Chromosome-level genome assembly of an agricultural pest Zeugodacus tau (Diptera: Tephritidae).

The fruit fly Zeugodacus tau (Diptera: Tephritidae) is a major pest of melons and other cucurbits in Southeast Asia. In this study, we used Illumina, Nanopore, and Hi-C sequencing technologies to assemble a reference genome of Z. tau at the chromosomal level. The assembled genome was 421.79 Mb and consisted of six chromosomes (one X-chromosome + five autosomes). The contig N50 was 4.23 Mb. We identified 20,922 protein-coding genes, of which 17,251 (82.45%) were functionally annotated. Additionally, we found 247 rRNAs, 435 tRNAs, 67 small nuclear RNAs, and 829 small RNAs in the genome. Repetitive elements accounted for 55.30 Mb (13.15%) of the genome. This high-quality genome assembly is valuable for evolutionary and genetic studies of Z. tau and its relative species.

Next-generation genetic sexing strain establishment in the agricultural pest Ceratitis capitata.

Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male-female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique.

RNAi-mediated knockdown of α-Spectrin depresses reproductive performance in female Bactrocera dorsalis.

The female reproductive potential plays a crucial role in reproduction, population dynamics and population maintenance. However, the function of endogenous genes in undifferentiated germ cells has been largely unknown in Bactrocera dorsalis. In this study, the conservative analysis showed that α-Spectrin shared a similarity in B. dorsalis and other dipteral flies. Further, the differential expression of α-Spectrin was examined in B. dorsalis by RT-qPCR, and the expression pattern of α-Spectrin protein was identified in female adult ovaries by using immunostaining. During the development of ovary, the change on the number of undifferentiated germ cells was also characterized and analyzed. To understand the function of α-Spectrin in B. dorsalis ovary, the RNAi-based knockdown was conducted, and the RNAi efficiency was examined by RT-qPCR, western blot and bioassay. The results revealed that the α-Spectrin dsRNA could strikingly decrease the expression level of α-Spectrin in ovaries and diminish oviposition and ovary size as a consequence of downregulation of α-Spectrin. Overall, our study facilitates reproductive research on the function of conservative genes in B. dorsalis ovary, which may provide a new insight into seeking novel target genes for pest management control.

Female semiochemicals stimulate male courtship but dampen female sexual receptivity.

Chemical communication plays a vital role in mate attraction and discrimination among many insect species. Here, we document a unique example of semiochemical parsimony, where four chemicals act as both aphrodisiacs and anti-aphrodisiacs in different contexts in Bactrocera dorsalis. Specifically, we identified four female-specific semiochemicals, ethyl laurate, ethyl myristate, ethyl cis-9-hexadecenoate, and ethyl palmitate, which serve as aphrodisiacs to attract male flies and arouse male courtship. Interestingly, these semiochemicals, when sexually transferred to males during mating, can function as anti-aphrodisiacs, inhibiting the receptivity of subsequent female mates. We further showed that the expression of elongase11, a key enzyme involved in the biosynthesis of these semiochemicals, is under the control of doublesex, facilitating the exclusive biosynthesis of these four semiochemicals in females and guaranteeing effective chemical communication. The dual roles of these semiochemicals not only ensure the attractiveness of mature females but also provide a simple yet reliable mechanism for female mate discrimination. These findings provide insights into chemical communication in B. dorsalis and add elements for the design of pest control programs.

Immature stages of Utetes anastrephae (Hymenoptera: Braconidae) developed in Anastrepha fruit fly larvae (Diptera: Tephritidae).

The morphology of the immature stages of Utetes anastrephae (Hymenoptera: Braconidae), a native parasitoid of larvae of flies of the Neotropical genus Anastrepha (Diptera: Tephritidae), is shown. This study aimed to characterize the immature stages and morphological changes in the development of the koinobiont endoparasitoid in two species of larval hosts, Anastrepha obliqua and Anastrepha ludens. The definition of structures and morphological changes during development was made through daily microscopic observations and photographs of dissected hosts. The immature development of the parasitoid corresponds to a holometabolous insect with three well-defined stages: egg (two days), larva with three larval instars (approximately eight days), and pupa (six days). Similar development times were obtained in the two host species. Males and females completed their cycle in 17 and 18 days, respectively. During egg-first instar development, host antagonistic activity through melanization and encapsulation as mortality factors was evident and frequent only in A. obliqua. These results serve as basic knowledge for the use of this parasitoid in the biological control of fruit flies.

