Carotenoids and retinoids in the gonad of brood-stock pikeperch: accumulation during vitellogenesis and influence on egg quality in farmed pikeperch Sander lucioperca.

Carotenoids are determinants of reproductive fitness and egg quality. Here we studied the accumulation of astaxanthin (AX), canthaxanthin (CA) zeaxanthin (ZX), lutein (LU), retinol (RX) and dehydroretinol (DR) during vitellogenesis comparing previtellogenic and vitellogenic pikeperch (Sander lucioperca) eggs (n = 5 each), as well as selected tissues (liver, fat and muscles) in first süawning females (1176-1450 g). Futhermore, we compared egg batches with high (88-99% hatching rate, n = 5) or low (40-67% hatching rate, n= 5) egg quality. Vitellogenic follicles revealed higher concentrations of DR, RX, ZX and LU compared to previtellogenic follicles. Neither CA nor AX was detectable. In parallel, DR and RX were mobilized in the liver. In adipose and muscle tissue, comparing previtellogenic and vitellogenic females, no significant differences in carotenoid/retinoid content were observed. In high quality egg batches, both DR and RX were increased. LU was lower in high quality than in low quality eggs. In a conclusion, the amount of retinoids seems suboptimal in low quality egg batches and increased DR and RX are desirable in pikeperch. Since hypervitaminosis of retinoids can be problematic though, supplementation of the food with carotenoids, which can serve as precursors for retinoids, has to be carried out carefully.

Misbalance of thyroid hormones after two weeks of exposure to artificial light at night in Eurasian perch Perca fluviatilis.

Artificial light at night (ALAN) can affect the physiology and behavior of animals because it alters the natural rhythm of light and darkness. Thyroid hormones (TH) are partially regulated by the light information of photoperiod and are involved in metabolic adjustments to daily and seasonal changes in the environment, such as larval and juvenile development, somatic growth and reproduction. ALAN can change photoperiodic information and might thereby lead to changes in thyroid metabolism, but so far research on this topic is scarce. Therefore, we tested in two different experiments the effects of nocturnal illumination at a wide range of light intensities on TH in plasma of Eurasian perch (Perca fluviatilis). Total 3,3',5-triiodo-L-thyronine (T3) was significantly affected by ALAN and reduced at the highest tested intensity of 100 lx after only two weeks of exposure. Although total L-thyroxine (T4) was not significantly affected, the ratio of T3 to T4 tended to slightly decrease at 100 lx. In a second low-light experiment ALAN did not have clear effects on T3, T4 or the ratio of T3 to T4 at intensities between 0.01 lx and 1 lx. The results show first signs of endocrine disruption in thyroid metabolism after a relatively short ALAN exposure of two weeks under high-intensity streetlight conditions. Misbalanced thyroidal status can have serious implications for metabolic rates as well as developmental and reproductive processes.

Impacts of the synthetic androgen Trenbolone on gonad differentiation and development - comparisons between three deeply diverged anuran families.

Using a recently developed approach for testing endocrine disruptive chemicals (EDCs) in amphibians, comprising synchronized tadpole exposure plus genetic and histological sexing of metamorphs in a flow-through-system, we tested the effects of 17ß-Trenbolone (Tb), a widely used growth promoter in cattle farming, in three deeply diverged anuran families: the amphibian model species Xenopus laevis (Pipidae) and the non-models Bufo(tes) viridis (Bufonidae) and Hyla arborea (Hylidae). Trenbolone was applied in three environmentally and/or physiologically relevant concentrations (0.027 µg/L (10-10 M), 0.27 µg/L (10-9 M), 2.7 µg/L (10-8 M)). In none of the species, Tb caused sex reversals or masculinization of gonads but had negative species-specific impacts on gonad morphology and differentiation after the completion of metamorphosis, independently of genetic sex. In H. arborea and B. viridis, mounting Tb-concentration correlated positively with anatomical abnormalities at 27 µg/L (10-9 M) and 2.7 µg/L (10-8 M), occurring in X. laevis only at the highest Tb concentration. Despite anatomical aberrations, histologically all gonadal tissues differentiated seemingly normally when examined at the histological level but at various rates. Tb-concentration caused various species-specific mortalities (low in Xenopus, uncertain in Bufo). Our data suggest that deep phylogenetic divergence modifies EDC-vulnerability, as previously demonstrated for Bisphenol A (BPA) and Ethinylestradiol (EE2).

