Xiudan Wang

Ig heavy chain genes and their locus in grass carp Ctenopharyngodon idella.

The cDNA and genomic sequences of IgD and IgZ were characterized in grass carp Ctenopharyngodon idella in the present study, and with the identification of a BAC clone covering zeta, mu, and delta genes, the IgH locus containing these Ig genes and other V, D, J genes was also illustrated in this fish. Secretory and membrane-bound IgZ were identified, with two transmembrane exons spliced within the CH4 exon, as reported in IgM of mammals and IgZ in other teleost fish. The first and second constant domains of IgZ shows more than 90% nucleotide identity with respective domains of grass carp IgM. The IgD has a structure of delta1-(delta2-delta3-delta4)(2)-delta5-delta6-delta7-TM-UTR, with the repeat of delta2-delta3-delta4; but intron was not found between the two repeat, i.e. between the first delta2-delta3-delta4 (delta2.1-delta3.1-delta4.1) and the second delta2-delta3-delta4 (delta2.2-delta3.2-delta4.2), and the intron between delta3.1 and delta4.1 was much shorter than the intron between delta3.2 and delta4.2. The genomic organization of the IgH locus has a pattern of Vn-Dn-Jn-Czeta-Dn-Jn-Cmu-Cdelta, as reported in other teleost fish. Thirteen V(H), fourteen D, and twelve J(H) genes were observed in this locus, with the similarity of three D segments and four J(H) segments being the same in the upstream of Czeta and Cmu. The transcriptional enhancer located at the mu-delta intergenic region was also analyzed and it seems possible that this enhancer is functional as verified in zebrafish and channel catfish.

Ocean acidification stimulates alkali signal pathway: A bicarbonate sensing soluble adenylyl cyclase from oyster Crassostrea gigas mediates physiological changes induced by CO2 exposure

Ocean acidification (OA) has been demonstrated to have severe effects on marine organisms, especially marine calcifiers. However, the impacts of OA on the physiology of marine calcifiers and the underlying mechanisms remain unclear. Soluble adenylyl cyclase (sAC) is an acid-base sensor in response to [HCO3-] and an intracellular source of cyclic AMP (cAMP). In the present study, an ortholog of sAC was identified from pacific oyster Crassostrea gigas (designated as CgsAC) and the catalytic region of CgsAC was cloned and expressed. Similar to the native CgsAC from gill tissues, the recombinant CgsAC protein (rCgsAC) exhibited [HCO3-] mediated cAMP-forming activity, which could be inhibited by a small molecule KH7. After 16days of CO2 exposure (pH=7.50), the mRNA transcripts of CgsAC increased in muscle, mantle, hepatopancreas, gill, male gonad and haemocytes, and two truncated CgsAC forms of 45kD and 20kD were produced. Cytosolic CgsAC could be translocated from the cytoplasm and nuclei to the membrane in response to CO2 exposure. Besides, CO2 exposure could increase the production of cAMP and intracellular pH of haemocytes, which was regulated by CgsAC (p<0.05), suggesting the existence of a [HCO3-]/CgsAC/cAMP signal pathway in oyster. The elevated CO2 could induce an increase of ROS level (p<0.05) and a decrease of phagocytic rate of haemocytes (p<0.05), which could be inhibited by KH7. The results collectively suggest that CgsAC is an important acid-base sensor in oyster and the [HCO3-]/CgsAC/cAMP signal pathway might be responsible for intracellular alkalization effects on oxidative phosphorylation and innate immunity under CO2 exposure. The changes of intracellular pH, ROS, and phagocytosis mediated by CgsAC might help us to further understand the effects of ocean acidification on marine calcifiers.

Soluble adenylyl cyclase mediates mitochondrial pathway of apoptosis and ATP metabolism in oyster Crassostrea gigas exposed to elevated CO2

