The expression of the proximodistal axis patterning genes Distal-less and dachshund in the appendages of Glomeris marginata (Myriapoda: Diplopoda) suggests a special role of these genes in patterning the head appendages

The genes Distal-less, dachshund, extradenticle, and homothorax have been shown in Drosophila to be among the earliest genes that define positional values along the proximal-distal (PD) axis of the developing legs. In order to study PD axis formation in the appendages of the pill millipede Glomeris marginata, we have isolated homologues of these four genes and have studied their expression patterns. In the trunk legs, there are several differences to Drosophila, but the patterns are nevertheless compatible with a conserved role in defining positional values along the PD axis. However, their role in the head appendages is apparently more complex. Distal-less in the mandible and maxilla is expressed in the forming sensory organs and, thus, does not seem to be involved in PD axis patterning. We could not identify in the mouthparts components that are homologous to the distal parts of the trunk legs and antennnae. Interestingly, there is also a transient premorphogenetic expression of Distal-less in the second antennal and second maxillary segment, although no appendages are eventually formed in these segments. The dachshund gene is apparently involved both in PD patterning as well as in sensory organ development in the antenna, maxilla, and mandible. Strong dachshund expression is specifically correlated with the tooth-like part of the mandible, a feature that is shared with other mandibulate arthropods. homothorax is expressed in the proximal and medial parts of the legs, while extradenticle RNA is only seen in the proximal region. This overlap of expression corresponds to the functional overlap between extradenticle and homothorax in Drosophila.     

Functional analyses in the hemipteran Oncopeltus fasciatus reveal conserved and derived aspects of appendage patterning in insects

The conservation of expression of appendage patterning genes, particularly Distal-less, has been shown in a wide taxonomic sampling of animals. However, the functional significance of this expression has been tested in only a few organisms. Here we report functional analyses of orthologues of the genes Distal-less, dachshund, and homothorax in the appendages of the milkweed bug Oncopeltus fasciatus (Hemiptera). This hemimetabolous insect has typical legs but highly derived mouthparts. Distal-less, dachshund, and homothorax are conserved in their individual expression patterns and functions in the legs of Oncopeltus, but their functions in other appendages are in some cases divergent. We find that specification of antennal identity does not require wild-type Distal-less activity in Oncopeltus as it does in Drosophila. Additionally, the mouthparts of Oncopeltus show novel patterns of gene expression and function, relative to other insects. Expression of Distal-less in the maxillary stylets of Oncopeltus does not seem necessary for proper development of this appendage, while dachshund and homothorax are crucial for formation of the mandibular and maxillary stylets. These data are used to evaluate hypotheses for the evolution of hemipteran mouthparts and the evolution of developmental mechanisms in insect appendages in general.     

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.     

Fine structure and functional morphology of the mouthparts of Bittacus planus and Terrobittacus implicatus (Insecta: Mecoptera: Bittacidae)

The Bittacidae are unique in Mecoptera for their adults being predaceous. However, their mouthparts have not been well documented for functional morphology to date. Here, we investigated the mouthpart morphology of the hangingflies Bittacus planus Cheng and Terrobittacus implicatus (Huang & Hua) using scanning electron microscopy. The mouthparts are of the mandibulate type and situated at the tip of an elongated rostrum. The labrum is greatly elongated, roughly twice as long as the subquadrate clypeus. The epipharynx is furnished with a row of basiconic sensilla arranged evenly as a median band extending from the apex to the base. The mandibles are slender and elongated, bearing a sharp lateral and a small mesal tooth. The maxillae are well developed, each consisting of a partially sclerotized cardo and a stipes, a hirsute galea and a lacinia, and a five-segmented maxillary palp. The sensillar pattern on the distal segment of the maxillary palp differs slightly between the two bittacid species. The labium is composed of a postmentum, a prementum, and a pair of two-segmented labial palps. The feeding mechanism of bittacids is briefly discussed in combination with the mouthpart morphology and their feeding habits.     

Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning.

