Instructions for determining the susceptibility or resistance of adult mosquitoes to organochlorine insecticides

WORLD HEALTH ORGAMZAHON \mcw'vecfiw. 581. ORGANISATl‘ON MONDlALE DE LA SANTE mm13“ mm . ’NDE::-- »~ 7,- INSTRUCTIONS FOR DEIERMlNING THE SUSCEP’FIBlLlTY GR RE‘SLSTANL‘S UF ADULT MOSQUITOS m ORGANOCEYLORINE INSECTICED‘ES“ 1. INTRODUCTION rPurpose and limitetions 0f the test This method measures the levels oE susceptibility ot a populatien of adult masqultos ta 3 given organochlorine insecticide,i The technique is detigmed tn detect the emergence at an insecticide-resistant strain if and when it appears. In many cases, the Eltst imdltation OE incipient resistance may be an increase in the number of mosquitee in sprayed hcuses. Nevertheless, it is desirable to detect resistance as early as gossihle, befmre it is widely established in the insect population. For this purpose, it is necessary {a} to establish the Susceptibility levels DE normal populatiens of mosquitos ef the species aceruedg to eatablish a “base-line" for comparison, and (b) to make subsequent routine checks of the susceptibility leVel, by use of a monitoring test at a "diagnestic concentration". Fer eeme oi the more impartant vector species (especially annphelines) base-line date have been attained fur laboratory attains of known susceptibility; and from theae, the diagnostic caneentrations estimated. These are given in a later paragraph and may assist in saving the time and expense of repeating all the work 0f establishing a susceptible base line. This test is specifically devised to detECt phyeiological resistance. However, the effect of resistance on the transmissian of dise33e and the advieebility 0E cantimuing ta use the same insecticide in a given area cannet be determined by resistance tests alone; general Epidemiological appraisal and entamnloglealiaeeesement by other means are necessary. The resistance test is not éesigfied t0 evaluate the effectiveness 9f insecticides in the field; for this purpose, other techniques must be used. Establishing the base line Batches of moequitos are exposed ta different concentrations of insecticide and the martality at each level is determined. It is suggested that a preliminary test he made at each concentration in the complete range provided, ueing the standard exposure of l huur. This will inéicate the general level of susceptibility; furthet tests should than be made on this basis. A series of 4 concentrations should be chosen, some of which will give partial mcrtalities (i.e. at least one of them aheuld give 100% mortality and one should give less than 50% mortality). Tests at these caucentrations should be repeated 4 times with samples from the same population of‘mosquitas. To reveal the full range of natural variation3 this set of tests should be made at several locations and at different seasons, so tar as this is practicable. Even if a true base line (i.e. for an untreated population) cannot be eetab« lished owing ta previous applications of insecticides for any purpose, the susceptibility level at the vetted: species should be determined immediately. Subsequent routine checks by diagnostic concentrations (a) Chpsing the dlaggeetic concentration when newtese~line data ate available The most scientific way ta select the diagnoetic concentration, is to use the data ébtained with the susceptible strain, plotted en logarithmic—probability paper, as described in Results (13,. 5) and select the tencventratlon corresponding to 99.92, mortality. The 1 Annex 1A (Wld filth Urg. techn. Rep. Ser., 1970, No. 443) has been revised ea a result 95 discussions held at the WHO Expert Committee an Resistance at Vectors and Reservoirs re Pestl~ tides which met in Geneva from 16 £0 23 September 1975. The issue of this document does nut constitute Ce dotument ne consume pats we puhhcatmn formal publication. it should not be reviewed. H m; dmt fairs; robgg: d‘aucun cgmptg renew 0L; abstracted m quoted withaut the agreement of resume m damn-me Citfilfim‘: sane I-amgn'smmn tie the World Hearth Organization, Authors alone I’Dggamsmmn Momialede {a game Len mummy. are responsible for views expressed in Slgned expgrimées dam-s vies animal; signe‘s nengagem arhtles. awe inurq autalwe WHO/VBC/75.581 page 2 . . l prinCLples are explained in Annex 1 Criteria and Meaning of Tests for Determining Suscepti- bility or Resistance of Insects to Insecticides. AS a 'rule Of thumb" method, however, it is usually adequate to use a concentration double the lowest concentration which has consistently given a complete kill in all the tests used to establish the base line. (b) Established