Toxicity of Bacillus thuringiensis Cry2Ab and the inheritance of Cry2Ab resistance in the Pink bollworm, Pectinophora gossypiella (Saunders).

Though the insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis (Bt) are effective against insect pests, evolution of resistance remains an issue of great concern. Here, we explored the ability of pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae), a key pest of cotton, to evolve resistance to Cry2Ab and characterized it in terms of inheritance. Sixteen day bioassay of Bt Cry2Ab toxin against 5-day old pink bollworm larvae showed median lethal concentration (LC(50)) ranging 0.16-1.44 µg/g diet for five different populations collected from Srivilliputtur (Tamil Nadu), Jalgaon (Maharashtra), Bharuch (Gujarat), New Delhi (Delhi) and Sri Ganganagar (Rajasthan). Selection of pink bollworm for evolution of Cry2Ab resistance led to the maximal of 37.75-fold resistance vis-à-vis the most susceptible strain. Further studies on inheritance using above parental populations showed autosomal and semidominant nature of Cry2Ab resistance, with dominance h values of 0.69 and 0.79 for two reciprocal crosses. The inheritance of Cry2Ab resistance appeared to be governed by multiple alleles/genes. Cry2Ab resistance was associated with fitness costs in terms of prolonged larval and pupal period when resistant parent population was reared on the diet without toxin. Fitness cost in terms of larval period appeared to be inherited in F(1), F(2) and backcross progenies. F(2) progeny also inherited these differences in larval and pupal periods. These studies clearly advocate rigorous monitoring of Cry2Ab resistance and compliance of resistance management in the pink bollworm for ensuring Bt cotton sustainability.

Identification and characterization of the Sudanese Bacillus thuringiensis and related bacterial strains for their efficacy against Helicoverpa armigera and Tribolium castaneum.

Forty-four isolates of Bacillus thuringiensis like bacteria from various sources in different locations from Sudan were tested for their insecticidal activity. The toxicity of these isolates ranged from 6.6 to 70% to the neonates of cotton bollworm, Helicoverpa armigera at 10 ppm concentration. The most effective ones are Kb-29, St-6 and Wh-1 comparable with HD-1. Toxicity of isolates to larvae of the red flour beetle, Tribolium castaneum ranged from 20 to 100%. Isolates St-2 and St-23 gave 100% larval mortality within 15 days of exposure and were at par with Ab-8, Ab-12, Kb-26, Kb-30, Om-4, Po-2, Po-5, Po-7, Sa-8 and Wh-5 and were also comparable with E. coli clone expressing Cry3 toxin. The most effective five isolates viz., Kb-29, St-2, St-6, St-23 and Wh-1 belonged to B. thuringiensis. The St-6 isolate, which also showed high toxicity to T. castaneum larvae, had cry1 genes along with coleopteran active cry28 genes, but not cry3 genes. Of the 25 isolates characterized with 16s DNA sequencing, seven belonged to Paenibacillus spp., one Lysinibacillus sphaericus, one Bacillus pumilus, four Bacillus spp., and rest 12 belonged to B. thuringiensis. Biochemical characterization in each species showed variation. The present study shows potential of some isolates like Kb-29, St-2, St-6, St-23 and Wh-1 as promising bioinsecticides.

A common HPLC-PDA method for amino acid analysis in insects and plants.

A common method for analysis of 17 amino acids from various insect species and plant parts was standardized using HPLC-PDA. Prior to hydrolysis, lyophilization of test samples was found indispensible to remove excess moisture, which interferes in hydrolysis and separation of amino acids. After the hydrolysis of plant and insect samples, 500 and 100 microL of boiling HCl, respectively for reconstitution, and 20 microL of hydrolyzed samples used for derivatization, provided best results. Gradient profile of mobile phase and run time up to 65 min were standardized to (i) overcome the problems related to eluting underivatized sample part, (ii) optimize the use of mobile phase and run time, and (iii) get better separation of different amino acids. Analysis of Chilo partellus larvae reared on sorghum seedling powder based artificial diet indicated that arginine and histidine quantities were on par in both samples. However, methionine was higher, and leucine, isoleucine, lysine, phenylalanine, threonine and valine were lower in sorghum seedlings than in C. partellus larvae, suggesting compensation of these amino acids by the insect through voracious feeding, as is being expected from artificial diet. This method was found highly sensitive, reproducible and useful for the analysis of amino acids for better understanding of insect-plant interactions.

