Roxanna M. Smolowitz

Cytochrome P4501A induction and inhibition by 3,3′,4,4′-tetrachlorobiphenyl in an Ah receptor-containing fish hepatoma cell line (PLHC-1)

The induction of cytochrome P4501A1 (CYP1A1) in rat hepatoma cells has been used by some investigators to determine ‘dioxin equivalents’ in environmental samples, including extracts of fish tissues. However, the relative potency of inducing compounds may vary between species, suggesting the need for taxon-specific model systems. In this paper we present an initial characterization of CYP1A induction in one such system, a teleost liver cell line (PLHC-1) derived from a hepatocellular carcinoma of Poeciliopsis lucida (Hightower, L.E. and Renfro, J.L., 1988. J. Exp. Zool. 248, 290). Specific binding of the photoaffinity ligand 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin([125I]N3Br2DD) to proteins in PLHC-1 cytosol indicated the presence of the Ah receptor, which is known to control CYP1A induction in mammals. 3,3′,4,4′-Tetrachlorobiphenyl (TCB) induced a microsomal protein in PLHC-1 cells that was recognized by monoclonal antibody (MAb) 1-12-3 to scup CYP1A1 (P450E) on immunoblots. Immunohistochemical staining of whole cells with MAb 1-12-3 showed specific recognition of CYP1A induced by TCB. No staining was seen in untreated or vehicle-treated cells. There was an excellent quantitative correlation between amounts of CYP1A protein detected immunohistochemically and in immunoblots of cell homogenates. In a dose response experiment, maximal induction of ethoxyresorufin O-deethylase (EROD) activity occurred at 0.1 μM TCB; at higher concentrations (1 and 10 μM), EROD activity was reduced as compared to the activity at 0.1 μM TCB. In contrast, immunoreactive CYP1A protein increased with increasing TCB concentration up to 10 μM. The loss of EROD activity at high concentrations of TCB did not result from changes in cell number or viability. The apparent inhibition or inactivation of CYP1A catalytic activity by the higher concentrations of halogenated biphenyls has been seen, but not generally recognized, both in vivo and in cultured cells from diverse vertebrate species. PLHC-1 cells may be a good model system for studying Ah receptor-mediated regulation of gene expression, for determining the fish-specific toxic or inducing potency of halogenated aromatic hydrocarbon congeners, and for investigating the mechanism of CYP1A inhibition or inactivation by environmental contaminants such as TCB.

Experimental and Histological Studies of Four Life- History Stages of the Eastern Oyster, Crassostrea virginica, Exposed to a Cultured Strain of the Dinoflagellate Prorocentrum minimum

Effects of the dinoflagellate Prorocentrum minimum (strain EXUV) upon four life-history stages of the eastern oyster--embryos, feeding larvae, newly set spat, and juveniles--were investigated in laboratory exposure studies. Embryonic development was not affected significantly by living, heat-killed, or sonicated cells, or by growth-medium extracts from P. minimum cultures. Feeding larvae, however, showed poor growth and poor development of the digestive system when fed P. minimum, as compared with larvae fed Isochrysis sp. (strain T-ISO). Growth of larvae fed mixed P. minimum + Isochrysis diets was intermediate. Larvae and newly set spat that had been fed a diet of 1/3 P. minimum + 2/3 Isochrysis exhibited distinctive changes in digestive-system anatomy. Spat showed an abnormal accumulation of lipid in the stomach epithelium. Absorptive cells in the digestive glands of both larvae and spat contained accumulation bodies, often with a laminated, fibrous appearance in preparations for transmission electron microscopy. These accumulation bodies were PAS (periodic acid-Schiff) positive and may correspond to autolysosomal bodies within P. minimum cells. Juvenile oysters developed the ability to digest P. minimum, but only after a refractory period of about 2 weeks, during which most P. minimum was filtered but rejected as pseudofeces. The linking of accumulation bodies within absorptive cells of oyster digestive diverticula with dinoflagellate autolysosomal bodies suggests a mechanism by which some dinoflagellates interfere with feeding in phytoplankton grazers.

Pathologic Cuticular Changes of Winter Impoundment Shell Disease Preceding and During Intermolt in the American Lobster, Homarus americanus

Cuticular lesions from twenty-four market sized lobsters (Homarus americanus) with winter impoundment shell disease were examined. Histological descriptions of cuticular lesions were correlated with the substage of molt for each lobster, because cuticle components and inflammatory mechanisms vary in each. A lesion severity grading system was developed and applied to four specific substages of the five-stage (A-E) molting cycle. Lesions present in substage C4, in which the membranous layer is deposited, and D0 (passive premolt) were divided into five grades, ranging from mild erosions (Grade I) to severe ulceration (Grade V) of the cuticle. Cuticular lesions from lobsters in C4/D0 were compared with cuticular lesions from lobsters in substages C2/C3. Defensive mechanisms exhibited by animals in all substages were epicuticle deposition, melanization, inflammatory cell infiltration, and pseudomembrane formation. In addition, animals in C4 and D0 showed proliferation of the membranous layer in affected foci. The lesion grading scheme presented in this paper can be used to describe and compare both inter- and intraspecies crustacean shell lesions.

CULTURE-DEPENDENT CHARACTERIZATION OF THE MICROBIAL COMMUNITY ASSOCIATED WITH EPIZOOTIC SHELL DISEASE LESIONS IN AMERICAN LOBSTER, HOMARUS AMERICANUS

Abstract Epizootic shell disease in the American lobster is an important factor affecting lobster fisheries in and around the Long Island Sound. It is a strictly dermal disease, because no correlation was observed between occurrence of epizootic shell disease and hemolymph infection. The culturability of bacteria from lesions was variable and averaged around 1.1%. The lesions contained two to four orders of magnitude more bacteria than healthy carapace surfaces of the same animal. Chitinoclastic bacteria comprised a very small fraction of bacteria present in the lesions, suggesting that their role in epizootic shell disease may be limited. Phylogenetic analysis of bacteria isolated from the lesions showed no typical bacterial pathogens of lobsters such as Aerococcus viridans or Vibrio fluvialis. Moreover, bacteria commonly associated with shell disease of other Crustacea or other forms of shell disease of the American lobster were not found. Two common groups of bacteria were isolated from lesions of all lobsters used in this research: one belonging to a species complex affiliated with the Flavobacteriaceae family and the second belonging to a series of closely related if not identical strains of Pseudoalteromonas gracilis. Bacteria isolated from only a few lobsters were related to Shewanella frigidimarina, Alteromonas arctica, Vibrio lentus, Shewanella fidelia, Pseudoalteromonas tunicata and Vibrio spp. Based on the analyses of culturable isolates, overall microbial communities found in lesions of lobsters from eastern Long Island Sound and Buzzards Bay appear to be similar to each other.

MODIFIED LABORATORY CULTURE TECHNIQUES FOR THE EUROPEAN CUTTLEFISH SEPIA OFFICINALIS

The cuttlefish Sepia officinalis Linnaeus, 1758, is an important model for a variety of biological and biomedical investigations (1). To introduce this organism to the North American research communities, and make it readily available, various methods have been used to maintain or culture the species. The most intensive efforts and successes have been achieved by Forsythe and colleagues at the University of Texas Medical Branch in Galveston (2,3). Their most noteworthy achievement was to culture seven consecutive generations in large-scale recirculating seawater systems. Recently, cuttlefish were brought to the Marine Resources Center (MRC) of the Marine Biological Laboratory, where they have also been cultured successfully through their life cycle. Presently the third laboratory generation is under culture. Unlike previous work on Sepia culture in the United States, our focus has been to use a mostly open (flow-through) seawater system modified to function well through a northern winter, and to develop feeding methods that are suitable for our locale.

Bacterial Communities Associated with Lesions of Shell Disease in the American Lobster, Homarus americanus Milne-Edwards

ABSTRACT Shell disease in Crustacea is a widely recognized syndrome having a polymicrobial etiology, and manifesting itself as lesions of the shell with a variable structure and shell location. We characterized major members of bacterial communities in epizootic shell disease lesions of the American lobster (Homarus americanus, Milne Edwards) and compared these communities with the ones found in study cases of impoundment and enzootic shell disease. Bacteria belonging to several Flavobacteriaceae genera (Aquimarina, Tenacibaculum, Polaribacter, Maribacter, Cellulophaga) within the phylum Bacteroidetes appear to have particular attraction to lobster lesions. The most prominent Bacteroidetes in lobster lesions were representatives of the genus Aquimarina sp., but only Aquimarina ‘homaria’ was detected in all analyzed lesions of epizootic, impoundment, and enzootic shell disease. It was found on 45% of surfaces unaffected by shell disease, but in smaller numbers compared with lesions. Alphaproteobacteria represent the most diverse class of proteobacteria found in both lesions and on unaffected surfaces. Three bacteria of this class appear to be ubiquitous in shell disease lesions, but only one specific alphaproteobacterium tentatively assigned to the genus Thalassobius (herein designated as ‘Thalassobius’ sp.) was present in all analyzed lesions of epizootic, impoundment, and enzootic shell disease. A ubiquitous gammaproteobacterium called ‘Candidatus Homarophilus dermatus’ was also prevalent in lesions, but just as commonly it was associated with surfaces unaffected by shell disease. The bacteria A. ‘homaria’ and ‘Thalassobius’ sp. are dominant and appear obligatory in lobster shell lesions, and are only occasionally detected on unaffected surfaces, which serve as intermediate reservoirs for the two potential pathogens. Therefore, these two bacteria stand out as potential shell-disease pathogens.

Exposures of Homarus americanus Shell to Three Bacteria Isolated from Naturally Occurring Epizootic Shell Disease Lesions

ABSTRACT Epizootic shell disease (ESD) is an emerging form of shell disease of the American lobster (Homarus americanus) that has had detrimental effects on the fishery in southern New England. Three bacteria commonly isolated from lesions of wild lobsters with ESD—a novel Aquimarina sp. (A. ‘homaria’ I32.4), a novel Rhodobacteraceae species (‘Thalassobius’ sp. I31.1) and a Pseudoalteromonas sp. (Pseudoalteromonas ‘gracilis’ ISA7.3)—were applied directly to normal and abraded juvenile lobster carapaces, and then monitored for persistence over time and for the development of shell-disease lesions at 3 different temperatures (10°C, 15°C, and 20°C). Without abrasion of the carapace, no lesions developed in the exposures. After abrasion and exposure with a pure culture of A. ‘homaria’ I32.4, lesions developed at all 3 temperature and A. ‘homaria’ was detected in the lesions of all animals tested. Surprisingly, ‘Thalassobius’ sp. I31.1 also colonized these lesions. A coexposure with all 3 bacteria also demonstrated lesion development and the persistence of A. ‘homaria’ I32.4 and ‘Thalassobius’ sp. I31.1. The bacterium P. ‘gracilis’ ISA7.3 was not able to persist in any of the challenged lesions. Abraded areas of the cuticle with no bacteria added directly were also colonized by A. ‘homaria’ and ‘Thalassobius’ sp., and moderate lesions developed; however, the directly exposed lesions were significantly more severe (P < 0.05). The bacterium A. ‘homaria’, but not ‘Thalassobius’ sp., was detected in spontaneous lesions that developed independent of any abrasion and/or bacterial exposures. A novel bacterium, ‘Candidatus Kopriimonas aquarianus’ was also detected in spontaneous lesions. This study shows that 2 bacteria isolated from ESD lesions of wild lobsters are able to persist in and act together as important components of lesion development on abraded surfaces of American lobsters. This indicates that they are likely major contributors to lesion development in the ESD polymicrobial infection and may represent significant pathogens of the American lobster.

Cellular alterations preceding neoplasia in Pseudopleuronectes americanus from Boston Harbor

Abstract Dysplastic and neoplastic liver disease has been described in adult winter flounder from Boston Harbor. In this study we examined the pathogenesis of the disease. Early changes, in fish of 100–300 mm, included biliary proliferation, abnormal vacuolation of biliary pre-ductular epithelial cells and macrophage aggregation. The identification of biliary pre-ductular epithelial cells was based on nuclear morphology, numerous cellular junctions with adjacent hepatocyte apices, lack of contiguity with sinusoidal structures and central location in the hepatic tubule. Fish longer than 300 mm had progressively worsening pan-tubular changes in cholangiocytes and hepatocytes which culminated in grossly visible foci of vacuolation and associated neoplasia. Flounder from less contaminated sites showed occasional macrophage aggregations, but none of the other changes noted above. We suggest that the initial vacuolar change in Boston flounder occurs predominantly in pre-ductular cells, which may be stem cells, extending later to fill entire hepatic tubules and intrahepatic portions of the biliary system. The involvement of the vacuolation process in the development of neoplasia is a question we are currently addressing.

INFLUENCE OF HOST GENETIC ORIGIN AND GEOGRAPHIC LOCATION ON QPX DISEASE IN NORTHERN QUAHOGS (=HARD CLAMS), MERCENARIA MERCENARIA

Abstract QPX (Quahog Parasite Unknown) a protistan pathogen of northern quahogs (=hard clams), Mercenaria mercenaria, has caused disease outbreaks in maritime Canada, and in Massachusetts, New York, New Jersey, and Virginia, USA. Although epizootics have occurred in wild hard clam populations, the parasite has most seriously affected cultured hard clams, suggesting that aquaculture practices may promote or predispose clams to the disease. In this investigation the influence of clam genetic origin and the geographic location at where they are grown on QPX disease susceptibility was examined in a common garden experiment. Aquaculture stocks were acquired from hatcheries in Massachusetts, New Jersey, Virginia, South Carolina, and Florida and spawned at a single hatchery in Virginia. All stocks were originally, although not exclusively, derived from wild hard clam populations from each state. The seed clams were deployed at two sites, New Jersey and Virginia, and evaluated during the subsequent 2.5 y for growth, survival, and QPX disease. At both sites, South Carolina- and Florida-derived clam stocks exhibited significantly higher QPX prevalence and lower survival than New Jersey and Massachusetts clam stocks. Levels in the Virginia stock were intermediate. In Virginia, mortality at the termination of the experiment was 78%, 52%, 36%, 33%, and 20% in the Florida, South Carolina, Virginia, Massachusetts, and New Jersey hard clam stocks, respectively. Mortality was significantly correlated with QPX prevalence. Maximum QPX prevalence in the South Carolina and Florida stocks ranged from 19% to 21% and 27% to 29%, respectively, whereas in the Virginia, New Jersey, and Massachusetts stocks prevalence was 10% or less. Similar trends were observed in New Jersey where mortality at the termination of the experiment was estimated to be 53%, 40%, 20%, 6%, and 4% in the Florida, South Carolina, Virginia, Massachusetts, and New Jersey clam stocks, respectively. QPX prevalence peaked at 18% in the Florida stock, 38% in the South Carolina, 18% in the Virginia, and 5% in the New Jersey and Massachusetts stocks. These results suggest that host genotype is an important determinant in susceptibility to QPX disease. As such, hard clam culturist should consider the genetic origin of clam seed stocks an important component of their QPX disease avoidance/management strategies.

Developmental Expression of the Nfe2-Related Factor (Nrf) Transcription Factor Family in the Zebrafish, Danio rerio

Transcription factors in the CNC-bZIP family (NFE2, NRF1, NRF2 and NRF3) regulate genes with a wide range of functions in response to both physiological and exogenous signals, including those indicating changes in cellular redox status. Given their role in helping to maintain cellular homeostasis, it is imperative to understand the expression, regulation, and function of CNC-bZIP genes during embryonic development. We explored the expression and function of six nrf genes (nfe2, nrf1a, nrf1b, nrf2a, nrf2b, and nrf3) using zebrafish embryos as a model system. Analysis by microarray and quantitative RT-PCR showed that genes in the nrf family were expressed throughout development from oocytes to larvae. The spatial expression of nrf3 suggested a role in regulating the development of the brain, brachia and pectoral fins. Knock-down by morpholino anti-sense oligonucleotides suggested that none of the genes were necessary for embryonic viability, but nfe2 was required for proper cellular organization in the pneumatic duct and subsequent swim bladder function, as well as for proper formation of the otic vesicles. nrf genes were induced by the oxidant tert-butylhydroperoxide, and some of this response was regulated through family members Nrf2a and Nrf2b. Our results provide a foundation for understanding the role of nrf genes in normal development and in regulating the response to oxidative stress in vertebrate embryos.