/. Embvycl. exp. Morph. Vol. 60, pp. 345-358, 1980
Printed in Great Britain © Company of Biologists Limited 1980
345
Ommatidium assembly and formation
of the retina-lamina projection in interspecific
chimeras of cockroach
By MARK S. NOWEL 1
From the Department of Zoology, University of Leicester
SUMMARY
By grafting operations, interspecific eye chimeras of the cockroaches Gromphadorhina
portentosa andLeucophaea maderae were produced. Mechanisms involved in the development
of both the compound eye and the retina-lamina projection have been studied. Most cell types
composing the eyes of these cockroaches are cytologically distinguishable in the chimera;
also, retinula axons forming the retina-lamina projection in the two species are of vastly
different lengths. At the border between host and graft eye tissue, individual ommatidia are
formed containing cells of both types. In particular, it is shov/n that the four cone cells can
be found in any of the possible combinations of the two cell types. This shows that the cone
cells within one ommatidium are not necessarily related by lineage. These results are in
agreement with the hypothesis that cells within an ommatidium are determined by position
rather than by a lineage mechanism. Furthermore, formation of mosaic ommatidia suggests
that mechanisms governing eye formation are similar in these two species. The formation
of the projection from donor retina to host lamina shows that axon elongation is not rigidly
programmed, but that the axons grow until they reach a suitable target at which point
connexions are made.
INTRODUCTION
By generating interspecific chimeras it is possible to investigate the question
of whether pattern-forming mechanisms in different organisms are similar. In
this study two species of cockroach, Gromphadorhina portentosa and Leucophaea
maderae, have been used to create chimeric compound eyes. This experimental
material enables us to investigate the specific question of cell lineage in development, because the eye cells in the two species are cytologically identifiable. It
also enables us to ask the general question of whether or not ommatidia in
different genera are formed by the same mechanisms. Finally, the compound
eye as a component of the nervous system has enabled us to examine questions
concerning nerve growth in a chimera.
Concerning the question of cell lineage, it has been possible to confirm the
observations of previous workers that there is no causal relationship between
lineage and determination in the formation of ommatidia (Yagi & Koyama,
1
Author's address: Department of Zoology, University of Leicester, Leicester LEI 7RH,
U.K.
346
M. S. NOWEL
1963; Hanson, Ready & Benzer, 1972; Ready, 1973; Benzer, 1973; Shelton &
Lawrence, 1974; Green & Lawrence, 1975; Ready, Hanson & Benzer, 1976;
Nardi, 1977; Shelton, Anderson & Eley, 1977; Lawrence & Green, 1979). In
particular, cone-cell lineages have been examined. At the borders of graft and
host tissues, ommatidia are found containing cells of both genotypes. The two
types of cells behave autonomously and mosaic ommatidia have normal
numbers of cells even though some are of one genotype and some are of the
other. Thus, whichever pattern-forming mechanism is responsible for ommatidium assembly, it is the same in these different genera.
Concerning nerve growth, the mode of establishment of connexions between
the eye and optic lobe has been examined. In the two species, retinula axons
growing into the optic lobe have to grow different distances to form their
connexions. In an interspecific chimera, L. maderae retinal tissue is three times
farther away from its target, the lamina of its G. portentosa host, than it would
be from its own lamina in the unoperated situation. Nevertheless, retinula
axons from the foreign retina can form connexions in the lamina of the underlying optic lobe. This provides valuable information on factors governing
nerve growth and target discrimination.
MATERIALS AND METHODS
Cultures of G. portentosa and L. maderae were maintained under conditions
of constant temperature (24 °C) and an alternating cycle of 12 h light/12 h
dark, and fed on a diet of rat pellets and water.
