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.     

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 10 mm 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.     

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.     

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.     

Mechanical aspects of heartbeat reversal in pupae of Manduca sexta

Pulsations of the dorsal vessel were investigated with new optocardiographic techniques based on the transmission and reflection of pulse-light through optic fibers. This noninvasive technique enabled simultaneous, in vivo multisensor recordings of the heartbeat without touching the pupal integument. There was a very regular heartbeat reversal with 3 distinctive phases: (a) a backward-oriented (retrograde) cardiac pulsation; (b) a forward-oriented (anterograde) pulsation with faster frequency; and (c) shorter or longer periods of temporary cardiac standstill that usually occurred after the termination of the anterograde phase. Occasionally, there were localized series of systolic cardiac contractions during the retrograde phase. Simultaneous recordings from the base and the tail of the abdomen revealed a reciprocal, "mirror image-like", quantitative relationship. The most intensive anterograde hemolymph flow occurred at the base while the most intensive retrograde flow occurred at the tail of the abdomen. The bi-directional switchovers of heartbeat (reversal) were occasionally associated with modifications during each of the unidirectional cardiac phases. Anterograde peristalsis showed a 2-fold higher frequency of pulsation in the thoracic aorta in comparison with the posterior parts of the heart. Thus, in addition to the "odd" peristaltic waves originating at the tail, there were intercallated "even" peristaltic waves originating in the middle of the abdomen. Both of them propagated hemolymph through the thoracic aorta into the head; the first waves took the hemolymph in from the distal end, while the second sucked it from the middle of the abdomen. The use of multiple optocardiographic sensors also enabled detection of cardiac pulsations on the opposite, ventral side of the body, within the ventral perineural sinus. The ventral side of the head showed only the presence of an anterograde pulse, whereas the ventral side of the tail exhibited a strong reciprocal retrograde phase and a very weak anterograde phase. These results explain why the existence of a periodic heartbeat reversal should be essential for circulatory functions at both extremities of the cylindrical insect body. In diapausing pupae, regular cycles of heartbeat reversal were substituted by prolonged periods of anterograde pulsation during the entire duration of bursts of CO2 release (average duration of the burst was 18-20 min, periodicity 5 to 18 h). The physiological nature of such feed-back correlation between heartbeat and metabolic CO2 production is not yet clear, because the anterograde heartbeat could be also induced by a number of nonspecific factors unrelated to CO2 (mechanical irritation, injury, injections, elevated temperature). During the postdiapause, developing pharate-adult stage, the correlation between CO2 and anterograde heartbeat completely disappeared. It has been concluded that regulation of insect heartbeat represents a highly coordinated, myogenic stereotype with inherent rhythmicity, which can be modified by a number of external and internal factors.     

Contraction of the ventral abdomen potentiates extracardiac retrograde hemolymph propulsion in the mosquito hemocoel

Background

Hemolymph circulation in mosquitoes is primarily controlled by the contractile action of a dorsal vessel that runs underneath the dorsal midline and is subdivided into a thoracic aorta and an abdominal heart. Wave-like peristaltic contractions of the heart alternate in propelling hemolymph in anterograde and retrograde directions, where it empties into the hemocoel at the terminal ends of the insect. During our analyses of hemolymph propulsion in Anopheles gambiae, we observed periodic ventral abdominal contractions and hypothesized that they promote extracardiac hemolymph circulation in the abdominal hemocoel.

