Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.

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Soybean flour and wheat germ are the two most important protein components of wheat germ-based insect artificial diets. The effect of modifying the proportion of these two ingredients in a Noctuidae-specific diet was investigated utilizing the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae), with the goal of developing a suboptimal diet that, without drastically affecting this insect's growth and reproductive rates, could manifest subtle negative effects in this insect. The original diet formula contained 2.51% protein. When the proportions of soybean flour and wheat germ were changed to 2.15% protein the net reproductive rate of the first generation was significantly lower. In the second generation, the net reproductive rate, development time, percent female survivorship, fertility, intrinsic rate of increase, finite rate of increase and female longevity were significantly lower in both the 2.15% and 2.26% protein diets. The survival rate of immatures to the adult stage was 1% in the 2.05% protein diet in the first generation. Interestingly, females exposed to these suboptimal diets produced a significantly higher number of eggs but the survival of their larvae was significantly reduced. It is evident from these results that modifications to the protein content and the nutrient composition profile of the original wheat germ-based insect artificial formula can be used to produce subtle negative effects on the growth of tobacco budworm.

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.

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.

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.