C The vascular anatomy of the Annelid worms has, in general, been little studied. This is especially true of marine worms of the class Polychaeta. The errent polychaetes (Errantia) seem to have received most of the attention; there are, for example, very adequate studies of the families Nephtyidae and Nereidae. The sedentary forms (Sedentaria) and those forms which cannot be easily placed into either grouping have generally escaped serious study. A prime example of this lack is the more or less sedentary family Cirratulidae. A detailed study of the vascular anatomy of Chartozone was done by Meyer in 1887; more general works exist on Audorina filagera and Cirratulus Setosa (Claparede, 1873), Cirratulus cirratus (Picton, 1898 and Courtney, 1958), Dodecaceria concharum, and Audorina tentaculata (Courtney, 1958). Cirriformia spirabrancha (Moore, 1904) is a member of this family in which the vascular anatomy has not been examined. In order to add to the limited knowledge of polychaete vascular anatomy, and also for the challenge and enjoyment of working with a completely unexamined species, g. spirabrancha was chosen for this study. The vascular organization, blood flow pattern, and blood vessel structure have been examined in detail. MATERIALS AND METHODS Specimens of C. spirabrancha were collected from shallow mud flats near the Municipal Pier, Monterey, California. The course of all the major vessels and most of the smaller branches was traced by dissection under a binocular dissecting microscope. Doubtful connections between vessels and the course of smaller ones were confirmed by the examination of serial sections. For dissection, the worms were lightly anesthetized in a solution of magnesium chloride and seawater. The region of dissection was then painted with 1% novocaine, permitting extensive surgery without excessive muscular stimulation. The anesthetized worms, when dissected, were allowed to revive in fresh seawater. The vessels thus exposed continued to exhibit what was assumed to be normal contractile behavior for periods of one hour or more. To determine flow patterns, india ink was injected into the vessels at various points. In most cases, the india ink quickly settled on the vessel wall near the point injected. However, a few specks would occasionally remain in solution, allowing observation of flow. For microscopic examination, the worms were fixed in Bouin's fluid and the sections stained with the Foote-Goldner modification of Masson's trichrome (Jones, 1950). RESULTS The vascular system in C. spirabrancha, as in most polychaetes, can be viewed as a combination of two general systems: a typical replicating segmental system, and a longitudinal system which is superimposed upon the segmental system. The segmental system generally consists of a series of contractile vessels which move blood through a roughly circular pathway within each segment. The major vessels of this system (see Figure 1,A) are the ventro-lateral vessels (VL), the dorso-lateral vessels (DL), the medio¬ lateral vessels (ML), the medio-ventral vessel (MV), and the branchia supply vessels (BS). The longitudinal system consists of five major vessels (see Figure 1,A): the dorsal vessel (DV), the supra-esophageal vessel (SV), the ventral vessel (VV), and paired lateral vessels (LV). These vessels communicate indirectly with each other through the segmental system except in the modified anterior segments and the pygidium, where the dorsal and ventral vessels communicate directly in the terminal anal segmental ring. Proceeding along the longitudinal axis of C. spirabrancha, variations appear in the vascular anatomy of both the longitudinal and segmental systems. These differences appear principally between the anterior, central, and posterior sections of the worm. For purposes of discussion, these sections are labelled I, II, and III. Section I refers to the prostomium and anterior segments 1-7. This section is characterized by the extensive modification of both the longitudinal and segmental vascular systems and by the presence of dorsally placed branchia on segment 6. Section II refers to the fully developed central portion of the worm, segments 10 through 200 (approximately). Despite minor variations in individual segments, the organization of the vascular anatomy here follows a consistent pattern. At the posterior end of this region, the longitudinal system is reduced by the joining of the dorsal and supra-esophageal vessels. Lateral branchia occur in almost every segment in this section. The posterior 100 (*) segments and the pygidium are referred to as Section III. Here, four longitudinal vessels are superimposed upon a segmental system which is similar to that of Section II. The major difference is a progressive simplification of the segmental system, proceeding posteriorly. A. Vascular Anatomy The dorsal vessel, suspended in a thin connective tissue mesentary below the dorsal muscle band, is continuous through¬ out Sections II and III. In Section II, a loose, spongy, intravasal organ commonly called the heart body is present. In a study of the heart body in polychaetes, Picton (1898) ascribed a mechanical function to the dorsal vessel in Cirratulids. It is not unreasonable to apply this conclusion to C. spirabrancha, since