Methodology/Principal Findings

We devised methods to simultaneously analyze both heart and abdominal contractions, as well as to measure hemolymph flow in the abdominal hemocoel. Qualitative and quantitative analyses revealed that ventral abdominal contractions occur as series of bursts that propagate in the retrograde direction. Periods of ventral abdominal contraction begin only during periods of anterograde heart contraction and end immediately following a heartbeat directional reversal, suggesting that ventral abdominal contractions function to propel extracardiac hemolymph in the retrograde direction. To test this functional role, fluorescent microspheres were intrathoracically injected and their trajectory tracked throughout the hemocoel. Quantitative measurements of microsphere movement in extracardiac regions of the abdominal cavity showed that during periods of abdominal contractions hemolymph flows in dorsal and retrograde directions at a higher velocity and with greater acceleration than during periods of abdominal rest. Histochemical staining of the abdominal musculature then revealed that ventral abdominal contractions result from the contraction of intrasegmental lateral muscle fibers, intersegmental ventral muscle bands, and the ventral transverse muscles that form the ventral diaphragm.

Conclusions/Significance

These data show that abdominal contractions potentiate extracardiac retrograde hemolymph propulsion in the abdominal hemocoel during periods of anterograde heart flow.     

Rhythms of passive and active ventilation, and circulation recorded in diapausing pupae of Mamestra brassicae using constant volume respirometry

Abstract.  The periodically occurring convective inflow of air into the tracheal system, or passive suction ventilation, together with the cyclic bursts of release of CO₂ and active ventilation, is recorded in diapausing pupae of Mamestra brassicae . A constant volume respirometer combined with an opto-cardiograph-actograph is used. In all pupae with a metabolic rate of 0.025–0.054 mL g⁻¹ h⁻¹, the bouts of almost imperceptible abdominal contractions are recorded during the bursts of carbon dioxide release and this mode of active ventilation is qualified as extracardiac haemocoelic pulsations. The pupae whose metabolic rate is 0.052–0.075 mL⁻¹ g⁻¹ h⁻¹ show more vigorous abdominal contractions. The results demonstrate that, in diapausing pupae, characterized with low metabolic rates, both passive suction ventilation, referred to also as passive suction inspiration, and active ventilation occurs. In approximately 50% of the pupae, each gas exchange microcycle during the interburst periods begins with a miniature PSI followed by a microburst of CO₂ release; in approximately 30% of the individuals, passive suction inspirations occur separately from CO₂ microbursts; in the remaining pupae, miniature ones without microbursts of CO₂ are recorded. A typical event is heartbeat reversion: in longer periods, the heart peristalses are directed forward (anterograde of heartbeat) and, in shorter periods, the heart peristalses are directed backward (retrograde of heartbeat). At 0 °C, the cyclic release of CO₂ and miniature passive suction inspirations during the interburst periods are preserved at lower frequencies but active ventilation is lost.     

Delayed pharmacological effects of proctolin and CCAP on heartbeat in pupae of the tobacco hornworm, Manduca sexta

Abstract. The effects in vivo of cardioactive peptides proctolin, CCAP and leucomyosuppressin (LMS) are investigated by means of noninvasive optocardiographic or thermographic techniques in postdiapause pupae of Manduca sexta. A constant pattern of heartbeat reversal in these pupae is manifested by regular alternations of the forward orientated (anterograde) and the backward orientated (retrograde) cardiac pulsations, with a periodicity of some 5–10 min. The heartbeat pattern is monitored continuously for several hours before and 24 h after injection of the investigated peptides. Injections of Ringer solution alone cause a slight, almost immediate increase of the rate of the pupal heartbeat (0–10%), which lasts only for 20–30 min. Injection of proctolin, CCAP or LMS does not show any immediate cardiostimulating effects (beyond those of Ringer) at concentrations up to 2 × 10⁻⁶ M (calculated from µg of the injected peptide and 70% pupal water content; 5–7 g pupal body mass). By contrast, injections of proctolin and CCAP in the range of 10^-9 − 10^-6 M concentrations cause delayed effects on the heartbeat, which are manifested only several hours after the injections. The delayed effects involve prolonged, or even continuous periods of unidirectional, more efficient and faster anterograde pulsations. Consequently, the flow of haemolymph through the head and thoracic parts of the pupal body increases. In the case of proctolin, the prolonged anterograde cardiac activity usually starts 5 h after the injections and the effect persists for 7–12 h. Using CCAP, the stimulation of anterograde activity starts 2.5–3 h after injections and lasts usually 7–8 h. Very small doses of peptides (10^-8 − 10^-9 M) do not change the latency period significantly, but they decrease the duration of the response. The frequency of the systolic contractions of the heart does not increase during the prolonged anterograde phase. Injections of LMS to produce a final concentration of 10⁻⁶ M in the pupa induce pathophysiological disturbances of heartbeat reversal and peristalsis. The effects start with a delay of some 1.5–2.5 h after the injections. By contrast to the effects of proctolin and CCAP, LMS does not produce delayed anterograde cardiac pulsations. These results show that the most intensively investigated cardiostimulating peptides in vitro, proctolin and CCAP, have no direct cardiostimulating activity under physiological conditions in vivo. It is concluded therefore that the delayed pharmacological effects of these peptides observed in the pupae of M. sexta, represent a secondary effect, resulting from stimulation of nonspecific, extracardiac myotropic or other physiological functions.     

Onset of heartbeat revealed by successive structural changes in the developing single embryonic heart of the waterstrider (Gerris paludum insularis)

Videofilm images of the heartbeat in the living embryos of the waterstrider, Gerris paludum insularis, were analyzed to demonstrate successive changes in the width of the contractile heart at the different developmental stages. This information is graphically represented and termed structural cardiogram. Onset of the embryonic heartbeat characterized by anteriorly spreading peristaltic movement of the heart wall occurs at about 55% HL (percent heart length) as early as at about 81 h after katatrepsis (K+81 h embryo). This peristaltic wave occurs almost always following swinging movement of abdominal tip observed exclusively at this stage. Similar peristaltic wave of the heart wall may also be observed at later stages, exclusively in the anterior two-fifth of the heart. Conduction velocity of the peristaltic wave estimated from structural cardiogram of K+81 h embryo was approximately 0.57 mm/s; it was approximately 1.33 mm/s in the K+102 h embryos. In the posterior three-fifth of the heart, however, rhythmic movement was not peristaltic. Development of heartbeat generator in the specific region of the heart was discussed in relation to the onset of embryonic heartbeat.     

Control of extracardiac haemolymph pressure pulses in Tenebrio molitor

The pupae of Tenebrio exhibit periodic pulsations in the haemolymph pressure which are independent of the heartbeat. Tensometric records of the extracardiac pulses show certain specific modifications caused by changes in either the internal or external environment. Chronological changes in the pulse pattern were associated with adult morphogenesis and ecdysis. The abdominal pump controlling extracardiac pressure pulsations is independent of the brain or of any other cephalic part of the nervous system. The nerve impulses controlling the pump arise only in the mesothoracic ganglion. They are carried by the connectives to certain abdominal ganglia from which they are further transmitted to the contracting intersegmental abdominal muscles. The extracardiac pulses in haemolymph pressure aid in the maintenance of water balance and respiration and assist with the circulation of haemolymph through the appendages.     

Is Embryonic Limulus Heart Really Myogenic?

Although the neurogenic nature of the heartbeat in adult Limulushas been well studied and is undisputed, we contest the reportsthat the embryonic heartbeat is myogenic. This notion, basedon histological, calorimetric, and drug studies, is challengedby evidence from transmission electron microscopy and intracellularrecording. The first, infrequent heartbeats occur at the timeof the third embryonic molt when only the anterior portion ofthe heart tube is formed and functional. Contractions extendfurther caudad concomitant with lumen formation in the rearheart segments. All lumen-containing heart sections that wehave examined, from the earliest on, have revealed neural elementsin a bundle at the dorsal midline of the heart. Axons 1/m orless in diameter are prevalent: vesicle-filled terminal-likeareas adjacent to muscle cells are often present as well, evenin the youngest beating hearts. Myocardial cells show excitatorypostsynaptic potentials as soon as heartbeat has begun, butthey often fail to summate in the earlier stages so that contractionsare few. Resting potentials remain at –65 to –70mV from the onset of heartbeat until well after the larva hashatched, but heartbeat frequency, regularity, depolarizationheight (never overshooting) and duration all increase as embryosget older, probably as innervation of muscle fibers increasesand coordination between pacemaker and follower neurons improves.We have found no evidence that embryonic Limulus heart passesthrough a myogenic phase and believe that it is neurally drivenfrom the beginning.     

Co-ordination of firing activity of neurosecretory cells with cardiac activity in the silkmoth Bombyx mori

Electrocardiograms and electrical action potentials of cerebral neurosecretory cells producing bombyxin (an insulin-related neuropeptide) were simultaneously recorded from male pupae of the silkmoth Bombyx mori. A pupa showed alternations in the flow of haemolymph due to a rhythmic heartbeat reversal: a train of retrograde heartbeat with a slow pulse rate followed a train of anterograde heartbeat with a higher pulse rate. Intervals of heartbeat reversals changed throughout the pupal period. At any stage of the pupal period, firing activity of a population of bombyxin-producing (BP) cells rapidly declined after the start of anterograde heartbeat and an inactive state of cells continued during an anterograde heartbeat period. Analyses of ultradian bursting rhythmicity of a single BP cell revealed that a bursting phase of the cell often delayed at a time when the anterograde cardiac activity occurred at the preceding inter-burst period of firing rhythm. The results support the postulation that firing (secretory) activity of an insect neurosecretory cell system may be co-ordinated with circulation of haemolymph for rapid and pulsatile delivery of the peptides released to target organs.     

Roles of aorta,ostia and tracheae in heartbeat and respiratory gas exchange in pupae of Troides rhadamantus Staudinger 1888 and Ornithoptera priamus L. 1758 (Lepidoptera,Papilionidae)

In non-diapausing pupae of the two birdwing butterfly species Troides rhadamantus and Ornithoptera priamus (Lepidoptera, Papilionidae) heart activity and CO₂ release rates were measured simultaneously within the initial half of pupal development. Heartbeat patterns in these pupae consist of three different types of activity: Continuous forward-pulse periods of different duration with a frequency range of about 0.25–0.52 s⁻¹, continuous backward-pulse periods with lower frequencies (0.15–0.29 s⁻¹) and intermittent backward-pulse periods when short series of three to 10 single heartbeats at frequencies of 0.12–0.35 s⁻¹ alternated with heart pauses of 2–10 min. CO₂ release was discontinuous (CFO-type) from about four to 12 days after pupation in Troides rhadamantus and from about four to 18 days in Ornithoptera priamus. Mean CO₂ release rates were very low in both species (10–30 nmol g⁻¹ min⁻¹). After this period, heart pauses occurred more frequently, probably indicating the onset of metamorphosis and the beginning partial histolysis of the heart. Infrared-optical and thermometrical measurements of heartbeat indicated that haemolymph transport within the dorsal vessel in forward direction is more effective than in backward direction. This is deduced from the higher heartbeat frequency and heartbeat amplitude of the forward pulsations. Results from ultrasonic doppler velocimetry suggest that haemolymph flow velocity is highest during the relatively long diastasis of 2–3 s (30–40 mm s⁻¹), while minimum particle speed (about 20 mm s⁻¹) is at the end of systole and the beginning of diastole. This would mean that haemolymph velocity is highest between two consecutive peristaltic waves. In contrast to the haemolymph velocity, the speed of the peristaltic wave measured with the infrared transmission technique was lower (about 8.4–22 mm s⁻¹ in Troides, 10–23 mm s⁻¹ in Ornithoptera) and remained constant during forward pulse periods. During backward beating the speed was lower (8–20 mm s⁻¹ in Troides, 9–17 mm s⁻¹ in Ornithoptera) and decreased during backward pulse periods. During day two to seven in Troides and day three to nine in Ornithoptera, spiracular opening periods coincided with changes in heartbeat direction from backward to forward pulsations. A possible influence is the more efficient convective haemolymph mixing in the haemocoel during forward heartbeat. The mixing allows to bring the haemolymph in close contact with the tracheal system where the discharge of CO₂ takes place. Heartbeat may therefore serve for shortening the diffusion pathways for a rapid transition into the tracheal system during the open period of the spiracles.     

Aminergic modulation of the myogenic heart in the branchiopod crustacean Triops longicaudatus

Although crustaceans typically have a neurogenic heart, the primitive crustacean Triops longicaudatus has a myogenic heart with the heartbeat arising from the endogenous rhythmic activity of the myocardium. In the present investigation, the effects of six biogenic amines, epinephrine, norepinephrine, dopamine, octopamine, serotonin and histamine, on the myogenic heart of T. longicaudatus were examined. Epinephrine, norepinephrine, dopamine and octopamine accelerated the heartbeat, increasing both the frequency and amplitude of the action potential of the myocardium in a concentration dependent manner. The ability of epinephrine and norepinephrine to produce the acceleratory effects was more potent than that of dopamine and octopamine; the threshold concentrations of epinephrine and norepinephrine were approximately 10(-10) M and those of dopamine and octopamine approximately 10(-7) M. Serotonin weakly inhibited the heartbeat, decreasing both the frequency and amplitude of the myocardial action potential in a concentration dependent manner with a threshold concentration of approximately 10(-6) M. Histamine exhibited no effect on the heartbeat. The results provide the first evidence for direct effects of amines on the crustacean myocardium and suggest neurohormonal regulation of the myogenic heart in T. longicaudatus.     

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.     

Haemozoin formation in the midgut of the blood-sucking insect Rhodnius prolixus

Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark-brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood-sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517-518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat-labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.     

Excitatory neural control of posterograde heartbeat by the frontal ganglion in the last instar larva of a lepidopteran, Bombyx mori

The frontal ganglion of the silkworm (Bombyx mori) gives rise to a visceral nerve, branches of which include a pair of anterior cardiac nerves and a pair of the posterior cardiac nerves. Forward-fill of the visceral nerve with dextran labeled with tetramethyl rhodamine shows the anterior cardiac nerves innervate the anterior region of the dorsal vessel. Back-fill of the anterior cardiac nerves with Co²⁺ and Ni²⁺ ions and the fluorescent dye reveals that the cell bodies of two motor neurons are located in the frontal ganglion. Injection of 5, 6-carboxyfluorescein into the cell body of an identified motor neuron shows that the neuron gives rise to an axon running to the visceral nerve. Unitary excitatory junctional potentials (EJPs) were recorded from a myocardial cell at the anterior end of the heart. They responded in a one-to-one manner to electrical stimuli applied to the visceral nerve, or to impulses generated by a depolarizing current injected into the cell body. EJPs induced by stimuli at higher than 0.5 Hz showed facilitation while those induced at higher than 2 Hz showed summation. Individual EJPs without summation, or a train of EJPs with summation, caused acceleration in the phase of posterograde heartbeat and heart reversal from anterograde heartbeat to posterograde heartbeat. It is likely that the innervation of the anterior region of the dorsal vessel by the motor neurons, through the anterior cardiac nerves is responsible for the control of heartbeat in Lepidoptera, at least in part.     

Cold-resistant changes in heartbeat of the Japanese spiny lobster

Heartbeat in Panulirus japonicus acclimated to 20°C is often augmented during cooling to 15^oC. Augmented contractions of the heart coincided with increasing amplitude of electrocardiogram. In cold saline, a pericardial hormone serotonin (10⁻⁷ M) increased both the amplitude and duration of the heartbeat while another hormone octopamine (10⁻⁶ M) slightly relieved the cold depression of heart rate despite a smaller increase in beat amplitude. In contrast, the application of the cold saline containing F1 (a FMRFamide-related peptide of pericardial hormones, 10⁻⁹ M) maintained the rate and amplitude of the heartbeat around the control level during cold exposure. This suggests that in the presence of F1, the lobster heart becomes cold resistant clearly. We previously reported that the pericardial organs of spiny lobsters are activated by a small fall in body temperature. The ligamental nerves, extensions of the pericardial organs, terminate in the heart beside the ostia and their ends remain in the isolated hearts. Therefore, the ligamental nerve ends might release their hormones into the ventricle with the fall in temperature even in the isolated hearts.     

Biogenic amines evoke heartbeat reversal in larvae of the sweet potato hornworm,Agrius convolvuli

The sweet potato hornworm, Agrius convolvuli, possesses a pair of anterior cardiac nerves innervating the dorsal vessel. The anterior cardiac nerves branch off the visceral nerve that arises posteriorly from the frontal ganglion. Heartbeat reversal from anterograde heartbeat to posterograde heartbeat is triggered by the anterior cardiac nerves. Application of octopamine (OA) during the anterograde heartbeat phase reverses the anterograde heartbeat to the posterograde heartbeat, while application of OA during the phase of posterograde heartbeat accelerates heartbeat. The heartbeat reversal from anterograde heartbeat to posterograde heartbeat evoked by stimuli applied to the visceral nerve is blocked by application of the octopaminergic antagonists, phentolamine and chlorpromazine. The results suggest that OA may be a neurotransmitter for the anterior cardiac nerve. The alary muscle of the second segment receives excitatory innervation from the posterior cardiac nerve and from the nerve which extends from the second abdominal ganglion. Activation of the alary muscle results in acceleration of posterograde heartbeat. Other neurotransmitters, besides OA, may take part in the resultant acceleration.     

Cardioacceleratory action of tachykinin-related neuropeptides and proctolin in two coleopteran insect species

Several cardioactive peptides have been identified in insects and most of them are likely to act on the heart as neurohormones. Here we have investigated the cardioactive properties of members of a family of insect tachykinin-related peptides (TRPs) in heterologous bioassays with two coleopteran insects, Tenebrio molitor and Zophobas atratus. Their effects were compared with the action of the pentapeptide proctolin. We tested the cardiotropic activity of LemTRP-4 isolated from the midgut of the cockroach Leucophaea maderae, CavTK-I and CavTK-II isolated from the blowfly Calliphora vomitoria. The semi-isolated hearts of the two coleopteran species were strongly stimulated by proctolin. We observed a dose dependent increase in heartbeat frequency (a positive chronotropic effect) and a decrease in amplitude of contractions (a negative inotropic effect). In both beetles the TRPs are less potent cardiostimulators and exert lower maximal frequency responses than proctolin. LemTRP-4 applied at 10(-9)-10(-6) M was cardiostimulatory in both species inducing an increase of heart beat frequency. The amplitude of contractions was stimulated only in Z. atratus. CavTK-I and CavTK-II also exerted cardiostimulatory effects in Z. atratus at 10(-9)-10(-6) M. Both peptides stimulated the frequency, but only CavTK-II increased the amplitude of the heart beat. In T. molitor, however, the CavTKs induced no significant effect on the heart.Immunocytochemistry with antisera to the locust TRPs LomTK-I and LomTK-II was employed to identify the source of TRPs acting on the heart. No innervation of the heart by TRP immunoreactive axons could detected, instead it is possible that TRPs reach the heart by route of the circulation. The likely sources of circulating TRPs in these insects are TRP-immunoreactive neurosecretory cells of the median neurosecretory cell group in the brain with terminations in the corpora cardiaca and endocrine cells in the midgut.In conclusion, LemTRP-4, CavTK-I and CavTK-II are less potent cardiostimulators than proctolin and also exert stimulatory rather than inhibitory action on amplitude of contractions. The differences in the responses to proctolin and TRPs suggest that the peptides regulate heart activity by different mechanisms.