A technique for the bioassay of insecticide deposits on leaf and bark samples in relation to tsetse control operations

Bull. Org. nwnd. Santi 11972, 47, 811-813Bull. Wld Hith Org. A technique for the bioassay of insecticide deposits on leaf and bark samples in relation to tsetse control operations C. RODRIGUES 1 & D. A. T. BALDRY 2 During tsetse control operations it may be necessary to determine the effective toxicity and persistence of insecticide residues on vegetation. A simple method for preparing samples of twig bark for bioassay and an exposure apparatus that has been found very satisfactory for exposing tsetse flies to residues on leaves and bark are described. The apparatus is easily manipulated and involves little handling of the test material. During tsetse control and eradication campaigns, and in insecticide field trials, it is often necessary to determine the initial toxicity of insecticide deposits on vegetation and to monitor their persistence. Since the results of chemical analysis of vegetation residues can be misleading, and because such data cannot be obtained quickly, tsetse control personnel usually rely on bioassay techniques, insects of the species against which the control programme is directed being used for the tests. Although various methods have been devised for exposing tsetse flies to leaf deposits of insecti- cide, the technique developed by Kernaghan & Johnston (1962) appears to have been most widely used for this purpose (Baldry, 1963, 1964; Riordan, 1966a, 1966b). The apparatus was designed for exposing testse to leaf deposits, but with some slight modification it could also be used for the bioassay of deposits on tree trunks. Thus Riordan (1966b) took slices of bark from Isoberlinia doka trees, trimmed them to circular disks, and fitted them on to the piston of the apparatus, while Baldry & Riordan 3 reported a more refined method that involves cutting uniformly sized disks of bark from tree trunks and branches by means of a cork borer. This communication describes a bioassay tech- 1 Field Zoologist, Veterinary Research Laboratories, P.O. Kabete, Kenya. 2 Project Scientist-Entomologist. Present address: WHO Regional Office for Africa, boite postale 6, Brazzaville, Peoples' Republic of the Congo. 'Baldry, D. A. T. & Riordan, K. (1965) Notes on ento- mology, In: WHO unpublished document PD/68.9, p. 207. nique, based on the same principle as that of Kerna- ghan & Johnston (1962), that has proved to be suitable for the bioassay of insecticide residues on both leaves and the type of small twig commonly used by Glossina as resting sites. MATERIALS AND METHODS Preparation of vegetation samples Leaves required no preparation since they were exposed intact. Pieces of twig 5 cm long by 1.3 cm in diameter were cut with sharp secateurs from shrubs and small trees inside a G. pallidipes thicket habitat in the Lambwe Valley in western Kenya. The area of bark surface on each twig sample was approximately 20 cm2. In the laboratory, the bark was removed from each twig sample by holding the cut ends between the thumb and finger of one hand, making a shallow full-length incision down one side of the twig, and then gently stripping off the bark layer. With some types of twig it was necessary to cut between the bark and underlying vascular tissue. Once the roll of bark had been freed, it was flattened by means of forceps on a piece of card measuring 9 x 6 cm and secured by staples at each end. The card-mounted samples were then ready to be tested in the exposure apparatus. The exposure apparatus The apparatus is easily constructed from the common laboratory materials listed below: 2969 -811 - C. RODRIGUES & D. A. T. BALDRY Plunger. A rubber or cork stopper 2.6 cm in diameter, a glass or wooden rod 13 cm long and 7 mm in diameter, and a circular metal washer 3.3 cm in diameter with a central hole 7 mm in diameter. Chamber. A 12-cm length of glass tubing, with an outer diameter of 3 cm and a 2.7-cm bore, and a 6 x 6-cm square piece of 8-mm thick plywood or plastic with a central circular hole 3.3 cm in diameter. Retaining plate. A 6 x 6-cm square piece of 1-mm thick alloy sheet and 2 rubber bands. These items are assembled and glued together as shown in Fig. 1. O 1 2 3 4cm Test sample Retaining plate A test sample is held flat and tight between the lower surface of the platform and the retaining plate, by means of the rubber bands passed around these parts. Bioassay exposure procedure The procedure for exposing tsetse to insecticide residues on leaves or bark in this apparatus is as follows. The plunger and retaining plate are re- moved from the apparatus and the test material (whole leaves or bark mounted on card) is placed between the platform and the retaining plate, with the residue-bearing surface facing the exposure chamber. The retaining plate is secured by means of the two rubber bands so that the test material is held flat and firmly in place. A tsetse fly is placed in the glass container and quickly confined by introducing the plunger a short distance. The apparatus is then rotated so that the retaining plate faces the light, thereby encouraging the fly to alight on the test surface at the bottom of the tube. When this happens, the time is noted and the plunger is fully inserted with the washer resting on the top of the glass tube. The fly is now confined in a small exposure chamber. The apparatus is placed upright on the retaining plate for the selected exposure period. At the end of the exposure period the apparatus is inverted and the plunger withdrawn a few centi- metres to dislodge the fly; sometimes a few light taps may be needed on the side of the chamber. The plunger is then withdrawn completely and the test insect is allowed to fly into a holding cage or other container. The test sample is replaced by another sample for further tests. When the bioassay tests have been completed, the apparatus is dis- mantled and cleaned with a suitable organic solvent, such as hexane or acetone, before being stored for re-use. WHO 20790 Fig. 1. Apparatus for exposing insects to insecticide residues on leaves or bark. The plunger should be freely moveable inside the tube and when the plunger washer rests on the top of the tube, the lower surface of the piston head is approximately 7 mm from the bottom of the tube. DISCUSSION The advantages of the bioassay technique des- cribed above are that the apparatus is more easily manipulated and the handling of residue-bearing test material is reduced to a minimum. Several series of exposure chambers were used successfully to expose several thousand G. pallidipes to residues of dieldrin invert emulsion on leaves and bark during the aerial spraying trials. 812 BIOASSAY OF INSECTICIDE DEPOSITS ON LEAF AND BARK SAMPLES 813 ACKNOWLEDGEMENTS The writers are indebted to the Director of Veterinary Services and Chief Zoologist, Veterinary Services Divi- sion, Ministry of Agriculture, Kenya, for providing facilities at the Lambwe Valley Field Station. R1]SUMt TECHNIQUE DE MESURE BIOLOGIQUE DE L'ACIIVITE, DE DEPOTS D'INSECTCIDE SUR DES ECHANTILLONS DE FEUILLE OU D'ECORCE DURANT LES OPERATIONS DE LUTTE CONTRE LA MOUCHE TSE-TSE Divers procedes ont ete mis au point pour evaluer I'ac- tivite initiale ou residuelle d'insecticides deposes sur la vegetation. On decrit ici une methode simple pour prelever l'ecorce des petites branches qui servent fr6quemment de lieux de repos A diverses especes de glossines. Apres avoir coupe des morceaux de branche de 5 cm de long et de 1,3 cm de diametre, on en IibWre l'6corce A l'aide d'un scalpel. Le fragment obtenu, d'une surface de 20 cm2 environ, est ensuite agrafe sur un support pouvant etre adapte au dispositif d'epreuve. Le dispositif utilise et decrit par les auteurs consiste en un tube de verre dans lequel glisse un piston. Ce tube repose par une de ses extremites sur la surface oti est fixe le materiel d'epreuve (feuille ou morceau d'ecorce). Apres avoir enleve le piston, on introduit l'insecte dans le tube. Le piston est ensuite remis en place et en le depla- Sant vers le bas on cree une petite chambre d'exposition. Cette technique differe de celles d'autres auteurs dans lesquelles le materiel a tester est fixe sur la surface du piston, ce qui oblige a lui donner des dimensions precises. Les auteurs ont utilise cette technique, simple et ef- ficace, avec des resultats tres satisfaisants lors de tests biologiques portant sur plusieurs milliers de Glossina pallidipes dans des r6gions trait6s par la dieldrine. REFERENCES Baldry, D. A. T. (1963) Bull. ent. Res., 54, 497 Baldry, D. A. T. (1964) Bull. ent. Res., 55, 49 Kernaghan, R. J. & Johnston, M. R. L. (1962) Bull. Wld Hlth Org., 26, 139 Riordan, K. (1966a) Bull. ent. Res., 56, 615 Riordan, K. (1966b) In: Proceedings of the Eleventh Meeting of the International Scientific Council for Trypanosomiasis Research, Nairobi, Lagos, Organization of African Unity, p. 133 (publication No. 100) 9