Leg length and bristle density, both necessary for water surface locomotion, are genetically correlated in water striders.

Access to hitherto unexploited ecological opportunities is associated with phenotypic evolution and often results in significant lineage diversification. Yet our understanding of the mechanisms underlying such adaptive traits remains limited. Water striders have been able to exploit the water-air interface, primarily facilitated by changes in the density of hydrophobic bristles and a significant increase in leg length. These two traits are functionally correlated and are both necessary for generating efficient locomotion on the water surface. Whether bristle density and leg length have any cellular or developmental genetic mechanisms in common is unknown. Here, we combine comparative genomics and transcriptomics with functional RNA interference assays to examine the developmental genetic and cellular mechanisms underlying the patterning of the bristles and the legs in Gerris buenoi and Mesovelia mulsanti, two species of water striders. We found that two duplication events in the genes beadex and taxi led to a functional expansion of the paralogs, which affected bristle density and leg length. We also identified genes for which no function in bristle development has been previously described in other insects. Interestingly, most of these genes play a dual role in regulating bristle development and leg length. In addition, these genes play a role in regulating cell division. This result suggests that cell division may be a common mechanism through which these genes can simultaneously regulate leg length and bristle density. We propose that pleiotropy, through which gene function affects the development of multiple traits, may play a prominent role in facilitating access to unexploited ecological opportunities and species diversification.

Parental RNA interference as a tool to study genes involved in rostrum development in the Neotropical brown stink bug, Euschistus heros.

In insects, the identity of body segments is controlled by homeotic genes and the knockdown of these genes during embryogenesis can lead to an abnormal development and/or atypical phenotypes. The main goal of this study was to investigate the involvement of labial (lab), deformed (dfd), sex comb reduced (scr), extradenticle (exd) and proboscipedia (pb) in rostrum development in the Neotropical brown stink bug Euschistus heros, using parental RNAi (pRNAi). To achieve this objective, 10-days-old adult females were first microinjected with double-stranded RNAs (dsRNA) targeting these five genes. Then, the number of eggs laid per female, the percentage of hatched nymphs with normal or abnormal phenotype and target gene silencing were evaluated. Except for the dsDfd-treatment, the number of eggs laid per female per day was not affected by the different dsRNA-treatments compared to the control (dsGFP). However, treatment with either dsLab, dsDfd, dsScr or dsExd caused a strong reduction in egg hatching. The dsExd-treatment caused no apparent change in phenotype in the nymphs while hatched nymphs from the dsDfd, dsScr and dsPb-treatment showed abnormalities in the rostrum. Particularly for the dsPb-treatment, 91% of the offspring displayed a bifurcated rostrum with a leg-like structure. Overall, these results indicate that these five genes are involved in E. heros embryonic development and that the knockdown of dfd, scr and pb leads to an abnormal development of the rostrum. Additionally, this study demonstrates the efficiency of pRNAi in studying genes involved in embryogenesis in E. heros, with clear phenotypes and a strong target gene silencing in the next generation, after treatment of the parent female adult with gene-specific dsRNA.

The multiple roles of caudal in early development of the milkweed bug Oncopeltus fasciatus.

The homeobox transcription factor Caudal has conserved roles in all Bilateria in defining the posterior pole and in controlling posterior elongation. These roles are seemingly similar and are difficult to disentangle. We have carried out a detailed analysis of the expression, function and interactions of the caudal ortholog of the milkweed bug, Oncopeltus fasciatus, a hemimetabolous insect with a conservative early development process, in order to understand its different functions throughout development. In Oncopeltus, caudal is not maternally deposited, but has a sequence of roles in the posterior of the embryos throughout early development. It defines and maintains a posterior-anterior gradient in the blastoderm and modulates the activity of segmentation genes in simultaneous segmentation during the blastoderm stage. It later defines the invagination site and the posterior segment addition zone (SAZ) in the germband. It maintains the posterior SAZ cells in an undifferentiated proliferative state, while promoting dynamic expression of segmentation genes in the anterior SAZ. We show that rather than being a simple posterior determinant, Caudal is involved in several distinct regulatory networks, each with a distinct developmental role.

