Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 30
Filtrar
1.
Fish Shellfish Immunol ; 153: 109840, 2024 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-39153579

RESUMEN

Infectious diseases have significantly impacted Atlantic salmon aquaculture worldwide. Modulating fish immunity with immunostimulant-containing functional feeds could be an effective strategy in mitigating disease problems. Previously, we characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida bacterin on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. A set of miRNA biomarkers of Atlantic salmon head kidney responding to pIC and/or bacterin immune stimulations was identified (Xue et al., 2019) [1]. Herein, we report a complementary qPCR study that investigated the impact of the pIC, bacterin and dietary CpG on the expression of immune-relevant mRNAs (n = 31) using the same samples as in the previous study (Xue et al., 2019) [1]. Twenty-six of these genes were predicted target transcripts of the pIC- and/or bacterin-responsive miRNAs identified in the earlier study. The current data showed that pIC and/or bacterin stimulations significantly modulated the majority of the qPCR-analyzed genes involved in various immune pathways. Some genes responded to both stimulations (e.g. tnfa, il10rb, ifng, irf9, cxcr3, campb) while others appeared to be stimulation specific [e.g. irf3, irf7a, il1r1, mxa, mapk3 (pIC only); clra (bacterin only)]. A. salmonicida bacterin stimulation produced a strong inflammatory response (e.g. higher expression of il1b, il8a and tnfa), while salmon stimulated with pIC showed robust interferon responses (both type I and II). Furthermore, the current data indicated significant down-regulation of immune-relevant transcripts (e.g. tlr9, irf5, il1r1, hsp90ab1, itgb2) by dietary immunostimulant CpG, especially among pre-injection and PBS-injected fish. Together with our prior miRNA study, the present research provided complementary information on Atlantic salmon anti-viral and anti-bacterial immune responses and on how dietary CpG may modulate these responses.


Asunto(s)
Adyuvantes Inmunológicos , Aeromonas salmonicida , Alimentación Animal , Dieta , ARN Mensajero , Salmo salar , Animales , Salmo salar/inmunología , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/administración & dosificación , Alimentación Animal/análisis , Dieta/veterinaria , ARN Mensajero/genética , ARN Mensajero/metabolismo , Aeromonas salmonicida/fisiología , Inmunidad Innata/efectos de los fármacos , Biomarcadores , Enfermedades de los Peces/inmunología , Suplementos Dietéticos/análisis , Oligodesoxirribonucleótidos/farmacología , Oligodesoxirribonucleótidos/administración & dosificación , MicroARNs/genética , Riñón Cefálico/inmunología , Poli I-C/farmacología , Poli I-C/administración & dosificación
2.
Int J Mol Sci ; 25(1)2023 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-38203361

RESUMEN

Micro RNAs (miRNAs) are short non-coding RNAs that act as post-transcriptional gene expression regulators. Genes regulated in vertebrates include those affecting growth and development or stress and immune response. Pikeperch (Sander lucioperca) is a species that is increasingly being considered for farming in recirculation aquaculture systems. We characterized the pikeperch miRNA repertoire to increase the knowledge of the genomic mechanisms affecting performance and health traits by applying small RNA sequencing to different developmental stages and organs. There were 234 conserved and 8 novel miRNA genes belonging to 104 families. A total of 375 unique mature miRNAs were processed from these genes. Many mature miRNAs showed high relative abundances or were significantly more expressed at early developmental stages, like the miR-10 and miR-430 family, let-7, the miRNA clusters 106-25-93, and 17-19-92. Several miRNAs associated with immune responses (e.g., slu-mir-731-5p, slu-mir-2188-5p, and slu-mir-8159-5p) were enriched in the spleen. The mature miRNAs slu-mir-203a-3p and slu-mir-205-5p were enriched in gills. These miRNAs are similarly abundant in many vertebrates, indicating that they have shared regulatory functions. There was also a significantly increased expression of the disease-associated miR-462/miR-731 cluster in response to hypoxia stress. This first pikeperch miRNAome reference resource paves the way for future functional studies to identify miRNA-associated variations that can be utilized in marker-assisted breeding programs.


