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EFFECT OF ORGANOPHOSPHATE INSECTCIDE CHLORPYRIFOS ON SOME BIOCHEMICAL COMPONENTS OF TROGODERMA GRANARIUM LARVAE EVERTS.

Byline: Khawaja Abdul Mujeeb, Maria Latif, Muhammad Ayaz Ilyas and Syed Shahid Ali

ABSTRACT: The stored grain pests are causing huge damage to stored grains world especially tropical regions. Different pesticides and fumigation are used for their control. In the present study, effect of an organophosphate, chlorpyrifos is being evaluated on Khapara bettle (Trogoderma granarium) which is a very serious pest in stored grain godones of Pakistan. For this purpose, the LC50 of chlorpyrifos against 4th instar larvae of four different strains of T. granarium collected from different cities of Punjab like Khanewal, Lahore, Muzaffargarh and Sheikhupura were determined by residual film method. The LC50 values shown by these strains were 1575.5, 2673.9, 1150.1 and 1790.0 ug /ml respectively. On the basis of LC50 Lahore strain considered as resistant strain against chlorpyrifos, whereas Muzaffargarh strain used as susceptible strain.

Larvae of these two strains of T. granarium were exposed to sub lethal dose (LC20) for the evaluation of toxic effects of the insecticide on glucose, total lipid, soluble protein and total protein. The biochemical analysis was carried out after 24, 48, 96 and 192 hours of chlorpurifos treatments. The following insecticide treatment showed significant increase in glucose content in Lahore (resistant) and Muzaffargarh (susceptible) strains throughout the treatment. Lipid content also showed similar change with highly significant increase in almost all treatment of chlorpyrifos in both strains. Soluble protein content depicted highly significantly increase in both the strains after 24, 96 and 196 hours except Lahore strain, which showed non-significant rise after 48 hour's treatment. The total protein content of resistant and susceptible strains showed highly significant increase in all treatments.

Key Words: Insecticide resistance, Chlorpyrifos, Biochemical contents, Khapra beetles.

INTRODUCTION

Pakistan is facing a major problem of insect pest development due to agro-ecological conditions of country [1]. A socioeconomic survey in Pakistan in 1984-85 confirmed that insect infestation was the most significant cause of loss of stored grains during storage. A preliminary review of public sector storage facilities in Pakistan confirmed that the loss due to insect infestation and mould growth was severe. Stored grain pest initiate or aggravate the process of deterioration of stored products as they feed upon them. They cause significant losses in quality and quantity of stored products and pose a major problem for the agriculture sector and food industry [2].

The major insect species known to infect wheat include Khapra beetle, Trogoderma granarium Everts; Lesser grain borer; Rhizopertha dominica (F); Rice weevil, Stitphilus oryzae (L.) and Red flour beetle, Tribolium castaneum (Hbst.) [3,4]. All these insects may be found extensively in most developing countries to different extremes. Khapra beetle is one of the serious pest of stored wheat in Indo-Pakistan causing tremendous loss [5, 6, 7, 8].

It is estimated that 5-10% of worlds grain losses may reach 50% in tropical countries where hot and humid and storage facilities are improper and inadequate. The development of resistance in this pest against conventional insecticides such as phosphine, malathion, actellic and some pyrethriods has further aggravated its economic importance [9].

Various measures have been adopted to control this insect pest. Most important method of controlling insect infestation is chemical control, by insecticides which are used, primarily, to control pest that infest cultivated crops, plants or to eliminate insect in specific area. Due to high toxicity most of them are not used for stored grains and there are certain recommended pesticides with very low mammalian toxicity, which have been allowed for use on stored grains.

On chemical basis, insecticides are classified as organochlorines (OC), Organophosphates (OP) carbamates and pyrethroids. Organophosphate and pyrethroids are now the largest and most widely used classes of insecticides. Chemicals currently used around the world to treat stored grain include chlorpyrifos methyl, lindane, malathion, piperonyl butoxide plus pyrethrins [10]. Others are primiphos methyl, methyl parathion, dichlorvos, diazinon etc. [11]. Pyrethriods are less toxic to non- target animals when compared with organochlorines and organophosphate insecticides and highly toxic for insects [12]. More than 120 plants and plant products are used to control Khapra beetle. Neem (Azadirachta indica) kernels are widely used in India. Garlic, ginrer, lemon leaves etc. are some examples, whose extracts are used to control stored grain pests. This is effective and cheap method to control the stored grain pests [13].

