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.     

Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua

Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae.     

A proteomic approach to study the mechanism of tolerance to Bt toxins in Ostrinia furnacalis larvae selected for resistance to Cry1Ab

A Cry1Ab-resistant population of Asian corn borer (ACB-AbR) exhibiting approximately 100 times greater resistance to activated Cry1Ab than a susceptible population (Ostrinia furnacalis; ACB-BtS), was previously shown to exhibit high levels of cross-resistance to Cry1Ah (131-fold), but no cross-resistance to Cry1Ie. It was suggested that the proposed mechanism of resistance was due to the alteration of specific receptors for Cry toxins in the midgut brush border membrane. In the present study a proteomic-based approach was used to identify proteins from brush border membrane vesicles (isolated from both resistant and susceptible Ostrinia furnacalis larvae) interacting with biotinylated Cry1Ab, Cry1Ah, and Cry1Ie. 2D-Electrophoresis in combination with ligand blots were employed and putative protein identities obtained using MALDI-ToF/ToF mass spectrometry. The V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins were identified as interacting with the Cry toxins tested in the ACB-AbR and ACB-BtS larvae. The biotinylated Cry toxins showed markedly stronger interactions with proteins in the resistant compared to the susceptible larvae, suggesting an up-regulation of the V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins in the resistant (ACB-AbR) larvae. Interestingly, Cry1Ie interactions with the V-type proton ATPase catalytic subunit A in the ACB-BtS larvae appeared to be absent.     

Genetic variation in fitness parameters associated with resistance to Bacillus thuringiensis in male and female Trichoplusia ni

Resistance of insects to insecticides is often associated with reduced fitness in the absence of selection. We examined fitness trade-offs associated with resistance to the microbial insecticide, Bacillus thuringiensis (Bt), across full-sib families in a resistant population of Trichoplusia ni. Significant genetic variation in and heritability of susceptibility to Bt occurred among the full-sib families. Male pupal weight was positively correlated with Bt susceptibility, indicating a potential fitness cost, but no such correlation occurred for females. Significant heritability for pupal weight was present for males but not females. A significant negative genetic correlation existed between development time and Bt susceptibility, indicating that resistant larvae developed more slowly than susceptible larvae. Selection for Bt resistance in T. ni resulted in changes in life-history traits that affected males more than females.     

Transgenic broccoli expressing aBacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F₁ hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F₁ larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.     

Susceptibility of larvae of Trichoplusia ni and Anticarsia gemmatalis to intrahemocoelic injections of conidia and blastospores of Nomuraea rileyi

The LD₅₀ for larvae of Trichoplusia ni injected with blastospores of Nomuraea rileyi was 4.30 ± 1.16 hyphal bodies/larva; the LD₅₀ for injected conidia was ca. 25,000 conidia/larva. The dose-mortality regression line for blastospores was Y = 4.6504 + 0.5487 X. Larval mortalities of Anticarsia gemmatalis and T. ni at 100 blastospores/larva were 0.4 ± 0.5% and 96.7 ± 1.9%, respectively. At a dosage of 25,000 conidia/larva, larval mortalities for A. gemmatalis and T. ni were 0.4 ± 0.5% and 43.1 ± 8.7%, respectively. Thus, larvae of A. gemmatalis were > 100 times and >200 times more resistant to injected conidia and blastospores, respectively, than were larvae of T. ni. Resistance of A. gemmatalis to N. rileyi may not be solely at the integumental barrier, as is often believed, but may also be a function of an internal physiological response.     

The infectivity of a nuclear polyhedrosis virus of the velvetbean caterpillar for eight noctuid hosts

Eight species of noctuid larvae were tested for susceptibility to a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis. Velvetbean caterpillar larvae were highly susceptible to crude preparations of polyhedral inclusion bodies (PIBs; LD₅₀ = 4.7 PIBs/larva), but preparations of purified polyhedra were much less effective against these larvae (LD₅₀ = 319.7 PIBs/larva). Of seven other noctuid species tested, only Heliothis virescens was as susceptible to the virus as A. gemmatalis. High dosages were required to kill Heliothis zea, Trichoplusia ni, Pseudoplusia includens, and Spodoptera ornithogalli. Plathypena scabra and Spodoptera frugiperda were not susceptible.     

