J. Embryo I. exp. Morph. Vol. 61, pp. 35-49, 1981
Printed in Great Britain © Company of Biologists Limited 1981
35
Blastokinesis in embryos of the bug,
Pyrrhocoris apterus.
A light and electron microscopic study 1. Normal blastokinesis1
By EUGENIE C. ENSLEE 1 AND LYNN M. RIDDIFORD 2
From the Biological Laboratories, Harvard University, Cambridge,
and Biology Department, Seton Hall University, South Orange, New Jersey
SUMMARY
In the bug, Pyrrhocoris apterus, blastokinesis (a reversal of the position of the embryo
within the egg) is seen to involve contraction of the serosa that is attached to the embryo's
head. As the serosal cells change from squamous to columnar in the course of blastokinesis,
a dense zone of microfilaments appears just under the apical surface. Many apical protrusions
develop above this zone. After the embryo is in its final position the zone disappears and
later the cells degenerate.
Laterally, the serosal cells are connected by belt desmosornes, septate junctions and gap
junctions. As blastokinesis progresses, more lateral surface is recruited below them from the
original basal surface.
Microtubules running parallel to the plasma membrane are seen near the apical microfilaments and along other surfaces of the cell. Secretory granules are evident both within
serosal cells and along the apical surface, probably providing a lubricant for movement against
the chorion. Yolk cells are common basal to the serosa, possibly mobilizing nutrients for it.
This study of blastokinesis in Pyrrhocoris provides a dramatic example of cell shape change
that is correlated with the appearance of microfilaments. In its details blastokinesis is comparable to morphogenetic events such as amphibian neural tube formation and ascidian
metamorphosis.
INTRODUCTION
Blastokinesis (katatrepsis phase) results in a reversal of the insect embryo's
position within the egg. Recent studies of Hemiptera have indicated that this
change involves the pulling of the embryo by a sheet of epithelial cells, the
serosa, which is attached to the head of the embryo (Cobban, 1968; Enslee &
Riddiford, 1977). During blastokinesis, this squamous epithelium contracts into
* This paper is a portion of a dissertation submitted to the graduate school of Harvard
University, in partial fulfilment of the requirements for the degree of Doctor of Philosophy
(Enslee, 1973) and has been presented in part at the American Society of Zoologists meeting
(Enslee & Riddiford, 1973).
1
Author's present address: Department of Anatomy, College of Medicine and Dentistry
of New Jersey, New Jersey Medical School, 100 Bergen Street, Newark, N.J. 07103, U.S.A.
2
Author's present address: Department of Zoology, University of Washington, Seattle,
Washington 98195, U.S.A.
36
E. C. ENSLEE AND L. M. RIDDIFORD
a small knob of columnar cells. This has been observed in numerous hemipterans
(Butt, 1949; Cobban, 1968; Mellanby, 1936; Seidel, 1924). A contractile pulling
function was proposed for the serosa-over a century ago (Brandt, 1869, cited
by Sander, 1976), but no attempt to relate cell structure and function has appeared
in the literature. In other orders of insects other parts of the egg, especially the
embryo, have been implicated, reflecting the different organization of the eggs
studied (reviewed by Enslee & Riddiford, 1977).
The following light and electron microscopic study of blastokinesis in embryos
of the linden bug, Pyrrhocoris apterus, indicates the appearance of an apical
zone of microfilaments in the serosal cells during this process. The relationship
of this zone to adjacent apical and lateral cell surfaces is consistent with a contractile role. This contraction would account for both the pulling action of the
serosa and the dramatic change in the shape of its constituent cells, comparable
to what has been found in many other systems (Baker & Schroeder, 1967;
Burnside, 1971; Cloney, 1966; Pollard & Weihing, 1974; Schroeder, 1976;
Wessells e* a/., 1971).
MATERIALS AND METHODS
For collection of timed batches of fertilized eggs, mating pairs of Pyrrhocoris
adults were kept under an 18 L:6D photoperiod at 30-5 °C, 60-70% relative
humidity (Enslee & Riddiford, 1977). Eggs at appropriate developmental stages
were selected by means of a brief immersion in 95 % ethanol which rendered
the chorion transparent. This treatment had no adverse effect on subsequent
embryonic development. For both light and electron microscopy, each stage was
represented by at least three individuals.
The eggs were dechorionated and then fixed in 3 % glutaraldehyde for light
microscopy serial sections as described previously (Enslee & Riddiford, 1977)
Thick sections (1-2 fim) of the material prepared for electron microscopy were
used for extensive supplemental observations. For electron microscopy, eggs
were fixed at room temperature according to the glutaraldehyde-H2O2 method
of Perrachia & Mittler (1972) using 6% glutaraldehyde in 2 mM-CaCl2, 0-1 M
sucrose and 0-1 M phosphate buffer (pH 7-4). For rapid fixation the chorion
of the submerged egg was pierced with a minutien and the hole was quickly and
carefully enlarged. As soon as the tissue surfaces were slightly rigid, the chorion
was removed. Final dissection and gross observation were done in the buffer
wash (2 mM-CaCl2, 0-616 M sucrose, 0-1 M phosphate buffer, pH 7-4) just prior
to the osmium tetroxide fixation step (2% OsO4 in 0-1 M phosphate buffer,
pH 7-4 with 2 rriM CaCy. Just before dehydration the eggs were stained en bloc
with 0-5% uranyl magnesium acetate in 0-9% NaCl for 2h. Then they were
embedded in fipon.
Sections were taken from the anterior polar region, in the median plane.
Some near-tangential sections were also prepared. Thin sections were stained
with 7-5% uranyl magnesium acetate for 3 h (Frasca & Parks, 1965) and with
Blastokinesis in embryo o/Pyrrhocoris apterus. /
A
B
37
C
Fig. 1. Semidiagrammatic longitudinal sections through embryos, showing changes
in position as blastokinesis progresses. Embyo (A) is typical of a stage shortly before
the process begins. Embryo (B) is turning around. Embryo (C) has reached its final
position. The anterior pole of the egg is always at the top. Eggs are approximately
1 mm in length, ab = abdomen; am = amnion; ap = appendage; asb = amnionserosa border; hd = head; s = serosa; yk = yolk. The germ band (embryo) is
stippled. (This figure is adapted from one in Enslee & Riddiford (1977). Permission
has been obtained from the publishers to reprint it.)
0-35% lead citrate for four minutes (Reynolds, 1963). All micrographs are of
double-fixed Epon-embedded specimens. Toluidine blue staining with phasecontrast optics and an orange (No. 23A) Wratten filter was used for photomicrographs and a Siemens Elmiskop I for electron micrographs.
RESULTS
Changes in the serosal cells during blastokinesis
The changing positions of the Pyrrhocoris embryo and serosa during blastokinesis are diagrammed in Fig. 1 (Enslee & Riddiford, 1977). The serosa changes
from a squamous (Fig. 2 A) to a low columnar epithelium (Fig. 2B) as the
embryo's head moves to the anterior pole. When the head is crossing the anterior
pole, the serosal cells are about five times as high as wide (Fig. 2C). When the
head is in its final position, the serosa invaginates into the yolk with its cells
becoming flask-shaped (Fig. 2 D). In this form it is called the dorsal organ (Cobban,
1968). Shortly afterward, the lateral surfaces of the embryo come together in
the dorsal midline (dorsal closure), and the dorsal organ degenerates.
Throughout blastokinesis the cytoplasm of serosal cells is metachromatic.
The nuclei have conspicuous nucleoli and little heterochromatin. Osmiophilic
yolk granules and clear vesicles are common features of these cells. As the cells
become columnar, the yolk granules are found more often in the basal halves
of the cells whereas small clear vesicles are more common just apical to the
centrally located nuclei (Fig. 2C). A new population of giant clear vesicles
dominates the mid-region of the cells after dorsal-organ formation (Fig. 2D).
With phase-contrast optics, a smooth dense line just below the apical surface
oftheserosa is evident during blastokinesis. (Fig. 2B). Above this line the surface
38
E. C. ENSLEE AND L. M. RIDDIFORD
"- TV v ir
Fig. 2. Light micrographs of serosa (A) just prior to and (B) about midway through
blastokinesis; (C) as the head of the embryo is moving across the anterior pole of the
egg and (D) as the serosa (now the dorsal organ) sinks into the yolk. Osmiophilic
yolk granules, clear vesicles and nuclei are visible in the cells. A dense apical zone
(bracketted in B) is present after blastokinesis begins. Bar 50 /«n. d = degenerating
cells; gb = germ band; s = serosa; y = yolk mass; yc — yolk cell; * = location
of Fig. 8.
of the cells is ruffled. The line is sharpest as the embryo nears the anterior pole;
then as the head moves across the pole and the serosa invaginates, interruptions
and ripples are seen in this zone (Fig. 2C, D). In order to elucidate more clearly
these changes, it was necessary to turn to a study of the ultrastructure of the cells.
Fine structure of the serosa during blastokinesis
1. Preblastokinesis
Just prior to blastokinesis the serosal cells are flat. Patches of punctate or
filamentous material are sometimes found in the apical cytoplasm (Fig. 3 A).
Microtubules lie parallel to all cell surfaces.
Adjacent cells are connected by three organelles. Near the apical surface is
a belt desmosome, with parallel membranes thickened on their cytoplasmic
surfaces and very thin intercellular bridges. Basal to the belt desmosomes is a
zone of septate junctions and occasional gap junctions (Fig. 3 A, B). The desmo-
Blastokinesis in embryo o/Pyrrhocoris apterus. /
39
some and junctional region are often convoluted and sometimes interrupted
by a vesicle-like intercellular space. (Junction terminology conforms to Satir &
Gilula, 1973).
2. Blastokinesis
During blastokinesis the apical surface became increasingly ruffled above the
dense line seen with the light microscope (Fig. 4 A, B). These cytoplasmic protrusions sometimes contain organelles such as mitochondria and rough endoplasmic
reticulum (RER). The dense zone itself is composed of two components: (1) a
layer of microfilaments and microtubules running parallel to the egg's surface;
and (2) convolutions in the belt desmosome on the cell's lateral plasma membranes (Fig. 4B). The folds of these lateral membranes nearest the filamentous
layer are distinctive in that they are almost parallel to the surface. The microfilaments appear to merge with the lateral membranes or their dense cytoplasmic
sides and also criss-cross the bases of apical protrusions. To ascertain the
orientation of microtubules and microfilaments within the planar apical zone,
sections were cut nearly tangential to the serosal surface. Some of the microtubules are aligned parallel to, or curved along, a lateral margin while others
appear to lie randomly (Fig. 5). Microfilaments sometimes occur in bundles or
lie in one dominant direction (Fig. 5), but no constant pattern was evident.
Perfectly tangential sections could not be obtained due to the large size of the
cells and the curvature of the egg but examination of many slightly oblique
sections indicated that microfilaments are present throughout most of the cell
apex. Possibly their density is variable.
The junctional zone continues to be prominent as the cells become more
cuboidal. However, extensive non-junctional surface appears laterally, and the
basal surface area decreases. No distinct boundary separates the basal and the
non-junctional lateral surfaces. The spaces between adjacent cells are irregular
and filled with a flocculent material (Fig. 4A).
In contrast to cell-surface configurations, the internal cytoplasmic organelles
undergo no dramatic changes during blastokinesis. The RER appears to become
more abundant and is occasionally seen in stacked cisternae, especially late in
the process. At all stages small granules, about 70 to 110 nm in diameter, are
seen throughout the cell in Golgi complexes and other vesicles (Fig. 6), and on
the apical surface of the cell, particularly among the protrusions (Fig. 4B).
Microtubules are common near the plasma membrane, tending to be parallel
to it, not exclusively in the apical zone.
3. Late blastokinesis
As the head starts to traverse the pole, the serosal cells become laterally
compressed (Fig. 2C). The junctions between cells, perpendicular to the egg
surface, become much longer. Below the often convoluted septate junction, the
facing membranes appear scalloped (Fig. 6). Most of the cell surface is now
40
E. C. ENSLEE AND L. M. RIDDIFORD
Blastokinesis in embryo o/Pyrrhocoris apterus. /
41
lateral as the basal surface has been shifted up and laterally. The apical surface
is ruffled above a somewhat less distinct zone of microfilaments and microtubules. In one-quarter to one-half of the sampled cells of a typical individual
undergoing blastokinesis, a zone that contains only free ribosomes is found
either lateral or basal to the nucleus; when the head is crossing the pole, it
appears to be only basal. In one egg, where the embryo's head was about threefourths of the way towards the anterior pole, filamentous material was seen in
this region (Fig. 7).
4. Formation of the dorsal organ
When the embryo's head is in position covering the pole of the egg, the serosa
assumes a concave form as it invaginated to form the dorsal organ. Its flaskshaped cells (Fig. 2D) are still connected in their apical halves by the convoluted
lateral zone, including belt desmosomes and septate junctions. An apical band
is distinctly seen by phase-contrast optics, but not in thin sections, possibly
because the extreme concavity of the apical surface precludes a section ideal for
displaying the components. Some of the giant clear vesicles seen in mid-cell in
Fig. 2D are found to be invaginations of extra-cellular space. Among and basal
to the flask-shaped cells are found disintegrating cells (Fig. 8). Somewhat later
during dorsal closure of the embryo, this dorsal organ region is occupied by
more-or-less spherical cells showing abundant evidence of degeneration pycnotic nuclei, swollen RER, dense inclusions, and membranous whorls (Fig.
9). In some, the cytoplasm is very condensed. Intercellular associations and
structural asymmetry are lacking or diminished. Adjacent to these cells are
various healthy cells of the developing embryo.
Yolk cells
Yolk cells are defined as any cell observed near the serosa but never in direct
contact with the egg surface and never joined with a serosal cell by a septate
junction or belt desmosome. Two types of yolk cells were observed: (1) a thin
FIGURES 3 AND 4
Fig. 3. Electron micrographs of serosa just prior to blastokinesis, showing adjacent
cell surfaces. (A) Gap junctions are sometimes found. (B) Near the apical surface
is a belt desmosome, basally, a septate junction. Breaks in membrane apparently
occur during removal of closely appressed chorion. Bar 0-5 /*m. bd = belt desmosome; gj = gap junction; mt — microtubule;^ = serosal cell; sj — septate junction; y = yolk cell.
Fig. 4. Electron micrographs of serosal cells when embryo's head was about threefourths of the way toward anterior pole (corresponds to Fig. 2B). (A) Overview,
(B) closeup of apical protrusions and apical zone of microfilaments, microtubules and
belt desmosomes (arrows in (A)). Bar 10/tm. ap = apical protrusions; bd = belt
desmosome; / = extracellular flocculent material; yg = yolk granule; circles
denote microtubules.
42
E. C. ENSLEE AND L. M. RIDDIFORD
Blastokinesis in embryo o/Pyrrhocoris apterus. /
43
cell which extends long and sometimes convoluted processes along the basal
surface of the serosa; it is a constant feature in pre- or early blastokinesis (Fig.
3B); (2) a thicker cell whose cytoplasm displaces the large osmiophilic yolk
granules (Fig. 2C). This latter cell type is often seen near the tip of the abdomen
before and during early blastokinesis. Then, as blastokinesis progresses several
are usually found directly under the thickening serosa (Fig. 2C). In electron
micrographs these cells display a distinctive fenestrated appearance (Figs. 4A,
7 and 8). The internalized membranes and spaces are judged to represent surface
on the basis of the similarity of flocculent material in them and in the intercellular
spaces; however, the surface of the yolk cell immediately below the serosa rarely
appear to have invaginations. Thus, the continuity of the internal and external
zones is uncertain. Mitochondria and RER are common in the cytoplasmic
trabeculae, and a Golgi apparatus is sometimes seen.
During blastokinesis, the spaces between the serosa and the yolk cells vary
from broad to very narrow, even in the same embryo. By the time the head is
moving across the pole, serosa and yolk cells are packed closely together (Fig. 7).
At no time is a basal lamina or other extracellular fibrous material visible.
DISCUSSION
A contractile function for the serosa
The following observations of our ultrastructural study are consistent with
the hypothesis that the serosa is an active participant in moving the embryo
through blastokinesis.
(a) An apical zone of microfilaments becomes conspicuous in serosal cells
when the head of the embryo is moving from the posterior toward the anterior
pole of the egg (see Fig. 10). The zone is remarkably taut, parallel to the surface
of the egg.
(b) Above this zone the apical surface is thrown into many protrusions.
(c) Microtubules are seen parallel to the surfaces of the cell at all stages.
FIGURES 5-7
Fig. 5. Section nearly tangential to the surface of a serosal cell in an egg where the
embryo's head is about half-way to the anterior pole. Both parallel and randomly
oriented microtubules and microfilaments are present. Bar 10/*m. ap = apical
protrusion; mf= microfilaments: arrows denote microtubules.
Fig. 6. Section through the serosa late in blastokinesis showing intercellular zones
below the septate junctions. Bar 10/tm. yg = yolk granule; sec = vesicle with
secretory granules.
