Phylogenetic relationships of two species of Haematoloechus (Trematoda: Haematoloechidae) infecting Euphlyctis spp. from the biodiversity hotspot, Western Ghats, India.

During a parasitological survey in the biodiversity hotspot, two species of the digenetic trematode, Haematoloechus from the lungs of Indian skipper frog Euphlyctis cyanophlyctis and the Indian green frog, E. hexadactylus inhabiting the biodiversity hotspot, Western Ghats, India, were found and described using morphological and molecular tools. Morphological examinations indicated that the two species are Haematoloechus almorai Pande, 1937 and H. (H.) variegatus capyristes (Klein, 1905; Ingles, 1932) Odening, 1958 as previously described in the original manuscripts but with some morphological differences. The ITS region (ITS1, 5.8S and ITS2) and 28S gene of ribosomal DNA sequences were performed for phylogenetic studies showed that the sequences of these species did not match with any of the previously reported sequences on GenBank database. Phylogenetic analysis placed H. almorai and H. (H.) variegatus capyristes in a well-supported clade under the genus Haematoloechus. This is the first molecular study of present Haematoloechus species from the Western Ghats, India and discussed their phylogenetic position.

Phylogenetic position of Tremiorchis ranarum Mehra and Negi, 1926 (Trematoda: Plagiorchiidae) with remark on this monotypic genus.

Tremiorchis is a monotypic genus of digenetic trematode (Plagiorchiidae: Plagiorchiinae), infecting the frogs Rana tigrina (Hoplobatrachus tigerinus) and R. cyanophlyctis (Euphlyctis cyanophlyctis). Metacercaria use to infect Rana tigrina (Hoplobatrachus tigerinus) and R. cyanophlyctis (Euphlyctis cyanophlyctis) as intermediate hosts, while the cercaria stage found from apple snail, Pila virens. Adults of T. ranarum harbor mature frogs of H. tigerinus and E. cyanophlyctis. Besides the frequent infection of Tremiorchis, no DNA sequence data are currently available for this monotypic genus. The present communication, deals with the sequence data for nuclear ribosomal genes, 18S, small internal transcribed spacers (ITS1-5.8S-ITS2) and 28S to molecularly characterize T. ranarum. Besides this, phylogenetic relationship among the members of the Plagiorchiida is also discussed in detail. An attempt has also been made to provide detailed molecular affinities of T. ranarum with other trematode genera.

First Report of Molecular Characterization of Oochoristica hemidactyli Johri, 1955 (Cestoda, Linstowiidae) from the Common Wall Lizard, Hemidactylus brooki Gray, 1845 (Reptilia: Gekkonidae) Along with Morphological Redescription.

INTRODUCTION: During an investigation, a rare cestode species belonging to the genus, Oochoristica were found in Meerut, UP, India, from the intestine of the host, Hemidactylus brookii Gray, 1845 (Reptilia: Gekkonidae). The tapeworms were found to be similar in their morphological characters with Oochoristica hemidactyli Johri, 1955 (Cestoda: Linstowiidae). MATERIALS AND METHODS: The host Hemidactylus brookii used in this analysis was caught from May to July 2018 from Department of Zoology, Chaudhary Charan Singh University, Meerut (UP), India. Parasites were examined by microscopy. 18S and ITS1-5.8S-ITS2 region of ribosomal RNA were sequenced and analyzed for the molecular study. RESULTS: This paper provides the first trustworthy confirmation about the occurrence of O. hemidactyli inferred with molecular data using ribosomal 18S and ITS1-5.8S-ITS2 gene cluster DNA sequences. Molecular analysis shows that the studied specimens were clustered separately and did not show a close relationship with any other cestode spp., represented in the GenBank. CONCLUSION: This is the foremost molecular characterization of any species of Oochoristica and of O. hemidactyli after 64 years of its original description that contributing to the systematic analysis of the helminthological biodiversity of reptilian hosts. These results provide a reference for future molecular study of species of Oochoristica.

First report on the molecular characterization of Diaphorocleidus armillatus Jogunoori et al. 2004 (Monogenea: Dactylogyridae) infecting the gills of introduced fish, Gymnocorymbus ternetzi in India.

During an investigation of the diversity of monogenean parasites in Meerut, U.P., India, the exotic freshwater fish Gymnocorymbus ternetzi was found infected with the gill dactylogyrid species of Diaphorocleidus Jogunoori et al. 2004. Using morphological study, the present monogeneans were found similar in morphology of anchors, bars and male copulatory organ with D. armillatus Jogunoori et al. 2004. This is the first genetic characterization of D. armillatus based on the analysis of 18S ribosomal RNA sequence (1800 bp) and associated with morphological redescription. Our study suggests that more molecular investigation is required on Diaphorocleidus species for deducing correct phylogenetic position of D. armillatus.

Molecular Characterization of Nippostrongylus brasiliensis (Nematoda: Heligmosomatidae) from Mus musculus in India.

Mus musculus (Rodentia: Muridae) has generally been infected with a rodent hookworm Nippostrongylus brasiliensis. In this report, we present morphological and molecular identification of N. brasiliensis by light and scanning electron microscopy and PCR amplification of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the protein sequences encoded by cox1 gene, respectively. Despite the use of N. brasiliensis in many biochemistry studies from India, their taxonomic identification was not fully understood, especially at the species level, and no molecular data is available in GenBank from India. Sequence analysis of cox1 gene in this study revealed that the present specimen showed close identity with the same species available in GenBank, confirming that the species is N. brasiliensis. This study represents the first record of molecular identification of N. brasiliensis from India and the protein structure to better understand the comparative phylogenetic characteristics.

Molecular Phylogeny of Nematodes (Oxyurida: Travassosinematidae) from Orthoptera (Gryllotalpidae) Inferred by Mitochondrial Cytochrome C Oxidase Subunit 1 Gene.

In this study, we sequenced mt Cox 1 gene sequences of five nematode spp. that were infective to arthropod, Gryllotalpa africana. The nematode belongs to Thelastomatoidea, a group of pinworms that parasitizes only invertebrates. Currently, in India spp. of this group are distinguished mainly on the basis of morphological characters that present possible confusions. Therefore, we identified the species through morphological and genetic analysis. We selected mt Cox 1 gene region to show their phylogenetic position with closely related spp. and confirmed their molecular validation. The present findings are important to confirm the phylogenetic position and relationship among five nematode spp. and avoid misidentification regarding their validation, as it is more necessary in that case when many species harbours the same host.

First Molecular Characterization of Aspiculuris tetraptera (Nematoda: Heteroxynematidae) from Mus musculus (Rodentia: Muridae) in India.

Mus musculus (Rodentia: Muridae) were infected by a nematode belonging to the order Oxyurida. According to external morphological characters it was found to be Aspiculuris tetraptera Schulz, 1924. Present communication deals with molecular characterization of A. tetraptera infecting intestine of Mus musculus in Meerut (U.P.), India. A partial sequence of 18S ribosomal and cytochrome c oxidase subunit 1 (Cox 1) mitochondrial DNA analyses were performed. Sequence corresponding to 18S rRNA and mt Cox 1 gene was identical to sequences reported for A. tetraptera on GenBank. These results confirm the taxonomic validation of A. tetraptera parasitizing Mus musculus. This is the first report of molecular study of A. tetraptera in India.

Functional dynamics observations of haptoral armature in Mizelleus indicus (Jain 1957) Pandey et al. 2003.

The negative effect of monogenea on piscine host may vary in the host species. Genus Mizelleus (Monogenea: Ancyrocephalidae) was reported for the first time from the freshwater shark, Wallago attu (Bl. and Schn.) at Lucknow by Jain (1957). Haptor or opisthohaptor of monogenea which is more or less discoid muscular structure situated posteriorly, is the chief adhesive organ. It is usually provided with anchors and marginal hooklets. Anchors, ventral transverse bar, together with the additional supporting dorsal bars (patches) are active elements of haptor while, marginal hooklets serve as secondary supportive participants, whereas, the dorsal transverse bar can be treated as an inactive element in the whole organ of attachment. Functional dynamics detail of haptoral armature is almost unattended. The anchors in case of M. indicus (Jain 1957) Pandey et al. (2003) are disposed one against the other and used to act like four lobed grapnel like a spreader. All components of haptoral armature contribute in one way or another in the process of functional process. Interestingly the worms do not follow the same pattern of sequences during their movements. Various elements and factors are involved in the process. The authors tried to pinpoint the changes in the activity of one major element i.e., dorsal anchor. The movement and change in position/orientation of various elements with reference to dorsal anchors were observed and summarized. Present paper is rare effort in unexplored aspect in monogenea biology.

Molecular characterization of Gastrothylax crumenifer (Platyhelminthes: Gastrothylacidae) from goats in the western part of India by LSU of nuclear ribosomal DNA.

The rumen parasite, Gastrothylax crumenifer (Platyhelminthes: Gastrothylacidae), is a highly pathogenic trematode parasite of goat (Capra hircus). It sucks blood that causes acute disease like anemia, and severe economic losses occur due to morbidity and mortality of the ruminant infected by these worms. The study of these rumen paramphistomes, their infection, and public health importance remains unclear in India especially in the western part of state Uttar Pradesh (U.P.), Meerut, India, where the goat meat consumption is very high. This paper provides the molecular characterization of G. crumenifer recovered from the rumen of Capra hircus from Meerut, U.P., India by the partial sequence of 28S rDNA. Nucleotide sequence similarity searching on BLAST of 28S rDNA from parasites showed the highest identity with those of G. crumenifer from the same host Capra hircus. This is the first report of molecular identification of G. crumenifer from this part of India.

Morphological redescription and molecular characterization of three species of Travassosinematidae (Nematoda: Oxyurida: Thelastomatoidea) from Gryllotalpa africana Beauv (Orthoptera: Gryllotalpidae).

Binema mirzaia (Basir, 1942a) Basir, 1956, Cameronia nisari (Parveen and Jairajpuri, 1985) Adamson and Van Waerebeke, 1992a and Mirzaiella meerutensis Singh and Malti, 2003 are redescribed morphologically along with molecular identification from the intestine of mole cricket Gryllotalpa africana. Molecular characterization was carried out using the D2-D3 expansion domains of the 18S ribosomal DNA region. This study first time presents molecular data for the above three nematode species.

Integrated circoviral rep-like sequences in the genome of cyprinid fish.

Recently a new group of circoviruses have been detected in tissues of Barbel fish and European catfish in Hungary. In our study circovirus genomes were screened in eight additional fish species for the detection and characterization of circoviruses. Two species of these bore circoviral sequences based on conventional PCR assay targeting the replication-associated protein coding gene fragments. Interestingly, the methods successfully used before failed to amplify other parts of the circular viral genome, suggesting the presence of partial, integrated genetic elements in the genome of the host. The successfully sequenced fragments of the Indian rohu (Labeo rohita) encoded mutations which may cause frameshifts or termination in the coding region described previously in other vertebrates. Phylogenetic analyses presumed that integration of the viral genetic elements might have progressed concurrently or following the diversification of cyprinid fish. Further studies on the nature of whole circovirus genomes and integrated elements may help to understand their potential role and evolution in different fish species.

Phylogenetic analysis of the Dactylogyroides longicirrus (Monogenea: Dactylogyridae) based on the 18S and ITS 1 ribosomal genes.

The present study describes the molecular phylogenetic analysis of Dactylogyroides longicirrus (Monogenea: Dactylogyridae) infecting the gill filaments of fish Puntius sophore from the site Guwahati, Assam, India. The parasite Dactylogyroides longicirrus (Tripathi, 1959) Gusev, 1976 from Northeast Indian region is presented based on sequence data of a 738 base-pair fragment of ribosomal 18S small subunit and first internal transcribed spacer (ITS 1). Phylogenetic relationships were inferred using neighbour joning and maximum parsimony methods and the results support the validation of D. longicirrus. The study is also supported by secondary structure model prediction by using minimum free energy which can be considered a promising tool for monogenean species identification. This is the first report of this parasite from Northeast region of India, with this, the 18S and ITS 1 rDNA region amplified in the study is also the first sequence of the genus Dactylogyroides.

Secondary structure and phylogenetic utility of the ribosomal large subunit (28S) in monogeneans of the genus Thaparocleidus and Bifurcohaptor (Monogenea: Dactylogyridae).

Present communication deals with secondary structure of 28S rDNA of two already known species of monogeneans viz., Bifurcohaptor indicus and Thaparocleidus parvulus parasitizing gill filaments of a freshwater fish, Mystus vittatus for phylogenetic inference. Secondary structure data are best used as accessory taxonomic characters as their phylogenetic resolving power and confidence in validity. Secondary structure of the 28S rDNA transcript could provide information for identifying homologous nucleotide characters, useful for cladistic inference of relationships. Such structure data could be used as taxonomic character. The study supports that species-level sequence variability renders 28S sequence as a unique window for examining the behavior of fast evolving, non-coding DNA sequences. Apart from this it also confirms that molecular similarity present in various species could be host-induced.

PCR-based molecular characterization, phylogenetic analysis and secondary structure of the 28S rDNA of Thaparocleidus wallagonius (Monogenea: Dactylogyridae) - the most primitive species of this genus from India.

Species of the monogenean genus Thaparocleidus are specific to freshwater siluriform fish. The infection caused by these gill parasites are a major health problem to fish. But, to focus the control strategies of these parasites, first it is important to establish an accurate discrimination by molecular methods. In the present study, phylogenetic and structural analysis of 28S region of ribosomal DNA of T. wallagonius species collected from fish Wallago attu from Meerut (U.P.), India, was carried out. In the first step, we amplified, sequenced 28S region of ribosomal DNA of T. wallagonius to establish the phylogenetic relationship with other species of this genus. T. wallagonius found on gill filaments of fish W. attu, is the most primitive parasite of this genus from India, was unequivocally discriminate from other species of the same genus in this study. A secondary-structure model of the large subunit rDNA was also predicted using a combined comparative and thermodynamic approach. Molecular morphometric and phylogenetic relationship of T. wallagonius are discussed in detailed that based on molecular analysis using bioinformatic tools.

Phylogenetic study of nine species of freshwater monogeneans using secondary structure and motif prediction from India.

The present study was performed to identify and validate monogenean species from different piscine hosts using molecular tools. Nine species of freshwater monogeneans were collected from gills and skin of freshwater fishes at Hastinapur, Meerut, India. After microscopic examination, molecular analysis was performed utilizing 28S gene marker. Phylogenetic analysis indicated the validation and systematic position of these nine different monogeneans belongs to the Dactylogyridae and Gyrodactylidae families. The findings also confirm that the 28S rDNA sequence is highly conserved and may prove to be useful in taxonomic studies of parasitic platyhelminthes. Besides this, the study is also supplemented by molecular morphometrics that is based on 28S secondary structure homologies of nine monogenean species. The data indicate that 28S motifs i.e., ≤ 50bp in size can also be considered a promising tool for monogenean species identification and their validation.