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The 70 kDa heat shock proteins (HSP70s) and the constitutive members of the HSP70 family (heat shock cognates; HSC70s) play essential roles in various biological processes. The number of hsp70/hsc70 in the database is rapidly increasing because of their importance and the automatic annotation of newly sequenced genomes. However, accumulating evidence indicates that neither hsp70 nor hsc70 forms a monophyletic gene family, raising the need to reconsider the annotation strategy based on the traditional concept of the inducible HSP70 and constitutive HSC70s. The main aim of this study is to establish a systematic scheme to annotate hsp70-like genes taking the latest phylogenetic insights into account. We cloned two hsp70s from the rotifer Brachionus plicatilis sensu stricto (s.s.), an emerging model in evolutionary genetics, and conducted a genome-wide screening of B. plicatilis s.s. hsp70s using the two sequences as queries. A total of 15 hsp70-like genes were found, and 7 of them encoded distant members of the HSP70 family, the function of which largely remains unknown. Eight canonical hsp70s were annotated according to a recently proposed nomenclature based on the molecular evolution: e.g., HSP70cA1/B1 for the cytosolic lineage, HSP70er1 for the endoplasmic reticulum lineage, and HSP70m1 for the mitochondrial lineage. The two cloned hsp70s, HSP70cB1 and HSP70cB2, ubiquitously increased their mRNA levels up to 7.5 times after heat treatment as assessed by semi-quantitative PCR, real-time PCR, and in situ hybridization. This systematic screening incorporating a reasonable update to the annotation strategy would provide a useful example for future HSP70 studies, especially those in non-traditional model organisms.

The reproductive biology of deep-sea fishes is largely unknown because of the difficulty of behavioral observations in this environment. In this study, the reproductive behavior of the deep-sea snailfish, Careproctus pellucidus, which lives at depths > 200 m, was observed in an aquarium. To the best of our knowledge, this is the first report to observe and describe the reproductive behavior of deep-sea fish under free-living conditions. Fishes were collected at a depth of approximately 300 m (36°70′ N; 141°00′ E) on July and August 2018 and kept for observation until March 2019. We observed that almost all spawnings took place between one male and one female. Males defended a spawning substrate against other males and exhibited body-wiggling behavior for both courtship and aggressive display. Females visited the male’s territory and spawned a demersal adhesive egg mass on the substrate. The territorial male then sought the spawned eggs using filamentous rays in the lower pectoral-fin lobe and released sperm when he located the eggs. Males remained near the spawning substrate after spawning but did not perform any parental care. Notably, sneaking behavior by a non-territorial male was observed in one case; this is the first report of this alternative reproductive strategy (or tactic) in a deep-sea fish. Our study reveals the unique reproductive biology of the deep-sea fish, C. pellucidus, which does not depend on visual information and uses other sensory modalities.

In this study, we determined the genomic DNA sequences of the mucosal galectin-encoding genes from all 19 species and subspecies of the genus Anguilla. The nucleotide sequences of the galectin genes were c. 2.3-2.5 kb long and the organisation of their four exons and three introns was conserved in all species. An unusual sequence was found in the fourth exon of Anguilla reinhardtii, resulting in a unique deduced amino-acid sequence at the C-terminus. All six amino-acid residues important for β-galactoside binding were conserved in three species, while one residue (R73 ) was substituted to K73 in the other 16 species-subspecies, including Anguilla marmorata. However, this substitution did not appear to affect the sugar-binding ability of galectins because the galectin of A. marmorata was previously shown to bind to lactose. We also discuss the molecular evolution of galectins among Anguilla spp. and the homologues previously identified in Conger myriaster.

Two new species of Bleekeria Günther, 1862 are described from specimens collected in the Andaman Sea off the coast of Myanmar during bottom surveys conducted by the R/V Dr Fridtjof Nansen in 2015 and 2018. They are distinguished from each other and from congeners by a combination of morphological and meristic characters as well as fin coloration and genetic variance. Bleekeria albicauda sp. nov. has pelvic fins, 40–41 dorsal-fin rays, 54–55 total vertebrae, no teeth in jaws, 4 scale rows between dorsal-fin origin and lateral line, a single row of about 10 scales on mid-upper part of opercle, scales on central part of body clearly shorter than their height, caudal fin with white upper and lower lobes when fresh (unique within the genus). Bleekeria nigrilinea sp. nov. has no pelvic fins, 37–39 dorsal-fin rays, 49–50 total vertebrae, 2½ scale rows between dorsal-fin origin and lateral line (the smallest count within the genus with B. estuaria of Mozambique brackish water), 5–6 scales on mid-upper part of opercle arranged in a single row, scales on central part of body clearly longer than their height, upper and lower margins of caudal fin black when fresh (unique within the genus). The COI gene sequences of the two new species showed clear genetic divergence (pairwise K2P, >10 %) from Bleekeria estuaria Randall & Ida, 2014 and Bleekeria mitsukurii (Jordan & Evermann, 1902). A key to the species of Bleekeria is provided.

