Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus.

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.

Identification and function analysis of an immune deficiency homolog in swimming crab, Portunus trituberculatus.

The immune deficiency (IMD) pathway is involved in both antiviral and antibacterial immune responses in Drosophila. IMD protein is the key adaptor to link the extracellular signal and the intracellular reaction to initiate the signal transduction in IMD pathway. In present study, the cDNA of the IMD (Pt-IMD) was identified from a marine crab, Portunus trituberculatus. The Pt-IMD is predicted to encode 170 amino acids with a death domain. Real-Time quantitative PCR analysis showed that Pt-IMD was constitutively expressed in hemocytes, intestine, gill, heart, muscle and hepatopancreas in normal crab. Moreover, the transcript of Pt-IMD in large-granule hemocytes is approximately 6-fold higher than semi-granular cells and agranular cells. Intracellular localization showed Pt-IMD was distributed mainly in the cytoplasm when it was over-expressed in Drosophila Schneider 2 (S2) cell. Functionally, over-expression of Pt-IMD could activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. Furthermore, Pt-IMD expression was also knock-down by RNAi to determine the function of Pt-IMD on regulation of the expression of different antimicrobial peptides (AMPs) in crab. In the primary cultured hemocytes challenged with or without Vibrio alginolyticus, after Pt-IMD was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), ALF3, crustin1, crustin3, arasin2, hyastatin1and hyastatin3 have been significantly inhibited in normal cell or bacterial infected cell, while the expression of lysozyme was normal in non-infected cells and was significantly induced in bacterial infected cells, which compared to the non-specific dsRNA treated cells.

Vibrio parahaemolyticus infection impaired intestinal barrier function and nutrient absorption in Litopenaeus vannamei.

The intestine is the primary target of pathogenic microbes during invasion. However, the interaction of Vibrio parahaemolyticus (V. parahaemolyticus) with intestinal epithelial cells and its effects on the intestinal function of Litopenaeus vannamei (L. vannamei) are poorly studied. Therefore, the aim of this study was to investigate the influence of V. parahaemolyticus infection on intestinal barrier function and nutrient absorption in L. vannamei. In the present study, a total of 90 shrimp were randomly divided into two groups including the control group and V. parahaemolyticus infection group (final concentration of 1 × 105 CFU/mL), with three replicates per group. The result showed that compared with the control group, V. parahaemolyticus infection increased (P < 0.05) serum diamine oxidase activity and endotoxin quantification, and down-regulated (P < 0.05) the mRNA levels of intestinal peroxinectin, integrin, midline fasciclin at 48 h and 72 h; V. parahaemolyticus infection decreased (P < 0.05) the mRNA expression of intestinal amino acid transporter (CAT1, EAAT3 and ASCT1) and glucose transporter (SGLT-1, GLUT) at 24 h, 48 h and 72 h, and increased (P < 0.05) serum glucose and amino acid (Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr, Phe, Lys, His and Arg) concentration at 24 h. The results indicated that V. parahaemolyticus infection increased intestinal permeability, inhibited absorption of glucose and amino acid in L. vannamei.

Characterization, subcellular localization and function analysis of myeloid differentiation factor 88 (Pt-MyD88) in swimming crab, Portunus trituberculatus.

Myeloid differentiation factor 88 (MyD88) is a universal and essential adaptor protein required for the Toll-like receptors (TLRs) pathway activation in invertebrates as well as in vertebrates. Herein, we characterized a MyD88 (Pt-MyD88) cDNA sequence in the swimming crab (Portunus trituberculatus). The Pt-MyD88 ORF is predicted to encode 469 peptides with an N-terminal death domain and a typical C-terminal TIR domain. Real-Time quantitative PCR analysis showed that the Pt-MyD88 transcriptions were constitutively expressed in hemocytes, gill, intestine, heart and muscle in normal crab. The expressions of Pt-MyD88 would be down-regulated by V. alginolyticus or LPS challenge, and be up-regulated by WSSV infection in hemocytes. Intracellular localization showed Pt-MyD88 was distributed mainly in the cytoplasm when it was over-expressed in human cell HEK293T or in Drosophila Schneider 2 (S2). Functionally, over-expression of Pt-MyD88 could either activate the NF-κB in HEK293T cells or activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. In primary cultured hemocytes of swimming crab, after Pt-MyD88 was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), hyastatin3, crustin1 and crustin3 have been significantly inhibited, while the expression of other AMPs is normal compared to non-specific dsRNA treated cells.

Hepatopancreas and ovarian transcriptome response to different dietary soybean lecithin levels in Portunus trituberculatus.

Ovaries (O) are specialized tissues that play critical roles in producing oocytes and hormones. The crustacean hepatopancreas (H) is a metabolic organ that plays important functions including absorption, storage of nutrients and vitellogenesis during growth and ovarian development. However, genetic information on the biological functions of the crustacean ovaries and hepatopancreas are limited. This study compared the transcriptome in the ovary and the hepatopancreas of female P. trituberculatus fed two different diets containing 0% (SL0) and 4% soybean lecithin (SL4), respectively during the growth and ovarian maturation stages by Illumina HiSeq4000 sequencer. The differences between ovary and hepatopancreas of P. trituberculatus were also compared at transcriptional level. A total of 55,667 unigenes were obtained with mean length of 962 bps across the four treatment groups (SL0_O, SL4_O, SL0_H and SL4_H). In ovary, there were 257 differentially expressed genes (DEGs) between SL0_O and SL4_O, with 145 down- and 112 up-regulated genes in the SL4_O group. Candidate genes involved in ovarian development were detected in SL4_H group. In hepatopancreas, 146 DEGs were found between SL0_H and SL4_H, including 43 down- and 103 up-regulated genes in the SL4_H group. The specific DEGs were mainly involved with lipid related metabolism pathways, including fat digestion and absorption, PPAR signaling pathway and insulin resistance. 14,725 DEGs were found in the comparison between SL0_O and SL4_H, including 7250 up- and 7475 down-regulated genes in the SL4_H group. The specific DEGs were mainly involved with lipid (fat digestion and absorption, linoleic acid metabolism), hormone (steroid hormone biosynthesis, ovarian steroidogenesis, etc), and amino acid (phenylalanine metabolism, arginine biosynthesis, tyrosine) related metabolism pathways. Crabs fed the SL4 diet exhibited higher gene expression of cryptocyanin 1 (cc1), cryptocyanin 2 (cc2) and neuroparsin 1 (np1) in hepatopancreas and ovarian than those fed the SL0 diet, however, crab fed SL4 diet showed higher gene expression of fatty acid-binding protein 1 (fabp1), vitellogenin (vtg) and Delta-6 desaturase-like protein (fadsd6) in hepatopancreas than those fed the SL0 diet. Moreover, crabs fed the SL0 diet had lower gene expression of vtg, extracellular copper­zinc superoxide dismutase (cuznsod) and estrogen sulfotransferase (ests) in ovary compared to those fed the diet containing 4% soybean lecithin. These results might provide important clues with respect to elucidating the molecular mechanisms underlying the regulation of phospholipid on the gonadal development and lipid metabolism of P. trituberculatus.

Dietary yeast hydrolysate and brewer's yeast supplementation could enhance growth performance, innate immunity capacity and ammonia nitrogen stress resistance ability of Pacific white shrimp (Litopenaeus vannamei).

An 8-week feeding trial was conducted to evaluate the effects of dietary yeast hydrolysate and brewer's yeast supplementation on growth, immune-related genes expression and ammonia nitrogen stress resistance of Pacific white shrimp (Litopenaeus vannamei). Three isonitrogenous and isolipidic practical diets were formulated to contain 0% (control diet), 1% yeast hydrolysate and 1% brewer's yeast, respectively. 360 juvenile L. vannamei with an initial weight (0.88 ±â€¯0.01 g) was randomly divided into 3 treatments in four replicates (30 shrimp per replicate). The results indicated that shrimp fed the diet containing 1% yeast hydrolysate had a significantly higher weight gain (WG), and specific growth rate (SGR) than that fed the control diet, and the lowest feed conversion ratio (FCR) was occurred in the 1% yeast hydrolysate supplementation group. Proximate composition in whole body and muscle among all treatments was not significantly influenced by the dietary yeast hydrolysate or brewer's yeast supplementation. The challenge test with ammonia nitrogen showed that lower cumulative survival was observed in those fed the control diet, and the highest cumulative survival was occurred at shrimp fed the 1% yeast hydrolysate supplementation. Shrimp fed the control diet had higher inflammation-related genes expression levels of tnf-α and il-1ß in the intestine than those fed the diets supplemented with 1% yeast hydrolysate or 1% brewer's yeast, however, there was no significant difference in expression level of alp in intestine among all treatments. The relative expression levels of mTOR signal pathway genes (eif4ebp, eif4e1a, eif4e2 and p70s6k) were significantly up-regulated in the shrimp fed the diets supplemented with 1% yeast hydrolysate, and the lowest gene expression levels of eif4ebp, eif4e1a, eif4e2 and p70s6k in the intestine were occurred at the control diet. The highest expression levels of the immune-related genes (dorsal, relish, and proPO) in the intestine were observed at shrimp fed the 1% yeast hydrolysate supplementation, and the lowest expression levels of these genes were occurred at shrimp fed the control diet, however, there was no significant difference in gene expression of lysozyme among all treatments. The expression levels of penaeidin3a, crustin, proPO, and IMD in the hepatopancreas were significantly influenced by the dietary yeast hydrolysate, brewer's yeast or no yeast product supplementation, shrimp fed the 1% yeast hydrolysate supplementation had higher expression levels of these genes than those fed the control diet. The present study indicated that dietary 1% yeast hydrolysate or brewer's yeast supplementation could improve growth performance, enhance innate immunity, and strengthen resistance of ammonia nitrogen stress, and dietary 1% yeast hydrolysate supplementation provides better immunostimulatory effects than brewer's yeast of L. vannamei.

