Laboratory rearing of S.damnosum complex in the eastern OCP

WORLD HEALTH ORGANIZATTON AFRICAT{ RECTON ORGANISATION MONDI.ALE DE LA SANTE REGION DE L'AFRIQUE@ oMS / ONCHO, B.P. 36 KARA, REPIJBLIQITE TOGOLAISE TEL. 60.60.7 51 60.06.26 (Fax.60.06.2O LABORATORY REARING OF S.DAIVINOSUM COMPLEX IN THE SOUTH EASTERN ZONE OF THE ONCHOCERCIASIS CONTROL PROGRAMME. AUGUST TO OCTOBER 1996 AND JANUARY TO FEBRUARY 1997 by Fiasorgbor Bit6ou Naniogud Adji N" 136 /OCP/KARA/ZONE EAST February 1997 roN G. S. S. A. ,\ .f !,o (.. rn '(') c) RATn-g \C'..r). t , I I 2LABORATORY REARING OF S.DAMNOSUM COMPLEX IN THE EASTERN OCP The DNA technique is applicable to both larval and adult stages. Studies are therefore intensified throughout the OCP to improve the technique and have it fully operational. To achieve this objective requires pure populations of the different members of the S.damnosum (s.1.) complex. Since it is at times difficult to obtain pure populations, the OCP, according to recommendation N" 6 of the Entomological and Insecticide Research Meeting of Bouake in May 1996 has resorted to series of laboratory rearing of the various species in an attempt to provide the DNA laboratory in Bouakd (C6te d'lvoire) with sufficient larval and adult samples for the study. In the Eastern OCP the laboratory rearing of the members of the S.damnosum complex started in 1995 in the Ou6m6 River Basin at M'Bit6koukou and Kaboua from where dark wing- tuft females were earlier identified by DNA probes (heteroduplex technique) as savanna species (S.sirbanum) a result which was contrary to cytotaxonomic finding which recorded the same populations as the Beffa form of S.soubrense. In 1996 the rearing work was repeated at the two sites in the Oudm6 basin (Benin) in addition to two new other sites, Tsatsadu Falls on R. Tsatsadu (Ghana) and Kouniohou on R. Ban-Houo (Togo). The actual work at the last site, Kouniohou was carried out in January/February 1997. Three species of S.damnosum (s.1.) complex have been successfully reared from egg to adult under laboratory conditions. These were S.soubrense (Beffa) from M'Bdtdkoukou and Kaboua situated on the Oudm6 and Okpara respectively in Benin ; S. vahense from Tsatsadu Falls in Ghana and S.squamosum from Kouniohou (R. Ban-Houo) in Togo. A _!tS sqme technique for rearing as described in our Mission Report No! | 509/OCP/Kara/Zone East December 1995 was again employed for the investigations in-1996/97. I L__ We report below the results of the study undertaken in the four different river basins (see Tables l-15). I. M'BETEKOUKOU (R OUEME) I 2 3 4 5 6 7 8 9 Date of collection No of females caught Date induced to oviposit N" of single egg batches obtained N" of egg batches pooled Date eggs hatched into larvae Date started feeding Date larvae started pupating Date of emergence of first adults : 15108196 :60 : 1818196 :5 :8 : 2018196 (morning) : 20/8196 (afternoon) :3018196 : l/9/96 10 ll 3 Date of emergence of last adults : 919196 Cycle of development from eggs to adults ranged from 18 to 26 days for the whole egg batches. Cytotaxonomic identification : 8l larvae examined 100% S.soubrense (Beffa) Morphological identification : I I females examined : 9 sou/squa (82 %) 2 sav. (18 o/o) 44 larttae examined 100 % S.soubrense (Beffa) 4l females examined 100 % sor-r/squa. Date of collection : 1819196 N" of flies caught : 7l Date induced to oviposit :2119196 N" of single egg batches obtained : 4 No of egg batches pooled : 6 Date eggs hatched into larvae :2319196 (morning) Date started feeding : 2319196 (afternoon) Date larvae started pupating :6110196 Date of emergence of first adults : 8110196 Date of emergence of last adults : 19110196 Cycle of development from eggs to adults ranged from 2l to 32 days for the whole egg batches. cytotaxonomic identification : 108 larvae examined l0o yo S.vahense Morphological identification : 20 females examined 100 oz S.yahense. 4. KOUNIOHOU (R. BAN-HOUO) 2. KABOUA (R. OKPARA) 1. Date of collection : 14181962. No of females caught :243. Date induced to oviposit : 17181964. N" single egg batches obtained : 55. N" of egg batches pooled : 06. Date eggs hatched into larvae : l9l8t96 (morning)7. Date started feeding : 1918196 (afternoon)8. Date larvae started pupating :29t81969. Date of emergence of first adults :3118t9610. Date of emergence of last adults : 9/9t9611. Cycleof developmentfromeggstoadultsrangedfrom 18 to27 daysforthewholeegg batches. Cytotaxonomic identifi cation Morphological identification 3. TSATSADU-FALLS (R. TSATSADU) t2 l3 12. 13. l. 2. 3. 4. 5. 6. 7. 8. 9. r0. 11. 12. 13. I 2 Date of collection N" of flies caught 22nt97 62 43. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Date induced to oviposit :2511197 N" of single egg batches obtained : 5 N" of egg batches pooled : 8 Date eggs hatched into larvae :2711197 (morning) Date started feeding :2711197 (afternoon) Date larvae started pupating :712197 Date of emergence of first adults : l0l2l97 Date of emergence of last adults : 19/2197 Cycle of development from eggs to adults ranged from 20 to 29 days for the whole egg batches. Cytotaxonomic identification : 70 larvae examined 100 % S.squamosum Morphological identification : 30 females examined 100 % sor.r/squa Results (See Tables I - 15) Table I : Gives details (labels or codes) of the 13 dark wing-tuft mother flies from M'B6tdkoukou. Table 2 : Gives details (labels or codes) of the 5 dark wing-tuft mother flies from Kaboua. Table 3 : Gives details (labels or codes) of the 1l dark wing-tuft mother flies from Tsatsadu-Falls. Table 4 : Gives details (labels or codes) of the pale wing tuft mother flies from Kouniohou Table 5 : Gives details of the total number of larvae collected from the 13 egg batches from M'Bitdkoukou for cytotaxonomic and DNA identifications. Table 6 : Gives details of the total number of larvae collected from the 5 single egg batches from Kaboua for cytotfionomic and DNA identifications. Table 7 : Gives details of the total number of larvae collected from the I I egg batches from Tsatsadu-Falls for Cytotaxonomic and DNA identifications. Table 8 : Gives details of the total number of larvae collected from the 13 egg batches from Kouniohou for cytotaxonomic and DNA identifications. Table 9 Gives the total number of reared adult simuliids from the 13 egg batches from M'Bdt6koukou. Table 10 : Gives the total number of reared adult simuliids collected from the 5 single egg batches from Kaboua. Table I I : Gives the total number of reared adult simuliids collected from the I I egg 52Table 12 Table 13 Table 14 Table 15 TABLE I TABLE 2 batches from Tsatsadu-Falls Gives the total number of reared adult simuliids collected from the 13 egg batches from Kouniohou. : Gives the overall total of the number of adult male and females obtained during the study. : Gives the final result of cytota,xonomic identifications of all the larvae collected for the study. : Gives the final result of morphological identification of adult females collected from the study. M'BETEKOUKOU FET\4ALES WITH DARK WING.TUFTS MORPHOLOGICALLY SCORED AS SO/SQ NUMBER OF MOTHER FLY SPECIES MBE N" I MBE N" 2 MBE N" 3 MBE N" 4 MBE N" 5 MBE (Pool = 8) forest lt ( I il il KABOUA FEIvIALES WITH DARK WING-TUFTS MORPHOLOGICALLY scoRED AS SO/SQ NUMBER OF MOTHER FLY SPECIES KAB KAB KAB KAB KAB Nol No2 No3 No4 No5 forest tl tt I ( I 6TABLE 3 TABLE 4 TSATSADU-FALLS FEMALES WITH DARK WING-TUFTS MORPHOLOGTCALLY SCORED AS YAHENSE NUMBER OF MOTHER FLY SPECIES TSA N" 1 TSA N' 2 TSA NO 3 TSA NO 4 TSA NO 5 TSA N" (Pool =6) forest il ll il il KOUNIOHOU FEMALES WITH PALE WINGS-TUFTS MORPHOLOGICALLY SCORED AS sou/sQ NUMBER OF MOTHER FLY SPECTES KOU NO I KOU N" 2 KOU N'3 KOU N" 4 KOU N'5 KOU (Pool : 8) forest I lt I il ll ll 7TOTAL NUMBER OF LARVAE COLLECTED FOR CYTOTAXONOMIC AND DNA DETERMINATIONS (abdomen for Cytotaxonomic Identification and head for DNA determination). TABLE 5 TABLE 6 M'BETEKOTJKOU : MBE NUMBER OF MOTHER FLY N' OF LARVAE COLLECTED MBE NO I MBE N" 2 MBE N" 3 MBE NO 4 MBE NO 5 MBE (Pool= 8) 10 l0 I 0 10 50 TOTAL 8l KABOUA = KAB NI.JMBER OF MOTHER FLY NO OF LARVE COLLECTED KAB IGB I(AB KAB KAB N"l No2 No3 No4 No5 10 l0 4 r0 10 TOTAL 44 8TABLE ? TABLE 8 REMARK Each larva was cut into two, the head for DNA determination and the abdomen for cytotaxonomic identifi cation. TSATSADU.FALLS = TSA NUMBER OF MOTHER FLY N' OF LARVAE COLLECTED TSA N" I TSA N' 2 TSA N' 3 TSA N" 4 TSA N" 5 TSA (Pool = 6) 10 5 0 57 0 36 TOTAL 108 KOUNIOHOU : KOU NUMBER OF MOTHER FLY N" OF LARVAE COLLECTED KOU NO 1 KOU NO 2 KOU NO 3 KOU NO 4 KOU NO 5 KOU (Pool: 8) l0 2 r0 6 10 32 TOTAL 70 9TABLE 9 TABLE 10 TABLE II TOTAL NUMBER OF ADULTS COLLECTED FOR MORPHOLOGICAL IDENTTFICATIONS AND HETERODUPLEX ANALYSIS M,BETEKOUKOU = MBE KABOUA = KAB TSATSADU.FALLS = TSA Number of mother Females Males Total oZ Females MBE N" I MBE N' 2 MBE N" 3 MBE N" 4 MBE NO 5 MBE (Pool = 8) 2 5 0 0 4 22 8 I 0 0 6 2t l0 6 0 0 l0 43 20 83 0 0 40 51 TOTAL 33 36 69 48 Number of mother fly Females Males Total oZ Females KAB KAB KAB KAB KAB No No NO No No 1 2 3 4 5 25 25 J t2 8 50 47 t4 l0 t4 75 72 t7 22 22 33 35 r8 55 36 TOTAL 73 135 208 35 Number of mother fly Females Males Total o/o Females TSA N" I TSA N" 2 TSA N" 3 TSA N" 4 TSA N" 5 TSA (Pool: 6) 28 0 0 68 0 52 t4 0 0 78 0 45 42 0 0 146 0 97 67 0 0 47 0 54 TOTAL 148 137 285 52 r0 TABLE 12 TABLE 13 TABLE 14 KOUNIOHOU = KOU N.B. : The rearing systems of troughs I to 4 stopped on their own in the night of l/2t97. As a result, no flies were obtained from them for the study. GRAND TOTAL CYTOTAXONOI/flC IDENTIF ICATION RESULTS Number of mother fly Females Males Total oZ Females KOU N" I KOU N" 2 KOU N" 3 KOU N" 4 KOU N' 5 KOU (Pool = 8) 47 33 55 35 t02 68 4; 49 TOTAL 80 90 170 47 LOCATION Females Males Total oZ Females M'BETEKOUKOU KABOUA TSATSADU.FALLS KOUNIOHOU 33 73 148 80 36 135 137 90 69 208 28s t70 48 35 52 47 TOTAL 334 398 732 46 LOCATION No IDENTIFIED CYTOSPECIES SO (Beffa) SQ YA M'BETEKOUKOU KABOUA TSATSADU-FALLS KOI.JNIOHOU 8l 44 108 70 8r o: 70 108 TOTAL 303 125 70 108 LOCATION N" IDENTTFIED MORPHOSPECIES % Sou/Squa % Sa % Ya Sou/Squa Sav Ya M'Bdtdkoukou Kaboua Tsatsadu-Falls Kouniohou ll 4t 20 30 9 4t 0 30 2 0 0 0 0 0 20 0 82 100 0 100 l8 0 0 0 0 0 100 0 Total r02 80 2 20 78 2 20 ll MORPHOLOGICAL IDENTIFICATION RESULTS TABLE 15 OBSERVATIONS / COMMENTS - A total of 732 adults was collected. This is made up of 334 females and 398 males. As already reported