Adult identification in simulium: apparent dependence of polyteny on food source in malpighian tubule and nurse cell nuclei during oogenesis: final report

Adult identification in SlOutlq4ql apparent dependence of polyteny on food gouree ln .olplghian tubuLe and nurse ceLL nuclei duri:lg oogeneale. Flnal Report l{lLliam S. Procunier W.H.O. COIJABORATING CENru f6r the studf ol SIMULIIDAE & PHLEBOTOMINAB rn relation to ONCHOCERCIASIS & LEISHMANIASIS Department of Entomology Briti.sh l'luseun (ttaturat Hletory), Londou, Unlted K5rtgdom, SU| lBD *^-rerary adviser to the OCP/OMS Program, Ouagadougou Burkino Faso, l{ay 1985. ,.sffir oT7t4'; ?Za 6tr :.?".q',--;} 1. This study vas undertaken to determile chromosone poJ.ytenizatioa and to asoees tbo parity determinations..in S. damnosum s.l. the opti.mum tloe 36j na.lplgblau pbysiological age of the fllcs for fhe optinurn time for malpighlan tubul.e "h"oro.ooe polytenization appeana to occur after a bloodneal for the ti.me period the oocarte eaters aeiosls up to rnd iacluding the karyosome stage (I'igures IB-LD). The nucl,el (karyoeome) becoaoa condensed and rernains at this etage until fertillsed, uhereupon the let and 8nd mei.oti.c divisions are cornpletod and the spern fuses nith the functioaa-1 oocytr to form a zygote, The malpighian nuclej- appearr to break down at the onset of vitellogenesis (laying dowu of yolk) (Figure IE). Onset of meioete varlee anong species and especially between aevann4$ anrl*forest llploe: optimum tLne - forest I-4 hrs; savarurah T-11 hrs. Previous to this study it was suggestod that chromosome polytenlzatlon appeared to be dependent on food Bource (Procunier 1984 and Procunler & poat L985) with the degree of polyteny being stage specific. I{ence by the tLme adult flice obtained a bLood meal moet ehould already be at stage II (Procunler 1984) polytcny. Most tissues i.n which high metabollc turnover is required duriag ontogenetle developnent appear to respoad to the demand either through eelectlve anpllflcatlon of genes and,/or the whoLe genone (Ashburner 198O). In the case of the whole gonome (chromosome conplenent) thls oay ta]<e the form of polyteny or endoduplicatloa (doubllng of cbromosome number). Siace ma-lpighian tubules function as an excretory organ which j.lvolves water ba.Lance (Wiggleswortb 1984) great demands would be put on tbis organ upon acqulriag r bl-oodrneal. Further, the time to synthesise DNA from a normal 2c value to 2ol4o value (tutt polyteny) would also requlre a certain length of tine. Consequently the observation that, the flias are already at stage II pollrEeay is consistent ultb tbe tj.rne constrainls put on the flles syetem j-n order to oalce ready the nucl"car oachiaery for high metabolic turnover in response to the bloodrneal. Eovever, littLe ig known about ceIJ, cycle in blackfliee ulth regard to DNA synthesj.s ti.me (except Bedo 1984 - does not glve actual length of tlror ln his paper) a.rrd hov long the celIs are in mitotic (melotic) divlslon. s G2 M G1 DNA synthesis 'Gap 2 mltosts (meiosis) Gap 1 ) ({, ^o.;; tq6L ) In DrosophiLa (Kfng I97O) the time required for meiosis up to karyosone etagc ts 27 hrs with the total time for egg maturation belag 4 days. In noequitoea this detailed j-nformation is not avaiLable coucerning aIL the atages of oogcacsla except the half gravid (yolk ]) etage can occur 12 hre aftar a bloodneal. Boncver, i:r mosquitoes readable chromosomes are frclm the nurse ce1ls. These organlaua provi.de some clues concerning duration of temporal events duri.:rg,^oogenesla. ft J.s important to establish that a particular r,:t;rge can be used consistantly aa a'. physiological marker of age. Cytologica] observationsfrom a nunber of differcnt species, !. yahenser g. ryttpqu.].i,9. glggg4ggr [. soubfense B (Procunier & Poet 1985), S. oyapockensy'roralmense anrl 3. Iimbatum (SheHey, Moraes, Luna Dtar & Procunier 1985) suggests that a relative tlrue perlod exists for coneistenbly