Effects of New Psychoactive Substance Esketamine on Behaviors and Transcription of Genes in Dopamine and GABA Pathways in Zebrafish Larvae.

Esketamine (ESK) is the S-enantiomer of ketamine racemate (a new psychoactive substance) that can result in illusions, and alter hearing, vision, and proprioception in human and mouse. Up to now, the neurotoxicity caused by ESK at environmental level in fish is still unclear. This work studied the effects of ESK on behaviors and transcriptions of genes in dopamine and GABA pathways in zebrafish larvae at ranging from 12.4 ng L- 1 to 11141.1 ng L- 1 for 7 days post fertilization (dpf). The results showed that ESK at 12.4 ng L- 1 significantly reduced the touch response of the larvae at 48 hpf. ESK at 12.4 ng L- 1 also reduced the time and distance of larvae swimming at the outer zone during light period, which implied that ESK might potentially decrease the anxiety level of larvae. In addition, ESK increased the transcription of th, ddc, drd1a, drd3 and drd4a in dopamine pathway. Similarly, ESK raised the transcription of slc6a1b, slc6a13 and slc12a2 in GABA pathway. This study suggested that ESK could affect the heart rate and behaviors accompanying with transcriptional alterations of genes in DA and GABA pathways at early-staged zebrafish, which resulted in neurotoxicity in zebrafish larvae.

The azole biocide climbazole induces oxidative stress, inflammation, and apoptosis in fish gut.

Climbazole is an azole biocide that has been widely used in formulations of personal care products. Climbazole can cause developmental toxicity and endocrine disruption as well as gut disturbance in aquatic organisms. However, the mechanisms behind gut toxicity induced by climbazole still remain largely unclear in fish. Here, we evaluate the gut effects by exposing grass carp (Ctenopharyngodon idella) to climbazole at levels ranging from 0.2 to 20 µg/L for 42 days by evaluating gene transcription and expression, biochemical analyses, correlation network analysis, and molecular docking. Results showed that climbazole exposure increased cyp1a mRNA expression and ROS level in the three treatment groups. Climbazole also inhibited Nrf2 and Keap1 transcripts as well as proteins, and suppressed the transcript levels of their subordinate antioxidant molecules (cat, sod, and ho-1), increasing oxidative stress. Additionally, climbazole enhanced NF-κB and iκBα transcripts and proteins, and the transcripts of NF-κB downstream pro-inflammatory factors (tnfα, and il-1ß/6/8), leading to inflammation. Climbazole increased pro-apoptosis-related genes (fadd, bad1, and caspase3), and decreased anti-apoptosis-associated genes (bcl2, and bcl-xl), suggesting a direct reaction to apoptosis. The molecular docking data showed that climbazole could form stable hydrogen bonds with CYP1A. Mechanistically, our findings suggested that climbazole can induce inflammation and oxidative stress through CYP450s/ROS/Nrf2/NF-κB pathways, resulting in cell apoptosis in the gut of grass carp.

Earliest Prepared core technology in Eurasia from Nihewan (China): Implications for early human abilities and dispersals in East Asia.

Organized flaking techniques to obtain predetermined stone tools have been traced back to the early Acheulean (also known as mode 2) in Africa and are seen as indicative of the emergence of advanced technical abilities and in-depth planning skills among early humans. Here, we report one of the earliest known examples of prepared core technology in the archaeological record, at the Cenjiawan (CJW) site in the Nihewan basin of China, dated 1.1 Mya. The operational schemes reconstructed from the CJW refit sets, together with shaping patterns observed in the retouched tools, suggest that Nihewan basin toolmakers had the technical abilities of mode 2 hominins, and developed different survival strategies to adapt to local raw materials and environments. This finding predates the previously earliest known prepared core technology from Eurasia by 0.3 My, and the earliest known mode 2 sites in East Asia by a similar amount of time, thus suggesting that hominins with advanced technologies may have migrated into high latitude East Asia as early as 1.1 Mya.

Screening androgen receptor agonists of fish species using machine learning and molecular model in NORMAN water-relevant list.