Future habitat changes of Bactrocera minax Enderlein along the Yangtze River Basin using the optimal MaxEnt model.

Background: Bactrocera minax (Enderlein, 1920) (Diptera: Tephritidae) is a destructive citrus pest. It is mainly distributed throughout Shaanxi, Sichuan, Chongqing, Guizhou, Yunnan, Hubei, Hunan, and Guangxi in China and is considered to be a second-class pest that is prohibited from entering that country. Climate change, new farming techniques, and increased international trade has caused the habitable area of this pest to gradually expand. Understanding the suitable habitats of B. minax under future climate scenarios may be crucial to reveal the expansion pattern of the insect and develop corresponding prevention strategies in China. Methods: Using on the current 199 distribution points and 11 environmental variables for B. minax, we chose the optimal MaxEnt model to screen the dominant factors that affect the distribution of B. minax and to predict the potential future distribution of B. minax in China under two shared socio-economic pathways (SSP1-2.6, SSP5-8.5). Results: The current habitat of B. minax is located at 24.1-34.6°N and 101.1-122.9°E, which encompasses the provinces of Guizhou, Sichuan, Hubei, Hunan, Chongqing, and Yunnan (21.64 × 104 km2). Under future climate scenarios, the potential suitable habitat for B. minax may expand significantly toward the lower-middle reaches of the Yangtze River. The land coverage of highly suitable habitats may increase from 21.64 × 104 km2 to 26.35 × 104 × 104 km2 (2050s, SSP5-8.5) ~ 33.51 × 104 km2 (2090s, SSP5-8.5). This expansion area accounts for 29% (2050s, SSP1-2.6) to 34.83% (2090s, SSP1-2.6) of the current habitat. The center of the suitable habitat was predicted to expand towards the northeast, and the scenario with a stronger radiative force corresponded to a more marked movement of the center toward higher latitudes. A jackknife test showed that the dominant variables affecting the distribution of B. minax were the mean temperature of the driest quarter (bio9), the annual precipitation (bio12), the mean diurnal range (bio2), the temperature annual range (bio7), and the altitude (alt). Discussion: Currently, it is possible for B. minax to expand its damaging presence. Regions with appropriate climate conditions and distribution of host plants may become potential habitats for the insects, and local authorities should strengthen their detection and prevention strategies. Climate changes in the future may promote the survival and expansion of B. minax species in China, which is represented by the significant increase of suitable habitats toward regions of high altitudes and latitudes across all directions but with some shrinkage in the east and west sides.

Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of white gene in melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) produced white eye phenotype.

Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNA to target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.

Chromosome-level genome assembly of an important wolfberry fruit fly (Neoceratitis asiatica Becker).

Tephritidae pests are significant agricultural pests with a notable impact on the economy, with a wide range of species and most having broad host ranges and strong reproductive abilities. However, the wolfberry fruit fly, Neoceratitis asiatica (Becker), is a Tephritidae fly which only harms wolfberry. Here, we assembled and annotated N. asiatica genome at the chromosome level and compared it with the genomic and transcriptomic information from other Tephritidae flies. The assembled genome of N. asiatica had a size of 563.8 Mb and achieved a completeness level of 99.1%, 18,387 genes were annotated totally. All contigs were assembled into 7 linkage groups with an N50 of 93.166 Mb assisted by the Hi-C technique. The high-quality genome developed here will provide a significant resource for exploring the genetic basis of the adaptive and reproductive differences among various Tephritidae pests, and provides an important theoretical basis for the prevention and control of Tephritidae pests.

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.

Unique and generic crossed metabolism in response to four sub-lethal environmental stresses in the oriental fruit fly, Bactrocera dorsalis Hendel.

