Bivalve transcriptomics reveal pathogen sequences and a powerful immune response of the Mediterranean mussel (Mytilus galloprovincialis)
Bivalves have colonized the interface between land and sea for the last 500 million years. Although bivalves lack an adaptive immune system, they are extraordinarily well adapted to adverse environmental conditions. Bivalves are valuable aquaculture resources worldwide and are used as sentinels for monitoring pollution. In this work, the immune transcriptomes of mussels (Mytilus galloprovincialis and edulis) and clam (Ruditapes decussatus) were sequenced. For comparative purposes, an already published transcriptome dataset of Ruditapes philippinarum was also included in the analyses. The 454 pyrosequencing of stimulated hemocytes resulted in more than 400,000 reads for each transcriptome. The percentage of annotated sequences ranges from 50% for mussels to 30–40% for clams. Considering the 28,061 non-redundant sequences from the four transcriptomes, the four species share 785 genes. Moreover, sequences related to different putative pathogens were found in the four bivalves. A high number of bivalve herpesvirus ORFs were found, which confirms the value of NGSs as tools to detect and quantify pathogen RNA. Based on an examination of the immune-enriched transcriptomes of these four species, we can conclude that bivalves present an immune system that differs from its conventional characterization as a simple innate immune response against invading pathogens. Enrichment analyses showed that species in the Mytilus genus, especially M. galloprovincialis, possesses a significantly higher number of sequences related to immune processes and killing molecules than species in the Ruditapes genus. This could be related to the broader ecological niche occupied by mussels and the scarcity of reported mussel mass mortalities compared to the high number of mass mortalities reported for clams.
This work has been funded by the EU Project REPROSEED (245119) and 10 PXIB 402 096 PR from Xunta de Galicia; and partially supported by Ministerio de Economía y Competitividad through Intramural 201640E024 and MYTIPEP (AGL2015-65705-R). We also acknowledge the support of Xunta de Galicia to our group (IN607B 2016/12). RM wishes to acknowledge the Spanish MICINN for her FPI Spanish research Grant (BES-2009-029765) and the EU H2020 funded Project VIVALDI (678589). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). We would also like to thank Ainhoa Blanco for the supply of the blue mussel, Mytilus edulis.
BN, LB and AF conceived and designed the project. PB and MM performed the annotation step. RM and PB made the functional annotation analyses. GFC did the BUSCO analyses. BN and RM made the analysis of pathogen sequences. RM wrote the manuscript. All listed authors revised, edited, read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Bower SM (2010) Synopsis of infectious diseases and parasites of commercially exploited shellfish. Fisheries and oceans Canada. http://www.dfo-mpo.gc.ca/science/aah-saa/diseases-maladies/index-eng.html. Accessed Nov 2017
- Carlsson J, Gauthier DT, Carlsson JEL, Coughlan JP, Dillane E, Fitzgerald RD, Keating U, McGinnity P, Mirimin L, Cross TF (2013) Rapid, economical single-nucleotide polymorphism and microsatellite discovery based on de novo assembly of a reduced representation genome in a non-model organism: a case study of Atlantic cod Gadus morhua. J Fish Biol 82:944–958PubMedCrossRefGoogle Scholar
- Dame RF (1993) The role of bivalve filter feeder material fluxes in estuarine ecosystems. In: Dame RF (ed) Bivalve filter feeders. Nato ASI Series (Series G: Ecological Sciences), vol 33. Springer, Berlin, Heidelberg, pp 245–269Google Scholar
- Fisher WS (1986) Structure and functions of oyster hemocytes. In: Brehélin M (ed) Immunity in invertebrates. Proceedings in life sciences. Springer, BerlinGoogle Scholar
- Gerdol M, De Moro G, Manfrin C, Milandri A, Riccardi E, Beran A, Venier P, Pallavicini A (2014) RNA sequencing and de novo assembly of the digestive gland transcriptome in Mytilus galloprovincialis fed with toxinogenic and non-toxic strains of Alexandrium minutum. BMC Res Notes 7:722PubMedPubMedCentralCrossRefGoogle Scholar
- Kinoshita S, Wang N, Inoue H, Maeyama K, Okamoto K, Nagai K, Kondo H, Hirono I, Asakawa S, Watabe S (2011) Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster. PLoS One 6:e21238PubMedPubMedCentralCrossRefGoogle Scholar
- Le Roux F, Wegner KM, Baker-Austin C, Vezzulli L, Osorio CR, Amaro C, Ritchie JM, Defoirdt T, Destoumieux-Garzón D, Blokesch M, Mazel D, Jacq A, Cava F, Gram L, Wendling CC, Strauch E, Kirschner A, Huehn S (2015) The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis. Front Microbiol 6:830PubMedPubMedCentralGoogle Scholar
- Leite RB, Milan M, Coppe A, Bortoluzzi S, dos Anjos A, Reinhardt R, Saavedra C, Patarnello T, Cancela ML, Bargelloni L (2013) mRNA-Seq and microarray development for the Grooved Carpet shell clam, Ruditapes decussatus: a functional approach to unravel host–parasite interaction. BMC Genom 14:741CrossRefGoogle Scholar
- Marteil L (1976) Shellfish culture in France. Part 2. Oyster and mussel biology. Original title: La conchyliculture française. Biologie de l’huître et de la moule. Revue des Travaux de l´institut des Pêches maritimes. France: IFREMER 40:149–345. http://archimer.ifremer.fr/doc/00000/1796/. Accessed Nov 2017
- Milan M, Coppe A, Reinhardt R, Cancela LM, Leite RB, Saavedra C, Ciofi C, Chelazzi G, Patarnello T, Bortoluzzi S, Bargelloni L (2011) Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring. BMC Genom 12:234CrossRefGoogle Scholar
- Moreira R, Balseiro P, Romero A, Dios S, Posada D, Novoa B, Figueras A (2012a) Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity. Dev Comp Immunol 36:140–149PubMedCrossRefGoogle Scholar
- Morton B (1981) The anomalodesmata. Malacologia 21:35–60Google Scholar
- Newell RIE (2004) Ecosystem influences of natural and cultivated populations of suspension-feeding bivalve molluscs: a review. J Shellfish Res 23:51–61Google Scholar
- Novoa B, Romero A, Álvarez ÁL, Moreira R, Pereiro P, Costa MM, Dios S, Estepa A, Parra F, Figueras A (2016) Antiviral activity of myticin C peptide from mussel: an ancient defence against herpesviruses. J Virol 90(7692):7702Google Scholar
- Pinkenburg O, Meyer T, Bannert N, Norley S, Bolte K, Czudai-Matwich V, Herold S, Gessner A, Schnare M (2016) The human antimicrobial protein bactericidal/permeability-increasing protein (BPI) inhibits the infectivity of influenza a virus. PLoS One 11:e0156929PubMedPubMedCentralCrossRefGoogle Scholar
- Riesgo A, Andrade SC, Sharma P, Novo M, Pérez-Porro AR, Vahtera V, González VL, Kawauchi GY, Giribet G (2012) Comparative description of ten transcriptomes of newly sequenced invertebrates and efficiency estimation of genomic sampling in non-model taxa. Front Zool 9:33PubMedPubMedCentralCrossRefGoogle Scholar
- Romero A, Dios S, Poisa-Beiro L, Costa MM, Posada D, Figueras A, Novoa B (2011) Individual sequence variability and functional activities of fibrinogen-related proteins (FREPs) in the Mediterranean mussel (Mytilus galloprovincialis) suggest ancient and complex immune recognition models in invertebrates. Dev Comp Immunol 35:334–344PubMedCrossRefGoogle Scholar
- Shagin DA, Rebrikov DV, Kozhemyako VB, Altshuler IM, Shcheglov AS, Zhulidov PA, Bogdanova EA, Staroverov DB, Rasskazov VA, Lukyanov S (2002) A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. Genome Res 12:1935–1942PubMedPubMedCentralCrossRefGoogle Scholar
- Wang J, Wang L, Yang C, Jiang Q, Zhang H, Yue F, Huang M, Sun Z, Song L (2013) The response of mRNA expression upon secondary challenge with Vibrio anguillarum suggests the involvement of C-lectins in the immune priming of scallop Chlamys farreri. Dev Comp Immunol 40:142–147PubMedCrossRefGoogle Scholar