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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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Belavilas-Trovas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

The olive fruit fly, Bactrocera oleae, is the most important pest for the olive fruit but lacks adequate transcriptomic characterization that could aid in molecular control approaches. We apply nanopore long-read RNA-seq with internal RNA standards allowing absolute transcript quantification to analyze transcription dynamics during early embryo development for the first time in this organism. Sequencing on the MinION platform generated over 31 million reads. Over 50% of the expressed genes had at least one read covering its entire length validating our full-length approach. We generated a de novo transcriptome assembly and identified 1768 new genes and a total of 79,810 isoforms; a fourfold increase in transcriptome diversity compared to the current NCBI predicted transcriptome. Absolute transcript quantification per embryo allowed an insight into the dramatic re-organization of maternal transcripts. We further identified Zelda as a possible regulator of early zygotic genome activation in B. oleae and provide further insights into the maternal-to-zygotic transition. These data show the utility of long-read RNA in improving characterization of non-model organisms that lack a fully annotated genome, provide potential targets for sterile insect technic approaches, and provide the first insight into the transcriptome landscape of the developing olive fruit fly embryo.

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.

Background: The olive fruit fly (Bactrocera oleae) is the most destructive pest of the olive cultivation worldwide causing significant production losses and olive fruit impoverishment, as its larvae feed exclusively on the olive fruit. Reproductive and sexual behavior, as well as host-plant recognition of the fly, are highly dependent on its chemosensory system. Therefore, exploring the role of genes that play a critical role in olfaction, could reveal potential molecular targets that determine species-specific features on chemical communication and could be used to impair sexual behavior., Results: In this study we identified the gene that encodes the conserved olfactory co-receptor Orco (Odorant receptor co-receptor), which interacts with all divergent insect odorant receptors, and investigated how disruption of its expression affects chemoreception. We initially searched the expression profile of Bo-Orco in both sexes during sexual maturation, as well as pre- and post-mating communication by relative quantitative real time PCR (qRT-PCR) analysis suggesting that Bo-Orco was abundantly expressed in sexually mature adults. We further investigated the functional role of Bo-Orco in mating and oviposition behavior via transient gene silencing that was performed through in vivo dsRNA hemolymph injections in sexually mature flies 7 days after eclosion. Orco-knockdown phenotypes in both sexes showed reduced copulation rates in mating competitiveness tests, possibly through impaired olfactory-mediated detection of sex pheromone. In addition, oviposition was significantly inhibited in dsRNA-Orco injected females in a post-mating behavior test., Conclusions: Our results demonstrate that Orco plays a crucial role in the reproductive behavior of the olive fruit fly, since pre- and post-mating processes were affected. This is the first report in the olive fruit fly that links the chemosensory pathway with the mating behavior and the reproductive potential at a molecular basis, rendering this gene a potential target for the improvement of the olive fruit fly population control techniques.

Insect pests can cause crop damage in yield or quality, resulting in profit losses for farmers. The primary approach to control them is still the use of chemical pesticides resulting in significant hazards to the environment and human health. Biological control and the sterile insect technique are alternative strategies to improve agriculture protection. However, both strategies have significant limitations. A newly introduced approach that could be both effective and species-specific is the RNA interference mechanism. One key point for the success of this strategy is the delivery method of double-strand RNA (dsRNA) to the insects. A method of dsRNA delivery to insects with potential use in the field is the oral delivery, feeding the insects engineered microorganisms that produce dsRNA. Here, we present the first protocol for dsRNA feeding using modified bacteria, in the olive fruit fly, the most important insect pest of cultivated olives. We chose to target the sex peptide receptor gene. The sex peptide receptor interacts with the sex peptide, a peptide that is responsible for the postmating behavior in the model organism, Drosophila melanogaster. Feeding the female olive fruit fly with bacteria that produced dsRNA for the sex peptide receptor gene resulted in the development of female insects with significantly lower oviposition rates. Administration of dsRNA producing bacteria in insect diet against target genes that lead to genetic sexing or female-specific lethality could be added in the armory of control methods., (© 2020 Wiley Periodicals, Inc.)

