New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Use of All-Male cyp17a1-Deficient Zebrafish (Danio rerio) for Evaluation of Environmental Estrogens.

Natural and synthetic environmental estrogens (EEs) are widespread and have received extensive attention. Our previous studies demonstrated that depletion of the cytochrome P450 17a1 gene (cyp17a1) leads to all-testis differentiation phenotype in zebrafish and common carp. In the present study, cyp17a1-deficient zebrafish with defective estrogen biosynthesis were used for the evaluation of EEs, as assessed by monitoring vitellogenin (vtg) expression. A rapid and sensitive assessment procedure was established with the 3-day administration of estradiol (E2), followed by examination of the transcriptional expression of vtgs in our cyp17a1-deficient fish. Compared with the control fish, a higher E2-mediated vtg upregulation observed in cyp17a1-deficient zebrafish exposed to 0.1 µg/L E2 is known to be estrogen receptor-dependent and likely due to impaired in vivo estrogen biosynthesis. The more responsive vtg expression in cyp17a1-deficient zebrafish was observed when exposed to 200 and 2000 µg/L bisphenol A (BPA) and perfluoro-1-octanesulfonate (PFOS). The estrogenic potentials of E2, BPA, and PFOS were compared and assessed by the feminization effect on ovarian differentiation in cyp17a1-deficient zebrafish from 18 to 50 days postfertilization, based on which a higher sensitivity of E2 in ovarian differentiation than BPA and PFOS was concluded. Collectively, through the higher sensitivity to EEs and the capacity to distinguish chemicals with different estrogenic potentials exhibited by the all-male cyp17a1-deficient zebrafish with impaired estrogen biosynthesis, we demonstrated that they can be used as an excellent in vivo model for the evaluation of EEs. Environ Toxicol Chem 2024;43:1062-1074. © 2024 SETAC.

IGFBP1a is a nutrient deficient response factor that can inhibit fish reproduction through the hypothalamus-pituitary-ovary axis†.

Reproduction is a high energy consuming process, so long-term malnutrition can significantly inhibit gonadal development. However, little is known about the molecular mechanism by which fasting inhibits reproduction. Our present study found that fasting could dramatically induce insulin-like growth factor binding protein 1 (IGFBP1) expression in the liver, hypothalamus, pituitary and ovaries of grass carp. In addition, IGFBP1a in the hypothalamus-pituitary-gonad axis could inhibit the development of gonads. These results indicated that fasting may participate in the regulation of fish gonadal development through the mediation of IGFBP1a. Further studies found that IGFBP1a could markedly inhibit gonadotropin-releasing hormone 3 expressions in hypothalamus cells. At the pituitary level, IGFBP1a could significantly reduce the gonadotropin hormones (LH and FSH) expression by blocking the action of pituitary insulin-like growth factor 1. Interestingly, IGFBP1a could also directly inhibit the expression of lhr, fshr, and sex steroid hormone synthase genes (cyp11a, cyp17a, and cyp19a1) in the ovary. These results indicated that IGFBP1a should be a nutrient deficient response factor that could inhibit fish reproduction through the hypothalamus-pituitary-ovary axis.

Formation of Different Polyploids Through Disrupting Meiotic Crossover Frequencies Based on cntd1 Knockout in Zebrafish.

Polyploidy, a significant catalyst for speciation and evolutionary processes in both plant and animal kingdoms, has been recognized for a long time. However, the exact molecular mechanism that leads to polyploid formation, especially in vertebrates, is not fully understood. Our study aimed to elucidate this phenomenon using the zebrafish model. We successfully achieved an effective knockout of the cyclin N-terminal domain containing 1 (cntd1) using CRISPR/Cas9 technology. This resulted in impaired formation of meiotic crossovers, leading to cell-cycle arrest during meiotic metaphase and triggering apoptosis of spermatocytes in the testes. Despite these defects, the mutant (cntd1-/-) males were still able to produce a limited amount of sperm with normal ploidy and function. Interestingly, in the mutant females, it was the ploidy not the capacity of egg production that was altered. This resulted in the production of haploid, aneuploid, and unreduced gametes. This alteration enabled us to successfully obtain triploid and tetraploid zebrafish from cntd1-/- and cntd1-/-/- females, respectively. Furthermore, the tetraploid-heterozygous zebrafish produced reduced-diploid gametes and yielded all-triploid or all-tetraploid offspring when crossed with wild-type (WT) or tetraploid zebrafish, respectively. Collectively, our findings provide direct evidence supporting the crucial role of meiotic crossover defects in the process of polyploidization. This is particularly evident in the generation of unreduced eggs in fish and, potentially, other vertebrate species.

Estrogen Signaling Inhibits the Expression of anti-Müllerian hormone (amh) and gonadal-soma-derived factor (gsdf) during the Critical Time of Sexual Fate Determination in Zebrafish.

The mechanism of fish gonadal sex differentiation is complex and regulated by multiple factors. It has been widely known that proper steroidogenesis in Leydig cells and sex-related genes in Sertoli cells play important roles in gonadal sex differentiation. In teleosts, the precise interaction of these signals during the sexual fate determination remains elusive, especially their effect on the bi-potential gonad during the critical stage of sexual fate determination. Recently, all-testis phenotypes have been observed in the cyp17a1-deficient zebrafish and common carp, as well as in cyp19a1a-deficient zebrafish. By mating cyp17a1-deficient fish with transgenic zebrafish Tg(piwil1:EGFP-nanos3UTR), germ cells in the gonads were labelled with enhanced green fluorescent protein (EGFP). We classified the cyp17a1-deficient zebrafish and their control siblings into primordial germ cell (PGC)-rich and -less groups according to the fluorescence area of the EGFP labelling. Intriguingly, the EGFP-labelled bi-potential gonads in cyp17a1+/+ fish from the PGC-rich group were significantly larger than those of the cyp17a1-/- fish at 23 days post-fertilization (dpf). Based on the transcriptome analysis, we observed that the cyp17a1-deficient fish of the PGC-rich group displayed a significantly upregulated expression of amh and gsdf compared to that of control fish. Likewise, the upregulated expressions of amh and gsdf were observed in cyp19a1a-deficient fish as examined at 23 dpf. This upregulation of amh and gsdf could be repressed by treatment with an exogenous supplement of estradiol. Moreover, tamoxifen, an effective antagonist of both estrogen receptor α and ß (ERα and Erß), upregulates the expression of amh and gsdf in wild-type (WT) fish. Using the cyp17a1- and cyp19a1a-deficient zebrafish, we provide evidence to show that the upregulated expression of amh and gsdf due to the compromised estrogen signaling probably determines their sexual fate towards testis differentiation. Collectively, our data suggest that estrogen signaling inhibits the expression of amh and gsdf during the critical time of sexual fate determination, which may broaden the scope of sex steroid hormones in regulating gonadal sex differentiation in fish.

Zebrafish gonad mutant models reveal neuroendocrine mechanisms of brain sexual dimorphism and male mating behaviors of different brain regions.

