Evolution of nubbin function in hemimetabolous and holometabolous insect appendages

Insects display a whole spectrum of morphological diversity, which is especially noticeable in the organization of their appendages. A recent study in a hemipteran, Oncopeltus fasciatus (milkweed bug), showed that nubbin (nub) affects antenna morphogenesis, labial patterning, the length of the femoral segment in legs, and the formation of a limbless abdomen. To further determine the role of this gene in the evolution of insect morphology, we analyzed its functions in two additional hemimetabolous species, Acheta domesticus (house cricket) and Periplaneta americana (cockroach), and re-examined its role in Drosophila melanogaster (fruit fly). While both Acheta and Periplaneta nub-RNAi first nymphs develop crooked antennae, no visible changes are observed in the morphologies of their mouthparts and abdomen. Instead, the main effect is seen in legs. The joint between the tibia and first tarsomere (Ta-1) is lost in Acheta, which in turn, causes a fusion of these two segments and creates a chimeric nub-RNAi tibia–tarsus that retains a tibial identity in its proximal half and acquires a Ta-1 identity in its distal half. Similarly, our re-analysis of nub function in Drosophila reveals that legs lack all true joints and the fly tibia also exhibits a fused tibia and tarsus. Finally, we observe a similar phenotype in Periplaneta except that it encompasses different joints (coxa–trochanter and femur–tibia), and in this species we also show that nub expression in the legs is regulated by Notch signaling, as had previously been reported in flies and spiders. Overall, we propose that nub acts downstream of Notch on the distal part of insect leg segments to promote their development and growth, which in turn is required for joint formation. Our data represent the first functional evidence defining a role for nub in leg segmentation and highlight the varying degrees of its involvement in this process across insects.     

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.     

even-skipped is not a pair-rule gene but has segmental and gap-like functions in Oncopeltus fasciatus, an intermediate germband insect

The pair-rule gene even-skipped is required for the initiation of metameric pattern in Drosophila. But Drosophila segmentation is evolutionarily derived and is not representative of most insects. Therefore, in order to shed light on the evolution of insect segmentation, homologs of the pair-rule gene even-skipped have been studied in several insect taxa. However, most of these studies have reported the expression eve but not its function. We report the isolation, expression and function of the homolog of Drosophila even-skipped from the intermediate germband insect Oncopeltus fasciatus. We find that in Oncopeltus, even-skipped striped expression initiates in a segmental and not pair-rule pattern. Weak RNAi suppression of Oncopeltus even-skipped shows no apparent pair-rule like phenotype, while stronger RNAi suppression shows deletion of nearly the entire body. These results suggest that in Oncopeltus, even-skipped is not acting as a pair-rule gene. In almost all insects, prior to its striped expression, even-skipped is expressed in a conserved broad gap-like domain but its function has been largely ignored. We find that this early broad domain is required for activation of the gap genes hunchback and Krüppel. Given the large RNAi deletion phenotype and its regulation of hunchback and Krüppel, even-skipped seems to act as an über-gap gene in Oncopeltus, indicating that it may have both upstream and downstream roles in segmentation.     

Larval-adult oviduct transformation in an insect (Oncopeltus fasciatus): cytological study

The lateral oviducts of the milkweed bug undergo a drastic larval-adult transformation during the last larval instar. This transformation is hormonally regulated and is connected with considerable morphological and cytological reorganization. Prior to transformation, the ducts are long and thin. The epithelial cells are fusiform with their long axes running cranial-caudal. They are tilted and slightly helically arranged. During transformation, the lateral oviducts shorten dramatically, the cells become columnar, and their long axes shift into the plane of cross sections. Whereas the cells appear cytologically uniform in the larval state, after transformation, the lateral oviducts are divided into a cranial secretory part, and a caudal part which exhibits a cuticular lining. Secretory processes and deposition of the cuticle start in the larva, immediately after transformation. Cuticle synthesis is first observed next to the common oviduct and progresses craniad. The cuticle-lined luminal surface reveals circular ridges and an intricate pattern of cteniform spines which are directed caudally. The novel mode of cell rearrangement during oviducal transformation and the functional significance of the two parts of the adult lateral oviducts are discussed.     

Functional analyses of tiptop and antennapedia in the embryonic development of Oncopeltus fasciatus suggests an evolutionary pathway from ground state to insect legs

In insects, selector genes are thought to modify the development of a default, or 'ground state', appendage into a tagma-specific appendage such as a mouthpart, antenna or leg. In the best described example, Drosophila melanogaster, the primary determination of leg identity is thought to result from regulatory interactions between the Hox genes and the antennal-specifying gene homothorax. Based on RNA-interference, a functional analysis of the selector gene tiptop and the Hox gene Antennapedia in Oncopeltus fasciatus embryogenesis is presented. It is shown that, in O. fasciatus, tiptop is required for the segmentation of distal leg segments and is required to specify the identity of the leg. The distal portions of legs with reduced tiptop develop like antennae. Thus, tiptop can act as a regulatory switch that chooses between antennal and leg identity. By contrast, Antennapedia does not act as a switch between leg and antennal identity. This observation suggests a significant difference in the mechanism of leg specification between O. fasciatus and D. melanogaster. These observations also suggest a significant plasticity in the mechanism of leg specification during insect evolution that is greater than would have been expected based on strictly morphological or molecular comparisons. Finally, it is proposed that a tiptop-like activity is a likely component of an ancestral leg specification mechanism. Incorporating a tiptop-like activity into a model of the leg-specification mechanism explains several mutant phenotypes, previously described in D. melanogaster, and suggests a mechanism for the evolution of legs from a ground state.     

Double membrane-bounded intestinal microvilli in Oncopeltus fasciatus

A double plasma membrane (DPM) surrounding intestinal microvilli of the migratory milkweed bug, Oncopeltus fasciatus, is described. Mutant and wild types of the phytophagous insect have been studied by conventional SEM and TEM procedures with the use of membrane-enhancing staining methods. Longitudinal and transverse sections revealed a DPM surrounding microvilli and continuing over the apical portions of the intestinal cell. The outer membrane of the DPM contributes to an intestinal lining or peritrophic membrane (PTM), which apparently accumulates in layers. SEM studies reveal a rugose intestinal surface and complete PTM in both starved and fed insects. Only rarely are exposed microvilli seen by SEM. SEM examinations also enable the observation of numerous blebs on the luminal side of the PTM apparently held in position by a neck-like attachment and apparently derived from the outer membrane of the DPM. Preliminary TEM studies of microvilli revealed unique microvesicle-like structures, lying just inside the inner membrane of the DPM, which may be of membrane origin based on their typical trilaminar appearance after en bloc staining with uranyl acetate. Highly ordered microfilaments were observed to occupy the most central aspect of the microvilli.     

Regeneration of appendages in the large milkweed bug, Oncopeltus fasciatus

An analysis was made of the regeneration of legs and antennae of Oncopeltus. Amputations were performed on first instar larvae within 24 hr after hatching, and on later instars within 24 hr after ecdysis. The resulting regenerates were then measured at each instar. When amputations were performed soon after hatching, there was no significant effect on the duration of any instar. The regenerate was usually visible after the second post-operative ecdysis, and was smaller than a normal appendage (hypomorphic). Removal of the three distal segments of the antenna usually resulted in regeneration of only one segment which was abnormally long and showed a combination of the bristle patterns characteristic of the two most distal segments of the control. In a few such cases a partial intersegmental membrane was present in the regenerated segment. Removal of the tarsus resulted in a structurally complete regenerate which was smaller than the control tarsus. The largest leg regenerates were obtained when amputation was performed through the tibia. With amputation through the femur, a decrease in length of the remainder of this segment was observed after the first ecdysis. This type of amputation and amputation through the trochanter in some cases resulted in the formation of a globular stump containing tarsal claws. The results indicate that amputation of part of an appendage in Oncopeltus does not stimulate an increased growth rate in the stump, but merely causes reorganization of the stump material which subsequently grows at the normal rate. Since even the most hypomorphic regenerates contained well-formed claws, even though proximal parts were missing, it appears that the reorganization process must begin at the most distal point and proceed proximally.     

Functions of the ecdysone receptor isoform-A in the hemimetabolous insect Blattella germanica revealed by systemic RNAi in vivo

The molecular basis of ecdysteroid function during development has been analyzed in detail in holometabolous insects, especially in Drosophila melanogaster, but rarely in hemimetabolous. Using the hemimetabolous species Blattella germanica (German cockroach) as model, we show that the ecdysone receptor isoform-A (BgEcR-A) mRNA is present throughout the penultimate and last nymphal instars in all tissues analyzed (prothoracic gland, epidermis and fat body). To study the functions of BgEcR-A, we reduced its expression using systemic RNAi in vivo, and we obtained knockdown specimens. Examination of these specimens indicated that BgEcR-A during the last nymphal instar is required for nymphal survival, and that reduced expression is associated with molting defects, lower circulating ecdysteroid levels and defects in cell proliferation in the follicular epithelium. Some BgEcR-A knockdown nymphs survive to the adult stage. The features of these specimens indicate that BgEcR-A is required for adult-specific developmental processes, such as wing development, prothoracic gland degeneration and normal choriogenesis.     

RNAi analysis of Deformed, proboscipedia and Sex combs reduced in the milkweed bug Oncopeltus fasciatus: novel roles for Hox genes in the hemipteran head

Insects have evolved a large variety of specialized feeding strategies, with a corresponding variability in mouthpart morphology. We have, however, little understanding of the developmental mechanisms that underlie this diversity. Until recently it was difficult to perform any analysis of gene function outside of the genetic model insects Drosophila melanogaster and Tribolium castaneum. In this paper, we report the use of dsRNA-mediated interference (RNAi) to dissect gene function in the development of the milkweed bug Oncopeltus fasciatus, which has specialized suctorial mouthparts. The Hox genes Deformed (Dfd), proboscipedia (pb) and Sex combs reduced (Scr) have previously been shown to be expressed in the gnathal appendages of this species. Strikingly, the milkweed bug was found to have an unusual expression pattern of pb. Here, by analyzing single and combination RNAi depletions, we find that Dfd, pb and Scr are used in the milkweed bug to specify the identity of the mouthparts. The exact roles of the genes, however, are different from what is known in the two genetic model insects. The maxillary appendages in the bug are determined by the activities of the genes Dfd and Scr, rather than Dfd and pb as in the fly and beetle. The mandibular appendages are specified by Dfd, but their unique morphology in Oncopeltus suggests that Dfd's target genes are different. As in flies and beetles, the labium is specified by the combined activities of pb and Scr, but again, the function of pb appears to be different. Additionally, the regulatory control of pb by the other two genes seems to be different in the bug than in either of the other species. These novelties in Hox function, expression pattern and regulatory relationships may have been important for the evolution of the unique Hemipteran head.     

Energy expenditure during embryonic development under constant versus variable temperatures Oncopeltus fasciatus (Dallas)

During embryonic development the rates of oxygen consumption and weight loss rise gradually. Total O₂ consumption, like total weight losses previously reported, rises at lower incubation temperatures, and at minimal incubation temperatures is nearly double that at an optimal temperature. The Q₁₀ for oxygen consumption rises from 3.0 at the start of development to 3.7 and then drops to 3.25 shortly before hatching. After hatching the rate of O₂ consumption rises 50% and the Q₁₀ drops to about 2.Eggs transferred from a minimal (15°) to an optimal (25°) temperature show a transitory overshoot in rate of O₂ consumption. Following such transfers, O₂ consumption rates are the same as for eggs being incubated under the same but constant temperatures.Calculations show that there is an unexpectedly large retardation of development at near minimal temperatures. Even a short daily exposure to a favorable temperature ameliorates the retarding and debilitating effects of incubation at minimal temperatures. As a result less O₂ is consumed and less weight lost than at corresponding constant temperatures. The significance of these data is discussed.

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Zusammenfassung Während der Embryonalentwicklung verläuft der Sauerstoffverbrauch streng gekoppelt mit der Gewichtsabnahme; beide steigern sich mit der Entwicklung allmählich. Wie die früher beschriebene Gewichtsabnahme, so nimmt auch der Gesamtsauerstoffverbrauch bei niederen Temperaturen zu und ist bei minimalen Inkubationstemperaturen fast doppelt so groß wie bei optimaler Inkubationstemperatur. Der Q₁₀ für den Sauerstoffverbrauch steigt von 3,0 bei Beginn der Entwicklung auf 3,7 und fällt dann auf ungefähr 3,25 kurz vor dem Schlüpfen. Nach dem Schlüpfen, wenn die Muskelaktivität eine größere Rolle spielt, steigt der O₂-Verbrauch auf etwa 50%, aber der Q₁₀ fällt auf etwa 2.Wenn die Eier von einer minimalen in eine optimale Temperatur übertragen werden, ergibt sich ein vorübergehender Überschuß in der O₂ Verbrauchsrate. Solchen Übertragungen (von 15° nach 25° und zurück) folgend, gleichen die Sauerstoff-Verbrauchsraten denen von Eiern, die konstant unter diesen Temperaturen gehalten wurden.Die verwirrende Frage, warum der Gesamtsauerstoffverbrauch und der Gesamtgewichtsverlust geringer sind, wenn die Eier bei minimaler Mitteltemperatur abwechselnd in hoher und niederer Temperatur gehalten werden, im Gegensatz zu konstanten Inkubationstemperaturen, wird auf Grund von Berechnungen interpretiert, die zeigen, daß in der Nähe minimaler Temperaturen eine unerwartet große Entwicklungsverzögerung eintritt. Es scheint, daß sogar ein kurzer täglicher Aufenthalt in einer günstigen Temperatur die verzögernden und abschwächenden Wirkungen minimaler Inkubationstemperaturen verbessert. Wahrscheinlich erfordem gewisse Stoffwechselprozesse Temperaturen von 20° oder mehr, aber die Entwicklung selbst kann bei beträchtlich niedrigeren Temperaturen vorwärtsschreiten; die Natur einer solcherart möglichen biochemischen Insuffizienz bleibt zu klären.Es scheint klar, daß die bei der Berechnung von mittleren Temperaturen und Tages-Grad-Anhäufungen angenommene Linearität nicht zutrifft, wenn sie auf die gekrümmten Wachstumskurven der Insekten angewandt werden.

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Paper No. 4808, Scientific Journal Series, Minnesota Agricultural Experiment Station, St. Paul 1, Minnesota.     

An energetic analysis of host plant selection by the large milkweed bug,Oncopeltus fasciatus

Summary The large milkweed bug, Oncopeltus fasciatus, is a specialized seed feeder that has been observed completing nymphal development in the field on only a small proportion of its potential host species within the genus Asclepias. In central Missouri only two of the six milkweed species studied, A. syriaca and A. verticillata, commonly supported nymphal O. fasciatus growth in the field. The seed of all six species, however, was equally suitable food for bugs reared in the laboratory. In laboratory preference tests, adult bugs chose to feed on the largest seeds, A. hirtella, but such a preference could not explain the observed field feeding patterns.One explanation to account for the observed host plant selection is based upon an energetic analysis. Only A. syriaca provided enough seed biomass for a clutch of O. fasciatus nymphs to develop on a single plant, and only A. verticillata grew in high enough density that a clutch could find sufficient food within the limited range of nymphal movement. These results illustrate a corollary of the resource concentration hypothesis: within a plant group whose members share similar secondary plant chemistries, the only species that will be viable hosts for a specialized herbivore are those that provide the minimal resource density necessary for the completion of nymphal development.In central Missouri, O. fasciatus has specialized on a critical resource density, not traits of individual Asclepias species. The appearance of host selection within the potential host plant spectrum is the result of a characteristic growth form, seed output, and dispersion pattern for each milkweed species that makes some species much more likely than others to produce sufficient seed resources.     

Growth and Reproduction of Leptomonas oncopelti in the Milkweed Bug, Oncopeltus fasciatus

SYNOPSIS. A laboratory colony of Oncopeltus fasciatus was found to be infected with Leptomonas oncopelti. Inasmuch as the parasite is transmitted from parent to offspring an opportunity presented itself to study the biology and transmission of this parasite under controlled laboratory conditions. An apparatus for observing individual bugs was designed and the presence or absence of flagellates in the feces determined. Flagellates were not shed until the bugs became adults after which they appeared in every defecation. Dissection of infected bugs revealed that flagellates were not present in the rectum until adulthood. Further studies indicated that in the midgut of the insect there is a departure from binary fission to budding. The nucleus divides and one of the newly formed nuclei migrates toward a newly formed kinetoplast. Rarely there is still another kinetoplast/nucleus division. In the event the new axoneme grows within the cytoplasmic sheath of the parent flagellum, smaller organisms produced by unequal cytokinesis remain attached. If the axoneme grows free, the smaller daughter organisms become free-swimming. Passage into the rectum of the adult bugs causes a rounding up of all parasites although the leishmaniform organisms continue to divide. It is presumed that infection of clean bugs is accomplished by the ingestion of leishmaniform organisms through a common water source. The reason for the presence of flagellates in the rectum of the adult but not in the nymphal insect and the mechanism responsible for the change from binary to unequal fission are not known.     

Epithelial reorganization events during late extraembryonic development in a hemimetabolous insect

As extra-embryonic tissues, the amnion and serosa are not considered to contribute materially to the insect embryo, yet they must execute an array of morphogenetic movements before they are dispensable. In hemimetabolous insects, these movements have been known for over a century, but they have remained virtually unexamined. This study addresses late extraembryonic morphogenesis in the milkweed bug, Oncopeltus fasciatus. Cell shape changes and apoptosis profiles are used to characterize the membranes as they undergo a large repertoire of final reorganizational events that reposition the embryo (katatrepsis), and eliminate the membranes themselves in an ordered fashion (dorsal closure). A number of key features were identified. First, amnion-serosa “fusion” involves localized apoptosis in the amnion and the formation of a supracellular actin purse string at the amnion-serosa border. During katatrepsis, a ‘focus’ of serosal cells undergoes precocious columnarization and may serve as an anchor for contraction. Lastly, dorsal closure involves novel modifications of the amnion and embryonic flank that are without counterpart during the well-known process of dorsal closure in the fruit fly Drosophila melanogaster. These data also address the long-standing question of the final fate of the amnion: it undergoes apoptosis during dorsal closure and thus is likely to be solely extraembryonic.     

Saugverhalten und Nahrungsaufnahme von Oncopeltus fasciatus Dallas (Heteroptera,Lygaeidae)

Zusammenfassung Das Verhalten von Oncopeltus fasciatus bei der Nahrungssuche und- aufnahme wurde untersucht. Durch Aufspeicheln und Aufsaugen von Speichelsekret wird ein Substrat auf Eignung für Einstich und Nahrungsaufnahme geprüft. Die histologische Bearbeitung des Saugortes in einer Pflanze (Vicia faba) zeigt eine deutliche Bevorzugung des Leitungssystems beim Einstich. Hier werden Speichelscheiden gebildet. Im Samen (Asclepias syriaca und Helianthus annuus) werden die reservestoffreichen Cotyledonen besaugt. Der Einstich erfolgt ohne Sekretion einer Speichelscheide. Die Funktion dieser Speichelscheide wird diskutiert. Unter extremen Bedingungen tritt bei der sonst phytophagen Wanze trotz ihres ausgeprägten Subsozialverhaltens Kannibalismus auf. Beim Durchstechen der Cuticula dürften überwiegend mechanische Kräfte wirksam sein. Im Speichel konnte kein Chitin-spaltendes Enzym nachgewiesen werden. Weiterhin wurde die Saugzeitenverteilung zwischen Pflanze (Wasseraufnahme) und Samen (Nahrungsaufnahme) über einen Tagesablauf bestimmt. Die Wanzen dürften an der Pflanze in kürzeren Saugphasen Sättigung erreichen als an den dehydratisierten Samen. Die beträchtliche Menge and Speichel (1,14 mg/Wanze/Std), die in einen Samen injiziert werden, emulgieren und suspendieren dessen Inhalt und bringen ihn so in eine für das Insekt aufsaugbare Form.

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Summary The feeding behaviour of Oncopeltus fasciatus has been investigated. Exploration of food materials is performed by means of watery saliva which is secreted onto surfaces of substrates and sucked back again. Histological tests indicate that the animal preferably pierces the conducting system of the Vicia faba plant. Oncopeltus fasciatus secretes sheath material which coagulates and forms a lining to the path of the stylets during plant feeding. Feeding on seeds (Asclepias syriaca and Helianthus annuus) is not accompanied by stylet sheath formation. The function of this stylet sheath is discussed. Under extreme conditions the phytophagous animal shows cannibalism in spite of its marked subsocial behaviour. Penetration of the cuticle seems to be effected mainly by mechanical forces. No chitin-splitting enzyme could be detected in the saliva. The feeding activity over a photoperiod of 12 hrs on plants (uptake of water) and seeds (uptake of nutritive materials) is determined. It is suggested that on a green plant the animal is saturated more rapidly than on dehydrated seeds. The considerable amounts of watery saliva emulsify and suspend the contents of the seeds. Into seeds of Asclepias syriaca saliva is injected at a rate of approximately 1.14 mg/animal/hr.

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Durchgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft im Rahmen einer Herrn Prof. Kloft gewährten Sachbeihilfe.