Safety assessment of transgenic Cry2Aa rice to a generalist predator, Paederus fuscipes Curtis (Coleoptera: Staphylinidae).

The insecticidal crystal proteins of Cry2A family from Bacillus thuringiensis (Bt) are important candidate proteins expressed in gene pyramiding Bt crops. A transgenic rice line (T2A-1) harboring a synthetic Cry2A* (Cry2Aa) gene showed effective resistance to some lepidopteran rice pests. As a generalist predator in rice ecosystems, the rove beetle (Paederus fuscipes) can prey on many rice insect pests such as planthoppers. Considering the possible exposure of Cry2Aa to P. fuscipes through tritrophic food chain, it is necessary to assess the potential risks of T2A-1 rice to this predator. In this study, a tritrophic experiment was conducted to assess the prey-mediated effects of Cry2Aa on P. fuscipes through the T2A-1 rice-Nilaparvata lugens-P. fuscipes food chain. After preying on N. lugens nymphs reared on T2A-1, no accumulated Cry2Aa could be detected in P. fuscipes adults, despite Cry2Aa being detected in N. lugens. In addition, no harmful effects were detected on the life table parameters of P. fuscipes in this tritrophic chain. Additionally, direct exposure to a high dose of purified Cry2Aa protein, representing the worst case scenario, showed no significant adverse effects on the development of P. fuscipes. These results showed that transgenic Cry2Aa rice had no harmful effects on P. fuscipes.

New insights into the infection of the American cockroach Periplaneta americana nymphs with Metarhizium anisopliae s.l. (Ascomycota: Hypocreales).

AIMS: To study the marked resistance of Periplaneta americana to entomopathogenic Metarhizium anisopliae. METHODS AND RESULTS: The low susceptibility of 4th instar nymphs applied topically with conidia seemed to be related to an active removal of conidia by the cockroach and to a disabled or retarded germination and subsequent development of conidia on the cuticle (up to 80% germination in the next 7 days after application). Inhibitions or delays of germination were related to the composition of the epicuticular fatty acids (30·1% w/w oleic, 28·3% w/w linoleic, 24·5% w/w palmitic and 11·7% w/w stearic acid) reported here. Propagules invading the nymphs through the cuticle took at least 3 days to reach the haemocoel, and no propagules were found after day 8 post-treatment. Strain IP 46 infected >50% of nymphs treated with doses ≥2 × 104  hyphal bodies (HB) nymph-1 and reduced the survival of nymphs ≤50%. Most nymphs (>70%) survived after injection of 6 × 103 and 2 × 103  HB nymph-1 . CONCLUSIONS: Findings emphasize a distinct resistance of nymphs of the American cockroach to infections by M. anisopliae. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings provide support for the development of biological control of this synanthropic cockroach pest.

Identification, characterization and expression of a receptor for the unusual myosuppressin in the blood-feeding bug, Rhodnius prolixus.

Myosuppressins are a family of the FMRFamide-like peptides. They have been characterized in many insects and shown to inhibit visceral muscle contraction. Rhodnius prolixus possesses an unusual myosuppressin in that the typical FLRFamide C-terminal motif is unique and ends with FMRFamide. In the present study, we isolated the cDNA sequence for the R. prolixus receptor for this unusual myosuppressin (RhoprMSR). Quantitative PCR indicates high relative transcript expression of RhoprMSR in the central nervous system and also supports the previously described physiological effects of RhoprMS on the digestive system, with expression of the RhoprMSR transcript in the midgut and hindgut. Expression of the RhoprMSR transcript was also found in the female and male reproductive system of 5th instar nymphs, with transcript expression greater in the female reproductive tissues. No expression was found in the salivary glands or Malpighian tubules. A functional receptor expression assay confirmed that the cloned RhoprMSR is indeed activated by RhoprMS (half maximum effective concentration = 42.7 nM). Structure-activity studies based upon both functional receptor assays and physiological assays showed the importance of the HVFMRFamide moiety, as further N-terminal truncation removed all activity.

Identification of the Alarm Pheromone of Hygia lativentris and Changes in Composition during Development.

Heteropteran insects produce a series of volatile compounds from their scent glands that protect them from predators and parasites. These compounds also play roles in chemical communication that elicit aggregation, dispersal, and mating behaviors. Hygia lativentris (Coreidae) adults frequently aggregate on host plants. When disturbed, they quickly disperse with the release of a sour smell, suggesting that these bugs possess an alarm pheromone in their secretions. This adult secretion-induced dispersal has been examined with a laboratory assay. Hexanal, the predominant component of the adult secretion was identified as a component of the alarm pheromone by evaluation of the adult bug's response time and escape distance from the chemical source. Physicochemical analyses with gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy revealed that secretory components differed between nymphs and adults, and also during adult aging. Nymphs produced two unsaturated compounds, (E)-2-hexenal and (E)-4-oxo-2-hexenal, together with hexanal and 1-hexanol, which were found in all developmental stages. In adults, hexyl acetate was the major component of secretions within 3 days of emerging, while the amount of this ester decreased and those of hexanal, hexanoic acid, and hexanal trimer increased with aging. The decomposition of hexyl acetate into hexanal via 1-hexanol was attributed to the presence of esterases and alcohol dehydrogenases specifically found in adult secretory glands. In contrast, the formation of a hexanal trimer may be due to a non-enzymatic reaction under acidic conditions.

Parent and halogenated polycyclic aromatic hydrocarbons in farmed cockroaches and implications for human exposure.

Medicinal insects have been widely used to cure human diseases for ages. Nevertheless, knowledge about the toxic chemicals accumulated in medicinal insects and their effects on human health was insufficient. In the present study, sixteen priority polycyclic aromatic hydrocarbons (PAHs) and nine halogenated PAHs (HPAHs) were determined in farmed medicinal cockroaches to address this issue. Total concentrations of PAHs in young nymphs, old nymphs, and adults ranged from 162 to 1025, 252 to 967, and 267 to 1168 ng/g, respectively. Levels of the sum of HPAHs varied from 0.84 to 9.17, 1.86 to 5.21, and 1.01 to 8.60 ng/g for young nymphs, old nymphs, and adults, respectively. The daily intake and excess cancer risk of PAHs and HPAHs were calculated for people who take cockroach-related drugs. Our results indicated that females and children have slightly higher exposure levels from the perspectives of gender and age, respectively. The estimated excess cancer risk of PAHs and HPAHs were both lower than the priority risk level (10(-4)), indicating a low potential carcinogenic risk with the medicinal cockroach consumption.

Cuticle Fatty Acid Composition and Differential Susceptibility of Three Species of Cockroaches to the Entomopathogenic Fungi Metarhizium anisopliae (Ascomycota, Hypocreales).

Differences in free fatty acids (FFAs) chemical composition of insects may be responsible for susceptibility or resistance to fungal infection. Determination of FFAs found in cuticular lipids can effectively contribute to the knowledge concerning insect defense mechanisms. In this study, we have evaluated the susceptibility of three species of cockroaches to the entomopathogenic fungi Metarhizium anisopliae (Metschnikoff) Sorokin by topical application. Mortality due to M. anisopliae was highly significant on adults and nymphs of Blattella germanica L. (Blattodea: Blattellidae). However, mortality was faster in adults than in nymphs. Adults of Blatta orientalis L. (Blattodea: Blattidae) were not susceptible to the fungus, and nymphs of Blaptica dubia Serville (Blattodea: Blaberidae) were more susceptible to the fungus than adults. The composition of cuticular FFAs in the three species of cockroaches was also studied. The analysis indicated that all of the fatty acids were mostly straight-chain, long-chain, saturated or unsaturated. Cuticular lipids of three species of cockroaches contained 19 FFAs, ranging from C14:0 to C24:0. The predominant fatty acids found in the three studied species of cockroaches were oleic, linoleic, palmitic, and stearic acid. Only in adults of Bl. orientalis, myristoleic acid, γ-linolenic acid, arachidic acid, dihomolinoleic acid, and behenic acid were identified. Lignoceric acid was detected only in nymphs of Bl. orientalis. Heneicosylic acid and docosahexaenoic acid were identified in adults of Ba. dubia.

Hot topic: Brown marmorated stink bug odor compounds do not transfer into milk by feeding bug-contaminated corn silage to lactating dairy cattle.

Brown marmorated stink bug (BMSB; Halyomorpha halys) is an emerging invasive species of grave concern to agriculture as a polyphagous plant pest with potential negative effects on the dairy industry. The purpose of this study was to determine the risk of including BMSB-contaminated silage in lactating dairy cow rations. First, 6 dairies, either highly infested (n=3; 30 to 100 bugs per stalk) or not infested (n=3), were sampled to assess the prevalence of bug secretion compounds tridecane (major component) and E-2-decenal (stink odor component) in silage and milk. Second, using wild BMSB, a mini-silo dose-response experiment (adding 100, 50, 25, 10, and 1 freshly crushed bugs/0.5kg of chopped corn) was conducted to assess the effect of ensiling on BMSB stink odor compounds. Finally, synthetic BMSB stink odor compounds (10g of tridecane and 5g of E-2-decenal) were ruminally infused twice daily over 3 d, and samples of milk, urine, and rumen fluid were collected to evaluate disposition. Bug stink odor compounds were sampled by solid-phase microextraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). Milk production and feed composition were unaffected when BMSB-contaminated silage was fed. Moreover, no E-2-decenal was detected in silage or milk (detection threshold = 0.00125µg/mL). The dose-response of tridecane in mini-silo samples exhibited a linear relationship (R(2)=0.78) with the amount of BMSB added; however, E-2-decenal was completely decomposed and undetectable in spiked mini-silos after ensiling. Both synthetic secretion compounds infused into rumen were undetectable in all milk and urine samples. E-2-Decenal was not detectable in rumen fluid, whereas tridecane was detected only at 15 min postinfusion but not present thereafter. Feed intake was unaffected by infusion treatment and BMSB secretion compounds (E-2-decenal and tridecane) were not observed in milk. E-2-Decenal and tridecane from the metathoracic gland of BMSB are not able to contaminate milk either due to the ensiling process or because of metabolism within the rumen. Concern over BMSB stink odor compounds contaminating the fluid milk supply, even on highly infested farms, is not warranted.

Defensive compounds and male-produced sex pheromone of the stink bug, Agroecus griseus.

Agroecus griseus is a serious corn pest in Brazil. Contents of the dorsal abdominal glands (DAGs) in nymphs, and the metathoracic gland (MTG) in adults of this species were characterized and quantified. Compounds found were similar to those of other Pentatomidae species and included aliphatic hydrocarbons, aldehydes, oxo-alkenals, and esters. However, two compounds were found in the MTG that have not been described previously for this family. Mass spectrometry, infrared spectroscopy, and gas chromatographic analysis using coinjection with authentic standards confirmed the identities of the compounds as enantiopure (S)-2-methylbutyl acetate and 3-methyl-2-butenyl acetate. The five nymphal instars showed significantly different ratios of components, mainly between those of the first and later instars. No significant differences were detected in MTG contents between sexes. Gas chromatography (GC) analysis of aeration extracts of males and females showed the presence of a compound released exclusively by males. Gas chromatography - electroantennographic detection (GC-EAD) assays indicated that the male-specific compound is bioactive in females, suggesting the presence of an attractant pheromone. The mass spectrum and infrared data for this compound matched with methyl 2,6,10-trimethyltridecanoate, a sex pheromone component previously detected in the stink bugs, Euschistus heros and E. obscurus. The synthetic standard coeluted with the natural pheromone on two different GC stationary phases, confirming the proposed structure. Y-tube olfactometer assays showed that the synthetic standard was strongly attractive to females, and GC-EAD tests produced responses with antennae from females similar to those of the natural pheromone.

Development of a mitochondrial 12S rDNA analysis for distinguishing Sciuridae species with potential to transmit Ehrlichia and Borrelia species to feeding Amblyomma americanum (Acari: Ixodidae).

Unique oligonucleotide probes were synthesized to distinguish among closely related vertebrate mitochondrial rDNA sequences present in residual bloodmeals in emergent Amblyomma americanum (L.) (Acari: Ixodidae) nymph life-stage ticks. Use of these probes enabled the identification of the Eastern gray squirrel as an important bloodmeal source in nymphs harboring Ehrlichia and Borrelia species. These results were confirmed by identifying these same bacterial genera in Eastern gray squirrel tissues.

Antibacterial activity of 4-oxo-(E)-2-hexenal from adults and nymphs of the heteropteran, Dolycoris baccarum (Heteroptera: Pentatomidae).

We identified 4-oxo-(E)-2-hexenal (4-OHE) as a common component of the secretion from both Dolycoris baccarum nymphs (66.5 ± 34.7 µg/bug) and adults (87.4 ± 48.0 µg/bug) by GC/MS. We also found that this compound inhibited the growth of bacteria starting at 10 µg. The stronger antibacterial activity of 4-OHE than that of (E)-2-hexenal and (E)-2-octenal might be explained by the reactivity of α,ß-unsaturated aldehydes with nucleophilic molecules.

