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AMER. ZOOL., 39:199-214 (1999) Evolution of the Cardiovascular System in Crustacea1 JERREL L. WILKENS 2 Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada SYNOPSIS. An attempt is made to explain the evolution of different cardiovascular morphologies of crustaceans on the basis of (1) changes in the development of the body plan of different species, (2) the advent in the malacostracans of segmental arteries that provided the circulatory potential for growth in body size and speciation, (3) the need for more powerful hearts to propel blood through larger bodies, and (4) the embryological substrate that would allow for the development of regional specialization. Electrophysiological evidence supports the hypothesis that the archetypal crustacean heart was myogenic, but in more advanced forms this pacemaking mechanism has become subservient to the neural drive from the cardiac ganglion. This transition may have been the result of the selective advantages to possessing a discrete cardiac ganglion, which itself was easily controlled by nervous inputs from the CNS and by circulating hormones. INTRODUCTION This essay on the evolution of the cardiovascular system (CVS) in the Crustacea is constructed on the recognition that evolutionary change in form must be functional. Functional requirements of the CVS of a crustacean are the adequate supply to all tissues of oxygen and nutrients and the removal of waste products, coordination (hormone distribution), and temperature regulation (insects and possibly in other thermoregulators such as terrestrial crabs). These requirements have resulted in some type of circulatory system in all metazoans above the flatworms. When animals are small enough or sedentary they may not need anything more than the ability to move coelomic fluid, but larger animals require a dedicated pump and a distribution system. It is assumed that a tubular heart without arteries is a plesiomorphic feature of the crustacean blood-vascular system. The early forms must have possessed a more-orless uniform segmentation and an extended tubular heart running from the head to the tail. The different CVSs that are found in many extant groups are viewed as apomorphic derivatives of the archetype. This discussion will be guided by three princi' Invited essay on circulation to commemorate the life and career of Charlotte Mangum. 2 E-mail: [email protected] pies: (1) "the interpretation of form must always be tempered with an understanding of function" (Schram, 1982); (2) generalized structures that appear to be primitive may in fact represent a secondary simplification and may not represent temporal priority (Gould, 1977); and (3) new structures and functions are more likely to have been layered on preexisting ones rather than to have arisen as de novo inventions. RELATIONSHIP BETWEEN PHYLOGENY AND CVS ANATOMY It is useful, as a first approach to understanding the evolution of the CVSs in the crustaceans, to consider the evolution of the major groups of the subphylum. Unfortunately, the fossil record for the Crustacea is uneven (Schram, 1982) and many of the supposedly primitive forms appear later than more advanced forms. For example, the earliest decapods are identified in the Permian (286-245 Ma), while the supposedly more primitive Cladocera and Copepoda first appear in the Cretaceous (144-65 Ma) and Tertiary (65-1.8 Ma), respectively (Benton, 1993). The late appearance in the fossil record of the supposedly more primitive representatives may be explained because they are small, delicate and might not have been preserved easily. On the other hand, the cladistic synthesis of the evolution of the Crustacea by Schram (1986) places the 199 200 JERREL L. WILKENS FIG. 1. Cladogram depicting the relationships of the selected classes to orders of Crustacea based on Schram (1986). The four classes are named along their respective lower branches of the tree. Orders are at the top of the cladogram except for the isopoda and amphiopoda which are suborders of the order Edriophthalma. The cardiovascular characters mapped onto the cladogram are: tubular heart extending the length of the body (1); reduced heart restricted to thorax (2); reduced heart restricted to abdomen (3); loss of heart (4); myogenic pacemaker (5); neurogenic pacemaker (6); segmental paired lateral arteries (7). decapods on an earlier branch than the Phyllopoda and Maxillopoda, and each of latter two groups contains supposedly primitive species. Further research may clarify these phyletic relationships. Since the fossil record is incomplete, I am working from the assumption that the major groups are related as illustrated in cladograms and taxonomic listing presented by Schram (1986) and illustrated in Fig. 1. Schram (1986) recognizes four classes of crustaceans as opposed to the six listed by most earlier authors (Bowman and Abele, 1982; Brusca and Brusca, 1990; Barnes and Harrison, 