BACKGROUND: Sexually dimorphic mating behaviors differ between sexes and involve gonadal hormones and possibly sexually dimorphic gene expression in the brain. However, the associations among the brain, gonad, and sexual behavior in teleosts are still unclear. Here, we utilized germ cells-free tdrd12 knockout (KO) zebrafish, and steroid synthesis enzyme cyp17a1-deficient zebrafish to investigate the differences and interplays in the brain-gonad-behavior axis, and the molecular control of brain dimorphism and male mating behaviors. METHODS: Tdrd12+/-; cyp17a1+/- double heterozygous parents were crossed to obtain tdrd12-/-; cyp17a1+/+ (tdrd12 KO), tdrd12+/+; cyp17a1-/- (cyp17a1 KO), and tdrd12-/-; cyp17a1-/- (double KO) homozygous progenies. Comparative analysis of mating behaviors were evaluated using Viewpoint zebrafish tracking software and sexual traits were thoroughly characterized based on anatomical and histological experiments in these KOs and wild types. The steroid hormone levels (testosterone, 11-ketotestosterone and 17ß-estradiol) in the brains, gonads, and serum were measured using ELISA kits. To achieve a higher resolution view of the differences in region-specific expression patterns of the brain, the brains of these KOs, and control male and female fish were dissected into three regions: the forebrain, midbrain, and hindbrain for transcriptomic analysis. RESULTS: Qualitative analysis of mating behaviors demonstrated that tdrd12-/- fish behaved in the same manner as wild-type males to trigger oviposition behavior, while cyp17a1-/- and double knockout (KO) fish did not exhibit these behaviors. Based on the observation of sex characteristics, mating behaviors and hormone levels in these mutants, we found that the maintenance of secondary sex characteristics and male mating behavior did not depend on the presence of germ cells; rather, they depended mainly on the 11-ketotestosterone and testosterone levels secreted into the brain-gonad regulatory axis. RNA-seq analysis of different brain regions revealed that the brain transcript profile of tdrd12-/- fish was similar to that of wild-type males, especially in the forebrain and midbrain. However, the brain transcript profiles of cyp17a1-/- and double KO fish were distinct from those of wild-type males and were partially biased towards the expression pattern of the female brain. Our results revealed important candidate genes and signaling pathways, such as synaptic signaling/neurotransmission, MAPK signaling, and steroid hormone pathways, that shape brain dimorphism and modulate male mating behavior in zebrafish. CONCLUSIONS: Our results provide comprehensive analyses and new insights regarding the endogenous interactions in the brain-gonad-behavior axis. Moreover, this study revealed the crucial candidate genes and neural signaling pathways of different brain regions that are involved in modulating brain dimorphism and male mating behavior in zebrafish, which would significantly light up the understanding the neuroendocrine and molecular mechanisms modulating brain dimorphism and male mating behavior in zebrafish and other teleost fish.

Cryo-electron tomography of intact cardiac muscle reveals myosin binding protein-C linking myosin and actin filaments.

Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430 Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region. MyBP-C regulates contraction in a phosphorylation dependent fashion that may be through binding of its N-terminal domains with myosin or actin. Understanding the 3D organisation of MyBP-C in the sarcomere environment may provide new light on its function. We report here the fine structure of MyBP-C in relaxed rat cardiac muscle by cryo-electron tomography and subtomogram averaging of refrozen Tokuyasu cryosections. We find that on average MyBP-C connects via its distal end to actin across a disc perpendicular to the thick filament. The path of MyBP-C suggests that the central domains may interact with myosin heads. Surprisingly MyBP-C at Stripe 4 is different; it has weaker density than the other stripes which could result from a mainly axial or wavy path. Given that the same feature at Stripe 4 can also be found in several mammalian cardiac muscles and in some skeletal muscles, our finding may have broader implication and significance. In the D-zone, we show the first demonstration of myosin crowns arranged on a uniform 143 Å repeat.

Novel pituitary actions of GnRH in teleost: The link between reproduction and feeding regulation.

Gonadotropin-releasing hormone (GnRH), as a vital hypothalamic neuropeptide, was a key regulator for pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the vertebrate. However, little is known about the other pituitary actions of GnRH in teleost. In the present study, two GnRH variants (namely, GnRH2 and GnRH3) and four GnRH receptors (namely, GnRHR1, GnRHR2, GnRHR3, and GnRHR4) had been isolated from grass carp. Tissue distribution displayed that GnRHR4 was more highly detected in the pituitary than the other three GnRHRs. Interestingly, ligand-receptor selectivity showed that GnRHR4 displayed a similar and high binding affinity for grass carp GnRH2 and GnRH3. Using primary culture grass carp pituitary cells as model, we found that both GnRH2 and GnRH3 could not only significantly induce pituitary reproductive hormone gene (GtHα, LHß, FSHß, INHBa, secretogranin-2) mRNA expression mediated by AC/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II pathways but also reduce dopamine receptor 2 (DRD2) mRNA expression via the Ca2+/CaM/CaMK-II pathway. Interestingly, GnRH2 and GnRH3 could also stimulate anorexigenic peptide (POMCb, CART2, UTS1, NMBa, and NMBb) mRNA expression via AC/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II pathways in grass carp pituitary cells. In addition, food intake could significantly induce brain GnRH2 mRNA expression. These results indicated that GnRH should be the coupling factor to integrate the feeding metabolism and reproduction in teleost.

Sexual dimorphic effects of igf1 deficiency on metabolism in zebrafish.

Insulin-like growth factor 1 (IGF1) is an essential effector of the growth hormone (GH)/IGF1 axis for somatic growth regulation in mammals. However, its functions have not been thoroughly investigated in zebrafish in vivo. In this study, the igf1-deficient zebrafish model was developed using the CRISPR/Cas9 technique. In this study all the results were performed on both male and female animals. The growth of both male and female igf1-deficient zebrafish were reduced. The igf1 deficiency leads to significant complementary up-regulation of transcriptional expression levels of insulin, igf2 and igf3. This suggested that igf2 and igf3 may act with redundant functions. While the upregulation of gh1 expression can only be detected in igf1-deficient females. At the same time, significant growth retardation, fatty liver, reduced activated levels of ribosomal S6 (S6) are seen only in igf1-deficient males. On the other hand, significant hyperglycemia, elevated transcriptional expression levels of phosphenolpyruvate carboxykinase (pepck) and levels of phosphorylated extracellular signal-regulated kinase (ERK1/2), with additional reduced hepatic lactate/pyruvate (L/P) ratios can only observed in igf1-deficient females. Impaired glucose uptake has been recorded in the primary cultured hepatocytes from igf1-deficient females, but not males. Intriguingly, exposure to 17beta-estroadiol (E2) can partially ameliorated the defects of fatty liver and activation of AKT/mTOR signaling in igf1-deficient males. Our studies demonstrate the significant functions of IGF1 on somatic regulation in zebrafish, with asymmetric gender-related consequences. Our data thus suggest that the zebrafish IGF1 is preferentially required for the activation of AKT/mTOR signaling in male zebrafish, but glucose uptake in females.

Comparative genome anatomy reveals evolutionary insights into a unique amphitriploid fish.

Triploids are rare in nature because of difficulties in meiotic and gametogenic processes, especially in vertebrates. The Carassius complex of cyprinid teleosts contains sexual tetraploid crucian carp/goldfish (C. auratus) and unisexual hexaploid gibel carp/Prussian carp (C. gibelio) lineages, providing a valuable model for studying the evolution and maintenance mechanism of unisexual polyploids in vertebrates. Here we sequence the genomes of the two species and assemble their haplotypes, which contain two subgenomes (A and B), to the chromosome level. Sequencing coverage analysis reveals that C. gibelio is an amphitriploid (AAABBB) with two triploid sets of chromosomes; each set is derived from a different ancestor. Resequencing data from different strains of C. gibelio show that unisexual reproduction has been maintained for over 0.82 million years. Comparative genomics show intensive expansion and alterations of meiotic cell cycle-related genes and an oocyte-specific histone variant. Cytological assays indicate that C. gibelio produces unreduced oocytes by an alternative ameiotic pathway; however, sporadic homologous recombination and a high rate of gene conversion also exist in C. gibelio. These genomic changes might have facilitated purging deleterious mutations and maintaining genome stability in this unisexual amphitriploid fish. Overall, the current results provide novel insights into the evolutionary mechanisms of the reproductive success in unisexual polyploid vertebrates.

Characterization of the Interrenal Gland and Sexual Traits Development in cyp17a2-Deficient Zebrafish.

Unlike the Cytochrome P450, family 17, subfamily A, member 1 (Cyp17a1), which possesses both 17α-hydroxylase and 17,20-lyase activities involved in the steroidogenic pathway that produces androgens and estrogens, Cytochrome P450, family 17, subfamily A, polypeptide 2 (Cyp17a2) possesses only 17α-hydroxylase activity and is known essential for the synthesis of cortisol. Besides with expressed in testes and ovaries, where the cyp17a1 is mainly expressed, cyp17a2 is also expressed in the interrenal gland in fish. Until now, the roles of cyp17a2 in fish, especially in sexual traits development and hypothalamic-pituitary-interrenal (HPI) axis, are poorly studied. To investigate the roles of Cyp17a2 in teleosts, the cyp17a2-null zebrafish was generated and analyzed by us. The significantly decreased cortisol concentration was observed both in the cyp17a2-deficient males and females at adult stage. The interrenal gland enlargement, increased pituitary proopiomelanocortin a (pomca) expression, decreased locomotion activity and response to light-stimulated stress were observed in cyp17a2-deficient fish. Intriguingly, the cyp17a2-deficient males were fertile and with normal breeding tubercles on the pectoral fin, but females were infertile, deficient in genital papilla and with decreased gonadosomatic index (GSI). The increased progesterone (P4), 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and 11-ketotestosterone (11-KT) in the cyp17a2-deficient males and females were observed. The increased concentration of testosterone (T) and estradiol (E2) was observed in cyp17a2-/- females and cyp17a2-/- males, respectively. By examining the ovaries development of cyp17a2-deficient fish at 3 months postfertilization (mpf), we observed that the oocytes were over-activated. Taken together, our findings demonstrate that Cyp17a2 is indispensable for production and physiology of cortisol, and cyp17a2-deficiency resulted in diminished cortisol but accumulated P4 and DHP, which may result in the over-activated oocytes in cyp17a2-deficient females.

Novel Pituitary Actions of TAC4 Gene Products in Teleost.

Tachykinin 4 (TAC4) is the latest member of the tachykinin family involved in several physiological functions in mammals. However, little information is available about TAC4 in teleost. In the present study, we firstly isolated TAC4 and six neurokinin receptors (NKRs) from grass carp brain and pituitary. Sequence analysis showed that grass carp TAC4 could encode two mature peptides (namely hemokinin 1 (HK1) and hemokinin 2 (HK2)), in which HK2 retained the typical FXGLM motif in C-terminal of tachyinin, while HK1 contained a mutant VFGLM motif. The ligand-receptor selectivity showed that HK2 could activate all 6 NKRs but with the highest activity for the neurokinin receptor 2 (NK2R). Interestingly, HK1 displayed a very weak activation for each NKR isoform. In grass carp pituitary cells, HK2 could induce prolactin (PRL), somatolactin α (SLα), urotensin 1 (UTS1), neuromedin-B 1 (NMB1), cocaine- and amphetamine-regulated transcript 2 (CART2) mRNA expression mediated by NK2R and neurokinin receptor 3 (NK3R) via activation cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3)/protein kinase C (PKC) and calcium2+ (Ca2+)/calmodulin (CaM)/calmodulin kinase-II (CaMK II) cascades. However, the corresponding stimulatory effects triggered by HK1 were found to be notably weaker. Furthermore, based on the structural base for HK1, our data suggested that a phenylalanine (F) to valine (V) substitution in the signature motif of HK1 might have contributed to its weak agonistic actions on NKRs and pituitary genes regulation.

Reproductive Regulation of PrRPs in Teleost: The Link Between Feeding and Reproduction.

Prolactin-releasing peptide (PrRP), a sort of vital hypothalamic neuropeptide, has been found to exert an enormous function on the food intake of mammals. However, little is known about the functional role of PrRP in teleost. In the present study, two PrRP isoforms and four PrRP receptors were isolated from grass carp. Ligand-receptor selectivity displayed that PrRP1 preferentially binds with PrRP-R1a and PrRP-R1b, while PrRP-R2a and PrRP-R2b were special receptors for PrRP2. Tissue distribution indicated that both PrRPs and PrRP-Rs were highly expressed in the hypothalamus-pituitary-gonad axis and intestine, suggesting a latent function on food intake and reproduction. Using grass carp as a model, we found that food intake could significantly induce hypothalamus PrRP mRNA expression, which suggested that PrRP should be also an anorexigenic peptide in teleost. Interestingly, intraperitoneal (IP) injection of PrRPs could significantly induce serum luteinizing hormone (LH) secretion and pituitary LHß and GtHα mRNA expression in grass carp. Moreover, using primary culture grass carp pituitary cells as a model, we further found that PrRPs could directly induce pituitary LH secretion and synthesis mediated by AC/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II pathways. Finally, estrogen treatment of prepubertal fish elicited increases in PrRPs and PrPR receptors expression in primary cultured grass carp hypothalamus cells, which further confirmed that the PrRP/PrRPR system may participate in the neuroendocrine control of fish reproduction. These results, taken together, suggest that PrRPs might act as a coupling factor in feeding metabolism and reproductive activities in teleost.

Functions of the Thyroid-Stimulating Hormone on Key Developmental Features Revealed in a Series of Zebrafish Dyshormonogenesis Models.

The hypothalamic-pituitary-thyroid (HPT) axis regulates many critical features in vertebrates. Utilizing TALENs and CRISPR/Cas9 techniques, thyroid-stimulating hormone subunit beta a (tshba), thyroglobulin (tg), and solute carrier family 16 member 2 (slc16a2) mutant zebrafish lines were generated. Among the three mutants, the earliest time point for the significantly altered T3 contents was observed in tshba mutants, which resulted in the most severe defects, including typical defects such as the retardation of inflated anterior swimming bladder (aSB), proper formation of fin ray and posterior squamation (SP), the larval-to-juvenile transition (LTJT) process, juvenile growth retardation, and mating failure. In tg mutants, which are actually compensated with an alternative splicing form, growth retardation was observed in the juvenile stage without LTJT and reproductive defects. The evident goiter phenotype was only observed in tg- and slc16a2 mutants, but not in tshba mutants. Other than goiters being observed, no other significant developmental defects were found in the slc16a2 mutants. Regarding the reproductive defects observed in tshba mutants, the defective formation of the secondary sex characteristics (SSCs) was observed, while no obvious alterations during gonad development were found. Based on our analyses, zebrafish at the 6-12 mm standard length or 16-35 days post-fertilization (dpf) should be considered to be in their LTJT phase. Using a series of zebrafish dyshormonogenesis models, this study demonstrated that the TSH function is critical for the proper promotion of zebrafish LTJT and SSC formation. In addition, the elevation of TSH levels appears to be essential for goiter appearance in zebrafish.

A Highly Contiguous Genome Assembly of a Polyphagous Predatory Mite Stratiolaelaps scimitus (Womersley) (Acari: Laelapidae).

As a polyphagous soil-dwelling predatory mite, Stratiolaelaps scimitus (Womersley) (Acari: Laelapidae), formerly known as Stratiolaelaps miles (Berlese), is native to the Northern hemisphere and preys on soil invertebrates, including fungus gnats, springtails, thrips nymphs, nematodes, and other species of mites. Already mass-produced and commercialized in North America, Europe, Oceania and China, S. scimitus will highly likely be introduced to other countries and regions as a biocontrol agent against edaphic pests in the near future. The introduction, however, can lead to unexpected genetic changes within populations of biological control agents, which might decrease the efficacy of pest management or increase the risks to local environments. To better understand the genetic basis of its biology and behavior, we sequenced and assembled the draft genome of S. scimitus using the PacBio Sequel platform II. We generated ∼150× (64.81 Gb) PacBio long reads with an average read length of 12.60 kb. Reads longer than 5 kb were assembled into contigs, resulting in the final assembly of 158 contigs with an N50 length of 7.66 Mb, and captured 93.1% of the BUSCO (Benchmarking Universal Single-Copy Orthologs) gene set (n = 1,066). We identified 16.39% (69.91 Mb) repetitive elements, 1,686 noncoding RNAs, and 13,305 protein-coding genes, which represented 95.8% BUSCO completeness. Combining analyses of genome family evolution and function enrichment of gene ontology and pathway, a total of 135 families experienced significant expansions, which were mainly involved in digestion, detoxification, immunity, and venom. Major expansions of the detoxification enzymes, that is, P450s and carboxylesterases, suggest a possible genetic mechanism underlying polyphagy and ecological adaptions. Our high-quality genome assembly and annotation provide new insights on the evolutionary biology, soil ecology, and biological control for predaceous mites.

Pituitary Actions of EGF on Gonadotropins, Growth Hormone, Prolactin and Somatolactins in Grass Carp.

