Little is known about the nature of bacteria associated with the surface and gut of marine copepods, either in laboratory-reared animals or in the natural environment. Nor is it known whether such animals possess a gut flora. The present report deals with studies of microorganisms isolated from healthy, laboratoryreared copepods of the species Acartia tonsa Dana, from several species of wild copepods collected from a marine or estuarine environment, and from laboratory dishes containing moribund copepods. Evidence for a unique gut flora in laboratory-reared animals is presented; the predominant bacteria were represented by the genus Vibrio. Other organisms such as Pseudomonas and Cytophaga were found less abundantly associated with the copepods and not specifically associated with the gut. Acartia tonsa Dana, an estuarine and neritic calanoid copepod, has proved to be useful as an experimental animal in standardized laboratory culture (25). However, little is known about the effects of associated microorganisms on such marine invertebrates, or, in fact, about the actual associations themselves, either in the natural environment or in laboratory cultures. Ecological relationships and changes in such relationships that might occur when wild copepods are brought into culture should be understood, especially if there is a unique association between copepods and bacteria, such as symbiosis or commensalism. Mass mortalities frequently observed in laboratory culture of copepods may be caused by specific pathogens or opportunistic bacteria, but little is known about bacterial pathogens infecting copepods. In addition, the gut flora of the copepod may confer protection on the animals, but until the existence of a commensal gut flora in the copepod is established, such an hypothesis cannot be proven. Research for the project reported here was initiated on a cruise in the Gulf of Mexico aboard the U.S.N.S. MIZAR. Several species of copepods were collected at four different stations, including sites in the open ocean, off the continental shelf, and in the Mississippi Delta. Samples were also collected in the Anclote River,

Production of a toxin by Vibrio parahaemolyticus Kanagawa-phenomenon negative strains was examined. Ammonium sulfate fractions of broth culture filtrates were dialyzed, concentrated by lyophilization, and tested for toxic effects by mouse intraperitoneal injection. One fraction, which we think is a toxin, was isolated from a broth culture filtrate of V. parahaemolyticus FC 1011 (a Kanagawaphenomenon negative strain) and consistently produced lethal effects in mice at high concentrations and diarrhea in lower concentrations. The toxin was assayed for mouse LD50 and ability to produce diarrhea via forced feeding in mice. V. parahaemolyticus FC 1011 toxin was found to be protein, to be inactivated by heat or trypsin hydrolysis, and to produce positive skin permeability reactions in rabbits. However, it failed to induce fluid accumulation in ligated ileal loops in rabbits.Prominent among symptoms recorded for victims of Vibrio parahaemolyticusassociated food poisoning are diarrhae, abdominal pain and vomiting, with chills, fever, headache and bloody stools less frequently reported (2,3,7,8,10,19,33,(36)(37)(38). V. parahaemolyticus illnesses have frequently been diagnosed as dysentery and non-typhoidal salmonellosis (1-3). Bacterial-induced diarrhea is currently thought to be mediated by at least three mechanisms : (i) cell-free protein enterotoxins, liberated by nonpenetrating organisms and initiating fluid accumulation in the gut, viz., Vibrio cholerae (12), Escherichia coli (29) and Clostridium perfringens (10) , (ii) invasion by organisms, such as Shigella (15) and E. coli (11), manifesting cytotoxic properties which interfere with host cell functions and cause diarrhea; and (iii) a combination of these. Because of the variety of symptoms reported by V. parahaemolyticus gastroenteritis victims and the conflicting pathology data, it is not clear whether the disease is a manifestation of effects of enterotoxin on the host, invasion of the causative agent via the gut, or to toxic components produced only by certain serotypes of V. parahaemolyticus. Another member of the genus Vibrio, V. cholerae, is known to produce an exo-enterotoxin, associated with symptoms elicited by the organism (12). Because of the strong similarity of V. parahaemolyticus-associated disease with cholera, there is reason to suspect that V. parahaemolyticus may produce an enterotoxin (6, 40).

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Biological properties of endotoxins prepared from three strains of Vibrio parahaemolyticus were compared with reference to commercially prepared Salmonella typhi endotoxin. Endotoxin assays performed in rabbits included dermal Shwartzman reactivity, pyrogenicity, heat stability, and ability to induce tolerance as well as cross-tolerance.Mice were used for endotoxin LD50 determinations. Results showed V. parahaemolyticus endotoxins were similar to that of S.typhi strain 0901.Induction of tolerance to V. parahaemolyticus strain 11590 endotoxin resulted in complete cross-tolerance to S. typhi endotoxin, and vice versa. Partial cross-tolerance to S. typhi endotoxin was demonstrated with rabbits rendered tolerant to endotoxin from V. parahaemolyticus strains Sak-3 and FC 1011. Absorption spectra, nitrogen, phosphorus and carbohydrate analyses revealed additional similarities between endotoxins from V. parahaemolyticus and endotoxin from a member of the Enterobacteriaceae.Known in Japan for two decades as a causative agent of large outbreaks of seafoodassociated poisoning, Vibrio parahaemolyticus has recently been found associated with similar outbreaks in the U.S.A. [1,4,7,8,14,26,40,42,43], and other countries [2,3]. Nevertheless, the mode of pathogenesis of V. parahaemolyticus is poorly understood. Strains isolated from clinical specimens and displaying hemolysis on selected media (Kanagawa-phenomenon positive) are considered to be enteropathogenic [30,39,49], whereas nonhemolytic (Kanagawa-phenomenon negative) seafood or seawater isolates are generally considered non-enteropathogenic [19,37,38]. The problem is further complicated by the frequent isolation of several different serotypes of V. parahaemolyticus during an outbreak of food poisoning [7,8].Biologically active components (hemolysins) from selected strains of V.parahaemolyticus have been isolated which do not, however, account for the spectrum of gastroenteritis symptoms observed in clinical cases [12,17,19,25,28,29,31,36,50,51]. A single case of endotoxin shock and leg gangrene due to infections by V. parahaemolyticus following an attack of gastroenteritis has been demonstrated [36].Endotoxins from V.parahaemolyticus have not previously been studied, although some chemical analyses ofO antigens [44,45] and K antigens have been reported [32,33]. The purpose of this research was to study the biological properties of V.parahaemolyticus endotoxins and to determine if they are similar to endotoxins classically prepared from selected members of the Enterobacteriaceae, such as Salmonella typhi.

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