Environment-Mediated Vertical Transmission Fostered Uncoupled Phylogenetic Relationships between Longicorn Beetles and Their Symbionts.

The Coleoptera Cerambycidae (longicorn beetles) use wood under different states (living healthy, freshly snapped, completely rot, etc.) in a species-specific manner for their larval diet. Larvae of some Cerambycidae groups have mycetomes, accessory organs associated with the midgut that harbor fungal symbiont cells. The symbionts are thought to improve nutrient conditions; however, this has yet to be shown experimentally. To deduce the evolutionary history of this symbiosis, we investigated the characteristics of the mycetomes in the larvae of longicorn beetles collected in Japan. Lepturinae, Necydalinae, and Spondylidinae are the only groups that possess mycetomes, and these three groups' mycetomes and corresponding fungal cells exhibit different characteristics between the groups. However, the phylogenetic relationship of symbiont yeasts does not coincide with that of the corresponding longicorn beetle species, suggesting they have not co-speciated. The imperfect vertical transmission of symbiont yeasts from female to offspring is a mechanism that could accommodate the host-symbiont phylogenetic incongruence. Some Lepturinae species secondarily lost mycetomes. The loss is associated with their diet choice, suggesting that different conditions between feeding habits could have allowed species to discard this organ. We found that symbiont fungi encapsulated in the mycetomes are dispensable for larval growth if sufficient nutrients are given, suggesting that the role of symbiotic fungi could be compensated by the food larvae take. Aegosoma sinicum is a longicorn beetle classified to the subfamily Prioninae, which does not possess mycetomes. However, this species contains a restricted selection of yeast species in the larval gut, suggesting that the symbiosis between longicorn beetles and yeasts emerged before acquiring the mycetomes.

Evolutionary analysis of two complement C4 genes: Ancient duplication and conservation during jawed vertebrate evolution.

The complement C4 is a thioester-containing protein, and a histidine (H) residue catalyzes the cleavage of the thioester to allow covalent binding to carbohydrates on target cells. Some mammalian and teleost species possess an additional isotype where the catalytic H is replaced by an aspartic acid (D), which binds preferentially to proteins. We found the two C4 isotypes in many other jawed vertebrates, including sharks and birds/reptiles. Phylogenetic analysis suggested that C4 gene duplication occurred in the early days of the jawed vertebrate evolution. The D-type C4 of bony fish except for mammals formed a cluster, termed D-lineage. The D-lineage genes were located in a syntenic region outside MHC, and evolved conservatively. Mammals lost the D-lineage before speciation, but D-type C4 was regenerated by recent gene duplication in some mammalian species or groups. Dual C4 molecules with different substrate specificities would have contributed to development of the antibody-dependent classical pathway.

The complement system of elasmobranches revealed by liver transcriptome analysis of a hammerhead shark, Sphyrna zygaena.

Comprehensive studies of the complement genes in basal vertebrates have revealed that cyclostomes have apparently primitive complement systems whereas bony fish have well-developed complement systems comparable to those of mammals. Here we have performed liver transcriptome analysis of a hammerhead shark, Sphyrna zygaeana, to elucidate the early history of vertebrate complement evolution. Identified genes were; one C1qB, one C1r, one C1s, one MASP-1/-3, one MASP-2, two factor B/C2, one C3, three C4, one C5, one C6, one C7, one C8A, three C8B, one C8G, one C9, two factor I and one S protein. No MBL, ficolin, C1qA or C1qC were found. These results indicate that the lectin, classical, alternative and lytic pathways were established in the common ancestor of jawed vertebrates. In addition to the absence of MBL and ficolin, the MASP transcripts lacked the serine protease domain, suggesting that the lectin pathway was lost in the hammerhead shark lineage.

Evolution of the complement system in protostomes revealed by de novo transcriptome analysis of six species of Arthropoda.

To elucidate the evolutionary history of the complement system in Arthropoda, de novo transcriptome analysis was performed with six species among the Chelicerata, Myriapoda, and Crustacea, and complement genes were identified based on their characteristic domain structures. Complement C3 and factor B (FB) were identified from a sea spider, a jumping spider, and a centipede, but not from a sea firefly or two millipede species. No additional complement components identifiable by their characteristic domain structures were found from any of these six species. These results together with genome sequence information for several species of the Hexapoda suggest that the common ancestor of the Arthropoda possessed a simple complement system comprising C3 and FB, and thus resembled the alternative pathway of the mammalian complement system. It was lost at least twice independently during the evolution of Arthropoda in the millipede lineage and in the common ancestor of Crustacea and Hexapoda.

