Architectonics of the Central Nervous System of Acoela ... - Springer Link

ISSN 00220930, Journal of Evolutionary Biochemistry and Physiology, 2008, Vol. 44, No. 1, pp. 95—108. © Pleiades Publishing, Ltd., 2008. Original Russian Text © Kotikova, Raikova, 2008, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2008, Vol. 44, No. 1, pp. 83—93.

MORPHOLOGICAL BASICS FOR EVOLUTION OF FUNCTIONS

Architectonics of the Central Nervous System of Acoela, Platyhelminthes, and Rotifera E. A. Kotikova and O. I. Raikova Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia Email: [email protected] Received June 27, 2007

Abstract—Based on the literature and own data, consecutive stages of development of the central nervous system (CNS) in the lower Bilateria are considered—separation of brain from parenchyma, formation of its own envelopes, and development of the trunk and orthogonal nervous system. Re sults of histochemical (cholinergic and catecholaminergic) and immunocytochemical (5HT and FMRFamid immunoreactive) studies of the CNS in representatives of Acoela, free living and par asitizing Platyhelminthes and Rotifera are considered. The comparative analysis makes it possible to describe development and complication of the initially primitive Bilateria plexus nervous system. A special attention will be paid to the Acoela phylogenesis, based on molecularbiology data and results of study of their nervous system. DOI: 10.1134/S002209300801012X Key words: nervous system, Acoela, Platyhelminthes, Rotifera.

Catenulida and Rhabditophora [6]. The latter in cludes both the major part of freeliving and all parasitic flatworms. Until now, no clear synapo morphies combining these two groups have been found. Rhabditophora apart from macrostomids form the Trepaxonemata group with a certain type of spermatozoid axoneme (the spiral central cyl inder) [6], which characterizes it as the monophyl etic group. For the last quarter of the XX century, his tochemical and immunocytochemical (ICC) methods have become predominant for study of the invertebrate nervous system by using fluorescent and confocal laser scanning microscopes. These techniques have made it possible to reveal parts of the nervous system with a certain ergity and im munoreactivity (IR), which expanded significant ly the results obtained using classic histological methods. Due to the recent revision of the Bilateria mac

INTRODUCTION Phylogeny of lower Bilateria has been essential ly revised for the last few years due to results of mo lecularbiological studies. The basic state of Acoela (acoelic turbellaria) among Bilateria was accepted by outstanding zoologists, particularly by our teacher Academician A.V. Ivanov. However, until recently, this group was placed among Platyhelm inthes (flatworms) and thereby the entire group of flatworms turned out to be in the base of the Bila teria tree. The current molecular phylogeny indi cates that Acoela is the most ancient branch of the Bilateria tree, which is independent of Platyhelm inthes [1–3]. Nowadays, Platyhelminthes are as cribed to the Lophotrochozoa group combining animals with trochoforelike larva (annelids, mol luscs, etc.) or with a lophophore [4]. Rotifera are also ascribed to this group [5]. Recently, Platyhel minthes have been subdivided into two groups: 95

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ACOELA

Fig. 1. Structure of the AChergic nervous system. (a) The acoelian Actinoposthia beklemishevi, (b) the turbel larian Gieyzstoria expedita.

rosystem, it seems essential to analyze the exten sive material on the nervous system structure and to evaluate morphologic data on the central ner vous system (CNS) including brain, longitudinal trunks, and their connecting commissures. CNS appeared in the flatworms. It is to trace the initial stages of improvement not only of all elements of this system separately, but also of the system as a whole, and to analyze pathways of its evolution in Acoela, Rhabditophora, and Rotifera. For perfor mance of the phylogenetic analysis, 150 species of Bilateria belonging to Acoela (24 species), Platy helminthes—Rhabditophora, including freeliving turbellaria (64 species), cestodes (22 species), trematodes (16 species), monogeneic flukes (9 spe cies), and rotifers (15 species) were studied. Cho linergic (AChergic), catecholaminergic (CAer gic) and 5HT and FMRFamide or GYRF amideimmunoreactive (IR) parts of the nervous system were examined. Peculiarities of exposure of all parts of the nervous system and their interac tion are an index of their developmental level, while the level is determined by analysis of the obtained data.

