Laboratory-measured grazing and ingestion rates of the salp, Thalia democratica Forskal, and the doliolid, Dolioletta gegenbauri Uljanin (Tunicata, Thaliacea)

Grazing and ingestion rates of laboratory-born Thalia democraticaaggregates and Dolioletta gegenbauri gonozooids, phorozooidsand oozooids were determined while fed Isochrysis galbana (4–5µm diameter) alone or in combination with Peridinium trochoideum(16–18 µm diameter) at concentrations of 0.15–0.70mm³ x 1^–1. Grazing rates (ml x zooid^–1 x 24 h ^–1)ranged from 10 to 355, and at zooid weights greater than 5 µgcarbon were in order oozooid > gonozooid > aggregate.Grazing rates increased exponentially with increasing zooidweight. Weight-specific grazing rates (ml x µgC^–1x 24 h^–1) were independent of the four-fold initial foodconcentration. Mean weight-specific grazing rates increasedlinearly with increasing zooid weight for the aggregates andoozooids, but gonozooid mean rates were independent of zooidweight. Aggregate and gonozooid ingestion rates (10⁶ µm³x zooid^–1 x 24 h^–1) ranged from 4 to 134 while oozooidrates ranged from 3 to 67. All ingestion rates were independentof the initial food concentration but increased linearly withincreasing zooid weight at similar rates. All mean weight-specificingestion rates (ml x µgC^–1 x 24 h^–1) wereindependent of zooid weight. The mean aggregate daily ration(µgC ingested x µg body C^–1) was 59% and themean doliolid ration was 132%. Field studies indicate that normalconcentrations of D. gegenbauri in the Georgia Bight clear theirresident water volume (1 m³) in about 4 months, but that highlyconcentrated, swarm populations which occur along thermohalinefronts clear their resident water volume in less than 1 day. ¹Current address: MacLaren Plansearch Ltd., P.O.Box 13250, sta.A.,St.John's, Nfld. A1B 4A5     

Electron-microscopic studies of iodine-binding and peroxidase activity in the endostyle of the larval amphioxus (Branchiostoma lanceolatum)

Summary The asymmetric endostyle in the larval amphioxus (Branchiostoma lanceolatum) was examined by light-and electron-microscopic cytochemistry (peroxidase; incubation in diaminobenzidine) and autoradiography (incubation in ¹²⁵I^-). Compared to the adult the same cellular zones were also found in the larval endostyle, with the exception of zone 1, which was absent. The corresponding adult and larval zones had a similar morphology. All cells in zones 5a, 5b, and 6 were reactive for peroxidase. A reaction product was also present in the lateral 2 to 3 cell rows of zone 3. The dense reaction product was located on the inner surface of membranes of the rough endoplasmatic reticulum, Golgi apparatus and vesicles, and multivesicular bodies as well as on the outer surface of the luminal plasma membrane. An incomplete row of granule-containing, peroxidase-negative cells was located between zones 5b and 6. After incubation of larvae in sea water containing ¹²⁵I^-, autoradiographic grains were selectively concentrated over the lumen at the apical surface of all peroxidase-positive zones. The highest grain density occurred in relation to zone 5a, which in the adult has been recognized as the iodination center. Few grains were located over the cytoplasm. Methimazole, an inhibitor of peroxidase, abolished the cytochemical reaction and the appearance of autoradiographic grains. The observations indicate that iodination in the larval endostyle takes place extracellularly and is catalyzed by peroxidase bound in the plasma membrane.     

Ultrastructural localization of the iodination centre in the endostyle of the adult amphioxus (Branchiostoma lanceolatum)