Asunto(s)
MicroARNs , Humanos , Animales , MicroARNs/genética , Agricultura , Acuicultura , Cruzamiento , Genómica
3.
Int J Mol Sci ; 23(19)2022 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-36232504

RESUMEN

Moritella viscosa is a bacterial pathogen causing winter-ulcer disease in Atlantic salmon. The lesions on affected fish lead to increased mortality, decreased fish welfare, and inferior meat quality in farmed salmon. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional regulation by guiding the miRNA-induced silencing complex to specific mRNA transcripts (target genes). The goal of this study was to identify miRNAs responding to Moritella viscosa in salmon by investigating miRNA expression in the head-kidney and the muscle/skin from lesion sites caused by the pathogen. Protein coding gene expression was investigated by microarray analysis in the same materials. Seventeen differentially expressed guide-miRNAs (gDE-miRNAs) were identified in the head-kidney, and thirty-nine in lesion sites, while the microarray analysis reproduced the differential expression signature of several thousand genes known as infection-responsive. In silico target prediction and enrichment analysis suggested that the gDE-miRNAs were predicted to target genes involved in immune responses, hemostasis, angiogenesis, stress responses, metabolism, cell growth, and apoptosis. The majority of the conserved gDE-miRNAs (e.g., miR-125, miR-132, miR-146, miR-152, miR-155, miR-223 and miR-2188) are known as infection-responsive in other vertebrates. Collectively, the findings indicate that gDE-miRNAs are important post-transcriptional gene regulators of the host response to bacterial infection.


Asunto(s)
MicroARNs , Moritella , Salmo salar , Animales , Riñón Cefálico/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero , Salmo salar/genética , Salmo salar/metabolismo
4.
Int J Mol Sci ; 23(15)2022 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-35955964

RESUMEN

Smoltification (parr-smolt transformation) is a complex developmental process consisting of developmental changes that lead to remodeling of the Atlantic salmon gill. Here, the expression changes of miRNAs and mRNAs were studied by small-RNA sequencing and microarray analysis, respectively, to identify miRNAs and their predicted targets associated with smoltification and subsequent sea water adaptation (SWA). In total, 18 guide miRNAs were identified as differentially expressed (gDE miRNAs). Hierarchical clustering analysis of expression changes divided these into one cluster of 13 gDE miRNAs with decreasing expression during smoltification and SWA that included the miRNA-146, miRNA-30 and miRNA-7132 families. Another smaller cluster that showed increasing expression consisted of miR-101a-3p, miR-193b-5p, miR-499a-5p, miR-727a-3p and miR-8159-5p. The gDE miRNAs were predicted to target 747 of the genes (DE mRNAs), showing expression changes in the microarray analysis. The predicted targets included genes encoding NKA-subunits, aquaporin-subunits, cystic fibrosis transmembrane conductance regulator and the solute carrier family. Furthermore, the predicted target genes were enriched in biological processes associated with smoltification and SWA (e.g., immune system, reactive oxygen species, stress response and extracellular matrix organization). Collectively, the results indicate that remodeling of the gill involves the post-transcriptional regulation of gene expression by the characterized gDE miRNAs.


Asunto(s)
MicroARNs , Salmo salar , Animales , Expresión Génica , Branquias/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Salmo salar/genética , Salmo salar/metabolismo , Agua de Mar
5.
J Fish Biol ; 98(4): 1172-1185, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33332611

RESUMEN

This study finds significant differences in hepatic fatty acid composition between four groups of Atlantic salmon (Salmo salar) consisting of offspring from families selected for high and low capacities to express the delta 6 desaturase isomer b and fed diets with 10% or 75% fish oil. The results demonstrated that hepatic lipid metabolism was affected by experimental conditions (diet/family). The fatty acid composition in the four groups mirrored the differences in dietary composition, but it was also associated with the family groups. Small RNA sequencing followed by RT-qPCR identified 12 differentially expressed microRNAs (DE miRNAs), with expression associated with family groups (miR-146 family members, miR-200b, miR-214, miR-221, miR-125, miR-135, miR-137, miR_nov_1), diets (miR-203, miR-462) or both conditions. All the conserved DE miRNAs have been reported as associated with lipid metabolism in other vertebrates. In silico predictions revealed 37 lipid metabolism pathway genes, including desaturases, transcription factors and key enzymes in the synthesis pathways as putative targets (e.g., srebp-1 and 2, Δ6fad_b and c, hmdh, elovl4 and 5b, cdc42). RT-qPCR analysis of selected target genes showed expression changes that were associated with diet and with family groups (d5fad, d6fad_a, srebp-1). There was a reciprocal difference in the abundance of ssa-miR-203a-3p and srebp-1 in one group comparison, whereas other predicted targets did not reveal any evidence of being negatively regulated by degradation. More experimental studies are needed to validate and fully understand the predicted interactions and how the DE miRNAs may participate in the regulation of hepatic lipid metabolism.


Asunto(s)
Alimentación Animal/análisis , Dieta/veterinaria , Grasas de la Dieta/análisis , Hígado/efectos de los fármacos , MicroARNs/metabolismo , Salmo salar/genética , Animales , Grasas de la Dieta/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Metabolismo de los Lípidos , Hígado/metabolismo , MicroARNs/genética , Salmo salar/metabolismo
6.
Int J Mol Sci ; 21(11)2020 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-32498303

RESUMEN

Macrophages are among the first cells to respond to infection and disease. While microRNAs (miRNAs) are involved in the process of monocyte-to-macrophage differentiation in mammals, less is known in teleost fish. Here, Atlantic salmon head kidney leukocytes (HKLs) were used to study the expression of miRNAs in response to in vitro culture. The morphological analysis of cultures showed predominantly monocyte-like cells on Day 1 and macrophage-like cells on Day 5, suggesting that the HKLs had differentiated from monocytes to macrophages. Day 5 HKLs also contained a higher percentage of phagocytic cells. Small RNA sequencing and qPCR analysis were applied to examine the miRNA diversity and expression. There were 370 known mature Atlantic salmon miRNAs in HKLs. Twenty-two miRNAs (15 families) were downregulated while 44 miRNAs (25 families) were upregulated on Day 5 vs. Day 1. Mammalian orthologs of many of the differentially expressed (DE) miRNAs are known to regulate macrophage activation and differentiation, while the teleost-specific miR-2188, miR-462 and miR-731 were also DE and are associated with immune responses in fish. In silico predictions identified several putative target genes of qPCR-validated miRNAs associated with vertebrate macrophage differentiation. This study identified Atlantic salmon miRNAs likely to influence macrophage differentiation, providing important knowledge for future functional studies.


Asunto(s)
Riñón Cefálico/citología , Macrófagos/citología , MicroARNs/genética , Monocitos/citología , Salmo salar/genética , Animales , Adhesión Celular , Diferenciación Celular , Análisis por Conglomerados , Perfilación de la Expresión Génica , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Pequeño no Traducido/genética , Estallido Respiratorio , Programas Informáticos , Regulación hacia Arriba
7.
BMC Genomics ; 18(1): 349, 2017 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-28472924

RESUMEN

BACKGROUND: MicroRNAs (miRNAs) control multiple biological processes including the innate immune responses by negative post-transcriptional regulation of gene expression. As there were no studies on the role(s) of miRNAs in viral diseases in Atlantic salmon, we aimed to identify miRNAs responding to salmonid alphavirus (SAV) infection. Their expression were studied at different time points post infection with SAV isolates associated with different mortalities. Furthermore, the genome sequences of the identified miRNAs were analysed to reveal putative cis-regulatory elements, and, finally, their putative target genes were predicted. RESULTS: Twenty differentially expressed miRNAs (DE miRNAs) were identified. The expression of the majority of these increased post infection with maximum levels reached after the viral load were stabilized or decreasing. On the other hand, some miRNAs (e.g. the miRNA-21 family) showed decreased expression at the early time points post infection. There were significant differences in the temporal expression of individual miRNA associated with different SAV isolates. Target gene prediction in SAV responsive immune network genes showed that seventeen of the DE miRNAs could target 24 genes (e.g. IRF3, IRF7). Applying the Atlantic salmon transcriptome as input 28 more immune network genes were revealed as putative targets (e.g. IRF5, IRF4). The majority of the predicted target genes promote inflammatory response. The upstream sequences of the miRNA genes revealed a high density of cis-regulatory sequences known as binding sites for immune network transcription factors (TFs). A high expression in the late phase could therefore be due to increased transcription promoted by immune response activated TFs. Based on the in silico target predictions, we discuss their putative roles as early promotors or late inhibitors of inflammation. We propose that the differences in expressions associated with different SAV isolates could contribute to their differences in mortality rates. CONCLUSIONS: This study represents the first steps in exploring miRNAs important in viral-host interaction in Atlantic salmon. We identified several miRNAs responding to SAV infection. Some likely to prohibit harmful inflammation while other may promote an early immune response. Their predicted functions need to be validated and further studied in functional assays to fully understand their roles in immune homeostasis.


