Conservation and diversification of gene function during mouthpart development in Onthophagus beetles

The evolutionary success of insects is in part attributable to the tremendous diversification of their mouthparts, which permitted insects to radiate into novel food niches. The developmental genetic basis of mouthpart development has been well studied in at least two insect taxa possessing derived mouthparts, the hemipteran Oncopeltus fasciatus and Drosophila. However, much less is known about the regulation of mouthpart differentiation of the presumed ancestral mandibulate type. Here we aim to extend current insights into the patterning of mandibulate mouthparts through a functional genetic analysis of three leg gap genes, homothorax (hth), dachshund (dac), and Distal-less (Dll), in the dung beetle Onthophagus taurus, a species whose mouthpart arrangement has in part retained, as well as diverged form, the ancestral mandibulate mouthpart type. We specifically include in this study a first functional genetic analysis of the adult labrum, an enigmatic mouthpart whose appendicular origin has been the subject of a long-standing debate. Our results support a functional role of all three patterning genes in the development of the labium, maxilla, as well as the labrum. In contrast, mandible development appeared to rely only on the patterning functions of hth and dac, but not Dll. Here, our results raise the possibility that evolutionary changes in the dac-patterning may have played an important role in the evolutionary transition from a short, triangular mandible adapted for chewing to the elongated, flat, and blade-like mandible of modern filter-feeding scarabaeine beetles. In general, our results contribute to a growing body of studies that suggest that basic patterning genes can contribute to morphological evolution of adult features while maintaining traditional patterning responsibilities at earlier developmental stages or in other body regions.     

Tomographic Reconstruction of Neopterous Carboniferous Insect Nymphs

Two new polyneopteran insect nymphs from the Montceau-les-Mines Lagerstätte of France are presented. Both are preserved in three dimensions, and are imaged with the aid of X-ray micro-tomography, allowing their morphology to be recovered in unprecedented detail. One–Anebos phrixos gen. et sp. nov.–is of uncertain affinities, and preserves portions of the antennae and eyes, coupled with a heavily spined habitus. The other is a roachoid with long antennae and chewing mouthparts very similar in form to the most generalized mandibulate mouthparts of extant orthopteroid insects. Computer reconstructions reveal limbs in both specimens, allowing identification of the segments and annulation in the tarsus, while poorly developed thoracic wing pads suggest both are young instars. This work describes the morphologically best-known Palaeozoic insect nymphs, allowing a better understanding of the juveniles’ palaeobiology and palaeoecology. We also consider the validity of evidence from Palaeozoic juvenile insects in wing origin theories. The study of juvenile Palaeozoic insects is currently a neglected field, yet these fossils provide direct evidence on the evolution of insect development. It is hoped this study will stimulate a renewed interest in such work.     

Mouthparts of flower-visiting insects

This review deals with the morphology and function of adult insect mouthparts modified to feed on nectar, pollen or petals. Specialization to nectar-feeding is evident in formation of proboscides of various lengths and designs. Proboscides of many Hymenoptera and Diptera function according to adhesion mechanisms that load nectar onto extensible apical mouthpart regions before fluid is conveyed along the food canal to the mouth by capillarity and suction. Predominantly suctorial proboscides evolved once in Lepidoptera, probably twice in Coleoptera, variously in some Hymenoptera and several times with similar design in Diptera. Many of them are particularly long and have sealed food tubes, specialized apical regions, new proboscis resting positions and modified feeding movements. Mouthparts of obligate pollen-feeding insects can be characterized by modified mandibles, specialized bristles for pollen manipulation and elaborate feeding movements. Often saliva is crucial for pollen retention and ingestion. In Coleoptera, intact pollen is gathered by sweeping movements of mouthparts; in Diptera, it is suspended in saliva prior to suction. Pollen is crushed by asymmetrical mandibles in aglossatan Lepidoptera and one group of basal Hymenoptera. Pollen-piercing mouthparts occur in Thysanoptera and one group of Diptera. Some butterflies and few Diptera extract nutrients from pollen by mixing it externally with saliva on their mouthparts. No mouthpart specializations to petal-feeding are reported in flower-visiting insects.     

The mouth apparatus of Lithodes maja (Crustacea: Decapoda) – form,function and biological role

Being able to utilize many different food resources is probably an important aspect of the success of decapod crustaceans which fill a wide range of various ecological niches worldwide. The phenomenon is facilitated by the complex mouth apparatus found in this group, whose representatives possess six pairs of mouthparts– mandibles, maxillae 1, and maxillae 2 and three maxillipeds, the first three pairs of thoracic appendages which are also specialized to food manipulation. These six pairs are able to perform a number of movements for transporting, aligning, crushing and cutting. Studies into the functional morphology of mouthparts have already been carried out in some decapod species. This study focuses on Lithodes maja, a species of the hitherto understudied king crabs (Lithodidae), chosen on the grounds of their remarkable evolutionary history as ‘derived hermit crabs’. Individuals were filmed while being presented with different kinds of food. To obtain structural information on the individual mouthpart elements as naturally arranged in relation to one another, the shape of the mouthparts was 3D‐reconstructed from micro‐CT scans. These data were complemented by scanning electron microscopy, to analyse the surface structures in detail. There is evidence that the various elements of the mouthparts of L. maja can be sorted into six functional groups: (i) transporting mouthpart elements, (ii) aligning/sorting mouthpart elements, (iii) clutching/holding mouthpart elements, (iv) tearing/cutting/crushing mouthpart elements, (v) current‐generating mouthpart elements and (vi) grooming mouthpart elements. According to our 3D reconstruction, there only seem to be minor differences in morphology and relative position between the mouthparts of L. maja and those of the closely related species, Pagurus bernhardus.     

Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum

Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. Here, we study the metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum, using RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, we tested the prediction that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, we tested hypotheses for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. Our data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal-distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles.     

A biomechanical analysis of prognathous and orthognathous insect head capsules: evidence for a many‐to‐one mapping of form to function

Insect head shapes are remarkably variable, but the influences of these changes on biomechanical performance are unclear. Among ‘basal’ winged insects, such as dragonflies, mayflies, earwigs and stoneflies, some of the most prominent anatomical changes are the general mouthpart orientation, eye size and the connection of the endoskeleton to the head. Here, we assess these variations as well as differing ridge and sclerite configurations using modern engineering methods including multibody dynamics modelling and finite element analysis in order to quantify and compare the influence of anatomical changes on strain in particular head regions and the whole head. We show that a range of peculiar structures such as the genal/subgenal, epistomal and circumocular areas are consistently highly loaded in all species, despite drastically differing morphologies in species with forward‐projecting (prognathous) and downward‐projecting (orthognathous) mouthparts. Sensitivity analyses show that the presence of eyes has a negligible influence on head capsule strain if a circumocular ridge is present. In contrast, the connection of the dorsal endoskeletal arms to the head capsule especially affects overall head loading in species with downward‐projecting mouthparts. Analysis of the relative strains between species for each head region reveals that concerted changes in head substructures such as the subgenal area, the endoskeleton and the epistomal area lead to a consistent relative loading for the whole head capsule and vulnerable structures such as the eyes. It appears that biting‐chewing loads are managed by a system of strengthening ridges on the head capsule irrespective of the general mouthpart and head orientation. Concerted changes in ridge and endoskeleton configuration might allow for more radical anatomical changes such as the general mouthpart orientation, which could be an explanation for the variability of this trait among insects. In an evolutionary context, many‐to‐one mapping of strain patterns onto a relatively similar overall head loading indeed could have fostered the dynamic diversification processes seen in insects.     

The development and evolution of insect mouthparts as revealed by the expression patterns of gnathocephalic genes

To understand better both the development and evolution of insect mouthparts, we have compared the expression pattern of several developmentally important genes in insects with either mandibulate or stylate-haustellate mouthparts. Specifically, we examined the expression of the proboscipedia (pb) and Distal-less (Dll) gene products as well as three regulators of pb, Sex combs reduced (Scr), Deformed (Dfd), and cap 'n' collar (cnc). These genes are known to control the identity of cells in the gnathal segments of Drosophila melanogaster and would appear to have similar conserved functions in other insects. Together we have made an atlas of gene expression in the heads of three insects: Thermobia domestica and Acheta domestica, which likely exemplify the mandibulate mouthparts present in the common insect ancestor, and Oncopeltus fasciatus, which has piercing-sucking mouth parts that are typical of the Hemiptera. At the earliest stages of embryogenesis, only the expression of pb was found to differ dramatically between Oncopeltus and the other insects examined, although significant differences were observed later in development. This difference in pb expression reflects an apparent divergence in the specification of gnathal identity between mandibulate and stylate-haustellate mouthparts, which may result from a "phylogenetic homeosis" that occurred during the evolution of the Hemiptera.     

