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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.

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. The white pupae (wp) phenotype has been used for decades to selectively remove females of tephritid species in genetic sexing, but the determining gene is unknown. Here, the authors show that wp phenotype is produced by parallel mutations in a Major Facilitator Superfamily domain containing gene across multiple species.

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.

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 ( tra2 ts ) 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 tra2 ts SNPs in Cctra2 . 100% female to male conversion was successfully achieved in flies homozygous for the tra2 ts2 mutation. However, it was not possible, to identify a permissive temperature for the mutation allowing the rearing of a tra2 ts2 homozygous line, as lowering the temperature below 18.5 °C interferes with regular breeding of the flies.

The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing vast economical damage in horticultural systems. A currently used control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. Transgenic approaches hold great promise to improve key aspects of a successful SIT program. However, there is strict or even prohibitive legislation regarding the release of genetically modified organisms (GMO), while novel CRISPR-Cas technologies might allow to develop genetically enhanced strains for SIT programs classified as non-transgenic. 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. Considering the possibility that CRISPR-induced alterations in organisms could be classified as a non-GMO in the US and Europe, our approach to homology-directed repair genome editing can be used to genetically improve strains for pest control systems like SIT without the need to struggle with GMO directives. Furthermore, it can be used to recreate and use mutations, found in classical mutagenesis screens, for pest control systems.

Flooding insect pest populations with huge numbers of sterilized males is an effective mean of biological control as they mate with, but cannot fertilize, wild females. The greatest challenge of the sterile insect technique (SIT) is the removal of unrequired factory reared females prior to sterilization and release. Spontaneous white-pupae (wp−) color mutations have been integrated as a dimorphic selectable marker into historical SIT strains for three major tephritid fruit fly pests, Bactrocera dorsalis, Ceratitis capitata and Zeugodacus cucurbitae. Here we identify parallel genetic mutations causing the phenotype in all three species using diverse experimental approaches. The B. dorsalis wp− locus was introgressed into a related species, Bactrocera tryoni, and whole-genome sequencing identified a 37 bp truncating mutation within a gene containing a Major Facilitator Superfamily domain (MFS). In C. capitata and Z. cucurbitae, cytogenetics, comparative genomics and transcriptomic analysis of strains carrying brown and white pupae phenotypes identified an 8,150 bp insertion of a putative transposon into the C. capitata MFS ortholog and a 13 bp deletion in the Z. cucurbitae ortholog. In B. tryoni CRISPR/Cas9-mediated knock-out of the putative Bt_wp developed mosaic white and brown puparium colors in G0, and G1 progeny with recessive white pupae phenotypes. In C. capitata, complementation crosses of CRISPR-induced wp− mutants to flies carrying the naturally occurring recessive wp− mutation confirmed the role of the gene. Gene editing technology carries the potential for engineering white pupae phenotypes and generating dimorphic SIT strains in other tephritids or insect pest species. Competing Interest Statement The authors have declared no competing interest.