Profiles of bacterial communities and environmental factors associated with proliferation of malaria vector mosquitoes within the Kenyan Coast.

Background: Since Anopheles mosquitoes which transmit and maintain the malaria parasite breed in the outdoor environment, there is an urgent need to manage these mosquito breeding sites. In order to elaborate more on the ecological landscape of mosquito breeding sites, the bacterial community structure and their interactions with physicochemical factors in mosquito larval habitats was characterised in Kwale County (Kenya), where malaria is endemic. Methods: The physical characteristics and water physicochemical parameters of the habitats were determined and recorded. Water samples were also collected from the identified sites for total metagenomic DNA extraction in order to characterise the bacterial communities within the breeding sites. Results and Discussion: Sites where mosquito larvae were found were described as positive and those without mosquito larvae as negative. Electrical conductivity, total dissolved solids, salinity and ammonia were lower in the rainy season than in the dry season, which also coincided with a high proportion of positive sites. Pseudomonadota was the most common phyla recovered in all samples followed by Bacteroidota and then Actinomycetota. The presence or absence of mosquito larvae in a potential proliferation site was not related to the bacterial community structure in the sampled sites, but was positively correlated with bacterial richness and evenness. Conclusion: Generally, the presence of Anopheles mosquito larvae was found to be positively correlated with rainy season, bacterial richness and evenness, and negatively correlated with electrical conductivity, total dissolved solids, salinity and ammonia. The findings of this study have implications for predicting the potential of environmental water samples to become mosquito proliferation sites.

Diversity and taxonomic revision of methanogens and other archaea in the intestinal tract of terrestrial arthropods.

Methane emission by terrestrial invertebrates is restricted to millipedes, termites, cockroaches, and scarab beetles. The arthropod-associated archaea known to date belong to the orders Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales, and in a few cases also to non-methanogenic Nitrososphaerales and Bathyarchaeales. However, all major host groups are severely undersampled, and the taxonomy of existing lineages is not well developed. Full-length 16S rRNA gene sequences and genomes of arthropod-associated archaea are scarce, reference databases lack resolution, and the names of many taxa are either not validly published or under-classified and require revision. Here, we investigated the diversity of archaea in a wide range of methane-emitting arthropods, combining phylogenomic analysis of isolates and metagenome-assembled genomes (MAGs) with amplicon sequencing of full-length 16S rRNA genes. Our results allowed us to describe numerous new species in hitherto undescribed taxa among the orders Methanobacteriales (Methanacia, Methanarmilla, Methanobaculum, Methanobinarius, Methanocatella, Methanoflexus, Methanorudis, and Methanovirga, all gen. nova), Methanomicrobiales (Methanofilum and Methanorbis, both gen. nova), Methanosarcinales (Methanofrustulum and Methanolapillus, both gen. nova), Methanomassiliicoccales (Methanomethylophilaceae fam. nov., Methanarcanum, Methanogranum, Methanomethylophilus, Methanomicula, Methanoplasma, Methanoprimaticola, all gen. nova), and the new family Bathycorpusculaceae (Bathycorpusculum gen. nov.). Reclassification of amplicon libraries from this and previous studies using this new taxonomic framework revealed that arthropods harbor only CO2 and methyl-reducing hydrogenotrophic methanogens. Numerous genus-level lineages appear to be present exclusively in arthropods, suggesting long evolutionary trajectories with their termite, cockroach, and millipede hosts, and a radiation into various microhabitats and ecological niches provided by their digestive tracts (e.g., hindgut compartments, gut wall, or anaerobic protists). The distribution patterns among the different host groups are often complex, indicating a mixed mode of transmission and a parallel evolution of invertebrate and vertebrate-associated lineages.

"Methanoplasmatales," Thermoplasmatales-related archaea in termite guts and other environments, are the seventh order of methanogens.

The Euryarchaeota comprise both methanogenic and nonmethanogenic orders and many lineages of uncultivated archaea with unknown properties. One of these deep-branching lineages, distantly related to the Thermoplasmatales, has been discovered in various environments, including marine habitats, soil, and also the intestinal tracts of termites and mammals. By comparative phylogenetic analysis, we connected this lineage of 16S rRNA genes to a large clade of unknown mcrA gene sequences, a functional marker for methanogenesis, obtained from the same habitats. The identical topologies of 16S rRNA and mcrA gene trees and the perfect congruence of all branches, including several novel groups that we obtained from the guts of termites and cockroaches, strongly suggested that they stem from the same microorganisms. This was further corroborated by two highly enriched cultures of closely related methanogens from the guts of a higher termite (Cubitermes ugandensis) and a millipede (Anadenobolus sp.), which represented one of the arthropod-specific clusters in the respective trees. Numerous other pairs of habitat-specific sequence clusters were obtained from the guts of other termites and cockroaches but were also found in previously published data sets from the intestinal tracts of mammals (e.g., rumen cluster C) and other environments. Together with the recently described Methanomassiliicoccus luminyensis isolated from human feces, which falls into rice cluster III, the results of our study strongly support the idea that the entire clade of "uncultured Thermoplasmatales" in fact represents the seventh order of methanogenic archaea, for which the provisional name "Methanoplasmatales" is proposed.

Metagenomic analysis of the microbiota in the highly compartmented hindguts of six wood- or soil-feeding higher termites.

BACKGROUND: Termites are important contributors to carbon and nitrogen cycling in tropical ecosystems. Higher termites digest lignocellulose in various stages of humification with the help of an entirely prokaryotic microbiota housed in their compartmented intestinal tract. Previous studies revealed fundamental differences in community structure between compartments, but the functional roles of individual lineages in symbiotic digestion are mostly unknown. RESULTS: Here, we conducted a highly resolved analysis of the gut microbiota in six species of higher termites that feed on plant material at different levels of humification. Combining amplicon sequencing and metagenomics, we assessed similarities in community structure and functional potential between the major hindgut compartments (P1, P3, and P4). Cluster analysis of the relative abundances of orthologous gene clusters (COGs) revealed high similarities among wood- and litter-feeding termites and strong differences to humivorous species. However, abundance estimates of bacterial phyla based on 16S rRNA genes greatly differed from those based on protein-coding genes. CONCLUSION: Community structure and functional potential of the microbiota in individual gut compartments are clearly driven by the digestive strategy of the host. The metagenomics libraries obtained in this study provide the basis for future studies that elucidate the fundamental differences in the symbiont-mediated breakdown of lignocellulose and humus by termites of different feeding groups. The high proportion of uncultured bacterial lineages in all samples calls for a reference-independent approach for the correct taxonomic assignment of protein-coding genes.