The circulatory system of phoronid larvae

Doklady Biological Sciences, Vol. 375, 2000, pp. 654–656. Translated from Doklady Akademii Nauk, Vol. 375, No. 5, 2000, pp. 712–714. Original Russian Text Copyright © 2000 by Temereva, Malakhov. GENERAL BIOLOGY The Circulatory System of Phoronid Larvae E. N. Temereva and Corresponding Member of the RAS V. V. Malakhov Received June 23, 2000 In most marine invertebrates, larvae possess many provisory structural characteristics which are adaptive for the larval stage and are not preserved in the adult animal. On the other hand, various heterochronies occur during the formation of the primordia of organs typical of adult forms: the formation of these primordia either occurs at early stages of larval development or is considerably delayed [1]. The circulatory system does not typically develop even in the largest larvae of marine invertebrates. Phoronidae is the only taxon of marine invertebrates in which the larvae have a circulatory system. The indications that a circulatory system is present in actinotrochs are found in the early studies on this taxon (studies by Metschnikoff [2], Caldwell [3], Roule [4], and Masterman [5]). Nevertheless, no complete anatomic reconstruction of the circulatory system of actinotrochs has been made to date. Moreover, the fine histological structure of the circulatory system of phoronids also remains unknown. The objective of this work was to reconstruct the anatomic organization of the circulatory system of phoronid larvae and to study the ultrastructure of this system. The object of this study was the larvae of two phoronid species at late developmental stages (immediately before metamorphosis): Phoronis ijimai (= Actinotrocha vancouverensis) and Phoronopsis harmeri (= Actinotrocha harmeri). The general anatomy of the circulatory system was studied by means of reconstruction based on a complete series of histological sections. The fine structure of vascular walls and erythrocytes was studied using a transmission electron microscope. For light microscopy, the larvae were fixed in 4% formalin, dehydrated, and impregnated with Paraplast. Then we made sections, mounted them, and stained them with hematoxylin. For electron microscopy, the larvae were fixed in a 2% glutaraldehyde solution based on a 0.1 M cacodilate buffer (pH 7.2) containing sucrose (100 mM). After washing, the larvae were additionally fixed with a 1% solution of osmium tetroxide and were stored in 70% ethanol. Then, the larvae were dehy- Department of Invertebrate Zoology, Faculty of Biology, Moscow State University, Vorob’evy gory, Moscow, 119899 Russia drated and impregnated with a mixture of araldites. Ultrathin sections were contrasted with uranyl acetate and lead citrate. The general anatomy and ultrastructure of the circulatory system of the larvae were very similar in both species studied. In all cases (if not specified in the text), the description given in this study refers equally to both species studied. The hemocoel, the central organ of the circulatory system in phoronid larvae, a circular cavity is situated between the second and first septs, i.e., the residue of the primary body cavity (blastocoel). The circle of hemocoel surrounds the esophagus; this is the place of separation of both radial tentacular blood vessels and three axial vessels: the odd dorsal vessel and paired ventrolateral vessels (Fig. 1). Tentacular vessels run along tentacles and are completely enclosed on all sides by the horseshoe-shaped (on cross sections) coelomic canal. The dorsal vessel is a tubular canal running along the dorsal wall of the stomach up to the approximate beginning of the posterior part of the stomach. Ventrolateral vessels are wide canals between the wall of the stomach and the coelothelial lining of the stomach’s surface (splanchnopleura). These canals run down to the approximate medial part of the stomach. In addition to the aforementioned main blood vessels, there are some irregularly arranged vessels forming the hemal plexus on the surface of the basal membrane of the stomach (not shown in Fig. 1). The wall of the blood vessels is formed by monociliate epithelial-muscular cells. The cells of the wall are arranged into one layer and are connected