Adult Male Fish Research Articles

An organizer controls the development of the “sword,” a sexually selected trait in swordtail fish

Male swordtail fish of the genus Xiphophorus (Poeciliidae) possess a "sword" that is composed of several colored elongated ventral fin rays of the caudal fin. The sword is a secondary sexual trait that evolved through sexual selection by female preference. To uncover the developmental mechanisms underlying the metamorphosis from a juvenile caudal fin to the sword, we have devised a transplantation protocol to assay the fate of single transplanted fin rays and their interactions with flanking rays. These experiments provide evidence for the existence of a previously unrecognized inductive signal that originates in those rays that develop into the two longest sword rays. This "sword organizer" causes adjacent fin rays to grow and become integrated into the sword and induces the development of an additional, typically pigmented sword in grafts to the dorsal part of the caudal fin. We show that the potential to develop a sword is restricted to certain parts of the caudal fin. Our findings suggest that the evolution of swords in swordtails required the acquisition of two developmental mechanisms: the establishment of signaling competence in prospective sword rays in the embryo or early larva, and its activation through androgen signaling in adult male fish.

Environmental estrogens suppress hormones, behavior, and reproductive fitness in male fathead minnows

This study explored the possibility that environmental estrogens in sewage effluent may reduce the reproductive fitness of adult male fish by suppressing their reproductive behaviors, including their ability to compete for nests and females. Male fathead minnows (Pimephales promelas) were exposed for three weeks to either blank control, effluent released by a sewage treatment plant (STPE), waterborne estradiol (E2), or a synthetic androgen (methyltestosterone [MT]). Afterward, fish were placed with females and a nest, and their behavior was monitored for 5 d in either the presence or the absence of a competing (unexposed control) male. Males exposed to either the STPE or E2 (approximately 50 ng/L, a level chosen to mimic the estrogenic content of the STPE) had elevated levels of circulating vitellogenin (p < 0.05) and lower levels of 11-ketotestosterone (KT; p < 0.05). Nearly all STPE- and E2-exposed males spawned successfully in the absence of a competing male, but in both cases, exposed males suffered nearly total reproductive failure when they had to compete. Conversely, males exposed to MT (approximately 50 ng/L) outcompeted control males. Behavioral observations suggested that subtle differences in agonistic behaviors, typically associated with circulating androgens (i.e., KT), were responsible. We speculate that male fish exposed to estrogenic effluent in the field are less likely to reproduce successfully within large populations of wild fish, thereby causing abnormal and potentially detrimental patterns of gene flow within those populations.

Comparative Effects of Direct Cadmium Contamination on Gene Expression in Gills, Liver, Skeletal Muscles and Brain of the Zebrafish (Danio rerio)

The effects of cadmium (Cd) on gene expression were examined in four organs (gills, liver, skeletal muscles and brain) of the zebrafish. Adult male fish were subjected to three different water contamination pressures over periods of 7 and 21 days: control medium (C(0): no Cd added) and two contaminated media (C(1): 1.9 +/- 0.6 microg Cd l(-1), and C(2): 9.6 +/- 2.9 microg Cd l(-1)). Fourteen genes involved in antioxidant defences, metal chelation, active efflux of organic compounds, mitochondrial metabolism, DNA repair and apoptosis were selected and their expression levels investigated by quantitative real-time PCR. Cadmium concentrations were determined in the four organs and metallothionein (MT) protein levels investigated in brain, liver and gills. Although skeletal muscle was a poor Cd-accumulating tissue, many genes were up-regulated at day 7: mt1, cyt, bax, gadd and rad51 genes. Three additional genes, c-jun, pyc and tap, were up-regulated in muscles at day 21 whereas bax, gadd and rad51 had returned to basal levels. Surprisingly, mt1 and c-jun were the only genes displaying a differential induction after 21 days in liver, although this organ accumulated the highest cadmium concentration. In brain, only mt1, mt2 and c-jun genes were up-regulated after 21 days. In gills, the highest response was observed after 7 days, featuring the differential expression of oxidative stress-response hsp70 and mitochondrial sod genes, along with genes involved in mitochondrial metabolism and metal detoxification. Then, after 21 days, the expression of almost every genes returned to basal levels while both mt1 and mt2 genes were up-regulated.

Energy Density of Lake Whitefish Coregonus clupeaformis in Lakes Huron and Michigan

We collected lake whitefish Coregonus clupeaformis off Alpena and Tawas City, Michigan, USA in Lake Huron and off Muskegon, Michigan USA in Lake Michigan during 2002–2004. We determined energy density and percent dry weight for lake whitefish from both lakes and lipid content for Lake Michigan fish. Energy density increased with increasing fish weight up to 800 g, and then remained relatively constant with further increases in fish weight. Energy density, adjusted for weight, was lower in Lake Huron than in Lake Michigan for both small (≤800 g) and large fish (>800 g). Energy density did not differ seasonally for small or large lake whitefish or between adult male and female fish. Energy density was strongly correlated with percent dry weight and percent lipid content. Based on data from commercially caught lake whitefish, body condition was lower in Lake Huron than Lake Michigan during 1981–2003, indicating that the dissimilarity in body condition between the lakes could be long standing. Energy density and lipid content in 2002–2004 in Lake Michigan were lower than data for comparable sized fish collected in 1969–1971. Differences in energy density between lakes were attributed to variation in diet and prey energy content as well as factors that affect feeding rates such as lake whitefish density and prey abundance.

Mutation in the Zebrafish Homolog of the RNA Helicase DHX8 Result in Hematopoietic Defects.

Hematopoiesis is a tightly regulated process that requires the coordinated efforts of many tissue-specific transcription factors. Lateral mesoderm tissue undergoes a series of restricted proliferation and differentiation that result in the production of the hematopoietic stem cells. These cells then differentiate to populate the different cell lineages. While some factors essential for this process have been defined, many remain unknown. Zebrafish offer a powerful model system in which it is possible to conduct forward phenotype-driven screening. We have undertaken a chemical mutagenesis screen using N-nitroso-N-ethylurea (ENU) in the zebrafish to uncover mutants in myelopoiesis and early hematopoiesis. Adult male fish were exposed to ENU in a protocol designed to mutagenize spermatagonia with a mutation frequency of approximately 1:1000. Mutagenized males were crossed to wild-type females to generate F1 females. Haploid embryos from F1 females were screened at 27 hours of development by RNA in situ hybridization using the myeloid-specific marker, leukocyte-specific plastin (l-plastin), and the stem cell marker, core binding factor b (cbfb). One mutant line identified from this screen, mummy, showed reduced cbfb expression and lack of l-plastin expression. Additional in situ staining has confirmed that mummy has a myeloid defect as other myeloid markers such as Lys.C, Mpo, and C/ebp1 were also significantly reduced. Mummy mutant also have a degreased expression of gata1 and bE1 globin (RBC markers) as well as the stem cell marker scl. These results suggested that the defect in mummy is at or up-stream of the HSC level. The mummy mutant embryos also suffer from widespread cell death and die around 36 hours post fertilization. Genetic mapping and positional cloning has identified the mutated gene as the zebrafish homolog of DHX8, which encodes an RNA helicase. The yeast homolog of dhx8, prp22, functions during mRNA splicing and RNA processing. Morpholino injection into wild-type embryos has confirmed that reduced expression of dhx8 can produce a similar phenotype. The wild-type zebrafish dhx8 cDNA but not the mutated dhx8 cDNA identified in mummy was able to partially rescue the mummy phenotype. Preliminary data from RT-PCR and microarray analysis suggest that mummy mutants have defects in the production of spliced transcripts of some genes but not others. Genes with decreased spliced transcripts in the mummy mutant include scl, cbfb, and gata1, which may explain the hematpoietic defects observed. Our results demonstrate that mutations in an RNA helicase involved in splicing could result in specific blockage of hematopoiesis.

Neuropeptide Y in the forebrain of the adult male cichlid fish Oreochromis mossambicus: Distribution, effects of castration and testosterone replacement

We studied the organization of the neuropeptide Y (NPY)-immunoreactive system in the forebrain of adult male cichlid fish Oreochromis mossambicus and its response to castration and testosterone replacement by using morphometric methods. Immunoreactivity for NPY was widely distributed in the forebrain, and the pattern generally resembled that in other teleosts. Whereas immunoreactivity was conspicuous in the ganglia of nervus terminalis (NT; or nucleus olfactoretinalis), a weak reaction was detected in some granule cells in the olfactory bulb and in the cells of area ventralis telencephali pars lateralis (Vl). Moderately to intensely immunoreactive cells were distinctly seen in the nucleus entopeduncularis (NE), nucleus preopticus (NPO), nucleus lateralis tuberis (NLT), paraventricular organ (PVO), and midbrain tegmentum (MT). NPY fibers were widely distributed in the forebrain. Castration for 10/15 days resulted in a drastic loss of immunoreactivity in the cells of NE (P<0.001) and a significant decrease (P<0.01) in their cell nuclear size. However, cell nuclei of the NT neurons showed a significant increase in size. A highly significant reduction in the NPY-immunoreactive fiber density (P<0.001) was observed in several areas of the forebrain. Although testosterone replacement reversed these changes, fibers in some areas showed supranormal responses. Immunoreactive cells in Vl, NPO, NLT, PVO, and MT and fiber density in some other areas did not respond to castration. We suggest that the NPY-immunoreactive elements that respond to castration and testosterone replacement may serve as the substrate for processing the positive feedback action of the steroid hormone.

Comparative Effects of Dietary Methylmercury on Gene Expression in Liver, Skeletal Muscle, and Brain of the Zebrafish (Danio rerio)

Effects of dietary methylmercury (MeHg) on gene expression were examined in three organs (liver, skeletal muscle, and brain) of the zebrafish (Danio rerio). Adult male fish were fed over 7, 21, and 63 days on three different diets: one control diet (C0: 0.08 microg of Hg g(-1), dry wt) and two diets (C1 and C2) contaminated by MeHg at 5 and 13.5 microg of Hg g(-1), dry wt. Total Hg and MeHg concentrations were determined in the three organs after each exposure duration, and a demethylation process was evidenced only in the liver. Thirteen genes known to be involved in antioxidant defenses, metal chelation, active efflux of organic compounds, mitochondrial metabolism, DNA repair, and apoptosis were investigated by quantitative real-time RT-PCR and normalized according to actin gene expression. Surprisingly, no change in the expression levels of these genes was observed in contaminated brain samples, although this organ accumulated the highest mercury concentration (63.5 +/- 4.4 microg g(-1), dry wt after 63 days). This lack of genetic response could explain the high neurotoxicity of MeHg. coxI and cytoplasmic and mitochondrial sod gene expressions were induced early in skeletal muscle and later in liver, indicating an impact on the mitochondrial metabolism and production of reactive oxygen species. Results demonstrated that skeletal muscle was not only an important storage reservoir but was also affected by MeHg contamination. The expression of the metallothionein mt2 and the DNA repair rad51 genes was up-regulated in liver between 21 and 63 days, whereas in skeletal muscle, mt2 remained uninduced, and gadd and rad51 were found to be repressed.

