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The Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), one of the most important invasive pests of fresh fruits and vegetables from the coastal Mediterranean habitats, is expanding its current geographic distribution to cooler more temperate areas of Europe. Every year since 2010 the fly is detected in the area of Vienna, Austria. However, whether it can establish permanent populations is not known. In this current paper, the capacity of C. capitata to overwinter in Vienna, Austria (48.1° northern latitude) was studied over 2 consecutive winter seasons (2020-2022). Overwintering trials with different life stages (larva, pupa, and adult) of C. capitata were performed in the open field and in the protected environment of a basement without a heating system. Control flies were kept under constant conditions in a climate chamber (25 °C, 60% RH, 14:10 L:D). Our data showed that no life stage of the Mediterranean fruit fly was able to survive the Austrian winter in the open field. However, in the protected environment C. capitata outlived the winter months in all studied life stages at least in small numbers and several surviving females were able to lay eggs at the time of the following fruiting season. Implications of these findings for the ongoing geographic range expansion of the pest in temperate European countries are discussed.

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In flies (Diptera), the ovary displays several distinct patterns of the follicular epithelium formation and diversification. Two main patterns have been identified in the true flies or Brachycera, namely the Rhagio type and the Drosophila type. These patterns align with the traditional division of Brachycera into Orthorrhapha and Cyclorrhapha. However, studies of the follicular epithelium morphogenesis in cyclorrhaphans other than Drosophila are scarce. We characterise the developmental changes associated with the emergence of follicle cell (FC) diversity in two cyclorrhaphans belonging to the family Tephritidae (Brachycera, Cyclorrhapha). Our analysis revealed that the diversification of FCs in these species shows characteristics of both the Rhagio and Drosophila types. First, a distinct cluster of FCs, consisting of polar cells and border-like cells, differentiates at the posterior pole of the ovarian follicle. This feature is unique to the Rhagio type and has only been reported in species representing the Orthorrhapha group. Second, morphological criteria have identified a significantly smaller number of subpopulations of FCs than in Drosophila. Furthermore, while the general pattern of FC migration is similar to that of Drosophila, the distinctive migration of the anterior-dorsal FCs is absent. In the studied tephritids, the migration of the anterior polar cell/border cell cluster towards the anterior pole of the oocyte is followed by the posterior migration of the main body cuboidal FCs to cover the expanding oocyte. Finally, during the onset of vitellogenesis, a distinct subset of FCs migrates towards the centre of the ovarian follicle to cover the oocyte's anterior pole. Our study also highlights specific actions of some FCs that accompany the migration process, which has not been previously documented in cyclorrhaphans. These results support the hypothesis that the posterior and centripetal migrations of morphologically unique FC subsets arose in the common ancestor of Cyclorrhapha. These events appear to have occurred fairly recently in the evolutionary timeline of Diptera.

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Bactrocera dorsalis and Bactrocera correcta are two invasive species that can cause major economic damage to orchards and the fruit import and export industries. Their distribution is advancing northward due to climate change, which is threatening greater impacts on fruit production. This study tested the rapid cold-hardening ability of the two species and identified the temperature associated with the highest survival rate. Transcriptome data and survival data from the two Bactrocera species' larvae were obtained after rapid cold-hardening experiments. Based on the sequencing of transcripts, four Hsp genes were found to be affected: Hsp68 and Hsp70, which play more important roles in the rapid cold hardening of B. dorsalis, and Hsp23 and Hsp70, which play more important roles in the rapid cold hardening of B. correcta. This study explored the adaptability of the two species to cold, demonstrated the expression and function of four Hsps in response to rapid cold hardening, and explained the occurrence and expansion of these two species of tephritids, offering information for further studies.

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Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into a specific genomic target site. Both strategies, however, often face limitations due to low transgenesis efficiencies. We aimed to enhance transgenesis efficiencies by utilizing capped mRNA as a source of transposase or recombinase instead of a helper plasmid. A systematic comparison of transgenesis efficiencies in Aedes mosquitoes, as models for hard-to-transform insects, showed that suppling piggyBac transposase as mRNA increased the average transformation efficiency in Aedes aegypti from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in Ae. albopictus with pBac mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of piggyBac transposase delivered as a plasmid did not improve the transformation efficiency in Ae. aegypti or the agricultural pest Drosophila suzukii. We believe that the use of mRNA has strong potential for enhancing piggyBac transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids.

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Sterile Insect Technique (SIT) is a biocontrol strategy that has been widely utilized to suppress or eradicate outbreak populations of insect pests such as tephritid fruit flies. As SIT is highly favored due to it being species-specific and environmentally friendly, there are constant efforts to improve the efficiency and efficacy of this method in particular at low pest densities; one of which is the use of genetically enhanced strains. Development of these desirable strains has been facilitated by the emergence of the CRISPR/Cas genome-editing technology that enables the rapid and precise genomic modification of non-model organisms. Here, we describe the manual microinjection of CRISPR/Cas9 reagents into tephritid pest Bactrocera tryoni (Queensland fruit fly) embryos to introduce ideal traits as well as the molecular methods used to detect successful mutagenesis.