Androgen receptor (AR) agonists have strong endocrine disrupting effects in fish. Most studies mainly investigate AR binding capacity using human AR in vitro. However, there is still few methods to rapidly predict AR agonists in aquatic organisms. This study aimed to screen AR agonists of fish species using machine learning and molecular models in water-relevant list from NORMAN, a network of reference laboratories for monitoring contaminants of emerging concern in the environment. In this study, machine learning approaches (e.g., Deep Forest (DF)), Random Forests and artificial neural networks) were applied to predict AR agonists. Zebrafish, fathead minnow, mosquitofish, medaka fish and grass carp are all important aquatic model organisms widely used to evaluate the toxicity of new pollutants, and the molecular models of ARs from these five fish species were constructed to further screen AR agonists using AlphaFold2. The DF method showed the best performances with 0.99 accuracy, 0.97 sensitivity and 1 precision. The Asn705, Gln711, Arg752, and Thr877 residues in human AR and the corresponding sites in ARs from the five fish species were responsible for agonist binding. Overall, 245 substances were predicted as suspect AR agonists in the five fish species, including, certain glucocorticoids, cholesterol metabolites, and cardiovascular drugs in the NORMAN list. Using machine learning and molecular modeling hybrid methods rapidly and accurately screened AR agonists in fish species, and helping evaluate their ecological risk in fish populations.

An azole fungicide climbazole damages the gut-brain axis in the grass carp.

Azole antifungal climbazole has frequently been detected in aquatic environments and shows various effects in fish. However, the underlying mechanism of toxicity through the gut-brain axis of climbazole is unclear. Here, we investigated the effects of climbazole at environmental concentrations on the microbiota-intestine-brain axis in grass carp via histopathological observation, gene expression and biochemical analyses, and high-throughput sequencing of the 16 S rRNA. Results showed that exposure to 0.2 to 20 µg/L climbazole for 42 days significantly disrupted gut microbiota and caused brain neurotoxicity in grass carp. In this study, there was an alteration in the phylum and genus compositions in the gut microbiota following climbazole treatment, including reducing Fusobacteria (e.g., Cetobacterium) and increasing Actinobacteria (e.g., Nocardia). Climbazole disrupted intestinal microbial abundance, leading to increased levels of lipopolysaccharide and tumor necrosis factor-alpha in the gut, serum, and brain. They passed through the impaired intestinal barrier into the circulation and caused the destruction of the blood-brain barrier through the gut-brain axis, allowing them into the brain. In the brain, climbazole activated the nuclear factor kappaB pathway to increase inflammation, and suppressed the E2-related factor 2 pathway to produce oxidative damage, resulting in apoptosis, which promoted neuroinflammation and neuronal death. Besides, our results suggested that this neurotoxicity was caused by the breakdown of the microbiota-gut-brain axis, mediated by reduced concentrations of dopamine, short chain fatty acids, and intestinal microbial activity induced by climbazole.

Ephedrine and cocaine cause developmental neurotoxicity and abnormal behavior in zebrafish.

Ephedrine (EPH) and cocaine (COC) are illegal stimulant drugs, and have been frequently detected in aquatic environments. EPH and COC have negative effects on the nervous system and cause abnormal behaviors in mammals and fish at high concentrations, but their mechanisms of neurotoxicity remain unclear in larvae fish at low concentrations. To address this issue, zebrafish embryos were exposed to EPH and COC for 14 days post-fertilization (dpf) at 10, 100, and 1000 ng L-1. The bioaccumulation, development, behavior, cell neurotransmitter levels and apoptosis were detected to investigate the developmental neurotoxicity (DNT) of EPH and COC. The results showed that EPH decreased heart rate, while COC increased heart rate. EPH caused cell apoptosis in the brain by AO staining. In addition, behavior analysis indicated that EPH and COC affected spontaneous movement, touch-response, swimming activity and anxiety-like behaviors. EPH and COC altered the levels of the neurotransmitters dopamine (DA) and γ-aminobutyric acid (GABA) with changes of the transcription of genes related to the DA and GABA pathways. These findings indicated that EPH and COC had noticeable DNT in the early stage of zebrafish at environmentally relevant concentrations.

Climbazole causes cell apoptosis and lipidosis in the liver of grass carp.

