Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

New genes helped acorn barnacles adapt to a sessile lifestyle

Nature Genetics (2024)Cite this article

Subjects

Abstract

Barnacles are the only sessile lineages among crustaceans, and their sessile life begins with the settlement of swimming larvae (cyprids) and the formation of protective shells. These processes are crucial for adaptation to a sessile lifestyle, but the underlying molecular mechanisms remain poorly understood. While investigating these mechanisms in the acorn barnacle, Amphibalanus amphitrite, we discovered a new gene, bcs-6, which is involved in the energy metabolism of cyprid settlement and originated from a transposon by acquiring the promoter and cis-regulatory element. Unlike mollusks, the barnacle shell comprises alternate layers of chitin and calcite and requires another new gene, bsf, which generates silk-like fibers that efficiently bind chitin and aggregate calcite in the aquatic environment. Our findings highlight the importance of exploring new genes in unique adaptative scenarios, and the results will provide important insights into gene origin and material development.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Chromosome-level genome assembly of A. amphitrite and the result of comparative genomic analysis.
Fig. 2: Key genes involved in cyprid settlement.
Fig. 3: Function and evolution of the transposon gene bcs-6.
Fig. 4: Microscopic observation, characterization and multi-omics of A. amphitrite shells.
Fig. 5: Identification and function of a barnacle-specific shell formation gene (bsf).
Fig. 6: The evolution and function of genes underlying acorn barnacle (A. amphitrite) adaptation to a sessile lifestyle.

Similar content being viewed by others

Data availability

The A. amphitrite genome and all of the sequence data have been deposited with BioProject under accession: PRJNA878644. The genome assembly and annotation files are available at figshare (https://doi.org/10.6084/m9.figshare.21310305)105. RNA-seq data of differential developmental stages were deposited with BioProject under accession: PRJNA877841. The induced and inhibited settlement of barnacles RNA-seq were deposited in the SRA database under accession: PRJNA878556. The mass spectrometry proteomics data have been deposited in the iProX integrated proteome resources under the accessions: PXD039109 and PXD039111. Source data are provided with this paper.

Code availability

The custom pipelines and scripts were deposited at Zenodo106 and GitHub (https://github.com/ZhaofangHan/Acorn-Barnacle-Genome)107.

References

  1. Love, A. C. Darwin and Cirripedia prior to 1846: exploring the origins of the barnacle research. J. Hist. Biol. 35, 251–289 (2002).

    Article  Google Scholar 

  2. Darwin, C. A Monograph on the Sub-class Cirripedia, With figures of all the Species. The Balanidae (or Sessile Cirrepedes); the Verrucidae, etc. Vol. 2, p. 684 (The Ray Society, 1854).

  3. Schultz, M., Bendick, J., Holm, E. & Hertel, W. Economic impact of biofouling on a naval surface ship. Biofouling 27, 87–98 (2011).

    Article  CAS  PubMed  Google Scholar 

  4. Callow, M. E. & Callow, J. A. Marine biofouling: a sticky problem. Biologist 49, 10–14 (2002).

    PubMed  Google Scholar 

  5. Fan, H., Wang, J. & Gong, J. P. Barnacle cement proteins‐inspired tough hydrogels with robust, long‐lasting, and repeatable underwater adhesion. Adv. Funct. Mater. 31, 2009334 (2021).

    Article  CAS  Google Scholar 

  6. Yuk, H. et al. Rapid and coagulation-independent haemostatic sealing by a paste inspired by barnacle glue. Nat. Biomed. Eng. 5, 1131–1142 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dreanno, C. et al. An α2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite. Proc. Natl Acad. Sci. USA 103, 14396–14401 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gohad, N. V. et al. Synergistic roles for lipids and proteins in the permanent adhesive of barnacle larvae. Nat. Commun. 5, 4414 (2014).

    Article  CAS  PubMed  Google Scholar 

  9. Fernández, M., Arias, J., Neira-Carrillo, A. & Arias, J. Austromegabalanus psittacus barnacle shell structure and proteoglycan localization and functionality. J. Struct. Biol. 191, 263–271 (2015).

