Bactrocera zonata is a highly invasive agricultural pest that causes extensive damage to fruit crops. The Sterile Insect Technique (SIT), a species-specific and environmentally friendly pest control method, significantly benefits from the availability of Genetic Sexing Strains (GSSs) that enable efficient mass production of males for sterile release. However, no GSS currently exists for B. zonata limiting SIT applications targeting this important invasive pest. Here, we report two key advancements toward GSS development in this species. First, we present a high-quality, chromosome-level genome assembly from male B. zonata, identifying two scaffolds derived from the Y chromosome, which represent potential targets for future male-specific genetic engineering. Second, we demonstrate the feasibility of CRISPR/Cas9 genome editing in B. zonata by generating stable, homozygous white-eye mutants through targeted disruption of the conserved white-eye gene. This visible, recessive phenotype serves as a proof-of-concept for developing selectable markers in this species. Together, these results provide foundational genomic and genetic tools to support the development of GSSs in B. zonata, advancing the potential for sustainable, genetics-based pest control strategies.

Anastrepha fraterculus is a cryptic species complex with at least eight morphotypes distributed across the Americas. Among them, A. fraterculus sp.1, present in Argentina, is a major pest impacting fresh fruit production. Integrated pest management strategies, including chemical control and trapping, are currently employed to mitigate its effects. Genetic sexing strains of A. fraterculus sp.1 are being evaluated for use in sterile insect technique programs. To support traditional and emerging control methods, this study aimed to enhance the genomic understanding of this morphotype. Individual female and male samples were sequenced using long- and short-read technologies. The female genome (760 Mb) was de novo assembled into 58 scaffolds and the male genome (750 Mb) into 68 scaffolds, with BUSCO completeness scores of 98.8% and 98.7%, respectively. Synteny analysis revealed complete scaffolds of the five autosomes and enabled near-complete reconstruction of the X and Y chromosomes. Gene prediction identified 17 751 and 16 535 protein-coding genes (for female and male genomes, respectively), with repetitive regions representing 46% of both genomes. Additionally, the mitochondrial genome was fully assembled and annotated. This comprehensive genomic resource reveals candidate genes for functional studies, including gene editing and RNA interference, as successfully applied in related tephritid species. These findings lay the foundation for innovative, complementary biocontrol tools against A. fraterculus.

The sterile insect technique (SIT) is a highly effective biologically-based method for the suppression of many insect pest populations. SIT efficacy could be improved by methods of male sterilization that avoid the use of irradiation that can result in diminished fitness and mating competitiveness. Alternative sterilization methods include conditional disruption of genes for male fertility, or using their sperm-specific promoters to drive the expression of genes for lethal effectors. Testing has begun for the testis-specific β2-tubulin gene, though additional male fertility genes are required for redundancy or replacement, and for species where the β2-tubulin isoform does not exist or is not testis-specific. Here we had the goal of identifying and characterizing the sequence and transcriptional expression of two genes in the caribfly, Anastrepha suspensa, that are cognates of D. melanogaster spermatocyte-specific male fertility genes. In Drosophila, wampa encodes a coiled-coil dynein subunit required for axonemal assembly essential to microtubule-based sperm motility, while Prosα6T is a proteasome subunit gene required for spermatid individualization and nuclear maturation. In A. suspensa a cognate to wampa exhibited testis-specific transcript expression, which was minimal in both male and female body tissue. A Prosα6T cognate was not apparent in A. suspensa, but its constitutive isoform, Prosα6, expresses in male testes, but also in male and female body tissue. Thus, for A. suspensa, wampa remains a strong candidate gene for male sterility strategies for SIT including a direct target for gene-editing knockout and use of its promoter for testis-specific toxicity or cell death in conditional expression systems.

