Maribel Portilla

Reduced Levels of Membrane-Bound Alkaline Phosphatase Are Common to Lepidopteran Strains Resistant to Cry Toxins from Bacillus thuringiensis

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.

Susceptibility of isofamilies of Spodoptera frugiperda (Lepidoptera: Noctuidae) to Cry1Ac and Cry1Fa proteins of Bacillus thuringiensis.

Abstract. The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the most important insect pests on the American continent. Its control has relied primarily on multiple applications of insecticides that can amount to 1,000 g of active ingredient per hectare on some of approximately 30 crops the insect damages. The use of genetically engineered crops that express Bacillus thuringiensis (Bt) Berliner toxins, Bt-corn, Zee meys L.; and Bt-cotton, Gossypium hirsutum L.; are other ways to control this insect. However, fall armyworm is one of the Lepidoptera species least susceptible to Bt proteins, and a case of high tolerance to Bt-corn has already being reported. We found the susceptibility to Cry1Ac and Cry1Fa proteins of Bt in 133 isofamilies from five regions of three countries was similar to the susceptibility of two Bt-susceptible laboratory colonies to these proteins. Four isofamilies from Puerto Rico were very tolerant to Cry1Fa and not so tolerant to Cry1Ac. Two of the four isofamilies were backcrossed with a Bt-susceptible laboratory colony and their progeny was as susceptible to both Bt proteins as was the Bt-susceptible colony, indicating that resistance to Bt is a recessive trait.

Soybean flour and wheat germ proportions in artificial diet and their effect on the growth rates of the tobacco budworm, Heliothis virescens

Abstract 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 insects 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.

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

Abstract 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.

Effect of Modification of the NI Artificial Diet on the Biological Fitness Parameters of Mass Reared Western Tarnished Plant Bug, Lygus hesperus

Abstract 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.

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.

Estimation of Median Lethal Concentration of Three Isolates of Beauveria bassiana for Control of Megacopta cribraria (Heteroptera: Plataspidae) Bioassayed on Solid Lygus spp. Diet.

The kudzu bug, Megacopta cribraria (F.), is an urban nuisance and significant agricultural pest. The median lethal concentrations of three strains of Beauveria bassiana (Balsamo), including the Mississippi Delta native strain (NI8) isolated from Lygus lineolaris (Palisot de Beauvois), the commercial strain BotaniGard® (GHA) (Victor, NY, USA), and the B. bassiana strain isolated from M. cribraria (KUDSC), were estimated on kudzu bug adults. A technique developed to evaluate B. bassiana against L. lineolaris was used. Younger adults (eight days after collection) were treated with NI8 and GHA and older adult (50 days after collection) were treated with NI8, GHA and KUDSC. Higher concentrations (n × 106, n × 107) of NI8 and GHA caused kudzu bug mortality two days after treatment in younger adults and similar concentrations of NI8, GHA, and KUDSC caused mortality one day after treatment in older adults. Lower concentrations (n × 104, n × 105) were not significantly different in mortality between strains. LS50 values of the KUDSC were significantly lower than NI8 and GHA values in older adults. This is the first available information on median lethal concentration of B. bassiana on kudzu bug adults bioassayed on artificial diet. It was determined that B. bassiana (KUDSC and NI8) are highly effective for young adults at very low doses (LC50 1.98–4.98 viable spores per mm2).

Simple Methods to Devitalize Eggs and Larvae of Heliothis virescens and Helicoverpa zea under Laboratory Conditions

Abstract. Heliothine moths are economically damaging insect pests, significantly affecting world crop production each year. Several species in the subfamily are commonly used in agricultural research laboratories. Research protocols should include methods to ensure that excess individuals are completely devitalized before being discarded, preventing further spread of the pests. Processes such as ultracold temperature freezing, autoclaving, or holding insects under adverse conditions for a long period of time are commonly used devitalization methods. Each method adds significant amounts of time and cost; also increasing competition for space in already crowded laboratories. While these frequently used methods might be necessary for some very resilient species, other species can succumb under less elaborate methods of devitalization. One hour of exposure to undiluted household bleach, brief exposure (>20 seconds) of wetted individuals to microwave radiation, storage in a household freezer for 48 hours, and contact with 70% isopropyl alcohol for 24 hours completely devitalized eggs and L1 larvae of corn earworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (Fab.). These very effective methods might also be applicable for other noctuid species.

Abridged Life Tables for Cephalonomia stephanoderis and Prorops nasuta (Hymenoptera: Bethylidae) Parasitoids of Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) Reared on Artificial Diet

Biological aspects and demographic parameters of Cephalonomia stephanoderis Betrem (Hymenoptera: Bethylidae) and Prorops nasuta Waterston (Hymenoptera: Bethylidae) parasitoids of the coffee berry borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae) were investigated using diet-reared CBB hosts. Developmental time from eggs to adults, oviposition, and postoviposition period were comparable for both parasitoids. However, P. nasuta had a considerably longer preoviposition and longevity period averaging 17.3 and 63.1 d, respectively. The reproductive rate for C. stephanoderis was 46.1 daughters per female with a mean generation time of 47.4 d, whereas P. nasuta had a reproductive rate of 18.3 daughters per female in a mean time of 58.6 d. Oviposition behavior was also different with C. stephanoderis typically ovipositing on CBB prepupae and pupae, while P. nasuta preferred prepupae and second-instar CBB larvae. An abridged cohort life table for both parasitoids was constructed for growth rates estimations.

Lethality of the Entomogenous Fungus Beauveria bassiana Strain NI8 on Lygus lineolaris (Hemiptera: Miridae) and its Possible Impact on Beneficial Arthropods

Abstract Bioassays were conducted to examine the pathogenicity of the fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales), strain NI8, against Lygus lineolaris (Palisot de Beauvois) and its impact on beneficial arthropods including Apis mellifera L., Crysoperla rufrilabris Burmeister, Orius insidiosus Say, Hippodamia convergens Guérin-Méneville, Harmonia axyridis (Pallas), Coleomegilla maculata De Geer, and field-collected Aranea spiders Salticidae and Thomisidae. Insects were treated with four concentrations of NI8 (3.9 × 104, 2.3 × 105, 4.2 × 106, and 1.5 × 107 spores/ml) directly via topical spray. Median lethal concentration (LC50), sporulation response, and resistance ratio were estimated for all species except for the two groups of spiders. No significant differences in mortality (10 d after application) were observed among L. lineolaris, A. mellifera, and C. rufilabris, and all three species were highly affected when exposed to the highest concentrations of B. bassiana with 99.0, 98.2, and 90.0% mortality, respectively. Between 35 and 45% of the tested populations of O. insidiosus, H. convergens, field-collected crab spiders, and C. maculata were killed at 1.5 × 107 spores/ml; whereas only 22 and 27%, respectively, of the field-collected jumping spiders and H. axyridis were killed at 10 d with the same concentration. No significant differences were found between the LC50s measured for L. lineolaris and C. rufilabris 10 d after application. Results suggested that C. rufilabris would be highly affected by the NI8 strain of B. bassiana when applied for control of L. lineolaris. In contrast, B. bassiana appeared to have lower effects on the other beneficial arthropods assayed at the concentrations targeted for L. lineolaris control.