Assessment of potential impacts due to unintentionally released Bt maize plants on non-target aphid Rhopalosiphum padi (Hemiptera: Aphididae)

Genetically modified maize crops expressing Bacillus thuringiensis (Bt) toxins (Bt maize) are increasingly cultivated worldwide, and large amounts of Bt maize have been imported to Korea. Before evaluating the environmental impacts of Bt maize of unknown origin on non-target insects, crystal (Cry) protein types in the imported Bt maize plants were identified. Because Cry1F was found in the tested Bt maize plants, Rhopalosiphum padi, a non-lepidopteran species, was selected as the non-target insect species. Additionally, a widely cultivated domestic maize strain was selected as an alternative control. No difference in survival rate, alata vivipara production, or host preference was observed between R. padi fed on the Bt maize and the control non-Bt maize, indicating that Bt maize plants had no sub-chronic adverse effects on R. padi. The average number of nymphs from Bt maize-fed aphids was 1.73-fold higher than that of non-Bt maize-fed aphids, implying that R. padi population density can increase after several generations in Bt maize fields. An enzyme-linked immunosorbent assay revealed that Cry1F toxin concentrations increased gradually in the body of R. padi when they were fed Bt maize, but that all ingested Cry toxins were excreted within 10 days after Bt-fed aphids were transferred to non-Bt maize, suggesting little possibility of Cry toxin exposure via R. padi to the endoparasitoids. However, the possibility still remains that Cry toxins can be transferred to predatory insects in higher trophic levels if they consume Bt maize-fed aphids.     

Characterization of entomopathogenic nematodes and symbiotic bacteria active against Spodoptera frugiperda (Lepidoptera: Noctuidae) and contribution of bacterial urease to the insecticidal effect

Entomopathogenic nematodes carrying symbiotic bacteria represent one of the best non-chemical strategies for insect control. Infective juveniles of Heterorhabditidae and Steinernematidae nematodes actively seek the host in the soil, penetrating through insect’s openings to reach the hemocoel where symbiotic bacteria in the genera Photorhabdus or Xenorhabdus, respectively, are released. The bacteria replicate and produce virulence factors that rapidly kill the insect host, providing nutrients for the nematodes development and reproduction within the insect cadaver. More studies are necessary to better understand the factors implicated in the nematode-bacteria association, particularly focusing the bacterial symbionts, the final effectors of the insect death. Our group has shown that ureases are lethal to some groups of insects and may contribute to the entomopathogenic properties of the symbiotic bacteria.The fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is one of the major insect pests in corn (Zea mays) crops in Brazil, with infestations resulting in reduction up to 39% yield and losses amounting US$ 500 million annually. Native strains of entomopathogenic nematodes active against S. frugiperda represent a promising alternative to the intensive use of chemical insecticides to control fall armyworm population in corn plantations.In this study we screened soil nematodes collected in the south region of Brazil for pathogenicity against S. frugiperda. Symbiotic bacteria associated with these nematodes were isolated and characterized. We also evaluated urease production by the symbiotic bacteria in vitro and along the course of infection in S. frugiperda and demonstrated that urease production correlated positively to their entomopathogenicity.     

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.     

Transgenic Bt Rice Does Not Challenge Host Preference of the Target Pest of Rice Leaffolder,Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Background

Transgenic Bt rice line T2A-1 expresses a synthesized cry2A gene that shows high resistance to Lepidoptera pests, including Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae). Plant volatile orientation cues and the physical characteristics of the leaf surface play key roles in host location or host-plant acceptance of phytophagous insects. These volatile compounds and physical traits may become altered in Bt rice and it is not known whether this influences the behavior of C. medinalis when searching for oviposition sites.

Results

The results of electronic nose analysis showed that the Radar map of Bt rice cultivars was analogous to the non- Bt rice cultivars at each growing stage. PCA analysis was able to partly discriminate between some of the Bt vs. non-Bt rice sensors, but could not to separate Bt cultivars from non-Bt cultivars. The total ion chromatogram between Bt and non-Bt rice cultivars at the seedling, booting and tillering stages were similar and 25 main compounds were identified by GC-MS. For most compounds, there was no significant difference in compound quantities between Bt and non-Bt rice cultivars at equivalent growth stages. The densities of the tubercle papicles and the trichomes on the upper and lower surfaces were statistically equal in Bt and non-Bt rice. The target pest, C. medinalis, was attracted to host rice plants, but it could not distinguish between the transgenic and the isogenic rice lines.

