Jianmin Yang

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus.

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca(2+) binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln(122)-Pro(123)-Asn(124)) in LvLectin-1, but QPD (Gln(128)-Pro(129)-Asp(130)) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P<0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P<0.01) and 12 h (P<0.05), but the expression level of LvLectin-1 down-regulated significantly (P<0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus.

Identification and transcriptional analysis of two types of lectins (SgCTL-1 and SgGal-1) from mollusk Solen grandis

C-type lectin and galectin are two types of animal carbohydrate-binding proteins which serve as pathogen recognition molecules and play crucial roles in the innate immunity of invertebrates. In the present study, a C-type lectin (designated as SgCTL-1) and galectin (designated as SgGal-1) were identified from mollusk Solen grandis, and their expression patterns, both in tissues and toward three pathogen-associated molecular patterns (PAMPs) stimulation were characterized. The full-length cDNA of SgCTL-1 and SgGal-1 was 1280 and 1466 bp, containing an open reading frame (ORF) of 519 and 1218 bp, respectively. Their deduced amino acid sequences showed high similarity to other members of C-type lectin and galectin superfamily, respectively. SgCTL-1 encoded a single carbohydrate-recognition domain (CRD), and the motif of Ca(2+)-binding site 2 was EPN (Glu(135)-Pro(136)-Asn(137)). While SgGal-1 encoded two CRDs, and the amino acid residues constituted the carbohydrate-binding motifs were well conserved in CRD1 but partially conserved in CRD2. Although SgCTL-1 and SgGal-1 exhibited different tissue expression pattern, they were both constitutively expressed in all tested tissues, including hemocytes, gonad, mantle, muscle, gill and hepatopancreas, and they were both highly expressed in hepatopancreas and gill. Furthermore, the mRNA expression of two lectins in hemocytes was significantly (P < 0.01) up-regulated with different levels after S. grandis were stimulated by lipopolysaccharide (LPS), peptidoglycan (PGN) or β-1,3-glucan. Our results suggested that SgCTL-1 and SgGal-1 from razor clam were two novel members of animal lectins, and they might function as pattern recognition receptors (PRRs) taking part in the process of pathogen recognition.

Critical roles of sea cucumber C-type lectin in non-self recognition and bacterial clearance

C-type lectin is one important pattern recognition receptor (PRR) that plays crucial roles in multiple immune responses. A C-type lectin from sea cucumber Apostichopus japonicus (AjCTL-1) was characterized in the present study. The amino acid sequence of AjCTL-1 shared high similarities with other C-type lectins from invertebrates and vertebrates. The C-type lectin domain (CTLD) of AjCTL-1 contained a Ca(2+)-binding site 2 and four conserved cysteine residues. AjCTL-1 mRNA expression patterns in tissues and after bacterial challenge were then analysed. Quantitative PCR revealed that AjCTL-1 mRNA was widely expressed in the tested tissues of healthy sea cucumber. The highest expression level occurred in gonad followed by body wall, coelomocytes, tentacle, intestinum and longitudinal muscle, and the lowest expression level was in respiratory tree. AjCTL-1 mRNA expression in coelomocytes was significantly induced by gram-negative Listonella anguillarum and gram-positive Micrococcus luteus, with different up-regulation patterns post-challenge. Recombinant AjCTL-1 exhibited the ability to bind peptidoglycan directly, agglutinate M. luteus, Staphylococcus aureus and Escherichia coli, in a Ca(2+)-dependant manner, and enhance the phagocytosis of coelomocytes against E. coli in vitro. The results indicated that AjCTL-1 could act as a PRR in Apostichopus japonicus and had critical roles in non-self recognition and bacterial clearance against invading microbes.

Cloning and transcriptional analysis of two sialic acid-binding lectins (SABLs) from razor clam Solen grandis

Sialic acid-binding lectin (SABL) plays crucial role in both innate and adaptive immune responses benefiting from its predominant affinity toward glycan. In the present study, two SABLs from razor clam Solen grandis (designated as SgSABL-1 and SgSABL-2) were identified, and their expression patterns, both in tissues and towards microorganism glycan stimulation, were then characterized. The cDNA of SgSABL-1 and SgSABL-2 was 988 and 1281 bp, containing an open reading frame (ORF) of 744 and 570 bp, respectively, and deduced amino acid sequences showed high similarity to other invertebrates SABLs. Both SgSABL-1 and SgSABL-2 encoded a C1q domain. SgSABL-1 and SgSABL-2 were found to be constitutively expressed in a wide range of tissues with different levels, including mantle, gill, gonad, hemocyte, muscle, and hepatopancreas, and both of them were highly expressed in hepatopancreas. SgSABL-1 and SgSABL-2 could be significantly induced after razor clams were stimulated by acetylated subunits-containing glycan LPS and PGN, suggesting the two SgSABLs might perform potential function of glycan recognition. In addition, SgSABL-2 could also be induced by β-1,3-glucan. All these results indicated that SgSABL-1 and SgSABL-2 might be involved in the immune response against microbe infection and contributed to the pathogens recognition.

