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Manzoor et al. / Pakistan Entomologist 2011, 33(2): 125-130 parasitoid female, such as: venom and polydnaviruses needs, ecological adaptation and developmental (PDVs) (Pennacchio and Strand, 2006). characteristics. It is used as a host in favor of many hymenoptera species (Burges, 1978; Smith, 1965; Haewoon The parasitic form of life is very prolific and has evolved in et al., 1995; Coskun et al., 2006; Dweck et al., 2010;) The copious orders of arthropods. In parasitic insects adults are present study was carried out with the aim to evaluate the free living and a few numbers of progeny develop per host. biological activity of the venom of the wasp. . The hosts are not survived during the encounters so many parasitic insects are different from other parasitic organisms (Vinson, 1975). MATERIALS AND METHODS Regulation of host by insect parasitoids is a multifaceted procedure which consequences in a common physiological condition, ultimately increasing the suitability of the host Rearing of host parasitoid culture (Vinson and Iwantsch, 1981). According to their origin and The ectophagous larval parasitic wasp Bracon hebetor (Say.) function, there are different categories of host regulation (Hymenoptera: Braconidae) was reared under laboratory factors. Adult parasitoid females produce maternal origin th conditions on the mature larvae (5 instar) of greater wax factors that are injected at the time of oviposition, while moth Galleria mellonella (Lepidoptera: Pyralidae) by factors belonging to embryonic origin are obtained from the following a slightly modified approach as described by Alserosal membrane or from the other tissues associated with Tememi, 2005; Khan et al., 2005; Ahmed and Ahmad, 2006. embryo. Host physiology is also regulated by the parasitoid The adults of the parasitoid, B. hebetor were collected directly larvae. from fodder Trifolim alexandrium L., located at the campus of The host-parasitoid association that was considered in the the University of Agriculture, Faisalabad, Pakistan. The present study is Bracon hebetor (Say.) (Hymenoptera: collected parasitoids were identified on the basis of their Braconiodae) and Galleria mellonella (L.) (Lepidoptera: morphological characters. This parasitoid has two Pyralidae). Bracon hebetor (Say.) is a gregarious, phenotypes, depending on the temperature. During summer arrhenotokous, idiobiont, ectoparasitoid wasp, attacking the season, the adults have brownish colour while, during winter larvae of lepidopterons (Tuncyurek, 1972; Cline et al., 1984; season, the adults have blackish colour (Ahmed et al., 1985). Gul and Gulel 1995; Heimpel et al., 1997; Darwish et al., The larvae, pupae and adults of the host, G. mellonella were 2003). It has been widely used in studies of host parasitoid collected from the infested bee hives located at the campus of interactions because of its high reproductive rate, short University of Agriculture, Faisalabad, Pakistan. The host and generation time and significant range of host species parasitoid cultures were maintained in two separate glass jars, o (Tuncyurek, 1972; Gul and Gulel, 1995). B. hebetor (Say) both were kept at 27-30 C±1, 65±5% temperature, relative completes its development on different species of humidity (RH) and 18 h light /6 h dark photoperiod. Lepidopteran larvae by using generally Pyralidae The culture of the parasitoid was reared on the larvae of the (Lepidoptera) larvae as host . Most of the species of Pyralidae greater wax moth G. mellonela as a host by using glass vials of (Lep.) are agricultural pests on some field and storage crops. (2×10 cm) in the insect molecular biology laboratory, Toxic bio-active peptides are present in the venom of the Department of Agri. Entomology, University of Agriculture, th B.hebetor which blocks the neuromuscular transmissions in Faisalabad. Each vial contains 2 to 3 of to 5 instar larvae of larvae of lepidopteran species by inhibiting exocytosis of pre the host and one female of the parasitoid, provided with cotton synaptic vesicles (Piek, 1966; Walther and Reinecke, 1983). swabs/pads soaked in 50% honey and water as food source for Its females have a preference to attack last (fifth) instar of host adult of B. hebetor. The females of the parasitoid start to larvae (Benson, 1973). The host is first injected with venom parasitize the larvae of the host by first injecting a small th that induces entire and everlasting paralysis within 15 quantity of paralyzing venom into mature (5 instar) larvae of minutes (Piek, 1966; Piek et al., 1974). After host-feeding, a the host before egg laying on them, which induces partial or female lays between 5 and 25 eggs per host depending upon complete paralysis and then deposit between 3 to 20 eggs on the size of the host, host-encounter pace, and physiological the outside of the host. After parasitism, the larvae of the host state of the ovipositing female (Benson, 1973; Hagstrum and become sluggish and later on, the females start egg laying on Smittle, 1977) . Females lay eggs on the host body and after these larvae. After 24 hours, the parasitized larvae of the host hatching the eggs, the larvae of the parasitoid produce silken were shifted from the vials to new sterilized glass jars (9×5 cocoons and pupate outside the body of the host. Adult wasps cm) by using brush, provided with wax, pollen and honey as emerge from pupae ~16 days after oviposition. food for developing host larvae by keeping at the same The greater wax moth, Galleria mellonella (L.) (Lepidoptera: environmental conditions stated as above and the females of th Pyralidae) is an economically vital pest of the honeybee and is the parasitoid were shifted to new vials containing 2-3 (4-5 one of the most devastating pests of wax in the world. Moth's instar) larvae of the host for further parasitization and this larval stages feed on wax, honey and pollen thus causing process remained continue until the last moment of the female serious economic damage as they tunnel through the comb. life. The eggs were hatched within 2-3 days into transparent The larvae of the wax moth, cause considerable damage to larvae, which were directly bored into the body of the host combs left unattended by bees (Caron, 1992). G. mellonella is larvae for feeding. These larvae, after completing their preferred in entomological studies, because of its nutritional feeding/development came out the host body for pupation. Cite this article as: Manzoor, A., Zain-ul-Abdin, M. Arshad, M.D. Gogi, H. Shaina, E. Mubarik, S.K. Abbas and M.A. Khan, 2011. Biological activity of the toxic peptides from venom of Bracon hebetor (Say.) (Hymenoptera: Braconidae). Pak. Entomol., 33(2): 125-130. 126
