Examination of the role of FMRFamide-related peptides in the circadian clock of the cockroach Leucophaea maderae

The accessory medulla, the circadian clock of the cockroach Leucophaea maderae, is abundant in neuropeptides. Among these neuropeptides are the FMRFamide-related peptides (FaRPs), which generally share the C-terminal RFamide. As a first step toward understanding the functional role of FaRPs in the circadian clock of the cockroach, immunocytochemistry with antisera against various FaRPs, MALDI-TOF mass spectrometry, and injections of two FaRPs combined with running-wheel assays were performed. Prominent FMRFamide-like immunoreactivity was found in maximally four soma clusters associated with the accessory medulla and in most neuropils of the protocerebrum. By MALDI-TOF mass spectrometry, various extended FMRFamides of the cockroach L. maderae were partially identified in thoracic perisympathetic organs, structures known to accumulate extended FMRFamides in insects. By mass match, several of these peptides were also detected in the accessory medulla. Injections of FMRFamide and Pea-FMRFa-7 (DRSDNFIRF-NH₂) into the vicinity of the accessory medulla caused time-dependent phase-shifts of locomotor activity rhythms at circadian times 8, 18, and 4. Thus, our data suggest a role for the different FaRPs in the control of circadian locomotor activity rhythms in L. maderae.     

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.     

Pigment-dispersing hormone (PDH)-immunoreactive neurons form a direct coupling pathway between the bilaterally symmetric circadian pacemakers of the cockroach Leucophaea maderae

Circadian locomotor activity rhythms of the cockroach Leucophaea maderae are driven by two bilaterally paired and mutually coupled pacemakers that reside in the optic lobes of the brain. Transplantation studies have shown that this circadian pacemaker is located in the accessory medulla (AMe), a small neuropil of the medulla of the optic lobe. The AMe is densely innervated by about 12 anterior pigment-dispersing-hormone-immunoreactive (PDH-ir) medulla (PDHMe) neurons. PDH-ir neurons are circadian pacemaker candidates in the fruitfly and cockroach. A subpopulation of these neurons also appears to connect both optic lobes and may constitute at least one of the circadian coupling pathways. To determine whether PDHMe neurons directly connect both accessory medullae, we injected rhodamine-labeled dextran as neuronal tracer into one AMe and performed PDH immunocytochemistry. Double-labeled fibers in the anterior, shell, and internodular neuropil of the AMe contralaterally to the injection site showed that PDH-ir fibers directly connect both accessory medullae. This connection is formed by three anterior PDHMe neurons of each optic lobe, which, thus, fulfill morphological criteria for a direct circadian coupling pathway. Our double-label studies also showed that all except one of the midbrain projection areas of anterior PDHMe neurons were innervated ipsilaterally and contralaterally. Thus, anterior PDHMe neurons seem to play multiple roles in generating circadian rhythms. They also deliver timing information output and perform mutual pacemaker coupling in L. maderae. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants STE 531/7-1, 2, 3, and Human Science Frontier     

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.     

The polypeptide structure of Vitellogenin and Vitellin from the cockroach,Leucophaea maderae

Vitellogenin (Vg) synthesized by the fat body of Leucophaea maderaeis made up of four polypeptides with molecular weights of 160,000, 105,000, 98,000, and 57,000. Other polypeptides previously reported as part of Vg are associated with other proteins. Vitellin (Vt), the yolk protein (YP) isolated from mature oocytes and from newly formed oothecae, is a protein with a sedimentation coefficient of 28s and consists of three polypeptides with molecular weights of 105,000, 85,000, and 57,000. During vitellogenesis, the YP of developing oocytes contains both Vt and a 14s component. The 14s component is made up of four polypeptides with molecular weights of 105,000, 90,000, 85,000, and 57,000. The data suggest that 14s may not be a discrete protein but rather a form in transition between Vg and Vt in which the 98,000 dalton polypeptide is converted to the 85,000 dalton polypeptide of Vt through a 90,000 dalton intermediate. The 160,000 dalton peptide of Vg does not appear to be a part of Vt. Under alkaline conditions, both the 14s component and Vt are reduced to a polypeptide with a lower sedimentation rate in sucrose gradients. When acid conditions are restored, a protein resembling 14s is obtained. This suggests that the YP is a loosely held aggregate of similar or identical proteins with a molecular weight of about 250,000.     

