The Genetic Basis of Scale-Loss Phenotype in the Rapid Radiation of Takifugu Fishes.

Rapid radiation associated with phenotypic divergence and convergence provides an opportunity to study the genetic mechanisms of evolution. Here we investigate the genus Takifugu that has undergone explosive radiation relatively recently and contains a subset of closely-related species with a scale-loss phenotype. By using observations during development and genetic mapping approaches, we show that the scale-loss phenotype of two Takifugu species, T. pardalis Temminck & Schlegel and T. snyderi Abe, is largely controlled by an overlapping genomic segment (QTL). A search for candidate genes underlying the scale-loss phenotype revealed that the QTL region contains no known genes responsible for the evolution of scale-loss phenotype in other fishes. These results suggest that the genes used for the scale-loss phenotypes in the two Takifugu are likely the same, but the genes used for the similar phenotype in Takifugu and distantly related fishes are not the same. Meanwhile, Fgfrl1, a gene predicted to function in a pathway known to regulate bone/scale development was identified in the QTL region. Since Fgfr1a1, another memebr of the Fgf signaling pathway, has been implicated in scale loss/scale shape in fish distantly related to Takifugu, our results suggest that the convergence of the scale-loss phenotype may be constrained by signaling modules with conserved roles in scale development.

Expression and presentation of immune-related membrane proteins of fish by a cell surface display platform using insect cells.

Cell surface display is a useful platform to examine the interactions between two proteins of interest, such as immune receptors and ligands. This technique is also useful for studies on the immune receptors of lower vertebrates and invertebrates. However, in many cases, the commonly used cell culture temperature is relatively high for proteins from such organisms. Since insect cells can be cultured at lower temperatures than many other cells, and since they are equipped with "quality control" system, which is advantageous for the presentation of properly folded proteins, we anticipated that the insect cell surface display system could be more suitable for that type of research. In the present study, multiple cloning site of the commercially available expression vector pIB/V5-His was modified, and whether this vector could be useful to present fish immune-related membrane proteins was investigated. Using this plasmid, fugu's CD8α and CC chemokine receptor 7 could be presented on the cell surface. The clones of the lamprey variable lymphocyte receptors obtained previously by the yeast surface display (YSD) system as hen's egg lysozyme (HEL) binders also could be presented on the cell surface and bound to HEL. These results suggest that functional immune-related membrane proteins can be presented on the insect cell surface, indicating that this system is useful for immunological studies on exothermal animals.

Identification of the sex-determining locus in grass puffer (Takifugu niphobles) provides evidence for sex-chromosome turnover in a subset of Takifugu species.

There is increasing evidence for frequent turnover in sex chromosomes in vertebrates. Yet experimental systems suitable for tracing the detailed process of turnover are rare. In theory, homologous turnover is possible if the new sex-determining locus is established on the existing sex-chromosome. However, there is no empirical evidence for such an event. The genus Takifugu includes fugu (Takifugu rubripes) and its two closely-related species whose sex is most likely determined by a SNP at the Amhr2 locus. In these species, males are heterozygous, with G and C alleles at the SNP site, while females are homozygous for the C allele. To determine if a shift in the sex-determining locus occurred in another member of this genus, we used genetic mapping to characterize the sex-chromosome systems of Takifugu niphobles. We found that the G allele of Amhr2 is absent in T. niphobles. Nevertheless, our initial mapping suggests a linkage between the phenotypic sex and the chromosome 19, which harbors the Amhr2 locus. Subsequent high-resolution analysis using a sex-reversed fish demonstrated that the sex-determining locus maps to the proximal end of chromosome 19, far from the Amhr2 locus. Thus, it is likely that homologous turnover involving these species has occurred. The data also showed that there is a male-specific reduction of recombination around the sex-determining locus. Nevertheless, no evidence for sex-chromosome differentiation was detected: the reduced recombination depended on phenotypic sex rather than genotypic sex; no X- or Y-specific maker was obtained; the YY individual was viable. Furthermore, fine-scale mapping narrowed down the new sex-determining locus to the interval corresponding to approximately 300-kb of sequence in the fugu genome. Thus, T. niphobles is determined to have a young and small sex-determining region that is suitable for studying an early phase of sex-chromosome evolution and the mechanisms underlying turnover of sex chromosome.

