BIOLOGY 468/568

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PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY

STUDENT NAME:

Charles Moffett

PROJECT TITLE:

Nematocyst Formation in the Jellyfish Aurelia aurita

Species Identification

• Kingdom: Animalia
  
  
• Phylum: Cnidaria
  
  
• Class: Scyphozoa
  
  
• Order:
  
  
• Family:
  
  
• Genus: Aureila
  
  
• Species: Aurita
  
  

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Key Words:

INTRODUCTION

A characteristic of all cnidarians are batteries of stinging cells called cnidocytes. Within the cnidocytes is the nematocyst which is an inverted tubule armed at one end with a harpoon exhibiting hooks and spines (Watson 1988). Exterior to the capsule projecting from the apical surface of the cnidocyte is the cnidocil, a modified flagellum portraying the typical 9+2 microtubule configuration. This structure acts as a mechanical trigger (Hickman et al., 1988), which when triggered, discharges the nematocyst everting the harpoon-like structure out of the capsule into the tissue of the prey (Brusca & Brusca 1990) (Figure 1). Although a number of ultrastructural studies have been made on the cnidocytes of individual life stages of cnidarians no studies have been conducted on these cells at the different life stages of the organism. The scyphozoan Aurelia aurita illustrates the classical cnidarian life cycle which includes both a benthic scyphistoma and a planktonic medusan stage (Figure 2). The purpose of the present study is to ascertain if there are any developmental differences in the ultrastructural organization of the cnidocytes within these two stages.

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MATERIAL AND METHODS

Medusae tentacles and intact scyphistomae were prepared for TEM by fixing for 45 minutes in phosphate-buffered, 5% glutaraldehyde (pH 7.4). They were then washed exhaustively in three changes of buffer prior to secondary fixation in 1% Osmium tetroxide (90 minutes). Following three twenty minutes washes in buffer, the fixed samples were dehydrated in a graded series of alcohols. The dehydrated samples were washed twice in propylene oxide prior to infiltration and embedding in Spurr epoxy resin.

Sections of a silver/gold interference color were cut on a Sorvall MT IIB ultramicrotome, stained with uranyl acetate (20 minutes) and lead citrate (5 minutes) and examined under a JEOL 1200 EXII TEM at an accelerating 80 kV.

Only medusae were used for SEM. Tenticular samples of tissue, incubated with Artemia to induce nematocyst discharge, were fixed overnight in 5% glutaraldehyde buffered in seawater. Following fixation, the samples were rinsed in seawater, dehydrated in a graded series of alcohols, and dried at 330C using Peldri II . The dried samples were sputter coated with Au/Pd prior to viewing on a digital Cambridge SEM.

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RESULTS

Scyphistomae

FIGURE 1.



The life cycle of Aurelia aurita. (after Brusca and Brusca, 1988).

FIGURE 2.



The four stages of the discharge of a typical cnidarian nematocyst hitting and perforating the cuticle of a prey an everting its filament into the target. (Tardent and Holstein, 1982).

FIGURE 3.



Photograph of schyphistomae polyps showing numerous tentacles funneling towards the mouth. A brownish colored strobila is also visible. Magnification x15.

FIGURE 4.



Cnidocyte showing an oblique section of a fully mature nematocyst. The capsule wall (Cw) appears as a laminated electron dense structure surrounded by a microtubule derived complex (Mt). The whole structure is approximately 9microns in diameter. The ridged filament thread can be clearly seen enclosed within the capsule. Isolated mitochondria and multivesicular bodies occur in the cytoplasm and the nucleus appear ablated.

FIGURE 5

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Nematocysts showing a highly irregular capsular wall (Cw). There is no signs of either lamination nor a microtubular derived coat. Tridactyl sub-structure is visible in some of the filaments (Fi). These filaments appear to be only loosely packaged. Cm=cell membrane of cnidocyte.

FIGURE 6.



High power magnification of a cnidocyte nucleus (Nu) showing signs of degeneration. Circular figures, possibly degenerating mitochondria (Mi) and multivesicular bodies are visible in the cell cytoplasm.


Medusae

FIGURE 7.



Adult medusa of Aurelia aurita.

FIGURE 8.



A mature nematocyst showing a rigid capsule wall (Cw). It is common to see the nucleolus (Nc) in a comparatively dense nucleus (Nu). The Golgi body (Go) from an adjacent nematocyte can also be seen. Fi= filament.

