Morphological and Molecular Characterization of Paragonimus Species Isolated from Freshwater Crabs in Southern Yunnan, China

Paragonimus species are highly prevalent in various regions of China. The study’s objective is to isolate and identify Paragonimus from natural habitats and compare the phylogenetic diversity of Paragonimus in southern Yunnan province, China. Metacercariae of Paragonimus was isolated from crabs, and morphologic identification was performed by microscopy. Metacercariae were injected into experimental Paragonimus free Sprague Dawley rats. After 114 days, adult worms and eggs were isolated from multiple organs. Morphologic identification confirmed the initial identification. DNA was extracted from 5 adult worms, and molecular characterization was performed by amplification and sequencing of CO1 and ITS2 regions, followed by phylogenetic analysis. Out of 447 crabs captured, 186 crabs were found to be infected. A total of 4 species of Paragonimus was observed from naturally infected crabs. Paragonimus microrchis (2), Paragonimus heterotremus (1), Paragonimus proliferus (1), and Paragonimus skrjabini (1) were isolated and identified. A total of 32 sequences were downloaded from the National Center for Biotechnology Information, and 5 sequences generated in the study were used for phylogenetic analysis. In the phylogenetic tree of the CO1 gene, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini were clustered with the same species, and the confidence values of their branches were >95%. A congruent phylogenetic relationship was observed with the ITS2 phylogenetic tree. In the phylogenetic tree constructed with the combined dataset of CO1 and ITS2 datasets, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini clustered with the same species, and their branch confidence values were >94%. Paragonimus microrchis clustered with Paragonimus bangkokensis in both datasets. Phylogenetic analysis revealed robustness of the double loci method as against the single-locus method with either CO1 or ITS2 alone. Paragonimus species isolated from the southern Yunnan province, China, was phylogenetically diverse, and the analysis revealed the clustering of multiple species of Paragonimus isolated from different geographic locations.

Surveys of Afrotemperate forests yields two new freshwater crabs (Decapoda: Potamonautidae: Potamonautes MacLeay, 1838) from South Africa

Sampling of remote inland aquatic habitats in South Africa has constantly been yielding novel endemic freshwater crab species (Potamonautes MacLeay,1838). During the present study, we report on the discovery and description of two new freshwater crab species (Potamonautes baziya sp. nov., and P. mariepskoppie sp. nov.) from Afrotemperate forested mountain regions in the Eastern Cape and Mpumalanga provinces of South Africa, respectively. Phylogenetic evidence derived from DNA sequence data of three partial mitochondrial loci (12S rRNA, 16S rRNA and cytochrome oxidase subunit one, COI) corroborates the evolutionary distinction of the two novel species. In addition, morphological and ecological data for the two new species further delineate their evolutionary distinction from congeneric sister species. A comparison of the taxonomically important gonopods 1 and 2 and carapace features among the sister species and other known freshwater crabs of South Africa was further used to provide evidence for the distinction of the two novel species. The discovery of two new species suggest that remote mountainous areas or unsampled regions in South Africa likely harbor several novel species, reiterating a call to document aquatic inland biodiversity in forested and mountainous regions of the country.

Freshwater crabs could act as vehicles of spreading avian influenza virus

Avian influenza virus (AIV) possessed significant risk to various animals and human health. Wild birds, especially waterfowls are considered to be the natural reservoir of AIVs. The ecology of AIV is still far from being fully understood. Freshwater crabs are nonnegligible biotic factor in AIV ecosystem. We analyzed the ability of freshwater crabs accumulate and spread AIV. We found that AIV remain infectious in water only for 36 h but persist in crabs for 48 h. Crabs could accumulate AIV in their gills and gastrointestinal tracts. The AIV titers in crabs were higher than the surrounding contaminated water. Crabs could accumulate AIV from contaminated water, carry the virus and spread to naïve crabs via surrounding water. Our study identified freshwater crab as a novel transmission vehicle in AIV ecosystem.

Redescriptions of three species of freshwater crabs from Angola, southern Africa (Brachyura: Potamoidea: Potamonautidae)

The taxonomy of three species of freshwater crabs from Angola, Potamonautes adeleae Bott, 1968, P. macrobrachii Bott, 1953, and P. kensleyi Cumberlidge & Tavares, 2006, is revised based on examination of the paratypes and new material. Updated diagnoses, illustrations, and distribution maps are provided for these three species, and their conservation status is discussed.

Morphological and Molecular Characterization of Paragonimus skrjabini Complex from Yunnan, China: A Brief Report

Purpose To perform environmental sampling and molecular identification of Paragonimus in endemic regions, which may help in minimizing transmission among humans. Methods Mountain crabs from the genus Potamiscus were collected and the encysted metacercariae were extracted and subjected to morphological identification, followed by animal inoculation in Sprague–Dawley (SD) rats. After 112 days of infection, animals were killed and adult worms were extracted from lungs and muscles. The morphology of adult worms was characterized by microscopy and molecular identification was done by polymerase chain reaction, followed by sequencing of cox1 and ITS2 genes. Phylogenetic analysis was done by maximum parsimony method. Results A total of 447 crabs were captured from the streams of Tongchang Town, Jinping County, Yunnan Province, China. The infection rate was found to be 41% (186 out of 447 crabs). The metacercariae of Paragonimus skrjabini was identified by the characteristics round or spherical encysted form measuring 410 to 460 × 400 to 460 µm. After animal infection in SD rats, adults were presumptively confirmed to be P. skrjabini, which was also confirmed by gene amplification and sequence analysis of cox1 and ITS2 regions. Paragonimus skrjabini clustered with previously reported P. skrjabini from Yunnan and Vietnam. The confidence values of their branches were > 95%. Phylogenetic analysis of the ITS2 region revealed two distinct clusters with distinct geographical grouping. Phylogenetic analysis with the combined data sets reiterated the geographical grouping with P. skrjabini from Yunnan clustering with strains from Vietnam. Conclusion Metacercariae of P. skrjabini was discovered in freshwater crabs in Yunnan province, China, and the strains were phylogenetically related to P. skrjabini from Vietnam.