Revist a de Saúde Pública
I SSN 0034- 8910 versão im pressaRev Saúde Pública 2003; 37(5)
Scanning electron microscopy of eggs of
Haemagogus
leucocelaenus (Diptera: Culicidae)
Jer onim o Alencara, An t h on y Ér ico Guim arãesa, Rubens Pint o Melloa, Cat ar ina Macedo Lopesa, Nicolas Dégallierb and Jacenir R Sant os - Malleta
a
Depart m ent of Ent om ology. Fundação I nst it ut o Osw aldo Cr uz ( Fiocr uz) . Rio de Janeir o, RJ, Br azil. bI nst it ut o de Pesquisa par a o Desenvolvim ent o [Developm ent al Research I nst it ut e] Brasília, DF, Brazil
ABSTRACT
Objective
To obser v e m or phological det ails of t he eggs of Haem agogus ( Conopost egus ) leucocelaenus, seen for t he fir st t im e by scanning elect r on m icr oscopy ( SEM) , w it h m or phom et r ic analy sis of t he m ain st r uct ur es.
Methods
7.2 sodium cacody lat e buffer , t hen dehy dr at ed in et hanol and dr ied using t he cr it ical point m et hod. Th is w as t h en set u p on m et allic suppor t s, cov er ed w it h gold and obser v ed using t he Jeol 5310 scanning elect r on m icr oscope. Measur em ent s w er e m ade w it h t he aid of t he Sem afor e analy sis soft w ar e cou pled t o t h e elect r on m icr oscope.
Results
The eggs pr esent ed ellipt ical out lines of approxim at ely 574 µm in lengt h and 169 µm in w idt h, w it h an egg in dex (l/ w rat io ) of 3.39 µm . The exochorion was ext rem ely regular and had ornam ent at ion t hat w as usually hex agonal but som et im es pent agonal. Tuber cles w er e obser v ed on t he chor ionic cells, sy m m et rically arranged in relat ion t o t he longit udinal axis. I nside t he cells, t here were sm aller, individualized t ubercles, som e arranged peripherally and ot hers grouped t o a great er or lesser ext ent in t he cent er . The sur face of t he chor ionic r et iculum did not pr esent r ugosit y. The m icr opylar apparat us w as form ed by a prom inent and cont inuous collar of 8.32 µm in t hickness, w it h a slight ly ir r egular sur face. The m icr opy lar disk w as v er y ev ident , and w as cont inuous w it h t he collar . The m icr opy le w as seen at t he cent er of t his disk , m easur ing 1.6 µm and wit h a m icropylar apparat us of 27. 3 µm in diam eter.
Conclusions
The or nam ent at ion of t he ex ochor ion pr esent s differ ences in r elat ion t o t he t uber cles of chor ionic cells and t he ext er nal chor ionic r et iculum bet w een t he eggs of Hg. Leucocelaenus, in com parison wit h t he eggs of Hg. j ant hinom y s and Hg. equinus, and also in r elat ion t o t hose of Aedes aegy pt i, Ae.
albopict us and Ae baham ensis. I n v ar ious aspect s, t he eggs of Hg. leucocelaenus have m ore
r esem blance t o t h ose of Hg. Equinus t han t hose of Hg . j ant hinom y s, w it h gr eat er differ ences pr esent ed in r elat ion t o t he eggs of Hg. spegazzinii and Hg. lucifer .
Keywords
Dipt er a, ult r ast r uct ur e. Culicidae, ult r ast r uct ur e. Eggs. Micr oscopy , elect r on, scanning. Haem ago gu s.
I N TRODUCTION
The Haem agogus genus pr esent s lar ge specific div er sit y and consist s of 32 species ( Ar nell,1 1973) . Many of t hem are ext rem ely im port ant from an epidem iological point of view be cause of t heir involvem ent in t he t r ansm ission of t he for est yellow fever vir us and ot her ar bovir uses, t her eby act ing in t he m aint enance of t he nat ur al cy cle of t hese zoonoses. The for est y ellow fev er t r ansm it t er s at present known in Brazil are exclusively m osquit oes and t he ver t ebr at e host s, w hich ar e m ainly prim at es, including m an ( Degallier et al,5 1992) .
Haem agogus ( Conopost egus) leucocelaenus is an essent ially for est species w hose pr efer r ed habit at is
( Con opost egus) leucocelaenus w as r ecent ly im plicat ed as a pr im ar y v ect or for for est y ellow fev er in
sout heast er n Br azil.* The ar bov ir uses Wy eom y ia, I lhéus, Maguar i, Tucunduba and Una hav e also been isolat ed fr om t his species ( Kar abat sos,1 1 1985; Hervé et al,8 1986) . I n t he labor at or y , it has been show n t o be a m or e efficient v ect or for t he y ellow fev er v ir us t han Aedes aegy pt i ( Waddell,1 5 1949) .
Hg . leu cocelaen u s has r ecent ly r eceiv ed special at t ent ion due t o it s incr easing m edical im por t ance
( Forat t ini & Gom es,6 198 8) . I t s geographical dist ribut ion ext ends from Trinidad t o sout hern Brazil and nor t her n Ar gent ina. I n Br azil, it occur s m ainly in t he st at es of t he sout her n, sout heast er n and w est er n - cent ral regions ( Consoli & Lourenço- d e- Oliveira,4 1988) .
* Pe r son a l com m u n ica t ion fr om Pr ofe ssor Pe dr o V a scon ce llos, Ar bov ir u s La bor a t or y of
I n st it u t o Ev a n dr o Ch a ga s.
The fir st st udy on m osquit o eggs using scanning elect r on m icr oscopy w as m ade by Mat suo & Kunou1 3 ( 1972) . St udies hav e only been m ade on t he eggs of four species of Haem agogus: Hg. spegazzinii Br èt hes, Hg. lucifer ( Howard, Dyar & Knab) ( Mat t ingly,1 4 1973) , Hg . equinus an d Hg. j ant hinom y s ( Linley & Chadee,1 0 1991) . The pr esent st udy had t he obj ect iv e of obser v ing t he eggs of Hg.
leu cocelaen u s using scanning elect r on microscopy ( SEM) and perform ing m orphom et ric analysis of t he
m ain st r uct ur es.
M ET HODS
Eggs of Hg. leucocelaenus w ere obt ained from fem ales capt ured in t he Biological Reserve of Tinguá, in t he m unicipalit y of Nov a I guaçu, St at e of Rio de Janeir o, at t he lat it ude of S 22º 28 - 22º 39’ and longit ude of W 43º 13’ - 43º 34’. These w er e capt ur ed w hen alr eady nat ur ally engor ged, using a m anual m out h aspir at or and t aken t o t he labor at or y on t he sam e day. Only fem ales in per fect condit ion w er e ut ilized. They w er e indiv id ually isolat ed in flat - bot t om ed glass t ubes m easuring 25 m m in diam et er and 50 m m in height t hat , at t he bot t om , cont ained a piece of dam pened cot t on w ool cov er ed w it h filt er paper . This had t he funct ion of ser v ing as a subst r at e for egg- laying ( Bat es & Ro ca-Gar cia,2 1945) . Ar ound 10 fem ales w er e ut ilized, and 20 eggs w er e obt ained. Thr ee of t hese w er e subm it t ed t o m orphom et ric analysis.
I m m ediat ely aft er being laid, t he eggs were fixed in 2.5% glut araldehyde and post fixed in 1% osm ium t et roxide, bot h in a 0.1M, pH 7.2 sodium cacody lat e buffer . Aft er w ashing in t he sam e buffer , t he eggs w er e dehy dr at ed in a ser ies of incr easing et hanol concent r at ions and subm it t ed t o t he cr it ical point drying m et hod, using superdry CO2 in Balzer’s apparat us. Following t his, t he m at er ial w as set up on m et allic suppor t s, cov er ed w it h gold and obser v ed using t he Jeol 5310 scanning elect r on m icr oscope.
The m easur em ent s w er e m ade dir ect ly fr om t he im ages obt ained, w it h t he aid of t he Sem afor e analy sis soft w ar e coupled t o t he m icroscope. The follow ing par am et er s w er e ut ilized: t ot al lengt h, t ot al widt h, t hickness of t he m icropylar collar and size of t he m icropyle. Maxim um s, m inim um s, av er ages and st andar d dev iat ions ar e cit ed in t he Table.
The t er m inology ut ilized for t he descr ipt ion of t he eggs follow s Har bach & Knight7 ( 1980) .
RESULT S
m icropyle and 61.9 µm in t he post erior region. The egg index, or in ot her w ords t he rat io bet w een lengt h and w idt h, w as calculat ed by using t he w idt h of t he cent r al r egion, t hus obt aining 3.39 µm.
Figures - Part 1 - Haemagogus leucocelaenus: whole egg. Scale 100 µm. OCT – External chorionic tubercle. Part 2 - Anterior region of the egg, micropyle and exochorion. Scale 20 µm. MiC – Collar of the micropyle. Part 3 - Structures of the chorionic cells. Scale 20 µm. OCR – External chorionic reticulum. OCC – External chorionic cell. OCT – External chorionic tubercle. Part 4 - Detail of the micropylar apparatus. Scale 10 µm. MiC – Collar of the micropyle. Mi – Micropyle. MiD – Disk of the micropyle.
The ex t er nal coat ing of t he eggs pr esent ed an ex t r em ely r egular ex ochor ion. The m aj or it y of t he exochor ion cells had or nam ent at ion w it h a hexagonal appear ance, but som et im es t his w as pent agonal ( Figure, part 2) .
On t he m ar gins of t hese chor ionic cells, t uber cles m easur ing 1.49 t o 6.32 µm in diam et er were observed, sym m et rically arranged in relat ion t o t he longit udinal axis. I nside t hem , t here were sm aller, individualized t ubercles, som e arranged per ipher ally and ot her s gr ouped t o a gr eat er or lesser ex t ent in t he cent er ( Figur e, par t 3) .
I n it s cent er, a very evident m icropylar disk was observed, m easuring around 7.29 µm in diam et er. I t was cont inuous wit h t he collar, in t he form of m em branous creases arranged uniform ly in a helical m anner . A v er y ev ident or ifice, t he m icr opy le, w as v iew ed at t he cent er of t his disk , m easur ing 1.6 µm . The t ot al ext er nal diam et er of t he m icr opylar appar at us w as 27.3 µm ( Figur e, par t 4) .
D ISCUSSION
The or nam ent at ion of t he exochor ion is an excellent par am et er for m aking com pa r isons bet w een species and r ev eals significant differ ences, especially in r elat ion t o t he pr esence of t uber cles in t he chor ionic cells. The eggs of Hg. j ant hinom y s and Hg. equinus exam ined by Linley & Chadee1 0 ( 1991) w er e differ ent iat ed by t he shapes of t he cells and dist r ibut ion of t hese t uber cles. I n Hg. j ant hinom ys, t hese aut hor s obser v ed chor ionic cells w it h a hex agonal appear ance and som et im es slight ly ov aloid. I n Hg. Equinus, t hese cells usually had a hexagonal appear ance, but som et im es w er e pent agona l, w hich w as also obser v ed in t he pr esent st udy , in Hg. leucocelaenus ( Figur e, par t 2) . Hg . equinus pr esent s sm all chor ionic t uber cles scat t er ed unifor m ly in t he cent er and per ipher y of t he cell, w hile in
Hg. j ant hinom ys t hey ar e ar r anged at t he cent er of t he cell. No sim ilar it y w as found in t he dist r ibut ion
of t hese t uber cles in Hg. leucocelaenus. I n Hg . Equinus, m any of t he t uber cles pr esent fused appear ance, w hile ot her s ar e clear ly scat t er ed and differ fr om Hg. leucocelaenus by present ing sm all nodules on t heir sur faces. The ex t er nal chor ionic r et iculum obser v ed in Hg. leucocelaenus has a sm oot h appear ance, t hus differ ing fr om w hat w as obser ved by Linley & Chadee1 0 ( 1991) in Hg.
j ant hinom y s and Hg. equinus. Linley9 ( 1989) obser v ed t hat t he ex t er nal chor ionic ret iculum in Aedes
aeg y p t i, Ae. albopict us an d Ae. baham ensis also pr esent ed r ugosit y , w it h a fine r et icular m esh. The
sam e aut hor r epor t ed t hat , in t hese species, t her e w as only one cent r al t uber cle inside t he chor ionic cells, t hus diverging from observat ions m ade regarding Haem agogus.
I n Hg. Spegazzinii, Mat t ingly1 4 ( 1973) did not view t he m icropyle in t heir observat ions under t he opt ical m icr oscope. Differ ing gr eat ly fr om t he eggs of Hg. spegazzinii and Hg. lucifer , w hich do not pr esent a cir cular apical collar , t h e eggs of Hg. leucocelaenus resem ble m ore t hose of Hg. equinus t han t hose of Hg. j ant hinom ys. The m icr opyle is also sm aller t han in t hose t w o species. The m icr opy lar disk , locat ed at t he cent er of t he m icr opy lar appar at us, differ s fr om w hat w as obser v ed in eggs of Hg. j ant hinom ys ( Linley & Chadee,1 0 1991) , since in t he lat t er t her e w as no cont inuit y of t he disk w it h t he collar .
Linley & Chadee1 0 ( 1991) r epor t ed t hat , in Hg. j ant hinom y s and Hg. Equinus, t he dor sal sur face of t he eg g w as f ou nd t o hav e adher ed t o t he subst r at e by m eans of filam ent ous t uber cles. These aut hor s point ed out t hat t hese st r uct ur es w ould hav e t he possible funct ion of m ak ing t he egg m or e secur e, t hereby im peding physical rem oval by predat ors, as well as allowing t hem t o float in r ainw at er . Such filam ent s w er e not obser v ed in Hg. leucocelaenus .
ACKNOWLED GEM ENT S
To t he Labor at ór io de Biologia Celular Her t ha Mey er of t he I nst it ut o de Biofísica Car los Chagas Filho of t he Universidade Federal do Rio de Janeiro, for allowing t he use of t he scanning elect r on m icr oscope.
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15. Waddell MB. Com par at iv e efficacy of cer t ain Sout h Am er ican Aedes and Haem agogus m osquit oes as labor at or y v ect or s of y ellow fev er . Am J Tr op Med 1949; 29: 567 - 75.
Address to correspondence
Jer onim o Alencar
Depart am ent o de Ent om ologia – Fiocruz Av. Brasil, 4365
21045 - 900 Rio de Janeiro, RJ, Brazil E- m ail: j alencar @ioc.fiocr uz.br
Received on 16/ 10/ 2002. Review ed on 1/ 4/ 2003. Approved on 8/ 5/ 2003.
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