Showing papers in "Marine Biotechnology in 2014"
TL;DR: The expansion and high diversity in gene families related to innate immunity, point to a complex defense system in the lophotrochozoan C. virginica, probably in adaptation to a pathogen-rich environment.
Abstract: As a benthic filter-feeder of estuaries, the eastern oyster, Crassostrea virginica, faces tremendous exposure to microbial pathogens. How eastern oysters without adaptive immunity survive in pathogen-rich environments is of fundamental interest, but studies on its immune system are hindered by the lack of genomic resources. We sequenced the transcriptome of an adult oyster with short Illumina reads and assembled 66,229 contigs with a N50 length of 1,503 bp. The assembly covered 89.4 % of published ESTs and 97.9 % of mitochondrial genes demonstrating its quality. A set of 39,978 contigs and unigenes (>300 bp) were identified and annotated by searching public databases. Analysis of the gene set yielded a diverse set of 657 genes related to innate immunity, including many pertaining to pattern recognition, effectors, signal transduction, cytokines, and apoptosis. Gene families encoding C1q domain containing proteins, CTLD, IAPs, Ig_I-set, and TRAFs expanded in C. virginica and Crassostrea gigas. Many key genes of the apoptosis system including IAP, BAX, BAC-2, caspase, FADD, and TNFR were identified, suggesting C. virginica posses advanced apoptosis and apoptosis-regulating systems. Our results show that short Illumina reads can produce transcriptomes of highly polymorphic genomes with coverage and integrity comparable to that from longer 454 reads. The expansion and high diversity in gene families related to innate immunity, point to a complex defense system in the lophotrochozoan C. virginica, probably in adaptation to a pathogen-rich environment.
135 citations
TL;DR: High biomass-producing strains that also have high lipid and high EPS yield may be better candidates for commercial production of biofuels and other coproducts.
Abstract: Heterotrophic growth of thraustochytrids has potential in coproducing biodiesel for transportation, as well as producing a feedstock for omega-3 long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), especially docosahexaenoic acid (DHA) for use in nutraceuticals In this study, we compared eight new endemic Australian thraustochytrid strains from the genera Aurantiochytrium, Schizochytrium, Thraustochytrium, and Ulkenia for the synthesis of exopolysaccharide (EPS), in addition to biodiesel and LC-PUFA Aurantiochytrium sp strains readily utilized glucose for biomass production, and increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased biomass yield by an average factor of 17 Ulkenia sp strain TC 010 and Thraustochytrium sp strain TC 033 did not utilize glucose, while Schizochytrium sp strain TC 002 utilized less than half the glucose available by day 14, and Thraustochytrium sp strain TC 004 utilized glucose at 4 % w/v but not 2 % w/v of the culture suggesting a threshold requirement between these values Across all strains, increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased total fatty acid methyl ester content by an average factor of 19 Despite an increasing literature demonstrating the capacity of thraustochytrids for DHA synthesis, the production of EPS from these organisms is not well documented A broad range of EPS yields was observed The maximum yield of EPS was observed for Schizochytrium sp strain TC 002 (299 mg/L) High biomass-producing strains that also have high lipid and high EPS yield may be better candidates for commercial production of biofuels and other coproducts
105 citations
TL;DR: It is revealed that the SbpAPX gene is a potential candidate, which not only confers abiotic stress tolerance to plants but also seems to be involved in plant growth.
Abstract: Salicornia brachiata Roxb., an extreme halophyte, is a naturally adapted higher plant model for additional gene resources to engineer salt tolerance in plants. Ascorbate peroxidase (APX) plays a key role in protecting plants against oxidative stress and thus confers abiotic stress tolerance. A full-length SbpAPX cDNA, encoding peroxisomal ascorbate peroxidase, was cloned from S. brachiata. The open reading frame encodes for a polypeptide of 287 amino acid residues (31.3-kDa protein). The deduced amino acid sequence of the SbpAPX gene showed characteristic peroxisomal targeting sequences (RKRAI) and a C-terminal hydrophobic region of 39 amino acid residues containing a transmembrane domain (TMD) of 23 amino acid residues. Northern blot analysis showed elevated SbpAPX transcript in response to salt, cold, abscisic acid and salicylic acid stress treatments. The SbpAPX gene was transformed to tobacco for their functional validation under stresses. Transgenic plants over-expressing SbpAPX gene showed enhanced salt and drought stress tolerance compared to wild-type plants. Transgenic plants showed enhanced vegetative growth and germination rate both under normal and stressed conditions. Present study revealed that the SbpAPX gene is a potential candidate, which not only confers abiotic stress tolerance to plants but also seems to be involved in plant growth.
93 citations
TL;DR: Findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture.
Abstract: Vibrio parahaemolyticus is a significant cause of gastroenteritis resulting from the consumption of undercooked sea foods and often cause significant infections in shrimp aquaculture. Vibrio virulence is associated with biofilm formation and is regulated by N-acylated homoserine lactone (AHL)-mediated quorum sensing. In an attempt to reduce vibrio colonisation of shrimps and mortality, we screened native intestinal bacilli from Indian white shrimps (Fenneropenaeus indicus) for an isolate which showed biofilm-inhibitory activity (quorum quenching) against the pathogen V. parahaemolyticus DAHP1. The AHL-lactonase (AiiA) expressed by one of these, Bacillus licheniformis DAHB1, was characterised as having a broad-spectrum AHL substrate specificity and intrinsic resistance to the acid conditions of the shrimp intestine. Purified recombinant AiiA inhibited vibrio biofilm development in a cover slip assay and significantly attenuated infection and mortality in shrimps reared in a recirculation aquaculture system. Investigation of intestinal samples also showed that AiiA treatment also reduced vibrio viable counts and biofilm development as determined by confocal laser scanning microscopy (CLSM) imaging. These findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture.
88 citations
TL;DR: The present study indicates the immunomodulatory role of the LAB L. lactis D1813 on the kuruma shrimp immune system and supports its potential use as an effective probiotic in shrimp aquaculture.
Abstract: Fifty-one lactic acid bacteria (LAB) strains were isolated and identified based on 16S ribosomal DNA sequence from the intestinal tracts of 142 kuruma shrimps (Marsupenaeus japonicus) collected from Kanmon Strait, Fukuoka and Tachibana Bay, Nagasaki, Japan. Cellular immunomodulatory function of 51 isolated LAB strains was assessed by measuring the level of interferon (IFN)-γ induction in mouse spleen cell culture. The strain Lactococcus lactis D1813 exhibited the highest amount of IFN-γ production and also bactericidal activity and was selected for testing its immunomodulatory role as a probiotic in kuruma shrimp. We also assessed the effect of dietary incorporation of this probiotic on resistance to Vibrio penaeicida infection in the kuruma shrimp. Our results demonstrate that probiotic L. lactis D1813-containing diet-fed (105 cfu g−1) shrimps displayed a significant up-regulation of lysozyme gene expressions in the intestine and hepatopancreas. However, insignificantly higher expression of anti-lipopolysaccharide factor, super oxide dismutase, prophenoloxidase, and toll-like receptor 1 was recorded in the intestine of shrimps fed the probiotic diet. Moreover, significantly increased (P < 0.01) resistance to the bacterial pathogen in term of better post-infection survival (61.7 %) was observed in the shrimps fed with the probiotic-incorporated diet compared with the control diet-fed group (28.3 %). The present study indicates the immunomodulatory role of the LAB L. lactis D1813 on the kuruma shrimp immune system and supports its potential use as an effective probiotic in shrimp aquaculture.
86 citations
TL;DR: The first application of diffusion growth chambers for the isolation of cultivable and previously uncultivated bacteria from sponges is described, indicating that the diffusion growth chamber approach can be successfully applied in a natural, living marine environment such as spongees.
Abstract: Marine sponges contain dense and diverse microbial communities, which are renowned as a source of bioactive metabolites. The biological activities of sponge-microbe natural products span a broad spectrum, from antibacterial and antifungal to antitumor and antiviral applications. However, the potential of sponge-derived compounds has not been fully realized, due largely to the acknowledged “supply issue.” Most bacteria from environmental samples have resisted cultivation on artificial growth media, and cultivation of sponge-associated bacteria has been a major focus in the search for novel marine natural products. One approach to isolate so-called “uncultivable” microorganisms from different environments is the diffusion growth chamber method. Here, we describe the first application of diffusion growth chambers for the isolation of cultivable and previously uncultivated bacteria from sponges. The study was conducted by implanting diffusion growth chambers in the tissue of Rhabdastrella globostellata reef sponges. In total, 255 16S rRNA gene sequences were obtained, with phylogenetic analyses revealing their affiliations with the Alpha- and Gammaproteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. Fifteen sequences represented previously uncultivated bacteria belonging to the Bacteroidetes and Proteobacteria (Alpha and Gamma classes). Our results indicate that the diffusion growth chamber approach can be successfully applied in a natural, living marine environment such as sponges.
67 citations
TL;DR: This study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.
Abstract: Plastids are ideal subcellular hosts for the expression of transgenes and have been successfully used for the production of different biopolymers, therapeutic proteins and industrial enzymes. Phaeodactylum tricornutum is a widely used aquatic feed species. In this study, we focused on developing a high-efficiency plastid expression system for P. tricornutum. In the plastid transformation vector, the site selected for integration was the transcriptionally active intergenic region present between the trnI and trnA genes, located in the IR (inverted repeat) regions of the plastid genome. Initially, a CAT reporter gene (encoding chloramphenicol acetyltransferase) was integrated at this site in the plastid genome. The expression of CAT in the transformed microalgae conferred resistance to the antibiotic chloramphenicol, which enabled growth in the selection media. Overall, the plastid transformation efficiency was found to be approximately one transplastomic colony per 1,000 microalgae cells. Subsequently, a heterologous gene expression cassette for high-level expression of the target gene was created and cloned between the homologous recombination elements. A TA cloning strategy based on the designed XcmI-XcmI sites could conveniently clone the heterologous gene. An eGFP (green fluorescent protein) reporter gene was used to test the expression level in the plastid system. The relatively high-level expression of eGFP without codon optimisation in stably transformed microalgae was determined to account for 0.12 % of the total soluble protein. Thus, this study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.
67 citations
TL;DR: The identification of genomic regions associated with grilsing or late sexual maturation provide an opportunity to incorporate this information into selective breeding programs that will enhance Atlantic salmon farming.
Abstract: In Atlantic salmon aquaculture, early sexual maturation represents a major problem for producers. This is especially true for grilse, which mature after one sea winter before reaching a desirable harvest weight, rather than after two sea winters. Salmon maturing as grilse have a much lower market value than later maturing individuals. For this reason, most companies desire fish that grow fast and mature late. Marker-assisted selection has the potential to improve the efficiency of selection against early maturation and for late sexual maturation; however, studies identifying age of sexual maturation-related genetic markers are lacking for Atlantic salmon. Therefore, we used a 6.5K single-nucleotide polymorphism (SNP) array to genotype five families from the Mainstream Canada broodstock program and search for SNPs associated with early (grilsing) or late sexual maturation. There were 529 SNP loci that were variable across all five families, and this was the set that was used for quantitative trait loci (QTL) analysis. GridQTL identified two chromosomes, Ssa10 and Ssa21, containing QTL related to grilsing. In contrast, only one QTL, on Ssa18, was found linked to late maturation in Atlantic salmon. Our previous work on these five families did not identify genome-wide significant growth-related QTL on Ssa10, Ssa21, or Ssa18. Therefore, taken together, these results suggest that both grilsing and late sexual maturation are controlled independently of one another and also from growth-related traits. The identification of genomic regions associated with grilsing or late sexual maturation provide an opportunity to incorporate this information into selective breeding programs that will enhance Atlantic salmon farming.
60 citations
TL;DR: Nine major QTL on seven chromosomes that were significant or highly significant with moderate to large effects of at least 13 % of the total phenotypic variance for BCWD resistance are identified in this study.
Abstract: Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture We previously detected genetic variation in survival following challenge with Flavobacterium psychrophilum (Fp), the causative agent of BCWD in rainbow trout (Oncorhynchus mykiss) A family-based selection program to improve resistance was initiated in 2005 at the USDA National Center for Cool and Cold Water Aquaculture Select crosses were made in 2007 and 2009 to evaluate family-based disease survival using Fp injection challenges From each putative F2/BC1 family generated in 2009, 200–260 fish were challenged in 4–7 replicates per family Whole genome QTL scans of three F2/BC1 families were conducted with about 270 informative microsatellite loci per family spaced at an average interval size of 6 cM throughout the rainbow trout genome Markers on chromosomes containing QTL were further evaluated in three additional F2/BC1 families The additional F2/BC1 families were sire or dam half-sibs (HS) of the initially genome scanned families Overall, we identified nine major QTL on seven chromosomes that were significant or highly significant with moderate to large effects of at least 13 % of the total phenotypic variance The largest effect QTL for BCWD resistance explaining up to 40 % of the phenotypic variance was detected on chromosome OMY8 in family 2009070 and in the combined dam HS family 2009069–070 The nine major QTL identified in this study are candidates for fine mapping to identify new markers that are tightly linked to disease resistance loci for using in marker assisted selection strategies
59 citations
TL;DR: A large-scale characterization of pm-miRNAs and their potential function in biomineralization, providing a foundation to understand shell formation is demonstrated.
Abstract: MicroRNAs (miRNAs) are short-nucleotide RNA molecules that function as negative regulators of gene expression in various organisms. However, miRNAs of Pinctada martensii have not been reported yet. P. martensii is one of the main species cultured for marine pearl production in China and Japan. In order to obtain the repertoire of miRNAs in P. martensii, we constructed and sequenced small RNA libraries prepared from P. martensii by Solexa deep sequencing technology and got a total of 27,479,838 reads representing 3,176,630 distinct sequences. After removing tRNAs, rRNAs, snRNAs, and snoRNAs, 10,596,306 miRNA reads representing 18,050 distinct miRNA reads were obtained. Based on sequence similarity and hairpin structure prediction, 258 P. martensii miRNAs (pm-miRNA) were identified. Among these pm-miRNAs, 205 were conserved across the species, whereas 53 were specific for P. martensii. The 3' end sequence of U6 snRNA was obtained from P. martensii by 3' rapid amplification of cDNA end PCR reaction and sequence-directed cloning. Eight conserved pm-miRNAs and two novel pm-miRNAs were validated by stem-loop quantitative real-time PCR with U6 snRNA as an internal reference gene. pm-miRNAs and the reported biomineralization-related genes were subjected to target analysis by using target prediction tools. Some of the pm-miRNAs, such as miR-2305 and miR-0046, were predicted to participate in biomineralization by regulating the biomineralization-related genes. Thus, this study demonstrated a large-scale characterization of pm-miRNAs and their potential function in biomineralization, providing a foundation to understand shell formation.
57 citations
TL;DR: This enhanced transcriptome has detected several transcripts that encode proteins with sequence similarity with previously described shell biomineral proteins including Shematrins and lysine-rich matrix proteins (KRMPs) not previously found in Mytilus.
Abstract: The common blue mussel, Mytilus edulis, has a bimineralic shell composed of approximately equal proportions of the two major polymorphs of calcium carbonate: calcite and aragonite. The exquisite biological control of polymorph production is the focus of research interest in terms of understanding the details of biomineralisation and the proteins involved in the process of complex shell formation. Recent advances in ease and availability of pyrosequencing and assembly have resulted in a sharp increase in transcriptome data for invertebrate biominerals. We have applied Roche 454 pyrosequencing technology to profile the transcriptome for the mantle tissue of the bivalve M. edulis. A comparison was made between the results of several assembly programs: Roche Newbler assembler versions 2.3, 2.5.2 and 2.6 and MIRA 3.2.1 and 3.4.0. The Newbler and MIRA assemblies were subsequently merged using the CAP3 assembler to give a higher consensus in alignments and a more accurate estimate of the true size of the M. edulis transcriptome. Comparison sequence searches show that the mantle transcripts for M. edulis encode putative proteins exhibiting sequence similarities with previously characterised shell proteins of other species of Mytilus, the Bivalvia Pinctada and haliotid gastropods. Importantly, this enhanced transcriptome has detected several transcripts that encode proteins with sequence similarity with previously described shell biomineral proteins including Shematrins and lysine-rich matrix proteins (KRMPs) not previously found in Mytilus.
TL;DR: It is suggested that fucosterol is a promising botanical agent to protect against skin photodamage by modulating AP-1 and TGF-β1 signaling and that MMP-1 activation is correlated with IL-6.
Abstract: Exposure to ultraviolet (UV) light causes matrix metalloproteinase (MMP) overexpression and extracellular matrix depletion, leading to skin photoaging. The activation of MMP is related to increased interlukin-6 (IL-6) and type I procollagen production, which is regulated by transforming growth factor-β1 (TGF-β1). Activator protein-1 (AP-1) activation induces MMP-1 production and reduces type I procollagen secretion. Fucosterol, which is extracted and purified from the brown algae Hizikia fusiformis, is a phytosterol. We assessed the effects of fucosterol on photodamage and investigated its molecular mechanism of action in UVB-irradiated normal human dermal fibroblasts by using enzyme-linked immunosorbent assay, Western blot analysis, and reverse transcription-polymerase chain reaction. Our results showed that fucosterol significantly decreased the UVB-induced expression of MMP-1, IL-6, p-c-Jun, and p-c-Fos. Additionally, fucosterol markedly increased the UVB-induced production of type I procollagen and TGF-β1. Our results indicate that fucosterol regulates MMP-1 and type I procollagen expression by modulating AP-1 and TGF-β1 signaling and that MMP-1 activation is correlated with IL-6. These data suggest that fucosterol is a promising botanical agent to protect against skin photodamage.
TL;DR: Dynamic but moderate changes in the expression of a broad range of immune-relevant features implying the gill’s involvement in pathogen defense strategies are uncovered.
Abstract: The fish gills represent a crucial organ for the communication with the aquatic environment. Transcriptional changes in gills of two hatchery rainbow trout strains in response to injection with the potent pathogen Aeromonas salmonicida were detected by global gene expression profiling using a 4×44K oligonucleotide microarray. Emphasis was placed on “day 3 postinfection” representing a decisive time point for the resolution of inflammation. The comparison of features and pathways differentially regulated in branchial tissues revealed that the local breeding strain BORN and imported American rainbow trout apply common and specific immune strategies. In gills of infected BORN trout, we observed a dynamic regulation of genes controlling NF-κB pathways and the induction of factors promoting the development of myeloid cells, whereas an increased expression of lysozyme and immunoglobulin genes was obvious in gills of infected import trout. In order to prove the relevance of the array-predicted candidates as well as well-known immune genes for gill immunity, a subsequent in vitro experiment was conducted. Altogether, we uncovered dynamic but moderate changes in the expression of a broad range of immune-relevant features implying the gill’s involvement in pathogen defense strategies.
TL;DR: Mapping of QTL for contents of different fatty acids is the first step towards improving the omega-3 content in the fillets of fish by using marker-assisted selection and is important for understanding the biology of fatty acid deposition.
Abstract: Omega-3 fatty acids are essential fatty acids for human health. Therefore, increasing both percentage of omega-3 and a better fatty acid profile in fish fillets is one of the breeding goals in aquaculture. However, it is difficult to increase the omega-3 content in fish fillets, as the phenotypic selection of these traits is not easily feasible. To facilitate the genetic improvement of the Asian seabass for optimal fatty acid profiles, a genome-wide scan for quantitative trait loci (QTL) affecting fatty acid level in the flesh of the Asian seabass was performed on an F2 family containing 314 offspring. All family members were genotyped using 123 informative microsatellites and 22 SNPs. High percentages of n-3 polyunsaturated fatty acids (PUFA), especially C22:6 (DHA 16.48 ± 3.09 %) and C20:5 (EPA 7.19 ± 0.86 %) were detected in the flesh. One significant and 54 suggestive QTL for different fatty acids and a water content trait were detected on the whole genome. QTL for C18:0b was located on linkage groups (LG) 5. QTL for total n-3 PUFA content in flesh were mapped onto LG6 and LG23 with the phenotypic variance explained ranging from 3.8 to 6.3 %. Four QTL for C22:6 were detected on LG6, LG23, and LG24, explaining 3.9 to 4.9 % of the phenotypic variance, respectively. Mapping of QTL for contents of different fatty acids is the first step towards improving the omega-3 content in the fillets of fish by using marker-assisted selection and is important for understanding the biology of fatty acid deposition.
TL;DR: The common QTL may be a major candidate region for disease resistance against V. anguillarum infection in Japanese flounder and explain more than 60 % of the phenotypic variance.
Abstract: A recent genetic linkage map was employed to detect quantitative trait loci (QTLs) associated with Vibrio anguillarum resistance in Japanese flounder. An F1 family established and challenged with V. anguillarum in 2009 was used for QTL mapping. Of the 221 simple sequence repeat (SSR) markers used to detect polymorphisms in the parents of F1, 170 were confirmed to be polymorphic. The average distance between the markers was 10.6 cM. Equal amounts of genomic DNA from 15 fry that died early and from 15 survivors were pooled separately to constitute susceptible bulk and resistance bulk DNA. Bulked segregant analysis and QTL mapping were combined to detect candidate SSR markers and regions associated with the disease. A genome scan identified four polymorphic SSR markers, two of which were significantly different between susceptible and resistance bulk (P=0.008). These two markers were located in linkage group (LG) 7; therefore, all the SSR markers in LG7 were genotyped in all the challenged fry by single marker analysis. Using two different models, 11-17 SSR markers were detected with different levels of significance. To confirm the associations of these markers with the disease, composite interval mapping was employed to genotype all the challenged individuals. One and three QTLs, which explained more than 60 % of the phenotypic variance, were detected by the two models. Two of the QTLs were located at 48.6 cM. The common QTL may therefore be a major candidate region for disease resistance against V. anguillarum infection.
TL;DR: Transgenic plants showed better seed germination and growth characteristics in both hyperionic and hyperosmotic stresses, and transgenic plants exhibited higher water content, membrane stability and less electrolyte leakage in stress conditions.
Abstract: Dehydration-responsive element binding (DREB) transcription factor (TF) plays a key role for abiotic stress tolerance in plants. Earlier, we have published the isolation and characterisation of an A-2-type SbDREB2A TF from an extreme halophyte Salicornia brachiata. The SbDREB2A protein lacks potential proline (P), glutamic acid (E), serine (S) and threonine (T) (PEST) sequence which is known to act as signal peptide for protein degradation. In this study, SbDREB2A TF was over-expressed in tobacco plants without any modification in polypeptide sequence. Transgenic plants showed better seed germination and growth characteristics in both hyperionic and hyperosmotic stresses. Transgenic plants exhibited higher water content, membrane stability and less electrolyte leakage in stress conditions. The transgenic plants accumulated less Na(+) and higher K(+) than wildtype (WT) plants. The transgenic plants revealed higher chlorophyll content, water use efficiency (WUE) and net photosynthesis rate. Transgenics exhibited higher level of proline and low amount of MDA and H2O2 under stress conditions. The real-time PCR of transgenics showed higher expression of downstream heat shock genes (Hsp18, Hsp26 and Hsp70), TFs (AP2 domain containing TF, HSF2 and ZFP), signalling components (PLC3 and Ca (2+) /calmodulin) and dehydrins (ERD10B, ERD10D and LEA5) under different abiotic stress treatments.
TL;DR: A genome scan to detect quantitative trait loci affecting resistance and survival to viral haemorrhagic septicaemia (VHS) and candidate genes related to disease resistance to improve turbot production are identified.
Abstract: One of the main objectives of genetic breeding programs in turbot industry is to reduce disease-related mortality. In the present study, a genome scan to detect quantitative trait loci (QTL) affecting resistance and survival to viral haemorrhagic septicaemia (VHS) was carried out. Three full-sib families with approximately 90 individuals each were genotyped and evaluated by linear regression and maximum likelihood approaches. In addition, a comparison between QTL detected for resistance and survival time to other important bacterial and parasite diseases affecting turbot (furunculosis and scuticociliatosis) was also carried out. Finally, the relationship between QTL affecting resistance/survival time to the virus and growth-related QTL was also evaluated. Several genomic regions controlling resistance and survival time to VHS were detected. Also significant associations between the evaluated traits and genotypes at particular markers were identified, explaining up to 14 % of the phenotypic variance. Several genomic regions controlling general and specific resistance to different diseases in turbot were detected. A preliminary gene mining approach identified candidate genes related to general or specific immunity. This information will be valuable to develop marker-assisted selection programs and to discover candidate genes related to disease resistance to improve turbot production.
TL;DR: Results show that targeted gene disruption using ZFN is efficient in rainbow trout but depends on the ZFN design, and it is concluded that L43 seems dispensable for SdY function.
Abstract: Gene targeting is a powerful tool for analyzing gene function. Recently, new technology for gene targeting using engineered zinc-finger nucleases (ZFNs) has been described in fish species. However, it has not yet been widely used for cold water and slow developing species, such as Salmonidae. Here, we present the results of successful ZFN-mediated disruption of the sex-determining gene sdY (sexually dimorphic on the Y chromosome) in rainbow trout (Oncorhynchus mykiss). Three pairs of ZFN mRNA targeted to different regions of the sdY gene were injected into fertilized rainbow trout eggs. Sperm from 1-year-old male founders (parental generation one or P1) carrying a ZFN-induced mutation in their germline were then used to produce F1 non-mosaic animals. In these F1 populations, we characterized 14 different mutations in the sdY gene, including one mutation leading to the deletion of leucine 43 (L43) and 13 mutations at other target sites that had different effects on the SdY protein, i.e., amino acid insertions, deletions, and frameshift mutations producing premature stop codons in the mRNA. The gonadal phenotype analysis of the F1-mutated animals revealed that the single L43 amino acid deletion did not lead to a male-to-female sex reversal, but all other mutations induced a clear ovarian phenotype. These results show that targeted gene disruption using ZFN is efficient in rainbow trout but depends on the ZFN design. We also characterized new sdY mutations resulting in male-to-female sex reversal, and we conclude that L43 seems dispensable for SdY function.
TL;DR: The inhibition of marine biofouling by the bromotyrosine derivative ianthelline, isolated from the Arctic marine sponge Stryphnus fortis, is described and shown to inhibit both marine micro- and macrofoulers with a pronounced effect on marine bacteria.
Abstract: The inhibition of marine biofouling by the bromotyrosine derivative ianthelline, isolated from the Arctic marine sponge Stryphnus fortis, is described. All major stages of the fouling process are investigated. The effect of ianthelline on adhesion and growth of marine bacteria and microalgae is tested to investigate its influence on the initial microfouling process comparing with the known marine antifoulant barettin as a reference. Macrofouling is studied via barnacle (Balanus improvisus) settlement assays and blue mussel (Mytilus edulis) phenoloxidase inhibition. Ianthelline is shown to inhibit both marine micro- and macrofoulers with a pronounced effect on marine bacteria (minimum inhibitory concentration (MIC) values 0.1–10 μg/mL) and barnacle larval settlement (IC50 = 3.0 μg/mL). Moderate effects are recorded on M. edulis (IC50 = 45.2 μg/mL) and microalgae, where growth is more affected than surface adhesion. The effect of ianthelline is also investigated against human pathogenic bacteria. Ianthelline displayed low micromolar MIC values against several bacterial strains, both Gram positive and Gram negative, down to 2.5 μg/mL. In summary, the effect of ianthelline on 20 different representative marine antifouling organisms and seven human pathogenic bacterial strains is presented.
