Product Citations and Abstracts

The specific purposes of the present work are to review the systematics and cytology of genus Lagenophrys and to report the results of investigations on morphologic, morphogenetic, and biologic aspects of Lagenophrys callinectes. New results reported and described herein are: (1) the first description of ultrastructure of the trophont of a species of Lagenophrys; (2) detailed patterns of the infraciliature of L. callinectes; (3) changes in the infraciliature associated with cytologic changes in the life cycle of the peritrich; (4) the structures involved in the physical and biological relationship between L. callinectes and the blue crab; and (5) host-ciliate interactions such as host-organ preference of the ciliate, seasonal intensities of infestations, seasonal prevalences, and host-specificity. Finally, the relationships that these results may have to the further elucidation of peritrich taxonomic affinities and phylogeny will be discussed.

Couch, John A. 1975. Histopathological Effects of Pesticides and Related Chemicals on the Livers of Fishes. In: Pathology of Fishes. William E. Ribelin and George Migaki, Editors. University of Wisconsin Press, Madison, WI. Pp. 559-584. (ERL,GB 152).

Evidence for the accumulation of pesticides in aquatic ecosystems is abundant. Nontarget species such as fishes from salt and fresh water have been monitored for pesticide contamination. Certain pesticides, e.g., organochlorines and their metabolites, accumulate in wild fish, particularly in liver and fatty tissues. Results of controlled laboratory exposures of fishes to pesticides and related chemicals reveal that the liver is often the organ with highest pesticide concentrations, and greatest damage or impairment. This information, combined with the general knowledge that the liver of vertebrates is a chief metabolic and detoxication organ, suggests that a review of the histopathology of the livers of fishes in reference to pesticide exposure would be of value.

Couch, John A., Lee A. Courtney and Steven S. Foss. 1981. Laboratory Evaluation of Marine Fishes as Carcinogen Assay Subjects. In: Phyletic Approaches to Cancer. EPA-600/D-80-039. Clyde J. Dawe, Editor. Japan Scientific Society Press, Tokyo. Pp. 125-139. (ERL,GB 171). (Avail. from NTIS, Springfield, VA: PB82-205824)

The U.S. Environmental Protection Agency (EPA) and the National Cancer Inst. (NCI) have major responsibilities for determining the fate and risks of carcinogenic agents in the natural environment. Under the auspices of EPA/NCI, the Carcinogen Research Team at the U.S. EPA Lab, Gulf Breeze, has a major role in investigating the fate, effects, and risks of carcinogenic agents in the aquatic portion of the biosphere. In regard to this role, there is a need for practical, experimental exposure systems for the short term, and long term exposure of fishes and invertebrates in order to evaluate their responses to environmentally significant carcinogens. We have designed and tested an adaptable, aquatic laboratory system for flowing water or static water assays of carcinogenic or suspect carcinogenic agents against marine fishes. We report here the design, results of long term tests, and the future uses of the system for determining the risks of carcinogenic agents in the aquatic environment, and as a system complementary to mammalian assay systems, but which permits the phylogenetic expansion of carcinogen assay methodology. A pilot test of the described system has been completed. This test utilized flowing filtered, estuarine water, controlled water temperature, controlled photo period, controlled nutrition of test species, oxygen concentration monitoring, and various life cycle stages of the test fish, the sheepshead minnow, Cyprinodon variegatus, and the suspect carcinogenic herbicide, trifluralin. Continuous exposures to 1 to 5 µg/l trifluralin were conducted with zygote, thru embryogenesis to adult stages of the fish.

Couch, John A. 1974. Pathological Effects of Urosporidium (Haplosporida) Infection in Microphallid Metacercariae. J. Invertebr. Pathol. 23(3):389-396. (ERL,GB 211).

Extensive pathological changes occur in Megalophallus metacercariae as a result of natural infections by the haplosproidan hyperparasite Urosporidium crescens. Infected and uninfected metaceriae, recovered from blue crabs from Chincoteague Bay, Maryland, were examined and compared histologically in regard to condition of metacercarial cyst wall, tegument, and specialized parenchymal cells. Changes from normal found in heavily infected metacercariae were (1) suppression and replacement of possible secretory and parenchymal cells by the hyperparasite, (2) lack of reticulin stromata, polysaccharides, and acid mucopolysaccharides, (3) reduction in thickness of cyst wall, tegumental, and connective tissue structures, and (4) loss of mobility and resistance to mechanical pressures. Though severe pathological changes occur in heavily infected metacercariae, most infected metacercariae remain viable within the blue crab and therby serve as a vector for Urosporidium until the death of the blue crab. At the time of the crab's death and disorganization, infected metacercariae rupture and release spores of the hyperparasite.

Couch, John A. 1974. Free and Occluded Virus, Similar to Baculovirus, in Hepatopancreas of Pink Shrimp. Nature. 247(5438):229-231. (ERL,GB 213).

A rod-shaped, free and occluded virus exists in a marine shrimp, indicating that marine crustacea are potential hosts for viruses similar to certain viruses infecting insects and mites. So far, the virus has been found only in shrimp taken from near Cedar Key and experimentally exposed to the toxic chemical, Aroclor® 1254 (PCB). The virus probably is a natural parasite, however, previously undetected, of estuarine and marine shrimp. Studies of possible interactions of the PCB and virus in pink shrimp may provide valuable information needed to clarify the relationship between natural infectious diseases and pollutant chemicals in the aquatic environment.

Couch, John A. 1974. Enzootic Nuclear Polyhedrosis Virus of Pink Shrimp: Ultrastructure, Prevalence, and Enhancement. J. Invertebr. Pathol. 24(3):311-331. (ERL,GB 215).

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 (PIB's). Histochemically and ultrastructrually, the shrimp PIB's 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 PIB's. 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.

Couch, John A. and DelWayne R. Nimmo. 1974. Ultrastructural Studies of Shrimp Exposed to the Pollutant Chemical, Polychlorinated Biphenyl (Aroclor 1254). Bull. Soc. Pharmacol. Environ. Pathol. 11(2):17-20. (ERL,GB 216).

Pink shrimp (Penaeus duorarum), exposed for 30 to 50 days to 3 µg of polychlorinated biphenyls (PCB) per liter in flowing sea water, accumulated up to 40 mg of PCB per kg in hepatopancreatic tissue. Fifty per cent or more of the experimental shrimp died after 20 to 30 days exposure. Light and electron microscopy of shrimp surviving 30-day exposure revealed two forms of hepatopancreatic cellular alterations that may reflect toxic responses. The most prevalent cytopathic alteration was the occurrence of small (20 to 50 nm.) and large (100 to 700 nm.) vesicles in nuclei of absorptive cells. Such vesicles were observed in 30 to 80 percent of cell profiles from exposed shrimp. These cells also presented abnormally proliferated endoplasmic reticulum and high numbers of free and attached ribosomes. The other significant difference between exposed and control shrimp was the presence of a baculovirus in 5 to 8 percent of the hepatopancreatic nuclei of exposed shrimp. This virus was associated with several nuclear and cytoplasmic alterations, and is the first Baculovirus to be reported in animals other than insects or mites. The significance of the virus infection and PCB exposure of the infected shrimp will be discussed.

Couch, John A. and D.R. Nimmo. 1974. Detection of Interactions Between Natural Pathogens and Pollutants in Aquatic Animals. In: Proceedings of the Regional Symposium on Diseases of Aquatic Animals. Louisiana State University. Center for Wetland Resources, Baton Rouge, LA. Pp. 261-268. (ERL,GB 219).

The purpose of this paper is to give two examples of the detection of possible interactions between natural pathogens and chemical pollutants in selected Gulf of Mexico estuarine animals. These will include results of both experimental laboratory work and field observations in the vicinity of Pensacola, Florida.

Couch, John A. 1973. Ultrastructural and Protargol Studies of Lagenophrys callinectes (Ciliophora: Peritrichida). J. Protozool. 20(5):638-647. (ERL,GB 220).

Ultrastructural and protargol studies reveal that the trophont of Lagenophrys callinectes, though highly specialized, generally conforms to the basic peritrich structural pattern. Features described for L. callinectes trophonts which are unique for the genus are the fine structure and arrangement of the lorica and lips, the attachment organelle of the peristomial cytoplasm, its attachment to the loricastome walls, and the arrangement of the aboral kinetosomes of the trophont. Lack of a distinct scopularized region, and of a ventral lorica wall also characterize L. callinectes trophonts. The 4-row terminal peniculus, as revealed by protargol staining, differs from the 6-row terminal peniculus of L. nassa suggesting that the patterns of infundibular structure, as revealed by protargol, should be useful in future taxonomic studies of Lagenophrys species.

Couch, John A. 1976. Attempts to Increase Baculovirus Prevalence in Shrimp by Chemical Exposure. In: Tumors in Aquatic Animals. Clyde J. Dawe, Dante G. Scarpelli, and Sefton R. Wellings, Editors. S. Karger, Basel. 20:304-314. (ERL,GB 240). (Progress in Experimental Tumor Research; v. 20)

Little information is available concerning interactions between pollutant chemicals and viruses in aquatic animals. Samples of pink shrimp (Penaeus duorarum) with various enzootic levels of a natural baculovirus infection were experimentally exposed to low levels of Aroclor 1254, a polychlorinated biphenyl (PCB), mirex, cadmium, and methoxychlor in the laboratory. No consistent pattern of increase in prevalence of virus was found, and no indication of tumor induction was detected.

Couch, John, George Gardner, John C. Harshbarger, M.R. Tripp and Paul P. Yevich. 1974. Histological and Physiological Evaluations in Some Marine Fauna. In: Marine Bioassays. Marine Technology Society, Washington, DC. Pp. 156-173. (ERL,GB 250).

The development of pathology, as applied to aquatic toxicology, depends heavily on the knowledge of normal histology and physiology if anomalies, due to pollutants or disease, are to be accurately defined. However, at present, knowledge of normal morphology or metabolic activities is either incomplete or lacking for most marine or coastal organisms. Obviously, well-coordinated efforts will be required to characterize normal ranges and interpret the morphological or physiological responses of aquatic organisms to various factors including pollutants. Most laboratories cannot justify well-defined pathological units, although the need often arises through governmental enforcement organizations and other activities. The techniques and examples thus presented are intended to offer some means of obtaining evaluations of aquatic organisms' well-being or disorders.

Couch, John A., Max D. Summers and Lee Courtney. 1975. Environmental Significance of Baculovirus Infections in Estuarine and Marine Shrimp. Ann. N.Y. Acad. Sci. 266:528-536. (ERL,GB 253).

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 were made of baculovirus-like particles and associated polyhedral inclusion bodies in a noninsect arthropod host. The new host was the pink shrimp, Penaeus duorarum, 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 promising biologic control agents for numerous insect pests. The insect baculoviruses have shown narrow host specificity, and all experimental attempts so far to infect noninsect species with insect baculoviruses have failed. The purpose of the present paper is to consider the significance of the shrimp virus in regard to the ecology of its crustacean host.

Couch, John. 1975. Discussion from Selected Papers Presented at EPA-USDA Working Symposium. In: Baculoviruses for Insect Pest Control: Safety Considerations. Max D. Summers, Rilo Engler, Louis A. Falcon, and P. Vail, Editors. American Society for Microbiology, Washington, DC. Pp. 58-62, 111-114. (ERL,GB 262).

In conclusion, I would like to point out the difficulty of working with some of these noninsect invertebrates. It is very hard to work with some of the marine invertebrates, for which there are no cell lines; they are not even amenable to culturing of the whole organism. One therefore can anticipate running into problems, extreme problems, in testing or applying tests of the NPVs and GVs to these organisms. This is a pioneering field with regard to aquatic organisms, but I think it has great promise. I would emphasize my original point that we should start close to the source from a conceptual point of view in testing some of the NPVs and GVs and look critically at the effect on other arthropods, particularly crustacea. . . The major new evidence that emerges here is that many groups of invertebrates are capable of harboring viruses that formerly were studied only in more obvious insect hosts. Thus, we may need to broaden our views on virus-host concepts and seek more widely for host-virus interactions.

Couch, John. 1975. Evaluation of In Vivo Specificity of Insect Viruses: Discussion. In: Baculoviruses for Insect Pest Control: Safety Considerations. Max Summers, Reto Engler, Louis A. Falcon, and Patrick V. Vail, Editors. American Society for Microbiology, Washington, DC. Pp. 60-62. (ERL,GB 262a).

In conclusion, I would like to point out the difficulty of working with some of these noninsect invertebrates. It is very hard to work with some of the marine invertebrates, for which there are no cell lines; they are not even amenable to culturing of the whole organism. One therefore can anticipate running into problems, extreme problems, in testing or applying tests of the NPVs and GVs to these organisms. This is a pioneering field with regard to aquatic organisms, but I think it has great promise. I would emphasize my original point that we should start close to the source from a conceptual point of view in testing some of the NPVs and GVs and look critically at the effect on other arthropods, particularly crustacea.

Couch, John. 1975. Evaluation of the Exposure of Fish and Wildlife to Nuclear Polyhedrosis and Granulosis Viruses: Discussion. In: Baculoviruses for Insect Pest Control: Safety Considerations. Max Summers, Reto Engler, Louis A. Falcon, Patrick V. Vail, Editor. American Society for Microbiology, Washington, DC. Pp. 111-114. (ERL,GB 262b).

The major new evidence that emerges here is that many groups of invertebrates are capable of harboring viruses that formerly were studied only in more obvious insect hosts. Thus, we may need to broaden our views on virus-host concepts and seek more widely for host-virus interactions.

Couch, John A. 1981. Viral Diseases of Invertebrates Other Than Insects. In: Pathogenesis of Invertebrate Microbial Diseases. EPA-600/D-80-055. Elizabeth W. Davidson, Editor. Allenheld, Osmun, Totowa, NJ. Pp. 127-160. (ERL,GB 274). (Avail. from NTIS, Springfield, VA: PB82-155292)

Considerable recent interest has focused on the discovery of viruses that cause diseases in non-insect invertebrates. Since Vago (1966) reported the first virus disease of a marine crustacean, viruses and/or viral diseases have been reorted for shrimps, crabs, daphnids, entoniscid isopods, oysters of several genera, octopods and squids, annelids, trematodes, nematodes, hydra, and a sponge. Most of the reports contained only brief descriptions of the virus with little information on pathogenesis in the hosts. A few reports have been more extensive and contain detailed descriptions of virus-related pathogenesis and virus-host cell relationships. This chapter will describe some aspects of the pathogenesis of better known virus diseases in several exemplary non-insect invertebrate hosts. Also included will be brief descriptions of some lesser known viruses in other invertebrates to demonstrate the range of relationships among viral groups and non-insect invertebrates. Available information on the histopathogenesis, cellular pathogenesis, physiological, and behavioral responses to select host-virus interactions will be discussed under the headings of virus/host types (Table 5-1)

Couch, John A. 1978. Diseases, Parasites, and Toxic Responses of Commercial Penaeid Shrimps of the Gulf of Mexico and South Atlantic Coasts of North America. EPA-600/J-78-072. U.S. Fish Wildl. Serv. Fish. Bull. 76(1):1-44. (ERL,GB 283). (Avail. from NTIS, Springfield, VA: PB-290 075)

A reference work and review of both infectious and noninfectious diseases of commercial penaeid shrimps of the Gulf and South Atlantic region of the United States is presented. Disease is second only to predation and periodic physical catastrophes in limiting numbers of penaeid shrimps in nature and second only to nutritional and reproductive requirements in limiting aquacultural successes with penaeid shrimps. Infectious agents causing disease in penaeid shrimps are a virus, bacteria, fungi, protozoa, helminthes, and nematodes. A well-described Baculovirus infects larval and adult shrimp and is associated with mortality, particularly in larval shrimp. Bacteria of the genera Vibrio, Beneckea, and Leucothrix are associated with disease in penaeid shrimps, but bacterial roles in mortality are unclear. The same is largely true for fungi with members of the genera Lagenidium and Fusarium causing pathogenesis in cultured shrimp. Lagenidium causes severe destruction of larval shrimp tissues. Of the many protozoan groups represented in and on penaeid shrimps as tissue parasites and commensals, the Microsporida of the genera Nosema, Thelohania, and Pleistophora are the most destructive. The ciliate protozoa Zoothamnium sp., Lagenophrys sp., and Parauronema sp. may cause dysfunction in shrimp. An undescribed apostome ciliate is associated with black gill disease. A suctoria, Ephelota sp., is an ectocommensal of larval shrimp, attaching to the cuticle. The six species of gregarines reported cause little or no pathogenesis, and a single reported flagellate species role in shrimp health is uncertain. Flatworms found in penaeid shrimps are metacercariae of a species of Microphallus in muscles and viscera, metacerariae of Opecoeloides fimbriatus in viscera, plerocercoid larvae of Prochristianella hispida in the hepatopancreas and hemocoel, and four other cestode developmental stages. Nematodes found are Thynnascares sp., Spirocamallanus pereirai, Leptolaimus sp., and Croconema sp. Noninfectious disease agents in penaeid shrimps are chemical pollutants, heavy metals, and environmental stresses. Organochlorine, organophosphate, and carbamate pesticides all have adverse effects in penaeids. Fractions of petroleum, particularly the naphthalenes, are very toxic to shrimp. Little other work has been done on the effects of petroleum on penaeid shrimps. Cadmium causes black gills in shrimp by killing gill cells. Mercury is accumulated by penaeids and may interfere with their osmoregulatory abilities. Many chemotherapeutic chemicals used routinely in treatment of fish diseases are toxic to shrimp at certain determined concentrations. Spontaneous pathoses found are a benign tumor, muscle necrosis, and gas bubble disease. "Shell disease" is discussed from points of view of possible causes. A syndrome of "broken backs" is reported in penaeid shrimps for the first time. An overview is presented for general needs in penaeid shrimp health research.

