iWORLD HEALTH ORGANIZATION AFRICAN REGION ORGANISATION MONDIALE DE LA SANTE REGION DE L'AITRIQUE ONCHOCTRCIASIS CONTROL PROGRAMME IN THT VOLTA RIVER BASIN ARTA PROGRAM}IL DE LUTTT CONTRI L'ONCHOCTRCOSI, DANS LA R[,GION DU BASSIN DE LA VOLTA Tdl 333 12- 329 10-3385?-Tdldx ONCHO 5241UV EXPERT ADVISORY COMMITTEE Second meeting ocP lEAC2.4 Geneva 12-16 October 1981 ORIGTNAL: FRENCH RESISTANCE TO TEMEPHOS IN THE SI}ruLII}I DAMNOSI]M COMPLEX URRENT SITUAT]ON (AUGUST, 1981) FORESEEABLE ENTOMOLOGICAL AND EPIDEUIOLOG]CAL CONSEQUENCES EFFECTS ON THE VECTOR CONTROL STRATEGY OF THE PROGRAMME 1. Historical Review Resistance to temephos in 1arva1 populations of the S. d:mnosum complex was diagnosedfor the first time in May, 1980 on the Lower Bandama River in Ivory Coast, in the vast complex of breeding sites ("Chutes Gauthier") located between the Taabo dam and the town of Tiassa16, after all other possible causes of the reappearance and maintenance of high biting densities since March had been eliminated. With the exception of an experimental series of six weekly treatment cycles, always spectacularly effective, performed in L976 as part of a study of reinvasion, this part of Bandama had only been treated regularly on a weekly basis since March, 1979. A11 during the year of L979, these treatments had a remarkable and constant effectiveness (dor.rn to a mean of less than one bite/man/day instead of several hundred to a few thousand). On the other hand, the many plantations which line the lovrer Bandama and cover the hinterland had already been the objects of intensive applications of various agricultural insecticides for many years. The causes proposed for this place were many: this unexpected and relatively early appearance at exactly geographic isolation of the 1ocal populations of Simuliidae, welI shor^m by the experiments of 1976 and reinforced in 1979 by the closure of the Taabo dam; selection of resistance by the early treatments either with temephos (1976) or with other chemicals with a cross-resistance to temephos (possibility not demonstrated); selection of resistance by under-dosing brought about by changes in the height-discharge relationship of stream gauges caused by the construction of the Taabo dam (hypothesis not confirmed), or because of the nature of the breeding site whose large area and many ramifying channels make it difficult to deliver a correct dose to every part of eyery breeding site, in spite of optimal treatment techniquel C { ocP /EAC?,4 Page 2 in the szrme way, the possible passage during several years of infinitesimal doses of temephos (or other larvicides) applied each week in the upper Bandama basin(essentially in the Nzi and Marahou6 Rivers, the Kossou Lake serving as a settling basin for the White Bandama River) has been evoked as a possible cause of the selection of resistancel possible changes in the mode and degree of action of temephos due to the changing of the physical-chemical composition of the water after the formation of the Taabo Lake have also been envisaged. Although all the other causes cannot be eliminated, it seems nol^/ to be accepted that the relative isolation of the vector populations, along with their specific identity, played a determining role in the selection of the resistance under intense insecticide pressure. Among the three species of the complex inhabiting the lower Bandama the two forest species were directly affected by the resistance; that is S. soubrense Vajime and Dunbar, 1975 and S. sanctipauli Vajime and Dunbar, t915. The first is almost exclusively I imited to the southern forest zone while the second is the form adapted to less humid surroundings and can develop in the guinea savanna zone and even in the southern sudan savanna, when ecological conditions are favourable, permanently or seasonally (rainy season). For a growing number of cytotaxonomists, these two forms constitute in fact a single species, which we will designate hereafter under the somewhat unorthodox name of S. soubrense- s anct ip au1 i . 2. Geographical extent of the phenomenon It seems now possible to accept that the propagation of the resistance occurs by migration of resistant adults step-by-step in a single hydrological basin or between adjacent basins, and not blr successive independent selections one after the other; the existence of heterogenous.dosage-mortality lines on Ehe lourer Corno6 in May 1981 (see 3.3.1'1) is significant in this respect. The history of the expansion of the phenomenon is thus that of the progressive contamination of the different foci of S. soubrense-s-gleliPauli which were treated with temePhos. After the lower Bandama the resistance to temephos was thus successively demonstrated on the lower Marahou6 (August, 1980), the Nzi and the Kan (November, 1980), the middle Sassandra and the upper Marahou6 (January 1981), the Graba (February), the White Bandama, the upper Sassandra and the Bou (March) , and the middle and lower Como6 (May). With the exception of the last case, diagnosed four months after the extension of temephos treatments on this stretch of river, no new focus of resistance to temephos was uncovered after March, in spite of a reinforced surveillance, and many of the rivers mentioned above were in reality suspected since August to September, 1980: Nzi, Kan, middle Sassandra and Bou. a Currently, the basins concerned with resistance the moment these limits do with those of the knovrn di of the rivers enumerated previously draw the limits of the foci inside the limits of the area treated by the Progranrne. For not exceed the frontiers of Ivory coast (see map) and coincide stribution of S. soubrense-sanctipauli However, it must be noted that the following river basins are not affected: the Black Volta in the Bui area (Ghana) and the basins of rivers Bago6 and Baou16 in Mali and Ivory Coast. In Bui some flies showing S. soubrense-sanctipauli rnorphological characters have been recently identified, but no larva of these species has yet been co11 Ma1i. S. soubrense-sanctipauli larvae were collected at the beginning of "rr.".pEl6Tfity t."ts carriea out then, after 1ocal failures of temephos t shown only normal susceptible S. sirbanum larvae (in the meantime the rei and an improved application of-lanricide allowed again looZ efficiency of ected. In outhern 1981 but the reatments, have nvasion had started) temephos treatments. I ocP /EAC?.4 page 3 3. Main characteristics of the resistance 3. 