Hemal Spaces Research Articles

Microscopic Anatomy of the Lining of Hemal Spaces in the Penaeid Shrimp, Sicyonia ingentis

The purpose of this paper is to present a morphological description of three different types of acellular material lining hemal spaces in a shrimp, providing a background for addressing future questions. The vasculature of the penaeid shrimp, Sicyonia ingentis, includes vessels leading from the heart into arteries which branch and expand into sinuses before returning hemolymph back to the heart. Early work showed that an endothelium was absent, and a basement membrane (BM) separated tissues from the hemolymph. Therefore, it was suggested that hemocytes could identify anything other than the BM as a “foreign” entity. This study demonstrates three major types of acellular material lining the hemal spaces of S. ingentis. Cardiomyocytes, digestive gland tubules, and abdominal muscle fibers are covered by BMs. Major arteries are lined by a fibrillin-like fibrous material. Finally, sheaths of collagenous connective tissues cover the heart and digestive gland as well as the outer surface of arteries, the gut, and gonad. Our understanding of hemocyte receptors and extracellular matrices in general have greatly expanded but the biochemical composition of the matrices lining crustacean hemal spaces, their role in regulating nutrient uptake, and the cells responsible for their deposition deserve further attention.

English

Shells of crabs are infected with a high prevalence of diseases than any other crustacean. Bacteria, viruses, fungi and several other pathogens influence higher percentage of shell disease in crabs. Infections in crab affected growth and metabolism and it may also lead to death. This work deals with the microscopic examination of shell diseased crabs with reference to its variation in hemocytes count, histopathology and biochemical estimation. The total heterotrophic bacterial (THB) population in normal shell was found to be 5.15 x 104 ± 0.09 Colony forming unit (CFU) ml-1 and THB load was noted to be slightly increased than in normal shell and the count was 5.95 x 104 ± 0.12  CFU ml-1 in the infected shell. The current findings suggest a positive correlation between bacterial shell disease and the secondary infection affecting hemolymph (total hemocyte count), proximate composition (protein, carbohydrate and lipid) and histopathology of the animal (gills, hepatopancreas and heart). The total hemocyte count and differential count were experimented which showed lower number of total hemocytes in infected crabs while no highly accountable difference was found in differential count. Hematology studies reported total hemocyte as 9.93 x106 ± 0.07 cells ml-1 in normal and 6.525 x 106 ± 0.12 cells ml-1in shell diseased hemolymph. The level of protein, carbohydrate and lipid contents suffered a mild alteration in crabs with bacterial shell disease. Histopathology of gills hadshown variations such as dilated lamellae and hemocyte accumulation (nodules), hepatopancreas showed mild difference with increased necrosis, appearance of hemocytic nodules in the hemal spaces and heart with very mere infection while no reportable changes were accomplished in muscle tissue.   Key words: Shell disease, bacteria, hematology, histopathology, biochemical composition.

The evolutionary origins of chordate hematopoiesis and vertebrate endothelia

The vertebrate circulatory system is the most complex vascular system among those of metazoans, with key innovations including a multi-chambered heart and highly specialized blood cells. Invertebrate vessels, on the other hand, consist of hemal spaces between the basal laminae of epithelia. How the evolutionary transition from an invertebrate-type system to the complex vertebrate one occurred is, however, poorly understood. We investigate here the development of the cardiovascular system of the cephalochordate amphioxus Branchiostoma lanceolatum in order to gain insight into the origin of the vertebrate cardiovascular system. The cardiac markers Hand, Csx (Nkx2-5) and Tbx4/5 reveal a broad cardiac-like domain in amphioxus; such a decentralized organization during development parallels that seen in the adult anatomy. Our data therefore support the hypothesis that amphioxus never possessed a proper heart, even transiently during development. We also define a putative hematopoietic domain, supported by the expression of the hematopoietic markers Scl and Pdvegfr. We show that this area is closed to the dorsal aorta anlages, partially linked to excretory tissues, and that its development is regulated by retinoic acid, thus recalling the aorta-gonads-mesonephros (AGM) area of vertebrates. This region probably produces Pdvegfr+ hemal cells, with an important role in amphioxus vessel formation, since treatments with an inhibitor of PDGFR/VEGFR lead to a decrease of Laminin in the basal laminae of developing vessels. Our results point to a chordate origin of hematopoiesis in an AGM-like area from where hemal Pdvegfr+ cells are produced. These Pdvegfr+ cells probably resemble the ancestral chordate blood cells from which the vertebrate endothelium later originated.

A METHOD FOR ASSESSING REMOVAL OF FOREIGN PARTICLES FROM THE BLOOD BY FIXED PHAGOCYTES OF THE AMERICAN LOBSTER, HOMARUS AMERICANUS

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.

The origin of the endothelial cells: an evo‐devo approach for the invertebrate/vertebrate transition of the circulatory system

Circulatory systems of vertebrate and invertebrate metazoans are very different. Large vessels of invertebrates are constituted of spaces and lacunae located between the basement membranes of endodermal and mesodermal epithelia, and they lack an endothelial lining. Myoepithelial differentation of the coelomic cells covering hemal spaces is a frequent event, and myoepithelial cells often form microvessels in some large invertebrates. There is no phylogenetic theory about the origin of the endothelial cells in vertebrates. We herein propose that endothelial cells originated from a type of specialized blood cells, called amoebocytes, that adhere to the vascular basement membrane. The transition between amoebocytes and endothelium involved the acquisition of an epithelial phenotype. We suggest that immunological cooperation was the earliest function of these protoendothelial cells. Furthermore, their ability to transiently recover the migratory, invasive phenotype of amoebocytes (i.e., the angiogenic phenotype) allowed for vascular growth from the original visceral areas to the well-developed somatic areas of vertebrates (especially the tail, head, and neural tube). We also hypothesize that pericytes and smooth muscle cells derived from myoepithelial cells detached from the coelomic lining. As the origin of blood cells in invertebrates is probably coelomic, our hypothesis relates the origin of all the elements of the circulatory system with the coelomic wall. We have collected from the literature a number of comparative and developmental data supporting our hypothesis, for example the localization of the vascular endothelial growth factor receptor-2 ortholog in hemocytes of Drosophila or the fact that circulating progenitors can differentiate into endothelial cells even in adult vertebrates.

Hepatopancreatic Alterations in Litopenaeus vannamei (Boone, 1939) (Crustacea: Decapoda: Penaeidae) Experimentally Infected with a Vibrio alginolyticus Strain

As in other countries, the presence of disease has affected and limited the development of shrimp culture in Venezuela. Vibriosis is one of the most prevalent, causing high mortality not only in larval cultures but also in shrimp production. The present work shows the histological changes in hepatopancreas of Litopenaeus vannamei experimentally induced by a strain of Vibrio alginolyticus. The shrimps were infected by the immersion technique, being exposed to a concentration of 5.2 × 107 bacteria/ml. As soon as their behavior indicated a moribund condition, the animals were sacrificed, and their hepatopancreata were fixed and processed for histological observation. Light microscopical observations revealed loss of the acinar structure of the digestive gland and sloughing off of cellular lining because of cytolisis. The acinar cellular content was dispersed within the lumina and among the acini. Clusters of hemocytes, in the majority loaded with bacteria, were observed under the connective tissue capsule surrounding the hepatopancreatic lobes and in the hemal spaces of the connective tissue sheath surrounding the tubules.

Internal Defenses of the Snail Biomphalaria glabrata: III. Observations on Tubular Helical Filaments Induced in the Hemolymph by Foreign Material

Injection of foreign particles (zymosan, latex beads, living bacteria) and substances (ferritine, horseradish peroxidase) in Biomphalaria glabrata induces the rapid appearance of tubular double-helical filaments in the hemolymph. This rapid induction may be due to the polymerization of precursor proteins of the hemolymph. Tubular helical filaments are also observed in normal snails, especially in the hemal spaces of the proximal kidney; they seem to belong to the extracellular matrix and form bundles of a few parallel units associated with fine fibrils. Hemocytes adhere to these bundles by means of the filopodia tip or by small areas of the cell surface; consequently, they may become temporarily sedentary in the hemal spaces. The deposition of helical filaments seems increased in ageing snails and in snails parasitized by Echinostoma caproni.

The Zooxanthellal Tubular System in the Giant Clam.

Giant clams (family Tridacnidae) are special in that they contain large numbers of symbiotic dinoflagellates, Symbiodinium sp., commonly called zooxanthellae which live in the clam's siphonal mantle (hypertrophied siphonal tissues) (1) and are important in its nutrition (2, 3). In 1946, Mansour (4) partially described a tubular system arising from the clam stomach, extending into the mantle and containing zooxanthellae. However, the eminent scientist Sir Maurice Yonge (5, 6) disputed its existence. Subsequently, Yonge's views appear to have suppressed further investigations of Mansour's observations. The zooxanthellae have been universally regarded as living in the hemal spaces of the mantle (2, 5, 7, 8, 9). This study, however, has conhrmed the presence of the tubular system indicated by Mansour and has shown that the zooxanthellae live within a branched, tubular structure that has no direct connection with the hemolymph. The existence of this tubular system has important implications for our understanding of the symbiosis between tridacnids and their symbiotic algae.