Couch, John A. 1977. Ultrastructural Study of Lesions in Gills of a Marine Shrimp Exposed to Cadmium. J. Invertebr. Pathol. 29(3):267-288. (ERL,GB 298).

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.

Couch, John A. and Lee Courtney. 1977. Interaction of Chemical Pollutants and Virus in a Crustacean: A Novel Bioassay System. EPA-600/J-77-140. Ann. N.Y. Acad. Sci. 298:497-504. (ERL,GB 300). (Avail. from NTIS, Springfield, VA: PB-290 036)

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 pathogen-host interactions.

Couch, John A. and Susan Martin. 1982. Protozoan Symbionts and Related Diseases of the Blue Crab, Callinectes sapidus Rathbun, from the Atlantic and Gulf Coasts of the United States. In: Proceedings of the Blue Crab Colloquium, Biloxi, Mississippi, October 16-19 1979. Harriet M. Perry and W.A. Van Engel, Editors. Gulf States Marine Fisheries Commission, Ocean Springs, MS. Pp. 71-80. (ERL,GB 325).

The blue crab (Callinectes sapidus Rathbun, 1896) supports valuable fisheries along the mid-Atlantic and Gulf coasts of the United States. Because the crab is an estuarine species, capable of ranging widely within its habitat, it is subject to the rigors of the euryhaline environment, as well as to the stresses caused by human activity along coastlines. It has been demonstrated that captive-crab populations are particularly susceptible to parasites and commensals, and to their associated disease and debilities. Within wild populations, extensive mortalities due to these factors are difficult to monitor, but do cause fluctuating losses to the crab fishery. This paper reviews existing knowledge on the more common protozoan symbionts and diseases of the blue crab found on the eastern and Gulf coasts. Available information on recognition and diagnosis of disease, site of infection, and pathogenicity is included. Data are presented on taxonomy, morphology, and life cycles of associated protozoan parasites and commensals of Callinectes sapidus.

Couch, John A., James T. Winstead and Larry R. Goodman. 1977. Kepone-Induced Scoliosis and Its Histological Consequences in Fish. EPA-600/J-77-077. Science. 197(4303):585-587. (ERL,GB 327). (Avail. from NTIS, Springfield, VA: PB-277 182)

Scoliosis in fish is caused by several diverse agents that possibly act on the central nervous system, neuromuscular junctions, or ionic metabolism. The organochlorine pesticide Kepone induces scoliosis in the sheepshead minnow. Some effects associated with Kepone-induced scoliosis in these fish are disruption of myotomal patterns, inter- and intramuscular hemorrhage, fractured centra of vertebrae, and death. The histological syndrome of Kepone poisoning in fish and the clinical syndrome in humans suggest that the nervous system is a primary target for Kepone and that scoliosis is a secondary effect of Kepone poisoning in fish.

Couch, John A., Lee A. Courtney, James T. Winstead and Steven S. Foss. 1979. American Oyster (Crassostrea virginica) as an Indicator of Carcinogens in the Aquatic Environment. In: Animals as Monitors of Environmental Pollutants. EPA-600/J-79-080. National Academy of Sciences, Washington, DC. Pp. 65-84. (ERL,GB 338). (Avail. from NTIS, Springfield, VA: PB80-18524)

The American oyster (C. virginica) was used as the experimental animal for chronic exposure to 3-methylcholanthrene (3-MC) and benzo[a]pyrene (BP) in an exposure system in which the carcinogens can be continuously injected into free flowing water at fixed rates ranging from 1 to 5 µg/l. Experiments designed to determine uptake and distribution of H3MC and H3BP showed that these are concentrated in oyster tissues in direct proportion to the dosage of carcinogen injected into the system. Residual concentrations as high as 84.4 µg/kg of MC and 36.4 µg/kg of BP were present in oysters as long as 6 months following exposure. Autoradiography showed intense localization of H3BP in distal portions of the tubules of the digestive gland and to a lesser extent in the gonadal tissues. Aryl hydrocarbon hydroxylase (AHH) activity was present in homogenates of hepatopancreas after 5.5 months of exposure to the carcinogens, in contrast to control animals in which AHH activity was quite low. In eight oysters exposed to MC, an infiltration of cells believed to be of hematopoietic origin was encountered in the mantle. Some appear to be identical in type to those which constitute sarcoma-like lesions encountered in feral oyster populations. However, it would be premature at this stage to assign any etiological significance to the experimental findings.

Couch, J.A., J.T. Winstead, D.J. Hansen and L.R. Goodman. 1979. Vertebral Dysplasia in Young Fish Exposed to the Herbicide Trifluralin. EPA-600/J-79-072. J. Fish Dis. 2(1):35-42. (ERL,GB 346). (Avail. from NTIS, Springfield, VA: PB80-177751)

Sheepshead minnows, Cyprinodon variegatus Lacepede, exposed to 5.5 to 31 µg/l of the herbicide trifluralin, throughout their first 28 days of life, developed a heretofore undescribed vertebral dysplasia. This dysplasia consisted of symmetrical hypertrophy of vertebrae (three to 20 times normal), characterized by foci of osteoblast and fibroblasts actively laying down bone and bone precursors. Effects of the abnormal vertebral development were dorsal vertebral growth into the neural canal, ventral compression of renal ducts, and longitudinal fusion of vertebrae. Fish, exposed for 51 days to 16.6 µg/l trifluralin and thereafter depurated for 41 days, showed no increase in vertebral dysplasia during depuration; however, residual spinal column damage was evident. Serum calcium concentrations were elevated in adult fish exposed for 4 days to 16.6 µg/l trifluralin. Fluorosis or mimicry of hypervitaminosis A are considered possible mechanisms for the osseous effect, but are not considered to be the only possible causes. The highly predictable nature of this disorder in experimental exposures strengthens the probability that young fish may serve as experimental models for determining effects of chemicals on early vertebrate ontogeny, particularly in regard to skeletal development.

Couch, John A. and James T. Winstead. 1979. Concurrent Neoplastic and Protistan Disorders in the American Oyster (Crassostrea virginica). EPA-600/J-79-099. Haliotis. 8(2):249-253. (ERL,GB 353). (Avail. from NTIS, Springfield, VA: PB80-199706)

One of 373 oysters examined as part of a histological survey of oysters from Apalachicola Bay, Florida, USA, had a concurrent blood cell proliferative disorder and a protistan infection. The neoplastic blood cells (leukocytes) were found throughout the vesicular connective tissues and blood spaces in sections of the oyster. These proliferating cells resembled the neoplastic cells described by Couch and Farley from other specimens of Crassostrea virginica from Chesapeake Bay, Maryland, USA. Mitotic figures were abundant in foci of the neoplastic tissue. Epithelial tissues of the gut of this oyster were infected by spore and schizogonic stages of Dermocystidium marinum, a presumed pathogenic protist of oysters. Spores of this protist and other proliferative stages were observed in connective tissues and blood spaces. There was no morphological evidence that the neoplastic blood cells were related to the protist life cycle stages.

Couch, John A. 1983. Diseases Caused by Protozoa. In: Biology of Crustacea, Vol. 6: Economic Aspects: Fisheries, Culture and Pathobiology. EPA-600/J-80-021. Anthony J. Provenzano, Editor. Academic Press, Inc., New York, NY. Pp. 79-111. (ERL,GB 380).

Crustacea serve as hosts to symbiotic, commensal, parasitic, and pathogenic representatives of all major taxa of Protozoa. Studies of microsporidian epizootics in shrimp, crayfish, and other decapod Crustacea, amoebic epizootics in crabs, and ciliate protozoan outbreaks in shrimps and crabs demonstrate the strong periodic and chronic impact of Protozoa. Mortalities ranging from 1 to 100% in natural and captive populations of Crustacea have been linked to protozoan etiologies. As pathogens of Crustacea, Protozoa have been more intensively studied than most viral, bacterial, fungal, or metazoan pathogens. Yet, there are huge gaps in our knowledge concerning life-histories, mechanisms of transmission, and pathogenesis of Protozoa associated diseases of Crustacea, even in the cases of long-known relationships. This review of representative Protozoan-Crustacean relationships emphasized disease-causing Protozoa and the related responses of their specific crustacean hosts. Examples of all major taxa of Protozoa occurring in or on Crustacea are examined. Surveys of Protozoa known to be associated with decapod Crustacea have been done but not for other orders of Crustacea. The predominant use of decapod Crustacea as exemplary hosts reflects the substantial disease research done on this taxon of Crustacea because of their commercial importance.

Couch, John A. 1982. Aquatic Animals as Indicators of Environmental Exposures. EPA-600/J-82-242. J. Environ. Sci. Health Part A Environ. Sci. Eng. 17(4):473-476. (ERL,GB 431). (Avail. from NTIS, Springfield, VA: PB83-131128)

Aquatic animals are useful as indicators of many kinds of pollutants in the aquatic environment. The presence of pollutants in the general environments of air, land and water is reflected in their accumulations in and effects on aquatic organisms because the aquatic portion of the biosphere is often the "sink" for human-generated pollutants. Aquatic animals lend themselves particularly well to the study of special phenomena, such as carcinogenesis and teratogenesis. The use of aquatic animals and systems adds an important dimension to research on those pollutants that may affect wildlife and human health.

