Two sets of candidate crustacean wing homologues and their implication for the origin of insect wings

Abstract

The origin of insect wings is a biological mystery that has fascinated scientists for centuries. Identification of tissues homologous to insect wings from lineages outside of Insecta will provide pivotal information to resolve this conundrum. Here, through expression and clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) functional analyses in Parhyale, we show that a gene network similar to the insect wing gene network (preWGN) operates both in the crustacean terga and in the proximal leg segments, suggesting that the evolution of a preWGN precedes the emergence of insect wings, and that from an evo-devo perspective, both of these tissues qualify as potential crustacean wing homologues. Combining these results with recent wing origin studies in insects, we discuss the possibility that both tissues are crustacean wing homologues, which supports a dual evolutionary origin of insect wings (that is, novelty through a merger of two distinct tissues). These outcomes have a crucial impact on the course of the intellectual battle between the two historically competing wing origin hypotheses.

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Fig. 1: Expression and functional analyses of vg in Parhyale.
Fig. 2: Expression and functional analyses of nub in Parhyale.
Fig. 3: Expression and functional analyses of apA in Parhyale.
Fig. 4: Expression of other wing patterning genes in Parhyale.
Fig. 5: The candidate wing homologues of Parhyale and the evolutionary relationship among wing homologues.

Data availability

The sequences of the cDNA fragments cloned in this study have been deposited in GenBank with the accession numbers MG703506 (Ph-vg), MG703508 (Ph-nub), MG703509 (Ph-apA), MG703510 (Ph-apB) and MG703507 (Ph-vvl).

References

  1. 1.

    Clark-Hachtel, C. M. & Tomoyasu, Y. Exploring the origin of insect wings from an evo-devo perspective. Curr. Opin. Insect Sci. 13, 77–85 (2016).

    PubMed  Google Scholar 

  2. 2.

    Quartau, J. A. An overview of the paranotal theory on the origin of the insect wings. Publicações do Inst. Zool. ‘Dr. Augusto Nobre’. Fac. Cienc. do Porto 194, 1–42 (1986).

    Google Scholar 

  3. 3.

    Kukalova-Peck, J. Origin of the insect wing and wing articulation from the arthropodan leg. Can. J. Zool. 61, 1618–1669 (1983).

    Google Scholar 

  4. 4.

    Tomoyasu, Y., Ohde, T. & Clark-Hachtel, C. M. What serial homologs can tell us about the origin of insect wings. F1000Research 6, 268 (2017).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Alexander, D. E. in On the Wing: Insects, Pterosaurs, Birds, Bats and the Evolution of Animal Flight 74–102 (Oxford Univ. Press, 2015).

  6. 6.

    Grodnitsky, D. L. Form and Function of Insect Wings: The Evolution of Biological Structures (The Johns Hopkins Univ. Press, 1999).

  7. 7.

    Hughes, C. L. & Kaufman, T. C. Hox genes and the evolution of the arthropod body plan. Evol. Dev. 4, 459–499 (2002).

    CAS  PubMed  Google Scholar 

  8. 8.

    Wagner, G. P. Homology, Genes, and Evolutionary Innovation (Princeton Univ. Press, 2014).

  9. 9.

    Ohde, T., Yaginuma, T. & Niimi, T. Insect morphological diversification through the modification of wing serial homologs. Science 340, 495–498 (2013).

    CAS  PubMed  Google Scholar 

  10. 10.

    Clark-Hachtel, C. M., Linz, D. M. & Tomoyasu, Y. Insights into insect wing origin provided by functional analysis of vestigial in the red flour beetle, Tribolium castaneum. Proc. Natl Acad. Sci. USA 110, 16951–16956 (2013).

    CAS  PubMed  Google Scholar 

  11. 11.

    Medved, V. et al. Origin and diversification of wings: insights from a neopteran insect. Proc. Natl Acad. Sci. USA 112, 15946–15951 (2015).

    CAS  PubMed  Google Scholar 

  12. 12.

    Elias-Neto, M. & Belles, X. Tergal and pleural structures contribute to the formation of ectopic prothoracic wings in cockroaches. R. Soc. Open Sci. 3, 160347 (2016).

    PubMed  PubMed Central  Google Scholar 

  13. 13.

    Linz, D. M. & Tomoyasu, Y. Dual evolutionary origin of insect wings supported by an investigation of the abdominal wing serial homologs in Tribolium. Proc. Natl Acad. Sci. USA 115, E658–E667 (2018).

    CAS  PubMed  Google Scholar 

  14. 14.

    Tomoyasu, Y. Evo–devo: the double identity of insect wings. Curr. Biol. 28, R75–R77 (2018).

    CAS  PubMed  Google Scholar 

  15. 15.

    Clark-Hachtel, C. M., Moe, M. R. & Tomoyasu, Y. Detailed analysis of the prothoracic tissues transforming into wings in the Cephalothorax mutants of the Tribolium beetle. Arthropod Struct. Dev. 47, 352–361 (2018).

    PubMed  Google Scholar 

  16. 16.

    Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A. & Patel, N. H. The crustacean Parhyale hawaiensis: a new model for arthropod development. Cold Spring Harb. Protoc. 4, (2009).

  17. 17.

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

    CAS  PubMed  Google Scholar 

  18. 18.

    Martin, A. et al. CRISPR/Cas9 mutagenesis reveals versatile roles of Hox genes in crustacean limb specification and evolution. Curr. Biol. 26, 14–26 (2016).

    CAS  PubMed  Google Scholar 

  19. 19.

    Sun, D. A. & Patel, N. H. The amphipod crustacean Parhyale hawaiensis: an emerging comparative model of arthropod development, evolution, and regeneration. Wiley Interdiscip. Rev. Dev. Biol. 8, e355 (2019).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    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).

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Williams, J. A., Bell, J. B. & Carroll, S. B. Control of Drosophila wing and haltere development by the nuclear vestigial gene product. Genes Dev. 5, 2481–2495 (1991).

    CAS  PubMed  Google Scholar 

  22. 22.

    Halder, G. et al. The Vestigial and Scalloped proteins act together to directly regulate wing-specific gene expression in Drosophila. Genes Dev. 12, 3900–3909 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Kim, J. et al. Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382, 133–138 (1996).

    CAS  PubMed  Google Scholar 

  24. 24.

    Baena-López, L. A. & García-Bellido, A. Genetic requirements of vestigial in the regulation of Drosophila wing development. Development 130, 197–208 (2003).

    PubMed  Google Scholar 

  25. 25.

    Niwa, N. et al. Evolutionary origin of the insect wing via integration of two developmental modules. Evol. Dev. 12, 168–176 (2010).

    CAS  PubMed  Google Scholar 

  26. 26.

    Averof, M. & Cohen, S. M. Evolutionary origin of insect wings from ancestral gills. Nature 385, 627–630 (1997).

    CAS  PubMed  Google Scholar 

  27. 27.

    Tomoyasu, Y., Arakane, Y., Kramer, K. J. & Denell, R. E. Repeated co-options of exoskeleton formation during wing-to-elytron evolution in beetles. Curr. Biol. 19, 2057–2065 (2009).

    CAS  PubMed  Google Scholar 

  28. 28.

    Ng, M., Diaz-Benjumea, F. J. & Cohen, S. M. nubbin encodes a POU-domain protein required for proximal-distal patterning in the Drosophila wing. Development 121, 589–599 (1995).

    CAS  PubMed  Google Scholar 

  29. 29.

    Cifuentes, F. J. & Garcia-Bellido, A. Proximo-distal specification in the wing disc of Drosophila by the nubbin gene. Proc. Natl Acad. Sci. USA. 94, 11405–11410 (1997).

    CAS  PubMed  Google Scholar 

  30. 30.

    Turchyn, N., Chesebro, J., Hrycaj, S., Couso, J. P. & Popadić, A. Evolution of nubbin function in hemimetabolous and holometabolous insect appendages. Dev. Biol. 357, 83–95 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Li, H. & Popadić, A. Analysis of nubbin expression patterns in insects. Evol. Dev. 6, 310–324 (2004).

    CAS  PubMed  Google Scholar 

  32. 32.

    Prakash, A. & Monteiro, A. apterous A specifies dorsal wing patterns and sexual traits in butterflies. Proc. R. Soc. B 285, 20172685 (2018).

    PubMed  Google Scholar 

  33. 33.

    Brook, W. J., Diaz-Benjumea, F. J. & Cohen, S. M. Organizing spatial pattern in limb development. Annu. Rev. Cell Dev. Biol. 12, 161–180 (1996).

    CAS  PubMed  Google Scholar 

  34. 34.

    Zecca, M. & Struhl, G. Control of Drosophila wing growth by the vestigial quadrant enhancer. Development 134, 3011–3020 (2007).

    CAS  PubMed  Google Scholar 

  35. 35.

    Browne, W. E., Price, A. L., Gerberding, M. & Patel, N. H. Stages of embryonic development in the amphipod crustacean, Parhyale hawaiensis. Genesis 42, 124–149 (2005).

    PubMed  Google Scholar 

  36. 36.

    Peel, A. The evolution of arthropod segmentation mechanisms. BioEssays 26, 1108–1116 (2004).

    CAS  PubMed  Google Scholar 

  37. 37.

    Patel, N. H. et al. Expression of engrailed proteins in arthropods, annelids, and chordates. Cell 58, 955–968 (1989).

    CAS  PubMed  Google Scholar 

  38. 38.

    Clark, E., Peel, A. D. & Akam, M. Arthropod segmentation. Development 146, dev170480 (2019).

    CAS  PubMed  Google Scholar 

  39. 39.

    Grimm, S. & Pflugfelder, G. O. Control of the gene optomotor-blind in Drosophila wing development by decapentaplegic and wingless. Science 271, 1601–1604 (1996).

    CAS  PubMed  Google Scholar 

  40. 40.

    Cook, O., Biehs, B. & Bier, E. brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila. Development 131, 2113–2124 (2004).

    CAS  PubMed  Google Scholar 

  41. 41.

    Pflugfelder, G. O., Eichinger, F. & Shen, J. T-box genes in Drosophila limb development. Curr. Top. Dev. Biol. 122, 313–354 (2017).

    CAS  PubMed  Google Scholar 

  42. 42.

    Kobayashi, Y. Formation of subcoxae-1 and 2 in insect embryos: the subcoxal theory revisited. Proc. Arthropod. Embryol. Soc. Jpn 48, 33–38 (2017).

    Google Scholar 

  43. 43.

    Mashimo, Y. & Machida, R. Embryological evidence substantiates the subcoxal theory on the origin of pleuron in insects. Sci. Rep. 7, 12597 (2017).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Snodgrass, R. E. in Principles of Insect Morphology 157–192 (Cornell Univ. Press, 1935).

  45. 45.

    Deuve, T. What is the epipleurite? A contribution to the subcoxal theory as applied to the insect abdomen. Ann. Soc. Entomol. Fr. 54, 1–26 (2018).

    Google Scholar 

  46. 46.

    Coulcher, J. F., Edgecombe, G. D. & Telford, M. J. Molecular developmental evidence for a subcoxal origin of pleurites in insects and identity of the subcoxa in the gnathal appendages. Sci. Rep. 5, 15757 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Bruce, H. S. & Patel, N. H. Insect wings and body wall evolved from ancient leg segments. Preprint at bioRxiv https://doi.org/10.1101/244541 (2018).

  48. 48.

    Kukalova-Peck, J. Phylogeny of higher taxa in Insecta: finding synapomorphies in the extant fauna and separating them from homoplasies. Evol. Biol. 35, 4–51 (2008).

    Google Scholar 

  49. 49.

    Franch-Marro, X., Martín, N., Averof, M. & Casanova, J. Association of tracheal placodes with leg primordia in Drosophila and implications for the origin of insect tracheal systems. Development 133, 785–790 (2006).

    CAS  PubMed  Google Scholar 

  50. 50.

    Moczek, A. P. & Rose, D. J. Differential recruitment of limb patterning genes during development and diversification of beetle horns. Proc. Natl Acad. Sci. USA 106, 8992–8997 (2009).

    CAS  PubMed  Google Scholar 

  51. 51.

    Fisher, C. R., Wegrzyn, J. L. & Jockusch, E. L. Co-option of wing-patterning genes underlies the evolution of the treehopper helmet. Nat. Ecol. Evol. 4, 250–260 (2020).

    PubMed  Google Scholar 

  52. 52.

    Shiga, Y. et al. Repeated co-option of a conserved gene regulatory module underpins the evolution of the crustacean carapace, insect wings and other flat outgrowths. Preprint at bioRxiv https://doi.org/10.1101/160010 (2017).

  53. 53.

    Carroll, S. B. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 134, 25–36 (2008).

    CAS  PubMed  Google Scholar 

  54. 54.

    Knoll, A. H. & Carroll, S. B. Early animal evolution: emerging views from comparative biology and geology. Science 284, 2129–2137 (1999).

    CAS  PubMed  Google Scholar 

  55. 55.

    Prokop, J. et al. Paleozoic nymphal wing pads support dual model of insect wing origins. Curr. Biol. 27, 263–269 (2017).

    CAS  PubMed  Google Scholar 

  56. 56.

    Requena, D. et al. Origins and specification of the Drosophila wing. Curr. Biol. 27, 3826–3836.e5 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Wagner, G. P. & Lynch, V. J. Evolutionary novelties. Curr. Biol. 20, R48–R52 (2010).

    CAS  PubMed  Google Scholar 

  58. 58.

    Monteiro, A. & Podlaha, O. Wings, horns, and butterfly eyespots: how do complex traits evolve? PLoS Biol. 7, e37 (2009).

    PubMed  Google Scholar 

  59. 59.

    Kuratani, S., Kuraku, S. & Nagashima, H. Evolutionary developmental perspective for the origin of turtles: the folding theory for the shell based on the developmental nature of the carapacial ridge. Evol. Dev. 13, 1–14 (2011).

    PubMed  Google Scholar 

  60. 60.

    Lyson, T. R., Bever, G. S., Scheyer, T. M., Hsiang, A. Y. & Gauthier, J. A. Evolutionary origin of the turtle shell. Curr. Biol. 23, 1113–1119 (2013).

    CAS  PubMed  Google Scholar 

  61. 61.

    Chuong, C. M., Chodankar, R., Widelitz, R. B. & Jiang, T. X. Evo-devo of feathers and scales: building complex epithelial appendages. Curr. Opin. Genet. Dev. 10, 449–456 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Widelitz, R. B. et al. Molecular biology of feather morphogenesis: a testable model for evo-devo research. J. Exp. Zool. B 298, 109–122 (2003).

    Google Scholar 

  63. 63.

    Chen, C.-F. et al. Development, regeneration, and evolution of feathers. Annu. Rev. Anim. Biosci. 3, 169–195 (2015).

    PubMed  Google Scholar 

  64. 64.

    Hu, Y., Linz, D. M. & Moczek, A. P. Beetle horns evolved from wing serial homologs. Science 366, 1004–1007 (2019).

    CAS  PubMed  Google Scholar 

  65. 65.

    Shubin, N., Tabin, C. & Carroll, S. Deep homology and the origins of evolutionary novelty. Nature 457, 818–823 (2009).

    CAS  PubMed  Google Scholar 

  66. 66.

    Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A. & Patel, N. H. Fixation and dissection of Parhyale hawaiensis embryos. Cold Spring Harb. Protoc. 4, (2009).

  67. 67.

    Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A. & Patel, N. H. In situ hybridization of labeled RNA probes to fixed Parhyale hawaiensis embryos. Cold Spring Harb. Protoc. 4 (2009).

  68. 68.

    Shippy, T. D., Coleman, C. M., Tomoyasu, Y. & Brown, S. J. Concurrent in situ hybridization and antibody staining in red flour beetle (Tribolium) embryos. Cold Spring Harb. Protoc. 4 (2009).

  69. 69.

    Bassett, A. & Liu, J.-L. CRISPR/Cas9 mediated genome engineering in Drosophila. Methods 69, 128–136 (2014).

    CAS  PubMed  Google Scholar 

  70. 70.

    Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A. & Patel, N. H. Injection of Parhyale hawaiensis blastomeres with fluorescently labeled tracers. Cold Spring Harb. Protoc. 4, (2009).

  71. 71.

    Gloor, G. & Engels, W. Single-fly DNA preps for PCR. Drosoph. Inf. Newsl. 1, (1991).

  72. 72.

    Grimaldi, D. & Engels, M. S. Evolution of the Insects (Cambridge Univ. Press, 2005).

  73. 73.

    Misof, B. et al. Phylogenomics resolves the timing and pattern of insect evolution. Science 346, 763–767 (2014).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank N. Patel and H. Bruce for technical assistance and sharing their preprint, and X. Franch-Marro for the Ph-vvl clone. We also thank the Center for Bioinformatics and Functional Genomics (CBFG) and the Center for Advanced Microscopy and Imaging (CAMI) at Miami University for technical support, S. Yi for technical assistance and T. Ohde, A. Fernándes, D. Linz, N. Patel, H. Bruce, A. Martin and other members of the Tomoyasu and Patel labs for helpful discussions. This work is supported by the Miami University Faculty Research Grants Program (CFR) (to Y.T.), the National Science Foundation (NSF) (IOS1557936 to Y.T.), an NSF Graduate Research Fellowship (to C.M.C.-H.) and EDEN: Evo-Devo- Eco Network (NSF-IOS0955517 to C.M.C.-H.).

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Contributions

C.M.C.-H. and Y.T. conceived the experiments. C.M.C.-H. performed the experiments. C.M.C.-H. and Y.T. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Yoshinori Tomoyasu.

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Extended data

Extended Data Fig. 1 Alignments of evolutionarily conserved protein domains.

a, Alignment for the Vg Tondu domain. b, c, POU domain (b) and homeodomain (c) alignments of Nub and Vvl. Both Nub and Vvl belong to the POU-homeodomain protein family; however, each class possesses some characteristic amino acids in the conserved domains (yellow and blue highlighted amino acids). d, Ap homeodomain alignment. Characteristic amino acids found among the two classes of Apterous (A and B) and the corresponding vertebrate homologs are highlighted. The presence of characteristic amino acids for A and B classes indicates that the emergence of these two arthropod Apterous classes preceded the divergence of crustaceans and hexapods. Amino acid sequences for Parhyale proteins were translated from the cloned cDNA sequences. In some cases, additional sequence was added from the published genome assembly (Ph-apA and Ph-apB) or additional cDNA clones (Ph-vvl)49 to obtain the longest conserved domain amino acid sequence possible. Amino acid sequences for other species were obtained from NCBI and OrthoDB except for Af-ApB homeodomain which was obtained from26. Species abbreviations: Af- Artemia franciscana, Am-Apis mellifera, Bg-Blattella germanica, Bm-Bombyx mori, Da-Daphnia magna, Dm- Drosophila melanogaster, Dr- Danio rerio, Mm- Mus musculus, Ph-Parhyale hawaiensis, Of-Oncopeltus fasciatus, Tc-Tribolium castaneum.

Extended Data Fig. 2 Deletions induced by vg and apA CRISPR/Cas9 KO.

a, vg KO2 alignments. b, vg KO3 alignments. c, apA KO2 alignments. d, apA KO3 alignment. The top line in each alignment shows the WT sequence with the targeted site (green) and the Protospacer adjacent motif (PAM) (blue highlight). Red in brackets with “Δ” indicates the number of base pairs deleted from that region and blue in brackets with “+” indicates the number of base pairs added to that region. Yellow nucleotides indicate SNPs.

Extended Data Fig. 3 T7 endonuclease I assay.

a, nub KO1. b, nub KO3. c, apA KO2. d, apA KO3. “M” refers to the sacrificed hatchling that exhibited visible mutant phenotypes, while “WT” with gene prefix indicates CRISPR/Cas9 injected individuals that lacked any visible abnormalities. “WT” with no gene prefix is the negative control, with the corresponding genomic region isolated from un-injected WT hatchlings. “+” and “-” indicate presence and absence of T7 endonuclease I. Asterisks indicate T7 endonuclease I digested bands.

Extended Data Fig. 4 Additional images for nub KO.

Individual with curled tergal phenotype (arrowhead).

Extended Data Fig. 5 Ph-apB expression pattern.

Arrow indicates strong expression of Ph-apB in the brain. Embryo is ~stage 21.

Extended Data Fig. 6 Insect wing gene network.

A simplified version of the wing gene network (WGN) described in Drosophila. The six genes investigated in this study are indicated in red.

Extended Data Fig. 7

Expression pattern for all genes analyzed in this study.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Video 1

Ph-vg expression pattern (confocal).

Supplementary Video 2

Confocal image for WT.

Supplementary Video 3

Confocal stack for WT.

Supplementary Video 4

Confocal image for Ph-vg KO.

Supplementary Video 5

Confocal stack for Ph-vg KO.

Supplementary Video 6

Ph-nub expression pattern (confocal).

Supplementary Video 7

Confocal image for Ph-nub KO.

Supplementary Video 8

Confocal stack for WT (T4).

Supplementary Video 9

Confocal stack for Ph-nub KO (T4).

Supplementary Video 10

Ph-apA expression pattern (confocal).

Supplementary Video 11

Confocal image for Ph-apA KO.

Supplementary Video 12

Confocal stack for Ph-apA KO.

Supplementary Video 13

Confocal stack for Ph-apA KO (severe).

Supplementary Video 14

Ph-ci expression pattern.

Supplementary Video 15

Ph-En visualization (confocal).

Supplementary Video 16

Ph-omb expression pattern (confocal).

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Clark-Hachtel, C.M., Tomoyasu, Y. Two sets of candidate crustacean wing homologues and their implication for the origin of insect wings. Nat Ecol Evol (2020). https://doi.org/10.1038/s41559-020-1257-8

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