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The genome size (C-value) of a species indicates the total amount of DNA included in its haploid chromosomes. It is one of the fundamental characteristics that relate closely to the evolution of organisms. Information on molluskan C-values is limited despite the abundance of morphological diversity. Therefore, to better understand molluskan genome evolution, we conducted a comprehensive survey of the C-values and AT content of 146 species in four molluskan classes by flow cytometry. The difference in AT content was found to be related to an increase in C-value in gastropods and cephalopods. For cephalopods, a positive correlation of AT content with the increase in chromosome number and C-value was observed from Nautilus pompilius and N. macromphalus (2n = 52) to the specialized groups of octopus (2n > 60) and squid (Sepiida and Teuthida; 2n = 92–112). In contrast, a negative correlation between C-value and AT content in gastropods was demonstrated from the limpet group Patellogastropoda (2n = 16–18) to the specialized group Neogastropoda (2n = 26–72). Our data suggest at a likely positive correlation between AT content and C-value in the cephalopods lineages but a negative correlation between them in the gastropod lineages. Furthermore, the reduction of the C-value in the Bivalvia lineage could not have been caused by the selective deletion of AT-rich regions. Our study adds valuable data to the currently limited knowledge base of molluskan C-values, which will provide important information for future genome projects in mollusks.

The Japanese common sea cucumber (Apostichopus japonicus) is a major marine product from Sanriku, Japan, but its populations were severely affected by the 2011 Tohoku earthquake, possibly decreasing its genetic diversity and increasing its extinction risk. In this study, we estimated the genetic structure and diversity of sea cucumbers from Touni and Yamada Bays of Sanriku over 4 years after the earthquake. The between-population genetic structure was estimated using two mitochondrial DNA regions (cytochrome c oxidase subunit I and 16S rDNA). Genetic differentiation (as measured by pairwise FST) was not significant between locations. Thus, even after the tsunami, gene flow and genetic diversity among the two sea cucumber populations were maintained. Our data also suggested that sea cucumbers in Sanriku experienced population expansion of about 0.20–0.24 million years ago, during the stable Mindel-Riss interglacial period. We conclude that A. japonicus populations in Sanriku could maintain their genetic structure throughout multiple disastrous tsunamis over the past several 1000 years. The high dispersal ability of planktonic larvae may enable the entrance of new recruits, thereby reducing risks associated with genetic structure and diversity changes stemming from mass die-offs in a given body of water from the past to the present.

The hemocytes of molluscs take various roles including immunity and wound healing. Oyster gill was found to undergo hematopoiesis and produce adult somatic progenitor cells. In gastropod molluscs, however, such special gill structure has not been reported. In the present study, we used Pacific abalone, a gastropod mollusc, and explored unique gill structures undergoing active proliferation. By immunofluorescence staining, two proliferation markers, proliferating cell nuclear antigen (PCNA) and phospho-histone H3 (H3P), were detected in marginal regions of the gill filaments, which were characterized by thick epithelium with cilia. In situ hybridization for Sox genes, the stem cell markers, also showed signals in the corresponding regions. BrdU, a DNA replication-detecting reagent, and nuclear-localized β-catenin, a putative epithelial–mesenchymal transition (EMT) marker, were also detected in the same regions. These results suggest that the marginal regions of gill filaments are highly proliferative, possess stemness, and likely undergoing EMT. Further immunofluorescence staining for hemocytes with PCNA, H3P, and β-catenin revealed that these cells also exhibited these markers, implying a possible link between the hemocytes and the gill cells. These results showed a structure containing actively proliferating cells in abalone gills, and points to the importance of classification of cell population in this tissue.

Abstract Yolk-consuming (lecithotrophic) embryos of oviparous animals, such as those of fish, need to make do with the maternally derived yolk. However, in many cases, yolk possesses little carbohydrates and sugars, including glucose, the essential monosaccharide. Interestingly, increases in the glucose content were found in embryos of some teleost fishes; however, the origin of this glucose has been unknown. Unveiling new metabolic strategies in fish embryos has a potential for better aquaculture technologies. In the present study, using zebrafish, we assessed how these embryos obtain the glucose. We employed stable isotope (13C)-labeled substrates and injected them to the zebrafish embryos. Our liquid chromatography-mass spectrometry-based isotope tracking revealed that among all tested substrate, glutamate was most actively metabolized to produce glucose in the zebrafish embryos. Expression analysis for gluconeogenic genes found that many of these were expressed in the yolk syncytial layer (YSL), an extraembryonic tissue found in teleost fishes. Generation 0 (G0) knockout of pck2, a gene encoding the key enzyme for gluconeogenesis from Krebs cycle intermediates, reduced gluconeogenesis from glutamate, suggesting that this gene is responsible for gluconeogenesis from glutamate in the zebrafish embryos. These results showed that teleost YSL undergoes gluconeogenesis, likely contributing to the glucose supplementation to the embryos with limited glucose source. Since many other animal lineages lack YSL, further comparative analysis will be interesting.

Cnidarians are considered ancestral metazoans and, therefore, are important taxa for studying animal evolution. However, little is known about the group’s genome size ( C value), which is an important parameter in whole-genome sequencing. To address this issue, we measured the C values of 27 cnidarian species from Japan, using flow cytometry, and found that they ranged from 0.26 to 3.56 pg. Excluding the results for Agalma elegans and Physalia physalis (order Siphonophorae), which had the highest C values among the species included in the present study, the C values for the cnidarians were 0.26–1.49 pg. In particular, we found that hydrozoans possessed relatively large and wide-ranging C values, indicating that evolution within the group involved considerable gains or losses of genomic content. Overall, the C values reported in the present study could be valuable for whole-genome sequencing, using next-generation sequencers, and for future research in cytogenetics.

Octopus ( Amphioctopus ) areolatus is an important marine cephalopod in Japan. We examined its diploid chromosome number, karyotype, and genome size ( C value), and performed fluorescence in situ hybridization (FISH) using a vertebrate telomeric probe. The diploid chromosome number was 60 in embryonic cells, with 24 pairs of metacentric chromosomes, four pairs of metacentric/submetacentric chromosomes, and two pairs of submetacentric chromosomes. The mean C value was estimated to be 5.47 pg. From these findings, and those reported previously for other octopus species, we suggest that the factor causing the quantum change of C value in O. ( A. ) areolatus was genome duplication, and not polyploidy. In telomeric FISH analysis, hybridization signals were clearly observed in the telomeric regions of the chromosomes. This is the first report of FISH analysis of cephalopod chromosomes, and our findings suggest that the telomere sequence of O. ( A. ) areolatus is (TTAGGG) n , which may allow gene mapping in the future.

Genome size ( C value) is an important index for phylogenetic studies. Haliotidae (abalones) includes many species widely distributed throughout the world’s oceans, which makes this family interesting for phylogenetic studies. To examine Haliotidae phylogeny, we determined the C value and adenine and thymine base pair content (AT %) of Haliotis discus hannai and H. diversicolor aquatilis by using flow cytometry. The C values of H. discus hannai and H. diversicolor aquatilis were 1.84 and 1.45 pg, with AT % of 62.3 and 66.3 %, respectively. These data represent the first report of abalones classified as Pacific Northwest ( H. discus hannai ) and Indo-Pacific ( H. diversicolor aquatilis ) groups, and provides new validation for previous theories related to Haliotidae phylogeny.

:  The telomere sequence type (TTAGGG)n is known to be distributed in various phyla in the Animalia and in Mastigophora (Protista). However, the telomere type of Porifera (sponges), a phylum comprising the lowest multicellular animals, has not been reported. In this study, we examined the three sponge species Leucetta chagosensis, Halichondria japonica, and Halichondria panicea for the presence of the telomere type (TTAGGG)n by fluorescence in situ hybridization (FISH). The oligonucleotide probe (TTAGGG)7 clearly displayed signals on the interphase nuclei of all three sponges. In contrast, the (TTAGG)7 probe, which has one base fewer than (TTAGGG)7, did not display the signals. These results suggest that the telomeres of the three sponges consist of (TTAGGG)n, which is identical to the sequence type found in many higher multicellular animals and in Mastigophora. Additionally, this is the first study to reveal a telomere sequence type for Porifera. Moreover, these results suggest that Porifera are phylogenetically related to Mastigophora, and supports the general theory that Porifera evolved from Mastigophora. Further, this study strongly suggests that the origin of the (TTAGGG)n telomere sequence is to be found in a common ancestor of either the Bilateria and Porifera, or the Protista.

:  In order to develop a highly efficient method for mass production of triploid Pacific abalone Haliotis discus hannai, caffeine treatment that is safe and inexpensive was optimized. To suppress the first meiotic division, fertilized eggs were exposed to either a 10‐ or 15‐mM caffeine solution for 24 min beginning at 12 min after fertilization. In most treated batches, the rates of cleaved eggs showed no significant difference from the control batches. However, in most treated batches, the rates of occurrence of normal larvae and the survival rates of the early juveniles were significantly lower than those of the controls. The triploid rates at 6 days to 11 months after settlement in all the treated batches were extremely high (91–100%). There was no significant difference in the mean triploid rates between 10‐ and 15‐mM caffeine treatments. These results suggest that both treatment conditions were conducive to triploid abalone production. One live 2n/3n mosaic specimen was found in the treated batches. However, since the frequency of mosaic was extremely low, the mosaicism would probably not have an adverse effect on the stable production of triploid abalones.