Investigating the potential of X chromosome shredding for mouse genetic biocontrol.

CRISPR-Cas9 technology has facilitated development of strategies that can potentially provide more humane and effective methods to control invasive vertebrate species, such as mice. One promising strategy is X chromosome shredding which aims to bias offspring towards males, resulting in a gradual and unsustainable decline of females. This method has been explored in insects with encouraging results. Here, we investigated this strategy in Mus musculus by targeting repeat DNA sequences on the X chromosome with the aim of inducing sufficient DNA damage to specifically eliminate X chromosome-bearing sperm during gametogenesis. We tested three different guide RNAs (gRNAs) targeting different repeats on the X chromosome, together with three male germline-specific promoters for inducing Cas9 expression at different stages of spermatogenesis. A modest bias towards mature Y-bearing sperm was detected in some transgenic males, although this did not translate into significant male-biasing of offspring. Instead, cleavage of the X chromosome during meiosis typically resulted in a spermatogenic block, manifest as small testes volume, empty tubules, low sperm concentration, and sub/infertility. Our study highlights the importance of controlling the timing of CRISPR-Cas9 activity during mammalian spermatogenesis and the sensitivity of spermatocytes to X chromosome disruption.

Improved reference quality genome sequence of the plastic-degrading greater wax moth, Galleria mellonella.

Galleria mellonella is a pest of honeybees in many countries because its larvae feed on beeswax. However, G. mellonella larvae can also eat various plastics, including polyethylene, polystyrene, and polypropylene, and therefore, the species is garnering increasing interest as a tool for plastic biodegradation research. This paper presents an improved genome (99.3% completed lepidoptera_odb10 BUSCO; genome mode) for G. mellonella. This 472 Mb genome is in 221 contigs with an N50 of 6.4 Mb and contains 13,604 protein-coding genes. Genes that code for known and putative polyethylene-degrading enzymes and their similarity to proteins found in other Lepidoptera are highlighted. An analysis of secretory proteins more likely to be involved in the plastic catabolic process has also been carried out.

The evolving story of sulfoxaflor resistance in the green peach aphid, Myzus persicae (Sulzer).

BACKGROUND: The green peach aphid, Myzus persicae (Sulzer), is one of the most economically important crop pests worldwide. Insecticide resistance in this pest was first detected over 60 years ago, with resistance in M. persicae now spanning over 80 active ingredients. Sulfoxaflor is a relatively new insecticide that is primarily used to control sap-feeding insects. In 2018 resistance to sulfoxaflor was discovered in field populations of M. persicae in Australia. This study aimed to determine the current distribution and phenotypic levels of sulfoxaflor resistance in Australian clones of M. persicae and to investigate how these patterns relate to clonal type. RESULTS: For the first time, we show there is low-level resistance (8-26-fold) distributed across Australia, with resistance being detected in aphids collected from approximately 20% of all M. persicae collected and screened. Furthermore, this study shows sulfoxaflor resistance is found in two M. persicae haplotypes, providing evidence that there have been multiple independent evolutionary events which have given rise to sulfoxaflor resistance in this species. CONCLUSION: These findings have important implications for the chemical control of M. persicae in Australia, especially when considering the broader genetic background of these aphids which are known to harbour a number of other insecticide resistance mechanisms. We recommend continuous monitoring of sulfoxaflor resistance in field populations of M. persicae (in Australia and elsewhere) and further research into the underlying genetic mechanisms conferring resistance to sulfoxaflor in both clonal haplotypes. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The redlegged earth mite draft genome provides new insights into pesticide resistance evolution and demography in its invasive Australian range.