In mammals, epidermal growth factor (EGF) plays a vital role in both pituitary physiology and pathology. However, the functional role of EGF in the regulation of pituitary hormones has rarely reported in teleost. In our study, using primary cultured grass carp pituitary cells as an in vitro model, we examined the effects of EGF on pituitary hormone secretion and gene expression as well as the post-receptor signaling mechanisms involved. Firstly, we found that EGF significantly reduced luteinizing hormone (LHß) mRNA expression via ErbB1 coupled to ERK1/2 pathway, but had no effect on LH release in grass carp pituitary cells. Secondly, the results showed that EGF was effective in up-regulating mRNA expression of growth hormone (GH), somatolactin α (SLα) and somatolactin ß (SLß) via ErbB1 and ErbB2 and subsequently coupled to MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways, respectively. However, EGF was not effective in GH release in pituitary cells. Thirdly, we found that EGF strongly induced pituitary prolactin (PRL) release and mRNA expression, which was mediated by ErbB1 and subsequent stimulation of MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways. Interestingly, subsequent study further found that neurokinin B (NKB) significantly suppressed EGF-induced PRL mRNA expression, which was mediated by neurokinin receptor (NK2R) and coupled to AC/cAMP/PKA signal pathway. These results suggested that EGF could differently regulate the pituitary hormones expression in grass carp pituitary cells.

Validation of the six-and-twelve criteria among patients with hepatocellular carcinoma and performance score 1 receiving transarterial chemoembolization.

BACKGROUND: Transarterial chemoembolization (TACE) is recommended for patients with intermediate hepatocellular carcinoma (HCC) according to treatment guidelines. However, a large number of patients with advanced HCC also receive TACE in clinical practice, especially for those with liver-confined HCC and Eastern Cooperative Oncology Group score (ECOG) 1. In view of previous studies, such patients have different prognoses from advanced HCC patients with macrovascular invasion or extrahepatic spread; therefore, patients with ECOG 1 alone might be classified into the intermediate stage and benefit from TACE treatment, but a study particularly focusing on such patients and exploring the effectiveness of TACE therapy is lacking. AIM: To investigate treatment outcomes of TACE in HCC patients with ECOG 1 alone and propose a specific prognostic model. METHODS: Patients from 24 Chinese tertiary hospitals were selected in this nationwide multicenter observational study from January 2010 to May 2016. Overall survival (OS) was estimated using Kaplan-Meier curves and compared by the log-rank test. Multivariate Cox regression was used to develop the potential prognostic models. The discriminatory ability of the models was compared and validated in various patient subgroups. The individual survival prediction for six-and-twelve (6&12) criteria, defined as the algebraic sum of tumor size (cm) and tumor number, was illustrated by contour plot of 3-year survival probability and nomogram. RESULTS: A total of 792 eligible patients were included. During follow-up, median OS reached 18.9 mo [95% confidence interval (CI): 16.9-21.0]. Three independent multivariate analyses demonstrated that tumor size, tumor number, α-fetoprotein level, albumin-bilirubin grade and total bilirubin were prognostic factors of OS (P < 0.05). The previously proposed 6&12 criteria was comparable or even better than currently proposed with the highest predictive ability. In addition, the 6&12 criteria was correlated with OS in various subgroups of patients. The patients were stratified into three strata with score ≤ 6, > 6 but ≤ 12, and > 12 with different median OS of 39.8 mo (95%CI: 23.9-55.7), 21.1 mo (95%CI: 18.4-23.8) and 9.8 mo (95%CI: 8.3-11.3), respectively (P < 0.001). CONCLUSION: TACE is effective for advanced HCC patients with ECOG 1 alone, and the 6&12 criteria may help with clinical decision-making.

Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration.

Adenosine triphosphate (ATP), the cellular energy currency, is essential for life. The ability to provide a constant supply of ATP is therefore crucial for the construction of artificial cells in synthetic biology. Here, we describe the bottom-up assembly and characterization of a minimal respiratory system that uses NADH as a fuel to produce ATP from ADP and inorganic phosphate, and is thus capable of sustaining both upstream metabolic processes that rely on NAD+, and downstream energy-demanding processes that are powered by ATP hydrolysis. A detergent-mediated approach was used to co-reconstitute respiratory mitochondrial complex I and an F-type ATP synthase into nanosized liposomes. Addition of the alternative oxidase to the resulting proteoliposomes produced a minimal artificial "organelle" that reproduces the energy-converting catalytic reactions of the mitochondrial respiratory chain: NADH oxidation, ubiquinone cycling, oxygen reduction, proton pumping, and ATP synthesis. As a proof-of-principle, we demonstrate that our nanovesicles are capable of using an NAD+-linked substrate to drive cell-free protein expression. Our nanovesicles are both efficient and durable and may be applied to sustain artificial cells in future work.

Validation and evaluation of clinical prediction systems for first and repeated transarterial chemoembolization in unresectable hepatocellular carcinoma: A Chinese multicenter retrospective study.

BACKGROUND: The treatment outcome of transarterial chemoembolization (TACE) in unresectable hepatocellular carcinoma (HCC) varies greatly due to the clinical heterogeneity of the patients. Therefore, several prognostic systems have been proposed for risk stratification and candidate identification for first TACE and repeated TACE (re-TACE). AIM: To investigate the correlations between prognostic systems and radiological response, compare the predictive abilities, and integrate them in sequence for outcome prediction. METHODS: This nationwide multicenter retrospective cohort consisted of 1107 unresectable HCC patients in 15 Chinese tertiary hospitals from January 2010 to May 2016. The Hepatoma Arterial-embolization Prognostic (HAP) score system and its modified versions (mHAP, mHAP2 and mHAP3), as well as the six-and-twelve criteria were compared in terms of their correlations with radiological response and overall survival (OS) prediction for first TACE. The same analyses were conducted in 912 patients receiving re-TACE to evaluate the ART (assessment for re-treatment with TACE) and ABCR (alpha-fetoprotein, Barcelona Clinic Liver Cancer, Child-Pugh and Response) systems for post re-TACE survival (PRTS). RESULTS: All the prognostic systems were correlated with radiological response achieved by first TACE, and the six-and-twelve criteria exhibited the highest correlation (Spearman R = 0.39, P = 0.026) and consistency (Kappa = 0.14, P = 0.019), with optimal performance by area under the receiver operating characteristic curve of 0.71 [95% confidence interval (CI): 0.68-0.74]. With regard to the prediction of OS, the mHAP3 system identified patients with a favorable outcome with the highest concordance (C)-index of 0.60 (95%CI: 0.57-0.62) and the best area under the receiver operating characteristic curve at any time point during follow-up; whereas, PRTS was well-predicted by the ABCR system with a C-index of 0.61 (95%CI: 0.59-0.63), rather than ART. Finally, combining the mHAP3 and ABCR systems identified candidates suitable for TACE with an improved median PRTS of 36.6 mo, compared with non-candidates with a median PRTS of 20.0 mo (log-rank test P < 0.001). CONCLUSION: Radiological response to TACE is closely associated with tumor burden, but superior prognostic prediction could be achieved with the combination of mHAP3 and ABCR in patients with unresectable liver-confined HCC.

Hyperandrogenism in POMCa-deficient zebrafish enhances somatic growth without increasing adiposity.

The endocrine regulatory roles of the hypothalamic-pituitary-adrenocortical axis on anxiety-like behavior and metabolic status have been found throughout animal taxa. However, the precise effects of the balancing adrenal corticosteroid biosynthesis under the influence of adrenocorticotrophic hormone (ACTH), a pro-opiomelanocortin (POMC)-derived peptide, on animal energy expenditure and somatic growth remain unknown. POMC has also been identified as one of the candidate loci for polycystic ovary syndrome, which features hyperandrogenism and some prevalence of obesity in patients. Here we show that zebrafish lacking functional POMCa exhibit similar phenotypes of stress response and body weight gain but not obesity as observed in mammalian models. In contrast with the impaired anorexigenic signaling cascade of melanocyte-stimulating hormones and leptin, which are responsible for their obesity-prone weight gain observed in various pomc mutant mammals, analyses with our pomca mutant series indicate that ACTH is the key regulator for the phenotype with enhanced somatic growth without obesity in pomca-deficient zebrafish. Hypocortisolism associated with hyperandrogenism has been observed in the pomca-deficient zebrafish, with enhanced activation of mammalian target of rapamycin complex 1; reutilization of amino acids and fatty acid ß-oxidation are observed in the muscle tissue of the pomca-deficient fish. After reducing hyperandrogenism by crossing our pomca mutant fish with a cyp17a1-deficient background, the phenotype of enhanced somatic growth in pomca-deficient fish was no longer observed. Thus, our work also demonstrated that the role of POMCa in stress response seems to be conserved in vertebrates, whereas its effect on adipostasis is unique to teleosts.