Abstract Ocean acidification (OA) has deleterious impacts on immune response and energy homeostasis status of Mollusca. In the present study, the apoptosis ratio of hemocytes and the adenosine triphosphate (ATP) allocation in gill tissues were determined after Pacific oysters Crassostrea gigas were exposed to elevated CO2 environment (pH = 7.50) for 16 days.The apoptosis ratio in CO2 exposure group (35.2%) was significantly higher (p < 0.05) than that in the control group, and the increased apoptosis ratio induced by elevated CO2 could be significantly inhibited (p < 0.05) by KH7, a specific inhibitor of a bicarbonate sensor soluble adenylyl cyclase (sAC). After CO2 exposure, sAC in oyster (CgsAC) was found to be clustered with mitochondria in the cytoplasm, and the pro‐caspase‐3 was cleaved into two small fragments. Moreover, the activities of caspase‐3 and caspase‐9 also increased post CO2 exposure and these increases could be inhibited by KH7. However, the activities of caspase‐8 did not change significantly compared with that in the control group. After CO2 exposure, the ATP content in the gill increased significantly (p < 0.05) and such increase could also be inhibited by KH7. The ATP content in purified gill mitochondria decreased significantly (p < 0.05) after CO2 exposure, which was also inhibited by KH7. These results implied that the elevated CO2 could activate the mitochondria‐CgsAC pathway of apoptosis and ATP metabolism in oyster, and this pathway played essential roles in maintaining the homeostasis and the balance of energy metabolism in response to OA. HighlightsCgsAC mediates the increase of apoptosis rate caused by CO2 exposure.The signal of CgsAC was clustered with mitochondria after CO2 exposure.CgsAC mediates the increase of Caspase‐3 and caspase‐9 activity caused by CO2 exposure.CgsAC mediates the increase of ATP in gill tissue caused by CO2 exposure.CgsAC mediates the decrease of ATP in gill mitochondria caused by CO2 exposure.

The versatile functions of LRR-only proteins in mollusk Chlamys farreri

Leucine-rich repeat (LRR)-only proteins are involved in the innate immune responses as they mediate protein-ligand interactions. In the present study, three novel LRR-only proteins, CfLRRop-4, CfLRRop-5 and CfLRRop-6, were identified and characterized from Zhikong scallop Chlamys farreri. They all contained LRR motifs with consensus signature sequences of LxxLxLxxNxL or LxxLxLxxCxxL. All the mRNA transcripts of three CfLRRops were high abundant in hepatopancreas, gills and gonads, and their mRNA transcripts in hemocytes could respond to the stimulations of different microbes, including Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. These three CfLRRops exhibited similar ligand binding and recognition characteristics as Toll-like receptors (TLRs) and NOD-like receptors (NLRs). The immune effectors, including tumor necrosis factor α, superoxide dismutase, catalase and lysozyme, varied significantly after the scallops were stimulated by recombinant LRR-only proteins. All these results indicated that LRR-only proteins are functionally differentiated and exhibit different immunomodulation activities on various downstream immune effectors.

A shell-formation related carbonic anhydrase in Crassostrea gigas modulates intracellular calcium against CO2 exposure: Implication for impacts of ocean acidification on mollusk calcification

Ocean acidification (OA) could decrease the shells and skeletons formation of mollusk by reducing the availability of carbonate ions at calcification sites. Carbonic anhydrases (CAs) convert CO2 to HCO3- and play important roles in biomineralization process from invertebrate to vertebrate. In the present study, a CA (designated as CgCA) was identified and characterized in Pacific oyster C. gigas. The cDNA of CgCA was of 927bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain. The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes. During the early development period, the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae. Elevated CO2 increased the mRNA transcripts of CgCA in muscle, mantle, hepatopancreas, gill and hemocytes significantly (p<0.05) and induced the translocation of CgCA in hemocytes and mantle. Moreover, elevated CO2 also caused the decrease of intracellular Ca2+ in hemocytes (p<0.05). The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca2+ in hemocytes (p<0.05). Besides, the decrease of intracellular Ca2+ content caused by Ca2+ reagent ionomycin could affect localization of CgCA in mantle tissue. The results indicated CgCA played essential roles in calcification and elevated CO2 accelerated the mutual modulation between calcium and CgCA, implying reduced calcification rate and dissolved shells under OA.

The immunomodulation of a maternal translationally controlled tumor protein (TCTP) in Zhikong scallop Chlamys farreri

