Pergamon

hr. I. insect Morph&.

& Embryol.. Vol. 24, No. 2, pp. 213-222, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved

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LARVAL INTEGUMENT AND ITS DIFFERENTIATIONS IN MICROPTERIX CALTHELLA L. (LEPIDOPTERA : MICROPTERIGIDAE): ANATOMY AND ULTRASTRUCTURE

Claude Hamon and Georges Chauvin

Laboratoire dEntomologie Fondamentale Avenue du GCntraI et AppliquCe, UniversitC de Rennes I, Campus Leclerc, 35042 Rennes CCdex, France de Beaulieu,

(Accepted 27 May 1994) Abstract-The outer layer of the integument of the larva of Micropferix calthella (Lepidoptera : Micropterigidae) shows numerous modifications shaped like crests, nipples, and cones. Sections made at the level of these modifications or structures show that internal and external layers of the exocuticle are separate and form intracuticular cavities filled with a fluid. Every segment on the thorax and abdomen bears 3 disc-shaped structures, one dorsally and 2 laterally. These discs correspond to muscle attachments. Leaf-shaped structures, measuring up to 180pm high in the last-instar larva, are located along longitudinal lines on all segments. Despite their great anatomical diversity, these structures have the same ultrastructural organization. Although larvae appear to lack spiracles, they have an internal tracheal system, which does not seem to be linked with the intracuticular cavities. Index descriptors (in addition to those in title): Cuticle, scanning transmission electron microscope, phyllogeny, integument. electron microscope,

INTRODUCTION

Zeugloptera (sub-order of Lepidoptera) are close to Trichoptera (Friedlander, 1983; Kristensen, 1984, 1985). The study of eggs and spermatozoa of some species suggests that the systematic position of Zeugloptera in the current classification should be modified (Chauvin and Chauvin, 1980; Jamieson, 1987; Sonnenschein and Hauser, 1990; Hamon and Chauvin, 1992). Studies of Zeuglopteran larvae (Lorenz, 1961; Luff, 1964) show the unique aspect of these insects both in their anatomy and in their life cycle, which is partially aquatic. The nutritional requirements of larvae have not been defined accurately, and rearing trials on moss, hepatica, or young leaves of various plants, have given poor results (Lorenz, 1961; Heath, 1976). Only the last instar of the larva of Microprerix calthellu has been fully described using a magnifying glass (Lorenz, 1961). It confirms earlier drawings by Chapman (1894) and Portier (1949). Lorenz (1961) noted that the head, with its long antennae, could retract fully into the thorax and that the first 8 abdominal segments possessed ventral legs. He also pointed out that instead of the usual 2 Postpedes present in other lepidopteran larvae, there was an outgrowth or trilobated appendix, which opened out and stuck to the ground like a sucker when the larva moved. He described the presence of a large number of leaf-shaped appendices, distributed on the entire body in longitudinal lines.
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He suggested that these appendices placed near each ventral leg and touching the ground, helped in stabilizing the larva when it moved. To our knowledge, only one study describes the ultrastructure of the integument in the Zeuglopteran larvae of Subatinca chalcophanes (Kristensen, 1990). He concluded that its integument is the most aberrant arthropod cuticle known because it bears numerous cuticular cavities. He hypothesized that this unique cuticular system is somehow related to maintaining the water balance of the larva in cases of temporary drying of their litter. Because of the particular characteristics of Zeugloptera, we thought it was important to study the ultrastructure of the integument of the larva Micropterix calthefla. In the present work, we describe the ultrastructure of the integument of larvae as well as some of its differentiations. MATERIAL AND METHODS

Larvae of M. calthellu were obtained from a site at Plestin-les-Greves near Lannion in the west of France. Larvae were extracted from the superficial layers of soil using the technique described by Vannier (1971). These larvae live only in damp soil, near brooks; and furthermore, they are scattered quite widely, which makes it difficult to obtain a large number of samples. First-instar larvae were obtained from eggs placed on damp moss in a Petri dish. For scanning electron microscopy (SEM), larvae were fixed for 5 min in 2% glutaraldehyde in a buffer solution of sodium phosphate (pH 7.2). Preparations were then dehydrated in acetone and gold coated. For thin and ultrathin sections, whole larvae were fixed for 1 hr in chilled 2.5% glutaraldehyde in cacodylate buffer (pH 7.2), and postfixed in 1% osmium tetroxide in buffer. They were then dehydrated, first in ethanol then in propylene oxide and embedded in an epon-araldite mixture. Thin sections were stained with toluidine blue. For transmission electron microscopy (TEM), sections were contrasted with uranyl acetate and lead citrate and observed in a Philips CM 12 microscope. The anatomical study was done on 5 first-instar larvae and 4 larvae, probably in their final stage of development. For the ultrastructural study, we used 5 first-instar larvae and one last-instar larva.

RESULTS
Anatomy

There are no fundamental differences between the anatomy of the first- and last-instar larvae. In its first stage, the larva is 0.8 mm long and 0.2 mm wide. In the presumed last stage, it is 2.5 mm long and 0.6 mm wide. No clear separation is visible between the segments of the thorax; however the abdominal segments are very distinct (Fig. 1A). The entire surface of the integument is invested with transverse crests. In the medio-dorsal region of each segment, they run along parallel lines, which can come closer like lips. Pointed expanses perpendicular to these crests give a prickly aspect to the whole surface of the larva (Fig. lB, D). All segments have a well-marked lateral bulge (Fig. 1A). At the level of the bulges, there is an alignment of cone-shaped structures (Fig. 1B). These structures bear straight vertical ribs that are spiked with short digitations (Figs 1B and 3A). On each of the 3 thoracic segments and first 8 abdominal segments, there are 3 circular structures of about 65 pm in diameter in the last-instar larva (Fig. lB, C, D). One of these discs is in the median plane, and the other 2 are located on the lateral bulges of the body (Fig. 1D). The circular structures appear to be placed along 3 parallel lines. Their surface is raised in a slight dome and their edge turned up in a crest. A peripheral groove and a ring raised above the surface join them to the integument

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Fig. 1. Anatomy of the larva (SEM). A. Dorsal view of a first-instar larva, with head protruding. B. Detailed view of the left dorso-lateral region showing integumentary folds covering the median discs, leaf-shaped structures, spiny integumentary folds and cone-shaped structures. C. Lateral view of the posterior end of last-instar larva showing type D I-s structures and lateral discs. D. Last-instar larva, with retracted head, showing I-s structures and median and lateral discs. a = antennae; co = cone-shaped structures; lb = lateral bulge; Id = lateral discs; 1s = I-s structures; md = median discs; sif = spiny integumentary folds; th = thorax.

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(Fig. lB, C, D). The dome sometimes has, at its center, a small outgrowth (Fig. 1D). Central discs are different from lateral discs; they are surrounded by integumentary crests that can partially conceal them (Fig. 1B). Although numerous observations have been made by SEM and TEM, spiracles have never been detected on the body of the larvae. The leaf-shaped (l-s) structures are set along longitudinal lines on the surface of the larva (Fig. 1A). Each segment has 8 l-s structures, easily seen in a dorsal view. The 4 dorsal l-s structures and the 2 latero-dorsal ones are placed ahead of the circular structures; the other 2 latero-abdominal ones are slightly more posterior (Fig. 1D). Despite variations in size or anatomy, all l-s structures have a common basic pattern. Each structure has a broad distal region more or less branching out and a thinned basal part enclosed in a short cylinder with smooth sides emerging from the integument. Ribs and broad folds radiate out from this basal region (Fig. 2A, B, C, D, E). The surfaces of l-s structures lack pores. The anatomy of l-s structures varies, from one segment to another and from one larval stage to another. However, it seems that the general shape is finer in first-instar larvae (Fig. 2A, B) than in older larvae (Fig. 2C,D,E). Four main types have been identified: type A, typical of young larvae, and types B, C and D, found in older larvae. A brief description of their anatomy follows. Type A (Fig. 2A, B), found on young larvae, has smaller dimensions than those of types B, C and D (approximately 25 pm in height and 15 pm in diameter). It has clear, definite ribs and deep folds, which join and smooth out at the level of insertion in the integumentary cylinder. The free apical end has 20-30 digitations, which are relatively long (6 pm). Ribs, which delimit the folds, form one or 2 hook-shaped points at their distal ends. Type B (Fig. 2C) measures about 100 pm in height and 110 pm in diameter. It is broader than it is high and has a stumpy shape. Ribs are smooth and have pointed tips that surround the distal part, which is shaped like a circular tray. The surfaces of folds bear small digitations giving a hairy appearance. Type C (Fig. 2D) is club-shaped, about 65 pm in height and 42 pm in diameter. Longitudinal ribs are smooth and well separated at the base, then they become regularly crested at their apical ends. They meet at the spherical end of the club. Type D (Fig. 2E) is cylindrical in shape. Its free end, slightly bent, bears short digitations. Longitudinal ribs and folds are poorly marked. It is about 180 pm in height and 50 pm wide. The dimensions of the integumentary ring where leaf-shaped structures insert, depend on the latters size: c. 3 pm in diameter and 3.5 pm in height for type A; c. 12 pm in diameter and 6 pm in height for types B, C, and D. The posterior region of the head, which invaginates when it retracts within the thorax, is covered with a supple integument without definite modifications.
Ulstrastructure

