Gut symbiotic bacteria are involved in nitrogen recycling in the tephritid fruit fly Bactrocera dorsalis.

BACKGROUND: Nitrogen is considered the most limiting nutrient element for herbivorous insects. To alleviate nitrogen limitation, insects have evolved various symbiotically mediated strategies that enable them to colonize nitrogen-poor habitats or exploit nitrogen-poor diets. In frugivorous tephritid larvae developing in fruit pulp under nitrogen stress, it remains largely unknown how nitrogen is obtained and larval development is completed. RESULTS: In this study, we used metagenomics and metatranscriptomics sequencing technologies as well as in vitro verification tests to uncover the mechanism underlying the nitrogen exploitation in the larvae of Bactrocera dorsalis. Our results showed that nitrogenous waste recycling (NWR) could be successfully driven by symbiotic bacteria, including Enterobacterales, Lactobacillales, Orbales, Pseudomonadales, Flavobacteriales, and Bacteroidales. In this process, urea hydrolysis in the larval gut was mainly mediated by Morganella morganii and Klebsiella oxytoca. In addition, core bacteria mediated essential amino acid (arginine excluded) biosynthesis by ammonium assimilation and transamination. CONCLUSIONS: Symbiotic bacteria contribute to nitrogen transformation in the larvae of B. dorsalis in fruit pulp. Our findings suggest that the pattern of NWR is more likely to be applied by B. dorsalis, and M. morganii, K. oxytoca, and other urease-positive strains play vital roles in hydrolysing nitrogenous waste and providing metabolizable nitrogen for B. dorsalis.

Transcriptome analysis uncovers different avenues for manipulating cold performance in Chrysomya megacephala (Diptera, Calliphoridae).

Temperature strongly impacts the rates of physiological and biochemical processes, which in turn can determine the survival and population size of insects. At low temperatures performance is limited, however, cold tolerance and performance at low temperature can be improved after short- or long-term acclimation in many insect species. To understand mechanisms underlying acclimation, we sequenced and compared the transcriptome of the blowfly Chrysomya megacephala under rapid cold hardening (RCH) and long-term cold acclimation (LCA) conditions. The RCH response was dominated by genes related to immune response, spliceosome, and protein processing in endoplasmic reticulum with up-regulation during recovery from RCH. In contrast, LCA was associated with genes related to carbohydrate metabolism and cytoskeleton branching and stabilizing. Meanwhile, mRNA levels of genes related to glycerophospholipid metabolism, and some heat shock proteins (Hsps) were collectively up-regulated by both RCH and LCA. There were more genes and pathway adjustments associated with LCA than RCH. Overall, the transcriptome data provide basic information of molecular mechanisms underpinning the RCH and LCA response. The partly independent molecular responses to RCH and LCA suggest that several avenues for manipulating cold performance exist and RCH might be more effective as it only triggers fewer genes and affects the general metabolisms less. These observations provide some appropriate methods to improve cold tolerance of C. megacephala, and hold promise for developing an extended use of mass-reared C. megacephala with better cold performance as a pollinator of crops at low temperatures.

Gut microbiota metagenomics and mediation of phenol degradation in Bactrocera minax (Diptera, Tephritidae).

BACKGROUND: Gut microbiota mediating insect-plant interactions have many manifestations, either by provisioning missing nutrients, or by overcoming plant defensive reactions. However, the mechanism by which gut microbiota empower insects to survive by overcoming a variety of plant secondary metabolites remains largely unknown. Bactrocera minax larvae develop in immature citrus fruits, which present numerous phenolic compounds that challenge the larvae. To explore the role of gut microbes in host use and adaptability, we uncovered the mechanisms of phenol degradation by gut microbes using metagenomic and metatranscriptomic analyses, and verified the degradation ability of isolated and cultured bacteria. Research on this subject can help develop potential strain for the environmental friendly pest management operations. RESULTS: We demonstrated the ability of gut microbes in B. minax larvae to degrade phenols in unripe citrus. After antibiotic treatment, coniferyl alcohol and coumaric aldehyde significantly reduced the survival rate, body length and body weight of the larvae. The metagenomic and metatranscriptomic analyses in B. minax provided evidence for the presence of genes in bacteria and the related pathway involved in phenol degradation. Among them, Enterococcus faecalis and Serratia marcescens, isolated from the gut of B. minax larvae, played critical roles in phenol degradation. Furthermore, supplementation of E. faecalis and S. marcescens in artificial diets containing coniferyl alcohol and coumaric aldehyde increased the survival rate of larvae. CONCLUSION: In summary, our results provided the first comprehensive analysis of gut bacterial communities by high-throughput sequencing and elucidated the role of bacteria in phenol degradation in B. minax, which shed light on the mechanism underlying specialist insect adaption to host secondary metabolites via gut bacteria. © 2024 Society of Chemical Industry.

Identification of a new gustatory receptor BminGR59b tuned to host wax in a specialist, Bactrocera minax (Diptera: Tephritidae).

Host location plays a pivotal role in the coevolution between insects and plants, particularly for specialist insect herbivores with a limited host range. However, how specialists precisely select the appropriate site for oviposition through gustatory system remains elusive. In this study, we investigated the effects of the gustatory system on the host plant selection of a devastating pest in Citrus spp., Bactrocera minax, by conducting behavioral assays. Through genomic and transcriptomic data analysis as well as RNAi technology, we identified a novel gustatory receptor, BminGR59b, highly expressed in the forelegs of female B. minax, which played a critical role in host plant selection before oviposition decision. Additionally, our results encompassing heterologous expression in Sf9 cells and oviposition behavior assay revealed that n-eicosane is the ligand for BminGR59b. Finally, employing the dual luciferase reporter system alongside yeast one-hybrid techniques and RNAi, we verified that the transcription factor BminCEBP regulated the up-regulation of BminGR59b in sexually matured adults. These findings offer new insights into the close-range host fruit recognition and selection for oviposition in a specialist tephritid fruit fly B. minax, which also sheds light on the transcriptional regulation mechanisms underlying the gustatory-mediated oviposition in specialist herbivores for the first time.

Yeast mediates the interspecific interaction between introduced Bactrocera dorsalis and indigenous Bactrocera minax.

BACKGROUND: Host plant-microbe associations mediate interspecific interactions amongst herbivorous insects. However, this theory has rarely been ecologically verified in tephritid fruit flies. Research on this subject can not only help predict tephritid species invasion and occurrence patterns, but also develop potential novel lures for the control of the tephritid fruit fly pests. Recently, we observed mixed infestation of Bactrocera minax and Bactrocera dorsalis larvae in citrus orchards, which prompted us to explore the underlying mechanism. RESULTS: Following oviposition by B. minax, the yeast Pichia kluyveri translocated to and proliferated inside the citrus fruit. The level of d-limonene released from citrus fruits containing P. kluyveri was 27 times higher than that released from healthy fruits. Mature B. dorsalis females were attracted to d-limonene and oviposited into fruits previously infested by B. minax. Furthermore, the interspecific interaction between B. dorsalis and B. minax within the same fruit significantly decreased the number of surviving larvae and pupal weight in B. dorsalis, but its effect on B. minax was weaker. CONCLUSION: In the studied interspecific interaction, B. minax occupies the dominant position, implying ecological significance for this species in terms of consolidating its own niche and inhibiting the invasion of exotic species. To our best knowledge, this is the first report from both ecological and physiological perspectives on a symbiotic yeast mediating the interaction between B. minax and B. dorsalis through altering fruit volatiles. © 2022 Society of Chemical Industry.

Identification and expression profiles of gustatory receptor genes in Bactrocera minax larvae (Diptera: Tephritidae): Role of BminGR59f in larval growth.

Insects employ various types of gustatory receptors (GRs) to identify nutrient-rich food and avoid toxic substances. The larval gustatory system is the critical checkpoint for food acceptance or rejection. As a specialist herbivore, the larvae of Bactrocera minax feed only on unripe citrus fruits. However, how larvae use GRs to check and adapt to the secondary metabolites in unripe citrus fruits remains unknown. In this study, we first performed developmental expression profiles showing that most BminGRs genes were highly expressed in 1st and 2nd instar larvae and that tissue-specific expression indicated high expression of most BminGRs genes in the mouthparts of 2nd instar larvae. Furthermore, we found that silencing BminGR59f by RNA interference (RNAi) affected the growth of 2nd instar B. minax larvae. Hesperidin and naringin were screened as ligands of BminGR59f via RNAi and cell calcium imaging, and the combination of these two flavones increased the body weight of larvae. In summary, we identified a novel gustatory perception pattern in B. minax for detecting hesperidin and naringin, which boosted the growth of B. minax larvae. These results shed light on how specialist herbivores detect and adapt to host metabolites in adverse environments depending on larval GRs.

Behavioral and genomic divergence between a generalist and a specialist fly.

The evolution of feeding habits leads to speciation in insects. Bactrocera true fruit flies display diverse feeding habits across species. We combine behavioral and functional genomic studies to probe the divergence between the specialist B. minax and the generalist B. dorsalis. We find that both vision and olfaction contribute to their respective host preferences, with a dominant effect of vision over the olfaction in short range. Correspondingly, host location-related genes are significantly enriched in the phototransduction pathway, of which the long-wavelength rhodopsin confers the color preference in both species and has been subject to selection in the specialist. We also find a massive expansion of olfactory receptors in the generalist, along with signatures of conditional expression and positive selection. The phylogenetic context suggests an ancestrally important role of vision in the host location of Bactrocera, as well as the increased performance and plasticity of olfaction alongside the arising of generalism.

Mutation of Bdpaired induces embryo lethality in the oriental fruit fly, Bactrocera dorsalis.

BACKGROUND: Pair-rule genes were identified and named for their role in segmentation in animal embryos. Paired, a homolog of mammalian PAX3, acts as one of several pair-rule genes and is key in defining the boundaries of future parasegments and segments during insect embryogenesis. RESULTS: We cloned the paired gene from the oriental fruit fly, Bactrocera dorsalis, and then applied CRISPR/Cas9-mediated genome editing to investigate its physiological function in the embryonic stage of this pest. We identified one transcript for a paired homolog in B. dorsalis, which encodes a protein containing a Paired Box domain and a Homeobox domain. Phylogenetic analysis confirmed that the paired gene is highly conserved and the gene was highly expressed at the 12-14 h-old embryonic stage. Knock-out of Bdpaired led to lack of segment boundaries, cuticular deficiency, and embryonic lethality. Sequence analysis of the CRISPR/Cas9 mutants exhibited different insertion and deletions in the Bdpaired locus. In addition, the relative expression of Wingless (Wg) and Abdominal A (Abd-A) genes were significantly down-regulated in the Bdpaired mutant embryos. CONCLUSION: These results indicate that Bdpaired gene is critical for the embryonic development of B. dorsalis, and could be a novel molecular target for genetic-based pest management practices to combat this serious invasive pest. © 2019 Society of Chemical Industry.