BioInvasions Records (2014) Volume 3, Issue 4: 297–300 Open Access doi: http://dx.doi.org/10.3391/bir.2014.3.4.12 © 2014 The Author(s). Journal compilation © 2014 REABIC Rapid Communication Molecular characterization of two non-native species of Dactylogyrus (Monogenea: Dactylogyridae) recovered from introduced hosts in India Haren Ram Chiary1*, Anshu Chaudhary1, Hridaya Shanker Singh1 and Umesh Chandra Goswami2 1Molecular Taxonomy Laboratory, Department of Zoology, University Road, C. C. S. University, Meerut, Uttar Pradesh - 250004, India 2Department of Zoology, Guwahati University, Guwahati, Assam - 781014, India E-mail: [email protected] (HRC), [email protected] (AC), [email protected] (HSS), [email protected] (UCG) *Corresponding author Received: 25 January 2014 / Accepted: 26 August 2014 / Published online: 2 October 2014 Handling editor: Vadim Panov Abstract Freshwater fish from the Guwahati in River Brahmaputra, Assam, India were screened for infection with Monogenea. The recovered parasites were diagnosed by PCR and sequencing the 18S and 28S rDNA subunit regions. Comparison of the obtained nucleotide sequences with sequences in GenBank identified the specimens as Dactylogyrus extensus Mueller and Van Cleave, 1932 and D. lamellatus Achmerow, 1952, recovered from Cyprinus carpio and Ctenopharyngodon idella (Cypriniformes: Cyprinidae) respectively. The obtained sequences differed by only 2-3 nucleotides from the sequences deposited in GenBank. The presence of D. extensus and D. lamellatus in India can result from the introductions of their respective host carrying their parasites with them. Key words: invasive species, Cyprinus carpio, Ctenopharyngodon idella, India Introduction to India many years back, to enhance the performance of seed production for pond culture A major risk associated with introductions of and for stocking in lakes and reservoirs. C. fish is the transmission of their parasitic fauna to carpio and C. idella have gained entry into native fish host that may be highly susceptible to various reservoirs either through accidental or infection (Kirk 2003; Taraschewski 2006; Kelly et deliberate introduction. Current Indian populations al. 2009). The transmission of non-native parasites of C. carpio originate from two introductions to to new geographical areas can have serious effects India; one in 1939 (“German” strain) and the on populations of native species. A classical second in 1957 (“Bangkok” strain) (Jhingran 1991). example is the invasive monogenean Gyrodactylus Cyprinus carpio was introduced to India for salaris Malmberg, 1957 that has reduced the aquaculture, due to its easy breeding, omnivorous average density of salmon parr in infected rivers feeding habits, warm water adaptability, good in Norway and caused losses to fisheries industries growth and hardy nature. C. idella was introduced and tourism (Bakke et al. 2007; Harris et al. 2011). to India in 1959 by import from Hong Kong and In addition, threats by non-native monogenean Japan. After this unauthorized introduction, this parasites has recently been documented in various fish became popular among the aquaculturists, studies (Christison and Baker 2011; Parker et al. proved to be a boon in aquaculture and acted as 2011; Ondrackova et al. 2011; Ellender and Weyl an instrument for yield optimization from ponds. 2014). The ecological impact of non-native mono- In India, the monogenean fauna of the Northeast genean parasites is particularly poorly studied in region is unexplored. Dactylogyrus Diesing, 1850 many geographical regions including India, and is a genus of monogeneans that are highly specific such studies are urgently needed. to freshwater fish of Cyprinidae and includes The cyprinids Cyprinus carpio Linnaeus, 1758 more than 900 nominal species (Gibson et al. (common carp) and Ctenopharyngodon idella 1996). It includes a group of ectoparasites of fish (Valenciennes, 1844) (grass carp) was introduced and is recognized by a four-lobed head with four 297 H.R. Chiary et al. eyespots. They are mainly morphologically iden- personal; Eppendorf). PCR amplification was tified on the basis of male copulatory organ and performed using the following protocol: after an the haptoral parts like anchors, bars and hooks initial denaturation at 94°C for 3 min, 35 cycles (Gussev 1985). It is a species rich genus and identi- at 94°C for 30 sec (denaturation), 55°C for 18S fied solely on the basis of morphological features and 59°C for 28S for 45 sec (annealing), 72°C for 1 is sometimes problematical for very closely min (elongation) with a final elongation at 72°C for related species. 