Molecular Cloning and Expression Analysis of the Myostatin Gene in Sea Perch (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>)

Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. However, few reports are available about the structure and function of MSTN in teleost. Here, the MSTN gene was cloned from sea perch (Lateolabrax japonicus) by homology cloning and genomic walking. In the 4873-bp genomic sequence, three exons, two introns, and 5′ and 3′ flanking sequences were identified. The sea perch MSTN gene encodes a 374-amino acid protein, including a signal peptide, conserved cysteine residues, and a RXXR proteolytic cleavage domain. Expression analysis of MSTN revealed that MSTN was highly expressed in eyes, brain, and muscle; intermediately in intestine; and weakly in gill, spleen, liver, and heart. It was demonstrated that MSTN mRNA was highly expressed in embryonic stem cell line (LJES1), but it was undetectable in several types of somatic cell lines from sea perch, including fibroblast-like cell, epithelioid cell, and lymphocyte-like cell. Further, it was demonstrated that the 5′ flanking region of the MSTN gene can drive the expression of green fluorescent protein (GFP) reporter gene in LJES1 cells and transgenic zebrafish (Danio rerio). This is the first report on the expression profile of MSTN gene in various types of cell cultures.     

Enhanced hyperplasia in muscles of transgenic zebrafish expressing <Emphasis Type="Italic">Follistatin1</Emphasis>

Myostatin is a member of the transforming growth factor-β (TGF-β) super-family and functions as a negative regulator of muscle growth. Binding of the specific receptor, Activin receptor IIB (Act RIIB), with myostatin or other related TGF-β members, could be inhibited by the activin-binding protein follistatin (Fst) in mammals. Overexpressing Fst in mouse skeletal muscle leads to muscle hypertrophy and hyperplasia. To determine if Fst has similar roles in fish, we generated transgenic zebrafish expressing high levels of zebrafish Fst1 using the promoter of the zebrafish skeletal muscle-specific gene, myosin, light polypeptide 2, skeletal muscle (Mylz2). Independent transgenic zebrafish lines exhibited elevated expression levels of myogenic regulatory genes MyoD and Pax7 in muscle cells. Adult Fst1 overexpressing transgenic zebrafish exhibited a slight body weight increase. The high level of Fst1 expression dramatically increased myofiber numbers in skeletal muscle, without significantly changing the fiber size. Our findings suggest that Fst1 overexpression can promote zebrafish muscle growth by enhancing myofiber hyperplasia.     

A Functional Study of Transforming Growth Factor-Beta from the Gonad of Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The transforming growth factor (TGF)-β superfamily is a group of important growth factors involved in multiple processes such as differentiation, cell proliferation, apoptosis and cellular growth. In the Pacific oyster Crassostrea gigas, the oyster gonadal (og) TGF-β gene was recently characterized through genome-wide expression profiling of oyster lines selected to be resistant or susceptible to summer mortality. Og TGF-β appeared specifically expressed in the gonad to reach a maximum when gonads are fully mature, which singularly contrasts with the pleiotropic roles commonly ascribed to most TGF-β family members. The function of og TGF-β protein in oysters is unknown, and defining its role remains challenging. In this study, we develop a rapid bacterial production system to obtain recombinant og TGF-β protein, and we demonstrate that og TGF-β is processed by furin to a mature form of the protein. This mature form can be detected in vivo in the gonad. Functional inhibition of mature og TGF-β in the gonad was conducted by inactivation of the protein using injection of antibodies. We show that inhibition of og TGF-β function tends to reduce gonadic area. We conclude that mature og TGF-β probably functions as an activator of germ cells development in oyster.     

Molecular characterization of growth differentiation factor 9 and its spatio-temporal expression pattern in gibel carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>)

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor β (TGF-β) superfamily with a key role in regulating follicle development. In this study, the GDF9 full-length genomic DNA and cDNA were isolated and characterized from the gibel carp ovary using rapid-amplification of cDNA ends (RACE) and LD-PCR. The full-length genomic DNA and cDNA sequences of GDF9 are 3979 and 2044 bp which code 428 amino acid residues with a specific RKKR protease cleavage site of TGF-β superfamily. Sequence analysis showed that gibel carp was similar to zebrafish and other fish species. Spatio-temporal expression analysis using real-time quantitative PCR revealed that GDF9 mRNA was largely expressed in ovary and testis. GDF9 is mainly present at stage I follicles indicating its important role in early follicles development. The same result was obtained in immunohistochemistry localization of GDF9 protein. Within the follicle, the follicle layer cells were barely expressed whereas GDF9 mRNA was mostly expressed in the oocytes. Supplemented with human chorionic gonadotropin (hCG) in isolated follicles, the expression of GDF9 mRNA was increased firstly and then decreased. The results of this study indicated that GDF9 gene played a role in fish during development of follicles, especially in the early stage follicles.     