Epididymal C4b-binding protein is processed and degraded during transit through the duct and is not essential for fertility.

C4b-binding protein (C4BP) is known as one of the circulating complement regulators that prevents excessive activation of the host-defense complement system. We have reported previously that C4BP is expressed abundantly in the rodent epididymis, one of the male reproductive organs connecting the testis and vas deferens, where immature spermatozoa acquire their motility and fertilizing ability during their transit through the duct. Epididymal C4BP (EpC4BP) is synthesized androgen-dependently by the epithelial cells, secreted into the lumen, and bound to the outer membrane of the passing spermatozoa. In this study, we found that EpC4BP is secreted as a large oligomer, similar to the serum C4BP, but is digested during the epididymal transit and is almost lost from both the luminal fluid and the sperm surface in the vas deferens. Such a processing pattern is not known in serum C4BP, suggesting that EpC4BP and serum C4BP might have different functional mechanisms, and that there is a novel function of EpC4BP in reproduction. In addition, the disappearance of EpC4BP from the sperm surface prior to ejaculation suggests that EpC4BP works only in the epididymis and would not work in the female reproductive tract to protect spermatozoa from complement attack. Next, we generated C4BP-deficient (C4BP-/-) mice to examine the possible role of EpC4BP in reproduction. However, the C4BP-/- mice were fertile and no significant differences were observed between the C4BP-/- and wild-type mouse spermatozoa in terms of morphology, motility, and rate of the spontaneous acrosome reaction. These results suggest that EpC4BP is involved in male reproduction, but not essential for sperm maturation.

Evolution of the complement system.

The mammalian complement system constitutes a highly sophisticated body defense machinery comprising more than 30 components. Research into the evolutionary origin of the complement system has identified a primitive version composed of the central component C3 and two activation proteases Bf and MASP in cnidaria. This suggests that the complement system was established in the common ancestor of eumetazoa more than 500 million years ago. The original activation mechanism of the original complement system is believed to be close to the mammalian lectin and alternative activation pathways, and its main role seems to be opsonization and induction of inflammation. This primitive complement system has been retained by most deuterostomes without major change until the appearance of jawed vertebrates. At this stage, duplication of the C3, Bf and MASP genes as well as recruitment of membrane attack components added the classical and lytic pathways to the primitive complement system, converting it to the modern complement system. In contrast, the complement system was lost multiple times independently in the protostome lineage.

Evolution of dimorphisms of the proteasome subunit beta type 8 gene (PSMB8) in basal ray-finned fish.

The proteasome subunit beta type 8 (PSMB8) gene encodes a catalytic subunit of immunoproteasome that plays a central role in the processing of antigenic peptides presented by major histocompatibility complex class I molecules. The A- and F-type alleles defined by the 31st amino acid residue determining cleaving specificity have been identified from ray-finned fish, amphibia, and reptiles. These two types show extremely long-term trans-species polymorphism in Polypteriformes, Cypriniformes, and Salmoniformes, suggesting the presence of very ancient lineages termed A and F. To elucidate the evolution of the PSMB8 dimorphism in basal ray-finned fish, we analyzed Pantodon buchholzi (Osteoglossiformes), seven species of Anguilliformes, and Hypomesus nipponensis (Osmeriformes). Both A and F lineage sequences were identified from P. buchholzi and H. nipponensis, confirming that these two lineages have been conserved by basal ray-finned fish. However, both the A- and F-type alleles found in Anguilliformes species belonged to the F lineage irrespective of their types. This apparently suggests that the A lineage was lost in the common ancestor of Anguilliformes, and recovery of the A type within the F lineage occurred in Anguilliformes. The apparent loss of the F lineage and recovery of the F type within the A lineage have already been reported from tetrapods and higher teleosts. However, this is the first report on the reverse situation and reveals the dynamic evolution of the PSMB8 dimorphism.

Dimorphisms of the proteasome subunit beta type 8 gene (PSMB8) of ectothermic tetrapods originated in multiple independent evolutionary events.

The proteasome subunit beta type 8 gene (PSMB8) encodes one of the beta subunits of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex class I molecules. Dimorphic alleles of the PSMB8 gene, termed A and F types, based on the deduced 31st amino acid residue of the mature protein have been reported from various vertebrates. Phylogenetic analysis revealed the presence of dichotomous ancient lineages, one comprising the F-type PSMB8 of basal ray-finned fishes, and the other comprising the A-type PSMB8 of these animals and both the F- and A-type PSMB8 of Xenopus and acanthopterygians, indicating that evolutionary history of the PSMB8 dimorphism was not straightforward. We analyzed the PSMB8 gene of five reptile and one amphibian species and found both the A and F types from all six. Phylogenetic analysis indicated that the PSMB8 F type was apparently regenerated from the PSMB8 A type at least five times independently during tetrapod evolution. Genomic typing of wild individuals of geckos and newts indicated that the frequencies of the A- and F-type alleles are not highly biased in these species. Phylogenetic analysis of each exon of the reptile PSMB8 gene suggested interallelic sequence homogenization as a possible evolutionary mechanism for the apparent recurrent regeneration of PSMB8 dimorphism in tetrapods. An extremely strong balancing selection acting on PSMB8 dimorphism was implicated in an unprecedented pattern of allele evolution.

Comprehensive analysis of medaka major histocompatibility complex (MHC) class II genes: implications for evolution in teleosts.

The major histocompatibility complex (MHC) class II molecules play central roles in adaptive immunity by regulating immune response via the activation of CD4 T cells. The full complement of the MHC class II genes has been elucidated only in mammalian species to date. To understand the evolution of these genes, we performed their first comprehensive analysis in nonmammalian species using a teleost, medaka (Oryzias latipes). Based on a database search, cDNA cloning, and genomic PCR, medaka was shown to possess five pairs of expressed class II genes, comprising one IIA and one IIB gene. Each pair was located on a different chromosome and was not linked to the class I genes. Only one pair showed a high degree of polymorphism and was considered to be classical class II genes, whereas the other four pairs were nonclassical. Phylogenetic analysis of all medaka class II genes and most reported teleost class II genes revealed that the IIA and IIB genes formed separate clades, each containing three well-corresponding lineages. One lineage contained three medaka genes and all known classical class II genes of Ostariophysi and Euteleostei and was presumed to be an original lineage of the teleost MHC class II genes. The other two lineages contained one nonclassical medaka gene each and some Euteleostei genes. These results indicate that multiple lineages of the teleost MHC class II genes have been conserved for hundreds of millions of years and that the tightly linked IIA and IIB genes have undergone concerted evolution.

Guideline for hereditary angioedema (HAE) 2010 by the Japanese Association for Complement Research - secondary publication.

This guideline was provided by the Japanese Association for Complement Research targeting clinicians for making an accurate diagnosis of hereditary angioedema (HAE), and for prompt treatment of the HAE patient in Japan. This is a 2010 year version and will be updated according to any pertinent medical advancements.

Long-lived dichotomous lineages of the proteasome subunit beta type 8 (PSMB8) gene surviving more than 500 million years as alleles or paralogs.

On an evolutionary time scale, polymorphic alleles are believed to have a short life, persisting at most tens of millions of years even under long-term balancing selection. Here, we report highly diverged trans-species dimorphism of the proteasome subunit beta type 8 (PSMB8) gene, which encodes a catalytic subunit of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex (MHC) class I molecules, in lower teleosts including Cypriniformes (zebrafish and loach) and Salmoniformes (trout and salmon), whose last common ancestor dates to 300 Ma. Moreover, phylogenetic analyses indicated that these dimorphic alleles share lineages with two shark paralogous genes, suggesting that these two lineages have been maintained for more than 500 My either as alleles or as paralogs, and that conversion between alleles and paralogs has occurred at least once during vertebrate evolution. Two lineages termed PSMB8A and PSMB8F show an A(31)F substitution that would probably affect their cleaving specificity, and whereas the PSMB8A lineage has been retained by all analyzed jawed vertebrates, the PSMB8F lineage has been lost by most jawed vertebrates except for cartilaginous fish and basal teleosts. However, a possible functional equivalent of the PSMB8F lineage has been revived as alleles within the PSMB8A lineage at least twice during vertebrate evolution in the amphibian Xenopus and teleostean Oryzias species. Dynamic evolution of the PSMB8 polymorphism through long-term persistence, loss, and regaining of dimorphism and conversion between alleles and paralogs implies the presence of strong selective pressure for functional polymorphism of this gene.

[Efficacy of indocyanine green videoangiography for carotid endarterectomy].

OBJECTIVE: The aim of the present study is to assess whether the technique of surgical microscope-based indocyanine green videoangiography (ICG-VA) and the FLOW800 system are efficient for hemodynamic evaluation during carotid endarterectomy (CEA). METHODS: 20 CEAs for 19 patients were performed from January to July 2011. Before and after endarterectomy, ICG was injected intravenously. After the procedure, ICG-VA was analyzed by FLOW800. Regions of interest were the common carotid artery, the plaque, the internal carotid artery, and the external carotid artery to evaluate changes in each intensity value. RESULTS: The distal end of carotid plaque and the proximal end were identified in 85% of CEAs and 78.9% of cases. In 5 cases (26.3%) with more severe stenosis (>90%), the decrease of blood flow intensity in the internal carotid artery was delayed before endarterectomy. After endarterectomy, the finding was improved in all the 5 cases. After the endarterectomy, the intensity values in the common carotid artery, the plaque, the internal carotid artery, and the external carotid artery had increased 162 ± 129, 337 ± 212, 139 ± 151 and 177 ± 143. Especially in the value of the plaque, there was a great improvement. CONCLUSIONS: ICG-VA provides information of the plaque location and the patency of the arterial vessel during CEA. Using FLOW800, semiquantitive information of hemodynamics was able to be acquired, especially for the case of severe stenosis with collapse of the internal carotid artery.

Highly divergent dimorphic alleles of the proteasome subunit beta type-8 (PSMB8) gene of the bichir Polypterus senegalus: implication for evolution of the PSMB8 gene of jawed vertebrates.

The proteasome subunit beta type-8 (PSMB8) gene encodes a catalytic subunit of the immunoproteasome, which is involved in the generation of peptides presented by MHC class I molecules. To date, highly diverged dichotomous alleles of PSMB8 have been reported in Oryzias species (actinopterygian teleosts) and Xenopus species (sarcopterygian amphibians). These dimorphic alleles share a similar substitution (A/V(31)F/Y) at the 31st position of the mature protein, which is most probably involved in formation of the S1 pocket. This substitution likely confers different cleavage specificities on the dimorphic PSMB8s. In addition, two paralogous PSMB8 genes possessing the A and F residues at the 31st position have been reported in sharks. Phylogenetic analysis indicated that the two types of PSMB8 of Oryzias, Xenopus, and sharks arose by independent evolutionary events. Here, we identified another pair of dimorphic alleles of PSMB8, which have the A and F residues at the 31st position of the mature protein, from bichir, Polypterus senegalus, a basal actinopterygian. The sequences of the mature proteins-encoding region of the dimorphic alleles of bichir PSMB8, the A and F types, showed only 72.7% and 77.5% identities at the nucleotide and the deduced amino acid levels, respectively. Their intronic sequences show almost no similarity, indicating that the dimorphic alleles of bichir PSMB8 have a very ancient origin. However, phylogenetic analysis showed that the dimorphisms of PSMB8 of bichir, Xenopus, and Oryzias arose by independent evolutionary events, suggesting the presence of a strong selective pressure for possessing the dimorphism.

Evolution of the thioester-containing proteins (TEPs) of the arthropoda, revealed by molecular cloning of TEP genes from a spider, Hasarius adansoni.

The thioester-containing protein (TEP) family of genes, found in most Eumetazoan genomes, is classified into two subfamilies: the alpha-2-macroglobulin (A2M) subfamily and the C3 subfamily. Many A2M subfamily members, including insect TEP (iTEP), have been reported from the Arthropoda, whereas the C3 subfamily members have been reported only from two horseshoe crab species thus far. To elucidate the evolution of these genes among the Arthropoda, TEP genes were isolated from a spider, Hasarius adansoni (Chelicerata), by reverse transcription polymerase chain reaction (RT-PCR) amplification using universal degenerate primers specific for the thioester region. Four different TEP genes were identified. Phylogenetic analysis using the entire amino acid sequences of these and various other TEP sequences from the Eumetazoa indicated that two of the spider genes are type C3 (HaadC3-1 and HaadC3-2), one is type A2M (HaadA2M) and the other is closely related to iTEP (HaadiTEP). These results suggest that the common ancestor of the Arthropoda possessed at least three TEP genes, C3, A2M and iTEP and that they were lost differentially in the Crustacean and Hexapodan lineages.

Molecular phylogeny of Japanese Catocala moths based on nucleotide sequences of the mitochondrial ND5 gene.

Phylogenetic relationships of 31 Japanese Catocala species were analyzed based on the partial nucleotide sequences of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene (762 bp). When several non-Catocala Noctuidae moths were designated as the outgroup, these Catocala species formed a monophyletic group. However, divergences between these Catocala species were very deep, and no close phylogenetic relationships were recognized among them except for that between the two recently separated species, C. xarippe and C. fulminea. The remote relationships implied for several pairs of species suggest that the color of the hindwings is a changeable characteristic, and does not reflect phylogenetic lineage. Continental specimens were analyzed in 20 of 31 Catocala species, and all of them showed a close relationship with their Japanese counterpart. However, the closeness of the nucleotide sequences between the Japanese and continental individuals of the same species varied from species to species, indicating that isolation between the Japanese and continental populations of these species occurred at many different times. The two analyzed species endemic to North America showed a close relationship with their morphologically inferred Japanese counterparts, indicating that the geographic separation and following speciation between these Eurasian and American species occurred much more recently compared with the speciation events among the Catocala species now found in Japan.

Retained orthologous relationships of the MHC Class I genes during euteleost evolution.

Major histocompatibility complex (MHC) class I molecules play a pivotal role in immune defense system, presenting the antigen peptides to cytotoxic CD8+ T lymphocytes. Most vertebrates possess multiple MHC class I loci, but the analysis of their evolutionary relationships between distantly related species has difficulties because genetic events such as gene duplication, deletion, recombination, and/or conversion have occurred frequently in these genes. Human MHC class I genes have been conserved only within the primates for up to 46-66 My. Here, we performed comprehensive analysis of the MHC class I genes of the medaka fish, Oryzias latipes, and found that they could be classified into four groups of ancient origin. In phylogenetic analysis using these genes and the classical and nonclassical class I genes of other teleost fishes, three extracellular domains of the class I genes showed quite different evolutionary histories. The α1 domains generated four deeply diverged lineages corresponding to four medaka class I groups with high bootstrap values. These lineages were shared with salmonid and/or other acanthopterygian class I genes, unveiling the orthologous relationships between the classical MHC class I genes of medaka and salmonids, which diverged approximately 260 Ma. This suggested that the lineages must have diverged in the early days of the euteleost evolution and have been maintained for a long time in their genome. In contrast, the α3 domains clustered by species or fish groups, regardless of classical or nonclassical gene types, suggesting that this domain was homogenized in each species during prolonged evolution, possibly retaining the potential for CD8 binding even in the nonclassical genes. On the other hand, the α2 domains formed no apparent clusters with the α1 lineages or with species, suggesting that they were diversified partly by interlocus gene conversion, and that the α1 and α2 domains evolved separately. Such evolutionary mode is characteristic to the teleost MHC class I genes and might have contributed to the long-term conservation of the α1 domain.

Transspecies dimorphic allelic lineages of the proteasome subunit beta-type 8 gene (PSMB8) in the teleost genus Oryzias.

The proteasome subunit ß-type 8 (PSMB8) gene in the jawed vertebrate MHC genomic region encodes a catalytic subunit of the immunoproteasome involved in the generation of peptides to be presented by the MHC class I molecules. A teleost, the medaka (Oryzias latipes), has highly diverged dimorphic allelic lineages of the PSMB8 gene with only about 80% amino acid identity, termed "PSMB8d" and "PSMB8N," which have been retained by most wild populations analyzed. To elucidate the evolutionary origin of these two allelic lineages, seven species of the genus Oryzias were analyzed for their PSMB8 allelic sequences using a large number of individuals from wild populations. All the PSMB8 alleles of these species were classified into one of these two allelic lineages based on their nucleotide sequences of exons and introns, indicating that the Oryzias PSMB8 gene has a truly dichotomous allelic lineage. Retention of both allelic lineages was confirmed except for one species. The PSMB8d lineage showed a higher frequency than the PSMB8N lineage in all seven species. The two allelic lineages showed curious substitutions at the 31st and 53rd residues of the mature peptide, probably involved in formation of the S1 pocket, suggesting that these allelic lineages show a functional difference in cleavage specificity. These results indicate that the PSMB8 dimorphism was established before speciation within the genus Oryzias and has been maintained for more than 30-60 million years under a strict and asymmetric balancing selection through several speciation events.

Proteolytic cascades and their involvement in invertebrate immunity.

Bacteria and other potential pathogens are cleared rapidly from the body fluids of invertebrates by the immediate response of the innate immune system. Proteolytic cascades, following their initiation by pattern recognition proteins, control several such reactions, notably coagulation, melanisation, activation of the Toll receptor and complement-like reactions. However, there is considerable variation among invertebrates and these cascades, although widespread, are not present in all phyla. In recent years, significant progress has been made in identifying and characterizing these cascades in insects. Notably, recent work has identified several connections and shared principles among the different pathways, suggesting that cross-talk between them may be common.

Evolution of thioester-containing proteins revealed by cloning and characterization of their genes from a cnidarian sea anemone, Haliplanella lineate.

To elucidate the evolutionary origin of genes encoding thioester-containing proteins (TEPs), TEP genes were isolated from a cnidarian, a sea anemone, Haliplanella lineate. Phylogenetic tree analysis of the four identified cnidarian TEP genes and various TEP genes of many metazoa, indicated that they could be classified into two subfamilies: the alpha-2-macroglobulin (A2M) subfamily encodining A2M, CD109 and insect TEPs, and the C3 subfamily encoding complement C3, C4 and C5. Two of the four cnidarian TEP genes belonged to the A2M subfamily, showing a close similarity to human A2M and CD109, respectively and thus were termed HaliA2M and HaliCD109. The other two genes belonged to the C3 subfamily, and were termed HaliC3-1 and HaliC3-2. Cnidarian TEPs retained the basic domain structure and functionally important residues for each molecule, and their mRNA were detected at different parts of the sea anemone body. These results suggest that gene duplication and subsequent functional differentiation among C3, A2M and CD109 were very ancient events predating the divergence of the cnidaria and bilateria.

Evolutionary analysis of two classical MHC class I loci of the medaka fish, Oryzias latipes: haplotype-specific genomic diversity, locus-specific polymorphisms, and interlocus homogenization.

The major histocompatibility complex (MHC) region of the teleost medaka (Oryzias latipes) contains two classical class I loci, UAA and UBA, whereas most lower vertebrates possess or express a single locus. To elucidate the allelic diversification and evolutionary relationships of these loci, we compared the BAC-based complete genomic sequences of the MHC class I region of three medaka strains and the PCR-based cDNA sequences of two more strains and two wild individuals, representing nine haplotypes. These were derived from two geographically distinct medaka populations isolated for four to five million years. Comparison of the genomic sequences showed a marked diversity in the region encompassing UAA and UBA even between the strains derived from the same population, and also showed an ancient divergence of these loci. cDNA analysis indicated that the peptide-binding domains of both UAA and UBA are highly polymorphic and that most of the polymorphisms were established in a locus-specific manner before the divergence of the two populations. Interallelic recombination between exons 2 and 3 encoding these domains was observed. The second intron of the UAA genes contains a highly conserved region with a palindromic sequence, suggesting that this region contributed to the recombination events. In contrast, the alpha3 domain is extremely homogenized not only within each locus but also between UAA and UBA regardless of populations. Two lineages of the transmembrane and cytoplasmic regions are also shared by UAA and UBA, suggesting that these two loci evolved with intimate genetic interaction through gene conversion or unequal crossing over.