Report on the scientific advisory panel working group on rearing simulium spp and establishment of a laboratory colony of simulium damnosum

tWORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE ONCHOCERCIASIS CONTROL PROGRAMME IN THE VOLTA RIVER BASIN AREA SCIENTIFIC ADVISORY PANEL (SAP) Geneva 20-23 October 1975 REPORT ON TTIE SCIENTIFIC ADVISORY PANEL WORK]NG GROUP ON REARING SIMULIUM SPP AND ESTABLISHMENT OF A IABORATORY COIONY OF SIMULIUM DAMNOSUM CONTENIS LIST OF PARTICIPANTS INTRODUCTION REARING SIMULIUM SPECIES OPEN SYSTEM TROUGH REARING - DR H. JAMNBACK CLOSED SYSTEM REARING OF S. DAMNOSUM ("KIBWEZI'' AND ''KISIWANI'' CYIOTYPES) IN EAST AFRICA - DR J. GRUNEWALD . CLOSED SYSTEM REARING OF SII-IULIUM SPECIES GEORGIA, USA - DR R. TAYIOR INCLUDING S. DAI'O{O SUM S .L. IN ATIANTA, 6 7 8 CIOSED AND OPM{ SYSTEM REARING OF SIMULIUM DISTRIBUTION OF SIMULIUM DAMNOSUM SPECIES GROUP IN THE VOLTA RTVER BASIN AREA - SPECIES ]N C,AIIADA AND S. DAI'T{OSUM S.L. AND OTHffi. SIMULIUM SPECIES IN I4TEST AFRICA - J. IIDKRY OCP/SAP/76. I ORIGINAL; ENGLISH Page 2 3 3 4 t 2 3 4 5 4 5 6 6 7 8 9 DR C. G. VAJI]'IE NATURAL REQUIREMENTS OF THE IMMAI'I]RE STAGES - PHYSICO-CHEMICAL PROPERTIES IN TITE WATER - DR J. Gts.I.]NEWALD 9. PROBLEI.{S ASSOCIATED WITI{ DEVELOPING A SELF-PERPETIJATING COIONY 10. RECOMMENDATIONS ANNEX: AGENDA 1l The rssue of thrs document does not constltute formal publrcation. lt should not be revtewed. abstracted or quoted wrthout the agreement of the World Health Organtzatron. Authors alone are responsrble for vrews expressed rn signed artrcles. Ce document ne constrtue pas une publrcation. ll ne doit farre I'objet d'aucun compte rendu ou r6sumd ni d'aucune crtation sans l'autonsatron de l'Organrsatron Mondiale de la Sant6. Les oprnions expnmdes dans les artrcles srgn6s n'engagent que leurs auteurs. ocP/sAP/76.L Page 2 LIST OF PARTICIPANTS Dr J. Grunewald, Tropenmedizinisches lnstitut der Universitat, Wilhemstrasse 11, 7400 IUbingen l, Federal Republic of Germany Dr H. Jamnback, Director, State Science Service, The University of the State of New York, The State Education Department, Albany, New York L2224, United States of America(Rapporteur ) Mr J. Mokry, Research Unit on Vector Pathology, Memorial University of Newfoundland, St. Johnrs, Newfoundland, Canada AlC 557 Dr R. Taylor, Scientist Director, Vector Biology and Control Division, Bureau of Tropical Di.seases, CenEer for Disease Control, Atlanta, Georgia 30333, United States of America WHO Secretariat Dr C. E. Curti-s, Geneticist, Division of Vector Biology and Control Chief, Simulium Control Operations, Onchocerciasis Control Programme,Dr R. Le Berre, Ouagadougou Mr J. D. M. Marr, Entomologist/Biologist, Onchocerciasis Control Progranrne, Support Unit, Geneva Mr G. Qu6lennec, Entomologist/Chemist, Onchocerciasi-s Control Programme, Support Unit, Geneva Dr C. G. Vajime, Cytotaxonomi-st, Onchocerciasi-s Control Progranune, Ouagadougou Dr A. R. Zahar, Entomologist, Division of Malaria and other Parasitic Diseases Dr B. O. L. Duke, Senior Medical Officer, Division of Malaria and other Parasitic Diseases and Dr E. Onori, Epidemiologist,Onchocerciasis Control Programme, Support Unit, Geneva attended part of the discussions ? OCPISAP/16.L Page 3 I. INTRODUCTION The establishment of a colony of one or more species of the Simulium damnosum complex would be a tremendous asset to the study of this vector and would oPen uP the possibility for many i-nvestigations to be pursued in the laboratory, rather than exclusively in the field, and also provide greater scope for research in institutions outside, as well as inside, the onchocerciasis endemic areas. It wouId, among other things, provide larvae for the laboratory testing of new insecticides and ne\,/ susceptibility testing methods, adults for observing the reaction to different attractants for the development of suitable trapping devices, and it could be a step towards establishinB a laboratory model, Lf a counterPart animal host can be found, for transmission studies, drug testing, etc. While a measure of success has been recorded with S. decorum and S. vittatum in America and B. erythrocePhala in Europe, no colony has been esEablished with a vector of onchocerciasis, although significant advances have been made with S. damnosum ("Kibwezil'cytotype) in East Africa. The development of single generations of the West African species of the S. damnosum complex, which goes part of the way towards the establishment of a colony, would assist in some aspects of this research, as well as providing related eggs, larvae, pupae and adults for species identification by morphological methods. It is, in fact, a matter of urgency to develop a satisfactory system for this purpose in early L976 to assist in the study of the S. damnosum s.l. appearing during the rainy season in the periphery of that zone of the Onchocerciasis Control Programme under treatment. One of the main problems to be overcome in evolving either systern is the collection, storage and transport of material, wheEher eggs, pupae or gravid females. The greater thedistance, the Sreater the risk, and, therefore, initial effort in the development of singlegenerati-ons must be Promoted in West Africa. Those institutions, however, already engagedin colony work elsewhere should be encouraged as much as possibre. The report of the second meeting of the Scientific and Technical Advisory CommiEtee (STAC) emphasized the need to pool knowledge of advances which had been made in these fields.Accordingly, a Scientific Advisory panel (Snp) working group was convened for that purpose j.nGeneva from 20 to 23 october 1975. This report of Ehat meeting reviews the work carried ourby some of the ParticiPants. rhe main problems are highlighted and recommendaEions made onthe most satisfactory manner in which research should be undertaken to solve them. Because of the differences, knovrn and apparent, beEween the va rious species of the the Onchocerciasis ControlS. damnosum complex, a resum6 of their distribution in relation EoProgramme is also included. 2. REARING SIMULIUM SPECIES There are basically thTo methods of rearing Simulium water that passes only once through a trougt, .o,EG[ sysEemrr, in which the \^/ater is recirculated. : an 'ropen sys temr' , i. e. running the developing stages, and a ilclosed In the open systen, water from a lake or pond runs into the Eroughs, preferably gravityfed. An advantage of this system is that the wat er contains nutrients so the Iarvae do notrequire feeding. Also, in this system which closely simulates natural conditions, there isdanger of losing larvae because of power failur e, pump failure, overfeeding, etc.noHowever, iE has the disadvantage that the \,nater quality is difficult to alter, a factor whichmay be of importance in rearing some species of the S. damnosum comp lex. A closed system, which invariably relies on electric pumps to operate it, requi-res nuchmore attent'ion in so far as the larvae must be fed the correct quantiE.y of food at establishedintervals, the v,ater quality must be mainEained, filters, if used, must be cleaned, etc., butit has the distinct advantage that E.he r"rater quality can be adjusted to meet the needs of anyparticular species. OCP/SAP/76.I Page 4 3. OPEN SYSTEM TROUGII RL{RIIiG - DR H. J,:rti-}!CK This system is used routinely at the Canbrroge, Ner.r York, Iaboratorr,. Trventy-fcur \,rooden troughs, each about 3 ft long, I ft wide and 8 in deep, are arranged in four parallel ro\r's of six each. Water from a spring-fed pool flows into one end of each trcugh t.hrcugh a fauceE and out over a lip 6 in rvide b5, 4 in long. This acts as a miniature spillway. Fiel-<i collected larvae, or larvae hatching fror,r eggs placed in the trough, mierate within a few hours to the lip of the outlet. Water florus over them in a thin film so that it is possible to observe in detail, through a binocular microscope, the actions of the larvae. This has proven to be especially useful in observing the different responses of larvae to various insecticides and formulations, and in determining the method of transmission of various Simulium parasites, The system requires no maintenance or handling to rear Large numbers of Simulium larvae other than pl-acing:he eggs or larvae in the trough. SYSTEM REARII'IG OF S. DAMNOSU}I (I'KIBWEZIt' AND ''KISIWANI'' CYTOTYPES) IN EAST - DR J. GRUNEWALD A rearing technique, with a closed sys:em in which the condition of the water may be regulated, has been developed and used very successfully for rearing B. erythroc epha la in Europe. The main feature of the methoC is that the pH value, the conductivil-y and the ionic balance of che culture \,rater are adjusted anc melntained at similar levels as occur in t'he natural breeding habitats. During the rearing process, the culture \rater is poliuted by metaboiic b1,-products of the larvae and by decaying food particles. Since most bLackfly larvae are l./err sensitive to L,ater pollution, especially to a high ammcnia ani nitrite content, the culture water req,uires purification. The apparatus therefore consists essentially of a culture chanber anci a purification system through which the waLer ls circula:ed. Water from the culture chamber passes thr:ough a gravel filter a:'ld an activated charcoal- filter before returning io the rearing vessel via a container r;ith the s.rc;lergei rccts of a Monstera species (Araceae), These roots renove various ions, such as excess phosohates r"ETiEQ"n "or"porr[- Ln the original design used for D. erythrocephala, a \rrater current ie a five litre jar is prociuced by a magnetic stirrer. This apparatus has proved to be successful for rearing Iarvae of certain East African species, i:rcluCl:g the "Sanje" form of the Simuliun da:li:csura ccrplex. However, using this apparatus, oni., :-i- c f the eggs of I'Kisirnzani'r could be reared to the adult stage. trKibwezi" larvae alvrays failed to develop in this system. Iarzestigations with these trvo Iatter sp ecres oi :he s. damnosum complex demonstrated the need ior ceriain modifications to provide the kind oi waier flow anci Eurbulence they require. A 'rodified apparatus, which uses a gutter anC a centrifugal pump (flow rate: 1.56 r3,/sec)i-- piace of the glass jar with the magnetic stirrer, has so far been used successfuili, with both "Kisirueni!! (ca. 55% adults from egzs) and "Kibr,,ezi" (ca. 23% adults from eggs). The "Kibi,zezi" form of the S. damnosum complex has novr been successfully induced to conplete every stage of .ts llfe-.y"IGEe ta-boratory (Raybould & Grunewald, Lg75).1 so far, it is the the S, damnosum complex knor"'n to nate in the laboratorv, not onlv in iarge cages ;:t aiso in small tubes. Nevertheless, certain problems remain: it rs onlv ruiLh iifficulty that feirales can be induced to blood feed on guineafowl, chicken or the ears of rabbits and : large proportion of the eggs laid fail to develop. The proportion of fed females vdried .lron 0 to 4O%. The feeding of iarales with a bLooci-sucrose solution gave better results and in il crials 96.L7" of the females took bloodr but again the eggs failed to develop. ?he establishment of a laboratory coloni'cfrrKibwezif'would seem to be a reai possibility in the not too distant future, provided difficulties connected with blood-feeding and egg-der.relopment can be overcome, but iL is not a knor,rn vector of onchocerciasis. CLOSED AFRICA c-. l1' form of I Raybou ld , J. N. & Grunewald, J. (L975) Tropenmed. Parasit., 26, 155-168 OCP/SAP/76. I Page 5 5. CLOSED SYSTEM REARING OF SIMULIUM SPECIES INCLUDING SIMULIUM DAMNOSUM S.L IN ATIANTA, GEoRGIA, USA - DR R. TAYLOR Preliminary studies with Ewo North American Simulium species, S. vittatum and S. decorum, in Atlanta, Georgia, USA, indicated E.hat these two species could be handled at least through one generation in a laboratory located there. Aquaria with airstones h,ere used for rearing the field collected late instar larvae to adults. S. vittatum fed on shaved stomachs of rabbits. Adults of this species placed in a mating cage after feeding laid egg masses and a few lst instar larvae emerged but did not survive. It was clear at this juncture that equipment for larval rearing would be required that would simulate normal stream flow. A recirculating aquarium with a 170 gal capacity(a "Iobster tankrr), was acquired and a trough to simulate a sE.ream bed was added \^ras used in this equipment because of availability at the time of acquisition. S. decorum Late instar larvae and pupa e of S. decorum were field collect.ed , aS \^7efg S vittatum This field material was placed in small caged aquaria and emerging adults were collecLed and placed in a mating cage with a small pan of r^rater aerated with an airstone. One inch plastic squares were placed in the water for egg laying. Adults were offered sucrose pads, heparinized beef blood sponges and fresh calfts