diagnostic concentrations It has been shown that with a few exceptions, 4% DDT with an exposure of 2 hours will kill susceptible anopheline mosquitos, so that the diagnostic concentration and exposure should be sought in this region. Similarly, it has been found that 0.4% dieldrin with 1 hour exposure will kill all the susceptible anophelines in the species tested (except, possibly members of the An. maculipennis complex which may require 0.8%). These data were obtained with unfed female mosquitos at 27°C. It is likely that they willalso apply to blood fed specimens though not for those entering hibernation. They are given for guidance and should be checked carefully under various local field conditions before final adoption. Use of diagnostic exposures Tests should be made periodically, preferably with five replicates of 15 mosquitos per tube. It is recognized, hoWever, that it may be difficult to obtain sufficient numbers to satisfy refined statistical requirements, especially when the insecticide is effectively reducing numbers, or in seasons when breeding is reduced. A warning of possible incidence of resistance is given when survivors regularly appear in tests with a correctly selected diagnostic exposure. Occasional survivors in such checks could be due to normal variation. But the regular occurrence of survivors in three successive tests constitutes a warning signal calling for further investigations. Condition of mosguitos Although there is seldom a large difference in susceptibility between two sexes, female mosquitos (preferably blood—fed) should be used exclusively in field tests. This is because they survive better and show lower control mortalities. If mosquitos are scarce, it is permissible to use a mixture of fed and unfed females provided the proportion of each is recorded. Mosquitos may be collected from sprayed and unsprayed premises in the zone, but their source should be reported on the form provided. In instances where it is not possible to collect a sufficient number of adult mosquitos for testing, these specimens may sometimes be provided by collecting the immature stages and rearing the adult therefrom. In some circumstances females without a blood meal may be used exclusively, e.g. those recently emerged from a collection of larvae. Conditions of test The experiments should be carried out indoors, if possible, in buildings free from insecticidal contamination and extremes of temperature, humidity, illumination and wind. Where possible, subsequent comparison tests should be made under similar conditions of temperature and humidity. Transport of insects to a base laboratory often results in mortality from causes other than the insecticide; this will be evident as high mortality in the controls. Report of the Expert Committee on Resistance of Vectors and Reservoirs to Pesticides (1975). WHOIVBC/75.58l page 3 2. QDMPGSITION 0? TEST KIT (a? 20 plastic tubes, 125 mm in 1ength and &fi mm in diameter; 8 0f these (with red den) are used for exposing the mQSQuitos ta the insecticide; 2 (with green dmt) are used far the central expcsure withsnL insecticide; 10 {with green flat) are used as hOLding—tubes for pre~ test sorting and pastaexposure observetian. Each tube is fitted at ans end with a Lé—mesh Screen. In order {a identify the cancentrations used with them, the red expasure tubes shouid be numbered 1 ta 8, the green control expasure tubes 9 and 10 and the holding tubes la ta 10a, ib} 10 Slide units, each with e screw-cap on either side and provided with a 20~mm filling hale. (c) (i) For diagnostic tesgg Two packages of papers impregnated with 4% DDT (p,p1 isomer) in mineral oil (Risella 17, Shell); twc packages; each of papers impregnated with HEOD (purified dieldrin} in mineral oil, at Concentrations of 0.41 and 4%; and one package of papers treated with ail only (each paekage contains eight papers). (c) (ii) For base—line data determinaciqn Five packages of papers impregnated with DDT (p,p'-isomer) in mineral oil (Risella 17, Shell) at concentrations of 0.25%,»G,SZ, 110%, 2.0% and 4.0%, respectively, and one package treated with ail unly; six packages cf papers impregnated with HEQD (purified dieldrin) in mineral oil, at cancentrations DE 0305%, 0.12, 0,21, O,4%, 0¢SX and 4.0% and One package treated with oil cnly. (d) Sheets of clean paper (12 x 15 cm) for lining the balding-tubes. {e3 20 spring«wire clips to holfi the papers in pesition against the walls of the tubes. The 12 silver clips shauld be uaed only far the holding-tubes and the central exposure tubeS; the eight capper clips should be used only fer the insecticide exposure tubes. (f) 2 glass aspirator tubes, 12 mm in internal diameter, tagethar with 60 cm of tubing‘ (3) 1 roll of self—adhesive plastic tape. (h) Instructinn sheets, a set of report farms (and where base—line data are being collected, three sheets ef logaprobability paper for plotting regression linea). 