The metazoan 70-kDa heat shock protein (HSP70) family contains several members localized in different subcellular compartments. The cytosolic members have been classified into inducible HSP70s and constitutive heat shock cognates (HSC70s), but their distinction and evolutionary relationship remain unclear because of occasional reports of “constitutive HSP70s” and the lack of cross-phylum comparisons. Here we provide novel insights into the evolution of these important molecular chaperones. Phylogenetic analyses of 125 full-length HSP70s from a broad range of phyla revealed an ancient duplication that gave rise to two lineages from which all metazoan cytosolic HSP70s descend. One lineage (A) contains a relatively small number of genes from many invertebrate phyla, none of which have been shown to be constitutively expressed (i.e., either inducible or unknown). The other lineage (B) included both inducible and constitutive genes from diverse phyla. Species-specific duplications are present in both lineages, and Lineage B contains well-supported phylum-specific clades for Platyhelminthes, Rotifera, Nematoda, Porifera/Cnidaria, and Chordata. Some genes in Lineage B have likely independently acquired inducibility, which may explain the sporadic distribution of “HSP70” or “HSC70” in previous phylogenetic analyses. Consistent with the diversification history within each group, inducible members show lower purifying selection pressure compared to constitutive members. These results illustrate the evolutionary history of the HSP70 family, encouraging us to propose a new nomenclature: “HSP70 + subcellular localization + linage + copy number in the organism + inducible or constitutive, if known.” e.g., HSP70cA1i for cytosolic Lineage A, copy 1, inducible.

Species-specific restriction fragment length polymorphism in the intron of the androgen receptor gene (ar5) was found in glass to silver-stage individuals of Anguilla japonica (n = 51) and A. marmorata (n = 21). The sequence analysis of 16S rDNA from 328 anguillid leptocephali collected in the North Equatorial Current of the western North Pacific Ocean revealed the specimens to be A. japonica (n = 194), A. marmorata (n = 128), A. bicolor pacifica (n = 5) and A. luzonensis (n = 1). All leptocephali of A. japonica and A. marmorata were monomorphic and did not share an allele at the ar5 locus, indicating that the two species are reproductively isolated.

To investigate the presence of non-native anguillid eels in Japanese waters, 141 eels were collected from seven sampling sites throughout the Tone River system. Genetic species identification showed an extraordinary dominance of the non-native European eel Anguilla anguilla in the uppermost site of the study area. Estimation of age from otoliths suggested that the European eels were introduced into the river in the 1990s, corresponding to previous reports from other Japanese water systems. Comparison of the von Bertalanffy growth curve parameters indicated that the European eels caught in the Tone River system appeared to have a similar or even higher growth rate than the same species in the original habitats in Europe. The long-term inhabitation and the normal development of European eels in Japanese waters suggest that regulations prohibiting the release of non-native eels and safeguards against accidental escape from culture ponds must be strictly maintained in order to ensure the conservation of the native Japanese eel.

Pseudapocryptes elongatus is one of the oxudercine gobies, which show varying degrees of amphibious behaviour and capacities to breathe air. There is little information on the early life history of P. elongatus, particularly of their morphology and larval habitat and duration. This study focused on the life history of larval and juvenile stages of P. elongatus investigated by genetic species identification, morphological observation and otolith analyses using specimens collected in June and October 2012 from estuaries in Bac Liêu Province, southern Vietnam (09°14′N 105°43′E). Genetically identified juvenile P. elongatus were characterized by (1) a slender body form, (2) the anterior edges of both the anal and second dorsal fins located at the midpoint along the body axis, (3) scarce chromatophores over the body surface, (4) melanophores in the parietal region between the eyes, and (5) a single row of melanophores along the base of the anal fin. Mean age at recruitment to estuaries was 38.0 ± 4.1 days, and otolith Sr/Ca ratios ranged from 8.9 to 9.9 mmol mol−1, suggesting that larval migration from their spawning sites requires more than 1 month in saline environments.

It is common to count the numbers of specified fish in the field after speciation of captured fish according to their morphology and to subject these counts to appropriate statistical analyses. In recent years, a non-invasive method to estimate the abundance of a particular fish species using environmental DNA (eDNA) has been developed. However, it is still difficult to determine accurate numbers of fish species using such method. We predict that the estimation of individuals of certain fish species in the field is more accurate and easier by using haplotypes of DNA in the fast evolutionary region. Therefore, we focused on the regulatory region (D-loop) in mitochondrial DNA, which is known to have a high genetic variation at the intraspecific level of the targeting eel. We investigated haplotype diversity in eel at first and then determined the number of D-loop haplotypes contained in their exfoliated cells in breeding water. Finally, we developed a novel analytical method, HaCeD-Seq, to estimate the number of individuals based on the abovementioned data.