Assessing the susceptibility of cruciferous Lepidoptera to Cry1Ba2 and Cry1Ca4 for future transgenic cruciferous vegetables.

Advances in transgenic plants expressing Bacillus thuringiensis (Bt) insecticidal gene(s) offer a promising alternative to traditional insecticides for control of lepidopteran pests on important cruciferous vegetable crops such as cabbage and cauliflower. A public-private partnership, the Collaboration on Insect Management for Brassicas in Asia and Africa (CIMBAA), was formed in 2005 with the goal of developing dual-gene Bt cauliflower and cabbage, initially for India, to replace the use of broad spectrum, traditional insecticides. As a first step in this effort, the major lepidopteran pests of cruciferous vegetable crops [Plutella xylostella (L.), Pieris rapae (L.), Pieris brassicae (L.), Crocidolomia binotalis (L.), Hellula undalis (F.), Diacrisia obliqua Walker, Spodoptera litura F., and Helicoverpa armigera (Hübner)] were collected over a large geographic area (India, Indonesia, Taiwan, China, Australia, and the United States) and tested against purified Cry1Ba2 and Cry1Ca4 toxins, the toxins proposed to be expressed in the CIMBAA plants. Our results demonstrate that Cry1Ba2 and Cry1Ca4 were effective against the primary target of the CIMBAA plants, P. xylostella, regardless of geographic location, and had LC50 values <1.3 ppm. Furthermore, one or both toxins were effective against the other major pest Lepidoptera, except for S. litura or H. armigera which were less susceptible. No cross-resistance has been found between Cry1Ba2 and Cry1Ca4, suggesting cry1Ba2+cry1Ca4 cauliflower and cabbage could be an effective and sustainable tool to control, P. xylostella, the key lepidopteran pest on cruciferous vegetable crops, as well as most other Lepidoptera. As the CIMBAA plants are being developed, further tests are needed to determine whether they will express these proteins at sufficient levels to control all the Lepidoptera. Sustainable use of the dual-gene plants also is discussed.

Helicoverpa armigera baseline susceptibility to Bacillus thuringiensis Cry toxins and resistance management for Bt cotton in India.

Transgenic cotton that produces insecticidal proteins from Bacillus thuringiensis (Bt), often referred to as Bt cotton, is widely grown in many countries. Bt cotton with a single cry1A gene and stacked also with cry2A gene has provided satisfactory protection against the damage by the lepidopteran bollworms, especially the cotton bollworm, Helicoverpa armigera (Hübner) which is considered as a key pest. The baseline susceptibility of the larvae of H. armigera to Cry1Ac and other toxins carried out in many countries has provided a basis for monitoring resistance. There is no evidence of development of field-level resistance in H. armigera leading to the failure of Bt cotton crop anywhere in the world, despite the fact that Bt cotton was grown on the largest ever area of 12.1 million hectares in 2006 and its cumulative cultivation over the last 11 years has surpassed the annual cotton area in the world. Nevertheless, the Bt resistance management has become a necessity to sustain Bt cotton and other transgenic crops in view of potential of the target insects to evolve Cry toxin resistance.

Development and mechanisms of resistance to Bacillus thuringiensis endotoxin Cry1Ac in the American bollworm, Helicoverpa armigera (Hübner).

The American bollworm, H. armigera, evolved 31-fold resistance to selection pressure of B. thuringiensis endotoxin Cry1Ac within six generations. The Cry1Ac selected larvae of H. armigera showed cross-resistance to Cry1Aa and Cry1Ab both in terms of mortality and growth reduction. Studies on mechanisms of resistance to Cry1Ac showed that proteases of resistant insects degraded Cry1Ac faster than those of susceptible insects, which led to the relative unavailability of toxin of about 58 kDa for binding and perforation of midgut epithelial membrane of the target insect. Besides, resistant and susceptible populations of H. armigera differed in the binding of their receptors with Cry1Ac toxin. These results suggest the possibility of both mechanisms existing in imparting resistance. These findings mandate the necessity of B. thuringiensis resistance management for usage of B. thuringiensis either as a conventional insecticide or through transgenic crops.