<Emphasis Type="Italic">Aeromonas salmonicida</Emphasis> infected fish transfer disease to healthy fish <Emphasis Type="Italic">via</Emphasis> water

Experimental studies of infection transmission via water from infected to healthy fish were conducted. The dark-brown bacterial colonies typical for Aeromonas salmonicida on tryptone soya agar (TSA) have been isolated and counted (from 3.0±0.6×10² to 3.5±0.5×10⁵ c.f.u. g⁻¹) from the internal organs of naturally infected (NI) and experimentally infected (EI) perch and sea trout. No significant differences in dark-brown bacterial counts were detected between EI perch and EI sea trout. The assessment and comparison of the alterations of the biological parameters of EI European perch and sea trout with bacterium Aeromonas salmonicida subsp. salmonicida with naturally infected perch were conducted. No mortality was recorded in groups of EI perch and sea trout. Whereas, the mortality of NI perch (collected from the main sites of outbreak of disease) was observed from the second day of the experiments. Changes in morphophysiological parameters of EI perch and sea trout were similar. Different alterations in blood cell parameters of EI fish were observed, and the most noticeable was the decrease (P≤0.01) in white blood cell count (WBC) of EI perch and sea trout. Based on these results it can be deduced that there is infection transmission of bacterium A. salmonicida from European perch via water to other fish species.     

Molecular cloning and daily variations of the <Emphasis Type="Italic">Period</Emphasis> gene in a reef fish <Emphasis Type="Italic">Siganus guttatus</Emphasis>

As the first step in understanding the molecular oscillation of the circa rhythms in the golden rabbitfish Siganus guttatus—a reef fish with a definite lunar-related rhythmicity—we cloned and sequenced a Period gene (rfPer). The rfPer gene contained an open reading frame that encodes a protein consisting of 1,452 amino acids; this protein is highly homologous to PER proteins of vertebrates including zebrafish. Phylogenetic analyses indicated that the rfPER protein is related to the zebrafish PER1 and PER4. The expression of rfPer mRNA in the whole brain, retina, and liver under light/dark (LD) conditions increased at 06:00 h and decreased at 18:00 h, suggesting that its robust circadian rhythm occurs in neural and peripheral tissues. When daily variation in the expression in rfPer mRNA in the whole brain and cultured pineal gland were examined under LD conditions, similar expression patterns of the gene were observed with an increase around dawn. Under constant light condition, the increased expression of rfPer mRNA in the whole brain disappeared around dawn. The present results demonstrate that rfPer is related to zPer4 and possibly zPer1. The present study is the first report on the Period gene from a marine fish.     

Identification and expression of amphioxus <Emphasis Type="Italic">AmphiSmad1/5/8</Emphasis> and <Emphasis Type="Italic">AmphiSmad4</Emphasis>

Smad family proteins are identified as intracellular signal mediators of the TGF-β superfamily. In this study, we identified two novel members of the Smad family, termed as AmphiSmad1/5/8 and AmphiSmad4, from Chinese amphioxus. Both AmphiSmad1/5/8 and AmphiSmad4 showed a typical domain structure of Smad proteins consisting of conserved MH1 and MH2 domains. Phylogenetic analysis placed AmphiSmad1/5/8 in the Smad1, 5 and 8 subgroup of the R-Smad subfamily, and AmphiSmad4 in the Co-Smad subfamily. The spatial and temporal gene expression patterns of AmphiSmad1/5/8 and AmphiSmad4 showed that they may be involved in the embryonic development of notochord, myotome and alimentary canal, and may help to establish the specification of dorsal-ventral axis of amphioxus. Moreover, AmphiSmad1/5/8 and AmphiSmad4 showed extensive distribution in all adult tissues examined, suggesting that these two genes may play important roles in the morphogenesis of a variety of tissues especially notochord and gonad.     

Observations of a wild polar bear (<Emphasis Type="Italic">Ursus maritimus</Emphasis>) successfully fishing Arctic charr (<Emphasis Type="Italic">Salvelinus alpinus</Emphasis>) and Fourhorn sculpin (<Emphasis Type="Italic">Myoxocephalus quadricornis</Emphasis>)

Polar bears, Ursus maritimus, throughout their range, are nutritionally dependent on ringed (Phoca hispida) and bearded seals (Erignathus barbatus), which are predominantly caught on the sea ice. Other marine prey species are caught and consumed, but less frequently. As the annual sea ice retreats, polar bears throughout their range are forced ashore, where they mostly live off their stored adipose tissue. However, while land-bound they have been observed catching birds and terrestrial mammals. Although polar bears evolved from brown bears (U. arctos), direct observations of polar bears diving for and catching fish have not been reported. Here, we document observations of a young male polar bear catching Arctic charr (Salvelinus alpinus) and Fourhorn sculpin (Myoxocephalus quadricornis) by diving in Creswell Bay, Nunavut. We recorded six search bouts, where six fish were caught during dives, which were preceded by a snorkel. The average dive and snorkel length was (mean ± SD) 13 ± 5 and 6 ± 2 s, respectively.     

Production of Bioactive Rockfish (<Emphasis Type="Italic">Sebastes schlegeli</Emphasis>) Myostatin-1 Prodomain in an <Emphasis Type="Italic">Escherichia coli</Emphasis> System

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth in mammalian species, and its activity is inhibited by MSTN prodomain, the N-terminal part of proMSTN cleaved during post-translational MSTN processing. In fish, MSTN also appears to suppress fish muscle growth with its activity being inhibited by prodomain. The objective of this study was to produce bioactive MSTN-1 prodomain of rockfish (S. schlegeli), a commercial aquaculture species in East Asia, in E. coli using maltose binding protein (MBP) as a fusion partner. Rockfish MSTN-1 prodomain (sMSTN1pro) cDNA was cloned into the pMALc2x vector, and proteins (MBP-sMSTN1pro) were expressed in Rosetta-gami 2(DE3)pLysS cells by IPTG induction. The MBP-sMSTN1pro was expressed in soluble forms, and affinity purified using amylose resin. The affinity purified MBP-sMSTN1pro suppressed MSTN activity in vitro. The results suggest that MBP is probably a useful fusion partner in producing bioactive MSTN prodomains of various animal species in E. coli.     

Life history responses of <Emphasis Type="Italic">Daphnia longispina</Emphasis> to mosquitofish (<Emphasis Type="Italic">Gambusia holbrooki</Emphasis>) and pumpkinseed (<Emphasis Type="Italic">Lepomis gibbosus</Emphasis>) kairomones

In the present study, the effect of chemical cues from two fish species (mosquitofish and pumpkinseed), at different concentrations, was tested in life history experiments with Daphnia longispina. The two fish species used represent the most abundant planktivores of many Mediterranean shallow lakes (SW Europe), where the indigenous fish communities have been replaced by such exotic assemblages. Results have shown a similar response of D. longispina to both fish species: kairomones stimulated daphnids to produce more offspring, which resulted in higher fitness (r), relatively to a fishless control. Fish presence also induced an earlier first reproduction, a smaller size at maturity of daphnids, and the production of smaller-sized neonates. Significant correlations with fish concentration (indirect measure of fish kairomone concentration) were found for size at maturity and neonate size, for both fish species. These results are in accordance to the “positive response” observed by other authors, which represents a defence mechanism to face losses caused by fish predators. The chemically mediated size reduction of mature females and neonates is an adaptive response to the size-selective predation exerted by fish. Pumpkinseed introduction is very recent in the lake of origin of the daphnids used in the experiments and its kairomone produced similar effects to mosquitofish in the life history of D. longispina. These results are contrary to the existence of a species-specific kairomone and support the hypothesis of a general fish kairomone. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

Molecular and cellular detection of expression of vitellogenin and <Emphasis Type="Italic">zona radiata</Emphasis> protein in liver and skin of juvenile salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) exposed to nonylphenol

