Anna Karenina as a promoter of microbial diversity in the cosmopolitan agricultural pest Zeugodacus cucurbitae (Diptera, Tephritidae).

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 ß 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.

RNA virus diversity and prevalence in field and laboratory populations of melon fly throughout its distribution.

Insects have a rich diversity of RNA viruses that can either cause acute infections or persist in host populations without visible symptoms. The melon fly, Zeugodacus cucurbitae (Tephritidae) causes substantial economic losses through infestation of diverse cucurbit and other crops. Of Indomalayan origin, it is now established in many tropical regions of the world. The virome diversity of Z. cucurbitae is largely unknown across large parts of its distribution, including the Indian subcontinent. We have analysed three transcriptomes each of one field-collected and one laboratory-reared Z. cucurbitae population from Bangalore (India) and discovered genomes of ten putative RNA viruses: two sigmaviruses, one chimbavirus, one cripavirus, one noda-like virus, one nora virus, one orbivirus, one partiti-like virus, one sobemovirus and one toti-like virus. Analysis of the only available host genome of a Hawaiian Z. cucurbitae population did not detect host genome integration of the detected viruses. While all ten viruses were found in the Bangalore field population only seven were detected in the laboratory population, indicating that these seven may cause persistent covert infections. Using virus-specific RNA-dependent RNA polymerase gene primers, we detected nine of the RNA viruses with an overall low variant diversity in some but not all individual flies from four out of five Indian regions. We then screened 39 transcriptomes of Z. cucurbitae laboratory populations from eastern Asia (Guangdong, Hainan, Taiwan) and the Pacific region (Hawaii), and detected seven of the ten virus genomes. We found additional genomes of a picorna-like virus and a negev-like virus. Hawaii as the only tested population from the fly's invasive range only had one virus. Our study provides evidence of new and high RNA virus diversity in Indian populations within the original range of Z. cucurbitae, as well as the presence of persistent covert infections in laboratory populations. It builds the basis for future research of tephritid-associated RNA viruses, including their host effects, epidemiology and application potential in biological control.

Two heat shock cognate 70 genes involved in spermatogenesis regulate the male fertility of Zeugodacus cucurbitae, as potential targets for pest control.

The melon fly Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agricultural quarantine pest threatening fruit and vegetable production. Heat shock cognate 70 (Hsc70), which is a homolog of the heat shock protein 70 (Hsp70), was first discovered in mice testes and plays an important role in spermatogenesis. In this study, we identified and cloned five Hsc70 genes from melon fly, namely ZcHsc70_1/2/3/4/5. Phylogenetic analysis showed that these proteins are closely related to Hsc70s from other Diptera insects. Spatiotemporal expression analysis showed that ZcHsc70_1 and ZcHsc70_2 are highly expressed in Z. cucurbitae testes. Fluorescence in situ hybridization further demonstrated that ZcHsc70_1 and ZcHsc70_2 are expressed in the transformation and maturation regions of testes, respectively. Moreover, RNA interference-based suppression of ZcHsc70_1 or ZcHsc70_2 resulted in a significant decrease of 74.61% and 63.28% in egg hatchability, respectively. Suppression of ZcHsc70_1 expression delayed the transformation of sperm cells to mature sperms. Meanwhile, suppression of ZcHsc70_2 expression decreased both sperm cells and mature sperms by inhibiting the meiosis of spermatocytes. Our findings show that ZcHsc70_1/2 regulates spermatogenesis and further affects the male fertility in the melon fly, showing potential as targets for pest control in sterile insect technique by genetic manipulation of males.

Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

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 ß-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.

BdTTLL3B-mediated polyglycylation is involved in the spermatogenesis in Bactrocera dorsalis.