To examine species composition and population structures in sand lance (Ammodytidae) along the northern Pacific coast of Japan, genetic analysis were carried out for specimens collected in 2014 from Otsuchi Bay, Iwate, Ishinomaki Bay, Miyagi, off Soma, Fukushima and Ise-Mikawa Bays, Aichi. The samples consisted of Ammodytes japonicus and Ammodytes heian, of which the latter is a recently described species. Neither species exhibited significant genetic differences among localities. Only A. japonicus was found in the most southern locality at Aichi, but it decreased northward to

Commercial exploitation of tropical Anguilla species has been expanding rapidly in Southeast Asia, even though their ecological status is unknown. In the present study, we investigated the species composition of anguillid glass eels recruited in southern Mindanao, the Philippines. The glass eel specimens were collected monthly between April 2014 and March 2015 in General Santos and identified by morphological and genetic analyses (n = 4745). Among the seven species/subspecies identified, A. marmorata was the dominant species (76.1 %), A. bicolor pacifica was the second most abundant (19.5 %), and A. celebesensis, A. interioris, A. luzonensis, A. borneensis, and A. japonica rarely occurred (0.02–3.0 %). In addition, caudal fin pigmentation, which is an important diagnostic trait at the glass eel stage, was found to be similar in the short-finned A. bicolor pacifica and the long-finned A. interioris and A. borneensis. The recruitment of Anguilla in the Philippine archipelago comprises seven anguillid species, including endangered, vulnerable, and data deficient species, according to the latest IUCN Red List assessment. The results of the present study provide essential information for future studies of tropical anguillid species, and might contribute to the development of strategies for the management of their stocks.

We developed a novel molecular genetic method using restriction fragment length polymorphisms (RFLPs) to identify the taxonomically debatable Japanese sand lances of the genus Ammodytes. Partial nucleotide sequences of the mitochondrial DNA cytochrome c oxidase subunit 1 (COI) gene of Ammodytes japonicus (n = 405), Ammodytes heian (n = 284), and Ammodytes hexapterus (n = 15) were determined and found to contain species-specific restriction sites for HindIII-HF and RsaI enzymes. The observed RFLP patterns from a combination of two enzymes were specific in each species except for specimens with rare haplotypes of A. japonicus (1.5 %) and A. heian (2.1 %), whereas detailed morphological analysis using 29 characters failed to discriminate the COI-identified specimens of these two species collected from the same locality. The developed molecular method will allow rapid and precise identification of the Japanese sand lances, especially for the sympatric A. japonicus and A. heian, thereby providing a reliable means for ecological research and stock management of these species.

Fish skin and its corresponding mucus are important defenses against infections from pathogenic organisms, and several types of defensive molecules, particularly lectins, are often found in mucus. Freshwater eels of the genus Anguilla are widely distributed throughout the world, and the genus currently contains 16 species plus 3 subspecies. In this work, genomic sequences of skin mucus C-type lectin-encoding genes from all 19 presently known species/subspecies of the genus Anguilla were determined. The nucleotide sequences were approximately 2 kbp long, and the genomic structure of five exons and four introns was common in all species. All of the encoded C-type lectins were considered functional because of the presence of calcium ion-binding and sugar-binding motifs as well as conserved cysteine residues. Phylogenetic analysis of the genus indicated that the C-type lectins in freshwater eels might be divided into two clusters, corresponding to their temperate and tropical growth habitats, suggesting that Anguilla species C-type lectin has evolved to adapt to microflora in each environment. This study is the first to comprehensively describe the immune-related genes in a complete fish genus.

Tropical species of anguillid glass eels have recently been exploited to be a substitute for the commercially important Anguilla japonica or A. anguilla, so information about their life histories is needed to facilitate conservation. To understand ecological aspects of glass eel recruitment patterns of tropical eels, Anguilla spp., monthly monitoring of species compositions at the mouth of the Cagayan River in northern Luzon in the Philippines was carried out at different time periods from May 2008 to September 2009 and from November 2011 to December 2012. Species identifications were made for 32,178 glass eels using morphology and/or genetics, and a total of five anguillid species, Anguilla bicolor pacifica, A. celebesensis, A. japonica, A. luzonensis, and A. marmorata, were found. In both time periods A. marmorata was dominant followed by A. luzonensis and A. bicolor pacifica, but the proportion of A. marmorata decreased from 55.1% in 2008–2009 to 41.2% in 2011–2012, with A. bicolor pacifica i...

