Filling in the gaps: A reevaluation of the Lygus hesperus peptidome using an expanded de novo assembled transcriptome and molecular cloning.

Peptides are the largest and most diverse class of molecules modulating physiology and behavior. Previously, we predicted a peptidome for the western tarnished plant bug, Lygus hesperus, using transcriptomic data produced from whole individuals. A potential limitation of that analysis was the masking of underrepresented genes, in particular tissue-specific transcripts. Here, we reassessed the L. hesperus peptidome using a more comprehensive dataset comprised of the previous transcriptomic data as well as tissue-specific reads produced from heads and accessory glands. This augmented assembly significantly improves coverage depth providing confirmatory transcripts for essentially all of the previously identified families and new transcripts encoding a number of new peptide precursors corresponding to 14 peptide families. Several families not targeted in our initial study were identified in the expanded assembly, including agatoxin-like peptide, CNMamide, neuropeptide-like precursor 1, and periviscerokinin. To increase confidence in the in silico data, open reading frames of a subset of the newly identified transcripts were amplified using RT-PCR and sequence validated. Further PCR-based profiling of the putative L. hesperus agatoxin-like peptide precursor revealed evidence of alternative splicing with near ubiquitous expression across L. hesperus development, suggesting the peptide serves functional roles beyond that of a toxin. The peptides predicted here, in combination with those identified in our earlier study, expand the L. hesperus peptidome to 42 family members and provide an improved platform for initiating molecular and physiological investigations into peptidergic functionality in this non-model agricultural pest.

Fusarium Hardlock Associated With Lygus lineolaris (Hemiptera: Miridae) Injury in Southeastern Cotton.

The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is an important insect pest in cotton that feeds on reproductive fruit, contributing to yield loss. Economically damaging infestations of L. lineolaris have doubled in Virginia since 2013. Escalation of L. lineolaris abundance may increase Fusarium hardlock disease observed in this region, compounding economic losses. Research has linked Fusarium hardlock with fungal species Fusarium verticillioides and F. proliferatum. Field and greenhouse experiments were performed to investigate (i) Fusarium hardlock occurrence in field plots managed and unmanaged for L. lineolaris, (ii) severity of F. verticillioides infection of cotton bolls with and without the presence of L. lineolaris feeding in a greenhouse setting, and (iii) Fusarium species composition and prevalence within field-collected L. lineolaris and cotton lint with and without insect feeding injury and hardlock symptoms present. Nearly twice the amount of hardlock (i.e., proportion of hardlocked locules) occurred in field-collected bolls with L. lineolaris feeding symptoms (0.40 ± 0.02) compared with bolls without (0.21 ± 0.01). Based on real-time quantitative PCR, cotton bolls exposed to F. verticillioides inoculum and caged with L. lineolaris adults had greater levels of F. verticillioides DNA compared with untreated bolls. F. proliferatum, F. verticillioides, and F. fujikuroi were isolated from field-collected L. lineolaris and hardlocked cotton lint at harvest. These findings suggest that the presence of L. lineolaris is associated with an increased risk of Fusarium hardlock in Southeastern cotton, and both should be carefully managed using timely insecticide applications and cultural control practices to minimize yield loss.

Expression Profiles of Digestive Genes in the Gut and Salivary Glands of Tarnished Plant Bug (Hemiptera: Miridae).

Host plant preference of agricultural pests may shift throughout the growing season, allowing the pests to persist on wild hosts when crops are not available. Lygus Hahn (Hemiptera: Miridae) bugs are severe pests of cotton during flowering and fruiting stages, but can persist on alternative crops, or on weed species. Diversity of digestive enzymes produced by salivary glands and gut tissues play a pivotal role in an organism's ability to utilize various food sources. Polyphagous insects produce an array of enzymes that can process carbohydrates, lipids, and proteins. In this study, the digestive enzyme repertoire of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by high-throughput sequencing followed by cDNA cloning and sequencing. This study identified 87 digestive genes, including 30 polygalacturonases (PG), one ß-galactosidase, three α-glucosidases, six ß-glucosidases, 28 trypsin-like proteases, three serine proteases, one apyrase-like protease, one cysteine protease, 12 lipases, and two transcripts with low similarity to a xylanase A-like genes. RNA-Seq expression profiles of these digestive genes in adult tarnished plant bugs revealed that 57 and 12 genes were differentially expressed in the salivary gland and gut (≥5-fold, P ≤ 0.01), respectively. All polygalacturonase genes, most proteases, and two xylanase-like genes were differentially expressed in salivary glands, while most of the carbohydrate and lipid processing enzymes were differentially expressed in the gut. Seven of the proteases (KF208689, KF208697, KF208698, KF208699, KF208700, KF208701, and KF208702) were not detected in either the gut or salivary glands.

A Molecular Approach for Detecting Stage-Specific Predation on Lygus hesperus (Hemiptera: Miridae).

A molecular gut analysis technique is described to identify predators of Lygus hesperus (Knight), a significant pest of many crops. The technique is unique because it can pinpoint which life stage of the pest was consumed. Sentinel egg masses designed to mimic the endophytic egg-laying behavior of L. hesperus were marked with rabbit serum, while third instar and adult L. hesperus were marked with chicken and rat sera, respectively. Then, the variously labeled L. hesperus life stages were introduced into field cages that enclosed the native arthropod population inhabiting an individual cotton plant. After a 6-h exposure period, the predator assemblage, including the introduced and native L. hesperus population, in each cage were counted and had their gut contents examined for the presence of the variously marked L. hesperus life stages by a suite of serum-specific enzyme-linked immunosorbent assays (ELISA). The whole-plant sampling scheme revealed that Geocoris punticpes (Say) and Geocoris pallens Stal (Hemiptera: Geocoridae) and members of the spider complex were the numerically dominant predator taxa in the cotton field. The gut content analyses also showed that these two taxa appeared to be the most prolific predators of the L. hesperus nymph stage. Other key findings include that Collops vittatus (Say) (Coleoptera: Melyridae) and Solenopsis xyloni McCook (Hymenoptera: Formicidae) appear to be adept at finding and feeding on the cryptic L. hesperus egg stage, and that L. hesperus, albeit at low frequencies, engaged in cannibalism. The methods described here could be adapted for studying life stage-specific feeding preferences for a wide variety of arthropod taxa.

A Laboratory Diet-Overlay Bioassay to Monitor Resistance in Lygus lineolaris (Hemiptera: Miridae) to Insecticides Commonly Used in the Mississippi Delta.

A laboratory, diet-overlay pesticide bioassay was developed using a susceptible population of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), to study its susceptibility to neonicotinoid, sulfoxamine, organophosphate, and pyrethroid insecticides (thiamethoxam, sulfoxaflor, acephate, and permethrin, respectively). The diet-overlay bioassay was compared to the traditional glass-vial surface residue bioassay. We measured LC50 values by feeding tarnished plant bug adults known doses of insecticides dispensed on top of diet in a 10% solution of honey water for thiamethoxam and 10% acetone in water solutions for permethrin, acephate, and sulfoxaflor. Both the diet-overlay and glass-vial bioassays used dose-response (mortality) regression lines to calculate LC50 values for each insecticide at 6-, 24-, 48-, and 72-h post-exposure. Data variability from the glass-vial bioassay was higher for permethrin, sulfoxaflor, and thiamethoxam than the diet-overlay bioassay, for all evaluation times. In contrast, there was lower variability among replicates to acephate in the glass-vial assay compared to the diet-overlay assay. Control mortalities observed on diet-overlay bioassay were lower (0-5%) than those observed on the glass-vial bioassay (4-27%). The use of green beans, floral-foam, rolling glass vials, and insect handling made the existing standard method tedious to manipulate and difficult to handle large numbers of individuals. The nonautoclaved solid diet provides an opportunity to significantly reduce cost and variability associated with procedures of other bioassay methods. In general, the baseline data provide a basis for future comparison to determine changes in resistance over time.

A Gut Analysis Technique for Pinpointing Egg-Specific Predation Events.

A universal food immunomarking technique (UFIT) is described for postmortem gut analysis detection of predation on the egg stage of Lygus hesperus Knight (Hemiptera: Miridae). Collops vittatus Say (Coleoptera: Melyridae) and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) were fed a single L. hesperus egg that was marked with rabbit and chicken sera proteins. The protein-marked egg remnants were detectable in the guts of the majority of the predators by each sera-specific enzyme-linked immunosorbent assay (ELISA) for 3 to 6 h after a feeding event. A novel technique was then developed to expose protein-marked eggs to predators that simulated the L. hesperus endophytic oviposition behavior. The procedure entailed embedding L. hesperus eggs in an artificial substrate that mimicked the stem of a plant. A predator feeding choice study was then conducted in cages that contained a cotton plant and artificial stems containing endophytic (concealed) and exophytic (exposed) egg patches. The endophytic and exophytic egg treatments were marked with chicken and rabbit protein, respectively. The gut analyses revealed that higher proportions of both predator populations contained remnants of the exophytic egg treatment and L. hesperus eggs were more vulnerable to C. vittatus than H. convergens. This study shows how the UFIT can be used to pinpoint stage-specific feeding activity on two distinct egg exposure treatments (endophytic and exophytic) of the same species.

