Bisphenol A Analogues Induce Neuroendocrine Disruption via Gut-Brain Regulation in Zebrafish.

There is epidemiological evidence in humans that exposure to endocrine-disrupting chemicals such as bisphenol A (BPA) is tied to abnormal neuroendocrine function with both behavioral and intestinal symptoms. However, the underlying mechanism of this effect, particularly the role of gut-brain regulation, is poorly understood. We exposed zebrafish embryos to a concentration series (including environmentally relevant levels) of BPA and its analogues. The analogue bisphenol G (BPG) yielded the strongest behavioral impact on zebrafish larvae and inhibited the largest number of neurotransmitters, with an effective concentration of 0.5 µg/L, followed by bisphenol AF (BPAF) and BPA. In neurod1:EGFP transgenic zebrafish, BPG and BPAF inhibited the distribution of enteroendocrine cells (EECs), which is associated with decreased neurotransmitters level and behavioral activity. Immune staining of ace-α-tubulin suggested that BPAF inhibited vagal neural development at 50 and 500 µg/L. Single-cell RNA-Seq demonstrated that BPG disrupted the neuroendocrine system by inducing inflammatory responses in intestinal epithelial cells via TNFα-trypsin-EEC signaling. BPAF exposure activated apoptosis and inhibited neural developmental pathways in vagal neurons, consistent with immunofluorescence imaging studies. These findings show that both BPG and BPAF affect the neuroendocrine system through the gut-brain axis but by different mechanisms, revealing new insights into the modes of bisphenol-mediated neuroendocrine disruption.

Life cycle assessment of hepatotoxicity induced by cyhalofop-butyl in environmental concentrations on zebrafish in light of gut-liver axis.

Cyhalofop-butyl (CB) poses a significant threat to aquatic organisms, but there is a discrepancy in evidence about hepatotoxicity after prolonged exposure to environmental levels. The aim of this study was to investigate long-term hepatotoxicity and its effects on the gut-liver axis through the exposure of zebrafish to environmental concentrations of CB (0.1,1,10 µg/L) throughout their life cycle. Zebrafish experienced abnormal obesity symptoms and organ index after a prolonged exposure of 120 days. The gut-liver axis was found to be damaged both morphologically and functionally through an analysis of histology, electron microscopy subcellular structure, and liver function. The disruption of the gut-liver axis inflammatory process by CB is suggested by the rise in inflammatory factors and the alteration of inflammatory genes. Furthermore, there was a noticeable alteration in the blood and gut-liver axis biochemical parameters as well as gene expression linked to lipid metabolism, which may led to an imbalance in the gut flora. In conclusion, the connection between the gut-liver axis, intestinal microbiota, and liver leads to the metabolic dysfunction of zebrafish exposed to long-term ambient concentrations of CB, and damaged immune system and liver lipid metabolism. This study gives another knowledge into the hepatotoxicity component of long haul openness to ecological centralization of CB, and might be useful to assess the potential natural and wellbeing dangers of aryloxyphenoxypropionate herbicides.

Effects of penthiopyrad on the hypothalamic-pituitary-thyroid (HPT) axis in zebrafish.

Exposure to specific pesticides has been demonstrated to alter normal thyroid function of aquatic vertebrates. This study aimed to investigate the impact of penthiopyrad (PO) on the thyroid function of zebrafish, further elucidating its toxic mechanisms on the early developmental stages of zebrafish. Exposure to sublethal doses of PO (0.3-1.2 mg/L) for 8 days from 2 h after fertilization resulted in a significant reduction in larval swim bladder size and body weight, accompanied by developmental abnormalities such as pigment deposition and abnormal abdominal development. Perturbations in the hypothalamic-pituitary-thyroid (HPT) axis in larvae manifested as a marked upregulation of crh, tg, ttr, and ugt1ab expression, alongside downregulation of trß expression, culminating in elevated thyroxine (T4) and triiodothyronine (T3) levels. Additionally, molecular docking results suggest that PO and its metabolites may disrupt the binding of thyroid hormones to thyroid hormone receptor beta (TRß), compromising the normal physiological function of TRß. These findings highlight the PO-induced adverse effects on the HPT axis of larvae under sublethal doses, eventually leading to abnormal development and growth inhibition.

