Abstract: Background and Objective: The cotton leafworm, Spodoptera littoralis (Lepidoptera: Noctuidae) is the most destructive pests and causing a great loss for several vegetables and field crops. So, the present study was aimed to evaluate and compare the effects of chlorantraniliprole and chlorfluazuron on certain behavioral and biological aspects of S. littoralis at different stages. Materials and Methods: Under laboratory conditions, the toxicity of chlorantraniliprole and chlorfluazuron against the egg masses, 2nd and 4th larval instars of S. littoralis were evaluated. The impact of tested insecticides on the feeding, oviposition of females and biological aspects of S. littoralis was also carried out. Results: Overall, chlorfluazuron was more toxic than chlorantraniliprole. According to repellency index (RI %), tested insecticides have a repulsive effect for the feeding of 2nd and 4th larval instars as well as for oviposition of females. Sublethal concentrations significantly reduced larval and pupal weight, adult survival, percent of pupation and adult emergence, female fecundity, fertility percentage, weight and protein content of ovaries. While, larval and pupal durations were increased. Conclusion: It was concluded that chlorantraniliprole and chlorfluazuron have insecticidal, behavioral and biological effects on S. littoralis stages and may be used as alternatives to conventional insecticides in IPM programs.
INTRODUCTION
The cotton leaf worm, Spodoptera littoralis (Lepidoptera: Noctuidae) is one of the most destructive pests, which causes a significant economic loss for many economically-important vegetables and field crops belonging to 44 families. It can be found year around in Egypt feeding on numerous crops, include 73 species1-4.
The excessive use of broad-spectrum insecticides, like pyrethroids, carbamates and organophosphates cause several environmental problems, destruction of natural enemy populations and development of resistance to different groups of insecticides5.
Among the most promising chemical control tactics, the biorational control agents such as synthetic insect growth regulators (IGRs) and anthranilic diamides, which have novel modes of action and claimed to be safer for beneficial organisms6,7. Many Synthetic growth regulating insecticides (IGRs) and anthranilic diamides have been successfully used in IPM programs against many lepidopterous insects such as S. littoralis and S. litura8-10.
Specific biochemical processes of certain insects is affected by synthetic growth regulating insecticides (IGRs), especially those which are essential for insect growth and development. The disruptions that IGRs can cause hormone regulations associated with insect metamorphosis can, ultimately, lead to insect death11. Chlorfluazuron (Group 15, IRAC) disrupts chitin synthesis during the molting process12 and it has been used as reproductive inhibitor13. Chlorfluazuron can impact oogenesis and ovarian development14,15, oviposition stimulating factors16, inhibiting protein content in both ovaries and testes in newly emerged adults17 and affect fecundity, fertility and hatchability.
Chlorantraniliprole (coragen®) is a new insecticide belonging to a newer class of selective insecticides (anthranilic diamides) (Groups 28, IRAC). This class of insecticides is effectively controlling lepidopterous insects, especially insects that have developed resistance to older classes of insecticides10. Anthranilic diamides bind to receptors in insect muscles (ryanodine) causing an uncontrolled release of calcium from internal stores in the sarcoplasmic reticulum, leading to feeding cessation, paralysis and death of target insects18,19. So far, there are few resistance cases to chlorfluazuron and chlorantraniliprole20. The development of alternative insect management strategies, allow the rational use of insecticides and adjust application timing are crucial in providing resistance management to various insecticides. This study aimed to evaluate and compare the effects of chlorantraniliprole and chlorfluazuron on certain behavioral and biological aspects of S. littoralis at different stages.
MATERIALS AND METHODS
Insect rearing: A laboratory strain of Spodoptera littoralis was reared in insect physiology lab, Department of Applied Entomology and Zoology, Faculty of Agriculture, Alexandria University, Egypt, on castor bean leaves, Ricinus communis L., under constant conditions of 27±2°C and RH 65±5%.
Tested insecticides: Two insecticides of different groups were tested for their efficacies, behavioral and biological effects on S. littoralis through the period from September, 2017-October, 2018. Chlorfluazuron (Topron® 5% EC) was produced by Agrochem Co., Alexandria. Chlorantraniliprole (Coragen® 20% SC) was produced by DuPont™ de Nemours Co.
Insecticidal activities of chlorantraniliprole and chlorfluazuron against S. littoralis at different life stages Ovicidal activity: Egg-masses of S. littoralis at 0-24 h ages were counted with the hand lens (10X) and leaf disks containing 100 eggs were dipped for 20 sec in different concentrations of the tested insecticides. Another set of egg-masses (100 eggs) on leaf disks were dipped in water to represent the control. Each concentration, including the control, was replicated three times. Treated and untreated egg-masses were left to dry and kept at 27±2°C, RH 65±5%. After maximum hatch, microscopic examination was made to calculate the hatchability for each treatment. The LC50 values were calculated using Biostat ver. (2.1) software for probit analysis21.
Larvicidal activity: The efficacy of the tested insecticides, chlorantraniliprole and chlorfluazuron, was assessed against the 2nd and 4th larval instars of S. littoralis. Serial concentrations of each insecticide were prepared using distilled water. The 2nd larval instar was tested at 0.1, 0.5, 1, 2 and 5 mg L–1. Whereas, the 4th larval instar was tested at 1, 5, 10, 20 and 30 mg L–1. Castor bean leaves, almost equal in size, were dipped in the tested concentrations for 10 sec then left to dry. A set of castor leaves were dipped in distilled water only for the control. Each treatment was replicated 3 times (20 larvae per each). The larvae were allowed to feed on treated leaves and the mortality percentages were recorded after 48 and 96 h, corrected according to Abbott’s formula22. The LC50 values were calculated as concentration-mortality regressions, which analyzed by Biostat ver. (2.1) software for probit analysis21.
Repulsive effects of the tested insecticides
Antifeedant activity: Antifeedant activities of chlorantraniliprole and chlorfluazuron were studied for each 2nd and 4th larval instars of S. littoralis using leave discs in no choice test. The larvae were kept without food for 4 h before treatment. Fresh castor leave discs were dipped in 0.1, 0.5, 1, 2 and 5 mg L–1 for 2nd instar and 1, 5, 10, 20 and 30 mg L–1 for 4th instar for both insecticides. The discs were left to dry, weighed before being provided to the larvae. Discs for untreated control were dipped in distilled water only. Each treatment was replicated three times with 20 larvae for each replicate. After 24 h, larvae were removed and the remaining leave discs reweighed. The feeding deterrence (FD %) was calculated using Isman et al.23 formula:
where, C was the consumption of the control discs and T was the consumption of the treated discs.
Anti-oviposition activity: A pair of male and female pupae was separately put in 500 mL plastic cups with a ball of cotton dipped in 10% sugar solution for feeding and changed daily till egg-mass depositions. The newly emerged adults were left to feed and mate. Leaves of tafla were treated with concentrations (0.5, 1, 2, 5 and 10 mg L–1) of tested insecticides and provided to adults for laying egg. Two days after the beginning of egg laying, adults were removed and the number of eggs laid on treated and control tafla leaves were counted using a binocular. Adult repellency and anti-oviposition effect were calculated according to Pascual-Villalobos and Robledo24:
where, C was the number of eggs in the control and T was the number of eggs in the treatment.
Effect of chlorantraniliprole and chlorfluazuron on biological aspects of S. littoralis: To assess the effects of tested insecticides on the biology of S. littoralis, fresh castor leaves were dipped in LC25 and LC50 equivalent concentrations of the two tested insecticides. The treated leaves were provided to newly hatched larvae as food. Another set of castor leaves dipped in distilled water were provided for untreated control. The leaves were changed daily. Three replicates were used for each treatment, with one hundred larvae were tested for each replicate. Larval weight (mg) was measure after 5, 10 and 15 days after treatment. Larval duration (days), percentage pupation, pupal duration (days), pupal weight (mg), percentage adult emergence, mean number of eggs laid each day over the course of the female lifespan (fecundity), the hatchability of laid egg (percent of fertility), weight and protein content of ovaries were recorded.
Effect of chlorantraniliprole and chlorfluazuron on ovarian weight and ovarian protein content of S. littoralis females: The effects of tested insecticides on ovarian weight and protein contents in newly emerged females were assessed by feeding the larvae on castor leaves treated with LC25 and LC50 equivalent concentrations of the tested insecticides. Castor leaves dipped in water were used as untreated control. Ovaries were weighed and dissected from newly emerged females. The ovaries were homogenized in 1 mL of Tris 50 mM buffered at pH 7. The homogenate was centrifuged at 2500 rpm for 10 min, then the supernatant was collected and used for protein estimation. The total protein was determined according to Bradford25 using bovine serum albumin (BSA) as a standard protein. Absorbance was measured at 595 nm.
Statistical analysis: Statistical analysis of the obtained data and all the probable comparison combinations were analyzed in factorial design using SAS procedure26 at probability level of 0.05. Means were compared using the least significant difference test (LSD).
RESULTS
Chlorantraniliprole and chlorfluazuron toxicity against S. littoralis stages: The efficacy of chlorantraniliprole and chlorfluazuron against different stages of S. littoralis was assessed based on LC50 values, which confirmed the high toxicity of chlorfluazuron against different stages compared to chlorantraniliprole (Table 1). Chlorfluazuron was the most effective against S. littoralis egg masses with LC50 value 1.24 mg L–1. Chlorfluazuron was more toxic than chlorantraniliprole against 2nd and 4th larval instars whether after 48 or 96 h, with LC50 values 0.19, 0.09 for 2nd instar and 5.98, 1.42 mg L–1 for 4th instar. The LC50 values decreased over time of insecticide exposure. These results also showed that the 2nd instar larvae were more sensitive to the tested insecticides.
Repulsive effects of the tested insecticides: Insects usually choose appropriate plant for feeding and/or laying egg.
| Table 1: | Toxicity of chlorantraniliprole and chlorfluazuron against different stages of S. littoralis |
*SE: Standard error | |
| Table 2: | Antifeedant activity of chlorantraniliprole and chlorfluazuron against the 2nd and 4th larval instars of S. littoralis after 48 h from exposure |
*Feeding deterrence | |
| Table 3: | Anti-oviposition activity of chlorantraniliprole and chlorfluazuron against S. littoralis females |
Therefore, dismissal strategy may be one of the most effective methods used in IPM programs or/and prevent insects from feeding or egg laying. Consequently, insects die and the number of off spring decreases. The two tested insecticides significantly affected the palatability of insects for food (Table 2). When comparing the repellent effects of the 1 and 5 mg L–1 concentrations on the 2nd and 4th larval instars, showed that chlorfluazuron seemed to prevent larval feeding, especially for the 4th instar, compared to chlorantraniliprole. Feeding deterrence percentages were 12.4 and 25.5% for the 2nd instar treated with chlorfluazuron compared to 20.9 and 33.7% for the 4th instar. These antifeedant effects of the tested insecticides may cause feeding cessation of larval stages, leading to protein deficiency in different stages and ultimate mortality.
Repellency index (RI %) showed the significant repulsive effect for egg laying of tested insecticides (Table 3). Like antifeedant effect, chlorfluazuron was more deterrence than chlorantraniliprole for females. This repulsive effect has a positive correlation with elevate in concentration. The highest repellency index 42% was recorded with 10 mg L–1 chlorfluazuron.
Impact of the tested insecticides on biological aspects of S. littoralis
Impact on larval and pupal weight: The tested insecticides reduced larval and pupal weight, especially when the larvae were exposed to chlorfluazuron LC50 (Fig. 1). The most affected stage was the 5 days larva, where its weight reduced from 52.6-27.3 mg, with percentage of reduction 48.19%.
| Fig. 1: | Effect of chlorantraniliprole and chlorfluazuron at LC25 and LC50 concentrations on the weight of larvae after 5, 10 and 15 days from treatment and pupae of S. littoralis |
| Fig. 2: | Effect of chlorantraniliprole and chlorfluazuron at LC25 and LC50 concentrations on larval duration, pupal duration and adult survival of S. littoralis |
On the contrary, the pupal stage was the lowest affected stage, where, the weight reduced from 292.1-252.4 mg, with percentage of reduction 13.6%. These results synchronized with the previously mentioned results of antifeedant effect of the tested insecticides.
Impact of the tested insecticides on survival of S. littoralis life stages: The larval, pupal and adult survival was significantly affected by the sublethal concentrations of chlorantraniliprole and chlorfluazuron (Fig. 2). The duration of larval and pupal stages was increased, especially when the larvae were exposed to LC50 concentration of chlorfluazuron (0.19 mg L–1), where it was 23.17 and 11.83 days for larval and pupal stages, respectively. On the contrary, adult survival was significantly decreased as a result of larval exposure to the tested insecticides. These results indicated that these two insecticides affect the survival of adults, decrease the available time for female to lay eggs and subsequently decrease female fecundity.
In addition to the decline effects of insecticides on stages duration, the application by chlorfluazuron caused malformations of produced stages as showed in Fig. 3-6. As compared with normal stages, treatments with the different concentrations of chlorfluazuron caused different degrees of abnormalities, in 2nd larvae (Fig. 3a-d), legless larva with huge head (Fig. 3b) curved larva with dark rings on the abdomen (Fig. 3c) and curved larva with a pupal cuticle on the dorsal region of the head and thorax (Fig. 3d). Also, the 4th instar larvae showed malformations due to the treatment (Fig. 4a-d), larva with distended thorax and sclerotized head (Fig. 4b) some malformations in head and thoracic legs (Fig. 4c) and distorted and sclerotized head (Fig. 4d). Application of early larval stage by chlorfluazuron produced abnormal pupae (Fig. 5a-d), partial molting of prepupae (Fig. 5b and c) and Larva-pupa intermediates (Fig. 5d).
| Fig. 3(a-d): | Some abnormalities of the 2nd instar larvae of S. littoralis as a result of chlorfluazuron applications, (a) Control and (b-d) Distorted larvae |
| Fig. 4(a-d): | Effect of chlorfluazuron applications on the 4th instar larvae of S. littoralis, (a) Control and (b-d) Some malformations |
| Fig. 5(a-d): | Effect of chlorfluazuron applications on S. littoralis pupae, (a) Control and (b-d) Abnormal pupae |
The adults of S. littoralis affected by the application where, adults failure to emerge from their pupal cuticle (Fig. 6a-d), the adult with short wings and unmolted mouthparts (Fig. 6b) and partial molting of pupae (Fig. 6c and d).
Percentages of pupation and adult emergence of S. littoralis as affected by tested insecticides: Chlorantraniliprole and chlorfluazuron significantly decreased percentages of pupation and adult emergence, especially when LC50 of chlorfluazuron was used in application (Fig. 7). The percent of pupation and adult emergence was 51.67 and 44.3%, respectively.
Effect of insecticides on fecundity and percentages of fertility of S. littoralis females: The exposure of larval stage to the tested insecticides significantly reduced fecundity and fertility of females (Fig. 8).
| Fig. 6(a-d): | Adult emergence of S. littoralis affected by chlorfluazuron applications, (a) Control and (B-d) Adults failure to emerge from their pupal cuticle |
| Fig. 7: | Effect of chlorantraniliprole and chlorfluazuron at LC25 and LC50 concentrations on percentages of pupation and adult emergence of S. littoralis after larval application |
| Table 4: | Effect of chlorantraniliprole and chlorfluazuron on ovarian weight and ovarian protein content |
Means within a column followed by different letters are significantly different (p<0.05) | |
Chlorfluazuron was more effective than chlorantraniliprole. The LC50 concentrations cause greater declines in fecundity and fertility compared to the effect of LC25 concentrations. The application by LC50 concentrations resulted in reduction of fecundity from 855-498 with reduction percentage 42% chlorfluazuron and from 855-572 with reduction percentage 33.1% for chlorantraniliprole. The percent of fertility reduced from 95.33-52 and to 74.33% for chlorfluazuron and chlorantraniliprole, respectively.
Impact of insecticides on ovarian weight and ovarian protein content of S. littoralis females: Treated females with chlorantraniliprole and chlorfluazuron at LC25 and LC50 significantly reduced both of ovarian weight and protein contents of the ovaries compared to the untreated control (Table 4). The ovarian weights were 55.87 mg and 64.77 mg after larval application with chlorfluazuron and chlorantraniliprole at LC50 concentrations, respectively, compared to control 71.93 mg.
| Fig. 8: | Effect of chlorantraniliprole and chlorfluazuron at LC25 and LC50 concentrations on fecundity and percentages of fertility of S. littoralis females after larval application |
There is no significant difference between the effect of chlorantraniliprole LC50 and chlorfluazuron LC25 on ovarian fresh weight or ovarian protein content.
DISCUSSION
The results confirmed the toxic effects of tested insecticides against the different stages of S. littoralis, with superiority of chlorfluazuron. These results were in agreement with those reported results of the highly toxicity of chlorfluazuron against S. littoralis larvae27,28. The application of chlorfluazuron (Topron®) found to reduce the cotton leafworm infestation about 85.7% and increased cotton yield29.
There were various reports of the effects of chlorfluazuron against other insects. Higher dosages of chlorfluazuron significantly reduced Spodoptera litura population14. Similar toxic effect was also recorded against Palpita indica eggs30, male pupae of Tribolium castaneum31 and larvae of Agrotis ipsilon in comparison with conventional insecticides32.
A good control of corn earworm in soybean by chlorantraniliprole was reported by Adams et al.33. The toxic effect of chlorantraniliprole was recorded against many insects such as apple maggot, blue berry maggot and cherry fruity34, eggs and larvae of Lobesia botrana35. Chlorantraniliprole also was recommended for controlling Tuta absoluta36 and showed better toxicity against S. litura population37, Helicoverpa armigera, A. ipsilon and S. litura38. The mortality of Amyelois transitella eggs was doubled by adding chlorantraniliprole39. Toxicity of chlorantraniliprole was also reported against 2nd instar larvae of Spodoptera cosmioides40. The 2nd larval instar was the most sensitive stage to insecticides. This trend previously recorded for chlorfluazuron against S. litura larvae41,42 and for chlorantraniliprole against S. littoralis larvae43.
The tested insecticides significantly affected the palatability of insects for food and oviposition with advantage of chlorfluazuron, as it was more deterrence for insects, as well. These findings were in agreement with those found that chlorantraniliprole caused repellency against Asian subterranean termites and Coptotermes gesteroi44. Chlorantraniliprole activates the feeding cessation and finally insect death18. Unlike, the repellency effect, chlorantraniliprole does not have behavioral effects as food repellent36.
Referring to the data of tested insecticides impacts on the biological aspects of S. littoralis, all treatments have a reduction effect on larval and pupal weight. By the same way, chlorfluazuron significantly decreased pupal weight of A. ipsilon45. On the contrary, S. cosmioides pupal weight increased by exposed larvae to sublethal concentrations of chlorantraniliprole40.
Also, the tested insecticides significantly increased the duration of larval and pupal stages, while, they decreased adult longevity. This effect was suggested previously, chlorfluazuron increased A. ipsilon larval and pupal duration32,45. Chlorantraniliprole increased the S. cosmioides larval and pupal stages40. The tested insecticides significantly shortened female’s longevity, hence the oviposition period was significantly reduced. These observations may explain the sharp decline in fecundity and fertility of females treated by these insecticides. Concerning the reduction effect on the adult longevity, it was reported for chlorfluazuron on P. indica30 and on A. ipsilon45. This result disagreed with the recorded delaying action of chlorfluazuron on Pectinophora gossypiella adult longevity46-48. Chlorantraniliprole also prolonged the longevity of S. cosmioides adults40.
All treatments with chlorfluazuron caused malformations of S. littoralis stages, this agreed with large extent with the recorded results that chlorfluazuron had the higher rates of deformed pupation of S. littoralis28. Chlorfluazuron also induced morphological abnormalities of A. ipsilon life stages32.
Chlorantraniliprole and chlorfluazuron, significantly decreased pupation and adult emergence percentages of S. littoralis as similar as chlorfluazuron lower concentrations, caused significant reduction in pupation and adult emergence of S. littoralis49. The LC50of chlorfluazuron significantly declined the pupation and adult emergence percentage of A. ipsilon45. On the contrary, chlorfluazuron had the higher rates of adult emergence for S. littoralis28.
Furthermore, fecundity and fertility of S. littoralis females significantly decreased when the larval stage exposed to the tested insecticides. This result was confirmed for chlorfluazuron which significantly reduced S. littoralis fertility, fecundity and hatchability28,49. The effect of chlorfluazuron has been previously illustrated also for other insects such as S. litura14, in P. gossypiella46,47, in P. indica30, A. ipsilon32,45 and in S. cosmioides for chlorantraniliprole40.
Tested insecticides significantly reduced ovarian weight and protein ovary contents. These results were indicated for chlorfluazuron against S. litura14 and A. ipsilon females45.
Although, the present study confirmed the effectiveness of chlorantraniliprole and chlorfluazuron against S. littoralis, further investigations may be needed in the field trial as well as using them in IPM programs for controlling this target pest in the future.
CONCLUSION
On the basis of overall findings, it was concluded that chlorantraniliprole and chlorfluazuron had impacts on the behavioral and biological aspects of S. littoralis. These tested insecticides provided a good control of S. littoralis and may be used as alternatives to conventional insecticides in IPM programs for controlling this target pest.
SIGNIFICANCE STATEMENT
This study suggested that the tested insecticides, regardless their toxic effect, disruptively affected the biological and behavioral aspects of S. littoralis. These effects are very important because offspring can then be reduced and as a consequence, the insect population can be maintained below a level of economic loss. This study will help the researcher to trend towards using other environmental safer insecticides from different classes and mode of actions which has become an unabated challenge in controlling cotton insect pests.