Abstract: Synergistic effect of W. calendulacea plant extract combined with Lamda cyhalothrin were demonstrated against red flour beetle T. castaneum in methanol extract. W. calendulacea plant extract offered synergistic action when used Lamda cyhalothrin. It was noted that plant extract indicates synergistic action from 1:1 to 1:5 ratio and above.
INTRODUCTION
Since 1940s increasingly large amount of insecticides and herbicides have been developed and marketed for insect pest and weed control. Resistance within or between whole classes of insecticides is a serious threat to control and management of many important insect pests. Pest control with pesticides is now a very important issue with different synthetic chemicals possessed several side effects. The tremendous difficulty and investment associated with the development of new, safe and cost-effective insecticides, there is a given need to preserve the efficacy of current insecticides. This may be achieved by the use of proper synergistic chemicals with insecticide[1].
Resistant strains of any insect pests were developed through the survival and reproduction of individuals after exposure to a given insecticide and as a result insect pests have now developed tolerance to all major classes of insecticides[2-4].
Development of insecticide resistance in stored grain pests is a global problem[5]. Resistance among population of T. castaneum is widespread throughout the world[6-11]. T. castaneum has been reported to develop great resistance against different chlorinated hydrocarbon (e.g. DDT, BHC/lindane) and organophosphates insecticides (e.g. malathion, fenetrothion, pirimiphos-methyl, dichliovos), carbamate (e.g. carbaryl and certain fumigants (e.g. methyl bromide, phosphine)[5, 9,12-17].
Synergists could play an important role in combating resistant population of T. castaneum and other insect pests, as well as reduction of insecticides application rates, in potential environmental contamination and improvement in the performance of integrated pest management programs. The presence of pesticides in the environment not only increases the non-target organism mortality, but also their levels can be reached where mammals ultimately can be seriously affected. Therefore the introduction of botanical pesticides or plant extract as synergist could be great beneficial, both economically and ecologically, especially since tests have shown that synergism increases in toxicity of insecticide is only towards insects and not mammals[18].
Wodelia calendulacea Less. (Asteraceae) is a traditional ayurvedic herbe which grow abundantly in the tropical and sub-tropical parts of the world. In ayurvedic medicine W. calendulacea is used as best drug for the treatment of liver cirrhosis and infective hepatitis, liver enlargement, jaundice and other ointments of the liver and gall bladder. The present study was aimed at standardizing a protocol for finding out insecticide or synergistic effect against red flour beetle T. castaneum Herbst.
MATERIALS AND METHODS
Local strain of the red flour beetle T. castaneum Herbst. were used in the present study which obtained from the stock culture maintained in the IPM Laboratory, Institute of Biological Sciences, University of Rajshahi, Bangladesh.
The whole plant Wedelia calendulacea Less. were collected and dried in oven at 40°C for 36 h and powdered in a morter and pastle separately. Extractions were done in a soxhlet apparatus. The powdered materials were put into the thimble of the soxhlet and extractions were carried out successfully with petroleum ether, ethyl acetate, acetone and methanol. The extracted materials were dried in rotary vacuum evaporator under reduced pressure.
Technical grade lambda-cyhalothrin 25 EC or 250 g L-1[(RS)-a-cyno-3-phenozybenyl(Z)-(1RS)-(2-Chloro-3,3,3-trifluropropenyl)-2-dimehyl-cyelopropene carboxylate], commercial available as karate[A product of ACI Pharmaceutical Company, Bangladesh Limited was used].
Bioassay procedures: Lamda cyhalothrin and plant extracts were dissolved in methanol to make required volume. The desired serial dilutions were prepared from the stock solutions using methanol. Various concentrations of each chemical were applied to thoracic notum of 5 days old adult T. castaneum using surface film contact method. Four concentrations were used in which six replications of beetles were treated. One batch of control was maintained in which only methanol was applied. Mortality of treated beetles was recorded after 24 h.
Analysis of data: The mortality data was corrected using Abbott’s formula[19], pr-po-pc, where, pr = corrected mortality%, po = observed, pc = control. Probit analyses were carried out as described by Busvine[20], using apple computer. Co-toxicity co-efficient were calculated as described by Sun and Johnson[21].
If the mixture gives a co-efficient significantly greater then 100, it indicates a synergistic action. On the other hand, when a mixture gives co-toxicity co-efficient less than 100, the effect of the mixture indicates an antagonistic action.