Conclusions

There were no significant differences between the Bt rice line, T2A-1 and the non-Bt rice for volatiles produced or in its physical characteristics and there were no negative impacts on C. medinalis oviposition behavior. These results add to the mounting evidence that Bt rice has no negative impact on the target insect oviposition behavior.     

Corethrellonema grandispiculosum n. gen., n. sp. and Aproctonema chapmani n. sp. (Nematoda: Tetradonematidae), Parasites of the Dipterous Insect Genera,Corethrella and Culicoides in Louisiana

Two new nematodes of the family Tetradonematidae, parasitic in aquatic dipterous insects in Louisiana, are presented. Corethrellonema grandispiculosum n. gen., n. sp., from the chaoborid fly, Corethrella brakeleyi Coquillett, and Aproctonema chapmani n. sp., from the sand fly, Culicoides arboricola Root and Hoffman, are described and illustrated. The biology and life histories of these nematodes show that the adults occur in the last larval instar of the insect host. The adult nematodes mate in the body cavity of the insect, and later the female nematode, replete with eggs, exits from the larval fly causing the death of the insect. Male nematodes usually remain in the insect cadaver.     

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.     

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.     

Long-Term Persistence of Yersinia pseudotuberculosis in Entomopathogenic Nematodes

Entomopathogenic nematodes (EPNs) are small worms whose ecological behaviour consists to invade, kill insects and feed on their cadavers thanks to a species-specific symbiotic bacterium belonging to any of the genera Xenorhabdus or Photorhabdus hosted in the gastro-intestinal tract of EPNs. The symbiont provides a number of biological functions that are essential for its EPN host including the production of entomotoxins, of enzymes able to degrade the insect constitutive macromolecules and of antimicrobial compounds able to prevent the growth of competitors in the insect cadaver. The question addressed in this study was to investigate whether a mammalian pathogen taxonomically related to Xenorhabdus was able to substitute for or “hijack” the symbiotic relationship associating Xenorhabdus and Steinernema EPNs. To deal with this question, a laboratory experimental model was developed consisting in Galleria mellonella insect larvae, Steinernema EPNs with or without their natural Xenorhabdus symbiont and Yersinia pseudotuberculosis brought artificially either in the gut of EPNs or in the haemocoel of the insect larva prior to infection. The developed model demonstrated the capacity of EPNs to act as an efficient reservoir ensuring exponential multiplication, maintenance and dissemination of Y. pseudotuberculosis.     

Plant resistance signalling hijacked by a necrotrophic fungal pathogen

The strategies used by necrotrophic fungal pathogens to infect plants are often perceived as lacking the sophistication of their haustorium producing, host defence suppressing, biotrophic counterparts. There is also a relative paucity of knowledge regarding how effective gene-for-gene based resistance reactions might function against necrotrophic plant pathogens. However, recent data has emerged from a number of systems which has highlighted that particular species of necrotrophic (and/or hemibiotrophic) fungi, have evolved very sophisticated strategies for plant infection which appear, in fact, to hijack the host resistance responses that are commonly deployed against biotrophs. Both disease resistance (R) protein homologues and mitogen-activated protein kinase (MAPK) cascades commonly associated with incompatible disease resistance responses; appear to be targeted by necrotrophic fungi during compatible disease interactions. These findings highlight an emerging sophistication in the strategies deployed by necrotrophic fungi to infect plants.Key words: Mycosphaerella graminicola, Septoria tritici, Triticum aestivum, mitogen-activated protein kinase, programmed cell death, fungal pathogen, disease resistance, disease susceptibility, toxin     

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence

We examined the influence of insect cadaver desiccation on the virulence and production of entomopathogenic nematodes (EPNs), common natural enemies of many soil-dwelling insects. EPNs are often used in biological control, and we investigated the feasibility of applying EPNs within desiccated insect cadavers. Desiccation studies were conducted using the factitious host, Galleria mellonella (Lepidoptera: Pyralidae, wax moth larvae) and three EPN species (Heterorhabditis bacteriophora ‘HB1’, Steinernema carpocapsae ‘All’, and Steinernema riobrave). Weights of individual insect cadavers were tracked daily during the desiccation process, and cohorts were placed into emergence traps when average mass losses reached 50%, 60%, and 70% levels. We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. We observed apparent differences in the desiccation rate of the insect cadavers among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than −2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. At the 60% mass loss level, however, desiccated insect cadavers from each of the three species successfully produced IJs when exposed to moist sand, suggesting that insect cadaver desiccation may be a useful approach for biological control of soil insect pests.     

