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.

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.

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.

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.

Identification of the western tarnished plant bug (Lygus hesperus) olfactory co-receptor Orco: expression profile and confirmation of atypical membrane topology.

Lygus hesperus (western tarnished plant bug) is an agronomically important pest species of numerous cropping systems. Similar to other insects, a critical component underlying behaviors is the perception and discrimination of olfactory cues. Consequently, the molecular basis of olfaction in this species is of interest. To begin to address this issue, we utilized homology-based PCR as a commonly accepted abbreviation but if necessary it is polymerase chain reaction methods to identify the L. hesperus olfactory receptor co-receptor (Orco) ortholog, a receptor that has been shown to be essential for olfaction. The L. hesperus Orco (LhOrco) shares significant sequence homology with known Orco proteins in other insects. Parallel experiments using the sympatric sister species, Lygus lineolaris (tarnished plant bug), revealed that the Lygus Orco gene was completely conserved. Surprisingly, a majority of the membrane topology prediction algorithms used in the study predicted LhOrco to have both the N and C terminus intracellular. In vitro immunofluorescent microscopy experiments designed to probe the membrane topology of transiently expressed LhOrco, however, refuted those predictions and confirmed that the protein adopts the inverted topology (intracellular N terminus and an extracellular C terminus) characteristic of Orco proteins. RT-PCR analyses indicated that LhOrco transcripts are predominantly expressed in adult antennae and to a lesser degree in traditionally nonolfactory chemosensory tissues of the proboscis and legs. Expression is not developmentally regulated because transcripts were detected in all nymphal stages as well as eggs. Taken together, the results suggest that LhOrco likely plays a critical role in mediating L. hesperus odorant perception and discrimination.

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.

Effect of modification of the NI artificial diet on the biological fitness parameters of mass reared western tarnished plant bug, Lygus hesperus.

The NI artificial diet is the only known successful diet for mass rearing the western tarnished plant bug, Lygus hesperus Knight (Hemiptera: Miridae). This diet has been used for more than a decade. However, because it contains cooked chicken egg, and thus requires laborious preparation (Cohen 2000), this diet is difficult to use. Three modifications (D1, D2, D3) of the NI diet were investigated in hopes of developing a more easily prepared diet that avoids the cooked egg and improves mass fitness parameters of L. hesperus. The modified D3 diet, containing autoclaved chicken egg yolk based component, had the highest egg/cage/day production (13120 ± 812 SE). This was significantly greater than diets D1, containing autoclaved dry chicken egg yolk based component (9027 ± 811 SE), D2, containing autoclaved chicken egg white based component (8311 ± 628 SE), and NI, which contained autoclaved chicken egg yolk + cooked egg diet (7890 ± 761 SE). Significant differences were observed in the weights of all developmental stages except for eggs and first instar nymphs. Higher rates of fertility, hatchability, and low mortality in nymphs during the first instar were also obtained in the modified D3 diet. The results clearly indicated that the D3 diet provided an opportunity to significantly reduce rearing cost by avoiding time-consuming issues with preparation of a cooked egg diet. This should result in an increase in production capacity and a reduction in production costs.

Oviposition and development of the tarnished plant bug (Heteroptera: Miridae) on field maize.

Reduced insecticide use in cotton, Gossypium hirsutum L., as a consequence of the Boll Weevil Eradication Program and the broad adoption of Bt cotton, have helped make the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), a consistent pest of cotton each year in the mid-south. Maize, Zea mays L., has been implicated as having a role in the season-long dynamics of tarnished plant bug infestations in cotton. To date, no published information exists describing the quality of maize as a host for tarnished plant bug. No-choice field studies indicated that adult tarnished plant bug females oviposited into maize leaves, tassels, and ears. Laboratory studies showed that first-instar tarnished plant bugs could successfully develop to the adult stage when fed maize silks at the R1 growth stage, tassels before (VT) and during (R1) pollen shed, and milk stage (R3) kernels from the tip and base of the ear. The proportion of nymphs surviving to the adult stage on these tissues was often similar to that of broccoli, Brassica oleracea L. Nymphs did not develop to adults when fed V5 or R1 maize leaves. However, survival of first instars to the adult stage was improved when nymphs fed on tassels with pollen for 6 d and then moved to silks or leaves. Another field study showed that tarnished plant bugs reproduced in maize mainly during the tassel (VE and VT) and the R1-R3 ear growth stages, and a single new generation was produced in maize during these stages. The highest population recorded during the study (24 June 2005) consisted mostly of nymphs and was estimated to be 29,600/ha (12,000/acre). These studies showed that maize is a suitable host for tarnished plant bug reproduction and development, and its production plays a significant role in the population dynamics of the tarnished plant bug in the mid-south.

Plant host effect on the development of Heliothis virescens F. (Lepidoptera: Noctuidae).

