Toxicity of chlorpyrifos, spinosad and abamectin on cotton bollworm, Helicoverpa armigera and their sublethal effects on fecundity and longevity


Cotton bollworm, Helicoverpa armigera Hubner is the most important pest of cotton, tomato and chickpea in Iran. The pest has been found to develop resistance against conventional insecticides. Using of insecticides with different mode of action may result an appropriate control of the pest and may delay insecticide resistance development. In this study, we attempted to investigate the lethal and sublethal effects of one conventional, two biorational insecticides on larvae at different stages of H. armigera, and its adult longevity and fecundity under the laboratory condition. The LC50 values of chlorpyrifos, spinosad and abamectin were 4.6, 62.26 and 460.5 ppm based on formulated materials. The results showed that chlorpyrifos and spinosad were more effective insec- ticides against 3rd instar larvae of cotton bollworm compared to abamectin based on formulated materials. Furthermore, abamectin and chlorpyrifos negatively affected longevity and fecundity of adults that emerged from treated third instars. But, spinosad had no significant effect on longevity and fecundity of the adults. All the tested insecticides seemed to be effective against cotton bollworm. Spinosad and abamectin might be preferred due to their environmental friendly impacts. Abamectin also markedly re- duced female fecundity and longevity for adults exposed as third instars larvae compared with spinosad. The larvicidal and repro- ductive effects of abamectin against the pest and its biorationality suggest that abamectin is suitable for integration into an IPM program for cotton bollworm.


Cotton bollworm, Helicoverpa armigera Hubner is the most destructive pest of cotton and tomato in Iran. The cotton  bollworm feeds  on  most  plant  parts  including stem, leave, flower buds, flower heads and fruits at dif- ferent larval development stages (Moral Garcia, 2006). This pest can survive in unstable habitats and colonize various agricultural ecosystems (Fitt, 1989). This pest with characteristics such as, ability to migrate, high fe- cundity, facultative diapause, polyphagy and ability to develop resistance to conventional insecticides causes considerable damages on the host plants (Fitt, 1989; MacCaffery, 1998). In Iran, the damage caused by the pest is around 20-25% of the yield and rises to 50-75% at times of outbreak of the pest (Mojeni et al., 2005). Similar to most of other cotton and tomato growing countries, insecticides are largely used for controlling cotton bollworm in Iran.

Endosulfan, profenofos and thiodicarb have been the commonly used insecticides for controlling H. armigera in recent years in Iran (Mo- sallanazhad et al., 2003). The use of widespread insecti- cides at high doses, as well as inappropriate timing have cause adverse effects on natural enemies and resulte in resurgence of cotton bollworm (Metcalf and Luckman, 1975). The pest has developed resistance against con- ventional insecticides such as chlorinated hydrocarbons, synthetic  pyrethroides  and  organophosphates  in  Pakistan (Ahmad et al., 1998a, 1998b, Ahmad et al., 1999), Spain (Torres-Vila et al., 2002a; 2002b) and Australia (Gunning et al., 1984; Gunning and Easton, 1994). De- spite no record of resistance of cotton bollworm against insecticides in Iran, establishment of resistance to con- ventional insecticides by this pest is not unexpected.

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Therefore, using insecticides with different mode of action may result an appropriate control of the pest and may delay insecticide resistance development. Chlor- pyrifos, spinosad and abamectin were selected for studying against H. armigera from different classes of the insecticides. Chlorpyrifos exerts its toxic action by inhibiting certain important enzymes of the nervous sys- tem, cholinsterases (ChE) (Ware and Whitacre, 2004). Mode of action of abamectin is blocking of the neuro- transmitter gamma aminobutyric acid (GABA), at the neuromuscular junction in insects and mites (Ware and Whitacre, 2004). Spinosad acts by disrupting binding of acetylcholine to nicotinic acetylcholine receptors at the postsynaptic cells (Salgado, 1997). Field studies were carried out to evaluate the efficacy of different insecti- cides such as chlorpyrifos, endosulfan, profenofos and spinosad against lentil pod borer (Helicoverpa spp.) and results showed that spinosad was the most effective in- secticide in reducing the population of lentil pod borer (Ahamad Memon et al., 2005).

