Abstract: Studies on mortality and rate of development of Helicoverpa armigera due to feeding of spore- -endotoxin complex of indigenous strain HD-695 (8,500 IU mg-1) and standard strain HD-1-S-1980 (16,000 IU mg-1) of Bacillus thuringiensis var. kurstaki revealed that the differences in potencies did not cause significant differences in retardation. Both the bacterial toxins induced similar biological effects (significant larval mortalities, marked larval retardation, reduction in pupation, emergence and fecundity, as well as inconsistent increase in pre-oviposition period and prolongation of generation period) of the test insect.
Introduction
Insect pests are a major cause of damage to the world’s commercially important agricultural crops. Helicoverpa armigera (Huebner) is one of the most devastating insect pest of many crops. Indiscriminate, continuous and excessive use of chemical insecticides have created a number of problems i.e., development of resistance in pest population biological and environmental hazards and water pollution etc. It is, therefore imperative on part of the agricultural scientists to use and recommend only those insecticides having no or least biological hazards. Bacillus thuringiensis Berliner (B.t.) is a naturally occurring, gram-positive, spore-forming soil-bacterium and preparations containing B.t are widely used as a biological insecticide in agriculture and forestry. (Deml et al., 1999). Owing to its specific mode of action, B.t. products are unlikely to pose any biological and environmental hazards (Anonymous, 1999). In order to evaluate properly the role of pathogen (B.t.) in management of insect pest, the study should be extend to trace the effect of B.t. on the survivors. Among the valuable contributions pertaining to this subject are those of Yamvrias (1962), Abdallah and Abul-Nasr (1970 a and b), Morris (1977), Dulmage et al. (1978), Khalique et al. (1982), Fast and Regniere (1984), Retnakaran et al. (1983), van Frankenhuzen (1987), Ajanta et al. (1999) Deml et al. (1999) and Khalique and Ahmed (2001 and 2002). Present study elaborates on the impact of spore-δ-endotoxin complex of indigenous strain (HD-695) of Bacillus thuringiensis and its comparison with spore-crystal complex of standard reference strain (HD-1-S-1980) on the mortality and biology of Helicoverpa armigera.
Materials and Methods
Rearing of the test insect, H. armigera was done on the artificial diet formula developed by Ahmed 1983 and neonate (1st instar) larvae were used in the bioassay. Two strains of Bacillus thuringiensis subsp. kurstaki, the indigenous strain namely HD-695 and reference standard strain HD-1-S-1980 having predetermined potency 8,500 IU mg–1 (Khalique and Ahmed, 2001) and 16,000 IU mg–1 (arbitrary potency and Versoi, 1981) respectively were used in the experiments.
Nine serial dilutions of both the strains were prepared in buffered solution pH 7.0 (Khalique and Ahmed, 2002). Ten ml of B. thuringiensis buffer suspension from No.1 was pipetted out and added to 90.0 ml of freshly prepared diet kept at 55-60°C in 250 ml beaker. This mixture was thoroughly mixed to obtain resulting concentration of 60.0 μg ml–1 diet. The rest of the toxin concentration from 45.0, to 3.75 μg ml–1 diet were prepared from suspension number 2 to 9, respectively.
After thorough mixing of the toxin into diet, the mixture was distributed into twenty five sterilized capsule vials so that each capsule vial contained approximately 4.0 ml of toxin mixed diet. In control only 10.0 ml buffer was added. The capsule vials containing treated and untreated diet were kept at room temperature for about 30 minutes for cooling and solidification of diet.
Each vial containing toxin mixed diet and without toxin (control) was infested with one neonate larva with the help of a sterilized soft camel hair brush. A separate brush was used for each concentration and control group. The infested vials were plugged with sterilized cotton wool. The capsule vials were kept upside down at room temperature, which ranged from 24±2.5°C and 65 to 85% RH. Each experiment was replicated four times in different days and 25 larvae were used for each replicate.
In these experiments, larvae were allowed to feed on spore-δ-endotoxin mixed diet for ten days and then transferred to toxin free diet and allowed to developed till pupation. To avoid any influence of the diet causing possible deterioration over long period of larval development, the fresh diet was placed in the capsule vials from time to time. Records were maintained on daily basis to determine larval mortality, larval period, pupation, pupal period and emergence of adults. All pupae from each treatment and control batches were weighed within 24 h of pupation and placed on a blotting paper in a wide mouth glass jar for adult emergence. The data on longevity of emerged adult, pre-oviposition period, sex ratio and fecundity of adult were also recorded. Normal adults from treated and untreated group were paired separately and released in glass lamp oviposition cages (2 pair/cage) (Khalique et al., 1982) and the cages were checked on alternate days till death of the pairs.
Results
Cumulative mortalities: The concentration-mortality response of H. armigera at various developmental stages for ten-days exposure to HD-695 and HD-1-S-1980 indicated (Table 1 and 2) that maximum mortalities were obtained within seven days. Further increase in mortalities between 7 days and pupation remained within the range of 3.0 to 13.0% for both the toxins in different concentrations. There was no pronounced difference in mortalities at pupation and at emergence for both the toxins. The mortalities recorded in control (common to both the strains) were 0.0, 1.0 and 10.0% at 7 days, at pupation and at emergence, respectively. (Table 1 and 2).
Effect of toxin on larval development and pupation: The average larval period of control larvae was 23.3±0.46 days (Table 3). On comparison with control, the average larval periods at the lowest concentration (3.75 μg ml–1) of HD-695 and HD-1-S-1980 were recorded to be 28.01±0.61 days and 27.13±0.68 days, respectively, indicating a marked difference between untreated and treated larvae. As the concentrations of the toxins increased, the larval time also increased. No marked difference in larval retardation was noted due to feeding of HD-695 and HD-1-S-1980. The highest retardation (34.6±2.76) in larval development was noted at 60.00 μg ml–1 of HD-695. The percentage of the larvae that survived and pupated was reduced to 6.0 and 5.0% for those larvae reared on diet containing 60.0 μg toxin ml–1 diet of HD-695 and HD-1-S-1980 respectively, as compared with 99.0% in the control. In both the strains, the percentage of larvae s ucceeded t o reach pupation, decreased with the increase in toxin concentration. (Table 3).
| Table 1: | Concentration-mortality response of H. armigera at different developmental stages after exposure to spore-δ-endotoxin of HD-695 |
| Table 2: | Concentration-mortality response of H. armigera at different developmental stages after exposure to spore-δ-endotoxin of HD-1-S-1980 |
| * Mortalities were corrected for the natural response rate (Abbott, 1925) | |
Effect of toxins on pupal weight, pupal duration and emergence: The average weight of pupae developed from untreated larvae was 0.377±0.090 g, (Table 4) while the average weight of pupae developed from those treated with different concentrations of HD-695 varied from 0.371±0.010 g to 0.384±0.057 g, indicating negligible difference in pupal weight among various concentrations. The average weight of the pupae developed from larvae treated with different concentrations of HD-1-S-1980 also varied from 0.362±0.051 g to 0.387±0.011 g. In general the weight of pupae developed from treated larvae was found more or less similar than that of untreated ones, showing no adverse effect of the toxins on the pupal weight. Overall the percentage of adult emergence was also found to be affected as it was 49 and 70% after larval exposure to the toxin of HD-1-S-1980 and HD-695 respectively at 3.75 μg ml–1 and decreased up to 4% at 60 μg toxin ml–1 of diet in both the strains, as compared to control emergence (90%).
| Table 3: | Influence of spore-δ-endotoxin of HD-1-S 1980 and HD-695 on the larval development Helicoverpa armigera |
| Table 4: | Influence of spore- δ- endotoxin of HD-1-S 1980 and HD-695 on the pupal weight of Helicoverpa armigera |
| *Including 95% confidence limit | |
The average pupal duration of untreated and treated group of HD-695 and HD-1-S-1980 (Table 5) showed that the average pupal period of control pupae was 17.23±0.37 days. A comparison of average pupal period of control pupae with those treated with various concentrations of HD-695, indicated that pupal period increased slightly, at the highest concentration (60 μg ml–1), the average pupal period was 19.00±1.63 days. Similarly a slight increase in pupal duration of the pupae obtained from different treatments of HD-1-S-1980 was also noted. The results indicated that irrespective of the difference in potencies, no marked effect appeared in pupal weight and pupal duration of the insect. (Table 4 and 5).
Effect of toxins on the sex ratio and pre-oviposition period: The results of the control group illustrated that the natural sex ratio was almost 1:1 (Table 6). A comparison of sex ratios of control adults with those of the treated ones indicated that spore-crystal-complex of HD-695 and HD-1-S-1980 had no significant effect on sex ratio but a slight deviation from the natural sex ratio was found at 3.5, 7.5 and 11.25 μg ml–1 of HD-1-S-1980. The average pre-oviposition period of control adults was 3.66±0.14 days. The results of treated group (HD-695) when compared to untreated one, showed that there occurred an irregular increase in pre-oviposition period in different concentrations of toxin. Similar results were obtained in pre-oviposition period of HD-1-S-1980 treated group with maximum period (8.42 days) in 15.0 μg ml–1 treatment. No significant difference was noted in pre-oviposition periods of adults treated with HD-695 and HD-1-S-1980. (Table 6).
Effect of toxins on longevity and fecundity of adults: The average longevity and fecundity of treated and untreated adults are given in Table 7. The average longevities of male and female adults in control group were 21.8±4.88 days and 18.30±3.31 days, respectively. No adverse effect in longevities was observed between the untreated and toxins (HD-1-S-1980 and HD-695) treated adults.
The average fecundity of adults in control was 969.25±255.81, while the fecundities of adults emerged from 3.75 μg ml–1 treatment of HD-695 and HD-1-S-1980 were 772.30±293.26 and 730.10±70.66 respectively. These results indicated that there was a marked difference in fecundity of treated and untreated adults. When the average fecundities of adults were compared among the treatments of HD-695 and HD-1-S-1980, there was no consistent response of the insect between the treatments and the reproductive potential of the treated adults. (Table 7).
Effect of toxin on survival time: The time of death of H. armigera larvae were observed at different intervals after exposing the larvae for 10 days to spore-δ-endotoxin complex of HD-695 and HD-1-S-1980. Both toxins caused larval mortalities in the first ten days of the exposure (Table 8). The larvae that survived after 10 days of toxin exposure showed very low mortality rate in the rest of the period (11-35 days) (Table 8).
| Table 5: | Influence of spore-δ- endotoxin of HD-1-S 1980 and HD-695 on the pupal duration of Helicoverpa armigera |
| Table 6: | Influence of spore- δ- endotoxin of HD-1-S 1980 and HD-695 on the sex ratio and pre-oviposition period of Helicoverpa armigera |
| Table 7: | Impact of spor-δ-endotoxin complex of HD-1-S-1980 and HD-695 on the longevity and fecundity of Helicoverpa armigera (Hubn) |
| * Including 95% confidence limit | |
| Table 8: | Time of death of Helicoverpa armigera (Hubn) larvae exposed to spor-δ-endotoxin complex of HD-1-S-1980 and HD-695 |
Discussion
The concentration-mortality response of H. armigera at different development stages indicated maximum mortalities with in 7 days. These results are in agreement with Ali and Young (1993) they reported most mortality (60-91%) of Helicoverpa zea and Heliothis virescens occurred at 7 days and pupation. In the field survival of both the species generally decreased as Bacillus thuringiensis rate increased.
The results on larval developmental period showed that all treatments of both the toxins adversely affected the larval development by causing significant delay. Alchanatis et al. (2000) used an image analysis system to test the effect of feeding Bacillus thuringiensis (Bt) transgenic cotton to H. armigera larvae; it reduced leaf consumption h-1 and increased the inter-meal time. This feeding profile suggests that Bt toxic protein suppressed feeding, because the longer resting times than those of controls were probably needed by the larvae to recover from the Bt toxicity. Karim et al. (2000) showed a correlation between toxin concentration and inhibitory response with Cry2A toxin and the lag time decreased with increasing concentration of toxin applied, which is evidence of dose response. The delayment of larval period corresponds to observations of Khalique and Ahmed (2001), they reported that increase in larval period (after 7 days exposure) was directly related to concentration of toxin present in the diet. No marked differences in larval retardation was noted between the survivors of HD-1-S-1980 and HD-695 treatments thereby indicating that the difference in potencies of these toxins did not contribute to any significant difference in larval period. These results also strengthenened by the earlier observations of Khalique and Ahmed (2002), they reported non-significant differences between the larval period of H. armigera exposed to strains (HD-1-S-1980 and HD-244) for seven days.
The average pupal weight of HD-695 treated group did not differ from the average weight of pupae reared on HD-I-S- 1980 toxin and there was also no difference in pupal weight of untreated and treated pupae. These results also fell in agreement with the observation recorded in the context of the seven days exposure effect of HD-694 (Khalique and Ahmed, 2001); HD-244 and HD-1-S-1980 (Khalique and Ahmed, 2002) on pupal weight indicating that H. armigera larvae have pronounced ability to recover from ten days exposure of B. thuringiensis and could pupate normally. The observations supported the results of Abdallah and Abul-Nasr (1970b), Fast and Regniere (1984) and Dulmage et al. (1978).
The average pupal duration of HD-695 and HD-1-S-1980 treated group showed non-significant difference after 10 days exposure, such observation were also reported by Soliman et al. (1970), Dulmage and Martinez (1978), Dulmage et al. (1978) and Salama et al. (1981).Cui and Xia et al. (1999) studied the effects of transgenic B.t. cotton on development and reproduction of Helicoverpa armigera. They observed that rate of pupation and emergence decreased by 48.2-87.5 and 66.7-100%, respectively. The difference in potencies of these strains did not show any impact on pupal duration and emergence. Justin et al. (1994) evaluated the interaction of Bacillus thuringiensis Berliner with NPV against H. armigera, they found an inverse co-relation between adult emergence with the dose of B.t. No marked difference due to bacterial toxins in sex ratio was observed other than slight deviation of sex ratio from the natural pattern in some treatments (3.75 and 7.50 μg ml–1 of HD-1-S-1980). Similar results were reported by Afify and Matter (1969) that out of three treatments one treatment showed a slight decrease in the ratio of females but gave no evidence of a definite effect of the pathogen on the ratio. McGaughy (1978) reported that B. thuringiensis (Dipel) formulation had no appreciable effects on the sex ratio of moths, E. cautella and P. interpunctella that survived. Soliman et al. (1970) reported that sex ratio was not changed to the side of females. The effect of toxins on the sex ratio did not significantly differ from each other. The observation of Morris (1976) holds good in support of present results with non-significant difference in sex ratio of C. fumiferana treated with Thuricide and Dipel.
The average pre-oviposition period of the adults developed from larvae treated with HD-695 and HD-1-S-1980 steadily increased in comparison with control. In case of HD-695, the increase in pre-oviposition period at higher concentrations was more pronounced than that at lower concentrations and similar trend of increase in pre-oviposition was observed in case of HD-1-S-1980. Afify and Matter (1969) reported non-significant difference in the pre-oviposition period between the control and the treated A. kühniella.
Abdallah and Abul-Nasr (1970a) observed delay and irregularity in oviposition of S. littoralis adults when larvae were allowed to feed on Biotrol sprayed cotton leaves at a concentration of 2.0% for different periods. They also observed that shortening of the moth life span was consistent feature of the treatment of B. thuringiensis. Salama et al. (1981) illustrated that the longevity of the moth of H. armigera and S. liltoralis was not affected as a result of larval treatment and showed no clear correlation with concentration of larval exposure to the toxin.
Present results indicated no considerable difference in longevity of HD-695 and HD-1-S-1980 treated groups at all concentrations in comparison with control. The substantial reduction, delay and irregularity in oviposition of Spodoptera littoralis due B. thuringiensis treatment was reported by Abdallah and Abul-nasr (1970a). A significant reduction in the fecundity of A. kuehniella, after exposure of B. thuringiensis was also observed by Afify and Matter (1969). The longevities of both the groups also differed non-significantly with each other. A comparison of control fecundity with those of HD-695 and HD-1-S-1980 treated batches showed considerable but inconsistent reduction in fecundity at almost all the B. thuringiensis treatments. Soliman et al. (1970) reported variations in longevity are of no significance with regard to the population density, as long as the sex ratio is not changed to the side of the females and the fertility of the females is adversely affected with larval treatment.
The results of the ten days exposure of the toxins (HD-695 and HD-1-S-1980) revealed that larval treatment mainly resulted in causing retardation in larval development period, decreasing pupation percentage and adult emergence, increase in pre-oviposition period, decrease in fecundity, no significant effect on pupal weight and pupal duration. The comparative study of two toxins (HD-695 and HD-1-S-1980) did not significantly differ from each other in causing a marked difference in disorientation in the development of H. armigera.