Autochthonous transmission of the Indomalayan parasite, Transversotrema patialense, in the Caribbean: Molecular, morphological, and experimental evidence.

The Asian freshwater snail Melanoides tuberculata has been established since the 1960s in the Americas, where it transmits cercariae of a small number of digenetic trematode species from its native range. In 2021-2022, 24 M. tuberculata were discovered shedding transversotrematid cercariae in Puerto Rico, where parasites of this snail have not been previously studied. Adult transversotrematids (in some cases, gravid) were found on field-caught fish and on fish exposed to shedding snails, including on fish species native to Puerto Rico. Adults and cercariae were identified as Transversotrema patialense (Soparkar, 1924), a species native to the Indomalayan region. Morphological identification was supported with 28S rDNA sequences closely matching that from unidentified transversotrematid cercariae in Thailand. The absence of T. patialense in snails collected prior to 2021, increasing prevalence of infection in snails collected thereafter, and lack of variation in parasite DNA sequences (28S, internal transcribed spacer 2, cytochrome c oxidase I) from three isolates are consistent with a recently introduced and possibly expanding parasite population. Transversotrema patialense has been recorded outside its native range before, but most studies (including a prior record in the Americas) reported the parasite from captive hosts from commercial sources such as pet shops. The present results thus provide the first demonstration of natural transmission of T. patialense in the Americas. Phylogenetic analysis of 28S but not of ITS2 show the transversotrematid genus Transversotrema Witenberg, 1944 is paraphyletic, with Crusziella Cribb, Bray and Barker 1992 nested within it.

Benchmarking assembly free nanopore read mappers to classify complex millipede gut microbiota via Oxford Nanopore Sequencing Technology.

Millipedes are key players in recycling leaf litter into soil in tropical ecosystems. To elucidate their gut microbiota, we collected millipedes from different municipalities of Puerto Rico. Here we aim to benchmark which method is best for metagenomic skimming of this highly complex millipede microbiome. We sequenced the gut DNA with Oxford Nanopore Technologies' (ONT) MinION sequencer, then analyzed the data using MEGAN-LR, Kraken2 protein mode, Kraken2 nucleotide mode, GraphMap, and Minimap2 to classify these long ONT reads. From our two samples, we obtained a total of 87,110 and 99,749 ONT reads, respectively. Kraken2 nucleotide mode classified the most reads compared to all other methods at the phylum and class taxonomic level, classifying 75% of the reads in the two samples, the other methods failed to assign enough reads to either phylum or class to yield asymptotes in the taxa rarefaction curves indicating that they required more sequencing depth to fully classify this community. The community is hyper diverse with all methods classifying 20-50 phyla in the two samples. There was significant overlap in the reads used and phyla classified between the five methods benchmarked. Our results suggest that Kraken2 nucleotide mode is the most appropriate tool for the application of metagenomic skimming of this highly complex community.