Mutations on M3 helix of Plutella xylostella glutamate-gated chloride channel confer unequal resistance to abamectin by two different mechanisms

Abamectin is one of the most widely used avermectins for agricultural pests control, but the emergence of resistance around the world is proving a major threat to its sustained application. Abamectin acts by directly activating glutamate-gated chloride channels (GluCls) and modulating other Cys-loop ion channels. To date, three mutations occurring in the transmembrane domain of arthropod GluCls are associated with target-site resistance to abamectin: A309V in Plutella xylostella GluCl (PxGluCl), G323D in Tetranychus urticae GluCl1 (TuGluCl1) and G326E in TuGluCl3. To compare the effects of these mutations in a single system, A309V/I/G and G315E (corresponding to G323 in TuGluCl1 and G326 in TuGluCl3) substitutions were introduced individually into the PxGluCl channel. Functional analysis using Xenopus oocytes showed that the A309V and G315E mutations reduced the sensitivity to abamectin by 4.8- and 493-fold, respectively. In contrast, the substitutions A309I/G show no significant effects on the response to abamectin. Interestingly, the A309I substitution increased the channel sensitivity to glutamate by one order of magnitude (∼12-fold). Analysis of PxGluCl homology models indicates that the G315E mutation interferes with abamectin binding through a steric hindrance mechanism. In contrast, the structural consequences of the A309 mutations are not so clear and an allosteric modification of the binding site is the most likely mechanism. Overall the results show that both A309V and G315E mutations may contribute to target-site resistance to abamectin and may be important for the future prediction and monitoring of abamectin resistance in P. xylostella and other arthropod pests.     

Tebufenozide resistance is associated with sex‐linked inheritance in Plutella xylostella

The diamondback moth (DBM), Plutella xylostella (L.), is a major pest of cruciferous crops. Tebufenozide, a novel nonsteroidal ecdysone agonist, exhibits good efficacy and has played an increasingly important role in the control of Lepidopteran pests in China. For its resistance management, the genetic basis of tebufenozide resistance was studied using a laboratory selected resistant strain of DBM (resistant ratio, RR = 268). A series of crosses with laboratory susceptible and resistant strains revealed that tebufenozide resistance in this pest was partially biased toward female heredity, with a large difference in RR for F₁ (RR = 29) and rF₁ progeny (RR = 147). The dominance calculated for these 2 cross progeny was ?0.788 and 0.09, respectively. Further analysis showed that the susceptible male and female larvae were similar in their sensitivity to tebufenozide, but the resistant female larvae showed significantly higher resistance than the resistant male larvae. The heredity of tebufenozide resistance in DBM might be linked with the W sex chromosome, which suggested that DBM has the ability to develop high levels of resistance to tebufenozide. This is the first report of sex‐linked inheritance of tebufenozide resistance in P. xylostella (L.).     

Abamectin resistance in strains of vegetable leafminer,Liriomyza sativae (Diptera: Agromyzidae) is linked to elevated glutathione S‐transferase activity

Abamectin resistance was selected in the vegetable leafminer, Liriomyza sativae (Blanchard) (Diptera: Agromyzidae) under laboratory conditions, and cross‐resistance patterns and possible resistance mechanisms in the abamectin‐resistant strains (AL‐R, AF‐R) were investigated. Compared with the susceptible strain (SS), strain AL‐R displayed 39‐fold resistance to abamectin after 20 selection cycles during 25 generations, and strain AF‐R exhibited 59‐fold resistance to abamectin after 16 selection cycles during 22 generations. No cross‐resistance to cyromazine was found in both abamectin‐resistant strains. However, we failed to select for cyromazine resistance in L. sativae under laboratory conditions by conducting 17 selection cycles during 22 generations. However, moderate levels of cross‐resistance to abamectin (6–9 fold) were observed in strains which received cyromazine treatments. Biochemical analysis showed that glutathione S‐transferase (GST) activity in both abamectin‐resistant strains (AL‐R, AF‐R) was significantly higher than in the susceptible strain (SS), suggesting metabolically driven resistance to abamectinin L. sativae. Recommendations of mixtures or rotation of cyromazine and abamectin should be considered carefully, as consecutive cyromazine treatments may select for low‐level cross‐resistance to abamectin.     

Preference of Bt‐resistant and susceptible Busseola fusca moths and larvae for Bt and non‐Bt maize

The sustainability of genetically engineered insecticidal Bacillus thuringiensis Berliner (Bt) maize, Zea mays L. (Poaceae), is threatened by the evolution of resistance by target pest species. Several Lepidoptera species have evolved resistance to Cry proteins expressed by Bt maize over the last decade, including the African maize stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae). The insect resistance management (IRM) strategy (i.e., the high‐dose/refuge strategy) deployed to delay resistance evolution is grounded on certain assumptions about the biology and ecology of a pest species, for example, the interactions between the insect pest and crop plants. Should these assumptions be violated, the evolution of resistance within pest populations will be rapid. This study evaluated the assumption that B. fusca adults and larvae select and colonize maize plants at random, and do not show any preference for either Bt or non‐Bt maize. Gravid female B. fusca moths of a resistant and susceptible population were subjected to two‐choice oviposition preference tests using stems of Bt and non‐Bt maize plants. Both the number of egg batches as well as the total number of eggs laid on each stem were recorded. The feeding preference of Bt‐resistant and susceptible neonate B. fusca larvae were evaluated in choice test bioassays with whorl leaf samples of specific maize cultivars. Although no differential oviposition preference was observed for either resistant or susceptible female moths, leaf damage ratings indicated that neonate larvae were able to detect Bt toxins and that they displayed feeding avoidance behaviour on Bt maize leaf samples.     

Silicon‐mediated resistance against specialist insects in sap‐sucking and leaf‐chewing guilds in the Si non‐accumulator collard

Soil amendment with Silicon (Si) can increase plant resistance against insect herbivores, but the underlying mechanisms remain unclear. The mechanical resistance hypothesis (MRH) states that Si accumulated in epidermal cells directly and passively protects against herbivores by creating a mechanical barrier. The physiological resistance hypothesis (PRH) states that Si enhances resistance by activating plant biochemical and physiological processes. We tested both hypotheses by manipulating Si fertilization of the Si non‐accumulator collard, Brassica oleracea L. cv. acephala (Brassicaceae). Then, we assessed functional and ultrastructural plant responses and the developmental and reproductive performance of the leaf‐chewing larvae of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), and the sap‐sucking cabbage aphid, Brevicoryne brassicae L. (Hemiptera: Aphididae). There was a 20% increase in leaf Si content. Silicon deposition in epidermal cells was identified by confocal microscopy and directly coincided with lower performance of P. xylostella, but did not affect B. brassicae. On the other hand, we found no unequivocal evidence that Si‐mediated changes in primary and secondary metabolism improved plant resistance against the insects. Negative mechanical effects of Si on the insects may have masked beneficial effects of increased water, nitrogen, and mineral contents in Si‐treated collards. Silicon did not change leaf contents of hemicellulose, cellulose, and lignin. Although Si‐mediated increases in leaf glucosinolates (GLS) correlated with lower larval performance and higher oviposition preference of P. xylostella, both P. xylostella and B. brassicae are highly specialized in overcoming such secondary metabolites. Thus, mechanical resistance may have impaired P. xylostella, rather than the Si‐mediated increase in GLS. We suggest that the PRH may depend on the degree of insect feeding specialization, so that toxic Si‐mediated defenses may be more efficient against unadapted polyphagous herbivores. For them, a toxic barrier may be added to the mechanical resistance.     

Downregulation of ubiquitin gene expression in the deltamethrin‐resistant diamondback moth (Plutella xylostella L.)

Plutella xylostella (L.) has long been regarded as a very important universal pest of cruciferous crops. It is therefore important to understand its mechanisms of insecticide resistance. In our previous study, we obtained a differently expressed sequence with relatively high homology to the ubiquitin (UBI) gene as assessed via cDNA representational difference analysis (RDA). To further study this product, we cloned UBI open reading frame (ORF) sequences identified from both sexes of the deltamethrin‐resistant strain (DRF and DRM) and the deltamethrin‐sensitive strain (SF and SM) of P. xylostella. The deduced protein sequence of DRM was different from the other samples. The expression level was measured using real‐time quantitative polymerase chain reaction (PCR) and Western blot. The results of both analyses revealed the expression levels, in order from highest to lowest, to be in SF, SM, DRF and DRM. This indicates that the long‐term use of deltamethrin on P. xylostella results in decreased expression of the UBI gene.     

