- Vol. 116, No. 1, January & February 2005 1

ULTRASTRUCTURE OF THE EGG CHORION OF SINUATA OLIVIER 1811 (: ) FROM TURKEY1

Selami Candan,"Zekiye Suludere,"Fatma A~ikgoz,"and Abdullah Hasbenli"

ABSTRACT: The ultrastructure of the egg chorion of Nemopteva sinuata Olivier 1811 was studied with scanning (SEM) and transmission (TEM) electron microscopy. Females were collected from Antalya, Finike, Turkey, and maintained under laboratory conditions. Eggs were laid singly in cotton batting. The eggs are spherical, snow-white, and lusterless; with a diameter of 0.82-0.91 mm; and with one micropyle. The chorion is highly sculptured with regular hexagonal convexities on the sur- face touching one another. The micropyle is in disk form; in profile it resembles a comet with no opening. In cross section, three layers of chorion are easily distinguished by transmission electron microscopy.

KEY WORDS: Neuroptera, Nemopteridae, Nemopteva sinuata, ultrastructure, chorion, micropylar process, scanning and transmission electron microscopy

From recent studies, it appears that the, surface structure of various chorionic modifications has morphological, physiological and taxonomic significance in various orders (Hinton, 1981; Downey and Allyn, 1984; Salkeld, 1983, 1984; Margaritis, 1985; Gaino et al., 1987; Sahlen, 1996; Lounibos et al., 1997; Suludere et al., 1999; Baker and Chandrapatya,,2001; Wolf and Reid, 2001). Scanning and transmission electron microscopy has become a useful tool for detailed description of surface morphology and ultrastructure in eggs (Mouzaki et al., 1991; Simiczyjew, 1994; Bundy and McPherson, 2000; Danielczok and Kocorek, 2003; Candan and Suludere, 2003; Candan et al. 2004). Hinton (1981) and Margaritis (1985) provide an extensive survey of respiratory and morpho- logical structures of insect eggs. Egg surface structure and ultrastructure of Neu- roptera species, including Nemopteridae, has been reported by many authors, however, accurate knowledge of the egg morphology is still lacking for many taxa (Hinton, 1981; Mazzini, 1976; Cutler, 1993; Monserrat, 1985, 1996; Shields and Pupedis, 1997). The ecology and egg of Nemoptera sinuata has been briefly described by Popov (2002) using light microscope. According to Popov (2002) adults of N. sinuata occur in meadows and open sunny places with Mediterranean and Sub Mediterranean vegetation near a shel- tered river valley and feed only on pollen. They are most active at noon between the middle of May and the end of June. N. sinuata is a diurnal insect. It flies, feeds and lays eggs only during the day.

I Received on June 24,2004. Accepted on November 12,2004. z Gazi University, Science and Arts Faculty, Department of Biology, 06500 Ankara, Turkey. E-mails: (SC) [email protected], (ZS) [email protected], (FA) [email protected], and (AH) [email protected].

ENTOMOLOGICAL NEWS 116 (1): 1, January & February 2005 Mailed on March 3 1, 2005 2 ENTOMOLOGICAL NEWS

The eggs are usually laid in the morning, by a female, with half open wings and a drooping abdomen, perched on blossoms or racemes of plants, e.g. Achilles. An egg is oviposited every two minutes, and after 4-6 eggs the female moves on to another raceme. Up to 70 eggs are laid by a female over a period of 10 days, during the total female life span of 20 days. Within the first five days the number gradually drops from 14 to 9 eggs per day. The eggs fall directly to the ground or onto dry vegetation. They are not adhesive, but elastic and bounce when coming into contact with a hard surface, as for example a piece of wood. Our study describes the ultrastructure of the egg of the Neuropteran, N. sinuata using scanning (SEM) and transmission electron microscopy (TEM).

METHODS Eggs were obtained from ovipositions by females of N. sinuata collected from Antalya, Finike, Turkey (16 June 2001). Females deposited eggs singly onto cot- ton batting in plastic jars. The eggs were prepared for SEM following the methodology described by Suludere (1988). Cleaned and dried eggs were mount- ed with double-sided tape on SEM stubs and coated with gold in a Polaron SC 502 Sputter Coater. They were examined with a Jeol JSM 5600 Scanning Elec- tron Microscope at 15-20 kV. Other eggs were fixed with 2.5% glutaraldehyde in a phosphate buffer (pH 7.2) for 2 hours and post fixed with 1% osmium tetrox- ide in a phosphate buffer (pH 7.2) for 1 hour. The samples were then embedded in Glauert's araldite medium and the ultra thin sections were stained with Rey- nold's lead citrate following uranyl acetate. These eggs were examined with a Zeiss EM 900 Transmission Electron Microscope at 80 kV.

RESULTS AND DISCUSSION From our observations; N. sinuata deposited eggs singly into cotton batting in the laboratory and egg number varied from 35 to 60 and were not adhesive. N. sinuata eggs are spherical, snow-white, opaque, and lusterless. Eggs average 0.82-0.91 mm (Fig. l,2). The genus Nemoptera is one of the few genera among all Neuroptera with spherical eggs. In the Chrysopidae, Brothidae, and Manti- spidae, eggs that are generally cylindrical, e.g. Mantispa sayi Banks (Manti- spidae) eggs are elongate and cylindrical with rounded ends. Each N. sinuata egg has a buttonlike micropylar process at one apex; at the opposite end a short, thin flexible stalk anchors the egg to the substrate by the egg stalk (Shields and Pupedis, 1997). Mazzini (1976) also stated that Chrysopa carnea Steph. (Chry- sopidae) has a cylindrical egg and the micropylar area of the egg is at the anteri- or pole, while the posterior pole has a peduncle which functions to attach the egg to the substrate. Monserrat (1996) reported that Lertha so$ae Monserrat (Ne- mopterinae) has an ovoid egg and the micropyle is conical with a discoidal and conspicuous apex. Surface of chorion covered with convex ovarian follicle cell impressions, irregularly hexagonal and bordered by subcylindrical crests. The color of the eggs is yellowish white when laid, rosaceous some days later, and Vol. 116, No. 1, January & February 2005 3 dark grayish prior to hatching. In addition the eggs of Nemoptera bipennis were described by Withycombe (1925) and Monserrat (1985). Eggs of N. bipennis are spherical, similar to a golf ball, prolonged in the cephalic apex by a truncated cone formation in whose end it is located micropyle. The chorion has hemi- spheric swellings more convex in the equatorial zone than in the poles. The cephalic pole has a truncated cone formation that in its base is limited by the hexagonal margins of the peripheral swellings. Micropyle, as a plate, is to circu- late, thicker in the external margins. Its surface shows a porous structure, with multitude of internal tubules that give a sponge aspect to it.

Fig. 1. Scanning electron micrograph (SEM) of Nemoptera sinuata egg, lateral view. Scale bar =I00 pm.

Fig. 2. SEM of egg of N. sinuata, end view with micropyle. Scale bar = 100 pm. 4 ENTOMOLOGICAL NEWS

In Neuroptera, including Nemopteridae, and in the Chrysopidae, eggs are laid unconcealed and usually not in an organized arrangement, although occasionally a straight row of 10-15 eggs may be found across a leaf or stem. Some species of Chvysopa lay single singly, e.g. C. rufilabris Burmesiter and C. chi Fitch (Smith, 1922). Each egg has a single stalk, but if the female is confined in a small space, the stalk of one egg may be attached part of the way up the stalk of another egg (Hinton, 1981). However, these three families are not closely related and have presumably independently evolved a stalked egg. The stalks of the eggs of the Mantispidae are only about twice the length of the egg, whereas in many Chrysopidae they may be over 10 times the length of the egg (Hinton, 1981; Shields and Pupedis, 1997). Nemoptera sinuata eggs do not have an egg stalk. In Neuropterans, the micropylar apparatus is usually conspicuous as a plate or knob raised well above the surface. In the Coniopterygidae the micropylar apparatus is on a conical projection at the anterior pole. In the Chrysopidae the knob is somewhat flattened and saucer-shaped. In the Myrmeleontidae and As- calaphidae, in which the micropyle is also in the form of a shallow saucer, there is a ring of perforations at the margin of saucer and from these perforations the canals pass inwards to an area at the centre. In both Myrmeleontidae and Ascalaphidae there appears to be a similar structure at the posterior pole and it has therefore been suggested that in these two families there are both anterior and posterior micropyles (Withycombe, 1925). Henry (1972) states that in the Ascalaphidae identical micropyles are present on both the anterior and poste- rior ends. In Mantispa sayi eggs, micropylar prominence is roughly dome- shaped and above the surface of the chorion. The basal periphery of the micropylar knob is divided into 8-10 scallops that project downward (Shields and Pupedis, 1997). In C. carnea the micropylar region appears circular and the margin of the micropylar area has 30 indentations each of which corre- sponds to a micropylar orifice (Mazzini, 1976). Also Monserrat (1996) stated that eggs of L. sofiae have a conical micropyle with a discoidal and conspicu- ous apex. In N. sinuata, there is one micropylar process at the anterior pole (Fig. 1-4). When seen from above, the micropyle has the form of a disk; in profile it looks like a cornet with no opening in it (Fig. 3). A polar lid with the micropyle in the center is opened during hatching (Fig. 4). The micropylar process has a central canal for the passage of sperm and has a porous structure which serves for respiratory interchange (Southwood, 1956; Cobben, 1968; Hinton 1981 ; Lambdin and Lu, 1984; Shuxhi, 1985; Javahery, 1994; Candan, 1997; Candan 1999; Candan and Suludere, 1999 a, b; Suludere et al. 1999; Candan and Suludere, 2003; Candan et al., 2004). Similar observations are presented in this study for N.sinuata. Vol. 116, No. 1, January & February 2005

Fig. 3. SEM of a closed micropyle (*) in unhatched egg of N.sinuata. Scale bar = 100 pm.

