Rearing and colonization of simulium damnosum complex and related investigations: January to June 1978

t-; .s_'F .v ', fr /stn /r, ,r* Subjectt Instit ut ionr Persorurel: Date of Submission; Per,iodl ooveredr Signatu:se of responsible offioel: E. The species compositionin Ghanar ONCEOCEBCIASIS CONIAOL PBOGAAI'{IIIE, RESEARCH REFONTING. Rearing and colonization of Simulium damnosum complex and reLated investigations. r{Ho/ocP JTN' RAYTOULD Ith July1 1978. January to Juno, L978. , -. -./y';, t - CONTENTS Ar Introduotion and Summaryr B, Rearing the progeny of mar-blting E. 3gggEg! u. reinvading females in western Ivory Coast. Cr Rearing other specLes. n, The species oomposition of lafvae oollectecl frrm of Sr 3glgggg ".1. at A 1.. includlng the field. sukawkaw I'erry . 1lggggggg1s.l.Fo Gr Hr Ir J. K. Lr T{, Nr 0r Pleltmlnary studies on melmithicls palasitising S Ovarla1. development of S . damnosum solr femaLes after b]-ood feeding. Supp1y of matelial to collabolating workers: 1r Speoimens for studles on morphological taxonomy. 2. Speoimens for age-grading investigations.t*3. Living material. Work cElried out by staff of the Pub]l.-o Health Laboratory at Akosombor Vleittng researoh worlcersr Tralnlngl Leotr:-res and demonsttations. Plesentation of lesults for publication. Attendance at meetingse Pulchase of apparatus o Research getablishments vl,slted. AO INTRODUCTIO{ ANT SIJM}&\RY For the first time since my Ieoluitment to QCP, I am presenting a combined report oovering two quarters. n maJor reason fnr this is that soon after my return from leave I nushed to Seguela in western Ivory Coast to establish a realing laboratory for investlgations on the reinvaslon problem. fn view of the urgency of these leinvasion studiess I oordd not delay my departure while f prepaled my report fol the perlocl January to I'Iaroh, In addition, I took my home leave during the first quarter of the year as well as prepallng a paper fnr publication and attending a meeting at Ouagatlouflour fhese actLvities reduoed the time avaiLable fol expelimental worlc clurtng the periocl ooncerned. My most important investigations during the past sir months have been the reinvasion studies at Seguela. lihle worlc will be oontinued in JuIy and it will be some tirne before all the reared larvae have been identifietl. Only an initial :repolt on this work is thereforo included here. r1 much more detailed aocount wirl be given in my report for the thlrd qualtet. Other studies calried out during the period under :seview include pleliminary investigaticns on mermithid palasites and on the gonotrophic cycle of s. damnosum s.r, femaros. Larvae for oytotaxonomlo tdenttfication were corleoted from various sites in Ghana and the rvory Coast, MateriaL was supplietl to oollaborating laboratories for studios on morphological taxonomyr age-grading inveetigationsl leali-ng work eto. Collabolative investigations by VoIta Biver rluthority staff at the pub}io Hsalth Labolatory, .Lkosombo, are proc€oding welI. A number of dlstingulshecl visitorE wele leceived during the perlod undol roview. These included scientists who speut a few days in Alcosombo obtaining speoialised infoilnatlon and others who remained hele for a longe:r periotl oompleting losearoh projects on onchoceroiasis vsctors. During my home leave the oppoltunity was taken to atton6 meetings, visit research establishrnents and purchase apparatus. I2 BI REANING IIIE PROGEfT OF MAN- NG S. DAMN0SUM S.L. I Considerable progless has refently been made in the morphologioal taxonomy of West African specios of the S. damnosurn compler (qgiff6v6r67 Sechan and Pendriezp L9771 Dang and Petersonl I9?8; Garms porsr oomm.)r Nsve:rthelessT lt ls not yet possibl-e to reliably identify individuals of all species within the complex by molphological means. Thls is espeoially true of old and rubbed reinvading females of unknown originc ft is still neoeggaly, thereforel to rea! tho larval progeny of reinvadlng fomales fol cytotaxonomic ldentifioation. Reoent extensions in the oontrol plogramme area have rosultecl in new aleag of reinvaiion beceming apparent in the western Ivoly Coast. I thelefore left Akosombo on }]th May for Seguela. Rearlng systems lrefe set up at Seguela sub-seotol offico using appalatus carrled from Akosombo. Iulethods G 0n the advise of Drs. Gavms and Davies biting femalos of E. damnosum EoIr wore collected from the fivo locations listed in Table 1. Larvae dosoribed we!e reared using threo difforent lcinds of rearing apparatusfin oallier qualterly reports: L. An apparatus using complossed air in whioh an aeration stone is p)-aoed in a nalrow tube (Quartelly Boport for Oclober to December t 1977). 2t An appalatus using an electlo-magnetio stirrer. l. An apparatus in which water is passed down an inclined trough and recirculated by a oentrifugal. pump (Quartorly Report fol July to September, L977). In aLl caeres cuLtures of algao wove usod as a souroe of food. [tre algae wero filtered from the culturo solution (Nakamurars metlium) and washed off the filtel-pape! into the roaling apparatus. The biggest diffloulty enoounterod in this r.rork was that of power f ai lule s. -J- @jE. Larvae developed extremely rapidly using the inclinod tlough teohnigue. The first lalvae were largo cnough to fix fol oytotaxonomio identification oight days after hatching and pupation oomnenced after nine or ten days. In the oase of material from Kongassor for example, 42 pupae wole obtained within 13 days of hatching evon though most larvae were kilIed fol fixation, Tho reared lalvae were identifiod, as alwqys, by Dr. Charles VaJime. Identificattons given in Table J1 are limited to larvae realed prlol to my departure from Seguera in earry June, (The statement rrrearing in progresstr in the Table also refers to that time). The continuing programrne at Seguola is now in the capable hancls of Mr.Barro Tele who received a training in the relevant techniques last yea.r and ls now an expelienced workel in this fieId, This wolk will be repeated Later in the rainy Beason wh.en control has been extended to include certain adtlitional major livers including the sassandra. complete resurts wirr therefore Le presentod in my next qualtorly report in which the signifioance of our flndings in relation to the reinvasion problem wilr also be considefed. Cr NG OT}IEB SPECIF"S S' unicornuturn (fitcu a numbor of other African simuliids that only require a fairry srow rate of wator frow) was found to be very easy to roire Rapid development to adults ocourred in simplo containels supplied with a compressed air flow and no special treatment lras rsguired, D. TITE SPECr ES COMPOSITION OF LARVAE COTLECTED PROM TTIE F'rELD During the period under roview larvae were colLocted frnom a few sites ln Ghana and a couple of widely sepalated loorll,oae't-n lbe Irory coast' This material was sent to Dr. vajime and hts identifioations are presentotl in TabIo 2, I * 4- In genelaI the specios identifiqd ale tn acoordance with earlier findings and thelefore to be expocted. 