Bactrocera dorsalis is a well-known invasive pest that causes considerable ecological and economic losses worldwild. Although it has a wide environmental tolerance, few studies have reported its mechanism of adaptation to multiple sub-lethal environmental stresses. In this study, 38, 41, 39 and 34 metabolites changed significantly in B. dorsalis under four sub-lethal stresses (heat, cold, desiccation and hypoxia), as found by the metabolomic method. Therein, lactic acid and pyruvic acid were induced, whereas metabolites in the tricarboxylic acid (TCA) cycle such as citric acid, α-ketoglutarate acid, malic acid and fumaric acid were reduced under at least one of the stresses. Enzyme activity and quantitative polymerase chain reaction (qPCR) analyses verified the repression of pyruvic acid proceeding into the TCA cycle. In addition, the levels of several cryoprotectants and membrane fatty acids in B. dorsalis were altered. The findings indicated that B. dorsalis has evolved shared metabolic pathways to adapt to heat, hypoxia and desiccation stresses, such as reducing energy consumption by activating the anaerobic glycolytic metabolism. Cryoprotectants and membrane fatty acids were produced to improve the efficiency of stress resistance. This study revealed the unique and generic crossed physiological mechanism of insects to adapt to various environmental stresses.

Growth inhibitory, immunosuppressive, cytotoxic, and genotoxic effects of γ-terpinene on Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae).

γ-Terpinene, a monoterpene widely present in essential oils of many medicinal and aromatic plants with numerous biological properties, was evaluated for its insecticidal activity against melon fruit fly, Zeugodacus cucurbitae (Coquillett). Different concentrations (5, 25, 125, 625, and 3125 ppm) of γ-terpinene along with control were fed to larvae of melon fly. The number of pupae formed and adults emerged declined significantly after treatment. Morphologically deformed adults and pupae were also observed. The developmental duration too prolonged in treated larvae. Food assimilated, mean relative growth rate, larval weight gain, and pupal weight also declined. In the larvae treated with LC30 and LC50 concentrations, there was a decline in the titers of phenoloxidase and total hemocyte count, and variations were observed in the differential hemocyte count, suggesting an immunosuppressive effect of γ-terpinene on melon fly. Both concentrations also led to an increase in the apoptotic and necrotic cells as well as decrease in the viable hemocytes in the circulating hemolymph of treated larvae. Comet parameters (tail length, % tail DNA, tail moment, and olive tail moment) of γ-terpinene fed larvae increased significantly. Given the observed effects of γ-terpinene on normal developmental and nutritional physiology, its immunosuppressive properties, and its potential for genome damage, it can be considered for incorporation into integrated pest management strategies for controlling Z. cucurbitae.

Population mixing mediates the intestinal flora composition and facilitates invasiveness in a globally invasive fruit fly.

BACKGROUND: Changes in population heterozygosity and genetic diversity play important roles in mediating life history traits of organisms; these changes often lead to phenotypic evolution in offspring, which become superior to their parents. In the present study, we examined phenotypic differentiation, the intestinal microbiome composition, and metabolism shift in the oriental fruit fly (Bactrocera dorsalis) by comparing an inbred (monophyletic) original population and an outbred (mixed) invasive population. RESULTS: The results showed that the outbred population of B. dorsalis had significantly higher biomass, adult longevity, and fecundity than the inbred population. Additionally, intestinal microflora analysis revealed that both Diutina rugosa and Komagataeibacter saccharivorans were significantly enriched in the outbred population with higher genetic heterozygosity. D. rugosa enrichment altered amino acid metabolism in the intestinal tract, and supplementing essential amino acids (e.g. histidine and glutamine) in the diet led to an increase in pupal weight of the outbred population. Additionally, transcriptome analysis revealed that the HSPA1S gene was significantly downregulated in the outbred population. HSPA1S was involved in activation of the JNK-MAPK pathway through negative regulation, caused the upregulation of juvenile hormone (JH), and led to an increase in biomass in the outbred flies. CONCLUSION: In conclusion, the outbred population had an altered intestinal microbe composition, mediating metabolism and transcriptional regulation, leading to phenotypic differentiation; this may be a potential mechanism driving the global invasion of B. dorsalis. Thus, multiple introductions could lead to invasiveness enhancement in B. dorsalis through population mixing, providing preliminary evidence that changes in the intestinal microbiome can promote biological invasion. Video Abstract.

Natural Oil Lure Outperforms Trimedlure in Capturing Males of the Mediterranean Fruit Fly, Ceratitis capitata (Diptera: Tephritidae).

Females of certain tephritid fruit fly species (Diptera: Tephritidae) pose an enormous agricultural threat, as they oviposit in commercially important fruits and vegetables. Trapping networks are often operated in fruit fly-free areas to detect incipient infestations. Trapping relies largely on male attractants, so-called male lures, with trimedlure (TML) being used to detect invasive Ceratitis spp. Operating large-scale surveillance programs incurs substantial costs for both supplies and labor, and the problem is exacerbated by the fact that trimedlure (as well as other male lures) is effective for relatively short intervals in the field (6-8 weeks). Because frequent servicing increases costs, there is considerable interest in modifying existing lures or developing new formulations to extend their effective field longevity. Here, we present results of a field study in Hawaii on a wild population of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), that compared male captures in traps baited with (i) fresh liquid TML, (ii) TML plugs, (iii) a novel controlled-release TML sachet, and (iv) a novel natural oil blend dispensed from a sachet. Catch was recorded weekly for 12 weeks and then at 16 and 20 weeks, with 12 traps deployed per treatment. The natural oil formulation, which contains the natural plant product α-copaene, was as effective as the fresh liquid TML even after weathering for 20 weeks. Future work will focus on developing a dispenser for this formulation that is compatible with standard trap design and deployment.

Immunomodulatory, cyto-genotoxic, and growth regulatory effects of nerolidol on melon fruit fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae).

Insects have evolved a robust immune system consisting of humoral and cellular branches and their orchestrated response enables insect to defend against exogenous stressors. Exploration of underlying immune mechanisms of insect pest under allelochemical stress can give us new insights on insect pest management. In this study, nerolidol, a plant sesquiterpene was evaluated for its insecticidal, growth regulatory, immunomodulatory, and cyto-genotoxic effects against melon fruit fly, Zeugodacus cucurbitae (Coquillett). First, second, and third instar larvae of Z. cucurbitae were fed on artificial diet containing different concentrations (5, 25, 125, 625, and 3125 ppm) of nerolidol. Results revealed a significant reduction in pupation and adult emergence as well as prolongation of developmental duration of treated larvae. Decline in growth indices showed remarkable growth inhibitory effects of nerolidol. Pupal weight and nutritional parameters viz. Larval weight gain, food assimilated, and mean relative growth rate declined after treatment. Immunological studies on second instar larvae depicted a drop in total hemocyte count and variations in proportions of plasmatocytes and granulocytes of LC30 and LC50 treated larvae. Phenoloxidase activity in nerolidol treated larvae initially increased but was suppressed after 72 h of treatment. The frequency of viable hemocytes decreased and that of apoptotic and necrotic hemocytes increased with both the lethal concentrations of nerolidol. Comet assay revealed a significant damage to DNA of hemocytes. The findings of the current study indicate that nerolidol exerts its insecticidal action through growth regulation, immunomodulation, and cyto-genotoxicity thus revealing its potential to be used as biopesticide against Z. cucurbitae.

Insecticidal potential of endophytic Streptomyces sp. against Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) and biosafety evaluation.