Mouthparts of the bark beetle (Ips acuminatus) as a possible carrier of pathogenic microorganisms

The bark beetle Ips acuminatus Gyllenhal (Coleoptera: Curculionidae: Scolytinae) has been recently reported as one of the most serious secondary pests of pine trees. Since the adult beetles chew the sapwood to create tunnels, they have developed effective drilling mouthparts enough to make galleries directly into the heartwood of the tree. The mouthparts of this bark beetle exhibit typical morphology of mycophagous coleopteran beetles and have those characteristics of chewing mouthparts. Both maxillary and labial palpi have the functions of directing the food to the mouth and holding it while the mandibles chew the food. Although this bark beetle did not have prothoracic mycangial cavities, yeast-like spores were concentrated at the invaginated surface of mouthparts where cuticular hairs are densely packed. In particular, the cuticular surface around the base segments of these palpi has sufficient spaces to accommodate microorganisms during the series of drilling or feeding processes. Therefore, this paper reports detailed observation of the cuticular structure of the mouthpart using the field emission scanning electron microscopy (FESEM) for the purpose of demonstrating its possible implication to act as external carriers of pathogenic microorganisms.     

CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: A genetic tool in an apterygote development model of wing evolution

Despite previous developmental studies on basally branching wingless insects and crustaceans, the evolutionary origin of insect wings remains controversial. Knowledge regarding genetic regulation of tissues hypothesized to have given rise to wings would help to elucidate how ancestral development changed to allow the evolution of true wings. However, genetic tools available for basally branching wingless species are limited. The firebrat Thermobia domestica is an apterygote species, phylogenetically related to winged insects. T. domestica presents a suitable morphology to investigate the origin of wings, as it forms the tergal paranotum, from which wings are hypothesized to have originated. Here we report the first successful CRISPR/Cas9-based germline genome editing in T. domestica. We provide a technological platform to understand the development of tissues hypothesized to have given rise to wings in an insect with a pre-wing evolution body plan.     

Utility of geometric morphometrics for inferring feeding habit from mouthpart morphology in insects: tests with larval Carabidae (Insecta: Coleoptera)

Feeding habits are important life‐history traits in animals; however, methods for their determination are not well established in many species. The larvae of the beetle family Carabidae are an example. The present study tested the utility of geometric morphometrics of mouthpart morphology to infer the feeding habits of carabid larvae. Using Pterostichus thunbergi as a model system, larval feeding habits were inferred using geometric morphometrics of mouthparts and the results were compared with those obtained from rearing experiments. The rearing experiments indicated that P. thunbergi larvae are carnivores that require snails as an essential part of the diet. Through geometric morphometrics, associations between mouthpart morphology and larval feeding habits were confirmed for species in which these two traits are known. A discriminant analysis using these associations classified P. thunbergi larvae as snail/slug feeders, which is a result compatible with the rearing experiments. Geometric morphometrics also revealed that morphological integration and ontogenetic shape change might play roles in the diversification of mouthpart morphology. Overall, these results demonstrate the utility of the geometric morphometrics of mouthparts to infer feeding habit and to clarify the mechanisms of mouthpart morphological diversification in the study group, and the results also serve as a basis for future studies of other insect groups.     

Hypothesis testing in evolutionary developmental biology: a case study from insect wings

Developmental data have the potential to give novel insights into morphological evolution. Because developmental data are time-consuming to obtain, support for hypotheses often rests on data from only a few distantly related species. Similarities between these distantly related species are parsimoniously inferred to represent ancestral aspects of development. However, with limited taxon sampling, ancestral similarities in developmental patterning can be difficult to distinguish from similarities that result from convergent co-option of developmental networks, which appears to be common in developmental evolution. Using a case study from insect wings, we discuss how these competing explanations for similarity can be evaluated. Two kinds of developmental data have recently been used to support the hypothesis that insect wings evolved by modification of limb branches that were present in ancestral arthropods. This support rests on the assumption that aspects of wing development in Drosophila, including similarities to crustacean epipod patterning, are ancestral for winged insects. Testing this assumption requires comparisons of wing development in Drosophila and other winged insects. Here we review data that bear on this assumption, including new data on the functions of wingless and decapentaplegic during appendage allocation in the red flour beetle Tribolium castaneum.     

Analysis of nubbin expression patterns in insects

Previous studies have shown that the gene nubbin (nub) exhibits large differences in expression patterns between major groups of arthropods. This led us to hypothesize that nub may have evolved roles that are unique to particular arthropod lineages. However, in insects, nub has been studied only in Drosophila. To further explore its role in insects in general, we analyzed nub expression patterns in three hemimetabolous insect groups: zygentomans (Thermobia domestica, firebrat), dyctiopterans (Periplaneta americana, cockroach), and hemipterans (Oncopeltus fasciatus, milkweed bug). We discovered three major findings. First, observed nub patterns in the ventral central nervous system ectoderm represent a synapomorphy (shared derived feature) that is not present in other arthropods. Furthermore, each of the analyzed insects exhibits a species-specific nub expression in the central nervous system. Second, recruitment of nub for a role in leg segmentation occurred early during insect evolution. Subsequently, in some insect lineages (cockroaches and flies), this original role was expanded to include joints between all the leg segments. Third, the nub expression in the head region shows a coordinated change in association with particular mouthpart morphology. This suggests that nub has also gained an important role in the morphological diversification of insect mouthparts. Overall, the obtained data reveal an extraordinary dynamic and diverse pattern of nub evolution that has not been observed previously for other developmental genes.     

Receptors of Garlic (Allium sativum) Lectins and Their Role in Insecticidal Action

Garlic (Allium sativum) lectins are promising candidate molecules for the protection against chewing (lepidopteran) as well as sap sucking (homopteran) insect pests. Molecular mechanism of toxicity and interaction of lectins with midgut receptor proteins has been described in many reports. Lectins show its effect right from sensory receptors of mouth parts by disrupting the membrane integrity and food detection ability. Subsequently, enter into the gut lumen and interact with midgut glycosylated proteins like alkaline phosphatase (ALP), aminopeptidase-N (APN), cadherin-like proteins, polycalins, sucrase, symbionin and others. These proteins play critical role in life cycle of insect directly or indirectly. Lectins interfere with the activity of these proteins and causes physiological disorders leading to the death of insects. Lectins further transported across the insect gut, accumulated in various body parts (like haemolymph and ovary) and interact with intracellular proteins like symbionin and cytochrome p450. Binding with cytochrome p450 (which involve in ecdysone synthesis) might interfere in the development of insects, which results in growth retardation and pre-mature death.     

Re‐establishment of biting mouthparts in desert‐living dung beetles (Scarabaeidae: Scarabaeinae) feeding on plant litter—Old structures reacquired or new ones evolved?

Evolution of mouthparts in adult dung beetles (Scarabaeidae: Scarabaeinae) for eating moist, fresh dung was linked with a loss of the ability to chew. However, the desert‐living genus Pachysoma, probably evolved from a wet‐dung feeding, Scarabaeus‐like ancestor, has switched to a diet of dry fecal pellets (of rodents or small ruminants) and plant litter that requires re‐establishment of chewing. Indeed, gut contents of a litter‐feeding Pachysoma species indicate efficient food comminution. Based on scanning electron microscopy, cutting and grinding mouthpart structures in six Pachysoma species, of two lineages and with different food preferences, are described and compared with homologous structures in wet‐dung feeding Scarabaeus species. In Pachysoma, cutting and breaking of large food items is probably performed by a clypeal scraper, a prominent epipharyngeal tooth and large maxillary galeal hooks. Further comminution is achieved by a large, grinding area evolved on the mandibular molae. Interspecific differences and the probable function and evolution of these structures are discussed. Particularly, the unique tools for cutting/breaking are completely novel structures and not results of some reacquisition of normal biting mouthparts. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

中华蚊蝎蛉成虫口器感器的超微结构

口器感器在昆虫取食活动中起着重要作用, 但蚊蝎蛉成虫口器上感器的种类和形态迄今未见报道。我们利用扫描电子显微镜, 观察了中华蚊蝎蛉Bittacus sinensis Walker成虫口器上的感器。结果显示: 中华蚊蝎蛉口器上共有8种感器, 分别为锥形、毛形、刺形、指形、掌状、钟形、柱状感器及Böhm氏鬃毛, 主要集中于内唇、 下颚须以及下唇须上。锥形感器和刺形感器数量最多; 毛形感器主要在下颚轴节、 茎节和下唇的亚颏和前颏有分布; 钟形感器和Böhm氏鬃毛只存在于下唇须和下颚须上。下颚须端节和下唇须端节的感器种类相同, 以锥形感器为主。高度骨化的上颚以及下颚内颚叶与外颚叶上未发现感器分布。简要讨论了口器感器在昆虫分类中的意义。     

Mouthparts and stylet penetration of the lac insect Kerria lacca (Kerr) (Hemiptera:Tachardiidae)

Hitherto less known aspects on mouthpart morphology and penetration mechanism of the lac insect Kerria lacca have been explored. Unique details of the mouthparts, i.e. morphology of labium and stylets and salivary sheath have been brought out. The gross morphology of the mouthparts though resembled other plant sucking homopterans; a two-segmented labium with symmetrically distributed six pairs of contact-chemoreceptors on its surface was distinct; the mandibular stylets had serrations on its extreme apical region, while the maxillary stylets had their external surface smooth with parallel longitudinal grooves on their inner surface. Formation of flanges, salivary sheath and penetration pathway observed along with probing and penetration of the stylets intracellularly up to the phloem cells, as illustrated herein, are the addition to the existing knowledge on the structural details of the mouthparts and the feeding behavior thereupon.     

Insect Leaf-Chewing Damage Tracks Herbivore Richness in Modern and Ancient Forests

The fossil record demonstrates that past climate changes and extinctions significantly affected the diversity of insect leaf-feeding damage, implying that the richness of damage types reflects that of the unsampled damage makers, and that the two are correlated through time. However, this relationship has not been quantified for living leaf-chewing insects, whose richness and mouthpart convergence have obscured their value for understanding past and present herbivore diversity. We hypothesized that the correlation of leaf-chewing damage types (DTs) and damage maker richness is directly observable in living forests. Using canopy access cranes at two lowland tropical rainforest sites in Panamá to survey 24 host-plant species, we found significant correlations between the numbers of leaf chewing insect species collected and the numbers of DTs observed to be made by the same species in feeding experiments, strongly supporting our hypothesis. Damage type richness was largely driven by insect species that make multiple DTs. Also, the rank-order abundances of DTs recorded at the Panamá sites and across a set of latest Cretaceous to middle Eocene fossil floras were highly correlated, indicating remarkable consistency of feeding-mode distributions through time. Most fossil and modern host-plant pairs displayed high similarity indices for their leaf-chewing DTs, but informative differences and trends in fossil damage composition became apparent when endophytic damage was included. Our results greatly expand the potential of insect-mediated leaf damage for interpreting insect herbivore richness and compositional heterogeneity from fossil floras and, equally promisingly, in living forests.     

Profound head modifications in Claviger testaceus (Pselaphinae,Staphylinidae, Coleoptera) facilitate integration into communities of ants

Clavigeritae is a group of obligate myrmecophiles of the rove beetle subfamily Pselaphinae (Staphylinidae). Some are blind and wingless, and all are believed to depend on ant hosts through feeding by trophallaxis. Phylogenetic hypotheses suggest that their ancestors, as are most pselaphines today, were free-living predators. Morphological alterations required to transform such beetles into extreme myrmecophiles were poorly understood. By studying the cephalic morphology of Claviger testaceus, we demonstrate that profound changes in all mouthpart components took place during this process, with a highly unusual connection of the maxillae to the hypopharynx, and formation of a uniquely transformed labium with a vestigial prementum. The primary sensory function of the modified maxillary and labial palps is reduced, and the ventral mouthparts transformed into a licking/‘sponging’ device. Many muscles have been reduced, in relation to the coleopteran groundplan or other staphylinoids. The head capsule contains voluminous glands whose appeasement secretions are crucial for the beetle survival in ant colonies. The brain, in turn, has been shifted into the neck region. The prepharyngeal dilator is composed of an entire series of bundles. However, the pharynx does not show any peculiar adaptations to taking up liquid food. We demonstrate that far-reaching cephalic modifications characterize C. testaceus, and that the development of appeasement glands and adaptation of the mouthparts to trophallaxis determine the head architecture of this extreme myrmecophile.