0f partioular interestl howeverl is the pregence of S. squamosum in Aimaso stream at Atimpoku n6a1 l\kosombo. For tho past few yoals smaLl streams near .ikosombo have not been frowing beoause of inadequate rainfalr' IIow that they are flowing again aftel heavy rainsr E. -EgE@QIllIEl a small stleam speciesl has been reoordcd for the ftrst time in the area. E. TIIE SPECIES COMPOSITION OF S. DAIINOSUI,I S.L. /rT ASUKATKAIJ FERNY IN GIIANA The oytotaxonornic identlfLcation of, Iarvao collected from the breeding site in the Asukawkaw Rive! at Asukawkaw Ferry r and of tho larval progeny of man-blting S. 3gry".1' f emales from tho same location, has suggestetl that both the brooding and man-biting populations are virtually pure S. sanctipauli . Apart fnom this spocics only a single specimen of S. soubrenso has boen recorded flom tho Blle&r (fne identificaiions refelred to wele made by Dr. Chartes Vajime and have been presented in earlier guarterly reports). Asukawlcaw tr'erry is therefore of partioular intelest to OCP in that it harbours the Purest Icnown population of Sr sanotipauli in Ghana. Dte. Pete!son and Dang of the Biosystematics Besearoh Instltutep 0ttawa, havo recently sent me their tdentifications (using morphoLogioal chalacters) of material flom the above Iooation. Their findings, presented in Table 11 also suggest that the S. lgry ".1. population concerned is composed main).y of ! . sanctipauli. llevertheless , their results suggest the presenoe of othor spocies ln small nurnbers. Howeverl tho presence of three marrbiting E. -geglesum s.I. fefialer is perhaps not surprising, in view of the faot that this species is known to bleed furthe:r upstream. The presence of the so ca1Ied trspotted speciesrrI is intelesting. Thls rtspecLesrt has aIso been recorded from Senchi Rapids in the River VoIta near,lkosorhbo. Its taxonomic status has not yet been determined. 5F. PRELI MINANY ES ON MEN}IITHIDS PARASITISINq S.DA MNOSUM S.L, Melmithidparasiteshavebgenfoundtobocommonin$.damnosumS.1. larYae in the Pawmpawm, '1krum and Worobong rivers which t'o' 1n1o the volta Lake on the southern shore of its Afram Rlver arm' Tho -8.'494I19S!E s'Ir population ln all three rlvers is comprised almost entirely of !"IeE@' The mermithids occur in two corour formsr greenish and off-white' At Boti Fal1s on the Pawmpawm whele adult dlssections are regularly in prof,ressl the whitish folm is known to ocour in the adult flies' For identiflcation pu]lposes adult wolms are lequired in addition to juvenilec that havo Just eme1Sed from the host' Postparasitic juveniles havo therefore been kept alive in sand until after their final moult and then sent to Dr. G.O. poinar at the Univelsl-ty of California. Specimens from the akrum Rivel, collected in Decombet ]-977r were ldontified by Dr. Poinar as Gastromermis Iebelrei Mondetl Polnar and Bepuadoa, As it seemE IikeIy that two species are in fact present at least in the Akrum and Pawmpawm rlvers and probably in the Worobong 6s well-' more material for ldentiflcation is being collected from all three sites' Information on mermithids palasitising s. dam[os[Ill solo is of interest to OCP not only because of their possible uso in biologioal oontrol (which is unlikely to become feasible in the foreseeable futuro), but also lecause these parasitos havo a restrlcted distribution and thel-r presence in reinvadlng female flies may provldo some olue as to the posstble plaoe of origin of the flies conoernedr G. OVABTAL MENT OF S, }AMNOSUM S.L. BLOOD EEEDING Following suggestions that information on the minimum duration of the gonatlophio oycle would be of value to OCPI somo preliminaryl albeit ratber superf,loial 1 investigations on this subjeot were cal1ied out' Females of E. ag!ry ".1. and s. sirbanum were allowed to engolge on a human bait subjeot and then kept out-of-doors in a shaded situation ln sepalate tubes. During the coulse of thls investigation minimum temperatures ranged fr,on 24-2BoC and ma:rimum temperatures from Z6-370C. Most of the egg follicles in flies dissected 615 hours after feeding had alroaful reached stage four of Christopherts olassification. Aftel 2{ hours they were at late sta6e IV to eartl-y V and wel.e apparontly fully formed after about J6 houre. 