Genetic diversity and species identification of Argulus parasites collected from major aquaculture regions of India using RAPD-PCR

Abstract

Argulus is one of the most important fish parasites that cause heavy economic loss to aquaculture industry. The present investigation was undertaken to study the genetic diversity of the Argulus sp. collected from 13 locations representing major aquaculture zones in India by RAPD analysis and to develop species-specific markers. Thirteen random decamer primers were used to amplify DNA fragments from three individual parasites of each location. Of the 172 bands scored by the primers, 168 were polymorphic. The per cent polymorphic loci and gene diversity values varied within a range of 8.14–43.02 and 0.0342–0.1727 respectively. Nei's genetic similarity between populations across all the primers ranged from 0.363 to 0.969. The dendrogram based on Nei's genetic distance showed two clusters; Bangalore and Mandi populations forming one cluster, and the rest in another cluster. The clusters also revealed strong correlation with the species identified as A. japonicus and A. siamensis respectively by morphological method. The study thus indicated A. siamensis as the major prevalent species in carp culture farms in India. Species-specific primers were designed from unique sequences cloned from RAPD fragments that could able to identify A. siamensis and A. japonicus separately.

Introduction

Argulosis, caused by an ectoparasite of genus, Argulus, is of major economic concern in all phases of the aquaculture industry from production to marketing. Argulus sp. (Crustacea: Branchiura) commonly known as fish lice is one of the most damaging ectoparasite of fish. Severe damage to fish stocks, both in trout (Menezes, Ramos & da Silva 1990) and carp farms (Rahman 1996) have been reported due to epidemics of argulosis. Argulus sp. is also reported from freshwater lakes (Valtonen, Holmes & Koskivaara 1997), riverine systems (Natarajan 1982) and fish farms (Buchmann & Uldal 1995; Grignard, Melard & Kestemon 1996) though its intensity is much higher in culture conditions than in natural environment.

Argulus sp. is cosmopolitan in distribution, found in all the continents except Antarctica (William 2008) and can survive in wide range of temperatures. About 129 species of Argulus have been reported worldwide (William 2008). It has a complex life cycle involving several metamorphic stages (Walker, Flik & Wendelaar Bonga 2004). Argulus species cause skin lesions on the host by their suckers and proboscis while feeding. These lesions often lead to secondary infections by bacteria and fungi (Walker et al. 2004). The infection also causes reduced appetite, weight loss and anaemia in fish. Argulus serves as a vector of some viruses and parasitic nematode larvae (Walker et al. 2004). Hence, argulosis is responsible for production loss, mortality, breeding failure, marketing problems and secondary infections. In spite of many studies going on, argulosis remains a major concern due to non-availability of any permanent control measures.

Genetic data and phylogeographical structure of a species provide the basic information required to understand their evolution and biogeographical history. Identification of parasites, particularly crustaceans, poses serious problem for the researchers throughout the world. Genome based approaches are now in use to identify parasitic species and study their diversity.

The phylogenetic studies of Argulus sp. are very limited. After the initial submission of a partial sequence of 28S rRNA of Argulus sp. to GenBank (Oakley & Cunningham 2001), Møller, Olesen, Avenant-Oldewage, Thomsen and Glenner (2008) presented the first molecular phylogeny of Branchiura, Pentastomida and other Maxillopoda based on mitochondrial 16S rRNA, nuclear 18S and 28S rRNA. Lavrov, Brown and Boore (2004) made comparative phylogenetic analysis of Pentastomida using cytochrome c oxidase subunit 1 (pcox1), cox2 and NADH dehydrogenase subunits (pnad4) and sequenced complete mitochondrial DNA (mt DNA) of Argulus americanus. In a recent study, Wadeh, Alsarakibi and Li (2010) investigated the genetic variability within fish louse Argulus japonicus from Africa, Middle East and Asia by examining sequence variability in three mtDNA regions, namely, cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4). However, there is no available data pertaining to phylogenetic study of Argulus sp. prevalent in India.