The SEM micrographs show that all surface modifications, in particular the cone-shaped ones, correspond to hollow structures (Fig. 3A, B). These integumentary cavities are filled with a fluid, which disappears during sample preparation. Only small amorphous concretions remain on the internal walls of cavities (Fig. 3B). Semi-thin sections confirm that surface modifications correspond to cavities (Fig. 3C). Sections

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Fig. 2. Anatomy of sensilla (SEM). A and B. Type A l-s structures characteristic of young larvae. The organ is asymmetrical and its ribs (ri) are well marked. The distal or apical end bears numerous digitations. C. Type B l-s structure in a young larva. The apical end is wide and the surface bears thin digitations. D. Type C l-s structure in an old larva; it is club-shaped and ribs are crested. E. Type D l-s structure in an old larva. Shape is cylindrical, ribs are crested or cut at their apices. The leaf-shaped structures inserts into an integumentary cylinder (ic).

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seen under TEM show that the ultrastructure of the integument is typical; it has an endocuticle overlaid by an exocuticle and an epicuticle (Fig. 3D, E). The endocuticle, about 1 pm thick, is made of several loose fibrous layers and is continuous with the underlying epithelium. More often, it is separated from the thin exocuticle (0.2 pm) (Fig. 3E). The epicuticle is a thin film (0.03-0.04pm) covering all integumentary modifications. Ovoid, electron-dense granules are seen incorporated within the epicuticle. Their structure is reminiscent of that of microorganisms such as algae or bacteria (Fig. 3E). Integumentary modifications such as crests, nipples, and cones are formed by the epicuticle and by the external layers of the exocuticle, which is 0.1 pm thick (Fig. 3E). Cuticular cavities result from the separation of external and internal layers of the exocuticle. These cavities extend to all integumentary modifications even within the small digitations or thorn-like structures that they bear (Fig. 3D, E). In TEM, the cavities appear empty, the substance they contained having probably been destroyed during sample preparation. In the integumentary modifications, the endocuticle and the underlying epithelium are not modified (Fig. 3E). Sections show that at the site of integumentary discs, there are no unusual structures; these sites correspond to muscle attachments. The thin end of the l-s structure penetrates inside a cuticular cylinder halfway up its height (Fig. 4A). Transverse sections show that the ribs that radiate out from the insertion point of the I-s structures, are filled with a fibrous tissue of exocuticular nature (Fig. 4B, C). The integumentary epicuticle, described above, uniformly covers the whole surface of the l-s structure. The internal cavity contains a single cell (Fig. 4B, C), whose cytoplasm (Fig. 4C, D) branches out into numerous microvilli, whose dense extremities are attached to a more or less circular peripheral envelope. Fibrils observed between cytoplasmic expanses and the cuticle are of skeletal nature, and the organ does not contain muscle fibers. The inner part of the cytoplasm of this cell is less electron-dense than the outer part (Fig. 4C), there being no membrane between these 2 parts. Inside the inner cytoplasm, there are electron-light and -dense vesicular structures. With the optical and electron microscopes, we have frequently observed tracheae that lack spiracles (Fig. 3D, E).
DISCUSSION AND CONCLUSION

The integument of the larva M. calthellu is characterized by numerous external modifications (crests, cones, nipples), whereas that of S. calcophanes, described by Kristensen (1990), is uniformly flat. However, the integuments of these 2 Zeuglopterans have the same original characteristic: intracuticular cavities. These juxtaposed cavities are limited by walls of exocuticular nature and filled with a fluid.

