A method for assessing removal of foreign particles from the blood by fixed phagocytes of the American lobster, Homarus americanus.

ABSTRACT The fixed phagocytes are noncirculating cells in the digestive gland (hepatopancreas) of the American lobster (Homarus americanus) and other decapod crustaceans. They are attached to the outer walls of the terminal hepatic arterioles and lie in the hemal spaces, where they are bathed in blood; they remove foreign particles from the blood as it circulates through the digestive gland. This study has developed and adapted methods for the in vivo assessment of the activity of the fixed phagocytes of lobsters by measuring the uptake of foreign particles from the blood. After fluorescent microspheres were injected into the blood, samples of the digestive gland were excised, arterioles were prepared for microscopy, confocal laser scanning micrographs were collected and microspheres and cells were counted. These methods may be used in larger-scale studies to assess the state of activity of this important part of the immune system of lobsters, and to compare immune activity in lobsters exposed to various environmental and anthropogenic stresses.

KEY WORDS: Homarus americanus, American lobster, phagocytosis, fixed phagocytes, cellular immunity, fluorescence microscopy, confocal microscopy, Long Island Sound

INTRODUCTION

This project is part of an effort to develop tools to assess the immune system of lobsters, specifically using the activity of the fixed phagocytes of the digestive gland as an indicator.

The digestive gland (hepatopancreas) (1) of the American lobster (Homarus americanus, H. Milne Edwards, 1837) is a large, complex organ (reviewed by Conklin 1995, and Factor 1995).

This organ synthesizes and secretes digestive enzymes, it is the site of final digestion of food and nutrient absorption, and it is involved in excretion, the molt cycle, storage of inorganic reserves, lipid and carbohydrate metabolism, and storage of lipids. The digestive gland consists of many small digestive tubules. On each side of the animal, the lumina of the digestive tubules coalesce into a large duct for each of the three lobes, which, in turn, join to form a single large duct that enters the digestive tube.

The digestive gland of Homarus americanus and its digestive tubules are supported by and invested in a network of connective tissue, and hemal sinuses of the open circulatory system are present among adjacent digestive tubules (Factor & Naar 1985). This massive organ is supplied with blood by the hepatic artery, which is subdivided into many terminal hepatic arterioles that are interspersed between and among the digestive tubules. The wall of the terminal arterioles is formed by flattened endothelial cells, and the fixed phagocytes are attached to the outer surface of the endothelium (Factor & Naar 1990). The terminal arterioles lie in the hemal sinuses, and their fixed phagocytes are bathed in the blood that circulates through the digestive gland. The fixed phagocytes are well situated to come into contact with foreign particles as the blood passes over them as it circulates through the small hemal spaces of the organ. The structure and arrangement of the fixed phagocytes also maximizes surface area and, therefore, contact with the blood (Factor & Naar 1990, Factor 1995).

The digestive gland has long been implicated in phagocytic uptake. Saint-Hilaire (1893) described the concentration of carmine particles and other substances injected into the blood of "l'ecrevisse" and noted their concentration in "le pancreas" associated with branches of the hepatic artery, but probably did not recognize the role of the fixed phagocytes. Cuenot described "l'organe phagocytaire" associated with the final branches (terminal arterioles) of the hepatic artery (1903), studied the concentration of injected ink in 43 species of European decapods (1905), and is generally recognized for discovery of the ubiquitous occurrence and importance of these fixed phagocytes of the digestive gland (Johnson 1987). The early work seems to have been forgotten for many years, and Johnson (1987) noted that the interpretation of uptake and clearance experiments were affected by this lack of awareness of the fixed phagocytes. Johnson (1987) reviewed fixed phagocytic and pinocytotic cells in decapod crustaceans and credited Hoover (1977) with the rediscovery of Cuenot's (1905) landmark paper.

Johnson et al. (1981) showed that the fixed phagocytes played a role in removing the gaffkemia bacterium (Aerococcus viridans var. homari) from the blood of infected American lobsters (Homarus americanus). Factor and Beekman (1990) injected carbon particles into the blood of Homarus americanus and demonstrated their sequestration in the pericellular space of the fixed phagocytes before phagocytosis. Recently, Shields and Behringer (2004) showed the aggregation of virions within the perforated membrane of the fixed phagocytes of the digestive gland of a spiny lobster (Panulirus argus) infected with the blood of diseased lobsters.

The relative importance of the fixed phagocytes of the digestive gland, compared with phagocytic cells in circulation and in the gills, has not been determined. Also, it has not been possible so far to estimate the number of fixed phagocytes in the digestive gland. The sheer massiveness of the digestive gland and the circulation pattern of blood through it, however, strongly suggest that the fixed phagocytes collectively play an important (yet mostly overlooked) role in removing foreign particles from the blood of Homarus americanus.

Physiological effects of several environmental stresses on Homarus americanus have been shown in recent studies. For example, Spees et al. (2002a, 2002b) showed alterations in gene expression of heat shock proteins and polyubiquitin in response to induced thermal and osmotic stresses. Dove et al. (2004) suggested that excretory calcinosis, a new fatal disease of Homarus americanus, may have resulted from anomalously high bottom temperatures in Long Island Sound (New York and Connecticut, USA) during the summer of 2002.

In clearance studies in the Atlantic blue crab (Callinectes sapidus), Holman et al. (2004) concluded that a combination of hypoxia (reduced [O.sub.2]) and hypercapnia (elevated C[O.sub.2]) impairs the ability to clear injected Vibrio campbellii bacterial cells from the blood. Tall et al. (2003) implicated a Vibrio fluvialis-like strain of bacterium in limp lobster disease in Homarus americanus.

De Guise et al. (2004) have shown that transient exposure of Homarus americanus to relatively small concentrations of the insecticide malathion (6-7 times lower than the L[C.sub.50]) reduced the rate of phagocytosis during in vitro tests of hemocytes. They concluded that American lobsters "appear extremely sensitive to the immunotoxic effects of malathion," that they are at the sensitive end of the spectrum of responses of aquatic animals, and that "it is likely that malathion-induced suppression of phagocytosis in lobsters could result in a significant decrease in lobster resistance to disease." Mullen et al. (2004) have reported that infection of a parasitic paramoeba was the primary cause of the mass mortality of American lobsters in western Long Island Sound (New York and Connecticut, USA) in 1999. Whereas De Guise et al. (2004) concluded that it is "not possible to determine with any certitude the role of malathion in the 1999 lobster die-off," the possible connection among environmental or anthropogenic stress, depressed immune function, and susceptibility to infectious agents remains intriguing.

This early evidence shows that environmental conditions may alter the activity of the immune system, and that the fixed phagocytes play an important (if not well understood) role in cellular immunity. Considering the need to study natural and anthropogenic environmental stressors, we conclude that additional tools are needed to analyze the status of various immune functions, including the impact of stressors on the fixed phagocytes. The goal of this project is to develop and adapt methods for in vivo assessment of the activity of the fixed phagocytes of the digestive gland of lobsters by measuring the uptake of foreign particles from the blood.

MATERIALS AND METHODS

The lobsters used in this study were purchased from commercial sources and maintained in a refrigerated, aerated aquarium, equipped with bacterial filters, at ~15[degrees]C, 29-30 ppt, and 5-6 mg [O.sub.2]/L.

Standardized foreign particles were used to challenge the immune system. Fluoresbrite carboxylate polystyrene microspheres, 1.0 [micro]m diameter, from Polysciences, Inc. (Warrington, Pennsylvania) were used. These are impregnated with the manufacturer's "yellow-green" fluorescent dye, which has an excitation maximum of 458 nm and an emission maximum of 540 nm.

The number of particles injected was standardized based on the weight of each lobster, following a formula adapted from Reade (1968) for experiments on uptake of carbon particles in a freshwater crayfish, used by Factor and Beekman (1990) for experiments on uptake of carbon particles in the American lobster, and adapted for these experiments on uptake of polystyrene microspheres. To determine the volume of microsphere suspension to be injected into each lobster, 0.0014 mL of microsphere suspension per g of body weight was used. This volume of microsphere suspension in water was mixed with an equal volume of crustacean physiological saline (Cavanaugh 1975, p. 70) that was mixed to double-strength; therefore, the final suspension of microspheres was in single-strength crustacean saline. The surfactant Tween 20 was added (1.0% v/v) to avoid clumping of the microspheres, the suspension was mixed in a vortex mixer, and introduced air bubbles were allowed to dissipate.

The microsphere suspension in saline was injected into the ventral hemocoel of the lobster through the thin cuticular membranes at the base of the pereiopods (walking legs) using a 22-gauge needle, with the total volume divided among several pereiopods. Because the microsphere suspension has a bright, yellow-green color, its circulation could usually be seen initially through the thin membranes on the ventral surface of the cephalothorax and abdomen of the lobster. After injection, lobsters were replaced in the aquarium for an uptake period of 90 min.

Twenty minutes before the 90-min uptake period was completed, each animal was cold-anesthetized in a freezer. The dorsal carapace was removed, and samples of the digestive gland were excised and fixed in vials of cold 3.0% glutaraldehyde in 0.2 M sodium cacodylate buffer that had been adjusted to pH 7.0 with 1.0 M HCl, with 30 mg/mL NaCl and 20 [micro]g/mL Ca[Cl.sub.2] added for osmotic balance. Tissue samples were fixed at least overnight, but generally much longer, and were stored until needed in the fixing solution in the refrigerator. When needed, samples of digestive gland were rinsed in water to remove the fixative, forced through a tissue sieve, and arterioles were separated manually from the slurry using very fine forceps.

BOBO-3 nuclear stain from Molecular Probes, Inc. (Eugene, Oregon) was applied to accentuate the fixed phagocyte ceils in the fluorescence microscope. Arterioles were mounted on microslides using either the Prolong Antifade mounting medium from Molecular Probes, Inc., or the Aqua Poly Mount mounting medium from Polysciences, Inc., both of which are designed for fluorescence microscopy.

Preliminary observations were made with a Zeiss Universal or a Nikon E400 light microscope using the FITC filter set. Confocal images were collected with a Noran Oz confocal laser scanning imaging system mounted on an Olympus IX-70 inverted microscope. Microspheres and cells were imaged in separate channels based on the spectral properties of the fluorescent dyes. In the example presented, a total of 81 optical sections were collected at a step size of 0.5 [micro]m. Image analysis (segmentation and quantification) was carried out with the 3-D Cell Segmentation & catFISH Analysis software v70 (Rensselaer Polytechnic Institute and University of Arizona). This software and approach to automated, 3-dimensional counting of fluorescing objects from confocal microscope image stacks is described by Chawla et al. (2004), and it incorporates new methods for image segmentation (Lin et al. 2003). Validation studies of automated counts using this software showed a 96.5% concordance with a consensus of manual counts by human expert observers (Chawla et al. 2004). Using the same data and parameters, repeated counts give identical results.

For scanning electron microscopy of tissues that bad been fixed and stored as described earlier, arterioles were separated with a tissue sieve, placed in small capsules with mesh ends, dehydrated in an ethanol series, and dried in an EMS 850 critical-point dryer, using C[O.sub.2] as the transitional fluid. Specimens were then mounted with double-coated tape, coated with gold/palladium in a Hummer Jr. sputter coater, and observed with an ISI SR-50 scanning electron microscope.