Randomly amplified polymorphic DNA (RAPD) is a simple and easy molecular method to determine genetic diversity, taxonomic identity and systemic details of various organisms (Welsh & McClelland 1990; Williams, Kubilik, Livak, Rafaski & Tingey 1990). This technique has been used to estimate genetic diversity in parasites (Todd, Walker, Wolff, Northcott, Walker, Ritchie, Hoskins, Abbott & Hazon 1997), penaeid shrimp (Shi, Kong, Liu, Hart, Zhuang & Deng 1999; Song, Xiang, Li, Liu & Liu 1999; Zhuang, Shi, Kong, Liu, Liu, Meng & Deng 2001; Mishra, Chaudhuri, Krishna, Kumar & Lakra 2009) and fish (Naish, Warren, Bardakci, Skibinski, Carvalho & Mair 1995; Dahle, Rahman & Eriksen 1997; Tassanakajon, Pongsomboon, Rimphanitchayakit, Jarayabhand & Boonsaeng 1997, 1998; Lakra, Goswami, Mohindra, Lal & Punia 2007).

In the present study, genetic diversity and genetic similarity within and among 13 Argulus populations were investigated using RAPD analysis and further species-specific markers were developed for two isolated species of Argulus found in Indian aquaculture systems. It provides baseline data for future work on population structure analysis of Argulus sp., as very limited information exist for this economically important species and will help in identification of these parasites.

Materials and Methods

Collection of parasites

Argulus parasites were collected from major freshwater aquaculture regions of India (Fig. 1) and also based on outbreak information during February 2008 to February 2010 and were used as 13 different populations in this study. The parasites were preserved in 4% neutral buffered formalin (NBF) (for morphological identification) and in absolute alcohol (for extraction of genomic DNA) after thorough washing with PBS and incubating in PBS for an hour before being transferred to fixatives. The alcohol preserved samples were stored at −20°C, until DNA was extracted. Host caudal fin samples were also collected in absolute alcohol for DNA extraction. The parasites were identified morphologically based on Meehean (1940), mostly observing the respiratory areas, secondary maxillae and swimming appendages of male specimens.

Figure 1. Map of India showing sampled locations of Argulus species. 1. CIFA, Khurda, Orissa, 2. Siula, Puri, Orissa, 3. Balasore, Orissa, 4. Jajpur, Orissa, 5. Kolkata Bheri, West Bengal, 6. Tura, Meghalaya, 7. Hessarghata, Karnataka, 8. Bangalore, Karnataka, 9. Mandi, Himachal Pradesh, 10. Vijayawada, Andhra Pradesh, 11. Jagatsinghpur, Orissa, 12. Naihati, West Bengal and 13. Nellore, Andhra Pradesh.

Isolation of genomic DNA

Genomic DNA was isolated from individual parasite or host by phenol-chloroform extraction method following the method of Sambrook and Russel (2001) with minor modifications. DNA from three individuals of each population of each location was used in this study. Individual parasite was crushed using a sterile mortar and pestle in the presence of 700 μL TEN buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl) and transferred to a 2.0 mL eppendorf tube. Proteinase K and SDS were added at a final concentration of 500 μg mL⁻¹ and 1% respectively. The mixture was mixed thoroughly and incubated overnight at 37°C. DNA was extracted thrice with phenol, phenol:chloroform:isoamyl alcohol (25:24:1) followed by chloroform:isoamyl alcohol (24:1), and was precipitated by adding 0.3 M sodium acetate and absolute alcohol. DNA pellet obtained by centrifugation was further washed with 70% ethanol. The pellet was air dried and dissolved in TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0). Extracted DNA was checked for its purity and quantity using NANODROP (ND1000, Thermo Scientific, Wilmington, DE, USA) and diluted with distilled water for a working concentration of 50 μg mL⁻¹.

