Substance P immunoreactivity in sensory structures and the central and pharyngeal nervous system of Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida)

Immunoreactivity (IR) obtained by monoclonal antibodies to substance P (SP) was studied in the asexually reproducing microturbellarians Stenostomum leucops and Microstomum lineare. The IR pattern was studied by confocal and ordinary fluorescence microscopy. In both species, IR occurs in the brain in peripheral cells, neuropilar fibres, in longitudinal cords and in the pharyngeal nervous system. The IR patterns reveal neuroanatomical details not observed with other neuroactive substances. In both species, immunopositive cells send fibers to the ciliary pits. In M. lineare, additional fibres run to more frontally located sensory structures. In S. leucops, two pharyngeal nerve rings are visualized. The pharyngeal nerve ring close to the surface associated with symmetrical immunopositive cell pairs is demonstrated for the first time, while the deeper-lying pharyngeal nerve ring has been previously demonstrated by antibodies to the molluscan cardioactive peptide FMRF-amide. Two cells with strong IR are connected by short fibres to the pharyngeal nerve ring in M. lineare. In the developing new individuals, i.e., the zooids of M. lineare, IR to SP is first revealed in nerve fibres growing out from parental lateral nerve cords towards the centre of the worm where the new brain commissure will appear. Immunopositive cells in the brain periphery and close to the developing ciliary pits appear later. Simultaneous staining by antibodies to SP and 5-HT shows that IR to SP appears later than IR to 5-HT.     

The organization of the nervous system in Plathelminthes. The neuropeptide F-immunoreactive pattern in Catenulida,Macrostomida, Proseriata

The organization of the nervous system of Archilopsis unipunctata Promonotus schultzei and Paramonotus hamatus (Monocelididae, Proseriata) and Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida) was studied by immunocytochemistry, using antibodies to the authentic flatworm neuropeptide F (NPF) (Moniezia expansa). The organization of the nervous system of the Monocelididae was compared to that of the nervous system of Bothriomolus balticus (Otoplanidae), a previously studied species of another family of the Proseriata. The results show that the main nerve cords (MCs), independent of lateral or ventral position in the Monocelididae and the Otoplanidae, correspond to each other. The study also confirms the status of the lateral cords as main cords (MCs) in S. leucops and M. lineare. Common for MCs in the members of the investigated taxa are the following features: MCs consist of many fibres, originate from the brain and are adjoined to 5-HT-positive neurons. In Monocelididae and Otoplanidae, the MCs additionally have the same type of contact to the pharyngeal nervous system. Also common for both proseriate families is the organization of the two lateral nerve cords, with weaker connections to the brain, and the pair of dorsal cords running above the brain. The organization of the minor cords differs. The Monocelididae have a pair of thin ventral cords forming a mirror image of the dorsal pair. Furthermore, an unpaired ventral medial cord connecting medial commissural cells was observed in P. schultzei. Marginal nerve cords, observed in Otoplanidae, are absent in Monocelididae. All minor nerve cords are closely connected to the peripheral nerve plexus. The postulated trends of condensation of plexal fibres to cords and/or the flexibility of the peripheral nerve plexus are discussed. In addition, the immunoreactivity (IR) pattern of NPF was compared to the IR patterns of the neuropeptide RFamide and the indoleamine, 5-HT (serotonin). Significant differences between the distribution of IR to NPF and to 5-HT occur. 5-HT-IR dominates in the submuscular and subepidermal plexuses. In the stomatogastric plexus of M. lineare, only peptidergic IR is observed in the intestinal nerve net. The distribution of NPF-IR in fibres and cells of the intestinal wall in M. lineare indicates a regulatory function for this peptide in the gut, while a relationship with ciliary and muscular locomotion is suggested for the 5-HT-IR occurring in the subepidermal and submuscular nerve, plexuses. In M. lineare, the study revealed an NPF- and RFamide-positive cell pair, marking the finished development of new zooids. This finding indicates that constancy of these cells is maintained in this asexually reproducing and regenerating species.     

Are there differences between proseriates and lower flatworms in ultrastructure of the nervous system?

The ultrastructure of the CNS (central nervous system) in three species of the Proseriata — Archiloa unipunctata (O. Fabricius), Bothriomolus balticus (Meixner), and Promonotus schultzei (Meixner) — was studied and compared to that of the lower flatworms Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida) in material available from our previous studies. Conventional electron microscopical fixation was used for A. unipunctata and B. balticus; the tannic-acid-incubation method (TARI), a method that reveals especially nonsynaptic exocytotic release of neuronal substances, was applied in study of P. schultzei.In general, the ultrastructural features of neurons and nerve fibers were similar in proseriates and lower flatworms. A striking feature common to both groups was the secretory appearance of all neurons. A significant difference between them is the occurrence, in the proseriates, of wrapping of neurons by glial cells. Heterogeneity in the population of neuronal vesicles and in structure of synapses in the proseriates are probably advanced characters; by contrast, S. leucops has relatively homogeneous vesicles and simple synapses. A gradual advancement from the state in the catenulids through that in the macrostomids to the proseriates seems to be reflected in the differentiation of synapses and the variability of neuronal vesicles. This probably reflects differences in functional demands but also evolutionary advancement.     

Growth factors in asexually reproducing catenulida and macrostomida (Plathelminthes)?

Summary The occurrence and localization of immuno-reactivity (IR) to epidermal growth factor receptor (EGF-r), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), revealed by the present study, indicate growth-factor-like substances in the asexually reproducing flatworms,Stenostomum leucops andMicrostomum lineare. IR to all three antibodies occurs in the nervous system. Differences in the number of positive cells and intensity of IR during asexual development of new individuals were observed. By confocal scanning laser microscopy, immunopositive growth cones of nerve fibres were seen in developing zooids, and weakly or unstained perikarya were observed in close contact with the positive nerve fibres. Antibodies to the growth factors EGF-r, EGF and bFGF as well as to the neuroactive substances 5-HT and RF-amide had a negative influence on the growth and asexual reproduction of culturedS. leucops. No significant differences in the influence of antibodies to growth factors and antibodies to the neuroactive substances were observed.     

Extracellular matrix in some microturbellarians

Electron microscopy was used to study the ECM (extracellular matrix) in the epidermis, brain, and parenchyma of the catenulid turbellarians Stenostomum leucops leucops (Dugès) and Stenostomum leucops aquariorum Luther and of the proseriates Archiloa unipunctata (Fabricius) and Promonotus schultzei Meixner. Specimens were fixed with a tannic-acid fixation that enhances staining of extracellular proteins (TARI method) or a conventional electron microscopical method. Differences between species were found in the amount of matrix and extracellular fibrils. In S. leucops leucops the ECM was very sparse, consisting of only a thin electron-dense sheath beneath the epidermis and surrounding neuropil. These sheaths were not found in S. leucops aquariorum which had only rarely encountered extracellular spaces. In P. schultzei and A. unipunctata ECM was more abundant. A. unipunctata had a distinct sheath surrounding the neuropil and a fibrillar subepidermal membrane; the same membranes in P. schultzei appeared only as patches of ECM. In A. unipunctata hemidesmosome-like structures attached the epidermal cells to the underlying membrane, and from these attachments, fibrils projected to the underlying muscle layer. The TARI method revealed exocytotic activity in the brain. The morphology of ECM in these and other flatworms indicate that it could be an internal medium for transport and exchange of substances among cells. Variability in the structure of ECM among species probably does not reflect evolutionary relationships.     

Stem cells in microturbellarians

Summary A combination of microscopical, immunocytochemical, and autoradiographic techniques were employed to study stem cells and their fates during asexual reproduction and regeneration in two microturbellarians,Microstomum lineare (Macrostomida) andStenostomum leucops (Catenulida). Special attention was paid to the development of the immunoreactivity (IR) to FMRF/RF-amide and 5-HT in differentiating nerve cells.Asexual reproduction inM. lineare andS. leucops occurs by paratomy, i.e., fragmentation after completed differentiation of the new organs. Regeneration, on the other hand, involves a combination of morphallactic and epimorphic processes without the formation of a regeneration blastema. The only cells incorporating tritiated thymidine ([³H]T) were the mesenchymal and gastrodermal neoblasts, which proliferate continuously replenishing the population of stem cells available for growth, asexual reproduction and regeneration. These proliferative cells occurred in two ultrastructurally different forms, differing from each other only by the presence or absence of ciliar basal bodies in the cytoplasm. Few differentiated cells were labeled in the head piece after completed regeneration. A greater amount of labeled differentiated cells were, however, observed postpharyngeally in the first zooid as well as in zooids having developed during the same time (i.e., 20–45 h after the treatment with [³H]T). Furthermore, many labeled cells were still undifferentiated at that time or just in the beginning of the differentiation process. It can therefore be concluded that neoblasts function both as reserve cells and as functional stem cells for all differentiated cell types in these worms. IR to FMRF/RF-amide neuropeptides was not observed in nerve cells differentiating from neoblasts until the occurrence of dense-core vesicles in their cytoplasm. Due to methodological difficulties only weak or no IR to 5-HT could be traced in the nervous system of the asexual and regenerating worms.Abbreviations ICC Immunocytochemical - IR immunoreactivity - [³H]T tritiated thymidine     

The turbellaria fauna of a small water reservoir in Central Russia

The Turbellaria fauna has been studied in the Uvod’ Reservoir. A total of 18 species have been found representing three orders: Catenulida, Macrostoimida, and Neorhabdocoela. The patterns of Turbellaria distribution in the reservoir have been analyzed. The most abundant species have been determined: Stenostomum leucops Duges, 1828, Microstomum lineare (O.F. Müller, 1774), Bothromesostoma essenii M. Braun, 1885, and Mesostoma lingua (Abildgaard, 1789).     

The catecholaminergic nerve plexus of Holothuroidea

Catecholamines have been extensively reported to be present in most animal groups, including members of Echinodermata. In this study, we investigated the presence and distribution of catecholaminergic nerves in two members of the Holothuroidea, Holothuria glaberrima (Selenka, 1867) (Aspidochirotida, Holothuroidea) and Holothuria mexicana (Ludwig, 1875) (Aspidochirotida, Holothuroidea), by using induced fluorescence for catecholamines on tissue sections and immunohistochemistry with an antibody that recognizes tyrosine hydroxylase. The presence of a catecholaminergic nerve plexus similar in distribution and extension to those previously reported in other members of Echinodermata was observed. This plexus, composed of cells and fibers, is found in the ectoneural component of the echinoderm nervous system and is continuous with the circumoral nerve ring and the radial nerves, tentacular nerves, and esophageal plexus. In addition, fluorescent nerves in the tube feet are continuous with the catecholaminergic components of the radial nerve cords. This is the first comprehensive report on the presence and distribution of catecholamines in the nervous system of Holothuroidea. The continuity and distribution of the catecholaminergic plexus strengthen the notion that the catecholaminergic cells are interneurons, since these do not form part of the known sensory or motor circuits and the fluorescence is confined to organized nervous tissue.     

The nervous system of Microstomum lineare (Turbellaria,Macrostomida)

Summary 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 densecore 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 neuro-secretory 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.     

