Laboratory and Field Investigations on Compatibility of Beauveria bassiana (Hypocreales: Clavicipitaceae) Spores With a Sprayable Bioplastic Formulation for Application in the Biocontrol of Tarnished Plant Bug in Cotton.

Two isolates of Beauveria bassiana (Balsamo) Vuillemin, including the commercial strain GHA and the Mississippi Delta native NI8 strain, and two emulsifiers, Tween-80 and a starch-based sprayable bioplastic, were evaluated in the laboratory and field for pathogenicity and infectivity against the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae). The effect on fruit damage based on within-season cotton plant mapping was also examined. The highest mortality 10 d after treatment was found with insects caged on cotton terminals sprayed with NI8 + Tween-80, followed by those exposed to NI8 + bioplastic. Similarly, sporulation was shown to be higher in NI8 + Tween-80 than in other treatments. Plots sprayed with B. bassiana showed at least a twofold decrease in tarnished plant bug adults 3 d after treatment compared with control plots. Little to no variation was observed in tarnished plant bug nymph populations between treated and untreated plots. Within-season plant mapping provided clear evidence of damage to cotton caused by tarnished plant bug. The highest percentage retention of all first position fruiting structures was observed in plots treated with NI8 + Tween-80 (93.41 ± 1.51) followed by NI8 + bioplastic (90.25 ± 1.52). Both treatments were significantly different when compared with GHA + Tween-80 (82.89 ± 2.26) and GHA + bioplastic (70.48 ± 3.19), and both GHA formulations did not differ from the control (63.61 ± 2.96). Overall, these results indicated that B. bassiana application resulted in >50% mortality of tarnished plant bug regardless of the isolates by direct spray or by contact. However, the superior performance of the Mississippi Delta native NI8 strain was observed in all treatment applications and evaluation times.

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.

Patterns of Tarnished Plant Bug (Hemiptera: Miridae) Resistance to Pyrethroid Insecticides in the Lower Mississippi Delta for 2008-2015: Linkage to Pyrethroid Use and Cotton Insect Management.

Populations of tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), from the Lower Mississippi Delta regions of Arkansas, Louisiana, and Mississippi were evaluated from 2008 through 2015 for susceptibility to pyrethroid insecticides using a diagnostic-dose assay with permethrin. Resulting data add to the compilation of pyrethroid susceptibility data carefully tracked in this pest since 1994 and provide continuing evidence of high frequencies of pyrethroid resistance in field populations of the tarnished plant bug. Resistance levels are variable, and some populations remain susceptible suggesting practical value in the continued use of the diagnostic-dose assays prior to pyrethroid treatments. Recent studies with dose-response models suggest that levels of pyrethroid resistance in some populations may still be evolving, with some populations requiring higher doses to reach levels of control comparable to those observed 10 yr ago. Concerns for frequent use of multiple classes of insecticides and possible selection for tarnished plant bugs with metabolic resistance mechanisms capable of detoxifying available insecticide chemistries warrant continued efforts to manage resistance in this important crop pest. Associations among measured pyrethroid resistance levels, published data on annual use of pyrethroid insecticides, and annual estimates of cotton insect losses and control costs were explored and summarized for the 8 yr of this investigation. Mortality of tarnished plant bugs at the diagnostic-dose of permethrin was negatively correlated with kilograms of pyrethroids applied per acre of harvested cropland.

Influences of acephate and mixtures with other commonly used pesticides on honey bee (Apis mellifera) survival and detoxification enzyme activities.

Acephate (organophosphate) is frequently used to control piercing/sucking insects in field crops in southern United States, which may pose a risk to honey bees. In this study, toxicity of acephate (formulation Bracket®97) was examined in honey bees through feeding treatments with sublethal (pollen residue level: 0.168 mg/L) and median-lethal (LC50: 6.97 mg/L) concentrations. Results indicated that adult bees treated with acephate at residue concentration did not show significant increase in mortality, but esterase activity was significantly suppressed. Similarly, bees treated with binary mixtures of acephate with six formulated pesticides (all at residue dose) consistently showed lower esterase activity and body weight. Clothianidin, λ-cyhalothrin, oxamyl, tetraconazole, and chlorpyrifos may interact with acephate significantly to reduce body weight in treated bees. The dose response data (LC50: 6.97 mg/L) revealed a relatively higher tolerance to acephate in Stoneville bee population (USA) than populations elsewhere, although in general the population is still very sensitive to the organophosphate. In addition to killing 50% of the treated bees acephate (6.97 mg/L) inhibited 79.9%, 20.4%, and 29.4% of esterase, Glutathione S-transferase (GST), and acetylcholinesterase (AChE) activities, respectively, in survivors after feeding treatment for 48 h. However, P450 activity was elevated 20% in bees exposed to acephate for 48 h. Even though feeding on sublethal acephate did not kill honey bees directly, chronic toxicity to honey bee was noticeable in body weight loss and esterase suppression, and its potential risk of synergistic interactions with other formulated pesticides should not be ignored.

