Ingeborg Werner

Effects of neurotoxic insecticides on heat-shock proteins and cytokine transcription in Chinook salmon (Oncorhynchus tshawytscha) ☆

This study investigated sublethal, molecular effects of two current-use insecticides, chlorpyrifos (CP) and esfenvalerate (EV) in juvenile Chinook salmon. Heat-shock protein (hsp60, hsp70, hsp90) expression was quantified by Western blotting in muscle, liver and gill, and transcription of four cytokines (TGF-beta, IL-1beta, IGF-1, Mx-protein) was measured by real-time TaqMan PCR in anterior kidney and spleen. Expression of hsp was increased in muscle and liver at 1.2 and 7.2 microg/L CP, and at 0.01 and 0.1 microg/L EV, respectively. Transcription of IL-1beta and TGF-beta was elevated in kidney at 1.2 microg/L CP, while EV had no effect. No changes in cytokine transcription were observed in the spleen. Our results show that these insecticides cause cellular effects at environmental concentrations, and that hsps are sensitive indicators of sublethal exposure to CP and EV. In addition, CP may exert immunotoxic effects by altering the transcription of important mediators of the fish immune system.

Expression of immune-regulatory genes in juvenile Chinook salmon following exposure to pesticides and infectious hematopoietic necrosis virus (IHNV)

Impairment of fish immune function as a consequence of polluted aquatic environments can result in changes in susceptibility to disease. In this study, we investigated the effects of two widely used insecticides, chlorpyrifos (CP) and esfenvalerate (EV), and a pathogen, infectious hematopoietic necrosis virus (IHNV), singly and in combination, on survival and cytokine (Mx protein, IL-1beta, TGF-beta and IGF-1) expression in juvenile Chinook salmon. Fish were exposed for 96 h to sublethal concentrations of CP (3.7 microg L(-1)) or EV (0.08 microg L(-1)), allowed to recover in clean water for seven days, then exposed to IHNV (6.4x10(5)TCID(50)mL(-1)) for 1.5h. Mortality was recorded daily, and spleen and anterior kidney samples were collected on day 4 (after CP or EV treatment), day 20 and day 60 (after CP or EV treatment and subsequent IHNV exposure) of the experiment. Significant mortality after 60 days was observed following exposure to EV (17%) or IHNV (20%), and prior insecticide exposure did not synergize the acute effects of pathogen treatment. By day 4, exposure to CP as well as EV led to a significant decrease of Mx protein and IL-1beta expression; by day 20, EV-exposed fish significantly overexpressed IL-1beta. Mx protein transcription was up-regulated in spleen and kidney of all IHNV-exposed fish groups by day 20. All but one treatment (EV) led to significantly decreased IGF-1 transcription in spleen on days 20 and 60, whereas a short-term increase was seen after CP exposure (day 4). In kidney, decreases of IGF-1 transcription were less pronounced. TGF-beta transcription was up-regulated in CP/IHNV and EV/IHNV exposure groups. Our results indicate that CP and EV alter the expression of cytokines, but this did not negatively affect the ability of fish to survive a subsequent exposure to IHNV. Induced TGF-beta transcription indicated that the combined stressors affected fish in a synergistic manner, but the consequences are unknown. Increased transcription of Mx protein was a reliable indicator of virus exposure.

Field validation of hsp70 stress proteins as biomarkers in Asian clam (Potamocorbula amurensis): is downregulation an indicator of stress?

The focus of this paper is to consider the applicability of the hsp70 stress protein response as a biomarker in field studies. Stress proteins (or heat shock proteins, hsp) of the hsp70 family are induced by sublethal concentrations of a variety of environmental pollutants. However, few studies have applied these proteins as biomarkers of environmental stress under field conditions. Our laboratory is investigating hsp70 proteins and other responses of Asian clam (Potamocorbula amurensis) as potential biomarkers in laboratory and field studies. Our efforts include two studies presently being conducted in northern San Francisco Bay: (1) monthly collection of clams from four sites along a cadmium contamination gradient; (2) 7 day in situ exposure of clams at two selected sites at Mare Island Naval Shipyard. Here we present results on hsp70 proteins in P. amurensis in field-collected and outplanted clams. Both field projects are ongoing, therefore the results presented here do not represent completed studies;...The focus of this paper is to consider the applicability of the hsp70 stress protein response as a biomarker in field studies. Stress proteins (or heat shock proteins, hsp) of the hsp70 family are induced by sublethal concentrations of a variety of environmental pollutants. However, few studies have applied these proteins as biomarkers of environmental stress under field conditions. Our laboratory is investigating hsp70 proteins and other responses of Asian clam (Potamocorbula amurensis) as potential biomarkers in laboratory and field studies. Our efforts include two studies presently being conducted in northern San Francisco Bay: (1) monthly collection of clams from four sites along a cadmium contamination gradient; (2) 7 day in situ exposure of clams at two selected sites at Mare Island Naval Shipyard. Here we present results on hsp70 proteins in P. amurensis in field-collected and outplanted clams. Both field projects are ongoing, therefore the results presented here do not represent completed studies; rather, they illustrate a portion of our experience. For this workshop, we illustrate weaknesses and strengths of these proteins as biomarkers, and we underscore where additional work is needed. In field-collected clams (study no. 1), site-specific differences in levels of two hsp70 proteins, hsp70 and hsp76, were measured in May and June 1997. Although an inverse correlation exists between cadmium tissue concentrations and hsp70 protein levels, differences detected may be reflective of a salinity gradient. Results from recent laboratory exposures to cadmium and a range of salinities are discussed. After in situ exposure for 7 days (study no. 2), both hsp70 and hsp76 levels were significantly reduced in clams from site R. However, given a brief heat-shock in the laboratory, hsp70 protein levels were significantly higher in clams from this site than in controls. Results indicate that downregulation as well as upregulation of hsp70 proteins may be indicators of stress in P. amurensis.

Pesticide and pathogen: heat shock protein expression and acetylcholinesterase inhibition in juvenile Chinook salmon in response to multiple stressors.

Rapid expression of heat shock protein (hsp) families in response to a variety of stressors has been demonstrated in many organisms, including fish. The present 60-d challenge study was designed to compare hsp induction in juvenile Chinook salmon following exposure to individual pesticides, virus, and both stressors combined. Heat shock protein expression patterns over time were monitored and related to the extent of virus infection and mortality. Acetylcholinesterase (AChE) inhibition and recovery in response to applied stressors were measured in brain. High enzyme inhibition levels have been correlated with imminent mortality, and other sublethal physiological effects have been observed in fish concurrent with depressed AChE activity. Mortality was elevated considerably in fish exposed to 0.08 microg/L of the pyrethroid esfenvalerate (EV). Mortality due to infectious hematopoietic necrosis virus (IHNV) was lower in groups previously treated with pesticides; however, these fish died sooner than individuals exposed to virus only. Both pesticides, EV and the organophosphate (OP) chlorpyrifos (CP), as well as virus exposure, induced hsp expression, but highest hsp levels were observed after the combined treatments, suggesting an additive effect between virus and pesticides. Highest virus titers were accompanied by strongest hsp induction, indicating a connection between virus concentration and hsp expression. In conclusion, the measurement of hsp expression appears to be a very sensitive, integrative indicator of stress. Esfenvalerate and IHNV did not affect AChE activity, and exposure to 3.7 microg/L CP led to significantly inhibited AChE for at least 20 d. The time required for complete recovery of AChE activity raises concern about deleterious behavioral effects.

Effects of dietary exposure to the pyrethroid pesticide esfenvalerate on medaka (Oryzias latipes)

The pyrethroid insecticide esfenvalerate is widely used on orchard crops throughout California. In the aquatic environment, this compound is likely to accumulate in sediments, food particles and benthic organisms due to its lipophilicity and environmental persistence. This pilot project tested the hypothesis that esfenvalerate is toxic to medaka (Oryzias latipes) when taken up with the diet. For 7 days fish were fed diets, which contained esfenvalerate in three different concentrations (4, 21, 148 mg/kg, measured). Endpoints measured were mortality, fecundity, fertilization and hatching success of embryos, viability of larvae and cellular stress protein (hsp60, hsp70, hsp90) levels. The toxicity of aqueous exposure of medaka to esfenvalerate was also determined. Whereas the 96-h LC50 in the aqueous exposure was <9.4 microg/l, the dietary exposure did not cause mortality. Possible effects of dietary esfenvalerate were seen on fertilization and hatching success and the number of non-viable larvae. Expression of hsp60 and hsp90 showed a dose-dependent response pattern.

Toxicity of methyl-tert-butyl ether to freshwater organisms

Increased input of the fuel oxygenate methyl-tert-butyl ether (MTBE) into aquatic systems has led to concerns about its effect(s) on aquatic life. As part of a study conducted by University of California scientists for the State of California, the Aquatic Toxicology Laboratory, UC Davis, reviewed existing literature on toxicity of MTBE to freshwater organisms, and new information was generated on chronic, developmental toxicity in fish, and potential toxicity of MTBE to California resident species. Depending on time of exposure and endpoint measured, MTBE is toxic to various aquatic organisms at concentrations of 57-> 1000 mg/l (invertebrates), and 388-2600 mg/l (vertebrates). Developmental effects in medaka (Oryzias latipes) were not observed at concentrations up to 480 mg/l, and all fish hatched and performed feeding and swimming in a normal manner. Bacterial assays proved most sensitive with toxicity to Salmonella typhimurium measured at 7.4 mg/l within 48 h. In microalgae, decreased growth was observed at 2400 and 4800 mg/l within 5 days. MTBE does not appear to bioaccumulate in fish and is rapidly excreted or metabolized. Collectively, the available data suggests that at environmental MTBE exposure levels found in surface waters (< 0.1 mg/l) this compound is likely not acutely toxic to aquatic life. However, more information is needed on chronic and sublethal effects before we can eliminate the possibility of risk to aquatic communities at currently detected concentrations.

Toxicity of storm‐water runoff after dormant spray application in a French prune orchard, Glenn County, California, USA: Temporal patterns and the effect of ground covers

Organophosphorous (OP) insecticides, especially diazinon, have been detected routinely in surface waters of the Sacramento and San Joaquin River watersheds, coincident with rainfall events following their application to dormant orchards during the winter months. Preventive best management practices (BMP) aim at reducing off-site movement of pesticides into surface waters. Two proposed BMPs are: The use of more hydrophobic pyrethroid insecticides believed to adsorb strongly to organic matter and soil and the use of various types of ground cover vegetation to increase the soils capacity for water infiltration. To measure the effectiveness of these BMPs, storm water runoff was collected in a California prune orchard (Glenn County, CA, USA) during several rainstorms in the winter of 2001, after the organophosphate diazinon and the pyrethroid esfenvalerate were applied to different orchard sections. We tested and compared acute toxicity of orchard runoff from diazinon- and esfenvalerate-sprayed sections to two species of fish (Pimephales promelas, Onchorhynchus mykiss) and three aquatic invertebrates (Ceriodaphnia dubia, Simocephalus vetelus, Chironomus riparius), and determined the mitigating effect of three ground cover crops on toxicity and insecticide loading in diazinon-sprayed orchard rows. Runoff from the esfenvalerate-sprayed orchard section was less toxic to waterflea than runoff from the diazinon-sprayed section. However, runoff from the orchard section sprayed with esfenvalerate was highly toxic to fish larvae. Samples collected from both sections one month later were not toxic to fish, but remained highly toxic to invertebrates. The ground cover crops reduced total pesticide loading in runoff by approximately 50%. No differences were found between the types of vegetation used as ground covers.

