Pigmentation and tunic cells in Cystodytes dellechiajei (Urochordata, Ascidiacea)

The tunic of Cystodytes dellechiajei (Poly- citoridae), a colony-forming species of the Ascidiacea that contains biologically active alkaloids, was investigated using light microscopy, laser-scanning microscopy and nuclear magnetic resonance techniques. The colonies contain numerous individual zooids, which are embedded in a common tunic. Each zooid is protected by a firm capsule of overlapping calcareous spicules. The colonies lack blood vessels in the tunic, but six morphologically different types of tunic cells were found: pigment cells, bladder cells, vacuolated filopodial cells, granular filopodial cells, morula cells and granular cells. Rod-like bacteria were found in the tunic matrix. Bladder cells and pigment cells could be identified as storage units for acid and pyridoacridine alkaloids, making the tunic inedible and repelling predators. Filopodial cells have long filopodia, which probably are connected to each other. They may be involved in transportation processes within the tunic tissue. The functions of the morula cells and the granular cells are unknown as yet. With its several specialised cells, the tunic of C. dellechiajei represents a dynamic living tissue containing biologically active compounds. Accepted: 20 September 2000     

Cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea) 2: demonstration of proteins

In a previous paper, cellulose fibres were demonstrated in the larval, the metamorphosing, and the juvenile tunics. In this paper we used cytochemical methods and X-ray microanalysis to obtain additional information on tunic morphogenesis in Halocynthia papillosa. The chemical composition of the tunic evolves with its structural complexity. The larval and juvenile fibres are shown to be structurally and chemically different. While neither proteins nor glycosaminoglycans seem to be associated with the larval fibres, the juvenile fibres consist of a cellulose core wrapped in a sheath of tannophilic proteins. Patches of glycosaminoglycans line their longitudinal axes. In the course of metamorphosis, the cuticle undergoes profound modifications in regions of spine morphogenesis. Granular material that was previously called fibro-granular material (Lübbering et al., 1993) is essential to the formation of cuticular plates and spines. During metamorphosis, this material accumulates in epidermal granules and is discharged into the tunic. It crosses the fundamental layer of the tunic and reaches the cuticle. Our results strongly suggest that this material consists of proteins rich in cysteine and hydrophobic amino acids.     

Population genetics, phylogeography and speciation of Cystodytes (Ascidiacea) in the western Mediterranean Sea

Several morphotypes that so far have been attributed to the allegedly cosmopolitan ascidian Cystodytes dellechiajei occur in the Mediterranean Sea. Colour variation is the difference most frequently reported. In this study, we addressed the genetic structure of this ascidian in relation to geographical location and colour morph. Partial sequences of the gene cytochrome  c oxidase subunit 1 (COI) were obtained from seven populations of the western Mediterranean, encompassing eight colour varieties. All population genetic analyses (exact test, pairwise F _ST, hierarchical analysis of molecular variance, multidimensional scaling, nested clade analysis) indicated clearly that differences between colour morphs are large enough to obscure any geographical differentiation when colours are combined within localities. When variance due to colour divergence was removed, however, a significant geographical variability between localities remained. The genetic divergence between the colour morphs analysed was significant in comparisons of the brown and purple forms with the others, but not among the green, blue, and white morphs. Phylogeographic analyses suggest that population fragmentation and range expansions have shaped the present-day distribution of the haplotypes. Taken together with existing chemotype information, our results indicate that several species are present in the area, and that a thorough revision of the genus is necessary.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 203–214.     

Ultrastructural localization of calcium, by electron-probe X-ray microanalysis, in the small intestine of suckling rats

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.     

Ovulation and egg segregation in the tunic of a colonial ascidian,Diplosoma listerianum (Tunicata,Ascidiacea)

Summary The process of egg segregation in the tunic of the ovoviviparous ascidian Diplosoma listerianum was studied by light and electron microscopy. One egg at a time was seen to mature in each zooid. The eggs had large yolk and grew on the ovary wall enveloped in four layers: (1) outer follicle cells (OFC), long and rich in RER (rough endoplasmic reticulum) and with dense granules in the Golgi region; (2) flat inner follicle cells (IFC); (3) a loosely fibrillar vitelline coat (VC); (4) test cells encased on the egg surface. The growing egg protrudes from the ovary wall and presses on the contiguous epidermis. Granulocytes enter the space between the epidermis and the egg and insinuate cytoplasmic protrusions, disrupting the continuity of the OFC layer. At ovulation, OFC and IFC are discharged and form a post-ovulatory follicle (corpus luteum). The epidermis shrinks and closes, possibly by activation of microfilaments, causing the egg to be completely surrounded by the tunic. In the zooid, the wound caused by the passage of the egg is repaired both by contraction of the epidermis and by phagocytic activity. Altered spermatozoans are found in phagocytosing cells in the lumen of the ovary. These are presumably remnants of those which entered to fertilize the egg before segregation.     

Tunic cells in pyrosomes (Thaliacea, Urochordata): cell morphology, distribution, and motility

Abstract. Cellular components of the tunic were histologically examined in 3 pyrosome species representing all 3 genera of the order: Pyrosoma atlanticum, Pyrosomella verticillata , and Pyrostremma spinosum . Three cell types are distributed in the tunic. Tunic amebocytes, irregularly shaped and motile, often contain granules and/or phagosomes. Spherical tunic cells contain many round vesicles with eosinophilic and acidic materials. Tunic net cells form a cellular network in which their long filopodia connect with one another. The net cells are densely distributed just beneath the tunic surface lining the common cloacal cavity and may produce tension to maintain the colony shape. The presence of net cells suggests a phylogenetic relationship between pyrosomes and some aplousobranch ascidians. Test fibers are multicellular cords that run in the tunic and connect the zooids. In P. atlanticum , they are attached to they are attached to the epidermal cells of the zooids, and transverse cloacal muscles are attached to the other (proximal) side of the epidermal cells. The test fibers may mediate coordination of the zooids and control muscle contraction.     

Characterization and supramolecular architecture of the cellulose-protein fibrils in the tunic of the sea peach (Halocynthia papillosa,Ascidiacea, Urochordata)

Summary— The cellulose-protein fibrils, which constitute by far the bulk of the fibrous fraction of the sea peach tunic (Halocynthia papillosa), were structurally and chemically characterized, either in situ or after extraction procedures, with the use of classical electron microscoy combined with diffraction contrast imaging and electron diffraction, histochemistry, affinity cytochemistry and chemical analysis. These fibrils exhibit a cross-sectional shape close to a parallelogram. The cyrstallites forming their core, with lateral dimensions ranging from roughly 5 to 20 nm, are composed of native cellulose of higher crystallinity than that of plant cellulose. They are associated with acid mucopolysaccharidés (amps) and proteins which form a coating material appearing as a continuous sheath enveloping the axial crystallite in the cuticular layer or as patches more-or-less periodically distributed around and along the fibre axis in the fundamental layer. Tunicin, the alkali-insoluble fibrous fraction, is not pure cellulose, yielding only 22–60% of its dry weight as glucose equivalents, depending on the tunical layer. It is suggested that in addition to the high degree of crystallinity of the tunical cellulose, the presence of a significant amount of coating material composed of amino acids and proteoglycans firmly linked to cellulose molecules contributes to tunicin's high resistance to hydrolysis.     

The dorsal strand of Polyandrocarpa misakiensis (Protochordata: Ascidiacea): a light and electron microscopic study

The dorsal strand of a budding ascidian, Polyandrocarpa misakiensis , was studied by light and electron microscopy. The length and morphology of this organ vary even among zooids in the same colony. After curving between the paired posterior nerve trunks, the strand turns left and terminates in a dead end. The dorsal strand of this species is one of the simplest types among ascidians, since there is no branching, no extensive nerve plexus, and no neuronal or non-neuronal endocrine cells surrounding it. The strand mainly consists of a simple cuboidal epithelium, but has several variations, such as multilayered regions, and protrusions of a cellular chain. The strand cells usually have mitochondria, free ribosomes and a Golgi complex of dictyosomal type. There are many mitotic cells in the strand epithelium. The presence, between the cerebral ganglion and dorsal strand epithelium, of cells with morphology intermediate between the strand and neuronal cells suggests that the dorsal strand might supply cellular components, such as neurones, to the cerebral ganglion in adult zooids. At the caudal end, the strand cells show ultrastructural features suggesting active protein synthesis and secretion. These cells appear to be liberated from the epithelium to release the content of their granules.     

Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Ascidiacea)

A morpho‐functional study of the colonial ascidian Botrylloides leachi haemocytes was carried out to propose their classification, relationships and specializations. This characterization was obtained by (i) investigations of both living and aldehyde‐fixed cells by light and electron microscopy; (ii) cytochemical and cytoenzymatic assays; (iii) lectin‐affinity assays; (iv) phagocytosis and haemagglutination assays; and (v) anti‐CD34 immunocytochemical assay for vertebrate haematopoietic stem cells. Results indicate that the haemoblast is a circulating stem cell and there are at least five haemocyte differentiation pathways, the last two of which have never been described in botryllids: (i) phagocytic line (hyaline amoebocytes and macrophage‐like cells) share ultrastructural features, the same hydrolytic enzymes and WGA lectin binding, and are involved in yeast phagocytosis and erythrocyte rosette formation; (ii) cytotoxic line (granular amoebocytes and morula cells) with vacuoles containing oxidative enzymes and polyphenolic substrates; (iii) vacuolated cell line (pigment cells and nephrocytes) involved in catabolite storage; (iv) compartment cell line (compartment amoebocytes and compartment cells) able to agglutinate erythrocytes and characterized by vacuoles with a moderately electron‐dense content, positive to arylsulphatase activity and binding DBA, UEA‐I, HPA lectins; and (v) granular cell line includes trophic cells, able to infiltrate the gut epithelium, showing a cytoplasm filled of PAS‐positive vacuoles with arylsulphatase, chloroacetylesterase and β‐glucuronidase activities.     

The genus Herdmania Lahille, 1888 (Tunicata, Ascidiacea) in Australian waters

Revision of material from Australian waters previously assigned to Herdmania momus ( Savigny, 1816 ) has resulted in the recognition of five species (including two new indigenous species). A tropical Indo-West Pacific range is recorded for the type species ( H. momus ), although it is not recorded from Western Australia. A circum-continental range for the third and largest species, H. grandis ( Heller, 1878 ) is confirmed. Herdmania pallida (Heller, 1878), with a wide range in the western Pacific and Indian Ocean including the north-eastern and north-western coasts of Australia, is a different species from the Atlantic Ocean species (formerly thought to be conspecific). Herdmania fimbriae , a new species with a geographical range from southern to north-eastern Australia, is distinct from H. mentula , also a new species, from the north-western coast. The structure of the gonads and their ducts and accessory membranes and the arrangement and number of body wall muscles distinguish the species. Species differences are supported by the maximum size of individuals, nature of their test, number of branchial folds, number of internal longitudinal vessels on the folds and between the dorsal lamina and dorsal fold, the size and form of the dorsal tubercle and dorsal lamina, and the shape of the ciliated opening of the neural duct, although these characters are affected to some extent by growth. A variety of accessory membranes associated with both male and female gonoducal apertures are found in the majority of species in this genus. © 2002 The Linnean Society of London. Zoological Journal of the Linnean Society , 134 , 359–374.     

Studies on the distribution of Na and CI in two species of lupin (Lupinus luteus and Lupinus angustifolius) differing in salt tolerance

The distribution of K, Na and CI in various tissues was studied in two species of lupin, Lupinus luteus L. (ex. Portugal) and L. angustifolius L. (cv. Kubesa) under conditions of NaCl-induced salinity stress. L. luteus appeared more tolerant to salt and less effective in excluding Na and Cl from its above-ground parts than L. angustifolius . Electron probe X-ray microanalysis of vacuolar contents of individual root cells of salt-treated L. luteus showed a decreasing gradient of Na and CI contents from the epidermis inwards, but in the inner cortical cells adjacent to the pericycle/endodermis, Na levels were again high while Cl remained low. L. luteus may be inefficient in restricting entry of Na into the shoot because of a limited capacity for Na storage in the vacuoles of root cells. In addition, this species appears to exclude Cl from the vacuoles of inner cortical root cells but not from its symplastic pathway. In the leaves of L. luteus , Cl levels were particularly high towards the lower surface (abaxial side), while Na accumulated in the adaxial side of the leaf. Likewise, different cells of the petiole appeared to place unequal demands on Na and Cl for osmotic adjustment. It is suggested that excess Na in the adaxial side of the leaf and the generally high levels of Na and CI in the foliage may explain the 50% fresh weight stimulation which was observed in L. luteus but not in L. angustifolius at 50 m M NaCl. However, the halophytic response of L. luteus is limited to moderate concentrations of NaCl since 100 m M NaCl caused severe necrosis and leaf abscission.     

The distribution of zinc, cadmium, lead and copper within the hepatopancreas of a woodlouse

The distribution of metals within the hepatopancreas of Oniscus asellus (Crustacea, Isopoda) from two uncontaminated sites, and two sites contaminated with zinc, cadmium and lead, has been studied by atomic absorption spectrophotometry, light microscopy, transmission and scanning electron microscopy and X-ray microanalysis. The hepatopancreas contains two types of intracellular granule. The first type, in the S cells, are spherical granules which contain copper, sulphur and calcium. In woodlice from contaminated sites, these ‘copper’ granules, also contain zinc, cadmium and lead. The second type, in the B cells, are flocculent deposits which contain iron. In woodlice from contaminated sites, these ‘iron’ granules also contain zinc and lead. Moribund woodlice from contaminated sites have large numbers of ‘copper’ and ‘iron’ granules in the hepatopancreas and a fine deposit of zinc and lead on the membranes of the cells. There are numerous microorganisms in close association with the microvillous border of the hepatopancreas of woodlice from all four sites. Within the microorganisms of Oniscus asellus from contaminated sites, there are deposits of material which contain zinc, lead, calcium and phosphorus ‘Copper’ and ‘iron’ granules could have evolved as storage sites for essential metals to be utilized when demand from the body exceeds uptake from the food. Woodlice in contaminated sites may be able to ‘detoxify’ potentially harmful amounts of essential and non-essential metals by storing them in a relatively insoluble form within these granules.     

The distribution of lead in duckweed (Lemna minor L.) root tip

While considerable information on lead distribution in the cells of terrestrial plants has been collected, little is known about lead localization in the cells of the aquatic plant. Lemna minor L. (duckweed) roots were examined using X-ray microanalysis. After 1-h treatment with lead, its concentration was the highest in small vacuoles. After 6 and 12 h, the lead content of cell walls gradually increased. The changes of lead level between vacuoles and cell walls may result from redistribution of this metal from symplast to cell walls or it may reflect increased apoplastic transport. Lead was not found in the ground cytoplasm of any variants of the experiments. This fact and presence of lead in small vesicles suggests that endocytosis may play the role in lead uptake in Lemna.     

Morphology, performance, behavior and ecology of three color morphs in males of the lizard Podarcis melisellensis

Males of a Croatian population of the lacertid lizard Podarcismelisellensis exhibit a striking polymorphism, with colorationof the throat and abdomen ranging from completely white, toyellow or orange. In a first attempt to explore the potentialecological and evolutionary significance of this polymorphism,we compared the three forms of males in aspects of their morphology,whole-animal performance, behavior, and ecology. Orange malesare, on average, larger in snout-vent length and have disproportionatelylarger heads than either white or yellow males. This is reflectedin orange males having higher bite force capacity and theoreticallyan increased access to harder prey. Residual limb length, maximalsprint speed and maximal exertion do not differ among colormorphs. Body temperatures in the field are similar in the threemorphs, but yellow males are caught at sites with slightly higherair temperatures than are orange and white males. Behavioralobservations show no differences in time budgets or in the timingof activities among morphs. Microhabitat use is also similarin the three color morphs, but orange males were more ofteninitially seen on rocky substrates. Our findings suggest thatthe observed polymorphism likely does not originate from a divergencein niche or use of resources, but possibly reflects an underlyingpolymorphism in mating tactics.     

Coloration affects heating and cooling in three color morphs of the Australian bluetongue lizard,Tiliqua scincoides

The color-mediated thermoregulation hypothesis predicts that dark body color (low reflectance) allows organisms to gain heat more efficiently than does pale coloration (high reflectance). This prediction is intuitive and widely assumed to be true, but has poor empirical support. We used rare, captive-bred, mutant melanistic, albino and wild-type Australian bluetongue lizards, Tiliqua scincoides to measure the effects of skin reflectance on the heating and cooling rates. We measured heating under an artificial radiant heat source and cooling rates in an ice-cooled box using live lizards in a room with still air. The effect of skin reflectance on heat transfer was clear, despite the substantial influence of body size. Melanistic T. scincoides showed low reflectance and gained heat faster than highly reflective albinos. Melanistic lizards also lost heat faster than albinos. Wild-type lizards were intermediate in reflectance, gained heat at rates indistinguishable from melanistic lizards, and lost heat at rates indistinguishable from albino lizards. This study system allowed us to control for variables that were confounded in other studies and may explain the inconsistent support for the color-mediated thermoregulation hypothesis. Our results provide clear evidence that skin reflectance influences the rate of heating and cooling in ectotherms.     

Loss of test cells leads to the formation of new tunic surface cells and abnormal metamorphosis in larvae of Ciona intestinalis (Chordata, Ascidiacea)

The larvae of the ascidian Ciona intestinalis from which the chorion with the test cells and follicle cells were removed developed normally without the test cells until the early tailbud stage. A number of round-shaped cells morphologically similar to the test cells but with different lectin affinities and autofluorescence, then appeared on the neck region of the demembranated embryos. The new cells had three different types: round, particulate, and granular, and these cells increased in number after the late tailbud stage. The morphology of the adhesive papillae, tunic layers and epidermis of the demembranated larvae was similar to that of control larvae; however, the affinity to lectins was different in the swimming period. Control larvae attached to the substratum after the swimming period, resorbed the tail completely and underwent rotation of the visceral organs. Conversely, rotation occurred before completion of tail resorption in the demembranated larvae. Furthermore, the metamorphic events progressed more slowly in the demembranated larvae. These results suggest that the test cells play important roles in normal development and morphogenesis of ascidian larvae. Received: 4 December 1998 / Accepted: 9 April 1999     

Ultrastructural and microspectrophotometric characterization of multiple species of cyanobacterial photosymbionts coexisting in the colonial ascidian Trididemnum clinides (Tunicata,Ascidiacea, Didemnidae)

Trididemnum clinides is a multi-photosymbiotic ascidian that inhabits shallow coral reef lagoons. Three types of cyanobacteria are harboured in the tunic of the ascidian colony; of these, two are unicellular coccoid cyanobacteria and the other is a multicellular filamentous type. They also differ in ultrastructure and distribution patterns within the host tunic. Microspectrophotometric analysis revealed the composition of photosynthetic pigments in each photosymbiont. One of the coccoid types is yellowish-green and is distributed under the colony surface. This photosymbiont cell preferentially absorbs red and blue light, and therefore the dominant colour in the inner tunic is green. The other two types of coexisting photosymbionts contain the green-light-absorbing R-phycoerythrin as the major photosynthetic pigment; they exploit the wavelengths of light not used by the first type of photosymbiont. In T. clinides, the outer and inner photosymbionts in the tunic have different photosynthetic pigments, which adapt to each microhabitat, thereby sharing the incident light resources effectively.