obtaiaing readable malpighian tubule chromogomes. Thle concLusion is firthcr supported by the observation that individaals which show dj-fferentia-L folllcular development (Fig. 2C aad 2D) remaln at stage I11 polytenizatioa independent of tine after a bLoodmeal (a6 long as aome follicles are at the right stage) rhtle those individuals uhich sbow gonotrophic concordance cannot be read after the ooclrte enters vitellogenesis. Tiaing of onset of meiosis, polfienlzation and cluration of oogenesla La undcr hormonal control. These processes seem t() be dependeut on the production of R'ddt$"1 o',,1 Je'tata llll ecdysone and its derivatives and,/or juvenile hormone ( , '.). lcccrtly it has been shown that Juveni.Le hormone can induce polyploldization la folllclo cerls (Lapoi nte et aI I 9g5 ) duri ng vi tet logeriesi ! (r'igie 28 ) 6. ,. The seguence of hormonally medlated evonts during oogenesis appear to bc a function of marry factors - eogo blood motabolism, excretory regulatioa tto.o tr'ormation of the peritrophic merobrane occurs very rapidLy after acquirenent of a bloodnea1 in many blackfliee vith the laylag doun of nenbrnnes varyiag ln ttnc Reirt + LeLare' and type ( 1981). Formatlon of the nembrane has been looked at ulth rcgard to understanding factors Il-triting the traasolssion.of onchocerca (PUf[ppon X977 ancl R"''<J*lqhcrt- r 198f). However, formation of the peritrophic meobrane tray arso bc a x.l,nttlng factor in deterrniniag the onset of meiosis and srlbeeouent vtteLlogerresLe. It ts interesting to note that my results for onset of meiosis (optinum tlne for chromosome polytenization) correlate poeitively with the ti:ne taken for fornatlon of membranes. Savannah species which have a thick membrana require 6 Ura? to lay down while forest species (again the idea of d,lscrealttr" periods for oarktng developmental events) which have a thin membrane requlre only I tlr. (PnUlppo\ :lg??). Further, differences in saturation (oogenesis) tlmes between species ui].J. certainly affect vector dynanicswith respect to transmission. Paraelteg havc evolved a growth time to L, staSe which correlate with egg development so that gonotrophic concordance will ensure paraeite survlval because it is infectlvc at the Jrd bloodmea-l. Ti.me for oogenesis would provlde a strong isolating nrclranlco for parasi*'e differentiation which could aJlow further selective procesaea to account for differences between savpnnah and Fores. :;;Ilr:;^tfiXi.:'itlii*',i"*u be that species found interfacing the two ecologlcal zones would exhibit ainilar maturation tlmes and hence could allow developnent of botb rrstrainsrr of paraeite. A series of photomicrographs depictlag the events of oogenesis is gtvea with appropriate notes discussing parlty and gonotrophlc concordaace (Flguree IA-LI and 2A-2G). Photomicrographs of tha adult poLytene chromosome complenent of 3 cpeclco is also given. It should be noted that lndividual"s whicb give mediocre prtparattoas (marginal) can be analyzed with appropriale photomicroscoplc set-up and hrvLng preknowledge of larval banding aequences. 5 i-udividuals of g. sirbanum wcrc alao positively identified; however, photomicrographs wore not takon because alldee uere made at Bamako. Research Recorunendations The f ol l owi ng stud'ies are envi saged: l) To determine if malipighian tubule nuclei will give consistently readablo chromosomes so as to be used for routine adult identification nov{ that the optlmum time for chromosome polytenization has been detenfitlned for 3 spccles ln !. damnosum s. I . 2) To determine if a techno'logytransfer in adult ident'ification techniques'ls possible using DNA probesand malpigh'ian tubule chromosomes from the same fly. Under (l) study sites and protocol for adult identificat'ion as outlined'in the previous advisorship (Procun'ier .l984) can be followed. Under 12) as'in (l) except the fly would be severed in two - with the abdom{nal part being used for chromosome'identification and the other half for DNA analyrls. Dr Post has 3 probes now wh'ich can differentiate 3 species pairs; pS03 : sancti paul i /soubrense B, pS01 'l - squamor,uT/yahense affi pSQl - si rbanug ( Post '1985 - Penn State). fhis techn'ique allows populations to be separated by quantitativa means based on the amount of bi nd'i ng ( radi oact'i vi ty ) the probe shows when anneaiing to the DNA of a particular species. However, the technique needs to be verified against a standard to ensure the quantitat'ive difference don't overltp especially for siblings in sympathy. If quantitative difference hold up the DNA method would have tremendous pract'ica1 value not on'ly for identifying the vector but also to determine relative vector potential (could probably process 200-500 f1 i es/day) . One of the study sites should chromosomes preparations were approximate time to look for the be at Tienfala, Mali because (l ) identifiable obtai ned f rom there (S 'indi vi dual s ) ; (21 the optimum.chromosome pglytenization in S. sirbanum 'i s known; (3 ) 0r Post al ready has a probe Consquently, th'is population gives the greatest 'i n i denti f i cati on techni ques . which can identify S. s'irbanum. prom'ise for a technology transfer A second s'ite coul d be Soubre where al though I arval popu'l ati ons of -S. yaheEe and sanctipauli are in allopatry the sites are close enough for the adults to be considered 'in sympatry. Again Dr Post has probes which can separate yahense from s.anctipauli. Alternatively, some other site where populations occur ln sympatry, especially damnosum s.s, (Neither one of us has looked at damnosum.) Another possibi'lity is to go where S. s'irbanum is zoophilic in its distribution. 3) A s'imilar study could be undertaken w'lth Dr Angela Philips. I st'lll do not know whether they have done the critical analysis to show'if peak dlfferences between 2 populations (single adult) are additive - this is necessary to differentiate between intra and interspec'ific variatjon. i.e. suppose the folIowing G.C. profiIes were obtained: I adult (sirbanum) 3mm I adult (yahense) does A + B give a peak height 4) Pari ty determi nat'ions shoul d be f easi b'le (.l984). However, an in-depth study should be follicular relicts are most easily discernible' 6mm A -/ UM 4.5mm A^^ using the techniques of Procunler done to verify the stage at whiqh References Ashburner, M. 1980. Some aspects of the structure and function of polytene. chromosomes of the Diptera. Symp. R. Entomol. Soc. London. I0. Blackwell Scientific Publicat'ions, Oxford, 20pp. Detinova, T.S. 1962. Age grouping methods in Diptera of medical importance. t.lHO monograph series No. 47. Geneva, Swrueer'lBnd. 216pp. Bedo, D. G. I 982. Patterns of polytene-chrornosome rep'l i cati on (Diptera: Simul i idae) . Gentica 59: 9-2.l . Cupp, E.I.l. & Colf ins, R.C. .l979. The gonotroph'ic cycie in Ann.J. Trop. Med. Hyg. 28: 422-426. 'ln Simu I I um ornaflf$. Simulium ochraceum. and parous rates of anthropophi'lic transmission of 0nchocerca Simul I um damnogum Garms, R. 1975. 0bservat'ions on filarial jnfections blackflies in Guatemala, with reference to the volvulus. frop. med. Parasit. ?6: .I69-182. King, R.C. .l970. 0varian development in Qrolophill lg&lfggasler. Academlc Press Inc. (London) Ltd., Berkeley Squaare House, London. 2?7pp. Lapointe, M.C. Koeppe, J.K. & Nair, K.K. .l985. 'Fo'llicle cel'l polyp'loldy in Leucophaea maderae: regulation by juvenile hormone. J. Insect. Physiol. 3l(2): I 87-t 94. Lewis, D.J. 1957. Aspects of the structure, biology and study of Ann. trop. Med. Parasit. 5l: 340-358. Madahar, D.P. 1967 " Cytologica'l studies on blackf'l'ies (Simuliidae: Diptera) Ph.D. thesis, University of Toronto, Toronto, Ontario, Canada. -2- Phil'ipp0n, B. 1977. Etude de la transmission d'0ncherca volvulus (Leuckart 1983) ( Nematoda, Simuliidae) (oRsroM). 0nchocercidae) par Simulium damnosum Theobald, '1903 (Dlptera: en Afrique trop'icale. Trav. Docum. Off . Rech. Sc'i. Tech. 53: l-308 Procunier, l,l.S. 1984. In Onchocerca/Simul'lum relat'ionships in regard to the transmi ss'ion of human onchocerc'iasis in West Ririca. l{HO/Fi 1 StlG document. ( In press ) . Procunier, I.l.S. .l975. A cytological study of two close-ly related blacltfly specles: Cnephia dacotensis and Cnephia or.nithophilia (Diptera: Simuliidae). Can , ,1. Zool. 53: I 622-1637. Procunier, W.S. (D'i ptera ) : and nurse Rejd, G.D.F. & simu'lijds, mi rgrat'ion & Post, R.J. '1985. Adu'lt identiflcation of simulium damnosum s.l. apparent dependence on polyteny on food source in malpigh'ian tubule cell nucle'i during oogenesis. Tropen. med. Paraslt. (subm.itted) Lehane, M.J. '1983. Peritrophic membrane formation'in three temperate Simulium ornatum, S. equjmum and S. lineatana, with respect to the of onchocercal microfilariae. Ann. Trop. Med. Parasit. 78(5): 527-539. Riddiford, L.M. & Truman, J.W. 1978. Btodhemi$try of rnsect hormones and Insect growth regu'lations. In Biochemistry of Insects. Academic Press Inc. l'11 Fifth Avenue, New York, New York'10003. Pp.308-355. shell€y, A.J., Luna Dias, A.P.A., Moraes, M.A.P. & Procunler, l.l.S.1985. The status of Simulium oyapoekense s.'l ./rora'imense and Simulium limbatum of the S.'---- -J -r amazonicum group as vectors of human onchocerc'ias'is ln lowland tropical forest land savannah areas of Brazil. Bull. Ent, Res. (submitted) l,{igglesworth, V.B. .l984. Insect physiology. 8th edn. Chapman & Hall, 733 Thlrd Avenue, New York, New York .l00.l7. lglpp. 0ogelesis ln Simulium Prev'ious attempts to elucidate the events of oogenes'ls in blackflies has boen part'ia11y restricted by available techniques applied ln preparing and stainlng material and the type of photomicroscopy used'in doEumenting this process. (Lewls 1958, Madahar 1967, Garms .l975, Cupp and Collins T$fgt. However.,'tillzatlon of different fixation and staining methods coupled with the use of interferenca microscopy has pnovided the means for understanding ovarian development more fully. Oogenesis in adult Slnuliun ; i,r.rdaranoaurn i.I. aadf€'. roraltrcDse Figure IA. Magnification XIOO. NeuJ.y emorged S. yahense adult: a-geraarlunl b - proxi.aal follicJ.er c - distaJ. folllcle. Nota nuree cells arc uou polyteni-zed (etage I - arrov f). Oocyte is aot dlsti-aguishable at thia stage. rf igure IB. fiagnification XIOO. 9. gSlgllgli tr.s. Tuo bours after bloodoeal. Nurse cells polytenized (stage II-III). Oocyte le apparent. Nucleus at pa.chtrrtene stage of meioels (arrorl c). figure lC. Magnificatioa XIOO. 9. sa-nctipiruli n.s. tlo hours after bloodncal. Precocious condensation of oocyte 'uucleua begindfifg (arror. e). Nurse celLa are at stage III. Fi.gure LD. Magnification Xl@. E. sanc-tipauli n.s. Four hours aftar blootlncel. Oocyte shows condensed nuclear material in the form of a karyoooe (arrou c)'. Yolk forrnation (arrow e). ViteJ"logeneeis appears to be starting. Figure LE. Magnification XIOO. g. EBBS!&gg}! tr.s. Elght hours after bloodneal. YoIk forrnation *. Karyosome preeent (arrow). Nurse cells po).lrtenized (stage III). FolLicuLar epi-h,eL1a1 ceJ-ls appear also to be partially polytealzed (arrov f). Cel1 volume increasing. figure LF. M,agnification Xj@. 5.. eangtiq+uIi a.q Trelve houre after bloodnoal. {) yoJJx formation. Karyosome stl}I vlslble. Nurse ce}ls EtilL stage III polyteny. r,ollicular epitheU.un partially polytenized (arrou g). CelI volnoc increasing. Fi.gure lG. Magnification X2OO. S. yahense. 22 hours after bloodmeal. 4/J yo].k forrnatlon. Ce11 volu.ne lacreased. Nurse ceJ.I etage I. Iuterfcrence microscopy shovs yolk readily. Karyosome etiLL vialble. Yolk doee not cover oocyte. Figr:re 18. !{agnificati.on X1@. g. Iglry. 24 hours after bLoodroeal. Oocyte sti11 vj.sibIe. YoLk covers nost of egg. Nurse cellspushed to one end. Karyosone appoara to be brokea doUn. FiSure )-I. Magnification XI)o. E. rorairr.nse' 48 hours after bloodreal' Interference microscopy. Chorion formatj.on around egg. Gernarluo and proximal, foJ-licle attached outside cborlon (a, b). Proximal fol1icLe appcara to be at a sinil.ar stage as that described for Fig. IB. Nuroe ceLls aro polytenized (arotr f). Paroue females may require less tj.ne to nature tlJlce proximal folLicle partial-ly developed. Figure IJ. Magnification X150. E. Loraimonse. 48 hours after bloodneal'. Interference microscopy. Chorlon present uith ce1ls eloughed off (arrow n). Probably nurse cells. Note also probable presencs of vitellln membranc (partiaJ.ly broken) with yolk underneath (amow v). w bc B Oogeuesis, Gonotrophlc Concordance and parity Figure 2A. Magnification XIOO. g. g!g,!gg. Sour hours aftar bloodneaL. Note the pre6ence of 2 darkly etalned heteropycnotlc bodj.ee ln each diataL fo]-licle (arro,rs e) i-naicati-ng that 2 karyosbmea are present per fol).lclc. th.is is u:r-like that seen in DrosophlLa, Toetse and mosquitoee, and hae aot bcc! (PrccuxiC." lq? 5 ) reported before in blackfli""A a - Sermariun, b - proximal follicle' c - distal foLlic1e. Figure 28. {agnification X!@. ,8. 933Siip39,ti Ir.8. Zfi yolk fornatloa sbowlng polytenized foll,icular epithella-L nuclei (arrow g) covertng the oocyte. Nurse ce1ls stilL partially visible. Fol.Ilc1e celLs are probably active in synthesis of viteLlin (membrans). Tlris stage corrgsPonds to stage III of Christopher for mosquitoes and stage ]OA of !ryIlilg. Figure 2C. Magnification )€oo, 9. su*Sgli n.s. T'relve hours Interference microscopy. Differential follicular development ig to presence of rod and circular like fun51 (arrow c). Fo1licles of development from uewly emerged (arow p) to 4/) yu.Lk forrnation (arrow 5). Figure 2D. Magnification X2OO. l. S9!3:!9Eli !.s. Same as figure 2C excapt differential follicular development probably under genic control. Not all' indj.viduals show gonotrophic concordance (wilI affect vector dynanics). rUrtL fetor4 io tnqr.,3rr.rrrort Parlty Figure 2E. Magnificatioa X2OO. 9. IgIgE. Four houre after bloodmeal. Relict egg (i) and d.eveloping oocytes (k). after bloodlncal. probably duo '6hou raJxgc Figr-rre 2F. Magnification X5@. ,8. eoubrense of a stalk (pedicle - arrou d) attached to a - gernariun, b - proxinaS. foJ.licLe. B. Non blood-fed. Note prceencc distal folIicIe (c); One bour after blood.rneal. Probable pro:rMhl folJ.icle, es- dLstal Figure 2G. Magnification fo].li.cular relict (d) ; foLIicle. X2OO. S. yabense. a-geraaf"luorb- #.0 r.* {i, Il-r's ,f "*$rh p I / ';,k** b Adult Si-ouliun sanctipaull tr.s. Magnification XIOOO. Diagnostic iaversions i.nclude I L P+Q ,lrd II-!;!Q!. At tbe Sassandra R. (Soubre) IT L 7 is polymorphic. Tb.is ladlvldual shora the II L-7 inverted sequence (the p.b. oits distaL and close to the cnd of chromosome) nhereas in the j:rdlvidual in the next flgure shoue fi L-7 sequence as standard (tUe p.b. sits more proxinal). IS IL IIs IIt urs III L short arm of chronosome one loag aro of chronosome one sbort arm of chromosome one long a:m of chromosome one short arm of chromosome ono long arn of chromosome one C - centromerer RB - ring of Balblani, db - double bubbLe, pb - para Balbiani, NO - nucleolar organizer. vf l-l frl l-i 19/ o^.o J F{FraFr /.x,r/ r i"{3 ",ffc hit ,t o,", 'L/t tl zt rn H-tH i,wEer\ --l*1rilr^'r-&:J ''sl."q3-- Uf. oroH -o llcr\55\- = 2' L, Adult Sirnulium sanctipauLi t1.8. ard, E. 13!g Maguification XIOOO. A, C, D show chromosome conplement of S. sancttbault n.s. which is*eharacterlzed by II t - 4.6A being fixed. Note II L-l sequence ie standard (conpare to other figure). B - cbromosome I of S. Yahense. yahense is fixed for the heavy Uana (H) in the centromere region, unlike that for i. gg*g!i!3gE n.s. compare banding betneen A and B. Preknowledge of diagnostic sequences greatly facilitate analysis of mediocre preparattone especially in areas of known cytotypeo' $na;= . irl'ilbSl* IL -e',{^ "r(S k *.b ttu NO "61 lr" ',.;,.,:{i -,. ,ii ,i F l 'il1l ._j il :\ i,1 c D 6r t lrw