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.

Geographical variation in larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) to Bacillus thuringiensis spore-crystal mixtures and purified crystal proteins and associated resistance development in India.

The susceptibility of larvae of the diamondback moth, Plutella xylostella Linnaeus to purified crystal proteins and spore-crystal preparations of Bacillus thuringiensis was investigated for 13 populations from seven states in India. The LC50 (microg ml(-1), 48 h) values of Cry proteins for different populations of P. xylostella ranged from 0.14-3.74 (Cry1Aa), 0.007-1.25 (Cry1Ab), 0.18-2.47 (Cry1Ac) and 0.12-3.0 (Cry1C). The LC50 (mg (ai) l(-1), 48 h) of spore-crystal preparations ranged from 0.02-0.98 (HD-1) and 0.06-2.14 (HD-73). Significantly higher LC50 values for all tested toxins and strains were obtained with populations collected from Iruttupallam and Ottanchathiram in the southern state of Tamil Nadu, whereas some of the populations collected from the northern part of India were more susceptible than the susceptible IARI 17-65 population. The high levels of resistance in the Iruttupallam and Ottanchathiram populations to Cry1Ab suggested selection pressure by Cry1Ab, which is the predominant toxin in B. thuringiensis formulations used in India. Cry1Ab was found to be more toxic than the other toxins. The population from Iruttupallam showed increased resistance following selection with Cry1Ab in the laboratory (LC50 from 1.25 to 4.31 microg ml(-1) over two generations) and also showed cross resistance to CrylAa and CrylAc. The resistance to Biobit in the field population from Iruttupallam declined slowly; requiring c. 33 generations for an overall 10-fold decline in LC50 when the insects were reared in the laboratory without exposure to B. thuringensis.

Changes in haemolymph constituents of American bollworm, Helicoverpa armigera (Hübner), infected with nucleopolyhedrovirus.

Six types of haemocytes viz., prohaemocytes, plasmatocytes (round, fusiform, vermiform and spindle shaped), granular cells, spherule cells, oenocytoids and adipohaemocytes were found in the haemolymph of larvae of American bollworm H. armigera. The total and differential haemocyte counts (THC and DHC) in H. armigera haemolymph were affected by nucleopolyhedrovirus (NPV) treatment. There was a general decrease in THC in response to NPV treatment in both young and old larvae. However the decrease was more apparent in 5 and 8 day old larvae than in 10 day old larvae. The differential haemocytes showed less of granular cells and more of spherule cells and prohaemocytes in the old larvae. Plasmatocytes and granular cells in 10 day old larvae initially phagocytosed polyhedra; however, disintegrated after 3 to 4 hr. The haemolymph of NPV treated larvae melanized slowly particularly in old larvae. Phenoloxidase (PO) activity decreased positively with granular cells and oenocytoids in 10 day old treated larvae. Cellular fraction had high level of PO activity, which was transferred to plasma in response to NPV infection in the older larvae. The role of NPV pathogenesis vis-à-vis immunity in insect is discussed.

Effect of sublethal concentration of Bacillus thuringiensis var. kurstaki on food and developmental needs of the american bollworm, Helicoverpa armigera (Hübner).

Effect of sublethal concentration of B. thuringiensis on the first, third, fourth and fifth instar larvae of the American bollworm, H. armigera was investigated to study their response to food consumption, digestion, utilization, and their development till adult formation. The young larvae surviving B. thuringiensis treatment in their first instar and third instar delayed larval period by two to three days, but did not consume more food as compared to control. However, they showed higher digestibility of food as compared to control, which was compensated by their reduced ability to utilize the digested food for body substance. Contrary to the effect on first and third instar larvae, the fifth instar larvae surviving B. thuringiensis treatment in its fourth instar consumed less food, showed less absorption efficiency in digesting food, but compensated by increase in the utilization of ingested and digested food into body substance. Insects surviving B. thuringiensis HD-1 sublethal toxicity adapted to normal larval growth when fed on untreated food, depending upon insect growth prior to treatment. The moths emerging from B.thuringiensis treated larvae had sex ratio favouring females, and adult pairs laid less fertile eggs than those from the untreated ones. The response of B. thuringiensis treated larvae to their food and developmental needs is discussed.