In butterflies, bees, flies and true bugs specific mouthparts are in close contact or even fused to enable piercing, sucking or sponging of particular food sources. The common phenomenon behind these mouthpart types is a complex composed of several consecutive mouthparts which structurally interact during food uptake. The single mouthparts are thus only functional in conjunction with other adjacent mouthparts, which is fundamentally different to biting–chewing. It is, however, unclear when structural mouthpart interaction (SMI) evolved since this principle obviously occurred multiple times independently in several extant and extinct winged insect groups. Here, we report a new type of SMI in two of the earliest wingless hexapod lineages—Diplura and Collembola. We found that the mandible and maxilla interact with each other via an articulatory stud at the dorsal side of the maxillary stipes, and they are furthermore supported by structures of the hypopharynx and head capsule. These interactions are crucial stabilizing elements during food uptake. The presence of SMI in these ancestrally wingless insects, and its absence in those crustacean groups probably ancestral to insects, indicates that SMI is a groundplan apomorphy of insects. Our results thus contradict the currently established view of insect mouthpart evolution that biting–chewing mouthparts without any form of SMI are the ancestral configuration. Furthermore, SMIs occur in the earliest insects in a high anatomical variety. SMIs in stemgroup representatives of insects may have triggered efficient exploitation and fast adaptation to new terrestrial food sources much earlier than previously supposed.
Hexapoda (insects in the broad sense) have evolved an astonishing diversity of mouthparts tailored to use different resources of food [1,2]. For example, dragonflies and crickets use biting–chewing motions of their mandibles to chop food particles, true bugs evolved piercing–sucking mouthparts to suck fluids from plants, flies evolved sponging mouthparts, and moths and butterflies evolved the unique, maxillary-derived proboscis to siphon mostly nectar of flowers [1,2]. Although mouthparts functionally interact in nearly all insects to process food, many winged insects evolved a structural interaction , sometimes even leading to mouthpart fusion, resulting in the formation of new mouthpart types such as the proboscis of many lineages. Corresponding morphological changes are radical, and morphologically very different to each other; a comparable diversity and complexity of mouthparts is not present in any other arthropod group. It is unclear when this major trend in insect evolution—structural mouthpart interaction (SMI)—evolved for the first time.
The general principle of SMI apparently evolved multiple times independently in several extant winged insect groups, and in other extinct groups such as Palaeodictyopterida. Current hypotheses assume the rolling–biting mouthpart type without any form of SMI as ancestral. This condition consists of mandibles attached with one or two joints to the head, as the potential groundplan in insects [3,4]. However, these hypotheses fall short in explaining the early evolution of insect mouthparts, as they neglect the mouthpart configuration and phylogenetic relationships of the earliest insects, the ancestrally wingless Protura, Collembola and Diplura.
These minute soil and leaf litter inhabitants are important decomposers of rotten organic material , also frequently feeding on the cell content of fungal hyphae , plant roots [2,7] and, in rare cases, on soft-bodied soil arthropods . They possess mouthparts (mandibles and maxillae) which are almost entirely hidden within the head capsule (entognathy) so that the head forms an external (functional) mouth opening (henceforth ‘oral opening’) in addition to the anatomical mouth opening. Piercing, and to a minor extent biting–chewing motions, through this narrow oral opening allows the insect to penetrate plant cell walls or soft animal tissue and suck out the contained liquids . Small blended food particles also are milled between the mandibles in many Collembola, while some species of Diplura prey on other soil organisms with their knife-like mandibles after hauling them towards the oral opening with their maxillae . A growing body of morphological [8–10] and transcriptome-based studies  supports a sistergroup relationship of the entognathous Diplura with ectognathous insects (Cercophora hypothesis), while Protura + Collembola (=Ellipura) are the sistergroup to Cercophora.
Owing to their entognathy and their small size, mouthpart morphology and movements during food uptake have been a long-standing enigma in Collembola and Diplura. In this study, we used three complementary synchrotron microCT (SR-µCT) imaging set-ups [12–14] to investigate the mouthpart anatomy of Collembola and Diplura at high magnifications while entirely preserving the spatial mouthpart arrangement. The mouthpart morphology of these ancestral insects displays an unusual type of SMI, based on homologous structures of the mandibles and maxillae.
2. Experimental procedures
Samples were critical-point dried (model E4850, BioRad) to avoid shrinking artefacts of the internal anatomy and mounted on beamline-specific specimen holders. High-resolution SR-µCT (HR SR-µCT) was performed at the Deutsches Elektronen Synchrotron (DESY; beamlines DORIS III/BW2 and PETRA III/IBL P05, operated by the Helmholtz-Zentrum Geesthacht, Hamburg, Germany) with a stable beam energy of 8 keV and high-density resolution in absorption contrast mode [12,15]. The field of view (FOV) was adjusted to each sample in order to maximize resolution. HR SR-µCT at the Swiss Light Source of the Paul-Scherrer Institut (PSI; Villigen, Switzerland; beamline TOMCAT ) was done using a stable beam energy of 10 keV in absorption-contrast mode and with an energy of 20 keV in phase-contrast mode. We used the 20× objective in order to realize a FOV of 0.83 × 0.70 mm with a resulting pixel size of 0.325 µm². HR SR-µCT at the Super Photon ring-8 GeV (SPring-8; Hyogo, Japan) was done at the beamline BL47XU  using a stable beam energy of 8 keV in absorption-contrast mode. The tomography system consists of full-field X-ray microscope with Fresnel zone plate optics . The FOV and effective pixel size are 0.11 mm2 and 0.0826 µm2, respectively.
The dipluran Campodea sp. and the collembolan Pogonognathellus flavescens were scanned at DESY as well as at PSI. All following species were investigated at PSI: Neanura muscorum, Thaumanura sp., Megaphorura arctica, Lepidocyrtus lignorum, Orchesella sp., Dicyrtoma sp., Triacanthella rosea, Neotropiella carli, Brachystomellides neuquensis, Podura aquatica, Sminthurinus niger, Isotoma sp., Desoria sp. (all Collembola), Campodea augens, Metriocampa sp., Lepidocampa weberi, Occasjapyx japonicus, Occasjapyx akiyamae, Catajapyx aquilionaris, Atlasjapyx cf atlas (all Diplura). The proturan Acerentomon sp. and the collembolan Megalothorax sp. were investigated at SPring-8. Collembola show a wide variety of mandible and maxilla forms , which is the reason for our coverage of all major groups within Collembola (see electronic supplementary material S2 for taxon coverage). However, we restrict our results to the biting–chewing mouthpart type for Collembola, as groups with this mouthpart type were repeatedly recovered as more ancestral with respect to collembolan phylogenetic relationships [17–19].
Subsequent segmentation of the reconstructed image stacks was accomplished with Reconstruct  and ITK-SNAP . We used manual segmentation as well as semi-automatic segmentation algorithms based on grey-value differences (‘Wildfire’ tool in Reconstruct), or the ‘region competition’ algorithm for fully automatic three-dimensional segmentation (in ITK). Rendering of the resulting mesh objects was done with Blender (blender.org). Objects were imported as wrl-files (Reconstruct) or stl-files (ITK) into Blender. The surface meshes were smoothed using the smoothing modifier of Blender. Vertex reduction of the meshes was done with the ‘remove doubles’ algorithm set to 0.03. No further processing was done, other than light and camera adjustment in order to minimize structure alteration. Figures were produced with Scribus; all programs used are distributed under the general public license. Three-dimensional models of the relevant mandible, maxilla and head structures of A. cf. atlas (electronic supplementary material S3) and P. flavescens (electronic supplementary material S4) can be viewed with the open-source program Meshlab. Other three-dimensional model viewing programs should also work, but were not tested by us. TIFF image stacks of the SR-µCT scans of Atlasjapyx and P. flavescens are provided as reduced film sequences in electronic supplementary material, movies S1a,b, and in full size (16 GB) upon request.
