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Keywords:

  • Helicoverpa armigera;
  • cantharidin;
  • sublethal;
  • population parameters;
  • abnormalities

Abstract

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS
  6. 4 DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Background

The cotton bollworm, Helicoverpa armigera (Hübner), is a serious and cosmopolitan pest of many economic crops. Its control has not been adequate owing to its resistance to many groups of insecticides. Toxicity of cantharidin on armyworm and diamondback moth has already been reported. However, its toxicity on H. armigera has not been investigated previously. In this study, lethal and sublethal effects of cantharidin on H. armigera under laboratory conditions are reported.

Results

Results showed gross abnormalities in the population parameters of H. armigera, ranging from larvae to adults. Reduction in larval weight and wing malformation were observed in the cantharidin-treated population cohort, and higher mortality at the larval, pupal and adult stages was observed in cantharidin-treated H. armigera compared with the control. Moreover, almost 5 times less fecundity was recorded in the treated population cohort. Fertility was also severely affected, and reduction in all population parameters was observed.

Conclusion

Cantharidin caused larval mortality and other serious abnormalities in H. armigera population parameters, and therefore may have positive implications for pest management decision-making process. More interestingly, the experiment revealed that cantharidin in sublethal dose mimicked insect growth regulator insecticides. Furthermore, cantharidin could be used as a precursor compound for the synthesis of new analogues and compounds to replace ineffective older compounds. © 2013 Society of Chemical Industry

1 INTRODUCTION

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS
  6. 4 DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Helicoverpa (Heliothis) armigera (Hübner) (Lepidoptera: Noctuidae) is a highly polyphagous agricultural pest. Host species for H. armigera come from a broad spectrum of families and include important agricultural crops such as cotton, maize, chickpea, pigeonpea, sorghum, sunflower, soya bean and groundnuts.[1]H. armigera is a cosmopolitan pest, and its presence has been recorded in Asia, Europe, Australia and Africa.[2] Global yield loss from this pest is estimated at $US 2 billion annually.[3]

The indiscriminate use of insecticides, particularly during the 1980s and 1990s, contributed to the emergence of the cotton bollworm, H. armigera, as a primary pest of cotton in recent years. Its control was not always adequate, probably owing to the development of resistance. A moderate to high level of resistance to pyrethroids and organophosphorus insecticides was recorded in field populations of H. armigera.[4]

Biopesticides with a mode of action different to that of conventional insecticides may reduce the risk of insecticide resistance and pest resurgence problems while being comparatively safe and ecologically acceptable. In early studies, cantharidin emulsifiable concentrate (EC) pesticide was found to have low toxicity against quail, ladybird beetles and soil microorganisms according to the results of safety evaluations against non-target organisms.[5]

The insecticidal and antifeedant activities of cantharidin are well established and have been reported on armyworm and diamondback moth.[6] However, its lethal and sublethal toxicity on population parameters has not yet been studied in H. armigera. Here, the focus will be on the sublethal toxicity of cantharidin owing to its great significance for low mammalian toxicity.

The main purpose of this study was to ascertain and explore the effect of cantharidin on the development, fecundity, fertility and other ecological parameters of H. armigera by cantharidin-incorporated diet in the laboratory.

2 MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS
  6. 4 DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

2.1 Cantharidin extraction and purification

Cantharidin was extracted and purified in the Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Yangling, Shaanxi Province, Northwest A&F University, from meloide beetles, commercially procured, using standard laboratory protocol.

2.2 Insect rearing

Helicoverpa armigera larvae were procured from Henan Jiyuan Baiyun Industry Co., Ltd., China, and reared until F1 for use in bioassays. Groups of 24 larvae were placed into 24-chamber plastic boxes and placed in an incubator at 22 ± 2 °C and 40 ± 5% RH with a 12 h photoperiod. After pupation, pupae were collected and placed in a plastic jar with cotton cloth attached on both sides and a vial 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.

2.3 Artificial diet

Cotton bollworms were reared in the laboratory on modified semi-synthetic diet.[7] The diet consisted of chickpea flour, sorbic acid, Wesson's salt, vitamin (ABDEC), ascorbic acid, brewer's yeast, choline chloride, agar, formaldehyde, streptomycin sulphate and methyl-p-hydroxy benzoate, under laboratory conditions of 22 ± 2 °C and 40 ± 5% RH with a 12:12 h light:dark photoperiod. A homogeneous stock of third-instar larvae was obtained for respective bioassay and induction treatments.

2.4 Bioassay and calculation of the lethal time (LT50)

Third-instar larvae of H. armigera were used to determine the lethal time, LT50. Healthy insects were introduced to artificial diet treated with 0.5 mg g−1 of cantharidin. Data were recorded at 12, 24, 48, 72 and 96 h after treatment and subjected to probit analysis for calculation of LT50 and LT90.[8]

2.5 Insect treatment

Cantharidin was extracted in the laboratory as mentioned earlier. More robust third-instar larvae were selected for this study. Insects were starved for 8 h before being given cantharidin-incorporated diet containing 0.025 mg g−1 of cantharidin for 3 days. Afterwards, larvae were provided with untreated artificial diet.

2.6 Development of life table data

Two cohorts of H. armigera first-instar larvae were randomly selected from the laboratory colony and placed individually in 24-cell insect culture racks until third instar. Third-instar larvae in the control group were fed 1 g of artificial diet with acetone and in the treatment group with 0.025 mg g−1 of cantharidin-treated artificial diet until 72 h, after which fresh artificial diet was substituted without cantharidin until pupation.

Developmental times, age-stage-specific survival rates (sij), age-stage-specific fecundity (fij), age-specific survival rate (lx) and age-specific fecundity (mx), where x is the age and j is the stage, were recorded daily until the death of all individuals. After adult emergence, females and males were paired and placed in transparent plastic containers of 0.5 L capacity with cotton cloth on top for oviposition to improve ventilation. Insects were fed 10% honey solution in water. Age-stage, two-sex life tables were constructed according to Chi.[9] Percentage fertility was calculated as (eggs hatched ÷ eggs laid) × 100. Effective fecundity was calculated as the actual number of viable eggs per female.

2.7 Statistical analysis and population parameters

Fecundity and fertility data from the females for each population cohort were subjected to an independent-sample t-test using SPSS-17.[10] A confidence level of P ≤ 0.05 was considered to be significant.

2.8 Population parameters estimated

Intrinsic rate of increase (r):

  • display math(1)

Net reproductive rate (R0):

  • display math(2)

Mean generation time (T):

  • display math(3)

The age-stage life expectancy (eij) was calculated according to Chi and Su.[11] The intrinsic rate of increase was estimated by using the iterative bisection method from the Euler–Lotka equation [equation (1)] with age indexed from 0.[12] Visual Basic-based TWOSEX life table computer software was used to calculate these parameters.[13] This software also includes a function for estimating the standard error of population parameters using the jackknife technique.[14] Data obtained as output text format were exported to MS Excel.[15] Differences in life history traits and population parameters between H. armigera treated and untreated with cantharidin were compared by t-tests.[16]

3 RESULTS

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS
  6. 4 DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Bioassay results showed that an artificial diet containing 0.5 mg g−1 of cantharidin caused larval mortality of 16, 29, 79, 95 and 100% after 12, 24, 48, 72 and 96 h respectively (Fig. 1). Lethal times LT50 and LT90 were found to be 26.62 and 44.98 h respectively.

image

Figure 1. Percentage mortality of H. armigera after different intervals, exposed at third larval instar to artificial diet containing 0.5 mg g−1 of cantharidin.

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The significant negative effect of cantharidin can be seen in the curves of age-stage survival rate (sij) (Fig. 2). The pupal mortality of the cantharidin-treated cohort was 20%, whereas in the control it was 0%. As a result, lower survival curves in adult stages were observed. The negative effect of cantharidin was observed during the adult stage of both male and female. The age-stage life expectancy in the cantharidin-treated cohort was shorter compared with the control cohort. The life expectancy of a new egg was 35 days in the cantharidin-treated cohort, whereas it was 41 days in the control cohort, a reduction of 6 days (Fig. 3).

image

Figure 2. Survival rate curves (sij) of cantharidin-treated and control population cohorts exposed to artificial diet containing 0.025 mg g−1 of cantharidin at third larval instar.

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image

Figure 3. Life expectancy of different stages (days) of cantharidin-treated and control population cohorts exposed to artificial diet containing 0.025 mg g−1 of cantharidin at third larval instar.

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The age-specific survival rate (lx) was also negatively affected by cantharidin (Fig. 4). The age-specific survival rate in the cantharidin-treated cohort decreased sharply after 8 days, whereas a slight reduction was seen in the control cohort after 10 days. The age-stage-specific mortality was also negatively affected by cantharidin (Fig. 5). A 20% larval and 8% pupal mortality was observed in the cantharidin-treated cohort, whereas only 4% larval mortality was observed in the control cohort. No mortality was observed at the pupal stage in the control cohort. By taking fertility into consideration, the negative effect of cantharidin was evident in age-specific fecundity (mx) (Fig. 6). The highest age-specific fecundity in the cantharidin-treated cohort was 13, whereas in the control cohort it was 45.

image

Figure 4. Age-specific survival rate (lx) of cantharidin-treated and control population cohorts exposed to artificial diet containing 0.025 mg g−1 of cantharidin at third larval instar.

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image

Figure 5. Age-stage-specific mortality of cantharidin-treated and control population cohorts exposed to artificial diet containing 0.025 mg g−1 of cantharidin at third larval instar.

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image

Figure 6. Age-specific fecundity (mx) of cantharidin-treated and control population cohorts exposed to artificial diet containing 0.025 mg g−1 of cantharidin at third larval instar.

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Significant reductions in larval and pupal weights were observed in the cantharidin-treated cohort (Figs 7 and 8). Mean reductions of 76% in larval weight and 29% in pupal weight were observed in the cantharidin-treated cohort.

image

Figure 7. Comparison of larval weight after 6 days of exposure at third larval instar to artificial diet containing 0.025 mg g−1 of cantharidin.

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image

Figure 8. Comparison of pupal weight after 6 days of exposure at third larval instar to artificial diet containing 0.025 mg g−1 of cantharidin. After 6 days, larvae were allowed to feed on fresh artificial diet until pupation.

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Cantharidin treatment also resulted in significant reductions in the population parameters (Table 1). The intrinsic rate of increase (r), the finite rate of increase (λ) and the net reproduction rate (R0) in the cantharidin-treated cohort showed reductions of 30, 4 and 78% compared with the control cohort, whereas the mean generation time (T) was extended by 4% in cantharidin treatment. Change in the mean time (days) of different life stages was also observed in the cantharidin treatment (Table 2). A noticeable change at the larval stage was observed with an increase in mean time by 2.72 days in the cantharidin-treated cohort, whereas a reduction of 1.46 days was observed at the adult stage. No noticeable change in mean time at the pupal stage was observed between the cantharidin-treated and control cohorts. The pre-oviposition period, fecundity and fertility were also significantly affected by cantharidin treatment (Table 3). Cantharidin treatment caused an increase of 0.79 days in the adult pre-oviposition period. Both fecundity and fertility were severely affected by cantharidin treatment. A reduction of 85% in fertility was recorded in the cantharidin-treated cohort.

Table 1. Ecological parameters of cantharidin-treated and control population cohorts of H. armigeraa at 22 ± 2 °C and 45–50% RH
Ecological parametersCantharidin± SEControl± SE
  1. a

    The jackknife method was used to obtain the mean ± SE from each population cohort.

Intrinsic rate of increase (r)0.09570.0070.1370.0043
Finite rate of increase (λ)1.09880.00791.14710.0046
Net reproductive rate (R0)45.9212.41207.6233.58
Mean generation rate (T)40.360.6338.970.33
Table 2. Mean time (days) for different stages of cantharidin-treated and control population cohorts of H. armigera at 22 ± 2 °C and 45–50% RH
Life stageCantharidin± SEControl± SE
Egg3030
Larva15.890.2713.170.21
Pupa14.240.2414.780.11
Adult10.800.1712.260.23
Table 3. Effect of cantharidin on the life history traits of cantharidin-treated and control population cohorts of H. armigeraa at 22 ± 2 °C and 45–50% RH
TreatmentPre-oviposition period (days)Fecundity (eggs female−1)Fertility (%)Effective fecundity (eggs female−1)
  1. a

    Means followed by different letters within the same column differ significantly between cantharidin and the control at P ≤ 0.05 using an independent-sample Student t-test.

Cantharidin    
x ± SE3.87 ± 0.06 a134.20 ± 0.81 a13.12 ± 0.41 a125 ± 1.20 a
n24242424
Control    
x ± SE3.08 ±0.05 b364.20 ± 1.77 b87.75 ± 0.61 b376.04 ± 2.85 b
n24242424

Also, cantharidin treatment caused malformation and abnormalities at the pupal and adult stages (Fig. 9). Most of the male moths in the cantharidin-treated cohort were observed to have crippled wings, whereas female moths had miniature wings. Malformation of pupae was also noticed in the cantharidin treatment.

image

Figure 9. Effect of cantharidin on different life stages of H. armigera: (A) female moth with crippled wings; (B) male moth with miniature crippled wings; (C) dark-brown dead larvae at fifth larval stage; (D) pupal malformation. E1, size of normal larvae; E2, size of cantharidin-treated larvae; F1, colour of normal pupa; F2 and F3, colour of dead pupae.

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4 DISCUSSION

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS
  6. 4 DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

In this study, cantharidin was found to have strong insecticidal activities. Similarly, insecticidal and antifeedant activities of cantharidin were reported on armyworm and diamondback moth.[6] However, its sublethal effects on population parameters have not yet been studied. Sublethal toxicity is of great practical importance from the management as well as from the ecological point of view.

Earlier studies showed that the bioactivity of cantharidin was bound to phosphoprotein 2A (PP2A).[17] Other than PP2A, detailed physiological and biochemical effects of cantharidin and its mechanism of action remain widely unknown.[18-22] In the present study, cantharidin treatment in lethal and sublethal doses caused significantly high mortality and reduced all population parameters. Dead larvae turned dark brown after death. However, to understand the mechanism of its lethal and sublethal effects, more extensive experimentation on biochemical and molecular levels is needed.

Cantharidin in the present study caused a reduction in larval and pupal weight (Fig. 9). Decrease in larval weight could be attributed to the decreased level of enzyme activity, indicating general disturbance in metabolism, in cantharidin-treated insects. Similar symptoms in previous studies suggested that the reduced level of digestive enzymes caused reduced phosphorus liberation for energy metabolism, a decreased rate of metabolism and a decreased rate of transport of metabolites in an investigation of azadirachtin effects on enzyme regulation.[23, 24] Cantharidin in the present study showed prolonged larval duration and caused other deformities at the pupal (Fig. 9) and adult stages. These deformities could be due to the decreased level of alkaline phosphatase (ALP) because the latter is an important synthesising enzyme of tyrosine, the precursor of dopamine and octopamine, which are known to take part in the control of levels of insect developmental hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E).[25-27]An earlier study showed that the level of ALP was significantly lower after cantharidin treatment, and the decrease was more profound with prolonged exposure time.[28]

Furthermore, crippled wings of both male and female (Fig. 9) were observed in the cantharidin-treated cohort population. This could cause flight and mating disruption under field conditions and consequently make the moths an easy prey, and consequently a reduction in field population could be achieved. As a matter of fact, all these morphological traits have a profound effect on the fitness of the population under field conditions. Decreasing population growth curves indicate a profound reduction in the forthcoming generation population. Previous studies 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.[29]

Typically, natural mortality of between 1 and 15% is regarded as normal during the immature stage (at first larval instar, generally), because this stage is the most sensitive one.[30] However, mortality recorded at later larval and pupal stages in the cantharidin-treated population cohort could be related to the chronic effects of cantharidin.

Another detrimental effect of cantharidin was seen on fecundity and fertility of H. armigera. In the present study, both the egg number and the duration of oviposition were reduced, as it is important to take into account the relevance of oviposition of the female in determining the future population growth.[31]

In light of the above-mentioned studies, it is concluded that cantharidin has significant effects on all population parameters. Among all the population parameters, fertility was the worst affected. Interestingly, cantharidin mimicked insect growth regulatrs (IGRs) in sublethal doses. In short, more research is needed to explore the mechanism and potential of cantharidin as insecticide for large-scale use. However, its use in low dose could be considered for pest management, and its scope as a synergist with other chemicals may be explored. Moreover, emphasis should also be placed on synthesising new cost-effective analogues while making them more effective and environmentally safer.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS
  6. 4 DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The authors sincerely appreciate the financial support of the Special Fund for the Public Interest (Agriculture) (200903052) by The Ministry of Science and Technology of China and The Ministry of Agriculture, China, and the ‘13115’ Sci-Tech Innovation Project of Shaanxi Province (2007ZDKG-14).

REFERENCES

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS
  6. 4 DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
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