Influence of batch-specific biochemical egg characteristics on embryogenesis and hatching success in farmed pikeperch.

Low and variable egg quality remains a major issue in aquaculture impeding a reliable and continuous supply of larvae, particularly in emerging species, such as pikeperch, Sander lucioperca. We assessed the influence of batch-specific egg parameters (fatty acid (FA) profiles, cortisol content) on embryo life-stages until hatching (survival at 2, 24, 48, 72 h post fertilization (hpf), hatching rate) in an integrated study under commercial hatchery conditions (44 egg batches). Embryo mortality was elevated until 48 hpf (average 9.8% mortality between 2 and 48 hpf). Embryos surviving until 48 hpf were very likely (98.5%) to hatch successfully. The inherent egg FA composition was variable in-between batches. Total FA content ranged form 66.1 to 171.7 µg/mg (dry matter) total FA. Whereas specific FA ,18 : 0 and 20 : 5(n-3) (eicosapentaenoic acid) of the polar fraction and the ratio of 22 : 6(n-3) (docosahexaenoic acid) to 20 : 5(n-3) within the neutral fraction, were significantly correlated with early embryo development, contents of the respective FA did not differ between high (>90% hatching rate), mid (70% to 90% hatching rate) and low (<70% hatching rate) quality egg batches. Late embryo development and hatching were relatively independent of the FA profiles highlighting stage-dependent influences especially during early embryogenesis. Cortisol levels ranged from 22.7 to 293.2 ng/ml and did not directly explain for mortalities. However, high cortisol was associated with a lower content of specific FA, in particular highly unsaturated FA. These results demonstrate the magnitude of inter-individual differences in the batch-specific biochemical egg composition under stable hatchery conditions and suggest a stress-mediated lack of essential FA, which in turn affects early embryo survival. Surprisingly, embryos are able to cope well with a broad range of inherent egg parameters, which limits their predictive potential for egg quality in general. Still, specific FA profiles of high quality egg batches have potential for formulating species-specific broodstock diets and improving reproductive management in pikeperch.

Application of low-frequency sonophoresis and reduction of antibiotics in the aquatic systems.

A major concern in aquaculture is the use of chemical therapeutics, such as antibiotics, because of their impact on the environment as well as on the fish product. As a potential tool for reducing antibiotic use, we tested the application of low-frequency ultrasound as a method for enhancing antibiotic uptake. Rainbow trout juveniles (Oncorhynchus mykiss) were exposed to two different concentrations of oxytetracycline (OTC), flumequine (FLU) and florfenicol (FLO), administered by bath after the application of ultrasound. After exposure, concentrations of these substances were measured in the liver and blood of treated fish. Results showed that the ultrasound treatment can significantly increase the uptake for all three antibiotics. The uptake of OTC for example, in fish exposed to an OTC concentration of 20 mg L-1 after prior treatment with ultrasound, was similar to the OTC concentrations in their liver and blood to fish exposed to 100 mg L-1 without sonication. For FLU and FLO, the use of ultrasound caused significant differences of uptake in the liver at high antibiotic concentrations. This suggests that the use of ultrasound as a technique to deliver antibiotics to fish can ultimately reduce the amount of antibiotics discharged into the aquatic environment.

Effects of the non-steroidal anti-inflammatory drug(NSAID) naproxen on gene expression of antioxidant enzymes in zebrafish (Danio rerio).

The aim of this study was to investigate the effects of naproxen on the gene expression of antioxidant enzymes in adult zebrafish. Surprisingly, after 2 weeks exposure no significant effect on the mRNA expression of the target genes was found in the liver. However, mRNA levels of three genes were altered significantly in the intestine. The expression of Ucp-2 decreased at the environmental concentration of 1µg/L while mRNA expression of GST p2 increased at the concentration of 100µg/L. The mRNA level for the antioxidant enzyme CAT was up-regulated significantly at both the concentrations used. Exposure to naproxen caused only moderate effects on the expression of antioxidant genes in the intestine rather than in the liver, which demonstrates that the intestine is more sensitive to waterborne naproxen exposure than the liver. Interestingly, the adverse side effects of NSAIDs occur in the gastrointestinal tract of humans. To our knowledge, this is the first study that has focused on transcriptional effects of naproxen on zebrafish.

The progestin levonorgestrel disrupts gonadotropin expression and sex steroid levels in pubertal roach (Rutilus rutilus).