1992). The CVS characterSubphylum Crustacea Class Remipedia Class Malacostraca Subclass Hoplocarida Order Stomatopoda, Squilla oratorio Subclass Eumalacostraca Order Syncarida Suborder Anaspidacea istics discussed in this paper are mapped onto the cladogram. Even though it is proposed that a myogenic pacemaker (character5) is plesiomorphic, this characteristic does not map at the root of the cladogram because the only form where the origin of the heart beat is known to be myogenic is Triops longicaudatus (Phyllopoda, Notostraca). The species on which there are physiological observations are included in the taxonomic listing that follows. In this list the major groups from which it is desirable to obtain more data are readily apparent, namely the Remipedia, Phyllocarida and Maxillopoda. CRUSTACEAN CARDIOVASCULAR EVOLUTION 201 Order Euphausiacea Order Amphionidacea Order Decapoda Suborder Dendrobranchiata Superfamily Penaeoidea, Metapanaeus ensis Suborder Reptantia Infraorder Astacidae, Procambarus clarkii, Homarus americanus Infraorder Palinura, Palinurus japonicus, Palinurus interuptus Order Mysida Order Edriophthalma Suborder Amphipoda Suborder Isopoda, Bathynomus giganteus, Ligia exotica Class Phyllopoda Subclass Phyllocarida Order Leptostraca Subclass Cephalocarida Subclass Sarsostraca Order Anostraca, Artemia sp. Subclass Calmanostraca Order Notostraca, Triops longicaudatus Order Cladocera, Daphnia sp. Class Maxillopoda Subclass Ostracoda Subclass Copepoda Subclass Thecostraca Order Cirripedia I will begin by reviewing the morphology of the CVSs of adult stages based on the descriptions presented by McLaughlin (1980, 1983) (Figs. 2 and 3). This is followed by functional considerations. The systems described demonstrate a clear progression from simple to complex morphologies. Even though this discussion presents the CVSs in a generalized manner, it is recognized that there is further variation possible in each group. The heart The CVS in the Remipedia (order Nectiopodia) consists of an elongate middorsal vessel running the length of the body (Schram, 1986). The CVS of the "primitive" Phyllopoda (orders Brachypoda, Anostraca and Notostraca) is also viewed as being antecedent to more derived systems. In these forms it consists of a middorsal heart tube that extends the length of the body (Fig. 2A). As there are no arteries, blood exits from the heart into the hemocoel through an anterior cardiac opening. Blood is probably channeled through the body in specific pathways before it reenters the heart through 14 to 18 pairs of segmental ostia. This simple contractile tube without arteries may represent the simplest plan. In the Malacostraca the heart remains an elongated tube in the mantis shrimps (order Stomatopods) (Fig. 3A), but there appears to have been a change from a tubular to a shorter heart in the eumalacostracans (Fig. 3B—D). Most authors group the Leptostraca (Phyllocarida) at the root of the Malacostraca, and these forms also have an elongated tubular heart. In the Eumalacostraca the heart is a shorter tube in the Syncarida and Edriophtalma amphipods, and a shorter organ in the Mysidacea, Decapoda and Edriophthalma isopods. There is a further trend toward shorter pumping organs within each of these two broad group of Eumalacostraca. Different portions of the heart tube have been retained because the heart is located in the thoracic region of mysids, euphausids, and decapods (Fig. 3B, D), but in the abdomen in most isopods (Fig. 3C). 202 JERREL L. WILKENS ostia heart B ostium ostium heart D anterior artery heart ostium FIG. 2. Drawings of the body plan showing the heart and, if present, the anterior aorta of representatives of the (A) Class Phyllopoda, Order Notostraca, (B) Phyllopoda, Order Cladocera, (C) Maxiilopoda, Ostracoda, (D) Class Maxiilopoda, Order Copepoda, and (E) Class Phyllopoda, Order Leptostraca (drawings modified from McLaughlin, 1980; classification from Schram, 1986). The heart of decapods is thought to represent the condensation of the first three segments of the ancestral heart (Wilkens et ai, \991b). The reduction in heart length seems to have been an irreversible occurrence even in very small species (see below for functional considerations). The class Phyllopoda contains orders that have primitive tubular hearts (Anostraca, Notostraca, Leptostraca, Brachypoda) (Fig. 1A, E) as well as the Cladocera (Fig. IB) where a globular heart is associated with reduction in number of body segments. In the Maxillipoda the living representa- tives of the ostracods and copepods possess simple CVSs that may represent apomorphic reductions from the plesiomorphic plan, again associated with a reduction in the number of body segments (Fig. 1C, D). All representatives are small, have a reduced number of somites, and the heart is a short organ that receives blood through one or two pair of ostia (Fig. 2B-D). The cirripeds and some small ostracods lack a true heart but possess a secondarily derived membranous blood pump that moves blood by cycles of compression and relaxation of the pumping structure by contractions of vis- 203 CRUSTACEAN CARDIOVASCULAR EVOLUTION B ant. artery ostium lat. artery heart abd. heart adorsal . lat. ant. med. ant. rter y artery artery ostium / lat. artery i sternal artery hepatic artery FIG. 3. Drawings of selected representatives of the Class Malacostraca with the components of the cardiovascular system labeled. (A) Order Stomatopoda; (B) Subclass Eumalacostraca, Order Amphipoda; (D) Subclass Eumalacostraca, suborder Isopoda; (E) Subclass Eumalacostraca, Infraorder Astacidae (drawings modified from McLaughlin, 1980; classification from Schram, 1986). ceral or somatic muscles. It is assumed that portions of the heart, derived from segmental blocks of mesoderm, were deleted as the number of somites was reduced. A single anterior artery leaves the heart in some ostracods, but in only some species of the cladocerans and copepods. If the reductions in size of the heart occurred in ancestors of these present day small species, the limitations in blood distribution may have been an important factor that has prevented representatives of the sub-malacostracan classes from attaining large body size, except in some sedentary cirripeds. 204 JERREL L. WILKENS phyllopodian Leptostraca possess a very rudimentary arterial system that consists of at most a single anterior artery leaving the heart, although this artery may branch. Malacostracans and Leptostraca differ dramatically in having a more or less elaborate segmental arterial system along the entire length of the body. The classification schemes that group the Leptostraca with the Malacostraca makes sense if we assume that the appearance of segmental arteries was a singular event that made possible the unique radiation displayed by the Malacostraca. On the other hand, segmental lateral arteries may have arisen independently more than once. In these two groups, anterior, posterior, and segmental arteries arise from the heart regardless of whether it is tubular or shortened. The advent of a segmental arterial supply to the limbs and other tissues remote from the heart may have been the evolutionary innovation that allowed some of the more advanced members of this class to become large. The simple squeeze and slosh circulatory dynamics of animals with a heart tube and no arteries would not have been able to move blood through the 2 meter long legs of spider crabs or to all parts of large animals such as American lobsters of 25 kg body weight. Once segmental arteries were developed, the advantage to the direct supply of blood to remote tissues would continue even if the heart tube were shortened. When we examine malacostracans with shortened or globular hearts we observe that several arteries leave the heart. This would occur if the remnant of the original heart tube had pulled along the segmental arteries from the somites from which it withdrew (Fig. 4). Following this argument, the anterior arteries (median anterior, lateral anterior, and hepatic arteries) of decapods are thought to be the original segmental lateral arteries to cephalic and anterior thoracic somites as are Vasculature found in the more primitive forms such as All representatives of the phylum Ar- the phyllocaridian Leptostraca and hoplothropoda possess an open circulatory sys- caridian Stomatopoda (mantis shrimps). tem where at least the "venous" return A second line of evidence supporting the pathways are through hemocoelomic tissue proposition that shortened hearts were despaces and sinuses, not in actual vessels. rived from tubular ancestors is found in the Arteries are present in some forms. All innervation of the heart in decapods and crustaceans except the Malacostraca and the isopods. The points of origin in the ventral Ostia Blood returns to the heart through valved ostia. In the most primitive condition a pair of ostia may occur in the heart tube in each body segment (see below for embryonic origin of ostia), but they may have been secondarily lost from variable numbers of segments of the tube (Anostraca, Notostraca, Stomatopoda, Decapoda). As the heart tube became shorter, possibly in parallel with reduction of number of body segments, many of the segmental ostia were lost. Any ostia present in an animal are probably derived from the ancestral segmental ones rather than de novo structures, given their embryonic origin. Thus, the number of pairs of ostia, unless they were secondarily lost, could indicate the number of segments of the ancestral heart tube that have been retained. By this argument, the heart of the cladocerans, ostracods and copepods should represent only one or two segments of the primitive heart tube. The heart of decapods must represent the fusion, and enlargement, of at least three segments of the heart tube because there are three pairs of ostia (Fig. 4C). In isopods the shortened heart retains one or two pairs of ostia and it appears that portions of the heart lack ostia. It has been argued that ostia may have been lost from segments of a heart that do not receive oxygenated blood (Burnett, 1984). Stomatopods have abdominal gills and the the abdominal portions of the heart tube have ostia. The caudal heart of isopods, and therefore the location of the ostia, is correlated with the respiratory function of the pleopods (Barnes and Harrison, 1992). In decapods the dorsal abdominal artery is thought to represent a remenant of the primitive heart tube (see below), but there are no abdominal gills and the dorsal abdominal artery does not possess ostia. CRUSTACEAN CARDIOVASCULAR EVOLUTION 205 AMA HA DAA FIG. 4. Schematic drawings of the cardiovascular systems of a primitive malacostracan, (A) based on stomatopods, (B) a hypothetical intermediate or transitional state, and (C) a decapod. Anterior to left,; anterior median artery (AMA), anterior lateral artery (ALA), hepatic artery (HA), sternal artery (SA), dorsal abdominal artery (DAA), non-muscular flap valve (flap). (From Wilkens et al., 1997b). nerve cord of the cardioregulatory nerves are similar if not homologous. One pair of inhibitory and two pairs of acceleratory neurons occur in each group. In isopods the cell bodies of the cardioinhibitory neurons are located in the first thoracic ganglion (Tanaka and Kuwasawa, 19916), and one pair of cardioacceleratory neurons occurs in both the second and third thoracic ganglia (Tanaka and Kuwasawa, 1991a; Sakurai and Yamagishi, 1998). In the decapod spiny lobster, Panulirus argus, where the thoracic and abdominal ganglia are concentrated into a single thoracic ganglion, the cardioinhibitor nerves arise from the anterior region of this ganglion and the two pairs of cardioaccelerators arise immediately posterior (Maynard, 1953). Although the cell bodies of these neurons have not been traced by dye injection, their points of origin appear to be homologous to those of the isopods. The heart itself sends an artery directly to each of the thoracic and abdominal appendages in the phyllocaridan leptostracans and some hoplocarids. This pattern is modified somewhat in the ediophtalmian iso- pods in that the anterior lateral artery branches to supply the first four thoracopods. The dorsal abdominal artery (modified ancestral heart tube) in the anaspids, mysids, euphausids and decapods supplies arteries directly to the pereopods, but in the thorax a single artery (descending or sternal artery) divides to send an artery into each pleopod and to the ventral nerve cord. In decapods, and perhaps in the other orders where it occurs, the sternal artery seemingly arose from one of the segmental lateral arteries (Fig. 5). The tendency for a single artery to branch to supply blood to several appendages may have occurred as the heart tube became shortened in length. Although it does not in itself suggest an evolutionary process, the present plan is much simpler than would have resulted if all of the ancestral segmental arteries had been retained. The dorsal abdominal artery It is proposed that the dorsal abdominal artery of prawns, spiny lobsters and chelate lobsters may represent the modified posterior remnant of the original heart tube (Bur- 206 JERREL L. WILKENS DAA Gut SegLA Flex VNC FIG. 5. Drawing of a stylized transverse section of the 2nd abdominal segment of a lobster, Homarus americanus, showing the dorsal abdominal artery (DAA), the pair of segmental lateral arteries (Seg. LA) and the asymmetric large medial branch on one side. It is proposed that the sternal or descending artery is derived from one of the segmental lateral arteries. Ext, abdominal extensor muscles; Flex, abdominal flexor muscles; VNC, ventral nerve cord. (From Wilkens et al., 1997b). nett, 1984; Martin et al., 1989; Wilkens et assist or assume the primary role of pumpal, 1997b). The lateral walls of the dorsal ing blood as mentioned above for barnacles abdominal artery contain bands of muscle and small copepods and ostracods. The cor whereas all other arteries lack muscle. The frontale of the median anterior artery of contractions of these muscles in Homarus decapods (Steinacher, 1979) is an example are modified by the neurohormone procto- of an auxiliary pump where the small dilin in similar manner to cardiac muscle ameter and length of an artery would result (Wilkens et al., 19976). No function has yet in high resistance to flow. The guiding prinbeen identified for this muscle. If the dorsal ciple here is that the muscular development abdominal artery is derived from the orig- of the heart, in particular its ability to geninal heart tube, the absence of segmental erate force, must be correlated with the reostia may have occurred in parallel with the sistance and afterload to blood outflow that loss of abdominal gills (see below). Further, arises from the cumulative length of the arthe bicuspid valves at the origin of each of terial system and its branches and the mean the segmental lateral arteries leaving the blood pressure. When the circulatory pathdorsal abdominal artery and of the arteries way is short, as in quite small organisms, leaving the heart appear to be homologous the seemingly low pressure and low veloc(Alexandrowicz, 1932). ity pumping action of a pulsatile tube is adequate. Contraction of the tubular heart in FUNCTIONAL MORPHOLOGY: RELATIONSHIP branchiopods occurs as a peristaltic wave in BETWEEN BODY SIZE, LIFE SYLE AND THE T. longicaudatus (Yamigishi et al., 1997) COMPLEXITY OF THE CVS and Artemia sp. (Doyle and McMahon, unHeart published observations). The hydrodynamThe heart is generally the pump for the ics of tubular hearts await study, but the circulatory system, but other pumping pressures developed by peristaltic waves of mechanisms such as accessory pumps may contraction may be lower than those of a CRUSTACEAN CARDIOVASCULAR EVOLUTION heart that contracts as a single unit. Those forms lacking a heart are able to move blood (coelomic fluid) through the hemocoel by movements of the body and gut (McLaughlin, 1980). From a functional perspective, one can ask why the primitive heart tube that runs from the head to tail regions has been shortened in some forms. The heart tube may have been shortened in the Cladocera, Ostracoda and Copepoda as body segments were fused and eliminated. A second possible reason for condensation may have been the need for a more powerful pump to propel blood though longer and more highly branched arteries. Moving from the more primitive and smaller phyllocaridans to the most advanced and often large malacostracan decapods, the heart has changed from a thin walled tube no more than one muscle cell layer thick to a thick walled highly muscular ventricle. The proposed folding and the incorporation of at least three body segmental components of the original heart tube to form the decapod heart (Wilkens et al., 1991 b) may have allowed for the development of a thicker walled heart, one composed of multiple layers of muscle vs. the single layer of muscle found in tubular hearts. This would have allowed the heart to generate greater force which in turn would have allowed the heart to pump blood into longer and more extensive vascular spaces. A powerful heart is clearly a necessity to move blood throughout larger crustaceans such as crabs and lobsters. The lobster heart generates systolic pressure of 1.6 kPa as it overcomes a total peripheral resistance of 1.93 kPa s ml"1 (Wilkens et al., 1997a). Ostia Whereas the number of pairs of ostia may indicate the number of segments of the primitive heart tube that are retained, it is recognized that ostia may be lost from portions of the heart. Ostia are present in those abdominal regions of the stomatopod heart tube that receive oxygenated blood from gills or related gas exchange structures (Burnett and Hessler (1973), but they are absent in these regions in decapods that lack abdominal gills. It appears that the loss 207 of gas exchange structures in a group of animals has been accompanied by the loss of ostia from the corresponding regions of the heart tube. It is adaptive for the heart to only pump oxygenated blood. Peripheral circulation Video recordings show the movement of hemolymph in radiating and anastomosing tissue spaces in the integument immediately below the carapace in myodocopid and podocopid ostracodes (Abe and Vannier, 1995). The short diffusion distance across the thin cuticle indicates that the integumentary circulation plays a respiratory function. These elaborate channel patterns occur on the shells of fossil ostracodes dating from the middle Cambrian (Vannier et al., 1997). These findings point to the elaboration of a circulatory system early in the evolution of the Crustacea. The circulatory paths in the animals in the classes other than the Malacostraca are relatively short, and even the anostracans and notostracans that attain lengths of 10 cm are small in cross section. The smallest of these animals often lack blood vessels. The only vessel leaving the heart in any of these animals is a single anterior artery supplying the head region. Either segmental lateral arteries were never present or, less likely, they were secondarily lost. The absence of extensive arterialization may account for the small size of animals in these five classes of crustaceans. An arterial system may be a requirement to allow growth in body size beyond some minimal limit, or, stated another way, increased body size may require more extensive arterial arborization for adequate perfusion of tissues remote to the blood pumping organ. Highly aerobic tissues such as the central nervous system require a dedicated blood supply particularly if they are large or are down stream of other metabolically active tissues. This seems to apply to the vascularization of the decapod central nervous system (Sandeman, 1967; Shivers, 1970; Abbott, 1971), particularly if that tissue is behind a well developed blood-brain barrier (Abbott, 1970; Brown and Sherwood, 1981). Circulatory development therefore is clearly related to the require- 208 JERREL L. WILKENS ments for minimal diffusion distances. On the other hand, arterial development may not be correlated closely with activity levels if the animal is small, sedentary, or when there is an alternate route to supply oxygen and remove CO2 There is relatively great flexibility in the degree of development of the peripheral vascular system. For example, the polychaete and oligochaete Annelida possess an extensive closed vascular system made up of mesodermally derived arterial and venous vessels, but in the Hirudinea hearts are secondarily derived (McMahon et al., 1997). Flexibility in vascular system development is also seen in mammals where blood vessels readily extend into expanding tissues such as into adipose tissue of obese individuals, into neoplastic tumors, and even into transplanted tissues. The growth in body size and appendage length in the large Malacostraca may have stimulated elongation of the arteries to supply the "new" tissues. PHYSIOLOGICAL EVOLUTION Origin of heart beat Evolution has been conservative when it comes to functionally important molecules and processes; notable examples that extend across the animal kingdom include photoreception (Randall et al., 1997) and voltage gated ion channels (Hille, 1992). The conservative nature of successful evolutionary processes predicts that, because of its vital importance, the control of the heart contractile rhythm throughout the Crustacea will also be conserved. Until the last few years crustaceans were thought to possess neurogenic hearts in which the pacemaker was a small ganglion of neurons, the cardiac ganglion, located on or in the heart itself, even though pharmacological experiments indicated that anostracan and cladoceran hearts might be myogenic (Maynard, 1960). The acceptance of the universality of a neurogenic mechanism in the Crustacea should have been questioned given that the hearts of annelids and molluscs are all myogenic (McMahon et al., 1997). On the assumption that myogenic mechanisms are the more primitive, is it possible to identify at what point the switch from myogenic to neurogenic pacemaking occurred in this sub-phylum? The heart beat pacemaker of the notostracan T. longicaudatus is myogenic throughout the entire life cycle (Yamagishi et al., 1997). The simple tubular heart does not contain nerve cells, and tetrodotoxin (TTX), the highly specific blocker of neuronal activity, does not affect the beat rhythm. The myocytes are electrically coupled so that the heart is a functional syncytium. The myogenic pacemaker rhythm consists of 20-30 mV slow waves of depolarization without spikes that sweep via gap junctions along the length of the heart. The wave of depolarization will cause a peristaltic wave of contraction. For a heart beat arising from spontaneous myogenic pacemakers to work, the myocytes need to be linked by gap junctions that will allow the wave of depolarization to sweep across the entire muscle sheet. Myocytes in T. longicaudatus are coupled electrically and they function as a single muscle oscillator (Yamagishi et al., 1997). Because the Notostraca are considered to have been among the earliest groups of crustaceans to appear, the peristaltic contraction of a myogenic heart is probably the most primitive. Gap junctions are retained in mantis shrimp (Irisawa and Hama, 1965) and lobster (Van der Kloot, 1970) hearts. Moving to the Malacostraca we find that the heart of the adult isopod Ligia (Megaligia) exotica displays both myogenic and neurogenic activity (Fig. 6A, Ai, 1966). The posterior portion of the heart, behind the level of the 6 neuron cardiac ganglion, displays spike potentials riding on slow waves of depolarization when innervated by the cardiac ganglion, but only slow waves after it is sectioned from the rest of the heart (Fig. 6B). These latter slow wave potentials are typical of a pacemaker potential. Slow pacemaker potentials are not recorded from the anterior innervated regions of the heart, but rather action potentials arise abruptly from a stable baseline as would be expected if myogenic pacemaking were subordinated to neural drive (Fig. 6C). The anterior regions still can display myo- CRUSTACEAN CARDIOVASCULAR EVOLUTION B A arteries CG 209 \ / < s —. a ostia ,.-— —— 7 8 9 10 III 11 \ / 12 13 h FIG. 6. (A) Schematic drawing of Ligia exotica heart (dorsal view), (B) two examples of potentials recorded from segment 12. In the intact heart a spike arises from a slow a slow diastolic depolarization (i), but this is lost when input from the cardiac ganglion is removed when the heart is sectioned at segment 9 (ii), (C) electromyograms from the anterior half of the heart (i) from the dorsal surface of the anterior half of the heart and (ii) from a region hear the ganglionic trunk (From Ai, 1996). genie rhythmicity when functionally denervated (see below). The heart beat of the embryo and early juvenile L. exotica is strictly myogenic (Yamagishi, 1996; Yamagishi and Hirose, 1997). In early juveniles less than 5 days old the rate of slow wave muscle potentials is unaffected by TTX, although sodium-de- pendent spike potentials arising from them are eliminated (Fig. 7A). The cardiac ganglion of these early juveniles innervates the myocytes, but does not yet produce bursts of action potentials. Stimulation of the ganglion at this stage produces excitatory synaptic junctional potentials (EJP) in the muscle that are able to reset and entrain the 210 JERREL L. WILKENS A 50 mV 01s 50 mV a> 3 I "- 05s 0.5 min 0 5s B 100 mg cr FIG. 7. Effects of tetrodotoxin (TTX) on the cardiac ganghonic and muscle activities in (A) early juvenile two days after hatching and (B) adult Ligia exotica (Isopoda) hearts. (Al) Simultaneous intracellular microelectrode recordings from the heart muscle (upper trace) and extracellular recordings from cardiac ganglion (lower trace). (A2) Effects of TTX on heart muscle activity recorded intracellularly (upper trace) and frequency of muscle burst discharge (Hz, lower trace). The arrowhead shows the time when the perfusion saline was changed to TTX-containing saline (10 6 M). (B) The impulse activity recorded from the cardiac ganglion (top trace), the mechanical contractions of the heart (middle trace) and the frequency of the heartbeat (bottom trace). TTX (10 6 M) was applied during the period indicated by the black bar. (From Yamagishi and Hirosi, 1997). myogenic rhythm. The onset of spontaneous, but irregular, EJP-like small depolarizing potentials from the cardiac ganglion occurs between days 5 to 12. These EJPs are blocked by TTX. When the cardiac ganglion begins producing regular bursts of action potentials in the motoneurons that innervate the myocardium the heart becomes neurogenic. However, even adult hearts display slower oscillatory (myogenic) potentials after the neural activity is blocked by TTX (Fig. 7B). Thus, the myogenic rhythm has become subordinated to, but not eliminated by, neurogenic drive. The ionic conductances responsible for the myogenic rhythm persist in adults. The anatomy of the CVS of the Stomatopoda is considered to reflect a primitive condition (McLaughlin, 1980); however the heart of Squilla oratorio is electrically silent when isolated from the cardiac ganglion neurons (Shibuya, 1961). By the hypothesis proposed here, the loss of myogenicity represents a derived condition involving the loss of expression of the ionic channels that produce pacemaker potentials by the myocytes. Heart beat in adult decapods appears to be purely neurogenic; however, embryonic hearts may be myogenic. In H. americanus the heart arises from segmentally derived lateral blocks of mesoderm, and myoblasts CRUSTACEAN CARDIOVASCULAR EVOLUTION and neuroblasts are both present in embryonic hearts at the onset of contractions (Burrage, 1978; Burrage and Sherman, 1978). These authors concluded that the initial twitches of the heart result from cardiac ganglion input because the sides of the, as yet incompletely formed, heart twitched synchronously. There is no evidence to indicate whether at this stage of development the two blocks of muscle are in direct communication with one another. The increase in heart rate and regularity in beating on the second day after the initial beats may represent the proliferation of neuromuscular synapses and the continued development in the neuropil of the cardiac ganglion. Embryonic hearts that have just begun to beat (1 to 2 contractions during a 5 minute interval) contain a 9 neuronal cardiac ganglion. At this stage the embryonic heart has fully developed contractile units and innervation with distinct synaptic junctions; however, these morphological observations do not allow us to evaluate whether the heart beats are myogenic or neurogenic. In the decapod shrimp Metapenaeus ensis, TTX does not prevent heart beat in early juveniles of less than 5 mg body weight, but it does arrest heart beat in larger ones (Doyle and McMahon, 1998). In larger juveniles, whose heart beat has been arrested by TTX, electrical stimulation still causes heart contractions. Because these observations were made on intact animals by video techniques, it is not known whether the early juvenile hearts exhibit any membrane potential oscillations or whether the beat rhythm can be reset as would be expected if the rhythm was myogenic. Histological evidence of the presence of cardiac neurons in these seemingly myogenic stages is wanting at this point. The ostial valve muscles of crustacean hearts are considered to be a model system for the study of the myocardium much as trabeculae are used in the study of mammalian hearts (Yazawa, Wilkens, ter Keurs and Cavey, unpublished observations). The isolated ostia from adult H. americanus hearts do not exhibit spontaneous changes in membrane potential or contraction, but they contract when electrically stimulated. As with Squilla, adult Homarus myocardi- 211 um appears to have lost its myogenic pacemaker; however, not all attributes of a myogenic system are lost since gap junctions are retained in both hearts (Irisawa and Hama, 1965; Van der Kloot, 1970, respectively). The available data suggest that the hearts of embryonic and early juvenile decapods have retained the 'primitive' myogenic contractile rhythmicity, but that the myogenic potential is lost as neurogenic drive becomes established. I advance the hypothesis that the earliest crustaceans possessed myogenically driven hearts, but in more advanced groups there is a shift toward neurogenic pacemaking arising from a group of neurons, the cardiac ganglion, found on (stomatopods, Alexandrowicz, 1934) or inside (decapods, Alexandrowicz, 1932) the heart. There may be a strong evolutionary advantage to neurogenicity since it is found in all Malacostroca that have been examined. It is not know whether the cardiac ganglion of stomatopods and decapods is homologous. The evolutionary pressures that might have driven this process are not known, but the evolutionary opportunities provided may be substantial. Two possible advantages are envisioned. In Squilla the cardiac ganglion causes simultaneous contraction of the entire elongated tubular heart (Shibuya, 1961) which will generate more force that would the peristaltic waves of contraction in myogenic hearts. Such synchronous contractions are able to move blood into all arteries including the posterior artery, something that would be more difficult in forms where the tube contracts peristaltically. Second, it appears more efficacious for the cardioregulatory nerves from the central nervous system to synapse on the cardiac ganglion than to innervate the complete myocardium. The advantage of rapid responses is obvious in the abrupt cardiac arrest that is used as part of a defensive strategy by decapods to avoid detection by preditors such as sharks that locate prey by electroreception (Wilkens et al, 1974). EMBRYOLOGY Even though it is highly variable across the subphylum, the embryonic development 212 JERREL L. WILKENS of the CVS may provide some clues to the differentiation reflects the fact that the orievolution of this system in the Crustacea. gin and early elaboration of these systems Embryology does not tell us much about lies within the early history of the Metazoa the origins and interrelationships of crusta- itself. The Hox genes are a highly concean classes, but there is an underlying uni- served homeobox that programs a characty in terms of fundamentally similar fate teristic sequence of morphological gene exmaps of the blastula or blastoderm and the pression (Averof and Akam, 1993; Osorio retention of a nauplius state in development et ah, 1997). Does a cluster of homeotic (Anderson, 1982). The development of the genes also mediate the expression of genes postnaupliar somites is similar from bran- that specify the CV system? If or when the chiopods to the malacostracans including genetic analysis of developing body plans the elaboration of the segmental mesoderm is extended to intra-arthropod diversity, we that produces the cardiac and perivisceral may learn whether Hox-type gene clusters hemocoel. The segmentally derived meso- account for the different CV systems, or dermal pericardial septum proliferates into whether the types of plans are developmenthe pericardial hemocoel a mass of cells tal consequences of different body plans. that grows toward the midline and forms ACKNOWLEDGMENTS the wall of the heart (Kume and Dan, 1968; Anderson, 1982). The ostia arise from inI thank Dr. B. Chatterton, Department of tersegmental spaces between the cardiac Geology and Geophysics, University of Alcomponents of successive somites. The se- berta, for providing paleontological guidrial repetition of this pattern would produce ance, and Dr. B. R. McMahon for helpful the heart tube with intersegmental ostia comments on the manuscript. This study along the entire length of the embryo. If the was supported in part by the Natural Scimorphology of the adult heart reflects ence and Engineering Research Council of changes in body plan and circulatory de- Canada. mands, study of the post-naupliar developREFERENCES ment of this system across the subphylum may be instructive. By this argument, the Abbott. N. J. 1970. Absence of blood-brain barrier in heart of cladocerans, ostracods and copea crustacean, Carcinus maenas L. Nature (London) 225:291-293. pods may represent the embryonic contributions of one or two somites and one in- Abbott, N. J. 1971. The organization of the cerebral ganglion in the shore crab, Carcinus maenas. II. tersegmental pair of ostia. The potential for The relation of intracellular blood vessels to other the embryonic production of a thoracic brain elements. Z. Zellforsch. 120:401-419. 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