Fine structure of the egg-shell in two humpbacked flies, Megaselia scalaris and Megaselia spiracularis (Diptera: Phoridae)

1~1. J. IPISW

Pergamon

Morphd

&Embryo/., Vol. 25, No. 3, pp. 289-294. 1996 Copyright J; 1996 Elsevier Science Ltd Prmted I” Great Bntam. All nghts reserved 002&7322,‘96 1615.00+0.00

PII: SOO20-7322(96)00001-3

FINE STRUCTURE FLIES, MEGASELIA

OF THE EGG-SHELL IN TWO HUMPBACKED SCALARIS AND MEGASELIA SPIRACULARIS (DIPTERA: PHORIDAE)

Klaus Werner tJohannes

Gutenberg-Universitat

iDepartment

of Plant Protection,

Mainz,

WolPl_ and Guanchun

Liu$

Institut

Shenyang

fur Anthropologie, Colonel-Kleinmann Weg 2a, 55128 Mainz, Germany Agricultural University, Shenyang, Liaoning 110161, P.R. China

(Accepted 14 February 1996) Abstract-The present study deals with the fine structure of the shell in the main body of deposited eggs of 2 humpbacked flies Megaselia scalaris and M. spiracularis (Diptera: Phoridae). Eggs deposited within a period of 3 h were embedded for transmission electron microscopy. In ultrathin sections, the overall architecture of the egg-shell was qualitatively similar to that of Drosophila melanogaster. There were, however, a series of quantitative differences. A prominent vitelline membrane bordered the plasma membrane. The chorion consisted of a thin inner layer, pillars, and a thin outer layer. There was a prominent external layer consisting of finely granular material. A 6-7 layered crystalline layer, and an amorphous electron-dense layer separated the vitelline membrane and the chorion. Hypochlorit treatment was used in order to determine the nature of this electron-dense layer. While all chorionic elements were removed, the layer in question was resistent to hypochlorit treatment, and therefore corresponds most probably to the wax layer found in Drosophila eggs. Copyright 0 1996 Elsevier Science Ltd Index descriptors (in addition

to those in the title): Chorion,

vitelline membrane,

wax layer.

INTRODUCTION

Flies of the genus Megaselia show a series of remarkable traits. In M. scalaris, sex determination is unusual. The location of a male epistatic sex-determining factor moves at a low rate between the individual chromosomes (Mainx, 1964). Recent molecular and genetic work confirmed this finding and showed that the change of the sex-determining factor does not involve the transfer of a larger DNA fragment (Traut and Willhoeft, 1990). M. scalaris possesses 3 homomorphic chromosome pairs, 1 telocentric and 2 metacentric, which are visible using light microscopy in chromosome spreads (Johnson et al., 1988). Fine structure work involving ultrathin serial sections of mitotic spindles revealed, in addition, the presence of typically 2 minute elements. These qualitatively resembled the centromeres of the regular chromosomes, but were devoid of chromosome arms. In male meiosis, the tiny elements were observed to pair, the prerequisite for regular segregation (Wolf et al., 1991). The presence of 2 minute elements has also been observed in serially sectioned spermatogonial spindles of another species, M. spiracularis (Wolf, unpublished data).

*Author

to whom correspondence

should be addressed. 289

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K. W. Wolf and G. Liu

These features render representatives of the Phoridae attractive experimental models, and the last few years have seen efforts in describing the structure of chromosomes, spindles and sperm in Megaselia scalaris (Curtis et al., 1989; Wolf, 1990a, b; Curtis and Benner, 199 1; Wolf et al., 1991, 1993, 1994a, b). The surface morphology of eggs has been studied in 2 representatives of the genus Megaselia using scanning electron microscopy (Furukawa and Kaneko, 1981), but information on the fine structure of eggs is missing for any humpbacked fly (for reviews on insect egg structure, see Hinton, 1981; Margaritis, 1985). In the present report, we concentrate on the fine structure of the shell in deposited eggs of M. scalaris and M. spiracularis. Additionally, we describe the effects of hypochlorit treatment on the egg-shell in both species. Hypochlorit treatment is usually employed in Diptera in order to render the egg-shell softer when it comes to microinjection and cell transplantation in embryos. The Megaselia species examined in the present study did not visibly differ from one another in the architecture of the egg-shell. It turned out that the egg-shell of both species was qualitatively very similar to that of Drosophila melanogaster in overall architecture. Individual components were, however, most often thicker.

MATERIALS

AND

METHODS

The animals Megaselia scalaris, strain “Wien”, and Megaselia spiracularis were kept in the laboratory in plastic tubes. The rearing conditions and the medium composition were identical for both species and have been described previously (Johnson et al., 1988). M. spiracularis was collected and identified by one of us (G.L.). The animals were found in Shenyang (P.R. China). Pupae were mailed to Liibeck (Germany) in the summer of 1994, and were held in the laboratory for the duration of the experiments (Johnson et al., 1988). EIrctron microscopy Adult animals, 2-3 days old, were transferred into culture vials containing food (Johnson et al., 1988). Using wetted forceps, deposited eggs were collected from the food surface or the vial wall after about 3 h. A batch of the eggs was embedded without pretreatment. Another batch was pretreated with a 1:2 dilution of a Na-hypochlorit solution (Carl Roth GmbH, Karlsruhe, Germany). The eggs were incubated at room temperature until the egg surface appeared smooth upon inspection with a dissecting microscope. Usually 10-15 min were needed. The further steps did not differentiate between treated and untreated eggs. They were transferred into cacodylate buffer (0.1 M, pH 7.2) containing 2.5% gluataraldehyde and 2% formaldehyde. The eggs were individually cut open using microscissors. Fixation was carried out overnight at 4°C. After rinsing in cacodylate buffer, the specimens were postfixed in 1 % 0~0, in this buffer (1 h). The specimens were rinsed again, dehydrated in ethanol and embedded in Epon 812. The main body of the eggs was located in semithin sections stained according to Jeon (1965). Ultrathin sections through the egg periphery were analysed with a Philips EM 400 transmission electron microscope operated at 80 kV. The magnification of the electron microscope was calibrated using a 1200 lines/mm replica grating.

Fig. 1. Survey micrograph of a longitudinal section through the untreated egg-shell in Megaselia scalaris. A portion of the peripheral cytoplasm is visible at the bottom. The succession of egg layer is: vitelline membrane (vm), wax layer (WI), innermost chorionic layer (icl), inner chorion (ic), pillars (p), outer chorion (oc), and external layer (ex). Note the prominent transparent space of varying thickness between the outer chorion and the external layer. Bar represents 1 pm. Fig. 2. Detail of the untreated egg-shell in Megaselia scalaris. The crystalline nature of the innermost chorionic layer (icl) is obvious, while the wax layer (WI) and the vitelline membrane appear homogeneous. The thin inner chorion (ic) is detectable. Bar represents 200 nm. Fig. 3. The hypochlorit-treated egg-shell of Megaselia scalaris. The vitelline membrane (vm) and the wax layer (WI) are visible. Bar represents 0.5 pm. Fig. 4. The hypochlorit-treated egg-shell of Megaselia spiracularis. Vitelline membrane (vm) and wax layer (WI) are preserved. Bar represents 0.5 pm.

The Egg-shell

in Megaselia Species

-.

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291

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K. W. Wolf and G. Liu RESULTS

Portions of the eggs selected for our fine structure analysis were in their main body. M. scalaris and M. spiracualaris were not visibly different from one another in their egg-shells. We used a terminology for elements of the egg-shell as in the review article of Margaritis (1985). A prominent vitelline membrane of low electron density followed the plasma membrane of the egg (Fig. 1). The thickness of this first extracellular layer varied slightly in our sample (0.48-0.76 pm). A thin (width about 27 nm), relatively electron-dense, layer was attached to the vitelline membrane (Figs. 1 and 2). It is interpreted as the wax layer (see below). The next layer was characterized by its horizontally striated organization. At high resolution, 6 to 7 thicker striations could be observed parallel to the egg surface. These were interspersed with relatively thin layers. The total width of this layer amounted to about 70 nm. The multilayered organization strongly indicated that it represented the innermost chorionic layer (compare Sweeny et al., 1970; Furneaux and Mackay, 1976; Margaritis et al., 1980). The inner and the outer portions of the chorion were thin (at most 18 nm). Pillars connected both layers at irregular intervals. The pillars had a loose appearance and consisted of threads and more compact portions of amorphous material of low electron density. The space between the pillars was very transparent and contained filamentous material in low frequency. The total width of the chorion amounted to approximately 1.6 pm. A transparent space of uneven width (0.340.9 pm) separated the chorion from the external layer. We do not know whether this represents the exochorion or a mucous layer. We use, therefore, the term external layer for the outermost element of the eggs under study. The external layer was about 0.76 pm thick and consisted of fine granular material. After treatment with hypochloric acid, both the external layer and the chorion, including the inner crystalline layer, were removed in both Megaselia species (Figs. 3 and 4). A dense layer at the outer face of the vitelline membrane persisted. This resistance suggests that we are dealing with the wax layer, known from Drosophila eggs.