Gut commensal bacteria promote host reproduction by modulating metabolism and nutrition, but the molecular mechanisms by which microbes regulate reproduction remain unclear. Here, we show that gut commensal bacteria promote host reproduction by providing the amino acid methionine, which controls the RNA m6A modification level of insulin receptor (InR) in the ovary of the invasive insect Bactrocera dorsalis. Antibiotic-treated B. dorsalis shows reduced RNA m6A methylation levels and methionine content, resulting in arrested ovarian development and decreased fecundity. The gut commensal bacterium Enterobacter hormaechei-derived metabolite methionine restores the decreased RNA m6A level and the reproductive defects. Notably, the knockdown of METTL3 and METTL14, two genes encoding the RNA m6A methyltransferases, reduces InR mRNA and protein levels and impairs ovarian development in B. dorsalis. Our findings further expand the functional landscape of RNA m6A modification to include nutrient-dependent control of ovarian development and highlight the essential role of epigenetic regulation in microbe-host interactions.

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.

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.

Genetic biocontrol approaches to insect pest management offer high species specificity and reduce insecticidal chemical pollution, but these approaches require the release of genetically modified organisms that must perform across a variety of environmental conditions. Because organisms often display phenotypic and gene expression variability when exposed to different environments, it is reasonable to expect that transgenic systems may be prone to environmentally mediated variation in expression and function. In this study, we examined the influence of two abiotic variables, temperature and nutrition, on the penetrance of an early embryonic Tet-off conditional lethality system in the vinegar fly, Drosophila melanogaster. We manipulated parental and offspring environments independently to determine the impact of life stage of exposure on transgene performance by estimating the probability of larval hatching and measuring transcript abundance of the transgenic system components. Our findings revealed that: 1) transgene performance has distinct norms of reaction to temperature and nutrition, 2) the effects of abiotic challenge are greatest when exposure occurs in the embryos expressing the transgene compared to parental exposure, and 3) stress exposures at the extreme limits of permissible conditions can dramatically decrease the penetrance of transgenic lethality. While variation in transcript abundance of the transgenic system was observed in some environments, these changes were not fully congruent with patterns of phenotypic penetrance, suggesting that the observed variation in lethality is likely driven by processes downstream of transcription.

The sterile insect technique (SIT) is a highly effective biologically-based method for the population suppression of highly invasive insect pests of medical and agricultural importance. The efficacy of SIT could be significantly enhanced, however, by improved methods of male sterilization that avoid the fitness costs of irradiation. An alternative sterilization method is possible by gene-editing that targets genes essential for sperm maturation and motility, rendering them nonfunctional, similar to the CRISPR-Cas9 targeting of β2-tubulin in the genetic model system, Drosophila melanogaster. However, since genetic strategies for sterility are susceptible to breakdown or resistance in mass-reared populations, alternative targets for sterility are important for redundancy or strain replacement. Here we have identified and characterized the sequence and transcriptional expression of two genes in a Florida strain of Drosophila suzukii, that are cognates of the D. melanogaster spermatocyte-specific genes wampa and Prosalpha6T. Wampa encodes a coiled-coil dynein subunit required for axonemal assembly, and the proteasome subunit gene, Prosalpha6T, is required for spermatid individualization and nuclear maturation. The reading frames of these genes differed from their NCBI database entries derived from a D. suzukii California strain by 44 and 8 nucleotide substitutions/polymorphisms, respectively, though all substitutions were synonymous resulting in identical peptide sequences. Expression of both genes is predominant in the male testis, and they share similar transcriptional profiles in adult males with β2-tubulin. Their amino acid sequences are highly conserved in dipteran species, including pest species subject to SIT control, supporting their potential use in targeted male sterilization strategies.

Cotesia flavipes Cameron is an important larval parasitoid exploited for the control of the spotted stem borer, Chilo partellus (Swinhoe). Several studies have evaluated the toxic effects of insecticides on C. partellus, however, little is known about non-target effects of insecticides on this parasitoid, when used to control C. partellus. This laboratory study evaluated the lethal and sublethal effects of twelve insecticides on C. flavipes. Residual toxicity tests showed that organophosphates (chlorpyrifos, triazophos and profenofos) exhibited highest contact toxicity to C. flavipes adults with a LC50 range from 0.63 to 1.05 mg a.i/l, while neonicotinoids (nitenpyram, acetamiprid and imidacloprid) were less toxic to C. flavipes with a LC50 range from 1.27 to 139.48 mg a.i/l. Sugar-insecticide feeding bioassays showed that organophosphates, pyrethroids (cypermethrin, bifenthrin and lambda-cyhalothrin) and carbamates (thiodicarb, carbaryl and methomyl) were highly toxic to C. flavipes adults and caused 100% mortality at 48 h of exposure, while imidacloprid caused 66% mortality at 48 h of exposure. Risk quotient analysis showed that among all tested insecticides, imidacloprid and acetamiprid were less toxic to C. flavipes adults with a risk quotient value of 0.88 and 1.6, respectively. Furthermore, exposure of immature C. flavipes through their host bodies significantly decreased the parasitism rate at their F1 and F2 generations. Risk quotient analysis of insecticides indicated that imidacloprid and acetamiprid were the least toxic to C. flavipes. This study provides important information that will be used in incorporating the most suitable insecticides in integrated pest management programs with reduced negative impacts on non-target beneficial arthropods.

The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. Unfortunately, irradiation in normal atmospheric conditions can be damaging for males, because it generates substantial oxidative stress that, combined with mass-rearing conditions, may reduce their sexual competitiveness and quality. In this study, oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate the role of oxidative stress and cellular antioxidants in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se , increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

One of the more effective means of controlling insect pest populations without the use of noxious agents has been the sterile insect technique (SIT) originally proposed by Knipling (1955). This method has proven effective in eradicating several insect pests, notably the screwworm fly from Mexico and the Mediterranean fruit fly from Mexico and Guatemala (Snow 1988). At present, programs are being evaluated to target other insects, including various tephritid fruitflies, mosquitoes, the boll weevil, and lepidopterans. In general, SIT takes advantage of the fact that for insect females that mate once or twice in a breeding season or lifetime, mating with a sterile male results in inviable progeny. Upon large-scale sterile-male release, indigenous pest populations can either be eradicated within several generations or finally eradicated when initially suppressed by insecticides.

The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. However, irradiation in normal atmospheric conditions can be damaging for males, because irradiation generates substantial biological oxidative stress that, combined with domestication and mass-rearing conditions, may reduce sterile male sexual competitiveness and quality. In this study, biological oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate their role in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited only a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se, increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

Genetically modified conditional lethal strains have been created to improve the control of insect pest populations damaging to human health and agriculture. However, understanding the potential for the genetic breakdown of lethality systems by rare spontaneous mutations, or selection for inherent suppressors, is critical since field release studies are in progress. This knowledge gap was addressed in a Drosophila tetracycline-suppressible embryonic lethality system by analyzing the frequency and structure of primary-site spontaneous mutations and second-site suppressors resulting in heritable survivors from 1.2 million zygotes. Here we report that F 1 survivors due to primary-site deletions and indels occur at a 5.8 × 10 −6 frequency, while survival due to second-site maternal-effect suppressors occur at a ~10 −5 frequency. Survivors due to inherent lethal effector suppressors could result in a resistant field population, and we suggest that this risk may be mitigated by the use of dual redundant, albeit functionally unrelated, lethality systems.

Regulation of male sexual differentiation by a Y chromosome-linked male determining factor (M-factor) is one of a diverse array of sex determination mechanisms found in insects. By deep sequencing of small RNAs from Bactrocera dorsalis early embryos, we identified an autosomal-derived microRNA, miR-1-3p, that has predicted target sites in the transformer gene ( Bdtra ) required for female sex determination. We further demonstrate by both in vitro and in vivo tests that miR-1-3p suppresses Bdtra expression. Injection of a miR-1-3p mimic in early embryos results in 87–92% phenotypic males, whereas knockdown of miR-1-3p by an inhibitor results in 67–77% phenotypic females. Finally, CRISPR/Cas9-mediated knockout of miR-1-3p results in the expression of female-specific splice variants of Bdtra and doublesex ( Bddsx ), and induced sex reversal of XY individuals into phenotypic females. These results indicate that miR-1-3p is required for male sex determination in early embryogenesis in B. dorsalis as an intermediate male determiner.