Longitudinal Measurements of Tarnished Plant Bug (Hemiptera: Miridae) Susceptibility to Insecticides in Arkansas, Louisiana, and Mississippi: Associations with Insecticide Use and Insect Control Recommendations.

Concentration-response assays were conducted from 2008 through 2015 to measure the susceptibility of field populations of Lygus lineolaris (Palisot de Beauvois) from the Delta regions of Arkansas, Louisiana, and Mississippi to acephate, imidacloprid, thiamethoxam, permethrin, and sulfoxaflor. A total of 229 field populations were examined for susceptibility to acephate, 145 for susceptibility to imidacloprid, and 208 for susceptibility to thiamethoxam. Permethrin assays were conducted in 2014 and 2015 to measure levels of pyrethroid resistance in 44 field populations, and sulfoxaflor assays were conducted against 24 field populations in 2015. Resistance to acephate and permethrin is as high or higher than that previously reported, although some populations, especially those exposed to permethrin, appear to be susceptible. Variable assay responses were measured for populations exposed to imidacloprid and thiamethoxam. Average response metrics suggest that populations are generally susceptible to the neonicotinoids, but a few populations from cotton fields experiencing control problems exhibited elevated LC50s. Efforts to associate variability in LC50s with recorded use of insecticides and estimated cotton insect losses and control costs suggest that intensive use of insecticides over several decades may have elevated general detoxifying enzymes in L. lineolaris and some field populations may be exhibiting resistance to multiple classes of insecticide. These results suggest that efforts should be made to manage these pests more efficiently with a reduced use of insecticides and alternative controls.

Feeding toxicity and impact of imidacloprid formulation and mixtures with six representative pesticides at residue concentrations on honey bee physiology (Apis mellifera).

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.

Synergistic toxicity and physiological impact of imidacloprid alone and binary mixtures with seven representative pesticides on honey bee (Apis mellifera).

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.

Estimation of Median Lethal Concentration of Three Isolates of Beauveria bassiana for Control of Megacopta cribraria (Heteroptera: Plataspidae) Bioassayed on Solid Lygus spp. Diet.

The kudzu bug, Megacopta cribraria (F.), is an urban nuisance and significant agricultural pest. The median lethal concentrations of three strains of Beauveria bassiana (Balsamo), including the Mississippi Delta native strain (NI8) isolated from Lygus lineolaris (Palisot de Beauvois), the commercial strain BotaniGard(®) (GHA) (Victor, NY, USA), and the B. bassiana strain isolated from M. cribraria (KUDSC), were estimated on kudzu bug adults. A technique developed to evaluate B. bassiana against L. lineolaris was used. Younger adults (eight days after collection) were treated with NI8 and GHA and older adult (50 days after collection) were treated with NI8, GHA and KUDSC. Higher concentrations (n × 106, n × 107) of NI8 and GHA caused kudzu bug mortality two days after treatment in younger adults and similar concentrations of NI8, GHA, and KUDSC caused mortality one day after treatment in older adults. Lower concentrations (n × 104, n × 105) were not significantly different in mortality between strains. LS50 values of the KUDSC were significantly lower than NI8 and GHA values in older adults. This is the first available information on median lethal concentration of B. bassiana on kudzu bug adults bioassayed on artificial diet. It was determined that B. bassiana (KUDSC and NI8) are highly effective for young adults at very low doses (LC50 1.98-4.98 viable spores per mm²).

Identification of Genes Potentially Responsible for extra-Oral Digestion and Overcoming Plant Defense from Salivary Glands of the Tarnished Plant Bug (Hemiptera: Miridae) Using cDNA Sequencing.