1 Organizatio! of suscepti!rl.LtJ lestin€ lee4! Following reconrnendations by the 1981 Informal Working Group on Temephos resistance (document OCP/I^JG/81.1) and to meet the need for susceptibility data in the Progranrne area, three teams have been trained to perform the tests. Each team is composed of a technician, two laboratory auxillaries and a driver and vehicle, These teams, operating under the general supervision of an entomologist, can be requested to perform tests anyrshere in the Progrannne area at a few dayst notice. To augment the number of tests in the extension study zones of Ghana and southern Togo, contracts have been signed with personnel of the Volta River Authority based in Akosombo, Ghana. This team has been trained in the Mouchet method as used by OCP and their larval age selection is checked by OCP personnel. In addition, sub-sector chiefs (technicians) in some key areas have been trained to do susceptibility tests and been provided with equipment. The tests are performed at the river side or in an air-conditioned room if the latter can be reached in about one hourts time. Depending on whether prospection is by foot, vehicle or helicopter the collection site may be a few kilometres up to 1OO kilometres away from the test site. It is not always easy to find larvae for tests, even when adult biting densities are high. In the rainy season (when the studies began) 1arval breeding is widely dispersed on trailing vegetation, access is only possible by boat and rapidly fluctuating water level-s often deeply submerge the best substrates. In some cases tests were done on l-arvae reared from field-collected eggs or blood-fed females. Augmenting natural substrates with tethered palm fronds is sometimes useful, but often requires several weeks for colonization. In treated rivers, the density of resistant larvae is often very 1ow. Sometimes treatments can be suspended to allow populations to build up. However, if susceptible larvae of savanna species are present this is not always operationally acceptable. A1so, with suspension, resistant larvae may be diluted by non-resistant flies colonizing the breeding site. In all cases of temephos treatment failure, serious efforts have been made to perform susceptibility tests before changing insecticides, although occasionally the only indicator available is a change in the morphological characters of the adult population. Final1y, all studies of resistance are hampered by the present inability to colonize S. damnosum s.1. Progress is rapidly being made in this fie1d, and it is hoped that studies of resistant strains will receive high priority when colonies are established 3.2 Standardization of test methodology The tests are carried out using the method described by Mouchet et aI. (1977) and recently adopted by the WHO as the standard method for S. damnosum s.1. A detailed protocol has been written for technicians performing the tests. The method has been significantly modified in the matter of selection of the larvae. Mouchet et a1. clearly showed that young larvae are more susceptible, and suggested the use of 4th and 5th instar larvae only. In practice, this creates two problems. First, limiting the tests to young larvae creates operational problems when results are needed rapidly for treatment decisions: if the larvae present are 6th and 7th instars it is not desirable to leave the river untreated long enough for a ne\,r generation to develop. Secondly, the morphological criteria suggested by Mouchet et al. are either rather vague or too difficult for use for thousands of larvae by numerous technicians working independently. After careful study of the larval morphology and the susceptibility of different ages of larvae it was decided to recognize t\to types of tests, one with "young" l-arvae(4th, 5th and optionally 3rd instars) and another with "o1d" larvae (1ate 6th and 7th instars) ocP |EACZ.4 Page 4 A pictorial key was devised for selection of the larvae. This key uses simple and relatively unequivocal morphological characters to separate the larvae into four groups (1) Very young larvae (approximately 3rd instar): these larvae are somewhat more susceptible than young larvae, but can be used when nothing else is available; they are difficult to manipulate and observel (2) Young larvae (approximately 4th and 5th instars): these larvae are recommended by l"louchet et al. They are more susceptible than older larvae and the slope of the dosage-mortality curve obtained with them is very sensitive to the development of resistance (see section 3.3); on the other hand they are more difficult to find than o1d larvae in treated rivers; a1so, they are more difficult to manipulate and cannot be identified cyrotaxonomically; knowledge of the species. cornposition of the population testeal must be based on independent samples of oltler Larvae taken-at the. same time which may not be representative; (3) Intermediate larvae (approximately early 6th instar) : these larvae should never be used for tests; it is during this stage of development that the larvae are changing rapidly physiologically between the very suscePtible state of young larvae and the roughly 1o times more tolerant state of old larvae; tests with such larvae will obviously be highlY variable; (4) O1d larvae (approximately late 6th and early 7th instars): these larvae are about 1O times more tolerant than the young larvael data obtaineil with them can only be compared with other tests done with old larvae; also the slopes of the dosage- mortality curves obtained with old larvae are less sensitive to the development of resistance than the same data for young larvae; on the other hand, o1d larvae are easier to find, easy to manipulate and can be identified individually by cytotaxonomy; for routine detection of developed resistance by diagnostic doses, o1d larvae are equally as useful as Young larvae. It should be noted that variability of results of these tests is such that a difference of up to 2 times in LC.^ or LC..is usually not significant. Even when consecutive tests are performed within "'Y., dayB)at the same test site by the same test team' a difference of 2x in these parameters can occur. Variations in physiological state of the larvae' differences between cytospecies and between populations of the same cytospecies all play a part in Ehis variation. 0n the part of the oPerator, sma11 changes in the method of larva1 age selection can produce a difference of 2x in the LCso:. Only.differences of at leastgi i' LC.^ and/or LC^. or decreases in the slope of th3uline (with or without changes in the tCro) caiube consideida to be significant (see 3'3'1)' 3.3 Srr*n"r, and discussion of susceptibility data gathered since the aPPearance of res istance In spite of the difficulties to find suitable larvae and necessarv adjustments of the technique, since June, 198O, about 1OO susceptibility tests have been carried out by OCP personnel in the Progranrne area, involving around 40 OOO larvae' A surmary of the tests is presented in Table I. The locations of most of the test sites are indicated on the map. The dose-mortality curves for certain tests are presented in figures 1-7, and detailed data for several key tests are presented in Tables II and III' 3.3. 1 Tests with temePhos 3.3. 1. 1 Changes in dosage-nortal ity dafa with resistance The first sign of resistance has always been the failure of treatment, particularly the presence of young s. damnosum larvae widely scattered in the breeding site. (the pr"."o"" of old r"il-iilil part of a breeding site more often indicates Poor coverage OCP IEACZ ,4 paee 5 by the treatment) In susceptibility tests, resistant populations always haye some survivors at the diagnostic doses of O.25 ng/l for young larvae and 2.5 ngll for o1d 1a1vae. The survival may be from a few percent to over 5OZ, but is usually less than 1OZ. In general the limit for 1002 mortality increases from 0.O5 ng,l 1 ro 1 .O mg/l for young larvae and from 0.5 mg/1 to 10 mg/l for o1d larvae. Resistant populations of young larvae have produced two types of dcsqge-mortality curves. fn sorne cases, the line is straight (homogenous) with a much lower slope than normal larvae (Fig. 1, lines 5, 5, J and 8; Fig. 2 lines 4,15, 6). ln other cases the line is definitely broken into segments (heterogenous by Chi'test): Fig.1, line 4; Fig. 3 and 4. The first case may be interpreted as an interbreeding population heterozygous for the resistance factor, which does not show complete dominance or is polygenic, Individual larvae show wide variations in suscepribility. The second type of curve may be said to represent a mixtr:re of resistant and non- resistant populations which are not interbreeding. The left-hand portion of such curves represents a susceptible population and the right-hand a homozygous resistant population,(i.e. there is 1itt1e individual variation in susceptibility), The two populations may be of the same cytospecies (Fig. 3 where all the larvae were S, soubrense) cr of a different cytospecies (F ig. 4 where about l5Z of the larvae were S,damnosLrm s.str. In theory the level of the horizontal parr-iTEETine representsand 852 S. soubrense) the percentage of the population which is susceptible; for example for line 4 in Fig, 1 the horizontal part of the line lies between 982 and 992 and there was 98.82 mortality at the diagnostic dose; for line I in Fig.4 the horizontal part of line lies between 9OZ aod 952 aod there was 882 mortality at the diagnostic dose. For o1d larvae, there is not as rm:ch dara. However, it seerqs that resistance in o1d larvae is not expressed by a change in slope of the dosage-mortality line but rather by a shift of the line to the right (Fie. 6), In terms of LC.r., and LCo., the first type of curve gives a slight or no in LC-^, and increadE of tC^'-'of about 10 rimes and a chanse in the LC--lLC- Z-l t3uto-zo. For exampI",vSt Chures Gauthier rhe mean fi[ures forci?5r"r?3 treatment (Gui11et, 1980) are: ].ncreas e ratio from be fore LC5o = o.o19i LCSS = 0.056; LC LC 2.96. For six tests after appearance of resistance: LCSO = o.o52i LC,S = 0.68i LCg5lLCro = 13.2 At Danangoro before the treatment for three tests (Mouchet et aL.,1977): LCSO = 0.031i LC9S = 0.081l' LC95/Lcro = 2.65 After appearance of resistance: LCro = 0.06; LCnr= 0.81; taSSltaSo = 13.5 The increase in the LCrr/LC'O ratio seems to be the most consistent indicator. With the heterogenous curves the LC.^ are difficult to estimate, By extending the vertical Parts of the curves (dotted findY), separate estimates for the resistant and normal populations can be made. For example, combining two tests with resistant larvae at Bac S6mien on the Sassandra River in Ivory Coast (Fig.4), for the susceptible oopulation LC.., = 0.015 mg/l, LCo. = O.026 and LCo./LC.^ = 1.73; for rhe resistant poputation LC:: = 0.46, LC^ - = O'.1. LC^- /LC-^ = l'.52. '\n rhis case rhe IC-, and LC, - are both incrBHsed by abo,lI)30 times ti? tr,3u.ropes are paralle1. Thi, i,.,3?"rt"" 95 that the resistant part of the population was more or less homogenous in susceptibility, and thus presumably homozygous for the resistant factor. 0559 (Fig.6). For the resistant larvae (Ti6bissou) LCsn = o,96, LC for the susceptible larvae (Asukawkaw) LCc,^ = 0.052, LCo. = 0. Here again both LC'O and LC* increase ab6[t equally and-the s 3.7. LC^.ltc.