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Susceptibilities of bollworm, Helicoverpa zea (Boddie) and tobacco budworm, Heliothis virescens (F.) to Cry1Ac were measured via a diet-incorporated assay with MPV II at the University of Arkansas during 2002-2004. Lethal concentration-mortality (LC50) estimates of five laboratory, seven laboratory-cross, and 10 field populations of H. virescens varied 12-fold. Pooled susceptibilities of H. virescens across all laboratory and field populations varied five-fold. The LC50 estimates for H. virescens were higher than those reported by previous research before the introduction of transgenic crops. However, the ratio of susceptibility of laboratory and field populations was similar, suggesting no change in overall species susceptibility. Individual LC50 estimates of five laboratory, nine laboratory-cross, and 57 field populations of H. zea varied over 130-fold. Pooled susceptibilities across laboratory and field populations varied widely. Among the field populations, colonies from non-Bacillus thuringiensis (Bt) crops were generally more susceptible than those from Bt crops. Across the Bt crops expressing Cry protein, colonies from Bollgard (Monsanto Company) cotton had lower susceptibility to CrylAc than those from Bt corn and those from non-Bt crops.

Imidacloprid is the most widely used insecticide in the world. In this study, we used spraying methods to simulate field exposures of bees to formulated imidacloprid (Advise® 2FL) alone and binary mixtures with seven pesticides from different classes. Synergistic toxicity was detected from mixtures of Advise (58.6 mg a.i./L imidacloprid)+Domark (512.5 mg a.i. /L tetraconazole), Advise+Transform (58.5 mg a.i./L sulfoxaflor), and Advise+Vydate (68 mg a.i./L oxamyl), and mortality was significantly increased by 20%, 15%, and 26% respectively. The mixtures of Advise+Bracket (88.3 mg a.i./L acephate) and Advise+Karate (62.2 mg a.i./L L-cyhalothrin) showed additive interaction, while Advise+Belay (9.4 mg a.i./L clothianidin) and Advise+Roundup (1217.5 mg a.i./L glyphosate) had no additive/synergistic interaction. Spraying bees with the mixture of all eight pesticides increased mortality to 100%, significantly higher than all other treatments. Except Bracket which significantly suppressed esterase and acetylcholinesterase (AChE) activities, other treatments of Advise-only and mixtures with other pesticides did not suppress enzyme activities significantly, including invertase, glutathione S-transferase (GST), and esterase and AChE. Immunity-related phenoloxidase (PO) activities in survivors tended to be more variable among treatments, but mostly still statistically similar to the control. By using specific enzyme inhibitors, we demonstrated that honey bees mainly rely on cytochrome P450 monooxygenases (P450s) for detoxifying Advise, while esterases and GSTs play substantially less roles in the detoxification. This study provided valuable information for guiding pesticide selection in premixing and tank mixing in order to alleviate toxicity risk to honey bees. Our findings indicated mixtures of Advise with detoxification-enzyme-inducing pesticides may help bees to detoxify Advise, while toxicity synergists may pose further risk to bees, such as the Bracket which not only suppressed esterase and AChE activities, but also increased toxicity to bees.

Imidacloprid is the most widely used insecticide in agriculture. In this study, we used feeding methods to simulate in-hive exposures of formulated imidacloprid (Advise® 2FL) alone and mixtures with six representative pesticides for different classes. Advise, fed at 4.3 mg/L (equal to maximal residue detection of 912 ppb active ingredient [a.i.] in pollen) induced 36% mortality and 56% feeding suppression after 2-week feeding. Treatments with individual Bracket (acephate), Karate (λ-cyhalothrin), Vydate (oxamyl), Domark (tetraconazole), and Roundup (glyphosate) at residue level had a mortality range of 1.3–13.3%, statistically similar to that of control (P>0.05). The additive/synergistic toxicity was not detected from binary mixtures of Advise with different classes of pesticides at residue levels. The feeding of the mixture of all seven pesticides increased mortality to 53%, significantly higher than Advise only but still without synergism. Enzymatic data showed that activities of invertase, glutathione S-transferase, and acetylcholinesterase activities in imidacloprid-treated survivors were mostly similar to those found in control. Esterase activity mostly increased, but was significantly suppressed by Bracket (acephate). The immunity-related phenoloxidase activity in imidacloprid-treated survivors tended to be lower, but most treatments were statistically similar to the control. Increase of cytochrome P450 activity was correlated with Advise concentrations and reached significant difference at 56 mg/L (12 ppm a.i.). Our data demonstrated that residue levels of seven pesticide in pollens/hive may not adversely affect honey bees, but long term exclusive ingestion of the maximal residue levels of imidacloprid (912 ppb) and sulfoxaflor (3 ppm a.i.) may induce substantial bee mortality. Rotating with other insecticides is a necessary and practical way to reduce the residue level of any given pesticide.

Summary 1.A high dose ⁄ refuge strategy has been adopted in the USA to manage the risk of Bacillus thuringiensis (Bt) resistance in target pests such as the cotton bollworm (CBW), Helicoverpa zea (Boddie) in transgenic Bt cotton Gossypium hirsutum L. Structured refuges, consisting of non-Bt cotton, have been a mandated part of this strategy to produce non-selected insects that are temporally and spatially synchronous with insects from the Bt crop, diluting Bt resistance alleles through mating. However, the bollworm is highly polyphagous and exploits a large number of crop and weedy hosts concurrently with Bt cotton. 2. A study was carried out in five major US cotton-producing states during 2002 and 2003 using the ratios of 13 C to 12 C in bollworm moths to estimate the proportions of the population originating from C 3 or C 4 plants. A separate study measured gossypol residues in moths from four states in 2005 and 2006, enabling the identification of moths whose natal hosts were cotton rather than other C 3 hosts. 3. C 4 hosts served as the principal source of bollworm moths from mid-to-late June to early September, depending on the state. Beginning in late August ⁄ early September and lasting 1-4 weeks, the majority of moths exhibited isotopic compositions characteristic of C 3 hosts. During this period, however, the minimum percentage of moths that developed as larvae on C 4 hosts was typically >25%. By mid-September and through October and November, the majority of the bollworm population exhibited C 4 isotopic compositions. 4. Between late June and early August, cotton-derived bollworm moths (moths with gossypol residues) comprised <1% of moths in all states, and remained below this level throughout the season in North Carolina. In other states, cotton-derived moths increased between early August and early September to peak at an average of 19AE1% of all moths. 5. Synthesis and applications. Data on 13 C ⁄ 12 C ratios and gossypol residues in CBW moths were used to assess the importance of structured non-Bt cotton refuges for the management of Bt resistance risk in H. zea. Weekly estimates of bollworm breeding on cotton, C 3 plants other than cotton and C 4 plants showed that, throughout the season, the majority of bollworm moths caught in pheromone traps adjacent to cotton fields did not develop as larvae on cotton. This result implies that management practices in cotton such as the use of structured cotton refuges will play a relatively minor role -particularly compared with maize Zea mays L. -in managing potential resistance to Bt cotton in populations of the CBW in the US Cotton Belt.

Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.

To combat an increasing abundance of sucking insect pests, >40 pesticides are currently recommended and frequently used as foliar sprays on row crops, especially cotton. Foraging honey bees may be killed when they are directly exposed to foliar sprays, or they may take contaminated pollen back to hives that maybe toxic to other adult bees and larvae. To assess acute toxicity against the honey bee, we used a modified spray tower to simulate field spray conditions to include direct whole-body exposure, inhalation, and continuing tarsal contact and oral licking after a field spray. A total of 42 formulated pesticides, including one herbicide and one fungicide, were assayed for acute spray toxicity to 4-6-d-old workers. Results showed significantly variable toxicities among pesticides, with LC50s ranging from 25 to thousands of mg/liter. Further risk assessment using the field application concentration to LC1 or LC99 ratios revealed the risk potential of the 42 pesticides. Three pesticides killed less than 1% of the worker bees, including the herbicide, a miticide, and a neonicotinoid. Twenty-six insecticides killed more than 99% of the bees, including commonly used organophosphates and neonicotinoids. The remainder of the 13 chemicals killed from 1-99% of the bees at field application rates. This study reveals a realistic acute toxicity of 42 commonly used foliar pesticides. The information is valuable for guiding insecticide selection to minimize direct killing of foraging honey bees, while maintaining effective control of field crop pests.

Selection pressure on bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), by cotton, Gossypium hirsutum (L.) (Malvaceae), that produces one or more Bacillus thuringiensis Berliner (Bt) proteins is reduced by plantings of non‐Bt refuge cotton that produce non‐selected individuals. However, the contributions of non‐Bt, non‐cotton crop hosts to the overall effective refuge for H. zea on Bt cotton have not been estimated. A 2‐year, season‐long study was conducted in five US cotton‐producing states to assess the spatial and temporal population dynamics and host use of H. zea. Helicoverpa zea larval estimates in commercial crop fields demonstrated that non‐cotton crop hosts, such as maize, Zea mays L. (Poaceae), grain sorghum, Sorghum bicolor (L.) Moench (Poaceae), peanut, Arachis hypogaea L. (Fabaceae), and soybean, Glycine max (L.) Merrill (Fabaceae), collectively support much larger larval populations than cotton throughout the season. Larval populations were almost entirely restricted to maize in the middle part of the season (June and portions of July), and were observed in non‐cotton crop hosts more frequently and typically in larger numbers than in cotton during the period when production would be expected in cotton (July and August). Numbers of H. zea larvae produced in replicated strip trials containing various crop hosts paralleled production estimates from commercial fields. In contrast, the number of H. zea adults captured in pheromone traps at interfaces of fields of Bt cotton and various crop hosts rarely varied among interfaces, except in instances where maize was highly attractive. With the exception of this early season influence of maize, moth numbers were not related to local larval production. These data demonstrate that H. zea adults move extensively from their natal host origins. Therefore, non‐cotton crop hosts, and even relatively distant hosts, contribute significantly to effective refuge for H. zea on Bt cotton. The results presented here demonstrate that substantial natural refuge is present for Bt‐resistance management of H. zea throughout the mid‐South and Southeast portions of the US cotton belt.