A protocol and training guidelines for mosquito sampling in remote areas with limited power supply.

Mosquito-borne diseases pose a significant threat in many Southeast Asian countries, particularly through the sylvatic cycle, which has a wildlife reservoir in forests and rural areas. Studying the composition and diversity of vectors and pathogen transmission is especially challenging in forests and rural areas due to their remoteness, limited accessibility, lack of power, and underdeveloped infrastructure. This study is based on the WHO mosquito sampling protocol, modifies technical details to support mosquito collection in difficult-to-access and resource-limited areas. Specifically, we describe the procedure for using rechargeable lithium batteries and solar panels to power the mosquito traps, demonstrate a workflow for processing and storing the mosquitoes in a -20 °C freezer, data management tools including microclimate data, and quality assurance processes to ensure the validity and reliability of the results. A pre- and post-test was utilized to measure participant knowledge levels. Additional research is needed to validate this protocol for monitoring vector-borne diseases in hard-to-reach areas within other countries and settings.

Occurrence of five distinct clades of mermithid nematodes (Nematoda: Mermithidae) infecting black fly larvae (Diptera: Simuliidae) in tropical streams in Malaysia.

Mermithids are the most common parasites of black flies and are associated with host feminization and sterilization in infected hosts. However, information on the species / lineage of black fly mermithids in Southeast Asia, including Malaysia requires further elucidation. In this study, mermithids were obtained from black fly larvae collected from 138 freshwater stream sites across East and West Malaysia. A molecular approach based on nuclear-encoded 18S ribosomal RNA (18S rRNA) gene was used to identify the species identity / lineage of 77 nematodes successfully extracted and sequenced from the specimens collected. Maximum likelihood and neighbor-joining phylogenetic analyses demonstrated five distinct mermithid lineages. Four species delimitation analyses: automated simultaneous analysis phylogenetics (ASAP), maximum likelihood Poisson tree processes with Bayesian inferences (bPTP_ML), generalized mixed yule coalescent (GMYC) and single rate Poisson tree processes (PTP) were applied to delimit the species boundaries of mermithid lineages in this data set along with genetic distance analysis. Data analysis supports five distinct lineages or operational taxonomic units for mermithids in the present study, with two requiring further investigation as they may represent intraspecific variation or closely related taxa. One mermithid lineage was similar to that previously observed in Simulium nigrogilvum from Thailand. Co-infection with two mermithids of different lineages was observed in one larva of Simulium trangense. This study represents an important first step towards exploring other aspects of host - parasite interactions in black fly mermithids.

Natural infections and distributions of parasitic mermithids (Nematoda: Mermithidae) infecting larval black flies (Diptera: Simuliidae) in tropical streams of Malaysia.

Mermithids are parasites of black flies that cause host mortality along with physical and behavioural changes in infected hosts. However, there is a lack of knowledge on the distribution of mermithids infecting black fly larvae and the factors that influence these distributions in Asia, including Malaysia. A total of 13,116 mid- to late-instar black fly larvae belonging to 42 species were collected from 138 streams across East and West Malaysia and screened for the presence of mermithid parasites. Overall, 121 mermithids were obtained from 107 (0.82%) larvae of nine (21.4%) black fly species. The average number of mermithids per black fly host was 1.10 ± 0.04 (SE), ranging from one to three mermithids per host. Mermithid infection was highest in Simulium trangense, with a frequency of occurrence of 6.5%, followed by S. cheongi (5.8%) and S. angulistylum complex (2.9%). Infection was lowest in S. brevipar and S. tahanense, with a frequency of occurrence of 0.7% each. Regression analysis indicated that mermithid infections in larval black flies were significantly associated with cooler and shallower streams with more canopy cover, dense riparian vegetation, high dissolved oxygen, and lower conductivity and complete pH. Forward logistic regression further indicated that infections in S. cheongi were associated with shaded, cooler, slightly acidic streams with higher conductivity and dissolved oxygen. These findings suggest that mermithid infections in larval black flies in Malaysia are not randomly distributed and are influenced by the breeding habitat of their hosts.

New Species of Termitomyces (Lyophyllaceae, Basidiomycota) from Sabah (Northern Borneo), Malaysia.

The genus Termitomyces (Lyophyllaceae, Basidiomycota) is often associated with fungus-feeding termites (Macrotermitinae) due to their strong symbiotic relationships. The genus is widely found exclusively in certain regions of Africa and Asia. They are recognized as edible mushroom within Southeast Asia as well. But it is often misidentified based on morphology by the local communities especially in Malaysia for Chlorophyllum molybdites which is a highly poisonous mushroom. Thus, it is necessary to study the genus for Malaysia with the synergy of using both morphological and molecular identification. In this study, we aim to describe another new species as an addition to the genus Termitomyces found within Sabah, Malaysia. We generated two new sequences (nrLSU and mtSSU) for the new species and a total of 28 nrLSU and mtSSU sequences were retrieved from GenBank for the phylogenetic analysis using maximum likelihood and Bayesian inferences. We identified that the new collection from Sabah province is a new species and named as Termitomyces gilvus based on the termites found in the mound. A phylogeny tree made from the concatenated genes of LSU and mtSSU suggests that T. gilvus is closely related to T. bulborhizus from China. According to our results, the combination of molecular and morphology proved to be a robust approach to re-evaluate the taxonomic status of Termitomyces species in Malaysia. Additional surveys are needed to verify the species diversity and clarify their geographic distribution.

Microfungi Associated with Pteroptyx bearni (Coleoptera: Lampyridae) Eggs and Larvae from Kawang River, Sabah (Northern Borneo).

Overlooking the importance of insect disease can have disastrous effects on insect conservation. This study reported the microfungi that infect Pteroptyx bearni eggs and larvae during ex-situ rearing project. Two different species of microfungi that infected the firefly's immature life stages were isolated and identified. Penicillium citrinum infected the firefly's eggs while Trichoderma harzianum infected the firefly during the larval stage. Both microfungi species caused absolute mortality once infection was observed; out of 244 individual eggs collected, 75 eggs (32.5%) were infected by Penicillium citrinum. All 13 larvae that hatched from the uninfected eggs were infected by Trichoderma harzianum. This study was the first to document the infection of Pteroptyx bearni's eggs and larvae by Penicillium citrinum and Trichoderma harzianum.