Physiological Entomology (2009) 34, 296–299 DOI: 10.1111/j.1365-3032.2009.00684.x

S H O R T C O M M U N I C A T I O N

Contributions on cardiac physiology in Diplopoda(Myriapoda)W I E L A N D H E R T E LInstitut fur Allgemeine Zoologie und Tierphysiologie, Biologisch-Pharmazeutische-Fakultat, Friedrich-Schiller-Universitat Jena,

Jena, Germany

Abstract. Investigations into cardiac physiology in Myriapoda are rare, but heart beatgeneration is not considered to be uniform throughout this taxon. Although cardiacautomatism in Chilopoda is neurogenic, superimposed onto a myogenic automatism,the present study reveals, on the basis of electrophysiological experiments includingelectrocardiograms and the first intracellular recordings from dorsal vessel muscle cellsof Archispirostreptus gigas Peters, 1855 (Diplopoda: Spirostreptida, Spirostreptidae),that heartbeat generation in Diplopoda is clearly myogenic. Experiments withtetrodotoxin confirm this result, and also show that proctolin, acetylcholine andoctopamine have no effect. The results are discussed from the perspective ofcomparative cardiac physiology in arthropods.

Key words. Electrocardiogram, heart, myogenic automatism, neurogenic automatism,pacemaker potential, proctolin, tetrodotoxin.

Introduction

Comprehensive studies of the arthropod taxa chelicerates, crus-taceans and insects are available with regard to the physiologyof the circulatory system (Richter, 1973; Jones, 1977; Miller,1985; Watson & Groome, 1989; Pass, 2000; McMahon, 2001;Hertel & Pass, 2002), whereas onychophorans and myriapodshave received much less attention to date. Although someinformation about the cardiac physiology of Onychophora andChilopoda is available (Sundara Rajulu, 1966, 1968; SundaraRajulu & Singh, 1969; Hertel et al., 2002), only one studyexists on Diplopoda (Cingalobolus bugnioni ) (Sundara Rajulu,1967), and Symphyla have not been investigated at all. Pau-ropoda, which include very small species only, do not developcirculatory organs.

Descriptions of both types of heart automatism are availablefor Myriapoda on the basis of pharmacological criteria, withstudies suggesting a myogenic automatism in Diplopoda anda neurogenic one in Chilopoda (Sundara Rajulu, 1966, 1967).Electrophysiological methods confirm a neurogenically gener-ated heartbeat in Chilopoda (Scolopendra cingulata, Lithobiusforficatus), although this neurogenic automatism is superim-posed over a basic myogenic-triggered heartbeat (Hertel et al.,2002).

As in onychophorans, insects and chilopods, the circulatorysystem in diplopods is highly reduced and consists largely of

Correspondence: Wieland Hertel, Institut fur Allgemeine Zoologieund Tierphysiologie, Friedrich-Schiller-Universitat Jena, Erbertstraße1, D-07743 Jena, Germany. Tel.: +493641949113; fax: +493641949102; e-mail: wieland.hertel@uni-jena.de

a dorsal heart vessel with two pairs of ostia in each diploseg-ment. Posteriorly, this dorsal heart ends blindly. It possessestwo pairs of lateral cardiac arteries in each segment. Morecomplex structures, such as a supraneural vessel, a maxillipedarch or mandibular arteries, as found in Chilopoda, are absentin Diplopoda, but a perineural sinus channels the haemolymphthrough the ventral body space (Leiber, 1935; Wirkner & Pass,2000, 2002). The heart beat itself is visible as a peristaltic wave.The myocardial cells of the heart wall of Diplopoda containonly one myofibril and display mitochondria-filled outpocket-ings of membrane (Seifert & Rosenberg, 1978). Thus, they bearthe most similarity to the cardiac cells of Insecta (Sanger &McCann, 1968). The dorsal heart of Diplopoda is innervated bya dorsal heart nerve (Leiber, 1935; Seifert & Rosenberg, 1978),as is the case in the other Arthropoda taxa, except Insecta. Inthe latter, the innervation is realized by two lateral heart nerves,which also contain neurosecretory elements. These nerves areregarded as a derived state of heart innervation in arthropods(Hertel & Pass, 2002).

The present study provides electrophysiological data regard-ing the function and automatism of the diplopod heart.

Materials and methods

The present study investigated the adult Archispirostreptusgigas Peters, 1855 (Diplopoda: Spirostreptida, Spirostreptidae).Experiments on the dorsal vessel were carried out on semi-isolated hearts or heart parts consisting of some segments,

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Cardiac physiology in Diplopoda 297

which involved removing a small band on the ventral side ofthe animal, including the leg pairs, the intestinal tract and otherorgans. The heart preparations were transferred into the testchamber without perfusion and were used for electrophysiolog-ical measurements or pharmacological experiments by a dropon method. Electrocardiogram (ECG) recordings were con-ducted extracellularly using varnish-isolated silver electrodes(diameter 40–100 μM) and AC amplification. Floating glassmicroelectrodes filled with 3 M KCl were used to investigateintracellular potentials by means of the conventional recordingtechnique. All experiments were carried out at room tempera-ture (22 ± 2 ◦C) in saline as previously described by Cook &Holman (1975).

Results and Discussion

The heart beat rate in A. gigas is 38 ± 3 min−1.The ECG of A. gigas is smooth with no oscillations

(Fig. 1). Intracellular recordings revealed action potentialsof 32 ± 2 mV with a smooth course, no oscillations and aduration of approximately 500 ms, measured at the point ofthe threshold value for the voltage-triggered action potential.Clear pacemaker activity of 4–6 mV is seen in the most cases(Fig. 2). The measured resting potential ranges between −30and −50 mV.

To examine the role of the nervous system in beat generationand to determine the type of automatism, tetrodotoxin (TTX)was applied to the heart. TTX at 2 × 10−4 mol l−1 has

absolutely no effect (Fig. 2). The pacemaker activity, heart beatrate and action potentials remain unchanged for ≥ 5 min andmore. The heart nerve is not found to influence beat generationin any way. We can thus conclude that a myogenic automatismis present in A. gigas.

With regard to the presumable heart beat modulation inDiplopoda, some possible mediators were tested. Thus, proc-tolin and octopamine, both important mediators in accessorypulsatile organs in insects (Hertel & Pass, 2002), as wellas acetylcholine, a transmitter for heart beat regulation indiplopods proposed by Sundara Rajulu (1967), were selected.Proctolin at 10−7 to 10−4 mol l−1 is completely ineffective.Furthermore, the heart of A. gigas does not react to octopamineat 10−6 to 10−2 mol l−1. No clear physiological reaction isnoted either when acetylcholine at 10−7 to 8 × 10−3 mol l−1 isapplied. Occasional heart accelerations up to 20% after appli-cation of acetylcholine at 8 × 10−6 to 8 × 10−4 mol l−1 arenot reproducible and, in most cases, the heart does not react toacetylcholine.

Heart automatism in Myriapoda is non-uniform. On the basisof results obtained using pharmacological methods, a neuro-genic automatism was considered to be present in Chilopodaand myogenic beat generation was proposed for Diplopoda(Sundara Rajulu, 1966, 1967). In the present study, the myo-genic automatism in diplopods is confirmed for A. gigas on thebasis of electrophysiological investigations. Clear pacemakeractivity for triggering and smooth action potentials of longduration are demonstrated, both of which are typical of the

Fig. 1. Electrocardiogram (ECG) of Archispirastreptus gigas (Diplopoda) compared with electrocardiograms of Periplaneta americana (Insecta)(original) and Araneus sp. (Chelicerata) (original) and a schema of the circulatory system of Diplopoda. Oscillations can be seen only in Araneus(neurogenic automatism). The schema is an original illustration reproduced with the permission of Dr Christian Wirkner (University of Rostock).

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298 W. Hertel

Fig. 2. Action potentials of myocardial fibres generated by myogenic automatism in Archispirostreptus gigas (Diplopoda). The resting potentialamounts to –50 mV. The action potentials are without oscillations and reveal smooth pacemaker activity (above). Application of tetrodotoxin (TTX)has no effect (middle and below).

myocardial cells of myogenically generated hearts. Compare,for example, the characteristics of myocardial cells in insects(Miller, 1971; Richter, 1971; Richter & Hertel, 1997). TheECG of A. gigas also exhibits a smooth wave form withoutoscillations, as is typical of myogenically triggered insect hearts(Richter, 1967; Hertel, 1971; Hertel et al., 1985). Under theexperimental conditions of parts of heart consisting of somesegments and after treatment with TTX, the heart beat of A.gigas remains unchanged, supporting the hypothesis that heartbeat automatism is myogenic in diplopods.

Heart beat generation in Euarthropoda is seen phyloge-netically to be primarily myogenic (Miller, 1985; Wilkens,1999; McMahon, 2001; Hertel & Pass, 2002; Hertel et al.,2002). The discovery of a myogenic heart beat automatismin Onychophora supports the assumption that this is the ple-siomorphic mode for Arthropoda in general (Hertel et al.,2002). In chelicerates and crustaceans, neurogenic generationis dominant in most taxa but, although the myogenic automa-tism is subordinated to the neurogenic one in these taxa, ithas not been eliminated (Wilkens, 1999). Thus, the primarymyogenic beat generation mechanism is present in embryonalstages in crustaceans and can be re-instated experimentallyin chelicerates and through the application of proctolin inLimulus (Richter & Sturzebecher, 1971; Watson & Groome,1989; Wilkens, 1999). The clearly myogenic automatism inDiplopoda together with the fact that the dominant neurogenicheart beat generation mechanism in Chilopoda is superimposed

over a basic myogenic one (Hertel et al., 2002) provides sup-port for the assumption that the primordial heart beat generationmechanism in the phylum Arthropoda is myogenic.

Acknowledgements

I am grateful to the Institute of Zoology, University of Mainz,for supplying the animals and Michael Richter, Dipl.-Ing.,Institute of General Zoology, University of Jena, for assistancewith technical equipment. I am indebted to Dr ChristianWirkner, Institute of Biosciences, University of Rostock, forgiving me permission to use his diagram of the Diplopodaheart.

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Accepted 23 March 2009

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Journal compilation © 2009 The Royal Entomological Society, Physiological Entomology, 34, 296–299