Sven Wuertz

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,20β-dihydroxy-4-pregnen-3-one (17,20β-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 LHβ and FSHβ 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.

Naturally-induced endocrine disruption by the parasite Ligula intestinalis (Cestoda) in roach (Rutilus rutilus).

Fish represent the most frequently used vertebrate class for the investigation of endocrine disruption (ED) in wildlife. However, field studies are complicated by exposure scenarios involving a variety of anthropogenic and natural influences interfering with the endocrine system. One natural aspect rarely considered in ecotoxicological studies is how parasites modulate host physiology. Therefore, investigations were carried out to characterise the impacts of the parasitic tapeworm Ligula intestinalis on plasma sex steroid levels and expression of key genes associated with the reproduction in roach (Rutilus rutilus), a sentinel species for wildlife ED research. Parasitisation by L. intestinalis suppressed gonadal development in both genders of roach and analysis of plasma sex steroids revealed substantially lower levels of 17beta-oestradiol (E2) and 11-ketotestosterone (11-KT) in infected females as well as E2, 11-KT, and testosterone in infected males. Consistently, in both, infected females and males, expression of the oestrogen dependent genes such as vitellogenin and brain-type aromatase in liver and brain was reduced. Furthermore, parasitisation differentially modulated mRNA expression of the oestrogen and androgen receptors in brain and liver. Most prominently, liver expression of oestrogen receptor 1 was reduced in infected females but not in males, whereas expression of oestrogen receptor 2a was up-regulated in both genders. Further, insulin-like growth factor 1 mRNA in the liver was increased in infected females but not in males. Despite severe impacts on plasma sex steroids and pituitary gonadotropin expression, brain mRNA levels of gonadotropin-releasing hormone (GnRH) precursors encoding GnRH2 and GnRH3 were not affected by L. intestinalis-infection. In summary, the present results provide basic knowledge of the endocrine system in L. intestinalis-infected roach and clearly demonstrate that parasites can cause ED in fish.

Expression of gonadotropin subunits in roach (Rutilus rutilus, Cyprinidae) infected with plerocercoids of the tapeworm Ligula intestinalis (Cestoda).

Plerocercoids of the tapeworm Ligula intestinalis (Cestoda: Bothriocephalidea) have been reported to inhibit gametogenesis of their intermediate fish hosts. However, mechanistic studies are rare and the proximate cues leading to impaired reproduction still remain unknown. In the present study we investigated the effects of infection by L. intestinalis on reproductive parameters of roach (Rutilus rutilus, Cyprinidae), a common fish host of this parasite. Field studies on roach demonstrated that in both genders infection prevented gonad development. As revealed by quantitative PCR, infection was accompanied by essentially lower pituitary expression of follicle-stimulating hormone beta-subunit (FSHbeta) and luteinizing hormone beta-subunit (LHbeta) mRNA compared with uninfected roach, providing clear evidence for gonadotropin-insufficiency as the cause of arrested gametogenesis. Under controlled laboratory conditions infected roach showed lower mRNA levels of FSHbeta but not of LHbeta, despite histology revealing similar gonad stages as in uninfected conspecifics. These findings indicate the involvement of FSH rather than LH in mediating effects of infection early during gonad development in roach. Moreover, the impact of L. intestinalis on reproductive parameters of roach appeared to be independent of the parasite burden. Together, these data provide valuable information on the role of FSH and LH as mediators of parasite-induced sterilization in a vertebrate and implicate the selective inhibition of host reproduction by L. intestinalis as a natural source of endocrine disruption in fish.

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.

Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay

Increasing lead contamination in Egyptian ecosystems and high lead concentrations in food items have raised concern for human health and stimulated studies on monitoring ecotoxicological impact of lead‐caused genotoxicity. In this work, the alkaline comet assay was modified for monitoring DNA strand breakage in sensitive early life stages of the African catfish Clarias gariepinus. Following exposure to 100, 300, and 500 μg/L lead nitrate, DNA strand breakage was quantified in embryos at 30, 48, 96, 144, and 168 h post‐fertilization (PFS). For quantitative analysis, four commonly used parameters (tail % DNA, %TDNA; head % DNA, %HDNA; tail length, TL; tail moment, TM) were analyzed in 96 nuclei (in triplicates) at each sampling point. The parameter %TDNA revealed highest resolution and lowest variation. A strong correlation between lead concentration, time of exposure, and DNA strand breakage was observed. Here, genotoxicity detected by comet assay preceded the manifested malformations assessed with conventional histology. Qualitative evaluation was carried out using five categories are as follows: undamaged (%TDNA ≤ 10%), low damaged (10% < %TDNA ≤ 25%), median damaged (25 < %TDNA ≤ 50%), highly damaged (50 < %TDNA ≤ 75%), and extremely damaged (%TDNA > 75%) nuclei confirming a dose and time‐dependent shift towards increased frequencies of highly and extremely damaged nuclei. A protective capacity provided by a hardened chorion is a an interesting finding in this study as DNA damage in the prehatching stages 30 h‐PFS and 48 h‐PFS was low in all treatments (qualitative and quantitative analyses). These results clearly show that the comet assay is a sensitive tool for the detection of genotoxicity in vulnerable early life stages of the African catfish and is a method more sensitive than histological parameters for monitoring genotoxic effects.

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.

Stage-dependent differences in RNA composition and content affect the outcome of expression profiling in roach (Rutilus rutilus) ovary.

The influence of changing composition and content of RNA on the results of expression profiling was studied in the group-synchronous ovaries of roach (Rutilus rutilus) over the course of their maturation. The highest yield of total RNA was detected in the primary growth and early cortical alveolus stages. The total RNA yield gradually decreased through the late cortical alveolus and late vitellogenic stages. In the primary growth and early cortical alveolus stages, total RNA was characterized by a low percentage of 18S and 28S rRNA and a high percentage of smaller-sized RNAs (tRNA, 5S and 5.8S rRNA), whereas 18S and 28S rRNA had increased by the late cortical alveolus stage and dominated by the late vitellogenic stage. The ratio of mRNA to total RNA was highest at the primary growth stage but decreased significantly in later ovarian stages. When total RNA was used for reverse transcription (RT), the shift in the mRNA/total RNA ratio influenced the results of qPCR expression profiling of several commonly used reference genes (ribosomal protein L8, elongation factor-1α, RNA polymerase-subunit B5, and β2-microglobulin) and of two target genes, gonad-type aromatase (cyp19a1a) and follistatin (fst). We conclude that the expression of target genes should be related to the mRNA pool using the same input of either mRNA to RT or cDNA to qPCR. Furthermore, gene expression was related to tissue-specific RNA yield per body mass (RNA yield x ovary mass x body mass⁻¹) thereby reflecting the massive increase in the size and cellular composition of the ovary during the reproductive cycle.

The European seabass (Dicentrarchus labrax) innate immunity and gut health are modulated by dietary plant-protein inclusion and prebiotic supplementation

ABSTRACT Inclusion of prebiotics in aqua feeds, though a costly strategy, has increased as a means to improve growth. Still, its effects on health improvement are not fully disclosed. Regarding their immunestimulatory properties, research has focused on carbohydrates such as fructooligosaccharides and xylooligosaccharides demonstrating their modulatory effects on immune defences in higher vertebrates but few studies have been done on their impact on fish immunity. Replacing fish meal (FM) by plant protein (PP) sources is a current practice in the aquaculture business but their content in antinutrients is still a drawback in terms of gut well‐functioning. This work intends to evaluate the short‐term effect (7 or 15 days feeding the experimental diets) on juvenile European seabass (Dicentrarchus labrax) immune status of dietary i) replacement of FM by PP sources; ii) prebiotics supplementation. Six isoproteic (46%) and isolipidic (15%) diets were tested including a FM control diet (FMCTRL), a PP control diet (PPCTRL, 30 FM:70 PP) and four other diets based on either FM or PP to which short‐chain fructooligosaccharides (scFOS) or xylooligosaccharides (XOS) were added at 1% (FMFOS, PPFOS, FMXOS, PPXOS). The replacement of FM by PP in the diets induced nitric oxide (NO) and lysozyme production, while immunoglobulins (Ig), monocytes percentage and gut interleukin 10 (IL10) gene expression were inhibited. Dietary scFOS supplementation inhibited total bactericidal activity and neutrophils relative percentage regardless protein source and increased plasma NO and thrombocytes percentage in fish fed FM‐based diets, while monocytes percentage was increased in PPFOS‐fed fish. XOS supplementation down‐regulated immune gene expression in the gut while it partly enhanced systemic response. Inconsistency among results regarding FM replacement by PP‐based ingredients exposes the need for further research considering both local and systemic responses. Distinct outcomes of prebiotic supplementation were highlighted reflecting sight‐specific effects with no clear interaction with protein source. HIGHLIGHTSThe immunomodulatory effects of prebiotics and plant protein sources were tested.Vegetable protein sources both activate and suppress different immune parameters.Xylooligosaccharides inhibited gut immune defences, regardless of protein source.Fructooligosaccharides effects suggest a slight stimulation of cell dynamics.

Up-regulation of gonadotropin mRNA-expression at the onset of gametogenesis in the roach (Rutilus rutilus): Evidence for an important role of brain-type aromatase (cyp19a1b) in the pituitary

The present study characterized changes in key parameters of reproduction in adult roach (Rutilus rutilus) from Lake Grosser Mueggelsee (Berlin, Germany) during natural gametogenesis. Fish of both sexes were sampled in monthly intervals between April and August in order to cover the onset of gametogenesis. Investigated parameters included gonad histology, plasma levels of 17β-oestradiol (E2), testosterone (T), 11-ketotestosterone (11-KT), and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) as well as the expression of gonadotropin subunits in the pituitary. Furthermore, the mRNA-expression of brain-type aromatase (cyp19a1b), androgen receptor (ar), and estrogen receptor isoforms was studied at the pituitary level. The onset of gametogenesis - as indicated by follicles with cortical alveoli in females and first spermatogonia B in males - was observed in July, accompanied by a significant up-regulation of follicle-stimulating hormone β (fshβ) mRNA in the pituitary in both sexes. On the other hand, luteinizing hormone β (lhβ) mRNA increased later on in August. In males, the increase of fshβ mRNA in July coincided with a rise in plasma 11-KT concentrations. In females, E2 in plasma increased later, not until August, shortly before true vitellogenesis (late cortical alveoli stage). Expression of sex steroid receptors in the pituitary revealed only minor seasonal fluctuations. Most pronounced, ar mRNA displayed the highest level pre-spawning in both sexes. Interestingly, cyp19a1b mRNA-expression in the pituitary increased in parallel with fshβ already before any changes in plasma E2 or T occurred. These data suggest an important role of pituitary FSH and aromatase at the onset of gametogenesis in the roach.

Impact of microcystin containing diets on physiological performance of Nile tilapia (Oreochromis niloticus) concerning stress and growth

Diets containing Microcystis with considerable amounts of the cyanotoxin microcystin-LR (MC-LR) were fed to determine their impact on the physiological performance of the omnivorous Nile tilapia (Oreochromis niloticus) with regard to stress and growth performance. Four different diets were prepared based on a commercial diet (control, MC-5% [containing 5% dried Microcystis biomass], MC-20% [containing 20% dried Microcystis biomass], and Arthrospira-20% [containing 20% dried Arthrospira sp. biomass without toxin]) and fed to female Nile tilapia. Blood and tissue samples were taken after 1, 7, and 28 d, and MC-LR was quantified in gills, muscle, and liver by using high-performance liquid chromatography (HPLC). Only in the liver were moderate concentrations of MC-LR detected. The stress hormone cortisol and glucose were analyzed from plasma, suggesting that all modified diets caused only minor to moderate stress, which was confirmed by analyses of hepatic glycogen. In addition, the effects of the different diets on growth performance were investigated by determining gene expression of hypophyseal growth hormone (GH) and hepatic insulin-like growth factor-I (IGF-I). For all diets, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) demonstrated no significant effect on gene expression of the major endocrine hormones of the growth axis, whereas classical growth data, including growth and feed conversion ratio, displayed slight inhibitory effects of all modified diets independent of their MC-LR content. However, no significant change was found in condition or hepatosomatic index among the various diets, so it seems feasible that dried cyanobacterial biomass might be even used as a component in fish diet for Nile tilapia, which requires further research in more detail.