FIGURE 9.



Longitudinal section of a nematocyte showing basally situated nucleus (Nu) beneath mature nematocyst. All the various components comprising the nematocyst including the capsule call (Cw), ornate harpoon (Ha), filament threads (Fi), opercular hatch (Op), cnidocil trigger (Cn) surrounded by a microvillar tuft (Mv). The nematocyst of the adjacent is torn from the section due to mineralization by calcium. An=adjacent nematocyst. Nc=nucleolus.

FIGURE 10.



High power shot of the apical region of the nematocyst. The opercular (Op) structure shows clear striations of alternating electron dense and electron lucent material. The apical surface of the operculum is covered by a amorphous coating of fibrillar material that is contiguous with the microtubule (Mt) derived coat surrounding the capsule. The capsule wall (Cw) is approximately 150 nm in thickness and has an electron dense inner and outer surface. The tridactyl structure of an inverted filament is clearly visible. The "9+2" microtubular structure together with the basal body and the ciliary rootlet of the cnidocil can be seen. The membrane of the cnidocil is contiguous with the amorphous coat of the operculum. Septate junctions separate adjacent cells.

FIGURE 11.



Low power SEM micrograph of medusa tentacle showing numerous cnidocils surrounded by tufts of microvilli. Inset: Higher power shot of a tuft of microvilli and the cnidocil.

FIGURE 12.



Discharged nematocyst lodged in the cuticle of Artemia prey. The harpoon and filament attached to the bulbous capsule can be clearly seen.

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DISCUSSION

No major differences were observed in the ultrastructure of the cnidocyte of the medusa and the scyphistoma. Although each stage performs a different role in the life cycle of the organism both stages have a similar diet of small crustaceans and larval fish (Moffett 1993). It would appear therefore that stage-specific specializations in nematocysts structure are unnecessary. The ultrastructural organization of the mature nematocyst of Aurelia is comparable to those reported for other Cnidarians. The major features are the cnidocil, microvillar tuft, capsular wall, operculum, harpoon and filaments. The observation of a laminated substructure to the operculum has not been previously recorded. The origin, formation, and function of the laminations is currently unknown, but it presumably facilitates the process of nematocyst discharge. The initial template for the development of the nematocyst capsule wall is thought to be from vesicles derived from the trans-cisternal face of the Golgi Apparatus (Watson 1988). These vesicles become later associated with a cage-like array of 50-150 microtubules derived from a centriole pair located near the Golgi which, in some unknown manner, orchestrate the addition of wall material in a location- specific pattern. Two theories have been proposed for the development of the harpoon and filament apparatus: 1) capsule is developed first and the apparatus is subsequently loaded into to it; 2) the apparatus is produced synchronously with the capsule. Figure 5 supports the latter hypothesis that the structures are formed simultaneously prior to the completion of development of the capsule wall.

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CITATIONS

Brusca, G. J. & Brusca, R. C. (1990). Invertebrates. Publ. Sinauer Assoc., Inc.: Mass.

Heeger, T. and Moller, H. (1987). Ultrastructural observations on prey capture and digestion in the Scyphomedusae Aurelia aurita. Mar. Biol. 96, 391-400.

Hickman, C.P., Hickman, F. & Roberts, L.S. (1988). Integrated Principles of Zoology. Publ. Mosby College, St. Louis, MO. pp 184-185.

Holstein, Tardent. (1982). History and current knowledge concerning discharge of cnidae. In: Biology of Nematocysts. Publ. Academic Press Inc., Inc. p. 321.

Moffett, C. W. (1993). The rearing of the Scyphozoan Aurelia aurita. Unpublished.

Schaadt, M. (1993). The life cycle and nematocyst roles in Aurelia aurita. Pers. comm.

Spangenburg, D. (1993). Nematocyst types of scyphistomae in Aurelia aurita.

Watson, G. M. (1988). Ultrastructure and cytochemistry of developing nematocysts. In: The Biology of Nematocysts. Publ. Academic Press Inc., p. 143-161.

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ACKNOWLEDGEMENTS

The authors would like to acknowledge the Mike Schaadt and the Cabrillo Marine Aquarium for the donation of Aurelia specimens. Thanks also go to Tom Douglas of the EMF at CSULB for his help and assistance in this project. This work was made possible by NSF Instrumentation Grant DIR 8820774.

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