Fruit flies of Tephritidae family pose a serious threat to cultivation of fruits and vegetables across the world. Among them, melon fruit fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) is a devastating pest of plants from Cucurbitaceae family. In a rising concern about the harmful effects associated with the use of chemical insecticides and development of resistance in pest insects, safer pest management strategies such as, use of biopesticides of microbial origin are being contemplated. Therefore, the present study aimed to evaluate the insecticidal potential of Streptomyces sp. SP5 protein extract against Z. cucurbitae. MTT assay, Ames mutagenicity, DNA nicking, and comet assay were conducted to determine the biosafety of protein extract. Second instar larvae of Z. cucurbitae were treated with various concentrations (1, 100, 200, 300, 400, and 500 µg/ml) of Streptomyces sp. SP5 protein extract. The protein extract showed significant larvicidal effects with LC50 value of 308.92 µg/ml. The percentage of adults emerged declined with increase in concentration. There was significant prolongation in developmental durations of the larvae. Various morphological aberrations in the form of deformed adults and pupae and decline in pupal weight were also observed. The nutritional physiology of the treated larvae was also adversely affected. The results from biosafety evaluation revealed antimutagenic and non-toxic nature of Streptomyces sp. proteins. This study indicates that Streptomyces sp. SP5 has the potential to be used as an ecologically safe biocontrol agent against Z. cucurbitae.

Differences in rectal amino acid levels determine bacteria-originated sex pheromone specificity in two closely related flies.

Sex pheromones are widely used by insects as a reproductive isolating mechanism to attract conspecifics and repel heterospecifics. Although researchers have obtained extensive knowledge about sex pheromones, little is known about the differentiation mechanism of sex pheromones in closely related species. Using Bactrocera dorsalis and Bactrocera cucurbitae as the study model, we investigated how the male-borne sex pheromones are different. The results demonstrated that both 2,3,5-trimethylpyrazine (TMP) and 2,3,5,6-tetramethylpyrazine (TTMP) were sex pheromones produced by rectal Bacillus in the two flies. However, the TMP/TTMP ratios were reversed, indicating sex pheromone specificity in the two flies. Bacterial fermentation results showed that different threonine and glycine levels were responsible for the preference of rectal Bacillus to produce TMP or TTMP. Accordingly, threonine (glycine) levels and the expression of the threonine and glycine coding genes were significantly different between B. dorsalis and B. cucurbitae. In vivo assays confirmed that increased rectal glycine and threonine levels by amino acid feeding could significantly decrease the TMP/TTMP ratios and result in significantly decreased mating abilities in the studied flies. Meanwhile, decreased rectal glycine and threonine levels due to RNAi of the glycine and threonine coding genes was found to significantly increase the TMP/TTMP ratios and result in significantly decreased mating abilities. The study contributes to the new insight that insects and their symbionts can jointly regulate sex pheromone specificity in insects, and in turn, this helps us to better understand how the evolution of chemical communication affects speciation.

Gut bacterial population and community dynamics following adult emergence in pest tephritid fruit flies.

Gut microbiota are important contributors to insect success. Host-microbe interactions are dynamic and can change as hosts age and/or encounter different environments. A turning point in these relationships the transition from immature to adult life stages, particularly for holometabolous insects where there is radical restructuring of the gut. Improved knowledge of population and community dynamics of gut microbiomes upon adult emergence inform drivers of community assembly and physiological aspects of host-microbe interactions. Here, we evaluated the bacterial communities of the pest tephritid species melon fly (Zeugodacus cucurbitae) and Medditeranean fruit fly (medfly, Ceratitis capitata) associated with the pupae life stage and timepoints immediately following adult eclosion. We used a combination of culturing to determine cultivatable bacterial titers, qPCR to determine 16S-rRNA SSU copy numbers, and 16S V4 sequencing to determine changes in communities. Both culturing and qPCR revealed that fly bacterial populations declined upon adult emergence by 10 to 100-fold followed by recovery within 24 h following eclosion. Titers reached ~ 107 CFUs (~ 108 16S rRNA copies) within a week post-emergence. We also observed concurrent changes in amplicon sequence variance (ASVs), where the ASV composition differed overtime for both melon fly and medfly adults at different timepoints. Medfly, in particular, had different microbiome compositions at each timepoint, indicating greater levels of variation before stabilization. These results demonstrate that tephritid microbiomes experience a period of flux following adult emergence, where both biomass and the makeup of the community undergoes dramatic shifts. The host-microbe dynamics we document suggest plasticity in the community and that there may be specific periods where the tephritid gut microbiome may be pliable to introduce and establish new microbial strains in the host.

Knockdown of the ecdysone receptor disrupts development and causes mortality in the melon fly, Zeugodacus cucurbitae.

Cucurbits are important economic plants that are attacked by numerous pests, among which the melon fly Zeugodacus cucurbitae is extremely problematic. New sustainable pest control strategies are necessary to replace chemical insecticides that are harmful to the environment, human health and nontarget species. The RNA interference (RNAi) technology is one of the most promising tools due to high efficiency and species specificity. We developed an RNAi strategy targeting the ecdysone receptor (ECR) of Z. cucurbitae, which plays an important role in moulting and reproduction. We identified, described and isolated the ECR gene of Z. cucurbitae and measured its expression pattern across developmental stages and tissues. ZcECR knockdown via dsZcECR ingestion caused a significant larval mortality and abnormal phenotypes in pupae and adults. About 68% of larvae fed with a dsZcECR-treated diet failed to enter the pupal stage and died. In addition, ZcECR knockdown dramatically reduced pupal weight (by 3.24 mg on average) and fecundity (by about 23%). RNAi targeting the ECR gene is therefore a promising method to control Z. cucurbitae, paving the way for the development of novel sustainable and highly specific control strategies.

Profiles of Volatiles Emitted from Orange Fruits Infested by Ceratitis capitata (Diptera: Tephritidae).

The profiles of volatile organic compounds (VOCs) emitted from orange fruits infested by Ceratitis capitata were examined. VOCs were collected from the headspace of oranges immediately, 24 h, 5 days, 8 days, and 13 days after oviposition and when larvae exit the fruit by applying either static or dynamic sampling techniques. A total of 32 and 47 compounds were detected in infested orange fruits when using static and dynamic techniques, respectively. Differences in the volatile profile of oranges were observed. Classification models were employed, showing that infested fruits emitted a chemical profile distinct from that of non-infested ones. Limonene was associated with cell disruption. (E)-ß-Ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene, hexyl butanoate, butyl hexanoate, and hexyl hexanoate were the VOCs that were correlated with the infestation of sweet oranges by C. capitata.

Functional analysis of an overexpressed glutathione S-transferase BdGSTd5 involved in malathion and malaoxon detoxification in Bactrocera dorsalis.

Glutathione S-transferases (GSTs) are one of the three detoxification enzyme families. The constitutive and inducible overexpression of GSTs genes plays an important role in insecticide resistance. Previous study showed that malathion resistance was polygenic, and elevated GSTs activity was one of the important factor participating in malathion resistance of Bactrocera dorsalis (Hendel), a serious economic pest worldwide. BdGSTd5 overexpression was inducible upon exposure to malathion. However, the involvement of BdGSTd5 in malathion resistance has not been clarified. In this study, we found that BdGSTd5 sequence harbored the conserved region of delta class GSTs, which were overexpressed in malathion resistant strain of B. dorsalis compared to malathion susceptible strain. The highest mRNA expression level of BdGSTd5 was found in 1-day-old adult, and the levels decreased with aging. The dsBdGSTd5 injection effectively silenced (73.4% reduction) the expression of BdGSTd5 and caused significant increase in susceptibility to malathion with a cumulative mortality increasing of 13.5% at 72 h post malathion treatment (p < 0.05). Cytotoxicity assay demonstrated that BdGSTd5 was capable of malathion detoxification. Molecular docking analysis further indicated the interactive potential of BdGSTd5 with malathion and its toxic oxide malaoxon. The recombinant BdGSTd5 exhibited glutathione-conjugating activity toward 1-chloro-2, 4-dinitrobenzene and malathion and malaoxon metabolic capacity with significant reduction (p < 0.05) of the peak areas by 90.0% and 73.1%, respectively. Taken together, the overexpressed BdGSTd5 contributes to malathion metabolism and resistance, which detoxify the malathion in B. dorsalis via directly depleting malathion and malaoxon.