    Article  PubMed  Google Scholar 

  10. Nousek, N. A. Shell formation and calcium transport in the barnacle Chthamalus fragilis. Tissue Cell 16, 433–442 (1984).

    Article  CAS  PubMed  Google Scholar 

  11. Fears, K. P., Orihuela, B., Rittschof, D. & Wahl, K. J. Acorn barnacles secrete phase‐separating fluid to clear surfaces ahead of cement deposition. Adv. Sci. 5, 1700762 (2018).

    Article  Google Scholar 

  12. Kamino, K. Mini-review: barnacle adhesives and adhesion. Biofouling 29, 735–749 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. So, C. R. et al. Oxidase activity of the barnacle adhesive interface involves peroxide-dependent catechol oxidase and lysyl oxidase enzymes. ACS Appl. Mater. Interfaces 9, 11493–11505 (2017).

    Article  CAS  PubMed  Google Scholar 

  14. Dickinson, G. H. et al. Barnacle cement: a polymerization model based on evolutionary concepts. J. Exp. Biol. 212, 3499–3510 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Liang, C. et al. Biochemistry of barnacle adhesion: an updated review. Front. Mar. Sci. 6, 565 (2019).

    Article  Google Scholar 

  16. Chen, S., Krinsky, B. H. & Long, M. New genes as drivers of phenotypic evolution. Nat. Rev. Genet. 14, 645–660 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tautz, D. & Domazet-Lošo, T. The evolutionary origin of orphan genes. Nat. Rev. Genet. 12, 692–702 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Yuan, J. et al. Convergent evolution of barnacles and molluscs sheds lights in origin and diversification of calcareous shell and sessile lifestyle. Proc. Biol. Sci. 289, 20221535 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Chan, B. K. et al. The evolutionary diversity of barnacles, with an updated classification of fossil and living forms. Zool. J. Linn. Soc. 193, 789–846 (2021).

    Article  Google Scholar 

  20. Walker, G. The adhesion of barnacles. J. Adhes. 12, 51–58 (1981).

    Article  CAS  Google Scholar 

  21. Aldred, N., Alsaab, A. & Clare, A. S. Quantitative analysis of the complete larval settlement process confirms Crisp’s model of surface selectivity by barnacles. Proc. Biol. Sci. 285, 20171957 (2018).

    PubMed  PubMed Central  Google Scholar 

  22. Matsumura, K. et al. Immunological studies on the settlement-inducing protein complex (SIPC) of the barnacle Balanus amphitrite and its possible involvement in larva–larva interactions. Proc. Biol. Sci. 265, 1825 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yamamoto, H., Tachibana, A., Matsumura, K. & Fusetani, N. Protein kinase C (PKC) signal transduction system involved in larval metamorphosis of the barnacle, Balanus amphitrite. Zool. Sci. 12, 391–396 (1995).

    Article  CAS  Google Scholar 

  24. Zhang, Y. et al. The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacle Balanus (= Amphibalanus) amphitrite. J. Exp. Biol. 215, 3813–3822 (2012).

    CAS  PubMed  Google Scholar 

  25. Lucas, M., Walker, G., Holland, D. & Crisp, D. An energy budget for the free-swimming and metamorphosing larvae of Balanus balanoides (Crustacea: Cirripedia). Mar. Biol. 55, 221–229 (1979).

    Article  Google Scholar 

  26. Fukushima, M. et al. Isolation and characterization of 2-nitroimidazole produced by Streptomyces species as an inhibitor of both carbonic anhydrase and shell formation in the barnacle Balanus amphitrite. Mar. Biotechnol. 4, 103–110 (2002).

    Article  CAS  Google Scholar 

  27. Matsubara, H. et al. Modulating effect of acorn barnacle C-type lectins on the crystallization of calcium carbonate. Fish. Sci. 74, 418–424 (2008).