RAPD-PCR

The PCR reaction was carried out using 25 ng of genomic DNA as template DNA in a total volume of 25 μL reaction mixture. The reaction conditions optimized for amplification of random fragments were 0.75 units Taq polymerase (Genei, India), 1× Taq buffer A, 100 mM dNTPs and 20 picomole RAPD primer per reaction. The amplification was carried out in a thermal cycler (MJ Research, Waltham, MA, USA) as per the following programme: initial denaturation at 94°C for 5 min, 40 cycles of 94°C for 1 min, 36°C for 1 min and 72°C for 2 min, followed by final extension at 72°C for 7 min. Thirteen random decamer primers (OPA04, OPA11, OPA14, OPH04, OPH08, OPH11, OPH15 (IDT, Milpitas, CA, USA) and OPC08, OPC11, OPC15, OPC19, OPY02, OPY20 (QIAGEN OPERON, Nattermannallee, Cologne, Germany) were selected from kit A, C, H and Y depending upon repeatability and reproducibility of amplified fragment patterns and were used for amplification of DNA by PCR. Samples without genomic DNA (no DNA controls) as well as fish genomic DNA (host DNA controls) were included in each PCR amplification run. Fish genomic DNA was taken to remove ambiguity for presence of any similar banding patterns for further downstream applications of parasitic DNA. The criteria followed for taking a fragment into further analysis were reproducibility and distinctness of fragments.

Agarose gel electrophoresis

Amplified products were separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide in 1× TBE buffer at 100 V for 2 h. To determine the molecular size, 100 bp DNA ladder was run alongside RAPD products. The gels were visualized in a UV gel documentation system (ALPHA INNOTECH, San Leandro, CA, USA).

Analysis of data

The bands observed in each lane were compared with all the other lanes on the same gel. The reproducible bands were scored visually as either presence (1) or absence (0). Scores with respect to all the primers were used for constructing a single data matrix. The data were analyzed using PopGene version 1.31 software (Yeh, Yang & Boyle 1999) for estimation of polymorphic loci, genetic diversity within populations, interpopulation genetic diversity and for construction of dendrogram among populations based on genetic distances (Nei 1972). The unweighted pair group method with arithmetic average (UPGMA) was used to construct the dendrogram and the robustness of the dendrogram was tested using 1000 bootstraps.

Development of species-specific markers

The unique bands obtained with a few primers based on morphologically identified Argulus species were excised from gels. Three bands/amplicons per species were gel purified, cloned into pGMET (Promega, Madison, WI, USA) vector and sequenced in triplicate clones using an ABI377 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) using Bigdye Terminator Chemistry) employing the universal primers. The consensus sequences generated for both species are submitted to GenBank (Accession numbers JF795591 and JF795592). Primers were subsequently designed from both the sequences using Primer Premier 3 software (Premier Biosoft, Palo Alto, CA, USA) to specifically amplify both species as identified morphologically to test the specificity and/or presence of any cross-amplification using the parasitic DNA collected from 13 different populations. Besides, samples without genomic DNA (negative controls), host DNA control, DNA from pool of zoo- and phytoplanktons and Macrobrachium rosenbergii (de Man) were included in each amplification run and in no case desired amplicons from these controls were detected. The PCR reaction conditions optimized for amplification were 0.75 units Taq polymerase (Genei, India), 1× Taq buffer A, 100 mM dNTPs and 10 pM of each forward and reverse primer per reaction. The amplification was carried out in a thermal cycler (MJ Research, UK) as per the following programme: initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 45 s, annealing temp (53°C for ArS1 and 54°C for ArJ1) for 45 s and 72°C for 1.5 min followed by final extension at 72°C for 7 min. Ten microlitre of each amplicon was examined by agarose gel electrophoresis as described earlier.

