BIOLOGY 468/568
PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY
Monday, 22-Apr-1996 15:34:31 PDT
STUDENT NAME:
Scot Hink.
PROJECT TITLE:
Comparative electron microscopic investigation of the mucous gland cells of two Spionidae, (Annelida, Polychaeta), with emphasis on their possible role in tube particle selection.
Species Identification
Kingdom: Animalia
Phylum: Annelida
Class: Polychaeta
Order: Errantia
Family: Spionidae
Genus: Polydora (bosc), Psuedopolydora (Czerniavsky)
Species:
Polydora ligni
(Webster), Pseudopolydora paucibranchiata (kuda)
ABSTRACT
The mucous gland cells of two different Spionidae,
Polydora lignii
and
Pseudopolydora paucibranchia
, were investigated by scanning and transmission electron microscopy and compared with respect to location and cellular morphology. The mucous glands may have some bearing on the particle size selection made by each species for tube construction.
The regular mucous gland cells were elliptical in shape and occurred scattered throughout the entire length of the organisms. They were filled with 0.05-3.5 micron mucous granules. Mucous gland cells were generally embedded between the epidermal cells and the basal lamina. Mucous granules of various sizes and densities were observed in both species and may simply represent stages in the growth of the cells.
The results of the mucous gland cell comparison indicate that both species share similar gland structures. While the results do not support the hypothesis that different mucous glands or granules from the worms body influence tube particle size selection, it also doesn't support the size model either since the worms used were of similar size. These results may simply indicate that tube particle selection is a function of the palps and the mucous glands contained within those structures, and not a function of the segmented mucous glands.
Key Words:
Polychaeta, Transmission Electron Microscopy, Scanning Electron Microscopy, Spionidae,
Polydora ligni
,
Pseudopolydora pausibranchia
INTRODUCTION
Spionid polychaetes are common inhabitants of shallow marine habitats and estuaries around the world. They are an important member of the benthic community and therefore an important food source for larger marine inhabitants. Although their importance is known, little is known about their ultrasructure and cellular morphology and its possible link to tube building behavior.
Tubes are essential for the survival of many species of polychaetes. This was first demonstrated in the lab by Mortenson (1944). While Mortenson provided a crude description of the tube building process of
Polydora lignii
, no exact mechanism was provided for the building process, only that the palps were a means of particle selection. In later studies, an elaborate mechanism for particle selection was suggested which was based primarily around the idea of particle size as the chief agent of particle selection (Dorsett, 1961). Dorsett suggested that the palps pick up the particles and they are directed toward the mouth. It is here that the particles are selected. The small particles pass directly into the gut and the other larger particles are used for tube building. These results are also supported by (Dauer
et. al.
, 1981). These results suggest a relation to the size of the species as an indicator to particle selection. In fact, other studies have shown that larger worms select larger particles (Whitlatch, 1974). The size of the worm is based on its length and the size difference may cause some morphological restraint such as mouth size or palp size. This restraint may regulate the particle sizes used in tube building (Whitlatch, 1974). The problem with this mechanism is that the average sizes of different worms such as
Polydora lignii
and Pseudopolydora paucibranchia overlap.
The average length of Polydora lignii is 3-10 mm (Hartman, 1936). The average length of
Pseudopolydora paucibranchia
is 4-7 mm (Light, 1978). If the previously stated mechanisms were to be used in regards to tube building, then one would expect a great deal of variance in the tube materials used for each worm depending on its maturity. If this is not the case then the mechanism for particle selection must be different. This is not to say that particle size is not the selective agent. Size selectivity in deposit feeders is unequivocal (Luckenbach
et. al.,
1988). This only means that something other than morphological size may be at work.
Another particle selection model was suggested in which the selection is done at the palps and is a function of the mucous adhesion strength (Jumars et. al., 1982). The results of this study show that selection can occur at the point of pick-up, without transporting the particle for later selection. Other studies have also shown results which support this model. The adhesive strength of the tentacular mucus of the spionid polychaete
Scoleleptis squamata
has been hypothesized to be critical in capturing and retaining particles (Dauer, 1983). The main significance of these studies is that it could indicate hat the mucous, and hence cellular morphology, of the species may be just as important with respect to particle selection as is the size or ultrastructure of the species.
The particle sizes used in tube construction must be selected, and they must also be held together. In order to hold the tubes together and maintain their integrity, the segmented mucous glands on the worms body are used to cement the particles together after they are placed on the rim of the tube (Dorsett, 1961). Dorsett also suggested that the segmented mucous glands were used to line the tube This finding was later supported by Dauer (1985). The significance of these findings is that the mucous from the segmented mucous glands on the body may also play a role in the determination of the tube particle size.
The intent of this research is to study the mucous glands of two different polychaetes of similar size, which build two different tubes, to discover if the morphology of the mucous glands or mucous itself might play a role in the particle selection for the tubes. At the same time this research is intended to further the knowledge of the processes of mucous synthesis and storage in mucous gland cells.
MATERIALS AND METHODS
The polychaetes
Pseudopolydora paucibranchiata
, and
Polydora lignii
were collected on November 15th, 1994 during low tide. Both species were collected in Los Angeles county from the southwest shore of Alamitos bay approximately 1.5 km from the entrance. The substrate selected was from a depth of approximately .5 meters below the surface. The substrate was filtered through a 1.0 mm mesh sieve and the remaining material was taken back to the lab.
At the lab the various tubes were placed in a petri dish and manipulated under a dissecting microscope. The worms were coaxed from their tubes and placed in petri dishes containing clean sea water. The worms were identified using Light (1978) and with the assistance of
Dr. Tom Douglass.
Eight worms of each species were collected and fixed whole in 10% (v/v) gluteraldehyde in Millonig's phosphate buffer (pH 6.7) for 1 hour (Douglass and Jones, 1991). Fixation was carried out in a 4 degree Celsius water bath. The worms were then rinsed 3 times in Millonig's phosphate buffer for 10 minutes each, post fixed in 1% (v/v) OsO for 1 hour and again rinsed 3 times for 10 minutes each (Douglass and Jones, 1991). The worms were dehydrated with a cold graded ethanol series and held in 100% cold ethanol for dissection (two of each species held for TEM preparation).
The dissection was done in cold 100% ethanol under a dissection microscope. Each worm was cut into three equal sections consisting of the prostomium section, the mid section and the pygidium section. During dissection the specimens were brought to room temperature and then taken through four 15 minute changes of 100% ethanol, followed by two 10 minute changes in propylene oxide. Infiltration consisted of three 8 hour changes in a 2:1 mixture of propylene oxide and Spurr resin, a 1:2 mixture, and 100% Spurr resin respectfully. The specimens were embedded using flat embedding molds with the sections being oriented for transverse cuts. The specimens were cured for 16 hours at 70 degrees Celsius in a vacuum oven.
Ultrathin sections were done using a thermal advance microtome. The sections were stained for 4 minutes each with lead citrate and uranyl acetate. Micrographs were taken using a Joel JEM 1200 EX2 transmission electron microscope.
Specimens for the scanning electron microscope were kept whole and taken directly from 100% ethanol into the critical point drying process. The whole specimens were then sputter coated and viewed on an Amray 1000 scanning electron microscope.
RESULTS
The scanning electron microscopic investigation focused primarily on obtaining and displaying the size differences of the tubes of each worm and also the size of the worms themselves. The tube constructed by
Pseudopolydora paucibranchiata
(fig. 1A) was found to contain material up to 50 microns in length, however the majority of the particles appear to be in the range of approximately 10 microns or less. The tube of
Polydora lignii
(fig. 1B) contains material up to 250 microns in length, with the majority of the particles in the 50-75 micron range. A crude estimate of particle size ratio was determined to be 5:1 in favor of the tube of
Polydora lignii
. Backscatter techniques were also employed to get a rough estimate of tube material used in both tubes. The results showed that although the
Pseudopolydora paucibranchiata
tube (fig. 1A) appeared to be constructed more of organic material, it was mostly silica based (observed). The
Polydora lignii
tube (fig 1B) was also observed with backscatter techniques and also found to be primarily silica based (observed).
The sizes of the worms were then compared to determine their ratio. The diameter of
Pseudopolydora paucibranchiata
was measured from figure 1C and was found to be 100 microns. The same measurement was also done on
Polydora lignii
using figure 2B and found to be 160 microns. This gives worm size ratio of 1.6:1 again favoring
Polydora lignii
. The length of the worms could not be determined in this same manner, but both worms were within the average ranges given by Hartmann (1936) and Light (1978), (observed).
The overall morphology of the two worms was quite similar as shown in figure 1C and figures 2A-C. Their setae and the mucous extrusion visible in figure 1C compares closely to that in figures 2B and 2C. Unfortunately the palps, important in tubebuilding, did not survive the fixation in the
Pseudopolydora paucibranchiata
specimen. This limited the comparison to only the segmented body in the scanning electron microscopic investigation and the mucous glands contained in them in the transmission electron investigation.
The cuticle of the worms was quite similar. Figures 3 and 4, which show the cuticle of
Pseudopolydora paucibranchiata
and
Polydora lignii
respectively, show only subtle differences. The microvilli of
Pseudopolydora paucibranchiata
(fig. 3) are approximately 0.8 microns in length and are densely packed (14-15 per micron). The cuticle of
Polydora lignii
(fig. 4) shows microvilli of slightly longer length, approximately 1.0 micron, and much less dense (10-12 per micron).
The mode of mucous delivery was similar in both worms. The most common delivery system was that of the apical sac. Variations of the apical sac were visible in both worms (fig. 6A-C). Pores in the epithelium were also present in both worms (observed). Figure 6D is representative of this formation.
Secretory granules were visible in the epithelium of both worms. The structure varied from granular to filament-like and from electron dense to almost transparent (plates 3-9). The granules varied in size from 0.05 micron (fig. 6B) to approximately 3.5 microns (fig. 8). The shapes of the secretory granules varied from worm to worm but also within the same worm (plates 3-9). Figure 8 shows the variation within the same worm. The secretory granules appeared scattered throughout the epidermal tissue but mostly near the surface in both worms (fig. 4-5). Mucous gland cells containing filament-like secretory products in close association with the golgi apparatus were found primarily in
Polydora lignii
(fig 7A-B). The vast majority of secretory granules found in
Pseudopolydora paucibranchiata
resemble the granules shown in figures 9A and 9B.
Transmission Electron Microscopy
Fig. 1. A-B. Contrasting tubes of the polychaetes. C, Full view of
Pseudopolydora paucibranchiata
.
(A) Section of the tube of
Pseudopolydora paucibranchiata
(455x);
(B) Section of the tube of
Polydora lignii
(195x);
(C) Full view of
P. paucibranchiata
showing setae (se) and mucous secretions (mu)from the segmented body. The prostomium (pr) is shown without palps and the pygidium (py) has been removed to show cross section of body (305x). 20kv operating voltage.
Fig. 2. Body segments of
Polydora lignii
.
(A) Prostomium segment showing tentacular palps (tp) with medial ciliated groove (cg) on palp (715x);
(B) Mid section showing setae (se) on segmented body (625x);
(C) Pygidium of
P. lignii
(925x). 20kv operating voltage.
Fig. 3.
Epithelial tissue of
Pseudopolydora paucibranchiata
. Observe the microvilli (mv) and the cilia (ci) projecting from the epidermal cells. Also observe the actin filaments (af) of the microvilli, the rostral rootlet (rr) of the cilia, the mitochondria (mt) and the scattered secretory granule (sg) within the epithelial tissue (48000x). 80kv operating voltage.
Fig. 4.
Epithelial tissue of
Polydora lignii
. Observe the microvilli (mv) and the cilia (ci) projecting from the epidermal cells. Also observe the actin filaments (af) of the microvilli, the rostral rootlet (rr) of the cilia, the mitochondria (mt) and the scattered secretory granule (sg) within the epithelial tissue (48000x). 80kv operating voltage.
Fig. 5.
Epithelial tissue of
Pseudopolydora paucibranchiata
. Observe the various scattered secretory granules (sg) and the apical sac (29000x). 80kv operating voltage.
Fig. 6. Extrusion of secretory products from the mucous glands.
(A)
Polydora lignii
. Observe the release of the mucous granules (mu) from the microvilli (mv) enclosed apical sac (as) of the mucous gland (29000x);
(B)
Pseudopolydora paucibranchiata
. Translucent secretory granules (sg) within the structure among the microvilli (mv) (60000x).
(C)
Polydora lignii
. Observe the relatively empty apical sac (as) and the secretory granules (sg) within the cell (6000x).
(D)
Pseudopolydora paucibranchiata
. Pore (po) in the epidermis (7250x). 60kv operating voltage for A and C. 80kv operating voltage for B and D.
Fig. 7. Secretory granules of
Polydora lignii
.
(A) Observe the various densities of the secretory granules (sg) and the close association with the golgi apparatus (ga) within the cell. Also observe the cell relative to the basal lamina (bl) (9500x).
(B) Observe the non-granular, filament like structure of a secretory granule (sg) from fig 7A (145000x). 60kv operating voltage for fig. 7A. 80kv operating voltage for fig. 7B.
Fig. 8.
Secretory granules of
Pseudopolydora paucibranchiata
. Observe the overall granular structure, and also observe the oblong shape of secretory granule (sg) b compared to secretory granule (sg) a. (12000x). 80kv operating voltage.
Fig. 9. Magnified images of the secretory granules from fig 8. Observe the filament structure (fi) surrounding each granule. Also observe the overall electron dense granular nature.
(A) (145000x).
(B) (72500x).
80kv operating voltage.
DISCUSSION
Mucous cell extrusion was observed and found to be similar in both species. The apical sac was the primary means of extrusion and is nearly the same as the apical sacs observed by Crawford and Chia (1974). Cuticular pores (fig. 6D) similar to those seen by Hausmann (1982) were also observed but most extrusions were from the apical sac. No transitory forms between the secretory product and secretory material were observed in either polychaete. This is in agreement with Storch and Welsch (1972).
The mucous cells and mucous granules with respect to location and morphology show a great diversity among the cells and granules. This diversity was apparent not only between the two spionids, but also within each worm itself (Hausmann, 1982; Storch and Welsch, 1972). No determination could be made as to whether different cells and granules were being observed, such as the different cells seen by Hausmann (1982) or the differences were merely stages of maturation, the conclusion drawn by Storch and Welsch (1972).
There were also no apparent characteristic arrangements of mucous cells or granules that would indicate a conclusive difference between various cells. The only exception was the mucous cells with the golgi associated filament-like granules that were observed mainly in
Polydora lignii
. This may indicate a difference in mucous granules, or it may be related to the limited observations made.
Limited observations of the ultrathin sections on the transmission electron microscope was of the problems encountered that could have influenced the results. This limitation was the product of the time limit in which to complete this study. With more time, more conclusive results could be obtained. Another problem involved literature research. The limited time meant less depth in research which proved costly. After preparing the specimens, new studies were found which indicated that the palps were the selective instruments (Jumars
et. al.,
1982). While this doesn't rule out the segmented mucous glands from playing a role in tube building, it does seem to indicate that the palps are the area to focus on.
The entire study did not prove inconclusive. The observations and results which show the tube size ratio (5:1) compared to the body size ratio (1.6:1) seems to indicate that body size may not be the only selective factor involved in tube particle selection. This is true provided that the palp size roughly corresponds to the body size. The disparity between the tube particle sizes of the similar sized polychaetes doesn't fully support the mechanical models of particle selection which state that size is the critical selective agent (Luckenbach
et. al.,
1988).
There is a need for future research to explain the discrepancy of tube particle sizes of similar polychaetes. The focus of the study should be on palp morphology of
Polydora lignii
and
Pseudopolydora paucibranchiata
, and the fine structure of the mucous glands contained in the contact area. It would also be beneficial to test the adhesion of the mucous obtained from the palps.
In conclusion, there is an obvious difference in tube construction with respect to particle size in the two polychaetes studied. With the morphology of the different worms being so similar, it seems as if the mucous cells may play a role in the tube particle selection. Due to the high degree of variation among the mucous cells, it may be difficult to determine the role even if the mucous cells of the palps are observed. However, since the mucous cells of the palps were not observed, no conclusion in regards to their role in selection can be determined. While the results do not support the role of the mucous glands in tube particle selection, the results also do not fully support the size model of particle selection either.
CITATIONS
Crawford, B.J. and Fu-Shiang Chia. (1974). Fine structure of the mucous ell in the sea pen,
Ptilosarcus guerneyi
, with special emphasis on the possible role of microfilaments in the control of mucous release. Canadian Journal of Zoology. 52:1427-1432.
Dauer, D.M. (1983). Functional morphology and feeding behavior of Scolelepis squamata (Polychaeta Spionidae). Marine Biology. 77: 279-288.
Dauer, D.M. (1985). Functional morphology and feeding behavior of
Paraprionospio pinnata
(Polychaeta Spionidae). Marine Biology. 85: 143-151.
Dauer, D.M., C.A. Maybury and R.M. Ewing. (1981). Feeding behavior and general ecology of several spionid polychaetes from the Chesapeake Bay. Journal of Experimental Biology and Ecology. 54:21-38.
Dorsett, D.A. (1961). The behavior of
Polydora ciliata
(Johnst.). Tube-building and burrowing. Journal of the Marine Biological Association U.K. 41:577-590.
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List of Abbreviations Used:
af = Actin Filament
as = Apical Sac
bl = Basal Lamina cg =
Ciliated Groove
ci = Cilia
ga = Golgi Apparatus
mu = Mucous
mv = Microvilli
po = Pore
pr = Prostomium
py = Pygidium
rr = Rostral Rootlet
se = Setae
sg = Secretory Granule
tp = Tentacular Palp
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