Acute toxicity of alpha-cypermethrin to guppy ( Poecilia reticulata, Pallas, 1859)

Alpha-cypermethrin is a synthetic pyrethroid insecticide used to control pests in domestic, industrial and agricultural situations. Adult male guppy fish ( Poecilia reticulata standard test species) were selected for the bioassay experiments. The experiments were performed in four series and the 96-h LC 50 value was determined for guppies. The acute toxicity experiments were carried out by static method and behavioral changes in guppies were determined for each alpha-cypermethrin (98% technical grade) concentration. Data obtained from the alpha-cypermethrin investigation were evaluated by the use of probit analysis statistical method and the 96-h LC 50 value for guppy was estimated as 9.43 μg/l.

Functional changes of the LH-immunoreactive cells in adenohypophysis of the common carp (Cyprinus carpio) from rivers contaminated with estrogenic chemicals.

The adverse effect of estrogenic chemicals on luteinizing hormone-immunoreactive (LH-ir) cells in the adenohypophysis of common carp (Cyprinus carpio) was examined using immunocytochemical and morphometric methods. Adult male fish were collected from two contaminated sites (Ishizu and Wada Rivers) and from a control pond at Agriculture, Food and Environmental Sciences, Research Center of Osaka Prefecture. The concentration of nonylphenol, bisphenol A and 17beta-estradiol in the Ishizu River was 3-4 times higher than that in the Wada River. The proportion and size of LH-ir cells were evaluated using the point-counting method by optical microscopy. In control carp, the proportion of LH-ir cells in the breeding season was significantly lower than in the pre- and post-breeding seasons. The same tendency was also found in Ishizu and Wada River carp, but without statistical significance. The proportion of LH-ir cells in Ishizu River carp was significantly lower than that of the control and the Wada River in all seasons. The LH-ir cells in control carp increased in size in the breeding season. LH-ir cells in Ishizu River carp were significantly (p<0.05) smaller than those in control fish, but not different from Wada River carp. A disturbance in the secretory function of LH-ir cells was found in carp from the Ishizu River; granulation and vacuolation were not in synchronization with those of control and Wada River fish. Our data suggest that the estrogenic chemicals in the Ishizu River interfere with functions of LH-ir cells directly or through the testis.

Fitness consequences of prey depletion for the common goby Pomatoschistus microps

Pomatoschistus microps (Pisces: Gobiidae), a predatory, benthic feeder, occurs in high densities in variable, shallow, muddy coastal waters where prey availability can be unpredictable. Here, we investigate the effect and consequences of prey depletion on the body condition and nest building of adult male fish using manipulative mesocosm experiments in which the comparative value of meiofaunal and macrofaunal prey was investigated. Fish maintained in mesocosms from which macrofauna had been removed from the sediment had a significantly reduced hepatosomatic index (HSI), and were less likely to build nests than fish in mesocosms containing macrofauna and meiofauna, suggesting an indirect link between prey availability and nest quality. Reduced resource availability, in the form of scarcity of macrofauna, appears to have profound implications for the fitness of natural field populations.

MNU Induction of Neoplasia in a Platyfish Model

Interspecific hybrid crosses between members of the fish genus Xiphophorus have been used for over 70 years to study the genetic aspects of melanoma formation. In the well-established “Gordon-Kosswig” cross, the platyfish X. maculatus is outcrossed to the swordtail X. helleri, and the resulting backcross segregants spontaneously develop melanoma. We recently produced a distinct cross between X. maculatus and another platyfish species, X. couchianus. X. maculatus strain Jp 163 A is homozygous for several X-linked pigment pattern genes, including the Spotted dorsal (Sd), Dorsal red (Dr), and Anal fin spot (Af). Af is a sex-limited trait, coding exclusively for melanophores distributed on the modified anal fin or “gonopodium” in the adult male fish. Within F1 and BC1 hybrids (to X. couchianus), the Sd pigment pattern is phenotypically suppressed, whereas Dr and Af are enhanced. We exposed BC1 hybrids to the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Treatment led to the development of schwannomas, fibrosarcomas, and retinoblastomas. In addition, numerous MNU-treated males that inherited Af developed a pronounced melanotic phenotype, with melanin-containing cells oftentimes totally covering the gonopodium and extending further to grow within the ventral regions of the fish. Genetic linkage analysis of the BC1 hybrids revealed a significant (p < 0.01) association between CDKN2X genotype and the phenotypic degree of melanization. Such an association is consistent with a locus within linkage group V playing a role in the development of melanosis and delineates three genetic preconditions and a carcinogenic scheme resulting in melanosis of the ventral regions of hybrid fish. The overall study further alludes to the potential of using Xiphophorus fish to study carcinogenic mechanisms for tumors other than melanoma (schwannoma, fibrosarcoma, and retinoblastoma) and should enable extensive pathologic and molecular genetic studies of derived neoplastic abnormalities.

Reproductive effects of long-term exposure to Bisphenol A in the fathead minnow (Pimephales promelas).

Bisphenol A (BPA), a high-volume chemical used to make polycarbonate plastic, epoxy resins, and other chemicals has been reported to be weakly estrogenic. To investigate the effects of long-term exposure to Bisphenol A, a multigeneration study was conducted in which fathead minnows (Pimephales promelas) were exposed to water concentrations of BPA in the range from 1 to 1280 micrograms/L. In this paper, we report the growth and reproductive effects of BPA on sexually mature adults in the F0 generation (after 43, 71, and 164 d of exposure) and the effects on hatchability in the F1 generation. Mean measured concentrations of BPA in the water for all doses, over a 164-d exposure period, were between 70% and 96% of nominal. An inhibitory effect of BPA on somatic growth (length and weight) occurred in adult male fish exposed to 640 and 1280 micrograms/L (after 71 and 164 d). BPA induced vitellogenin synthesis (VTG; a biomarker for estrogen exposure) in males at concentrations of 640 and 1280 micrograms/L after 43 d and 160 micrograms/L after 71 d. In females, plasma VTG concentrations were elevated above controls only after 164-d exposure to 640 micrograms/L. Inhibition of gonadal growth (as measured by the gonadosomatic index) occurred in both males and females at concentrations of 640 and 1280 micrograms/L after 164 d. In males, a concentration of 16 micrograms/L altered the proportion of sex cell types in the testis, suggesting inhibition of spermatogenesis. Concentrations of BPA that induced VTG synthesis and affected gonadal development were lower than those that resulted in discernible effects on reproductive output. Egg production was inhibited at a BPA concentration of 1280 micrograms/L, and hatchability in the F1 generation was reduced at a BPA concentration of 640 micrograms/L (there were not enough eggs spawned in the 1280 micrograms/L group for hatchability studies to be conducted). The results demonstrate that BPA acts as a weak estrogen to fish when administered via the water, with effects on breeding at and above 640 micrograms/L.

Effects of p‐nonylphenol, methoxychlor, and endosulfan on vitellogenin induction and expression in sheepshead minnow (Cyprinodon variegatus)

AbstractTemporal and dose‐responserelationshipsofvitellogenin(VTG) mRNA nductionand subsequent plasma VTG accumulation were established for sheepshead minnows (Cyprinodon variegatus) treated with p‐nonylphenol (an alkylphenol) and the organochlorine pesticides methoxychlor and endosulfan. Thirty‐two adult male fish per treatment were continuously exposed to measured concentrations of 0.64, 5.4, 11.8, 23.3, and 42.7 μg/L p‐nonylphenol; 1.1, 2.5, 5.6, 12.1, and 18.4 μg/L methoxychlor; and in two separate tests, 15.9, 36.3, 68.8, 162, 277, 403, 590, and 788 ng/L endosulfan using an intermittent flow‐through dosing apparatus. Separate triethylene glycol (50 (μl/L) and 17β‐estradiol (65.1 ng/L) treatments served as the negative and positive controls, respectively. Four fish were randomly sampled from each test concentration on days 2, 5, 13, 21, 35, and 42 of exposure, and levels of hepatic VTG mRNA induction and serum VTG accumulation were determined for each individual. Overall, fish exposed to p‐nonylphenol or methoxychlor demonstrated a rapid, dose‐dependent synthesis of VTG mRNA up to day 5 of exposure, followed by a relatively constant dose‐dependent expression through day 42. Both chemicals showed a dose‐dependent increase in plasma VTG over the entire time course of exposure, with significantly elevated VTG levels by the fifth day of exposure to p‐nonylphenol at concentrations of 5.4 μg/L or greater and to methoxychlor at concentrations of 2.5 μg/L or greater. Exposure to 0.64 μg/L p‐nonylphenol resulted in highly variable plasma VTG levels of less than 6 mg/ml. Exposures with endosulfan failed to induce measurable levels of either hepatic VTG mRNA or serum VTG at the chemical concentrations tested. Our results demonstrate that the sheepshead minnow bioassay is a suitable estuarine/marine teleost model for in vivo screening of potentially estrogenic substances.

Effects of p-nonylphenol, methoxychlor, and endosulfan on vitellogenin induction and expression in sheepshead minnow (Cyprinodon variegatus).

Temporal and dose-response relationships of vitellogenin (VTG) mRNA induction and subsequent plasma VTG accumulation were established for sheepshead minnows (Cyprinodon variegatus) treated with p-nonylphenol (an alkylphenol) and the organochlorine pesticides methoxychlor and endosulfan. Thirty-two adult male fish per treatment were continuously exposed to measured concentrations of 0.64, 5.4, 11.8, 23.3, and 42.7 micrograms/L p-nonylphenol; 1.1, 2.5, 5.6, 12.1, and 18.4 micrograms/L methoxychlor; and in two separate tests, 15.9, 36.3, 68.8, 162, 277, 403, 590, and 788 ng/L endosulfan using an intermittent flow-through dosing apparatus. Separate triethylene glycol (50 microliters/L) and 17 beta-estradiol (65.1 ng/L) treatments served as the negative and positive controls, respectively. Four fish were randomly sampled from each test concentration on days 2, 5, 13, 21, 35, and 42 of exposure, and levels of hepatic VTG mRNA induction and serum VTG accumulation were determined for each individual. Overall, fish exposed to p-nonylphenol or methoxychlor demonstrated a rapid, dose-dependent synthesis of VTG mRNA up to day 5 of exposure, followed by a relatively constant dose-dependent expression through day 42. Both chemicals showed a dose-dependent increase in plasma VTG over the entire time course of exposure, with significantly elevated VTG levels by the fifth day of exposure to p-nonylphenol at concentrations of 5.4 micrograms/L or greater and to methoxychlor at concentrations of 2.5 micrograms/L or greater. Exposure to 0.64 microgram/L p-nonylphenol resulted in highly variable plasma VTG levels of less than 6 mg/ml. Exposures with endosulfan failed to induce measurable levels of either hepatic VTG mRNA or serum VTG at the chemical concentrations tested. Our results demonstrate that the sheepshead minnow bioassay is a suitable estuarine/marine teleost model for in vivo screening of potentially estrogenic substances.

Early life‐stage mortality in zebrafish (Danio rerio) following maternal exposure to polychlorinated biphenyls and estrogen

AbstractIn the present study, specific polychlorinated biphenyl (PCB) congeners were examined for embryo and early life stage mortality in zebrafish (Danio rerio). A set of eight PCBs and two hydroxylated PCBs and 17β‐estradiol were tested. Of the compounds tested, 4′‐OH‐PCB30 (hydroxylated polychlorinated biphenyl) and PCB104 were found to be highly toxic to embryos following maternal exposure and transfer to the oocyte. It was also observed that 17β‐estradiol exposure resulted in a high incidence of embryo mortality. Analysis of estrogen receptor levels during embryonic development showed increased mRNA (ribonucleic acid) levels from the 1K stage to 50% epiboly. Following injection of the different compounds, the estrogen receptor mRNA levels were also analyzed in adult male fish to determine if there was a correlation between embryo mortality and estrogenicity of the studied PCBs. The two PCBs that were highly embryo toxic were observed to be estrogenic.

Fathead minnow ( Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds

The egg yolk precursor protein, vitellogenin (VTG), was purified from blood plasma of 17β-estradiol (E 2)-treated male fathead minnows ( Pimephales promelas) by anion-exchange chromatography on DEAE-agarose. A rabbit antiserum was raised against their blood plasma and then adsorbed with plasma from untreated (control) males to render the antiserum specific to VTG. The adsorbed antiserum was used to detect fathead minnow VTG (fVTG) in Western and dot blotting experiments and in an enzyme-linked immunosorbent assay (ELISA). The antiserum recognised fVTG as a ∼156 kDa protein in plasma from vitellogenic females and E 2-injected males but not untreated males. Its identity was confirmed by analysis of: (1) amino acid composition; (2) an internal amino acid sequence; (3) reactivity to the homologous antiserum; and (4) recognition by monoclonal antibodies prepared against the VTG from common carp ( Cyprinus carpio) and brown bullhead ( Ameiurus nebulosus). Specificity of the homologous antiserum to fVTG was confirmed by Western blotting of serially diluted plasma from vitellogenic females. Utility of the antiserum and purified fVTG for detecting exposure of male fathead minnows to estrogenic compounds was verified using a dot blotting immunoassay of fVTG and detected by chemiluminescence. Adult male fish were exposed to various concentrations of E 2 (10 −8, 10 −9 and 10 −10 M) in their rearing water and plasma assayed for the presence of VTG at different time points (2, 7, 14 and 21 days). A competitive, antibody-capture, quantitative ELISA was then developed based on the purified fVTG and its respective antiserum. The ELISA was validated by demonstrating parallel binding slopes of dilution curves prepared with plasma from E 2-injected males, vitellogenic females, and aqueous egg extracts as compared with purified fVTG standard. Plasma concentrations of VTG as low as 3 ng ml −1 were detected in the ELISA, for which inter- and intra-assay coefficients of variation were both less than 5%. Furthermore, plasma from control males was unreactive with the fVTG antiserum. The VTG ELISA could be useful for the detection of estrogenic properties associated with certain compounds and could be easily incorporated into standard laboratory toxicity assays using this species.

Fall and spring somatic energy content for Alaskan Pacific herring ( Clupea pallasi Valenciennes 1847) relative to age, size and sex

During the fall of 1995 and spring of 1996, the whole body energy content (WBEC) of Pacific herring ( Clupea pallasi Valenciennes 1847) from Prince William Sound, Alaska, was examined. Somatic energy (kJ·g −1 wet wt.) exhibited a wide range of values relative to fish length. In the fall young of the year (YOY) recruits had an average of 5.7 kJ·g −1 wet wt. for whole body samples vs. 8.0 for age 1 and 9.4–10.2 kJ·g −1 for fish of ages 2 to 7. The following spring the 1995 year class which had just survived their first winter averaged 4.4 kJ·g −1 wet wt. for somatic samples, and age 1 fish had similar values, while herring ages 2 to 7 had WBEC>5 kJ·g −1. The difference in somatic energy content between adult male and female fish captured in fall and again in the spring just prior to spawning, was about 4 kJ·g −1 wet wt., or about a 40% change. The difference in mean values for somatic energy content for YOY herring was only about 1.4 kJ·g −1 suggesting either the recruits ate more food during the winter than the adults, had lower energetic needs, or only those with higher than average energy stores survived the winter. The fall measures of WBEC showed the YOY and age 1 fish stored markedly less energy to over-winter than older herring. Thus, energetically the recruiting year class, and those entering their second winter, are the most at risk of over-winter mortality.

Modulation of glycoprotein hormone α- and gonadotropin IIβ-subunit mRNA levels in the pituitary gland of mature male African catfish, Clarias gariepinus

The cDNAs encoding the glycoprotein hormone α-subunit (GPα) and the gonadotropin IIβ-subunit (GTH IIβ) were cloned from the pituitary gland of the African catfish, Clarias gariepinus. Using RNase protection analysis, we studied the steady-state mRNA levels of GPα as well as GTH IIβ in the pituitary gland of adult male catfish. Castration of adult male catfish resulted in a significant decrease of GTH IIβ mRNA levels, whereas there was no change in the GPα mRNA levels. Treatment of intact males with a single dose of 11-ketotestosterone (11-KT) resulted in dose-dependent increases in mRNA levels of both GPα and GTH IIβ. We conclude that 11-KT, a prominent, non-aromatizable teleost androgen, has a stimulatory effect on the pituitary mRNA levels of GPα and GTH IIβ of adult male fish.

Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate

In Drosophila melanogaster and Caenorhabditis elegans, the elucidation of developmental mechanisms has relied primarily on the systematic induction and isolation of mutations in genes with specific functions in development. Such an approach has not yet been possible in a vertebrate species, owing to the difficulty of analyzing and keeping a sufficiently high number of mutagenized lines of animals. We have developed the methods necessary to perform large-scale saturation screens for mutations affecting embryogenesis in the zebrafish, Danio (Brachydanio) rerio. Firstly, a new aquarium system was developed to raise and keep large numbers of strains of genetically different fish safely and with little maintenance care. Secondly, by placing adult male fish in water containing the chemical mutagen, ethylnitrosourea, we induced point mutations in premeiotic germ cells with a rate of one to three mutations per locus per 1,000 mutagenized haploid genomes. This rate, which is similar to the mutagenesis rates produced by ethylmethanesulfonate in Drosophila, was determined for alleles at four different pigmentation genes. Finally, in a pilot screen in which mutagenized fish were inbred for two generations and scored for embryonic mutants, we isolated 100 recessive mutations with phenotypes visible in the homozygous embryos. The high rate of induction and recovery of point mutations, in addition to an efficient aquarium system to house large numbers of mutagenized lines, means that it is now possible to perform saturation mutagenesis screens in a vertebrate, similar to those done in invertebrates.

Detection of γ-ray-induced DNA damages in malformed dominant lethal embryos of the Japanese medaka ( Oryzias latipes) using AP-PCR fingerprinting

Adult male fish of the medaka HNI strain exposed to 9.5 Gy or 19 Gy (0.95 Gy/min) of γ-rays were mated with non-irradiated female fish of the Hd-rR strain. Genomic DNA was prepared from malformed individual embryos which were expected to be dominant lethal and used for AP-PCR fingerprinting. By the use of a part of the T3 promoter sequence (20 mer), which to our knowledge, is not found in the medaka genome as an arbitrary primer, we found polymorphisms in genomic fingerprints which could distinguish the parental strains. On the other hand, we found that the fingerprints of F 1 hybrids were the sum of those of their parents. Based on those findings, we analyzed the fingerprints of genomic DNA of each severely malformed embryo, because we expect that radiation-induced genomic damages resulting in severe malformation and eventually in dominant lethals should be detected as changes in paternal fingerprints of F 1 hybrids. Indeed, we succeeded in detecting changes in genomic DNA as loss of some paternal bands in fingerprints of malformed embryos. One of 10 malformed embryos obtained from 9.5 Gy γ-irradiated males had lost one band of the paternal origin and 4 of 12 malformed embryos obtained from 19 Gy γ-irradiated males had lost 5 bands. These results indicated a possibility that quantitative as well as qualitative estimation of γ-ray-induced DNA damages can be made by this method which does not require the functional selection based on a specific target gene.