Conservation of shibire and RpII215 temperature-sensitive lethal mutations between Drosophila and Bactrocera tryoni.

The sterile insect technique can suppress and eliminate population outbreaks of the Australian horticultural pest, Bactrocera tryoni, the Queensland fruit fly. Sterile males mate with wild females that produce inviable embryos, causing population suppression or elimination. Current sterile insect releases are mixed sex, as the efficient removal of unrequired factory-reared females is not yet possible. In this paper, we assessed the known Drosophila melanogaster temperature-sensitive embryonic lethal alleles shibire (G268D, shits1) and RNA polymerase II 215 (R977C, RpII215ts) for potential use in developing B. tryoni genetic sexing strains (GSS) for the conditional removal of females. Complementation tests in D. melanogaster wild-type or temperature-sensitive genetic backgrounds were performed using the GAL4-UAS transgene expression system. A B. tryoni wild-type shibire isoform partially rescued Drosophila temperature lethality at 29°C by improving survivorship to pupation, while expressing B. tryoni shits1 failed to rescue the lethality, supporting a temperature-sensitive phenotype. Expression of the B. tryoni RpII215 wild-type protein rescued the lethality of D. melanogaster RpII215ts flies at 29°C. Overexpressing the B. tryoni RpII215ts allele in the D. melanogaster wild-type background unexpectedly produced a dominant lethal phenotype at 29°C. The B. tryoni shibire and RpII215 wild-type alleles were able to compensate, to varying degrees, for the function of the D. melanogaster temperature-sensitive proteins, supporting functional conservation across species. Shibire and RpII215 hold potential for developing insect strains that can selectively kill using elevated temperatures; however, alleles with milder effects than shits1 will need to be considered.

CRISPR/Cas9 Mutagenesis to Generate Novel Traits in Bactrocera tryoni for Sterile Insect Technique.

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.

Patterns of sperm use by twice-mated female Queensland fruit flies.

Multiple mating by females, polyandry, is common in insects, including in tephritid fruit flies. Female insects that remate commonly store sperm of multiple males. How the sperm of different males contribute to paternity is an important element of sexual selection. Sexual behavior and reproduction of the Queensland fruit fly (Qfly), Bactrocera tryoni, has been extensively investigated both in relation to understanding this economically important species' reproductive biology and in relation to implications for Sterile Insect Technique (SIT), whereby sterile flies are released to constrain reproduction of pest populations. Despite numerous studies of pre- and postcopulatory sexual selection in Qfly, there have been no direct studies of paternity patterns in polyandrous female Qflies. We used two morphologically distinguishable lines to investigate patterns of sperm use in Qfly. The two lines showed comparable mating performance evidenced by similar mating and remating frequency, copula duration, and proportion of second mate paternity (P2) between reciprocal crosses. The mechanism of sperm usage, with P2 close to 0.5 immediately after the second mating followed by gradual decrease of P2 as females aged, is most consistent with stratification or repositioning of sperm. Patterns observed in the present study are compared with the available information from other tephritid fruit flies, and are discussed in relation to this species' reproductive biology, known patterns of sperm storage, and SIT.

Lessons from Drosophila: Engineering Genetic Sexing Strains with Temperature-Sensitive Lethality for Sterile Insect Technique Applications.

A major obstacle of sterile insect technique (SIT) programs is the availability of robust sex-separation systems for conditional removal of females. Sterilized male-only releases improve SIT efficiency and cost-effectiveness for agricultural pests, whereas it is critical to remove female disease-vector pests prior to release as they maintain the capacity to transmit disease. Some of the most successful Genetic Sexing Strains (GSS) reared and released for SIT control were developed for Mediterranean fruit fly (Medfly), Ceratitis capitata, and carry a temperature sensitive lethal (tsl) mutation that eliminates female but not male embryos when heat treated. The Medfly tsl mutation was generated by random mutagenesis and the genetic mechanism causing this valuable heat sensitive phenotype remains unknown. Conditional temperature sensitive lethal mutations have also been developed using random mutagenesis in the insect model, Drosophila melanogaster, and were used for some of the founding genetic research published in the fields of neuro- and developmental biology. Here we review mutations in select D. melanogaster genes shibire, Notch, RNA polymerase II 215kDa, pale, transformer-2, Dsor1 and CK2α that cause temperature sensitive phenotypes. Precise introduction of orthologous point mutations in pest insect species with CRISPR/Cas9 genome editing technology holds potential to establish GSSs with embryonic lethality to improve and advance SIT pest control.

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.

Identification of Y-chromosome scaffolds of the Queensland fruit fly reveals a duplicated gyf gene paralogue common to many Bactrocera pest species.

Bactrocera tryoni (Queensland fruit fly) are polyphagous horticultural pests of eastern Australia. Heterogametic males contain a sex-determining Y-chromosome thought to be gene poor and repetitive. Here, we report 39 Y-chromosome scaffolds (~700 kb) from B. tryoni identified using genotype-by-sequencing data and whole-genome resequencing. Male diagnostic PCR assays validated eight Y-scaffolds, and one (Btry4096) contained a novel gene with five exons that encode a predicted 575 amino acid protein. The Y-gene, referred to as typo-gyf, is a truncated Y-chromosome paralogue of X-chromosome gene gyf (1773 aa). The Y-chromosome contained ~41 copies of typo-gyf, and expression occurred in male flies and embryos. Analysis of 13 tephritid transcriptomes confirmed typo-gyf expression in six additional Bactrocera species, including Bactrocera latifrons, Bactrocera dorsalis and Bactrocera zonata. Molecular dating estimated typo-gyf evolved within the past 8.02 million years (95% highest posterior density 10.56-5.52 million years), after the split with Bactrocera oleae. Phylogenetic analysis also highlighted complex evolutionary histories among several Bactrocera species, as discordant nuclear (116 genes) and mitochondrial (13 genes) topologies were observed. B. tryoni Y-sequences may provide useful sites for future transgene insertions, and typo-gyf could act as a Y-chromosome diagnostic marker for many Bactrocera species, although its function is unknown.