Background Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. Results Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. Conclusions These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

BackgroundThe hopper hAT-family transposable element isolated from the Oriental fruit fly, Bactrocera dorsalis, is distantly related to both the Drosophila hobo element and the Activator element from maize. The original 3120bp hopper(Bd-Kah) element isolated from the Kahuku wild-type strain was highly degenerate and appeared to have a mutated transposase and terminal sequences, while a second 3131bp element, hopper(Bd-we), isolated from a white eye mutant strain had an intact transposase reading frame and terminal sequences consistent with function.ResultsThe hopper(Bd-we) element was tested for function by its ability to mediate germline transformation in two dipteran species other than B. dorsalis. This was achieved by creating a binary vector/helper transformation system by linking the hopper(Bd-we) transposase reading frame to a D. melanogaster hsp70 promoter for a heat-inducible transposase helper plasmid, and creating vectors marked with the D. melanogaster mini-white(+) or polyubiquitin-regulated DsRed fluorescent protein markers.ConclusionsBoth vectors were successfully used to transform D. melanogaster, and the DsRed vector was also used to transform the Caribbean fruit fly, Anastrepha suspensa, indicating a wide range of hopper function in dipteran species and, potentially, non-dipteran species. This vector provides a new tool for insect genetic modification for both functional genomic analysis and the control of insect populations.

: The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most devastating and highly invasive agricultural pests world-wide, resulting in severe economic loss. Thus, it is of great interest to understand the transcriptional changes that occur during the activation of its zygotic genome at the early stages of embryonic development, especially the expression of genes involved in sex determination and the cellularization processes. In this study, we applied Illumina sequencing to identify B. dorsalis sex determination genes and early zygotic genes by analyzing transcripts from three early embryonic stages at 0–1, 2–4, and 5–8 h post-oviposition, which include the initiation of sex determination and cellularization. These tests generated 13,489 unigenes with an average length of 2185 bp. In total, 1683, 3201 and 3134 unigenes had significant changes in expression levels at times after oviposition including at 2–4 h versus 0–1 h, 5–8 h versus 0–1 h, and 5–8 h versus 2–4 h, respectively. Clusters of gene orthology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed throughout embryonic development to better understand the functions of differentially expressed unigenes. We observed that the RNA binding and spliceosome pathways were highly enriched and overrepresented during the early stage of embryogenesis. Additionally, transcripts for 21 sex-determination and three cellularization genes were identified, and expression pattern analysis revealed that the majority of these genes were highly expressed during embryogenesis. This study is the first assembly performed for B. dorsalis based on Illumina next-generation sequencing technology during embryogenesis. Our data should contribute significantly to the fundamental understanding of sex determination and early embryogenesis in tephritid fruit flies, and provide gene promoter and effector gene candidates for transgenic pest-management strategies for these economically important species.

CRISPR/Cas9-mediated gene-editing, using injected Cas9 protein, was achieved in the Caribbean fruit fly, Anastrepha suspensa, by initially targeting an exogenous transgene, polyubiquitin-regulated EGFP (PUb-EGFP), for heritable non-homologous end-joining (NHEJ) knock-outs using an individual sgRNA. Multiple deletion mutations, ranging from two to five nts proximal to the target site, were identified phenotypically by the loss of green fluorescence in transgenic flies that were also marked with PUb-DsRed. This represented a relatively high efficiency rate of 29% for germ-line mutations. Similar conditions were then used to target an endogenous sex-determination gene, As-transformer-2 (Astra-2), using two sgRNAs that targeted independent exon sequences 671 bp apart. Somatic mutations were identified phenotypically in G(0) adult flies at a frequency of 81% based upon intersexual genital morphology, expected to occur only in XX females since Astra-2 knock-outs by dsRNA do not have a phenotypic effect in XY males. Consistent with this expectation, twelve types of short indels, ranging from -15 nts to +5 nts, were identified proximal to the 5' sgRNA-1 target site in intersexual adults. However, the 3' sgRNA-2 target was only associated with a single 774 bp deletion extending from the sgRNA-1 target site to 100 bp downstream of the sgRNA-2 target. This is encouraging for the eventual use of dual target sites for homology-directed repair (HDR) insertions, but suggests that the sgRNA-2 target site tested may not be optimal for Astra-2 HDR modification.