The impact of fruit fly gut bacteria on the rearing of the parasitic wasp <i>Diachasmimorpha longicaudata</i>

Area-wide integrated pest management strategies against tephritid fruit flies include the release of fruit fly parasitic wasps in the target area. Mass rearing of parasitic wasps is essential for the efficient application of biological control strategies. Enhancement of fruit fly host fitness through manipulation of their gut-associated symbionts might also enhance the fitness of the produced parasitic wasps and improve the parasitoid rearing system. In the current study, we added three gut bacterial isolates originating from Ceratitis capitata (Wiedemann) and four originating from Bactrocera oleae (Rossi) (both Diptera: Tephritidae) to the larval diet of C. capitata and used the bacteria-fed larvae as hosts for the development of the parasitic wasp Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae). We evaluated the effect of the bacteria on wasp life-history traits and assessed their potential use for the improvement of D. longicaudata rearing. Enterobactersp. AA26 increased fecundity and parasitism rate and accelerated parasitoid emergence. Providencia sp. AA31 led to faster emergence of both male and female parasitoids, whereas Providencia sp. 22 increased the production of female progeny. Bacillus sp. 139 increased parasitoid fecundity, parasitism rate, and production of female progeny. Serratia sp. 49 accelerated parasitoid emergence for both males and females and increased production of female progeny. Klebsiella oxytoca delayed parasitoid emergence and Enterobacter sp. 23 decreased parasitoid fecundity and parasitism rate. Our findings demonstrate a wide range of effects of fruit fly gut symbionts on parasitoid production and reveal a great potential of bacteria use towards enhancement of parasitic wasp rearing

Manipulation of insect gut microbiota towards the improvement of <i>Bactrocera oleae</i> artificial rearing

Bactrocera oleae (Rossi) (Diptera: Tephritidae) is the main pest of olive trees (Olea europaeaL.), causing major damages in olive crops. Improvement of mass rearing is a prerequisite for the successful development of large-scale sterile insect technique (SIT) applications. This can be achieved through the enrichment of artificial diets with gut bacteria isolates. We assessed the efficiency of three gut bacteria previously isolated fromCeratitis capitata(Wiedemann), and four isolated fromB. oleae, as larval diet additives in both live and inactivated/dead forms. Our results showed that deadEnterobactersp. AA26 increased pupal weight, whereas both live and dead cells increased pupal and adult production and reduced immature developmental time, indicating that its bacterial cells serve as a direct nutrient source. LiveProvidenciasp. AA31 improved pupal and adult production, enhanced male survival under stress conditions, and delayed immature development. DeadProvidenciasp. AA31, however, did not affect production rates, indicating that live bacteria can colonize the insect gut and biosynthesize nutrients essential for larval development. Live and deadBacillussp. 139 increased pupal weight, accelerated immature development, and increased adult survival under stress. Moreover, liveBacillussp. 139 improved adult production, indicating thatBacilluscells are a direct source of nutrients. DeadSerratiasp. 49 increased pupal and adult production and decreased male survival under stress conditions whereas live cells decreased insect production, indicating that the live strain is entomopathogenic, but its dead cells can be utilized as nutrient source.Klebsiella oxytoca,Enterobactersp. 23, andProvidenciasp. 22 decreased pupal and subsequent adult production and were harmful forB. oleae. Our findings indicate that deadEnterobactersp. AA26 is the most promising bacterial isolate for the improvement ofB. oleaemass rearing in support of future SIT or related population suppression programs

Isolation and characterization of microsatellite markers from the olive fly, Bactrocera oleae, and their cross-species amplification in the Tephritidae family