Field Evaluation of Potential Pheromone Lures for Lygus lineolaris (Hemiptera: Miridae) in the Mid-South.

Plant bugs (Hemiptera: Miridae) are phytophagous pests of cultivated plants around the world. In the mid-South region of the United States, Lygus lineolaris (Palisot de Beauvois) is a primary pest of cotton, and causes economic damage. Previously published research about the volatiles produced by members of the genus Lygus, and other closely related groups, indicated that they produce blends of hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal. Varying ratios of the three compounds were loaded into pipette tips, and screened in combination with non-UV white sticky cards for attractiveness to field populations of L. lineolaris in Mississippi. Field screening indicated that a lure expressing a ratio of 4:10:7 was the most effective at collecting L. lineolaris, and collected similar numbers of individuals to those reported in other studies using traps baited with live virgin insects over a similar period of time. Availability of a synthetic pheromone usable in the climate of the mid-South will enable broader scale landscape level monitoring for populations of L. lineolaris before movement into cotton fields and resulting economic damage.

RNA interference-mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus.

Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus.

ECDYSTEROID AND CHITINASE FLUCTUATIONS IN THE WESTERN TARNISHED PLANT BUG (Lygus hesperus) PRIOR TO MOLT INDICATE ROLES IN DEVELOPMENT.

Vital physiological processes that drive the insect molt represent areas of interest for the development of alternative control strategies. The western tarnished plant bug (Lygus hesperus Knight) is a pest of numerous agronomic and horticultural crops but the development of novel control approaches is impeded by limited knowledge of the mechanisms regulating its molt. To address this deficiency, we examined the fundamental relationship underlying the hormonal and molecular components of ecdysis. At 27°C L. hesperus exhibits a temporally controlled nymph-adult molt that occurs about 4 days after the final nymph-nymph molt with ecdysteroid levels peaking 2 days prior to the final molt. Application of exogenous ecdysteroids when endogenous levels had decreased disrupted the nymphal-adult molt, with treated animals exhibiting an inability to escape the old exoskeleton and resulting in mortality compared to controls. Using accessible transcriptomic data, we identified 10 chitinase-like sequences (LhCht), eight of which had protein motifs consistent with chitinases. Phylogenetic analyses revealed orthologous relationships to chitinases critical to molting in other insects. RT-PCR based transcript profiling revealed that expression changes to four of the LhChts was coordinated with the molt period and ecdysteroid levels. Collectively, our results support a role for ecdysteroid regulation of the L. hesperus molt and suggest that cuticle clearance is mediated by LhCht orthologs of chitinases that are essential to the molt process. These results provide the initial hormonal and molecular basis for future studies to investigate the specific roles of these components in molting.

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.

Identification and characterization of a sex peptide receptor-like transcript from the western tarnished plant bug Lygus hesperus.

Lygus hesperus females exhibit a post-mating behavioural switch that triggers increased egg laying and decreased sexual interest. In Drosophila melanogaster, these changes are controlled by sex peptide (SP) and the sex peptide receptor (DmSPR). In Helicoverpa armigera, SPR (HaSPR) also regulates some post-mating behaviour; however, myoinhibiting peptides (MIPs) have been identified as the SPR ancestral ligand, indicating that SPR is a pleiotropic receptor. In the present study, we identified a transcript, designated L. hesperus SPR (LhSPR), that is homologous to known SPRs and which is expressed throughout development and in most adult tissues. LhSPR was most abundant in female seminal depositories and heads as well as the hindgut/midgut of both sexes. In vitro analyses revealed that fluorescent chimeras of LhSPR, DmSPR and HaSPR localized to the cell surface of cultured insect cells, but only DmSPR and HaSPR bound carboxytetramethylrhodamine-labelled analogues of DmSP21-36 and DmMIP4. Injected DmSP21-36 also failed to have an effect on L. hesperus mating receptivity. Potential divergence in the LhSPR binding pocket may be linked to receptor-ligand co-evolution as 9 of 13 MIPs encoded by a putative L. hesperus MIP precursor exhibit an atypical W-X7 -Wamide motif vs the W-X6 -Wamide and W-X8 -Wamide motifs of Drosophila MIPs and SP.

