Branchiostoma Belcheri Research Articles

Identification and expressional analysis of green fluorescent protein genes in amphioxus

Endogenous green fluorescent proteins (GFPs) have recently been found in amphioxus, where different characteristics of light production in different development stages and between different individuals have manifested. We identified the GFP genes in an amphioxus species Branchiostoma belcheri and monitored real time fluorescence signals of GFPs during different developmental stages to provide an insight on the GFP function in amphioxus. We found there are at least 12 endogenous GFP genes in amphioxus genome, and fluorescent expression changes in body position during different developmental stages. Additionally, GFP expression after metamorphosis development differed significantly among different amphioxus individuals, suggesting that there are multiple GFP homologous genes responsible for fluorescent expression. Expression levels of these genes varied significantly during different developmental stages, indicating that different GFP genes may have their unique functions in the development of amphioxus during specific phases of growth.

HTS-PEG: A Method for High Throughput Sequencing of the Paired-Ends of Genomic Libraries

Second generation sequencing has been widely used to sequence whole genomes. Though various paired-end sequencing methods have been developed to construct the long scaffold from contigs derived from shotgun sequencing, the classical paired-end sequencing of the Bacteria Artificial Chromosome (BAC) or fosmid libraries by the Sanger method still plays an important role in genome assembly. However, sequencing libraries with the Sanger method is expensive and time-consuming. Here we report a new strategy to sequence the paired-ends of genomic libraries with parallel pyrosequencing, using a Chinese amphioxus (Branchiostoma belcheri) BAC library as an example. In total, approximately 12,670 non-redundant paired-end sequences were generated. Mapping them to the primary scaffolds of Chinese amphioxus, we obtained 413 ultra-scaffolds from 1,182 primary scaffolds, and the N50 scaffold length was increased approximately 55 kb, which is about a 10% improvement. We provide a universal and cost-effective method for sequencing the ultra-long paired-ends of genomic libraries. This method can be very easily implemented in other second generation sequencing platforms.

Consecutive Spawnings of Chinese Amphioxus, Branchiostoma belcheri, in Captivity

Cephalochordate amphioxus is a promising model animal for studying the evolutionary and developmental mechanisms of vertebrates because its unique phylogenetic position, simple body plan and sequenced genome. However, one major drawback for using amphioxus as a model organism is the restricted supply of living embryos since they are available only during spawning season that varies from a couple of days to several months according to species. Therefore we are aiming to develop methods for obtaining viable amphioxus embryos in non-spawning season. In the current study, we found that Branchiostoma belcheri could develop their gonads and spawn consecutively in the laboratory when cultured in a low density at a high temperature (25–28°C) supplied with sufficient food and proper cleanness. Among the approximate 150 observed animals, which spawned spontaneously between November and December 2011, 10% have spawned twice, 10% three times, and 80% four times, through April 2012. The quality and quantity of the gametes reproduced in the consecutive spawning have no obvious difference with those spawned once naturally. Spawning intervals varied dramatically both among different animals (from 1 to 5 months) and between intervals of a single individual (from 27 to 74 days for one animal). In summary, we developed a method with which, for the first time, consecutive spawnings of amphioxus in captivity can be achieved. This has practical implications for the cultivation of other amphioxus species, and eventually will greatly promote the utilization of amphioxus as a model system.

Induction of phenoloxidase and other immunological activities in the humoral fluids of amphioxus Branchiostoma belcheri challenged with Lipopolysaccharide (LPS)

The knowledge concerning the humoral immunity is scarce in amphioxus Branchiostoma belcheri. This study measured the humoral parameters including phenoloxidase (PO) activity, lysozyme activity, antimicrobial activity, microbial agglutinin, and hemagglutinin in amphioxus humoral fluids before and after lipopolysaccharide (LPS) challenge. Humoral fluids from unchallenged Branchiostoma belcheri (B. belcheri) had PO activity, lysozyme, antimicrobial, microbial agglutinating, and hemagglutinating activities, which may represent part of the baseline level of innate immunity in this organism. After challenge with LPS, many humoral parameters were all increased significantly including the PO activity, lysozyme activity, growth-inhibiting activities against Gram-negative bacteria Escherichia coli (E. coli) and Vibrio alginolyticus (V. alginolyticus), growth-inhibiting activities against Gram-positive bacteria Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis), microbial agglutinating activities against Micrococcus lysodeikticus (M. lysodeikticus), B. subtilis, and S. aureus, and hemagglutinating activities against rabbit and human A and O erythrocytes. In contrast, the agglutinating activities against V. harveyi and E. coli and the hemagglutinating activity against human B erythrocytes in the humoral fluids were reduced in response to LPS challenge. It appears that the humoral fluids of B. belcheri contain components that are able to differentiate different microbes and different human blood cell types.

Proteomic characterization and evolutionary analyses of zona pellucida domain-containing proteins in the egg coat of the cephalochordate, Branchiostoma belcheri

BackgroundZona pellucida domain-containing proteins (ZP proteins) have been identified as the principle constituents of the egg coat (EC) of diverse metazoan taxa, including jawed vertebrates, urochordates and molluscs that span hundreds of millions of years of evolutionary divergence. Although ZP proteins generally contain the zona pellucida (ZP) structural modules to fulfill sperm recognition and EC polymerization functions during fertilization, the primary sequences of the ZP proteins from the above-mentioned animal classes are drastically different, which makes it difficult to assess the evolutionary relationships of ZP proteins. To understand the origin of vertebrate ZP proteins, we characterized the egg coat components of Branchiostoma belcheri, an invertebrate species that belongs to the chordate subphylum Cephalochordata.ResultsFive ZP proteins (BbZP1-5) were identified by mass spectrometry analyses using the egg coat extracts from both unfertilized and fertilized eggs. In addition to the C-terminal ZP module in each of the BbZPs, the majority contain a low-density lipoprotein receptor domain and a von Willebrand factor type A (vWFA) domain, but none possess an EGF-like domain that is frequently observed in the ZP proteins of urochordates. Fluorescence in situ hybridization and immuno-histochemical analyses of B. belcheri ovaries showed that the five BbZPs are synthesized predominantly in developing eggs and deposited around the extracellular space of the egg, which indicates that they are bona fide egg coat ZP proteins. BbZP1, BbZP3 and BbZP4 are significantly more abundant than BbZP2 and BbZP5 in terms of gene expression levels and the amount of mature proteins present on the egg coats. The major ZP proteins showed high polymorphism because multiple variants are present with different molecular weights. Sequence comparison and phylogenetic analysis between the ZP proteins from cephalochordates, urochordates and vertebrates showed that BbZP1-5 form a monophyletic group and share no significant sequence similarities with the ZP proteins of urochordates and the ZP3 subtype of jawed vertebrates. By contrast, small regions of homology were identifiable between the BbZP and ZP proteins of the non-jawed vertebrate, the sea lamprey Petromyzon marinus. The lamprey ZP proteins were highly similar to the ZP1 and ZP2 subtypes of the jawed vertebrates, which suggests that the ZP proteins of basal chordates most likely shared a recent common ancestor with vertebrate ZP1/2 subtypes and lamprey ZP proteins.ConclusionsThe results document the spectra of zona pellucida domain-containing proteins of the egg coat of basal chordates. Particularly, the study provides solid evidence for an invertebrate origin of vertebrate ZP proteins and indicates that there are diverse domain architectures in ZP proteins of various metazoan groups.

Cloning and characterization of a COMMD4 gene from amphioxus (Branchiostoma belcheri): An insight into the function and evolution of COMMD4

The recently discovered COMM domain-containing (COMMD) protein family has ten members (COMMD1–10). A COMMD protein contains a carboxyl-terminal COMM (copper metabolism gene MURR1) domain which provides an interface for protein–protein interactions. However, no COMMD4 gene has been identified in amphioxus. In this study, we report the identification and characterization of a COMMD4 gene from the amphioxus (designated as AmphiCOMMD4). First, we cloned the full length of AmphiCOMMD4 gene and found that the deduced amino acid sequence of AmphiCOMMD4 has 55.5–82.5% similarity and 38.8–64.0% identity to those from other species. Second, the genomic structure analysis of AmphiCOMMD4 gene showed that COMMD4 underwent intron loss in amphioxus. Third, we found that the AmphiCOMMD4 was ubiquitously and differentially expressed in five investigated tissues (muscles, gills, intestine, hepatic cecum and gonad). Finally, we found that the expression level of AmphiCOMMD4 gene was induced by LPS stimulation. In conclusion, our study elaborates the COMMD4 in amphioxus and provides an insight into the innate immunity and the evolution of the COMMD family.