The development process and seasonal changes of the gonad in Octopus ocellatus Gray off the coast of Qingdao, Northeast China

Little information exists on the gonadal development and maturity scale of Octopus ocellatus, an economically important octopus species commonly found in the west Pacific Ocean. A total of 171 O. ocellatus samples were collected monthly off the coast of northeast China (Qingdao, Shandong) from December 2011 to November 2012. Total body weight, mantle length, gonadosomatic index, and reproductive tract parameters of the samples were analyzed in males and females. The process of gonadal development in O. ocellatus was defined based on gonadal histological examination. Nine stages of oocyte development were identified during oogenesis, and a maturity scale of five stages (immature, developing, maturing, mature, and spent) was proposed for both ovary and testis. Additionally, the maturation status and dominant germ cell types of O. ocellatus in relation to monthly variation were identified. The seasonal changes showed that O. ocellatus off the coast of Qingdao is a fast-growing species with a short life span, which reproduces only once in a lifetime and then dies. Its reproductive cycle can be divided into four periods, beginning with the breeding period from April. Males become sexually mature in a smaller size than females, indicating that the mating behavior occurs early with sperm stored in the oviductal glands of the females before fertilization and spawning. This work contributes to the understanding of the maturation process in O. ocellatus, laying a foundation for further study on the mechanism of sperm storage in female octopuses and providing a reference for artificial breeding and fisheries impact assessment of this species.

Construction of a full-length cDNA library of Solen grandis Dunker and identification of defense- and immune-related genes

The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the ‘switching mechanism at the 5′-end of the RNA transcript’ (SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL−1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes (E-value < 1e−5; percent identity >80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.

Construction of a normalized full-length cDNA library of cephalopod Amphioctopus fangsiao and development of microsatellite markers

Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450 (25%) were assigned into 33 Gene Ontology terms, 576 (31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275 (15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

An Inhibitor κB Homolog from the Bivalve Mollusc Solen grandis that Responds to Immune Challenge

ABSTRACT The nuclear factor-&kgr;B (NF-&kgr;B) signaling pathway has been studied extensively in mammalians and insects but has been less well investigated in marine molluscs. Inhibitor of &kgr;B (I&kgr;B), an important component of the NF-&kgr;B signaling pathway, serves as a crucial mediator of the innate immune system. A homolog of I&kgr;B was identified in a razor clam (Solen grandis), designated as SgI&kgr;B, and its messenger RNA expression was detected both in tissues and towards pathogen-associated molecular patterns. Full-length complementaryDNAof SgI&kgr;B is 2,232 bp, containing a 181-bp 5′ untranslated region (UTR) and a 970-bp 3# UTR with a poly (A) tail. The open reading frame is 1,080 bp, encoding a 359-amino acid polypeptide with a predicted molecular weight of 40.1 kDa and an isoelectric point of 4.88. A potential PEST motif (E2SNDLEMDTCPLEMDS17) and the I&kgr;B degradation motif (ES44GYKS48) are located at the N-terminus, and 2 conserved casein kinase II phosphorylation sites (S337DEE340 and S346YDD349) exist at the C terminus. The presence of 6 conserved ankyrin repeats in SgI&kgr;B and its close phylogenetic relationship with other I&khgr;Bs strongly suggest that SgI&kgr;B belongs to the I&khgr;B superfamily. Messenger RNA of SgI&kgr;B is expressed constitutively in various tissues of healthy S. grandis, with the greatest expression in gill and hepatopancreas, followed by gonad, mantle, hemocyte, and muscle in descending order. Messenger RNA expression of SgI&kgr;B in hemocytes is upregulated significantly to varying degrees (P < 0.01) on stimulation with lipopolysaccharide, peptidoglycan, and &bgr;-1,3-glucan. The results indicate the existence of a NF-&kgr;B signaling pathway in S. grandis and provide evidence for possible regulatory mechanisms during an immune challenge.

Role of scavenger receptor from Octopus ocellatus as a co-receptor of Toll-like receptor in initiation of TLR-NF-κB signaling during anti-bacterial response

&NA; Scavenger receptors are crucial for innate immunity owing to their prominent role in clearance of harmful endogenous factors, immune recognition, and more importantly, as co‐receptors of Toll‐like receptors (TLRs) to initiate downstream responses. At present, invertebrate scavenger receptors, especially their role in immune mechanisms, are largely unknown. We report here that scavenger receptors form a diverse superfamily in Octopus ocellatus, including at least five different members with distinct tissue expression patterns. Two members, OoSR‐B and OoSR‐I, are grouped into class B and I scavenger receptors, respectively. OoSR‐B and OoSR‐I are located on the hemocyte membrane, and both recombinant scavenger receptors could serve as pattern recognition receptors to bind a broad range of pathogen‐associated molecular patterns. Although OoSR‐B and OoSR‐I expression was induced by bacterial stimulation, only OoSR‐B promoted hemocyte phagocytosis. Moreover, OoSR‐B, but not OoSR‐I, could act as a co‐receptor of TLR to activate TLR‐NF‐&kgr;B signaling and initiate TNF‐&agr; production during anti‐bacterial response. As the first report on an invertebrate scavenger receptor acting as a co‐receptor of TLR, our study reveals the immune mechanism mediated by scavenger receptors in O. ocellatus, and provides new insight into the evolution of this important receptor family.