Results

Based on the morphological characters, the parasites of Mandi and Bangalore were identified as A. japonicus, whereas all other specimens obtained from rest 11 locations were A. siamensis. The representative RAPD profiles of 39 individual Argulus samples from all the 13 populations generated by primers OPC 19 and OPH 11 are depicted in Fig. 2a and b. It was clearly evident from these figures as well as data generated using other primers, that the RAPD profiles of Bangalore and Mandi populations were clearly distinct. RAPD profiles of Argulus sp. obtained by 13 random primers are summarized in Table 1. From all the 13 primers, 172 bands were scored, of which 168 bands (97.67%) were polymorphic (Table 1). Number of the scored fragments varied from 2 to 11 with a size range of 188–2719 bp (Table 1). Genetic diversity within populations varied from 0.0342 ± 0.1187 to 0.1727 ± 0.2092. The Jajpur, Orissa population was found to have maximum genetic diversity with 43.02% polymorphic loci and the Nellore population from Andhra Pradesh had a minimum with 8.14% polymorphic loci (Table 2). The RAPD banding patterns obtained using host DNA were clearly distinct from that of parasites of all locations.

Figure 2. RAPD profile of 39 individual Argulus samples from 13 different locations by OPC 19 (a) and OPH11 (b) primers. Lane M- 100 bp DNA ladder, Lanes 1–39- three samples from each location (Lanes 1–3, CIFA; 4–6, Siula; 7–9, Balasore; 10–12, Jajpur; 13–15, Kolkata Bheri; 16–18, Tura; 19–21, Hessarghata; 22–24, Bangalore; 25–27, Mandi; 28–30, Vijayawada; 31–33, Jagatsinghpur; 34–36, Naihati and 37–39, Nellore).

Table 1. RAPD profiles of Argulus sp. obtained by 13 random primers

Primer	Sequence (5′ to 3′)	No. of bands scored	Size of fragments (bp)	Total no. of bands	No. of polymorphic bands	Polymorphic bands (%)
OPA4	AATCGGGCTG	3–9	294–1241	14	14	100
OPA11	CAATCGCCGT	2–6	322–2719	10	10	100
OPA14	TCTGTGCTGG	5–11	406–1705	14	14	100
OPC8	TGGACCGGTG	6–11	387–2659	17	16	94.1
OPC11	AAAGCTGCGG	3–8	283–1945	11	11	100
OPC15	GACGGATCAG	2–10	380–1448	12	12	100
OPC19	GTTGCCAGCC	4–8	479–1546	14	14	100
OPH4	GGAAGTCGCC	3–9	242–1608	10	9	90
OPH8	GAAACACCCC	5–10	312–1518	15	14	93.3
OPH11	CTTCCGCAGT	3–10	549–1888	11	11	100
OPH15	AATGGCGCAG	5–9	274–2142	14	13	92.8
OPY2	CATCGCCGCA	3–7	249–1812	14	14	100
OPY20	AGCCGTGGAA	3–9	188–2292	16	16	100
Total				172	168	97.67

Table 2. Number and percentage of polymorphic loci and gene diversity values within populations of Argulus sp

Population	Polymorphic loci		Gene diversity (Mean ± SD)
	No.	%
A. siamensis, CIFA, Khurda, Orissa	27	15.7	0.0684 ± 0.1642
A. siamensis, Siula, Puri, Orissa	23	13.37	0.0748 ± 0.1640
A. siamensis, Balasore, Orissa	27	15.7	0.0648 ± 0.1540
A. siamensis, Jajpur, Orissa	74	43.02	0.1727 ± 0.2092
A. siamensis, Kolkata Bheri, West Bengal	28	16.28	0.0637 ± 0.1517
A. siamensis, Tura, Meghalaya	20	11.63	0.0574 ± 0.1514
A. siamensis, Hessarghata, Karnataka	18	10.47	0.0358 ± 0.1171
A. japonicus, Bangalore, Karnataka	15	8.72	0.0362 ± 0.1219
A. japonicus, Mandi, Himachal Pradesh	32	18.6	0.0712 ± 0.1540
A. siamensis, Vijayawada, Andhra Pradesh	51	29.65	0.1371 ± 0.2071
A. siamensis, Jagatsinghpur, Orissa	16	9.3	0.0403 ± 0.1234
A. siamensis, Naihati, West Bengal	28	16.28	0.0569 ± 0.1325
A. siamensis, Nellore, Andhra Pradesh	14	8.14	0.0342 ± 0.1187