The aim of the present study was to investigate the effects of the synthetic progestin levonorgestrel (LNG) on the reproductive endocrine system of a teleost fish, the roach (Rutilus rutilus). Pubertal roach were exposed for 28 days in a flow-through system to four concentrations of LNG (3, 31, 312, and 3124 ng/l). Both males and females treated with 3124 ng/l LNG exhibited the upregulated levels of vitellogenin and oestrogen receptor 1 mRNA in the liver. At the same concentration, LNG caused a significant upregulation of the mRNA expression of the gene encoding luteinising hormone ß-subunit (lhß) and the suppression of the mRNA expression of the gene encoding follicle-stimulating hormone ß-subunit (fshß) in the pituitary of both male and female roach. A lower LNG concentration (312 ng/l) suppressed mRNA expression of fshß in males only. Females treated with 3124 ng/l LNG exhibited significantly lower plasma 11-ketotestosterone (11-KT) and oestradiol (E2) concentrations, whereas their testosterone (T) level was higher compared with the control. Females exposed to 312 ng/l LNG presented significantly lower plasma E2 concentrations. Males exposed to ≥31 ng/l LNG exhibited significantly reduced 11-KT levels. As determined through a histological analysis, the ovaries of females were not affected by LNG exposure, whereas the testes of males exposed to 31 and 312 ng/l LNG exhibited a significantly higher percentage of spermatogonia B compared with the control. The results of the present study demonstrate that LNG disrupts the reproductive system of pubertal roach by affecting the pituitary gonadotropin expression and the sex steroid levels. This disruption was determined to occur in males after exposure to an environmentally relevant concentration (31 ng/l). Moreover, the highest tested concentration of LNG (3124 ng/l) exerted an oestrogenic effect on fish of both sexes.

Nutritional status and gene expression along the somatotropic axis in roach (Rutilus rutilus) infected with the tapeworm Ligula intestinalis.

The tapeworm Ligula intestinalis inhibits gametogenesis of its fish host, the roach (Rutilus rutilus). We investigated whether L. intestinalis infection makes significant demands on nutritional resources and consequently manipulates the endocrine somatotropic axis of roach. Two groups of naturally infected and uninfected roach were studied: a field group (natural feeding) and a laboratory group (ad libitum food supply). In females, no significant impact of parasitization on storage substrates (glycogen, lipids, and protein) was detected, whereas in males, either lipid content of the liver (field group) or lipid of the muscle and glycogen of the liver (laboratory group) were slightly decreased. Except for the females of the field group, higher mRNA expression of growth hormone (gh) in the pituitary of infected fish was observed. Furthermore, the expression of hypophyseal somatolactin α and ß (slα, slß) was up-regulated in infected females of the field and laboratory group, respectively. In liver and muscle, mRNA expression of insulin-like growth factors (igf1, igf2) and igf receptor (igfr) remained either unchanged or were up-regulated with infection. Parasitization showed inconsistent effects on gh receptor 1 (ghr1) expression in liver and muscle, whereas ghr2 mRNA was mostly not influenced by infection. In general, the expression profile of genes involved in the somatotropic axis as well as the content of storage substances in infected roach did not resemble that of food-deprived fish either under natural or ad libitum feeding. In conclusion, the present study does not indicate starvation of L. intestinalis infected roach, and it is suggested that the inhibition of reproduction attenuated the nutritional demand of parasitization.

Risks of seawater ozonation in recirculation aquaculture--effects of oxidative stress on animal welfare of juvenile turbot (Psetta maxima, L.).

Ozone is frequently used for water treatment and disinfection in recirculating aquaculture systems. However, due to the fragmentary data on chronic toxicity of ozone produced oxidants (OPO) and its safe concentrations, the daily application of ozone in aquaculture is challenging. To evaluate the chronic effects of sublethal OPO concentrations, juvenile turbot (Psetta maxima, L.) were exposed to OPO concentrations of 0.06, 0.10 and 0.15 mg/l for 21 days. Gills were analysed for histopathological alterations and mRNA expression of heat shock protein 70 (hsp70), hsp90 as well as glutathione-S-transferase (gst) were determined in the gills and the liver after 1d, 7d and 21 d. Histopathologic findings confirmed adverse effects at 0.10-0.15 mg/l, but these (necrosis, lamellar clubbing, hypertrophy, hyperplasia) could only be observed after an extended exposure (mostly 21 d), and were considered as irreversible tissue damage. Hsp70 expression in the gills was only significantly increased at the highest OPO concentration (0.15 mg/l) on 1d and 7d, and returned to basic levels until day 21. Hsp90 mRNA was already increased at 0.10mg/l after 1 and 7 days of exposure, and again was comparable to the control group on day 21. In contrast, elevated gst mRNA expression was only observed on day 7 at 0.10mg and 0.15 mg/l. Although similar trends were observed in the liver for all markers, differences were only significant in exceptional cases due to the high individual variation observed. Thus, mRNA expression in the gills rather than in the liver is recommended as a marker to characterize OPO-induced oxidative stress in turbot. It has to be noted that mRNA expression returned to basic levels on day 21 regardless the actual OPO concentration, suggesting a collapse of adaptive mechanisms as a possible explanation for the observed tissue damage.

