The effect of synthetic female sex pheromone on the transmission of the fungus Metarhizium brunneum by male Agriotes obscurus click beetles.

Autodissemination techniques can potentially be used to distribute insecticides, including microbial insecticides, to cryptic pests. This approach is reliant on the target insect either passing the pathogen passively to other insects or the pathogen cycling within the population after the initial host dies. Here we examine, in small scale experiments, whether male Agriotes obscurus click beetles passively transmit the spores of the fungus Metarhizium brunneum directly, or indirectly via the environment, and whether this is influenced by exposure to synthetic female pheromone. We found that the beetles did not avoid M. brunneum spores and that this behaviour was not affected by pheromone. Exposure to pheromone increased beetle movement and uptake of spores, but this did not result in an increase in infected beetles under our conditions. Beetles were able to transfer spores at high levels via environmental contamination. However, contamination of the environment declined rapidly after exposure to the spores. The results are discussed in the context of developing an autodissemination strategy for click beetles.

Effect of Collection Month, Visible Light, and Air Movement on the Attraction of Male Agriotes obscurus L. (Coleoptera: Elateridae) Click Beetles to Female Sex Pheromone.

Elaterid female sex pheromone, while currently used for monitoring the adult life stage (click beetle), has only recently been explored as a potential management tool. Consequently, there is little understanding of how abiotic and biotic conditions influence the response of click beetles to the pheromone. We examined whether the response of male Agriotes obscurus L. (Coleoptera: Elateridae) beetles to a cellulose-based formulation of female sex pheromone ('pheromone granules') is influenced by air movement, presence of visible light, and month of beetle collection. In addition, we investigated the distance from which beetles were attracted to the pheromone granules. Click beetle response was determined by measuring movement parameters in free-walking arena experiments. The response to pheromone was not affected by the presence or absence of visible light. We found that beetles collected earlier in the season had increased activity and interaction with pheromone under moving air conditions, compared to beetles collected later. When controlling for storage time, we confirmed that individuals collected in May were less active than beetles collected in March and April. In the field, beetles were recaptured from up to 14 m away from a pheromone granule source, with over 50% being recovered within 4.4 h from a distance of 7 m or less. Understanding how abiotic and biotic factors affect pest response to pheromone can lead to more effective and novel uses of pheromone-based management strategies.

Modification of reproductive schedule in response to pathogen exposure in a wild insect: Support for the terminal investment hypothesis.

Trade-offs in the time and energy allocated to different functions, such as reproductive activities, can be driven by alterations in condition which reduce resources, often in response to extrinsic factors such as pathogens or parasites. When individuals are challenged by a pathogen, they may either reduce reproduction as a cost of increasing defence mechanisms or, alternatively, modify reproductive activities so as to increase fecundity thereby minimizing the fitness costs of earlier death, a behaviour consistent with the terminal investment hypothesis (TIH). The TIH predicts that individuals with decreased likelihood of future reproduction will maximize current reproductive effort, which may include shifts in reproductive timing. We examined how wild, adult female click beetles (Agriotes obscurus) responded after exposure to the fungal pathogen Metarhizium brunneum. Field-collected beetles exposed to a high concentration of M. brunneum died earlier and in greater numbers than those exposed to a low concentration. Using a multivariate approach, we examined the impact of pathogen challenge on lifespan and a suite of reproductive traits. Stepdown regression analysis showed that only female lifespan differed among the fungal treatments. Fungal-induced reductions in lifespan drove changes in the reproductive schedule, characterized by a decrease in preoviposition period. Moving the start of egg laying forward allowed the females to offset the costs of a shortened lifespan. These changes suggest that there is a threshold for terminal investment, which is dependent on strength of the survival threat. From an applied perspective, our findings imply that exposing adult click beetles to M. brunneum to reduce their population density might not succeed and is an approach that needs further investigation.

Resistance of Trichoplusia ni populations selected by Bacillus thuringiensis sprays to cotton plants expressing pyramided Bacillus thuringiensis toxins Cry1Ac and Cry2Ab.

Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops.

Effect of low levels of Bacillus thuringiensis exposure on the growth, food consumption and digestion efficiencies of Trichoplusia ni resistant and susceptible to Bt.

It is becoming increasingly clear that the impact of low doses of an environmental stressor, such as Bacillus thuringiensis, can often not be predicted from high dose experiments, and the impact of these effects on the evolution of resistance has received little attention. In the present study, we examined the effect of low levels of B. thuringiensis exposure on the growth, food consumption and digestion efficiencies of Trichoplusia ni resistant and susceptible to Bt. Larvae were fed on specified Bt concentrations continually for 3 days. Resistant larvae exhibited increases in diet consumption, weight gain and conversion of ingested food to biomass in response to feeding continually on some of the Bt concentrations. The positive effect of feeding on low levels of Bt on the growth of resistant larvae was modulated by initial larval size and the amount of food consumed. In contrast, susceptible larvae exhibited reductions in growth and frass production at all tested Bt concentrations. Further studies are needed to determine the role of accelerated growth on the evolution of Bt resistance in T. ni populations and to evaluate the importance of life-history responses to sublethal concentrations in the context of insecticide resistance management.

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.

Modified Bacillus thuringiensis toxins and a hybrid B. thuringiensis strain counter greenhouse-selected resistance in Trichoplusia ni.

Resistance of greenhouse-selected strains of the cabbage looper, Trichoplusia ni, to Bacillus thuringiensis subsp. kurstaki was countered by a hybrid strain of B. thuringiensis and genetically modified toxins Cry1AbMod and Cry1AcMod, which lack helix alpha-1. Resistance to Cry1AbMod and Cry1AcMod was >100-fold less than resistance to native toxins Cry1Ab and Cry1Ac.

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.

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 cost of resistance to Bacillus thuringiensis varies with the host plant of Trichoplusia ni.

Selection for resistance to insecticides, diseases and parasitoids is assumed to be costly and often requires tradeoffs with reproductive fitness. The costs of resistance, however, are often difficult to measure. Cabbage looper, Trichoplusia ni, a generalist Lepidopteran herbivore, has become highly resistant following the extensive use of the microbial insecticide, Bacillus thuringiensis kurstaki (Bt) in vegetable greenhouses. We compared the growth rate, pupal size and survival of resistant, susceptible and hybrid T. ni larvae fed on tomato, bell pepper and cucumber. Performance was best on cucumber and worst on pepper, and the magnitude of fitness costs associated with Bt resistance increased with declining host plant suitability. This supports the hypothesis that in this system, resistance costs are condition dependent and are greatest in the most stressful environment. Management strategies that rely on the presence of fitness costs to reduce the frequency of resistance genes must consider this variation and should be more successful on crops that are less suitable food plants. In general, condition dependence should be considered in studies designed to measure the costs of resistance.

Inheritance of resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni.

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F(1) reciprocal crosses and backcrosses between F(1) larvae and resistant (P(R)) and susceptible (P(S)) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F(1) crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F(1) larvae was slightly higher than that for P(S), suggesting that resistance is partially recessive. The responses of both backcross progeny (F(1) x P(R), F(1) x P(S)) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F(1) x P(R) backcross but decreased the correspondence with the F(1) x P(S) backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.

Inheritance of resistance to Bacillus thuringiensis Cry1Ac toxin in a greenhouse-derived strain of cabbage looper (Lepidoptera: Noctuidae).

A population of cabbage looper, Trichoplusia ni (Hübner), collected from commercial greenhouses in the lower mainland of British Columbia, Canada, in 2001 showed a resistance level of 24-fold to Dipel, a product of Bacillus thuringiensis (Bt) subspecies kurstaki. This population was selected with Cry1Ac, the major Bt Cry toxin in Dipel, to obtain a homogenous population resistant to Cry1Ac. The resulting strain of T. ni, named GLEN-Cry1Ac, was highly resistant to Cry1Ac with a resistance ratio of approximately 1000-fold. The larvae from the GLEN-Cry1Ac strain could survive on Cry1Ac-expressing transgenic broccoli plants that were highly insecticidal to T. ni and diamondback moth, Plutella xylostella (L.). The inheritance of Cry1Ac resistance in this T. ni strain was autosomal and incompletely recessive. The degree of dominance of the resistance was -0.402 and -0.395, respectively, for the neonates in reciprocal crosses between the GLEN-Cry1Ac and a laboratory strain of T. ni. Using chi2 goodness-of-fit test, we demonstrated that the inhibition of larval growth resulting from testing 12 toxin doses in the progeny of the backcross fit the predicted larval responses based on a monogenic inheritance model. Therefore, we conclude that the inheritance of the resistance to Cry1Ac in the T. ni larvae is monogenic.

Rapid evolution and the cost of resistance to Bacillus thuringiensis in greenhouse populations of cabbage loopers, Trichoplusia ni.

The microbial insecticide Bacillus thuringiensis (Bt) has become the mainstay of non-chemical control of Lepidopteran pests, either as sprays or through the incorporation of Bt toxins into transgenic crops. Given the wide use of Bt, it is striking that currently only one pest species, Plutella xylostella, has been reported to have developed significant resistance to Bt outside the laboratory. By contrast, we report here the frequent and rapid development of resistance to B. thuringiensis kurstaki (Dipel, Abbott) in populations of cabbage loopers, Trichoplusia ni, in commercial greenhouses. Resistance to Bt appears to be costly and there is a rapid decline of resistance in populations collected from greenhouses and maintained in the laboratory without selection. Management of pests resistant to Bt in vegetable greenhouses will require sporadic use of Bt-based sprays or alternatively use of sprays containing other Bt toxins.

Enhanced fumigant toxicity of p-cymene against Frankliniella occidentalis by simultaneous application of elevated levels of carbon dioxide.

The fumigant toxicity of the essential oil component p-cymene was assessed against Western Flower Thrips, Frankliniella occidentalis. F occidentalis adult females, first- and second-instar larvae and eggs were exposed for 2, 24 and 48 h to combinations of three p-cymene doses and two carbon dioxide levels (ambient, 10%). Additional experiments were conducted on F occidentalis adult females with lower carbon dioxide levels (ambient, 2%, 4%, 6%) applied in combination with p-cymene. Combined applications of p-cymene and carbon dioxide were found to increase significantly the fumigant toxicity of p-cymene against both adult female and larval thrips, but not thrips eggs. An increase in exposure time also led to an increase in adult and larval mortalities in both the p-cymene alone and combined treatments. These results indicate that by combining applications of the essential oil component, p-cymene, with increased carbon dioxide, it may be possible to achieve toxicity levels similar to those of standard chemical fumigants.