Amazonian (Trichechus inunguis) and West Indian (Trichechus manatus) manatees are aquatic mammals vulnerable to extinction found in the Amazon basin and the coastal western Atlantic. Toll-like receptors (TLR) play a key role in recognizing pathogen-associated molecular patterns using leucine-rich repeats (LRRs). We described the diversity of TLR4 and TLR8 genes in these two species of manatee. Amazonian manatee showed seven SNPs in TLR4 and the eight in TLR8, while West Indian manatee shared four and six of those SNPs, respectively. In our analysis, TLR4 showed one non-conservative amino acid replacement substitution in LRR7 and LRR8, on the other hand, TLR8 was less variable and showed only conserved amino acid substitutions. Selection analysis showed that only one TLR4 site was subjected to positive selection and none in TLR8. TLR4 in manatees did not show any evidence of convergent evolution compared to species of the cetacean lineage. Differences in TLR4 and TLR8 polymorphism may be related to distinct selection by pathogens, population reduction of West Indian manatees, or an expected consequence of population expansion in Amazonian manatees. Future studies combining pathogen association and TLR polymorphism may clarify possible roles of these genes and be used for conservation purposes of manatee species.

This study investigated the RNA virome associated with the phlebotomine sand fly Pintomyia fischeri using a metatranscriptomic approach applied to the Meta29 library, composed of 21 specimens collected at the São Paulo Zoo, Brazil. Read-based taxonomic analysis revealed a diverse viral community composed of viruses associated with the families Iflaviridae, Narnaviridae, Partitiviridae, Reoviridae, Solemoviridae, Tombusviridae, Totiviridae, and Tymoviridae, in addition to highly abundant unclassified RNA viruses related to the ShiM 2016 group. Assembly and annotation analyses enabled the characterization of four viral RNA genomes associated with the families Partitiviridae (PfPartitiV-1a-SP, PfPartitiV-1b-SP, and PfPartitiV-2-SP) and Narnaviridae (PfNarnaV-1-SP). Sequence comparisons revealed low amino acid identity relative to currently available reference viral sequences, supporting the classification of these sequences as highly divergent and potentially novel viral lineages. Structural modeling of the RNA-dependent RNA polymerase (RdRp) proteins identified the viral polymerase-specific catalytic motifs A, B, and C. Phylogenetic analyses further supported the evolutionary divergence of the identified viruses relative to currently described taxa. Overall, these findings expand current knowledge of viral RNA diversity associated with Neotropical sand flies and highlight the value of metatranscriptomic approaches for the detection and characterization of previously undescribed viruses associated with medically important insect vectors.