Fig. 4. Micropyle opening (arrow) of a hatched egg of N.sinua- ta. Scale bar = 100 pm.

In the Chrysopidae, Mantispidae, and other Neuroptera in which the surface of the chorion is densely set with projections, a film of air is trapped by these when the eggs are flooded. This is an example of plastron respiration projections as in some dipterous eggs with rather similar projections e.g. Anopheles (Culici- dae) and some Syrphidae. The projections protect the general surface against the effects of turbulence, and they establish a boundary layer of air creating a humid- ity gradient that reduces the loss of water (Hinton, 1981). Fig. 5. Hexagonal pattern on the chorion surface of N. sinuata. Scale bar = 100 ym

Fig. 6. High magnification of the hexagonal pattern on the chori- on surface of N.sinuata egg. Scale bar = 10 ym.

The surface of the egg chorion of Mantispa sayi Banks appears to be devoid of these features, but closer examination reveals a meshwork of interconnecting ridges on the surface of the chorion and the micropylar surface except at the region of stalk attachment (Shields and Pupedis, 1997). The surface of the egg chorion of C. carnea also shows a uniform surface sculpturing at low magnifi- cation and near the peduncle the surface appears smooth. At higher magnifica- tion the surface sculpturing of the chorion appears as raised, irregular projections joined by narrow bridges (Mazzini 1976). In L. sojiae eggs, the surface of the chorion is covered with convex ovarian follicle cell impressions, irregularly hexagonal and bordered by sub-cylindrical crests. Vol. 116, No. 1, January & February 2005 7

The egg chorion of N. sinuata is highly sculptured, with many regular hexag- onal convexities on the surface, which touch one another and these hexagonal boundaries are well defined. The surface of the hexagonal boundaries are de- pressed and rough (Fig. 5,6). Popov (2002) stated that about 30 convexities have been counted on the periphery of the egg and about 180 convexities on the whole egg surface. According to our observations, N. sinuata eggs have similar chori- on morphology with L. sojiae and N. bipennis but L. sojiae has different egg shapes. The egg shape in L. sojiae is ovoid but in N. sinuata and N, bipennis it is spherical. After the 15th or 16th day the egg becomes light pink and then grey on one side. The embryo lies in the form of a semicircle in the egg. A polar lid with the micropyle in the centre is opened during hatching. The lid is cut off by an egg- breaker on the larval clypeus and the eggshell breaks by pressure from the dor- sal surface of the larva. The split becomes an almost complete circle and the lid separates without breaking from the egg (Fig. 7). The newly hatched larva is 1.9-2.2 mm long including the jaws. They are dorsally grey, with an oblong, transverse, dark spot on both sides of the median line of every thoracic and abdominal segment. There is a large, almost black, spot on the head and the body is densely covered with long and short setae (Fig. 8). Egg hatching and first instar larvae of N. sinuata from Bulgaria (Popov, 2002) and N. bipennis IIliger reared by Monserrat (1996) are very similar to those we observed.

Fig. 7. Hatched egg of N. sinuata. Scale bar = 100 pm. 8 ENTOMOLOGICAL NEWS

Fig. 8. The first instar larva of N. sinuata. Scale bar = 100 ym.

Fig. 9. Cross section of the egg-chorion of N. sinuata (TEM). Endochorion (I), exochorion (2), extrachorion (3) X 10,000.

Examination of a cross section of the chorion shows that it is composed of three layers. The basal endochorion has layers of fibers. Under this there are air cavities of different sizes which are thought to function in plastron respiration. The exochorion appears as a large and homogenous layer. Above the exochorion is the extrachorion, which is composed of electron dense and electron light lay- ers extending to the exochorion layer (Fig. 9). Mazzini (1976) reported that the egg of C. carnea has two layers, the endochorion and exochorion. The endo- Vol. 116, No. 1, January & February 2005 9 chorion consists of plurilaminar layers, each periodically arranged and parallel to the egg surface. The exchorion consists of numerous projections, separated from one another by spaces. Cutler (1993) observed an inner chorion and pillars that formed a continuous air space in M. interrupta Say eggs. Insect eggs require a large surface area in order to supply the oxygen required by the developing embryo. Because of the presence of air spaces as seen in the cross sec- tion under TEM, the eggs of N. sinuata are able to perform plastron respiration, thus supplying enough oxygen without significant moisture loss. As suggested by Hinton (1969), the chorion is probably so structured so that water loss is kept to a minimum.

ACKNOWLEDGEMENTS We wish to thank Kirikkale University Research Centre for providing SEM facilities.

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Bibliography of the Neuropterida Reference number (r#): 11582

Reference Citation: Candan, S.; Suludere, Z.; Açikgöz, F.; Hasbenli, A. 2005 [2005.??.??]. Ultrastructure of the egg chorion of Nemoptera sinuata Olivier 1811 (Neuroptera: Nemopteridae) from Turkey. Entomological News, Philadelphia 116:1-10.

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