in 1995 males were again the most numerous during the first few days of emergence. The females were later predominant. - Apart from the 5 advantages stated in the 1995 report, larvae reared under standard conditions could be used for susceptibility to insecticides, and the adults, initially free from infection with filarial parasites, could be used in transmission experiments. - lnformation on the length of larval life of the simulium vectors of Onchocerciasis (duration of immature stages) is of fundamental importance in the planning of control campaigns. CONCLUSION - The work was effectively carried out in perfect condition and the objective seems to be achieved. Cytotaxonomic identification of 125 larvae of S.damnosum reared from females with dark wing-tufu in the Ou6md river basin (M'Bitdkoukou and Kaboua) gave 100 oZ result as S.soubrense (Beffa), a forest cytotype. However, 52 adult females from the same reared samples were morphologically scored as 50 (96 %) forest and 2 (4 %) savanna species. - Results of all the 108 larvae and 20 adult females from Tsatsadu-Falls came out cytologically and morphologically as 100 oZ S.yahense. - Results of all 70 larvae and 30 adult females from Kouniohou came out cytologically and morphologically as 100 7o S.squamosum. t2 RECOMMENDATION - Chromosomal differences in the larvae remain the most reliable taxonomic characters available. However, it is the adult females coming to bite man which the Programme needs to have identified. For this reason, the laboratory maintenance and rearing of S.damnosum complex species, combined with the cytotaxonomic determination of reared larvae, has proved to be an important research tool for OCP. t.j E{ ax -oc *oc $;i$EiFd €aeF F+* "l ! u9.u *Fg e -ata t TEF 11.il -8!t E sp 6 Fa e a EE ss 685e:2EE tail rg (, g>3oLz e.9 eq tt af, &- <o 9>f= \=v qq 5= €G e.E 6; no tq G,z1 -lCot:z t{ P2 i9 90 ;9 So <E Dvt -le 'xre!rE>OclCO>e b9 6G>agt >clzno= :ztr Eg5Cl x3 x8 EOFSoy EE =.-ttl ,vO1o "E s?6= a=6: a€l :a oo \oI .D oe oq0 SEqebq ir\ D)E?u106 oo ?(! E ? D)(D !!ITlo AA zE< .ES ur lctsf1E.lAAoo zE< EE (1 Firle3 EFE -Ff (1 Bi5Ela!eeE EFEcFf >z oO E= -=OO oOGG<;' 6'!.e$iq !gri z'z 60 != 60 ooGtG<;' Fc.e$Ei €P_$* GI F:Z-) -lCOU? ttl P2ol 9Ei9 ;9 "tP<E >'u, -lC4vU,)>a -l>U,)>atr>F9 't! \->Ert->rt-V'F >u,ZF'tJ t'lxxooCEzz oo ooCC EE>a ,z xo b9 Ea =s I o.rar,a s 1;a; a 3a a a 3'. IgFg aafi oolt3ltrlrlGI ! u9.u *r8 FtEqB .i rsF 11.d - (I 8.3 IoccZ ^9 oc oooo ss?? ee ss?? ca a3 CCBB T t s t5A 8 s (J1.fe c9 co da:=3. =.a5 e6 9= :6s;- e.E E! (! OQGIf;EoQhai? F6-T?!c(!(!o EF11lc(! oq ll12aoAA oOoo zE< E=E=(t; q D!t1IDrlAA{{60 z .! .<- fE o FiECtt eE i*6={r6 o FEEO!a .=! EiF -Ff <3 oO E= -J60;t cT. Fq.6!.d9 E=t* <=oo EE -E606-a aT. Fgeqei €EE: 2z>Y I rI o tcon a -'1 lo r . PROGRAIvI ME t TOGO ,"1 ONCHO -BENIN "l t5 <. 1 N/G ERg" t? (o BURKINA FASO rJ I$ h t(on di a t," ) e, ri q' ngou t{kki \ g rO o ( v' ; I I I I I d \ q l.{ $ t a no o A.R,/r g i t t eo ldr KAB OUA TE KO U KOU i o .e i ifo o ,q It o o o 50 l0o km t Porlo lt{ovo ''l a coToNorJ 31 I 16 -)/\ L- t -?. oBuRr< t'.J o \t a- \ Q t o( 7 ) t q" rrrq i c ! .o a. io5 B E $ Tornole c N *o 00 I ACCRA Sites for the reorang of S domnosum complex A coTorlou LOME LOCATI ONS 1. TSATSADU FALLS 2. KoUNIoHoU 3.M,BETE KOUKOU 4. K AB OUA filole G H A olzotl o oq o o e :' N N o Pru / d --, 5eo€ \ I l I Koro rirou o: o I I ) / 3 I o c "(o 0 s T .J\, 2 1 I I urn ,o d o a. S e kondl rn the Sou th-Eostern Zone of the O C p(t996) ( oo