Teratological cases of the antennae in the family Aradidae (Hemiptera: Heteroptera).

Teratological cases of the antennae in the family Aradidae (Hemiptera: Heteroptera) are widely described for the first time. Four hundred seventy-six specimens of flat bugs were studied, and antennal malformations were found in 14 of them (2.94%) (belonging to eight species and three subfamilies: Aradinae, Aneurinae and Mezirinae). All of the teratologies were observed using optical microscopy; moreover, in order to determine any compensatory regeneration, selected cases were also studied using a scanning electron microscope. In almost all of the specimens, the successful regeneration of the sensory organs to various degrees was observed. Additional results were the discovery of a previously unrecognized type of sensillum in flat bugs - a campaniform sensillum as well as significant differences in the distribution of the sensilla depending on the systematic affiliation.

Shifting roles of Drosophila pair-rule gene orthologs: segmental expression and function in the milkweed bug Oncopeltus fasciatus.

The discovery of pair-rule genes (PRGs) in Drosophila revealed the existence of an underlying two-segment-wide prepattern directing embryogenesis. The milkweed bug Oncopeltus fasciatus, a hemimetabolous insect, is a more representative arthropod: most of its segments form sequentially after gastrulation. Here, we report the expression and function of orthologs of the complete set of nine Drosophila PRGs in Oncopeltus Seven Of-PRG-orthologs are expressed in stripes in the primordia of every segment, rather than every other segment; Of-runt is PR-like and several orthologs are also expressed in the segment addition zone. RNAi-mediated knockdown of Of-odd-skipped, paired and sloppy-paired impacted all segments, with no indication of PR-like register. We confirm that Of-E75A is expressed in PR-like stripes, although it is not expressed in this way in Drosophila, demonstrating the existence of an underlying PR-like prepattern in Oncopeltus These findings reveal that a switch occurred in regulatory circuits, leading to segment formation: while several holometabolous insects are 'Drosophila-like', using PRG orthologs for PR patterning, most Of-PRGs are expressed segmentally in Oncopeltus, a more basally branching insect. Thus, an evolutionarily stable phenotype - segment formation - is directed by alternate regulatory pathways in diverse species.

Dynamics of growth zone patterning in the milkweed bug Oncopeltus fasciatus.

We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited.

Blastoderm segmentation in Oncopeltus fasciatus and the evolution of insect segmentation mechanisms.

Segments are formed simultaneously in the blastoderm of the fly Drosophila melanogaster through a hierarchical cascade of interacting transcription factors. Conversely, in many insects and in all non-insect arthropods most segments are formed sequentially from the posterior. We have looked at segmentation in the milkweed bug Oncopeltus fasciatus. Posterior segments are formed sequentially, through what is probably the ancestral arthropod mechanism. Formation of anterior segments bears many similarities to the Drosophila segmentation mode. These segments appear nearly simultaneously in the blastoderm, via a segmentation cascade that involves orthologues of Drosophila gap genes working through a functionally similar mechanism. We suggest that simultaneous blastoderm segmentation evolved at or close to the origin of holometabolous insects, and formed the basis for the evolution of the segmentation mode seen in Drosophila We discuss the changes in segmentation mechanisms throughout insect evolution, and suggest that the appearance of simultaneous segmentation as a novel feature of holometabolous insects may have contributed to the phenomenal success of this group.

Dynamic BMP signaling polarized by Toll patterns the dorsoventral axis in a hemimetabolous insect.

Toll-dependent patterning of the dorsoventral axis in Drosophila represents one of the best understood gene regulatory networks. However, its evolutionary origin has remained elusive. Outside the insects Toll is not known for a patterning function, but rather for a role in pathogen defense. Here, we show that in the milkweed bug Oncopeltus fasciatus, whose lineage split from Drosophila's more than 350 million years ago, Toll is only required to polarize a dynamic BMP signaling network. A theoretical model reveals that this network has self-regulatory properties and that shallow Toll signaling gradients are sufficient to initiate axis formation. Such gradients can account for the experimentally observed twinning of insect embryos upon egg fragmentation and might have evolved from a state of uniform Toll activity associated with protecting insect eggs against pathogens.

Oviposition behaviors and ontogenetic embryonic characteristics of the western tarnished plant bug, Lygus hesperus.

The western tarnished plant bug, Lygus hesperus Knight (Hemiptera: Miridae) is a key pest of fruit, vegetable, and field crops in the western United States, but many aspects of L. hesperus ecology are poorly documented. A sound understanding of oviposition behavior and characterization of the phases of embryonic development would be useful in studies of interactions between L. hesperus and its hosts, and in efforts to better understand the developmental consequences of low temperatures. Because L. hesperus insert their eggs into the host, most of the egg is obscured from view, and some aspects of oviposition and subsequent egg development cannot be observed directly. A novel observational method which took advantage of the propensity for L. hesperus to oviposit in semi-transparent sheets of agarose was used to observe oviposition and subsequent embryonic development. Lygus hesperus females stylet-probed prospective oviposition sites and during oviposition the ovipositor followed the path of the final probe. Oviposition, from insertion to withdrawal of the ovipositor, required ∼30 seconds. Identifiable phases of embryo development included egg swelling, katatrepsis, appearance of body segments and appendages, development of red pigmentation in the eyes and terminal antennal segments, and formation of the 3(rd) embryonic cuticle. These phases were observed at about 0.3, 0.4, 0.5, 0.6, and 0.8 of the total duration between oviposition and hatch, respectively. Infertile eggs did not exhibit any of these phases. Our descriptions of embryonic development will facilitate the study of L. hesperus egg biology and ecology, and permit estimates of egg population age structure and prediction of egg hatch.

Respiratory morphology of the Abedus herberti Hidalgo egg chorion (Hemiptera: Belostomatidae).

Although giant water bugs (Hemiptera: Belostomatidae) are large, aquatic insects known for their obligate paternal egg brooding behaviors, little research has focused on the structure of their eggs. The respiratory requirements of developing embryos likely created selection for brooding, so a thorough understanding of the respiratory morphology of belostomatid eggs could help explain how brooding behaviors facilitate embryonic gas exchange. This study used scanning electron microscopy to document the respiratory microstructure of the eggs of Abedus herberti, a back brooding giant water bug. The exochorion is similar to that of other belostomatids in texture and organization except that the respiratory region is confined to the uppermost quarter of the egg. This is the area exposed to the atmosphere by encumbered males. A plastron network made up of densely packed vertical projections demarcates the boundary between the respiratory and nonrespiratory regions of the chorion. The internal chorion is composed of alternate air-filled and denser layers that likely facilitate the movement of oxygen from the aeropyles at the top of the eggs to the developing embryonic tissues. J. Morphol., 2011. © 2011 Wiley-Liss, Inc.

Using microscopy to assess chorion structural integrity and parasitoid oviposition sites on stink bug (Hemiptera: Pentatomidae) eggs.

Previous efficacy studies found that many insecticides used by growers could be having an adverse effect on egg parasitoids (Telenomus podisi) developing in the eggs of the brown stink bug (Euschistus servus), while unhatched stink bugs experienced lower levels of mortality. One plausible explanation for this was that insecticides might enter parasitized eggs more readily via oviposition wounds. Parasitized E. servus eggs, as well as nonparasitized stink bug (Acrosternum hilare, E. servus, Murgantia histrionica, and Podisus maculiventris) eggs, were examined using electron microscopy. Egg response to perforation by a tungsten probe served as a control. Microscopy images depicted the chorion surface as characterized by a matrix of ridges and micropylar processes in a ring around the margin of the operculum. Observations of oviposition sites showed a "scab" formed where the ovipositor penetrated the chorion, and at sites penetrated by the probe. These formations appeared to be the result of fluids from inside the egg leaking out, drying, and hardening after oviposition or probe perforation, suggesting that the response was not due to substances secreted by the parasitoid. Further, no open wounds or holes were seen to increase the possibility of insecticides entering parasitized eggs.

