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Cabbage looper, Trichoplusia ni (Hübner) is an important lepidopteran pest on many vegetable and greenhouse crops, and some field crops. Although there are no commercial transgenic Bt vegetable or greenhouse crops, T. ni is a target of Bollgard II cotton, which produces Cry1Ac and Cry2Ab. We expand on previous work that examined the effect of Bt crops on parasitoids using Bt-resistant lepidopteran populations as hosts. Cry1Ac/Cry2Ab-resistant T. ni larvae were used to eliminate host quality effects and to evaluate the direct effects of Bt cotton on the parasitoids Copidosoma floridanum (Ashmead) and Cotesia marginiventris (Cresson). These tri-trophic studies confirm that Bt cotton had no significant impact on development, success of parasitism, survival and adult longevity of C. marginiventris when using Bt-resistant T. ni fed on Bt cotton. Similarly, this Bt cotton had no significant impact on the development, mummy weight and the number of progeny produced by C. floridanum. Our studies verified that lyophilized Bt crop tissue maintained its insecticidal bioactivity when incorporated into an artificial diet, demonstrating that hosts and parasitoids were exposed to active Cry proteins. The egg-larval parasitoid C. floridanum, or similar species that consume their entire host, should be considered useful surrogates in risk assessment of Bt crops to non-target arthropods.

Laboratory studies were conducted to investigate tritrophic transfer of insecticidal Cry proteins from transgenic cotton to an herbivore and its predator, and to examine effects of these proteins on the predator's development, survival, and reproduction. Cry1Ac and Cry2Ab proteins from the bacterium Bacillus thuringiensis (Bt) produced in Bollgard-II (BG-II, Event 15985) cotton plants were acquired by Thrips tabaci Lindeman (Thysanoptera: Thripidae), an important sucking pest of cotton, and its generalist predator, Orius insidiosus (Say) (Hemiptera: Anthocoridae). The average protein titers in BG-II cotton leaves were 1,256 and 43,637 ng Cry1Ac and Cry2Ab per gram fresh leaf tissue, respectively. At the second trophic level, larvae of T. tabaci reared on BG-II cotton for 48-96 h had 22.1 and 2.1% of the Cry1Ac and Cry2Ab levels expressed in leaves, respectively. At the third trophic level, O. insidiosus that fed on T. tabaci larvae had 4.4 and 0.3% of the Cry1Ac and Cry2Ab protein levels, respectively, expressed in BG-II plants. O. insidiosus survivorship, time of nymphal development, adult weight, preoviposition and postoviposition periods, fecundity, and adult longevity were not adversely affected owing to consumption of T. tabaci larvae that had fed on BG-II cotton compared with non-Bt cotton. Our results indicate that O. insidiosus, a common predator of T. tabaci, is not harmed by BG-II cotton when exposed to Bt proteins through its prey. Thus, O. insidiosus can continue to provide important biological control services in the cotton ecosystem when BG-II cotton is used to control primary lepidopteran pests.

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is an important pest of maize in the United States and many tropical areas in the western hemisphere. In 2001, Herculex I(®) (Cry1F) maize was commercially planted in the United States to control Lepidoptera, including S. frugiperda. In 2006, a population of S. frugiperda was discovered in Puerto Rico that had evolved resistance to Cry1F maize in the field, making it the first well-documented case of an insect with field resistance to a plant producing protein from Bacillus thuringiensis (Bt). Using this resistant population, we conducted tri-trophic studies with a natural enemy of S. frugiperda. By using resistant S. frugiperda, we were able to overcome possible prey-mediated effects and avoid concerns about potential differences in laboratory- or field-derived Bt resistance. We used the Cry1F-resistant S. frugiperda to evaluate effects of Cry1F on Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae), a larval endoparasitoid of S. frugiperda, over five generations. Our results clearly demonstrate that Cry1F maize does not affect development, parasitism, survivorship, sex ratio, longevity or fecundity of C. marginiventris when they parasitize Cry1F maize-fed S. frugiperda. Furthermore, the level of Cry1F protein in the leaves was strongly diluted when transferred from Bt maize to S. frugiperda and was not detected in larvae, cocoons or adults of C. marginiventris. Our results refute previous reports of C. marginiventris being harmed by Bt proteins and suggest that such results were caused by prey-mediated effects due to using Bt-susceptible lepidopteran hosts.

Geocoris punctipes (Say) and Orius insidiosus (Say) are generalist predators found in a wide range of crops, including cotton (Gossypium hirsutum L.) and maize (Zea mays L.), where they provide important biological control services by feeding on an array of pests, including eggs and small larvae of caterpillars. A high percentage of cotton and maize in the United States and several other countries are transgenic cultivars that produce one or more of the insecticidal Cry proteins of Bacillus thuringiensis Berliner (Bt). Here we quantify effects of three Cry proteins on the life history of these predators over two generations when they are exposed to these Cry proteins indirectly through their prey. To eliminate the confounding prey quality effects that can be introduced by Bt-susceptible prey, we used Cry1Ac/Cry2Ab-resistant Trichoplusia ni (Hübner) and Cry1 F-resistant Spodoptera frugiperda (J.E. Smith) in a series of tri-trophic studies. Survival, development, adult mass, fecundity, and fertility were similar when predators consumed larvae feeding on Cry1Ac/Cry2Ab cotton or Cry1 F maize compared with prey feeding on isogenic or near-isogenic cotton or maize. Repeated exposure of the same initial cohort over a second generation also resulted in no differences in life-history traits when feeding on non-Bt- or Bt-fed prey. Enzyme-linked immunosorbent assay showed that predators were exposed to Bt Cry proteins from their prey and that these proteins became increasingly diluted as they moved up the food chain. Results show a clear lack of effect of three common and widespread Cry proteins on these two important predator species. The use of resistant insects to eliminate prey quality effects provides a robust and meaningful assessment of exposure and hazard.

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.

Spodoptera frugiperda (JE Smith) represents the first documented case of field-evolved resistance to a genetically engineered crop expressing an insecticidal protein from Bacillus thuringiensis (Bt). In this case it was Cry1F-expressing maize (Mycogen 2A517). The ladybird beetle, Coleomegilla maculata, is a common and abundant predator that suppresses pest populations in maize and many other cropping systems. Its larvae and adults are polyphagous, feeding on aphids, thrips, lepidopteran eggs and larvae, as well as plant tissues. Thus, C. maculata may be exposed to Bt proteins expressed in genetically engineered crops by several pathways. Using Cry1F-resistant S. frugiperda larvae as prey, we evaluated the potential impact of Cry1F-expressing maize on several fitness parameters of C. maculata over two generations. Using Cry1F resistant prey removed any potential prey-mediated effects. Duration of larval and pupal stages, adult weight and female fecundity of C. maculata were not different when they were fed resistant S. frugiperda larvae reared on either Bt or control maize leaves during both generations. ELISA and insect-sensitive bioassays showed C. maculata were exposed to bioactive Cry1F protein. The insecticidal protein had no effect on C. maculata larvae, even though larvae contained 20-32 ng of Cry1F/g by fresh weight. Over all, our results demonstrated that the Cry1F protein did not affect important fitness parameters of one of S. frugiperda's major predators and that Cry1F protein did not accumulate but was strongly diluted when transferred during trophic interactions.

Transgenic cotton that produced one or more insecticidal proteins of Bacillus thuringiensis (Bt) was planted on over 15 million hectares in 11 countries in 2009 and has contributed to a reduction of over 140 million kilograms of insecticide active ingredient between 1996 and 2008. As a highly selective form of host plant resistance, Bt cotton effectively controls a number of key lepidopteran pests and has become a cornerstone in overall integrated pest management (IPM). Bt cotton has led to large reductions in the abundance of targeted pests and benefited non-Bt cotton adopters and even producers of other crops affected by polyphagous target pests. Reductions in insecticide use have enhanced biological control, which has contributed to significant suppression of other key and sporadic pests in cotton. Although reductions in insecticide use in some regions have elevated the importance of several pest groups, most of these emerging problems can be effectively solved through an IPM approach.