New self-sexing Aedes aegypti strain eliminates barriers to scalable and sustainable vector control for governments and communities in dengue-prone environments.

For more than 60 years, efforts to develop mating-based mosquito control technologies have largely failed to produce solutions that are both effective and scalable, keeping them out of reach of most governments and communities in disease-impacted regions globally. High pest suppression levels in trials have yet to fully translate into broad and effective Aedes aegypti control solutions. Two primary challenges to date-the need for complex sex-sorting to prevent female releases, and cumbersome processes for rearing and releasing male adult mosquitoes-present significant barriers for existing methods. As the host range of Aedes aegypti continues to advance into new geographies due to increasing globalisation and climate change, traditional chemical-based approaches are under mounting pressure from both more stringent regulatory processes and the ongoing development of insecticide resistance. It is no exaggeration to state that new tools, which are equal parts effective and scalable, are needed now more than ever. This paper describes the development and field evaluation of a new self-sexing strain of Aedes aegypti that has been designed to combine targeted vector suppression, operational simplicity, and cost-effectiveness for use in disease-prone regions. This conditional, self-limiting trait uses the sex-determination gene doublesex linked to the tetracycline-off genetic switch to cause complete female lethality in early larval development. With no female progeny survival, sex sorting is no longer required, eliminating the need for large-scale mosquito production facilities or physical sex-separation. In deployment operations, this translates to the ability to generate multiple generations of suppression for each mosquito released, while being entirely self-limiting. To evaluate these potential benefits, a field trial was carried out in densely-populated urban, dengue-prone neighbourhoods in Brazil, wherein the strain was able to suppress wild mosquito populations by up to 96%, demonstrating the utility of this self-sexing approach for biological vector control. In doing so, it has shown that such strains offer the critical components necessary to make these tools highly accessible, and thus they harbour the potential to transition mating-based approaches to effective and sustainable vector control tools that are within reach of governments and at-risk communities who may have only limited resources.

Human papillomavirus (HPV) detection in vaginal self-samples: evaluation of eNat® as an alternative suspension medium to ThinPrep®PreservCyt® for vaginal swabs.

Human Papillomavirus (HPV) testing on self-collected samples allows for improved coverage rates of cervical cancer (CC) screening programs. ThinPrep®PreservCyt® (HOLOGIC®, USA) medium is widely used for the suspension of cervical and vaginal self-samples. However, this medium is costly, toxic, and flammable, involving special handling procedures which make its use difficult in screening programs, particularly in low- and middle-income countries. This pilot study aimed to evaluate the analytical performance of eNat ® (Copan SpA), an alternative non-alcohol-based suspension medium, compared to ThinPrep®PreservCyt® (HOLOGIC®) for high-risk HPV (hrHPV) detection in vaginal self-collected swabs using three different real-time polymerase chain reaction (RT-PCR) HPV assays: Anyplex™II HPV28 (Seegene, Korea), Papilloplex® High Risk HPV (GeneFirst, UK), and HPV OncoPredict (Hiantis, Italy). 30 women, referred to colposcopy, were enrolled in this observational, prospective pilot study and asked to collect two vaginal self-taken samples, which were suspended in 5 mL of ThinPrep®PreservCyt® or eNat®. Nucleic acids were extracted from 200 µL using Microlab Nimbus platform (Seegene, Korea) and tested with the three different RT-PCR full-genotyping high-risk HPV assays. The HPV results of vaginal samples resuspended in the two different media were compared to those obtained from the reference clinician-collected cervical sample from the same woman. hrHPV detection in vaginal self-samples suspended in both media demonstrated a substantial agreement with cervical samples with the three assays under-investigation (0.667

Engineered expression of the invertebrate-specific scorpion toxin AaHIT reduces adult longevity and female fecundity in the diamondback moth Plutella xylostella.

BACKGROUND: Previous genetic pest management (GPM) systems in diamondback moth (DBM) have relied on expressing lethal proteins ('effectors') that are 'cell-autonomous', that is, they do not leave the cell in which they are expressed. To increase the flexibility of future GPM systems in DBM, we aimed to assess the use of a non-cell-autonomous, invertebrate-specific, neurotoxic effector - the scorpion toxin AaHIT. This AaHIT effector was designed to be secreted by expressing cells, potentially leading to effects on distant cells, specifically neuromuscular junctions. RESULTS: Expression of AaHIT caused a 'shaking/quivering' phenotype that could be repressed by provision of an antidote (tetracycline): a phenotype consistent with the AaHIT mode-of-action. This effect was more pronounced when AaHIT expression was driven by the Hr5/ie1 promoter (82.44% of males, 65.14% of females) rather than Op/ie2 (57.35% of males, 48.39% of females). Contrary to expectations, the shaking phenotype and observed fitness costs were limited to adults in which they caused severe reductions in mean longevity (-81%) and median female fecundity (-93%). Quantitative polymerase chain reactions of AaHIT expression patterns and analysis of piggyBac-mediated transgene insertion sites suggest that restriction of the observed effects to the adult stages may be due to the influence of the local genomic environment on the tetO-AaHIT transgene. CONCLUSION: We demonstrated the feasibility of using non-cell-autonomous effectors within a GPM context for the first time in Lepidoptera, one of the most economically damaging orders of insects. These findings provide a framework for extending this system to other pest Lepidoptera and to other secreted effectors. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Engineered female-specific lethality for control of pest Lepidoptera.

The sterile insect technique (SIT) is a pest control strategy involving the mass release of radiation-sterilized insects, which reduce the target population through nonviable matings. In Lepidoptera, SIT could be more broadly applicable if the deleterious effects of sterilization by irradiation could be avoided. Moreover, male-only release can improve the efficacy of SIT. Adequate methods of male-only production in Lepidoptera are currently lacking, in contrast to some Diptera. We describe a synthetic genetic system that allows male-only moth production for SIT and also replaces radiation sterilization with inherited female-specific lethality. We sequenced and characterized the doublesex (dsx) gene from the pink bollworm (Pectinophora gossypiella). Sex-alternate splicing from dsx was used to develop a conditional lethal genetic sexing system in two pest moths: the diamondback moth (Plutella xylostella) and pink bollworm. This system shows promise for enhancing existing pink bollworm SIT, as well as broadening SIT-type control to diamondback moth and other Lepidoptera.

Engineered repressible lethality for controlling the pink bollworm, a lepidopteran pest of cotton.

The sterile insect technique (SIT) is an environmentally friendly method of pest control in which insects are mass-produced, irradiated and released to mate with wild counterparts. SIT has been used to control major pest insects including the pink bollworm (Pectinophora gossypiella Saunders), a global pest of cotton. Transgenic technology has the potential to overcome disadvantages associated with the SIT, such as the damaging effects of radiation on released insects. A method called RIDL (Release of Insects carrying a Dominant Lethal) is designed to circumvent the need to irradiate insects before release. Premature death of insects' progeny can be engineered to provide an equivalent to sterilisation. Moreover, this trait can be suppressed by the provision of a dietary antidote. In the pink bollworm, we generated transformed strains using different DNA constructs, which showed moderate-to-100% engineered mortality. In permissive conditions, this effect was largely suppressed. Survival data on cotton in field cages indicated that field conditions increase the lethal effect. One strain, called OX3402C, showed highly penetrant and highly repressible lethality, and was tested on host plants where its larvae caused minimal damage before death. These results highlight a potentially valuable insecticide-free tool against pink bollworm, and indicate its potential for development in other lepidopteran pests.

Use of a regulatory mechanism of sex determination in pest insect control.

The sexual development of an insect is defined through a hierarchical control of several sex determining genes. Of these genes, transformer (tra) and doublesex (dsx) are well characterized and functionally conserved, especially dsx. Both genes are regulated at the transcriptional level through sex-specific alternative splicing. Incorporation of a genetically engineered sexspecific splicing module derived from these genes in transgenic systems, such as RIDL (release of insects carrying a dominant lethal), would allow the production of male-only insects for control programmes without any physical intervention.

Effect of chemotherapeutic treatment on cytokine (IFN-gamma, IL-2, IL-4, IL-5, IL-10) gene transcription in response to specific antigens in Brugia malayi-infected Mastomys coucha.

Cytokine (interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10) gene transcription in response to filarial antigens was determined in peripheral blood mononuclear cells of Brugia malayi-infected Mastomys coucha in the course of untreated and chemotherapeutically abbreviated infections. Transcript levels in infected untreated animals suggest particular time courses for the various cytokines with ongoing parasite development and differing efficacies of female, male, microfilarial, and L3 antigens in inducing cytokine gene transcription. Gene transcription of both of Th1- and Th2-associated cytokines were initiated in the course of the infection in a manner that does not fit in a simple Th1-Th2 paradigm. IFN-gamma and IL-4 gene transcripts prevailed during prepatency. In case of the other cytokine genes considered in the study, transcription in general peaked around beginning of patency. During the phase of increasing microfilaremia (approximately 120-180 days p. i.) cytokine gene transcription was generally decreased. Later on, when the parasitemia had leveled off, except IFN-gamma, transcript levels often tended to increase. In chemotherapeutically treated animals, the outcome varied with the different efficacies of the drugs employed. The highly microfilaricidal cyclodepsipeptide BAY 44-4400 eliminated circulating microfilariae and partially sterilized adult worms without killing them. This kind of treatment hardly affected cytokine responses. In contrast, the therapy with Flubendazole, a selectively macrofilaricidal benzimidazole, and particularly the application of CGP 20376, a highly efficient microfilaricidal and macrofilaricidal benzthiazole, resulted in enhanced transcription of the Th1-associated IFN-gamma and IL-2 genes as well as of the Th2-associated IL-5 gene 2-3 months after treatment. IL-10 gene transcription seemed transiently increased after 1 month. There was no effect of any treatment on the IL-4 gene transcription.

Genetic sexing through the use of Y-linked transgenes.

Sterile insect technique (SIT)-based pest control programs rely on the mass release of sterile insects to reduce the wild target population. In many cases, it is desirable to release only males. Sterile females may cause damage, e.g., disease transmission by mosquitoes or crop damage via oviposition by the Mediterranean fruit fly (Medfly). Also, sterile females may decrease the effectiveness of released males by distracting them from seeking out wild females. To eliminate females from the release population, a suitable sexual dimorphism is required. For several pest species, genetic sexing strains have been constructed in which such a dimorphism has been induced by genetics. Classical strains were based on the translocation to the Y chromosome of a selectable marker, which is therefore expressed only in males. Recently, several prototype strains have been constructed using sex-specific expression of markers or conditional lethal genes from autosomal insertions of transgenes. Here, we describe a novel genetic sexing strategy based on the use of Y-linked transgenes expressing fluorescent proteins. We demonstrate the feasibility of this strategy in a major pest species, Ceratitis capitata (Wiedemann), and discuss the advantages and disadvantages relative to other genetic sexing methods and potential applicability to other species.

Female-specific insect lethality engineered using alternative splicing.

The Sterile Insect Technique is a species-specific and environmentally friendly method of pest control involving mass release of sterilized insects that reduce the wild population through infertile matings. Insects carrying a female-specific autocidal genetic system offer an attractive alternative to conventional sterilization methods while also eliminating females from the release population. We exploited sex-specific alternative splicing in insects to engineer female-specific autocidal genetic systems in the Mediterranean fruit fly, Ceratitis capitata. These rely on the insertion of cassette exons from the C. capitata transformer gene into a heterologous tetracycline-repressible transactivator such that the transactivator transcript is disrupted in male splice variants but not in the female-specific one. As the key components of these systems function across a broad phylogenetic range, this strategy addresses the paucity of sex-specific expression systems (e.g., early-acting, female-specific promoters) in insects other than Drosophila melanogaster. The approach may have wide applicability for regulating gene expression in other organisms, particularly for combinatorial control with appropriate promoters.

Transposon-free insertions for insect genetic engineering.

Methods involving the release of transgenic insects in the field hold great promise for controlling vector-borne diseases and agricultural pests. Insect transformation depends on nonautonomous transposable elements as gene vectors. The resulting insertions are stable in the absence of suitable transposase, however, such absence cannot always be guaranteed. We describe a method for post-integration elimination of all transposon sequences in the pest insect Medfly, Ceratitis capitata. The resulting insertions lack transposon sequences and are therefore impervious to transposase activity.

A dominant lethal genetic system for autocidal control of the Mediterranean fruitfly.

The Sterile Insect Technique (SIT) used to control insect pests relies on the release of large numbers of radiation-sterilized insects. Irradiation can have a negative impact on the subsequent performance of the released insects and therefore on the cost and effectiveness of a control program. This and other problems associated with current SIT programs could be overcome by the use of recombinant DNA methods and molecular genetics. Here we describe the construction of strains of the Mediterranean fruit fly (medfly) harboring a tetracycline-repressible transactivator (tTA) that causes lethality in early developmental stages of the heterozygous progeny but has little effect on the survival of the parental transgenic tTA insects. We show that these properties should prove advantageous for the implementation of insect pest control programs.