Influence of host phylogeographic patterns and incomplete lineage sorting on within-species genetic variability in Wigglesworthia species, obligate symbionts of tsetse flies.

Vertical transmission of obligate symbionts generates a predictable evolutionary history of symbionts that reflects that of their hosts. In insects, evolutionary associations between symbionts and their hosts have been investigated primarily among species, leaving population-level processes largely unknown. In this study, we investigated the tsetse (Diptera: Glossinidae) bacterial symbiont, Wigglesworthia glossinidia, to determine whether observed codiversification of symbiont and tsetse host species extends to a single host species (Glossina fuscipes fuscipes) in Uganda. To explore symbiont genetic variation in G. f. fuscipes populations, we screened two variable loci (lon and lepA) from the Wigglesworthia glossinidia bacterium in the host species Glossina fuscipes fuscipes (W. g. fuscipes) and examined phylogeographic and demographic characteristics in multiple host populations. Symbiont genetic variation was apparent within and among populations. We identified two distinct symbiont lineages, in northern and southern Uganda. Incongruence length difference (ILD) tests indicated that the two lineages corresponded exactly to northern and southern G. f. fuscipes mitochondrial DNA (mtDNA) haplogroups (P = 1.0). Analysis of molecular variance (AMOVA) confirmed that most variation was partitioned between the northern and southern lineages defined by host mtDNA (85.44%). However, ILD tests rejected finer-scale congruence within the northern and southern populations (P = 0.009). This incongruence was potentially due to incomplete lineage sorting that resulted in novel combinations of symbiont genetic variants and host background. Identifying these novel combinations may have public health significance, since tsetse is the sole vector of sleeping sickness and Wigglesworthia is known to influence host vector competence. Thus, understanding the adaptive value of these host-symbiont combinations may afford opportunities to develop vector control methods.

The tsetse fly Glossina fuscipes fuscipes (Diptera: Glossina) harbours a surprising diversity of bacteria other than symbionts.

Three different bacterial species are regularly described from tsetse flies. However, no broad screens have been performed to investigate the existence of other bacteria in this medically and agriculturally important vector insect. Utilising both culture dependent and independent methods we show that Kenyan populations of Glossina fuscipes fuscipes harbour a surprising diversity of bacteria. Bacteria were isolated from 72% of flies with 23 different bacterial species identified. The Firmicutes phylum dominated with 16 species of which seven belong to the genus Bacillus. The tsetse fly primary symbiont, Wigglesworthia glossinidia, was identified by the culture independent pathway. However, neither the secondary symbiont Sodalis nor Wolbachia was detected with either of the methods used. Two other bacterial species were identified with the DNA based method, Bacillus subtilis and Serratia marcescens. Further studies are needed to determine how tsetse flies, which only ever feed on vertebrate blood, pick up bacteria and to investigate the possible impact of these bacteria on Glossina longevity and vector competence.

Challenging the Wigglesworthia, Sodalis, Wolbachia symbiosis dogma in tsetse flies: Spiroplasma is present in both laboratory and natural populations.

Profiling of wild and laboratory tsetse populations using 16S rRNA gene amplicon sequencing allowed us to examine whether the "Wigglesworthia-Sodalis-Wolbachia dogma" operates across species and populations. The most abundant taxa, in wild and laboratory populations, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. The species richness of the microbiota was greater in wild than laboratory populations. Spiroplasma was identified as a new symbiont exclusively in Glossina fuscipes fuscipes and G. tachinoides, members of the palpalis sub-group, and the infection prevalence in several laboratory and natural populations was surveyed. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse-associated Spiroplasma, present in G. f. fuscipes and G. tachinoides. Spiroplasma density in G. f. fuscipes larva guts was significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density was higher in testes than ovaries, and was significantly higher density in live versus prematurely deceased females indicating a potentially mutualistic association. Higher Spiroplasma density in testes than in ovaries was also detected by fluorescent in situ hybridization in G. f. fuscipes.