Synergistic effects of esfenvalerate and infectious hematopoietic necrosis virus on juvenile chinook salmon mortality.

Sublethal concentrations of pollutants may compromise fish, resulting in increased susceptibility to endemic pathogens. To test this hypothesis, juvenile chinook salmon (Oncorhynchus tshawytscha) were exposed to sublethal levels of esfenvalerate or chlorpyrifos either alone or concurrently with infectious hematopoietic necrosis virus (IHNV). Three trials were performed with fish exposed to concentrations of IHNV between 0.8 x 10(2) and 2.7 x 10(6) plaque-forming units/ml and to 5.0 microg/L of chlorpyrifos or 0.1 microg/L of esfenvalerate. The presence and concentration of IHNV in dead fish were assayed by virus isolation and plaque assay techniques, respectively. Among groups exposed to both esfenvalerate and IHNV, 83% experienced highly significant (p < 0.001) mortality, ranging from 20 to 90% at 3 d post-virus exposure, and cumulatively died from 2.4 to 7.7 d sooner than fish exposed to IHNV alone. This trend was not seen in any other treatment group. Virus assays of dead fish indicate a lethal synergism of esfenvalerate and IHNV. Chlorpyrifos had no observed effect on total mortality or IHNV susceptibility. The present results suggest that accepted levels of pollutants may be seemingly nonlethal to fish but, in fact, be acting synergistically with endemic pathogens to compromise survivorship of wild fish populations through immunologic or physiologic disruption.

Heat Shock Proteins in Juvenile Steelhead Reflect Thermal Conditions in the Navarro River Watershed, California

Abstract This study examined expression levels of two heat shock proteins, hsp72 and hsp78, in white muscle of steelhead (anadromous rainbow trout Oncorhynchus mykiss) parr collected between 25 July and 4 August 2000 at 11 sites in the Navarro River watershed, California. The goal was to determine whether site-specific thermal conditions were causing cellular stress responses in resident fish. The results demonstrated a highly significant sigmoidal relationship of the inducible isoform hsp72 and a linear relationship of the constitutive isoform hsp78 with water temperatures. Laboratory experiments showed that hsp72 was induced in fish from coldwater as well as warmwater sites by exposure to water at 25°C. Stream temperatures above which significantly elevated hsp72 levels were detected in field-collected fish were 18–19°C in terms of both short- and long-term averages and 20–22.5°C in terms of daily maximum averages. The highest hsp72 levels were measured at warmwater sites with the largest diurnal temper...

Spatial profiles of hsp70 proteins in Asian clam (Potamocorbula amurensis) in northern San Francisco Bay may be linked to natural rather than anthropogenic stressors

Multi-year investigations in northern San Francisco Bay by United States Geological Survey have linked reduced condition indices in populations of Asian clam (Potamocorbula amurensis) with elevated cadmium tissue concentrations. Our study seeks to determine whether levels of hsp70 proteins in P. amurensis can be correlated with these findings, and/or are related to histopathologic alterations and concentrations of metallothionein-like proteins. Here we present our results on stress proteins in clams collected monthly from four field stations between July 1996 and January 1998. In addition, animals were exposed in the laboratory to a range of salinities. Stress proteins were analyzed by Western blotting using monoclonal antibodies. Hsp70 protein levels in field-collected clams were significantly higher at the seaward (high salinity/low cadmium) stations (12.5, 8.1) than at the landward (low salinity/high cadmium) stations (6.1, 4.1). Laboratory studies showed that clams exposed to 0.1 ppt salinity had markedly lower hsp70 levels than clams exposed to higher salinities. In view of our previous laboratory studies showing that cadmium induces hsp70 in P. amurensis, our present results indicate that reduced hsp70 protein levels in field-collected clams may be linked to salinity effects rather than cadmium tissue concentrations.