WHO onchocerciasis control programme research reporting: rearing and colonization of simulium damnosum complex and related investigations: April 1980 - June 1980

WHO OI{GIOCENCIASIS CONTNOL PNOGRAIflIE RESEABSI BPONTING. Subj oot I Instituti ont Personnel: Date of submission: Period oovered! Signature of responsibl e officerl Searing and oolonization of Simuliurn dalnnosum wHo/ocP. J.N. R3ybould. 1st Augustp 1980. April- 1980 - June 1980. ./"/.;/."i- .fiird-'.t)ta'a a a a a rra a a . a a a a a a a A. B. CONIENTS rN[nolxrcrr 0N aND srrMllanY. [qA IUPNOVEMEIII AND S/AIUABION OF REARING TECHNIq'ES. lt Eearing single sgg batches. 2. F\rrther evaluation of techniques for overooming the effect of powel failures. 3. Sstab1ishmsnt of a rsaring laboratory on a pontoon on the Volt a l,ake. 4. Bearing worlc at Natitingou and Bobo-Ilioulasso. 5, Continuing searoh for othsl Isboratory mating populations of S:-,gggEgg s. r. 6. The problem of pool egg developrnent. sI'tJutE oN THE WINC'.TUpT Co[oURATroN 0F S. SqrAMosuu. '1. Investigatione on rea!ed fIios. 2. Investigations in the field. INSEGIIoIIIE SUSCEFTIBTIIIY TESTS ON BAI{DAI{A AND COMOE, NIVENS. S. DAI{NOSJU S.L. LAEIIAE T'NOU fiIE G. E. UTSTRIH'rION OF X'IE[,T COLLECIED }TAI8RIAI VIITH PANTICUIAA ns'EnENcE r0 !:_EgIEgE3. F" IIIVBSTIGATIONS BY. V.R.A. SIAF'F AT AKOSOMBO CARRIED OUT IN CONSUI,EATION WIIH OCP. f. Studies on the wing-tuft colouration of S...Fqyj4.qFgm at Boti X'alIs. 2. The reoolonization of Boti E'alIs by $.---sEreflo-F9.{ eftel the resumption of river fIow. 3. Natulal- oontlol of fl:_ _{apno_*g breeding sites at the Senohi and Kpong Bapids. G. SUPruY 0F IIIAT&1RIAL T0 COLLABOBATING WOBKERS. H, TNAINING AND VISITONS. I, UEE,TINCS ATTENDM" J. ilCffi PLANNSD FoR NEm qJAmER. K, ACKNOWtmqmlU,ITS. ns'EBENCSi. TABLES. A' INTNOIIICTION Bearing resul.ts during the period under review have been the best to date. In same instances 9O/" of hatched larvas have survivecl througlr to the atlult sta8o. Growth has also frequent}y been rapid with pupation from nine claJs (afte! hatchlng) orwardsr llhese improved results have been obtained at Natitingou as wel-l- as Alcosoobo. Technigies developed to overoome the effeot of power failures have proved to be successful in praotice at Akosombo anil elsewhere. Better rearing vesults have facilitated investigations on the inheritarce of the wing:tuft colour character in !:_.gSEE9!g. A rearing laboratory established on the VoIta Lakee using water pumped directly from the Lal<e has given rathel ilisappointing results. This ooulil be related to a turn-over of the lake whioh killect S. damnoEum 1&rv,?s in nearly breeding sites. Susoeptibility teste carried out at Bouake'on from the lower Bandamas oonfirrned the presence of a Popul ation. Tetetou (on the Rivel rno) for It is hopetl that a naer slte'o? for tbis purp,rse S. soubrense has at Iast leaohed an adequate density at S. sanotipauli I esi stant use in attsnpted colonization work. the Biver Pra wll1 also'yiel.C S. soubrense Jr Br IHE I}{PNOVE[I{EN3 AN3 5{IALUAT.ION.OF BSARING TECIINIq]E5 Realing sinile egg batches. Studies on the wing-tuft colouration of Sr.scfl:ralllosum (see section c) have invoLved tre learing of single egg batohes' [his plovided an opportunity for the fulther evaluation of rearing techniqtres to whicb oertain modifications have recentlJ been made. The results obtained during the periocl uncler reviell have been the best to date. 2- sulvival rates in the labolatory are difficult to measulo beoause manJ laboratoly laicl eggs fail to develop, possibly because they are not fertilized. It is therefore necer,saly to lcnow how many eBBs hatchr 1.athel than the numbet of eg€s added to the leaIin6 gystdn. Howevel , in one oacie where arr atiernpt w3s made to count the very ealLy staSe lalvaer a figule between sixty and seventy was obtained. Fifty-eight of the larvae ooncerned were suooessfully xeared to adults and six others developecl fully but failed to emerge from the pupa. A second e88 batch was not added to a lea1ing systsn until the number of developing eggs hail been oounted as aocuratery as possible (a plocsflure rendered diffioult by the fact that e88s are sometimes laicl on top of eaoh other) and a figure of about 127 was obtained. The number of pupae subsequentLy leared was 118 r all but foul of wiiioh gnerged as adults. These figures suggest a survival rate from egg to atlult of about )U/, wtth additional individuaLs beooming fully developed but failing to emerge from the PuPao As well as good survivslr deivelopment has been rapidl with the first pupations sometimes ocourring on the 9th day after hatchinS' t{herr adoquate food has been providedl development has also been higbl-y synchronous with all adults emerging witliin a few days of each other' The r easons f or the imploved r esul ts ar e not entir eJ-y o1 ear but are pregumably tslsted to good nutrition. Ttre aIga] oultur" ussd waE mirture of chlorell-a enrersonii ( oulture 211/8h) r .aI51SE9SSSSSE oonvolutusandaspeciesofGcillatoriatogetherwithmisoellaneous Protozoa. Ttre use of wooden rearing trouglrs Bay also have been benefioial (see below). &cellent rearing results have also been obtained at Natitingou ( seetion 84). -3- 2. Further eva,luation of techni 6s fol overcomin the effect oI powel IalluIeE. The valious teohniques designed to overcome the effect of power fail.ures and described in my ptevious reportr have been fulthel tested tluling the period under raviow. Wooden rearing trouglrs hsve been found tc not only dry out Iess rapidly than plastic materiaLs when the water flow gtops during a power failurel brlt also to support a more profuse surface growth of microorganisms. [he lattel charactoristic m4y result in better larval nutrition than is thc oase when Flastio mateliaIs are rsed. A remaining problem, howevor, is that hard dark woods mako it difficult to see the larVae. Convelsolyr Light coloured soft woods rot rapidly i.n water. We are therefore searoliing for a wood whioh is hald and resistant but light in oolout. The automatic switch-over 6ysterl has been found to worlc extremely well except in the case of exceptionally 1ong power failures lrhich may run-down the battery. Circuit troalds inoorporating the swith-ovel :ystem have been supplied to both Natitingou and Bobo-Dioulasso. The 'rautomatic floodi.ngtt method has proved to wolk very well exoept, ouce againr in the case of exoeptional-Iy long powe! failures when plolonged Iaok of watsl flow msy cause high mortalities. 3. Establishnent of a rearing Iabolqle1y on-a pontoan on the In spite of greatly improved resuLts with our Iaboratory realing systemsr it is not yet possible to completely standardise oonditions. 1Ihere are often oonsiclerable differenceE between the results obtained from two separate learing systems in the same Iaboratory 2t the same time. In 3n stte'npt to obtain the saltte conditions in different realing systons operating simultaneouslyr - 4- a raaring laboratorJ has been established on the Volta Lake so that vrater 6sn be pumped direotly from the lahe to the rearing qystems which overflow back into the Iake. Initial results have been disappointi.ngt giving slower development thsn we usually aohieve in the laboratory. It is alifficult to interprst these results, howevor, in visw of reoent radical chsn5es in the chemis-'ry of the lake water (see section F.3). F\rlthel investi6;ations will be oarried out uhen the lalce leturns to aolrnaL r 4. Rearing work at Natilingou-and Bobo-Dioulasso. Rearing work in conneotion with the reinvasion studies has been oarnied out on a congiderable scale at both Natitingou and Bobo-Dioulasso by Messrg. T. Barro and B. Sawadogo respectiveLy. At ths time of writing l-arvae 11srn three leoations have been !eared at Bobo-nioulasso and results at Natitingou have been exoellent. Larval development has been rapicl with sone pupation only nine days after hatching. Up to about 3OO late instal larvae have been obtainod per rearing system and on one oocaslon 22O Larvae were obtained from only thres 'batotleO, llh.e automatic flooding msthod has given partioularly good results, Mr. BarIo has been feeding with a mixture of algae and rrf st,33rt a vegetable diet for tropical fish. ,. Continuing search fg:;>Lqgl*+3loratory mating pop S. soubrense is the only lr'iest Afrioan membel of the S. damnosum complex hnown to mate (albeit in small number) in the l-aboratory' Al-though most ottrel msmbers of the oomplex have been investigated for Iaboratory mating many times without positive rosultsr it is possible that this oharactelistic may be confined to certain populations within the speoi€s Gorlc€ro€do - 5- Dl, Grunewald of Tubingen university has volunteered to collabolate in a continuing searoh for laboratoly mating populations of &-1}E48833 s.1. in li{est Afrioa. Pupae oollected from the fie}'l are plaoed in msting cageg snfl the emelged adults given time to mate' llhey ars tiisn placed in aloohol and later examined for spermatophores and for sperm in the spermath,:oa. species recentlJ sent to Tubingen for examination in this respect have inoluded S' sEla'nesumt S. damnosulll sr Sr and S. sanctipauli' 6. The ploblem of poor egg de\,919!gsg!' }'lhen members of tir" E-SryEg! complex ard other simuliids are artificially induced to oviposit in captivity, mapy of the eggs obtained fail to develop. Tho reason for this is unkncwn but noE- fertilizationcouldbetheC&llsorEggshavethereforebeenfixed just aftel oviposi,tion for oytologioal elcalnination by }I. R. Post of the Liverpool ScirooL of Tlopical Medioine' 3r' Post hopes to determine whether or not *.", of the eggs are unfeltilized' I' Investi€ations o4-rser-e'l fIi es' .- ['his investigation is being carlied out in collaborations with Dl. Kurtak. As was discusse'l (in more detail) in my pr eviou report t the variable wing-tuft colouration of lLg^ggggoould be normal valiationwitninasinglespeciesolduetothepresenoeofmorethan on species. To throw f r:lthel Ii 6}rt on thi s matt el r the separate realing of single egg batches of .E:--ESgggg from Boti FaLls has continued. fhe wing-tuft colouration of both the fsnale Palent and her labolatory reared progeny, has been scored using the following sYstem: C. strrrsrEs oN lrlE lrrNC-!!u--99LNAATr9I -6- A = aLI Pale hairs B = pale with up to ) dark hairs C = mixed pale and dark hairs D = dallc with uP to 5 Pale hairs E = aLI dalk. Besults obtained by the end of the period under review are given belowl Parent No. of ProgenY Male ProgenY Ferna} e progeny I II III IV v \rI vII 3 5 4 16 64 14 B A B B E A B B 2& 4ts zfi 1A,6E 12Ar7 A-Br 10B,{C 4E 7E 1B jB,1I) 1A-Br 1E 2Cr 1Dr6E 26A12a-Bt2Bt 7C 1A-Br 43tZCt 1nr 2E 1E In addition to the above, 118 progeny have been reared from an s. souamosum fgnale from Fazao in Togo but they hsve not yet been 6coled. -E-&- Althouglr final interpletation of the results is best left until the investigation is complete it is already clear (as was mentioned in my previous repolt) that the iliff erent types of wing-tuft can oocur in the offspring of a single f]y and do not Iep1esent separate populstions of the oytospecies. It is also evident tirat the wing-tuft oharaoter is expressed diffelently in males and females' In view of the possibility tirat water temperature (or some othel onvironmental factor) mqy influence wing-tuft colourationr !:-i.!B34.9.gllE is now being reared at diffelent temperatures' -7 - 2,. Biting oatches at Boti Falls by &Ir. John Adiamah and his tean from the Publio Health Laboratory at Akosombo have continued to dater As has been pointed out in earli:rr repoltsr the purpose of this work is to investiga,te po ,sib1e seasonal clranges in wing-tuft colouration. A ploblem that has baoome app rent, howeverl is that in oertain seasons the number of biting females is too low for the results to be statistioal-1y significant. In addition to reguJ-ar Larval oolleotions frorn Botir therefore, we ale ma-ting monthly collections of pupae which are usually available in laI8er nurnbcrs than biting females' [he adults emerging from the pupae are being sent to Dr. Kurtak for studies on the wing-tufts. An advantage of t,,is technique is that information is obtained on the males as well as the femaLes. Ihe results obtained eilould relate to the field situation unlus6 the temperature to which the pupae zre subjeoted influencesthe wing-tuft oolouration assumed. Repeated f ailures of oontrol of rS. dalnnosum s.I. in the lower Bandarna River neoessitated urgent investigations on the insecticide susceptibility of the population oonoerned,. Tests vrsre therefore oarried out on larvae flom chutes Gauthier in the Bandamal whsls the oontrol failuree hail been ";rpeliencedl and (for comparison) on IaIvae from A16p6:.r, the River Comoel The Iarvae were tlansported to Boualce' by helicoptel and tssted in th€ 0.C.P. premises there. This work was carriecl out in collaboration with Ml. M. Ouedraogo' These tests suppLement investi5ations by Dr' P'" Guillet 3t the qsstltut de Becherohes sur IrOnchooe!co6€rr' The technique employed was based on that of Mouohet et a1r ($11) in whioh a three hour exposule period is used,. Beoause earlier tests by Gui-Ilet hacl suSgested N. INSECTICIDE SUSCEPTIBILITY ON 3Atr{NOSUM S.L. I,ARVAE -B- a lower resistanoe Ieval thsn thst apparent in the fielcll an atlditional holcling period was added to the test to determine whether thole was any subseguent leoovery of moribund Iarvae. Tests wore carried out using alcohol solutions of Abate flom a WilO insecticide susceptibility test-hit. A few Preliminary tests were also perfoxmed using dilutions of an ernulsifiable conoentrate of Chlorfoxim taken from a treatment drum. In the time available it Has only possible to do one rePlicate of most tgs{s. fhis work has already been presented in a separate report to V.C.U. which detaile the methods used. The results showed considelable difference in susceptibiLity Ievel between the larvae from the two sitsg (fatfe I & 2). The concentration-mortality legression line (presented in my original report) obtained for the Conroe'Larvae was ohalaoteristic of a susoeptible population w:rile that fol the Banclama larvae indioateal a resistant population. Cytotaxonomic identifications of the Iarvae (falf e 3) show that the population concelned i" Lggllilggli. Moltalities at the end of the nine hourg.rolding psrlod (not detailed in this sunmary) were much hiBher tnan those reoorded immedi:te1y after exposure providing no evidenoe of subsequent rooovery ofmribund Iarvae. AI1 ooncentrations of Chlorfoxim tested gave lOVl mortality. The results of the few tests perfolmed su8rsest that therrMouohet methodtt is both adequate and convenient in its original folm without the addition of aeration systemsl holding peliod stc. White 8lass bowls readily available in Boualce'were found to be partioulalIJ suitable for thetests. Mortalities with Grlorfoxim were muoh higher than expectedr possibly beoause dilutions of the emulsifiable -9- ooncentrate wele used and not the :.WHO test-ki.t al oohoL solution. Nevertherlessl the lesult grlowed that the Chlorfoxim tested was stil} potent; the assessment of which was the purpose of the test. 1It. = E #H#+ffii#t#h*-ffisdetiffi TFrospection in searci: of &_SlglgSSX breeding sites have continued as adequate supplies of this