Neural System Impairment and Involved Microglia-Neuron Regulation of Broflanilide in Zebrafish Larvae.

Broflanilide is widely used to control pests and has attracted attention due to its adverse effects on aquatic organisms. Our previous study showed that broflanilide has a negative impact on the central nervous system (CNS) at lethal dosages; however, its neural effects under practical situations and the underlying mechanisms remain unknown. To elucidate how broflanilide affects the CNS, we exposed zebrafish larvae to broflanilide at 16.9 and 88.0 µg/L (the environmentally relevant concentrations) for 120 h. Zebrafish locomotion was significantly disturbed at 88.0 µg/L, with a decreased moving distance and velocity accompanied by an inhibited neurotransmitter level. In vivo neuroimaging analysis indicated that the nerves of zebrafish larvae, including the axons, myelin sheaths, and neurons, were impaired. The number of neurons was significantly reduced after exposure, with an impaired morphological structure. These changes were accompanied by the abnormal transcription of genes involved in early CNS development. In addition, an increased total number of microglia and an elevated proportion of amoeboid microglia were observed after 88.0 µg/L broflanilide exposure, pointing out to an upstream role of microglia activation in mediating broflanilide neurotoxicity. Meanwhile, increased inflammatory cytokine levels and brain neutrophil numbers were observed, implicating significant inflammatory response and immune toxicity. Our findings indicate that broflanilide interferes with microglia-neuron regulation and induces neurodevelopmental disorders.

Research into the correlation between positional skull deformation and motor performance of infants aged under 4 months.

OBJECTIVE: To investigate the correlation between positional skull deformation (PD) and motor performance of infants under 4 months of age. METHODS: Infants aged under 4 months were enrolled in the children's healthcare and the premature infants follow-up Clinic of the Second Affiliated Hospital of Army Military Medical University. The cranial vault asymmetry (CVA) and cephalic index (CI) were calculated in all infants, and the infant motor performance test (TIMP) was used to evaluate the infant motor performance. The motor performances of infants with different types and degrees of PD were compared, so were the incidences of PD in infants with different motor performance levels. RESULTS: Overall, 2118 infants were recruited and divided according to the types of PD and TIMP scores. The comparison of TIMP scores within different types of PD at different months of age showed that, regardless of the types of PD, TIMP scores of infants with PD were lower than those of normal infants. In particular, the difference in TIMP scores was statistically significant (P < 0.05) in infants with dolichocephaly, plagiocephaly,dolicho-plagiocephaly and brachy-plagiocephy. In addition, the comparison of CVA values of infants with different TIMP score levels at different months of age showed that the CVA values of the extremely low-level group were significantly higher than those of the medium-level and high-level group, especially in the 3-month-old and 4-month-old groups, which showed significant statistical differences (P < 0.05). CONCLUSIONS: PD and motor performance of infants aged under 4 months seem to interact and influenc each other. The more serious the severity of PD were,the worse the motor performance of infants. Conversely, the incidence of PD increased in infants with poor motor performance.

Effects of virtual reality on preoperative anxiety in children: A systematic review and meta-analysis of randomised controlled trials.

AIMS: To synthesise and evaluate the effectiveness of virtual reality interventions in preoperative children. BACKGROUND: Children consider operations as a predictable threat and stressful event. Children's anxiety before an operation increases as the time draws closer. Children could understand the operating room environment and process before the operation using virtual reality, which may reduce their anxiety before an operation. DESIGN: A systematic review and meta-analysis of randomised controlled trials following the Cochrane method were conducted. METHOD: CINAHL, Cochrane Library, Embase, Joanna Briggs Institute, MEDLINE and PubMed databases were searched for randomised controlled trials published before February 2021. A random-effects model meta-analysis to calculate pooled prevalence and 95% confidence intervals was performed. Conduction of the review adheres to the PRISMA checklist. RESULTS: Of 257 articles screened, six interventions involving 529 participants aged 4-12 years were included in the analysis. All study evidence levels were B2/Level 2, the quality was medium to high on the modified Jadad scale, with a low risk of bias. The results revealed that virtual reality significantly reduced preoperative anxiety in children (SMD: -0.91, 95% CI: -1.43 to -0.39, p = .0006). Furthermore, virtual reality significantly improved children's compliance with anaesthesia (SMD: 3.49, 95% CI: 1.32 to 9.21, p = .01). CONCLUSION: Children who used virtual reality before an operation felt more familiar with the operating room environment and understood the preoperative preparation procedures. Virtual reality effectively reduced children's anxiety and improved their compliance with anaesthesia. RELEVANCE TO CLINICAL PRACTICE: This systematic review and meta-analysis investigated the effect of virtual reality on preoperative anxiety in children and the findings supported its positive effects. The results could provide a reference for incorporating virtual reality into preoperative preparation guidelines.

