Sperm-derived sperm motility-initiating substance from amphioxus Branchiostoma belcheri

The sperm of amphioxus, Branchiostoma belcheri, were immotile when excised from the testis and suspended in seawater. The sperm became motile upon spawning in natural seawater, suggesting mechanisms that triggered sperm motility during spawning. When a male amphioxus that underwent spawning was transferred to a cup containing a small amount of natural seawater, and then the seawater containing the spawned sperm was centrifuged, the supernatant caused motility initiation in the immotile sperm from the testis. This sperm motility-initiating activity was also found in the supernatant of seawater in which immotile sperm from the testis were incubated overnight. These suggest that in the amphioxus, a sperm motility-initiating substance resides in the sperm, and upon spawning, the substance is transformed into a free and active form to activate the sperm. Partial purification of the substance revealed it as a small and heat-stable substance with maximum UV absorbance at 234 nm.     

Purification and characterisation of phenoloxidase from amphioxus Branchiostoma belcheri tsingtauense

Phenoloxidase (PO) from the humoral fluid of amphioxus B. belcheri tsingtauense was purified using a sequential combination of ammonium sulphate precipitation, Sephadex G-200 chromatography and DEAE Sepharose Fast Flow chromatography. In PAGE, the purified enzyme exhibited a single band of 150 kDa under non-reducing conditions, and was resolved to three bands with molecular masses of 72, 46 and 44 kDa, respectively, under reducing conditions, suggesting that the PO in amphioxus humoral fluid seems to be a heterotrimer of three polypeptides held together by disulphide bonds. The substrate specificity and inhibition characteristics both indicate that the PO isolated from amphioxus humoral fluid is a tyrosinase-type enzyme. In addition, mouse antisera against the purified PO were prepared, and their specificity was confirmed by Western blotting, facilitating the future determination of the origin of PO in the humoral fluid and the distribution of PO-synthesising tissues in amphioxus.     

Presence and characterization of complement-like activity in the amphioxus Branchiostoma belcheri tsingtauense

The humoral fluid of Branchiostoma belcheri tsingtauense was examined for the presence of complement-like activity. The humoral fluid showed hemolytic activity for rabbit erythrocytes and those from species representing mammals, birds, amphibians and fish, but not sensitized sheep erythrocytes. There was no relationship between phylogeny of the target erythrocytes and degree of hemolysis. The hemolytic activity was optimally assayed at 20 degrees C, at pH 7.5, and in the presence of 10 mM Mg2+. The hemolytic activity was Mg2+-dependent and heat-sensitive, and was abrogated by treatment with rabbit anti-human C3 serum, zymosan, methylamine, hydrazine, and phenylmethylenesulfonyl fluoride. In addition, Western blotting and titration by turbidimetric immunoassay (TIA) revealed that amphioxus humoral fluid contained C3 component, and its concentration is about 1.17 mg/ml, which is comparable to C3 concentration in human or dog sera. These suggest that the hemolytic activities displayed by amphioxus humoral fluid appear to represent the vertebrate complement system probably operating via the alternative pathway.     

Characterization and expression of p23 gene in the amphioxus Branchiostoma belcheri

The cDNA AmphiP23, encoding an amphioxus p23, was identified from the gut cDNA library of amphioxus Branchiostoma belcheri. It contains a 513 bp open reading frame corresponding to a deduced protein of 170 amino acids. Phylogenetic analysis shows that vertebrate and invertebrate p23/p23-like proteins are each grouped together, with AmphiP23 falling at the base of vertebrate p23/p23-like clade, suggesting that the divergence of vertebrate and invertebrate p23 genes probably occurs prior to the split of invertebrate/vertebrate from a common ancestor around 550 million years ago. Northern blotting reveals a ubiquitous expression pattern of AmphiP23 in all adult tissues examined, while whole mount in situ hybridization demonstrates a tissue- and stage-specific expression pattern of AmphiP23 in developing embryos and larvae. Presumably, the ubiquitous expression pattern of AmphiP23 in adult amphioxus represents the ancestral type of p23 gene prior to its split to human paralogs p23 and tsp23, while the tissue- and stage-specific expression pattern during early embryonic development implicates a role of AmphiP23 in anterior/posterior patterning.     