RESULTS

Separation of the terminal hepatic arterioles from the digestive gland tissue by sieving results in segments of isolated arterioles surrounded by their attached fixed phagocytes (Fig. 1a). Except for being broken into segments, the arterioles appear to be otherwise intact; the various components, including the fixed phagocytes and the perforated membrane, remain in place. Uptake of the injected 1.0-[micro]m diameter polystyrene microspheres can be seen in scanning electron micrographs of fixed phagocytes from these arteriole segments. The outline of several microspheres can clearly be seen lying under the perforated membrane (Fig. 1b), in the pericellular space between the cell surface membrane and the perforated membrane.

[FIGURE 1 OMITTED]

Compound light microscopy has only very limited depth of field. Using the x40 objective lens of the compound light microscope allows only a single layer of the three-dimensional arteriole to be focused at a time (Fig. 2a). Using fluorescence microscopy, only those microspheres in the thin plane of focus appear as distinct objects (Fig. 2b). Most of the microspheres that have been taken up by the fixed phagocytes are out of focus and collectively appear as fluorescent clouds. It is impossible to obtain counts of microspheres from such images.

[FIGURE 2 OMITTED]

Confocal laser scanning microscopy allows the collection of multiple images from a single field of view, with each image representing a single layer that is isolated from the others by an optical plane of section. The images can be built up into a three-dimensional stack of images or viewed individually.

Using a fluorescent microsphere that excites and emits at one set of wavelengths, and a fluorescent nuclear stain that excites and emits at a different set of wavelengths, it was possible to image the microspheres in one channel and the fixed phagocyte cells in another channel. The excitation maximum of the microspheres is 458 nm (microspheres were excited at 488 nm) and the emission maximum is 540 nm (emission of 525-550 nm was collected). The excitation maximum of the nuclear stain is 570 nm (cells were excited at 568 nm) and the emission maximum is 602 nm (emission of 605-655 nm was collected).

Confocal images in a layered stack could be viewed individually, or the data from all of the horizontal layers in a stack could be viewed together as maximum intensity projections. Projections could be made either from the microsphere channel alone (Fig. 3a), or from the cell channel alone (Fig. 3b), or from the two channels combined (Fig. 3c).

[FIGURE 3 OMITTED]

Uptake of microspheres could also be quantified using confocal images. Figure 3d shows the result of segmentation of an image from the microsphere channel; it represents a single layer from the three-dimensional stack of 81 layers. Figure 3e shows the result of segmentation of an image from the same layer, but from the cell channel. In both, the microspheres and cells are outlined (in cyan) and labeled (in purple) as they are counted. In this example, the analysis of the entire three-dimensional stack for both channels resulted in counts of 320 microspheres and 137 cells, for an average of 2.33 microspheres per cell.

DISCUSSION

The in vitro and in vivo uptake of fluorescent particles by circulating phagocytic hemocytes can readily be evaluated using flow cytometry, which allows high-speed counting of particles from a large number of cells (e.g., De Guise et al. 2004). The uptake of particles by large numbers of fixed phagocytes of the digestive gland cannot be analyzed in this way, as the manual technique for separating the terminal arterioles from the digestive gland tissue is laborious, and methods for separating the individual fixed phagocyte cells from the arterioles and perforated membranes are not available.

The methods developed and adapted in this study allow the injection of standardized fluorescent microspheres, fixing tissues, and separating and mounting arterioles for microscopy. The use of confocal laser scanning microscopy, combined with the collection of microsphere images in one channel and cell images in another channel, makes it possible with appropriate software to count both the microspheres and the cells present in a field of view.

This approach will allow the in vivo quantification of uptake and removal from the blood by the fixed phagocytes of the digestive gland of standardized fluorescent microspheres injected into the blood. These methods may be used in larger-scale studies to assess the state of activity of this important, but not very well understood, part of the immune system of lobsters, and to compare immune activity in lobsters exposed to various environmental and anthropogenic stresses.