The progestin norethisterone affects thyroid hormone-dependent metamorphosis of Xenopus laevis tadpoles at environmentally relevant concentrations.

Previously, levonorgestrel (LNG) has been shown to be an endocrine disruptor of the amphibian thyroid system. In the present study, we investigated whether anti-thyroidal effects are a common property of progestins other than LNG. Premetamorphic Xenopus laevis tadpoles were exposed to norethisterone (NET) and dienogest DIE (each at 0.1-10nM) and LNG (10nM) until completion of metamorphosis. LNG and NET at all concentrations caused a significant developmental retardation whereas DIE did not impair time to metamorphosis. In LNG and 10nM NET exposed animals, tsh mRNA levels increased considerably later than the developmental delay occurred and thyroid histopathology showed no signs of TSH-hyperstimulation. Instead, thyroid glands from these treatments appeared inactive in producing thyroid hormones. Thyroidal transcript levels of dio2 and dio3 were increased by treatments with LNG and NET at 1nM and 10nM, whereas iyd mRNA was reduced by LNG and 10nM NET. Expression of slc5α5 was not changed by any treatment. Effects of DIE differed from those induced by LNG and NET. No developmental delay was measurable; however, tshß and dio2 mRNAs were increased in pituitary glands of tadpoles exposed to 1.0nM and 10nM DIE. Thyroid histopathology displayed no abnormalities and thyroidal mRNA expression of the genes analyzed (slc5α5, iyd, dio2, dio3) was not changed by DIE. Overall, our results provide evidence that the anti-thyroidal effects already known from LNG are also present in another progestin, namely NET, even at environmentally relevant concentrations. In conclusion we suggest that progestins do not only pose an environmental risk in terms of their impact on reproductive success of aquatic vertebrates, but also with respect to their anti-thyroidal properties affecting amphibian metamorphosis.

Compensatory Cell Movements Confer Robustness to Mechanical Deformation during Embryonic Development.

Embryonic development must proceed despite both internal molecular fluctuations and external perturbations. However, mechanisms that provide robustness to mechanical perturbation remain largely uncharacterized. Here, we use light-sheet microscopy, comprehensive single-cell tracking, and targeted cell ablation to study the response of Caenorhabditis elegans embryos to external compression. Compression changes the relative positions of many cells and causes severe distortions of the embryonic axes. A large-scale movement of cells then corrects this distortion. Only a few specific cells are required for these compensatory movements, and one cell, ABarppap, appears to generate force, dramatically changing as it moves to its correct local cellular environment. During these movements, we also observed "egressions", cells moving out onto the surface, and lineages that undergo both ingression and egression. In total, our work describes how the embryo responds to a major mechanical deformation that can occur during the early development in situ and puts forward a model to explain how the response is coordinated.

The plasticizer bisphenol A affects somatic and sexual development, but differently in pipid, hylid and bufonid anurans.

Due to their terrestrial habitats and aquatic reproduction, many amphibians are both very vulnerable and highly suitable bioindicators. The plasticizer bisphenol A (BPA) is one of the most produced chemical substances worldwide, and knowledge on its impacts on humans and animals is mounting. BPA is used for the industrial production of polycarbonate plastics and epoxy resins and found in a multitude of consumer products. Studies on BPA have involved mammals, fish and the fully aquatic anuran model Xenopus laevis. However, our knowledge about the sexual development of non-model, often semi-terrestrial anuran amphibians remains poor. Using a recently developed experimental design, we simultaneously applied BPA to two non-model species (Hyla arborea, Hylidae; Bufo viridis, Bufonidae) and the model X. laevis (Pipidae), compared their genetic and phenotypic sex for detection of sex reversals, and studied sexual development, focusing on anatomical and histological features of gonads. We compared three concentrations of BPA (0.023, 2.28 and 228 µg/L) to control groups in a high-standard flow-through-system, and tested whether conclusions, drawn from the model species, can be extrapolated to non-model anurans. In contrast to previous studies on fish and Xenopus, often involving dosages much higher than most environmental pollution data, we show that BPA causes neither the development of mixed sex nor of sex-reversed individuals (few, seemingly BPA-independent sex reversals) in all focal species. However, environmentally relevant concentrations, as low as 0.023 µg/L, were sufficient to provoke species-specific anatomically and histologically detectable impairments of gonads, and affected morphological traits of metamorphs. As the intensity of these effects differed between the three species, our data imply that BPA diversely affects amphibians with different evolutionary history, sex determination systems and larval ecologies. These results highlight the role of amphibians as a sensitive group that is responsive to environmental pollution.

