Pectoral Fin Anomalies in tbx5a Knockdown Zebrafish Embryos Related to the Cascade Effect of N-Cadherin and Extracellular Matrix Formation.

Functional knockdown of zebrafish tbx5a causes hypoplasia or aplasia of pectoral fins. This study aimed to assess developmental pectoral fin anomalies in tbx5a morpholino knockdown zebrafish embryos. The expression of cartilage-related genes in the tbx5a morphant was analyzed by DNA microarray, immunostaining, and thin-section histology to examine the detailed distribution of the extracellular matrix (ECM) during different pectoral fin developmental stages. Chondrogenic condensation (CC) in the tbx5a morpholino knockdown group was barely recognizable at 37 h postfertilization (hpf); the process from CC to endoskeleton formation was disrupted at 48 hpf, and the endoskeleton was only loosely formed at 72 hpf. Microarrays identified 18 downregulated genes in tbx5a-deficient embryos, including 2 fin morphogenesis-related (cx43, bbs7), 4 fin development-related (hoxc8a, hhip, axin1, msxb), and 12 cartilage development-related (mmp14a, sec23b, tfap2a, slc35b2, dlx5a, dlx1a, tfap2b, fmr1, runx3, cdh2, lect1, acvr2a, mmp14b) genes, at 24 and 30 hpf. The increase in apoptosis-related proteins (BAD and BCL2) in the tbx5a morphant influenced the cellular component of pectoral fins and resulted in chondrocyte reduction throughout the different CC phases. Furthermore, tbx5a knockdown interfered with ECM formation in pectoral fins, affecting glycosaminoglycans, fibronectin, hyaluronic acid (HA), and N-cadherin. Our results provide evidence that the pectoral fin phenotypic anomaly induced by tbx5a knockdown is related to disruption of the mesoderm and ECM, consequently interfering with mesoderm migration, CC, and subsequent endoskeleton formation.

Anti-apoptotic effects of IGF-I on mortality and dysmorphogenesis in tbx5-deficient zebrafish embryos.

BACKGROUND: Tbx5 deficiency in zebrafish causes several abnormal phenotypes of the heart and pectoral fins. It has been reported that exogenous human growth hormone can enhance expression of downstream mediators in the growth hormone and insulin-like growth factor I (IGF-I) pathway and partially restore dysmorphogenesis in tbx5 morphants. This study aimed to further evaluate the effects of IGF-I on cell apoptosis and dysmorphogenesis in zebrafish embryos deficient for tbx5. RESULTS: Among the five studied groups of zebrafish embryos (wild-type embryos [WT], tbx5 morphants [MO], mismatched tbx5 morpholino-treated wild-type embryos [MIS], IGF-I-treated wild-type embryos [WTIGF1], and IGF-I-treated tbx5 morphants [MOIGF1]), the expression levels of the ifg1, igf1-ra, ifg-rb, erk1, and akt2 genes as well as the ERK and AKT proteins were significantly reduced in the MO group, but were partially restored in the MOIGF1 group. These expression levels remained normal in the WT, MIS, and WTIGF1 groups. Exogenous human IGF-I also reduced the incidence of phenotypic anomalies, decreased the expression levels of apoptotic genes and proteins, suppressed cell apoptosis, and improved survival of the MOIGF1 group. CONCLUSIONS: These results suggest that IGF-I has an anti-apoptotic protective effect in zebrafish embryos with tbx5 deficiency.

The complete mitochondrial genome of Hymenocera picta (Malacostraca: Decapoda: Hymenoceridae).

In this study, the complete mitochondrial genome sequence of the Hymenocera picta is reported for the first time. The length of genome is 15,786 bp, including 13 protein-coding genes, two ribosomal RNA genes, and 21 transfer RNA genes. Nucleotide composition of the whole mitogenome was 37.26% A, 28.42% T, 21.92% C, and 12.40% G. The AT and GC skewness of mitogenome sequence was 0.135 and 0.277, respectively. The reconstructed phylogenetic relationships of 22 Decapoda species based on 13 protein-coding genes were highly supported and the clade of all Palaemonoidea shrimps included had a high support value. Our results shall provide a better understanding in the evolutionary histories of the Decapoda.