Rotifers have been used as a model in aging studies due to their several advantages such as short lifespan, ease of culture, and asexual reproduction that enables clonal culture. Recent molecular phylogenetic analyses revealed that rotifers belong to the superphylum Lophotrochozoa and phylogenetically distant from major invertebrate models. Taking advantage of this unique phylogenetic position and development of molecular technologies, rotifers are now recognized to be a traditional but also emerging model in aging studies. This chapter summarizes current knowledge of rotifer aging in association with some traditional aging theories and future prospects. Reevaluation of past works, as well as future advances in rotifer studies, would fill the taxonomic gaps and help us to understand aging mechanisms from a broad perspective.

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.

The Japanese eel Anguilla japonica is an important aquaculture fish species in the East Asian countries of Japan, China, Korea, and Taiwan. All glass eel fry are captured from the wild and understanding the recruitment patterns of the glass eel is important. The larvae of A. japonica are passively transported to the East Asian Continental Shelf by the North Equatorial Current, the Kuroshio, the Kuroshio intrusion currents, and coastal currents. In each location, recruitment time is diverse: It is November in Taiwan and April in the Yalu River. How the glass eels reach recruitment areas remains poorly understood. Here, we combine information from larval ages based on otolith increments, simulated drifting paths on the East Asian Continental Shelf, and main fishing seasons in each location of East Asia. We identify five main recruitment blocks: (1) The main Kuroshio, (2) The Taiwan Strait Warm Current, (3) The Taiwan Warm Current, (4) The Yellow Sea Warm Current and (5) The branch of Yellow Sea Warm Current. The counted age of the glass eels is significantly underestimated for the later recruits, possibly due to the cessation of the otolith edge growth under low water temperatures. This study clarifies the eel&rsquo, s larval characteristics and transport mechanisms in the East Asia Continental Shelf, providing important information for its recruitment dynamics in the marine stage.

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Two types of lactose-specific lectins, galectin (AJL-1) and C-type lectin (AJL-2), were previously identified in the mucus of adult Anguilla japonica. Here, we compared the expression profiles of these two homologous lectins at the adult and juvenile stages between the tropical eel Anguilla marmorata and the temperate eel A. japonica. Only one lectin, predicted to be an orthologue of AJL-1 by LC-MS/MS, was detected in the mucus of adult A. marmorata. We also found that an orthologous gene to AJL-2 was expressed at very low levels, or not at all, in the skin of adult A. marmorata. However, we detected the gene expression of an AJL-2-orthologue in the skin of juvenile A. marmorata, and a specific antibody also detected the lectin in the juvenile fish epidermis. These findings suggest that expression profiles of mucosal lectins vary during development as well as between species in the Anguilla genus.

A new anguillid eel species, Anguilla luzonensis, was recently discovered on Luzon Island of the northern Philippines, but little is known about its life history. DNA identification was used to determine that five leptocephali of this species (29.2–51.2 mm) were collected offshore in the western North Pacific (13°–17.5° N, 125°–141° E) in April, June, and July between 2002 and 2009. One leptocephalus was caught west of Luzon and four were close to the spawning areas of Anguilla japonica and Anguilla marmorata. Otolith microstructure showed that the leptocephali were up to 103–138 days old. Glass eels from northern Luzon in previous studies that were thought to be Anguilla celebesensis now appear likely to have been A. luzonensis, and they were estimated by otolith analysis to have long larval durations similar to those of A. marmorata. Estimated hatching dates of these glass eels and the A. luzonensis leptocephali were both in the February to May season. Offshore presence of leptocephali of A. l...

To determine the actual status of the recruiting glass eel stock of Anguilla japonica, we conducted a continuous monthly monitoring program for 2 years between November 2009 and October 2011 at the Sagami River estuary, Japan. A total of 114 and 372 A. japonica glass eels were observed in the 2009–2010 and 2010–2011 seasons, respectively. Recruitment patterns were the same in both years, starting in early winter (December in 2009 and November in 2010), increasing slightly until March, decreasing in April, but attaining the largest peak in June, after which no glass eels were observed during August–October. The Japanese eel has been known to spawn mainly in the summer, and glass eels recruit to their freshwater growth habitats during the winter to early spring. Our results clearly demonstrate an unexpected late arrival of glass eels in the early summer for two recent consecutive year classes. The summer recruitment found in our study indicates the unusual phenology of the Japanese eel, which may be a possible response to recent climate change.

Chum salmon (Oncorhynchus keta) fry were reared in freshwater (FW), mimicking the hatchery enhancement program, and in seawater (SW) to determine the effect on growth. The fry groups in SW showed exponential growth, whereas those in FW grew in a linear manner in two experiments over 49 and 70 days. Fry adapted to SW for 49 days survived well after returning to FW for 7 days. Gill Na+, K+-ATPase activity in the SW group was higher than that of the FW group. The chloride cells in gill filaments increased in number and developed larger in size in SW fry. Pituitary growth hormone (GH) mRNA levels increased 14 days after transfer to SW. The mRNA levels of GH receptor (GHR) and insulin-like growth factor I (IGF-I) in the liver and gill increased in the fry reared in SW.

We assayed the effects of a variety of antiprotozoal compounds against trophonts of Cryptocaryon irritans , the causative agent of ‘white spot disease of marine fish’ in vitro using the double layered media that we developed previously for the culture of the parasite. In the assay, ionophores, particularly sodium salinomycin, showed apparent killing and growth-suppression effects against the parasite. As there was no mortality in Japanese flounder Paralychthys olivaceus that were fed a diet containing sodium salinomycin (200 ppm) for two weeks, we evaluated the efficacy of 200 ppm sodium salinomycin against C. irritans in Japanese flounder. We fed Japanese flounders a medicated diet for 5 d prior to and 3 d after challenge with C. irritans. In the experimental group, the number of protomonts recovered from the fish and the size of tomonts that were transformed from the protomonts were significantly reduced, when compared to the control group. Furthermore, in a different experiment, the fish that were fed a diet medicated with sodium salinomycin survived longer than those fed an unmedicated diet after challenge. Sodium salinomycin can be a good candidate drug for chemotherapy and control of Cryptocaron irritans infection.

Eggs of the Japanese eel Anguilla japonica collected in the western North Pacific were identified by onboard species-specific polymerase chain reaction (PCR) and DNA nucleotide sequencing after the cruise. Fish eggs of various species were collected by large plankton net tows at 12 stations along the southern part of the West Mariana Ridge on 19–25 May 2009. A total of 43 fish eggs were distinguished morphologically as possibly being of A. japonica. Thirty-one of those were analyzed by PCR, which included 15 eggs collected at 12°50–55′N, 141°15–20′E (in 5 tows) that showed positive results. The 16S ribosomal RNA (rRNA) gene sequences of eggs determined after the cruise indicated that 31 A. japonica eggs had been collected. The remaining eggs were of mesopelagic eel species (Serrivomeridae and Derichthyidae), or unidentified species. The morphological characteristics of the A. japonica eggs were consistent with those of artificially spawned eggs, except they had a slightly larger diameter. The egg diameter range did not overlap with those of mesopelagic eels of the Serrivomeridae, which often spawn in the same area as A. japonica. These results suggest that egg diameter and embryo shape can be used to morphologically identify naturally spawned A. japonica eggs.

The distribution of all larval stages of the Japanese eel, Anguilla japonica, were examined using historical catch records and original data in the western North Pacific (WNP) to evaluate existing information about the larval distribution and migration of this species. A total of 148 preleptocephali, 2547 leptocephali, 6 metamorphosing larvae, and 21 glass eels were collected during 37 cruises over a 52-year period (1956–2007). Sampling effort was spatio-temporally biased in latitude/longitude among seasons with sampling effort being concentrated near the western margin of the subtropical gyre near Taiwan in the winter season and extensive effort occurring near the spawning area to the east near the seamount chain of the West Mariana Ridge in summer during the spawning season. The distribution of preleptocephali (4.2–8.7 mm) was limited to a narrow area around 14°N, 142°E just west of the southern part of the seamount chain, while leptocephali (7.7–62.0 mm) were widely distributed at increasing size westward in the North Equatorial Current (NEC) to the region east of Taiwan. Metamorphosing larvae (52.7–61.2 mm) were collected only in the area 21–26°N, 121–129°E to the east of Taiwan, while glass eels (51.3–61.2 mm) occurred only within or west of the Kuroshio. These distributions suggest that leptocephali begin to metamorphose within or just east of the Kuroshio, then after completion of metamorphosis the glass eels detrain from the current and migrate inshore. The relationship between catch date and body size of leptocephali suggested that the spawning season is from April to August, but further sampling is needed to eliminate possible effects of sampling bias. This analysis is consistent with the existing hypothesis that Japanese eel larvae born near the West Mariana Ridge are transported westward in the NEC and then transfer to the Kuroshio to recruit to East Asia, although more sampling effort is needed for later stage larvae in the NEC bifurcation region to help understand the larval migration in relation to the possible impacts of ocean–atmosphere changes.