Susceptibility of the cabbage looper, Trichoplusia ni, and the velvetbean caterpillar, Anticarsia gemmatalis, to several isolates of the entomopathogenic fungus Nomuraea rileyi

Cabbage looper larvae, Trichoplusia ni, were equally susceptible to isolates of the entomogenous fungus Nomuraea rileyi from Missouri, Mississippi, and Brazil; an isolate from Florida was 7–17 times less active than the other isolates. Velvetbean caterpillars, Anticarsia gemmatalis, were either only slightly susceptible or not susceptible to the isolates of N. rileyi from Missouri, Florida, and Mississippi. In contrast, larvae of A. gemmatalis cultured from three locations (Missouri, Florida, and Brazil) all were equally susceptible to a Brazilian isolate of N. rileyi.     

Baculovirus-resistant Anticarsia gemmatalis responds differently to dietary rutin

The velvetbean caterpillar, Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae), the major soybean [Glycine max (L.) Merrill (Fabaceae)] defoliator pest in Brazil can be controlled by a specific and virulent nuclear polyhedrosis virus (AgMNPV). Flavonoids such as rutin (quercetin 3‐O‐rhamnosylglucoside) were identified in soybean; it is known that this compound plays an important role in plant defense against lepidopteran pests. Studies were carried out to evaluate the biological and physiological activity of rutin (0.65 and 1.30%) on populations of A. gemmatalis resistant and susceptible to AgMNPV. Larvae from the resistant population were more negatively influenced by rutin, in comparison to larvae of the susceptible population, even with the addition of the lowest level of the flavonoid (0.65%) to the insect diet. The highest mortality (98%) was observed in the resistant population, when larvae fed on the diet containing 1.30% of rutin. Elongation of the feeding time, smaller initial larval weight, and pupal weight was observed on the virus‐resistant and ‐susceptible populations after adding 0.65 and 1.30% rutin to the diet. Larvae of the resistant population to AgMNPV fed on diet plus rutin 0.65% were also less efficient in the conversion of ingested and digested food into biomass.     

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.     

Risk assessment system establishment for evaluating the potential impacts of imported Bacillus thuringiensis maize on a non-target insect,Tenebrio molitor

Large amounts of genetically modified grains producing Bacillus thuringiensis (Bt) toxins have been imported to Korea. Therefore, the establishment of a risk assessment system for evaluating the potential impacts of imported Bt maize on non-target insects is important. Before evaluating the environmental impacts of Bt grains of unknown origin, Cry protein types must first be identified in test Bt grains. Cry toxins of imported Bt maize grains were analyzed by ELISA. Because all tested Bt maize grains contained Cry1A, Tenebrio molitor, a non-lepidopteran species, was selected as the non-target insect species. A domestic maize strain that showed few differences in nutritional composition compared to the Bt maize grain was used as the alternative non-Bt control. Slightly increased survival rate and head capsule width of Bt maize-fed T. molitor were observed, indicating that Bt maize has no sub-chronic adverse effects on T. molitor. An ELISA test revealed that concentrations of Cry1A toxins slowly increased in the body of T. molitor when the insects were fed Bt maize. Such substantial amounts of Cry toxins remaining in the alimentary tract of larvae indicate that Cry toxins can be transferred to the higher trophic level of predatory insects. However, no Cry proteins were detected in the hemolymph of the Bt maize-fed larvae, suggesting that there is little possibility of Cry toxin exposure via T. molitor to the higher endoparasitoids. The risk assessment strategies and protocols established in this study may also be applicable to other imported Bt crops in Korea.     

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.     

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.     