RESULTS AND DISCUSSION
The separation of different compounds from the whole plant of W. calendulacea was done by extraction with four solvents. Methanol extract separates oil, fats and fatty acids whereas ethyl acetate separates terpenes, alkaloids, flavonoids and steroids. Acetone separated chlorophylls, dyes and other alkaloids and methanol extracts separates all the remaining alkaloids and acidic compounds.
| Table 1: | Effect of Lamda cyhalothrin and Wedelia calendulacea plant extract against Tribolium castaneum (Herbst.) after 24 h of exposure |
| (-) indicates no sensitivity | |
Extracts in other solvents were found to be non-toxic to T. castaneum (Table 1) though methanolic extract was used for further investigation. Dose mortality experiment was done with five different doses of lambda-cyhalothrin (1800, 900, 450, 225 and 112.50 μg cm-2) applied on five days old beetles. The LD50 was calculated as 219.0014 μg cm-2) (Table 1) with 95% confidence limit as 147.224 to 325.7723 μg cm-2. The regression equation (Fig. 1) showed a good fit in significant χ2-value (0.729 at 3 df).
Different doses (750.00, 375.00, 187.50, 93.95, 46.873 μg cm-2) of methanol extract was shown wide range of mortality after 24 h. The LD50 was calculated as 89.928 μg cm-2 with 95% confidence limit 62.3025 to 129.387. From significant value of the χ2 (0.73206 at 3 df) it is evident that the regression line is fitted well (Fig. 2). Ethyl acetate, acetone and petroleum ether extract has shown no toxicity (Table 1).
The combined doses of Lambda cyhalothrin and methanol extract at different ratio (Table 2) were applied to 5 days old T. castaneum and the LD50 value has been calculated as 179.012, 106.654, 63.0494, 33.5282 μg cm-2 at a ratio of 1:1, 1:2, 1:5 and 1:10, respectively. The regression line is presented in Fig. 3.
| Table 2: | Toxicity and Co-toxicity Co-efficient of combined doses of Lamda cyhalothrin mixture of Wedelia calendulacea plant extract in methanol to adult Tribolium castaneum (Herbst.) |
| Fig. 1: | Regression line of probit mortality of T. castaneum on log dose of Lambda cyhalothrin |
Hewlett[22] has been investigated mechanism of synergism in insecticides. Sun and Johnson[23] stated that the insect is able to metabolize some or most of the insecticides to non-toxic compounds or to compound less insecticides in the absence of synergist but synergists reduce this metabolism and thus a greater portion of the insecticides exert it's toxic effect. The sites of detoxification and action appear in general to be separate and thus the synergism does not depend on knowledge of the mode of toxic action of the insecticides. Moreover, the same enzyme complex, within the insect appears to be responsible for oxidative detoxification of insecticides of different chemical structures. Synergists inhibit the enzymes responsible for toxicants degradation[24,25]. Otaki et al.[26], Otaki and Williums[27] showed that the insect body contains enzymes for the degradation of hormones like the molting hormone (MH) that may be mode of action of plant extractions.
| Fig. 2: | Regression line of probit mortality of T. castaneum on log dose of methanol extract of W. calendulacea |
Another possible explanation was advanced by Walker and Thomson[28] who found that simultaneous application of MH and Juvenile Hormone (JH) caused an increase in MH activity. A hypothesis for the mode of action of MH and JH when applied together was that the MH activated to synthesis of RNA and JH simultaneously by induced a duplication of DNA. This process causes such a severe disturbance in insect that it leads to its death because the DNA and RNA synthesis are mutually and perhaps exclude other.
It was observed in this investigation that lambda-cyhalothrin when used in conjunction with W. calendulacea plant extract offered significant synergism resulting low LD50 values for lambda cyhalothrin. From the co-toxicity co-efficient value (Table 2) it may be noticed that the W. calendulacea plant extract in methanol behaves as a synergist from 1:1 to 1:5 ratios and above.
| Fig. 3: | Probit mortality of log doses of Lambda cyhalothrin and methaolic extract of W. calendulacea in the ratio of 1:1, 1:2, 1:5, 1:10 |
The synergistic action of the plant extractions used in this investigation is to some extent similar to the results of Dyte and Rowlands[29] who reported higher mortality of T. castaneum adults in combined doses of insecticides (eg. fenetrithion, bromoxon and maloxon and synergists (Sesamex, SKF, 525A and PBO-1) in comparison with the mortality due to individual action of chemicals. The result is also similar to that of Ishaaya et al.[30] who reported higher mortality of T. castaneum in combined doses of insecticides (eg. trans and cis Cypermethrin) and synergist (Pyperonyl butoxide) in comparison with the mortality due to individual action of the chemicals. Mondal[31] observed the same results using insecticides (pirimiphos-methyl) and synergist (methyl quinone) on T. castaneum. Khalequzzaman and Islam[32] also reported the same results using insecticide (methacrifos) and synergist (leaf and seed extract) of Datura metal on same insect.
ACKNOWLEDGMENT
The authors would like to thank Director, Institute of Biological Sciences and Chairman, Department of Pharmacy, University of Rajshahi for providing necessary laboratory facilities.