Jo Ellen Hose

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.

Elevated circulating erythrocyte micronuclei in fishes from contaminated sites off Southern California

Abstract Frequencies of circulating erythrocyte micronuclei in two marine fish species from contaminated areas off southern California were elevated relative to fishes from less contaminated sites. Micronuclei frequencies from contaminated sites were four times higher in white croaker ( Genyonemus lineatus ) and eleven times higher in kelp bass ( Paralabrax clathratus ). The increased micronuclei frequency was related to previously determined environmental concentrations of chlorinated hydrocarbons (DDTs and PCBs) and polycyclic aromatic hydrocarbon metabolites. However, micronuclei frequency was only weakly correlated to individual body burdens of chlorinated hydrocarbons in white croaker as determined in this study. Applications and limitations of piscine micronucleus measurements 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.

Reproductive impairment in a fish inhabiting a contaminated coastal environment off Southern California.

White croaker (Genyonemus lineatus), collected from a highly contaminated site in San Pedro Bay and from a reference site 80 km away (Dana Point), were induced to spawn in the laboratory. Forty-one per cent of San Pedro Bay females and 54% of Dana Point females spawned. Examination of the ovaries of non-spwaning females revealed that spawning was imminent in the remainder of Dana Point fish but only in 16% of the San Pedro Bay fish. The remainder of the San Pedro Bay fish (43%) contained only immature, yolky oocytes. No croakers containing more than 3.8 ppm ovarian total DDT could be induced to spawn whereas 36% of a contemporaneous San Pedro Bay sample had ovarian total DDT residues in excess of 4 ppm. This suggests that the inability to induce spawning in white croaker may be associated with an ovarian total DDT threshold of about 4 ppm. These data, coupled with observed decreases in fecundity (32%), fertility (14%), and early oocyte loss (30%) relative to reference fish, could partially explain the population declines observed for many southern California fishes since the 1940s.

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.

Contaminant concentrations and toxicity of sea-surface microlayer near Los Angeles, California

Abstract Sea-surface microlayer samples were collected from six nearshore areas receiving different amounts of anthropogenic inputs. The samples were analyzed for selected trace metals, chlorinated hydrocarbons, and polycyclic aromatic hydrocarbons. The relative toxicities of the samples were determined with fish embryo bioassays. Contaminant concentrations generally increased from offshore to the inshore stations. Contaminant concentrations were several orders of magnitude higher in microlayer samples from the highly industrialized Los Angeles and Long Beach harbors compared to samples from a site 15km offshore. Microlayer samples from the inshore stations were significantly more toxic, and induced significantly more developmental abnormalities and chromosome aberrations, than samples from the offshore stations.

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.

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.

Field applications of the piscine anaphase aberration test: lessons from the Exxon Valdez oil spill

Several large-scale genotoxicity assessments have been performed in coastal marine areas that have demonstrated either localized or widespread genetic effects resulting from human activity. One common assessment method is the anaphase aberration test, a measurement of abnormal chromosome division, using embryolarval fishes. It can be used to detect the presence of mutagens within a poorly characterized complex mixture or monitor specific genotoxins and is easily adapted for laboratory screening. One comprehensive marine genotoxicity assessment was conducted using Pacific herring (Clupea pallasi) following the Exxon Valdez oil spill (EVOS) in Prince William Sound (PWS), AK in late March 1989. In early May, genetic damage was detected at many sites within the oil trajectory and was correlated with concentrations of polycyclic aromatic hydrocarbons characteristic of Exxon Valdez oil (EVO) in intertidal mussels. Effects were related spatially and temporally to oil exposure. Anaphase aberration rates decreased throughout May and June 1989, and by 1991, genotoxicity was undetectable. The abundance of the 1989 herring year class in PWS is significantly reduced; this is the first reported example linking genotoxicity to subsequent population level effects. This review describes the methodology for the anaphase aberration test using fish eggs, its applications for large-scale assessments and supportive laboratory studies, and its limitations for prediction of higher level effects on populations.