The role of peritrophic membrane in the resistance of Anticarsia gemmatalis larvae (Lepidoptera: Noctuidae) during the infection by its nucleopolyhedrovirus (AgMNPV)

The aim of this study was to analyze morphologically the peritrophic membrane (PM) of Anticarsia gemmatalis larvae resistant (RL) and non-resistant (susceptible) (SL) to the A. gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV), in the presence of viral infection. Also, in this investigation the results between SL and RL were compared to improve the understanding of the resistance mechanisms to the virus. The PM of SL of A. gemmatalis was less efficient as a barrier against the viral infection since it was found to be more fragile than the PM of RL. The lower chitin content as seen from weaker fluorescent staining in SL as well as the abundance of non-solubilized vesicular materials in the ectoperitrophic space, would cause the malformation of this membrane, facilitating the passage of the virus toward the epithelium of the midgut. On the other hand, in RL, the intensity of WGA (wheat germ agglutinin)-conjugated FITC (fluorescein) reaction of the PM was greater than in SL, making this insect more resistant to infection. We can conclude that the effectiveness of the PM in protecting against pathogens is dependent on the integrity of the epithelial cells of the midgut and of the structural preservation of the PM, being directly implicated in the resistance of A. gemmatalis larvae to AgMNPV.     

Effect of the interaction between Anticarsia gemmatalis multiple nucleopolyhedrovirus and Epinotia aporema granulovirus,on A. gemmatalis (Lepidoptera: Noctuidae) larvae

The bean shoot borer Epinotia aporema Wals. (Lepidoptera: Tortricidae) and the velvet bean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) are key pests of soybean and other legume crops in South America. They are often found simultaneously in certain regions. A. gemmatalis nucleopolyhedrovirus (AgMNPV) is widely used to control A. gemmatalis. More recently, E. aporema granulovirus (EpapGV) has been characterized and evaluated as a bioinsecticide for E. aporema. In order to increase its potential use and to design optimized strategies for the management of lepidopteran pests, we evaluated the interaction between EpapGV and AgMNPV on third instar A. gemmatalis larvae. Larvae fed with 50 AgMNPV OBs/larva showed an increase in the mortality rates (from 42% to 81%) and a decrease in the median survival time (from 7.7 days to 5.7 days) when these OBs were mixed with 6000 EpapGV OBs/larva. When 300 AgMNPV OBs/larva were used alone or in combination with EpapGV OBs no changes in biological parameters were observed. No mortality was detected in A. gemmatalis larvae treated with EpapGV alone. In larvae fed with the viral mixtures, only AgMNPV DNA was detected by PCR. A. gemmatalis peritrophic membranes (PMs) examined by SDS–PAGE and scanning electron microscopy showed signs of damage. Notably, we found the presence of spheroidal bodies associated with damaged areas in the PMs of larvae fed with EpapGV but not in those that were given AgMNPV alone. These results show that EpapGV increases the viral potency of AgMNPV, and thus the insecticidal efficiency, suggesting that the use of formulations including both viruses might be a valuable tool for pest management.     

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.     

Consumption of Bt Rice Pollen Containing Cry1C or Cry2A Protein Poses a Low to Negligible Risk to the Silkworm Bombyx mori (Lepidoptera: Bombyxidae)

By consuming mulberry leaves covered with pollen from nearby genetically engineered, insect-resistant rice lines producing Cry proteins derived from Bacillus thuringiensis (Bt), larvae of the domestic silkworm, Bombyx mori (Linnaeus) (Lepidoptera: Bombyxidae), could be exposed to insecticidal proteins. Laboratory experiments were conducted to assess the potential effects of Cry1C- or Cry2A-producing transgenic rice (T1C-19, T2A-1) pollen on B. mori fitness. In a short-term assay, B. mori larvae were fed mulberry leaves covered with different densities of pollen from Bt rice lines or their corresponding near isoline (control) for the first 3 d and then were fed mulberry leaves without pollen. No effect was detected on any life table parameter, even at 1800 pollen grains/cm² leaf, which is much higher than the mean natural density of rice pollen on leaves of mulberry trees near paddy fields. In a long-term assay, the larvae were fed Bt and control pollen in the same way but for their entire larval stage (approximately 27 d). Bt pollen densities ≥150 grains/cm² leaf reduced 14-d larval weight, increased larval development time, and reduced adult eclosion rate. ELISA analyses showed that 72.6% of the Cry protein was still detected in the pollen grains excreted with the feces. The low exposure of silkworm larvae to Cry proteins when feeding Bt rice pollen may be the explanation for the relatively low toxicity detected in the current study. Although the results demonstrate that B. mori larvae are sensitive to Cry1C and Cry2A proteins, the exposure levels that harmed the larvae in the current study are far greater than natural exposure levels. We therefore conclude that consumption of Bt rice pollen will pose a low to negligible risk to B. mori.