Screening of multiple hormonal activities in water and sediment from the river Nile, Egypt, using in vitro bioassay and gonadal histology.

In Egypt, until yet no records are available regarding possible multiple hormonal activities in the aquatic systems and especially in the river Nile. In this paper, in vitro yeast estrogen screen (YES) and yeast androgen screen (YAS) were used to assess (for the first time) the multiple hormonal activities in surface waters and sediments of the river Nile. This study aimed to determine whether river Nile water can cause changes in gonadal histology of Nile tilapia (Oreochromis niloticus niloticus). All water samples exhibited extremely low levels of estrogenicity. Estrogenicity was nearly not detected in any of the sediment samples. Unlike the estrogenicity, significant androgenic activities were recorded in the water and sediment samples along the course of the river Nile. The present study reports for the first time quantification anti-estrogenic and anti-androgenic activities with high levels in both water and sediment of the river Nile. The greatest anti-estrogenic and anti-androgenic activities were observed in samples from downstream river Nile. These results indicated that the anti-estrogenic and anti-androgenic activities along the Nile course were great and the pollution of the sites at downstream was more serious than the upstream sites due to industrial and anthropogenic activities at these sites. Good correlations were observed among some hormonal activities, suggesting coexistence of these contaminants in the environmental matrices. There were no signs of sexual disruption in any of the gonads analyzed from either male or female Nile tilapia, demonstrating that no hormonal activity present along the Nile course was sufficient to induce adverse effects on reproductive development. Further investigation is necessary to identify the compounds responsible for the hormonal activities in the river Nile and to examine effects of very low levels of hormonally active compounds on gonadal histology, as well as in the development of more sensitive biomarkers.

Comprehensive single cell-resolution analysis of the role of chromatin regulators in early C. elegans embryogenesis.

Chromatin regulators are widely expressed proteins with diverse roles in gene expression, nuclear organization, cell cycle regulation, pluripotency, physiology and development, and are frequently mutated in human diseases such as cancer. Their inhibition often results in pleiotropic effects that are difficult to study using conventional approaches. We have developed a semi-automated nuclear tracking algorithm to quantify the divisions, movements and positions of all nuclei during the early development of Caenorhabditis elegans and have used it to systematically study the effects of inhibiting chromatin regulators. The resulting high dimensional datasets revealed that inhibition of multiple regulators, including F55A3.3 (encoding FACT subunit SUPT16H), lin-53 (RBBP4/7), rba-1 (RBBP4/7), set-16 (MLL2/3), hda-1 (HDAC1/2), swsn-7 (ARID2), and let-526 (ARID1A/1B) affected cell cycle progression and caused chromosome segregation defects. In contrast, inhibition of cir-1 (CIR1) accelerated cell division timing in specific cells of the AB lineage. The inhibition of RNA polymerase II also accelerated these division timings, suggesting that normal gene expression is required to delay cell cycle progression in multiple lineages in the early embryo. Quantitative analyses of the dataset suggested the existence of at least two functionally distinct SWI/SNF chromatin remodeling complex activities in the early embryo, and identified a redundant requirement for the egl-27 and lin-40 MTA orthologs in the development of endoderm and mesoderm lineages. Moreover, our dataset also revealed a characteristic rearrangement of chromatin to the nuclear periphery upon the inhibition of multiple general regulators of gene expression. Our systematic, comprehensive and quantitative datasets illustrate the power of single cell-resolution quantitative tracking and high dimensional phenotyping to investigate gene function. Furthermore, the results provide an overview of the functions of essential chromatin regulators during the early development of an animal.

The synthetic gestagen Levonorgestrel disrupts sexual development in Xenopus laevis by affecting gene expression of pituitary gonadotropins and gonadal steroidogenic enzymes.

In the present study, Xenopus laevis tadpoles were chronically exposed to four concentrations of the synthetic gestagen Levonorgestrel (LNG; 10(-11), 10(-10), 10(-9), and 10(-8)M) starting at Nieuwkoop and Faber (NF) stage 48 until completion of metamorphosis. At NF 58 and 66, brain-pituitary and gonad samples were taken for gene expression analyses of gonadotropins and gonadal steroidogenic enzymes. Exposure to 10(-9) and 10(-8)M LNG until NF 58 repressed messenger RNA (mRNA) expression of luteinizing hormone (LH) ß in both genders. This decrease was persistent after further treatment until NF 66 in the 10(-8)M LNG treatment. Expression of follicle-stimulating hormone (FSH) ß was affected sex-specifically. No effect was present in NF 58 females, whereas LNG at 10(-9) and 10(-8)M significantly increased FSHß mRNA levels in males. In NF 66 females, 10(-9)M LNG treatment increased FSHß gene expression, whereas a decrease was observed in NF 66 males exposed to 10(-8)M LNG. In gonads, expression of steroid-5-alpha-reductase was affected sex-specifically with increased mRNA levels in females but repressed levels in males. Gene expression of further gonadal steroidogenic factors was decreased by 10(-8)M LNG in both genders at NF 66. Assessment of gonad gross morphology and histology revealed poorly developed testes in the 10(-8)M LNG treatment. Our results reveal considerable effects of chronic LNG exposure on sexual development of amphibians. The persistent inhibition of LHß expression concomitant with decreased mRNA levels of gonadal steroidogenic enzymes is suggested to result in the disruption of reproduction in adult amphibians.

Agricultural land-use affects the nutritional quality of stream microbial communities.

We investigated how the lipid composition (fatty acids and sterols) of benthic microbial mats, which represent an important basal food resource for stream food webs, differs between tropical streams located in protected pristine and agricultural Cerrado savannah areas. The total microbial biomass and lipid composition differed significantly between pristine and agricultural streams in parallel with differences in water quality and hydrodynamic characteristics. Agricultural streams exhibited lower total biomass of benthic microbial mats than pristine streams. However, the higher concentrations of essential polyunsaturated fatty acids, such as linoleic acid (LIN, 18:2ω6), α-linolenic acid (ALA, 18:3ω3), and eicosapentaenoic acid (EPA, 20:5ω3), that were observed in agricultural streams suggest enhanced lipid complexity and a higher nutritional quality of the microbial community relative to pristine streams. Meanwhile, pristine stream microbial communities had higher total concentrations of saturated fatty acids and cholesterol than those of agricultural streams, reflecting their heterotrophic microbial communities. Moreover, stream morphotype and associated differences in the hydrodynamic characteristics affected the community composition and thereby also the lipid composition of microbial mats. Land-use-induced changes in the total biomass and lipid composition of microbial communities may affect the trophic transfer of energy in stream food webs, leading to changes in the composition and productivity of primary consumers and their predators, and thereby affecting stream ecosystem functioning.

Enrichment and cultivation of pelagic bacteria from a humic lake using phenol and humic matter additions.

Individual bacterial populations are known to respond differently toward substrate availability. To test how the availability of either pure phenol or natural humic matter (HM) selects for specific pelagic bacteria phylotypes from a humic lake (Lake Grosse Fuchskuhle, northeastern Germany), we used culture-dependent and -independent approaches. Using a batch approach, the bacterial community composition (BCC) differed depending on both the quantity and the quality of added substrates. Using a dilution-to-extinction approach, distinct BCC were detected by eliminating less abundant species. Most bacteria that were common in the lake were favored by phenol, and yet different subsets of the native BCC were enriched by HM. Specific bacterial groups with different growth requirements were consistently present, negatively influenced, or positively enriched following substrate additions. This study comprises the first explicit demonstration that bacteria such as Methylobacterium, Methylophilus, and Methylosinus spp. can be enriched on phenol or HM. Our isolation approaches led to the successful cultivation of a variety of native bacteria from the lake, such as Novosphingobium (Alphaproteobacteria) and Flexibacter (Bacteroidetes), or phenol-utilizing bacteria such as members of Actinobacteria or Burkholderia (Betaproteobacteria). Enrichment and cultivation on phenol and HM as substrates revealed highly specialized bacterial communities that resemble those found in many HM-rich lakes.

Changes in pelagic bacteria communities due to leaf litter addition.

In many limnetic systems, the input of allochthonous organic matter, e.g., leaf litter, is a substantial source of dissolved organic carbon (DOC) for pelagic bacteria, especially in fall and winter when autochthonous DOC production is low. However, relatively little is known about community changes of pelagic lake bacteria due to leaf litter input which includes both the release of leaf leachates and microorganisms from the leaf litter into the surrounding water. Therefore, we have experimentally studied the effects of different types of leaf litter (Betula pendula, Fagus silvatica, and Pinus silvestris) on the pelagic bacterial community composition by adding leaves to different treatments of epilimnic water samples (unfiltered, 0.2 µm and 5.0 µm-pre-filtered) from humic Lake Grosse Fuchskuhle (Northeastern Germany). The addition of leaf litter led to a significant increase in DOC concentration in lake water, and each leaf litter type produced significantly different amounts of DOC (p = <0.001) as well as of specific DOC fractions (p = <0.001), except of polysaccharides. DGGE banding patterns varied over time, between types of leaf litter, and among treatments. Bacteria belonging to known bacterial phylotypes in the southwest basin of Lake Grosse Fuchskuhle were frequently found and even persisted after leaf litter additions. Upon leaf litter addition, α-proteobacteria (Azospirillum, Novosphingobium, and Sphingopyxis) as well as ß-proteobacteria (Curvibacter and Polynucleobacter) were enriched. Our results indicate that supply of leaf litter DOM shifted the bacterial community in the surrounding water towards specific phylotypes including species capable of assimilating the more recalcitrant DOC pools. Statistical analyses, however, show that DGGE banding patterns are not only affected by DOC pools but also by treatment. This indicates that biological factors such as source community and grazing may be also important for shifts in bacterial community structure following leaf litter input into different lakes.

Cytochrome P450-dependent metabolism of PCB52 in the nematode Caenorhabditis elegans.

There are 75 full length cytochrome P450 (CYP) genes known in the genome of the nematode Caenorhabditis elegans. The individual biological functions of the vast majority are mostly as yet unknown. Here the impact of cytochrome P450 isoforms on the metabolism of PCB52, an ortho-substituted, non-coplanar 2,2',5,5'-tetrachlorbiphenyl, as a model PCB of these worldwide distributed pollutants is investigated. Organic extracts, isolated from treated worms and analyzed by GC/MS, contained two obvious PCB52-derived products which have been identified as C3-, C4- and/or C6-hydroxy-PCB52. Moreover, these hydroxylase reactions strictly required the functional expression of the NADPH-dependent cytochrome P450 reductase (CPR) encoding emb-8 gene, which was recently shown to be essential also for several other cytochrome P450-dependent enzymatic reactions. Multiple and subsequent single RNAi-gene silencing experiments, as well as the use of cyp-mutant strains, identified members of the CYP-14A subfamily and CYP-34A6 as the major isoforms contributing to PCB52 metabolism in C. elegans. In the gene-silenced worms and mutants, the reduction in formation of hydroxylated products ranged from 55% to 78%. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent PCB metabolites and may thus facilitate future studies on biotransformation.

Reduction in vegetative growth of the water mold Saprolegnia parasitica (Coker) by humic substance of different qualities.

Humic substances (HS) account for 50-80% of the dissolved organic matter in non-eutrophicated freshwater ecosystems. HS are not inert, but are taken up by and may interact with aquatic organisms. However, at present no information is available on the interaction of HS with fungi, for instance, the fish-pathogenous species Saprolegnia parasitica. To fill this gap, we tested effects of HS on S. parasitica growth in-vitro using 25-500mgL(-1) carbon of HS on GY-agar. We investigated 20 HS including natural organic matter (NOM) samples, two lignite derived HS, and one synthetic HS. The aim was (1) to find out, if there are inhibiting effects and (2) if potential effects can be explained by humic matter properties by structure activity relationships. The growth of S. parasitica was related to the growth on HS-free agar controls. Characterization of HS and NOMs included elemental analysis, high-pressure size exclusion chromatography (HPSEC), UV/VIS, FTIR-, and EPR-spectroscopy in order to obtain information on elemental and structural composition including various metals, molecular weights of the HS fraction, aromaticity, free organic radicals, and functional groups. NOMs with high moieties of high-molecular carbohydrates supported the growth of S. parasitica, all other HS and NOMs reduced it. However, no inhibition of the development of the sporangia and primary zoospores was found. Therefore, the impact of the HS on S. parasitica has to be classified as fungistatic, rather than fungicidal. Synthetic and lignite-derived HS were among the most efficient HS sources. Growth inhibition was correlated (p<0.05) with the molecular weights of the HS-fraction, sUVa, COOH groups, C and H. Our results suggest that especially HS with higher molecular weights and aromaticity which contain a high number of organic radicals are the most efficient in reducing fungal growth. Furthermore, highly functionalized HS seem to be important for the observed effect. The development of internal oxidative stress could be a mechanism explaining the observed growth inhibition of S. parasitica.

Retinol-binding protein as a biomarker to assess endocrine-disrupting compounds in the environment.

Endocrine-disrupting compounds (EDC) are predominantly investigated with respect to their ability to mimic or block estrogenic actions. However, it is well-known that EDC can act as agonists or antagonists of androgen- and estrogen-response systems. For that reason, there is an obvious need for bioassays providing the possibility of detecting (anti-)estrogenic and (anti-)androgenic effects. The retinol-binding protein (RBP) seems to be a useful molecular biomarker for assessing all modes of action of EDC, because it is regulated by sex steroid hormones. This study was conducted to establish RBP as a biomarker for determination of (anti-)estrogenic and (anti-)androgenic effects of EDC using a Xenopus laevis primary hepatocyte culture system. It could be shown that RBP mRNA expression in X. laevis hepatocytes was stimulated by estrogens in a dose-dependent manner whereas a combination of estrogen and androgen or estrogen and anti-estrogen treatment suppressed estrogenic stimulating effects. Androgens testosterone and dihydrotestosterone were able to reduce RBP mRNA expression and the anti-androgen vinclozolin could abolish the mRNA synthesis-suppressing activity of the androgen dihydrotestosterone. These results clearly demonstrated that RBP mRNA expression patterns in Xenopus laevis hepatocytes have different modes of (anti-)estrogenic and (anti-)androgenic action and can be used for examination of suspected EDC. Moreover, water samples from sewage-treatment plant effluents were applied to liver cells and expression levels of RBP and estrogen receptor mRNA (a known estrogenic biomarker) were detected. These samples had high estrogenicity but caused low to moderate induction of RBP mRNA synthesis, leading to the conclusion that RBP levels represent the sum of all possible effects (estrogenic and other effects) of EDC in environmental samples.

Bisphenol A induces feminization in Xenopus laevis tadpoles.

To evaluate possible estrogenic effects of bisphenol A (BPA) in an amphibian model, Xenopus laevis tadpoles were exposed to BPA and 17beta-estradiol (E2) during larval development. After metamorphosis, the gonadal phenotype was determined by gross morphology, and testes were further examined histologically to validate the results. BPA treatment altered the normal sex ratio toward females depending on the BPA concentrations added. Chemical analysis showed a time-dependent decline of BPA during semistatic exposure, indicating that BPA is taken up and metabolized to some extent by tadpoles. In addition, tadpoles were exposed to BPA and E2 for 2 weeks during sensitive stages of sexual differentiation. Afterward, the expression of an estrogenic biomarker, estrogen receptor (ER) mRNA, was assessed by semiquantitative RT-PCR. Both BPA and E2 up-regulated ER mRNA significantly. In conclusion, these results show clear evidence that BPA induces feminization in X. laevis tadpoles, revealing an estrogenic potency of BPA that influences sexual development in amphibians.