An immunocytochemical method for histamine: application to the planarians

Histamine-immunoreactivity was investigated in the planarians Dugesia tigrina and Polycelis nigra. Specific antisera against a histamine-protein conjugate were used, and 1-ethyl—3 (3-dimethyl-aminopropyl) carbodiimide was used both as coupling agent to prepare the antigen and as a tissue fixative. In D. tigrina, histamine-immunoreactivity was restricted to photoreceptor cells in the cerebral eye. In P. nigra, nerve fibers were found in the ventral nerve cord and nerves running laterally from these. The epidermal eyes did not display histamine-immunoreactivity. The results suggest that histamine may be a transmitter in some of the most primitive animals. They also suggest that the distribution of histamine may differ in planarians.     

GABA in the nervous system of the planarian Polycelis nigra

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in vertebrates and it has a similar inhibitory role in several invertebrate taxa. The transmitters serotonin, octopamine, catecholamines and histamine are present in flatworms while evidence for GABA is still lacking. Therefore, we have studied the occurrence of GABA-like immunoreactivity (IR) in the planarian nervous system. Specimens of Polycelis nigra were fixed in 4% 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide with 2% paraformaldehyde. The GABA-antiserum was raised in rabbits against GABA conjugated to keyhole limpet hemocyanin. Preabsorption with GABA-ovalbumin conjugate abolished all IR. The results were further confirmed with an monoclonal antibody and high pressure liquid chromatography (HPLC). In P. nigra GABA-like IR was seen as long, often varicose, sparsely distributed fibers in the ventral longitudinal nerve cords. IR was also located in a few cell somata in the brain and in the neuropil of the brain. The IR was restricted to the central nervous system and was absent in peripheral nerves and plexuses. The HPLC analysis supported the presence of GABA.Our results suggest that GABA is an interneuronal transmitter in P. nigra. The results also suggest a phylogenetically old origin of GABAergic neurotransmission.     

Geographical distribution of Polycelis (Seidlia) auriculata in Japan

Polycelis (Seidlia) auriculata is endemic to mountain districts of Japan, from the central part of Honshû to the area of the Daisetsu Mts of Hokkaidô. In northern Japan, it sometimes occurs in cold-water biotopes of lowland areas. The progenitor of P. auriculata appears to have been the oldest immigrant into northern Japan among the Japanese Polycelis species, entering through a northern route as a preglacial faunal element. P. auriculata now shows a discontinious distribution in northern Japan. By virtue of its geographical and vertical distribution, ecological niche, variation in anatomy of the copulatory apparatus, and cytodemes, this species appears to be in the process of transformation.     

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.     

Triclads (Turbellaria: Tricladida) as predators of lake-dwelling stonefly and mayfly nymphs

It has been demonstrated by using a serological technique that Polycelis nigra (Müller) and P. tennis (Tjima) feed on mayfly and stonefly nymphs in unproductive lakes. Such feeding is less in productive lakes where these insects form a smaller proportion of the invertebrate fauna of the stony littoral zone. These nymphs form a significantly larger part of the diet of P. nigra than of P. tenuis; both triclad species feed more on stoneflies than mayflies.     

The food niches of the invasive Dugesia tigrina (Girard) and indigenous Polycelis tennis Ijima and P. nigra (Müller) (Turbellaria; Tricladida) in a Welsh lake

The invasion of Llyn Coron in North Wales by the American immigrant Dugesia tigrina resulted in the almost entire displacement of the native Polycelis tenuis and P. nigra populations. Because competition for food is the most important factor controlling and regulating populations of British lake-dwelling triclads, the diets of the invasive and native triclads in the lake were examined in an attempt to explain the successful invasion.A serological technique, the precipitin test, identified the gut contents of field-collected triclads. Niche breadth, electivity and niche overlap indices were used in analysis of the data. A broad food niche was recorded for all the triclad species. Oligochaetes predominated in the diet of D. tigrina followed by Asellus and chironomids, in almost equal proportions, snails and caddisflies, with mayflies scarcely eaten. Both Polycelis species fed heavily on oligochaetes followed by Asellus. P. tenuis ate slightly higher proportions of chironomids and caddisflies than snails and mayflies, whilst P. nigra consumed similar proportions of these four prey taxa. Gammarus, scarce in Llyn Coron, and cladocerans were not eaten by the Polycelis species and by only a few D. tigrina.To-date there has been no apparent shift in the diet of the Polycelis species as a consequence of the invasion by D. tigrina. In the absence of any evidence for the partitioning of food on the basis of prey size or condition, the considerable overlap in the diets of D. tigrina and the Polycelis species suggests the potential for severe inter-specific competition for food. The superior exploitation by D. tigrina of the available food resource requires explanation.     

