Proactive resistance management for sustaining the efficacy of RNA interference for pest control.

Biopesticides based on RNA interference (RNAi) took a major step forward with the first registration of a sprayable RNAi product, which targets the world's most damaging potato pest. Proactive resistance management is needed to delay the evolution of resistance by pests and sustain the efficacy of RNAi biopesticides.

Early Growth Patterns of Bacillus cereus on Potato Substrate in the Presence of Low Densities of Black Soldier Fly Larvae.

Bacillus cereus is a common and ubiquitous bacterium that can cause foodborne illnesses in humans and other animals. Common methods of contact between foodborne pathogens and their victims include exposure through contaminated food or food containment products. Using larvae of black soldier flies, Hermetia illucens, for biological conversion of wastes into components of animal feeds is a rapidly growing technology. However, contamination of larval biomass with pathogenic microorganisms may challenge its use on an industrial scale. We conducted laboratory experiments to test the effects of the black soldier fly larvae developing on simulated potato waste substrate on B. cereus abundance. We observed a general increase in the number of colony-forming units and concentration of hblD - gene when the larvae were present in the substrate, although the effect was modulated by larval densities and time since inoculation. It is possible that starch breakdown by black soldier fly larvae may provide a beneficial environment for B. cereus. Our results differ from the suppression in the presence by black soldier fly larvae reported for several other bacterial species and highlight the importance of taking proper food safety measures when using this technology.

Effects of Low Doses of a Novel dsRNA-based Biopesticide (Calantha) on the Colorado Potato Beetle.

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) is a destructive pest of the cultivated potato, Solanum tuberosum. Members of this species are well-suited to agricultural habitats because of a suite of physiological adaptations and their ability to evolve resistance to multiple insecticides. Recently, a novel double-stranded RNA (dsRNA) insecticide (Calantha, active ingredient ledprona) has been demonstrated as an effective tool to manage Colorado potato beetle populations through RNA interference (RNAi). Previous studies have demonstrated the lethality of the high doses of ledprona but had not assessed possible effects of low doses that may happen due to product degradation in the environment, incomplete spray coverage, and foliage growth. Exposure of fourth instar larvae to low concentrations of ledprona interfered with their pupation. Exposure of adults significantly reduced their mobility after seven days, as well as their fertility. Reproductive effects were stronger in females, especially when exposed before reaching sexual maturity. The observed effects of low doses of ledprona may aid in the overall management of Colorado potato beetles by reducing the size of resident populations, inhibiting beetle movement within and between fields, and reducing the population growth rate.

Suppression of Methicillin-Resistant Staphylococcus aureus and Reduction of Other Bacteria by Black Soldier Fly Larvae Reared on Potato Substrate.

Larvae of black soldier flies, Hermetia illucens, are increasingly used for biological conversion of animal and plant wastes into ingredients of animal feeds on an industrial scale. The presence of pathogenic microorganisms in harvested larvae may be a serious problem for wide-scale adoption of this technology. Fortunately, black soldier fly larvae may have some antimicrobial properties. Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium associated with various environments that can be pathogenic to humans and farmed animals. We tested whether black soldier fly larvae suppress MRSA on potato substrate. Autoclaved potatoes containing black soldier fly larvae (P+BSFL), potatoes inoculated with MRSA and containing black soldier fly larvae (P+MRSA+BSFL), and potatoes inoculated with MRSA (P+MRSA) were incubated in glass jars. Substrate samples were taken after 3 and 7 days of incubation and plated on Trypticase soy agar (TSA) and Staphylococcus medium 110 agar (SA) to quantify total bacteria and MRSA, respectively. DNA was extracted from potato substrates on both days and sequenced to assess bacterial and fungal diversity using 515F/806R and internal transcribed spacer (ITS) 1/2 primers, respectively, and QIIME 2.0 software. Both total bacterial and MRSA-specific CFU were reduced in the presence of black soldier fly larvae, with a larger reduction for the latter. Twenty-five bacterial genera and 3 fungal genera were detected. Twenty bacterial genera were shared among the treatments and the days, but their relative abundances often varied. Among the most abundant genera, only Enterococcus and Lactococcus were universally present. Our findings confirm antimicrobial properties of black soldier fly larvae. IMPORTANCE Larvae of black soldier flies, Hermetia illucens, may be used to provide an environmentally sustainable and economically viable method for biological conversion of animal and plant wastes into ingredients of animal feeds on an industrial scale. However, contamination of harvested larvae by pathogenic microorganisms inhabiting decaying substrates may be a serious problem for wide-scale adoption of this technology. Fortunately, black soldier fly larvae may have some antimicrobial properties, including suppression of several common pathogens. Our study showed that such a suppression applies to methicillin-resistant Staphylococcus aureus, which is a ubiquitous bacterium pathogenic to animals (including humans).

Characterization of molecular and kinetic properties of two acetylcholinesterases from the Colorado potato beetle, Leptinotarsa decemlineata.

The molecular and biochemical properties of two acetylcholinesterases (LdAChE1 and LdAChE2) from the Colorado potato beetle, Leptinotarsa decemlineata, were investigated in this study. Polyacrylamide gel electrophoresis in conjunction with western blotting with LdAChE1- or LdAChE2-specific antibodies suggested that LdAChE1 exists in a soluble form, whereas LdAChE2 exists in both soluble and amphiphilic forms with a glycophosphatidylinositol anchor. Both LdAChEs exist as homodimers with each monomer connected with a disulfide bond. LdAChE1 was the most highly expressed in the thorax followed by the head, leg, and abdomen, whereas LdAChE2 was the most highly expressed in the head, followed by the thorax, leg, and abdomen. The overall expression levels of LdAChE1, however, were higher than those of LdAChE2 in all examined tissues. Kinetic analysis using recombinant LdAChE1 and LdAChE2 showed that LdAChE2 has a 4.8-fold higher catalytic efficiency toward acetylthiocholine iodide compared to LdAChE1. LdAChE2 was more sensitive to organophosphorus and carbamate insecticides than LdAChE1. The addition of irreversibly phosphorylated LdAChE1 via paraoxon titration significantly reduced LdAChE2 inhibition by insecticides and glycoalkaloids, suggesting a sequestration role of soluble LdAChE1 in the chemical defense against xenobiotics. Taken together, LdAChE2 may be the main enzyme for synaptic transmission, thus serving as a toxicologically more relevant target, whereas the soluble LdAChE1 may function as a bioscavenger.

Toxicity of a novel dsRNA-based insecticide to the Colorado potato beetle in laboratory and field trials.

BACKGROUND: The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) is one of the most notorious pests of the potato, Solanum tuberosum. Potato beetles are capable of developing resistance to various insecticides in relatively few generations. Novel and effective means of controlling Colorado potato beetle populations are constantly required to protect potato crops and prevent loss of yield. The knockdown of gene function through ribonucleic acid interference has been demonstrated in Colorado potato beetles, suggesting the use of this technology as a means of beetle management. A novel double-stranded RNA-based insecticide with the active ingredient, ledprona, has been tested in variable dose laboratory bioassays, followed by field studies. RESULTS: Exposure to ledprona resulted in both increased beetle mortality and decreased foliage consumption in all four instars and adult beetles. Effects decreased from earlier to later life stages. No ovicidal activity was detected. Onset of mortality was slower compared with existing chemical insecticides. Nevertheless, field applications of formulated ledprona to potato plots resulted in their protection comparable with that provided by spinosad and chlorantraniliprole. CONCLUSION: Based on the results of this study, formulated ledprona has attributes to become a useful tool in controlling Colorado potato beetle populations that is likely to be a good fit in integrated pest management protocols. © 2022 Society of Chemical Industry.

First Sprayable Double-Stranded RNA-Based Biopesticide Product Targets Proteasome Subunit Beta Type-5 in Colorado Potato Beetle (Leptinotarsa decemlineata).

Colorado potato beetle (CPB, Leptinotarsa decemlineata) is a major pest of potato and other solanaceous vegetables in the Northern Hemisphere. The insect feeds on leaves and can completely defoliate crops. Because of the repeated use of single insecticide classes without rotating active ingredients, many chemicals are no longer effective in controlling CPB. Ledprona is a sprayable double-stranded RNA biopesticide with a new mode of action that triggers the RNA interference pathway. Laboratory assays with second instar larvae fed Ledprona showed a dose-response where 25×10-6g/L of dsPSMB5 caused 90% mortality after 6days of initial exposure. We also showed that exposure to Ledprona for 6h caused larval mortality and decreased target messenger RNA (mRNA) expression. Decrease in PSMB5 protein levels was observed after 48h of larval exposure to Ledprona. Both PSMB5 mRNA and protein levels did not recover over time. Ledprona efficacy was demonstrated in a whole plant greenhouse trial and performed similarly to spinosad. Ledprona, currently pending registration at EPA, represents a new biopesticide class integrated pest management and insecticide resistance management programs directed against CPB.