Nervous apparatus of the acoelic turbellaria is characterized by unusual diversity and gives exam ples of incomplete state that is usually interpreted as initial for all Bilateria [7]. We called the acoelic nervous system the trunk nervous system [8] and the brain—the commissural brain [9]. Histochem ical and ICC studies showed that Acoela brain looks like a ring, arch, or several commissural rings connected with longitudinal connectives [9–11]. It has no its own membrane and is penetrated by muscle fibers and ducts of frontal glands [12, 13]. Some longitudinal AChergic trunks run from the brain; they lose strong longitudinal orientation at the posterior end of the body and fuse with the sub muscular nervous plexus twining round the whole body of acoelic turbellaria [14]. The dorsal part of the Acoela nervous system is always developed bet ter than the ventral one regardless of the brain shape and the umber of trunks (Fig. 1a). In Platy helminthes Rhabditophora and most Spiralia, the dorsal nervous trunks are never dominating (Fig. 1b)1. Both brain and the nervous trunks demonstrate selective localization of 5HT and FMRF(GYIRF) amid IR without colocalization of polypeptides and monoamines in the same cell. In the brain area, many small and several pairs of large 5HTIR neu rons are found (Fig. 2a), mainly in the sites of in tersection of connectives and commissures. The FMRF(GYIRF)amid IR reveals symmetric clus ters or 1–2 symmetric pairs of large peptidergic neurons (Fig. 2b) [11]. Our studies have confirmed the dual (orthogon + endon) nature of the brain [15]. The 5HT and FMRFamide IR elements correspond to the brain “orthogonal” part (Figs. 1a, 1b). However, these IR elements were not found in the other, endonal, 1 Abbreviations for figures. AA—Anterior arch; AC—anteri

or commissure; C—commissure; CC—circular commis sure; DC—dorsal connective; DLN—dorsolateral neuron; DLS—dorsolateral trunk; DN—dorsal neuron; DS—dor sal trunk; E—eyes; ES—elliptical structure; B—brain; BN—brain neuron; LC—lateral connective; LS—lateral trunk; N—neuron; Ne—neuropil; PC—posterior commis sure; S—statocyst; VLN—ventrolateral neuron; VLT— ventrolateral trunk; VN—ventral neuron; VT—ventral trunk.

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 44 No. 1 2008

ARCHITECTONICS OF THE CENTRAL NERVOUS SYSTEM

brain part connected with statocyst. Earlier, it was suggested that the endonal brain (the statocyst brain) is homologous to the cerebral ganglion of Platyhelminthes [15]. The absence of the 5HT or FMRF(GYIRF)amide IR in the statocyst gan glion of Acoela casts doubt on this homology, as the cerebral ganglion of Catenulida and Platyhel minthes Rhabditophora is characterized by a pro nounced 5HT and FMRFamide IR, which is related to the central neuropil and to its surround ing brain neurons [10]. The current taxonomy of Acoela, which only begins to be elaborated [16], takes into account new signs obtained by current techniques of electron microscopy, ICC, and data of molecular biology. Some stages of evolution of the Acoela nervous apparatus can be traced in representatives of the lower and higher families by taking new signs into account. The lower Acoela are characterized by the 9 + 2 pattern of spermatozoid axonemes and by the presence of cortical cytoplasmic microtubules [17, 18]. Among these families are Paratomellidae, Diopisthoporidae, Haploposthiidae, Solenofilo morphidae, Actinoposthiidae, and Isodiameth tridae. The higher Acoela characterized by modi fied axonemes of spermatozoid and axial and dis tal cytoplasmic microtubules include Mecynosto midae, Childiidae, Convolutidae, Sagittiferidae, and Anaperidae. In Diopisthoporus longitubus, ICC revealed a sub muscular brain ring at the anterior end of the body and extensive superficial plexus. A similar brain structure, according to electron microscopy and traditional histology, is seen in Paratomellidae and Solenofilomorphidae. In Haploposthia viridis and H. lactomaculata, ICC revealed a small superficial (but submuscular) commissural brain. According to data of classical histology, H. brunea [19] and Kuma albiventer [20] have a welldeveloped diffuse nervous plexus in the covering layer and the stato cyst ganglion. Thus, within the limits of the Hap loposthiidae family, several stages of immersion of the nervous system elements into parenchyma un der the musculature are traced [12, 15]. Actinopo sthia beklemishevi has a commissural brain with one or two ringshaped commissures with 8 longitudi nal connectives converted into longitudinal ner vous trunks (Figs. 1a, 2a, and 2b). Faerlia fragilis and F. glomerata among Isodiametridae are ob

