Post-larval development of compound eyes and stemmata of Chaoborus crystallinus (De Geer, 1776) (Diptera : Chaoboridae): Stage-specific reconstructions within individual organs of vision

Int. J. Insect Morphol. & Embryol. Vol. 23, No. 3, pp. 261 274, 1994

Pergamon

Copyright© 1994ElsevierScienceLtd Prinled in Great Britain. All rights reserved 0020~7322/94 $7.00 +0.00

POST-LARVAL DEVELOPMENT OF COMPOUND EYES AND STEMMATA OF C H A O B O R U S C R Y S T A L L I N U S (DE GEER, 1776) (DIPTERA" CHAOBORIDAE): STAGE-SPECIFIC RECONSTRUCTIONS WITHIN INDIVIDUAL ORGANS OF VISION

R . R . M E L Z E R * a n d H . F . PAULUSJ" Institut fiir Biologie I (Zoologie) der Universitfit, Albertstr.21a, D-79104 Freiburg, F.R.G. (Accepted 29 June 1993)

Abstract

During metamorphosis, the dioptric apparatus of the larval compound eye of

Chaoborus crvstallinus (Diptera : Nematocera) is radically reconstructed. The thin larval

cornea of the ommatidia is replaced by strongly curved corneal lenses, and the eucone larval cone is replaced by an imaginal cone of the acone type. Curvature of the future lens is already apparent in very young pupae, in which the cornea consists only of a thin epicuticle with corneal nipples. Fibrillary cuticle is secreted by cone and primary pigment cells throughout pupal development. Lens formation is accompanied by movement of the nuclei of the accessory pigment cells. The larval cone disintegrates unexpectedly late in young imagos. During late pupal development, 7 cone cell projections emerge. In contrast to the dioptric apparatus, the retinula cells and rhabdom remain almost unchanged during metamorphosis. The main refractive element of the larval ommatidium appears to be the cone, while that of the imaginal ommatidium is the corneal lens. In addition to the compound eyes, the pairs of stemmata are retained during the whole post-larval development. Pupal stemmata show no structural differences from the larval stemmata. The stemmata are still present in 2-day-old imagos ("retained s t e m m a t a ' ) , but the primary stemma loses its dioptric apparatus and is proximally relocated to the basal region of the compound eye. The reconstructions in the visual system of Chaoborus, which occur during ontogeny, are probably connected with the change from aquatic living larvae to aerial adults, and appear to fulfill stage-specific needs of vision.

Index descriptors (in addition to those in title): cornea formation, eucone and acone crystallire cones, change of dioptric apparatus, retained stemmata.

INTRODUCTION

THE COMPOUND eyes

of Chaoboridae and Culicidae are already well developed in the aquatic larvae of these groups (Constantineanu, 1930; Haas, 1956); this means that the l a r v a e o f C h a o t ) o r u s n o t o n l y h a v e s t e m m a t a ( l a t e r a l o c e l l i ) a s all o t h e r H o l o m e t a b o l a a l s o d o , b u t p o s s e s s , in a d d i t i o n , c o m p o u n d e y e s w i t h a f u l l y d e v e l o p e d r h a b d o m a n d

Present addresses: *Zoologisches Institut der Universitfit, Luisenstr. 14, D-80333 Miinchen, F.R.G. *lnstitut ffir Zoologie der Universitfit Wien, Abteilung Evolutionsbiologie, Althanstr.14, A-1090 Wien, Austria. 261

