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.

A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations.

Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.

Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test a CRISPR-based homing gene drive for Culex quinquefasciatus, and show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes.

AePUb promoter length modulates gene expression in Aedes aegypti.

Molecular tools for modulating transgene expression in Aedes aegypti are few. Here we demonstrate that adjustments to the AePUb promoter length can alter expression levels of two reporter proteins in Ae. aegypti cell culture and in mosquitoes. This provides a simple means for increasing or decreasing expression of a gene of interest and easy translation from cells to whole insects.

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti.

Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects' biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos. Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard. Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control.

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.

Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test the first CRISPR-based homing gene drive for Culex quinquefasciatus, demonstrating the possibility of using this technology to control Culex mosquitoes. Our results show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes.

Accentuating the positive and eliminating the negative: Efficacy of TiO2 as digestibility index marker for poultry nutrition studies.

Inert digestibility index markers such as titanium dioxide are universally accepted to provide simple measurement of digestive tract retention and relative digestibility in poultry feeding trials. Their use underpins industry practice: specifically dosing regimens for adjunct enzymes added to animal feed. Among these, phytases, enzymes that degrade dietary phytate, inositol hexakisphosphate, represent a billion-dollar sector in an industry that raises ca. 70 billion chickens/annum. Unbeknown to the feed enzyme sector, is the growth in cell biology of use of titanium dioxide for enrichment of inositol phosphates from extracts of cells and tissues. The adoption of titanium dioxide in cell biology arises from its affinity under acid conditions for phosphates, suggesting that in feeding trial contexts that target phytate degradation this marker may not be as inert as assumed. We show that feed grade titanium dioxide enriches a mixed population of higher and lower inositol phosphates from acid solutions. Additionally, we compared the extractable inositol phosphates in gizzard and ileal digesta of 21day old male Ross 308 broilers fed three phytase doses (0, 500 and 6000 FTU/kg feed) and one inositol dose (2g/kg feed). This experiment was performed with or without titanium dioxide added as a digestibility index marker at a level of 0.5%, with all diets fed for 21 days. Analysis yielded no significant difference in effect of phytase inclusion in the presence or absence of titanium dioxide. Thus, despite the utility of titanium dioxide for recovery of inositol phosphates from biological samples, it seems that its use as an inert marker in digestibility trials is justified-as its inclusion in mash diets does not interfere with the recovery of inositol phosphates from digesta samples.

Manipulating Insect Sex Determination Pathways for Genetic Pest Management: Opportunities and Challenges.

Sex determination pathways in insects are generally characterised by an upstream primary signal, which is highly variable across species, and that regulates the splicing of a suite of downstream but highly-conserved genes (transformer, doublesex and fruitless). In turn, these downstream genes then regulate the expression of sex-specific characteristics in males and females. Identification of sex determination pathways has and continues to be, a critical component of insect population suppression technologies. For example, "first-generation" transgenic technologies such as fsRIDL (Female-Specific Release of Insects carrying Dominant Lethals) enabled efficient selective removal of females from a target population as a significant improvement on the sterile insect technique (SIT). Second-generation technologies such as CRISPR/Cas9 homing gene drives and precision-guided SIT (pgSIT) have used gene editing technologies to manipulate sex determination genes in vivo. The development of future, third-generation control technologies, such as Y-linked drives, (female to male) sex-reversal, or X-shredding, will require additional knowledge of aspects of sexual development, including a deeper understanding of the nature of primary signals and dosage compensation. This review shows how knowledge of sex determination in target pest species is fundamental to all phases of the development of control technologies.

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.

Toward a CRISPR-Cas9-Based Gene Drive in the Diamondback Moth Plutella xylostella.

Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipteran insects, yeast, and mice for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and single guide RNA (sgRNA) expression in the diamondback moth (DBM), Plutella xylostella, and test the first split gene drive system in a lepidopteran. The DBM is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable Cas9-medated germline cleavage as well as maternal and paternal Cas9 deposition were observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drives or other Cas9-based genetic control strategies in DBM and other lepidopterans.

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.

Genetic pest management and the background genetics of release strains.

Genetic pest management (GPM) methods involve releasing modified versions of a pest species to mate with wild pests in the target area. Proposed for a wide range of applications in public health, agriculture and conservation, most progress has been made with pest insects. Offspring of the released modified insects and wild pests carry the modification-which might be transgenes, artificially introduced Wolbachia or genetic damage from radiation, for example-but they also carry a complete haploid genome from their laboratory-reared parent, as well as one from their wild parent. Unless these F1 hybrids are completely unable to reproduce, further mating will lead to introgression of DNA sequences from the release strain into the wild population. We discuss issues around strain selection and the potential consequences of such introgression. We conclude that such introgression is probably harmless in almost all circumstances, and could, in theory, provide specific additional benefits to the release programme. We outline population monitoring approaches that could be used, going forward, to determine how background genetics may affect GPM. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.

Transmission efficiency drives host-microbe associations.

Sequencing technologies have fuelled a rapid rise in descriptions of microbial communities associated with hosts, but what is often harder to ascertain is the evolutionary significance of these symbioses. Here, we review the role of vertical (VT), horizontal (HT), environmental acquisition and mixed modes of transmission (MMT), in the establishment of animal host-microbe associations. We then model four properties of gut microbiota proposed as key to promoting animal host-microbe relationships: modes of transmission, host reproductive mode, host mate choice and host fitness. We found that: (i) MMT led to the highest frequencies of host-microbe associations, and that some environmental acquisition or HT of microbes was required for persistent associations to form unless VT was perfect; (ii) host reproductive mode (sexual versus asexual) and host mate choice (for microbe carriers versus non-carriers) had little impact on the establishment of host-microbe associations; (iii) host mate choice did not itself lead to reproductive isolation, but could reinforce it; and (iv) changes in host fitness due to host-microbe associations had a minimal impact upon the formation of co-associations. When we introduced a second population, into which host-microbe carriers could disperse but in which environmental acquisition did not occur, highly efficient VT was required for host-microbe co-associations to persist. Our study reveals that transmission mode is of key importance in establishing host-microbe associations.

Expanding the CRISPR Toolbox in Culicine Mosquitoes: In Vitro Validation of Pol III Promoters.

CRISPR-Cas9-based "gene drive" technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to date-and an expanded molecular toolbox with which to build them-will be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives.

Contribution of maternal effects to dietary selection in Mediterranean fruit flies.

Individual responses to dietary variation represent a fundamental component of fitness, and nutritional adaptation can occur over just a few generations. Maternal effects can show marked proximate responses to nutrition, but whether they contribute to longer term dietary adaptation is unclear. Here, we tested the hypotheses that maternal effects: (i) contribute to dietary adaptation, (ii) diminish when dietary conditions are constant between generations, (iii) are trait-specific and (iv) interact with high- and low-quality food. We used experimental evolution regimes in the medfly (Ceratitis capitata) to test these predictions by subjecting an outbred laboratory-adapted population to replicated experimental evolution on either constant high calorie sugar ('A') or low-calorie starch ('S') larval diets, with a standard adult diet across both regimes. We measured the contribution of maternal effects by comparing developmental and adult phenotypes of individuals reared on their own diet with those swapped onto the opposite diet for either one or two generations (high and low maternal effect conditions, respectively), both at the start and after 30 generations of selection. Initially, there were strong maternal effects on female body mass and male mating success but not larval survival. Interestingly, the initial maternal effects observed in female body mass and male mating success showed sex-specific interactions when individuals from high calorie regimes were tested on low calorie diets. However, as populations responded to selection, the effects of maternal provisioning on all traits diminished. The results broadly supported the predictions. They show how the contribution of maternal effects to dietary responses evolves in a context-dependent manner, with significant variation across different fitness-related traits. We conclude that maternal effects can evolve during nutritional adaptation and hence may be an important life history trait to measure, rather than to routinely minimize.

Testing for Assortative Mating by Diet in Drosophila melanogaster.

Experimental studies of the evolution of reproductive isolation in real time are a powerful way to reveal the way that fundamental processes, such as mate choice, initiate divergence. Mate choice, while frequently described in females, can occur in either sex, and can be affected by the genetics or environment of an individual. Here we describe simple protocols for assessing mating outcomes in fruit flies, which in this context can be used to assess reproductive isolation derived from rearing on different diets over multiple generations.

Diet, Gut Microbes and Host Mate Choice: Understanding the significance of microbiome effects on host mate choice requires a case by case evaluation.

All organisms live in close association with microbes. However, not all such associations are meaningful in an evolutionary context. Current debate concerns whether hosts and microbes are best described as communities of individuals or as holobionts (selective units of hosts plus their microbes). Recent reports that assortative mating of hosts by diet can be mediated by commensal gut microbes have attracted interest as a potential route to host reproductive isolation (RI). Here, the authors discuss logical problems with this line of argument. The authors briefly review how microbes can affect host mating preferences and evaluate recent findings from fruitflies. Endosymbionts can potentially influence host RI given stable and recurrent co-association of hosts and microbes over evolutionary time. However, observations of co-occurrence of microbes and hosts are ripe for misinterpretation and such associations will rarely represent a meaningful holobiont. A framework in which hosts and their microbes are independent evolutionary units provides the only satisfactory explanation for the observed range of effects and associations.

Recent advances in threshold-dependent gene drives for mosquitoes.

Mosquito-borne diseases, such as malaria, dengue and chikungunya, cause morbidity and mortality around the world. Recent advances in gene drives have produced control methods that could theoretically modify all populations of a disease vector, from a single release, making whole species less able to transmit pathogens. This ability has caused both excitement, at the prospect of global eradication of mosquito-borne diseases, and concern around safeguards. Drive mechanisms that require individuals to be released at high frequency before genes will spread can therefore be desirable as they are potentially localised and reversible. These include underdominance-based strategies and use of the reproductive parasite Wolbachia Here, we review recent advances in practical applications and mathematical analyses of these threshold-dependent gene drives with a focus on implementation in Aedes aegypti, highlighting their mechanisms and the role of fitness costs on introduction frequencies. Drawing on the parallels between these systems offers useful insights into practical, controlled application of localised drives, and allows us to assess the requirements needed for gene drive reversal.