Effectiveness and residual activity of four common insecticides used in the Mississippi Delta to control tarnished plant bugs in cotton.

The tarnished plant bug, (TPB) Lygus lineolaris Palisot de Beauvois (Hemiptera: Miridae) is a key pest of cotton in the midsouth region and some areas of the eastern United States. Its control methods have been solely based on chemical insecticides which has contributed to insecticidal resistance and shortened residual periods for control of this insect pest. This study was conducted over a two-year period and examined the efficacy and residual effect of four commercial insecticides including lambda-cyhalothrin (pyrethroid), acephate (organophosphate), imidacloprid (neonicotinoid), and sulfoxaflor (sulfoxamine). The effectiveness and residual effects of these insecticides were determined by application on cotton field plots on four different dates during each season using three different concentrations (high: highest labeled commercial dose (CD), medium: 1/10 of the CD, low: 1/100 of the CD) on field cotton plots. Four groups of cotton leaves were randomly pulled from each treated plot and control 0-, 2-, 4-, 7-, and 9-days post treatment (DPT) and exposed to a lab colony of TPB adults. One extra leaf sample/ plot/ spray /DPT interval (0-2-4-7-9-11) during 2016 was randomly collected from the high concentration plots and sent to Mississippi State Chemical Laboratory for residual analysis. Mortality of TPB adults was greatest for those placed on leaves sprayed with the organophosphate insecticide with mortalities (%) of 81.7±23.4 and 63.3±28.8 (SE) 1-day after exposure (DAE) on leaves 0-DPT with the high concentration for 2016 and 2017, respectively, reaching 94.5±9.5 and 95.4±7.6 6-DAE each year. Mortality to all insecticides continued until 9 and 4-DPT for high and medium concentrations, respectively. However, organophosphate (39.4±28.6) and pyrethroid (24.4±9.9) exhibited higher mortality than sulfoxamine (10.6±6.6) and the neonicotinoid (4.0±1.5) 7-DAE on 9-DPT leaves with the high concentration. Based on our results using the current assay procedure, TPB adults were significantly more susceptible to contact than systemic insecticides and due to its residual effect, organophosphate could kill over 80% of the TPB population 7-DPT.

Sublethal effects of a commercial Bt product and Bt cotton flowers on the bollworm (Helicoverpa zea) with impacts to predation from a lady beetle (Hippodamia convergens).

Insecticidal Bacillus thuringiensis Berliner (Bt) toxins produced by transgenic cotton (Gossypium hirsutum L.) plants have become an essential component of cotton pest management. Bt toxins are the primary management tool in transgenic cotton for lepidopteran pests, the most important of which is the bollworm (Helicoverpa zea Boddie) (Lepidoptera: Noctuidae) in the United States (U.S.). However, bollworm larvae that survive after consuming Bt toxins may experience sublethal effects, which could alter interactions with other organisms, such as natural enemies. Experiments were conducted to evaluate how sublethal effects of a commercial Bt product (Dipel) incorporated into artificial diet and from Bt cotton flowers impact predation from the convergent lady beetle (Hippodamia convergens Guérin-Méneville) (Coleoptera: Coccinellidae), common in cotton fields of the mid-southern U.S. Sublethal effects were detected through reduced weight and slower development in bollworm larvae which fed on Dipel incorporated into artificial diet, Bollgard II, and Bollgard 3 cotton flowers. Sublethal effects from proteins incorporated into artificial diet were found to significantly alter predation from third instar lady beetle larvae. Predation of bollworm larvae also increased significantly after feeding for three days on a diet incorporated with Bt proteins. These results suggest that the changes in larval weight and development induced by Bt can be used to help predict consumption of bollworm larvae by the convergent lady beetle. These findings are essential to understanding the potential level of biological control in Bt cotton where lepidopteran larvae experience sublethal effects.

Evaluating Acheta domesticus (Orthoptera: Gryllidae) for the reduction of fumonisin B1 levels in livestock feed.

Mycotoxins that contaminate grain can cause the devaluation of agricultural products and create health risks for the consumer. Fumonisins are one such mycotoxin. Produced primarily by Fusarium verticillioides (Hypocreales: Nectriaceae) (Nirenberg, 1976) on corn, fumonisins' economic impact can be significant by causing various diseases in livestock if contaminated corn is not monitored and removed from animal feed. Finding safe alternatives to the destruction and waste of contaminated grain and restoring its economic value is needed for a sustainable future. Safe reintroduction into the farm food web may be possible through a consumable intermediary such as insects. This study demonstrates the suitability of the house cricket, Acheta domesticus L., as an alternative protein source in domestic animal feed by quantifying fumonisin B1 (FB1) levels in their subsequent insect meal and frass. Small colonies of 2nd instar A. domesticus were reared to 5th instar adults on nutrient-optimized corn-based diets treated with 4 levels of FB1 from 0 to 20 ppm. Increasing levels of FB1 had no adverse effects on the survivorship or growth of A. domesticus. Insect meals prepared from A. domesticus had significantly lower levels of FB1, at 3%-5% of their respective diets, while frass did not differ significantly from their diet. The successful rearing to adulthood of A. domesticus on fumonisin-contaminated diet paired with lower levels of FB1 in their processed insect meal supports the idea that more sustainable agricultural practices can be developed through remediation of low-value mycotoxin-contaminated grain with safer, higher-value insects as livestock feed components.

A new software for automated counting of glistenings in intraocular lenses in vivo.

AIM: To assess the performance of a bespoke software for automated counting of intraocular lens (IOL) glistenings in slit-lamp images. METHODS: IOL glistenings from slit-lamp-derived digital images were counted manually and automatically by the bespoke software. The images of one randomly selected eye from each of 34 participants were used as a training set to determine the threshold setting that gave the best agreement between manual and automatic grading. A second set of 63 images, selected using randomised stratified sampling from 290 images, were used for software validation. The images were obtained using a previously described protocol. Software-derived automated glistenings counts were compared to manual counts produced by three ophthalmologists. RESULTS: A threshold value of 140 was determined that minimised the total deviation in the number of glistenings for the 34 images in the training set. Using this threshold value, only slight agreement was found between automated software counts and manual expert counts for the validating set of 63 images (κ=0.104, 95%CI, 0.040-0.168). Ten images (15.9%) had glistenings counts that agreed between the software and manual counting. There were 49 images (77.8%) where the software overestimated the number of glistenings. CONCLUSION: The low levels of agreement show between an initial release of software used to automatically count glistenings in in vivo slit-lamp images and manual counting indicates that this is a non-trivial application. Iterative improvement involving a dialogue between software developers and experienced ophthalmologists is required to optimise agreement. The results suggest that validation of software is necessary for studies involving semi-automatic evaluation of glistenings.

Combining visual cues and pheromone blends for monitoring and management of the tarnished plant bug Lygus lineolaris (Hemiptera: Miridae).

BACKGROUND: The tarnished plant bug Lygus lineolaris (Palisot de Beauvois) is considered the most damaging pest of cotton (Gossypium hirsutum L.) in the mid-southern United States. Previous studies have reported the role of different ratios of volatile metathoracic gland components such as hexyl butyrate, (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal in eliciting low-level attraction of L. lineolaris. In this study, we tested different visual cues (colored sticky cards) in combination with olfactory cues (pheromone blends) to optimize the attraction and capture of L. lineolaris in the field. RESULTS: Red-colored sticky cards were more attractive to L. lineolaris adults than white, blue or yellow cards. Red sticky cards combined with blends of three potential pheromone components attracted significantly more L. lineolaris adults than sticky cards without a blend added. Traps baited with a blend of hexyl butyrate, (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal in 4:10:7 ratio, respectively, caught a significantly higher number of L. lineolaris than those baited with 10:4:2 or 7:10:4 blends or an unbaited control in the first week of the experiment. CONCLUSIONS: Combining visual cues (red color) with olfactory cues (pheromone blends) significantly increased the capture of L. lineolaris in the field. This device or a future iteration could contribute towards sustainable and environmentally appropriate early-season monitoring and management of L. lineolaris in the field. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Practical resistance to Cry toxins and efficacy of Vip3Aa in Bt cotton against Helicoverpa zea.

BACKGROUND: Crops genetically engineered to make insect-killing proteins from Bacillus thuringiensis (Bt) have revolutionized management of some pests. However, the benefits of such transgenic crops are reduced when pests evolve resistance to Bt toxins. We evaluated resistance to Bt toxins and Bt cotton plants using laboratory bioassays and complementary field trials focusing on Helicoverpa zea, one of the most economically important pests of cotton and other crops in the United States. RESULTS: The data from 235 laboratory bioassays demonstrate resistance to Cry1Ac, Cry1Fa, and Cry2Ab occurred in most of the 95 strains of H. zea derived from Arkansas, Louisiana, Mississippi, Tennessee, and Texas during 2016 to 2021. Complementary field data show efficacy decreased for Bt cotton producing Cry1Ac + Cry1Fa or Cry1Ac + Cry2Ab, but not Cry1Ac + Cry1Fa + Vip3Aa. Moreover, analysis of data paired by field site and year shows higher survival in bioassays was generally associated with lower efficacy of Bt cotton. CONCLUSIONS: The results confirm and extend previous evidence showing widespread practical resistance of H. zea in the United States to the Cry toxins produced by Bt cotton and corn, but not to Vip3Aa. Despite deployment in combination with Cry toxins in Bt crops, Vip3Aa effectively acts as a single toxin against H. zea larvae that are highly resistant to Cry toxins. Furthermore, Vip3Aa adoption is increasing and previous work provided an early warning of field-evolved resistance. Thus, rigorous resistance management measures are needed to preserve the efficacy of Vip3Aa against this highly adaptable pest. © 2022 Society of Chemical Industry.

Field studies on the deterioration of microplastic films from ultra-thin compostable bags in soil.

In recent years, some countries have replaced single-use plastic bags with bags manufactured from compostable plastic film that can be used for collecting food wastes and composted together with the waste. Because industrial compost contains undeteriorated fragments of these bags, application to field soil is a potential source of small-sized residues from these bags. This study was undertaken to examine deterioration of these compostable film microplastics (CFMPs) in field soil at three different localities in Italy. Deterioration of CFMPs did not exceed 5.7% surface area reduction during the 12-month experimental period in two sites located in Northern Italy. More deterioration was observed in the Southern site, with 7.2% surface area reduction. Deterioration was significantly increased when fields were amended with industrial compost (up to 9.6%), but not with home compost. Up to 92.9% of the recovered CFMPs were associated with the soil fungus Aspergillus flavus, with 20.1%-71.2% aflatoxin-producing isolates. Application of industrial compost resulted in a significant increase in the percentage of CFMPs associated with A. flavus. This observation provides an argument for government regulation of accumulation of CFMPs and elevation of hazardous fungi levels in agricultural soils that receive industrial compost.

Genetic Knockouts Indicate That the ABCC2 Protein in the Bollworm Helicoverpa zea Is Not a Major Receptor for the Cry1Ac Insecticidal Protein.

Members of the insect ATP binding cassette transporter subfamily C2 (ABCC2) in several moth species are known as receptors for the Cry1Ac insecticidal protein from Bacillus thuringiensis (Bt). Mutations that abolish the functional domains of ABCC2 are known to cause resistance to Cry1Ac, although the reported levels of resistance vary widely depending on insect species. In this study, the function of the ABCC2 gene as a putative Cry1Ac receptor in Helicoverpa zea, a major pest of over 300 crops, was evaluated using CRISPR/Cas9 to progressively eliminate different functional ABCC2 domains. Results from bioassays with edited insect lines support that mutations in ABCC2 were associated with Cry1Ac resistance ratios (RR) ranging from 7.3- to 39.8-fold. No significant differences in susceptibility to Cry1Ac were detected between H. zea with partial or complete ABCC2 knockout, although the highest levels of tolerance were observed when knocking out half of ABCC2. Based on >500-1000-fold RRs reported in similar studies for closely related moth species, the low RRs observed in H. zea knockouts support that ABCC2 is not a major Cry1Ac receptor in this insect.

Early Warning of Resistance to Bt Toxin Vip3Aa in Helicoverpa zea.

Evolution of resistance by pests can reduce the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Because of the widespread resistance of Helicoverpa zea to crystalline (Cry) Bt toxins in the United States, the vegetative insecticidal protein Vip3Aa is the only Bt toxin produced by Bt corn and cotton that remains effective against some populations of this polyphagous lepidopteran pest. Here we evaluated H. zea resistance to Vip3Aa using diet bioassays to test 42,218 larvae from three lab strains and 71 strains derived from the field during 2016 to 2020 in Arkansas, Louisiana, Mississippi, Tennessee, and Texas. Relative to the least susceptible of the three lab strains tested (BZ), susceptibility to Vip3Aa of the field-derived strains decreased significantly from 2016 to 2020. Relative to another lab strain (TM), 7 of 16 strains derived from the field in 2019 were significantly resistant to Vip3Aa, with up to 13-fold resistance. Susceptibility to Vip3Aa was significantly lower for strains derived from Vip3Aa plants than non-Vip3Aa plants, providing direct evidence of resistance evolving in response to selection by Vip3Aa plants in the field. Together with previously reported data, the results here convey an early warning of field-evolved resistance to Vip3Aa in H. zea that supports calls for urgent action to preserve the efficacy of this toxin.

Termite Presence and Feeding on Loblolly Pine Wood Differs Among Four Root-Infecting Bluestain (ophiostomatoid) Fungal Species.

Bark beetles and root weevils can impact forests through tree death on landscape scales. Recently, subterranean termites have been linked to these beetles via the presence of bluestain fungi (Ascomycota: Ophiostomataceae), which are vectored to trees by beetles. However, only a small subset of bluestain species have been examined. Here, we tested whether termite-bluestain association patterns in the field reflect termite feeding preference in laboratory choice trials. We documented the presence of four bluestain fungi (Leptographium procerum (W.B. Kendr.), L. terebrantis (Barras & Perry), Grosmannia huntii (Rob.-Jeffr.), and G. alacris (T.A. Duong, Z.W. de Beer & M.J. Wingf.) in the roots of 2,350 loblolly pine trees in the southeastern United States and whether termites were present or absent on these roots and paired this with laboratory choice feeding trials. Termites were found 2.5-fold on tree roots with at least one bluestain fungus present than tree roots without bluestain fungi. Although termites in this study and others were associated with L. procerum, L. terebrantis, and marginally G. huntii, termites only showed preferential feeding on wood inoculated with G. huntii in laboratory trials. This suggests that increased termite presence on wood with bluestain fungi may be driven by factors other than increased wood palatability. Termites could thus disproportionately affect wood turnover rates for specific pools (e.g., bark beetle and root weevil attacked trees) and in some cases (e.g., G. huntii) accelerate wood decomposition. This study supports the growing evidence that the association between subterranean termites and bluestain fungi is spatially and taxonomically widespread.

Landscape Effects on Native Bees (Hymenoptera: Anthophila) Captured in Pheromone Traps for Noctuid Crop Pests (Lepidoptera: Noctuidae).

Noctuid pests, including tobacco budworm (Chloridea virescens (Fab.)) and bollworm (Helicoverpa zea (Boddie)), are significant pests of southern row crops including cotton (Gossypium hirsutum L.), corn (Zea mays L.), and soybean (Glycine max (L.) Moench.). This pest complex is seasonally monitored through Hartstack traps that are baited with synthetic lepidopteran pheromones across the southern United States. We examined bycatch from the noctuid traps deployed across the Mississippi Delta in 2015, 2016, and 2017 for the presence of bees. The most abundant species collected were honey bees (Apis mellifera L.), bumble bees (Bombus spp.), and long-horned bees (Melissodes spp.); these three genera accounted for 82.4% of specimens collected. We also evaluated the proportion of local- and landscape-level habitats on the abundance and richness of the bees caught as bycatch. The proportion of natural and semi-natural habitat affected the abundance and richness of bees collected at the landscape level, but not at more local scales. Additional research is needed to better understand these interactions between bycatch and landscape factors to minimize non-target collections.

Temporal Occurrence of Plusiinae on Soybean in the Mississippi River Delta.

The subfamily Plusiinae of the moth family Noctuidae is made up 400 species worldwide. Two species of the subfamily, the soybean looper, Chrysodeixis includens (Walker), and the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), are important defoliating insect pests of various field crops and have been the subject of previous Plusiinae surveys in the Southern U.S. Soybean fields were sampled in the Mississippi Delta from 2010 to 2012 to determine the temporal occurrence of various Plusiinae species on soybean. As in previous surveys, C. includens was the most common Plusiinae species in soybean during the 3-yr survey, especially in late season collections (July-September). Rachiplusia ou (Guenée) (Lepidoptera: Noctuidae) was the predominant species observed in early season collections (May-early July). Populations of R. ou during the first sample dates during 2010 were much higher than those observed during the other years of the survey. Only three collected larvae successfully developed into T. ni adults, one each collected during May, June, and July. Although R. ou was not commonly reported in previous studies on soybean, it occurred in low numbers during June and July during the 3-yr study. The temporal occurrence and species composition followed a predictable pattern in all 3 yr of the study.