^ = LC^ - ILC:'! = 2',.'69 .95 5U- -are paraIIeI. ocP /EACZ.4 page 6 With o1d larvae, not as much data is available, but one can compare the test of 31/08/81 at Ti6bissou with the test of 11/05/81 on the Asukawkaw River, (Ghana) T4, lopes damnosum 3.85. and There is thus always a part of the population which remains susceptible, even when the treatments are failing. However, some of these results may be artifacts in the sense that treatments were often suspended some time before tests. The repeated tests at Chutes Gauthier have shorrn that the resistant larvae do not disappear when treatments are stopped or another insecticide used. Line 4 on Fig.l was obtained after lC weeks of chlorphoxim treatment and three weeks of suspension Line 8 on the same Fig. was obtained after an additional 15 weeks of suspension. 3.3.1.2 Cytospecies involved in resistance In 41 tests involving resistant larvae, 3O have involved populations composed exclusively of S. soubrense-sanctipauli. In ten other cases S. damnosr;m s. str. and/ or S. sirbanum have been present in the general samples, but have never been found among the survivors at a diagnostic dose of temephos. In one case (Test.ro 112 at Ass6r6kro in Ivory Coast) larvae of S. s. str were found among the survivors of 2.5 ngll temepho (8OZ) as in the general samples. A test previous to this and two afterwards 1n" 125 and 128) have only produced S. s in about the same proPortron at the same site (n" 89) soubrense among the survivors although S. damnosum s. str. was always present in the general population. There is no other evidence that resistance to temephos occurs outside of the cytospec les pair of S. soubrense-sanctipauli. It is not possible to say if one member of the pair is more resistant than the other. Initially, Guillet et a1. (1980) reported a slightly higher proportion of S. san",l:lpauli among the survivors than in the general population (8OZ vs 652). However, this has not been conf irmed in other tests. In fact, S. sanctipauli has only been found Ermong the survivors at one other site, Ass6r6kro on the Kan river(fests no L24 and 126). At that site there was no increase in the proportion of S. sanctipauli among the survivors. At Chutes Gauthier itself, the proportion of cytospecies in the general samples has changed from 35 soubrense/55 sanctipauli in May, 1980 to 55 soubrense/45 sanctipauliin February, 1981 to 88 soubrense/ 3/sanctipauli/ 4 damnosum s. i" llay, tS8t. At the same time the susceptibility has not changed a great deal. s tr. This implies a fair degree of genetic mixing between these two "species" At sites further North, resistant larvae have always been identified as S. soubrense It is well known that this species, although considered a "forest" species, can occupy breeding sites as far North as the sudan savanna, especially in the wet season S. sanctipauli , on the other hand, is rarely found outside the forest zone. AI has unf sanctiP though the possible "marker" inversion mentioned by Guillet et al. (1980) ortunately not been consistently found in resistant poPulations, soubrense/ auli hybrid chromosome arrangements do seem to be unusually cortrnon in resistant populations Resistant populations of S. soubrense have existed in the same breeding sites with populations of S. sirbanum and S. damnosum s. str. No hybrid chromosome combinations have Ue.r-"e6-a.ra, ,ith_ff,E-ffi!-tion noted above, larvae of the two savanna species have never been found among survivors of diagnostic doses. This confirms their gene tic isolation from S. soubrense. ocP /EAC?.4 page 7 3.3.7 Tests with other insecticides i^lith the appearance of resistance to temephos, chlorphoxim has been used intermittently as a replacement insecticide in all the rivers affected by resistance(see map and 2.1). Numerous checks of susceptibility to chlorphoxim have been carried out. No evidence of any change in susceptibility has been found (Fig. 5). However treatment failures with chlorphoxim will continue to be investigated as possible cases of cross-resistance. In terms of other insecticides, tests are being carried out as resistant larvae are available. The curves obtained so far are shovm in Fig. 7. In the absence of published data gathered by the Mouchet method, it is not easy to interpret these resulfs until data from non-resistant S. soubrense-sanctipauli populations are available. This is also being done as quickly as possible. Certainly, however, the curves for DDT and methoxychLor do not show the plateau shape demonstrated by Guillet et al.(1977) in Mali for S. damnosum complex larvae resistant to DDT. 0n the other hand resistant larvae do have a slightly higher l00Z mortality threshold (0.L25 ngll : Test no 120) than larvae of S. soubrense-sanctipauli tested in an untreated river in Ghana (0.0625 mg/l : Test no-16rJl--This "ariation can however occur in two tests ofthe same population. Efforts will continue to determine the spectrum of resistance. 4. Consequences of temephos resistance 4.L Introduction of chlorphoxim on arr operational scale From 1O June 1980, at Chutes Gauthier, temephos was replaced by chlorphoxim, the only replacement product then available and tested (weekly applications at 0.025 mg/l/10 Mn). The results were spectacul.ar, captures falling from 2 4OO to one female/man/day in five weeks. With capture figures worsening in the Taabo region, it was decided to extend larviciding upstreaml however, stocks of chlorphoxim being limited and the firm of Bayer producing the larvicide only to order, it became necessary to suspend the chlorphoxim treatments at Chutes Gauthier. Wide disparities also became apparent in the efficacy of different drums of chlorphoxim, some having clearly deteriorated with the storage time (three years). Fina11y, the initial chlorphoxim applications on the lower Bandama had to be suspended at the end of August for lack of larvicide, this being the first episode in an all too long series of supply failures. These applications could not be resumed until the end of October owing to a build-up of delays in delivery of the new order. This time all the Ivory Coast watercourses, where resistance to temephos had meanwhile been diagnosed and temephos applications had been progressively suspended (end of August on the lower Bandama, September on the Nzi and the Marahou6, October on the l.