/. Embryol. exp. Morph. Vol. 27, 2, pp. 367-379, 1972
Printed in Great Britain
367
Regeneration, post-embryonic
induction and cellular interaction in the eye
of Periplaneta americana
By CORNELIA ANNE TUTEN HYDE 1
From the Department of Biology, Georgia Southern College
SUMMARY
1. The eye grows by the addition of new ommatidia rather than by an increase in size of
existing ones.
2. New ommatidia develop as a result of induction of epidermal cells by functional
ommatidia.
3. The eye has great regenerative ability, but following removal of the entire eye and the
growth zone that surround it regeneration does not occur.
4. Transplanted eye cells or epidermal cells from normal-eyed or from lavender-eyed
mutants resulted in the development of normal eye pigmentation in a mutant that lacked any
pigmentation in the eye. Only the eye to which the graft had been made developed pigments.
The development of pigments was a result of tissue contact, not of a blood-borne factor.
5. When epidermal tissues from the prothorax of normal-eyed roaches were placed above
the developing eye of a mutant with no pigment in the eye they were eventually incorporated
into the eye as it grew. When these cells became eye cells, normal eye pigments were synthesized.
6. Growth of the eye is by a constant recruitment of epidermal cells that express their own
genetic capability when they become eye cells.
INTRODUCTION
Periplaneta americana L., the American cockroach, has been studied by many
investigators, but phenomena related to normal development of the compound
eye and its capacity for regeneration have apparently not been the subject of
careful investigation. These phenomena and the related one of the genetics of
pigmentation in the eye are the subject of this paper.
In the exopterygotes investigated in embryonic and nymphal life (AphisWitlaczil, 1884; Rhodnius prolixus-Mellanby, 1937; Notonecta glauca-LMtke,
1940) the compound eye begins to differentiate at the posterior margin and
differentiation progresses toward the future anterior margin of the eyes (Lew,
1933).
There are apparently only three previous experimental studies related to
regeneration of the eye of P. americana. Bodenstein (1962) transplanted imaginal
eyes to the prothoracic shields of nymphs and found that they molted in syn1
Author's address: Department of Biology, Georgia Southern College, Statesboro,
Georgia 30458, U.S.A.
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C. A. T. HYDE
chrony with the host and that a new cornea was formed. In a single paragraph
Penzlin (1963) says that the eye is capable of some regeneration, but does not go
into detail. Parabiotic experiments by Bodenstein (1959) in which the white-eyed
mutant (also known as pearl- Ross, Cochran & Smyth, 1964) was joined to a
normal-eyed nymph failed to change the eye color of either partner. A similar
experiment in which the lavender-eyed mutant was joined to the normal (Ross
et al. 1964) failed to change the eye color of either partner.
Ross et al. found that in crosses between the white-eyed {pearl) and lavender
that the Fx hybrids had normally pigmented eyes as did hybrids between either
lavender or pearl and normal-eyed roaches. They concluded that both pearl and
lavender are autosomal recessives and are inherited independently.
Mutants of the strains studied by Bodenstein and Ross et al. were used in this
study.
MATERIALS AND METHODS
The experimental insects were kept at 24 °C, were fed commercial dog food
and fresh apple and provided with water.
Since the two mutations had been shown to be autosomal by Ross et al.
(1964), no selections were made on the basis of sex. In the first experiments,
surgery was performed 4 days after an ecdysis, in order to be certain that the
insect was well past the extreme sensitivity to the anesthetic that is characteristic
just before and after ecdysis. In later experiments familiarity with the appearance of the eye during the intermolt made it possible to select animals that were
far from an ecdysis.
In making the transplants, great care was exercised so that the graft and the
tissue of the host fitted closely together. If the edge of the graft protrudes beneath the tissue of the host the epidermis of the latter tends to cover the graft and
the grafted tissue heals independently.
Melted soft paraffin was used as a bandage. This bandage covered the area of
the operation completely so that the grafts would be held firmly in position.
Tissues for histological examination were prepared by standard techniques.