An offspring signal of quality affects the timing of future parental reproduction.

Solicitation signals by offspring are well known to influence parental behaviour, and it is commonly assumed that this behavioural effect translates into an effect on residual reproduction of parents. However, this equivalence assumption concerning behavioural and reproductive effects caused by offspring signals remains largely untested. Here, we tested the effect of a chemical offspring signal of quality on the relative timing and amount of future reproduction in the European earwig (Forficula auricularia). We manipulated the nutritional condition of earwig nymphs and exposed females to their extract, or to solvent as a control. There were no significant main effects of exposure treatment on 2nd clutch production, but exposure to extracts of well-fed nymphs induced predictable timing of the 2nd relative to the 1st clutch. This result demonstrates for the first time that an offspring signal per se, in the absence of any maternal behaviour, affects maternal reproductive timing, possibly through an effect on maternal reproductive physiology.

Nutritional metabolic bone disease in juvenile veiled chameleons (Chamaeleo calyptratus) and its prevention.

Nutritional metabolic bone disease (NMBD) is one of the most frequently observed pathological conditions in herpetoculture. To develop guidelines for NMBD prevention in growing veiled chameleons (Chamaeleo calyptratus), 56 hatchlings were divided into 6 groups [group UV, with UVB exposure; group No: no supplements; group CaAUV: with calcium (Ca), vitamin A, UVB; group CaA: with Ca, vitamin A; group CaADUV: with Ca, vitamin A, cholecalciferol, UVB; and group CaAD, with Ca, vitamin A, cholecalciferol] and reared for 6 mo on locust-based diets. The nutrient composition of the locusts' diet and the locust-based diet for the chameleons was determined. The diagnosis included the detailed description of clinical findings, histopathology, measurements of serum Ca, 25-hydroxycholecalciferol (25-OHD(3)), liver 25-OHD(3), vitamin A, bone mineral density, and bone mineral concentration. Chameleons that received no dietary supplementation of Ca, vitamin A, and cholecalciferol developed NMBD. When Ca and vitamin A were supplemented, the chameleons did not develop NMBD, independently of additional UVB and dietary cholecalciferol. The best prevention for NMBD was achieved by chameleons that received locusts gut-loaded with 12% Ca and dusted with 250,000 IU/kg (75 mg/kg) vitamin A and 25,000 IU/kg (0.625 mg/kg) cholecalciferol plus provision of long (10 h/d), low irradiation exposure (3-120 µW/cm(2)) to UVB. Chameleons that were fed diets low in vitamin A, cholecalciferol, and Ca were diagnosed with fibrous osteodystrophy. We noticed an interaction of vitamin A and cholecalciferol supplementation in the storage of vitamin A in the liver and formation of colon calcifications. From these findings, recommendations for the rearing of juvenile chameleons were derived.

The juvenile hormone described in Rhodnius prolixus by Wigglesworth is juvenile hormone III skipped bisepoxide.

Juvenile hormones (JHs) are sesquiterpenoids synthesized by the corpora allata (CA). They play critical roles during insect development and reproduction. The first JH was described in 1934 as a "metamorphosis inhibitory hormone" in Rhodnius prolixus by Sir Vincent B. Wigglesworth. Remarkably, in spite of the importance of R. prolixus as vectors of Chagas disease and model organisms in insect physiology, the original JH that Wigglesworth described for the kissing-bug R. prolixus remained unidentified. We employed liquid chromatography mass spectrometry to search for the JH homologs present in the hemolymph of fourth instar nymphs of R. prolixus. Wigglesworth's original JH is the JH III skipped bisepoxide (JHSB3), a homolog identified in other heteropteran species. Changes in the titer of JHSB3 were studied during the 10-day long molting cycle of 4th instar nymph, between a blood meal and the ecdysis to 5th instar. In addition we measured the changes of mRNA levels in the CA for the 13 enzymes of the JH biosynthetic pathway during the molting cycle of 4th instar. Almost 90 years after the first descriptions of the role of JH in insects, this study finally reveals that the specific JH homolog responsible for Wigglesworth's original observations is JHSB3.

Ixodes scapularis saliva components that elicit responses associated with acquired tick-resistance.

Ticks and tick-borne diseases are on the rise world-wide and vaccines to prevent transmission of tick-borne diseases is an urgent public health need. Tick transmission of pathogens to the mammalian host occurs during tick feeding. Therefore, it is reasoned that vaccine targeting of tick proteins essential for feeding would thwart tick feeding and consequently prevent pathogen transmission. The phenomenon of acquired tick-immunity, wherein, repeated tick infestations of non-natural hosts results in the development of host immune responses detrimental to tick feeding has served as a robust paradigm in the pursuit of tick salivary antigens that may be vaccine targeted. While several salivary antigens have been identified, immunity elicited against these antigens have only provided modest tick rejection. This has raised the possibility that acquired tick-immunity is directed against tick components other than tick salivary antigens. Using Ixodes scapularis, the blacklegged tick, that vectors several human pathogens, we demonstrate that immunity directed against tick salivary glycoproteins is indeed sufficient to recapitulate the phenomenon of tick-resistance. These observations emphasize the utility of tick salivary glycoproteins as viable vaccine targets to thwart tick feeding and direct our search for anti-tick vaccine candidates.

Whole animal matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of ticks - Are spectra of Ixodes ricinus nymphs influenced by environmental, spatial, and temporal factors?

In the recent years matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a useful tool to characterize arthropod species and their different stages of development. It was reported for sand flies and mosquitoes at immature stages and also assumed for ticks that geographic location can have a subtle influence on MALDI-TOF mass spectra which allows the discrimination of animals with specific local variations of the MALDI-TOF MS phenotype. It is so far uncertain, however, if these mass-spectrometric differences are based on genetic variation or on spectral features which depend on environmental or temporal features. The aim of this study was to analyze the influence of the geographic location, environmental factors and the season of the year on the MALDI-TOF mass spectra of Ixodes (I.) ricinus nymphs and if spectral variation would allow to draw conclusions with respect to the tick's provenience or conditions that influence the tick life cycle. Application of multivariate statistical models on spectra of ticks collected in different seasons and different habitats and locations within Germany showed that the impact of the location seemed to be small while season and habitat seemed to have stronger impact on the MALDI-TOF mass spectra. Possibilities and limitations of MALDI-TOF mass spectra to draw conclusions on the tick life cycle are discussed.

Antibacterial/Antifungal Activity of Extracted Chitosan From American Cockroach (Dictyoptera: Blattidae) and German Cockroach (Blattodea: Blattellidae).

We investigated bactericidal and fungicidal properties of chitosan extracted from adults and nymphs from both German cockroach, Blattella germanica (Blattodea: Blattellidae) and American cockroach, Periplaneta americana (Dictyoptera: Blattidae). The cuticle of adults and nymphs extracted from both cockroaches were dried and ground. The powders were demineralized and deproteinized followed by deacetylation using NaOH. Finally, the chitosan yields were examined for antibacterial and antifungal activities. The degree of deacetylation (DD) was different between adults and nymph stages. The antimicrobial effect of American cockroach chitosan (ACC) and German cockroach chitosan (GCC) was tested against four bacteria and four fungi. The extracted chitosans from American cockroach, Periplaneta americana and German Cockroach, Blattella germanica suppressed the growth of Gram-negative/positive bacteria except Micrococcus luteus. The growth of Aspergillus flavus and Aspergillus niger were notability inhibited by the extracted chitosans. The antimicrobial effect of the chitosan depended on the cockroach species, with chitosan of the American cockroach showing more inhibitory effect. This difference may be due to differences in the structure of chitin between the two cockroach species.

Cultivar and insecticide applications affect the physiological development of the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

Nilaparvata lugens (Stål) (Hemiptera:Delphacidae) is a serious migratory pest of rice in Asia. Pest resurgence often occurs because of insecticide overuse. Using both susceptible (TN1) and moderately resistant (Xieyou 963) cultivars, we studied the effect of foliar insecticides on the percentage of brachypterous adults, female reproductive rate, and crude fat and soluble sugar contents in third- and fifth-instar nymphs and adults. The percentage of brachypterous adults and reproductive rate of adult females developed from nymphs that fed on insecticide-treated plants varied significantly with rice cultivar, type of insecticide, and its concentration. Feeding on susceptible plants increased the percentage of brachypterous adults and reproductive rate of adult females. Also, deltamethrin increased brachypterous production relative to imidacloprid and triazophos. The highest reproductive rate was on plants treated with triazophos. All insecticide treatments in both cultivars resulted in increase of soluble sugar contents in third- and fifth-instar nymphs and adults developed from nymphs feeding on insecticide-treated rice plants. This effect was stronger on the susceptible cultivar. Changes of crude fat content after N. lugens feeding on insecticide-treated plants were related to its feeding duration. Crude fat content in adult developed from nymphs feeding on treated plants was significantly higher that on control plants. These studies showed that plant and insecticide influences on physiological ecology of this planthopper will influence its population dynamics under commercial production of rice in Asia.

