Changes in Behavior and Diel Melatonin Secretion Toward Estivation in Western Sand Lance, Ammodytes japonicus (Uranoscopiformes, Ammodytidae).

Western sand lance, Ammodytes japonicus, is known to have an estivation period, in which they cease feeding and stay in the sand from early summer to late autumn, followed by gonadal maturation. During the feeding period prior to estivation, they swim in daytime and spend the night in the sand. Before they start swimming, they show a typical behavior of head-exposing from the sand, which is likely to be related to foraging and predation avoidance. Our previous study revealed that melatonin regulates such diel behavior of this species. To elucidate the mechanisms of behavioral regulation throughout the life cycle of this sand lance, the present study examined the changes in behavior and melatonin secretion toward the estivation period. Both head-exposing and swimming behaviors were frequently observed at the transition period toward estivation. On the other hand, occurrence of these behaviors was suppressed just before entering estivation. Subsequently, it was found that plasma melatonin concentration was about three times higher at night than in daytime in the non-estivation period, while it was retained at high levels throughout the day in the estivation period. These results indicate that diurnal swimming behavior of sand lance from the feeding to estivation periods is associated with the daily cycle of melatonin secretion.

Circadian Rhythm and Endocrinological Control on the Swimming and Sand Burrowing Behaviors of Japanese Sand Lances Ammodytes spp. (Uranoscopiformes, Ammodytidae).

In diurnal and nocturnal organisms, daily activity is regulated by the perception of environmental stimuli and circadian rhythms, which enable organisms to maintain their essential behaviors. The Japanese sand lances genus Ammodytes are coastal marine fish that exhibit unique nocturnal sand burrowing behavior. To elucidate the extrinsic and intrinsic regulation of this behavior and its endocrinological basis, we conducted a series of rearing experiments under various light conditions and hormone administrations. Under a light-dark photoperiod, the fish showed three types of behavior: sand buried, head-exposed from sand, and swimming/feeding. During the transition from dark to light periods, the fish first showed head exposure, followed by swimming and foraging, and buried themselves in the sand immediately after shifting to the dark period. Under constant light conditions, fish exhibited swimming behavior during the period corresponding to the acclimated light period. In addition, swimming did not occur under constant dark conditions but head exposure was observed at the time of the dark-light transition during acclimation. These observations indicate that the essential behavior of sand lances is regulated by both light and circadian rhythms. Subsequently, a melatonin-containing diet promoted the onset of burrowing in 10 to 120 min in a dose-dependent manner at 0.3-128 µg/g-diet, suggesting the direct behavioral regulation by this hormone. These findings suggest that the behavior of sand lances is strictly regulated by an intrinsic mechanism and that melatonin is a regulatory endocrine factor that induces burrowing behavior.

Migration Behavior of Anguilla celebesensis Silver Eels within their Tomini Bay Spawning Area.

The tropical Celebes eel, Anguilla celebesensis, has a short migration between its spawning and growth habitats. Its spawning areas were hypothesized to be in Tomini Bay and the Celebes Sea after collecting their small leptocephali. However, there is no information about the silver eel oceanic spawning migration behavior of A. celebesensis. To better understand their short-distance spawning migration behavior, four large female silver eels (Eel 1-4) were equipped with pop-up satellite archival tags (PSATs) and released near the mouth of the Poso River in Tomini Bay of Sulawesi Island on 22 February (Eel 1-3) and 11 March 2010 (Eel 4). All PSATs ascended in Tomini Bay and transmitted their data. Eel 3 and 4 provided clear records of consistent diel vertical migration (DVM: eight days-Eel 3, 13 days-Eel 4) with daytime dives to mean depths of 444.7 m (Eel 3) and 539.0 m (Eel 4), where mean temperatures were 9.1°C (Eel 3) and 7.7°C (Eel 4), and nighttime ascents to mean depths of 132.8 m (Eel 3) and 112.4 m (Eel 4), where mean temperatures were 20.6°C (Eel 3) and 23.4°C (Eel 4). Eel 3 and 4 started to dive to deeper water around nautical dawn and swam up to shallower water around sunset. During nighttime, both eels swam in deeper and colder water during nights with moonlight than during nights without moonlight, and there was a negative linear relationship between experienced water temperatures with the moon in the sky and the lunar age for the eels. The A. celebesensis daily rhythm of DVM behaviors was similar to spawning-migration DVM behaviors of other anguillid species. Essential life history characteristics of A. celebesensis appear to be a short migration between freshwater growth habitat and ocean spawning habitat, and high GSI values with advanced gonadal development in downstream-migrating silver eels.

A genome-wide screening of the 70 kDa heat shock protein (HSP70) genes in the rotifer Brachionus plicatilis sensu stricto with a characterization of two heat-inducible HSP70 genes.

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 complex evolution of the metazoan HSP70 gene family.

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.

Description of two new species of sandlances, genus Bleekeria (Perciformes, Ammodytidae) from the Andaman Sea (northeastern Indian Ocean).

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.

HaCeD-Seq: a Novel Method for Reliable and Easy Estimation About the Fish Population Using Haplotype Count from eDNA.

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.

Mucosal galectin genes in all freshwater eels of the genus Anguilla.

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 ß-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.

Whole-Genome Sequencing of 84 Japanese Eels Reveals Evidence against Panmixia and Support for Sympatric Speciation.

