Mission report: laboratory rearing of S. damnosum complex in the Ouémé river bassin in Bénin

1MISSTON REPORT Laboratory Rearing of S. damnosum complex in the Ou6m6 River Basin in Benin Introduction Dark wing-tuft adult females of the S.damnosum (Theobald complex were identified by DNA probes as savanna cytospecies (S.damnosum s.s. 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 form of ,S-gqgbrense, believed to have penetrated into the OCP area from Nigeria. ,,' ,' 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 Control Programme in West Africa. After exhaustive discussions and analysis by the VCU hierarchy, it was finally decided to investigate the situation by Rearing Wild-Caught Adult Females of the S.damnosum complex direct from the Ou6m6 River and also from R. Okpara. the main tributary of the Ou6md 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 wingtufts in the Ou6m6 fuver Basin Area. The Study The study was undertaken by members of the Applied Research and Environmental Monitoring (AREM) Team from Kara, Togo reinforced by an Assistant Entomologist from Bouak6 in C6te d'Ivoire. A field Laboratory was temporarily established at the Sub-Sector of Bohicon in Benin on August 17 1995. Thirty-two (32) wild-caught females were collected in plastic tubes from M'BETEKOUKOU on August 18 1995 and another thirty-two (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 representing34.3 %o of the total sixty-four (64) flies captured have been successfully induced to oviposit by immersion in water. The remaining 42 flies (65.6 %) were either dead or refused to oviposit. Of the 22flies which have been induced to lay eggs, 7 from M'Bdt6koukou and 6 from Kaboua were reared separately as single egg batches up to the adult stage. Three (3) females with zdark wing-tufts from Kaboua gave very little eggs. The 3 egg batches were pooled and reared together up to the adult stage. Six (6) other flies (3 from M'B6t6koukou and 3 from Kaboua) which were identified as females with pale wing-tufts were also pooled and reared together up to adult stage. The Rearing System Larval development requires suitable conditions of water-flow and turbulence and correct chemical conditions for the cytospecies concerned. As such, different techniques for rearing simuliids, especially under artificial conditions in the field, provide the conditions of flow or turbulencel and other factors for rearing simuliids. Tle apparatus used in this present study is composed o!,a very simple wide-mouthed 2 litre bott'es and I litre beakers filled with river water. These cc.rtainers were connected to a Hy Flow Air Pomp by means of plastic pipe tubings with diffusers and passing through a much bigger plastic tuber 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 Rearins As already stated above the collection of the wild-caught adult females took place at M'B6t6koukou and Kaboua on 1 8 and 1 9 August I 995 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 captured individually in plastic tubes, packed together 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 little wad of mosquito netting and filled with 5 ml of river water. It was these nettings bearing the egg batches which were removed and plunged into the rearing system described above for the larval rearing. The first stage 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 the grew bigger. The last instar larvae (7th stage) began to pupate after 7 days. These also took another 3 - 4 days to emerge into adults, first the males and later the females. A pice of mosquito netting with a hole was mounted on a wire and 3fixed to the top of the rearing apparatus immediately the larvae began to pupate. This made the collection of the emerging adults very easy. The adult simuliids were preserved in 70 - 80 % alcohol. LABLING All the 22 adult females (16 with dark wing-tufts and 6 with pale wing-tufts) used for the rearing were labelled as MBE for M'B6t6koukou 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 points during the first day of capture in the field. The same codes were used throughout the study as below : Table I M'BETEKOUKOU KABOUA MBE 1 BBE 3 MBE 7 MBE 9 MBE IO IVIBE 12 MBE 13 Females with Dark Wing-tufts reared as single Egg Batches. KAB 2 KAB 3 KAB 5 KAB 6 KAB 7 ICAB 18 Females with Dark Wing-tufts Reared as single egg Batches. KAB 4 KAB 9 KAB 11 Females with Dark Wing-tufts. (Pooled) MBE 2 MBE 8 MBE 11 KAB 1 KAB 13 KAB 15 Females with Pale Wing-tufts from M'B6tdkoukou and Kaboua pooled and Reared together. 4Sampling of S.damnosum (s.1.) Larvae and Adults for Cytotaxonomic, Morpho lo gic/Tvlorphometric and DNA Determinations. larvae 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 larva with a scapel just betow the histoblast. - Put the head in 80 % alcohol and abdomen in carnoy's fixative. - Label each sample - Keep the samples in the refrigerator (Not in the freezing apartment). - Make permanent lides of all the larvae cytologically identified. The Adults - Collect the adults and preserve them in 80 %o alcohol. - Label each sample. - For morphologic, morphometric 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 eppendorf tube. - Keep the fly in 80 % alcohol in a catching tube. RESULTS (SeeTablesl-5) Table I : Gives details (labels or codes) of the 22 Ail:Jt Femeles successfully used for the study. Table 2 : Gives the Number of S.damnosum larvae collected for cytotaxonomic and DNA determinations. : Gives the total Number of Adult Females collected for Morphology I Morphometric identifications and Direct Heteroduplex Analysis (DHDA). Table 4 Gives the final result of cytotaxonomic identification as 100 % S.soubrense (Beffa). Gives the final result of Morphological identification as 96 % sou/squa (forest) and 4 o% as savanna species. Tables 3 Table 5 5TOTAL NUMBER OF LARVAE COLLECTED TABLE 2 M'BETEKOUKOU: MBE KABOUA KAB KAB POOL : Three dark wing-tuft flies with few eggs pooled 04.09.9s KAB : 4,9, ll t2 MBE and KAB POOL : Eggs of six Pale Wing-tuft females Pooled 03.09.9s MBE : 2,8, 11 KAB : 1, 13, 15 23 GRAND TOTAL 262 DATE OF COLLECTION No OF MOTHER FLY No OF LARVAE COLLECTED 03.09.9s 02.09.95 02.09.95 02.09.95 04.09.9s 31.08.9s 03.09.95 MBE 1 MBE 3 MBE 7 MBE 9 MBE 10 MBE 12 MBE 13 4 l0 2t 10 t6 l0 10 7 TOTAL 81 03.09.9s 0s.09.95 05.09.95 06.09.95 31.08.95 09.09.95 KAB KAB KAB KAB KAB KAB 2 J 5 6 7 18 10 10 18 31 10 6l TOTAL 146 6REMARK Each larva was cut into two, the head for DNA determination and the abdomen for Cytotaxonomic identifi cation. TOTAL NUMBER OF ADULTS COLLECTED TABLE 3 KAB POOL : Three dark wing-tuft flies with few eggs pooled. 04.09.9s Nos.4,9,11 19 22 4t 46 DATE No MO/THER ,FLY FEMAL ES MALES TOTAL o//o F'EMALES 06.09.95 07.09.95 08.09.95 08.09.95 04.09.95 MBE 1 MBE 3 MBE 7 MBE 10 MBE 12 0 1 6 15 4 I I 4 13 J I 2 10 28 7 0 50 60 54 58 TOTAL 26 22 48 54 05.09.9s 06.09.95 08.09.95 08.09.9s 05.09.9s 10.09.95 KAB 2 KAB 3 BAK 5 KAB 6 KAB 7 KAB 18 58 l9 20 40 46 16 40 JJ 40 66 88 21 98 52 60 106 134 37 59 37 33 38 34 43 TOTAL 218 310 528 4t MBE AND KAB (POOL) : Eggs of Six Pale Wing-tuft females pooled. 703.09.9s MBE: 2,8, 7l KAB: 1, 13, 15 6 l5 2t 29 GRAND TOTAL 269 369 638 42 CYTOTAXONOMIC,IDENTIFICATION RESULTS TABLE 4 MORPHOLOGICAL IDENTIFICATION RESULTS TABLE 5 DATE LOCATION No IDENTIFIED CYTOSPECIES So (Beffa) 31.08.95 31.08.9s M'BETEKOUKOU KABOUA 64 94 64 94 (100 %) (100 %) TOTAL ls8 rs8 (100 %) DATE LOCATION No IDENT IFIED MORPHOSPECIE S o//o sou/sQU A o/ ,/o SOU / SQUA SAV 2-8.9.9s 3-14.9.95 M'BETEKOUKO U KABOUA 2t 60 20 58 1 2 95 97 5 J TOTAL 8l 78 3 96 4 8OBSERVATIONS / 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-tufts (3 from M'B6t6koukou 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. - Laboratory rearing of S.damnosum (s.1.) requires regular supply of electricity. In the present study, there was no iy'terruption and everything went on well with the ent/re rearing system. - An aerated river water is needed at all times to change the water in the rearing system once a day for the bigger 2 I botlle and twice a day for the small I I bottle. The water must not be fetched from the rivers under larviciding treatment. 