Miniaturized QuEChERS based methodology for multiresidue determination of pesticides in odonate nymphs as ecosystem biomonitors.

The impacts of the modern, agrochemicals based agriculture that threatens the overall systems sustainability, need to be monitored and evaluated. Seeking for agroecosystems monitors, the present article focus in the occurrence and abundance of aquatic macroinvertebrates, that have been frequently used as bioindicators of water quality due to their relationship with land use. Some of these organisms are on the top of the food chain, where bioaccumulation and biomagnification processes can be observed, and they can turn into secondary pollution sources of systems and terrestrial organisms as well. Odonate nymphs, which belong to the functional group of predators, were selected for this study. A methodology to determine 73 pesticide residues in odonate nymphs by LC-MS/MS and GC-MS/MS was developed. A QuEChERS sample preparation strategy was adapted. As it is complex to obtain samples especially in disturbed ecosystems, the method was minimized to a sample size of 200mg of fresh nymphs. The method was validated and good recoveries (71-120%) with RSDs below 20% for the majority of the studied pesticides at least at two of the assayed levels 1, 10 and 50µgkg-1 were obtained. For 32 analytes the limit of quantitation was 1µgkg-1 and 10µgkg-1 for the others. The lineal range was observed between 1-100µgkg-1 in matrix-matched and solvent calibration curves for most of the assessed pesticides. LC-MS/MS matrix effects were evaluated, 40% of the analytes presented low or no signal suppression. Only flufenoxuron presented high matrix effects. The obtained methodology is adequate for pesticide multiresidue analysis in aquatic macroinvertebrates (odonates) aiming to contribute to the ecological state evaluation of freshwater ecosystems.

Interaction between two aggregation chemical signals in Triatoma infestans (Hemiptera: Reduviidae).

The nymphs and adults of Triatoma infestans spend much of their time aggregated among themselves within narrow and dark shelters. The search for a suitable shelter depends in part on the recognition of chemical signals coming from the feces and the cuticle of the other individuals who use the refuge. The aim of this study was determine the possible interaction between the chemical signals associated to the feces and to the cuticle of T. infestans. The results showed that the insects remained significantly more time on the feces that had contact with legs and the feces plus footprints than feces or footprints alone, demonstrating the interaction between evaluated signals. These results demonstrates also that feces extracted a chemical stimulus from the legs. Understanding the interaction feces-legs as an interaction feces-cuticle of legs, the results suggest that the feces could extract some cuticular compound with activity on the behavior of the insects. This is the first report of the interaction between the two aggregation signals recognized in T. infestans and of the increase in the behavioral response of insects exposed to feces that had contact with a cuticular structure.

Transfer of Cry1Ac and Cry2Ab proteins from genetically engineered Bt cotton to herbivores and predators.

With the cultivation of Bt cotton, the produced insecticidal Cry proteins are ingested by herbivores and potentially transferred along the food chain to natural enemies, such as predators. In laboratory experiments with Bollgard II cotton, concentrations of Cry1Ac and Cry2Ab were measured in Lepidoptera larvae (Spodoptera littoralis, Heliothis virescens), plant bugs (Euschistus heros), aphids (Aphis gossypii), whiteflies (Bemisia tabaci), thrips (Thrips tabaci, Frankliniella occidentalis), and spider mites (Tetranychus urticae). Tritrophic experiments were conducted with caterpillars of S. littoralis as prey and larvae of ladybird beetles (Harmonia axyridis, Adalia bipunctata) and lacewings (Chrysoperla carnea) as predators. Immunological measurements (ELISA) indicated that herbivores feeding on Bt cotton contained 5%-50% of the Bt protein concentrations in leaves except whiteflies and aphids, which contained no or only traces of Bt protein, and spider mites, which contained 7 times more Cry1Ac than leaves. Similarly, predators contained 1%-30% of the Cry protein concentration in prey. For the nontarget risk assessment, this indicates that Bt protein concentrations decrease considerably from one trophic level to the next in the food web, except for spider mites that contain Bt protein concentrations higher than those measured in the leaves. Exposure of phloem sucking hemipterans is negligible.