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

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 LD50S 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.

Alarm pheromones and the influence of pupal odor on the aggressiveness of Polybia paulista (Ihering) (Hymenoptera: Vespidae)

Field bioassays were used to demonstrate that aggressive behavior of Polybia paulista (Ihering) workers is elicited by alarm pheromones present in the venom reservoirs of nest defenders and that the brood care pheromone (pupal odor) produced by the young inside the nest also plays an important defensive role. Pupal odor was extracted from the surface of pupa bodies with methanol. When bioassayed alone, the pupal odor elicited onlyu attractiveness of workers towards the odor source, but no stinging attacks were observed. However, in the presence of alarm pheromones, the brood care pheromone potentiated the effect caused by the pupal odors, increasing the number of stinging attacks during an action of colony defense. Thus, the presence of pupae within the nest not only releases brood care but also enhances the aggressiveness of workers in P. paulista colonies.

Insects as biological models to assay spider and scorpion venom toxicity

This study was undertaken to develop an experimental protocol using insects as biological models to assay venom toxicity of the following spiders Loxosceles gaucho, Phoneutria nigriventer, Nephilengys cruentata and Tityus serrulatus scorpion. Three different insect species were bioassayed: Apis mellifera (Hymenoptera), Grillus assimilis (Orthoptera), and Diatraea saccharalis (Lepidoptera). Venoms were injected into the hemocele of insects with a microsyringe at concentrations that caused dose/weight-dependent effects; doses causing either paralysis (ED50) or death (LD50) were recorded for each venom and insect test-species. T. serrulatus and L. gaucho venoms were lethal to all tested species, while P. nigriventer venom caused paralysis and death, and N. cruentata venom caused only paralysis at the doses assayed. A comparison between the insect test species described above revealed that A. mellifera was highly sensitive to all venoms tested; even a tiny amount of N. cruentata non-lethal venom caused a change in the walking pattern leading to transient paralysis. D. saccharalis larvae were very resistant to all four venoms.