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Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments

Nature Ecology & Evolution volume 4, pages 1703–1712 (2020)Cite this article

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Abstract

The origin of insect wings has long been debated. Central to this debate is whether wings are a novel structure on the body wall resulting from gene co-option, or evolved from an exite (outgrowth; for example, a gill) on the leg of an ancestral crustacean. Here, we report the phenotypes for the knockout of five leg patterning genes in the crustacean Parhyale hawaiensis and compare these with their previously published phenotypes in Drosophila and other insects. This leads to an alignment of insect and crustacean legs that suggests that two leg segments that were present in the common ancestor of insects and crustaceans were incorporated into the insect body wall, moving the proximal exite of the leg dorsally, up onto the back, to later form insect wings. Our results suggest that insect wings are not novel structures, but instead evolved from existing, ancestral structures.

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Fig. 1: Crustacean and insect legs.
Fig. 2: Knockout phenotypes of leg patterning genes.
Fig. 3: Comparison of the expression of ara and pnr in Tribolium and Parhyale.
Fig. 4: Evidence for a precoxal leg segment in Parhyale.
Fig. 5: The lateral body wall of insects may be composed of two incorporated leg segments and two exites, the wing and the lobe.
Fig. 6: Proposed leg segment homologies.

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Data availability

All of the data that support the findings of this study are available in the main text or the Supplementary Information.

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Acknowledgements

We thank C. Clark-Hachtel and Y. Tomoyasu for sharing results before publication. We thank E. Jarvis for the Parhyale phalloidin muscle stains and A. Pomerantz for the Oncopeltus image and whole adult Parhyale image. We thank C. Wolff, T. Deuve, Y. Kobayashi, F. Schram and J. W. Shultz for helpful discussion. We thank J. Shen for confirming that spalt RNAi reduces both the wing and lobe in Locusta. We thank G. Mardon for the image of the Drosophila dac knockout adult leg. This work is supported by the National Science Foundation (NSF; IOS-1257379 to N.H.P.) and NSF Graduate Research Fellowship (to H.S.B.).

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Authors and Affiliations

  1. University of California, Berkeley, Berkeley, CA, USA

    Heather S. Bruce

  2. Marine Biological Laboratory, Woods Hole, MA, USA

    Heather S. Bruce & Nipam H. Patel

  3. Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL, USA

    Nipam H. Patel

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  1. Heather S. Bruce
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H.S.B. and N.H.P. conceived the experiments. H.S.B. performed the experiments, conceived the model and wrote the manuscript. N.H.P. edited and revised the manuscript.

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Correspondence to Heather S. Bruce.

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Extended data

Extended Data Fig. 1 Expression of leg gap genes in whole embryos and dissected third thoracic legs (T3).

a–d, Dll-e. e–h, Sp6-9. i–l, dac2. m–p, exd. q–t, hth. Embryonic expression data for Dll-e (Browne 2005; Serano 2015), Sp6-9 (Schaeper 2010), and exd and hth (Prpic 2008) have been previously characterized, but not at the level of individual leg segments. (d) Dll-e is expressed in leg segments 1–5; in the interior of the tergal plate (Tp), coxal plate (Cp), and gill (G). (h) Sp6-9 is expressed in leg segments 1–6. (l) dac2 is expressed in leg segments 3–5. Expression in segment 3 may be stronger at other time points. (p) exd is expressed in the body wall through leg segment 3. Exd is expressed only in the base of the gill (distal gill not visible here). (t) hth is expressed in the body wall through leg segment 5. Hth is expressed only in the base of the gill. Note that both insects and Parhyale share a peculiar disparity between hth expression and function, wherein hth knockout deletion extends one more leg segment further than the hth expression domain. Whole embryo scale bars = 100 µm. Dissected leg scale bar = 25 µm. a, n = 5; b, n = 6; c, n = 7; e, n = 6; f, n = 8; g, n = 8; i, n = 4; j, n = 4; k, n = 5; m, n = 3; n, n = 6; o, n = 4; q, n = 3; r, n = 3; s, n = 8.

Extended Data Fig. 2 T7 endonuclease assay to confirm CRISPR-Cas9 mutagenesis.

For each gene, one or two wild type (WT) hatchlings were assayed, and one, two, or three KO hatchlings were assayed. T7 endonuclease was either added (+) or not added (–) to the heteroduplex mixture. In brief, a ~1 kb region flanking the CRISPR-Cas9 target site by at least 300 bp to either side was amplified by PCR from either WT or KO hatchlings. The purified PCR products were denatured, then slowly cooled to allow WT DNA and mutant DNA with indels to anneal, resulting in a ‘bubble’ of unpaired DNA (heteroduplex) at the target site. T7 endonuclease was added to the (+) samples, incubated, and run on a 1.5% agarose gel. KO animals are mosaic, so if the target site was cut, the indels will cause heteroduplexes when annealed with either a WT strand, or a different indel. When a single deletion is present, each half of the cut heteroduplex adds up to approximately 1 kb (see Sp6-9 KO 1 and 2). Some deletions are large enough to be seen without the T7 endonuclease assay (see Dll-e KO), and some hatchlings had multiple deletions which produced multiple bands when cut with T7 (see exd KO 1, hth KO 2, dac2 KO). In hatchlings that have a phenotype but only the WT band (Exd KO 2 and Hth KO 1), the deletion may be so large that one or both of the primer sites may have been deleted.

Extended Data Fig. 3 CRISPR-Cas9 knockout phenotypes in thoracic legs 4 and 5 (T4, T5).

Note that T4 and T5 are morphologically and molecularly indistinguishable, and are treated as interchangeable here. a, Wild type T4 leg. b, Sp6-9 KO T4/T5 leg. c, dac KO T4/T5 leg. The fused and nearly deleted remnant of leg segments 3–5 are in gray. Gill is unaffected, but became oriented upward during specimen mounting. d, Dll KO T4/T5 leg. Scale bar = 50 µm.

Extended Data Fig. 4 Exd and Hth phenotypes.

Body segment fusions are due to the interaction of exd and hth with engrailed during segmentation (Kobayashi 2003). a, Body segment fusions/deletions in Exd knockout whole hatchling. Confocal of unilaterally affected hatchling, dorsal view, anterior at bottom, posterior at left. Left side of animal (L) appears WT. The foreshortening of only the right (R) half twists the body laterally into a nearly spiral shape. The tissue where the eye (E) would have been located is deleted, leaving a recess. Left first antenna (An1), left and right telson (tL, tR). White brackets compare the length of the body segments in right fused and left unfused segments. b, WT T4 leg. c, WT T3 leg, same as Fig. 2a. d, Exd KO T4 leg. Loss of exd deletes/fuses leg segments 1–4 and proximal 5, while the distal half of leg segment 5, and all of leg segments 6 and 7 are WT, because the joint between leg segments 5 and 6 is normal, but there is no joint on the proximal side of leg segment 5, indicated by the proximally faded cyan shading. exd KO causes transformation of remaining T3 leg segments towards a T4/5 identity: compare blue leg segment 6 of WT T3 (Fig. 2) and T4 legs to that of exd KO T3 (Fig. 2) and T4 legs. e,f, Exd and hth KOs produce similar body segment fusions/deletions and proximal thoracic leg segment fusions/deletions. Colours in leg segments are as in Fig. 2.

Extended Data Fig. 5 Drosophila dachshund KO leg.

dac4/dac4 homozygote from dac mutant lines created by Professor Iain Dawson (Mardon 1994). The trochanter through proximal tarsus (leg segments 3–5, and proximal tarsus) are affected, forming a single, fused tissue. Figure adapted with permission from Graeme Mardon.

Extended Data Fig. 6 Parhyale precoxa forms a true, muscled joint and extends musculature to another leg segment.

Confocal images. a, Phalloidin stain of muscle in right half of Parhyale hatchling. Contrast simple, anterior-posterior body muscles to orthogonal, complexly arranged leg muscles. No muscles cross the coxa-basis joint, as noted by Boxshall 1997. Note that all three plates (tergal, coxal, and basal) form contiguous cuticle with their leg segment, that is there is no distinguishing suture between leg segment and exite. b, Optical section showing superficial muscles of right half. Cuticle in grey, muscle in pink. c, Confocal of dissected left half, medial view. Coxal plate and basis partially cut. The precoxa forms a joint with two articulations with the coxa: an anterior, bifurcated, load-bearing hinge articulation (arrowhead), and a posterior gliding articulation (arrow). Orthogonal muscles visible as striations on T4 precoxa. d, Close-up of left T4, medial-anterior view, showing bifurcated hinge articulation. In a-d, the precoxa forms two articulations with the coxa: an anterior, bifurcated, load-bearing hinge articulation (arrowhead), and a posterior gliding articulation (arrow). Coxa is red (coxal plate not shaded, to focus on joints), basis is orange, precoxa is magenta pink. The proximal-most region of the legs brace against each other at (<) and (*). The length of the protrusion is twice that of the coxa: compare the beginning (< and *) and end of the protrusion where it forms a joint (arrow and arrowhead) with the coxa (most visible in b). Muscles in green insert on the precoxa-coxa joint, indicating that this is a true joint, and not merely a point of flexure in the exoskeleton (annulation; Boxshall 2004, 2013; Shultz 1989). The shorter, anterior muscle originates in the protruding precoxa to insert on the rim of the next leg segment, the coxa. This muscle is therefore an intrinsic muscle, a hallmark of a true leg segment (Boxshall 2004, 2013; Shultz 1989). Panels a,b adapted with permission from Erin Alberstat.

Extended Data Fig. 7 Exites can be split into anterior and posterior lobes in crustaceans and insects.

a–d, Ectopic wing formation on T1 following Scr RNAi in Tribolium. Mildly affected individuals have wing tissue emerging from unconnected anterior and posterior regions of the body wall. In more severe phenotypes, these anterior and posterior tissues are fused into a more completely transformed wing. e–j, Split exites in crustaceans and insects. Anterior lobe (red outline or arrow), posterior lobe (blue outline or arrow). e, Split coxal plates in Parhyale. f, A malacostracan crustacean, Anaspides, with split anterior and posterior exites. g, Decapod with split anterior and posterior exites (arthrobranchs). h, Oncopeltus with split anterior and posterior supracoxal lobes. i,j, Gin traps require the wing genes vg and ap, consistent with an exite identity. Gin traps have split anterior and posterior jaws. Images adapted with permission from ref. 8, PNAS (a–d); from Erin Alberstat (e); from ref. 39, John Wiley and Sons (f); from ref. 38, Arthropod Systematics & Phylogeny (g); from Aaron Pomerantz (h); and from ref. 78, PNAS (i,j).

Extended Data Fig. 8 Proximal gene expression in later Parhyale and Tribolium embryos.

a, pannier is expressed in the dorsal-most tissue in late Parhyale embryos during dorsal closure. b, Tribolium embryo, vestigial marks the future wing region adjacent to the spiracle. The three domains of araucan expression remain distinct even at later stages.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Tables 1–12 and Discussion.

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Supplementary Video 1

Supracoxal lobe of a cricket nymph.

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Bruce, H.S., Patel, N.H. Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments. Nat Ecol Evol 4, 1703–1712 (2020). https://doi.org/10.1038/s41559-020-01349-0

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