Complete Genome Sequence of Streptococcus iniae 89353, a Virulent Strain Isolated from Diseased Tilapia in Taiwan.

Streptococcus iniae 89353 is a virulent strain isolated from diseased tilapia in Taiwan. The full-genome sequence of S. iniae 89353 is 2,098,647 bp. The revealed genome information will be beneficial for identification and understanding of potential virulence genes of Streptococcus iniae and possible immunogens for vaccine development against streptococcosis.

The complete mitochondrial genome of Babylonia borneensis (Gastropoda: Neogastropoda: Buccinidae).

The complete mitochondrial genome sequence of the Babylonia borneensis is reported for the first time in this study. The length of genome was 15 556 bp, including 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. The nucleotide composition of the mitogenome showed AT-rich feature, with the AT content of 68.2%. Comparison of the identity of the B. borneensis mitogenome with B. areolata, B. lani and B. lutosa was 87.5%, 87.4% and 86.9%, respectively. The construction of phylogenetic tree showed high bootstrap support value. Babylonia borneensis grouped together with other Babylons and the lineages of Buccinidae was strongly supported. In this study, our results could provide a further understanding in the phylogenetic relationships of the Neogastropoda.

The complete mitochondrial genome of Poecilia formosa (Actinopterygii: Cyprinodontiformes: Poeciliidae).

The complete mitochondrial genome sequence of the Poecilia formosa is first reported in this study. The length of genome is 16 550 bp, including 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. Congeneric mitogenome sequence identity is 87.7% with P. reticulate and 93.1% with P. sphenops. The reconstructed phylogenetic relationships of 16 Cyprinodontiformes species based on 13 protein-coding genes were highly supported and the clade of all Poecilia fishes included had a support value of 100%. Our results shall provide a better understanding in the evolutionary histories of the Cyprinodontiformes.

Inhibition of the P2X7 receptor reduces cystogenesis in PKD.

The P2X7 receptor participates in purinergic signaling, which may promote the progression of ADPKD. We examined the effects of a P2X7 receptor antagonist and a P2X7 receptor agonist on cyst development in a zebrafish model of polycystic kidney disease in which we knocked down pkd2 by morpholinos. We used live wt-1b pronephric-specific GFP-expressing zebrafish embryos to directly observe changes in the pronephros. Exposure of pkd2-morphant zebrafish to a P2X7 receptor antagonist (oxidized ATP [OxATP]) significantly reduced the frequency of the cystic phenotype compared with either exposure to a P2X7 receptor agonist (BzATP) or with no treatment (P < 0.01). Histology confirmed improvement of glomerular cysts in OxATP-treated pkd2 morphants. OxATP also reduced p-ERK activity and cell proliferation in pronephric kidneys in pkd2 morphants. Inhibition of P2X7 with an additional specific antagonist (A-438079), and through morpholino-mediated knockdown of p2rx7, confirmed these effects. In conclusion, blockade of the P2X7 receptor reduces cyst formation via ERK-dependent pathways in a zebrafish model of polycystic kidney disease, suggesting that P2X7 antagonists may have therapeutic potential in ADPKD.

Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish.

Myostatin belongs to the transforming growth factor (TGF)-beta superfamily and is a potent negative regulator of skeletal muscle development and growth. We utilized microinjection of an antisense RNA-expressing vector to establish a hereditarily stable myostatin gene knockdown zebrafish strain with a double-muscle phenotype. Real-time PCR and immunostaining revealed that the myostatin messenger (m)RNA and protein levels in homozygous transgenic zebrafish were 33% and 26% those of the non-transgenic controls, respectively. Also, the mRNA levels of myogenic regulatory factor markers such as MyoD, myogenin, Mrf4, and Myf5 were dramatically elevated in myostatin-suppressed transgenic fish compared to the non-transgenic controls. Although there was no significant difference in body length, homozygous transgenic zebrafish were 45% heavier than non-transgenic controls. Histochemical analysis showed that the cross-sectional area of the muscle fiber of homozygous transgenic fish was twice as large as that of non-transgenic controls. This is the first model zebrafish with a hereditarily stable myostatin-suppressed genotype and a double-muscle phenotype.

