Mitochondrial genome of a Japanese placozoan.

Placozoans are marine invertebrates found in tropical and subtropical waters. Their body plan is among the simplest of free-living animals. The present study determined the mitochondrial genome sequence of a placozoan collected on the coast of Shirahama, Wakayama, Honshu, Japan, and compared it with those of Trichoplax adhaerens from the Red Sea and of three strains from the Caribbean Sea. The sequences of mitochondrial respiratory chain of the Japanese placozoan genes are very similar to those of the BZ49 strain from the Caribbean Sea. However, there are distinct differences in gene arrangement, such as the location of two open reading frames. This Japanese placozoan is therefore distinguishable from the other strains. Based on current knowledge of placozoan 16S diversity our 'Shirahama' strain most likely represents the H15 lineage, known from the Philippines. In the mitochondrial genome of placozoans, substitution rates are slower than in bilaterians, whereas the rate of rearrangements is faster.

Distinction of cell types in Dicyema japonicum (phylum Dicyemida) by expression patterns of 16 genes.

Dicyemids (phylum Dicyemida) are endoparasites, or endosymbionts, typically found in the renal sac of benthic cephalopod molluscs. The body organization of dicyemids is very simple, consisting of only 9 to 41 somatic cells. Dicyemids appear to have no differentiated tissues. Although categorization of somatic cells, to some types, is based on differences in the pattern of cilia and their position in the body, whether or not these cells are functionally different remains to be revealed. To provide insight into the functional differentiation, we performed whole mount in situ hybridization (WISH) to detect expression patterns of 16 genes, i.e., aquaglyceroporin, F-actin capping protein, aspartate aminotransferase, cathepsin-L-like cysteine peptidase, Ets domain-containing protein, glucose transporter, glucose-6-phosphate 1-dehydrogenase, glycine transporter, Hsp 70, Hsp 90, isocitrate dehydrogenase subunit alpha, Rad18, serine hydroxymethyltransferase, succinate-CoA ligase, valosin-containing protein, and 14-3-3 protein. In certain genes, regional specific expression patterns were observed among somatic cells of vermiform stages and infusoriform larvae of dicyemids. The WISH analyses also revealed that the Ets domain-containing protein and Rad18 are molecular markers for agametes.

Phylogenetic analysis of dicyemid mesozoans (phylum Dicyemida) from innexin amino acid sequences: dicyemids are not related to Platyhelminthes.

Dicyemid mesozoans are endoparasites, or endosymbionts, found only in the renal sac of benthic cephalopod molluscs. The body organization of dicyemids is very simple, consisting of usually 10 to 40 cells, with neither body cavities nor differentiated organs. Dicyemids were considered as primitive animals, and the out-group of all metazoans, or as occupying a basal position of lophotrochozoans close to flatworms. We cloned cDNAs encoding for the gap junction component proteins, innexin, from the dicyemids. Its expression pattern was observed by whole-mount in situ hybridization. In adult individuals, the innexin was expressed in calottes, infusorigens, and infusoriform embryos. The unique temporal pattern was observed in the developing infusoriform embryos. Innexin amino acid sequences had taxon-specific indels which enabled identification of the 3 major protostome lineages, i.e., 2 ecdysozoans (arthropods and nematodes) and the lophotrochozoans. The dicyemids show typical, lophotrochozoan-type indels. In addition, the Bayesian and maximum likelihood trees based on the innexin amino acid sequences suggested dicyemids to be more closely related to the higher lophotrochozoans than to the flatworms. Flatworms were the sister group, or consistently basal, to the other lophotrochozoan clade that included dicyemids, annelids, molluscs, and brachiopods.

Squid vascular endothelial growth factor receptor: a shared molecular signature in the convergent evolution of closed circulatory systems.

The highly specialized cephalopod cardiovascular system has long been considered a valuable model for understanding the evolution of circulatory systems. Despite the number of studies devoted to this topic, the developmental regulatory mechanisms remain largely unexplored. Here, we focus on the vascular endothelial growth factor receptor (VEGFR). This factor is known to mediate levels of endothelial growth factor that is involved in hematopoiesis and vasculogenesis including multichambered heart development in vertebrates. We found a squid VEGFR ortholog that is expressed in the developing blood vessels, notably in the sheet-like endothelial cells of the systemic and branchial hearts. The highly restricted localization of VEGFR in the vascular endothelial cells and its shared expression pattern in the developing hearts of cephalopods and vertebrates suggest a shared molecular signature of closed circulatory systems that has been independently elaborated during evolution.

Unique genome of dicyemid mesozoan: highly shortened spliceosomal introns in conservative exon/intron structure.

Dicyemids are enigmatic endoparasites, or endosymbionts, living in the renal sac of benthic cephalopod molluscs. The body of dicyemids consists of only 9-41 cells, with neither extracellular matrices nor differentiated tissues. Due to the unusually simple body organization, dicyemids have long been the subject of phylogenetic controversy. Molecular evidences suggest dicyemids are lophotrochozoans that have secondarily lost many morphological characters. We studied 40 genes of the dicyemid Dicyema japonicum and found that their spliceosomal introns are very short (mean length=26 bp). This size was shorter than that of introns of animals, such as Fugu rubripes and Oikopleura dioica which possess compact genome and introns. In the intron size, the dicyemid was nearly equal to the chlorarachniophyte Bigelowiella natans nucleomorph (18-21 bp) which has the shortest introns of any known eukaryote. Despite the short introns, the intron density (5.3 introns/gene) of the dicyemid is similar to that in model invertebrates. In addition, the exon/intron structure of the dicyemid is more similar to vertebrates than to the model invertebrates. These results suggest that the positions of the introns are possibly conserved under functional constraints.

The expression of tubulin and tektin genes in dicyemid mesozoans (Phylum: Dicyemida).

Dicyemid mesozoans (Phylum Dicyemida) are endoparasites (or endosymbionts) that typically are found in the renal sac of benthic cephalopod mollusks such as octopuses and cuttlefishes. Adult dicyemids likely adhere to the renal appendage of hosts via cilia of calotte peripheral cells. These cilia seem to be continuously worn away in the interaction between the dicyemids and the epidermal cells of host renal appendages. We cloned 4 cDNAs and genes, alpha-tubulin, beta-tubulin, tektin B, and tektin C, which are thought to play a key role in ciliogenesis, from Dicyema japonicum, and studied expression patterns of these genes by whole-mount in situ hybridization. We detected coexpression of these genes in the calotte peripheral cells, but not in the trunk peripheral cells. This suggests that regeneration and turnover of cilia continuously occur in the calotte. In vermiform and infusoriform embryos, we also detected coexpression patterns of these genes, which might correlate with ciliogenesis during the embryogenesis. We also predicted the secondary structure and the coiled-coil regions of dicyemid tektins.

Cloning of chitinase-like protein1 cDNA from dicyemid mesozoans (Phylum: Dicyemida).

Dicyemid mesozoans are endoparasites found in the renal sacs of benthic cephalopods. Adult dicyemids insert the distinct anterior region, termed a "calotte," into renal tubules of the host. We cloned cDNA encoding chitinase-like protein from the dicyemid Dicyema japonicum (Dicyema-clp 1), and also cloned the gene fragment corresponding to the cDNA. Dicyema-clp1 has the hydrophobic amino acid-rich region, but not the chitin-binding domains at the C terminus. Analyses using the SignalP prediction program suggest this hydrophobic amino acid-rich region is the anchor sequence to plasma membranes. The putative catalytic site in glyco18 domain exhibited 1 substitution from aspartic acid to asparagine. The gene fragment had short 9 introns (22-26 bp), and the coding sequence consisted of 10 exons (30-233 bp). Specific and strong expression of Dicyema-clpl was detected in the calotte of vermiform stages by whole mount in situ hybridization. N-acetyl-D-glucosamine was detected on the outer surface of both peripheral cells of dicyemids and epidermal cells of host renal appendages. Dicyema-clp appears to be associated with N-acetyl-D-glucosamine in the interface between dicyemid peripheral cells and epidermal cells of the host renal appendage, and possibly aids in adhering the calotte to host epidermal cells.

Phylogeny of selected Sepiidae (Mollusca, Cephalopoda) based on 12S, 16S, and COI sequences, with comments on the taxonomic reliability of several morphological characters.

Phylogenetic relationships among 11 species of sepiids from Japanese waters and Sepia officinalis from Mediterranean were studied using partial sequences of the mitochondrial 12S rRNA, 16S rRNA, and cytochrome c oxidase subunit I genes. These three genes had been analyzed in an Atlantic species S. elagans and was obtained from database. In the two-gene set analysis (16S+COI), sequence data of another 4 species were added from database. We also studied morphological characters of radulae, tentacular clubs, and cuttlebones. The molecular phylogeny was not congruent with relationships detected by the number of rows in radulae and the arrangement of suckers on the tentacular club. As to the cuttlebone shape, the molecular phylogeny suggests the separation of two groups, Doratosepion species with a lanceolate cuttlebone and the others with a broad cuttlebone. Our molecular phylogenetic study revealed these sepiids are separated into four clades. The first clade includes Sepia officinalis, S. hierrendda, S. bertheloti, S. pharaonis and Sepiella japonica. The second clade consists of S. latimanus and Metasepia tullbergi from sub-tropical waters. The third clade includes Sepia esculenta, S. madokai, S. aculeata and S. lycidas, which have a cuttlebone with a prominent spine. The fourth clade consists of Doratosepion species complex, S. kobiensis, S. lorigera, S. pardex, S. peterseni, and S. sp., which are characterized by a narrow cuttlebone with a distinct outer cone at the posterior end. The lack of membranous structures in the cuttlebone is a synapomorphy for this clade. S. elegans did not clearly belong to any of these clades and might represent the fifth clade.

Phylogenetic relationships among major species of japanese coleoid cephalopods (Mollusca: Cephalopoda) using three mitochondrial DNA sequences.

Phylogenetic relationships among 36 species of major coleoid cephalopods from Japanese waters were studied using partial sequences of three mitochondrial genes, 16S rDNA, 12S rDNA, and cytochrome c oxidase subunit I gene. Octopoda and Decapoda were monophylic groups. Within Sepioidea, Sepiadariidae and Sepiolidae were not closely related to Sepiidae, but rather related to Teuthoidea. Sepiidae with a distinct calcareous shell formed a single cluster. Myopsida was closely related to Oegopsida. Within Octopoda, Opisthoteuthis depressa and Argonauta argo diverged earlier than Octopodiidae. The common octopuses in Japanese waters were separated into three clusters. The first cluster occupied a basal position, and includes large-sized octopuses, such as Enteroctopus dofleini and Octopus (Paroctopus) conispadiceus from the continental shelf and upper slope. The second cluster consisted of long-armed octopuses, such as O. ornatus, O. minor, and O. sasakii. The third cluster contained small- to medium-sized octopus, such as Amphioctopus fangsiao, A. areolatus, O. cyaneus, and O. vulgaris, in which several species possess ocelli on the web. The second cluster formed the sister group to the third cluster.

Renal organs of cephalopods: a habitat for dicyemids and chromidinids.

The renal organs of 32 species of cephalopods (renal appendage of all cephalopods, and renal and pancreatic appendages in decapods) were examined for parasite fauna and for histological comparison. Two phylogenetically distant organisms, dicyemid mesozoans and chromidinid ciliates, were found in 20 cephalopod species. Most benthic cephalopods (octopus and cuttlefish) were infected with dicyemids. Two pelagic cephalopod species, Sepioteuthis lessoniana and Todarodes pacificus, also harbored dicyemids. Chromidinid ciliates were found only in decapods (squid and cuttlefish). One dicyemid species was found in branchial heart appendages of Rossia pacifica. Dicyemids and chromidinids occasionally occurred simultaneously in Euprymna morsei, Sepia kobiensis, S. peterseni, and T. pacificus. The small-sized cephalopod species, Idiosepius paradoxus and Octopus parvus, harbored no parasites. Comparative histology revealed that the external surface of renal organs varies morphologically in various cephalopod species. The small-sized cephalopod species have a simple external surface. In contrast, the medium- to large-sized cephalopod species have a complex external surface. In the medium- to large-sized cephalopod species, their juveniles have a simple external surface of the renal organs. The external surface subsequently becomes complicated as they grow. Dicyemids and chromidinids attach their heads to epithelia or insert their heads into folds of renal appendages, pancreatic appendages, and branchial heart appendages. The rugged and convoluted external surface provides a foothold for dicyemids and chromidinids with a conical head. They apparently do not harm these tissues of their host cephalopods.

Cell number and cellular composition in infusoriform larvae of dicyemid mesozoans (Phylum Dicyemida).

Cell numbers and cellular composition were examined in infusoriform larvae of 44 species of dicyemid mesozoans belonging to 6 genera; Conocyema, Dicyema, Dicyemennea, Dicyemodeca, Microcyema, and Pseudicyema. In addition, literature on infusoriform larvae of another 20 species was reviewed. Infusoriform larvae consist of a constant cell number which is species-specific. Small interspecific variations are found in total cell numbers, 35, 37, 39, 41 and 42. The most frequent cell number encountered in infusoriform larvae studied is either 37 or 39. Infusoriform larvae with 35 cells are found in three genera. Infusoriform larvae with 37 cells are found in four genera. Infusoriform larvae with 39 cells are found in four genera. Most differences in total cell numbers are due to the absence or presence of particular ventral cells. In all infusoriform larvae, the lateral, dorsal and caudal areas are cell constant, whereas in the apical and ventral areas a distinct and variable configuration of cells are present. In cellular composition, a total of 29 cells (15 cell types) were recognized in all infusoriform larvae examined. Additional cell types are characteristic of a relatively few species. Even in infusoriform larvae with the same total cell numbers, cellular composition varies by species. Thus, there are 7 variations of cellular composition in infusoriform larvae with 37 cells. Differences in larval cell numbers and types do not warrant traditional generic separation of dicyemids.

Biology of dicyemid mesozoans.

We reviewed recent advances of some aspects on the biology of dicyemid mesozoans. To date 42 species of dicyemids have been found in 19 species of cephalopod molluscs from Japanese waters. The body of dicyemids consists of 10-40 cells and is organized in a very simple fashion. There are three basic types of cell junction, septate junction, adherens junction, and gap junction. The presence of these junctions suggests not only cell-to-cell attachment, but also cell-to-cell communication. In the development of dicyemids, early stages and cell lineages are identical in vermiform embryos of four genera, Conocyema, Dicyema, Microcyema, and Pseudicyema. Species-specific differences appear during later stages of embryogenesis. In the process of postembryonic growth in some species, the shape of the calotte changes from conical to cap-shaped and discoidal. This calotte morphology appears to result from adaptation to the structure of host renal tissues and help to facilitate niche separation of coexisting species. In most dicyemids distinctly small numbers of sperms are produced in a hermaphroditic gonad (infusorigen). The number of eggs and sperms are roughly equal. An inverse proportional relationship exists between the number of infusorigens and that of gametes, suggesting a trade-off between them. Recent phylogenetic studies suggest dicyemids are a member of the Lophotrochozoa.

Fine structure of dicyemid mesozoans, with special reference to cell junctions.

The fine structure of the dicyemid mesozoan, Dicyema acuticephalum, from Octopus vulgaris, was studied with special attention to intercellular junctional complexes between various kinds of cells. Two types of intercellular junction, namely, adherens junctions and gap junctions, were found in both vermiform stages and in infusoriform embryos. Adherens junctions were classified into two types. Zonulae adherentes-like junctions were observed between adjacent peripheral cells at vermiform stages, between adjacent external cells of infusoriform embryos, and between members of groups of internal cells that covered the urn in infusoriform embryos. Maculae adherentes-like junctions were seen between a peripheral cell and an axial cell at vermiform stages. In infusoriform embryos, these junctions were observed between various types of cells, excluding urn cells. Gap junctions were found between adjacent peripheral cells at vermiform stages, whereas in infusoriform embryos these junctions were located between various types of cells excluding urn cells. Dicyemids might be the most primitive multicellular animals to possess these basic types of cell junctions. Ciliary rootlet systems at vermiform stages and in infusoriform embryos were unique in structure compared with those of other primitive multicellular animals. J Morphol 231:297-305, 1997. © 1997 Wiley-Liss, Inc.

The cell lineages of two types of embryo and a hermaphroditic gonad in dicyemid mesozoans.

The present review summarizes the patterns of cell division and the cell lineages of the two types of embryo, namely, the vermiform embryo and infusoriform embryo, of the dicyemid mesozoan Dicyema japonicum. The infusoriform embryo develops from a fertilized egg. The early cleavages are holoblastic and spiral. At around the 20- to 24-cell stage, cleavages become asynchronous and the cleavage pattern changes from spiral to bilateral. The fully formed infusoriform embryo consists of 37 cells and exhibits bilateral symmetry. These 37 cells are produced after only four to eight rounds of cell division. The vermiform embryo develops asexually from an agamete. Cell division proceeds spirally in the early stages, becoming bilateral from the fifth cell division onward. The fully formed vermiform embryo consists of 23 cells and exhibits bilateral symmetry. The 23 cells are produced after only four to six rounds of cell division. During the development of both types of embryo, a few cells consistently undergo extremely unequal divisions; the much smaller daughter cells eventually degenerate and ultimately disappear during embryogenesis. This disappearance may be an example of programmed cell death. The cell lineages appear to be invariant among embryos, apart from the derivation of the lateral cells of infusoriform embryos. In addition to a description of embryogenesis, the present review includes details of development of the functionally hermaphroditic gonad, the infusorigen, and gametogenesis. The infusorigen is formed from an agamete. The first spermatogonium is produced by the second division and the first oogonium is produced by the third division. Oogonia occupy the outer surface of the axial cell of the infusorigen, while spermatogonia are incorporated into the cytoplasm of the axial cell of the infusorigen. The infusorigen consists finally of approximately 20 cells.