In vivo treatment with progestogens causes immunosuppression of carp Cyprinus carpio leucocytes by affecting nitric oxide production and arginase activity.

In this study, carp Cyprinus carpio were injected with various steroid compounds, including synthetic and natural progestogens and the glucocorticoid cortisol, to investigate effects on leucocytes isolated from their kidneys. Injection of cortisol led to an increased spleeno-somatic index (I(S)) on day 21 post-injection (pi) and immunosuppressive effects measured as decreased nitric oxide (NO) production and increased arginase activity in isolated leucocytes on days 14 and 21 pi, respectively. Moreover, reduced NO production was also observed after injection of the synthetic progestogens, levonorgestrel (LEV) and medroxyprogesterone acetate. In addition, LEV influenced arginase activity in head kidney cells on day 14 and day 21 pi. This study is the first demonstration in fishes that the application of these steroid compounds in vivo affects NO production and arginase activity of isolated leucocytes.

Influence of temperature on puberty and maturation of pikeperch, Sander lucioperca.

Among external factors, temperature is known to exhibit a prominent role in reproduction of temperate fish species. Here, temperature related induction of puberty in pikeperch Sander lucioperca was investigated. For the first time the key factors of the pikeperch brain-pituitary-gonad axis, targeting the mRNA expression of the luteinising hormone (LH) and the follicle stimulating hormone (FSH), as well as the plasma sex steroids estradiol (E2), testosterone (T), 11-ketotestosteron (11-KT) and 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) were addressed in the experiment. Concomitant the maturational stages were described histologically. After 3 months, female pikeperch kept at 12°C revealed significant increases in the GSI and plasma E2 concentration and 90% of the females were mid-vitellogenic. After 5 months, females kept between 9 and 15°C exhibited significant up-regulation of E2 and GSI as well as comparable histological outcome. At 6 and 23°C in nearly all females stagnation of oogenesis was recorded. Congruently, T was increased at 12 and 15°C. Expression analysis revealed a significant up-regulation of LHß and FSHß mRNA in females from early-vitellogenesis, and from mid-spermatogenesis in males, correlated to elevated plasma concentrations of steroids (except for E2 in males). In conclusion, moderate temperatures (12-15°C for) for at least 3 months were required to proceed with first maturation in juvenile pikeperch. The most efficient effect was observed at 12°C, while high (23°C) or low (6°C) temperatures prevented gonadal maturation. So temperature was identified as a prime factor in the induction of puberty in pikeperch, as revealed by histological as well as endocrine parameters.

Developmental regulation of gene expression in the thyroid gland of Xenopus laevis tadpoles.

Thyroid hormones (TH) are the primary morphogen regulating amphibian metamorphosis. However, knowledge about molecular mechanisms regulating thyroid gland activity in anuran tadpoles is very scarce. In this study, we characterized gene expression profiles in thyroids of Xenopus laevis tadpoles during spontaneous metamorphosis. Using real-time PCR, elevated expression of slc5a5, tpo, tshr, and sar1a mRNAs was detected at late prometamorphic and climax stages. For dio2 and dio3 but not dio1, developmental regulation of thyroidal expression was evident from a strong up-regulation at late stages. Conversely, expression of the DNA replication markers mcm2 and pcna declined at climax stages. The presence of functional feedback mechanisms at premetamorphic stages was examined in two experiments. Stage 52 tadpoles were exposed for 72 h to 1.0 microg/l thyroxine (T4). This treatment caused reduced mRNA expression of slc5a5, tpo, and dio2, whereas no significant changes were detectable for tshr expression in thyroids and tshb expression in the pituitary. In another experiment, stage 46 tadpoles were treated with 20 mg/l sodium perchlorate (PER) for 5 and 10 days. Within this period of time, control tadpoles developed to stages 50 and 52, respectively. PER treatment resulted in up-regulation of slc5a5, tpo, and tshr mRNAs at both time points and increased dio2 mRNA expression at day 10. Effects of PER on thyroid histology were only apparent on day 10. Together, our analyses of thyroidal gene expression demonstrate a marked developmental regulation for functional markers of thyroid activity, two deiodinases as well as for DNA replication markers. Expression patterns detected in PER- and T4-treated tadpoles indicate that functional feedback signaling controlling thyroid activity is already active during premetamorphosis.