Reverse transcription loop-mediated isothermal amplification for rapid and sensitive detection of nervous necrosis virus in groupers.

The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is a sensitive nucleic acid diagnostic method that can amplify rapidly a target template; it can be applied for the diagnosis of viral disease in grouper aquaculture. In this study, two outer and two inner primers were designed from nervous necrosis virus (NNV) coat protein gene sequence. The reaction temperature and time for the detection of NNV were optimized at 65 degrees C for 60min. The detection limit of RT-LAMP is 10(-6) NNV-RNA from infected groupers, and more sensitive than the one-step RT-PCR and nested RT-PCR. The combination of RNA rapid extraction and RT-LAMP, the process can be completed within 2h. Thus, the RT-LMAP is a rapid, sensitive, specific and efficient method for detection of NNV in groupers.

Cascade effect of cardiac myogenesis gene expression during cardiac looping in tbx5 knockdown zebrafish embryos.

Zebrafish tbx5 expresses in the heart, pectoral fins and eyes of zebrafish during embryonic development. In zebrafish, injection of tbx5 morpholino antisense RNA caused changes of heart conformation, defect of heart looping, pericardium effusion, dropsy of ventral position and decreased heart rate. We suggested that cardiac myogenesis genes might be responsible for this phenomenon. Morpholino antisense RNA which against the initiation site of tbx5 gene was designed in order to knockdown the expression of tbx5, and the results were analyzed by whole-mount in situ hybridization and quantitative real-time PCR. Expression of cardiac myogenesis genes amhc, vmhc and cmlc2 were expressed constantly at the early embryonic development and reached its highest rate right before cardiac looping initiated. These cardiac myogenesis genes showed insufficient expressions within different heart defect embryos. Moreover, vmhc showed ectopic expression in addition to heart looping defect in heart defective embryos at 36 hpf. Our data suggests that the heart failure caused by the knockdown of tbx5 gene might result from the down-regulation of cardiac myogenesis genes.

Overexpression of Myostatin2 in zebrafish reduces the expression of dystrophin associated protein complex (DAPC) which leads to muscle dystrophy.

Myostatin, a member of the TGF-beta superfamily, is a potent negative regulator of skeletal muscle and growth. Previously, we reported Mstn1 from zebrafish and studied its influence on muscle development. In this study, we identified another form of Myostatin protein which is referred to as Mstn2. The size of Mstn2 cDNA is 1342 bp with 109 and 132 bp of 5' and 3'-untranslated regions (UTRs), respectively. The coding region is 1101 bp encoding 367 amino acids. The identity between zebrafish Mstn1 and 2 is 66%. The phylogenetic tree revealed that the Mstn2 is an ancestral form of Mstn1. To study the functional aspects, we overexpressed mstn2 and noticed that embryos became less active and the juveniles with bent and curved phenotypes when compared to the control. The RT-PCR and in situ hybridization showed concurrent reduction of dystrophin associated protein complex (DAPC). In cryosection and in situ hybridization, we observed the disintegration of somites, lack of transverse myoseptum and loss of muscle integrity due to the failure of muscle attachment in mstn2 overexpressed embryos. Immunohistochemistry and western blot showed that there was a reduction of dystrophin, dystroglycan and sarcoglycan at translational level in overexpressed embryos. Taken together, these results indicate the suitability of zebrafish as an excellent animal model and our data provide the first in vivo evidence of muscle attachment failure by the overexpression of mstn2 and it leads to muscle loss which results in muscle dystrophy that may contribute to Duchenne syndrome and other muscle related diseases.

Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages.

