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The sub-lethal toxicological effects of cantharidin were tested to evaluate its toxicity to Helicoverpa armigera Hub. (Lepidoptera: Noctuidae). The objectives of our studies were to ascertain the long term chronic effects of cantharidin on the population dynamics of H. armigea under laboratory conditions. The data were analyzed by TWOSEX life table software. Our results showed gross abnormalities in population parameters of H. armigera, ranging from larvae to adults. Reduction in larval weight and wings malformation was observed in treated population cohort. Comparatively, higher mortality at larval, pupal and adult stages was recorded in treated, compared to untreated. Almost five times less fecundity was recorded in treated population cohort. Fertility was also severely affected. In short, reduction in all population parameters was registered. It is beyond the scope of our research to imagine the effect of cantharidin in the field populations of the pest. However, use of cantharidin in the field could also cause serious abnormalities in H. armigera population parameters and may have implications for pest management decision making process. More interestingly our experiment revealed that cantharidin in sub-lethal dose mimicked IGRs insecticides.
American bollworm, Helicoverpa armigera Hübner, Lepidoptera; Noctuidae is considered one of the major pest of cotton, legumes and more than hundred other plant species (Bhatnagar et al., 1982). Helicoverpa armigera is a cosmopolitan pest and its presence has been recorded in Asia, Europe, Australia and Africa (EPPO, 2006). Global yield loss from this pest is amounting to US $ 2 billions annually (ICRISAT, 2003)
The indiscriminate use of insecticides, particularly during 80s and 90s contributed to the emergence of the cotton bollworm, Helicoverpa armigera as a primary pest of cotton, in recent years. Control of this pest was not always adequate probably due to the development of resistance. Moderate to high level of resistance to pyrethroides and organophosphorus insecticides were recorded in field population of H. armigera (Ahmed et al., 1995).
Biopesticides with mode of action other than the conventional insecticides may reduce the risk of insecticide resistance and pest resurgence problems while comparatively safe and ecologically acceptable. In early studies, our candidate insecticide, cantharindin EC pesticide had been found to have low toxicity against quail, ladybird beetles and soil microorganisms by (Feng-Li et al., 2009) while studying on its safety evaluation against non-target organisms.
The insecticidal and anti-feedant activities of cantharidin are well established fact as elucidated by (Yalin et al., 2003) on armyworm and diamond moth. However, its sub-lethal toxicity on population parameter has not yet been studied. Here we put emphasis on sub-lethal toxicity of cantharidin as it is of great significance in terms of low mammalian toxicity.
The main purpose of this study was to ascertain and explore the effect of cantharidin on development, fecundity, fertility and other ecological parameters of H. armigera Hub. by cantharidin incorporated diet, in the laboratory.
Materials and Methods:
Cantharidin extraction and purification:
Cantharidin was extracted and purified in the "Key laboratory of plant protection resources and pest management, Ministry of Education, Yangling 712100, Shaanxi Province, Northwest A & F University" from meloide beetles, commercially procured, using standard laboratory protocol.
Helicoverpa armigera larvae procured from Henan Jiyuan Baiyun Industry Co., Ltd. China and reared until F1 for use in bioassay. Group of 24 larvae were placed into 24 chamber plastic box obtained from the company. The boxes were placed in an incubator at 22Â±2â-‹C and 40Â±5% RH with 12 hrs photoperiod. After pupation pupae were collected and placed in plastic jar having cotton cloth on both sides and vial was placed in the middle with 10% sugar solution dispensed through cotton. Eggs were collected on the lower and upper cotton cloth and placed in transparent plastic bags for emergence.
Cotton bollworms were reared in the laboratory on modified semi-synthetic diet (Ahmed and McCaffery,1991), consisting of Chickpea flour, Sorbic acid, Wesson's salt, Vitamin (ABDEC), Ascorbic acid, Yeast (Brewer's), Choline chloride, Agar agar, formaldehyde, streptomycin sulphate and methyl-p-hydroxy benzoate, under laboratory condition of 22Â±2Â°C, 40Â±5% RH and 12:12 hrs Light: Dark. A homogeneous stock of third instar larvae was obtained for respective bioassay and induction treatments
Bioassay and calculation of Lethal time (LT50):
Third instar larvae of H. armigera were used to determine lethal time, LT50. Healthy insects were introduced to artificial diet treated with 0.5mg g-1 cantharidin. Data were recorded after 12, 24, 48, 72 and 96 hrs after treatment. Data were subjected to probit analysis for calculation of LT50 (Finney, 1971).
Cantharidin used in our studies was extracted in the laboratory as mentioned above. Third instar larvae, being more robust were selected for this study. Insects were kept hungry for 8 hours before their introduction to cantharidin's treated diet. Insects were introduced to diet containing 0.05 mg g-1 of cantharidin for three days. Afterwards, larvae were provided with untreated artificial diet.
Development of life table data:
Two cohorts of Helicoverpa armigera first instar larvae were randomly selected from laboratory colony and placed individually in 24 cells insect culture racks until third instar. Third instar larvae in control group were fed artificial diet with acetone and larvae in treatment group were fed with 0.05mg g-1 cantharidin treated artificial diet until 72 hours and replaced with artificial diet without cantharidin until pupation.
Developmental times, age-stage specific survival rates (sij), age stage specific fecundity (fij), age specific survival rate (lx), where x is the age and j is the stage, and age-specific fecundity (mx) were recorded daily until the death of all individuals. After adult emergence, females and males were paired and placed in plastic container of 0.5 liter capacity with cotton cloth for oviposition and on the lid to improve ventilation and were fed 10% honey solution in water. Age-stage, two-sex life tables were constructed following (Chi, 1988). Percent fertility was calculated as: (eggs hatched ÷ eggs laid) x 100. Effective fecundity was calculated as eggs laid female-1.
Statistical analysis and population parameters:
Fecundity and fertility data from the female for each population cohort were subjected to independent sample t-test using SPSS-17 (SPSS Inc., 2007). Confidence level of Pâ‰¤0.05 was considered significant.
Population parameters of each cohort were calculated by:
The age-stage life expectancy (eij) was calculated according to Chi and Su (2006). The intrinsic rate of increase was estimated by using the iterative bisection method from Euler-Lotka equation (Eq. 1) with age indexed from 0 (Goodman, 1982). The visual basic based TWOSEX life table computer software was used to calculate these parameters (Chi, 2008). This software also includes function for the estimation of standard error of population parameters using Jackknife technique (Meyer et al., 1986). Data obtained as output text format were exported to MS Excel (Microsoft, 2003). Differences in life history traits and population parameters between H. armigera treated and untreated to cantharidin were compared by t-tests (Zar, 1996).
Bioassay results showed profound toxicity of cantharidin on third instar larvae of H. armigera (Fig. 1). Lethal time LT50 and LT90 were found to be 26.62 and 44.98 hrs, respectively.
Cantharidin showed short-term as well as long term effects on H. armigera different life stages. Short-term effects include reduced larval size, anorexia, whereas long term effects include pupal, moth malformation as well as reduced pupal weight and moth size. Development time for larvae to pupae in cantharidin treated cohort population was longer as compared to untreated control.
Negative effect of cantharidin can also be seen in age stage survival rate curves (Fig. 2). High mortality at larval stage was observed in cantharidin treated cohort population as compared to untreated control. Pupal mortality was also observed in cantharidin treated cohort, whereas no mortality was observed in untreated control. Consequently, lower survival curves at larval, pupal and adult stages were obtained. Severe negative impact of cantharidin was observed on larval and pupal stage. The age stage specific life expectancy in cantharidin treated cohort was shorter for larvae and pupae as compared to untreated control cohort (Fig. 3.). Age specific survival rate showed step wise reduction pattern in cantharidin treated cohort as compared to untreated control cohort (Fig. 4). Age specific mortality can be seen more pronounced in cantharidin treated as compared to untreated control (Fig. 5). Considering fertility of cohort, cantharidin negatively affected on reproduction was obvious in age specific cohort fecundity (Fig. 6). Reduced larval and pupal weights were obtained in cantharidin treated in comparison with untreated (Fig. 7, 8). In our experiment more or less reduction was seen in all population parameters (Table 1). The intrinsic rate of increase (r) and net reproductive rate (R0) of cantharidin treated cohort was 0.09 and 1.09, whereas 0.13 and 1.14 in untreated control, respectively. Table 2 shows increased larval and reduced adult duration in cantharidin treated cohort. Mean generation time remained 40.36 and 38.97 in treated and untreated, respectively. Fecundity, fertility and effective fertility in cantharidin treated cohort remained 134.2, 13.12 and 125, whereas 364.20, 87.75 and 376.04, respectively. All the parameters remained significantly different at Pâ‰¤0.05 (Table 3).
In present studies cantharidin was found to have strong insecticidal activities. Like wise, the insecticidal and anti-feedant activities of cantharidin as well established fact was elucidated by (Yalin et al., 2003) on armyworm and diamond moth. However, its sub-lethal effects on population parameters have not been studied so far. Sub-lethal toxicity is of great practical importance from the management as well as ecological point of view.
Earlier studies showed that the toxicity of cantharidin has been its binding to phosphoprotein 2A (PP2A). Other than PP2A detailed physiological and biochemical effects of cantharidin and its mechanism of action remains widely unknown (Graziano and Casida, 1987; Kawamura et al, 1990; Graziano et al, 1987; Decker 1968; Bagatell et al, 1969). In our experiment cantharidin treatment of sub-lethal dose significantly reduced all population parameters.
Cantharidin in our study caused reduction in larval weight. Decrease in larval weight could be because of the decreased level of enzymes activity and indicated general disturbance in metabolism, in cantharidin treated insects. Other relevant studies suggested that reduced level of digestive enzymes suggests reduced phosphorus liberation for energy metabolism, decreased rate of metabolism, as well as decreased rate of metabolites and could be the direct effect of cantharidin on enzyme regulation (Huang et al, 2004; Nathan et al, 2005). Furthermore, crippled wing of both male and female were observed in cantharidin treated cohort population. This could cause flight disruption and mating under field conditions and consequently make them liable to be an easy prey and consequently reduction in field population could be achieved. As a matter of fact, all these morphological traits have profound effect on fitness of population under field conditions. Decreasing population growth curves indicates the profound reduction in forth coming generation population. In previous studies, Sharma et al., 2006 reported similar symptoms by treating H. armigera larvae with azadirachtin-A and tetrahydroazadirachtin-A concentrates that caused growth inhibition, malformation and mortality in a dose-dependent manner.
Typically, natural mortality between 1% and 15% is regarded normal during immature stage (at first larval instar, generally), because this stage is the most sensitive one (Carvalho et al., 1998). However, mortality recorded at later larval and pupal stage in cantharidin treated population cohort could be related to chronic effects of cantharidin.
Another detrimental effect of cantharidin was seen on fecundity and fertility of H. armigera. In our studies both the egg number and duration of oviposition was reduced as it is important to take into account the relevance of oviposition of female in determining the future population growth (Lewontin, 1965).
In light of the above studies we may conclude that cantharidin has deep profound effects on all population parameters. Amongst all the population parameters effected, fertility was worst effected. Interestingly, cantharidin mimicked IGRs in sub-lethal doses. In short, more research is needed to explore the mechanism and potential of cantharidin and its analogues for future pest management.