Cytotaxonomy of S.damnosum complex: January- December 1995

.)' i'. li. a T .+ t WOR.LD HEALTH ORGANIZATION AFRICAi{ nEGION SUBJECT PERSONNEL ORGAI{ISATION MONDTALE DE LA SAI{TE REGION DETAFruQUE 1995. OMS ' ONCHO, B.P. 36 KARA, REPUBLIQUE DU TOGO TEL.60.60.75 Cytotaxonomy of S.damnosum complex. George K. Fiasorgbor (Technical Officer) S6dou P. Naniogde (Laboratory Technician) I DecerfiberiPERIOD COVERED : January - SIGNATURE OF RESPONSIBI# OFFICER 1,oe,3L -4 1qr,* 2. 3. 4. 5. 6. DATE OF SUBMISSION CONTENTS 1. INTRODUCTION CYTOLOGICAL IDENTI FICATIONS MATERIALS / METHODS RESULTS SEASONAL DISTRIBUTION AND MOVEMENT OF THE DIFFERENT CYTOSPECIES THE DIFFERENT METHODS OF IDENTIFYING THE VECTORS OF HUMAN ONCHOCERCIASIS IN THE OCP. I.ABORATORY REARING OF S.DAMNOSUM COMPLEX IN THE OUEME RIVER BASIN AREA IN BENIN. 8. TRAINING 9. MEETINGS 10. OBSERVATIONS / COMMENTS 11. ACKNOWLEDMENTS 7 ( l. , A'q? r', ?,., \ i,ilr f: q,'.,'. l or ,A_ "Jitr^ t, \P) \- u'r'-1 ;cr-r il I .t'.' -T>, 1. INTRODUCTION This report embodies the results of all activities carried out by the cytotaxonomic laboratory at Kara Oogo) in 1995. Few results of samples collected in 1994 but identified in 1995 were also contained in the present report. Rainfall : Throughout the entire Eastern Operation Area, the rains started very early and continued up to November 1995. This was contrary to the situation in 1994 when the rains began late and finished quite early in October. As a result of the sufficient rains in 1995, several potential breeding sites especially on the tributaries and dams becamer productive for the aquatic stages of S.damnosum (s.1.) and larvae were received from many important breeding sites for cytologicql analysis in the course of the year. I --..- -- -'--.-e--/ - -' .t .a All results presented in thjs report have already been communicated to those who have contributed materials for cytological identification and to those scientists involved in operational research, larviciding and surveillance activities. 2. CYTOLOGICAL IDENTIFICATIONS Routine identifications were carried out on the larvae of S.damnosum (s.1.) from ilttne river basins making up the Eastern Operation Area of the OCP. ftrese include the Bas-Bandama, Como6, Volta, Mono, Asukawkaw, Sio, Ou6m6 and the Niger. ln 1995 a total of Four Thousand Seven Hundred and Ninety-Eight (4798) larvae of S.damnosum (s.1.) were processed for cytological identification. Out of this number Four Thousand Six Hundred and Sixty-Eigh (4668) representing g7 % of the total sample received were successfully identified. The remaining 3 o/o of the larvae not identified produced poor chromosomes and therefore unreadable. 3. MATERIALS ' METHODS MATERIALS : Specimens on which this report is based came from three sources : (i) specimens from Routine checks by the sectors and sub-sectors(ii) Specimens from Susceptibility Tests(iia) Reared Specimens from the Ou6m6 River Basin. METHODS Larvae of the S.damnosum species complex were collected frbm all the main rivers tributaries and dams in the Eastern Operation Area. The larvae were preserved --j--) 1 -!t 1ri l- 2 in the field in carnoy's fixative (3 parts absolute alcohol I part glacial acetic acid). The samples were stained and chromosome preparations made as described by Dunbar (19721and Boakye (1988). ldentifications were made using the chromosome maps of Vajime and Dunbar (1975) ; Meredith et al. (1986) and Surtees et al. (1988). 4. RESULTS GYTOLOGTCAL TDENT|F|CATIONS (1 995) Tn, BASIN sntra6r-e stzE TOTAL IDENTIFIED o/o I IDENTIFIED BAS BANDAMA ASUKA\A'KAW PRA /TANO COMOE VOLTA OUEME NIGER MONO SIO 1567 529 821 128 509 825 355 I 85 1551 811 119 493 734 345 522 I 84 97 89 99 97 99 100 99 93 99 TOTAL 4798 4668 97 a'-. --r.-, 3 5. SEASONAL DISTRIBUTION AND MOVEMENT OF THE CYTOSPECIES As in the previous years, the savanna cytospecies (S.damnosum (s.s.) and S. sirbanum) were more widely distributed in 1995 than the forest species (S. squamosum, S. yahense, S. sanctipauli and S. soubrense(Beffa). S. damnosum (s.sl (Map 2) As usual, in 1995, S. damnosum (s.s) was found mainly in the transition zone between the forest and the gavanna. The species was recorded from allthe mpjor rivers and their tributaries. lt is fhe most predominant of the two savanna cyto$ecies (9. damnosum (s.s.) and S. sirbanum) found in the forest areas of C6te d'lvoire, Ghana, Togo and Benin. S. damnosum (s.s.) makes dynamic movements towards the north and south in the rainy season than in the dry season. S. sirbanum (Map 3) The distribution range of S. sirbanum in 1995 was almost the same as that of S. damnosum(s.s) in the Eastern OCP. However, its proportion in the total larvae of S. damnosum (s.1.) examined was very little. S. sirbanum occurred predominantly in the savanna Zones and its presence in the forest areas was observed mainly in the rainy season. S. squamosum (Map 4) S. squamosum was largely confined to the Asukawkaw, Dayi, Sio and the tributaries of the Mono : all in the mountainous forest on the Ghana / Togo border. ln the wet season, S. squamosum makes some incursions into benin. Two cases were recorded in Benin in 1995. The first was on the River Zou at Tob6 and the second was registered at Tch6lenga on R. Binah, a small tributary of the K6ran River. S. squamosum was recorded from four breeding sites on the West Bank of the Volta Lake in Ghana. These were Yabraso (R. Oyoko) and Kintampo Falls (R. Punpum) both tributaries of the Black Volta. The remaining two other cases were from Jerusalem on River Fia and Manso on R. Tanfi, both tributaries of the Pru River. S. yahense (Map 5) S.yahense was identifified in 1995 in larvalsamples from Menusci on R. Menu and Abrubruwa on R. Labo, both tributaries of R. Asukawkaw in Ghana. a,-ti +'- S. sanctipauli (s.s.) (Map 6) in 1995, S. sanctioauli (s.s.) was registered on the Como6 from Amouakoukro and Koutoukro. The only case recorded on the bas Bandama at N'Zinoua as shown on the map was collected in December 1994. The Sassandra collection from Kop6ragui came to Kara by mistake as this river belongs to Zone West. S. soubrcnse (Beffa) (Map 7) , The presence of S. soubrense (Beffa) in lthe Eastern OCP is largely due to periodic migrations (rainy seasons) from Nigeria. lr1 1995 the species was recorded from five collecting points : Three from routine samples/rom Agolin - Pahou and Gba in Benin '' and Landa-Mono in Togo. The other two records of S. soubrense (Beffa) from M'b6t6koukou and Kaboua were the results of S. damnosum (s.1.) rearing work undertaken by the AREM Team from Kara as part of the special study of adult females with dark wing-tufts in the Ou6m6 River Basin Area in Benin. This study was initiated by the Vector Control Unit of the OCP. 4 II 'T>.'7.- 5 6. THE DIFFERENT METHODS OF IDENTIFYING THE VECTORS OF HUMAN ONCHOCERCIASIS IN THE OCP. A. CYTOTAXONOMY Cytotaxonomic identification is based on the micro morphological differences in the banding patterns of the polythene chromosomes obtained from the larval salivary glands. This method allows for the lunambiguous identification of all the species r members of the S.damnosum compfex. lt has therefore remained the reference t taxonomic standard for the identificltion of S.damnosum s.l. against which other / techniques are validated. However, the technique can only be applied to the larval stage' of the Vector and not to the adglt fly that is implicated in transmission studies. B. MORPHOLOGY AND MORPHOMETRY Classical morphological identifications of adult flies in the OCP is based essentially, on colour variations of certains characters (wing tufts, fore coxa and antenna) and antenna structure. This permits the separation of adult female S.damnosum (s.1.) ihto "savanna" and "forest" flies and S. yahense. Unfortunately, the colour characters are not uniform over a species distribution range which leads to some misidentifications especially, in the transition zones between the forest and savanna areas. Notwithstanding this setback, classical morphological classification has the advantage of being simple, fast and easily applicable by all technicians. Recent improvements in morphotaxonomy involve the use of metric characters such as the length of the antenna and thorax. These measurements are analysed using multivariate statistics to separate the members of the S.damnosum (s.1.).The technique requires a more specialised training and therefore cannot be easily adapted for use by all technicians. C. ELECTROPHORESIS Phosphoglucomutase (PGM) and trehalase (TRE) isoenzyme electrophoresis on cellulose acetate gel plates has been used in the OCP to separate members of the S. damnosum (s.1.) into three categories : "savanna/S. sanctipauli (s.1.), S. squamosum and S. yahense. lmprovements in this technique makes it possible to be used in field situations. However, specialised training is required for electrophoresis which limits it's applicability as a routine technique over the entire OCP area. lt is advantageous for specifific studies in areas where difficulties exist in separating "savanna" flies from S.squamosum by classical morphological identifications. ri D. DHDA (DNA ' PCR BASED TDENT|F|CAT|ON TECHNTQUE HETERODUPLEX The identification of members of the S. damnosum (s.1.) using molecularbiological techniques depends on the differential migration of homoduplex and heteroduplex hybrids in an agarose gel electrophoresis. lt is the most recent identification technique in the OCP. The standard DNA sequence for hybridisation (ND4) is from S. damnosum (s.s.). This sequen@ when hybridised to an identical sequence obtained any S. damnosum (s.s.) forms a homoduplex but a heteroduplex is formed when the hybridisation is to a different sequence (that is sequences ftorp other members of the S. damnosum (s.1.) a heteroduplex hybrid is formed. // The technique is applicable to both larval and adult stages allows for the specific identification of S. damnosum (s.s.); S. sirbanum ; S. leonense ; S. sanctipauli and S. squamosum. DNA sequences for other species such as S. yahense have not been developed due to the initial absence of pure populations of these species. Some of the identifications were also contrary to those from classical morphology especially flies with dark wing-tufts and fore coxa. These results need to be confirmed through a validation of the heteroduplex technique by comparing the results with those obtained by cytotaxonomy. The techniques can only be undertaken in speciallised laboratories. However, when validated it will be the most reliable method for adult species identification. 