Deep orange gene editing triggers temperature-sensitive lethal phenotypes in Ceratitis capitata.

BACKGROUND: The Mediterranean fruit fly, Ceratitis capitata, is a significant agricultural pest managed through area-wide integrated pest management (AW-IPM) including a sterile insect technique (SIT) component. Male-only releases increase the efficiency and cost-effectiveness of SIT programs, which can be achieved through the development of genetic sexing strains (GSS). The most successful GSS developed to date is the C. capitata VIENNA 8 GSS, constructed using classical genetic approaches and an irradiation-induced translocation with two selectable markers: the white pupae (wp) and temperature-sensitive lethal (tsl) genes. However, currently used methods for selecting suitable markers and inducing translocations are stochastic and non-specific, resulting in a laborious and time-consuming process. Recent efforts have focused on identifying the gene(s) and the causal mutation(s) for suitable phenotypes, such as wp and tsl, which could be used as selectable markers for developing a generic approach for constructing GSS. The wp gene was recently identified, and efforts have been initiated to identify the tsl gene. This study investigates Ceratitis capitata deep orange (Ccdor) as a tsl candidate gene and its potential to induce tsl phenotypes. RESULTS: An integrated approach based on cytogenetics, genomics, bioinformatics, and gene editing was used to characterize the Ccdor. Its location was confirmed on the right arm of chromosome 5 in the putative tsl genomic region. Knock-out of Ccdor using CRISPR/Cas9-NHEJ and targeting the fourth exon resulted in lethality at mid- and late-pupal stage, while the successful application of CRISPR HDR introducing a point mutation on the sixth exon resulted in the establishment of the desired strain and two additional strains (dor 12del and dor 51dup), all of them expressing tsl phenotypes and presenting no (or minimal) fitness cost when reared at 25 °C. One of the strains exhibited complete lethality when embryos were exposed at 36 °C. CONCLUSIONS: Gene editing of the deep orange gene in Ceratitis capitata resulted in the establishment of temperature-sensitive lethal mutant strains. The induced mutations did not significantly affect the rearing efficiency of the strains. As deep orange is a highly conserved gene, these data suggest that it can be considered a target for the development of tsl mutations which could potentially be used to develop novel genetic sexing strains in insect pests and disease vectors.

Gene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata.

Homing-based gene drives are recently proposed interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate how CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way.

Next-generation genetic sexing strain establishment in the agricultural pest Ceratitis capitata.

Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male-female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique.

Development of a novel genetic sexing strain of Ceratitis capitata based on an X-autosome translocation.

Genetic sexing strains (GSS), such as the Ceratitis capitata (medfly) VIENNA 8 strain, facilitate male-only releases and improve the efficiency and cost-effectiveness of sterile insect technique (SIT) applications. Laboratory domestication may reduce their genetic diversity and mating behaviour and hence, refreshment with wild genetic material is frequently needed. As wild males do not carry the T(Y;A) translocation, and wild females do not easily conform to artificial oviposition, the genetic refreshment of this GSS is a challenging and time-consuming process. In the present study, we report the development of a novel medfly GSS, which is based on a viable homozygous T(XX;AA) translocation using the same selectable markers, the white pupae and temperature-sensitive lethal genes. This allows the en masse cross of T(XX;AA) females with wild males, and the backcrossing of F1 males with the T(XX;AA) females thus facilitating the re-establishment of the GSS as well as its genetic refreshment. The rearing efficiency and mating competitiveness of the novel GSS are similar to those of the T(Y;A)-based VIENNA 8 GSS. However, its advantage to easily allow the genetic refreshment is of great importance as it can ensure the mass production of high-quality males and enhanced efficacy of operational SIT programs.

Genomic and cytogenetic analysis of the Ceratitis capitata temperature-sensitive lethal region.

Genetic sexing strains (GSS) are an important tool in support of sterile insect technique (SIT) applications against insect pests and disease vectors. The yet unknown temperature-sensitive lethal (tsl) gene and the recently identified white pupae (wp) gene have been used as selectable markers in the most successful GSS developed so far, the Ceratitis capitata (medfly) VIENNA 8 GSS. The molecular identification of the tsl gene may open the way for its use as a marker for the development of GSS in other insect pests and disease vectors of SIT importance. Prior studies have already shown that the tsl gene is located on the right arm of chromosome 5, between the wp and Zw loci (tsl genomic region). In the present study, we used genomic, transcriptomic, bioinformatic, and cytogenetic approaches to characterize and analyze this genomic region in wild-type and tsl mutant medfly strains. Our results suggested the presence of 561 genes, with 322 of them carrying SNPs and/or insertion-deletion (indel) mutations in the tsl genomic region. Furthermore, comparative transcriptomic analysis indicated the presence of 32 differentially expressed genes, and bioinformatic analysis revealed the presence of 33 orthologs with a described heat-sensitive phenotype of Drosophila melanogaster in this region. These data can be used in functional genetic studies to identify the tsl gene(s) and the causal mutation(s) responsible for the temperature-sensitive lethal phenotype in medfly, and potentially additional genes causing a similar phenotype.

Unveiling biogeographical patterns in the worldwide distributed Ceratitis capitata (medfly) using population genomics and microbiome composition.

Invasive species are among the most important, growing threats to food security and agricultural systems. The Mediterranean medfly, Ceratitis capitata, is one of the most damaging representatives of a group of rapidly expanding species in the family Tephritidae, due to their wide host range and high invasiveness potential. Here, we used restriction site-associated DNA sequencing (RADseq) to investigate the population genomic structure and phylogeographical history of medflies collected from six sampling sites, including Africa (South Africa), the Mediterranean (Spain, Greece), Latin America (Guatemala, Brazil) and Australia. A total of 1907 single nucleotide polymorphisms (SNPs) were used to identify two genetic clusters separating native and introduced ranges, consistent with previous findings. In the introduced range, all individuals were assigned to one genetic cluster except for those in Brazil, which showed introgression of an additional genetic cluster that also appeared in South Africa, and which could not be previously identified using microsatellite markers. Moreover, we assessed the microbial composition variations in medfly populations from selected sampling sites using amplicon sequencing of the 16S ribosomal RNA (V4 region). Microbiome composition and structure were highly similar across geographical regions and host plants, and only the Brazilian specimens showed increased diversity levels and a unique composition of its microbiome compared to other sampling sites. The unique SNP patterns and microbiome features in the Brazilian specimens could point to a direct migration route from Africa with subsequent adaptation of the microbiota to the specific conditions present in Brazil. These findings significantly improve our understanding of the evolutionary history of the global medfly invasions and their adaptation to newly colonized environments.

A multiplex PCR assay for the identification of fruit flies (Diptera: Tephritidae) of economic importance in South Africa.

The fruit fly (Diptera: Tephritidae) species, Ceratitis capitata, Ceratitis cosyra, Ceratitis rosa, Ceratitis quilicii, and Bactrocera dorsalis are of economic importance in South Africa. These agricultural pests cause extensive damage to a range of commercially produced fruit, primarily for export. These pests are of phytosanitary significance, and their presence in fruit-producing regions in South Africa has led to restrictions in export trade of fresh produce. Accurate identification of these flies, particularly at immature stages intercepted in fruit consignments originating from South Africa, is essential but remains an ongoing challenge. A rapid and accurate identification assay to differentiate these five species is needed for inspection and pest surveillance. High throughput sequencing data were generated for each of the five fruit fly species, and five sets of species-specific primers were designed for use in a multiplex PCR. Each primer set amplifies an amplicon of a different size for each species allowing for accurate identification. PCR sensitivity tests demonstrate that the limit of detection for this assay is 10 ng and 4 ng of DNA when extracted from larvae and adult specimens, respectively. The assay developed can be applied in fruit inspection and survey activities within the country and at ports of entry.

Characterisation of the symbionts in the Mediterranean fruit fly gut.

Symbioses between bacteria and their insect hosts can range from loose associations through to obligate interdependence. While fundamental evolutionary insights have been gained from the in-depth study of obligate mutualisms, there is increasing interest in the evolutionary potential of flexible symbiotic associations between hosts and their gut microbiomes. Understanding relationships between microbes and hosts also offers the potential for exploitation for insect control. Here, we investigate the gut microbiome of a global agricultural pest, the Mediterranean fruit fly (Ceratitis capitata). We used 16S rRNA profiling to compare the gut microbiomes of laboratory and wild strains raised on different diets and from flies collected from various natural plant hosts. The results showed that medfly guts harbour a simple microbiome that is primarily determined by the larval diet. However, regardless of the laboratory diet or natural plant host on which flies were raised, Klebsiella spp. dominated medfly microbiomes and were resistant to removal by antibiotic treatment. We sequenced the genome of the dominant putative Klebsiella spp. ('Medkleb') isolated from the gut of the Toliman wild-type strain. Genome-wide ANI analysis placed Medkleb within the K. oxytoca / michiganensis group. Species level taxonomy for Medkleb was resolved using a mutli-locus phylogenetic approach - and molecular, sequence and phenotypic analyses all supported its identity as K. michiganensis. Medkleb has a genome size (5825435 bp) which is 1.6 standard deviations smaller than the mean genome size of free-living Klebsiella spp. Medkleb also lacks some genes involved in environmental sensing. Moreover, the Medkleb genome contains at least two recently acquired unique genomic islands as well as genes that encode pectinolytic enzymes capable of degrading plant cell walls. This may be advantageous given that the medfly diet includes unripe fruits containing high proportions of pectin. The results suggest that the medfly harbours a commensal gut bacterium that may have developed a mutualistic association with its host and provide nutritional benefits.

Expanding the Medfly Virome: Viral Diversity, Prevalence, and sRNA Profiling in Mass-Reared and Field-Derived Medflies.

The Mediterranean fruit fly (medfly), Ceratitis capitata, is an agricultural pest of a wide range of fruits. The advent of high-throughput sequencing has boosted the discovery of RNA viruses infecting insects. In this article, we aim to characterize the RNA virome and viral sRNA profile of medfly. By means of transcriptome mining, we expanded the medfly RNA virome to 13 viruses, including two novel positive ssRNA viruses and the first two novel dsRNA viruses reported for medfly. Our analysis across multiple laboratory-reared and field-collected medfly samples showed the presence of a core RNA virome comprised of Ceratitis capitata iflavirus 2 and Ceratitis capitata negev-like virus 1. Furthermore, field-collected flies showed a higher viral diversity in comparison to the laboratory-reared flies. Based on the small RNA sequencing, we detected small interfering RNAs mapping to all the viruses present in each sample, except for Ceratitis capitata nora virus. Although the identified RNA viruses do not cause obvious symptoms in medflies, the outcome of their interaction may still influence the medfly's fitness and ecology, becoming either a risk or an opportunity for mass-rearing and SIT applications.

Probiotic based-diet effect on the immune response and induced stress in irradiated mass reared Ceratitis capitata males (Diptera: Tephritidae) destined for the release in the sterile insect technique programs.

Ceratitis capitata (medfly) is one of the most devastating crop pests worldwide. The Sterile Insect Technique (SIT) is a control method that is based on the mass rearing of males, their sterilization, and release in the field. However, the effectiveness of the technique depends on the quality of the released males and their fitness. We previously isolated and selected a probiotic bacteria (Enterobacter sp.), from wild-caught medflies, according to criteria that improved biological quality traits of reared medfly males.We firstly evaluated the impact of the irradiation on the expression of different immune and stress genes in the medfly sterile males. Expression was measured at differents time points ranging from 0 to 168 h after irradiation to capture the response of genes with distinct temporal expression patterns. Then, we supplemented the larval diet with previously isolated Enterobacter sp.strain, live and autoclaved at various concentrations to see whether the probiotic treatments affect, through their protective role, the gene expression level, and quality traits. The irradiation had significant effect on the genes attacin, cecropin, PGPR-LC, hsp23, and hsp70 level expression. The expression of attacin and PGPR-LC was up-regulated while that of cecropin was down-regulated. Hsp genes showed decreased levels between 0 and 18 h to peak at 72 h. However, the supplementation of the probiotic strain, either live or autoclaved, was statistically significant only for attacingene. However, significant interaction time x probiotic was noticed for attacin, cecropin, hsp23 and hsp70. The probiotic treatments also improved the quality control parameters like pupal weight. From this work we can conclude that a consortium of parabiotics (autoclaved probiotics) treatment will be recommended in insectaries considering both the beneficial effects on mass reared insects and its general safety for insectary workers and for environment.

Engineered sex ratio distortion by X-shredding in the global agricultural pest Ceratitis capitata.

BACKGROUND: Genetic sex ratio distorters are systems aimed at effecting a bias in the reproductive sex ratio of a population and could be applied for the area-wide control of sexually reproducing insects that vector disease or disrupt agricultural production. One example of such a system leading to male bias is X-shredding, an approach that interferes with the transmission of the X-chromosome by inducing multiple DNA double-strand breaks during male meiosis. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. RESULTS: Here, we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long- and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome-specific sequence repeats. When targeted by gRNAs in conjunction with Cas9, cleavage of these repeats yielded a significant and consistent distortion of the sex ratio towards males in independent transgenic strains, while the combination of distinct distorters induced a strong bias (~ 80%). CONCLUSION: We provide a first demonstration of CRISPR-based sex distortion towards male bias in a non-model organism, the global pest insect Ceratitis capitata. Although the sex ratio bias reached in our study would require improvement, possibly through the generation and combination of additional transgenic lines, to result in a system with realistic applicability in the field, our results suggest that strains with characteristics suitable for field application can now be developed for a range of medically or agriculturally relevant insect species.

White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp- mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp- mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

Targeting the autosomal Ceratitis capitata transformer gene using Cas9 or dCas9 to masculinize XX individuals without inducing mutations.

BACKGROUND: Females of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs. RESULTS: With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults. CONCLUSIONS: Our data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination.

Genetic structure and symbiotic profile of worldwide natural populations of the Mediterranean fruit fly, Ceratitis capitata.

BACKGROUND: The Mediterranean fruit fly, Ceratitis capitata, is a cosmopolitan agricultural pest of worldwide economic importance and a model for the development of the Sterile Insect Technique (SIT) for fruit flies of the Tephritidae family (Diptera). SIT relies on the effective mating of laboratory-reared strains and natural populations, and therefore requires an efficient mass-rearing system that will allow for the production of high-quality males. Adaptation of wild flies to an artificial laboratory environment can be accompanied by negative effects on several life history traits through changes in their genetic diversity and symbiotic communities. Such changes may lead to reduced biological quality and mating competitiveness in respect to the wild populations. Profiling wild populations can help understand, and maybe reverse, deleterious effects accompanying laboratory domestication thus providing insects that can efficiently and effectively support SIT application. RESULTS: In the present study, we analyzed both the genetic structure and gut symbiotic communities of natural medfly populations of worldwide distribution, including Europe, Africa, Australia, and the Americas. The genetic structure of 408 individuals from 15 distinct populations was analyzed with a set of commonly used microsatellite markers. The symbiotic communities of a subset of 265 individuals from 11 populations were analyzed using the 16S rRNA gene-based amplicon sequencing of single individuals (adults). Genetic differentiation was detected among geographically distant populations while adults originated from neighboring areas were genetically closer. Alpha and beta diversity of bacterial communities pointed to an overall reduced symbiotic diversity and the influence of the geographic location on the bacterial profile. CONCLUSIONS: Our analysis revealed differences both in the genetic profile and the structure of gut symbiotic communities of medfly natural populations. The genetic analysis expanded our knowledge to populations not analyzed before and our results were in accordance with the existing scenarios regarding this species expansion and colonization pathways. At the same time, the bacterial communities from different natural medfly populations have been characterized, thus broadening our knowledge on the microbiota of the species across its range. Genetic and symbiotic differences between natural and laboratory populations must be considered when designing AW-IPM approaches with a SIT component, since they may impact mating compatibility and mating competitiveness of the laboratory-reared males. In parallel, enrichment from wild populations and/or symbiotic supplementation could increase rearing productivity, biological quality, and mating competitiveness of SIT-important laboratory strains.

Improving the Phenotypic Properties of the Ceratitis capitata (Diptera: Tephritidae) Temperature-Sensitive Lethal Genetic Sexing Strain in Support of Sterile Insect Technique Applications.

The genetic sexing strain (GSS) of the Mediterranean fruit fly (Ceratitis capitata (Wiedemann)) Vienna 8D53- is based on a male-linked translocation system and uses two selectable markers for male-only production, the white pupae (wp) and the temperature sensitivity lethal (tsl) genes. In this GSS, males emerge from brown pupae and are resistant to high temperatures while females emerge from white pupae, are sensitive to high temperatures. However, double homozygous females (wp tsl/wp tsl) exhibit a slower development rate compared to heterozygous males (wp+tsl+/wp tsl) during the larval stage, which was attributed to the pleiotropic effects of the tsl gene. We present the first evidence that this slower development is due to a different gene, here namely slow development (sd), which is closely linked to the tsl gene. Taking advantage of recombination phenomena between the two loci, we report the isolation of a novel temperature sensitivity lethal strain using the wp mutation as a morphological marker, which showed faster development (wp tsl FD) during the larval stage and increased in its temperature sensitivity compared with the normal tsl strain. Moreover, the introgression of this novel wp tsl FD combined trait into the Vienna 8D53- GSS, resulted in a novel Vienna 8D53- FD GSS, where females showed differences in the thermal sensibility, larval development speed, and productivity profiles. The modification of these traits and their impact on the mass rearing of the GSS for sterile insect technique applications are discussed.

Female-to-male sex conversion in Ceratitis capitata by CRISPR/Cas9 HDR-induced point mutations in the sex determination gene transformer-2.

The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating. Critical for all SIT programs is a conditional sexing strain to enable the cost-effective production of male-only populations. Compared to current female-elimination strategies based on killing or sex sorting, generating male-only offspring via sex conversion would be economically beneficial by doubling the male output. Temperature-sensitive mutations known from the D. melanogaster transformer-2 gene (tra2ts) induce sex conversion at restrictive temperatures, while regular breeding of mutant strains is possible at permissive temperatures. Since tra2 is a conserved sex determination gene in many Diptera, including the major agricultural pest Ceratitis capitata, it is a promising candidate for the creation of a conditional sex conversion strategy in this Tephritid. Here, CRISPR/Cas9 homology-directed repair was used to induce the D. melanogaster-specific tra2ts SNPs in Cctra2. 100% female to male conversion was successfully achieved in flies homozygous for the tra2ts2 mutation. However, it was not possible, to identify a permissive temperature for the mutation allowing the rearing of a tra2ts2 homozygous line, as lowering the temperature below 18.5 °C interferes with regular breeding of the flies.

Host Suitability and Fitness-Related Parameters in Coptera haywardi (Hymenoptera: Diapriidae) Reared on Irradiated Ceratitis capitata (Diptera: Tephritidae) Pupae Stemming From the tsl Vienna-8 Genetic Sexing Strain.

Coptera haywardi (Ogloblin) is a pupal endoparasitoid of tephritid flies with great potential as a biological control agent worldwide as it does not attack other Diptera. To reach its full potential, its mass rearing needs to be enhanced lowering costs. Here, we focused on the use of irradiated pupae of Ceratitis capitata (Wiedemann) stemming from the temperature-sensitive lethal (tsl) Vienna-8 genetic sexing strain (= CcVienna-8), which is mass-produced in the San Juan Medfly and Parasitoid Mass Rearing Facility in Argentina. Exposure of 1- to 2-d-old CcVienna-8 pupae irradiated at 90 Gy to 6- to 8-d-old C. haywardi females at a 10:1 host/parasitoid ratio for 24 h turned out to be highly successful for the rearing of this parasitoid. High radiation doses (90-100 Gy) did not adversely influence fitness parameters of C. haywardi offspring F1, namely lifetime reproductive rates, adult life expectancy, and survival time. Demographic parameters in C. haywardi F1 from irradiated CcVienna-8 young pupae were improved compared to those values recorded from parasitoid originated from nonirradiated CcVienna-8 pupae. These findings will help to enhance parasitoid mass rearing for augmentative releases against medfly in Argentinean fruit-producing regions.

Field detection and predicted evolution of spinosad resistance in Ceratitis capitata.

BACKGROUND: The sustainable control of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), is compromised by the development of resistance to malathion and lambda-cyhalothrin in Spanish field populations. At present, field populations remain susceptible to spinosad. However, the resistant strain JW-100s has been obtained under laboratory selection with spinosad, and resistance has been associated with the presence of different mutations causing truncated transcripts of the α6 subunit of the nicotinic acetylcholine receptor (nAChRα6). RESULTS: An F1 screen assay followed by the molecular characterization of surviving flies has been used to search for spinosad-resistant alleles in field populations. Two different resistant alleles giving rise to truncated isoforms of Ccα6 have been identified, which corresponds to an estimated allelic frequency of at least 0.0023-0.0046. The fitness values of the resistant nAChRα6 alleles found in the laboratory strain JW-100s were estimated to be 0.4 for RR and 0.2 for SR. Mathematical modelling predicted that spinosad-resistant alleles will rapidly decline over time in field populations if their fitness cost was the same as estimated for laboratory-resistant alleles. However, they are predicted to increase in the field if their fitness cost is lower and resistance management strategies are not implemented. CONCLUSION: Spinosad-resistant alleles have been detected in field populations for the first time. Our modelling simulations indicate that the best option to delay the appearance of spinosad resistance would be its rotation with other insecticides without cross-resistance. The integrated F1 screen/molecular genetic analysis presented here can be used for future monitoring studies. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Development and Validation of Real-Time PCR Method to Estimate Stored Sperm in the Spermathecae of Ceratitis capitata (Diptera: Tephritidae).

The development of polymerase chain reaction (PCR) markers to identify the Y chromosome of Ceratitis capitata Wiedemann has permitted the detection of sperm transferred to females during mating. However, a molecular technique to quantify the sperm transferred has not yet become available. The current method to quantify the amount of sperm has been the direct counting of sperm heads. Thus, the purpose of this research was to develop and validate an accurate molecular method of diagnosis based on the application of an absolute quantitative real-time PCR, which allows the assessment of the quantity of sperm stored in the spermathecae. For this, Y-specific sequences were used to re-design and test distinct sperm markers. From the amplification product of samples detected as strong positives in conventional PCR, a cloning process of the target sequence was carried out to build the required standard curve. A series of known dilutions of this standard material was prepared for the absolute quantification process. A Roche Lightcycler 480 Real-Time PCR System and SYBRGreen fluorescent dye were used to quantify the sperm contained in the spermathecae of 4-d-old mated females and virgins. Wild-type and Vienna-8 strain sterile males were used to quantify the sperm transferred at four mating durations (10, 30, 60, and 90 min) under laboratory conditions. To validate the reported quantitative method, our results were compared by counting sperm heads under a fluorescent microscope using the same experimental design. In addition, DNA samples were also evaluated and compared by conventional PCR.

Worldwide Phylogeography of Ceratitis capitata (Diptera: Tephritidae) Using Mitochondrial DNA.

The Mediterranean fruit fly, Ceratitis capitata (Weidemann), is one of the most economically important tephritid species worldwide. It has spread across six geographic regions as a result of successful invasions and continues to cause substantial losses to agricultural communities. Our study examined 1,864 flies originating from 150 localities, using mitochondrial DNA sequencing methods. We tested for population structure and revealed the genetic diversity for 1,592 specimens gathered from 144 wild fly collections from 46 countries representing the entire geographic range for this species. We also include in this study 272 Sterile Insect Technique (SIT) specimens from four SIT facilities. We recovered 202 haplotypes from the current sampling and updated previously published work to reveal a total of 231 haplotypes for this pest. These data show population structure at and below the regional level for these collections, shedding light on the current demographics for this species. We observed four common haplotypes, seen among 62% of the samples sequenced that have worldwide distribution. Three haplotypes were seen in SIT flies, with one seen as the predominant haplotype. Our work showed that two of the haplotypes were private to SIT flies, not present among wild fly collections. However, a third haplotype common among wild fly collections was also seen in one SIT facility but at a low frequency based on the current sampling. We provide guidance on the interpretation of these methods for the source estimation of current and future infestations.