    Article  CAS  Google Scholar 

  28. Zhang, G. et al. Chemical component and proteomic study of the Amphibalanus (= Balanus) amphitrite shell. PLoS ONE 10, e0133866 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Pastuzyn, E. D. et al. The neuronal gene arc encodes a repurposed retrotransposon gag protein that mediates intercellular RNA transfer. Cell 172, 275–288 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Percharde, M. et al. A LINE1-nucleolin partnership regulates early development and ESC identity. Cell 174, 391–405 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ong-Abdullah, M. et al. Loss of Karma transposon methylation underlies the mantled somaclonal variant of oil palm. Nature 525, 533–537 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chuong, E. B., Elde, N. C. & Feschotte, C. Regulatory activities of transposable elements: from conflicts to benefits. Nat. Rev. Genet. 18, 71–86 (2017).

    Article  CAS  PubMed  Google Scholar 

  33. Servais, T. & Harper, D. A. The great Ordovician biodiversification event (GOBE): definition, concept and duration. Lethaia 51, 151–164 (2018).

    Article  Google Scholar 

  34. Kumar, S., Stecher, G., Suleski, M. & Hedges, S. B. TimeTree: a resource for timelines, timetrees, and divergence times. Mol. Biol. Evol. 34, 1812–1819 (2017).

    Article  CAS  PubMed  Google Scholar 

  35. Mateo, L. J. et al. Visualizing DNA folding and RNA in embryos at single-cell resolution. Nature 568, 49–54 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Muszbek, L., Bereczky, Z., Bagoly, Z., Komáromi, I. & Katona, É. Factor XIII: a coagulation factor with multiple plasmatic and cellular functions. Physiol. Rev. 91, 931–972 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Okazaki, Y. & Shizuri, Y. Structures of six cDNAs expressed specifically at cypris larvae of barnacles, Balanus amphitrite. Gene 250, 127–135 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. Babcock, G. T. & Wikström, M. Oxygen activation and the conservation of energy in cell respiration. Nature 356, 301–309 (1992).

    Article  CAS  PubMed  Google Scholar 

  39. Fernie, A. R., Carrari, F. & Sweetlove, L. J. Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Curr. Opin. Plant Biol. 7, 254–261 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Høeg, J. T., Maruzzo, D., Okano, K., Glenner, H. & Chan, B. K. Metamorphosis in balanomorphan, pedunculated, and parasitic barnacles: a video-based analysis. Integr. Comp. Biol. 52, 337–347 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Khalifa, G. M., Weiner, S. & Addadi, L. Mineral and matrix components of the operculum and shell of the barnacle Balanus amphitrite: calcite crystal growth in a hydrogel. Cryst. Growth Des. 11, 5122–5130 (2011).

    Article  CAS  Google Scholar 

  42. Andersen, S. O. Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer. Insect Biochem. Mol. Biol. 30, 569–577 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. Martin, S. et al. Genomic adaptations to an endoparasitic lifestyle in the morphologically atypical crustacean Sacculina carcini (Cirripedia: Rhizocephala). Genome Biol. Evol. 14, evac149 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Serano, J. M. et al. Comprehensive analysis of Hox gene expression in the amphipod crustacean Parhyale hawaiensis. Dev. Biol. 409, 297–309 (2016).

    Article  CAS  PubMed  Google Scholar 

  45. Kapusta, A. et al. Transposable elements are major contributors to the origin, diversification, and regulation of vertebrate long noncoding RNAs. PLoS Genet. 9, e1003470 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Zhang, X. et al. Penaeid shrimp genome provides insights into benthic adaptation and frequent molting. Nat. Commun. 10, 356 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Zhang, G. et al. The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490, 49–54 (2012).

    Article  CAS  PubMed  Google Scholar 

  48. So, C. R. et al. Sequence basis of barnacle cement nanostructure is defined by proteins with silk homology. Sci. Rep. 6, 36219 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Zhong, C. et al. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nat. Nanotechnol. 9, 858–866 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Gan, K. et al. Adhesive materials inspired by barnacle underwater adhesion: biological principles and biomimetic designs. Front. Bioeng. Biotechnol. 10, 870445 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Liu, S., Cerione, R. A. & Clardy, J. Structural basis for the guanine nucleotide-binding activity of tissue transglutaminase and its regulation of transamidation activity. Proc. Natl Acad. Sci. USA 99, 2743–2747 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Chen, L., DeVries, A. L. & Cheng, C.-H. C. Evolution of antifreeze glycoprotein gene from a trypsinogen gene in Antarctic notothenioid fish. Proc. Natl Acad. Sci. USA 94, 3811–3816 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Baalsrud, H. T. et al. De novo gene evolution of antifreeze glycoproteins in codfishes revealed by whole genome sequence data. Mol. Biol. Evol. 35, 593–606 (2018).

