Whole-body Homogenates Research Articles

Whole-body corticosteroid and plasma cortisol concentrations in larval and juvenile Atlantic cod Gadus morhua L. following acute stress

Methods were developed to assess whole-body immunoreactive corticosteroid concentrations (IRC) and plasma levels of cortisol in Atlantic cod subjected to several common, acute stressors. A measurable corticosteroid stress response was observed at the first sampling in whole bodies of 8-day post-hatch (dph) larvae. Two groups of juveniles (∼5 and 30 g) were subjected to a 30 s net stressor and whole-body IRC and plasma cortisol levels were determined. Post-stressor IRC in smaller fish rose approximately 14-fold, peaked at 1 h, were sustained for 3–6 h and returned to pre-stressor levels within 24 h. Post-stressor plasma cortisol levels in larger fish rose approximately 18-fold, peaked at 0.5–1 h, were sustained for 1–3 h and then returned to near pre-stressor levels after 24 h. Immunoreactive corticosteroid concentrations appeared to remain elevated longer than plasma cortisol levels suggesting that steroids other than cortisol were contributing to total immunoreactivity in homogenates. Juveniles exposed to either a grading procedure or high density transport had maximal IRC and plasma cortisol levels within 90 min which returned to pre-stressor levels within 24–72 h. A reduction in water salinity (20 g L−1) did not moderate the corticosteroid response in juveniles. The results show that Atlantic cod respond to common, acute stressors in a manner similar to other teleosts. Whole-body homogenates can be used to identify changes in IRC in response to acute stress in cod with the caveat that recovery IRC may differ from plasma cortisol concentrations.

Sex steroids in the female zebrafish ( Danio rerio) : Effects of cyproterone acetate and leachate-contaminated sediment extract

In Molnbyggen, a leachate-contaminated lake in Sweden, effects on the reproductive system of perch included a decreased frequency of sexually mature female perch, reduced gonadosomatic index and decreased plasma levels of androstenedione and testosterone, but the contaminants responsible for these effects have not yet been identified. A biomarker-directed fractionation approach could be used to narrow the search for the compound(s) responsible for the adverse effects in Molnbyggen female perch. Thus, it is important to develop and test a suitable biomarker, which can be used in this type of approach. We evaluated if decreased concentrations of sex steroids could work as an in vivo end-point in female zebrafish fed an organic extract of bottom sediments from Molnbyggen. The responsiveness of zebrafish to endocrine alteration was evaluated using a positive control substance, cyproterone acetate (CA), which is known to inhibit steroid biosynthesis. After the 21-day exposure period the concentrations of testosterone (T) and estradiol (E2) were successfully determined by HRGC/HRMS in whole-body homogenates of individual fish. Median T concentrations were 1.7 pmol/g fish in controls and were decreased after exposure both to CA (0.55 pmol/g fish) and Molnbyggen sediment extract (1.2–1.3 pmol/g fish). Median E2 concentrations were 5.3 pmol/g fish in controls and were decreased after CA exposure (2.3 pmol/g fish) and after exposure to Molnbyggen sediment extract (4.0–4.5 pmol/g fish). This suggests that sex steroid concentrations in zebrafish are responsive to endocrine alteration and indicates that compounds with endocrine altering potency are present in Molnbyggen sediments.

Comparison of Vitellogenin Induction, Sex Ratio, and Gonad Morphology Between Zebrafish and Japanese Medaka After Exposure to 17α-Ethinylestradiol and 17β-Trenbolone

The pharmaceutical estrogen 17alpha-ethinylestradiol (EE2) and the anabolic androgen 17beta-trenbolone (Tb) can interfere with the endocrine and reproductive systems of fish. The potency of these chemicals in zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) was assessed using the core end points vitellogenin (Vtg) concentration at 38 days post-hatch and sex ratio and gonad morphology at 60 days post-hatch. Vtg concentrations were measured in fish whole-body homogenate samples using enzyme-linked immunosorbent assay. Increased Vtg concentration and feminization of fish after exposure to 10 ng/L EE2, as well as masculinization after exposure to 50 ng/L of Tb, were observed in zebrafish. Intersex was observed in medaka exposed to EE2. A decrease in Vtg production after Tb exposure (50 ng/L) was measured in both zebrafish and medaka. Analyses of gonad morphology revealed increased testicular area and sperm percentage in Tb-exposed zebrafish, whereas increased sperm percentage was measured in Tb-exposed medaka. The higher sensitivity of zebrafish compared with medaka to both EE2 and Tb was revealed in the study.