Proximodistal patterning in the Drosophila leg is elaborated from the circular arrangement of the proximal domain expressing escargot and homothorax, and the distal domain expressing Distal-less that are allocated during embryogenesis. The distal domain differentiates multiply segmented distal appendages by activating additional genes such as dachshund. Secreted signaling molecules Wingless and Decapentaplegic, expressed along the anterior-posterior compartment boundary, are required for activation of Distal-less and dachshund and repression of homothorax in the distal domain. However, whether Wingless and Decapentaplegic are sufficient for the circular pattern of gene expression is not known. Here we show that a proximal gene escargot and its activator homothorax regulate proximodistal patterning in the distal domain. Clones of cells expressing escargot or homothorax placed in the distal domain induce intercalary expression of dachshund in surrounding cells and reorient planar cell polarity of those cells. Escargot and homothorax-expressing cells also sort out from other cells in the distal domain. We suggest that inductive cell communication between the proximodistal domains, which is maintained in part by a cell-sorting mechanism, is the cellular basis for an intercalary mechanism of the proximodistal axis patterning of the limb.     

<Emphasis Type="Italic">Proboscipedia</Emphasis> represses distal signaling in the embryonic gnathal limb fields of<Emphasis Type="Italic"> Tribolium castaneum</Emphasis>

Orthologs of the Hox genes Sex combs reduced ( Scr) and proboscipedia ( pd) are active in the developing labial appendages of all insect species tested. The remarkable variation among insect gnathal structures, particularly in the distal podomeres, suggests two Hox genes may enhance the adaptive potential of gnathal appendage morphology. Functional studies in the fruitfly Drosophila melanogaster, the flour beetle Tribolium castaneum and the milkweed bug Oncopeltus fasciatus show that cooperation between Scr and pb has been generally conserved, but specific mechanisms have been altered during evolution. Cross-regulation of pb by Scr is evident in Drosophila and Tribolium, the more closely related of the three species, but not in Oncopeltus. In all three species, pb function is restricted to the distal podomeres, but details are only known for Drosophila and Oncopeltus, two species exhibiting specialized stylate-haustellate mouthparts. Drosophila pb is required for distal Scr expression, and to repress the appendage patterning genes dachshund and Distal-less ( Dll). Oncopeltus pb has the novel capacity to specify leg fates. Little is known about distal functions of Tribolium pb. Hypomorphic mutations of the Tribolium pb ortholog maxillopedia can be arranged in a graded phenotypic series of palp to leg transformations along both the proximodistal and dorsoventral axes. Mid-embryonic expression profiles of Tribolium pb, Scr, wingless ( wg) and Dll genes were examined in maxillopedia hypomorphic and null mutant backgrounds. Levels of pb and Scr are significantly reduced in the distal appendage field. Tribolium pb therefore positively regulates distal Scr expression, a role that it has in common with Drosophila pb. Tribolium wg is normally down-regulated in the distal domain of the embryonic gnathal appendage buds. It becomes activated distally in maxillopedia hypomorphs. Repression of wg by pb has not been reported in the labial imaginal discs of Drosophila. Alterations of Tribolium Scr and wg expression occur in Dll-expressing cells, however, unlike in Drosophila labial imaginal discs, Dll expression appears unaffected in pb hypomorphic backgrounds. We conclude that the Hox genes Sex combs reduced and proboscipedia control an appendage organizer and cell autonomous fate determination during embryonic labial palp development in Tribolium.     

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。     

The establishment of segmentation in the Drosophila leg.

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. I demonstrate that segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and I define the sequence in which the initial segmental subdivisions arise. I further demonstrate that the proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished.     

Functional Morphology and Sexual Dimorphism of Mouthparts of the Short-Faced Scorpionfly Panorpodes kuandianensis (Mecoptera: Panorpodidae)

Mouthparts are closely associated with the feeding behavior and feeding habits of insects. The features of mouthparts frequently provide important traits for evolutionary biologists and systematists. The short-faced scorpionflies (Panorpodidae) are distinctly different from other families of Mecoptera by their extremely short rostrum. However, their feeding habits are largely unknown so far. In this study, the mouthpart morphology of Panorpodes kuandianensis Zhong et al., 2011 was investigated using scanning electron microscopy and histological techniques. The mandibulate mouthparts are situated at the tip of the short rostrum. The clypeus and labrum are short and lack distinct demarcation between them. The epipharynx is furnished with sublateral and median sensilla patches. The blade-shaped mandibles are sclerotized and symmetrical, bearing apical teeth and serrate inner margins. The maxilla and labium retain the structures of the typical pattern of biting insects. The hirsute galea, triangular pyramid-shaped lacinia, and labial palps are described in detail at ultrastructural level for the first time. Abundant sensilla are distributed on the surface of maxillary and labial palps. The sexual dimorphism of mouthparts is found in Panorpodes for the first time, mainly exhibiting on the emargination of the labrum and apical teeth of mandibles. Based on the features of mouthparts, the potential feeding strategy and feeding mechanism are briefly discussed in Panorpodes.     