Structural insight into the mechanism of streptozotocin inhibition of O-GlcNAcase

Despite decades of its use in diabetes research, the mechanism of cytotoxicity of streptozotocin (STZ) toward pancreatic β-islet cells has remained a topic of discussion. Although STZ toxicity is likely a function of its capacity to promote DNA alkylation, it has been proposed that STZ induces pancreatic β-cell death through O-GlcNAcase inhibition. In this report, we explore the binding mode of STZ to a close homolog of human O-GlcNAcase, BtGH84 from Bacteroides thetaiotaomicron. Our results show that STZ binds in the enzyme active site in its intact form, without the formation of a covalent adduct, consistent with solution studies on BtGH84 and human O-GlcNAcase, as well as with structural work on a homolog from Clostridium perfringens. The active site of the BtGH84 is considerably deformed upon STZ binding and as a result the catalytic machinery is expelled from the binding cavity.     

Mating and Sexual Communication by Steinernema carpocapsae (Nemata: Steinernematidae)

Entomopathogenic nematodes are lethal insect parasites that reproduce exclusively inside their hosts in nature. Infection decisions made by the free-living infective-stage juveniles have an impact on reproductive success, but it is likely that mating decisions are made by adults while inside their host. We investigated sexual communication between male and female adult stages of Steinernema carpocapsae (Rhabditida: Steinernematidae) to assess whether mating is chemically mediated during the adult stage or results from incidental encounters between adults inside the insect host. To assess chemical communication, we measured the behavioral response of adult male S. carpocapsae to several different potential sources of chemical information. Male S. carpocapsae responded to virgin females only and were not influenced by mated conspecific females, conspecific males, or heterospecific females. These results show that species-specific communication takes place between adult entomopathogenic nematodes within the host cadaver just prior to mating.     

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.     

Resistance evolution to the first generation of genetically modified Diabrotica-active Bt-maize events by western corn rootworm: management and monitoring considerations

Western corn rootworm (Diabrotica virgifera virgifera; WCR) is a major coleopteran maize pest in North America and the EU, and has traditionally been managed through crop rotation and broad-spectrum soil insecticides. Genetically modified Bt-maize offers an additional management tool for WCR and has been valuable in reducing insecticide use and increasing farm income. A concern is that the widespread, repeated, and exclusive deployment of the same Bt-maize transformation event will result in the rapid evolution of resistance in WCR. This publication explores the potential of WCR to evolve resistance to plant-produced Bt-toxins from the first generation of Diabrotica-active Bt-maize events (MON 863 and MON 88017, DAS-59122-7 and MIR604), and whether currently implemented risk management strategies to delay and monitor resistance evolution are appropriate. In twelve of the twelve artificial selection experiments reported, resistant WCR populations were yielded rapidly. Field-selected resistance of WCR to Cry3Bb1 is documented in some US maize growing areas, where an increasing number of cases of unexpected damage of WCR larvae to Bt-maize MON 88017 has been reported. Currently implemented insect resistance management measures for Bt-crops usually rely on the high dose/refuge (HDR) strategy. Evidence (including laboratory, greenhouse and field data) indicates that several conditions contributing to the success of the HDR strategy may not be met for the first generation of Bt-maize events and WCR: (1) the Bt-toxins are expressed heterogeneously at a low-to-moderate dose in roots; (2) resistance alleles may be present at a higher frequency than initially assumed; (3) WCR may mate in a non-random manner; (4) resistance traits could have non-recessive inheritance; and (5) fitness costs may not necessarily be associated with resistance evolution. However, caution must be exercised when extrapolating laboratory and greenhouse results to field conditions. Model predictions suggest that a 20 % refuge of non-Diabrotica-active Bt-maize can delay resistance evolution in WCR under certain conditions. This publication concludes that further research is needed to resolve the remaining scientific uncertainty related to the appropriateness of the HDR in delaying resistance evolution in WCR, resistance monitoring is essential to detect early warning signs indicating resistance evolution in the field, and that integrated pest management reliant on multiple tactics should be deployed to ensure effective long-term corn rootworm management and sustainable use of Bt-maize.