Molecular and Morphological Study of a New Species Pallisentis Anandai N

Trends1540 in Biosciences 10(7), Print : ISSN 0974-8431,Trends 1540-1543, in Biosciences 2017 10 (7), 2017

Molecular and Morphological Study of A New Species Pallisentis anandai n. sp. (Quadrigyridae, Van Cleve, 1920) of Channa punctatus NEELAM KUMARI GAUTAM, MANOJ KUMAR UPADHYAY#, RAMAKANT MAURYA, SANJEEV KUMAR VERMA AND A. M. SAXENA* Department of Zoology, University of Lucknow, Lucknow, Uttar Pradesh #Biotech Park, Sector G, Jankipuram, Kursi Road,, Lucknow, Uttar Pradesh *email: [email protected], [email protected],

ABSTRACT Polyacanthocephala (Bullock, 1969; Amin, 1985; Garcý´a- Pallisentis (Quadrigyridae, Van Cleve, 1920) are Varela et al., 2002; Garcý´a-Varela and Nadler, 2005; endoparasites of vertebrates and arthropods. In the present Taraschewski, 2005), in which majority of 62.7% of the species mainly infecting aquatic hosts (Kennedy, 2006). study a new species Pallisentis anandai n. sp. was recorded first time by morphological and molecular phylogenetic Acanthocephala belong to the Palaeacanthocephala (Kennedy, 2006) with the two orders Echinorhynchidaand, method from the intestine of Channa punctatus. The species were found morphologically distinct; proboscis Polymorphida with around 57% species richness and also armed with 4 circles of 10 hooks each. Lemniscus of left show the highest species diversity and are the most common side and right side was almost equal in size and shape. acanthocephalans of marine teleost fish. Collar spines were 15 transvers circles each with 20 Past systematic efforts have focused on creating spines. Neck was small. There were 29 circles of spines classifications with little consideration of phylogenetic in trunk, each with 12 spines. Cement gland was single, methods. In this study Acanthocephalans were collected syncytial and cylindrical mass with 24 nuclei. Saefftigen’s from fresh water fishes, identified morphological as well as pouch elongated sac like. Morphological distinction also molecular method using 18s rRNA. supported by genetical evidences. Obtained sequences of MATERIALS AND METHODS 18s rRNA region were analyzed by using Maximum Likelihood (ML), Neighbour-joining (NJ) and UPGMA SPECIMEN COLLECTION method. The tree length was found 215. The consistency Specimens of Pallisentis sp. were collected from the index (0.698113), the retention index (0.529412), and the intestine of C. punctatus, from the local market and fresh composite index was 0.490014 (0.369589) for all sites water river of Uttar Pradesh, India. Hosts were dissected and parsimony-informative sites. and examined. Parasites flattened and fixed in AFA fixative, stained with acetoalum carmine, dehydrated in ascending Keyword Acanthocephala, Endoparasite, fish, grades of ethanol, cleared in xylene and mounted in DPX. Pallisentis. Figures were drawn with Camera Lucida, attached to Phase Contrast Microscope (Olympus CX-41).The parasites were The phylum Acanthocephala consists of identified using conventional morphological criteria endoparasites of arthropods and vertebrates (Nickol and (Bhattacharya, 2007). Crompton 1985), commonly mentioned as a thorny-headed MOLECULAR ANALYSIS worms, among the most basal tripoblasts (Hyman, 1951; Brusca and Brusca, 1990; Winnepenninckx et al., 1995; Genomic DNA isolated with Qiagen’s DNeasy Tissue Wallace et al., 1996). Kit. The 18S rRNA genes were amplified from the genomic DNA samples by the polymerase chain reaction (PCR): Earlier taxonomic groups of Acanthocephala based forward primer 58-AGATTAAGCCATGCATGCGTAAG-38, on traditional classification such as morphological features reverse primer 58- TGATCCTTCTGCAGGTTCACCTAC-38. and host characteristics (Bullock, 1969). The most For each reaction mixture 100 ng of genomic DNA was used. classification of Acanthocephala is mostly based on the Thermal cycling used an initial denaturation period of 5 location of the lacunar canals, the persistence of ligament min at 94°C, annealing 1 min at 94°C, 56°C for 1min and sacs in females, the number and type of cement glands in then 72 °C at 1 min followed by 30 cycle, and final extension males, the number and size of proboscis hooks, and the at 72°C for 10 min. The PCR products were evaluated in host taxonomy and ecology (Bullock 1969; Amin 1985). On 1.5% agarose gels. The sequencing was carried out by the the basis of morphological evidence an earlier study method of chain termination by dideoxynucleotides (Sanger hypothesized that Acanthocephala is the sister taxon of et al., 1977). the rotifer (Lorenzen, 1985). Nielsen (1995) and Wallace et al. (1996) have both proposed a sister relationship between PHYLOGENETIC ANALYSIS Rotifera and Acanthocephala, leaving each phylum In addition to the 18S ribosomal gene sequence of monophyletic. species of Pallisentis, other available sequences of The phylum Acanthocephala is divided into three Acanthocephalan were obtained from GenBank. The classes Archiacanthocephala, Palaeacanthocephala and sequences were initially aligned using the Clustal X Eoacanthocephala (Amin 1987). Recent classifications algorithm. Phylogenetic relationships were determined by differentiate the four classes Archiacanthocephala, maximum-likelihood analyses (ML), Neighbour-joining (NJ), Eoacanthocephala, Palaeacanthocephala and Maximum parsimony (MP), and UPGMA trees were also GAUTAM et al., Molecular and Morphological Study of A New Species Pallisentis anandai n. sp. 1541

