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Although sex determination and differentiation are key developmental processes in animals, the involvement of non-coding RNA in the regulation of this process is still not clarified. The tiger pufferfish ( Takifugu rubripes ) is one of the most economically important marine cultured species in Asia, but analyses of miRNA and long non-coding RNA (lncRNA) at early sex differentiation stages have not been conducted yet. In our study, high-throughput sequencing technology was used to sequence transcriptome libraries from undifferentiated gonads of T. rubripes . In total, 231 (107 conserved, and 124 novel) miRNAs were obtained, while 2774 (523 conserved, and 2251 novel) lncRNAs were identified . Of these, several miRNAs and lncRNAs were predicted to be the regulators of the expression of sex-related genes (including fru-miR-15b/ foxl2 , novel-167, novel-318, and novel-538/ dmrt1 , novel-548/ amh , lnc_000338, lnc_000690, lnc_000370, XLOC_021951, and XR_965485.1/ gsdf ). Analysis of differentially expressed miRNAs and lncRNAs showed that three mature miRNAs up-regulated and five mature miRNAs were down-regulated in male gonads compared to female gonads, while 79 lncRNAs were up-regulated and 51 were down-regulated. These findings could highlight a group of interesting miRNAs and lncRNAs for future studies and may reveal new insights into the function of miRNAs and lncRNAs in sex determination and differentiation.

Background Takifugu rubripes is an economically valuable fish species in Asia. The implementation of all-male culture for T. rubripes is highly anticipated in aquaculture. Aromatase inhibitor (AI, letrozole) treatment was found to be an efficient method to induced masculinization in T. rubripes , as reported in our previous study. Here, to further explore the underlying regulation mechanism of AI-induced masculinization, a whole-transcriptome analysis comparing was conducted between AI-induced masculinized XX (AI-XX) gonads and control (Con) gonads in T. rubripes . Results In Con-XX/Con-XY comparison, 1,172 differential expression (DE) mRNAs, 129 DEmiRNAs, 210 DElncRNAs, and 4 DEcircRNAs were identified. In the Con-XX/AI-XX comparison, 1,329 DEmRNAs, 174 DEmiRNAs, 6 DEcircRNAs and 280 DElncRNAs were found. Con-XX/Con-XY and Con-XX/AI-XX comparisons shared 690 DEmRNAs, 50 DEmiRNAs, 3 DEcircRNAs, and 105 DElncRNAs. The analyses of protein-protein interaction (PPI) and competitive endogenous RNA (ceRNA) network identified interactions among these shared DERNAs. Kcnh2b , trim27 , cnnm2b , reln , cckbra , pkd1l2 , steap4 , gsg1l , hamp , and foxg1c were predicted as the top ten of hub genes. miRNAs included miRNA-27 family and miRNA-489 family showed targeting relationship with hub genes. GO and KEGG functional enrichment analysis showed that the targeted genes were mainly enriched in GO:0065008 regulation of biological quality and TGF-beta signaling pathway. qPCR validation confirmed the differential expression of selected mRNAs, and ncRNAs. Conclusions This research comprehensively reveals the potential regulatory effects of ncRNAs on cellular motility, fate regulation, and hormonal regulation during gonadal masculinization in T. rubripes . It may provide significant insights into the regulation mechanisms underlying sex reversal in fish.

Background As the critical tissue of the central nervous system, the brain has been found to be involved in gonad development. Previous studies have suggested that gonadal fate may be affected by the brain. Identifying brain-specific molecular changes that occur during estrodiol-17β (E 2 ) -induced feminization is crucial to our understanding of the molecular control of sex differentiation by the brains of fish. Results In this study, the differential transcriptomic responses of the Takifugu rubripes larvae brain were compared after E 2 treatment for 55 days. Our results showed that 514 genes were differentially expressed between E 2 -treated-XX (E-XX) and Control-XX (C-XX) T. rubripes , while 362 genes were differentially expressed between E 2 -treated-XY (E-XY) and Control-XY (C-XY). For example, the expression of cyp19a1b , gnrh1 and pgr was significantly up-regulated, while st, sl, tshβ, prl and pit-1, which belong to the growth hormone/prolactin family, were significantly down-regulated after E 2 treatment, in both sexes. The arntl1 , bhlbe , nr1d2 , per1b , per3 , cry1 , cipc and ciart genes, which are involved in the circadian rhythm, were also found to be altered. Differentially expressed genes (DEGs), which were identified between E-XX and C-XX, were significantly enriched in neuroactive ligand-receptor interaction, arachidonic acid metabolism, cytokine-cytokine receptor interaction and the calcium signaling pathway. The DEGs that were identified between E-XY and C-XY were significantly enriched in tyrosine metabolism, phenylalanine metabolism, arachidonic acid metabolism and linoleic acid metabolism. Conclusion A number of genes and pathways were identified in the brain of E 2 -treated T. rubripes larvae by RNA-seq. It provided the opportunity for further study on the possible involvement of networks in the brain-pituitary-gonadal axis in sex differentiation in T. rubripes .

Background The regulatory roles of non-coding RNAs (ncRNAs) during sex differentiation in teleosts have received widespread attention recently. Mifepristone (RU486, a progesterone antagonist), which acts as an endocrine disruptor, can affect reproduction and sex differentiation in teleosts. Results The expression of ncRNAs in the gonads of tiger puffer ( Takifugu rubripes ) during RU486 (500 µg/g diet) induced masculinization process was examined by RNA-sequencing. A total of 4,381 long non-coding RNAs (lncRNAs), 309 circular RNAs (circRNAs), and 1,020 microRNAs (miRNAs) were identified. The expression of 41 differentially expressed (DE) lncRNAs and 20 DE miRNAs, which showed sexual dimorphic expression patterns in genetic female gonads in the control group (C-XX) vs. genetic male gonads in the control group (C-XY), were altered in genetic female gonads in the RU486 treated group (RU-XX). The genes targeted by DE ncRNAs were mainly enriched in sex-related pathways, such as calcium signaling, ovarian steroidogenesis, and cortisol synthesis and secretion. The results of co-expression and competing endogenous RNA (ceRNA) network analysis indicated that miRNAs (e.g., miR-205-z and fru-miR-122) and lncRNAs (including XR_003890915.1 and XR_003885862.1) may have pivotal roles, and lncRNAs (including XR_003890295.1, MSTRG.11750.1, and XR_003888827.1) may act as miRNA sponges, involved in the competition between miRNAs and sex-related genes during tiger puffer masculinization process. Dual luciferase reporter assay results identified that ovarian steroidogenesis related gene hsd17b1 is a downstream target of fru-miR-122. The expression of 4 lncRNAs, 4 circRNAs, and 6 miRNAs were validated by qPCR, indicating the accuracy and dependability of RNA-Seq. Conclusions This study provided the evidence that ncRNAs may participate in RU486-induced masculinization in T. rubripes , and may enhance our understanding of the regulatory network of sex differentiation in fugu.

Light is a key environmental parameter known to influence fish throughout various stages of their life, from embryonic development to sexually mature adults. In a recent study, the effects of different light conditions on the growth of Dicentrarchus labrax larvae were investigated using light-emitting diodes (LEDs) as a light source. Here, pathological examinations were carried out to assess whether variations in light affected the visual system of the larvae, including any negative impacts on the retina or the growth rate. Although light did not affect the total thickness (TT) of the retina, the thickness of the retinal pigment epithelium layer (PRE), photoreceptor layer (PRos/is), outer nuclear layer (ONL), and inner nuclear layer (INL), and the PRE/TT and ONL/TT ratios were all significantly higher in larvae exposed to blue light than in larvae exposed to white light. Additionally, the thickness of PRE and the outer nuclear layer and the RPE/TT and ONL/TT ratios of larvae exposed to 2.0 W m−2 were significantly lower than in larvae exposed to 0.3 W m−2. By contrast, the INL/TT ratio in larvae exposed to 2.0 W m−2 was significantly higher than in larvae exposed to 0.3 W m−2. Additionally, the INL and ganglion cell layer nuclei density of larvae exposed to 2.0 W m−2 were significantly higher than in those exposed to 0.3 W m−2 (p < 0.05). Transmission electron microscopy revealed different levels of abnormalities in the photoreceptor layers in all treatment groups. Considering the growth of the larvae, the results of the study suggest that continuous LED exposure induced damage to photoreceptor cells but was not relevant to the growth performance of D. labrax larvae. Moreover, the results obtained here also support the high plasticity of retinal development in response to altered environmental light conditions.