Evolution of CUT class homeobox genes: insights from the genome of the amphioxus, Branchiostoma floridae

CUT class homeobox genes, including CUX/CASP, ONECUT, SATB and COMPASS family genes, are known to exhibit diverse features in the homeodomain and the domain architecture. Furthermore, the intron/exon organization of CUX/CASP is different between vertebrates and protostomes, and SATB genes are only known for vertebrates, whereas COMPASS genes have only been found in protostomes. These observations suggest a complex evolutionary history for the CUT class homeobox genes, but the evolution of CUT class homeobox genes in the lineage to vertebrates remained largely unknown. To obtain clearer insights into this issue, we searched the genome of amphioxus, Branchiostoma floridae, a lower chordate, for CUT class homeobox genes by extensive BLAST survey and phylogenetic analyses. We found that the genome of Branchiostoma floridae encodes each single orthologue of CUX/CASP, ONECUT, and COMPASS, but not the SATB gene, and one atypical CUT gene likely specific to this species. In addition, the genomic structure of the amphioxus CUX/CASP gene turned out to be protostome-type, but not vertebrate-type. Based on these observations, we propose a model in which SATB is suggested to evolve at the expense of COMPASS and this change, together with the structural change in CUX/CASP, is supposed to take place in the lineage to vertebrates after divergence of the amphioxus and vertebrate ancestors. The present study provides an example of dramatic evolution among homeobox gene groups in the vertebrate lineage and highlights the ancient character of amphioxus, retaining genomic features shared by protostomes.     

雌雄文昌鱼同工酶的表型差异

本文应用聚丙烯酰胺凝胶电泳结合生化染色方法分析了雌雄文昌鱼中苹果酸酶、苹果酸脱氢酶、酸性磷酸酶和酯酶四种同工酶的酶谱。首次发现苹果酸酶、苹果酸脱氢酶和酸性磷酸酶表型在文昌鱼雌性和雄性个体之间存在差异 ,而在同一性别不同个体之间无差异。酯酶表型较复杂 ,不但在不同性别个体之间而且在同一性别不同个体之间都出现一定差异     

Whole‐genome resequencing reveals the pleistocene temporal dynamics of Branchiostoma belcheri and Branchiostoma floridae populations

Global climatic fluctuations governed the ancestral demographic histories of species and contributed to place the current population status into a more extensive ecological and evolutionary context. Genetic variations will leave unambiguous signatures in the patterns of intraspecific genetic variation in extant species since the genome of each individual is an imperfect mosaic of the ancestral genomes. Here, we report the genome sequences of 20 Branchiostoma individuals by whole‐genome resequencing strategy. We detected over 140 million genomic variations for each Branchiostoma individual. In particular, we applied the pairwise sequentially Markovian coalescent (PSMC) method to estimate the trajectories of changes in the effective population size (N_e) of Branchiostoma population during the Pleistocene. We evaluated the threshold of sequencing depth for proper inference of demographic histories using PSMC was ≥25×. The PSMC results highlight the role of historical global climatic fluctuations in the long‐term population dynamics of Branchiostoma. The inferred ancestral N_e of the Branchiostoma belcheri populations from Zhanjiang and Xiamen (China) seawaters was different in amplitude before the first (mutation rate = 3 × 10^?9) or third glaciation (mutation rate = 9 × 10^?9) of the Pleistocene, indicating that the two populations most probably started to evolve in isolation in their respective seas after the first or third glaciation of the Pleistocene. A pronounced population bottleneck coinciding with the last glacial maximum was observed in all Branchiostoma individuals, followed by a population expansion occurred during the late Pleistocene. Species that have experienced long‐term declines may be especially vulnerable to recent anthropogenic activities. Recently, the industrial pollution and the exploitation of sea sand have destroyed the harmonious living environment of amphioxus species. In the future, we need to protect the habitat of Branchiostoma and make full use of these detected genetic variations to facilitate the functional study of Branchiostoma for adaptation to local environments.     

Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 degrees C to 35 degrees C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.     

Verification and tissue-specific expression of nifU-like gene from the amphioxus Branchiostoma belcheri

A nifU-like gene exhibiting similarity to nifU of nitrogen fixation gene cluster was identified for the first time from the gut cDNA library of amphioxus Branchiostoma belcheri. Both RT-PCR and Northern blotting as well as in situ hybridization histochemistry verified that the cDNA represents an amphioxus nifU-like gene rather than a microbial contaminant. The nifU-like gene encodes a protein of 164 amino acid residues including a highly conserved U-type motif (C-X26-C-X43-C), and shares 66-86% identity to NifU-like proteins from a variety of species including vertebrates, invertebrates and microbes. It is expressed in a tissue-specific manner in the digestive system including epipharyngeal groove, endostyle, hepatic caecum and hind-gut and in the gill, ovary and testis. Taken together, it is highly likely that NifU-like protein plays some tissue-dependent and critical role in amphioxus.     

Verification and tissue-specific expression of nifU-like gene from the amphioxus Branchiostoma belcheri.

A nifU-like gene exhibiting similarity to nifU of nitrogen fixation gene cluster was identified for the first time from the gut cDNA library of amphioxus Branchiostoma belcheri. Both RT-PCR and Northern blotting as well as in situ hybridization histochemistry verified that the cDNA represents an amphioxus nifU-like gene rather than a microbial contaminant. The nifU-like gene encodes a protein of 164 amino acid residues including a highly-conserved U-type motif (C-X(26)-C-X(43)-C), and shares 66-86% identity to NifU-like proteins from a variety of species including vertebrates, invertebrates and microbes. It is expressed in a tissue-specific manner in the digestive system including epipharyngeal groove, endostyle, hepatic caecum and hindgut and in the gill, ovary and testis. Taken together, it is highly likely that NifU-like protein plays some tissue-dependent and critical role in amphioxus.     

Epidermal receptor development and sensory pathways in vitally stained amphioxus (Branchiostoma floridae)

Chloromethyl (CM) DiI was applied to the exterior of living embryos, larvae, and metamorphic juveniles of amphioxus. This fluorescent dye is taken up preferentially (but not highly selectively) by epidermal receptors and often stains sensory axons to their full extent. Type I primary receptors in the epidermis first become morphologically detectable along the rostrocaudal axis of the 2.5 day larva when their epidermal perikarya extend unbranched axons to the nerve cord. These axons run posteriorly or anteriorly within the nerve cord, depending on whether their perikarya are located, respectively, rostral or caudal to the most posterior pharyngeal slit. In later larvae, axons of type I receptors are organized into a dorsal and a subdorsal sensory tract on either side of the nerve cord. In the epidermis of metamorphic juveniles, CM-DiI also stains type II receptors (which are axonless, secondary receptors) and ventral pit cells (which may not be receptors). It is probable, but not yet conclusively demonstrated, that peripheral neurites from Retzius bipolar cells (primary intramedullary sensory neurones) synapse with type II secondary epidermal receptors or ramify freely among the other epidermal cells. The discussion considers homologies among epidermal sensory receptors in chordates.     

Identification and tissue-specific expression of amphioxus GM2 activator protein gene from amphioxus Branchiostoma belcheri.

An amphioxus cDNA, AmphiGM2AP, encoding GM2 activator protein was isolated from the gut cDNA library of Branchiostoma belcheri. It is 907 bp long, and its longest open reading frame codes for a precursor protein consisting of 242 amino acid residues with a signal peptide of 14 amino acids. The deduced amino acid sequence includes a conserved domain typical of GM2APs between residues 53 and 224, a single N-linked glycosylation site at position 65 and 8 conserved cysteines. Phylogenetic analysis showed that amphiGM2AP forms a club together with invertebrate GM2APs, indicating that AmphiGM2AP is evolutionarily closely related to invertebrate GM2APs rather than vertebrate ones. Both Northern blotting and in situ hybridization histochemistry analyses revealed a tissue-specific expression pattern of AmphiGM2AP in adult amphioxus with the strongest expression in the digestive system, which is in contrast to the widespread expression pattern of human, mouse and sheep GM2AP genes. It is suggested that AmphiGM2AP is possibly involved in the take-in of digested food components like lipid molecules.     

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-Pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.