Life cycle of Calyptospora funduli (Apicomplexa: Calyptosporidae).

The taxonomic status of the extraintestinal piscine coccidium Calyptospora funduli is based in part on its requirement of an intermediate host (the daggerblade grass shrimp Palaemonetes pugio). In the present study, grass shrimp fed livers of Gulf killifish (Fundulus grandis) infected with sporulated oocysts of C. funduli exhibited numerous sporozoites suspended in the intestinal contents when fresh squash preparations were examined by light microscopy. Using this method, sporozoites were not seen in intestinal epithelial cells of the grass shrimp or in any other cell types. Ultrastructural examination, however, revealed sporozoites in the cytoplasm of the gut basal cells. Cross-sections of 1-13 sporozoites were seen within a single cell, and those sporozoites each appeared to be situated in individual membrane-bound vesicles, rather than in a single parasitophorous vacuole. These ultrastructural observations indicate that in the grass shrimp intermediate host, sporozoites that develop into an infective stage probably undergo that development in gut mucosal basal cells. Prior studies revealed that these sporozoites modified their structure over 4-5 days and that before that time, they were not infective to the fish host. Following ingestion of an infected shrimp by a killifish, the infective sporozoites apparently reach the liver of their killifish definitive hosts through the bloodstream. Sporozoites were seen in blood smears from the longnose killifish, Fundulus similis, 4 hr after fish were fed experimentally infected grass shrimp. Additionally, coccidian trophozoites and early meronts were seen in hepatocytes from several longnose killifish at 48, 72, and 96 hr postinfection. This study, in conjunction with previous findings, clearly confirms that a true intermediate host is required in the life cycle of C. funduli, that a developmental period of about 5 days in grass shrimp is necessary for sporozoites to become infective to killifishes, and that sporozoites do occur intracellularly in gut basal cells of the grass shrimp.

Proliferative lesions in swimbladder of Japanese medaka Oryzias latipes and guppy Poecilia reticulata.

Thirteen cases of proliferative lesions of the swimbladder were encountered in Japanese medaka Oryzias latipes and guppy Poecilia reticulata from about 10,000 medaka and 5000 guppies used in carcinogenicity tests and histologically examined. Two of the 4 cases from medaka and 8 of the 9 from guppies occurred in untreated control specimens. The lesions affected the gas gland epithelium and included hyperplasia, adenoma, and adenocarcinoma. One medaka had hyperplasia of the gas gland epithelium and in 1 guppy the gland was enlarged with an increase in the number of epithelial layers. Gas gland adenomas, 3 cases in medaka and 1 in the guppy, were typically larger than the hyperplastic lesions, formed expansive masses up to 1 mm in greatest dimension, and exhibited a solid or glandular growth pattern and mild cellular pleomorphism. Adenocarcinoma was the most advanced lesion and all 7 cases occurred in guppies. Adenocarcinomas sometimes filled the entire swimbladder and measured up to 2.5 mm in diameter. Cells of adenocarcinomas were highly pleomorphic, with atypical nuclei, and an elevated mitotic activity. Because most of these tumors occurred in fish from control groups or in tests with noncarcinogenic compounds, the lesions observed here are probably spontaneous rather than chemically induced. Their rare occurrence, however, makes swimbladder proliferative lesions in small-fish carcinogenesis models sensitive indicators of compounds that might target cells of the gas gland.

Evaluation of bleached kraft mill process water using Microtox(R), Ceriodaphnia dubia, and Menidia beryllina toxicity tests.

To determine whether a 7- to 10-d embryo toxicity/teratogenicity test with the inland silverside fish, Menidia beryllina, is a sensitive indicator for evaluation of bleached kraft mill effluents, we compared this test with the Microtox(R) 15-min acute toxicity test and the Ceriodaphnia dubia 7-d chronic toxicity test. Water samples used in each test were collected from three areas in a bleached kraft pulp and paper mill using a 100% chlorine dioxide bleaching process: 1) river water prior to use in the mill; 2) the combined acid/base waste stream from the pulping process prior to biological treatment in the aerated stabilization basin (ASB); and 3) the effluent from the ASB with a retention time of approximately 11 d. Relative toxicity determined by the three tests for each water sampling location was compared. All three toxicity tests were predictive indicators of toxicity; however, the C. dubia and M. beryllina tests were the more similar and sensitive indicators of toxicity. Process water (ASB influent) prior to biological treatment in the ASB was toxic at all concentrations using the Microtox(R) and C. dubia tests. The fish embryo test showed no toxicity at 1% concentrations, slight toxicity at 10%, and acute toxicity at the 100% ASB influent concentration. Tests with biologically-treated ASB effluent indicated a substantial reduction in observed toxicity to Microtox(R) bacteria, C. dubia, and M. beryllina. No toxic responses were observed in any test at a 1% ASB effluent concentration which was the approximate effluent concentration in the receiving river following mixing. No relationship was found among any toxicological response and effluent levels of adsorbable organic halides, polychlorinated phenolic compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, total suspended solids, color, chemical oxygen demand, or total organic carbon.

Diagnostic criteria for degenerative, inflammatory, proliferative nonneoplastic and neoplastic liver lesions in medaka (Oryzias latipes): consensus of a National Toxicology Program Pathology Working Group.

Diagnostic criteria are presented for degenerative, inflammatory, nonneoplastic proliferative, and neoplastic lesions in the liver of medaka (Oryzias latipes), a small fish species frequently used in carcinogenesis studies. The criteria are the consensus of a Pathology Working Group (PWG) convened by the National Toxicology Program. The material examined by the PWG was from Medaka exposed to N-nitrosodiethylamine for 28 days, removed to clean water, and sacrificed 4, 6, or 9 mo after initiation of exposure. Degenerative lesions included hepatocellular intracytoplasmic vacuolation, hepatocellular necrosis, spongiosis hepatis, hepatic cysts, and hepatocellular hyalinization. Inflammatory lesions consisted of granulomas, chronic inflammation, macrophage aggregates, and focal lymphocytic infiltration. Nonneoplastic proliferative lesions comprised foci of cellular alteration (basophilic focus, eosinophilic focus, vacuolated focus, and clear cell focus) and bile duct hyperplasia. Neoplastic lesions included hepatocellular adenoma, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma. Two lesions composed mainly of spindle cells were noted, hemangiopericytoma and spindle cell proliferation. Rather than being an exhaustive treatment of medaka liver lesions, this report draws from the published literature on carcinogen-induced liver lesions in medaka and other fish species and attempts to consolidate lesion criteria into a simplified scheme that might be useful to pathologists and other researchers using medaka lesions for risk assessment or regulatory purposes.

Exocrine pancreatic neoplasms in the mummichog (Fundulus heteroclitus) from a creosote-contaminated site.

A high prevalence of exocrine pancreatic neoplasms occurred in mummichog, Fundulus heteroclitus, from a creosote-contaminated site in the Elizabeth River, Virginia. A total of 20 neoplasms were found in a group of about 1,300 fish obtained at this site over a 2-yr period. Of 240 fish collected during October 1991, 3.3% had pancreatic neoplasms. Adjusted total lesion prevalence for large adult fish (Size Class III: total length = 75-85 mm; Size Class IV: total length > 85 mm) was 6.7%. Pancreatic neoplasms were not observed in 234 fish collected at this site during May 1991, nor were they found in 420 fish obtained during fall 1991 from 1 uncontaminated and 6 moderately contaminated localities. Lesions involved both mesenteric and intrahepatic exocrine pancreas and ranged from well-differentiated acinar cell adenomas to poorly differentiated acinar cell carcinomas. One fish had an atypical acinar cell focus. All specimens with pancreatic neoplasms also had hepatocellular lesions. This epizootic of exocrine pancreatic neoplasia is the first to be reported in a wild fish population. Based on chemical characterization of the site and limited experimental data on chemically induced pancreatic carcinogenesis in other small fish species, the neoplasms were probably caused by exposure of the mummichog to chemical carcinogens in their environment.

Ultrastructure of normal and neoplastic exocrine pancreas in the mummichog, Fundulus heteroclitus.

The ultrastructure of exocrine pancreatic neoplasms occurring in the mummichog, Fundulus heteroclitus, from a creosote-contaminated site in the Elizabeth River, Virginia, is described and related to nonneoplastic exocrine pancreas. Normal mummichog pancreas was an anastomosing tubular gland, with parenchymal cells organized as branched, anastomosing tubules around a central ductular system. The pancreatic ductular system consisted sequentially of terminal canalicular lumens lined by acinar cells, pancreatic preductules formed by an acinar and a centroductular cell, pancreatic ductules lined by 2 centroductular cells, and pancreatic ducts lined by cuboidal or columnar epithelial cells resting on a basal lamina and stromal sheath. Acinar cell adenomas retained the normal tubular organization and relationship between acinar and centroductular cells. Tumor cells exhibited nuclear pleomorphism but contained a full complement of normal zymogen granules and rough endoplasmic reticulum (RER). Some adenomas exhibited necrosis and cellular degeneration. Acinar cell carcinomas ranged from well to poorly differentiated. They exhibited loss of cell polarity, moderate to severe nuclear pleomorphism, extensive variation in size, shape, and number of zymogen granules, variability in RER content, and cellular degeneration. Acinar cell neoplasms in the mummichog were similar to those induced chemically in other fishes and certain mammals, suggesting that this fish population has been exposed to potent chemical carcinogens and that the species may be an effective indicator of polluted estuarine environments.