at systole, the lumen is completely obliterated by the contraction of the walls against the heart body. Kennedy and Dales (1958) examined the heart body in a related species, Audorina tentaculata, by long paper chromatography of extracts and other methods. They concluded that in addition to the possible mechanical function, the heart body is also a haematopoetic organ. The dorsal vessel is not continuous through Section I. It tapers somewhat in segments 9, 8, and 7, and in segment 6, the lateral vessels (LV) branch off from it. The dorsal vessel itself then bifurcates to form the dorsal branchia vessels (DBV). In its course through Section II, the dorsal vessel is about 5 mm. in diameter, narrowing to 2 mm. when passing through a septa. It is directly connected to the supra-esophageal vessel (SV) in each segment by a thin-walled vessel just posterior to the septa. At the posterior end of Section II, the dorsal vessel joins vessel SV and, throughout Section III, communicates directly in each segment with the capillary net about the gut and with vessel DL (see Figure 1,B). Vessel SV, lying just ventral to the dorsal vessel and directly above the gut is continuous throughout Section II. In each segment, one, two, or three small vessels branch from it and supply a capillary network about the gut. On the posterior side of each septa, the medio-lateral vessels (ML) branch off from vessel SV and proceed laterally, joining vessel DL. In Section I (see Figure 2), vessel SV continues beyond the terminus of the dorsal vessel. In segment 4, two small vessels branch from it and enter the dorsal muscle mass. In segment 1, vessel SV branches, again, the dorsal branch bifurcating to form the dorsal prostomial vessels (DPV); the ventral branch forming the supra- pharyngeal vessel (SPV). Vessels DPV supply the extensive capillary network in the body wall of the prostomium. Vessel SPV opens into the capillary net surrounding the pharynx. The circum-oral vessels (CO) drain these capillary networks and in segment 1, join to form the ventral vessel (VV). In segments 1-5, the ventral vessel receives blood from the lateral branchia through the latero-ventral vessels (LV). These branchia are supplied from a small vessel (ST) which runs along the exterior wall of the pharynx and drains the capillary network surrounding it. In segment 2, a branch proceeds dorsally from the ventral vessel, then bifurcates to form two smaller vessels which run to the individual nephridia. After the lateral vessels (LV) branch from the dorsal vessel in segment 6, they proceed ventrally to the lateral midline and then continue posteriorly throughout Sections II and III. These vessels usually terminate in blind endings approximately five segments anterior to the pygidium. In each segment, a branch from the lateral vessel (LV) either enters a branchia, if one is present, or ends abruptly at the body wall. As mentioned above, the segmental vascular organization throughout Section II repeats itself in each segment (see Figure 1,A). The medio-lateral vessels (ML) run from vessel SV to vessels DL which lie just inside the dorsal muscle band. Vessels DL connect dorsally to vessels PV, a pair of small vessels which run the length of the worm. Vessels PV supply both the dorsal muscle band and the dorsal body wall through a series of small branches. Vessels DL run ventrally from their connections with vessels ML, joining vessels LV and the efferent branchial vessels in small bulbs near the lateral body walls. Vessels LV run from these bulbs to the ventral vessel (VV) with small branches supplying both the ventral and lateral muscle bands. In each segment, the gut capillary plexus is supplied from vessel SV and drains into the sub-esophageal vessel (SIV) which runs directly to the ventral vessel. In Section III, the segmental vascular pattern is repeated as above, but there appears to be a general gradation of development from the fully developed segments of Section II posteriorly to the pygidium. Figure 1,B is a diagram of a section through a typical posterior segment. Here the segmental vessels connect directly to the dorsal vessel and vessel ML connects directly to vessel PV. In the most 0 posterior segments of this section, both vessels PV and DL are lacking; vessels ML connect directly to vessels LV. Ventrally, vessel SIV is not present; it is replaced by a direct connection between the gut plexus and the ventral vessel. B. Flow Patterns and Vascular Activity The general pattern of flow through the vascular system of C. spirabrancha is a partial interdigitation of two sub-patterns. The intra-segmental pattern of flow is generally circular. Blood moves ventrally from vessel SV through the gut plexus to the ventral vessel, then flows dorsally through vessel LV and vessel DL. Flow is then latero-medial through vessel ML back to vessel SV. Superimposed upon this pattern is the rather sluggish dorsal-anterior to ventral-posterior longitudinal flow pattern. The actual volume displacement, and in some vessels the direction of flow, is dependent upon the activity of the animal. The motivating force for blood flow is supplied by local contractions of muscular vessels walls. The activity, spreading along a vessel as a contractile wave, causes displacement of a volume of