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retinula cells, indicative of visual functioning (Melzer and Paulus, 1991). As opposed to the imaginal ommatidia of other Nematocera, the larval ommatidia of C h a o b o r u s have several very unusual features, such as the presence of a eucone crystalline cone and the lack of a corneal lens (Meizer and Paulus, 1991). Generally, the ommatidia of Nematocera possess acone (or pseudocone) cones and strongly curved corneal lenses (Brammer, 1970; Seifert et al., 1985; O'Grady and Mclver, 1987; Kreuzmann et al., 1989). Using light microscopy, earlier investigators of ommatidial development in Chaoboridae and Culicidae, observed that the corneal lenses are first formed during pupal development, and that the eucone larval cones disappear (Constantineanu, 1930; Haas, 1956). This suggests that the unusual characters of the larval ommatidia are stage-specific adaptations to particular visual needs of the larvae that change during metamorphosis when the imaginal characters of the ommatidium are formed (Melzer and Paulus, 1991). In order to provide a better understanding of these stage-specific changes, the first aim of this study was to elucidate the fine structure and the post-larval development of the compound eyes. Knowledge of late ommatidial development may provide insight into the plasticity of ommatidial structure and may supply further indirect evidence for the actual function of the larval compound eyes. Direct evidence for this is not yet available, although Duhr (1955) conducted behavioral experiments by painting over the compound eyes of larvae. The second aim of this study was to examine the fate of the stemmata during metamorphosis. C h a o b o r u s larvae possess 2 pairs of lateral ocelli, which are situated caudal (primary stemmata) and dorsal to the compound eyes (accessory stemmata; Melzer and Paulus, 1991). Constantineanu (1930) observed that these stemmata are still found in pupae and freshly hatched adults, and thus might be retained during metamorphosis as "imaginal stemmata", as has also been described for some Coleoptera (Schultz et al., 1984; Mischke, 1986) and Trichoptera (Hagberg, 1986). Further extraocular photoreceptors, which are situated in the neighborhood of the compound eyes or optic lobes, and therefore might also represent retained stemmata, are found in representatives of the following groups: Coleoptera (Bott, 1928; Wachmann, 1981), Lepidoptera (Umbach, 1934; Ehnbohm, 1948), Trichoptera (Ehnbohm, 1948), Strepsiptera (R6sch, 1913; Kinzelbach, 1971; Wachmann, 1972) and Brachycera (Dietrich, 1909; Hofbauer and Buchner, 1989). They are also found in Formicidae (Eric Meyer pers. commun.), and according to Ast's (1919) illustrations possibly also in imagos of Megaloptera. Therefore, the question arises: are retained stemmata common to all imagos of Holometabola? To decide if such extraocular photoreceptors are always retained stemmata, or if another type of lateral eye exists in insects, the late ontogeny of stemmata must be examined in various Holometabola groups. Information used to answer the above questi~)ns is also valuable in determining the homology and development of Bolwig's organ, ..which is the derived stemma of Brachycera (Melzer and Paulus, 1989, 1990) and possibly has an imaginal counterpart (Hofbauer and Buchner, 1989; N/issel et al., 1988; for a review see Meinertzhagen and Hanson, in press, 1993).

M A T E R I A L AND M E T H O D S Pupae and imagos of Chaoborus crystallinus were raised under natural light conditions at room temperature. All specimens were identically prepared for electron microscopy to avoid any influence

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263

resulting from different states of light adaptation. The light/dark cycle was approximately 15.5 hr/8.5 hr. Specimens were prepared 5~5 hr after sunrise. Good fixation of the pupae was achieved by removing the pupal cuticle from the future imaginal heads. In addition, it was necessary to open the heads dorsally and caudally, and cut off the antennae. The heads were fixed according to Franke et al. (1969) and embedded in Glycidether 100. Ultrathin sections were cut on a Reichert OM-U3 with a diamond knife, double-stained on a LKB 2168 grid stainer and examined with a Zeiss EM9-S2. The following 5 developmental stages were investigated (in parenthesis: percentage of pupal development completed): (1) fresh pupae (0-15%), (2) 2-day-old pupae (3~45%), (3) fully differentiated, 4-6-day pupae (80-100%), (4) fresh imagos and (5) 2- or 3-day-old imagos. RESULTS