Fig. 1. Pallisentis anandai n. sp. (A) entire view of Male; (B) enlarged view of proboscis. inferred using MEGA 6.06.Generated sequence was size and shape, 1.94 mm in length and 0.11 mm in width. deposited to the Gen Bank database. Collar spines were 15 transvers circles each with 20 spines. RESULTS AND DISCUSSION Space of collar recorded 0.12 mm long and 0.038 mm wide. Neck was small, 0.26 mm long and 0.20 mm wide. There were MORPHOLOGICAL CHARACTERIZATION 29 circles of spines in trunk, each with 12 spines. Cement The specimen of Pallisentis from the intestine of C. gland was single, syncytial and cylindrical mass with 24 punctaus was collected from Tadikhana, Lucknow, India nuclei, size was 0.49 mm long and 0.22 mm wide. (26°842 653 N, 80°942 673 E). The isolated specimen was Cement gland reservoir recorded 0.32 mm in length and 0.18 found new species. Morphological features of Pallisentis mm in width. Anterior testis was 0.38 mm in length and 0.20 anandai n. sp. (Fig. 01): Body was 6.68 mm long and 0.53 mm in width, posterior was 0.41mm in length and 0.20 mm in mm wide. Proboscis armed with 4 circles of 10 hooks each, width. Seminal vesicles were 0.48 mm in length and 0.15 mm size of proboscis was 0.26 mm long and0.23 mm wide, in width. Saefftigen’s pouch elongated sac, 0.20 X 0 proboscis receptacle was 0.64 mm long and 0.21 mm wide. .01mm. Lemniscus of left side and right side was almost equal in 1542 Trends in Biosciences 10 (7), 2017

Fig. 2. Tree topology of Pallisentis anandai n. sp. using 18S rRNA regions, following Neighbour-joining (NJ) method. Bootstrap values calculated 1000 replicates.

Fig. 3. Tree topology of Pallisentis anandai n. sp. using 18S rRNA regions, following Maximum likelihood (ML) method. Bootstrap values calculated 1000 replicates.

Molecular characterization were found that was some resemblance with P. nagpurensis (Bhalerao, 1931) consisting of 20 – 30 nuclei. Anterior testis Molecular analysis of P. anandai n. sp. (Accession Number KR149270) was performed, approximately 865 bp was 0.60-0.65 X 0.10-0.12 and 0.729-0.748 X 0.095-0.205, of small subunit sequences of 18sr RNA was successfully posterior testis 0.62-0.68 X 10.00-15.00 and 0.654-0.841 X amplified. Amplified 18S genes were searched at NCBI 0.093- 0.392 in P. mehrai and P. garuai respectively but nucleotide BLAST for similarity index. Pallisentis anandai anterior testis is 0.38 X 0.20 mm, posterior 0.41 X 0.20 mm n. sp. showed 90% identity with Neoechinorhynchus sp. was found in P. anandai n. sp. Cement gland reservoir was GL-2015 (KU363972).The nucleotide variability of 18S rRNA not mentioned in P. mehrai and P. garuai. was Adenine 237, Thymine 230, Guanine 230 and Cytosine Two well supported clade were forms, P. anandai n. 178. Total GC content was found 47.2%. sp. forms sister clade with Acanthocephaloides propinquus, Acanthocephalus dirus, Echinorhynchus PHYLONGENETIC ANALYSIS truttae and Acanthocephalus lucii with bootstrap value The cladograms of 18S rDNA regions have similar supports 47/100 that is poorly significance. topology according to NJ (Fig. 02) and ML methods (Fig. ACKNOWLEDGMENTS 03). The evolutionary history was inferred using the Maximum Parsimony method. Tree 1 out of 2 most We thank the University Grant Commission New Delhi parsimonious trees (length = 215) is shown. The for the financial assistance through a fellowship Rajeev consistency index was (0.698113), the retention index was Gandhi National Fellowship (F1-17.1/2013-14/RGNF-2013- (0.529412), and the composite index was 0.490014 (0.369589) 14-SC-UTT-43684 /(SAIII/Website). for all sites and parsimony-informative sites (in LITERATURE CITED parentheses). The percentage of replicate trees in which Agarwal, S. C. 1958. A new species of the genus Pallisentis the associated taxa clustered together in the bootstrap test (Acanthocephala). Current Science 27:107. (1000 replicates) was shown next to the branches. Amin, O. M.1985.Classification. In: Crompton DWT, Nickol BB, Pallisentis anandai n. sp. were found closely related eds. Biology of the Acanthocephala, Cambridge University Press, to genus Pallisentis in having proboscis armed with 4 circles London, 27–72. of hooks each with 10 hooks (Bhalerao, 1931; Sarkar, 1956; Amin, O. M. 1987. Key to the families and subfamilies of Agarwal, 1958; Tadros, 1966; Sahay et al., 1967; Rai, 1967; Acanthocephala with the erection of a new class Gupta and Fatma, 1985; Gupta and Sinha, 1991) that’s why (Polyacanthocephala) and a new order (Polyacanthorhynchida). we placed him in genus Pallisentis. Pallisentis anandai n. Jounal of Parasitolongy, 73:1216–1219. sp. differs from all known species of genus Pallisentis Bhalerao, G. D. 1931. On a new species of acanthocephala from except P. nagpurensis (Bhalerao, 1931); P. garuai (Sahay et Ophiocephalus striatus. The Annals and magazine of natural al., 1971) and P. mehrai (Gupta and Fatma, 1985) showed history, 24:861-868. some resemblance with P. mehrai (Gupta and Fatma, 1985) Bullock, W. L. 1969. Morpholongical features as tool and pitfall in trunk spines consist of 20 – 30 circles and P. garuai (Sahay acanthocephalan systematics. In: Schmidt GD (eds) Problems in et al., 1971) with 28 – 32 circles and extended up to posterior systematics of parasites. University Park Press, Baltimore, 9– but in P. anandai n. sp. not extended up to posterior end, 45. only 29 circles of trunk spines were found with 12 spines in Brusca, R. C. and Brusca, G. J. 1990. Invertebrates. Sinauer, Sunderland, each circle. There was 10 – 15 nuclei mentioned in P. mehrai 922. (Gupta and Fatma, 1985), but in P. anandai n. sp. 24 nuclei Bhattacharya, S.B. 2007. Handbook on Indian Acanthocephala. GAUTAM et al., Molecular and Morphological Study of A New Species Pallisentis anandai n. sp. 1543

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Received on 08-02-2017 Accepted on 15-02-2017