Interaction of 17α-ethinylestradiol and methyltestosterone in western mosquitofish (Gambusia affinis) across two generations.

The interactions between estrogen and androgen in aquatic animals remain largely unknown. In this study, two generations (F0 and F1) of western mosquitofish (Gambusia affinis) were continuously exposed to 17α-ethinylestradiol (EE2, 10 ng/L), methyltestosterone (MT, 10 ng/L (MTL); 50 ng/L (MTH)), and mixtures (EE2+MTL and EE2+MTH). Various endpoints, including sex ratio (phenotypic and genetic), secondary sex characteristics, gonadal histology, and transcriptional profile of genes, were examined. The results showed that G. affinis exposed to MTH and EE2+MTH had a > 89.7 % of phenotypic males in F1 generation, with 34.5 and 50.0 % of these males originated from genetic females, respectively. Moreover, females from F0 and F1 generations exposed to MTH and EE2+MTH exhibited masculinized anal fins and skeletons. The combined effect of MT and EE2 on most endpoints was dependent on MT. Furthermore, significant transcriptional alterations in certain target genes were observed in both the F0 and F1 generations by EE2 and MT alone and by mixtures, showing some degree of interactions. These findings that the effects of EE2+MTH were primarily on the phenotypic sex of G. affinis in offspring generation suggest that G. affinis under chronic exposure to the binary mixture contaminated water could have sex-biased populations.

Evaluation of the toxicity of clozapine on the freshwater diatom Navicula sp. using the FTIR spectroscopy.

Clozapine is an often prescribed neuroactive pharmaceutical and frequently detected in the aquatic environments. However, its toxicity on low trophic level species (i.e., diatoms) and associated mechanisms are seldom reported. In this study, the toxicity of clozapine on a widely distributed freshwater diatom Navicula sp. was evaluated using the FTIR spectroscopy along with biochemical analyses. The diatoms were exposed to various concentrations of clozapine (0, 0.01, 0.05, 0.10, 0.50, 1.00, 2.00, 5.00 mg/L) for 96 h. The results revealed that clozapine reached up to 392.8 µg/g in the cell wall and 550.4 µg/g within the cells at 5.00 mg/L, suggesting that clozapine could be adsorbed extracellularly and accumulated intracellularly in diatoms. In addition, hormetic effects were displayed on the growth and photosynthetic pigments (chlorophyll a and carotenoid) of Navicula sp., with a promotive effect at concentrations less than 1.00 mg/L while an inhibited effect at concentrations over 2 mg/L. Clozapine induced oxidative stress in Navicula sp., accompanied by decreased levels of total antioxidant capacity (T-AOC) (>0.05 mg/L), in which, the activity of superoxide dismutase (SOD) (at 5.00 mg/L) was increased whereas the activity of catalase (CAT) (>0.05 mg/L) was decreased. Furthermore, FTIR spectroscopic analysis showed that exposure to clozapine resulted in accumulation of lipid peroxidation products, increased sparse ß-sheet structures, and altered DNA structures in Navicula sp. This study can facilitate the ecological risk assessment of clozapine in the aquatic ecosystems.

Levonorgestrel and dydrogesterone affect sex determination via different pathways in zebrafish.

Levonorgestrel (LNG) and dydrogesterone (DDG) are two commonly used synthetic progestins that have been detected in aquatic environments. They could affect fish sex differentiation, but the underlying mechanisms remain unknown. Here we investigated the effects of LNG (5 ng L-1 and 50 ng L-1), DDG (100 ng L-1) and their mixtures on gonadal differentiation and sex determination in zebrafish at transcriptomic and histological levels from 2 hours post-fertilization (eleutheroembryos) to 144 days post-fertilization (sexual maturity). Germ cell development and oogenesis pathways were significantly enriched in LNG and the mixture of LNG and DDG treatments, while insulin and apoptosis pathways in the DDG treatment. LNG and the mixture of LNG and DDG strongly decreased transcripts of germ cell development and oogenesis related genes, while DDG increased the transcripts of insulin and apoptosis related genes at 28 days post fertilization (dpf) and 35 dpf. Furthermore, DDG caused ∼ 90% males, and LNG and the mixture of LNG and DDG resulted in 100% males on all sampling dates. Specifically, most males in LNG and the mixture of LNG and DDG treatments were "Type I" males without juvenile oocytes at 28 dpf and 35 dpf, while those in DDG treatment were "Type II" and "Type III" males with a few juvenile oocytes. These results indicated that LNG and DDG promoted testicular differentiation via different pathways to cause male bias. LNG and DDG mixtures have similar effect on testicular differentiation to LNG alone. The findings from this study could have significant ecological implications to fish populations.

Medroxyprogesterone acetate affects eye growth and the transcription of associated genes in zebrafish.

Medroxyprogesterone acetate (MPA) is a widely used synthetic progestin in contraception pills and hormone replacement therapy. However, its effects on eye growth and development and function were largely unknown. In this study, the transcription of genes in the Notch signaling pathway and the visual cycle network were evaluated after chronic MPA exposure at 4.32 (L), 42.0 (M), and 424 (H) ng L-1 for 120 days in zebrafish. Meanwhile, the histology of the eyes was also examined. Transcriptional results showed that MPA at all three concentrations significantly increased the transcription of notch1a, dll4, jag1a, ctbp1 and rbpjb (key genes in the Notch signaling pathway) in the eyes of females. The up-regulation of noth1a, ctbp1 and kat2b was also observed in the eyes of males exposed to MPA at 424 ng L-1. In the visual cycle pathway, MPA increased the transcription of opn1sw1, opn1sw2, arr3a and rpe65a in the eyes of females from the M and H treatments. Histopathological analysis showed that exposure to 42.0 ng L-1 of MPA increased the thicknesses of inner nuclear layer in females and outer segment in males. Moreover, exposure to 424 ng L-1 of MPA increased the lens diameter in females. These results indicated that chronic MPA exposure affected the transcription of genes in the Notch signaling and in the visual cycle pathways, resulting in overgrowth of the eyes and interference of the eye functions. This study suggests that MPA pose a risk to fitness and survival of zebrafish in areas where MPA contamination exists.

Endocrine disruption in western mosquitofish from open and closed aquatic ecosystems polluted by swine farm wastewaters.

Swine farm wastewaters (SFWs) are generally discharged either directly into nearby rivers or into fish ponds as a source of fertilizer/food for aquaculture in China. SFWs contain various contaminants including steroid hormones. However, there is an extreme paucity of data on their effects in fish populations. Here we investigated the endocrine disrupting effects of SFWs in G. affinis from 2 rivers (7 sites) and 2 fish ponds (2 sites) receiving SFWs and a reference site in Guangdong Province, China. In this study, a total number of 3078 adult western mosquitofish (Gambusia affinis) were collected and the sex ratio was determined. In addition, secondary sexual characteristics were examined and the transcriptional levels of target genes were analyzed. The results showed the mosquitofish populations had a significant increase in male-to-female ratio from 7 sites (including 2 fish ponds) among the 9 sampling sites. The hemal spines of females were masculinized at most sites while the hemal spines of males were feminized at approximately half of the sites (including 2 fish ponds). Significant reduction in vitellogenin (Vtg) mRNA expression was observed in females from 2 sites (including RS7) while elevated Vtg mRNA expression was noticed in males from 2 sites along the rivers (including RS7). Redundancy analysis showed that androgens in the water samples were closely related with male-to-female ratio in the mosquitofish populations and the masculinized hemal spines of females. The findings from this study demonstrated that discharge of SFWs could result in occurrence of both masculinized females and feminized males in mosquitofish population.

Dydrogesterone affects the transcription of genes in visual cycle and circadian rhythm network in the eye of zebrafish.

Dydrogesterone (DDG) is a synthetic progestin used in contraception and hormone replacement therapy. Our previous transcriptome data showed that the response to light stimulus, photoperiodism and rhythm related gene ontology (GO) terms were significantly enriched in the brain of zebrafish after chronic exposure to DDG. Here we investigated the effects of DDG on the eye of zebrafish. Zebrafish were exposed to DDG at three concentration levels (3.39, 33.1, and 329 ng L-1) for 120 days. Based on our previous transcriptome data, the transcription of genes involved in visual cycle and circadian rhythm network was examined by qPCR analysis. In the visual cycle network, exposure to all concentrations of DDG significantly decreased transcription of grk7a, aar3a and guca1d, while increased the transcription of opn1mw4 and opn1sw2 at the low concentration. Importantly, exposure to all concentrations of DDG down-regulated the transcription of rep65a that encodes a critical enzyme to catalyze the conversion from all-trans-retinal to 11-cis-retinal in the eye of male zebrafish. In the circadian rhythm network, DDG enhanced the transcription of clocka, arntl2 and nifil3-5 at all three concentrations, while it decreased the transcription of cry5, per1b, nr1d2b and si:ch211.132b12.7. In addition, DDG decreased the transcription of tefa in both males and females. Moreover, histological analysis showed the exposure to 329 ng L-1 of DDG decreased the thickness of retinal ganglion cell in the eye of male zebrafish. These results indicated that DDG exposure could affect the transcription of genes in visual cycle and circadian rhythm network in the eyes of zebrafish. This suggests that DDG has potential negative impact on the normal eye function.

Endocrine disrupting effects in western mosquitofish Gambusia affinis in two rivers impacted by untreated rural domestic wastewaters.

Domestic wastewaters are an important source of endocrine disrupting chemicals in the receiving aquatic environment. Most rural domestic wastewaters (RDWs) in China have been directly discharged into the aquatic environment without any treatment. Here we studied the effects of RDWs on the western mosquitofish (Gambusia affinis) from two rural rivers receiving untreated RDWs. Mosquitofish samples were collected at 5 sampling sites along two rivers during dry and wet seasons. Sex ratios, secondary sex characteristics and transcriptional levels of target genes related to the endocrine system in adult females and males were determined. In parallel, various pollutants including steroid hormones, phenolic compounds, pesticides, polycyclic aromatic hydrocarbons (PAHs) and heavy metals were measured in the water samples at all sites. The results showed that the androgenic effects in the fish were evidenced by significant increase in male to female ratio in fish populations at two sampling sites and by the presence of modified hemal spines in females at four sampling sites when compared to the reference site. The males from the two rivers had increased Vtg mRNA expressions with a maximal 6.2-fold increase relative to the reference site and a delayed development of hemal spines. The redundancy analysis (RDA) showed that some physiological parameters were related to steroid hormones, phenolic compounds and PAHs. The findings from this study suggest that RDWs can lead to masculinization in females and feminization in males.

Medroxyprogesterone acetate affects sex differentiation and spermatogenesis in zebrafish.

Medroxyprogesterone acetate (MPA) is a widely used synthetic progestin and it has been frequently detected in aquatic environments. However, its effects on aquatic organisms remain largely unknown. Here we investigated the chronic effects of MPA on sex differentiation and gonad development in zebrafish. Zebrafish larvae at 20 days post fertilization (dpf) were exposed to 4.32, 42.0, and 424 ng L-1 of MPA until they reached 140 dpf. The results showed that chronic exposure to 42.0 ng L-1 of MPA caused 60% proportion of males as well as significant up-regulation of dmrt1 (˜1.79 fold) and hsd17b3 (˜1.92 fold). Histological analysis showed MPA significantly increased the frequency of immature spermatocytes accompanied with the increased transcription of dmrt1 (˜2.06 fold) and ar (˜1.73 fold) in the testes. Meanwhile, MPA exposure significantly increased the transcription of lhb at all exposure concentrations in the males. In contrast, it significantly suppressed the transcription of lhb (˜-8.06-fold) and fshb (˜-6.35-fold) at 42.0 ng L-1 in the females. Collectively our results demonstrated that MPA had androgenic activity, and could affect sex differentiation and spermatogenesis in zebrafish at environmentally relevant concentrations. The findings from this study suggest that MPA in the aquatic environment may pose potential androgenic risks to fish populations.

[Combined effects of fluoxetine and triclosan on Pseudorasbora parva.] / 氟西汀与三氯生复合暴露对麦穗鱼的毒性效应.

We examined the effects of binary mixture of two different pharmaceutical and personal care products (PPCPs) on aquatic organisms. The topmouth gudgeon Pseudorasbora parva, native to northeast China, was used to study the combined effects of fluoxetine (FLX) and triclosan (TCS) on its different organs. After acute (4 h) and chronic (42 d) treatments, the combined effects of FLX/TCS on phase I and phase II detoxification enzymes, nervous system, digestive system and antioxidant system were evaluated. The results showed that the AChE activity in the brain of P. parva was transiently inhibited, whereas the activity of EROD in the liver was continuously inhi-bited. The activity of α-Glu in the intestine was induced after acute exposure but was inhibited after chronic exposure. Meanwhile, the level of lipid peroxidation in the liver was elevated after chronic exposure. In conclusion, the mixture of fluoxetine and triclosan produced toxic effects on multiple organs of P. parva, which could be alleviated after prolonged exposure, indicating an acclimation. However, the mechanisms of this acclimation needed further study.

Horizontal transmission of Paranosema locustae (Microsporidia) in grasshopper populations via predatory natural enemies.

BACKGROUND: Paranosema locustae Canning, 1953 (Microsporidia) provides effective control of grasshoppers. Horizontal transmission of P. locustae is known to occur, and evidence for the mechanism of this transmission via predatory natural enemies has been found. We conducted a 3-year laboratory and field study to assess the potential impact of feces from both grasshoppers Locusta migratoria L. and their natural enemies on the persistence of P. locustae. RESULTS: We found that P. locustae persisted among grasshopper populations in treated areas and in adjacent untreated areas for up to 2 years, and the density of grasshoppers decreased in both areas. We showed that healthy grasshoppers could be infected by eating food contaminated by feces from their natural enemies. Grasshopper predators retained a large number of spores acquired from eating grasshoppers infected with P. locustae. Spores in the feces of the main natural enemy, the beetle Pterostichus gebleri Dejean 1828, in the treated area showed clear viability. CONCLUSION: These results demonstrate that predatory natural enemies are important vectors for this microsporidian disease, and suggest that sustainable transmission and continuing population suppression might be achieved by horizontal transmission via natural enemies, which should be maximized to increase the effectiveness of P. locustae. © 2018 Society of Chemical Industry.

Modulation of transcription of genes related to the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes in zebrafish (Danio rerio) embryos/larvae by androstenedione.

This study aimed to determine the effects of Androstenodione (AED) on the transcriptional expression of genes involved in the hypothalamic-pituitary-gonadal (HPG) and the hypothalamic-pituitary-adrenal (HPA) axes in the zebrafish embryos/larvae. Zebrafish embryos were exposed to 0, 4.0, 45.0, 487.0, and 980.0 ng/L of AED from the day of fertilization to 144 h post fertilization (hpf), during which the transcriptional profiles of key genes related to the HPG and HPA axes were examined daily using quantitative real-time PCR. The AED exposure significantly up-regulated several receptor signaling pathways and the key genes involved in various steps of the steroidogenic pathways were also affected. In addition, the AED exposure could significantly modulate the transcriptional profiles of the other target genes related to hypothalamic and pituitary hormones. The findings of this study suggest that AED, at environmentally relevant concentrations, affects the adrenal endocrine systems and the reproduction of zebrafish by interrupting the HPG and HPA axes.

Efficacy of Bt maize producing the Cry1Ac protein against two important pests of corn in China.

Ostrinia furnacalis (Guenée) and Helicoverpa armigera (Hübner) are the most important pests of maize in China. A laboratory study and a 2-year field study on the efficacy of transgenic maize expressing the Cry1Ac protein BT38 against O. furnacalis and H. armigera were performed. We found that the husks, kernels, and silks of BT38 showed significant efficacy against larvae of O. furnacalis and H. armigera. In the field, when neonate larvae of O. furnacalis and H. armigera were on plants at different growth stages and when levels of leaf-damage or number of damaged silks were used to score efficacy, we found that BT38 showed significant insecticidal efficacy against O. furnacalis and H. armigera, but the non-Bt maize did not show significant efficacy against either pest. These results suggest that the insecticidal efficacy of Bt maize expressing the Cry1Ac protein could be useful in the integrated pest management of these key maize pests.

Forebrain-specific loss of synaptic GABAA receptors results in altered neuronal excitability and synaptic plasticity in mice.

Mutations that result in the defective trafficking of γ2 subunit containing GABAA receptors (γ2-GABAARs) are known to reduce synaptic inhibition. Whether perturbed clustering of non-mutated GABAARs similarly reduces synaptic inhibition in vivo is less clear. In this study we provide evidence that the loss of postsynaptic γ2-GABAARs upon postnatal ablation of gephyrin, the major scaffolding protein of inhibitory postsynapses, from mature principal neurons within the forebrain results in reduced induction of long-term potentiation (LTP) and impaired network excitability within the hippocampal dentate gyrus. The preferential reduction in not only synaptic γ2-GABAAR cluster number at dendritic sites but also the decrease in γ2-GABAAR density within individual clusters at dendritic inhibitory synapses suggests that distal synapses are more sensitive to the loss of gephyrin expression than proximal synapses. The fact that these mice display behavioural features of anxiety and epilepsy emphasises the importance of postsynaptic γ2-GABAAR clustering for synaptic inhibition.

The mechanism for microsporidian parasite suppression of the hindgut bacteria of the migratory locust Locusta migratoria manilensis.

Locusts aggregate into bands of nymphs and swarms of adults that can pose a major threat to crop. Previous studies have shown that infection by the microsporidian parasite Paranosema locustae prevents locust aggregation behavior and we show that gut bacteria, which produce components of locust aggregation pheromones, are substantially reduced in locusts infected with P. locustae. We found that P. locustae could reduce the diversity, abundance and community composition of Locusta migratoria's gut bacteria. The parasite infection was also shown to interrupt the peroxidase activity of locust hindgut. Genome-wide expression analysis showed that the parasite infection suppressed peroxidase mRNA relative expression of locust hindgut, but had no effects on attacin expression and superoxide dismutase at 16 d post-inoculation with 20,000 P. locustae spores. Our findings reveal the mechanisms by which P. locustae impairs bacterial diversity and community structure of Locusta migratoria's gut bacteria.

[Mechanisms of Cr (VI) toxicity to fish in aquatic environment: A review].

With increasing consumption and applications of chromium in metallurgy, electroplating, tanning process and stainless steel industry, chromium contamination has become a global environmental problem. In general, Cr(VI) has higher permeability across the cell membrane than Cr(III) and hence is considered more toxic than Cr(III). Oxidative stress could be induced following reactive oxygen species (ROS) normally produced in fish under Cr(VI) exposure due to its variable valences. Furthermore, the intermediates of Cr, e.g. Cr(V) and Cr(IV) , produced by cellular reduction processes can bind with DNA and result in mutagenic effects. These combined effects will threaten the growth, development and population structure of different fish species. In this paper, we reviewed published results on the toxic effects of Cr(VI) in fish at levels of molecules, tissues, organs and individuals. The mechanisms of toxicity and detoxification of Cr(VI) in various aspects were discussed. In addition, we also put forward perspectives on the toxicity of chromium in aquatic organisms.

Malignant transformation of 293 cells induced by ectopic expression of human Nanog.

Tumor development has long been known to resemble abnormal embryogenesis. The ESC self-renewal gene NANOG is purportedly expressed in some epithelial cancer cells and solid tumors, but a casual role in tumor development has remained unclear. In order to more comprehensively elucidate the relationship between human Nanog and tumorigenesis, the hNanog was ectopically expressed in the 293 cell line to investigate its potential for malignant transformation of cells both in vitro and in vivo. Here we provide compelling evidence that the overexpression of hNanog resulted in increased cell proliferation, anchor-independent growth in soft agar, and formation of tumors after subcutaneous injection of athymic nude mice. Pathologic analysis revealed that these tumors were poorly differentiated. In analysis of the underlying molecular mechanism, two proteins, FAK and Ezrin, were identified to be upregulated in the hNanog expressing 293 cells. Our results demonstrate that hNanog is a potent human oncogene and has the ability to induce cellular transformation of human cells.

[Multipotential differentiation and potential applications of adipose-derived stem cells].

Adipose tissue contains a population of multipotent cells called adipose-derived stem cells (ADSCs). With the similar properties of marrow-derived mesenchymal stem cells, ADSCs have the ability to differentiate differentiate towards adipogenic, osteogenic, chondrogenic, myogenic, endothelial, hematopoietic, hepatic, islet, and neurogenic cell lineages. As adipose tissue in harvested in large amounts with minimal morbidity, it can be widely used in tissue engineering, organ repair and gene therapy. This paper focused on the plasticity of ADSCs and reviewed the new advances of this field. Finally, the problems and prospect for application was also discussed.

[The targeting study of human serum albumin gene].

To generate transgenic porcine which expresses human serum albumin (HSA), the HSA gene targeting vector was constructed with HSA cDNA as the gene of interestand partial porcine serum albumin (PSA) gene as homologous arms which respectively were 7.2 kb 5' regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron. The resistant gene neo was inserted into intron 1 and tk was ligated to the 3' end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.

[The synthesis and function analysis of omega-3 fatty acid desaturase gene from Caenorhabditis briggssae].

Omega-3 polyunsaturated fatty acids (PUFAs) have been broadly investigated and shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. In mammals, the level of omega-3 PUFAs is relatively too low compared with omega-6 PUFAs, which metabolically and functionally distinct from omega-3 PUFAs and often have important opposing physiological functions. Either the inefficiency of omega-3 PUFAs or the excess of omega-6 PUFAs will cause many healthy problems. So methods have been sought to increase the amount of omega-3 PUFAs and to improve the omega-6/omega-3 ratio in body. In this study, the sFat-1 gene, which putatively encodes a omega-3 fatty acid desaturase, was chemically synthesized according to the sequence from Caenorhabditis briggssae (with codon usage modified), and constructed into a mammal expression vector pcDNA3. 1-sFat1-EGFP. This vector was introduced into CHO cells by lipid-mediated transfection, and it's expression quickly and effectively elevated the cellular omega-3 PUFAs (from 18-carbon to 22-carbon) contents and dramatically improved the ratio of omega-6/omega-3 PUFAs. Cellular lipids extracts from stably selected cells were analyzed with GC-MS and the results showed that amount of total omega-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29% (in sFat-1 cells), whereas the amount of total omega-3 PUFAs increased from 7.86% to 24.02%, respectively. The omega-6/omega-3 ratio also dropped from 6.23 to 1.47. These data demonstrates the Caenorhabditis briggssae omega-3 Fatty Acid Desaturase gene, sFat-1, was synthesized successfully and can produce omega-3 PUFAs by using the corresponding omega-6 PUFAs as substrates, which shows its potential for use in the production of omega-3 PUFAs in transgenic animals.

[Synthesis of total sFat-1 gene by PCR-restrict enzyme ligation method].

Gene synthesis is very important in life science research, and it becomes a technique in common use. It is difficult for long gene synthesis, because the mismatches and mutations of DNA sequence in nucleotide fragments assembling. This study established a new method for long gene synthesis, which was referred to as PCR-restrict enzyme ligation method. With this method, a omega-3 fatty acid desaturase gene, sFat-1, from Caenorhabditis briggssae, was successfully assembled from 27 synthesized nucleotide fragments (60 ~ 68 bp for each fragment ) following 3 rounds of PCR (7 reactions) and 2 rounds of restrict enzyme ligation (3 reactions). This shows that the PCR-restrict enzyme ligation method is an effective method for long gene synthesis.