ACKNOWLEDGMENTS

This research and publication was supported by the National Sea Grant College Program of the US Department of Commerce's National Oceanic and Atmospheric Administration under award #R/FTD-7 to Dr. Jan Factor and the Research Foundation of State University of New York for New York Sea Grant. Supplemental support was provided by Texaco Undergraduate Research Support Awards to the Natural Sciences at Purchase College, SUNY. The authors thank Dr. James Turner, Wadsworth Center, Light Microscopy Core Facility, New York State Department of Health, for use of the confocal laser scanning microscope, and Prof. Badrinath Roysam, Electrical, Computer, and Systems Engineering, Rensselaer Polytechnic Institute, for use of the 3-D Cell Segmentation & catFISH Analysis v70 software (Rensselaer Polytechnic Institute, University of Arizona). The image analysis software was developed in the laboratory of Prof. Badrinath Roysam at Rensselaer Polytechnic Institute under support from National Institutes of Health grants #AG18230 and AG023309, and by the Center for Subsurface Sensing and Imaging Systems, under the Engineering Research Centers Program of the National Science Foundation (Award #EEC-9986821), of which Prof. Roysam is an Associate Director. We also thank Dr. Linda Bastone, Purchase College, for helpful discussions. The views expressed herein do not necessarily reflect the views of any of these organizations.

LITERATURE CITED

Cavanaugh, G. M. editor. 1975. Formulae and Methods VI of the Marine Biological Laboratory Chemical Room. Woods Hole, MA: The Marine Biological Laboratory. 84 pp.

Chawla, M. K., G. Lin, K. Olson, A. Vazdarjanova, S. N. Burke, B. L. McNaughton, P. F. Worley, J. F. Guzowski, B. Roysam & C. A. Barnes. 2004. 3D-catFISH: a system for automated quantitative three-dimensional compartmental analysis of temporal gene transcription activity images by fluorescence in situ hybridization. J. Neurosci. Meth. 139:13-24.

Conklin, D. E., 1995. Digestive physiology and nutrition. In: J. R. Factor, editor. Biology of the lobster Homarus americanus. San Diego, CA: Academic Press. pp. 395-440.

Cuenot, L. 1903. L'organe phagocytaire des Crustaces Decapodes. C.R. Acad. Sci., Paris 137:619-620.

Cuenot, L. 1905. L'organe phagocytaire des Crustaces Decapodes. Arch Zool. Exp. Gen., Ser.4, 3:1-16, pl. i.

De Guise, S., J. Maratea & C. Perkins. 2004. Malathion immunotoxicity in the American lobster (Homarus americanus) upon experimental exposure. Aquat. Toxicol. 66:419-425.

Dove, A. D. M., C. LoBue, P. Bowser & M. Powell. 2004. Excretory calcinosis: A new fatal disease of wild American lobsters Homarus americanus. Dis. Aquat. Org. 58:215-221.

Factor, J. R. 1995. The digestive system. In: J. R. Factor, editor. Biology of the lobster Homarus americanus. San Diego, CA: Academic Press. pp. 395-440.

Factor, J. R. & J. Beekman. 1990. The digestive system of the lobster, Homarus americanus: III. Removal of foreign particles from the blood by fixed phagocytes of the digestive gland. J. Morphol. 206:293-302.

Factor, J. R. & M. Naar. 1985. The digestive system of the lobster, Homarus americanus: I. Connective tissue of the digestive gland. J. Morphol. 184:311-321.

Factor, J. R. & M. Naar. 1990. The digestive system of the lobster, Homarus americanus: II. Terminal arterioles of the digestive gland. J. Morphol. 206:283-291.

Holman, J. D., K. G. Burnett & L. E. Burnett. 2004. Effects of hypercapnic hypoxia on the clearance of Vibrio campbellii in the Atlantic blue crab, Callinectes sapidus Rathbun. Biol. Bull. 206:188-196.

Hoover, K. L. 1977. The effect of a virus infection on the hemocyte population in Carcinus maenas. Sc.D. Thesis, Johns Hopkins University.

Johnson, P. T. 1987. A review of fixed phagocytic and pinocytotic cells of decapod crustaceans, with remarks on hemocytes. Dev. Comp. Immunol. 11:679-704.

Johnson, P. T., J. E. Stewart & B. Arie. 1981. Histopathology of Aerococcus viridans var homari infection (gaffkemia) in the lobster Homarus americanus, and a comparison with histological reactions to a gram-negative species, Pseudomonas perolens. J. Invertebr. Pathol. 38:127-148.

Lin, G., U. Adiga, K. Olson, J. F. Guzowski, C. A. Barnes & B. Roysam. 2003. A hybrid 3D watershed algorithm incorporating gradient cues and object models for automatic segmentation of nuclei in confocal image stacks. Cytometry 56A:23-36.

Milne Edwards, H. 1837 Histoire Naturelle des Crustaces. Comprenant l'Anatomie, la Physiologie et la Classification de ces Animaux. Vol. II. Librairie Encyclopedique de Roret, Paris.

Mullen, T. E., K. S. Russel, M. T. Tucker, J. L. Maratea, T. G. Burrage, C. Koerting, L. Hinckley, C. R. Perkins, S. De Guise, S. Frasca, Jr. & R. A. French. 2004. Paramoebiasis associated with mass mortality of American lobster (Homarus americanus) in Long Island Sound, USA. J. Aq. Animal Health 16:29-38.

Reade, P. C. 1968. Phagocytosis in invertebrates. Aust. J. exp. Biol. Med. Sci. 46:219-229.

Saint-Hilaire, C. 1893. La fonction phagocytaire des vaisseaux hepatiques de l'ecrevisse. Viestnik Estestroznaeia (Rev. Sci. Nat. St.-Petersbourg), 4e annee:347-348.

Shields, J. D. & D. C. Behringer, Jr. 2004. A new pathogenic virus in the Caribbean spiny lobster Panulirus argus from the Florida Keys. Dis. Aquat. Org. 59:109-118.

Spees, J. L., S. A. Chang, M. J. Snyder & E. S. Chang. 2002a. Thermal acclimation and stress in the American lobster, Homarus americanus: equivalent temperature shifts elicit gene expression patterns for molecular chaperones and polyubiquitin. Cell Stress Chaperones 7:97-106.

Spees, J. L., S. A. Chang, M. J. Snyder & E. S. Chang. 2002b. Osmotic induction of stress-responsive gene expression in the lobster Homarus americanus. Biol. Bull. 203:331-337.

Tall, B. D., S. Fall, M. R. Pereira, M. Ramos-Valle, S. K. Curtis, M. H. Kothary, D. M. T. Chu, S. R. Monday, L. Kornegay, T. Donkar, D. Prince, R. L. Thunberg, K. A. Shangraw, D. E. Hanes, F. M. Khambaty, K. A. Lampel, J. W. Bier & R. C. Bayer. 2003. Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease. Appl. Environ. Microbiol. 69:7435-7446.

van Weel, P. B. 1974. Hepatopancreas? Comp. Biochem. Physiol. 47S:1-9.

JAN ROBERT FACTOR, (1) * KELLY ORBAN, (1) DONALD H. SZAROWSKI, (2) GANG LIN, (3) TIMOTHY LAROCCA, (1) AMANDA BECKER (1) AND KAREN JACOFF-KAPUSTA (1)

(1) Natural Sciences Bldg., Purchase College, State University of New York, 735 Anderson Hill Rd., Purchase, New York 10577; (2) Wadsworth Center, Light Microscopy Core Facility, New York State Department of Health, Albany, New York; (3) Electrical, Computer, and Systems Engineering, Rensselaer Polytechnic Institute, Troy, New York 

* Corresponding author. E-mail: [e-mail]

(1) The term digestive gland is used here for the organ often called midgut gland or hepatopancreas; see van Weel ('74) for a discussion of the various terms applied to this organ.