An enzyme-linked immunosorbent assay for rare minnow ( Gobiocypris rarus) vitellogenin and comparison of vitellogenin responses in rare minnow and zebrafish ( Danio rerio)

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine vitellogenin (Vtg) in rare minnow ( Gobiocypris rarus) based on the separation and purification of rare minnow Vtg (r-Vtg) as well as the production of polyclonal antibody against r-Vtg in rabbits. Three different ELISAs for measuring r-Vtg were then compared: (1) indirect ELISA with the antibody against carp ( Cyprinus carpio) Vtg (c-Vtg) (IC-ELISA); (2) competitive ELISA with the antibody against c-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CC-ELISA); (3) competitive ELISA with the antibody against r-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CR-ELISA). The result showed that the homologous CR-ELISA was the most sensitive among the three assays for quantifying r-Vtg. The sensitivities to 17α-ethinylestradiol (EE 2) of rare minnow and zebrafish ( Danio rerio) were compared upon the establishment of homologous competitive ELISA. The lowest observed effect concentrations (LOECs) to induce Vtg were found to be 0.8 ng EE 2 l − 1 for rare minnow and 4 ng EE 2 l − 1 for zebrafish respectively. Afterwards, CR-ELISA was applied to measure Vtg concentration in whole body homogenate (WBH) of juvenile rare minnow fed by three diets (tubifex from wastewater treatment plant, Artemia nauplii and commercial pellet food), and the agreements between bioassay and GC–MS analysis demonstrated that rare minnow was a sensitive fish model for assessing estrogenic effects of endocrine disrupting compounds in aquatic environment.

Trials to Induce Protective Immunity in Mice and Sheep by Application of Protoscolex and Hydatid Fluid Antigen or Whole Body Antigen of Echinococcus granulosus

In the current study, soluble proteins prepared from 200 mature Echinococcus granulosus and protoscolices of sheep hydatid cysts were applied to immunize sheep and mice respectively. The samples were mechanically homogenized in a blender, sonicated and the final yield was maintained at -20 degrees C until analysis. Hydatid fluid was isolated from liver or lung of sheep under sterile conditions. In the first experiment, 15 mice were randomly allocated to three groups of five mice each. Each mouse in groups 1 and 2 was immunized with 100 microg of hydatid fluid and protoscolex proteins in 100 microl of phosphate-buffered saline (PBS) and emulsified with an equal volume of Freund's complete adjuvant (FCA) respectively. The mice of group 3 were immunized with adjuvant in PBS. The mice were boosted 4 weeks after the first vaccination with the same preparation except that FCA was replaced by Freund's incomplete adjuvant (FIA). In the second experiment, eight male or female lambs 4-6 months of age, were allocated to two groups of four lambs each. Each lamb in the test group was vaccinated subcutaneously in the neck with a 2-ml dose of vaccine (1 mg of whole body protein of E. granulosus dissolved in 1 ml of PBS plus 1 ml of FCA). Control lambs were vaccinated with adjuvant in PBS. Lambs were boosted the same way as in the first experiment. Three weeks after the second vaccination, each mouse and lamb received a challenge infection with 2000 protoscolices intraperitoneally and each lamb additionally received 10 gravid E. granulosus. All mice and sheep were killed after 7 months and examined for hydatid cysts. In these studies, protective immunity was induced in mice with protoscolex protein and with hydatid fluid, and in sheep with whole-body homogenate of E. granulosus and the levels of protection afforded were found to be 72.1, 82.6 and 90.9% respectively.

Diet and weaning age affect the growth and condition of Dover sole (Solea solea L.)

The effect of diet type (frozen Artemia biomass and two inert diets: micro-bound [MB] and micro-extruded [ME]) and two weaning ages (early weaning and late weaning, 50 and 64 days after hatching, respectively) were studied in Solea solea larvae. The experiment lasted 56 and 42 days for early and late weaning, respectively. The mortality results showed the highest values for late weaning (39%) in the Artemia treatment. No significant differences in mortality were observed between the inert diets. The final dry weight values were higher for late weaning than for early weaning. At both weaning ages, fish receiving the same treatments had similar tendencies for dry weight and standard length. Fish fed with MB presented significantly higher dry weight and standard length, followed by ME, while the lowest values at both weaning ages were recorded for the Artemia treatment. Similar amounts of highly unsaturated fatty acid fractions among the inert diets were reflected by the absence of significant differences in the susceptibility to oxidation (thiobarbituric acid reactive substances testing); however, significant differences were found in carbohydrate, protein and lipid contents of whole-body homogenates for both early and late weaning. At the end of the experiment no significant differences in biochemical contents were observed between the two inert diets. The results of this study suggest that weaning starting on day 50 (early weaning), using a good quality inert diet, leads to higher survival, growth and fish condition.