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'B6t6koukou were insufficient as compared to those from Kaboua. - The engorged simuliids from M'B6t6koukou and Kaboua were induced to oviposit on27 artd 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 : Provision of material for cytotaxonomic determination, morphological identification and DNA analysis. Species identification of reinvading flies Distribution of biting populations of S.damnosum complex. Host preferences of species within the complex. Vector potential of S.damnosum complex species for Onchocerca volvulus and Onchocerca Ochengi. I 2 J 4 5 9CONCLUSION The work was effectively carried 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 Ou6m6 River basin (M'B6t6koukou and Kaboua) gave 100 % result as S.so-ubrense (Beffa), a forest cytotype. Eighty-One (81) adult females from the same reared r*pf"r were morphologically determined as 96 oZ forest and 4 oZ savanna species. The two results seem to be almosi in concordance to prove that the simulium damnosum populations with dark wing-tufts in the Out/m6 River Basin Area belong more to the forest grogp than to the savanna species as shown by DNA determinations. 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 established in Kara, Togo. RECOMMENDATION - The 4%o finding by simple morphological identification of reared females with dark wing- tufts as savanna species has to be further investigated. This is likely to be the same case as S.souamosum in the Southern Extension Zone (Ghana and Togo) where some populations were f**O Uy the same identification method (Morphology) to have possessed both dark and pale wing-tufts. - The 60 % finding of hybrid populations of S.damnosum (s.s.) and S.soubrense (Beffa) also calls for thorough invesiigation. This is very important because it will provide useful information as to the vectorial role oi these hybrid populations and the exact strain 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 statistic analysis, we recommend that the Rearing Work be repeated next rainy season with much emphasis on the rearing of females with pale wing-tufts. 10 ACKNOWLEDGEMENTS We wish to expres our sincere thanks to Mr. Barro T616 for helping to set up the rearing system at Bohicon and to Mr. Sedou Naniogu6 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 thier encouragement and valuable support. We are especially indebted to Dr. Laurent Toe for the DNA determina-tions ; to Dr. A.K. Diallo, the OCP Sector Chief of Parakou and Dr. L. Assogba, 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 in the field. ,' I -7- l- l- AI\IIYEX 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. t2. 13. 14. 15. 16. 17. 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 Jerrycans Plastic tubes (Small size- 0.8 C) Plastic tubes (Medium - 1.2 C) l Generator (As standby for electricid "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) Propsection glass tubes (25 ml) supply) Epandorf plastic tubes Washing bottles (For holding alcohol) Washing bottles (For holding water) Rubber bands (1 Pkt) Pipettes Rubber-teats Machine oil Plastic bags @n o o {pto '1 . PROGRAMME +-. ON CHO -BEN IN +.- AURKINA FAS O U , Port<i t{wo KAB OUA TEKOU KOU NIGER \ I t* ll o ro \ l(ondi O lo i (l i (rrgotJ TOGO rcP \ k l.rJ $ qJ t i i I i i o ( <, \ AR/r o !, ac S{o .e s.ro f o5 ,o o lo g o C oJ 30 50 tOOkm