10 min. PCR products were examined on 1.5% Molecular analyses provide an alternative for agarose–TBE (Tris–borate–EDTA) gels, stained identification of monogenean species. Most with ethidium bromide and visualized under studies have been established using molecular ultraviolet light. Amplification products were markers of 18S and 28S region of ribosomal purified by PurelinkTM Quick Gel Extraction Kit DNA to identify and distinguish monogenean (Invitrogen) following the manufacturer’s species (see e.g. Chisholm et al. 2001; instruction. Gel-purified PCR products were Desdevises 2001; Simkova et al. 2004; Wu et al. sequenced using a Big Dye Terminator version 3.1 2007; Chaudhary and Singh 2012, 2013). For cycle sequencing kit in ABI 3130 Genetic Analyser, Dactylogyrus species several studies have been done Applied Biosystems with the same primers. using molecular analyses (Simkova et al. 2004, A BLAST N search (Zhang et al. 2000) of the 2007; Wu et al. 2007). The present study describes 18S and 28S rDNA sequences was performed the results of a parasitological investigation of C. and the best hits were retrieved. carpio and C. idella in India with focus on their monogenean parasites. Results Methods During our parasitological investigation of Brahmaputra River near Guwahati, Assam, India, The two Dactylogyrus species included in this monogeneans belonging to the genus Dactylogyrus study were taken from the two fish species were found. Microscopic analysis revealed that namely common carp (Cyprinus carpio) and grass C. carpio and C. idella were infected with two carp (Ctenopharyngodon idella). About 20 fish Dactylogyrus species respectively. Based on from each species were caught from the river morphological features of the shape of the anchors, Brahmaputra with a local fish grocer present at connective bar and the shape of the marginal the site Guwahati (26° 11′ 0″ N; 91° 44′ 0″ E), hooks, the specimens most closely resembeled D. Assam, India. Immediately after captured, fish extensus and D. lamellatus respectively. were killed by a sharp blow to the head, The selected rDNA regions were successfully dissected and monogeneans collected from gill amplified and sequenced from of D. extensus and filaments. Morphology and molecular study of D. lamellatus specimens. Sequences are deposited parasites were performed as described by in GenBank with accession numbers JQ926197, Chaudhary and Singh (2013). The slides have JQ926198 (D. extensus) and JQ926199, JQ926200 been deposited in the museum of the Department (D. lamellatus). The 18S sequence sizes were of Zoology (voucher number HS/Monogenea/2012/ 741, 750 base pairs and for 28S they were 730, 01 and HS/Monogenea/2012/02), Chaudhary Charan 737 base pairs respectively. The BLAST N Singh University, Meerut, UP, India. search showed that the obtained 18S sequences For this study, primers were designed based of D. extensus and D. lamellatus had no on the sequence of 18S (forward, 5’-CGGTTG difference in K2P-distance to sequences from CAATTTTTATGTGG-3’ and reverse, 5’- other geographical isolates. The 28S rDNA GAGTGATCCACCACTTGCAG-3’) and 28S sequences, however, showed a difference of (forward, 5’-TCTAGTAACGGCGAGTGAACG- 0.047 (K2P-distance) for D. extensus, while D. 3’ and reverse, 5’-GGTGGAAGGTCTACCTCA lamellatus showed a difference of only 0.002. GC-3’) using the software Primer3 (Rozen and Skaletsky 2000). Each amplification reaction Discussion was performed in a final volume of 25 µl containing 3 µl of lysate, 10x PCR buffer, 1U Taq The results from the present study showed the polymerase (Biotools), 0.4mM deoxyribonucleotide presence of D. extensus and D. lamellatus in triphosphates (dNTP) and 10 pM of each primer India. These parasites might have been introduced to pair in a thermocycler (Eppendorf Mastercycler India, long time ago together with their respective 298 Molecular characterization of two non-native species of Dactylogyrus hosts, but remained unrecorded due to a lack of colonize within this range and could be affecting studies. The Dactylogyrus monogenean parasites, the species-rich ecosystems. This represents a D. extensus Mueller & Van Cleave, 1932 and D. considerable threat to aquatic biodiversity and lamellatus Achmerow, 1952 were recorded from fishery development of country within a relatively the gills of C. carpio and C. idella respectively. short time scale. Therefore, detailed monitoring Identification was first by examination of the of establishment, expansion and impact of non- copulatory organ and the haptoral parts. Because native fauna are urgently needed for proper the morphological studies alone may be not management and possible eradication in the Indian sufficient for identification of Dactylogyrus region. To prevent new parasite introductions in species, additional molecular analyses were this region, there is a need to emphasize monitoring performed to allow an accurate