Expression and Functional Analysis of the Metallothionein and Metal-Responsive Transcription Factor 1 in Phascolosoma esculenta under Zn Stress.

Metallothioneins (MTs) are non-enzymatic metal-binding proteins widely found in animals, plants, and microorganisms and are regulated by metal-responsive transcription factor 1 (MTF1). MT and MTF1 play crucial roles in detoxification, antioxidation, and anti-apoptosis. Therefore, they are key factors allowing organisms to endure the toxicity of heavy metal pollution. Phascolosoma esculenta is a marine invertebrate that inhabits intertidal zones and has a high tolerance to heavy metal stress. In this study, we cloned and identified MT and MTF1 genes from P. esculenta (designated as PeMT and PeMTF1). PeMT and PeMTF1 were widely expressed in all tissues and highly expressed in the intestine. When exposed to 16.8, 33.6, and 84 mg/L of zinc ions, the expression levels of PeMT and PeMTF1 in the intestine increased first and then decreased, peaking at 12 and 6 h, respectively, indicating that both PeMT and PeMTF1 rapidly responded to Zn stress. The recombinant pGEX-6p-1-MT protein enhanced the Zn tolerance of Escherichia coli and showed a dose-dependent ABTS free radical scavenging ability. After RNA interference (RNAi) with PeMT and 24 h of Zn stress, the oxidative stress indices (MDA content, SOD activity, and GSH content) and the apoptosis indices (Caspase 3, Caspase 8, and Caspase 9 activities) were significantly increased, implying that PeMT plays an important role in Zn detoxification, antioxidation, and anti-apoptosis. Moreover, the expression level of PeMT in the intestine was significantly decreased after RNAi with PeMTF1 and 24 h of Zn stress, which preliminarily proved that PeMTF1 has a regulatory effect on PeMT. Our data suggest that PeMT and PeMTF1 play important roles in the resistance of P. esculenta to Zn stress and are the key factors allowing P. esculenta to endure the toxicity of Zn.

Screening of Reference Genes for Quantitative Real-Time PCR Analysis in Tissues and during Testis Development, and Application to Analyze the Expression of kifc1 in Hemibarbus labeo (Teleostei, Cypriniformes, Cyprinidae).

The selection of proper reference genes is vital for ensuring precise quantitative real-time PCR (qPCR) assays. This study evaluates the stability of the expression of nine candidate reference genes in different tissues and during testicular development in H. labeo. The results show that eef1a is recommended as a reference gene for qPCR analysis in tissues and during testicular development. Furthermore, we evaluated the optimal number of reference genes needed when calculating gene expression levels using the geomean method, revealing that two reference genes are sufficient. Specifically, eef1a and rps27 are recommended for analysis of gene expression in tissues, whereas eef1a and actb are advised for evaluating gene expression during testicular development. In addition, we examined the expression pattern of kifc1, a kinesin involved in the reshaping of spermatids. We detected peak expression levels of kifc1 in testes, with its expression initially increasing before decreasing throughout testicular development. The highest expression of kifc1 was observed in stage IV testes, the active period of spermiogenesis, suggesting a possible role for kifc1 in the regulation of the reshaping of spermatids and hence testicular development. This study represents the first investigation of reference genes for H. labeo, providing a foundation for studying gene expression patterns and investigating gene expression regulation during testicular development.

Histological and Transcriptomic Insights into the Ovary Development of Hemibarbus labeo Injected with Spawn-Inducing Hormones.

Naturally, the ovaries of many farmed fish can only develop to stage IV (mainly including stage IV oocytes, known as full-grown postvitellogenic oocytes). Therefore, spawn-inducing hormone injections are used to promote ovary development and oocyte maturation, facilitating reproduction in the aquaculture industry. The study of spawn-inducing hormones and their underlying neuroendocrine mechanisms has been a recent focus in fish reproductive biology. However, the intra-ovarian regulatory mechanisms of ovary development and oocyte maturation after hormone injection require further investigation. In this study, we explored the histological and transcriptomic map of the ovary of Hemibarbus labeo after hormone injection to reveal changes in the ovary. The gonad index significantly increased after hormone injection for 5.5 h, after which no significant change was observed. Histological analysis showed that the nuclei had moved to one side of the oocytes at 5.5 h after hormone injection. Moreover, the volume of the oocytes increased and their yolk membranes thickened. Oocytes then underwent their first meiotic division at 5.5-11 h after hormone injection. Subsequently, the follicular membrane was ruptured, and ovulation was completed at 11-16.5 h after hormone injection. In addition, we identified 3189 differentially expressed genes (DEGs) on comparing the transcriptomes at different time points after hormone injection. These DEGs were significantly enriched in the GO terms of nervous system process, molecular transducer activity, and extracellular region, and the KEGG pathways of TNF signaling and cytokine-cytokine receptor interaction; these may play important roles in ovary development and oocyte maturation. Within these pathways, genes such as apoe, creb3, jun, junb, il11, and il8 may play important roles in steroid hormone synthesis and ovulation. Conclusively, our results show detailed sequential dynamics of oocyte development and provide new insights into the intra-ovarian regulatory mechanisms of ovarian development and oocyte maturation in H. labeo. These findings may be important for research on improving egg quality and reproduction in aquaculture.

Functional insights of digestion, absorption, and immunity in different segments of the intestine in Hemibarbus labeo from transcriptomic analysis.

The intestine is an important organ for food digestion and absorption and body immunity in fish. In this study, we investigated the abundance of transcripts from different segments of the intestinal tract using transcriptome sequencing technology in Hemibarbus labeo, to provide functional insights into digestion, absorption, and immunity in the anterior intestine (AI), middle intestine (MI), and posterior intestine (PI). We found 5646 differentially expressed genes (DEGs), which were significantly enriched to GO terms of carbohydrate metabolic process, transmembrane transport, iron ion binding, lipid metabolic process, and KEGG pathway of fat digestion and absorption, mineral absorption, protein digestion and absorption, vitamin digestion and absorption, indicating that the digestion and absorption function of food is different in AI, MI, and PI. In practice, most genes, enriched in the KEGG pathway for digestion and absorption of nutrients, are upregulated in AI and MI, indicating stronger roles for food digestion and absorption in these segments. Furthermore, we found that genes involved in the KEGG pathway of lysosome and endocytosis pathway are upregulated in PI, suggesting stronger antigen-presenting capabilities in PI. However, some cytokine receptor genes, including ccr4, cxcr2, tnfrsf9, il6r, csf3r, and cxcr4, are highly expressed in AI, reflecting the regional immune specialization in different segments. This study provides functional insights into digestion, absorption, and immunity in different segments of the intestine and supports the regional functional specialization within different segments of the intestine in H. labeo.

Expression dynamics indicate the involvement of SPG7 in the reproduction and spermiogenesis of Phascolosoma esculenta.

Spastic paraplegia 7 (SPG7) is an m-AAA protease subunit involved in mitochondrial morphology and physiology. However, its function in animal reproduction is yet to be evaluated. In this study, its molecular features, subcellular localization, and expression dynamics were investigated to analyze its potential function in the reproduction of male Phascolosoma esculenta, an economically important marine species in China. The full-length cDNA of P. esculenta spg7 (Pe-spg7) measures 3053 bp and encodes an 853-amino acid protein (Pe-SPG7). Pe-SPG7 includes two transmembrane domains, an AAA domain and a proteolytic domain. Amino acid sequence alignment revealed that SPG7 was conserved during evolution. The mRNA and protein expression of spg7 indicated its involvement in reproduction. Its expression was the highest in coelomic fluid, where spermatids develop, and it was significantly higher in the breeding stage than in the nonbreeding stage. SPG7 was mainly found in the mitochondria of spermatids in the coelomic fluid, indicating that it functions in this organelle in spermatids. Immunofluorescence experiments showed that SPG7 was expressed and colocalized in the mitochondria during spermiogenesis, suggesting its involvement in P. esculenta spermiogenesis. Therefore, SPG7 may participate in spermiogenesis by functioning in the mitochondria and regulate the reproduction of male P. esculenta. This study provided insights into the function of SPG7 in animal reproduction and P. esculenta gametogenesis.

The Effect of Prohibitins on Mitochondrial Function during Octopus tankahkeei Spermiogenesis.

Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic expression characteristics of Ot-PHBs, observed the colocalization of Ot-PHB1 with mitochondria and polyubiquitin, and studied the effect of phb1 knockdown on mitochondrial DNA (mtDNA) content, reactive oxygen species (ROS) levels, and apoptosis-related gene expression in spermatids. Our aim was to explore the effect of Ot-PHBs on mitochondrial function during the spermiogenesis of Octopus tankahkeei (O. tankahkeei), an economically important species in China. The predicted Ot-PHB1/PHB2 proteins contained an N-terminal transmembrane, a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin domain), and a C-terminal coiled-coil domain. Ot-phb1/phb2 mRNA were widely expressed in the different tissues, with elevated expression in the testis. Further, Ot-PHB1 and Ot-PHB2 were highly colocalized, suggesting that they may function primarily as an Ot-PHB compiex in O. tankahkeei. Ot-PHB1 proteins were mainly expressed and localized in mitochondria during spermiogenesis, implying that their function may be localized to the mitochondria. In addition, Ot-PHB1 was colocalized with polyubiquitin during spermiogenesis, suggesting that it may be a polyubiquitin substrate that regulates mitochondrial ubiquitination during spermiogenesis to ensure mitochondrial quality. To further investigate the effect of Ot-PHBs on mitochondrial function, we knocked down Ot-phb1 and observed a decrease in mtDNA content, along with increases in ROS levels and the expressions of mitochondria-induced apoptosis-related genes bax, bcl2, and caspase-3 mRNA. These findings indicate that PHBs might influence mitochondrial function by maintaining mtDNA content and stabilizing ROS levels; in addition, PHBs might affect spermatocyte survival by regulating mitochondria-induced apoptosis during spermiogenesis in O. tankahkeei.

Toward Better Structure and Constraint to Mine Negative Sequential Patterns.

Nonoccurring behavior (NOB) studies have attracted the growing attention of scholars as a crucial part of behavioral science. As an effective method to discover both NOB and occurring behaviors (OB), negative sequential pattern (NSP) mining is successfully used in analyzing medical treatment and abnormal behavior patterns. At this time, NSP mining is still an active and challenging research domain. Most of the algorithms are inefficient in practice. Briefly, the key weaknesses of NSP mining are: 1) an inefficient positive sequential pattern (PSP) mining process, 2) a strict constraint of negative containment, and 3) the lack of an effective Negative Sequential Candidate (NSC) generation method. To address these weaknesses, we propose a highly efficient algorithm with improved techniques, named sc-NSP, to mine NSP efficiently. We first propose an improved PrefixSpan algorithm in the PSP mining process, which connects to a bitmap storage structure instead of the original structure. Second, sc-NSP loosens the frequency constraint and exploits the NSC generation method of positive and negative sequential patterns mining (PNSP) (a classic NSP mining method). Furthermore, a novel pruning strategy is designed to reduce the computational complexity of sc-NSP. Finally, sc-NSP obtains the support of NSC by using the most efficient bitwise-based calculation operation. Theoretical analyses show that sc-NSP performs particularly well on data sets with a large number of elements and items in sequence. Comparison and extensive experiments along with case studies on health data show that sc-NSP is 10 times more efficient than other state-of-the-art methods, and the number of NSPs obtained is 5 times greater than other methods.

Transcriptomes of Testes at Different Developmental Stages in the Opsariichthys bidens Predict Key Genes for Testis Development and Spermatogenesis.

Testis development is a complex process involving multiple genes, and the molecular mechanisms underlying testis development in Opsariichthys bidens remain unclear. We performed transcriptome sequencing analysis on a total of 12 samples of testes from stages II, III, IV, and V of O. bidens and obtained a total of 79.52 Gb clean data, as well as 288,573 transcripts and 116,215 unigenes. Differential expression analysis showed that 22,857 differentially expressed genes (DEGs) were screened in six comparison groups (III vs. II, IV vs. II, V vs. II, IV vs. III, V vs. III, and V vs. IV). Kyoto Encyclopedia of Genes and Genomes enrichment analysis of DEGs showed that six comparison groups were significantly enriched for a total of 20 significantly up- or down-regulated pathways, including six pathways related to signal transduction, three pathways related to energy metabolism, five pathways related to disease, and two pathways related to ribosomes. Furthermore, our investigation revealed that DEGs were enriched in several important functional pathways, such as Huntington's disease signaling pathway, TGF-ß signaling pathway, and ribosome signaling pathway. Protein-protein interaction network analysis of DEGs identified 63 up-regulated hub genes, including 9 kinesin genes and 2 cytoplasmic dynein genes, and 39 down-regulated hub genes, including 13 ribosomal protein genes. This result contributes to the knowledge of spermatogenesis and testis development in O. bidens.

Expression Dynamics Indicate Potential Roles of KIF17 for Nuclear Reshaping and Tail Formation during Spermiogenesis in Phascolosoma esculenta.

Kinesin family member17 (KIF17), a homologous dimer of the kinesin-2 protein family, has important microtubule-dependent and -independent roles in spermiogenesis. Little is known about KIF17 in the mollusk, Phascolosoma esculenta, a newly developed mariculture species in China. Here, we cloned the open reading frame of Pe-kif17 and its related gene, Pe-act, and performed bioinformatics analysis on both. Pe-KIF17 and Pe-ACT are structurally conserved, indicating that they may be functionally conserved. The expression pattern of kif17/act mRNA performed during spermiogenesis revealed their expression in diverse tissues, with the highest expression level in the coelomic fluid of P. esculenta. The expressions of Pe-kif17 and Pe-act mRNA were relatively high during the breeding season (July-September), suggesting that Pe-KIF17/ACT may be involved in spermatogenesis, particularly during spermiogenesis. Further analysis of Pe-kif17 mRNA via fluorescence in situ hybridization revealed the continuous expression of this mRNA during spermiogenesis, suggesting potential functions in this process. Immunofluorescence showed that Pe-KIF17 co-localized with α-tubulin and migrated from the perinuclear cytoplasm to one side of the spermatid, forming the sperm tail. Pe-KIF17 and Pe-ACT also colocalized. KIF17 may participate in spermiogenesis of P. esculenta, particularly in nuclear reshaping and tail formation by interacting with microtubule structures similar to the manchette. Moreover, Pe-KIF17 with Pe-ACT is also involved in nuclear reshaping and tail formation in the absence of microtubules. This study provides evidence for the role of KIF17 during spermiogenesis and provides theoretical data for studies of the reproductive biology of P. esculenta. These findings are important for spermatogenesis in mollusks.

Mitochondrial Features and Expressions of MFN2 and DRP1 during Spermiogenesis in Phascolosoma esculenta.

Mitochondria can fuse or divide, a phenomenon known as mitochondrial dynamics, and their distribution within a cell changes according to the physiological status of the cell. However, the functions of mitochondrial dynamics during spermatogenesis in animals other than mammals and fruit flies are poorly understood. In this study, we analyzed mitochondrial distribution and morphology during spermiogenesis in Sipuncula (Phascolosoma esculenta) and investigated the expression dynamics of mitochondrial fusion-related protein MFN2 and fission-related protein DRP1 during spermiogenesis. The mitochondria, which were elliptic with abundant lamellar cristae, were mainly localized near the nucleus and distributed unilaterally in cells during most stages of spermiogenesis. Their major axis length, average diameter, cross-sectional area, and volume are significantly changed during spermiogenesis. mfn2 and drp1 mRNA and proteins were most highly expressed in coelomic fluid, a spermatid development site for male P. esculenta, and highly expressed in the breeding stage compared to in the non-breeding stage. MFN2 and DRP1 expression levels were higher in components with many spermatids than in spermatid-free components. Immunofluorescence revealed that MFN2 and DRP1 were consistently expressed and that MFN2 co-localizes with mitochondria during spermiogenesis. The results provide evidence for an important role of mitochondrial dynamics during spermiogenesis from morphology and molecular biology in P. esculenta, broadening insights into the role of mitochondrial dynamics in animal spermiogenesis.

Characterization of Copper/Zinc Superoxide Dismutase Activity on Phascolosoma esculenta (Sipuncula: Phascolosomatidea) and Its Protection from Oxidative Stress Induced by Cadmium.

Phascolosoma esculenta, an economically important species inhabiting the high tide areas of the intertidal zone, is particularly sensitive to water pollution. Considering its potential as a bioindicator, studies on the ecotoxicology of P. esculenta are imperative. The toxic effects of cadmium (Cd) were analyzed by exposing P. esculenta to different concentrations of Cd (6, 24, 96 mg/L). In this study, the changes in the antioxidative indexes of total superoxide dismutase (T-SOD), glutathione s-transferase (GST), reduced glutathione (GSH), and microscale malondialdehyde (MDA) were recorded. Copper/zinc superoxide dismutase (Cu/Zn SOD) is one of the most important free radical scavenging members. To reveal the antioxidative function of P. esculenta, an important member of the antioxidative system, designated Pe-Cu/Zn SOD, was cloned and analyzed. Phylogenic analysis revealed that Pe-Cu/Zn SOD was located in the invertebrate evolutionary branch of intracellular Cu/Zn SOD (icCu/Zn SOD). The quantitative real-time polymerase chain reaction results showed that Pe-Cu/Zn SOD messenger ribonucleic acid was widely expressed in all tissues examined. The highest expression levels in coelomic fluid after Cd exposure indicated its function in the stress response. Using a prokaryotic expression system, we obtained a Pe-Cu/Zn SOD recombinant protein, which enhanced the heavy metal tolerance of Escherichia coli. In vivo assays also confirmed that the Pe-Cu/Zn SOD recombinant protein had an antioxidative and free radical scavenging ability. A Cd toxicity experiment, in which purified Pe-Cu/Zn SOD protein was injected into the body cavities of P. esculenta, showed that the reactive oxygen species content in the coelomic fluid of the experimental group was significantly lower compared with the control group. These results suggest that Pe-Cu/Zn SOD played a role in Cd detoxification by chelating heavy metal ions and scavenging reactive oxygen free radicals, and that P. esculenta could be used as a bioindicator to evaluate heavy metal pollution.

Comparative Transcriptome Analysis of Head Kidney of Aeromonas hydrophila-infected Hypoxia-tolerant and Normal Large Yellow Croaker.

The large yellow croaker (Larimichthys crocea) is one of the most economically important marine fish on the southeast coast of China and much of its yield is usually lost by hypoxia. To address this problem and lay a foundation for culturing a new strain of large yellow croaker with hypoxia tolerance, our research group screened a hypoxia-tolerant population of L. crocea. Surprisingly, we also found that hypoxia-tolerant population exhibited higher survival when infected with pathogens compared to the normal population during the farming operation. In order to understand the mechanism underlying the higher survival rate of the hypoxia-tolerant population and enrich the head kidney immune mechanism of L. crocea infected with pathogens, we compared and analyzed the head kidney transcriptome of the hypoxia-tolerant and normal individuals under Aeromonas hydrophila infection. We obtained 159.68 GB high-quality reads, of which more than 87.61% were successfully localized to the reference genome of L. crocea. KEGG analysis revealed differentially expressed genes in the signaling pathways involving immunity, cell growth and death, transport and catabolism, and metabolism. Among these, the toll-like receptor signaling pathway, Nod-like receptor signaling pathway, cytokine-cytokine receptor interaction, phagosome, apoptosis, and OXPHOS pathways were enriched in both groups after infection compared to before, and were enriched in infected tolerant individuals compared to normal individuals. In addition, we found that the expression of hif1α and its downstream genes were higher in the hypoxia-sensitive group of fish than in the normal group. In conclusion, our results showed some signaling pathways and hub genes, which may participate in A. hydrophila defense in the head kidney of two populations, and may contribute to the higher survival rate in the hypoxia-tolerant population. Overall, these findings increase our understanding of the defense mechanism within the head kidney of L. crocea under A. hydrophila infection, and suggest a preliminary hypothesis for why hypoxia-tolerant individuals may exhibit a higher survival rates after infection. Our study provides scientific evidence for the breeding of a new hypoxia-tolerant strain of L. crocea for aquaculture.

Characterization of Mitochondrial Prohibitin in Opsariichthys bidens and Its Potential Functions in Spermatogenesis.

Spermatogenesis is the intricate and coordinated process by which spermatogonia develop into haploid differentiated spermatozoa. Mitochondria are essential for spermatogenesis, and prohibitin (PHB) is closely associated with mitochondrial structure and function during spermatogenesis. Although PHB has been implicated in spermatogenesis in some taxa, its roles in Opsariichthys bidens have not been determined. In this study, the expression patterns and potential functions of PHB in spermatogenesis in O. bidens were characterized using histological microscopic observations, PCR cloning, real-time quantitative PCR (qPCR), Western blotting (WB) and immunofluorescence (IF). The full-length cDNA of Ob-phb was 1500 bp encoding 271 amino acids. A sequence alignment demonstrated that the PHB protein is conserved among different animals. qPCR revealed that phb mRNA is widely distributed in O. bidens and highly expressed in the testes at stages IV and V. WB revealed that Ob-PHB is located in the mitochondria of testes. IF revealed the colocalization of PHB signals and mitochondria. Signals were detected around nuclei in spermatogonia and spermatocytes, gradually moving to the tail region during spermiogenesis, and finally aggregating in the midpiece. These results indicate that Ob-PHB was expressed in the mitochondria during spermatogenesis. In addition, this study proposed Ob-PHB may participate in the degradation of mitochondria and cell differentiation during spermatogenesis.

The potential function of KIF17 in large yellow croaker (Larimichthys crocea) spermatid remodeling: molecular characterization and expression pattern during spermiogenesis.

KIF17, which belongs to the kinesin-2 protein family, plays an indispensable role in mammalian spermiogenesis. However, the role of KIF17 in fish spermatid remodeling during spermiogenesis remains poorly understood. Therefore, we aimed to study the role of KIF17 in spermatid remodeling during Larimichthys crocea (L. crocea) spermiogenesis. The kif17 cDNA sequence, 3247 bp in length, was cloned from L. crocea testis, which consisted of a 347-bp 5'-untranslated region (UTR), 413-bp 3' -UTR, and 2487-bp open reading frame. Bioinformatic analyses revealed that KIF17 obtained from L. crocea (Lc-KIF17) exhibited a high sequence identity compared with those from other teleosts and possessed the structural features of other kinesin-2 proteins. Based on structural similarity, we speculate that the role of Lc-KIF17 may be similar to that of KIF17 in other animals. Lc-kif17 mRNA was diffusely expressed in L. crocea tissues and was highly expressed in the testis, especially at stage IV testicular development. Immunofluorescence analysis revealed that Lc-KIF17 signals colocalized with ß-tubulin signals and migrated from the perinuclear cytoplasm to the side of the nucleus where the tail forms during spermiogenesis. These findings revealed that KIF17 may be involved in L. crocea spermiogenesis. In particular, KIF17 may participate in spermatid remodeling by interacting with perinuclear microtubules during L. crocea spermiogenesis. Collectively, this study contributes to an improved understanding of the mechanism underlying L. crocea spermiogenesis and provides a basis for further research on L. crocea reproduction and development.

Molecular Cloning of Dynein Heavy Chain and the Effect of Dynein Inhibition on the Testicular Function of Portunus trituberculatus.

Dynein is a motor protein with multiple transport functions. However, dynein's role in crustacean testis is still unknown. We cloned the full-length cDNA of cytoplasmic dynein heavy chain (Pt-dhc) gene and its structure was analyzed. Its expression level was highest in testis. We injected the dynein inhibitor sodium orthovanadate (SOV) into the crab. The distribution of Portunus trituberculatus dynein heavy chain (Pt-DHC) in mature sperm was detected by immunofluorescence. The apoptosis of spermatids was detected using a TUNEL kit; gene expression in testis was detected by fluorescence quantitative PCR (qPCR). The expression of immune-related factors in the testis were detected by an enzyme activity kit. The results showed that the distribution of Pt-DHC was abnormal after SOV injection, indicating that the function of dynein was successfully inhibited. Apoptosis-related genes p53 and caspase-3, and antioxidant stress genes HSP70 and NOS were significantly decreased, and anti-apoptosis gene bcl-2 was significantly increased. The activities of superoxide dismutase (SOD) and alkaline phosphatase (AKP) were significantly decreased. The results showed that there was no apoptosis in testicular cells after dynein function was inhibited, but the cell function was disordered. This study laid a theoretical foundation for the further study of apoptosis in testis and the function of dynein in testis and breeding of P. trituberculatus.

Genetics Responses to Hypoxia and Reoxygenation Stress in Larimichthys crocea Revealed via Transcriptome Analysis and Weighted Gene Co-Expression Network.

The large yellow croaker (Larimichthys crocea) is an important marine economic fish in China; however, its intolerance to hypoxia causes widespread mortality. To understand the molecular mechanisms underlying hypoxia tolerance in L. crocea, the transcriptome gene expression profiling of three different tissues (blood, gills, and liver) of L. crocea exposed to hypoxia and reoxygenation stress were performed. In parallel, the gene relationships were investigated based on weighted gene co-expression network analysis (WGCNA). Accordingly, the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that several pathways (e.g., energy metabolism, signal transduction, oxygen transport, and osmotic regulation) may be involved in the response of L. crocea to hypoxia and reoxygenation stress. In addition, also, four key modules (darkorange, magenta, saddlebrown, and darkolivegreen) that were highly relevant to the samples were identified by WGCNA. Furthermore, some hub genes within the association module, including RPS16, EDRF1, KCNK5, SNAT2, PFKL, GSK-3ß, and PIK3CD, were found. This is the first study to report the co-expression patterns of a gene network after hypoxia stress in marine fish. The results provide new clues for further research on the molecular mechanisms underlying hypoxia tolerance in L. crocea.

Hif-1α/Hsf1/Hsp70 signaling pathway regulates redox homeostasis and apoptosis in large yellow croaker ( Larimichthys crocea) under environmental hypoxia.

