Structure of adult helmet
The helmet of the adult Antianthe expansa covers most of the dorsum of the body [7] (Fig. 2a-d) and has a pair of lateral projections (humeral horns) at its anterior end (Fig. 2e). The helmet is connected to the main body only at its anterior margin (Fig. 2c, C’), i.e., covering over the mesothorax, metathorax, and abdomen with no other connections (Fig. 2c, d). Thus, the venter of the helmet is actually external to the body.
In the posterior half of the helmet, there is a wall-like structure that bridges the left and right sides (Fig. 2c-f). This structure is known as the septum, and was first named by Buckton and Poulton in 1903 [9, 12]. Detailed observation of the helmet using paraffin sections indicated that the helmet is composed of a bi-layered cuticle (Fig. 3B’, B″, C′, C″). The space between the two layers is connected to the body. Some unstructured tissues and cells were observed between these layers (Fig. 3B’, B″, C′, C″). The apical area (median carina) (light blue arrows in Fig. 3a-c) and the bottom of the helmet, beginning at the base of humeral horns and extending posteriorly (orange arrows in Fig. 3a-c), are enlarged (with an interlayer space) to form tubular structures. These tubular structures have also been described as median carina and lateral carinae in a previous study [9]. The median carina connects to the body at the prothorax, just posterior to the head, and the lateral carinae connect at the lateral projection (Fig. 3d). Since the carinae have a thicker cuticle than the other areas of the helmet (Fig. 3b, B’), they are likely to function as a framework for the helmet. The carinae also function as pathways for body fluid that is pumped at the imaginal molt in order to extend the densely folded helmet primordium. The septum is also composed of two layers, both of which are connected to the lower layer (also known as the ventral lobe [9]) of the helmet (Fig. 3C’, C″). In other words, the lower layer is folded inside with the inner folded layers attached to each other to form the septum (Fig. 3c). These structures (bi-layer helmet, median carina, lateral carinae, and septum) were observed in a previously studied species, Stictocephala bisonia, which also has roof-like helmet [9]. One difference between Antianthe and Stictocephala is that the Antianthe has two pairs of lateral carinae (Fig. 3b-d) while the Stictocephala has just one pair.
Structure of nymphal pronotum
In the last nymphal instar the dorsal area of the pronotum extends to the posterior part of mesothorax forming a “helmet sheath” that contains part of the folded adult helmet (Fig. 4a, b). This helmet sheath is a single layer and the inner surface of the sheath is connected to the body (Fig. 4b). As Stegmann (1998) mentioned in his study [9], the nymphal pronotum is a large, single-layered outgrowth of integument connecting with the body cavity. On the helmet sheath, there is a pair of thorn-like horns projecting laterally (Fig. 4a, b, c arrow); however, these are missing in the adult stage. At the imaginal molt, the developed adult helmet formed inside the helmet sheath extends into a large roof-like helmet (Fig. 4d-i). This extension of the folded helmet likely occurs by pumping hemolymph (body fluid) into the median and lateral carinae, similar to the extension of wings by pumping hemolymph into wing veins. This expansion process occurs during a short time period. Also, the less-elastic cuticle layers have already formed on the surface of helmet at this stage. Therefore, cytological activities, such as cell division, are not likely to be involved in this transition, similar to the transformation of a densely folded horn primordium into a long bifurcated horn seen in rhinoceros beetles [5]. Thus, there are two transitions: 1) structural transition from a monolayer sheath-like structure into bi-layer plywood-like structure, and 2) an increase in the size of the helmet. Preparations for these transitions are complete before the final molt. To investigate how these two transitions are achieved inside the nymphal helmet sheath, we next analyzed the development of the helmet during the last nymphal stage.
Morphogenetic processes of helmet during nymphal stage
In order to investigate the developmental trajectory of the helmet, we observed the inner developing helmet structure of the final instar nymph by using a micro-CT scan (Fig. 5). As we could not keep living treehoppers in the laboratory due to permission restrictions, we collected dozens of final instar nymphs in the field and immediately fixed them. The chronological order was reconstructed based on the development of their wings and flight muscles. In the young final instar, i.e. prior to adult helmet morphogenesis, the epithelia of the pronotum including the helmet sheath were a monolayer covered by nymphal cuticle (Fig. 5b). Adult helmet morphogenesis begins with apolysis (detachment of the epithelial sheet from old cuticle) of the helmet epithelium. Apolysis likely occurs at the most posterior part (Fig. 5c), where the lower layer first detaches from the nymphal cuticle (Fig. 5c, white arrow). Along with the apolysis process, the anterior upper layer also detached from the cuticle (Fig. 5d, yellow arrows). At this stage, the helmet epithelium appears to degenerate like a deflated balloon, that is, the upper and lower layer come together and the internal space of the developing helmet mostly disappears (Fig. 5d). This is a remarkable transition from a single to a double layer (Fig. 5d). The entire newly formed helmet then shrank and apolysis was complete (Fig. 5e). After this stage, the helmet grew larger (Fig. 5f) and finally filled the entire space of the helmet sheath and other areas of the pronotum (Fig. 5g). As the helmet grew, many folded structures were also formed (Fig. 5f, g).
Next, we observed the outer morphology and topological structures of the developing helmet after shrinking (corresponding to Fig. 5e) and the fully developed helmet (corresponding to Fig. 5g) by using scanning electron microscopy (SEM) and paraffin sections. We found that the shape of the developing helmet after shrinking was very similar to that of the adult helmet, although the total helmet size was still much smaller at this stage (Fig. 6a). From the ventral side, the septal structure can also be observed (green highlighted area in Fig. 6b). By observing a cross-section of the helmet, it is apparent that the lower and upper layers have become attached to form bi-layer structure (Fig. 6c). In this paraffin section the median carina and bi-layer septum were also clearly recognized (Fig. 6c). Thus, most of the characteristic components of the adult helmet structure (bi-layer roof shape, septum and median carina) have already appeared by this stage, so this developing helmet can be referred to as a “miniature” of the adult helmet. In other words, the structural transition from a single-layered, sheath-like structure to double-layered roof-shaped structure, one of the two transitions mentioned above, is nearly complete at this stage.
After this stage the miniature helmet grows larger to its final folded structure. As the helmet grows many folded furrows are formed on its surface (Fig. 6d, e, f). This growth with furrow formation is likely responsible for the size transition from a small nymphal helmet sheath to a large adult one. There are at least two kinds of furrows. The first type consists of deep furrows formed by bending the bi-layer epithelial sheet (hereafter macro furrow). The most apparent macro furrow runs along the anterior posterior axis on both sides of the helmet (indicated by a yellow dashed line in Fig. 6d and by arrowheads in Fig. 6e and f). The second type consists of superficial dense furrows seen in most of the surface of the helmet (hereafter micro furrow). Most of these micro furrows are irregular “zigzag” in form (highlighted in pink in Fig. 6e) which enable the helmet to become broadened in every direction. In some specific areas, such as the surface of the median and lateral carina, the micro furrows are regular and parallel (highlighted in blue in Fig. 6e), which enable the tubular structures to expand in one direction. Both macro and micro furrows might be responsible for the enlargement of the helmet and determine the rate and direction of expansion.
Although the cytological contribution to helmet shrinking and its re-growth with folding is still unknown, we suggest that various cell activities (proliferation, shape change, migration and cell death) are involved in those processes. In particular, extensive cell proliferation is highly likely to be involved in the growth of the miniature helmet.
In conclusion, helmet development of Antianthe expansa occurs as follows. A sheath-like, monolayer epithelial sheet undergoes apolysis and shrinks into a miniature helmet by the lower and upper layer coming together (structural transition from monolayer to bi-layer). The miniature helmet then grows larger while forming both macro and micro furrows (size transition from small to large) (Fig. 5h).
Developmental similarities between helmet and wing
As we have shown, the treehopper develops densely folded primordia before the molt and extends them at molting, thereby forming a complex 3D structure. This developmental pattern is also seen in the development of the rhinoceros beetle horn [5] (Fig. 7). However, there are critical differences between the treehopper helmet and the beetle horn. In the beetle horn, the primordium is a single layered, sac-like structure and is extended by body fluid pressure filling the inside of the sac (Fig. 7). On the other hand, the treehopper helmet is a double layered, plywood-like structure and is extended by body fluid pressure through the tubular carinae (Fig. 7).
In its transformation from a monolayer to a bi-layer structure, the treehopper helmet shows similarity with wing development (Fig. 7). In hemimetabolous insects, the wing develops inside a sac-like wing bud as a monolayer structure, but transforms into a double layered, flat 2D structure during development [2] (Fig. 7). Initially, the bi-layer wing is a small miniature of the adult wing (Fig. 7), but subsequent cell proliferation enables the wing to grow by forming dense furrows via bending the bi-layer sheet (Fig. 7). Even in wing development in the highly-derived Drosophila, dorsal and ventral epithelia come together to form a double layered, flat miniature wing during development [13]. Moreover, the furrows are extended by pumping hemolymph through tubular structures in both treehopper helmet (carina) and wing (vein) (Fig. 7). Although anatomical homology between helmet and wing has not been supported [14, 15], recent molecular developmental studies suggest that the wing and helmet share molecular networks. That is, important wing developmental genes expressed in developing helmets [10] and the transcriptomic profile of developing helmets show more similarity to the developing wing than to other body regions (e.g. legs and tergites) [11]. Developmental similarity between helmet and wing shown in this study also supports this idea.