BIOLOGY 468/568
PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY
Monday, 22-Apr-1996 15:34:09 PDT
STUDENT NAME:
Roberta Maricich
PROJECT TITLE:
Ultrastructural Comparison of Irridescent and Orange Scales of the Heliconian Species
Dione vanillae
and Observations on Scale Development.
Species Identification
Kingdom: Animalia
Phylum: Arthropoda
Class: Insecta
Order: Lepidoptera
Family: Heliconidae
Genus: Dione
Species: vanillae
ABSTRACT
The metallic scales in
Dione vanillae
have a different structure from the orange scales. The metallic scales have lamellae which act as a thin-film iridescence. This is known as a Morpho type scale. The height of the ridges in the metallic scales (0.53 microns) was greater than that of the orange scales (0.3 microns). The distance between the ribs was less in the metallic scale (0.6 microns) than in the orange scale (1.08 microns). Although the distances between the top of the ridges was approximately the same (1.85 microns metallic, 1.8 microns orange). Extremely small development of the pupal stage scale was seen on day two, but the ridge on day 4 was more developed with a distinct layer of cuticle.
Key Words:
TEM,
Dione vanillae
, scale development
INTRODUCTION
Much work has been done on the morphology and ultrastructure of butterfly scales. Each scale of a butterfly develops from a single specialized epidermal cell into a flattened cuticle. The scale cell then continues to develop into a standard scale structure (Fig 1b). These scales provide a wide spectra of color for the butterflies either through pigmentation and/or structure.
It is the variation in this standard structure of the scale which gives
Dione vanillae
of the Heliconidae Lepidoptera its metallic silver spots on the underside of their wings (Fig. la). The type of structural differences compared to a standard orange pigmented scale along with development of these scales is the subject of this study. The metallic silver color in the scale is considered a white structural color. This color is obtained when the light is reflected off the colorless cuticle having specific pattern of structures in the scale. This may be due to thin-film interference from lamellae, microrib, or a number of other variations in the structures of the scales.
This investigation will identify the type of structure responsible for the metallic color in the adult scales, compare this structure to that of the orange pigmented scales of this species and address the formation of the scales in pupal stage from two to four days.
MATERIALS AND METHODS
Larvae of
Dione vanillae
were reared and observed for pupation. After pupation they were allowed to develop for two and four day periods. At this time, the developing wings were dissected in 3% glutaraldehyde in phosphate buffer on ice to obtain rapid tissue fixation (Greenstein 19'71). Sections of an adult wing were also prepared by carefully scrapping the scales off one side of the wing leaving either the desired metallic or pigmented scales.
The specimens were then fixed for one hour in a primary fixative of 3% glutaraldehyde in Phosphate buffer followed by fixation in 1% phosphate buffered osmium on ice. Specimens were then washed and dehydrated with a series of washes with 30%, 50%, 75%. and 95% ethanol. This was followed with a longer rinse in 100% ethanol and two ten minute changes in propylene oxide. The specimens were then infiltrated using a series of rinses. First with a 2:1 mixture of propylene oxide:Spurr resin, second a 1:2 mixture of propylene oxide:Spurr resin and finally two periods of four hours in 100% Spurr resin. The specimens were then placed in embedding molds with Spurr (1969) resin. Some manipulation was performed at this stage to obtain the desired orientation of the adult scales. The molds were left in the vacuum for two days. Ultrathin sections were cut using an ultramicrotome and glass knives. Celloidin coated grids were carbon stabilized and used when working with ultrathin sections from the adult scales due to the scales tendency to pull away from the resin. The grids were stained in uranyl acetate for 3.5 minutes and lead citrate for 2.5 minutes. They were then observed on a JEOL-1200EXII TEM at 80kv.
RESULTS
The cross section of the adult metallic and orange scales showed characteristic features of typical lepidopteran scales. (Figs. 2 and 3). These structures include a lower and upper lamina which are connected by trabeculae that are found under the ribs. The ribs are part of the upper lamina and are connected perpendicularly to the ridges that run longitudinal to the scale axis. The lower lamina is flat.
The adults metallic scales were found to differ in a few ways from that of the orange pigmented scales. It should be noted that the pigment granules did not show in the orange scales due to their tendency not to fix well with standard fixation (Ghiradella 1974). The metallic scale is much larger in appearance to that of the orange scale (Fig. 2 and Fig. 3) The distance from ridge to ridge is approximately the same in the metallic scale (1.85 microns) and orange scale (1.8 microns). However, the distance of the ribs between ridges is a shorter distance in the metallic scale (0.6 microns) while much larger in the orange scale (1.08 microns). The height of the metallic scale ridge (0.53 microns) is shorter than that of the orange scale (0.3 microns).
The most significant difference found was the features on the side of the ridge of the scale. The silver had a great many lamellae (Fig. lc and Fig. 5). The trabeculae between the lower and upper lamina of the silver metallic scale also had many more trabeculae remnants than did the orange scale. The pupal scales at two days do not show an obvious development of the scale. There is a shadow of an outline for the start of a cuticle layer at this stage (Fig. 7). The pupal stage at the fourth day shows a very defined line of lower and upper cuticle layer (Fig. 8). The development of a ridge is much more apparent.
Explanation of Figures.
FIG 1.
FIG1a. The underside of the hindwing of
Dione Vanillae
showing the approximate placement of the metallic scales. FIG1b. A single specialized cell represents a scale. The scale is composed of an upper lamina (UL), lower lamina (LL), the ridge (R), ribs (RI), and trabeculae (T). (c) The lamella of the ridge which give the aetallic scale its thin-film iridescence.
FIG 2.
Shows the cross section of an orange scale. Note the same general structure as seen in the metallic scale. The ridges in this scale do not contain lamellae as in the metallic scale. X12,000. Bar = 0.5 microns.
FIG 3.
Shows the cross section of a metallic scale. This shows the upper lamina (UL) and lower lamina (LL). On the upper lamina are the ridges (R) and the ribs (RI) . On the ridges are the lamellae (L) which make the interference reflector responsible for the metallic color. The trabeculae (T) can be seen between the upper and lower lamina. X12,000. Bar = 0.5 microns.
FIG 4.
Shows a transverse section of a metallic scale. X20,000. Bar = 0.2 microns.
FIG 5.
Shows detail of a ridge of a metallic scale. Notice the many lamellae (L) on the side of the ridge. X30,000. Bar = 0.2 microns
FIG 6.
Shows detail of a ridge of an orange scale. The ridge (R) on this scale is much less complex than the metallic scale ridge. X30,000. Bar = 0.2 mcirons.
FIG 7.
Day 2 of the pupal stage. Shows a section through the very start of a ridge. The darker lines on top and bottom show the f irst sign of a cuticle layer (CU) developing. X40,000. Bar = 0.2 microns.
FIG 8.
Day 4 of the pupal stage. Shows a section through a ridge along in further development. The cuticle layer (CU) is even more apparent. X20,000. Bar = 0.2 microns.
DISCUSSION
The metallic scales of the adult
Dione vanillae
, differ structurally from the orange scales giving them their characteristic metallic color. The presence of parallel ridges called lamellae on the main ridges of the scale makes an interference reflector. This is known as thin-film iridescence and is not found on the pigmented orange scales. The larger size of the metallic scale may also have something to do with this white metallic color. When the scales were removed by scrapping it was noticed the metallic scales needed slightly more pressure and effort to remove them from the wing. Perhaps this was due to the size difference in the scales.
The metallic scale differs from the orange in distance between the ridges where the ribs start, but the two scales are approximately the same distance from the tops of the ridges. This shows much more space is used by the metallic ridge. This would appear most likely due to the need for space from the lamellae present on the ridges. The trabeculae of the silver metallic scale also seemed to have more remnants thus perhaps a greater structure of the trabeculae is present then in the orange scale. This could also reflect light adding to the formula for the metallic color.
The development of the wino scales in
Dione vanillae
from day two to day f our proceeds rather rapidly. The cuticle layer and the start of a ridge can be seen in day two of the pupal stage (Fig. 7). The cuticle layer and start of the ridge is much more apparent on the fourth day of pupal development (Fig. 8). These examples of development, however, are not known to represent either a thin-film iridescent scale or a standard pigmented scale. The pupal wing was sectioned at random. The observation of development to the fourth day of the scale shows rapid development in these scales as compared to other Lepidopteran scale (Greenstein 1972).
It would have been fascinating to compare the development of the two different types of scales. This would entail a delicate dissection of the pupal wing. Perhaps the outline of the veins of the wing could be used to approximate the location of the developing reflective scales. There was no obvious difference in appearance on the pupal wings at day two and four. This suggest that the differentiation of the scale happens towards the end of development in the scales (Ghiradella 1974).
CITATIONS
Ghiredella H., 1974. Development of UV reflection butterfly scales how to make an interference filter. J Morph., 142: 395-409.
Ghiradella H. and Radigan W. 1976. Development of butterfly scales Part 2. Struts lattices and surface tension. J Morph., 150: 279-295.
Greenstein, M. E. 1971. The ultrastructure of developing wings in the giant silkmoth, Hyalophora cecropis I. Generalized epidermal cells. J. Morph., 136: 1-22.
Greenstein, M. E. 1972 .The ultrastructure of developing wings in the giant silkmoth, Hyalophora cecropis. II. Scale forming and socket-forming cells. J. Morph., 136: 23-52.
Spurr, A. 1969. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastructure Res. 26: 31-43.