3. Results and discussion
The mouthparts of Collembola and Diplura are hidden within the head, which is defined as an entognathous condition. Only the tips of the mouthparts protrude from the oral opening, which is generated by the fused head capsule and labium . Collembolan and dipluran mouthparts show a specific type of SMI, based on an articulatory point between the mandible and the maxilla. This contact point has the same structural basis and is present in all studied taxa, although Collembola show a wide variety of mandible forms .
(a) Anatomy of the structural mouthpart interaction in Diplura
In Diplura, the contact point is formed by an articulatory stud of the maxillary stipes (STST; figure 1a,b,d). This stud is a short, upraised prominence, and is supported by two internal stipital strengthening ridges (SR, figure 1d). The dorsal ridge originates at the base of the stud and extends along the inner stipital wall to fuse with the ventral stipital ridge. The tip of the stud is in contact with a slight concavity at the posterior outer wall of the mandible. At the contact point, a further strengthening ridge is present inside the mandible continuing from the dorsal to the ventral mandibular rim (SRmd; figure 1d).
Two other formerly described contact points  are present in Diplura, which interact with the mandible during food uptake. One is composed of a spine-like anterior part of the hypopharynx (APHY; figure 1c), which is in contact with the ventral base of the incisival area of the mandible. The other contact point is formed by a spine-like apodeme of the head capsule (APHC; figure 1b,c), which is in contact with the dorsal base of the incisival area right opposite the APHY. The APHC and APHY are connected to each other at the inner mandibular rim, thus forming an arch enclosing the mandible (figure 1c). The mandible of Diplura is moved by nine muscles; most of these serve double functions depending on the location of the mandible during movement (electronic supplementary material S2).
Mandible movement in Diplura is a combination of protraction–adduction with retraction–abduction as countermovements (electronic supplementary material, movie S1a). The three interaction points serve as crucial guiding structures for the mandibles during food uptake. The stipital stud prevents a lateral evasion of the posterior part of the mandible, while the apodemes of head and hypopharynx collectively serve as ‘splints’ preventing dorsal and ventral sheering out of the mandibles during movement (electronic supplementary material, movie S1a).
(b) Anatomy of the structural mouthpart interaction in Collembola
The newly found SMI in Collembola also is composed of a stipital stud (STST) that originates at the median anterior (dorsal) wall of the stipes (figure 1e–g). As in Diplura, this stud is supported by two strengthening ridges, one spanning from the base of the stud to the opposite side of the maxillary stipes where it fuses with the second strengthening ridge (SR; figure 1g). The stipital stud articulates with the posterior mandibular wall during mouthpart movement (electronic supplementary material, movie S1b).
The movement of the mandible is further stabilized by three other contact points. One articulation point is composed of a thickening of the lateral parts of the clypeus (THCL) close to the base of the labrum (figure 1f), which interacts with the mandibles at the anterior transition zone between mola and incisivi (electronic supplementary material, movie S1b). The other two contact points are an anterior part of the hypopharynx (APHY) interacting with the mandible at the inner wall dorsal of the mola [4,22,23] and a spine-like apodeme of the head capsule (APHC) in contact with the anterior (dorsal) mandibular wall, as in Diplura (figure 1f,g; electronic supplementary material, movie S1b).
Mandible movement in Collembola is characterized by rotation around the dorsoventral axis in addition to protraction/abduction and retraction/adduction (electronic supplementary material, movie S1b). The rotation is executed during and after protraction in order to shear off food particles or to mill these fragments between the mandibular molar areas [4,22]. As Koch  and Manton  previously pointed out, collembolan mandibles are distally supported during this movement by the THCL, and the APHC and APHY, which hold the mandibles like fingers a pencil. However, it was unclear how proximal (or posterior) support of the mandibles could be achieved with this configuration and how the mandibular main body is prevented from moving posterad. The STST of the maxillae fulfils both of these functions by stabilizing mandible movement in the transversal plane. During biting, mandibles and maxillae move synchronously (electronic supplementary material, movie S1b; ) with the effect that the maxillae are stabilizing the entire movement sequence of the mandibles.
(c) The evolution of structural mouthpart interactions in insects
In current hypotheses, it is postulated that the rolling–biting mouthpart type without any form of SMI, but composed of mandibles attached with one or two joints to the head, is the ancestral condition in insects (groundplan) [3,4]. However, these hypotheses inadequately explain the evolution of insect mouthparts as they fail to account for the mouthpart configuration and phylogenetic relationships of the earliest insects, the ancestrally wingless Protura, Collembola and Diplura.
Although the stipital studs of the maxillae in Collembola and Diplura support slightly different functions during mouthpart movement, we consider them homologous. In both groups, these articulatory studs are located on the dorsal side of the stipes and are supported by a conspicuous configuration of two strengthening ridges at the inner side of the stipes that show the same arrangement. These ridges reinforce the stipital stud to counter loads imposed on it and the maxilla during mandibular movement, as is the case for other ridges in the insect head [25–28]. The two modes of mandible–maxilla interaction in Collembola and Diplura are thus examples of a structural mouthpart interaction in ancestrally wingless insects.
We further propose that the APHC and APHY interactions with the mandibles are also homologous in Collembola and Diplura . In both lineages, the apodeme of the head capsule originates at the same location lateral to the labral base and above the oral opening, and continues inwards alongside the dorsal base of the incisival area of the mandibles. Likewise, the ventral part of the hypopharyngeal fulturae extends along the ventral base of the incisival area and is connected to the APHC in both Collembola and Diplura. The location, structural basis, origin, contact with the mandibles and spatial arrangement to other structures are the same in Collembola and Diplura.
The homology of the SMIs in Collembola and Diplura has important implications for our understanding of insect mouthpart evolution (figure 2). Currently available morphological [9,33] and molecular evidence  strongly suggests that Collembola and Diplura do not form a monophyletic group. Instead Diplura are the sistergroup to ectognathous insects (bristletails, silverfish and winged insects) and Collembola the sistergroup to Protura (Ellipura hypothesis; figure 2). Additionally, SMIs of any type are not known to occur in crustaceans [34,35]. Thus, the presence of a homologous SMI in Collembola and Diplura implies the presence of this basic principle in stemgroup representatives of the entire Hexapoda.
Although the SMI in Collembola and Diplura is based on homologous structures, the general principle is morphologically variable among early insects (figure 2). Recently, another SMI type was discovered in the ectognathous Archaeognatha , but its structural basis is different: the maxillary palpus instead of the stipes interacts via a protuberance with the anterior side of the mandible and prevents an anterior sheering out of the mandible during movement. The SMI in entognathous Protura is formed by the so-called fulcro-tentorium  as well as the anterior portion of the stipital body, both of which act as a ‘sheath’ guiding the mandible during protraction (figure 2; electronic supplementary material, three-dimensional model S5 and ). Owing to these morphological differences, the SMIs of Archaeognatha and Protura appear not homologous to the SMI in Collembola and Diplura.
It has been suggested that patterns of Hox gene expression are correlated with the evolution of novel mouthpart morphologies [37–39]. In Protura, Collembola and Diplura, Hox gene expression in developing mouthparts has not been studied yet; therefore, the influence of these regulatory mechanisms on the general principle is unclear. It could be rewarding to investigate gene expression patterns in Protura, Collembola and Diplura in order to clarify the evolution of gene expression patterns characteristic for structurally interacting mouthparts.
It remains equally unclear until which point SMIs are exhibited in the stemline of early insects before this principle occurs again in more derived insects (figure 2). The enormous, winged Palaeodictyopterida, the only major insect group to have become extinct, had stylet-like paired mandibles and maxillae, and a hypopharynx . These were interlocked with each other [2,40], thus representing the basic principle of SMI. Evidence suggests that their diet consisted mainly of plant fluids [2,32]. The phylogenetic placement of Palaeodictyopterida is a matter of debate (figure 2). They are placed as sistergroup to modern dragonflies (Odonata ), as sistergroup to Palaeoptera (Ephemeroptera + Odonata ) or even as sistergroup to Neoptera .
Regardless of the position of Palaeodictyopterida, the biting–chewing mouthpart type with mandibles connected to the head via articulations, as shown in Archaeognatha, can no longer be attributed as the plesiomorphic condition in insects. Rather, the entognathous piercing–sucking functional mouthpart unit with elongated, distally pointed mandibles appears ancestral. Exposed, structurally uncoupled mouthparts are a groundplan apomorphy of silverfish and winged insects.
It is assumed that mouthpart diversification enabled the exploration of new food sources [43,44], and accordingly was probably triggered by the evolution of vascular plants (approx. 415 Ma ) and trees (approx. 380 Ma ), the radiation of seed plants (approx. 340–280 Ma ) and the radiation of flowering plants (approx. 120–70 Ma [48,49]). Likewise, a correlation of the piercing–sucking functional mouthpart unit with the appearance of non-symbiotic terrestrial fungi [29,50] or bryophytes  in the mid-Ordovician is a likely scenario given the potential origin and diversification of Collembola and Diplura at the same time as indicated by recent dating analyses (figure 2) . In this scenario, the mandibles of the earliest insects are protracted and retracted through a narrow oral opening (entognathy), and they are stabilized by the maxillae and hypopharynx to penetrate the thin cell walls of fungi and plants.
All research was conducted under appropriate licences for collecting insects (personal licence 45/7/3).
All electronic supplements are deposited in the Dryad repository at http://dx.doi.org/10.5061/dryad.gf643.
A.B., R.M., M.S., P.V., F.W., K.U. and B.M. designed the experiments; A.B., P.T.R., R.M., P.V., K.U. and F.W. conducted the experiments; A.B., P.T.R., R.M. and B.M. analysed the data; and A.B., R.M. and B.M. wrote the manuscript. All authors read and approved the final version of the manuscript.
We declare we have no competing interests.
The study received funding from DESY (ID: I-20120065), PSI (ID: 20110069 & 20140056) and SPring-8 (ID: 2014B1046) for the beamline experiments. A.B. was supported by the Japanese Society for the Promotion of Science (JSPS, ID: P14071) and R.M. by a Grant-in-Aid from the JSPS (Scientific Research C, ID: 25440201). A.B. and P.T.R. were additionally partly supported by the joint research program of the DAAD and the University of Tsukuba (ID 57060275).
Cyrille A. D'Haese provided specimens of Megaphorura arctica, Lepidocyrtus lignorum, Orchesella sp., Dicyrtoma sp., Triacanthella rosea, Neotropiella carli and Brachystomellides neuquensis. Karen Meusemann helped to collect Podura aquatica, Alex Böhm collected Catajapyx aquilionaris and Kaoru Sekiya provided Occasjapyx japonicus, O. akiyamae and Lepidocampa weberi. Atlasjapyx cf. atlas was kindly provided by Thomas Wesener. Felix Beckmann and Julia Herzen (both Helmholtz-Zentrum Geesthacht) helped during the experiments at DESY beamlines DORIS III/BW2 and PETRA III/IBLP05. We furthermore thank Sina David, Karen Meusemann, Björn von Reumont, Katrin Wittig, Susanne Düngelhoef, Thorsten Klug, Makiko Fukui, Toshiki Uchifune and Yoshie Jintsu-Uchifune for their help during the beamline experiments. Nikola Szucsich gave useful advice during the preparation of the manuscript. Hans Pohl and Torsten Wappler did the vector drawings of insects in figure 2.
- Received May 1, 2015.
- Accepted June 26, 2015.
- © 2015 The Author(s)
Published by the Royal Society. All rights reserved.