Field Suppression of Spotted Wing Drosophila (SWD) (Drosophila suzukii Matsumura) Using the Sterile Insect Technique (SIT).

Drosophila suzukii (spotted wing drosophila­SWD) is an economically important pest of soft and stone fruit worldwide. Control relies on broad-spectrum insecticides, which are neither fully effective nor environmentally sustainable. The sterile insect technique (SIT) is a proven, effective and environmentally friendly pest-management tool. Here, we investigated, for the first time, the potential of using SIT to control D. suzukii in field conditions without physical barriers that limit insect invasion. A proprietary method of rearing and irradiation with X-rays was used to obtain males that were > 99% sterile. Sterile males were released twice per week from April to October 2021 on a site in Kent, UK, where everbearing strawberries were grown in open polytunnels. The infestation of wild female D. suzukii was monitored weekly using red sticky traps with dry lure at the treated site and at two similar control sites that did not receive sterile male releases. Releases of sterile males suppressed the wild female D. suzukii population by up to 91% in comparison with the control sites. We thus demonstrated the feasibility of SIT to achieve season-long control of D. suzukii using early, sustained and dynamically targeted releases of sterile males. This provides a promising environmentally friendly method to control this important pest.

Diamide insecticide resistance in transgenic Drosophila and Sf9-cells expressing a full-length diamondback moth ryanodine receptor carrying an I4790M mutation.

BACKGROUND: Resistance to diamide insecticides in Lepidoptera is known to be caused primarily by amino acid changes on the ryanodine receptor (RyR). Recently, two new target site mutations, G4946V and I4790M, have emerged in populations of diamondback moth, Plutella xylostella, as well as in other lepidopteran species, and both mutations have been shown empirically to decrease diamide efficacy. Here, we quantify the impact of the I4790M mutation on diamide activation of the receptor, as compared to alterations at the G4946 locus. RESULTS: I4790M when introduced into P. xylostella RyR expressed in an insect-derived Sf9 cell line was found to mediate just a ten-fold reduction in chlorantraniliprole efficacy (compared to 104- and 146-fold reductions for the G4946E and G4946V variants, respectively), whilst in the field its presence is associated with a ≥150-fold reduction. I4790M-mediated resistance to flubendiamide was estimated to be >24-fold. When the entire coding sequence of P. xylostella RyR was integrated into Drosophila melanogaster, the I4790M variant conferred ~4.4-fold resistance to chlorantraniliprole and 22-fold resistance to flubendiamide in the 3rd instar larvae, confirming that it imparts only a moderate level of resistance to diamide insecticides. Although the I4790M substitution appears to bear no fitness costs in terms of the flies' reproductive capacity, when assessed in a noncompetitive environment, it does, however, have potentially major impacts on mobility at both the larval and adult stages. CONCLUSIONS: I4790M imparts only a moderate level of resistance to diamide insecticides and potentially confers significant fitness costs to the insect.

Evidence for the Involvement of the Chemosensory Protein AgosCSP5 in Resistance to Insecticides in the Cotton Aphid, Aphis gossypii.

It has been speculated that insect chemosensory proteins (CSPs) may have additional roles beyond olfaction. In this study, the phylogenetic and genomic analyses of the CSPs of the cotton aphid, Aphis gossypii, revealed the presence of gene gain-and-loss among different aphid field populations. Differential expressions of eight CSP genes were demonstrated after treatments with insecticides of different modes of action. The expression of AgosCSP5 was significantly upregulated by the insecticide treatments in a dose-dependent manner. The Drosophila flies overexpressing AgosCSP5 were significantly less susceptible to the insecticides, omethoate, imidacloprid and cypermethrin but not to deltamethrin and tau-fluvalinate, compared with control flies. The transgenic Drosophila flies exhibited an LC50 resistance ratio of 2.6 to omethoate, compared with control flies. Likewise, the mortality of the transgenic flies to imidacloprid and cypermethrin was significantly lower than that of the control flies (p < 0.01). Homology modelling, molecular docking and dynamic simulation supported the interactions and revealed a higher stability of AgosCSP5/insecticide complexes than AgosCSP5/semiochemical complexes. Our study demonstrates for first time the in vivo evidence for the involvement of CSP genes in insecticide resistance of crop insect pests and provides new insights of the newly discovered CSP-mediated insect resistance mechanism to insecticides.

Laboratory Selection, Cross-Resistance, Risk Assessment to Lambda-Cyhalothrin Resistance, and Monitoring of Insecticide Resistance for Plant Bug Lygus pratensis (Hemiptera: Miridae) in Farming-Pastoral Ecotones of Northern China.