{hite Bandama), urere put under chlorphoxim until the end of December (southern part of the zone) and the beginning of January (northern part of the zone). Apart from six applications of chlorphoxim in February-March on the middle Sassandra, following demonstration of resistance to temephos on that stretch, further delays in delivery made it impossible to resume larviciding unril mid-May over the whole extent of the Bandama and Sassandra basins and the end of May on the middle and lower Como6. This resumption and the routine use of chlorphozim in the foci of resistance to temephos had been agreed to by a working group on resistance to temephos which met in Geneva in March 1981, and this approval was confirmed by the Ecological Group at its meeting in Ouagadougou in May 1981, conditional upon judicious utilization over a limited time and area. ocP {EACZ.4 Page 8 From mid-May to the end of July the normal sequence of chlorphoxim applications u/as several times upset by stock shortages resulting from very late deliveries which necessitated postponement of treatments, dosage reductions, changes in treatment circuits and, final1y, untimely suspensions; only on the lower Bandama and lower Como6 could larviciding be properly carried out throughout this period. What is more, the chlorphoxim formulation used during this period proved very faulty and unsuitable compared with previous batchesl in particular, its inadequate range of action necessitated much rescheduling of treatment circuits and helicopter overtime. Some resumptions of temephos larviciding after a spe11 of chlorphoxim treatment in foci of temephos resistance occurred, either because they were necessitated by events - exhaustion of chlorphoxim stocks simultaneously with the need to neutralize potential sources of reinvasion by savanna females - or because they were deliberately decided upon with a view to selecting resistant popu lations of S. soubrense-sanctipauli for specific investigations on the nature and origin of resistance. The former category includes the temephos treatments of the Sassandra and upper basins of the I,'rhite Bandama, Marahou6 and Nzi during the dry season and in May and Junel into the latter fa1I the applications made in July and August on the upper basins of the Marahou6 and Sassandra and on the Kan. In both cases resistant populations of S. soubrense-sanctipauli recolonized the foci within two to five weeks. Since August regular weekly chlorphoxim treatment has been applied to the basins of the Bandama (White Bandama, Marahou6, Nzi, Bandama and tributaries), of the Sassandra (Bagb6-Sassandra and tributaries) and of the Como6 (including the L6raba): see maP. It appears that the present formulation, though better than the one used in May, is not as satisfactory as the 1973 and 1977 formulations, especially as regards range of action, though operational conditions and emergency situations have precluded strict comparative studies in this regard. Such an investigation is progranrned for October: it will consist in comparisons between the ranges of action of various batches of chlorphoxim and Abate 2OO CE Procida in different types of river with different discharge rates and aL concentrations of 0.025 mg and O.O5 mg/I/10 mn. Because of the inconsistency of the quality of chlorphoxim formulations, the nr.rmber of weekly treatment series required on a given stfetch of river before a period of suspension cannot be realistically predicted. lloreover, it has several times been necessary to double the dose from 0.O25 nglU 1O mn, particularly on the lower basins of the Como6 and Bandama. 4.2 Search for other reP lacement larvicides The need to intensify this search has been stressed by all the Programne I.lorking Groups that have met in 198O and 1981 ' In the past, the difficulties encountered in the screening of new products and new formulations stef,med mainly from lack of eagerness on the Part of firms to produce new specific larvicides on an experimental basis. This year the bottleneck was rather the fact that the test teams of OCCGE (IRTO) and the VCU were fully occupied with certain products offered in many different formulations, such as Bacillus thuringiensis serotype H-14 and microencapsulated chlorpyrifos-methyl; since 1980 IRTO and VCU have been sharing the testing between them (stages 4 and 5 for IRTO and stages 6 and 7 for VCU, though the stages 4 and 5 testing B.t. H-14 is not being done under OCCGE-OCP agreements). In 1981 the supply of chemically different new products remained practically nil At stages 4 and 5, none of the microencapsulated pyrimifos-methyl formulations gave promising results, but several varieties of microcapsule remain to be tested. ocP lEAC2 .4 page 9 Similarly, the new tests of diflubenzuron (growth inhibitor) hav despite several replicates the tests of formulations of Teknar richer in active ingredient have not kept their initial promise experimental formulations of B.t. H-14 tested by IRTO was given testing at the subsequent stag"" lSolvay's BL 59 SA). e-been disappointing;R " -'(SandozWIDC) and only one of the its approval for At stages 6 and 7, after the good performances obtained with the very high water Ievels in September 1980 on the Marahou6 with Teknar (I9 km total effective range and 15 km partial range), the product was successfully tested at low Trater on a stretch of the L6raba several dozen kilometres long for six consecutive weeks under particularly difficult operational conditions (very 1ow discharge rates and complex of Gr6chan breeding sites). These stage 7 trials showed that (a) the formulation is effective at 1ow water; (b) it can be used with the Progranrnets normal aircraft and larviciding equipment subject to a slight modification of the latterts filtersl(c) despite some impairments it can remain sufficiently effective after over eight monthst storage in tropical conditions; and (d) the addition of water (2OZ) to the larvicide can be done up to one week before utilization. The chief difficulty now remaining is the 1ow active ingredient content (0.82) which, added to the need for prior dilution with at least 2OZ of water, put its transport and application beyond the capacity of the Progranrners present aircraft at highwater time. New stage 6 tests at intermediate discharge rates are planned for October with this formulation, as also with BL 59 SA and a Scandinavian formulation of methoxychlor containing a higher concentration of active ingredient than the usual formulations. But worrying delays are already occurring in the delivery of these new products. 4.3 Strengthening of aquatic surveillance With the decision to apply regular chlorphoxim treatment to the Ivory Coast hydrological basins affected by temephos resistance, all the specialized working groups have also strongly emphasized the need for increased aquatic surveillance, which wasput into effecE for rnonitoring the invertebrate fauna from the beginning of 1981 under the supervision of the ORSTOM hydrobiology laboratory at Bouak6. The unavoidable suspension of operations during the dry season was followed by substantial repopulation which was not much affected by the resumption of larviciding, the batch of chlorphoxim put into use in l"lay exhibiting an ineffectiveness which was fortunately as great against the non-target fauna as against blackf1y. It is still too early to analyse the effects on the invertebrate fauna of the new batch started in Augus t . Fish monitoring of rivers under chlorphoxim treatment was also strengthened during the year under the supervision of the specialized team of the Abidjan University Institute of Tropical Ecology. It is sti11 too soon to observe whether there has been any effect on the fish populations exposed to the chlorphoxim. With this strengthening of aquatic monitoring the recruitment of the VCU hydrobiologist has become more vitally necessary than ever and constitutes the number one priority in the Unit's recruitment activities. 4.4 Research on the vectorial capaci ty of S.souhrense-sanctipauli Because of the resistance it is capable of rapidly developing of its ability to colonize savanna regions, where it can transmit of 0. volvulus, S. soubrense has become one of the most dangerous Progrannne. There is as yet no proof that this species has taken advantage of the disappearance of temephos-susceptible species to extend its area of distribution, and all locations where it has been reported since the advent of resistance belong to to temephos and the 1oca1 strains species for the ocP |EACZ.4 page 10 its initial area of distribution The difference is that, through the effect of now accounts for up to 10OZ of the 1arva1 represented only IOZ at most; but there is no as been accompanied by an absolute growth in selection by temeP,hos, S. soubrense populations, whereas originall yit proof that this relative increase h numbers. When S. soubrense manages to survive through the dry season in a sudan-type savanna .uElor,-(l-er.t, and southern Mali, for example) the numbers of biting females captured on human bait do not seem to be corTrlnensurate with the pre-adu1t population densities. Under such dry-season conditions, in February 1981, appropriate larviciding (B. r. H-14) rid the L-eraba of S.soubrense for about two months. I,,rhen S. soubrense finds itself in comp etition with larvae of S. sirbanum (for examp Ie in south-western the pre-adultMali from May to August owing to the reinvasion , the balance of populations tilts very rapidly in favour of the savanna species. The prel S. soubrense tend to show iminary analyses conducted on the natural rates o populations by larvae morphologically indistingui that these rates progressively decline with incre f infestation of shable from O. volvulus asing distance from the zones usually colonized in abundance by S' soubrense (forest' pre-forest sector and guinea savanna), i.e. that they decline as one goes from south to north. These are sti11 only very preliminary data, based on approximate identification of S. soubrense and on small numbers of representatives of that species. More conclus ve resu1ts are expected from dissection operations underway in both the western part (Phase IV) and the area. They should make it Possi transmission entailed bY the Pre eastern part (extension study zones) of the Prograurne b1e to assess at the various seasons the risk of :S€DC€ of S. soubrense in marginal savanna foci where this is not the natural majority species. 4.5 Research on the mechan isms of resistance develoPment 4.5.7 Tests with synergists Although they do not offer a solution to the oPerational problems caused by resistance, synergists may help to increase knowledge of the enzyme systems involved in resistance. Specifically, the following synergists have been selected for tests with temephos: -pipe;rpnylbutoxide:inhibitorofmuttifunctionoxydasesl - ;ei'B) (triburyl phosphorotrithioate) : inhibitor of hydrolytic esterase - TpP (triphenyl phosphate) : inhibitor of carboxyesterases. preliminary experiments indicated that the maximum doses of these materials which do not cause mortality to resistant larvae are l.O mg/I for old larvae and O.625 mg/1 for young larvae. Very preliminary results indicater5{rat Piperonyl butozide has no effect or even a negative potentiation whilerwhen DEF@o. ipp were mixed with 0.O625 ngll temephos the mortality was 852 versus 5OZ for temephos alone' 4.5,2 Enzyme studies An investigation is underway of the phosphate esterases of adult flies from resistant and non-resistant areas and from Ivory coast before resistance occurred' This enzym.e system is likely to be involved in resistance. Preliminary results with electrophoresis indicate possible differences in both the types of isoenzymes Present and their activity' It is hoped that a sirnplified may come from the study. ocP lEAC2.4 page 11 staining technique for diagnosis of resistance 4.6 Impact of resistance on transmission of onchocerciasis In 1980 resistance resulled, wherever it appeared, in a flare-up of onchocerciasis transmission measured by the Annual Transmission Potential (ATP): e.g. on the lower Bandama in May and June (700 and 1 200 larvae/man/month at Chutes Gauthier), the Nzi (1 200 larvae from September to November at Ga16goua, nearly 900 aE F6t6kro) and the middle Marahou6 (f 300 larvae in September) at the end of the rainy season. These flare-ups were due to the fact that the resistant populations could not be reduced inrnediately, either because this was the first manifestation of the phenomenon (as on the lower Bandama in May and June) or because the replacement larvicide was not available in sufficient time (on the Nzi and Marahou6 in September- October). They were reflected in a universal and sometimes spectacular increase in the ATP, between L979 and 1980, on the lower Bandama (from 1OO to over 2 7OO at Chutes Gauthier and from 20 to over 3OO at Taabo), on the lower Nzi (from 3O to over 1 2OO at Gal6goua) and the middle Nzi (from 40 to over I O0O at F6t6kro), on the middle Marahou6 (from 20 to over 1 5OO at Danangoro) and on the lower part of the hrfrite Bandama (from 3 to over 2OO at Niakaramandougou), The other basins (middle and upper Sassandra, upper Marahou6, upper reaches of the White Bandama, upper Como6, and L6raba) have been little affected by the rise in ATP levels, which in 1981 signified a return to hyperendemic transmission on the lower Bandama, the lower and middle Nzi and the middle Marahou6, and a recurrence of mesoendemic transmission on the lower waters of the White Bandama. Note, however, that these changes in ATP have a clear epidemiological import only in the savanna foci (White Bandama, Marahou6 and middle Nzi), for in forest foci such as those of the lower Bandama and lower Nzi there exists not even a rough scale of concordance between the ATP values and the severity of the disease. In 1981 the flare-ups in intensity of transmission were due to several causes: total suspensions of larviciding due to exhaustion of chlorphoxim sEocks; the main such interruplion was from February to May and resulted in a renewal of transmission, fortunately attenuated by the dry season but difficult to overcome and perceptible in May on the upper Marahou6, the upper basin on the Sassandra and the Boul - temporary 1ocal suspensions for the purpose of economizing chlorphozim in period of shortage: F6t6kro and S6mien in July; - interruptions of chlorphoxim larviciding (in periods of shortage) followed by resumptions of temephos treatment to neutralize sources of reinvasion (upper basins of the Marahou6 and Sassandra in June and July) or to select temephos- resistant populations for research purposes (the same basins, plus Kan, in August and September); the very low efficacy of the batch of chlorphoxim used from May to July; lower Bandama, upper Marahou6, middle and upper Sassandra, Bou; the appearance of new foci of resistance which could not be irmnediately neutralized for lack of suitable larvicide: L6raba (January), lower Como6 (May). ocP lEACz.4 page 12 The effects of these various causes were very unequal and in reality only those locations subjected to the combined action of several of them showed a marked rise in their ATP values between 1980 and 1981 (up to Ehe end of August only): Kongasso on the upper Marahou6 (from 60 to nearly 7OO larvae), Bac S6mien on the middle Sassandra(from 45 to over 9O0 larvae), Vialadougou on the upper basin of the Sassandra(from 120 to over 5OO larvae), F6t6kro on the middle Nzi (which received during the single month of July 1981 the equivalent of two-thirds of the 1980 ATP). Ttre ATP doubled on the Bou (from 100 to 2OO larvae approximately) but the other reaches exhibited either good results similar to those for 1980 (upper Marahou6, White Bandama, middle and upper Como6) or spectacular reductions in the ATP: from 2 5OO to 25o at Chutes Gauthier, from 230 to 3o at Taabo, from 1 2oo to 9 at Ga16goua, from 1 5OO to 1OO at Danangoro (middle Llarahou6) despite several monthsr complete suspension of larviciding, and from over 200 to 20 on the lower waters of the l,trhite Bandama (Niakaramandougou). Note that the ATP on the L6raba in 1981 (141), like the t98O figure (172), is mainly attributable to the reinvasion, the difference corresponding exactly to the slight recrudescence of transmission which followed the advent of resistance in January. Putting together the results for l98O and 1981, the foci affected in at least one of the two years by a return to a transmission level considered as hyperendemic are the following: the lower Bandama, the lower and middle Nzi, the middle Marahou6 (upst.ream as far as Kongasso inclusive), and the middle and upper Sassandra: it must be stressed that in this lasE-named basin the ATP had never been brought below the threshold of tolerability of 1OO larvae/man /year owing to the proximity of untreated foci further to the west. The foci where transmission rose in at least one year from the hypoendemic level reached ia L979 to a mesoendemic level are: the lower waters of the White Bandama, the Bou and the L6raba (mainly because of reinvasion in the case of the L6raba). The foci where transmission remained throughout at a satisfactory and tolerable level are: the upper Baou16 and upper Bago6 (not affected by resistance), the upPer basin., of the Marahou6, the middle and upper hhite Bandama, and the middle and upper Como6.