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What physiological and neuro-molecular changes control the female oviposition behavior post-mating in insects? The molecular changes that occur in a gravid female insect are difficult to dissect out considering the distinct behavioral patterns displayed by different insect groups. To understand the role of the brain center in Oriental fruit fly, Bactrocera dorsalis oviposition, egg-laying behavior was analyzed in γ-octalactone exposed, decapitated mated B. dorsalis females. Interestingly, the females displayed a possible urge to oviposit, which suggests a natural instinct to pass on the gene pool. Expression analysis of certain genes involved in oviposition behavior was also carried out in these insects to explore the molecular aspects of such behavior. This study tries to assess the involvement of brain center in egg-laying and also explore the role of certain neurotransmitter-related receptors in decapitated B. dorsalis oviposition behavior. Our results indicate that B. dorsalis oviposition behavior could potentially have a bypass route of neuronal control devoid of the brain. The study reported here establishes that decapitation in gravid females fails to abolish their ability to sense ovipositional cues and also to oviposit.

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BACKGROUND: Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. RESULTS: Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. CONCLUSIONS: We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.

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The olive fruit fly, Bactrocera oleae, causes great damage to the quality and quantity of olive production worldwide. Pest management approaches have proved difficult for a variety of reasons, a fact that has brought about a need for alternative tools and approaches. Here we report for the first time in B. oleae the development of the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing tool, using the well-known eye colour marker gene scarlet. Two synthetic guide RNAs targeting the coding region of the scarlet gene were synthesized and shown to work efficiently in vitro. These reagents were then microinjected along with purified Cas9 protein into early-stage embryos. Successful CRISPR-induced mutations of both copies of the scarlet gene showed a striking yellow eye phenotype, indicative of gene disruption. Multiple successful CRISPR events were confirmed by PCR and sequencing. The establishment of an efficient CRISPR-based gene editing tool in B. oleae will enable the study of critical molecular mechanisms in olive fruit fly biology and physiology, including the analysis of insecticide resistance mechanisms and the discovery of novel insecticide targets, as well as facilitate the development of novel biotechnology-based pest control strategies.

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Tephritidae is a large family that includes several fruit and vegetable pests. These organisms usually harbor a variegated bacterial community in their digestive systems. Symbiotic associations of bacteria and fruit flies have been well-studied in the genera Anastrepha, Bactrocera, Ceratitis, and Rhagoletis. Molecular and culture-based techniques indicate that many genera of the Enterobacteriaceae family, especially the genera of Klebsiella, Enterobacter, Pectobacterium, Citrobacter, Erwinia, and Providencia constitute the most prevalent populations in the gut of fruit flies. The function of symbiotic bacteria provides a promising strategy for the biological control of insect pests. Gut bacteria can be used for controlling fruit fly through many ways, including attracting as odors, enhancing the success of sterile insect technique, declining the pesticide resistance, mass rearing of parasitoids and so on. New technology and recent research improved our knowledge of the gut bacteria diversity and function, which increased their potential for pest management. In this review, we discussed the diversity of bacteria in the economically important fruit fly and the use of these bacteria for controlling fruit fly populations. All the information is important for strengthening the future research of new strategies developed for insect pest control by the understanding of symbiotic relationships and multitrophic interactions between host plant and insects.

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In the six decades since 1960, the oriental fruit fly, Bactrocera dorsalis (Hendel), has been announced successfully eradicated in California by the U.S. Department of Agriculture a total of 564 times. This includes eradication declarations in one city a total of 25 different years, in 12 cities 8-19 different years, and in 101 cities 2-7 different years. We here show that the false negatives in declaring elimination success hinge on the easily achieved regulatory criteria, which have virtually guaranteed the failure of complete extirpation of this pest. Analyses of the time series of fly detection over California placed on a grid of 100-km2 cells revealed (1) partial success of the eradication program in controlling the invasion of the oriental fruit fly; (2) low prevalence of the initial detection in these cells is often followed by high prevalence of recurrences; (3) progressively shorter intervals between years of consecutive detections; and (4) high likelihood of early-infested cells also experiencing the most frequent outbreaks. Facing the risk of recurrent invasions, such short-term eradication programs have only succeeded annually according to the current regulatory criteria but have failed to achieve the larger goal of complete extirpation of the oriental fruit fly. Based on the components and running costs of the current programs, we further estimated the efficiency of eradication programs with different combinations of eradication radius, duration, and edge impermeability in reducing invasion recurrences and slowing the spread of the oriental fruit fly. We end with policy implications including the need for agricultural agencies worldwide to revisit eradication protocols in which monitoring and treatments are terminated when the regulatory criteria for declaring eradication are met. Our results also have direct implications to invasion biologists and agriculture policy makers regarding long-term risks of short-term expediency.

