-banded sea krait or Chinese sea snake (Laticauda semifasciata) as the specialty of the house.Moreover,this species has become the target for smoked sea snake meat in the Philippines[15].

Harvesting of sea snake has been going on essentially unnoticed by national and international conservation organizations for more than a decade.The enforcement oflaws aimed at managing the trade in widely harvested terrestrial snakes such as various cobra species,may have the unintended consequence ofincreasing the market for sea snakes [3].No sea snake species are currently listed under the Convention on International Trade in Endangered Species of Flora and Fauna (CITES) [8].However,a research team has reported in the journal of Conservation Biology that fishermen have noticed the decline in sea snake population since 2009.The researchers now want to understand if this is due to overfishing or other factors including pollution[3].Scientific knowledge of sea snakes is limited,lacking detailed information on distribution,abundance,ecology,and physiology.Available data are dispersed among published and unpublished sources.Although,there have been several studies on the conservation of terrestrial snakes,the exploitation of sea snakes has received very little attention apart from a few and limited reports[16].

In recent years,incidences of meat fraud have raised significant social attention [17-19].The adulteration and substitution of meat are a serious concern for consumers.The examination of the meat components in food industries is important for religious,quality assurance,food safety,public health,and legal concerns[20,21].Street food vending is a common feature of most cities in developing countries through the provision of ready-made meals at relatively inexpensive prices.The quest for profit maximization by the vendors or the need to make street foods affordable for the consumers,make some vendors patronize cheap and unsafe ingredients that may be detrimental to the health of the consumers[22,23].

A brief,PCR-RFLP protocol with a set selected enzyme for rapid identification of common domestic animals have been reported[24,25].The first report of using PCR-RFLP for terrestrial venomous snakes identification in Thailand has previously been published[26].However,no one has ever been interested in sea snake identification using molecular tools.We successfully developed PCR-RFLP mitochondrial marker to distinguish between sea snake species.The target source for sea snake identification comes from blood,shed skin,fresh meat and cooked meat.Cooking meat samples at various temperatures with holding period 30 min did not any adverse effects of amplification of species-specific markers for sea snake species.However,it was seen that Cyt b could not be amplified from all samples of sea snakes in extreme conditions of heat process by autoclaving.It was probably due to disintegration of DNA during the autoclaving process.To avoid the destruction effect of the high temperature,the small size of the DNA fragment should be amplified.PCR-RFLP pattern of restriction enzymes used in this study was highly specific and the specificity did not vary with the processing temperatures of the meat samples.This technique could be applied with equal efficiency to blood,shed skin,fresh and processed meats.However,the limitation of PCR-RFLP is its inapplicability to detect minor components present at less than 40%in binary mixtures between sea snake meats and commercial meats(fish and pork).Hence,this technique could be recommended for quick identification as a DNA marker for sea snake contaminationin meat products of seafood market and street food business which cause serious social effects.

5.Conclusions

PCR-RFLP was developed in this study could be useful in identifying the species of sea snakes from blood,skin,fresh or cooked sea snake meats.The process ofidentification is simple,rapid,reliable and straightforward to trace meat adulteration and could differentiate species of sea snakes presented in binary mixtures between sea snake meats and commercial meats (fish and pork).Hence,it could be routinely applied for species identification and verification the quality control of seafood industrial and street food business.This is a preliminary effort in sea snakes to generate markers considering funds and high throughput equipment in the lab are not available.Hopefully this technique will not only improve sea snake species identification,but also strengthen the law enforcement for overharvesting of sea snakes.The information from these studies may assist conservation and management ofillegal trade in sea snake species in Thai waters,especially the Gulf of Thailand.

Conflict ofinterest

The authors have no conflict ofinterest to declare in this study.

Acknowledgements

The authors would like to thank the staffs of snake farm,Queen Saovabha Memorial Institute for collecting snake samples.This work was supported by a grant from The Thai Red Cross Society,Bangkok,Thailand(Grant Number QSMI 5809).