    Article  CAS  PubMed  Google Scholar 

  54. Ruan, J. & Li, H. Fast and accurate long-read assembly with wtdbg2. Nat. Methods 17, 155–158 (2020).

    Article  CAS  PubMed  Google Scholar 

  55. Chin, C.-S. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10, 563–569 (2013).

    Article  CAS  PubMed  Google Scholar 

  56. Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Dudchenko, O. et al. De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds. Science 356, 92–95 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Durand, N. C. et al. Juicebox provides a visualization system for Hi-C contact maps with unlimited zoom. Cell Syst. 3, 99–101 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Parra, G., Bradnam, K. & Korf, I. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23, 1061–1067 (2007).

    Article  CAS  PubMed  Google Scholar 

  60. Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov, E. M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31, 3210–3212 (2015).

    Article  PubMed  Google Scholar 

  61. Stanke, M., Diekhans, M., Baertsch, R. & Haussler, D. Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics 24, 637–644 (2008).

    Article  CAS  PubMed  Google Scholar 

  62. Majoros, W. H., Pertea, M. & Salzberg, S. L. TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders. Bioinformatics 20, 2878–2879 (2004).

    Article  CAS  PubMed  Google Scholar 

  63. Korf, I. Gene finding in novel genomes. BMC Bioinformatics 5, 59 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Alioto, T., Blanco, E., Parra, G. & Guigó, R. Using geneid to identify genes. Curr. Protoc. Bioinformatics 64, e56 (2018).

    Article  PubMed  Google Scholar 

  65. Burge, C. B. & Karlin, S. Finding the genes in genomic DNA. Curr. Opin. Struct. Biol. 8, 346–354 (1998).

    Article  CAS  PubMed  Google Scholar 

  66. Slater, G. S. C. & Birney, E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics 6, 31 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Haas, B. J. et al. Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. Nucleic Acids Res. 31, 5654–5666 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Haas, B. J. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the program to assemble spliced alignments. Genome Biol. 9, R7 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  70. Bernot, J. P. et al. Chromosome-level genome assembly, annotation, and phylogenomics of the gooseneck barnacle Pollicipes pollicipes. GigaScience 11, giac021 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Cui, Z. et al. The Chinese mitten crab genome provides insights into adaptive plasticity and developmental regulation. Nat. Commun. 12, 2395 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Tang, B. et al. Chromosome-level genome assembly reveals the unique genome evolution of the swimming crab (Portunus trituberculatus). GigaScience 9, giz161 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Kao, D. et al. The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion. eLife 5, e20062 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  74. Kang, S. et al. The genome of the Antarctic-endemic copepod, Tigriopus kingsejongensis. GigaScience 6, 1–9 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Barreto, F. S. et al. Genomic signatures of mitonuclear coevolution across populations of Tigriopus californicus. Nat. Ecol. Evol. 2, 1250–1257 (2018).

    Article  PubMed  Google Scholar 

  76. Colbourne, J. K. et al. The ecoresponsive genome of Daphnia pulex. Science 331, 555–561 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Chipman, A. D. et al. The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima. PLoS Biol. 12, e1002005 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Li, L., Stoeckert, C. J. & Roos, D. S. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 13, 2178–2189 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  80. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Yang, Z. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput. Appl. Biosci. 13, 555–556 (1997).

    CAS  PubMed  Google Scholar 

  82. Mendes, F. K., Vanderpool, D., Fulton, B. & Hahn, M. W. CAFE 5 models variation in evolutionary rates among gene families. Bioinformatics 36, 5516–5518 (2021).

    Article  PubMed  Google Scholar 

  83. Zhang, Z. KaKs_Calculator 3.0: calculating selective pressure on coding and non-coding sequences. Genomics Proteomics Bioinformatics 20, 536–540 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    Article  CAS  PubMed  Google Scholar 

  85. Bolger, A., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Pertea, M. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 33, 290–295 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550–550 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  89. Wu, T. et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation 2, 100141 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, 599 (2008).

    Article  Google Scholar 

  91. Lu, S. et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 48, D265–D268 (2020).

    Article  CAS  PubMed  Google Scholar 

  92. Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Yan, Y., Tao, H., He, J. & Huang, S.-Y. The HDOCK server for integrated protein–protein docking. Nat. Protoc. 15, 1829–1852 (2020).

    Article  CAS  PubMed  Google Scholar 

  94. Lowe, T. M. & Eddy, S. R. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Ernst, J. & Bar-Joseph, Z. STEM: a tool for the analysis of short time series gene expression data. BMC Bioinformatics 7, 191 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  96. Tamura, K., Stecher, G. & Kumar, S. MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38, 3022–3027 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Nunez, J. C. et al. Footprints of natural selection at the mannose-6-phosphate isomerase locus in barnacles. Proc. Natl Acad. Sci. USA 117, 5376–5385 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. He, J., Mo, D., Chen, J. & Luo, L. Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae. Nat. Protoc. 15, 3361–3379 (2020).

    Article  CAS  PubMed  Google Scholar 

  99. Castro-Mondragon, J. A. et al. JASPAR 2022: the 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 50, D165–D173 (2022).

    Article  CAS  PubMed  Google Scholar 

  100. Ioannidou, Z. S., Theodoropoulou, M. C., Papandreou, N. C., Willis, J. H. & Hamodrakas, S. J. CutProtFam-Pred: detection and classification of putative structural cuticular proteins from sequence alone, based on profile hidden Markov models. Insect Biochem. Mol. Biol. 52, 51–59 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Addadi, L., Moradian, J., Shay, E., Maroudas, N. & Weiner, S. A chemical model for the cooperation of sulfates and carboxylates in calcite crystal nucleation: relevance to biomineralization. Proc. Natl Acad. Sci. USA 84, 2732–2736 (1987).

  102. Bahn, S. Y., Jo, B. H., Choi, Y. S. & Cha, H. J. Control of nacre biomineralization by Pif80 in pearl oyster. Sci. Adv. 3, e1700765 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  103. Zhang, G., He, L.-S., Wong, Y. H., Yu, L. & Qian, P.-Y. siRNA transfection in larvae of the barnacle Amphibalanus amphitrite. J. Exp. Biol. 218, 2505–2509 (2015).

    PubMed  Google Scholar 

  104. Wu, B.-K., Mei, S.-C., Chen, E. H., Zheng, Y. & Pan, D. YAP induces an oncogenic transcriptional program through TET1-mediated epigenetic remodeling in liver growth and tumorigenesis. Nat. Genet. 54, 1202–1213 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  105. Han, Z. Genome assembly and annotation of the acorn barnacle Amphibalanus amphitrite. figshare https://doi.org/10.6084/m9.figshare.21310305.v1 (2024).

  106. Han, Z. Acorn barnacle genome. Zenodo https://doi.org/10.5281/zenodo.10825185 (2024).

  107. Han, Z. Acorn-barnacle-genome. GitHub github.com/ZhaofangHan/Acorn-Barnacle-Genome (2024).

Download references

Acknowledgements

We thank Y. Wu for assistance with sample collection, Y. Zou for help with data submission and G. Duan and M. Han for their support in making the schematic drawings of barnacles, all from Xiamen University. Z.F. acknowledges funding from the National Natural Science Foundation of China (32102775). Y.-Y.Z. acknowledges funding from the National Natural Science Foundation of China (32071485). D.F. acknowledges funding from the National Natural Science Foundation of China (42376090), the National Key Research and Development Program of China (2022YFC3106004 and 2022YFC3103904), the Project of Fujian Ocean Synergy Alliance (FOCAL2023-0302), the Science and Technology Project of Fujian Province (2022H0003) and the MEL-RLAB Program (202101).

Author information

Author notes

  1. These authors contributed equally: Zhaofang Han, Zhixuan Wang.

Authors and Affiliations

  1. State Key Laboratory of Mariculture Breeding, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China

    Zhaofang Han, Pei Su, Miaoqin Huang & Danqing Feng

  2. State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China

    Zhixuan Wang, Zekun Huang, Liying Chen & Caihuan Ke

  3. Division of Marine Science and Conservation, Nicholas School of the Environment, Duke University, Beaufort, NC, USA

    Daniel Rittschof

  4. State-Province Joint Engineering Laboratory of Marine Bioproducts and Technology, Xiamen University, Xiamen, China

    Huanhuan Hao, Shanshan Yao & Danqing Feng

  5. Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, China

    Yuan-Ye Zhang

Authors
  1. Zhaofang Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhixuan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel Rittschof
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zekun Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Liying Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Huanhuan Hao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shanshan Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pei Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Miaoqin Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yuan-Ye Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Caihuan Ke
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Danqing Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.F., C.K., Y.-Y.Z. and Z. Han conceived and designed the scientific objectives. Z.W. and P.S. collected samples for sequencing DNA and RNA. Z. Han assembled the barnacle genomes and contributed to all of the data analyses. Z.W. conducted settlement experiments and performed the RNA pull-down, ISH, EMSA and Dual-Luciferase reporter assay and analyzed the resulting data. Z. Han conducted an analysis of the transcriptomic and proteomic data of shell. Z.W. performed SEM, I-SEM and Raman microscopy to validate shell structures. Z.W. performed in vivo RNAi of bcs-6 and in vitro crystallization and binding assays to validate bsf functions. Z. Huang, L.C., H.H., S.Y. and M.H. provided suggestions for the data analysis. Z. Han, Z.W., and Y.-Y.Z. drafted the first version of the manuscript and D.F., C.K., Y.-Y.Z., Z. Han, Z.W. and D.R. contributed to the revision of the manuscript.

Corresponding authors

Correspondence to Yuan-Ye Zhang, Caihuan Ke or Danqing Feng.

Ethics declarations

Competing interests

The authors declare that they do not have any competing interests.

Peer review

Peer review information

Nature Genetics thanks Rafael Acemel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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

Extended data

Extended Data Fig. 1 Genome survey and Hi-C interaction heatmap of Amphibalanus amphitrite.

a, Genome size was estimated by dividing the total number of effective k-mers by the number of homo-peaks. Genome-wide heterozygosity rate and repetitive content were estimated by GenomeScope using the k-mer 17 histogram. b, The upper panel shows the Hi-C interaction heatmap of A. amphitrite across all chromosomes. The genome was split into 100 kb bins, and the colors represent the interaction frequencies between individual chromosomes. The lower panel shows an example of a region on Chr11. Below the heatmap is a view of the first eigenvector (PC1) used to determine the A (positive) and B (negative) compartments, the directionality index (DI) used to call topologically associating domains (TADs) at a 40kb resolution, and gene density of the reference sequence (RefSeq). The black arrows in the TAD triangular map indicate boundaries. c, Size distribution of topological domains (TADs) and their boundaries (d), and gene densities in different chromatin domains (e).

Extended Data Fig. 2 Comparative genomic analysis of barnacles and other arthropods.

a, The distribution of divergence of transposable elements (TEs) of eight crustaceans. SINE/LINE: short/long interspersed nuclear element; LTR: long terminal repeat; DNA: DNA transposon. b, The expanded (red) and contracted (blue) gene families, and the frequency distribution of synonymous substitution rates (Ks) between pairwise paralogues for nine crustaceans and the outlier species. c, The 604 expanded families in the common ancestor of barnacles were filtered, where families with a total gene count across these species of less than five were excluded, resulting in 26 gene families. The top nine gene families are listed in Fig. 1d. Abbreviations: Grik2, glutamate receptor ionotropic, kainate 2; mGluR, metabotropic glutamate receptor; nAChRs, nicotinic acetylcholine receptors; Tuba, tubulin alpha chain; Tubb, tubulin beta chain; FT, cadherin-related tumor suppressor; STS, steryl-sulfatase; SPR, sex peptide receptor; Tmem2, transmembrane protein 2; Setd1, histone-lysine N-methyltransferase; Tret1, facilitated trehalose transporter; FucTC, alpha-(1,3)-fucosyltransferase C; WFDC3, WAP four-disulfide core domain protein 3.

Extended Data Fig. 3 Loss of Hox3 and abd-A in barnacle genomes.

a, TAD structures around Hox genes at a 40 kb resolution. The anterior genes (lab, pb, Dfd, Scr, ftz and Antp) and posterior genes (Ubx and Abd-B) were grouped into two TADs. The black arrows stand for TAD boundaries. b, Alignments of short and long reads to genomic regions of the two absent homeobox genes (Hox3 and abd-A). c, Numbers of transposable elements (TEs) enriched across the homeobox gene region. d, The alignment of Hox3 homeodomain (represented in red) in six arthropod species, namely, Parhyale hawaiensis, Litopenaeus vannamei, Portunus trituberculatus, Eriocheir sinensis, Daphnia pulex and Strigamia maritima.

Extended Data Fig. 4 Transcriptomic analysis of settled and unsettled samples in experiments I and II.

a, Sample legends of the two experiments. b, Cluster and PCA analysis of samples, and GO enrichment of differentially expressed genes (DEGs) for experiment II. The color scale indicates the dissimilarity between pairwise samples in gene expression profiles, and a darker color indicates that the gene expression profiles are more similar. One replicate was discarded due to being an outlier. If the replicate were included in the analysis, there would be limited consistent results between the two experiments, that is, many fewer overlapping terms. Only significantly enriched terms are displayed in the right panel. c, Cluster and PCA of samples, and GO enrichment of DEGs for experiment I. d, Venn diagram of DEGs in the two experiments, and Gene Ontology (GO) analysis results for the 91 overlapping DEGs between the two experiments. e, Weighted gene coexpression network analysis (WGCNA) of experiment I. The upper panel on the left displays a cluster analysis based on the dissimilarity between pairwise gene expression patterns of experiment I. The upper panel on the right shows the eigengene expression pattern of the Blue Module. The eigengene represents the gene expression profiles of a module. The lower panel illustrates the correlation between the eigengene expression pattern and phenotypes (settled vs. non-settled) for different modules, with the Blue Module exhibiting the highest correlation. In b, c and d, the P values in enrichments analysis were calculated using the hypergeometric test and adjusted for multiple comparisons with clusterProfiler. Padj < 0.05 and q value < 0.05 were considered significant enrichments.

Extended Data Fig. 5 Domain structures and sequences of proteins involved in cyprid settlement, and immunofluorescence analysis results for TGase.

a, The nine genes encode proteins that contain Tryp_SPc (trypsin) domains. The protein domains were predicted using a web server (http://pfam-legacy.xfam.org). b, The sequence alignment of Tryp_SPc domains of Homo sapiens thrombin and nine proteins. c, Ribbon drawings showing the structural similarity between Tryp_SPc domains of H. sapiens thrombin and that of nine proteins. Note that the comparison between the Tryp_SPc domain of one gene (AaSB) and H. sapiens thrombin has already been shown in Fig. 2e. d, Ribbon drawings showing the structural similarity between A. amphitrite transglutaminase (TGase, AlphaFold identifier: AF-A0A6A4WL68-F1) and different H. sapiens TGase. TGase1 (PDB: 1GGT) and TGase3 (PDB: 1L9M). e, Negative control of immunofluorescence analysis of TGase expression in the attachment disc of an A. amphitrite juvenile (representative images of n = 2 independent experiments).

Source data

Extended Data Fig. 6 Alignment of bcs-6 transposon in A. amphitrite genome, and the location and expression of bcs-6 in P. pollicipes genome.

a, Sequence alignments of the U3 region in the 5′ LTR of 26 intact bcs-6 gene copies in the A. amphitrite genome. Cis-regulatory elements (CREs) were predicated by the web server AliBaba2.1. b, The locations of three intact bcs-6 gene copies and their expression levels in the P. pollicipes genome. The expression levels were calculated with published RNA-seq data (NCBI, no. PRJNA394196) from pooled larvae and adults of P. pollicipes and visualized by a tool in Integrative Genomics Viewer (IGV). c, The schematic diagrams of shedding cyprid shells of an acorn barnacle and a stalked barnacle, modified from video-based observations under the Creative Commons CC-BY-NC license40.

Extended Data Fig. 7 Function analysis of A. amphitrite bcs-6.

a, The visualization of siRNA in the knockdown experiment after treating cyprids with control agents and cy3-labeled siRNA for 12 hours (representative images of n = 2 independent experiments). NC-1: negative control, 2 μL ml−1 LipoHigh; NC-2: negative control, cy3-labeled siRNA against gfp; siRNA-1: cy3-labeled siRNA-1 against bcs-6; siRNA-2: cy3-labeled siRNA-2 against bcs-6. Red channel signals were extracted to reveal the precise interfering location of siRNA in the cyprids. b, Fluorescence in situ hybridization (FISH) analysis revealed subcellular localization of overexpressed bcs-6 (the experiment was performed once). c, KEGG enrichment of co-expressed genes with bcs-6 across different developmental stages. d, SDS-PAGE and silver staining of the RNA pull-down eluents (representative images of n = 2 independent experiments). e, Transcriptional pattern of 24 adjacent genes within a range of 10 kb upstream and downstream of bcs-6, among which five exhibited a coexpression pattern with bcs-6 (shown in red).

Source data

Extended Data Fig. 8 Chemical and proteomic analysis of barnacle shell plates.

a, The Raman spectrum was employed to determine the types of crystals produced by in vitro mineralization. Mineralization was conducted in CaCl2 solution, with the addition of BSA or BSF (protein products of rctg452.10). b, The Venn plot of proteomic analysis, including three technical replicates per plate type. c, Transcriptional levels of all of the genes identified in the nine expanded gene families with functions related to shell formation (Fig. 1d).

Extended Data Fig. 9 Predicted protein dimer of ctg452.10, and motif comparison of ctg452.10 and ctg452.9.

a, The predicted structure of the ctg452.10 protein dimer was generated using HDOCK software (http://hdock.phys.hust.edu.cn/). The presence of lysine (K) and glutamine (Q) in the ctg452.10 protein suggests the potential formation of amide bonds. b, The motifs of proteins encoded by ctg452.10 and ctg452.9 in A. amphitrite, as well as the corresponding homologous sequence in P. pollicipes, were predicted using MEME (https://meme-suite.org). c, The predicted motifs were aligned using ClustalW for sequence comparison.

Supplementary information

Supplementary Information

Supplementary Notes 1–3 and Supplementary Tables 1–17.

Reporting Summary

Peer Review File

Supplementary Data

Supplementary Data 1: The mass spectrometry of shell proteomics data. Supplementary Data 2: The mass spectrometry of bcs-6-binding proteins in the RNA pull-down assay.

Source data

Source Data Figs. 1–5 and Extended Data Figs. 1–9

Statistical source data.

Source Data Fig. 2

Barnacle photos and immunofluorescence.

Source Data Fig. 3

EMSA gel and whole-mount in situ hybridization.

Source Data Fig. 4

Microscopy scans and immuno-electron microscopy (IEM).

Source Data Fig. 5

Microscopy scans, coomassie staining gel and binding blot.

Source Data Extended Data Fig. 5

Immunofluorescence images.

Source Data Extended Data Fig. 7

FISH and RNA pull-down gel.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Han, Z., Wang, Z., Rittschof, D. et al. New genes helped acorn barnacles adapt to a sessile lifestyle. Nat Genet (2024). https://doi.org/10.1038/s41588-024-01733-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41588-024-01733-7

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research