Genetic diversity of the tsetse fly microbiome from Campo, Southern Cameroon: implications in new strategies to control trypanosomiases

Abstract

The interest of vector control in the fight against African Trypanosomiases has been reinforced in recent years by the development of small insecticide-impregnated screens, known as "Tiny Targets". In addition, impairing the development of the parasite in the vector and therefore reducing the vectorial competence of tsetse flies was shown to be an alternative or additive method to stop disease transmission. To improve knowledge in developing this new vector control strategies, study on the characterisation of bacterial communities hosted by tsetse flies, their association with trypanosome establishment and their potential implication in the flies’ fitness during vector control with "Tiny Targets" was carried out in Campo in Southern Cameroon. The study site was divided into two areas, the South-West experiment area with vector control and the eastern control area. Tsetse flies were collected in December 2018 and July 2019 as baseline data prior to the Tiny Targets installation. Then, around 2000 targets were deployed in the South-West area and replaced every six months covering 2-year periods. Post-intervention surveys were conducted every six months and apparent tsetse densities were calculated, compared, and mapped. The different trypanosome species and the origin of Glossina palpalis palpalis flies blood meals were identified by PCR. Amplification of the highly variable V3-V4 region of the 16S rRNA bacterial gene and sequencing with Illumina Miseq® platform were carried out, followed by meta-genomic analysis to identify the different bacterial communities, to determine the impact of certain taxa in flies’ infectivity or fitness. Our study revealed the presence of four species of trypanosomes in Glossina palpalis palpalis. From two collection periods (December 2018 and July 2019), a total of 18.52% tsetse was infected with at least one trypanosome species respectively. Specifically, 13.75% were infected by Trypanosoma congolense, 4.51% by T. brucei s.l., 1.51% by T. vivax and 0.32% by T. simiae. From all the flies infected with T. brucei s.l., 4 were infected with Trypanosoma brucei gambiense subspecies which is the parasite responsible for sleeping sickness. Blood meal analysis revealed 2 out of 85 tsetse flies analysed fed on humans. Following the tsetse control intervention, tsetse densities decreased by 61% after six months and up to 73% after twelve months (pre-intervention: 2.48 flies/trap/day, 95%CI [1.92-3.14]; 12-months post-intervention: 0.66 tsetse/trap/day, 95%CI [0.42-0.94]). This decrease was not sustained after 18 months where the 12-months density doubled, and 24 months where the density still increased by 17% (18 months: 1.45 tsetse/trap/day, 95%CI [1.07-1.90] and 24 months: 1.71 tsetse/trap/day, 95%CI [1.27-2.24]). In the control area, variation of tsetse densities was observed during the two years, with a general increase from 2.43 [0.73-5.77] to 3.64 [1.47-7.70] tsetse/trap/day. In addition, trypanosome infection rates dropped by 75% in both areas (P-value < 0.001) from 21.20% to 5.06% and from 13.14% to 3.45% in intervention and control areas respectively. Characterisation of the bacterial flora of the tsetse examined shown a total of 4 phyla and 31 bacterial genera present in tsetse flies. The primary symbiont Wigglesworthia was present in almost all specimens with an overall relative abundance of 47.29%. However, this symbiont seems to be replaced by Serratia or Burkholderia in some flies of the species G. tachinoides. Overall, significant differences were observed in bacterial diversity between tsetse species (p<0.001), between teneral and non-teneral flies (p<0.001) and between flies with and without mature trypanosome infections (p<0.031). Moreover, differential abundance tests shown some bacteria taxa associated with trypanosome maturation in tsetse flies, the most important being Dechloromonas (log2 Fold Change = -21.44; p<0.001), Ralstonia (log2 Fold Change = -10.59; p<0.001), Listeria (log2 Fold Change = -5.41; p<0.001), Serratia (log2 Fold Change = 3.39; p<0.001) et Staphylococcus (log2 Fold Change = 5.48; p= 0.003). Significant increase was observed in bacteria diversity and composition in tsetse flies during the vector control (Before vector control, Shannon H = 0.45; six months H = 0.44; twelve months H = 1.24; eighteen months H = 0.99; p = 4,9 x 10-05). Assessment of the relative abundances of the different phyla before and after the installation of Tiny targets showed a significant reduction in the abundance of Firmicutes (p < 0.02) and a concomitant increase in Proteobacteria (p < 0.001). In addition, differential abundance testing showed several bacteria genera contributing to differences between tsetse flies collected before and after 18 months of vector control. Phylogenetic analyses of bacterial genera represented by several operational taxonomic units (OTUs) showed that these OTUs exhibited some degree of intra-generic genetic polymorphism, strengthening the hypothesis that vector competence may be related to given bacteria genotypes. This study shown bacteria taxa associated with trypanosome infection maturation in flies, which therefore need further studies for an understanding of their mechanism of action and alternatively, transformed and used to block trypanosome development in tsetse flies.