Characterization of the first mitochondrial genome of a catenulid flatworm: Stenostomum leucops (Platyhelminthes)

The first complete mitochondrial genome of a catenulid, Stenostomum leucops, was characterized. Illumina sequencing and 90 813 reads were utilized in the assembly, producing one contig with an average coverage of 1118×. The length of this genome is 15 742 bp with 12 protein‐coding, two rRNA and 22 tRNA genes. Although the atp8 gene is absent in other Platyhelminthes, a highly divergent putative atp8 gene was found in S. leucops. In contrast to other Platyhelminthes, the mitochondrial genes of S. leucops are encoded on both strands. The gene order in the S. leucops mitogenome is very divergent from those observed in other Platyhelminthes, showing only small blocks of synteny. With AAA as the codon for lysine S. leucops shows the probable plesiomorphic condition, whereas Rhabditophora possess the derived GAA. This evolutionary transition is correlated with changes in the respective anticodons in trnK. It remains unclear whether the absence of the D arm and loop in trnS1 is a convergence in Catenulida and Neodermata.     

Neuropeptides in free-living and parasitic flatworms (Platyhelminthes). An immunocytochemical study

The nervous systems of the turbellarians Microstomum lineare and Polycelis nigra and of the cestodes Diphyllobothrium dendriticum and Schistocephalus solidus were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the neuropeptides FMRF-amide, vasotocin, leu-enkephalin, met-enkephalin, neurotensin, somatostatin, and VIP, and to the bioamine serotonin. Anti-FMRF-amide positive perikarya and fibers occurred in all species, while the occurrence of the vertebrate brain-gut peptides and serotonin varied between the species. Anti-somatostatin and anti-VIP gave a negative result. Anti-FMRF-amide and anti-vasotocin positive immunoreactivity was found in the brain and gut of M. lineare, and in the CNS and the peripheral nerve net of the cestodes. We suggest that the brain-gut peptides of free-living flatworms act on the subtegumental region in the cestodes, which lack a gut but absorb their nutrients directly through the tegument.     

On the number and spatial distribution of the catecholamine-containing (GA-positive) neurons in some higher and lower turbellarians — a comparison

Original data on the distribution of catecholamines in Provortex karlingi Ax (Rhabdocoela), Microstomum sp. (Macrostomida), and three species of Acoela are presented. Three groups of homologous CA-ergic (catecholaminergic) neurons are discussed: (1) pharyngeal neurons, (2) L-neurons lying postcerebrally in the lateral body regions, and (3) brain neurons. The pharyngeal neurons constitute a synapomorphy of the Rhabditophora. The systematic distribution of the L- and brain neurons, together with the character concerning number of longitudinal nervous trunks, implies that the Catenulida and Rhabditophora are more closely related to each other than to Acoelomorpha. Though these conclusions need confirmation, they show that knowledge of the distribution of neurons containing a given transmitter may be useful for elucidating phylogenetic relationships of lower plathelminths.     

The importance of non-pollen plant parts as food sources for the common blossom thrips, Frankliniella schultzei

Like other flower thrips, Frankliniella schultzei Trybom (Thysanoptera: Thripidae) feeds on pollen. However, the influence of a pollen diet on the life history of F. schultzei may not be as significant as reported for other thrips species. Frankliniella schultzei was reared successfully and with low mortalities (20%) on Wax Mallow, (Malvaviscus arboreus Cav.) plant part diets. Development times and fecundity on a petal diet were not significantly different from that on a pollen diet. Fecundity on a diet combining M. arboreus pollen, petal and leaf tissues was significantly higher than those individually containing these tissues. In laboratory choice tests, F. schultzei females encountered petal most often of the three plant parts. Pollen and leaf were encountered with similar frequencies. A significantly higher proportion of petal encounters (0.8) resulted in feeding than did pollen encounters (0.5). Few leaf encounters (0.1) were followed by feeding. Adult and larval F. schultzei were found in M. arboreus flowers but not on leaves. All parts of the flower were inhabited and not just the pollen-bearing petal apices and anthers. We propose that feeding on pollen within M. arboreus flowers is just one of many influences on the life history of F. schultzei and suggest that this may extend to other thrips species/host-plant combinations.     

