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.

Experimental and Numerical Studies on the Behaviors of Autoclaved Aerated Concrete Panels with Insulation Boards Subjected to Wind Loading.

Autoclaved aerated concrete panels (AACP) are lightweight elements in civil engineering design. In this paper, experiments and numerical analyses were conducted to study the flexural behavior of an enclosure system that consisted of AACPs and a decorative plate. A full-scale test was conducted to investigate the behavior of the enclosure system under wind suction. Load-deflection curves and load-strain relationships under different wind pressures were recorded and discussed. The effects of thickness, reinforcement ratio, and strength grade on the flexural behavior of AACPs were numerically investigated. Based on the numerical results, we found that the flexural behavior of AACPs can be improved by increasing the thickness or the reinforcement ratio. A comparison of finite element and theoretical results calculated using American and Chinese design formulae was conducted, and the results indicated the existing design formulae can conservatively estimate the major mechanical indices of AACPs.

The Whole-Genome Sequencing and Hybrid Assembly of Mytilus coruscus.

The hard-shelled mussel (Mytilus coruscus) is an economically important shellfish that has been cultivated for the last decade. Due to over-exploitation, most mussel stocks have dramatically declined. Efforts to study this species' natural distribution, genetics, breeding, and cultivation have been hindered by the lack of a high-quality reference genome. To address this, we produced a hybrid high-quality reference genome of M. coruscus using a long-read platform to assemble the genome and short-read, high-quality technology to accurately correct for sequence errors. The genome was assembled into 10,484 scaffolds, a total length of 1.90 Gb, and a scaffold N50 of 898 kb. Ab initio annotation of the M. coruscus genome assembly identified a total of 42,684 genes. This accurate reference genome of M. coruscus provides an essential resource with the advantage of enabling the genome-scale selective breeding of M. coruscus. More importantly, it will also help in deciphering the speciation and local adaptation of the Mytilus species.

Gene Identification and Characterization of Correlations for DEPs_DEGs Same Trend Responding to Salinity Adaptation in Scylla paramamosain.

Scylla paramamosain is a commercially important species distributed along the coast of southern China and other Indo-Pacific countries. Sudden salinity drop exceeding the adjustment capability of S. paramamosain can result in damage or even mortality. In our previous study, we had analyzed the mechanism of adapting sudden drop in salinity from the level of transcriptomics and proteomics, respectively. This study performed a correlation analysis of RNA sequencing transcriptomics and iTRAQ proteomics in order to investigate the adaptation mechanisms to sudden salinity drop from 23‰ to 3‰. There were 3954 correlations and a total of 15 correlations for differentially expressed proteins (DEPs) and differentially expressed genes (DEGs) from proteomics and transcriptomics. The correlation between DEPs and DEGs was 0, and the Spearman correlation coefficient of the same trend correlation for DEPs and DEGs was the highest, reaching 0.9080. KEGG pathway enrichment correlation revealed that protein digestion and absorption (Ko04974), proximal tubule bicarbonate (Ko04964), and bile secretion (Ko04976) played important roles in Na+/H+ and Na+/K+ exchange. In addition, important genes related to osmoregulation, such as ion transport and carbonic anhydrase, were also detected in the correlation analysis for same trend DEPs_DEGs. In conclusion, the proteome and transcriptome correlation results from this study indicate that ion transport plays a critical role in the adaptation of S. paramamosain to sudden reduction in salinity.

Identification and characterization of miRNAs in the gills of the mud crab (Scylla paramamosain) in response to a sudden drop in salinity.

BACKGROUND: The mud crab (Scylla paramamosain) is a euryhaline and commercially important species. MiRNAs participate in the regulation of many physiological activities. RESULTS: The miRNA transcriptome of the gills of S. paramamosain was used to investigate the expression profiles of miRNAs in response to a sudden drop in salinity. In total, seven known miRNAs and 43 novel miRNAs were identified, with 18 differentially expressed small RNAs. Fourteen thousand nine hundred fifty-one differentially expressed miRNAs target genes were screened by prediction. GO analysis of differentially expressed miRNAs target genes indicated that 578 genes associated with cellular processes, 523 associated with metabolic processes, and 422 associated with single-organism processes were the most strongly affected by a sudden drop in salinity from 23‰ to 3‰. KEGG pathway analysis showed 14 pathways were related to amino acid metabolism, which plays an important role in osmoregulation. Besides, several pathways were associated with starch and sucrose metabolism (ko00500), glycosaminoglycan degradation (ko00531), and galactose metabolism (ko00052). CONCLUSIONS: S. paramamosain regulated osmotic pressure and energy balance by regulating target genes to adapt to a sudden changes in salinity. These results provided a basis for further investigations of miRNA-modulating networks underlying the osmoregulation of S. paramamosain.

A proteomics of gills approach to understanding salinity adaptation of Scylla paramamosain.

Scylla paramamosain (Crustacea) is a commercially important euryhaline species distributed along the coast of southern China and other Indo-Pacific countries. However, a sudden variation in salinity will cause injury or even death to S. paramamosain. In this paper, we simulated a sudden decrease in salinity due to heavy precipitation in crab ponds. Comparison of gill microstructures of individuals in the control group and decreased salinity group showed gills became shorter and thicker, while the top of the filaments became swollen and then returned to normal after 120 h. A total of 3962 proteins were identified by proteomic sequencing of gills after 120 h under conditions of decreased salinity. 845 proteins were differentially expressed: 371 up-regulated and 474 down-regulated. Of the enriched KEGG pathways, 20 were up-regulated and 14 were down-regulation (P < 0.05). Among the significantly enriched up-regulated pathways, six were associated with amino acid metabolism and three were associated with Na+-K+-ATPase enzymatic activities. Pathways associated with redox metabolism and energy metabolism were identified. These results showed that in response to a decrease in salinity, S. paramamosain could adapt to the environment after 120 h. Molecular mechanism of this adaptation involved amino acid metabolism and Na+-K+-ATPase ion transport. Meanwhile, energy metabolism and redox metabolism were critical to the adaptation to a sudden decrease in salinity. This study, for the first time at the protein level, revealed the molecular mechanisms underlying salinity adaption of S. paramamosain and provides theoretical guidelines for the cultivation of S. paramamosain and other marine crustaceans.

Transcriptomic analysis of adaptive mechanisms in response to sudden salinity drop in the mud crab, Scylla paramamosain.

BACKGROUND: Scylla paramamosain (Crustacea: Decapoda: Portunidae: Syclla De Hann) is a commercially important mud crab distributed along the coast of southern China and other Indo-Pacific countries (Lin Z, Hao M, Zhu D, et al, Comp Biochem Physiol B Biochem Mol Biol 208-209:29-37, 2017; Walton ME, Vay LL, Lebata JH, et al, Estuar Coast Shelf Sci 66(3-4):493-500, 2006; Wang Z, Sun B, Zhu F, Fish Shellfish Immunol 67:612-9, 2017). While S. paramamosain is a euryhaline species, a sudden drop in salinity induces a negative impact on growth, molting, and reproduction, and may even cause death. The mechanism of osmotic regulation of marine crustaceans has been recently under investigation. However, the mechanism of adapting to a sudden drop in salinity has not been reported. METHODS: In this study, transcriptomics analysis was conducted on the gills of S. paramamosain to test its adaptive capabilities over 120 h with a sudden drop in salinity from 23 ‰ to 3 ‰. RESULTS: At the level of transcription, 135 DEGs (108 up-regulated and 27 down-regulated) annotated by NCBI non-redundant (nr) protein database were screened. GO analysis showed that the catalytic activity category showed the most participating genes in the 24 s-tier GO terms, indicating that intracellular metabolic activities in S. paramamosain were enhanced. Of the 164 mapped KEGG pathways, seven of the top 20 pathways were closely related to regulation of the Na+ / K+ -ATPase. Seven additional amino acid metabolism-related pathways were also found, along with other important signaling pathways. CONCLUSION: Ion transport and amino acid metabolism were key factors in regulating the salinity adaptation of S. paramamosain in addition to several important signaling pathways.

Molecular cloning of a C-type lectin from Portunus trituberculatus, which might be involved in the innate immune response.

C-type lectin plays an important role in the innate immune response of crustaceans including Portunus trituberculatus which is an important marine species. In the present study, we cloned the full length of a C-type lectin (designated as PtCTL4) from P. trituberculatus via 3'RACE. The full length of the nucleic acid sequence has a length of 654 bp including an open reading frame (ORF) of 480 bp. PtCTL4 possesses conserved CTL features, while containing a CRD domain with Ca2+ binding site 2 and six conserved cysteine residues. Quantitative RT-PCR analysis showed that PtCTL4 expression level was highest in the hepatopancreas, while it was relatively low in other tissues such as hemocytes, eyestalk, muscle, and gonad. The expression level of PtCTL4 reached a maximum at 3 h after challenge with Vibrio alginolyticus, then decreased to the lowest level at 12 h, and returned to normal level at 48 h. Hemagglutination analysis showed that the recombinant PtCTL4 (rPtCTL4) can agglutinate rabbit erythrocyte. The rPtCTL4 can agglutinate Gram-positive bacteria (Bacillus aquimaris, Micrococcus lysodeik, and Staphylococcus aureus) and Gram-negative bacteria (Aeromonas hydrophila, V. alginolyticus, and Chryseobacterium indologenes) in the presence of Ca2+. This study indicated that PtCTL4 acts as a pattern recognition receptor in the innate immune response of P. trituberculatus.

Characterization and functional analysis of a novel C-type lectin from the swimming crab Portunus trituberculatus.

C-type lectins (CTLs) are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a novel C-type lectin (designated as PtCTL1) was identified and characterized from Portunus trituberculatus. The full-length cDNA of PtCTL1 was of 702 bp, containing a 5' untranslated region (UTR) of 91 bp, a 3' UTR of 110 bp with a poly (A) tail, and an open reading frame (ORF) of 501 bp encoding a polypeptide of 166 amino acids with a putative signaling peptide of 21 amino acids. A C-type lectin carbohydrate-recognition domain (CRD) containing four conserved cysteines was identified in the amino acid sequence of PtCTL1. The cDNA fragment encoding the mature peptide of PtCTL1 was recombined into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame and expressed in Escherichia coli Origami (DE3). The recombinant PtCTL1 (rPtCTL1) can agglutinate all the tested bacteria, including three Gram-positive bacterial strains and three Gram-negative bacterial strains. In addition, erythrocyte agglutination and LPS-binding activity were observed in a Ca2+-dependent manner. The erythrocyte agglutination was inhibited by EDTA, indicating that PtCTL1 was Ca2+-dependent. The mRNA transcripts of PtCTL1 were detected mainly in the tissues of hepatopancreas and hemocytes and its levels were significantly up-regulated in hemocytes following Vibrio alginolyticus challenge. These results indicate that PtCTL1 may function as a pattern recognition receptor (PRR) for protecting P. trituberculatus from bacterial infection. Moreover, such findings also provide evidence for further understanding the innate immunology of invertebrate.