John A. Couch

Cytological changes during progression of neoplasia in selected fish species

Abstract Cytological changes during progression of experimentally induced hepatic neoplasia in fishes were reviewed with emphasis on recent findings in Cyprinodon variegatus and Oryzias latipes. Hepatocytes are particularly sensitive to toxic changes during early phases of response to carcinogens reflecting both lethal and sublethal alterations. In these degenerative lesions, enzyme histochemical studies reveal marked deficiency of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase and adenosine triphosphatase. Surviving hepatocytes are either enlarged, encircled by cells with small nucleus to cytoplasm ratios, and have altered nuclear morphology suggestive of an inability to divide, or are smaller, apparently rapidly dividing, and have basophilic cytoplasm. In both species, development of spongiosis hepatis occurred following cytotoxic phases. This lesion apparently provides abundant space for cellular remodeling during neoplastic progression leading to eventual multinodular change. Foci of altered hepatocytes included basophilic, eosinophilic (both species) and clear cell (Cyprinodon variegatus only). Enzyme alterations preceded other tinctorial, morphologic alterations and were seen in cells composing foci and tumors, suggesting lineage of phenotypic alteration. Cytologic changes within other resident cell populations during neoplastic progression were reviewed.

An enzootic nuclear polyhedrosis virus of pink shrimp: Ultrastructure, prevalence, and enhancement☆

Abstract A nuclear polyhedrosis virus exists in pink shrimp, Penaeus duorarum , from waters of the northern Gulf of Mexico. This virus is rod-shaped, 269 nm long, and possesses an outer envelope surrounding its nucleocapsid. The nucleocapsid is 50 nm in diameter. The virus occurs in nuclei of host hepatopancreatic and midgut cells, and is both free in the nucleus and occluded within pyramidal-shaped polyhedral inclusion bodies (PIBs). Histochemically and ultrastructurally, the shrimp PIBs appear to be ribonucleoprotein and in fine structure bear close resemblance to polyhedral inclusion bodies of Baculovirus species from insects. However, the lattice line-to-line spacing is greater than that usually reported for insect PIBs. Crowding and chemical stress of shrimp in aquaria may enhance and increase the virus infection and prevalence. In limited experiments, shrimp fed heavily infected hepatopancreatic tissues had much higher mortality than controls fed only fish. The virus appears to be enzootic in pink shrimp in nature. Cytopathological changes in infected cells of shrimp appear similar to those in insects infected with certain species of Baculovirus . The name Baculovirus penaei n.sp. is proposed for the shrimp virus.

Ultrastructural study of lesions in gills of a marine shrimp exposed to cadmium1

Pathologic black gills of pink shrimp, Penaeus duorarum , exposed to 763 μg/l of cadmium chloride for 15 days were studied with transmission electron microscopy and were compared with normal gills of control pink shrimp. Local as well as extensive areas of cell death and necrosis were found in the distal gill filaments of black gills from cadmium-exposed shrimp. It is proposed that necrosis of specialized epithelial cells and septum cells in black gill filaments and contiguous nonblack gill tissue could cause osmoregulatory, detoxifying, and respiratory dysfunction in crustacea, particularly in individuals undergoing environmental stress such as salinity fluctuation.

Architectural pattern, tissue and cellular morphology in livers of fishes: Relationship to experimentally-induced neoplastic responses

The teleost liver is one of the most sensitive organs to show alteration in biochemistry, physiology and structure following exposure to various types of environmental pollutants. Despite the importance of this organ to environmental toxicology and to ecotoxicology where biomarkers of exposure and of deleterious effect are found, the architectural pattern is not well known. This chapter reviews an architectural plan for teleost liver and compares that to the often cited mammalian pattern. Hepatic tubules composed principally of hepatocytes and biliary epithelial cells are in close proximity to lacunae which are of mesodermal origin. As is described, the tubule and lacunae concepts provide a means to better interpret morphologic alterations following exposure. These concepts are used to illustrate features of the chronic toxicity following exposure to proven carcinogens.

Spongiosis Hepatis: Chemical Induction, Pathogenesis, and Possible Neoplastic Fate in a Teleost Fish Model*

Spongiosis hepatis (SH), first reported as a distinct lesion associated with certain forms of hepatic neoplasia in rats, has also been induced with chemicals, in a predictable fashion, in small teleost fishes being studied as carcinogenesis research models. The sheepshead minnow (Cyprinodon variegattis), exposed to N-nitrosodiethylamine (DENA) in sea water, provided the model for this study. The fish developed SH and presented a spectrum of developmental or progressive stages of the lesion over a 140 week holding period following a 6 week exposure to @ 57 mg/L DENA. The origin of SH in the fish model is homologous to that in the rat model, both species having the perisinusoidal cell (stellate cells of Ito) in the space of Disse as the cell of origin. Light (LM) and electron microscopy (EM) studies characterized the different pathogenetic stages of SH in liver of the sheepshead minnow and revealed a possible late transition of SH to putative polymorphic cell neoplasms. The possible preneoplastic or neoplastic nature of SH from its time of origin in chemically exposed fish to time of appearance of associated presumptive neoplasms is discussed. SH may be a bioindicator of exposure to certain chemicals in some vertebrate species, from fishes to mammals.

INTERACTION OF CHEMICAL POLLUTANTS AND VIRUS IN A CRUSTACEAN: A NOVEL BIOASSAY SYSTEM*

A large group of shrimp, 23.3% of which had light patent Baculovirus infections, was divided equally into two groups. One group was exposed to the chemical stressor Aroclor 1254 (a polychlorinated biphenyl) at 0.7 ppb for 35 days in flowing seawater. The other group was maintained as a control group in flowing seawater. Viral prevalence in exposed shrimp samples increased with time at a significantly greater rate than did viral frequency in control shrimp. Viral prevalence in Aroclor-exposed shrimp survivors was 75% after 35 days, whereas in control shrimp, only 45.7% had patent viral infections. This finding suggests an interaction among chemical stressor (Aroclor 1254), host, and virus. The nature or mechanism of this interaction has not been defined, but the shrimp-virus system shows, promise for future bioassays of influence of low concentrations of pollutants on natural pathogenhost interactions.

Autographa californica Nuclear Polyhedrosis Virus Efficiently Enters but Does Not Replicate in Poikilothermic Vertebrate Cells

The host range of the insect virus Autographa californica nuclear polyhedrosis virus (AcMNPV) was examined. AcMNPV could not initiate a productive infection in frog, turtle, trout, or codling moth cell lines. After exposure to AcMNPV, neither viral DNA nor RNA synthesis could be detected in these cell lines as assayed by nucleic acid probe hybridization. Entry of AcMNPV nucleocapsids into the cytoplasm and viral DNA into the nucleus, however, was as efficient in the nonpermissive cell lines as it was in a permissive insect cell line. The data suggest that the block in AcMNPV infection in these nonpermissive cell lines is at a stage subsequent to viral DNA entry into the nucleus.

Ultrastructural Studies of Shrimp Exposed to the Pollutant Chemical Polychlorinated Biphenyl (Aroclor 1254)

In this study, hepatopancreatic tissue was selected as the tissue of choice for monitoring cellular effects because of the functional significance of the hepatopancreas to crustacea. The hepatopancreas is a complex, gland-like organ that functions in digestion, secretion, absorption, and storage of nutrients (Fig. 3). The hepatopancreas consists of masses of acini or digestive tubules which branch off of two hepatopancreatic ducts that Fig. 1. Polychlorinated biphenyl (Aroclor) molecule.

ENVIRONMENTAL SIGNIFICANCE OF BACULOVIRUS INFECTIONS IN ESTUARINE AND MARINE SHRIMP

Certain enveloped, rod-shaped DNA viruses have long been known as pathogens of insects under the descriptive term “nuclear polyhedrosis viruses.” These viruses have been extensively and intensively studied since Berghold’s early reports in 1947. Subsequent to Berghold’s classic early studies, many rod-shaped viruses associated with polyhedral inclusion bodies of a crystalline nature have been described from different species of insects that represent several orders of Insecta. At present, The International Committee on Nomenclature of Viruses places the nuclear polyhedrosis viruses of arthropods in subgroup A under the genus or group name Baculovirus.’ Prior to 1973, there were no reports of viruses that resemble baculoviruses in animals other than insects or mites. In 1973 and 1974, the first reports 3, were made of baculovirus-like particles and associated polyhedral inclusion bodies in a noninsect arthropod host. The new host was the pink shrimp, Penaeus duorurum, from Florida waters of the northern Gulf of Mexico. These reports indicated for the Baculovirus group a host range extension into the arthropod class Crustacea. In regard to specific characterization and identification of the shrimp virus, it is pertinent to report that not all of Koch’s postulates have been satisfied. Koch’s postulates, however, were meant to be used to show specificity of a microorganism as an etiologic agent for a disease condition and not specifically to determine phylogenetic affinity or identity of the microorganism. The latter task (identification) includes determination of biologic, morphologic, chemical, and physical characteristics. Much of our effort has gone into these determinations for the shrimp virus. The first of Koch’s postulates (that of association or presence of a microorganism with a disease condition) has been satisfied for patent virus infections in shrimp; that is, inclusion bodies and virions are present in all patent infections that exhibit cytopathologic characteristics. The second of Koch’s postulates (that of isolation and pure culture of the microorganism) has not been satisfied for the shrimp virus and poses a severe problem because of the lack of continuous cell cultures of crustacean tissues in which to isolate and grow the virus. At present, we are attempting to use established insect cell lines in which to grow the shrimp virus. The baculoviruses have attracted much attention in recent years largely because some microbiologists and entomologists consider these viruses to be

Rôle of pathobiology in experimental marine biology and ecology

Abstract The roles that pathobiology may play in the advance of experimental marine science and ecology are diverse. As a multidisciplinary science, pathobiology will permit the investigation of structural and functional phenomena that influence marine systems, particularly in reference to cause-effect relationships. Pathobiology focuses on disease and dysfunction, as natural biological responses and processes, rather than as just clinical syndromes or histopathological endpoints (structural lesion). Many opportunities exist for cooperative investigations among pathobiologists and scientists from other backgrounds in determining health requirements and limits for populations and species of estuarine and marine organisms. Specific topics of research of high present interest are: (1) toxicological pathology of coastal species; (2) pathophysiology of aquatic forms; (3) novel virological studies in marine species; and (4) development of functional and mode-of-action models among estuarine-marine species.