Oysters in the Classroom: Hunting for Hemocytes

forms, function, and microscope techniques

Introduction:

Mollusks, like many other invertebrates, have an open circulatory system that does not confine hemolymph to traditional vessels like veins, arteries, and capillaries. Instead, an open circulatory system will circulate hemolymph through a number of cavities and sinuses in various parts of the organism. In an oyster, the hemolymph is circulated in this way and can be readily found in the pericardial cavity that contains the heart. Within the hemolymph of the oyster there are three basic types of hemocytes that perform a wide variety of functions from defense to nutrient transport.

A readily found hemocyte known as a granulocyte can make a very impressive showing under oil immersion light microscopy. These hemocytes appear to be filled with small "grains" and have long pseudopodia that extend from the outer surface of the cell. The pseudopodia are used for mobility and the capture of foreign bodies and disease causing organisms like dermo, Perkinsus marinus.

Oyster hemocyte (400x) with pseudopodia extended.

The procedure for harvesting hemocytes (and the video clips) were developed in collaboration with Dr. Gerardo Vasta of the UMBI Center of Marine Biotechnology in Baltimore along with Dr. Eric Schott, Dr. Jose A.F. Robledo, and Wolf Pecher. Their assistance has been an invaluable part of the translation of these practical techniques. The procedure is simple and requires only a few pieces of equipment. Viewing hemocytes is best accomplished under oil immersion with a 100x objective lens. Lower magnification will allow you to see them but not in great detail. Follow the procedure and you will be able to see these cells live on a glass slide.

Materials:

Compound light microscope with 100x oil immersion objective
Glass slides and cover slips
Fresh live oysters
Oyster knife for shucking
Glass capillary pipette, micropipet, or needle (19 gauge) & syringe

Procedure:

Carefully shuck the oyster taking care not to damage any tissues. For help, see step 11 of the Oyster Anatomy: Internal Laboratory.
Remove the right valve and drain off any excess fluid from the oyster. The oyster should be lying in the left valve or "on the half shell".
Locate the pericardial cavity which contains the heart. See step 17 of the Oyster Anatomy: Internal Laboratory, on the web for the location of the heart.
Using the pipette or the needle and syringe, remove 1 or 2 ml of the hemolymph from the pericardial cavity (Figure 1).
Place a drop of the hemolymph on a glass slide and let it sit undisturbed for 5-10 minutes. This will give the hemocytes time to settle onto the slide and spread their pseudopodia (Figure 2).
Place a cover slip on the drop of hemolymph and focus on the sample under low power. Step up the magnification and focus on an area with a few hemocytes (happy hunting).
Place a drop of oil on the coverslip before using the 100x objective. Focus on the hemocytes and observe their pseudopodia in action (Figure 3).

Videos:

	Preparing the Hemolymph				Hemocytes in Action
			Quicktime				Quicktime RealPlayer

Wrap-Up:

Compare and contrast the hemocytes with your own white blood cells and how the immune system of the oyster is different from that of a mammal.

References:

The Eastern Oyster. Victor S. Kennedy, Roger I.E. Newell, and Albert F. Eble, Eds. Maryland Sea Grant College, University System of Maryland, College Park, MD. 1996.

Oyster Anatomy Laboratory, interactive lesson on oyster anatomy and how to properly "shuck" an oyster.

Personal Communcation, Extracting hemocytes from the hemolymph of oysters, 2000, Drs. Gerardo Vasta, Eric Schott, Jose A.F. Robledo, and Wolf Pecher.

Research by Dr. Gerardo Vasta:

Assessing the Presence and Virulence of Dermo Disease in the Environment Using a PCR-Based Diagnostic Assay for the Extrachromosomal Plastid Genome of Perkinsus marinus. 1996-1997. Gerardo Vasta and Adam G. Marsh. Center of Marine Biotechnology. University of Maryland Biotechnology Institute.

The Role of Iron and Host-Derived Growth-Factors in Regulating Gene Expression in the Oyster Parasite Perkinsus marinus: Strategies for Inhibiting Proliferation. 1996-1997. Gerardo Vasta and Adam G. Marsh. Center of Marine Biotechnology. University of Maryland Biotechnology Institute.

The Molecular Basis for the Etiology of the Oyster Dermo Disease: Gene Regulation Events Susceptible to Chemical Inhibition. 1996-1997. Gerardo Vasta and Adam G. Marsh. Center of Marine Biotechnology. University of Maryland Biotechnology Institute.

A Molecular Approach to Environmental Studies on Perkinsus marinus. Transmission Dynamics of Infection in Chesapeake Bay. 1999-1999. Gerardo Vasta. Center of Marine Biotechnology. University of Maryland Biotechnology Institute.