NORMAL GROWTH AND DEVELOPMENT
P. americana L. is an exopterygote insect that passes through a variable
(10-12) number of instars. During the course of this investigation the nymphal
instars varied in duration from about 2 weeks (first one) to 2 months (last
instars). One year passed from hatching to adulthood. This slow development
makes the gradual changes taking place in the eye comparatively easy to follow.
More than 200 nymphs, including several individuals of each instar, were studied
in this experiment.
At the time of hatching the eye is triangular in shape. This triangle is nearly
equilateral, with its apex at a point near the dorsal edge of the base of the antenna
and the base of the triangle extending from the posterodorsal corner of the eye
Development of the eye of Periplaneta
369
Fig. 1. Shape and dimensions of the area of mature ommatidia and the maturation
zone of the compound eye of representative nymphal stages and the newly molted
imago (I). (Camera lucida drawings.) O, Ommatidia; Mz, maturation zone. Line
indicates 1 mm.
to the ventral tip of the eye which is slightly below the ventral edge of the border
of the antenna. An unpigmented zone that is approximately equilateral in its
dimensions completely surrounds the pigmented, functional ommatidia of the
eye. The posterior edge of the eye has large ommatidia and the dorsal, anterior'
and ventral tip edges of the pigmented area show a pattern of smaller and more
numerous ommatidia.
As the nymph grows, the growth of the eye is more extensive dorsally, anteriorly and ventrally than it is along the posterior edge. The eye of the adult more
nearly approximates an isosceles triangle, the base of which is the dorsal edge of
the eye with the apex of the triangle at the ventral tip. The eye of the adult
(Fig. 4) is strongly curved anteriorly and extends almost half-way around the
base of the antenna both dorsally and ventrally.
The eye of the adult covers a much greater proportion of the head than does
that of the young nymph. In the first instar the eye extends dorsally and ventrally only slightly above and below the dorsal and ventral margins of the base of
370
C. A. T. HYDE
the antenna, but in the adult both the gena and vertex are greatly reduced in
relation to the size of the eye.
The width of the unpigmented zone of the eyes of nymphs of various stages
varies and there is a maturation zone around the eye of the newly ecdysed adult
(Fig. 1). The insects used for Fig. 1 were selected at random before the exoskeleton had hardened and darkened following an ecdysis. The drawings were made
24 h later, after hardening and darkening were complete.
The maturation of the ommatidia is a continuous process in this insect. Observation of the eye of a newly ecdysed nymph of any instar (Fig. 5) as well as the
newly molted adult reveals that the margin of the area of pigmented ommatidia
appears to be serrate with that half of an ommatidium immediately adjacent to
the mature area pigmented and its distal half unpigmented. As growth continues,
these ommatidia become progressively pigmented and the margin of the mature
area becomes smooth in outline. This process is first noticeable along the
posterior margin of the eye at the point where the eye is narrowest - that is, a
little below the mid-posterior edge. This is apparent by about 10 days following the ecdysis. The progressive pigmentation of the other edges of the eye
continues slowly until the next ecdysis.
With the approach of the next molt the dorsal, anterior, and ventral tip
sections of the area that was unpigmented previously are darkly pigmented.
There remains a thin white band along the posterior edge. Growth is less
extensive there at every instar than in the other areas.
The dimensions of the unpigmented zone just after a molt added to those of
the mature ommatidia can be taken as a rough estimate of the dimensions of the
mature area of ommatidia of the next instar. This cycle of maturation is repeated from instar to instar and includes the imago. The imaginal eye is not
completely pigmented at the time of the larva-adult ecdysis, but the unpigmented zone surrounding the eye of the newly molted adult becomes pigmented
over a period of not less than 2 weeks following ecdysis (Fig. 1).
Granular intracellular pigments can be seen beyond the original dimensions
of the unpigmented band along the dorsal edge of the eye in nymphs of fifth and
later instars. Appearance of these pigments indicates that the molt will take
place within approximately 2 days and can be used for predicting the next molt.
As mentioned, the dimensions of the unpigmented zone of a given area change
from one instar to the next. This area is widest in those areas which are growing
most rapidly. The changes in the dimensions of the unpigmented zone are
reflected in the progressive changes of the shape of the mature area of the eye.
