Edmundo Casillas

Suppression of B-cell mediated immunity in juvenile chinook salmon (Oncorhynchus tshawytscha) after exposure to either a polycyclic aromatic hydrocarbon or to polychlorinated biphenyls

Juvenile chinook salmon (Oncorhynchus tshawytscha) were injected intraperitoneally with either the polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA)1 or with the commercial polychlorinated biphenyl (PCB) mixture, Aroclor 1254, to assess effects on the B-cell mediated immune response. B-cell mediated immunity was assessed by examination of the primary and secondary plaque-forming cell (PFC) responses of anterior kidney and splenic leukocytes to a T-independent antigen, TNP-keyhole limpet hemocyanin (TNP-KLH). Salmon exposed to DMBA at dosages of 20% or 1% of the 96 hr LD50 (12.7 mg and 0.6 mg/kg of salmon, respectively) or to PCBs at a dosage of 20% of the 96 hr LD50 (54.0 mg/kg of salmon) exhibited a suppressed PFC response. The secondary PFC response of anterior kidney and splenic leukocytes to both antigens and the primary splenic PFC response to TNP-LPS were suppressed in salmon exposed to either DMBA or PCBs. However, only the primary PFC response of anterior kidney leukocytes to TNP-LPS was suppressed in salmon exposed to PCBs and no suppression of this response was observed in salmon exposed to DMBA. Neither anterior kidney or splenic leukocytes from salmon exposed to DMBA or PCBs showed an altered primary PFC response to the T-dependent antigen, TNP-KLH. These results suggest that B-cell mediated immunity in salmon is suppressed by known mammalian immunosuppressants and that suppression of the PFC response observed previously in salmon from an urban estuary may be due to contaminant exposure.

Inducibility of spawning and reproductive success of female english sole (parophrys vetulus) from urban and nonurban areas of puget sound, Washington

Abstract Vitellogenic female English sole were sampled from four areas in Puget Sound that varied in the nature and degree of chemical contamination. The fish were then injected with an analogue of Luteinizing Hormone Releasing Hormone (LHRH) in the laboratory to induce spawning. Ability to spawn, time to spawn, larval viability, and initial concentrations of plasma estradiol and vitellogenin [measured as alkaline-labile protein associated phosphate (ALP)] were assessed. In general, low initial plasma estradiol and ALP concentrations and subsequent reproductive impairment were most common in English sole from sites where levels of sediment contaminants [e.g. polycyclic aromatic hydrocarbons and polychlorinated biphenyls (PCB)] and measures of contaminant exposure in fish (hepatic PCB concentrations and aryl hydrocarbon hydroxylase activity) were highest, and where pollution-associated liver lesions in fish (i.e. neoplasms, foci of cellular alterations, specific degeneration/necrosis, and storage disorders) were most prevalent. Additionally, the time to spawn was found to be inversely correlated with initial plasma estradiol concentrations. Spawning success was found to be positively correlated with initial plasma estradiol and ALP concentrations as determined by logistic regression analysis; nearly 62% of the variability in the spawning response could be accounted for by these factors. Fertilization success was also found to be positively correlated with initial ALP concentrations, whereas fish captured from contaminated sites produced lower proportions of normal larvae. Overall, these findings suggest that contaminant exposure may result in poor reproductive success of female English sole. This may be related to a hormone imbalance or to slower ovarian development (non-synchronous timing) of female English sole from contaminated sites.

Increased Susceptibility of Juvenile Chinook Salmon to Vibriosis after Exposure to Chlorinated and Aromatic Compounds Found in Contaminated Urban Estuaries

Abstract Saltwater-adapted juvenile chinook salmon Oncorhynchus tshawytscha exposed to aromatic and chlorinated compounds, representative of contaminants found in urban estuaries in Puget Sound, have a higher susceptibility to vibriosis than do fish exposed only to the solvent vehicle. Susceptibility to vibriosis was assessed by examining the percent cumulative mortality of the salmon after exposure to the bacterial pathogen Vibrio anguillarum. The aromatic and chlorinated compounds examined consisted of a sediment extract from the Hylebos Waterway that was enriched in butadienelike compounds (chlorinated-enriched Hylebos Waterway sediment extract (CHWSE)), a model mixture of polycyclic aromatic hydrocarbons (PAHs), a polychlorinated biphenyl mixture (Aroclor 1254), hexachlorobutadiene (HCBD), and 7,12-dimethylbenz(a)anthracene (DMBA). Two trials were conducted. In trial l, the percent cumulative mortality of juvenile chinook salmon exposed to V. anguillarum after receiving either CHWSE, HCBD, or the mode...

Suppression of immunological memory in juvenile chinook salmon (Oncorhynchus tshawytscha) from an urban estuary

In this present study, juvenile chinook salmon were collected from a contaminated urban estuary, the Duwamish Waterway, as well as from a non-urban estuary, the Nisqually River estuary, to determine if exposure to toxic chemicals affects immunocompetency. Juvenile chinook salmon were also sampled from the two hatcheries that release salmon into these estuaries. The ability of anterior kidney (AK) and splenic (SP) leucocytes from primed and unprimed juvenile salmon from the Duwamish Waterway, Nisqually River estuary, and their respective hatcheries to produce a primary and secondary in vitro plaque-forming cell (PFC) response to the hapten, trinitrophenyl (TNP) was examined. Trinitrophenyl-keyhole limpet haemocyanin (TNP-KLH) was added in vitro to AK leucocytes or as TNP-lipopolysaccharide (TNP-LPS) to both AK and SP leucocytes. The primary AK and SP plaque-forming cell response to TNP in salmon from the estuaries or hatcheries was not significantly different. Primed AK leucocytes from salmon collected from the hatcheries and the non-urban estuary were able to produce a heightened secondary response to TNP-KLH; in contrast, primed AK leucocytes from salmon collected from the urban estuary were unable to produce a secondary PFC response to TNP-KLH. Anterior kidney leucocytes from salmon collected from all four areas were able to produce a heightened secondary PFC response to TNP-LPS. However, the AKs plaque-forming cell response generated in primed chinook salmon collected from the urban estuary to TNP-LPS was significantly lower than that produced in salmon from the hatchery. This suppressed PFC response in primed AK cells to TNP-LPS was not observed in primed AK leucocytes from salmon collected from the non-urban estuary and its hatchery. These results suggest that the cells involved in generating immunological memory to TNP-KLH and TNP-LPS were affected in salmon exposed to contaminants in the urban estuary, with cells which generate memory to TNP-KLH being more affected. The PFC responses of primed SP leucocytes from salmon collected from the Duwamish Waterway and Nisqually River estuaries, stimulated in vitro with TNP-LPS were not significantly different. The consequence of suppressed immunological memory in disease resistance of juvenile salmon is currently unknown.

Cumulative Effects of Natural and Anthropogenic Stress on Immune Function and Disease Resistance in Juvenile Chinook Salmon

Abstract Previous studies have shown that juvenile chinook salmon Oncorhynchus tshawytscha exposed in the field or the laboratory to polychlorinated biphenyls (PCBs), an anthropogenic stressor, are immunosuppressed. It is not known whether simultaneous exposure to natural stressors can increase this immunosuppression. To examine the effects of natural and anthropogenic stressors on immune function, we infected juvenile chinook salmon with metacercariae of the trematode Nanophyetus salmincola by exposing the fish to infected freshwater snails Juga plicifera. Infected (>300 metacercariae per fish) and noninfected salmon were then injected with either the commercial PCB mixture Aroclor 1254 or an acetone–emulphor carrier. B cell function was examined by in vitro hemolytic plaque-forming cell (PFC) assay. Nanophyetus salmincola infection resulted in significantly lower anterior kidney primary PFCs and lower splenic secondary PFCs. The combination of N. salmincola infection and Aroclor 1254 exposure caused a l...

Genetic Analyses Provide Insight into the Early Ocean Stock Distribution and Survival of Juvenile Coho Salmon off the Coasts of Washington and Oregon

Abstract Estimating the stock proportions of mixed-stock fishery samples by means of genetic stock identification has played an important role in the management of salmon fisheries. In addition, stock identification of individual fish has applications for population studies, forensic cases, and management issues. We examined 11 microsatellite DNA loci in 84 populations of coho salmon Oncorhynchus kisutch sampled at 78 locations from southern British Columbia to northern California to construct a database of microsatellite allele frequencies. We then evaluated the applicability of the database for estimating stock proportions in a mixed fishery and assigning individuals to their regions of origin. The loci were highly polymorphic: observed heterozygosity ranged from 0.754 to 0.943. Using genetic distance calculations, we identified six major geographic regions and 15 smaller subregions into which the populations grouped. Computer simulations and a sample of 143 coho salmon with known origins showed that th...

Bioindicators of contaminant exposure and sublethal effects in benthic fish from Puget Sound, WA, USA

Abstract A suite of chemical and biochemical parameters was measured in three species of benthic flatfish (English sole, Parophrys vetulus; rock sole, Lepidopsetta bilineata; and starry flounder, Platichthys stellatus) sampled from up to five sites in Puget Sound, WA, USA, to assess the sensitivity of the parameters to differences in levels of contaminant exposure and the relative merit of the use of a suite of indices for assessing exposure and sublethal effects. The indices examined were hepatic levels of polychlorinated biphenyls (PCBs) and biliary fluorescent aromatic-compound concentrations, hepatic activities of aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase, and levels of total hepatic GSH and hydrophobic DNA-xenobiotic adducts; the last-mentioned were determined by using the 32P-postlabeling assay. The results showed that the indices examined could discriminate among sites exhibiting different degrees of chemical contamination; however, species differences in the range of response of some indices were observed. Additionally, the use of multiple indices appeared to enhance the assessment of contaminant exposure and sublethal effects.

Evaluation of Bioindicators of Contaminant Exposure and Effects in Coastal Ecosystems

The issue of chemical contamination of the coastal environment is gaining increasing importance in the balance between the need to use chemicals and the desire to conserve and protect the ecosystem. In grappling with the complexity of contaminant interactions in the aquatic environment (Fig. 28.1), it becomes evident, although not universally accepted, that to properly assess the impact of chemical pollution on the health of ecosystems, a better understanding of processes that are vital to the normal functioning of various components (e.g. individuals, populations, or communities) of the system must be developed. Accordingly, we have focused our investigations on contaminant interactions at subcellular, cellular, and tissue levels of marine organisms to identify indices that assess alterations in critical physiological and biological processes (Fig. 28.2), with the aim that some of these measurements may serve as early warning signals of individual, population, or community effects. The logic underlying such an approach is that for the pollutantinduced perturbations to be evident in the ecosystem, the first level of interaction has to be at the individual level. Moreover, cause and effect relationships between chemical contamination and observed changes at the population or community level are more difficult to establish than they are at the individual level.

Enhanced superoxide anion production in activated peritoneal macrophages from english sole (Pleuronectes vetulus) exposed to polycyclic aromatic compounds

Abstract As in mammals, macrophages play a vital role in the destruction of infective organisms in fish. The current study was done to determine if exposure to polycyclic aromatic compounds (PACs), a group of chemical contaminants commonly found in the sediments of urban marine areas, alters the ability of peritoneal macrophages (MOs) from English sole (Pleuronectes vetulus) to produce cytotoxic reactive oxygen intermediates (ROIs). Initially, assay conditions including concentration of MOs, type of in vitro stimulant, tissue culture media, and incubation time were optimized to measure production of superoxide anion (O2−), the progenitor ROI, in English sole MOs. English sole were then injected with an organic solvent extract of a PAC-contaminated sediment equivalent to 20 g sediment (about 860 μg selected PACs) per kg fish, via their dorsal lymphatic sinus. Peritoneal MOs were harvested on days 1, 3, 7, and 14 post-injection. Elicited peritoneal MOs from English sole injected with the sediment extract produced significantly more O2− after stimulation in vitro with either opsonized zymosan on days 3 and 7 after exposure, or phorbol myristate acetate on day 7 when compared to the vehicle-injected or uninjected fish. Macrophages of fish injected with the vehicle responded comparably to those from uninjected individuals. No differences in the basal amounts of O2− production from activated peritoneal MOs were observed among the treatment groups. This study demonstrates that exposure of English sole to PACs altered macrophage production of O2−. Although the direct effects of the enhanced production of this ROI are unknown, the higher levels of superoxide anion production within peritoneal macrophages may contribute to immunodysfunction and oxidative damage in P. vetulus.

Characteristics of Peripheral Blood Cells from Rainbow Trout Evaluated by Particle Counter, Image Analysis, and Hemocytometric Techniques

Abstract Hematological assessments of peripheral blood cells from rainbow trout Oncorhynchus mykiss were compared by using a hemocytometer, image analysis, and an electronic particle counter. Although statistical differences were noted, the diameters of white blood cells (WBCs) were comparable among the methods used, but lengths and widths of red blood cells (RBCs) were remarkably different among the three methods. Red blood cells were significantly smaller when the particle counter was used compared with sizes obtained by using the hemocytometer or image analysis. Counts of whole blood and isolated RBC concentrations were similar between the hemocytometer and the particle counter in the automatic calculation mode; yet they were significantly different with the manual calculation method, which used the raw data from the particle counter. White blood cell counts from peripheral whole blood could not be obtained with the particle counter; it converted all particle volumes into spherical equivalents and, thu...