Multiple neuropeptides produced by sex-specific neurons control activity of the male accessory glands and gonoducts in the silkworm Bombyx mori.

The male accessory glands (AG) and gonoducts of moths develop during metamorphosis and are essential for successful fertilization of females. We found that these reproductive organs are innervated by a sex-specific cluster of peptidergic neurons in the posterior 9th neuromere of the terminal abdominal ganglion (TAG). This cluster of ~20 neurons differentiate during metamorphosis to innervate the accessory glands and sperm ducts. Using immunohistochemistry and in situ hybridization (ISH) we showed that these neurons express four neuropeptide precursors encoding calcitonin-like diuretic hormone (CT-DH), allatotropin (AT) and AT-like peptides (ATLI-III), allatostatin C (AST-C), and myoinhibitory peptides (MIPs). We used contraction bioassay in vitro to determine roles of these neuropeptides in the gonoduct and accessory gland activity. Spontaneous contractions of the seminal vesicle and AG were stimulated in a dose depended manner by CT-DH and AT, whereas AST-C and MIP elicited dose dependent inhibition. Using quantitative RT-PCR we confirmed expression of receptors for these neuropeptides in organs innervated by the male specific cluster of neurons. Our results suggest a role of these neuropeptides in regulation of seminal fluid movements during copulation.

Risk factors for failure of continuous positive airway pressure treatment in patients with ostructive sleep apnoea.

BACKGROUND AND AIMS: Obstructive sleep apnoea is a potentially serious sleep disorder associated with the risk of cardiovascular disease. It is treated with continuous airway pressure (CPAP) but this is not always successful. Unsuccessful cases should be treated by bilevel positive airway pressure (BiPAP). The aim of this study was to determine whether common respiratory parameters and/or body mass index (BMI) can be used to predict the probability CPAP failure and hence start such patients on BiPAP from the outset. METHODS: A sample of patients treated by CPAP for OSAS was evaluated a retrospective cohort study. The data measured in sleep monitoring of the successfully treated group and of the group where CPAP had failed were compared. Subsequently, the predictive abilities of BMI, Apnoea Index (AI), Apnoea-Hypopnea Index (AHI), percentage of sleep time in less than 90% oxygen saturation (T90), average oxygen saturation over the duration of sleep (SaO2) and average desaturation per hour of sleep (ODI) were assessed with respect to CPAP failure, both individually and in combination. RESULTS: A sample of 479 patients was included in the study. All of the recorded variables except AI were significantly associated with failure of CPAP and their ability to predict the failure ranged from poor to moderate. Since there was significant correlation among all the variables measured a two-variable prediction model combining T90 and BMI produced no significant improvement in the quality of CPAP failure prediction. CONCLUSIONS: BMI was a significant predictor of CPAP failure although it was slightly less predictive than T90. The set of monitored variables included in our study does not allow for CPAP failure to be predicted with clinically relevant reliability.

Hypermetabolic Conversion of Plant Oil into Water: Endothermic Biochemical Process Stimulated by Juvenile Hormone in the European Firebug, Pyrrhocoris apterus L.

The physiological and biochemical mechanisms that enable insects to feed on dry food to secure enough water for larval growth were investigated. The study was carried out with a plethora of physiological methods, ranging from the simple volumetric determination of O2 consumption and water intake to more advanced methods such as scanning microrespirography and thermovision imaging of insect's body temperature. The experiments were done on the European firebug, Pyrrhocoris apterus, which feeds exclusively on dry linden seeds. In order to survive, it needs to drink water or suck a sap from plants occasionally. It was found that the young larval instars compensate the occasional water deficiency by the increased production of metabolic water. The juvenile hormone (JH)-dependent production of metabolic water, which was previously found in other species consuming dry food, was achieved in P. apterus by total metabolic combustion of the dietary lipid (neutral seed oil). The water-producing, hypermetabolic larvae were heated from inside by endothermic energy released from the uncoupling of oxidation from oxidative phosphorylation. The "warm", hypermetabolic larvae burning the dietary oil into CO2 and water showed the increased rates of respiratory metabolism. Microrespirographic recording of these larvae revealed the ratio of the respiratory quotient (RQ, CO2/O2) of 0.7, which indicated the breakdown of a pure triglyceride. The warm hypermetabolic larvae could be easily spotted and distinguished from the "cold" larvae on the screen of a thermovision camera. The last instar larvae lacking the JH were always only cold. They metabolized a carbohydrate substrate exclusively (RQ = 1.0), while the dietary lipid was stored in the fat body. In comparison with the hypermetabolic larvae of some other species fed on dry food, which exhibited the highest rates of O2 consumption ever recorded in a living organism (10-20 mL O2/g per hour), the metabolic difference between the warm and cold larvae of P. apterus was only some 30% (not a reported 10-fold difference), which was presumably due to their ability to drink. We conclude that a very important, though still largely neglected, epigenetic biochemical role of insect JH depends on switchover between the utilization of dietary lipid (+JH; production of metabolic water) and carbohydrate (-JH; lipid storage in the fat body). The hypermetabolic water supply in insects fed on dry food, which is associated with enormous rates of O2 consumption, liberates endothermic energy that heats the body and potentially influences the insect thermoregulation. A possibility that the JH-dependent lipolytic hormone stimulates the total metabolic breakdown of nutritional lipids may be absolutely different from the currently known adipokinetic peptides that have been emphasized.