Laboratory studies on the biology ofSpalangia nigra [Hym.: Pteromalidae]

At 21 °C,Spalangia nigra Latreille (Hymenoptera: Pteromalidae) averaged 29.3 days between exposure and emergence of 1^st progeny from host house flies,Musca domestica L. (Diptera: Muscidae). At 27 °C, the average developmental time to 1^st emergence was reduced to 26.6 days, and a majority of adult wasps emerged from host house fly puparia between 29 and 40 days postoviposition. The sex ratio of progeny ranged from 1.4 to 1.8 female-to-male, but all progeny of virgin females were male. Male wasps lived from 6.8–15 and females 11–17.8 days at 27 °C; honey as a food source increased longevity. No significant differences in parasitism byS. nigra were associated with host house fly pupal densities ranging from 1 to 200 pupae per female-male pair of wasps, but average percent parasitism decreased at host densities greater than 50. House fly pupae exposed to parasitism at ages ranging from 4 to 96 h did not differ in subsequent production of adult flies.S. nigra did not demonstrate preference for house flies or stable flies,Stomoxys calcitrans (L.) (Diptera: Muscidae) as hosts. The results of these studies indicate thatS. nigra may contribute significantly to previously unexplained mortality of house flies and stable flies.      

The responses to current flow of two stream dwelling triclads,Crenobia alpina (Dana) and Polycelis felina (Dalyell)

Summary An apparatus is described which allows triclads to choose between the fast and slow regions of a stirred tank. In the absence of a stone substratum Crenobia alpina preferred or tolerated higher current speeds than Polycelis felina, this effect was enhanced when a stone substratum was added to the apparatus. These results indicate that the absence of Polycelis felina from the steep gradient regions of streams could be accounted for by its preference for slower micro-currents. Conversely the presence of Crenobia alpina in steep gradient regions could be accounted for by its preference for or tolerance to higher micro-currents.     

Studies on the speciation of Japanese freshwater planarian Polycelis auriculata based on the analysis of its karyotypes and constitutive proteins

Various kinds of chromosomal polymorphisms or karyotypic variations are found in the Japanese freshwater planarian Polycelis auriculata. Within this species, there are found worms whose chromosome numbers are 2n = 6, 10, 11, 12 and others, and 3x = 6 and 9. There are some which have cells with triploidy and tetraploidy complements (3x = 6 & 4x = 8), and others which have cells with triploidy and hexaploidy complements (3x = 6 & 6x = 12). These worms with such varied karyotypes are usually found in separate habitats, though occasionally they occur together. Electrophoretic analysis of the proteins extracted from the karyotipically different worms which belong to three different local populations shows some dissimilarity in the constitutive proteins according to their karyotypic differences. The results obtained suggest that this species is still in the process of speciation or chromosomal evolution.     

Effects of collembola (arthropoda) and relative germination date on competition between mycorrhizalPanicum virgatum (Poaceae) and non-mycorrhizalBrassica nigra (Brassicaceae)