Aromatase mRNA expression in the brain of adult Xenopus laevis exposed to Lambro river water and endocrine disrupting compounds.

Aromatase P450 (P450 arom; Cyp19) is a key enzyme for vertebrate reproduction and brain development that catalyzes the conversion of androgens to estrogens. The aim of this study was to improve the knowledge on EDC effects by analysing their potential impact on brain P450 arom in adult Xenopus laevis exposed for 4 weeks to an environmental sample, the water of the river Lambro (LAM), the most polluted tributary of the Po river in North Italy. Other groups were exposed to individual compounds 10(-8) M tamoxifen (TAM), ethinylestradiol (EE2), flutamide (FLU) and methyldihydrotestosterone (MDHT) known for their (anti)estrogenic and (anti)androgenic modes of action. Expression of CYP19 was evaluated in brain extracts by quantitative RT-PCR, using a pair of primers located in the open reading frame (ORF) that allowed the simultaneous amplification of all transcripts (Aro-ORF) and a pair of primers specific for brain aromatase (Aro-B). Significant increase in Aro-ORF and Aro-B mRNA levels were observed in both females and males exposed to LAM. Different changes were observed for the model compounds using two pairs of primers. Aro-ORF mRNA expression was significantly increased in EE2 and MDHT exposed males and in FLU-exposed females, while it was significantly decreased in TAM exposed females. Aro-B mRNA was significantly increased in both sexes exposed to FLU and decreased in TAM exposed females. In conclusion, aromatase mRNA in the brain of X. laevis was regulated differentially in a gender specific manner by certain (anti)estrogenic and (anti)androgenic EDCs, supporting previous hypotheses that diverse compounds present in the river Lambro may induce feminization and demasculinization effects.

Expression profiles of LHbeta, FSHbeta and their gonadal receptor mRNAs during sexual differentiation of Xenopus laevis tadpoles.

The gonadotropins, luteinising hormone (LH) and follicle stimulating hormone (FSH), are important hormones regulating reproductive biology in vertebrates, especially the processes of steroidogenesis and gamete maturation. Despite the role of gonadotropins during the reproductive cycle in amphibians is well established, much less is known about the functional maturation of the hypothalamus-pituitary-gonad axis during larval development. Therefore, the present study aimed to analyze the expression profiles of hypophyseal LHbeta and FSHbeta mRNA and of their corresponding gonadal receptors (LH-R, FSH-R) in Xenopus laevis tadpoles during their ontogeny and sexual differentiation. The first significant elevation of LHbeta and FSHbeta mRNA was observed at late premetamorphosis. A clear raise of LHbeta mRNA was present during prometamorphic stages especially in males, while the LH-R only slowly increased during ontogeny with highest levels during metamorphic climax. In contrast, FSHbeta mRNA expression only slightly increased during ontogeny, however in both sexes the FSH-R mRNA was considerably elevated at prometamorphosis and further at metamorphic climax. Our results suggest that LHbeta and LH-R mRNA expression might be involved in initial maturation events of gametes, at least in males, while the gradually increase of FSH-R mRNA coincided with the advancing process of gamete maturation in both sexes. The present study provides for the first time evidence based on expression of gonadotropins and their corresponding gonadal receptors that the hypothalamus-pituitary-gonad axis evolves already at early stages of ontogeny and sexual differentiation in amphibians.

Exposure of Xenopus laevis tadpoles to finasteride, an inhibitor of 5-alpha reductase activity, impairs spermatogenesis and alters hypophyseal feedback mechanisms.

Sexual steroids have major regulatory functions in gonadal development, maturation of gametes and sexual differentiation in vertebrates. Previous studies in amphibians provided evidence that dihydrotestosterone and activity of 5-alpha reductases might play a significant role in androgen-mediated reproductive biology. To test the involvement of 5-alpha reductases in maturation of gametes in amphibians, Xenopus laevis was exposed to finasteride (FIN), a known inhibitor of 5-alpha reductase enzyme activity. In a long-term exposure from stage 46 to 66, severe disruption of spermatogenesis was observed in histological analysis of testes as detected by occurrence of empty spermatocysts, while ovaries remained unaffected. Real-time PCR analyses of male and female brain revealed an increase of LHbeta mRNA and a decrease of FSHbeta mRNA in males, suggesting a signalling on testes that could result in increased steroidogenesis and reduced Sertoli cell proliferation. Accordingly, the mRNA expression of P450 side chain cleavage enzyme and 5-alpha reductase type 2 was increased in testes, while no effects could be observed on steroidogenic genes in ovaries. A short-term exposure to testosterone, FIN and testosterone+FIN showed that transient effects of FIN targeted males selectively and, in particular, interfered with the hypothalamus-pituitary-gonad axis. Furthermore, a negative feedback of testosterone on LHbeta was observed on males and females. This study provides evidence that exposure of X. laevis to FIN, an inhibitor of 5-alpha reductases, impaired spermatogenesis and involved sex-specific hypophyseal feedback mechanisms.

Perchlorate and ethylenethiourea induce different histological and molecular alterations in a non-mammalian vertebrate model of thyroid goitrogenesis.

Despite evidence for a conserved role of thyroid-stimulating hormone (TSH) in regulating vertebrate thyroid function, molecular data on thyroid responses to TSH are mainly limited to mammalian species. In this study, we examined histological and molecular changes in the thyroid of Xenopus laevis tadpoles during a 12-day treatment with 20mg/l perchlorate (PER) and 50mg/l ethylenethiourea (ETU). Inhibition of thyroid hormone (TH) synthesis by PER and ETU was evident from developmental retardation, reduced expression of TH-regulated genes and up-regulation of tshb-A mRNA. Thyroid histopathology revealed goiters with strikingly different follicular morphologies following PER and ETU treatment. Using real-time PCR, we analyzed thyroids sampled on day 12 for differential expression of 60 candidate genes. Further temporal analyses were performed for a subset of 14 genes. Relative to the control, PER and ETU treatment modulated the expression of 51 and 49 transcripts, respectively. Particularly genes related to TH synthesis and protein metabolism were similarly affected by PER and ETU. However, several genes were differentially expressed in PER- and ETU-treated tadpoles. Specifically, goiter formation in the PER treatment was associated with low expression of genes related to DNA replication but high expression of negative growth regulators. Results from this work provide for the first time a characterization of gene expression profiles during goitrogenesis in a non-mammalian vertebrate model. Overall, our data suggest that, in addition to TSH over-stimulation, further mechanisms related to the mode of goitrogen action contribute to the regulation of thyroid gene expression.

Effect of housefly maggot meal (magmeal) diets on the performance, concentration of plasma glucose, cortisol and blood characteristics of Oreochromis niloticus fingerlings.

A 56-day feeding trial was conducted to access the effect of housefly maggot meal (magmeal) diets on the performance, concentration of plasma glucose, cortisol and blood characteristics of Oreochromis niloticus fingerlings. Seven feeds formulated to contain 36% protein and 20 kJ g(-1) gross energy (dry matter basis), were prepared by replacing fish meal with magmeal. Fifteen fingerlings (initial average weight 2.0 +/- 0.1 g) stocked per experimental tank were fed in triplicates at 5% body weight in two portions per day (a level previously established). Growth and food conversion ratio were adequate and comparable without any significant differences (p < 0.5) between feeding groups. Mean values for haematocrit and plasma glucose were not significantly different (p < 0.05) among the feeding groups. Fish group fed control diet (containing highest inclusion level of fish meal and without magmeal) gave the lowest haemoglobin concentration (5.96 +/- 0.22 g dl(-1)). This value was significantly different from other feeding groups. Stressful conditions in fish and in mammals are associated with decreased growth, haematocrit (packed cell volume) and haemoglobin values, increased whole blood glucose (hyperglycaemia) and plasma cortisol concentrations. No such physiological changes were observed in this study. Results suggest that feeding O. niloticus fingerling with magmeal diets did not cause any form of physiological stress. Magmeal can be used as a good alternative protein source in tilapia diets.

