Skip to main content
SpringerLink
Log in

Temporal Gene Expression Signature of Plasma Extracellular Vesicles-MicroRNAs from Post-Smolt Coho Salmon Challenged with Piscirickettsia salmonis

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

Piscirickettsiosis is the most important bacterial disease in the Chilean salmon industry, which has borne major economic losses due to failure to control it. Cells use extracellular vesicles (EVs) as an inter-cellular communicators to deliver several factors (e.g., microRNAs) that may regulate the responses of other cells. However, there is limited knowledge about the identification and characterization of EV-miRNAs in salmonids or the effect of infections on these. In this study, Illumina sequencing technology was used to identify Coho salmon plasma EV-miRNAs upon Piscirickettsia salmonis infection at four different time points. A total of 118 novels and 188 known EV-miRNAs, including key immune teleost miRNAs families (e.g., miR-146, miR-122), were identified. A total of 245 EV-miRNAs were detected as differentially expressed (FDR < 5%) in terms of control, with a clear down-regulation pattern throughout the disease. KEGG enrichment results of EV-miRNAs target genes showed that they were grouped mainly in cellular, stress, inflammation and immune responses. Therefore, it is hypothesized that P. salmonis could potentially benefit from unbalanced modulation response of Coho salmon EV-miRNAs in order to promote a hyper-inflammatory and compromised immune response through the suppression of different key immune host miRNAs during the course of the infection, as indicated by the results of this study.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

Availability of data and material

Sequence data file has been deposited in the Sequence Read Archive (SRA), accession number National Center for Biotechnology Information (NCBI) SRA# PRJNA627938.

References 

  • Abels ER, Breakefield XO (2016) Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake. Front Immunol 36:301–312

    CAS  Google Scholar 

  • Abu-Jamous B, Kelly S (2018) Clust: automatic extraction of optimal co-expressed gene clusters from gene expression data. Genome Biol 19:172

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Andreassen R, Høyheim B (2017) miRNAs associated with immune response in teleost fish. Dev Comp Immunol 75:77–85

    Article  CAS  PubMed  Google Scholar 

  • Barría A, Christensen KA, Yoshida GM, Correa K, Jedlicki A, Lhorente JP, et al. (2018) Genomic predictions and genome-wide association study of resistance against Piscirickettsia salmonis in Coho salmon (Oncorhynchus kisutch) using ddRAD sequencing. G3 8:1183-1194

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Barría A, Doeschl-Wilson AB, Lhorente JP, Houston RD, Yáñez JM (2019) Novel insights into the genetic relationship between growth and disease resistance in an aquaculture strain of Coho salmon (Oncorhynchus kisutch). Aquaculture 511:734207

    Article  Google Scholar 

  • Bhatnagar S, Schorey JS (2007) Exosomes released from infected macrophages contain Mycobacterium avium glycopeptidolipids and are proinflammatory. J Biol Chem 282:25779–25789

    Article  CAS  PubMed  Google Scholar 

  • Bravo S, Midtlyng PJ (2007) The use of fish vaccines in the Chilean salmon industry 1999–2003. Aquaculture 270:36–42

    Article  CAS  Google Scholar 

  • Campoy E, Colombo MI (2009) Autophagy in intracellular bacterial infection. Bba-Mol Cell Res 1793:1465–1477

    CAS  Google Scholar 

  • Cassat JE, Skaar EP (2013) Iron in infection and immunity. Cell Host Microbe 13:509–519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chan PP, Lowe TM (2016) GtRNAdb 2.0: An expanded database of transfer RNA genes identified in complete and draft genomes. Nucl Acids Res 44:D184–D189

    Article  CAS  PubMed  Google Scholar 

  • Chandan K, Gupta M, Sarwat M (2020) Role of host and pathogen-derived MicroRNAs in immune regulation during infectious and inflammatory diseases. Front Immunol 10:3081

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Chatterjee A, Roy D, Patnaik E, Nongthomba U (2016) Muscles provide protection during microbial infection by activating innate immune response pathways in Drosophila and zebrafish. Dis Model Mech 9:697–705

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chu Q, Sun Y, Cui J, Xu T (2017) Inducible microRNA-214 contributes to the suppression of NF-κB-mediated inflammatory response via targeting myd88 gene in fish. J Biol Chem 292:5282–5290

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chu Q, Yan X, Liu L, Xu T (2019) The inducible microRNA-21 negatively modulates the inflammatory response in teleost fish via targeting IRAK4. Front Immunol 10:1623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cui L, Hu H, Wei W, Wang W, Liu H (2016) Identification and characterization of MicroRNAs in the liver of blunt snout bream (Megalobrama amblycephala) infected by Aeromonas hydrophila. Int J Mol Sci 17:1972

    Article  PubMed Central  CAS  Google Scholar 

  • Das K, Garnica O, Dhandayuthapani S (2016) Modulation of host miRNAs by intracellular bacterial pathogens. Front Cell Infect Microbio 6:79

    Google Scholar 

  • da Silva Duran BO, Fernandez GJ, Mareco EA, Moraes LN, Salomão RAS, Gutierrez De Paula T et al (2015) Differential microRNA expression in fast- and slow-twitch skeletal muscle of Piaractus mesopotamicus during growth. PLoS One 10:e0141967

    Article  CAS  Google Scholar 

  • Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS (2003) MicroRNA targets in Drosophila. Genome Biol 5:R1

    Article  PubMed  PubMed Central  Google Scholar 

  • Evensen Ø (2016) Immunization strategies against Piscirickettsia salmonis infections: review of vaccination approaches and modalities and their associated immune response profiles. Front Immunol 7:482

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Friedländer MR, Chen W, Adamidi C, Maaskola J, Einspanier R, Knespel S et al (2008) Discovering microRNAs from deep sequencing data using miRDeep. Nat Biotechnol 26:407–415

    Article  PubMed  CAS  Google Scholar 

  • Fryer JL, Hedrick RP (2003) Piscirickettsia salmonis: A Gram-negative intracellular bacterial pathogen of fish. J Fish Dis 26:251–262

    Article  CAS  PubMed  Google Scholar 

  • Gao K, Jin J, Huang C, Li J, Luo H, Li L et al (2019) Exosomes derived from septic mouse serum modulate immune responses via exosome-associated cytokines. Front Immunol 10:1560

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hartjes TA, Mytnyk S, Jenster GW, van Steijn V, van Royen ME (2019) Extracellular vesicle quantification and characterization: common methods and emerging approaches. Bioengineering 6:7

    Article  CAS  PubMed Central  Google Scholar 

  • Herkenhoff ME, Oliveira AC, Nachtigall PG, Costa JM, Campos VF, Hilsdorf AWS et al (2018) Fishing into the microRNA transcriptome. Front Genet 9:88

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Hu Y hua, Zhang B cun, Zhou H zhen, Guan X lu, Sun L (2017) Edwardsiella tarda-induced miRNAs in a teleost host: global profile and role in bacterial infection as revealed by integrative miRNA–mRNA analysis. Virulence 8:1457–1464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang Y, Shen X-J, Zou Q, Wang S, Tang S, Zhang G (2010) Biological functions of microRNAs: a review. J Physiol Biochem 67:129–139

    Article  PubMed  CAS  Google Scholar 

  • Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J (2005) Repbase Update, a database of eukaryotic repetitive elements. Cytogenet Genome Res 110:462–467

    Article  CAS  PubMed  Google Scholar 

  • Kang JY, Park H, Kim H, Mun D, Park H, Yun N et al (2019) Human peripheral blood-derived exosomes for microRNA delivery. Int J Mol Med 43:2319–2328

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kertesz M, Iovino N, Unnerstall U, Gaul U, Segal E (2007) The role of site accessibility in microRNA target recognition. Nat Genet 39:1278–1284

    Article  CAS  PubMed  Google Scholar 

  • Kirschner MB, Edelman JJB, Kao SCH, Vallely MP, Van Zandwijk N, Reid G (2013) The impact of hemolysis on cell-free microRNA biomarkers. Front Genet 4:94

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lagos L, Tandberg J, Kashulin-Bekkelund A, Colquhoun DJ, Sørum H, Winther-Larsen HC (2017) Isolation and characterization of serum extracellular vesicles (EVs) from Atlantic salmon infected with Piscirickettsia salmonis. Proteomes 5:34

    Article  PubMed Central  CAS  Google Scholar 

  • Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Leiva F, Rojas M, Reyes D, Bravo S, Garcia K, Moya J, Vidal R (2020) Identification and characterization of miRNAs and lncRNAs of Coho salmon (Oncorhynchus kisutch) in normal immune organs. Genomics 112:45–54

    Article  CAS  PubMed  Google Scholar 

  • Lhorente JP, Gallardo JA, Villanueva B, Carabaño MJ, Neira R (2014) Disease resistance in Atlantic salmon (Salmo salar): coinfection of the intracellular bacterial pathogen Piscirickettsia salmonis and the sea louse Caligus rogercresseyi. PLoS One 9:e95397

    Article  PubMed  PubMed Central  Google Scholar 

  • Magnadóttir B, Uysal-Onganer P, Kraev I, Dodds A, Gudmundsdottir S, Lange S (2020) Extracellular vesicles, deiminated protein cargo and microRNAs are novel serum biomarkers for environmental rearing temperature in Atlantic cod (Gadus morhua L.). Aquac Rep (16):100245

    Article  Google Scholar 

  • Marshall SH, Tobar JA (2014). In: Gudding R, Lillehaug A, Øystein E (eds) Vaccination against piscirickettsiosis. Wiley-Blackwell, USA

    Chapter  Google Scholar 

  • Maudet C, Mano M, Eulalio A (2014) MicroRNAs in the interaction between host and bacterial pathogens. FEBS Lett 588:4140–4147

    Article  CAS  PubMed  Google Scholar 

  • Mauel MJ, Miller DL (2002) Piscirickettsiosis and piscirickettsiosis-like infections in fish: a review. Vet Microbiol 87:279–289

    Article  PubMed  Google Scholar 

  • Nachtigall PG, Dias MC, Carvalho RF, Martins C, Pinhal D (2015) MicroRNA-499 expression distinctively correlates to target genes sox6 and rod1 profiles to resolve the skeletal muscle phenotype in Nile tilapia. PLoS ONE 10:e0119804

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Nahid MA, Yao B, Dominguez-Gutierrez PR, Kesavalu L, Satoh M, Chan EK (2013) Regulation of TLR2-mediated tolerance and cross-tolerance through IRAK4 modulation by miR-132 and miR-212. J Immunol 190:1250–1263

    Article  CAS  PubMed  Google Scholar 

  • Nawrocki EP, Burge SW, Bateman A, Daub J, Eberhardt RY, Eddy SR et al (2015) Rfam 12.0: updates to the RNA families database. Nucleic Acids Res 43:D130–D137

    Article  CAS  PubMed  Google Scholar 

  • Ni B, Rajaram MVS, Lafuse WP, Landes MB, Schlesinger LS (2014) Mycobacterium tuberculosis decreases human macrophage IFN-γ Responsiveness through miR-132 and miR-26a. Immunol 193:4537–4547

    CAS  Google Scholar 

  • Olsen AB, Melby HP, Speilberg L, Evensen Håstein T (1997) Piscirickettsia salmonis infection in Atlantic salmon Salmo salar in Norway — epidemiological, pathological and microbiological findings. Dis Aquat Organ 31:35–48

    Article  Google Scholar 

  • Palazzo AF, Lee ES (2015) Non-coding RNA: What is functional and what is junk? Front Genet 6:2

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Pulgar R, Hödar C, Travisany D, Zuñiga A, Domínguez C, Maass A et al (2015) Transcriptional response of Atlantic salmon families to Piscirickettsia salmonis infection highlights the relevance of the iron-deprivation defence system. BMC Genomics 16:495

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P et al (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucl Acids Res 41:D590–D596

    Article  CAS  PubMed  Google Scholar 

  • Ramírez R, Gomez FA, Marshall SH (2015) The infection process of Piscirickettsia salmonis in fish macrophages is dependent upon interaction with host-cell clathrin and actin. FEMS Microbiol Lett 362:1–8

    Article  PubMed  CAS  Google Scholar 

  • Ranganathan K, Subramanian K, Pachiappan P (2013) The multitudinous role of microRNAs in various biological systems. J Pharm Res 6:679–683

    CAS  Google Scholar 

  • Rao X, Huang X, Zhou Z, Lin X (2013) An improvement of the 2ˆ(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath 3:71–85

    PubMed  PubMed Central  Google Scholar 

  • Roberts TC, Coenen-Stass AML, Wood MJA (2014) Assessment of RT-qPCR normalization strategies for accurate quantification of extracellular microRNAs in murine serum. PLoS One 9:e89237

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140

    Article  CAS  PubMed  Google Scholar 

  • Rodrigues M, Fan J, Lyon C, Wan M, Hu Y (2018) Role of extracellular vesicles in viral and bacterial infections: pathogenesis, diagnostics, and therapeutics. Theranostics 8:2709–2721

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rozas M, Enríquez R (2014) Piscirickettsiosis and Piscirickettsia salmonis in fish: a review. J Fish Dis 37:163–188

    Article  CAS  PubMed  Google Scholar 

  • Rozas-Serri M, Peña A, Arriagada G, Enríquez R, Maldonado L (2018) Comparison of gene expression in post-smolt Atlantic salmon challenged by LF-89-like and EM-90-like Piscirickettsia salmonis. J Fish Dis 41:539–552

    Article  CAS  PubMed  Google Scholar 

  • Rozas-Serri M, Peña A, Maldonado L (2018) Transcriptomic profiles of post-smolt Atlantic salmon challenged with Piscirickettsia salmonis reveal a strategy to evade the adaptive immune response and modify cell-autonomous immunity. Dev Comp Immunol 81:348–362

    Article  CAS  PubMed  Google Scholar 

  • Samanta S, Rajasingh S, Drosos N, Zhou Z, Dawn B, Rajasingh J (2018) Exosomes: new molecular targets of diseases. Acta Pharmacol Sin 39:501–513

    Article  CAS  PubMed  Google Scholar 

  • Schmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schorey JS, Cheng Y, McManus WR (2021) Bacteria- and host-derived extracellular vesicles — two sides of the same coin? J Cell Sci 134:jcs256628

    Article  CAS  PubMed  Google Scholar 

  • Sedgwick AE, D’Souza-Schorey C (2018) The biology of extracellular microvesicles. Traffic 19:319–327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shabalina SA, Spiridonov NA (2004) The mammalian transcriptome and the function of non-coding DNA sequences. Genome Biol 5:105

    Article  PubMed  PubMed Central  Google Scholar 

  • Signore A (2013) About inflammation and infection. EJNMMI Res 3:8

    PubMed  Google Scholar 

  • Simeone P, Bologna G, Lanuti P, Pierdomenico L, Guagnano MT, Pieragostino D et al (2020) Extracellular vesicles as signaling mediators and disease biomarkers across biological barriers. Int J Mol Sci 21:2514

    Article  CAS  PubMed Central  Google Scholar 

  • Sohel MH (2016) Extracellular/circulating microRNAs: release mechanisms, functions and challenges. Achiev Life Sci 10:175–186

    Google Scholar 

  • Spies D, Renz PF, Beyer TA, Ciaudo C (2019) Comparative analysis of differential gene expression tools for RNA sequencing time course data. Brief Bioinform 20:288–298

    Article  PubMed  CAS  Google Scholar 

  • Spencer N, Yeruva L (2021) Role of bacterial infections in extracellular vesicles release and impact on immune response. Biomed J 44:157–164

    Article  PubMed  Google Scholar 

  • Storey JD, Xiao W, Leek JT, Tompkins RG, Davis RW (2005) Significance analysis of time course microarray experiments. Proc Natl Aca Sci USA 102:12837–1242

    Article  CAS  Google Scholar 

  • Tacchi L, Bron JE, Taggart JB, Secombes CJ, Bickerdike R, Adler MA et al (2011) Multiple tissue transcriptomic responses to Piscirickettsia salmonis in Atlantic salmon (Salmo salar). Physiol Genomics 43:1241–1254

    Article  CAS  PubMed  Google Scholar 

  • Tahamtan A, Teymoori-Rad M, Nakstad B, Salimi V (2018) Anti-inflammatory microRNAs and their potential for inflammatory diseases treatment. Front Immunol 9:1377

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tam S, Tsao MS, McPherson JD (2015) Optimization of miRNA-seq data preprocessing. Brief Bioinform 16:950–963

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tancini B, Buratta S, Sagini K, Costanzi E, Delo F, Urbanelli L et al (2019) Insight into the role of extracellular vesicles in lysosomal storage disorders. Genes 10:510

    Article  CAS  PubMed Central  Google Scholar 

  • Tidball JG, Villalta SA (2010) Regulatory interactions between muscle and the immune system during muscle regeneration. Am J Physiol Regul Integr Comp Physiol 298:R1173–R1187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Valenzuela-Miranda D, Gallardo-Escárate C (2016) Novel insights into the response of Atlantic salmon (Salmo salar) to Piscirickettsia salmonis: interplay of coding genes and lncRNAs during bacterial infection. Fish Shellfish Immunol 59:427–438

    Article  CAS  PubMed  Google Scholar 

  • Valenzuela-Miranda D, Valenzuela-Muñoz V, Farlora R, Gallardo-Escárate C (2017) MicroRNA-based transcriptomic responses of Atlantic salmon during infection by the intracellular bacterium Piscirickettsia salmonis. Dev Comp Immunol 77:287–296

    Article  CAS  PubMed  Google Scholar 

  • Wery M, Kwapisz M, Morillon A (2011) Noncoding RNAs in gene regulation. Wiley Interdiscip Rev Syst Biol Med 3:728–738

    Article  CAS  PubMed  Google Scholar 

  • Xie W, Li M, Xu N, Lv Q, Huang N, He J et al (2013) miR-181a regulates inflammation responses in monocytes and macrophages. PLoS One 8:e58639

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu L, Yang BF, Ai J (2013) MicroRNA transport: a new way in cell communication. J. Cell Physiol 228:1713–1719

    Article  CAS  PubMed  Google Scholar 

  • Xu T, Chu Q, Cui J, Zhao X (2018) The inducible microRNA-203 in fish represses the inflammatory responses to Gram-negative bacteria by targeting IL-1 receptor-associated kinase 4. J Biol Chem 293:1386–1396

    Article  CAS  PubMed  Google Scholar 

  • Yamamura S, Imai-Sumida M, Tanaka Y, Dahiya R (2018) Interaction and cross-talk between non-coding RNAs. Cell Mol Life Sci 75:467–484

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This research was supported by CORFO-INNOVA Chile 12IDL2-16192 and DICYT 022043VS, Vicerrectoría de Investigación, Desarrollo e Innovacion. K.K.G thank the Fellowship USA 1899 from the Universidad de Santiago de Chile.

Author information

Authors and Affiliations

  1. Laboratory of Molecular Ecology, Genomics and Evolutionary Studies, Department of Biology, Universidad de Santiago de Chile, Santiago, Chile

    Francisco Leiva, Scarlet Bravo, Killen Ko Garcia & Rodrigo Vidal

  2. Benchmark Animal Health Chile, Santa Rosa 560 Of.26, Puerto Varas, Chile

    Javier Moya

  3. IDEVAC SpA, Francisco Bilbao 1129 Of. 306, Osorno, Chile

    Osiel Guzman

Authors
  1. Francisco Leiva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Scarlet Bravo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Killen Ko Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Javier Moya
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Osiel Guzman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rodrigo Vidal
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.L. and R.V designed research; S.B., K. K. G., and F.L., developed experimental protocols and bioinformatics pipelines; J.M. conducted the sampling; and R.V., F.L., and K. K. G, wrote the paper. All authors reviewed the manuscript.

Corresponding author

Correspondence to Rodrigo Vidal.

Ethics declarations

Ethics Approval

The totality of the animal experiments in this study performed in agreement with the Institutional Ethics Committee of the Universidad de Santiago (USCH-1) and AVMA- 2019 guidelines.

Competing Interests

The authors declare no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (XLSX 7444 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Leiva, F., Bravo, S., Garcia, K.K. et al. Temporal Gene Expression Signature of Plasma Extracellular Vesicles-MicroRNAs from Post-Smolt Coho Salmon Challenged with Piscirickettsia salmonis. Mar Biotechnol 23, 602–614 (2021). https://doi.org/10.1007/s10126-021-10049-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-021-10049-0

Keywords

Search

Navigation