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The satyr of Greek mythology was half‐man, half‐goat, with an animal persona signifying immoderate sexual appetites. In biology, satyrization is the disruption of reproduction in matings between closely related species. Interestingly, its effects are often reciprocally asymmetric, manifesting more strongly in one direction of heterospecific mating than the other. Heterospecific matings are well known to result in female fitness costs due to the production of sterile or inviable hybrid offspring and can also occur due to reduced female sexual receptivity, lowering the likelihood of any subsequent conspecific matings. Here we investigated the costs and mechanisms of satyrization in the Drosophila melanogaster species subgroup of fruitflies. The results showed that D. simulans females experienced higher fitness costs from a loss of remating opportunities due to significantly reduced post‐mating sexual receptivity than did D. melanogaster females, as a result of reciprocal heterospecific matings. Reciprocal tests of the effects of male reproductive accessory gland protein (Acp) injections on female receptivity in pairwise comparisons between D. melanogaster and five other species within the melanogaster species subgroup revealed significant post‐mating receptivity asymmetries. This was due to variation in the effects of heterospecific Acps within species with which D. melanogaster can mate, and significant but nonasymmetric Acp effects in species with which it cannot. We conclude that asymmetric satyrization due to post‐mating effects of Acps may be common among diverging and hybridising species. The findings are of interest in understanding the evolution of reproductive isolation and species divergence., Seminal Fluid Proteins (Sfps) are transferred with the ejacuate during mating, conferring a variety of post‐mating effects on females. We found asymmetries in the strength and effects of these Sfps in reciprocal cross‐species comparisons between closely related Drosophila species.

CRISPR‐based gene drives bias inheritance in their favour by inducing double‐stranded breaks (DSBs) at wild‐type homologous loci and using the drive transgene as a repair template—converting drive heterozygotes into homozygotes. Recent studies have shown that alternate end‐joining repair mechanisms produce cut‐resistant alleles that rapidly induce drive failure. Multiplexing—simultaneously targeting multiple sites at the wild‐type locus—is commonly assumed to overcome this issue since resistance would need to develop at all target sites for the system to fail. This may work for some population suppression drives targeting essential (e.g. viability or fertility) genes if careful design can ensure cut‐resistant alleles themselves have low fitness. However, here, models are used to demonstrate that this approach will be ineffective when targeting neutral loci. We then go on to compare the performance of four alternative population‐level multiplexing approaches with standard individual‐level multiplexing. Two of these approaches have mechanisms preventing them from becoming linked, thus avoiding multiple simultaneous DSBs and giving a large improvement. Releasing multiple unlinked drives gives a modest improvement, while releasing multiple drives that may become linked over time produces a decrease in performance under the conditions tested here. Based on performance and technical feasibility, we then take one approach forward for further investigation, demonstrating its robustness to different performance parameters and its potential for controlling very large target populations.

Recently, a novel sex‐determination system was identified in the silkworm (Bombyx mori) in which a piwi‐interacting RNA (piRNA) encoded on the female‐specific W chromosome silences a Z‐linked gene (Masculinizer) that would otherwise initiate male sex‐determination and dosage compensation. Masculinizer provides various opportunities for developing improved genetic pest management tools. A pest lepidopteran in which a genetic pest management system has been developed, but which would benefit greatly from such improved designs, is the diamondback moth, Plutella xylostella. However, Masculinizer has not yet been identified in this species. Here, focusing on the previously described ‘masculinizing’ domain of B. mori Masculinizer, we identify P. xylostella Masculinizer (PxyMasc). We show that PxyMasc is Z‐linked, regulates sex‐specific alternative splicing of doublesex and is necessary for male survival. Similar results in B. mori suggest this survival effect is possibly through failure to initiate male dosage compensation. The highly conserved function and location of this gene between these two distantly related lepidopterans suggests a deep role for Masculinizer in the sex‐determination systems of the Lepidoptera., We have identified a Masculinizer homologue (PxyMasc) in the diamondback moth, Plutella xylostella.As in Bombyx mori, this gene controls doublesex splicing and male viability suggesting deep conservation of this sex‐determination mechanism.PxyMasc functions early (3–6 h) in embryonic development to initiate male‐form doublesex splicing, but its transcript is rapidly (6–24 h) eliminated in females.

The insertion of exogenous genetic cargo into insects using transposable elements is a powerful research tool with potential applications in meeting food security and public health challenges facing humanity. piggyBac is the transposable element most commonly utilized for insect germline transformation. The described efficiency of this process is variable in the published literature, and a comprehensive review of transformation efficiency in insects is lacking. This study compared and contrasted all available published data with a comprehensive data set provided by a biotechnology group specializing in insect transformation. Based on analysis of these data, with particular focus on the more complete observational data from the biotechnology group, we designed a decision tool to aid researchers' decision-making when using piggyBac to transform insects by microinjection. A combination of statistical techniques was used to define appropriate summary statistics of piggyBac transformation efficiency by species and insect order. Publication bias was assessed by comparing the data sets. The bias was assessed using strategies co-opted from the medical literature. The work culminated in building the Goldilocks decision tool, a Markov-Chain Monte-Carlo simulation operated via a graphical interface and providing guidance on best practice for those seeking to transform insects using piggyBac.

Genetic control strategies offer great potential for the sustainable and effective control of insect pests. These strategies involve the field release of transgenic insects with the aim of introducing engineered alleles into wild populations, either permanently or transiently. Their efficacy can therefore be reduced if transgene-associated fitness costs reduce the relative performance of released insects. We describe a method of measuring the fitness costs associated with transgenes by analyzing their evolutionary trajectories when placed in competition with wild-type alleles in replicated cage populations. Using this method, we estimated lifetime fitness costs associated with two repressible female-lethal transgenes in the diamondback moth and olive fly as being acceptable for field suppression programs. Furthermore, using these estimates of genotype-level fitness costs, we were able to project longer-term evolutionary trajectories for the transgenes investigated. Results from these projections demonstrate that although transgene-associated fitness costs will ultimately cause these transgenes to become extinct, even when engineered lethality is repressed, they may persist for varying periods of time before doing so. This implies that tetracycline-mediated transgene field persistence in these strains is unlikely and suggests that realistic estimates of transgene-associated fitness costs may be useful in trialing 'uncoupled' gene drive system components in the field.

The diamondback moth, Plutella xylostella, is one of the most economically important agricultural pests. The larvae of this moth cause damage by feeding on the foliage of cruciferous vegetables such as cabbage, broccoli, cauliflower and rapeseed. Control generally comprises chemical treatment; however, the diamondback moth is renowned for rapid development of resistance to pesticides. Other methods, such as biological control, have not been able to provide adequate protection. Germline transformation of pest insects has become available in recent years as an enabling technology for new genetics-based control methods, such as the Release of Insects carrying a Dominant Lethal (RIDL(®) ). In the present study, we report the first transformation of the diamondback moth, using the piggyBac transposable element, by embryo microinjection. In generating transgenic strains using four different constructs, the function of three regulatory sequences in this moth was demonstrated in driving expression of fluorescent proteins. The transformation rates achieved, 0.48-0.68%, are relatively low compared with those described in other Lepidoptera, but not prohibitive, and are likely to increase with experience. We anticipate that germline transformation of the diamondback moth will permit the development of RIDL strains for use against this pest and facilitate the wider use of this species as a model organism for basic studies.

The sterile insect technique (SIT), when used for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), generally relies on the release of sterile flies of only the male sex. Male selection is achieved through the use of a genetic sexing strain (GSS) in which females are killed by heat treatment in the generation prior to release. Transgenic sexing strains (TSS) have been developed that perform the same function of female-lethality, this time by withholding tetracycline (or related compounds) from the larval diet. The use of TSS may allow for certain problems associated with conventional GSS, such as strain instability and reduced productivity in mass-rearing, to be avoided. The performance, and principally the sexual competitiveness, of released male flies is important for the success of an SIT control programme. This study describes field cage experiments in which the competitiveness of males from a TSS (OX3376B) was compared with that of a conventional GSS (VIENNA-8) and two wild-type strains (TOLIMAN and ARG). When competing for female mates with wild-type males, OX3376B male performance was acceptable. When OX3376B males competed directly for mates with VIENNA-8 males, VIENNA-8 slightly outperformed the TSS males. Parallel tests, in which wild-type males competed with either OX3376B or VIENNA-8 males, showed that males from both sexing strains were highly competitive with wild-type males. These results suggest that OX3376B in particular, and TSS in general, show sufficiently good mating competitiveness to merit further research into their suitability for eventual use in SIT programmes.

Germ-line transformation of a major agricultural pest, the Mexican fruit fly (Anastrepha ludens Loew, Mexfly), was achieved using composite piggyBac transposable elements marked with green, yellow and red fluorescent proteins (CopGreen, PhiYFP and J-Red). We also investigated the possibility of generating transposon-free insertions, in order to address potential concerns relating to proposed field use of transgenic Mexfly. We describe a highly efficient method for transforming Mexfly, compare efficiency of piggyBac terminal sequences for transformation and also describe the derivation of a transposon-free insertion line. The development of an efficient transformation system for Mexfly holds great promise for improved applications of the sterile insect technique, a major component of the present control measures for this economically important pest species.

Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germ-line transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species.