Genetic similarity among populations ranged from 0.363 to 0.969, the highest similarity was found between Argulus population collected from CIFA and Siula, whereas the least similarity was found between Mandi and Jagatsingpur population (Table 3). UPGMA dendrogram (Fig. 3) based on Nei's genetic distance for Argulus sp. of 13 populations (Table 3) showed that Bangalore, Karnataka and Mandi, Himachal Pradesh populations formed a distinct cluster, being clearly separated from other populations. All other populations were grouped in the major branch and formed the main cluster. Taking into account that all individuals of rest 11 locations gathered into one clade, it was noticed that all populations were distributed in different subclades with high bootstrap value. However, samples collected based on geographical distance showed their closeness, although having high bootstrap value.

Figure 3. UPGMA dendrogram based on Nei's genetic distance for Argulus sp. collected from 13 locations in India by RAPD analysis.

Table 3. Nei's unbiased genetic identity (above diagonal) and genetic distance (below diagonal) values between populations of Argulus sp

Population	CIFA, Khurda, Orissa	Siula, Puri, Orissa	Balasore, Orissa	Jajpur, Orissa	Kolkata bheri, West Bengal	Tura, Meghalaya	Hessarghata, Karnataka	Bangalore, Karnataka	Mandi, Himachal Pradesh	Vijayawada, Andhra Pradesh	Jagatsinghpur, Orissa	Naihati, West Bengal	Nellore, Andhra Pradesh
CIFA, Khurda, Orissa	****	0.9690	0.9516	0.8048	0.9365	0.9170	0.9337	0.4070	0.4059	0.9141	0.9388	0.9425	0.9170
Siula, Puri, Orissa	0.0315	****	0.9460	0.8052	0.9185	0.9078	0.9140	0.3798	0.3698	0.9048	0.9337	0.9495	0.9170
Balasore, Orissa	0.0497	0.0555	****	0.8041	0.9373	0.8781	0.9084	0.4076	0.3892	0.8850	0.9135	0.9354	0.9324
Jajpur, Orissa	0.2172	0.2166	0.2180	****	0.7959	0.7742	0.8152	0.4545	0.4576	0.8384	0.7904	0.8112	0.7966
Kolkata bheri, West Bengal	0.0656	0.0850	0.0648	0.2283	****	0.8948	0.9096	0.4172	0.3890	0.8717	0.9060	0.9265	0.8995
Tura, Meghalaya	0.0867	0.0967	0.1299	0.2560	0.1111	****	0.9097	0.4252	0.4281	0.8610	0.8996	0.9032	0.8619
Hessarghata, Karnataka	0.0686	0.0900	0.0960	0.2043	0.0948	0.0946	****	0.4035	0.4072	0.8839	0.9162	0.9231	0.8891
Bangalore, Karnataka	0.8989	0.9681	0.8975	0.7885	0.8851	0.8553	0.9076	****	0.9243	0.4357	0.3637	0.3733	0.3744
Mandi, Himachal Pradesh	0.9017	0.9948	0.9437	0.7818	0.9441	0.8483	0.8986	0.0787	****	0.4640	0.3630	0.3666	0.3665
Vijayawada, Andhra Pradesh	0.0898	0.1001	0.1222	0.1762	0.1373	0.1497	0.1234	0.8308	0.7679	****	0.9220	0.9122	0.8827
Jagatsinghpur, Orissa	0.0632	0.0685	0.0905	0.2352	0.0987	0.1058	0.0857	1.0114	1.0134	0.0812	****	0.9624	0.9174
Naihati, West Bengal	0.0592	0.0518	0.0667	0.2092	0.0764	0.1018	0.0801	0.9854	1.0035	0.0919	0.0384	****	0.9287
Nellore, Andhra Pradesh	0.0867	0.0867	0.0699	0.2274	0.1104	0.1487	0.1176	0.9824	1.0038	0.1248	0.0863	0.0740	****

