BIOLOGY 468/568

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PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY

STUDENT NAME:

Bridget Zepp

PROJECT TITLE:

Ultrastructural analysis of the pharyngeal basket of the tunicate Styela montereyensis and the alimentary canal of the endosymbiotic amphipod Leucothoe alata, with emphasis on relationship.

Species Identification:

• Kingdom Animalia
  
  
• Phylum Crustacea
  
  
• Class Malacostraca
  
  
• Subclass Eumalacostraca
  
  
• Superorder Peracarida
  
  
• Order Amphipoda
  
  
• Suborder Gammaridea
  
  
• Family Leucothoidae
  
  
• Genus Leucothoe
  
  
• species alata
  
  
• Kingdom Animalia
  
  
• Phylum Chordata
  
  
• Subphylum Urochordata
  
  
• Order Stolidobranchia
  
  
• Genus Styela
  
  
• species montereyensis

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ABSTRACT

Leucothoe alata is a symbiotic amphipod living in the pharyngeal basket of the tunicate Styela montereyensis. The amphipod alimentary canal was examined by means of: 1) Light Microscopy to distinguish food content, and 2) Transmission Electron Microscopy to determine whether cell structure resembles that of previously studied parasitic and nonparasitic Copepoda. Also, the pharyngeal basket integrity of S. montereyensis and the anterior region of L. alata were both examined using Scanning Electron Microscopy. By comparing the results with previous studies, the amphipod-tunicate relationship is concluded to be nonparasitic.

KEY WORDS:

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INTRODUCTION

Sessile, marine invertebrates that create their own feeding and respiratory currents such as sponges and ascidiaceans are often exploited as hosts by a variety of other animals (Gotto,1959). For instance, the amphipodLeucothoe spinicarpa is known to inhabit the sponge Spheciospongia vesparia. L. spinicarpa is sheltered within S. vesparia and may feed on either the suspended organic particles contained in the water flow or on the host tissue itself (Westinga and Hoetjes, 1981). A similar type of relationship is found between the amphipod Leucothoe alata and the tunicate Styela montereyensis in Alamitos Bay, California.

Although L. alata is known to inhabit S. montereyensis, the precise ecological relationship between symbiont and host is obscure. Specifically, the amphipod nutritional source, alimentary canal morphology, and mode of symbiosis are uncertain. Cytological studies on the alimentary canal are extremely limited in number. Schmitz and Scherrey (1983) used both Light Microscopy and staining procedures to describe the digestive tract of the freshwater amphipod Hyalella azteca. Their results reveal that the digestive tract is differentiated into a foregut, midgut, and hindgut. Due to the limitations of the techniques used, the ultrastructure of each digestive region was not described. Furthermore, their study did not define the nutritional source of H. azteca.


The present study utilizes both Light Microscopy and Transmission Electron Microscopy to clarify the nutritional source and ultrastructure of the alimentary canal of L. alata. Subsequently, a Scanning Electron Microscopy analysis on the pharyngeal basket of S. montereyensis is performed in order to determine wall integrity. The anterior region, particularly the mouth parts, antennules, and antennae, of L. alata was analyzed by Scanning Electron Microscopy to elucidate feeding strategies. Correlations with previous studies may bring light into the amphipod-tunicate relationship.

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MATERIALS AND METHODS

Preparations of samples for Light Microscopy and Transmission Electron Microscopy.

The tunicates Styela montereyensis were collected from Alamitos Bay, Long Beach, California on November 30, 1994 by basic snorkeling techniques. The tunicates were dissected to recover the amphipod Leucothoe alata from the pharyngeal basket. Once recovered, the amphipods alimentary canal was carefully removed. Amphipods were fixed for 1 hr. at 4 C in a mixture containing 2% gluteraldehyde, .12 M Millonig buffer, and 20% sea water, thus bringing the osmolarity to 1200 MOSM and pH 6.8. Subsequently, the amphipods were rinsed for 1 hr. in the same buffer. Then, specimens were post-fixed for 2hr. at 4 C in a solution of 1% osmium tetroxide and .12M Millonig buffer, and rinsed for another 1 hr. in the same buffer. Specimens were dehydrated in a graded ethanol and propylene oxide series. Samples were embedded in a firm Spurr low viscosity resin. Sections were cut with a Sorvall MT2 ultramicrotome.

For Light Microscopy, 1µ sections were stained with Toluidine blue to examine the foregut. Additionally, Light Microscopy was employed to observe dissected mouthparts of L. alata.

For Transmission Electron Microscopy, 1 anstrums sections were placed onto 200 mesh copper grids stained with 2% uranyl acetate followed by lead citrate, and examined in a JEOL 1200 EXII transmission electron microscope.

Preparation of samples for Scanning Electron Microscopy

Styela montereyensis were injected through the oral siphon with 2.5% gluteraldehyde and .2 M sodium cacodylate buffer. Representative samples of the pharyngeal basket with and without associate (control) were then dissected and pinned out in fresh fixative for 1 hr. Specimens were rinsed with buffer and dehydrated in a graded ethanol series. Samples were critical point dried in a LADD 2800 at 1250 psi and 42 C. The specimens were mounted on metal stubs with double sided adhesive tape followed by 3 minutes of gold palladium sputter coat in a Humer I sputter system.


From the pharyngeal basket with associate, a specimen of L .alata was removed and prepared using similar fixation processes as the pharyngeal baskets. Finally, the pharyngeal baskets and L. alata were observed and photographed with a MR1000 Scanning Electron Microscope.

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RESULTS

The alimentary canal ofL. alata is comprised of a straight tube divided into three distinct regions: a foregut, midgut, and hindgut. The foregut was examined through Light Microscopy to determine if the nutritional source contains suspended particles carried in with the water currents created by S. montereyensis. Analysis showed that the foregut was empty. Light Microscopy of L. alata mouth parts showed an elaborate set of mandibles, maxillules, maxillae, and maxillipeds.

The midgut was examined by Transmission Electron Microscopy. Presumably, absorption and digestion takes place in the midgut. The midgut cell structure is composed of microvilli, mitochondria, vesicles, and smooth endoplasmic reticulum (Plate 1 and 2). Microvilli increases the surface area of the gut lumen, thereby aiding in food uptake. Numerous vesicles present throughout the midgut evolved from pinocytic absorption. However, the nature of the ingested material was not resolvable under high magnification.

The pharyngeal baskets, with associate and without associate, were examined by Scanning Electron Microscopy to determine the integrity of the wall structure. Upon examination, the pharyngeal basket is composed of longitudinal vessels and transverse vessels. Both pharyngeal baskets, with associate and without associate, revealed no damage to the wall structure (Plate 3 and 4). However, numerous amounts of debris were present on the pharyngeal basket with associate.

The oral region ofL. alata was also examined by Scanning Electron Microscopy (Plate 5). The Scanning Electron Microscope shows well developed maxillipeds and gnathopods 2. In addition, the antennules and antennae of L. alata are reduced in length as compared to free living amphipods.





PLATE 1 -2. Midgut cross section of Leucothoe alata showing microville (Mv), mitochondria (M), vesicles (V), and smooth endoplasmic reticulum (Ser) Plate 1 X20,000 and Plate 2 X80,000.



PLATE 3. Styela montereyensis pharyngeal wall structure X110.



PLATE 4. Styela montereyensis pharyngael basket wall structure with associate X80.



PLATE 5. Anterior region of amphipod,Luecothoe alata,showinng antennae (A), annetenule (An), gnathopod 2 (G), and maxillapeds (Mx) X350.

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DISCUSSION

Results from the Transmission Electron Microscopy of the alimentary canal and Scanning Electron Microscopy of the pharyngeal basket revealed Leucothoe alata to be nonparasitic. If the relationship would have been parasitic, then the pharyngeal wall would show signs of damage (e.g. tearing by L. alata mouthparts). Scanning Electron Microscopy of the pharyngeal basket with associate indicate no mastication whatsoever. Furthermore, midgut cell morphology observed here resemble those of previous studies on nonparasitic Copepoda (Arnaud, 1978).

Light microscopy of the foregut and Transmission Electron Microscopy of the midgut did not reveal precisely whether L. alata feeds passively on the food particles brought in by the water currents created by S. montereyensis. Nevertheless, L. alatamust accomplish feeding while inside the host. The mucus secreted by S. montereyensis may provide one nutritional source for L. alata. The copious mucus secretions of the host provide a high quality food source and are especially important for smaller symbionts such as amphipods and copepods (Vader, 1983). Possibly, the indeterminate food content within the contractile vacoules may be mucus considering the fact that mucus can be easily absorbed into pinocytotic vesicles. Nonetheless, the nutritional source for L. alata remains unclear.

A full complement of mouthparts were revealed under Light Microscopy. Subsequently, Scanning Electron Microscopy revealed well developed maxillipeds, as well as a size reduction in both antennules and antennae. The maxillipeds are nonconical in shape which further supports the idea of nonparasitism since most families of amphipods known to be parasitic have piercing mouthparts arranged in a large conical bundle (Thomas and Taylor, 1981). Similar to amphipods living in sea anemones, the reduced antennules and antennae may be morphological adaptations for endosymbiosis by reducing entanglement in the mucus (Vader, 1983). Scanning Electron Microscopy also revealed large amounts of debris within the pharyngeal basket with associate. The debris was not distinguishable under high magnification and may be amphipod fecal matter. S. montereyensis may obtain additional nutritional value from the fecal matter of L. alata. I hasten to add, large amounts of debris may clog the filter feeding apparatus.


Further studies need to be performed in order to distinguish the precise nutritional source ofL. alata. For instance, radiolabeled phytoplankton may be fed to S. montereyensis to see whether L. alata actually feeds on the phytoplankton brought in by water currents. Or the mucus lining the pharyngeal basket may be dyed in order to determine if L. alata feeds on the mucus secretions itself.

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CITATIONS

Arnaud, J., Brunet, M., and Mazza J. 1978. Studies on the Midgut of Centropages typicus (Copepoda, Calanoid). Cell Tiss. Res. 187: 333-353.

Gotto, R. V. 1959. An ascidian "hotel". The Irish naturalist' journal 13:99.

Schmitz, E. H. and Scherrey, P. M. 1983. Digestive Anatomy of Hyalella azteca (Crustacea, Amphipoda). J. Morph. 175: 91-100.

Thomas, J. D. and Taylor, G. W. 1981. Mouthpart morphology and feeding strategies of the commensal amphipod, Anamixis hanseni Stebbing. Bull. Mar. Science 31(2): 462-467.

Vader, W. 1983. Associations between amphipods (Crustacea: Amphipoda) and sea anemones (Anthozoa, Actiniaria). Mem. austr. Mus. 18: 141-153.


Westinga, E. and Hoetjes, P. C. 1981. Intrasponge Fauna of Spheciospongia vesparia (Porifera, Demospongiae) of Curacao and Bonaire. Mar. Biol. 62: 139-150.

Acknowledgements

List of Abbreviations Used