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo-we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene-specific single-guide RNAs. Gene disruption of Bo-we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo-we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control., (© 2020 Wiley Periodicals, Inc.)

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.

Agricultural pest control using genetic-based methods provides a species-specific and environmentally harmless way for population suppression of fruit flies. One way to improve the efficiency of such methods is through self-limiting, female-eliminating approaches that can alter an insect populations' sex ratio toward males. In this microreview, we summarize recent advances in synthetic sex ratio distorters based on X-chromosome shredding that can induce male-biased progeny. We outline the basic principles to guide the efficient design of an X-shredding system in an XY heterogametic fruit fly species of interest using CRISPR/Cas gene editing, newly developed computational tools, and insect genetic engineering. We also discuss technical aspects and challenges associated with the efficient transferability of this technology in fruit fly pest populations, toward the potential use of this new class of genetic control approaches for pest management purposes., (© 2019 Wiley Periodicals, Inc.)

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y ( MoY ), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Y chromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Real-time quantitative-PCR has been a priceless tool for gene expression analyses. The reaction, however, needs proper normalization with the use of housekeeping genes (HKGs), whose expression remains stable throughout the experimental conditions. Often, the combination of several genes is required for accurate normalization. Most importantly, there are no universal HKGs which can be used since their expression varies among different organisms, tissues or experimental conditions. In the present study, nine common HKGs (RPL19, tbp, ubx, GAPDH, α-TUB, β-TUB, 14-3-3zeta, RPE and actin3) are evaluated in thirteen different body parts, developmental stages and reproductive and olfactory tissues of two insects of agricultural importance, the medfly and the olive fly. Three software programs based on different algorithms were used (geNorm, NormFinder and BestKeeper) and gave different ranking of HKG stabilities. This confirms once again that the stability of common HKGs should not be taken for granted and demonstrates the caution that is needed in the choice of the appropriate HKGs. Finally, by estimating the average of a standard score of the stability values resulted by the three programs we were able to provide a useful consensus key for the choice of the best HKG combination in various tissues of the two insects.

Sex chromosomes have many unusual features relative to autosomes. The in depth exploration of their structure will improve our understanding of their origin and divergence (degeneration) as well as the evolution of genetic sex determination pathways which, most often are attributed to them. In Tephritids, the structure of Y chromosome, where the male-determining factor M is localized, is largely unexplored and limited data concerning its sequence content and evolution are available. In order to get insight into the structure and organization of the Y chromosome of the major olive insect pest, the olive fly Bactrocera oleae, we characterized sequences from a Pulse Field Gel Electrophoresis (PFGE)-isolated Y chromosome. Here, we report the discovery of the first olive fly LTR retrotransposon with increased presence on the Y chromosome. The element belongs to the BEL-Pao superfamily, however, its sequence comparison with the other members of the superfamily suggests that it constitutes a new family that we termed Achilles. Its ~7.5 kb sequence consists of the 5'LTR, the 5'non-coding sequence and the open reading frame (ORF), which encodes the polyprotein Gag-Pol. In situ hybridization to the B. oleae polytene chromosomes showed that Achilles is distributed in discrete bands dispersed on all five autosomes, in all centromeric regions and in the granular heterochromatic network corresponding to the mitotic sex chromosomes. The between sexes comparison revealed a variation in Achilles copy number, with male flies possessing 5-10 copies more than female (CI range: 18-38 and 12-33 copies respectively per genome). The examination of its transcriptional activity demonstrated the presence of at least one intact active copy in the genome, showing a differential level of expression between sexes as well as during embryonic development. The higher expression was detected in male germline tissues (testes). Moreover, the presence of Achilles-like elements in different species of the Tephritidae family suggests an ancient origin of this element.