species are required for in".,esti€ations directs6 towards collonization. It m3y be seen from table {, that at Tetetou on tne Mcno, the few lr,rv2s found fluring a visit in March were mainly S. .d.aqlissum. Since that time the number of Iarvae pnd pupae present have increased greatly as has the proportion of S. soubrense in the population (fatte 4). Unfortunately S. soubrense has not returned to the Senchi Rapids near Altosombo nor has it beooute numerous in the Sio fiiver near Palime. For the time being, thelefolBr we!e are dependent on msterial from Tetetou for worlc directed towards colonization, S. soubrense being the only known laboratorJ mating species in }iest Africa. Atternpts to ooll:ot from the Rivel Pla in hresteln Grana have hitherto f ail ecl because the breeding sites, al.thougb extansiver ?r€ away from major roads. Eowever, uith the help of detailed maps some accessible sites have now b,.en l"ocatecl and cot l ections made from near the villaee of Hsmsng. The larvae have been dispatched for gytotaxonomic identification. It is hoped tbat S. soubrense wilI be found to be present. The speoies composition of reoently identified (although not necessarily recently colLeo:sfl) material from various locations in Grana and fogol is givea tn Table 4. 0f some interest is the fincling thst many breeding sites near Atakpaoe (Amouoblol Bzirnel Ckpahoe) have pure populations of Sr seua{nosumo fr 2. _-10 _ Fr INVJSTIGATICNS BY VHA STiFF rrT AK0S0MB0 CARRIED OUf IN CONSIJLTi)TI0N l,lITH OCP. Studie.s on the wir-rA-tu{X ggloqrStion of S. Squaros6'r'I-FEiia-- See under secticn C 2r The recolonizati.on of Boti FaIls UV S. S-gggggsg af ter the resumption of-river flofi. So s{*amosurn eg€is and lalvae wore found about erght days afts! the resumption cf rivsr flow. The si ze of the larvae suggested that they hatched from eggs laiC abou-t four days earliel. This supports last Jearrs observations that recolonization by g_lggglgsug occurs very soon aftcr the river starts to flow. Nevorthelgss the subssqusnt buitd up in nurnbers of biting females toot place rathor sI ow] Y. 3. Ielqfq! control of S. damnosum complex brcodtng:sites at the During the monti, of June biting densities of &_3gIIg!jI s.I. built up to a higr leveI. Whgn the rapj.ds wcrc inspeoted on 30 June e886r larvae and pupae wele found to be pr esent in enormous numbers. Subsequent to that timel however, the biting density feII rapidly and a biting catch carried out a fow clays lsterl o1o:e to the rapidsr yielded very few f1ics. tvhen the rapids were again visited on tth Julyr no pupa'e and only a few dead Ialvao and some 0gg6 were found. fhis phenomenon is be1ieved to irxvc been due to a turnover of the lake which also resulted in very high fish mortalities. The destruction of the immature stages of S. damnosurn s.1. in this manner hay Ue mo!o. comthon than is Tsalis^C and c.ul d b^o'firo increasin6ly as morr'd?,mE.are built. -fi, - guPl tY 0F MATERIAL T0 CCllr:BoRriTB[G I{oRKERS Now that my assi stant l,{rr Freeman Mordzif a i s f uIIy conversant with allroutine laborstory and Ji-e]-d activitiesr it is prssible to dispatch m3terial to colLaborating workcrs witliout usi.ng too much of my own time. So dafirrosurn eg€,s have bcen dispatched to Cornel 1 and Idassachusretts Universitissr the London SohcoI of Tropical ldedicine and the Institute of Tropical Itledicinc at Tubingen. Good relring lesults (using the e€{s supplicd) havc bcen obtained by Dr. Grunewald at tubingen and cxceptional progross has been made in Dr. Cupprs departqrent at Cornell University. Coll ections iof So 6guamo6um h-rve b,-en given to Dr. H. Townson during his consultartsh{{l with 0.C.P. I and varlous blackflies (indtuain8 some rare species) have bocn sent to the NaturaL.Histoly Mrseum in l,ondon. LarVae fol oytota:rcncmic studies have been sent to the liverpool Sohool of Tropical Medicine. H. TAJ.IIIINC ,\N} VISfORS Mr. .1. 