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Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata , and Zeugodacus cucurbitae , a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp − mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni . The conserved phenotype and independent nature of wp − mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests. The white pupae (wp) phenotype has been used for decades to selectively remove females of tephritid species in genetic sexing, but the determining gene is unknown. Here, the authors show that wp phenotype is produced by parallel mutations in a Major Facilitator Superfamily domain containing gene across multiple species.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap “n” collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to β-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance. Graphical Abstract Graphical Abstract

Many organisms enter a dormant state in their life cycle to deal with predictable changes in environments over the course of a year. The timing of dormancy is therefore a key seasonal adaptation, and it evolves rapidly with changing environments. We tested the hypothesis that differences in the timing of seasonal activity are driven by differences in the rate of development during diapause in Rhagoletis pomonella, a fly specialized to feed on fruits of seasonally limited host plants. Transcriptomes from the central nervous system across a time series during diapause show consistent and progressive changes in transcripts participating in diverse developmental processes, despite a lack of gross morphological change. Moreover, population genomic analyses suggested that many genes of small effect enriched in developmental functional categories underlie variation in dormancy timing and overlap with gene sets associated with development rate in Drosophila melanogaster. Our transcriptional data also suggested that a recent evolutionary shift from a seasonally late to a seasonally early host plant drovemore rapid development during diapause in the early fly population. Moreover, genetic variants that diverged during the evolutionary shift were also enriched in putative cis regulatory regions of genes differentially expressed during diapause development. Overall, our data suggest polygenic variation in the rate of developmental progression during diapause contributes to the evolution of seasonality in R. pomonella. We further discuss patterns that suggest hourglass-like developmental divergence early and late in diapause development and an important role for hub genes in the evolution of transcriptional divergence

An autosomal chromosome pair in Drosophila , the dot chromosome, is shown to have evolved from an ancestral X chromosome: these findings explain several previously puzzling aspects of dot chromosome biology and challenge the view that differentiated sex chromosomes represent a terminal evolutionary stage. A former sex chromosome unmasked Sex-determination mechanisms are known to evolve frequently in species with identical sex chromosomes, but distinct sex chromosomes (such as X and Y) are seen as a terminal evolutionary stage resulting from chromosome-specific adaptations such as dosage compensation or an accumulation of sex-specific mutations. Using whole-genome sequencing and comparative genomics, Beatriz Vicoso and Doris Bachtrog show that an autosomal pair in Drosophila , the dot chromosome, evolved from an X chromosome. Their results explain several previously puzzling aspects of the biology of the Drosophila dot chromosome as remnants of its former life as a sex chromosome. Although transitions of sex-determination mechanisms are frequent in species with homomorphic sex chromosomes 1 , 2 , 3 , heteromorphic sex chromosomes are thought to represent a terminal evolutionary stage owing to chromosome-specific adaptations such as dosage compensation or an accumulation of sex-specific mutations 1 , 4 . Here we show that an autosome of Drosophila , the dot chromosome, was ancestrally a differentiated X chromosome. We analyse the whole genome of true fruitflies (Tephritidae), flesh flies (Sarcophagidae) and soldier flies (Stratiomyidae) to show that genes located on the dot chromosome of Drosophila are X-linked in outgroup species, whereas Drosophila X-linked genes are autosomal. We date this chromosomal transition to early drosophilid evolution by sequencing the genome of other Drosophilidae. Our results reveal several puzzling aspects of Drosophila dot chromosome biology to be possible remnants of its former life as a sex chromosome, such as its minor feminizing role in sex determination 5 or its targeting by a chromosome-specific regulatory mechanism 6 . We also show that patterns of biased gene expression of the dot chromosome during early embryogenesis, oogenesis and spermatogenesis resemble that of the current X chromosome. Thus, although sex chromosomes are not necessarily evolutionary end points and can revert back to an autosomal inheritance, the highly specialized genome architecture of this former X chromosome suggests that severe fitness costs must be overcome for such a turnover to occur.

Gut microbial communities are critical in determining the evolutive success of fruit fly phytophagous pests (Diptera, Tephritidae), facilitating their adaptation to suboptimal environmental conditions and to plant allelochemical defences. An important source of variation for the microbial diversity of fruit flies is represented by the crop on which larvae are feeding. However, a “crop effect” is not always the main driver of microbial patterns, and it is often observed in combination with other and less obvious processes. In this work, we aim at verifying if environmental stress and, by extension, changing environmental conditions, can promote microbial diversity in Zeugodacus cucurbitae (Coquillett), a cosmopolitan pest of cucurbit crops. With this objective, 16S rRNA metabarcoding was used to test differences in the microbial profiles of wild fly populations in a large experimental setup in Eastern Central Tanzania. The analysis of 2,973 unique ASV, which were assigned to 22 bacterial phyla, 221 families and 590 putative genera, show that microbial α diversity (as estimated by Abundance Coverage Estimator, Faith’s Phylogenetic Diversity, Shannon-Weiner and the Inverse Simpson indexes) as well as β microbial diversity (as estimated by Compositional Data analysis of ASVs and of aggregated genera) significantly change as the species gets closer to its altitudinal limits, in farms where pesticides and agrochemicals are used. Most importantly, the multivariate dispersion of microbial patterns is significantly higher in these stressful environmental conditions thus indicating that Anna Karenina effects contribute to the microbial diversity of Z . cucurbitae . The crop effect was comparably weaker and detected as non-consistent changes across the experimental sites. We speculate that the impressive adaptive potential of polyphagous fruit flies is, at least in part, related to the Anna Karenina principle, which promotes stochastic changes in the microbial diversity of fly populations exposed to suboptimal environmental conditions.

Background True fruit flies (Diptera: Tephritidae) are among the most destructive pests of fruit and vegetables worldwide and are on the top of quarantine pest lists. To respond effectively to a fruit fly invasion, we need to identify the species rapidly and reliably to understand its biological features and guide response decisions. Molecular techniques have been used to improve the diagnostic ability circumventing many difficulties of morphological identification. However, the commonly used Cytochrome Oxidase I ( COI ) gene lacks sufficient variation to distinguish species within Bactrocera species complexes. Here we conducted mitochondrial genome sequencing to identify additional genetic markers that could aid diagnosis of Bactrocera fruit fly species. Results We assembled 82 complete mitochondrial genomes from 16 Bactrocera species, including 13 species for which no mitochondrial genome data were previously available, as well as one species each from Dacus aneuvittatus , Dirioxa pornia and Zeugodacus gracilis . Phylogenetic analysis of the Tephritidae family confirmed the monophyly of the Bactrocera genus but could not properly resolve species within species complexes. Comparative mitochondrial genome analysis revealed that intergenic spacer and NADH dehydrogenase genes, specifically ND2 and ND6 , harbour enough variations for new specific real-time PCR assays. Based on these findings, six TaqMan-based real-time PCR assays targeting ND2 , COI , and CO3 genes were successfully designed and assessed for their specificity and sensitivity in detecting Bactrocera curvipennis , a member of the B. tryoni complex. Of these, one real-time PCR assay targeting the ND2 gene proved to be the most specific and sensitive. It detects B. curvipennis specifically at the level of 1 copy/µL of target DNA. Conclusions Mitochondrial sequence analysis and comparative studies indicate that mitochondrial genomes offer valuable genetic markers for accurate diagnosis of Bactrocera fruit flies. The successful development of the B. curvipennis real-time PCR assay highlights the importance of having additional genetic markers to advance the molecular diagnostics in economically important Bactrocera species.

Background The Tephritidae family, commonly referred to as true fruit flies, comprises of a substantial group within order Diptera. Numerous species within this family are major agricultural pests, with a tendency to infest a wide array of fruits and vegetables in tropical and sub- tropical regions, leading to considerable damage and consequent reductions in the market value of the crops. Methods and results The current study was aimed to propose a promising solution to the menace posed by fruit flies by offering rapid, accurate and reliable species identification by using character-based DNA barcode methodology. The Tephritid specimens were collected from Cucurbitaceous plants of southern parts of West Bengal, India, and a total of eight species from Tephritidae family were obtained belonging to three genera, namely Bactrocera (Macquart, 1835), Dacus (Fabricius, 1805) and Zeugodacus (Hendel, 1927). Their morphological features were meticulously studied based on available literature, along with genetic analysis based on mitochondrial COI and ND1 gene sequences. A total of 30 uniquely variable sites at nucleotide position 42,48,51,60,66,72, 105,111,144,198,207,243, 273,297,307,318,345,357, 375,378,381,387,399,400, 402,436,444,450,453 and 460 in COI gene were discerned among Tephritid species in the present study. Conclusions The character-based DNA barcode holds the potential to differentiate closely related species of fruit flies and morphologically look-a-like ones. The novel method will be very significant in terms of rapid, precise and reliable species identification and might be extremely essential for early detection during pest outbreaks by facilitating timely intervention strategies to mitigate crop damage.

The guts of metazoans are in permanent contact with the microbial realm that includes beneficial symbionts, nonsymbionts, food-borne microbes and life-threatening pathogens. However, little is known concerning how host immunity affects gut bacterial community. Here, we analyze the role of a dual oxidase gene ( BdDuox ) in regulating the intestinal bacterial community homeostasis of the oriental fruit fly Bactrocera dorsalis . The results showed that knockdown of BdDuox led to an increased bacterial load, and to a decrease in the relative abundance of Enterobacteriaceae and Leuconostocaceae bacterial symbionts in the gut. The resulting dysbiosis, in turn, stimulates an immune response by activating BdDuox and promoting reactive oxygen species (ROS) production that regulates the composition and structure of the gut bacterial community to normal status by repressing the overgrowth of minor pathobionts. Our results suggest that BdDuox plays a pivotal role in regulating the homeostasis of the gut bacterial community in B. dorsalis .

Tephritidae is an economically important family among Diptera that also exhibits high diversity, biogeographical distribution, and different lifestyles. Despite the recent release of genomes and mitochondrial genome sequences of various species of the family, the evolutionary history of the group and the origin of host adaptation within it remain poorly understood. We undertook a whole-mitochondrial-genome study covering molecular variation at the mitochondrial level by analyzing 10 new mitochondrial genomes obtained from genomic data reported and downloaded from the SRA database from NCBI, analyzed in FastQC and assembled through MITGARD, and 44 mitogenomes available in the Organelle—Refseq database, in total representing 4 subfamilies, 9 tribes, 13 genera, and 54 species. We determined compositional asymmetry and codon usage patterns across the different subfamilies analyzed by using DNASp6 and CAICal. We found high evolutionary rates in the NADH genes, which could play an important role in the adaptation of species to different hosts and environmental variation. By using maximum likelihood phylogenetic reconstruction obtained by IQTREE and ModelFinder, and lifestyle and distribution data of the included species, we considered a generalist feature, explained as possible predominant adaptation in some members of the family. This study in Tephritidae tries to demonstrate possible patterns among molecular variability in mitogenomes, adaptations, and lifestyles. Our findings suggest that selection pressures on certain NADH genes may be linked to host specificity in some Tephritidae species, providing evolutionary insights into how molecular evolution drives ecological adaptation or biogeographical diversity, probably in response to changing environmental conditions and host–parasite co-evolution across taxa.

Abstract Bactrocera dorsalis (Hendel, 1912), a major invasive pest, survives under extreme climates through molecular and tissue-specific cold stress adaptations. In this study, we investigated the tissue-specific impacts of cold stress on the survival and molecular response of B. dorsalis. Results showed that cold stress had a significant effect on survival rates. The Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that signaling and metabolic pathways were activated by cold stress in the head and fat body during a transcriptome analysis. Under cold stress, 184 and 365 genes were differentially expressed in the head and fat body, respectively. RNA interference (RNAi)-mediated knockdown of transposon Ty3-I Gag-Pol polyprotein (Ty3-I) and Ty3-G Gag-Pol polyprotein (Ty3-G) in the head and fat body, significantly reduced the larval survival. Relative expression analysis revealed that expression of the Ty3-I and Ty3-G Gag-Pol polyprotein was greatly reduced in the head of cold treated larvae relative to controls (dsGFP) and that the expression level of Ty3-I Gag-Pol polyprotein in the fat body was not significantly reduced by cold stress. These results highlight the tissue-specific response of Ty3-I and Ty3-G Gag-Pol polyproteins in mediating cold stress responses and aid in understanding their importance in survival and stress adaptation. Additionally, the identification of important stress-responsive genes provides a foundation for the development of RNAi-based strategies for pest control using the targeted disruption of stress adaptation gene pathways for more effective control of B. dorsalis populations.