The rapid growth of commercial shrimp mariculture has created a need for shrimp disease research. With close confinement production disease problems exist. To assist in developing diagnostic procedures and to assist in the monitoring of the health of Penaeus vannamei, fifty P. vannamei were used for hemocyte characterization studies. We observed three distinguishable types of circulating hemocytes; hyalinocytes (HC), eosinophilic granulocytes (EG), and intermediate granulocytes (IG). Hyal inocytes were the smallest hemocytes with a large central nucleus and a fringe of cytoplasm. Eosinophilic granulocytes were normally the largest of the three cell types with a small, sometimes eccentric, nucleus. The cytoplasm contained an abundance of large eosinophilic granules. The intermediate granulocytes were intermediate in size and contained small granules in an abundant cytoplasm. The percentage of each hemocyte was HC (10-25), EG (5-10) and IG (65-75). Another cell, the cyanocyte, which has a large eosinophilic inclusion body, was abundant in the loose connective tissue but did not occur in the circulating hemolymph. The examination of hemocytes present in sections of embedded tissues was successfully accomplished with formalin fixed tissues. Hemocytes were readily observed in a hemal sinus of the gill, in the sternal sinus, or adjacent to the hepatopancreas. The hemopoietic tissue was consistently located in three sites: 1) in the base of the second maxilliped 2) dorsolateral to the cardiac stomach and 3) adjacent to and enveloping the ophthalmic artery.

Introduction

Shrimp is the most valuable fishery in the U.S. with a yearly value of over $500 million. U.S. per capita consumption of shrimp has been increasing steadily from 1.9 pounds in 1980 to 2.7 pounds in 1986, an increase of about 10% per year. Commercial shrimp farms can produce 1.5 crops of shrimp per year with a gross value of $2,000 to $4,000 per acre, but with close confinement production, disease problems exist. Because high density, confined rearing is unnatural and produces stress, some shrimp disease agents become prominent, diminishing profits by 5-20% or greater. We hope to offset these detrimental effects by developing technology for a more accurate, early diagnosis of disease.

Several of the penaeid shrimp are the focus of efforts to develop profitable methodologies for raising shrimp in mass culture. Pacific white shrimp, Penaeus vannamei, which is native to the western coast of the United States and Mexico, is of particular interest because of its hardiness in culture, and is currently the focus of research on large-scale shrimp farming for the expanding seafood market.

The primary purpose of this research was to describe and classify the hemocytes of Penaeus vannamei prior to assessing the hemocyte response associated with environmental stress and disease and prior to assessing the efficiency of the differential count in monitoring the health of P. vannamei. The classification of hemocytes of P. vannamei constitutes component of the current shrimp diseases research program at Texas A&M University. Hemocytes are the crustacean "blood cells," with a wide variety of known and postulated functions. They are known, for example, to participate in clotting reaction in injury, and in the defense against disease-producing agents. They are also involved in an inflammatory reaction (Johnson, 1980; Fontaine and Lightner, 1975; Lightner and Redman, 1977) and in ecdysis (Sewell, 1955).

The involvement of the hemocyte in most of the histopathologic conditions suggests the importance and extent of involvement of the hemocyte in the internal defense mechanisms of crustacea (Sindermann, 1971; Sis et al., 1980; Lee et al., 1985). The hemocytes are basic and important parts of the defense mechanism and also must have several roles to play during normal function (Johnson, 1980).

Hemocytic infiltration, apparently in response to adverse environmental factors, was reported by Sis et al. (1980) in the Muscle, hepatopancreas, gut, gonad, antennal gland, gill, and heart of penaeid shrimp imp. (Lee et al., 1985) observed hemocytic infiltration in the hepatopancreas, foregut wall, and gill. Elevated numbers of eosinophilic granulocytes have been seen in the hepatopancreas, gut , gonad, and gill of penaeid shrimp (Sis et al., 1980).

Classification and morphologic characterization of the three hemocyte types of Procambarus clarkii as described by Lee (1984) were assessed in our laboratories. This provided a foundation for further work on the role of hemocytes in crustacean pathology and immunology. Differences in the differential count values for the three types of hemocytes suggested a seasonal variation rather than a variation due to water quality parameters of calcium, temperature, or ammonia.

Exposure of hemolymph to a fungus caused encapsulation of hyphae with eventual melanization of the encapsulating hemocytes in shrimp (Hose et al., 1984). Changes were also seen in several hemolymph parameters such as alkaline phosphatase, serum glutamine oxaloacetic transaminase (SGOT), glucose, total protein, hemocyte count, and hemopoietic tissue mitotic index. Hemolymph from severely infected shrimp was hypoproteinemic, contained lower numbers of circulating hemocytes, and frequently failed to coagulate (Hose et al., 1984).

Authors do not report all the same information in classifying crustacean hemocytes. Relatively few workers have agreed on common terminology. Parameters used to classify the hemocytes have been based on the ratio of cytoplasm to the nucleus, and on size and staining reaction of the cells containing granules. The normal structure of hemocytes from penaeid shrimp described by Martin and Graves (1985) as either the agranular (hyaline cells) , small granule (semi-granular cells), or large granule (granulocytes). Agranular hemocytes were the smallest, lack granules, and composed 12% of the circulating population. Small granule hemocytes composed 78% of all hemocytes, were non-refractile, and contained 1-40 granules 0.4 alm in diameter. Large granule hemocytes composed 9% of the hemocytes, were filled with 0.8 µm granules, were highly refractile, and were electron dense using electron microscopy. Another classification of the cellular portion of hemolymph is according to its staining properties. The hyaline cell is clear to light blue with a dark nucleus. The eosinophilic granulocyte has a smaller dark blue nucleus and eosinophilic granules. Semigranular cells have qualities of the other two cells making it difficult to differentiate (George and Nichols, 1948).

The study of crustacean hemopoietic tissue began in the early 1800's. It, has been referred to as lymphogenic, lymphocytogenic and leukopoietic tissue or organ (Johnson, 1980). Following the research of Allen (1893), a French scientist by the name of Cuenot (1895) successfully searched for hemopoietic tissue in several decapods. Cuenot found the tissue to be located next to the opthalmic artery and assembled a theory explaining cell origin and differeciation. His theory implies that all crustacean circulatiny cell types (hyaline, semigranulated, granulated) are all derived from each other through their maturation process. In 1978, Bodammer used the crab species Calnetes sapidus and confirmed Cuenots theory. Fontaine (1978) used white shrimp Penaeus setiferis in which he observed hemopoietic tissue at the base of each pereiopod, cephalothorax and at the base of the maxilla. Fontaine also agreed with Cuenots theory.

Another theory has also been suggested by Kollmann (1908). He claimed that all blood cells are derived from a single cell line which proliferate in the basophilic nodules. In the nodules, cells called hemoblasts are formed and then evolve into four types of hemocytes or cyanoblasts and cyanocytes. Ghiretti-Magaldi et al., (1988) found hemopoietic tissue nodules of the gizzard wall of the common shore crab. They suggested a similar pattern for hemopoiesis of Crustacea Decapods. Whether each cell type is derived from one stem cell or whether each cell type represents different evolutionary stages of a single cell line has not been definitely settled. Many investigators believe that circulating hemocytes form a developmental series beginning with the hyaline and ending with a mature fully granulated cell (Cuenot,1895; Cornick and Stewart,1978 and others). Sternshein and Burton (1980) stated that each hemocyte possessed its own unique characteristics with regard to morphology and behavior and that there was no indication that hyaline cells are immature granulocytes. Considering the importance of the hemocyte, it is necessary to characterize the normal cellular composition of hemocytes for P. vannamei. This classification can be used to build a base of information on how the numbers and proportions of hemocytes vary in response to certain disease conditions and how they can be utilized as indicators of the animal's state of health.

Materials and Methods

Fifty shrimp (Penaeus vannamei)were used for characterization studies. They were obtained from the Texas A&M University shrimp research facility in Corpus Christi, Texas and maintained in controlled aquatic systems at the Aquatic Animal Medicine Laboratory, College of Veterinary Medicine, Texas A&M University. Hemolymph. A drop of hemolymph bled from a cut antennae was allowed to fall into a drop of 10% buffered formal in on a glass slide. The two were gently mixed with a circular motion of a needle or small glass rod. The fixed cells were air dried and stained by immersion in a Giemsa stain, which had been diluted with a working buffer (Harleco Buffered water), for 40 minutes. The percentages of each of the three types of hemocytes in 100 cells were determined. Three differential counts were made from each sample. Histologic studies: A buffered 10% formalin solution (0.3 to 0.6 ml depending on the size) was injected into the hepatopancreas of the shrimp. The lateral carapace was removed from over the gills and pereiopods were removed with scissors. The shrimp was cut transversely, caudal to the cephalothorax, and cranial to the 6th abdominal segment. The cephalothorax was then cut longitudinally, in a midsagittal plane. The sagittal section of 112 of the cephalothorax and a cross section of the 6th abdominal segment were placed in tissue capsules and then into a 10% formalin fixative. Routine paraffin histologic methods were used in section preparation. The medial surface of the sagittal section was presented to the knife. The slides were stained with a standard Harris hematoxylin eosin series.

Results

We found three types of distinguishable hemocytes: hyal inocytes, intermediate (semi-granular or small-granule) granulocytes and eosinophilic granulocytes. A fourth type of cell, cyanocyte, was found only in sectioned tissue and not in hemolymph removed from the shrimp.

Hyalinocytes were the smallest (5-7 um) of the cell types with a large central nucleus (4-6um) surrounded by a scanty basophilic cytoplasm. The shape of the cell was either round or oval. The round nucleus was either homogeneous or granular. These cells contained few or no visible cytoplasmic granules in the fixed and stained light microscopy sections. They comprised 10-25% of the three cell types.

Intermediate granulocytes were intermediate in size between hyalinocytes and granulocytes. The nucleus was central or eccentric, spherical or lobed, often reniform or indented. The nucleus is somewhat smaller than a nucleus of a hyalinocyte but definitely larger than a nucleus of a granulocyte; often the granules in the cytoplasm are not visible at magnifications less than 1,000x. The granules were small and sparsely dispersed in the abundant cytoplasm. They were the most abundant cell type with a count of 65-75%. Eosinophilic granulocytes were normally the largest of the three types of cells with a small, sometimes eccentric, reniform nucleus. The identifying characteristic was the abundance of large eosinophilic granules in the cytoplasm. Eosinophil ic granulocytes are easily identifiable on light microscopic sections. They were the least abundant (5-10%) cell type present. The large granules ranged in size from .5 to 1.5 um. The number of granules present in the cytoplasm ranged from 10 to 48. The cell measured an average of 11.5 um in length and 4-6 um in width.

The examination of hemocytes present in sections of paraffin embedded tissue can be successfully accomplished with formalin fixed tissues . Although we found Davidson's fixative superior for tissue c1arity , formalin fixative was more desirable for hemocyte classification. Hemocytes were most easily observed for classification in the hemal sinus of the gill, in the sternal sinus adjacent to the pereiopods or along the surface of the hepatopancreas. Occasionally hemocytes adhere to the walls of the hernial sinuses or to each other resulting in clumping of the cells.

Cyanocytes were easily located and abundant within the loose connective tissue of the rostrum and along the dorsal midline of the abdominal segments. They can also be found bordering the epidermis and are more abundant along the dorsal midline and the ventral body wall between the nerve cord and the epidermis. They are also located along the epidermis of the pereiopods and epipodites. The individual cells are two to three times as large as a granulocyte and contain one large eosinophilic inclusion body which moves the small nucleus to an eccentric position. A limited number of small cyanocytes were found in the interstitial connective tissue of the hepatopancreas. Cyanocytes were not seen within the hemal sinuses of the gill or in hemolymph removed from the shrimp.

Hemopoietic tissue in P. vannamei was consistently located in three sites: 1) in the base of the second maxilliped 2) dorsolateral to the cardiac stomach and 3) adjacent to and enveloping the ophthalmic artery. It consisted of a sheet of tissue, composed of many small lobules. Each lobule of hemopoietic tissue is enveloped by a thin sheath of fibrous connective tissue and is easily identifiable on histological sections.

Discussion

The decreasing nucleo-cytoplasmic ratio as well as an increasing number and size of the granules observed within granulocytes in our study tend to correspond with those authors who favor the hypothesis of a continuous differentiation series starting with the hyalinocyte and progressing to the mature granulocyte. Hyalinocytes have been shown to be discharged from hemopoietic nodules (Ghiretti-Magaldi et al., 1977), suggesting that they are the basic cells from which the other cells develop (Bauchau, 1981).

Sternshein and Burton (1980) found no evidence to suggest that the cell types found in hemolymph are functional and/or developmental stages in the life history of one basic cell type. They found no indication that hyalinocytes are immature granulocytes, a concept originally proposed by Cuenot (1895).

Martin and Graves (1985), using two species of penaeid shrimp based their classification scheme solely on the absence or presence and relative size of granules. In addition to their basis we have added nuclear-cytoplasmic ratio and the staining properties of the eosinophilic granulocyte. This does not change their classification scheme because the agranular cells always had a large nucleus and scant, cytoplasm. The eosinophilic granulocyte corresponds to their large-granule cells because all of the large granule cells in our study exhibited eosinophilic granules. All other cells (Intermediate granulocyte) conveniently contained small granules and abundant cytoplasm, corresponding to their small-granule hemocyte.

Dumont et al. (1966) observed, with phase contrast optics, the disappearance of granules and the subsequent formation of vacuoles. They stated that the granules are dispersed in the plasma where they presumably contribute to the formation of gelatinous clot. Bauchau and De Brouwer (1972) said that hyaline and semigranulated cells both released material from the cytoplasm during the course of gel formation, Unestam and Nylund (1972) observed the extrusion of granules from fully granulated cells. We also observed many cells that appeared to have released their granules. It was not uncommon to find granules surrounding the hemocyte. These explosive granulocytes may represent different stages in different decapods (Johnson, 1980).

Cyanocytes are included in this report because Fahrenbach (1970) considered cyanocytes to be a special type of hemocyte. Sewell (1955) and others believed that these cells were derived from hyaline hemocytes. Johnson (1980) states that there has been no experimental evidence produced to support the idea that cyanocytes (her reserve inclusion cells) are derived from hemocytes.

Ghiretti-Magaldi et al. (1977) presented ultrastructural evidence supporting an origin in hemopoietic tissue. Ghiretti-Magaldi et al., 1973, established that hemocyanin (Hcy) is stored and probably produced in "agranular eosinophils" in loose connective tissue. Sewell ( 1955 ) reported that these cells provided the material used in producing the new cuticle.

Cyanocytes store Hcy and other materials that can be used by the blue crab during the stress of starvation or disease (Johnson, 1980). Cuenot (1893) recognized that "protein cells" (cyanocytes) were common in well-fed carcinus maenas but absent in starving crabs. Cuzon and Ceccaldi (1971) said that Hcy, which decreases in the shrimp Penaeus kerathurus during starvation, has the function of a reserve protein.

The large cells containing a single large cytoplasmic inclusion, and found in loose connective tissue in all decapods, have been termed protein cells by Cuenot (1893), nephrophagocytes by Bruntz (1907), spherule cells by Kollmann (1908), oval reserve cells by Travis (1955), lipoprotein cells by Sewell (1955), cyanocytes by Fahrenbach (1970) and Ghiretti-Magaldi et al. (1977), agranular eosinophils by Ghiretti-Magaldi et al.(1973), adipohemocyte by Ravindranath (1974), and reserve inclusions by Johnson (1980). Although all of the above terms are good descriptive terms, only one term, lipo­protein hemocyte, was reported three times; the terms cyanocyte and adipohemocyte were each used twice: and the other terms used only once in the literature. These cells with a large eosinphilic cytoplasmic granule, found in our P. vannamei tissues, appear to be the same cell reported by all of the above authors. We chose to call the cell a cyanocyte in this report; however, like Ghiretti-Magaldi et al. (1973, 1977) we could change our mind. In an attempt to produce and use an objective, consistent system for classifying penaeid hemocytes, and after observing these cells in our laboratories, it seemed logical for us to use the criteria adopted by Mix and Sparks (1980). Hyalinocytes with their dark large nuclei and scanty cytoplasm, eosinophilic granulocytes with their small nuclei, and numerous eosinophilic granules are easily and consistently identified. All other cells are intermediate: the size of the granules and the size of the nuclei are intermediate. The overall size of the cell is normally intermediate, but not always. Hyalinocytes were normally smaller and eosinophilic granulocytes were normally larger; however, hemocytes of all three types can be the same size. There is a strong desire to know whether the cell types are each unique (deriving from a stem cell), whether there is a continuous maturation and differentiation, from a stem cell, or whether there is a reverse evolution series brought on by degranulation. The question of cell lineage is of academic importance but may not need to be answered to achieve our diagnostic objectives because the hyal inocyte, intermediate granulocyte and eosinophilic granulocyte permit a rapid identification that can be used for histopathologic determinations and for differential hemocyte counts.

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