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.

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.

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.

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.

The complete genome sequence of a single-stranded RNA virus from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois).

The complete genome sequence of a single-stranded RNA virus infecting the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by sequencing cDNA prepared from insects collected from the Mississippi Delta. The 9655 nucleotide positive-sense single-stranded RNA genome of the L. lineolaris single-stranded RNA virus (LyLV-1) contained a single open reading frame of 8958 nucleotides encoding a 2986 amino acid genome polypeptide. The open reading frame was flanked by untranslated regions of 603 and 69 nucleotides at the 5'- and 3'- ends of the genome, respectively. Database searches and homology based modeling was used to identify four capsid proteins (VP1-VP4), helicase/AAA-ATPase, cysteine protease (C3P), protease 2A, and the RNA-directed RNA polymerase (RdRp). In addition, a region with weak similarity to the eukaryotic structural maintenance of chromosome (SMC) domain was identified near the amino-terminal of the polyprotein and adjacent to the VP1 domain. The amino acid sequence of LyLV-1 was approximately 44.4% similar to that of sacbrood virus (SBV) of the honey bee. The genomic organization of both viruses showed remarkable similarity with the exception of highly divergent amino acid regions flanking fairly conserved structural and non-structural polypeptide regions. High similarity to the SBV genome and similarities in the genome organization and amino acid sequence with the viruses of the family Iflaviridae suggested that LyLV-1 was a novel member of this family. Virus particles were 39 nm in diameter and appeared to transmit vertically via eggs. Although this virus may only cause covert infections under normal conditions, the potential for using this virus in biological control of L. lineolaris is discussed.