Aberrant splicing of a nicotinic acetylcholine receptor alpha 6 subunit is associated with spinosad tolerance in the thrips predator Orius laevigatus.

Susceptibility to insecticides is one of the limiting factors preventing wider adoption of natural enemies to control insect pest populations. Identification and selective breeding of insecticide tolerant strains of commercially used biological control agents (BCAs) is one of the approaches to overcome this constraint. Although a number of beneficial insects have been selected for increased tolerance to insecticides the molecular mechanisms underpinning these shifts in tolerance are not well characterised. Here we investigated the molecular mechanisms of enhanced tolerance of a lab selected strain of Orius laevigatus (Fieber) to the commonly used biopesticide spinosad. Transcriptomic analysis showed that spinosad tolerance is not a result of overexpressed detoxification genes. Molecular analysis of the target site for spinosyns, the nicotinic acetylcholine receptor (nAChR), revealed increased expression of truncated transcripts of the nAChR α6 subunit in the spinosad selected strain, a mechanism of resistance which was described previously in insect pest species. Collectively, our results demonstrate the mechanisms by which some beneficial biological control agents can evolve insecticide tolerance and will inform the development and deployment of insecticide-tolerant natural enemies in integrated pest management strategies.

UDP-glycosyltransferases act as key determinants of host plant range in generalist and specialist Spodoptera species.

Phytophagous insects have evolved sophisticated detoxification systems to overcome the antiherbivore chemical defenses produced by many plants. However, how these biotransformation systems differ in generalist and specialist insect species and their role in determining insect host plant range remains an open question. Here, we show that UDP-glucosyltransferases (UGTs) play a key role in determining the host range of insect species within the Spodoptera genus. Comparative genomic analyses of Spodoptera species that differ in host plant breadth identified a relatively conserved number of UGT genes in generalist species but high levels of UGT gene pseudogenization in the specialist Spodoptera picta. CRISPR-Cas9 knockouts of the three main UGT gene clusters of Spodoptera frugiperda revealed that UGT33 genes play an important role in allowing this species to utilize the poaceous plants maize, wheat, and rice, while UGT40 genes facilitate utilization of cotton. Further functional analyses in vivo and in vitro identified the UGT SfUGT33F32 as the key mechanism that allows generalist S. frugiperda to detoxify the benzoxazinoid DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), a potent insecticidal phytotoxin produced by poaceous plants. However, while this detoxification capacity is conserved in several generalist Spodoptera species, Spodoptera picta, which specializes on Crinum plants, is unable to detoxify DIMBOA due to a nonfunctionalizing mutation in SpUGT33F34. Collectively, these findings provide insight into the role of insect UGTs in host plant adaptation, the mechanistic basis of evolutionary transitions between generalism and specialism and offer molecular targets for controlling a group of notorious insect pests.

A cytochrome P450 insecticide detoxification mechanism is not conserved across the Megachilidae family of bees.

Recent work has demonstrated that many bee species have specific cytochrome P450 enzymes (P450s) that can efficiently detoxify certain insecticides. The presence of these P450s, belonging or closely related to the CYP9Q subfamily (CYP9Q-related), is generally well conserved across the diversity of bees. However, the alfalfa leafcutter bee, Megachile rotundata, lacks CYP9Q-related P450s and is 170-2500 times more sensitive to certain insecticides than bee pollinators with these P450s. The extent to which these findings apply to other Megachilidae bee species remains uncertain. To address this knowledge gap, we sequenced the transcriptomes of four Megachile species and leveraged the data obtained, in combination with publicly available genomic data, to investigate the evolution and function of P450s in the Megachilidae. Our analyses reveal that several Megachilidae species, belonging to the Lithurgini, Megachilini and Anthidini tribes, including all species of the Megachile genus investigated, lack CYP9Q-related genes. In place of these genes Megachile species have evolved phylogenetically distinct CYP9 genes, the CYP9DM lineage. Functional expression of these P450s from M. rotundata reveal they lack the capacity to metabolize the neonicotinoid insecticides thiacloprid and imidacloprid. In contrast, species from the Osmiini and Dioxyini tribes of Megachilidae have CYP9Q-related P450s belonging to the CYP9BU subfamily that are able to detoxify thiacloprid. These findings provide new insight into the evolution of P450s that act as key determinants of insecticide sensitivity in bees and have important applied implications for pesticide risk assessment.

Overexpression of the UDP-glycosyltransferase UGT34A23 confers resistance to the diamide insecticide chlorantraniliprole in the tomato leafminer, Tuta absoluta.

The tomato leafminer, Tuta absoluta, is an invasive crop pest that has evolved resistance to many of the insecticides used for its control. To facilitate the investigation of the underpinning mechanisms of resistance in this species we generated a contiguous genome assembly using long-read sequencing data. We leveraged this genomic resource to investigate the genetic basis of resistance to the diamide insecticide chlorantraniliprole in Spanish strains of T. absoluta that exhibit high levels of resistance to this insecticide. Transcriptomic analyses revealed that, in these strains, resistance is not associated with previously reported target-site mutations in the diamide target-site, the ryanodine receptor, but rather is associated with the marked overexpression (20- to >100-fold) of a gene encoding a UDP-glycosyltransferase (UGT). Functional expression of this UGT, UGT34A23, via ectopic expression in Drosophila melanogaster demonstrated that it confers strong and significant resistance in vivo. The genomic resources generated in this study provide a powerful resource for further research on T. absoluta. Our findings on the mechanisms underpinning resistance to chlorantraniliprole will inform the development of sustainable management strategies for this important pest.

The genome sequence of the Neotropical brown stink bug, Euschistus heros provides insights into population structure, demographic history and signatures of adaptation.

The Neotropical brown stink bug, Euschistus heros, is a major pest of soybean in South America. The importance of E. heros as a pest has grown significantly in recent times due to increases in its abundance and range, and the evolution of insecticide resistance. Recent work has begun to examine the genetic diversity, population structure, and genetic mechanisms of insecticide resistance in E. heros. However, to date, investigation of these topics has been hampered by a lack of genomic resources for this species. Here we address this need by assembling a high-quality draft genome for E. heros. We used a combination of short and long read sequencing to assemble an E. heros genome of 1.4 Gb comprising 906 contigs with a contig N50 of 3.5 MB. We leveraged this new genomic resource, in combination with genotyping by sequencing, to explore genetic diversity in populations of this species in Brazil and identify genetic loci in the genome which are under selection. Our genome-wide analyses, confirm that there are two populations of E. heros co-occurring in different geographical regions in Brazil, and that, in certain regions of the country these populations are hybridizing. We identify several regions of the genome as under selection, including markers associated with putative insecticide resistance genes. Taken together, the new genomic resources generated in this study will accelerate research into fundamental aspects of stinkbug biology and applied aspects relating to the sustainable control of a highly damaging crop pest.

Adaptation of Helicoverpa armigera to Soybean Peptidase Inhibitors Is Associated with the Transgenerational Upregulation of Serine Peptidases.

Molecular phenotypes induced by environmental stimuli can be transmitted to offspring through epigenetic inheritance. Using transcriptome profiling, we show that the adaptation of Helicoverpa armigera larvae to soybean peptidase inhibitors (SPIs) is associated with large-scale gene expression changes including the upregulation of genes encoding serine peptidases in the digestive system. Furthermore, approximately 60% of the gene expression changes induced by SPIs persisted in the next generation of larvae fed on SPI-free diets including genes encoding regulatory, oxidoreductase, and protease functions. To investigate the role of epigenetic mechanisms in regulating SPI adaptation, the methylome of the digestive system of first-generation larvae (fed on a diet with and without SPIs) and of the progeny of larvae exposed to SPIs were characterized. A comparative analysis between RNA-seq and Methyl-seq data did not show a direct relationship between differentially methylated and differentially expressed genes, while trypsin and chymotrypsin genes were unmethylated in all treatments. Rather, DNA methylation potential epialleles were associated with transcriptional and translational controls; these may play a regulatory role in the adaptation of H. armigera to SPIs. Altogether, our findings provided insight into the mechanisms of insect adaptation to plant antiherbivore defense proteins and illustrated how large-scale transcriptional reprograming of insect genes can be transmitted across generations.

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.

The effect of DNA methylation on bumblebee colony development.

BACKGROUND: Although around 1% of cytosines in bees' genomes are known to be methylated, less is known about methylation's effect on bee behavior and fitness. Chemically altered DNA methylation levels have shown clear changes in the dominance and reproductive behavior of workers in queen-less colonies, but the global effect of DNA methylation on caste determination and colony development remains unclear, mainly because of difficulties in controlling for genetic differences among experimental subjects in the parental line. Here, we investigated the effect of the methylation altering agent decitabine on the developmental rate of full bumblebee colonies. Whole genome bisulfite sequencing was used to assess differences in methylation status. RESULTS: Our results showed fewer methylated loci in the control group. A total of 22 CpG loci were identified as significantly differentially methylated between treated and control workers with a change in methylation levels of 10% or more. Loci that were methylated differentially between groups participated in pathways including neuron function, oocyte regulation and metabolic processes. Treated colonies tended to develop faster, and therefore more workers were found at a given developmental stage. However, male production followed the opposite trend and it tended to be higher in control colonies. CONCLUSION: Overall, our results indicate that altered methylation patterns resulted in an improved cooperation between workers, while there were no signs of abnormal worker dominance or caste determination.

The Euphausia superba transcriptome database, SuperbaSE: An online, open resource for researchers.

Antarctic krill (Euphausia superba) is a crucial component of the Southern Ocean ecosystem, acting as the major link between primary production and higher trophic levels with an annual predator demand of up to 470 million tonnes. It also acts as an ecosystem engineer, affecting carbon sequestration and recycling iron and nitrogen, and has increasing importance as a commercial product in the aquaculture and health industries. Here we describe the creation of a de novo assembled head transcriptome for E. superba. As an example of its potential as a molecular resource, we relate its exploitation in identifying and characterizing numerous genes related to the circadian clock in E. superba, including the major components of the central feedback loop. We have made the transcriptome openly accessible for a wider audience of ecologists, molecular biologists, evolutionary geneticists, and others in a user-friendly format at SuperbaSE, hosted at http://www.krill.le.ac.uk.

A comparison of right ventricular non-apical defibrillator lead position with traditional right ventricular apical position: a single centre experience.

BACKGROUND: Implantable cardioverter defibrillator (ICD) leads have traditionally been placed at the right ventricular apex (RVA). An important minority of patients with an ICD may develop a future requirement for bradycardia support. Pacing from the RVA may be detrimental, promoting heart failure and mortality. Increasingly non-apical right ventricular (RVNA) lead positions have been suggested as an alternative pacing site. METHODS: A retrospective review of 512 patients who received an ICD at our institution between 1999 and 2011 was conducted. A comparison of lead performance characteristics was performed between RVNA sites and those at RVA. Data were collated from chart review and the pacing database. RESULTS: The mean follow-up period in the RVNA cohort was 40.4±25.9 months and in the RVA cohort it was 38±31.8 months (p=0.43). The RVNA cohort consisted of 144 leads and 368 leads in the RVA cohort. The groups had similar baseline clinical characteristics. No significant difference was detected in the proportion of patients receiving an appropriate ICD defibrillation (RVNA 10.4% vs. RVA 16.8%; p=0.07), inappropriate defibrillation (RVNA 7.6% vs. RVA 7.6%; p=0.99) or an unsuccessful defibrillation (RVNA 0% vs. RVA 1.7%; p=0.12). There was also no significant difference in the proportion of patients receiving successful anti-tachycardia pacing (ATP) (RVNA 13.2% vs. RVA 17.4%; p=0.49) or failed ATP (RVNA 2.7% vs. RVA 4.1%; p=0.25). There was no significant difference in lead impedance (p=0.99), sensing (p=0.59) and pacing threshold (p=0.34). CONCLUSION: In this large retrospective study, RVNA ICD lead had similar stability and therapy efficacy compared to the traditional RVA position. This potentially has important implications for the suitability of RVNA as an alternative site for ICD leads.