<p>Abstract</p> <p>Background</p> <p>The Tephritidae family of insects includes the most important agricultural pests of fruits and vegetables, belonging mainly to four genera (<it>Bactrocera, Ceratitis, Anastrepha </it>and <it>Rhagoletis</it>). The olive fruit fly, <it>Bactrocera oleae</it>, is the major pest of the olive fruit. Currently, its control is based on chemical insecticides. Environmentally friendlier methods have been attempted in the past (Sterile Insect Technique), albeit with limited success. This was mainly attributed to the lack of knowledge on the insect's behaviour, ecology and genetic structure of natural populations. The development of molecular markers could facilitate the access in the genome and contribute to the solution of the aforementioned problems. We chose to focus on microsatellite markers due to their abundance in the genome, high degree of polymorphism and easiness of isolation.</p> <p>Results</p> <p>Fifty-eight microsatellite-containing clones were isolated from the olive fly, <it>Bactrocera oleae</it>, bearing a total of sixty-two discrete microsatellite motifs. Forty-two primer pairs were designed on the unique sequences flanking the microsatellite motif and thirty-one of them amplified a PCR product of the expected size. The level of polymorphism was evaluated against wild and laboratory flies and the majority of the markers (93.5%) proved highly polymorphic. Thirteen of them presented a unique position on the olive fly polytene chromosomes by <it>in situ </it>hybridization, which can serve as anchors to correlate future genetic and cytological maps of the species, as well as entry points to the genome. Cross-species amplification of these markers to eleven Tephritidae species and sequencing of thirty-one of the amplified products revealed a varying degree of conservation that declines outside the <it>Bactrocera </it>genus.</p> <p>Conclusion</p> <p>Microsatellite markers are very powerful tools for genetic and population analyses, particularly in species deprived of any other means of genetic analysis. The presented set of microsatellite markers possesses all features that would render them useful in such analyses. This could also prove helpful for species where SIT is a desired outcome, since the development of effective SIT can be aided by detailed knowledge at the genetic and molecular level. Furthermore, their presented efficacy in several other species of the Tephritidae family not only makes them useful for their analysis but also provides tools for phylogenetic comparisons among them.</p

De novo assembly of the olive fruit fly (Bactrocera oleae) genome with linked-reads and long-read technologies minimizes gaps and provides exceptional Y chromosome assembly

Background: The olive fruit fly, Bactrocera oleae, is the most important pest in the olive fruit agribusiness industry. This is because female flies lay their eggs in the unripe fruits and upon hatching the larvae feed on the fruits thus destroying them. The lack of a high-quality genome and other genomic and transcriptomic data has hindered progress in understanding the fly’s biology and proposing alternative control methods to pesticide use. Results: Genomic DNA was sequenced from male and female Demokritos strain flies, maintained in the laboratory for over 45 years. We used short-, mate-pair-, and long-read sequencing technologies to generate a combined male-female genome assembly (GenBank accession GCA_001188975.2). Genomic DNA sequencing from male insects using 10x Genomics linked-reads technology followed by mate-pair and long-read scaffolding and gap-closing generated a highly contiguous 489 Mb genome with a scaffold N50 of 4.69 Mb and L50 of 30 scaffolds (GenBank accession GCA_001188975.4). RNA-seq data generated from 12 tissues and/or developmental stages allowed for genome annotation. Short reads from both males and females and the chromosome quotient method enabled identification of Y-chromosome scaffolds which were extensively validated by PCR. Conclusions: The high-quality genome generated represents a critical tool in olive fruit fly research. We provide an extensive RNA-seq data set, and genome annotation, critical towards gaining an insight into the biology of the olive fruit fly. In addition, elucidation of Y-chromosome sequences will advance our understanding of the Y-chromosome’s organization, function and evolution and is poised to provide avenues for sterile insect technique approaches