blood from one portion of the system to another. These contractile waves are irregular and frequently conflict with one another. Injury or irritation to a vessel at any point will set up contractile waves in both directions. This may cause flow to cease or to be reversed. The dorsal longitudinal vessel is the main contractile vessel. The peristaltic waves which pass along it originate in the segments just anterior to the terminal anal segmental ring and sweep cephalad. In Section II, both the supra¬ esophageal vessel (SV) and the dorsal vessel are involved, contracting simultaneously. As the contractile wave reaches the junction of vessels LV and DBV with the dorsal vessel, blood flows into both the dorsal branchia and the lateral vessels. In vessel SV, the wave of contraction ceases near segment 9 and only slight contractions are observed anterior to this. In an unanesthetized worm which has been secured to a dissecting pan in such a way that only limited movements are possible, blood flow in the non-contractile ventral vessel is scarcely perceptible. Flow here is probably dependent almost exclusively on body movements. In the lateral vessels (LV), contractile waves have not been observed, and blood flow here is probably also dependent upon body movements. The flow into and out of the lateral branchia appears to result from the extension and contraction of these organs. When the branchia are severed close to the body wall, blood flow from the body through the open vessels is minimal; however, when the distal end of the organ is removed, blood flows quite readily. 2 C. Histology of the Blood Vessels With the exceptions of the ventral and lateral vessels, the blood vessels of C. spirabrancha are all contractile. Hanson (1949) stated that the blood vessel walls of Polychaetae and Oligochaetae are composed of three layers: (1) an endothelium occasionally appearing as a continuous layer, (2) a layer of collagenous connective tissue, and (3) an outer peritoneal layer, either differentiated into a muscle coat, or, more often, with contractile fibers in the tails of stellate cells. Most of the blood vessels in this species follow this basic plan. Stellate cells occur on the walls of all segmental vessels and on the walls of the vessels within the branchia. A heavy muscular coat surrounds both the lateral vessels and the dorsal vessel. A thin band of connective tissue and a thin endothelium occur in the dorsal vessel; a thick layer of connective tissue occurs in the lateral vessels. The walls of the supra-esophageal vessel are composed of only two distinct layers: a connective tissue layer and a peritoneal layer of occasional stellate cells with contractile fibers radiating from them. DISCUSSION Although the blood vascular system of C. spirabrancha follows the general pattern of closed circulatory systems found in the class Polychaetae, it provides several anatomical surprises. The lateral vessels, found only in certain other 8 Cirratulids, are especially interesting. Superimposed upon the general dorsal-anterior, ventral-posterior longitudinal flow pattern with its segmental interconnections, these vessels make up a "sub-system" concerned specifically with providing the lateral branchia with a constant blood supply. The efferent vessels in these branchia lead directly into vessel LV. Here the blood is able to mix with blood moving dorsally in the segmental system. This provides a reasonably efficient system for the supply of oxygenated blood to the various tissues. This "sub-system" does not appear homologous to any of the branchial supply systems found in the other polychaetes reviewed. Another interesting feature of the vascular anatomy of C. spirabrancha is the anterior extension of the supra¬ esophageal vessel which supplies the capillary network of the pharynx and prostomium. In a closely related species, A. tentaculata, Courtney (1958) described a direct connection of the supra-esophageal vessel with the dorsal vessel in segment 7. The dorsal vessel, rather than terminating as in C. spirabrancha, proceeds anteriorly to supply these capillary networks. Due to the cursory nature of Courtney's paper, this connection may have been misinterpreted in A. tentaculata; however, anatomical differences of this sort do exist in closely related species of other families of polychaetes. Although there are some 70 (f) branchia present in 14 an individual worm, only five to ten of these are exposed above the substrate surface at any one time. No particular ratio of exposure could be established between the two different types. This presents an interesting problem, since these two types of branchia appear to differ only in vascular anatomy. No special experiments were performed to determine if any functional differences existed. When the worm is exposed to an anaerobic environment, however, it has been observed that the lateral branchia become turgid with blood, while the dorsal branchia maintain their normal ebb and flow. This would seem to indicate that the lateral branchia are more efficient respiratory organs, since a much greater amount of blood is thus exposed for gas exchange. Considered from a functional standpoint, the total circulation in C. spirabrancha does not present itself as a very efficient process. The lack of valvular structures in the connectives of the lateral vessels (LV) and the dorsal branchial vessels (DBV) with the dorsal vessel is particularly interesting. Blood flow into both the lateral vessels and the dorsal branchia must be very nearly random. Blood returning to the dorsal vessel from the dorsal branchia may flow into the lateral vessels, back into the dorsal vessel, or even return to the dorsal branchia. Flow into the lateral vessels is also uncontrolled. Since the lateral vessels are probably important in respiration, this is not particularly favorable. 18 Blood is supplied to the prostomium and pharynx from the supra-esophageal vessel. This is a remarkedly inefficient means of providing freshly oxygenated blood to these tissues, since no direct connectives exist between any of the branchia and this vessel. It is likely that this problem is solved by a rapid movement of blood into this area. Evidence for this conclusion is limited, but it has been observed that when the prostomium is removed from intact worms, profuse bleeding occurs. An attempt was made to compare the vascular anatomy of C. spirabrancha to that of other members of the family Cirratulidae. The literature on the family is quite limited and the works which do exist are generally cursory and incomplete. Therefore, although the vascular anatomy exhibits certain similarities to that described for both A. tentaculata and C. cirratus, the dissimilarities between these three species and the others studied are such that no general scheme for the family can be described. 3. 4. SUMMARY 1. The anatomical organization of the vascular system of g. spirabrancha has been studied in detail. Blood flow patterns and the fine structure of the blood vessels were worked out. The most striking observation was the discovery of a very specialized branchial supply system. This system consists of a pair of non-contractile lateral vessels which branch from the dorsal vessel in segment 6 and run posteriorly throughout the entire worm. In each segment, branches of this vessel run into the lateral branchia. No mechanical heart or hearts were found. The circulation of blood appears to occur as a result of a combination of three factors: contractile waves moving cephalad in the dorsal and supra-esophageal vessels, local contractions moving along individual vessels, and movements involving the entire worm. The main flow patterns are shown to consist of an interdigitation of two circulatory systems, one a primitive segmental type, and the second a posterior-anterior flow involving the whole worm. Neither system is independent of the other and no valvular structures were found. The result is that actual flow is often sluggish and quite inefficient in most of the worm. 5. An intravasal structure called the "heart body" was found 16 6. in the dorsal vessel. Similarities in the structure of this organ in C. spirabrancha and in related species in which the pigments from this organ have been studied lead to the conclusion that this may be a haematopoetic organ. The histology of the contractile blood vessels has shown them to be similar in structure to the contractile vessels in other polychaetes. ACKNOWLEDGEMENTS This work is supported in part by the Undergraduate Research Participation Program of the National Science Foundation, Grant GY-4369. Special thanks are extended to the staff of the Hopkins Marine Station of Stanford University for the assistance and quidance afforded me during my research. LITERATURE CITED Claparéde, E., 1873. Recherches sur la Structure des Annélides Sédentaires. 199 p. Geneve: Georg. Courtney, W.A.M., 1958. Certain Aspects of the Biology of the Cirratulid Polychaetes. PhD Thesis, University of London. Hanson, Jean, 1949. The Histology of the Blood System in Oligochaeta and Polychaeta. Biol. Reviews, Cambridge Philos. Soc., 24;2: 127-173. Jones, Ruth M., ed., McClung's Handbook of Microscopic Technique, 3rd edition, Hafner Pub. Co., N.Y., p. 249. Kennedy, G.Y. and Dales, R.P., 1958. The Function of the Heart-body in Polychaetes. J. Mar. Biol. Ass'n. U.K., 37, 15-31. Meyer, E., 1887. Studien über den Körperbau der Anneliden. Mitt. Zool. Sta. Nepal, Bd. 7:592-741. Picton, L.J., 1898. The Heart-body and Coelomic Fluid of Certain Polychaeta. Quat. J. Mier. Sci., 41: 263-302. C B. Dy GP/ WW - . A. (Fgase 4) D 768 4 2 .. : ..... AANV . . S.p th NP .... DBV HB ..— .. he 7. 4.. . LDPV 2 FIGURE LEGENDS Figure 1. A. Gross section of a typical segment in Section II. Semi-diagrammatic. DV, dorsal vessel; PV, small longitudinal vessels; ML, medio-lateral vessel; SV, supra¬ esophageal vessel; DL, dorso-lateral vessel; BS, branchial supply vessels; LV, lateral vessel; SIV, sub-intestinal vessel; VV, ventral vessel; LB, lateral branchia; GP, gut capillary plexus. B. Cross section of a typical segment in Section III. Vessels marked as in A. Figure 2. Semi-diagrammatical sketch of the anterior portion of C. spirabrancha (Section I). Vessels marked as in Figure 1 except for: MV, medio-ventral vessel; DBV, dorsal branchia supply vessels; SPV, supra-pharyngeal vessel; DPV, dorsal¬ prostomial vessel; CO, circum-oral vessel; ST, small vessel supplying anterior lateral branchia; M, mouth; DB, dorsal branchia; HB, heart body; NP, area in which the nephridia lie.