Neural control of the dorsal vessel in lepidopterous insects

Introduction: Arthropod circulatory systems and the relationship discussion

Günther Pass

Institut für Zoologie, Universität Wien, A-1090 Vienna, Austria

Arthropods exhibit in their circulatory system great variation in functional morphology, physiological control and biological functions. This refers especially to the design of the vascular system and to the structure and function of the circulatory pumps. With respect to cardiac control we find both hearts with neurogenic and with myogenic automaty. Despite the multiplicity and complexity of the biological tasks of the circulatory system, the pumping organs and the peripheral vessels are relatively simply organized. Therefore this organ system seems in arthropods especially suited for the analysis of its evolutionary origin, transformations and physiological adaptations.
Induced by molecular data new discussions on arthropod relationships recently flared up providing several conflicting and unorthodox phylogenetic hypotheses. This situation requires a reevaluation of data from all relevant research areas. Contributions to this symposium will review the morphologies and physiologies of the circulatory systems of the various arthropods in the light of these hypotheses. Aims are to find out what research on the circulatory system can contribute to the phylogenetic problem, but also what the evolutionary perspective may contribute to an integrative and holistic understandig of organ physiology.

Physiology of the crustacean cardiovascular system viewed from an evolutionary perspective

Jerrel Wilkens

Biological Sciences, University of Calgary, Calgary, AB, Canada

The heart may be the most primitive component of the crustacean circulatory system. It varies from a simple longitudinal tube in the Anastraca (brine shrimps) to the abdominal heart in isopods and the thoracic heart in decapods. All hearts seem to be derived from the original longitudinal tube and all beat autogenically. Adult stomatopods (mantis shrimp) and decapods (crayfish, crabs and lobsters) possess neurogenic hearts and the myocardium is inactive in the absence of the cardiac ganglion; however, myogenicity may be the more primitive condition. The myogenic branchiopod (Triops) heart lacks nerves, the isopod heart continues to beat after the cardiac ganglion is blocked and at least the embryonic and early larval heart of decapods appears to beat myogenically. There may be a selective advantage for neurogenicity, yet the origin of the cardiac ganglion is unknown.

There are no arteries in the small 'primitive' forms, or at most a simple anterior artery, and blood is pumped toward the anterior ganglia. Arteries may have been induced by increase in body size or conversely it may have preceded and allowed increased size. With arterialization, and maybe in taxa without arteries, there is control of blood distribution to different organs and tissues by the cardio-arterial valves and the regulation of arterial peripheral resistance. The crustacean open circulatory system does not possess capillaries or veins, yet in all forms the hemocoelic (venous) flow pathways are orderly. Venous blood perfuses the gas exchange surfaces. Blood returns to the heart through valved ostia. The location of ostia is variable, but they appear to be restricted to venous pathways that deliver oxygenated blood to the pericardial sinus and heart.

Evolutionary morphology and physiology of the circulatory organs in insects - a review

Wieland Hertel 1) and Günther Pass 2)

1) Institut für Allgemeine Zoologie und Tierphysiologie, Friedrich-Schiller-Universität Jena, D-07743 Jena, Germany
2) Institut für Zoologie, Universität Wien, A-1090 Vienna, Austria

An overview is given on the research of the two last decades on insect circulatory organs in an evolutionary context. With respect to functional morphology it became obvious that the dorsal vessel („heart") flow has changed during evolution of hexapods. In all apterygotes and in mayflies it is bidirectional. In most pterygote insects, however, it is unidirectional. In some insect groups the flow direction alternates. This heart beat reversal is a derived condition which probably occurred several times during insect evolution. Special reference is given to the hemolymph supply of body appendages. While in ancestral hexapods they are supplied by arteries, accessory pulsatile organs (APO) exist in most higher insects. These autonomous auxiliary pumps are evolutionary innovations. They evolved by recruitment of building blocks from various organ systems which were assembled to new functional units.
All pulsatile circulatory organs in insects investigated so far are characterized by spontaneous rhythmical contractions which are generated by a myogenic automatism. This myogenity is considered a primary property in insects. Despite the basic myogenic trigger insect hearts have a neuronal control in addition. Numerous modulators, especially neuropeptidergic factors, can influence the heart contraction. The pumping muscles of the APOs have similar physiological properties despite their different evolutionary origin. In antenna-hearts the pacemaker activity is comparable with that of the heart, but the neuronal modulators may be strongly different. The data are discussed in the light of different hypotheses on the evolution of arthropods.

Insect abdomen - heartbeat manager in insects

Tartes U.¹⁾, Vanatoa A.¹), Kuusik, A.²)

1) Institute of Zoology and Botany, Estonian Agricultural University, Tartu, Estonia
2) Institute of Plant Protection, Estonian Agricultural University, Tartu, Estonia

Many types of reflex cardiac response to a variety of stimuli have been described in insects (reviewed in Ai & Kuwasawa, 1995). Although the insect heart has inherent properties of myogenicity, it is largely controlled by the central nervous system (Miller, 1997). Close coordination between heartbeats and body movements suggests a nervous control of the dorsal vessel but also of the pupal skeletal muscles.
In Leptinotarsa decemlineata heart activity periods are initialised by abdominal strokes, although heartbeat and abdominal movement frequencies are different (Kuusik, et al., in print). An tactile stimulus evoked abdominal movements followed soon afterwards by contractions of the heart. Repeated stimuli evoked body rotating movements at any time with heartbeats starting at the first movement.
We hypothesize that periodically occurring abdominal movements play an active role in synchronization of the periods of heartbeat and pupal movements. We conclude from our results that one possible mechanism for regulating heartbeat periodicity in pupae of L. decemlineata is based on cardiac reflexes triggered by rhythmic body movements.
Similar phenomenon can be observed in Galleria mellonella and Tenebrio molitor pupae. Although there is no correlation between heartbeat and abdominal movements in diapausing Pieris brassicae pupae.

References
AI, H. and KUWASAWA, K. (1995) Neural pathways for cardiac reflexes triggered by external mechanical stimuli in larvae of Bombyx mori. J. Insect Physiol., 41 , 1119-1131
MILLER, T.A. (1997). Control of circulation in insects. General Pharmacology, 29, 23-38.

Myoactive peptides and their role in cardio-regulation in Drosophila.

Ruthann Nichols

University of Michigan, Biological Chemistry Department, Ann Arbor, MI, USA 48109-1048, nicholsr@umich.edu

Peptides transmit and modulate numerous physiological parameters. Comparison of structures, expression patterns, and activities between species provides insight into peptide functions. Drosophila melanogaster FDDY(SO3H)GHMRFamide (drosulfakinin I or DSK I), TDVDHVFLRFamide (dromyosuppressin or DMS), SDNFMRFamide, and pEVRFRQCYFNPISCF (flatline or FLT) represent four structurally distinct peptide families. DSK I has a high degree of identity to other sulfakinins with the consensus structure -XDY(SO3H)GHMRFamide, where X is E or D. DMS has a high degree of identity to other myosuppressins with the consensus structure XDVDHVFLRFamide, where X is pE, P, or T. SDNFMRFamide has a high degree of identity to other FMRFamide-containing peptides at the C terminus, but a distinct N-terminal extension. FLT has a high degree of identity to other Manduca allatostatin (Mas AS)-like peptides with the consensus structure pEVRXRQCYFNPISCF, where X is F or Y.
DMS, SDNFMRFamide, and FLT decrease heart rate albeit with different magnitudes and time-dependent responses. DMS and FLT are expressed in the gastrointestinal tract and affect gut motility; however, SDNFMRFamide expression and effect on the gut has not been reported. These data suggest the peptides have different roles in physiology. The peptides have non-overlapping cellular expression patterns, which suggests different mechanisms regulate their synthesis and release. The conserved structures, expression patterns, and activities of these four myotropins suggest they have important but different roles in cardiovascular biology and different signaling pathways.

Neural Control of Heart Reversal in Manduca sexta.

Norman T. Davis and David Dulcis

Division of Neurobiology, University of Arizona, Tucson, AZ, 85721-0077, USA.
( ntd@neurobio.arizona.edu)

The heart of adult Manduca sexta reverses between phases of anterograde and retrograde contractions, and so adult hearts have an anterograde pacemaker at the terminal chamber and a retrograde pacemaker located anteriorly on the aorta. Stimuli can initiate a cardiac reversal reflex, but only during the anterograde phase, indicating neural control during this phase of contraction. The anterograde phase disappears when neuronal input to the heart is blocked by tetrodotoxin. The terminal chamber of the heart is innervated by a bilateral pair of neurons extending through dorsal nerves-eight. These cells are motor neurons-one of neuromere-eight (MN1₈) of the terminal abdominal ganglion and are serial homologs of motor neurons-one of other abdominal ganglia. During metamorphosis, the target of MN1₈ is respecified from larval skeletal muscles to cardiac muscles of the terminal chamber. Transection of dorsal nerves-eight in vitro results in cardiac reversal to continuous retrograde contractions. When dorsal nerve eight is stimulated, the heart reverses to anterograde contractions; when stimulation is stopped, the heart reverts to retrograde contractions. Reversal to anterograde contractions occurs when the anterograde pacemaker receives bursts of impulses from MN1₈, making this pacemaker dominant. In the absence of input from MN1₈, the retrograde pacemaker becomes dominant and the heart reverses to retrograde contractions. We propose that cells MN1₈ are pacemaker neurons, that their period of bursting activity results from a slow potassium current (pacemaker potential), and that their periods of inactivity results from a system of reciprocal inhibition or inhibition through the cardiac reversal reflex.

Interaction of circulation and respiration in insects

Stefan K. Hetz

Dept. Animal Physiology, Humboldt Universität zu Berlin, D-10115 Berlin, Germany

On the basis of their well developed tracheal gas exchange system, insects are generally considered to lack a circulatory system especially designed for respiratory gas transport. Although most insect species do not possess any pigments for oxygen transport, there is founded evidence for a significant interaction of circulatory and respiratory function as pointed out by Wasserthal (1996). Direct interaction of haemolymph circulation and tracheal ventilation (Hetz et al. 1999) may be supported by activity of intersegmental muscles, by the coelopulse system (Slama 2000) and further accessory pulsatile organs (as reviewed by Pass 2000). Accordingly, during specific conditions haemolymph convection may represent an important factor for respiratory gas transport. Recently, new techniques better suited for small specimens have been developed for the study of aspects related to circulation-mediated respiratory gas exchange. An overview of current techniques and recent data is presented and possible future developments are discussed by this paper.

References
WASSERTHAL, L. T. (1996). Interaction of circulation and tracheal ventilation in holometabolous insects. ADVANCES IN INSECT PHYSIOLOGY 26, 298-351.
HETZ, S. K., PSOTA, E. and WASSERTHAL, L. T. (1999). 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). INTERNATIONAL JOURNAL OF INSECT MORPHOLOGY & EMBRYOLOGY 28, 131-144.
SLAMA, K. (2000). Extracardiac Versus Cardiac Haemocoelic Pulsations in Pupae of the Mealworm (Tenebrio molitor L.). JOURNAL OF INSECT PHYSIOLOGY 46, 977-992.
PASS, G. (2000). Accessory pulsatile organs: evolutionary innovations in insects. ANNUAL REVIEW OF ENTOMOLOGY 45, 495-518.

Effect of temperature on Ca²⁺ cycling in atrial myocytes of rainbow trout.

H.A. Shiels^{1, 2}, M. Vornanen², A.P. Farrell¹

¹ Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada
² Department of Biology, University of Joensuu, Joensuu, Finland

Rainbow trout inhabit eurythermal environments and thus we tested the idea that they have evolved mechanisms to cope with acute changes in temperature. We used the whole-cell patch-clamp technique combined with fura-2 Ca²⁺ transients to study the effects of acute, physiologically relevant temperature change on (1) the electrophysiological properties of the L-type Ca²⁺ channel and (2) the temperature dependency of sarcoplasmic reticulum Ca²⁺ loading in rainbow trout atrial myocytes. Using physiologically relevant action potentials as the voltage-clamp stimulus waveform, the temperature dependency of ICa had a Q₁₀ ~ 2, but the charge carried by ICa was unaffected by temperature. Similarly sarcoplasmic reticulum Ca²⁺ loading was found to be temperature-independent provided cells were loaded using a physiological action potential waveform applied at a physiologically relevant frequency for the given experimental temperature. The cardiac action potential plays a pivotal role in maintain fairly constant Ca²⁺ flux during acute temperature changes in rainbow trout.

Neural control of the dorsal vessel in lepidopterous insects

Kazuyuki Uchimura¹, Tomoko Matsushita², Hiroyuki Ai³ and Kiyoaki Kuwasawa¹

¹Deapartment of Biological Sciences, Tokyo Metropolitan University, 1-1 Minami-osawa, Hachioji-shi, Tokyo 192-0397, Japan.
²Department of Oral Physiology, Ohu University, Mitsumido 31-1, Tomita-machi, Koriyama-shi, Fukusima, 963-8611.
³Div. of Biology, Dept. of Earth System Sciences, Fukuoka Univ.,8-19-1 Nanakuma, Jonan-ku, Fukuoka city, Fukuoka 814-0180, Japan.

We have previously reported that several types of reflexes of the dorsal vessel are caused by external stimuli applied to various regions of the body in lepidopterous insects (Bombyx mori and Agrius convolvuli). Among the reflexive responses of the dorsal vessel, The junction of the aorta and anterior end of the heart is the initiating site for reflex induction of posterograde heartbeat and thus heartbeat reversal. The anterior cardiac nerve branches off the visceral nerve extending from the frontal ganglion and innervates the junctional region of the dorsal vessel (Ai and Kuwasawa, 1995). (The visceral nerve is known as the recurrent nerve.) Activation of the anterior cardiac nerve triggered posterograde heartbeat even during a period of anterograde heartbeat.
We have now found another nerve branching off the nerve from the corpus cardiacum and innervating the junctional region of the dorsal vessel. This nerve inhibited retrograde heartbeat when electrical pulses were applied at the rostral cut-stump of the nerve. The inhibitory function of this nerve may explain a previous observation. In Manduca sexta, transection of the 3rd cardiacal nerve, extending from the corpus cardiacum, resulted in disinhibition of the pacemaker for the posterograde heartbeat (Davis et al., 2000). These results may indicate that the cardiacal nerve contains inhibitory axons to the anterior region of the dorsal vessel, responsible
for cardiac inhibition of posterograde heartbeat.

The pericardial neurohemal organ in the abdomen of the tsetse fly and other cyclorraphan flies.

Shirlee Meola, Peter Langley and Helga Sittertz-Bhatkar

Institutions: USDA, ARS, Knipling-Bushland Insect Research Laboratory, Kerrville TX; University of Wales, Cardiff,UK; Texas A&M University, College Station,TX

The ventral surface of the tubular heart of cyclorraphan flies is supported by a dorsal (pericardial) septum that is anchored to the
sclerites by alary muscles. Unlike other insects, the pericardial septum of these flies contains a central band of longitudinal muscles. A
histological study of the abdominal aorta in several species of cyclorraphan flies, revealed this longitudinal muscle is present in adults, but not the larval stage and extends beneath the heart from the 1st to the 5th abdominal segment. Four pairs of alary muscles insert on the ventrolateral surface of the longitudinal muscle, resulting in a pericardial sinus separate from the hemocoel. An ultrastructural study of the pericardial septum and sinus of the tsetse fly, Glossina morsitans, as well as the stable fly, Stomoxys calcitrans and another muscid, Orthellia caesarion, determined that neurosecretory fibers arising from the segmental nerves not only innervate the fibers of the longitudinal muscle, but extend between the muscle fibers, into the pericardial sinus where they release their products. In the tsetse fly, in addition to pericardial release, the neurosecretory fibers extend between the fibers of the myocardium and terminate in the lumen of the aorta. Thus the pericardial sinus of the adult flies was found to be a large neurohemal site in which the counter contractions of the circular muscles of the heart and the longitudinal muscle of the pericardial septum provide a forceful means of distributing neurosecretory material throughout the abdomen of the reproductive stage of these insects.

Myoactive peptides and their role in cardio-regulation: conservation of activity and structure.

Janna Merte and Ruthann Nichols

University of Michigan, Biological Chemistry Department, Ann Arbor, MI, USA 48109-1048, nicholsr@umich.edu

To understand the roles and mechanisms of actions of myotropins, we are elucidating their structures, distributions, and activities. Drosophila melanogaster FDDY(SO3H)GHMRFamide (sulfakinin I, DSK I), TDVDHVFLRFamide (myosuppressin, DMS), SDNFMRFamide, and pEVRYRQCYFNPISCF (flatline, FLT) represent four structurally distinct myotropic peptide families.
DSK I, DMS, and SDNFMRFamide all contain a RFamide C terminus and, thus, by definition are FMRFamide-related peptides (FaRPs). The FaRP superfamily can be divided into subgroups including sulfakinins, myosuppressins, and FMRFamide-containing peptides. Sulfakinins have the consensus structure -XDY(SO3H)GHMRFamide, where X is E or D. Likewise, myosuppressins have a high degree of structure identity with the consensus structure XDVDHVFLRFamide, where X is pE, P, or T. In contrast, FMRFamide-containing peptides, like SDNFMRFamide, have a common C-terminal FMRFamide, but distinct N-terminal extension. The high degree of structure identity for sulfakinins and for myosuppressins suggests their N-terminal structures are critical for activity and justifies the subdivision of FaRPs.
FLT and other Manduca allatostatin (Mas AS)-like peptides have the consensus structure pEVRXRQCYFNPISCF, where X is F or Y. FLT structure is significantly different from DSK I, DMS, and SDNFMRFamide. FLT is not a FaRP and does not contain a C-terminal amide. These myotropins have non-overlapping cellular distributions in the central nervous system, which suggests different regulatory mechanisms affect their synthesis and release. Myotropins affect heart rate albeit with different magnitudes and different time-dependent responses. The differences among these myotropin structures, distributions, and activities suggest they have different signaling pathways and correspondingly different biological functions.

Regulation of circulation in insect by pumps, diaphragms and the coelopulse system.

Thomas A. Miller

Entomology Department, University of California, Riverside, CA

Insects have evolved mechanisms and strategies to ensure complete circulation of the hemolymph (Miller, 1997). The extent of the innovations in accessory pulsatile organs that perfuse appendages in insects is evident in the recent article by Pass (2000). The appendages in most insect pupae are not supplied by special circulatory organs, instead the hemocoelic pressure system (Slama, 2000) appears to assist in perfusion of hemolymph in all parts of the body. The pressure pulses appear to play a key role in respiration as well (Slama, 1999). The fact that respiration and circulation in insects are functionally connected was made clear by the work of Wasserthal (1996). Both ventilation movements and circulation movements are probably controlled by an autonomic nervous system (the coelopulse). The components of this system are only just being described, but probably are capable of great sophistication in directing hemolymph flow.

Miller, T.A. (1997) Control of circulation in insects. Gen. Pharmacol. 29: 23-38.
Pass, G. (2000) Accessory pulsatile organs: evolutionary innovations in insects. Ann. Rev. Entomol. 45: 495-518.
Slama, K. (2000) Extracardiac versus cardiac haemocoelic pulsations in pupae of the mealworm (Tenebrio molitor L.). J. Insect Physiol. 46: 977-992.
Slama, K. (1999) Active regulation of insect respiration. Ann. Entomol. Soc. Am. 92: 916-929.
Wasserthal, L. T. (1996) Interaction of circulation and tracheal ventilation in holometabolous insects. Adv. Insect Physiol. 26: 297-351.

Central ganglionic vascularization of the isopod crustacean, Bathynomus doederleini.

Kosuke Tanaka¹, Yoko F.-Tsukamoto², and Kiyoaki Kuwasawa²

¹Department of Biology, Kyorin University School of Medicine, Mitaka, Japan, ²Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Japan
ktanak@kyorin-u.ac.jp

Decapods have a rich capillary system within all the central ganglia (Sandeman, 1967) although decapods have an open circulatory system. We studied the vascular system for the central ganglia of an isopod Bathynomus doederleini. There are thirteen arteries arising from the heart, three anterior arteries and five pairs of lateral arteries. The anterior median artery runs to the cephalon, where the cerebral ganglion is supplied arterioles from the cor-frontale. Arterioles from the first lateral artery (LA1) extend to the second to fifth thoracic ganglion (TG2-TG5). Arterioles from LA2, LA3 and LA4 extend to TG6, TG7 and TG8, respectively.
We performed anatomical and histological studies of arterioles supplying the central ganglia, by means of injection of inks through the heart or arteries and then making paraffin sections of the central nervous system. In the cerebral ganglion, the lumen between periganglionic sheath and epiganglionic sheath and channels extruding into the neuropil were filled with injected ink. In the thoracic ganglia, the lumen between the periganglionic sheath and epiganglionic sheath was filled with injected ink, but channels filled with ink were not observed in the neuropil of the ganglion. These results suggest that, in Bathynomus, arterioles open in the lumen between the periganglionic sheath and epiganglionic sheath and there are some blood channels extending into neuropil in the cerebral ganglion. Indeed, when the thoracic ganglia were perfused through these arterioles with saline containing humoral substances, the perfusates effectively exerted influence on neurons in the thoracic ganglia.

Cardiac pacemaker mechanism in the ostracod crustacean Vargula hilgendorfii

Yukiko Ishii and Hiroshi Yamagishi

Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
( yamagishi@biol.tsukuba.ac.jp)

To explore diversity of cardiac pacemaker mechanism in crustaceans, the heartbeat of the ostracod V. hilgendorfii, one of the lower orders within the Crustacea was examined electrophysiologically. The heart is single chambered and composed of a single layer of myocardial cells characterized by localization of myofibrils at the epicardial site. A single neuron situated on the outer surface of the dorsal heart wall sends an axon into the heart wall and the axon is branched widely forming many neuromuscular junctions on the myocardial cells. The frequency of the heartbeat changes widely and each heartbeat follows a myocardial action potential composed of a spike and plateau potential. The myocardial cells couple electrically and fire almost synchronously. By application of 1 µM TTX, the action potential of the myocardium disappeared, the myocardial membrane depolarized and oscillatory slow potentials often appeared. The frequency of the action potential was almost unchanged during injection of a depolarizing or hyperpolarizing current pulse into the myocardium. When the myocardial membrane was depolarized by the current the oscillatory slow potentials appeared in addition to the action potentials and its frequency was higher with stronger intensities of the current. The results suggest that, though the myocardial cell has conditional oscillatory properties, the heartbeat of V. hilgendorfii is basically neurogenic with the single motor neuron in the heart acting as a pacemaker.

The circulatory organs in myriapods: comparative morphology and physiology

Wirkner C.S.¹, Hertel W.², Pass G.¹

¹Institut für Zoologie, Universität Wien, A-1090 Vienna, Austria
²Institut für Allgemeine Zoologie und Tierphysiologie, Friedrich-Schiller-Universität Jena, D-07743 Jena, Germany

Within the recent debate on arthropod phylogeny myriapods play a crucial role. The present paper deals with the circulatory system of all high-rank taxa of myriapods, with exception of Pauropoda which lack circulatory organs at all. The morphologies of the circulatory organs were investigated in representative species by means of semithin sections and combined with data from the literature.
Among myriapods, the vascular system exhibits a wide range of complexity. In chilopods, the circulatory system consists of two longitudinal central vessels: the dorsal vessel and the ventral vessel. In the first body segment, the maxilliped arch connects these two vessels. From the longitudinal vessels peripheral arteries branch off which supply long body appendages or sinuses. In Scutigeromorpha, the circulatory system is closely linked with respiratory tasks by physiological and morphological features (e.g. hemocyanin, close spatial relation of heart and tracheal lungs, high-performance heart). In diplopods, there is only a longitudinal dorsal vessel. Ventrally, the blood flows in a perineural sinus. Peripheral vessels branchig off the dorsal vessel supply the antennae and sinuses. In symphylans, both a dorsal and a ventral vessel occur which are connected by an unpaired vessel. Peripheral arteries supply long body appendages and sinuses.
Physiological literature on myriapod circulatory organs is very scarce and we present the first electrophysiological data from the dorsal vessel of chilopods. Results are discussed from phylogenetic as well as from general evolutionary points of view.