Is oxidative stress the mechanism of blue sac disease in retene‐exposed trout larvae?

Retene (7-isopropyl-1-methylphenanthrene) causes blue sac disease (BSD) in early life stages of fish, an effect similar to that of 2,3,7,8-tetrachlorodibenzo(p)dioxin. The signs of BSD include cytochrome P450 (CYP1A) induction, edema, hemorrhaging, and craniofacial deformities, indicating membrane damage, circulatory failure, and impaired development. To test if the underlying cause was oxidative stress, rainbow trout (Oncorhynchus mykiss) larvae were exposed to waterborne retene or to known prooxidants (paraquat, t-butyl hydroperoxide, and carbon tetrachloride) in the presence or absence of vitamin E, an antioxidant. Fish exposed to retene showed an increased prevalence of BSD, reduced tissue concentrations of vitamin E and total glutathione, and a lower percentage of glutathione in a reduced form. Coexposure to vitamin E reduced the prevalence of BSD and restored tissue concentrations of vitamin E, but it did not affect retene uptake or tissue concentrations of glutathione. These responses are consistent with oxidative stress as a mode of action of retene. However, retene did not affect whole-body lipid peroxide concentrations, and prooxidants did not affect the prevalence of BSD and had only minimal effects on tissue glutathione and vitamin E. Possible explanations for these conflicting results include prooxidant exposures were insufficient to generate oxidative stress; lipid peroxidation may not be measurable in whole-body homogenates of retene-exposed fish if effects are localized to endothelial cells, where CYP1A enzymes are most induced; or retene may have an alternate mode of action (e.g., adduction of retene metabolites to lipids, protein, or DNA).

The Chinese rare minnow ( Gobiocypris rarus) as an in vivo model for endocrine disruption in freshwater teleosts: a full life-cycle test with diethylstilbestrol

Chinese rare minnow ( Gobiocypris rarus), a freshwater teleost, was exposed to diethylstilbestrol (DES) at 0.05, 0.5, 1 and 5 μg/L from fertilized eggs for up to mature period under flow-through condition. Several endpoints that related to development, reproductive fitness and transgenerational effects were evaluated. It was found that body length and body weight were significantly reduced and vitellogenin (Vtg) levels were significantly increased for fish exposed to DES. Histological examination showed that the sex ratios of F 0 fish skewed to female and about 2% of the fish exposed to 0.05 μg/L DES developed testes–ova. The reproductive success, as determined from data on egg production, was reduced in female fish exposed to 0.05, 0.5, 1 and 5 μg/L DES. The lowest-observed-effect concentrations (LOEC) for changes of sex ratios, reproductive success and histology alteration of F 0 are 0.05 μg/L. In the offspring, transgenerational effects on egg hatching rate, egg fertilization and Vtg levels of juvenile individuals were not observed. However, survival of F 1 generation fry significantly declined. The analysis of sex steroid levels revealed a significant decrease of testosterone (T) in the whole body homogenates (WBH) of male progeny and somewhat elevation of estradiol (E 2) in the WBH of female offspring. These findings indicate that exposure to DES causes a variety of developmental, reproductive and transgenerational effects.

Xenoestrogenic effects of ethinylestradiol in zebrafish (Danio rerio).

To assess the estrogenic effects of ethinylestradiol on zebrafish, zebrafish at different developmental stages (embryos, juveniles, and adults) were exposed to the synthetic hormone ethinylestradiol (EE2) in concentrations of 1, 10, and 100 ng/L for up to 33 days. Survival, hatching, length, weight, growth, condition, hepatosomatic index, gonadosomatic index, and vitellogenin (VTG) production were examined. Exposure of zebrafish juveniles and embryos to 100 ng EE2/L for up to 33 days had significant effects on survival, growth, and hatching. Two VTG fragments with molecular weights of approximately 140 and 170 kDa were detected with protein electrophoresis and Western blotting in the blood of exposed males and exposed and unexposed females, as well as in whole-body homogenates of exposed and unexposed juveniles. Significantly higher VTG concentrations (compared to controls) were measured in adults exposed to 10 and 100 ng EE2/L for 14 days, but not in fish exposed to 1 ng EE2/L. This study demonstrated that (1) zebrafish juveniles, larvae, and embryos are sensitive to the toxic effects of the endocrine disrupter EE2; (2) the effects on VTG production in adults are detected after exposure to environmentally relevant concentrations of EE2; (3) unexposed juvenile zebrafish produce measurable concentrations of VTG.