Heliothis virescens F. is an important polyphagous pest that can develop on >100 plant species, including 20 economic crops. Populations of this insect are believed to be locally maintained on a few crops and weed hosts in Washington County, MS. To find the intrinsic value of these plants for the development of H. virescens populations, we fed different laboratory and wild colonies with fresh and lyophilized plant tissue under a constant temperature. Development time of this insect under laboratory conditions varied up to 10 d between plant hosts and was dependent on the type of plant tissue provided: fresh or lyophilized. Life table parameters such as net reproductive rate, finite rate of increase, and generation time indicated that Trifolium repens, a wild host growing around agricultural fields year round, could be one of the most suitable local plant hosts for the development of H. virescens. Two species of Geranium, previously reported as the source of the first H. virescens generation in the region, had lower intrinsic value as a food source than did T. repens. Gossyipium hirsutum, perhaps the most important crop source of H. virescens in the region, produced low net reproductive rate and finite rate of increase parameters. Sampling conducted in agricultural fields during 2006 and 2007 found no larvae on the above mentioned wild hosts as it was previously reported. Results indicated that H. virescens populations in this region were not supported by the wild plant species growing around agricultural fields during the time when the survey took place.

Enhanced esterase gene expression and activity in a malathion-resistant strain of the tarnished plant bug, Lygus lineolaris.

Extensive use of insecticides on cotton in the mid-South has prompted resistance development in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois). A field population of tarnished plant bugs in Mississippi with 11-fold higher resistance to malathion was used to examine how gene regulation conferred resistance to this organophosphate insecticide. In laboratory bioassays, synergism by the esterase inhibitors S,S,S,-tributylphosphorotrithioate (DEF) and triphenylphosphate (TPP) effectively abolished resistance and increased malathion toxicity by more than 80%. Esterase activities were compared in vitro between malathion susceptible and resistant (selected) strains. More than 6-, 3- and 10-fold higher activities were obtained with the resistant strain using alpha-naphthyl acetate, beta-naphthyl acetate, and p-nitrophenyl acetate, respectively. Up to 95% and 89% of the esterase activity in the susceptible and resistant strains, respectively, was inhibited by 1 mM DEF. Inhibition of esterase activity up to 75% and 85% in the susceptible and resistant strains, respectively, was obtained with 0.03 mM TPP. Esterase activities in field populations increased by up to 5.4-fold during the fall season. The increase was synchronized with movement of the insect into cotton where exposure to pesticides occurred. Esterase cDNA was cloned and sequenced from both malathion susceptible and resistant strains. The 1818-nucleotide cDNA contained a 1710-bp open reading frame coding a 570 amino acid protein which was similar to many insect esterases conferring organophosphate resistance. No amino acid substitution was observed between susceptible and resistant strains, indicating that esterase gene mutation was not involved in resistance development in the resistant strain in Mississippi. Further examination of esterase gene expression levels using quantitative RT-PCR revealed that the resistant strain had a 5.1-fold higher level of esterase mRNA than the susceptible strain. The results of this study indicated that up-regulation of the esterase gene appeared to be related to the development of resistance in the tarnished plant bug.

Effect of ULV malathion use in boll weevil (Coleoptera: Curculionidae) eradication on resistance in the tarnished plant bug (Heteroptera: Miridae).

Tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), from regions 1, 2, and 3 of the boll weevil, Anthonomous grandis Boheman, eradication program in Mississippi were collected from wild hosts and tested for malathion resistance during the spring and fall of 2000 and 2001. Plant bugs were also tested in region 1 in late-July and October of 1999, just before and after multiple applications of ultra-low-volume (ULV) malathion were used for reproduction-diapause control of boll weevils in August and September. Regions 1 (north Delta), 2 (south Delta), and 3 (hills) began boll weevil eradication in 1999, 1998, and 1997, respectively. A glass-vial bioassay was used to determine resistance in plant bugs to malathion by comparing LC50 values against an LC50 value obtained for susceptible plant bugs. Comparison of the LC50 value obtained for plant bugs at a location in the spring was also made with the LC50 value obtained in the fall at the same location. After multiple applications of malathion made for reproduction-diapause boll weevil control in region 1 in August and September, malathion resistance increased by 4.9-, 6.5-, and 20.8-fold in plant bug populations from the three test locations. Results from testing bugs from all three eradication regions were similar. Malathion resistance usually increased significantly from spring to fall and then declined significantly from fall to spring of the next year. Despite reduced use of malathion in all three eradication regions for boll weevils in 2001, resistance to malathion in plant bugs still increased significantly from spring to fall at all test locations in regions 1 and 2 (the Delta). Malathion resistance did not increase significantly in plant bug populations in region 3 (the hills) in 2001 from spring to fall at three of four test locations in this year. Possible causes for the higher malathion resistance found in plant bugs in the Delta are discussed. Overall test results showed that the use of malathion in boll weevil eradication in cotton probably contributed to increases in resistance to malathion in plant bug populations in the eradication areas. However, the expression of this resistance was usually rapidly lost by spring of the following year. Boll weevil eradication did not seem to produce a permanent increase in the expression of malathion resistance in tarnished plant bug populations found in the eradication regions.