A penaeidin family, mo-penaeidin was cloned from the haemocytes of tiger shrimp Penaeus monodon using genomic polymerase chain reaction (PCR) by gene specific primers. Analysis of nucleotide sequence revealed that this mo-penaeidin consists of 1348 bp containing one intron (680 bp) and two exons (210 and 458 bp). It has an open reading frame (ORF) of 222 p, which encodes a protein of 74 amino acids including a signal peptide of 19 amino acids. The calculated molecular mass of the mature protein (55 amino acids) is 6.059 kDa with an estimated pI of 9.3. The deduced amino acid sequence of mo-penaeidin has similarity to that of penaeidin from Fenneropenaeus chinensis (73%), Farfantepenaeus paulensis (66%), Litopenaeus schmitti (53-67%), L. stylirostris (50-67%), L. setiferus (50-62%), L. vannamei (44-66%), and Marsupenaeus japonicus (33%), respectively. Phylogenetic tree analysis indicated that penaeidin (including mo-penaeidin, penaeidin, and penaeidin 5, 2, 3k, 3c1) of P. monodon is distinct from penaeidin 1, penaeidin 2, penaeidin 3 and penaeidin 4 of other penaeid shrimps. The mo-penaeidin mRNA was detected in various tissues including ovary and mandibular organ. The mo-penaeidin mRNA was present in one cell to postlarva stage with higher level at nauplius I (9h post hatching) and higher expression during the intermoult stage indicating an early innate immunity and different immunity at moulting stage.

Cloning and characterisation of a prophenoloxidase from the haemocytes of mud crab Scylla serrata.

A prophenoloxidase (proPO) cDNA was cloned from the haemocytes of mud crab Scylla serrata using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has a full length of 2663bp, with an open reading frame of 2019bp, a 124-bp 5'-untranslated region, and a 520-bp 3'-untranslated region containing a poly A signal. It encodes a protein of 673 amino acids with a predicted molecular weight of 77.5kDa and with an estimated pI of 5.96. It contains two putative tyrosinase copper-binding motifs with six histidine residues (copper A, 185, 189, 211, and copper B, 346, 350, 386). The proPO has thiol-ester-like motif (GCGWPQHM), which showed similar structural features of proPOs from other decapod crustaceans. It also contains five possible glycosylation sites, and a conserved C-terminal region common to all known proPOs. Sequence comparison showed that the proPO-deduced amino acid of mud crab S. serrata has an overall similarity of 78%, 57%, 56%, 51-55%, 54%, 53%, 52%, 52%, and 52% to that of Dungeness crab Cancer magister, American lobster Homarus americanus, European lobster Homarus gammarus, kuruma prawn Marsupenaeus japonicus, crayfish Pacifastacus leniusculus, white shrimp Litopenaeus vannamei, tiger shrimp Penaeus monodon, green tiger shrimp Penaeus semisulcatus, and giant freshwater prawn Macrobrachium rosenbergii, respectively. The proPO was strongly expressed in haemocytes, but not in heart, eyestalk, gill, muscle, ovary, hepatopancreas, stomach, and intestine. The proPO transcript of mud crab S. serrata increased significantly in 12 and 24h post-lipopolysaccharide (LPS) injection, but returned to the original values in 72h post injection.

Molecular cloning of myostatin gene and characterization of tissue-specific and developmental stage-specific expression of the gene in orange spotted grouper, Epinephelus coioides.

In this article we report the molecular cloning and characterization of a nonmammalian myostatin (growth and differentiation factor-8, MSTN) homolog from the orange spotted grouper (Epinephelus coioides) by polymerase chain reaction (PCR) cloning. The grouper MSTN gene consists of two introns [Intron I (363 bp) and Intron II (811 bp)] flanked by three exons [Exon I (379 bp), Exon II (371 bp) and Exon III (381 bp)]. A full-length cDNA clone (2608 bp) of the MSTN gene (GenBank DQ493889, nucleotide sequence in the coding region identical to GeneBank AY856860) was also isolated. This cDNA encodes a polypeptide of 376 amino acid residues that showed 25% to 96% homology with MSTNs of molluscan, teleostean, avian, and mammalian species. Phylogenetic analysis of the grouper MSTN polypeptide confirmed the evolutionary relationships of this MSTN with other known MSTNs. Results of reverse transcription (RT)-PCR analysis of the total RNA extracted from different tissues revealed that MSTN gene is expressed not only in the skeletal muscle, but also in other tissues. MSTN mRNA was also detected in different embryonic developmental and larval stages. Because the tissue-specific expression of MSTN gene in grouper is different from that in mammals, it might suggest that MSTN gene may possess additional functions other than regulating muscle growth in fish.