The compound eye Basic steps in ommatidial development. To help understand the structural modifications that will be described, we include a brief survey of the basic steps of ommatidial development. In Chaoborus, the development of larval ommatidia starts a few days after hatching, and continues until the larvae are fully grown (4th larval instar), because new ommatidia are successively formed on the anterior edge of the c o m p o u n d eye. This means that some larval ommatidia are completed when others are just beginning to differentiate or are still in the embryonic stage. Fully differentiated larval ommatidia have a complete inventory of cells, the final form and structure of the rhabdom, as well as a crystalline cone of the eucone type (for details see Melzer and Paulus, 1991). After the complete number of larval ommatidia has been attained, development ceases until metamorphosis. This halt can last for several months in hibernating larvae of the 4th instar. The ommaditia of young pupae undergo changes which signal a new developmental mode. During larval development, the ommatidia differentiate in succession, in the wake of the anteriorly progressing wavefront. In pupae and young imagos, structural modifications occur simultaneously in all ommatidia across the entire c o m p o u n d eye. This means that ommatidia with "pupal" characters were never observed as neighbors of ommatidia that still showed "larval" characters. Formation of new ommatidia at the anterior edge of the compound eye was not observed within the examined developmental stages. Therefore it is possible that young pupae already have the complete number of ommatidia ~f the adult. Retinula cells and rhabdom. The ommatidia of pupae and imagos each have 8 retinula cells: 6 peripheral cells (R1-R6) and 2 central cells, R7 and R8, which form the central " t a n d e m " rhabdomeres (Fig. 1B-D). As with the larvae, a large extracellular space between the rhabdomeres is lacking, and the rhabdomeres of R 1 - R 6 are laterally fused. Therefore, the rhabdom belongs to the fused type rather than to the open rhabdom type as found in Brachycera (see also Brammer, 1970). Apart from an increase in the rhabdom diameter (up to 30% was observed), the arrangement of the retinula cells and their rhabdomeres does not differ significantly from that of the larvae (Fig. 1A). Further detailed description is therefore not necessary, and we refer to our study of the ommatidium of the larva (Melzer and Paulus, 1991). The nuclei of the retinula cells begin to move distally toward the basement m e m b r a n e in 2-3-day-old imagos. The nucleus of R7 is always the most distal nucleus (Fig. 1B) and the first to be found distal to the basement membrane. Occasionally, the R7 nucleus is found in its distal position already in the larvae (see Melzer and Paulus, 1991).

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FIG. 1. Schematic reconstruction of post-larval ommatidial development in Chaoborus; A. fully developed larval ommatidium, as found in older larvae (after Melzer and Paulus, 1991); B. ommatidium of a 2-day-old imago, longitudinal section; C,D. as B cross-sections at levels indicated by lines; E - G . development of dioptric apparatus and pigment cells; E. larva; F. young pupa; G. older pupa; H. young imago. A P C = accessory pigment cell; BL = basement membrane; C = Cornea; CC = crystalline cone; PPC = primary pigment cell; R7,R8 = central retinula cells; SC = Semper cell; SCP = cone cell process; small arrow = corneal nipples; large arrow = nucleus of an accessory pigment cell within the proximal pigment-filled sac.

Post-larval Development in Chaoborus crystallinus

FIG. 2. Post-larval d e v e l o p m e n t of the ommatidia of Chaoborus. A - D formation of the corneal lenses; E - I reconstruction of the cone. A. cornea formation in a young pupa; arrowhead = epicuticle and corneal nipples; arrows = microvilli within the cuticle deposition zone; B. as in A; arrowheads = epicuticle; arrows = microvilli; C. older pupa; large arrowhead = epicuticle and corneal nipples; arrow = cuticle deposition zone; small arrowheads = termination sites of pore canals; D. 2-day-old imago, fibrillary cuticle of a corneal lens; E. youn~ pupa, eucone crystalline cone; arrow = regular a r r a n g e m e n t of cone granules in the neighborhood of Semper cell m e m b r a n e s ; F. 2-day-old imago, Semper cell (SC) with cone cell projection (arrow); G. dioptric apparatus of a freshly hatched imago; arrowhead = proximal part of the cone with "vesicular" plasma; H. 2-day-old imago, acone cone; I. 2-day-old imago; cone cell projection (asterisk) s u r r o u n d e d by 2 peripheral retinula cells. 1-4 = cone sectors situated within the 4 Semper cells; C = Cornea; CC = crystalline cone; D = d e s m o s o m e ; E X = exocuticle; E N = endocuticle; M = ring of mitochondria; N = corneal nipples; PC = pigment cell; PPC = primary pigment cell; R = retinula cell; SC = Semper cell.

265

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R . R . MELZER and H. F. PAULUS

FIG. 3. Post-larval d e v e l o p m e n t of the s t e m m a t a A. y o u n g p u p a , p r i m a r y s t e m m a ; a r r o w = s t e m m a unit with l i n e a r r h a b d o m ; a r r o w h e a d = cuticle; B. o l d e r p u p a , a c c e s s o r y s t c m m a ; a r r o w s = r h a b d o m e r e s with high e l e c t r o n d e n s i t y ; C. o l d e r p u p a , optic n e r v e of a c c e s s o r y s t e m m a ; D. 2-day-old i m a g o , r e t a i n e d s t e m m a unit with b r a n c h e d r h a b d o m ; E. 2 - d a y - o l d i m a g o , s t e m m a n e r v e s (asterisks) e n t e r i n g the optic lobes; F. o l d e r p u p a , a c c e s s o r y s t e m m a ; a r r o w h e a d - cuticle; C C - single-cell crystalline cone; E = e p i d e r m i s ; NRV = n u c l e u s of refractive cell ( R F ) ; NE = nucleus of e p i d e r m a l cell; R = r e t i n u l a cell.

Cornea. During metamorphosis, the flat larval cornea is replaced by numerous, strongly curved corneal lens units (Fig. 1F-H). The surface structure of the curved cornea is already fully developed in very young pupae (Figs 1F and 2A). Contrary to the situation in the larvae, numerous tiny, spherical corneal nipples are found having a diameter of 100-150 nm at their base and a maximal diameter of approximately 200 nm (Fig. 2A-C). They are composed of a distal epicuticle (30nm) and a proximal subcuticle, which still does not show any fibrillary structure (Fig. 2A,B). This means that the formation of exocuticle or endocuticle is just beginning at the earliest stage we

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examined. At this very young stage, a subcuticle or cuticle deposition zone (up to 0.5tzm) is found adjacent to the epicuticle. Numerous microvilli with plasma membrane plaques project into this zone (Fig. 2A,B). The microvilli originate from both Semper and primary pigment cells and not from secondary pigment cells. Although the very thin cuticle appears to have no stabilizing function, the final shape of the corneal lens has already been attained in young pupae (Figs 2A and 3F). The cuticle of each ommatidium is arched in a hemisphere and encloses the distal parts of the Semper and primary pigment cells. During later cornea formation, a fibrillary cuticle is continuously added (Figs ] F - H and 2C). Beginning with this stage, the cornea is divided into an endocuticle (lesser electron density, Fig. 2C) and an exocuticle (greater electron density, Fig. 2C). Distal layers are formed prior to the proximal layers. The fibrillary cuticle is composed of numerous lamellae that have a characteristic helicoidal pattern of fibers (Fig. 2D). The lamellae are approximately 0.3/xm thick and are arranged in alternating layers of greater and lesser electron density. Numerous small pore canals (Fig. 2C), which pass through the cuticle, originate close to electron-dense vesicles within the plasma of the Semper or pigment cells. The canals terminate between the cornea nipples (Fig. 2C). The hemisphere, which originally had only a thin cuticular covering, is completely filled with lens material in older pupae (Figs 1G and 2(;). In the last phase of its development, the cornea also becomes proximally arched, thereby constituting a biconvex lens. Semper and primary pigment cells are displaced proximally by the developing cornea (Fig. 1H). Cone. The eucone crystalline cone of the larval ommatidium, which is situated within the plasma of the Semper cells, is retained throughout the entire pupal development (Figs 2E,G and 1F,G). It is noteworthy that the crystalline-like arrangement of granules in the center of the cone is also retained during the first developmental steps of the pupa. Co:ae granules are regularly arranged at least in the neighborhood of junctional structures connecting the cell membranes of the 4 cone cells (Fig. 2E). The packing density c f granules may decrease in some almost completely developed pupae (Fig. 2G). Also, 'Lhe form of the crystalline cone and the Semper cells is modified. The proximal region of the Semper cells narrows and the part of the cone with the dense arrangement of granules is more distally displaced (Fig. 2G), leaving a "vesicular" proximal plasma (Fig. 2G) which is later found throughout the Semper cells (see below). Shortly before the imago hatches, the cone granules and the surrounding layer of mitochondria bel;in to disintegrate (Fig. 2G). In young imagos, the granules are arranged more and more loosely until they finally disappear (Fig. 2F,H). The mitochondria become equally distributed with Semper cell plasma. This means that the eucone cone is completely reduced during the first hours of the imaginal stage. After reduction, the plasma of the 4 Semper cells has a low electron density, which is characterized by numerous vesicles, ER-cisternae, microtubules and large mitochondria ("acone cone;" Figs 1B,H and 2F,H). At the same time as the eucone cone is reduced, cone cell projections grow out of the proximal region of the Semper cells (Figs 1G,H and 2F,I). The projections have a plasma of low electron density and contain bundles of microtubules projecting apicobasally. From the Semper cell base, the projections run between the 6 peripheral retinula cells (Fig. 2I) and the centrally projecting part of R7. Thus, a total of 7

268

R.R. MZI~ZERand H. F. PAut,us

projections is present. Proximal to the rhabdomere of R7, only 6 projections remain, in accordance with the number of retinula cells participating in rhabdom formation. We could not determine if the 2 projections in the neighborhood of R7 fuse proximal to the rhabdomere, or if one of them terminates. The projections follow along the edge of the zonulae adherentes, which connect adjacent retinula cells (Figs 1C,D and 2I). Pigment cells. The participation of the primary pigment cells in the formation of the corneal lens has been described above. Modifications in form or fine structure of these cells do not occur during post-larval development. A movement of the nuclei was observed within the accessory pigment cells. During larval development, the nuclei are situated within the distal plasma (Fig. 1A,E). In freshly molted pupae, in which cornea formation is just beginning, the nuclei of the accessory pigment cells are found proximally within the pigment-filled sacs near the basement membrane of the compound eyes (Fig. IF). With progressive cornea formation or thickness, the nuclei move distally (Figs 1G and 2G). At the end of pupal development and in imagos, they are found again at their starting-point lateral to the primary pigment cells (Fig. 1H). Accessory pigment cells in larvae have pigment granules which are distinctly smaller than granules within retinula or primary pigment cells (Fig. 1A,E). This character has already disappeared in young pupae which possess large granules like the other pigmented cells (Fig. 2G). Primary stemma The organization of the primary stemmata of larval Chaoborus remains unchanged during the entire pupal development. As with the larvae, they are composed of 3 partial stemmata, and possess a dioptric apparatus which is composed of a single-cell crystalline cone and refractive cells (Fig. 3A). Modifications, however, take place first at the end of pupal development and in newly hatched imagos, during which the stemma relocates inward to the level of the nuclear region of the compound eye between the basement membrane and the optic lobes. Although the stemma loses contact with the cuticle, it retains its position at the compound eye's posterior margin. During relocation, the dioptric apparatus of the primary stemma is completely reduced. Adjacent to the former stemma cornea are normal epidermal cells which show no characteristics of the Semper and refractive cells previously located there. The fate of the dioptric apparatus cannot be determined in detail. The sunken group of retinula cells in 2-day-old imagos still shows the characteristic division of partial stemmata, each with its respective rhabdom arrangement (Fig. 3D). Accessory stemma No modifications of retinula cell arrangement and in the form of the rhabdom were noticed in the accessory stemma of Chaoborus throughout the entire observed period of ontogeny (Fig. 3B,F). This means that both primary and accessory stemma maintain their larval organization during pupal development. The 2 accessory stemmata of both pupae and imagos are situated at the upper side of the head, dorsal to the optic lobes. They are composed of some 40 retinula cells, which form a complex and large rhabdom. Several rhabdomeres are relatively electron dense (Fig. 3B). Pigment granules are absent. The epidermal cells located between stemma and cuticle did not form a distinct dioptric apparatus (Fig. 3F).

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Optic nerve Chaoborus larvae possess an optic nerve that is composed of retinula axons from both the compound eyes and the stemmata. The pupae and imagos, however, lack a common nerve for all visual organs. Originating from the compound eye, bundles of 8 axons each from an ommatidium project directly into the cell body rind of the optic neuropils, which are situated in close vicinity to the compound eyes. Projecting from the primary and accessory stemmata are 2 distinct stemma nerves connecting the stemmata with lhe brain. These nerves coming from 2 different directions first approach each other when they enter the optic lobes (Fig. 3E). The dorsal nerve projects from the' accessory stemma, and the caudal nerve from the primary stemma. As with the larvae, each pupal or imaginal stemma nerve contains 40 retinula axons. The stemmata of larvae, pupae and imagos therefore have a constant number of retinula cells. DISCUSSION Post-larval develepment of the ommatidium The basic plan of ommatidial development in the compound eye of Chaoboridae does not differ significantly from that of Culicidae, which are regarded as their sister group (Hennig, 1973; Wood and Borkent, 1989). In both taxa, ommatidia are formed successively during larval development at the anterior edge of the compound eye (Constantineanu, 1930; Sato, 1950, 1951; Haas, 1956; White, 1961, 1963), a pattern which is found in all compound eyes (Meinertzhagen, 1973). Beginning with the onset of the pupal stage, the time between the development of neighboring ommatidia is shortened so that all ommatidia of a compound eye are at the same developmental stage anatomically. A similar relaxation of the temporal wave of development occurs in Brachycera (Meinertzhagen, 1973). Formation of ~:he corneal lens is the most important reconstruction during pupal development. Cuticle formation in Chaoborus is very similar to that of many other insects (for review see Weis-Fogh, 1970). It is interesting, however, that the extremely thin cornea of young pupae is already arched in the same manner as the later corneal lens. This meanr~ that the cornea does not grow outwards, but appears to fill a previously established "casting mold" (see also Altner and Prillinger, 1980). Nuclear migration in the accessory pigment cells coincides with this mode of cornea formation. These nuclei are found within the proximal pigment sacs only in young pupae and only when the cornea is already arched, but still very thin. While the cornea thickens, the nuclei return to their initial position distally. Therefore nuclear migration and the formation of the cornea appear to be synchronized. This synchronization might be a secondary effect of morphogenetic processes within the pigment cells that induce cornea shape and formation. Migration of nuclei with a possibly morphogenetic function also occurs in the retinula cells. In larval Chaoboridae and Culicidae, the retinula cell nuclei are situated proximal to the basement membrane (Melzer and Paulus, 1991; Haas, 1956). As in Aedes (Brammer, 1970), the nucleus of R7 is the most distally found nucleus, and it appears to be the first that relocates to a position distal to the basement membrane. In adult Aedes (Brammer, 1970) the nucleus of R8 is the only nucleus still found proximal to the

