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 ...

The University of Tokyo

University of Houston-Victoria

the university of tokyo

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.

We cloned a full-length cDNA encoding vitellogenin (VTG) from a marine teleost, the Japanese sillago Sillago japonica. The cloned sillago VTG contained signal peptide, lipovitellin heavy chain, phosvitin, lipovitellin light chain, and beta'-component in the order from the N-terminus. An exposure to 17beta-estradiol significantly increased the levels of plasma VTG, but not hepatic VTG mRNA in males. Neither plasma VTG nor hepatic VTG mRNA levels were affected by the exposure to 4-tert-octylphenol. Hepatic VTG mRNA levels in males increased at 1 day after intraperitoneal administration of 17beta-estradiol but decreased in the subsequent 5 days. However, plasma VTG levels remained high for 5 days after administration, suggesting that the accumulation period of plasma VTG is longer than that of hepatic VTG mRNA in males. Therefore, VTG mRNA may be a suitable indicator of temporal exposure to estrogenic chemicals in the environment, whereas plasma VTG is useful to detect consecutive exposure.

Molecular characterization of Japanese sillago vitellogenin and changes in its expression levels on exposure to 17beta-estradiol and 4-tert-octylphenol.

A monogonont rotifer Brachionus plicatilis has been widely used as a model organism for physiological, ecological studies and for ecotoxicology. Because of the availability of parthenogenetic mode of reproduction as well as its versatility to be used as live food in aquaculture, the population dynamic studies using the rotifer have become more important and acquired the priority over those using other species. Although many studies have been conducted to identify environmental factors that influence rotifer populations, the molecular mechanisms involved still remain to be elucidated. In this study, gene(s) differentially expressed by calorie restriction in the rotifer was analyzed, where a calorie-restricted group was fed 3 h day(-1) and a well-fed group fed ad libitum. A subtracted cDNA library from the calorie-restricted rotifer was constructed using suppression subtractive hybridization (SSH). One hundred sixty-three expressed sequence tags (ESTs) were identified, which included 109 putative genes with a high identity to known genes in the publicly available database as well as 54 unknown ESTs. After assembling, a total of 38 different genes were obtained among 109 ESTs. Further validation of expression by semi-quantitative reverse transcription-PCR showed that 29 out of the 38 genes obtained by SSH were up regulated by calorie restriction.

Identification of genes differentially expressed by calorie restriction in the rotifer (Brachionus plicatilis).

Although energy metabolism in mammals is critically regulated by adipokines from adipocytes, it is unclear whether this is the case in fish as well. In this study, over 30,000 expressed sequence tags (ESTs) were obtained from adipose tissue in rainbow trout Oncorhynchus mykiss peritoneal cavity and searched for genes possibly related to lipid metabolism. Large numbers of ESTs encoded digestive enzymes and hormones usually found in the pancreas in higher vertebrates, consistent with the fact that pancreatic cells are dispersed in the adipose tissue. Many ESTs encoded apolipoprotein C-I, fatty acid-binding proteins and lymphocyte G0/G1 switch protein 2, which function in lipid transport, fatty acid accumulation and adipocyte differentiation, respectively. None of the ESTs encoded adipokines. We therefore obtained a cDNA encoding adiponectin, an adipokine that regulates oxidation of glucose and lipids in peripheral tissues, using rainbow trout ESTs in the public database. Real-time RT-PCR analyses revealed that its transcript levels were high in muscle and quite low in adipose tissue. These results strongly suggest that adipocytes of rainbow trout and possibly other fish species, unlike those of mammals, are not involved in the production of adipokines.

EST analysis on adipose tissue of rainbow trout Oncorhynchus mykiss and tissue distribution of adiponectin.

Glutamine synthetase (GS) has been described as one of the oldest functioning genes and thus a good molecular clock protein. GS is diverged into three distinct forms, type I (GSI), type II (GSII) and type III (GSIII), the last type of which is a member of the most recently discovered family among GSs and thus has been reported from a limited number of prokaryotes. In the present study, we determined the full-length sequence of GSIII from the marine diatom Chaetoceroscompressum. The 3' untranslated region of the diatom GSIII gene was composed of a polyadenylation signal followed by a poly (A)(+) tail, clearly demonstrating that its mRNA is transcribed from the eukaryotic genome. We also screened available genome databases and identified full-length GSIII sequences from 5 eukaryotic species. These eukaryotic GSIIIs specifically contained regions A-D and a long additional sequence flanking region V toward the C-terminal site, both being specific to GSIII. Phylogenic analysis revealed that eukaryotic GSIIIs are not within a monophyletic relationship with the possible occurrence of lateral gene transfer in GSIII during evolution.

