Genetic Resistance to Bacillus thuringiensis Alters Feeding Behaviour in the Cabbage Looper,Trichoplusia ni

Evolved resistance to xenobiotics and parasites is often associated with fitness costs when the selection pressure is absent. Resistance to the widely used microbial insecticide Bacillus thuringiensis (Bt) has evolved in several insect species through the modification of insect midgut binding sites for Bt toxins, and reports of costs associated with Bt resistance are common. Studies on the costs of Bt-resistance restrict the insect to a single artificial diet or host-plant. However, it is well documented that insects can self-select appropriate proportions of multiple nutritionally unbalanced foods to optimize life-history traits. Therefore, we examined whether Bt-resistant and susceptible cabbage loopers Trichoplusia ni differed in their nutrient intake and fitness costs when they were allowed to compose their own protein:carbohydrate diet. We found that Bt-resistant T. ni composed a higher ratio of protein to carbohydrate than susceptible T. ni. Bt-resistant males exhibited no fitness cost, while the fitness cost (reduced pupal weight) was present in resistant females. The absence of the fitness cost in resistant males was associated with increased carbohydrate consumption compared to females. We demonstrate a sex difference in a fitness cost and a new behavioural outcome associated with Bt resistance.     

Is decreased generalized immunity a cost of Bt resistance in cabbage loopers Trichoplusia ni?

We studied the immune response to Bacillus thuringiensis kurstaki (Btk) in susceptible (Bt-RS) and resistant (Bt-R) Trichoplusia ni after exposure to low doses of Btk and injection with Escherichia coli. We measured the levels of resistance, the expression profiles of hemolymph proteins, the phenoloxidase (PO) activity, and the differential number of circulating hemocytes in resistant and susceptible individuals. Individuals from the Bt-RS line became more resistant following a previous exposure to sub lethal concentrations of Btk, but the resistance to Btk of the Bt-R line did not change significantly. Similarly the Bt-R strain showed no significant changes in any of the potential immune responses, hemolymph protein levels or PO activity. The number of circulating hemocytes was significantly lower in the Bt-R strain than in the Bt-RS strain. Exposure to Btk decreased the hemocyte counts and reduced PO activity of Bt-RS larvae. Hemolymph protein concentrations also declined significantly in the susceptible larvae continually exposed to Btk. Seven peptides with antibacterial activity were identified in the hemolymph of Bt-RS larvae after exposure to Btk and five were found in the Bt-R larvae. When exposed to a low level Bt challenge the susceptible strain increases in tolerance and there are concomitant reductions in hemolymph protein concentrations, PO activity and the number of circulating hemocytes.     

Dietary Mechanism behind the Costs Associated with Resistance to Bacillus thuringiensis in the Cabbage Looper,Trichoplusia ni

Beneficial alleles that spread rapidly as an adaptation to a new environment are often associated with costs that reduce the fitness of the population in the original environment. Several species of insect pests have evolved resistance to Bacillus thuringiensis (Bt) toxins in the field, jeopardizing its future use. This has most commonly occurred through the alteration of insect midgut binding sites specific for Bt toxins. While fitness costs related to Bt resistance alleles have often been recorded, the mechanisms behind them have remained obscure. We asked whether evolved resistance to Bt alters dietary nutrient intake, and if reduced efficiency of converting ingested nutrients to body growth are associated with fitness costs and variation in susceptibility to Bt. We fed the cabbage looper Trichoplusia ni artificial diets differing in levels of dietary imbalance in two major macronutrients, protein and digestible carbohydrate. By comparing a Bt-resistant T. ni strain with a susceptible strain we found that the mechanism behind reduced pupal weights and growth rates associated with Bt-resistance in T. ni was reduced consumption rather than impaired conversion of ingested nutrients to growth. In fact, Bt-resistant T. ni showed more efficient conversion of nutrients than the susceptible strain under certain dietary conditions. Although increasing levels of dietary protein prior to Bt challenge had a positive effect on larval survival, the LC₅₀ of the resistant strain decreased when fed high levels of excess protein, whereas the LC₅₀ of the susceptible strain continued to rise. Our study demonstrates that examining the nutritional basis of fitness costs may help elucidate the mechanisms underpinning them.     

Resistance to Bacillus thuringiensis in the cabbage looper (Trichoplusia ni) increases susceptibility to a nucleopolyhedrovirus

Cabbage loopers, Trichoplusia ni, are pests in many agricultural settings including vegetable greenhouses in British Columbia (Canada), where microbial insecticides based on Bacillus thuringiensis (Bt) toxins are commonly used. Frequent use of these insecticides has led to resistance in some populations. An alternative microbial control is the multiple nucleopolyhedrovirus of the alfalfa looper (Autographa californica), AcMNPV which occurs naturally, but at low frequencies in T. ni populations. Bioassays show that T. ni resistant to Bt were twice as susceptible to AcMNPV as were individuals from the Bt-susceptible strain and AcMNPV could be complementary in a resistance management program for T. ni.     

Characterizing indirect prey-quality mediated effects of a Bt crop on predatory larvae of the green lacewing, Chrysoperla carnea

There is increasing evidence that insecticidal transgenic crops can indirectly cause detrimental effects on arthropod predators or parasitoids when they prey on or parasitize sublethally affected herbivores. Our studies revealed that Chrysoperla carnea is negatively affected when fed Bt-susceptible but not Cry1Ac-resistant Helicoverpa armigera larvae that had fed Bt-transgenic cotton expressing Cry1Ac. This despite the fact that the predators ingested 3.5 times more Cry1Ac when consuming the resistant caterpillars. In order to detect potential differences in the nutrient composition of prey larvae, we evaluated the glycogen and lipid content plus the sugar and free amino acid content and composition of caterpillars fed non-Bt and Bt cotton. The only change in susceptible H. armigera larvae attributable to Bt cotton feeding were changes in sugar concentration and composition. In case of the Cry1Ac-resistant H. armigera strain, feeding on Bt cotton resulted in a reduced glycogen content in the caterpillars. The predators, however, appeared to compensate for the reduced carbohydrate content of the prey by increasing biomass uptake which caused an excess intake of the other analyzed nutritional compounds. Our study clearly proves that nutritional prey-quality factors other then the Bt protein underlie the observed negative effects when C. carnea larvae are fed with Bt cotton-fed prey. Possible factors were an altered sugar composition or fitness costs associated with the excess intake of other nutrients.     

A recombinant immunosuppressive protein from Pimpla hypochondriaca (rVPr1) increases the susceptibility of Lacanobia oleracea and Mamestra brassicae larvae to Bacillus thuringiensis

The precise mechanisms underlying Bacillus thuringiensis-mediated killing of pest insects are not clear. In some cases, death may be due to septicaemia caused by Bt and/or gut bacteria gaining access to the insect haemocoel. Since insects protect themselves from microbes using an array of cellular and humoral immune defences, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) could increase the susceptibility of two pest Lepidoptera (Lacanobia oleracea and Mamestra brassicae) to Bt. Bio-assays indicated that injection of 6 μl of rVPr1 into the haemocoel of both larvae caused similar levels of mortality (less than 38%). On the other hand, the LD_30-40 of Bt for M. brassicae larvae was approximately 20 times higher than that for L. oleracea larvae. Furthermore, in bio-assays where larvae were injected with rVPr1, then fed Bt, a significant reduction in survival of larvae for both species occurred compared to each treatment on its own (P < 0.001); and for L. oleracea larvae, this effect was more than additive. The results are discussed within the context of insect immunity and protection against Bt.     

A Comprehensive Assessment of the Effects of Bt Cotton on Coleomegilla maculata Demonstrates No Detrimental Effects by Cry1Ac and Cry2Ab

The ladybird beetle, Coleomegilla maculata (DeGeer), is a common and abundant predator in many cropping systems. Its larvae and adults are predaceous, feeding on aphids, thrips, lepidopteran larvae and plant tissues, such as pollen. Therefore, this species is exposed to insecticidal proteins expressed in insect-resistant, genetically engineered cotton expressing Cry proteins derived from Bacillus thuringiensis (Bt). A tritrophic bioassay was conduced to evaluate the potential impact of Cry2Ab- and Cry1Ac-expressing cotton on fitness parameters of C. maculata using Bt-susceptible and -resistant larvae of Trichoplusia ni as prey. Coleomegilla maculata survival, development time, adult weight and fecundity were not different when they were fed with resistant T. ni larvae reared on either Bt or control cotton. To ensure that C. maculata were not sensitive to the tested Cry toxins independent from the plant background and to add certainty to the hazard assessment, C. maculata larvae were fed artificial diet incorporated with Cry2Ab, Cry1Ac or both at >10 times higher concentrations than in cotton tissue. Artificial diet containing E-64 was included as a positive control. No differences were detected in any life-table parameters between Cry protein-containing diet treatments and the control diet. In contrast, larvae of C. maculata fed the E-64 could not develop to the pupal stage and the 7-d larval weight was significantly negatively affected. In both feeding assays, the stability and bioactivity of Cry proteins in the food sources were confirmed by ELISA and sensitive-insect bioassays. Our results show that C. maculata is not affected by Bt cotton and is not sensitive to Cry2Ab and Cry1Ac at concentrations exceeding the levels in Bt cotton, thus demonstrating that Bt cotton will pose a negligible risk to C. maculata. More importantly, this study demonstrates a comprehensive system for assessing the risk of genetically modified plants on non-target organisms.     

The impact of strain diversity and mixed infections on the evolution of resistance to Bacillus thuringiensis

Pesticide mixtures can reduce the rate at which insects evolve pesticide resistance. However, with live biopesticides such as the naturally abundant pathogen Bacillus thuringiensis (Bt), a range of additional biological considerations might affect the evolution of resistance. These can include ecological interactions in mixed infections, the different rates of transmission post-application and the impact of the native biodiversity on the frequency of mixed infections. Using multi-generation selection experiments, we tested how applications of single and mixed strains of Bt from diverse sources (natural isolates and biopesticides) affected the evolution of resistance in the diamondback moth, Plutella xylostella, to a focal strain. There was no significant difference in the rate of evolution of resistance between single and mixed-strain applications although the latter did result in lower insect populations. The relative survivorship of Bt-resistant genotypes was higher in the mixed-strain treatment, in part owing to elevated mortality of susceptible larvae in mixtures. Resistance evolved more quickly with treatments that contained natural isolates, and biological differences in transmission rate may have contributed to this. Our data indicate that the use of mixtures can have unexpected consequences on the fitness of resistant and susceptible insects.     

Inheritance of Cry1Ac resistance and associated biological traits in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

The analysis of reciprocal genetic crosses between resistant Helicoverpa armigera strain (BH-R) (227.9-fold) with susceptible Vadodara (VA-S) strain showed dominance (h) of 0.65-0.89 and degree of dominance (D) of 0.299-0.782 suggesting Cry1Ac resistance as a semi-dominant trait. The D and h values of F₁ hybrids of female resistant parent were higher than female susceptible parent, showing maternally enhanced dominance of Cry1Ac resistance. The progeny of F₂ crosses, backcrosses of F₁ hybrid with resistant BH-R parent did not differ significantly in respect of mortality response with resistant parent except for backcross with female BH-R and male of F₁ (BH-R × VA-S) cross, suggesting dominant inheritance of Cry1Ac resistance. Evaluation of some biological attributes showed that larval and pupal periods of progenies of reciprocal F₁ crosses, backcrosses and F₂ crosses were either at par with resistant parent or lower than susceptible parent on treated diet (0.01 μg/g). The susceptible strain performed better in terms of pupation and adult formation than the resistant strain on untreated diet. In many backcrosses and F₂ crosses, Cry1Ac resistance favored emergence of more females than males on untreated diet. The normal larval period and the body weight (normal larval growth) were the dominant traits associated with susceptible strain as contrast to longer larval period and the lower body weight (slow growth) associated with resistance trait. Further, inheritance of larval period in F₂ and backcross progeny suggested existence of a major resistant gene or a set of tightly linked loci associated with Cry1Ac sensitivity.     

The genetic basis for mating-induced sex differences in starvation resistance in Drosophila melanogaster

Multiple genetic and environmental factors interact to influence starvation resistance, which is an important determinant of fitness in many organisms, including Drosophila melanogaster. Recent studies have revealed that mating can alter starvation resistance in female D. melanogaster, but little is known about the behavioral and physiological mechanisms underlying such mating-mediated changes in starvation resistance. In the present study, we first investigated whether the effect of mating on starvation resistance is sex-specific in D. melanogaster. As indicated by a significant sex × mating status interaction, mating increased starvation resistance in females but not in males. In female D. melanogaster, post-mating increase in starvation resistance was mainly attributed to increases in food intake and in the level of lipid storage relative to lean body weight. We then performed quantitative genetic analysis to estimate the proportion of the total phenotypic variance attributable to genetic differences (i.e., heritability) for starvation resistance in mated male and female D. melanogaster. The narrow-sense heritability (h²) of starvation resistance was 0.235 and 0.155 for males and females, respectively. Mated females were more resistant to starvation than males in all genotypes, but the degree of such sexual dimorphism varied substantially among genotypes, as indicated by a significant sex × genotype interaction for starvation resistance. Cross-sex genetic correlation was greater than 0 but less than l for starvation resistance, implying that the genetic architecture of this trait was partially shared between the two sexes. For both sexes, starvation resistance was positively correlated with longevity and lipid storage at genetic level. The present study suggests that sex differences in starvation resistance depend on mating status and have a genetic basis in D. melanogaster.     

Effect of dietary selenium supplementation on resistance to baculovirus infection

We have examined the effects of dietary selenium (Se) supplementation on larval growth and immunocompetence of the lepidopteran pest, the cabbage looper, Trichoplusia ni. Supplementation of the diet of T. ni larvae with 10–20 ppm Se resulted in a 1 day delay in pupation. The effects of the addition and/or removal of dietary Se on total Se bioaccumulation and sequestration were determined by neutron activation analysis of pupae. Early penultimate instar larvae moved from selenium containing diet to basal diet lost total pupal Se content down to the level of those fed basal diet. Conversely, larvae moved from basal diet to diet containing additional Se rapidly attained pupal Se levels comparable to larvae fed Se throughout larval development. Therefore, dietary Se is rapidly accumulated or lost during larval development, but significant amounts are sequestered from diet into pupae. Larvae were reared on diet supplemented with 5 or 10 ppm Se until the onset of the penultimate instar then infected per os with increasing concentrations of the fatal baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Larvae fed Se in the penultimate and ultimate instars were more resistant to viral infection than larvae not fed Se in the final instars. This study indicates that dietary Se levels rapidly impact Se assimilation and sequestration and that tissue Se levels are an important factor in resistance to AcMNPV infection in larval T. ni.     

Genetic effects on flight capacity in the beet armyworm,Spodoptera exigua (Lep., Noctuidae)

The beet armyworm, Spodoptera exigua, is an important migratory insect pest in tropical and subtropical regions worldwide. The current study investigated genetic variation in the flight capacity of both female and male moths, using a quantitative genetics approach. The offspring–parent regression showed that parents had a significant influence on the flight duration of offspring, and the heritability estimated as 0.302. The upward selection increased mean flight duration from 123.7 to 284.6 min in females and from 113.9 to 254.0 min in males during 8 h of flight test; by contrast, downward selection decreased it from 123.7 to 65.6 min in females and from 113.9 to 29.8 min in males, while it did not change significantly in either females or males of the control line over eight generations. The mean realized heritability was estimated as 0.432 based on upward selection but 0.130 on downward selection, suggesting the asymmetry of response to selection on flight capacity. Reciprocal crosses between the two selected lines confirmed the dominance of ‘long‐flying genes’ in the inheritance of flight capacity. A positive genetic correlation was found between increased flight duration and pupal weight. The presence of such additive genetic variance and covariance for flight capacity and the fitness trait, pupal weight, in the population of S. exigua may have underpinned the evolution of its migratory behaviour.     

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops.     

Quantitative genetics of adult behavioral response and larval physiological tolerance to permethrin in diamondback moth (Lepidoptera: Plutellidae)

We investigated the genetic basis of adult behavioral response and larval physiological tolerance to permethrin within two diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), populations from Wooster and Celeryville, OH, with different average levels of larval tolerance. The adult behavioral response was measured as oviposition site preference and was investigated using full-sib design and parent-offspring regression. Additive genetic variance (0.134 +/- 0.02) and the heritability (h2 = 0.31 +/- 0.08) for the behavioral response was significant for the Celeryville population, suggesting that in this population, a high proportion of phenotypic variation for adult behavioral response to permethrin was heritable genetic variation. The larval physiological response was measured with a topical application bioassay and was investigated using a half-sib design. Significant additive genetic variances and heritabilities for physiological tolerance to permethrin were detected in both populations. The genetic correlation between adult behavioral response and larval physiological tolerance to permethrin were negative, but significant only in the Celeryville population; indicating that adults from this population that are more behaviorally responsive produced offspring that are more susceptible to permethrin. Our findings have implications for the evolution and management of insecticide resistance in the diamondback moth. The adult behavioral response can lower the exposure of larvae to the insecticide, lowering selection pressure for physiological resistance in larvae. Furthermore, to the extent that the adult behavioral response increases fitness, it can indirectly select for larval susceptibility because of the negative correlation between the two traits.     

Genetic variation in resistance, but not tolerance, to a protozoan parasite in the monarch butterfly

Natural selection should strongly favour hosts that can protect themselves against parasites. Most studies on animals so far have focused on resistance, a series of mechanisms through which hosts prevent infection, reduce parasite growth or clear infection. However, animals may instead evolve tolerance, a defence mechanism by which hosts do not reduce parasite infection or growth, but instead alleviate the negative fitness consequences of such infection and growth. Here, we studied genetic variation in resistance and tolerance in the monarch butterfly (Danaus plexippus) to its naturally occurring protozoan parasite, Ophryocystis elektroscirrha. We exposed 560 monarch larvae of 19 different family lines to one of five different parasite inoculation doses (0, 1, 5, 10 and 100 infective spores) to create a range of parasite loads in infected butterflies. We then used two proxies of host fitness (adult lifespan and body mass) to quantify: (i) qualitative resistance (the ability to prevent infection; also known as avoidance or anti-infection resistance); (ii) quantitative resistance (the ability to limit parasite growth upon infection; also known as control or anti-growth resistance); and (iii) tolerance (the ability to maintain fitness with increasing parasite infection intensity). We found significant differences among host families in qualitative and quantitative resistance, indicating genetic variation in resistance. However, we found no genetic variation in tolerance. This may indicate that all butterflies in our studied population have evolved maximum tolerance, as predicted by some theoretical models.     

Does multiple paternity influence offspring disease resistance?

It has been suggested that polyandry allows females to increase offspring genetic diversity and reduce the prevalence and susceptibility of their offspring to infectious diseases. We tested this hypothesis in wild‐derived house mice (Mus musculus) by experimentally infecting the offspring from 15 single‐ and 15 multiple‐sired litters with two different strains of a mouse pathogen (Salmonella Typhimurium) and compared their ability to control infection. We found a high variation in individual infection resistance (measured with pathogen loads) and significant differences among families, suggesting genetic effects on Salmonella resistance, but we found no difference in prevalence or infection resistance between single‐ vs. multiple‐sired litters. We found a significant sex difference in infection resistance, but surprisingly, males were more resistant to infection than females. Also, infection resistance was correlated with weight loss during infection, although only for females, indicating that susceptibility to infection had more harmful health consequences for females than for males. To our knowledge, our findings provide the first evidence for sex‐dependent resistance to Salmonella infection in house mice. Our results do not support the hypothesis that multiple‐sired litters are more likely to survive infection than single‐sired litters; however, as we explain, additional studies are required before ruling out this hypothesis.     

Mechanism of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Greenhouse Population of the Cabbage Looper, Trichoplusia ni

The cabbage looper, Trichoplusia ni, is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouse-evolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni. This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut.     

The influence of nutritional and genetic factors on larval performance of the cinnabar moth, Tyria jacobaeae

In a laboratory experiment using full-sibs, 60% of the variation in pupal weight of the monophage Tyria jacobaeae L. (Lepidoptera, Arctiidae) could be explained by variation in the nitrogen concentration of the food plant, Senecio jacobaea L. and only 4% by variation in sugar concentration. Larval weight and growth rates of young and old larvae were also positively correlated with nitrogen and sugar concentration. Developmental time was negatively correlated with nitrogen concentration. In a second experiment full-sib families differed significantly in larval weight at day 7, mortality, growth rate and developmental time. Pupal weight did not differ significantly among families, but was positively correlated with nitrogen concentration of Senecio. Larval performance was not significantly influenced by concentrations of sugars or alkaloids. We conclude that larval performance of Tyria during most of the larval period is mainly determined by genetic factors, but pupal weights are primarily determined by nitrogen concentration of the food plant.