In developing bioassays for estrogenic effects, vitellogenin (Vtg) induction and zona radiata protein (Zr-protein) induction in males and juveniles of oviparous vertebrates have been used as sensitive biomarkers for estrogenicity. Nonylphenol (NP) produces similar and parallel expression patterns of Vtg and Zr-protein levels in plasma and surface mucus of salmon, the response being concentration- and time-dependent. We have explored the potential mechanisms of Vtg and Zr-protein expression in surface mucus by comparative molecular and cellular approaches. Liver, skin, blood, and surface mucus samples were collected from fish exposed to a single waterborne concentration of NP (10 and 60 μg/l), 3, 7, and 10 days post-exposure, for gene expression analysis (liver and skin; quantitative real-time polymerase chain reaction) and protein analysis (blood and surface mucus; enzyme-linked immunosorbent assay). Protein expression was localized by immunohistochemistry. NP produced concentration- and time-dependent increases of hepatic estrogen receptors (ERα and ERβ), Vtg, and Zr-protein mRNA and plasma protein levels. These responses paralleled cellular detection of Vtg and Zr-protein in the liver with unique expression patterns in the cytoplasm of hepatocytes, hepatic sinusoids, and endothelial cells. ERα, Vtg, and Zr-protein mRNA were detectable in the skin. ERβ was the only skin response that was NP-concentration-dependent, especially at day 10 post-exposure. Immunohistochemistry for Vtg and Zr-protein in skin showed unique expression patterns in mucus vacuoles, epidermal cells, and scales in an NP-concentration- and time-specific manner. Thus, analysis of skin mRNA levels for xenoestrogen biomarker responses is a less-promising approach than protein analysis. The immunohistochemical localization of Vtg and Zr-protein levels in the skin further validates surface mucus as a sensitive biomarker source for estrogenic compounds. These responses represent an improvement for the detection of endocrine-disrupting compounds and related pollutants in the environment. The Norwegian Research Council (NFR) financially supported this study.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

<Emphasis Type="Italic">Decapentaplegic</Emphasis> (<Emphasis Type="Italic">dpp</Emphasis>) regulates the growth of a morphological novelty,beetle horns

Studies focusing on the development of morphological novelties suggest that patterning genes underlying traditional appendage development (i.e. mouthparts, legs, and wings) also play important roles in patterning novel morphological structures. In this study, we examine whether the expression and function of a member of the TGF-β signaling pathway, decapentaplegic (dpp), promotes development of a morphologically novel structure: beetle horns. Beetle horns are complex secondary sexual structures that develop in the head and/or prothorax, lack obvious homology to other insect outgrowths, and vary remarkably between species and sexes. We studied dpp expression through in situ hybridization, performed functional analyses with RNA interference, and gathered allometric measurements to determine the role of dpp during both pronotal and head horn development in both sexes of two morphologically dissimilar species in the Onthophagus genus, Onthophagus binodis and Onthophagus sagittarius. Our findings show that in addition to affecting growth and patterning of traditional appendages, dpp regulates beetle horn growth and remodeling.     

BAHD superfamily of acyl-CoA dependent acyltransferases in <Emphasis Type="Italic">Populus</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis>: bioinformatics and gene expression

Plant acyl-CoA dependent acyltransferases constitute a large specific protein superfamily, named BAHD. Using the conserved sequence motifs of BAHD members, we searched the genome sequences of Populus and Arabidopsis, and identified, respectively, 94- and 61-putative genes. Subsequently, we analyzed the phylogeny, gene structure, and chromosomal distribution of BAHD members of both species; then, we profiled expression patterns of BAHD genes by “in silico” northern- and microarray-analyses based on public databases, and by RT-PCR. While our genomic- and bioinformatic- analyses provided full sets of BAHD superfamily genes, and cleaned up a few existing annotation errors, importantly it led to our recognizing several unique Arabidopsis BAHD genes that inversely overlapped with their neighboring genes on the genome, and disclosing a potential natural anti-sense regulation for gene expressions. Systemic gene-expression profiling of BAHD members revealed distinct tissue-specific/preferential expression patterns, indicating their diverse biological functions. Our study affords a strong knowledge base for understanding BAHD members’ evolutionary relationships and gene functions implicated in plant growth, development and metabolism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning and expression of <Emphasis Type="Italic">Tenebrio molitor</Emphasis> antifreeze protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded β-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.     

Authentic seed-specific activity of the <Emphasis Type="Italic">Perilla</Emphasis> oleosin 19 gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.