Molecular cloning and characterization of an IKK homologue from amphioxus (Branchiostoma belcheri)

The IκB kinase (IKK) plays a crucial role in activation of the transcription factor nuclear factor kappa B (NF-κB) by phosphorylating the Inhibitory of NF-κB (IκB), which triggers the subsequent polyubiquitylation and degradation of IκBα through the 26S proteasome. In this study, we reported the cloning of an IKK homologue cDNA (designated as AmphiIKK) from amphioxus, Branchiostoma belcheri. The full-length cDNA consists of 3,087 bp with an ORF that encoded a predicted protein of 777 amino acid residues. The putative amphioxus IKK protein possesses the characteristic organization of the mammalian IKK proteins, which consists of a serine/threonine kinase domain, a leucine zipper motif and a putative helix-loop-helix motif. And the deduced amino acid of AmphiIKK shared 43.8-64.0 % similarity and 23.6-43.3 % identified with the IKKs from other species. Real-time PCR (RT-PCR) analysis indicated that AmphiIKK was ubiquitously expressed in all tissues and we also discovered that the expression of AmphiIKK was affected after lipopolysaccharides stimulation. Our findings help to highlight a potential important regulatory pathway in the innate immune response in the amphioxus and the evolution of the IKK family.

Isolation and functional analysis of the promoter of the amphioxus Hsp70a gene

Amphioxus is a promising laboratorial model animal for studying the evolutionary and developmental mechanisms that appeared during the invertebrate-chordate to vertebrate transition. However, the main drawback for the use of amphioxus as a model organism is the lack of well-developed technical approaches. Conditional gene expression, as performed with thermal control, is a very useful strategy in gene function studies. To make this method possible in amphioxus studies, here we report the isolation and characterization of an amphioxus Hsp70 gene (Hsp70a) and its promoter in Chinese amphioxus (Branchiostoma belcheri). Hsp70a showed very low expression at normal temperatures but was robustly induced in animals upon heat shock. The basal cis-acting elements (CAAT and TATA), as well as four heat shock elements (HSEs), were found within the regulatory region (−1031 to −11 upstream from the start codon), but surprisingly most of the elements were located in the 5′UTR region (−252 to −10). Reporter constructs, including sequences from both the transcription start site (TSS) and ATG were tested for transient expression in EPC cells and microinjected zebrafish embryos. Results suggested that the 5′UTR region, which includes a TATA box at −92bp, a CAAT box at −152bp, and three HSE elements (−212 to −106), represents the core hsp promoter sequence of the B. belcheri Hsp70a gene. Therefore in this study we identified an effectively thermo-inducible promoter in amphioxus that could be used for the establishment of a conditional gene expression system in which the target gene can be regulated in a temporal- or tissue-specific way in amphioxus.

Identification, expression of a glycoprotein hormone receptor homolog in the amphioxus Branchiostoma belcheri with implications for origin of vertebrate GpHRs

Amphioxus is phylogenetically located at the most primitive position of the chordate clade. Its endocrine system has been extensively studied; however, key information about the glycoprotein hormone (GpH) and its receptor (GpHR) in the endocrine system remained to be elucidated. In this study, a GpHR homologous gene, BbGpHR-like, has been identified in the amphioxus Branchiostoma belcheri. It contains a 1377bp open reading frame that corresponds to a deduced protein of 458 amino acid residues with a predicted molecular mass of approximately 50kDa. Phylogenetic analysis shows that BbGpHR-like is located at the position between vertebrate and invertebrate, indicating amphioxus B. belcheri is in an evolutionary transitional state between vertebrate and invertebrate in the GpHR lineage. BbGpHR-like has been successfully expressed in Pichia pastoris. Western blot analysis confirmed that the receptor produced a cross-immunoreactivity with human thyrotropin receptor (TSHR), the luteinizing hormone receptor (LHR) and the follicle-stimulating hormone receptor (FSHR). In situ hybridization and immunohistochemical analyses revealed that the BbGpHR-like transcripts and proteins were strongly distributed in the ovaries, testes and/or endostyle. These findings suggest that BbGpHR-like possibly plays an ancient and fundamental role in the control of thyroid hormone synthesis and gametogenesis in chordates. Presumably, the amphioxus BbGpHR-like represents the ancestral form of the GpHR gene prior to its split to the vertebrate paralogs gonadotropin receptor and thyrotropin receptor.