Fig. 7. Slightly oblique section showing the base of the serosa in an egg where the
embryo's head was three-fourths of the way to the anterior pole. A zone of free
ribosomes (r) is evident. Small arrows mark a filamentous mat. Large arrows denote
the overall orientation of the base (b) of the serosa. Bar 20/*m. / = extracellular
flocculent material; s = serosal cell; yc = yolk cell.
44
E. C. ENSLEE AND L. M. R I D D I F O R D
^^WQlvvJ.jft
Fig. 8. Basal portions of bottle-shaped serosal cells (s) in an invaginated dorsal
organ. Degenerating cells (dc) are present within and outside of the dorsal organ
in the yolk cell (yc) region. Arrows denote general orientation of bases (b) of serosal
cells. Bar 10 /tin. dse = dorsal surface of the embryo.
Fig. 9. Degenerating serosal cells in an embryo which is completing dorsal closure.
The loss of asymmetry is a prominent feature of these cells. Bar 10 /tm. pn =
pycnotic nucleus.
(d) Laterally the cells are joined by belt desmosomes and septate and gap
junctions. Prior to blastokinesis, the entire lateral surface consists of these
specialized zones. During blastokinesis, unspecialized basal surface is shifted
laterally to a position below the junctional zones.
These features of the serosa bear a striking resemblance to several other
systems. During tail resorption in certain ascidians, cells of the tail epidermis
become contractile. These cells develop a zone of apical microfilaments which
are oriented primarily in the axis of contraction - parallel to the tail axis (Cloney,
1966). In the folding neural plate of amphibians (Baker & Schroeder, 1967;
Burnside, 1971), morphogenesis is correlated with the tapering of the constituent
cells as the microfilaments appear in a ring in the cells' apices. Some of the
serosal microfilaments of Pyrrhocoris run in parallel arrays and others describe
Blastokinesis in embryo o/Pyrrhocoris apterus. /
45
During blastokinesis
Fig. 10. Diagrammatic representation of changes in the serosal cell during blastokinesis. Arrows denote location of diagrammed cells. Magnification of the two
cells is the same except that the maximum width of the preblastokinesis cell would
probably be greater than what is shown. As the cell elongates, the apical surface is
thrown into protrusions (ap) above an apparently contractile band of microfilaments (m/). Cells are held together by a lateral zone consisting of a belt desmosome
(bd), a septate junction (sj) and gap junctions (not shown).
small arcs. Some are seen in the bases of apical protrusions as in the ascidian
tail epithelium. The protrusions are consistent with the existence of very small,
irregular fields in the apical surface, delimited by contractile elements which are
connected to the plasma membrane. The serosal cell apex may be covered with
such fields, each microfilament bundle being a side of one field or of two adjacent
fields. After contraction had occurred, an apical protrusion would have bulged
upward as the field perimeter narrowed around its base.
The apical protrusions seen in the amphibian cells are not very conspicuous.
The ascidian tail epithelial cells, where the microfilament zone is throughout the
apex, develops many protrusions like those in the serosa, except that they are
somewhat more elongate.
Desmosomes are commonly associated with microfilaments and apparently
serve as anchoring sites (Satir & Gilula, 1973; Burnside, 1971). In the Pyrrhocoris
46
E. C. ENSLEE AND L. M. RIDDIFORD
embryos a fold in the serosal belt desmosome usually is oriented in line with the
apical microfilament zone. Thus the microfilaments seem to be actively pulling
against it.
Additional support for the intrinsic contractile nature of the serosa comes
from the cases of juvenile-hormone-exposed embryos described by Enslee &
Riddiford (1977). In such embryos the serosa commonly breaks during blastokinesis but contracts anyway, even though the embryo does not complete
blastokinesis. Thus the embryo is not compressing it.
But contraction of the apical microfilaments of the serosa may not be the
only force pulling on the embryo, especially in the later stages of blastokinesis.
The amnion or the amnion-serosa junction may be especially important then.
Invagination, which the serosa undergoes at the end of blastokinesis, is the
expected result of continued apical contraction (Lewis, 1947). But if only the
cell apices were contracting, the invagination would occur at the centre of the
anterior pole and the head of the embryo would not move across the pole.
Instead, the head does move across the pole, changing its position relative to
the extraembryonic membranes (Enslee & Riddiford, 1977). A band of filamentous material was detected in the basal region of a serosal cell as the head
approached the pole (Fig. 7), but its role and that of the amnion-serosa junction
in these complex movements remain to be clarified.
Microtubules appear to maintain a constant relationship to the plasma
membrane throughout blastokinesis and probably serves a cytoskeletal function.
Apically they always were associated with the microfilaments which may indicate
an interaction between the two during movement.
The septate and gap junctions seen between the serosal cells are indicative
of intercellular communication (Satir & Gilula, 1973; Lowenstein, Kanno &
Socolar, 1978). Combined with desmosomes, these are typical of insect epidermis
(Caveny, 1976; Poodry & Schneiderman, 1970), follicle cells (Mahowald, 1972),
and salivary glands (Oschman & Berridge, 1970; Lowenstein, 1975); hence, it is
not surprising to find them in this important extraembryonic membrane as well.
The terminal stage of blastokinesis is marked by cell death in the serosa (Figs.
8 and 9). It is likely, then, that some of the synthetic activity evident in the serosa
cells (active nucleus and abundant polyribosomes) was directed toward forming
hydrolytic enzymes to be activated when the cells had carried out their primary
function (Novikoff, Essner & Quintana, 1964; Wattiaux, 1969).
The environment of the serosal cell
Secretory granules are found both within and at the apical surfaces of the
serosal cells, and flocculent material is seen in the basal and non-junctional
lateral regions. The latter may have come from either the serosa or the yolk
cells. The function of both of these materials may possibly be lubrication. Since
the serosa was moving against the inside of the chorion under considerable
pressure of the egg contents, a lubricant would facilitate progress.
Blastokinesis in embryo 0/Pyrrhocoris apterus. /
47
Another possible function for the basal flocculent material is nutrition for the
serosa. The yolk cells are similar, though not identical, to cells of the perineurium
in insect ganglia (Smith, 1967). The perineurium cells have been credited with
transporting food to the cells of the ganglion which are remote from the blood
(Wigglesworth, 1960). Like the yolk cells they are fenestrated and have processes
which they send into the tissue they are feeding. The absence of basal lamina
beneath the serosa is consistent with this idea. Johannsen & Butt (1941) assert
that the function of the yolk cells, or vitellophages as they are sometimes called,
is to transform the yolk so it can be assimilated by the embryo. In the hemipteran,
Gem's paludum insularis, these vitellophages have also been recognized as progenitors of the midgut epithelium (Mori, 1976). The absence of a basal lamina
is of further interest because in the ascidian tadpole metamorphosis (Cloney,
1966) the basal lamina split off from the epithelial cells at the start of tail resorption, and remained with the underlying muscle cells as the epithelium moved
away. In contrast, the basal lamina was gradually forming as neurulation
progressed in the amphibian Taricha torosa (Burnside, 1971).
Significance of contractile serosa
The serosa has been shown to consist of contractile cells containing a taut
apical zone of microfilaments whose time and site of appearance correspond to
the presence of a constricting force. It thus resembles other epithelia in which
cells are changing shape (Schroeder, 1976), especially in a short time. Like the
epidermis in the collapsing tail of the ascidian tadpole (Cloney, 1966), it is
characterized by very rapid and extreme cell shape changes. The ascidian
Amaroucium completes tail resorption in about six minutes. The major posteriorto-anterior movement of blastokinesis takes less than an hour (Enslee & Riddiford, 1977). In contrast, the remodelling of the amphibian neural plate requires
many hours (Burnside & Jacobson, 1968), and cell shape changes are not so
dramatic.
Contraction of microfilament zones is a fundamental process in morphogenesis. In speed and degree of cell shape change, serosal contraction in Pyrrhocoris and ascidian tail resorption appear to represent extreme expressions of
the phenomenon.
We wish to thank Dr Oscar AuerbacH for the use of his electron microscopy facilities and
supplies at the Veterans Administration Hospital, East Orange, N.J., and his colleagues,
especially Dr Julio Frasca and Terry Parks for invaluable assistance; Dr Christopher Woodcock for technical guidance early in the research; Dr Beth Burnside for photomicroscopy
facilities; and Drs Richard Cloney, John Edwards and Peddrick Weis for reading the
manuscript.
This research was supported by funds from the Milton Fund, Harvard University and from
the National Science Foundation to L. M.R., and Biology department funds at Seton Hall
University to E.C.E.
48
E. C. ENSLEE AND L. M. R I D D I F O R D
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{Received 30 April 1980, revised 4 June 1980)