A. Cone-shaped structures (co) seen under SEM in a Fig. 3. Structure of the integument. last-instar larva. Ribs are spiked with microdigitations (m). B. A fracture in the integument observed under SEM. Cone-shaped structures correspond to cuticular cavities (cc), their walls being lined with small granules (g). C. Transverse section of a first-instar larva observed under the optical microscope. Cavities (cc) correspond to integumentary cone-shaped structures. A leafshaped structure (Is) of type A and a lateral disc (Id) can be seen. D. Ultrastructure of the integument between cone-shaped structures of a first-instar larva. E. Cuticular cavity of the ep = epicuticle; ec = epithelial cell; en = endocuticle; ex = exocuticle; integument. ilex = internal layers of exocuticle; n = nucleus; elex = external layers of exocuticle: tr = trachea.

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In contrast to what Kristensen (1990) observed in Sabatinca, there does not seem to be a direct relationship between the distribution of cuticular cavities and the underlying epithelial cells in M. calthella. In M. calthellu, there are 8 leaf-shaped structures per segment. The absence of muscle fibers within these organs indicates that they have a very limited involvement with movement, contrary to what Lorenz thought (1961). Despite their various shapes, these organs contain only one cell, lack pores, and are made of a stalk inserted in a raised cuticular ring. Their features are common to sensory organs, such as sensilla chaetica (mechanoreceptors) defined by Altner (1977). In none of the observed cases have dendrites been detected in the cavity of l-s structures. An electrophysiological study is necessary to prove that the leaf-shaped structures have a sensory function. They have very small digitations at their free end, and might play a role as hygrophobic structures. The external anatomy of the integument of M. calthellu is close to that of the integument of Nymph& nymphaeatu described by Reichholf (1976). This author noted a change in the appearance of the cuticular surface depending on the larval stage and its physiology. In the hydrophilic stage, represented by young larvae living in water and breathing through their integument, there are no small round structures such as the crests and cones observed in M. calthella. In the hydrophobic stage, when older larvae have a tracheal system, there are, in addition to the small round modifications, other leaf-shaped protuberances. Young larvae utilize the oxygen dissolved in water, but old larvae breathe atmospheric air. In Nymphulu, as in M. calthellu, the small integumentary modifications (2-3 pm in height) are numerous, whereas leaf-shaped structures (10-1.5 pm in height) are few. As in M. calthellu, hygrophobic outgrowths have deep longitudinal ribs. Our observations made by SEM on last-instar larvae of Nymphula show, however, that structures described by Reichholf (1976) do not appear to be sense organs. Not only does their shape differ from that of sensilla chaetica, but they are attached to the integument through a pointed wedge. In M. calthellu, the different modifications, as well as the l-s structures, can create a thin layer of air on the surface of the body when immersed in water. We were able to verify this by dipping live larvae in water. The layer of air would provide a kind of respiratory plastron, as defined by Hinton (1958), around the body of the larva, which would allow it to breathe in the event of the flooding of its habitat. The integumentary anatomy is therefore related to the subaquatic way of life of the larva. The fact that we have seen internal tracheae without being able to observe spiracles, makes it difficult to understand how this insect breathes. A study is currently under way to investigate this phenomenon.

Fig. 4. Structure of leaf-shaped structures. A. Longitudinal section of a type A I-s structure, observed under the optical microscope in a first-instar larva. The thinned base of the I-s structure inserts into an integumentary cylinder (ic). A cell (C) fills the l-s structure. B. Transverse section across an I-s structure (TEM). The integument delimits a cavity filled by a cell (C) whose plasma membrane forms microvilli (mv). A fibrous tissue becomes electron dense at their contact. C. Transverse section at the base of an l-s structure (TEM). Ribs contain a fibrous tissue. The ends of microvilli are attached to a more or less circular limiting envelope. Arrowheads indicate the electron-light and -dense vesicles. D. Detail of the contact zone between microvilh and limiting envelope. ep = epicuticle; ex = exocuticle; ft = fibrous tissue; icp = inner cytoplasmic part; le = limiting envelope; mv = microvilli; ri = ribs.

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Acknowledgements-We thank Miss Germaine Boguais for carrying out the embedding and sectioning TEM, and also Mr Joseph Le Lannic of the Centre de Microscopic & Balayage de IUniversitC de Rennes. also thank Mrs Maryvonne Rault for typing the manuscript.

for We

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