0nly one out of ten flies laid (a very few) eggs when immersed, in water about l! hours after blood-feeding but four out of ten oviposited when thus stimulatod about )O hours aftey engor6,ement. fhese very limited results suggest the possibility t.hat oviposition in nature may sometimes occull on the second day afte:r engorS,ement. H, SUPPLY OF I,TATERIAL TC COf,LIIBONAIING WONKERS 1. Specimons fol studies in morpholog ical taronom.y. I4aterial dispatohe<l to Dts. Peterson and Dang of the Biosystematics Research Institutoe Ottawa, Canadae duling the period under reviow has included alcohol preservod adults of S. damnosum s.1. and S. sirbanum and a few pinned and freeze-dried speclmens of the same species and of S. sanctipaull. The main problem still outstanding in the rocognition of morphologioal differences betweon the speoies (within the S . d,amnosum complex) concefns S. soubrense. As was pointod out in my previous quarterly reportl adults obtained from pupae colleoted flom the Komoe River near iibengorlrou, bolieved to be a pure S. soubrense breeding sitet were found to resemble S, .vahense from elsewhere. The Komoe material was found to be quite different from flies initlalJ.y believed to be S. soubrense from the River Volta near Akosombo. To help solve this taxonomio conundrums tho l(omoe Biver was again visitod on two ocoasions! 12th May anal 4th trune. Unfortr:natelyl ):reeding had not been resumed in the lapids near ltalcro which is considered to be the purost soubronse breoding site known in the aro&o }lowevere large collections of pupae were made from Amoulcoukro about 60 l{xn upstroam- Larvab colleoted sirnultaneously were lator identified as 29 .E. g.4445g!! s.1. and 22 S. soubrense. IDrs. Peterson and lang apparently stiIl Iequile identified S. sirbanum rnales and S. sanot ipauli females realed from single egg batches a.nd an attempt will be made to fu]fil this requilement in the neal future. Laboratory emerged adults of s. squamosum were Eupplied to DIs. Garms and Davies for testing Dang and Petersonrs prelimlnary key' These wele obtained flom what is probably the purest known breeding site of the speoies: Boti Fa1]s in the Volta Region of Ghana. 2' Spe cimens for a ae-erading invest ieations. A mole precise knowledge of the age of leinvading !.349899-"'l' females would be of great valuo to the Plogramme' The PoLovodova a8o- 6rading technique has only been used suocessfully for separating parous flom nulliparous individuals and not to determine how many timos they ale palousr It is alsc possible that reinvading femaleE may have difficulty in feerling regularly which might prolon5 the gonotrophic cycl e. The caLendar age of maqy medically important Diptera can be determined by counting tho daily 5rowth layels of skeletal apodelllesr It is not yet known whethor or not suoh layers are formsd in simuliids. For this reason, a large collection of S. 34ggg ".I. femaLes of known age was sent tc )r. D. Schlein of the Hebrew Univelsity Medical School in Jorusalem, who is an expert on this toohniquo. The flies concerned were kopt alive fol know perlods from their time of emergonoe in the laboratory. Larg;e numbers of { and 8 day otd flies were sent and smallor numbers of older indivlduals. Unfortunately tho consignment never a:rrived and a second consignment has therefore been dispatched. If daily gror^rth layers are f ound tc be prosent in simuliidsl then fulther collaboration is onvisaged. -8- 3. livlng materlal. Fgrthel ccnsignments of eggs have been dispatohed to nr.J.Glunewald at the Institute of Troptoal Pledioine at Tubingen. Some of these have been suocessfully learod through to adults but for unknown reasons larval development at Tubingen has been much slowor than in Wost Aflioa. .ldults posted to Eubingen failed to survivel but 30 out of 33 females carried in a f}aslc with ice by a member of the Tubingen staff survlved although the journey took three days. S. damnosum s.1. egg,s wero cleliverod at the London Schoo1 of Trcpical Medicine when I returned on leave. These wele used for tiesue culturs work by Dr, M. Pudney and for learing erperiments by Mr.I Eeld. I, WORK CANRIED OIJT BY STIIFE' OF TITE PUBLIC HEALTH LABORAEORY n-f-xmB6ffi Sinoe January this year, Mrr Tony Opoku and his assistants have been working in collabolation with OCP staff from Tamale in the dissection of biting catohos at .ihamarrsu, Dodi-papasey rlsu-lcawkaw and Dodo-fie ln the Volta Region of Ghana. Now that a new sub-seotor office has been openod at Hohoe, Mr. Opolcurs oontribution in this particular respect will probably no longer be required but the experience gained will bo valuable for the future. Mr. John.ldiama}rs investigations on the biting behaviour and vector and Boti Fallspotential. cf S. yahense and S. squamosu,m at Tsatsadu Fal1s/respectively commenoed in Marchl following a preliminary training peliod with the OCP Tamale toam. Since that time biting oatches have proglessively increased. The lesults obtained up to the time of my nert quarterly report will be analysed and presented by l,Ir.Atliamah as a separate annex in that report. -9- J, VTSTTTNG RESEiRqiI_IlIoilcERs_ Dr. M.t{. Seruice; head of the Departmont of Mecllss} EYltomology at titc Liverpool School of Tropioal Medioine, spent the period from 7th June to 5ttr ,luly carlying out a research pro61amme at Akosombo. The investigations were all on subjeots cf intelost to the OCP and an effort was made to faoilitate his work as fav as posslblo. the Dr. Service invostigated;luse of a redesigned and improved version of the Monks l{ocil J-i6{nt trap for sampling S.r darrnosum soI. aud also tested traps using a chemical light sourG€o In additionr he suocessfully sollected material for the doteotion of pledators of the aquatic stages of the S. damnosum compler using the ELIST| s:rological teohniquo. Dr. f. Gregory, Pllnctpal Research Entomologist cif the Nigo:rian fnstitute for Trypanosomiasis Resoarohl spent a fow days at Akosombo duling the lattor half of June to obtain information on blaokfiy learinS, techniques and to discuss possible future collaboration in oertain aspeots of researoh on onchooeroiasis vectors. Ko IRAINING, LECTUREE AND DEMONEIEATIONE !1r, Bamo TeIe assisted with my rearing worlc at Seguela and then continued the work after my departure. The opportunity was taken to familiarise him with new techniques developed since hls training period last year. MrrE.M. Podersen of DANfDA visited Akosombo between 21st April and 2nd l{ay. He was glven a thorough briefing with partioular referenoe to onchooerciasis yeotr.rrs in Tanzania where he will be worklng. Lecturss a,nd demonstrations were given to Aocra Branoh of the Ghana Soienoe Assooiation and to Uinneba Seoondary School. ,-10- L. PNESENTTTION OF RESULTS FON PUBLICATIOi'I The following paper was prcpared during the period undor r eview: Baybould, J.N.1 Vajimoe C.G. and Quil16v6l6 , D. The laboratory maintenance and rearing of Simulium damnosum complex species as a resealoh tool for OCP. 0the4 papers on onchooeloiasis vectors were compl'oted in col-laboration with various colleagues duling my leave but these wsre mostLy concerned with work carried out beforo I joined OCP. MI /ITTEIID,INCE AT MEETI}IGS The following meetings were attended eithsl in l{est Africa or while on leave in Englandt Meeting on trCoorditration of Research in iiolminthology and Onchocelclasis transmissionrr, Ouagadougour 27 February to 2 l{arch, 1978. Symposium on the Kpong, Hydloelectlic Projectl Ghana Ecolcgioal Society, Aocra, 1Oth ;1priI, I978. Spling meeting of the British Sooioty for Parasitologyl University of Kentl Canterbury, T7 .ilpril, 1978. r a PUNCHAgE OF APPARATUS Drring my leave in England the opportunity was taken to obtain apparatus fvom London and Liverpool. Certain otirel itoms wele obtained during, two visits to Lom6. O. BESEAACH ESTaBLIS}i}MITS VISITE} Research establishments visited during my home leave fol discusslons with professional coll-eaguos included the British Musoum (Uatural llistory), the Liverpcol Schocl of Tropical l,lerlicino and the Zoology Department of Livor^pooJ. ijnlvelsity. -11 - P. VISITONS fn additlon to those mentioned under sections J and Kr tho following visitors were reoeived:- t/lrc Mr. H.P. Wirtz Miss M. Napp Instituto of Tropical Medioiuer$ubingen. 28/thT H t/z/ta Group of 20 Scientists -Ghana Soienoe /lssooiation (Aoora Blanch). Lr/zh\ Large Brgup of pupils "od teaohers, Dr. I. Ladnyi Dr. S.K' Litvinov Dr. C,Ro Jones I'Ir. Elliot Berg Mr. Henk Koppen Ml. Vann Molyvann MI. N.S. Belo-Giwa Dr. Fergus McCullough Drr Seulier Dr. HoSchul z-Key - tlinneba Secondary School. - Assistant Director-Generalr WHOlGoneva. - WHO1 Accra. - WIICl Accla. - IBnDr Ouoho. Mission. - IBRD1 Washingtonr D.C, - WE, Lausanne - National Oncho' Socrotari;rt1 Aocra. - Ecv/vBc, llilo, Genova. - Schlsto. trlesearoh Projeot s Accra. - Institute of Hygienel Lom6. $/z/ta t/ t/te 4/7 /78 il il It ir It il n r ll zz/z/ta ll D!. Dr. Dr. Dr. Dr. M.S. Zak R. Laryea P:rinoe T. Croley 0.Osbcrne - USAIDT Accra. - USAIDT Washington. - Consultant to USAfD tt il ll It lt ,t Q. WOBK PLIIiII{ED FOR NEXT QUAATEB I Iioaring wcrk on reirrvading S . damnosum females at Seguola, Ivory Coast,a will be oontinued and the results analysod. 2t Studies on Onohccerca ochengi in ocllaboyation with !rs.Omar and Denk6 will coiltnBrrcor 3. New and improved learing teohniques recently developed will be quantitatively evaluat ed. 4. Fulther attempts tc find a laboratory mating spocies wiII be marle. 5. An attempt will be made to roar Sr sirbanum and Se sanotipauli from single egg batches tc provide identified adults for, etudies on morphological taxonomy. - 1) - Q' WONK PLAII}TED FoR I[mT QUARTER (Contd.). s Iriolk on mermithids will continue.a 7. Compalative studies r-.n f oeding behaviour and onohocelciasis transhission by !. yahsnse and Sr Sg5mosum will continue. This work is being oalried out by I[r.JrH. +diama]r and his assistants in this labolatoly (i.". rlkosombo PubIic IIeaIth Laboratory) in ce.Ilabolaticn with OCP. B, Mr. A.K, 0poku and his assistants in this laboratory wtlI oommenoe new programmes in placo of their work with famal-e Seotor which has been di econtinuod. BT ACKNOITLDDGE}ImITS I am grateful to Dr. C. Vajime who was responsible for all the cytotaxonomio identifica.ticns; to Mr. B, TeIe for hie consoientious and invaluable assistanoe with the r€aling worlc at Soguola and to Mr.J.Fo Walsh and Drs. R. Garms and J.B. Davies for useful sug,"ostions and discuesion. I would also like tc thank the staff of the Public Health Laboratory at ,Ikosombo "nd of Seguela su}-sector for help in various wo]6r NEFERE}TCES QuiII6v6r6l D.e Sechan, Y anrl Pondriez, B. (tgtt). Dtude du complere SimuLirm damncsum en Afriquo de It Ouest V. Identification morphologiquo clee femelles en Cote drfvoire, Troperunedr Parasit .1 28_r 244-253. Dang, PrTo and Petersonl BrV. (fgZA). Preliminary koy to the sir West Afrioan specios of the Simulium damnosum oompler. Unpublished mlmeoglaphed plovisional dooument, Biosystematics Rosoaroh Instituto, Ottawar Canada. oa v) 2oH E{ H() E] P"a r-I-tr-\o a f F{ op. o F{ p> Fl H o P E{d , t .t. H o H >> <! H .r{ h r1 o a i{ o o f{p{r ooE F{ ,lO 'r) Ed aoa .Fl q) tH F{ .r{ ErodosA() a aOF{9trl r-i hF{P 'r{Fq) durdoE P '.{ (Du)r{ @o7 thoa .qlfo ,{q-{ Pod o O q< .r{ ,"1 +{: oo od r{ ..{ or{ Er{ *+ a H F( E a() =!ro E Hpq oaa .ri(5 o !4 z E] H t-]a o pq l-f frlb t5 t-la E<zop{ * t'l() Fl P" IFI t-lH EaEfrl t+ -cE<E{Eo>z .p<Eloe<Q(nE}lF{v F--\o e-F- fi{oEl 'P{o57tu OJsf rf)r{ f'I etr <Filo>Fl oF(.{O<E]tlrE a(JA7, trIHF(FiE(5 <Hot-] P(piA a(5a HEE ZrJ<{HO E-'l F.F6tr{ (nu(nZ f.lHp(M 2<,<Hotr1 xEP. oo Fl aa(5trI ZFiHC5Ft? o. <HFItrl p{ F< o\ @ trl!f trt(5 dE1ZH>iHtotrii4Fl <()0rF]TqEF( E{ trl F FElaaf4 nifqHP{< =o c) e trlatnEl aqciH Boo(J atrl H(JF?P()o ZMHE{ A Fq Ht5Fl if()o HH A f{latat'lEFq HHo() eHzH Fl() zH EF c tr a a F,l()R Er E-l <HEtrlrt oC)Fr O C' FEIEF]O N r-l .4E{zaz Fr{ a ,1 f*'A N -l r- tr- ,-l F E] E{() F]f4 FlE{ Fl<on() CCF l'r\ o OJ € o\ -1 co I- r.r\ o\ c\J co D- r-a\ c-- Fl @r* tr\ @ ?1 @r- rn r.)(v tr\ r{ @c- r-a\ lr\ A d aa BI 3t EI EI 'f(5 H E{H Fq Iz s$ -a >t ta FrHE{ or/jc)(,L) >{tsl Eo OH f"{ A F4e E-]t-] E{F(A '{ t'lF1 =E EF"l o E Ff tr]Fl OE + fs] o H EJHao o.Eo c) aHHo f+'l P{a E] E< a F F]Fl rrl F I I@N t-la r-l H aa 2oH E<Haog o() a f-lH() f*Ip{ a zH a E't E{H U' rJ HAFl tr]E FA Eofr F{ e F]g.() Fl .FJ E{dtno4oo () F'I H ,J t-] rE<Fl oQaz<HI :rl .{'t/rl 2 ol< EI E -41 elt ' E-jl a?0la Fqo a 0) E 't{ 'r (U UI o -{F{(Il .q() a E{ trl c)f) a) 4 (U E o F{ o B ul tr o .J {t(t o .d(i< .rl f( oa .rl r{ rJ .-{ * I |-t <OZcoE-tM.{F!f, . E'\-mG rnHo\E Fc{ Pi '{'E{Ot.'1 &,o H FiHao Hoo a raH(> t-l P.UI t-l H }{E{ cqan o<>oH()(5 H Erap o() Ff, <1 H il F]o Eo m a N H rJ Fq E{ c\l rn CV .+ f'Ia f-lHpq !)oa aa a Ia (\' c\I Er 2" <I Fql H1al a "rl (\I .$ Ia aa EF(n o € A aa o\ r{ Ntrl o\(\I o H E{() c);l arfHOAFq4Er:<oHAH hr OCO!(O|4tr-()><-\ Z .r.lfrl o Pi\.AHA.t) @r-I\O\O .ri \\ lc{6J \ndFt .1 kl D()Fl>{ co <:= F rD -< ---E{> \o <<-\!O>1 rriH< cli l I <oE'l tclFiEE{()aa oco oMr.- m <\_ E r\-HIEC\J4tsrl o ODFiosdEI(5DOEcoO E frlr-H O frl'\Pi4<lr\O r-'\E rEN 'i H ZFI I Ju-\ Hot'lAa A Fq Er E{oAa a H oa I{s +l 0) P. o H .o (C R a a A trl p oG d e oj o 3 a H( .e + cr o .il +{ d+ H od +{ r{ rl <:i rt P{D A{ u F"IZAH.{ErEHtq Fq F. U trlaiH<F{EHrq Fq Fq oH EiHao Ar =o(J a E] Ho Fl A{a () zHTrrH<AZ1< -4 <5 triOE >{E]Etro o< x14 -sBE{< 14l-l i.r<a()<i H()Eoo}lEOE P{.EFIO.ELl BEItnlAol BEI< -{t Fc nl *2.O cDl HE{ t)H2AHOFO.HH E'lOEOE EEHFI()<i E{()AOfct Fl trte Er< trtf{H frrH E'l E{arz < f'lE{Ft2H F Fq E{(J Fl f'l()FJ <llE{orr,();t EI b-o r{ o+ E @ C\l r-l ro ta aa E5ao a F aa HFfi DI<l O.i F.tl E{1ol a\ .{l(nl rl ,l st $. b\<<r F- \o N f'l E{ e I> F\ F{ t- r-- F- N F{ @ r'l l'-' t---. N r{ @ .{ axo oi txo FqAp. (n tr]E{}l}J< BE < f-i tn f-IFrFi}l<{DEEFq 'i tr{ ccl E+Ho rn F.l tEl E'IDX n fr. El <o ul I I