Khapra beetle is one of the world's most feared stored-product pests. In fact, it has been nominated as one of the 100 worst invasive species worldwide. Established infestations are difficult to control because of the beetle's ability to live without food for long periods of time and to survive on foods of low moisture content, its habit of crawling into tiny cracks and crevices and remaining there for long periods, and its relative tolerance to many surface insecticides and fumigants. Therefore, it is important to prevent the khapra beetle's introduction into un-infested areas [14]. Some fumigants give control at high dosages, even though this beetle is more resistant to fumigants than most stored product pests. High concentrations of fumigant must be maintained over the fumigation period to allow penetration into all cracks and crevices. In an eradication program, both fumigants and surface sprays are used in combination with preventive measures.

High tolerance of phosphine was observed among larvae of khapra beetle held in crevices for 2-3 months before fumigation at 20deg. Tolerance was related to larval density during rearing as well as larval age. The effect of ten natural powdered foods on the susceptibility of full-grown larvae of khapra beetle to four common fumigants was evaluated. In general, methyl bromide was found to be the most toxic fumigant against khapra beetle from all foods, followed in a decreasing order of toxicity by ethylene dibromide, carbon disulphide and ethylene dichloride [15]. Resistance to fumigants has been frequently reported in insect pests of stored products and is one of the obstacles in controlling these pests. Ten populations of T. castaneum, nine populations of R. dominica and seven populations of O. surinamensis were resistant to phosphine [16]. In Pakistan khapra beetle from sindh and Punjab showed high level of resistance against different insecticides [17].

Due to excessive use of insecticides, insects have developed resistant against them [18,19]. Resistance is defined as a decreased response of population of animals and plants to a pesticide/insecticide as a result of previous exposure to the toxicant. Khapra beetle shows signs of resistance to some common chemicals such as phosphine and malathion. Populations of khapra beetle were able to multiply following fumigation with phosphine even when a higher than normal dose of 60 g/tones was used [20]. Larval mortality in a resistant strain averaged 5-12% compared to 84-94% in a susceptible strain in India when treated with phosphine fumigant. Phosphine resistance can be easily selected in mature larvae, resulting in four-fold increase in resistance after two generations [21]. Resistance has also been reported in various stored grain pests against phosphine in various parts of the world including Pakistan [22, 23].

The objective of the present study is to evaluate insecticide resistance with levels of different biochemical parameters and to assess the effect of organophosphate (chlorpyrifos) on Khapara (T. granarium). This work is expected to help in understanding the chemical control mechanism of stored grain pests.

MATERIALS AND METHODS

Five strains of T. granarium Everts. were used in this study. Fresh culture was collected from wheat godowns of Lahore and Sheikhupura. The old strains were obtained from Department of Zoology University of the Punjab, which were collected ten years ago from Lahore, Khanewal and Muzaffargarh. The culture of T. granarium Everts. was maintained in the culture room of Zoology Department of University of the Punjab Lahore. The temperature was maintained at 31+10C and 35-40% humidity. Crushed wheat was used as a supporting medium. Wheat was initially fumigated with phosphine to kill the insects if any present in wheat. Following fumigation wheat was spread in fresh air for 4-5 hours. The wheat was placed in oven overnight at 60 0C, and then wheat was shifted into sterilized jars for culture rearing. The jars were filled 1/4th with wheat and 40 beetles were added to it. The jars were covered with muslin cloth to prevent escape of beetles and entry of mites, moths and other small organisms.

The beetles were transferred to next jars after 2 days, to maintain the age of larvae for experimental purposes. Wheat containing eggs was placed back in the same jars, in which the 4th instar larvae obtained after 27+1, days. The 4th instar larvae were used for toxicological studies.

Toxicants Used

Chlorpyrifos is an organophosphate with a broad range of insecticidal activity and is effective by contact, ingestion and vapour action but is not systemic. It is used to control flies, household pests, mosquitoes (larvae and adult), household pests, and of various crop pests in soil and foliage; also used for control of ectoparasite on cattle and sheep. Its volatility is great enough to form insecticidal deposits on nearby untreated surface. It is not persistent in soil. It is non-phytotoxic at insecticidal concentrations. It is degraded in soil, initially to 3, 5, 6-trichloropyridin-2-ol which is subsequently degraded to organochlorines compounds and carbon dioxide. It persists in soil for 60-120 days.

For determination of LC50, residual film method was used for which serial dilutions of chlorpyrifos were prepared in acetone. LC50 of insecticide was determined against different strains separately. For this purpose three sets, each of three Petri plates, for different doses were used. These doses were applied on the center of glass Petri plates (size 130 cm) and rotated manually to make a thin film. 1 ml of insecticide solution was sufficient to spread as a thin film on entire surface of Petri plates. In the control Petri plates, only acetone was applied. After the dishes were air dried and acetone evaporated, ten healthy insects were introduced in different Petri plate and then covered. After 24 hours, mortality was recorded. Larvae showing no movement after pressing with brush were considered dead. The criterion of mortality used in this study was the one described by Lloyd [24].

The mortality data were, thereafter, subjected to probit analysis by Minitab software. LC50 values were derived from this analysis and expressed in ug insecticide for 4th instar larvae of T. granarium.

Experimental Procedure

Two strains of T. granarium have selected after the determination of LC50 for the study of effect of insecticide. Muzaffargarh strain has considered being susceptible strain and the Lahore strain treated as resistant strain. The sub-lethal doses (LC20) shown in Table-I were used to determine the effects of insecticides against these strains of T. granarium. Three sets, each of three Petri plates, both for control as well as experiment were used. First set was used for Muzaffargarh strain, second set for Lahore strain which we consider as resistant strain and third set of Petri dish with acetone alone served as a control. The doses were prepared in acetone and spread on the Petri plates as described above. After the acetone evaporated, thirty larvae were introduced in different Petri plates in the absence of food. The larvae were exposed to insecticide for a period of 24 hours. Live larvae from each Petri dish were then weighed and used for the estimation of glucose, total lipid, and proteins contents.

Tabe I: LC50 and LC20 (sub-lethal) doses of insecticides used against Lahore, Sheikhupura, Khanewal and Muzaffargarh strains of T. granarium

Insecticide###Strains###LC50###LC20

###Khanewal###1575.5###1083.1

###Lahore###2673.9###2131.2

Chlorpyrifos###Muzaffargarh###1150.1###615.1

###Sheikhupura###1790.0###1407.3

Thirty larvae of each strain of T. granarium were crushed separately in 2 ml of their respective buffers and saline solutions in teflon glass homogenizers for 2 minutes. The homogenates were centrifuged at 2200 rpm for 30 minutes at 4degC. The supernatants were used for the estimation of proteins, total lipid and glucose contents, the saline soluble proteins and total protein were estimated according to Lowry [25], total lipid estimation was performed according to the method mentioned by Zollner and Kirsch [26] and glucose was estimated by o-toluidine method of Hartal [27].

RESULT

Lethal Concentration (LC50)

Khanewal, Lahore, Muzaffargarh and Sheikhupura strains of T. granarium had LC50 1575.5, 2673.9, 1150.1 and 1790.0 ug/ml respectively. Based on their susceptibility to chlorpyrifos insecticide the different strains of T. granarium can be arranged in the order Muzaffargarh greater than Khanewal greater than Sheikhupura greater than Lahore.

When LC50 of other strains were compared with that of Lahore, it required more concentration of insecticide for mortality of insects, so it is consider as resistant strain against chlorpyrifos, whereas Muzaffargarh strain used as susceptible strain because it required lower dose of chlorpyrifos insecticide for LC50. The larvae of T. granarium were analyzed after treatment of sub-lethal dose (LC20) of chlorpyrifos which were 615.11 ug/ml for Muzafargarh and 2131.2 ug/ml for Lahore strain.

Biochemical Estimation

The effect of chlorpyrifos insecticide has been ascertained in term of estimation of glucose, total lipid, soluble protein and total protein contents of 4th instar larvae of T. granarium and these effects was determined after 24, 48, 96 and 192 hours (Table II to V).

Biochemical Analysis after 24 Hours

A control glucose contents of susceptible Muzaffargarh strain of T. granarium was 86.50 +3.66, whereas as resistant Lahore strain showed 58.13 +4.00, respectively. After insecticide treatment Muzaffargarh strain showed significant increase of glucose, whereas Lahore strain depicted non-significant increase. The average lipid content of Muzaffargarh and Lahore strains were 1.62+- 0.22, and 2.66 +- 0.24 g/l, respectively. In both the strains moderate significant increase observed after 24 hours treatment of insecticide. The average soluble protein contents of both Muzaffargarh and Lahore strains were 123.6+- 2.52 and 272+- 1.77, respectively. After treatment highly significant increase was observed in Lahore strain, whereas Muzaffargahr strain showed significant increase. The average control total protein contents were 308+-10.3 and 175+- 9.3, respectively in Muzaffergarh and Lahore strain, after treatment both the strains depicted highly significant increase (Table-II).

Table II: Biochemical analysis of Muzaffargarh and Lahore strains of T. granarium after 24 hours

Parameter###Muzaffargarh strain###Lahore strain

###Control###Treated###Control###Treated

Glucose(mg/dl)###86.50a+- 3.66###86.50+- 3.66###58.13a +4.0###67.25 +1.70

Lipid (g/l)###1.62 a+- 0.22###2.66 +-0.24###2.66a +0.24###3.9 +0.08

Soluble protein###123.6a+- 2.52###140.3 +-3.3###272a +1.77###427 +7.6

(ug/mg)###

Total protein###308.0 a+-10.3###469 +- 12.45###175a +9.30###427 +7.6

(ug/mg)

Biochemical Analysis After 48 Hours

The control glucose contents of Muzaffargarh and Lahore old strains were 89.32 +- 5.5 and 65.9 +- 5.72 mg/dl, respectively. After treatment glucose content increased significantly in both the strains. The average lipid content of Muzaffargarh and Lahore old strains were 1.8 +- 0.1 and 2.94 +- 0.17 g/l, respectively. Muzaffargarh strain showed highly significant increase, while moderate significant increase in lipid content of Lahore strain observed after treatment of insecticide.

The average soluble protein content of Muzaffargarh strain was 113.1 +3.8 ug/mg and of Lahore old strain was 30.72 +0.77 ug/mg, respectively. There was no significant difference in soluble protein content observed until 48 hours in both Muzaffargarh and Lahore strain. The total protein content of Muzaffargarh strain and Lahore strains were 346.5 +8.6 and 182 +9.3 ug/mg, respectively. The total protein content showed highly significant increase after 48 hours in both strains (Table-III).

Table III: Biochemical analysis of Muzaffargarh and Lahore strains of T. granarium after 48 hours

Parameter###Muzaffargarh strain###Lahore strain

###Control###Treated###Control###Treated

Glucose(mg/dl###89.32a+- 5.5###113.9**+-1.89###65.9a +5.72###100.17* +7.9

Lipid (g/l)###1.80a+- 0.1###2.96***+-0.02###2.94a +0.17###4.4** +0.12

Soluble protein###113.1a+- 3.8###145.8** +-0.95###30.72a +0.2###45.3+7.56

(ug/mg)###

Total protein###346.5a+-8.6###563.5*** +- 7.6###182a +9.3###469*** +12.5###

(ug/mg)

Biochemical analysis after 96 hours

The average glucose content of Muzaffargarh susceptible strain and Lahore resistant strains were 88.1 +1.75 and 87.67+14.01 mg/dl, respectively. After 96 hours of insecticide treatment highly significant increase of glucose was observed in both the strains. The average lipid content of Muzaffargarh and Lahore old strains were 1.9+0.06 and 3.64+-0.24, respectively. After treatment both the strains showed highly significant increase in lipid content.

The control soluble protein content of Muzaffargarh susceptible and Lahore resistant strains were 133+- 4.36 and 37.3+-1.95, respectively. Insecticide treatment showed highly significant increase in soluble protein content after 96 hours. The average total protein content of Muzaffargarh and Lahore strains were 385 +20.6 and 217 +8.30, respectively. Both the strains showed highly significant increase after treatment of insecticide (Table-IV).

Biochemical analysis after 192 hours

The average glucose content of both Muzaffargarh susceptible and Lahore resistant strains had 88.1 +1.75and 95.0 +14.3 mg/dl, respectively. After treatment of insecticide both Muzaffargarh and Lahore strains showed highly significant increase in glucose content. The control lipid content of both Muzaffargarh and Lahore strain were 2.0 +0.01 and 3.58+0.05 respectively. Highly significant increase of lipid content was observed in both the strains of T. granarium after 196 hours treatment of insecticide.

The average soluble protein content of both Muzaffargarh and Lahore strain were 97.00 +2.16 and 39.66 +1.8, respectively. Both Muzaffargarh and Lahore strains depicted highly significant increase of soluble protein content after insecticide treatment. The total protein content were at least three to six fold higher then soluble protein which were 339.5 +7.56 and 217.0 +7.30, respectively. Like soluble protein both the strains showed highly significant increase after treatment with insecticide (Table-V).

DISCUSSION

The development of resistance to chemical insecticides in arthropod pests constitute worldwide economic problem [28]. The khapra beetle (T. granarium) is a common insect pest of bulk grain, oil seed and warehouse facilities throughout the world. It is one of the most important pests of household, commercial food processing establishments and flour mills in Pakistan. There are numerous reports from various countries on the widespread resistance in several stored-product insect to the OP and fumigants [29]. Resistance to chlorpyriphos-methyl, pirimiphos-methyl, and malathion was detected in lesser grain borer, Rhyzopertha dominica (F), collected from 8 sites in the sites of Minas Gerais and Sao Paulo in Brazil and from 7 sites of northeast Kansas. Apparently chlorpyrifos resistance has evolved as a result of selection to other OP insecticide in Brazil [30].

Present study revealed that glucose contents increased throughout experimental study except in Lahore strain after 24 hours. In T. castaneum elevation in glucose contents and deplection of glycogen contents was reported that 48 hours treatment of Talcord when compared with their respective control. Glycogen content was utilized drastically whereas glucose content showed elevation possibly due to interconversion of polysaccharides to monosaccharide [31]. Ripcord treatment also increased glucose, fructose, total lipids and cholesterol contents, while glycogen content was decreased tremendously, when Tribolium castaneum treated larvae were compared with their respective controls [32].

In all experiments lipid and soluble protein contents were increased in both Muzaffargarh and Lahore strain, so elevation of lipid and soluble protein contents could be attributed to their possible conversion under chlorpyrifos stress conditions. Talcord insecticide treatment on T. castaneum showed in increased lipid, cholestrol, soluble protein, uric acid and urea contents than their respective controls. Raised level of soluble protein may be related increased activities of various enzymatic activities [31, 32]. Various insecticide induce abnormalities have also been reported in susceptible and resistant strain of T. castaneum [33, 34]. Changes in metabolism and adverse affects on the behavior and reproductive performance in insects also reported [35].

In this study T. granarium larvae showed significant increase in total protein contents throughout experiments. Similar results also depicted that the increase in glycogen and total lipid in T. castaneum provide primary source of energy after 4 days treatment of Ripcord while total protein provided secondary source of energy as it showed increase in the first two days of treatment [32].

Although fewer macromolecular contents in this study increase throughout the experiment with the treatment of sublethal dose of insecticide, but prolonged use of this insecticide might be develop molecular abnormalities which could be sufficient to play an important role in the pest control programme.

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Author:Mujeeb, Khawaja Abdul; Latif, Maria; Ilyas, Muhammad Ayaz; Ali, Syed Shahid
Publication:Science International
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Date:Sep 30, 2011
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