Hemocyte production in trematode-infectedLymnaea truncatula

In trematode-infected Lymnaea truncatula, as in other lymnaeids, hemocytes are formed in the connective tissue. Mitoses are found singly in blood vessels or connective tissue or occur in hemocyte nodules, developing along the mantle epithelium or associated with blood sinuses. The so-called hemocyte-producing organ in L. truncatula is not equivalent to that in Biomphalaria glabrata, but rather involves the proximal part of the kidney sac. It has a dual structure: the main part, containing podocytes and broad hemal spaces, is adapted for hemolymph filtration; the apical portion, adhering to the mantle and pericardial epithelia, has a thicker connective-tissue frame in which hemocyte nodules may develop. The role of this region of the kidney in hemocyte formation is discussed.

The digestive system of the lobster, Homarus americanus: II. Terminal hepatic arterioles of the digestive gland.

The digestive gland (midgut gland, hepatopancreas) of the American lobster, Homarus americanus (Decapoda: Nephropidae), is supplied with blood by the hepatic artery. Numerous branches, ending in terminal hepatic arterioles, permeate the organ and discharge blood into the hemal sinuses. Histological and ultrastructural studies show the arterioles to be elongate tubes that lie in the hemal spaces, interspersed among the digestive tubules of the digestive gland. The terminal hepatic arterioles comprise three distinct populations of cells: endothelial cells form the wall of the blood vessel; hemocytes circulate through the lumen of the vessel; and fixed phagocytes are attached to the outer surface of the endothelium. In addition, extracellular membranes form an endothelial intima that lines the lumen and a perforated membrane that covers the outer surface of the arteriole.

The site of ultrafiltration in the kidney sac of the pulmonate gastropod Biomphalaria glabrata

Ultrastructural observation of the renopericardial region in B. glabrata revealed the presence of a site for ultrafiltration located in the proximal region of the kidney sac, in the vicinity of the pericardium. This region comprises podocyte-like cells covering large hemal spaces supplied by a renal artery. Extracellular material observed in spaces between podocyte foot processes resembled small septate junctions rather than diaphragms such as observed in various kidneys. The effective ultrafiltration barrier may be the thick basal lamina of the podocytes. The hemal spaces have a connective tissue frame rich in fixed phagocytes and migrating hemocytes which may play a role in the internal defence system of the snail.

A review of fixed phagocytic and pinocytotic cells of decapod crustaceans, with remarks on hemocytes

Clearance of foreign materials from the hemocoel of decapod crustaceans involves several distinct kinds of cells. Proteins, and possibly viruses below 30 nm diameter, are known to be removed by the branchialpodocytes, which are specialized pinocytotic nephrocytes located in the gills. These cells, and podocytes of the antennal gland (excretory organ) which may also be of importance in clearance, are similar to podocytes of Bowman's capsule of the vertebrate kidney. Phagocytic clearance of particulate material (with possible exception of the small viruses) is accomplished by three kinds of cells, one free and the other two fixed to tissues facing hemal spaces. The hemocytes, free cells suspended in the hemolymph, are the most abundant and generalized of these phagocytic cells. Phagocyticreservecells are applied to myofibers in the heart of penaeid and palaemonid shrimp, and probably the same as reserve cells found throughout the hemocoel of all decapods. They are minimally involved in clearance of injected carbon and carmine, but are not known to phagocytize naturally occurring particles such as bacteria or viruses. The fixedphagocytes are very important in the clearance of some substances. They are located on the exterior surfaces of arterioles in hemal spaces of the hepatopancreas. They are highly specialized cells designed to sequester large amounts of various types of particulate matter, not only through phagocytosis but by trapping and retaining vast numbers of particles within a net- or sievelike layer of granular material which surrounds the free surface of the fixed phagocyte. Derivation, morphology, development, and function of the above cell types are described and discussed.

The vasculature of the white bodies in the ommastrephid squid Illex illecebrosus (LeSueur, 1821): a proposed route for the dissemination of newly formed hemocytes

The vasculature of the white bodies within Illex illecebrosus (LeSueur, 1821) was investigated by employing arterial injections of latex and RENOgrafin-60 (a radiopaque dye) and light, electron, and scanning electron microscopy. The information obtained from these various methods indicates that the white bodies are well supplied with blood. Blood flows to a white body by way of the anterior and posterior ophthalmic arteries; passes through its tissue via small vessels, sinuli, and hemal spaces; and eventually leaves the tissue to enter the enveloping optic sinus (and hence, the venous circulation) by way of hemooscula, here described for the first time. These data have been employed to determine the route for the dissemination of the newly formed hemocytes, which evidence suggests originate in the white bodies. Furthermore, this elucidation of the vasculature provides a means of assessing both the multilobate configuration of the white bodies and the rationale for the latter's position on the medial surface of the squid's eye.

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

Histological response of lobsters to injection of Aerococcus viridans var. homari, cause of gaffkemia, was followed over a 14-day period. Salient features in infected lobsters, Homarus americanus, were: aggregations of hemocytes occurring in hemal spaces throughout the tissues and increasing in number and size with time; the early phagocytosis of bacteria by the system of fixed phagocytes (FPs) present in hemal spaces of the hepatopancreas; and premature release of differentiating hemocytes from the hemopoietic tissue, so that by 14 days that tissue consisted mainly of large stem cells. Mass release of differentiating hemocytes presumably occurred to replace hemocytes lost from the circulation by their incorporation into aggregations or by lysis of individual cells ruptured through the pressure of phagocytized bacteria that were multiplying in them. Bacteria and their remains were present in FPs at 2 days but not visible in single or aggregated hemocytes until 6 days, when free bacteria were also present in the hemolymph. By 6 days, all bacteria, whether phagocytized or free, appeared normal and were surrounded by nonstaining halos that extended well beyond the stainable capsular material. As predicted earlier in physiological studies, gaffkemia is a nontoxic, noninvasive bacteremia. There was hemal stasis and consequent injury in the antennal gland due to free and aggregated hemocytes that occluded hemal spaces of that organ, but other tissues and organs appeared normal except for depletion of glycogen. Aggregations of hemocytes were present in lobsters 2 and 12 days after injection of a nonpathogenic, Gram-negative bacterium, Pseudomonas perolens. Unlike the case with gaffkemia, necrotic hemocytes were common in the aggregations, presumably in response to damage by endotoxin. A further difference was that aggregations were common in the heart of P. perolens-injected lobsters but rare in the heart of gaffkemic lobsters. Bacteria were not seen in hemolymph, hemocytes, or other cells of P. perolens-injected lobsters.

Encapsulation of Aspidogaster conchicola (Trematoda: Aspidogastrea) by unionid mussels

Of 334 mussel specimens representing 13 species, 143 individuals of seven species were found infected with Aspidogaster conchicola; encapsulation of worms was seen in six molluscan species. Encapsulated worms were most abundant anterior to the pericardium and were surrounded by inner fibroblastic and outer fibrocytic/fibrous walls, with occasional adjoining compressed host connective tissues. The inner wall contained acid mucins and phospholipids, while the outer wall contained reticulum fibers, neutral mucins, and phospholipids. Capsule structure was compared to the molluscan encapsulation classification system of T. C. Cheng and E. Rifkin (1970, Amer. Fish. Soc. Spec. Pub., No. 5, pp. 443–496) and to G. B. Pauley and C. D. Becker's (1968, J. Parasitol., 54, 917–920) account of A. conchicola encapsulation. Capsule contents included living or moribund adult worms, viable eggs or empty egg shells produced by disintegrated worms, juveniles hatched from eggs deposited by encapsulated adults, and host cells of which “brown cells” were most abundant. Because of the high frequency of encapsulated moribund worms observed in this study (more than 60%), we infer that this host reaction probably contributed to the parasite's death. Worm eggs individually encapsulated by hemocytes in hemal spaces also were observed. Life cycle implications of juvenile A. conchicola within digestive gland diverticula are discussed.

Paramoebiasis in the Blue Crab, Callinectes sapidus

Paramoeba pernicgosa occurs throughout the year in tissues of blue crabs from Chincoteague Bay, Virginia. P. perniciosa is a parasite of the connective tissues and hemal spaces, invading the circulating blood within blood vessels only when the infection is terminal. An early to mid-summer epizootic is marked by the appearance of amoebae in the circulating blood and mortality due to paramoebiasis. Pathological changes caused by large numbers of amoebae include tissue displacement, probable lysis of some types of tissue including hemocytes, and depletion of reserve nutrients within the host. Most infected crabs react in some manner to the presence of amoebae. Phagocytosis of amoebae by hemocytes is the most common defense mechanism, but encapsulation of amoebae by hemocytes and destruction of amoebae by humoral factors also occur, and on occasion blue crabs successfully overcome infection with P. perniciosa . The transmission of P. perniciosa , by inoculation of infected hemolymph, is described.