The pattern of the maturation of ommatidia of different sizes, as outlined
above, is maintained throughout the growth stages. Smaller and more numerous
ommatidia are formed along the dorsal, anterior, and ventral tip margins; larger
and fewer ommatidia are formed along the mid-posterior edge.
Studies of frontal sections cut across the center of the eyes of newly molted
sixth- or seventh-instar nymphs (Fig. 6) show that the functional ommatidia of
Development of the eye of Periplaneta
Operation
MI
371
Ml
Fig. 2. Regeneration in the compound eyes of nymphs from which a part of the
mature area of ommatidia and the growth and maturation zones has been removed.
(Semidiagrammatic.) Ml, First post-operative molt; M2, second post-operative
molt. Stippled area represents regenerated area.
the wild-type eye are densely packed with large pigmented granules that are
particularly numerous along the length of the rhabdom and just underneath the
cornea. Pigmented granules are located, as well, along the nerve fibers leading to
the optic ganglion. The pigmented exocuticle of the head becomes gradually
thinner as it approaches the cornea.
The margin of the eye shows ommatidia in various stages of maturation.
Those nearest the densely pigmented and presumably functional ommatidia have
lenses and are pigmented on one side only, the side nearest the mature ommatidia. Farther from the mature ommatidia can be seen tightly packed elongated
cells, the long axes of which are oriented at right angles to the body surface.
These cells have large elongate nuclei. The proximal ends of the cells curve
beneath the functional part of the eye.
Farther away from the eye, occasional mitotic figures which are oriented so
that the division products will be at right angles to the body surface, can be seen.
These divisions can be seen in newly molted nymphs as well as in those later in
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C. A. T. H Y D E
Donor
Host
Result
D
iJ
§
Fig. 3. For legend see facing page.
Development of the eye of Periplaneta
Donor
Host
373
Result
K
Fig. 3 (cont.)
Fig. 3. Transplantations between the wild-type and the lavender- and pearl-eyed
mutants. (Semidiagrammatic.) Column 1, donor and source of graft (in rectangle).
Column 2, host and location of graft (in rectangle). Column 3, result. Black eye represents wild type. Vertical bars represent lavender eye. No shading represents pearl
eye. Stipples indicate development of wild-type pigmentation in the pearl grafts
or eye in epidermis that was induced to become a part of the eye. In row A the graft
is to the head of a wild-type nymph from which the eye had been removed.
E M B 27
374
C. A. T. HYDE
Development of the eye of Periplaneta
375
the instar, but have not been observed in areas far from the eye in newly molted
nymphs (Fig. 6).
The mitotic region surrounding the eye will be called the growth zone, and the
unpigmented zone seen in the living animal will be designated the zone of
maturation.
EYE REGENERATION EXPERIMENTS
In order to test the capacity of the eye to regenerate and in order to see
whether the pattern of growth as outlined above could be interrupted, a series of
experiments involving removal of one-half of the mature ommatidia of the eye
was performed. In every case, whether or not the maturation zone was left intact,
extirpation was followed in the succeeding molt by a rapid intrusion of epidermis,
followed, in succeeding ecdyses, by a gradual return of the eye to its normal
shape and size, with ommatidia of normal sizes being formed as outlined above
(See Figs. 2 and 7).
These extirpations were made from the dorsal one-half, the ventral one-half
and the anterior and posterior halves. The only major distinction in the pattern
of returning to normal was that at the first post-operative ecdysis there was a
wider maturation zone at the anterior edge than obtained as a result of the other
operations.
In the event that a few intact pigmented cells were left after an operation, a
new maturation zone formed completely around these cells and an additional
eye developed as a result. (See Fig. 8.)
The eye therefore has great capacity for regeneration, but in an additional
experiment the removal of the entire eye, mature ommatidia, maturation zone,
FIGURES 4-9
Fig. 4. Normal imaginal eye of Periplaneta americana showing the anatomic relationships between the compound eye, the unpigmented ocellus, and the base of the antenna. A maturation zone surrounds the eye of this recently molted specimen, x 10.
Fig. 5. A part of the maturation zone at the dorsal edge of the eye of a newly molted
third- or fourth-stage wild-type nymph showing the spread of pigment granules
into the maturation zone. From a whole mount, x 200.
Fig. 6. Frontal section of eye of newly molted wild-type nymph showing the arrangement of pigment granules along the rhabdom and underneath the cornea, (a)
Growth zone, (b) maturation zone, (c) mature ommatidia.
Fig. 7. Eye of an imago from which the dorsal half of the mature ommatidia was
removed at the third or fourth instar. x 10.
Fig. 8. Eye of an imago from which the dorsal half of the mature ommatidia was
removed at the third or fourth instar and that developed two small dorsal areas of
ommatidia. One of these was posterodorsal, and the other anterodorsal. The anterodorsal one became incorporated into the eye. x 10.
Fig. 9. Right side of the head of an imago from which the entire eye was removed
at the third or fourth instar. x 10.
24-2
376
12
C. A. T. HYDE
Development of the eye of Periplaneta
377
and growth zone was followed by the side of the head being covered completely
with epidermis and exoskeleton typical of the head and there was no regeneration
of the eye (See Fig. 9).
TRANSPLANTATION EXPERIMENTS
The experiments are summarized diagrammatically in Fig. 3. In the first
experiment the eye was entirely removed from animals with normally pigmented eyes. They were allowed to ecdyse twice in order to be certain that the eye
was entirely absent and then a rectangle of tissue from the center of the eye of a
pearl nymph was placed into the location of the original eye. These grafts grew,
gradually changed shape to approach the normal and became pigmented
normally. The pigment appeared first near the edge of the graft and gradually
spread inward. (See Figs. 3 A and 10.)
As a further test, tissues from the eye were placed on the prothorax. These
grafts grew, gradually widening toward the anterior end of the host, and in every
case where a pearl graft was placed into either a lavender or a normal-eyed host
the graft developed pigments. The eyes of pearl hosts receiving grafts of either
lavender or normal tissues did not develop pigments, and lavender eye tissues did
not develop normal pigments, regardless of the host.
An additional test was the exchange of one-half of the eye, including the
growth and maturation zone, between similarly sized individuals of the different
genetic combinations. In every case the pearl half of the eye, whether host or
FIGURES
10-14
Fig. 10. Grafted eye of a wild-type imago from which the eye was completely removed
at the third or fourth instar. After the nymph had molted twice a rectangular graft
from the center of the eye of a late instar pearl imago was placed into the area from
which the eye had been removed. The graft grew, changed shape, and developed
wild-type pigmentation, x 50. (See also Fig. 3 A.)
Fig. 11. The dorsal one-half of the right eye is from a graft from a wild-type nymph.
The wild-type pigment has begun to develop in the pearl half of the eye. The left
eye is unpigmented. x 10. (See also Fig. 3E.)
Fig. 12. Lateral view of a lavender-eyed imago. The dorsal half of the eye was a
graft from a pearl-eyed nymph. The pearl portion of the reconstructed eye has developed wild-type pigmentation which is spreading dorsally. Neither the lavender
portion of the host's eye nor the other eye of the host developed wild-type
pigmentation, x 20. (See also Fig. 3 G.)
Fig. 13. Lateral view of the right eye of apearl imago. The dorsal half of the eye was a
graft from a lavender-eyed nymph when both insects were fifth or sixth instar. The
pearl half of the eye has developed wild-type pigmentation. The lavender portion
of the eye has remained lavender. The other eye of the pearl host did not develop
pigment. x20. (See also Fig. 3F.)
Fig. 14. Late instar pearl nymph that had received a graft of epidermis from the prothorax of a wild-type nymph. The graft was placed far above the eye. Following
the third post-operative molt pigment has begun to develop in the eye of the host
and the line of the graft can be seen, x 20. (See also Fig. 3 M.)
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C. A. T. HYDE
graft, developed normal pigments. The other eye of a pearl host was never
affected. See Figs. 11-13.
The final experiments were the placement of tissues from the prothorax far
above the eye and into the center of the eye. As the eye grew, these tissues were
eventually incorporated into the eye, and when a pearl eye grew into lavender or
normal tissue and this tissue became part of the eye, normal pigments developed.
(See Fig. 14.)
DISCUSSION
It can be readily observed that the eye of P. americana is constantly growing
and maturing since there is a constant maturation of ommatidia all around the
periphery of existing ommatidia. Further indication of this can be seen following
the removal of tissue anywhere around the periphery. New growth and maturation zones appear by the time of the ecdysis succeeding the operation with no
evidence of a scar.
Following complete removal of the eye, no eye regenerates from the remaining
head epidermis, much of which would eventually have been replaced by eye
tissue had the eye remained in place. This epidermis is not determined in its
developmental fate at the time of hatching.
That there is progressive recruitment of cells from the epidermis by the mature
ommatidia is indicated by the fact that epidermal tissue from a normal eyed or
lavender-eyed mutant placed far above the eye of a pearl-eyed mutant is eventually incorporated into the eye and synthesizes pigments that are typical pigments
characteristic of the normal-eyed insect. There is no synthesis of these pigments
until the cells become eye cells. Epidermis that is placed directly within the eye
will heal the wound and is also induced to become eye tissue after which it
synthesizes pigments.
The work of Ross et al. (1964) established the fact that pearl and lavender are
inherited independently as autosomal recessives. The Fx hybrids between them or
with either one crossed with the normal eyed roach had normally pigmented
eyes. It is of interest here that putting eye tissues of these two mutants in conjunction results in the synthesis of normal eye pigments in the pearl mutant tissue.
This plainly indicates the co-operative action of genes in different cells. That this
is a result of direct contact is indicated by the fact that the other eye of a pearleyed mutant is never affected by these grafts.
Two substances, each acting only over short distances by direct tissue contact,
are indicated here: one that induces the epidermal cells to develop as eye tissue,
and a second one that results in the synthesis of the pigments of the eye.
Part of a dissertation presented to the faculty of the University of Virginia. I would like to
thank my major professor, Dr Dietrich Bodenstein, and Drs James Dent and Howard
Hamilton of the University and Dr Horton H. Hobbs of the U.S. National Museum.
I would also like to thank Drs Donald Cochran and Mary Ross, Virginia Polytechnic
Institute, who provided the mutants used in this study.
Development"of the eye of Periplaneta
379
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BODENSTEIN, D. (1959). Contributions to the problem of eye pigmentation in insects: studied
by means of intergeneric organ transplantation in Diptera. Smithsonian Misc. Coll. 137,
23-41.
BODENSTEIN, D. (1962). Humoral Conditions and Cellular Interactions in the Development of the
Insect Eye. Proc. of the Twenty-third Biology Symposium, Oregon State University Press.
LEW, G. T.-W. (1933). Head characters of the Odonata with special reference to the development of the compound eye. Entomologia am. 14, 41-97.
LUDTKE, H. (1940). Die Embryonale und Postembryonale Entwicklung des Auges bei Notonecta
glauca (Hemiptera-Heteroptera). Z. Morph. Okol. Tiere 37, 1-37.
MELLANBY, H. (1937). The later embryology of Rhodniusprolixus. Q. Jlmicrosc. Sci. 79,1-42.
PENZLIN, H. (1963). Uber die Regeneration bei Schaben (Blattaria). I. Das Regenerationsvermogen und die Genese des Regenerat. Wilhelm Roux Arch. EntwMech. 154, 434-465.
Ross, M. H., COCHRAN, D. G. & SMYTH, T., JR. (1964). Eye-color mutation in the American
cockroach, Periplaneta americana. Ann. ent. Soc. Am. 57, 790-792.
WARD, C. L. & HAMMEN, C. S. (1957). New mutations affecting tryptophan-derived eye
pigments in three species of insects. Evolution 11, 60-64.
WITLACZFL, E. (1884). Entwicklungschichte der Aphiden. Z. wiss. Zool. 40, 559-596.
{Manuscript received 19 May 1971, revised 20 October 1971)