Saliva is known to play a crucial role in tarnished plant bug (TPB, Lygus lineolaris [Palisot de Beauvois]) feeding. By facilitating the piercing, the enzyme-rich saliva may be used for extra-oral digestion and for overcoming plant defense before the plant fluids are ingested by TPBs. To identify salivary gland genes, mRNA was extracted from salivary glands and cDNA library clones were sequenced. A de novo-assembling of 7,000 Sanger sequences revealed 666 high-quality unique cDNAs with an average size of 624 bp, in which the identities of 347 cDNAs were determined using Blast2GO. Kyoto Encyclopedia of Genes and Genomes analysis indicated that these genes participate in eighteen metabolic pathways. Identifications of large number of enzyme genes in TPB salivary glands evidenced functions for extra-oral digestion and feeding damage mechanism, including 45 polygalacturonase, two α- amylase, one glucosidase, one glycan enzyme, one aminopeptidase, four lipase, and many serine protease cDNAs. The presence of multiple transcripts, multigene members, and high abundance of cell wall degradation enzymes (polygalacturonases) indicated that the enzyme-rich saliva may cause damage to plants by breaking down plant cell walls to make nutrients available for feeding. We also identified genes potentially involved in insect adaptation and detoxifying xenobiotics that may allow insects to overcome plant defense responses, including four glutathione S-transferases, three esterases, one cytochrome P450, and several serine proteases. The gene profiles of TPB salivary glands revealed in this study provides a foundation for further understanding and potential development of novel enzymatic inhibitors, or other RNAi approaches that may interrupt or minimize TPB feeding damage.

Complete Mitochondrial Genome of Helicoverpa zea (Lepidoptera: Noctuidae) and Expression Profiles of Mitochondrial-Encoded Genes in Early and Late Embryos.

The mitochondrial genome (mitogenome) of the bollworm, Helicoverpa zea (Boddie), was assembled using paired-end nucleotide sequence reads generated with a next-generation sequencing platform. Assembly resulted in a mitogenome of 15,348 bp with greater than 17,000-fold average coverage. Organization of the H. zea mitogenome (gene order and orientation) was identical to other known lepidopteran mitogenome sequences. Compared with Helicoverpa armigera (Hübner) mitogenome, there were a few differences in the lengths of gaps between genes, but the lengths of nucleotide overlaps were essentially conserved between the two species. Nucleotide composition of the H. zea mitochondrial genome was very similar to those of the related species H. armigera and Helicoverpa punctigera Wallengren. Mapping of RNA-Seq reads obtained from 2-h eggs and 48-h embryos to protein coding genes (PCG) revealed that all H. zea PCGs were processed as single mature gene transcripts except for the bicistronic atp8 + atp6 transcript. A tRNA-like sequence predicted to form a hammer-head-like secondary structure that may play a role in transcription start and mitogenome replication was identified within the control region of the H. zea mitogenome. Similar structures were also found within the control regions of several other lepidopteran species. Expression analysis revealed significant differences in levels of expression of PCGs within each developmental stage, but the pattern of variation was similar in both developmental stages analyzed in this study. Mapping of RNA-Seq reads to PCG transcripts also identified transcription termination and polyadenylation sites that differed from the sites described in other lepidopteran species.

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.

Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1.

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.

Spray Toxicity and Risk Potential of 42 Commonly Used Formulations of Row Crop Pesticides to Adult Honey Bees (Hymenoptera: Apidae).

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.

Evidence of multiple/cross resistance to Bt and organophosphate insecticides in Puerto Rico population of the fall armyworm, Spodoptera frugiperda.

Fall armyworm (FAW) is a damaging pest of many economic crops. Long-term use of chemical control prompted resistance development to many insecticide classes. Many populations were found to be significantly less susceptible to major Bt toxins expressed in transgenic crops. In this study, a FAW strain collected from Puerto Rico (PR) with 7717-fold Cry1F-resistance was examined to determine if it had also developed multiple/cross resistance to non-Bt insecticides. Dose response assays showed that the PR strain developed 19-fold resistance to acephate. Besides having a slightly smaller larval body weight and length, PR also evolved a deep (2.8%) molecular divergence in mitochondrial oxidase subunit II. Further examination of enzyme activities in the midgut of PR larvae exhibited substantial decreases of alkaline phosphatase (ALP), aminopeptidase (APN), 1-NA- and 2-NA-specific esterase, trypsin, and chymotrypsin activities, and significant increases of PNPA-specific esterase and glutathione S-transferase (GST) activities. When enzyme preparations from the whole larval body were examined, all three esterase, GST, trypsin, and chymotrypsin activities were significantly elevated in the PR strain, while ALP and APN activities were not significantly different from those of susceptible strain. Data indicated that multiple/cross resistances may have developed in the PR strain to both Bt toxins and conventional insecticides. Consistently reduced ALP provided evidence to support an ALP-mediated Bt resistance mechanism. Esterases and GSTs may be associated with acephate resistance through elevated metabolic detoxification. Further studies are needed to clarify whether and how esterases, GSTs, and other enzymes (such as P450s) are involved in cross resistance development to Bt and other insecticide classes.

Altered gene regulation and potential association with metabolic resistance development to imidacloprid in the tarnished plant bug, Lygus lineolaris.

BACKGROUND: Chemical spray on cotton is almost an exclusive method for controlling tarnished plant bug (TPB), Lygus lineolaris. Frequent use of imidacloprid is a concern for neonicotinoid resistance in this key pest. Information of how and why TPB becomes less susceptible to imidacloprid is essential for effective monitoring and managing resistance. RESULTS: Microarray analysis of 6688 genes in imidacloprid-selected TPB (Im1500FF) revealed 955 upregulated and 1277 downregulated (≥twofold) genes in Im1500FF, with 369 and 485 of them annotated. Five P450 and nine esterase genes were significantly upregulated, and only one esterase gene and no P450 genes were downregulated. Other upregulated genes include helicases, phosphodiesterases, ATPases and kinases. Pathway analyses identified 65 upregulated cDNAs that encode 51 different enzymes involved in 62 different pathways, including P450 and esterase genes for drug and xenobiotic metabolisms. Sixty-four downregulated cDNAs code only 17 enzymes that are associated with only 23 pathways mostly related to food digestion. CONCLUSIONS: This study demonstrated a significant change in gene expression related to metabolic processes in imidacloprid-selected TPB, resulting in overexpression of P450 and esterase genes for potential excess detoxification and cross/multiple resistance development. The identification of these and other enzyme genes establishes a foundation to explore the complicity of potential imidacloprid resistance in TPB.

A novel bioassay to evaluate the potential of Beauveria bassiana strain NI8 and the insect growth regulator novaluron against Lygus lineolaris on a non-autoclaved solid artificial diet.

A non-autoclaved solid diet was used to evaluate the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) strain NI8 and the insect growth regulator novaluron (Diamond® 0.83EC insecticide) for control of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae). The diet was composed of toasted wheat germ, ground lima bean meal, soy flour, yolk of chicken eggs, inhibitor, and agar. It was prepared in one step by blending the ingredients in boiling water. The diet was used to bioassay L. lineolaris from the second instar to the adult stage. Fourth and fifth instars and adults of L. lineolaris were more susceptible than second and third instars to infection by B. bassiana, whereas second, third, and fourth instars had higher mortality than fifth instars 10 days after exposure to novaluron. No effects on longevity were observed in adults treated with novaluron when compared with the control, but longevity was significantly different from that of adults exposed to B. bassiana. Adults of L. lineolaris were maintained for over a month without changing the diet. The non-autoclaved diet is semi-liquid before it cools, which facilitates the mechanics of diet packaging similar to food packaging or lepidopteran diet preparation. This solid artificial diet for Lygus bugs provides improved research capacity for studying the ecology and susceptibility of Lygus spp. to a number of different control agents, including beneficial organisms, insect pathogens, and insecticidal toxins being developed for transgenic technologies.

Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and Cry1Ab-resistant strains of sugarcane borer, Diatraea saccharalis.

Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is responsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin-like proteinases were sequenced from Cry1Ab-SS and Cry1Ab-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, including signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab-SS and Cry1Ab-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab-SS and Cry1Ab-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab-SS and Cry1Ab-RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly associated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.

Helicoverpa zea and Bt cotton in the United States.

