Genetic characterization and phylogenetic relationships of Phyllodistomum parasites in Indian subcontinent: insights from freshwater fish and shrimp hosts.

Phyllodistomum is the large digenean group of fish parasites, with 25 species described so far in the Indian subcontinent. Here, we redescribed two adult species of Phyllodistomum (P. srivastava Rai 1964 and P. parorchium Jaiswal 1957) collected from freshwater fish Heteropneustes fossilis Bloch, 1974 and Glossogobius giuris Ham, 1822, respectively, and an unknown Phyllodistomum metacercaria from shrimp (Macrobrachium dayanum Henderson, 1893). These parasites were genetically characterized using 28S and first and second internal transcribed spacers (ITS1 and ITS2) regions of the nuclear ribosomal DNA and CoxI region of the mitochondrial (mt) DNA to establish the link between metacercaria and adult. Morphologically, both the unknown metacercaria in shrimp and adult Phyllodistomum srivastava in fish, resembled in terms of crenulated margin of hind body, arrangement of diagonal testes, bipartite seminal vesicle, and compact paired vitelline masses. The two adult parasite species, P. srivastava from P. parorchium, were different in terms of shape and size of the body, ratio of suckers, the absence of crenulated margin of hind body, a single chambered seminal vesicle, and deeply lobed paired vitelline masses, in the former species. Comparison of the 28S, ITS, and mtCoxI sequence data suggested P. srivastava and Phyllodistomum metacercaria belong to the same species, and supported the distinction between P. srivastava and P. parorchium. Exploring the potential impact of Phyllodistomum infection on host behaviour and health would be prospective areas for future research.

Characterization of Clinostomum (Digenea: Clinostomidae) spp. in India.

Platyhelminths belonging to the family Clinostomidae (Digenea) have a worldwide distribution and are known to infect piscivorous birds through their intermediate hosts, usually fish species. In the present study, clinostome metacercariae were collected from fish hosts, including Channa punctata (Bloch 1793) (n = 25) and Trichogaster fasciata Bloch and Schneider 1801 (n = 25), from a freshwater system in India. The experimental infection of cattle egrets, Ardea (Bubulcus) ibis Linnaeus 1758, with some of the live metacercariae found in the present study was successful. Live adult parasites were obtained from the buccal cavity of the birds. Both metacercaria and adult specimens were subjected to molecular studies to obtain the sequences of 28S, ITS1, and ITS2 (nuclear rDNA) regions. The parasites were found to belong to three species, Clinostomum giganticum Agarwal 1959; C. piscidium Southwell and Prashad 1918; and Euclinostomum heterostomum (Rudolphi 1809). Phylogenetic analyses of the sequences obtained from the adults and metacercariae established a link between the metacercariae in the fish hosts and adults in the avian host, which is essential to elucidate their partial life cycle and specify morphological characteristics in the metacercarial stage.

Morphological and molecular characterization of six Indian Tetracotyle type metacercariae (Digenea: Strigeidae Railliet, 1919), using ribosomal and mitochondrial DNA.

Strigeids have a cup-shaped fore body, containing a holdfast organ with two lobes and hind body. The species diversity of strigeids remains incomplete, especially in the Indian sub-continent. Here, we described six Tetracotyle type metacercariae (T. muscularis, T. fausti, T. lucknowensis, T. xenentodoni, T. mathuraensis and T. glossogobii) from five fresh water fish, collected at Lucknow (India). Next, we characterized these metacercariae using ribosomal (18S, 28S, ITS2) and mitochondrial DNA (COI) to determine their systematic and phylogenetic position. Molecular identification using inter-specific variation range for all four molecular markers revealed 1.9-4.9% (18S); 3.3-8.8% (28S); 6.8-12.9% (ITS2), and 3.5-9.4% (COI) among six Tetracotyle type metacercariae. In phylogenetic analysis, constructed by neighbour-joining (NJ) and maximum likelihood (ML) methods, T. fausti, T. glossogobii, T. xenentodoni, T. lucknowensis and T. mathuraenis nested as sister groups with the member of strigeids for all four markers used; T. muscularis, however, formed a basal clade. Furthermore, phylogenetic placement states the monophyly of the Tetracotyle type of metacercariae in all the markers (18S, 28S, COI), except ITS2.