Effects of Broflanilide on Oxidative Stress and Expression of Apoptotic Genes in Zebrafish (Danio rerio) Gill.

Broflanilide exerted negative impacts on the gill of zebrafish. Thus, in this study, zebrafish gill was used to assess the apoptosis toxicity of broflanilide by determining the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) and apoptosis-related genes. The results found that the minimum threshold for the content and time of broflanilide affecting enzyme content and gene expression was 0.26 mg/L after 24 h exposure. After 96 h exposure, broflanilide could cause apoptosis and exerted significantly increased contents of ROS and MDA, while inhibiting the activities of SOD, CAT, and GPx at 0.26 and 0.57 mg/L. Broflanilide also had adverse effects on apoptosis-related genes, such as tumor protein p53 (p53), associated × (Bax), B-cell lymphama-2 (Bcl-2), caspase-3, caspase-9, and apoptotic protease activating factor-1(apaf-1), at 0.26 mg/L and 0.57 mg/L after 96 h exposure, respectively. These results provide new insight into the potential toxicity mechanisms of broflanilide in zebrafish gills.

Bisphenol F Impaired Zebrafish Cognitive Ability through Inducing Neural Cell Heterogeneous Responses.

The central nervous system (CNS) is a sensitive target for endocrine-disrupting chemicals, such as bisphenol analogues. Bisphenol A (BPA) usage is associated with the occurrence of many neurological diseases. With the restricted use of BPA, bisphenol F (BPF) has been greatly introduced for industrial manufacture and brings new hazards to public CNS health. To understand how BPF affects the neural system, we performed a cognitive test for zebrafish that are continuously exposed to environmentally relevant concentrations (0.5 and 5.0 µg/L) of BPF since embryonic stage and identified suppressed cognitive ability in adulthood. Single-cell RNA sequencing of neural cells revealed a cell composition shift in zebrafish brain post BPF exposure, including increase in microglia and decrease in neurons; these changes were further validated by immune staining. At the same time, a significant inflammatory response and increased phagocytic activity were detected in zebrafish brain post BPF exposure, which were consistent with the activation of microglia. Cell-specific transcriptomic profiles showed that abnormal phagocytosis, activated brain cell death, and apoptosis occurred in microglia post BPF exposure, which are responsible for the neuron loss. In addition, certain neurological diseases were affected by BPF in both excitatory and inhibitory neurons, such as the movement disorder and neural muscular disease, however, with distinctly involved genes. These findings indicate that BPF exposure could lead to an abnormal cognitive behavior of zebrafish through inducing heterogeneous changes of neural cells in brain and revealed the dominating role of microglia in mediating this effect.

Protective effects of 5-aminolevulinic acid against toxicity induced by alpha-cypermethrin to the liver-gut-microbiota axis in zebrafish.

To explore whether and how 5-aminolevulinic acid (ALA) can relieve the toxicity to the liver-gut-microbiota axis caused by alpha-cypermethrin (α-CP), adult zebrafish were exposed to α-CP (1.0 µg L-1) with or without 5.0 mg L-1 ALA supplementation. In the present work, the calculated LC50 of α-CP+ALA was 1.15 µg L-1, increasing about 1.16-fold compared with that of α-CP group (0.99 µg L-1), which indicated that ALA can alleviate the toxicity of α-CP. ALA also alleviated the histopathological lesions in the liver and gut induced by α-CP. Transcriptome sequencing of the liver showed that ALA rescues the differential expression of genes involved in the oxidation-reduction, heme metabolism, and complement activation pathways associated with dysfunctions induced by α-CP, and these findings were verified by RT-qPCR analysis and detection of the activities of enzymes in the liver-gut axis. The gut microbiota 16S rRNA sequencing results showed that α-CP alone induced gut microbial dysbiosis, which was efficiently antagonized by ALA due to decreasing the relative abundances of Cetobacterium and 3 major pathogens, and increasing the relative abundances of beneficial genera. Taken together, the results indicate that ALA might be a promising candidate for attenuating the adverse effects caused by pesticide-induced environmental pollution.

Quizalofop-P-ethyl induced developmental toxicity and cardiotoxicity in early life stage of zebrafish (Danio rerio).

Quizalofop-P-ethyl (QpE), a highly efficient selective herbicide, has good control effect on annual and perennial weeds. However, its excessive use will pose a threat to the ecological environment. QpE has been proven harmful to aquatic organisms, but there is little evidence on the adverse effects of QpE in the early life of aquatic organisms. In this work, zebrafish (Danio rerio) embryos were treated with 0.10, 0.20, 0.30, 0.40, and 0.50 mg/L of QpE for 120 h. The findings revealed that the LC50 value of QpE to zebrafish embryos was 0.23 mg/L at 96 hpf. QpE exposure significantly increased the mortality rate, decreased the hatching rate and caused morphological defects during zebrafish embryonic development, with a concentration dependent manner. QpE also caused severe morphological changes in the cardiovascular system, as well as resulted in a dysfunction in cardiovascular performance. Meanwhile, both histopathological examination and neutrophil observations showed inflammatory response occurred in the heart. Furthermore, several genes associated with heart development and inflammation were significantly altered following QpE exposure. A protein-protein interaction (PPI) network analysis proved that there was a connection between the changed heart development-relevant and inflammation-related genes. Taken together, our findings suggest that QpE causes cardiotoxicity in zebrafish embryos by altering the expression of genes in the regulatory network of cardiac development, which might be aggravated by inflammatory reactions, thereby affecting embryo development. These findings generated here are useful for in-depth assessment of the effects of QpE on early development of aquatic organisms and providing theoretical foundation for risk management measures.

Parental exposure to propiconazole at environmentally relevant concentrations induces thyroid and metabolism disruption in zebrafish (Danio rerio) offspring: An in vivo, in silico and in vitro study.

Propiconazole is used against fungal growth in agriculture and is released into the environment, but is a potential health threat to aquatic organisms. Propiconazole induces a generational effect on zebrafish, although the toxic mechanisms involved have not been described. The aim of this study was to investigate the potential mechanisms of abnormal offspring development after propiconazole exposure in zebrafish parents. Zebrafish were exposed to propiconazole at environmentally realistic concentrations (0.1, 5, and 250 µg/L) for 100 days and their offspring were grown in control solution for further study. Heart rate, hatching rate, and body length of hatched offspring were reduced. An increase in triiodothyronine (T3) content and the T3/T4 (tetraiodothyronine) ratio was observed, indicating disruption of thyroid hormones. Increased protein level of transthyretin (TTR) in vivo was consistent with the in silico molecular docking results and T4 competitive binding in vitro assay, suggests higher binding affinity between propiconazole and TTR, more than with T4. Increased expression of genes related to the hypothalamus-pituitary-thyroid (HPT) axis and altered metabolite levels may have affected offspring development. These findings emphasizes that propiconazole, even on indirect exposure, represents health and environmental risk that should not be ignored.

Analysis of cranial type characteristics in term infants: a multi-center study.

BACKGROUND: Positional head deformity (PHD) is defined as a change in the shape of an infant's skull due to an external force. In certain cases, it can lead to cosmetic deformities or even neurological issues due to its impact on the developing nervous system. Therefore, we conducted this study to investigate the incidence and characteristics of PHD in term infants in China and preliminarily establish a localized diagnostic reference standard. METHODS: Overall, 4456 term infants from three medical institutions in Chongqing were and divided and analyzed according to their age. Cranial vault asymmetry (CVA) and cephalic index (CI) were calculated in all infants. The current international diagnostic criteria were used to understand PHD incidence and analyze the CVA and CI distribution. RESULTS: According to the current international standards, the total detection rate of PHD in Chongqing's term infants was 81.5%, with brachycephaly alone being the most frequent (39.4%), followed by brachycephaly with plagiocephaly (34.8%) and plagiocephaly alone (6.2%). The detection rates of dolichocephaly were low: alone, 0.9% and combined with plagiocephaly, 0.2%. According to age, plagiocephaly (44.5%) and brachycephaly (82.0%) were the most frequent in the 2-3-month group. The 75th/90th/97th and 3rd/10th/25th/75th/90th/97th percentiles of CVA and CIs were 0.4/0.7/1.0 and 76.4/78.8/82.3/91.1/94.6/99.2%, respectively. CONCLUSIONS: According to the current international standards, the PHD detection rate among term infants in Chongqing was high. Therefore, a new diagnostic standard for Chinese infants was proposed where CVA ≥ 0.4 cm indicates plagiocephaly, CI ≥ 91% indicates brachycephaly, and CI ≤ 82% indicates dolichocephaly.

Environmentally relevant concentrations of tralopyril affect carbohydrate metabolism and lipid metabolism of zebrafish (Danio rerio) by disrupting mitochondrial function.

Tralopyril (TP), an antifouling biocide, is widely used to prevent heavy biofouling, and can have potential risks to aquatic organisms. However, there is little information available on the toxicity of tralopyril to aquatic organisms. In this study, the effect of TP on carbohydrate and lipid metabolism, and related mechanisms were evaluated in zebrafish (Danio rerio) larvae. Adverse modifications in carbohydrate metabolism were observed in larvae: hexokinase (HK) activity, succinate dehydrogenase (SDH) activity, and adenosine triphosphate (ATP) content were significantly decreased; and transcript expression of genes (GK, HK1, and PCK1) was also significantly changed. Changes of TG content, FAS activity and transcript expression of genes (ACO, ehhadh, and fas) indicate that TP disrupt lipid metabolism in zebrafish larvae. The change in expression of genes (ndufs4, Sdhα, and uqcrc2) involved in the mitochondrial respiratory complexes, and genes (polg1 and tk2) involved in the mitochondrial DNA replication and transcription indicates that these adverse effects on carbohydrate and lipid metabolism are caused by mitochondrial dysfunction.

[Interpretation of the international expert recommendations of clinical features to prompt referral for diagnostic assessment of cerebral palsy].

Under the guidance and support of national policies in recent years, the community medical system has been developed rapidly, among which primary child healthcare is carried out routinely in community hospitals, greatly alleviating the pressure of specialized pediatric hospitals and departments of pediatrics in secondary and tertiary general hospitals. However, due to the lack of professional training for primary child healthcare personnel in community medical institutions, early symptoms of children with cerebral palsy cannot be identified and so children with cerebral palsy are often unable to receive early diagnosis and intervention, which may affect their prognosis. An article about international expert consensus and recommendations on early identification and referral of cerebral palsy in community medical institutions was published in Development Medicine and Child Neurology in 2020. It proposed six clinical features that should prompt referral and two warning signs that warrant enhanced monitoring, as well as five recommendations for referral to medical experts and other healthcare professionals for the diagnosis of cerebral palsy. The recommendations may help primary child healthcare personnel in community medical institutions to early identify the children at high risk of cerebral palsy, thus reducing the delay of referral and intervention. This article gives an interpretation of the recommendations based on the actual situation in China, in order to improve the ability of primary child healthcare personnel in community medical institutions to early identify high-risk signals of cerebral palsy and conduct reasonable referral. This will help to achieve the early identification, early diagnosis, and early intervention to improve the prognosis of children with cerebral palsy.

Exposure to Boscalid Induces Reproductive Toxicity of Zebrafish by Gender-Specific Alterations in Steroidogenesis.

Boscalid is a succinate dehydrogenase inhibitor fungicide and is frequently detected in surface water. Due to the frequent detection of boscalid, we evaluated its impact on the reproduction of adult zebrafish following a 21 d exposure to 0, 0.01, 0.1, and 1.0 mg/L. Following exposure to boscalid, the fertility of female zebrafish and fertilization rate of spawning eggs were reduced in a concentration-dependent manner up to a respective 87% and 20% in the highest concentration. A significant 16% reduction in the percentage of late vitellogenic oocytes was noted in ovaries, and a significant 74% reduction in the percentage of spermatids in testis was also observed after treatment with 1.0 mg/L. 17ß-Estradiol (E2) concentrations decreased significantly in females (34% decrease) but significantly increased in males (15% increase) following 1.0 mg/L boscalid treatment. The expression of genes (such as era, er2b, cyp19a, and cyp19b) related to the hypothalamus-pituitary-gonad-liver (HPGL) axis was significantly altered and positively correlated with E2 concentrations in female and male zebrafish (p < 0.05). Molecular docking results revealed that the binding modes between boscalid and target proteins (ER and CYP19) of zebrafish were similar to that of the reference compounds and the target proteins. The binding energies indicate that boscalid may have a weak estrogen-like binding effect or CYP19 inhibition, potentially altering the HPGL axis, thereby reducing E2 concentrations and fecundity in females. In contrast, boscalid caused significant induction of E2 steroidogenesis and subsequent feminization of gonads in males, indicating gender-specific adverse outcome pathways.

Chronic exposure of zebrafish (Danio rerio) to flutolanil leads to endocrine disruption and reproductive disorders.

The pesticide flutolanil has been detected in fish and aquatic environments, but its potential impact on the endocrine function is unknown. In this study, two-month zebrafish were exposed to the environmentally relevant concentrations of flutolanil for 60 days to examine the reproductive endpoints on the gonad endocrine system. Increased 17 beta-estradiol (17ß-E2) content and 17ß-E2/T ratio and decreased testosterone (T) in male suggested that flutolanil produces the estrogenic effect. In support of this view, vitellogene (vtg1, vtg2) and cytochrome P450 aromatase 19a (cyp19a) expression were up-regulated in the male liver. The levels of global DNA methylation were increased in ovary. Parental zebrafish exposure to different concentrations of flutolanil affected the offspring development as shown by short body length, and increased mortality. Thus, these results demonstrate that flutolanil exposure results in gonad endocrine disruption, decreased reproduction, and developmental toxicity in F1, highlighting the importance of assessing the potential environmental risks of flutolanil application.

Computational and experimental investigations on the interactions of aryloxy-phenoxy-propionate herbicides to estrogen receptor alpha in zebrafish.

When the amount of pesticide exceeds the self-purification ability of the environment, it will be enriched in the human body through the atmosphere, soil, water circulation, etc., threatening human health. Aryloxy-phenoxy-propionate (APP) herbicides are a class of acetyl-CoA carboxylase (ACCase) inhibitor herbicides, widely used in field-weeding of soybean, cabbage, peanut and other crops. However, due to the water circulation, surface runoff and the agronomic practices such as watering irrigation, APP herbicides have the risk of polluting water and destroying the living environment of aquatic organisms. In this paper, a multistep framework combining homology modeling, molecular docking and molecular dynamic simulations were adopted to explore the interactions between APP herbicides and zebrafish estrogen receptor α (ERα) to investigate the estrogenic activities of the herbicides. The structure of zebrafish ERα was modeled by homology modeling, using the human's estrogen receptor α (PDB ID:2YJA) as the template. Then, eight typical APP herbicides were selected to dock with the zebrafish ERα, and it was determined that there were clear interactions between the herbicides and the receptor. The binding patterns of Quizalofop-P-ethyl (QPE), Clodinafop-propargyl (CP) and Haloxyfop-P (HP) with ERα were further investigated by molecular dynamics and binding free energy calculation. The results showed the van der Waals force and electrostatic force were the main driving forces for maintaining the stability of the complex system. In order to verify the theoretical prediction, an exposed experiment was conducted to study the effects of different concentrations of herbicides on VTG level of zebrafish in vivo and the results were consistent with the computational method. The results of this study revealed the mechanism of the action between APP herbicides and zebrafish estrogen receptors, and also provided ideas for optimizing the herbicides.

Transcriptome analysis of Spodoptera litura reveals the molecular mechanism to pyrethroids resistance.

Spodoptera litura is a destructive agricultural pest and has evolved resistance to multiple insecticides, especially pyrethroids. At present, the resistance mechanism to pyrethroids remains unclear. Four field-collected populations, namely CZ, LF, NJ and JD, were identified to have high resistance to pyrethroids comparing to pyrethroid-susceptible population (GX), with resistant ratio ranging from 11.5- to 9123.5-fold. To characterize pyrethroid resistance mechanism, the transcriptomes between two pyrethroid-resistant (LF and NJ) and a pyrethroid-susceptible (GX) populations were compared by RNA-sequencing. Results showed that multiple differentially expressed genes were enriched in metabolism-related GO terms and KEGG pathways. 35 up-regulated metabolism-related unigenes were selected to verify by qRT-PCR and 15 unigenes, including 4 cytochrome P450s (P450s), 5 glutathione S-transferase (GSTs), 1 UDP-glycosyltransferase (UGT), 4 carboxylesterases (COEs) and 1 and ATP-binding cassette transporters (ABC), were all up-regulated in the four pyrethroid-resistant populations. The expression levels of CYP3 and GST3, which were annotated as CYP6A13 and GSTE1, respectively, showed positive correlation with their pyrethroid resistance levels among the four pyrethroid-resistant populations. While the expression levels of CYP5, CYP12, COE4 and ABC5 showed good correlation with their pyrethroid resistance levels in at least three populations. UGT5 had the highest expression level among the tested UGT genes in the four pyrethroid-resistant populations. RNAi mediated silencing of CYP6 increased the cumulative mortality treated by beta cypermethrin and cyhalothrin significantly, while silencing of GST3 increased the cumulative mortality treated by fenvalerate significantly. CYP3, CYP5, CYP6, CYP12, GST3, COE4, UGT5 and ABC5 play important roles in pyrethroid resistance among the four pyrethroid-resistant populations. Our work provides a valuable clue for further study of pyrethroid resistance mechanisms in S. litura.

Chronic Toxic Effects of Flutolanil on the Liver of Zebrafish ( Danio rerio).

Flutolanil is a broad-spectrum amide fungicide that is widely used to prevent fungal pathogens in agriculture. However, its usage may have a potential environmental impact on organisms. So far, few literatures have investigated the chronic toxicity of flutolanil at concentrations relevant to environmental conditions in the nontarget aquatic organisms. This study was aimed at evaluating whether the long-term exposure of flutolanil affects oxidative stress, immune response, and apoptosis in the liver of zebrafish ( Danio rerio). The results showed that the activity of catalase (CAT) was significantly decreased in the liver in all flutolanil-treated groups. Interestingly, the malondialdehyde (MDA) contents were remarkably increased following the flutolanil exposure. Deoxyribonucleic acid (DNA) damage was increased with a concentration-dependent manner. The transcription level of genes involved in apoptosis and the immune system were significantly altered following flutolanil chronic exposure in zebrafish liver. Furthermore, the caspase-3 enzyme activity was significantly increased. Taken together, this study demonstrated that the resulting effects on oxidative stress, immune toxicity, and apoptosis may be responsible for the pathological alterations in zebrafish liver after flutolanil exposure at concentrations relevant to environmental conditions, advancing the knowledge of pesticide environmental risk assessment.

Long-Term Exposure to Environmental Concentrations of Azoxystrobin Delays Sexual Development and Alters Reproduction in Zebrafish ( Danio rerio).

The strobilurin fungicide azoxystrobin (AZO) can induce adverse effects in aquatic organisms, but data are lacking on endpoints associated with sexual development and reproduction following chronic exposure to AZO. In this study, zebrafish embryos (F0) at 2-4 h postfertilization (hpf) were exposed to 0.2, 2.0, and 20.0 µg/L AZO until 120 d postfertilization (dpf). Decreased male ratio and increased intersex ratio were observed by 20.0 µg/L AZO at 42 and 60 dpf, but this effect disappeared at 120 dpf. AZO at 20.0 µg/L inhibited growth, retarded gonadal development, and disrupted sex hormone and vitellogenin in females at 60 and 120 dpf and in males at 42, 60, and 120 dpf. These effects were associated with altered expression of cyp19a, cyp19b, hsd3b, hsd17b, vtg1, and vtg2. Exposure to 2.0 µg/L AZO altered mRNA levels of these transcripts in females at 120 dpf and in males at 60 and 120 dpf. Reproduction ability was reduced by 20.0 µg/L AZO at 120 dpf. Developmental defects were observed after F1 embryos from exposed parents of 20.0 µg/L were reared in AZO-free water at 96 hpf. Overall, these data provide new understanding of fish sexual development and reproduction following chronic exposures to AZO.