TL;DR: It appears that photoperiod, lifestyle, and posttranscriptional regulation are all important drivers of RBCL expression in this ecologically important dinoflagellate.
Abstract: Although the importance of anthozoan-dinoflagellate (genus Symbiodinium) endosymbioses in the establishment of coral reef ecosystems is evident, little is known about the molecular regulation of photosynthesis in the intra-gastrodermal symbiont communities, particularly with respect to the rate-limiting Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). In this study, we analyzed rubisco mRNA (rbcL) and protein (RBCL) concentrations over the diel cycle in both cultured and endosymbiotic Symbiodinium samples. In the former, rbcL expression increased upon illumination and decreased during the dark, a pattern that was upheld under continual dark incubation. A different trend in rbcL expression was observed in endosymbiotic Symbiodinium residing within sea anemone (Aiptasia pulchella) tissues, in which illumination gradually led to decreased rbcL mRNA expression. Unexpectedly, RBCL protein expression did not vary over time within anemone tissues, and in neither cultured nor endosymbiotic samples was a correlation between gene and protein expression documented. It appears, then, that photoperiod, lifestyle, and posttranscriptional regulation are all important drivers of RBCL expression in this ecologically important dinoflagellate.
TL;DR: The first comprehensive transcriptome dataset for the genus Echinolittorina is reported, indicating that there were overrepresented amount of genes enriched in a broad spectrum of biological processes and pathways, including those associated with cytoskeleton organization, developmental regulation, signaling transduction, infection, and cardiac function.
Abstract: Echinolittorina snails inhabit the upper intertidal rocky shore and face strong selection pressures from thermal extremes and fluctuations. Revealing the molecular processes of adaptive significance is greatly obstructed by the scarcity of genomic resource for these taxa. Here, we reported the first comprehensive transcriptome dataset for the genus Echinolittorina. Using Illumina HiSeq 2000 platform, about 52 M and 54 M paired-end clean reads were, respectively, generated for the control and heat-stressed libraries. Totally, 115,211 unique transcript fragments (unigenes) were assembled, with an average length of 453 bp and a N50 size of 492 bp. Approximately one third of the unigenes could be annotated according to their homology matches against the Nr, Swiss-Prot, COG, or KEGG databases, and they were found to represent 23,098 non-redundant genes. Gene expression comparison revealed that 1,267 and 6,663 annotated genes were, respectively, up- and downregulated with at least twofold changes upon heat stress. Gene Ontology and KEGG pathway analyses indicated that there were overrepresented amount of genes enriched in a broad spectrum of biological processes and pathways, including those associated with cytoskeleton organization, developmental regulation, signaling transduction, infection, and cardiac function. In addition, a transcriptome-wide search for polymorphic loci yielded a total of 11,228 simple sequence repeats (SSRs) from 9,938 unigenes and 138,631 single nucleotide polymorphism (SNP) and insertion/deletion (INDEL) sites among 22,770 unigenes. The large number of transcript sequences acquired, the biological pathways identified, and the candidate microsatellite and SNP/INDEL loci discovered in the study will serve as valuable resources for further investigations of genetic differentiation and thermal adaptation among populations.
TL;DR: Results show that mitochondria are among the first responders to environmental and nutritional stress stimuli in gilthead sea bream, and functional phenotyping of this cellular organelle is highly promising to obtain reliable markers of growth performance and well-being in this fish species.
Abstract: The effects of nutrient availability on the transcriptome of cardiac and skeletal muscle tissues were assessed in juvenile gilthead sea bream fed with a standard diet at two feeding levels: (1) full ration size and (2) 70 % satiation followed by a finishing phase at the maintenance ration. Microarray analysis evidenced a characteristic transcriptomic profile for each muscle tissue following changes in oxidative capacity (heart > red skeletal muscle > white skeletal muscle). The transcriptome of heart and secondly that of red skeletal muscle were highly responsive to nutritional changes, whereas that of glycolytic white skeletal muscle showed less ability to respond. The highly expressed and nutritionally regulated genes of heart were mainly related to signal transduction and transcriptional regulation. In contrast, those of white muscle were enriched in gene ontology (GO) terms related to proteolysis and protein ubiquitination. Microarray meta-analysis using the bioinformatic tool Fish and Chips ( http://fishandchips.genouest.org/index.php ) showed the close association of a representative cluster of white skeletal muscle with some of cardiac and red skeletal muscle, and many GO terms related to mitochondrial function appeared to be common links between them. A second round of cluster comparisons revealed that mitochondria-related GOs also linked differentially expressed genes of heart with those of liver from cortisol-treated gilthead sea bream. These results show that mitochondria are among the first responders to environmental and nutritional stress stimuli in gilthead sea bream, and functional phenotyping of this cellular organelle is highly promising to obtain reliable markers of growth performance and well-being in this fish species.
TL;DR: The results demonstrate that a biofilm-based PBR that minimizes hydrodynamic shear forces is applicable to technical-scale cultivation of dinoflagellates and may foster biotechnological applications of these abundant marine protists.
Abstract: Products from phototrophic dinoflagellates such as toxins or pigments are potentially important for applications in the biomedical sciences, especially in drug development. However, the technical cultivation of these organisms is often problematic due to their sensitivity to hydrodynamic (shear) stress that is a characteristic of suspension-based closed photobioreactors (PBRs). It is thus often thought that most species of dinoflagellates are non-cultivable at a technical scale. Recent advances in the development of biofilm PBRs that rely on immobilization of microalgae may hold potential to circumvent this major technical problem in dinoflagellate cultivation. In the present study, the dinoflagellate Symbiodinium voratum was grown immobilized on a Twin-Layer PBR for isolation of the carotenoid peridinin, an anti-cancerogenic compound. Biomass productivities ranged from 1.0 to 11.0 g m−2 day−1 dry matter per vertical growth surface and a maximal biomass yield of 114.5 g m−2, depending on light intensity, supplementary CO2, and type of substrate (paper or polycarbonate membrane) used. Compared to a suspension culture, the performance of the Twin-Layer PBRs exhibited significantly higher growth rates and maximal biomass yield. In the Twin-Layer PBR a maximal peridinin productivity of 24 mg m−2 day−1 was determined at a light intensity of 74 μmol m−2 s−1, although the highest peridinin content per dry weight (1.7 % w/w) was attained at lower light intensities. The results demonstrate that a biofilm-based PBR that minimizes hydrodynamic shear forces is applicable to technical-scale cultivation of dinoflagellates and may foster biotechnological applications of these abundant marine protists.
TL;DR: A genetic linkage map of the large yellow croaker was constructed using type II microsatellite markers and expressed sequence tag (EST)-derived microsatellites in two half-sib families to identify quantitative trait locis (QTLs) that can be applied in marker-assisted selection programs to improve the growth traits.
Abstract: Large yellow croaker (Larimichthys crocea) is an important maricultured species in China. A genetic linkage map of the large yellow croaker was constructed using type II microsatellites and expressed sequence tag (EST)-derived microsatellites in two half-sib families (two females and one male). A total of 289 microsatellite markers (contained 93 EST-SSRs) were integrated into 24 linkage groups, which agreed with the haploid chromosome number. The map spanned a length of 1,430.8 cm with an average interval of 5.4 cm, covering 83.9 % of the estimated genome size (1,704.8 cm). A total of seven quantitative trait locis (QTLs) were detected for growth traits on five linkage groups, including two 1 % and five 5 % chromosome-wide significant QTLs, and explained from 2.33 to 5.31 % of the trait variation. The identified QTLs can be applied in marker-assisted selection programs to improve the growth traits.
TL;DR: Long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible in the Nile tilapia Oreochromis niloticus.
Abstract: Germ cell transplantation offers promising applications in finfish aquaculture and the preservation of endangered species. Here, we describe an intraperitoneal spermatogonia transplantation procedure in the Nile tilapia Oreochromis niloticus. Through histological analysis of early gonad development, we first determined the best suitable stage at which exogenous germ cells should be transplanted into the recipients. For the transplantation procedure, donor testes from a transgenic Nile tilapia strain carrying the medaka β-actin/enhanced green fluorescent protein (EGFP) gene were subjected to enzymatic dissociation. These testicular cells were then stained with PKH26 and microinjected into the peritoneal cavity of the recipient fish. To confirm colonization of the donor-derived germ cells, the recipient gonads were examined by fluorescent and confocal microscopy. PKH26-labeled cells exhibiting typical spermatogonial morphology were incorporated into the recipient gonads and were not rejected within 22 days posttransplantation. Long-term survival of transgenic donor-derived germ cells was then verified in the gonads of 5-month-old recipients and in the milt and vitelogenic oocytes of 1-year-old recipients, by means of PCR using EGFP-specific primers. EGFP-positive milt from adult male recipients was used to fertilize non-transgenic oocytes and produced transgenic offspring expressing the donor-derived phenotype. These results imply that long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible. Upon further improvements in the transplantation efficiency, this intraperitoneal transplantation system could become a valuable tool in the conservation of genetic resources for cichlid species.
TL;DR: This is the first report of hemocyanin cDNA (FCHc) cloned from Fenneropenaeus chinensis and recombinant expression of two C-terminal fragments and an inhibition assay showed that FCHc-C1 and FCHC-C2 were anionic AMPs with antifungal and antibacterial activities.
Abstract: Peptides derived from shrimp hemocyanin have antimicrobial properties. This is the first report of hemocyanin cDNA (FCHc) cloned from Fenneropenaeus chinensis and recombinant expression of two C-terminal fragments. Based on sequence analysis of Fenneropenaeus chinensis hemocyanin FCHc, we subcloned two FCHc fragments by designing special primers. Two antimicrobial peptides (AMPs) were derived from FCHc (FCHc-C1 and FCHc-C2). The recombinant sequence of FCHc-C1 consisted of 207 bp encoding 69 amino acids and the recombinant sequence of FCHc-C2 consisted of 120 bp encoding 40 amino acids. The results of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that recombinant FCHc-C1 and FCHc-C2 peptides (rFCHc-C1 and rFCHc-C2) were expressed successfully. An inhibition assay showed that FCHc-C1 and FCHc-C2 were anionic AMPs with antifungal and antibacterial activities.
TL;DR: While fatty acid and biomass productivity may be inseparable in macroalgae, cultivation conditions have a significant carryover effect in the post-harvest delivery of high-quality bio-oils, and there was an interactive effect of stocking density and drying technique.
Abstract: Biomass productivity was quantified for the marine macroalga Derbesia tenuissima cultivated outdoors at seven stocking densities from 0.25 to 8 g L−1 for 5 weeks. Total lipids and fatty acid quantity and quality was measured from samples that were freeze-dried, dried by oven (75 °C), food dehydrator (60 °C), or outdoor in the sun (40 °C) or shade (38 °C). Stocking densities of 0.25 to 2 g L−1 yielded the highest biomass productivities (>20 g dry weight m−2 day−1) with no effect on total lipid quantity (11 %), or fatty acid quantity (5.3 %) or quality at any density tested. However, there was an interactive effect of stocking density and drying technique, with a decrease of up to 40 % in polyunsaturated fatty acids in sun-dried compared to freeze-dried biomass. Notably, while fatty acid and biomass productivity may be inseparable in macroalgae, cultivation conditions have a significant carryover effect in the post-harvest delivery of high-quality bio-oils.
TL;DR: The results demonstrate that application of transgenic technology of humanized fat1 and fat2 in farmed fishes can largely improve the n-3 LC-PUFA production.
Abstract: Omega-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential nutrients for human health. However, vertebrates, including humans, have lost the abilities to synthesize EPA and DHA de novo, majorly due to the genetic absence of delta-12 desaturase and omega-3 desaturase genes. Fishes, especially those naturally growing marine fish, are major dietary source of EPA and DHA. Because of the severe decline of marine fishery and the decrease in n-3 LC-PUFA content of farmed fishes, it is highly necessary to develop alternative sources of n-3 LC-PUFA. In the present study, we utilized transgenic technology to generate n-3 LC-PUFA-rich fish by using zebrafish as an animal model. Firstly, fat1 was proved to function efficiently in fish culture cells, which showed an effective conversion of n-6 PUFA to n-3 PUFA with the n-6/n-3 ratio that decreased from 7.7 to 1.1. Secondly, expression of fat1 in transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.8- and 2.4-fold, respectively. Third, co-expression of fat2, a fish codon-optimized delta-12 desaturase gene, and fat1 in fish culture cell significantly promoted n-3 PUFA synthesis with the decreased n-6/n-3 ratio from 7.7 to 0.7. Finally, co-expression of fat1 and fat2 in double transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.7- and 2.8-fold, respectively. Overall, we generated two types of transgenic zebrafish rich in endogenous n-3 LC-PUFA, fat1 transgenic zebrafish and fat1/fat2 double transgenic zebrafish. Our results demonstrate that application of transgenic technology of humanized fat1 and fat2 in farmed fishes can largely improve the n-3 LC-PUFA production.
TL;DR: The potential benefits of increasing dietary VK levels in larval diets are highlighted, and new insights on the mechanisms mediating the positive effects observed on larval performance and skeletal development are brought.
Abstract: Nutritional factors strongly influence fish larval development and skeletogenesis, and may induce skeletal deformities. Vitamin K (VK) has been largely disregarded in aquaculture nutrition, despite its important roles in bone metabolism, in γ-carboxylation of Gla proteins, and in regulating gene expression through the pregnane X receptor (Pxr). Since the mechanisms mediating VK effects over skeletal development are poorly known, we investigated the effects of VK-supplementation on skeletal development in Senegalese sole larvae, aiming to identify molecular pathways involved. Larvae were fed live preys enriched with graded levels of phylloquinone (PK) (0, 50, and 250 mg kg−1) and survival rate, growth, VK contents, calcium content and incidence of skeletal deformities were determined, revealing an improvement of larval performance and decreasing the incidence of deformities in VK-supplemented groups. Comparative proteome analysis revealed a number of differentially expressed proteins between Control and Diet 250 associated with key biological processes including skin, muscle, and bone development. Expression analysis showed that genes encoding proteins related to the VK cycle (ggcx, vkor), VK nuclear receptor (pxr), and VK-dependent proteins (VKDPs; oc1 and grp), were differentially expressed. This study highlights the potential benefits of increasing dietary VK levels in larval diets, and brings new insights on the mechanisms mediating the positive effects observed on larval performance and skeletal development.
TL;DR: Results of the current studies confirmed the possibility of producing disease-resistant homozygous rainbow trout strains by transgenesis of cecropin P1 or CF-17 gene and followed by androgenesis.
Abstract: Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit anti-bacterial and anti-viral characteristics in aquaculture important fish species, we produced transgenic rainbow trout expressing cecropin P1 or a synthetic cecropin B analog, CF-17, transgene by sperm-mediated gene transfer method. About 30 % of fish recovered from electroporation were shown to carry the transgene as determined by polymerase chain reaction (PCR) amplification assay. Positive P1 transgenic fish were crossed to non-transgenic fish to establish F1 transgenic founder families, and subsequently generating F2, and F3 progeny. Expression of cecropin P1 and CF-17 transgenes was detected in transgenic fish by reverse transcription (RT)-PCR analysis. The distribution of body sizes among F1 transgenic fish were not significantly different from those of non-transgenic fish. Results of challenge studies revealed that many families of F2 and F3 transgenic fish exhibited resistance to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). All-male homozygous cecropin P1 transgenic families were produced by androgenesis from sperm of F3 heterozygous transgenic fish in one generation. The resistant characteristic to A. salmonicida was confirmed in progeny derived from the outcross of all-male fish to non-transgenic females. Results of our current studies confirmed the possibility of producing disease-resistant homozygous rainbow trout strains by transgenesis of cecropin P1 or CF-17 gene and followed by androgenesis.