Visceral muscles and myogenic activity in the hindgut of the cockroach,Leucophaea maderae

Summary The hindgut of the Madeira cockroach contains an intricate network of longitudinal and circular muscles that are distinctive for each region. In the rectum, the longitudinal muscles are symmetrically arranged in 6 distinct bands, while the circular muscles appear as a uniform layer over the rectal pads. In the colon, the muscle fibers are arranged in an irregular lattice with the longitudinal fibers generally superimposed on the circular ones but with an evident weaving between the layers. In addition to these muscle layers, a delicate, superficial network of muscle-like fibers covers many portions of the colon and rectum.In spite of the bewilderingly complex motile activity of deganglionated hindguts, all activity could be classified under 4 basic types after cinematographic analysis: segmentation, compression, peristalsis, or reverse peristalsis or a combination thereof. Although much of the activity that occurred was seemingly random, there was an evident rhythmicity that spontaneously arose and ended in several types of motility during the course of observations. The defined modes of activity seemed to be completely myogenic in nature, as all 4 categories were readily observed in hindguts 30 min after treatment with tetrodotoxin (10^–6 g/ml). Each region of the hindgut seemed to have its own particular rhythm.Action potentials were recorded both intracellularly and extracellularly from all regions of the hindgut; amplitude usually ranged between 10 and 20 mV for intracellular recordings, and such spike potentials were often preceded by a slow depolarizing pre-potential. Generally, however, the depolarization was abrupt. Transmembrane potentials from the visceral muscle fibers were never truly at rest. Slow, continuous fluctuations (3–8 mV) were common. At times, plateau-type action potentials were recorded, but generally the repolarization contour was almost linear with time. Contractions were evoked by action potentials but not by the slow, rhythmic fluctuations in the membrane potential.No particular region or structure in the hindgut showed an exclusive pacemaker function. However, there was an evident gradient of increased excitability progressing in an caudal direction from the ileum.In a sodium-free saline, the amplitude of action potentials was remarkable enhanced from 5 to 10 min after the initial change. Even after a 20 min exposure, action potentials were still often present although their frequency and amplitude dropped. Tetrodotoxin (10^–6g/ml) had no. pronounced effect on frequency or amplitude of action potentials. However, spike potentials ceased within 1.5 min after exposure to a sodium and calcium-free saline. When such preparations were re-exposed to a sodium-free saline containing normal calcium, the action potentials reappeared, suggesting that calcium might be a current-carrying ion. Although action potentials in a calcium-free medium showed variability, we generally saw a marked reduction in amplitude of potentials within 5 min. We further observed that 2 mM manganous ion completely abolished action potentials within 2 min. Thus, it seems likely that sodium is not the sole current-carrying ion in cockroach hindgut muscle.The authors express their indebtedness to Ms. Susan Swann, Mr. Gerald Holt, Mr. David Owens, and Ms. Mary Strand for their competent technical assistance.     

Immunocytochemical localization of leucomyosuppressin-like peptides in the CNS of the cockroach,Leucophaea maderae

Immunocytochemistry was used to determine sites of synthesis and pathways for the transport of the neuropeptide, Leucomyosuppressin (pQDVDHVFLRFamide) in the cockroach,Leucophaea maderae. This study led to identification of neurons in the brain and thoracic ganglia reactive to polyclonal antibodies raised against this peptide. No immunoreactive cells were found in the subsophageal or abdominal ganglia. Although the corpus cardiacum contained no intrinsic cells immunoreactive to LMS antibodies, the periphery of this organ and that of the nervi corporis allati contain an abundance of LMS-reactive terminals.     

Isolation and partial characterization of a second myotropic peptide from the hindgut of the cockroach,Leucophaea maderae

• 1.1. Proctolin and a second myotropic peptide were extracted from the hindgut of the cockroach Leucophaea maderae with methanol-water-acetic acid (90:9:1). The two peptides were easily separated by HPLC on a μ-Bondapak-phenyl column.
• 2.2. Like proctolin, the second peptide was heat stable and was inactivated by the exopeptidases aminopeptidase M and carboxypeptidase Y.
• 3.3. The response of the isolated hindgut to the new peptide was distinguishable from the response to proctolin by the following features: (a) a longer interval following application (1–4 min) to reach a maximum contraction, and (b) a much larger amplitude for single phasic contractions. Like proctolin, the new peptide could cause a protracted stimulation of the hindgut for more than 2 hr.

     

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.     

Localization of leucokinin VIII in the cockroach,Leucophaea maderae,using an antiserum directed against an achetakinin-I analog

An antiserum against an achetakinin analog selectively localized leucokinin VIII (LKVIII) in the CNS ofLeucophaea maderae. Preabsorption studies of the achetakinin antiserum with either preimmune serum or LKVIII prevented a positive reaction in both ELISA and immunocytochemical procedures. LKVIII immunoreactive neurons were found in the brain, frontal, and subesophageal ganglion, all 3 thoracic ganglia and the terminal ganglion. Nerves originating from the thoracic and terminal abdominal ganglia contain LKVIII material. Lateral and medial neurosecretory cells synthesizing LKVIII-like products contribute axons to the nervi corporis cardiaci that terminate in neurohemal sites in the corpora cardiaca and nervi corporis allati. Thus, leucokinin VIII, like leucokinin I (LKI) and leucomyosuppressin (LMS), appears to have both a neurohemal and neurotransmitter mode of regulating target cells inL. maderae.     

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)     

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.     

Comparative effects of leucomyosuppressin on the visceral muscle systems of the cockroach Leucophaea maderae.

1. Leucomyosuppressin (LMS) did not inhibit the spontaneous contractions of visceral muscles of the cockroach Leucophaea maderae uniformly as a group but rather showed a selective suppression of activity in the foregut and hindgut. The threshold of LMS inhibition for these organs was 10(-11) M for the foregut and 3 x 10(-11) M for the hindgut. The maximum response for each organ was generally recorded at 2.4 x 10(-8) M. 2. Both the heart and the oviduct were 100-1000 times less sensitive to LMS than either the foregut or the hindgut. Although the responses of the heart to LMS (10(-9) to 10(-8) M) were somewhat inconsistent, the myocardium showed a reduction in either the amplitude or frequency of contractions in 75% of the preparations tested. The oviduct showed the lowest level of responsiveness of all the muscles tested. Even at a concentration of 10(-7) M LMS, the amplitude and frequency of contractions showed no more than a 58% inhibition. 3. Desensitization to LMS was observed in three of the four muscle types tested. The phenomenon occurred in 37% of the foreguts, 34% of the hindguts and 54% of the heart preparations tested. The results of this study show that each visceral muscle type has its own unique response profile to LMS and support the idea that peptides may be multifunctional regulators.     

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.     

Studies on the cellular defense reactions of the Madeira cockroach, Leucophaea maderae: nodule formation in response to injected bacteria

Nodules were formed in the Madeira cockroach, Leucophaea maderae, in response to injections of low doses (3 x 10(4) bacteria/insect) of three strains of Bacillus cereus and Escherichia coli K12 D31. The most pathogenic strain of bacteria used, B. cereus B1, produced the greatest cellular response, while the least pathogenic, E. coli K12 D31, injected at the same dose, caused little nodule formation. Similarly, nodules were generally found to be larger following injection of pathogenic bacteria such as B. cereus B1 than to the weak pathogen, E. coli K12 D31. There was, however, no difference in the extent of nodule formation with the four bacterial strains/species if they were heat killed prior to injection. Histologically, the nodules formed in response to all bacterial species employed were similar, with a central necrotic core enclosing cell debris and occasional bacteria, and an outer, thin sheath of plasmatocyte-like hemocytes. Possible reasons for the enhanced cellular reactivity observed in L. maderae to pathogenic bacteria are discussed.     

Studies on the cellular defense reactions of the madeira cockroach, Leucophaea maderae: in vitro phagocytosis of different strains of Bacillus cereus and their effect on hemocyte viability

Monolayers of Leucophaea maderae hemocytes, consisting of mainly plasmatocytes and coagulocytes, were incubated with three strains of Bacillus cereus of differing pathogenicities, and the levels of phagocytosis and hemocyte viability were determined. Incubation with viable B. cereus strains NCTC 2599, NCIB 3329, and B1 resulted in a significant drop in hemocyte viability after 60 min of incubation compared with the saline-only controls. The greatest effect, however, resulted from incubation with B. cereus B1 which is the most pathogenic of the three strains studied. The killing effect of the three B. cereus strains was abolished following their UV irradiation. Incubation of monolayers with viable B. cereus B1 resulted in a level of phagocytic activity at all time periods lower than that with the other two strains. The highest levels of phagocytosis were achieved with UV-killed B. cereus, although no significant differences were found in these values between the three strains at any of the incubation times. Phospholipase C, a lytic enzyme shown to be released by all three strains of B. cereus, although in varying amounts, also caused hemocyte death following its incubation with the monolayers. These data suggest that the hemocyte killing and resistance to phagocytosis by B. cereus are caused by the release of phospholipase C, or some other related toxin(s) by the bacteria.     

Study on the extraction, purification and quantification of jasmonic acid, abscisic acid and indole-3-acetic acid in plants

Introduction  – Jasmonic acid (JA), abscisic acid (ABA) and indole‐3‐acetic acid (IAA) are important plant hormones. Plant hormones are difficult to analyse because they occur in small concentrations and other substances in the plant interfere with their detection. Objective  – To develop a new, inexpensive procedure for the rapid extraction and purification of IAA, ABA and JA from various plant species. Methodology  – Samples were prepared by extraction of plant tissues with methanol and ethyl acetate. Then the extracts were further purified and enriched with C₁₈ cartridges. The final extracts were derivatised with diazomethane and then measured by GC‐MS. The results of the new methodology were compared with those of the Creelman and Mullet procedure. Results  – Sequential elution of the assimilates from the C₁₈ cartridges revealed that IAA and ABA eluted in 40% methanol, while JA subsequently eluted in 60% methanol. The new plant hormone extraction and purification procedure produced results that were comparable to those obtained with the Creelman and Mullet's procedure. This new procedure requires only 0.5 g leaf samples to quantify these compounds with high reliability and can simultaneously determine the concentrations of the three plant hormones. Conclusion  – A simple, inexpensive method was developed for determining endogenous IAA, ABA and JA concentrations in plant tissue. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis,characterization, antifungal evaluation and 3D-QSAR study of phenylhydrazine substituted tetronic acid derivatives

A series of 3-(1-(2-(substituted phenyl)hydrazinyl)alkylidene)furan-2,4(3H,5H)-diones were designed and prepared using two synthetic routes. Their structures were confirmed by FT-IR, ¹H NMR, ¹³C NMR, MS, elemental analysis and single-crystal X-ray diffraction. Their bioactivity was evaluated against Botrytis cinerea in vitro. Most target compounds exhibited remarkable antifungal activity. Two compounds 7f and 7h were highly effective and their EC₅₀ values were 0.241 μg/mL and 0.167 μg/mL, respectively, close to that of the control drug procymidone. 3D-QSAR studies of CoMFA and CoMSIA were carried out. Models with good predictive ability were generated with the cross validated q² values for CoMFA and CoMSIA being 0.565 and 0.823. Conventional r² values were 0.983 and 0.945, respectively. The results provided a practical tool for guiding the design and synthesis of novel and more potent tetronic acid derivatives containing substituted phenylhydrazine moiety.     

Contribution of anaerobic protozoa and methanogens to hindgut metabolic activities of the American cockroach, Periplaneta americana.

The ciliate Nyctotherus ovalis occurs in high numbers in the hindgut of the American cockroach (Periplaneta americana) and harbors methanogenic bacteria as endosymbionts. The contribution of these hindgut microorganisms to metabolic and developmental processes of P. americana was studied by comparing cultures of cockroaches in which the composition of the hindgut microbial population was altered in various ways. Rearing the insects protozoan free resulted in increased insect generation time, decreased adult body weight, and absence of methane production. After feeding of protozoan-free adult cockroaches with a hindgut suspension containing N. ovalis and methanogens, methane increased to normal values and insect body weight was restored during the development of the second generation of insects. Feeding the protozoan-free cockroaches a hindgut suspension which was made free of N. ovalis resulted in an increase in methane production to only about 20% of the normal methane production level. This suggests that the methanogenic endosymbionts of N. ovalis are the major source of methane production in the hindgut. Inhibition of methanogens by addition of bromoethanesulfonic acid to the drinking water of a normal cockroach culture resulted in a reduction of methane production to about 2% of the normal level. No effects on insect body weight or the number of N. ovalis organisms were observed, but the fermentation pattern in the hindgut was shifted towards a relative increase in propionate levels. Similar results were obtained for in vitro cultures of hindgut microorganisms treated with bromoethanesulfonic acid. The results suggest a major role for hindgut protozoa in cockroach metabolic activities, especially during the insect growth period. The relatively large amounts of methane produced by cockroaches and by other methane-producing xylophagous insects suggest a major contribution by insects to global methane production.     

3-Hydroxy-3-phenylpropanoic acid is an intermediate in the biosynthesis of benzoic acid and salicylic acid but benzaldehyde is not

Stable-isotope-labelled (²H₆,¹⁸O) 3-hydroxy-3-phenylpropanoic acid, a putative intermediate in the biosynthesis of benzoic acid (BA) and salicylic acid (SA) from cinnamic acid, has been synthesized and administered to cucumber (Cucumis sativus L.) and Nicotiana attenuata (Torrey). Analysis of the products by gas chromatography-mass spectrometry revealed incorporation of labelling into BA and SA, but not into benzaldehyde. In a separate experiment, 3-hydroxy- 3-phenylpropanoic acid was found to be a metabolite of phenylalanine, itself the primary metabolic precursor of BA and SA. These data suggest that cinnamic acid chain shortening is probably achieved by β-oxidation, and that the proposed “non-oxidative” pathway of side-chain degradation does not function in the biosynthesis of BA and SA, in cucumber and N. attenuata. Received: 10 February 2000 / Accepted: 18 April 2000