Based on the morphological classifications, a couple of bands observed very common and typical to both the species were gel purified, cloned and sequenced to design further primer sets to amplify correctly the two major species obtained. Based on the results (data not shown), two bands were further narrowed down for subsequent analysis. A fragment of ~ 280 bp obtained using OPH 04 was sliced from the gel, purified, cloned and sequenced. The sequence information is given in Fig. 4. One set of primer designed from this sequence was able to amplify a single specific band for all the populations (11 populations) of the main cluster (Fig. 6a). Similarly another primer was designed from the sequence (Fig. 5) obtained from a fragment generated by OPY 02 that amplified a single specific band for the two populations of other minor cluster only, i.e., Banglore, Karnataka population and Mandi, Himachal Pradesh populations (Fig. 6b).

Figure 4. Nucleotide sequence of the common band for design of specific primer for A. siamensis.

Figure 5. Nucleotide sequence of the common band for design of specific primer for A. japonicus.

Figure 6. PCR reaction showing specific amplification of A. siamensis (a) and A. japonicus (b) DNA by the developed species-specific primers.

Discussion

The present study shows genetic variation within and between two Argulus species collected from 13 different locations of India during a period of 2 years survey. This is the first report regarding the genetic variation of Argulus species collected from freshwater aquaculture systems of India. As this parasite is causing heavy loss to the carp aquaculture industry and about 129 sp. of Argulus repoted worldwide, identification of the prevalent and dominant species from aquaculture systems was highly essential for control and prevention of this disease. Development of species-specific molecular markers would help in easy diagnosis and hence are useful tools in aquatic health management in general and for parasitic disease in particular.

The carp culture forms the main stay of Indian aquaculture and Indian major carps (IMCs) contribute to more than 90% of the total aquaculture production. Argulosis is very common and responsible for major economic loss in carp culture farms in India (Saurabh, Sahoo, Mohanty, Mohanty, Jena, Mukherjee & Sarangi 2010). However, no major breakthrough has been achieved to prevent this parasitic disease. Understanding of the species prevalence or dominance in various culture systems of carps in India could be of great importance for the future development of prophylactic measures to prevent this disease and also for biogeographical inferences. This study seems to be novel and indicated Argulus siamensis (Wilson) as the major and only prevalent species to be associated with IMCs culture to target further investments in developing immunoprophylactics specifically towards this species.

Genetic variation within and among populations is essential for a species to survive in various environmental situations (Ryman, Utter & Laikre 1995). This study further confirmed that RAPD could successfully be used for measurement of variation within and between populations (Caccone, Allegrucci, Fortunato & Sbordoni 1997; Todd et al. 1997; Hassanien 2008).

Among all the populations Jajpur, Orissa population showed maximum intrapopulation genetic diversity, i.e., 0.1727 with 43.02% loci being polymorphic. Although the present data are unable to explain this high degree of variation, it was noticed that there was repeated outbreak of argulosis in that particular location even after repeated use of drugs.

Highest similarity observed between Argulus population collected from CIFA and Siula population may be attributed to close geographical location and also are parasites of common host species. On the other hand, Mandi population and Jagatsingpur population shared least similarity, which are collected from very distant locations and also have different host species. In a study concerning intraspecific genetic variation in Japanese loach (Misgurnus anguillicaudatus), Alam and Khan (2001) suggested that populations showing higher intra-population similarity and lower frequency of polymorphic loci are likely to have less heterozygosity than those showing less intra-population similarity. In other words, populations having higher similarity are more homogeneous groups.