6 7MISSION REPORT LABORATORY REARING OF S. DAMNOSUM COMPLE){ IN TIIE OUEME RTVER BASIN IN BEIYIN Introduction Dark wing-tuft adutt females of the S.damnosum (Theobald complex were identified by DNA probes asi savanna cytospecies (S.aannosum ss. and S.sirbanum) in the Ou6m6 river basin area in the Republic of Benin. Hitherto, the same populations were morphologically scored as forest flies of the Beffa fgrm of S.soubrense, believed to have peneEated into thq OCP area fromNigeria. I I This DNA discovery puts-the criteria being used by OCP for morphological and morphometric identifications of the wild-caught adult females of the S.damnosum complex into discordance. The phenomenon therefore calls for thorough investigation and confirmation so as to pave the way for the necessary changes to be made in all existing entomological records for the Ou6m6 River Basin Area of the Onchocerciasis Confiol Programme in West Africa. After exhaustive discussions and analysis by the VCU hierarchy, it was finally decided to investigate the sinration by rearing wild-caught adult females of the S.damnosum complex direct from the Oudmd river and also from river Okpar4 the main tributary of the Oudmd river. The objective was to provide samples for cytotaxonomic, morphologic/morphometric and DNA determinations to confirm once and for all the actual identity of these biting adult females with dark wingtufu in the Oudmd River Basin Area. The Study The study was undertaken by members of the Applied Research and Environmental Monitoring (AREM) team from Karq Togo reinforced by an Assistant Entomologist from Bouaki in C6te d'Ivoire. A field Laboratory was temporarily established at the Sub-Sector of Bohicon in Benin on August 17 1995. Thirry-nro (32) wild-caught females were collected in plastic tubes from M'BETEKOUKOU on August l8 1995 and another thirty-nvo (32) from KABOUA on August 19 1995. They were all allowed to feed in the field on human baits and then maintained until gravid in the laboratory. Twenty-two (22) females representing 34.3 % of the total sixty-four (64) flies captured have been successfully induced to oviposit by immersion in water. The remaining 42 flies (65.6 o/o) were either dead or refused to oviposit. Of the 22 flies which have been induced to lay eggs, 7 with dark wing-tufts from M'B6tdkoukou (Table l) and 6 with dark wing-tufts from Kaboua (Table 2) were reared 8separately as single egg batches up to the adult stage. Three (3) femates with dark wing-tufu from Kaboua (Table 3) gave very little eggs. The 3 eggbatches were pooled and reared together up to the adult stage. Six (6) other flies (3 from M'Bdtdkoukou and 3 from Kaboua) which were identified as females with pale wing-tufu were also pooled and reared together up to adult stage. (Table 4). The Rearine System Larval development requires suitable conditions of water-flow and turbulence and correct chemical conditions for the cytospecies concemed. As such, different techniques for rearing simuliids, especially under artificial conditions in the field, provide the conditions of flow or turbulence, and other factors for rearing simulids. The apparatus used in this present stu.6y is composed of a very simple wide-mouthed 2 litre bottles and I litre beakers filled with river water. These containers were connected to a Hy Flow Air pomp by means of plastic pipe tubings with diffusers and passing through a much bigger plastic tube before ending in the rearing bottle or beaker. The Hy Flow Air pomp was generated by electricity throughout the entire period of the study (approximately four weeks). The Actual Rearing As already stated above the collection of the wild-caught adult females took place at M'Bdtikoukou and Kaboua on 18 and 19 August 1995 respectively. Contrary to the method used by the OCP catching teams, where the flies are caught before they can take any blood-meal, simuliids intended for this research were allowed to be fully fed on human blood. They were then captnred individually in plastic tubes, packed toBether in wet cotton wool and kept in ice-chest during transportation to the field laboratory at Bohicon. In the laboratory, the flies were transfered to new catching tubes prepared with a piece of blotting paper cut