Angiotensin I-converting enzyme-like activity in a cephalochordate

The presence of angiotensin I-converting enzyme-like activity (ACELA) was investigated in whole body homogenates of Branchiostoma lanceolatum using a highly sensitive fluorimetric assay. The measured enzyme activity was inhibited by the two angiotensin converting enzyme inhibitors, captopril and enalapril, suggesting a biochemical pathway that could generate the angiotensins observed in the central nervous system of amphioxus.

Characterization and cDNA cloning of a clofibrate-inducible microsomal epoxide hydrolase in Drosophila melanogaster.

In order to understand the roles of the epoxide hydrolases (EHs) in xenobiotic biotransformation in insects, we examined the induction of EHs by exogenous compounds in Drosophila melanogaster third instar larvae. Among the chemicals tested, clofibrate, a phenoxyacetate hypolipidermics drug, increased EH activity towards cis-stilbene oxide approximately twofold in larval whole-body homogenates. The same dose of clofibrate also induced glutathione S-transferase activity. The effect of clofibrate on EH induction was dose-dependent and the highest activity occurred with a 10% clofibrate application. Three other substrates conventionally used in EH assays (trans-stilbene oxide, trans-diphenylpropene oxide and juvenile hormone III) were poorly hydrolysed by larval homogenates, with or without clofibrate administration. Because the increased EH activity was localized predominantly in the microsomal fraction, we synthesized degenerate oligonucleotide primers with sequences corresponding to conserved regions of known microsome EHs from mammals and insects in order to isolate the gene. The 1597 bp putative cDNA of D. melanogaster microsomal EH (DmEH) obtained from a larval cDNA library encoded 463 amino acids in an open reading frame. Northern blot analysis showed that the transcription of DmEH was increased in larvae within 5 h of clofibrate treatment. Recombinant DmEH expressed in baculovirus hydrolysed cis-stilbene oxide (23 nmol.min-1.mg protein-1) and was located mainly in the microsomal fraction of virus-infected Sf9 cells. There was no detectable EH activity toward juvenile hormone III. These observations suggest that DmEH is involved in xenobiotic biotransformation, but not in juvenile hormone metabolism, in D. melanogaster.

Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening.

The yolk protein precursor vitellogenin (Vtg) in plasma has proved to be a simple and sensitive biomarker for assessing exposure of fish to environmental estrogens. Within international bodies such as the Organization for Economic Cooperation and Development (OECD) work is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals, and in the focus of this development are the fish test species common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). In this study we have developed quantitative enzyme linked immunosorbent assays (ELISAs) for Vtg in common carp/fathead minnow, zebrafish and Japanese medaka. The assays were developed using a combination of monoclonal and polyclonal fish Vtg antibodies in a sandwich format, using stabilized Vtg from the test species as a standard. The carp Vtg ELISA has a working range of 1-63 ng/mL, a minimal detection limit of 0.6 ng/mL, and may also be used for quantification of Vtg in fathead minnow. In fathead minnow whole-body homogenate samples, the practical detection limit is 400 ng/mL due to the matrix effect. The zebrafish Vtg ELISA has a working range of 0.5-63 ng/mL, a minimal detection limit of 0.4 ng/mL, and a practical detection limit of 200 ng/mL in whole-body homogenate samples. The medaka Vtg ELISA has a working range of 0.25-16 ng/mL, a minimal detection limit of 0.1 ng/mL, and a practical detection limit of 125 ng/mL in whole-body homogenate samples. The intra- and inter-assay variations were below 20% for all assays. The assays were evaluated with sets of representative samples spanning the wide dynamic range of Vtg-levels found in fish exposed to environmental estrogens, and all three assays are currently undergoing international inter-laboratory validation.