Particularity and universality of a putative Gram-negative bacteria-binding protein (GNBP) gene from amphioxus (Branchiostoma belcheri): Insights into the function and evolution of GNBP

Gram-negative bacteria-binding proteins (GNBPs) are important pattern recognition proteins (PRPs), which can initiate host defense in response to pathogen surface molecules. The roles of GNBP in innate immunity of arthropods and molluscs have recently been reported. However, the GNBP gene has not been characterized in the species of higher evolutionary status yet. In this study, we identified and characterized an amphioxus GNBP gene (designated as AmphiGNBP). First, we identified and cloned the AmphiGNBP and found that the AmphiGNBP encodes a putative protein with 558 amino acids, which contains a conserved β-1, 3-glucan recognizing and binding domain. Second, we found that the AmphiGNBP encodes two extra WSC (cell Wall integrity and Stress response Component) domains, which are unique in AmphiGNBP protein. The two WSC domains of AmphiGNBP protein coupled with the expansion of amphioxus immunity repertoire might undergo intensive domain shuffling during the age of the Cambrian explosion. Finally, we found that the AmphiGNBP was mainly expressed in immune tissues, such as hepatic cecum and intestine, and the expression of AmphiGNBP was affected after LPS stimulation. In conclusion, our findings disclose the particularity and universality of AmphiGNBP and provide profound insights into the function and evolution of GNBP.

Molecular cloning, characterization and expression analysis of tumor necrosis factor receptor-associated factor 3 (TRAF3) from pearl oyster Pinctada fucata

TRAF3 is a highly versatile regulator that negatively regulates JNK and alternative nuclear factor-κB signalling, but positively controls type I interferon production. To investigate TRAF3 function in innate immune responses among invertebrate especially mollusk, we characterized TRAF3 (PfTRAF3) from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfTRAF3 cDNA is 2261 bp with an open reading frame of 1623 bp encoding a putative protein of 541 amino acids. The deduced PfTRAF3 contains a RING finger domain, two TRAF domains with zinc finger domains and a conserved C-terminal meprin and TRAF homology (MATH) domain. Comparison and phylogenetic analysis revealed that PfTRAF3 from mollusk shared a higher identity with Ciona intestinalis TRAF3 from urochordata, Branchiostoma belcheri TRAF3 from cephalochordate, and even TRAF3 from vertebrate than with insect homologues. Furthermore, gene expression analyses suggested that PfTRAF3 was involved in the immune response to Vibrio alginolyticus.

HaploMerger: reconstructing allelic relationships for polymorphic diploid genome assemblies.

Whole-genome shotgun assembly has been a long-standing issue for highly polymorphic genomes, and the advent of next-generation sequencing technologies has made the issue more challenging than ever. Here we present an automated pipeline, HaploMerger, for reconstructing allelic relationships in a diploid assembly. HaploMerger combines a LASTZ-ChainNet alignment approach with a novel graph-based structure, which helps to untangle allelic relationships between two haplotypes and guides the subsequent creation of reference haploid assemblies. The pipeline provides flexible parameters and schemes to improve the contiguity, continuity, and completeness of the reference assemblies. We show that HaploMerger produces efficient and accurate results in simulations and has advantages over manual curation when applied to real polymorphic assemblies (e.g., 4%–5% heterozygosity). We also used HaploMerger to analyze the diploid assembly of a single Chinese amphioxus (Branchiostoma belcheri) and compared the resulting haploid assemblies with EST sequences, which revealed that the two haplotypes are not only divergent but also highly complementary to each other. Taken together, we have demonstrated that HaploMerger is an effective tool for analyzing and exploiting polymorphic genome assemblies.

Identification and characterization of a putative lipopolysaccharide-induced TNF-α factor (LITAF) gene from Amphioxus (Branchiostoma belcheri): An insight into the innate immunity of Amphioxus and the evolution of LITAF