Ilver. Mating occurred and egg batches were collected, counted and placed in the sirulated stream bed of the large aquarium. Aged tap water in this aquarium had been inoculated over a four-week period with approximately 15 gal of water from the field collection site. Finely ground dog t'chowrr (of low fat content) and lab 'rchowr' (of medium fat content) were added daily beginning four days after egg set, at which time lst instar larvae were observed. Feeding continued until pupae began to appear approximately 10 days after egg set. Adults began emerging on day 11 and conEinued through day L4. A I: I ratio was observed in the nearly 400 adults collected. These adults were placed in the mating cage, offered the same feeding media as before and eggs were again collected, set up in Ehe large aquarium and reared through in the same manner as before. Eggs from these approximately I8O adults produced only one male and one female. Two shipments of S. darnnosum s. I. eggs were placed in the large aquaritln for rearing The first shipment t.oi:t "-T6.ra-n-6 area of lvory CoasE was treated as was S. decorum and yielded approximately 80 adults in a l4-day period, the sex ratio being I:1, males to females These died within 72 hours although offered the same conditions as S. decorum adults. The second shipment of 42OO eggs from the Ouagadougou area of Upper Volta did not develop although transported from field to laboratory in under 24 hours. They were found to have collapsed after a few days in the aquarium. Ihe above Simulium rearing equipment held a balanced pH of 7.5 and a temperature of 24'-25oc or". "lffiEof seven months and showed no evidence of fouling. While otherhydrochemical factors were not studied in detail, it appears that they remained within the growing limits of both S. decorum and at least one species of the S. damnosum complex. It therefore seems teasiUtffiGiffnue with this equipment to test tf,;-;;;tinued rearing potential of cycled S. decorum and first generation S. damnosum s.1. until Ehis tar get species is cycled. Subsequent studies with this equipmenE should test larval loading Several 4t' x 1.4't stream bed aquaria have been constructed to resemble the L4tt x 72tt large aquarium stream bed. Preliminary studies indicate that larvae will attach and react much the same way as in the large aquarium, Fouling occurred within 12-16 hours, however, and no adults were reared through in these smaller units. Reservoir, filtration, pumping and plumbing equipment which is planned should make it possible to operate up to 48 of these units in groups of three or four from the same reservoir. This may make possible the rearing of from 12 to 16 different Simulium species concurrenEly. These units may be adapEed for field use as they are lightweight and portable. 6ocPlsAP/76.L Page 6 CLOSED AND OPEN SYSTEM REARING OF SIMULIUM SPECIES OTIIER SIMULIUM SPECIES IN i,JEST AIRICA - J. }'trOKRY IN CANADA AND S. DAMNOSUM S.L. AND A. Canada Although bubblers have been found useful in studying certain asPects of the larval biology of S. venustum such as number of instars, effects of temperature on grow-th and effects of populatiol de.sity, the system is unsuitable for colony work because of low iurvivaL (2L%), sma11 numbers of larvae/adults per cylinder and because adults emerge over an extended period. A trough has been constructed which is suitable for mass-rearing attemPts but the weak link is retiance upon a pump and a constant supply of electricity. Larvae have been reared from egg to adult in large numbers but no precise data are available at this Eime. l,trating of the adults in 250 ml jars with a L/2" Layer of water-soaked plaster has been observed in Prosimulium mixtum-fuscum. Females were provided wirh liver and apple slices. The use of mating cages and liver proved unsuccessful in inducing nating in L_g1!1g, S. decorum S. vittatuff, S: verecundum and S. tuberosum. Blood-feeding was successfully accomplished with aIl of the above Simulium species when offered the ear of a rabbit. S. vittatum gave especially good and consistent resulLs. All takefailed to feed on the shaved sEomach of a guinea pig. Ovarian development was seen Eo place in fed females routinely dissected to examine the spermatheca for matinS. B. Africa An rropen-endt' trough using water from a tap has been constructed in Bouak6 and has shovrn some promise. First, second and third instar larvae of S. unicornutum, S. alcocki and S. adersi have been observed to moult and increase in size with a mi-nimum of drift. The t.o"gtr tr"r 25 small barri-ers along its two metres length which serve as convenient atEachment sitesfortheIarvae.Ihesystem'however,remainstobetes!edon@s.l.(Tap warer analysis proved to be hydrochemically very similar to breeding site r^/ater. ) A technique was established for obtaining viable eggs from field collected, blood-fed S. damnosum s.1. adults which showed very marked improvements both in the carriage and holding of females over the use of small tubes, The blood-fed females were transPorted to the laboratory in 250 m1 containers with a wet cottonwool lining. Females were held in the laboratory in a large (80 cm x 25 cm x 25 cm) black cloth cage wetted daily with a 5% sucrose solution. A petri dish half filled with water and with strips of filter Paper was placed in the cage after five days and females (13 out of 63) oviposited naturally on these. Furthermore, females were left in a condition sui.table for further study or for subsequent blood feeding. Plans are being implemented for combining this system with the trough system described above. 7. DISTRIBUTION OF SIMULIUM DAMNOSUM SPECIES GROUP IN T}IE VOLTA RIVER BASIN AREA - DR C. G. VAJIME Eight species are known in and around the Onchocerciasis Control Progranrne area are listed below \^rith their distribution: These S. squamosum S. yahense S. soubrense S. sanctipauli S. damnosum S. dieguerense S. sirbanum ("Billet') ( t'Yah" ) (rrsoubrer') (rrBandamarr )("NiIe") (I'Dieguera") ("sirba") ("sirba"I ) ) ) Predominate in the forest S. sudanense PredominaEe in the savanna ) ) ) ) ) ocP/sAP/76.L Page 7 S. dieguerense and S. sudanense are relatively rare and do not need to be considered at the momenE. This distribution of the S. damnosum species grouP is, to a very large extent, related to the two main climatic regions, i.e. forest and savanna' In nature these regions are type condition in the forest zon region. In the same waY, it wo ones are known exclusivelY from associated with the forest onlY not absolutely deunrcated. There are pockeEs of savanna- e and pockets of forest-type environment in the savanna uld seem, neither the forest-loving species nor the savanna the reg ions they are associated with. Among the sPecies S. squamosr:m, S. soubrense and S. damnosum have been found in the savanna, within the control area. In the control area, S. damnosum and S. sirbanum are the dominant sPe S. damnosum extends n forested area is not affinity for such drY a rcies, in the north as far north as evident in the reas and clearly and south respectivel-y with a considerable overlap' Koni and Bandiagara in Ma1i, but its high density i north. It is S. sirbanum that shows a Persistent predominates in the northern Part of the control area. A11 the species exceP t S. yahense breed in large rivers. S. yabenle breedsS. sirbanum shows a strong Preference for in creeks extrerneand small tributaries of large rivers savanna condit.ions. The correlation of known foci of Ehe species with endemic areas of onchocerciasis suggests veryStrong1ythatS.squamosum,9@andS.sirbanumaregoodvectors.This observatio., reqrire?-a- direct .onfirmition. It is also imPortant Eo determine the vectoral capacities of the remaining species, namely S. yahense , S. s4ne tipauli and S. soubrense. If it can be proven that Ehey are definitely non-vectors of onchocerciasis, any future migration of such flies into the control area would cause no concern. In this regard among others, the breeding of the S. damnosum species group under controlled conditions is of uEmost, practical importance. Then it should be possible to define Ehe vectors more precisely. Some areas, where apparently unmixed larval populations of these species occur, are listed below: Species Location S. squamosum S. damnosum S sirbanum S. sanctipauli S. soubrense Kint.ampo, Ghana Asukawkaw, Ghana Bandama/La f igue con f luence, Korho go Ivory Coast Vea dam, Ghana Nagbangre dam, Upper Volta Sotuba dam, I,IaIi St PauI River, Liberia Sassandra River at Soubre Ivory Coast NATURAL REQUIREMENTS OF THE IMMATURE STAGES DR J. Gts.UNEWALD PHYSICO-CHEMICAL PROPERTIES ]N TIiE WATER - In the hope of establishing a colony of a species of the Simulium damnosum complex, Ehe water conditions were first. investigated in a range of breeding sites in West. and East Africa The investigation has revealed a number of notable differences amongsE these breeding sites, 8 ocP/sAP/76.1 page 8 particularly with regard to the pH va1ue, the conductivity and the concentration of certain ions. 0n the basis of differences in pH and conductivity alone, the various species can be divided into three main groups. Group I sanctipauli, S. yahense and ":S Mengenserrcytotype These three have been found in water courses of the West African tropical rain forest zone. Their breeding sites are characterized by an acid water with a pH value below 7 and a conductivity al$rays below 50 /rmhos. Regarding the ionic composi.tion, S. sanctipauli andS. yahense larvae were alwa ys found in water with a high pot.assium sodium sulfate content 'rMengense" was found in a magnesium carbonate water. Group II: S s irbanum S. sudanense S. damnosum S. squamosum and the rrSan j er' , "Nkusi" , rrNyamagasanitr, rrJoviil cytotypes This group includes species and cytotypes of the tropical rain forest zone, of the savanna zone and of mountai.n areas. The reaction of the water of the breeding sites of these species was neutral, weakly acid or weakly alkaline. The conductivity ranged from 50 to l5O7umhos. There were few differences in ionic composition: S. squamosum occurred in r^7ater high in magnesium carbonate. The water of the breeding sites of S. sirbanum S. sudanense and S. damnosum was similar but with a proportion of calcium to magnesium of nearly one to one. The East African cytotypes I'Nkusirr, rrNyamagasanitr, 'rSanjerrandrrJovirrwere found in r^7aEer with a high concentration of potassium sodium carbonate, withrrSanjetrsites having also a similarly high concentration of calcium magnesium chloride. Group IlI: I'Kibwezi" r t'Kisi.wanirr cytotypes The hydrochemical conditions of the breeding sites of these two East African species were very distinctive. The breeding sites of both are characterized by high alkalinity with pH values between 8 and 9, and very high conductivity values between 4OO and 5O0fmhos. The ionic composition of the sites of these t\,/o species, however, was very distinctive. While "Kibwezi" was found in potassium sodium carbonate water, rrKisi-wanirroccurred in sites with potassium sodium chloride. 9, PROBLEMS ASSOCIATED WITH DEVELOPING A SELF-PERPETUATING COLONY Although every stage of the life cycIe of S. damnosum I'Kibwezirr cyto type has been reared l;l the laboratory, and B. erythrocephala reared thr ough several generati-ons a self-perpetuating colony of any S. damnosum complex species or cytotype has yet to be achieved. The obstacles lie boEh in obtaining stable hydrochemical conditions and in inducing the adults to feed on -riood, mate and lay eggs in captivity. Factors to be considered in developing suitable and stable hydrochemical conditionsjnclude a Iarge regenerative capacity. This includes large reservoirs, charcoal, gravel or other kinds of filters, etc. It is necessary to remove ammonium nitrites and nitrates p-roduced by the larvae, as these in high concenErations are toxic. As the larvae are filter feeders, a current must be set up. In the case of S. damnosum species and cytotypes, high florv rates are usually required in the order of 1.5 m/sec. The conductivity, which is a measure of the concentration of ions present, should be in the range of 50-150 micro-pmhos. The pH for most species and cytotypes is near neutral in the 6.5-7.5 range. Water hardness is also a factor, but the limits have not been closely defined. Less important is temperature, which should be in ilne 22"-28oC range for S. damnosum complex species. Light ;na-v be a factor, but it has not been sysLematically studied. It should be noted that some plastics used in rearing systems may be toxic to the larvae, e.g. PVC with softeners. Collection, storage, transportation, rearing and maintenance of the different stages also pose problems. It is usually not difficult to collect eggs Iaid in breeding sites in tl-re field. They should be collected and cooled inrnediately after they are laid, usually at i,-r sk , in the case of some S. damnosum complex species. Methods for storage and transPortation OCP/SAP/ 76. 1 PaSe 9 require further study, especially the possibility of freezing or cooling eggs' to inhibit or stop their development temporarily. The use of CO2, nitrogen or aEmospheres low in oxygen might also inhibit development. Larvae although sensitive to unfavourable conditions and difficulE to store or transport, can readily be reared either in open systems where the water passes through once, or enclosed systems where the water is recycled. As mentioned above, some species may require a high rate of flow. Feeding is not a problem. Ordinary dog meal or laboratory foods, finely ground liver powder or yeast, have been used successfully. It is imporEant, however, not to provide too much food as this witl change the hydrochemical conditions of the water and will tend to develop mould which, in turn, can attack the larvae. The pupal stage has not been employed extensively in transporting Simulium species for subsequent colonization. However, they may be useful for this purpose as the pupa is resistanE Eo adverse conditions and some species have been kept in the refrigerator for as long as four weeks at I.7'C, emergence taking place three or four days after they are removed from the refrigerator. Adults can be used as source material for a self-perpetuaEing colony but are difficult to transport long distances. They can be maintained in the laboratory either in cages or small vials if they are provided with a source of sugar. Apple slices work particularly well. l"Iany workers report thaE these adults live longer in the dark. In all cases, handling of adult Simulium should be minimized to avoid high mortality rates. Some species will feed directly on drops of blood, or blood on sponges. Others will feed on blood mixed with sucrose solution. S. vittatum, B. erythrocephala and S. 4erES!"m rrKibwezirrand "Kisi-wanirrcyEotypes, will feed on rabbit ears, but others rarely can be induced to feed on blood in any form in captivity. Blood-fed females of some species will lay eggs in small vials or cages on moist filter paper or small pieces of ptastic floating in water. Sorne females that will not lay eggs normally will do so if decapitated or exposed to C02. One of the chief difficulties in developing a self-perpetuating colony is mating. Although adults mate freely in nature, j-n capEivity many species are reluctant to do so. The adults of S. damnosum ItKibwezi-t' cytotype are cage copulating in the laboratory. Exposure to S. decorumC02 gas may also induce mating. Fresh bloody calf's liver is fed upon readily by and appears to be a fact.or in inducing mating. It will probably be necessary to take these facEors into account and test thern in various combinations in order to develop self-perpetuating colonies of one or more of the different species or cytotypes of the S. danpoqqm complex. 10. RECOMMENDATIONS The Onchocerciasis Control Programme requires information pertaining to the idenEification of species of the S. damnosum complex, development of insecticide resistance and factors influencing the effectiveness of insecticide formulations. It also needs a laboratory model for Eransmission studies and for Eesting the effectiveness of rranti-volvulus" drugs. These aII require rearing of Simulium species in the laboratory. To achieve these objectives, the Working Group makes the following proposals: (1) In regard to obtaining eggs for rearing, the Group recouunends that blood-fed females of the West African S. damnosum species be collected in the field and procedures developed for obtaining eggs from these adults This, together with the collection of eggs laid in natural breeding sites, is a necessary first step for reari-ng larvae for taxonomic identification and for rearing adults for morphological comparison with other species of the S. damnosum complex The Working Grolp recon*ends that th , precedence being given to 9. qq,faqo!9a, S. oncho cerc i.a si s. damnosum and S. sirbanum which are implicated in the transmission of ocP/sLPl76.L page 10 (2) In regard to S. damnosum breeding habitats, the Group recommends a detailed investigation of water quality in breeding sites in West Afr19.a popr.rlaqed by Ll@ s.I., as it relates to species distribution. The hydrochemical characteristics of water in which S. damnosum s.l. vectors breed should be duplicated in laboratory rearing systems. The Working Group recommends that this surve be rnade as soon as ssible (3) In regard to rearing single generations of the S. damnosum complex, the Group reconrnends that a field station be established in West Africa to rear immature stages so that particular S. damnosum species associated with the control areas can be identified. Although the adults cannot be separated at this time, Iarvae reared from eggs obtained from blood-engorged females can be identified cytogenetically. The field station should be located outside present and projected control areas, but accessible to them, and situated so that \"rater from a large Iake or river can be fed to it by gravitl'. Initially, an oPen system should be used for rearing. However,a separate closed system (preferably portable) should be developed for use in cases where special T.rater characteristics are required. The field station should begin operation during the first quarter of L976. be put in charge. The l,rlorking Group reconrnends that an experienced research worker Once established, this field station could be used to provide larvae for susceptibility testinq. It is important to have such tests carried out at one location to assure ccrnparabilit;'of data. In addition, the field station could later be used for testing rnsecticicie formulations, parasite transmission, marking methods for flight range studies, etc. (+) in regard to transportation of eggs, pupae and adults of S. damnosum s.1., the Group recomrnends an investigation of methods whereby these stages can be maintained alive for eriods sufficient to ermit their safe shi t to collaborat laboratories involved in research on laborat colonization. The Wor Gro recommends that this sEud be irnp lemented immediately, (5) Ln regard to Simulium behaviour in captivit the Group reconrnends studies of longevity nating, blood-feeding and oviposition, it being recogni-zed that some species of the S, dannosum complex may require special procedures for colonizatLorl (5t In regarC to establishing a S. damnosum s.l. colony, the Group notes that recomnendations(1) to (5) are alI elements supporting this objective. Meanwhile, live S. damnosum s.l. staqes should be shipped on a continuing basis to collaborating research agencies to expedite develcpment of such a colony. Laboratory colonization will make it possible to apply selection pressure for studying insecticide resistance (including cross-resistance) and will aid i:r predicting ruhen, how or if resistance will develop during the course of the Orlc'hocerciasis Control Programme. Rearing would assist in research on marking methods for iiieht range studies. Colonization, among other things, may also make possible development of a iaboratory model of onchocerciasis transmission, useful in research on chemotherapeutic agents; an effective and safe drug would reduce the duration of the 0nchocerciasis Control Prograrme. Other research on biological parameters directly applicable to the control canpaiqn cculd be carried out in research laboratories where colonies are established. IOCP/SAP/76.L PaSe 1l ANNEX AGENDA 1. Rearing of Simulium spp. in America - Dr H. Jamnback 2, Experience in rearing Simulium in Canada and Africa - Mr J. Mokry 3 The use of a lobster tank in developing a colony of S. decorum and experiences with S damnosum s 1. - Dr R. Taylor 4. The development of a laboratory colony of S. damnosum (ttKibwezi.rt and rrKisiwanirl cytotypes) in East Africa and experience with West African cyLoEypes - Dr J. Grunewald 5. Distribution and characteristics of different cytotyPes of S. damnosum - Dr C. Vajime Natural requirernents of the lnurature stages - physico-chemical propert.ies of the water, speed of current, nutrition, etc. - Dr J. Grunewald 7, Problems to be overcome: maintaining stable water conditions in rearing tank; collection, storage and transport of eggs, pupae and gravid fanrales; feeding of larvae; maintenance of adults - captivity; feeding of adults; mating; ovipo siting 8. Systems reconrnended to be tried 9. Cytotypes of S. damnosum or other Simulium sp. - order of preference 10. Rearing of single generations only 11. Prograrune reconrnended { 6 a