Mucosal IgM Antibody with d-Mannose Affinity in Fugu Takifugu rubripes Is Utilized by a Monogenean Parasite Heterobothrium okamotoi for Host Recognition.

How parasites recognize their definitive hosts is a mystery; however, parasitism is reportedly initiated by recognition of certain molecules on host surfaces. Fish ectoparasites make initial contact with their hosts at body surfaces, such as skin and gills, which are covered with mucosa that are similar to those of mammalian guts. Fish are among the most primitive vertebrates with immune systems that are equivalent to those in mammals, and they produce and secrete IgM into mucus. In this study, we showed that the monogenean parasite Heterobothrium okamotoi utilizes IgM to recognize its host, fugu Takifugu rubripes Oncomiracidia are infective larvae of H. okamotoi that shed their cilia and metamorphose into juveniles when exposed to purified d-mannose-binding fractions from fugu mucus. Using liquid chromatography-tandem mass spectrometry analysis, proteins contained in the fraction were identified as d-mannose-specific IgM with two d-mannose-binding lectins. However, although deciliation was significantly induced by IgM and was inhibited by d-mannose or a specific Ab against fugu IgM, other lectins had no effect, and IgM without d-mannose affinity induced deciliation to a limited degree. Subsequent immunofluorescent staining experiments showed that fugu d-mannose-specific IgM binds ciliated epidermal cells of oncomiracidium. These observations suggest that deciliation is triggered by binding of fugu IgM to cell surface Ags via Ag binding sites. Moreover, concentrations of d-mannose-binding IgM in gill mucus were sufficient to induce deciliation in vitro, indicating that H. okamotoi parasites initially use host Abs to colonize host gills.

Identification and characterization of pufflectin from the grass pufferfish Takifugu niphobles and comparison of its expression with that of Takifugu rubripes.

Pufflectin found in Takifugu rubripes (Tr pufflectin) is the first animal lectin reported to show sequence similarity to monocotyledonous plant lectins. In the present study, we identified and characterized an orthologous lectin from Takifugu niphobles (Tn pufflectin), a species closely related to T. rubripes. Tn pufflectin exhibits 86% identity to Tr pufflectin with two conserved mannose-binding domains. Tn pufflectin was mainly expressed in the skin, gills, brain, and muscles; however, it was expressed at a lower level in the other examined tissues. Recombinant Tn pufflectin, expressed by Escherichia coli, exhibited binding activity specific for d-mannose. The expression of pufflectin in the gills was much lower in T. niphobles than in T. rubripes; notably, the former and latter are resistant and susceptible, respectively, to the monogenean parasite Heterobothrium okamotoi, which parasitizes gills. This suggests that pufflectin might be utilized by the parasite for host recognition.

Influences of radiation on carp from farm ponds in Fukushima.

A massive release of artificial radionuclides from the Fukushima Dai-ichi Nuclear Power Plant caused radioactive contamination of farms as well as of aquatic products. Carp in small ponds in the highly radiocontaminated area of Iitate Village, Fukushima Prefecture, have been confined to the ponds since the accident, and it is thought that the carp may have suffered health issues as a result. Therefore, I investigated the health condition of the carp in order to elucidate the effects of radiation.Blood neutrophil, monocyte and lymphocyte counts in the carp from three ponds in Fukushima were lower than those in carp from a non-polluted pond in Tochigi Prefecture. Histological observations indicated abnormal hyperplasia of macrophages in the spleen, kidney, liver and pancreas of carp in Fukushima. Although there are likely to have been deleterious effects on carp health due to the radiation in Fukushima, this has not yet been confirmed because only one control pond was available for comparison, and I was not able to find any symptoms in the carp that correlated with internal cesium concentration. Further research is now being conducted to investigate the effects of radiation on carp.

Genetic Basis Underlying Behavioral Correlation Between Fugu Takifugu rubripes and a Closely Related Species, Takifugu niphobles.

Correlated suits of behaviors (behavioral syndrome) are commonly observed in both inter- and intraspecific studies. In order to understand the genetic basis of such a correlation between species, we compared ten behaviors classified into five categories (acclimation, feeding, normal swimming, reaction to a novel object and activity in a novel environment) between two pufferfish species, Takifugu rubripes and T. niphobles. The two species showed consistent differences in nine behaviors with a significant correlation among behaviors. Quantitative trait locus (QTL) analysis using second generation hybrids revealed that different sets of small effect QTL are associated with the observed interspecific behavioral disparity. This indicates that correlations in temperament traits between them are governed by many genes with small effects, and each behavior has been selected to form particular combination patterns. One of the QTL showing small pleiotropic effect includes the Drd4 gene known for its association with behavioral traits in some animal taxa including mammals.

Screening of candidate genes encoding proteins expressed in pectoral fins of fugu Takifugu rubripes, in relation to habitat site of parasitic copepod Caligus fugu, using suppression subtractive hybridization.

Caligus fugu is a parasitic copepod specific to the tetraodontid genus Takifugu including the commercially important Takifugu rubripes. Despite the rapid accumulation of knowledge on other aspects of its biology, the host and settlement-site recognition mechanisms of this parasite are not yet well understood. Since the infective copepodid stage shows preferential site selection in attaching to the fins, we considered it likely that the copepodid recognizes chemical cues released or leaking from the fins, and/or transmembrane protein present on the fins. To isolate molecules potentially related to attachment site specificity, we applied suppression subtractive hybridization (SSH) PCR by identifying genes expressed more highly in pectoral fins of T. rubripes than in the body surface skin. We sequenced plasmid DNA from 392 clones in a SSH library. The number of non-redundant sequences was 276, which included 135 sequences located on 117 annotated genes and 141 located in positions where no genes had been annotated. We characterized those annotated genes on the basis of gene ontology terms, and found that 46 of the identified genes encode secreted proteins, enzymes or membrane proteins. Among them nine showed higher expression in the pectoral fins than in the skin. These could be candidate genes for involvement in behavioral mechanisms related to the site specificity shown by the infective copepodids of C. fugu.

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

BACKGROUND: In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. RESULTS: In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries. CONCLUSIONS: These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

Genomic regions of pufferfishes responsible for host specificity of a monogenean parasite, Heterobothrium okamotoi.

The genetic mechanisms underlying host specificity of parasitic infections are largely unknown. After hatching, the larvae of the monogenean parasite, Heterobothrium okamotoi, attach to the gill filaments of hosts and the post-larval worms develop there by consuming nutrients from the host. The susceptibility to H. okamotoi infection differs markedly among fish species. While this parasite can grow on tiger pufferfish (also called fugu), Takifugu rubripes, it appears to be rejected by a close congener, grass pufferfish, Takifugu niphobles, after initial attachment to the gills. To determine the genetic architecture of the pufferfish responsible for this host specificity, we performed genome-wide quantitative trait loci analysis. We raised second generation (F2) hybrids of the two pufferfish species and experimentally infected them with the monogenean in vivo. To assess possible differences in host mechanisms between early and later periods of infection, we sampled fish three h and 21days after exposure. Genome scanning of fish from the 3h infection trial revealed suggestive quantitative trait loci on linkage groups 2 and 14, which affected the number of parasites on the gill. However, analysis of fish 21days p.i. detected a significant quantitative trait locus on linkage group 9 and three other suggestive quantitative trait loci on linkage groups 7, 18 and 22. These results indicated the polygenic nature of the host mechanisms involved in the infection/rejection of H. okamotoi. Moreover the analyses suggested that host factors may play a more important role during the growth period of the parasite than during initial host recognition at the time of attachment. Within the 95% confidence interval of the linkage group 9 quantitative trait locus in the fugu genome, there were 214 annotated protein-coding genes, including immunity-related genes such as Irak4, Muc2 and Muc5ac.

