Comparative immunocytochemical study of release sites of insulin,glucagon and AKH-like products in Locusta migratoria,Periplaneta americana,and Carausius morosus

Summary Insulin, glucagon and adipokinetic hormone antisera were applied to the corpora cardiaca, perisympathetic organs, neurohemal areas and peripheral neurosecretory cells of three insect species, the locust Locusta migratoria, the cockroach Periplaneta americana, and the stick insect Carausius morosus. The neurohemal part of the corpora cardiaca was shown to be immunoreactive to both insulin and glucagon antisera while the glandular cells reacted to adipokinetic hormone antisera. The perisympathetic organs seem to be devoid of these three substances, but certain peripheral neurohemal areas contained AKH and glucagon immunoreactive products. The latter were found to originate in the peripheral neurosecretory cells.     

Further characteristics of adipokinetic and hyperglycaemic factor(s) of stick insects

When an extract of the corpora cardiaca/corpora allata from two species of wingless stick insects, Carausius morosus and Cuniculina impigra, which cause no adipokinetic or hyperglycaemic effect when injected into the donor insects themselves, is injected into adult Locusta migratoria it resulted in an increase in the haemolymph lipid concentration. The lipid elevation was time dependent, with a maximum effect about 90–180 min after injection, and was also dose-dependent. About 0.001–0.002 (C. morosus) and 0.01 (C. impigra) gland equivalents were needed to produce a significant increase; a maximal effect was reached with approx. 0.075 (C. morosus) and 0.25 (C. impigra) gland equivalents. Carausius extract was also able to elevate carbohydrate concentration in the haemolymph of Periplaneta americana. However, the effect was weak and no maximal response was reached even with a dose of 0.5 gland equivalents. Adipokinetic hormone activity was present in CC/CA extracts of larval Carausius; the activity was about 30 times lower in 1-day-old 2nd instar individuals, and approx. 5 times less at the beginning of the 6th instar than that found in adults. In both stages the hormone levels increased gradually from the beginning to the end of the instar. No age-related changes were observed during the adult stage. Further studies on the lipid-mobilising factor of C. morosus revealed that it was stored entirely in the CC and not in other nervous tissue, e.g. brain, CA, suboesophageal ganglion, thoracic and abdominal cord. The factor was heat stable for at least 1 hr at 100°C and retained its adipokinetic activity after incubation with trypsin and the exopeptidases such as carboxypeptidase A and leucine aminopeptidase. However, activity was abolished when incubated with thermolysin and α-chymotrypsin. From these experiments a close resemblance to the locust AKH, a blocked decapeptide, is suggested.     

Effects of acetylcholine,physostigmine, and hemicholinium-3 on spontaneous electrical activity of neurosecretory nerves in Carausius and Rhodnius

The neurohaemal, lateral branch of the median nerve in Carausius morosus and the neurohaemal, ventral motor nerve of Rhodnius prolixus are stimulated by acetylcholine to increase the frequency of action potentials recorded via extracellular electrodes from isolated nerves. Physostigmine potentiates electrical activity in both insects and hemicholinium-3 has a depressant effect on Carausius preparations. These results suggests a cholinergic mechanism associated with neurosecretory activity.     

Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH

Summary An immunocytochemical method was used to differentiate between immunoreactive substances in glandular cells in the corpora cardiaca (CC) and in certain cerebral neurons in 2 insect species, Locusta migratoria migratorioides and Periplaneta americana. The staining properties of antisera raised to different parts of the decapeptide adipokinetic hormone (AKH) were compared and their specificity was determined by preabsorption with AKH and related peptides. Antibodies raised to the N-terminal part of AKH (serum 433) and the central and C-terminal part (serum 241) were found to have different staining properties.In the CC of the locust both antisera show a strong immunoreactivity with glandular cells, we therefore suggest that at least one of the compounds revealed is AKH. Some of the glandular cells in the locust and large numbers of glandular cells in the CC of the cockroach are revealed by the N-terminal specific antiserum. On the other hand, neurons in the central nervous system are revealed only by the C-terminal specific antiserum. The possible identity of the various substances revealed by these two antisera is discussed.     

Ultrastructural study of cholecystokinin-like immunoreactivity in endocrine cells of the insect midgut

Summary Electron-microscopic immunocytochemistry for the demonstration of CCK-like material in basal endocrine cells of the midgut of Aeschna cyanea, Locusta migratoria, Carausius morosus and Periplaneta americana was performed by use of the peroxidase-antiperoxidase procedure and the colloidal gold method. Immunoreactive cells appeared scattered among digestive and regenerative cells of the epithelium. Immunoreactivity was specifically detected over round to oval electron-dense granules whose size appeared rather different from species to species. Thus, the average size of 30% (d30) of the largest granules ranged from 312 nm in Periplaneta americana to 159 nm in Carausius morosus with intermediate values in Aeschna cyanea (d30=195 nm) and Locusta migratoria (d30=225 nm).     

Effects of starvation and dehydration on osmotic and ionic balance in Carausius morosus

This study describes the effects that prolonged desiccation has on osmotic and ionic balance in Carausius morosus, an insect with a rather unusual haemolymph composition (it is rich in divalent cations and has a low sodium content). After 7 days at 0% r.h., there is a 50 per cent decrease in water content of the animal. This loss is largely confined to the haemolymph, which declines in volume to about one-seventh of its previous value. In spite of these drastic changes, the osmotic concentration of the haemolymph increases only by about 20 per cent while the concentrations of the main blood ions increase by similar or smaller amounts. So, as with other insects so far examined, Carausius turns out to have very effective control of the osmotic and ionic concentrations of its internal milieu in the face of severe stress upon it.     

Influence of aminergic and peptidergic substances on heart beat frequency in the stick insect Carausius morosus (Insecta,Phasmatodea)

The dorsal heart of the Indian stick insect, Carausius morosus, is responsible for the anterograde flow of hemolymph to the aorta and into the body cavity. The contraction frequency of the insect heart is known to be influenced by several substances of neural source. Here, a semi‐exposed heart assay was employed to study the effect of an aminergic substance (octopamine) and three neuropeptides (C. morosus hypertrehalosemic hormone [Carmo‐HrTH], crustacean cardioactive peptide [CCAP], and proctolin) on heart contraction. The contraction frequency was measured as beats per minute in adults ligated between the head and the prothorax. All three investigated neuropeptides had a stimulatory effect on heart contraction that lasted approximately 6 min, after which the normal heart beat rate was restored. Proctolin and CCAP stimulated the rate of heart beat also in unligated stick insects, whereas Carmo‐HrTH was active only in ligated insects. The latter could suggest that when the stick insect is not ligated, a competing substance may be released from the head of C. morosus; the competing substance is, apparently, not physiologically active but it binds or blocks access to the receptor of Carmo‐HrTH‐II, thereby rendering the HrTH peptide “not active.” In ligated stick insects, 6.7 × 10^?8 M Carmo‐HrTH‐II significantly increased the heart beat rate; higher doses resulted in no further increase, suggesting the saturation of the HrTH receptor. Octopamine inhibited the rate at which the heart contracted in a dose‐dependent manner; inhibition was achieved with 10^?4 M of octopamine.     

Vitellogenin uptake and vitellin localization in insect follicles examined using monoclonal antibodies and confocal scanning microscopy

Summary

Confocal scanning immunofluorescent microscopy and monoclonal antibodies were used to examine the route of uptake of vitellogenin (VG) by vitellogenic follicles and the ooplasmic localization of vitellin (VN) in the cricket, Acheta domesticus, and the stick insect, Carausius morosus. Uptake and cytoplasmic regionalization of a non-vitellogenic sulfated protein, sp 157/85, by C. morosus oocytes were also examined. By indirect immunofluorescence VG in both species and sp 157/85 were visualized in spaces between follicle cells and in peripheral yolk spheres. One cricket VG polypeptide had a regionalized distribution in the folliclular epithelium, and VN polypeptides in both species and sp 157/85 in C. morosus had regionalized distributions within the ooplasm. Localization of sp 157/85 to the anterior pole of the oocyte appeared to be stage-specific.     

Antisera against AKHs and AKH precursors for experimental studies of an insect neurosecretory system

Three different oligopeptides based on the amino acid sequence of Schistocerca gregaria AKH I and AKH II were designed and synthesized. They were used to raise rabbit antisera capable of differentiating between AKH I and II and of recognizing the identified precursors of the AKHs (P1 and P2). Analogue I (Lys-Tyr-Thr-Pro-Asn-Trp-Gly-Thr-NH₂) generated AKH I specific antisera, analogue II (Lys-Tyr-Leu-Asn-Phe-Ser-Thr-Gly-Trp-NH₂) generated AKH II specific antisera. A non-amidated analogue of AKH I called analogue III (Lys-Tyr-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-OH) generated antisera capable of recognizing both the P1 and P2 AKH precursors. The antiserum recognizing the precursor (antiserum IIIa) can be used to monitor experimentally induced changes in precursor levels in the locust corpus cardiacum. The antisera described here are discussed in terms of their utility in studying the insect neurosecretory system.     

Stick insects (Phasmida) as prey of spiders: size,palatability and defence mechanisms in feeding tests

Summary The maximum body length of the phasmid Carausius morosus which the ctenid spider Cupiennius salei can overwhelm is 2–3x larger than the length of crickets. Eight phasmid species were offered to Cupiennius salei in feeding tests. Among them, Carausius morosus was raised on 4 different food plants. All species of prey items were accepted by the spider predator very readily (>80%). This indicates that phasmids are a highly palatable prey group for spiders. The possible importance of chemical defence and its obvious lack under laboratory conditions are discussed.     

Mono- and polyclonal antibodies as probes to study vitellin processing in embryos of the stick insect Carausius morosus

During embryonic development, insect vitellins (Vt) are degraded by limited proteolysis to yield a number of lower-molecular weight polypeptides. The aim of the present study was to identify these polypeptides in the embryo and to verify how they relate to Vt polypeptides deposited in the oocyte during vitellogenesis. To this end a panel of poly- and monoclonal antibodies (Pab, Mab) was raised against Vt polypeptides and employed by immunoelectrophoresis and immunoblotting on embryos belonging to different developmental stages. Through this approach three major staining patterns were observed. First, Mab 4 reacts with both polypeptides B₁ and E₂₀, suggesting that polypeptide B₁ is gradually trimmed to yield polypeptide E₂₀ in late embryos. Second, Mab 12 is specific for polypeptide A₃ which is retained unchanged throughout embryogenesis. Third, Pab anti-A₂ and Mab 13 show that polypeptide A₂ is processed to yield polypeptide E₉ through limited proteolysis. In conclusion, the staining patterns reported in this study show that Vt polypeptides in developing embryos of the stick insect Carausius morosus undergo at least two major processing events concerning polypeptides B₁ and A₂.     

A tryptophan-substituted member of the AKH/RPCH family isolated from a stick insect corpus cardiacum.

A neuropeptide, Cam-HrTH-I, has been isolated from the corpus cardiacum of the Indian stick insect Carausius morosus. The peptide causes hyperlipaemia in Locusta migratoria and hypertrehalosaemia in Periplaneta americana and is related to the previously isolated Cam-HrTH-II (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) by substitution, apparently by a hexose, on the Trp residue. This appears to be the first example of such substitution on a Trp residue.     

Co-localization of the adipokinetic hormones I and II in the same glandular cells and in the same secretory granules of corpus cardiacum of Locusta migratoria and Schistocerca gregaria

Summary The immunocytochemical reactivity of the glandular cells of the corpus cardiacum (CCG-cells) of Locusta migratoria and Schistocerca gregaria was investigated at the electron-microscopic level, using the protein A-gold method, with three antisera against fragments of the adipokinetic hormones AKH I and AKH II. This combination of antisera permitted discrimination between anti-AKH I and anti-AKH II immunoreactivity. Fixation in a mixture of 2% glutaraldehyde and 2% formaldehyde, in combination with low-temperature embedding in Lowicryl K₄M, produced the highest and most consistent selective immunogold labelling of the secretory and ergastoplasmic granules. All secretory granules in all CCG-cells investigated possessed a distinct anti-AKH I-immunopositive reaction, whereas most secretory granules showed a weaker anti-AKH II immunoreaction. Ergastoplasmic granules reacted similar to the secretory granules. The average immunolabelling of the secretory granules was higher in the processes than in the cell bodies of the CCG-cells. The results in Schistocerca gregaria were essentially similar to those in Locusta migratoria. It is concluded that (i) the individual CCG-cells synthesize AKH I as well as AKH II; (ii) these hormones coexist in the same ergastoplasmic and secretory granules; and (iii) these granules contain a higher content of AKH I than AKH II.     

Die Redoxpigmente von Carausius morosus und ihre Bedeutung für den morphologischen Farbwechsel

Summary From the epidermis of Carausius morosus two ommochromes were isolated, and identified by various means as Xanthommatine and Ommine. Their amount was determined by photometry for animals of different colour.Morphological colour change results mainly from changes in ommochrome content.If the lower part of their compound eyes is blackened, green specimens become brown by an increase of ommochrome production.This colour change can be evoked within a single larval instar.It can also be evoked in adult specimens, although to a small degree only.Dark specimens with unblinded eyes become paler under normal illumination in consequence of an increase of bright pigments and of the increase in bodysize, while the amount of ommochrome increases only very slightly.A decrease of ommochrome content or a loss of ommochrome by faeces or offshed cuticles was never observed.At high temperature (28° C) both, ommochrome production in the epidermis and melanin formation in the cuticle are increased.Implantation of supernumerous Corpora allata causes the ommochrome content to increase. After extirpation of the Corpora allata no decrease of ommochrome content is found but the green pigment, insectoverdin vanishes. Apparently in Carausius ommochrome may deposited but is never removed from the integument, which may explain the similar coloration after both experiments.     

Structural differentiation in the mid-gut epithelium of the phasmid Carausius morosus Brunner

The epithelial cells of the anterior central and posterior regions of the midgut of Carausius morosus are described. Possible functions of the cells, indicated by structural differentiation, are discussed.     

An invertebrate [hydroxyproline]-modified neuropeptide: further evidence for a close evolutionary relationship between insect adipokinetic hormone and mammalian gonadotropin hormone family

An octapeptide of the adipokinetic hormone (AKH) peptide family is identified in the corpora cardiaca of the stink bug, Nezara viridula, by ESI-MS^N (electrospray ionization multistage MS). This is the second AKH in N. viridula and it has a hydroxyproline residue at position 6, whereas the major AKH (known as Panbo-RPCH) has Pro as the sixth amino acid residue. The correct sequence assignment of [Hyp⁶]-Panbo-RPCH is confirmed by retention time and MS spectra of the synthetic peptide. Various extraction procedures were followed to ascertain whether the hydroxylation is an artefact of extraction, or whether it is due to a true post-translational modification at the prohormone level. The proline hydroxylation is unique for invertebrate neuropeptides, while it has been described in the vertebrate gonadotropin-releasing hormone (GnRH). The current finding is another piece of evidence that AKH and GnRH form a peptide superfamily and are closely related evolutionarily. Biologically, [Hyp⁶]-Panbo-RPCH is active in vivo as an AKH, causing hyperlipaemia in the stink bug at low doses, indicating again that it is an endogenous, mature and functional hormone in this insect species.     

Regulation of the body-substrate-distance in the stick insect: Step responses and modelling the control system

The feed back mechanism subserving the regulation of the body-substrate-distance in the stick insect Carausius morosus has been investigated by means of step-like stimuli. Based on the results obtained in open-loop experiments a model is developed which describes the results obtained under closed loop conditions. When the experimental animal is pushed or pulled in dorso-ventral direction an initial fast and a subsequent very slow change of the body height z over the substrate are observed. The late slow response is a nearly linear function of time and can last for more than one hour if the animal is pulled with moderate forces. It withstands less effectively if pushed. During the slow phase of the response sudden changes of z and of the slope of the z(t)-curves occur, presumably due to corresponding changes of the amplification within the feed back loop.     

Sequencing and biological effects of an adipokinetic/hypertrehalosemic peptide in the stick insect, Baculum extradentatum

The corpora cardiaca of the Vietnamese stick insect, Baculum extradentatum, contain a member of the adipokinetic hormone/red pigment-concentrating hormone/hypertrehalosemic hormone (AKH/RPCH/HrTH) family of peptides whose sequence is identical to that originally described for the Indian stick insect, Carausius morosus. This decapeptide, Carmo-HrTH-II (pELTFTPNWGTa), has both hypertrehalosemic and cardioacceleratory activity in B. extradentatum, and hyperlipaemic activity in locusts. Reversed-phase high performance liquid chromatography (RP-HPLC) of corpora cardiaca extract followed by MALDI-TOF MS/MS also revealed a novel modification of a second peptide in B. extradentatum: the tryptophan residue at position 8 is post-translationally modified to kynurenine.     

The control of body position in the stick insect (Carausius morosus), when walking over uneven surfaces

An investigation has been made of the way, in which the height of the body of an insect (Carausius morosus) is controlled when walking over an uneven terrain. The animals have been filmed from the side while walking over different types of irregularity (step up, step down, obstacle, ditch). A frame by frame analysis of the height of the three thoracic segments of the insect has been performed. A computer model has been set up, which is able to describe the experimental results within the exactness of measurement. This model consists of three independent height controllers, each having a unique characteristic. The coupling of these three controllers is performed mechanically. One possible interpretation of this model is that the height of each segment is controlled by a closed loop mechanism with a proportional element as a controller.Supported by the Deutsche Forschungsgemeinschaft