Molecular characterisation of the twisted wing endoparasitoid Dipterophagus daci (Strepsiptera) and its interactions with Wolbachia and multiple tephritid fruit fly host species

Australia hosts over 300 species of tephritid fruit flies, including the Queensland fruit fly, Bactrocera tryoni, Australia’s most significant horticultural pest. Australian tephritid fruit fly species have previously been found to be infected by two strains of the common insect endosymbiont Wolbachia. Wolbachia are maternally inherited endosymbionts in about 50% of insect species and can affect host reproduction and fitness. The phylogenetic incongruence of Wolbachia and their hosts indicates that horizontal transfer between species can also happen. Previously reported Wolbachia infections in Australian tephritid fruit fly species were unusual because they were detected in only seven out of 24 tested species (29 %) at low prevalence and titres and were restricted only to individuals of tropical Australia. This PhD thesis investigated these Wolbachia infections further by performing whole genome sequencing (WGS) of the field-caught Wolbachia-positive flies. This revealed an unexpected presence of almost complete mitochondrial genomes (mitogenomes) of another insect, in addition, to the expected fruit fly mitogenome in the genomic libraries of fruit fly. The additional mitogenomes belonged to a twisted-wing endoparasitoid, Dipterophagus daci (Strepsiptera), suggesting a possible link between Wolbachia and the presence of D. daci in tephritid fruit flies. This study therefore investigated the host-endoparasitoid-endosymbiont interaction between tephritid fruit fly species, Dipterophagus daci and Wolbachia. Overall, this study investigated and resolved the enigma of Wolbachia infections previously detected in tephritid fruit flies and presented evidence that the presence of Wolbachia in tephritid fruit flies was due to concealed early stages of parasitisation by D. daci. This reveals that the detection of Wolbachia in host taxa could be due to concealed parasitisation by strepsipterans or other endoparasitoids, and this could lead to incorrect assignment of Wolbachia to a wrong host. Additionally, it suggests that Australian tephritid fruit flies may not be naturally infected by Wolbachia therefore making them amenable for control using Wolbachia-based incompatible insect technique in the future. Furthermore, this study presents six D. daci mitogenomes and nine fruit fly mitogenomes which will be a useful source for future studies, in particular of the biology and ecology of the unique strepsipteran D. daci, and its impact on fruit fly population dynamics. This work also presents the first microbiome of a strepsipteran, which is a valuable contribution to the parasite microbiome studies

Substantial rearrangements, single nucleotide frameshift deletion and low diversity in mitogenome of Wolbachia‑infected strepsipteran endoparasitoid in comparison to its tephritid hosts

Insect mitogenome organisation is highly conserved, yet, some insects, especially with parasitic life cycles, have rearranged mitogenomes. Furthermore, intraspecific mitochondrial diversity can be reduced by fitness-affecting bacterial endosymbionts like Wolbachia due to their maternal coinheritance with mitochondria. We have sequenced mitogenomes of the Wolbachia-infected endoparasitoid Dipterophagus daci (Strepsiptera: Halictophagidae) and four of its 22 known tephritid fruit fly host species using total genomic extracts of parasitised flies collected across > 700 km in Australia. This halictophagid mitogenome revealed extensive rearrangements relative to the four fly mitogenomes which exhibited the ancestral insect mitogenome pattern. Compared to the only four available other strepsipteran mitogenomes, the D. daci mitogenome had additional transpositions of one rRNA and two tRNA genes, and a single nucleotide frameshift deletion in nad5 requiring translational frameshifting or, alternatively, resulting in a large protein truncation. Dipterophagus daci displays an almost completely endoparasitic life cycle when compared to Strepsiptera that have maintained the ancestral state of free-living adults. Our results support the hypothesis that the transition to extreme endoparasitism evolved together with increased levels of mitogenome changes. Furthermore, intraspecific mitogenome diversity was substantially smaller in D. daci than the parasitised flies suggesting Wolbachia reduced mitochondrial diversity because of a role in D. daci fitness

Bacterial Communities Are Less Diverse in a Strepsipteran Endoparasitoid than in Its Fruit Fly Hosts and Dominated by Wolbachia

Microbiomes play vital roles in insect fitness and health and can be influenced by interactions between insects and their parasites. Many studies investigate the microbiome of free-living insects, whereas microbiomes of endoparasitoids and their interactions with parasitised insects are less explored. Due to their development in the constrained environment within a host, endoparasitoids are expected to have less diverse yet distinct microbiomes. We used high-throughput 16S rRNA gene amplicon sequencing to characterise the bacterial communities of Dipterophagus daci (Strepsiptera) and seven of its tephritid fruit fly host species. Bacterial communities of D. daci were less diverse and contained fewer taxa relative to the bacterial communities of the tephritid hosts. The strepsipteran’s microbiome was dominated by Pseudomonadota (formerly Proteobacteria) (> 96%), mainly attributed to the presence of Wolbachia, with few other bacterial community members, indicative of an overall less diverse microbiome in D. daci. In contrast, a dominance of Wolbachia was not found in flies parasitised by early stages of D. daci nor unparasitised flies. Yet, early stages of D. daci parasitisation resulted in structural changes in the bacterial communities of parasitised flies. Furthermore, parasitisation with early stages of D. daci with Wolbachia was associated with a change in the relative abundance of some bacterial taxa relative to parasitisation with early stages of D. daci lacking Wolbachia. Our study is a first comprehensive characterisation of bacterial communities in a Strepsiptera species together with the more diverse bacterial communities of its hosts and reveals effects of concealed stages of parasitisation on host bacterial communities

Comprehensive transcriptome of the maize stalk borer, Busseola fusca, from multiple tissue types, developmental stages, and parasitoid wasp exposures

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Host-endoparasitoid-endosymbiont relationships: concealed Strepsiptera provide new twist to Wolbachia in Australian tephritid fruit flies

Wolbachia are widespread endosymbionts that affect arthropod reproduction and fitness. Mostly maternally inherited, Wolbachia are occasionally transferred horizontally. Previously, two Wolbachia strains were reported at low prevalence and titres across seven Australian tephritid species, possibly indicative of frequent horizontal transfer. Here, we performed whole-genome sequencing of field-caught Wolbachia-positive flies. Unexpectedly, we found complete mitogenomes of an endoparasitic strepsipteran, Dipterophagus daci, suggesting that Wolbachia in the flies are linked to concealed parasitisation. We performed the first genetic characterisation and detected D. daci in Wolbachia-positive flies not visibly parasitised, but most Wolbachia-negative flies were D. daci-negative, presumably reflecting polymorphism for the Wolbachia infections in D. daci. We dissected D. daci from stylopised flies and confirmed that Wolbachia infects D. daci, but also found Wolbachia in stylopised fly tissues, likely somatic, horizontally transferred, non-heritable infections. Furthermore, no Wolbachia cif and wmk genes were detected, and very low mitogenomic variation in D. daci across its distribution. Therefore, Wolbachia may influence host fitness without reproductive manipulation. Our study of 13 tephritid species highlights that concealed early stages of strepsipteran parasitisation led to the previous incorrect assignment of Wolbachia co-infections to tephritid species, obscuring ecological studies of this common endosymbiont and its horizontal transmission by parasitoids. This article is protected by copyright. All rights reserved