Aromatase, steroid-5-alpha-reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny.

The key enzymes involved in the production of endogenous sex steroids are steroid-5-alpha-reductase and aromatase converting testosterone (T) into dihydrotestosterone (DHT) and into estradiol (E2), respectively. To gain more insights into the molecular mechanisms of sexual differentiation of amphibians, we determined the mRNA expression of steroid-5-alpha-reductase type1 (Srd5a1), type2 (Srd5a2) and aromatase (Aro) during ontogeny starting from the egg and ending after completion of metamorphosis in Xenopus laevis. Expression of all three enzymes was measured by means of semi-quantitative RT-PCR, determining for the first time Srd5a1 and Srd5a2 mRNA expression in amphibians. mRNA was analyzed in whole body homogenates from stage 12 to 48, while brain and gonads with kidney were studied separately from stage 48 to 66. Different ontogenetic mRNA expression patterns were observed for all genes analyzed, revealing early mRNA expression of Srd5a1 already in the egg at stage 12 whereas Srd5a2 and Aro was detected at stage 39. Sex-specific mRNA expressions of Srd5a2 and of Aro were determined in the gonads with kidney but not in brain. Srd5a2 was two-fold higher expressed in testes than in ovaries while Aro mRNA was ten-fold higher in ovaries. No gender-specific mRNA expression was observed for Srd5a1 in gonads and in brain. The ontogenetic patterns of Aro, Srd5a1 and Srd5a2 suggest that these genes are involved in sexual differentiation of gonads and brain already in early developmental stages. Especially in gonads Srd5a2 seems to be important for physiological regulation of testis development while Aro is associated with the development of ovaries.

Expression of IGF-I and IGF-I receptor in male and female sterlet, Acipenser ruthenus--evidence for an important role in gonad maturation.

Recent in vitro studies suggest that insulin-like growth factor I (IGF-I) is involved in cell differentiation and steroidogenesis in the gonad and could therefore function as an important trigger in vivo. In this study, sensitive real-time RT-PCR assays were used to determine IGF-I and the IGF-I receptor (IGF-IR) mRNA expression in maturing male and female sterlet (Acipenser ruthenus) over a period of two years: In the first year, females entering vitellogenesis (maturing female group, MFG) revealed an increase of IGF-I expression in the ovaries in contrast to females that did not enter vitellogenesis (non-maturing female group, NMFG). Congruently, IGF-IR expression was elevated in females at the onset of vitellogenesis (MFG), decreased towards the first winter, and increased to similar levels at late vitellogenesis in the second winter just prior to spawning. In the second year, NMFG reached the onset of vitellogenesis. Here, IGF-I and IGF-IR reached similar levels as previously observed in the first year in MFG. In males, low and constant IGF-I expression was observed in the testis, whereas IGF-IR was expressed at a constant high level comparable to those of females entering vitellogenesis. These findings suggest an involvement of IGF-I as an important paracrine regulator of gonad maturation, particularly in the ovary.

The role of the IGF-I system for vitellogenesis in maturing female sterlet, Acipenser ruthenus Linnaeus, 1758.

Transition from previtellogeneic to vitellogenic oocyte growth is a critical phase for folliculogenesis in sturgeon and may often be postponed for several years. Recent findings on the involvement of insulin-like growth factor I (IGF-I) in cell differentiation processes of oocyte follicle and ovarian steroidogenesis of teleosts in vitro led to the hypothesis that paracrine IGF-I could function as a potential trigger in vivo. For the first time, IGF-I and its corresponding receptor (IGF-IR) were identified in a non-teleostean fish. Real-time PCR assays for IGF-I and IGF-IR mRNA were established, normalising mRNA expression of the target genes to beta-microglobulin (beta2m). We clearly show that expression of IGF-I in the gonad is a substantial source for IGF-I-mediated effects in follicles compared to liver, brain, muscle and adipose tissue. Among these tissues, IGF-IR mRNA was highest in the gonad. With regard to different cohorts of coexisting follicles, highest expression of IGF-I and IGF-IR were met in developing follicles, indicating that IGF-I functions as an intraovarian modulator of follicle faith. Comparing previtellogenic follicles in females that matured within two years with non-maturing females f the same age, revealed an increases of 2.3-fold for IGF-I and 2.8-fold for IGF-IR mRNA expression in maturing females. These findings implicate an important role of paracrine IGF-I in early vitellogenesis and identify it as candidate vitellogenesis inducing factor (VIF), determining the faith of the follicle.