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basement m e m b r a n e . These findings appear to contradict the fact that in Drosophila, the nucleus of R7 is the last to undergo morphogenetic m o v e m e n t s (Tomlinson and Ready, 1986; Ready, 1989). In the 4-cone cell stage of Drosophila, however, the relative position of the R7 nucleus is also distal, and that of the R8 nucleus proximal. Therefore, it is suggested that the nuclear movements, described here for Chaoborus, start from a similar situation, and change the absolute, but not the relative, position of the nuclei. Final organization of the imaginal ommatidium occurs in older pupae and substantially in young imagos. The primary change in this period is the reduction of the eucone crystalline cone, which has also been observed by light microscopy in Culicidae (Constantineanu, 1930; Haas, 1956). Both the crystalline-like structure of the cone and the surrounding ring of mitochondria disintegrate. This results in a condition, which satisfies Grenacher's (1879) definition of an acone crystalline cone. An acone cone has no refractive specializations; it functions as a vitreous body surrounded by a pigment cell aperture (for review see Nilsson, 1989). It follows that the main refractive elements of the dioptric apparatus are exchanged during ontogeny. The larva has no corneal lens, but a eucone cone, while the imago has a strongly curved lens and an acone cone. It is interesting that the acone Semper cells of the imago show a fine structure very similar to the refractive cells of the primary stemma (Melzer and Paulus, 1991). The homology of the refractive cells is unsolved, and it is possible that they represent Semper cells of the acone type, which are present, in addition, to the single-cell crystalline cone. Cone cell projections are first formed when the cone disintegrates. These projections occur also in Culicidae ( B r a m m e r , 1970) and numerous other insects. Pigment-filled sacs at the base of these projections are present in Culicidae ( B r a m m e r , 1970). Possibly, they are present only in Chaoborus imagos that are older than those we examined. Nevertheless, and in contrast to the larval stage, imaginal ommatidia of Chaoborus are very similar to the ommatidia of Culicidae and other " N e m a t o c e r a " (e.g. B r a m m e r , 1970; Seifert and Smola, 1990). It must be emphasized that the reconstructions described above occur within an individual ommatidium. The terms +'larval" and "imaginal" o m m a t i d i u m do not imply that different ommatidia replace each other as occurs in hemimetabolous insects such as dragonflies (Mouze, 1984). On the contrary, a single o m m a t i d i u m undergoes stage-specific modifications during ontogeny. This plasticity in ommatidial structure must be taken into account when structural characteristics of c o m p o u n d eyes are analyzed from an evolutionary point of view. Our results suggest that a change between different cone types may occur intraindividually. During ontogeny, most of the characters described above do not diverge from the developmental pathway leading to imaginal organization. They might simply be transitional stages that would also be present even if no larval vision existed. The larval crystalline cone of the eucone type, however, is formed specifically for the larval stage and is reduced later during metamorphosis. This means that it is a specific larval character or caenogeny (Osche, 1982: "transitorisches LarvalmetaphS.n"). It follows that the ommatidia of Chaoborus pass successively through 2 adaptive stages, a larval and an imaginal stage. The complex characters c o m m o n to the eucone cone of Chaoborus and other insects suggest that they are homologous and have not evolved independently. Since the eucone cone of larval Chaoborus is unique among the Diptera, it follows that the capacity to form

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such a cone must be genetically present in the basic plan of Diptera, although not phenotypically e'cpressed (Osche, 1965: "latente Potenz").

Functional morpi~ology Although the optics of the larval and imaginal ommatidia of Chaoborus have not yet been examined, our morphological findings allow some remarks on the putative function of the ornmatidia's dioptric apparatus. First, the formation and actual function of the eucone cone for aquatic stages can be explained only by its usefulness for vision. Its presence is thus indirect evidence for the functioning of the larval compound eye. Second, the po,;t-larval reconstruction of the dioptric apparatus indicates that a stage-specific change takes place between 2 different types of image formation. Vision requirements of the larvae and imagos are different, because the former are aquatic and the latter terrestrial. It follows that the larval dioptric apparatus should be adapted for vision below the water surface, and the imaginal one for vision in air. As inferred by Exner (1891), curved corneal lenses are useless beneath the water surface, because water and the arthropod cuticle have similar refractive indices. It follows that aquatic arthropods should possess lens cylinders with a gradient of different refracti~le indices and not normal convex lenses (see also Land, 1981; Nilsson, 1989). Apart from this, a situation very similar--and presumably c o n v e r g e n t - - t o Chaoborus is found in the apposition compound eye of the brine shrimp, Artemia, which also has a flat cornea and a marked crystalline cone. Elofsson and Odselius (1975) and Nilsson and Odselius (1981) have shown that the cornea is optically inactive and that the cone functions as a "glycogen" lens. These conditions are most likely also fulfilled by the dioptric apparatus of larval Chaoborus. It follows that the larval and imaginal optic systems of Chaoborus differ primarily in the location of the main refractive power. Lens function is achieved by the corneal lens in imagos and by the cone in larvae. Pupal and imaginal stemmata As indicated by the complete rhabdom structure and innervation from the optic lobes, the stemmata are functional during all development stages. The loss of the dioptric apparatcts of the primary stemma during late metamorphosis indicates that its functions may change or be restricted. It was not possible to decide whether refractive and cone cells are reduced completely or re-differentiated into epidermal cells. Our findings suggest that a relocation of the stemmata during metamorphosis, as evidenced in many Holometabola (see above), may also be common in Diptera. Characters of the stemmata can be reduced in different ways during the relocation process. In Chaoborus, only the dioptric apparatus is modified. In other Holometabola, the arrangement of retinula cells and rhabdom may also change. A highly derived situation might be found in Coleoptera (Wachmann, 1981) and Lepidoptera (Umbach, 1934; Ehnbohm, 1948) where the extraocular photoreceptors are not only relocated to the base of the compound eyes, but appear to be lowered to the optic lobes. A common feature of most ttolometabola is that retained stemmata lose contact with the cuticle. As shown in Melzer and Paulus (1990), the stemmata of larval Chaoborus and an imaginal photoreceptor cells cluster, which is located at the posterior compound eye bases in Drosopk, ila (Hofbauer and Buchner, 1989) and other flies (N~issel et al., 1988),

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have a similar, a n d p r o b a b l y h o m o l o g o u s , n e u r o n a l e q u i p m e n t . T h e r e t a i n e d s t e m m a o f i m a g i n a l C h a o b o r u s , as e v i d e n c e d in this p a p e r , also r e p r e s e n t s a c l u s t e r of r e t i n u l a cells a n d has the s a m e p o s i t i o n in r e l a t i o n to the c o m p o u n d e y e s as the p h o t o r e c e p t o r s m e n t i o n e d a b o v e . A p a r t f r o m this, the p r o j e c t i o n p a t t e r n of the s t e m m a a x o n s within the optic l o b e s as d e s c r i b e d for the larva ( M e l z e r a n d Paulus, 1990) is still f o u n d in t h e i m a g o (in p r e p a r a t i o n ) . This s u p p o r t s the h y p o t h e s i s t h a t t h e p h o t o r e c e p t o r clusters f o u n d in a d u l t flies m i g h t also be r e m n a n t s o f a s t e m m a , i.e. B o l w i g ' s o r g a n ( M e l z e r a n d P a u l u s , 1989, 1990). A s s h o w n in M e i n e r t z h a g e n a n d H a n s o n (1993), h o w e v e r , the f o l l o w i n g findings m a k e it i m p o s s i b l e to solve t h e s e q u e s t i o n s at p r e s e n t : First, B o l w i g ' s o r g a n a p p e a r s to d e g e n e r a t e d u r i n g m e t a m o r p h o s i s (Tix et al., 1989). S e c o n d , at t h e p o s t e r i o r e d g e o f t h e c o m p o u n d e y e is a n o t h e r g r o u p of p h o t o r e c e p t o r s which is p r e s e n t w h e n B o l w i g ' s o r g a n is still in its " l a r v a l " p o s i t i o n n e a r the c e p h a l o p h a r y n g e a l a p p a r a t u s ( T o m l i n s o n a n d R e a d y , 1987). M e i n e r t z h a g e n a n d H a n s o n (1993) suggest that s t e m m a t a m i g h t b e split into a g r o u p t h a t is r e l o c a t e d with the e y e i m a g i n a l disk, a n d a s e c o n d g r o u p that is r e l o c a t e d to t h e p h a r y n g e a l a p p a r a t u s ( B o l w i g ' s o r g a n ) . In this c o n n e c t i o n , it is i n t e r e s t i n g t h a t t h e a c c e s s o r y s t e m m a of C h a o b o r u s a n d o t h e r n e m a t o c e r a n s has, in fact, a fine s t r u c t u r e similar to B o l w i g ' s o r g a n as f o u n d in p r i m i t i v e flies ( M e l z e r a n d P a u l u s , 1991). A p a r t f r o m this, b o t h s t e m m a t a i n n e r v a t e t h e s t e m m a l a m i n a , which is s i t u a t e d at the p o s t e r i o r e d g e of t h e c o m p o u n d e y e ' s n e u r o p i l . O n l y o n e o f the 2 s t e m m a t a can be h o m o l o g o u s with B o l w i g ' s o r g a n , while t h e fate o f t h e o t h e r s t e m m a is as yet u n k n o w n . Acknowledgements--We thank Renate R/Sssler (Freiburg) for technical assistance, K. Vogt (Freiburg) for

helpful discuss!ons, E. P. Meyer (Ztirich) for sharing unpublished data, and J. D. Plant (Vienna) for correcting the English. This study was partly supported by a grant from the Landesgraduiertenf6rderung Baden-Wtirttemberg given to R. R. Melzer.

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