Measurement of Survival Time in <em>Brachionus </em>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 ...

Kitasato University

Kitasato university

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.

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

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.

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

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.

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

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.

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 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.

Oceanic spawning ecology of freshwater eels in the western North 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.

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

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.

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

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.

Tatsuki Yoshinaga

School of Marine Biosciences,
Department of Marine Biosciences,
School of Marine Biosciences, Department of Marine Biosciences

Kitasato University

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

Tatsuki Yoshinaga

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Marine Biosciences,Department of Marine Biosciences,School of Marine Biosciences, Department of Marine Biosciences

Kitasato University

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

School of Marine Biosciences,
Department of Marine Biosciences,
School of Marine Biosciences, Department of Marine Biosciences