Experimental studies were conducted by injecting or feeding white spot syndrome virus (WSSV) derived from infected shrimp, Penaeus monodon (Fabricius), collected from the south‐east coast of India, to five species of shrimp, two species of freshwater prawns, four species of crabs and three species of lobsters. All species examined were susceptible to the virus. Experimental infections in the shrimp had the same clinical symptoms and histopathological characteristics as in naturally infected P. monodon. A cumulative mortality of 100% was observed within 5–7 days in shrimp injected with WSSV and 7–9 days in shrimp fed with infected tissue. Two species of mud crab, Scylla sp., survived the infection for 30 days without any clinical symptoms. All three species of lobsters, Panulirus sp., and the freshwater prawn, Macrobrachium rosenbergii (De Man), survived the infection for 70 days without clinical symptoms. However, bioassay and histology using healthy P. monodon revealed that crabs, prawns and lobsters may act as asymptomatic carriers/reservoir hosts of WSSV. This is the first report to suggest the carrier/reservoir capacity of these hosts through histological and bioassay evidences. Ultrastructural details of the virus in experimentally infected shrimp, P. vannamei, (Boone), were also studied.

An immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies cells in all active phases of the cell cycle. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to three small fish species, medaka (Oryzias latipes), guppy (Poecilia reticulata), and western mosquitofish (Gambusia affinis) that are used in carcinogenesis bioassays and environmental sentinel studies. Our study showed that PCNA can be identified in routinely processed, paraffin embedded specimens of these fishes. Optimum staining conditions were dependent on fixative, primary antibody, antigen retrieval processing, and protein blocking reagent. Best results were achieved using 10% neutral buffered formalin as the fixative, clone PC10 as the primary antibody, and a combination of powdered milk and bovine serum albumin as a protein block. Except for medaka specimens, antigen retrieval was not required for specimens preserved in 10% neutral buffered formalin, but was required for the other fixatives tested. In whole fish specimens, PCNA marked cells in normally proliferating tissues such as testis, ovary, primary filament epithelium of the gill, hematopoietic tissues, thymus, retina and alimentary tract. The study demonstrated the successful application of mammalian-based PCNA technology to these aquatic species. Further applications of the assay will aid in understanding the role of cell proliferation in normal, diseased, and toxicant-affected tissues of aquatic animals.

Infections by multiple species of bacteria occurred in hepatopancreatic epithelial cells of cultured Pacific white shrimp (Penaeus vannamei). Grossly, hepatopancreases of moribund shrimp were pale white. Light microscopically, hepatopancreatic tubules appeared atrophied and were associated with granulomas. Examination by scanning and transmission electron microscopy revealed heavy cytoplasmic infections by three forms of microorganisms: (1) a rickettsia-like bacterium, (2) a helical form of a mollicute-like bacterium, and (3) a filamentous mollicute-like bacterium. The rod-shaped rickettsia (900 nm long by 300 nm wide) appeared to be free in the cytoplasm and had both a plasma membrane and a cell wall. Neither form of mollicute possessed a cell wall. The helical mollicute was blunt at its wide end (about 260 nm in diameter) where it contained electron-lucent bodies. Helical turns along its tapered axis resembled those of a spiroplasma (the only helical form of mycoplasma in the class Mollicutes) or a spirochete. The helical bacterium did not possess periplasmic flagella characteristic of spirochetes, which lends support to its being a type of spiroplasma. The filamentous mollicute consisted of masses of short, branched filaments 60 nm wide with intermittent spherical dilations and terminal blebs on the branches. The presumed mollicutes have not been reported previously in crustaceans. Each bacterium, or concurrent infections of the bacteria, are pathogenic to cultured shrimp, could impact culture operations and thus deserve more study.

A need exists for whole animal toxicity, mutagenesis, and carcinogenesis models that are alternative to the traditional rodent test models and that are economical, sensitive, and scientifically acceptable. Among small fish models, the Japanese medaka (Oryzias latipes) is preeminent for investigating effects of carcinogenic and/or toxic waterborne hazards to humans. The guppy (Poecilia reticulata), although less widely used, is valuable as a comparison species. Both species are easy to maintain and handle in the laboratory and there is a large body of background information on their responsiveness to a range of classes of carcinogens. There are considerable data on the occurrence of background diseases and on spontaneous neoplastic lesions, both of which occur relatively rarely. With few modifications, the medaka and guppy are amenable to carcinogenicity testing under the rigid standards established by the National Toxicology Program (NTP) for rodent tests. The advantages of the small fish in carcinogenesis studies are best realized in long-term studies that involve environmentally realistic exposures. Studies to identify chronic effects can be conducted in about 12 months, near the life span of medaka in our laboratory. Practically, 9-month studies are optimal but shorter study cycles and a variety of exposure/growout and initiation/promotion scenarios are available. Studies on 3 compounds tested in medaka under NTP protocols are under review and preliminary analysis indicates that chronic carcinogenicity bioassays with medaka, guppy, and potentially with other small fish species are feasible and scientifically valid.

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