Nirmal and Manjit (2008) reported that spinosad and chlorpyrifos had higher efficacy against third instar lar- vae of cotton bollworm compared with endosulfan, ace- phate and cypermetrin. Abamectin and spinosad are bio- rational insecticides that are known as antibiotics (Ware and Whitacre, 2004). Abamectin is produced by the soil bacterium, Streptomyces avermitilis Burg (White et al., 1997).  Spinosad  is  derived  from  soil  actinomycetes,

Saccharopolyspora spinosa Mertz et Yao, containing a naturally occurring mixture of spinosyn A and spinosyn D. Spinosad had high toxicity to H. armigera and was a safe insecticide to some of natural enemies (Rafiee Dast- jerdi et al., 2008; Amalendu, 2010). But it seems that sublethal doses of pesticides may affect physiology and behavior of pests and theirs natural enemies (Johnson and Tabashnik,  1999).  It  has  been  reported  that  sublethal doses of insecticides may reduce and or increase longev- ity, fertility or fecundity of pests. Therefore, the study of sublethal effects of insecticides may show ecological as- pects of insecticide applications and provides a suitable pest management program. In this study, we investigated the effects of lethal and sublethal effects of two biora- tional and one conventional insecticide on longevity and fecundity of an important insect pest of cotton and tomato H. armigera under laboratory conditions.

Materials and methods


The eggs of cotton bollworm were taken from De- partment of Plant Protection of the University of Tabriz, Iran. When the eggs were hatched, the larvae were transferred  to  plastic  containers  containing  artificial diet. The artificial diet was made using cowpea powder 205 gr, powdered agar 14 g, ascorbic acid 3.5 g, sorbic acid 1.1 g, methyl-p-hydroxybenzoate 2.2 g, yeast 35 g, wheat germ 30 g, formaldehyde 37% 2.5 ml, vegetable oil 5 ml and distilled water 650 ml (Shorey and Hale,1965). In order to prevent cannibalism, the larvae were individualy transferred into a 33 ml glass vials at third instar. The vials were maintained in an insectarium at 25 ± 1 °C, 70 ± 5% RH and a photoperiod of 16:8 (L:D) until pupation. After emergence, 15 pairs of adult moths were released into 18× 20 cm plastic container with 1:1 sex ratio for mating and egg-laying. The adults were fed on a 10% honey solution.


Insecticides tested were spinosad (SpinTor® 24SC, Dow  AgroSciences,  India),  chlorpyrifos  (Dursban® 48EC, DowElanco, England) and abamectin (Gyamec- tin® 1.8EC, Gyah, Iran).


The toxicity of insecticides was assessed on 3rd instar larvae of the cotton bollworm. Third instar larvae of the pest were exposed to spinosad by mixing the insecticide dilutions with artificial diet and exposed to abamectin and chlorpyrifos by residue contact method. Oral bioas- say method was chosen for spinosad because although it exhibits good activity via topical or contact application, better activity is observed by oral application or by in- jection (Sparks et al., 1997; 1998) suggesting that pene- tration through the insect cuticle may be comparatively slow (Salgado and Sparks, 2010). The ranges of concen- trations were determined for the insecticides by prelimi- nary dose-setting tests. The ranges of used concentra- tions were 30-100, 3-7, and 200-800 ppm for spinosad, chlorpyrifos and abamectin, respectively. The main bioassay tests were conducted with six different concentra- tions based on logarithmic intervals for each insecticide. Each concentration involved 3 replications and each bioassay test was replicated three times. After applying the insecticides, 20 third instar larvae were transferred into Petri dishes (9 cm diameter) for each concentration in both methods. The Petri dishes were kept in incubator at 25 ± 1 °C, 70 ± 5% RH and a photoperiod of 16:8 (L:D). Mortality was recorded after 24 h in all experi- ments.

Determination of sublethal effects of H. armigera

About 100 third instar larvae of cotton bollworm were treated with LC30 of each insecticide by the same meth- ods used for bioassay tests for them. After 24 hours, the survivors were collected and kept in glass vials indi- vidually on artificial diet until pupation. The pupal weight and life span of pupal were recorded. For fecun- dity and longevity study, each pair of emerging male and female adults were collected and kept in cylindrical containers. The adults were fed on 10% honey solution. The experiments were carried out with 10 replications for all tested insecticides and control. The number of eggs laid by each female was recorded daily until the female died. The longevity of female adult was recorded as  well.  Experiment  were  conducted  at  25  ±  1  °C, 70 ± 5% RH and a photoperiod of 16:8 (L:D).

Statistical analysis

The data were  analyzed using probit procedures of SAS program (SAS Institute, 2002). To compare toxic- ity of the same insecticide in different bioassay meth- ods, as well as the toxicity of different chemicals with each other, the ratios of the LC50 values and their related 95% confidence limits were calculated (Robertson and Preisler, 1992).



The LC90, LC50  and LC30  values of the tested insecti- cides against third instar larvae of H. armigera are pre- sented in table 1. Based on LC50  values, chlorpyrifos was the most toxic insecticide on third instar larvae of cotton bollworm followed by spinosad and abamectin. The toxicity of insecticides tested was significantly dif- ferent (table 1).

Sublethal effects on pupae

Sublethal effects of LC30  of the tested insecticides on pupal weight and period are shown in table 2. The pupal periods and pupal weight of cotton bollworm in treat- ment with abamectin and chlorpyrifos were significantly the longer and lighter than the control (pupal periods; F = 11.4, df = 3, P < 0.0001: pupal weight; F = 4.4, df = 3, P = 0.01). However, spinosad did not affect the pupal period and pupal weight significantly.

Sublethal effects on longevity and fecundity of adults Sublethal effects of LC30 of spinosad, chlorpyrifos and abamectin on longevity and fecundity of female cotton bollworm treated with LC30 of the insecticides at 3rd in- star larvae are shown in table 3. Abamectin and chlo- ryrifos reduced the mean number of eggs oviposited by cotton bollworm female significantly compared to con- trol and spinosad (F = 6.02; df = 3; P = 0.001). Female fecundity was reduced by 36.5, 34.5 and 9.9% in abamectin, chloryrifos and spinosad treatments com- pared to control, respectively.

Treatments of third instar larvae with abamectin and chlorpyrifos  reduced  longevity  of  adults  significantly (F = 3.84; df = 3; P = 0.015). However, spinosad did not affect  adult  longevity,  significantly.  Abamectin  and chlorpyrifos reduced the longevity by 18.5 and 17.5% compared with control, respectively. There were non- significant differences between abamectin and chlor- pyrifos effects on adult longevity. And also adult longevity was not affected significantly by spinosad.

Sublethal effect on adult oviposition

Oviposition by females in different treatments during the days after adult emergence had the same pattern and is shown in figure 1. Maximum number of oviposited eggs was in the days 4th-9th after adult emergence in all treatments.


Results obtained from acute toxicity assays of the tested insecticides showed that chlorpyrifos and spinosad were the most toxic chemicals against cotton bollworm. Simi- lar results were found by Aslam et al. (2004), who have reported that chlorpyrifos was the most effective insec- ticide for controlling cotton bollworm among tested insecticides. Nirmal and Manjit (2008) reported that the LC50 values of spinosad and chlorpyrifos were 0.04 and 0.66 ppm, respectively. Differences in LC50  values of these two studies would be due to differences in the duration of exposure to the insecticide and larval instar. Therefore, according to our results and the mentioned studies spinosad and chlorpyrifos have high potential for controlling cotton bollworm.

Chlorpyrifos had the highest slope among the test in- secticides (table 1). The high slope indicates that a slight increase in insecticide concentration will lead to high mortality compared with the other insecticides. How- ever, the higher slope indicates that there will be in- creased selection pressure on the population. Thus, there will be a greater risk of selection of resistant individuals compared with the other insecticides especially in cases of continued use of the same insecticide.

The  current  study demonstrated  that  abamectin  and chlorpyrifos significantly reduced the pupal weight, lon- gevity and fecundity of adult cotton bollworm. Spinosad did not affect the pupal period, pupal weight and fecun- dity of the adults. Furthermore, spinosad had no effect on longevity in compared with control. The results of present study are in agreement with those of Nirmal and Manjit (2008) who reported that LC30  and LC50 values of chlorpyrifos significantly reduced the means of oviposition of cotton bollworm compared with control. Furthermore, they have reported that LC50 values of spinosad did not affect the longevity of cotton boll- worm, but LC30 values of spinosad significantly reduced the oviposition compared with control.  In contrast with our results, Wang et al. (2009) reported that 0.04 and 0.16 mg kg-1 of spinosad increased the pupal periods and reduced pupal weight. It also reduced longevity and fe- cundity of adult cotton bollworms. Sublethal dose of spinosad  increased  the  fecundity  of  Orius  insidious

(Say) compared with control, but this increase was not significant (Elzen, 2001). Pineda et al. (2007) reported that spinosad reduced the fecundity and fertility of Spo- doptera littoralis (Boisduval) adults when treated orally and residually. The type of formulation of the insecti- cides, differences among the populations used and ex- posure methods may account for the different results in our study and those of others.

In addition, it was demonstrated that chlorpyrifos and spinosad had high acute toxicity against cotton bollworm. Abamectin and clorpyrifos negatively affected the longevity and fecundity of the pest. These impacts are very important for practical management of the pest, be- cause these effects may lead to the reduction of the pest population to a lower level even under economic injury level. Therefore, the authors propose that both lethal and sublethal effects of the insecticides should be considered in developing of a pest management program.


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Authors’ addresses:

Moosa SABER (corresponding author, e-mail:,, Samad VOJOUDI (, Department of Plant Protec- tion, College of Agriculture, University of Maragheh, Maragheh, Iran; Mir Jalil HEJAZI (, Department of Plant Protection, College of Agriculture, Uni- versity of Tabriz, Tabriz, Iran; Reza TALAEI HASSANLOUI (, Department of Plant Protection, College of Agriculture, University of Tehran, Karaj, Iran


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