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.     

Ultrastructure of pigment-dispersing hormone-immunoreactive neurons in a three-dimensional model of the accessory medulla of the cockroach<Emphasis Type="Italic"> Leucophaea maderae</Emphasis>

Locomotor activity rhythms of the cockroach Leucophaea maderae are orchestrated by two bilaterally symmetric, mutually coupled, circadian pacemakers. They lie in the optic lobes of the brain and are confined to the accessory medulla (AMe), ventro-medially to the medulla. The AMe is innervated by approximately 12 pigment-dispersing hormone (PDH)-immunoreactive anterior medulla neurons (PDHMe), which are circadian pacemaker candidates in the fruitfly and the cockroach. We have developed a three-dimensional computer model of the AMe and associated structures as a framework for neuroanatomical studies. Our greatly improved understanding of this structure in space has allowed us further to subdivide the anterior PDHMe into three subgroups, i.e., large, medium-sized, and small anterior PDHMe. The synaptic connections of two of these subgroups have been examined within subcompartments of the AMe by light and electron microscopy. The large, intensely staining, anterior PDHMe contain medium-sized dense-core vesicles and form input and output synapses with profiles densely filled with clear vesicles primarily in the anterior and shell neuropil of the AMe. The medium-sized anterior PDHMe contain large dense-core vesicles and constitute input and output synapses either with profiles being densely filled with clear vesicles, or with profiles containing granular dense-core vesicles. The small, weakly staining anterior PDHMe belong to a morphological group different from the large and medium-sized PDHMe and cannot be further identified at the electron-microscopic level because of their weak PDH immunoreactivity.This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants STE 531/7-1, 2, 3, and Human Science Frontier     

The oviduct musculature of the cockroach Leucophaea maderae and its response to various neurotransmitters and hormones

The musculature of the oviduct consists of an outer, irregular layer of longitudinal muscle and an inner layer of circular muscle. The four basic modes of activity—compression, segmentation, peristalsis, and reverse peristalsis—were evident in the isolated oviduct. These spontaneous events often occurred in an organized sequence. In fact eggs could be transported down the lateral oviducts by this myogenic activity once the sphincter between the common oviduct and vagina was severed. Myographic recordings were made of only the contractions of the longitudinal muscles. L-glutamate caused a distinct phasic contraction at 2.2 × 10^?5 M. The response became larger and more complex as the concentration of the amino acid was increased. Acetylcholine (1.6 × 10^?5 M) caused either a phasic or tonic response, or a combination thereof. By contrast, 5HT and tyramine simply increased the frequency of small phasic contractions, although in some preparations both monoamines caused an inhibition. The ecdysones, a juvenile hormone analogue (1 × 10^?6 M), and prostaglandin E₂ had no effect on oviduct activity. Initially high KCI solutions (162 mM) without Ca⁺⁺ induced a strong contraction but subsequent additions failed to do so. However, when a high KCI solution (158 mM) with 2 mM Ca⁺⁺ was added to the preparation the response was partially restored. Also the potent calcium antagonist Mn⁺⁺ (2mM) can suppress spontaneous activity.     

Structural and functional classification of antennal sensilla of the cockroach,Leucophaea maderae

Summary Antennal sensilla of Leucophaea maderae were investigated electrophysiologically, labeled and then examined with the scanning or transmission electron microscope. The sensilla can be classified into morphological types according to their external shape and the structure of their hair wall. Sensilla showing similar reaction spectra of their cells can be cathegorized into physiological groups. The morphological classification corresponds to the physiological grouping: one morphological type of sensillum comprises one or several groups of physiologically similar sensilla. In many of these groups constant combinations of physiologically different cells occur. The possible functional significance of the relationships found between the structural features of the sensilla and the physiological properties of their sensory cells is discussed.Supported by the Deutsche Forschungsgemeinschaft (Scha 292/1)     

Localization of leucomyosuppressin in the brain and circadian clock of the cockroach <Emphasis Type="Italic">Leucophaea maderae</Emphasis>

The myosuppressins (X1DVX2HX3FLRFamide), which reduce the frequency of insect muscle contractions, constitute a subgroup of the FMRFamide-related peptides. In the cockroach Leucophaea maderae, we have examined whether leucomyosuppressin (pQDVDHVFLRFamide) is present in the accessory medulla, viz., the circadian clock, which governs circadian locomotor activity rhythms. Antisera that specifically recognize leucomyosuppressin stain one to three neurons near the accessory medulla. MALDI-TOF mass spectrometry has confirmed the presence of leucomyosuppressin in the isolated accessory medulla. Injections of 1.15 pmol leucomyosuppressin into the vicinity of the accessory medulla at various circadian times have revealed no statistically significant effects on the phase of circadian locomotor activity rhythms. This is consistent with the morphology of the myosuppressin-immunoreactive neurons, which restrict their arborizations to the circadian clock and other optic lobe neuropils. Thus, leucomyosuppressin might play a role in the circadian system other than in the control of locomotor activity rhythms.     

Social Interactions and the Circadian Rhythm in Locomotor Activity in the Cockroach Leucophaea maderae

The role of social interactions in entrainment has not been extensively studied in the invertebrates. Leucophaea maderae is a gregarious species of cockroach that exhibits extensive social interactions. Social interactions associated with copulation between the sexes have been shown to be regulated by the circadian system. We show here that social interactions between males are also under circadian control. We examined the question of whether or not these rhythmic social contacts could function as zeitgebers capable of regulating circadian phase and period. Animals initially in phase that were housed as groups or pairs of single sex or mixed sex in constant darkness for 2–7 weeks were found to drift out of phase. Their behavior was not significantly different from individual animals maintained in isolation. Further, animals that were initially out of phase by 12 h housed as groups or pairs were not significantly different in phase from animals that were isolated. The results show the circadian clocks of cockroaches are remarkably insensitive to the extensive social interactions that occur between individuals.     

Evidence for two vitellogenin-related genes in Leucophaea maderae: the protein primary structure and its processing

We previously reported a cDNA for vitellogenin (Vg) from the cockroach, Leucophaea maderae (Lm). In the present study, we identified another cDNA encoding a second Vg (Vg2) having stretches of amino acid sequences different from the first one, Vg1, reported earlier. The complete nucleotide sequence of Vg2 consisted of 5,915 bp, which encoded a primary protein of 1,911 residues including a 16-residue putative signal peptide. The regions different in both Vg precursors (Pro-Vg1 and pro-Vg2) were four in number, and two, relatively longer, existed at the carboxy terminal. The presence of two Vg-related cDNAs was confirmed by sequencing of RT-PCR products generated using primers designed based on the common sequences flanking the regions different in amino acid sequences. Both forms were transcribed since they could be amplified on mRNA from fat bodies of different individual females. Southern blot analysis of digested genomic DNA revealed the existence of two Vg-related genes in L. maderae indicating that each Vg cDNA originated from a separate gene. Also, the immunoblot analysis using antibodies generated against peptides unique to both Vg1 and Vg2 probed the same antigen in the same individual, suggesting LmVg to be a product coded by two different Vg precursors. Both Vg primary products showed 96% similarity at an amino acid level. Compared to other insect Vgs, Vg2 showed a slightly higher (1-2%) similarity than Vg1. We previously reported, based on amino-terminal sequence analysis, that L. maderae pro-Vg was cleaved into four subunit polypeptides (112-, 100-, 92-, and 55-kD), which were deposited in the egg as four respective vitellin (Vn) polypeptides. We show now based on immunoblot analysis that the 112-kD polypeptide is further cleaved, near the C-terminus, to an 87-kD polypeptide before it is secreted into the hemolymph. Both the L. maderae Vgs were compared with each other and with other insect Vgs and the processing pattern is discussed.     