to one another by interdigitation contacts (the outgrowths of a cell enter the notches of another; Figs. 2a, 2b). Specialized desmosome-like contacts were found both on the side directed to the lumen of the vessel and on the side facing the body cavity. The cells of the vascular wall are separated from the lumen of the vessel by a 100- to 200-nm-thick layer of extracellular matrix. Single thick fibers (80 to 100 nm in diameter), which are oriented along the axis of the vessel, are included in the thickness of the extracellular matrix (Fig. 2a). In some sections, the cell surface facing the vessel’s inside appeared as numerous evaginations, where the layer of extracellular matrix was shaped similarly to the curves of these evaginations (Fig. 2a). In other cases, the internal surface of the vessel was smooth (Fig. 2b). Proba- 0012-4966/00/1112-0654$25.00 © 2000 MAIK “Nauka /Interperiodica” THE CIRCULATORY SYSTEM OF PHORONID LARVAE 655 cc c hem bb sr mf ex tf lv bms (a) c cc tbv mf vbv lv am dbv ex bms (b) Fig. 1. A scheme of the circulatory system of a phoronid larva at late developmental stages. The number of tentacles is deliberately reduced. Designations: dbv, dorsal blood vessel; hem, hemocoel; tbv, tentacular blood vessel; vbv, ventrolateral blood vessel. bb sr (c) bly, this depends on the degree to which the vessel is filled with liquid. Contractile filaments are located in the basal (i.e., facing the lumen of the vessel) part of cells and can be oriented either longitudinally, transversally, or at different angles to the axis of the vessel (Fig. 2b). The apical (facing the coelom) cell surface is supplied with cilia. The base of a cilium is submerged into the pit on the cell surface, and a short streaked rootlet goes from the basal body (Figs. 2a, 2b). The basal body and the rootlet are supplied with the lining formed by the cisterns of the Golgi apparatus. Vascular walls have different widths, which also vary depending on the degree to which the vessel is filled. Nevertheless, the walls of lateral and tentacular vessels are, as a rule, formed by flattened cells, whereas the wall of the dorsal vessel consists of elevated, bulbshaped cells (Fig. 2b). In the latter case, the nucleus is situated in the widened apical part of the cell. The cells DOKLADY BIOLOGICAL SCIENCES Vol. 375 2000 Fig. 2. Ultrastructure of the elements of the circulatory system in a phoronid larva: (a) a structural scheme of the wall of the ventral blood vessel; (b) a structural scheme of the wall of the dorsal blood vessel; (c) aggregation of erythrocytes (the scale marker is equal to 1.4 µm). Designations: am, amoebocyte; bb, basal body; c, cilium; cc, coelothelial cells of vascular wall; bms, basal membrane of the stomach; ex, extracellular matrix; lv, lumen of vessel; mf, myofilaments; sr, streaked rootlet; tf, thick fibers in the extracellular matrix. of the vascular wall have ovate or flattened nuclei; the chromatin is arranged as large lumps. The nuclei contain one or two nucleoli. The cytoplasm includes the channels of rough endoplasmic reticulum, mitochondria, and vacuoles with loose content (Figs. 2a, 2b). The lumen of vessels contains loose granular material. Single amoebocytes occur in some vessels (especially in the vessels of the preintestinal plexus). These 656 TEMEREVA, MALAKHOV are the cells that have irregular shapes and outgrowths (Fig. 2b). In these cells, the nucleus has a large nucleolus. In the cytoplasm of amoebocytes, we found the vesicles of smooth reticulum, which vary in size, and vacuoles with loose content. Erythrocytes form round-shaped aggregations in the cavity of the circular hemocoel, which are easily discernible in live larvae, owing to the presence of hemoglobin, a bright red respiratory pigment. Ph. ijimai larvae have two such aggregations; in Ph. harmeri larvae, four aggregations are typically found. Larval erythrocytes are ovate or irregularly shaped cells; their diameter is 5 µm (Fig. 2c). They are arranged close to each other; however, we did not found any specialized cellular contacts between them. The nuclei of erythrocytes are ovate and have one or two nucleoli. The cytoplasm of erythrocytes appears electron-dense because of abundant microgranular material, which is likely to represent the respiratory protein (hemoglobin). Sparse mitochondria have a dense internal matrix. Transparent vesicles of various size are found in the cytoplasm of erythrocytes. An unusual feature of larval erythrocytes is the presence of rudimentary cilia (Fig. 2c). These rudiments are represented by a basal body which is situated near the surface of an erythrocyte and has a short streaked rootlet. The basal body and the rootlet have a lining of packed cisterns of the Golgi apparatus (Fig. 2c). We did not observe the undulapodia of cilium in any section studied. Larvae and adult forms of Phoronidae differ considerably in the general anatomy of the circulatory system. This fact is in good agreement with the fundamental difference in the structural pattern of larvae and adults. The entire phoronid body is known to be formed in the course of metamorphosis from the ventral metasomal outgrowth; this is why the main axes of the larval and adult forms do not coincide [6]. The change that the circulatory system of phoronid larvae undergoes during metamorphosis remains poorly studied. We can only assume the presence of some accordance between the larval and definitive circulatory systems. For example, the circular hemocoel of larvae is likely to bring the circular vessels of the lophophore into origin. The radially directed tentacular larval vessels can be considered the primordia of the blind tentacular vessels of adult forms. The dorsal vessel that runs along the intestine displays very fine correspondence with the medial vessel of adult phoronids. Probably, two short lateral larval vessels correspond to the primordia of lateral vessels of adult forms; however, this question requires a special study. A comparison between the fine structure of vessels in larvae and adult forms is equally interesting. Emig [7] distinguished four types of the organization of blood vessels in adult phoronids. Strictly speaking, larval vascular walls do not completely correspond to any of these types. Most of all, they are similar to type 2 in Emig’s classification [7]. This type is characterized by the presence of a uniform cellular wall (which is formed by coelomic myoepithelium), extracellular matrix, and a discontinuous intravascular endothelial layer. Thus, the only difference in the structure of the vascular wall of larvae from that of adult forms is the lack of endothelium. However, appendiculate amoebocytes, which are particularly abundant in the intestinal plexus and in the dorsal vessel, may fulfill the function of endothelium. The erythrocytes of larvae considerably differ from those of adult forms in the general shape of cells (erythrocytes are spherical in adult phoronids and irregularly shaped in larvae), density of cytoplasmic hemoglobin (in adults, the cytoplasm of erythrocytes is characterized by very high electron density), and the presence of rudimentary cilia in larval erythrocytes. The latter feature suggests that erythrocytes have originated from the ciliate cells of the coelomic epithelium. Taken together, these data indicate that, the circulatory system of phoronid larvae is characterized by a high anatomic complexity and a relatively high level of cell differentiation. Probably, the early development of the larval circulatory system is related to the important role of the circulatory system in vital functions of phoronids. In fact, having a relatively low level of differentiation of other systems of organs, phoronids are characterized by very complicated, voluminous, and, moreover, closed circulatory system. REFERENCES 1. Ivanova-Kazas, O.M., Evolyutsionnaya embriologiya zhivotnykh (Evolutionary Morphology of Animals), St. Petersburg: Nauka, 1995. 2. Metschnikoff, E., Z. Wiss. Zool., 1871, vol. 21, pp. 286– 313. 3. Caldwell, W.H., Proc. R. Soc. London, 1882, vol. 34, pp. 371–383. 4. Roule, L., C. R. Acad. Sci. Paris, 1896, vol. 122, pp. 1343–1345. 5. Masterman, A.T., Quart. J. Microscop. Sci., 1897, vol. 40, pp. 281–339. 6. Beklemishev, V.N., Osnovy sravnitel’noi anatomii bespozvonochnykh (Basic Comparative Anatomy of Invertebrates), vol. 1: Promorfologiya (Promorphology), Moscow: Nauka, 1964. 7. Emig, C.C., Mar. Biol., 1982, vol. 19, pp. 2–90. DOKLADY BIOLOGICAL SCIENCES Vol. 375 2000