Short-term postoperative distress associated with open vs. transoral robotic surgery (TORS) in patients with T1-T2 carcinomas of the tongue base and supraglottis.

BACKGROUND: This study compared the adverse effects of open surgery (OS) including lateral pharyngotomy and supraglottic laryngectomy vs. transoral robotic surgery (TORS) in the treatment of stage T1 and T2 carcinomas of the tongue base and supraglottis. METHODS: A retrospective study involving a 49 (13 female and 36 male) patients with untreated T1 or T2 carcinomas. Twenty two were operated on using TORS and 27 underwent conventional OS. The indicators for comparison were: total blood loss during surgery, post-operative pain measured with the Visual Analog Scale (VAS); global, emotional and physical post-operational states assessed with the standardized M.D. Anderson Dysphagia Inventory (MDADI) and psychosocial distress (PD) questionnaire. Apart from blood loss, subjective symptoms were evaluated 1 and 6 weeks and 6 months after surgery. The differences in indicators between groups were analyzed using Fisher's Least Significant Difference (LSD) test at the 5% significance level. RESULTS: Mean general OS and TORS associated blood loss were 405 and 29 ml, respectively. The mean MDADI score in TORS vs. OS patients one week, six weeks and six months postoperatively was 60.01 vs. 44.93, 91.01 vs. 62.19 and 94.18 vs. 93.56. The mean VAS score in the TORS vs. OS group at the same time intervals were 5.09 vs. 5.56, 2.09 vs. 3.11 and 1.27 vs. 1.33. All differences between TORS and OS were statistically significant with the exception of 6 month values for particular scores. The mean PD score in TORS vs. OS patients in one week, six weeks and 6 months was 26.82 vs. 25.11, 39.95 vs. 29.22 and 44.73 vs. 44.52. Only the six week distinctions were significant. The both methods were comparable in terms of the risk of locoregional tumour recurrence. CONCLUSIONS: The study confirmed the assumption of the TORS as a minimally invasive procedure significantly reducing the intraoperative blood loss, pain, swallowing and psychosocial distress as late as 6 weeks postoperatively in patients with early staged carcinomas of the tongue base and supraglottis.

Comprehensive physiology and toxicology of ecdysogens--The metabolically activated porphyrin-ecdysteroid complexes in insects.