ABSTRACT Translationally controlled tumor protein (TCTP) is initially described as a highly conserved protein implicated in cell growth, and it is subsequently confirmed to play important roles in mediating the innate immune response, especially the inflammatory. In the present study, the full‐length cDNA sequence of a TCTP from Zhikong scallop Chlamys farreri (designed as CfTCTP) was cloned by rapid amplification of cDNA ends (RACE) technique based on the expression sequence tag (EST) analysis. It was of 1230 bp with an open reading frame (ORF) of 543 bp encoding a polypeptide of 180 amino acids. The deduced amino acid sequence contained a conserved TCTP signature sequence (from I47 to E58) and it shared 26.1%–48.9% similarities with previously identified TCTPs. CfTCTP was clustered with the TCTP from Argopectehs irradias in the phylogenetic tree and was designated into a single branch of mollusk with TCTP from Ruditapes philippinarum. The mRNA transcripts of CfTCTP were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas, kidney and gonad, with the highest expression level in hepatopancreas. The mRNA expression level of CfTCTP in oocytes and fertilized eggs kept at a higher level, and was down‐regulated from 2‐cell embryos to the lowest level in gastrula. Then it was up‐regulated in trochophore and dropped down in the late veliger larvae to the similar level as that in oocytes. After pathogen‐associated molecular patterns (PAMPs) stimulation, the expression of CfTCTP mRNA in haemocytes was increased at 3 or 6 h, and fall down to the normal level at 24 h. The recombinant protein of CfTCTP could induce the release of histamine from BT‐549 cells. All these results indicated that CfTCTP was a pro‐inflammatory factor and it could be maternally transferred from female gonad to oocytes and offspring, and play pivotal role in the embryonic development and immune protection of scallops. HIGHLIGHTSA TCTP was identified from Zhikong scallop Chlamys farreri.CfTCTP was maternally transferred to oocytes and began to synthesis from trochophore.CfTCTP took part in the response to different challenges in the early phase of infection in scallops.rCfTCTP mediated histamine‐releasing from BT‐549 cells.

The modulation role of serotonin in Pacific oyster Crassostrea gigas in response to air exposure

ABSTRACT Serotonin, also known as 5‐hydroxytryptamine (5‐HT), is a critical neurotransmitter in the neuroendocrine‐immune regulatory network and involved in regulation of the stress response in vertebrates and invertebrates. In the present study, serotonin was found to be widely distributed in the tissues of Pacific oyster Crassostrea gigas, including haemolymph, gonad, visceral ganglion, mantle, gill, labial palps and hepatopancreas, and its concentration increased significantly in haemolymph and mantle after the oysters were exposed to air for 1 d. The apoptosis rate of haemocytes was significantly declined after the oysters received an injection of extra serotonin, while the activity of superoxide dismutase (SOD) in haemolymph increased significantly. After the stimulation of serotonin during air exposure, the apoptosis rate of oyster haemocytes and the concentration of H2O2 in haemolymph were significantly decreased, while the SOD activity was significantly elevated. Furthermore, the survival rate of oysters from 4th to 6th d after injection of serotonin was higher than that of FSSW group and air exposure group. The results clearly indicated that serotonin could modulate apoptotic effect and redox during air exposure to protect oysters from stress. HighlightsNeurotransmitter 5‐HT existed extensively in the tissues of oyster C. gigas.The level of 5‐HT in haemolymph and mantle increased significantly against air exposure stress.5‐HT could reduce apoptotic effects of haemocytes and modulate redox in haemolymph after air exposure.5‐HT could decrease the mortality of oysters during air exposure, and protect them from stress.

Two novel LRR and Ig domain-containing proteins from oyster Crassostrea gigas function as pattern recognition receptors and induce expression of cytokines

ABSTRACT Leucine‐rich repeat (LRR) domain and immunoglobulin (Ig) domain are both competent immune recognition modules, and the immunological roles of LRR and Ig domain containing‐ proteins (LRRIGs) are speculated to be multifunctional and worth investigating. In the present study, two novel LRRIGs, CgLRRIG‐1 and CgLRRIG‐2, were identified and characterized from oyster Crassostrea gigas. Both of them contained an N‐terminal LRR region, an Ig domain, a transmembrane region, and a C‐terminal cytoplasmic tail. The mRNA transcripts of CgLRRIG‐1 and CgLRRIG‐2 were constitutively expressed in muscle, gill, hepatopancreas, mantle, gonad and hemocytes with the highest expression level in hepatopancreas. Their mRNA expression levels in hemocytes were significantly up‐regulated after the stimulations with four PAMPs including peptidoglycan (PGN), lipopolysaccharide (LPS), glucan (GLU) and polyinosinic‐polycytidylic acid (poly I:C) and one bacteria Vibrio anguillarum. The recombinant proteins, rCgLRRIG‐1 and rCgLRRIG‐2, could bind to PGN, LPS, GLU and poly I:C, and rCgLRRIG‐2 exhibited higher binding affinity. Additionally, rCgLRRIG‐1 and rCgLRRIG‐2 could significantly induce the expression of CgTNF‐1 and CgIL17‐5 in cultured oyster hemocytes, and the activity of rCgLRRIG‐2 was higher than that of rCgLRRIG‐1. All these results indicated that CgLRRIG‐1 and CgLRRIG‐2 could function as immune effectors or pro‐inflammatory factors as well as PRRs in oyster. HighlightsTwo LRR and Ig domain containing proteins with different structure and activity were identified from Crassostrea gigas.CgLRRIGs were expressed universally with the highest levels in hepatopancreas.CgLRRIGs could be induced by PAMPs and bacteria stimulations, and displayed bind high affinities towards PAMPs.CgLRRIGs could induce the expression of CgTNF‐1 and CgIL17‐5 in hemocytes.

