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The microturbellarians Stenostomum leucops (Catenulida), Microstomum lineare (Macrostomida), Promonotus schultzei (Proseriata) and Gyratrix hermaphroditus (Rhabdocoela) were subjected to the glyoxylic-acid-induced-fluorescence method to reveal catecholaminergic parts of their nervous systems. Histofluorescence indicative of catecholamines was evident in nerve cells and fibers of all investigated species. The patterns of distribution were compared to those of other neuroactive substances. The catecholaminergic fluorescence was at least partly confined to separate neuronal subsets in S. leucops and M. lineare and probably also in P. schultzei. In S. leucops, the presence of dopa and dopamine was demonstrated by high-pressure liquid chromatography. The occurence of catecholamines in these representatives of four orders of microturbellarians suggests that catecholamines arose early in the evolution of flatworms and underscores the importance of dopaminergic substances for the function of early nervous systems.

Specific antibodies against histamine were used to demonstrate the occurrence and cellular distribution of histamine-like immunoreactivity in three species of flatworms (phylum Platyhelminthes). In the parasitic cestode Diphyllobothrium dendriticum, histamine-reactivity was found in neurons of the main nerve cords, and in cells lining the central and peripheral excretory ducts. In the free-living microturbellarian Microstomum lineare and in the planarian Polycelis nigra, histamine-immunoreactivity was restricted to cells and fibres of the nervous system. The occurrence of histamine or a related substance in the nervous system of flatworms, which represent primary bilateria, indicates the importance of this neuroactive substance in the animal kingdom.

The nervous system of the adult pike-tapeworm Triaenophorus nodulosus was studied to identify nerve cells and fibers immunoreactive to serotonin (5-HT) and RFamide (RF) on whole-mount preparations and frozen sections. Neurons immunoreactive to 5-HT were seen solely in the central nervous system, while those immunoreactive to RF occurred in the peripheral nervous system as well as in the central nervous system. In the scolex, both types of nerve fibers were found. While the gonads were not innervated by either fiber type, the reproductive tract showed RF-immunoreactive nerves. On the ultrastructural level, five types of neurons and three types of release sites and a neuromuscular junction could be distinguished. Levels of 5-HT, measured spectrofluorimetrically, were found to be lower in the tapeworm than in the tissues where it resides in its host, indicating a possibility that the parasite absorbs this bioamine from its environment. Additional key words: 5-HT, RFamide, neuron types, neurotransmitter release sites, spectrofluorimetry During the last two decades, knowledge of the neuroanatomy of flatworms has grown (for review, see Reuter & Gustafsson 1995). For the parasitic flatworms, however, ultrastructure of neuronal elements is still rather poorly known, and information on the nature and number of neuronal mediators in flatworms in general is sparse. To fill part of this gap, we have studied the nervous system (NS) of the pike-tapeworm Triaenophorus nodulosus (PALLAS 1781) with immunocytochemical, ultrastructural, and spectrofluorimetric methods. T. nodulosus parasitizes the intestine of pike as an adult and parasitizes other economically important fishes, such as burbot, as a plerocercoid, the last larval stage. The first parasitic larval stage, the procercoid, develops in fresh-water crustaceans. Apart from the studies of Michajlow (1932, 1933, 1934) little information is available on the histology, especially the neuroanatomy, of the Triaenophoridae. Gustafsson (1973), investigating cellular composition in the neck region of plerocercoids of T. nodulosus, a To whom correspondence should be addressed. Phone: 358 21 2654603; FAX: 358 21 2654748; E-mail: magustaf@ra.abo.fi noted that the cells surrounding the main nerve cords constitute as much as 19% of the total cell population. In light microscopical studies of the general neuroanatomy of T. nodulosus, Kotikova & Kuperman (1977) monitored acetylcholinesterase activity during ontogenesis and documented formation of nerve plexuses and cholinergic nervous elements in the scolex and an increase in the number of longitudinal nerve cords at each successive stage of development; Biserova et al. (1991) used the glyoxylic-acid method to study the aminergic NS of adults and showed evidence of serotonin in ganglia of the brain, connecting commissure, and main nerve cords. Ultrastructure of sensory structures in the scolex was investigated by Biserova et al. (1991); and ultrastructure of the integument, frontal glands, and excretory system was examined by Kuperman (1973, 1988) and Kuperman & Davydov (1982a,b). We have extended the study of adults of T. nodulosus by comparing patterns of peptidergic and aminergic nerve elements, distinguishing types of nerve cells and neurotransmitter-release sites, and measuring serotonin (5-HT) concentrations. The nervous system of flatworms can be subdivided into central and peripheral elements (see Reuter & This content downloaded from 157.55.39.35 on Thu, 01 Sep 2016 04:58:52 UTC All use subject to http://about.jstor.org/terms Biserova, Gustatsson, Reuter, & Terenina Gustafsson 1995 and Reuter et al., in press, for discussion of terminology). The central nervous system of T. nodulosus consists of the bilobed brain and the two main nerve cords (MC's). The peripheral nervous system comprises all the other longitudinal cords (the minor cords), ring commissures in the cortical parenchyma, and nervous structures associated with the genital organs.

