Govind T. Gujar

Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India.

BACKGROUND The pink bollworm is one of the most destructive pests of cotton. Transgenic cotton producing Bt toxin Cry1Ac or a combination of Cry1Ac and Cry2Ab2 has been used effectively against this pest. However, some other insects have evolved resistance to Bt toxins in the field. During the 2007-2008 and 2008-2009 seasons, pink bollworm populations in India were surveyed to evaluate their responses to Cry1Ac and seed powder containing Cry1Ac and Cry2Ab2. RESULTS The results provide evidence that resistance to Cry1Ac had evolved by 2008 in a population sampled from non-Bt cotton in the Amreli district of Gujarat in western India. The median lethal concentration of Cry1Ac for five-day-old larvae (LC50 ) was significantly higher for insects derived in 2008 from Amreli than for any of the other field populations tested from four locations in India. For Cry1Ac, the mean LC50 for the strain derived from Amreli in 2008 was 44 times higher than for the most susceptible population. However, for seed powder of Bollgard II containing primarily Cry2Ab2, the 2008 Amreli population was only slightly less susceptible than the most susceptible population. CONCLUSIONS The data reported here constitute the first evidence of field-evolved resistance of pink bollworm to Cry1Ac. This initial evidence spurred more extensive evaluations during the 2009-2010 growing season, which confirmed field-evolved resistance to Cry1Ac in Amreli. The lack of cross-resistance to Cry2Ab2 suggests that plants producing this toxin are likely to be more effective against resistant populations than plants producing only Cry1Ac.

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera).

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.

Inheritance of Cry1Ac resistance and associated biological traits in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae).

The analysis of reciprocal genetic crosses between resistant Helicoverpa armigera strain (BH-R) (227.9-fold) with susceptible Vadodara (VA-S) strain showed dominance (h) of 0.65-0.89 and degree of dominance (D) of 0.299-0.782 suggesting Cry1Ac resistance as a semi-dominant trait. The D and h values of F(1) hybrids of female resistant parent were higher than female susceptible parent, showing maternally enhanced dominance of Cry1Ac resistance. The progeny of F(2) crosses, backcrosses of F(1) hybrid with resistant BH-R parent did not differ significantly in respect of mortality response with resistant parent except for backcross with female BH-R and male of F(1) (BH-RxVA-S) cross, suggesting dominant inheritance of Cry1Ac resistance. Evaluation of some biological attributes showed that larval and pupal periods of progenies of reciprocal F(1) crosses, backcrosses and F(2) crosses were either at par with resistant parent or lower than susceptible parent on treated diet (0.01 microg/g). The susceptible strain performed better in terms of pupation and adult formation than the resistant strain on untreated diet. In many backcrosses and F(2) crosses, Cry1Ac resistance favored emergence of more females than males on untreated diet. The normal larval period and the body weight (normal larval growth) were the dominant traits associated with susceptible strain as contrast to longer larval period and the lower body weight (slow growth) associated with resistance trait. Further, inheritance of larval period in F(2) and backcross progeny suggested existence of a major resistant gene or a set of tightly linked loci associated with Cry1Ac sensitivity.

Host crop influence on the susceptibility of the American bollworm, Helicoverpa armigera, to Bacillus thuringiensis ssp. kurstaki HD‐73

Studies on the susceptibility of F1 neonates of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) collected from chickpea in Delhi and cotton in Punjab, Haryana and Rajasthan in northern India, to Bacillus thuringiensis ssp. kurstaki HD‐73, and the impact of host crop diets on insect susceptibility, were carried out by diet incorporation bioassays. The susceptibility of F1 neonates of H. armigera to Bacillus thuringiensis ssp. kurstaki HD‐73 ranged from twofold (LC50 96 h, 84.5–164.2 µg (ai) l−1) for chickpea to about fivefold (LC50 96 h, 51.1–247.7 µg (ai) l−1) for cotton. The F1 neonates of insects collected from pearl millet were twice as tolerant as those collected from cotton and sunflower at Sirsa to B. thuringiensis ssp. kurstaki HD‐73, suggesting that there was an influence of host crops on insect susceptibility. Insects originally collected from cotton fields at Bhatinda and reared for four generations on a chickpea‐based meridic diet were used to initiate host‐specific colonies of H. armigera. These host‐specific colonies were allowed to complete one generation on meridic diets prepared with different hosts, viz., cabbage, cauliflower, chickpea, green pea, pearl millet, and pigeon pea. Larvae of H. armigera were heaviest on the 15th day, and had a higher growth rate on a pigeon pea‐based diet than all other host diets. The larval period was shorter on chickpea and pigeon pea, with higher percentage pupation than all other host‐diets. The pupal weight of H. armigera was greater on chickpea and pigeon pea diets than on other host diets. The growth and development of larvae was significantly poorer on pearl millet diet than on other host diets. The F1 neonates of H. armigera belonging to cabbage, cauliflower, and pearl millet host‐specific colonies were more susceptible than those belonging to chickpea, green pea, and pigeon pea host‐specific colonies to B. thuringiensis ssp. kurstaki HD‐73, suggesting the importance of proteinaceous nutrients in tolerance. The F1 neonates of the pearl millet colony of H. armigera grown on a chickpea‐diet for 4 days were significantly more tolerant to B. thuringiensis ssp. kurstaki HD‐73 than those reared on the pearl millet‐based diet. These studies show the impact of the host diet of H. armigera on tolerance to B. thuringiensis.

Differential toxicity of Bacillus thuringiensis strains and their crystal toxins against high-altitude Himalayan populations of diamondback moth, Plutella xylostella L.

BACKGROUND Diamondback moth, Plutella xylostella (L.), is a major insect pest of crucifers in the biodiversity-rich north-western Indian Himalayan hills. The present investigation was aimed at determining the susceptibility pattern of P. xylostella populations collected from different locations of this region to autochthonous and standard Bacillus thuringiensis strains. RESULTS Among the reference as well as indigenous B. thuringiensis strains tested, sub spp. kurstaki HD-1, kurstaki HD-73, galleriae HD-8, local galleriae/colmeri strain BtOa1 and some of their Cry1 class toxins were found to be highly toxic. Surprisingly, the sub sp. tolworthi HD-125, local tolworthi strain BtHa1 and Cry9 class toxins were found to be non-toxic. Midgut homogenate from fourth-instar larvae was found to activate 130 kDa protoxin from the local tolworthi strain BtHa1 into 68 kDa toxin, but failed to exert any larval mortality, probably owing to lack of receptor binding. CONCLUSION The present study provides valuable baseline susceptibility data for the deployment of B. thuringiensis-based control methods, as well as for future monitoring of development of resistance in P. xylostella to B. thuringiensis in this ecologically sensitive region.

Hemocyte diversity of the American bollwormHelicoverpa armigera

The hemocytes of the American bollwormHelicoverpa armigera (Hübner) were studied in the last instar larvae by phase contrast microscopy to investigate the differences among 14 populations, collected from ten locations, spread over a distance of approximately 3000 km in six different states of India. The cluster analysis of differential hemocytes showed as many as 11 clusters of populations at 5% and six clusters at 10% homogeneity level. The cluster analysis of hemocytes of seven populations from cotton crops showed five and three clusters at 5% and 10% homogeneity levels, respectively. Diversity of hemocytes of four populations from chickpea was wider than that from cotton. The differences in hemocytes of various populations may be due to space, time, host plant, insecticide use, other agroecological conditions, and insect genetic variabilityper se. The field populations ofH. armigera heavily treated with insecticides appeared to contain more spherulocytes at the expense of plasmatocytes and granulocytes. The treatment of 6-day-old larvae ofH. armigera with cypermethrin decreased plasmatocytes and granulocytes, and increased prohemocytes and spherulocytes in the hemolymph of last instar larvae. Thus, differential hemocyte counts may serve as an indicator of insecticide exposure and the diversity of insect populations.