Molecular cloning and characterization of cDNA of penaeidin-like antimicrobial peptide from tiger shrimp (Penaeus monodon).

We report the molecular cloning and characterization of a penaeidin-like antimicrobial peptide (AMP) complementary DNA from the hemocytes of tiger shrimp (Penaeus monodoni). A tiger shrimp AMP cDNA containing the entire coding region of the peptide, determined by rapid amplification of cDNA 5' and 3' ends and polymerase chain reaction of the messenger RNA isolated from hemocytes of the shrimp, is 683 bp in length with an open reading frame of 222 bp. The deduced amino acid sequence of this antimicrobial peptide consists of 55 amino acid residues of the mature peptide and a signal peptide of 19 amino acid residues. The mature peptide contains a proline-rich domain at the N terminus and 6 cysteine residues at the C terminus, and it shares less than 50% amino acid sequence identity with the mature penaeidins of Litopenaeus vannamei. To demonstrate the bactericidal activity of this peptide, a synthetic peptide was prepared according to the amino acid sequence deduced from the cDNA of tiger shrimp penaeidin-like AMP. By the minimal inhibitory concentration MIC assay, the synthetic peptide was shown to exert bactericidal activity against Escherichia coli, Vibrio harveyi, Vibrio alginolyticu, and Aerococcus viridans. It also inhibited the growth of 2 filamentous fungi, Fusarium pisi and Fusarium oxysporum. Tiger shrimp penaeidin-like AMP mRNA was detected in hemocytes, gills, intestines, eyestalks, hepatopancreas, and muscles of the tiger shrimp by reverse transcriptase-polymerase chain reaction assay. Although the highest level was detected in hemocytes, challenging tiger shrimp with V. harveyi did not result in a significant increase of the mRNA level in hemocytes.

Production of transgenic silver sea bream (Sparus sarba) by different gene transfer methods.

We have been interested in developing convenient mass gene transfer methods for producing strains of silver sea bream (Sparus sarba) with superior genetic traits for aquaculture. A transgene construct carrying rainbow trout growth hormone (rtGH) complementary DNA driven by a common carp b-actin promoter was introduced into silver sea bream by electroporating the sperm with the rtGH transgene and using the treated sperm to fertilize eggs stripped from mature females. The presence of the GH transgene in presumptive transgenic individuals was detected by polymerase chain reaction (PCR) analysis. Between 56% and 70% of the animals carried the GH transgene. We refer to this method as sperm-mediated gene transfer (SMGT). Since the handling stress of stripping gametes from female sliver sea bream brood fish could cause severe mortality, an alternative gene transfer method would be highly desirable. We developed a liposome-based method to transfer the GH transgene into the fish. This method, referred as testis-mediated gene transfer (TMGT), involves injecting the liposome-transgene mixture into the gonads of male sea bream at least 48 hours before spawning. The males were mated to reproductively active females, and fertilized eggs were collected for further incubation. Between 59% and 76% of the hatched fry were found by PCR analysis to carry the rtGH transgene. The efficiency of gene transfer was improved more than 80% by injecting multiple doses of the liposome-transgene mixture into the gonads of treated males. Results of Southern blot analysis of DNA isolated from PCR-positive animals showed that the transgene was integrated into the host genome and could be transmitted to its offspring. The rtGH transgene was expressed in many of the rtGH-transgenic fish. Several P1 GH-transgenic silver sea bream exhibited significant growth enhancement compared with nontransgenic controls. Our studies showed that faster-growing silver sea bream could be produced by a variety of mass gene transfer technologies. These gene transfer technologies would be of great value to aquaculture.