aT. MINISTERIO DE AGRICULTURA PESCA Y AUMENTACION INSTITUTO NACIONAL DE INVESTIGACIONES AGRARIAS

FINE STRU CTURE OF THE EGG OF CERA Tl TlS CA P/TA TA (WIE DEMANN) (DIPTERA: TE PHRIT IDAE)

l. MAYO , E. M. ROBLES-CHI LLIDA , M. MUÑIZ , M. ANDER SON

INVESTIGACION AGRARIA

Producción y Protección Vegetales

Vol. 4 (2) 1989 Separata n úm. 9

FINE STRUCTURE OF THE EGG OF CERA TlTl5 CAPlTATA (WIEDEMANN)

(DIPfERA: TEPHRITIDAE)

l. MAYO E.M. ROBLF.S-CHJLLIDA

Ce ntro de Investigacion es Biológicas . e.S. I.e. Ve lázq uez , 144. 28006 Madrid .

M.MUÑIZ

Instituto de Edafolog ía y Biología Vegetal. e. S.I. e. Serra no. 115. 28006 Madri d.

M.ANDERSON

Dpt . of Zoology and Co mpa ra tive Physiology. Un iversity of Birmin gham . P.O . Box 363 . Birm ingham B 15 2IT. E ngland .

SUMMARY

T he chorion o f the eggs of Ceratitis capitata, have bee n studied with scanning and tr ansmis­sion e lectron microscop y. Th is study rcveals some srruc tura l d iffer cnccs bet ween the microp ylar area and main body of the egg. Th e cho rion consists of three laye rs: Exoc horion, e ndo chorio n and innermost cho rionic layer. At the micro pylar area , the whole thickness o f cori on is five times thicker than is o n the body of the egg . Th e cavities o f endochorion are mor e ab undant in the microp ylar arca. The e ndoc ho rio n of the main body has two interco nnected tra becular layers. The re is an intermed iate zone, where the cavi ties o f two trabecula r layer s are not conn ected to each oth er , simila r to the end ochorionic layer of the micropylar a rea . T he mor ­phology of the egg she ll of C. capitata is discussed in relation to the egg' resistence to varying concc nt ra tions o f electrolytes a nd its permea bility, compa red with ot he r Di pter a .

KEY WORD S: Ceratltis capitata, ultra structure , egg, cho rio n. resisten ce , perm eability.

INTRODUCTION

Th ere have been a number of structura l stud ies ca rried ou t int o th e de-

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taiJed form of the egg shell of a selection of Díptera of socioeconomic irn­portance, as Drosophila melanogaster (Hinton, 1959; Quattropani, Ander­son, 1969; Klug et al. , 1974; Margaritis et al ., 1976, 1980), Calliphora eryt­hrocephala (Hinton, 1963) , Culex pipiens (Hinton , 1968) , Dacus oleae (Ro­bles-Chillida et al. , 1971), Musca domestica (Cummings, O'Halloran , 1974) and Aedes aegypti (Mathew, Rai , 1975) . Likewise certain studies have done on the structure of the egg membranes in certain Diptera looking particu­lady at their influence on water absorption , permeability to electrolytes and respiratory processes (Robles-Chillida et al. , 1970; Limbourg , Zalokar, 1973; Hinton, 1960a, 1967, 1969, 1971).

Recent work by Margaritis (1983) on Dacus olea e and Ceratit is capitata , has shown peroxidase activity, related to the crosslinks of the chorion pro­teins, in the endochorion and in the crystalline innermost chorionic layer. Although Margaritis (1983, 1985) and Dallai et al. (1985) de scribed sorne characteristics of the egg shell in Ceratitis capitata , there is very little infor­mation concerning the ultrastructure of the egg shells in the micropylar area and surface structure of the entire egg. The present work concerns itself with a detailed ultrastructural study of the protective envelope surrounding the egg of Ceratitis capitata with special reference being paid to the chorionic shells .

MATERIAL AND METHODS

Specimens of Ceratitis capitata were obtained from a population reared at the Instituto de Edafología y Biología Vegetal, on a new larval diet that includes Hansenula anomala as a protein source (Muñíz, Andrés, 1983; Andrés, Muñíz, 1984).

For examination by scanning (SEM) and transmission (TEM) electron microscopy, the eggs were prepared in the following way.

For SEM , the unfixed eggs were attached directly onto specimen stubs , and coated with gold . The samples were examined at 20 kv. using an ISI OS 130 electron microscope.

For TEM , the collacted eggs were fixed in 2,5 p . 100 phosphatebuffered glutaraldehyde at a molarity of 0,25M, and left in this fixative for 3hrs, at 4°C. Postfixation in 1 p . 100 phosphatebuffered osmium tetroxide (0,25M , 2 hrs ., 4°C) , was followed by dehydration in a graded ethanol series and trans­ference to 1,2 epoxypropane. The eggs were then kept in a 50:50 mixture of 1,2 epoxypropane and Epon resin for 24 hrs . at room temperature and ern­bedded in fresh Epon resino Ultrathin sections were cut with a diamond knife on a Reichert OM U2 ultramicrotome and collected on Formvar­coated copper grids. They were double-stained with uranyl acetate and lead citrate before being observed at 80 kv. in a Philips EM 300 electron micro­scope.

265 EGG OF CERA TJ TlS CA f'ITA TA

RESULTS

SEM.-The egg of Ceralitis capitata is oblong in shape , about 950 J1.m long and 200 J1.m in diameter. The anterior pole of the egg or micropylar area (Fig. 1) consists of a chorionic protrusion with severa1 papiliform for­mations form a circular surface about 10 J1.m in diameter (Fig. 2) . The micropyle is about 4-5 J1.m in diameter (Fig . 3) and forms the orifice of a large tube which penetra tes the interior o f the oocyte after traversing the chorion.

Extending from the papiliform formations is the chorionic microscup­ture; it consists of several polygonal plated (hexagon generally) of about 30 J1.m long and 8 J1.m in width , irregulars and lengthened along the longitudinal axis of the egg (Fig . 1); the polygonal surface shows several crateriforrn undulations about 0,6-0,8 J1.m in diameter (Fig . 4) which presumably repre­sent impressions left by sloughed off follicle cells. Each polygonal plate is

Fig. I.-SEM of ante rior po le o f the egg, sho wing the papiliform formations (pf ) a nd polygon al plates (pp) . X 250.

MEB del polo anterior del hu evo, mostrando las formaciones papiliform es (pf) y las placas poligonales (pp). X 250.

Fig. 2.-Delail of the papiliform ío rrnations sur rounding the micropyle (M ). X 750.

Detalle de las formaciones papiliformes que rodean al micropilo (M). X 750.

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tailed form of the egg shell of a selection of Diptera of socioeconomic im­portance, as Drosophila melanogaster (Hinton, 1959; Quattropani, Ander­son, 1969; Klug et al., 1974; Margaritis et al., 1976, 1980), Calliphora eryt­hrocephala (Hinton, 1963), Culex pipiens (Hinton, 1968), Dacus oleae (Ro­bles-Chillida et al., 1971), Musca domestica (Cummings, O'Halloran, 1974) and Aedes aegypti (Mathew, Rai, 1975). Likewise certain studies have done on the structure of the egg membranes in certain Diptera looking particu­lady at their infiuence on water absorption, permeability to electrolytes and respiratory processes (Robles-Chillida et al., 1970; Limbourg, Zalokar, 1973; Hinton, 1960a, 1967, 1969, 1971).