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The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing enormous economic damage in horticultural systems. A successful and environment-friendly control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. However, the SIT is not readily applicable to each pest species. While transgenic approaches hold great promise to improve critical aspects of the SIT to transfer it to new species, they are suspect to strict or even prohibitive legislation regarding the release of genetically modified (GM) organisms. In contrast, specific mutations created via CRISPR-Cas genome editing are not regulated as GM in the US, and might thus allow creating optimal strains for SIT. Here, we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring. Based on the achieved efficiency, this approach could also be used to introduce mutations which do not produce a screenable phenotype and identify positive mutants with a reasonable workload. Furthermore, CRISPR-Cas HDR would allow to recreate mutations formerly identified in classical mutagenesis screens and to transfer them to related species to establish new (SIT-like) pest control systems. Considering the potential that CRISPR-induced alterations in organisms could be classified as non-GM in additional countries, such new strains could potentially be used for pest control applications without the need to struggle with GMO directives.

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The Mediterranean fruit fly, Ceratitis capitata, is a major agricultural pest worldwide. The development of genetic sexing strains (GSSs) for this species that allows male-only sterile insects releases has boosted the effectiveness of the environmental friendly pest control method known as the sterile insect technique. The last generation of these strains, the VIENNA 7 and VIENNA 8, are currently used in all mass rearing facilities worldwide and are considered as models for such pest control applications. The sterile insect technique depends on the rearing of sufficient numbers of adequate "biological quality" laboratory flies to be released in the field. Currently, there is an increasing amount of studies focusing on the characterization of the symbiotic communities and development of probiotic diets. In our study, two bacterial isolates, an Enterobacter sp. (strain AA26) and a Klebsiella oxytoca strain, were used as probiotics in larval and adult diet. These strains have been shown to be beneficial, affecting several aspects related to the rearing efficiency and biological quality of the medfly VIENNA 8D53+ GSS. Our results demonstrate the effect of K. oxytoca on the developmental duration of the immature stages and, to some extent, on flight ability. On the other hand, our study does not support the presence of any beneficial effect of (a) K. oxytoca on pupal and adult recovery and adults' survival under stress conditions when provided as a larval diet supplement and (b) K. oxytoca and Enterobacter sp. AA26 on mating competitiveness when provided as adult diet supplements. Possible explanations for inconsistencies with previous studies and the need for universalizing protocols are discussed. Our findings, combined with previous studies can support the sterile insect technique, through the improvement of different aspects of mass rearing and biological properties of laboratory reared insect pests.

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Fruit flies are considered important pests of world fruit production, causing significant losses in yield and limiting the free movement of fruit due to the restrictions imposed by importing countries. Thus, the objective of the study was to investigate the infestation rates and faunal analysis to characterize the fruit fly assemblage in an orchard located in Bambuí, MG, Brazil. To this end, samples were taken weekly with fly traps hung in the trees, at a height of about 1.60 m height. Faunal analysis and study of infestation rates were conducted. The captured species were Ceratitis capitata, Anastrepha grandis, A. fraterculus, A. pseudoparallela, A. obliqua, A. dissimilis, A. zenildae, A. sororcula, A. similis, A. aczeli and A. bistrigata. The species varied throughout the studied period, but only C. capitata reached an economic threshold requiring better management of these insects in areas with citrus, acerola and fig. The predominant species was C. capitata.

As moscas-das-frutas são consideradas importantes pragas da fruticultura mundial, causando perdas significativas à produção e limitando o livre trânsito de frutas devido às restrições impostas pelos países importadores. Dessa forma, teve-se por objetivo estudar os índices de infestação e faunísticos para caracterização da assembleia de moscas-das-frutas em pomar localizado na região de Bambuí, MG, Brasil. Para tanto, foram realizadas coletas semanais com armadilhas caça-moscas penduradas nas copas das árvores, a uma altura de aproximadamente 1,60 m de altura. Foi realizada análise faunística e índices de infestação. As espécies capturadas foram Ceratitis capitata, Anastrepha grandis, A. fraterculus, A. pseudoparallela, A. obliqua, A. dissimilis, A. zenildae, A. sororcula, A. similis, A. aczeli e A. bistrigata. As espécies encontradas variaram durante todo o período estudado, porém apenas C. capitata atingiu nível de dano econômico necessitando de um melhor manejo desses insetos nas áreas com citros, acerola e figo. A espécie predominante foi C. capitata.

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Comparison of the genomes of different Drosophila species has shown that six different chromosomes, the so-called ''Muller elements," constitute the building blocks for all Drosophila species. Here, we confirm previous results suggesting that this conservation of the Muller elements extends far beyond Drosophila, to at least tephritid fruit flies, thought to have diverged from drosophilids 60-70 mYr ago. Less than 10 percent of genes differ in chromosome location between the two insect groups. Within chromosomes, however, the order is highly scrambled, as expected from the comparison between Drosophila species. The data also support the notion that the sex chromosomes of tephritid flies originated from an ancestor of the dot chromosome 4 of Drosophila. Overall, therefore, no new chromosome has been created for perhaps a billion generations over the two evolutionary lines. This stability at the chromosome level, which appears to extend to all Diptera including mosquitoes, is in stark contrast to other groups such as mammals, birds, fish and plants, in which chromosome numbers and organization vary enormously among species that have diverged over much fewer generations.

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