Early patterning and blastodermal fate map of the head in the milkweed bug Oncopeltus fasciatus.

The process of head development in insects utilizes a set of widely conserved genes, but this process and its evolution are not well understood. Recent data from Tribolium castaneum have provided a baseline for an understanding of insect head development. However, work on a wider range of insect species, including members of the hemimetabolous orders, is needed in order to draw general conclusions about the evolution of head differentiation and regionalization. We have cloned and studied the expression and function of a number of candidate genes for head development in the hemipteran Oncopeltus fasciatus. These include orthodenticle, empty spiracles, collier, cap 'n' collar, and crocodile. The expression patterns of these genes show a broad conservation relative to Tribolium, as well as differences from Drosophila indicating that Tribolium + Oncopeltus represent a more ancestral pattern. In addition, our data provide a blastodermal fate map for different head regions in later developmental stages and supply us with a "roadmap" for future studies on head development in this species.

Mutual regulatory interactions of the trunk gap genes during blastoderm patterning in the hemipteran Oncopeltus fasciatus.

The early embryo of the milkweed bug, Oncopeltus fasciatus, appears as a single cell layer - the embryonic blastoderm - covering the entire egg. It is at this blastoderm stage that morphological domains are first determined, long before the appearance of overt segmentation. Central to the process of patterning the blastoderm into distinct domains are a group of transcription factors known as gap genes. In Drosophila melanogaster these genes form a network of interactions, and maintain sharp expression boundaries through strong mutual repression. Their restricted expression domains define specific areas along the entire body. We have studied the expression domains of the four trunk gap gene homologues in O. fasciatus and have determined their interactions through dsRNA gene knockdown experiments, followed by expression analyses. While the blastoderm in O. fasciatus includes only the first six segments of the embryo, the expression domains of the gap genes within these segments are broadly similar to those in Drosophila where the blastoderm includes all 15 segments. However, the interactions between the gap genes are surprisingly different from those in Drosophila, and mutual repression between the genes seems to play a much less significant role. This suggests that the well-studied interaction pattern in Drosophila is evolutionarily derived, and has evolved from a less strongly interacting network.

Late extraembryonic morphogenesis and its zen(RNAi)-induced failure in the milkweed bug Oncopeltus fasciatus.

Many insects undergo katatrepsis, essential reorganization by the extraembryonic membranes that repositions the embryo. Knockdown of the zen gene by RNA interference (RNAi) prevents katatrepsis in the milkweed bug Oncopeltus fasciatus. However, the precise morphogenetic defect has been uncertain, and katatrepsis itself has not been characterized in detail. The dynamics of wild type and zen(RNAi) eggs were analyzed from time-lapse movies, supplemented by analysis of fixed specimens. These investigations identify three zen(RNAi) defects. First, a reduced degree of tissue contraction implies a role for zen in baseline compression prior to katatrepsis. Subsequently, a characteristic 'bouncing' activity commences, leading to the initiation of katatrepsis in wild type eggs. The second zen(RNAi) defect is a delay in this activity, suggesting that a temporal window of opportunity is missed after zen knockdown. Ultimately, the extraembryonic membranes fail to rupture in zen(RNAi) eggs: the third defect. Nevertheless, the outer serosal membrane manages to contract, albeit in an aberrant fashion with additional phenotypic consequences for the embryo. These data identify a novel epithelial morphogenetic event - rupture of the 'serosal window' structure - as the ultimate site of defect. Overall, Oncopeltus zen seems to have a role in coordinating a number of pre-katatreptic events during mid embryogenesis.

Diverging functions of Scr between embryonic and post-embryonic development in a hemimetabolous insect, Oncopeltus fasciatus.

