Author Correction: A transgenic approach for controlling Lygus in cotton.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Quantitative Differences in Feeding Behavior of Lygus lineolaris (Hemiptera: Miridae) on Transgenic and Nontransgenic Cotton.

Lygus lineolaris (Palisot de Beauvois) is one of the most important pests on cotton in the United States. Previous research showed that transgenic cotton plants expressing the Bacillus thurigiensis (Bt) crystalline protein Cry51Aa2.834_16 (designated MON 88702) have insecticidal effects on nymphal L. lineolaris. The present study is the first to examine effects of a Bt-expressing cotton on feeding by a heteropteran like L. lineolaris. We compared stylet probing behaviors of third-instar nymphs on pin-head squares (i.e., buds <3 mm wide) of MON 88702 cotton versus nontransgenic (control) DP393 plants using AC-DC electropenetrography. Waveforms were quantified based on appearances previously characterized and correlated with adult L. lineolaris feeding behaviors; nymphal and adult waveforms had the same appearance. Generalized third-instar feeding included maceration of tissues during cell rupturing (waveform CR), tasting/testing during a waveform called transition (T), and ingestion (I); all were similar between MON 88702 and DP393 plants. However, the number of events and duration of each waveform were different between treatments. Relative to nymphs on DP393, those on MON 88702 spent more time overall in stylet probing, due to increased number of maceration events per probe and longer durations of tasting/testing, per waveform event, per probe, and per insect; yet, ingestion events were shorter and more frequent. These findings support that MON 88702 cotton plants were less palatable and/or preorally digestible to L. lineolaris nymphs than DP393, suggesting antixenosis for MON 88702. Transgenic cotton antixenosis could positively affect cotton pest management by reducing feeding of L. lineolaris nymphs and protecting crop yield.

First transgenic trait for control of plant bugs and thrips in cotton.

BACKGROUND: Plant bugs (Lygus spp.) and thrips (Thrips spp.) are two of the most economically important insect pest groups impacting cotton production in the USA today, but are not controlled by current transgenic cotton varieties. Thus, seed or foliar-applied chemical insecticides are typically required to protect cotton from these pest groups. Currently, these pests are resistant to several insecticides, resulting in fewer options for economically viable management. Previous publications documented the efficacy of transgenic cotton event MON 88702 against plant bugs and thrips in limited laboratory and field studies. Here, we report results from multi-location and multi-year field studies demonstrating efficacy provided by MON 88702 against various levels of these pests. RESULTS: MON 88702 provided a significant reduction in numbers of Lygus nymphs and subsequent yield advantage. MON 88702 also had fewer thrips and minimal injury. The level of control demonstrated by this transgenic trait was significantly better compared with its non-transgenic near-isoline, DP393, receiving insecticides at current commercial rates. CONCLUSION: The level of efficacy demonstrated here suggests that MON 88702, when incorporated into existing IPM programs, could become a valuable additional tool for management of Lygus and thrips in cotton agroecosystems experiencing challenges of resistance to existing chemical control strategies. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Certain applied electrical signals during EPG cause negative effects on stylet probing behaviors by adult Lygus lineolaris (Hemiptera: Miridae).

This study is the first to fully evaluate whether electrical signals applied to large insects during electropenetrography (EPG; also called electrical penetration graph) negatively affect insect behavior. During EPG, electrical signals are applied to plants, and thus to the gold-wire-tethered insects feeding on them. The insect completes an electrical circuit whose changes in voltage reflect the insect's stylet probing/penetration behaviors, recorded as waveform output. For nearly 50 years of EPG science, evidence has supported that there are no or negligible effects on tiny insects from applied electricity during EPG. Recently however, EPG studies of large-bodied hemipterans such as heteropterans and sharpshooter leafhoppers have been published. The wider stylet diameters of such large insects cause them to have lower inherent resistances to applied signals compared with smaller insects, conveying more electrical current. The present study asked whether such increased currents would affect insect stylet probing, by comparing Lygus lineolaris behaviors on pin-head cotton squares using an AC-DC electropenetrograph. Effects of AC or DC applied signals were separately examined in two factorial studies, each comparing four input resistor (Ri) levels (106, 107, 108 and 109 Ω) and four applied voltage levels (2, 60, 150 and 250 mV). Results showed that changes in both probing and non-probing behaviors were indeed caused by changing signal type, Ri level, or applied voltage. Negative effects on feeding were numerically greater overall for DC than AC applied signals, perhaps due to muscular tetany from DC; however, AC versus DC could not be statistically tested. Results strongly support the need for flexible Ri and applied voltage levels and types, to tailor instrument settings to the size and special needs of each insect subject. Our findings will facilitate further EPG studies of Lygus spp., such as host plant resistance or insecticidal assays/bioassays to assess mode of action and appropriate dosage. It is hoped that this study will also inform EPG studies of similar, large heteropterans in the future.

Correlation of Electropenetrography Waveforms From Lygus lineolaris (Hemiptera: Miridae) Feeding on Cotton Squares With Chemical Evidence of Inducible Tannins.

Probing behavior of Lygus lineolaris (Palisot de Beauvois) has previously been characterized with electropenetrography (EPG). Cell rupturing (CR) and ingestion (I) EPG waveforms were identified as the two main stylet-probing behaviors by adult L. lineolaris. However, characterization and identification of EPG waveforms are not complete until specific events of a particular waveform are correlated to insect probing. With the use of EPG, histology, microscopy, and chemical analysis, probing behavior of L. lineolaris on pin-head cotton squares was studied. Occurrences of waveforms CR and I were artificially terminated during the EPG recording. Histological samples of probed cotton squares were prepared and analyzed to correlate specific types and occurrences of feeding damage location and plant responses to insect feeding. Both CR and I occurred in the staminal column of the cotton square. Cell rupturing events elicited the production of dark-red deposits seen in histological staining that were demonstrated via chemical analysis to contain condensed tannins. We hypothesize that wounding and saliva secreted during CR triggered release of tannins, because tannin production was positively correlated with the number of probes with single CR events performed by L. lineolaris. Degraded plant tissue and tannins were removed from the staminal column during occurrence of waveform I. These results conclude the process of defining CR and I as probing waveforms performed by L. lineolaris on pin-head cotton squares. These biological definitions will now allow EPG to be used to quantitatively compare L. lineolaris feeding among different plant treatments, with the goal of improving pest management tactics against this pest.

Characterization of the Activity Spectrum of MON 88702 and the Plant-Incorporated Protectant Cry51Aa2.834_16.

The spectrum of insecticidal activity of Cry51Aa2.834_16 protein targeting hemipteran and thysanopteran insect pests in cotton was characterized by selecting and screening multiple pest and non-pest species, based on representation of ecological functional groups, taxonomic relatedness (e.g. relationship to species where activity was observed), and availability for effective testing. Seven invertebrate orders, comprising 12 families and 17 representative species were screened for susceptibility to Cry51Aa2.834_16 protein and/or the ability of the protein to protect against feeding damage in laboratory, controlled environments (e.g. greenhouse/growth chamber), and/or field studies when present in cotton plants. The screening results presented for Cry51Aa2.834_16 demonstrate selective and limited activity within three insect orders. Other than Orius insidiosus, no activity was observed for Cry51Aa2.834_16 against several groups of arthropods that perform key ecological roles in some agricultural ecosystems (e.g. pollinators, decomposers, and natural enemies).

A transgenic approach for controlling Lygus in cotton.

Lygus species of plant-feeding insects have emerged as economically important pests of cotton in the United States. These species are not controlled by commercial Bacillus thuringiensis (Bt) cotton varieties resulting in economic losses and increased application of insecticide. Previously, a Bt crystal protein (Cry51Aa2) was reported with insecticidal activity against Lygus spp. However, transgenic cotton plants expressing this protein did not exhibit effective protection from Lygus feeding damage. Here we employ various optimization strategies, informed in part by protein crystallography and modelling, to identify limited amino-acid substitutions in Cry51Aa2 that increase insecticidal activity towards Lygus spp. by >200-fold. Transgenic cotton expressing the variant protein, Cry51Aa2.834_16, reduce populations of Lygus spp. up to 30-fold in whole-plant caged field trials. One transgenic event, designated MON88702, has been selected for further development of cotton varieties that could potentially reduce or eliminate insecticide application for control of Lygus and the associated environmental impacts.

Efficacy of Bauveria bassiana for red flour beetle when applied with plant essential oils or in mineral oil and organosilicone carriers.

The carriers mineral oil and Silwet L-77 and the botanical insecticides Neemix 4.5 and Hexacide were evaluated for their impacts on the efficacy of Beauveria bassiana (Balsamo) Vuillemin conidia against red flour beetle, Tribolium castaneum (Herbst), larvae. The dosages of liquid treatments were quantified by both conidia concentration in the spray volume and conidia deposition on the target surface. The latter approach allowed comparison with dry, unformulated conidia. The median lethal concentrations of B. bassiana in 0.05% Silwet L-77 solution or without a carrier were approximately double that for conidia in mineral oil. Carriers had highly significant effects on the efficacy of B. bassiana. The lower efficacy of conidia in aqueous Silwet L-77 may have been the result of conidia loss from the larval surface because of the siloxane's spreading properties. Neemix 4.5 (4.5% azadirachtin) delayed pupation and did not reduce the germination rate of B. bassiana conidia, but it significantly reduced T. castaneum mortality at two of four tested fungus doses. Hexacide (5% rosemary oil) caused significant mortality when applied without B. bassiana, but it did not affect pupation, the germination rate of conidia, or T. castaneum mortality when used in combination with the fungus.

Diatomaceous earth increases the efficacy of Beauveria bassiana against Tribolium castaneum larvae and increases conidia attachment.

This research tested the suppressive ability of Beauveria bassiana (Balsamo) Vuillemin alone and in combination with diatomaceous earth against the red flour beetle, Tribolium castaneum (Herbst). Adults did not show a dose response to B. bassiana, and the addition of diatomaceous earth (DE) did not result in a significant increase in mortality. Against larvae, however, DE at 190 mg/kg grain enhanced the efficacy of B. bassiana at all concentrations ranging from 33 to 2,700 mg of conidia per kilogram of grain. The presence of DE resulted in 17- and 16-fold decreases in the median lethal concentration of B. bassiana at 56 and 75% RH, respectively. No significant differences in larval mortality in response to B. bassiana and diatomaceous earth alone or in combination were found between 56 and 75% RH. Conidial attachment to larvae was significantly greater with 190 mg/kg DE than without it. The partial analysis of lipids taken up by DE from the larvae revealed the removal of phospholipids and long-chain fatty acids. These results support the hypothesis that diatomaceous earth enhances the efficacy of B. bassiana against larval T. castaneum, at least in part by damaging the insect cuticle, thus increasing conidial attachment and making nutrients more available to conidia for their germination.