A group of locusts was fed on grass which varied with the external weather conditions and water was supplied once a day in the form of soaked cotton pads.
J. Environ. Sci. – Special addition for the researches of the first Arab conference 24-26/02/2007 on environmental studies. Volume 3: 210-239.
PHYSIOLOGICAL CHANGES IN THE DESERT LOCUST SCHISTOCERCA GREGARIA IN RESPONSE TO DIET AND INJECTION WITH BACILLUS THURINGIENSIS BARAKAT, E. M. S. (1) and MESHRIF, W.S. (2) 1) Faculty of Science, Ain Shams University. 2) Faculty of Science, Tanta University.
ABSTRACT This study was done to determine variation of body weight, water content, haemolymph volume, density and pH as well as total haemolymph proteins of the desert locust Schistocerca gregaria (Forskal) with changing the rearing diet and the injection of Bacillus thuringiensis kurstaki Berliner (BT). Natural diet fed locusts were found to be more resistant to BT than artificial diet fed locusts. The estimated LD50 values were 5.73X105 and 9.52X104 cells/insect for natural diet fed and artificial diet fed locusts, respectively. This indicates a high level of resistance towards the bacteria. Natural diet fed locusts showed an increase in body water accompanied by a decrease of dry weight compared with artificial diet fed locusts. Bacterial treatment did not affect the dry weight, water content or haemolymph volume but increased haemolymph density and pH in natural diet fed locusts, while it increased the dry weight, haemolymph density and pH and decreased the water content and haemolymph volume in artificial diet fed locusts. These observed changes may be attributed to the induction of bacterial metabolites and the intensive loss of tissue water. Natural diet fed locusts accumulated higher haemolymph protein contents than artificial diet fed locusts. The bacterial treatment caused a significant decrease in the haemolymph protein content at 6 hr for the natural diet fed locusts and at 12 hr postinjection for artificial diet fed locusts. This may be due to the elimination of some proteins and/or their involvement in defense reactions.
INTRODUCTION Infections with bacteria are rather common in insects. The association of bacteria with insects has been recorded since Pasteur’s time and many species of pathogenic bacteria have been described. Since the description of Bacillus thuringiensis (B. t.) by Berliner in 1911, perhaps more is known of its mechanism of pathogenicity than of any other invertebrate bacterial pathogen. Several authors reviewed the toxicity of this organism, in addition to the developmental studies by Rogoff et al. (1969) which led to its use as a biological control agent for insect pests. The high interest in biological means of controlling insects intensifies the need for investigating the response of insects to disease organisms and foreign proteins. The haemolymph, in particular, offers a readily accessible criterion of response. For
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work to be meaningful, the desert locust, Schistocerca gregaria (Forskal) was used as an experimental animal in this investigation, because it represents a relatively important group of plant-feeding insects and it can be reared easily in the laboratory throughout the year. The haemolymph is a tissue made up of fluids and more than half a dozen of different types of cells. It can undergo quantitative changes to an extent virtually unknown for other tissues. The main functions of the haemolymph are phagocytosis and encapsulation of foreign materials, coagulation, storage/distribution of nutrients, and protein synthesis (Heusden, 1996). Insect haemolymph is influenced at least on the level of its physical properties such as volume, density and pH by several factors, among them are: age, diet, temperature and disease (Carrel et al., 1990). Changes in body weight and water content did not attract the immunologists in the past, although it gives integrated picture with haemolymph volume and density about effect of treatment. Haemolymph usually contains, in addition to a large amount of water, protein which is the main nitrogenous constituent of all living materials. A considerable attention has been paid to the characterization of haemolymph proteins and understanding their role in defense reactions. Pathogenic infection produces drastic changes in the haemolymph protein content of the infected host (Beard, 1945; Abdeen, 1986: El-Kattan, 1995). Due to the successful use of B. t. formulations in the Egyptian fields against Lepidoptera and Diptera, it was necessary to evaluate the use of this pathogen against the most destructive orthopteran pest, S. gregaria. The present work aimed to obtain information about the improvement of using entomopathogenic agents in pest management. Accordingly, the pathogenicity of B. t and the effect of changing the type of food (diet) on the total body weight and body water content, haemolymph volume, density, pH and haemolymph proteins of normal and B. t.-injected insects were investigated.
