Carruthers, L. V. | Search bioRxiv

bioRxiv2024

How should we sample snail microbiomes?

BackgroundThe microbiome is increasingly recognized to shape many aspects of its host biology and is a key determinant of health and disease. The microbiome may influence transmission of pathogens by their vectors, such as mosquitoes or aquatic snails. We previously sequenced the V4 region of the bacterial 16S rRNA gene from the hemolymph (blood) of Biomphalaria spp. snails, vectors of the human blood fluke schistosome. We showed that snail hemolymph harbored an abundant and diverse microbiome. This microbiome is distinct from the water environment and can discriminate snail species and populations. As hemolymph bathes snail organs, we then investigated the heterogeneity of the microbiome in these organs. ResultsWe dissected ten snails for each of two different species (B. alexandrina and B. glabrata) and collected their hemolymph and organs (ovotestis, hepatopancreas, gut, and stomach). We also ground in liquid nitrogen four whole snails of each species. We sampled the water in which the snails were living (environmental controls). Sequencing the 16S rRNA gene revealed organ-specific microbiomes. These microbiomes harbored a lower diversity than the hemolymph microbiome, and the whole-snail microbiome. The organ microbiomes tend to cluster by physiological function. In addition, we showed that the whole-snail microbiome is more similar to hemolymph microbiome. ConclusionsThese results are critical for future work on snail microbiomes and show the necessity of sampling individual organ microbiomes to provide a complete description of snail microbiomes.

microbiology↗

bioRxiv2020

Diminazene resistance in Trypanosoma congolense is linked to reduced mitochondrial membrane potential and not to reduced transport capacity

Trypanosoma congolense is one of the principal agents causing livestock trypanosomiasis in sub-Saharan Africa. This wasting disease is costing these developing economies billions of dollars and undermining food security. Only two old drugs, the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induce diminazene resistance in T. congolense laboratory strain IL3000 in vitro. Resistance was stable and not deleterious to in vitro growth. There was no cross-resistance with the phenanthridine drugs, with melaminophenyl arsenicals, with two promising new oxaborole trypanocides, nor with other diamidine trypanocides such as pentamidine, except the close structural analogues DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3H]-diminazene was also partly inhibited by folate, as well as by competing diamidine drugs, albeit at quite high concentrations, and uptake of tritiated diminazene and pentamidine was slow and low affinity. Uptake of [3H]-folate was in turn partly inhibited by diminazene, and inhibition of diminazene uptake by folate and pentamidine appeared to be additive, indicating multiple low affinity transport mechanisms for the drug. Expression of the T. congolense folate transporters TcoFT1-3 in diminazene-resistant T. b. brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3H]-diminazene uptake was not different in T. congolense IL3000 and its diminazene resistant clones and RNAseq and whole-genome sequencing of multiple resistant clones did not reveal major changes in folate transporter sequence or expression. Instead, flow cytometry revealed a strong and stable reduction in the mitochondrial membrane potential {Psi}m in all resistant clones. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters and that resistance is the result of a reduction in {Psi}m that limits mitochondrial accumulation of the drug.

microbiology↗