Structural and Functional Characterization of One Unclassified Glutathione S-Transferase in Xenobiotic Adaptation of Leptinotarsa decemlineata.

Arthropod Glutathione S-transferases (GSTs) constitute a large family of multifunctional enzymes that are mainly associated with xenobiotic or stress adaptation. GST-mediated xenobiotic adaptation takes place through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. To date, the roles of GSTs in xenobiotic adaptation in the Colorado potato beetle (CPB), a notorious agricultural pest of plants within Solanaceae, have not been well studied. Here, we functionally expressed and characterized an unclassified-class GST, LdGSTu1. The three-dimensional structure of the LdGSTu1 was solved with a resolution up to 1.8 Å by X-ray crystallography. The signature motif VSDGPPSL was identified in the "G-site", and it contains the catalytically active residue Ser14. Recombinant LdGSTu1 was used to determine enzyme activity and kinetic parameters using 1-chloro-2, 4-dinitrobenzene (CDNB), GSH, p-nitrophenyl acetate (PNA) as substrates. The enzyme kinetic parameters and enzyme-substrate interaction studies demonstrated that LdGSTu1 could catalyze the conjugation of GSH to both CDNB and PNA, with a higher turnover number for CDNB than PNA. The LdGSTu1 enzyme inhibition assays demonstrated that the enzymatic conjugation of GSH to CDNB was inhibited by multiple pesticides, suggesting a potential function of LdGSTu1 in xenobiotic adaptation.

Selection for high levels of resistance to double-stranded RNA (dsRNA) in Colorado potato beetle (Leptinotarsa decemlineata Say) using non-transgenic foliar delivery.

Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI ß; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.

Geographic Variation in Dominance of Spinosad Resistance in Colorado Potato Beetles (Coleoptera: Chrysomelidae).

Eastern New York State is frequently the site of Colorado potato beetle (Leptinotarsa decemlineata, Say) populations with the highest observed levels of insecticide resistance to a range of active ingredients. The dominance of a resistant phenotype will affect its rate of increase and the potential for management. On organic farms on Long Island, L. decemlineata evolved high levels of resistance to spinosad in a short period of time and that resistance has spread across the eastern part of the Island. Resistance has also emerged in other parts of the country as well. To clarify the level of dominance or recessiveness of spinosad resistance in different parts of the United States and how resistance differs in separate beetle populations, we sampled in 2010 beetle populations from Maine, Michigan, and Long Island. In addition, a highly resistant Long Island population was assessed in 2012. All populations were hybridized with a laboratory-susceptible strain to determine dominance. None of the populations sampled in 2010 were significantly different from additive resistance, but the Long Island population sampled in 2012 was not significantly different from fully recessive. Recessive inheritance of high-level resistance may help manage its increase.

Dose-Dependent Retention of Omega-3 Fatty Acids by Black Soldier Fly Larvae (Diptera: Stratiomyidae).

Black soldier fly larvae, Hermetia illucens (L.), are used to convert organic waste streams into insect-based animal feeds. We tested their ability to retain alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) from feeding substrates, which has important implications for their use in aquaculture. When supplementing a chicken feed diet with increasing concentrations of salmon oil (0-42%) over an increasing number of days (0-8), the concentrations of the three omega-3 acids in larvae increased significantly. Larval survival and biomass accumulation were not affected. Supplementing a chicken feed diet with increasing concentrations (0-14%) of Tetraselmis chui Butcher (Chlorodendrales: Chlorodendraceae) microalgae paste also significantly increased ALA and EPA contents of the harvested larvae. However, microalgae also decreased survival, harvested biomass, and individual growth of larvae feeding on the diet with the highest supplement concentration (14%). DHA was not detected in any microalgae diet or subsequent larval tissue samples. All three omega-3 polyunsaturated fatty acids tested in this study were accumulated in dose-dependent manner, with quadratic, and occasionally linear, equations providing the best description of the observed relationships. There were significant negative correlations between several fatty acids, indicating that they may replace one another in living larvae. Our findings confirm that black soldier fly larvae can retain ingested fatty acids and change fatty acid profiles in their tissues accordingly. However, optimizing nutrient content of harvestable larvae is likely to be more complicated than simply enriching their diets with omega-3 fatty acids.

Tolerance of Immature Black Soldier Flies (Diptera: Stratiomyidae) to Cold Temperatures Above and Below Freezing Point.

Black soldier flies, Hermetia illucens (L.), consume decaying organic materials at the larval stage and can be used for recycling a variety of biogenic wastes into value-added products. Black soldier flies are normally found in subtropical and warm temperate regions. Cold temperatures may prevent their establishment in colder areas, thus alleviating a concern of their becoming an invasive species. Potentially, cold temperatures can also be used to manipulate the rate of black soldier fly development, which may be needed for timing certain life stages for mass-production needs. In the present study, immature black soldier flies were highly susceptible to freezing. Their survivorship decreased as time spent at -12°C increased from 10 to 60 min. Only ca. 2% of eggs, <1% of larvae, and no pupae survived after 60 min of exposure. Chilling at 4°C also had a significant negative effect that became more pronounced as duration of exposure increased from 24 to 72 h. Only ca. 2% of eggs and second instars and ca. 23% of pupae survived after 72 h. In the same time, >80% of third instars and >90% of fifth instars were still alive following 72 h of exposure. Chilling fifth instars resulted in smaller adults but freezing them for 48 h resulted in bigger adults. Based on these results, black soldier fly is unlikely to establish in areas with long periods of subfreezing winter temperatures. Low temperatures may be used to manipulate development of the late instars, but at a cost of higher mortality.

Gender-Specific Acaricidal Properties and Sexual Transmission of Spirotetramat in Two-Spotted Spider Mite (Tetranychidae: Acariformes).

Two-spotted spider mite, Tetranychus urticae Koch (Tetranychidae: Acariformes), is one of the most important agricultural pests in the world. Their populations have a tendency of rapidly developing resistance to acaricides, making it necessary to have a variety of active ingredients for sustainable chemical control of this pest. We investigated acaricidal properties of a relatively new insecticide spirotetramat using its commercial formulation, Movento Energy. Spirotetramat applied at concentrations equivalent to the field rates of 78 and 90 g ai/ha had a strong negative effect on the survival of the treated T. urticae, with ca. 95% of females and ca. 65% of males dying after the treatment. Spirotetramat appeared to interfere with female reproductive system. Many of the dead treated females had eggs stuck in the oviduct and protruding from their bodies. Surviving treated females did not lay eggs. Furthermore, untreated females that mated with treated males did not produce female offspring and displayed the symptoms of spirotetramat poisoning. Toxic effects continued manifesting themselves after female mites were transferred from treated onto untreated plant culture. Contrary to previous studies, contact toxicity was also detected. None of the treated immature stages survived to adulthood. Based on these results, spirotetramat may be a good option for integrated pest management in crops that are simultaneously affected by sucking insects and spider mites and in pesticide rotation sequences that are a part of integrated resistance management programs.

Lethal and Sublethal Effects of Mineral Oil on Potato Pests.

Mineral oil is a product used to reduce Potato Virus Y transmission in potato fields. However, there is little information available about other effects that oil may have on insect pests of potato. To better understand how mineral oil affects potato pests, we performed a series of experiments testing the effects of oil on mortality, behavior, and development of potato aphids, Macrosiphum euphorbiae (Thomas) (Hemiptera: Aphididae), green peach aphids, Myzus persicae (Sulzer) (Hemiptera: Aphididae), and Colorado potato beetles, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). All three species showed negative behavioral responses to oil-treated potato foliage. Oil treatment also increased aphid mortality. Colorado potato beetle mortality was not affected, but developing on oil-treated potato plants resulted in prolonged development and smaller adults. Additionally, oil acted synergistically with the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae); Colorado potato beetle larvae were killed more rapidly when sprayed with both products compared with when sprayed with B. bassiana alone. Based on these results, mineral oil has the potential for expanded use in potato IPM programs.

Adaptation to toxic hosts as a factor in the evolution of insecticide resistance.

Insecticide resistance is a serious economic problem that jeopardizes sustainability of chemical control of herbivorous insects and related arthropods. It can be viewed as a specific case of adaptation to toxic chemicals, which has been driven in large part, but not exclusively, by the necessity for insect pests to tolerate defensive compounds produced by their host plants. Synthetic insecticides may simply change expression of specific sets of detoxification genes that have evolved due to ancestral associations with host plants. Feeding on host plants with more abundant or novel secondary metabolites has even been shown to prime insect herbivores to tolerate pesticides. Clear understanding of basic evolutionary processes is important for achieving lasting success in managing herbivorous arthropods.

Adult cannibalism in an oligophagous herbivore, the Colorado potato beetle.

Cannibalism, or intraspecific predation, can play a major role in changing individual fitness and population processes. In insects, cannibalism frequently occurs across life stages, with cannibals consuming a smaller or more vulnerable stage. Predation of adult insects on one another is considered to be uncommon. We investigated adult cannibalism in the Colorado potato beetle, Leptinotarsa decemlineata (Say), which is an oligophagous herbivore specializing on plants in family Solanaceae, and an important agricultural pest. Under laboratory conditions, starvation and crowding encouraged teneral adults to feed upon each other, which reduced their weight loss during the period of starvation. However, pupae were attacked and consumed before adults. Injured beetles had a higher probability of being cannibalized than intact beetles. Males were more frequently attacked than females, but that appeared to be a function of their smaller size rather than other gender-specific traits. Cannibalizing eggs at a larval stage did not affect beetle propensity to cannibalize adults at an adult stage. When given a choice between conspecific adults and mealworms, the beetles preferred to eat conspecifics. Cannibalistic behavior, including adult cannibalism, could be important for population persistence in this species.

Heat-induced mortality and expression of heat shock proteins in Colorado potato beetles treated with imidacloprid.

The Colorado potato beetle is an important pest of solanaceous plants in the Northern Hemisphere. Better understanding of its physiological responses to temperature stress and their interactions with still-prevalent chemical control has important implications for the management of this insect. We measured mortality and expression of the Hsp70 heat shock proteins in the Colorado potato beetle larvae exposed to sublethal concentration of the commonly used insecticide imidacloprid, and to supraoptimal temperatures. Both turned out to be significant stress factors, although induction of Hsp70 by imidacloprid observed in the present study was low compared to its induction by the heat. The two factors also interacted with each other. At an extreme temperature of 43 °C, exposure to a sublethal dose of imidacloprid resulted in a significant rise in larval mortality, which was not observed at an optimal temperature of 25 °C. Heat-stressed larvae also failed to respond to imidacloprid by producing more Hsp70. These findings suggest that when field rates of insecticides become insufficient for killing the exposed beetles under optimal temperature conditions due to the evolution of resistance in beetle populations, they may still reduce the probability of resistant beetles surviving the heat shock created by using propane flamers as a rescue treatment.

The Red Queen in a potato field: integrated pest management versus chemical dependency in Colorado potato beetle control.

Originally designed to reconcile insecticide applications with biological control, the concept of integrated pest management (IPM) developed into the systems-based judicious and coordinated use of multiple control techniques aimed at reducing pest damage to economically tolerable levels. Chemical control, with scheduled treatments, was the starting point for most management systems in the 1950s. Although chemical control is philosophically compatible with IPM practices as a whole, reduction in pesticide use has been historically one of the main goals of IPM practitioners. In the absence of IPM, excessive reliance on pesticides has led to repeated control failures due to the evolution of resistance by pest populations. This creates the need for constant replacement of failed chemicals with new compounds, known as the 'insecticide treadmill'. In evolutionary biology, a similar phenomenon is known as the Red Queen principle - continuing change is needed for a population to persevere because its competitors undergo constant evolutionary adaptation. The Colorado potato beetle, Leptinotarsa decemlineata (Say), is an insect defoliator of potatoes that is notorious for its ability to develop insecticide resistance. In the present article, a review is given of four case studies from across the United States to demonstrate the importance of using IPM for sustainable management of a highly adaptable insect pest. Excessive reliance on often indiscriminate insecticide applications and inadequate use of alternative control methods, such as crop rotation, appear to expedite evolution of insecticide resistance in its populations. Resistance to IPM would involve synchronized adaptations to multiple unfavorable factors, requiring statistically unlikely genetic changes. Therefore, integrating different techniques is likely to reduce the need for constant replacement of failed chemicals with new ones.