0poku of th e Instrtut e of riquati c Biology at .\chirnota has been seconded tc t.liis laboratory for most of the period unC,err roviewo 0nchocerciasis voctors for trainin6 and educational purposes have boen given to the Zoology Dept. of Legon Univelbity and the I.,i.B. at .lchimota. G. vi n!. B. J. a number int er est -12 - sitors included Dr. and I{rs. R.P. Lennon of USiID and post of the Liverpor;I SchocL of Tropical Medicine who spent of wceks in ttris labolatc-ry ldorking on investigaticns of to O. C. P. I. MCETINGS .\TTMtr}EiD I attended the reinvasion meeting at Lama-I(ara' J. W0RK PI:)IINED Fon NH(T qli,liE! 'lo Investigations dil ectcd towald6 the cclonization of S. soubr 9rl s €o Continuati,:n of search for oth"l f3boratoly mating DaDulaticns of S. damnosum s'Lr Studi es on So sQus.mosulll wingtuft oolouration including the possible effeot of temper4ture' AcKNOIdLEnGEMSI{[q I am grateful to Mr. F. llordzifa for his enthusiastio assistanoe loth in the laboratory and in the field and to Messrs' lI' Barrc and R. Sawadcgc fol roaring work at Nstitingou and Bobo-Dioulasso respectivoly; toDr.J.B.Daviesforusefuldiscussi(lnandadvite;toDrs'S'Mereditht R.PostandC.Vaiimeandl{r.G'Fiasorgb'lrforcytotaxonomiciCentifi^ cations; tc Drs. D. Kurtak, R. Post and Jr Grunewald and Mr' IU' ouedraogo for tireir collaboration and to fir. J.H. Adiamah and others in the Akosombc' PubligHea}thlaboratoryforcarrying,outinvcstigati.lllsofinterest to 0. C. P. Tho V;'1ta Rivcr iluthority and VoI ta L^1.e Hesearch and Devclcpment Prcject have kindly oontinued to provids ]3bor3tDltr' aocommodation and celtein othor facilities without wbt,.oh this wcllc oouLd not havo been calried out. I am indcbtod to Dr. L.K.A.Derban and Dr. E.K. Obeng-4samoa for their continuing support in this resPecto 2. 3. Mouchet r 'f3' NEFEHENCES J., frrelennec, C., Berlr D.r Sechan Y. and GrebautrS' (1977). Iicthodologie peur tester Ia sensibilite' des ls,rvss de Simul-ium 3gg!.9Sgg s.I. Cah. g.iJ.S.T.0rlII., 56r -Ent. mcd. et Ppr"sit.. t 15r 5>66. oo lr\(\l on o\O I R \o o ra\ C\.,\ooo I o rr\o C\] ooo\ oN oo(\J o tf'l a\o o l= o o rftr\c) oJ cqr E:{oa oQoo ooo oooo o ocorrrr \O l'r\tn I- O OC! 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H a rr) tqrl E FtJ "a QF F1 5a rq t{Eif t'lt/:t :=O F-r o.E{xH C) oC) FAF] H() H Ei() frttn zH I I trl lrl to .llpi >lI) ctrlC,ll\'ilrro ill o o\oN E{ o@ \o ra\ "Fl sf, zo Eotr{ E) F-Ia ,rI M{FI <n E{a E"] E{ >4 B{ l{r F1H Fq H Ftr O{ ET() a ll'a f'f FI H() H ErC'f'I <t) H Fqo (a Ei Fl5af'I F( ? r{ F{ Fq E{ T.:BLE 2. LC5s IND LcaS !:-njS!![I s. t. COMOO' ilIVIliS. VTILUES ( PPr) L.rRV"tE Fr!0I'{ OF iB.:lTE l:G.LIlTST nIE B lll}-:l'ir. .lliD *-- SOUNCE tc50 ( PP*) LcaS (pp*) GIUIS GI:UTHIEB B. 3"i113XIv1.\ ;rl EP5' B. COMCE o.o7 1.6 0.0.1 0.0 48 T-IBLE J. CYf oT .XO IO11I C I DISITIFI C :1TION6-*- OF L il?V'lE FnCl'! S liUE cori:cTroNS ^,S Ti{o51 TES'-'ED' 3.TE SOURCE NO. IU s"tMPLE ,S. SANGTIPAULI S. .SOSBNENSE UNIDB\]TI-FIABLE 4.6.80 CHUTES GAUTHIEN B. BANDAMA 4653 4. 6.80 lliPs' .R. COMOE 43341 * These identi.fications were madg bY Mr. G. I'iascrgbol' TABLE 4. THE SPIICI]1S OOI,IPOSITICN OF 35CM[TLY 00t[IcT'jD g, D-1i],nI0sljl,t s.L. FnoII ID'TATTIFISD FI5T,D GIIAII;r t\ND TOCO*' SP,CIXS CCI'IPOSITICN s0uncg DAIX sqda ya R. MONO T3T],TOU 21 / 3iBo 20/ 5/so n/t /8o 15 24 1 I 1B 13 E. SIO I(ATI BNI DGE D/ 5/8oo/t /8o 3 1 1 5 f0co F. SIO MESSIOBT g/>/Eo o/t lBo 4 4 R. AMOU AMOU-OBI,O n/>/Be fl/6/80 21 alL sq R. outg fiZTMfi fl/ 51Bo 11 R. irlt{0UTCHOU OKP.lHOE 7/ z/Bo z6 D/ z/eo 1l t/ tlta 15 SE'ICHI BAPIDS AIMASU N. ATI}IPOKU fi/1/18 $/g/t g 3 all sq SIANA BOTI F TLLS t t/t /tg tt /t /tg zd/ tt/tg q/ z/ao 41 t5 18 11 * Identifi catirns were and Mr. Fiasorgbor. made by Drs. Vajimer Meredith and Post