Extracardiac versus cardiac haemocoelic pulsations in pupae of the mealworm (Tenebrio molitor L.)

Pulsations in mechanical pressure of the pupal haemocoele were investigated by means of simultaneous recording from multiple sensors. It has been determined that cardiac and extracardiac haemocoelic pulsations are each regulated by substantially different and quite independent physiological mechanisms. At the beginning and in the middle of the pupal interecdysial period the anterograde heartbeat and extracardiac pulsations occur in similar, but not identical periods. During the advanced pharate adult stage, there appear almost uninterrupted pulsations from different sources: cardiac, extracardiac, intestinal, and the ventral diaphragm.Extracardiac pulsations are associated with pressure peaks of 200-500 Pa, occurring at frequencies of 0.3-0.5 Hz. The effect of heartbeat on haemocoelic pressure is very small, 100- to 500-fold smaller, comprising only some 1 or 2 Pa during the vigorous anterograde systolic contractions. Accordingly, extracardiac pulsations are associated with relatively large abdominal movements from 30-90 μm whereas heartbeat produces movements of only 100-500 nm. This shows that extracardiac pulsations can be easily confused with the anterograde heartbeat. It does not seem realistic to assume that the relatively weak insect heart, and not the 100- to 500-fold more powerful extracardiac system of abdominal pump, could be at all responsible for selective accumulation of haemolymph in anterior parts of the body, for inflation of wings or enhancement of tracheal ventilation.It has been established that thermography from the pericardial region is not specific for the heartbeat. It records subepidermal movement of haemolymph resulting from the actions of both dorsal vessel and extracardiac pressure pulses as well. Shortly before adult eclosion the cardiac and extracardiac pulsations occasionally strike in concert, which profoundly increases the flow of haemolymph through pericardial and perineural sinuses. The relatively strong extracardiac pulsations cause passive movements of various visceral organs, tissue membranes, or tissue folds, giving thus a false impression of an authentic pulsation of tissues. In addition, extracardiac pulsations cause rhythmical movements of haemolymph between various organs, thus preventing haemolymph occlusion at the sites where the heart does not reach. It has been emphasized, finally, that the function of the autonomic nervous system (coelopulse), which integrates extracardiac pulsations, depends on homeostatic moderation of excessive or deficient conditions in insect respiration and haemolymph circulation.     

Tracheal compression in pupae of the beetle Zophobas morio

Insects that are small or exhibit low metabolic rates are considered to not require active ventilation to augment diffusive gas exchange. Some pupae with low metabolic rates exhibit abdominal pumping, a behaviour that is known to drive tracheal ventilation in the adults of many species. However, previous work on pupae suggests that abdominal pumping may serve a non-respiratory role. To study the role of abdominal pumping in pupa of the beetle Zophobas morio, we visualized tracheal dynamics with X-rays while simultaneously measuring haemolymph pressure, abdominal movement, and CO₂ emission. Pupae exhibited frequent tracheal compressions that were coincident with both abdominal pumping and pulsation of pressure in the haemolymph. However, more than 63% of abdominal pumping events occurred without any tracheal collapse and hence ventilation, suggesting that the major function of the abdominal pump is not respiratory. In addition, this study shows that the kinematics of abdominal pumping can be used to infer the status of the spiracles and internal behaviour of the tracheal system.     

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.     

Monitoring the course of bacterial infections by hemolymph pressure pulses in insects

Changes in rhythmic pulsations of hemolymph pressure have been continuously monitored from several hours before injection of the pathogenic bacteria until death of the infected Tenebrio pupae. Lethality induced by five different species of entomopathogenic bacteria was associated with specific changes in the hemolymph pressure pattern. During the initial stages of infection (incubation period) the pupae continued to produce regular and synchronized series of normal pulsations. The pathophysiological symptoms of the acute disease became manifested by successive desynchronisation of the frequency and simultaneous depression of the amplitude of the pulsations. This irreversibly proceeded, with species specific modifications, until death of the pupae when all peaks in hemolymph pressure disappeared. Duration of the incubation period was inversely proportional to the injected dose, while the later period characterized by development of the pathophysiological symptoms had more or less constant course in infection with each bacterial species. Certain toxic enzymes, protease and phospholipase C, had similar action on hemolymph pressure pulses. Application of the tensometric method to pathophysiological studies in insects has been discussed.     

Abdominal movements, heartbeats and gas exchange in pupae of the Colorado potato beetle, Leptinotarsa decemlineata

The rhythms of abdominal movements, heartbeats and gas exchange in the pupae of Leptiontarsa decemlineata (Say) were recorded simultaneously using an electrolytic respirometer and infrared gas analyser, both combined with contact thermography. Abdominal pulsations and heartbeat occurred periodically with little variance among individuals. The abdominal pulsations and heartbeat pauses varied individually within large limits, with the frequency of abdominal pulsations being six to seven times lower than that of the heart pulses. A proportion of the pupae (20%) showed discontinuous gas exchange with large, actively ventilated CO₂ bursts, whereas others (≈ 25%) exhibited continuous regular microcycles (flutter) with abrupt intake of air into the tracheae before discrete microbursts of carbon dioxide. The abdominal pulsations exerted only a minor influence on ventilation during the microcycles. More than 90% of the bursts of abdominal movement coincided with a series of forward directed heartbeats, but interspersed between the bouts of abdominal movement commonly two to three heartbeat pulses were observed that were not associated with abdominal movements. A period of abdominal movement associated with a heartbeat pulse was commonly initiated by one or two vigorous strokes of abdominal rotation.     

Active regulation of respiration and circulation in pupae of the codling moth (Cydia pomonella)

Regulation of autonomic physiological functions has been investigated by means of multisensor electronic methods, including electrocardiographic recording of heartbeat, strain-gauge recording of extracardiac hemocoelic pulsations (EHPs), anemometric recording of air passage through spiracles and respirographic recording of O(2) consumption and CO(2) output. Pupae of Cydia exhibit continuous respiration without remarkable bursts of CO(2). The dorsal vessel of these pupae exhibited regular heartbeat reversals characterized by shorter intervals of faster (forward oriented or anterograde) pulsations and longer intervals of slower (backward oriented or retrograde) peristaltic waves. The periodically repeated EHPs were present during the whole pupal interecdysial period. The internal physiological mechanisms regulating the cardiac (heartbeat) and extracardiac (EHP) pulsations were completely independent for most of the pupal instar. Simultaneous multisensor analysis revealed that the anterograde heartbeat of the dorsal vessel had similar but not identical frequency with EHPs. During advanced pharate adult development, frequency of cardiac and extracardiac pulsation periods profoundly increased until almost uninterrupted pulsation activity towards adult eclosion. At this time, the cardiac and extracardiac pulsations occasionally performed in concert, which enhanced considerably the efficacy of hemolymph circulation in pharate adults with high metabolic rates. The fastest hemolymph flow through the main body cavity was always associated with EHPs and with anterograde heartbeat. Simple physical diffusion of O(2) and CO(2) through spiracles (diffusion theory of insect respiration) does not play a significant role in pupal respiration. Instead, several kinds of regulated, mechanical ventilations of the tracheal system, including EHPs are responsible for effective tracheal ventilation.     

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.     

Eclosion behavior in tsetse (Diptera: Glossinidae): Extrication from the puparium and expansion of the adult

The tsetse adult extricates itself from the puparium and surrounding substrate by a series of muscular contractions that generate a Stereotypic pattern of changes in hemolymph pressure. The digging action of the fly can be distinguished from a second pattern of hemocoelic pulsations that is used to remove obstacles from its path. When the fly is restrained extrication behavior will persist for over 10 h. If the adult's legs are freed while the remainder of the body remains encased in the puparium, the fly fails to engage in extrication behavior, a result which suggests that freedom of the legs switches off extrication behavior and permits the onset of expansion of the body to its final adult size and shape. Expansion behavior includes walking, grooming, pumping air into the gut, and contracting the abdominal muscles to generate rhythmic pulses of hemocoelic pressure. A barographic record of internal pressure changes reflects the dynamics of this morphogenetic process. Results from tsetse are compared with previous observations recorded in flesh flies.     

Water balance in Collembola and its relation to habitat selection: Water content,haemolymph osmotic pressure and transpiration during an instar

In the two collembolan species Orchesella cincta and Tomocerus minor the water content, haemolymph osmotic pressure and transpiration rate fluctuate with the feeding rhythm during each instar. The changes in water content, however, are due to changes in dry weight, because the absolute water weight stays constant during the instar.The intake of food is probably the cause of the increase in haemolymph osmotic pressure. Increase of osmotically active substances in the blood and/or blood volume reduction may be responsible for the rise in osmotic pressure. This change in osmotic pressure in turn may affect the responsiveness of the animals to water as well as their feeding behaviour.Changes in the epicuticle and in epidermal cell membranes may cause changes in the rate of transpiration. The high rate observed during ecdysis and during the mid-instar may explain the behaviour of the animals in varied water conditions.Dehydration during the instar causes an equivalent rise in osmotic pressure for both Tomocerus minor and Orchesella cincta. The water loss appears to involve the haemolymph. The physiological state of the animal does not influence the rise in osmotic pressure. There are no signs of any osmoregulation in the two species.     

Water balance and osmoregulation in a free-ranging tenebrionid beetle, Onymacris unguicularis, of the Namib Desert

Onymacris unguicularis, a fog-basking tenebrionid beetle of the Namib Desert, has mean water influx rates of 49.9 mg H₂O/g.d and mean efflux rates of 41.3 mg H₂O/g.d with mean mass gain being 10.7 mg/g.d. If only steady-state conditions are considered (no mass change), and passive vapour input subtracted, drinking accounts for 50% of water input. Active beetles must drink in order to maintain water balance, while inactive beetles can maintain water balance either eating seeds or by simply metabolizing fat. Little change is observed in ratios of haemolymph and total body water to dry mass when fogs occur, while significant changes in haemolymph osmotic pressure are associated with fog occurrence.For short periods, O. unguicularis can tolerate dehydration with only slight changes in the ratio of total body water and haemolymph to dry body mass and to haemolymph osmotic pressure. For longer periods however, active beetles must have access to fog water for water balance maintenance. This is probably necessary for reproduction.     

Damping of Pressure Pulsations in Mobile Hydraulic Applications by the Use of Closed Cell Cellular Rubbers Integrated into a Vane Pump

The present study evaluates the potential of a bio-inspired pulsation damper in a vane pump used in mobile hydraulic applications.Pressure pulsations caused by such positive displacement pumps can lead to malfunctions and noise in a hydraulic system.A common measure to reduce pressure pulsations is the integration of pressure pulsation dampers downstream of the pump.This type of damping measure can also be found in biology as e.g.in the human blood circulatory system.Such working principles found in living organisms offer a high potential for a biomimetic transfer into technical applications.The newly developed bio-inspired damper consists of cellular rubbers with non-linear viscoelastic material properties.In order to evaluate the new damping method,pressure pulsations were measured at two different back pressures and at a wide engine speed range of the vane pump.For further assessment,different setups,varying the stiffness of the cellular rubber materials and the damper volume,were tested.Within the tested back pressures,the pressure pulsations could be reduced by up to 40％.The developed integrated pulsation damper offers a high potential to dampen pressure pulsations of positive displacement pumps used in mobile hydraulic applications operating below 10 bar.     

Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamorphosis

Phenylalanine and tyrosine concentrations were measured in the haemolymph, fat body, and abdominal integument of the American cockroach, Periplaneta americana, during the pre- and post-ecdysial periods of cuticle formation and sclerotization.Gas-liquid chromatography of trimethylsilyl derivatives of phenylalanine, tyrosine, and their metabolites provided a very sensitive and rapid method for determining those amino acids in small haemolymph and tissue samples.Haemolymph tyrosine increased in two stages: initially near apolysis and 16 to 25 hr pre-ecdysis, reaching its highest concentration at ecdysis (3·5 μg tyrosine/mg haemolymph). During that time, total haemolymph tyrosine increased by approximately 700 μg/insect. Fat body and abdominal integument began to accumulate tyrosine near apolysis. Fat body tyrosine peaked between ecdysis and 3·3 hr post-ecdysis whereas abdominal integument tyrosine peaked at ecdysis. Maximum concentrations were 6·0 μg and 4·1 μg tyrosine/mg wet wt. of tissue, respectively. Between ecdysis and 24 hr post-ecdysis, the period of maximum sclerotization, total tyrosine in haemolymph and fat body decreased by approximately 600 μg and 420 μg/insect, respectively. Phenylalanine concentrations did not change significantly in the haemolymph, fat body, or abdominal integument during the pre- and post-ecdysial periods.The cockroach apparently does not store free phenylalanine or tyrosine in the fat body during larval development as compared to tyrosine storage in some Diptera. The rapid increase of haemolymph, fat body, and integument tyrosine just prior to ecdysis suggests another form of storage for this important amino acid.     

The source and action of head factors regulating juvenile hormone esterase in larvae of the cabbage looper, Trichoplusia ni

Brain (median or lateral regions) or suboesophageal ganglion (SOG) homogenates of Day 1 fifth instar larvae of Trichoplusia ni induced the appearance of haemolymph juvenile hormone esterase (JHE) when injected into Day 1, Day 2 or early Day 4 fifth instar ligated hosts. Brain and SOG homogenates of late fourth instars also induced JHE when injected into Day 1 hosts, whole late fifth instar and pupal tissue did not. The pattern of JHE induction by early fourth through Day 3 fifth instar brain and SOG homogenates correlated with natural haemolymph JHE activity occurring at these times. Implantation of late fourth and Day 1 fifth instar brains and/or SOG into similar age hosts similarly induced JHE activity while prothoracic and abdominal ganglia did not. The relative levels of induction following implantation were SOG<brain<brain+SOG. JHE activity which appears in the haemolymph following injection of brain homogenates appears to be largely due to a single enzyme which has an isoelectric point indistinguishable from that of the natural haemolymph enzyme. Evidence is presented which suggests that inhibitory as well as stimulatory brain factors are involved in JHE regulation.     

Ionic and osmotic regulation of the haemolymph of the dragonfly, Libellula quadrimaculata (Odonata : Libellulidae)

Larvae of the widespread dragonfly, Libellula quadrimaculata, were adapted to a series of salt solutions, and the osmotic pressure, and sodium, potassium and chloride concentrations in the haemolymph measured. The regulation of potassium is extremely efficient over the range 0–50 m-mole/l. external concentration. Above this, larvae die. Sodium and chloride are regulated to a lesser extent, the larvae being able to withstand considerable changes in the concentration of these ions in the haemolymph. However, at higher external concentrations, the haemolymph concentration of these ions is maintained below that of the external medium. The osmotic pressure is regulated in parallel with sodium concentration over most of the range tested. However, in higher salinities, the osmotic pressure of the haemolymph does not fall below that of the external medium. This is seen as a strategy to limit the amount of drinking in saline media. Overall, the osmoregulatory system of L. quadrimaculata resembles that of brackish-water insects, rather than that of the more strictly freshwater dragonflies that have been studied.     

The effects of 20-hydroxyecdysone on haemolymph pressure pulsations in Tenebrio molitor

Changes in haemocoelic pressure have been studied after the injection of exogenous 20-hydroxyecdysone, using a special tensometric method. Application of the hormone before the endogenous peak of ecdysteroid causes an acceleration of the progressive changes in the pulsation pattern. When given during the endogenous ecdysteroid peak, 20-hydroxyecdysone produces a retention of the existing type of pulsation. Also, administration of the hormone after the endogenous peak induces a retardation in the developmental programme of the pulsations. Shortly before ecdysis, the exogenous hormone does not affect the pulsation programme or the ecdysis. These changes may represent an elegant example of a homeostatic function of ecdysteroids in insect development. Involvement of 20-hydroxyecdysone in regulation of the basic haemolymph pressure is discussed.     

Effects of hypoxia,hypercapnia, and pH on ventilation rate in Nauphoeta cinerea

Injecting 50 μl of a phosphate-buffered Ringer's solution with adjusted oxygen pressure (PO₂), Carbon dioxide pressure (PCO₂) or hydrogen ion concentration (pH) stimulates ventilation in the cockroach, Nauphoeta cinerea. When the pH of the buffered Ringer's is held at 7.08, the observed pH of our cockroach haemolymph, neither a reduced PO₂ nor an elevated PCO₂ produced responses that could account for the observed changes in ventilation in insects during exercise or when exposed to elevated ambient CO₂. Ventilation changes produced by altering the pH of the injected saline are comparable to those observed in active insects and in insects exposed to elevated ambient CO₂. We show that the effect of an elevated ambient CO₂ on ventilation can be accounted for by changes in haemolymph pH.     

Effect of elevated temperature on osmotic and ionic regulation in a salt-tolerant caddisfly from Death Valley,California

The effects of temperature (8–10 or 20°C) on regulation of haemolymph osmotic and ionic concentrations were investigated over a range of salinities (0–25‰) in fifth-instar larvae of the Death Valley caddisfly Limnephilus assimilis. At low temperatures, levels of chloride and sodium in the haemolymph are regulated over a wide range of salinities corresponding to the salinities at which larvae occur in nature and at which they can complete development into adults. In contrast, haemolymph osmolality is constant at low salinities (<14‰) but approaches conformity with the medium at higher salinities. High temperature reduces the larva's ability to maintain low chloride concentrations in its haemolymph and also leads to a reduction in haemolymph osmotic pressure; thus, at high temperatures ions account for more of the haemolymph osmotic concentration than at low temperatures. These data suggest that the absence of larvae from thermal pools and from all Death Valley waters in summer can be explained by the effects of high water temperatures on hydromineral regulation.     

Rhythmic pulses of haemolymph pressure associated with parturition and ovulation in the tsetse fly,Glossina morsitans

Abstract Expulsion of the tsetse larva from the uterus of the female is preceded by 1–2 h of rhythmic pulses of haemolymph pressure that can be detected using a barographic technique. At first baseline pressure is maintained and all pulses are positive in relation to baseline. Then, about 1 h before parturition, baseline pressure increases, pulse intensity increases, and the pulses become both positive and negative in relation to baseline. Each pulse correlates with ‘bobbing’ action of the female's proboscis, the only external indication of this internal activity. A single large pressure pulse is observed at parturition, and thereafter the pressure level returns to the original baseline and pulsing action ceases. Around the presumptive time of ovulation, 1–2 h after parturition, another series of pressure pulses is observed. The pulses are the likely consequence of coordinated waves of muscular contraction that are essential preparation for successful parturition and ovulation.     

Variations de la pression interne au cours de l'émergence et de l'expansion des ailes chez Bombyx mori et Pieris brassicae

During the final stage of development the haemolymph of Lepidoptera does not seem to undergo great variations of pressure. At most, sudden rises of 10 to 15 mm Hg were noted during violent movements or at ecdysis.With an improved and very sensitive apparatus, a pressure eighteen times higher than the pressure measured normally was recorded. This sudden variation of blood pressure corresponds to the erectile stage of the extension of the wings. The study under different conditions has shown the importance of the part played by the abdominal muscles in the pressure of the haemolymph.     

Neurogenic pacemakers in the legs of Opiliones

ABSTRACT. After autotomy, the legs of all the species of Opiliones examined, and of a Kenyan Pholcid spider, twitched spontaneously at the femoro-patellar and tibio-basitarsal joints, for periods of up to an hour. These joints lack extensor muscles, extension being achieved at the femoro-patellar joint probably by haemolymph pressure, but at the tibio-basitarsal joint of Opiliones by a cuticular spring which can extend the joint fully. Comparable twitching activity could be evoked without autotomy if the central nervous system was burnt, or by asphyxiation. Electromyograms from the femur or tibia of an isolated twitching leg showed regular motor bursts which accompanied flexions, and sensory activity during extension. Forced movements of the joints did not perturb the rhythm of the motor bursts. An isolated proximal half of a femur could still generate the same bursting pattern whereas no other region showed this activity after its isolation. Bursts recorded in the tibia were shown to be dependent on the integrity of the femur. By stimulation of the femur with 1 -ms current pulses it was possible to reset the rhythm. Stimulation with 1-s pulses caused an acceleration or inhibition of the rhythm according to the direction of the current. Spontaneous bursts could be evoked in silent isolated legs, or in intact quiescent legs, by similar 1-s current pulses. It is postulated that the femur contains independent neurogenic pacemakers which are activated by injury current from the damaged leg nerve; they produce regular bursts of motor impulses without the interplay of proprioceptive loops, and are responsible for the movements observed.