Asunto(s)
Infecciones por Alphavirus/veterinaria , Enfermedades de los Peces/metabolismo , MicroARNs/metabolismo , Salmo salar/metabolismo , Infecciones por Alphavirus/genética , Infecciones por Alphavirus/metabolismo , Animales , Secuencia de Bases , Enfermedades de los Peces/genética , Enfermedades de los Peces/virología , Inmunidad Innata/genética , MicroARNs/genética , Miocardio/metabolismo , Interferencia de ARN , Salmo salar/genética , Salmo salar/virología , Análisis de Secuencia de ARN , Transcriptoma , Carga Viral
8.
BMC Genomics ; 14: 482, 2013 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-23865519

RESUMEN

BACKGROUND: MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the posttranscriptional level. They play important roles in multiple biological processes by regulating genes that control developmental timing, growth, stem cell division and apoptosis by binding to the mRNA of target genes. Despite the position Atlantic salmon (Salmo salar) has as an economically important domesticated animal, there has been little research on miRNAs in this species. Knowledge about miRNAs and their target genes may be used to control health and to improve performance of economically important traits. However, before their biological function can be unravelled they must be identified and annotated. The aims of this study were to identify and characterize miRNA genes in Atlantic salmon by deep sequencing analysis of small RNA libraries from nine different tissues. RESULTS: A total of 180 distinct mature miRNAs belonging to 106 families of evolutionary conserved miRNAs, and 13 distinct novel mature miRNAs were discovered and characterized. The mature miRNAs corresponded to 521 putative precursor sequences located at unique genome locations. About 40% of these precursors were part of gene clusters, and the majority of the Salmo salar gene clusters discovered were conserved across species. Comparison of expression levels in samples from different tissues applying DESeq indicated that there were tissue specific expression differences in three conserved and one novel miRNA. Ssa-miR 736 was detected in heart tissue only, while two other clustered miRNAs (ssa-miR 212 and132) seems to be at a higher expression level in brain tissue. These observations correlate well with their expected functions as regulators of signal pathways in cardiac and neuronal cells, respectively. Ssa-miR 8163 is one of the novel miRNAs discovered and its function remains unknown. However, differential expression analysis using DESeq suggests that this miRNA is enriched in liver tissue and the precursor was mapped to intron 7 of the transferrin gene. CONCLUSIONS: The identification and annotation of evolutionary conserved and novel Salmo salar miRNAs as well as the characterization of miRNA gene clusters provide biological knowledge that will greatly facilitate further functional studies on miRNAs in this species.


Asunto(s)
Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/genética , Salmo salar/genética , Animales , Secuencia de Bases , Secuencia Conservada , Evolución Molecular , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Familia de Multigenes/genética , Especificidad de Órganos
9.
Biology (Basel) ; 11(1)2022 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-35053128

RESUMEN

MicroRNAs (miRNAs) are endogenous small RNA molecules involved in the post-transcriptional regulation of protein expression by binding to the mRNA of target genes. They are key regulators in teleost development, maintenance of tissue-specific functions, and immune responses. Lumpfish (Cyclopterus lumpus) is becoming an emergent aquaculture species as it has been utilized as a cleaner fish to biocontrol sea lice (e.g., Lepeophtheirus salmonis) infestation in the Atlantic Salmon (Salmo salar) aquaculture. The lumpfish miRNAs repertoire is unknown. This study identified and characterized miRNA encoding genes in lumpfish from three developmental stages (adult, embryos, and larvae). A total of 16 samples from six different adult lumpfish organs (spleen, liver, head kidney, brain, muscle, and gill), embryos, and larvae were individually small RNA sequenced. Altogether, 391 conserved miRNA precursor sequences (discovered in the majority of teleost fish species reported in miRbase), eight novel miRNA precursor sequences (so far only discovered in lumpfish), and 443 unique mature miRNAs were identified. Transcriptomics analysis suggested organ-specific and age-specific expression of miRNAs (e.g., miR-122-1-5p specific of the liver). Most of the miRNAs found in lumpfish are conserved in teleost and higher vertebrates, suggesting an essential and common role across teleost and higher vertebrates. This study is the first miRNA characterization of lumpfish that provides the reference miRNAome for future functional studies.

10.
Biology (Basel) ; 11(5)2022 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-35625416

RESUMEN

Optimal smoltification is crucial for normal development, growth, and health of farmed Atlantic salmon in seawater. Here, we characterize miRNA expression in liver to reveal whether miRNAs regulate gene expression during this developmental transition. Expression changes of miRNAs and mRNAs was studied by small-RNA sequencing and microarray analysis, respectively. This revealed 62 differentially expressed guide miRNAs (gDE-miRNAs) that could be divided into three groups with characteristic dynamic expression patterns. Three of miRNA families are known as highly expressed in liver. A rare arm shift was observed during smoltification in the Atlantic salmon-specific novel-ssa-miR-16. The gDE-miRNAs were predicted to target 2804 of the genes revealing expression changes in the microarray analysis. Enrichment analysis revealed that targets were significantly enriched in smoltification-associated biological process groups. These included lipid and cholesterol synthesis, carbohydrate metabolism, protein metabolism and protein transport, immune system genes, circadian rhythm and stress response. The results indicate that gDE-miRNAs may regulate many of the changes associated with this developmental transition in liver. The results pave the way for validation of the predicted target genes and further study of gDE-miRNA and their targets by functional assays.

11.
Int J Legal Med ; 125(5): 629-36, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20552217

RESUMEN

Because of their sensitivity and high level of discrimination, short tandem repeat (STR) maker systems are currently the method of choice in routine forensic casework and data banking, usually in multiplexes up to 15-17 loci. Constraints related to sample amount and quality, frequently encountered in forensic casework, will not allow to change this picture in the near future, notwithstanding the technological developments. In this study, we present a free online calculator named PopAffiliator ( http://cracs.fc.up.pt/popaffiliator ) for individual population affiliation in the three main population groups, Eurasian, East Asian and sub-Saharan African, based on genotype profiles for the common set of STRs used in forensics. This calculator performs affiliation based on a model constructed using machine learning techniques. The model was constructed using a data set of approximately fifteen thousand individuals collected for this work. The accuracy of individual population affiliation is approximately 86%, showing that the common set of STRs routinely used in forensics provide a considerable amount of information for population assignment, in addition to being excellent for individual identification.


Asunto(s)
Computadores/legislación & jurisprudencia , Genética Forense/instrumentación , Genética Forense/legislación & jurisprudencia , Marcadores Genéticos/genética , Genética de Población/legislación & jurisprudencia , Genotipo , Repeticiones de Microsatélite/genética , Grupos de Población/genética , Inteligencia Artificial , Frecuencia de los Genes/genética , Humanos
12.
Noncoding RNA ; 7(4)2021 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-34698276

RESUMEN

Complete 3'UTRs unambiguously assigned to specific mRNA isoforms from the Atlantic salmon full-length (FL) transcriptome were collected into a 3'UTRome. miRNA response elements (MREs) and other cis-regulatory motifs were subsequently predicted and assigned to 3'UTRs of all FL-transcripts. The MicroSalmon GitHub repository provides all results. RNAHybrid and sRNAtoolbox tools predicted the MREs. UTRscan and the Teiresias algorithm predicted other 3'UTR cis-acting motifs, both known vertebrate motifs and putative novel motifs. MicroSalmon provides search programs to retrieve all FL-transcripts targeted by a miRNA (median number 1487), all miRNAs targeting an FL-transcript (median number 27), and other cis-acting motifs. As thousands of FL-transcripts may be targets of each miRNA, additional experimental strategies are necessary to reduce the likely true and relevant targets to a number that may be functionally validated. Low-complexity motifs known to affect mRNA decay in vertebrates were over-represented. Many of these were enriched in the terminal end, while purine- or pyrimidine-rich motifs with unknown functions were enriched immediately downstream of the stop codon. Furthermore, several novel complex motifs were over-represented, indicating conservation and putative function. In conclusion, MicroSalmon is an extensive and useful, searchable resource for study of Atlantic salmon transcript regulation by miRNAs and cis-acting 3'UTR motifs.

13.
Front Genet ; 12: 656334, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33986770

RESUMEN

Atlantic salmon (Salmo salar) is a major species produced in world aquaculture and an important vertebrate model organism for studying the process of rediploidization following whole genome duplication events (Ss4R, 80 mya). The current Salmo salar transcriptome is largely generated from genome sequence based in silico predictions supported by ESTs and short-read sequencing data. However, recent progress in long-read sequencing technologies now allows for full-length transcript sequencing from single RNA-molecules. This study provides a de novo full-length mRNA transcriptome from liver, head-kidney and gill materials. A pipeline was developed based on Iso-seq sequencing of long-reads on the PacBio platform (HQ reads) followed by error-correction of the HQ reads by short-reads from the Illumina platform. The pipeline successfully processed more than 1.5 million long-reads and more than 900 million short-reads into error-corrected HQ reads. A surprisingly high percentage (32%) represented expressed interspersed repeats, while the remaining were processed into 71 461 full-length mRNAs from 23 071 loci. Each transcript was supported by several single-molecule long-read sequences and at least three short-reads, assuring a high sequence accuracy. On average, each gene was represented by three isoforms. Comparisons to the current Atlantic salmon transcripts in the RefSeq database showed that the long-read transcriptome validated 25% of all known transcripts, while the remaining full-length transcripts were novel isoforms, but few were transcripts from novel genes. A comparison to the current genome assembly indicates that the long-read transcriptome may aid in improving transcript annotation as well as provide long-read linkage information useful for improving the genome assembly. More than 80% of transcripts were assigned GO terms and thousands of transcripts were from genes or splice-variants expressed in an organ-specific manner demonstrating that hybrid error-corrected long-read transcriptomes may be applied to study genes and splice-variants expressed in certain organs or conditions (e.g., challenge materials). In conclusion, this is the single largest contribution of full-length mRNAs in Atlantic salmon. The results will be of great value to salmon genomics research, and the pipeline outlined may be applied to generate additional de novo transcriptomes in Atlantic Salmon or applied for similar projects in other species.

14.
Front Immunol ; 12: 709910, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34484211

RESUMEN

The Atlantic salmon (Salmo salar) is an economically important fish, both in aquaculture and in the wild. In vertebrates, macrophages are some of the first cell types to respond to pathogen infection and disease. While macrophage biology has been characterized in mammals, less is known in fish. Our previous work identified changes in the morphology, phagocytic ability, and miRNA profile of Atlantic salmon adherent head kidney leukocytes (HKLs) from predominantly "monocyte-like" at Day 1 of in vitro culture to predominantly "macrophage-like" at Day 5 of culture. Therefore, to further characterize these two cell populations, we examined the mRNA transcriptome profile in Day 1 and Day 5 HKLs using a 44K oligonucleotide microarray. Large changes in the transcriptome were revealed, including changes in the expression of macrophage and immune-related transcripts (e.g. csf1r, arg1, tnfa, mx2), lipid-related transcripts (e.g. fasn, dhcr7, fabp6), and transcription factors involved in macrophage differentiation and function (e.g. klf2, klf9, irf7, irf8, stat1). The in silico target prediction analysis of differentially expressed genes (DEGs) using miRNAs known to change expression in Day 5 HKLs, followed by gene pathway enrichment analysis, supported that these miRNAs may be involved in macrophage maturation by targeting specific DEGs. Elucidating how immune cells, such as macrophages, develop and function is a key step in understanding the Atlantic salmon immune system. Overall, the results indicate that, without the addition of exogenous factors, the adherent HKL cell population differentiates in vitro to become macrophage-like.


Asunto(s)
Perfilación de la Expresión Génica , Leucocitos/inmunología , Macrófagos/fisiología , Salmo salar/inmunología , Animales , Células Cultivadas , Metabolismo de los Lípidos , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción/fisiología
15.
BMC Genomics ; 11: 706, 2010 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-21159188

RESUMEN

BACKGROUND: Single nucleotide polymorphisms (SNPs) represent the most widespread type of DNA variation in vertebrates and may be used as genetic markers for a range of applications. This has led to an increased interest in identification of SNP markers in non-model species and farmed animals. The in silico SNP mining method used for discovery of most known SNPs in Atlantic salmon (Salmo salar) has applied a global (genome-wide) approach. In this study we present a targeted 3'UTR-primed SNP discovery strategy that utilizes sequence data from Salmo salar full length sequenced cDNAs (FLIcs). We compare the efficiency of this new strategy to the in silico SNP mining method when using both methods for targeted SNP discovery. RESULTS: The SNP discovery efficiency of the two methods was tested in a set of FLIc target genes. The 3'UTR-primed SNP discovery method detected novel SNPs in 35% of the target genes while the in silico SNP mining method detected novel SNPs in 15% of the target genes. Furthermore, the 3'UTR-primed SNP discovery strategy was the less labor intensive one and revealed a higher success rate than the in silico SNP mining method in the initial amplification step. When testing the methods we discovered 112 novel bi-allelic polymorphisms (type I markers) in 88 salmon genes [dbSNP: ss179319972-179320081, ss250608647-250608648], and three of the SNPs discovered were missense substitutions. CONCLUSIONS: Full length insert cDNAs (FLIcs) are important genomic resources that have been developed in many farmed animals. The 3'UTR-primed SNP discovery strategy successfully utilized FLIc data to detect novel SNPs in the partially tetraploid Atlantic salmon. This strategy may therefore be useful for targeted SNP discovery in several species, and particularly useful in species that, like salmonids, have duplicated genomes.


Asunto(s)
Regiones no Traducidas 3'/genética , Biología Computacional/métodos , Cartilla de ADN/metabolismo , Polimorfismo de Nucleótido Simple/genética , Salmo salar/genética , Animales , Mutación INDEL/genética , Sistemas de Lectura Abierta/genética
16.
Genes (Basel) ; 11(9)2020 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-32911670

RESUMEN

Smoltification and early seawater phase are critical developmental periods with physiological and biochemical changes in Atlantic salmon that facilitates survival in saltwater. MicroRNAs (miRNAs) are known to have important roles in development, but whether any miRNAs are involved in regulation of gene expression during smoltification and the adaption to seawater is largely unknown. Here, small RNA sequencing of materials from head kidney before, during smoltification and post seawater transfer were used to study expression dynamics of miRNAs, while microarray analysis was applied to study mRNA expression dynamics. Comparing all timepoints, 71 miRNAs and 2709 mRNAs were identified as differentially expressed (DE). Hierarchical clustering analysis of the DE miRNAs showed three major clusters with characteristic expression changes. Eighty-one DE mRNAs revealed negatively correlated expression patterns to DE miRNAs in Cluster I and III. Furthermore, 42 of these mRNAs were predicted as DE miRNA targets. Gene enrichment analysis of negatively correlated target genes showed they were enriched in gene ontology groups hormone biosynthesis, stress management, immune response, and ion transport. The results strongly indicate that post-transcriptional regulation of gene expression by miRNAs is important in smoltification and sea water adaption, and this study identifies several putative miRNA-target pairs for further functional studies.


Asunto(s)
Proteínas de Peces/metabolismo , Riñón Cefálico/metabolismo , MicroARNs/genética , ARN Mensajero/metabolismo , Salinidad , Salmo salar/genética , Transcriptoma/efectos de los fármacos , Adaptación Fisiológica/genética , Animales , Proteínas de Peces/genética , Regulación de la Expresión Génica , Branquias/efectos de los fármacos , Branquias/crecimiento & desarrollo , Branquias/metabolismo , Riñón Cefálico/efectos de los fármacos , Riñón Cefálico/crecimiento & desarrollo , ARN Mensajero/genética , Salmo salar/crecimiento & desarrollo , Agua de Mar/análisis , Análisis de Secuencia de ARN
17.
Front Immunol ; 11: 2113, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33013890

RESUMEN

Infectious pancreatic necrosis virus (IPNV) infection has been a major problem in salmonid aquaculture. Marker-assisted selection of individuals with resistant genotype at the major IPN quantitative trait locus (IPN-QTL) has significantly reduced mortality in recent years. We have identified host miRNAs that respond to IPNV challenge in salmon fry that were either homozygous resistant (RR) or homozygous susceptible (SS) for the IPN-QTL. Small RNA-sequenced control samples were compared to samples collected at 1, 7, and 20 days post challenge (dpc). This revealed 72 differentially expressed miRNAs (DE miRNAs). Viral load (VL) was lower in RR vs. SS individuals at 7 and 20 dpc. However, analysis of miRNA expression changes revealed no differences between RR vs. SS individuals in controls, at 1 or 7 dpc, while 38 "high viral load responding" miRNAs (HVL-DE miRNAs) were identified at 20 dpc. Most of the HVL-DE miRNAs showed changes that were more pronounced in the high VL SS group than in the low VL RR group when compared to the controls. The absence of differences between QTL groups in controls, 1 and 7 dpc indicates that the QTL genotype does not affect miRNA expression in healthy fish or their first response to viral infections. The miRNA differences at 20 dpc were associated with the QTL genotype and could, possibly, contribute to differences in resistance/susceptibility at the later stage of infection. In silico target gene predictions revealed that 180 immune genes were putative targets, and enrichment analysis indicated that the miRNAs may regulate several major immune system pathways. Among the targets of HVL-DE miRNAs were IRF3, STAT4, NFKB2, MYD88, and IKKA. Interestingly, TNF-alpha paralogs were targeted by different DE miRNAs. Most DE miRNAs were from conserved miRNA families that respond to viral infections in teleost (e.g., miR-21, miR-146, miR-181, miR-192, miR-221, miR-462, miR-731, and miR-8159), while eight were species specific. The miRNAs showed dynamic temporal changes implying they would affect their target genes differently throughout disease progression. This shows that miRNAs are sensitive to VL and disease progression, and may act as fine-tuners of both immediate immune response activation and the later inflammatory processes.


Asunto(s)
Infecciones por Birnaviridae/veterinaria , Enfermedades de los Peces/genética , Interacciones Huésped-Patógeno/genética , Virus de la Necrosis Pancreática Infecciosa/fisiología , MicroARNs/genética , Salmo salar/genética , Animales , Secuencia de Bases , Infecciones por Birnaviridae/genética , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/virología , Simulación por Computador , Progresión de la Enfermedad , Resistencia a la Enfermedad , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Genotipo , Interacciones Huésped-Patógeno/inmunología , Sitios de Carácter Cuantitativo , ARN Viral/análisis , RNA-Seq , Salmo salar/crecimiento & desarrollo , Salmo salar/inmunología , Salmo salar/virología , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Análisis de Matrices Tisulares , Carga Viral
18.
Front Immunol ; 11: 587931, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33262769

RESUMEN

Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily "monocyte-like" at Day 1 to primarily "macrophage-like" at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.


Asunto(s)
Vesículas Extracelulares/inmunología , Macrófagos/inmunología , MicroARNs , Monocitos/inmunología , Salmo salar/genética , Salmo salar/inmunología , Animales , Células Cultivadas , Vesículas Extracelulares/genética , Riñón Cefálico/citología
19.
BMC Genomics ; 10: 502, 2009 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-19878547

RESUMEN

BACKGROUND: Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs) are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP), the number of sequences where the full-length of the cDNA insert has been determined has been small. RESULTS: High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91%) of the transcripts were annotated using Gene Ontology (GO) terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS). The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. CONCLUSION: This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS). This suggests that the remaining cDNA libraries generated by SGP represent a valuable cCDS FLIc source. The conservation of 7-mers in 3'UTRs indicates that these motifs are functionally important. Identity between some of these 7-mers and miRNA target sequences suggests that they are miRNA targets in Salmo salar transcripts as well.


Asunto(s)
ADN Complementario/genética , Salmo salar/genética , Animales , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Sistemas de Lectura Abierta , Poliadenilación , Isoformas de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Salmo salar/crecimiento & desarrollo , Regiones no Traducidas/genética
20.
Cells ; 8(1)2019 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-30641951

RESUMEN

MicroRNAs (miRNAs) are important post-transcriptional gene expression regulators. Here, 448 different miRNA genes, including 17 novel miRNAs, encoding for 589 mature Atlantic salmon miRNAs were identified after sequencing 111 samples (fry, pathogen challenged fry, various developmental and adult tissues). This increased the reference miRNAome with almost one hundred genes. Prior to isomiR characterization (mature miRNA variants), the proportion of erroneous sequence variants (ESVs) arising in the analysis pipeline was assessed. The ESVs were biased towards 5' and 3' end of reads in unexpectedly high proportions indicating that measurements of ESVs rather than Phred score should be used to avoid misinterpreting ESVs as isomiRs. Forty-three isomiRs were subsequently discovered. The biological effect of the isomiRs measured as increases in target diversity was small (<3%). Five miRNA genes showed allelic variation that had a large impact on target gene diversity if present in the seed. Twenty-one miRNAs were ubiquitously expressed while 31 miRNAs showed predominant expression in one or few tissues, indicating housekeeping or tissue specific functions, respectively. The miR-10 family, known to target Hox genes, were highly expressed in the developmental stages. The proportion of miR-430 family members, participating in maternal RNA clearance, was high at the earliest developmental stage.


Asunto(s)
MicroARNs/metabolismo , Salmo salar/embriología , Salmo salar/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Análisis de Secuencia de ARN/métodos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA