Chromosome Preparation and Preliminary Observation of Two Amphioxus Species in Xiamen

Amphioxus has an important evolutionary position as a result of their phylogenetic position relative to vertebrates. Understanding their chromosomes would provide key points in the study of evolutionary biology and comparative genomics. The difficulty in preparing amphioxus chromosomes currently provides a significant hurdle in this research. In the current study, we describe an improved method for metaphase preparation from amphioxus embryos and methodology for preparing metaphase spreads from regenerative somatic cells. Chromosomes of two amphioxus species from Xiamen waters in China are also observed. The diploid chromosome number was found to be 40 in Branchiostoma belcheri, while B. japonicum has 36, confirming the two are distinct species from cytotaxonomic viewpoint.

Hepatic caecum of amphioxus and origin of vertebrate liver

Liver is characteristic of all vertebrates.As a critical hub for many physiological processes including metabolism,innate immunity,protein synthesis and detoxification,its evolutionary origin was largely underappreciated in history,and only received due attention in recent decades.It has been suggested by morphological,ultrastructural and immunohistochemical studies that the hepatic caecum of amphioxus is homologous to the liver of vertebrate species.Molecular biology studies demonstrated that amphioxus hepatic caecum expresses plenty of vertebrate liver-specific genes.Our functional studies revealed significant similarities between amphioxus hepatic caecum and vertebrate liver.We also found that the functions of hepatic caecum are subjected to the regulation of pituitary hormones just as the liver does.These provide solid evidences supporting the notion that the hepatic caecum is the homologue of liver,which may represent the first stage in chordate evolution,laying a foundation for the subsequent formation of the liver as we know it in vertebrates.Further studies on the specification and morphogenesis of hepatic caecum in amphioxus will shed more lights on the origin and evolution of vertebrate liver.

Developmental Expression of an Amphioxus (Branchiostoma belcheri) Gene Encoding a GATA Transcription Factor

GATA factors are evolutionarily conserved and play crucial roles during embryonic development in both vertebrates and invertebrates. Vertebrate GATAs can be divided into two subgroups, the GATAI/2/3 and the GATA4/5/6 classes. Through genomic analysis, we have identified three GATA factors, representing the GATA1/2/3 and GATA4/5/6 subfamilies respectively, and one GATA like protein in the genome of the basal chordate amphioxus （Branchiostoma floridae, cephalochordata）. Partial sequence of GATA123 in the amphioxus Branchiostoma belcheri （BbGATA123） was cloned and its expression pattern during early embryonic development was studied. Expression of BbGATA123 is first detected in the mesendoderm during gastrulation. Interestingly, in the late neurula and early larva stages, it is expressed strongly in the cerebral vesicle and the mid gut region. Its expression is compared to Otx, a gene known crucial for the development of anterior structures. Our observations suggest that GATA123, together with Otx, might play an important role in the development of amphioxus cerebral vesicle, the counterpart of the vertebrate brain.

LABORATORY OBSERVATION ON SPAWNING,FECUNDITY AND LARVAL DEVELOPMENT OF AMPHIOXUS (BRANCHIOSTOMA BELCHERI TSINGTAUNESE)

Although amphioxus is widespread in temperate and tropical seas,its population is diminishing be-cause of environmental pollution.To keep the population of this evolutionarily important animal fromdiminishing,study on its reproduction and development is necessary.The main findings in this study onthe spawning and fecundity of the amphioxus reared in laboratory and its larval development are as fol-lows.1.Water temperature markedly affected the spawning.It spawned only when water temperature reached 21℃.2.Spawning of the amphioxus in laboratory was markedly extended.Initially,the amphioxus spawned at about 7:00 PM,but spawning time was postponed as spawning days went on.3.The number of eggs produced by a female ranged from 1400 to 12800,average of 5800.This al-so represents the fecundity of the amphioxus because it shedded all eggs within the ovary at a time.4.During the first few months of life of the amphioxus,its growth rate changed seasonally.Thegrowth rate in summer and fall was greater than that

MORPHOLOGICAL AND FUNCTIONAL STUDIES ON THE EPIDERMAL CELLS OF AMPHIOXUS (BRANCHIOSTOMA BELCHERI TSINGTAUENSE) AT DIFFERENT DEVELOPMENTAL STAGES

Epidermal Cells of amphioxus at different developmental stages were investigated by electron microscopy and colloidal carbon tracing experiments.Amphioxus epidermal cells showed different ultrastructural characteristics at larval and adult stages. The epidermal cells at all larval stages studied (24-96 h) had numerous usicles containing elatron dense materials in their apical Cytoplasm. In tracing experiments, carbon particles were found in apical vesicles and intercellular spaces. Under scanning electron microscope,many crater-like protrusions were observed on the surface of the cells. These results indicated that amphioxus larva epidermal cells may be capable of endocytosis. The epidermal cells of 3-month and adult amphioxus un obviously secretory ones characterized by well -developed peripheral filaments, a prominent Golgi apparatus and abundant apical secretory vesicles. This study also showed that adult amphioxus body surfare mucus contained lectin that could aggutinate human red blood cels. The authors propose that the epidermal cells of amphioxus larva and adult may contribute to the immune defense of the animal by different means.

PRODUCTION OF FERTILE EGGS AND SPERMS IN LABORATORY-REARED AMPHIOXUS BRAN- CHIOSTOMA BELCHERI TSINGTAUNESE

Although amphioxus has been studied embryologically for over 130 years,little is known concerning the gonadal development of the laboratory reared animal.The present study in which the amphioxus were successfully maintained in the laboratory for more than one year showed that they could produce fertile eggs and sperms like their wild counterparts.

Expansion of the Actin Gene Family in Amphioxus

Actins are a small family of ubiquitous proteins that are essential cytoskeletal components and are highly conserved during evolution. Actins are usually divided into two classes, the cytoplasmic and muscle actins, which have different functional roles. Here we systematically analyzed the actin genes in the genome of the primitive chordate amphioxus （Branchiostoma floridae）. We found that amphioxus contains more than 30 actin genes, many of which are linked. Phylogenetic analysis suggests the amphioxus actin genes have clearly undergone extensive expansion through tandem duplications. The actin genes＇ structure also varies a lot, containing 2 to 7 exons. We also cloned two muscle type of actin genes from the amphioxus （B. belcheri） and compared their expression patterns during early development. The slight difference in their expression suggests functional diversification of these actin genes. Our results shed light on the evolution both of actin genes themselves and their functional roles in chordate development.

Localization of nitric oxide synthase in the developing gonads of amphioxus Branchiostoma belcheri tsingtauense

The localization of nitric oxide synthases （NOS） is dealed with in the developing gonads of amphioxus Branchiostoma belcheri tsingtauense. It was found by NADPH-diaphorase staining that （1） NOS activity was present in the nuclear membranes of germinal vesicles during the entire period ofoocyte development; （2） NOS was localized in both the nuclear membranes and the perinuclear region of cytoplasm in the vitellogenetic oocytes; （3） NOS was relocated in the cortical layer in the mature egg; （4） NOS activity was present in spermatocytes, but not in the spermatogonia in the middle of October; （5） NOS was detected in both spermatozoa and spermatids as well as spermatocytes during the breeding season. This is the first report on the distribution pattern of NOS in the developing gonads in protochordates. These results suggest a role for NOS in the functioning of the nuclear membranes and yolk synthesis during oogenesis and in cell division and differentiation during spermatogenesis.

Ultrastructural study of oogenesis in Xiamen amphioxus

Using electron microscopic technique, ultrastructural characters of the oogonia and oocyte at the dif ferent phases of amphioxus are observed in detail in the present study.The squeezed out nucleolus, nucleolus-like bodies and yolk nucleus at the side of nucleus are the characters in the early egg cells of the first and secondary phase.The nucleus and cytoplasm of oocyte change obviously their morphology ad structure from large growth stage to mature stage (from Stage Ⅲ to Stage V). As a result, the dense distribution of nuclear pore and the exten sion and depression of nuclear envelope are observable.Mitochondria,Golgi complex,rough endoplasm reticular and annulate lamellae,and others in the cytoplasm join in the formation of yolk granular.Their morphology and number also change correspondingly with the development and maturation of the oocyte.These results will provide a whole base for the reproductive physiology and artificial propagation as well as the resource management of amphioxus.

INDUCTION OF ECTODERMAL CELLS FROM VEGETALENDODERMAL BLASTOMERES OF AMPHIOXUS(BRANCHIOSTOMA BELCHERI TSINGTAUNESE) EMBRYOS BY THE CALCIUM IONOPHORE A23187

Timing of vegetal-endodermal cell determination in amphioxus embryos remains uncertain. We tentatively testal effects of A23187, the calcium ionophore, on the deveopment of vegetal blastomeres isolated at the 16-cell stage. It was found that when vegetal blastomres committed to endodermwere treated with A23187 prior to gastrulation, they were transformed into ectodermal cells as evidenced by the cell morphology and function characteristic of epidermis. Howver, the developmental fate of the sam blastomeres untreated or treated with DMSO at the same stage or of those treated with A23187 after gastrulation remained unchanged. Thus, vegetal-endodermal cells in amphioxus embryos are not irreversibly deermined before the gastrula stage, and artificial incarease in intracelluar Ca2+ concentration can induce transdetermination of the predetermined endodermal cells into ectodermal cells.

Expression analysis of eight amphioxus genes involved in the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in the embryonic development of metazoans. Although the pathway has been studied extensively in many model animals, its function in amphioxus, the most primitive chordate, remains largely uncharacterized. To obtain basic data for functional analysis, we identified and isolated seven genes （Lrp5/6, Dvl, APC, Ckla, CklS, Gsk3β, and Gro） of the Wnt/β-catenin signaling pathway from the amphioxus （Branchiostoma floridae） genome. Phylogenetic analysis revealed that amphioxus had fewer members of each gene family than that found in vertebrates. Whole-mount in situ hybridization showed that the genes were maternally expressed and broadly distributed throughout the whole embryo at the cleavage and blastula stages. Among them, Dvl was expressed asymmetrically towards the animal pole, while the others were evenly distributed in all blastomeres. At the mid-gastrula stage, the genes were specifically expressed in the primitive endomesoderm, but displayed different patterns. When the embryo developed into the neurula stage, the gene expressions were mainly detected in either paraxial somites or the tail bud. With the development of the embryo, the expression levels further decreased gradually and remained only in some pharyngeal regions or the tail bud at the larva stage. Our results suggest that the Wnt/β-catenin pathway might be involved in amphioxus somite formation and posterior growth, but not in endomesoderm specification.

Antioxidant enzyme activities in different genders and tissues of amphioxus Branchiostoma belcheri tsingtauense

Information regarding antioxidant enzymes in amphioxus remains lacking, and this study was carried out to examine the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in different genders and tissues of amphioxus Branchiostoma belcheri tsingtauense. Results show that (1) CuZn-SOD, CAT and GPX activities in the whole amphioxus B. belcheri tsing- tauense were basically at the same levels in male and female amphioxus, whereas both T-SOD and Mn-SOD activities in male amphioxus were significantly higher than that in the female (P<0.05); (2) The testis had significantly higher T-SOD and CuZn-SOD activities than the ovary (P<0.05); (3) CuZn-SOD activity was undetectable in the guts of male and female amphioxus; (4) For both male and female am- phioxus, the activities of CAT and GPX in the gonads including testis and ovary were the lowest (P<0.05) among the tissues examined; (5) The gut and gill had the same level GPX activities while the gut had a higher CAT activity; (6) There was no clear difference in CAT and GPX activities in the corresponding tissues between male and female amphioxus. The study on SOD, CAT and GPX activities in different genders and tissues of the protochordate provides data for future comparison of amphioxus antioxidant enzymes with those of invertebrates and vertebrates.

Spatiotemporal variations of benthic macrofaunal community in the Xiamen Amphioxus Nature Reserve, eastern South China Sea

In order to realize the spatiotemporal variations of benthic macrofaunal communities at the"Amphioxus Sand"habitat,six surveys including four seasons and three consecutive summers(i.e.,2014,2015 and 2016)were conducted in two core sites,i.e.,Huangcuo(HC)and Nanxian-Shibaxian(NX),in the Xiamen Amphioxus Nature Reserve in China.A total of 155 species of macrofauna were recorded,therein,polychaetes were dominant in terms of species number and density.Significant spatiotemporal variations of macrofaunal communities were observed.The density of polychaetes and the biomass of molluscs in the HC site were higher than those in the NX site.Macrofauna were more diverse and abundant in the cold seasons(winter and spring)than that in the warm seasons(summer and autumn).The annual variations of macrofaunal communities may be attributed to the changes in sediment texture among the three years of the survey.The variations in macrofaunal communities were mainly related to the proportion of polychaetes within the community.In addition,the density of amphioxus(include Branchiostoma japonicum and B.belcheri)was negatively correlated to that of polychaetes,bivalves,and crustaceans.Amphioxus was less likely to be found in the sediments with higher silt and clay content.Five biotic indices including Margaref’s richness index(d),Peilou’s evenness index(J′),Shannon-Wiener diversity index(H′),AMBI and M-AMBI were calculated in the present study.AMBI seems suitable in assessing benthic health at the"Amphioxus Sand"habitat,and a potential risk of ecological health in Xiamen Amphioxus Nature Reserve should be aware.

Annual change of the gonadal development of the amphioxus in Xiamen

The study was made by observing the annual change of the gonadal development of amphioxus in Xiamen cohtinuously for two years from November, 1986 to November, 1988. We determined the gonadal diameter, gonadosomatic index of each amphioxus, and observed the histological characteristic of the gonadal development of the female and male by cut section, which provided the basis for dividing the stages gonads and the determining level of the gonadal development of amphioxus in each month.

Interplay Between Amphioxus Complement with Sea Bass Macrophages: Opsonic Activity of Amphioxus Humoral Fluids

Previous studies have shown the existence of a complement system in the amphioxus Branchiostoma japonicum.How-ever,whether it has an opsonic activity similar to that of vertebrates remains unknown.We demonstrated that the humoral fluid(HF)of amphioxus promoted the phagocytosis of yeast cells with sea bass(Lateolabrax japonicus)macrophages,whereas the C3-depleted and heated HF significantly lost the phagocytosis-promoting capacity.In addition,the precipitation of factor B(Bf)led to a marked loss of opsonic activity.Moreover,C3 fragments in the HF were found to bind to yeast cell surfaces.The results indicate that the amphioxus complement system is an important element involved in the opsonic activity,which promotes the sea bass macrophage phagocytosis by tagging yeast cells with C3 fragments via the activation of alternative complement pathway.

Expression, purification and polyclonal antibody generation of p23, an Hsp90 cochaperone, in the amphioxus Branchiostoma belcheri

The cDNA of amphioxus p23, a highly conserved co-chaperone for Hsp90, was cloned into a bacterial expression vector pGEX - 6P - 1 and the GST-tagged fusion protein was produced in Eschherichia coli cells. The recombinant p23 was purified by affinity purification, and its molecular mass was estimated to be approximately 22 kDa by sodium dedecyl sulfate-polyacrylamide gel electrephoresis. The N-terminus of purified p23 was sequenced, and the resulting amino acid sequence matches exactly the predicted residues deduced from the amphioxus p23 gene. Besides, pelyclonal antibodies against the recombinant p23 were generated, and these antibodies not only recognized specifically the fusion protein GST - p23 from induced E. coli cells, purified GST - p23 and p23 protein, but also reacted with the total protein extracted fi＇om the adult amphioxus and formed a single positive band. These results pave the way for identifying its tissue and subcellular localization, and may open the door to clarifying its structure and mechanisms of biological role.

Cryopreservation of amphioxus (Branchiostoma belcheri) spermatozoa

Amphioxus, Branchiostoma belcheri, a closest relative of vertebrates, is at a high risk of extinction due to a combination of low effective population size, altered native habitats and environmental pollution, yet little is known about cryopreservation of its gametes. This study deals with the cryopreservation of amphioxus senlen. The main findings are that （1） the extender of Yao et al. is the best one among the four extenders examined; （2） the appropriate ratio of semen to extender of Yao et al. plus cryoprotectant is from 1：5 to 1：7; （3） dimethyl sulfoxide （DMSO） and methanol are the better cryoprotectants than glycerol, with DMSO giving the best results; （4） the eggs fertilized with post-thaw spermatozoa are capable of developing to at least hatching stage, and the highest hatching rate is （12.4±3.0）%. This is the first report on freezing and thawing of amphioxus spermatozoa, providing a simple and practical protocol for cryopreservation of amphioxus spermatozoa and laying a foundation for safeguarding this endangered species.

Developmental expression amphioxus Branchiostoma pattern of calmodulin gene in belcheri tsingtauense

We investigated the developmental expression pattern of AmphiCaM in cephalochordate amphioxus （Branchiostoma belcheri tsingtauense）. We cultured and sampled the animals at different developmental stages （eggs and larvae）, and used in-situ hybridization and northern blotting to document the spatial and temporal changes in AmphiCaM expression. The alimentary tract dominates the development from the late neurula stage to the adult stage. AmphiCaM expression increased significantly in the alimentary tract during the late neurula stage and remained elevated in the adults. Our results indicate that AmphiCaM is involved in the differentiation of the alimentary tract in amphioxus; and furthermore, provide an insight into the change in function of CaM genes during evolution.

ARTIFICIAL CULTURE OF AMPHIOXUS (BRANCHIOSTOMA BELCHERI TSINGTAUENSE)

Mature amphioxus ( Branchiostoma belcheri tsingtauense ) collected by trawl in June, 1994 from Qingdao Shazikou sea area bottom sands, were cultured under controlled indoor conditions, where they spawned and their eggs were fertilized and hatched into larvae which gradually settled at the bottom and dug into the sands after 40-50 days culture in a water trough outdoors. The total survival rate of the larvae(LSR)was 5.5% before they went into the sands, 1.9% in 4 months, 0.7% in 10 months. The survival rate of the young fish which had dug into the sands (YSR) was 35.6% in 4 months, and 12.6% in 10 months. The amphioxus number tended to be constant from the 11th month on. In the first five months after incubation, the amphioxus body length increased by an average of about 1.5 mm a month, and about 0.4 mm a month from Dec. to May of next year. The maximum length after ten months was 24 mm; the average was about 11 mm. After two years culture in the water trough, the maximum length could reach 34-35 mm, when the amphioxus gonads began to develop.

Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

A full length amphioxus cDNA, encoding a novel phosducin-like protein （Amphi-PhLP）, was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame （ORF） of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind grit and testis, hepatic caecum, gill, endostyle, and epipbaryngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary （CHO） cells transfected with the expression plasmid pEGFP-NI/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.