BIOLOGY 468/568

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PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY

STUDENT NAME:

Michelle Johnson and Misty Borja

PROJECT TITLE:

An Ultrastructural Study on the Transportation and Intracellular Incorporation of Coelenterate Nematocysts by the Predatory Nudibranch Mollusc Hermissenda crassicornis.

Species Identification

• Kingdom:Animalia
  
  
• Phylum: Mollusca
  
  
• Class: Gastropoda
  
  
• Subclass: Opisthobranchia
  
  
• Order: Nudibranchia
  
  
• Suborder: Facelinidae
  
  
• Family: Facelinidae
  
  
• Genus: Hermissenda
  
  
• Species: crassicornis

Key Words:

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INTRODUCTION

Unlike most other marine mollusks which use either shells or cryptic coloration to provide protection against potential predators, the nudibranch Hermissenda crassicornis utilizes nematocysts for defense. Nematocysts are intracellular structures that contain harpoon-like devices that can be everted rapidly into the predator upon appropriate stimulation (Behrens, 1980). The most unusual aspect of this defense system is that the nudibranch cannot synthesize its own nematocysts but must rely upon acquiring them from its consumed prey which is composed mainly of coelenterates.

Very little is known about the process by which functional nematocysts are selectively transferred from the digested prey into the highly colored, projecting cerata on the lateral and dorsal surface of the mollusc. The prey is swallowed and transported through the gut and into the digestive gland (Wertheim, 1984). During the digestion process, the nematocysts of the prey remain undischarged and are transported via an undetermined mechanism through interconnecting tubules from the digestive diverticula into a sac like structure at the tip of the cerata called the cnidosac.

This research project aims to determine the ultrastructural process by which nematocyst capsules are transported through the digestive system of the nudibranch Hermissenda crassicornis and deposited in the cnidosac of the cerata.

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MATERIALS AND METHODS

A total of twenty individual Hermissends crassicornis were collected from the intertidal zone off Palos Verdes, California in March of 1995. They were transported in four air filled plastic bags, each containing five nudibranchs, to the marine laboratories at California State Long Beach where they were transferred to a 30 gallon glass tank containing aerated seawater. Each nudibranch was isolated within the tank in an inverted finger bowl to deter cannibalism. The animals were contained at a constant temperature of 15o C and were exposed to a 12 hour day/night light regime.

To study the transfer of nematocysts to the cnidosac, the nudibranchs were fed polyps of the moon jelly Aurelia aurita. Preliminary experiments showed these prey items to be suitable for these feeding experiments (Renton, 1994). Polyps of Aurelia aurita were supplied from a tank at Cabrillo Marine Aquarium, San Pedro, California, on 12 x 8 cm Plexiglas blocks. The polyps were maintained at 15o C in aerated seawater and fed weekly with freshly hatched Artemia brine shrimp. The twenty nudibranchs were divided into four treatment groups and a control group each containing four H. crassicornis. Prior to feeding, the cerata of all nudibranchs, excluding the control group, were stroked manually with a finger to stimulate discharge of the resident nematocysts and promote the incorporation of nematocysts from the prey items.

Individual cerata were simultaneously removed from all the nudibranchs 2, 4, 24, and 48 hours after feeding for ultrastructural evaluation. They were fixed immediately upon removal using 10% (v/v) glutaraldehyde in Millionigs phosphate buffer (pH 6.7) for one hour. For the first five minutes, fixation was carried at room temperature (23o C) after which the specimen bottles were placed in a 4o C water bath. Following primary fixation, the ceras were rinsed in 3 x 10 minute changes of phosphate buffer. They were then secondarily fixed in 1% osmium tetroxide in phosphate buffer for one hour. Excess fixature was removed by rinsing in 3x10 min changes of buffer. After dehydration with a cold graded ethanol series, the specimens were returned to room temperature, taken through four fifteen minute changes of 100% ethanol, two fifteen minute changes of propylene oxide, and then infiltrated with Spurr resin epoxy (Spurr, 1966) for a period of thirty-six hours. The cerata were then oriented laterally in flat embedding molds and cured overnight in a vacuum oven set at 65o C. Thick sections (ca. 0.4 (m) for light microscopy and thin sections for transmission electron microscopy were prepared using both glass and diamond knives on either a Porter-Blum MT-2 or a LKB Nova ultramicrotome. The former were stained with freshly filtered 0.5% (v/v) toluidine blue in 1% borax, the latter with lead citrate and uranyl acetate. Bright-field and NIC photomicrographs were taken with Olympus BHS microscope. Transmission electron micrographs were taken using a JEOL 1200 EX II transmission electron miscoscope operated at 60 or 80KV.

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RESULTS

For sake of brevity, the results from the various light and electron micrographs with accompanying legends are summerized in figures 1-16.




Figure 1-3.



Color photographs showing the gross morphology and anatomy of Hermissenda crassicornis (figure 1) and the adult (figure 2) and polyp (figure 3) phases of its prey, Aurelia aurita.

Figure 4a. c.


Schematic drawing of a typical cerata showing the connection of the digestive diverticula, intermediary tubule, and cnidosa

Figure 4b.



High power photograph of a cerata

Figure 5.



Light micrograph showing the longitudinal organization of a cerata of Hermissenda crassicornis. The structure is a celomic filled sac that contains a single tubular branch of the digestive diverticula and the cnidosac.




Figure 6.



Light micrograph of the cnidosac of the nudibranch Hermissenda crassicornis. The cnidosac is found in the apical region of the cerata and is comprised of a pear-shaped, sack-like structure, approximately 100 microns in diameter . Nematocysts can be observed as dark structures.

Figure 7.



Light micrograph of an experimentally fed nudibranch showing the presence of undischarged nematocyts from the consumed Aurelia aurita polyps in the lumen of a digestive tubule 48 hours after feeding.





Figure 8.



Light micrograph of a transverse section across the intermediary tubule connecting the digestive diverticulum to the cnidosac. Nematocyst, apparent as "holes in the section" can be seen within vacuolated cells of the tubule. The nematocysts appear to be acquired through the process of phagocytosis.




Figure 9.



Light micrograph of a longitudinal section of the tubule connecting the cnidosac with the digestive diverticulum.





Figure 10.



Low power transmission electron micrograph of outer mantle epithelium covering the cnidosac. The epithelium is composed primarily of ciliated and mucous cells.





Figure 11.



High power transmission electron micrograph of two cilia showing the longitudinal organization of the microtubule filaments.


Figure 12.



High power transmission electron micrograph of mucous cells showing mucous at various stages of formation and maturation.





Figure 13.



The cnidosac houses numerous vacuolated cells termed cnidocytes which contain the nematocysts.

Figure 14.



Each cnidocyte can contain numerous nematocysts

Figures 15 and16.


Transmission electron micrographs showing both transverse and longitudinal sections across nematocysts in the mature cnidocytes illustrating the typical ultrastructural architecture with an outer capsule, a harpoon, a tether and an operculum




Figure 17.



SEM micrograph of outer mantle epithelium showing clumped cilia.

Figure 18.



SEM of cryofractured cnidosac showing the apical region thought to contain the pore through which the nematocysts are discharged.

Figure 19.



SEM of cryofractured cnidosac showing the nematocysts in the cnidocytes of the cnidosac.

Figure 20

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SEM of cryofractured cnidosac showing the various layers comprising the sac.

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DISCUSSION

This is the first ultrastructural study, involving transmission electron microscopy, to visualize the appropriation of nematocysts by the cnidosac of a sea-slug from its digested prey. Several studies have described the gross anatomy and histology of the relevant structures, epithelia and cell types and in most respects my light micrographs corroborate these earlier findings.

Light micrographs show the cerata of Hermissenda crassicornis to be a celomic filled sac that contains a single tubular branch of the digestive diverticula that communicates apically, via a short ciliated canal, to the cnidosac. The pear shaped sac houses numerous nematocysts which are contained in vacuolated cells termed cnidocytes. The entrance of the sac channel is guarded by a muscular sphincter that can apparently control the passage of material from the digestive gland. At the apical tip of the sac the muscles again converge to form another sphincter or opening that communicates to the exterior and is presumably the route of expulsion of the nematocysts upon discharge. Light microscope evaluations of the time series of the experimentally fed organisms showed the presence of undischarged nematocysts from the consumed Aurelia aurita polyps in the lumen of the digestive tubules of Hermissenda 48 hours after feeding. Transmission electron micrographs of these nematocysts show that they are now ìacellularî and have been digested away from the coelenterate cnidocyte in which they were originally retained. This observation is contrary to the conclusions of Kepner (1943) who noted no evidence of nematocysts being ingested by the digestive gland of the ceras of the aeolid sea-slug, Aeolis pilata.

The incorporation of the nematocysts from the gut lumen into the cnidosac appeared to involve a number of steps. The initial phase of this process involved the cells lining the base of the tubule connecting the digestive diverticulum to the cnidosac. Intact nematocysts were observed within these cells in light micrographs, and, although the structures were removed during thin sectioning, a peri-nematocyst membrane could be observed delineating the nematocyst implying that it was of extracellular origin and had been internalized, presumably by a process such as phagocytosis. Cytoplasmic vacuolation was observed in these newly formed molluscan cnidocytes, the degree of which was correlated to their location in the tubule. Thus, the cells towards the digestive diverticular opening showed the least vacuolation while those at the proximal end, towards the cnidosac, showed the highest degree of vacuolation. This progression is interpreted as being indicative of a process which involves the development of the immature cnidocyte as it migrates proximally into the lumen of the cnidosac.

Electron micrographs of the nematocysts in the mature cnidocytes of the cnidosac show the typical ultrastructural architecture with a harpoon, a tether and an operculum. The similarity between the structure in the prey and host imply that little or no morphological modification has taken place during transportation and incorporation into the molluscan cell. It can be presumed, therefore, that the structures are still capable of discharge and therefore provide for both a defensive and predatory role in its newly acquired host.

The mechanisms which allow for the selective incorporation of the nematocyts by these progenitor cnidocytes in the connecting tubule is unknown at this time. Presumably, the ciliated cells in the epithelium lining the tubule and the muscular sphincters guarding the openings of the tubule play major pivotal roles in this process. Collectively, they could facilitate the directional movement of fluids from the lumen of the digestive diverticulum into the cnidosac. However, this alone, cannot account for the selectivity of uptake. All ultrastructural evidence to date supports the view that the digestion of the host material in the digestive diverticulum is essentially complete and that the only structures refractory to digestion are the nematocysts protected by their hardened capsule shell. If this proposition is true, then the apparent selectivity shown during cnidocyte incorporation may therefore be simply due to the removal of all other particles of suitable macromolecular dimensions and characteristics for phagocytotic uptake. Further studies involving electron dense markers with comparable dimensions to the nematocysts are necessary to resolve the issue.

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ACKNOWLEDGEMENTS

I would like to thank Dr. T. Douglas whose expertise and technical assistance were invaluable. Finally, I would like to thank Dr. A. Mason for his extraordinary patience and support with this project. His talent and skill accompanied by openness and kindness were an inspiration and a blessing.

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CITATIONS

ABBREVIATIONS USED