Life cycle of the parasitoid ranged from 9 to 12 days, through micro injection bioassay. Injections of venom (Heat depending on the rearing conditions (Temperature and and protease treated) were performed on non-parasitized humidity). Finally, these pupae were collected singly in hosts, on day 1 fourth and fifth instars, which were glass vials plugged with cotton swab/pad soaked with 50% synchronized by following the approach as described by honey and water as food source upon emergence of Pennacchio et al. (1995). Experimental insects were naive/virgin parasitoid adults which were separated by anaesthetized by placing in refrigerator or on ice and sexes: morphologically the females were distinguished injections containing 0.5-µl aliquots of treated venom were from the males by their pointed/conical abdomen performed into the pronotum of the larvae as stated by Quistad (ovipositor). The collected parasitoids were frozen at et al., 1996 and by following a slightly modified protocol of −80°C. Digilio et al. (1998, 2000) by using a glass microcapillary Venom extraction tube with a very fine tip/Micro-sampling syringe, 0.5 µl BioBasics (Cat.No. SR.031). Host Larvae receiving saline Females of B. hebetor of mixed ages were collected directly injections were acted as control. After injection, larvae were from the rearing cages singly in glass vials and were o kept at 30 C±1, 65±5% relative humidity (RH) and 18 h light anaesthetized by putting vials at 4°C in refrigerator for 30 /6 h dark photoperiod and data was scored for mortality at 24 2 min. The whole reproductive tract of adult females, hrs. A χ test was used to compare the recorded frequencies. previously anaesthetized was pulled out by grasping the ovipositor tip with a fine forceps, while holding and keeping the abdomen of the female with another forceps in a drop of Pringle's saline solution (Pringle, 1938) placed on a Petri dish RESULTS under microscope. The venom glands and reservoirs obtained Biological activities of the venom of B. hebetor and from each female were subsequently separated from the determination of its biochemical nature ovaries and other unnecessary tissues by using fine micro dissecting needles and transferred to a 20-µl drop of ice-cold The venom of B. hebetor showed considerable biological Pringle's solution. The venom reservoirs were gently opened activity against Galleria mellonella (Table 1). with fine needles to allow the diffusion of venom in a drop of Pringle's solution. Up to 20 venom reservoirs were pooled per Table 1 each 20-µl drop of Pringle's solution. At the end all drops of Mortality of the G. mellonella caused by the venom of B. Pringle's solution containing gently opened/ruptured venom hebetor reservoirs were transferred to 1.5 ml sterile eppendorf tube by Mortality Treatment Replications sucking only the liquid part of the drop with micropipette and % leaving behind the ruptured tissues of the reservoirs in the Petri dish, in order to ensure the maximum purity of the Crude venom 50 70 venom. The resulted crude extract was centrifuged at 5000 g Control ( Saline injections) 50 2 for 5 min, at 4°C. to remove any fragments of tissue and the supernatant was recovered and transferred to a sterile eppendorf tube which was finally be diluted with appropriate Micro injections of crude venom in the host species G. saline solution in order to obtain the needed concentration of mellonella, caused immediate paralysis, feeding disruptions female equivalents (number of females processed as and death occurs within 72 hours, but in some cases the venom described above)/µl. Extracted venom was stored at −80°C. has an immediate paralysis leading to death within 24 hours, the death rates recorded in the host species were high, Venom inactivation treatments reaching 70% of mature larvae of G. mellonella. Bioassays Heat treatment were performed to evaluate the bioactivity of the venom after digestion with protease and heat treatment, it was deduced To assess the sensitivity of the venom extract to heat, an that the active components of the venom were proteins, as inactivation protocol was adopted as described by Digilio et they lost their effectiveness after heat and protease treatments al. (2000). The heat inactivating treatment was performed by (Table 2). boiling the extract for 5 min. After a 5 min centrifugation at 12000g, the supernatant was recovered and stored at−80°C. Table 2 Activities of the venom of B. hebetor after treatment with Protease treatment protease and heat The enzymatic digestion treatment was carried out by using active proteases Protinase K (21.4mg/ml) (Fermentas Life Sciences, catalog # E00492), which was incubated with the venom extract, at 37°C, for 1.5 h. Untreated venom extract acted as control. Biological activity of venom Biological activity of total venom of B. hebetor was assessed Cite this article as: Manzoor et al. / Pakistan Entomologist 2011, 33(2): 125-130 Treatment Venom treated with heat Venom treated with proteases (Protinase K) Control ( Saline injections) Replications Mortality (%) of G. mellonella 50 6 50 4 50 2 Manzoor, A., Zain-ul-Abdin, M. Arshad, M.D. Gogi, H. Shaina, E. Mubarik, S.K. Abbas and M.A. Khan, 2011. Biological activity of the toxic peptides from venom of Bracon hebetor (Say.) (Hymenoptera: Braconidae). Pak. Entomol., 33(2): 125-130. 127
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