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.     

Prey-mediated effects of mCry51Aa2-producing cotton on the predatory nontarget bug Orius majusculus (Reuter)

Genetically engineered (GE) cotton, MON 88702, is protected against certain sucking pests, such as plant bugs and thrips, by producing mCry51Aa2, a modified protein from Bacillus thuringiensis (Bt). Predatory pirate bugs (Orius spp.), natural enemies contributing to biological pest control, are also sensitive to the insecticidal protein when exposed continuously to high concentrations. We evaluated effects of MON 88702 on Orius majusculus when fed prey types with different mCry51Aa2 concentrations. When neonates were provided exclusively Tetranychus urticae spider mites reared on MON 88702 (high mCry51Aa2 content), adverse effects on predator survival and development were confirmed, compared with specimens fed prey from near-isogenic non-Bt cotton. When fed a mixture of T. urticae and Ephestia kuehniella eggs (mCry51Aa2-free), predator life table parameters were similar to the treatment where eggs were fed exclusively. When mCry51Aa2-containing spider mites were provided for a limited time at the beginning or the end of juvenile development, effects were less pronounced. While pirate bug nymphs showed similar consumption rates for prey from Bt and non-Bt cotton, choice experiments revealed a preference for E. kuehniella eggs over spider mites. Lepidopteran larvae (Spodoptera littoralis, high mCry51Aa2 content) or cotton aphids (Aphis gossypii, mCry51Aa2-free) reared on MON 88702 as alternative prey did not result in adverse effects on O. majusculus. Our study suggests limited risk of mCry51Aa2-producing cotton for O. majusculus, because its sensitivity for the Bt protein is relatively low and its natural food consists of diverse prey species with varying concentrations of Bt protein.     

Pest species diversity enhances control of spider mites and whiteflies by a generalist phytoseiid predator

To test the hypothesis that pest species diversity enhances biological pest control with generalist predators, we studied the dynamics of three major pest species on greenhouse cucumber: Western flower thrips, Frankliniella occidentalis (Pergande), greenhouse whitefly, Trialeurodes vaporariorum (Westwood), and two-spotted spider mites, Tetranychus urticae Koch in combination with the predator species Amblyseius swirskii Athias-Henriot. When spider mites infested plants prior to predator release, predatory mites were not capable of controlling spider mite populations in the absence of other pest species. A laboratory experiment showed that predators were hindered by the webbing of spider mites. In a greenhouse experiment, spider mite leaf damage was lower in the presence of thrips and predators than in the presence of whiteflies and predators, but damage was lowest in the presence of thrips, whiteflies and predators. Whitefly control was also improved in the presence of thrips. The lower levels of spider mite leaf damage probably resulted from (1) a strong numerical response of the predator (up to 50 times higher densities) when a second and third pest species were present in addition to spider mites, and (2) from A. swirskii attacking mobile spider mite stages outside or near the edges of the spider mite webbing. Interactions of spider mites with thrips and whiteflies might also result in suppression of spider mites. However, when predators were released prior to spider mite infestations in the absence of other pest species, but with pollen as food for the predators, we found increased suppression of spider mites with increased numbers of predators released, confirming the role of predators in spider mite control. Thus, our study provides evidence that diversity of pest species can enhance biological control through increased predator densities.     

Interactions in the biological control of western flower thrips Frankliniella occidentalis (Pergande) and two-spotted spider mite Tetranychus urticae Koch by the predatory bug Orius insidiosus Say on beans

An omnivore shows preference to its preys and thus its control efficiency could be altered in different mix infestation system. The efficiency of Orius insidiosus for biocontrol of either Frankliniella occidentalis or Tetranychus urticae alone or for the two pests in combinations was studied on beans. When only mites or thrips were offered as prey, 1 or 2 O. insidiosus could considerably suppress pest populations at an initial density of 20, 40, and 80 adult female mites, and 100 and 160 thrips larvae, respectively. A single O. insidiosus was able to reduce mite populations by 52.9, 38.7, and 25.8% at initial densities of 20, 40, and 80 mites, respectively, two bugs achieved control levels of 60.6, 63.1, and 38.4%. Releases of 1 and 2 O. insidiosus resulted in corrected mortalities of 62.5 and 87.9%, and 46.3 and 71.9% in F. occidentalis at initial larval densities of 100 and 160, respectively. When two pests were simultaneously offered, the efficiency of O. insidiosus in controlling T. urticae markedly decreased. Furthermore, mite control decreased with increasing T. urticae densities and was also affected by the density of O. insidiosus. The presence of mites at initial densities of 20–80 females did not significantly influence thrips control by O. insidiosus. The presence of F. occidentalis resulted in higher oviposition by O. insidiosus females than the presence of mites, indicating that thrips are a more suitable resource than T. urticae for O. insidiosus. The implications for biocontrol of F. occidentalis and T. urticae are discussed.     

Preliminary studies of an integrated control programme for cucumber pests and an evaluation of methods of introducing Phytoseiulus persimilis Athias-Henriot for the control of Tetranychus urticae Koch

Preliminary investigations suggest that, when Phytoseiulus persimilis is used to control Tetranychus urticae, the major pest of cucumbers, the control of other pests and powdery mildew can be effected by means of Encarsia formosa for the control of whitefly (Trialeurodes vaporariorum), bed drenches of gamma-BHC for thrips control and dimethirimol for the control of cucumber mildew (Erysiphe cichoracearum). The parasite Aphelinus flavipes failed to control an infestation of Aphis gossypii in one trial. A comparison of different methods of introducing Phytoseiulus confirmed that the best results were obtained when an even, artificial infestation of Tetranychus urticae was established on the crop before the introduction of the predator.     

Amount and time course of ingestion of plant subcellular fractions by two thrips and one reference mite species

Thrips are known as cell-feeding sucking pests, but little information is available on which and how much of each subcellular fraction they ingest. In this study, the ingested amounts of nuclei and plastids in two representative thrips species (the western flower thrips, Frankliniella occidentalis, and the garden thrips, Frankliniella intonsa) along with two reference sucking pests (the two-spotted spider mite, Tetranychus urticae, a cell feeder, and the brown planthopper, Nilaparvata lugens, a sap feeder) were quantified by quantitative PCR using phytoene desaturase and rubisco as respective marker genes following feeding. The ingested amounts of plastids were significantly greater than those of nuclei in the thrips and mite species. In the thrips species, however, the fold differences in ingested amount between the two fractions were substantially lower than their original ratio in intact plant cells, suggesting that thrips ingest nuclei more selectively than plastids. Unlike the thrips species, the ratio between nuclei and plastids increased in T. urticae. In contrast to these cell-feeding insects, no subcellular fraction was detected in N. lugens. These findings suggest that transgenic expression of foreign hairpin RNA in the nucleus would deliver a substantial amount of target molecules to cell-feeding sucking pests, but not sap-feeding pests, when employing ingestion RNA interference-based control strategies.     

Olfactory Cues Used in Host Selection by Frankliniella occidentalis (Thysanoptera: Thripidae) in Relation to Host Suitability

Olfactory responses of Frankliniella occidentalis (Pergande) to odors from six vegetable host plants-cabbage (Brassica oleracea), lettuce (Lactuca sativa), cucumber (Cucumis sativus), eggplant (Solanum melongena), celery (Apium graveolens) and garlic (Allium sativum) - were measured in choice tests in a Y-tube olfactometer, while host suitability of these same plants was assessed based on life table parameters-development time, survival rates, fertility, offspring sex ratio, female longevity, and an index of population change (I) - measured on leaf disks. Olfactory response was measured on both undamaged and mechanically-damaged plants. Regardless of plant damaged, female F. occidentalis responded positively to the odors of cabbage, lettuce, cucumber, and eggplant, but showed negative responses to celery and garlic, when offered a choice of plant odor or clean air. When female thrips were offered choices between each possible pairing of undamaged plants, or pairings of mechanically-damaged plants, the order of preference was cabbage = lettuce = cucumber > eggplant > celery = garlic. Male thrips responded differently, males responded positively only to undamaged lettuce and eggplant compared to clean air, while among mechanically-damaged plants only cucumber elicited a positive response. Given a choice between all possible pairings of undamaged plants, male thrips preferred lettuce to celery, and eggplant to garlic, whereas among pairings of mechanically-damaged plants male thrips preferred lettuce to celery, and cucumber to eggplant. The order of suitability of the six host plants based on life table parameters for F. occidentalis was essentially the same as the order of preference by female thrips. These results indicate that host plant odor is an important cue for female F. occidentalis in recognizing hosts suitable for oviposition. Information on plant attraction and repellency may be useful in thrips pest management. In addition, the correspondence of preference and performance that we found suggests that evolution of local host adaptation and, perhaps, host specialization, is possible.     

Isolation and Characterization of Cuticular Polyester from the Leaf of Nicotiana tabacum

Cuticular membranes (ca.0.04mg/cm²) were isolated from fresh and flue-cured tobacco leaves (Nicotiana tabacum) as clear, colorless and thin films by oxalic ammonium treatment and successive cellulase-pectinase digestion. GC-MS analyses, after hydrogenolysis and trimethylsilylation, revealed that the membranes are mainly built up of 10,16-dihydroxy hexadecanoic acid in a polyester network structure which survives postharvest-treatments.

The fruit skins of solanaceaes; sweet pepper (Capsicum annuum), eggplant (Solanum melongena) and tomato (Solanum lycopersicum), have similar membranes which are essentially composed of 9,16- and 10,16-dihydroxy hexadecanoic acid.     

Transfer of fungal tolerance through interspecific somatic hybridisation between Solanum melongena and S. torvum

Tolerance to Verticillium wilt is found in a wild relative of eggplant, Solanum torvum. To transfer this tolerance to eggplant (S. melongena), protoplast fusions between eggplant and irradiated S. torvum protoplasts were performed. Putative hybrids were regenerated and evaluated for Verticillium tolerance and field value. A total of 12 plants were eventually selected to be incorporated into the breeding program, as they showed a high degree of Verticillium tolerance under field conditions combined with the morphological characters of eggplant, including normal seed set. DNA analysis showed one of these tolerant plants to have a changed pattern, with some changes corresponding to specific pattern characteristics of S. torvum. Received: 12 April 1998 / Revision received: 9 September 1998 / Accepted: 29 November 1998     

Attraction of a generalist predator towards herbivore-infested plants

The occurrence and strength of interactions among natural enemies and herbivores depend on their foraging decisions, and several of these decisions are based on odours. To investigate interactions among arthropods in a greenhouse cropping system, we studied the behavioural response of the predatory bug Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) towards cucumber plants infested either with thrips (Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)) or with spider mites (Tetranychus urticae Koch (Acari: Tetranychidae)). In greenhouse release-recapture experiments, the predatory bug showed a significant preference for both thrips-infested plants and spider mite-infested plants over clean plants. Predatory bugs preferred plants infested with spider mites to plants with thrips. Experience with spider mites on cucumber leaves prior to their release in the greenhouse had no effect on the preference of the predatory bugs. However, this experience did increase the percentage of predators recaptured. Y-tube olfactometer experiments showed that O. laevigatus was more attracted to odours from plants infested with spider mites than to odours from clean plants. Thus, O. laevigatus is able to perceive odours and may use them to find plants with prey in more natural conditions. The consequences of the searching behaviour for pest control are discussed.     

Insecticidal properties of nonactic acid and homononactic acid,the precursors of macrotetrolide antibiotics

(±)-Nonactic acid (1) and (±)-homononactic acid (2), the non-antibiotic precursors of macrotetrolide antibiotics, showed significant insecticidal effects (comparable with those of the commercial synthetic pesticide Metathion) onLeptinotarsa decemlineata, Epilachna varivestis andEuproctis chrysorrhoea. Anisoplia austriaca, Aphis fabae andCalandra granaria were less sensitive in this respect. A low acaricidal activity againstTetranychus urticae (not reaching that of the commercial miticide Acarition) was also found. Both1 and2 exhibited growth-stimulating properties for plants.     

Altered host plant preference of Tetranychus urticae and prey preference of its predator Phytoseiulus persimilis (Acari: Tetranychidae, Phytoseiidae) on transgenic Cry3Bb-eggplants

The behavior of the two-spotted spider mite, Tetranychus urticae Koch and the predatory mite Phytoseiulus persimilis A.-H. was investigated in laboratory experiments with transgenic Bt-eggplants, Solanum melongena L., producing the Cry3Bb toxin and corresponding isogenic, non-transformed eggplants. In bitrophic experiments, dual-choice disc tests were conducted to reveal the effects of transgenic eggplants on host plant preference of T. urticae. Adult spider mite females were individually placed on leaf discs (2 cm diameter) and were observed during five days. Females occurred significantly more frequently on transgenic halves on which also significantly more T. urticae eggs were found. The effects of a Cry3Bb-eggplant fed prey on the feeding preference of P. persimilis were investigated in tritrophic experiments. Sixteen spider mite females, eight of which had been taken from transgenic and eight from isogenic eggplants, were offered to well-fed females of P. persimilis and numbers of respective spider mites consumed were registered 12 h later when the predators were offered new spider mites again. This procedure was repeated six times. The results revealed that predatory mites consumed significantly less Bt-fed spider mites than prey that had been raised on control eggplants. These results indicate that eggplants expressing the Cry3Bb toxin for resistance against the Colorado potato beetle are more preferred by spider mites but are less preferred by their predator P. persimilis. Possible consequences of these findings for biological control of spider mites on eggplants are discussed.     

Effects of Ocimum basilicum and Ruta chalepensis hydrosols on Aphis gossypii and Tetranychus urticae

Hydrosols are by‐products derived during the extraction of essential oils and are produced in a high amount and at very low cost. Their effects on crop pests have been little studied, although recent studies have shown their insecticidal properties. In this work, the effects of hydrosols derived from Ocimum basilicum (sweet basil) and Ruta chalepensis (rue) were investigated on the serious crop pests Aphis gossypii and Tetranychus urticae. The main compounds of basil hydrosol were linalool (66.5%), eugenol (18.9%) and eucalyptol (7.1%), while rue's were 2‐nonanone and 2‐undecanone at 77.0% and 8.9%, respectively. The mortality and fecundity of A. gossypii and T. urticae were recorded for 7 days after application of hydrosols. Apterous adult aphids and female mites were sprayed on cucumber leaves in special structures. Both hydrosols caused significant mortality rates, which fluctuated between 46.0% and 64.0%. The negative control (deionized water) caused no mortality, while for the positive controls (synthetic pesticides) was 100%. The fecundity of A. gossypii and T. urticae was significantly reduced after the application of the hydrosols (17.1 nymphs of A. gossypii and 22.8 eggs for T. urticae had been produced per female after treated with the hydrosol of R. chalepensis and O. basilicum, in comparison with 41.6 and 49.5 in the negative controls, respectively). In conclusion, the hydrosols from both plants presented an important effect on the mortality and fecundity of both pests.     

Leaf cuticular n-alkanes as markers in the chemotaxonomy of the eggplant (Solanum melongena L.) and related species

The complex of species formed by eggplant (Solanum melongena L.) and its wild and weedy relatives (mainly S. incanum L. and S. insanum L.) is characterised by an extreme morphological divergence that is not always associated with genetic variation. The taxonomy of so‐called ‘spiny Solanum’ species (subgenus Leptostemonum) is therefore extremely unclear. Cultivated eggplant lacks resistance to pests that frequently occur among the wild forms and species. As these wild plants are a potential gene pool for improvement of eggplant cultivars, knowledge of the characteristics of taxonomic relations between plants of different origin is crucial. We suggest using the leaf cuticular n‐alkane chain length distribution pattern as an alternative taxonomic marker for eggplant and related species. The results are in good agreement with current knowledge of the systematics of these plants; at the same time, the method developed here is useful for verifying plant identification based on morphological traits. Analysis of 13 eggplant cultivars, five accessions of S. incanum and two lines of S. macrocarpon enabled the intraspecific variation within eggplant to be assessed as low. There was wide variability among S. incanum accessions, probably because plants described as S. incanum are members of a number of different species. Some Asian accessions (sometimes described as S. insanum) were found to be almost identical to S. melongena, while a truly wild African S. incanum plant showed extensive similarity. The usefulness of the chemotaxonomic approach in dealing with the S. melongena–S. incanum complex is discussed.     

Population growth potential of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) on tomato,potato, and eggplant

The influence of host plant on population dynamics of an invasive pest, Tuta absoluta was studied on three economically important solanaceous crops. Experiments were conducted in laboratory (29 ± 0.5°C, 75 ± 5% RH and a photoperiod of 14:10 hr [L:D]) using tomato (Solanum lycopersicum L.), potato (Solanum tuberosum L.) and eggplant (Solanum melongena L.). Results indicated that intrinsic rate of increase (r), finite rate of increase (λ) and net reproductive rate (R₀) were higher, and mean generation time (T) was the shortest on tomato. Results suggested that T. absoluta developed on all the three plants, and tomato plant was most preferred one. Results suggested that T. absoluta has a potential to become a serious pest on potato and even on eggplant under favourable conditions. We used the life tables of 0.025th and 0.975th percentiles of bootstraps to project the uncertainty of population growth, a new concept.     

Biological control of broad mites (<Emphasis Type="Italic">Polyphagotarsonemus latus</Emphasis>) with the generalist predator <Emphasis Type="Italic">Amblyseius swirskii</Emphasis>

The broad mite is a serious pest of a variety of crops worldwide. Several phytoseiid mites have been described to control these mites. However, broad mites are still one of the major pest problems on greenhouse pepper in South-eastern Spain. The generalist predatory mite A. swirskii is widely used against other pests of pepper plants such as thrips and whiteflies, the latter being a vector of broad mites. We assessed the potential of A. swirskii to control broad mites. The oviposition rate of A. swirskii on a diet of broad mites was lower than on a diet of pollen, but higher than oviposition in the absence of food. Population-dynamical experiments with A. swirskii on single sweet pepper plants in a greenhouse compartment showed successful control of broad mites.     

Gut content analysis to study predatory efficacy of Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) by molecular methods

Nesidiocoris tenuis is considered to make significant contributions to the control of greenhouse pests such as whiteflies, thrips, leafminers, lepidopterans, and spider mites, although there is little information based on direct observation of the predation of N. tenuis on these target pests. We developed a method to perform gut content analysis of N. tenuis based on DNA in which specific PCR primers were designed to detect the DNA of target pests. By means of gut‐content analysis, we found that the percentage of N. tenuis preying on Bemisia tabaci and Thrips palmi was approximately 40% in the field.     

Salivary proteins of spider mites suppress defenses in Nicotiana benthamiana and promote mite reproduction

Spider mites (Tetranychidae sp.) are widely occurring arthropod pests on cultivated plants. Feeding by the two‐spotted spider mite T. urticae, a generalist herbivore, induces a defense response in plants that mainly depends on the phytohormones jasmonic acid and salicylic acid (SA). On tomato (Solanum lycopersicum), however, certain genotypes of T. urticae and the specialist species T. evansi were found to suppress these defenses. This phenomenon occurs downstream of phytohormone accumulation via an unknown mechanism. We investigated if spider mites possess effector‐like proteins in their saliva that can account for this defense suppression. First we performed an in silico prediction of the T. urticae and the T. evansi secretomes, and subsequently generated a short list of candidate effectors based on additional selection criteria such as life stage‐specific expression and salivary gland expression via whole mount in situ hybridization. We picked the top five most promising protein families and then expressed representatives in Nicotiana benthamiana using Agrobacterium tumefaciens transient expression assays to assess their effect on plant defenses. Four proteins from two families suppressed defenses downstream of the phytohormone SA. Furthermore, T. urticae performance on N. benthamiana improved in response to transient expression of three of these proteins and this improvement was similar to that of mites feeding on the tomato SA accumulation mutant nahG. Our results suggest that both generalist and specialist plant‐eating mite species are sensitive to SA defenses but secrete proteins via their saliva to reduce the negative effects of these defenses.     

Biological control of thrips and whiteflies by a shared predator: Two pests are better than one

We studied the capacity of one species of predator to control two major pests of greenhouse crops, Western flower thrips (Frankliniella occidentalis (Pergande)) and the greenhouse whitefly (Trialeurodes vaporariorum (Westwood)). In such a one-predator–two-prey system, indirect interactions can occur between the two pest species, such as apparent competition and apparent mutualism. Whereas apparent competition is desired because it brings pest levels down, apparent mutualism is not, because it does the opposite. Because apparent competition and apparent mutualism occurs at different time scales, it is important to investigate the effects of a shared natural enemy on biological control on a time scale relevant for crop growth. We evaluated the control efficacy of the predatory mites Amblyseius swirskii (Athias-Henriot) and Euseius ovalis (Evans) in cucumber crops in greenhouse compartments with only thrips, only whiteflies or both herbivorous insects together. Each of the two predators controlled thrips, but A. swirskii reduced thrips densities the most. There was no effect of the presence of whiteflies on thrips densities. Whitefly control by each of the two predators in absence of thrips was not sufficient, yet better with E. ovalis. However, whitefly densities in presence of thrips were reduced dramatically, especially by A. swirskii. The densities of predators were up to 15 times higher in presence of both pests than in the single-pest treatments. Laboratory experiments with A. swirskii suggest that this is due to a higher juvenile survival and developmental rate on a mixed diet. Hence, better control may be achieved not only because of apparent competition, but also through a positive effect of mixed diets on predator population growth. This latter phenomenon deserves more attention in experimental and theoretical work on biological control and apparent competition.