ABSTRACT
Iridophores in Sepisteuthis lessanians were studied bu light
microscopic and ultrestructural techniques in order to identifu the
component of the cell responsible for iridescence. The thin film
interference was considered as a possible explanation for iridescence. This
hypothesis was rejected when the source of iridescence was isolated and
identified as a diffraction grating. These results suggest that the dorsal
iridophores could vary their degree of iridescence by changing the
conformation or spacing of the grooves making up the grating.
INTRODUCTION
The skin of the squid, Sepisteuthis lessanians, displays a wide range
of body patterns. These colorful patterns are thought to be useful in
camouf lage and visual communication (Hanlon, 1982). Iridophores are cells
that work in conjunction with chromatophores to change the outward
appearance of the squid mantle. As their name indicates, iridophores are
iridescent: theg are capable of giving off colors. This rainbow color effect
seems to be caused by the organization of platelet-like structures of high
refractive index within the cell (Brocco and Cloneg, 1980).
Iridophore cells are found all over the squid mantle in the third
dermal lager. They exist in two types: Physiologically active iridophores
found on the dorsal side of the squid are capable of varging the wavelength
of the color theg iridesce. Theg can be stimulated in vitroby acetylcholine
or by the Catt ionophore 423 187 (Cooper and Hanlon, 1986). Cells on the
ventral side, however, are incapable of such modulation and are
subsequently called inactive.
Although some work has been done in elucidating the mechanism by
which iridescence is modified (Cooper et al, 1990), few studies have been
aimed at explaining "iridescence" as such. This paper describes a studg of
the structure in iridophore cells responsible for this phenomenon.
HATERIALS AND HETHODS
Cell tissue preperstions Sepiateuthis lessonisnswere obtained from
the University of Texas Medical Branch at Galveston and kept in a 120 liter
tank with 21-22°C water. The squid were fed dailg and maintained in
healthy condition until they were used. Unless specified skin preparations
were made in the following manner: Live squid were anesthetized in a small
dish containing sea water and 1-22 EtOH solution. The squid were
decapitated, and an incision was made along the middle of the ventral side
of the mantle. The internal organs were removed, and the skin was pinned
out on a sylgard covered petri dish (chromatophores up). The preparation
was immersed in cold artificial seawater (450mM Nacl, 1OmM Caclz, 1Om
KCI, 5OmM NgC12,1OmM HEPES, pH 7.8) to prevent the skin from desiccating.
The first two dermal lagers including the chromatophore lager were
removed with forceps and the iridophore lager was used.
Morphologicel studies. All the iridophore tissue observed was treated
for at least 15 min with 18 Triton X-100 in KGE (KGE: 3M potassium
gluconate, 33M glycine, 2mM magnesium chloride, 2mM EGTA, pH 6.8).
Unused cell tissue was preserved at sub-zero temperature KGE with 508
glycerol and 18 Triton. Both preserved and fresh tissue were examined
under transmitted light and incident light. Pictures were taken using a
35mm Olympus or a Nikon camera mounted directly to the microscope.
Iridophore cells were also video taped as theg were illuminated from
different angles (optical fibers were used). Video taped data was analyzed
and printed using a MegaVision image processor.
Folerized light snalysis. Iridophore cells and platelets were observed
under a polarized light microscope. The 40X lens was used.
Differentisl Interference Centrast. Iridophore cells were examined
with DIC under a 40X lens. DIC was set on max extinction and then on either
side of max extinction.
Staining Cell tissue was washed with cold KGE and stretched out on
a microscope slide. The tissue was stained with the nuclear stain DAPI for
3 min, cover-slipped and observed under the fluorescence microscope. Some
tissue was also stained for actin with rhodamine phalloidin.
Frotesse digestion 12 solutions of trypsin, collagenase tupe A1,
papain, pronase, and dispase were prepared in Eppendorf tubes. Tissue
samples pretreated with 12 Triton X-100 were digested at room
temperature for two hours. Pictures were taken on the inverted microscope
at various intervals.
Effect of calcium concentration Iridophore cells pretreated with
Triton X-100 were washed with KGE (EGTA is a calcium chelating agent). A
piece of the tissue was then glued to a microscope slide and cover-slipped.
The cells were illuminated with incident light coming from a fixed lamp.
The cells were filmed as a high calcium (Cat2 was 1OuM) solution of KGE
was perfused through the preparation. Video taped data was analuzed on the
MegaVision image processor.
Platelet isolation. A piece of iridophore tissue, preserved by the
method described above, was washed with KGE and spread out on a
microscope slide. Platelets were mechanically teased awag under the
microscope. The KGE solution containing iridophore platelets was
centrifuged. Platelets were dehgdrated by resuspending and centrifuging in
302, 402, 502, 602, 702, 802, 902, 952, and finally 100 EtOH. The
platelets were then centrifuged once more and resuspended in
hexmethgldisilazane. Dehgdrated platelets were examined under scanning
electron microscopq.
RESULTS
Cell morphology. Microscopical examination of the iridophore lager
revealed elongated, football-shaped cells about 170um long and 50um wide
(Figure 1). Within these cells there were rodlike structures parallel to the
long axis of the cell. Platelets were approximatelg Soum long, but they
varied in length. When incident light was shone on the same piece of tissue
from different directions cells iridesced onlg if they were oriented with
their long axis perpendicular to the direction of illumination (Figure 2).
Freservation methad Iridescent cells were very well preserved in
KGE with 18 Triton X-100 and 508 glycerol.
Petergent Extrectien Triton X-100 extraction dissolved enough of
the membranous components to improve microscopical observation.
Extended treatment (more than two hours) of iridophores with detergent
caused the platelets to curl up. Eventually the platelets completely lost
their organization but in themselves appeared to remain intact. Such
platelets were still iridescent.
Frotesse digestions Platelets were fragmented upon digestion while
the control platelets kept in the same conditions without protease remained
the same. Trypsin, pronase (Figure 3), and collagenase tupe A1 were most
effective at desintegrating the platelets into smaller pieces whereas papain
and dispase were least effective. When observed under incident light, the
digested platelets were still reflecting light (Figure 4) and the reflection
corresponded to pieces of platelets.
Polerized light anslysis Both the connective tissue and individual
platelets were birefringent.
Differential Interference Contrast DIC revealed different indexes of
refraction within the Triton-treated cell (Figure 5).
Steining DAPI staining showed that the center of the cell, which
appeared devoid of platelets, is the site of the cell's nucleus (Figure 1).
Rhodamine phalloidin stain identified filamentous actin in the connective
tissue below the iridophores cells (Figure 6). Platelets did not stain for
actin.
Effect of incressing calcium concentration Increasing calcium
concentration did not appear to have ang effect on the platelet shape. The
degree of reflected light appeared to have remained unchanged (Figure 7).
Scanning electren microscepu Scanning electron microscopy of
individual platelets revealed ribbon-like structure. The platelet is about
50-70um wide with regularlg spaced grooves on one side. The groove
spacing was bimodal, with intervals of .73um or 1.34um (Figure 8).
DISCUSSION
Iridescence in squid and other cephalopods was thought to come as a
result of thin film interference of incident light rags (Denton and Land
1971) or of a diffraction grating: where light diffracts from the edges of
parallel platelets that are on edge relative to the skin surface (Brocco and
Cloneg 1980; Cloneg and Brocco 1983). In this studg evidence suggest that a
diffraction grating is indeed the source of iridescence, but on a smaller
scale than that described by Brocco and Cloney--namely within each
individual platelet (figure 9).
Simple examination of the iridophore under incident light is
indicative of a highlg organized cell. The cells were found to iridesce only
if light came from a specific direction. Besides, observation of tissue with
polarized light revealed that components in the cell were birefringent. This
suggests that whatever is responsible for iridescence must be arranged in a
very particular wag.
The platelets, almost alwags parallel to the long axis of the cell,
were assumed to be the elements responsible for the directional specificitu
Attempts to destrog iridescence by disorganizing the platelets within the
cell failed. Roger Hanlon already noticed that platelets curl up, or
disorgänize themselves upon extended exposure to Triton X-100. Howeyer
results of this studg show that such cells still give off rainbow colors; thet
still iridesce. Furthermore digestion of detergent treated cells with
proteases disintegrated the platelets to an even greater degree. This
confirms Hanlon's statement that the platelets are made up of proteinaceous
material. Surprisingly, the digested material was also iridescent (Figure 4).
It became clear that what was necessary for iridescence was the materia
of which the platelets are made and not the platelets as a whole or their
organization.
Examination of an individual platelet revealed a ribbon-like structure
with regular striations on only one of its sides. This is highly reminiscent
of a conventional diffraction grating and leaves little doubt as to the source
of iridescence. The platelet thickness was plotted versus its relative
frequency (graph 1). The ribs of the grating appear to come in two preferred
widths: .7Zum and 1.34um. Using the diffraction grating formula, a sins=m
(a=space between grooves, 8-angle of incidence with respect to the normal,
m=interference maxima, and A=wavelength) and substituting .73 and 1.34
for a, 1 for m, the range of 0 for which visible light (400-700nm) is given
off is 17-31° and 33-72° respectivelg. These values confirm that the
structures observed under scanning electron microscopy are capable of
creating visible light of different wavelength. This calculation predicts the
absence of iridescence when light is shone from directlg above and when De
17°. Transmitted light (light coming from directly above) fails to produce
iridescence which provides additional support for the diffraction grating
model.
The general approach in this studg consisted of breaking up the
components in the cell in order to identify the component responsible for
iridescence. The question remains to be answered: How are these striated
ribbon-like structures put together and arranged in a three dimensional
cell? The structures are 50-70um wide and can can attain manq times that
size in length. The cell, we will recall, is 170um long and about 5Oum wide.
It is possible that the ribbon-like structures weave up and down and back
up, with the strietions parallel to the long axis of the cell. This would
explain the 50um (notice that this is also the width of striated ribbon) the
rod-like structures—termed platelets. The platelets would actually be part
of a larger convoluted ribbon. This would also be supported by the fact that
the cell is only iridescent if incident light is coming from a direction
parallel to the short axis of the cell. Diffraction gratings work best when
the light is perpendicular to the grooves. Thus such arrangement could be
possible but more evidence is needed to confirm it.
How can squid regulated their iridescence? They mag have developed
a method of changing the spacing between the grooves of its diffraction
grating. It has been reported that platelets change thickness upon
stimulation with acetylcholine (Cooper et al. 1990). In that report tissue
was observed with transmission electron microscopg which mag have
obscured three-dimentional information. The changes in thickness of
platelets and space between platelets could be interpreted as a stretching
and contracting of the diffraction grating. This report suggests that the
distances between the grooves of the grating range between.73 and 1.34um
The space between the platelets reported by Cooper et al. was about .25 um.
Considering that the second value was made on fixed cells, which mag have
undergone some artifactual changes, it is possible that these two
measurements refer to the same thing-to the variation of the separation
between the grooves. The stretching and contracting of the squid
diffraction grating could then possibly involve an acto-myosin contractile
system as is suggested by the presence of filamentous actin in the
connective tissue underneath the platelets. All these questions and mand
more remain to be answered in further investigations.
ACKNOWLEDGEMENTS.
I would like to thank my advisor, Dr. Stuart Thompson all for his help
Special thanks also to Dr. Daniel Mazia for his advice and encouragement
throughout the quarter. I would also like to thank Bruce Hopkins for defying
the extinction of squid in the Monterey Bay and providing me with some
specimens. Many thanks also to als members of the Thompson lab: to Sam
Wang for his good choice of ambiance music, to Chris Mathes for sense of
humor, and of course to my crazy lab friends Mandy Schivell, Shari Gelber,
and William Timmins. I am also grateful to Roger Hanlon for his support and
advice, to all the Hopkins Marine Station faculty for making this class
possible, and to my dear housemates for a wonderful house atmosphere
think.. that's it.
FIGURES.
Figure 1. Iridophore cell treated with 18 Triton X-100 solution and stained
with DAPI; bar: 12.5um.
Figure 2. Iridophore tissue illuminated with (from left to right): transmitted
light, incident light from north, east, south, and west respectively; bar:
sooum.
Figure 3. Iridophores pre-treated with Triton X-100 after 2 hour digestion
with pronase; bar: 25um.
Figure 4. Same tissue as fig. 3 but with incident lighting.
Figure 5. Iridophores pre-treated with Triton X-100 under DIC; bar: 100um.
Figure 6. Iridophore tissue stained with rhodamine phalloidin; bar: 100um.
Figure 7. Iridophore tissue with (from left to right): no Ca+ 1OuM Ca+for
min, sOum Ca+ for 20min; bar: 100um.
Figure 8. Isolated platelets under SEM. Left: smooth side. Right: striated
side; bar: 5um.
Figure 9. Isolated platelet closeup; bar: 5um.
O
frequency
GRAPH
Spacing Between Platelet Grooves
20
o1

04
12 1.3 1.4 1.5 1.6 1.7 1.8 1.9
space between grooves (microns).
WORKS CITED.
Brocco, S. L. and R. A. Cloney. 1980. Reflector Cells in the Skin of Otopus
Defleini Cell Tissue Res. 205: 167-186.
Cloney, R. A., Brocco, S. L. 1983. Chromatophore Organs, Reflector Cells,
Iridocytes and Leucophores in Cephalopods. Am. Zool 23: 581-592.
Cooper, K. M., Hanson, R. T. 1986. Correlation or Iridescence Changes in
Iridophore Platelet Ultrastructure in the Squid Lolinguncula brevis
Exp. Biol. 12 1: 451-455.
Cooper, K. M., Hanlon, R. T., Budelmann, B. U. 1990. Physiological color
change in squid iridophores. Cell and Tissue Res. 259: 15-24.
Hanlon, R. T. 1982. The Functional organization of chromatophores and
iridescent cells in the body patterning of Loligo Plei(Cephalopoda
Myopsida). Malacologia 23(1): 89-119.
FIGURE
FIGURE
FIGURE 3
FIGURE 4
FIGUF
FIGURE 6
FIGURE 7
FIGURE 8
FIGURE 9