Polyglycylation is a post-translational modification that generates glycine side chains in the C-terminal domains of both α- and ß-tubulins. To date, the patterns and significance of polyglycylation across insect species remain largely unknown. The TTLL3B was thought to be a polyglycylase and be essential for polyglycylation in dipteran insects. In this study, the TTLL3B of Bactrocera dorsalis (BdTTLL3B) was identified and characterized. The BdTTLL3B expressed remarkably higher in adult males, especially in testes. The spatio-temporal patterns of polyglycylation were consistent with that of BdTTLL3B. Along with spermatogenesis, the intensity of polyglycylation was enhanced steadily and concentrated in elongated flagella. The expression of recombinant BdTTLL3B in Hela cells, which are genetically deficient in polyglycylation, catalyzed intracellular polyglycylation, validating the identity of BdTTLL3B as a polyglycylase. Knockout of BdTTLL3B significantly suppressed polyglycylation in testes and impaired male fertility, probably due to abnormal morphology of mitochondrial derivatives and over-accumulation of paracrystalline. Taken together, these findings indicated that the BdTTLL3B-mediated polyglycylation is involved in the spermatogenesis and play an important role in fertility of adult B. dorsalis. Therefore, the BdTTLL3B can be considered as a candidate target gene for the management of B. dorsalis, such as developing gene silencing/knockout-based sterile insect technology (SIT).

HSP gene superfamily in Aspongopus chinensis Dallas: unravelling identification, characterisation and expression patterns during diapause and non-diapause stages.

Aspongopus chinensis Dallas 1851, an insect of important economic value, faces challenges in artificial breeding due to mandatory diapause and limited access to wild resources. Heat shock proteins (Hsps) are thought to influence diapause in insects, but little is known about their role in A. chinensis during diapause. This study used genomic methods to identify 25 Hsp genes in A. chinensis, including two Hsp90, 14 Hsp70, four Hsp60 and five small Hsp genes, were located on seven chromosomes, respectively. The gene structures among the same families are relatively conserved. Meanwhile, the motif compositions and secondary structures of A. chinensis Hsps (AcHsps) were predicted. RNA-seq data and fluorescence quantitative PCR analysis showed that there were differences in the expression patterns of AcHsps in diapause and non-diapause stages, and AcHsp70-5 was significantly differentially expressed in both analysis, which was enriched in the pathway of response to hormone. All the results showed that Hsps play an important role in the diapause mechanism of A. chinensis. Our observations highlight the molecular evolution of the Hsp gene and their effect on diapause in A. chinensis.

Transcriptomic Identification and Characterization of Trehalose-6-Phosphate Synthase in Fat Body of the Oriental Fruit Fly, Bactrocera dorsalis.

The destructive agricultural pest oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), has been causing huge damage to the fruits and vegetable industry. Although many pertinent studies have been conducted on B. dorsalis, the functions of fat body still remain largely unknown. To this end, the comparative transcriptome analysis between fat body and carcass was performed in an attempt to provide insights into functions of fat body of B. dorsalis in the present study. A total of 1431 upregulated and 2511 downregulated unigenes were discovered in the fat body vs carcass comparison, respectively. The enrichment analysis of differentially expressed genes (DEG) revealed that most of the enriched pathways were related to metabolism. The reliability of DEG analysis was validated by qRT-PCR measurements of 12 genes in starch and sucrose metabolism pathway, including the trehalose-6-phosphate synthase (BdTPS) which was highly expressed in eggs, 5 d-old adults, and fat body. The RNAi of BdTPS significantly affected trehalose and chitin metabolism, larval growth, and larva-pupa metamorphosis. Collectively, the findings in this study enriched our understanding of fat body functions in metabolism and demonstrated the indispensable roles of BdTPS in trehalose-related physiological pathways.

Mitochondrial coding genes mediate insecticide tolerance in the oriental fruit fly, Bactrocera dorsalis (Hendel).

