Bacillus thuringiensis plants expressing Cry1Ac,Cry2Ab and Cry1F are not toxic to the assassin bug,Zelus renardii

Cotton‐ and maize‐producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non‐target organisms, including natural enemies that suppress pest populations. Here, we used Cry1F‐resistant Spodoptera frugiperda (J.E. Smith) and Cry1Ac and Cry2Ab‐resistant Trichoplusia ni (Hübner) as prey for the assassin bug, Zelus renardii (Kolenati), a common predator in maize and cotton fields. In tritrophic studies, we assessed several fitness parameters of Z. renardii when it fed on resistant S. frugiperda that had fed on Bt maize expressing Cry1F or on resistant T. ni that had fed on Bt cotton expressing Cry1Ac and Cry2Ab. Survival, nymphal duration, adult weight, adult longevity and female fecundity of Z. renardii were not different when they were fed resistant‐prey larvae (S. frugiperda or T. ni) reared on either a Bt crop or respective non‐Bt crops. ELISA tests demonstrated that the Cry proteins were present in the plant at the highest levels, at lower levels in the prey and at the lowest levels in the predator. While Z. renardii was exposed to Cry1F and Cry1Ac and Cry2Ab when it fed on hosts that consumed Bt‐transgenic plants, the proteins did not affect important fitness parameters in this common and important predator.     

Response of multiple generations of beet armyworm,Spodoptera exigua (Hübner), feeding on transgenic Bt cotton

Development, reproduction and food utilization of three successive generations of beet armyworm, Spodoptera exigua (Hübner), fed on transgenic and non‐transgenic Bt cotton were examined. Significantly longer larval life‐span and lower pupal weight were observed in three successive generations of S. exigua fed on transgenic Bt cotton compared with non‐transgenic Bt cotton. Significantly higher survival rate and adult fecundity of S. exigua were found in three successive generations of S. exigua fed on transgenic Bt cotton compared with non‐transgenic Bt cotton. The survival rate and adult fecundity of S. exigua were occurred significant increase in the third generation compared with the first generation after feeding on transgenic Bt cotton. Significantly lower consumption, frass and relative growth rate (RGR) were observed in three successive generations of S. exigua fed on transgenic Bt cotton compared with non‐transgenic Bt cotton. Cotton variety significantly affected all indices of larval consumption and utilization in three successive generations of S. exigua, except for efficiency of conversion of ingested food. However, beet armyworm generation only significantly affected RGR of S. exigua. The results of this study indicated food quality on the diet‐utilization efficiency of S. exigua was different along with beet armyworm generation. Measuring multigenerational development and food utilization of S. exigua at individual and population level in response to Bacillus thuringiensis (Bt) can provide a more meaningful evaluation of long‐term population dynamics than experiments on a single generation. It is imperative to develop an appropriate multigenerational pest management tactic to monitor the field population dynamics of non‐target pests (e.g., beet armyworm) in agricultural Bt cotton ecosystem.     

Feeding behaviour of Helicoverpa armigera larvae on insect-resistant transgenic cotton and non-transgenic cotton

Abstract: Feeding behaviour of Helicoverpa armigera Hübner (Lep.; Noctuidae) larvae on non‐transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum L.), Zhong 30, and transgenic cowpea trypsin inhibitor (CpTI)‐Bt cotton, SGK 321, and non‐transgenic cotton, Shiyuan 321, was investigated in both choice tests and no‐choice tests. The results of choice tests suggested that neonates have the ability to detect and avoid transgenic cotton. In the choice tests of neonates with both transgenic and non‐transgenic cotton leaves, a significantly greater proportion of larvae and higher consumption were observed on non‐transgenic cotton than on the transgenic Bt or CpTI‐Bt cotton. In the choice tests with leaves of two transgenic cotton lines, the proportion of neonates on leaf discs of the two lines was not significantly different, but there was significantly higher consumption on CpTI‐Bt transgenic cotton than that on Bt transgenic cotton. In addition, significantly more neonates were found away from the leaf discs, lower consumption and higher mortality were achieved in the choice test with two transgenic cotton leaves than in the choice tests containing non‐transgenic cotton leaves. Leaves and buds were examined in choice tests of fourth instars. It appeared that fourth instars were found in equal numbers on transgenic and non‐transgenic cotton, except when larvae were exposed to leaves for 3 h. However, the total consumption on transgenic cotton was lower than that of the non‐transgenic cotton, so fourth instars may still have the capacity to detect transgenic cotton and reduce feeding on it, although they showed no preference on either transgenic or non‐transgenic cotton. More larvae were found off diet in the treatments with leaves than that of buds, and the number of injured leaf discs by per fourth instar was significantly higher than that of buds in choice tests, suggesting that leaf is a less preferred organ for H. armigera larvae, elicited more larval movements. Similarly, in no‐choice tests of fifth instars, significantly fewer feeding time and more moving time occurred on leaf than that of bud, boll and petal. When cotton line was considered, compared with non‐transgenic cotton, significantly lower feeding time and higher resting time occurred on the two transgenic cottons. Overall, H. armigera larvae have the ability to detect the transgenic Bt and CpTI‐Bt cottons or the less preferred organs and selectively feed more on the non‐transgenic cotton or the preferred organs, especially the neonates, which have a high capacity for avoiding transgenic cotton.     

Species-specific differences in oak foliage affect preference and performance of gypsy moth caterpillars

Laboratory feeding experiments using two transgenic Bacillus thuringiensis (Bt) rape cultivars (Bt‐Westar and Bt‐Oscar) both expressing the Cry1Ac protein, and the corresponding untransformed lines, were carried out to study the effects of transgenic Bt rape on the non‐target herbivore Athalia rosae (L.) (Hymenoptera: Tenthredinidae). Furthermore, Cry1Ac protein concentration in Bt rape leaves, A. rosae larvae fed Bt rape, their faeces, eonymph instars, pupae, and adults were quantified using an enzyme‐linked immunosorbent assay (ELISA). There were no significant differences in mortality, larval development, and weight between transgenic Bt rape and non‐transgenic rape fed A. rosae. Additionally, we did not detect any significant differences in the fecundity and fertility of adult females either fed as larvae with transgenic Bt or with non‐transgenic rape. However, results of the ELISA indicated that Cry1Ac protein was detectable in larvae and faeces (Bt‐Westar 1.1 ± 0.2 and Bt‐Oscar 0.3 ± 0.2 µg Cry1Ac protein/g fresh weight) although this was less than in the leaf material, where concentrations were 2.2 ± 0.8 µg Cry1Ac protein/g fresh weight for Bt‐Westar and 7.5 ± 2.9 µg Cry1Ac protein/g fresh weight in Bt‐Oscar. In contrast, Cry1Ac protein could not be detected in eonymphs, pupae, or adults of A. rosae. Our results suggest that Cry1Ac protein in Bt rape does not have a significant effect on the herbivore A. rosae but the protein is still detectable after ingestion and excretion by these herbivores, thus providing the possibility of exposure to organisms other than herbivores.     

Fitness of Bt‐resistant cabbage loopers on Bt cotton plants

Development of resistance to the insecticidal toxins from Bacillus thuringiensis (Bt) in insects is the major threat to the continued success of transgenic Bt crops in agriculture. The fitness of Bt‐resistant insects on Bt and non‐Bt plants is a key parameter that determines the development of Bt resistance in insect populations. In this study, a comprehensive analysis of the fitness of Bt‐resistant Trichoplusia ni strains on Bt cotton leaves was conducted. The Bt‐resistant T. ni strains carried two genetically independent mechanisms of resistance to Bt toxins Cry1Ac and Cry2Ab. The effects of the two resistance mechanisms, individually and in combination, on the fitness of the T. ni strains on conventional non‐Bt cotton and on transgenic Bt cotton leaves expressing a single‐toxin Cry1Ac (Bollgard I) or two Bt toxins Cry1Ac and Cry2Ab (Bollgard II) were examined. The presence of Bt toxins in plants reduced the fitness of resistant insects, indicated by decreased net reproductive rate (R₀) and intrinsic rate of increase (r). The reduction in fitness in resistant T. ni on Bollgard II leaves was greater than that on Bollgard I leaves. A 12.4‐day asynchrony of adult emergence between the susceptible T. ni grown on non‐Bt cotton leaves and the dual‐toxin‐resistant T. ni on Bollgard II leaves was observed. Therefore, multitoxin Bt plants not only reduce the probability for T. ni to develop resistance but also strongly reduce the fitness of resistant insects feeding on the plants.     

Effects of elevated CO2 and transgenic Bt cotton on plant chemistry, performance, and feeding of an insect herbivore, the cotton bollworm

Effects of elevated atmospheric CO₂ (double‐ambient CO₂) on the growth and metabolism of cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), fed on transgenic Bacillus thuringiensis (Berliner) (Bt) cotton [Cry1A(c)], grown in open‐top chambers, were studied. Two levels of CO₂ (ambient and double‐ambient) and two cotton cultivars (non‐transgenic Simian‐3 and transgenic GK‐12) were deployed in a completely randomized design with four treatment combinations, and the cotton bollworm was reared on each treatment simultaneously. Plants of both cotton cultivars had lower nitrogen and higher total non‐structural carbohydrates (TNC), TNC:Nitrogen ratio, condensed tannin, and gossypol under elevated CO₂. Elevated CO₂ further resulted in a significant decrease in Bt toxin level in GK‐12. The changes in chemical components in the host plants due to increased CO₂ significantly affected the growth parameters of H. armigera. Both transgenic Bt cotton and elevated CO₂ resulted in a reduced body mass, lower fecundity, decreased relative growth rate (RGR), and decreased mean relative growth rate in the bollworms. Larval life‐span was significantly longer for H. armigera fed transgenic Bt cotton. Significantly reduced larval, pupal, and adult moth weights were observed in the bollworms fed elevated CO₂‐grown transgenic Bt cotton compared with those of bollworms reared on non‐transgenic cotton, regardless of the CO₂ level. The efficiency of conversion of ingested food and of digested food of the bollworm were significantly reduced when fed transgenic Bt cotton, but there was no significant CO₂ or CO₂× cotton cultivar interaction. Approximate digestibility of larvae reared on transgenic cotton grown in elevated CO₂ was higher compared to that of larvae fed non‐transgenic cotton grown at ambient CO₂. The damage inflicted by cotton bollworm on cotton, regardless of the presence or absence of insecticidal genes, is predicted to be more serious under elevated CO₂ conditions because of individual compensatory feeding on host plants caused by nitrogen deficiency.     

Harmonia axyridis (Coleoptera: Coccinellidae) Exhibits No Preference between Bt and Non-Bt Maize Fed Spodoptera frugiperda (Lepidoptera: Noctuidae)

A recent shift in managing insect resistance to genetically engineered (GE) maize consists of mixing non-GE seed with GE seed known as “refuge in a bag”, which increases the likelihood of predators encountering both prey fed Bt and prey fed non-Bt maize. We therefore conducted laboratory choice-test feeding studies to determine if a predator, Harmonia axyridis, shows any preference between prey fed Bt and non-Bt maize leaves. The prey species was Spodoptera frugiperda, which were fed Bt maize (MON-810), expressing the single Cry1Ab protein, or non-Bt maize. The predators were third instar larvae and female adults of H. axyridis. Individual predators were offered Bt and non-Bt fed prey larvae that had fed for 24, 48 or 72 h. Ten and 15 larvae of each prey type were offered to third instar and adult predators, respectively. Observations of arenas were conducted at 1, 2, 3, 6, 15 and 24 h after the start of the experiment to determine the number and type of prey eaten by each individual predator. Prey larvae that fed on non-Bt leaves were significantly larger than larvae fed Bt leaves. Both predator stages had eaten nearly all the prey by the end of the experiment. However, in all combinations of predator stage and prey age, the number of each prey type consumed did not differ significantly. ELISA measurements confirmed the presence of Cry1Ab in leaf tissue (23–33 µg/g dry weight) and S. frugiperda (2.1–2.2 µg/g), while mean concentrations in H. axyridis were very low (0.01–0.2 µg/g). These results confirm the predatory status of H. axyridis on S. frugiperda and that both H. axyridis adults and larvae show no preference between prey types. The lack of preference between Bt-fed and non-Bt-fed prey should act in favor of insect resistance management strategies using mixtures of GE and non-GE maize seed.     

Selective Feeding of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Hübner) and <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Fabricius) on Meridic Diet with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Toxins

Laboratory experiments were conducted to evaluate the behavior of Helicoverpa armigera (Hübner) and Spodoptera litura (Fabricius) larvae on meridic diet with different concentrations of Bt spray formulation Delfin or isolated Cry1Ac protein or the foliage and bolls from transgenic cotton, Bollgard hybrid RCH-317 Bt. Both insect species selectively fed on nontreated diet compared with the diet treated with Delfin. While H. armigera exhibited concentration response with Cry1Ac, this protein did not affect S. litura larvae. In general Helicoverpa selected diet with low concentrations (EC₂₀ and EC₅₀ levels) of Cry1Ac compared with higher concentrations of Cry1Ac. In order to develop appropriate management strategies, a thorough understanding of the behavioral mechanisms leading to the responses of insects to the proteins in transgenic varieties is required. Thus, based on results of the insects fed individually on the leaf discs or bolls from transgenic cotton plants alone or under choice situation with meridic diet revealed that H. armigera larvae preferred meridic diet to transgenic leaves or bolls expressing Cry1Ac protein. H. armigera larvae preferred meridic diet to plant material; more than 70% larvae were seen on the meridic diet, and average larval weight gain was in the range of 121.7–130.5 mg. However, in case of S. litura the larvae showed no significant discrimination between meridic diet and the leaf discs. In fact more than 60% larvae preferred leaf discs for feeding, though Cry1Ac expression in leaf discs was in the range of 0.9–2.18 μg/g. Thus differences in behavioral response could potentially impact the level of efficacy of crop cultivars that have been genetically engineered to produce these proteins.     

Stay or move: how Bt‐susceptible Helicoverpa armigera neonates behave on Bt cotton plants

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae occasionally have been reported to survive at management threshold levels in fields of Bollgard II^® cotton, Gossypium hirsutum L. (Malvaceae). The pattern and degree of larval survival is not easily predicted but depends on the ability of first instars to establish on host plants. Experiments were conducted with Bacillus thuringiensis Berliner (Bt)‐susceptible and Bt‐resistant larvae of H. armigera to understand how physiologically Bt‐susceptible H. armigera survive on Bt cotton plants, and examine how their first meal influences survival rates. In assays using cotton plant parts, both strains of larvae displayed similar tendencies to drop‐off specific plant parts of Bt and non‐Bt cotton. However, significantly more Bt‐susceptible larvae dropped off young leaves, mature leaves, and squares of Bt cotton compared to non‐Bt cotton plants. Egg cannibalism significantly improved the survival of Bt‐susceptible H. armigera larvae on Bt cotton plants. Larvae were more likely to eat live aged eggs, than newly laid or dead eggs. Survival significantly improved when larvae cannibalized eggs before feeding on Bt leaves. The behavior of Bt‐susceptible larvae with respect to drop‐off and egg cannibalism may help enhance their survival on Bt cotton plants.     

Processing of the maize Bt toxin in the gut of Mythimna unipuncta caterpillars

Mythimna unipuncta Haworth (Lepidoptera: Noctuidae) is a well‐known moth species whose larvae can cause devastating damage to some Poaceae crops, including maize (Zea mays L.). The low susceptibility to the Bacillus thuringiensis Berliner (Bt) toxin observed in L6 larvae of this species has been the object of several studies. This study aimed to clarify whether the toxin eliminated from the content of the peritrophic membrane is degraded or excreted and whether the effects of the Bt toxin depend on the doses ingested. To this end, L6 larvae were fed on diets with different amounts of lyophilized Bt or non‐Bt maize leaves. The effect of the Bt concentrations on larval development was measured and the fate of the toxin in the larval tissues was tracked. Results indicated that the larvae of M. unipuncta fed on the various diets showed few differences in weight gain, duration of development, or pupal weight between sublethal Bt concentrations. The larvae rapidly excreted a large part of the toxin ingested, whereas inside the peritrophic membrane the toxin was eliminated, degraded, or sequestered at a rate that increased with the dose and the duration of feeding. As a consequence, little toxin reached the midgut epithelium and therefore the binding sites of the toxin. Moreover, larvae fed on the Bt toxin recovered quickly when they were transferred to a non‐Bt diet.     

Rapid selection and characterization of Cry1F resistance in a Brazilian strain of fall armyworm

Transgenic maize (Zea mays L., Poaceae) event TC1507, producing the Cry1F protein of Bacillus thuringiensis Berliner, has been used for management of the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), in Brazil since 2009. A strain of S. frugiperda, obtained from field collections of larvae in TC1507 maize in Minas Gerais state in 2010, was selected in the laboratory for resistance to Cry1F using leaves of TC1507 maize in two selection regimes. Continuous exposure of larvae to Cry1F was more effective than exposure for 6, 8, and 10 days in the selection of resistant S. frugiperda individuals. With only four generations of laboratory selection, a strain with high levels of resistance to Cry1F was obtained, as indicated by the survival of insects reared on leaves of TC1507 maize plants and by the more than 300‐fold resistance level measured in bioassays with the purified Cry1F protein. Importantly, reciprocal crosses between control and the Cry1F‐selected strains revealed that the resistance is autosomal and incompletely recessive, and the response obtained in the backcross of the F₁ generation with the resistant strain was consistent with simple monogenic inheritance. Additionally, there were no apparent fitness costs associated with resistance either for survival or larval growth on non‐Bt maize leaves. Our findings provide experimental evidence for rapid evolution of Cry1F resistance in S. frugiperda in the laboratory and further reinforce the potential of this species to evolve field resistance to the TC1507 maize as previously reported. The resistant strain isolated in this study provides an opportunity to estimate the resistance allele frequency in the field and to determine the biochemical and molecular basis of the resistance, which should provide further information to assist in the resistance management of S. frugiperda on transgenic maize producing B. thuringiensis proteins.     

Bacillus thuringiensis protein transfer between rootstock and scion of grafted poplar

Bacillus thuringiensis (Bt) Cry1Ac protein is a toxin against different leaf‐eating lepidopteran insects that attack poplar trees. In the present study, the mode of migration of the Bt‐Cry1Ac protein within poplar grafts was investigated. Grafting was done using Pb29 (transgenic poplar 741 with cry1Ac genes), CC71 (transgenic poplar 741 with cry3A genes), non‐transgenic poplar 741 and non‐transgenic Populus tomentosa, either as scion or as rootstock. In order to detect migration of Bt‐Cry1Ac protein from one portion of the graft union to different tissues in the grafted plant, ELISA analysis was employed to assess the content of Bt‐Cry1Ac protein in the phloem, xylem, pith and leaves of the grafted poplar. To further verify migration of Bt‐Cry1Ac protein, Clostera anachoreta larvae, which are susceptible to Bt‐Cry1Ac protein, were fed leaves from the control graft (i.e., graft portion that originally did not contain Bt‐Cry1Ac protein). The results showed that Bt‐Cry1Ac protein was transported between rootstock and scion mainly through the phloem. Migration of Bt‐Cry1Ac protein in the grafted union was also evidenced in that the leaves of the control graft did have a lethal effect on C. anachoreta larvae in laboratory feeding experiments.     

Survival of Helicoverpa armigera larvae on and Bt toxin expression in various parts of transgenic Bt cotton (Bollgard II) plants

There is no conclusive evidence that Helicoverpa spp. (Lepidoptera: Noctuidae) in Australia have evolved significant levels of resistance to Bollgard II^® cotton (which expresses two Bt toxin genes, cry1Ac and cry2Ab). However, there is evidence of surviving larvae on Bollgard II cotton in the field. The distribution and survival of early‐instar Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae were examined on whole Bollgard II and non‐Bt cotton plants in greenhouse bioassays. The expression of Cry toxins in various parts of Bollgard II plants was compared to the survival of larvae in those locations. Only 1% of larvae survived after 6 days on greenhouse‐grown Bollgard II plants compared to 31% on non‐Bt cotton plants. Overall, and across all time intervals, more larvae survived on reproductive parts (squares, flowers, and bolls) than on vegetative parts (leaves, stems, and petioles) on Bollgard II plants. The concentration of Cry1Ac toxin did not differ between plant structures, whereas Cry2Ab toxin differed significantly, but there was no relationship between the level of expression and the location of larvae. This study provides no evidence that lower expression of Cry toxins in the reproductive parts of plants explains the survival of H. armigera larvae on Bollgard II cotton.     

Impacts of elevated CO2 on Bemisia tabaci infesting Bt cotton and its parasitoid Encarsia formosa

Atmospheric carbon dioxide concentration is expected to rise in the coming decades. Rising atmospheric CO₂ levels may alter plant‐insect‐parasitoid associations due to the indirect effects of CO₂ enrichment on phytochemicals important for herbivore and parasitoid nutrition. Tritrophic effects of elevated CO₂ on Bt cotton (GK‐12) and non‐transgenic (Simian‐3, or S3) cotton [Gossypium hirsutum L. (Malvaceae)], Bemisia tabaci (Gennadius) biotype B (Hemiptera: Aleyrodidae), and its parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae), were examined in open‐top chambers. Significantly, longer egg‐adult developmental duration and higher mortality of nymphs were observed under elevated CO₂ concentrations on both cotton cultivars during three successive generations. However, no significant differences were found in adult longevity, offspring sex ratio, and the number of eggs laid per female adult of B. tabaci fed on transgenic (GK‐12) or non‐transgenic cotton (S3) grown under elevated CO₂. Abundance of B. tabaci adults increased from 10 to 120 per plant and then decreased to 40 per plant through the growing season, but no significant differences in density occurred between CO₂ treatments and between cultivar treatments. Similarly, no significant differences were found in the developmental duration, parasitization rate, and adult emergence rate of E. formosa after parasitizing B. tabaci for three successive generations. Our results showed that the effects of transgenic Bt cotton did not significantly affect the development, survivorship, life span, or fecundity of B. tabaci and its parasitoids. Moreover, interactions between B. tabaci and E. formosa were not significantly affected by elevated CO₂. These results suggest that the biological control of B. tabaci by E. formosa would not be influenced by transgenic Bt cotton and/or elevated CO₂, indicating that the current risk management strategy regarding B. tabaci outbreaks and biocontrol by E. formosa will remain effective if the atmospheric CO₂ level continues to rise.     

Development of a novel‐type transgenic cotton plant for control of cotton bollworm

The transgenic Bt cotton plant has been widely planted throughout the world for the control of cotton budworm Helicoverpa armigera (Hubner). However, a shift towards insect tolerance of Bt cotton is now apparent. In this study, the gene encoding neuropeptide F (NPF) was cloned from cotton budworm H. armigera, an important agricultural pest. The npf gene produces two splicing mRNA variants—npf1 and npf2 (with a 120‐bp segment inserted into the npf1 sequence). These are predicted to form the mature NPF1 and NPF2 peptides, and they were found to regulate feeding behaviour. Knock down of larval npf with dsNPF in vitro resulted in decreases of food consumption and body weight, and dsNPF also caused a decrease of glycogen and an increase of trehalose. Moreover, we produced transgenic tobacco plants transiently expressing dsNPF and transgenic cotton plants with stably expressed dsNPF. Results showed that H. armigera larvae fed on these transgenic plants or leaves had lower food consumption, body size and body weight compared to controls. These results indicate that NPF is important in the control of feeding of H. armigera and valuable for production of potential transgenic cotton.     

Silicon application promotes rice growth and negatively affects development of Spodoptera frugiperda (J. E. Smith)

Silicon (Si) has been reported to enhance plant resistance against biotic and abiotic stressors and also benefit plant growth. These effects are more pronounced in grass species, especially with soil‐applied Si. This study investigated the effects of Si application on rice resistance to Spodoptera frugiperda development and plant vegetative growth. Effects of Si on rice were assessed via soil and foliar applications and compared with untreated plants (control). Si was soil‐ and foliar‐applied as 1% silicic acid solution at a dosage equivalent to 1.4 t Si per ha. After application, leaf material was collected from Si‐treated and untreated plants and placed in Petri dishes with individual S. frugiperda neonate larvae, where development was followed to adult emergence and biological parameters recorded. Vegetative growth parameters recorded in rice plants were the height, chlorophyll content, fresh and dry weights of shoots, and shoot Si content. No effects of Si application were observed on the durations of larval and pupal stages, larval and pupal survival, and sex ratio of S. frugiperda. Insects fed leaves from Si‐treated plants exhibited lower leaf consumption, larval and pupal weights, longevity of males and females, number of eggs, and egg viability. The negative effects were correlated with higher rice Si content. Si application to rice increased plant height, chlorophyll content and dry weight. Our study demonstrates that foliar‐applied Si is as efficient as soil‐applied Si in negatively affecting S. frugiperda development and providing beneficial effects on rice plant growth.     

Oviposition and feeding avoidance in Helicoverpa armigera (Hübner) against transgenic Bt cotton

Helicoverpa armigera (Hübner), the major target pest of transgenic Bacillus thuringiensis (Bt) cotton, remains susceptible to Bt cotton in China at present. Behavioural avoidance by ovipositing females might lead to reduced exposure to Bt cotton and minimize selection for physiological resistance. We examined the behavioural responses of H. armigera to Bt and non‐Bt cottons to determine whether behavioural avoidance to Bt cotton may be present. In oviposition choice tests, the number of eggs on non‐Bt cotton plants was significantly higher than on Bt cotton plants. Similarly, in no‐choice tests, Bt cotton plants attracted significantly fewer eggs compared with non‐Bt cotton plants. H. armigera neonates showed higher dispersal and lower establishment on Bt cotton than on non‐Bt cotton. First instars were found to feed consistently on non‐Bt cotton leaves, creating large feeding holes, but only produced tiny feeding holes on Bt cotton leaves. The H. armigera population used in this study showed avoidance of oviposition and feeding on Bt cotton. Our results provide important insights into one possible mechanism underlying the durability of Bt cotton resistance and may be useful for improving strategies to sustain the effectiveness of Bt crops.     

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

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

Sublethal effects of Bt toxin and chlorpyrifos on various Spodoptera exigua populations

Bt cotton (Cry1Ac) has been commercially grown in China since 1997, saving China's cotton production from attack by Bt‐target pests and also tremendously reducing pesticide usage. In recent years, however, Bt cotton, with 4.2 million ha of cultivation, has suffered from a secondary target pest, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). In China, growers have even had to re‐adopt conventional pesticides to control the pest, and this practice has already caused serious pesticide residue. In order to clarify the sublethal effects of chemical pesticide, the responses of a Bt‐susceptible and a Bt‐tolerant (Bt10) S. exigua strain to three treatment combinations were examined, including Bt toxin, sublethal chlorpyrifos, and Bt + sublethal chlorpyrifos. The susceptible and the Bt10 strain responded differently to dual pressure. Bt toxin + sublethal chlorpyrifos treatment lowered larval mortality and stimulated population increase of the susceptible S. exigua, whereas it delayed growth and development of the Bt10 strain. Under dual pressure, although larvae of the Bt10 strain developed faster than larvae of the susceptible strain, the Bt10 population experienced higher larval mortality, prolonged pupal duration, decreased pupal weight, decreased emergence rate, and shortened adult longevity. Compared with the susceptible strain, the Bt10 strain was deleteriously affected by sublethal chlorpyrifos. The Bt‐tolerant/resistant S. exigua population was more vulnerable to chemical pesticides like chlorpyrifos regardless of whether it was exposed to Bt toxin or not. Our study provides a reference for increasing the efficacy of control of S. exigua in Bt‐cotton planting areas.     

Orientation Behavior,Development and Survival of <Emphasis Type="Italic">Trichoplusia ni</Emphasis> (Lepidoptera: Noctuidae) Larvae on Cotton Expressing Cry1Ac and Cry2Ab and Conventional Cotton

This study was conducted to determine the effects of Bt cotton leaves (Bollgard II), non-Bt cotton leaves, and a mixture of Bt+non-Bt cotton leaves on larval orientation behavior, survival and development of Trichoplusia ni in the laboratory. Results indicate that in a no-choice test, more first and fifth instars remained on Bt leaves than the third instars. All larvae that remained on the leaves gradually moved to leaf edge. In the choice between a Bt and a non-Bt leaf, more first instars moved to non-Bt leaves, whereas the third and fifth instars did not show significant difference in the first 8 h, but eventually more moved to non-Bt leaves. More first instars fed non-Bt leaves than third instars and fifth instars. When larvae fed Bt leaves, 100% of first instars, 92.7% of third instars and 51.1% of fifth instars died in 108 h. Once larvae pupated, >90% developed to adults. First and third instars that fed Bt leaves developed slower but their pupae developed faster than those on Bt+non-Bt leaves, whereas fifth instars developed similar on the three types of leaves. First and third instars that fed Bt+non-Bt leaves resulted in less heavy pupae than those fed non-Bt leaves; whereas the fifth instars that survived on Bt leaves produced lighter pupae.