A microinjection technique using Drosophila melanogaster for bioassay-guided isolation of neurotoxins in arthropod venoms

Abstract

Modern analytical techniques permit isolation and structural determination of neurotoxins at the picomole level. However, bioassay-guided fractionation of the sample often relies on simple injection assays using insects, vertebrates or crustaceans of a fairly large size, thus consuming quite a large amount of the samples being investigated. In order to investigate samples of very small size, we have devised an insect microinjection method using glass micropipettes and Drosophila melanogaster adults as test insects. The validity of the method was tested with a series of six buthoid scorpion venoms ( Androctonus australis, Buthotus judaicus, Buthus tamulus, Centruroides sculpturatus, Leiurus quinquestriatus hebraeus, Tityus serrulatus) and one chactoid scorpion ( Scorpio maurus palmatus) as standards. The LD 50S of the venoms were determined using both the microinjection method and a classical injection assay with crickets ( Gryllus bimaculatus) as test insects. Results demonstrated that the new method can successfully be applied to the study of insect neurotoxic activity in arthropod venoms. The Gryllus: Drosophila ratio in amount of sample utilized is 100. However, for all Buthoid venoms tested, except L. quinquestriatus, Drosophila showed less sensitivity, thus reducing the gain by a factor of 2–10. Drosophila were several times more sensitive to the only chactoid venom tested. These results clearly demonstrate the advantage of using this microtechnique, when limited amounts of material are available for both chemical and biological work.