Morphological and molecular characterisation of three Indian Neascus-type metacercariae (Digenea: Diplostomidae).

Diplostomid digeneans are important parasites whose larval stages infect freshwater snails and fish as first and second intermediate hosts respectively. Diplostomid digeneans as adults are parasitic in many fish-eating birds and mammals. Our understanding of the species diversity of diplostomid digeneans remains incomplete, especially in the Indian sub-continent. Here, we describe three Neascus-type metacercariae (N. hanumanthai, N. gussevi, and N. xenentodoni) from freshwater fish specimens of Channa punctata (Bloch 1793), Trichogaster fasciata (Bloch and Schneider 1801) and Xenentodon cancila (Hamilton, 1822) respectively, collected in India. Next, we characterised these metacecariae using nuclear (28S and ITS1) and mitochondrial DNA (cox1) to determine their systematic and phylogenetic position. Molecular identification using interspecific variation for all three molecular markers revealed a closer relationship between N. hanumanthai and N. gussevi (1.9%-2.4%) than either of N. hanumanthai and N. gussevi to N. xenentodoni (3.1%-3.7% and 4.4%-4.0% respectively). In phylogenetic analyses, estimated by neighbour-joining (NJ) and maximum likelihood (ML) methods, N. gussevi and N. hanumanthai nested as sister groups of Posthodiplostomum Dubois, 1936 for all three markers used; N. xenentodoni, however, nested as a sister taxa of all other diplostomes when using 28S and ITS1 and nested as a sister taxa of Bolbophorus, Alaria and Neodiplostomum using cox1. These findings suggest that N. hanumanthai and N. gussevi are members of Posthodiplostomum, but that N. xenentodoni belongs to a separate and unknown genus. Similarly, by proteomics, we found that the cox1 protein sequences and structures were similar between N. hanumanthai and N. gussevi but distinct for N. xenentodoni.

Threading: A novel insilico indagation method for genetic characterization of some diplostomoid metacercariae (Digenea:Diplostomidae Poirier, 1886).

The protein encoding zone of Mitochondrial DNA region (inherited from single lineage) seems most suitable and effective for taxonomic, systematic, ecological, evolutionary, DNA barcoding, cryptic species and population studies, exploiting nucleotide/amino acid datasets (1D/2D/3D conformational level). Nowadays, expeditious computerized methods are in trend for analyzing genetic material to demonstrate variations at various levels of protein structures. Structural proteomics have implemented here for genetic identification, differentiation and relationship of species from information rich data of mt COI gene of the family Diplostomidae with inclusion of molecular tools. Various aspects have been utilized herein for re-validation and infallible discrimination of Trematode diplostomoid metacercariae (Tetracotyle lucknowensis Pandey, 1971; T. xenentodoni Chakrabarti, 1970; T. fausti Rai and Pande, 1969; T. muscularius Chakrabarti, 1970 and Diplostomulum minutum Pandey, 1968), the infective stage in the life cycle, causing severe damage to fish host, whose adults are found mainly in fish eating birds and mammals.

Management of mandibular sub condylar and condylar fractures using retromandibular approach and assessment of associated surgical complications.

PURPOSE: To evaluate surgical complications associated with open reduction and internal fixation of condylar fractures using retromandibular approach in terms of intraoperative and postoperative complications. METHOD: Ten patients with displaced unilateral/bilateral condylar fractures were selected for the study. Intraoperative complications were evaluated in the form of haemorrhage due to damage to retromandibular vein and damage to marginal mandibular branch of facial nerve. Postoperative complications like presence of infection, signs of Frey's syndrome, parotid fistula formation, facial nerve palsy, and discrepancy in occlusion and functions of temporomandibular joint were evaluated at intervals of 24 h, one week, six weeks and three months postoperative. Radiographically, the approximation of fracture fragments, plate fracture and screw loosening on orthopantomograph and Reverse Towne's view were evaluated at intervals of 24 h, six weeks and three months postoperatively. RESULTS: None of the patients suffered from any major complication intra and post operatively. CONCLUSION: Open reduction and internal fixation should be given due consideration in the management of displaced mandibular condylar fractures and is associated with minimal morbidity using retromandibular approach.