Grafts of eye margin and adjacent vertex epidermis were exchanged between
young, newly moulted G. portentosa and L. maderae nymphs. The animals were
anaesthetized by cooling in ice for 10-20 min and restrained with strips of
Plasticine on a bed of moulded Plasticine. Operations were carried out under a
dissecting microscope. Excisions of the integument were made using a razor
blade fragment (Gillette francais) supported in a pin vice, and grafts were
transferred from donor to host site using tungsten needles. After positioning
the graft in the host site which had been kept moist with insect saline (Hoyle,
1953), a small droplet of insect wax (Krogh & Weis-Fogh, 1951) was used to
seal the operation site. Operated animals were allowed to grow to the imago.
To map areas of neural projection from graft-derived ommatidia in the
chimeric eye into the lamina neuropile of the optic lobe, small localized lesions
were made within the graft-derived eye tissue of the adults. After immobilizing
the anaesthetized cockroach as for a grafting operation, a silver earthing wire
was inserted into the head through a hole cut in the cuticle at a posterior medial
point. A tungsten microelectrode was connected to a function generator set to
deliver 2 /iA at a frequency of 1 MHz. After removal of narrow strips of the
overlying cornea, the microelectrode was inserted into the exposed ommatidia to
be electrolytically destroyed, and left in each point of insertion for 10 sec. The
Cockroach compound-eye development
347
wounds were covered with insect wax. Sixteen hours later the animal was
killed and the eye and optic lobe were fixed in a mixture of glutaraldehyde and
paraformaldehyde (Karnovsky, 1965) in a 0-1 M-phosphate buffer (Hayat, 1970)
at pH 7-4 for 2-4 h. The tissue was post-fixed in phosphate-buffered 1 %
osmium tetroxide for 2-12 h, then dehydrated in an acetone series, cleared in
propylene oxide, and embedded in Spurr's resin following a long period of
infiltration.
The optic lobe was serially sectioned in 1 ^m horizontal sections with a
Huxley Ultramicrotome and glass knives, mounted in order on subbed slides,
stained with toluidine blue, and examined with a Zeiss compound microscope
for degenerating retinula axon terminals which appear dark blue following
such treatment (Geisert & Altner, 1974).
Chimeric eyes were sectioned perpendicular to the ommatidial long axis at
the graft/host border. Serial semithin (1 ju,m) sections were collected in order
on subbed slides and stained with toluidine blue. Ultrathin (80-100 nm)
sections cut on a Dupont diamond knife were collected on collodion films
(Pease, 1964), placed on to slot grids, stained with uranyl acetate and Reynolds'
(1963) lead citrate, and examined with an AE1-802 Electron Microscope or a
Siemens 102 Elmiskop.
For wax histology, quarter heads were fixed for at least 3 h in alcoholic
Bouin's fluid (Dubosq-Brasil) (Pantin, 1969) and left in 70 % isopropyl alcohol
for periods of several days to several weeks to wash out the fixative and to
soften the cuticle. After dehydration and embedding in paraffin wax, 10 /*m
thick horizontal sections were cut on a Cambridge rocking microtome, dried
on to albuminized slides and stained using a modification of the MalloryAzan technique (Schiimperli, 1977).
Experimental animals were photographed using a Zeiss Tessovar Photomacrographic Zoom system. Sectioned material was photographed on a Zeiss
Photomicroscope II.
RESULTS
Identification of cell genotypes in G. portentosa and L. maderae
Sections through ommatidia in adult G. portentosa and L. maderae at the
level of the crystalline cones are shown in Figs. 1 and 2. Cone cells in G. portentosa have a denser granularity and are considerably larger than those of L.
maderae: crystalline cones (composed of four Semper's or cone cells) in G.
portentosa measure approximately 35-40 pm in diameter at their bases with a
base to apex length of 60 [im, while those of L. maderae measure 25 /im in
diameter with a base to apex length of 35-40 /*m. In the chimeric situation
(Figs. 5-11) the relative sizes of not only the cone cells (Figs. 5-8) but also the
pigment granules which provide distinguishing features between both the
retinula and primary pigment cells of the two species (Figs. 7, 10) are easily
seen. In the primary pigment cells, pigment grains of G. portentosa are large
348
M. S. NOWEL
FIGURES 1 AND 2
Semithin sections through the compound eyes of G. portentosa (Fig. 1) and L.
maderae (Fig. 2) cut perpendicular to the ommatidial long axis at the level of the
crystalline cones (cc). The Semper's cells comprising the crystalline cones may be
distinguished on the basis of their relative sizes: cones of G. portentosa are composed of four large cells, while those of L. maderae are made up of four smaller
cells. Each crystalline cone is surrounded by two primary pigment cells (pp) and
numerous secondary pigment cells (sp). (The basis of the differential staining of cone
cells within the same cone, often observed, is not understood.) Bars represent
10/*m.
Cockroach compound-eye development
349
(1-3-1-7/mi) while those of L. maderae are slightly smaller (1-0-1-2/mi).
Pigmentation of the retinula cells is similarly distinguishable: larger (0-71-0 /mi) pigment grains in G. portentosa cells, smaller (0-35-0-5 /mi) grains in
those of L. maderae. Secondary pigment cells are not easily distinguishable.
Interspecific mosaic ommatidia
Of approximately 400 grafting operations, approximately 20 resulted in
interspecific chimeras. Following such operations, L. maderae eye tissue appears
to grow at a slightly faster rate and G. portentosa at a slightly slower rate than
does their host eye tissue. This gives the compound eye of each sort of chimera a
characteristic appearance (Figs. 3, 4).
Three chimeric eyes containing G. portentosa and L. maderae ommatidia were
sectioned and examined for the presence of ommatjdia composed of cells of
both sources, especially those whose crystalline cones were mosaics of cells of
the donor and host. Such ommatidia were common and readily identifiable at
the border between graft- and host-derived eye tissue (Fig. 5).
Several mosaic cones of the three possible combinations of constituent cells
(one G. portentosa cell with three L. maderae cells; one L. maderae with three
G. portentosa cells; or two cells from each source) were found (Figs. 6-8).
Mosaic ommatidia appear at random intervals along the graft/host border,
and occasionally two are found alongside each other. It is concluded that there
is no fixed clonal relationship between cells of a single crystalline cone.
Mosaic ommatidia in which each of the two primary pigment cells is from a
different source (one exhibiting donor-specific pigmentation and the other
exhibiting host-specific pigmentation) are found (Fig. 7). Ommatidia in which
the constituent retinula cells come from different sources are also seen (Figs. 5,
9). In Fig. 9 it can be seen that a single retinula cell has G. portentosa-spQcific
characteristics while the other seven retinula cells (as well as all four Semper's
cells and both primary pigment cells) are from L. maderae. These observations
confirm the conclusions of previous investigators that retinula cells and primary
pigment cells within a single ommatidium are not determined by sequence of
determinative cell divisions, and argue in favour of lineage independence of
3-5 (see page 350).
Fig. 3. Interspecific chimeric eye of a G. portentosa (G) host with a graft of L.
maderae (L.) eye margin and head epidermis. Bar represents 0-25 mm.
Fig. 4. Interspecific chimeric eye of a L. maderae (L) host with a graft of G.
portentosa (G) eye margin and head epidermis. Bar represents 0-25 mm.
Fig. 5. Semithin section through the graft/host border of a chimera similar to that
shown in Fig. 3. G. portentosa (G) host ommatidia have very large cones, while
L. maderae (L) graft ommatidia have small cones. Note two ommatidia (arrows)
with mosaic crystalline cones (shown again in Figs. 6 and. 7); * indicates an ommatidium (shown again in Fig. 9) with a mosaic sensory retinula. Bar represents 10 /im.
FIGURES
23
EMB 60
350
M. S. NOWEL
&
M
For legend see page 349.