Host specificity of Calyptospora funduli (Apicomplexa: Calyptosporidae) in atheriniform fishes.

Calyptospora funduli has a broad host specificity, infecting at least 7 natural and 10 additional experimental definitive hosts, all atheriniform fishes within 5 families, but most in the genus Fundulus. Barriers, apparently innate ones, prevent any development of C. funduli in perciform fishes but allow incomplete or abnormal development of the parasite in a few unnatural atheriniform hosts. In the freshwater species Fundulus olivaceus and Fundulus notti, these abnormalities consisted of asynchronous development, degeneration of the parasite in early stages of development, and the formation of numerous macrophage aggregates. Rivulus marmoratus has the ability to eliminate infections with a granulomatous inflammatory response. Additional barriers that limit natural infections of C. funduli in other hosts include feeding behavior, environmental conditions, and geographic isolation.

Unusual eosinophilic granule cell proliferation in coho salmon (Oncorhynchus kisutch).

Whitish, opaque, coalescing masses were observed coating the visceral organs of numerous adult coho salmon when they were dissected for artificial spawning in the autumn of 1983 at the Puntledge River hatchery, British Columbia. One affected fish was observed in November 1991. Histological examination of 10 of these fish (nine in 1983 and one in 1991) revealed that the lesions consisted of unusual proliferations of eosinophilic granule cells (EGCs) with some characteristics of neoplasia. The proliferative lesions extended throughout the gastrointestinal tract and infiltrated through all layers of the gut. Pancreatic tissue and adipose tissues associated with the pyloric caeca were also infiltrated and replaced by the proliferating EGCs. A mild diffuse infiltration was also observed in the spleen, liver and kidney. Histochemical analysis and ultrastructural examination showed the proliferating cells to be essentially identical with the EGCs which are usually concentrated in the stratum granulosum of the intestine in normal salmonid fishes.

Adenocarcinoma of the retinal pigment epithelium in the guppy Poecilia reticulata Peters.

A single case of adenocarcinoma of the retinal pigment epithelium occurred in a guppy, Poecilia reticulata, Peters. This is the first such tumour reported from fishes. The left eye of the affected fish was severely exophthalmic because of a large intraocular tumour mass. The tumour, which displaced normal retina anteriorly, consisted mainly of melanin-containing epithelial cells. Neoplastic cells were bilayered and arranged in a tubular pattern. The tumour was confined to the orbit. Although the specimen was from a group exposed to a mixture of halogenated organic compounds, the lesion was not considered to have been chemically induced because of its rare occurrence within the group as a whole.

Hepatic neoplasms in the mummichog Fundulus heteroclitus from a creosote-contaminated site.

High prevalences of idiopathic hepatic lesions were found in mummichog, Fundulus heteroclitus, from a site in the southern branch of the Elizabeth River, VA, contaminated with polycyclic aromatic hydrocarbons. Grossly visible hepatic lesions occurred in a total of 93% of the individuals from this site and 33% of these fish had hepatocellular carcinomas. Hepatic lesions were not detected in fish from two less contaminated sites. Lesions included foci of cellular alteration, hepatocellular adenoma, early and advanced hepatocellular carcinomas, and cholangiocellular proliferative lesions. Advanced carcinomas exhibited several distinct cellular patterns and some livers contained multiple neoplasms occupying up to 80% of the hepatic parenchyma. Sediments from the contaminated site contained extremely high concentrations (2200 mg/kg dry sediment) of polycyclic aromatic hydrocarbons, which are believed to originate from an adjacent wood treatment facility that has used creosote. Concentrations were 730- and 35-fold higher than those at the two other sites. These findings indicate a strong positive association between exposure to creosote-contaminated sediments and the high prevalence of hepatic neoplasms in a feral population of mummichog and support the putative role of polycyclic aromatic hydrocarbons in fish hepatocarcinogenesis. Additionally, they suggest that the mummichog may be a useful indicator of exposure to carcinogens in aquatic environments.

Goussia girellae n. sp. (Apicomplexa: Eimeriorina) in the opaleye, Girella nigricans.

Goussia girellae n. sp. is described from the opaleye fish, Girella nigricans. Merogonic stages were observed in the apices of intestinal epithelial cells, in the lamina propria, and in extra-intestinal sites including liver, gills, and spleen. Gamonts were observed in the intestinal epithelial cells. Only unsporulated oocysts were detected in the intestine, and sporulation occurred when feces containing oocysts were incubated for 48 h in seawater at 21 degrees C. Oocysts are elongated (24.8 x 14.7 micron) with a wall about 200 nm thick and have no residuum, micropyle, or polar granule. Sporocysts are ellipsoid (8.5 x 4.5 micron), have a thin two-layered wall approximately 30 nm thick, and consist of two valves joined by a suture. Although moribund opaleye were also infected with Gyrodactylus sp., Cryptobia sp., Cardicola sp., and epitheliocystis organisms (chlamydia), all fish were heavily infected with G. girellae and morbidity was thus attributed to the coccidium.

Exocrine pancreatic neoplasms induced by methylazoxymethanol acetate in the guppy Poecilia reticulata.

Exocrine pancreatic neoplasms developed in the guppy Poecilia reticulata following a single brief exposure to methylazoxymethanol acetate [(MAM-Ac) CAS: 592-62-1]. Fish 6-10 days old were exposed to concentrations of MAM-Ac up to 100 mg/liter for 2 hours. Exposed specimens were transferred to carcinogen-free water and sampled periodically for tumor development. Pancreatic neoplasms occurred in approximately 9% of histologically examined individuals exposed to 10 mg MAM-Ac/liter or less. Neoplastic lesions were not found in 122 control specimens. The neoplasms included 6 cases diagnosed as adenoma, 7 cases diagnosed as acinar cell carcinoma, and 2 cases diagnosed as adenocarcinoma. Adenomas consisted mainly of well-differentiated acinar cells that were filled with zymogen granules. Two adenomas also contained foci of atypical, less-differentiated acinar cells possessing basophilic, fibrillar cytoplasm. Acinar cell carcinomas occurred in several cellular patterns that ranged from well-differentiated to more anaplastic lesions; however, none exhibited areas of ductular proliferation. Adenocarcinomas, on the other hand, exhibited a glandular growth pattern and contained numerous ductlike structures. Both types of carcinomas appear to arise from primary acinar cells. Thus lesions probably progress from adenomas to acinar cell carcinomas and adenocarcinomas. The findings of carcinogen-induced pancreatic neoplasms in guppies further strengthen the usefulness of small fish species in carcinogen testing and provide an additional model for pancreatic tumors.

Intraocular neoplasms induced by methylazoxymethanol acetate in Japanese medaka (Oryzias latipes).

Intraocular neoplasms developed in the Japanese medaka, a small fish species, following a single brief exposure to methylazoxymethanol acetate [(MAM-Ac) CAS: 592-62-1]. Specimens 6-10 days old were exposed to doses of MAM-Ac up to 100 mg/liter for 2 hours and then transferred to carcinogen-free water for "grow-out." Of 218 exposed fish examined, 98 (45%) had neoplastic lesions in various stages of development. Of those exposed to 30 mg/liter or more, 57% had the lesions. No lesions were found in eyes or other tissues of 95 control specimens. Early and advanced neoplastic lesions were recognized. Early lesions were characterized by complexes of neoplastic retinal epithelium and tubes that consisted of cells of the sensory retina. Areas of mitotically active, heterogeneous cells associated with such complexes gave rise to advanced neoplasms. We considered the advanced neoplasms to be medulloepitheliomas, which differentiated into three principal cellular patterns: 1) solid masses of unpigmented cells, which frequently showed photoreceptor differentiation as well as ductular formation; 2) heavily pigmented cuboidal to columnar cells resembling retinal epithelium that formed adenomatous patterns; and 3) teratoid medulloepitheliomas. Teratoid medulloepitheliomas, which we considered the most advanced and malignant lesions, consisted of heterogeneous, highly mitotic, invasive cells and contained heteroplastic elements including striated muscle, undifferentiated mesenchymal tissues, and hyaline cartilage. We suggest that MAM-Ac induces hyperplasia of retinal cells followed by establishment of aberrant growth zones containing miscoded cells that give rise to medulloepitheliomas.

Development of aquarium fish models for environmental carcinogenesis: tumor induction in seven species.

For small fish species to be utilized as models for carcinogenicity testing they should be capable of developing neoplasms, preferably in multiple tissues, when exposed to known carcinogens. Seven species of small fish were exposed to methylazoxymethanol acetate (MAM-Ac) and tumor development was monitored. Specimens 6-10 days old were exposed to nominal concentrations of MAM-Ac up to 100 mg 1(-1) for 2 h, then transferred to carcinogen-free water. Hepatic neoplasms developed in the Japanese medaka, guppy, sheepshead minnow, Gulf killifish, inland silverside, rivulus, and fathead minnow. Additionally, neoplasms occurred in other organs and tissues of the medaka (retina, various mesenchymal tissues, exocrine pancreas, kidney, and nervous tissue), guppy (mesenchymal tissue, exocrine pancreas, and kidney), and sheepshead minnow (choroid gland, mesenchymal tissues, and nervous tissue). All tumors were diagnosed in specimens within 1 year post-exposure. Early signs of liver tumors appeared in medaka and guppy at about 1 month post-exposure. These studies show that both medaka and guppy would be good models because they appear sensitive to carcinogens, develop tumors in multiple tissues and are easy to breed and maintain. Certain other small fish species also may prove to be good models because of habitat preferences, breeding strategies, or genetic attributes.