Enhancing Biological Control Efficacy: Insights into the Feeding Behavior and Fitness of the Omnivorous Pest Lygus lineolaris.

Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), a true omnivorous insect, poses a significant threat to agriculture in the Neartic region. Understanding the feeding behavior of L. lineolaris is crucial for developing integrated pest management strategies. This study aimed to evaluate the effects of different diets on the fitness of L. lineolaris, with a focus on the diet source, feeding regime (phytophagy, zoophagy, and phytozoophagy), and number of diet items. The experimental design in the laboratory investigated the impacts of strawberry, canola and buckwheat flowers, as well as spider mites and aphids to explore relationships found in a conventional strawberry field. Results reveal that diet source, feeding regime, and the number of diet items influence L. lineolaris performance (i.e., survivorship rate, developmental time, and adult weight and length). Improvements in fitness are indicated by higher nymphal survival, shorter developmental time, and larger adults. Immature stages of L. lineolaris show improved fitness when provided with diets rich in canola compared to strawberry flowers and spider mites. Furthermore, the inclusion of multiple diet items in phytozoophagous regimes enhances insect performance. The findings emphasize the significance of understanding L. lineolaris' nutritional requirements and the biodiversity of target ecosystems for modeling energy flows and designing effective IPM strategies against this pest. This research contributes to the knowledge base for biological control programs targeting L. lineolaris in agricultural systems.

Microarray and Functional Pathway Analyses Revealed Significantly Elevated Gene Expressions Associated with Metabolic Resistance to Oxamyl (Vydate) in Lygus lineolaris.

The tarnished plant bug (TPB, Lygus lineolaris) remains a major pest for a variety of crops. Frequent sprays on row crops, especially cotton, prompted resistance development in field populations. To maintain chemical control as an effective tool against the pest, knowledge of global gene regulations is desirable for better understanding and managing the resistance. Novel microarray expressions of 6688 genes showed 685 significantly upregulated and 1382 significantly downregulated genes in oxamyl-selected TPBs (Vyd1515FF[R]) from a cotton field. Among the 685 upregulated genes (participated in 470 pathways), 176 genes code 30 different enzymes, and 7 of the 30 participate in 24 metabolic pathways. Six important detoxification pathways were controlled by 20 genes, coding 11 esterases, two P450s, two oxidases, and three pathway-associated enzymes (synthases, reductase, and dehydrogenase). Functional analyses showed substantially enhanced biological processes and molecular functions, with hydrolase activity as the most upregulated molecular function (controlled by 166 genes). Eleven esterases belong to the acting on ester bond subclass of the 166 hydrolases. Surprisingly, only one GST showed significant upregulation, but it was not involved in any detoxification pathway. Therefore, this research reports a set of 20 genes coding 6 enzyme classes to detoxify a carbamate insecticide oxamyl in Vyd1515FF. Together with three previous reports, we have obtained the best knowledge of resistance mechanisms to all four conventional insecticide classes in the economically important crop pest. This valuable finding will greatly facilitate the development of molecular tools to monitor and manage the resistance and to minimize risk to environment.

A chromosome scale assembly of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), genome.

OBJECTIVE: The tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), is a pest damaging many cultivated crops in North America. Although partial transcriptome data are available for this pest, a genome assembly was not available for this species. This assembly of a high-quality chromosome-length genome of TPB is aimed to develop the genetic resources that can provide the foundation required for advancing research on this species. RESULTS: The initial genome of TPB assembled with paired-end nucleotide sequences generated with Illumina technology was scaffolded with Illumina HiseqX reads generated from a proximity ligated (HiC) library to obtain a high-quality genome assembly. The final assembly contained 3963 scaffolds longer than 1 kbp to yield a genome of 599.96 Mbp. The N50 of the TPB genome assembly was 35.64 Mbp and 98.68% of the genome was assembled into 17 scaffolds larger than 1 Mbp. This megabase scaffold number is the same as the number of chromosomes observed in karyotyping of this insect. The TPB genome is known to have high repetitive DNA content, and the reduced assembled genome size compared to flowcytometric estimates of approximately 860 Mbp may be due to the collapsed assembly of highly similar regions.