MATERIALS AND METHODS Insects The desert locust, S. gregaria used in the present study was originated from Aswan (Upper Egypt) and established in the Locust and Grasshopper Research Department, Plant Protection Research Institute, Agricultural Research Center, Cairo,
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Egypt. The locusts were held at 30 ± 2˚C, a photoperiod of 16:8 (Light : Dark) and the relative humidity varied between 60 and 80%. Two different diets were used for feeding the locusts for several generations. A group of locusts was fed on grass which varied with the external weather conditions and water was supplied once a day in the form of soaked cotton pads. This group was referred to as natural diet fed locusts (NDFL). Another group of locusts was kept under the same conditions, but fed on an artificial diet as a dry mixture of; 2 bran: 2 dried whole milk: 2 wheat: 1 dried brewer’s yeast (parts by volume) according to methods described by Huxham et al. (1989). This group was referred to as artificial diet fed locusts (ADFL). All experiments outlined below were carried out with adults (both sexes) being within 2– 4 days after ecdysis. Preparation of bacterial pathogen The bacterium, B. thuringiensis kurstaki (3200 IU/mg, AGERIN- wettable powder) produced by the Agricultural Genetic Engineering Research Institute at the Ministry of Agriculture, Giza, Egypt, and was grown aerobically at 28 ± 2˚C in nutrient broth tubes for 48 hr. To obtain solitary pure colonies, nutrient agar plates were prepared and cultured with inoculates of the grown bacteria in the nutrient broth using the streaking dilution method. The plates were incubated at 28 ± 2ºC for 48 hr. After growth, only solitary colonies were selected, cultured on nutrient agar slants and incubated at 28 ± 2ºC for 48 hr, and then kept in the refrigerator at 4ºC until used. These slants were regenerated monthly. Prior to use, the pure isolate made from the bacterial sample previously prepared was transferred to a nutrient agar medium and incubated at 28 ± 2ºC for 24 hr. The grown bacteria were harvested by suspending in a physiological saline (0.5% NaCl). The bacterial suspension was centrifuged at 6000 rpm for 30 min. The resultant pellet of bacteria was washed three times with a sterile saline solution and centrifuged again at the same rate till the saline solution become completely clear. The obtained bacterial pellet was then stored at –18ºC until required. Injection technique Injection of insects was made with a 10 µl Hamilton micro-syringe fitted with a 26-gauge needle according to Miranpuri and Khachatourians (1993). Prior to injection, the locusts were chilled on ice for 20 min and the site of injection was swabbed with 70% ethanol. Injection was made through petroleum jelly, which helped to seal the wound and prevent excessive haemolymph loss following injection. Ten µl 212
of the bacterial suspension were injected into each insect in the last coxal corium. If any insect bled or the bacterial suspension was lost during or after injection, the locust was discarded. Control insects were injected only with equivalent volumes of 0.5% saline. The treated insects were maintained in separate cages at 30 ± 2ºC. Another negative control group of insects were not injected. Susceptibility of locusts to the bacterial pathogen To determine the effect of food on the susceptibility level of S. gregaria adults to the bacterium, B. t., groups of insects, each containing 10 individuals, were injected with four doses: 2.2X103; 2.2X104; 2.2X105 and 2.2X106 cells per insect. Doses were expressed as number of bacterial cells/insect. Final mortality percentages were scored 48 hr post-injection. A stock suspension of a dose of B. t. that produces 50% mortality was injected into the haemocoel of the locusts for investigating the influence of pathogenic infection on the various parameters studied. Determination of body weight and water content of the locusts In order to determine the effect of food on total body weight and to find out the relation between body water content and haemolymph volume after bacterial infection, the body water content was determined in normal and bacterial-treated locusts after 6, 12, 24 and 48 hr (along with saline-injected controls) by the method of Lee (1961). The body weight of locusts was determined gravimetrically for each individual insect. The locusts were weighed on electronic balance (BRAINWEIGH B100, OHAUS Scale, CORP. USA). The body water content of locusts was determined as the difference between fresh (total) body weight and body weight after drying for 2-3 days at 80ºC in an oven to constant weight (dry weight). The measurements were replicated 10 times. Collection of haemolymph Normal (un-injected) and injected locusts after 6, 12, 24 and 48 hr along with saline-injected controls were removed from the rearing cages and weighed individually. Locusts were submerged in hot water bath at 60ºC for 2-5 min and then, allowed to dry on paper towel. The heat-killed insects were amputated at the hind coxa with fine scissors. Gentle pressure was applied to the thorax until a drop of haemolymph appeared at the point of amputation. Estimation of haemolymph volume
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The haemolymph volume was determined for normal and bacterial-injected insects, along with their controls, following the method described by Yeager and Munson (1950) and modified by Lee (1961). Haemolymph from two individual insects was never combined. The tested insects were weighed individually and injected with 10 µl of 0.2% amaranth red dye (20 mg/ml of 0.5% NaCl), and left for 5 min for the dye to mix thoroughly with the blood. This time was often sufficient for the dye to become visible through the insect intersegmental membranes of the body and legs). Then, 10 µl of blood were extracted and diluted to 1 ml with 0.5% saline solution. A blank solution was made up from 10 µl of blood, taken before injection and diluted to 1 ml as previously described to minimize the errors due to blood inclusions. The optical density was measured spectrophotometrically by Nova spec, (Pharmacy Biotech.) at 515 nm using 1 ml cuvette against the standard solution, prepared by diluting 10 µl of the dye solution in 1 ml of saline solution. The blood volume (V) was calculated from the following equation: V = (dg1/g2) – a. Where: (g1) is the weight of dye injected, (g2) the weight of dye in the sample, (d) volume of sample and (a) volume of saline injected with the dye. Haemolymph volume was determined for 10 times at each of the different time intervals. A series of saline solutions containing 0.004–0.2 mg of amaranth dye were made to carry out a standard calibration curve. The absorbency of dyed solutions was measured at 515 nm against the blank prepared previously. The weight of amaranth dye in unknown sample was calculated from the equation obtained from the standard calibration curve. Weight of dye = Absorbency + 0.0038/4.629
(mg)
Estimation of haemolymph density The haemolymph densities were determined in un-injected and injected NDFL and ADFL following the method described by Carrel et al. (1990). The heat-fixed locusts were amputated in the hind leg with a scissors. The oozed blood was withdrawn directly into micro-capillary tubes calibrated at 1µl and pre-weighed using an electronic balance. The filled tube was quickly re-weighed. The haemolymph density was expressed as mg/µl. The measurements were replicated 10 times at each of the different time intervals for each determination. Estimation of haemolymph pH
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The haemolymph pH was determined for normal and injected locusts according to the method described by Heimpel (1955). The hind leg of the locust was amputated with a fine scissors. The bulb of the microelectrode (Model 671, pH meter, Extech., USA) was brought into contact with a drop of oozed haemolymph. Determination of the haemolymph pH was completed within a maximum of 15 sec, to reduce the possibility of altering the pH value by absorption of carbon dioxide. All measurements were accomplished at 28 ± 2ºC and samples were replicated 10 times at each time interval. Estimation of the total haemolymph protein The total protein content of the haemolymph was estimated in normal and injected locusts according to the method described by Bradford (1976) with bovine serum albumin (BSA) as the standard protein solution. 0.1ml of each haemolymph sample was pipetted into a test tube, then 5 ml of Coomassie brilliant blue G-250 (CBB) solution were added, and the contents of the tubes were mixed by vortexing. The absorbency at 595 nm was measured. This procedure was repeated 3 times for each determination. The total protein content was estimated as mg/ml using the following formula derived from the standard calibration curve: Protein concentration = Absorbency + 0.0049/0.8139
(mg/ml)
Data analysis The corrected results of susceptibility tests were represented graphically as probitlog regression line. Statistical analysis of data was made using the chi2 test. The different lethal concentrations and the confidence limits of LD50 value were done according to the method of Litchfield and Wilcoxon (1949). Data of the rest tests were expressed as the mean ± standard error (SE). Levels of significance for differences of the means were determined using the Student’s “t- test” for paired samples. The level of significance for each experiment was set at P < 0.05.
RESULTS Susceptibility of locusts to the bacterial pathogen Data obtained from the susceptibility tests of S. gregaria adults fed on natural or artificial diet to the injected B. t. are shown in Table1. The mortality percentages were recorded after 48 hr post-injection. The estimated LD50 values at 95%
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probability were 5.73X105 and 9.52X104 cells/insect for the NDFL and ADFL, respectively. These doses were used in the subsequent tests. Effect of diet and B. t. injection on body weight and water content The dry body weight and body water content of S. gregaria adults, fed on natural or artificial diet are shown by Fig. 1. In NDFL, the mean value of the dry body weight was 555.4 ± 52.6 mg and the body water content was 1367.6 ± 964.5 mg, representing 70.18 ± 1.3 % of the fresh body weight. In the ADFL, the mean value of the dry body weight was 733.4 ± 58.7 mg and the water content was 1311.3 ± 738 mg, representing 64.4 ± 1.0 % of the fresh body weight. Bacterial treatment did not affect the dry body weight and water content in NDFL (P> 0.05) at all time pionts postinjection compared with controls. In contrast, the dry body weight of ADFL increased significantly (P 0.05), but ADFL possessed a significant decrease (P 0.05) at 48 hr post-injection compared with control locusts. However, a significant increase (P