Couch, John A. and W. Peter Schoor. 1982. Effects of Carcinogens, Mutagens, and Teratogens on Non-Human Species (Aquatic Animals). In: Proceedings of the Second NCI/EPA/NIOSH Collaborative Workshop: Progress on Joint Environmental and Occupational Cancer Studies, September 9-11, 1981, Rockville, MD. Herman F. Kraybill, Ingeborg C. Blackwood, and Nancy B. Freas, Editors. U.S. Government Printing Office, Washington, DC. Pp. 688-726. (ERL,GB 433). (May, 1980 - October, 1981)

Aquatic systems and organisms are under examination in order to develop indicator, screening, and modeling capabilities for detection of carcinogens, mutagens and teratogens. In the third year of the program, several advances were made in the development of assays, utilizing sheepshead minnows (liver lesions via benzidine and aflatoxin exposure), rainbow trout (liver tumors via benzo(a)pyrene exposures), and freshwater catfish (papillomatous-like lesions via chlorinated effluent exposures). Results of long-term exposures of fish to the herbicide trifluralin show that induced boney growths are accompanied by histopathologic changes of the pituitary. These and other studies continue to reveal that fish have metabolic pathways similar to mammals for disposition of certain carcinogens. A significant number of cooperative agreements with principal investigators support an extramural complemental and supplemental effort in the identification of aquatic species and systems that may serve as early warning mechanisms.

Couch, J.A. 1984. Histopathology and Enlargement of the Pituitary of a Teleost Exposed to the Herbicide Trifluralin. EPA-600/J-82-152. J. Fish Dis. 7(2):157-163. (ERL,GB 438). (Avail. from NTIS, Springfield, VA: PB84-175306)

Pituitaries of sheepshead minnows, Cyprinodon variegatus, exposed for 19 months to 1-5 µg/l trifluralin in the laboratory exhibited enlargement, pseudocysts, congestion of blood vessels and oedema. Most of the fish with enlarged pituitaries also had induced diffuse and/or focal vertebral hyperostosis and other dysplastic vertebral changes. Several speculative mechanistic paths are suggested for the mode of effect of trifluralin on the vertebral and pituitary tissues. Abnormal enlargement or variation in sizes of pituitary from fish of the same age, along with histopathological findings may be related to the environmental quality of the fish's habitat, and provide indications of stress within individuals resulting from toxicant exposure.

Couch, J.A., S.M. Martin, G. Tompkins and J. Kinney. 1984. Simple System for the Preliminary Evaluation of Infectivity and Pathogenesis of Insect Virus in a Nontarget Estuarine Shrimp. EPA-600/J-84-134. J. Invertebr. Pathol. 43(3):351-357. (ERL,GB 460). (Avail. from NTIS, Springfield, VA: PB85-124618)

Biological control agents (biorationals) are increasingly important in pest control concepts. Certain insect viruses, particularly the baculoviruses (nuclear polyhedrosis viruses), are considered to have potential as biological pesticides and could be used widely in the environment. Therefore, test animals must be selected and methods and laboratory systems developed to evaluate the safety of these agents to nontarget species. A simple laboratory system has been designed and used to determine risks of infectivity and pathogenicity of an insect Baculovirus, originally isolated from the Alfalfa looper, Autographa cailfornica, to a nontarget arthropod, the grass shrimp, Palaemonetes vulgaris, by dietary exposure. This laboratory method also permits evaluation of other microbial biorationals against nontarget aquatic species, and provides an inexpensive standardized procedure of safety testing. Results from this study indicated that histopathological, ultrastructural and serological methods used provided no evidence that experimental exposure to the virus in our test system caused viral infection or related pathogenicity in the grass shrimp.

Couch, J.A. and W.J. Hargis, Jr. 1984. Aquatic Animals in Toxicity Testing. EPA-600/J-84-199. J. Am. Coll. Toxicol. 3(6):331-336. (ERL,GB 501).

Aquatic animals provide useful models for toxicological evaluations that bridge the gap between real world and laboratory problems. Select aquatic organisms are adaptable to laboratory experimentation in areas such as acute toxicity testing and chronic sublethal risks evaluation, including such phenomena as carcinogenesis, mutagenesis, and teratogenesis. General and specific examples of how aquatic animals are useful to toxicologists, as well as theoretical bases for their use, are discussed in this paper.

Couch, John A. and Lee A. Courtney. 1985. Attempts to Abbreviate Time to Endpoint in Fish Hepatocarcinogenesis Assays. In: Water Chlorination: Chemistry, Environmental Impact and Health Effects, Vol. 5. EPA-600/D-84-229. Robert L. Jolley, Editor. Lewis Publishers, Chelsea, MI. Pp. 377-398. (ERL,GB 518). (Avail. from NTIS, Springfield, VA: PB84-246297)

In recent years, the use of freshwater and marine fishes in carcinogen research and in environmental carcinogen monitoring has grown substantially. Several advances must be made with selected species to make fishes advantageous and practical as assay subjects. Some of these advances should be (1) precise characterization of neoplastic endpoints and progression in experimentally exposed fishes, (2) abbreviation of length in time needed for risk evaluation of carcinogens or suspect agents in fishes, and (3) correlation of endpoints for carcinogen effects in fishes with those in other more-routine test species such as rodents (mammals). Because we believe that fishes, as a phyletic group, have much to teach us about neoplasia and environmental carcinogenesis, we are studying the experimental induction, progression, and fate of neoplasms in the liver of a marine coastal fish, the sheepshead minnow (Cyprinodon variegatus). The agent used to induce liver lesions in these studies was N-nitrosodiethylamine (DEN). The sheepshead minnow has been used for several years as a toxicological and carcinogen assay subject in our laboratories.5,6 The objectives of this study were to (1) characterize liver neoplastic development and (2) reduce or abbreviate times to endpoints in liver carcinogen assays using the sheepshead minnow with histological, ultrastructural, and enzyme histochemical endpoints.

Couch, John A. and John C. Harshbarger. 1985. Effects of Carcinogenic Agents on Aquatic Animals: An Environmental and Experimental Overview. EPA/600/J-85/123. J. Environ. Sci. Health Part C Environ. Carcinog. Rev. 3(1):63-105. (ERL,GB 540). (Avail. from NTIS, Springfield, VA: PB86-100286/AS)

A major underlying motivation for seriously studying carcinogenesis in aquatic animals is the concept of utilizing selected lower animal species as models in understanding neoplasia and the neoplastic process. Numerous examples may be cited which illustrate the contribution that ectothermic animals, as models, have made to the principles of pathology, physiology, biomedicine, and now, perhaps, oncology. The purpose of this paper is to provide an overview of the present status of bivalve molluscs and teleost fishes in environmental and experimental studies of carcinogens and cancer.

Couch, J.A. and L.A. Courtney. 1987. N-Nitrosodiethylamine-Induced Hepatocarcinogenesis in Estuarine Sheepshead Minnow (Cyprinodon variegatus): Neoplasms and Related Lesions Compared with Mammalian Lesions. EPA/600/J-87/188. J. Natl. Cancer Inst. 79(2):297-321. (ERL,GB 589). (Avail. from NTIS, Springfield, VA: PB88-161799)

Groups of estuarine sheepshead minnows (Cyprinodon variegatus) were exposed to 50-60 mg/l N-nitrosodiethylamine (DENA) for five to six weeks. Exposure was stopped and the fish were then transferred to clean, flowing seawater. Induced liver lesions were studied in periodic samples of fish taken during the next 67 weeks of holding. Most of these lesions were compared to their counterpart lesions in the rat. Certain lesions such as hepatocellular carcinomas, cholangiolar carcinomas, spongiosis hepatis (SH), and cholangiofibrosis in our fish have apparent similar cellular origins and morphogenesis to those lesions in rats, and perhaps in other mammals. SH in the sheepshead minnow apparently arises from perisinusoidal cells and may be a neoplasm of this cell type. The general similarity of response to DENA in sheepshead minnows and rats suggests that this fish has promise as an assay subject for identifying some hepatocarcinogens, and as a sentinel organism for detecting hepatocarcinogens in contaminated coastal waters.

Couch, John A. 1986. Diseases and Parasites of Marine Fishes (Book Review). Estuaries. 9(3):229. (ERL,GB 601).

Recent and up-to-date review of the literature, outstanding illustrations and photomicrographs, and clarity in writing make this book a useful reference to students and researchers of marine fish diseases. The volume covers infectious, parasitic, and noninfectious causes of marine fish diseases in a systematic approach. Considerable emphasis is placed on the microbial and metazoan parasites of fishes, with a fairly general review of diseases associated with environmental and/or pollution problems. Over 200 photographs, many in color, are presented with good communicative value, and the line drawings are adequate to excellent in the sections on parasites. This book should be very useful to fish pathologists, parasitologists, microbiologists, aquatic ecologists, and environmental specialists. It could serve as a partial text for both undergraduate and graduate courses in the disciplines just mentioned. Because of the diversity of disciplines and subject matter covered, some of the discussion of cause and effect relationships in disease, and roles of parasites in the ecology of marine fishes are quite brief. Perhaps in future efforts it will be necessary to separate the pure parasitology and microbiology of marine fishes from their environmental- and pollution-related disorders in order to provide adequate discussion of each subject. Generally, this first attempt by two young authors is quite successful and provides another needed reference for investigators and students concerned with marine fishes' infectious and noninfectious diseases.

Couch, J.A. and S.S. Foss. 1990. Potential Impact of Microbial Insecticides on the Estuarine and Marine Environments. In: Safety of Microbial Insecticides. EPA/600/D-87/331. Marshall Laird, Lawrence A. Lacy and Elizabeth W. Davidson, Editors. CRC Press, Boca Raton, FL. Pp. 85-97. (ERL,GB 622). (Avail. from NTIS, Springfield, VA: PB88-125448)

This chapter presents an overview of safety tests of microbial insecticides to estuarine and marine organisms that have been performed to date. Approaches and experimental design, species of MPCAs tested, systems used, and endpoints and results evaluated for determiniation of risks of MPCAs to nontarget marine species are reviewed. The review order is by exemplary microbial agents, as follows: viruses, bacteria, fungi and protozoa. The studies presented describe relative simple procedures for exposing single species nontarget hosts to MPCAs. They incorporate a positive control bioassay to confirm the infectivity of the MPCA and are relatively inexpensive and reliable. Studies are usually based on the null hypothesis that infection and relative effects of nontarget host will not occur. To date, this null hypothesis has not been rejected based on results obtained following experimental exposure of nontarget estuarine species to relatively high concentrations of MPCAs.

Couch, John A. 1988. Role of Pathobiology in Experimental Marine Biology and Ecology. EPA/600/J-88/110. J. Exp. Mar. Biol. Ecol. 118:1-6. (ERL,GB 631). (Avail. from NTIS, Springfield, VA: PB88-251665)

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.

Couch, John A. 1991. Nuclear Polyhedrosis Viruses of Invertebrates Other Than Insects. In: Atlas of Invertebrate Viruses. EPA/600/D-88/068. Jean R. Adams and J.R. Bonami, Editors. CRC Press, Boca Raton, FL. (ERL,GB 633). (Avail. from NTIS, Springfield, VA: PB88-195201)

The occluded baculoviruses from non-insect hosts are described and reviewed for morphology, morphogenesis, cytopathology, and composition. The two known baculoviruses from crustacea are Baculovirus penaei and Penaeus monodon; the first exists worldwide and the second only in the Pacific. Both are viruses of shrimps or prawns and can cause lethal infections. The structure and morphogenesis of each virus is described, and the role of virus controlled events in cellular pathology is discussed.

Couch, John A. 1990. Toxicological Pathology: Introductory Remarks. In: Pathology in Marine Science. Frank O. Perkins and Thomas C. Cheng, Editors. Academic Press, San Diego, CA. Pp. 343-345. (ERL,GB 665).

This introduction describes the discipline of toxicological pathology, which deals with effects of toxins and toxicants in causing disease or influencing disease processes in organisms. The role of toxicological pathology is defined as follows: (1) to determine causes of specific tissue/cellular effects by specific chemicals; (2) to identify, characterize, and understand lesion and/or disease syndromes linked to toxins and toxicants; (3) to identify etiological agents, i.e., biological, infectious agents vs. chemical agents; (4) to understand relationships between and among biological and chemical injurious agents; and (5) to prescribe a preventative or therapeutic treatment.

Couch, John A. 1989. Membranous Labyrinth in Baculovirus-Infected Crustacean Cells: Possible Roles in Viral Reproduction. EPA/600/J-89/423. Dis. Aquat. Org. 7(1):39-53. (ERL,GB 669). (Avail. from NTIS, Springfield, VA: PB90-245747)

The origins and morphogenesis of the membranous labyrinth (ML) in Baculovirus penaei (BP) infected cells of penaeid shrimps (Crustacea:Decapoda) are described. The ML is usually a highly ordered, complex system of membranes arranged as a labyrinth or in concentric form in the cytoplasm near or against the nuclear envelope. The ML originates from dilated Golgi and ER vesicles and from the outer nuclear envelope. It grows apparently from proliferation of the cellular membranes of these systems, and its development may be correlated with the stages of BP infection. It is hypothesized that, because of the close parallel and concurrent development of the ML and virus reproduction, and other evidence, the ML is virus induced and controlled and may play at least three roles in the virus reproductive strategy: 1) provides a conduit or transport system for viral or occlusion body precursors from cytoplasm to nucleus; 2) provides increased membrane surface and volume for increased ATPase activity (related to energy demand for virus reproduction and transport of viral products); and 3) provides a mechanism for cell collapse and release of virus and occlusion bodies at end of virus reproduction period. Possible experimental methods with which to determine the functional role of the ML are discussed and implications for such a system for nuclear, DNA, viruses are considered.

Couch, John A. 1990. Pericyte of a Teleost Fish: Ultrastructure, Position, and Role in Neoplasia as Revealed by a Fish Model. EPA/600/J-90/366. Anat. Rec. 228(1):7-14. (ERL,GB 676). (Avail. from NTIS, Springfield, VA: PB91-163808)

The morphology and position of the pericyte, a periendothelial cell, is described for a teleost fish, Cyprinodon variegatus. This cell was found attached to the abluminal surfaces of capillaries, venules, and arterioles of the submucosa of the midgut of the fish. The cell was encompassed by a thin basal lamina, possessed numerous plasmalemmal vesicles, a "sole region" which contained thinner actin-like filaments and possibly thicker myosin-like filaments, and ranged in form from ovoid to stellate, with long cytoplasmic extensions that partially covered the endothelium of the associated microvessel. The pericyte of C. variegatus has been shown to give rise to hemangiopericytomas (experimentally induced with diethylnitrosamine) and possibly to pericytomas. In this regard and in regard to its normal ultrastructural morphology, and anatomical position, in relationship to microvasculature in this fish, the cell is very similar to other vertebrate pericytes. Limited evidence suggests that small fish species may be excellent study models for further elucidation of pericyte form, function, and role in disease.

Couch, John A. 1991. Spongiosis Hepatis: Chemical Induction, Pathogenesis, and Possible Neoplastic Fate in a Teleost Fish Model. EPA/600/J-92/213. Toxicol. Pathol. 19(3):237-250. (ERL,GB 699). (Avail. from NTIS, Springfield, VA: PB92-195700)

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 variegatus) 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 in the space of Disse as the cell of origin. LM and EM studies characterized the different progressive stages of SH in liver of the sheepshead minnow and revealed a probable late progression of SH to pericytoma-like lesions. The possible preneoplastic or neoplastic nature of SH from its time of origin in chemically exposed fish to time of appearance of pericytoma is discussed. SH may be a bioindicator of exposure to certain chemicals in some vertebral species, from fishes to mammals.

Couch, John A. 1993. Observations on the State of Marine Disease Studies. In: Pathobiology of Marine and Estuarine Organisms. EPA/600/A-93/136. John A. Couch and John W. Fournie, Editors. CRC Press, Boca Raton, FL. Pp. 511-530. (ERL,GB 780). (Avail. from NTIS, Springfield, VA: PB93-204170)

State of marine disease studies is described. Perhaps the greatest area of success in the last 20 years has been in the identification and characterization of viruses, bacteria, fungi, protozoan and metazoan disease agents. Opening of new areas of investigation such as that of interactions between pollutants and infectious agents or non-infectious syndromes such as neoplasia have provided challenges to younger, better equipped investigators in recent efforts. These successes, though not complete in themselves, provide an impetus to understanding complex disease issues. Long standing enigmas, such as complete understanding of the complex life-cycles of devastating pathogens such as protozoans of shellfish, and roles of certain toxicants in fish diseases remain to be better understood.

Couch, John A. and John W. Fournie, Editors. 1993. Pathobiology of Marine and Estuarine Organisms. EPA/600/R-92/232. CRC Press, Boca Raton, FL. 552 p. (ERL,GB 810). (Avail. from NTIS, Springfield, VA: PB93-131217)

This book is an up-to-date compendium of scientific findings related to diseases of marine and estuarine organisms. The information was presented at the Gulf Breeze Symposium on Marine and Estuarine Disease Research sponsored by the U.S. Environmental Protection Agency (EPA) Environmental Monitoring and Assessment Program (EMAP) held in October 1990 on Pensacola Beach, Florida. Authors review the current state of the science and recommend research for future studies of the impact of xenobiotics and other anthropogenic stress factors on disease processes in marine and estuarine organisms.

Couch, John A. 1993. Light and Electron Microscopic Comparisons of Normal Hepatocytes and Neoplastic Hepatocytes of Well-Differentiated Hepatocellular Carcinomas in a Teleost Fish. EPA/600/J-94/011. Dis. Aquat. Org. 16(1):1-14. (ERL,GB 841). (Avail. from NTIS, Springfield, VA: PB94-140696)

Well-differentiated hepatocellular carcinomas (HCC's) induced in the sheepshead minnow Cyprinodon variegatus with N-nitrosodiethylamine permitted light microscopical and ultrastructural comparisons of normal hepatocytes and adjacent HCC cells. Normal hepatocytes contained typical organelles with their intracellular distribution similar to that described for other teleosts. These cells revealed a strong compartmentalization of organelles consisting of restricted perinuclear cytoplasm containing rough endoplasmic reticulum (RER) and mitochondria; most of the rest of the cell's cytoplasm volume was filled with Beta-glycogen particles. Adjacent HCC cells at the border of the neoplasm interdigitated (invaded) between normal hepatocytes and replaced normal hepatocytes close to the edge and adjacent to the bile canaliculi. Within the HCC, cells had little intercellular space between them and atypical bile canaliculi were found occasionally between adjacent hepatocytes. The HCC cells appeared to differ mainly in a quantitative, rather than in a qualitative fashion, and in the distribution of organelles from normal hepatocytes. HCC cells were not compartmentalized; their organelles (RER and mitochondria) were distributed throughout the cytoplasm, and only small amounts of glycogen remained diffusely distributed or in small masses or foci. Mitochrondrial intermembrane myelin bodies (MMB's) were found frequently in the HCC cells but less frequently in the normal hepatocytes. Some cells at the edge of the HCCs appeared to be intermediate between inner HCC cells and normal hepatocytes in terms of abundance and distribution of organelles and glycogen. The possible significance of the MMB's and intermediate, edge cells is discussed, as well as the need to better characterize features of well-differentiated hepatic neoplasms in order to assure their proper inclusion in neoplasm incidence/prevalence data in fish carcinogenesis assays and field studies.

Couch, John A. 1995. Invading and Metastasizing Cardiac Hemangioendothelial Neoplasms in a Cohort of the Fish Rivulus marmoratus: Unusually High Prevalence, Histopathology, and Possible Etiologies. EPA/600/J-95/371. Cancer Res. 55(11):2438-2447. (ERL,GB 893).

An unusually high, unprecedented prevalence of cardiac hemangioendotheliomata, including hemangiomas, hemangioendotheliomas, and hemangioendotheliosarcomas, was found in a laboratory cohort of the small, teleost fish Rivulus marmoratus. The neoplasms occurred in 51 of 204 fish (25%) used in a carcinogenicity study of butylated hydroxyanisole fed in a lyophilized chicken liver diet for up to 9 months. The cardiac neoplasms occurred in approximately equal numbers of both control (fed lyophilized chicken liver but not exposed to butylated hydroxyanisole) and exposed (fed 0.8% butylated hydroxyanisole in chicken liver) fish. The neoplasms occurred in the bulbus arteriosus and ventricle (accompanied by an intense epicarditis), and in some cases, in the gills. At least one case of hemangioma was characterized by cavernous vessels in the bulbar wall. Hemangioendothelioma cases consisted of pleomorphic endothelial cells that formed continuous tracts and anastomosing, typical vascular channels of varying sizes. The hemangioendotheliosarcomas consisted of atypical, spindle, polygonal, or round endothelial cells that formed solid tumor masses that contained abnormal vessels with atypical endothelium tufting into their lumina. Some fish had neoplasm cases containing varying regions with mixtures of the above features. Incipient neoplasms occurred on/in the semilunar valves, and their cells appeared to invade the adjacent walls of the bulbus and/or the ventricle. The gill lesions represented possible metastatic neoplasms, probably formed by atypical endothelial cells that exfoliated from the edges of cardiac neoplasms into the lumina of the ventricle or bulbus and were then pumped to the arterioles of the gills via the ventral aorta. Fish examined from the original colony in the laboratory and from the wild had no neoplasms. Possible causes are discussed, and studies are under way in an attempt to determine the etiology of the neoplasm and to evaluate Rivulus marmoratus as a possible model organism for study of these types of cardiac neoplasms.

Couch, John A. 1996. Metastatic Gill Lesions Secondary to Cardiac Hemangioendotheliosarcomas in the Teleost Fish Mangrove Rivulus. J. Aquat. Anim. Health. 8(4):325-331. (ERL,GB 938).

Gill neoplasms have been infrequently described for teleost fishes. Nine of 51 cases of primary cardiac endothelial cell neoplasms of a teleost fish species, mangrove rivulus Rivulus marmoratus, also possessed endothelial cellneoplasms in their gills. These neoplasms occurred in the afferent arterioles of the gill filaments. They were made up of round cells or spindle cells that had nuclei similar to typical endothelial cell nuclei. The large gill neoplasms, with more anaplastic cells, penetrated the walls of the gill arterioles and invaded adjacent gill tissues. Supporting evidence for the metastatic nature of these gill neoplasms includes the following: (1)they were secondary in timing, numbers and size to the advanced sarcomas of the heart; (2)cell phenotypes of both gill and cardiac neoplasms were similar in cytoplasmic and nuclear features; (3)the cardiac neoplasms appeared to be shedding neoplastic endothelial cells into the lumen of the bulbus arteriosus with a direct route via the circulation of the ventral aorta to the afferent arterioles of the gills; and (4)direct evidence that the gill neoplasms arose from gill endothelium was lacking.

Couch, John A. 1996. Continuing Development of Fish Species as Additional Experimental Models in Cancer Research (Commentary). Toxicol. Pathol. 24(5):602. (ERL,GB R521).

Dr. Bunton's review begins with a summary of the salient historical steps in the development of chemical carcinogenesis concepts with fishes. Her interpretation of the early findings about responses of fishes as carcinogen assay or test organisms in relation to both environmental and mammalian health concerns leads to discussion of the more recent findings of molecular and genetic features derived from experimental fish carcinogenesis studies.

Couch, John A. 1979. Shrimps (Arthropoda: Crustacea: Penaeidae). In: Pollution Ecology of Estuarine Invertebrates. EPA-600/J-79-083. C.W. Hart, Jr. and L.H. Samuel, Editors. Academic Press, Inc., New York, NY. Pp. 235-258. (ERL,GB X046). (Avail. from NTIS, Springfield, VA: PB80-185259)

The majority of reports available about pollution and penaeid shrimps concerns studies involving the commercially valuable penaeid shrimps of the U.S. Atlantic states and Gulf Coast. Therefore, most of the information presented here will be related to the following three species: Penaeus duorarum (pink shrimp), Penaeus aztecus (brown shrimp), and Penaeus setiferus (white shrimp), all Atlantic and Gulf of Mexico species. Reference to other species of penaeid and some nonpenaeid crustacea will be made when specific studies contribute significantly to our understanding of pollution ecology of shrimps. This chapter will cover the following pollutant categories and situations: organic chemicals other than petroleum, petroleum and related compounds, heavy metals, biological agents, and interactions of pollutants and other factors. Under each of these divisions toxicity and specific tissue, organismic, population, and ecological effects will be reviewed when known. Further, the uptake, transport, and fate of pollutants will be discussed as they may affect the ecology of penaeid shrimps

Couch, John A., Frank G. Lowman and Ford A. Cross. 1980. Biomonitoring of Coastal Waters--An Overview. In: Biological Monitoring for Environmental Effects. Douglas L. Worf, Editor. D.C. Heath and Company, Lexington, MA. Pp. 93-95. (ERL,GB X156).

Estuarine and coastal waters of the United States not only serve as prime habitat for a significant fraction of commercial and recreational marine fisheries, but also receive industrial and municipal wastes from a rapidly expanding coastal economy. In order to ensure that levels of contaminants in coastal ecosystems do not affect either public health or fisheries resources adversely, both state and federal agencies are conducting a variety of biomonitoring programs. In addition, generic research projects are underway at several university, state, and federal laboratories to determine which environmental and physiological factors regulate the body burden of contaminants in marine organisms. This information can then be used to help interpret data obtained in biomonitoring programs and to allow more precise predictions of contaminant levels in biota prior to discharge.

Couch, John A. and W. Peter Schoor. 1981. Effects of Carcinogens, Mutagens and Teratogens on Non-Human Species-Aquatic Animals. In: Proceedings of the First NCI/EPA/NIOSH Collaborative Workshop: Progress on Joint Environmental and Occupational Cancer Studies. H.F. Kraybill, Ingeborg C. Blackwood, and Nancy B. Freas, Editors. U.S. Government Printing Office, Washington, DC. Pp. 333-376. (ERL,GB X332).

A research program using aquatic system and organisms to study the fate and effects of carcinogens in the aquatic environment has been underway during the FY's 78 and 79 at the Gulf Breeze EPA Laboratory. The two major investigative, disciplinary areas have been in pathobiology and biochemistry. A fish-carcinogen assay system has been developed that involves laboratory controlled long-term exposure of fish to suspect carcinogens followed by histopathology and physiology of exposed fish, induced growths, and related disorders. This system appears adequate for carcinogen tests for fish. A two year field study is underway of tumor, disease prevalence, and carcinogen residue or metabolites in fish and shellfish in variously polluted estuaries along the northern Gulf of Mexico. It is too early to predict the significance of the findings in this study, but several new tumor types have been discovered in fish. Biochemical studies have revealed that fish may respond in enzymatic reactions in ways similar to mammals exposed to the same carcinogens, and that fish may prove to be adequate supplemental, biological monitors of carcinogens in the environment.

Few studies of cephalopod molluscs have been made in situ. Mission 8-50, TEKTITE II, made possible a three-week study (July-August) of octopods and squids in Greater Lameshur Bay, St. John, U.S. Virgin Islands. A cross sectional study revealed the presence of at least three species of Octopus: O. briareus; O. macropus; and O. vulgaris. Observations on activity periods, territoriality, feeding, and general behavior of these octopods were made. Three different genera and species of squid were observed: Sepioteuthis sepioidea; Pickfordiateuthis pulchella; and Loligo plei. Observations on schooling behavior, space requirements, mating, and feeding were made on these squid.

Parrish, Patrick R., John A. Couch, Jerrold Forester, James M. Patrick, Jr. and Gary H. Cook. 1974. Dieldrin: Effects on Several Estuarine Organisms. In: Proc. 27th Annu. Conf. Southeast. Assoc. Game Fish Comm. Pp. 427-434. (ERL,GB 178).

Tests were conducted to determine (1) the acute toxicity of dieldrin in flowing sea water to American oysters (Crassostrea virginica), pink shrimp (Penaeus duorarum), grass shrimp (Palaemonetes pugio) and sheepshead minnows (Cyprinodon variegatus) and (2) the rate of dieldrin uptake and depuration by spot (Leiostomus xanthurus). Acute (96-hour) EC50's were: oysters, 12.5 µg/l; pink shrimp, 0.9 µg/l; grass shrimp, 11.4 µg/l; and sheepshead minnows 23.6 µg/l. Spot exposed to 0.0135, 0.075, 0.135, 0.75 or 1.35 µg/l for 35 days accumulated the chemical with maximum concentrations attained in 11 to 18 days. Maximum whole-body residue (wet-weight) was 6,000 X the concentration in test water. Spot contained no detectable dieldrin residues at the end of a 13-day depuration period in dieldrin-free water. Tissue alterations, such as subepithelial edema in gill lamellae and severe lysis and sloughing of the small intestine epithelium, occurred in spot exposed to 1.35 µg/l for four days.

Courtney, Lee A. and John A. Couch. 1981. Aspects of the Host-Commensal Relationship Between a Palaemonid Shrimp (Pontonia domestica) and the Pen Shell (Atrina rigida). EPA-600/J-81-041. Northeast Gulf Sci. 5(1):49-54. (ERL,GB 249). (Avail. from NTIS, Springfield, VA: PB82-158171)

Sixty pen shells (Atrina rigida), were collected and examined to determine the prevalence and morphometrics of the commensal shrimp (Pontonia domestica). The spatial and sexual distribution within the shell of the host, and the reproductive states of the shrimp were also investigated.

Winstead, James T. and John A. Couch. 1981. Proctoeces Sp. (Trematoda: Digenea) in the American Oyster, Crassostrea virginica. EPA-600/J-81-042. Trans. Am. Micros. Soc. 100(3):296-305. (ERL,GB 278). (Avail. from NTIS, Springfield, VA: PB82-205816)

Histological examination of over 6,600 individuals of the American oyster, Crassostrea virginica, inhabiting northern Gulf Coast estuaries revealed unencysted juvenile and possible adult stages of digenetic trematode, Proctoeces sp., infecting the gonadal ducts of the mollusc. Infection is the highest in oysters from Mississippi Sound near Pascagoula, Mississippi with prevalence peaking in later summer and early winter. The worm did not provoke a significant hemocytic response from the oyster. Oyster hemocytes were observed inside the digestive caeca of worms but there was no evidence of significant germinal feeding or gonadal impairment to the oyster. The hooked mussel, Brachidontes recurvus, found attached to shells of oysters may serve as first intermediate host and thus be significant in supplying cercariae for worms' opportunistic exploitation of C. virginica as surrogate intermediate host or final host.

Goodman, Larry R., David J. Hansen, John A. Couch and Jerrold Forester. 1978. Effects of Heptachlor and Toxaphene on Laboratory-Reared Embryos and Fry of the Sheepshead Minnow. In: Proc. 30th Annu. Conf. Southeast. Assoc. Game Fish Comm., Oct. 24-27, 1976, Jackson, MS. EPA-600/J-76-086. Wilmer Rogers, Editor. Southeastern Association of Game and Fish Commissioners, Frankfort, KY. Pp. 192-202. (ERL,GB 297). (Avail. from NTIS, Springfield, VA: PB-290 072)

Flow-through seawater bioassays of 28-days duration were conducted with the organochlorine pesticides heptachlor and toxaphene to determine their toxicity to and bioconcentration by embryos and fry of the sheepshead minnow (Cyprinodon variegatus). At technical heptachlor measured concentrations of 4.3, 3.5, 2.2, 2.0, and 1.2 µg/l (ppb), test animal survival was 1, 5, 61, 79, and 88% respectively. At toxaphene measured concentrations of 2.5, 1.1, 0.6, 0.3, and 0.2 µg/l, test animal survival was 10, 85, 79, 88, and 80% respectively. Average standard length of fry continuously exposed from fertilization to heptachlor concentrations of 4.3 and 3.5 µg/l was significantly reduced (a =0.01). Concentration factors (concentration in fish/measured concentration in water) for heptachlor averaged 3,600 and for trans-chlordane averaged 8,600. Heptachlor epoxide and cis-chlordane were also present in the fish. Concentration factors for toxaphene in fry averaged 9,800. Various histopathological characteristics not seen in control fish were observed in the liver, kidney, pancreas, and intestine of the few fish that survived 4.3 and 3.5 µg/l of heptachlor.

Middaugh, Douglas P., John A. Couch and Allan M. Crane. 1977. Responses of Early Life History Stages of the Striped Bass, Morone saxatilis to Chlorination. Chesapeake Sci. 18(1):141-153. (ERL,GB 304). (Avail. from NTIS, Springfield, VA: PB-269 932)

The toxicity of total residual chlorination (TRC) to early life stages of the striped bass, Morone saxatilis, was determined using percent embryo hatchability, incipient LC50 bioassays, histopathology, and avoidance responses. Beginning 8 to 9 hours after fertilization, developing embryos were exposed continuously to TRC in flowing water at 1.0-3.0 ppt salinity and 18 ± 1 C. Fifty-six percent of the control group (no TRC exposure) hatched. None of the embryos exposed to a measured TRC concentration of 0.21 mg/l hatched. Only 3.5 percent of the embryos exposed to 0.07 mg/l TRC and 23 percent of those exposed to 0.01 mg/l TRC hatched. Incipient LC50 bioassays were used to determine the sensitivity of 2-, 12- and 30-day-old striped bass to concentrations of TRC in flowing water (1.0-3.0 ppt salinity at 18 ± 1 C). The estimated incipient LC50 was 0.04 mg/l TRC for 2-day-old prolarvae, 0.07 mg/l for 12-day-old larvae and 0.04 mg/l for 30-day-old juveniles. Histological examination of 30-day-old juveniles which survived exposure in the incipient LC50 bioassay indicated gill and pseudobranch damage for fish exposed to 0.21 to 2.36 mg/l TRC. Statistical analysis of avoidance tests conducted at 1.0-3.0 ppt salinity and 18 ± 1 C with 24-day-old larvae showed significant (X2, p < 0.05) and reproducible avoidance responses to measured TRC concentrations of 0.79-0.82 mg/l and 0.29-0.32 mg/l. No avoidance was indicated at TRC concentrations of 0.16-0.18 mg/l.

Middaugh, Douglas P., Louis E. Burnett and John A. Couch. 1980. Toxicological and Physiological Responses of the Fish, Leiostomus xanthurus, Exposed to Chlorine Produced Oxidants. Estuaries. 3(2):132-141. (ERL,GB 378). (Avail. from NTIS, Springfield, VA: PB81-126559)

The sublethal and lethal effects of chlorine produced oxidants (CPO) on juveniles of the estuarine teleost, Leiostomus xanthurus, were investigated in flowing water tests conducted at 30 ± 1°C and 26 to 31 ppt salinity. Short-term LT50 tests were conducted at two nominal concentrations of NaOCl, 1.0 and 1.4 mg/l (respective measured CPO concentrations 0.09 and 0.12 mg/l) which were sublethal in 2,880 minute exposures; and three nominal concentrations, 1.6, 1.8 and 3.2 mg/l NaOCl (respective measured CPO concentrations 0.13, 0,20 and 0.37 mg/l) which were acutely toxic. Opercular ventilation rates in exposed spot were much higher than in control fish, but returned to rates only slightly above those of controls during the latter portion of the 2,880 minute exposure to the two sublethal CPO concentrations. Opercular rates at the three acutely toxic CPO concentrations remained much higher than control rates until the exposed fish died. Blood pH after 2,880 minutes of exposure to the sublethal concentrations of CPO; or at the respective estimated LT50 for lethal concentrations, showed significant decreases (X as low as 6.84) compared to controls (X 7.35). No significant changes in the percent methemoglobin were observed. Oxygen uptake by spot was depressed at all of the measured concentrations of CPO tested. Histopathological examination showed that gill respiratory epithelial tissues sloughed away from the underlying pillar cells. Complete denudation of circulatory tissues and hemangiectic secondary lamellae were observed in gill tissues from fish exposed to the highest CPO concentration of 0.37 mg/l.

Schoor, W. Peter and John A. Couch. 1979. Correlation of Mixed-Function Oxidase Activity with Ultrastructural Changes in the Liver of a Marine Fish. EPA-600/J-79-087. Cancer Biochem. Biophys. 4(2):95-103. (ERL,GB 382). (Avail. from NTIS, Springfield, VA: PB80-184278)

Specimens of mullet (Mugil cephalus), a marine fish, were given single doses of 3-methylcholanthrene intraperitoneally and the activity of the microsomal mixed-function oxygenase system in the liver was measured by the metabolism of benzo(a)pyrene. The enzyme system was found to be inducible with concomitant ultrastructural changes in the hepatocytes. The specific activity and the metabolic profile approximate those of the rat.

Courtney, Lee A. and John A. Couch. 1984. Usefulness of Cyprinodon variegatus and Fundulus grandis in Carcinogenicity Testing: Advantages and Special Problems. EPA-600/J-84-063. Natl. Cancer Inst. Monogr. 65:83-96. (ERL,GB 442). (Avail. from NTIS, Springfield, VA: PB84-226513)

Cyprinodon variegatus and Fundulus grandis, two species of cyprinodontid fishes extensively studied and used in toxicological and biological investigations, are compared as laboratory test animals. Their ecology, general biology, and suitability for various types of experimentation are examined. A laboratory system for exposure of these species at critical life stages (embryos, fry, juveniles) to suspect carcinogens is described. A discussion of the use, findings, and potential of these species in oncological studies and carcinogen assays is presented, particularly in regard to responses to 3 known or suspect carcinogens (trifluralin, benzidine, and aflatoxin). Finally, advantages and disadvantages or special problems in using the species as carcinogen test animals are reviewed.

Dawe, Clyde J. and John A. Couch. 1984. Debate: Mouse Versus Minnow: The Future of Fish in Carcinogenicity Testing. EPA-600/D-83-039. Natl. Cancer Inst. Monogr. 65:223-235. (ERL,GB 472).

The text is part of a debate and advocates the use of small fish species as laboratory test organisms in detecting and determining potency and activity of suspected carcinogens. The usefulness of fish species is compared and contrasted with the usefulness of the more conventional assay species, such as rodents.

Patton, John S. and John A. Couch. 1984. Can Tissue Anomalies That Occur in Marine Fish Implicate Specific Pollutant Chemicals?. In: Concepts in Marine Pollution Measurements. EPA/600/D-85/122. Harris H. White, Editor. University of Maryland, College Park, MD. Pp. 511-538. (ERL,GB 474). (Avail. from NTIS, Springfield, VA: PB85-212405)

The advantage of using tissue abnormalities in wild fish as a measure of fish health is that the abnormality, unlike sensitive biochemical anomalies, cannot be said to have occurred during capture or transport. This paper is concerned with factors (chemical, physical, or biological) in polluted waters that cause pathology.

Hinton, David E. and John A. Couch. 1984. Pathobiological Measures of Marine Pollution Effects. In: Concepts in Marine Pollution Measurements. EPA/600/D-85/123. Harris H. White, Editor. University of Maryland, College Park, MD. Pp. 7-32. (ERL,GB 475). (Avail. from NTIS, Springfield, VA: PB85-211217)

This paper reviews major categories of pathobiological research, citing strengths and weaknesses of each. Where appropriate, examples from the aquatic toxicology literature are cited. Finally, the application of the pathobiological approach to field studies and the importance of findings in aquatic species to other species including man are discussed.

Fournie, John W., Steven S. Foss and John A. Couch. 1988. Multispecies System for Evaluation of Infectivity and Pathogenicity of Microbial Pest Control Agents in Nontarget Aquatic Species. EPA/600/J-88/284. Dis. Aquat. Org. 5(1):63-70. (ERL,GB 640). (Avail. from NTIS, Springfield, VA: PB89-209654)

Microbial pest control agents (MPCAs-viruses, bacteria, protozoa, and fungi) are being used as biological pesticides and herbicides. Many of these agents are considered potential MPCAs and could be used widely in the environment. Therefore, test animals must be selected and laboratory systems developed to evaluate safety of such agents to nontarget species. A simple, multispecies laboratory system has been designed and used to determine risks of infectivity and pathogenicity of Colletotrichum gloeosporioides (Collego®), a registered postemergent mycoherbicide, to nontarget freshwater and estuarine plant and animal species. Test organisms included a freshwater and an estuarine fish, crustacean, bivalve mollusc, and plant. These multispecies systems also permit evaluation of other MPCAs against nontarget aquatic species and provide a standardized procedure for safety testing. Results from this study have shown these multispecies test systems to be viable, inexpensive, and reliable. Histopathological methods used indicated no evidence that experimental exposure to the fungal MPCA in our test system caused infection or related pathogenicity in the selected nontarget species.

Winstead, James T. and John A. Couch. 1988. Enhancement of Protozoan Pathogen Perkinsus marinus Infections in American Oysters Crassostrea virginica Exposed to the Chemical Carcinogen N-nitrosodiethylamine (DENA). EPA/600/J-88/352. Dis. Aquat. Org. 5(3):205-213. (ERL,GB 645). (Avail. from NTIS, Springfield, VA: PB89-237184)

American oysters Crassostrea virginica exposed to high concentrations (600 mgl-1) of n-nitrosodiethylamine (DENA) during winter (February to May) showed significant enhancement of an epizootic apicomplexan parasite, Perkinsus marinus. The parasite reproduced and caused atypical lesions in exposed oysters in water temperatures at its lower range (20°C). The reasons for this enhancement are not clear but may reflect damage to the oysters' nonspecific, cellular defense mechanisms by the DENA without concomitant negative effects on the parasite.

Harshbarger, John C., Elliot R. Jacobson, Charlie E. Smith and John A. Couch. 1980. Hematopoietic Neoplasms in Invertebrates and Cold-Blooded Vertebrates. In: Advances in Comparative Leukemia Research, 1979. David S. Yohn, Boris A. Lapin, and James R. Blakeslee, Editors. Elsevier North-Holland, New York, NY. Pp. 223-225. (ERL,GB X158).

Many excellent examples of hematopoietic neoplasms occur in invertebrate and cold-blooded vertebrate animals. Since these groups comprise over 99 per cent of the animal kingdom and contain an enormous diversity compared to warm-blooded animals, they could undoubtly provide models optimally suited for testing specific hypotheses. I recommend cancer researchers consider this potential in planning future experiments.

Hinton, David E., John A. Couch, Swee J. Teh and Lee A. Courtney. 1988. Cytological Changes During Progression of Neoplasia in Selected Fish Species. Aquat. Toxicol. 11(1,2):77-112. (ERL,GB X539).

Cytological changes during progression of 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. 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 nuclear to cytoplasmic rations, 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 cells (Cyprinodon variegatus only). Enzyme alterations preceded other morphologic alterations and were seen in cells of foci and tumors suggesting lineage of phenotypic alteration. Cytologic changes within other resident cell populations during neoplastic progression were reviewed.

Grizzle, John M., Marshall R. Putnam, John W. Fournie and John A. Couch. 1988. Microinjection of Chemical Carcinogens into Small Fish Embryos: Exocrine Pancreatic Neoplasm in Fundulus grandis Exposed to N-Methyl-N'-Nitro-N-Nitrosoguanidine. EPA/600/J-88/282. Dis. Aquat. Org. 5(2):101-105. (ERL,GB X571). (Avail. from NTIS, Springfield, VA: PB89-208698)

Gulf killifish, Fundulus grandis, embryos were injected with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Retention of carcinogen in the egg immediately after injection was highly variable. Hatching percentages of MNNG-injected and uninjected embryos were similar, but survival of MNNG-injected fish during the first week after hatching was less than half of the survival of controls. One of three MNNG-exposed fish examined after 20 weeks had a pancreatic acinar cell carcinoma, a type of neoplasm seldom found in fish. No neoplasms were found in controls nor in MNNG-exposed fish examined after 34 weeks. The neoplasm was invasive and had a high mitotic index, but no metastases were found. The acinar cell origin of the tumor was confirmed by ultrastructural examination of tissue reprocessed from paraffin into epoxy resin.