Recent work by Margaritis (1983) on Dacus oleae and Ceratitis capitata, has shown peroxidase activity, related to the crosslinks of the chorion pro­teins, in the endochorion and in the crystalline innermost chorionic layer. AJthough Margaritis (1983, 1985) and Dallai et al. (1985) described sorne characteristics of the egg shell in Ceratitis capitata, there is very little infor­mation concerning the ultrastructure of the egg shells in the micropylar area and surface structure of the en tire egg. The present work concerns itself with a detailed ultrastructural study of the protective envelope surrounding the egg of Ceratitis capitata with special reference being paid to the chorionic shells.

MATERIAL AND METHODS

Specimens of Ceratitis capitata were obtained from a population reared at the Instituto de Edafología y Biología Vegetal, on a new larval diet that includes Hansenula anomala as a protein source (Muñíz, Andrés, 1983; Andrés, Muñíz, 1984).

For examination by scanning (SEM) and transmission (TEM) electron microscopy, the eggs were prepared in the following way.

For SEM, the unfixed eggs were attached directly onto specimen stubs, and coated with gold. The samples were examined at 20 kv. using an ISI OS 130 electron microscope.

For TEM, the collacted eggs were fixed in 2,5 p. 100 phosphatebuffered glutaraldehyde at a moJarity of 0,25M, and left in this fixative for 3hrs, at 4°C. Postfixation in 1 p. 100 phosphatebuffered osmium tetroxide (0,25M, 2 hrs., 4°C), was followed by dehydration in a graded ethanol series and trans­ference to 1,2 epoxypropane. The eggs were then kept in a 50:50 mixture of 1,2 epoxypropane and Epon resin for 24 hrs. at room temperature and ern­bedded in fresh Epon resino Ultrathin sections were cut with a diamond knife on a Reichert OM U2 ultramicrotome and collected on Formvar­coated copper grids. They were double-stained with uranyl acetate and lead citrate before being observed at 80 kv. in a Philips EM 300 electron micro­scope.

265 EGG OF CERA 7J T /S CAP/TATA

RESULTS

SEM.-The egg of Cera litis capiiata is oblong in shape, about 950 p.m long and 200 JLm in diameter . The anterior pole of the egg or micropylar area (Fig. 1) consists of a chorionic protrusion with several papiliform for­mations form a circular surface about 10 p.m in diameter (Fig. 2). The micropyle is about 4-5 p.m in diameter (Fig. 3) and forms the orifi ce of a large tube which penetrates the interior o f the oocyte after traversing the choríon .

Extending from the papiliform formations is the chorionic microscup­ture; it consists of several polygonal plated (hexagon generally) of about 30 p.m long and 8 p.m in width, irregulars and lengthened along the longitudinal axis of the egg (Fig. 1); the polygonal surface shows several crateriform undulations about 0,6-0,8 p.m in diameter (Fig. 4) which presumably repre­sent impressions left by sloughed off follicle cells. Each polygonal piate is

Fig. I .-SEM of ant er ior pole of the egg, showin g the papiliform fo rmations (pf ) and polygon al plat es (pp). X 250.

MEB del polo anterior del huevo, mostrando las formaciones papilijormes (pf ) y las placas poligon ales (pp ). X 250.

Fig. 2.- De laíl of the papiliform form ations surround ing the microp yle (M). X 750.

Detalle de las forma ciones papilijormes que rodean al micropilo (M) . X 750.

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266 1. M A YO e l al .

"

4

Fig . 3.-5EM uf single micropyle 1:-'1). X 2.2:;0.

MEB del micropilo 1.\11. X 2.250.

Fig. 4.-SEM of poly gonal plates showing severa l crateri form undulations and acro py tcs (a). X 3.350.

MEB de las placas poligonales mostran do ondulaciones craterifo rmes y aer áfilos (a). X 3.350.

surrounding by a ridge approximately 0,25 J.tm thick. At the junction of several polygonal plates , there can be seen several circula r hol es 0 ,6-0 ,7 J.tm in diameter ; these holes are more abundant in the anterior region of the egg (Fig .4).

TEM.-The study of the chorion of the egg by TEM, reveals some dif­ferences between the main body and micropylar area of the egg .

In the micropylar area of the egg , ultrastructure observations have re­vealed three distinctly layers (Fig. 5) .

The outermost layer consists of an homogeneous material which corres­ponds to exochorion. At the surface of exochorion we have observed some gro oves and ridges which probably correspond to the papiliform formations surrounding the micropyle, as des cribed in the SEM studies . Sometimes, the outer surface of exochorion is cove red by a thin layer of a mucous-like sub stance . The thickness of the exochorion is abo ut 0 ,2-0 ,3 J.tm . Under the exochorion, can be distinguised the endochorion. It con sists of several

267 CGG üF CE RA Tl Tl S CA PITA TA

cavines and tr abecules of different sizes . The cav ities ar e ernpty and they form air spaces. Th e thickness of the e ndocho rion is ab out 4-5 ,um. The endoc horion is limit ed in the lower regio n to a thin layer o f a bo ut 0 ,01-0 ,02 ,um thick ; this layer is markedly electro n-dense and it is made up of am orph­ous mat erial. It may co rrespo nd to th e inne rmos t chori onic laye r.

In the main body of the egg , the chorion exhibited three sim ilar layers to those de scr íbed in the micropylar ar ea (F íg. 6), but the total th ickn ess of cho rion decreases gradua lly from the ante rior pol e (Fig. 7) .

The exocho rio n is about 0,05 ,um thick and is so metimes covered by a muc ou s (ayer . In the endoch ori on we ha ve been able to mak e out two trabecular, air-filled layers connected to each other. Th e inner trabec ular layer constitutes to severa l oval caviti es arra nge d lon gitud inall y. Th ey are

m

m

i C

.x - - __

..6

Fig. 5.-TE M of mieropylar area of the egg , sho wing the exoehorion (e x) wit h mu eus (m), endoe horion (en) with eav ities (e). and inne rrnost eho rionie layc r. X 17.700.

MET del área micropila r del huevo, mostrand o el exocorion (ex) con mucus (m), el endocorion (en) con sus cavidades (e) y la capa eorióniea más interna. X 17.700.

Fig. 6.- TEM o f the main bod y o f the egg . showing the exo ehorion (ex) eov ered by mu eus (m) . endoe ho rion (e n) with inne r (it) and o ute r (o t) tr abeeular layer. and inner most ehorionie

layer (ie) . No te the dee rease o f tot al th iekness o f cho rio n . X 24.000 .

M ET de la zona media del huevo , mostrando el exocorio n (ex) cubierto de mucus (m), el endocorion (en) con la capa trabeeular interna (it) y externa (o i), y la capa corionica m ás interna

(ie). Notar la dism inución en el grosor IOtal del corion. X 24.000.

lnvcst . Agr.. I'lOd . r ~()1. vcg. Vo l. 4 (2 ) 198')

268 1. MAYO ~ I a l.

also connected laterally and perpendicularly to the other cavities which form the outer trabecular layer. The cavities of the outer trabecular layer cross the main body of the endochorion, reaching its superior edge . The remain­der of endochorion is compact and homogeneous, showing several ir­regularities in its superior zone , which is in touch with the exochorion. These small irregularities probably correspond to crateriform undulations on the polygonal plates described in the SEM section. Obligue sections of the main body of the egg, reveal the perforated endochorion. The endochor­ion is about 1,02 JLm in thickness, at this level. The innermost chorionic layer is very thin and markedly electron-dense, it abuts the endochorion and is around 0,02 JLm.

The whole thickness of the chorion at the micropylar area is five times greater than it is on the main body of the egg.

There is an intermediate zone , situated between the micropylar area and main body of the egg (Fig. 8); in this area, the endochorion consists of two trabecular layers like the main body, but they have a different morphology . The cavities of the inner trabecular layer are enlarged longitudinally, and the cavities of the outer trabecular layer are circular in shape. There is not contact between the two trabecular layers in this intermediate zone of the egg.

DISCUSSION

The outer surface of the egg has been described in several species of Diptera by several authors (Hinton, 1959, 1960a, 1960b, 1960c, 1962a, 1962b, 1963, 1968; Hinton, Service, 1969; Horsfall et al. , 1970; Klug et al., 1974; Leopold et al., 1978; Margaritis et al ., 1980; Margaritis, 1985; Dallai et al. , 1985) .

The presence of structures such as polygonal plates, aeropyles, etc . on chorionic microscupture of Ceratitis capitata, is not a fundamentally specific characteristic for the egg of this insect; however, their size, location and number may constitute interesting data, not only from a possible taxonomic viewpoint (Giano, Mazzini, 1984), but also they are critical for correct em­bryonic development.

The polygonal plates of Ceratitis capitata are very similar to those ob­served in the lepidopterans Monopis rusticella (Chauvin, Barbier, 1972) and Galleria mellonella (Barbier, Chauvin, 1974), in the beetle Acanthoscelides obtectus (Biemont et al ., 1981) and in Drosophila melanogaster (Klug et al. , 1974).

The circular holes situated preferentially on the anterior zone of the egg of Ceratitis capitata, have the same shape and disposition, between the poly­gonal plated, as aeropyles observed in Galleria mellonella (Barbier , Chauvin, 1874) and on the convex surface of the eggs of Phalera bucephala, Acrolepia assectella and Plutella maculipennis (Chauvin et al., 1974).

269 EGG O F CE RA T/ T/5 CA P/ TA TA

8

Fig. 7.-TEM o f cho rion sho wing the microp ylar a rca (ma) a nd the intermedi are zone (iz) . X 5.600.

MET del corion mostrando el área micropilar (ma¡ y la ZO l1a intermedia (iz}. X 5.600.

Fig. 8.- TEM of the inte rrned iatc zon e o f th c cgg. situatc d betwccn the mierop ylar arca and main body. showing th e endoch ori on with inner (i t) and o utcr (0 1) trabccul ar layer s. X 11.200.

MET de la zo na intermedia. situada entre el área micropilar y la ::0110 media del hu evo , mostran­do el endocorion con la capa trabecular interna (in y la capa trabecular externa ( 0 1) , X 11.200.

Th e three layers of the chorion of Ceratitis capitata showed many differ­ences between the main body and micropylar area of the egg , as is seen in Drosophila melanogaster (Margaritis et al., 1980).

A trabecular endochorion enclosing air spaces is also found in Calliphora erythrocepgala (Wigglesworth, Salpeter, 1962), Musca domestica (Hinton, 1967), Drosophila melanogaster (M arg aritis, 1983), and severa l other species of Diptera . These cavities of the trabecular layer are thought to be air -filled and undoubtedly facilitate respiration .

The eggs shells not only allow respiration through specialized structures (aeropyles , pseudomicropyles, plastrons, etc. ) , but also the y avoid dehydra­tion of embryo and protect it against adverse en vironmental conditions.

Re sistance of insect egg to low relative humidity seems to be primarly related to the formation of a compact vitelline membrane, but the chorion

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itself may also play an important role in this (Biemont et al. , 1981) . Accord­ing to Margaritis (1983), the eggs of Ceratitis capitata have a thick vitelline membrane (0,6 JLm), but when they were exposed to low relative humidity, they were rapidly desiccated . However, the eggs of Dacus oleae have a very thin vitelline membrane and they were able to withstand this exposure for longer periods of time. For this reason, the resistance of the eggs to desicca­tion is influenced by other several factors , besides the existence of vitelline mebrane .

In order to establish the relationship between mortality of tephritidae eggs and several physical-chemical agents (pH, osmotic pressure, concentra­tion of electrolytes, etc.), we have carried out sorne preliminary investiga­tions on Ceratitis capitata (Muñíz , 1973; Robles-Chillida, 1975; Muñíz, 1976a, 1976b; Caamaño, Muñíz, 1980, Gil, 1981) . According to these inves­tigations, the eggs of Ceratitis capitata can survive concentrations of electro­Iytes seven times higher than those of Dacus oleae, and the chorion of the eggs of Ceratitis capitata is approximately 0 ,5 JLm thicker than that of Dacus oleae. For this reason, we think this characteristic may also have an ínflu­ence in attenuating or minimizing the effects of an adverse environment.

CONCLUSIONS

The chorion of Ceratitis capitata reveals sorne differences between the main body and micropylar area of the egg: In the micropylar area, the exochorion is about 0,2-0,3 JLm; the endochorion is 4-5 JLm thick, it has several cavities or trabecules and they are not connected to each other; the innermost chorionic layer is about 0,01-0,02 JLm.

In the main body, the exochorion is abou 0,05 JLm ; the endochorion is about 1,02 JLm, it has two trabecular layers connected to each other; the innermost chorionic layer is around 0,02 JLm .

The thickneed of chorion decreases gradually from the mícropylar area lo main body of the egg, and the whole thickness of the chorion al the micropylar area is five times greater than it ís on the maín body .

The thickness of chorion may play an important role in the resistance of the insect egg to adverse environment.

RESUMEN

Estructura fina del huevo de Ceratitis capitata (Wledemann) (Dlptera: Tepbrltldae)

El carian del huevo de Ceratitis capitata fue estudiado a microscopía electrónica de transmi­sión y barrido . Este estudio revel ó algunas diferen cias ultraestructurales entre el áre a micropi­lar y la zona med ia del huevo. El ca ria n está constituido por tres cap as: Exocorion, endoco rion y capa corión ica más interna . En el área micropil ar , e l grosor del carian es cinco veces superior al de la zona media del huevo. Las cavidades del endocorion son más abundantes en el área

271 EGG OF CERAT/T/5 CAP/TATA

micropilar. El endocorion de la zona media del huevo. Las cavidades del endocorion son más abundantes en el área micropilar. El endocorion de la zona media posee dos capas trabeculares interconectadas. Existe una zona intermedia, donde las cavidades de las dos capas trabeculares no se conectan entre sí, al igual que ocurre en el endocorion del área micropilar . Se discute la morfología de las cubiertas del huevo en relación con la resistencia del mismo , frente a varia­ciones en la concentración de electrolitos del medio y su permeabilidad, comparándola con la de otros Dípteros.

PALABRAS CLAVE: Ceratitis capitata, ultraestructura, huevo, corion , res istencia permea­bilidad .

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BIEMONT J.c., CHAUVIN G ., HAMON C., 1981. Ultrastructure and resistance to water loss in eggs of Acanthoscelides obtectus Say (Coleoptera: Bruchidae). J. Insect Physiol., 27, 667-679.

CAAMAÑO B., MUÑIZ M., 1980. Relación entre mortalidad y actividad media molal de electrolitos en huevos de Ceratitis capitata Wied . Graellsia, 34, 237-248.

CUMMINGS M.R. , O 'H'ALLORAN T .R, 1974. Polar aeropyles in the egg of the housefly Musca domestica (D iptera : Muscidae) . Trans. Amer. Microsc , Soc ., 93 , 277-280.

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HINTON H.E., 1967. The respiratory system of the egg-shell of the common housefly. 1. Insect Physiol., 13,647-651.

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MARGARITIS L.H., 1983. Structural heterogeneity of the egg-shell between Ceratitis capitata and Dacus oleae : Evidence for the participation of peroxidase in the crosslinking of chorion proteins . In: Fruit Flies of Ecomomic Importance. Cavalloro, R ., ed . A .A. Balkerna/ Rotterdam, pp. 114-121.

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Invest. Agr. : Prod . Prot . veg. Vol. 4 (2) 1989