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.     

Supernumerary regeneration in the large milkweed bug Oncopeltus fasciatus.

As an approach to the problem of pattern formation in the insect appendage, various graft combinations were studied in the legs of the large milkweed bug Oncopeltus fasciatus. Metathoracic legs of fourth instar larvae were amputated through the tibia within 24 hr after ecdysis and grafted back onto the stumps. The orientation of the graft was altered by rotation through 90 or 180° and/or by exchanging right and left stumps and grafts, yielding seven possible orientations in addition to the control. Many of these grafts resulted in the production of one or two supernumerary regenerates of the distal segments, which appeared at the graft junction after the second postoperative ecdysis. When two supernumerary regenerates resulted, one appeared to be produced from the stump and the other from the graft. When one regenerate was present, it appeared to be a composite of material produced from both the stump and the graft. In contrast to the results obtained in cockroaches, the external face of the leg appeared to be the only one capable of giving rise to a supernumerary regenerate.     

Effects of precocene II on female-specific hemolymph polypeptides in Oncopeltus fasciatus

Using polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, two major polypeptides of 200,000 and 170,000 daltons were detected in the hemolymph of mature female Oncopeltus fasciatus, but they were not found in the hemolymph of males or newly emerged females. Those polypeptides constituted the two major bands of early vitellogenic oocytes; however, they were absent from the yolk of mature eggs. The slower-migrating band (200,000 daltons) appears to correspond to a vitellogenic protein already identified in O. fasciatus, whose synthesis has been suggested to be independent of juvenile hormone (JH). Treatment of newly emerged adult females with the corpus allatum cytotoxin precocene II prevented the appearance of the female-specific bands and induced an important accumulation of other proteins in the hemolymph. Yolk deposition was also inhibited in those animals. Topical application of JH to precocene-treated females restored the appearance of the 200,000 and 170,000 dalton polypeptides in the hemolymph. These results suggest that JH is required for the synthesis of female-specific polypeptides in O. fasciatus.     

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.     

Characterization of an isoxanthopterin binding protein from Oncopeltus fasciatus.

Oncopeltus embryos contain a relatively large amount of isoxanthopterin, a small part of which is tightly bound to a high molecular weight (～500,000) protein. This causes a characteristic shift in the excitation and fluorescence maxima of isoxanthopterin from 348–409 nm to 355–420 nm, which provides a ready assay for the complex. Partial purification of the complex has been achieved, and some of its properties are described.     

Intercalary leg regeneration in the large milkweed bug Oncopeltus fasciatus.

Grafting operations were performed on the metathoracic legs of fourth instar Oncopeltus fasciatus within 24 hr after ecdysis. Different levels along the tibia were combined so that a lengthened tibia, approximately 1.5 times the normal length, or a shortened tibia, about half the normal length, were created. Intercalary regeneration occurred between the graft and stump in both combinations and the extra tissue was visible on the adult leg. The intercalary regenerate produced by the lengthened tibia showed reversed bristle polarity, while that produced in the shortened segment showed normal polarity. It is suggested that a regenerate with reversed polarity represents a mirror image duplication of the graft and might originate from the graft, whereas a regenerate with normal polarity may originate, as in normal regeneration, from the stump. It appears that each level in the appendage has the developmental capacity to produce only more distal structures. This conclusion is supported by the results of a grafting operation in which a portion of the tibia was grafted back on to the stump with its proximo-distal axis reversed. Regeneration of appropriate distal structures proceeded from the free proximal surface of the grafted tibia.     

Oncopeltus fasciatus zen is essential for serosal tissue function in katatrepsis

Unlike most Hox cluster genes, with their canonical role in anterior-posterior patterning of the embryo, the Hox3 orthologue of insects has diverged. Here, we investigate the zen orthologue in Oncopeltus fasciatus (Hemiptera:Heteroptera). As in other insects, the Of-zen gene is expressed extraembryonically, and RNA interference (RNAi) experiments demonstrate that it is functionally required in this domain for the proper occurrence of katatrepsis, the phase of embryonic movements by which the embryo emerges from the yolk and adjusts its orientation within the egg. After RNAi knockdown of Of-zen, katatrepsis does not occur, causing embryos to complete development inside out. However, not all aspects of expression and function are conserved compared to grasshopper, beetle, and fly orthologues. Of-zen is not expressed in the extraembryonic tissue until relatively late, suggesting it is not involved in tissue specification. Within the extraembryonic domain, Of-zen is expressed in the outer serosal membrane, but unlike orthologues, it is not detectable in the inner extraembryonic membrane, the amnion. Thus, the role of zen in the interaction of serosa, amnion, and embryo may differ between species. Of-zen is also expressed in the blastoderm, although this early expression shows no apparent correlation with defects seen by RNAi knockdown.     

Food level reaction norms in size-selected milkweed bugs (Oncopeltus fasciatus)

Abstract. 1. The hypothesis that the small size of nonmigratory milkweed bugs from Puerto Rico (relative to large migratory bugs from Iowa) might be an adaptation to low food levels was tested with bugs selected for large and small size as the consequence of positive genetic correlation between body size and wing length. The test involved examining reaction norms of life history traits to different food levels.
2. When nymphs were reared on 3 milkweed seeds per nymph from hatch to adult eclosion, large bugs were adversely affected with only 50% and 60% in each of two replicates reaching adulthood. Control (unselected) and small bugs, however, were unaffected at this food level. None of the lines was affected at levels of 6 and 12 seeds per nymph.
3. When adults were reared from eclosion on 12, 18 or 24 milkweed seeds per bug, large bugs were adversely affected relative to control and small bugs at all three food levels. At each food level fewer large bugs produced eggs than either small or control bugs which did not differ.
4. The evidence from the food level reaction norms suggests that larger bugs will be selected against during periods of food shortage of the sort likely to occur in Puerto Rico.     

Effect of ecdysterone on ethyl trimethyl dodecadienoate juvenile hormone action in Oncopeltus fasciatus

Ethyl trimethyl dodecadienoate exhibited a strong juvenile hormone action in large milkweed bug larvae (Oncopeltus fasciatus) whereas ecdysterone caused minimal juvenilizing effects. Topically applied ethyl trimethyl dodecadienoate was not synergized by simultaneously injected ecdysterone.     

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.     

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

SUMMARY 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.     

Identification of the adipokinetic hormone of the large milkweed bug Oncopeltus fasciatus

Abstract The adipokinetic hormone (AKH) of the large milkweed bug Oncopeltus fasciatus is isolated from an acidified methanolic extract of 200 corpora cardiaca, purified by single step reversed phase high‐performance liquid chromatography (HPLC) and N‐terminally deblocked using pyroglutamate aminopeptidase. The sequence is identified by Edman degradation and matrix assisted laser desorption/ionization‐time of flight mass spectroscopy as pGlu‐Leu‐Asn‐Phe‐Ser‐Pro‐Asn‐Trp amide. This structure is confirmed by chemical synthesis and coelution of native and synthetic peptide on HPLC. The AKH of O. fasciatus is identical to Tenmo‐HrTH, a member of the adipokinetic/red pigment‐concentrating hormone peptide family that had been isolated earlier from several tenebrionid beetles. Tenmo‐HrTH causes a significant rise in the concentration of haemolymph lipids when injected into adult male and female O. fasciatus, but displays no hyperglycaemic activity. There is no indication of the presence of other AKHs in O. fasciatus. The large milkweed bug represents the first member of the seed bugs (Lygaeidae) for which the endogenous AKH has been identified.     

Ultrastructural Analysis of the Neuroendocrine Apparatus of Oncopeltus fasciatus (Heteroptera)

• 1 In Oncopeltus fasciatus, the A-cells of the pars intercerebralis and their tracts are stainable in situ with the performic acid-victoria blue (PAVB) method. The axons from these cells, after traversing the corpus cardiacum, terminate in the anterior part of the aorta which thus serves as the neurohemal organ.
• 2 Ultrastructurally, four types of secretory neurons are distinguishable in the pars intercerebralis region: pic-I with granules measuring 1000–3000 Å in diameter; pic-II with granules of irregular size and shape, the elongate ones showing mean dimensions of 2400 × 1400 Å; pic-III with less electron-dense granules measuring 1000–2700 Å in diameter; pic-IV, present not only in the pars intercerebralis but also in adjacent regions of the brain, with variable proportions of granules measuring 700–1800 A and dense-cored vesicles measuring 1000–2400 Å.
• 3 The nervi corporis cardiaci contain at least three types of neurosecretory axons, based on granule content, presumably representing pic-I, pic-II and pic-III neurons.
• 4 The wall of the aorta contains endings of at least three distinct types, again representing pic-I, pic-II and pic-III neurons, and thus provides the neurohemal site for these three types of protocerebral neurosecretory cells. Axons of pic-IV neurons appear to enter the cerebral neuropil.
• 5 The corpus cardiacum is composed of two types of parenchymal secretory cells, with electron-dense granules measuring 1300–3000 Å and 1000–2300 Å in diameter, respectively. The corpus cardiacum also contains interstitial cells and some axons of extrinsic origin, with and without granules.
• 6 The corpus allatum may be paired or median, and receives a small number of at least two types of axons. The corpora allata of some reproducing females show a large number of PAVB-stainable inclusions which appear to be modified cytoplasmic organelles, but are definitely not neurosecretory material.
• 7 The hypocerebral ganglion is composed of two types of secretory-appearing neurons and glial cells. The two neuronal types contain secretory granules, 1000–3000 Å and 900–2100 Å in diameter, respectively. Axons of the recurrent nerve also may contain occasional granules.
• 8 In this heteropteran insect, the two principal functions of the corpus cardiacum appear to be spatially separated: the neurohemal function is subserved by the aortic wall, which permits release of material into both the aortic lumen and the hemocoel, and the intrinsic endocrine function is possessed by the parenchymal cells.

     

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.     

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.

---

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.

---

---

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