Helicoverpa zea (Boddie), the bollworm or corn earworm, is the most important lepidopteran pest of Bt cotton in the United States. Corn is the preferred host, but the insect feeds on most flowering crops and wild host plants. As a cotton pest, bollworm has been closely linked to the insecticide-resistance prone Heliothis virescens (F.), tobacco budworm. Immature stages of the two species are difficult to separate in field environments. Tobacco budworm is very susceptible to most Bt toxins, and Bt cotton is considered to be "high dose." Bollworm is less susceptible to Bt toxins, and Bt cotton is not "high dose" for this pest. Bt cotton is routinely sprayed with traditional insecticides for bollworm control. Assays of bollworm field populations for susceptibility to Bt toxins expressed in Bt cotton have produced variable results since pre-deployment of Bt cottons in 1988 and 1992. Analyses of assay response trends have been used by others to suggest that field resistance has evolved to Bt toxins in bollworm, but disagreement exists on definitions of field resistance and confidence of variable assay results to project changes in susceptibility of field populations. Given historical variability in bollworm response to Bt toxins, erratic field control requiring supplemental insecticides since early field testing of Bt cottons, and dramatic increases in corn acreage in cotton growing areas of the Southern US, continued vigilance and concern for resistance evolution are warranted.

Microarray analysis of gene regulations and potential association with acephate-resistance and fitness cost in Lygus lineolaris.

The tarnished plant bug has become increasingly resistant to organophosphates in recent years. To better understand acephate resistance mechanisms, biological, biochemical, and molecular experiments were systematically conducted with susceptible (LLS) and acephate-selected (LLR) strains. Selection of a field population with acephate significantly increased resistance ratio to 5.9-fold, coupled with a significant increase of esterase activities by 2-fold. Microarray analysis of 6,688 genes revealed 329 up- and 333 down-regulated (≥2-fold) genes in LLR. Six esterase, three P450, and one glutathione S-transferase genes were significantly up-regulated, and no such genes were down-regulated in LLR. All vitellogenin and eggshell protein genes were significantly down-regulated in LLR. Thirteen protease genes were significantly down-regulated and only 3 were up-regulated in LLR. More than twice the number of catalysis genes and more than 3.6-fold of metabolic genes were up-regulated, respectively, as compared to those down-regulated with the same molecular and biological functions. The large portion of metabolic or catalysis genes with significant up-regulations indicated a substantial increase of metabolic detoxification in LLR. Significant increase of acephate resistance, increases of esterase activities and gene expressions, and variable esterase sequences between LLS and LLR consistently demonstrated a major esterase-mediated resistance in LLR, which was functionally provable by abolishing the resistance with esterase inhibitors. In addition, significant elevation of P450 gene expression and reduced susceptibility to imidacloprid in LLR indicated a concurrent resistance risk that may impact other classes of insecticides. This study demonstrated the first association of down-regulation of reproductive- and digestive-related genes with resistance to conventional insecticides, suggesting potential fitness costs associated with resistance development. This study shed new light on the understanding of the molecular basis of insecticide resistance, and the information is highly valuable for development of chemical control guidelines and tactics to minimize resistance and cross-resistance risks.

Microarray analysis of global gene regulation in the Cry1Ab-resistant and Cry1Ab-susceptible strains of Diatraea saccharalis.

BACKGROUND: Extensive adoption of transgenic Bt corn in recent years for stalk borer control has increased risk of resistance evolution in the target pest populations. A Bt-resistant strain of the sugarcane borer, Diatraea saccharalis, was approximately 100-fold more tolerant to Cry1Ab toxin than the susceptible counterpart. To gain a better understanding of the molecular mechanisms of Bt resistance, the Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains of D. saccharalis were subjected to a microarray analysis. RESULTS: Results showed that the expression levels of many genes were significantly different between the Cry1Ab-RR and Cry1Ab-SS strains. Microarray analysis of 7145 cDNAs revealed 384 differentially expressed genes. A total of 273 genes were significantly upregulated 2-51.6-fold, and 111 genes were significantly downregulated 2-22.6-fold in the Cry1Ab-RR strain. The upregulation of three potential resistance-related genes, coding for a glutathione S-transferase (GST), a chymotrypsin-like protease (CHY) and a lipase (LP), was confirmed using real-time PCR, indicating a reproducibility of the microarray data. Ontology analysis revealed that more than twice the number of metabolic-related genes were upregulated compared with downregulated genes with the same biological function. Up to 35.2% of the upregulated genes in the resistant strain were associated with catalytic activity, while only 9.5% of the downregulated genes were related to the same catalytic molecular function. CONCLUSION: The large portion of metabolic- or catalytic-related genes with significant upregulations indicated a potential large increase in metabolic or catalytic activities in the Cry1Ab-RR strain. This cDNA microarray gene expression data could be used to characterize and identify new genes that may be associated with Bt resistance in D. saccharalis.