3. TEST PROCEDURE (3) Intn each ef the holdingntubes, insert a piece of clean white paper rolled inte a cylinder ta line the wall and fasten it in pasition with a spring-wire clip (silver), Attaflh the slides to the tubes. (b) Gellect approximately 200 temale mesquitos with the aspirator pravided (Fig. l, A). Damage reSulting from careless handling oE mosquitos during collecting may produce misleadingly high mortalities. Mosquitos should be callected in lots of not more than 10 (Fig. l, B} and gently transferred t9 the holaing—tubes through the filling—hale in each side (Fig. 1, C) to give 15 :0 25 pet cube. Any departure firom these figures may impair the reliability of the results. (c) A pre—test holding period may‘be neaessary t0 guard against including damaged SpeCimEflS in the test. For this purpaSe, Lhe holding—tubes are set upright, screen and up, for 1 hour or motee At the end of this time the damaged insects are remaved. WHO/VEC/TS . 681 page # FIG. 1. METHOD FDR DETERMINING THE SUSCEPTIBILITY OR RESISTAECE OF ADULT MOSQUITOS TO ORGANOCHLORINE INSECTICIDES ‘ ' , HOLDING“:33? was .. - * .. _ .. ' WHUIVBEI?§.53L §age 5 (d) Into each of the exposure tubes zntruduce a sheet of impregnatud papa:f ratiad inuu a cylinder to line the wall and fastemeé inta pusitian wick aa apprugriate syringawire cLiy. (2) Introduce the mosquitos intu the expugure tube by attaching it to the vanana scraw~to§ in the slide (Fig. l, D). The slide should be pulled out to‘a paint beyumd the Eiilingnhqle so that am part cf it acnludas the tuba upenings; the masquites are then blown gefic§y down inca the exposurg tuba. iIf necfissaryg the smalL safety knab an the a1iua may be Eileu dour to fiasilitate this operation.} Close the slide. Detach the halding-tube and set it asida. (f) Leave the exPesure tubes szanding upright with scream and up for 1 hour (Fig. I, E} undar conditipns of medutate, diffuse illuminatien. (g) At the en& cf the exposure period, transfer the mosquitns to the holdingmtubes by reversing pracedure (e). When some masquitos have been knocked dawn in the course ufi an exposure, the sxpusure tubes shuuid be held horizuntally and tapped to disiodga tbs iuaects from the slide before tha Latter is withdraWn. Attach the holding—tubeq span the slide, and gently blow the mosquitos into the holding—tube; Close the slide and remnve the expasure tube. Then Set the halding~tuba so that it stands an the slide and place a pad‘of Wat cottonswool an the screen (Fig. 1, F7. cardbaard catcens or cups er‘othet suitable cuntainers may he used instead of the holding—tubes, gravided thafi they are used consistently; (h) Kaep the holding«tubes for 24 hours in a secluded, shaued place, where the temperature dues not exceed 30°C. Wherever feasible, the maximum anu minimum temperaturu of the roam during the 24 hours shpuld be recarded. If necessary, the tubes should be protected from ants by placing them on a platform standing in a pan cf watar. If conditions are very hot and dry, a moist-chamber may be pregared by suspending damp towelling in a cantainer. (1) Mortality ecunts are made after 24 hours. Remove the dead mosquicas by gently detaching the slide and cautiously moving the tube aside. Affected specimens that are unable to walk 7 are to ba counted a8 dead. As an aid :0 counting the living specimans, thay are stunned by a sharp jerk uf the tube ur stupafiefl by chlaroform or ether. (The anaesthetics sheuld not be allowed to acme into‘direct contact with the plastic tube ana screw cap, which are soluble in these compounds.) The results should be recorded on the forms pravided‘ Cepies of completed forms shauld ha distributed in accordance with.the instructions an page 6, (j) (i) Fbgfidia postiq‘tuggs Four replicate tests should be dune with each of thu diagnostie concentratiens and} preférably, four controls with tha oil—treaLed papers. (11) Fur basg—}iagadetermination Fan: replicate tests shauld be dune with eaah of the different concentrations of either DDT Dr dieldrin and, preferably, four controls with the oilatreated papers. (k) Tests with central mortality in excaas of 20%, thuugh unsatisfactory, shuuld be securdefl. An invesrigatien into the cause$ Bf cuntral mortality should be made and steps taken to avuid it. A possible cause may he the cellection of mosquitos frum sprayed dweilings. In this case, it mgy be necessary to collect specimens from unsprayed ones, or to rest adults rearad from aquatic stages. 4. GENERAL REMARKS (a) Each impregnated paper‘may be used up :0 20 times; and up ta 3 wgeks after ramavak fram the package, ptuvided all possible precautions Bra taken against evapuration of the ail. to this and, the papers should be left in the tubes, with the npen end well wrapped, and placed in the kit baa, which in turn shauld be kupt in a cool place. No paper should be used more than 3 weeks after remeval from the packagfi, waol'vac/75 . 581 page 6 (b) 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 too low a temperature may cause crystallization in the higher insecticidal concentrations, Prolonged storage at high temperatures should be avoided. Papers should not be used after the expiry date Shown on the box. Chlorinated hydrocarbon papers have a useful life of,5 years from the date of impregnation, prGVided the packages are kept sealed at all times. (c) If the species of mosquito concerned is exceptionally insensitive the axPosure period may be increased to 2, 4, or 8 hours. In all cases, the 1—hour exPOSure period ShOUId be used first and the result recorded. To decide on a suitable exPosure period, conduct tests with the highest cencentration available until complete mortality is obtained. The complete range of concentration is then employed to give a concentration mortality regression line. 5. RESULTS (3) For base line data determinations, the user may desire to construct the dosage—mortality regression line from the results obtained in quadruple tests at the chosen concentrations. For this purpose, the results should be plotted on the logarithmic—probability paper provided. The regression line may be fitted by eye and concentrations expected to kill various percentages can he read from its The concentration to kill 30% is known as’LC5o; that for 95% kill, is L095, etc. The curve can be extended to estimate the LC99'9 (though it must be realised that this is very approximate). For accurate methods of computing various LC estimates, see Swaroop. (b) If the control mortality is between 5% and 20%, the percentage mortalities should be corrected by Abbott's tormula: 1 test mortality -‘Z control mortality X 100 100 - Z control mortality (c) Results obtained where control mortalities exceed 20% should be recorded but not corrected. The accuracy of the interpretation of results depends on the reliability of the data obtained. Utilizing the maximum number of specimens per tube (25) decreases the effect of individual différences in response. Regression lines based on similar numbers of specimens are more reliable. 6. INTERPRETfiTION OF RESULTS See Annex 1:2 Criteria and meaning of tests for determining the susceptibility of resistance of insects to insecticides. 7. DISTRIBUTION OF REPORTS It is of considerable importance to WHD'to receive copies of results obtained from the use of this test kit. it is therefore requested that copies of all reports be sent to the Eollowe ing addresses: 1 Swaroop, 3. Statistical methods in malaria eradication, Geneva, World Health ‘Organization, 1966 (Monograph Series, No. 51). 2 See Annex 1: Report of WHO Expert Committee on Resistance of Vectors and Reservoirs to Pesticides (1975)» WHO/VBCHS . 58 1 page 7 For anogheline agecies: 1. World Health OrganizatiOn, Division of Malaria Eradication, 1211 Geneva, Switzerland 2, The appropriate WHO regional office; 1 and 3. Project headquarters. The fourth copy should be retained by the investigator. For non-anopheline species 1. World Health Organization, Vector Biology and Control, 1211 Geneva, Switzerland; and 2. The appropriate WHO Regional Office.1 The third and fourth copies should be retained by the investigator. Addresses of WHO regional offices are as follows: World Health Organization, Regional Office for Africa, P.O. Box No. 6, Brazzaville, Congo World Health Organization, Regional Office for the Eastern Mediterranean, P.0. Box 1517, Alexandria, Egypt World Health Organization, Regional Office for South—East Asia, World Health House, Indraprastha Estate, Ring Road, New Delhi — 1, India World Health Organization, Regional Office for the Americas/Pan American Sanitary Bureau, 525, 23rd Street, N.W., Washington, D.C., 20037, United States of America World Health Organization, Regional Offica for Europe, 8 Scherfigavej, DK-ZlOO Copenhagen O, Denmark World Health Organization, Regional Office for the Western Pacific, P.O. Box 2932, 12115 Manila, Philippines. WEE? TEST FEE hflfiEflTfiIfllflE fiEfitfiTflNEE‘ m ABET? 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