Immunoreactions (IR) in the nervous systems (NS) of species representing the platyhelminthic taxa Acoela, Proseriata, Tricladida and Rhabdocoela (Dalyellioida, Typhloplanoida, Kalyptorynchia) have been studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method and the whole mount immunofluorescence (FITC) method. Antisera to the molluscan cardioactive neuropeptide FMRF-amide, to the bioamine 5-HT and to the vertebrate neuropeptides bovine pancreatic peptide (BPP), leu-enkephalin, substance P and human growth hormone releasing factor (GRF) were used. Perikarya and fibres positive to FMRF-amide antiserum (anti-FMRF-amide) appear in the NS of all investigated species. Since IR to anti-FMRF-amide has been obtained in worms representing taxa of all flatworm classes it is suggested that substances with similar sequences have been inherited from the stem form of Platyhelminthes. IR to 5-HT antiserum (anti-5-HT) observed in the NS of all the investigated flatworms confirm the importance of this bioamine, which may have appeared early in platyhelminth evolution. IR to GRF antiserum was found in the acoel, the triclad and the rhabdocoel species, IR to leu-enkephalin antiserum in the triclad and rhabdocoel species and IR to substance P antiserum in the rhabdocoels.

The subsarcolemmal cisternae of the sarcoplasmic reticulum form peripheral couplings with the sarcolemma. The junctional gap is crossed by periodic densities called junctional processes. Desmosomes provide mechanical coupling between the myofibres. Hemidesmosomes connect the myofibre with a well developed connective tissue sheath.

The innervation of the division zone in the asexually reproducing Microstomum lineare (Turbellaria) was studied by light and electron microscopy. Nerve fibres presumed to be peptidergic form a circumferential belt and longitudinal nerve fibres and nerve swellings join the circumferential belt from opposite directions. Peptidergic nervous influence on asexual reproduction by paratomy is indicated by (1) synaptoids and (2) omega-shaped configurations. These release sites are observed at muscles of the division zone and in the vicinity of neoblast cells.

The integrative mechanisms in phylum Platyhelminthes are of considerable phylogenetic interest since ancestral flatworms are believed to be progenitors to all "higher" metazoans. The phylum consists of free-living and parasitic worms. In this group of early bilateral animals, the nervous system is condensed and consists of a bilobed brain and longitudinal nerve cords. Endocrine glands and a circulatory system are still lacking. The present review deals mainly with microturbellarians and parasitic worms. Three main types of neurons, all containing vesicles, occur in the worms: neurons containing dense-core vesicles, peptidergic neurons and sensory neurons. All types of neurons react positively to antisera towards invertebrate and vertebrate neuropeptides. Neurons containing dense-core vesicles are the most frequent type. The dense-core vesicles occur both in the cell soma and in their long processes. Neurons of this type form synaptocrine and paracrine release sites. We ask: Is the neuron containing dcv an archaic type of neuron in which the characteristics of the stem cells for neurons have been preserved?