Innate immunity defenses against infectious agent in all multicultural organisms. TNF-α is an important cytokine that can be stimulated by Lipopolysaccharide (LPS) to regulate the innate immunity. The lipopolysaccharide-induced TNF-α factor (LITAF) functions as a transcription factor for regulating the expression of TNF-α as well as various inflammatory cytokines in response to LPS stimulation. The physiological significance of LITAF gene in the innate immunity of various animals has recently been reported. However, no LITAF gene has yet been identified in amphioxus, which is the best available stand-in for the proximate invertebrate ancestor of the vertebrates. In this study, we identified and characterized an amphioxus LITAF gene (designated as AmphiLITAF). First, we identified the AmphiLITAF from the amphioxus and found that AmphiLITAF gene with ∼1.6kb in length has a 827bp cDNA transcription product which encodes a putative protein with 127 amino acids containing conserved LITAF-domain, and the deduced amino acid of AmphiLITAF shared 37–60% similarity with the LITAFs from other species; second, we uncovered the spatial distribution of the LITAF in different tissues, the expression level of AmphiLITAF mRNA was the highest in hepatic cecum and intestine, moderate in muscles, gills and gonad, and the lowest in notochord. Our findings provide an insight into the innate immune response in the amphioxus and the evolution of the LITAF family.

The distribution and habitat use of <I>Branchiostoma belcheri</I> at the mouth of Jiaozhou Bay, Qingdao in Autumn

The distribution and habitat use of <I>Branchiostoma belcheri</I> at the mouth of Jiaozhou Bay, Qingdao in Autumn

Sialome analysis of the cephalochordate Branchiostoma belcheri, a key organism for vertebrate evolution

Sialic acid, a common terminal substitution of glycoconjugates, has been so far consistently identified in all vertebrates as well as in a growing number of bacterial species. It is assumed to be widely distributed among animal species of the deuterostome phylum, based on its identification in few echinoderm and all vertebrate species. However, whole sections of deuterostome, especially those intermediate species between invertebrates and vertebrates including cephalochordates, urochordates and hemichordates, are still unexplored in term of sialylation capacities. The discovery of functional sialic acid machinery in some of these species may shed new light onto the evolution of glycosylation capacities in deuterostome lineage. In a first approach, we investigated the sialylation pattern of a cephalocordate species, Branchiostoma belcheri, which occupies a strategic phylogenetic position to understand the transition of invertebrates toward vertebrates. Structural analysis of B. belcheri glycoconjugates established that this organism synthesizes large quantities of various sialic acids, some of which present rare or novel structures such as methylated sialic acids. These sialic acids were shown to be mainly associated with mono- and disialylated core 1-type O-glycans. Moreover, screening of the animal organs revealed the existence of exquisite tissue specificity in the distribution of sialic acids. Description of sialylation profiles was then correlated with the expression patterns of key enzymes involved in the biosynthesis of major forms of sialic acids, which provides the first complete overview of the sialylation patterns in cephalochordates.

Microsatellite DNA marker development and genetic diversity of Branchiostoma belcheri in Xiamen waters

Amphioxus occupies a transitional position between invertebrates and vertebrates and is therefore a promising laboratory model animal. As a powerful molecular marker in population genetics studies, microsatellite DNA is required for both the investigation of genetic diversity of a wild amphioxus population and strain breeding of laboratory cultured amphioxus. In the present study, we constructed a microsatellite-enriched DNA library of amphioxus Branchiostoma belcheri to develop this molecular marker. After screening the library and combining it with the data obtained from a public database, we successfully developed 21 pair of primers that could effectively amplify the microsatellite loci of amphioxus. Of those primer pairs, seven were in deviation of Hardy–Weinberg equilibrium, mostly caused by null alleles. The others had no linkage disequilibrium, indicating they are applicable for the genetic studies. Using those primer pairs, we further detected the genetic diversity of the wild amphioxus population in Xiamen waters. The results revealed that the number of alleles of each microsatellite locus was between three and 18 in the population, and observed and expected heterozygosities of the loci ranged from 0.415 to 0.947 and from 0.300 to 1.000, indicating very high genetic diversity of the population.

The conservation and uniqueness of the caspase family in the basal chordate, amphioxus

BackgroundThe caspase family, which plays a central role in apoptosis in metazoans, has undergone an expansion in amphioxus, increasing to 45 members through domain recombination and shuffling.ResultsIn order to shed light on the conservation and uniqueness of this family in amphioxus, we cloned three representative caspase genes, designated as bbtCaspase-8, bbtCaspase-1/2 and bbtCaspase3-like, from the amphioxus Branchiostoma belcheri tsingtauense. We found that bbtCaspase-8 with conserved protein architecture is involved in the Fas-associated death domain-Caspase-8 mediated pro-apoptotic extrinsic pathway, while bbtCaspase3-like may mediate a nuclear apoptotic pathway in amphioxus. Also, bbtCaspase-1/2 can co-localize with bbtFADD2 in the nucleus, and be recruited to the cytoplasm by amphioxus apoptosis associated speck-like proteins containing a caspase recruitment domain, indicating that bbtCaspase-1/2 may serve as a switch between apoptosis and caspase-dependent innate immune response in invertebrates. Finally, amphioxus extrinsic apoptotic pathway related caspases played important roles in early embryogenesis.ConclusionsOur study not only demonstrates the conservation of bbtCaspase-8 in apoptosis, but also reveals the unique features of several amphioxus caspases with novel domain architectures arose some 500 million years ago.

Molecular Cloning and Sequence Analysis of Peroxiredoxin cDNA from American White Shrimp (Litopenaeus vannamei)

Peroxiredoxin (Prx) is an antioxidant protein, which protects organisms against various oxidative stresses. In this study, we isolated Peroxiredoxin cDNA from the muscle tissues of American white shrimp, Litopenaeus vannamei, using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA consists of 962-bp, which includes a 49-bp 5′-untranslated region (UTR), a 316-bp 3′-untranslated region, and a 597-bp open reading frame (ORF) encoding 198 amino acids. The signal peptide sequence was not found in this cDNA. We aligned the deduced amino acid sequence with the known amino acid sequences of Fenneropenaeus indicus, Fenneropenaeus chinensis, Marsupenaeus japonicus, Scylla serrata, Drosophila melanogaster, Bos taurus, Branchiostoma belcheri, Anoplopoma fimbria, and Rattus norvegicus, and the sequence similarities scores were found to be 97%, 96%, 95%, 83%, 72%, 70%, 80%, 81%, and 75%, respectively. We also found 2-cysteine (Cys) residues in this peroxiredoxin sequence. The RT-PCR analysis revealed that the peroxiredoxin mRNA was expressed in the gills, hepatopancreas, muscles, intestine, and hemocytes. Studies using this newly cloned peroxiredoxin gene from Litopenaeus vannamei will add to the existing knowledge base on the physiological role of peroxiredoxin in shrimp species.

Identification of up-regulated genes in amphioxus neurula and the expression of AmphiFABP

Amphioxus is a good model organism for understanding the origin and developmental mechanism of vertebrates owing to its important evolutionary position. During the developmental process of amphioxus embryo, the neurula is a crucial stage because of neural tube and notochord formation as well as somite emergence at this stage. In order to isolate genes up-regulated at the neurula stage, we constructed an 11-hour neurula subtracted cDNA library of amphioxus Branchiostoma belcheri and sequenced 204 ESTs representing 82 contigs. Comparative analysis revealed that 55% of those contigs were homologous to various known genes while 45% of them had no significant similarity to any known genes. Those observations imply that the un-identified ESTs might contain some new genes which are involved in the development of amphioxus neurula. Real-time quantitative PCR (RTqPCR) indicated that the expression levels of 14 genes are up-regulated after gastrulation among 20 assayed genes. Of those up-regulated genes, we further cloned and sequenced the full-length of fatty acid binding protein gene (AmphiFABP). The deduced protein sequence was similar to that of vertebrate brain FABP and heart FABP, and in situ hybridization displayed that AmphiFABP, similar to their vertebrate cognates, was expressed not only in nervous system but also in embryonic somite and gut, hinting a multifunctional property of AmphiFABP in amphioxus.

Gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri

To understand the role of calcium-binding proteins of invertebrates in immunological response, amphioxus sarcoplasmic calcium-binding protein (SCP) was investigated in the present study. Following gene cloning, recombinant protein expression and purification and antibody preparation, the expression and alteration of SCP in the response to bacterial challenge were detected using Western blotting. SCP was not detected in the branchia, humoral fluid, gonad or in the gut of wounded animals, but it was abundant in muscle and appeared in the gut of healthy animals using Vibrio parahaemolyticus immunization and challenge. Furthermore, whether gut SCP possessed anamnestic response was investigated using cross-immune challenge between Gram-positive and –negative bacteria. Gut SCP showed stronger anamnestic activity or pattern-recognition in response to Gram-negative bacterium V. parahaemolyticus than Gram-positive bacterium Staphylococcus aureus. The response was faster and more species-specific to V. parahaemolyticus, whereas it was slower and longer to S. aureus. The reason why the response showed significant difference between Gram-positive and -negative bacteria awaits investigation. These results indicate that gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri.