TISSUE & CELL 1974 6 (3) 463-470 Published by Longman Group Ltd. Printed in Great Britain

JOSEPH C. DICKENS* and JOHN L. EATON?

FINE STRUCTURE OF OCELLI IN SPHINX MOTHS

ABSTRACT. The internal ocelli of sphinx moths have receptor cells with a rhabdomere structure that is unique for insects. The rhabdomere consists of an invagination of a single receptor cell membrane to produce a cavity lined with microvilli and containing small lumen.

Introduction

DORSAL ocelli are simple photoreceptors generally present in nymphal hemimeta- bolous and adult holometabolous and hemimetabolous insects (Goodman, 1970). Although Berlese (1909) located ocelli within the dorsal protocerebrum of the sphingid moth, Sphinx convoluvi, sphingid moths and some other Lepidoptera have been con- sidered to be anocellate (Goodman, 1970; Borror and Delong, 1971; Wigglesworth, 1965; Hoyle, 1955; Mazokhin-Porshnyakov, 1969). Eaton (1971), however, described the basic morphology of internal ocelli and indicated their presence in several families of anocellate moths and recently Dickens and Eaton (1973) reported internal ocelli in a butterfly as well as external ocelli in anocellate moths and butterflies. Therefore, a two-part ocellus exists in these moths. The two structures together probably represent the dorsal ocelli of other insect species but have become secondarily separate.

The presence of rhabdomeres (Ruck, 1964) in the internal ocellus could not be confirmed by Eaton (1971), but an electrical response in the form of a slow positive wave was recorded. Further study now leads us to believe that this wave represents the receptor

* Present address: Department of Entomology, Texas A & M University, College Station, TX., U.S.A. 77843.

t Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, Va. 24061.

Received 5 September 1973. Revised 8 January 1974.

cell generator potential which was recorded with reversed polarity due to the position of the recording electrode on the ocellar nerve (Eaton, unpublished data; Ruck, 1961). The purpose of this study was to determine whether rhabdomeres were present in the internal ocellus, and if so, to describe their structure.

Materials and Methods

Light microscopy

Adult sphingid moths obtained from light traps or from Manduca sexta pupae were light-adapted prior to several hours of fixation in alcoholic Bouin’s solution (Drake and McEwen, 1959). Paraffin sections were stained with either Mallory’s triple stain (Gray, 1964) or a modification of Holme’s silver stain (Larsen, 1960).

Transmission electron microscopy

Light-adapted adult moths were decapitated and their heads immersed in a cold glutaral- dehyde fixative (Lute, 1966; modified by Fleming and Saacke, 1972). Within 1 min after immersion, the vertex was removed to facilitate the penetration of the fixative to the internal ocelli. After an overnight wash in phosphate buffer the internal ocelli were excised from the brain. Post-fixation was in cold buffered 1 .O% osmium tetroxide. Dehydration was carried out through a graded series of ethanols and propylene oxide. Embedding was in Epon-812. Thin sections were cut using a Porter-Blum MT-l

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ultramicrotome with a glass knife. Sections were then picked up on copper grids and stained with uranyl acetate and lead citrate (Venable and Coggleshall, 1965; Watson, 1958). Grids were viewed with an RCA EMU-3H transmission electron microscope.

For orientation purposes, adjacent thick sections (1 p thick) were cut at intervals and stained with a buffered Azure II solution @eon, 1965).

Results

The term internal ocellus will be used to refer to the structure described by Eaton (1971). Table 1 lists lepidopterous insects

DICKENS AND EATON

previously reported to be anocellate having internal ocelli structurally similar to the sphingids.

The overall shape of the internal ocellus is variable. In Mund~ca sexta, a stalk, con- sisting primarily of nerve fibres, rises from the dorsal protocerebrum and extends approxi- mately 75-100 p to a retinular cell bulb some 50-60 p in diameter. The ocelli are enclosed within an acellular sac-like sheath which covers the entire dorsal protocerebrum to form an air space beneath the vertex of the head into which the ocelli protrude (Eaton, 1971; Fig. 1).

Beneath this sheath, the internal ocellus is surrounded by an acellular, electron dense neurilemma and a cellular perineurium. The outer amorphous neurilemma is cu. 3 p thick; beneath the neurilemma the cellular perineurium is several microns thick (Figs. 1, 2).

