Amphioxus Branchiostoma Belcheri Research Articles

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 °C to 35 °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 Ca 2+, 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.

Non-synchronous spawning behavior in laboratory reared amphioxus Branchiostoma belcheri Gray

Spontaneous spawning in amphioxus ( Branchiostoma belcheri Gray) was observed in a tank and the characteristics of spawning were analyzed by video recording under infrared light conditions during summer of 2001. Everyday when spawning was observed, without exception a male first swam up from the bottom and released sperm near the water at the surface of the tank. The initiation time of the male spawning gradually became earlier as days passed. Spawning males and females individually swam up at various intervals and released gametes. However, at the population level, the spawning pattern of amphioxus was considered to be synchronous because both males and females intensively spawned around 90 min after the spawning of the first male. The act of releasing eggs or sperms of individuals was shorter than 10 min in most of the spawning animals.

Two classic cadherin-related molecules with no cadherin extracellular repeats in the cephalochordate amphioxus: distinct adhesive specificities and possible involvement in the development of multicell-layered structures

We previously reported the existence of Bb-cadherin, a molecule related to classic cadherin, in the cephalochordate amphioxus (Branchiostoma belcheri). The structure of Bb-cadherin is unique in that it lacks the cadherin extracellular repeats, although its cytoplasmic domain shows close similarities to those of typical classic cadherins. The extracellular region of Bb-cadherin consists of laminin globular domains and a cysteine-rich EGF-like domain that are similar to domains in nonchordate classic cadherins. In this study, we identified a second amphioxus cadherin. It was designated Bb2-cadherin (Bb2C) while the previously reported cadherin has been renamed Bb1-cadherin (Bb1C). Bb2C is very similar to Bb1C in its overall structure and amino acid sequence. Genomic BLAST searches and phylogenetic analyses suggested that these two amphioxus genes have been generated through a gene duplication that occurred after separation of the cephalochordates from the other animals. They also bear distinct adhesive specificities. Immunohistochemical analyses showed that Bb1C and Bb2C, together with beta-catenin, appear to function as adherens junction constituents in the epithelia of different germ layers of the amphioxus embryo. Differential expression of the two cadherins was also observed in the developing, multicell-layered notochord. These observations suggest that, despite their unique structures, the functions and developmental roles of Bb1C and Bb2C are comparable to those of the classic cadherins characterized to date in other animal groups, such as the vertebrate E- and N-cadherins and the Drosophila DE- and DN-cadherins. The possible involvement of Bb1C and Bb2C in the development of multicell-layered structures characteristic of the cephalochordate body plan is presented.

C-banding pattern and nucleolar organizer regions of amphioxus Branchiostoma belcheri tsingtauense Tchang et Koo, 1936.

Chromosome banding study remains lacking in amphioxus heretofore. This paper reports the C-band pattern of the metaphase chromosomes of amphioxus Branchiostoma belcheri tsingtauense, the first description of banded karyotype in cephalochordate. The C-banding showed that around 50% of the second chromosome pair examined was heteromorphic, while the remaining ones were homomorphic. In contrast, the other 17 pairs were all homomorphic. These broadly support our previous suggestion that the second chromosome pair is a pair of sex-chromosomes. The C-banding stained positively about 54.3% of the chromosome surface. Most of the centromeric and terminal regions in many chromosomes had positive C-bands, and some interstitial C-bands were also observed in some chromosomes. It seems clear that chromosomes of amphioxus are highly heterochromatinized. In addition, silver staining revealed a single pair of nucleolar organizer regions (NORs) located in the telomeric regions of medium-sized chromosomes. It is suggested that the number and location of NORs in amphioxus represent the primitive condition for chordates.