The occurrence of eukaryotic type III glutamine synthetase in the marine diatom Chaetoceros compressum.

Insulin-like growth factors (IGFs) are well known to play essential roles in enhancement of myogenic differentiation. In this report we showed that initial IGF-I signal activation but long-term IGF-1 signal termination are required for myogenic differentiation. L6 myoblast stably transfected with myc-epitope tagged insulin receptor substrate-1, myc-IRS-1 (L6-mIRS1) was unable to differentiate into myotubes, indicating that IRS-1 constitutive expression inhibited myogenesis. To elucidate the molecular mechanisms underlying myogenic inhibition, IGF-I signaling was examined. IGF-I treatment of control L6 cells for 18 h resulted in a marked suppression of IGF-I stimulated IRS-1 association with the p85 PI 3-kinase and suppression of activation of Akt that correlated with a down regulation of IRS-1 protein. L6-mIRS1 cells, in contrast, had sustained high levels of IRS-1 protein following 18 h of IGF-I treatment with persistent p85 PI 3-kinase association with IRS-1, Akt phosphorylation and phosphorylation of the downstream Akt substrate, Foxo1. Consistent with Foxo1 phosphorylation, Foxo1 protein was excluded from the nuclei in L6-mIRS1 cells, whereas Foxo1 was localized in the nuclei in control L6 cells during induction of differentiation. In addition, L6 cells stably expressing a dominant-interfering form of Foxo1, Δ256Foxo1 (L6-Δ256Foxo1) were unable to differentiate into myotubes. Together, these data demonstrate that IGF-I regulation of Foxo1 nuclear localization is essential for the myogenic program in L6 cells but that persistent activation of IGF-1 signaling pathways results in a negative feedback to prevent myogenesis.

Constitutive expression of insulin receptor substrate (IRS)-1 inhibits myogenic differentiation through nuclear exclusion of Foxo1 in L6 myoblasts.

Lipid content is one of the major determinants of the meat quality in fish. However, the mechanisms underlying the species-specific distribution of lipid are still poorly understood. The present study was undertaken to investigate the mechanisms associated with lipid accumulation in two species of fish: torafugu (a puffer fish) and red seabream. The lipid content of liver and carcass were 67.0% and 0.8% for torafugu, respectively, and 8.8% and 7.3% for red seabream, respectively. Visceral adipose tissue was only apparent in the red seabream and accounted for 73.3% of its total lipid content. Oil red O staining confirmed this species-specific lipid distribution, and further demonstrated that the lipid in the skeletal muscle of the red seabream was mainly localized in the myosepta. We subsequently cloned cDNAs from torafugu encoding lipoprotein lipase 1 (LPL1) and LPL2, important enzymes for the uptake of lipids from blood circulation system into various tissues. The relative mRNA levels of peroxisome proliferator-activated receptor gamma (PPARγ) and the LPLs of torafugu were determined by quantitative real-time PCR together with their counterparts in red seabream previously reported. The relative mRNA levels of PPARγ and LPL1 correlated closely to the lipid distribution of both fish, being significantly higher in liver than skeletal muscle in torafugu, whereas the highest in the adipose tissue, followed by liver and skeletal muscle in red seabream. However, the relative mRNA levels of LPL2 were tenfold lower than LPL1 in both species and only correlated to lipid distribution in torafugu, suggesting that LPL2 has only a minor role in lipid accumulation. In situ hybridization revealed that the transcripts of LPL1 co-localized with lipids in the adipocytes located along the myosepta of the skeletal muscle of red seabream. These results suggest that the transcriptional regulation of PPARγ and LPL1 is responsible for the species-specific lipid distribution of torafugu and red seabream.

Differences in lipid distribution and expression of peroxisome proliferator-activated receptor gamma and lipoprotein lipase genes in torafugu and red seabream.

Pufferfish accumulate tetrodotoxin (TTX) at high levels in liver and ovary through the food chain. However, the mechanisms underlying TTX toxification in pufferfish have been poorly understood. In order to search gene candidates involved in TTX accumulation in the torafugu pufferfish Takifugu rubripes, a custom 4x44k oligonucleotide microarray slide was designed by the Agilent eArray program using oligonucleotide probes of 60 bp in length referring to 42,724 predicted transcripts in the publicly available Fugu genome database. DNA microarray analysis was performed with total RNA samples from the livers of two toxic wild specimens in comparison with those from a nontoxic wild specimen and two nontoxic cultured specimens. The mRNA levels of 1108 transcripts were more than 2-fold higher in the toxic specimens than in the nontoxic specimens. The levels of 613 transcripts were remarkably high, and 16 transcripts encoded by 9 genes were up-regulated more than 10-fold. These genes included those encoding forming structural filaments (keratins) and those related to vitamin D metabolism and immunity. It was also noted that the levels of the transcripts encoding serpin peptidase inhibitor clade C member 1, coagulation factor X precursor, complement C2, C3, C5, C8 precursors, and interleukin-6 receptor were high in the toxic liver samples.

DNA microarray analysis on gene candidates possibly related to tetrodotoxin accumulation in pufferfish.

Adipose tissue is a lipid storage organ characterized by the pronounced accumulation of adipocytes. Although adipose tissues are found in various parts of the vertebrate body, it is unclear whether these tissues have a common ancestral origin or have evolved in several phylogenetic lineages by independent adipocyte accumulation events. To gain insight into the evolutionary history of vertebrate adipose tissues, we determined the distribution of adipocytes by oil red O staining in skeletal muscle of 10 teleost species spanning eight orders: Tetraodontiformes, Pleuronectiformes, Spariformes, Salmoniformes, Clupeiformes, Beloniformes, Osmeriformes, and Cypriniformes. Accumulation of adipocytes in the myoseptum was observed in many species, including red seabream, rainbow trout, Pacific herring, Pacific saury, zebrafish and giant danio. We also found some order-, species-, and swimming mode-specific distribution patterns of adipocytes: 1) almost complete absence of intramuscular adipocytes in the order Tetraodontiformes (torafugu and spotted green pufferfish), 2) clear adipocyte accumulation in the inclinator muscles of fin in Japanese flounder, 3) a large intramuscular adipose tissue at the root of the dorsal fin in ayu, and 4) thick lipid layers consisting of subcutaneous adipose tissue and red muscle lipids in pelagic migratory fish (Pacific herring and Pacific saury). Of note, Pacific herring and Pacific saury are phylogenetically distinct species sharing a similar niche and swimming mode, suggesting that their analogous adipocyte/lipid distribution patterns are the consequence of convergent evolution. The potentially heterogeneous origin of adipose tissues has significant implications for the interpretation of their functional diversity.

Diversity of Lipid Distribution in Fish Skeletal Muscle.

Growth hormone (GH) increases phosphorylation and mRNA levels of hormone-sensitive lipase (HSL) in the livers of some marine teleosts. The hepatic GH-HSL axis appears to play important roles in fasting-induced lipolysis. However, it is not known whether GH exerts similar effects on HSL in fish adipose tissues. Functional differentiation of two fish-specific HSL isoforms (HSL1 and HSL2) also remains unclear. The present study seeks to address two unanswered questions about fish lipolysis using red seabream (Pagrus major): 1) Does GH increase phosphorylation and HSL mRNA levels in adipose tissue? 2) How do GH and fasting affect mRNA levels of two HSL isoform genes in the liver and adipose tissue? To this end, we first cloned HSL1 and HSL2 cDNAs and investigated their tissue distribution. Transcripts of both HSLs and HSL1 proteins were abundant in the visceral adipose tissue, gonads, and liver, suggesting its important role in adipose tissue lipolysis. HSL2 transcript levels were 20%-65% those of HSL1 except in the skin, and HSL2 proteins were not detected by our in-house antisera. Ex vivo administration of GH increased HSL phosphorylation, non-esterified fatty acid (NEFA) release, and levels of HSL1 and HSL2 mRNA in both the liver and visceral adipose tissue. Hepatic HSL2 mRNA was particularly sensitive to GH administration and sometimes exceeded HSL1 mRNA levels with up to 13-fold induction. In contrast, fasting for 4 and 7 d increased HSL1 mRNA levels, but had only marginal effects on HSL2 mRNA levels in both adipose tissue or liver. We concluded that GH does increase HSL mRNAs during adipose tissue lipolysis in red seabream; however, GH and fasting result in different induction ratio of two HSL isoform genes, suggesting that other hormone(s) also contributes to fasting-induced lipolysis.

Gen Kaneko

Department of Aquatic Bioscience,
Graduate School of Agricultural and Life Sciences,
Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences

The University of Tokyo

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

Gen Kaneko

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Aquatic Bioscience,Graduate School of Agricultural and Life Sciences,Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences

The University of Tokyo

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

Department of Aquatic Bioscience,
Graduate School of Agricultural and Life Sciences,
Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences