Abstract.-Two different bioassay methods; larval dip bioassay and surface diet bioassay were tested for the contact/ingestion toxicity and to see the advantage of one method over the other using spinosad against second instar Helicoverpa armigera. Little difference was observed in LC 50 values which were 170 and 130ppm for both the methods, however ingestion/contact toxicity (surface diet bioassay) as compared to contact toxicity only (larval dip bioassay) remained comparatively but not significantly different. Results also proved that surface diet bioassay had advantage over larval dip bioassay as it provided better estimate of potential toxicity

Ahmed Rashid

And Khan

Fatima Farhat

Jamil

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Pakistan J. Zool., vol. 39(2), pp.133-136, 2007. 
 
Estimation of Toxicity of Spinosad Using Two Different 
Bioassay Methods Against Cotton Bollworm, 
Helicoverpa armigera (Hub.) 
 
RASHID AHMED KHAN AND FARHAT FATIMA JAMIL 
Plant Protection Division, Nuclear Institute for Agriculture and Biology (NIAB),  
P.O. Box No. 128, Jhang Road, Faisalabad 
 
 Abstract.- Two different bioassay methods; larval dip bioassay and surface diet bioassay were tested for the 
contact/ingestion toxicity and to see the advantage of one method over the other using spinosad against second instar 
Helicoverpa armigera. Little difference was observed in LC50 values which were 170 and 130ppm for both the 
methods, however ingestion/contact toxicity (surface diet bioassay) as compared to contact toxicity only (larval dip 
bioassay) remained comparatively but not significantly different. Results also proved that surface diet bioassay had 
advantage over larval dip bioassay as it provided better estimate of potential toxicity. 
 
Key words: Spinosad, Bioassay, LC50, H. armigera. 
 
INTRODUCTION 
 
 Cotton is attacked by a number of sucking 
and chewing pests in Pakistan; amongst the latter, 
cotton bollworm, Helicoverpa armigera is currently 
a very important pest. It is currently the most 
important in economic terms and highly 
polyphagous agriculture pest. Host plants of H. 
armigera belongs to a broad spectrum of plant fauna 
and include important agricultural crops such as 
cotton, maize, chickpea, pigeonpea, sorghum, 
sunflower, soybean and groundnuts (Fitt, 1989). 
Due to wider host range, multiple generations, 
migratory behavior, high fecundity and existing 
insecticide resistance this became a difficult pest to 
tackle (Hussain et al., 1991; Khan et al., 1993; 
Ahmad et al., 2000). Like other cotton producing 
countries, Pakistan largely relied on chemical 
control of this pest. The indiscriminate use of 
insecticides, particularly during 80s and 90s 
contributed to the emergence of insecticide 
resistance in H. armigera. 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  
 
0030-9923/2007/0002-0133 $ 8.00/0 
Copyright 2007 Zoological Society of Pakistan. 
insecticides were recorded in field population of H 
armigera (Ahmad et al., 1995). 
 Hence newer molecules with novel mode of 
action are currently essential for the management of 
pyrethroid resistant of H. armigera. Spinosad is a 
reduced risk insecticide based on metabolites of a 
soil bacterium Saccharopolyspora spinosad (Mertz 
and Yao, 1990). This bacterial insecticide has a 
unique mode of action with a very low mammalian 
toxicity compared with other insecticides (Bert et 
al., 1997; Salgado, 1997; Thompson et al., 1997). In 
the present study the toxicity of spinosad was 
studied against H. armigera using two bioassay 
methods and also to see suitability of bioassay 
method for better estimates of results. 
 
MATERIALS AND METHODS 
 
Test insects 
 Cotton bollworms were reared in the 
laboratory on modified semi-synthetic diet (Ahmad 
and McCaffery, 1991), consisting of Chickpea flour, 
Sorbic acid, Wesson's salt, Vitamin (ABDE), 
Ascorbic acid, Yeast (Brewer's), Choline chloride, 
Agar agar, Formaldehyde, Streptomycin sulphate 
and Methyl para hydroxy benzoate, under laboratory 
R. AHMED ET AL. 
 
134 
conditions of 27±2°C, 65±5% RH and 14:10 L:D h.  
 
Larval dip bioassay 
 Aqueous dilutions of formulated (emulsion 
concentrates) insecticide were prepared and batches 
of second instar larvae were submerged for 5 sec. as 
described by Watkinson et al. (1984). A group of 50 
larvae were dropped into 100 ml of the appropriate 
dilution in 500 ml beaker and gently swirled for 5 
sec. to ensure complete wetting. The solution plus 
larvae were then poured through a fine nylon mesh 
suspended over an empty beaker. The solution was 
decanted and larvae separated by this process. After 
shade drying for about 5 min. the treated larvae 
were then transferred individually into semi 
synthetic diet. Control insects were treated with 
water alone. 
 
Surface-treated diet bioassay 
 The surface-treated diet bioassay methods 
were similar to those described by (Joyce et al., 
1986). Three ml of a chickpea based artificial diet 
was pipetted into 30 ml vials and allowed to cool at 
room temperature for approximately 1 h. For each 
insecticide tested, serial dilutions of formulated 
material (100 µl aliquots) were pipetted onto the 
diet surface, agitated to distribute evenly, and 
allowed to dry for approximately 30 min. Second 
instar larvae were placed into a series of vials that 
contained 4 different concentrations of formulated 
insecticide viz., 100, 200, 300, and 500 ppm along 
with untreated controls to determine the LC50 for a 
given insecticide. Each vial contained one larva. A 
minimum of 50 larvae per dose were tested for each 
insecticide concentration. Larvae were considered 
dead if no movement was observed after prodding 
with blunt forceps for 10s. Control vials were 
treated with distilled water only.  
 
Laboratory conditions and test insecticides 
 Bioassays were carried out at 26±1°C under 
approximately 12:12 (L:D) h photoperiod. Mortality 
was recorded at 48 h interval. A serial dilution of 
the formulated test insecticide was prepared as ppm 
of the active ingredient. 
 
Statistical analysis 
 Larval mortalities were assessed after 48 h. 
Results were expressed as percentage mortalities. 
Data were analyzed using computer based probit 
analysis programme (Finney, 1971). 
 
RESULTS AND DISCUSSION 
 
 Results of bioassays revealed non-significant 
variation in response of H. armigera to spinosad 
using two different bioassay methods in Table I. 
The LC50 values of spinosad for larval dip bioassay, 
surface diet bioassay on H. armigera with 95% 
confidence limit (lower and upper limit) are also 
presented in Table I. After 48 hrs of application of 
spinosad LC50 value was found as 170 ppm using 
larval dip bioassay, whereas 130 ppm for surface 
diet bioassay. Chi-square values shows that there 
was insignificant difference of mortality rate of H 
armigera among the concentrations of spinosad. 
The R2 value and regression equation for log dose 
and probit mortality has been shown in Figure 1. 
The R2 value and regression equation for larval dip 
bioassay was found as 0.93 and Y=140 x +1.85, 
whereas Y=1.82 x +1.14 for surface diet bioassay. 
In both the above mentioned cases the slopes were 
positive. Similar to our findings Ramasubramanian 
and Regupathy (2004) recorded low LD50 against 
early instar larvae of H. armigera. 
 
 
 
 Fig. 1. Relationship of log doses and 
probit mortalities of spinosad using two 
bioassay methods on second instar larvae of H. 
armigera. 
 
 Spinosad's high effectiveness is even against 
pyrethroid resistant strains is attributed to its novel 
mode of action as mentioned by (Watson, 2001) 
With the increasing resistance that H. armigera is 
TOXICITY OF SPINOSAD AGAINST COTTON BOLLWORM 
 
135 
exhibiting towards a wide range of insecticides 
especially pyrethroids (Ahmad et al., 1997) the need 
 
Table I.- Toxicity of spinosad against Helicoverpa armigera using two different methods of bioassay. 
 
Insecticide 
conc. 
(ppm) 
Log conc. Sample  
size (n) 
Number  
dead  
(Larvae) 
Mortality 
(%) 
Corrected 
mortality 
(%) 
LC50 (ppm)  
(95% FL of LC50) 
Slope  
(SE) 
Chi- 
square 
(df) 
         
Surface diet bioassay 
100 2.0 50 19 38 35.42 170.23 1.39 0.94 (2) 
200 2.30 50 28 56 54.17 (36.42-275.48) (0.37)  
300 2.47 50 35 70 68.75    
500 2.69 50 36 72 70.83    
         
Larval dip bioassay 
100 2.0 50 21 42 40.82 129.91  
(81.23-168.34) 
1.81 (0.38) 0.42 (2) 
200 2.30 50 32 64 63.27    
300 2.47 50 39 78 77.55    
500 2.69 50 42 84 83.67    
         
 
for the use  of  more  effective  insecticides is 
increasing. Spinosad in our studies showed more 
toxicity for surface diet bioassay probably because 
of both contact and ingestion action of spinosad. 
Tabashnik and Cushing (1987) also proved that leaf 
residue bioassay which is similar to surface diet 
bioassay in our experiment has advantage over 
topical application, as it is time and labour saving 
method and provide more accurate estimates. 
 It is concluded from the results that spinosad 
is effective against H. armigera as direct and 
indirect application. Furthermore, the use of surface 
diet bioassay provides better estimates of potential 
toxicity for laboratory bioassay studies. 
 
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(Received 28 September 2006, revised 14 November 2006) 
 


Estimation of Toxicity of Spinosad Using Two Different Bioassay Methods Against Cotton Bollworm, Helicoverpa armigera (Hub.)

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Estimation of Toxicity of Spinosad Using Two Different Bioassay Methods Against Cotton Bollworm, Helicoverpa armigera (Hub.)

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