The oriental fruit fly, Bactrocera dorsalis (Hendel), an invasive insect pest infesting fruits and vegetables, possesses a remarkable capacity for environmental adaptation. The investigation of behind mechanisms of the stress adaptability in B. dorsalis holds significantly practical relevance. Previous studies on the molecular mechanism underlying stress resistance in B. dorsalis have predominantly focused on nuclear-coding genes, with limited exploration on organelle-coding genes. In this study, we assessed alterations in the mitochondrial physiological parameters of B. dorsalis under exposure to malathion, avermectin, and beta-cypermethrin at LD50 dosages. The results showed that all three insecticides were capable of reducing mitochondrial complex IV activity and ATP content. Expression patterns of mitochondrial coding genes across different developmental stages, tissues and insecticide exposures were analyzed by RT-qPCR. The results revealed that these mitochondrial coding genes were expressed in various tissues and at different developmental stages. Particularly noteworthy, atp6, cox2, and cytb exhibited substantial up-regulation in response to malathion and avermectin treatment. Furthermore, RNAi-mediated knockdown of atp6 and cox2 resulted in the increased toxicity of malathion and avermectin against B. dorsalis, and cox2 silencing was also associated with the decreased complex IV activity. These findings suggest that atp6 and cox2 most likely play pivotal roles in mediating tolerance or resistance to malathion and avermectin in B. dorsalis. Our results provide novel insights into the role of mitochondrial coding genes in conferring tolerance to insecticides in B. dorsalis, with practical implications for controlling this pest in the field.

Genome report: chromosome-scale genome assembly of the West Indian fruit fly Anastrepha obliqua (Diptera: Tephritidae).

The West Indian fruit fly, Anastrepha obliqua, is a major pest of mango in Central and South America and attacks more than 60 species of host fruits. To support current genetic and genomic research on A. obliqua, we sequenced the genome using high-fidelity long-read sequencing. This resulted in a highly contiguous contig assembly with 90% of the genome in 10 contigs. The contig assembly was placed in a chromosomal context using synteny with a closely related species, Anastrepha ludens, as both are members of the Anastrepha fraterculus group. The resulting assembly represents the five autosomes and the X chromosome which represents 95.9% of the genome, and 199 unplaced contigs representing the remaining 4.1%. Orthology analysis across the structural annotation sets of high quality tephritid genomes demonstrates the gene annotations are robust, and identified genes unique to Anastrepha species that may help define their pestiferous nature that can be used as a starting point for comparative genomics. This genome assembly represents the first of this species and will serve as a foundation for future genetic and genomic research in support of its management as an agricultural pest.

Suppressing the expression of glutathione S-transferase gene GSTd10 increases the sensitivity of Zeugodacus cucurbitae against ß-cypermethrin.

Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agriculturally and economically important pest worldwide that has developed resistance to ß-cypermethrin. Glutathione S-transferases (GSTs) have been reported to be involved in the detoxification of insecticides in insects. We have found that both ZcGSTd6 and ZcGSTd10 were up-regulated by ß-cypermethrin induction in our previous study, so we aimed to explore their potential relationship with ß-cypermethrin tolerance in this study. The heterologous expression of ZcGSTd6 and ZcGSTd10 in Escherichia coli showed significantly high activities against 1-chloro-2,4-dinitrobenzene (CDNB). The kinetic parameters of ZcGSTd6 and ZcGSTd10 were determined by Lineweaver-Burk. The Vmax and Km of ZcGSTd6 were 0.50 µmol/min·mg and 0.3 mM, respectively. The Vmax and Km of ZcGSTd10 were 1.82 µmol/min·mg and 0.53 mM. The 3D modelling and molecular docking results revealed that ß-cypermethrin exhibited a stronger bounding to the active site SER-9 of ZcGSTd10. The sensitivity to ß-cypermethrin was significantly increased by 18.73% and 27.21%, respectively, after the knockdown of ZcGSTd6 and ZcGSTd10 by using RNA interference. In addition, the inhibition of CDNB at 50% (IC50) and the inhibition constants (Ki) of ß-cypermethrin against ZcGSTd10 were determined as 0.41 and 0.33 mM, respectively. The Ki and IC50 of ß-cypermethrin against ZcSGTd6 were not analysed. These results suggested that ZcGSTd10 could be an essential regulator involved in the tolerance of Z. cucurbitae to ß-cypermethrin.

Distribution analysis of TRH in Bactrocera dorsalis using a CRISPR/Cas9-mediated reporter knock-in strain.

Although the study of many genes and their protein products is limited by the availability of high-quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene-editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene-targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology-directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single-stranded oligodeoxynucleotide donor. This one-step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate-limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin-mediated behaviour of B. dorsalis.

Odorant Binding Protein Expressed in Legs Enhances Malathion Tolerance in Bactrocera dorsalis (Hendel).