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served to have a poorly developed superficial brain with several ringshaped commissures [10, 11]. At exit from brain the nervous trunks are lost in the superficial nervous plexus. At the same time, Ava gina incola (Isodiametridae) is characterized by the clearly pronounced bilaterally symmetric dorsal brain immersed deeply into parenchyma [11]. Among the higher Acoela, the nervous system in representatives of the Childiidae family, such as in 7 species of the Childia genus has been studied in details by using ICC methods [9]. Simultaneous ly, the monophyletic origin has been confirmed using a phylogenetic analysis of 18 S rDNA. Closely related species, despite significant differences, nev ertheless turned out to have common pattern of the nervous system structure without random varia tions. Evolution of the nervous system was traced in Childiidae from the primitive state (C. cyclo posthium) with the barrellike brain and several ringshaped commissures and up to the most de veloped bilaterally symmetric dorsal brain in C. brachiposthium, C. macroposthium, and C. crassum. Structure and evolution of the nervous system in side the Childiidae family was found to be in agree ment with the phylogenetic tree based on molecu lar data. Some synapomorphies have been revealed for some phylogenetic branches. Simultaneously, some cases of parallel evolution were identified. The obtained data allow stating with certainty that evolution of the nervous system goes in parallel and independently in different phylogenetic branches within the limits of each family, i.e., it cannot be said that representatives of the lower Acoela as a whole have the more primitive nervous system than representatives of the higher Acoela. In each fam ily, both primitive and the higher developed rep resentatives can be found. Development of the nervous system in different branches of acoelic flatworms starts from a diffuse superficial nervous plexus and then immersion of its elements deeply into the parenchyma occurs; these processes run in parallel and independently in different families. In primitive species, trunks are irregular and poorly distinguishable from the plexus. In the majority of Acoela, we consider four pairs of longitudinal trunks as a primary state. There are forms with an additional nonpaired dorsal trunk (6 species Childia [21] as well as Bal talimania sp. [22]). In some families (Haploposthi


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Fig. 2. Immunoreactivity in the brain area. CLSM. The acelian Actinoposthia beklemishevi: (a) 5HT IR, (b) FMRF amide IR; the turbellarian Castrella truncate: (c) 5HT IR, (d) FMRFamide IR; the wheel animalcule Asplanchna herricki: (e) 5HT IR, (f ) FMRFamide IR. Scale bar (μm): (a)–(c) 50, (e), (f ) 20.

idae, Convolutidae, and Sagittiferidae), an oligo merization of trunks to 3 pairs occurs; different

trunks are retained during oligomerization, al though a trend for predominant development of



dorsal trunks is observed. In large forms (Ana peridae), an additional pair of lateral trunks ap pears and the number of trunks reaches 5 pairs. PLATYHELMINTHES—CATENULIDA CNS of the catenulide Stenostomum leucops [10], like in all higher taxa of Platyhelminthes, con sists of the doubleblade brain with 5HT and FMRFamide IR and distinct fibrillar neuropil, pair of the main longitudinal nervous trunks asso ciated with 5HTlabeled neurons and some poorly developed pairs of minor trunks associated with nervous plexus. Colocalization of the 5HT and FMRFamide IR has not been found. The FMRF amide IR is dominating in ventral trunks, while in other trunks the 5HT IR nervous fibers are pre dominant. PLATYHELMINTHES—RHABDITOPHORA Our study of the Rhabditophora group includes freeliving turbellaria of Macrostomida, Proseria ta, Lecithoepiteliata, Prolecithophora, Polycladia, Temnocephalida, and Rhabdocoela orders, Typhloplanida, Dalyellioida, and Kalyptorhynchia suborders, while cestodes, monogeneic flukes and trematodes were taken for study from the parasitic Rhabditophora. In all the abovementioned ani mals, we described the orthogonal nervous trunk, which we subdivided to 8 orthogon types as follows: regular frequent, regular rare, irregular, uneven, concentrated, cellular, radiated, and longitudinally polymerized [8]. Four orthogon types are found in turbellaria of the Temnocephalida order and Typhloplanida suborder and in Trematoda of par asitic worms. So far, only one orthogon type was found in Lecithoepitheliata. Without going into details, it should be emphasized that one orthogon type can be seen both in two closely related groups (e.g., in preseriates and prolecithophores with reg ular rare orthogon) and representatives of two types, like this occurs in Rhabdocoela and Rotifera with concentrated orthogon. Turbellaria. Shape of turbellarian brain varies depending on taxonomic state of individual group, body shape and sizes as follows: arcshaped (Mi crostomum lineare—Macrostamida, Graffila graffi—Dalyellioida); ringshaped (Anthobotrium

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cornucopia—Cestoda); ellipsoidal (Archiloa uni punctata—Proseriata), butterflylike (Macrorhin chus crocea—Kalyptorhynchia, Prolecithophora sp.—Prolecithophora); dumbbelllike (Anoplodium mediale—Dalyellioida, Scutariella georgica—Tem nocephalida); trapezoid (Thalassovortex tyrrheni cus—Dalyellioida); doubleblade (Gyratrix her maphroditus—Kalyptorhynchia, Pterastericola ve dotovi—Dalyellioida); horseshoelike (Geocentro phora wagani—Lecithoepiteliata). Brain of most flatworms, like in Acoela, does not have its own membrane. Thus, among neurons of planaria, processes of other cells also are detected [23]. However, there are several species with en capsulated brain: these are prolicethphores [24– 25], lecithoepitheliates [26], proseriates [27, 28], and polyclades [29]. The presence of a protecting capsule is undoubtedly a progressive feature. Electron microscopic, histochemical, and ICC studies of the brain cell composition of the studied groups are few and very fragmentary, but even these scarce data demonstrate significant qualitative and quantitative variations inside the studied group. Thus, for example, several types of neurons were revealed in the brain of the turbellarian Microsto mum lineare [30, 31], while brain of the polyclade Notomata contains 4 types of neurons [32]; among them there are extremely neurons that resemble vertebrate neurons that are absent in other flat worms. Catecholaminergic brain cells of different tur bellaria most often are unipolars, although bipo lars can also be found, while multipolars are very rare. Cell sizes vary from 5 to 12 μm. The number of neurons also varies, for instance, from 4 in Prox enetes flabellifer, Typhloplanida, to 10 in Gyratrix hermaphroditus, Kalyptorhinchia (Fig. 3a), with the different number of CAergic neurons observed both the same order and in the same family. The CAergic neurons were mapped in 5 Rhabdocoela orders: three species of typhloplanids—Mesostoma lingua, Botromesostoma essennii, and Rhynchome sostoma rostratum and two daliellids Castrella trun cata and Gieysztoria cuspidata [33]. Despite essen tial variety of their orthogons, the number of brain neurons varied insignificantly—from 4 to 5 pairs with three geometrical patterns of localization. The first pattern is as follows: the bladeshaped out growth of neuropil has neurons on each side (B.


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Fig. 3. Structure of the CAergic system. (a) The tur bellarian Gyratryx hermaphroditus, (b) the wheel ani malcule Platyias quadricornis.

essennii, M. lingua, G. cuspidata); the second pat tern is a fanshaped localization in C. truncata with archlike expanded brain; the third pattern is found in R. rostratum with threestore distribution of neu rons and the coneshaped proboscis. On the other hand, the equal number of neurons was found in different families, e.g., Macrostomidae (M. curvi tuba) and Typhloplanidae (Trigonostomum breifus si) have 4 CAergic neurons in the brain. The max imal number of CAergic neurons (6–7 pairs) was revealed in the encapsulated brain of the proleci thophore Pseudostomum quadriculatum [34]. We carried out the ICC study of distribution of 5HT and FMRFamide IR brain neurons in three Rhabdocoela species: C. truncata (Figs. 2c, 2d), B. essennii, and M. lingua. These brain neu rons are located on the dorsal and lateral sides of the densefibrous archshaped neuropil. The num ber of the neurons varies from 3 to 9 pairs with cor responding dimensions from 4 to 12 μm [35, 36]. In dalyellids, the center of neuropil has an FMRFamide IR fibrous ellipsoid structure sur rounded on the dorsal side by 7 pairs of neurons (in C. truncata, as an example) (Fig. 2d). A constituent of the CNS are longitudinal trunks and their connecting commissures. As a rule, Rhabditophora has 3 pairs of trunks. There are dif ferent patterns of the trunk connection with brain. Most developed are ventral or ventrolateral trunks. The main, ventral trunks, as a rule, run directly

from the brain (Figs. 1a and 3a), while lateral and dorsal trunks are connected with the brain through 1–3 pairs of roots. In Rhabdocoela, ventral and lateral trunks have a common root (Gieysztoria ex pedita, Dalyelloida) (Fig. 1b) and this also is occa sionally observed in the ventral and dorsal trunks (B. essennii, Typhloplanida) [34]. Some species show maximal concentration of trunks and all three pairs run directly from the brain as the common root (Provortex karlingi, Dalyellioida). Apart from the main longitudinal trunks, turbellaria with a deep immersion of the brain and proximal areas of the trunks into parenchyma have some additional structures, more often in the form of anterior branches of the ventral and dorsal trunks (Geocen trophora levanidorum, Lecithoepitheliata; Frid maniella bargusinica, Prolecithophora) [37]. We revealed a specific structure in large lecithoepithe liats of the Geocentrophora genus. Very thin sub epithelial strands go over the ventral and dorsal submuscular trunks. The trunks and strands are connected with each other by many regularly dis tributed processes. Undoubtedly, this is a second ary new formation that is to be considered as the initial stage of formation of a novel longitudinal trunk or a way of enhancement of the conductive apparatus in a large turbellarian. In all studied Rhabdocoela species, longitudi nal fibers and CAergic, 5HT and FMRFamide IR neurons are distributed along all longitudinal trunks. The number of these neurons is small, but it is always constant—from 1 to 5 pairs in different combinations. Thus, C. truncata along the lateral trunks has 5 pairs of CAergic and 2 pairs of 5HT and FMRFamide IR neurons each, along the ven tral trunks—5 pairs of 5HT and 3 pairs of FMRF amide IR neurons; and along the dorsal trunks— 1 pair of 5HTIR neurons. Most pronounced in Mesostoma and Bothromesostomata are proximal and distal pharyngeal ganglia located in the sites of intersection of ventral trunks with transversal cir cular commissures. These sites have one pair of FMRFamide IR neurons and 3 pairs of 5HT IR neurons and 4 pairs of 5HT IR and 3 pairs of FMRFamideIR neurons, respectively, in the two studied species. Fibers of different transmitter specificity and IR run in parallel and FMRFamide IR fibers always are located under the 5HT IR fi bers.



Longitudinal trunks are connected with com missures, whose number and shape diversity char acterize type of orthogon peculiar to the turbellar ian species. All trunks are connected to the single whole by a submuscular nervous plexus looking like a cellular network with the network fibers lying at once above the trunks. The more developed Rhab docoela have stable distribution pattern both of trunks and of commissures, including cases of for mation in their intersection of a diamond or a tra pezium (Provortex karlingi and Thalassovortex tyr rhenicus—Dalyellioida) [38]. Morphological diver sity is typical of structure of the turbellarian ner vous system. Comparison of the groups according to the character of their morphologic diversity con tributes to reveal a picture of morphologic radia tion. Such approach was used by Mamkaev [39] who checked it on a great many of facts and for mulated the principle of morphologic radiation. Monogenea. The lower monogenea (Tetraon chidae, Dactylogyridae, and Ancyrocephalidae families) are characterized by regular rare orthogon with an archshaped brain and 3 pairs of longitu dinal trunks, of which the ventral and lateral ones trunks outgo as the common root from brain, while the dorsal trunks—from their specific characteris tic frontal semiring located above the brain. Only a few commissures are revealed between the trunks [40]. A peculiar group of the lower monogenea, Acanthocotilidae—Acanthocotile verrilli has a semilunar brain with a narrowed medial area [41]. Ventral and dorsal trunks outgo from the latero posterior horns as the common root, while the dor sal trunk roots run forward. Frontal semiring lo cated in front of the brain has lateral parts almost as powerful as the ventral trunks. Running from the semiring are additional dorsal nerves going in par allel to the main dorsal trunks till the posterior end. A pair of additional nervous strands that we call the additional lateral and medial nerves are locat ed along each ventral trunk. Lateral trunks lay on the abdominal side, slightly apart from its lateral edges. Ventral trunks run close to each other at a short distance of the proximal area and then di verse again. Two circular and one dorsal commis sures are located at the equal distance behind the cerebral area. In plexus of the 2.5–3 mm long worms, the thickest cords are predominantly of transverse orientation, while in largest worms, up

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to 4.5 mm in length, the plexus has a more chaotic structure with the plexus fibers ending by a small AChpositive terminal distributed near the body surface. In the depth of the worm, crosssections demonstrate welldistinguishable fibers of the in ternal plexus formed predominantly by dorsoven trally oriented fibers. Besides, large AChpositive neurons were revealed in the worm body behind the pharynx. The most part of these cells lay in the close vicinity to the main commissural elements. Additional longitudinal nerves have thickness comparable with that of dorsal trunks and, most probably, are a result of concentration of threads of the submuscular plexus. Anlagen of the nervous system in monogeneans (Diplozoidae family) start at the larval stage, which is called diporpa in higher monogeneans. In Para diplozoon rutili, brain, all major trunks and com missures revealed in larva are also found in adults [42]. Dorsal trunks without direct connection with brain are located above it and can be traced in di porpas of all ages only till the first pair of valves regardless of their total number; lateral trunks reach the most posterior pair of valves, while the ventral trunks become significantly thicker in their poste rior part and, as going through the center of the disc, give lateral branches to each valve. All trunks are connected with uniformly distributed circular commissures whose number increases with growth of diporpa and reaches 17–18 in the largest larvae. In adult worms this number reaches 20–22 com missures, although regularity of their distribution is somewhat impaired by asymmetric development of additional commissural connections. Structure of the nervous system in the monoge nean Gyrodactylus salaries, studied by 5HT and FMRFamide immunocytochemistry coincides with the classic description of the nervous system of the lower monogenea [43]. Fibers of different IR were revealed in the brain, they are running in parallel to each other along the longitudinal trunks and rare commissures. Differences are found only in distribution of neurons. In the higher monogenean Diclidophora merlan di, FMRFamide IR was revealed in brain, 3 pairs of longitudinal trunks, and multiple transverse commissures. FMRFamide IR is localized in densecore vesicles occupying most axons of the CNS [44].


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Cestoda (tapeworms). Development of the ner vous apparatus has been studied in the tapeworm Triaenophorus nodulosus (Cestoda, Pseudophyl lidea) in ontogeny [45]. Cholinesterase (ChE) in coracidium was found in oncosphere. Accumulation of the enzyme was observed in the area of crochets and is of diverse shape. The presence of the enzyme seems to indi cate the site of anlagen of the nervous system. Nervous system of the next procercoid stage is characteristically of orthogonal type represented by three pairs of longitudinal trunks connected with transverse commissures (from 3 to 7 in the num ber, depending on size). Most developed lateral trunks (which we call the main lateral trunks) are linked to each other at the posterior end. We suc ceeded to trace the dorsal and ventral trunks only till the middle of the body. The 10day old plerocercoids have the nervous system of orthogonal type and have 3 pairs of trunks connected with ringshaped commissures distrib uted uniformly along the entire length. At the pos terior end, the most developed lateral trunks are adjacent to two other pairs. The number of circu lar commissures is higher than in procercoid. The 20day old plerocercoid already has 5 pairs of longitudinal trunks. This stage is typical for ap pearance of accompanying trunks distributed lat erally to the main trunks and outgoing from a slightly thickened anterior ends of the latter. This suggests that formation of novel longitudinal trunks start at the anterior end. The completely formed plerocercoids able to infect their principal host have 5 pairs of longitu dinal trunks in scolex and 2 extra trunks in the body (accessory dorsal and ventral trunks appear) and all of them are linked with a dense network of a rough submuscular plexus. Besides, we described in tapeworms for the first time the internal nervous plexus connecting two main lateral trunks and run ning via the medullary parenchyma. It consists of rare nervous fibers with a thickness corresponding to loops of the submuscular plexus. In mature tapeworms T. nodulosus, the main nervous trunks in the anterior part of scolex are slightly enlarged and are adjoining to a pair of ac companying dorsal and ventral trunks. They are added in strobila by accessory dorsal and ventral trunks. All trunks lay in the cortical parenchyma

at the same level and are linked with a dense net work of fibrous plexus. This plexus looks like an irregular network, in which it is hard to trace lon gitudinal trunks and there are no regular transverse commissures. This is the most primitive state of the nervous apparatus in tapeworms, as already in rep resentatives of other families of the Pseudophyl lidea order the main lateral trunks are immersed into medullary parenchyma. Results of study of ontogenetic development of the nervous apparatus of tapeworms Eubothrium (Amphycotilidae family) [46] are in agreement with those obtained in T. nodulosus, which gives grounds to accept the welldeveloped orthogon with 3 pairs of longitudinal trunks as the initial state. Since this state is typical for early develop mental stages, it can be considered as recapitula tion. If this is the case, it is reasonable to suggest that their ancestor species had 3 pairs of longitudi nal trunks. Nervous system of Schistocephalus solidus (Li gulidae, Pseudophyllidea) is completely formed at the plerocercoid stage. It has no welldeveloped scolex, therefore the growth of the main lateral trunks and their proximal parts and connection of these parts by dorsal and ventral commissures is evident. The trunks here are 4–5 times thicker than in the strobila area. Remaining 12 pairs of longitu dinal trunks come near the main trunks and there fore they represent a part of CNS. The number of accessory trunks gradually increases towards the posterior end of strobila up to 40 pairs in plerocer coid and up to 50 pairs in adult tapeworm with the 10mm width of proglottis. These accessory trunks already are a part of the peripheral nervous system, as they have no direct contacts either with brain or with proximal areas of the main nervous trunks. As they run caudally (nearly the posterior end), the number of the trunks decreases gradually and in the caudal area with the 0.80mm width their num ber decreases to 8 pairs. A pair of the main lateral trunks is immersed into the central parenchyma, while all other trunks lay at the border of peripheral and central parenchy ma. One circular commissure is located in the pos terior area of each proglottis. 3 plexuses are distin guishable in tapeworms: one thin superficial (sub epithelial) and two others consisting of thicker fi bers, the rough (submuscular) and interior plexus.



All of them form the common network system. Histochemical and ICC studies of the nervous system of several tapeworm species revealed dis tribution of ChE, 5HT, histamine, catechola mines, and 12 polypeptides (see rev. [4748]). All together they form an essential part of the nervous system of the worm and fibers of different trans mitter specificity run in parallel to each other along the main elements of the tapeworm conductive system. The most complete picture of architecton ics of the nervous system was obtained by using study only of the cholinergic part of the nervous system. The cholinergic part of the nervous system of 22 species of tapeworms of 6 orders revealed sev eral stages of improvement of the brain: from the nonformed one with weakly developed ganglia (Caryophyllaeus) through overgrowth of the up per parts of longitudinal trunks (Diphyllobothri um) to circular (Eubothrium), then, to distinctly paired with two ganglia (Tetraahynchobothrium) and, finely, to the brain formed by two pairs of gan glia (Paricterotaenia). The number of longitudinal trunks is very variable. Thus, Pseudophyllidea has from 5 to 60 pairs, Tetraphyllidea and Proteoceph alidea—5 pairs, and the higher Cyclophyllidea— from 5 to 1 pairs. Despite significant variation, a trend to establishing of 5 pairs can be seen in all studied orders regardless of their taxonomic state [49]. The ringshaped commissures unite longitu dinal trunks of jointed tapeworms to the single whole. The posterior area of each proglottis as a rule contains one ringshaped commissure and only Tetraphyllidea has three commissures. Trematoda. The cholinergic nervous system of 14 trematode species and 2 cercarian species looks like a dumbbellshaped cerebral ganglion with two lobes connected with a short wide bridge. The gan glion as a rule gives 3 pairs of trunks, with the ven tral one being the most developed. Differentiation of body of most trematodes to locomotor and gen ital parts, put forward by Oshmarin [50], is reflected in structure of their nervous system, as longitudi nal trunks in the anterior (locomotor) area are en larged and connected with circular commissures, while in the posterior (genital) part the trunks are weakly developed and commissures are absent. In species without such a differentiation, the ring shaped commissures are uniformly distributed

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along the body [51]. On the whole, diversity of the trematode nervous system structure is due mainly to the commissural elements [52], which allow us to identify 5 variants on the basis of four types of orthogons [8]. The first variant is characterized by the presence of three circular commissures locat ed till the ventral sucker. This is a nonuniform or thogon described in Sphaerostoma bramae, Fello distomum fellis, etc. [51]. The second variant ap pears as the ventral sucker shifts to the posterior end of the body (Diplodiscus subclavatis, cercari um Microphallus pygmeus); this is a rare regular orthogon. The third variant has been found in Leu cochloridium sp. [49] whose ringshaped commis sures were revealed also behind the ventral sucker. In reality, according to its architectonics, this is a regular thick orthogon. The fourth structural vari ant is expressed in the large Fasciola hepatica that has 3 semicircular commissures without the ven tral part in the anterior region of the worm (till the ventral sucker). Lateral trunks do not run to the posterior part, while the ventral and dorsal trunks arborize and anastomose by forming a dense cel lular plexus. This is an example of a cellular or thogon. Orthogon of the Dicrocoelium dendriticum should be attributed to the fifth variant with 3 cir cular commissures in the anterior part and single cords in the posterior part, which go from dorsal and ventral trunks laterally. On the other hand, Podocotyle atomon has three commissures of com plicated configuration in the anterior part of the body, with some zones of the commissures (dou bled somewhere) being distributed at different lev els. These are examples of the irregular orthogon. All these variants were inherited from freeliving ancestors and appeared as early as in parasitic trem atodes. Large trematodes express accessory ante rior branches of ventral and dorsal trunks (Opis thorchis felineus [53], Fasciola hepatic [54]), and, what is most important, an interior plexus can be revealed and cholinergic neurons can be mapped. Among the latter, some unipolars and many bi and multipolar cells are seen. By the present time, many works have been pub lished on the trematode nervous system to estab lish it to have not only acetylcholine, but also CA (dopamine), serotonin, and a set of polypeptides (see rev. [47, 48]). However, in this case, only dis tribution of ChE gives the complete picture of the


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nervous apparatus architectonics, while other transmitters are revealed in brain and along some trunks and commissures. ROTIFERA Rotifers, or wheel animalcules, are character ized by eutely. Total number of cells in the body of wheel animalcules has been estimated to reach 1000 [55], with 20 % of them related to the brain complex. These data have been confirmed by a threedimensional reconstruction of neurons of the brain complex in one of the largest wheel animal cules, Asplanchna brightwelli [56]. 200 bilaterally symmetric brain neurons have been revealed. The catecholaminergic nervous system was stud ied in 12 species belonging to 2 subclasses and 5 or ders. It has the same structural pattern: suprapha ryngeal cerebral ganglion and the pair of ventro lateral trunks. Major differences are found in ge ometry of distribution of brain neurons. It can be Xshaped, archshaped, or ringshaped. Thus, the brain complex of Philodina sp. and Rotaria tardi grada (Bdelloida) includes 7 neurons oriented lon gitudinally [57, 58]. Neurons lay in three layers forming an Xshaped structure. In the upper lay er, the pair of diffusely placed large scaphoid neu rons (7 μm) is revealed. The middle layer includes the pair of polar perikarya with approached pro cesses (6 μm). The third, lower layer contains three rounded neurons (5 μm). Longitudinal ventrolat eral trunks originate from the lateral neurons of the last layer. One pair of neurons is found along the trunks in the posterior half of the body. In Manfre dium eudactilotum, Transversiramida, brain neu rons have an Xshaped distribution and lay in 4 lay ers. Their sizes vary from 3 to 6 μm. In two other species, Platyias quadricornis [59] (Fig. 3b) and Euchlanis dilatata, from 6 to 7 brain neurons have an archshaped distribution and, finally, in Bra chiomus quadridentatus and Lecane arcuata, 6 brain neurons (4–8 μm) have a circular distribution and 2 pairs of neurons are placed along the trunks. As planchna herricki and A. priodonta have 6 neurons (2–5 μm) in the archshaped brain and the pair of trunk neurons. In Notommata sp., Saeptiramida, 6 brain neurons are distributed semicircularly (6– 8 μm) and, finally, in Dicranophorus forcipatus, Antrorsiramida, the ringshaped brain has 11 neu

rons (2–7 μm) and the pair of neurons was revealed along the trunks [60]. Similar geometry of distri bution of brain neurons in representatives of 2 or ders or even 2 subclasses (Platyias, Asplanchna and Philidina, Manfredium) indicates independent and parallel development of these structures in ro tifers. In the lower Bdelloida, one type of distribu tion of brain neurons has been described, while higher organized rotifers—all three types. Immunohistochemical studies of 5HT and FMRFamide immunoreactivity. Platyias patulus. Six 5HTIR brain neurons have an Xshaped distribution in three layers, while12 FMRFamide IR neurons—an arch shaped distribution. Along trunks, 2 and 4 neurons, respectively, were revealed [61]. Euchlanis dilatata. Four 5HTIR and FMRF amide IR brain neurons have an archshaped distribution, although FMRFamide IR neurons demonstrate a more curved arch. Each of the prox imal and distal trunk portions has 4 neurons. Asplanchna herricki. Different geometry of dis tribution of brain neurons. The 5HTIR neurons form an archshaped figure (4 neurons), while the FMRFamide IR neurons—a ring of 14 neurons. Sizes of the IR neurons vary from 2 to 8 μm (Figs. 2e, 2f ). The total number of CAergic, 5HT and FMRFamide IR brain neurons accounts for from 3 to 8% neurons of the brain complex. There are no literature data on mapping of neurons in roti fers. CONCLUSION Analysis of the considered material allows con cluding that Platyhelminthes and Rotifera have CNS that includes brain, longitudinal trunks, transverse commissures and, probably, the sub muscular nervous plexus located at the level of trunks and commissures and completing formation of the single conducting system. Stages of improvement of the brain are estab lished from the commissural brain without its own membranes and not separated by anything from parenchyma in Acoela to separation of brain and formation of its own membranes in Rhabditopho ra. Shape diversity of brain is found, up to the com pact doubleblade brain in some turbellaria and



dumbbellshaped brain in parasitic trematodes. Longitudinal trunks whose number varies from 12 to 1 pairs (most often, there are 3 pairs in tur bellaria and 5 in tapeworms) either outgo directly from the brain or are connected with it by brain roots. Centralization of longitudinal trunks is re alized through formation of common roots, more often ventral and lateral trunks or, more seldom, of ventral and dorsal trunks, and only in the most highly organized Rhabdocoela, formation the common root for all three pairs of trunks is noted. Rhabditophora and Rotifera have the orthogo nal nervous system composed of 8 types of or thogons. The most developed are ventral or later al, but never dorsal trunks. The number of circular commissures and their distribution determine the orthogon type. Oligomerization as the final result of concentration reduces the number of commis sures to one pair or to their complete disappear ance (Rotifera), which is characteristic of the con centrated orthogon. Diversity of orthogon types in Rabditophora, the independent and parallel devel opment of the same orthogon type in the long re mote species is an indicator of morphologic diver sity within the limits of this group. The basic position of Acoela among Bilateria remains unchanged. The commissural brain and the trunk nervous system with the most developed elements on the dorsal side are revealed in Acoela. There is no evidence of orthogon as well as of 5 HT and FMRFamide IR cerebral ganglion (en don) characteristic of Platyhelminthes. These morphologic peculiarities can be an additional ar gument in favor of separation of Acoela from the Platyhelminthes taxon (as indicated by current molecular phylogeny) and of considering them as the most primitive of the nowadays living Bilate ria. The ancestor of Bilateria had seemed to have an intraepithelial nervous network without central ization or with the nervous ring at the anterior body end. The trunk and orthogon nervous system pecu liar to these groups appears in them independently and in parallel and reaches its maximal degree of concentration in some Rhabdocoela and all wheel animalcules. This is the first level of complication of the initially primitive plexus system of Bilateria and the end of the first stage of the CNS evolution. Data on mapping of neurons in rotifers have

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shown the single plan of their nervous system con struction. In the brain, CAergic, 5HT and FMRFamide IR neurons accounting for from 3 to 8% of the total number of the brain complex neurons have been revealed. Along the trunks, 3– 5 pairs of neurons of different ergity and IR are lo cated. Elements of cholinergic, CAergic nervous sys tem, 5HT and FMRFamide IR are oriented in parallel in the brain and along the main longitudi nal trunks. Neurons of different ergity and IR are located in certain brain areas and lay side by side along the trunks, but are never colocalized in one cell. This is the initial step for establishment of dif ferent transmitters in the simple nervous systems and their collegiality that with transition on to an other, higher level of development undoubtedly will be differentiated and, hence, will be morpho logically separated. Our performed analysis of data on Acoela, Platy helminthes, and Rotifera, although shows signifi cant variations within the limits of the studied groups, but, on the whole, their simple nervous sys tems are at the same level of development, which is illustrated by the total plan of the CNS estab lishment. REFERENCES 1.

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