The ultrastructure of and the presence of lytic activity in aminergic neurons from zooids representing different developmental stages were compared. The observations indicate clear differences between neurons of developing and mature zooids, and show that lytic activity occurs in the neurons of this turbellarian species. The differences in ultrastructure, size and nucleoplasms (NP) index motivate a division of the aminergic neurons of the zooids into three categories: (1) small aminergic neurons (SN) which occur in developing zooids; (2) flocculent aminergic neurons (FN) characterized by a network of flocculent splittings in the cytoplasm; (3) vacuolated aminergic neurons (VN) with large membrane limited vacuoles occupying a large part of the perikaryon. Slight acid phosphatase (AcPase) activity occurs in GERL of SN. An increased amount of AcPase reaction products in the Golgi apparatus, primary lysosomes and autophagosomes are observed in FN. Typical for VN are prominent lead deposits in the large vacuoles. The development and functional significance of the large vacuole are discussed.

The body surface of Microstomum lineare, Bothriomolus balticus and Archilopsis unipunctata was examined in scanning electron microscope. The threedimensional appearance of adhesive duo-gland organs, receptor cilia, globular bodies and pistil-like projections is described. The adhesive duo-gland papillae of Bothriomolus and Archilopsis appear as bouquetlike structures on either side of the tail as well as on the cilia covered body. In Microstomum the papillae occur as single projections among the ciliary body fur. Supplementary TEM investigation revealed crystalline bodies in the cytoplasm of viscid gland cells.

A whole mount immunocytochemical (ICC) method has been used for the investigation of immunoreactivity (IR) to the molluscan cardioactive peptide FMRF-amide, to 9 vertebrate neuropeptides—leu-enkephalin, growth hormone-releasing factor (GRF), urotensin I, urotensin II, bovine pancreatic peptide (BPP), β-endorphin, substance P, secretin and insulin—and to the bioamine 5-HT in the nervous system (NS) of the trematode Gyrodactylus salaris belonging to the taxon Monogenea. Positive results were obtained using antisera to FMRF-amide, leu-enkephalin, urotensin I, GRF and to 5-HT. The present results are the first documentation of the presence of neuroregulatory peptides and a bioamine in the nervous system (NS) of Monogenea. Differences in the distribution pattern of the IR to the different antisera indicate that different subsets of neurons are revealed. In addition, details of the basic neuroanatomical pattern in monogeneans are confirmed by the whole mount ICC method used in this study. Negative results were obtained with antisera to urotensin II, substance P, β-endorphin, secretin, insulin and bovine pancreatic polypeptide (BPP).

The distribution of cells preparing for proliferation during asexual reproduction by paratomy in Microstomum lineare (Turbellaria) was investigated using the technique of exogenous tritiated thymidine ([3H]T) labelling and routine electron microscopy. The subsequent fates of labelled cells in developing and mature zooids were followed by fixing tissues at various intervals. The only labelled cells are mesenchymal and gastrodermal neoblasts, occurring along the whole worm without any special growth zones or axial gradient. Organ primordia develop as a result of locally dividing as well as of migrating cells.

A whole mount immunofluorescence method was used for the localization of immunoreactivity (IR) to four regulatory peptides and the bioamine serotonin in the nervous system of Stenostomum leucops (Turbellaria, Platyhelminthes). The flatworm S. leucops belongs to the taxon Catenulida which, according to the new phylogenetic system by Ax [2], forms a key group between the coelenterates and more advanced flatworm species. Positive IR was obtained using antisera against FMRF-amide, β-endorphin, growth hormone releasing factor (GRF), substance P, and serotonin. The distribution patterns of these neuropeptide-like immunoreactivities differ significantly from each other. Antisera against Leu-enkephalin, bovine pancreatic polypeptide (BPP), bombesin, cholecystokinin (CCK-8), neurotensin, somatostatin, growth hormone (GH), secretin, and neurophysin II gave negative results. This primitive flatworm shows similarities with hydra in the lack of IR to anti-somatostatin, anti-Leu-enkephalin, and anti-BPP. These antisera give positive IR in more advanced flatworm species, indicating a later convergent evolution of vertebrate-like peptides within the phylum Platyhelminthes.

The epidermis of Gyratrix hermaphroditus can be described as semi-syn-cytial. Its ultrastructure is characterized by microvilli and cilia with two strong rootlets perpendicular to each other. The apical part of the epithelium contains mitochondria and vacuoles. The basal synthesizing layer is provided with cell boundaries, at least between the type II penetrating receptors in the anterior and posterior end of the worm. Four different types of sensory receptors are described. The type I receptor has a protruding cilium-bearing process and is found all over the body. The type II receptor is found in the anterior and posterior end and has a retracted process with a kinocilium surrounded by eight stereocilia. The type III receptor bears a balloon-shaped modified cilium and is located at the anterior end. The type IV receptor has a short cilium with an unstable ciliary membrane and occurs in the proboscis epithelium as well as in the pharynx epithelium. Phylogenetical aspects of the semi-syncytial epithelium and functional aspects of the sensory receptors are discussed.

The gastrodermal cell types of Microstomum lineare (Turbellaria, Macrostomida) were studied by electron microscopy. Their immunoreactivity (IR) to bovine pancreatic peptide (BPP), FMRF-amide and vasotocin, somatostatin, neurotensin, ACTH, CCK, bombesin, secretin, gastrin/CCK and insulin antisera was tested by light microscopic immunocytochemical methods. In addition to granular club cells and phagocytic cells, neurons and neoblasts occur in the gastroderm of this turbellarian species. This is the first observation of neurons in the gastroderm of a flatworm. Dense-core vesicles (70–100 nm diameter), electron lucent cytoplasm and numerous Golgi complexes characterize the neurons. Unpolarized two-way synapses, neuromuscular junctions and polarized chemical synapses can be observed in the gastroderm. Neoblasts with large nuclei and scanty cytoplasm and differentiating cells containing clusters of basal bodies occur next to the basal lamina of the gastroderm. BPP-like, FMRF-amide-like and vasotocin-like immunoreactivity is demonstrated in the gastroderm. Both BPP and FMRF-amide IR is restricted to the basal cytoplasm of the granular club cells, while a different location for IR to vasotocin antiserum is observed. The status of the neuronal cell in the gastroderm of M. lineare is discussed in relation to endocrine (paracrine) cells and neurons in the gastroderm of invertebrates.

The fine structure of the nerve cells in the brain of Gyratrix hermaphroditus is studied as well as the structure of the neuropile. General features of all nerve cells are a prominent neucleus with a small nucleolus, a scanty layer of cytoplasm containing mitochondria, free ribosomes, few profiles of granular endoplasmic reticulum, smooth endoplasmic reticulum, subsurface cisternae, well developed Golgi complexes, multivesicular bodies and numerous vesicles diagnostic for different nerve cell types. On the basis of the vesicle content four nerve cell types can be distinguished. The following types of vesicles are observed. Dense cored vesicles (50–80 nm), dense vesicles (60–120 nm), small lucent (synaptic) vesicles (40–50 nm), large lucent vesicles (50–200 nm) and lysosome-like vesicles (200–500 nm). The significance of the variations in the interior electron dense material of the multivesicular bodies is discussed.

During an ultrastructural examination, viruslike particles were observed in the turbellarian Gyratrix hermaphroditus. This is the first time viruslike particles have been found in a noncultivated platyhelminth species. The particles are 70 nm in diameter and have a capsidlike outer layer and an inner core measuring 40–50 nm in diameter. They occur in a crystalline arrangement in the nucleus as well as in the cytoplasm. Numerous cytoplasmic abnormalities were seen in connection with the particles. The occurrence of the particles in different tissues and their significance for the host are discussed.

The nervous system (NS) of Microstomum lineare (Turbellaria, Macrostomida) was studied by electron and light microscopy, combined with fluorescence histochemistry (Falck-Hillarp method for biogenic monoamines). The NS is primitively organized, with a bilobed brain, two lateral nerve cords lacking commissures, and peripheral nerve cells scattered along the nerve cords. The stomatogastric NS, with a pharyngeal nerve ring, is joined to the central NS by a pair of connective ganglia. A green fluorescence in all parts of the NS indicates catecholaminergic neurons as the dominant neuron type. Ultrastructurally, two types of neurons were identified on the basis of their vesicle content: 1. Aminergic (catecholaminergic) neurons containing dense-core vesicles of varying electron-density and size, i.e., small dense-core vesicles (diameter 50--100 nm), vesicles with a highly electron-dense core (60--140 nm), and vesicles with an eccentric dense-core. 2. Presumed peptidergic neurosecretory neurons containing large granular vesicles (diameter about 200 nm) in the stomatogastric NS and peripheral parts of the central NS. In light microscopy, paraldehyde-thionin stained neurons were observed in the same areas.

The eyespots of Microstomum lineare were studied by electron microscopy, light microscopy, and fluorescence microscopy. Each eyespot consists of two ciliary photoreceptor cells shielded by pigment cells and additional sensory cells. The photoreceptor cells are characterized by a distal intracellular cavity lined with 50-100 interwoven cilia. The other sensory cells are of two ultrastructurally different types, one with long cilia predominating and the other with balloonlike cilia. The pigment cells, which envelop processes of the sensory cells, contain pigment vacuoles varying in size and content and give a bright red fluorescence by the Falck-Hillarp method. The eyespots are suggested to perform a dual function as photoreceptors and chemoreceptors. The evolutionary significance of ciliary photoreceptors in Turbellaria is discussed.

A whole mount immunofluorescence method is used for an investigation of immunoreactivity (IR) to anti-(a-) 5-HT and anti-(a-)FMRF-amide in the nervous system (NS) of Microstomum lineare (Turbellaria, Macrostomida, Platyhelminthes). New details of the organization of the NS are demonstrated, differences in 5-HT and FMRF amide IR are revealed, and new information on the development of the NS in zooids is obtained. In contrast to previous reports of a reduction (one pair of nerve cords without transverse processes) of the basic turbellarian plan, IR to both antisera reveals three pairs of longitudinal nerve cords, and features of the orthogonal organization, characterized by transverse commissures. The lateral pair of nerve cords is the most prominent. The following differences in the patterns of 5-HT and FMRF-amide IR are observed: 1. Perikarya positive for a-5-HT and a-FMRF-amide in the brain show different localizations. 2. Perikarya positive for a-5HT occur along the main lateral nerve cords, while the cords visualized by FMRF-amide IR look double-stranded and lack associated perikarya. 3. 5-HT IR is observed in a postpharyngeal commissure, which is absent in the a FMRF-amide-stained preparations. 4. In developing zooids 5-HT IR is first observed in the postpharyngeal commissure and later on in an increasing number of perikarya and in the neuropile. The first FMRF-amide IR in developing zooids appears in the cerebral commissure and in two perikarya in front of this commissure.

The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.

The ultrastructure of release sites of neurochemical messenger substances in the microturbellarian Microstomum lineare was examined. Aminergic neurites form conventional synapses and synapse-like structures (SLS). Variants of true synapses include: "single" synapses with symmetric pre- and postsynaptic densities, "shared" synapses, i.e., contacts between 1 pre- and 2 postsynaptic fibres, en passant synapses between parallel axonal membranes, and synapses without thickenings having only clustered vesicles in the presynaptic terminal. SLS on a nerve cell soma or facing an intercellular stromal channel near muscles are described. Peptidergic neurites containing large granular vesicles (LGV) from synaptoids and signs of putative neurosecretory release. Synaptoids between neurites and between neurite and muscle have lucent vacuoles (about 100 nm) and dense material at the contact site. In en passage synaptoids dense-core vesicles are embedded in electron-dense material at the contact site. Putative signs of release of neurosecretory material other than "typical" exocytosis have been observed.