in a rectangular size. This was to facilitate easy movement of the insects in captivity. The lids of the tubes were cut with a small hole and covered with a piece of mosquito netting to permit air and also to feed the flies. After this operation, the simuliids were kept under an optimum temperature (22 - 27 degrees C) in an air-conditioned room and at times in the open air when the temperature outside was found to be not too high. The flies were fed on sugar solution 24 hours after blood meal and stimulated to oviposit 72 hours after capture. The tubes in which the eggs were layed were lined up with a tittle wad of mosquito netting and filled with 5 ml of river water. [t was these nettings bearing the egg batches which were removed and plunged into the rearing system described above for the larval rearing. The first srage larvae hatched from the eggs after 3 days. The young larvae were fed once a day on "Tetra" fish food and twice a day as they grew bigger. The las instar larvae (7th stage) began to pupate after ? days. These also took another 3 - 4 days to emerge into adults, first the 9males and later the females. A piece of mosquito netting with a hole was mounted on a wire and fixed to the top of the rearing apparatus immediately the lawae began to pupate. This made the collection of the emerging adults very easy. The adult simuliids were preserved in 70 - 80 % alcohol. Lablinp All the 22 adr:/rt females (16 with dark wing-tufu and 6 with pale wing-tufu) used for the rearing were labelled as MBE for M'Bitdkoukou and KAB for Kaboua. This code of three letters with their corresponding serial or alphabetical numbers originated from the number attributed to the mother simuliids at the two catching poins during the first day of capture in the field. The sarqe codes were used throughout the study as below : 7tt ,,/ Table I Table 2 M'BETEKOUKOU : MBE KABOUA: KAB Table 3 Table 4 MBE No I MBE No 3 MBE No 7 MBE No 9 MBE No l0 MBE No 12 MBE No 13 Females with dark wing-tufu reared as single egg batches. I(AB No 2 Females with dark I(AB No 3 wing-tufts reared KAB No 5 as single egg KAB No 6 batches. I(AB No 7 KAB No 18 KAB No 4 KAB No 9 KAB No 1l Females with dark wing-tufu pooled together MBE No 2 MBE No 8 MBE No l1 KAB No I I(AB No 13 I(AB No 15 Females with pale wing-tufts from M'B6tdkoukou and Kaboua pooled and reared together I -r- 10 SAMPLING O[' S.DAMNOSUM 6.L..1LARVAE AND ADULTS T'OR CYTOTAXON0MIC, MORPH0LOGIC/MORPHOMETRIC AI\[D DNA DETERMINATION Lantae realised from the study were shared with the heads for DNA probes determination and the abdomens for cytological identification. The procedure is as described below : The larvae : - Cut off the head of each lanra with a scapel just below the hisoblast. - Put the head in 80 o/o alcohol and,abdomen in carnoy's fixative. - Label each sample I - Keep the samples in the refrigerdtor (not in the freezing apartment). - Make permanent slides of all the larvae cytologically ideniifred. The adults : - Collect the adults and preserve them in g0 % alcohol. - Label each sample. - For morphologic, morphomefric and "Direct Heteroduplex Analysis" (DHDA) remove the 3 right legs from the same fly during the first morphologic identification and preserve them in 80 % alcohol in an eppendorftube. - Keep the fly in 80 % alcohol in a catching tube. Results (See Tables I - 16) Table I Gives details (labels or codes) of the 7 dark wing-tuft mother flies from M'Bdtdkoukou. Table 2 : Gives details (labels or codes) of the 6 dark wing-tuft mother flies from Kaboua. : Gives details (labels or codes) of the 3 dark wing-tuft mother flies from Kaboua which egg batches were pooled and reared together. Table 4 : Gives details (labels or codes) of the 6 pale wing-tuft mother flies-3 from M'B6t6koukou and 3 from Kaboua which egg batches were pooled and reared together. Table 5 : Gives details of the total number of larvae harvested from the 7 single egg batches from M'Bdtdkoukou for cytotaxonomic and DNA identifications. : Gives details of the total number of larvae harvested from the 6 single egg batches from Kaboua for cytotaxonomic and DNA identifications. Tables 3 Table 6 Table 7 Table 8 Table 9 Table l0 Table I I Table 12 Table 13 Table 14 Table 15 Table 16 11 Gives details of the total number of lawae harvested from the 3 egg batches from Kaboua (Nos 4, 9, I l) which were pooled and reared together. : Gives details of the number of larvae harvested from the 6 females with pale wing-tufts - 3 from M'Bitdkoukou and 3 from Kaboua. : Gives the total number of reared adult simuliids collected from 5 successful single egg batches from females with dark wing-tufts from M'Bet6koukou. : Gives the total number of reared adult simuliids collected from 6 successful single egg batches from females with dark wing-tufts from Kaboua. : Gives the total number ot r"*fa,adult simuliids collected from 3 egg batches from Kaboua (Nos. 4,9, ll) which were pooled and