The genetic architecture of growth rate in juvenile Takifugu species.

Closely related species have often evolved dramatic differences in body size. Takifugu rubripes (fugu) is a large marine pufferfish whose genome has been sequenced, whereas T. niphobles is the smallest species among Takifugu. We show that, unsurprisingly, the juvenile growth rate of T. rubripes is higher than that of T. niphobles in a laboratory setting. We produced F(2) progenies of their F(1) hybrids and found one quantitative trait locus (QTL) significantly associated with variation in juvenile body size. This QTL region (3.5 Mb) contains no known genes directly related to growth phenotype (such as IGFs) except Fgf21, which inhibits growth hormone signaling in mouse. The QTL in Takifugu spp. is distinct from the region previously known to control body size variations in stickleback or tilapia. Our results suggest that in the fish tested herein, genomic regions underlying body size evolution might have different genetic origins. They also suggest that many diverse traits in Takifugu spp. are amenable to genetic mapping.

Profiles of mRNA expression for prolactin, growth hormone, and somatolactin in Japanese eels, Anguilla japonica: The effect of salinity, silvering and seasonal change.

For understanding the functions of the growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family of hormones, we examined pituitary mRNA expression of these hormones in anguillid eels in relation to salinity difference, silvering, and seasonal change. Female Japanese eels (Anguilla japonica) were collected in the brackish Hamana Lake and its freshwater rivers from July to December. To clarify the effect of salinity, the habitat use history of the eels were determined using otolith microchemistry. Expression levels of mRNA of each hormone were determined using real time PCR. Although GH and PRL have been known to be osmoregulatory hormones, there were no consistent differences in expression levels of these hormones between different salinity habitats. In contrast, SL mRNA expression was higher in eels from freshwater rivers than from the brackish lake. GH mRNA expression clearly decreased during silvering, whereas PRL and SL mRNA expression did not change. We also showed that PRL mRNA and SL mRNA decreased in the brackish lake and PRL mRNA increased in freshwater rivers from autumn to early winter. These findings provide basic knowledge for a further understanding of the role of these hormones.

A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (fugu).

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.

A new type of lectin discovered in a fish, flathead (Platycephalus indicus), suggests an alternative functional role for mammalian plasma kallikrein.

A skin mucus lectin exhibiting a homodimeric structure and an S-S bond between subunits of ~40 kDa was purified from flathead Platycephalus indicus (Scorpaeniformes). This lectin, named FHL (FlatHead Lectin), exhibited mannose-specific activity in a Ca(2+)-dependent manner. Although FHL showed no homology to any previously reported lectins, it did exhibit ~20% identity to previously discovered plasma kallikreins and coagulation factor XIs of mammals and Xenopus laevis. These known proteins are serine proteases and play pivotal roles in the kinin-generating system or the blood coagulation pathway. However, alignment analysis revealed that while FHL lacked a serine protease domain, it was homologous to the heavy-chain domain of plasma kallikreins and coagulation factor XI therefore suggesting that FHL is not an enzyme but rather a novel animal lectin. On the basis of this finding, we investigated the lectin activity of human plasma kallikrein and revealed that it could indeed act as a lectin. Other genes homologous to FHL were also found in the genome databases of some fish species, but not in mammals. In contrast, plasma kallikreins and coagulation factor XI have yet to be identified in fish. The present findings suggest that these mammalian enzymes may have originally emerged as a lectin and may have evolved into molecules with protease activity after separation from common ancestors.