Innervation of the foregut of the cockroach Leucophaea maderae and inhibition of spontaneous contractile activity by callatostatin neuropeptides

Abstract. The innervation of the gut of the cockroach Leucophaea madera (F.) has been studied by means of wholemount immunocytochemistry with antisera raised against Leu-callatostatin, a cockroach allatostatin homologue identified from neuropeptide isolation and gene studies in the blowfly Calliphora vomitoria. Leu-callatostatin-imunoreactive neurones in the brain, with axon trajectories in the stomatogastric nervous system, innervate the foregut and midgut. Neurones in the last abdominal ganglion supply the hindgut and the midgut via the proctodeal nerve. In addition to a rich callatostatin-immunoreactive nerve supply, the midgut, including the midgut caeca, contain numerous callatostatin-immunoreactive endocrine cells. Physiological studies show that the spontaneous contractile activities of the foregut, but not the hindgut, are inhibited by callatostatin neuropeptides. Leu-callatostatin 3 was the most potent of the range of Leu-and Met-callatostatins tested, with a dose-dependent response between 10^-13 and 10^-7 M. This is similar to the results obtained with the previously identified myoinhibitory peptide of L. maderae , leucomyosuppressin. However, this peptide, with a different type of structure to the allatostatins, inhibits both foregut and hindgut motility equally. Experiments with a series of analogues of the Met-callatostatins showed that the free acid (as opposed to the carboxyamidated peptide) and N-terminally truncated peptides were inactive. These morphological and physiological results are thought to be representative of the, as yet unidentified, naturally occurring allatostatin homologues of L. maderae. This family of peptides should be added to the increasing list of insect gut myoinhibitory substances.     

Pigment-dispersing hormone-immunoreactive neurons in the cockroach Leucophaea maderae share properties with circadian pacemaker neurons

Neurons immunoreactive with antisera against the crustacean peptide -pigment dispersing hormone fullfill several anatomical criteria proposed for circadian pacemakers in the brain of the cockroach Leucophaea maderae. These include position of somata, projections to the lamina and midbrain and possible coupling pathways between the two pacemakers through commissural fibers. In behavioral experiments combined with lesion studies and immunocytochemical investigations we examined whether the presence of pigment-dispersing hormone-immunoreactive arborizations in the midbrain of the cockroach correlates with the presence of circadian locomotor activity. No rhythm was detected after severing both optic stalks in any animal for at least 12 days. Within the same time pigment-dispersing hormone-immunoreactive fibers in the midbrain disappeared. Two to seven weeks after the operation some of the cockroaches regained circadian locomotor activity, while others remained arrhythmic. In all cockroaches which regained rhythmic behavior pigment-dispersing hormone-immunoreactive fibers had regenerated and had largely found their original targets within the brain. In all arrhythmic cockroaches either none or very little regeneration had occurred. The period of the regained circadian activity inversely correlated with the number of regenerated immunoreactive commissural fibers. These data provide further evidence for the involvement of pigment-dispersing hormone-immunoreactive neurons in circadian clocks of orthopteroid insects.Abbreviations DD constant darkness - PDH pigment-dispersing hormone - PDHLI pigmentdispersing hormone-like immunoreactivity - PDFL a pigment-dispersing factor containing cells in the lamina - PDFMe pigment-dispersing factor containing cells in the medulla - QV quantification value     

The cockroach Leucophaea maderae needs more than juvenile hormone, vitellogenin and reserves to make a yolky egg

For the cockroach Leucophaea maderae the developmental profile of lipophorin (Lp) concentrations in the hemolymph was determined through the entire vitellogenic period. At mid-vitellogenesis the concentrations of Lp had risen to 6 times the level at emergence and then declined to 2/3 of such high values at ovulation. The racemic 10R,10S-JH-III bound to Lp with an affinity of K(d) = 5.76 nM and the natural enantiomer 10R-JH-III with a K(d) = 1.60 nM. Injections of anti-Lp into mated females caused a significantly reduced rate of oocyte growth and a substantial degree of oosorption. Injections of gamma-globulin did not significantly reduce oocyte growth and caused only a small number of oocytes to resorb. Starvation after mating had similar effects as treatment with anti-Lp. Because of the high affinity of JH to Lp and since Lp occurs in micromolar concentrations during vitellogenesis one can assume that practically all JH is bound and not available for hydrolysis by the JH esterases. Lp appears to function as an inhibitor of JH metabolism by the JHEs through substrate depletion. One may conclude that a normal rate of egg growth is only achieved when titers of Lp exceed those of JH and remove major portions of this substrate from degradation by the JHEs.     