Effects of infection with Anguillicola crassus and simultaneous exposure with Cd and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) on the levels of cortisol and glucose in European eel ( Anguilla anguilla ).

To investigate whether the stress response of European eels infected with Anguillicola crassus is influenced by environmental pollutants, experimentally infected eels were exposed to Cd and/or to 3,3', 4,4', 5-pentachlorobiphenyl (PCB 126). Serum cortisol and glucose concentrations of these eels were monitored over a period of 103 days and were compared with data from infected, unexposed eels as well as with data from uninfected eels. Additionally, the levels of cortisol were correlated with concentrations of Anguillicola-specific antibodies. All eels showed an initial increase of the cortisol levels until day 63. This general elevation of plasma cortisol is most likely due to handling stress, as all eels were repeatedly netted and afterwards inoculated with a feeding tube. At the end of the exposure period eels which were infected and those which were infected and simultaneously exposed to Cd and PCB showed significantly higher levels than the controls. The general course of serum glucose levels in eels resembled that of cortisol. Accordingly, Spearman correlation analysis revealed that an increase in serum cortisol concentrations is correlated with rising levels of glucose. With respect to immune-endocrine interactions a significant negative correlation between cortisol and anti-A. crassus antibodies was found. Our data show that A. crassus is the most potent stressor for European eels among the treatments tested within this study. This is important in terms of ecotoxicological studies as the main effects are caused by parasites rather than chemicals. Accordingly, effects of parasites on the physiological homeostasis of organisms must be considered in ecotoxicology. From the parasitological point of view our results suggest that probably as part of an unbalanced host-parasite interaction A. crassus evokes a strong cortisol response in A. anguilla, thereby suppressing the immune response which in turn enables the parasite to establish. The parasite-induced stress response in the newly adopted European eel might be one of the factors which contributes to the extremely effective colonizing strategy of A. crassus.

Luteinizing hormone, follicle stimulating hormone, and gonadotropin releasing hormone mRNA expression of Xenopus laevis in response to endocrine disrupting compounds affecting reproductive biology.

Environmental pollutants can interfere with the endocrine system of a variety of animals and are suggested to contribute to the worldwide decline of amphibians. In this study, the effects of endocrine disrupting compounds (EDC) on the hypothalamus-pituitary-gonad axis, regulating reproduction, were investigated in Xenopus laevis by determining their potential impact on gene expression of gonadotropin releasing hormone (GnRH), luteinizing hormone beta-subunit (LHbeta) and follicle-stimulating hormone beta-subunit (FSHbeta) in brain and pituitary using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). One environmental sample and four model compounds, ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT), and flutamide (FLU), corresponding to (anti)estrogenic and (anti)androgenic modes of action were used at 10(-8)M during a four weeks exposure of adults of both sexes. In general, males had a higher LHbeta mRNA level compared to females, while the mRNA expression of FSHbeta and GnRH did not differ between both sexes. EE2 and MDHT treatment decreased LHbeta mRNA expression in the brain of male X. laevis, while only EE2 but not MDHT reduced LHbeta mRNA in females indicating classical negative feed-back mechanisms on hypophyseal gonadotropin expression. TAM increased LHbeta mRNA and FSHbeta mRNA expression in female X. laevis while none of the other treatments showed an effect on FSHbeta mRNA expression. GnRH expression was not changed by any treatment and exposure of X. laevis to Lambro river water had no significant effect on any of the genes examined. It is reported for the first time in amphibians that gonadotropin mRNA expression is differentially regulated by (anti)estrogenic and (anti)androgenic EDC and that gender-specific patterns of gene expression exist.

Environmental agent susceptibility assessment using existing and novel biomarkers as rapid noninvasive testing methods.

This study is part of a project aimed at developing and validating novel noninvasive methods for the detection of biomarkers of endocrine disrupters (EDs) directly in the mucus of aquatic species, to identify novel functional biomarker(s) for EDs, and to verify their applicability for field studies. The multidisciplinary approach chosen aims at the development of an integrated testing strategy utilizing in vitro protocols to identify water and sediment fractions with potential endocrine-disrupting activity; the identification, characterization, and measurement of new biomarker(s) for EDs; the development and validation of a dipstick-based test method; and the development of (computer-assisted) predictive models. Some results of the first year of the project are presented here.