The roles of growth regulation and appendage patterning genes in the morphogenesis of treehopper pronota

Abstract

Treehoppers of the insect family Membracidae have evolved enlarged and elaborate pronotal structures, which is hypothesized to involve co-opted expression of genes that are shared with the wings. Here, we investigate the similarity between the pronotum and wings in relation to growth. Our study reveals that the ontogenetic allometry of the pronotum is similar to that of wings in Membracidae, but not the outgroup. Using transcriptomics, we identify genes related to translation and protein synthesis, which are mutually upregulated. These genes are implicated in the eIF2, eIF4/p70S6K and mTOR pathways, and have known roles in regulating cell growth and proliferation. We find that species-specific differential growth patterning of the pronotum begins as early as the third instar, which suggests that expression of appendage patterning genes occurs long before the metamorphic molt. We propose that a network related to growth and size determination is the more likely mechanism shared with wings. However, regulators upstream of the shared genes in pronotum and wings need to be elucidated to substantiate whether co-option has occurred. Finally, we believe it will be helpful to distinguish the mechanisms leading to pronotal size from those regulating pronotal shape as we make sense of this spectacular evolutionary innovation.

Keywords: Entylia carinata; growth; membracidae; morphogenesis; pronotum; treehopper.

Figure 1.

Treehopper species displaying variation in size and shape of the pronotum. (a–c, e–g) Adult treehopper species used in this study, including the outgroup (a) Aetalion reticulatum (Family: Aetalionidae), in which the pronotum, outlined in dashes (yellow in online version), is a small domed plate just behind the head, and five Membracidae, (b) Membracis mexicana, (c) Metheisa lucillodes, (e) Ennya chrysura, (f) Polyglypta costata and (g) Entylia carinata. The pronota of (h) 5th and (i) 4th instars of (g) Ent. carinata are much smaller than they are in the adult, but display the distal margin shape, an anterior horn and medial crest, characteristic of the species. (d) Melizoderes variegata from the family Melizoderidae, closely related to the Membracidae, does not have a posterior process and the exaggerated pronotal size. The pronotum, however, develops antereodorsally, diverging from the gently domed plate seen in A. reticulatum and most other hemipterans. (Online version in colour.)

Figure 2.

Ontogenetic allometry of pronotum and wing size relative to body size across six morphologically disparate treehopper species. The scaling relationships of the membracid pronotum and wing relative to body size in the 4th, 5th and adult stages, are noticeably similar compared to that of the outgroup, Aetalion reticulatum. There are large changes in the relative sizes of the pronotum in all membracids, with slopes (allometric coefficients) >2. These were similar to the relative size changes of the wing, with slopes >2. (Online version in colour.)

Figure 3.

Dorsal surface shape comparisons of the pronotal tissue in 4th instars, 5th instars, and adults across five membracid species. (a) Morphospace displaying PC1 and PC2 emphasize that there is little to no overlap in shapes across species, within a developmental stage (size of symbol). (b) Qualitative shape variation across developmental stages and species shows differences emerge early and continue to diverge with progressive stages. (c) Results of Procrustes ANOVA show a significant difference in shape between the 4th and the 5th, which for some species is larger than the shape difference between the 5th and the adult. There are still significant shape differences between the 5th and the adult for several species, but this was not a consistently observed pattern across all species. (Online version in colour.)

Figure 4.

Results from comparative transcriptomic analyses of the pronotum (anterior and posterior), wings and legs: overlapping significantly differentially expressed (DE) genes shared in the anterior and posterior pronotum with (a) wings and (b) legs respectively (FDR < 0.05) are shown in the Venn diagram. (a) There were 116 mutually significantly DE genes in the wings, anterior and posterior pronotal tissues compared to legs and (b) 40 mutually significantly DE genes in the legs, anterior, and posterior pronotal tissues compared to legs (see the electronic supplementary material, tables S3 and S4 for full list of genes). (c) The top significantly enriched canonical pathways in wings from ingenuity pathway analysis (IPA) show several pathways mutually enriched in the pronotum (anterior and posterior) and wings. Positive z-scores (purple in online version) indicate an upregulation of the pathway relative to legs. Negative z-scores (red in online version) indicate a downregulation of the pathway relative to legs. Four significantly enriched canonical pathways did not have enough data to determine whether the pathway was upregulated or downregulated in the pronotum and wings relative to legs (grey in online version). (Online version in colour.)