Comparison of ELISAs for detecting vitellogenin in the fathead minnow (Pimephales promelas)

Measurement of vitellogenin (VTG) concentrations in the fathead minnow (Pimephales promelas) is currently being considered and evaluated for screening of endocrine active substances. One of the proposed methods, an enzyme-linked immunosorbent assay (ELISA) based on VTG from carp (Cyprinus carpio), was recently evaluated in an inter-laboratory ring test using whole body homogenates from juvenile fathead minnows. The objective of the current study was to compare the performance of three different ELISAs for measuring fathead minnow VTG: (1) a heterologous carp VTG (cVTG) ELISA used in the ring test, (2) a homologous fathead minnow VTG (fVTG) ELISA, and (3) a hybrid ELISA with the antibody developed for cVTG, but using fVTG for coating the plates and preparing standard curves. VTG was measured in whole body homogenates from juvenile fathead minnows exposed to 17alpha-ethynylestradiol (EE(2); 10 ng/l) and whole body homogenates and plasma from adult fathead minnows exposed to 17beta-estradiol (E(2); 5 mg/kg; i.p.). The cVTG assay showed lower specificity for fathead minnow VTG in whole body homogenates and plasma from treated fish, compared to the fVTG assay. VTG concentrations in juvenile fathead minnow homogenates from the EE(2)-exposed group were approximately 50-fold higher when measured using the fVTG method compared to the cVTG method. Use of the homologous fVTG in the hybrid cVTG assay yielded VTG concentrations in the range of the fVTG assay but the low specificity persisted. The homologous fVTG assay is recommended to achieve accurate quantification of VTG levels in fathead minnows.     

Underwater Video Reveals Strong Avoidance of Chemical Alarm Cues by Prey Fishes

A diversity of fishes release chemical cues upon being attacked by a predator. These cues, commonly termed alarm cues, act as sources of public information warning conspecifics of predation risk. Species which are members of the same prey guild (i.e. syntopic and share predators) often respond to one another's alarm cues. The purpose of this study was to discriminate avoidance responses of fishes to conspecific alarm cues and cues of other prey guild members from responses to unknown damaged fish odours and novel odours. We used underwater video to measure avoidance responses of freshwater littoral species, namely fathead minnows (Pimephales promelas), finescale dace (Chrosomus neogaeus), and brook stickleback (Culaea inconstans), to both injured fish cues and novel non‐fish odours. The cyprinids (minnows and dace) showed significant avoidance of minnow cues over swordtail cues, morpholine, and the control of distilled water and tended to avoid fathead cues over cues of known prey guild members (stickleback). Cyprinids also significantly avoided cues of stickleback over unknown heterospecific cues (swordtail) and tended to avoid stickleback cues over morpholine and the distilled water control. Stickleback significantly avoided fathead minnow extract over the distilled water and tended to avoid stickleback and swordtail over distilled water. We conclude that fishes in their natural environment can show dramatic changes in behaviour upon exposure to alarm cues from conspecifics and prey guild members. These responses do not represent avoidance of cues of any injured fish or any novel odour.     

Substrate mobilization and hormonal changes in rainbow trout (Oncorhynchus mykiss,L.) and common carp (Cyprinus carpio,L.) during deep hypoxia and subsequent recovery

Common carp (at 20°C) and rainbow trout (at 15°C) were fitted with an indwelling cannula in the dorsal aorta. The fish were exposed to a controlled decline of waterpO₂ followed by 90 min deep hypoxia at 0.3 kPa (carp) or 4.8 kPa (trout). Thereafter, normoxic recovery was monitored in both species for 48 h. At regular intervals blood samples were analysed for glucose, lactate, free fatty acids, adrenaline, noradrenaline and cortisol. The oxygen restriction was maximal in both species and resulted in a significant increase of plasma lactate levels. In carp, adrenaline, noradrenaline and cortisol levels increased to 2, 50, and 753 ng·ml^-1 respectively during anoxia, whereas in trout these hormones increased to 12, 8 and 735 ng·ml^-1 respectively during hypoxia. In hypoxic trout, the plasma levels of glucose (3 mol·l^-1) were increased modestly whereas levels of free fatty acids (0.25 mmol·l^-1) were decreased to 0.15 mmol·l^-1. In carp, however, a marked and prolonged hyperglycaemia (from 5 to 10 mmol·l^-1) and a significant continuous depression of plasma levels of free fatty acids (from 0.4 to 0.2 mmol·l^-1) were observed indicating a difference in metabolic organization. It is suggested that hyperglycaemia is likely to be the result of hepatic glycogenolysis, stimulated by circulating catecholamines and a stimulation of gluconeogenesis by cortisol during recovery. The mechanism for the decline of plasma levels of free fatty acids is most probably a reduction of lipolytic activity, which appears to be an adaptation to hypoxia.     

Effects of handling on endocrinology and reproductive performance of the fathead minnow

Anaesthesia with MS-222 followed by intra-peritoneal (ip) injection (with a 10% ethanol in corn oil carrier) of fathead minnow either as one or three (weekly) treatments did not affect survival, behaviour or secondary sexual characteristics of the fish. Fecundity of the fish, as indicated by fertility and hatching success, was also unaffected. Gonadal condition (relative gonad mass, histopathology) was not altered in either sex. Male and female plasma sex steroids (β;-oestradiol, testosterone, 11-ketotestosterone) and male vitellogenin concentrations were not significantly affected by the treatments. Females subjected to either ip treatment regime had significantly higher plasma vitellogenin concentrations than control females. However, based on previous data, this difference did not appear to be treatment-related. Overall, exposure of fathead minnows to chemicals via the ip route should not confound the interpretation of toxicity tests with potential endocrine disrupting chemicals.     

Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas)

The fathead minnow (Pimephales promelas) has been proposed as a model species for assessing the adverse effects of endocrine-disrupting chemicals (EDCs) on reproduction and development. The purpose of these studies was to develop baseline reproductive biology and endocrinology data for this species to support interpretation of tests with potential EDCs. Pairs of reproductively-active fathead minnows (n=70) were evaluated with respect to reproductive cyclicity in terms of spawning interval and fecundity. The mode and mean (+/-SE) spawning intervals for the fish in this study were 3.0 and 3.7+/-0.1 days, respectively. The mean number of eggs produced per spawn was 85+/-2.8. Animals were sacrificed at periodic intervals during the established spawning cycle and measurements made of gonadal condition (gonadosomatic index [GSI], histopathology) and plasma concentrations of vitellogenin and sex steroids (beta-estradiol, testosterone, 11-ketotestosterone). The GSI in females varied significantly as a function of spawning interval, with the largest values occurring day 2 post-spawn, just prior to the interval of maximum spawning activity. Plasma beta-estradiol concentrations in females also varied significantly relative to peak values in the GSI and spawning activity. Vitellogenin concentrations in the female, and male GSI and steroid concentrations did not vary significantly relative to position in the spawning cycle. Concentrations of beta-estradiol in females and 11-ketotestosterone in males were positively correlated with testosterone concentrations.     

Assessing the efficacy of corn-based bait containing antimycin-a to control common carp populations using laboratory and pond experiments

Strategic use of oral toxicants could allow for practical and sustainable control schemes for the invasive common carp (Cyprinus carpio, or ‘carp’) if a toxicant selectively targeted carp and not native species. In this study, we incorporated antimycin-a (ANT-A), a known fish toxicant, into a corn-based bait and conducted a series of experiments to determine its toxicity, leaching rate, and species-specificity. Our results showed that ANT-A was lethal to carp at doses ≥ 4 mg/kg and that the amount of ANT-A that leached out of the bait in 72 h was not lethal to carp or bluegill (Lepomis macrochirus). Species-specificity trials were conducted in 227 L tanks, in which carp were stocked with three native species representing families that occur sympatrically with carp in our study region: the fathead minnow (Pimephales promelas), yellow perch (Perca flavescens) and bluegill. These trials showed high mortality of carp (46%) and fathead minnows (76%) but no significant mortality of perch or bluegill. Finally, a pond study, which used the same species composition except for fathead minnows, resulted in 37% morality among adult carp and no mortality among perch or bluegill. Our results suggest that corn-based bait that contains ANT-A could be used to selectively control carp in ecosystems dominated by percids or centrarchids, such as lakes across the Great Plains ecoregion of North America, where carp are especially problematic.     

Acquired Recognition of Chemical Stimuli from Pike,Esox lucius,by Brook Sticklebacks,Culaea inconstans (Osteichthyes,Gasterosteidae)

In this study we test whether brook sticklebacks (Culaea inconstans) can acquire predator recognition through releaser-induced recognition learning, i.e. simultaneous exposure to aversive ('releasing') stimuli and neutral stimuli causing learned aversion to the neutral stimuli. We exposed wild-caught pike-naive brook sticklebacks (collected from a creek containing fathead minnows, Pimephales promelas, but not pike, Esox lucius) to chemical stimuli from pike that were mixed with brook stickleback skin extract, fathead minnow skin extract, or a control of distilled water. In subsequent tests 2 d later, when only pike stimuli were presented, sticklebacks conditioned with stickleback skin extract and fathead minnow skin extract exhibited antipredator behaviour (i.e. increased schooling and movement toward the substrate), while those conditioned with distilled water did not. Sticklebacks conditioned with stickleback skin extract responded to pike with a more intense response, in terms of movement toward the substrate, than those conditioned with fathead minnow skin extract, suggesting that conspecific skin extract may be a stronger stimulus than heterospecific skin extract for learning recognition of predators. To our knowledge this is the first study to demonstrate that an acanthopterygian fish can acquire predator recognition through the pairing of conspecific alarm pheromone with the cue of a predator. Furthermore, our results are the first to demonstrate that fish can acquire predator recognition through the pairing of a heterospecific alarm pheromone with the cue of a predator. These results suggest that brook sticklebacks will benefit by being in close proximity to fathead minnows. Acquired predator recognition has long-term consequences in mediating predator-prey interactions.     

Impact of cyprinids on zooplankton and algae in ten drainable ponds

To study the impact of cyprinids on algae, zooplankton and physical and chemical water quality, ten drainable ponds of 0.1 ha (depth 1.3 m) were each divided into two equal parts. One half of each pond was stocked with 0 + cyprinids (bream, carp and roach of 10–15 mm), the other was free of fish. The average biomass of the 0 + fish at draining of the ponds was 466 kg ha^–1, to which carp contributed about 80%.The fish and non-fish compartments showed significant differences. In the non-fish compartments the density of Daphnia hyalina was 10–30 ind. l^–1 and that of Daphnia magna 2–4 ind. l-^–1, whereas in the fish compartments densities were c. 1 ind. l^–1. Cyclopoid copepods and Bosmina longirostris, however, showed higher densities in the fish compartments. The composition of algae in the two compartments differed only slightly, but the densities were lower in the non-fish compartments. The significant difference in turbidity was probably caused by resuspension of sediment by carp. No significant difference in nutrient concentration between the compartments was found.     

Identification of four ovarian receptor proteins that bind vitellogenin but not other homologous plasma lipoproteins in the rainbow trout,Oncorhynchus mykiss

Membrane proteins from ovarian follicles, testis and somatic tissues of rainbow trout, Oncorhynchus mykiss, were extracted by ultracentrifugation, separated on sodium dodecyl sulphate gels and isolated on polyvinyl difluoride membranes. Vitellogenin receptor proteins were visualised using protein staining and hybridisation with ¹²⁵I-vitellogenin Four follicle-membrane proteins, with molecular masses of 220, 210, 110 and 100 kDa, showed a strong affinity for vitellogenin and were specific to the ovary. Other homologous lipoproteins (very low density lipoprotein, low density lipoprotein and high density lipoprotein) had a very limited ability to displace ¹²⁵I-vitellogenin from its receptor, indicating that the ovarian receptor proteins were fairly specific for vitellogenin. Proteins with an affinity for very low density lipoprotein and low density lipoprotein were visualised in liver, spleen and muscle, eluting on sodium dodecyl sulphate gels with molecular masses of about 150 kDa. Peptides generated from trypsin digests of the receptor proteins with a high affinity for vitellogenin showed sequence homology with receptors in the lipoprotein family, including a sequence that is believed to act as the internalisation signal [Phe-Asp-Asn-Phe-Tyr-] and, a sequence identity with the recently characterised chicken vitellogenin/very low density lipoprotein receptor [Ser-Glu-Leu-Tyr-Glu-Pro-Ala-]. Together, the ligand blotting and peptide sequence data support the contention that the four ovarian membrane proteins isolated are receptor proteins specific for vitellogenin and they do not bind other plasma lipoproteins to any significant degree.Abbreviations BSA bovine serum albumin - HDL high density lipoprotein - LDL low-density lipoprotein - HPLC high performance liquid chromatograph - PVDF polyvinylidene difluoride - RIA radioimmunoassay - rt-VTG rainbow trout vitellogenin - SDS sodium dodecyl sulphate - VLDL very low density lipoprotein - VTG vitellogenin - VRP-1,-2,-3 or -4 vitellogenin receptor proteins     

Predicting the Effects of Endocrine Disrupting Chemicals on Fish Populations

This study evaluates the applicability and sensitivity of fish population dynamics modeling in assessing the potential effects of individual chemicals on population sustainability and recovery. Fish reproductive health is an increasingly important issue for ecological risk assessment following international concern over endocrine disruption. Life-history data from natural brook trout and fathead minnow populations were combined with effects data from laboratory-based studies, mainly concerning species other than brook trout and fathead minnows, to assess the likely impact of nonylphenol (NP) and methoxychlor (MXC) on brook trout (Salvelinus fontinalis) and fathead minnow (Pimephales promelas) population size. A delay differential equation (DDE) model with a 1-day timestep was used to predict the population dynamics of the brook trout and fathead minnows. The model predicts that NP, could enhance populations by up to 17% at a concentration of 30?µg l^?1 based on the results of reduction in survival and increased fecundity from life-cycle toxicity tests, however attempting to allow for growth reduction and its effect on fecundity results in a prediction of a 28% reduction in population numbers. For fathead minnows the DDE model predicts that the same concentration of NP could cause a population reduction of 21%. The differences in these predictions are related to these two species having different life history strategies, which are considered in the parameterization of the model. Post-application concentrations of MXC may peak around 300?µg l^?1 and then decline rapidly with time. Predictions show that such applications could cause a reduction of up to 30% in brook trout populations if the application occurs at the peak of the spawning season on successive years but that the effect would be less than 1% if the spawning season is avoided. Effects on the fathead minnow population size are predicted to be smaller (<4%) even if application occurs during the spawning period. Risk based statistics generated by the population dynamics models, such as interval decline risk or quasiextinction risk and predicted time to recovery complement traditional effects parameters such as LC50 and LOEC and may ultimately prove to be more useful in risk assessment.     

Swimming performance and energy homeostasis in juvenile laboratory raised fathead minnow (Pimephales promelas) exposed to uranium mill effluent

Research at the Key Lake uranium mill (Saskatchewan, Canada) suggests effluent discharged from the mill affects energy stores of resident fish, but the mechanisms by which energy homeostasis is affected and the subsequent effects on swimming performance are unknown. In the present study larvae were collected from laboratory raised adult fathead minnow (Pimephales promelas) exposed to 5% diluted uranium mill effluent or control (dechlorinated municipal) water, and reared in the same treatments to 60 days post hatch (dph). Critical swimming speed (U_crit) was significantly lower in effluent exposed 60 dph fish compared to control fish. Fish used in tests were considered fatigued and compared to fish without swim testing (non-fatigued). There were no differences in whole body glycogen or triglyceride concentrations between effluent exposed versus control fish. However, fatigued fish from both treatments had significantly lower triglycerides, but not glycogen, compared to non-fatigued fish from the same treatment. Whole body β–hydroxyacyl coenzymeA dehydrogenase activity was similar in fish from both treatments, but citrate synthase activity was significantly lower in effluent exposed fish. Our results suggest uranium mill effluent exposure in the laboratory affects aerobic energy metabolism and swimming performance in juvenile fathead minnow, which could affect wild fish survivability.     

Plasma vitellogenin in landlocked Atlantic salmon (Salmo salar Ouananiche): isolation, homologous radioimmunoassay and immunological cross-reactivity with vitellogenin from other teleosts

Vitellogenin was isolated by affinity chromatography and gel filtration from landlocked Atlantic salmon plasma. Vitellogenin was labelled with iodine-131 using iodogen and an homologous radioimmunoassay was developed. There was poor immunological cross-reactivity with vitellogenin or plasma from other teleosts. Parallelism of the vitellogenin standard to the displacement by plasma of vitellogenic salmon allowed the assay to be used to evaluate the seasonal concentration profile of vitellogenin in female adult salmon. Extracts of liver or ovary from female Atlantic salmon also yielded displacements parallel to the vitellogenin standard in the assay.     

Carp (Cyprinus carpio) vitellogenin: purification and development of a simultaneous chemiluminescent immunoassay

Vitellogenin (Vg) was purified from the serum of vitellogenic female carp (Cyprinus carpio) by hydroxylapatite column chromatography and gel filtration. Vg had an apparent molecular mass of 490 kDa and appeared as two bands corresponding to 190 and 156 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against carp lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. The amino acid composition of carp Vg was similar to previous reports of cyprinids. The chemiluminescent immunoassay (CLIA) for carp Vg was developed to quantify serum Vg using purified carp Vg and anti-Lv. Its measurable range was from 1.95 to 1000 ng/ml. The dilution curve in the CLIA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Furthermore, the assay had cross-reactivity with the sera of other female cyprinids (crucian carp and Japanese dace). In fish diets-experiments, Vg was detected in all fish in the fish meal containing soybean (20%) group, but was not detected in almost all of the fish in the fish meal-group. This suggests that a soybean based-diet may induce Vg production in the serum of cultivated carp.     

Characterization of vitellogenin from white sturgeon,Acipenser transmontanus

Sturgeon are an ancient family (Acipenseridae) of fishes that lie close to the divergence of fish that eventually evolved into terrestrial animals and those that evolved into modern teleost species. Therefore, white sturgeon vitellogenin sequences fill a gap in the current understanding of the functional domains of this protein family. Vitellogenin cDNA was sequenced and used to investigate gene expression in white sturgeon, Acipenser transmontanus. Estrogen-induced vitellogenin mRNA was detected in the livers of both males and females and was also detected in undifferentiated gonads of estrogen-treated fish. Low levels of vitellogenin mRNA were also detected in the testis of both control and estrogen-treated males. The cDNA encoded a 186-kDa protein that was missing only six to seven of the amino-terminal amino acids. Comparisons to silver lamprey, Xenopus, and chicken vitellogenin sequences indicate that the overall structure of the yolk protein domains were highly conserved. There was considerable homology in three regions of the lipovitellin I domain. These conserved sequences are likely to be involved in vitellogenin receptor binding. The phosvitin domain of white sturgeon vitellogenin contained fewer and shorter serine repeats as predicted from yolk protein phosphate content of fish compared to Xenopus and chicken. However, the vitellogenin of white sturgeon had a lower serine content as compared with silver lamprey, indicating that an increased serine content is not strictly a function of evolutionary age. Correspondence to: C. A. Bidwell     

Fatty acid composition of vitellogenin from four teleost species

The fatty acid compositions of vitellogenin and liver from cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus) and wolffish (Anarhichas lupus) were determined. Vitellogenin was isolated from plasma of estradiol-17-treated fish by precipitation with EDTA-Mg²⁺ and distilled water or by high-performance ion-exchange chromatography. In all investigated species, vitellogenin contained 16–18% (w/w) lipid, in which polyunsaturated fatty acids, predominantly 20:5 (n-3) and 22:6 (n-3), comprised about 50% of the total fatty acids. The proportions of saturated, monounsaturated, polyunsaturated and (n-3) fatty acids in vitellogenin of the different species were generally similar, although the relative content of specific fatty acids was distinctive for each species. The distribution of fatty acids in total lipids of vitellogenin was highly consistent among individual females of each species. In contrast, liver fatty acid composition varied considerably, both within and between species. Altogether, the differences in the fatty acid composition of vitellogenin and liver from each species indicate that a specific selection of fatty acids occurs during the lipidation of vitellogenin.Abbreviations BHT butylated hydroxytoluene - E-17 estradiol-17 - EDTA ethylenedinitrilo tetra-acetic acid disodium salt dihydrate - FA fatty acids - FAME fatty acid methyl esters - HDL high density lipoproteins - PUFA polyunsaturated fatty acids - SD standard deviation - TLC thin-layer chromatography - VHDL very high density lipoproteins - VLDL very low density lipoproteins - v/v volume per volume - w/v weight per volume - w/w weight per weight     

Distribution of surface soil mercury of Wuda old mining area,Inner Mongolia,China

In this study, mercury (Hg) concentrations in dustfall and topsoil were investigated. Three hundred forty-four samples were collected, including dustfall and topsoil samples, across an area of ～180 km² in Wuda, China. Dustfall Hg concentration in Wuda ranged from 10 to 6453 ng·g^?1_, with an average of 305 ng·g^?1, and topsoil Hg concentration ranged from 3 to 1537 ng·g^?1, with an average value of 135 ng·g^?1. The average dustfall Hg concentrations in the coalfield, industrial park, and urban areas were 289 ng·g^?1, 809 ng·g^?1, and 160 ng·g^?1, respectively, and the corresponding average topsoil Hg concentrations were 216 ng·g^?1, 242 ng·g^?1, and 91 ng·g^?1. Hg concentrations were significantly higher in the coalfield, industrial park, and urban areas compared with background values for Wuda and China tide soil. The coal Hg concentrations ranged from 273 to 346 ng·g^?1, with an average value of 317 ng·g^?1. Comparison of the Hg concentrations of Wuda coal with other regions and countries, indicated that Hg concentrations were significantly enriched in coal, highlighting that coal is the primary Hg source for Wuda District. While coal fires provided the primary source of Hg, some higher Hg values were caused by factors such as gangue hills and coalfield topography, the use of activated carbon with HgCl₂ as a catalyst in the industrial park, and several coal washeries in wasteland areas. In addition to atmospheric Hg, underground coal seam emissions served as another potential Hg source in the coalfield. The surface soil Hg in the coalfield and industrial park areas should be given closer attention in the future.     

Growth and production of O+ bream (Abramis brama), O+ roach (Rutilus rutilus) and O+ carp (Cyprinus carpio) in 10 drainable 0.1 Ha ponds

In ten 0.1 ha drainable ponds O⁺ cyprinids were stocked to assess their impact on the aquatic community. Bream and roach were stocked in early June, 1986, and carp in early July, 1986. The ponds were drained mid November, 1986. The relationship between the fish community and its food resource was estimated in terms of production and consumption with use of the data on growth and mortality of the fish. The average gross production of the fish species in the ten ponds (48.4 kg during the experiment) was dominated by the carp (58.5%). A minimum estimate of the food consumption of the O⁺ cyprinids is 760 kJ.m^–2. The average production of bream and roach was much lower than that of carp due to a higher mortality and a more specialized feeding pattern. The gross productions of carp, bream and roach in the ten ponds were not significantly related, although stomach analysis showed diet overlap between the carp and the bream.     

Cyprinid assemblages,and the physical and chemical characteristics of small northern Ontario lakes

Synopsis The cyprinid species, and physical and chemical characteristics were recorded from 58 small lakes in the Algoma district of northern Ontario. A group of typical stream-dwelling lithophilous species, which included common shiner, Notropis cornutus, creek chub, Semotilus atromaculatus, and blacknose dace, Rhinichthys atratulus, tended to occupy lakes of larger than average drainage areas. Phoxinus spp., pearl dace, Semotilus margarita, and fathead minnow, Pimephales promelas, occurred commonly in lakes with small drainage areas, and the average drainage area of lakes they occupied was near or below the overall average. Lakes without stream cyprinids were discriminated from lakes with them on a combination of chemical variables, including pH, alkalinity and calcium, which indicated that they were more susceptible to anthropogenic acidification.     

New methods for the purification of vertebrate vitellogenin.

Two procedures are described for the isolation of vitellogenin from plasma. Vitellogenin can be selectively precipitated from the plasma of estrogen-treated animals by Mg²⁺ in the presence of EDTA. The vitellogenin thus obtained is >99% pure and is relatively undegraded. In the second procedure, vitellogenin can also be isolated by DEAE-cellulose chromatography, resulting in preparations that contain <2% albumin. In combination, these two isolation procedures yield vitellogenin preparations of very high purity.