A draft Diabrotica virgifera virgifera genome: insights into control and host plant adaption by a major maize pest insect.

BACKGROUND: Adaptations by arthropod pests to host plant defenses of crops determine their impacts on agricultural production. The larval host range of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is restricted to maize and a few grasses. Resistance of D. v. virgifera to crop rotation practices and multiple insecticides contributes to its status as the most damaging pest of cultivated maize in North America and Europe. The extent to which adaptations by this pest contributes to host plant specialization remains unknown. RESULTS: A 2.42 Gb draft D. v. virgifera genome, Dvir_v2.0, was assembled from short shotgun reads and scaffolded using long-insert mate-pair, transcriptome and linked read data. K-mer analysis predicted a repeat content of ≥ 61.5%. Ortholog assignments for Dvir_2.0 RefSeq models predict a greater number of species-specific gene duplications, including expansions in ATP binding cassette transporter and chemosensory gene families, than in other Coleoptera. A majority of annotated D. v. virgifera cytochrome P450s belong to CYP4, 6, and 9 clades. A total of 5,404 transcripts were differentially-expressed between D. v. virgifera larvae fed maize roots compared to alternative host (Miscanthus), a marginal host (Panicum virgatum), a poor host (Sorghum bicolor) and starvation treatments; Among differentially-expressed transcripts, 1,908 were shared across treatments and the least number were between Miscanthus compared to maize. Differentially-expressed transcripts were enriched for putative spliceosome, proteosome, and intracellular transport functions. General stress pathway functions were unique and enriched among up-regulated transcripts in marginal host, poor host, and starvation responses compared to responses on primary (maize) and alternate hosts. CONCLUSIONS: Manual annotation of D. v. virgifera Dvir_2.0 RefSeq models predicted expansion of paralogs with gene families putatively involved in insecticide resistance and chemosensory perception. Our study also suggests that adaptations of D. v. virgifera larvae to feeding on an alternate host plant invoke fewer transcriptional changes compared to marginal or poor hosts. The shared up-regulation of stress response pathways between marginal host and poor host, and starvation treatments may reflect nutrient deprivation. This study provides insight into transcriptomic responses of larval feeding on different host plants and resources for genomic research on this economically significant pest of maize.

Enhanced metabolism and selection of pyrethroid-resistant western corn rootworms (Diabrotica virgifera virgifera LeConte).

Western corn rootworm (WCR) pyrethroid resistance has been previously reported in the United States (US) western Corn Belt, and cross-resistance and synergism studies suggested that both target site insensitivity and enhanced metabolism may be conferring WCR resistance to pyrethroids. The present study aimed to investigate the potential mechanisms of WCR pyrethroid resistance and to estimate the heritability of the resistance trait. Biochemical assays using model substrates and spectrophotometry revealed 2-4-fold higher activity of P450s and esterases in pyrethroid-resistant WCR populations, whereas the biological activity of glutathione S-transferase was similar between populations tested. No mutation in the voltage-gated sodium channel was detected in pyrethroid-resistant WCR individuals by sequencing PCR products containing the para-homologous L1014, T929, and M918 amino acid positions that are commonly associated with target site mutations in other pyrethroid-resistant insects. A pilot estimation of pyrethroid resistance heritability obtained during laboratory selection of a WCR population suggested a major genetic component of the resistance trait and predicted a 10-fold increase in WCR bifenthrin resistance within ~7 generations of insecticide lethal exposure. Results support earlier indirect evidence that enhanced metabolism may be contributing to WCR resistance to pyrethroids and illustrates the potential of WCR pyrethroid resistance evolution.

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.

RNAi-induced knockdown of white gene in the southern green stink bug (Nezara viridula L.).

The southern green stink bug (SGSB) Nezara viridula L. is one of the most common stink bug species in the United States and can cause significant yield loss in a variety of crops. A suitable marker for the assessment of gene-editing tools in SGSB has yet to be characterized. The white gene, first documented in Drosophila, has been a useful target to assess the efficiency of introduced mutations in many species as it controls pigmentation processes and mutants display readily identifiable phenotypes. In this study we used the RNAi technique to investigate functions and phenotypes associated with the white ortholog in the SGSB and to validate white as a marker for genetic transformation in this species. This study revealed that white may be a suitable marker for germline transformation in the SGSB as white transcript knockdown was not lethal, did not impair embryo development and provided a distinguishable phenotype. Our results demonstrated that the white ortholog in SGSB is involved in the pathway for ommochrome synthesis and suggested additional functions of this gene such as in the integument composition, management of hemolymph compounds and riboflavin mobilization.

The Use of Insecticides to Manage the Western Corn Rootworm, Diabrotica virgifera virgifera, LeConte: History, Field-Evolved Resistance, and Associated Mechanisms.

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Dvv) is a significant insect pest of maize in the United States (U.S.). This paper reviews the history of insecticide use in Dvv management programs, Dvv adaptation to insecticides, i.e., field-evolved resistance and associated mechanisms of resistance, plus the current role of insecticides in the transgenic era. In the western U.S. Corn Belt where continuous maize is commonly grown in large irrigated monocultures, broadcast-applied soil or foliar insecticides have been extensively used over time to manage annual densities of Dvv and other secondary insect pests. This has contributed to the sequential occurrence of Dvv resistance evolution to cyclodiene, organophosphate, carbamate, and pyrethroid insecticides since the 1950s. Mechanisms of resistance are complex, but both oxidative and hydrolytic metabolism contribute to organophosphate, carbamate, and pyrethroid resistance facilitating cross-resistance between insecticide classes. History shows that Dvv insecticide resistance can evolve quickly and may persist in field populations even in the absence of selection. This suggests minimal fitness costs associated with Dvv resistance. In the transgenic era, insecticides function primarily as complementary tools with other Dvv management tactics to manage annual Dvv densities/crop injury and resistance over time.

Molecular characterization of western corn rootworm pyrethroid resistance.

BACKGROUND: Western corn rootworm (WCR) pyrethroid resistance has been confirmed in the western US Corn Belt. Toxicological and biochemical studies indicated that multiple mechanisms of resistance might be involved in the resistance trait, such as enhanced metabolism and/or kdr target-site mutation(s) in the voltage-gated sodium channels. To characterize the mechanisms of WCR pyrethroid resistance at the molecular level, pairwise comparisons were made between RNA-Seq data collected from pyrethroid-resistant and -susceptible WCR populations. Gene expression levels and sodium channel sequences were evaluated. RESULTS: Seven transcripts exhibited significantly different expression (q ≤ 0.05) when comparing field-collected pyrethroid-resistant (R-Field) and -susceptible (S-Field) WCR populations. Three of the differentially expressed transcripts were P450s overexpressed in R-Field (9.2-26.2-fold). A higher number (99) of differentially expressed transcripts was found when comparing laboratory-derived pyrethroid-resistant (R-Lab) and -susceptible (S-Lab) WCR populations. Eight of the significant transcripts were P450s overexpressed in R-Lab (2.7-39.8-fold). This study did not detect kdr mutations in pyrethroid-resistant WCR populations. Other differentially expressed transcripts that may play a role in WCR pyrethroid resistance are discussed. CONCLUSION: This study revealed that P450-mediated metabolism is likely to be a major mechanism of WCR pyrethroid resistance, which could affect the efficacy of other insecticides sharing similar metabolic pathways. Additionally, results suggested that although laboratory selection of a pyrethroid-resistant WCR population may help to characterize resistance mechanisms, a field-selected population provided rare and perhaps major variants corresponding to the resistance trait.

Field efficacy of soil insecticides on pyrethroid-resistant western corn rootworms (Diabrotica virgifera virgifera LeConte).

BACKGROUND: Field-evolved pyrethroid resistance has been confirmed in western corn rootworm (WCR) populations collected from the United States (US) western Corn Belt. Resistance levels of WCR adults estimated in lab bioassays were confirmed to significantly reduce the efficacy of foliar-applied bifenthrin. The objective of the present study was to investigate the impact of WCR pyrethroid resistance levels on the performance of common soil-applied insecticide formulations (23.4% tefluthrin, 17.15% bifenthrin, and 0.1% cyfluthrin + 2.0% tebupirimphos). Field trials were conducted in 2016 and 2017 in three Nebraska, US, counties (Saunders, Clay, and Keith) where distinct levels of WCR susceptibility to pyrethroids (susceptible, moderately resistant, and highly resistant) had been previously reported in adult and larval bioassays. RESULTS: All soil insecticide treatments effectively protected maize roots from a pyrethroid-susceptible WCR population at Saunders. In contrast, the efficacy of bifenthrin and tefluthrin soil insecticides was significantly reduced at Clay and Keith, where pyrethroid-resistant WCR populations were reported. At Keith, where an additional failure of the cyfluthrin + tebupirimphos soil insecticide was observed, WCR laboratory dose-response bioassays showed a consistent ∼5-fold resistance level to the active ingredients bifenthrin, tefluthrin, and cyfluthrin. CONCLUSION: The efficacy of common soil insecticides used in the US for WCR management was significantly reduced in populations exhibiting relatively low levels of WCR pyrethroid resistance. Using a multitactical approach to manage WCR within an integrated pest management framework may mitigate resistance evolution and prolong the usefulness of WCR insecticides within the system. © 2019 Society of Chemical Industry.

Western corn rootworm pyrethroid resistance confirmed by aerial application simulations of commercial insecticides.

The western corn rootworm (Diabrotica virgifera virgifera LeConte) (WCR) is a major insect pest of corn (Zea mays L.) in the United States (US) and is highly adaptable to multiple management tactics. A low level of WCR field-evolved resistance to pyrethroid insecticides has been confirmed in the US western Corn Belt by laboratory dose-response bioassays. Further investigation has identified detoxification enzymes as a potential part of the WCR resistance mechanism, which could affect the performance of insecticides that are structurally related to pyrethroids, such as organophosphates. Thus, the responses of pyrethroid-resistant and -susceptible WCR populations to the commonly used pyrethroid bifenthrin and organophosphate dimethoate were compared in active ingredient bioassays. Results revealed a relatively low level of WCR resistance to both active ingredients. Therefore, a simulated aerial application bioassay technique was developed to evaluate how the estimated resistance levels would affect performance of registered rates of formulated products. The simulated aerial application technique confirmed pyrethroid resistance to formulated rates of bifenthrin whereas formulated dimethoate provided optimal control. Results suggest that the relationship between levels of resistance observed in dose-response bioassays and actual efficacy of formulated product needs to be further explored to understand the practical implications of resistance.

Bifenthrin Baseline Susceptibility and Evaluation of Simulated Aerial Applications in Striacosta albicosta (Lepidoptera: Noctuidae).

Striacosta albicosta (Smith) is a maize pest that has recently expanded its geographical range into the eastern United States and southeastern Canada. Aerial application of pyrethroids, such as bifenthrin, has been a major practice adopted to manage this pest. Reports of field failure of pyrethroids have increased since 2013. Striacosta albicosta populations were collected in 2016 and 2017 from maize fields in Nebraska, Kansas, and Canada and screened with bifenthrin active ingredient in larval contact dose-response bioassays. Resistance ratios estimated were generally low in 2016 (1.04- to 1.32-fold) with the highest LC50 in North Platte, NE (66.10 ng/cm2) and lowest in Scottsbluff, NE (50.10 ng/cm2). In 2017, O'Neill, NE showed the highest LC50 (100.66 ng/cm2) and Delhi, Canada exhibited the lowest (6.33 ng/cm2), resulting in a resistance ratio variation of 6.02- to 15.90-fold. Implications of bifenthrin resistance levels were further investigated by aerial application simulations. Experiments were conducted with a spray chamber where representative S. albicosta populations were exposed to labeled rates of a commercial bifenthrin formulation. Experiments resulted in 100% mortality for all populations, instars, insecticide rates, and carrier volumes, suggesting that levels of resistance estimated for bifenthrin active ingredient did not seem to impact the efficacy of the correspondent commercial product under controlled conditions. Results obtained from this research indicate that control failures reported in Nebraska could be associated with factors other than insecticide resistance, such as issues with the application technique, environmental conditions during and/or after application, or the insect's natural behavior. Data generated will assist future S. albicosta resistance management programs.

Cross-resistance and synergism bioassays suggest multiple mechanisms of pyrethroid resistance in western corn rootworm populations.

Recently, resistance to the pyrethroid bifenthrin was detected and confirmed in field populations of western corn rootworm, Diabrotica virgifera virgifera LeConte from southwestern areas of Nebraska and Kansas. As a first step to understand potential mechanisms of resistance, the objectives of this study were i) to assess adult mortality at diagnostic concentration-LC99 to the pyrethroids bifenthrin and tefluthrin as well as DDT, ii) estimate adult and larval susceptibility to the same compounds as well as the organophosphate methyl-parathion, and iii) perform synergism experiments with piperonyl butoxide (PBO) (P450 inhibitor) and S,S,S-tributyl-phosphorotrithioate (DEF) (esterase inhibitor) in field populations. Most of the adult field populations exhibiting some level of bifenthrin resistance exhibited significantly lower mortality to both pyrethroids and DDT than susceptible control populations at the estimated LC99 of susceptible populations. Results of adult dose-mortality bioassays also revealed elevated LC50 values for bifenthrin resistant populations compared to the susceptible control population with resistance ratios ranging from 2.5 to 5.5-fold for bifenthrin, 28 to 54.8-fold for tefluthrin, and 16.3 to 33.0 for DDT. These bioassay results collectively suggest some level of cross-resistance between the pyrethroids and DDT. In addition, both PBO and DEF reduced the resistance ratios for resistant populations although there was a higher reduction in susceptibility of adults exposed to PBO versus DEF. Susceptibility in larvae varied among insecticides and did not correlate with adult susceptibility to tefluthrin and DDT, as most resistance ratios were < 5-fold when compared to the susceptible population. These results suggest that both detoxifying enzymes and target site insensitivity might be involved as resistance mechanisms.