Assessing cultivar resistance to Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae) using a phenotyping method under semi‐field conditions

The orange wheat blossom midge, Sitodiplosis mosellana (Géhin), can significantly reduce wheat yield. Growing resistant wheat cultivars is an effective way of managing this pest. The assessment of cultivar resistance in field trials is difficult because of unequal pressure of S. mosellana caused by differences in cultivar heading dates relative to the flight period of S. mosellana adult females and huge variations of egg laying conditions from 1 day to another. To overcome these hurdles and to expose all cultivars homogeneously to the pest, an assessment method of cultivar resistance was developed under semi‐field conditions. In 2015, the resistance of 64 winter wheat cultivars to S. mosellana was assessed. Few or no larvae developed in the ears of resistant cultivars, but in susceptible cultivars, large numbers of larvae developed. Seventeen cultivars proved to be resistant, whereas 47 were susceptible. The identification of new resistant cultivars offers more opportunities to manage S. mosellana. The phenotyping method is easy, cheap, efficient and reliable. It can be used to guide the breeding of new resistant wheat cultivars. Using specific midge populations, this method could also be used in research on new resistance mechanisms in winter wheat or in other cereal species.     

Tritrophic Choice Experiments with Bt Plants,the Diamondback Moth (Plutella xylostella) and the parasitoid Cotesia plutellae

Parasitoids are important natural enemies of many pest species and are used extensively in biological and integrated control programmes. Crop plants transformed to express toxin genes derived from Bacillus thuringiensis (Bt) provide high levels of resistance to certain pest species, which is likely to have consequent effects on parasitoids specialising on such pests. A better understanding of the interaction between transgenic plants, pests and parasitoids is important to limit disruption of biological control and to provide background knowledge essential for implementing measures for the conservation of parasitoid populations. It is also essential for investigations into the potential role of parasitoids in delaying the build-up of Bt-resistant pest populations. The diamondback moth (Plutella xylostella), a major pest of brassica crops, is normally highly susceptible to a range of Bt toxins. However, extensive use of microbial Bt sprays has led to the selection of resistance to Bt toxins in P. xylostella. Cotesia plutellae is an important endoparasitoid of P. xylostella larvae. Although unable to survive in Bt-susceptible P. xylostella larvae on highly resistant Bt oilseed rape plants due to premature host mortality, C. plutellae is able to complete its larval development in Bt-resistant P. xylostella larvae. Experiments of parasitoid flight and foraging behaviour presented in this paper showed that adult C. plutellae females do not distinguish between Bt and wildtype oilseed rape plants, and are more attracted to Bt plants damaged by Bt-resistant hosts than by susceptible hosts. This stronger attraction to Bt plants damaged by resistant hosts was due to more extensive feeding damage. Population scale experiments with mixtures of Bt and wildtype plants demonstrated that the parasitoid is as effective in controlling Bt-resistant P. xylostella larvae on Bt plants as on wildtype plants. In these experiments equal or higher numbers of parasitoid adults emerged per transgenic as per wildtype plant. The implications for integrated pest management and the evolution of resistance to Bt in P. xylostella are discussed.     

Next‐generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant ‘pyramids’ producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross‐resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double‐stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH‐binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt‐resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt‐resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non‐transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.     

Insecticide resistance in populations of Tuta absoluta (Lepidoptera: Gelechiidae)

1 Control failures of insecticides used against the tomato leafminer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) in Brazil led to the investigation of the possible occurrence of resistance of this insect pest to abamectin, cartap, methamidophos and permethrin. 2 The insect populations were collected from seven sites in the states of Minas Gerais, Rio de Janeiro, and São Paulo. These populations were subjected to concentration–mortality bioassays using insecticide‐impregnated filter papers. 3 We were unable to obtain a single population which provided a susceptibility standard for all insecticides tested. Therefore, the resistance levels were estimated in relation to the most susceptible population to each insecticide. Resistance to abamectin and cartap were observed in all populations when compared with the susceptible standard population, with resistance ratios ranging from 5.2‐ to 9.4‐fold and from 2.2‐ to 21.9‐fold for abamectin and cartap, respectively. Resistance to permethrin was observed in five populations with resistance ratios ranging from 1.9‐ to 6.6‐fold, whereas resistance to methamidophos was observed in four populations with resistance ratios ranging from 2.6‐ to 4.2‐fold. 4 The long period and high frequency of use of these insecticides against this insect pest suggest that the evolution of insecticide resistance on them has been relatively slow. Alternatively, the phenomenon might be widespread among Brazilian populations of T. absoluta making the finding of suitable standard susceptible populations difficult and leading to an underestimation of the insecticide resistance levels in this pest. 5 Higher levels of resistance to abamectin, cartap and permethrin are correlated with greater use of these compounds by growers. This finding suggests that local variation in insecticide use was an important cause of variation in susceptibility.     

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth,Plutella xylostella

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field‐evolved indoxacarb resistance has been reported in several lepidopteran pests, including the diamondback moth Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong Province of China in 2012, was 750‐fold more resistant to indoxacarb and 70‐fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of complementary DNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the sixth segment of domain IV of the PxNa_v protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among 10 field‐collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34‐ to 870‐fold) and metaflumizone (1‐ to 70‐fold). The two mutations were never found to co‐exist in the same allele of PxNa_v, suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species.     

Trade‐off between thermal tolerance and insecticide resistance in Plutella xylostella

Fitness costs associated with resistance to insecticides have been well documented, usually at normal temperature conditions, in many insect species. In this study, using chlorpyrifos‐resistant homozygote (R_R) and chlorpyrifos‐susceptible homozygote (S_S) of resistance ace1 allele of Plutella xylostella (DBM), we confirmed firstly that high temperature experience in pupal stage influenced phenotype of wing venation in insecticide‐resistant and insecticide‐susceptible Plutella xylostella, and S_S DBM showed significantly higher thermal tolerance and lower damages of wing veins under heat stress than R_R DBM. As compared to S_S DBM, R_R DBM displayed significantly lower AChE sensitivity to chlorpyrifos, higher basal GSTs activity and P450 production at 25°C, but higher inhibitions on the enzyme activities and P450 production as well as reduced resistance to chlorpyrifos under heat stress. Furthermore, R_R DBM displayed significantly higher basal expressions of hsp69s, hsp72s, hsp20, hsp90, Apaf‐1, and caspase‐7 at 25°C, but lower induced expressions of hsps and higher induced expressions of Apaf‐1, caspase‐9, and caspase‐7 under heat stress. These results suggest that fitness costs of chlorpyrifos resistance in DBM may partly attribute to excess consumption of energy caused by over production of detoxification enzymes and hsps when the proteins are less demanded at conducive environments but reduced expressions when they are highly demanded by the insects to combat environmental stresses, or to excess expressions of apoptotic genes under heat stress, which results in higher apoptosis. The evolutionary and ecological implications of these findings at global warming are discussed.     

Effects of Rag1 aphid‐resistant soybean on mortality,development, and preference of brown marmorated stink bug

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), poses a new threat to soybean, Glycine max (L.) Merrill (Fabaceae), production in the north central USA. As H. halys continues to spread and increase in abundance in the region, the interaction between H. halys and management tactics deployed for other pests must be determined. Currently, the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is the most abundant and damaging insect pest of soybean in the region. Aphid‐resistant soybean, mainly with the Rag1 gene, is commercially available for management of A. glycines. Here, experiments were performed to evaluate the effects of Rag1 aphid‐resistant soybean on the mortality, development, and preference of H. halys. In a no‐choice test, mortality of H. halys reared on Rag1 aphid‐resistant soybean pods was significantly lower than when reared on aphid‐susceptible soybean pods (28 vs. 53%). Development time, adult weight, and proportion females of surviving adults did not differ when reared on Rag1 aphid‐resistant or aphid‐susceptible soybean pods. In choice tests, H. halys exhibited a preference for Rag1 aphid‐resistant over aphid‐susceptible soybean pods after 4 h, but not after 24 h. Halyomorpha halys exhibited no preference when tested with vegetative‐stage or reproductive‐stage soybean plants. The preference by H. halys for Rag1 aphid‐resistant soybean pods and the decreased mortality when reared on these pods suggests that the use of Rag1 aphid‐resistant soybean may favor this emerging pest in the north central USA.     

Functional and genetic characteristics of Chlorantraniliprole resistance in the diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae)

The diamondback moth (Plutella xylostella) is a globally distributed and important economic pest, and it has developed resistance to all conventional insecticide classes used in the field. Chlorantraniliprole is a new chemical class of insecticide that acts as a conformation‐sensitive activator of the insect ryanodine receptor (RyR). In the present study, a field strain (16.3‐fold resistance to chlorantraniliprole) was collected in Korea and lab‐selected with chlorantraniliprole for more than one year. The resulting strain presented 2,157‐fold resistance to chlorantraniliprole. A point mutation (G4946E) in the RyR gene was observed at a high frequency in the resistant strain. Enzyme assays indicated that glutathione S‐transferase (GST) and P450 activity in the resistant strain were 2.4‐ and 1.96‐times higher than that of the susceptible strain, respectively. The expression of the RyR, GST (sigma, omega, and zeta) and CYP321E1 gene was higher in the resistant strain than in the susceptible strain. The F₁ progeny resulting from reciprocal crosses did not reveal maternal effects or a diamide‐susceptible phenotype, which suggests an autosomal nearly recessive mode of inheritance. In addition, we surveyed the susceptibility to 13 insecticides (3 diamides, 2 synthetic pyrethroids, 2 spinosyns, 1 organophosphate, 1 oxadiazine, 1 avermectin, and 3 others) in the chlorantraniliprole‐resistant strain. The resistant strain exhibited high cross‐resistance to flubendiamide (5,910 fold) and showed no cross‐resistance to spinetoram, spinosad, indoxacarb, and metaflumizone. These results can serve as an important basis for guiding the use of insecticides in the field.     

Resistant cabbage cultivars change the susceptibility of Plutella xylostella to Bacillus thuringiensis

1 Laboratory studies demonstrated that the susceptibility of larvae of the lepidopteran crucifer pest Plutella xylostella to the insect pathogen Bacillus thuringiensis (Bt) was influenced by the host plant. 2 Larvae reared on the resistant cabbage cultivars Minicole F₁ and Red Drumhead were significantly more susceptible to Bt (the LC₅₀ fell to one half) than larvae fed leaves of susceptible cultivars. 3 However, a third resistant cultivar, Aquarius F₁, had no synergistic effect on Bt‐related mortality. 4 Actual uptake of Bt was monitored in the bioassays, as a preliminary experiment showed that the plant resistance reduced consumption of Bt‐treated leaf discs. However, differences in feeding rate did not explain the observed differences in mortality.     

Effects of abamectin selection on the genetic differentiation within Plutella xylostella (Lepidoptera: Plutellidae) based on amplified fragment length polymorphism

Abstract Diamondback moth, Plutella xylostella (L.) is one of the most serious insect pests for its remarkable ability to develop resistance to virtually every insecticide that has been used against it. In the present study, amplified fragment length polymorphism (AFLP) is used to study the genetic differentiation as well as the effects of abamectin-selection on population genetic differentiation for P. xylostella. A dendrogram was constructed from the matrix of genetic distances using the STATISTICA software (Version 4.5) and unweighted pair-group method with arithmetic averages (UPGMA). The data demonstrated that compared to the susceptible strain (ABM-s), the heterozygosity in the abamectin-resistant strain (ABM-r) decreased with the increased selection pressure and resistant level. When the resistance ratio was below 4.3, there was no significant differentiation at the genome DNA level. When the resistance ratio reached 5.8, a fairly significant differentiation began to appear, and when the resistance ratio reached and exceeded 8.1, there appeared a significant genetic differentiation. The results suggest that abamectin selection is associated with increased genetic polymorphism in P. xylostella.     

Temperature‐sensitive fitness cost of insecticide resistance in Chinese populations of the diamondback moth Plutella xylostella

Alleles conferring a higher adaptive value in one environment may have a detrimental impact on fitness in another environment. Alleles conferring resistance to pesticides and drugs provide textbook examples of this trade‐off as, in addition to conferring resistance to these molecules, they frequently decrease fitness in pesticide/drug‐free environments. We show here that resistance to chlorpyrifos, an organophosphate (OP), in Chinese populations of the diamondback moth, Plutella xylostella, is conferred by two mutations of ace1 – the gene encoding the acetylcholinesterase enzyme targeted by OPs – affecting the amino acid sequence of the corresponding protein. These mutations were always linked, consistent with the segregation of a single resistance allele, ace1R, carrying both mutations, in the populations studied. We monitored the frequency of ace1R (by genotyping more than 20 000 adults) and the level of resistance (through bioassays on more than 50 000 individuals) over several generations. We found that the ace1R resistance allele was costly in the absence of insecticide and that this cost was likely recessive. This fitness costs involved a decrease in fecundity: females from resistant strains laid 20% fewer eggs, on average, than females from susceptible strains. Finally, we found that the fitness costs associated with the ace1R allele were greater at high temperatures. At least two life history traits were involved: longevity and fecundity. The relative longevity of resistant individuals was affected only at high temperatures and the relative fecundity of resistant females – which was already affected at temperatures optimal for development – decreased further at high temperatures. The implications of these findings for resistance management are discussed.