The plant bug Lygus pratensis Linnaeus (Hemiptera: Miridae) is an important insect pest of alfalfa in grassland farming in northern China. A field population of L. pratensis was selected in the laboratory for 14 consecutive generations with lambda-cyhalothrin to generate 42.555-fold resistance. Selection also induced low cross-resistance to imidacloprid and beta-cypermethrin, and medium cross-resistance to deltamethrin. Realized heritability (h2) of lambda-cyhalothrin resistance was 0.339. Susceptible baselines of L. pratensis were established for five insecticides using the glass-vial method, the values of which were 6.849, 3.423, 8.778, 3.559, and 117.553 ng/cm2 for phoxim, methomyl, imidacloprid, lambda-cyhalothrin, and avermectin, respectively, along with the calculated LC99 diagnostic doses. This resistance risk assessment study suggests that a high risk of lambda-cyhalothrin resistance exists in the field. In addition, a 5-year field investigation of resistance monitoring of L. pratensis was conducted in seven alfalfa regions in farming-pastoral ecotones in northern China. The resistance levels of most populations were very low for phoxim, methomyl, and avermectin, with an upward trend for lambda-cyhalothrin resistance in the DK (Dengkou County), TKT (Tuoketuo County), XL (Xilinhot), and LX (Linxi County) populations during 2015-2019, and medium resistance level to imidacloprid in the TKT population in five years we sampled. The study provided information on chemical control, lambda-cyhalothrin resistance development, baseline susceptibility, and the status of resistance to five commonly-used insecticides against L. pratensis. These results could be used to optimize pyrethroid insecticide use as part of a pest integrated resistance management strategy against this key insect pest of alfalfa.

Fly-Tox: A panel of transgenic flies expressing pest and pollinator cytochrome P450s.

There is an on-going need to develop new insecticides that are not compromised by resistance and that have improved environmental profiles. However, the cost of developing novel compounds has increased significantly over the last two decades. This is in part due to increased regulatory requirements, including the need to screen both pest and pollinator insect species to ensure that pre-existing resistance will not hamper the efficacy of a new insecticide via cross-resistance, or adversely affect non-target insect species. To add to this problem the collection and maintenance of toxicologically relevant pest and pollinator species and strains is costly and often difficult. Here we present Fly-Tox, a panel of publicly available transgenic Drosophila melanogaster lines each containing one or more pest or pollinator P450 genes that have been previously shown to metabolise insecticides. We describe the range of ways these tools can be used, including in predictive screens to avoid pre-existing cross-resistance, to identify potential resistance-breaking inhibitors, in the initial assessment of potential insecticide toxicity to bee pollinators, and identifying harmful pesticide-pesticide interactions.

Odorant binding proteins promote flight activity in the migratory insect, Helicoverpa armigera.

Migratory insects are capable of actively sustaining powered flight for several hours. This extraordinary phenomenon requires a highly efficient transport system to cope with the energetic demands placed on the flight muscles. Here, we provide evidence that the role of the hydrophobic ligand binding of odorant binding proteins (OBPs) extends beyond their typical function in the olfactory system to support insect flight activity via lipid interactions. Transcriptomic and candidate gene analyses show that two phylogenetically clustered OBPs (OBP3/OBP6) are consistently over-expressed in adult moths of the migrant Old-World bollworm, Helicoverpa armigera, displaying sustained flight performance in flight activity bioassays. Tissue-specific over-expression of OBP6 was observed in the antennae, wings and thorax in long-fliers of H. armigera. Transgenic Drosophila flies over-expressing an H. armigera transcript of OBP6 (HarmOBP6) in the flight muscle attained higher flight speeds on a modified tethered flight system. Quantification of lipid molecules using mass spectrometry showed a depletion of triacylglyerol and phospholipids in flown moths. Protein homology models built from the crystal structure of a fatty acid carrier protein identified the binding site of OBP3 and OBP6 for hydrophobic ligand binding with both proteins exhibiting a stronger average binding affinity with triacylglycerols and phospholipids compared with other groups of ligands. We propose that HarmOBP3 and HarmOBP6 contribute to the flight capacity of a globally invasive and highly migratory noctuid moth, and in doing so, extend the function of this group of proteins beyond their typical role as chemosensory proteins in insects.

Evolutionary trade-offs of insecticide resistance - The fitness costs associated with target-site mutations in the nAChR of Drosophila melanogaster.

The evolution of resistance to drugs and pesticides poses a major threat to human health and food security. Neonicotinoids are highly effective insecticides used to control agricultural pests. They target the insect nicotinic acetylcholine receptor and mutations of the receptor that confer resistance have been slow to develop, with only one field-evolved mutation being reported to date. This is an arginine-to-threonine substitution at position 81 of the nAChR_ß1 subunit in neonicotinoid-resistant aphids. To validate the role of R81T in neonicotinoid resistance and to test whether it may confer any significant fitness costs to insects, CRISPR/Cas9 was used to introduce an analogous mutation in the genome of Drosophila melanogaster. Flies carrying R81T showed an increased tolerance (resistance) to neonicotinoid insecticides, accompanied by a significant reduction in fitness. In comparison, flies carrying a deletion of the whole nAChR_α6 subunit, the target site of spinosyns, showed an increased tolerance to this class of insecticides but presented almost no fitness deficits.

Identification and functional characterisation of a novel N-cyanoamidine neonicotinoid metabolising cytochrome P450, CYP9Q6, from the buff-tailed bumblebee Bombus terrestris.

Recent work has shown that two bumblebee (Bombus terrestris) cytochrome P450s of the CYP9Q subfamily, CYP9Q4 and CYP9Q5, are important biochemical determinants of sensitivity to neonicotinoid insecticides. Here, we report the characterisation of a third P450 gene CYP9Q6, previously mis-annotated in the genome of B. terrestris, encoding an enzyme that metabolises the N-cyanoamidine neonicotinoids thiacloprid and acetamiprid with high efficiency. The genomic location and complete ORF of CYP9Q6 was corroborated by PCR and its metabolic activity characterised in vitro by expression in an insect cell line. CYP9Q6 metabolises both thiacloprid and acetamiprid more rapidly than the previously reported CYP9Q4 and CYP9Q5. We further demonstrate a direct, in vivo correlation between the expression of the CYP9Q6 enzyme in transgenic Drosophila melanogaster and an increased tolerance to thiacloprid and acetamiprid. We conclude that CYP9Q6 is an efficient metaboliser of N-cyanoamidine neonicotinoids and likely plays a key role in the high tolerance of B. terrestris to these insecticides.

Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation.

We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation, and that these modifications regulate enzyme activity by preventing the formation of the active tetrameric form. The biotin-switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358, and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80, 100, and 140% activity, respectively, compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast, the Cys326Ser mutant exists predominantly in monomeric form. A biotin-switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK, confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer-tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.

An overview of functional genomic tools in deciphering insecticide resistance.

In this short review, we highlight three functional genomic technologies that have recently been contributing to the understanding of the molecular mechanisms underpinning insecticide resistance: the GAL4/UAS system, a molecular tool used to express genes of interest in a spatiotemporal controlled manner; the RNAi system, which is used to knock-down gene expression; and the most recently developed gene editing tool, CRISPR/Cas9, which can be used to knock-out and knock-in sequences of interest.

An analysis of variability in genome organisation of intracellular calcium release channels across insect orders.

Using publicly available genomic data, combined with RT-PCR validation, we explore structural genomic variation for two major ion channels across insect classes. We have manually curated ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP3R) ORFs and their corresponding genomic structures from 26 different insects covering major insect orders. We found that, despite high protein identity for both RyRs (>75%) and IP3Rs (~67%), the overall complexity of the gene structure varies greatly between different insect orders with the simplest genes (fewest introns) found in Diptera and the most complex in Lepidoptera. Analysis of intron conservation patterns indicated that the majority of conserved introns are found close to the 5' end of the channels and in RyR around the highly conserved mutually exclusive splice site. Of the two channels the IP3Rs appear to have a less well conserved organisation with a greater overall number of unique introns seen between insect orders. We experimentally validated two of the manually curated ORFs for IP3Rs and confirmed an atypical (3799aa) IP3R receptor in Myzus persicae, which is approximately 1000 amino acids larger than previously reported for IP3Rs.

Unravelling the Molecular Determinants of Bee Sensitivity to Neonicotinoid Insecticides.

The impact of neonicotinoid insecticides on the health of bee pollinators is a topic of intensive research and considerable current debate [1]. As insecticides, certain neonicotinoids, i.e., N-nitroguanidine compounds such as imidacloprid and thiamethoxam, are as intrinsically toxic to bees as to the insect pests they target. However, this is not the case for all neonicotinoids, with honeybees orders of magnitude less sensitive to N-cyanoamidine compounds such as thiacloprid [2]. Although previous work has suggested that this is due to rapid metabolism of these compounds [2-5], the specific gene(s) or enzyme(s) involved remain unknown. Here, we show that the sensitivity of the two most economically important bee species to neonicotinoids is determined by cytochrome P450s of the CYP9Q subfamily. Radioligand binding and inhibitor assays showed that variation in honeybee sensitivity to N-nitroguanidine and N-cyanoamidine neonicotinoids does not reside in differences in their affinity for the receptor but rather in divergent metabolism by P450s. Functional expression of the entire CYP3 clade of P450s from honeybees identified a single P450, CYP9Q3, that metabolizes thiacloprid with high efficiency but has little activity against imidacloprid. We demonstrate that bumble bees also exhibit profound differences in their sensitivity to different neonicotinoids, and we identify CYP9Q4 as a functional ortholog of honeybee CYP9Q3 and a key metabolic determinant of neonicotinoid sensitivity in this species. Our results demonstrate that bee pollinators are equipped with biochemical defense systems that define their sensitivity to insecticides and this knowledge can be leveraged to safeguard bee health.

Identification of S-Nitrosothiols by the Sequential Cysteine Blocking Technique.

Here, we describe a procedure for the identification of S-nitrosothiols that has been used in our laboratory to study the roles of protein S-nitrosylation in the immune responses of Arabidopsis thaliana and other organisms. It employs a modified version of the biotin-switch technique, which we termed the sequential cysteine blocking technique, encompassing the sequential redox-blocking of recombinant proteins followed by LC-MS/MS analysis.