Early Season Monitoring of Tarnished Plant Bug, Lygus lineolaris, in Wild Hosts Using Pheromone Traps.

The tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae), has a wide host range of over 700 plant species, including 130 crops of economic importance. During early spring, managing the field edges with weeds and other wild hosts is important in preventing early-season infestations of L. lineolaris in cotton to prevent damage to the squares and other fruiting structures. Scouting fields for L. lineolaris is time- and labor-intensive, and end-user variability associated with field sampling can lead to inaccuracies. Insect traps that combine visual cues and pheromones are more accurate, sustainable, and economically feasible in contrast to traditional insect detection methods. In this study, we investigated the application of red or white sticky cards baited with the female-produced sex pheromone to monitor overwintering L. lineolaris populations in early spring. Field experiments demonstrated that the red sticky cards baited with a pheromone blend containing hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal in 4:10:7 ratio are highly effective in trapping L. lineolaris adults in early spring before the row crops are planted, and in monitoring their movement into a cotton crop. The monitoring of L. lineolaris should help growers to make judicious decisions on insecticide applications to control early pest infestations, thereby reducing economic damage to cotton.

Resistance risk assessment of six pyrethroids and acephate toward the resistant adult tarnished plant bug, Lygus lineolaris.

Due to rapidly developed resistance, pest management relies less on pyrethroids to control economically damaging infestations of the tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois) in cotton fields of Mississippi. Yet, pyrethroid resistance remains prevalent in TPB populations. This study assessed the resistance levels in adult TPB to six common pyrethroids and acephate. Resistant TBPs were collected from wild host plants in late October after harvest in the Mississippi Delta region of the United States. Based on LC50 values, the field-resistant TPBs displayed higher resistance to permethrin, esfenvalerate, and bifenthrin (approximately 30 fold) and moderate resistance to λ-cyhalothrin, ß-cyfluthrin, ζ-cypermethrin, and acephate (approximately 15 fold). Further investigations showed that the inhibitors of three detoxification enzyme, triphenyl phosphate (TPP), diethyl maleate (DEM), and piperonyl butoxide (PBO) had synergistic effects on permethrin, λ-cyhalothrin, and bifenthrin in resistant TPBs. Furthermore, elevated esterase, GST, and P450 activities were significantly expressed in field-resistant TPBs. Additionally, GST and esterase were reduced after 48 h exposure to certain pyrethroids at LC50 dose. The synergistic and biochemical assays consistently indicated that P450 and esterase were involved in pyrethroid detoxification in TPBs. This study provides valuable information for the continued use of pyrethroids and acephate in controlling TPBs in cotton fields in the Mississippi Delta region of the United States.

Population Dynamics of Insect Pests and Beneficials on Different Snap Bean Cultivars.

Snap bean is an important crop in the United States. Insecticides are commonly used against pests on snap bean, but many pests have developed resistance to the insecticides and beneficials are threatened by the insecticides. Therefore, host plant resistance is a sustainable alternative. Population dynamics of insect pests and beneficials were assessed on 24 snap bean cultivars every week for six weeks. The lowest number of sweetpotato whitefly (Bemisia tabaci) eggs was observed on cultivar 'Jade', and the fewest nymphs were found on cultivars 'Gold Mine', 'Golden Rod', 'Long Tendergreen', and 'Royal Burgundy'. The numbers of potato leafhopper (Empoasca fabae) and tarnished plant bug (Lygus lineolaris) adults were the lowest on cultivars 'Greencrop' and 'PV-857'. The highest numbers of adults were found in Week 1 (25 days following plant emergence) for B. tabaci and Mexican bean beetle (Epilachna varivestis); Week 3 for cucumber beetle, kudzu bug (Megacopta cribraria), and E. fabae; Weeks 3 and 4 for thrips; Week 4 for L. lineolaris; and Weeks 5 and 6 for bees. Temperature and relative humidity correlated with B. tabaci, E. varivestis, bee, and predator ladybird beetle populations. These results provide valuable information on the integrated pest management of snap beans.

The mechanisms of metabolic resistance to pyrethroids and neonicotinoids fade away without selection pressure in the tarnished plant bug Lygus lineolaris.