Oxygen is an essential molecule for animal respiration, growth, and survival. Unlike in terrestrial environments, contamination and climate change have led to the frequent occurrence of hypoxia in aquatic environments, thus impacting aquatic animal survival. However, the adaptative mechanisms underlying fish responses to environmental hypoxia remain largely unknown. Here, we used large yellow croaker ( Larimichthys crocea) and large yellow croaker fry (LYCF) cells to investigate the roles of the Hif-1α/Hsf1/Hsp70 signaling pathway in the regulation of cellular redox homeostasis, and apoptosis. We confirmed that hypoxia induced the expression of Hif-1α, Hsf1, and Hsp70 in vivo and in vitro. Genetic Hsp70 knockdown/overexpression indicated that Hsp70 was required for maintaining redox homeostasis and resisting oxidative stress in LYCF cells under hypoxic stress. Hsp70 inhibited caspase-dependent intrinsic apoptosis by maintaining normal mitochondrial membrane potential, enhancing Bcl-2 mRNA and protein expression, inhibiting Bax and caspase3 mRNA expression, and suppressing caspase-3 and caspase-9 activation. Hsp70 suppressed caspase-independent intrinsic apoptosis by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and disturbed extrinsic apoptosis by inactivating caspase-8. Genetic knockdown/overexpression of Hif-1α and dual-luciferase reporter assay indicated that Hif-1α activated the Hsf1 DNA promoter and enhanced Hsf1 mRNA transcription. Hsf1 enhanced Hsp70 mRNA transcription in a similar manner. In summary, the Hif-1α/Hsf1/Hsp70 signaling pathway plays an important role in regulating redox homeostasis and anti-apoptosis in L. crocea under hypoxic stress.

Effects of acute low temperature stress on the hormones and gene expression of glucocorticoid receptor of large yellow croaker Larimichthys crocea.

The neuroendocrine system of fish responds to low temperature via regulating hormones. To explore the adaptability of Larimichthys crocea to low temperature, the levels of the plasma cortisol, thyroid stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), total cholesterol (TC), and glucose were determined after exposure to low temperature and during subsequent rewarming. Furthermore, the mRNA expression of the glucocorticoid receptor (GR) gene was analyzed under the stress. We found that the levels of the plasma cortisol, TSH, T3, glucose, and TC increased under the low temperature stress, suggesting that elevated hormones may be conducive to promoting the mobilization of the glucose and lipid in L. crocea exposed to low temperature. During the rewarming period, the plasma cortisol level decreased, whereas the T3 level was still significantly higher than that in the control group. Notably, the plasma T4 level was unaffected by the temperature changes. Furthermore, the sequence alignment and phylogenetic tree analysis revealed that the GR protein of L. crocea had high homology and a similar protein structure with those from other teleosts. Under the low temperature stress, the GR mRNA expression increased in the brain and head kidney, whereas it basically returned to the control level following rewarming. These findings revealed the changes of the hormones and the potential function of the GR gene in L. crocea following exposure to low temperature, providing some insights into breeding low temperature-resistant varieties of L. crocea.

Ultrastructure evidence for vesicles and double-membrane structures involved in cytoplasmic elimination during spermiogenesis in large yellow croaker, Larimichthys crocea (Teleostei, Perciformes, Scienidae).

Spermatids eliminate excess cytoplasm to form streamlined sperm during spermiogenesis, which mechanism is insufficiently elucidated in fish. In this study, we investigated the cytoplasmic elimination procedure in spermatid during spermiogenesis in the large yellow croaker (Larimichthys crocea) using transmission electron microscopy. The early spermatid is subrotund with a centrally located nucleus. With further development, nucleus polarizes into one side of the cell while the cytoplasm with numerous vesicles near the membrane migrates to the caudal region. Furthermore, exocytosis-like structures were detected in middle spermatid. In late spermatid, the vesicles are reduced and rarely observed. These findings indicate that vesicles may be involved in cytoplasmic elimination possibly via exocytosis. In the later spermatid, a double-membrane, autophagosome-like structure envelopes the cytoplasm, which may develop into a single-membrane structure, and gets discarded from the cell as a residual body from the caudal region. This suggests its potential functions in the formation of residual body and cytoplasmic elimination. Overall, our results revealed that polarized development of spermatid causes polarized distribution of cytoplasm necessary for cytoplasmic elimination. Moreover, they provide ultrastructure evidence for vesicles and double-membrane structures involved in discarding spermatid cytoplasm in large yellow croaker, thus offering novel insights into cytoplasmic elimination during spermiogenesis in fish.