Detailed methodology is described for the reproducible preparation of collagen--glycosaminoglycan (GAG) membranes with known chemical composition. These membranes have been used to cover satisfactorily large experimental full-thickness skin wounds in guinea pigs over the past few years. Such membranes have effectively protected these wounds from infection and fluid loss for over 25 days without rejection and without requiring change or other invasive manipulation. When appropriately designed for the purpose, the membranes have also strongly retarded wound contraction and have become replaced by newly synthesized, stable connective tissue. In our work, purified, fully native collagen from two mammalian sources is precipitated from acid dispersion by addition of chondroitin 6-sulfate. The relative amount of GAG in the coprecipitate varies with the amount of GAG added and with the pH. Since coprecipitated GAG is generally eluted from collagen fibers by physiological fluids, control of the chemical composition of membranes is arrived at by crosslinking the collagen--GAG ionic complex with glutaraldehyde, or, alternately, by use of high-temperature vacuum dehydration. Appropriate use of the crosslinking treatment allows separate study of changes in membrane composition due to elution of GAG by extracellular fluid in animal studies from changes in composition due to enzymatic degradation of the grafted or implanted membrane in these studies. Exhaustive in vitro elution studies extending up to 20 days showed that these crosslinking treatments insolubilize in an apparently permanent manner a fraction of the ionically complexed GAG, although it could not be directly confirmed that glutaraldehyde treatment covalently crosslinks GAG to collagen. By contrast, the available evidence suggests strongly that high-temperature vacuum dehydration leads to formation of chemical bonds between collagen and GAG. Procedures are described for control of insolubilized and "free" GAG in these membranes as well as for control of the molecular weight between crosslinks (Mc). The insolubilized GAG can be controlled in the range 0.5--10 wt. % while "free" GAG can be independently controlled up to at least 25 wt. %; Mc can be controlled in the range 2500--40,000. Studies by infrared spectroscopy have shown that treatment of collagen--GAG membranes by glutaraldehyde or under high-temperature vacuum does not alter the configuration of the collagen triple helix in the membranes. Neither do these treatments modify the native banding pattern of collagen as viewed by electron microscopy. Collagen--GAG membranes appear to be useful as chemically well-characterized, solid macromolecular probes of biomaterial--tissue interactions.

Design of an artificial skin. II. Control of chemical composition.

The data available in the literature concerning fish thrombocytes, lymphocytes, monocytes, macrophages and granulocytes is discussed and the dearth of reliable information concerning the functional identity of most fish leucocytes is underlined. The methods of applying nomenclatures to fish leucocytes encountered in the literature is discussed in some detail and found to be unsatisfactory. An attempt is made to simplify the confused and conflicting reports by applying the concepts which have recently arisen in the field of mammalian immunology. Nomenclatures are assigned to cells on a functional and morphological basis where sufficient data exists.

Thrombocytes are responsible for clot formation and are considered to be distinct, separate and unrelated to lymphocytes. Lymphocytes are recognised as immuno-competent cells though as yet little information exists on their role in immune mechanisms. Recent work is furnishing interesting results in this direction, especially in the origin of lymphocyte populations and their co-operative responses in immunity. Previous references to monocytes and macrophages in fish are confused and the literature is discussed in the light of the new classification of the Mononuclear Phagocyte System.

Very few experimental data exist concerning granulocytic leucocytes in fish, indeed the presence of such cells with granulocytic functions still requires proof. Cells with morphological similarity to mammalian granulocytes do exist in fish but information essential for an understanding of their role in defence mechanisms is lacking. The state of the literature concerning the eosinophil, basophil and mast cell in fish is so confused that an entirely new approach to a study of these cells is warranted. It is hoped that this review will supply guidelines for future research.

The leucocytes of fish: A review

Currently available crosslinking agents used in fixing bioprostheses are all highly (or relatively highly) cytotoxic, which may induce an adverse inflammatory reaction in vivo. It is therefore desirable to provide a crosslinking agent that is of low cytotoxicty and may form stable and biocompatible crosslinked products. To achieve this goal, a naturally occurring crosslinking agent-genipin-was used by our group to fix biological tissues. Genipin may be obtained from its parent compound, geniposide, which may be isolated from the fruits of Gardenia jasminoides Ellis. In our previous studies, it was found that the cytotoxicity of genipin is significantly lower than both glutaraldehyde and an epoxy compound. Also, it was shown that genipin can form stable and biocompatible crosslinked products. The present study further investigates the crosslinking characteristics and mechanical properties of a genipin-fixed bovine pericardium. Fresh and glutaraldehyde- and epoxy-fixed counterparts were used as controls. It was found that the denaturation temperatures of the glutaraldehyde- and genipin-fixed tissues were significantly greater than the epoxy-fixed tissue, although their fixation indices were comparable. The mechanical properties of fresh bovine pericardium are anisotropic. However, fixation tended to eliminate tissue anisotropy. The tendency in the elimination of tissue anisotropy for the genipin-fixed tissue was more remarkable than for the glutaraldehyde- and epoxy-fixed tissues. In addition, the genipin-fixed tissue had the greatest ultimate tensile strength and toughness among all the fixed tissues. Distinct patterns in rupture were observed in the study: The torn collagen fibers of the genipin- and glutaraldehyde-fixed tissues appeared to be bound together, while those of fresh and the epoxy-fixed tissues stayed loose. The results obtained in the study suggests that tissue fixation in glutaraldehyde, epoxy compound, and genipin may produce distinct crosslinking structures. The differences in crosslinking structure may affect the crosslinking characteristics and mechanical properties of the fixed tissues.

Crosslinking characteristics and mechanical properties of a bovine pericardium fixed with a naturally occurring crosslinking agent

A new and updated checklist of the fishes of the Red Sea is presented. A total of 1078 species belonging to154 families, 25 orders and two classes are listed. The number of species is considerably lower than that given in the last checklist (CLOFRES II, Goren and Dor, 1994) which included all records, "quotations" and distribution maps without distinguishing between substantiated and unsubstantiated records. In addition, an annotated list is provided for all those species that were recorded unjustifiably and were included in CLOFRES II and in subsequent publications.

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The Fishes of the Red Sea—Reappraisal and Updated Checklist

Fishes, amphibia and reptiles, the ectothermic vertebrates, are hosts for a variety of intraerythrocytic parasites including protists, prokaryotes, viruses and structures of uncertain status. These parasites may experience host temperature fluctuations, host reproductive strategies, population genetics, host habitat and migratory behaviour quite unlike those of endothermic hosts. Few blood infections of fishes, amphibia and reptiles have proven pathogenicity, in contrast to the many intraerythrocytic parasites of mammals and some birds which harm their hosts. Although not given the attention afforded to intraerythrocytic parasites of endotherms, those of ectotherms have been studied for more than a century. This review reports on the diversity, general biology and phylogeny of intraerythrocytic parasites of ectotherms. The existence of taxonomic confusion is emphasized and the main taxonomic features of most of the 23 better characterized genera, particularly the kinetoplastid and apicomplexan protists, are summarized. Transmission of protistan infections of aquatic ectotherms is also discussed. Leeches can transfer sporozoties or merozoites to the vertebrate host during feeding. Dormant sporozoites of Lankesterella may permit transmission of species of this genus between vertebrates by predation. The fish haemogregarine, Haemogregarina bigemina, probably has gnathiid isopods, rather than leeches, as its definitive hosts. Hepatozoon spp. in aquatic hosts, and Progarnia of caiman, may also use invertebrate hosts other than leeches. Protistan infections of terrestrial or semi-terrestrial hosts are transmitted by a variety of arthropods, or, in some cases, leeches, contaminated paratenic hosts, or sporocysts free in water. Transfer of protists between vertebrates by predation and congenitally may also occur. The biology of the host cells of these infections, the red blood cells of ectotherm vertebrates, is summarized and compared with that of mammalian erythrocytes. Erythropoiesis, the nature of the surface molecules (especially the possible existence of a major histocompatibility complex), the haemoglobins, and the shape and size of erythrocytes are discussed. The exoerythrocytic sites in which protists, prokaryotes, viruses and structures of uncertain status exist before erythrocyte entry are described. Tissue merogony, tissue cysts and invasion of the white cell series occur in a variety of protistan infections. Intraerythrocytic stages of protistan infections are also discussed, including modes of entry to erythrocytes, survival mechanisms, and multiplication. The impact of infection on host populations is difficult to assess, in part because there is no agreement in the literature on the criteria used to evaluate parasite-induced cost to the host. Almost all studies have been on haemogregarine and Plasmodium infections in, mainly, lizards, but also fishes and snakes. Some infections may be responsible for mortality in their hosts, but hosts themselves may be short-lived, or have a limited ability to recover from infection.

The biology of some intraerythrocytic parasites of fishes, amphibia and reptiles.

Differential Blood Cell Counts of 121 Species of Marine Fishes of Puerto Rico

A Survey of the Blood Parasites in the Fishes of the Red Sea

Heart or arterial blood smears were made from 1,340 marine fishes of 126 species, collected from the coastal waters of Puerto Rico. The only blood parasites found were haemogregarines. Haemogregarina bigemina was present in 11 fishes belonging to the following eight genera: Hemiramphus brasiliensis (Linnaeus), Lutjanus apodus (Walbaum), Ocyurus chrysurus (Bloch), Caranx bartholomaei Cuvier, C. hippos (Linnaeus), Haemulon aurolineatum Cuvier, Scarus croicensis (Bloch), Sparisoma aurofrenatum (Valenciennes), and Auxis thazard (Lacepede). All except Lutjanus apodus, Ocyurus chrysurus, and Caranx hippos are believed to be new records of infection by any haemogregarine. Haemogregarines (Protozoa: Sporozoa: Coccidia: Adeleidae) are the most common blood parasites found in marine fishes. None of their vectors is known and, therefore, as yet no complete life history of them has been worked out. The effect they may have upon the health of fishes, and whether or not they may be a factor in the periodic disappearance of commercially important species from a given area, are also unknown. The first haemogregarines of marine fishes in North America were reported from eastern Canada by Fantham et al. (1942). Laird (1961) also described a new species, Haemogregarina irkalukpiki, from northern Canada in the sea-run arctic char, Salvelinus alpinus. Saunders (1955, 1958, 1959, 1960, 1964) has described the presence of H. bigemina, and several new species of this genus, from the marine fishes of Florida, Bermuda, Bimini in the Bahamas, and the Red Sea. This paper gives the results of a general survey of the blood parasites of marine fishes from the coastal waters of Puerto Rico. As has been the case with a number of similar surveys elsewhere, only haemogregarines were encountered. The incidence of infection among the Puerto Rican fishes was very light, as has been true in every area the author has studied except the Red Sea (1960). It was hoped that in the Puerto Rican material one or more host species would be found which had a high enough incidence of parasitism by haemogregarines to make possible future experiments upon their vectors and methods of transmission. MATERIALS AND METHODS As is shown in Table I, 1,340 fishes of 126 species, belonging to 47 families and 12 orders, were examined for blood parasites. They were collected from early January through late April; and from Montalva Bay near Guanica as the easternmost point, westward through the La Parguera area to Cabo Rojo, and northward from there along the western coastline of Puerto Rico at El Combate, Mayagiiez 1This research was supported by grant GB2699 from the National Science Foundation. The author's deep thanks for aid in the collecting and identification of fish species go to the Staff of the Institute of Marine Biology of the University of Puerto Rico at Mayagiiez, especially to former Director John E. Randall, Drs. Luis R. Almadovar and Gilbert W. Bane, and Mr. Jorge Rivera L6pez; also to Mr. Donald S. Erdman, Fishery Biologist under Federal Aid to Sport Fish Restoration F-I-R, Department of Agriculture, P.R.; and to Capt. Tom Gifford of the sport-fishing boat "Princess Vista Mar" of the Clay Jackson Enterprises of Loiza, P.R. Living quarters and laboratory facilities were made available at the field station of the Institute of Marine Biology of the University of Puerto Rico on Isla Magueyes near La Parguera. TRANS. AMER. MICROSC. Soc. 85(2): 193-199. 1966. This content downloaded from 157.55.39.104 on Mon, 20 Jun 2016 06:04:11 UTC All use subject to http://about.jstor.org/terms DOROTHY CHAPMAN SAUNDERS

Dorothy Chapman Saunders

Papers

Differential Blood Cell Counts of 121 Species of Marine Fishes of Puerto Rico

A Survey of the Blood Parasites in the Fishes of the Red Sea

A survey of the blood parasites of the marine fishes of Puerto Rico.

Variations in thrombocytes and small lymphocytes found in the circulating blood of marine fishes.

Blood Parasites of Marine Fish of Southwest Florida, including a New Haemogregarine from the Menhaden, Brevoortia tyrannus (Latrobe)