Instructions for determining the susceptibility or resistance of Reduviid bugs to organochlorine insecticides

WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE WHO/VBC/75, 587 ENGLISH ONLY INDEXED INSTRUCTIONS FOR DETERMINING THE SUSCEPTIBILITY OR RESISTANCE OF REDUVIID BUGS TO ORGANOCHLORINE INSECTICIDES 1 1. INTRODUCTION This method measures the levels of susceptibility of a population of reduviid bugs to a given insecticide, The technique is designed to detect the emergence of an insecticide- resistant strain if and when it appears. For this purpose, it is necessary (a) to establish the susceptibility levels of normal reduviid bugs of the species concerned, and (b) to make subsequent routine checks of the susceptibility levels at periodic intervals, This test is devised specifically to detect physiological resistance. It is stressed that the test is not designed to evaluate the effectiveness of deposits of insecticides in the field, for which other entomological techniques must be used, Establishing the base-line Batches of reduviid bugs are exposed to different concentrations of insecticide and the mortality at each level is determined, It is suggested that a preliminary test be made at each concentration in the complete range provided, at the standard exposure of 1 day for organophosphorus compounds and 2 days for dieldrin. This will indicate the general level of susceptibility; further tests should then be made on this basis, A series of 4 co~cen- trations should be chosen, some of which give partial mortalities (i.e., at least one of them should give 100% mortality and one less than 50% mortality). Tests at these concentrations should be repeated 4 times with samples from the same population of reduviid bugs, To reveal the full range of natural variation, this set of tests should be made at several locations and at different seasons, so far as this is practicable. Subsequent routine checks Monitoring checks to detect emerging resistance are made by the use of a diagnostic concentration, which is expected to kill all susceptible reduviid bugs of a given species. The concentration is chosen (as described in Annex 12) from the nearest concentration available to that corresponding to 99.9% kill on the regression line established in the base- line tests. Alternatively, an approximation which can be used is the concentration double the lowest concentration that has consistently given 100% mortality in the 4 successive tests, These should be made periodically with 2-5 replicates, The occasional appearance of survivors in such checks may be due to normal variation, However, the regular occurrence of survivors (e.g., on 3 successive occasions) constitutes a signal calling for further investi- gations, Such investigations should include 4 tests at each of the concentrations used in establishing the original base-line. In this way, it is possible to detect the presence of physiological resistance and to determine the approximate proportion of resistant reduviid bugs in the population. 1 Annex 6 (Wld Hlth Org. techn. Rep. Ser., 1970, No. 443) has been revised as a result of discussions held at the WHO Expert Committee on Resistance of Vectors and Reservoirs to Pesticides which met in Geneva from 16 to 23 September 1975. 2 Report of the WHO Expert Committee on Resistance of Vectors and Reservoirs to Pesticides (1975). The issue of this document does not constitute formal publication. It should not be reviewed, abstracted or quoted without the agreement of the World Health Organization. Authors alone are responsible for views expressed in signed articles. Ce document ne constitue pas une publication. II ne doit faire l'objet d'aucun compte rendu ou resume ni d'aucune citation sans l'autorisation de !'Organisation Mondiale de la Sante. Les opinions exprimees dans les articles signes n'engagent que leurs auteurs. WHO/VBC/75.587 page 2 Experiments with laboratory colonies of reduviid bugs have been made to determine the concentrations and exposures necessary to kill normally susceptible specimens. The tests were made with Vth instar nymphs, 5 days after a blood meal, maintained at 27°c. Younger instars and adults should be more susceptible; also unfed or starved individuals. On the basis of this work, the following diagnostic treatments are suggested (which should, however, be checked under local environmental conditions): 4% dieldrin for 2 days, for Rhodnius prolixus and Triatoma maculata; 4% dieldrin for 5 days, for Triatoma infestans. Condition of reduviid bugs Last-stage nymphs should preferably be used (if adults are used, the results should be kept separate from those for nymphs). Conditions of test Tests should be carried out in a room free from insecticidal contamination. The reduviid bugs are exposed and held at a temperature between 20°C and 30°C and at a relative humidity above 25%. Some uniformity in these conditions can be secured by the use of the kit box. 2. COMPOSITION OF TEST KIT (a) 1 package containing 40 papers (2.4 cm x 15 cm) impregnated with HEOD (purified dieldrin) in mineral oil at a concentration of 0.4%, 0.8%, 1.6% and 4.0% and 1 package treated with oil only. (b) 6 glass test-tubes, 15 cm in length and 2.4 cm in internal diameter. (c) Test-tube rack, plastic covered, holding 6 tubes, together with a black foam plastic mat. (d) 15 pieces of fine-mesh gauze to fit over the tubes. (e) 25 rubber bands. (f) 2 pairs of collecting forceps. (g) 1 holding container. (h) 1 roll of self-adhesive plastic tape. (i) Instruction sheets, a set of report forms, and 3 sheets of log-probability paper for plotting regression lines. (j) 40 sheets of clean typing paper. 