Skip to main content
Log in

Analysis of novel immune–related genes and microsatellite markers in the transcriptome of Paphia undulata

  • Biology
  • Published:
Journal of Oceanology and Limnology Aims and scope Submit manuscript

Abstract

Increasingly, exogenous stressors such as pathogen infections, variable water conditions, and pollution are resulting in high mortality of Paphia undulata, deleteriously affecting the quality of clam harvests. The foot is a burrowing organ in clams. Physical damage and constant contact with the external environment cause the foot to be highly sensitive to pathogen invasion and water condition variation. In the present study, the foot tissue transcriptome was analyzed to identify genes involved in immune and stress responses. The P. undulata transcriptome included 5 286 668 078 bp reads generated by Illumina Hiseq2000 sequencing and were assembled into 1 785 226 contigs by de novo method. The contigs were clustered into 99 339 transcripts and further grouped into 60 201 unigenes. Of them, 22 260 unigenes were successfully annotated using public databases. Twelve genes that were response to immune and stress were identified with abundant expression levels, including heat shock protein 70, cold shock protein, complement C3, cathepsin L, ubiquitin carboxyl–terminal hydrolase L5, and translationally controlled tumor protein. Furthermore, 566 unigenes were found homologous to genes involved in the immune response systems of pathogen discrimination, signal transduction, and immune effector, such as lectins, toll–like receptors, complement pathway, toll–like receptor signaling pathway, heat shock proteins, antioxidant enzymes, lysozymes, and mucins, indicating that P. undulata could have a complete set of innate immune mechanisms. In addition, 4 270 microsatellite markers (SSRs) were identified from 60 201 unigenes, of which trinucleotide repeats were most abundant and 16 SSRs were tested to be polymorphic. The present study provides a new insight into innate immunity and stress response mechanisms in P. undulata.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Aguirre J, Ríos–Momberg M, Hewitt D, Hansberg, W. 2005. Reactive oxygen species and development in microbial eukaryotes. Trends Microbiol., 13(3): 111–118.

    Article  Google Scholar 

  • Akira S. 2003. Toll–like receptor signaling. J. Biol. Chem., 278(40): 38 105–38 108.

    Article  Google Scholar 

  • Altschul S F, Madden T L, Schäffer A, Zhang J H, Zhang Z, Miller W, Lipman D J. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res., 25(17): 3 389–3 402.

    Article  Google Scholar 

  • An H S, Lee J W. 2012. Development of microsatellite markers for the Korean mussel, Mytilus coruscus (Mytilidae) using next–generation sequencing. Int. J. Mol. Sci., 13(8): 10 583–10 593.

    Article  Google Scholar 

  • Bachali S, Jager M, Hassanin A, Schoentgen F, Jollès P, Fiala–Medioni A, Deutsch J S. 2002. Phylogenetic analysis of invertebrate lysozymes and the evolution of lysozyme function. J. Mol. Evol., 54(5): 652–664.

    Article  Google Scholar 

  • Bahia A C, Kubota M S, Tempone A J, Pinheiro W D, Tadei W P, Secundino N F C, Traub–Csekö Y M, Pimenta P F P. 2010. Anopheles aquasalis infected by Plasmodium vivax displays unique gene expression profiles when compared to other malaria vectors and plasmodia. PLoS One, 5 (3): e9795.

    Article  Google Scholar 

  • Beaz–Hidalgo R, Balboa S, Romalde J L, Figueras M J. 2010. Diversity and pathogenecity of Vibrio species in cultured bivalve molluscs. Environ. Microbiol. Rep., 2(1): 34–43.

    Article  Google Scholar 

  • Bettencourt R, Pinheiro M, Egas C, Gomes P, Afonso M, Shank T, Santos R S. 2010. High–throughput sequencing and analysis of the gill tissue transcriptome from the deep–sea hydrothermal vent mussel Bathymodiolus azoricus. BMC Genomics, 11: 559.

    Article  Google Scholar 

  • Botstein D, White R L, Skolnick M, Davis R W. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet., 32(3): 314–331.

    Google Scholar 

  • Brun N T, Bricelj V M, MacRae T H, Ross N W. 2008. Heat shock protein responses in thermally stressed bay scallops, Argopecten irradians, and sea scallops, Placopecten magellanicus. J. Exp. Mar. Biol. Ecol., 358(2): 151–162.

    Article  Google Scholar 

  • Carroll M C. 2004. The complement system in regulation of adaptive immunity. Nat. Immunol., 5(10): 981–986.

    Article  Google Scholar 

  • Chen X M, Li J K, Xiao S J, Liu X D. 2016. De novo assembly and characterization of foot transcriptome and microsatellite marker development for Paphia textile. Gene, 576(1): 537–543.

    Article  Google Scholar 

  • Clark M S, Thorne M A S, Vieira F A, Cardoso J C R, Power D M, Peck L S. 2010. Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing. BMC Genomics, 11: 362.

    Article  Google Scholar 

  • Conesa A, Götz S, García–Gómez J M, Terol J, Talón M, Robles M. 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21(18): 3 674–3 676.

    Article  Google Scholar 

  • Cronin S J, Nehme N T, Limmer S, Liegeois S, Pospisilik J A, Schramek D, Leibbrandt A, Simoes R D M, Gruber S, Puc U, Ebersberger I, Zoranovic T, Neely G G, von Haeseler A, Ferrandon D, Penninger J M. 2009. Genome–wide RNAi screen identifies genes involved in intestinal pathogenic bacterial infection. Science, 325(5938): 340–343.

    Article  Google Scholar 

  • Delanghe J R, Speeckaert R, Speeckaert M M. 2014. Complement C3 and its polymorphism: biological and clinical consequences. Pathology, 46(1): 1–10.

    Article  Google Scholar 

  • Deng Y W, Lei Q N, Tian Q L, Xie S H, Du X D, Li J H, Wang L Q, Xiang Y X. 2014. De novo assembly, gene annotation, and simple sequence repeat marker development using Illumina paired–end transcriptome sequences in the pearl oyster Pinctada maxima. Biosci. Biotechnol. Biochem., 78(10): 1 685–1 692.

    Article  Google Scholar 

  • Dunkelberger J R, Song W C. 2010. Complement and its role in innate and adaptive immune responses. Cell Res., 20(1): 34–50.

    Article  Google Scholar 

  • Feldmeyer B, Wheat C W, Krezdorn N, Rotter B, Pfenninger M. 2011. Short read Illumina data for the de novo assembly of a non–model snail species transcriptome (Radix balthica, Basommatophora, Pulmonata), and a comparison of assembler performance. BMC Genomics, 12: 317.

    Article  Google Scholar 

  • Feng B B, Dong L L, Niu D H, Meng S S, Zhang B, Liu D B, Hu S N, Li J L. 2010. Identification of immune genes of the Agamaki clam (Sinonovacula constricta) by sequencing and bioinformatic analysis of ESTs. Mar. Biotechnol., 12(3): 282–291.

    Article  Google Scholar 

  • Gendler S J, Spicer A P. 1995. Epithelial mucin genes. Annu. Rev. Physiol., 57: 607–634.

    Article  Google Scholar 

  • Gerdol M, Manfrin C, De Moro G, Figueras A, Novoa B, Venier P, Pallavicini A. 2011. The C1q domain containing proteins of the Mediterranean mussel Mytilus galloprovincialis: a widespread and diverse family of immune–related molecules. Dev. Comp. Immunol., 35(6): 635–643.

    Article  Google Scholar 

  • Gourdine J P, Smith–Ravin E J. 2007. Analysis of a cDNAderived sequence of a novel mannose–binding lectin, codakine, from the tropical clam Codakia orbicularis. Fish Shellfish Immunol., 22(5): 498–509.

    Article  Google Scholar 

  • Grieshaber M K, Hardewig I, Kreutzer U, Pörlner H O. 1994. Physiological and metabolic responses to hypoxia in invertebrates. In: Reviews of Physiology, Biochemistry and Pharmacology. Springer, Berlin, Heidelberg. p.43–147.

    Google Scholar 

  • Gueguen Y, Cadoret J P, Flament D, Barreau–Roumiguière C, Girardot A L, Garnier J, Hoareau A, Bachère E, Escoubas J M. 2003. Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria–challenged oyster, Crassostrea gigas. Gene, 303: 139–145.

    Article  Google Scholar 

  • Haas B J, Papanicolaou A, Yassour M, Grabherr M, Blood P D, Bowden J, Couger M B, Eccles D, Li B, Lieber M, MacManes M D, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey C N, Henschel R, LeDuc R D, Friedman N, Regev A. 2013. De novo transcript sequence reconstruction from RNA–seq using the Trinity platform for reference generation and analysis. Nat. Protoc., 8(8): 1 494–1 512.

    Article  Google Scholar 

  • Huan P, Wang H X, Liu B Z. 2012. Transcrip tomic analysis of the clam Meretrix meretrix on different larval stages. Mar. Biotechnol., 14(1): 69–78.

    Article  Google Scholar 

  • Iwanaga S, Lee B L. 2005. Recent advances in the innate immunity of invertebrate animals. J. Biochem. Mol. Boil., 38(2): 128–150.

    Google Scholar 

  • Iwasaki A, Medzhitov R. 2004. Toll–like receptor control of the adaptive immune responses. Nat. Immunol., 5(10): 987–995.

    Article  Google Scholar 

  • Janeway C A Jr, Medzhitov R. 2002. Innate immune recognition. Annu. Rev. Immunol., 20: 197–216.

    Article  Google Scholar 

  • Jensen K T, Castro N F, Bachelet G. 1999. Infectivity of Himasthla spp. (Trematoda) in cockle (Cerastoderma edule) spat. J. Mar. Biol. Assoc. UK, 79(2): 265–271.

    Article  Google Scholar 

  • Jeong E, Lee J Y. 2011. Intrinsic and extrinsic regulation of innate immune receptors. Yonsei Med. J., 52(3): 379–392.

    Article  Google Scholar 

  • Jiang WN, Hon Y, Inouye M. 1997. CspA, the major coldshock protein of Escherichia coli, is an RNA chaperone. J. Biol. Chem., 272(1): 196–202.

    Article  Google Scholar 

  • Kim J Y, Adhya M, Cho S K, Choi K S, Cho M. 2008. Characterization, tissue expression, and immunohistochemical localization of MCL3, a C–type lectin produced by Perkinsus olseni–infected Manila clams (Ruditapes philippinarum). Fish Shellfish Immunol., 25(5): 598–603.

    Article  Google Scholar 

  • Lauckner G. 1983. Diseases of Mollusca: Bivalvia. In: Kinne O ed. Diseases of Marine Animals. Biologische Anstalt Helgoland, Hamburg, Germany. p.477–961.

    Google Scholar 

  • Leethochavalit S, Chalermwat K, Upatham E S, Choi K S, Sawangwong P, Kruatrachue M. 2004. Occurrence of Perkinsus sp. in undulated surf clams Paphia undulata from the Gulf of Thailand. Dis. Aquat. Org. 60(2): 165–171.

    Google Scholar 

  • Li H J, Liu W D, Gao X G, Zhu D, Wang J, Li Y F, He C B. 2011. Identification of host–defense genes and development of microsatellite markers from ESTs of hard clam Meretrix meretrix. Mol. Biol. Rep., 38(2): 769–775.

    Article  Google Scholar 

  • Loker E S, Adema C M, Zhang S M, Kepler T B. 2004. Invertebrate immune systems–not homogeneous, not simple, not well understood. Immunol. Rev., 198(1): 10–24.

    Article  Google Scholar 

  • Matsubara H, Ogawa T, Muramoto K. 2006. Structures and functions of C–type lectins in marine invertebrates. Tohoku J. Agric. Res., 57 (1–2): 71–86.

    Google Scholar 

  • Moreira R, Balseiro P, Planas J V, Fuste B, Beltran S, Novoa B, Figueras A. 2012. Transcriptomics of in vitro immunestimulated hemocytes from the Manila clam Ruditapes philippinarum using high–throughput sequencing. PLoS One, 7 (4): e35009.

    Article  Google Scholar 

  • Mouritsen K N, Poulin R. 2003. The risk of being at the top: foot–cropping in the New Zealand cockle Austrovenus stutchburyi. J. Mar. Biol. Assoc. UK, 83(3): 497–498.

    Article  Google Scholar 

  • Niu D H, Jin K, Wang L, Feng B B, Li J L. 2013b. Molecular characterization and expression analysis of four cathepsin L genes in the razor clam, Sinonovacula constricta. Fish Shellfish Immunol., 35(2): 581–588.

    Article  Google Scholar 

  • Niu D H, Wang L, Sun F Y, Liu Z J, Li J L. 2013a. Development of molecular resources for an intertidal clam, Sinonovacula constricta. using 454 transcriptome sequencing. PLoS One, 8 (7): e67456.

    Google Scholar 

  • O’Connell–Milne S, Poulin R, Savage C, Rayment W. 2016. Reduced growth, body condition and foot length of the bivalve Austrovenus stutchburyiq in response to parasite infection. J. Exp. Mar. Biol. Ecol., 474: 23–28.

    Article  Google Scholar 

  • Pallavicini A, del Mar Costa M, Gestal C, Dreos R, Figueras A, Venier P, Novoa B. 2008. High sequence variability of myticin transcripts in hemocytes of immune–stimulated mussels suggests ancient host–pathogen interactions. Dev. Comp. Immunol., 32(3): 213–226.

    Article  Google Scholar 

  • Parcellier A, Gurbuxani S, Schmitt E, Solary E, Garrido C. 2003. Heat shock proteins, cellular chaperones that modulate mitochondrial cell death pathways. Biochem. Biophys. Res. Commun., 304(3): 505–512.

    Article  Google Scholar 

  • Paul–Pont I, de Montaudouin X, Gonzalez P, Soudant P, Baudrimont M. 2010. How life history contributes to stress response in the Manila clam Ruditapes philippinarum. Environ. Sci. Pollut. Res., 17(4): 987–998.

    Article  Google Scholar 

  • Peng M X, Niu D H, Wang F, Chen Z Y, Li J L. 2016. Complement C3 gene: Expression characterization and innate immune response in razor clam Sinonovacula constricta. Fish Shellfish Immunol., 55: 223–232.

    Article  Google Scholar 

  • Philipp E, Pörtner HO, Abele D. 2005. Mitochondrial ageing of a polar and a temperate mud clam. Mech. Ageing Dev., 126(5): 610–619.

    Article  Google Scholar 

  • Ruddell R G, Hoang–Le D, Barwood J M, Rutherford P S, Piva T J, Watters D J, Santambrogio P, Arosio P, Ramm G A. 2009. Ferritin functions as a proinflammatory cytokine via iron–independent protein kinase C zeta/nuclear factor kappaB–regulated signaling in rat hepatic stellate cells. Hepatology, 49(3): 887–900.

    Article  Google Scholar 

  • Sambrook J, Fritsch E F, Maniatis J. 1989. Molecular Cloning: A Laboratory Manual. 2 nd edn. Cold Spring Harbor Laboratory Press, New York. 1625p.

    Google Scholar 

  • Shi M J, Lin Y, Xu G R, Xie L P, Hu X L, Bao Z M, Zhang R Q. 2013. Characterization of the Zhikong scallop (Chlamys farreri) mantle transcriptome and identification of biomineralization–related genes. Mar. Biotechnol., 15(6): 706–715.

    Article  Google Scholar 

  • Song L S, Wang L L, Qiu L M, Zhang H A. 2011. Bivalve immunity. In: Söderhäll K ed. Invertebrate Immunity. Springer, Boston, MA. p.44–65.

  • Song X Y, Zhang H, Zhao J M, Wang L L, Qiu L M, Mu C K, Liu X L, Qiu L H, Song L S. 2010. An immune responsive multidomain galectin from bay scallop Argopectens irradians. Fish Shellfish Immunol., 28(2): 326–332.

    Article  Google Scholar 

  • Takeda K, Akira S. 2005. Toll–like receptors in innate immunity. Int. Immunol., 17(1): 1–14.

    Article  Google Scholar 

  • Tatusov R L, Galperin M Y, Natale D A, Koonin E V. 2000. The COG database: a tool for genome–scale analysis of protein functions and evolution. Nucleic Acids Res., 28(1): 33–36.

    Article  Google Scholar 

  • Thieringer H A, Jones P G, Inouye M. 1998. Cold shock and adaptation. Bioessays, 20(1): 49–57.

    Article  Google Scholar 

  • Thomas F, Renaud F, de Meeûs T, Poulin R. 1998. Manipulation of host behaviour by parasites: ecosystem engineering in the intertidal zone? Proc. Roy. Soc. B: Biol. Sci., 265(1401): 1 091–1 096.

    Article  Google Scholar 

  • Underhill D M, Ozinsky A. 2002. Toll–like receptors: key mediators of microbe detection. Curr. Opin. Immunol., 14(1): 103–110.

    Article  Google Scholar 

  • van de Wetering J K, van Golde L M G, Batenburg J J. 2004. Collectins: players of the innate immune system. FEBS J., 271(7): 1 229–1 249.

    Google Scholar 

  • Venier P, Varotto L, Rosani U, Millino C, Celegato B, Bernante F, Lanfranchi G, Novoa B, Roch P, Figueras A, Pallavicini A. 2011. Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis. BMC Genomics, 12: 69.

    Article  Google Scholar 

  • Wang A M, Wang Y, Gu Z F, Li S F, Shi Y H, Guo X M. 2011. Development of expressed Sequence tags from the pearl oyster, Pinctada martensii Dunker. Mar. Biotechnol., 13(2): 275–283.

    Article  Google Scholar 

  • Wang L L, Qiu L M, Zhou Z, Song L S. 2013. Research progress on the mollusc immunity in China. Dev. Comp. Immunol., 39 (1–2): 2–10.

    Article  Google Scholar 

  • Wang L L, Song L S, Zhao J M, Qiu L M, Zhang H, Xu W, Li H L, Li C H, Wu L T, Guo X M. 2009. Expressed sequence tags from the Zhikong scallop (Chlamys farreri): discovery and annotation of hostdefense genes. Fish Shellfish Immunol., 26(5): 744–750.

    Article  Google Scholar 

  • Weber M H W, Fricke I, Doll N, Marahiel M A. 2002. CSDBase: an interactive database for cold shock domaincontaining proteins and the bacterial cold shock response. Nucleic Acids Res., 30(1): 375–378.

    Article  Google Scholar 

  • Wu X W, Tan J, Cai M Y, Liu X D. 2014. Molecular cloning, characterization, and expression analysis of a heat shock protein (HSP) 70 gene from Paphia undulata. Gene, 543(2): 275–285.

    Article  Google Scholar 

  • Yang C Y, Wang L L, Siva V S, Shi X W, Jiang Q F, Wang J J, Zhang H, Song L S. 2012. A novel cold–regulated cold shock domain containing protein from scallop Chlamys farreri with nucleic acid–binding activity. PLoS One, 7 (2): e32012.

    Article  Google Scholar 

  • Yeh F C, Yang R, Boyle T J, Ye Z, Xiyan J M. 2000. PopGene32, Microsoft Windows–based freeware for population genetic analysis, version 1.32. Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, Alberta, Canada.

    Google Scholar 

  • Yeh H Y, Klesius P H. 2010. Characterization and tissue expression of channel catfish (Ictalurus punctatus Rafinesque, 1818) ubiquitin carboxyl–terminal hydrolase L5 (UCHL5) cDNA. Mol. Biol. Rep., 37(3): 1 229–1 234.

    Article  Google Scholar 

  • Zhang H, Song L S, Li C H, Zhao J M, Wang H, Qiu L M, Ni D J, Zhang Y. 2008. A novel C1q–domain–containing protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity. Fish Shellfish Immunol., 25(3): 281–289.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ziniu Yu.

Additional information

Supported by the Open Program of Key Laboratory of Cultivation and High–value Utilization of Marine Organisms in Fujian Province (No. 2015fjscq05), the Guangdong Province Program, China (Nos. 2014B020202011, 2016A020208011, A201601A04, 2016B020233005, 2016TQ03N905), the Science and Technology Planning Project of Guangdong Province, China (No. 2017B030314052), and the China Agriculture Research System (No. CARS–49)

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wu, X., Liu, X. & Yu, Z. Analysis of novel immune–related genes and microsatellite markers in the transcriptome of Paphia undulata. J. Ocean. Limnol. 37, 1301–1316 (2019). https://doi.org/10.1007/s00343-019-8154-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00343-019-8154-5

Keyword

Navigation