Entomol. Journal Page Setting, all articles - Pakistan Entomologist

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The host mortality caused by the heat and protease treated venom was almost similar to that mortality observed in control, which suggest that heat and protease treatments of venom have inactivated the proteins found in the venom blend of the wasp, confirming that the active components of the venom are proteins which are thermolabile and protease sensitive. Table 3 Crude venom vs saline injections Total No. of injected host larvae Manzoor et al. / Pakistan Entomologist 2011, 33(2): 125-130 Dead Alive % age of Mortality Mortality (%) Venom injected larvae 50 35 15 70 Saline injected larvae 50 1 49 2 (χ 2 = 50.173, P ≥ 0.05) df=1 Table 4 Venom treated with Heat vs saline injections Total No. of injected host larvae Dead Alive % age of Mortality Fig. 2 Mortality of the host with heat treated venom and saline injected larvae of G. mellonella Venom treated with Heat 50 3 47 6 Saline injected larvae 50 1 49 2 χ 2 =1.041 (χ 2 =1.041, P ≤ 0.05) df=1 Table 5 Venom treated with proteases vs Saline injections Total No. of injected host larvae Venom treated with Heat 50 Dead 2 Alive 48 % age of Mortality 4 Mortality (%) Saline injected larvae 50 χ 2 = 0.343 (χ 2 =0.034, P ≤ 0.05)df=1 1 49 2 Mortality (%) Fig. 1 Mortality of the host with crude venom and saline injected larvae of G. mellonella Cite this article as: Fig. 3 Mortality of the host with protease treated venom and saline injected larvae of G. mellonella DISCUSSION This preliminary study of functional analysis of the maternal secretions (venom) injected by ectoparasitoid B. hebetor in its host G. mellonella at the time of oviposition has allowed us to understand the mechanism of parasitization in host regulation process along with the chemical nature of the female secretion like venom to be involved in successful parasitization of the host. The venom of B. hebetor, is produced by the venom glands attached to the reproductive system of the female of the parasitoid which is responsible for immediate paralysis and death of the host, or at least stop the development of the host. Venom is a complex mixture of protinaceous compounds which usually cause particularly in Microbracon species 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. 128
flaccid paralysis. Our experimental evidence showed that the (Parkinson et al., 2002), Asobara Tabida (Moreau and active components of the venom are proteins, which usually Guillot, 2005) and Cotesia rubecola (Asgari et al., 2003, lost their activities upon treatment with heat and protinase K. Zhang et al., 2004). Many of these molecules would be to The venom, as well as the toxins, of Microbracon hebetor have neurotoxic and cytolytic function (Moreau and Guillot, causes a blockage of presynaptic glutamatergic excitatory 2005). transmission. The venom has no effect on the inhibitory As mentioned previously, paralysis was found in the larvae of transmission. The venom of Microbracon is slow acting G. mellonella after injection of the venom of B.hebetor could (Beard, 1952). The paralysis persists over a long period of be due to a dose of venom injected for this host. Our results are time without any apparent change in the ultrastructure of the in accordance with the dose dependent effect of the venom nerve endings being caused (Piek et al., 1974; Rathmayer and which was also observed in other parasitoids as Euplectrus Walther, 1976). A dose of venom equivalent to one venom plathypenae (Nakamatsu and Tanaka, 2004) and Euplectrus gland has the same effect as the mass-collected venom of 100 kuwanae (Uematsu and Sakanoshita, 1987). wasps. The 'gland-extract venom' as well as the 'mass- The next step was to fractionate venom components, to isolate collected venom' have a paralysing effect on G. mellonella cDNA encoding major proteins of the venom and then larvae which is apparently identical to the paralysis observed expression of the isolated cDNA in eukaryotic cells for more after a G. mellonella larva has been stung by Microbracon. rapid and abundant production of these proteins, expression in Only a few representatives of the family Braconidae behave eukaryotic cells should ensure the maintenance of postas ectoparassitoid (koinobiont), regulating the physiology of translational modifications that often can have an important the host without interrupting its growth (Pennacchio and role for biological activity associated with the protein. In this Strand 2006). Also in Bracon hebetor, venom is responsible way, we can make more easily the necessary functional for the paralysis and death of parasitized hosts, these effects studies. have been attributed to the active protein components in the One of the advantages of biotechnology to control insect pests venom confirmed by the bioassays of heat and protease is just to expand the potential use of parasitoids, not only as treatment of the venom and this is in similar with our findings living organisms, but also using genes and molecules derived The physiological effects of the venom of B. hebetor were from parasitoid which are involved in the process of assessed on the mature larvae of host species G. mellonella increasing their success in programs to control pests. through microinjections. The effects of the venom on the host In conclusion, the possibilities offered by biotechnology for species were quite significant. The larvae of G. mellonella, the application of the genes and molecules derived from immediately after injection, at least at the doses we used, parasitoids in the control of insect pests are increasing, the interrupting the feeding activity and there were actual signs of biotechnology industry looks forward to growing this type of paralysis. Thus death, takes over 24-48 hours, has always molecules, as shown in some places of patents components of been associated with paralysis. We can not conclude at this the venom of the parasitoid Bracon hebetor (Quistad and stage that the observed effects of venom on the host are the Leisy, 1996; Windass et al., 1996, Johnson et al., 1996, 1999). result of a synergistic effect of proteins occurring in the It is imperative to undertake a solid programme of research venom of the wasp because sometimes a single protein found which will address such issues, namely the identification of in the venom of certain parasitoids is not sufficient to induce new molecules with insecticidal activity from the indigenous paralysis and death of the host and it requires many toxic fauna of insect parasitoids for development of sustainable proteins for successful parasitization. Further molecular pest insect control strategies. studies are required to fractionate the venom blend of the wasp through HPLC and SDS-PAGE and then bioassays ACKNOWLEDGEMENT would be performed with chromatographic fractions. Concerning the function of the proteins occurring in the Thanks to Higher Education Commission (HEC), venom of the wasp, we can only make assumptions because Government of Pakistan and Pakistan Science Foundation) we did not isolate the proteins yet and its N-terminal (PSF), for providing financial support for this work, Vide sequence, therefore, we can make assumption only based on notification No. HEC/20-1455/R&D/09/223, PSF/Res/Pthe previous studies that the similarity of the proteins of the AU/Agr (405). venom may be associated with “trypsin-like serine proteases” with digestive functions, as evidenced from the feeding REFERENCES activity of the host species and it is also found in E. plathypenae, in which the larvae have digestive enzymes Ahmed, S. and M. Ahmad, 2006. Toxicity of some insecticides on belonging to the family of serine proteases similar to trypsin, Bracon hebetor under laboratory conditions. Phytoparasitica, 34 (4): 401-404. with which they digest the host's body (Nakamatsu and Al-Tememi, N.K., 2005. Integrated pest management of Helicoverpa Tanaka, 2004). Regarding the active components (Proteins) armigera (Hobner) (Lepidoptera: Noctuidae) on cotton by using bioof the venom of the wasp, we can not say anything about its control agents and selective insecticides. Ph.D thesis. Deptt. putative function because their cDNAs have not yet been Agri.Entomol. Univ. Agric. Faisalabad, Pakistan. obtained in the course of this work, but in general the Asgari, S., R. Zareie, G. Zhang and O. Schmidt, 2003. Isolation and literature contains few examples of such proteins, among characterization of a novel venom protein from an endoparasitoid, Cotesia rubecula (Hym; Braconidae). Arch. Insect. Biochem. which there are small peptides present in the venom of E. Physiol., 53: 92-100. orientalis (Doury et al., 1997), Pimpla hypochondriac Bale, J.S., J.C. van Lenteren and F. Bigler, 2008. Biological control and Cite this article as: Manzoor et al. / Pakistan Entomologist 2011, 33(2): 125-130 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. 129
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