Bactrocera dorsalis is a highly invasive species and is one of the most destructive agricultural pests worldwide. Organophosphorus insecticides have been widely and chronically used to control it, leading to the escalating development of resistance. Recently, odorant binding proteins (OBPs) have been found to play a role in reducing insecticide susceptibility. In this study, we used RT-qPCR to measure the expression levels of four highly expressed OBP genes in the legs of B. dorsalis at different developmental stages and observed the effect of malathion exposure on their expression patterns. The results showed that OBP28a-2 had a high expression level in 5 day old adults of B. dorsalis, and its expression increased after exposure to malathion. By CRISPR/Cas9 mutagenesis, we generated OBP28a-2-/- null mutants and found that they were more susceptible to malathion than wild-type adults. Furthermore, in vitro direct affinity assays confirmed that OBP28a-2 has a strong affinity for malathion, suggesting that it plays a role in reducing the susceptibility of B. dorsalis to malathion. Our findings enriched our understanding of the function of OBPs. The results highlighted the potential role of OBPs as buffering proteins that help insects survive exposure to insecticides.

The hAT family hopper transposon exists as highly similar yet discontinuous elements in the Bactrocera tephritid fly genus.

The hAT family transposable element, hopper, was originally discovered as a defective 3120-bp full-length element in a wild-type strain of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), and subsequently a functional 3131-bp element, hopperBdwe, was isolated from a white eye mutant strain. The latter study showed that closely related elements exist in melonfly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), a closely related subgenus, suggesting that hopper could have a widespread presence in the Bactrocera genus. To further understand the distribution of hopper within and beyond the B. dorsalis species complex, primer pairs from hopperBdwe and its adjacent genomic insertion site were used to survey the presence and relatedness of hopper in five species within the complex and four species beyond the complex. Based on sequence identity of a 1.94 kb internal nucleotide sequence, the closest relationships were with mutated elements from B. dorsalis s.s. and species synonymized with B. dorsalis including B. papayae, B. philippinensis and B. invadens, ranging in identity between 88.4% and 99.5%. Notably, Bactrocera carambolae (Drew & Hancock) (Diptera: Tephritidae), which is most closely related to B. dorsalis beyond the synonymized species, shared hopper identities of 97.3%-99.5%. Beyond the B. dorsalis complex, Z. cucurbitae, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) and Bactrocera zonata (Saunders) (Diptera: Tephritidae) shared identities of 83.1%-97.1%, while hopper was absent from the Bactrocera oleae (Gmelin) (Diptera: Tephritidae) strain tested. While the functional autonomous hopperBdwe element was not detected in these species, another closely related hopper element isolated from a B. dorsalis genetic sexing strain has an uninterrupted transposase open reading frame. The discontinuous presence of hopper in the Bactrocera genus has implications for its use for genomic manipulation and understanding the phylogenetic relationship of these species.

A transposon-based genetic marker for conspecific identity within the Bactrocera dorsalis species complex.

Here we describe a molecular approach to assess conspecific identity that relies on the comparison of an evolved mutated transposable element sequence and its genomic insertion site in individuals from closely related species. This was explored with the IFP2 piggyBac transposon, originally discovered in Trichoplusia ni as a 2472 bp functional element, that was subsequently found as mutated elements in seven species within the Bactrocera dorsalis species complex. In a B. dorsalis [Hendel] strain collected in Kahuku, Hawaii, a degenerate 2420 bp piggyBac sequence (pBacBd-Kah) having ~ 94.5% sequence identity to IFP2 was isolated, and it was reasoned that common species, or strains within species, should share the same evolved element and its precise genomic insertion site. To test this assumption, PCR using primers to pBacBd-Kah and adjacent genomic sequences was used to isolate and compare homologous sequences in strains of four sibling species within the complex. Three of these taxa, B. papayae, B. philippinensis, and B. invadens, were previously synonymized with B. dorsalis, and found to share nearly identical pBacBd-Kah homologous elements (> 99% nucleotide identity) within the identical insertion site consistent with conspecific species. The fourth species tested, B. carambolae, considered to be a closely related yet independent species sympatric with B. dorsalis, also shared the pBacBd-Kah sequence and insertion site in one strain from Suriname, while another divergent pBacBd-Kah derivative, closer in identity to IFP2, was found in individuals from French Guiana, Bangladesh and Malaysia. This data, along with the absence of pBacBd-Kah in distantly related Bactrocera, indicates that mutated descendants of piggyBac, as well as other invasive mobile elements, could be reliable genomic markers for common species identity.

Bursicon regulates wing expansion via PKA in the oriental fruit fly, Bactrocera dorsalis.

BACKGROUND: Bursicon is a heterodimeric neuropeptide that is involved in many physiological activities such as cuticle tanning, wing expansion, reproduction and immunity in insects. In this study, the role of bursicon in the wing expansion was investigated in Bactrocera dorsalis, an important invasive insect pest in agriculture. RESULTS: The cDNA sequences and deduced amino acids of bursicon genes (named BdBurs-α and BdBurs-ß) were determined, and two proteins typically contained 11 cysteine residues in conserved positions that were highly conserved in other insect species. The spatiotemporal expressions of bursicon genes showed that higher expression occurred at the pupal, early adult stage and ovaries, and lower expression at the late larval stage and in wing tissue (8-day-old pupae). Dysfunction of bursicon genes by dsRNA microinjection into 5-day-old pupae reduced PKA (a downstream component of the bursicon pathway) activity and resulted in malformed adult wings. PKA inhibitor injection into 5-day-old pupae also resulted in similar phenotypes. Hematoxylin & eosin staining of the adult wing showed that RNAi and PKA inhibitor treatment reduced the thickness of the wing cuticle, which wing cuticle thickness were ≈50% thinner than in the control. Furthermore, the expression of hedgehog (Bdhh) (one of 10 tested genes related to wing development) was significantly upregulated after RNAi and PKA inhibitor application. CONCLUSION: The results indicate that bursicon plays a crucial role in the wing expansion of B. dorsalis, suggesting bursicon genes have potential to be the targets for B. dorsalis control. © 2023 Society of Chemical Industry.

Genomic signals of local adaptation across climatically heterogenous habitats in an invasive tropical fruit fly (Bactrocera tryoni).

Local adaptation plays a key role in the successful establishment of pest populations in new environments by enabling them to tolerate novel biotic and abiotic conditions experienced outside their native range. However, the genomic underpinnings of such adaptive responses remain unclear, especially for agriculturally important pests. We investigated population genomic signatures in the tropical/subtropical Queensland fruit fly, Bactrocera tryoni, which has an expanded range encompassing temperate and arid zones in Australia, and tropical zones in the Pacific Islands. Using reduced representation sequencing data from 28 populations, we detected allele frequency shifts associated with the native/invasive status of populations and identified environmental factors that have likely driven population differentiation. We also determined that precipitation, temperature, and geographic variables explain allelic shifts across the distribution range of B. tryoni. We found spatial heterogeneity in signatures of local adaptation across various climatic conditions in invaded areas. Specifically, disjunct invasive populations in the tropical Pacific Islands and arid zones of Australia were characterised by multiple significantly differentiated single nucleotide polymorphisms (SNPs), some of which were associated with genes with well-understood function in environmental stress (e.g., heat and desiccation) response. However, invasive populations in southeast Australian temperate zones showed higher gene flow with the native range and lacked a strong local adaptive signal. These results suggest that population connectivity with the native range has differentially affected local adaptive patterns in different invasive populations. Overall, our findings provide insights into the evolutionary underpinnings of invasion success of an important horticultural pest in climatically distinct environments.

Molecular and functional characterization of an antenna-enriched glutathione S-transferase BminGSTd3 involved in undecanol degradation in the citrus fruit fly, Bactrocera minax (Enderlein) (Diptera Tephritidae).

