Development Genes and Evolution

, Volume 226, Issue 5, pp 339–348 | Cite as

Genomic identification, rapid evolution, and expression of Argonaute genes in the tilapia, Oreochromis niloticus

  • Wenjing Tao
  • Lina Sun
  • Jinlin Chen
  • Hongjuan Shi
  • Deshou Wang
Original Article


Argonaute proteins are key components of the small RNA-induced silencing complex and have multiple roles in RNA-directed regulatory pathways. Argonaute genes can be divided into two subfamilies: the Ago (interacting with microRNA/small interfering RNA) and Piwi subfamilies (interacting with piwi-interacting RNAs (piRNAs)). In the present study, genome-wide analyses firstly yielded the identification of different members of Agos and Piwis in the tilapia, coelacanth, spotted gar, and elephant shark. The additional teleost Ago3b was generated following the fish-specific genome duplication event. Selective pressure analysis on Agos and Piwis between cichlids and other teleosts showed an accelerated evolution of Piwil1 in the cichlid lineages, and the positive selected sites were located in the region of PIWI domain, suggesting that these amino acid substitutions are adapt to targeted cleavage of messenger RNA (mRNA) in cichlids. Ago1 and Ago4 were detected at higher levels at 5 days after hatching (dah) in both ovaries and testes compared with other stages, supporting the previously reported requirement of Ago-mediated pathways to clear the maternal mRNAs during the early embryogenesis. The Piwis were abundantly expressed in tilapia testes, indicating their essential roles in male germline, especially in spermatogenesis. Notable expression of Piwis was also detected in skeletal muscle, indicating that piRNA pathway may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. The expression of Piwil1 and Piwil2 was examined by quantitative PCR (qPCR) and in situ hybridization (ISH) to validate the spatial and temporal expression profiles. Taken together, these results present a thorough overview of tilapia Argonaute family and provide a new perspective on the evolution and function of this family in teleosts.

Key words

Argonaute family Positive selection Expression profile Tilapia 



This work was supported by grants 31502147, 91331119, 31030063, and 31572609 from the National Natural Science Foundation of China; grant 2012CB723205 from the National Basic Research Program of China; grant 20130182130003 from the Specialized Research Fund for the Doctoral Program of Higher Education of China; grants cstc2013kjrc-tdjs80003 and cstc2014jcyjA80001 from the Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission; and grants XDJK2016A003, XDJK2016B011, and XDJK2014B040 from the Fundamental Research Funds for the Central Universities.

Authors’ contributions

This study was designed by DSW and WJT and organized by DSW. JLC managed the experimental fish, HJS and LNS dissected the fish gonads, and WJT and JLC carried out RNA extractions. WJT, JLC, and LNS carried out the bioinformatics analyses. WJT, DSW, and HJS wrote the manuscript, and all authors critiqued the manuscript for important intellectual content. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

427_2016_554_MOESM1_ESM.xls (38 kb)
Table S1 Accession numbers and genomic locations of vertebrate Argonaute genes. (XLS 38 kb)
427_2016_554_MOESM2_ESM.xlsx (11 kb)
Table S2 Accession numbers and genomic locations of cichlid Argonaute genes. (XLSX 11 kb)
427_2016_554_Fig7_ESM.gif (50 kb)
Fig. S1

Synteny map comparing the orthologs of the Piwil1 gene locus and the genes flanking it in human, Coelacanth, chicken, clawed frog, spotted gar, fugu, tilapia and zebrafish. This map has been obtained by using the genome browser Genomicus. The genes (indicated by arrows) on the first line are from the reference species tilapia. The direction of arrows indicates the gene orientation only in this species. Orthologs of each gene in other species are shown in the same column. (GIF 49 kb)

427_2016_554_MOESM3_ESM.tif (399 kb)
High resolution image (TIFF 398 kb)
427_2016_554_Fig8_ESM.gif (457 kb)
Fig. S2

Full amino acid sequences of Piwil. Solid lines indicate PIWI and PAZ domains. (GIF 457 kb)

427_2016_554_MOESM4_ESM.tif (2.4 mb)
High resolution image (TIFF 2.39 mb)


  1. Al-Janabi O, Wach S, Nolte E, Weigelt K, Rau TT, Stohr C, Legal W, Schick S, Greither T, Hartmann A et al (2014) Piwi-like 1 and-4 gene transcript levels are associated with clinicopathological parameters in renal cell carcinomas. Cancer Res 74Google Scholar
  2. Amores A, Force A, Yan YL, Joly L, Amemiya C, Fritz A, Ho RK, Langeland J, Prince V, Wang YL, et al. (1998) Zebrafish hox clusters and vertebrate genome evolution. Science 282:1711–1714CrossRefPubMedGoogle Scholar
  3. Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, Landgraf P, Iovino N, Morris P, Brownstein MJ, Kuramochi-Miyagawa S, Nakano T, et al. (2006) A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442:203–207PubMedGoogle Scholar
  4. Aryal R, Jagadeeswaran G, Zheng Y, Yu QY, Sunkar R, Ming R (2014) Sex specific expression and distribution of small RNAs in papaya. BMC Genomics 15Google Scholar
  5. Bohne A, Heule C, Boileau N, Salzburger W (2013) Expression and sequence evolution of aromatase cyp19a1 and other sexual development genes in East African cichlid fishes. Mol Biol Evol 30:2268–2285CrossRefPubMedPubMedCentralGoogle Scholar
  6. Brawand D, Wagner CE, Li YI, Malinsky M, Keller I, Fan SH, Simakov O, Ng AY, Lim ZW, Bezault E, et al. (2014) The genomic substrate for adaptive radiation in African cichlid fish. Nature 513:375CrossRefPubMedPubMedCentralGoogle Scholar
  7. Carmell MA, Girard A, van de Kant HJG, Bourc’his D, Bestor TH, de Rooij DG, Hannon GJ (2007) MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline. Dev Cell 12:503–514CrossRefPubMedGoogle Scholar
  8. Carmell MA, Xuan ZY, Zhang MQ, Hannon GJ (2002) The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev 16:2733–2742CrossRefPubMedGoogle Scholar
  9. Castaneda J, Genzor P, Bortvin A (2011) piRNAs, transposon silencing, and germline genome integrity. Mutat Res 714:95–104CrossRefPubMedGoogle Scholar
  10. Castillo DM, Mell JC, Box KS, Blumenstiel JP (2011) Molecular evolution under increasing transposable element burden in Drosophila: a speed limit on the evolutionary arms race. Bmc Evol Biol 11Google Scholar
  11. Cerutti H, Casas-Mollano JA (2006) On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50:81–99CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cheng YY, Tao WJ, Chen JL, Sun LN, Zhou LY, Song Q, Wang DS (2015) Genome-wide identification, evolution and expression analysis of nuclear receptor superfamily in Nile tilapia, Oreochromis niloticus. Gene 569:141–152CrossRefPubMedGoogle Scholar
  13. Coggill P, Finn RD, Bateman A (2008) Identifying protein domains with the Pfam database. Curr Protoc Bioinformatics Chapter 2, Unit 2 5Google Scholar
  14. Cook MS, Blelloch R (2013) Small RNAs in germline development. Curr Top Dev Biol 102:159–205CrossRefPubMedGoogle Scholar
  15. Deshpande G, Calhoun G, Schedl P (2005) Drosophila argonaute-2 is required early in embryogenesis for the assembly of centric/centromeric heterochromatin, nuclear division, nuclear migration, and germ-cell formation. Genes Dev 19:1680–1685CrossRefPubMedPubMedCentralGoogle Scholar
  16. Fablet M, Akkouche A, Braman V, Vieira C (2014) Variable expression levels detected in the Drosophila effectors of piRNA biogenesis. Gene 537:149–153Google Scholar
  17. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  18. Giraldez AJ, Mishima Y, Rihel J, Grocock RJ, Van Dongen S, Inoue K, Enright AJ, Schier AF (2006) Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312:75–79CrossRefPubMedGoogle Scholar
  19. Girard A, Sachidanandam R, Hannon GJ, Carmell MA (2006) A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442:199–202PubMedGoogle Scholar
  20. Halic M, Moazed D (2009) Transposon silencing by piRNAs. Cell 138:1058–1060CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hall TMT (2005) Structure and function of argonaute proteins. Structure 13:1403–1408CrossRefPubMedGoogle Scholar
  22. Hedges SB (2002) The origin and evolution of model organisms. Nat Rev Genet 3:838–849CrossRefPubMedGoogle Scholar
  23. Houwing S, Kamminga LM, Berezikov E, Cronembold D, Girard A, van den Elst H, Filippov DV, Blaser H, Raz E, Moens CB, et al. (2007) A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in zebrafish. Cell 129:69–82CrossRefPubMedGoogle Scholar
  24. Kobayashi T, Kajiura-Kobayashi H, Nagahama Y (2000) Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus. Mech Dev 99:139–142CrossRefPubMedGoogle Scholar
  25. Kocher TD (2004) Adaptive evolution and explosive speciation: the cichlid fish model. Nat Rev Genet 5:288–298CrossRefPubMedGoogle Scholar
  26. Kolaczkowski B, Hupalo DN, Kern AD (2011) Recurrent adaptation in RNA interference genes across the drosophila phylogeny. Mol Biol Evol 28:1033–1042CrossRefPubMedGoogle Scholar
  27. Li MH, Wu FR, Gu Y, Wang TR, Wang H, Yang SJ, Sun YL, Zhou LY, Huang XG, Jiao BW, Cheng HK, Wang DS (2012) Insulin-like growth factor 3 regulates expression of genes encoding steroidogenic enzymes and key transcription factors in the Nile tilapia gonad. Biol Reprod 86(5):163, 1–10. doi: 10.1095/biolreprod.111.096248
  28. Lim SL, Tsend-Ayush E, Kortschak RD, Jacob R, Ricciardelli C, Oehler MK, Grutzner F (2013) Conservation and expression of PIWI-interacting RNA pathway genes in male and female adult gonad of amniotes. Biol Reprod 89:136CrossRefPubMedGoogle Scholar
  29. Liu G, Luo F, Song Q, Wu LM, Qiu YX, Shi HJ, Wang DS, Zhou LY (2014) Blocking of progestin action disrupts spermatogenesis in Nile tilapia (Oreochromis niloticus). J Mol Endocrinol 53:57–70CrossRefPubMedGoogle Scholar
  30. Liu J, Luo MJ, Sheng Y, Hong Q, Cheng HH, and Zhou RJ (2015) Dynamic evolution and biogenesis of small RNAs during sex reversal. Sci Rep-Uk 5Google Scholar
  31. Liu JD, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305:1437–1441CrossRefPubMedGoogle Scholar
  32. Louis A, Muffato M, Roest Crollius H (2013) Genomicus: five genome browsers for comparative genomics in eukaryota. Nucleic Acids Res 41:D700–D705CrossRefPubMedGoogle Scholar
  33. Louis A, Nguyen NT, Muffato M, Roest Crollius H (2015) Genomicus update 2015: KaryoView and MatrixView provide a genome-wide perspective to multispecies comparative genomics. Nucleic Acids Res 43:D682–D689CrossRefPubMedGoogle Scholar
  34. Ma JB, Yuan YR, Meister G, Pei Y, Tuschl T, Patel DJ (2005) Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434:666–670CrossRefPubMedPubMedCentralGoogle Scholar
  35. McFarlane L, Svingen T, Braasch I, Koopman P, Schartl M, Wilhelm D (2011) Expansion of the Ago gene family in the teleost clade. Dev Genes Evol 221:95–104CrossRefPubMedGoogle Scholar
  36. Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349CrossRefPubMedGoogle Scholar
  37. Meyer A, Van de Peer Y (2005) From 2R to 3R: evidence for a fish-specific genome duplication (FSGD). BioEssays 27:937–945CrossRefPubMedGoogle Scholar
  38. Morgan CC, Loughran NB, Walsh TA, Harrison AJ, O’Connell MJ (2010) Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins. BMC Evol Biol 10:39CrossRefPubMedPubMedCentralGoogle Scholar
  39. Murchison EP, Kheradpour P, Sachidanandam R, Smith C, Hodges E, Xuan Z, Kellis M, Grutzner F, Stark A, Hannon GJ (2008) Conservation of small RNA pathways in platypus. Genome Res 18:995–1004CrossRefPubMedPubMedCentralGoogle Scholar
  40. Obbard DJ, Jiggins FM, Halligan DL, Little TJ (2006) Natural selection drives extremely rapid evolution in antiviral RNAi genes. Curr Biol 16:580–585CrossRefPubMedGoogle Scholar
  41. Parker JS, Roe SM, Barford D (2004) Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J 23:4727–4737CrossRefPubMedPubMedCentralGoogle Scholar
  42. Peters L, Meister G (2007) Argonaute proteins: mediators of RNA silencing. Mol Cell 26:611–623CrossRefPubMedGoogle Scholar
  43. Platt RN, Vandewege MW, Kern C, Schmidt CJ, Hoffmann FG, Ray DA (2014) Large numbers of novel miRNAs originate from DNA transposons and are coincident with a large species radiation in bats. Mol Biol Evol 31:1536–1545CrossRefPubMedGoogle Scholar
  44. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, et al. (2012) The Pfam protein families database. Nucleic Acids Res 40:D290–D301CrossRefPubMedGoogle Scholar
  45. Sasaki T, Shiohama A, Minoshima S, Shimizu N (2003) Identification of eight members of the Argonaute family in the human genome. Genomics 82:323–330CrossRefPubMedGoogle Scholar
  46. Shekar PC, Naim A, Sarathi DP, Kumar S (2011) Argonaute-2-null embryonic stem cells are retarded in self-renewal and differentiation. J Biosci 36:649–657CrossRefPubMedGoogle Scholar
  47. Shen XY, Cui JZ, Gong QL (2011) Fox gene loci in Takifugu rubripes and Tetraodon nigroviridis genomes and comparison with those of medaka and zebrafish genomes. Genome 54:965–972CrossRefPubMedGoogle Scholar
  48. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefPubMedPubMedCentralGoogle Scholar
  49. Tao WJ, Yuan J, Zhou LY, Sun LN, Sun YL, Yang SJ, Li MH, Zeng S, Huang BF, Wang DH (2013) Characterization of gonadal transcriptomes from Nile tilapia (Oreochromis niloticus) reveals differentially expressed genes. PLoS One 8Google Scholar
  50. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25:1105–1111CrossRefPubMedPubMedCentralGoogle Scholar
  51. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2014) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and cufflinks (vol 7, pg 562, 2012). Nat Protoc 9:2513–2513CrossRefGoogle Scholar
  52. Williams RW, Rubin GM (2002) ARGONAUTE1 is required for efficient RNA interference in drosophila embryos. Proc Natl Acad Sci U S A 99:6889–6894CrossRefPubMedPubMedCentralGoogle Scholar
  53. Winter J, Jung S, Keller S, Gregory RI, Diederichs S (2009) Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol 11:228–234CrossRefPubMedGoogle Scholar
  54. Yan Z, Hu HY, Jiang X, Maierhofer V, Neb E, He L, Hu YH, Hu H, Li N, Chen W, et al. (2011) Widespread expression of piRNA-like molecules in somatic tissues. Nucleic Acids Res 39:6596–6607CrossRefPubMedPubMedCentralGoogle Scholar
  55. Yang ZH, Nielsen R, Goldman N, Pedersen AMK (2000) Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics 155:431–449PubMedPubMedCentralGoogle Scholar
  56. Yang ZH, Wong WSW, Nielsen R (2005) Bayes empirical Bayes inference of amino acid sites under positive selection. Mol Biol Evol 22:1107–1118CrossRefPubMedGoogle Scholar
  57. Yao QY, Xia EH, Liu FH, Gao LZ (2015) Genome-wide identification and comparative expression analysis reveal a rapid expansion and functional divergence of duplicated genes in the WRKY gene family of cabbage, Brassica oleracea var. capitata. Gene 557:35–42CrossRefPubMedGoogle Scholar
  58. Yi MH, Chen F, Luo MJ, Cheng YB, Zhao HB, Cheng HH, Zhou RJ (2014) Rapid evolution of piRNA pathway in the teleost fish: implication for an adaptation to transposon diversity. Genome Biol Evol 6:1393–1407CrossRefPubMedPubMedCentralGoogle Scholar
  59. Yigit E, Batista PJ, Bei YX, Pang KM, Chen CCG, Tolia NH, Joshua-Tor L, Mitani S, Simard MJ, Mello CC (2006) Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell 127:747–757CrossRefPubMedGoogle Scholar
  60. Yuan J, Tao WJ, Cheng YY, Huang BF, Wang  DS (2014) Genome-wide identification, phylogeny, and gonadal expression of fox genes in Nile tilapia, Oreochromis niloticus. Fish Physiol Biochem 40:1239–1252Google Scholar
  61. Yuan YR, Pei Y, Ma JB, Kuryavyi V, Zhadina M, Meister G, Chen HY, Dauter Z, Tuschl T, Patel DJ (2005) Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol Cell 19:405–419CrossRefPubMedPubMedCentralGoogle Scholar
  62. Zhang XB, Wang H, Li MH, Cheng YY, Jiang DN, Sun LN, Tao WJ, Zhou LY, Wang ZJ, Wang DS (2014) Isolation of doublesex- and Mab-3-related transcription factor 6 and its involvement in spermatogenesis in tilapia. Biol Reprod 91(6):136CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life ScienceSouthwest UniversityChongqingPeople’s Republic of China

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