ß-actin gene expression is variable among individuals and not suitable for normalizing mRNA levels in Portunus trituberculatus.

The housekeeping gene encoding ß-actin appears to be the most widely-used internal reference for gene expression studies in experimental animals or their cell lines. However, the effectiveness of ß-actin to normalize mRNA levels expression in many crustacean species is still object of debate. To date, it is still unclear if ß-actin is suitable to be utilized as the internal reference in qualitative real-time gene expression study in crab species. To address this concern, we evaluated 5 candidate reference genes encoding ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A, elongation factor 1-α (EF1-α), and 18 S ribosomal RNA (18 S rRNA) in the swimming crabs (Portunus trituberculatus) models. Our data showed that the ß-actin gene expression varied significantly across individual swimming crab individuals in gills or hemocytes and the expression of 18 S rRNA, EF1-α, cyclophilin or GAPDH gene were relatively stable compared to that of ß-actin. Moreover, the expression stability of the reference genes among different tissues in normal crabs or after WSSV challenge was also tested by geNorm and NormFinder software. Among tissues, 18 S rRNA was most stably expressed in different tissues, followed by cyclophilin A and EF1-α, compared to ß-actin and GAPDH. Upon to viral simulation, GAPDH was found to be the most stable internal control gene in gills and cyclophilin A was ranked as the most stable gene in hemocytes.

Molecular and functional characterisation of two elovl4 elongases involved in the biosynthesis of very long-chain (>C24) polyunsaturated fatty acids in black seabream Acanthopagrus schlegelii.

Elongation of very long-chain fatty acid (Elovl) 4 proteins are important fatty acyl elongases that participate in the biosynthesis of long-chain (C20-24) and very long-chain (˃C24) polyunsaturated fatty acids (LC-PUFA and VLC-PUFA, respectively) in teleost fish, especially in marine species. Moreover, knowledge of Elovl4 and other elongases such as Elovl2 has contributed to an advanced understanding of the LC-PUFA biosynthetic pathway in marine fish. In the present study, elovl4a and elovl4b were cloned from black seabream Acanthopagrus schlegelii and functionally characterised using recombinant expression in yeast. The elovl4a and elovl4b cDNA sequences included open reading frames (ORF) of 969 and 918 base pairs (bp), encoding proteins of 322 and 315 amino acids (aa), respectively. The functional characterisation of A. schlegelii Elovl4 proteins showed they were able to utilise all assayed C18-22 PUFA substrates except 22:6n-3. Moreover, it was particularly noteworthy that both A. schlegelii Elovl4a and Elovl4b proteins had the ability to elongate 20:5n-3 and 22:5n-3 to 24:5n-3, which can be potentially desaturated and ß-oxidised to 22:6n-3. Tissue transcript abundance analysis showed the highest expression of elovl4a and elovl4b in brain and eye, respectively, suggesting these tissues were major sites for VLC-PUFA biosynthesis in black seabream. The functions of the A. schlegelii Elovl4-like elongases, Elovl4a and Elovl4b, characterised in the present study, along with those of the Elovl5 and fatty acyl desaturase (Fads2) proteins of A. schlegelii characterised previously, provided evidence of the biosynthetic pathways of LC-PUFA and VLC-PUFA in this teleost species.