DISCUSSION

The overall morphology of the shell in deposited eggs of M. scalaris and M. spiracularis was similar to that of D. melanogaster. There were, however, some quantitative differences that are listed in Table 1, and we did not detect elements corresponding to the roof network of the D. melanogaster egg-shell in our material. The comparison between D. melanogaster and Megaselia species revealed that, with the exception of the inner and the outer layers of Table

1. Comparison

of quantitative (Margaritis,

parameters in egg-shell layers between 1985) and Megaselia species (this study)

Drosophila melanogaster

Layer

Drosophila melanogaster

Megaselia species

Vitelline membrane Wax layer Innermost chorionic

0.3 pm thick 5510 nm thick 4 to 5 sublayers total thickness: 40 nm thick 0.2 pm thick 0.74 pm thick 0.3 pm thick

0.48-0.76 nm thick 27 nm thick 6 to 7 sublayers total thickness: 70 nm around 18 nm thick around 18 nm thick 1.6 pm thick 0.76 pm thick

Inner chorion Outer chorion Chorion (total) Exochorion/external

layer

layer

40 nm

The Egg-shell

in Megaselia Species

293

all components were thicker in Megaselia. This suggests that the egg-shell in species is highly resistent to environmental hazards, namely dryness and bacterial attack. It is important to know that the larvae of both Megaseliu species examined in this study live on carrion in nature (e.g. Benner and Ostermeyer, 1980). The adults deposit their eggs on material most probably contaminated with many kinds of microorganisms. Under these conditions, an egg-shell equipped to persist in an unfavorable environment represents an advantage. In fact, differences in environmental factors during egg-laying determine the appearance of the egg-shell in individual insects (Margaritis, 1985) and this explains the similarity of shell architecture in our two Megaselia species. Far-reaching systematic deliberations based on shell morphology are hardly possible. Some qualitative differences in egg-shell morphology between D. melanogaster and our Megaseliu species should be pointed out. First, the fibrils forming the exochorion in D. melunogaster were missing in Megaselia species. The outer layer of the egg in these flies consisted of finely granular material. Thus, it is possible that an exochorion is missing in the Megaseliu species studied and that the outermost element is a mucous layer. The outer layer interacts directly with the environment and the granular texture may be interpreted as an adaptation to deposition on carrion. Secondly, the pillars of the chorion showed a loose organization in Megaselia species, whereas they were compact in D. melanogaster. The entire chorion was thicker in Megaselia species and the loose architecture of their pillars may be understood in terms of saving material and weight in the egg-shell. The nature and meaning of filamentous material located in the space between the pillars in Megaselia species is not known. As far as we are aware, the effect of hypochlorit treatment has not been assessed at the fine structural level in flies. It turned out in our Megaseliu species-and on account of the overall similarity to D. melunogaster, it probably applies also to this fly-that brief hypochlorit treatment removes all chorionic parts of the egg-shell. An electron-dense lamina lies at the egg surface after this treatment. The layer most probably represents the wax layer of D. mefunogaster, which is hydrophobic (Margaritis, 1985) and therefore resistant to agents in an aqueous medium. the chorion, Megaselia

Acknowledgement-We K.W.W. (Wo 394/3-l).

gratefully

acknowledge

the funding

of the “Deutsche

Forschungsgemeinschaft”

to

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