Climbazole, an azole, is widely used in personal care products, pharmaceuticals, and pesticides and is frequently detected in surface water. Climbazole has showed endocrine-disrupting effects. However, the effects of climbazole in fish are still largely unclear. In this study, grass carp (Ctenopharyngodon idella) and liver cell lines (L8824 cells) were treated with climbazole at concentrations ranging from 0.2 to 20 µg/L for 42 days in vivo and 24 h in vitro to evaluate the effects on the liver, respectively. Pathological, biochemical, and gene transcription and expression analyses were conducted to examine the hepatotoxicity. Our results showed that climbazole significantly decreased the hepatosomatic index, caused cell apoptosis in vivo and in vitro, and finally accumulated lipids in the liver. Beside, climbazole increased ROS levels, reduced Nrf2 and Keap1 mRNA and protein levels, and further decreased transcription of Nrf2-dependent downstream antioxidant enzyme genes, causing oxidative stress. Moreover, climbazole increased transcription and protein levels of apoptosis-related genes. Finally, climbazole damaged mitochondrial function and structure, disrupted liver lipid metabolism. Overall, climbazole caused hepatotoxicity, leading to a high ecological risk for aquatic organisms.

Chronic Paternal/Maternal Exposure to Environmental Concentrations of Imidacloprid and Thiamethoxam Causes Intergenerational Toxicity in Zebrafish Offspring.

Imidacloprid (IMI) and thiamethoxam (THM) are ubiquitous in aquatic ecosystems. Their negative effects on parental fish are investigated while intergenerational effects at environmentally relevant concentrations remain unclear. In this study, F0 zebrafish exposed to IMI and THM (0, 50, and 500 ng L-1) for 144 days post-fertilization (dpf) was allowed to spawn with two modes (internal mating and cross-mating), resulting in four types of F1 generations to investigate the intergenerational effects. IMI and THM affected F0 zebrafish fecundity, gonadal development, sex hormone and VTG levels, with accumulations found in F0 muscles and ovaries. In F1 generation, paternal or maternal exposure to IMI and THM also influenced sex hormones levels and elevated the heart rate and spontaneous movement rate. LncRNA-mRNA network analysis revealed that cell cycle and oocyte meiosis-related pathways in IMI groups and steroid biosynthesis related pathways in THM groups were significantly enriched in F1 offspring. Similar transcriptional alterations of dmrt1, insl3, cdc20, ccnb1, dnd1, ddx4, cox4i1l, and cox5b2 were observed in gonads of F0 and F1 generations. The findings indicated that prolonged paternal or maternal exposure to IMI and THM could severely cause intergenerational toxicity, resulting in developmental toxicity and endocrine-disrupting effects in zebrafish offspring.

Norethindrone suppress the germ cell development via androgen receptor resulting in male bias.

Progestins are widely used and detected in surface waters, and can affect gonad development and sexual differentiation in fish. However, the toxicological mechanisms of sexual differentiation induced by progestins are not well understood. Here, we investigated the effects of norethindrone (NET) and androgen receptor (AR) antagonist flutamide (FLU) on gonadal differentiation in zebrafish from 21 dpf (days post-fertilization) to 49 dpf. The results showed that NET caused male bias, while FLU resulted in female bias at 49 dpf. The NET and FLU mixtures significantly decreased the percentage of males compared to the NET single exposure. Molecular docking analysis showed that FLU and NET had similar docking pocket and docking posture with AR resulting in competitively forming the hydrogen bond with Thr334 of AR. These results suggested that binding to AR was the molecular initiating event of sex differentiation induced by NET. Moreover, NET strongly decreased transcription of biomarker genes (dnd1, ddx4, dazl, piwil1 and nanos1) involved in germ cell development, while FLU significantly increased transcription of these target genes. There was an increase in the number of juvenile oocytes, which was consistent with the female bias in the combined groups. The bliss independence model analysis further showed that NET and FLU had antagonistic effect on transcription and histology during gonadal differentiation. Thus, NET suppressed the germ cell development via AR, resulting in male bias. Understanding the molecular initiation of sex differentiation in progestins is essential to provide a comprehensive biological basis for ecological risk assessment.

Directional editing of self-supported nanoarray electrode for adaptive paired-electrolysis.

Anodic oxidation assisted hydrogen production under mild conditions powered by renewable electricity represents a sustainable approach to energy conversion systems. Here, we fabricated a versatile and universal self-supported nanoarray platform that can be intelligently edited to achieve adaptive electrocatalysis for alcohol oxidation reactions and hydrogen evolution reaction (HER). The obtained self-supported nanoarray electrocatalysts exhibit excellent catalytic activity due to the integration of multiple merits of rich nanointerface-reconstruction and self-supported hierarchical structures. Particularly, the membrane-free pair-electrolysis system coupling HER and ethylene glycol oxidation reaction (EGOR) required an applied voltage of only 1.25 V to drive the current density of 10 mA cm-2, which is about 510 mV lower than that of the overall water splitting, showing the capability to simultaneously produce H2 and formate with high Faradic efficiency and stability. This work demonstrates a catalytic self-supported nanoarray platform for energy-efficient production of high-purity H2 and value-added chemicals.

Dydrogesterone disrupts lipid metabolism in zebrafish brain: A study based on metabolomics and Fourier transform infrared spectroscopy.

Brain is a potential target for neuroprogestogens and/or peripheral progestogens. Previous studies reported that expression of genes about steroidogenesis, reproduction, cell cycle, and circadian rhythm in zebrafish brain could be affected by progestogens. However, there are limited information from metabolites or biomacromolecules aspects, leaving an enormous gap in understanding toxic effects of progestogens on fish brain. In this study, we exposed zebrafish embryos to 2.8, 27.6, and 289.8 ng/L dydrogesterone (DDG, a synthetic progestogen) until sexual maturity (140 days). LC-MS and GC-MS based untargeted metabolomics and Fourier-transform infrared (FTIR) spectroscopy were then performed to investigate the metabolic profiles and macromolecular changes of brain of these zebrafish. The results from multivariate statistical analysis of metabolite features showed a clear separation between different treatment groups of both female and male zebrafish brains. DDG exposure increased the levels of cholesterol, saturated fatty acids, and nucleoside monophosphates, but decreased the contents of polyunsaturated fatty acids (PUFAs), lysophosphatides, and nucleosides in dose-dependent manner. FTIR results indicated that DDG exposure led to accumulation of saturated lipids, reduction of nucleic acids and carbohydrates, and alteration of protein secondary structures. The findings from this study demonstrated that DDG could affect contents of metabolites and biomacromolecules of zebrafish brain, which may finally lead to brain dysfunctions.

A Time-course Transcriptional Kinetics of Genes in Behavior, Cortisol Synthesis and Neurodevelopment in Zebrafish Larvae Exposed to Imidacloprid and Thiamethoxam.

Agricultural use of neonicotinoid insecticides, neuroactive nitroguanidine compounds, has been detected everywhere in the global, posing significant hazard to nontarget organisms. This work studied the developmental neurotoxicity of zebrafish larvae exposed to imidacloprid (IMI) and thiamethoxam (THM), ranging from 0.05 µg L- 1 to 50 µg L- 1 for 35 days. Transcriptions of genes belonging to the behavior, neurodevelopment and cortisol synthesis in zebrafish larvae were monitored. The qPCR data demonstrated that with exposure time increased, the transcription of behavior related genes was down-regulated in both IMI and THM groups, such as macf1, cdh6 and syt10. Additionally, IMI and THM significantly up-regulated the transcriptions of actha, and down-regulated il1rapl1b and pi4k2a at 35 dpf. Importantly, IMI markedly enhanced the transcripiton of gfap, shha, nkx2.2a and nestin in a time dependent manner. This work provided the foundation for understanding zebrafish larvae's neurotoxicity induced by IMI and THM.

Enzyme-Inspired Microenvironment Engineering of a Single-Molecular Heterojunction for Promoting Concerted Electrochemical CO2 Reduction.

Challenges remain in the development of novel multifunctional electrocatalysts and their industrial operation on low-electricity pair-electrocatalysis platforms for the carbon cycle. Herein, an enzyme-inspired single-molecular heterojunction electrocatalyst ((NHx )16 -NiPc/CNTs) with specific atomic nickel centers and amino-rich local microenvironments for industrial-level electrochemical CO2 reduction reaction (eCO2 RR) and further energy-saving integrated CO2 electrolysis is designed and developed. (NHx )16 -NiPc/CNTs exhibit unprecedented catalytic performance with industry-compatible current densities, ≈100% Faradaic efficiency and remarkable stability for CO2 -to-CO conversion, outperforming most reported catalysts. In addition to the enhanced CO2 capture by chemisorption, the sturdy deuterium kinetic isotope effect and proton inventory studies sufficiently reveal that such distinctive local microenvironments provide an effective proton ferry effect for improving local alkalinity and proton transfer and creating local interactions to stabilize the intermediate, ultimately enabling the high-efficiency operation of eCO2 RR. Further, by using (NHx )16 -NiPc/CNTs as a bifunctional electrocatalyst in a flow cell, a low-electricity overall CO2 electrolysis system coupling cathodic eCO2 RR with anodic oxidation reaction is developed to achieve concurrent feed gas production and sulfur recovery, simultaneously decreasing the energy input. This work paves the new way in exploring molecular electrocatalysts and electrolysis systems with techno-economic feasibility.

Dydrogesterone and levonorgestrel at environmentally relevant concentrations have antagonist effects with rhythmic oscillation in brain and eyes of zebrafish.

Synthetic progestins levonorgestrel (LNG) and dydrogesterone (DDG) are frequency detected in surface water. Combined effects of LNG and DDG on gonad differentiation are similar to LNG single exposure in juvenile zebrafish. However, LNG and DDG mixtures have stronger effects on spermatogenesis in testes of adult zebrafish, which show variable at different life stage. Effects of LNG and DDG mixtures on eyes and brain remain unknown. Here we investigated effects of LNG, DDG and their mixtures on eyes and brain. Zebrafish were exposed to LNG, DDG and their mixtures from 2 hpf to 144 dpf. Rhythm and vision related biological processes were enriched in eyes and brain in LNG and DDG treatments, which indicated rhythmic oscillation in eyes and brain. The qPCR data revealed that both LNG and DDG decreased transcription of arntl2 and clocka, while increased transcription of per1a, per1b, rpe65a and tefa in eyes and brain. However, DDG and LNG mixtures had slight effect on transcription of genes related to rhythm and vision. In addition, LNG and DDG reduced the thickness of inner nuclear layer in the eyes. Bliss independent model revealed that LNG and DDG had antagonist effects on transcription and histology in eyes and brain. Moreover, LNG and DDG formed the same hydrogen bonds with green-sensitive opsin-4 and rhodopsin kinase GRK7a. Taken together, LNG and DDG competed with each other for the same binding residues resulting in antagonist effect in their mixtures treatments, and have significant ecological implications to assess combined effects of progestins mixtures on fish in different organs.

Transgenerational effects of androstadienedione and androstenedione at environmentally relevant concentrations in zebrafish (Danio rerio).

Androgens androstadienedione (ADD) and androstenedione (AED) are predominant steroid hormones in surface water, and can disrupt the endocrine system in fish. However, little is known about the transgenerational effects of ADD and AED in fish. In the present study, F0 generation was exposed to ADD and AED from 21 to 144 days post-fertilization (dpf) at nominal concentrations of 5 (L), 50 (M) and 500 (H) ng L-1, and F1 generation was domesticated in clear water for 144 dpf. The sex ratio, histology and transcription in F0 and F1 generations were examined. In the F0 generation, ADD and AED tended to be estrogenic in zebrafish, resulting in female biased zebrafish populations. In the F1 generation, ADD at the H level caused 63.5% females, while AED at the H level resulted in 78.7% males. In brain, ADD and AED had similar effects on circadian rhythm in the F0 and F1 generations. In the F1 eleutheroembryos, transcriptomic analysis indicated that neuromast hair cell related biological processes (BPs) were overlapped in the ADD and AED groups. Taken together, ADD and AED at environmentally relevant concentrations had transgenerational effects on sex differentiation and transcription in zebrafish.

Integrated 3D Open Network of Interconnected Bismuthene Arrays for Energy-Efficient and Electrosynthesis-Assisted Electrocatalytic CO2 Reduction.

Electrocatalytic CO2 reduction reaction (CO2 RR) toward formate production can be operated under mild conditions with high energy conversion efficiency while migrating the greenhouse effect. Herein, an integrated 3D open network of interconnected bismuthene arrays (3D Bi-ene-A/CM) is fabricated via in situ electrochemically topotactic transformation from BiOCOOH nanosheet arrays supported on the copper mesh. The resulted 3D Bi-ene-A/CM consists of 2D atomically thin metallic bismuthene (Bi-ene) in the form of an integrated array superstructure with a 3D interconnected and open network, which harvests the multiple structural advantages of both metallenes and self-supported electrodes for electrocatalysis. Such distinctive superstructure affords the maximized quantity and availability of the active sites with high intrinsic activity and superior charge and mass transfer capability, endowing the catalyst with good CO2 RR performance for stable formate production with high Faradaic efficiency (≈90%) and current density (>300 mA cm-2 ). Theoretical calculation verifies the superior intermediate stabilization of the dominant Bi plane during CO2 RR. Moreover, by further coupling anodic methanol oxidation reaction, an exotic electrolytic system enables highly energy-efficient and value-added pair-electrosynthesis for concurrent formate production at both electrodes, achieving substantially improved electrochemical and economic efficiency and revealing the feasibility for practical implementation.

A space-sacrificed pyrolysis strategy for boron-doped carbon spheres with high supercapacitor performance.

Targeting the potential application of morphological carbon in electrode materials, a space-sacrificed pyrolysis strategy was applied for the preparation of boron-doped carbon spheres (B-CSs), using commercial triphenyl borate (TPB) as carbon and boron co-source. The unique structure of TPB play an important role in the sacrificed space, and has notable effect on the surface area of B-CSs. The as prepared B-CSs possess a high surface area and boron content with uniform boron atoms distribution and high surface polarity, which contributes to the improvement of pseudo-capacitance. The sizes, specific surface areas, and boron contents of B-CSs can be easily regulated by varying the experimental parameters. The optimal sample has a boron content of 1.38 at%, surface area of 560 m2 g-1 and specific capacitance of 235F g-1. We can believe that this work would provide a flexible and extensible preparation technique of B-CSs for electrochemical applications.

Atomically Structural Regulations of Carbon-Based Single-Atom Catalysts for Electrochemical CO2 Reduction.

The electrochemical carbon dioxide reduction reaction (CO2 RR) converting CO2 into value-added chemicals and fuels to realize carbon recycling is a solution to the problem of renewable energy shortage and environmental pollution. Among all the catalysts, the carbon-based single-atom catalysts (SACs) with isolated metal atoms immobilized on conductive carbon substrates have shown significant potential toward CO2 RR, which intrigues researchers to explore high-performance SACs for fuel and chemical production by CO2 RR. Especially, regulating the coordination structures of the metal centers and the microenvironments of the substrates in carbon-based SACs has emerged as an effective strategy for the tailoring of their CO2 RR catalytic performance. In this review, the current in situ/operando study techniques and the fundamental parameters for CO2 RR performance are first briefly presented. Furthermore, the recent advances in synthetic strategies which regulate the atomic structures of the carbon-based SACs, including heteroatom coordination, coordination numbers, diatomic metal centers, and the microenvironments of substrates are summarized. In particular, the structure-performance relationship of the SACs toward CO2 RR is highlighted. Finally, the inevitable challenges for SACs are outlined and further research directions toward CO2 RR are presented from the perspectives.

Imidacloprid and thiamethoxam affect synaptic transmission in zebrafish.

Imidacloprid (IMI) and thiamethoxam (THM) are two commonly applied neonicotinoid insecticides. IMI and THM could cause negative impacts on non-target organisms like bees. However, the information about neurotoxicity of IMI and THM in fish is still scarce. Here we investigated the effects of IMI and THM on locomotor behavior, AChE activity, and transcription of genes related to synaptic transmission in zebrafish exposed to IMI and THM with concentrations of 50 ng L-1 to 50,000 ng L-1 at 14 day post fertilization (dpf), 21 dpf, 28 dpf and 35 dpf. Our results showed that IMI and THM significantly influenced the locomotor activity in larvae at 28 dpf and 35 dpf. THM elevated AChE activity at 28 dpf. The qPCR data revealed that IMI and THM affected the transcription of marker genes belonging to the synapse from 14 dpf to 35 dpf. Furthermore, IMI and THM mainly affected transcription of key genes in γ-aminobutyric acid, dopamine and serotonin pathways in larvae at 28 dpf and 35 dpf. These results demonstrated the neurotoxicity of IMI and THM in zebrafish. The findings from this study suggested that IMI and THM in the aquatic environment may pose potential risks to fish fitness and survival.

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.