Identification of the ribosomal proteins s15a and L19 from the amphioxusbranchiostoma belcheri tsingtauense

Abstract The complete cDNA and deduced amino acid sequences of the ribosomal protein S15a (AmphiS15a) and L19 (AmphiL19) of the amphioxus Branchiostoma belcheri tsingtauense were identified in the present study. The AmphiS15a cDNA is 540 nucleotides in length and encodes a 130 amino acid protein with a calculated molecular mass of 149 kDa. It shares 70-85% sequence identity to its known counterparts in the eukaryotes including human, rat, sea urchin, fruit fly and yeast. The AmphiL19 cDNA comprises 728 nucleotides and encodes a 197 amino acid protein with a calculated molecular mass of 23.4 kDa. It shows 59-79% sequence identity to the eukaryotic L19 proteins from human, rat, mouse, fruit fly, Caenorhabditis elegans and yeast Both AmphiS15a and AmphiL19 are highly basic and contain putative nucleolar localization signals. As house-keeping genes, they may both be useful to normalize gene expression in characterization of various genes from the amphioxus.

Gonadal state of wild amphioxus populations and spawning success in captive conditions during the breeding period in Japan.

Adult individuals of amphioxus (Branchiostoma belcheri) were collected by dredging from a research vessel at selected stations in two areas off the coast of Japan in July 2000: Deyama and Takamatsu so named by local fishermen in the Enshu Nada Sea. The number of males collected exceeded that of females at all the stations in Takamatsu and at four of five stations in Deyama. The over all sex ratio (males : females) of the collected animals was 1.2 : 1. The animals showed various maturational stages of the gonad, and approximately 70% had mature gonads. However, post-spawning animals were identified only at two stations in Takamatsu. Mature animals were placed in laboratory tanks. These animals remained in good conditions for about two months, and many animals spontaneously spawned in the tanks. This is the first report of spontaneous spawning of B. belcheri in Japan.

Origin of mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 involved in the lectin complement pathway traced back to the invertebrate, amphioxus.

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.

Cloning and phylogenetic analysis of an amphioxus myogenic bHLH gene AmphiMDF

In this study, a member of the MyoD gene family, AmphiMDF, was isolated from the embryos of amphioxus by degenerate PCR, followed by rapid amplification of cDNA ends (RACE). Southern blot analysis confirmed that only a single myogenic bHLH gene was present in the genome of amphioxus Branchiostoma belcheri tsingtauense. Sequence and phylogenetic analyses indicated that AmphiMDF falls at the base of its vertebrate homologs. The amino acid sequence of AmphiMDF was almost equally similar to those of the four clusters of the vertebrate MyoD family. This suggests that AmphiMDF is not only the sister but also the archetype of the vertebrate myogenic bHLH genes. The scenarios to explain the origin of the vertebrate MyoD gene family from the ancestral myogenic bHLH gene like AmphiMDF are also discussed.

The karyotype of amphioxus Branchiostoma belcheri tsingtauense (Cephalochordata)

The chromosome number and karyotype of amphioxus Branchiostoma belcheri tsingtauense were studied using embryonic cells. The diploid chromosome number (2n) of B. belcheri tsingtauense is 36, and its karyotype 2n=36, 2st+34t, FN=36. This is the first report on the karyotype of the cephalochordate. Evidence suggesting the possible presence of a pair of sex-chromosomes in the amphioxus has been provided.

Histochemical localization of constitutive nitric oxide synthases in amphioxus Branchiostoma belcheri tsingtauense

The present study demonstrated histochemically that the enzyme activity was present in the cerebral vesicle, epidermis, muscles, endostyle and anus of amphioxus Branchiostoma belcheri tsingtaunese. This is the first noted report on localization of constitutive nitric oxide synthases in a celphalochordate.

A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny.

Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic-binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell-cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister-groups vertebrates and urochordates.

Amphi-Eomes/Tbr1: an amphioxus cognate of vertebrate Eomesodermin and T-Brain1 genes whose expression reveals evolutionarily distinct domain in amphioxus development.

A cDNA for a novel T-box containing gene was isolated from the amphioxus Branchiostoma belcheri. A molecular phylogenetic tree constructed from the deduced amino acid sequence of the isolated cDNA indicates that this gene belongs to the T-Brain subfamily. In situ hybridization reveals that the expression is first detected in the invaginating archenteron at the early gastrula stage and this expression is down-regulated at the neurula stage. In early larvae, the expression appears again and transcripts are detected exclusively in the pre-oral pit (wheel organ-Hatschek's pit of the adult). In contrast to the vertebrate counterparts, no transcripts are detected in the brain vesicle or nerve cord throughout the development. These results are interpreted to mean that a role of T-Brain products in vertebrate forebrain development was acquired after the amphioxus was split from the lineage leading to the vertebrates. On the other hand, comparison of the tissue-specific expression domain of T-Brain genes and other genes between amphioxus and vertebrates revealed that the pre-oral pit of amphioxus has several molecular features which are comparable to those of the vertebrate olfactory and hypophyseal placode.

C6-like and C3-like molecules from the cephalochordate, amphioxus, suggest a cytolytic complement system in invertebrates.

The mammalian immune system has cytotoxic mechanisms, both cellular and humoral, that destroy the membrane integrity of target cells. The main effector molecules of these cytolytic mechanisms-perforin, used by killer lymphocytes, and the membrane attack complex (MAC) components of the complement system-share a unique module called the MAC/perforin module. Until now, both immunological cytotoxicity and the MAC/perforin module have been reported only in jawed vertebrates. Here, we report the identification of a protein containing the MAC/perforin module from the invertebrate cephalochordate, amphioxus ( Branchiostoma belcheri), using expressed sequence tag (EST) analysis of the notochord. The deduced amino acid sequence of this molecule is most similar to the primary structure of human complement component C6 and is designated AmphiC6. AmphiC6 shares a unique modular structure, including the MAC/perforin module, with human C6 and other MAC components. Another EST clone predicts the presence of a thioester-containing protein with the closest structural similarity to vertebrate C3 (therefore designated AmphiC3). AmphiC3 retains most of the functionally important residues of vertebrate C3 and is shown by phylogenetic analysis to be derived directly from the common ancestor of vertebrate C3, C4, and C5. Only opsonic activity has been assigned to the invertebrate complement system until now. Therefore, this is the first molecular evidence for complement-mediated immunological cytotoxicity in invertebrates.

Presence and induction by bacteria of D-galactoside-specific lectins in the humoral fluids of amphioxus Branchiostoma belcheri tsingtauense

The presence of lectins in the humoral fluids of amphioxus, an animal transitional from invertebrates to vertebrates, has thus far not been reported. It was shown here that lectins are present in the humoral fluids of amphioxus. The lectin activity in the humoral fluids of amphioxus is SH group-dependent and is specifically inhibited by various D-galactosides. Also, the humoral fluids obtained from E. coli-injected amphioxus showed increased agglutinating activity against human B and O, rabbit, grass carp and toad erythrocytes, suggesting that a form of adaptive response might exist in amphioxus.

Calcitonin‐immunoreactive cells of the digestive tract of the amphioxus are distributed concentrically in a restricted region of the mid‐gut

AbstractIn amphioxus (Branchiostoma belcheri), calcitonin‐immunoreactive cells of the digestive tract were distributed concentrically in a 1‐mm region of the latter half of the mid‐gut. In the mid‐gut caecum these cells were also present, but were dispersed throughout the length. The total number of cells in both parts ranged from 280 to 1157, varying from individual to individual, although the number in the mid‐gut caecum was only 100–200. These cells were morphologically typical gut endocrine cells. Considering their morphology and the characteristics of their distribution pattern, these cells may be related to some phenomena of digestive processes.

Temporal and Spatial Profiles of Alkaline Phosphatase Activity During Embryogenesis of Amphioxus Branchiostoma belcheri tsingtaunese

Temporal and spatial profiles of alkaline phosphatase (ALP) activity during the development of amphioxus have not been fully documented and thus the present study investigated this activity spectrophotometrically, electrophoretically and histochemically. The following results were observed: (1) spectrophotometrically, ALP activity increased markedly at the late gastrula stage and reached a plateau at 15 hr postfertilization; (2) the electrophoretic pattern of ALP isozymes changed dramatically during development; (3) ALP activity was initially localized in the posterior wall of the primitive gut and the anterior 5 to 6 somites at about the 15 hr larva stage, and then in the notochord and all the somites at about the 18 hr larva stage; (4) in 1-day larvae, ALP activity decreased in the posterior wall of the primitive gut and in the anterior 5 to 6 somites which had ALP activity at 15 hr, but it appeared in the newly formed somites, especially in the myosepta, the crevices cut in between adjoining somites; (5) in 2-day larvae, ALP activity was no longer visible in somites but became highly evident in most of the notochord except for its rostral region; and (6) when the lateral plate mesoderm pushed down ventrally on either side of the intestine and conjoined beneath the intestine, ALP activity was also detected in the conjoining lateral plate mesoderm. Apparently, two types of ALP exist in amphioxus larvae, the transient endodermal ALP and the constant mesodermal ALP, and the spatial and temporal correlation of ALP activity with the developing mesoderm, including the notochord, suggests that it plays a role in the differentiation of mesodermal structures during the development of amphioxus.

Phenoloxidase, a marker enzyme for differentiation of the neural ectoderm and the epidermal ectoderm during embryonic development of amphioxus Branchiostoma belcheri tsingtaunese

The development of phenoloxidase during amphioxus embryogenesis was spectrophotometrically and histochemically studied for the first time in the present study. It was found that (1) PO activity initially appeared in the general ectoderm including the neural ectoderm and the epidermal ectoderm at the early neurula stage but not in the mesoderm or the endoderm, and (2) PO activity disappeared in the neural plate cells but remained unchanged in the epidermal cells when the neural plate was morphologically quite distinct from the rest of the ectoderm. It is apparent that PO could serve as a marker enzyme for differentiation of the neural ectoderm from the epidermal ectoderm during embryonic development of amphioxus.

Genomic organization and evolution of actin genes in the amphioxus Branchiostoma belcheri and Branchiostoma floridae.

We previously described the cDNA cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203-215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon-intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages, and gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates.

Editorial comment

The distribution of ghrelin-like immunoreactive cells in amphioxus (Branchiostoma belcheri) was investigated by using immunohistochemical staining with rabbit antiserum against synthetical mammalian ghrelin. The results showed that ghrelin-like immunoreactive cells were distributed widely in the nervous system, Hatschek’s pit, wheel organ, digestive tract and gonads (ovary and testis). In nervous system, ghrelin-like immunoreactive neurons and their protrusions were distributed specifically on the dorsal side, ventral side and funnel part of brain vesicle, with a few dispersive immunoreactive nerve cells and their fibers in nerve tube. Ghrelin-like immunoreactivities were also detected in Hatschek’s pit epithelial cells and wheel organ cells, with positive substance located along cell membrane. In digestive tract, ghrelin-like immunoreactive cells existed in hepatic diverticulum, anterior and posterior region of midgut, and could be classified into two types, closed- and opened-type endocrine cells. The number of positive cells was most in hepatic diverticulum, secondary in posterior region of midgut and least in anterior region of midgut. In gonads, ghrelin-like immunoreactive substance was detected in oogonia, oocytes and follicle cells in ovary at the small and large growth stages and in early spermatogenic cells and Sertoli cells in testis. The extensive distribution of ghrelin-like cells in amphioxus suggested that these kinds of cells are conservative in evolution and diversified in function. At the same time, we found for the first time that ghrelin-like immunoreactive cells existed in brain vesicle and Hatschek’s pit, which provided new morphological evidence for the existence of an activation pathway between brain vesicle and Hatschek’s pit for the regulation of growth hormone excretion.

PARACRYSTALS OF AMPHIOXUS PARAMYOSIN

The present article recommends briefly a method for extracting amphioxus paramyosin.Ethanol-dehydrated amphioxus (Branchiostoma belcheri, Gray) when treated with a molar KCl solution followed by dialysis against water, will produce large quantities of paracrystals of paramyosin in a few hours. Under the electron microscope, paramyosin crystals of amphioxus showed cross striations with a periodicity of 14.5-15 and 5-7 nm.