Recovering individual haplotypes and a contiguous genome assembly from pooled long-read sequencing of the diamondback moth (Lepidoptera: Plutellidae).

The assembly of divergent haplotypes using noisy long-read data presents a challenge to the reconstruction of haploid genome assemblies, due to overlapping distributions of technical sequencing error, intralocus genetic variation, and interlocus similarity within these data. Here, we present a comparative analysis of assembly algorithms representing overlap-layout-consensus, repeat graph, and de Bruijn graph methods. We examine how postprocessing strategies attempting to reduce redundant heterozygosity interact with the choice of initial assembly algorithm and ultimately produce a series of chromosome-level assemblies for an agricultural pest, the diamondback moth, Plutella xylostella (L.). We compare evaluation methods and show that BUSCO analyses may overestimate haplotig removal processing in long-read draft genomes, in comparison to a k-mer method. We discuss the trade-offs inherent in assembly algorithm and curation choices and suggest that "best practice" is research question dependent. We demonstrate a link between allelic divergence and allele-derived contig redundancy in final genome assemblies and document the patterns of coding and noncoding diversity between redundant sequences. We also document a link between an excess of nonsynonymous polymorphism and haplotigs that are unresolved by assembly or postassembly algorithms. Finally, we discuss how this phenomenon may have relevance for the usage of noisy long-read genome assemblies in comparative genomics.

Self-limiting fall armyworm: a new approach in development for sustainable crop protection and resistance management.

BACKGROUND: The fall armyworm, Spodoptera frugiperda, is a significant and widespread pest of maize, sorghum, rice, and other economically important crops. Successful management of this caterpillar pest has historically relied upon application of synthetic insecticides and through cultivation of genetically engineered crops expressing insecticidal proteins (Bt crops). Fall armyworm has, however, developed resistance to both synthetic insecticides and Bt crops, which risks undermining the benefits delivered by these important crop protection tools. Previous modelling and empirical studies have demonstrated that releases of insecticide- or Bt-susceptible insects genetically modified to express conditional female mortality can both dilute insecticide resistance and suppress pest populations. RESULTS: Here, we describe the first germline transformation of the fall armyworm and the development of a genetically engineered male-selecting self-limiting strain, OX5382G, which exhibits complete female mortality in the absence of an additive in the larval diet. Laboratory experiments showed that males of this strain are competitive against wild-type males for copulations with wild-type females, and that the OX5382G self-limiting transgene declines rapidly to extinction in closed populations following the cessation of OX5382G male releases. Population models simulating the release of OX5382G males in tandem with Bt crops and non-Bt 'refuge' crops show that OX5382G releases can suppress fall armyworm populations and delay the spread of resistance to insecticidal proteins. CONCLUSIONS: This article describes the development of self-limiting fall armyworm designed to control this pest by suppressing pest populations, and population models that demonstrate its potential as a highly effective method of managing resistance to Bt crops in pest fall armyworm populations. Our results provide early promise for a potentially valuable future addition to integrated pest management strategies for fall armyworm and other pests for which resistance to existing crop protection measures results in damage to crops and impedes sustainable agriculture.

Exposure to genetically engineered olive fly (Bactrocera oleae) has no negative impact on three non-target organisms.

Bactrocera oleae (Diptera: Tephritidae) remains a major pest of olive fruit production worldwide. Current pest management programs largely depend on chemical insecticides, resulting in high economic and environmental costs. Alternative pest control approaches are therefore highly desirable. We have created a conditional female-specific self-limiting strain of B. oleae (OX3097D-Bol) that could be applied for sustainable pest control. OX3097D-Bol olive fly carries a fluorescent marker (DsRed2) for identification and a self-limiting genetic trait that is repressed by tetracycline. In the absence of tetracycline, the tetracycline transactivator (tTAV) accumulates, resulting in female death at larvae and early pupal stages. The aim of this study was to evaluate the impact of genetically engineered OX3097D-Bol olive fly on three non-target organisms that either predate or parasitize olive flies, one from the guild of parasitoids (Psyttalia concolor) and two from the guild of predators (Pardosa spider species and the rove beetle Aleochara bilineata). No significant negative effect was observed on life history parameters, mortality and reproductive capacity of the non-target organisms studied. These results suggest that potential exposure to DsRed2 and tTAV gene products (e.g. mRNA and encoded proteins) would have a negligible impact on on-target organisms in the guilds or predators and parasitoids.

Pest control and resistance management through release of insects carrying a male-selecting transgene.

BACKGROUND: Development and evaluation of new insect pest management tools is critical for overcoming over-reliance upon, and growing resistance to, synthetic, biological and plant-expressed insecticides. For transgenic crops expressing insecticidal proteins from the bacterium Bacillus thuringiensis ('Bt crops') emergence of resistance is slowed by maintaining a proportion of the crop as non-Bt varieties, which produce pest insects unselected for resistance. While this strategy has been largely successful, multiple cases of Bt resistance have now been reported. One new approach to pest management is the use of genetically engineered insects to suppress populations of their own species. Models suggest that released insects carrying male-selecting (MS) transgenes would be effective agents of direct, species-specific pest management by preventing survival of female progeny, and simultaneously provide an alternative insecticide resistance management strategy by introgression of susceptibility alleles into target populations. We developed a MS strain of the diamondback moth, Plutella xylostella, a serious global pest of crucifers. MS-strain larvae are reared as normal with dietary tetracycline, but, when reared without tetracycline or on host plants, only males will survive to adulthood. We used this strain in glasshouse-cages to study the effect of MS male P. xylostella releases on target pest population size and spread of Bt resistance in these populations. RESULTS: Introductions of MS-engineered P. xylostella males into wild-type populations led to rapid pest population decline, and then elimination. In separate experiments on broccoli plants, relatively low-level releases of MS males in combination with broccoli expressing Cry1Ac (Bt broccoli) suppressed population growth and delayed the spread of Bt resistance. Higher rates of MS male releases in the absence of Bt broccoli were also able to suppress P. xylostella populations, whereas either low-level MS male releases or Bt broccoli alone did not. CONCLUSIONS: These results support theoretical modeling, indicating that MS-engineered insects can provide a powerful pest population suppressing effect, and could effectively augment current Bt resistance management strategies. We conclude that, subject to field confirmation, MS insects offer an effective and versatile control option against P. xylostella and potentially other pests, and may reduce reliance on and protect insecticide-based approaches, including Bt crops.

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.