Quanxin Gao

Transcriptome and analysis on the complement and coagulation cascades pathway of large yellow croaker (Larimichthys crocea) to ciliate ectoparasite Cryptocaryon irritans infection

Large yellow croaker (Larimichthys crocea) is one of the most valuable marine fish in southern China. Given to the rapid development of aquaculture industry, the L. crocea was subjected to ciliate ectoparasite Cryptocaryon irritans. It therefore is indispensable and urgent to understand the mechanism of L. crocea host defense against C. irritans infection. In the present study, the extensively analysis at the transcriptome level for Cryptocaryoniasis in L. crocea was carried out. These results showed that 15,826,911, 16,462,921, and 15,625,433 paired-end clean reads were obtained from three cDNA libraries (A: 0 theronts/fish, B: 12,000 theronts/fish, and C: 24,000 theronts/fish) of the L. crocea immune-related tissues by Illumina paired-end sequencing technology. Totally, 30,509 unique transcript fragments (unigenes) were assembled, with an average length of 1715 bp. In B/A, C/A, and C/B pairwise comparison, 972, 900, and 1126 genes showed differential expression respectively. Differently expressed immune-related genes (DEIGs) were scrutinized, in B/A pairwise comparison, 48 genes showed differential expression, including 26 up-regulated genes and 22 down-regulated genes in B; in C/A pairwise comparison, there were 39 DEIGs, including 7 up-regulated genes and 32 down-regulated genes in C; in C/B pairwise comparison, 40 genes showed differential expression, including 11 up-regulated genes and 29 down-regulated genes in C. There were 16 DEIGs enriched KEGG pathways, in which the complement and coagulation cascades pathway was the top most DEIGs enriched pathway (B:A = 42; C:A = 28; C:B = 42). The coagulation and fibrinolytic system was in a highly active state after infected by C. irritans with non-lethal concentration; the alternative complement pathway may play an important role in the early stages of C. irritans infection. These results demonstrated that low-concentration infection can significantly induce the immunological response in fishes, however, when fishes were in fatal conditions, the immunity was suppressed.

Effects of Cryptocaryon irritans infection on the survival, feeding, respiratory rate and ionic regulation of the marbled rockfish Sebastiscus marmoratus.

To clarify the effects of a Cryptocaryon irritans infection on the physiological functions of the marbled rockfish Sebastiscus marmoratus, this study utilized C. irritans at concentrations of 2500; 5000; 7500; 10,000; 20,000; and 30,000 theronts/fish to infect marbled rockfish weighing 45 ± 3 g. The survival rate, food intake, respiratory rate, serum ion concentrations and gill Na+/K+-ATPase activity were determined. With the increase of the infection concentration and the passage of time, the survival rate of the rockfish gradually decreased. The groups infected with more than 5000 theronts/fish had stopped feeding within 4 days. The respiratory rates of the fish in the groups infected with 2500 and 5000 theronts/fish initially increased and then decreased. In contrast, the respiratory rate of the fish in the groups infected with more than 7500 theronts/fish was elevated to levels significantly higher than the control group after 12 h. The Na+/K+-ATPase activity and serum Na+ and Cl- concentrations increased with increasing infection concentration. In conclusion, the physiological functions of the fish infected with low concentrations of C. irritans can be effectively restored, whereas a high concentration infection induced severe stress. The declined food intake and accelerated respiratory rate could be useful for an early warning system as important indicators.

Growth, feed intake and immune responses of orange-spotted grouper (Epinephelus coioides) exposed to low infectious doses of ectoparasite (Cryptocaryon irritans)

To explore the effect of low-dose Cryptocaryon irritans infection on growth, feeding and antiparasitic immunity of orange-spotted grouper (Epinephelus coioides), this study utilized C. irritans at concentrations of 5500 theronts/fish (Group I, 1/10 of 96 h LC50) or 11,000 theronts/fish (Group II) to infect E. coioides weighing 38 g on average at week 0, 2 and 4, respectively. Food consumption was recorded daily; the fish were weighed weekly; serum immobilizing titer (SIT), and acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), lysozyme (LZM) activity were recorded every 2 weeks; the fish were treated with lethal dose (70,000 theronts/fish) of C. irritans in the 8th week and death number were recorded. The result shows that in the 1st week after the first infection, the fishs weight gain (WG), length gain (LG), and specific growth rate (SGR) dropped as parasite dose increased, and WG, SGR values were negative; while, after the 2nd and the 3rd infection, no significant differences were detected among the three groups. These results indicated that the 1st infection affected the fish most, while the following infections were protected by some immunity. In the 3rd, 7th, and 8th week, condition factor (CF) increased with the increased infectious dose, indicating that the parasite affected body length more than body weight. As the experiment went on, accumulated food consumption (AFC) of all three groups steadily grew (control > Group I > Group II). But on the 2nd day after the first infection, daily food consumption (DFC) of Group I and II significantly dropped, the decline of Group II was greater than that of Group I, DFC recovered in the following week, with Group I earlier than Group II. After the 2nd infection, DFC of Group I and II dropped again, Group II still dropped more than Group I, and both groups recovered on the 3rd day after infection. The 3rd infection caused no significant difference in week food consumption (WFC). These results indicated that a higher dose of infection causes a greater drop in FC and a slower recovery. Weekly feed conversion ratio (WFCR) values of Group I and II in the 1st week was negative; in the 2nd week, WFCR was lower in the group infected by a higher dose of parasite; while in the 3rd and following weeks, no significant pattern was observed. Accumulate feed conversion ratio (AFCR) dropped as the infectious dose increased (control > Group I > Group II), AFCR of Group I and II reached above 0 in the 2nd and 4th week, respectively. From the 4th week on, the inter-group AFCR of the 3 groups still took on a declining trend with the increased infectious dose but the gap became smaller. One week after the first infection, SIT of Group I and Group II were 0; one week after the 2nd infection, SIT reached up to 8 (Group I) and 16 (Group II) respectively; and after the 3rd infection, SIT further increased and peaked in the 7th week. When challenged by lethal dose of C. irritans, fish of all 3 groups began to die since the 3rd day after infection, and the final deaths were 14, 12 and 8 for the control group, Group I and Group II, respectively. ACP activity in the 1st, 5th, 7th but the 3rd week was higher in the experiment group than that in the control group, but no significant difference was detected between Group I and II throughout the experiment. AKP activity increased as the infectious dose increased, but the difference among the three groups gradually became less obvious in latter infections, and no significant difference can be detected in the end. SOD activity increased with infection dose at each time point, while both group I and group II had their SOD activities first increased and then decreased as times of infection increased. The LZM activity of the two infection groups increased as the infectious times increased. Combining the results on growth and feeding, we speculated that the fishs physiological condition stabilized after 3 rounds of infection. To sum up, low-dose infection by C. irritans can induce the fishs immunity, but at the cost of decreasing food intake, decreased food conversion, and lagged growth.

Induction of apoptosis in HT-29 cells by quercetin through mitochondria-mediated apoptotic pathway

Abstract With increasing use of natural plant-derived components, exploring the anti-proliferative effects of phytochemicals is increasingly gaining importance in designing anticancer drugs. Quercetin, a natural constituent abundantly present in food products, is capable of inducing apoptosis in tumor cells. However, little is known about its biological effect on colon cancer cells and molecular mechanism leading to this event. The aim of this study was to investigate the effect of quercetin on the apoptotic pathway in human colon carcinoma cells (HT-29 cells). The results indicated that quercetin induced suppression of cell viability and apoptosis in HT-29 cells in a dose-dependent manner. This involved characteristic changes in nuclear morphology, activation of caspases-3 and caspases-9, collapse of Mitochondrial membrane potential (ΔΨm), upregulation of pro-apoptotic Bax, and downregulation of anti-apoptotic Bcl-2 and Bcl-XL. Quercetin, which exerts anti-proliferative effect though different signaling pathways, is a classic candidate for anti-colon cancer drug design.

Diverse and abundant antibiotic resistance genes from mariculture sites of China's coastline

With the rapid development of mariculture in China, large amounts of antibiotics are being discharged into the aquatic environment. Little information is available regarding antibiotics and corresponding antibiotic resistance genes (ARGs) associated with maricultural environments in China. Sediments from eleven typical mariculture areas along the whole coastline of China were collected, and the sediment in Meijijiao in southern China was used as a non-mariculture control. The results revealed that antibiotics and their corresponding ARGs were widely distributed in most maricultural sediments, and present at low concentrations in samples from Meijijiao. The sulfonamide-resistance genes were prevalent, and the sul1 and sul2 in Penglai were the highest detected by using quantitative PCR. Moreover, remarkable differences in ARGs among different sites were observed. Due to the limited availability of primers to detect ARGs, illumina high-throughput sequencing was also used for profiling ARGs, and the results showed that the abundance of bacA in all samples was the highest compared to other ARGs, followed by mexF and mexB. This is the first study to comprehensively investigate the antibiotic resistance profile in typical mariculture areas along the whole coast of China. This study provides insights into the impacts of mariculture on the profiles of bacterial and ARG compositions in China.

Cloning, characterization, and function of MyD88 in silvery pomfret (Pampus argenteus) in response to bacterial challenge

Myeloid differentiation factor 88 (MyD88) is a key and universal downstream adapter for most Toll-like receptors (TLRs) and plays an important role in both the innate and adaptive immune response. In this study, the full-length cDNA of MyD88 (PAMyD88) from silvery pomfret (Pampus argenteus) was cloned and characterized. The PAMyD88 cDNA is 1545bp in length and contains an 876bp open reading frames (ORF). Multiple sequence alignment and phylogenetic tree analyzes revealed that the amino acid sequence of PAMyd88 was homologous to a variety of previously MyD88 molecules characterized from other species. The quantitative real-time reverse transcription-PCR analysis showed that the PAMyD88 mRNA was broadly expressed in all examined tissues, with higher levels observed in the immune-relevant organs. The results showed a significant up-regulation of the TLR2 and PAMyD88 transcript levels in response to L. plantarum and C. butyricum and a substantial expression level of TLR4 and PAMyD88 induced by V. anguillarum. Additionally, a challenge with V. anguillarum resulted in significant apoptosis, whereas the L. plantarum and C. butyricum induced only low levels of apoptosis. These data provide insight into the roles of PAMyD88 in the TLR signaling pathway in response to probiotic and pathogenic bacteria in silvery pomfrets.

Influence of dietary n-3 LC-PUFA on growth, nutritional composition and immune function in marine fish Sebastiscus marmoratus

A 60-day feeding experiment was conducted to investigate the influence of dietary omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) on growth, nutritional composition and immune function of marine fish Sebastiscus marmoratus. Five diets containing 3.6, 10.2, 18.2, 26.5, or 37.0 g/kg n-3 LC-PUFA were prepared. The results reveal significant influences of dietary n-3 LC-PUFA on the final weight, weight gain, specific growth rate, feed conversion ratio, and condition factor. As dietary n-3 LCPUFA increased, weight gain and specific growth rate increased and were significantly higher in groups fed 18.2, 26.5 and 37.0 g/kg than in groups fed 3.6 and 10.2 g/kg (P<0.05); there was no significant difference between groups fed 18.2, 26.5, or 37.0 g/kg (P>0.05). With increasing dietary n-3 LC-PUFA, eicosapentaenoic acid and docosahexenoic acid content in muscle and liver increased significantly, immunoglobulin class M content gradually increased from 9.1 to 14.8 μg/L, and lysozyme activity content increased from 1 355 to 2 268 U/mL. Broken line model analysis according to weight gain indicated that a dietary n-3 LC-PUFA level of 18.2 g/kg is essential for normal growth at a fat level of 125 g/kg. Therefore, appropriate dietary n-3 LC-PUFA not only promote growth and improve the n-3 LC-PUFA content, but also enhance immune function in S. marmoratus.

Characterization of TLR5 and TLR9 from silver pomfret (Pampus argenteus) and expression profiling in response to bacterial components