Hemimetabolous insects undergo an ancestral mode of development in which embryos hatch into first nymphs that resemble miniature adults. While recent studies have shown that homeotic (hox) genes establish segmental identity of first nymphs during embryogenesis, no information exists on the function of these genes during post-embryogenesis. To determine whether and to what degree hox genes influence the formation of adult morphologies, we performed a functional analysis of Sex combs reduced (Scr) during post-embryonic development in Oncopeltus fasciatus. The main effect was observed in prothorax of Scr-RNAi adults, and ranged from significant alterations in its size and shape to a near complete transformation of its posterior half toward a T2-like identity. Furthermore, while the consecutive application of Scr-RNAi at both of the final two post-embryonic stages (fourth and fifth) did result in formation of ectopic wings on T1, the individual applications at each of these stages did not. These experiments provide two new insights into evolution of wings. First, the role of Scr in wing repression appears to be conserved in both holo- and hemimetabolous insects. Second, the prolonged Scr-depletion (spanning at least two nymphal stages) is both necessary and sufficient to restart wing program. At the same time, other structures that were previously established during embryogenesis are either unaffected (T1 legs) or display only minor changes (labium) in adults. These observations reveal a temporal and spatial divergence of Scr roles during embryonic (main effect in labium) and post-embryonic (main effect in prothorax) development.

The role of the pupal determinant broad during embryonic development of a direct-developing insect.

Metamorphosis is one of the most common, yet dramatic of life history strategies. In insects, complete metamorphosis with morphologically distinct larval stages arose from hemimetabolous ancestors that were more direct developing. Over the past century, several ideas have emerged that suggest the holometabolous pupa is developmentally homologous to the embryonic stages of the hemimetabolous ancestor. Other theories consider the pupal stage to be a modification of a hemimetabolous nymph. To address this question, we have isolated an ortholog of the pupal determinant, broad (br), from the hemimetabolous milkweed bug and examined its role during embryonic development. We show that Oncopeltus fasciatus br (Of'br) is expressed in two phases. The first occurs during germ band invagination and segmentation when Of'br is expressed ubiquitously in the embryonic tissues. The second phase of Of'br expression appears during the pronymphal phase of embryogenesis and persists through nymphal differentiation to decline just before hatching. Knock-down of Of'br transcripts results in defects that range from posterior truncations in the least-affected phenotypes to completely fragmented embryonic tissues in the most severe cases. Analysis of the patterning genes engrailed and hunchback reveal loss of segments and a failure in neural differentiation after Of'br depletion. Finally, we show that br is constitutively expressed during embyrogenesis of the ametabolous firebrat, Thermobia domestica. This suggests that br expression is prominent during embryonic development of ametabolous and hemimetabolous insects but was lost with the emergence of the completely metamorphosing insects.

RNAi analysis of nubbin embryonic functions in a hemimetabolous insect, Oncopeltus fasciatus.

Although the expression of the POU homeodomain gene nubbin (nub) has been examined in several arthropod species, its function has been studied only in Drosophila. Here, we provide the first insight into functional roles of this gene in a hemimetabolous insect species, Oncopeltus fasciatus. The analysis of its function using RNAi resulted in the altered morphology of antennae and labial tubes in the head, legs in the thorax, and, most notably, the growth of ectopic appendages originating from abdominal segments A2-A6. This change in the morphology of the abdomen can largely be attributed to the altered expression patterns of two hox genes, Ubx and abd-A, in RNAinub embryos. First, abd-A expression is completely abolished in A3-A6. Second, weak Ubx expression expands posteriorly to encompass novel domains in A2 and A3. Concomitant with these changes, limbs on A2 and A3 are small and less developed, whereas limbs on A4-A6 are large thoracic-like legs. These results show that nub function is necessary for normal abd-A expression and thus plays a critical role in suppressing leg formation on the abdomen. The loss of this regulation leads to upregulation of Distal-less, and subsequent development of appendages. In Drosophila, however, abd-A expression is unaffected in a nub-depleted background, indicating that no such regulatory relationship exists between these two genes in the fruit fly. These differences reveal that variation exists in the genetic mechanisms that maintain an ancient insect feature, the limbless abdomen.