A flattened layer of glial cells surrounds each photoreceptor unit to separate it from the perineurium and other photoreceptor units in the area of the rhabdomere (Figs. 2, 3, 4A). Glial cell cytoplasm is scarce but mitochondria are scattered throughout. The numerous microtubules within the glial cell cytoplasm are packed closely together for retinular cell support (Figs. 3, 4A, B).

The basic structure of the photoreceptor unit is similar to other insect photoreceptors (Ruck, 1964). Numerous loosely organized microvilli about 800 A to 900 A in width arise from the surface of the receptor cell to form the rhabdomere (Figs. 3, 4A). Generally the photoreceptor structure is composed of the microvilli from a single receptor cell, but in some cases two or possibly three cells may contribute microvilli to form a rhabdom. The loosely organized microvilli are continuous at their bases with the retinular cell cytoplasm (Fig. 3).

The distal end of the retinular cell is characterized by a rhabdomere surrounding a relatively large lumen containing a homo- geneous electron dense substance (Fig. 3). Numerous multivesicular bodies surround the rhabdomere and at various levels open toward the microvilli. Vesicles resembling those found in the multivesicular bodies are present within the microvilli (Fig. 3). Glia may penetrate the receptor cell in this area. Mitochondria, though sparse in the cyto- plasm immediately surrounding the rhabdo-

Table 1. Lepidoptera previously reported to be anocellate having internal ocelli

Heterocera Sphingidae

Calasymbolus amyntor C. excaecatu (Eaton, 1971) C. myops (Eaton, 1971) Darapsa myron D. ~hohs (Eaton. 1971) D. iersicoior (Eaton, 1971) Herse cingulata Hyloicus chersis H. drupiflrarum (Eaton, 1971) Lapara coniferarum Manduca quinquemaculata (Eaton, 1971) M. rustica M. sexta (Eaton, 1971) Pholus achemon (Eaton, 1971) P. satellitia (Eaton, 1971) Spectrum lineata (Eaton, 1971) Sphinx jamaiciensis

gaturniidae Actias luna Automeris io Telea polyphemus (Eaton, 1971)

Citheronidae Anisota rubicunda Cifheronta regalis C. sepulchralis Eacles imperialis (Eaton, 1971)

Arctiidae Lithosiinae

Hypoprepia miniata

Rhopalocera Hesperiidae

Epargyreus clarus Thorybes pylades

OCELLI IN SPHINX MOTHS 465

Fig. I. Light micrograph of a 6 p section stained with Mallory’s Triple Stain of internal ocellus showing cross-sections of receptor cells; nl, neurilemma; n, receptor cell nucleus; r, rhabdomere. x 2000.

mere shown here, are numerous in the receptor cell cytoplasm outside the penetrat- ing glial cell area.

The receptor cell nucleus is located in the proximal portion of the cell (Fig. 4A). Here the rhabdomere is surrounded by mito- chondria, although mitochondria are also present in the more peripheral cytoplasm. Multivesicular bodies are present near the base of the microvilli. The receptor cell nucleus is large and multilobed. The peri- pheral cytoplasm contains free ribosomes as well as smooth and rough membranes. Cell junctions often occur where glial cell mem- branes penetrate the retinular cell cytoplasm and between adjacent retinular cells in this area.

Beneath the rhabdomere-bearing portion, the receptor cell narrows to form a terminal process still surrounded by glia (Fig. 4B). The cytoplasm of the terminal process contains spaced neurotubules as well as scattered mitochondria and multivesicular bodies.

Discussion

Electron microscopic studies of insect ocelli have been performed on only two paleop- terous insects, LibelIda pulchella (Ruck and Edwards, 1964) and Anax junius (Chappell and Dowling, 1972), two orthopteroid species, Peripianeta americana (Ruck, 1957) and Schistocerca gregaria (Goodman, 1970) and two neuropteroid species, Apis mellifeva

466 DICKENS AND EATON

(Link, 1909), this is the first study utilizing the electron microscope.

The structure of insect ocelli studied so far has been consistent. Rhabdoms are present and are formed by the apposition of rhabdomeres composed of straight lateral microvilli of two to five retinular cells (Mazokin-Porschnyakov, 1969). Opaque pig- ment granules or a reflecting tapetum are generally present to limit the entrance of light into the receptor cells (Chapman, 1970). The convergence of the terminal processes of numerous retinular cells on to relatively few second order fibres precludes attributing form vision to these structures (Goldsmith, 1964).

The structure of the internal ocellus described here is similar to that of other ocelli in that receptor cell synaptic processes do converge on to relatively few second order fibres and a microvillar photoreceptor structure is present. However, the loosely organized microvilli and the fact that the photoreceptor structure is generally com- posed of a circular rhabdomere formed by the invagination of the external membrane of a single receptor cell is unique among photoreceptor structures previously des- cribed for insects. Also, the lack of screening pigment in an ocellus has only been reported in Periplaneta where reflecting urate granules were present (Ruck, 1958).

Similar photoreceptor cell types have been described in another animal phylum and in another class of arthropods. In the median ocellus of Limulus rhabdoms composed by the apposition of rhabdomeres from adjacent retinular cells are present, but some cells possess structures referred to as ‘self- rhabdoms’ formed by the invagination of the

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Fig. 2. Diagram of longitudinal section of receptor cell reconstructed from cross-sections in following figures. Levels 1, 2 and 3 correspond to Figs. 3, 4A and 4B, respectively; er, endoplasmic reticulum; g, glial cell; gn, glial cell nucleus; m, mitochondria; mv, microvilli; mvb, multivesicular body; rn, recep- tor cell nucleus.

(Yanase and Katoaka, 1963) and Boettche- risca peregrina (Toh et al., 1971). Although light microscopic studies of lepidopteran ocelli have been performed on several species

Fig. 3. Cross-section through distal end of receptor cell showing association of multivesicular bodies with microvilli and vesicles within microvilli; 1, lumen: mt, microtubules; mv, microvilli; v, vesicle. Arrows indicate continuity of microvilli with receptor cell cytoplasm. x 22,000.

Fig. 4. A. Cross-section through proximal end of rhabdomere; g, glial cell; mv, microvilli; mvb, multivesicular body; rn, receptor cell nucleus. x 10,000. B. Cross- section through receptor cell terminal process; gn, glial cell nucleus; mt, microtubules; nt, neurotubules; rc, receptor cell. x 20,000.

OCELLI IN SPHINX MOTHS

retinular cell microvilli (Jones et al., 1971). In the phylum Annelida, the photoreceptor cell structure consists of a circular array of microvilli within a single cell. This structure has been referred to as a phaosome and was thought to be separate from the extracellular space. The phaosome has been described in the leech (Hansen, 1962; Rohlich and Torok, 1964; Clark, 1967) and the earth- worm (Rohlich et al., 1970). Recently the phaosome of the leech photoreceptor cell was shown by lanthanum deposition to be connected to the extracellular space (White and Walther, 1969). The loosely organized microvilli of the circular phaosome of the earthworm photoreceptor cell (Rohlich et a/.) is quite similar to the rhabdomeric structure in the internal ocellus.

As is the case with more typical dorsal ocelli, form vision is not possible for the internal ocellus. The lack of form vision is especially clear in this instance since the internal ocellus is located at some distance from the external cornea on the vertex (Dickens and Eaton, 1973). The detection of the plane of polarized light suggested as

469

function of fleshfly ocelli by Wellington (1953) is equally improbable. Toh et al. (1971) found that the microvilli of the dorsal ocelli in the fleshfly (a different species than that studied by Wellington) encircle the periphery of each retinular cell thus making sensitivity to the plane of light polarization impossible according to the hypothesis of Waterman and Horsch (1966). Similarly, the circular rhabdoms of the internal ocellus preclude this as a possible function.

At this time we do not know the function of the internal ocellus, but work is continuing to learn more about this unique photo- receptor.

Acknowledgements

We thank Dr A. H. Baumhover of the Oxford Tobacco Research Station, USDA, Oxford, North Carolina, for providing some of the tobacco hornworms used in this study and Janet Queisser for technical assistance with Fig. 2. This study was supported in part by a grant (GB-35541) from the National Science Foundation.

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