- Comparing two years 1980 and 1981 (from March 1980 to August 1981 inclusive), it is clear that the Progrartrnets activities have everywhere paid off whenever it has been possible to treat the foci concerned punctually and regularly with an efficacious formulation of chlorphoxim; the fai.lures recorded in 1981 are the result of deficiencies in the availability and in the efficacy of this replacement larvicide. This proves the appropriateness and effectiveness of the Programmers treatment strategy 5. Outlook for the future Apparently resistance to temephos is a phenomenon now well established in the proprarmre region, genetically well adapted to the environmental conditions and capable of persisting even in the absence of continuous insecticide pressure. 1 It hardly needs stating that only the Ivory Coast foci are considered here, resistance not having yet been reported beyond the boundaries of that country ocP |EAC?.4 page 13 If this resistance remains restricted to S. soubrense-sanctipauli and if this species does not colonize new habitats opened up by the disappearnace of the savanna species susceptible to temephos (as seems to have been the case so far), the theoretical limits of its extension within the treated area would be those of the present distribution of S. soubrense-sanctipaulil in that case they would already have been attained i. ri6-iT-TIEE"t tt"t-ttr" smaIl northern foci must be considered as potential foci of resistance where the S. soubrense populations are too smal1 and too ephemeral for the phenomenon to have yet been demonstrated. It is difficult to predict how resistance will spread and develop westward and eastward in the absence of selective treatments. If, on the other hand, routine larviciding with temephos is instituted in the foci of these regions where S. soubrense-sancti pauli is well represented, the risk that these treatments may shortly lose their effectiveness is real. Resistance does not seem to have spread to other species and the fact that phenomenon has not developed in the savanna forms after more than six years of intensive larviciding might imply that the risk of its appearing among them is within the initial Prograrnme area. Nevertheless, the finding recently recorded the Phase IV area on the Kan at Ass6r6kro (Test No. LLZ; see section 3.3.1.2) that extreme prudence and vigilance are required. the s I ight in shows In any case, it is clearly more than ever necessary to continue: - monitoring of susceptibility to temephos and other insecticides, in accordance with the protocol instiruted in 1981; - research on larvicides, which needs to be considerably intensified and extended in regard to screening of new formulations and new products, mode of absorption and action, and mechanisms of onset and development of resistance. To this end, a specialized team is being constituted within the Programmers Vector Control Unit; the distribution of insecticide research assignments will have as soon as possible to be redetermined with VBC, TDR, OCCGE and any other specialized bodies concerned. A very special effort will have to be undertaken with a view to the improvement of B.t. H-14 formulationsl - research on the vectorial capacity of S. soubrense-sanctipauli in the different bioclimatic conditions prevailing in tr,u Zi-r,. or r""istarrc" to temephos. rt would seem at present that the more northerly the latitude, the lower the vectorial capacity of the species (owing to the smallness and instability of the populations, their apparent zoophilia and their low natural rates of parasitization by 0. volvulus). These preliminary data must, however, be supplemented and followed up regularly by specialized teams of the Programme, in cooperation with those of IRTO, in view of the fact that the situation has certainly not attained a state of equilibrium in terms either of ecclogy or of host-parasite-vector adaptation. The stability of the resistance and experience in 1981 make it illusory to think in terms of a strategy based on alternation between temephos and a replacement larvicide in a focus of temephos resistance. At the same time, experience in 1980 and 1981 show that when an effective replacement larvicide is available, neutralization of the resistance is perfectly feasible. In 1981 the strategy was based so1e1y on the use of chlorphoxim as replacement larvicide. But apart from the risks of adverse side-effects on the aquatic environment, it has become clear that the formulation now produced is far less active than the previous formulations. ocP /EAC}.4 page 14 The need to have another replacement product available has therefore become particularly urgent. The only one that looks at a1l promising at present is B.t. H-14, and it is planned to use it operationally in all foci of resistance in the Ivory Coast starting next dry season. Unfortunately, the active ingredient content of the only active formulation at presenE available industrially is insufficient to make its utilization practicable in the rainy season with the aerial logistic means at Present available to the Prograrrne. Unless in the meantime a B.t. H-14 formulation richer in active material were to be developed, it *rst thereToE be anticipated that larviciding in the 1982 rainy season, as in 1981, will have to depend on the use of a chlorphoxim formulation whose shortcomings are manifest. There is hence a simultaneous need to intensify, diversify and accelerate research on B.t. H-14, to strengthen the logistics of aerial operations in the rainy season so as to achieve optimum effectiveness, and to maintain and strengthen monitoring of the aquatic invertebrate and fish fauna of chlorphoxim-treated watercourses rl 6l o{ '-l 1! Otr'^ ,!, o olD llLH 2lrrlol tr! ll a 4l . =t-al -!, .l !.(,l ft qi ! 0.i .i oB 0Jo o 0,)0Jilr cd o.E 0.) OJlL a)o'o!r! .d (d a oE o. 0le o) +J o! O) .ior i0J .iE -o c) ! AO o+l Uoo AQ d $o liooc)z& <a o d a I 0) a tr 0)! o a U) qi o a o .i ! -o, .i ti! a .i X ! oti o -E U E ]J B 0) d 0) ti u a ti q) I at I tr ot (.)xtr0,o Nqi o> !, EO ! 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