Discussion
Our study experimentally evolved pesticide resistance in flour beetles (T. castaneum ) to investigate the combined interactive effects of pesticide resistance and pesticide exposure on immunity and host-pathogen interactions. Pesticide-resistant insects and insects exposed to Btt both developed more slowly than their counterparts, indicating sub-lethal fitness costs to resistance and infection, respectively. We found that Pyr-resistant insects exhibited dampened transcriptional immune responses to Btt infection relative to their control-regime counterparts, although overall survival and development did not differ among OP- and Pyr-resistant and susceptible insects after Btt exposure. While the evolution of resistance did not substantially influence host infection phenotypes, exposure to pesticides did. Specifically, we observed that larvae were more likely to survive OP exposure if they were also orally exposed toBtt , with gene expression patterns suggesting dampened transcriptional stress responses in those beetles. Taken together, our data suggest that evolved resistance, pesticide exposure, and bacterial infection all exert contrasting effects on life history parameters that could ultimately influence the demography of insect populations in complex ways. The factorial nature of our study design, moreover, provides a new window into the physiological basis and fitness consequences of host responses to overlapping environmental stressors.
Insects, including T. castaneum , can rapidly evolve resistance to pesticides through mechanisms that enhance metabolic detoxification and cuticular modifications . Here, populations selected for pesticide resistance evolved it within 6-8 generations , demonstrating increased survival and faster development after pesticide exposure compared to control-selected populations. Differential gene expression between pesticide- and control- selection regimes suggested that OP resistance is associated with constitutive changes in cuticle gene expression while Pyr resistance is associated with oxidoreductase activity, heme binding, and cuticular modifications, consistent with previous studies in OP and Pyr resistant insects . Thus, while both OP and Pyr resistance involved changes in cuticle gene expression, Pyr resistance was also associated with changes in metabolic detoxification.
Oral Btt infection did not decrease survival in our study but was associated with slower development in T. castaneum larvae, indicating a direct cost of infection or an indirect cost from mounting an immune response, as previously observed in the same system and in other insects challenged with Bt toxins . Previously documented variation in resistance in T. castaneum against Cry3 proteins effective against other Tenebrionid beetles may explain why Btthad negative effects on development but did not instigate high mortality . Here, exposure to Btt induced the upregulation of AMPs (attacins and defensins) and one pathogenesis-related protein as well as differential expression of detoxification genes, including P450s and a CEH, GST, an ABC transporter, and several cuticle and development related genes. Previous transcriptomic studies of oral Bt infection inT. castaneum have identified similar transcripts, including attacin and defensin AMPs, a pathogenesis-related protein, a P450, and a GST , supporting the assumption that Btt oral exposure in our experiments presented an immune challenge despite the lack of infection-induced mortality.
Previous work has suggested that exposure to pesticides can reduce defenses against pathogens and stimulate an array of changes in humoral, cellular, and oxidative stress components of immunity . Our results reveal a strong interaction effect between Btt and OP exposure that manifests at both phenotype and gene expression levels. For example, dual oral exposure to Btt and OP improved larval survival and partially rescued development time relative to OP exposure alone, without exacerbating Btt -induced mortality. At the same time, Btt co-exposure modified the differential expression of 175 genes relative to OP exposure alone (Fig. 3B). In contrast, Pyr exposure did not influence survival with Btt infection, but larvae exposed to both Pyr and Btt also partially rescued development time compared to those exposed only to pesticides. However, in contrast to the effects on gene expression observed with dual OP and Bttexposure, Btt co-exposure with Pyr had negligible effects on gene expression with the overexpression of only one cuticle-related transcript. It is possible that the positive effects on development time seen with pesticide and Btt co-exposure compared to single exposure to pesticides are part of an advantageous response to increased risk (Roth & Kurtz, 2008) as has been seen in virus-infected caterpillars reared on more toxic diets .
There is also the potential for indirect antagonistic interactions between OP and Btt , e.g. through induction of cross-protective host defenses. For example, while Btt exposure induced upregulation of attacins, cytochrome P450s, and cuticle-related transcripts, combined OP and Btt exposure dampened the expression of these transcripts. It is possible that this dampened expression of immune and detoxification genes may underlie reductions in immunopathology or ROS damage corresponding to increased fitness, or the benefits to fitness may be due to differences in resource allocation when faced with dual pressures . Interestingly, single exposure to OP orBtt induced downregulation of an ABC transporter but this transcript was upregulated with co-exposure to OP and Btt . ABC transporters have been identified as receptors for Bt Cry proteins and aid in membrane pore formation , and the additional function of ABC transporters as xenobiotic pumps may help to mediate Bt toxicity . It is also possible that OP and Btt interact directly, as would be the case if Btt actively degrades OP for energy or if OP facilitatesBtt germination rates. Our in vitro data suggest thatBtt vegetative cells in liquid medium replicate faster in the presence of OP, lending credence to a direct interaction. On the other hand, there was no difference in germinated spore counts between control-diet and OP-diet disks, suggesting that direct interactions, if they matter in vivo , are not relevant until after the host ingests the diet.
Supporting the idea of localized cross-protective mechanisms, a previous study of oral vs. septic Btt infection in T. castaneumdemonstrated that oral infection induced greater changes in cuticle-related gene expression compared to septic infection . In our study, OP-exposed individuals were not protected from greater mortality upon septic Btt infection, suggesting that infection route has important implications for pesticide-pathogen interactions. We found cuticle-associated gene expression changes induced by OP exposure, and cuticular modifications of gut tissues may provide an additional layer of protection from oral Bt infection . Future work dissecting the relative importance of these direct and indirect interactions will facilitate our understanding of organismal adaptations to multiple stressors and will be crucial for refining integrative pest management (IPM) strategies employing both chemical and biopesticides.
The impact of evolved pesticide resistance on immunity is still poorly understood , and in the absence of pesticides we did not observe any significant interactions between pesticide resistance and Bt exposure on survival or development. Few genes were differentially expressed between OP resistant and susceptible beetles after Btt exposure, butBtt exposure in Pyr-resistant populations was associated with dampened upregulation of several genes, including two defensins, a histidine-rich glycoprotein, and a pathogenesis-related protein, that were more strongly upregulated in Pyr-susceptible populations afterBtt exposure. It is also possible that Pyr-resistant populations had constitutively higher immune gene expression resulting in reduced immune activation upon Bt infection. Moreover, an apolipophorin-III transcript, previously shown to be important in immunity against coleopteran-specific Bt toxins , was upregulated in Pyr-resistant populations. In contrast, cuticle-related protein and neuropeptide transcripts were downregulated in Pyr-regime larvae exposed toBtt . These immune gene changes associated with Pyr resistance may have important effects on interactions with other pathogens, and future research into Pyr resistance interactions with other bacterial or viral pathogens may yield important insights.
While we did not observe any phenotypic differences with pesticide selection regime and Btt infection here, we also did not observe mortality from Btt alone and it is possible that the immune challenge was not strong enough to elicit an interaction with resistance mechanisms. However, the differential expression of immune and cuticle functions in Pyr-regime larvae exposed to Btt could have important implications for insect interactions with other pathogens or xenobiotic pressures by modifying detoxification capabilities or providing an additional barrier against infection or toxins . Furthermore, changes in oxidoreductase activity observed after Pyr selection are likely to alter the status of reactive oxygen species (ROS) within the host and may impact immunity against different pathogens . Finally, it is possible that adaptation to pesticides could constrain host evolutionary responses to pathogens, or vice versa. Future experiments could simultaneously select for dual resistance to both pesticides and pathogens to see if evolutionary trajectories are similar or constrained relative to selection under a single stressor.