In this experiment the separate and interactive effects of grazing of vesicular-arbuscular mycorrhizal hyphae by collembola and of relative germination date on competition between the mycorrhizal perennial grassPanicum virgatum and the non-mycorrhizal annual cruciferBrassica nigra were investigated. In the absence of competition,P. virgatum mass and P uptake were not affected by collembola grazing; grazing did reduce tissue N concentration and root: shoot ratio. Competition fromB. nigra plants of the same age/size (“simultaneous competition”) significantly reducedP. virgatum total, root, and shoot mass relative to control plants not subject to competition. In contrast, when in competition,B. nigra plants did not differ in biomass fromB. nigra controls grown without competition. Simultaneous competition also reduced N and P uptake byP. virgatum, but not byB. nigra. Grazing by collembola during simultaneous competition increased the differences in nutrient uptake and tended to shift the competitive balance further toward the non-mycorrhizalB. nigra. WhenP. virgatum plants were subjected to competition fromB. nigra plants which germinated three weeks later (“offset competition”) the situation was reversed: offsetB. nigra plants were negatively affected by competition while the larger, olderP. virgatum plants were not. Thus, relative germination date is important in determining the relative competitive ability of these two species. Grazing by collembola did not affect offset competition. The grazing of VAM hyphae by collembola appears to increase N availability in this experimental system. Under simultaneous competition, this N is taken up by the more extensive root system ofB. nigra; under offset conditions, the root system of the smallerB. nigra plants is insufficient to take advantage of the added resources. Thus, we suggest that grazing-induced transient changes in nutrient availability and the differential abilities of the two species to make use of these added resources constitute the mechanisms by which relative germination date and collembola grazing influence competition.     

Feeding on gastropods by lake-dwelling Polycelis in the absence and presence of Dugesia polychroa (Turbellaria, Tricladida)

SUMMARY. Using a serological technique, the extent of feeding upon snails by Polycelis tenuis was compared in habitats lacking and containing Dugesia polychroa which is the major triclad predator of snails. It was found that P. tenuis ate significantly more snails when D. polychroa was absent. It was concluded that in the field interspecific competition for snails between these two triclad species was a more powerful process than any social feeding by P. tenuis on snails captured by D. polychroa .
Feeding on snails as a group by P. tenuis and P. nigra in the habitat where the two triclads co-existed was of a similar magnitude and this was also true of their feeding on the common snail species and genera. Approximately 10% of the triclads gave a positive reaction to snail antisera.
Feeding on snails by P. nigra in the three habitats lacking D. polychroa was similar in extent and did not differ from that of P. tenuis . It was concluded that P. nigra would be similarly affected by the presence and absence of D. polychroa as shown for P. tenuis .
No definitive seasonal pattern was detected in the incidence of feeding upon snails by either Polycelis species but this may have been obscured by seasonal changes in the detection period of snail antigens due to temperature effects. There was some evidence that such feeding increased during the period when small snails were present in the population.     

Extracellular Matrix in Platyhelminths,with Special Reference to the Presence of Fibronectin

The extracellular matrix (ECM) was studied in the turbellarians Polycelis nigra and Microstomum lineare, the cestode Diphyllobothrium dendriticum and the trematode Dicrocoelium dendriticum. Routine LM staining methods for connective tissue gave positive results only in P. nigra. Positive staining reactions were observed in the subepithelial basal lamina, around the pharynx and as strings in the tissues. Peroxidase-anti-peroxidase and immunofluorescence methods were used to identify fibronectin. Positive results were obtained in all species. Positive reactivity to anti-fibronectin was observed in the subepithelial basal lamina and as a thin network between the cells of the tissues. Some intracellular reactivity occurred, but the cell type was not identified. In M. lineare a strong positive reactivity was observed in the regenerating area.     

Ventilatory and blood gas adjustments in exercising isothermic ducks

Summary The free-living planarianPolycelis nigra has a complete sequence of glycolytic and tricarboxylic acid cycle enzymes together with an active betaoxidation sequence. Neither octopine dehydrogenase nor any other of the pyruvate: amino acid-linked dehydrogenases was present inP. nigra. The lactate dehydrogenase of this planarian was, however, unusual in being activated by fructose-1,6-bisphosphate.The steady state contents of the glycolytic and tricarboxylic acid cycle intermediates were measured in quick frozenP. nigra. A comparison of the mass action ratios with the equilibrium constants for the glycolytic reactions showed that phosphoglucomutase, glucosephosphate isomerase, aldolase, triosephosphate isomerase, phosphoglyceromutase and phosphopyruvate hydratase reactions are all near equilibrium, whilst phosphofructokinase and pyruvate kinase reactions are displaced from equilibrium. No phosphagen or phosphagen phosphotransferase activity could be detected inP. nigra but it is possible that the high levels of 3-phosphoglycerate could function as an alternative store of high energy phosphate.Under anaerobic conditionsP. nigra produces lactic acid; there is no evidence for the production of succinate, acetate or propionate, acids characteristically produced by parasitic platyhelminths.     