Wide distributions and cryptic diversity within a Microstomum (Platyhelminthes) species complex

Microstomum lineare is a common species of fresh and brackish waters found worldwide. Three genes (18S, CO1 and ITS) were sequenced from specimens of M. lineare collected from four countries, and the levels of cryptic diversity and genetic structuring were assessed. Results showed M. lineare has very wide haplotype distributions suggesting higher than expected dispersal capabilities. In addition, three new species were described on the basis of molecular taxonomy: Microstomum artoisi sp. nov., Microstomum tchaikovskyi sp. nov. and Microstomum zicklerorum sp. nov.     

Histochemical localization of FMRFamide, serotonin and catecholamines in embryonic Crepidula fornicata (Gastropoda, Prosobranchia)

 Recent reports indicate that neuronal elements develop in early larval stages of some Gastropoda from the Pulmonata and Opisthobranchia prior to the appearance of any ganglia of the future adult central nervous system (CNS). The present study describes similar early neuronal elements in Crepidula fornicata. A posterior FMRFamide-like immunoreactive (LIR) cell with anteriorly projected fibers was observed in the trochophore stage. Additional FMRFamide-LIR and serotonin-LIR cells and fibers were found in the apical organ in the trochophore and early veliger stages. FMRFamide-LIR and serotonin-LIR projections to the velum and foot were also detected at this time. As the veliger developed, peripheral FMRFamide-LIR and later catecholaminergic cells were located in the foot region. Also during this stage, catecholaminergic cells and processes were observed near the mouth. In addition, this study tentatively identified the first serotonin- and FMRFamide-LIR cells and fibers within the developing ganglia of the adult CNS, which appeared in close proximity to the earlier developing elements. These observations are consistent with the hypothesis that, in addition to its presumed role in the control of larval behaviors, the larval nervous system guides the development of the adult CNS. Larvae from the class Bivalvia and other invertebrate phyla also have neuronal elements marked by the presence of FMRFamide, serotonin, and catecholamines, and, therefore, this study may provide additional insights into phylogenetic relationships of the Gastropoda with other representatives of the Mollusca and different invertebrate phyla. Accepted: 10 February 1999     

Role of Catecholamines in Regulation of the Functional State of Vasopressinergic Cells of the Rat Hypothalamus

In in vivo and in vitro experiments there have been shown different mechanisms of regulation of hypothalamic vasopressinergic neurons, including regulation due to changes of activity level of brain catecholaminergic and NPY-ergic neurons innervating hypothalamic vasopressinergic cells. We demonstrated in in vitro experiments that dopamine and noradrenaline had no effects on vasopressin expression, but inhibited its release from cell perikarya in supraoptic and paraventricular nuclei of hypothalamus. Besides, activity of vasopressinergic neurons might probably be regulated via activation of synthesis of these neurotransmitters in vasopressinergic cells themselves in the supraoptic and paraventricular nuclei. To activate synthesis of various neurotransmitters, in our case, catecholamines and NPY, in vasopressinergic neurons, different stimuli adequate to trigger or activate synthesis of these substances are required. Synthesis of catecholamines in vasopressinergic cells of supraoptic and paraventricular nuclei was revealed after immobilization stress and adrenalectomy. NPY is synthesized in neurons of hypothalamic neurosecretory centers in norm, and its synthesis increases at disturbances of NPY-ergic innervation of vasopressinergic cells.     

Mating Behavior and Species Status of Host-Associated Populations of the Polyphagous Thrips, <Emphasis Type="Italic">Frankliniella schultzei</Emphasis>