3. PROCEDURE (a) Having located infested houses, collect the former) as possible (approximately 100). as many last-stage nymphs or adults (preferably The area from which collections are made should given series. Place the reduviid bugs in the The specimens are then brought to the be restricted as far as possible for tests in a holding tubes, taking care not to damage them. locality of the test. (b) Set up sufficient test-tubes to include 2 replicates 9f each concentration. Into each tube place 1 of the treated .papers, folded once lengthwise into the shape of a letter "V". Two papers impregnated with each of the concentrations of insecticide provided should be used and 1 control paper impregnated with oil alone. WHO/VBC/75. 587 page 3 (c) Transfer 10 bugs to each t-ube and close with the fine-mesh gauze. Set the tubes upright in the holding rack in the box. Close the box for the necessary exposure time. For most reduviids, 2 days exposure to dieldrin is adequate; but some species (e.g. Triatoma infestans) require a longer exposure in order to obtain 100% kill at the highest concentration available. (d) At the end of the appropriate exposure time, examine the reduviid bugs and record the mortality (count _bugs incapable of any movement, or unable to cling to the test paper, as dead). At the conclusion of the test, discard the test papers, destroy the bugs, and cleanse the tubes thoroughly. (e) Tests with a control mortality in excess of 20% are unsatisfactory and should be repeated. (f) When the test has been repeated 4 times with the same population of bugs, adequate data should be available for constructing a base-line of susceptibility as described in section 5. The results should be recorded on the forms provided. 4. GENERAL REMARKS After an impregnated paper has been removed, the package should be resealed carefully with the plastic tape provided. The packages should be kept in a cool place, but not in a refrigerator, as storage at too low a temperature may cause crystallization in the higher concentrations, Prolonged storage at high temperatures should be avoided. .Papers should not be used after the expiry date shown on the box. Dieldrin papers have a useful life of 5 years from the date of impregnation, provided the package is kept sealed at all times. Additional papers are available on request. 5. RESULTS (a) The user may desire to construct the dosage-mortality regression line from the results obtained in the quadruplicate tests at the chosen concentrations and, in many cases, from those of the single preliminary test as well. For this purpose, the results should be plotted on the logarithmic-probability paper orovided. The regression line may be fitted by eye and the concentrations expected to kill various percentages can be read from it. The concentration to kill 50% is known as LC50 ; that for 95% kill at Lc95 , etc. The curve can be extended to estimate the LC99.9 (though it must be realized that this is very approximate). For accurate methods of computing various LC estimates, see Swaroop.l (b) If the control mortality is between 5% and 20%, the percentage mortalities should be corrected by Abbott's formula: % test mortality - % control mortality x 100 100 - % control mortality 1 Swaroop, S. Statistical methods in malaria eradication, Geheva, World Health Organization, 1966 (Monograph Series, No. 51). WHO/VBC/75.587 page 4 6. INTERPRETATION OF RESULTS See Annex 1: 1 Criteria and meaning of tests for determining the susceptibility or resistance of insects to insecticides. 7. DISTRIBUTION OF REPORTS One copy of each completed report form should be sent to: World Health Organization, Vector Biology and Control, 1211 Geneva, Switzerland, and one copy to the appropriate WHO Regional Office.2 (World Health Organization Regional Office for the Americas/Pan American Health Organization, 525 23rd Street N.W., Washington, D.C. 20037, USA). 1 Report of the WHO Expert Conunittee on Resistance of Vectors and Reservoirs to Pesticides (1975). WHO/VBC/75. 587 page 5 WHO TEST FOR INSECTICIDE RESISTANCE IN ADULT OR LARVAL INSECTS REPORT FORM FOR RESPONSE TO DIAGNOSTIC DOSE OR DETERMINATION OF LC. 50 1-3 Study number 181 o I c I 4 - 8 Record number : I I I I 9 -32 Investigator : I I I I I I I I I I I I I I I I f I I I I I I I I I 33 -35 Country : Code: I I I I 36-47 Area: 48-59 Locality: I I I I I I I I I I I I I I I I I I I I I I I I I I 60 - 83 History of insectide usa : [ I I I I I I I I I I I I I I I I I I I I I I I I 84-87 Date of Test : []] []] month year I I I 11 I I I 88 - 95 I nsacticide tested : Code: 96 - 104 Insect species: Code: I I I I I I 11 I 105 Stage: 106 Sex: 107 Where collected : 108 Condition of insect : Larval OL Male 01 Sprayed shelters 01 Unfed 01 Nymph ON Female 02 Unsprayed shelters 02 Blood fed 02 Adult OA Unsexed OJ Field OJ Sugar fed 03 Laboratory reared 04 Gravid 04 Other 05 Unspecified 05 109 - 111 Average temperature (OC): I I I I 112 - 114 Average humidity ( %) : I I I I 115-117 Exposure period : D [D[IJ 120 Number of concentrations used : D days hours mins (excluding control) Concentration of Insecticide Number of Responsas Total Number Tested Control o I o I 121 -123 I I 124 - 126 I 127 -132 l 133 -135 I I 136 -138 I 139 -144 I 145-147 I 148 - 150 151 - 156 I 157-159 I 160 -162 163 - 168 I 169 - 171 I 172 - 174 175 -180 I 181 -183 I 184 - 186 187 - 192 I I 193 - 195 I I I 196 - 1981 - 199 - 204 I I 205 - 207 I I I 208 - 210 I ' a 211 -213 Probable Insecticide Resistance Status : I I I I WHO 5282 VBC (10/75) • 10000 I I I I I I