The polyhydroxylated derivatives of 6-keto,7-dehydrocholesterol (ecdysone, ecdysteroids, Ecd) are natural compounds widely distributed in plants. They exhibit strong anabolic, vitamin D-like, pharmacological effects in vertebrate animals and in the human body. In the larval stages of insects, injections of pure Ecd cause serious pathophysiological, "hyperecdysonic" syndromes associated with neuromuscular paralysis, premature cuticular apolysis and complete inhibition of ecdysis. Ecds do not penetrate insect cuticle. For this reason, all previous attempts to induce ecdysone responses by topical applications of Ecd failed. In this work, we tried to induce the topical effects of Ecd by preparation of more lipophilic complexes, with 2 or 4 molecules of 20-hydroxyecdysone (E20) attached to a relatively large nucleus of the porphyrin. The resulting porphyrin-E20 complexes (ecdysogens) have been subjected to standardised assays for ecdysone activity in the ligatured larvae ("dauerlarvae") of the greater waxmoth (Galleria mellonella). Similarly like the free E20 alone, porphyrin-E20 complexes had no effect when applied on the body surface or administered in the larval diet. When injected, however, they exhibited delayed effects, but the adverse ("hyperecdysonic") pathophysiological syndromes were reduced or abolished. It is concluded, therefore, that the replacement of pathophysiological, precocious or "hyperecdysonic" moults by the larval-pupal transformation, was due to successive metabolic liberation of the biologically active, free E20 from the porphyrin-E20 complex. The biological status of Ecd does not agree with their definition as the prothoracic gland (PG) hormone of insects, nor with the assumptions about a growth hormone of plants. A possibility that the most important status of Ecd may depend on the pharmacological properties of a sterolic D6 vitamin has been discussed.

Respiratory metabolism of salivary glands during the late larval and prepupal development of Drosophila melanogaster.

During the late larval period, the salivary glands (SG) of Drosophila show a cascade of cytological changes associated with exocytosis and the expectoration of the proteinaceous glue that is used to affix the pupariating larva to a substrate. After puparium formation (APF), SG undergo extensive cytoplasmic vacuolation due to endocytosis, vacuole consolidation and massive apocrine secretion. Here we investigated possible correlations between cytological changes, the puffing pattern in polytene chromosomes and respiratory metabolism of the SG. The carefully staged SG were explanted into small amounts (1 or 2µl) of tissue culture medium. The respiratory metabolism of single or up to 3 pairs of glands was evaluated by recording the rate of O2 consumption using a scanning microrespirographic technique sensitive to subnanoliter volumes of the respiratory O2 or CO2. The recordings were carried out at times between 8h before pupariation (BPF), until 16h APF, at which point the SG completely disintegrate. At the early wandering larval stage (8h BPF), the glands consume 2nl of O2/gland/min (=2500µl O2/g/h). This relatively high metabolic rate decreases down to 1.2-1.3nl of O2 during the endogenous peak in ecdysteroid concentration that culminates around pupariation. The metabolic decline coincides with the exocytosis of the proteinaceous glue. During and shortly after puparium formation, which is accompanied cytologically by intense vacuolation, O2 consumption in the SG temporarily increases to 1.6nl O2/gland/min. After this time, the metabolic rate of the SG decreases downward steadily until 16h APF, when the glands disintegrate and cease to consume oxygen. The SG we analyzed from Drosophila larvae were composed of 134 intrinsic cells, with the average volume of one lobe being 37nl. Therefore, a single SG cell of the wandering larva (with O2 consumption of 2nl/gland/min), consumes each about 16pl of O2/cell/min. A simultaneous analysis of the rate of protein and RNA synthesis in the SG shows a course similar to that found in respiratory metabolism.

Transitions in the heartbeat pattern during pupal diapause and adult development in the flesh fly, Sarcophaga crassipalpis.

The heartbeat of diapausing pupae of the flesh fly Sarcophaga crassipalpis was investigated using electrocardiographic methods including gravimetry, thermography, and optocardiography. During deep diapause, characterized by minimum metabolic activity, the heart exhibited discontinuous bouts of exclusively unidirectional, anterograde pulsations (40-60 contractions/min) that lasted only a few seconds. These bouts of cardiac pulsations were separated by periods of rest lasting 5-30 min. During infradian peaks of metabolic activity (4-day cycles) that occur throughout diapause, periods of rest were shortened and frequency of the anterograde heartbeat increased more than two-fold. Throughout diapause, the heart consistently exhibited a simple, bi-phasic pattern of pulsations generated by bouts of anterograde heartbeats interspersed with periods of cardiac rest. When the fly broke diapause and initiated pharate adult development, a new tri-phasic pattern was observed: the new pattern incorporated heartbeat reversal, as noted by the appearance of retrograde pulsations that directed hemolymph in a posterior direction. These retrograde heartbeats occurred exclusively in the abdominal portion of the dorsal vessel and were not observed in the head or thorax. The transition to pharate adult development was also accompanied by the appearance of extremely strong extracardiac pulsations that served a respiratory function. Although these pulsations made it more challenging to record heartbeat patterns in pharate adults, we observed progressive shortening of the resting periods and a continual increase in the rate of both anterograde and retrograde pulsations, a trend that was further magnified in the adult fly (anterograde contractions up to 300/min and retrograde contractions approximately 125/min). These results imply that the circulatory function of the heart is homeostatically regulated and is responsive to developmental changes and the diverse metabolic rate demands of larvae, diapausing pupae, pharate adults and adult flies.

A new look at the comparative physiology of insect and human hearts.

Recent electrocardiographic (ECG) studies of insect hearts revealed the presence of human-like, involuntary and purely myogenic hearts. Certain insects, like a small light-weight species of hoverfly (Episyrphus balteatus), have evolved a very efficient cardiac system comprised of a compact heart ventricle and a narrow tube of aorta, which evolved as an adaptation to sustained hovering flights. Application of thermocardiographic and optocardiographic ECG methods revealed that adult flies of this species use the compact muscular heart chamber (heart ventricle) for intensive pumping of insect "blood" (haemolymph) into the head and thorax which is ringed all over with indirect flight musculature. The recordings of these hearts revealed extremely high, record rates of forward-directed, anterograde heartbeat (up to 10Hz), associated with extremely enhanced synchronic (not peristaltic) propagation of systolic myocardial contractions (32.2mm/s at room temperature). The relatively slow, backward-directed or retrograde cardiac contractions occurred only sporadically in the form of individual or twinned pulses replacing occasionally the resting periods. The compact heart ventricle contained bi-directional lateral apertures, whose opening and closure diverted the intracardiac anterograde "blood" streams between the abdominal haemocoelic cavity and the aortan artery, respectively. The visceral organs of this flying machine (crop, midgut) exhibited myogenic, extracardiac peristaltic pulsations similar to heartbeat, including the periodically reversed forward and backward direction of the peristaltic waves. The tubular crop contracted with a periodicity of 1Hz, both forwards and backwards, with propagation of the peristaltic waves at 4.4mm/s. The air-inflated and blindly ended midgut contracted at 0.2Hz, with a 0.9mm/s propagation of the peristaltic contraction waves. The neurogenic system of extracardiac haemocoelic pulsations, widely engaged in the regulation of circulatory and respiratory functions in other insect species, has been replaced here by a more economic, myogenic pulsation of the visceral organs as a light-weight evolutionary adaptation to prolonged hovering flight. Striking structural, functional and even genetic similarities found between the hearts of Episyrphus, Drosophila and human hearts, have been practically utilised for inexpensive testing of new cardioactive or cardioinhibitory drugs on insect heart.

Myogenic nature of insect heartbeat and intestinal peristalsis, revealed by neuromuscular paralysis caused by the sting of a braconid wasp.

Larvae of the greater waxmoth (Galleria mellonella) become paralysed by the venom of the braconid wasp (Habrobracon hebetor) a few minutes after intoxication. The profound neuromuscular paralysis, which may last for several weeks, includes all somatic muscles that are innervated through neuromuscular transmission. The peristaltic contractions of the heart and intestine, which are regulated by the depolarisation potentials of the myocardium or intestinal epithelial muscles, remain unaffected and fully functional. Heartbeat patterns and intestinal pulsations were monitored in the motionless, paralysed larvae by means of advanced electrocardiographic recording methods (contact thermography, pulse-light optocardiography). The records revealed more or less constant cardiac pulsations characterised by 20-25 systolic contractions per minute. The contractions were peristaltically propagated in the forward (anterograde) direction, with a more or less constant speed of 10mm per second (23-25°C). Additional electrocardiographic investigations on larvae immobilised by decapitation revealed the autonomic (brain independent) nature of heartbeat regulation. Sectioning performed in the middle of the heart (4th abdominal segment) seriously impaired the pacemaker rhythmicity and slowed down the rate of heartbeat in the anterior sections. By contrast, the functions of the posterior compartments of the disconnected heart remained unaffected. These results confirmed our previous conclusions about the existence of an autonomic, myogenic, pacemaker nodus in the terminal part of an insect heart. They show an analogy to the similar myogenic, sinoatrial or atrioventricular nodi regulating rhythmicity of the human heart. Peristaltic contractions of the intestine also represent a purely myogenic system, which is fully functional in larvae with complete neuromuscular paralysis. Unlike the constant anterograde direction of the heartbeat, intestinal peristaltic waves periodically reversed anterograde and retrograde directions. A possibility that the functional similarity between insect and human hearts may open new avenues in the field of comparative cardiology has been discussed.

Juvenile hormone-stimulated synthesis of acyl-glycerols and vitamin E in female accessory sexual glands of the fire bug, Pyrrhocoris apterus L.

Secretory cells of the female accessory sexual glands (AG) of P. apterus grow and produce yellow oily exocrine secretion in response to stimulation by endogenous juvenile hormone (JH) or exogenous treatments by JH analogues. The secretion determines the property of future egg shells by coating the chorion surface of the oocytes that are passing individually through the common uterus during oviposition. Diapausing females with a physiologically inhibited endocrine system or females with artificially removed hormonal sources show inactive ovaries and empty AG without the secretory products. Ovary-ectomised females with the intact neuroendocrine system develop hypertrophic AG loaded with the oily secretion. This shows that there is no direct dependence between formation of the oily secretion in AG and ovarian growth. Chemical analysis of the secretory products revealed the presence of acetylated glycerols, with the most abundant stearoyl-diacetyl-glycerol, stearoyl-acetyl-propionyl-glycerol, and the corresponding derivatives of arachidonic acid. In addition to this, the JH-activated secretory cells of AG also produced gamma- and delta-tocopherols. The possible antioxidant or antimutagenic action of these vitamin E compounds in insect reproduction has been emphasized.

Effect of corazonin and crustacean cardioactive peptide on heartbeat in the adult American cockroach (Periplaneta americana).

Changes in the frequency of cardiac pulsations have been monitored in the decapitated body of adult P. americana before and 5 h after the injections of [Arg(7)]-corazonin and CCAP, using newly invented touch-free, noninvasive optocardiographic methods. Relatively large dosages of these peptides (10(-6) M concentrations in the body) had no effect on the rate of the heartbeat beyond the Ringer control limits. It has been concluded, therefore, that Corazonin and CCAP, which are currently cited in the literature as "the most potent cardiostimulating peptides" in insects, have no effect on the physiological regulation of cardiac functions in the living body.

Heartbeat patterns during the postembryonic development of Drosophila melanogaster.

Pulsations of the dorsal vessel were recorded in vivo during the whole postembryonic development of D. melanogaster, by means of a newly invented, pulse-light opto-cardiographic method. The young larvae of the 1st and 2nd instars submerged in the feeding medium exhibited extremely high rates of heartbeat, 7Hz at room temperature. These values are among the highest rates of heartbeat ever recorded in the animal kingdom. The fully grown larvae of the 3rd instar showed approximately half of the maximum heartbeat rate (3.5-4Hz), which became stabilized after pupariation to 2.5-2.7Hz. The larval heartbeat was always uni-directional, in the forward-oriented or anterograde direction and it was almost continuous. The slowly disintegrating, old larval heart used to beat at the constant frequency of 2.5-2.7Hz until complete cessation of all cardiac functions in 1-day-old puparium. In spite of the persisting constant heartbeat frequency, the transformation process of the larval heart was associated with successively decreasing amplitude of the systolic contractions and with the prolongation of the resting periods. The newly formed heart of the pupal-adult structure exhibited a qualitatively new pattern of heartbeat activity, which was manifested by periodic reversal of the heartbeat with the faster anterograde and slower retrograde phases. The frequencies of both of these reciprocal cardiac pulsations gradually increased during the advanced pharate adult period, reaching the values of 4-5Hz at the time of adult eclosion. Adult males and females also exhibited a perfect pattern of heartbeat reversal, with still very high rates of the anterograde heartbeat, in the range of 5-6Hz. In addition to the cardiac functions, we have recorded several kinds of extracardiac pulsations, which often interfered severely with the recordings of the heartbeat. There were strong, irregular extracardiac pulsations of a neurogenic nature (somatic muscles, oral armature) and relatively slow extracardiac pulsations of a myogenic nature (intestinal peristaltics, 0.2-0.3Hz). The extracardiac and cardiac pulsations were independent, their functions were not correlated. A possibility of creating new challenges in combination of molecular biology with the functional physiology of the heart have been discussed.