FIGURES
6-8
Three electron micrographs showing mosaic cones in interspecific chimeras. Such mosaic cones show that neither the ommatidium
nor the crystalJine cone is produced from a single ommatidial or cone mother cell. Bars represent 5 /*m.
Fig. 6. The crystalline cone is composed of one G. portentosa cell (G) and three L. maderae cells (L).
Fig. 7. The crystalline cone is composed of two G. portentosa cells (G) and two L. maderae cells (L). In addition, each of the two
primary pigment cells comes from a different source (G and L).
Fig. 8. The crystalline cone is composed of three G. portentosa cells (G) and one L. maderae cell (L).
352
M. S. NOWEL
<i ~
r/7
1CL
tfk*
%
Cockroach compound-eye development
353
any particular cell component of an ommatidium from other cells within that
structure.
Projection of graft-derived retinula axons to host lamina in interspecific chimera
In the adult cockroach, the projection of axons from the basement membrane
of the retina to the outer optic anlage is approximately 0-5-0-6 mm in G.
portentosa, and 0-2 mm in L. maderae (Figs. 12, 13). These differences are
apparent during larval development at the time that the connexions are originally established. At the larval stages during which the operations were performed, the separation between retina and outer optic anlage was approximately
0-2 mm in G. portentosa and 0-1 mm in L. maderae. Following interspecific
transplant operations, if G. portentosa retinula axons are to make connexions
in the lamina neuropile of their L. maderae hosts, they must be able to stop
elongating after growing only one half to one third of their normal length.
For retinula axons of L. maderae to make connexions in the lamina of their
G. portentosa hosts, they must double or treble the distance they have to grow
before reaching the target outer optic anlage.
Of three G. portentosa eyes (with a graft of L. maderae tissue) examined,
one showed degenerating retinula terminals in the host lamina neuropile
following microcautery of a set of graft-derived ommatidia (Fig. 14). Of seven
L. maderae eyes containing grafts of G. portentosa tissue which were examined
in the same way, three showed degenerating retinula axon terminals in the host
lamina neuropile (Fig. 15).
DISCUSSION
Ommatidium assembly
Bernard (1937) had proposed that all cells forming a particular ommatidium
were the division products of a single ommatidium mother cell. The presence of
mosaic ommatidia disproves this mechanism's involvement in compound-eye
development.
FIGURES
9-11
Fig. 9. Electron micrograph of a mosaic ommatidium lying on the graft/host border
of an interspecific chimera, taken at the level of the sensory retinula. There is the
normal compliment of eight retinula cells (numbered arbitrarily). The larger
pigment grains of the host G. portentosa retinula cell (6) clearly distinguishes it
from the seven graft-derived L. maderae cells (1-5, 7, 8). rh rhabdom.
Fig. 10. Electron micrograph through an interspecific mosaic ommatidium at the
level of the sensory retinula. The retinula cell of G. portentosa (G) origin has
larger pigment grains than those of L. maderae (L). Note the desmosome (arrow)
joining the adjacent cells of different specific origin at the periphery of the rhabdom
(rh). cp, Cone-cell process.
Fig. 11. Electron micrograph through an interspecific mosaic cone. Cone cells of
L. maderae (L) are smaller with a denser granularity than those of G.portentosa (G).
pp, primary pigment cell. Bars represent 2/*m.
354
^V 'r » V H !
M. S. NOWEL
Cockroach compound-eye development
355
Lawrence & Green (1979) have generated clones of red-pigmented cells in
white Drosophila eyes. Extremely small clones (even clones composed of only
two scorable cells) can contain both pigment and retinula cells. It is clear that
these different cell types composing the ommatidium are not derived by separate
cell lineages and determinative divisions. As a result of the present experiments,
which include the cone cells as well as the pigmented retinula and primary
pigment cells, it may now be said that any ommatidial cell may be unrelated by
lineage to any other cell component of that ommatidium. This reaffirms the
hypothesis that spatial considerations, or the position of cells within the
developing ommatidium, are determinative in the generation of particular cell
types (Shelton & Lawrence, 1974; Lawrence & Green, 1979).
How cell position within a developing pre-ommatidial cell cluster is assessed
is a matter for speculation. Certainly, information exchange is possible at the
time of cluster formation when gap junctions are widely distributed within the
undifferentiated cells of the eye margin of the locust (Eley & Shelton, 1976).
Another suggestion is that particular retinula cells are determined with respect
to their positional relationship with the five cone-cell processes in the locust
(Wilson, Garrard & McGinness, 1978).
The ability of a group of cells coming from two different genera to interact
and form a perfect ommatidium (four cone cells, two primary pigment cells,
eight retinula cells, plus secondary pigment cells) argues in favour of the
similarity or equivalence of the ommatidium-forming mechanisms in these
two different insects.
FIGURES
12-15
Fig. 12. Horizontal section through the compound eye (ce) and optic lobe of an
adult L. maderae. Note the relatively short (approximately 0-2 mm) retina-lamina
projection of retinula axons (ra). In, Lamina neuropile; mn, medulla neuropile. Bar
represents 01 mm.
Fig. 13. Horizontal section through the compound eye (ce) and optic lobe of an adult
G. portentosa shown at the same magnification as Fig. 12. Note the relatively long
(approximately 0-5-0-6 mm) retina-lamina projection of retinula axons (ra). In,
Lamina neuropile. Bar represents 01 mm.
Fig. 14. Lamina neuropile (In) of G. portentosa adult in which there was a graft of
L. maderae tissue. Following the introduction of a small lesion in the grafted eye
tissue, the appearance of degenerating terminals (arrow) in the lamina indicates that
axons project to the lamina from the (wounded) graft ommatidia. Bar represents
10/un.
Fig. 15. Lamina neuropile (In) of L. maderae adult in which there was a graft of
G. portentosa tissue. Following the introduction of a small lesion in the grafted eye
tissue, the appearance of degenerating terminals (arrow) in the lamina indicates that
axons project to the lamina from the (wounded) graft ommatidia. Bar represents
10/mi.
356
M. S. NOWEL
Axon projection
Cotman & Banker (1974) describe synapse formation as a two-step process:
axon elongation to approach the general vicinity of its set of target cells followed
by the establishment of synaptic contacts with a limited number of cells within
this region. The signal to the growing nerve fibres to stop elongating and form
connexions poses an interesting problem of developmental mechanisms: are
fibre tips directed to stop at a particular site and there form connexions, or do
they grow until they make connexions and then stop elongating?
Swisher & Hibbard (1967) have removed the target sites of Mauthner's fibres
in Xenopus embryos by removing the tails of two individuals and grafting them
together with their heads pointing in opposite directions. The Mauthner's
fibres pass caudally down the spinal cord of one embryo and, not encountering
their sites of termination (in the excised tail), continue into the spinal cord of
the grafted embryo rostrally into the second brain.
Altman's (1972, 1973) studies on the rat cerebellar cortex show that if the
normal migration of target neurons is retarded, projecting fibres are capable of
further elongation. The fibres bypass other (inappropriate) neurons without
making extensive synaptic contacts with these cells, and terminate on their
appropriate target cells. Having made these proper connexions, both the
elongation of the projecting axons and the migration of the target cells cease.
In the present studies, it is clear that the developmental programme for the
advancing retinula axons of the cockroach does not include instructions to
grow a particular distance and then form connexions. Rather, the variations
introduced by experimental manipulations of the projection distance indicate
that fibres grow until they reach cells which are recognized as prospective
target sites, possibly bypassing inappropriate (i.e. already-differentiated) lamina
ganglion cells to reach the target, the outer optic anlage. Apparently these
ganglion cells are deemed unsuitable despite their location in a more typical
projection distance for the presynaptic axons. In Daphnia, growing retinula tips
bypass differentiated ganglion cells to reach the proliferation zone of the lamina
(LoPresti, Macagno & Levinthal, 1973). Once suitable target cells are contacted,
synaptogenesis begins.
Several things remain unknown: (a) How adjustable is the axon's capacity
for elongation ? What restraints are imposed on the extent of axon elongation ?
(b) What is the nature of the signal to stop fibre elongation ? Is it the presence of
the target cell itself, or is it some property associated with the outer optic
anlage ?
CONCLUSIONS
In hemimetabolous insects, new retinal cells are generated by cell proliferation
within a budding zone of the eye margin (Nowel & Shelton, 1980). The results
of the present experiments, namely (a) the generation of inter-specific chimeric
Cockroach compound-eye
development
357
retinae; (b) the formation of mosaic ommatidia; (c) the formation of mosaic
crystalline cones; (d) the formation of mosaic pigment cell sleeves, and (e) the
formation of mosaic sensory retinulae, including an example in which only
one retinula cell differs in origin from the other seven as well as from all the
other scored cells in the ommatidium indicate that new ommatidia - and also
parts of new ommatidia - are not determined by a cell-lineage mechanism
but by some sort of interactive mechanism within the cell clusters forming at
the margin of the compound eye. This leads to determination and differentiation
of specific cell types making up the ommatidium.
With regard to axon projection to its target, the present experiments allow
certain conclusions to be drawn:
(a) Axons from receptor cells of one species of cockroach can grow into the
optic lobe of a host animal of a different species; they can apparently form
connexions with second-order cells and otherwise be integrated into the host
neuropile. Under normal circumstances, interspecific connexions between these
receptor and second-order cells would never be made, and species-specific cell
surface properties must exist. Nevertheless, successful establishment of connexions can occur between cells of different specific origins. Presumably the
affinities of retinula terminals for lamina ganglion cells override any interspecific differences.
(b) It is significant that the graft retinula cells allow apparently normal
development of the lamina (Figs. 14, 15). This implies that the stimulus for the
development of lamina ganglion cells and neuropile are similar in different
species.
(c) Retinula neurons of G. portentosa are able to curtail axon extension
prematurely to form connexions with second-order cells, apparently upon
reaching a particular signal which is met sooner in this experimental situation
than in a normal situation.
{d) Retinula neurons of L. maderae are able to grow further than their normal
programme of growth would have allowed. In the experimental situation (in
which they are growing in a G. portentosa host), axons apparently receive a
particular signal to cease elongation and begin synaptogenesis later than in a
normal situation.
My sincere thanks go to Dr Peter M. J. Shelton for supervising this work and for his
encouragement. We are grateful to the Science Research Council for its grant to P. M. J.
Shelton.
REFERENCES
ALTMAN, J. (1972). Postnatal development of the cerebellar cortex in the rat. III. Maturation
of the components of the granular layer. /. comp. Neurol. 145, 465-514.
ALTMAN, J. (1973). Experimental reorganization of the cerebellar cortex. III. Regeneration
of the external germinal layer and granule cell ectopia. /. comp. Neurol. 149, 153-180.
BENZER, S. (1973). Genetic dissection of behaviour. Scient. Am. 229 (12), 24-37.
358
M. S. NOWEL
F. (1937). Recherches sur la morphogenese des yeux composes d'arthropodes.
Bull. biol. Fr. Belg. Suppl. 23, 1-162.
COTMAN, C. W. & BANKER, G. A. (1974). The making of a synapse. In Reviews of Neuroscience, vol. I (ed. S. Ehrenpreis & I. J. Kopin), pp. 1-62. New York: Raven Press.
ELEY, S. & SHELTON, P. M. J. (1976). Cell junctions in the developing compound eye of the
desert locust Schistocerca gregan'a. J. Embryol. exp. Morph. 36, 409-423.
GEISERT, B. & ALTNER, H. (1974). Analysis of the sensory projection from the tarsal sensilla
of the blow-fly (Phormia terraenovae Rob.-Desv., Diptera). Experimental degeneration of
primary fibers. Cell Tiss. Res. 150, 249-259.
GREEN, S. M. & LAWRENCE, P. A. (1975). Recruitment of epidermal cells by the developing
eye of Oncopeltus (Hemiptera). Wilhelm Roux1 Archiv. devl Biol. Ill, 61-65.
BERNARD,
HANSON, T. E., READY, D. F. & BENZER, S. (1972). Use of mosaics in the analysis of pattern
formation in the retina of Drosophila. Rep. Div. Biol. Cal. hist. Tech. 37, 40.
A. (1970). Principles and Techniques ofElectron Microscopy: Biological Application,
vol. i, pp. 342-343. London: Van Nostrand Reinhold.
HOYLE, G. (1953). Potassium ions and insect nerve muscle. J. exp. Biol. 30, 121-135.
KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use
in electron microscopy. / . Cell Biol. 27, 137 A.
KROGH, A. & WEIS-FOGH, T. (1951). The respiratory exchange of the desert locust {Schistocerca gregan'a) before, during and after flight. / . exp. Biol. 28, 344-357.
LAWRENCE, P. A. & GREEN, S. M. (1979). Cell lineage in the developing retina of Drosophila.
Devi Biol. 11, 142-152.
LOPRESTI, V., MACAGNO, E. R. & LEVINTHAL, C. (1973). Structure and development of
neuronal connections in isogenic organisms: cellular interactions in the development of
the optic lamina of Daphnia. Proc. natn. Acad. Sci. U.S.A. 70, 433-437.
NARDI, J. B. (1977). The construction of the insect compound eye: The involvement of cell
displacement and cell surface properties in the positioning of cells. Devi Biol. 61, 287-298.
NOWEL, M. S. & SHELTON, P. M. J. (1980). The eye margin and compound-eye development in
the cockroach: evidence against recruitment. / . Embryol. exp. Morph. 60, 329-343.
PANTTN, C. F. A. (1969). Notes on Microscopical Technique for Zoologists. Cambridge University Press.
PEASE, D. C. (1964). Histological Techniques for Electron Microscopy. London: Academic
Press.
READY, D. F. (1973). Pattern formation in the retina of Drosophila. Rep. Div. Biol. Cal. hist.
Tech. p. 203.
READY, D. F., HANSON, T. E. & BENZER, S. (1976). Development of the Drosophila retina, a
neurocrystalline lattice. Devi Biol. 42, 211-221.
REYNOLDS, E. W. (1963). The use of lead citrate at high pH as an electron-opaque stain in
electron microscopy. / . Cell Biol. 17, 208-212.
SCHUMPERLI, R. A. (1977). Adaptation of the Mallory-Azan staining method to insect
nervous tissue. Stain Technol. 52, 55-56.
SHELTON, P. M. J., ANDERSON, H. J. & ELEY, S. (1977). Cell lineage and cell determination
in the developing compound eye of the cockroach, Periplaneta americana. J. Embryol. exp.
Morph. 39, 235-252.
SHELTON, P. M. J. & LAWRENCE, P. A. (1974). Structure and development of ommatidia in
Oncopeltus fasciatus. J. Embryol. exp. Morph. 32, 337-353.
SwrsHER, J. E. & HIBBARD, E. (1967). The course of Mauthner axons in Janus-headed
Xenopus embryos. /. exp. Zool. 165, 433-439.
WILSON, M., GARRARD, P. & MCGINNESS, S. (1978). The unit structure of the locust compound eye. Cell Tiss. Res. 195, 205-226.
YAGI, N. & KOYAMA, N. (1963). The Compound Eye of Lepidoptera. Tokyo: Maruzen.
HAYAT, M.
{Received 15 May 1980, revised 18 June 1980)