The Japanese eel (Anguilla japonica), European eel (Anguilla anguilla), and American eel (Anguilla rostrata) are migratory, catadromous, temperate zone fish sharing several common life cycle features. The population genetics of panmixia in these eel species has already been investigated. Our extensive population genetics analysis was based on 1400 Gb of whole-genome sequence (WGS) data from 84 eels. It demonstrated that a Japanese eel group from the Kuma River differed from other populations of the same species. Even after removing the potential adapted/selected single nucleotide polymorphism (SNP) data, and with very small differences (fixation index [Fst] = 0.01), we obtained results consistently indicating that panmixia does not occur in Japanese eels. The life cycle of the Japanese eel is well-established and the Kuma River is in the center of its habitat. Nevertheless, simple reproductive isolation is not the probable cause of non-panmixia in this species. We propose that the combination of spawning area subdivision, philopatry, and habitat preference/avoidance accounts for the non-panmixia in the Japanese eel population. We named this hypothesis the &ldquo, reproductive isolation like subset mapping&rdquo, (RISM) model. This finding may be indicative of the initial stages of sympatric speciation in these eels.

Summary 1 Extension of life span by food shortage, often mimicked by calorie restriction (CR) in the laboratory, is one of the most common life-history alterations in eukaryotes. Although the life-history of offspring often changes in response to the parental environment, it has remained ambiguous whether or not CR-induced longevity is transmitted to the next generation. 2 Here, we investigated the effects of CR on life span, oxidative stress resistance and the expression levels of two antioxidant enzymes, catalase and manganese superoxide dismutase (Mn SOD), in the parthenogenetic rotifer Brachionus plicatilis during two consecutive generations. 3 Rotifers under CR lived 50% longer than those fed ad libitum (AL) in association with enhancement of oxidative stress resistance and increased mRNA levels of catalase and Mn SOD. 4 The daughters from the CR-treated mothers lived 20% longer than those from the mothers fed AL regardless of food-rich and CR conditions for the daughter. Furthermore, the daughters from the CR-treated mothers were endowed at birth with a higher ability to resist oxidative stress and the increased mRNA levels for catalase, but not for Mn SOD. In agreement with the mRNA expression patterns, CR increased the protein levels of catalase and Mn SOD in eggs and the whole body of mothers, respectively. 5 Our results for these asexually reproducing rotifers provide the first evidence that the longevity and oxidative stress resistance resulting from CR can be passed on to subsequent generations.

Minegishi, Y., Yoshinaga, T., Aoyama, J., and Tsukamoto, K. 2009. Species identification of Anguilla japonica by real-time PCR based on a sequence detection system: a practical application to eggs and larvae. – ICES Journal of Marine Science, 66: 1915–1918. To develop a practical method for identifying Japanese eel Anguilla japonica eggs and larvae to species by a sequence detection system using a real-time polymerase chain reaction (PCR), we examined (i) the sensitivity of the system using samples at various developmental stages, and (ii) influences of intra- and interspecific DNA sequence variations in the PCR target region. PCR amplifications with extracted DNA solution at 7.0 ng µl−1 or lower were efficient at distinguishing A. japonica from other anguillids. A single egg at the gastrula or later developmental stages could also be identified. Two sequence variations in the PCR target region were observed in 2 out of 35 A. japonica collected from three localities, and from four year classes at a single locality. These mutations, however, did not affect the result of species identification achieved by A. japonica-specific PCR primers and probe. The accuracy of this PCR-based method of species identification will help in field surveys of the species.

Extensive collections were made of the larvae of the temperate Japanese eel Anguilla japonica and the tropical giant mottled eel Anguilla marmorata in an overlapping area of the North Equatorial Current region of the western North Pacific Ocean. Collections of 189 A. marmorata and > 2500 A. japonica larvae during nine surveys from 1991 to 2007 showed that these two anguillid eels have similar spawning areas just west of the southern West Mariana Ridge. In July to August 2006 and August 2007, morphologically and genetically identified A. marmorata preleptocephali were mainly collected between 14.5-15 degrees N and 142-142.5 degrees E, where A. japonica preleptocephali were also caught in some of the same net tows. Fewer A. marmorata preleptocephali, however, were collected (n = 31) compared to those of A. japonica (n = c. 165), and fewer small larvae of A. marmorata were collected per tow than A. japonica (n = 1-10 and 1-294, respectively), suggesting relatively smaller spawning aggregations of A. marmorata. The distribution of preleptocephali and small larvae was wider in longitude in A. marmorata (131- 143 degrees E) than in A. japonica (137-143 degrees E), while the latitudinal range was almost the same (12-17 degrees N). Although spawning by these two species overlaps both spatially and temporally, the tropical eels of the North Pacific population of A. marmorata probably have a much longer spawning season with fewer spawners, at least in summer, and recruit to a much wider latitudinal range of growth habitats.

To investigate the effect of oxidative stress on the survival of two Brachionus species of rotifer, we examined the effective dose of juglone, which generates reactive oxygen species, and compared survival times between the two species. First, we observed that juglone affected survival in the rotifers in a dose-dependent manner between 0.02 to 20 μM, and that treatment at above 2 μM showed acute toxicity causing the animals to die within a few hours. Next, we found the difference in survival time between the two species exposed to 20 μM juglone: B. rotundiformis was significantly more tolerant against the substance than the allied species B. plicatilis. The findings suggest that exploring the basis of species-specific abilities to persist under oxidative stress will be of great interest to uncover the underlying mechanisms governing the stress resistance of the rotifer, as well as contributing to their stable mass production for aquaculture.

Changes in expression of gill Na+/K+ -ATPase (NKA) on a short-term (96 h) time-course following hyposmotic shock (direct transfer to fresh water) of the euryhaline, marine milkfish were studied on gene, protein, and cell levels in this paper. Plasma osmolality and [Na+] responded with rapid declines in 3 h post-transfer yet, thereafter, remained constant. Plasma [Cl-] gradually fell to a significantly lower level at 6 h post-transfer. Gills responded to hyposmotic shock by a dual phase enhancement of NKA activity and protein abundance; (a) Before 24 h: NKA activity increased as early as 3 h and reached a maximum level from 6 to 12 h post-transfer coincided with the sustained lower levels of plasma osmolality, [Na+], and [Cl-] since 3 h post-transfer. This was followed by a gradual rise in alpha-subunit protein levels that peaked at 12 h post-transfer. Meanwhile, alpha-mRNA of NKA did no show significant change. (b) After 24 h: NKA activity as well as the amounts of alpha-subunit mRNA and protein increased significantly. Direct freshwater transfer induced a prompt and significant decrease of NKA immunoreactive (NKIR) cell abundance in filaments before 24 h, followed by a significant increase after 24 h due to their development in filaments and lamellae. Increased number of NKIR cells after 24 h of hyposmotic shock may occur in conjunction with rise of NKA activity as well as alpha-subunit mRNA and protein abundance. In conclusion, milkfish is able to avoid an excessive drop in plasma ions immediately upon hyposmotic shock and maintain plasma ions on a marginal lower level in fresh water. Notably, the initial increase in NKA activity (adjustive phase; 3-12 h) and delayed increase in NKA mRNA and protein abundance (regulatory phase; 48-96 h) indicate the importance of a higher level of the gill enzyme in milkfish upon hyposmotic shock.

To compensate for the limited number of morphological characteristics of fish eggs and larvae, we established a convenient and robust method of species identification for eggs of the Japanese eel (Anguilla japonica) using a real-time polymerase chain reaction (PCR) that can be performed onboard research ships at sea. A total of about 1.2 kbp of the mitochondrial 16S ribosomal RNA gene sequences from all species of Anguilla and 3 other anguilliform species were compared to design specific primer pairs and a probe for A. japonica. This real-time PCR amplification was conducted for a total of 44 specimens including A. japonica, A. marmorata, A. bicolor pacifica, and 6 other anguilliform species. Immediate PCR amplification was only observed in A. japonica. We then tested this method under onboard conditions and obtained the same result as had been produced in the laboratory. These results suggest that real-time PCR can be a powerful tool for detecting Japanese eel eggs and newly hatched larvae immediately after onboard sampling during research cruises and will allow targeted sampling efforts to occur rapidly in response to any positive onboard identification of the eggs and larvae of this species.

The rotifer Brachionus plicatilis is a widely-used model for population dynamics studies. During the population growth of B. plicatilis, life history parameters such as reproduction and lifespan change widely, and determine the balance between birth and death rates that regulates the population fluctuations. The lifespan of B. plicatilis was extended 30% by inhibiting a phosphatidylinositol-3-OH kinase involved in an insulin/insulin-like growth factor (IGF) signal transduction pathway that regulates the reproduction and lifespan in nematodes. Subsequently, we cloned a cDNA encoding Mn-superoxide dismutase (SOD), which may function downstream of the IGF pathway. Real-time reverse-transcription polymerase chain reaction analysis revealed that the expression level of Mn-SOD mRNA was higher in B. plicatilis with longer lifespans than those with shorter lifespans. In addition, stress proteins may also influence population dynamics as molecules regulating lifespan and molecular chaperones to maintain the cellular integrity. Accordingly, we cloned two stress protein genes encoding HSP70 and GRP94, and found that their expression changed during the population growth of rotifers. Thus, this novel approach of integrating population ecology and molecular biology has potential use in investigation the detailed mechanisms of rotifer population dynamics.

To examine the life history response and age-specific tolerance to starvation in the rotifer Brachionus plicatilis O.F. Muller, we carried out two series of individual culture experiments. In the first experiment, rotifers were fed until each of the ages of 1–4 days, and were then starved during the rest of their lifetimes. The control group was fed throughout their lifespans. Rotifers stopped active reproduction just after the onset of food deprivation, and showed shorter subsequent survival times when they were starved at older ages. The finding that the larger the number of offspring produced before food deprivation, the shorter the subsequent lifetime under starvation, appeared to reflect a trade-off with the cost of reproduction. In the second experiment, newborns were starved until each of the ages of 1–5 days, and were fed thereafter. The lifespans of the rotifers starved up to the age of 3 days were not statistically different from those that were not starved. Although the starved rotifers began to reproduce once fed again, their lifetime fecundity decreased significantly from that of the non-starved group. Based on these results, it was suggested that the reproductive suppression caused by starvation would cause rotifers to have a longer lifespan to allow for future reproduction.

We amplified three kinds of cDNA fragments encoding stress proteins from the rotifer Brachionus plicatilis by polymerase chain reaction in order to investigate the molecular mechanisms underlying mediation of its lifespan. The stress proteins were heat shock protein 70 (HSP70), glucose regulated protein 94 (GRP94) and ubiquitin-conjugating enzyme, which have been suggested to extend the lifespans of fruitfly and yeast. The isolated clones consisted of 579, 776 and 257 bp in the above order, respectively, and their deduced amino acid sequences showed 81, 59 and 48% identities, respectively, with corresponding sequences from the nematode Caenorhabditis elegans. As rotifers in the stationary phase can extend their lifespan we subsequently performed northern blot analysis on rotifers both in the exponential and stationary phases. The mRNA levels of HSP70 and GRP94 in the exponential growth phase were 2.5 and 1.6 times higher, respectively, than those in the stationary phase, whereas those of ubiquitin-conjugating enzyme did not differ between rotifers in the two phases. These results suggest that stress proteins are not directly responsible for the extension of rotifer lifespan, but another factor(s) is possibly involved. Our results, however, demonstrated that these genes are useful as molecular markers for monitoring the population growth of rotifer.

放射光蛍光X線を用いて, 動物プランクトンの一種であるシオミズツボワムシ (Brachionus plicatilis) 1個体に含まれる微量元素の2次元分析, 定量分析を行った. その結果, 4日齢単性生殖雌の2次元元素分布からは, 胃にFe, Zn, 生殖器にはFe, Zn, Cu, Ca, 頭部にMnが局所的に濃集していることが分かった. これらの元素は, 餌であるクロレラに含まれていることから, 餌を摂取することによりワムシ体内に蓄積されることがICP-MSの定量分析結果からも得られた. 放射光蛍光X線によるMn, Cu, Znの定量分析の結果は, 複数個体を用いて硝酸分解後ICP-MS測定により求めて平均化した値と同じ挙動を示した. 放射光を用いることで, ワムシ1個体の微量元素分析が可能であることが本研究により実証できた.

Background There is an increasing pressure on tropical species of the freshwater eel genus Anguilla for use in aquaculture, so species identification methods are needed to monitor these activities and to facilitate conservation efforts. A method was developed to genetically distinguish two subspecies of the Indonesian short-finned eel, Anguilla bicolor, based on an allelic discrimination technique. Results A single DNA nucleotide substitution in the mitochondrial DNA 16S rRNA gene was employed to identify the two subspecies where Anguilla bicolor bicolor and Anguilla bicolor pacifica possessed adenine and guanine, respectively. This substitution was highly conserved at 100% in A. bicolor bicolor (108/108) and 99.9% in A. bicolor pacifica (181/182), and the misidentification rate was estimated to be 0.34%. Subsequently, fluorescent-labeled oligo probes and PCR primers were designed and succeeded to clearly distinguish the two subspecies. Further, the other ten anguillid species that may be sympatrically distributed with A. bicolor showed negative results. Conclusions The method developed in this study is useful to accurately identify the two subspecies of A. bicolor and can contribute to ecological studies, stock management, and conservation.

To estimate the changes in the life history of the rotifer Brachionus plicatilis O.F. Müller under starvation, we carried out an individual culture and determined the effects of periodical food deprivation on its asexual reproductive characteristics such as lifespan, reproductive period, age at first egg and offspring production, and lifetime fecundity (total number of offspring produced in her lifetime). Rotifers were fed for 1-3 h daily, and were then starved until the next day. Control animals were fed throughout their lifespan. Starved rotifers matured and produced their first offspring at an older age than the control animals. The periodical starvation resulted in a decrease in the lifetime fecundity to less than half that of the non-starved control. The reproductive period and lifespan were 2-3 times longer in the starved animals than in the control animals. The negative relationship between lifespan and lifetime fecundity is interpreted as a trade-off in an alternative life-history strategy of rotifers under starved conditions. The great decrease in fecundity and extension of lifespan enables rotifers to compensate to keep the population in equilibrium.