Recent Advances in Insect Embryology in I apan and Poland
Edited by H. Ando and Cz. Iura
Arthropod. EmbryoL. Soc. Ipn.
(ISEBU Co. Ltd., Tsukuba) 1987

281

Ultrastructure of the Attachment Discin Perla sp. (Plecoptera) Egg

Elzbieta ROSCISZEWSKA
Synopsis
The attachment disc of eggs of Perla sp. (Plecoptera) is a specialized structure. It serves tofix the eggs to stones under water. The preliminary data, basing on scanning and transmission
electron microscopic observations, concerning the morphology of attachment disc are reported.

Introduction
Plecoptera are insects which are distributed all over the world, common insubmontane regions of the temperate climate zone. They can be found in theneighbourhood of streams and small rivers with a distinct water current, because cleanwater high in oxygen, is indispensable for their development.
One of many features adaptive to the aquatic habitat is that newly laid eggs of
Plecoptera stick strongly to stones, to gravel or to sand grains in the river bed. Becauseof this they remain in place in spite of water movement. The specialized surface
structre responsible for this permanent attachment of an egg is the so calledattachment disc (Hinton, 1981). In his book Hinton (1981) made only a few remarks
about this interesting structure.
The presentation of preliminary investigation data on the attachment disc in Perla
sp. is the aim of the present paper.

Materials and Methods
Adult females of Perla sp. were collected in the Bieszczady mountains (south-east
part of Poland) close to stream banks during their egg laying period. The insects weredecapitated, then the dissected ovaries and egg clusters were fixed. The material wasexamined under the phase-contrast light microscope, with a scanning electron

282

E. Rosciszewska

microscope (SEM) and with a transmission electron microscope (TEM).
1. For phase-contrast light microsc,ope studies, egg clusters were fixed in 2.5 %glutaraldehyde on 0.1 M phosphate buffer, pH 7.4 for 72 hr. Then the eggs wereisolated from clusters, rinsed in distilled water, placed in a droplet of insect Hoyle
solution and examined under a Peraval Interphaco Carl Zeiss Jena phase-contrast
microscope.
2. For SEM studies egg clusters were fixed in glutaraldehyde (for details see point no.
I), then eggs were isolated from clusters, dehydrated with acetone, air dried and coatedwith gold. After this they were examined with a Tesla BS 300 scanning electron
microscope.
3. For TEM studies after preliminary fixation in glutaraldehyde (for details see pointno. 1) ovaries were rinsed several times in phosphate buffer, pH 7.4 with the additionof sucrose, and fixed for 2 hr with 2 % OS04 in 0.1 phosphate buffer, pH 7.4. The
specimens were then rinsed several times in distilled water, dehydrated in a gradedseries of alcohol and acetone, and embedded in Epon 812. After this they were vacuum
dried for 2 hr. Blocks were cut on the Tesla BS 490 A ultramicrotome.
Semithin epon sections were stained with solution of metylene blue in 1 % borax.
Ultrathin sections contrasted with uranyl acetate and lead citrate were examined inTesla BS 500 electron microscope.

Results
The yellowish-brown oval egg of Perla-sp. is 0.4 mm long and 0.2 mm wide. The
surface of the chorion obtained by SEM shows a regular hexagonal sculpture similar inappearance to a honey-comb. In the centre of each hexagon there is a semisphericalstructure of unknown composition and origin (Figs. 1, 2, 4).
The posterior end of the egg (in an ovariole oriented towards the opening of theoviduct) is covered by a cap like attachment disc (Figs. 1, 2, 3) with similar hexagonal
sculpture. Each of the hexagons, however, displays a granular structure (Fig. 4) andcontains not one but from three to six of the semispherical structures mentioned above(Figs. 2, 4).
The phase-contrast microscope reveals the existence of a stem, sunk in thechorion, which anchors the attachment disc. The second finding is that semisphericalstructures are in turn supported and/or connected to tiny rod like structures (Fig. 3).
The attachment disc development was investigated as well. While examining
semi thin sections it was found that immediately above the already formed chorion, atthe egg end oriented towards the opening of the oviduct, there is cap like layer of aformless substance which in turn is covered by a layer of follicular cells. Then at theborder between follicular cells and the attachment disc and in the attachment discitself, some dense stained spots appear (Fig. 5 a), i. e., droplets of unknown origin andcomposition.
From the TEM studies it appears, that during formation the attachment disc ismade of medium electron density spongy material (Fig. 5). The space between thefollicular cells and the attachment disc is filled by similar material, which however is
not quite so condensed. This space is confined on one side, by the outer part of theAttachment disc in Perla

283

Fig. 1. SEM micrograph of the egg of Perla sp. X 160.
Fig. 2. The attachment disc (enlarged fragment of Fig. I). X 570.
Fig. 3. Phase-contrast micrograph of an egg. X 170.
Fig. 4. Enlarged fragment of Fig. 2. X 5,500.
s, stem; asterisk, semispherical structure; double arrow, rod like structures; single arrow,attachment disc.
attachment disc, which is a layer of much heavily condensed material, and on the otherside by the follicular cell's apical zone furnished with numerous microvilli (Figs. 5, 6).
In the space described one can also notice regularly distributed fields composed ofmaterial similar to that of the attachment disc.
The densely stained spots (droplets) observed in semithin sections were found to
be composed of a substance similar to that of the attachment disc but much more284

E. Rosciszewska

heavily condensed. The droplets on the side closest to the attachment disc are covered
with a thin electron dense layer (Fig. 5).

Discussion
Most species of Plecoptera lay between 100 and 500 eggs, usually in several
batches (Hinton, 1981). The females of Perla sp. carry clusters of eggs on their.
abdomens. After being laid the clusters disperse in the water, and single eggs cling
onto surface of stones or grains of gravel, fixed on by the attachment disc. This
structure, according to Miller (1939, cited after Hinton, 1981) is sticky. It is also elastic
and can be stretched even 10 times of its normal height before it tears apart.
From the preliminary investigation in Perla sp. it appears that the attachment discdevelops from a modified chorion. The shape is that of a cap which is anchored to theegg by a stem. The stem is elastic and can undergo much elongation (Rosciszewska,unpublished) which is in agreement with Miller'!, observations.
In the SEM studies it was found that the surface of the chorion and theattachment disc itself display a honey-comb like sculpture which according to the dataof Margaritis and collaborators (1980), seems to be follicular cells imprints.
Each hexagon is decorated by one (rarely one or three) and in the case of theattachment disc by three to six semispherical structures of unknown composition (seeResults), A reasonable assumption is that the structures are directly responsible for"glueing" the attachment disc (to a smaller extent the other parts of the egg surfacealso) to the stone under water. Certainly, this could be considered as an adaptive feature in Plecoptera.
The process of development of the attachment disc has not yet been described inliterature. From the present investigation on Prtr1a sp. it appears that the structure develops on, the chorion (Fig. 5 a) with the evident involvement of follicular cells. Facts
supporting this supposition are as follows: numerous ribosomes and elements of roughendoplasmic reticulum occur in their cytoplasm and numerous follicular cell microvilli
project towards the attachment disc. Furthermore, the subs~ance contained within thespace between follicular cells and the attachment disc seem~··.:o represent the precursorof the material building up the attachment disc.
Regularly distributed fields, which can be seen in this space, probably represent a
surface layer of the chorion possesing a characteristic honey-comb sculpture.
At the present stage of the investigation it is difficult to elucidate the role of thedensely stained droplets within the attachment disc (and within the space under follicular cells), however, it seems probable that they are connected with the formation of
the semispherical structures occuring on the hexagones. The possibility that the droplets are local reservoirs of a sticky (polymer? ) substance forming these structures on
the egg surface, can not be excluded. Furthermore, it might be suggested that thissticky substance could be transported to the surface of the chorion through tiny rodlike structures observed within the disc in phase-contrast microscope.

Attachment disc in Perla

285

Fig. 5. Electron micrograph ot a fragment on the attachment disc. X 30 , 000.
Fig. 5 a. Semi thin section of not fully developed attachment disc. X 960.
Fig. 6. Electron micrograph of regularly placed fields on the developing attachment disc.
X 25,000.
D, fragment of attachment disc; Fc, foIlicular cell; mv, microvilli; R, ribosomes; S, space between attachment disc and follicular cells; asterisk, droplet of unknown composition; blackarrow, outer part of attachment disc; bold white arrow, layer covering droplet; white arrow,regularly placed fields.

286

E. Rosciszewska

Acknowledgements
The author would like to thank sincerely to Dr. Sz. Bilinski for his encouragement
and interest in this work. Thanks are also due to MSc. E. Grz~da-Knutelska and MSc.
E. Kisiel for skilled technical assistance.

References
Hinton, H. E., 1981. Biology of Insect Eggs. Vol. 2. Pergamon Press, Oxford, New York.
Kishimoto, T. and H. Ando, 1985. External features of the developing embryo ,of the stonefly,
Kamimuria tibialis (Pictet) (Plecoptera, Perlidae). j. Morphol. 183: 311-326.
Margaritis, L. H., F. C. Kafatos, and W. H. Petri, 1980. The egg shell of Drosophila melanogaster.
I. Fine structure on the layers and regions on the wild-type egg shell. J. Cell Sci. 43: 1-33.

Addendum
Since this paper was submitted for publication, the author found an excellent. paper of Kishimoto and Ando (1985) on the embryology of a plecopteran Kamimuria
tibialis, including descriptions of the attachment disc. The shape and structure of theattachment disc of K. tibialis are in good agreement with those of Perla sp.
Author's adress: Dr. E. Rosciszewska
Department of Systematic Zoology,
Institute of Zoology, Jagiellonian
University, ul. Karasia 6, 30-060
Krak6w, Poland