Origin and diversification of wings: Insights from a neopteran insect

Abstract

Winged insects underwent an unparalleled evolutionary radiation, but mechanisms underlying the origin and diversification of wings in basal insects are sparsely known compared with more derived holometabolous insects. In the neopteran species Oncopeltus fasciatus, we manipulated wing specification genes and used RNA-seq to obtain both functional and genomic perspectives. Combined with previous studies, our results suggest the following key steps in wing origin and diversification. First, a set of dorsally derived outgrowths evolved along a number of body segments including the first thoracic segment (T1). Homeotic genes were subsequently co-opted to suppress growth of some dorsal flaps in the thorax and abdomen. In T1 this suppression was accomplished by Sex combs reduced, that when experimentally removed, results in an ectopic T1 flap similar to prothoracic winglets present in fossil hemipteroids and other early insects. Global gene-expression differences in ectopic T1 vs. T2/T3 wings suggest that the transition from flaps to wings required ventrally originating cells, homologous with those in ancestral arthropod gill flaps/epipods, to migrate dorsally and fuse with the dorsal flap tissue thereby bringing new functional gene networks; these presumably enabled the T2/T3 wing's increased size and functionality. Third, "fused" wings became both the wing blade and surrounding regions of the dorsal thorax cuticle, providing tissue for subsequent modifications including wing folding and the fit of folded wings. Finally, Ultrabithorax was co-opted to uncouple the morphology of T2 and T3 wings and to act as a general modifier of hindwings, which in turn governed the subsequent diversification of lineage-specific wing forms.

Keywords: RNA-seq; Sex combs reduced; Ultrabithorax; vestigial; wing origins.

Fig. 1.

Gene expression [mean log₂ (normalized read count + 0.01)] in the three wing libraries compared with the same gene in T1 body wall. Genes undetectable or nearly so in T1 body wall but up-regulated >fourfold in T2 wings are colored pink. Genes down-regulated >fourfold in T2 wings are shown in green.

Fig. 2.

Least-square means (+ SE) of normalized read counts of (A) a set of wing specification genes (vg, sd, wg, and ap) equally expressed in all wing types, and (B) the ventral origin genes nub and tracheal genes (trh, vvl, sal, and stat92E) that are missing in the T1 Scr RNAi ectopic wing. These means are from a model that included “gene” and “tissue” as independent variables. (C) Transcriptome-wide mean expression difference between T2 wing and T1 Scr RNAi ectopic wing for genes involved in development of dorsal closure, ventral furrow, or neither.

Fig. S1.

Expression in T1 Scr RNAi ectopic wing (A) and T3 (B) wings compared with T2 wings for 109 wing specific genes (undetectable or nearly so in T1 body wall and at least fourfold higher than minimal expression in T2 wing). Solid line is the line of identity (slope = 1, intercept = 0) and the flat dashed line (A) shows genes (open symbols) missing from the T1 ectopic wing. Note that these same genes, with one exception, are expressed in T3 wing at levels indistinguishable from T2 wing. One gene very highly expressed in all wing types (Moesin) is excluded from this plot because its inclusion necessitates rescaling the axes and reducing clarity of the key result.

Fig. S2.

Effects of vg, sd, and nub RNAi on the prothorax (T1) of Oncopeltus fasciatus. (A–D) The fronto-dorsal view of: wild type (A), vg RNAi (B), sd RNAi (C), and nub RNAi (D). (A′) The outline of the wild-type pronotum. (B′–D′) The overlay of vg RNAi, sd RNAi, and nub RNAi, respectively (gray), onto wild type (black). (E–H) The ventral view of T1 sternum in: wild type (E), vg RNAi (F), sd RNAi (G), and nub RNAi (H). (E′) The outline of wild-type sternum. (F′–H′) The overlay of vg RNAi, sd RNAi, and nub RNAi, respectively (gray), onto wild type (black).

Fig. S3.

(A) Fronto-ventral view of T1 ventral plate. (B–E) close-up of region outlined in A in a wild-type, vg RNAi, sd RNAi, and nub RNAi, respectively. (F) The lateral view of T1 plate. (G–J) The magnified detail of the outlined region in wild-type (G), vg RNAi (H), sd RNAi (I), and nub RNAi (J), respectively. The asterisk in J shows a notch created in the nub RNAi treatment.

Fig. S4.

The effect of wing specification genes on the length and width of the T1 notum (n = 10 for each gene).

Fig. 3.

The functions of wing genes on the mesothorax (T2) of Oncopeltus. (A) Dorsal morphology of the adult T2 segment; arrowheads point to the clavus of the forewings. (A′) The dissected dorsal T2 plate of a fifth nymph. The dotted line denotes the proximal-most boundary of wing primordia. (B) Wild-type fore- and hindwings. (C–E) The effects of vg RNAi, sd RNAi, and nub RNAi on adult wing morphology, respectively. (F) Dissected dorsal T2 notum of wild-type adult. (G–I) The effect of vg RNAi, sd RNAi, and nub RNAi, respectively. (J–M) The alignment of the scutellum and clavus of the forewing in wild-type (J), vg RNAi (K), sd RNAi (L), and nub RNAi (M) adults, respectively. In K–M, this alignment is disturbed causing scutellum and clavus to lose their close contact to one another (the created open space is artificially colored in green). scu, scutellum.

Fig. S5.

(A) Dorsal view of a T2 wing pad in wild-type fifth nymph. (B) Final adult T2 morphology with the focus on scutellum. (C) The heat lesions administered to the central region or laterally on the wing primordia of fifth nymphs (green highlighted regions in A) resulted in (respectively) malformation of the scutellum (D–D2) or wings (E–E2) in adults.

Fig. S6.

(Left) The dissected wild-type wing pad in fifth nymph, showing a central region (CR) and wing primordia (wg). (Right) RT-PCR showing expression of vg in the CR and wg regions, whereas nub is only expressed in the wg region.

Fig. 4.

Effects of Scr, Scr/vg, and Scr/nub RNAi on the T1 morphology. (A–D) Dorsal and (A′–D′) frontal view of wild-type T1 plate (A and A′), Scr RNAi T1 plate (B and B′), Scr/vg RNAi T1 plate (C and C′), and Scr/nub RNAi T1 plate (D and D′). To better distinguish the shape of the ectopic scutellum, its outer edges are outlined with a dotted white line in D.

Fig. S7.

Effects of tio RNAi on adult morphology. (A) Wild-type adult Oncopeltus showing the wings folded flat on the body. (B) Close-up of wild-type scutellum. (C) Close-up of wild-type T2 hinge showing the sclerite (black arrowhead) and connecting membrane (white arrow) and (D) wild-type forewing. (E) tio RNAi adult showing the misfolding wings. (F) Close-up of tio RNAi scutellum. (G) Close-up of tio RNAi T2 hinge and (H) forewing.

Fig. 5.

The role of Ubx in the Oncopeltus metathorax. (A and A′) Dorsal views of the wild-type and Ubx RNAi adults. (B) Wild-type fore- and hindwing display distinct morphologies with regards to their shape, color, and size. (B′) Although forewings look the same, hindwings are transformed into forewings in Ubx RNAi adults. (C) A side view of the dorsal plates in the wild-type showing that the T2 segment features a well-developed scutellum. (C′) A side view of Ubx RNAi adult indicates a presence of an ectopic scutellum on T3 (black arrow). (D) Close-up view focused on wild-type scutellum. (D′) In Ubx RNAi adults, a second ectopic scutellum forms beneath the T2 scutellum (black arrow). (E) Dissected T3 plate of wild-type adult. (E′) Dissected T3 plate of an Ubx RNAi adult. (F) A close-up view of T2 and T3 ventral plates in wild-type. The ventral T2 has a triangular shape, whereas the T3 is oval. (F′) In Ubx RNAi adults, the ventral T3 is transformed into a ventral T2 sternum. Abbreviations: Scu, scutellum; T2, mesothoracic segment; T3, metathorax thoracic segment.

Fig. 6.

Major events in the divergence and segmental diversification of insect wings. Note the presence of a T1 winglet in fossil insects, which persisted until after T2 and T3 wings elongated. Additionally, the divergence of T2/T3 wing morphology occurs also in some Paleoptera (Ephemeroptera), which if mediated by Ubx would place this feature at a more basal location than depicted here. Illustration by Daorong Fang.

Fig. S8.

RT-PCR analysis of vg,sd and nub mRNA expression levels. Lane 1 shows high levels of vg (A), sd (B), and nub (C) in wild-type Oncopeltus fifth nymphs; lane 2 shows greatly reduced levels of expression in the corresponding RNAi nymphs. 18S mRNA levels were used for controls for all three genes.