Histophysiological studies on the corpus allatum of Leucophaea maderae

Summary The structural differences between active and inactive corpora allata, visible under the light microscope, become more pronounced under the electron microscope. Aside from differences in cellular and nuclear diameters, and nuclearcytoplasmic ratios, there are qualitative characteristics in ultrastructural organization.The cytoplasm of active corpus allatum cells contains numerous sinuous mitochondria, distinct Golgi elements, ergastoplasmic units with a tendency to form whorls, agranular cytomembranes, and free ribosomes. Pleomorphic inclusion bodies resembling lysosomes are more or less numerous. The plump, ovoid nuclei frequently show two prominent nucleoli whose components may form a meshwork harboring chromatin.The marked reduction in the amount of cytoplasm occurring during the organ's return to inactivity is accompanied by a decrease in the number, and a change in the appearance, of some cytoplasmic organelles. The mitochondria tend to be smaller, and the ergastoplasm is reduced to scattered wisps of ribosome-studded membranes. Nuclei of inactive cells have smaller diameters than those of active ones.In all stages of activity, cell boundaries are clearly visible. As a result, the corpus allatum cell can now be characterized as a discrete unit of epithelioid character and rather complex shape. The plasma membrane may become folded when the cellular content shrinks to the inactive level. Aside from changing outlines, all corpus allatum cells have long, gradually thinning processes. These penetrate deeply into the parenchyma where they interlock with those of other cells; many processes eventually seem to reach the surface of the gland where the secretory products are released into the hemolymph. These have to pass through an acellular connective tissue layer that shares tinctorial and ultrastructural properties with those of a boundary (or basement) membrane.This stromal element forms a sheath and branching processes that extend into and permeate the parenchyma. It seems to represent a system of channels, not only for the release of secretory and other cellular products, but for the entry of nutrients and perhaps chemical messenger substances.Neurosecretory material can be observed in the form of structurally distinctive elements, i.e., as electron-opaque granular inclusions within axon terminals that become contiguous with corpus allatum cells.No definite statement can be made on the basis of the present study about the nature of the corpus allatum hormone or hormones, except that the ultrastructural criteria indicative of proteinaceous secretion, such as the appearance of secretory granules in spatial relation with Golgi elements, seem to be missing in the corpora allata of Leucophaea.Supported by Research Grants A-3984 and B-2145 from the U.S.P.H.S.     

Histophysiological studies on the corpus allatum of Leucophaea maderae

Summary On the basis of the occurrence, at the light microscopic level, of alkaline and acid phosphatases, the pigment epithelium covering the posterior surface of the iris in the albino rabbit can be divided into two zones not previously described, viz. a central zone close to the pupil, approximately corresponding to the area occupied by the iridic sphincter muscle, and a peripheral zone extending to the ciliary body. The central zone which is in intimate relation with the lens was found to have a high content of both phosphatases. At the fine structural level it exhibits a marked pinocytotic activity in the epithelium at the interdigitations between adjacent cells. Electron microscopy revealed that acid phosphatase is localized to the walls of the pinocytotic vesicles. Alkaline phosphatase is in evidence at the surface membrane folds and at microvillous processes between the epithelial cells and the adjoining muscle cells. Unlike the distribution of the acid phosphatase, that of the alkaline phosphatase does not differ fundamentally in the two zones at the fine structural level.In a series of dehydrogenases studied, staining with a view to succinic-, isocitric- and glucose-6-phosphate dehydrogenases revealed an evenly distributed content of enzyme throughout the epithelium. As to the lactic- and -hydroxybutyric dehydrogenases, contents seem to be lower in the pupillary than in the peripheral zone.     

Histophysiological studies on the corpus allatum during prepupal diapause in Monema flavescens (Lepidoptera)

The appearance of the corpus allatum, the central endocrine gland of diapause, was examined histologically in the slug moth prepupae, Monema flavescens (Lepidoptera) Before beginning of diapause, the secretory cells of the corpus allatum increase in size with the enrichment of the cytoplasm in quantity and in number of organelles, while RNA synthesis also starts. As diapause progresses, the secretory cells have many large unstained vacuoles in the cytoplasm, which were demonstrated to contain some substances of lipoidal nature. It is not clear that the substance is juvenile hormone itself or a material related to juvenile hormone. Agranular ER is the most characteristic organelle connected with mitochondria and situated around the vacuoles during diapause. Electron micrographs show that agranular ER and mitochondria have an essential role for the production of juvenile hormone. The function of the corpus allatum during diapause and the relationship between juvenile hormone and diapause are discussed.     

Immunoreactive material resembling vertebrate neuropeptides in the corpus cardiacum and corpus allatum of the insect Leucophaea moderae

Summary The presence and differential distribution of substances antigenically related to known vertebrate neuropeptides demonstrated within the corpus cardiacum of the insect Leucophaea are as follows: Of ten mammalian antisera tested, six yielded substantial immunoreactive deposits resembling oxytocin, somatostatin, Substance P, met-enkephalin, bombesin, and neurotensin, respectively. In the remaining four, the reaction was moderate (vasopressin, -endorphin) or marginal (LH-RF, calcitonin). With regard to their regional distribution, these biochemically distinct reaction products seem to fall into two groups: (1) Materials resembling oxytocin, vasopressin, met-enkephalin, -endorphin (and presumably also neurotensin and LH-RF) predominate in the central release area of the organ and are considered to be of extrinsic (cerebral) origin. (2) Substances localized primarily in areas rich in intrinsic glandular cells of the corpus cardiacum, and revealed by antisera raised against somatostatin, Substance P, and bombesin, are judged to be synthesized and stored within this organ. In peptidergic fibers entering the adjacent corpora allata, thus far Substance P-, -endorphin-, and LH-RF-like immunoreactivities have been demonstrated. Some of these new neuropeptides may be contained in classical neurosecretory neurons, formerly identified by less specific methods, others must be assigned to additional peptidergic neurons heretofore unknown.Supported by NSF grant BMS 74-12456 (B.S.). The excellent technical assistance of Mrs. Sarah Wurzelmann is gratefully acknowledged     

Neurosecretion. XIII. The ultrastructure of the corpus cardiacum of the insect Leucophaea maderae

Summary The corpora cardiaca of Leucophaea maderae contain two classes of intrinsic elements, parenchymal and interstitial cells. The parenchymal cells produce a secretory material first visible in the Golgi zones of the perikarya in the form of distinct electron-opaque granules. These undergo changes (gradual loss of electron-density, emergence of internal structure) as they accumulate in cellular processes.The parenchymal cells are best classified as neuroglandular elements since, in addition to secretory inclusions, they possess characteristics of ganglion cells such as axonlike processes, neurotubules, and sheaths. These covers are provided by branches of the second type of intrinsic elements, the interstitial cells. They are non-glandular structures of considerable morphological complexity. In the manner of glial elements, they permeate the entire organ and encapsulate not only the perikarya of parenchymal cells but cellular processes as well.The cytoplasmic processes include a) relatively short ones belonging to parenchymal cells, and b) long axons whose cell bodies lie within the central nervous system. Many of the latter contain electron-opaque granules of the kind found in electron-micrographs of typical neurosecretory cells. These extrinsic granules represent the second category of secretory products stored within the corpora cardiaca. By comparison with the product of the intrinsic gland cells, the neurosecretory granules seem to be fairly stable. Neither type seems to pass through the connective tissue sheath of the corpus cardiacum in the form of discrete granular entities.This sheath, which sends branches into the interior of the corpora cardiaca, has the properties of a basement membrane. It represents a pathway for the exchange of substances between the cellular components of the corpus cardiacum and the surrounding hemocoele.The dual character of the corpus cardiacum, namely that of a storage and release center for extrinsic neurosecretory substances and of an endocrine organ in its own right, is herewith established beyond doubt. The number of secretory products discernible on the basis of their morphology and localization (two size categories of extrinsic and one intrinsic type of granules) does not match the variety of physiologically active principles known at present. The assignment of specific functions to discrete morphological elements must await further studies.Supported by Research Grants A-3984 and B-2145 from the U.S.P.H.S.     

Antennal sense organs of the cockroach, Leucophaea maderae

Adult and nymphal antennae of the cockroach, Leucophaea maderae, contain nine or more different morphological types of sense organs. There is no outwardly apparent sexual dimorphism in adult antennae. Nymphs are dificient in gross numbers of sensilla. Sense organs are classified morphologically by their similarity to known types of sensila and are assigned functions on this basis and preliminary electrophysiological data: Sensilla chaetica (A), thick-walled mechanoreceptive hairs in groups on the antennal base; S. chaetica (B), thick-walled setae which are tactile and probably chemoreceptive, occurring in the antennal base and flagellum; S. trichodea (A), thin-walled chemoreceptive hairs of the flagellum; S. trichodea (B), minute hairs on the scape and pedicel; S. basiconica, thin-walled chemoreceptive pegs, and S. coeloconica (?pit-pegs”?) of the flagellum; S. campaniformia and scolopidia, mechanoreceptors in the base and flagellum; plus Johnston's organ and/or connective chrodotonal organs in the pedicel. Calculations based on absolute counts of sensilla and their known innervation yield an estimate of about 3.3 × 10⁴ sensilla and 10⁵ cells per antenna.     

Azadirachtin affects the circadian rhythm of locomotion in Leucophaea maderae

Abstract

Azadirachtin shortens the period length of the locomotor activity rhythm in the circadian rhythm of Leucophaea maderae and induces splitting of this rhythm in two components in about 40% of the animals. The phase relationship between the two components is 180°. Both shortening of period and splitting are more pronounced in animals possessing longer periods before the injection of azadirachtin.     

Neural organization of the circadian system of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac- tive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of γ-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.     

Orcokinin immunoreactivity in the accessory medulla of the cockroach Leucophaea maderae

The accessory medulla is the master circadian clock in the brain of the cockroach Leucophaea maderae and controls circadian locomotor activity. Previous studies have demonstrated that a variety of neuropeptides are prominent neuromediators in this brain area. Recently, members of the orcokinin family of crustacean neuropeptides have been identified in several insect species and shown to be widely distributed in the brain, including the accessory medulla. To investigate the possible involvement of orcokinins in circadian clock function, we have analyzed the distribution of orcokinin immunostaining in the accessory medulla of L. maderae in detail. The accessory medulla is densely innervated by approximately 30 orcokinin-immunoreactive neurons with cell bodies distributed in five of six established cell groups in the accessory medulla. Immunostaining is particularly prominent in three ventromedian neurons. These neurons have processes in a median layer of the medulla and in the internodular neuropil of the accessory medulla and send axonal fibers via the posterior optic commissure to their contralateral counterparts. Double-labeling experiments have revealed the colocalization of orcokinin immunostaining with immunoreactivity for pigment-dispersing hormone, FMRFamide, Mas-allatotropin, and γ-aminobutyric acid in two cell groups of the accessory medulla, but not in the ventromedian neurons or in the anterior and posterior optic commissure. Immunostaining in the ventromedian neurons suggests that orcokinin-related peptides play a role in the heterolateral transmission of photic input to the pacemaker and/or in the coupling of the bilateral pacemakers of the cockroach.This study was supported by the Deutsche Forschungsgemeinschaft, grant HO 950/9.     

Effects of leucomyosuppressin on the excitation-concentration coupling of insect Leucophaea maderae visceral muscle

1. Leucomyosuppressin (LMS) inhibited neurally evoked contractions of the hindgut of the cockroach Leucophaea maderae. The threshold for this inhibition of LMS was in the range of 1 × 10⁻¹⁰ M.2. LMS caused a sharp reduction in both l-glutamate and proctolin induced contractions. Dose-response profiles of the effect of LMS (held constant at 10⁻⁸M) on variable amounts of proctolin showed an inhibitory effect at 10⁻⁹ M proctolin and below, but at 5 × 10⁻⁹ M proctolin and above, LMS caused no inhibition.3. Potassium (158 mM) depolarized hindguts treated with LMS (10⁻⁸ M) showed a marked reduction (76% ± 2.1) in the proctolin (10⁻⁸ M) response.4. When calcium depleted preparations were returned to normal calcium levels (2 mM) in the presence of proctolin (10 ⁻⁸ M) a contraction occurred that was 45% ± 4 of the maximum in normal saline solution. However, LMS (10⁻⁸ M) reduced this response to only 28% ± 2 of the maximum.5. Proctolin (10⁻⁸ M) induced contractions in the presence of the manganous ions (2mM) fell to 63% ± 4 of the maximum but on the addition of LMS (10⁻⁸M), such responses fell to only 16% ± 5 of the maximum.6. These results offer evidence for a non-synaptic site of action for LMS and a perturbation of key calcium dependent events in the excitation-contraction coupling sequence of visceral muscle by this peptide.     

Immunocytochemical characterization of the accessory medulla in the cockroach Leucophaea maderae

Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect brain.     

Comparative pharmacological actions of leucokinins V-VIII on the visceral muscles of Leucophaea maderae

1. Leucokinins V-VIII (Lem-K-V to VIII) did not activate visceral muscles of the cockroach Leucophaea maderae uniformly as a group but rather showed a selective action on the muscles of the hindgut. This organ showed a contractile response to all of the leucokinins at 3 x 10(-10) M that was 2-20% above the mean level of spontaneous activity. The maximum response for each peptide was recorded at 2.1 x 10(-7) M. 2. Both the foregut and the oviduct were 100- to 1000-fold less sensitive than the hindgut, and each of the former required more than 10(-8) M to elicit a detectable excitation. The heart, by comparison, did not respond to any of these peptides. 3. The leucokinins caused a protracted excitation of contractile events in the hindgut that lasted for more than 60 min. Moreover, all four peptides evoked contractions from hindguts after membrane depolarization with 158 mM potassium. These results suggest that nonsynaptic receptors for the peptides exist in visceral muscle.     

Density-gradient centrifugation isolation of hormone-containing neurosecretory granules from the cockroach,Leucophaea maderae

Neurosecretory granules (NSG) containing hindgut-stimulating neurohormone (HSN) from Leucophaea maderae were isolated by densitygradient centrifugation of cockroach brain homogenates.High concentrations of HSN were consistently found in isolates containing large numbers of NSG. HSN was measured by bioassay and the NSG were identified by electron microscopy.     

The colon of Leucophaea maderae: fine structure and physiological features

The colon of L. maderae consists of a single columnar epithelium covered with a cuticle and of a musculo-connective sheath. The apical plasma membranes form a system of leaflets with numerous mitochondria inserted in association with microfilaments. Lateral plasma membranes are linked together by junctional complexes consisting of a zonula adherens and a long convoluted septate junction of the pleated type. In the basal region of the cell, numerous membrane infolds and scattered scalariform junctions with associated mitochondria are present. These cell specializations are typical of arthropod transporting organs, being distinctive features of ion and fluid transporting epithelia. The isolated colon exhibited a transepithelial electrical potential difference (PD) of about 100 mV, lumen side positive with respect to the haemolymph side. The PD was almost abolished by metabolic inhibitors, it was reduced by acetazolamide and SITS, and it was unaffected by ouabain. These effects suggest that HCO3- and Cl- are involved in the genesis of the PD, whereas Na+ is not directly responsible of the PD. Measurements of Na+ and Cl- fluxes across the colon wall confirm that Na+ moves following the PD across the tissue, while Cl- movement occurs against an electrochemical potential difference. The electrical profile of the epithelial cells is of the well type and it suggests that the primary or secondary active step for Cl- transport across the epithelium should be located at the mucosal border of the cell.     

Circadian rhythms in the mating behavior of the cockroach, Leucophaea maderae

Mating behavior of small populations of virgin males and females of the cockroach Leucophaea maderae were continuously monitored via time-lapse video recording in controlled laboratory conditions. The time of onset of copulation was found to be rhythmic in a light cycle of 12 h light alternated with 12 h of darkness, with the peak of mating behavior occurring near the light to dark transition. This rhythm persisted in constant dim red illumination and constant temperature. In constant conditions, the period of the rhythm was slightly less than 24 h, with a peak of copulation during the late subjective day. These data demonstrated that mating behavior is gated by a circadian clock. When males and females were taken from light cycles that were 12 h out of phase, a bimodal rhythm was observed with one peak in the males' late subjective day and a second peak of equal amplitude in the late subjective day of females. The results indicated that circadian systems in both males and females contribute to the circadian rhythm in copulation. Bilateral section of the optic tracts (OTX) of both males and females abolished the rhythm, but the rhythm persisted when OTX females were paired with intact males or when OTX males were paired with intact females. Furthermore, when OTX males or OTX females were paired with intact animals that were 12 h out of phase, a bimodal rhythm was still observed. These results suggested that the circadian pacemaker in the optic lobes of both male and female cockroaches participates in the control of mating, but that a pacemaker outside the optic lobes is also likely involved. Finally, it was shown that the female's olfactory response (measured by electroantennogram) to components of the male sex pheromone exhibited a circadian rhythm, but the data suggested the peripheral olfactory rhythm is not likely to be involved in the rhythm of mating behavior.