Integration of the genetic map and genome assembly of fugu facilitates insights into distinct features of genome evolution in teleosts and mammals.

The compact genome of fugu (Takifugu rubripes) has been used widely as a reference genome for understanding the evolution of vertebrate genomes. However, the fragmented nature of the fugu genome assembly has restricted its use for comparisons of genome architecture in vertebrates. To extend the contiguity of the assembly to the chromosomal level, we have generated a comprehensive genetic map of fugu and anchored the scaffolds of the assembly to the 22 chromosomes of fugu. The map consists of 1,220 microsatellite markers that provide anchor points to 697 scaffolds covering 86% of the genome assembly (http://www.fugu-sg.org/). The integrated genome map revealed a higher recombination rate in fugu compared with other vertebrates and a wide variation in the recombination rate between sexes and across chromosomes of fugu. We used the extended assembly to explore recent rearrangement events in the lineages of fugu, Tetraodon, and medaka and compared them with rearrangements in three mammalian (human, mouse, and opossum) lineages. Between the two pufferfishes, fugu has experienced fewer chromosomal rearrangements than Tetraodon. The gene order is more highly conserved in the three teleosts than in mammals largely due to a lower rate of interchromosomal rearrangements in the teleosts. These results provide new insights into the distinct patterns of genome evolution between teleosts and mammals. The consolidated genome map and the genetic map of fugu are valuable resources for comparative genomics of vertebrates and for elucidating the genetic basis of the phenotypic diversity of ~25 species of Takifugu that evolved within the last 5 My.

Dynamics of reproductive hormones during downstream migration in females of the Japanese eel, Anguilla japonica.

The profiles of sex steroids (estradiol-17ß, testosterone and 11-ketotestosterone) and the mRNA levels of gonadotropins (luteinizing hormone and follicle-stimulating hormone) were investigated before and after downstream migration in females of the Japanese eel species Anguilla japonica, which were collected in the brackish Hamana Lake and its inlet freshwater rivers. Eels were separated into three groups using otolith microchemistry: 'migrants' that grew in the inlet rivers and then made a downstream migration to Hamana Lake mainly in October and November; 'non-migrant' yellow eels caught in rivers during the same season; and 'residents,' which were yellow eels caught in rivers in August. Sex steroid levels, especially those of testosterone and 11-ketotestosterone, were higher in migrants than in non-migrants and residents. Real-time quantitative PCR analysis indicated that mRNA levels of luteinizing hormone (LH) ß-subunits were significantly higher in migrants than in other groups, whereas those of follicle-stimulating hormone ß-subunits did not show significant changes during downstream migration. The high levels of these hormones during downstream migration raise the question about if they also play a role in motivating the migratory behavior of eels.

Conservation of characteristics and functions of CD4 positive lymphocytes in a teleost fish.

The presence of helper and cytotoxic T cells in fish has been suggested, although T cell subsets have yet to be identified at the cellular level. In order to investigate the functions of CD4 and CD8α positive T cells we attempted to produce and characterize monoclonal antibodies (mAbs) against teleost CD4 and CD8α. Here we report the successful production of mAbs against CD4 and CD8α in clonal ginbuna crucian carp Carassius auratus langsdorfii and the function of CD4 positive T cells. In this study we demonstrate the presence of teleost CD4- and CD8α-positive T cell subsets with morphology, tissue distribution and gene expression similar to those of mammalian CD4- and CD8-positive T lymphocytes. Using mAbs we found that CD4/CD8 double positive T cells are only present in the thymus, suggesting that it is the site of T cell development. We further demonstrated in vitro proliferation of CD4 positive T cells by allogeneic combination of mixed leukocyte culture and antigen-specific proliferation of CD4 positive T cells after in vitro sensitization with OVA. In our previous study we showed that CD8α-positive lymphocytes are the primary cell type showing specific cytotoxicity against allogeneic targets. Collectively, these findings suggest that CD4 and CD8α positive T cells in ginbuna are equivalent to helper and cytotoxic T lymphocytes (CTL) in mammals, respectively. This is the first report to show the characteristics and functions of CD4 positive T cells in fish and these findings shed light into the evolutionary origins and primordial functions of helper T cells.