Summary The site of iodination in the endostyle of the adult amphioxus was examined by light-and electron-microscopic autoradiography. In accordance with previous studies, light-microscopic autoradiography showed a distinct accumulation of autoradiographic grains at the apical end of epithelial cells in the lateral part of the endostyle. In the electron microscope two distinct cellular zones were identified in an approximate position of the light-microscopic zone 5. Zone 5a, not previously recognized, was adjacent to zone 4 and consisted of six to nine rows of cells free of characteristic granules. Cells in zone 5b contained large mucous granules and had, in previous ultrastructural studies, been identified as belonging to the typical zone 5. Four or less incomplete rows of granule-containing cells, not observed in previous studies, marked the border between zones 5b and 6. After incubation in ¹²⁵I for 5 min, electron-microscopic autoradiography showed a selective concentration of label to zone 5a, which, thus, corresponds to the iodination centre seen in the light microscope. The grains were associated with cilia and microvilli in the lumen. After longer incubation times (30, 60, 90 min) grains were still concentrated at the surface of zone 5a but were also associated with the surface of zones 5b and 6. Grains were also located over the cytoplasm of all three zones. They were associated with vesicles and lysosome-like structures, suggesting secondary uptake of labelled products by endocytosis. Methimazole, an inhibitor of peroxidase, abolished the autoradiographic reaction. In conclusion, the site of iodination in the endostyle of amphioxus is located in zone 5a, which has not previously been ultrastructurally defined. Iodination in the endostyle is an extracellular process, but secondary uptake by endocytosis appears to occur.     

Effect of temperature on the filtration rate and percentage of assimilation ofSalpa fusiformis Cuvier (Tunicata: Thaliacea)

The effect of temperature on filtration and ingestion rates ofSalpa fusiformis Cuvier was determined over the temperature range 13–22°C at a constant algal concentration. Filtration and ingestion rates increased with temperature up to an optimum (19.7 to 20.2°C) beyond which they decreased. Food assimilation for this salp species was determined while fed onPhaeodactylum tricornutum Bohlin orHymenomonas elongata Stein at different algal concentrations. The percent assimilation varied between 28 and 39% (mean: 32%) with the diatomP. tricornutum as food, and between 39 to 81% (mean: 64%) with the flagellateH. elongata. Assimilation did not seem to depend on the algal concentration but it was greater with the flagellate than with the diatom, the latter having a higher ash content.     

The haemocytes of the salp Thalia democratica (Tunicata,Thaliacea): an ultrastructural and histochemical study in the oozoid

In comparative immunology and evolution of the chordate immune system, tunicates hold an important phylogenetic position as sister group of vertebrates. However, knowledge of the tunicate immune system is limited to the class Ascidiacea, in which some species are now considered model organisms. In the class Thaliacea, represented by fragile pelagic species, the few studies on their haemocytes go back to several decades ago and do not consider comparative aspects with ascidian haemocytes. In this study, we identified various haemocyte types and their distribution in the common salp Thalia democratica by comparative observations under light and electron microscopy and by histochemical, histoenzymatic and immunohistochemical techniques. By comparing specialisations with those of ascidian haemocytes, we detected an undifferentiated cell type (lymphocyte‐like cell) and three categories with four cell types, that is, (i) phagocytic line (hyaline amoebocyte and amoebocyte with large vacuoles), (ii) mast cell‐like line (granular cell) and (iii) storage cells (nephrocyte). Both phagocytes and granular cells appear to migrate in the tunic. Phagocytes adhere to the tunic which internally covers the oral siphon, where they probably function as sentinel cells of the pharynx. Results show the variety of haemolymph cells in the salp similar to phlebobranch ascidians.     

Structure and ultrastructure of eyes and brains of Thalia democratica (Thaliacea,Tunicata, Chordata)

Salps are marine planktonic chordates that possess an obligatory alternation of reproductive modes in subsequent generations. Within tunicates, salps represent a derived life cycle and are of interest in considerations of the evolutionary origin of complex anatomical structures and life history strategies. In the present study, the eyes and brains of both the sexual, aggregate blastozooid and the asexual, solitary oozooid stage of Thalia democratica (Forskål, 1775 ) were digitally reconstructed in detail based on serial sectioning for light and transmission electron microscopy. The blastozooid stage of T. democratica possesses three pigment cup eyes, situated in the anterior ventral part of the brain. The eyes are arranged in a way that the optical axes of each eye point toward different directions. Each eye is an inverse eye that consists of two different cell types: pigment cells (pigc) and rhabdomeric photoreceptor cells (prcs). The oozooid stage of T. democratica is equipped with a single horseshoe‐shaped eye, positioned in the anterior dorsal part of the brain. The opening of the horseshoe‐shaped eye points anteriorly. Similar to the eyes of the blastozooid, the eye of the oozooid consists of pigment cells and rhabdomeric photoreceptor cells. The rhabdomeric photoreceptor cells possess apical microvilli that form a densely packed presumably photosensitive receptor part adjacent to the concave side of the pigc. We suggest correspondences of the individual eyes in the blastozooid stage to respective parts of the single horseshoe‐shaped eye in the oozooid stage and hypothesize that the differences in visual structures and brain anatomies evolved as a result of the aggregate life style of the blastozooid as opposed to the solitary life style of the oozooid.     

Junctional complexes of the branchia and gut of the tunicate,Pyrosoma atlanticum (Pyrosomatida,Thaliacea)

Summary The branchia of pyrosomes has been found to be like that of ascidians in that it shares with the latter, besides the presence of peribranchial chambers, stigmata bordered by clusters of seven rows of flattened cells, each bearing a single row of long cilia. The intercellular junctions between ciliated cells of the branchial basket and between the cells lining the gut, in pyrosomes, have been studied in thin sections and by freeze-fracture. In both tissues tight and gap junctions are present, but the former are much more extensive in the gut. The gap junctions in the branchial basket exhibit some atypical features. Moreover, although there are extensive zonulae adhaerentes between the ciliated cells of the branchial basket, there are none between the epithelial cells of the intestinal tract. This feature of the branchiae, together with the alignment of their cells into highly ordered rows of seven cells, are similar to those found in some groups of ascidians. The evolutionary relationships between pyrosomes and the aplousobranch ascidians are considered in the light of these results.     

Distribution of peroxidase and iodination activity in the endostyles of Oikopleura albicans and Oikopleura longicauda (Appendicularia,Chordata)

Summary Oikopleura albicans and O. longicauda belong to the two subgenera Vexillaria and Coecaria, respectively. The morphology and ultrastructure of their endostyles were investigated with conventional microscopic procedures as well as with DAB cytochemistry and ¹²⁵I autoradiography at both light- and electron-microscopic levels. As expected, the general morphology of these endostyles is similar to all hitherto examined endostyles. They possess a ventral portion consisting of alternating glandular and ciliated cell zones, probably serving food capture, and a dorsal region, the corridor. Autoradiographic grains were found mainly in the corridor lumen associated with the apical surface of the two central rows of corridor cells. The same cells also gave strong positive reactions for peroxidase, the iodinating enzyme. Peroxidase activity was found in the apical plasma membrane as well as in the nuclear envelope, rough endoplasmic reticulum, Golgi area and cytoplasmic vesicles. Definitive conclusions concerning an apical uptake and subsequent release into the body fluid of iodinated material could not be made from the present experiments. Our investigations indicate that the two central rows of corridor cells in both subgenera of oikopleurids constitute the protothyroid region, possibly homologous to the vertebrate thyroid gland.     

Junctional diversity in two regions of the epidermis of Oikopleura dioica (Tunicata,Larvacea)

Summary A simple continuous epithelium surrounds the body of the pelagic larvacean. It consists of two zones of cells: oikoplast cells and flattened cells. The oikoplast cells are columnar and produce a thick extracellular house that ensheathes the body of the organism. These cells are joined laterally by wide tight junctions (zonulae occludentes). The tail of the animal is surrounded by exceedingly thin cells which are joined by narrow tight junctions under which lie intermediate junctions (zonulae adhaerentes) and gap junctions. A web of fibrous material inserts into the intermediate junctions. The transitional cells between the two epithelial zones have one lateral border with a wide tight junction, and the other lateral border with a narrow tight junction and a wide intermediate junction. In freeze-fracture replicas, the wide tight junction has a number of anastomosing ridges, in comparison with the narrow tight junction, which usually consists of only a single row of intramembranous particles. In replicas, the thin epithelial cells show unusual parallel arrays of particles in clusters on their apical plasma membranes. This simple epithelium, therefore, exhibits striking differences between the two cellular zones, in the structural characteristics of both the lateral borders and the apical membrane.     

Iodination activity in Eisenia foetida (Annelida,Oligochaeta)

Summary In the nervous system of the earthworm Eisenia foetida, we have previously found thyroglobulin-like immunoreactive neurons. In the present study iodination activity was investigated by injecting worms or incubating them in vivo with radioiodine; animals treated with methimazole (MMI), an inhibitor of peroxidase-catalyzed iodination, served as controls. Radioiodinated proteins were identified in soluble extracts from ¹²⁵I-incubated animals; the sedimentation pattern of soluble proteins from cephalic segments showed a peak of radioactivity in the 3–4 S region. In animals pretreated with 10^-3 M MMI for 48 h, ¹²⁵I-incorporation into soluble proteins from cephalic segments was drastically reduced. Light-microscopic autoradiographic studies showed silver-grains selectively concentrated in the brain and ventral nerve cord of ¹²⁵I-injected animals. The highest grain-density occurred in the cerebral ganglion beginning 5 min after tracer injection; the reaction product was mainly distributed between the neurosecretory cells and neuropile fibres in the zone of the presumptive plexiform neurohaemal complex. In MMI-pretreated animals the reaction product was not visible in either cerebral ganglion or ventral nerve cord. Of interest, the setae appeared consistently positive with or without MMI treatment. These observations indicate that protein iodination involving peroxidase occurs in the nervous system of earthworms and suggest that iodination mechanisms, other than peroxidase-catalyzed, may be operating in some scleroprotein structures such as setae.     

A morphological and immunohistochemical study of programmed cell death in Botryllus schlosseri (Tunicata,Ascidiacea)

The blastogenic cycle of the colonial ascidian Botryllus schlosseri concludes in a phase of selective cell and zooid death called takeover. Every week, all asexually derived parental zooids synchronously regress over a 30-h period and are replaced by a new generation. Here we document the sequential ultrastructural changes which accompany cell death during zooid degeneration. The principal mode of visceral cell death during takeover occurred by apoptosis, the majority of cells condensing and fragmenting into multiple membrane-bounded apoptotic bodies. Cytoplasmic organelles (mitochondria, basal bodies, striated rootlets) within apoptotic bodies retained ultrastructural integrity. Dying cells and fragments were then swiftly ingested by specialized blood macrophages or intraepithelial phagocytes and subsequently underwent secondary necrotic lysis. Certain organs (stomach, intestine) displayed a combination of necrotic and apoptotic changes. Lastly, the stomach, which demonstrated some of the earliest regressive changes, exhibited intense cytoplasmic immunostaining with a monoclonal antibody to ubiquitin at the onset of takeover. Affinity-purified rabbit antiserum against sodium dodecyl sulfate-denatured ubiquitin detected a characteristic 8.6-kDa mono-ubiquitin band by Western blot analysis. Collectively, these findings raise the possibility that cell death during takeover is a dynamic process which requires active participation of cells in their own destruction.     

Morphological aspects of fertilization in Phallusia (Ascidia) nigra (Ascidiacea,Tunicata)

The spermatozoa of Phallusia (Ascidia) nigra have an elongated head (approximately 5 m in length) in which a nucleus and a single mitochondrion are located side by side. There is no midpiece. The apex of the head is wedge-shaped. Acrosomal vesicles (approximately 55–65 nm in diameter) and moderately electron-dense material (MEDM) are present between the plasmalemma and the nuclear membranes in the anterior tip of the head. The MEDM occupies a central position and three or four acrosomal vesicles are seen in a line alongside it. The acrosomal vesicles disappear as the sperm makes contact with the surface of the chorion. Gamete fusion most likely occurs between a small process extending from the peripheral margin of the sperm apex and the egg surface, resulting in incorporation of the sperm into the egg from the anterior region of its head.     

Effect of monosodium glutamate on the neural complex of Ciona intestinalis (Tunicata)

Summary Short-term treatment of the ascidian (tunicate) Ciona intestinalis with monosodium glutamate produces a transient decrease in methionine-enkephalin-like immunoreactivity of neurones in the nervous ganglion. Moreover, it causes vacuolisation of the cells in the neural complex, particularly in the neural gland. Similar damages occur after ovariectomy. These results suggest that the ovary exerts an indirect influence on the neural gland via the nervous ganglion, and that the methionine-enkephalin-like substance could be the responsible neuromediator.A portion of these results has been presented as a poster at the 10th International Symposium on Comparative Endocrinology, Copper Mountain, Colorado, USA (July 1985).     

Neuronal organization of the ascidian (Urochordata) branchial basket revealed by cholinesterase activity

Summary Innervation of the ascidian branchial basket and other structures is demonstrated by staining for cholinesterase. Cholinesterase activity is not restricted to synaptic sites but is present throughout the neurons. Primary and secondary axonal bundles form a bilaterally symmetric innervation pattern around the large dorsal visceral nerve. These bundles continue to split into progressively smaller bundles as they course throughout the basket. Axons are suspended in a fibrous matrix and run within the blood sinuses on the atrial side of the basket. Stigmatal ciliated cells of the branchial basket are innervated by highly branched distal portions of neurons, whose cell bodies are located in the ganglion. Synaptic boutons, containing electron-lucent vesicles, are found at nearly all stigmatal ciliated cells. NiCl₂backfills of the visceral nerve reveal a distinct population of central neurons, some of which presumably control ciliary arrest.     

Quantitative X-ray microanalysis of the morula cell of the blood of the ascidian Halocynthia papillosa (Stolidobranchiata)

Summary The elemental composition of the morula cell of Halocynthia papillosa blood was studied by X-ray microanalysis with respect to the possible iron accumulation in this cell type. We found various amounts of Na, Mg, P, S, Cl, K, Ca, Fe and Br in the cytoplasm, nucleus and vacuoles. With the exception of a few cells, Ca, Fe and Br were not detected. Thus, the morula cells of the studied species are not iron-rich cells.     

Angiotensin II binding to tissues of the rainbow trout,Oncorhynchus mykiss,studied by autoradiography

Summary Tissue slices from seawater-adapted and freshwater-adapted rainbow trout, Oncorhynchus mykiss, were exposed to ¹²⁵I-angiotensin II (1.01·10^-9 M) and binding sites located by light-microscopic autoradiography. Binding/uptake was significantly inhibited by excess (10^-5 M) unlabelled angiotensin II, suggesting specific binding/uptake of angiotensin II to the ventral and dorsal aorta (smooth muscle), urinary bladder (smooth muscle and epithelial lining), glomeruli and proximal tubules, the gill (lamellae and central filament), skin (epithelium), intestine and oesophagus (mucosal epithelium), liver, heart (ventricular myocytes), adrenocortical tissue and brain (cerebellum and medulla oblongata). The specific binding/uptake of angiotensin II to tissues of freshwater- and seawater-adapted animals were generally similar. However, binding/uptake by the proximal tubules was significantly higher in freshwater-adapted trout than seawater-adapted trout. Specific binding/uptake of angiotensin II by the smooth muscle of the bladder was significantly higher in trout adapted to seawater than trout adapted to freshwater.     

Improvements in the determination of manganese peroxidase activity

The existing method of determining the activity of manganese peroxidase (MnP), produced by Phanerochaete chrysosporium, was improved. 2,6-Dimethoxyphenol at 80 mM was used as a substrate and, after the decolorization of the reaction mixture, H₂O₂ was added and the initial reaction rate was used to determine MnP activity.     

The binding of lectins to carbohydrate moieties on haemocytes of the insects,Blaberus craniifer (Dictyoptera) and Extatosoma tiaratum (Phasmida)

Summary The specificity and distribution of carbohydrate moieties, which act as receptors for lectins on the haemocytes of two insect species, Blaberus craniifer and Extatosoma tiaratum, were investigated. Four peroxidase-labelled lectins were utilised as probes: wheat-germ agglutinin, Ricinus communis (120) agglutinin, concanavalin A and Lens culinaris agglutinin, and binding visualised by reaction with DAB/H₂O₂. The lectin-binding studies indicated that Blaberus and Extatosoma plasmatocytes, and Extatosoma spreading granular cells possess surface N-acetyl-D-glucosamine, D-galactose and mannose moieties; Extatosoma cystocytes have D-galactose and mannose; whilst Blaberus granular cells/cystocytes and Extatosoma fine granular cells have mannose determinants.