Amir H. Tourani

True bugs harbour diverse microbial symbionts and are models of host–microbe interactions. Yet, most studies focus on bacterial partners of Holarctic‐Oriental species, leaving bacterial and fungal communities of Australasian taxa unexplored. We conducted the first comparative analysis of bacterial and fungal microbiota inhabiting the crypt‐bearing midgut region of Australian stink bugs by focussing on citrus‐associated Biprorulus bibax and Poecilometis strigatus (Pentatomidae), and Lyramorpha rosea and Musgraveia sulciventris (Tessaratomidae). Using high‐throughput amplicon sequencing, we found consistently low‐diversity bacterial communities dominated by host‐specific gammaproteobacterial symbionts within the Pantoea – Erwinia complex, although L. rosea and P. strigatus also harboured several other low‐abundance taxa. Fungal amplicon sequencing recovered low abundance taxa and unevenly represented communities, including Cladosporium , Eremothecium and unassigned Ascomycota, suggesting they were mostly environmentally acquired rather than conserved fungal associates. Despite host specificity, bacterial symbiont phylogeny was incongruent with host phylogeny, indicating host switches and decoupled host‐symbiont evolution. Egg‐surface sterilisation in B. bibax revealed vertical transmission of its dominant Pantoea ‐like symbiont via egg smearing, and its role in host fitness, with aposymbiotic offspring showing reduced survival, delayed development, shorter longevity and lower fecundity. Overall, our findings extend established patterns of stink bug symbiosis to understudied Australian taxa, providing new insights into host–microbe associations, particularly for Tessaratomidae.

BACKGROUND: Maternally transmitted endosymbionts like Wolbachia and Cardinium are common in arthropods including many pest species. By inducing cytoplasmic incompatibility (CI), they can support sustainable pest management via population‐suppression or replacement strategies. However, models underlying these approaches often neglect female remating and the pre‐ and post‐copulatory mechanisms that influence sperm use and compatibility. In many species, females mate multiple times and may encounter both compatible and incompatible males, potentially altering CI expression and endosymbiont spread. We therefore tested how remating and mating order affect CI and reproductive fitness in Kelly's citrus thrips, Pezothrips kellyanus , a haplodiploid citrus pest naturally carrying either Cardinium alone (C) or both Cardinium and Wolbachia (CW). RESULTS: We found that CI depended strongly on mating history. When a C female first mated with an incompatible CW male, CI was strong, and a second mating with a compatible C male only restored approximately 21% of female offspring production. Conversely, when a C female first mated with a compatible C male and then with an incompatible CW male, CI was substantially weaker, consistent with first‐male sperm precedence. Female fitness also varied with mating history; C females mated twice with CW males had the lowest fecundity, highest embryonic and post‐embryonic mortality, and shortest survival. CONCLUSION: First‐male sperm precedence limits restoration of female offspring production by a compatible mating following an incompatible mating. This may increase the fitness costs of accepting incompatible males in hosts with pre‐copulatory mate discrimination. More broadly, remating and mate sequence can alter CI strength, affecting models of endosymbiont spread and endosymbiont‐based pest management. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Males can influence future mating interactions of females after copulation by changing female signals that subsequent males will encounter. In insects, such effects commonly involve cuticular hydrocarbons (CHCs), but whether heritable microbial symbionts contribute to post-mating chemical signalling remains largely unknown. Kelly’s citrus thrips, Pezothrips kellyanus , provides an ideal system to address this question because reproductive compatibility is shaped by common arthropod endosymbionts. Across P. kellyanus populations, Cardinium occurs in almost all individuals whereas Wolbachia varies in prevalence and appears to spread by cytoplasmic incompatibility (CI). Yet, females with only Cardinium (C) avoid incompatible males carrying both Cardinium and Wolbachia (CW), and this discrimination is linked to the distinct CHC profile of CW males. Here, we tested whether this endosymbiont-associated male perfume persists on females beyond copulation by altering the female CHC profile and subsequent male mating behaviour. Using behavioural assays and GC–MS-based CHC profiling, we found that, independent of female endosymbiont association, females first mated with CW males received fewer antennal contacts and mating attempts from subsequent C males. Furthermore, mating remodelled female CHC profiles, while mating with CW males produced a distinctive post-mating chemical signature. Most notably, tridecane, previously detected only in CW males, occurred exclusively in females mated with CW males. Our findings show that endosymbionts can alter mated female CHCs and influence future sexual communication between male and female hosts. These findings reveal a previously unrecognised post-mating route through which endosymbionts reshape sexual communication, with potential consequences for reproductive compatibility and symbiont transmission dynamics.

Maternally transmitted endosymbionts of arthropods are common and phylogenetically diverse. Several bacteria, including Wolbachia and Cardinium, have independently evolved the ability to induce cytoplasmic incompatibility (CI) limiting the reproduction in females lacking the endosymbionts carried by their mates. While promoting endosymbiont spread, CI is costly to endosymbiont-free females. Such host-endosymbiont conflicts are expected to affect host mating preferences, yet this has scarcely been studied in hosts carrying multiple, potentially competing, endosymbionts. We investigated mate choice and chemical communication in a significant pest of citrus, Kelly’s citrus thrips (Pezothrips kellyanus), naturally carrying CI-inducing Cardinium and Wolbachia. Unlike females with both endosymbionts (CW) that had no preference for males with particular endosymbiont associations, females with only Cardinium (C) preferred compatible C and endosymbiont-free males over incompatible CW males. In contrast, endosymbiont-free females showed no preference, despite experiencing similar CI risks when facing incompatible C and CW males. Male mating success, however, mostly depended on female receptivity and not on endosymbiont association. Furthermore, chemical analyses revealed that males with different endosymbiont associations had distinctly different cuticular hydrocarbon (CHC) profiles, with the CHC profile of CW males markedly including tridecane, a compound known to influence animal behavior. The results indicate that Cardinium enables females to avoid Wolbachia-induced CI based on the distinct chemical cues of incompatible males. Our findings highlight the role of common endosymbionts and their interactions in sexual selection through their effects on chemical and behavioral traits of hosts, emphasizing the importance of these factors in endosymbiont and host population dynamics, as well as endosymbiont-based pest control strategies.

Many arthropods carry maternally inherited endosymbionts that cause cytoplasmic incompatibility (CI), manifested as embryonic mortality in matings of infected males with uninfected females. Infected females, however, do not suffer this cost. Therefore, in populations with mixed endosymbiont infections, selection is expected to favour mechanisms that enable hosts to avoid or mitigate CI. This may include changes in mating behaviour, such as reduced female receptivity to mating and/or remating when approached by incompatible males. Here, we investigated mating behavioural traits in haplodiploid thrips naturally associated with two CI-inducing endosymbionts, Cardinium and Wolbachia . Compared with females with both endosymbionts, those with only Cardinium showed reduced receptivity to males carrying both. However, surprisingly, females without endosymbionts were not less receptive to incompatible males. Furthermore, in contrast to females without endosymbionts, females with Cardinium were far less likely to remate with incompatible than compatible males irrespective of the compatibility type of the first mating. Our results suggest that endosymbiont-specific sexual selection processes occur, whereby females carrying only Cardinium recognize Wolbachia in coinfected males to avoid CI. This may hinder a CI-driven Wolbachia spread. Endosymbiont-mediated mating behaviours may be crucial for the dynamics of CI-inducing endosymbionts and their application in pest management strategies.