BACKGROUND: Heavy selection pressure prompted the development of resistance in a serious cotton pest tarnished plant bug (TPB), Lygus Lineolaris in the mid-southern United States. Conversely, a laboratory resistant TPB strain lost its resistance to five pyrethroids and two neonicotinoids after 36 generations without exposure to any insecticide. It is worthwhile to examine why the resistance diminished in this population and determine whether the resistance fade away has practical value for insecticide resistance management in TPB populations. RESULTS: A field-collected resistant TPB population in July (Field-R1) exhibited 3.90-14.37-fold resistance to five pyrethroids and two neonicotinoids, while another field-collected TPB population in April (Field-R2) showed much lower levels of resistance (0.84-3.78-fold) due to the absence of selection pressure. Interestingly, after 36 generations without exposure to insecticide, the resistance levels in the same population [laboratory resistant strain (Lab-R)] significantly decreased to 0.80-2.09-fold. The use of detoxification enzyme inhibitors had synergistic effects on permethrin, bifenthrin and imidacloprid in resistant populations of Lygus lineolaris. The synergism was more pronounced in Field-R2 than laboratory susceptible (Lab-S) and Lab-R TPB population. Moreover, esterase, glutathione S-transferase (GST), and cytochrome P450-monooxygenases (P450) enzyme activities increased significantly by approximately 1.92-, 1.43-, and 1.44-fold in Field-R1, respectively, and 1.38-fold increased P450 enzyme activities in Field-R2 TPB population, compared to the Lab-S TPB. In contrast, the three enzyme activities in the Lab-R strain were not significantly elevated anymore relative to the Lab-S population. Additionally, Field-R1 TPB showed elevated expression levels of certain esterase, GST and P450 genes, respectively, while Field-R2 TPB overexpressed only P450 genes. The elevation of these gene expression levels in Lab-R expectedly diminished to levels close to those of the Lab-S TPB populations. CONCLUSION: Our results indicated that the major mechanism of resistance in TPB populations was metabolic detoxification, and the resistance development was likely conferred by increased gene expressions of esterase, GST, and P450 genes, the fadeaway of the resistance may be caused by reversing the overexpression of esterase, GST and P450. Without pesticide selection, resistant gene (esterase, GST, P450s) frequencies declined, and detoxification enzyme activities returned to Lab-S level, which resulted in the recovery of the susceptibility in the resistant TPB populations. Therefore, pest's self-purging of insecticide resistance becomes strategically desirable for managing resistance in pest populations. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

A Dynamic Threshold Approach for Tarnished Plant Bug (Hemiptera: Miridae) Management in the Midsouthern U.S. Cotton.

One of the most economically important pests of cotton, Gossypium hirsutum L., in the midsouth region of the United States is the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois, Hemiptera: Miridae). Tarnished plant bug populations across the region have exhibited widespread resistance to numerous insecticide classes. To minimize late season resistance development, reducing unwarranted applications during the late flowering period can aid in resistance management and potentially reduce input costs. Trials were conducted during 2019 and 2020 to evaluate the impacts of tarnished plant bug populations in the later flowering period of cotton by modifying or terminating threshold regimes during the later weeks of bloom. Results showed that dynamic thresholds altered at the fourth week of bloom or later can reduce the number of late season applications made with no penalty to yield. Additionally, when utilizing a week of bloom termination approach, no significant yield losses were seen when terminating applications after the fourth week of bloom. These data may offer an alternative method to managing tarnished plant bug populations during the later flowering period of midsouth cotton.

Sampling Optimization and Crop Interface Effects on Lygus lineolaris Populations in Southeastern USA Cotton.

Tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae), is an economically damaging pest in cotton production systems across the southern United States. We systematically scouted 120 commercial cotton fields across five southeastern states during susceptible growth stages in 2019 and 2020 to investigate sampling optimization and the effect of interface crop and landscape composition on L. lineolaris abundance. Variance component analysis determined field and within-field spatial scales, compared with agricultural district and state, accounted for more variation in L. lineolaris density using sweep net and drop cloth sampling. This result highlights the importance of field-level scouting efforts. Using within-field samples, a fixed-precision sampling plan determined 8 and 23 sampling units were needed to determine L. lineolaris population estimates with 0.25 precision for sweep net (100 sweeps per unit) and drop cloth (1.5 row-m per unit) sampling, respectively. A spatial Bayesian hierarchical model was developed to determine local landscape (<0.5 km from field edges) effects on L. lineolaris in cotton. The proportion of agricultural area and double-crop wheat and soybeans were positively associated with L. lineolaris density, and fields with more contiguous cotton areas negatively predicted L. lineolaris populations. These results will improve L. lineolaris monitoring programs and treatment management decisions in southeastern USA cotton.