Patterning of the branched head appendages in Schistocerca americana and Tribolium castaneum

Much of our understanding of arthropod limb development comes from studies on the leg imaginal disc of Drosophila melanogaster. The fly limb is a relatively simple unbranched (uniramous) structure extending out from the body wall. The molecular basis for this outgrowth involves the overlap of two signaling molecules, Decapentaplegic (Dpp) and Wingless (Wg), to create a single domain of distal outgrowth, clearly depicted by the expression of the Distal-less gene (Dll). The expression of wg and dpp during the development of other arthropod thoracic limbs indicates that these pathways might be conserved across arthropods for uniramous limb development. The appendages of crustaceans and the gnathal appendages of insects, however, exhibit a diverse array of morphologies, ranging from those with no distal elements, such as the mandible, to appendages with multiple distal elements. Examples of the latter group include branched appendages or those that possess multiple lobes; such complex morphologies are seen for many crustacean limbs as well as the maxillary and labial appendages of many insects. It is unclear how, if at all, the known patterning genes for making a uniramous limb might be deployed to generate these diverse appendage forms. Experiments in Drosophila have shown that by forcing ectopic overlaps of Wg and Dpp signaling it is possible to generate artificially branched legs. To test whether naturally branched appendages form in a similar manner, we detailed the expression patterns of wg, dpp, and Dll in the development of the branched gnathal appendages of the grasshopper, Schistocerca americana, and the flour beetle, Tribolium castaneum. We find that the branches of the gnathal appendages are not specified through the redeployment of the Wg-Dpp system for distal outgrowth, but our comparative studies do suggest a role for Dpp in forming furrows between tissues.     

Distal-less and homothorax regulate multiple targets to pattern the Drosophila antenna

The Drosophila antenna is a highly derived appendage required for a variety of sensory functions including olfaction and audition. To investigate how this complex structure is patterned, we examine the specific functions of genes required for antenna development. The nuclear factors, Homothorax, Distal-less and Spineless, are each required for particular aspects of antennal fate. Coexpression of Homothorax, necessary for nuclear localization of its ubiquitously expressed partner Extradenticle, with Distal-less is required to establish antenna fate. Here we test which antenna patterning genes are targets of Homothorax, Distal-less and/or Spineless. We report that the antennal expression of dachshund, atonal, spalt, and cut requires Homothorax and/or Distal-less, but not Spineless. We conclude that Distal-less and Homothorax specify antenna fates via regulation of multiple genes. We also report for the first time phenotypic consequences of losing either dachshund or spalt and spalt-related from the antenna. We find that dachshund and spalt/spalt-related are essential for proper joint formation between particular antennal segments. Furthermore, the spalt/spalt-related null antennae are defective in hearing. Hearing defects are also associated with the human diseases Split Hand/Split Foot Malformation and Townes-Brocks Syndrome, which are linked to human homologs of Distal-less and spalt, respectively. We therefore propose that there are significant genetic similarities between the auditory organs of humans and flies.     

Microstructure of mandibulate mouthparts in the greater rice weevil,Sitophilus zeamais (Coleoptera: Curculionidae)

The greater rice weevil, Sitophilus zeamais, is a serious pest of stored grain. It chews a hole in the grain and deposits an egg inside. For the purpose of excavating a tunnel, these weevils are equipped with effective mandibulate mouthparts for penetrating and boring holes. The mouthparts of the weevil are a long slender snout, which consist of a labrum, a pair of mandibles, a pair of maxillae and a labium. Mothparts exhibit typical morphology of phytophagous coleopteran beetles and have characteristics of chewing mouthparts. Mandibles are connected to the head capsule through dicondyous articulation, which allows movement along one single rotating axis. Both labrum and labium are fused to the snout and form the upper and lower lips, respectively. Along the depressed surface of the snout, a number of short sensory hairs are sparsely arranged. The distal apexes of the maxillary and labial palpi are deeply depressed into a sensillar field, and only one type of the basiconic sensilla, which function as chemo‐ or gustatory receptors, occurs in both sexes.     

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.     

Fine structure and sensory apparatus of the mouthparts of the pear psyllid,Cacopsylla chinensis (Yang et Li) (Hemiptera: Psyllidae)

The pear psyllid, Cacopsylla chinensis (Yang et Li) (Hemiptera: Psyllidae), is one of the most significant economic pests of pear in China, causing direct damage through feeding by the highly specialized piercing–sucking mouthparts. The ultrastructural morphology and sensory apparatus of the mouthparts of the adult were examined using scanning and transmission electron microscopy. The piercing–sucking mouthparts of C. chinensis are composed of a three-segmented labium with a deep groove in the anterior side, a stylet fascicle consisting of two mandibular and two maxillary stylets, and a pyramid-shaped labrum. Proximal to the labium, the stylet fascicle forms a large loop within a membranous crumena. Mandibles, with more than ten teeth on the external convex region, can be seen on the distal extremity. Smooth maxillary stylets are interlocked to form a larger food canal and a smaller salivary canal. One dendritic canal housing 2 dendrites is also found in each mandible. Two types of sensilla trichodea, four types of sensilla basiconica, single as well as groups of sensilla campaniformia, and oval flattened sensilla occur in different locations on the labium, whereas a kind of sensilla basiconica is at the junction of the labrum and anteclypeus. Sensilla trichodea and sensilla campaniformia, always present with denticles, are present on the middle labial segment. Three types of sensilla basiconica, two types of sensilla trichodea and two oval flattened sensilla are located on the distal labial segment. The mouthpart morphology and abundance of sensilla located on the labium in C. chinensis are illustrated, along with a brief discussion of their taxonomic and putative functional significance.     

Development of the mouthparts in embryos of Haplothrips verbasci (Osborn) (Insecta,Thysanoptera, Phlaeothripidae)

The asymmetric “punch and suck” mouthparts of larval Haplothrips verbasci develop from paired appendages in the late, post-anatrepsis embryo similar to those of other insects. Later, the labrum flexes ventrally over the stomodaeum, the right mandibular appendage degenerates, the maxillary appendages divide into inner (lacinial) and outer (stipital) lobes, and the hypopharynx arises from the venters of the mandibular and maxillary segments. All cephalic segments consolidate anteriorly prior to katatrepsis, their appendages flex ventrally, and the labial appendages fuse medially to form the labium and the primordia of the salivary glands and valve. The left mandible and the lacinial lobes of the maxillae invaginate into the head during and after katatrepsis to form the mandibular and maxillary stylet-secreting organs and these later deposit the cuticle of their respective stylets. Cuticle of the mandibular lever is deposited by labral cells at the apex of the mandibular sheath during and after hatching. That of each maxillary lever is secreted simultaneously into the lumen of a ventrally-directed diverticulum developing from stipital cells at the apex of each maxillary sheath. Shortly after katatrepsis, the maxillary and labial palpi originate respectively from cells in the outer wall of each stipital lobe and at the apex of the labium. Muscles of the mouthparts arise after katatrepsis from cephalic mesoderm and are fully-differentiated before cuticle of the mandibular and maxillary levers has been deposited. Gnathal morphogenesis in embryos of H. verbasci resembles that occurring in bug embryos and provides additional evidence that Thysanoptera and Hemiptera evolved from a common psocopteroid stem species having small, paired, biting and chewing mandibles and well developed lacinial stylets.     

Fate map of the distal portion of Drosophila proboscis as inferred from the expression and mutations of basic patterning genes

The late-third-instar labial disc is comprised of two disc-proper cell layers, one representing mainly the ventral half of the anterior compartment (L-layer) and the other, the dorsal half of the anterior compartment and most, if not all, of the posterior compartment (M-layer). In the L-layer, Distal-less represses homothorax whereas no Distal-less-dependent homothorax repression occurs in the M-layer where Distal-less is coexpressed with homothorax. In wild-type labial discs, clawless, one of the two homeobox genes expressed in distal cells receiving maximum (Decapentaplegic+Wingless) signaling activity in leg and antennal discs, is specifically repressed by proboscipedia. A fate map, inferred from data on basic patterning gene expression in larval and pupal stages and mutant phenotypes, indicates the inner surface of the labial palpus, which includes the pseudotracheal region, to be a derivative of the distal portion of the M-layer expressing wingless, patched, Distal-less and homothorax. The outer surface of the labial palpus with more than 30 taste bristles derives from an L-layer area consisting of dorsal portions of the anterior and posterior compartments, each expressing Distal-less. Our analysis also indicates that, in adults and pupae, the anterior-posterior boundary, dividing roughly equally the outer surface of the distiproboscis, runs along the outer circumference of the inner surface of distiproboscis.     

Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta

The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.