Randall Luttrell

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

ABSTRACT 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.

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.

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.

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.

Temporal and Spatial Genetic Variability Among Tarnished Plant Bug (Hemiptera: Miridae) Populations in a Small Geographic Area

ABSTRACT The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is an important pest of cotton that also feeds on other crops and many wild hosts. In the mid-South, where tarnished plant bugs in cotton are controlled exclusively with synthetic insecticides, infestations resulted in >29% of all yield loss attributed to insect damage and the control costs exceeded US

Effect of Food and Temperature on Emergence from Diapause in the Tarnished Plant Bug (Hemiptera: Miridae)

10 per acre in 2013. They have developed resistance to the most commonly used insecticides. Estimations of gene flow and migration are important to understand the spread of resistance in tarnished plant bug populations. Here, we analyzed tarnished plant bugs collected from July to September, 2006, to estimate population genetic parameters using 13 microsatellite markers. Our data indicated that tarnished plant bug populations in the study area had undergone a population bottleneck and all loci deviated from Hardy—Weinberg equilibrium in one or more collections. Bayesian simulations and factorial correspondence analysis indicated the presence of two genetic clusters in the tarnished plant bug populations in the study area. Proportions of insects separated into the two genetic clusters changed from July to September. Genetic differentiation and reduced gene flow between populations were also observed. We postulate that while random genetic drift and gene flow may have contributed to the seasonal variations observed in the study populations, selection by insecticide sprays in cotton during 2005–2006 as well as other extrinsic factors could also have played a significant role in the temporal variation in genetic structure observed in the tarnished plant bug populations.

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

ABSTRACT Tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), overwinter as diapausing adults in North America. Overwintering adults were collected near Stoneville, MS from blooming henbit, Lamium amplexicaule L., and from plant debris during December and January and dissected to determine their reproductive status. Averaged over four winters, male and female tarnished plant bugs collected from henbit terminated diapause at a significantly higher rate than males and females from plant debris during each week of December and the first week of January. Both sexes in each habitat were nearly all reproductive by the end of January. Adults overwintering in plant debris terminated diapause during January in the absence of a food stimulus in all 5 yr studied. This emergence was thought to be controlled by an internal clock. Laboratory and field studies showed that emergence from diapause could be influenced by food, sex, and temperature. Adults overwintering on a suitable food source, blooming henbit, terminated diapause during December in the 4 yr studied, and males terminated diapause more rapidly than females. Food quality was important in emergence from diapause, and females on blooming henbit terminated diapause at a significantly higher rate than females on nonblooming mustard, Brassica juncea (L.) Cosson. Laboratory tests showed that diapausing adults reared in the laboratory and held at a diapause-maintaining photoperiod of 10:14 (L:D) h could be terminated from diapause by using food and temperature stimuli. The lower thermal threshold for development to reproductive adults was found to be near 10°C. The ability of diapausing adults to respond to food and temperature stimuli in December can enable tarnished plant bugs to take advantage of warm winters and winter hosts to produce a new generation earlier.

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

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.

Lethal and Sub-Lethal Effects of Beauveria bassiana (Cordycipitaceae) Strain NI8 on Chrysoperla rufilabris (Neuroptera: Chrysopidae)

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.

Demographic Parameters of Nezara viridula (Heteroptera: Pentatomidae) Reared on Two Diets Developed for Lygus spp.

Abstract A Mississippi Delta native strain (NI8 ARSEF8889) of Beauveria bassiana (Bals.-Criv.) Vuill. (Cordycipitaceae), isolated from Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), was tested on green lacewings, Chrysoperla rufilabris (Burmeister) (Neuroptera: Chrysopidae) at 4 spray concentrations (7.02 × 104, 105, 106, and 107 spores per mL) to evaluate effects on reproductive rates and adult life expectancy of this insect predator. The application method simulated atomized spray, and concentrations tested were similar to those used to measure impacts of the fungus on L. lineolaris. Significant effects of B. bassiana on C. rufilabris adults were found, and the severity of impact depended on the concentrations tested. Beauveria bassiana impacted all demographic measurements of C. rufilabris reproduction and survival. Intrinsic and finite rates of increase and gross and net reproductive rates of adults treated with the highest concentrations tested were significantly decreased, whereas doubling time increased for adults treated with the lowest test concentrations. Based on these observations, C. rufilabris will be affected by sprays of B. bassiana targeted at L. lineolaris if adults are present at the time and location of treatment. The measured lethal concentration, LC50, of 2.11 viable spores per mm2 compares to an LC50 of 2.75 spores per mm2 determined previously for L. lineolaris. Higher concentrations of spores per mm2 were required for sporulation (SR50) of the entomopathogenic fungus on C. rufilabris (13.60 viable spores per mm2) than concentrations required for mortality (LC50).