&NA; Toll like receptor (TLR) 5 and 9 are important members of the TLR family that play key roles in innate immunity in all vertebrates. In this study, paTLR5 and paTLR9 were identified in silver pomfret (Pampus argenteus), a marine teleost of great economic value. Open reading frames (ORFs) of paTLR5 and paTLR9 are 2646 and 3225 bp, encoding polypeptides of 881 and 1074 amino acids, respectively. Sequence analysis revealed several conserved characteristic features, including signal peptides, leucine‐rich repeat (LRR) motifs, and a Toll/interleukin‐I receptor (TIR) domain. Sequence, phylogenetic and synteny analysis revealed high sequence identity with counterparts in other teleosts, confirming their correct nomenclature and conservation during evolution. Quantitative real‐time PCR revealed that the that both TLRs were ubiquitously expressed in all investigated tissues, most abundantly in liver, kidney, spleen, intestine and gill, but lower in muscle and skin. In vitro immunostimulation experiments revealed that Aeromonas hydrophila lipopolysaccharide (LPS) and Vibrio anguillarum flagellin induced higher levels of paTLR9 and paTLR5 mRNA expression in isolated fish intestinal epithelial cells (FIECs) than Lactobacillus plantarum lipoteichoic acid (LTA), but all increased the secretion of IL‐6 and TNF‐&agr; and induced cell apoptosis and necrosis. Together, these results indicate that paTLR5 and paTLR9 may function in the response to bacterial pathogens. Our findings enhance our understanding of the function of TLRs in the innate immune system of silver pomfret and other teleosts. HighlightsORFs of paTLR5 and paTLR9 are 2646 and 3225 bp, encoding polypeptides of 881 and 1074 amino acids, respectively.Both TLRs were expressed in all investigated tissues, most abundantly in liver, kidney, spleen, intestine and gill, but lower in muscle and skin.A. hydrophila LPS and V. anguillarum flagellin induced high levels of paTLR9 and paTLR5 mRNA expression.L. plantarum LTA induced mild apoptosis, whereas V. anguillarum flagellin and A. hydrophila LPS induced severe apoptosis.TLR5 and TLR9 may play an important role in the inflammatory response induced by V. anguillarum and A. hydrophila.

Time-series transcriptomic analysis of the kelp grouper Epinephelus moara in response to low salinity stress

ABSTRACT The Kelp grouper Epinephelus moara is one of the most widely consumed and economically important marine fish in China. The species can tolerate a wide range of salinity, but genomic resources are not available, and the molecular mechanisms underlying adaptation to salinity at the transcriptomic level remain largely unclear. In this study, the transcriptomic responses of the liver of E. moara under low salinity were investigated using the Illumina digital gene expression system. After de novo assembly, 499,356 transcripts were generated and contributed 445,068 unigenes. A total of 14, 19, 33 and 3101 genes were differentially expressed following exposure to low salinity stress for 2, 6, 24 and 48 h, respectively. Only two genes were differentially expressed in all groups. Four genes related to metabolism and ambient salinity adaption were randomly selected to validate the differentially expressed genes (DEGs) by real-time PCR. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to analyse the functional significance of DEGs, including those responding to salinity through diverse biological processes, cellular components, molecular functions, and pathways associated with metabolic and osmotic responses. This work provides new insight into the response to salinity challenges in E. moara, and the findings expand our knowledge of the molecular basis of metabolic regulation mechanisms in this species. Additionally, the transcriptional data provide a valuable resource for future molecular and genetic studies on E. moara.

Effects of salinity on serum osmolality,catalase and gill ion-regulatory enzyme activities in silver pomfret

The juvenile silver pomfret were cultured in water with salinity 14,25 and 36,respectively,for 96h. And their serum osmolality,catalase( CAT) and gill V-H+-ATPase( VHA),Ca2 +-ATPase,carbonic anhydrase( CA) activities w ere tested in different salinity over time in this experiment. In this study,the serum osmolality w hich fluctuated w as positively correlated to salinity,and its variation range is 271- 379mOsm / kg. The serum osmolality of different salinity w ould return to normal at 96 h. CAT activities of salinity 36 and 14 returned to normal after they rose significantly( P 0. 05); the highest point of them w as salinity 36 at 8 h. VHA activities rose,then recovered in salinity 14,kept stable in control salinity,fell dow n then recovered in salinity 36. Ca2 +-ATPase activities increased significantly w hen salinity changed( P 0. 05),and the variation trend in salinity 14 w as more identifiable than that in salinity 36. CA activities in salinity 14 and 36 both increased significantly( P 0. 05),and the highest point w as found in salinity 36 at24 h. Through analysis of the data,the serum osmolality w as significantly negatively correlated to gill VHA activity( P 0. 01),and it w as significantly negatively correlated to CA activity( P 0. 01) in low salinity adaptation( 14 and 25). It also illustrated a contrary trend betw een serum osmolality and Ca2 +-ATPase activity in salinity 14. This research proved that serum osmolality w hich changes w ith salinity,w ill activate CAT w hen silver pomfret adapts to different salinity. And VHA,Ca2 +-ATPase and CA are playing important roles in osmoregulation.