An experimental study of the role of gradient and substratum in the distribution of two stream-dwelling triclads, Crenobia alpina (Dana) and Polycelis felina (Dalyell) in North Wales

A laboratory stream is described which has simulated steep- and shallow-gradient bottoms whose gradients can be varied. It was found that Polycelis felina, the principal triclad of shallow-gradient streams had a much reduced ability to colonize the simulated steep-gradient bottoms when compared to Crenobia alpina, which is the principal triclad of the steep-gradient bottoms in the field. P. felina is unable to move in the steep-gradient bottoms to any great extent, while C. alpina showed considerable ability to do this. When a reduction in flow was made this did not increase the proportion of P. felina to be found in the steep-gradient regions. The triclads were also tested for possible acclimatory responses and it was found that their responses were more fundamentally based. On the basis of the above evidence it is suggested that the absence of P. felina from the steep-gradient regions of streams in North Wales is due to a behavioural inability to tolerate the current regimes present in the steep-gradient bottoms.     

Sambucus nigra agglutinin is located in protein bodies in the phloem parenchyma of the bark

The bark of some young woody stems contains storage proteins which are subject to an annual rhythm: they accumulate in the autumn and are mobilized in the spring. We show here that the bark phoem-parenchyma cells of Sambucus nigra L. contain numerous protein bodies, and that the bark lectin (S. nigra agglutinin) which undergoes an annual rhythm is localized in these protein bodies. The protein bodies in the cotyledons of legume seeds also contain lectin, indicating that lectins may be storage compounds themselves or may have a function in storage and-or mobilization processes.Abbreviations PBS phosphate-buffered saline - IgG immunoglobulin - SNA Sambucus nigra agglutinin     

Application of genetic markers to the discrimination of European Black Poplar (Populus nigra) from American Black Poplar (P. deltoides) and Hybrid Poplars (P. x canadensis) in Switzerland

European Black Poplar (Populus nigra) is considered a rare and endangered tree species because of severe reduction of its natural riverine habitat and potential hybridisation with the related non-indigenous taxa P. deltoides and P. x canadensis. As it is difficult to distinguish these taxa solely based on their morphology, we applied a PCR-based assay with an easy-to-use and robust molecular marker set (cpDNA trnL-trnF/RsaI RFLP, nDNA win3 and nDNA POPX/MspI RFLP) in order to identify pure P. nigra. Different plant tissues could be used for fast and standardised DNA extraction. The application of the three marker types was tested on a number of different Populus taxa, and they were also used for the verification of pure P. nigra in a sample of 304 putative P. nigra individuals from Switzerland. Cross-checking of the DNA data with those using a traditional allozyme approach resulted in complete agreement. The availability of molecular identification methods is an important prerequisite for the conservation of European Black Poplar, because pure, non-introgressed plant material can then be used in restoration projects of European floodplains.     

Considerations on the neoblasts of planarians on the basis of certain karyological evidence

The problem of the sexual differentiation of the neoblasts of planarians is approached on the basis of karyological evidence assembled by the author and by his collaborators. In the polyploid and pseudogamic biotypes of Dugesia benazzii, D. lugubris and Polycelis nigra, the transformation of the neoblasts into oogonia or spermatogonia is accompanied by variations in the chromosome set. These variations may be considered to be dependent upon the gradient of sexuality — that is, of the territorial influences that determine the evolution in a female or a male direction. Significant evidence is also inferred from the chromosome cycle of hybrids between amphigonic diploid biotypes and polyploid biotypes. A new direction of research, based on the comparison between anterior and posterior regenerated segments of experimental polyploids with variable chromosome sets aims at establishing possible regional differences in the set of the neoblasts.Dedicated to Prof. Dr. J. Seiler on the occasion of his eightieth birthday.     

Parvalbumin-immunoreactive proteins in the nervous system of planarians

Summary 1. Using immunological methods, we have identified parvalbumin-like material in the triclade flatworms,Polycelis nigra, Polycelis auriculata, Crenobia alpina, Dugesia tahitiensis, andDugesia polychroa.2. Western immunoblot analysis of these five species revealed heat stable parvalbumin-immunoreactive proteins between 32 kD and 44 kD.3. Proteins at 19 and 32 kD revealed intense labeling with⁴⁵Ca²⁺.4. Double immunodiffusion of planarian supernatants showed complete fusion of precipitates, indicating immunological relatedness of the parvalbuminlike material among the species investigated.5. Immunocytochemical studies exhibit parvalbumin-immunoreactive material exclusively in neurons supporting the notion of an early evolutionary appearance of these proteins in the nervous system.     

A Quantitative Study of the Comparative Distribution of Non-parasitic Leeches and Triclads in the Stony Littoral of British Lakes

For each of the three species of leeches and four species of triclads inhabiting the stony littoral of 100 British lakes of diverse trophic status, numbers, biomass and mean weight were correlated against a wide range of physical, chemical and other variables. Very few significant correlations were found between the various biological variables and the non-chemical variables. Significant, positive correlations were obtained for all leech and triclad variables against all of the chemicals, with the exception of Erpobdella octoculata numbers, Helobdella stagnalis mean weight and all of the Polycelis nigra variables which did not correlate with any. Relationships between biological variables and calcium content are given special consideration. Over the range of lakes investigated, total triclad numbers exceeded leech numbers but the reverse was usually true for total biomass. Correlations between the various leech and triclad variables are examined.     

Observations on the comparative ecology of lake-dwelling triclads in southern Sweden,Finland and northern Britain

Summary There are more lake-dwelling species of triclad in northerm Britain (10) than in southern Sweden (7) and the distribution of some differ in the two countries over approximately comparable types of lake. Dendrocoelum lacteum (Müller) occurs in a wide range of lake types in s. Sweden but is restricted to more productive lakes in n. Britain. Polycelis nigra (Müller) (absent from Sweden) has a similar, wide occurrence in Britain. Planaria torva (Müller) is more common in s. Sweden in lakes of all types but particularly in the less productive lakes. The Polycelis species P. tenuis (Ijima) and P. hepta E. H. and Y. Melander, also the Dugesia lugubris species complex show much the same distribution in both areas. Polycelis felina (Dalyell) does not occur in Sweden. The small nuber of Finnish lakes sampled confirm the data for s. Sweden. There is significant correlation between the total abundance of triclads and both the hardness and dissolved matter contents of the Swedish lake waters. Quantitatively the s. Swedish triclad populations compare most closely with those of Mid-Scotland in the n. Britain area.

---

Zusammenfassung Es gibt im Norden Britanniens eine grössere Anzahl von Tricladen (10) die Süsswasserseen bewohnen, als in Süd-Schweden (7) und einige Sorten verbreiten sich in ungefähr gleichen Arten von Seen in verschiedenem Maasse in den beiden Ländern. Dendrocoelum lacteum (Müller) erscheint in einer grossen Reihe verschiedenartiger Seen in Süd-Schweden, beschränkt sich aber im Norden Britanniens auf die Seen mit grösserem Reichtum an Lebewesen. Polycelis nigra (Müller) — in Schweden nicht vorhanden-, hat eine ähnlich weite Verbreitung in Britannien. In Süd-Schweden ist in Seen aller Art ein reicherer Bestand an PLanaria torva (Müller), besonders in den Seen mit geringerem Bestand an Lebewesen. Die Polycelis Art P. tenuis (Ijima) und P. hepta E. H. und Y. Melander und auch der Dugesia lugubris Arten-Komplex zeigen dieselbe Verbreitung in beiden Gegenden. Polycelis felina (Dalyell) kommt in Schweden nicht vor. Die kleine Zahl finnischer Seen, denen Proben entnommen wurden, bestätigen die Befunde von Süd-Schweden. Wesentlicher Zusammenhang besteht zwischen der gesamten Reichhaltigheit von Tricladen und dem Gehalt an Kalk und aufgelöster Substanz bei den schwedischen Süsswasserseen. Mengenmässig ist die Verbreitung von Tricladen in Süd-Schweden der im Norden Britanniens am echesten zu verleichen, soweit es sich um Mittel-Schottland handelt.