The plasmablast-like leukocyte in the kidney of fugu (Takifugu rubripes).

In teleosts, the kidney is the major immune organ. From the kidney of fugu (Takifugu rubripes), we isolated a unique leukocyte population. This population shows properties similar to those of mammalian plasmablasts. First, adherent cells expressing IgM protein on their surface were obtained from the fugu kidney. Flow cytometry (FCM) showed that these cells were mainly composed of two cell populations: IgM+CD8α⁻ cells and IgM+CD8α+ cells. Further characterization of the IgM+CD8α⁻ population by RT-PCR demonstrated that the cells expressed secretory-type IgM as well as Bcl-6 and Blimp-1, developmental marker genes for the B cell lineage. Western blotting also showed that the cells secreted IgM protein. These results indicate that the IgM+CD8α⁻ cells are similar to cells at the plasmablast stage in mammals. This is the first report isolating plasmablast-like leukocytes in fish species. Our data also suggests that the teleosts kidney is a organ where B cells terminally differentiate into the plasma cells.

Teleost B7 expressed on monocytes regulates T cell responses.

In mammals, professional APCs induce adaptive immunity via the activation of T cells. During this process, B7 family molecules present upon APCs are known to play crucial roles in optimal T cell stimulation. In contrast, the confirmation of APCs in a nonmammalian vertebrate has yet to be achieved. To obtain further insights into the evolutionary origin of APCs, we have identified three members of the B7 family in the teleost Takifugu rubripes (fugu): B7-H1/DC, B7-H3, and B7-H4. The three fugu B7s were expressed on the surface of blood monocytes. The B7(+) monocytes, which are composed of at least two distinct populations, expressed the MHC class II component gene. The fugu B7 molecules bound to activated T cells, indicating that putative B7 receptors were expressed upon T cells. Fugu B7-H1/DC inhibited T cell proliferation concomitant with increasing levels of both IL-10 and IFN-gamma expression, whereas both B7-H3 and B7-H4 promoted T cell growth following IL-2 induction and the suppression of IL-10. These observations indicate that fugu B7s regulate T cell responses via receptors upon T cells. We suggest that fish B7(+) monocytes are APCs and that a costimulatory system has already developed in fish via the evolutionary process.

Regulator of complement activation (RCA) gene cluster in Xenopus tropicalis.

Genome and expressed sequence tag information of Xenopus tropicalis suggested that short-consensus repeat (SCR)-containing proteins are encoded by three genes that are mapped within a 300-kb downstream of PFKFB2, which is a marker gene for the regulator of complement activation (RCA) loci in human and chicken. Based on this observation, we cloned the three cDNAs of these proteins using 3'- or 5'-RACE technique. Since their primary structures and locations of the proximity to the PFKFB2 locus, we named them amphibian RCA protein (ARC) 1, 2, and 3. Expression in human HEK293 or CHO cells suggested that ARC1 is a soluble protein of Mr approximately 67 kDa, ARC2 is a membrane protein with Mr 44 kDa, and ARC3 a secretary protein with a putative transmembrane region. They were N-glycosylated during maturation. In human and chicken RCA clusters, the order in which genes for soluble, GPI-anchored, and membrane forms of SCR proteins are arranged is from the distant to proximity to the PFKFB2 gene. However, the amphibian ARC1, 2, and 3 resembled one another and did not reflect the same order found in human and chicken RCA genes. This may be due to self-duplication of ARCs to form a family, and it evolved after the amphibia separated from the ancestor of the amniotes, which possessed soluble, GPI-anchored, and membrane forms of SCR protein members. Taken together, frog possesses a RCA locus, but the constitution of the ARC proteins differs from that of the amniotes with a unique self-resemblance.