©2017 Institute of Parasitology, SAS, Košice DOI 10.1515/helm-2017-0010 HELMINTHOLOGIA, 54, 1: 68 – 76, 2017 Research Note Procamallanus spiculogubernaculus Agarwal, 1958 (Nematoda: Camallanidae) from Stinging catfi sh, Heteropneustes fossilis in India: morphological characterization and molecular data A. CHAUDHARY1*, C. VERMA1, V. TOMAR2, H. S. SINGH1 1Molecular Taxonomy Laboratory, Department of Zoology, Chaudhary Charan Singh University, Meerut (U.P.), India – 250004, *E-mail: [email protected]; 2Department of Zoology, Dyal Singh College, University of Delhi, Delhi, India – 110003 Article info Summary Received September 7, 2016 The nematode, Procamallanus spiculogubernaculus Agarwal, 1958 was found from the Stinging Accepted November 23, 2016 catfi sh, Heteropneustes fossilis (Bloch, 1794) from Ghazipur, Delhi, India. Morphological character- ization, including scanning electron microscope observation supplemented with DNA sequences is provided. Specimens recently found are characterized by the presence of an unlined buccal capsule having a small basal ring, esophagus muscular and glandular, vulva position is slightly post-equato- rial, tail conical, long, and ending in three digit-like processes, phasmids present at about mid-length and cloaca located at the posterior end. In this study the species P. spiculogubernaculus is validated on the basis of molecular data after 47 years from its original description. In the scanning electron mi- croscope examination, the topology of mouth and sensory pits in anterior portion, while the phasmids and digit like processes in posterior portion is clearly observed. Molecular data of the 18S ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. Molecular phy- logenetic analyses supported the validity of Procamallanus spiculogubernaculus and confi rmed the paraphyletic status of the members of Procamallanus, Spirocamallanus, Camallanus and Paraca- mallanus. Taxonomic status of members of the family Camallanidae are briefl y discussed along with the results of the systematic evaluation of P. spiculogubernaculus based on molecular data. Keywords: Nematode; Procamallanus; Scanning electron microscopy; Molecular analyses; 18S; cox1; India Introduction Procamallanus species is based on characters such as esopha- gus structure, presence of spicules, and gubernaculum in males, The genus Procamallanus Baylis, 1923 (Nematoda: Camallani- while in females it is based on the conical tail with digit-like tail dae) includes species that parasitize freshwater, brackish-water processes, vulva position and shape of the eggs. The rich diversi- and marine fi shes, less often amphibians. More than 20 species ty of the species of Procamallanus from H. fossilis in India made of this genus have been described from Bangladesh, India and Sri the species identifi cation diffi cult, as many descriptions are inade- Lanka (Sood, 1989) from Heteropneustes fossilis (Bloch, 1794). quate for diagnosis. Due to the lack of proper morphological identi- H. fossilis have high economic importance as food fi sh and for fi cation and descriptions, species of this genus remain unclear (De medicinal value (Talwar & Jhingran, 1991; Sherwani & Parwez, & Moravec 1980). Therefore, morphological identifi cation needs 2000; Acharya & Mohanty, 2014). Morphological identifi cation of the use of molecular analyses to supplement the proper identifi ca- * – corresponding author 68 tion and validation of species. Molecular tools are frequently used electron microscopy (SEM) studies, nematodes were fi xed in 4 % in nematode identifi cation and systematics (Gasser, 2001; Nadler formaldehyde, post-fi xed in 1 % osmium tetraoxide, dehydrated et al., 2005; Mattiucci & Nascetti, 2008; Borges et al., 2012; Jones through ascending grades of ethanol series, dried using the critical et al., 2012; Liu et al., 2013; Pekmezci et al., 2014; Di Azevedo et point dryer and sputter-coated with gold. The specimens were ex- al., 2015; Goswami et al., 2015, 2016). amined with a JEOL Neoscope JCM5000 scanning electron micro- In this study, supporting morphological and molecular information scope (Tokyo, Japan) at an accelerating voltage of 10 kV. is provided for P. spiculogubernaculus Agarwal, 1958 from the Samples for molecular analysis were fi xed in 95 % ethanol. freshwater fi sh H. fossilis collected from Ghazipur, Delhi, India. Genomic DNA extraction from female worm was conducted using the Qiagen DNeasyTM tissue kit (Qiagen, Germany) according to Materials and Methods the manufacturer’s instruction. PCR amplifi cation for partial 18S (900bp) and cox1 (574bp) gene regions were performed using A total of 25 specimens of H. fossilis (body length 14 – 19 cm) two different sets of the primers, Nem 18SF (5’-CGCGAATRGCT- were collected during January 2013 to March 2014 from Ghazipur CATTACAACAGC-3’) and Nem 18SR (5’-GGGCGGTATCT- (28.7342° N, 77.2728° E), Delhi, India for examination of parasites, GATCGCC-3’) (Floyd et al. 2005); LCO1490 (5’-GGTCAACAAAT- from which 14 fi sh were found infected. Host specimens were kept CATAAAGATATTGG-3’) and HC02198 (5’-TAAACTTCAGGGT- alive in aerated jars at 24 °C until dissection. Nematodes (7 female GACCAAAAAATCA-3’) (Folmer et al. 1994) respectively. Each specimens) from the small intestine of fi sh were collected in 0.6 % 25 μl PCR amplifi cation reaction contained 3 μl of DNA, 2.5 μl of saline solution and then stored in 70 % ethanol until processing 10× PCR buffer, 0.9 μl of each primer, 3.4 μl of deoxyribonucle- for morphology. For the light microscopy study, nematodes were otide triphosphates and 1.5 U of Taq DNA polymerase (Biotools, cleared and mounted with glycerin. Drawings were made with the Spain). The amplifi cation was carried out in an Eppendorf Mas- aid of a camera lucida. All measurements were taken in mm from tercycler (Eppendorf, Germany) with the following cycling profi le: the camera lucida diagram. Photos were taken with a light micro- 94 °C for 3 min (hot start), followed by 35 cycles of denaturation at scope equipped with differential interference contrast and digital 94 °C for 50 s, annealing for 1 min on 54 °C for 18S and 48 °C for image analysis system (Nikon, Eclipse 50i, Kawasaki, Japan). cox1, followed by 1 min extension at 72 °C and a fi nal extension Specimens were deposited in the Museum d’ Histoire Naturelle, at 72 °C for 5 min. The PCR products were electrophoresed on Geneva, Switzerland (MHNG-INVE-91853) and the Museum, De- 1.5 % agarose in TAE buffer stained with ethidium bromide and partment of Zoology, Chaudhary Charan Singh University, U.P., visualized under UV transilluminator. For sequencing, the PCR India under the voucher number Nem/2015/02. For scanning product was purifi ed using PurelinkTM Quick Gel Extraction and Table 1. Comparative morphometric data of P. spiculogubernaculus female specimens P. spiculogubernaculus P. spiculogubernaculus P. spiculogubernaculus Agarwal, 1958 (De & Moravec, 1980) present study Infecting: Host Heteropneustes fossilis H. fossilis H. fossilis Organ Intestine Stomach Intestine Body: Length 4.84 3.56 – 7.49 (3.44) 4.71 (3.25 – 5.31) Width 0.088 0.090 – 0.189 (0.096) 0.090 – 0.189 (0.096) Buccal capsule: Length 0.08 0.069 – 0.084 (0.063) 0.069 – 0.084 (0.063) Width 0.048 0.048 – 0.069 (0.057) 0.048 – 0.069 (0.057) Length of basal ring: – 0.009 0.009 Muscular oesophagus: Length 0.032 0.264 – 0.333 (0.282) 0.264 – 0.333 (0.282) Width – 0.057 – 0.066 (0.057) 0.057 – 0.066 (0.057) Glandular oesophagus: Length 0.046 0.318 – 0.450 (0.336) 0.318 – 0.450 (0.336) Width – 0.042 – 0.072 (0.048) 0.042 – 0.072 (0.048) Nerve ring distance from anterior end: – 0.129 – 0.156 (0.126) 0.129 – 0.156 (0.126) Vulva distance from posterior end: 2.2 1.58 – 3.48 (1.46) 3.26 (2.98 – 3.57) Tail length: 0.1 0.093 – 0.138 (0.090) 0.06 (0.05 – 0.07) Tail process length: – 0.003 0.002 69 PCR purifi cation Combo Kit (Invitrogen, Germany) following man- es and Hasegawa-Kishino-Yano model (HKY) for mtDNA cox1 ufacturer’s instructions. Purifi ed PCR products were sequenced were selected. The phylogenetic trees were inferred by Maximum (in both directions) using the Big Dye Terminator vr. 3.1 cycle se- Likelihood (ML) using MEGA vr. 6 (Tamura et al., 2013) with boot- quencing kit in ABI 3130 Genetic Analyzer, Applied Biosystems strap analysis based on 1000 replicates. Bayesian Inference (BI) (Foster City, USA) with the same primers cited above. analyses were also carried out in Topali 2.5 (Milne et al., 2009) Obtained sequence fragments of 18S rRNA and mt cox1 gene using MCMC chains searches on two simultaneous runs of four were assembled using MEGA V6.06 (Tamura et al., 2013). Am- chains over 1,000,000 generations with every 100th tree saved. biguous bases were clarifi ed using their corresponding ABI chro- The fi rst 25 % of the sampled trees were discarded as ‘burn in’. matograms. The 18S rDNA and mt cox1 gene sequences were The members belong to the family Physalopteridae, Dracunculi- compared for similarities with other available sequences from dae, Philometridae and Micropleuridae and Dracunculus insignis GenBank using BLAST (www.ncbi.nlm.nih.gov) and the related (EU646538) were used as out-groups for 18S rDNA and cox1 mt sequences based on nucleotide similarities were aligned with the DNA phylogenetic reconstructions respectively. software CLUSTAL W (Thompson et al., 1994). DNA pairwise dis- tances for cox1 and 18S rDNA were calculated in MEGA V6.06 Results and Discussion using the p-distance model. The dataset was tested prior to phy- logenetic analysis for the nucleotide substitution model of best fi t A total of 7 P. spiculogubernaculus specimens were collected from using ModelTest (Posada & Crandall, 1998) and the best-fi tting 14 infected host fi sh, H. fossilis. All specimens were found in the model was chosen using the Akaike Information Criterion (AIC). small intestine, none was recovered from the stomach, liver or The model GTR + G + I (general time reversible) for 18S sequenc- body cavity.