Persistence in soil of microplastic films from ultra-thin compostable plastic bags and implications on soil Aspergillus flavus population.

An increasing number of states and municipalities are choosing to reduce plastic litter by replacing plastic items, particularly single-use ones, with same-use products manufactured from compostable plastics. This study investigated the formation and persistence of compostable film microplastic particles (CFMPs) from ultra-thin compostable carrier bags in soil under laboratory conditions, and the potential impact of CFMPs on Aspergillus flavus populations in the soil. During a 12-month incubation period, compostable film samples in soils with small, medium or large populations of indigenous A. flavus, underwent 5.9, 9.8, and 17.1% reduction in total surface area, respectively. Despite the low levels of deterioration, the number of CFMPs released increased steadily over the incubation period, particularly fragments with size < 0.05 mm. Up to 88.4% of the released fragments had associated A. flavus and up to 68% of isolates from CFMPs produced aflatoxins. A. flavus levels associated with CFMPs increased rapidly during the initial part of the 12-month incubation period, whereas the percent aflatoxigenicity continued to increase even after A. flavus density leveled off later. During 12 months incubation, A. flavus DNA amounts recovered from CFMPs increased in soils with all levels of indigenous A. flavus, with the largest increases (119.1%) occurring in soil containing the lowest indigenous A. flavus. These results suggest that burying compostable film in soil, or application of compost containing CFMPs, may reduce soil quality and increase risk of adverse impacts from elevated aflatoxigenic A. flavus populations in soil.

First documentation of major Vip3Aa resistance alleles in field populations of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in Texas, USA.

The corn earworm, Helicoverpa zea, is a major target pest of the insecticidal Vip3Aa protein used in pyramided transgenic Bt corn and cotton with Cry1 and Cry2 proteins in the U.S. The widespread resistance to Cry1 and Cry2 proteins in H. zea will challenge the long-term efficacy of Vip3Aa technology. Determining the frequency of resistant alleles to Vip3Aa in field populations of H. zea is critically important for resistance management. Here, we provided the first F2 screen study to estimate the resistance allele frequency for Vip3Aa in H. zea populations in Texas, U.S. In 2019, 128 H. zea neonates per isofamily for a total of 114 F2 families were screened with a diagnostic concentration of 3.0 µg/cm2 of Vip3Aa39 protein in diet-overlay bioassays. The F2 screen detected two families carrying a major Vip3Aa resistance allele. The estimated frequency of major resistance alleles against Vip3Aa39 in H. zea in Texas from this study was 0.0065 with a 95% CI of 0.0014-0.0157. A Vip3Aa-resistant strain (RR) derived from the F2 screen showed a high level of resistance to Vip3Aa39 protein, with a resistance ratio of >588.0-fold relative to a susceptible population (SS) based on diet-overlay bioassays. We provide the first documentation of a major resistance allele conferring high levels of Vip3Aa resistance in a field-derived strain of H. zea in the U.S. Data generated from this study contribute to development of management strategies for the sustainable use of the Vip3Aa technology to control H. zea in the U.S.

Degradation of microplastic seed film-coating fragments in soil.

Encapsulating fungicides and/or insecticides in film-coatings applied to agronomic seeds has become a widely accepted method for enhancing seed germination and overall seedling health by protecting against many diseases and early-season insect pests. Despite advancements in seed film-coating technologies, abrasion of the seed coating can occur during handling and mechanical planting operations, resulting in variable amounts of detached fragments entering the soil. The present study investigated the degradation in soil of these plastic-like, small-sized fragments, referred to here as microplastic coating fragments. Degradation of microplastic coating fragments in soil was found to be highly variable. The lowest degradation rate (≤48 days) was observed in fragments detached from seeds coated with a commercial polymer mixture, while fragments from a biodegradable plastic formulation degraded completely within 32 days. When spores of the plant growth-promoting bacterium, Bacillus subtilis, were incorporated into the bioplastic, degradation was even more rapid (≤24 days). The fragment degradation rate was unaffected by incorporating two commonly used neonicotinoid insecticides, imidacloprid or thiacloprid, into either coating formulations, but insecticide dissipation rates in soil were more rapid when added associated with seed coating fragments than when spiked in directly. Half-lives of these two insecticides were reduced by up to 27% in fragments from bioplastic-coated seeds. These results are consistent with variable and not easily predicted soil degradation rates for seed coating fragments, with enhanced dissipation of coating-entrapped pesticides and with a higher degradation rate for biodegradable seed coating incorporating selected microbial strains.

Differences between two populations of bollworm, Helicoverpa zea (Lepidoptera: Noctuidae), with variable measurements of laboratory susceptibilities to Bt toxins exposed to non-Bt and Bt cottons in large field cages.

Interpreting variable laboratory measurements of Helicoverpa zea Boddie susceptibility to toxins from Bacillus thuringiensis Berliner (Bt) has been challenging due to a lack of clear evidence to document declining field control. Research that links laboratory measurements of susceptibility to survival on Bt crops is vital for accurate characterization and any subsequent response to the occurrence of an implied H. zea resistance event. In this study, H. zea survival and the resultant damage to plant fruiting structures of non-Bt, Bollgard II, and Bollgard III cottons from two insect colonies with differing levels of laboratory susceptibility to Bt toxins were evaluated in large field cages. Laboratory bioassays revealed resistance ratios of 2.04 and 622.14 between the two H. zea colonies for Dipel DF and Cry1Ac, respectively. Differences between the two H. zea colonies measured via bioassays with Bollgard II and Bollgard III cotton leaf tissue in the laboratory were not statistically discernable. However, there was 17.6% and 5.3% lower larval mortality in Bollgard II and Bollgard III for the feral relative to the laboratory colony of H. zea, respectively. Although H. zea larval numbers in cages infested with the laboratory susceptible colony did not differ between the two Bt cottons, there were fewer larvae per 25 plants in Bollgard III than in Bollgard II cotton in cages containing tolerant insects. Cages infested with tolerant H. zea moths had higher numbers of total larvae than those containing the laboratory susceptible colony in both Bollgard II and Bollgard III cottons. Bollgard II and Bollgard III cottons received 77.4% and 82.7% more total damage to total plant fruiting structures in cages infested with tolerant insects relative to those containing the laboratory susceptible colony. The damage inflicted to fruiting structures on Bollgard III cotton by a feral H. zea colony with decreased measurements of laboratory susceptibility to Dipel DF and Cry1Ac indicate that the addition of Vip3A to third generation Bt cottons may not provide sufficient control in situations where infestations levels are high.

Comparison of Three Bioassay Methods to Estimate Levels of Tarnished Plant Bug (Hemiptera: Miridae) Susceptibility to Acephate, Imidacloprid, Permethrin, Sulfoxaflor, and Thiamethoxam.

A laboratory colony of tarnished plant bugs reared solely on a meridic diet was exposed to acephate, imidacloprid, permethrin, sulfoxaflor, and thiamethoxam in dose-response experiments using floral-foam, glass-vial, and dipped-leaf assays. Results indicated that different assay methods produced different relative results across the different insecticides. Dose- and time-response regression models also indicated that length of exposure of tarnished plant bugs to insecticide-treated plant tissue is important. Time of exposure required to reach an LC90 at estimated recommended field rates suggested that the recommended lower field rate of acephate (0.56 kg ai/ha) would reach an LC90 of exposed tarnished plant bugs between 48 and 96 h post initial exposure. An LC90 of tarnished plant bugs exposed to permethrin (0.11 kg ai/ha) was not predicted from the regression modes over the 168-h observation; lower recommended application rates of imidacloprid (0.053 kg ai/ha), sulfoxaflor (0.053 kg ai/ha), and thiamethoxam (0.042 kg ai/ha) reached projected LC90s between 96 and 168 h of exposure. Collectively, the results of this study corroborate current existing procedures for tracking tarnished plant bug resistance to insecticides, but also illustrate the importance of additional field studies that empirically associate assay results to projected field control.

Effects of transgenic Bacillus thuringiensis cotton on insecticide use, heliothine counts, plant damage, and cotton yield: A meta-analysis, 1996-2015.

The primary management tactic for lepidopteran pests of cotton in the United States of America (USA) is the use of transgenic cotton that produces Bacillus thuringiensis Berliner (Bt) toxins. The primary target pests of this technology are Helicoverpa zea (Boddie) and Heliothis virescens (F.) in the eastern and central Cotton Belt of the USA. Concerns over the evolution of resistance in H. zea to Bt toxins and scrutiny of the necessity of Bt crops has escalated. We reviewed published and unpublished data from field trials of Bt cotton in the eastern and central Cotton Belt of the USA through 2015 to evaluate the effectiveness of Bt cotton (Bollgard, Bollgard II, WideStrike, WideStrike 3, and TwinLink). Bt cotton reduced insecticide usage, reduced heliothine pest numbers and damage, and provided a yield benefit, but Bollgard II and WideStrike efficacy declined in the Midsouth over the period evaluated. In the Southeastern region, heliothine damage remained constant through 2015, but yield benefits declined from 2010 until 2015. Resistance of H. zea to several Bt toxins is the most plausible explanation for the observed changes in Bt cotton efficacy. The introduction of new Bt toxins such as found in Widestrike 3 and Twinlink may preserve the benefits of Bt crops. However, while both Widestrike 3 and Twinlink had less damage than Widestrike, damage levels of both were similar to Bollgard II.

CRISPR/Cas9 mediated high efficiency knockout of the eye color gene Vermillion in Helicoverpa zea (Boddie).

Among various genome editing tools available for functional genomic studies, reagents based on clustered regularly interspersed palindromic repeats (CRISPR) have gained popularity due to ease and versatility. CRISPR reagents consist of ribonucleoprotein (RNP) complexes formed by combining guide RNA (gRNA) that target specific genomics regions and a CRISPR associated nuclease (Cas). The gRNA targeting specific gene sequences may be delivered as a plasmid construct that needs to be transcribed or as a synthetic RNA. The Cas nuclease can be introduced as a plasmid construct, mRNA, or purified protein. The efficiency of target editing is dependent on intrinsic factors specific to each species, the target gene sequence, and the delivery methods of CRISPR gRNA and the Cas nuclease. Although intrinsic factors affecting genome editing may not be altered in most experiments, the delivery method for CRISPR/Cas reagents can be optimized to produce the best results. In this study, the efficiency of genome editing by CRISPR/Cas system in the bollworm, Helicoverpa zea (Boddie), was evaluated using ribonucleoprotein (RNP) complexes assembled by binding synthetic gRNA with purified Cas9 nuclease engineered with nuclear localization signals to target the vermillion (eye color) gene. Mutation rates of adults emerging from embryos microinjected with 1, 2, or 4 µM RNP complexes were compared using replicated experiments. Embryos injected with 2 or 4 µM RNP complexes displayed significantly higher mutation rates (>88%) in surviving adults compared to those injected with 1 µM. The hatch rate in embryos injected with RNP complexes and with injection buffer only (mock injections) was reduced by 19.8(±5.2)% compared to noninjected control embryos, but did not differ significantly between injected embryos. Evaluation of potential off-target sites in H. zea genome did not identify any mutations. This study demonstrates that in vitro assembled synthetic RNP complexes can be used to obtain high genome editing rates in a reproducible manner in functional genomics or genetic manipulation studies.

Baseline Susceptibility of Lygus lineolaris (Hemiptera: Miridae) to Novaluron.

Tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), populations were collected from field locations in the Mississippi River Delta of Arkansas, Louisiana, and Mississippi. Third-instar F(1) nymphs from each field location, in addition to a laboratory colony, were screened for susceptibility to novaluron. Both a glass vial bioassay and a diet-incorporated bioassay used dose-response regression lines to calculate LC(50) and LC(90) values for novaluron. Mean LC(50s) for glass vial bioassays ranged from 44.70 ± 3.58 to 66.54 ± 4.19 µg/vial, while mean LC(50s) for diet-incorporated bioassays ranged from 12.10 ± 0.77 to 17.63 ± 2.42 µg/200 ml of artificial diet. A comparison of LC(50) values from the same field population screened using both bioassay methods failed to show a relationship. LC(50) values from field locations were compared with a historically susceptible population from Crossett, AR. Results indicated that considerable variability in susceptibility to novaluron exists within field populations of tarnished plant bugs across the Delta, including some locations with lower LC(50) values than a historically susceptible population.