Bactrocera minax is a disastrous pest of citrus crops in China. Numerous studies focused on the molecular mechanism of odorant perception of B. minax, but the molecular mechanism of odorant degradation remains unclear. Glutathione S-transferases (GSTs) are considered as a class of odorant-degrading enzymes involved in degrading odorant molecules in insects' olfactory system. Here, we identified a delta-class GST gene, BminGSTd3, from B. minax. It was predominantly expressed in adult's olfactory organ antennae. The bacterially expressed recombinant BminGSTd3 was able to catalyze the conjugation of glutathione (GSH) with 2, 4-dinitrochlorobenzene (CDNB). Spectrophotometric analysis showed that undecanol can inhibit catalytic activities of BminGSTd3. Metabolic assays exhibited that undecanol can be depleted by BminGSTd3. Undecanol is believed to be an important B. minax sex pheromone component. The other components of the pheromone remain unclear. To understand how BminGSTd3 specifically recognizes undecanol, a 3D model of BminGSTd3 was constructed by homology modeling. Molecular docking based on this model revealed that E64 and S65 are the key amino acids recognizing undecanol, and this was proven by site-directed mutagenesis and intrinsic fluorescence assays. We suggest that BminGSTd3 is an undecanol metabolizing GST in B.minax, and E64 and S65 may serve as the key binding sites.

Comparative metatranscriptomics reveals effect of host plant on microbiota gene expression of Anastrepha obliqua (Diptera: Tephritidae) larvae.

The microbiota associated with phytophagous insects perform several functions that help insects exploit plant resources. Thus, microorganisms contribute to the dispersal of phytophagous species to new host plants, thereby promoting diversification. In this study, metatranscriptomic analysis was used to compare the gene expression of the microbiome of Anastrepha obliqua Macquart larvae feeding on 3 of its host plants: Spondias purpurea L (red mombin), Mangifera indica L (mango), and Averrhoa carambola L (starfruit). To identify differential gene expression in relation to the host plant, transcript abundance was compared. The results of the taxonomic and functional beta-diversity analysis showed that there were significant differences in the structures and activities of the microbial communities depending on the infested plant. Among the microorganisms, bacteria and fungi were active components of the microbiota. Differential expression analyses showed that the different active genes in each of the plants analyzed were mainly grouped into categories related to carbohydrate and amino acid metabolism, with some of these genes coding for cytochrome o ubiquinol oxidase, cytochrome c, and the enzyme isocitrate dehydrogenase. The microbiota of A. carambola larvae differed more at the level of community structure and gene function, possibly due to the different nutritional composition of the A. carambola and the presence of a set of secondary metabolites specific to the family Oxalidaceae. In conclusion, the transcriptional activity of the microbiota of A. obliqua larvae is influenced by diet, which is important because it could influence the performance of the insect on each of its different host plants.

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.

Overexpression of ABCB transporter genes confer multiple insecticide tolerances in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

Bactrocera dorsalis is a notable invasive pest that has developed resistance to several commonly used insecticides in the field, such as avermectin, beta-cypermethrin and malathion. Investigating the mechanisms of insecticide resistance in this pest is of paramount importance for ensuring its effective control. The ATP-binding cassette transporter subfamily B (ABCB) genes, responsible for encoding transmembrane efflux transporters, represent a potential source of insecticide detoxification activity or transportation that remains largely unexplored in B. dorsalis. In this study, seven BdABCB genes were identified and comprehensive analyzed based on the latest genome and transcriptome dataset. Subsequently, we characterized the expression profiles of these genes across different development stages and tissues, as well as under different insecticide exposures. The results showed that the BdABCB genes were expressed at all stages in B. dorsalis, with BdABCB2 and BdABCB7 being highly expressed in the pupal stage, while BdABCB5 and BdABCB6 were highly expressed in the larval stage. Besides, the BdABCBs were highly expressed in the detoxification metabolic tissues. Among them, BdABCB5 and BdABCB6 were significantly overexpressed in the midgut and Malpighian tubules, respectively. Furthermore, with the exception of BdABCB6, the expression levels of the other six BdABCBs were significantly up-regulated following induction with avermectin, beta-cypermethrin and malathion. Six BdABCBs (BdABCB1-5 and BdABCB7) were knocked down by RNA interference, and the interference efficiencies were 46.58%, 39.50%, 45.60%, 33.74%, 66.37% and 63.83%, respectively. After injecting dsBdABCBs, the mortality of flies increased by 25.23% to 39.67% compared to the control upon exposure to the three insecticides. These results suggested that BdABCBs play crucial roles in the detoxification or tolerance of B. dorsalis to multiple insecticides.