Localisation of somatostatin- and gastrin-like immunoreactivity in the gastrointestinal tract of Ciona intestinalis L.

Summary Somatostatin- and gastrin-like immunoreactivity has been found by immunofluorescence in cells of the stomach and intestinal epithelia of Ciona intestinalis L. The cells containing the peptide immunoreactive to mammalian anti-gastrin can be restained with the Grimelius' technique for argyrophilia.     

The occurrence of argyrophilic and argentaffin cells in the gut of Ciona intestinalis L.

Summary Argyrophilic and argentaffin cells occur in the stomach and intestinal epithelium of the sea-squirt, Ciona intestinalis L.. These cells are characterized by their basal swelling which contains the nucleus surrounded by small secretory granules and by a filamentous cell-apex which reaches the gut lumen. The cells are scattered unevenly within the epithelium. Their number decreases rapidly towards the lower part of the intestine. The localization, size of granules and their shape are features which differentiate these cells from other secretory cells in the gut epithelium such as mucous cells. These cells are thought to possess an endocrine function.The excellent technical assistance of Mrs. R. Sprang is gratefully acknowledged     

Nerve cells and synaptic connections in the intestinal nerve of the snail,Helix pomatia L.

Summary The ultrastructure of nerve cells and the finestructural organization of synaptic contacts have been investigated in the intestinal nerve in the snail Helix pomatia. Three types of nerve cells, occurring singly or in groups, can be distinguished on the basis of the ultrastructure of their perikaryon and content of granules. The peripheral output of these nerve cells has been verified by retrograde CoCl₂ and NiCl₂ staining. Both axosomatic and axo-axonic specialized synaptic contacts occur in the intestinal nerve. Presynaptic elements of these synaptic contacts contain 100–120 nm granular vesicles or 120–200 nm neurosecretory-like granules. Following intracellular horseradish peroxidase (HRP) labelling of identified central neurons responsible for peripheral regulatory processes, several labelled axons running toward the periphery can be followed throughout the branches of the intestinal nerve. These labelled axon processes (either primary axon or small collaterals) form specialized synaptic contacts, inside the intestinal nerve, and are always in a postsynaptic position. The occurrence of peripheral axo-somatic and axo-axonic synapses provides a morphological basis for integrative processes taking place in the intestinal nerve (peripheral nervous system) of Helix pomatia.     

Substance P-, neurotensin- and bombesin-like immunoreactivities in the gill epithelium of Ciona intestinalis L.

Summary Substance P-, neurotensin- and bombesin-like immunoreactivities were localised in some gill epithelial cells in the pharynx of Ciona intestinalis L. No immunoreactivity was obtained with antisera to gastrin, glucagon, insulin, pancreatic polypeptide or calcitonin. Some of the epithelial cells of the gills were shown to be argyrophilic with the Grimelius technique.     

Isolation and characterization of ferritin from the hepatopancreas of the musselMytilus edulis

Summary The main iron-binding protein in the hepatopancreas of the musselMytilus edulis, which had been previously iron-loaded by exposure to carbonyl iron (spheres of elemental iron less than 5 m diameter), has been isolated to electrophoretic purity and identified as ferritin. This ferritin hasM _r, of 480000, pI of 4.7–5.0 and is composed of two subunits,M _r 18500 andM _r 24600. Under the electron microscope, it appears as electron-dense iron cores of average diameter 5 nm surrounded by a polypeptide shell to a final average overall diameter of 11 nm. The purified protein contains, on average, 200 iron atoms/molecule protein. On immunodiffusion,M. edulis hepatopancreas ferritin gives a partial cross-reaction with antiserum to horse spleen ferritin and lamprey (Geotria australis) liver ferritin but does not react with antiserum to chiton (Acanthopleura hirtosa) haemolymph ferritin.     

Blue-green algalike cells associated with the tunic of Ciona intestinalis L.

Summary Certain organisms resembling blue-green algae embedded in the tunic of the solitary ascidian Ciona intestinalis L. are described. Their probable symbiotic role as related to the peculiar habitat of this ascidian is suggested.     

Selective ingestion of phytoplankton by the bivalvesMytilus edulis L. andCerastoderma edule (L.)

Blue mussles (Mytilus edulis) and cockles (Cerastoderma edule) were collected in the Oosterschelde estuary (SW Netherlands) and exposed to diets consisting of the diatomPhaeodactylum tricornutum and silt. Algal concentrations were kept constant (20.10³ cells.ml^–1), and silt concentrations were varied between 3–80 mg.l^–1 (mussel experiments) and 20–120 mg.l^–1 (cockle experiments). Chlorophyll-a concentrations in the pseudofaeces were reduced compared to the diets, indicating selective ingestion of the algae. It was estimated that the mussels increase the ingestion of chlorophyll-a 2.0 times, and the cockles increase the chlorophyll-a ingestion 2.8 times, compared to the ingestion rate without selection. The selection coefficients were not affected by the SPM concentrations used. Phaeophytin-a concentrations in the faeces showed an increase as a consequence of the digestion of the ingested algae. Digestive efficiencies of chlorophyll-a varied between 36–92% The digestive efficiencies decreased with increasing SPM concentrations.Communication no. 541 of the Delta Institute for Hydrobiological Research.     

Ultrastructure and migration of screening pigments in the retina of Pieris rapae L. (Lepidoptera,Pieridae)

Summary The retinal morphology of the butterfly, Pieris rapae L., was investigated using light and electron microscopy with special emphasis on the morphology and distribution of its screening pigments. Pigment migration in pigment and retinula cells was analysed after light-dark adaptation and after different selective chromatic adaptations. The primary pigment cells with white to yellow-green pigments symmetrically surround the cone process and the distal half of the crystalline cone, whilst the six secondary pigment cells, around each ommatidium, contain dark brown pigment granules. The nine retinula cells in one ommatidium can be categorised into four types. Receptor cells 1–4, which have microvilli in the distal half of the ommatidium only, contain numerous dark brown pigment granules. On the basis of the pigment content and morphology of their pigment granules, two distal groups of cells, cells 1, 2 and cells 3, 4 can be distinguished. The four diagonally arranged cells (5–8), with rhabdomeric structures and pigments in the proximal half of the cells, contain small red pigment granules of irregular shape. The ninth cell, which has only a small number of microvilli, lacks pigment. Chromatic adaptation experiments in which the location of retinula cell pigment granules was used as a criterium reveal two UV-receptors (cells 1 and 2), two green receptors (cells 3 and 4) and four cells (5–8) containing the red screening pigment, with a yellow-green sensitivity.     

Innervation of the anterior byssal retractor muscle in Mytilus edulis L.

Summary Studies on the intrinsic innervation of the anterior byssal retractor muscle (ABRM) in Mytilus edulis L. were continued at the ultrastructural level. Electron micrographs show nerve processes ensheathed by glio-interstitial cells running between muscle fibers. The glio-interstitial cells may represent all the types of osmiophilic cells previously described by the light microscopic ZIO technique in the anterior byssal retractor muscle.     

Evaluation of the sample size used for the rapid bioassessment of rivers using macroinvertebrates

Experiments were designed to investigate selective predation by medium (40–55 mm carapace width: CW) and large (55–70 mm CW) Carcinus maenas when feeding on four bivalves of contrasting shell morphology. Size-selection was examined by presenting individual crabs with a wide size range of Mytilus edulis, Ostrea edulis, Crassostrea gigas and Cerastoderma edule. Medium-sized crabs preferred mussels 5–15 mm shell length (maximum shell dimension: SL) and cockles 5–10 mm SL, whereas large crabs preferred mussels 15–25 mm and cockles 10–20 mm SL. Crabs generally showed no preference for any particular size of either oyster species. Species-selection was examined by presenting individual crabs with paired combinations of the four bivalves in various proportions. When offered mussels and oysters simultaneously, both size categories of crabs consistently selected mussels, and food choice was independent of prey relative abundance. By contrast, C. maenas selected mussels and cockles as expected by the frequency in which each size category of crab encountered the preferred size ranges of prey. Crab preference clearly paralleled the rank order of prey profitability, which in turn was mainly determined by prey biomass, suggesting that active selection takes place at some point of the predation cycle. Experiments with epoxy resin models showed that initial reluctance of crabs to attack oysters was not associated with the ultimate energy reward. Moreover, they suggest that foraging decisions are partly based on evaluations of overall prey shape and volume, and that the minimum dimension of the shell constitutes an important feature which crabs recognise and associate with prey value.     

Localization of somatostatin-, substance P- and calcitonin-like immunoreactivity in the neural ganglion of Ciona intestinalis L. (Ascidiaceae)

Summary Indirect immunofluorescence studies using antisera to synthetic somatostatin, human calcitonin and substance P indicate, in the neural complex of the sea-squirt, Ciona intestinalis L., that these polypeptides are present in large perikarya situated at the periphery of the cerebral ganglion as well as in some smaller perikarya in the medulla. In the medullary and transitional zone, there are nerve fibres that cross-react positively with anti-calcitonin and antisubstance P.     

Distribution of GABA-like immunoreactivity during post-metamorphic development and regeneration of the central nervous system in the ascidian Ciona intestinalis

During regeneration of the neural ganglion in Ciona intestinalis, the pattern of reappearance of some peptidergic cells is similar to the ontogenetic patterns exhibited by these cell types during normal post-metamorphic development. Using a specific antiserum to gamma-aminobutyric acid (GABA), we describe here the appearance of GABA-ergic cells in Ciona during both post-metamorphic development and regeneration of the neural ganglion following total ablation. Post-metamorphic animals were divided into the categories: 1, 3–5, 6–10, 11–15 and 23–27 mm in body length. Regeneration was monitored at 12, 15, 18, 21, 28 and 56 days post ablation. The first appearance of GABA-like immunoreactive cells during normal development were at the 3 to 5-mm stage where they were seen as discrete cells, without processes, evenly distributed in the cortical region throughout the ganglion. Fibres were first seen at the 6 to 10-mm stage. As development proceeded, GABA-like immunoreactive cells became more concentrated near the nerve root exits and along the dorsal rind of the ganglion. In regenerating ganglia, GABA was first detected at 18–21 days post ablation, in cells that lacked any obvious processes and that were distributed in all regions of the ganglion. At 28 days post ablation, processes could be detected in the neuropile, and after 56 days GABA cells were found predominantly in the same regions as in the normally developing adult ganglion. Although the overall pattern reflects that in a normal adult, a few differences were detectable. For example, rather more GABAergic cells were concentrated ventrally in the ganglion close to the neural gland.     

Secondary production of an intertidal mussel (Mytilus edulis L.) population in the Eastern Scheldt (S.W. Netherlands)

We monitored an intertidal mussel (Mytilus edulis L.) population between June 1981 and June 1982 in the Eastern Scheldt estuary (S.W. Netherlands). Density and biomass of the population remained relatively constant over the study period. The shell length growth was described by a Gompertz growth curve. The parameters of this equation were estimated from a log-log-modified Ford-Walford plot of the growth-ring data. The slope of the relationship between animal weight and shell length is season-dependent, mainly due to the spawning cycle in larger mussels.Secondary production is estimated with the growth rate method. In the calculated growth rates the change in slope of the length-weight relationship is incorporated, as well as differences in length growth rates between summer and winter. Secondary production amounts to 156 g AFDW m^–2a ^–1 (expressed per m² of mussel bank). P:B is 0.50 a^–1. The mussel productivity is probably a limiting factor for the density of overwintering Oystercatchers (Haematopus ostralegus).     

Neuroepithelial bodies in the lung of the tree frog,Hyla arborea L.

Summary The endocrine-like cells (ELC), which together with nerve endings form the neuroepithelial bodies, are located on primary and secondary septa in the non-ciliated epithelium of the lung of Hyla arborea. ELC protrude markedly toward the lumen of the lung and are surrounded by pneumocytes, which separate ELC from the lumen by thin cytoplasmic processes. ELC possess a light cytoplasm containing two types of granules: (i) numerous small granules, 50–110 nm in diameter, and (ii) single large granules, 290–860 nm in diameter. Numerous nerve fibers, often forming synaptic junctions, can be observed in contact with ELC.This study was supported by a grant No. 476/II from the Polish Academy of Sciences     

Further cytochemical studies of the mucopolysaccharides of the test cells of Ciona intestinalis L.

Summary A morphological and cytochemical study (mucopolysaccharides, proteins and DNA) was made of the test cells of oocytes of Ciona intestinalis collected in February, April, July and September.The tests revealed an increase in the number of PAS-positive test cells in oocytes of 60–80 and of 90–100 during February and April and a parallel increase during these periods of mitotic activity in the cells in question. The findings obtained in this study, a continuation of earlier investigations, are discussed in the light of the possible links that might be established between the vic-glycol groups of the neutral mucopolysaccharides and the proteins.This investigation was carried out in part at the Zoological Station, Naples, using a study post of the National Research Council of Italy.     

In vitro culture of adult and juvenile bud explants of Passiflora species

Cultivar E23, an F1 hybrid of P. edulis and P. edulis f. flavicarpa is usually propagated by shoot-tip grafting. Various media were tested to evaluate the potential of E23 for in vitro propagation. Adult tissue was difficult to culture and did not respond to media containing low (<10 µM) concentrations of growth regulators. Growth of adult buds on intact stem sections was promoted by 1 week of dark incubation on MS basal medium plus 150 µM 2iP, 200 µM adenine sulphate and 17.1 µM IAA (3 mg l^–1), and further developed into shoots on MS medium plus 4.9 µM 2iP (1 mg l^–1) and 5.7 µM IAA (1 mg l^–1). By contrast, juvenile shoots of E23, and Passiflora species: edulis f. flavicarpa, edulis, alata, caerulea, mollissima, coccinea, herbertiana and suberosa grew rapidly on MS medium plus 10 µM kinetin and 5 µM IAA. Rapid multiplication was achieved on MS plus 20 µM BA, 10 µM kinetin, 5 µM IAA, and roots initiated on MS plus 5 µM IAA.Abbreviations IAA indole-3-acetic acid - 2iP N6-iso pentenyl adenine - BA N6-benzyl adenine     

Stylochus tauricus,a predator of the barnacle Balanus improvisus in the Black Sea

The flatworm Stylochus tauricus Jacubova has been found associated with the barnacle Balanus improvisus Darwin, on which it feeds. The predation rate (the number of barnacles eaten by one polyclad in a month) ranges between 5–10. Inside the empty shells of B. improvisus some egg-plates of S. tauricus were observed. Pelagic Götte's larvae aged 2–3 days possess 4 lobes while those aged 7–8 days have 5 lobes. Flatworms can prey on the young of another species Balanus eburneus Gould, whereas predation on the mussels Mytilus galloprovincialis Lam. is rare. There is a direct correlation between predator abundance and prey ingested.     

The hypothalamo-hypophysial system of the wild carp,Cyprinus carpio L.

Summary The posterior neurohypophysis (PNH)-pars intermedia complex of the wild and pond carp, Cyprinus carpio L., has been studied by light, fluorescence and electron microscopy. Gomori-positive neurosecretory fibres are abundant in the main trunk of the neurohypophysis as well as its roots penetrating the pars intermedia. Terminals of these fibres are in contact with capillaries of the general circulation and with glandular cells of the pars intermedia. Monoaminergic fibres with a weak green fluorescence, somewhat increasing after injection of nialamide into the pond carp, have largely the same distribution. Three types of neurosecretory fibres and their terminals have been recognized in the PNH-pars intermedia complex. Types-A₁ and -A₂ fibres, containing granules of 140–180 nm and 100–160 nm in diameter respectively, are peptidergic Gomori-positive. Type-A₂ fibres predominate in the PNH. The least frequent monoaminergic type-B fibres have granules of 60–100 nm in diameter. Numerous peptidergic and few monoaminergic neurosecretory terminals make contact with the capillaries located within the roots of the PNH as well as at the border between them and the pars intermedia. Both peptidergic and monoaminergic terminals make direct synaptoid contacts with the gland cells or end close to connective tissue septa, basal lamina or pituicytes. The PAS-positive gland cells and to a lesser degree the leadhaematoxylin-positive gland cells show these relationships with neurosecretory terminals. The question concerning the mode of interaction between peptidergic and monoaminergic structures in the dual control of the gland cells of the pars intermedia of teleosts is discussed.     

Excrétion de phosphatase acide par les cellules mucipares de la branchie de Mytilus edulis L.

Résumé Dans toutes les cellules mucipares de la branchie de Mytilus edulis L., quelques grains de secretion montrent une activité phosphatasique acide, décelée par la méthode cytochimique de Gomori appliquée au microscope électronique. A la proximité de ces grains se trouvent des dictyosomes golgiens où une activité phosphatasique se décèle dans les parties latérales des cisternes et dans les vésicules qui en émanent. Au moment de l'excrétion, tous les grains de sécrétion confluent et la phosphatase acide active est incluse dans l'amas de mucus expulsé dans la cavité palléale. Le rôle digestif de ces amas — et non purement mécanique — peut ainsi être considéré comme hautement probable. De telles manifestations enzymatiques sont totalement absentes dans le tapis muqueux ainsi que dans les cellules qui le constituent.

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Excretion of acidic phosphatase by the goblet-cells of the gill of Mytilus edulis L.An electron microscopic study

Summary Acidic phosphatase activity detected by the Gomori method applied to E.M., has been found in a few secretion granules of each goblet cell of the gill of Mytilus edulis L. In the close proximity of those granules golgian dictyosomes always occur, with acidic phosphatase activity in the lateral parts of the cisternae and in the vesicles arising from the latter. When the mucus is extruded, active and non active secretion granules are mixed and thus, acidic phosphatase activity is regularly found in the mucous tufts extruded into the palleal cavity. Therefore the digestive role — and not merely mechanic — of the mucous tufts may be considered as highly probable. Conversely, the secretion granules from which arises the continuous mucous cover of the epithelium and this cover also, are entirely devoid of any acidic phosphatase activity.

     

The proteomic response of sea squirts (genus Ciona) to acute heat stress: a global perspective on the thermal stability of proteins

Congeners belonging to the genus Ciona have disparate distributions limited by temperature. Ciona intestinalis is more widespread with a cosmopolitan distribution ranging from tropical to sub-arctic zones, while Ciona savignyi is limited to temperate-latitudes of the northern Pacific Ocean. To compare the heat stress response between congeners, we quantified changes in protein expression using proteomics. Animals were exposed to 22 °C, 25 °C, and 28 °C for 6 h, then recovered at a control temperature (13 °C) for 16 h (high heat stress experiment). In a second experiment we exposed animals to lower levels of heat stress at 18 °C, 20 °C, and 23 °C, with a 16 °C control. A quantitative analysis, using 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry (with a 69% and 93% identification rate for Ciona intestinalis and Ciona savignyi, respectively), showed changes in a number of protein functional groups, including molecular chaperones, extracellular matrix proteins, calcium-binding proteins, cytoskeletal proteins and proteins involved in energy metabolism. Our results indicate that C. intestinalis maintains higher constitutive levels of molecular chaperones than C. savignyi, suggesting that it is prepared to respond faster to thermal stress. Systematic discrepancies between estimated versus predicted molecular masses of identified proteins differed between protein families and were more pronounced under high heat conditions, suggesting that thermal sensitivities are lower for cytoskeletal proteins and ATP-synthase than for any other protein group represented on 2D gels.