Mechanisms of hormonal regulation of the peripheral circadian clock in the colon

Colonic function is controlled by an endogenous clock that allows the colon to optimize its function on the daytime basis. For the first time, this study provided evidence that the clock is synchronized by rhythmic hormonal signals. In rat colon, adrenalectomy decreased and repeated applications of dexamethasone selectively rescued circadian rhythm in the expression of the clock gene Per1. Dexamethasone entrained the colonic clock in explants from mPer2^Luc mice in vitro. In contrast, pinealectomy had no effect on the rat colonic clock, and repeated melatonin injections were not able to rescue the clock in animals maintained in constant light. Additionally, melatonin did not entrain the clock in colonic explants from mPer2^Luc mice in vitro. However, melatonin affected rhythmic regulation of Nr1d1 gene expression in vivo. The findings provide novel insight into possible beneficial effects of glucocorticoids in the treatment of digestive tract-related diseases, greatly exceeding their anti-inflammatory action.     

A circadian rhythm in neural activity can be recorded from the central nervous system of the cockroach

Summary Evidence presented in this paper indicates that a robust circadian rhythm in the frequency of neural activity can be recorded from the central nervous system of intact cockroaches, Leucophaea maderae. This rhythmicity was abolished by optic lobe removal. Spontaneous neural activity was then used as an assay to demonstrate that the optic lobe is able to generate circadian oscillations in vitro. These results provide direct evidence that the cockroach optic lobe is a self-sustained circadian oscillator capable of generating daily rhythms in the absence of neural or hormonal communications with the rest of the organism.Abbreviations CNS central nervous system - DD constant dark - LD light/dark cycle - SCN suprachiasmatic nucleus - ZT Zeitgeber time     

Organization of the Circadian System in Insects

The circadian systems of different insect groups are summarized and compared. Emphasis is placed on the anatomical identification and characterization of circadian pacemakers, as well as on their entrainment, coupling, and output pathways. Cockroaches, crickets, beetles, and flies possess bilaterally organized pacemakers in the optic lobes that appear to be located in the accessory medulla, a small neuropil between the medulla and the lobula. Neurons that are immunoreactive for the peptide pigment-dispersing hormone (PDH) arborize in the accessory medulla and appear to be important components of the optic lobe pacemakers. The neuronal architecture of the accessory medulla with associated PDH-immunoreactive neurons is best characterized in cockroaches, while the molecular machinery of rhythm generation is best understood in fruit flies. One essential component of the circadian clock is the period protein (PER), which colocalizes with PDH in about half of the fruit fly's presumptive pacemaker neurons. PER is also found in the presumptive pacemaker neurons of beetles and moths, but appears to have different functions in these insects. In moths, the pacemakers are situated in the central brain and are closely associated with neuroendocrine functions. In the other insects, neurons associated with neuroendocrine functions also appear to be closely coupled to the optic lobe pacemakers. Some crickets and flies seem to possess central brain pacemakers in addition to their optic lobe pacemakers. With respect to neuronal organization, the circadian systems of insects show striking similarities to the vertebrate circadian system. (Chronobiology International, 15(6), 567-594, 1998)     

Light affects the branching pattern of peptidergic circadian pacemaker neurons in the brain of the cockroach Leucophaea maderae

Pigment-dispersing factor-immunoreactive neurons anterior to the accessory medulla (aPDFMes) in the optic lobes of insects are circadian pacemaker neurons in cockroaches and fruit flies. The authors examined whether any of the aPDFMes of the cockroach Leucophaea maderae are sensitive to changes in period and photoperiod of light/dark (LD) cycles as a prerequisite to adapt to changes in external rhythms. Cockroaches were raised in LD cycles of 11:11, 13:13, 12:12, 6:18, or 18:6 h, and the brains of the adults were examined with immunocytochemistry employing antisera against PDF and orcokinin. Indeed, in 11:11 LD cycles, only the number of medium-sized aPDFMes specifically decreased, while it increased in 13:13. In addition, 18:6 LD cycles increased the number of large- and medium-sized aPDFMes, as well as the posterior pPDFMes, while 6:18 LD cycles only decreased the number of medium-sized aPDFMes. Furthermore, PDF-immunoreactive fibers in the anterior optic commissure and orcokinin-immunoreactive fibers in both the anterior and posterior optic commissures were affected by different lengths of light cycles. Thus, apparently different groups of the PDFMes, most of all the medium-sized aPDFMes, which colocalize orcokinin, respond to changes in period and photoperiod and could possibly allow for the adjustment to different photoperiods.