Gary G. Martin

A Decapod Hemocyte Classification Scheme Integrating Morphology, Cytochemistry, and Function

We have examined the hemocytes of three decapod crustaceans (Homarus americanus, Panulirus interruptus, and Loxorhynchus grandis) and propose a classification of these cells based on morphology, cytochemistry, and studies of cell functions. In all species, hyaline cells and granulocytes were identified. Although we have retained the widely used names for these cells, we show that traditional morphological features alone do not accurately differentiate between these categories. Historically, the term hyaline cell refers to hemocytes that contain no or only a few cytoplasmic granules, whereas granulocytes contain abundant granules. However, the size and number of granules in hyaline cells vary greatly between species and therefore are not useful criteria for identifying these cells. Since morphological identification alone is inadequate and misleading, especially with regard to hyaline cells, a combination of morphological, cytochemical and functional methods is necessary to identify decapod hemocytes. Features of hyaline cells include: a higher nucleocytoplasmic ratio than that of granulocytes, the presence of abundant small ({approx}50 nm), round, electron-dense deposits in the cytoplasm, and their accumulation of trypan blue dye prior to cytolysis. Granulocytes do not take up trypan blue or lyse during a 5-min incubation, and they contain prophenoloxidase and hydrolases. Hyaline cells are involved in the initiation of hemolymph coagulation whereas granulocytes are involved in defense against foreign material by phagocytosis and encapsulation. We propose that these criteria be applied to other crustacean species and expect that they will facilitate our understanding of the physiological roles of their hemocytes.

Fine structure and classification of shrimp hemocytes

The structure of hemocytes from two species of penaeid shrimp was examined by light and electron (TEM) microscopy. Hemocytes from the two species are indistinguishable and are classified as either agranular, small‐granule, or large‐granule hemocytes. Agranular hemocytes are the smallest of the hemocytes, lack granules, compose only 5–10% of the circulating hemocytes, and are nonrefractile when examined by light microscopy. Small‐granule hemocytes are the most abundant type of hemocyte (75% of all hemocytes), appear nonrefractile, and contain a variable number (1–40) of granules (0.4 μm diameter). Large‐granule hemocytes compose 10–20% of the hemocytes. They are filled with granules (0.8 μm in diameter) that are highly refractile when examined by light microscopy and are electron‐dense when examined by TEM. Our classification scheme is based solely on the absence or presence and relative size of granules. Features used by other researchers, such as cell size, shape, and staining properties, were not used because these features are subtle and/or subjective. The proposed classification is compared with schemes developed for other decapods, and its usefulness and limitations are discussed. This scheme will serve as a basis for further studies on the maturation and physiological function(s) or crustacean hemocytes.

Localization and roles of coagulogen and transglutaminase in hemolymph coagulation in decapod crustaceans

Abstract 1. 1. Of several anticoagulants tested, only N-ethyl maleimide (NEM) prevents lysis of shrimp hemocytes and maintains their normal morphology. 2. 2. Coagulogen, the clotting protein, is a plasma protein with molecular mass of ≈400 kDa. 3. 3. Transglutaminase, the enzyme that cross links coagulagen to form a visible clot, is more abundant in the hemocytes than in plasma. 4. 4. Hemocytes can be separated into two bands by differential centrifugation. The upper band is enriched wity hyaline cells, which initiate clot formation, and contains most of the transglutaminase activity. 5. 5. The mechanisms of hemolymph coagulation are discussed.

CYTOCHEMICAL FEATURES OF SHRIMP HEMOCYTES

Morphological studies suggest that there are several types of decapod hemocytes; however, distinguishing criteria based on conventional staining techniques are often subtle or ambiguous. Cytochemical features of ridgeback prawn (Penaeidae: Sicyonia ingentis) hemocytes were studied using specific stains for lysosomes, cytoplasmic contents, and granule enzymes. This approach facilitates the differentiation of cell types in the ridgeback prawn and provides information on the functions of and relation ships among different cell types. Agranular hemocytes and a subgroup of small granule hemocytes contain extensive cytoplasmic glycoprotein deposits which display smudgy, intense staining with Sudan black B. As previously shown, coagulogen-the clotting material in decapods-stains with Sudan black B when extracted from lysed hemocytes. Other hemocyte types display light staining limited to granule membranes. Lysosomes are not observed in agranular cells and are rarely present in small granule hemocytes with glycoprotein deposits. Small granule hemocytes without deposits and large granule hemocytes contain numerous lysosomes as shown by the presence of acid phosphatase, β-glucuronidase, and nonspecific esterase. Acid phosphatase is observed in the Golgi body of these cells, within small vesicles, and in small granules. The granules in large granule hemocytes rarely show acid phosphatase reaction, yet small acid phosphatase-positive vesicles fuse with the large granules. The acid phosphatase in the large granules may exist in an inactive form. Prophenoloxidase activity is localized only in large granules. The physiological significance of hemocyte cytochemistry is also discussed.

Defense functions of granulocytes in the ridgeback prawn Sicyonia ingentis

Abstract Classification of crustacean hemocytes has usually been based on subtle morphological features. We use morphological, cytochemical, and functional criteria to classify these cells in the ridgeback prawn, Sicyonia ingentis . Two major categories of hemocytes are found, hyaline cells and granulocytes. We previously demonstrated that only hyaline cells initiate coagulation and that two types of granulocytes may be distinguished using morphology and cytochemistry. This study shows that only granulocytes are involved in in vitro phagocytosis and encapsulation of foreign materials. Phagocytosis of the Gram-negative marine bacterium ( Cytophaga sp.) was accomplished primarily by small granule hemocytes, rarely by large granule hemocytes, and never by hyaline cells. Phagocytosis was enhanced by prior opsonization of bacteria with cell-free shrimp hemolymph. These results support previous cytochemical observations in which lysosomal enzymes are more abundant in small granule hemocytes than in large granule hemocytes and absent in hyaline cells. In addition, both large and small granule hemocytes, but not hyaline cells, attach to and establish capsules around hyphae of the fungus Fusarium solani . This role for granulocytes is supported by our demonstration of prophenoloxidase in these cells. The results of this study allow us to present a classification of shrimp hemocytes combining morphological, cytochemical, and functional criteria.

Patterns of Hemocyte Production and Release Throughout the Molt Cycle in the Penaeid Shrimp Sicyonia ingentis

The production and release of hemocytes was evaluated throughout the molt cycle in the shrimp Sicyonia ingentis. Hematopoiesis occurs in paired epigastric hematopoietic nodules (HPN) which consist of an extensive network of vessels. Hemocytes are produced within the walls of these tubules and released into the vessel lumens. During molt stage C (intermolt), few cells were present in the tubule wall; most of these were hematopoietic stem cells. Elevated mitotic rates during stages C to D1-2 (2-4%) led to the production and rapid release of individual hemocytes, primarily granulocytes. Although the mitotic rate progressively declined from stage D3-4 until after ecdysis (stage A1), the maturing hemocytes accumulated within the tubule walls. Around ecdysis, production of hyaline hemocytes exceeded that of granulocytes. Large groups of these hemocytes were channeled into the vessel lumens immediately after molting. Mitotic rates increased again during stages A2 and B with the number of hemocytes in the tubules reaching seven times that of stage C. Morphological stages in the transition of hematopoietic stem cells into hyaline hemocytes and granulocytes are described, and a model of decapod hemocyte maturation is presented.

Morphology of hemocyte lysis and clotting in the ridgeback prawn, Sicyonia ingentis

SummaryCoagulation of hemolymph in the shrimp Sicyonia ingentis was studied using light and electron microscopy. Differential counts of unclotted hemolymph show that 54% of the hemocytes are deposit cells characterized by a high nucleocytoplasmic ratio, a few granules, and cytoplasm filled with distinctive deposits. The remaining hemocytes have numerous large or small granules filling the cytoplasm. Examination of clotted hemolymph to which trypan blue had been added shows that deposit cells lyse, whereas the granulocytes exclude the dye, attach to slides, and extend filopodia. This suggests that deposit cells, not granulocytes, initiate coagulation. Ultrastructural changes in deposit cells were studied at specific times after mixing hemolymph and seawater. Deposit cells fixed immediately after removal from shrimp were shaped like elliptical discs and contained abundant, ∼ 50 nm diameter cytoplasmic deposits. After 30 s in seawater, deposit cells displayed several cytoplasmic blebs, and had aggregated the deposits. Cytolysis occurred by 45 s. Linear arrays of deposit appeared to extend through breaks in the plasma membrane, forming filamentous strands that hydrated to produce the clot. At 1 min after withdrawal, spheres of clotted hemolymph were seen, each surrounding a lysed deposit cell. Granulocytes remained relatively unchanged and trapped between adjacent expanding clots. Coagulation via hemocyte lysis is compared with other clotting mechanisms observed in various crustaceans and arthropods.

ELIMINATION OF SEQUESTERED MATERIAL FROM THE GILLS OF DECAPOD CRUSTACEANS

Ink particles injected into the hemolymph of the American lobster (Homarus americanus), spiny lobster (Panulirus interruptus), crayfish (Procambarus clarkii), and ridgeback prawn (Sicyonia ingentis) were rapidly removed from circulation, and most were sequestered within nodules in the gills. The morphology of the gills and the nodules were examined from the time of injection until the following molt. The process by which ink was cleared from the gills was the same in all four species. Nodules formed within 10 min after injection and were composed of hemocytes loosely attached to one another and binding small quantities of ink. Within one week, nodules became spherical and more compact with accumulations of ink surrounded by layers of flattened hemocytes. By one month, hemocytes in the nodules had degenerated leaving melanized masses which lay between the gill epithelium and the exoskeleton. Following molting, the gills of both trichobranchiate and dendrobranchiate species were clean or had very reduced numbers of nodules, and melanized masses were seen attached to the inner surface of the shed exoskeletons. A similar mechanism for cleaning the gills has been reported in crustaceans infected with parasites and in necrotic gill tissue caused by exposure to toxic heavy metals. We, therefore, suggest that the ability of the gill epithelium to wall off foreign material so that it is lost during the following molt is a general mechanism to prevent occlusion of the gill and maintain its role in ion regulation and respiration.

Structure of hematopoietic nodules in the ridgeback prawn, Sicyonia ingentis: Light and electron microscopic observations

The architecture and fine structure of the epigastric hematopoietic nodules of the ridgeback prawn, Sicyonia ingentis, are described. The nodules consist of a highly branched series of tubules that contain the maturing hemocytes within a connective tissue stroma. Hemocytes can exit the hematopoietic nodules by penetrating through fenestrations in the endothelial cell layer into the central hemal space or by migrating through the outer later of capsular cells and associated collagen fibrils. Four hemocyte categories were observed: agranular, small granule with cytoplasmic deposits, small granule without cytoplasmic deposits, and large granule hemocytes. This classification was based upon the presence, size, and type of cytoplasmic granules and the presence of cytoplasmic deposits. Only agranular cells and small granule hemocytes without cytoplasmic deposits appeared capable of division. Intermediate stages were observed between agranular hemocytes and small granule hemocytes with deposits and between small granule hemocytes without deposits and large granule hemocytes, suggesting existence of two distinct hemocyte lines.

Dynamic Adaptation of Liver Mitochondria to Chronic Alcohol Feeding in Mice BIOGENESIS, REMODELING, AND FUNCTIONAL ALTERATIONS

Background: Mitochondrial respiration plays an important role in alcohol metabolism by regenerating NAD+ needed for alcohol/acetaldehyde metabolism. Results: Chronic alcohol feeding caused many mitochondrial alterations, such as increased mitochondrial respiration, that enhanced acetaldehyde metabolism. Conclusion: Mitochondria in the liver adapt to the metabolic stress of alcohol. Significance: Mitochondrial alterations may play a role in many vital functions of the liver. Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD+ and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD+, the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver.