Thermal performance of the electron transport system Complex III in seven Alabama fishes.

Management of fish populations for conservation in thermally variable systems requires an understanding of the fish's underlying physiology and responses to thermal stress. Physiological research at the organismal level provides information on the overall effects of stressors such as extreme temperature fluctuations. While experiments with whole organisms provide information as to the overall effects of temperature fluctuations, biochemical assays of thermal stress provide direct results of exposure that are both sensitive and specific. Electron transport system (ETS; Complex III) assays quantify a rate-limiting step of respiratory enzymes. Parameters that can be estimated via this approach include optimum thermal temperature (Topt ) and optimal breadth of thermal performance (Tbreadth ), which can both be related to organismal-level temperature thresholds. We exposed enzymes of seven fish species (native fish chosen to represent a typical community in Alabama streams) to temperatures in the range 11-44°C. The resultant enzymatic thermal performance curves showed that Topt , the lower temperature for enzyme optimal thermal performance (Tlow ), the upper temperature for enzyme optimal thermal performance (Tup ), and Tbreadth differed among species. Relationships between enzymatic activity and temperature for all fish followed a pattern of steadily increasing enzyme activity to Topt before gradually decreasing with increasing temperature. A comparison of our enzyme optimum and upper-temperature limit results versus published critical thermal maxima values supports that ETS Complex III assays may be useful for assessing organismal-level thermal tolerance.

Functional differentiation of two lhx8 paralogs and possible regulatory role of lhx8a in Japanese flounder (Paralichthys olivaceus).

Lhx8, belonging to the LIM-Homebox family, is involved in the tooth, nervous system, and primordial follicles development in mammals. However, little is known about the regulatory roles of lhx8 in teleosts. In this study, two lhx8 duplicates were identified in Paralichthys olivaceus, termed Polhx8a and Polhx8b, respectively. Bioinformatic analysis showed that Polhx8a was more likely to be a teleost-specific paralog. According to expression analysis, Polhx8a transcripts were almost exclusively concentrated in the oocytes, while Polhx8b was weakly expressed in the spleen, gill, and some facial organs, indicating sub-functionalization of this gene pair during evolution. Furthermore, Polhx8a mRNA level elevated from perinucleolar oocyte (PNO) stage to vitellogenic oocyte (VO) stage transition and changed after exogenous hormone stimulation, proving that Polhx8a was involved in the oocyte development and could be regulated by sex hormones. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) experiments captured the positive protein interactions between PoLhx8a and the other two oocyte-specific transcription factors: PoFigla and PoNobox. After knocking down lhx8a in embryos or adult ovaries in vivo, the expression of oocyte-associated genes was significantly down-regulated (P < 0.05). Our findings suggest the evolution and functional differentiation of lhx8 genes, and shed light on the potential role of lhx8a in protein interactions and gene regulation in teleosts.

Effects of crowding stress on the hypothalamo-pituitary-interrenal axis of the self-fertilizing fish, Kryptolebias marmoratus.

We tested whether crowding stress affects the hypothalamo-pituitary-interrenal (HPI) axis of the self-fertilizing fish, Kryptolebias marmoratus, which is known to be aggressive in the laboratory conditions but sometimes found as a group from a single land crab burrow in the wild. The projection of corticotropin-releasing hormone (CRH) neurons to the adrenocorticotropic hormone (ACTH) cells in the pituitary was confirmed by dual-label immunohistochemistry; CRH-immunoreactive (ir) fibers originating from cell bodies located in the lateral tuberal nucleus (NLT) of the hypothalamus were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Then, fish were reared solitary or in pairs for 14 days, and the number of CRH-ir cell bodies in the NLT of the hypothalamus and cortisol levels in the body without head region were compared. The number of CRH-ir cell bodies and cortisol levels were significantly higher in paired fish. These results indicate that crowding stress affects the HPI axis in K. marmoratus which thrive in small burrows with limited water volume.

The osmotic response capacity of the Antarctic fish Harpagifer antarcticus is insufficient to cope with projected temperature and salinity under climate change.

Over the last decades, climate change has intensified. Temperatures have increased and seawater has become "fresher" in Antarctica, affecting fish such as Harpagifer antarcticus. Thus, this study aimed to evaluate changes in the osmoregulatory response of the Antarctic notothenioid fish Harpagifer antarcticus and evaluate how it will cope with the future climate change and environmental conditions in the Antarctic, and in the hypothetical case that its geographical distribution will be extended to the Magellanes region. The present study was undertaken to determine the interaction between temperature and salinity tolerance (2 °C and 33 psu as the control group, the experimental groups were 5, 8, and 11 °C and 28 and 23 psu) and their effect on the osmoregulatory status of H. antarcticus. We evaluated changes in gill-kidney-intestine NKA activity, gene expression of NKAα, NKCC, CFTR, Aquaporins 1 and 8 in the same tissues, muscle water percentage, and plasma osmolality to evaluate osmoregulatory responses. Plasma osmolality decreased with high temperature, also the gill-kidney-intestine NKA activity, gene expression of NKA α, NKCC, CFTR, Aquaporins 1, and 8 were modified by temperature and salinity. We demonstrated that H. antarcticus can not live in the Magallanes region, due to its incapacity to put up with temperatures over 5 °C and with over 8 °C being catastrophic.

Overexpression of the V-ATPase c subunit gene from Antarctic notothenioid fishes enhances freezing tolerance in transgenic Arabidopsis plants.

Theoretical and experimental studies have demonstrated that temperature is an important environmental factor that affects the regional distribution of plants. However, how to modify the distribution pattern of plants in different regions is a focus of current research. Obtain the information of cold tolerance genes from cold tolerance species, cloning genes with real cold tolerance effects is one of the most important ways to find the genes related to cold tolerance. In this study, we investigated whether transferring the VHA-c gene from Antarctic notothenioid fishes into Arabidopsis enhances freezing tolerance of Arabidopsis. The physiological response and molecular changes of VHA-c overexpressing pedigree and wildtype Arabidopsis were studied at -20 °C. The results showed that the malondialdehyde (MDA) and membrane leakage rates of WT plants were significantly higher than those of VHA-c8 and VHA-c11 plants, but the soluble sugar, soluble protein, proline and ATP contents of WT plants were significantly lower than those of VHA-c8 and VHA-c11 plants under -20 °C freezing treatment. The survival rate, VHA-c gene expression level and VHA-c protein contents of WT plants were significantly lower than those of VHA-c8 and VHA-c11 plants under -20 °C freezing treatment. Correlation analysis showed that ATP content was significantly negatively correlated with MDA and membrane leakage rate, and positively correlated with soluble sugar, soluble protein and proline content under -20 °C freezing treatment. These results demonstrated that overexpression of the VHA-c gene provided strong freezing tolerance to Arabidopsis by increasing the synthesis of ATP and improved the adaptability of plants in low temperature environment.

Dead-end (dnd) protein in fish-a review.

Dead end (dnd) is a germ plasm-specific maternal RNA discovered in zebrafish and then in other vertebrates. Dnd protein is essential for migration and motility of primordial germ cells (PGCs), only cells destined to transfer genetic information to offspring. PGCs arise far from somatic cells of developing gonads and they must migrate to their site of function. Migration of PGCs follows complex path by various developing tissues as their disruption impacts on the fertility. Recently, it has been found that dnd is not required for survival of PGCs and dnd-deficient zebrafish PGCs transdifferentiate into the somatic cells. In fish, targeting dnd causes removal of PGCs that ultimately affects sex differentiation. Sterility in various fish species can be achieved by knockdown or knockout of dnd. In our review, we have discussed dnd as a germ cell-specific molecular marker in fish, its interaction with miRNAs, and its use in aquaculture and fish conservation.

Transcriptional regulation mechanism of sterol regulatory element binding proteins on Δ6 fatty acyl desaturase in razor clam Sinonovacula constricta.

The razor clam, Sinonovacula constricta, contains high levels of long-chain PUFA (LC-PUFA), which are critical for human health. In addition, S. constricta is the first marine mollusc demonstrated to possess Δ6 fatty acyl desaturase (Fad) and complete LC-PUFA biosynthetic ability, providing a good representative to investigate the molecular mechanism of sterol regulatory element binding proteins (SREBP) in regulating Δ6 Fad for LC-PUFA biosynthesis in marine molluscs. Herein, S. constricta SREBP and Δ6 Fad promoter were cloned and characterised. Subsequently, dual luciferase and electrophoretic mobility shift assays were conducted to explore the SREBP binding elements in the core regulatory region of S. constricta Δ6 Fad promoter. Results showed that S. constricta SREBP had a very conservative basic helix-loop-helix-leucine zipper motif, while S. constricta Δ6 Fad promoter exhibited very poor identity with teleost Fads2 promoters, indicating their differentiation during evolution. A 454 bp region harbouring a core sequence in S. constricta Δ6 Fad promoter was predicted to be essential for the transcriptional activation by SREBP. This was the first report on the regulatory mechanism of LC-PUFA biosynthesis in marine molluscs, which would facilitate optimising the LC-PUFA biosynthetic pathway of bivalves in further studies.

Proteomics of Osmoregulatory Responses in Threespine Stickleback Gills.

The gill proteome of threespine sticklebacks (Gasterosteus aculeatus) differs greatly in populations that inhabit diverse environments characterized by different temperature, salinity, food availability, parasites, and other parameters. To assess the contribution of a specific environmental parameter to such differences it is necessary to isolate its effects from those of other parameters. In this study the effect of environmental salinity on the gill proteome of G. aculeatus was isolated in controlled mesocosm experiments. Salinity-dependent changes in the gill proteome were analyzed by Liquid chromatography/Tandem mass spectrometry data-independent acquisition (DIA) and Skyline. Relative abundances of 1691 proteins representing the molecular phenotype of stickleback gills were quantified using previously developed MSMS spectral and assay libraries in combination with DIA quantitative proteomics. Non-directional stress responses were distinguished from osmoregulatory protein abundance changes by their consistent occurrence during both hypo- and hyper-osmotic salinity stress in six separate mesocosm experiments. If the abundance of a protein was consistently regulated in opposite directions by hyper- versus hypo-osmotic salinity stress, then it was considered an osmoregulatory protein. In contrast, if protein abundance was consistently increased irrespective of whether salinity was increased or decreased, then it was considered a non-directional response protein. KEGG pathway analysis revealed that the salivary secretion, inositol phosphate metabolism, valine, leucine, and isoleucine degradation, citrate cycle, oxidative phosphorylation, and corresponding endocrine and extracellular signaling pathways contain most of the osmoregulatory gill proteins whose abundance is directly proportional to environmental salinity. Most proteins that were inversely correlated with salinity map to KEGG pathways that represent proteostasis, immunity, and related intracellular signaling processes. Non-directional stress response proteins represent fatty and amino acid degradation, purine metabolism, focal adhesion, mRNA surveillance, phagosome, endocytosis, and associated intracellular signaling KEGG pathways. These results demonstrate that G. aculeatus responds to salinity changes by adjusting osmoregulatory mechanisms that are distinct from transient non-directional stress responses to control compatible osmolyte synthesis, transepithelial ion transport, and oxidative energy metabolism. Furthermore, this study establishes salinity as a key factor for causing the regulation of numerous proteins and KEGG pathways with established functions in proteostasis, immunity, and tissue remodeling. We conclude that the corresponding osmoregulatory gill proteins and KEGG pathways represent molecular phenotypes that promote transepithelial ion transport, cellular osmoregulation, and gill epithelial remodeling to adjust gill function to environmental salinity.

Influence of dietary replacement of fish meal with fish soluble meal on growth and TOR signaling pathway in juvenile black sea bream (Acanthopagrus schlegelii).

An 8-week feeding trial was conducted to evaluate the effect of replacement of fish meal (FM) with fish soluble meal (FSM) on growth performance, feed utilization and expression of genes involved in TOR signaling pathway for juvenile black sea bream (Acanthopagrus schlegelii). Six isonitrogenous (41%) and isolipidic diets were prepared to contain graded levels of FSM which replaced 0% (control diet), 10%, 20%, 30%, 40% and 60% protein from FM. Triplicate groups of 20 fish with initial weight 0.51 ± 0.01 g were fed with experimental diets twice daily to apparent satiation. The results showed significant differences in growth performance and feed utilization among all treatments, final body weight (FBW), percent weight gain (PWG), specific growth rate (SGR) and protein efficiency ratio (PER) significantly increased with dietary replacement levels of FM with FSM increasing from 0% to 40% (P < 0.05), PWG, SGR and PER were significantly reduced when replacement of FM with FSM further increased from 40% to 60%. Based on PWG against replacement levels of FM with FSM, A two-slope broken-line model analysis indicated that the optimal replacement of FM with FSM is to be 42.59%. Moreover, the lowest feed conversion ratio (FCR) was observed in fish fed the 40% FSM replacement diet. Muscle amino acid profile in muscle revealed that total essential amino acids, arginine and threonine were significantly influenced by replacement levels of FSM, while there was no significant difference in NEAA among all treatments. The hematological indices were not affected by the replacement levels of FM with FSM. The relative expression levels of irs-1, pi3k, akt, igf-1, s6k1 and tor were up-regulated when replacement levels of FM with FSM increased from 0% to 40%, and higher values were observed in fish fed with 40% FSM replacement diet compared to those fed the other diets. However, relative expression of 4e-bp2 was down-regulated when replacement levels of FM with FSM increased from 0% to 40% (P < 0.05). In summary, the results of present study indicated that FSM could be a viable alternative protein source for black sea bream, dietary FSM supplementation could improve growth and up-regulate the relative expression of irs-1, pi3k, akt, igf-1, s6k1 genes related to TOR signaling pathway in liver of juvenile black sea bream.

Dietary selenium enhances the growth and anti-oxidant capacity of juvenile blunt snout bream (Megalobrama amblycephala).

Sodium selenite was added to basal diet at five levels (0.10, 0.42, 0.67, 1.06 and 1.46 mg Se/kg) and fed fish for 8 weeks. The dietary selenium requirement of juvenile blunt snout bream (Megalobrama amblycephala) was quantified. Dietaryseleniums at 0.67-1.06 mg Se/kg improved weight gain rate, specific growth rate and feed efficiency. The optimal amount was 0.96 mg/kg, for which the specific growth rate was 1.798%/day and the weight gain rate was 173.852% (p < 0.05). Se deposition in muscle was increased (p < 0.05) at ≥0.67 mg/kg, but moisture, protein, lipid and ash content were not affected. Physiological status and lipid metabolism were improved by 1.06-1.46 mg/kg dietary selenium based on total protein and albumin in plasma, and total cholesterol and triglycerides (p < 0.05). Activities of hepatic anti-oxidant enzymes catalase, total superoxide dismutase, glutathione peroxidase and reduced glutathione were enhanced at Se1.06 (p < 0.05). However, malondialdehyde content was lowered at Se1.06 (p < 0.05). Expression of anti-inflammatory cytokines, nuclear factor erythroid 2-related factor 2 (Nrf2) and kelch-like ECH-associated protein 1 (Keap-1) in liver were elevated at Se1.06 (p < 0.05), as were mRNA levels of glutathione peroxidase, copper zinc superoxide dismutase and catalase. Expression of pro-inflammatory cytokines, interleukin 8, tumour necrosis factor-α and transforming growth factor-ß were inhibited at 0.67-1.46 mg/kg (p < 0.05). In general, 0.96 mg/kg was optimal, and optimal selenium enhanced antioxidant stress tolerance and anti-inflammatory ability.

Effect of pterois volitans (lionfish) venom on cholinergic and dopaminergic systems.

Pterois volitans venom induces muscular fibrillation, which results from nerve transmission caused by the presence of acetylcholine (ACh). It also has cardiovascular effects that are due to its actions on muscarinic and nicotinic cholinergic receptors. In this study, we characterized the effects of P. volitans venom on nicotinic acetylcholine receptors (nAChRs) and dopaminergic neurons. After exposure to P. volitans venom, acetylcholinesterase (AChE) mRNA levels and the expression of the α2 subunit of nAChR increased in zebrafish embryos (15-20 somites). In addition, the lionfish venom blocked zebrafish α2 nAChR subunit functional expression and the ACh-induced response of human neuronal α3ß2 receptors. The latter receptor was blocked by a protein fraction named F2, which was isolated from P. volitans venom using reversed phase high performance liquid chromatography (RP-HPLC). This venom causes death in dopaminergic neurons, and affects the cholinergic system. The effect of these two systems may result in retarded embryonic development of zebrafish, since the two systems function in a related manner to control growth hormone secretion.

Conserved function of Pacific cod Caspase-3 in apoptosis.

Apoptosis plays a significant role in the cellular immune responses against infections, especially those related to viruses. Across various species, Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. However, its role remains relatively unknown in cod fish. In this study, we aimed to reveal the role of Pacific cod Caspase-3 (GmCasp3) in apoptosis and its evolutionary position. Our results showed that the GmCasp3 cDNA contains an open reading frame of 864 nucleotides; that codes for 287 amino acids long protein with a molecular weight of 32.03 kDa. The sequence alignments and 3-D model indicated that GmCasp3 contained highly conserved domains, such as "QACRG", "GSWFI" and "HG" active sites, however, the phylogenetic tree analysis revealed that both GmCasp3 and Atlantic cod caspase-3 clustered together are far from the high vertebrate branch, indicating they are at a lower position in vertebrate evolution. Red fluorescent labeling vector pDsRed2-C1-GmCasp3 was constructed and it was transfected into EPC cell lines. The result showed that GmCasp3 protein was distribute in the protoplasm and expressed in apoptotic cell debris. Moreover, the GmCasp3 enzyme activity increased with the increased post-transfection analysis time, while the genome DNA was visibly fragmented at 36 h post transfection. Flow cytometry analysis showed that the proportion of apoptosis cells increased from 12 h to 24 h post transfection. In conclusion, the conserved functions of GmCasp3 in apoptosis indicated that Pacific cod has the similar apoptotic characteristics as other animals.

Molecular characterization of a MOSPD2 homolog in the barbel steed (Hemibarbus labeo) and its involvement in monocyte/macrophage and neutrophil migration.

Motile sperm domain containing 2 (MOSPD2) is a single-pass membrane protein to which until recently little function had been ascribed. Although its mammalian homologs have been identified, the status of the mospd2 gene in lower vertebrates is still unknown. In the present study, cDNA of the mospd2 gene of barbel steed (Hemibarbus labeo) was cloned and sequenced to characterize its potential involvement in the innate immune system of this fish. Sequence analysis revealed that the predicted barbel steed MOSPD2 protein contained an N-terminal extracellular portion composed of a CRAL-TRIO domain, a motile sperm domain, and a transmembrane domain, as well as a short C-terminal intracellular domain. Phylogenetic tree analysis indicated that barbel steed MOSPD2 is closely related to that of zebrafish. Barbel steed mospd2 transcripts were detected in a wide range of tissues, with the highest level being found in the gill. In response to lipopolysaccharide (LPS) treatment or Aeromonas hydrophila infection, mospd2 gene expression was significantly altered in the head kidney, spleen, and mid-intestine. The expression of mospd2 gene was detected in monocytes/macrophages (MO/MФ), neutrophils, and lymphocytes, and was found to be mainly expressed in MO/MФ. At the same time, using flow cytometry, we also confirmed that MOSPD2 protein is located on MO/MФ, neutrophil, and lymphocyte membranes. Following treatment with LPS or A. hydrophila, MOSPD2 protein expression was induced in these immune cells. The migration of MO/MФ and neutrophils decreased significantly upon MOSPD2 blockade with anti-MOSPD2 IgG in a dose-dependent manner, whereas this treatment had no significant effect on lymphocytes migration. To the best of our knowledge, our study, for the first time, provides evidence that MOSPD2 mediates the migration of MO/MФ and neutrophils in a fish species.

Morphological features of mucous secretory organ and mucous secretion of loach Misgurnus anguillicaudatus skin for friction drag reduction.

We examined the functional morphology of loach Misgurnus anguillicaudatus skin by using synchrotron X-ray micro-computed tomography (SR-µCT) and high-contrast staining using osmium tetroxide or phosphotungstic acid (PTA), which enhances the image contrast of soft tissues. The captured high-spatial resolution images revealed that the surface ornamentations were stuck in the basement membrane of the loach scales. The ornamentations consisting of grooves (radii) and ridges (circuli) that can move freely and bend flexibly. The cross-sectional lateral microstructures of flat, concave and convex loach skins were observed from a live image of loach skin obtained through dark-field optical coherence tomography (OCT) imaging. The thickness of loach skin was changed with varying empty space between the mucous-cell layer and the scales by bending motion of loach. In addition, through direct measurement of drag reduction of loach skin, the mucous layer was found to have a strong influence on the reduction of skin friction. The present results enhance the understanding of the functional morphologies of mucous layer of loach to secrete mucus for skin friction reduction.

Accelerated evolution and positive selection of rhodopsin in Tibetan loaches living in high altitude.

Rhodopsin (RH1), the temperature-sensitive visual pigment, attained cold adaptation by functional trade-offs between protein stability and activity. Recent studies suggested convergent selection pressures drove cold adaptation of rhodopsin in high altitude catfishes through nonparallel molecular mechanisms. Here, we tested whether the similar shift occurred in RH1 of Tibetan loaches on the Qinghai-Tibet Plateau (QTP) by investigating the molecular evolution and potential effect on function of RH1. We sequenced RH1 from 27 Triplophysa species, and four lowland loaches and combined these data with published sequences. Tests using a series of models of molecular evolution resulted in strong evidence for accelerated evolution and positive selection in Triplophysa RH1. Three positively selected sites were near key functional domains modulating nonspectral properties of rhodopsin, substitutions of which were likely to compensate for cold-induced decrease in rhodopsin kinetics in cold environments. Moreover, although accelerated evolutionary rates in Tibetan loaches was convergent with those in high altitude catfishes, the sites under positive selection were nonoverlapping. Our findings provide evidence for convergent shift in selection pressures of RH1 in high altitude fish during the ecological transition to cold environment of the QTP.

IGFs Potentiate TAC3-induced SLα Expression via Upregulation of TACR3 Expression in Grass Carp Pituitary Cells.

In mammals, the tachykinin 3 (TAC3)/tachykinin receptor 3 (TACR3) systems have been confirmed to play an important role in the regulation of puberty onset. Using grass carp pituitary cells as the model, our recent study found that the TAC3 gene products could significantly induce somatolactin α (SLα) synthesis and secretion via TACR3 activation. In the present study, we seek to examine if pituitary TACR3 can serve as a regulatory target and contribute to TAC3 interactions with other SLα regulators. Firstly, grass carp TACR3 was cloned and tissue distribution showed that it could be highly detected in grass carp pituitary. Using HEK293 cells as the model, functional expression also revealed that grass carp TACR3 exhibited ligand binding selectivity and post-receptor signaling highly comparable to its mammalian counterpart. Using grass carp pituitary cells as the model, TACR3 mRNA expression could be stimulated by insulin-like growth factor (IGF)-I and -II via the IGF-I receptor coupled to phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways. Interestingly, IGF-I/-II cotreatment could also significantly enhance TAC3-induced SLα mRNA expression and the potentiating effect was dependent on TACR3 expression and activation of adenylate cyclase (AC)/cAMP/protein kinase A (PKA), phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3)/protein kinase C (PKC), and Ca2+/calmodulin (CaM)/calmodulin-dependent protein kinase II (CaMK-II) cascades. Besides, IGF-I-induced Akt phosphorylation but not MEK, extracellular signal-regulated kinase (ERK1/2), and P38MAPK phosphorylation was notably enhanced by TACR3 activation. These results, as a whole, suggest that the potentiating effect of IGFs on TAC3 gene products-induced SLα mRNA expression was mediated by TACR3 upregulation and functional crosstalk of post-receptor signaling in the pituitary.

Citric acid mitigates soybean meal induced inflammatory response and tight junction disruption by altering TLR signal transduction in the intestine of turbot, Scophthalmus maximus L.

A 12-week feeding trial was conducted to investigate the effect of citric acid on the involvement of TLRs in the soybean meal induced inflammatory response and tight junction disruption in the distal intestine of juvenile turbot (Scophthalmus maximus L.). Four isonitrogenous and isolipidic practical diets were formulated: fish meal-based diet (FM); 40% fish meal protein in FM replaced with soybean meal protein (SBM); SBM + 1.5% citric acid and SBM + 3% citric acid. Compared to the FM, diet SBM significantly increased the gene expression of TLRs (TLR2, TLR3, TLR5b, TLR9, TLR21, TLR22) and MyD88, as well as TLR related molecules (NF-κB, IRF-3, p38 and JNK), which were remarkably reduced by dietary citric acid. Similarly, citric acid supplementation in SBM markedly depressed gene expression of pro-inflammatory cytokines (TNF-α and IFN-γ) and pore-forming tight junction protein Claudin-7, and enhanced gene expression of the anti-inflammatory cytokine TGF-ß1 and TJ proteins related to the decrease in paracellular permeability (Claudin-3, Claudin-4, Occludin, Tricellulin and ZO-1). Compared to the SBM, the concentration of IgM and C4 in serum was significantly reduced by dietary citric acid. In brief, dietary citric acid could synchronously inhibit TLRs-dependent inflammatory response regulated by NF-κB and IRF3, as well as cause TLRs-dependent tight junction disruption modulated by p38 and JNK. Therefore, citric acid could function on mitigating soybean meal induced enteropathy in the distal intestine of juvenile turbot.

Gene knockout analysis reveals essentiality of estrogen receptor ß1 (Esr2a) for female reproduction in medaka.

In vertebrates, sex steroids play crucial roles in multiple systems related to reproduction. In females, estrogens and their receptor estrogen receptor (ER or Esr) play indispensable roles in the negative sex steroid feedback regulation of pituitary gonadotropin secretion, which prevents excessive development of ovarian follicles. However, the mechanism of this feedback regulation of a gonadotropin, follicle stimulating hormone (FSH), which is essential for folliculogenesis throughout vertebrates, is poorly understood. In the present study, we generated knockouts of all subtypes of nuclear estrogen receptors in a model teleost medaka, which is suitable for the study of endocrine control and behavioral assays, and analyzed fertility, behavior and functionality of estrogen feedback in each knockout line. Among the estrogen receptors, we revealed that an estrogen receptor Esr2a plays an essential role in this feedback regulation. In addition to this, we also found that esr2a-/- females showed oviduct atresia, which causes complete infertility. Interestingly, esr2a-/- females showed apparently normal sexual behavior but without oviposition in response to male courtship. This phenotype indicates that physical readiness and motivation of sexual behavior is independently controlled.

Expression of Ice-Binding Proteins in Caenorhabditis elegans Improves the Survival Rate upon Cold Shock and during Freezing.

Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth at freezing temperatures. IBPs are also thought to stabilize the cell membrane at non-freezing temperatures near 0 °C. These two effects have been assumed to reduce cold- and freezing-induced damage to cells and tissues. However, knowledge regarding the effects of IBP on the living animals is limited. Here, we characterized the relationship between the IBP effects and the physiological role by using the nematode Caenorhabditis elegans. The expression of fish (NfeIBPs)- and fungus-derived IBPs (AnpIBPs and TisIBP8) in C. elegans improved its survival rate during exposure to 0 and -2 °C (cold shock) and -5 °C (freezing). The observed cold tolerance of C. elegans after cold shock is attributable to the stabilization of cell-membrane lipids with IBPs, and the freezing tolerance at -5 °C can be attributed to the inhibition of ice-crystal growth by the IBPs. Significantly, the survival rate of C. elegans at -5 °C was improved by expression of wild-type AnpIBP and maximized by that of TisIBP8, whereas it was lowered when a defective AnpIBP mutant was expressed. These results suggest that the ice-binding ability of IBP has a good correlation with the survival rate of C. elegans during freezing.

Molecular characterization and expression profiles provide new insights into GATA5 functions in tongue sole (Cynoglossus semilaevis).

GATA5 is a member of the GATA transcription factor family, which serves essential roles in varieties of cellular functions and biological processes. In this study, we have accomplished the molecular cloning, bioinformatic analysis and preliminary function study of C. semilaevis GATA5. The full-length cDNA nucleotide sequence is 1955 bp, with a coding sequence of 1167 bp, which encodes a polypeptide of 388 amino acids. Homology, phylogenetic, gene structure and synteny analysis showed that C. semilaevis GATA5 was highly conserved among vertebrates. Tissue distribution pattern exhibited that C. semilaevis GATA5 was significantly expressed in heart, intestine, liver, kidney and gonad, with a sexual dimorphic feature observed in testis and ovary. Embryonic development expression profiles showed that C. semilaevis GATA5 transcripts increased at the blastula stage, and peaked at the heat-beating period. Strong signals were detected at spermatids of male testis and stage III oocytes of female ovary by ISH. The expression of C. semilaevis GATA5 was regulated by 17α-MT and E2 after hormone stimulation to the ovary. Together, all the results pointed out that GATA5 might play a vital role during gonadal maturation and the reproductive cycle of C. semilaevis. This study lays the foundation for further researches on the sex control breeding in tongue sole.