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control

Decoding the Reproductive System of the Olive Fruit Fly, Bactrocera oleae

In most diploid organisms, mating is a prerequisite for reproduction and, thus, critical to the maintenance of their population and the perpetuation of the species. Besides the importance of understanding the fundamentals of reproduction, targeting the reproductive success of a pest insect is also a promising method for its control, as a possible manipulation of the reproductive system could affect its destructive activity. Here, we used an integrated approach for the elucidation of the reproductive system and mating procedures of the olive fruit fly, Bactrocera oleae. Initially, we performed a RNAseq analysis in reproductive tissues of virgin and mated insects. A comparison of the transcriptomes resulted in the identification of genes that are differentially expressed after mating. Functional annotation of the genes showed an alteration in the metabolic, catalytic, and cellular processes after mating. Moreover, a functional analysis through RNAi silencing of two differentially expressed genes, yellow-g and troponin C, resulted in a significantly reduced oviposition rate. This study provided a foundation for future investigations into the olive fruit fly’s reproductive biology to the development of new exploitable tools for its control

Development of Toehold Switches as a Novel Ribodiagnostic Method for West Nile Virus

West Nile virus (WNV) is an emerging neurotropic RNA virus and a member of the genus Flavivirus. Naturally, the virus is maintained in an enzootic cycle involving mosquitoes as vectors and birds that are the principal amplifying virus hosts. In humans, the incubation period for WNV disease ranges from 3 to 14 days, with an estimated 80% of infected persons being asymptomatic, around 19% developing a mild febrile infection and less than 1% developing neuroinvasive disease. Laboratory diagnosis of WNV infection is generally accomplished by cross-reacting serological methods or highly sensitive yet expensive molecular approaches. Therefore, current diagnostic tools hinder widespread surveillance of WNV in birds and mosquitoes that serve as viral reservoirs for infecting secondary hosts, such as humans and equines. We have developed a synthetic biology-based method for sensitive and low-cost detection of WNV. This method relies on toehold riboswitches designed to detect WNV genomic RNA as transcriptional input and process it to GFP fluorescence as translational output. Our methodology offers a non-invasive tool with reduced operating cost and high diagnostic value that can be used for field surveillance of WNV in humans as well as in bird and mosquito populations

FISH with the BoR300 probe on mitotic and polytene nuclei of <i>Bactrocera oleae.</i>

<p>Chromosomes were counterstained with DAPI (blue). Female (a) and male (b) metaphase showing strong hybridization signals (red) on the centromeres of chromosomes 4 and 5. Polytene complement (c) showing strong hybridization signals (red) on the centromeric heterochromatic blocks (C) of chromosomes III and IV (arrows). Bar  = 3 µm (a, b), 20 µm (c).</p

Long non-coding RNAs regulate Aedes aegypti vector competence for Zika virus and reproduction.

Long non-coding RNAs (lncRNAs) play critical regulatory roles in various cellular and metabolic processes in mosquitoes and all other organisms studied thus far. In particular, their involvement in essential processes such as reproduction makes them potential targets for the development of novel pest control approaches. However, their function in mosquito biology remains largely unexplored. To elucidate the role of lncRNAs in mosquitoes' reproduction and vector competence for arboviruses, we have implemented a computational and experimental pipeline to mine, screen, and characterize lncRNAs related to these two biological processes. Through analysis of publicly available Zika virus (ZIKV) infection-regulated Aedes aegypti transcriptomes, at least six lncRNAs were identified as being significantly upregulated in response to infection in various mosquito tissues. The roles of these ZIKV-regulated lncRNAs (designated Zinc1, Zinc2, Zinc3, Zinc9, Zinc10 and Zinc22), were further investigated by dsRNA-mediated silencing studies. Our results show that silencing of Zinc1, Zinc2, and Zinc22 renders mosquitoes significantly less permissive to ZIKV infection, while silencing of Zinc22 also reduces fecundity, indicating a potential role for Zinc22 in trade-offs between vector competence and reproduction. We also found that silencing of Zinc9 significantly increases fecundity but has no effect on ZIKV infection, suggesting that Zinc9 may be a negative regulator of oviposition. Our work demonstrates that some lncRNAs play host factor roles by facilitating viral infection in mosquitoes. We also show that lncRNAs can influence both mosquito reproduction and permissiveness to virus infection, two biological systems with important roles in mosquito vectorial capacity