RAPD analysis uncovered varying degree of polymorphism from 13 primers and revealed that nuclear DNA variation in the populations is very high. UPGMA dendrogram based on Nei's genetic distance for Argulus sp. of 13 populations (Fig. 3) grouped Bangalore, Karnataka and Mandi, Himachal Pradesh populations in a separate cluster from rest of the populations. These two populations were collected from goldfish and common carp respectively, whereas all other populations were from IMCs. As per morphological identification Bangalore, Karnataka and Mandi, Himachal Pradesh populations were identified as Argulus japonicus (Thiele) where as other populations were identified as Argulus siamensis (results not shown). Hence, the dendrogram of Argulus sp. is consistent with the morphological classification. This result was consistent with other reports which investigated the Branchiuran genetic variability of the same species from different geographical locations based on mtDNA markers (Wadeh et al. 2010). Otranto, Testini, De Luca, Hu, Shamsi and Gasser (2005) proposed that population genetic structures of parasites were associated with high level of inbreeding, low intra-population genetic variability and large genetic differentiation among population from different geographical regions and/or hosts. The convergence of two individuals of A. japonicus from Bangalore and Mandi farms as close sisters whereas other isolates/populations being in different sub-clades could be explained as a very moderate genetic drift where these two individuals were being associated with common carp species. In general, this was the first constructed phylogenetic tree of A. siamensis individuals collected from major aquaculture regions of India along with A. japonicus isolates obtained from goldfish and common carp varieties. It is difficult to compare with previous work as the earlier studies were based on mtDNA or rRNA sequences (Abele, Kim & Felgenhauer 1989; Mallatt, Garey & Shultz 2004; Møller et al. 2008; von Reumont, Meusemann, Szucsich, Ampio, Gowri-Shankar, Bartel, Simon, Letsch, Stocsits, Luan, Wagele, Pass, Hadrys & Misof 2009; Wadeh et al. 2010) of Argulus species other than A. siamensis and was mostly to classify Crustaceans. This study further confirmed that RAPD-PCR could used as a tool for species differentiation of Argulus, besides population genetic studies.

Our study shows that about 97.6% bands are polymorphic. Higher polymorphism was observed because the populations belonged to different species though all belonged to the genus Argulus.

The traditional method used for identification of Argulus sp. is still based on morphological distinctions. However, these characteristics can only be distinguished in adult, mostly male specimens live/well-preserved by the experienced taxonomists. This method poses serious problems in case of closely related species. Primers generated to amplify the unique sequences directed to polymorphic DNA lead to amplification of DNA fragments that can compare and distinguish at the species or strain level. The primers developed for A. siamensis (main cluster) and A. japonicus (second cluster) did not cross amplify each other and might be species-specific. However, further specificity could be checked taking into consideration of other Argulus sp. In the absence of classical taxonomists these primers will help in identification of these parasites.

However, further analysis using co-dominant molecular markers like mitochondrial and microsatellite markers will further enhance the genetic makeup of the obtained populations.

Acknowledgments

This work is supported by the National Fund for Basic and Strategic Research in Agricultural Sciences (NFBSRA-AP20) of Indian Council of Agricultural Research, New Delhi. The authors thank the Director, CIFA for providing necessary facilities during this study. Thanks are extended to Dr P. Pradhan, NBFGR, Lucknow; Dr K. Anbarasu, Bharathidasan University, Tiruchirappalli; Dr P. P. Chakrabarty and Dr A. Dutta, RRC of CIFA, Rahara, West Bengal; Dr B. S. Giri and Dr P. V. Rangacharylu, RRC of CIFA, Vijayawada; Dr P. Venugopal, CIFE centre, Kakinada; Dr P. Haribabu, College of Fisheries, Nellore, and to all who helped us in collection of the specimens.