Stress protein response and catalase activity in freshwater planarian Dugesia ( Girardia) schubarti exposed to copper

The hsp60 expression pattern and catalase activity in the freshwater planarian Dugesia schubarti exposed to copper under laboratory conditions were investigated. In the hsp60 induction experiments, planarians were exposed to a range of copper concentrations (0–960 μg Cu/L) for 4 or 24 h, to concentrations of 50 or 100 μg Cu/L for 2, 4, 8, and 24 h at 19°C, and to heat shock at 27°C for 24 h. The concentrations of hsp60 in whole-body homogenates were determined immunochemically by Western blotting. Stress protein induction was detected only after 24 h treatment at 27°C. The tissue concentration of hsp60 remained unaltered in Cu-exposed planarians under the experimental conditions used. Catalase activity was significantly induced at concentrations of 40, 80, and 160 μg Cu/L after 24 h exposure. Our results suggest that catalase levels in planarians could represent biomarkers of interest for the estimation of copper effects in freshwater ecosystems.

Persistent organochlorine residues and their bioaccumulation profiles in resident and migratory birds from North Vietnam

Concentrations of persistent organochlorines (OCs), such as polychlorinated biphenyls (PCBs), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its metabolites (DDTs), hexachlorocyclohexane isomers (HCHs), hexachlorobenzene (HCB), and chlordane compounds (CHLs), were determined in whole-body homogenates of resident and migratory birds collected from the Red River estuary, North Vietnam, during March and October 1997. Contamination pattern was in the order of DDTs > PCBs > HCHs > CHLs > HCB in both resident and migratory birds. Residue concentrations, according to the feeding habit, showed little variability, which may reflect relatively similar trophic levels of the bird species analyzed. Resident birds accumulated greater concentrations of DDTs as compared to migrants. In contrast, HCH residues were greater in migratory species. Higher proportions of p,p'-DDT to total DDT concentrations were found in many species of residents and migrants, indicating recent exposure to technical DDT in northern Vietnam. Congener-specific PCB analysis showed the predominance of penta- and hexachlorobiphenyls in all the species analyzed. Estimation of hepatic microsomal enzyme activities suggested higher metabolic capacity for PCB congeners in shore birds from Vietnam as compared to higher-trophic predator birds and marine mammals. Comparison of OC residues in avian species in Asia-Pacific revealed that DDT residues in resident birds in North Vietnam are among the highest values reported for the countries surveyed, suggesting recent usage of DDT in Vietnam. Available data for birds, fish, and bivalves from the recent Asia-Pacific Mussel Watch Program suggested that Vietnam might be a potential source of DDT contamination in Asian developing countries. To our knowledge, this is the first study of the OC accumulation in avian species from Vietnam.

Development and validation of an enzyme‐linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio)

In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to quantify vitellogenin (Vtg) in zebrafish (Danio rerio). Zebrafish Vtg (zf-Vtg) was purified from whole-body homogenates of estradiol-exposed zebrafish, and polyclonal antibodies against zf-Vtg were raised. Using purified zf-Vtg as a standard and anti-zf-Vtg antibodies (DR-264), a competitive ELISA method was set up and validated. The working range of the assay is from 1 to 30 ng/ml (20-80% binding), and the detection limit is 0.4 ng/ml for purified zf-Vtg. In whole-body homogenates samples, the practical detection limit is higher than that for purified Vtg (40 ng/ml) due to matrix effect. The intra- and interassay variations were 4.7% and 14%, respectively, at 50% binding (n = 36). Its usefulness to detect changes in Vtg concentration in other cyprinid fish was also tested. In addition, the assay was used to assess Vtg induction in male zebrafish exposed to 17beta-estradiol (E2). Exposure of male zebrafish to 0.1, 1, 10, and 100 microg/L of E2 for 7 d led to a Vtg induction from the lowest concentration. The results show the suitability of the developed ELISA to quantify Vtg inductions in zebrafish, the cross-reactivity of DR264 antibodies with commonly used cyprinids, and the potential of zf-Vtg induction as a sensitive biochemical endpoint that could be used to detect estrogenic properties of chemical substances.

Utility of a juvenile fathead minnow screening assay for detecting (anti‐)estrogenic substances

The European Chemical Industry's aquatic research program for endocrine disrupters includes the development of an in vivo juvenile fathead minnow (Pimephales promelas) screening assay. Working within the Organization for Economic Cooperation and Development's (OECD, Paris, France) tiered approach to endocrine disrupter evaluation in fish, the juvenile fish screening protocol was adapted from the OECD test guideline 204. Six chemicals, with different (anti-)estrogenic potencies, were used to develop the in vivo juvenile fish screening protocol: diethylstilbestrol, 17alpha-ethynylestradiol, genistein, methoxychlor, 4-tert-pentylphenol, and ZM189,154 (a novel pharmaceutical antiestrogen). Mixed-sex juvenile fathead minnows were exposed to individual chemicals (with chemical analyzes) and sampled after 4, 7, 14, and 21 d of exposure. Wet weight, total length, condition factor, and whole-body homogenate concentrations of vitellogenin (VTG) were determined. Estrogens and antiestrogens were detected in this screen by virtue of the VTG response (an elevation or suppression, respectively) after 14 d. The study showed that the use of VTG concentrations in mixed-sex juvenile fish provides a sensitive and robust assay for the detection of both estrogenic and antiestrogenic chemicals, with widely divergent potencies.

Iodine species and the endocrine system: thyroid hormone levels in adult Danio rerio and developing Xenopus laevis.

Recently a new approach for the analysis of iodinated organic species in human serum has been developed using liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS). This method enables quantification of iodide, T4 and T3, as well as reverse T3 (rT3) and the synthetic precursors of TH, monoiodotyrosine (MIT), and diiodotyrosine (DIT) in a single injection. In this work, the LC-ICP-MS approach was used to analyze whole-body homogenates of adult male and female zebrafish (Danio rerio) and tadpoles of the African clawed frog (Xenopus laevis) at two different developmental stages (NF58 and 61) according to Nieuwkoop and Faber. The data demonstrate that the LC-ICP-MS method was successful at measuring I-, MIT, DIT, T4, T3, and rT3 in these two species. Furthermore, the method also detected five additional iodinated compounds which are currently unidentified.

Development of an ELISA for vitellogenin in whole body homogenate of zebrafish ( Danio rerio)

The yolk protein, lipovitellin (Lv) was purified from ovaries of mature female zebrafish ( Danio rerio) by gel filtration and anion exchange chromatography. Polyclonal antibodies against Lv were raised in rabbits. Anti-Lv IgG was purified by affinity chromatography. SDS-PAGE followed by Western blotting was performed to analyse the specificity of the antibody and the immunological similarities between Lv and vitellogenin (Vtg). Anti-Lv IgG was used to develop a direct non-competitive sandwich ELISA to measure Vtg concentrations of whole body homogenate (WBH) in zebrafish. The intra- and interassay variabilities were 5.8% and 10.4%, respectively. The sensitivity was 0.2 ng Vtg ml −1 and the practical detection limit was 40 ng Vtg g −1 fish (wet weight). Adult male zebrafish were exposed to a nominal water concentration of 10 ng l −1 of ethinylestradiol (EE 2) in a semi-static exposure system for 7 days. Compared with the control group, exposure to 10 ng EE 2 l −1 induced a 200-fold increase in Vtg levels.

Characterization of Proteases in the Skin Mucus of Atlantic Salmon (Salmo salar) Infected with the Salmon Louse (Lepeophtheirus salmonis) and in Whole-Body Louse Homogenate

Characterization of Proteases in the Skin Mucus of Atlantic Salmon (Salmo salar) Infected with the Salmon Louse (Lepeophtheirus salmonis) and in Whole-Body Louse Homogenate

Characterization of proteases in the skin mucus of Atlantic salmon (Salmo salar) infected with the salmon louse (Lepeophtheirus salmonis) and in whole-body louse homogenate.

As part of an investigation of the biochemical interactions between the salmon louse Lepeophtheirus salmonis and Atlantic salmon Salmo salar, we characterized protease activity in the skin mucus of noninfected Atlantic salmon and Atlantic salmon infected with L. salmonis and in an L. salmonis whole-body homogenate. Zymography revealed that mucus from infected salmon contained a series of low-molecular-mass (17-22 kDa) serine proteases that were not present in the mucus of noninfected salmon. Based on molecular mass, inhibition studies, and affinity chromatography, the series of proteases was identified as being trypsin-like. Similar proteases were observed in the L. salmonis homogenate and in mucus from noninfected Atlantic salmon following a 1-hr incubation with live L. salmonis. An antibody raised against Atlantic salmon trypsin failed to recognize any proteases in the mucus of noninfected salmon or infected salmon or in the L. salmonis homogenate. Collectively, these findings suggest that the trypsin-like proteases present in the mucus of infected Atlantic salmon were produced by L. salmonis, possibly to aid in feeding and evasion of host immune responses.