Transcriptome sequencing reveals the involvement of reactive oxygen species in the hematopoiesis from Chinese mitten crab Eriocheir sinensis

ABSTRACT Reactive oxygen species (ROS) produced in vivo during various electron transfer reactions are generally kept at a certain level since they are harmful to cells. However, it can sensitize hematopoietic progenitors to differentiation, and plays a signaling role in the regulation of hematopoietic cell fate. In the present study, the transcriptomes of crab HPT and hemocytes were sequenced using the Ion Torrent Proton sequencing platform. A total of 51,229,690 single end reads were obtained from six single‐end libraries, which were assembled into 31346 unireads as reference. After mapping and transcript assembling, 362 differently expressed genes were identified and 301 of them were deemed to be more abundant in HPT. GO annotation revealed that they were mostly implicated in DNA, RNA and protein synthesis, cell division, mitochondria activities and energy metabolism. The expression level of mitochondrial complexes I (mitochondrial NADH‐ubiquinone oxidoreductase) which was the main natural producers of mitochondrial ROS was found to be 8.6‐fold (p < 0.01) higher in HPT than that in hemocytes. In hemocytes, the proteinase genes associated with proPO activation from the 61 up‐regulated genes in hemocytes were the main up‐regulated genes which might be the potential markers for mature hemocytes. ROS level in HPT cells was relatively higher which was confirmed with the high expression level of mitochondria related genes identified by transcriptome sequencing. After the ROS level was depressed by N‐acetyl‐l‐cysteine (NAC), the production of hemocytes from HPT was inhibited, and the recovery of the total hemocytes counts was delayed. These results collectively indicated that the genes in redox system were more active in HPT, and ROS could function as an important modulator in the hematopoiesis of crab and promote the production of hemocytes from HPT. HighlightsROS level was higher in HPT cells than that in hemocytes of E. sinensis.NAC would inhibit the production of hemocytes through decreasing ROS level.The expression level of mitochondrial complexes I in HPT was 8.6‐fold higher than that in hemocytes.EsproPO and proteinases associated with proPO activation were more abundant in hemocytes.

The B-cell translocation gene 1 (CgBTG1) identified in oyster Crassostrea gigas exhibit multiple functions in immune response

Abstract B‐cell translocation gene 1 (BTG1) is a member of the anti‐proliferative gene family, which plays important roles in regulation of cell cycle. In the present study, a B‐cell translocation gene 1 molecule homologue (designed CgBTG1) are identified and characterized in oyster Crassostrea gigas. CgBTG1 contains a conserved BTG domain with Box A and Box B motifs, and it shares high similarities with both BTG1 and BTG2 proteins in vertebrates. CgBTG1 mRNA is predominantly expressed in hemocytes, and its expression level in hemocytes is significantly up‐regulated at 6 h (5.40‐fold, p < 0.01) post Vibrio splendidus stimulation. The apoptosis rate of oyster hemocytes is significantly decreased (p < 0.05) after CgBTG1 interfered by dsRNA (dsCgBTG1). This is indicated that CgBTG1 participated in the regulation of oyster hemocytes apoptosis. Furthermore, CgBTG1 could also induce the apoptosis of cancer cells (HeLa, A549 and BEL7402) in vitro. Compared with normal oysters, both vessel‐like structures and muscle fibers in CgBTG1 interfered oysters are severely damaged after V. splendidus challenge in paraffin section, considering that CgBTG1 possessed an analogous feature of angiogenesis for maintenance of vessel‐like structures in adductor muscle of oyster. The results suggests that CgBTG1 is a multi‐functional molecule involved in the immune response of C. gigas against pathogen infection, which provides more clues for intensive studies of BTG family proteins in invertebrates. HighlightsCgBTG1 is a B‐cell translocation gene 1 homologue identified in Crassostrea gigas.CgBTG1 could prompt the apoptosis in oyster hemocytes and vertebrate cancer cells.CgBTG1 possessed an analogous function of angiogenesis in adductor muscle of oyster.CgBTG1 exhibited multiple functions in immune response of C. gigas.