Larvae of this fluke are novel causes of granulomatous eye disease in children.

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Members of the family Gastrothylacidae (Trematoda: Digenea: Paramphistomata) are parasitic in ruminants throughout Africa and Asia. In north-east India, five species of pouched amphistomes, namely Fischoederius cobboldi, F. elongatus, Gastrothylax crumenifer, Carmyerius spatiosus and Velasquezotrema tripurensis, belonging to this family have been reported so far. In the present study, the molecular phylogeny of these five gastrothylacid species is derived using the second internal transcribed spacer (ITS2) sequence and secondary structure analyses. ITS2 sequence analysis was carried out to see the occurrence of interspecific variations among the species. Phylogenetic analyses were performed for primary sequence data alone as well as the combined sequence-structure information using neighbour-joining and Bayesian approaches. The sequence analysis revealed that there exist considerable interspecific variations among the various gastrothylacid fluke species. In contrast, the inferred secondary structures for the five species using minimum free energy modelling showed structural identities, in conformity with the core four-helix domain structure that has been recently identified as common to almost all eukaryotic taxa. The phylogenetic tree reconstructed using combined sequence-structure data showed a better resolution, as compared to the one using sequence data alone, with the gastrothylacid species forming a monophyletic group that is well separated from members of the other family, Paramphistomidae, of the amphistomid flukes group. The study provides the molecular characterization based on primary sequence data of the rDNA ITS2 region of the gastrothylacid amphistome flukes. Results also demonstrate the phylogenetic utility of the ITS2 sequence-secondary structure data for inferences at higher taxonomic levels.

Understanding the pattern of biodiversity along environmental gradients helps in identifying diversity hotspot areas that can be prioritized for conservation. While the elevational distribution of several taxa has been studied, responses of the subgroups within a taxon to elevation and its associated factors are not properly understood. Here we study species richness and butterfly density along an elevation gradient in Sikkim, Eastern Himalaya, India and explore the underlying causes of the patterns. We sampled butterflies using a fixed-width point count method in 16 elevational bands (150-200 m intervals), between a range of 300 and 3300 m a.s.l. We categorized butterflies into various subgroups based on family, range size, biogeographic affinity, and host-plant specialization. We recorded 3603 individuals and 253 species of butterflies after the completion of 1860 point counts. Overall, species richness in the majority of the subgroups (except for Riodinidae and Palearctic species) declines with elevation, as does the density of almost all the subgroups. From a selection of environmental factors, annual actual evapotranspiration has the strongest effect on the species richness pattern of butterflies as well as on the density of the overall butterfly community, especially the Lycaenidae family. The richness and density of butterfly groups display varied responses to the richness and density of trees and shrubs. The conducive climatic conditions and diverse habitats in the lower valleys of the Eastern Himalaya support a high diversity of butterflies (with majority of small range species) and thus warrants conservation attention. Highlights • The distribution of biodiversity along mountain elevational gradients has been well studied, but the disparity in patterns between various organisms and associated factors makes it difficult to develop a universal model for explaining the variation of biodiversity. • We report elevational patterns of species richness and density of butterflies in the Eastern Himalaya, both at whole group and subgroup levels. • A general decline in species richness and density with elevation is not supported for Riodinidae and for Palearctic species. • A measure of water energy balance, annual actual evapotranspiration, is the best correlated variable with species richness trends in butterflies. • Our paper highlights the importance of low elevation forests for butterfly diversity in the Eastern Himalaya.

Among the digenetic trematodes, paramphistomes are known to be the causative agent of "amphistomiasis" or the stomach fluke disease of domestic and wild animals, mainly ruminants. The use of 28S (divergent domains) and 18S rRNA for phylogenetic inference is significantly warranted for these flukes since it is as yet limited to merely the exploration of the second internal transcribed spacer (ITS2) region. The present study intended to explore the divergent domains (D1-D3) of 28S rRNA and simultaneously equate the phylogenetic information with 18S rRNA in paramphistomes. Divergence of the 28S rRNA domains was evident amongst the divergent (D) domains, where D1 domain emerged as the most variable and D2, the most robust domain, since the latter could provide a higher resolution of the species. D2 was the only domain that comprised compensatory mutations in the helices of its structural constraints; this domain is thus well suited for species distinction and may be considered a potential DNA barcode complementary to mitochondrial DNA. 28S (D1 + D2 + D3) rRNA provided a significant resolution of the taxa corroborating with the taxonomy of these flukes and thus proved to be more robust as a phylogenetic marker for lower levels than 18S rRNA. Phylogenetic inferences of paramphitomes are still scarcely explored; additional data from other taxa belonging to this family may estimate better the biodiversity of these flukes.

Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create an NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest trematode mitochondrial genome sequenced to date. The noncoding control regions are separated into two parts by the tRNA-Gly gene and don’t contain either tandem repeats or secondary structures, which are typical for trematode control regions. The gene content and arrangement are identical to that of F. hepatica. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The mtDNA for the intestinal fluke is reported herein for the first time by our group that would help investigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data. More so, it would serve as a resource for comparative mitochondrial genomics and systematic studies of trematode parasites.

Of paramphistomid trematodes, three species viz., Homalogaster paloniae, Calicophoron calicophorum and Orthocoelium streptocoelium are commonly prevalent in bovine hosts in Northeast India. The aim of the present study was to genetically characterise these species using rDNA second internal transcribed spacer (ITS2) so as to supplement the morphological criteria substantiated by molecular findings. The annotated ITS2 region from H. paloniae, C. calicophorum and O. streptocoelium were found to be 289 bp, 288 bp and 288 bp long, respectively. On comparison, the Indian isolates of the three species were observed to have a maximum identity of 99% with each of their respective counterparts from Japan. The secondary structure models were inferred using minimum free energy modelling algorithms. The paramphistomes displayed the typical four helix ITS2 secondary structure and differed from each other due to minor nucleotide differences. The consensus ITS2 secondary structure model revealed the presence of conservative motifs GACGAGGGUG and GCGGUAGAGUC in helix III. Monophyly is well supported for a clade consisting of the Japanese and Indian paramphistomes with significant bootstrap values.

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