reared together. : Gives the total number of reared adult simuliids collected from 6 egg batches - 3 from M'Bdtikoukou and 3 from Kaboua - females with pale wing-tufts. Gives the overall total of the number of adult males and females obtained during the study. : Gives the final result of cytota(onomic identification of all the larvae of females with dark wing-tufts as 100 o/o S.soubrense (Beffa). Gives the final result of cytotaxonomic identification of all the larvae of females with pale wing-tufu as 6 hydrids of S.damnosum (.s.s.) and S.soubrense (Beffa) and 4 S.damnosum (s.s.) : Gives the final result of morphological identification as 78 (96 %) sou/squa (forest) and 3 (4 %) as savanna species. t2 TABLE5 TABLE 6 Table 7 TOTAL NUMBER OF LARVAE COLLECTED M'BETEKOUKOU = MBE KABOUA KAB DATE OF COLLECTION No OF MOTHER FLY (dark wing tufts) SPECIES No OF LARVAE COLLECTED 03.09.95 02.09.9s 02.09.9s 02.09.9s 04.09.95 31.08.95 03.09.95 MBE No 1 MBE No 3 MBE No 7 MBE No 9 MBE No l0 MBE lrlo 12 MBE ,'No l3 Forest Forest Forest Forest Forest Forest Forest 4 r0 2t l0 16 10 10 TOTAL 7 8r Date of collection No of mother fly (dark wing tufts) Species No of larvae collected 03.09.9s 05.09.95 05.09.9s 06.09.95 31.08.95 09.09.9s KAB KAB KAB KAB KAB KAB No2 No3 No5 No6 No7 NolS Forest Forest Forest Forest Forest Forest I 0 10 18 31 l0 67 TOTAL 6 146 KAB Pool = Three dark wing-tuft flies with few eggs pooled together Date of collection No of mother fly (dark wing tufts) Species No of larvae collected 04.09.95 KAB : 4,9, ll Forest t2 13 Table 8 REMARK Each larva was cut into two, the head for DNA determination and the abdomen for Cytotaxonomic identifi cation. TOTAL NTJMBER OF ADULTS COLLECTED Table 9 - M'B6t6koukou MBE and I(AB (Pooled together) = Eggs of six pale wing-tuft females pooled together Date of collection No of mother fly (pale wing tuffs) Species No of larvae collected 03.09.9s MBE : 2, 8, l1 KAB : 1, 13, 15 sav. sav. 23 GRAND TOTAL 6 I zez Date Number mother fly Females Males Total o/o Females 06.09.95 07.09.95 08.09.95 08.09.95 04.09.95 MBE No I MBE No 3 MBE No 7 MBE No l0 MBE No 12 0 I 6 t5 4 I I 4 l3 3 I 2 10 28 7 0 50 60 54 58 TOTAL 26 22 48 54 Date No mother fly Females Males Total o/o Females 05.09.95 06.09.9s 08.09.95 08.09.95 05.09.95 r0.09.95 KAB KAB KAB KAB KAB KAB No2 No3 No5 No6 No7 Nol8 58 t9 20 40 46 16 40 33 40 66 88 2t 98 52 60 r06 t34 37 59 37 33 38 34 43 TOTAL 218 3r0 lszs 4t 14 Table 10 - Kaboua Table 1l Table 12 NB. The trough holding these egg batches produced only 6 females and 15 males because the rearing system was not complete. There was shortage of diffusers and plastic tubes (Medium - l.2c). As a result many larvae were lost each time the water has to be changed. I(AB (pool) : Three dark wing-tuft flies with few eggs pooled together Date No mother fly Females Males Total o/o Females 04.09.95 Nos.4, 9, 11 19 22 4t 46 Grand Total (Kaboua) 237 332 569 42 MBE and KAB (pooled) = Eggs of six pale wing-tuft females pooled together Date Number mother fly Females Males Total 7o Females 03.09.9s MBE: 2,8, ll KAB: 1, 13, 15 6 15 2t 29 t5 Table 13 Table 14 Table 15 CYTOTAXONOMIC IDENTIFICATION RESULTS (Larvae of females with darf wing-tufts) FEMALES MALES TOTAL % FEMALES GRAND TOTAL (IIBE and KAB) 269 369 638 42 DATE LOCATION No IDENTIFIED CYTOSPECIES So (Beffa) 31.08.9s 31.08.95 M'BETEKOUKOU KABOUA 64 94 64 (100%) 94 (100%) TOTAL 158 rs8 (r00%) DATE LOCATION (mother flies with pale wingt tuffs) No IDENTIFIED CYTOSPECIES Hybrid of S.da(s.s.) and S.so (Beffa) da (s.s.) 3.9.9s MBE: 2,8, ll KAB: l, 13, 15 r0 6 4 16 MORPHOLOGICAL IDENTIFICATION RESULTS (Adult flies) Table 16 N.B. Of the 269 reared, simuliids from females with dark wing-tufts (preiumed to be forest flies), 8l were examined morphologically as 78 sou/squa (forest) and 3 scored as savarrna (Table 16). All the 269 reared females were sent to Bouak6 for morphometric and DNA identifications. DATE LOCATION No IDENT IF'IED MORPHOSPECIES ot,o sou/sQUA o,to SavSOU / SQUA SAV 2-8.9.9s 3-14.9.95 M'BETEKOUKOU KABOUA 2t 60 20 58 I 2 95 97 5 3 TOTAL 8r 78 3 96 4 t7 OBSERVATIONS / COMMENTS - Morphological observation of the reared larvae of simuliids with dark wing-tufts reveals no presence of dorsal abdominal conical bumps (tubercles). According to current literature or criteria these should be classified into the S.sanctipauli and S.soubrense sub-group. - The reared larvae of all the six flies with pale wing-tufu (3 from M'Bitdkoukou and 3 from Kaboua) which were pooled and reared together, wore short abdominal tubercles and therefore belong to the sub-species group of S.damnosum (s.s.) and S.sirbanum. present system. Laboratory rearing of S.damnosum (s.1.) requires regular supply of electricity. In the .?Or, there was no intemrption and everything went of well with the entire rearing - An aerated river water is ngeded at all times to change the water in the rearing system once a day for the bigger 2 I bottle and twice a day for the small I I bottle. The water must not be fetched from the rivers under larviciding treafinent. If there is no alternative, the water must be fetched some 4-5 days after treatment. This precaution is very important in order to avoid contamination thus retarding the development of the larvae or killing them altogether. - The egg batches obtained from M'Bdtdkoukou were insufficient as compared to those from Kaboua. - The engorged simuliids from M'Bitdkoukou and Kaboua were induced to oviposit on 21 and' 22 August 1995 and the first neonates (mostly males) began to emerge on the 2 and 3 september 1995 respectively, thus taking a total of 13 days in both cases to complete the cycle of development, from eggs to adults. - The laboratory rearing of S.damnosum complex is useful to the OCP in the following fields of investigation : Provisionofmaterial forcytotaxonomic determination, morphological identification and DNA analysis. Species identification of reinvading flies Distribution of biting populations of S.damnosum complex species. Host preferences of species within the complex. Vector potential of S.damnosum complex species for Onchocerca volvulus and Onchocerca ochengi. I 2. 3 4. 5 18 CONCLUSION The work was effectively canied out in a very good condition and the objective seems to be achieved. The rearing in Benin went on smoothly without any hindrance. Electricity supply was regular throughout. Cytotaxonomic identification of 158 larvae of S.damnosum reared from females with dark wing-tufts in the Oudm6 River basin (M'Bdt6koukou and Kaboua) gave IOO % result as S.soubrense (Beffa), a forest cytotype. Eighty-One (81) adult females from the same reared samples were morphologically determined as 96 o/o forest and 4 oZ savanna species. The two results seem to be almost in concordance to prove that the Simulium damnosum populations with dark wing-tufts in the Ou6m6 River TBasin area belong more to the forest group than to the savanna species as shown by DNA d6terminations. t However, sufficient reared samples (larvae and adult females) originating from the same study were despatched to the Research Laboratory in Bouakd for DNA analysis and morphometric identifications to confirm or invalidate the cytotaxonomic and morphological findings estabtished in Kara, Togo. RECOMMENDATION - The 3 presumed savanna species by simple morphological identification (Table 16) recorded among a total of 81 reared adult females calls for further investigation. This is likety to be the same case as S.squamosum in the Southern Extension Zone (Ghana and Togo) where some populations were found by the same identification method (morphology) to have possessed both dark and pale wing-tufts. - The 6 larvae found as hybrids of S.damnosum (s.s.) and S.soubrense (Beffa) - (table 15) also calls for thorough investigation. This is very important because it will provide useful information as to the vectorial role of these hybrid populations and the exact stain of Onchocerciasis they transmit (Savanna or forest) if infact they are potential vectors. - The hybrid samples were obtained from females with pale wing-tufts. The present record of 6 hybrids between S.damnosum (s.s.) and S.soubrense (Beffa) and 4 S.damnosum (s.s.) is not sufficient from scientific point of view to draw any meaningful conclusion. In order to obtain a reasonable data for thorough statistical analysis, we recommend that the rearing work be repeated next rainy season with much emphasis on the rearing of females with pale wing-tufts. 19 ACKNOWLEDGEMENTS We wish to expres our sincere thanks to Mr. Barro Tdl6 for helping to set up the rearing system at Bohicon and to Mr. Sedou Naniogud for his conscientious technical assistance throughout. We are particulary grateful to the initiators of the study Dr. J.M. Hougard (Chief VCU), Dr. H. Agoua (CAT Zone East), and Dr. C. Back (Chief AREMflRU) for their encouragement and valuable support. We are especially indebted to Dr. Laurent Toe for the DNA determinations ; to Dr. A.K. Diallo, the OCP Sector Chief of Parakou and Dr. L. Assogb4 the National Coordinator of Benin for their visit and useful discussions ; to Mr. P. Gbaguidi, the Sub- Sector Chief of Bohicon and his entire staff for their cooperation; and finally to our two drivers Messrs. Agoli Kossi and Gbandi Assoumanou for their devoted services and immediate response to our needs inrthe field./ usru. I a 20 AI{NEX List of materials needed for S.damnosum (s.1.) rearins) l. 2. 3. 4. 5. 6. 7. 8. 9. 10. I t. 12. 13. t4. 15. 16. t7. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. Air pomp (Hy Flo) Diffusers Troughs (2 I Wide-Mouthed Bottles) Troughs (l I Beakers) Plastic, Plastid Jerrycans tubes (Small size- 0.8 C) Plastic tubes (Medium - 1.2 C) Generator (As standby for electricity supply) "Tetra" Fish food Thermometer Graduated pipette (10 ml) Graduated tubes (45 ml) Extension Line (For electricity) Scapel Mosquito netting Scissors (Medium-size) Scissors (Small-size) Forceps (Hard) Forceps (Soft) Long Forceps (With Hook end) Rubber-tubing for Air pomp) Clips (for fastening) Cotton wool Plastic catching tubes Plastic-tray Aluminium plate Plastic petri dishes Scotch tape (for labelling) Prospection glass tubes (25 ml) Ependorf plastic tubes Washing bottles (for holding alcohol) Washing bottles (for holding water) Rubber bands (1 pkt) Pipettes Rubber-teats Machine oil Plastic bags 2l d A OULT I I \ oaAar'rl Aqul t rc EGG d:\ I \ PUPA (ia cocoan) d: I I t I ilt tv rj / o vl vll 51 FGtg LARVAL Figurc l. Life cvcle of rhe fanrilv Sinruliidae (Diptera). The spccies chosen for illustrative [)urp()ses is Sinu/r,ilan damnosum,carrier of human onchocerciasis ('rirer blindness') in l\'esr\fric;r Lan,al stages (instars) shor.'the general larval. groNth: their proportions are not ne,rssaril;' exac( and not all spccies have seven insrars t ,gb -/ I CLASSIFICATI0T{ 0F THE SIX SPECIES 0F THE S: DAI{N0SUI.| COTIPLEX IN THE EASTERN ZONE OF THE OCP. Very prominent tubercles and large scales (Pr6sence de trEs longs Eubercules eE de larges 6cail1es) gEosEo .d Et"" Very liEEle Eubercles and large scales (Pr6sence de Eubercules un peu longs eE de larges 6caitles) I 22 Figure 2 I 2. j.i ..4 -t Cgaa@a sl sd tlrtanon td@ d rrrtiPall Ab sence ( Absence of de dorsal cubercles and no large scales tubercules dorsaux eE d'6cai11es moins lar8es)' S. SOUBREI{SE (BEFFA) BELOT{GS TO THIS GROUP.NB 3 a I aFigure 3. .rlfrr^,rrtr , !. dornnosum (s. s.)T $i II F S. soubrense ( Bzffo) Gi?. :' Larvalrchromosomes of S.damnosum (s.s.) and S.sooubrense (Beffa) 21 tsls Otl+ IIL- c/c # \\$\\t$1 t$ .:e ill *s.*#s rl [6/*u-..1t4- Uc/zq . ,s/A 4 ru t I It I qrltidLrrl+ ..1.S.tft + II L- 4 .6/.4.6 :j III ,D LI&u ,rt ;*r I r'.* -., --1., _ -.. .';;"^{ co ol =-l'lo-to J.o, 't=(ll,avto=,EtO = =-teFd.Ooo=a=(+ oc,=l,-toOJ. =-:El, .hO, -.O(O O .=t d- <o A-o -t c,r(, o< .ro =oo{ Cf Vto= t ocn N5 o crc)th doo,= =(1 EA,F Joo -: =.F -o, o . 'r<o A c,, o11OF otrro-tco ={cF=oAO1 AE d IA c c Ag =o ahE = ET c,o - = =(.I o tJ1 C C oe =o tE trr oo - 'n (o C -l o 5 ) ,.y' II !a N) t- "^ [.\ tr('N. I $N) I t'r-' = =GI arE ct (.)I E' o aro !, c, o ft \ \ Lt o.- Or J.1= -(a E oo E' o o o I I II (., 8. 25 TRAINING TRAINING PROGRAMME FOR TECHNICIANS IN ZONE EAST OF THE OCP 1995 1. ln view of recommandation No 2 of the last AREM Meeting in Bouak6 (01 - OG May 1995) the retraining and checking of the technical knowledge of technicians (Assistant Entomologists, Sub-Sector Chiefs and LabbratoryAssistants) of Zone East did not come on in 1995. This will be organized in 1996 to correspond with the two-yearly period stipulated in the above quoted recommendation. lt Hov/ever, dissection codes were assigned to 5 LaboratfryAssistants after passing a test. They were : - Awadi - Naniogu6 - Gouni - Boukari - Anthony Adji :S6dou : Bidjdtana : Loukoumane :Tefteh : AREM I Kara, Togo. AREM I Kara, Togo. Sous-Secteur / Kara, Togo. Sous-secteur / Parakou, B6nin. Sous-Secteur / Hohoe, Ghana. 2. From 24 July to 12 August 1995, Messr : George K. Fiasorgbor and Soulemana M. Bil6ou (AREM I Kara) undenarent three weeks training course at Bouak6 to permit them to acquire the necessary knowledge in the new technique of S. damnosum (s.1.) rearing in the laboratory. The principal objective of this training is to undertake the rearing of Simulium flies with dark wing-tufts at Kaboua (R.Okpara) and M'B6t6koukou(R. Ou6m6) in Benin. This work was carried out with great success from 17 August to 14 September 1995. MEETINGS The Senior Author of this report participated in the following meetings in the course of the Year 1995 : 9. (i) (ii) (i ii) (iv) (v) Aerial Contract Bidders'Meeting, Kara 1 - 2 March 1gg5. Bi-Annual Sector Meeting, Kara 25 - 26 April 1gg5. Research (AREM) Meeting, Bouak6 01 - 06 May 1g95. Bi-Annual Sector Meeting, Kara 28 - 29 September 1gg5. East Zonal VCU Meeting, Kara 30 October - 04 November 1gg5 26 10. OBSERVATIONS / COMMENTS - All the six main cytospecies of the S. damnosum complex (S. squanosum, S. yahense, S. damnosum (s.s.), S. sirbanum, S. sanctipauli (s.s.) and S. soubrense) have been registered in the course of the year. - The "Nile" form of S. damnosum (s.s.) was identified among samples from Naboulgou (R.Koumongou), sarakawa (R. Kara), Kaboua (R. okpara) and Bagan(R.M6). Tdutio - The monomorphic form of S. sirbanum nga (R. Oti) and Porga (R. Pendjari). fas recorded from Bec (R. Oti), - Six cases of hybrids between S. damnosum (s.s.) and S. soubrense (Beffa) were recorded from Kaboua I M'B6t6koukou in September 1995 among laboratory reared larvae of females with pale wing-tufts. Chromosomes I and lll have inversions typical of S.damnosum (s.s.) whilst chromosome lll wears inversions of S. soubrense (Beffa). - All male populations of S. squamosum recorded West of the Volta Lake were heterozygous for the inversion 3 CS. Those of the Eastern bank of the Lake were homozygous. 11. ACKNOWLEDGEMENTS We are sincerely grateful to Dr J.M. Hougard (Chief VCU), Dr H. Agoua (CAT Zone East), Dr L. Yam6ogo (CEEH), Dr C. Back (Chief AREM/IRU), Dr B.K. Akpoboua(CAT Zone West), Dr M. Kassambara (Sector Chief lKara), DrA.K. Diallo (Sector Chief / Parakou), Mr S.A. Sowah (Sector Chief / Tamal6), Mr M. Sarr (Sector Chief / Bouak6), The Assistant Entomologists, Laboratory Research Assistants, Laboratory Assistants, and the entire staff of the two aerobases at Kara and Odi6nn6 including all the Evergreen personnels who have contributed materials or assisted in one wiy or the other to make this work a success. otI1 QO0e9QQOCtCtCtC'CtCrOOc'.,CtC,CtCrC'CtCt.tCtCrctOC'iOOCt-CtCtCrC,C,CtoCtl, -t r -aa -| -(a -\r -o { o a o o I a 5 a e { ! o (I o (, o (' { (,l o aa N o N r Io N ts d N N (! - N N (.6 r.l : E t.r o c- o F E A N > > rr a e > : e =>nr >> =ru r'r E E E rr o A>E E'>>Eo > rrr oIil O { -- -(' { N !a N O O N O O .r Gl 6! a O N l\' O O ol G a O O N r N ti' O aa { O } Ct O lo (I Ct .a tai --.E-o-.oEE'Gob'E'E'E'Eo-E'€-oo-o=Eo G boE'EE'E'cE'oE'G-'cc-'EEo-G-'oIt (' tD or or (, (t q o o' (' (' or ot (I l, or (, o[ (, (I or (I ol (, Ot (,l ol q ot ol a ol Ot O,l ol o O ol O 6 ot gr = tt m o o o o oqr m ooooo oooctaooooooooaoooooooooooooooo oooooooobbbbbbbbeoooooooooooooooeooooooooooooooooooooooo !!eeo!!@tr@(!@@oE@oo@o@@o@o@@@oo@oE@@@o@Eooo@o@r! =2z- -Z- =Zz z 2 2 - - zz z z - - - - z - z2- 222 2222r2 z -2222 zI g i E i i I i i B B E B B E B 9 B 9 9 99 B 9 9 F 9 9 B B 9 BE 9 9 B 9 B 9 F B 9 9 F ==3==-3-=3..1..=.--3.=G.-=3=--c--t-c------== a, ctt an = --==iz? 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Sirbonum O S dom nosurr{sp ) O s. squomosum O -s Yo hensc tr S. Soubrensc(beffo) I S Sonctioouti(ss ) 2 Distribution of the Cytotypcs ol the S.domnosurn Complcx EASTERN OPERATION AREA (EOA) !o t t \ ) l0 A 5'l I 12 o q' 1995 \L _t- / CYTOTAXONOMY LrsT oF coLLECTtNG SITES (1995) 40 - R As*all<ar - R. Gban-Hou 1. B6riaboukro 2. N'd6nou 3. Kravassou 4. Dimbokro 5. Ass6r6kro 6. Badadougou 7. Gossiamandara 8. Chutes Dienkoa 9. Tountoun 10. Amouakoukro 11. Amiankouassikro 12. Koutoukro 13. Tagadi 14. Akanyakrom 15. Yabraso 16. Kintampo Falls 17. Manso 18. Jesusalem 19. Asubende 20. Yayonyiri 21. Porga 22. Toutionga 23. Titira 24. Landa-Pozanda 25. Kpayando 26. Aleh6ride 27. Bagan 28. Kpassa 29. Kechebi 30. Tinkiro - R.Bas Bandama - R.Bas bandama - R.N'4i - R.N7i - R.Kan - R.Como6 - R.Sissisoa - R.Dienkoa - Volta Noire - R.Como6 - R.Como6 - R.Como6 - Volta Noire - Volta Noire - R.Oyoko - R.Punpum - R.Tanfi - R. Fia - R. Pru - R. Kulpawn - R. Pendjari - R. Oti - R. K6ran - R. Kara - R. Kpaya - R. Bouale -R.M6 - R. Bassa - R. Chai - R. Asukawkaw 31. Asukawkaw-Bge 32. Djodji 33. Menuso 34. Dayi-Hotel 35. Kudzra 36. Kati 37. Amou-Oblo 38. Ayagba 39. Yotokop6 40. Kpoba 41. Tet6tou 42. Adedakope 43. Konigbo 44. Blifta 45. Aou-Losso 46. Landa-Mono 47. Kassena 48. Tobe 49. Gba 50. Agolin-Pahou 51. M'B6t6koukou 52. Kaboua 53. Bassa 54. Tchakparou 55. Kpessourou 56. Kali 57. Tufoi 58. Sutri Rapids 59. Nsawora 60. Ntrotroso - R. Menq - R. DaYir - R. Dayi - R. Sio - R. Amou - R. Gban-Hou - R. Yoto - R. Mono - R. Mono - R. Amou - R. Ani6 - R. Anie - R. Aou - R. Mono -R.Na - R. Zou - R. Ou6m6 - R. Ouem6 - R. Ou6me - R. Okpara - R. Okpara - R. Oudora - R. Ounamono - R. Ori - R. Pra - R. Tano - R. Tano - R. Tano 4L DISTRIBUTION OF THE DIFFERENT CYTOSPECIES IN THE EASTERN ZONE OF THE OCP 1995 - Bas Bandama and its tributaries : S.damnosum (s.s.), S.sirbanum and S.sanctipauli (s.s.). - Como6 : S.damnosum (s.s.), S.sirbanum and S.sanctipauli (s.s.) - Volta Noire and tributaries :f.damnosum (s.s.), S.sirbanum and S.sQuamosuy - Volta Blanche and tributariei : S.sirbanum. - Oti /Pendjari and tributaries : S.damnosum (s.s.), S.sirbanum and S.squamosum - Asukawkaw and tributaries : S.damnosum (s.s.), S.sirbanum, S.squamosum and S.yahense. - Dayi : S.damnosum (s.s.), S.sirbanum and S.squamosum. - Sio : S.damnosum (s.s.), S.sirbanum and S.squamosum. - Mono and tributaries : S.damnosum (s.s.), S.sirbanum, S.squamosum and S.soubrense (Beffa). - ouem6 and tributaries : s.damnosum (s.s.), s.sirbanum, s.squamosum and S.soubrense (Beffa). - Niger tributaries : S.damnosum (s.s.) and S.sirbanum. - Tano : S.sanctipauli (s.s.), S.yahense. - Pra : S.sanctipauli (s.s.). ! :o oT ouI o B= .O =Hi o? - 3 2* -r J J o tn tr 3 o ? f, m! n -{ { z Il-jL/ '(i; m{@ FI;HPJJ r:l ctao=1'J o a? mz .J(o(o(tl = -o l\) 5N , i I 'oI, o!o , 'ul>o o@ 7zv a,Fti o; r o ZC,{. 9.n cr 0, ? 3 n m!t> ,fr { I -ohz cn El{m U?mg. r-+ ctE o=, '93 mz ..1(o(o(tl - !(, s ! , oTo ,I1ro>zv_ -oH oa z'I att 'tl tr!, 3 oo tr 3 oJo f, m 'g n + { >6 _z ni9 a tt.1@ Uomf FoIr -) .-J(Ja o6!= JJ mz J(o(o(tl - ! 5 5s ! , o .E oI,E{>7ia> !-oH 1a o J o o o r> oJo F m! fr { ofz rl1IgH @ UIm< o - IIt- "OO3gE mz J(o(o(rl =!(, s Tuo! oE -{ _o;z ,o tmoc, Zo{o =o *. 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