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

Nature Ecology & Evolution volume 4, pages 1694–1702 (2020)Cite this article

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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.

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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).

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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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  1. Courtney M. Clark-Hachtel

    Present address: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Authors and Affiliations

  1. Department of Biology, Miami University, Oxford, OH, USA

    Courtney M. Clark-Hachtel & Yoshinori Tomoyasu

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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.

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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 4, 1694–1702 (2020). https://doi.org/10.1038/s41559-020-1257-8

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