Genomic data provide valuable insights into pest management issues such as resistance evolution, historical patterns of pest invasions and ongoing population dynamics. We assembled the first reference genome for the redlegged earth mite, Halotydeus destructor (Tucker, 1925), to investigate adaptation to pesticide pressures and demography in its invasive Australian range using whole-genome pool-seq data from regionally distributed populations. Our reference genome comprises 132 autosomal contigs, with a total length of 48.90 Mb. We observed a large complex of ace genes, which has presumably evolved from a long history of organophosphate selection in H. destructor and may contribute towards organophosphate resistance through copy number variation, target-site mutations and structural variants. In the putative ancestral H. destructor ace gene, we identified three target-site mutations (G119S, A201S and F331Y) segregating in organophosphate-resistant populations. Additionally, we identified two new para sodium channel gene mutations (L925I and F1020Y) that may contribute to pyrethroid resistance. Regional structuring observed in population genomic analyses indicates that gene flow in H. destructor does not homogenize populations across large geographic distances. However, our demographic analyses were equivocal on the magnitude of gene flow; the short invasion history of H. destructor makes it difficult to distinguish scenarios of complete isolation vs. ongoing migration. Nonetheless, we identified clear signatures of reduced genetic diversity and smaller inferred effective population sizes in eastern vs. western populations, which is consistent with the stepping-stone invasion pathway of this pest in Australia. These new insights will inform development of diagnostic genetic markers of resistance, further investigation into the multifaceted organophosphate resistance mechanism and predictive modelling of resistance evolution and spread.

Leveraging a natural murine meiotic drive to suppress invasive populations.

Invasive rodents are a major cause of environmental damage and biodiversity loss, particularly on islands. Unlike insects, genetic biocontrol strategies including population-suppressing gene drives with biased inheritance have not been developed in mice. Here, we demonstrate a gene drive strategy (tCRISPR) that leverages super-Mendelian transmission of the t haplotype to spread inactivating mutations in a haplosufficient female fertility gene (Prl). Using spatially explicit individual-based in silico modeling, we show that tCRISPR can eradicate island populations under a range of realistic field-based parameter values. We also engineer transgenic tCRISPR mice that, crucially, exhibit biased transmission of the modified t haplotype and Prl mutations at levels our modeling predicts would be sufficient for eradication. This is an example of a feasible gene drive system for invasive alien rodent population control.

Insects Co-opt Host Genes to Overcome Plant Defences.

Insect pests of plants, such as whiteflies, cause immense economic damage both through direct feeding and by transmitting viruses. In a major breakthrough, a paper by Xia et al.1 shows that some whiteflies have co-opted a gene from their plant host that has helped them neutralize a key component of the plant's defense. Plants produce a range of toxins as part of their defense against insect predation, and Xia et al. 1 show that, through a horizontal gene transfer (HGT) event from plant to insect, some whiteflies have acquired a gene whose original function was to protect the plants themselves from such damaging toxins through chemical modification that converts them to less harmful forms. Targeting of this gene in whiteflies using RNAi technology provided effective resistance in this ground-breaking study, which should lead others interested in crop protection to explore genes that have been transferred from plants to insects.

Overexpression of UDP-glucuronosyltransferase and cytochrome P450 enzymes confers resistance to sulfoxaflor in field populations of the aphid, Myzus persicae.

The green peach aphid, Myzus persicae, is a highly damaging, globally distributed crop pest that has evolved multiple resistance to numerous insecticides. It is thus imperative that insecticides that are not strongly compromised by pre-existing resistance are carefully managed to maximise their effective life span. Sulfoxaflor is a sulfoximine insecticide that retains efficacy against M. persicae clones that exhibit resistance to older insecticides. In the current study we monitored the efficacy of sulfoxaflor against M. persicae populations collected in Western Australia, following reports of control failures in this region. We identified clones with low (4-23-fold across multiple independent bioassay experiments), but significant, levels of resistance to sulfoxaflor compared with a reference susceptible clone. Furthermore, we demonstrate that sulfoxaflor resistance can persist after many months of culturing in the laboratory in the absence of insecticide exposure. Resistance was not conferred by known mechanisms of resistance to neonicotinoid insecticides, that act on the same target-site as sulfoxaflor, i.e. the R81T mutation or overexpresssion of the P450 gene CYP6CY3. Rather, transcriptome profiling of multiple resistant and susceptible clones identified the P450 CYP380C40 and the UDP-glucuronosyltransferase UGT344P2 as highly overexpressed (21-76-fold and 6-33-fold respectively) in the resistant clones. Transgenic expression of these genes demonstrated that they confer, low, but significant, levels of resistance to sulfoxaflor in vivo. Taken together, our data reveal the presence of low-level resistance to sulfoxaflor in M. persicae populations in Australia and uncover two novel mechanisms conferring resistance to this compound. The findings and tools generated in this study provide a platform for the development of strategies that aim to slow, prevent or overcome the evolution of more potent resistance to sulfoxaflor.

Fitness Costs Associated with Pyrethroid Resistance in Halotydeus destructor (Tucker) (Acari: Penthaleidae) Elucidated Through Semi-field Trials.

Pyrethroid resistance in the redlegged earth mite, Halotydeus destructor (Tucker), is primarily attributed to a kdr (knockdown resistance) mutation in the parasodium channel gene. To assess fitness costs associated with this resistance, adult resistant and susceptible populations were mixed in different proportions in microcosm tubs and placed in a shade-house simulating field conditions. Three separate experiments were undertaken whereby parental mites were collected from the field and offspring were followed for two to three generations. The association between fitness costs and kdr-mediated resistance was investigated by examining differences in mite numbers and changes in resistant allele frequencies across generations. In two (of the three) experiments, the population fitness measure of mites was significantly lower in microcosms containing a higher proportion of resistant individuals compared with treatments containing susceptible mites. No differences in mite fitness were observed between treatments in the third experiment; in this instance, the starting proportion of individuals homozygous for the resistant mutation was much lower (~40%) than in the other experiments (>90%). In all three experiments, a decrease in the resistant allele frequency across mite generations was observed. These findings indicate a potential deleterious pleiotropic effect of the kdr mutation on the fitness of H. destructor and have implications for resistance management strategies aimed at this important agricultural pest. Further experiments investigating fitness costs directly in the field are warranted.

Learnings from over a decade of increasing pesticide resistance in the redlegged earth mite, Halotydeus destructor (Tucker).

BACKGROUND: The redlegged earth mite, Halotydeus destructor (Tucker), is a destructive and economically important pest of winter grain crops and pastures in Australia. It is largely controlled by pesticides, but this mite has evolved resistance to pyrethroid and organophosphate chemicals. A national Resistance Management Strategy has been developed for pro-active management to delay further resistance evolution, though its success is reliant on a detailed understanding of the incidence, patterns of spread, current distribution and the nature of resistance in the field. Here, we report on a long-term resistance surveillance programme undertaken between 2006 and 2019 informed by resistance risk forecasting. RESULTS: By mapping the Australian distribution of resistance through time, we show that resistance is present across three Australian states and covers more than 3000 km. This current range includes a recently identified population exhibiting organophosphate resistance representing the most easterly location of resistance in H. destructor. Using field history information, we identify associations for the first time between crop management practices employed by farmers and the presence of pyrethroid resistance. Management strategies that could minimize the risk of further resistance include limiting local spread of resistance through farm hygiene practices, crop rotations and reducing pesticide usage. CONCLUSION: This study highlights the challenges of resistance in H. destructor but also indicates how quantitative resistance risk analysis can be developed to target field surveillance and delay further resistance. The management strategies highlighted in this study can help maintain the effectiveness of control options but will depend on farmer engagement and adoption. © 2021 Society of Chemical Industry.

Adaptive responses of free-living and symbiotic microalgae to simulated future ocean conditions.

Marine microalgae are a diverse group of microscopic eukaryotic and prokaryotic organisms capable of photosynthesis. They are important primary producers and carbon sinks but their physiology and persistence are severely affected by global climate change. Powerful experimental evolution technologies are being used to examine the potential of microalgae to respond adaptively to current and predicted future conditions, as well as to develop resources to facilitate species conservation and restoration of ecosystem functions. This review synthesizes findings and insights from experimental evolution studies of marine microalgae in response to elevated temperature and/or pCO2 . Adaptation to these environmental conditions has been observed in many studies of marine dinoflagellates, diatoms and coccolithophores. An enhancement in traits such as growth and photo-physiological performance and an increase in upper thermal limit have been shown to be possible, although the extent and rate of change differ between microalgal taxa. Studies employing multiple monoclonal replicates showed variation in responses among replicates and revealed the stochasticity of mutations. The work to date is already providing valuable information on species' climate sensitivity or resilience to managers and policymakers but extrapolating these insights to ecosystem- and community-level impacts continues to be a challenge. We recommend future work should include in situ experiments, diurnal and seasonal fluctuations, multiple drivers and multiple starting genotypes. Fitness trade-offs, stable versus plastic responses and the genetic bases of the changes also need investigating, and the incorporation of genome resequencing into experimental designs will be invaluable.

Separating two tightly linked species-defining phenotypes in Bactrocera with hybrid recombinant analysis.

BACKGROUND: Bactrocera tryoni and Bactrocera neohumeralis mate asynchronously; the former mates exclusively around dusk while the latter mates during the day. The two species also differ in the colour of the post-pronotal lobe (callus), which is predominantly yellow in B. tryoni and brown in B. neohumeralis. We have examined the genetic relationship between the two characters in hybrids, backcrosses and multigeneration hybrid progeny. RESULTS: Our analysis of the mating time of the parental species revealed that while B. tryoni mate exclusively at dusk, B. neohumeralis females pair with B. neohumeralis males during the day and with B. tryoni males at dusk. We found considerable variance in mating time and callus colour among hybrid backcross individuals of both sexes but there was a strong although not invariant trend for callus colour to co-segregate with mating time in both sexes. To genetically separate these two phenotypes we allowed the interspecific F1 hybrids to propagate for 25 generations (F25) without selection for mating time or callus colour, finding that the advanced hybrid population had moved towards B. tryoni phenotypes for both traits. Selection for day mating in replicate lines at F25 resulted in significant phenotypic shifts in both traits towards B. neohumeralis phenotypes in F26. However, we were unable to completely recover the mating time profile of B. neohumeralis and relaxation of selection for day mating led to a shift back towards dusk mating, but not yellow callus colour, by F35. CONCLUSION: We conclude that the inheritance of the two major species-defining traits is separable but tightly linked and involves more than one gene in each case. It also appears that laboratory conditions select for the B. tryoni phenotypes for mating time. We discuss our findings in relation to speciation theory and the likely effects of domestication during the generation of mass release strains for sterile insect control programmes.

Climate stress resistance in male Queensland fruit fly varies among populations of diverse geographic origins and changes during domestication.

BACKGROUND: The highly polyphagous Queensland fruit fly (Bactrocera tryoni Froggatt) expanded its range substantially during the twentieth century and is now the most economically important insect pest of Australian horticulture, prompting intensive efforts to develop a Sterile Insect Technique (SIT) control program. Using a "common garden" approach, we have screened for natural genetic variation in key environmental fitness traits among populations from across the geographic range of this species and monitored changes in those traits induced during domestication. RESULTS: Significant variation was detected between the populations for heat, desiccation and starvation resistance and wing length (as a measure of body size). Desiccation resistance was correlated with both starvation resistance and wing length. Bioassay data for three resampled populations indicate that much of the variation in desiccation resistance reflects persistent, inherited differences among the populations. No latitudinal cline was detected for any of the traits and only weak correlations were found with climatic variables for heat resistance and wing length. All three stress resistance phenotypes and wing length changed significantly in certain populations with ongoing domestication but there was also a strong population by domestication interaction effect for each trait. CONCLUSIONS: Ecotypic variation in heat, starvation and desiccation resistance was detected in Australian Qfly populations, and these stress resistances diminished rapidly during domestication. Our results indicate a need to select source populations for SIT strains which have relatively high climatic stress resistance and to minimise loss of that resistance during domestication.

Genome-wide patterns of differentiation over space and time in the Queensland fruit fly.

The Queensland fruit fly, Bactrocera tryoni, is a major pest of Australian horticulture which has expanded its range in association with the spread of horticulture over the last ~ 150 years. Its distribution in northern Australia overlaps that of another fruit fly pest to which some authors accord full species status, Bactrocera aquilonis. We have used reduced representation genome-wide sequencing to genotype 359 individuals taken from 35 populations from across the current range of the two taxa, plus a further 73 individuals from six of those populations collected 15-22 years earlier. We find significant population differentiation along an east-west transect across northern Australia which likely reflects limited but bidirectional gene flow between the two taxa. The southward expansion of B. tryoni has led to relatively little genetic differentiation, and most of it is associated with a move into previously marginal inland habitats. Two disjunct populations elsewhere in Australia and three on Melanesian islands are each clearly differentiated from all others, with data strongly supporting establishment from relatively few founders and significant isolation subsequently. Resequencing of historical samples from one of the disjunct Australian populations shows that its genetic profile has changed little over a 15-year period, while the Melanesian data suggest a succession of 'island hopping' events with progressive reductions in genetic diversity. We discuss our results in relation to the control of B. tryoni and as a model for understanding the genetics of invasion and hybridisation processes.

Genetic Biocontrol for Invasive Species.

Invasive species are increasingly affecting agriculture, food, fisheries, and forestry resources throughout the world. As a result of global trade, invasive species are often introduced into new environments where they become established and cause harm to human health, agriculture, and the environment. Prevention of new introductions is a high priority for addressing the harm caused by invasive species, but unfortunately efforts to prevent new introductions do not address the economic harm that is presently manifested where invasive species have already become established. Genetic biocontrol can be defined as the release of organisms with genetic methods designed to disrupt the reproduction of invasive populations. While these methods offer the potential to control or even eradicate invasive species, there is a need to ensure that genetic biocontrol methods can be deployed in a way that minimizes potential harm to the environment. This review provides an overview of the state of genetic biocontrol, focusing on several approaches that were the subject of presentations at the Genetic Biocontrol for Invasive Species Workshop in Tarragona, Spain, March 31st, 2019, a workshop sponsored by the OECD's Co-operative Research Program on Biological Resource Management for Sustainable Agricultural Systems. The review considers four different approaches to genetic biocontrol for invasive species; sterile-release, YY Males, Trojan Female Technique, and gene drive. The different approaches will be compared with respect to the efficiency each affords as a genetic biocontrol tool, the practical utility and cost/benefits associated with implementation of the approach, and the regulatory considerations that will need to be addressed for each. The opinions expressed and arguments employed in this publication are the sole responsibility of the authors and do not necessarily reflect those of the OECD or of the governments of its Member countries.

An RNAi supplemented diet as a reverse genetics tool to control bluegreen aphid, a major pest of legumes.

Aphids are important agricultural pests causing major yield losses worldwide. Since aphids can rapidly develop resistance to chemical insecticides there is an urgent need to find alternative aphid pest management strategies. Despite the economic importance of bluegreen aphid (Acyrthosiphon kondoi), very few genetic resources are available to expand our current understanding and help find viable control solutions. An artificial diet is a desirable non-invasive tool to enable the functional characterisation of genes in bluegreen aphid and discover candidate target genes for future use in RNA interference (RNAi) mediated crop protection against aphids. To date no artificial diet has been developed for bluegreen aphid, so we set out to develop a suitable diet by testing and optimising existing diets. Here, we describe an artificial diet for rearing bluegreen aphid and also provide a proof of concept for the supplementation of the diet with RNAi molecules targeting the salivary gland transcript C002 and gap gene hunchback, resulting in bluegreen aphid mortality which has not yet been documented in this species. Managing this pest, for example via RNAi delivery through artificial feeding will be a major improvement to test bluegreen aphid candidate target genes for future pest control and gain significant insights into bluegreen aphid gene function.

Locally Fixed Alleles: A method to localize gene drive to island populations.

Invasive species pose a major threat to biodiversity on islands. While successes have been achieved using traditional removal methods, such as toxicants aimed at rodents, these approaches have limitations and various off-target effects on island ecosystems. Gene drive technologies designed to eliminate a population provide an alternative approach, but the potential for drive-bearing individuals to escape from the target release area and impact populations elsewhere is a major concern. Here we propose the "Locally Fixed Alleles" approach as a novel means for localizing elimination by a drive to an island population that exhibits significant genetic isolation from neighboring populations. Our approach is based on the assumption that in small island populations of rodents, genetic drift will lead to alleles at multiple genomic loci becoming fixed. In contrast, multiple alleles are likely to be maintained in larger populations on mainlands. Utilizing the high degree of genetic specificity achievable using homing drives, for example based on the CRISPR/Cas9 system, our approach aims at employing one or more locally fixed alleles as the target for a gene drive on a particular island. Using mathematical modeling, we explore the feasibility of this approach and the degree of localization that can be achieved. We show that across a wide range of parameter values, escape of the drive to a neighboring population in which the target allele is not fixed will at most lead to modest transient suppression of the non-target population. While the main focus of this paper is on elimination of a rodent pest from an island, we also discuss the utility of the locally fixed allele approach for the goals of population suppression or population replacement. Our analysis also provides a threshold condition for the ability of a gene drive to invade a partially resistant population.

Extracellular endonucleases in the midgut of Myzus persicae may limit the efficacy of orally delivered RNAi.

Myzus persicae is a major pest of many crops including canola and Brassica vegetables, partly because it vectors plant viruses. Previously it has been reported that double-stranded RNA delivered to aphids by injection, artificial diet or transgenic plants has knocked down target genes and caused phenotypic effects. While these studies suggest that RNA interference (RNAi) might be used to suppress aphid populations, none have shown effects sufficient for field control. The current study analyses the efficacy of dsRNA directed against previously reported gene-targets on Green peach aphid (Myzus persicae) strains. No silencing effect was observed when dsRNA was delivered in artificial diet with or without transfection reagents. dsRNA produced in planta also failed to induce significant RNAi in M. persicae. Transcriptome analyses of the midgut suggested other potential targets including the Ferritin heavy chain transcripts, but they also could not be knocked down with dsRNA. Here we show that dsRNA is rapidly degraded by midgut secretions of Myzus persicae. Analysis of the transcriptome of the M. persicae midgut revealed that an ortholog of RNases from other insects was abundant.

Detoxifying enzyme complements and host use phenotypes in 160 insect species.

We use the genomes of 160 insect species to test the hypothesis that the size of detoxifying enzyme families is greater in species using more chemically diverse food resources. Phylogenetically appropriate contrasts in subsamples of the data generally support the hypothesis. We find relatively high numbers of cytochrome P450, glutathione S-transferase and carboxyl/choline esterase genes in omnivores and herbivores feeding on chemically complex tissues and relatively low numbers of these genes in specialists on relatively simple diets, including plant sap, nectar and pollen, and blood. Among Lepidoptera feeding on green plant tissue and Condylognatha feeding on sap we also find more of these genes in highly polyphagous species, many of which are major agricultural pests. These genomic signatures of food resource use are consistent with the hypothesis that some taxa are preadapted for insecticide resistance evolution.

Additive and epistatic interactions between AKR and AIN loci conferring bluegreen aphid resistance and hypersensitivity in Medicago truncatula.

Aphids, including the bluegreen aphid (BGA; Acyrthosiphon kondoi), are important pests in agriculture. Two BGA resistance genes have been identified in the model legume Medicago truncatula, namely AKR (Acyrthosiphon kondoi resistance) and AIN (Acyrthosiphon induced necrosis). In this study, progeny derived from a cross between a resistant accession named Jester and a highly susceptible accession named A20 were used to study the interaction between the AKR and AIN loci with respect to BGA performance and plant response to BGA infestation. These studies demonstrated that AKR and AIN have additive effects on the BGA resistance phenotype. However, AKR exerts dominant suppression epistasis on AIN-controlled macroscopic necrotic lesions. Nevertheless, both AKR and AIN condition production of H2O2 at the BGA feeding site. Electrical penetration graph analysis demonstrated that AKR prevents phloem sap ingestion, irrespective of the presence of AIN. Similarly, the jasmonic acid defense signaling pathway is recruited by AKR, irrespective of AIN. This research identifies an enhancement of aphid resistance through gene stacking, and insights into the interaction of distinct resistance genes against insect pests.