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 "reproductive isolation like subset mapping" (RISM) model. This finding may be indicative of the initial stages of sympatric speciation in these eels.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions.

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.

Differential expression of skin mucus C-type lectin in two freshwater eel species, Anguilla marmorata and Anguilla japonica.

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.

Spawning sites of the Japanese eel in relation to oceanographic structure and the West Mariana Ridge.

The Japanese eel, Anguilla japonica, spawns within the North Equatorial Current that bifurcates into both northward and southward flows in its westward region, so its spawning location and larval transport dynamics seem important for understanding fluctuations in its recruitment to East Asia. Intensive research efforts determined that Japanese eels spawn along the western side of the West Mariana Ridge during new moon periods, where all oceanic life history stages have been collected, including eggs and spawning adults. However, how the eels decide where to form spawning aggregations is unknown because spawning appears to have occurred at various latitudes. A salinity front formed from tropical rainfall was hypothesized to determine the latitude of its spawning locations, but an exact spawning site was only found once by collecting eggs in May 2009. This study reports on the collections of Japanese eel eggs and preleptocephali during three new moon periods in June 2011 and May and June 2012 at locations indicating that the distribution of lower salinity surface water or salinity fronts influence the latitude of spawning sites along the ridge. A distinct salinity front may concentrate spawning south of the front on the western side of the seamount ridge. It was also suggested that eels may spawn at various latitudes within low-salinity water when the salinity fronts appeared unclear. Eel eggs were distributed within the 150-180 m layer near the top of the thermocline, indicating shallow spawning depths. Using these landmarks for latitude (salinity front), longitude (seamount ridge), and depth (top of the thermocline) to guide the formation of spawning aggregations could facilitate finding mates and help synchronize their spawning.

Offshore spawning of Conger myriaster in the western North Pacific: evidence for convergent migration strategies of anguilliform eels in the Atlantic and Pacific.

The spawning area of the common Japanese conger, Conger myriaster, had remained unknown because spawning adults or its newly hatched larvae were never collected. Using genetic identification, we determined that C. myriaster spawns far offshore in the western North Pacific, just west of the spawning area of the Japanese eel, Anguilla japonica. In June 2008, six newly hatched C. myriaster larvae, 5.6-6.9 mm, were collected at the eastern edge of where many small unidentified Conger leptocephali (7-20 mm) were collected previously. The offshore spawning location of C. myriaster is analogous to that of the American conger eel, Conger oceanicus, and the American eel, Anguilla rostrata, in the Sargasso Sea, suggesting that convergent evolution of large-scale reproductive migration strategies in both anguillid and conger eels has occurred in the north Atlantic and Pacific subtropical gyres. The realization that two anguillids, A. rostrata and A. japonica, and two congers, C. oceanicus and C. myriaster, have evolved almost identical migration strategies in widely separated ocean basins suggests that natural selection for larval survival and recruitment success has resulted in long offshore spawning migrations in two phylogenetically distant taxa of anguilliform eels.

Oceanic spawning ecology of freshwater eels in the western North Pacific.

The natural reproductive ecology of freshwater eels remained a mystery even after some of their offshore spawning areas were discovered approximately 100 years ago. In this study, we investigate the spawning ecology of freshwater eels for the first time using collections of eggs, larvae and spawning-condition adults of two species in their shared spawning area in the Pacific. Ovaries of female Japanese eel and giant mottled eel adults were polycyclic, suggesting that freshwater eels can spawn more than once during a spawning season. The first collection of Japanese eel eggs near the West Mariana Ridge where adults and newly hatched larvae were also caught shows that spawning occurs during new moon periods throughout the spawning season. The depths where adults and newly hatched larvae were captured indicate that spawning occurs in shallower layers of 150-200 m and not at great depths. This type of spawning may reduce predation and facilitate reproductive success.

Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water.

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na(+)/K(+)-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35 per thousand), and hypersaline water (HSW 60 per thousand) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff's reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA alpha subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA alpha and beta subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.

cDNA cloning and expression analysis of thyroid hormone receptor in the coho salmon Oncorhynchus kisutch during smoltification.

Two types of cDNA clones encoding thyroid hormone receptor (TR) genes, TRalpha and TRbeta, were isolated from the coho salmon Oncorhynchus kisutch. The two genes each consists of multiple isoforms, derived from different loci or by an alternative splicing. RT-PCR revealed a broad localization of the TRalpha and TRbeta transcripts that both expressed in brain, gill, liver, muscle, kidney, ovary, skin and eyeball. During the early stages of the smoltification period, the mRNA level of the TR transcripts showed a large individual variability at the maximum of 10-fold. In contrast, as the plasma thyroid hormone level elevated at the middle of the period, the variation in the expression level became much smaller at 2-fold. After the completion of the smoltification, the mRNA accumulation level of the TR genes rapidly decreased. These findings suggest that the dynamics of the receptor (TR) should be taken account in addition to the availability of the hormone ligands (T3 and T4) to understand the underlying mechanisms of the smoltification causing numerous physiological and morphological changes in salmonids.

A quick method for species identification of Japanese eel (Anguilla japonica) using real-time PCR: an onboard application for use during sampling surveys.

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 molecular mechanisms of life history alterations in a rotifer: a novel approach in population dynamics.

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.