The thrips taxon, Frankliniella schultzei Trybom, could possibly comprise a complex of unrecognised cryptic species. We therefore made a range of reproductive behavioral observations on F. schultzei individuals from different host-associated populations to investigate their species status. We first described the mating behavior of F. schultzei taken from flowers of a primary host species, Malvaviscus arboreus. The pattern of male-female interaction was complex and protracted, with several behavioral steps being performed. These steps were similar for all mating pairs. Most females responded to male presence before physical contact, suggesting that male pheromones may be involved. We then compared mating behaviors, brood sizes and sex ratios of F. schultzei pairs derived from different host plant species. All pairs in crosses regardless of their original host plant species produced female offspring, indicating successful mating. Frequencies of behaviors were similar for all pairs. Only those crosses involving F. schultzei individuals from Erythrina crista-galli flowers differed from the general pattern in pre-copulation and copulation times. The females from E. crista-galli also produced significantly fewer offspring than females from other crosses. Finally we used traps baited with males from two populations, but set among flowers of one host plant species, to test for differential attraction of females. We found no evidence that the different host-associated populations investigated differ from one another functionally in their sex pheromone system. However, these results do not yet discount the possibility of cryptic species within the taxon F. schultzei. Populations of F. schultzei on many other host species need to be investigated from this perspective.     

The significance of prey in the diet of the phytophagous thrips, Frankliniella schultzei

1. Patterns of mite egg consumption by the phytophagous thrips Frankliniella schultzei Trybom were investigated. Although F. schultzei predation is somewhat similar to that of F. occidentalis (Pergande), the understanding of predation by these two phytophagous thrips was extended, allowing the functional significance of flower thrips’ predatory behaviour to be reinterpreted. 2. Second-instar larvae consumed significantly more eggs than any other life-stage, and the daily intake of eggs by second-instar larvae declined significantly with each successive day of the 4-day duration of instar two. 3. Mite eggs that had had their silken webbing removed were consumed at a significantly greater rate than those with their webbing intact. 4. Frankliniella schultzei immatures developed successfully both on diets containing cotton (Gossypium hirsutum L.) leaf tissue plus mite eggs and on cotton leaf tissue alone. Supplementing a leaf tissue larval diet with mite eggs lowered the developmental time from egg to adult significantly, as well as lowering the percentage mortality. Continuation of the mite egg supplement beyond adult eclosion increased fecundity significantly and extended life span over that achieved on a leaf diet alone. 5. In laboratory choice tests, mite eggs and pollen of Wax Mallow (Malvaviscus arboreus Cav.), the usual host of F. schultzei in Brisbane, were encountered with similar frequencies. Furthermore, the mean proportion of encounters with pollen grains that resulted in consumption of pollen did not differ significantly from the encounter : consumption rate for mite eggs. 6. Frankliniella schultzei, like F. occidentalis, does not seem to be specifically adapted for preying on mite eggs, even though such predation enhances performance and reproductive output of F. schultzei when constrained on cotton leaves. Comparison of performance results with those published for F. schultzei when reared on the floral parts of one of its primary hosts (M. arboreus) (Milne et al., 1996), indicates that mite egg predation does not make up completely for a deficient adult or larval diet.     

Relationships between Tospovirus Incidence and Thrips Populations on Tomato in Mendoza, Argentina

The objectives of this work were to estimate the capability of local populations of thrips as vectors of groundnut ringspot virus (GRSV) and tomato spotted wilt virus (TSWV), and to determine the species composition of vectors in tomato crops. Transmission assays were performed. Incidence of tospoviruses was estimated in commercial crops. Random samples of flowers were taken from tomato for identification of thrips. Of the five species of thrips tested, Frankliniella gemina (first record), F. occidentalis and F. schultzei transmitted GRSV and TSWV. F. schultzei was a significantly more efficient vector of GRSV than F. occidentalis under controlled assay conditions. The thrips were identified on flowers from six surveyed tomato crops. F. occidentalis was the most frequently identified species (43.0%), followed by F. schultzei (35.6%) and Thrips tabaci (10.1%). The incidence of tospoviruses was low (1.1–2.8%) in crops planted during August–September and greater (9.5–61.1%) in crops planted in December. GRSV was prevalent (85%) over TSWV (11%).     

Immunocytochemical localization of histamine in flatworms

Summary 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-immuno-reactivity 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.