Quantifying the expression of mRNAs in the gonads at the critical stage of molecular sex differentiation stage might help to clarify the regulatory network during early sex differentiation and provide new information on the role of sex-related genes in gonadal function. In this study, transcriptomic analysis of sex-related genes expression profiles in fugu gonads at 60 and 90 days after hatching (dah) was conducted firstly, and a total of 112,504,991 clean reads, encompassing 28.35 Gb of sequences were retrieved. Twenty-three thousand eight hundred ten genes were found to be expressed in juvenile fugu gonads, and we mainly focused on the differentially expressed genes that have the potential to be involved in the gonadal sex differentiation. For 60-dah juveniles, we identified 1014 genes that were upregulated in the ovary and 1570 that were upregulated in the testis. For 90-dah juveniles, we identified 1287 genes that were upregulated in the ovary and 1500 that were upregulated in the testis. The dimorphic expression patterns of 15 genes in gonads at 30 and 40 dah were further investigate using qPCR. Cyp11b and star were expressed at higher levels in XY than in XX, while cyp11a1 and cyp19a1a were expressed at higher levels in XX than in XY at 30 dah. At 40 dah, the levels of gsdf, dmrt1, dmrt3, cyp11c1, star, and hsd3b expression were higher in XY, while the levels of foxl2, cyp19a1a, wnt9b, and foxD4 expression were higher in XX. Sox9, cyp11a1, cyp17a1, cyp17a2, and nr5a2 were expressed at similar levels in XX and XY at 40 dah. This is the first report of gonadal transcriptome of fugu at early sex differentiation stage, and our results provide an archive for further study on molecular mechanism underlying sex differentiation in this species.

As the critical tissue of the central nervous system, the brain has been found to be involved in gonad development. Previous studies have suggested that gonadal fate may be affected by the brain. Identifying brain-specific molecular changes that occur during estrodiol-17[beta] (E.sub.2) -induced feminization is crucial to our understanding of the molecular control of sex differentiation by the brains of fish. In this study, the differential transcriptomic responses of the Takifugu rubripes larvae brain were compared after E.sub.2 treatment for 55 days. Our results showed that 514 genes were differentially expressed between E.sub.2-treated-XX (E-XX) and Control-XX (C-XX) T. rubripes, while 362 genes were differentially expressed between E.sub.2-treated-XY (E-XY) and Control-XY (C-XY). For example, the expression of cyp19a1b, gnrh1 and pgr was significantly up-regulated, while st, sl, tsh[beta], prl and pit-1, which belong to the growth hormone/prolactin family, were significantly down-regulated after E.sub.2 treatment, in both sexes. The arntl1, bhlbe, nr1d2, per1b, per3, cry1, cipc and ciart genes, which are involved in the circadian rhythm, were also found to be altered. Differentially expressed genes (DEGs), which were identified between E-XX and C-XX, were significantly enriched in neuroactive ligand-receptor interaction, arachidonic acid metabolism, cytokine-cytokine receptor interaction and the calcium signaling pathway. The DEGs that were identified between E-XY and C-XY were significantly enriched in tyrosine metabolism, phenylalanine metabolism, arachidonic acid metabolism and linoleic acid metabolism. A number of genes and pathways were identified in the brain of E.sub.2-treated T. rubripes larvae by RNA-seq. It provided the opportunity for further study on the possible involvement of networks in the brain-pituitary-gonadal axis in sex differentiation in T. rubripes.

We assessed the effects of light intensity and spectrum on the growth and survival of Takifugu rubripes larvae from 30 to 69 days after hatching. Five lighting regimes were applied using 0.5, 1.5, and 3.0 W m−2 full spectrum white (W0.5, W1.5, W3.0), 0.5 W m−2 yellow (Y0.5), and 0.5 W m−2 blue light (B0.5). At the end of the experiment, body length, wet weight, and specific growth rate from day 0 to day 39 were significantly greater in larvae reared under W3.0 than under B0.5 (P ˂ 0.05). No significant differences were observed among W0.5, W1.5, and W3.0, or among W0.5, Y0.5, and B0.5 (P > 0.05). Survival rate was significantly higher in larvae reared under W1.5 than W0.5 (P ˂ 0.05), but no significant differences were observed among W0.5, Y0.5, and B0.5 (P > 0.05). Additionally, light conditioning did not affect the total thickness of the retina. Although the ratio of the thickness of the retinal pigment epithelium layer/total thickness (TT) was significantly higher in larvae exposed to W3.0 compared with those exposed to other light conditions, and the thickness of the outer nuclear layer/TT was significantly lower in larvae exposed to W3.0 compared with those exposed to W0.5 (P < 0.05), no relationship was confirmed between the structure of the retina and the growth performance of the T. rubripes larvae. Expression patterns of two stress-related and seven growth-related genes were also compared with the biometric parameters investigated in the experimental groups. No significant differences in the aanat1a, crh, ss1, igf1, or igf2 expression were observed among the five treatments. Pomc expression was significantly lower in larvae exposed to W1.5 than the larvae exposed to W0.5, and it was significantly lower in larvae exposed to Y0.5 than in larvae exposed to W0.5 or B0.5 (P < 0.05). Significant differences were also found in the expression of gh, with the highest levels being observed under W3.0, while the lowest levels were observed in B0.5 (P < 0.05). Ghrh expression was significantly higher in W3.0 (P < 0.05). These results should be considered when designing rearing protocols for fugu larvae in aquaculture systems.

We report epidemiological and clinical investigations on ten pediatric SARS-CoV-2 infection cases confirmed by real-time reverse transcription PCR assay of SARS-CoV-2 RNA. Symptoms in these cases were nonspecific and no children required respiratory support or intensive care. Chest X-rays lacked definite signs of pneumonia, a defining feature of the infection in adult cases. Notably, eight children persistently tested positive on rectal swabs even after nasopharyngeal testing was negative, raising the possibility of fecal–oral transmission. Children infected with the COVID-19 outbreak coronavirus, SARS-CoV-2, show mild symptoms but prolonged shedding of viral RNA in feces, suggesting that the fecal–oral route might play a role in virus transmission.

Urban sewer conditions assessment is important for the proper conveyance of sanitary water to wastewater treatment plants prior to environmental discharge. An effective approach to address this important process needs to be developed. This paper presents a data-driven methodology for sewer condition assessment with gridding-based chemical markers measurement in combination with a Bayesian chemical mass balance (CMB) model. A field study was performed in an urban sewer in Nanjing, China, to test the robustness of the developed methodology. In this site, data library of chemical markers (total nitrogen, phosphate, chloride, and total hardness) for source flows, including domestic wastewater, commercial wastewater and groundwater, was established. Meanwhile, a gridding-based measurement of these chemical markers in sewer flows was performed along the assessed sewer. Then, the CMB model with Bayesian inference and parallel Markov Chain Monte Carlo simulations was developed to quantify source contributions in sewer flows based on the chemical markers data of source and sewer flows. Accordingly, the proportion of clean water infiltration into the sewer and associated sewer defect level can be assessed. The Bayesian CMB model presented that groundwater contributed 11~14% of the sewer flow, indicating a neglectable sewer defect condition. The sewer assessment result was further verified by on-site physical inspection with distributed temperature sensing of in-sewer flows, proving the reliability of the developed methodology. Using this data-driven approach, a preliminary screening of the high-risk sub-catchments with severe sewer defect levels can be made for the following targeted sewer defects locations, optimizing the labor-intensive, system-wide physical inspections. Therefore, the proposed approach offers a cost-effective solution for system-wide sewer inspections.