The connection between spinal cord and notochord in Amphioxus (Branchiostoma lanceolatum)

Summary Silver impregnation of nerves, histochemical reactions for acetylcholinesterase, and electron microscopy reveal an efferent innervation of the notochord in amphioxus. Extensions of the notochordal lamellae end in groups (the notochordal horns) just below the ventro-lateral surface of the spinal cord where they are opposed to large nerve terminals originating as short collaterals of axons running longitudinally in the nerve cord. This neurochordal junction resembles an ordinary neuro-muscular junction in several respects and is interpreted as a part of the muscular system found in the notochord itself. Acknowledgement. The author is indebted to the staffs at the Marine laboratory in Plymouth and the Biological station at Helgoland for supply of material. The expert advices and criticism of Q. Bone, Ph. D., Plymouth, and Dr. U. Welsch, Kiel, are also greatly appreciated.     

The infundibular organ of the lancelet (Branchiostoma lanceolatum,Acrania): an immunocytochemical study

Reissner's fibers are secretions produced by different ependymal areas of the chordate brain, viz., in adult vertebrates, by the dorsal subcommissural organ, and in all stages of cephalochordates (Branchiostoma lancelets), by the ventral infundibular organ. Fibers produced by these different organs are seemingly identical and the two fiber sources also share some immunocytochemical and lectin-binding properties. The secretions in these two glands are, however, not identical; the infundibular organ cells are strongly reactive with antibodies against vertebrate Reissner's fibers, but they do not react with antibodies raised against the source of the vertebrate fibers, viz., the subcommissural organ. The results support the possibility that, in adult vertebrates, the Reissner's fibers are composed of material not only from the subcommissural organ, but also from another, not yet identified, source that is identical or equivalent to the infundibular organ of the lancelet. There are indications that the infundibular organ is immunocytochemically closely akin to some secretory cells in the vertebrate embryonic brain and also to those that produce the juvenile vertebrate Reissner's fibers, viz., secretory cells in the flexural organ.     

The fine structure of lamellate cells in the brain of amphioxus (Branchiostoma lanceolatum,Cephalochordata)

Summary The lamellate cells of amphioxus have round nuclei, and cytoplasm with many mitochondria and a large amount of glycogen. Each of these cells projects a highly modified, branched cilium into the central canal, where it characteristically forms lamellar structures. Primary branches and secondary lamellae often contain accessory microtubules that are not derived from the axonema. The functional and evolutionary significance of this cell type is discussed in relation to the ciliary photoreceptors found in other chordates.This work is dedicated to Professor A. Carrato, Universidad Complutense, on the occasion of his 80th birthday     

Immunohistochemical localization of polypeptide hormones in endocrine cells of the digestive tract of Branchiostoma lanceolatum

Summary The digestive tract of the cephalochordate Branchiostoma lanceolatum was investigated with regard to occurrence and distribution of endocrine cells. By the use of the peroxidase-antiperoxidase (PAP) technique, cells in the gut epithelium reacting with antisera against 8 different mammalian polypeptide hormones were localized. Positive reactions were obtained with antisera against the four mammalian islet hormones (insulin, glucagon, pancreatic polypeptide, somatostatin) and against secretin, vasoactive intestinal polypeptide, pentagastrin and neurotensin. No immunoreactivity was found with antisera against members of the lipotropin family (ACTH, met-enkephalin, -endorphin), against big-gastrin, cholecystokinin, substance P and moulin. The exact mapping of the different polypeptide immunoreactive cells throughout the digestive tract of Branchiostoma lanceolatum is presented.     

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.     

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.     

Vergleichende histophysiologische Tracer-Untersuchungen an verschiedenen Organsystemen von Branchiostoma lanceolatum (Cephalochordata) und Brachydanio rerio (Teleostei)

Zusammenfassung In vergleichend autoradiographischen Untersuchungen wurde der Einbau von percutan applizierten 3H-Uridin, 3H-Histidin und 3H-Glucose in die wichtigsten Organsysteme (Epidermis, ZNS, Muskeln, Chorda, Leber, Kiemendarm, Darm) von Branchiostoma lanceolatum (Acranier) und Brachydanio rerio (Teleosteer) nach Inkorporationszeiten von 11 min bis zu 7 Tagen verfolgt. Der Stoffwechsel der markierten Substanzen in den einzelnen morphologisch miteinander zu homologisierenden Organen war bei den beiden Spezies sehr ähnlich, bei Branchiostoma allerdings (mit Ausnahme des ZNS) 4–5mal stärker als bei Brachydanio. Bei dem letzteren wurde außerdem eine zeitliche Verzögerung in der Tracer-Aufnahme (lag-Phase) beobachtet. Insbesondere der ZNS-Stoffwechsel von Acraniern zeigte ähnliche Charakteristika wie der von Vertebraten: Verbleib des Hauptanteils der neusynthetisierten RNS im Perikaryon, axonalen Protein-Transport, Vorwiegen der Glykogensynthese in den Nervenfaserendformationen. Allerdings fanden sich im ZNS von Branchiostoma niedrigere Stoffwechselraten als im ZNS von Brachydanio.

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Comparative histophysiological investigations of different organs in Branchiostoma lanceolatum (Cephalochordata) and Brachydanio rerio (Teleostei)

Summary Incorporation of 3H-uridine, 3H-histidine and 3H-glucose into some organs (epidermis, CNS, muscles, spinal cord, notochord, liver, gills, intestine) of Branchiostoma lanceolatum (Acrania) and Brachydanio rerio (Teleostei) was investigated by means of comparative autoradiograms following incorporation periods of 11 min to 7 days. The metabolism of the labeled substances in the various homologous organs examined was quite similar, although it was 4 to 5 times higher in Branchiostoma than in Brachydanio; in the latter there was also a delay of tracer incorporation of about 3 hrs, a so-called lag-phase. In particular the metabolism of the CNS of Branchiostoma showed the same characteristics as the CNS of vertebrates, e. g. storage of neuronally synthesized RNA in the neuronal perikarya, axonal flow of proteins, glycogen synthesis in nerve endings. However, metabolic activity of the CNS was lower in Branchiostoma.

Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Glycoconjugate profiles of the lancelet (Branchiostoma lanceolatum) ovary: a lectin histochemical study by laser confocal microscopy

The presence and the distribution of carbohydrate moieties in ripe lancelet (Branchiostoma lanceolatum) oocytes (mean diameter 130 microm) was studied by lectin histochemistry in combination with enzyme and chemical treatments. Binding sites for eight lectins with specificities towards different glycan moieties were studied on sections of the whole body of mature female lancelets. Only three of the lectins tested reacted positively. Concanavalin-A (ConA)-binding glycoconjugates were localized in the cytoplasm, namely in yolk granules, whereas Artocarpus integrifolia (AIA) and Ricinus communis (RCA) agglutinins bound strongly to extracellular coats of the oocyte identified as the jelly coat and vitelline layer. No other tissues of the lancelet body were found to be positive to any lectin tested, except gut enterocytes which reacted strongly with AIA. Reactivity to ConA was abolished by pretreatment of sections with N-glycosidase F but not by mild alkaline hydrolysis, confirming that the glycoconjugates were of the N-linked type. On the contrary, chemical removal of O-linked chains by mild alkaline hydrolysis abolished AIA and RCA reactivity but had no effect on ConA positivity.     

A histochemical study of the distribution of lectin binding sites in the developing oocytes of the lancelet Branchiostoma belcheri

The distribution of carbohydrate moieties in lancelet (Branchiostoma belcheri) oocytes has been studied at different stages of development, using a peroxidase-labeled lectin incubation technique, the PAS-reaction and Alcian Blue staining. Binding sites of 5 lectins, indicating the presence of different sugar moieties (Wheat germ agglutinin (WGA) for N-acetylglucosamine, Concanavalin A (Con A) for glucose/mannose, Helix pomatia agglutinin (HPA) for N-acetyl-D-galactosamine, Ricinus communis agglutinin (RCA-I) for galactose and Ulex europaeus agglutinin (UEA-I) for fucose), were identified and were shown to undergo considerable variation during oocyte development. In the previtellogenic stage, HPA, RCA-I and UEA-I were not identified on the oocyte surface, but WGA and Con A gave strongly positive reactions at this site. In the cytoplasm, 4 lectins (Con A, HPA, RCA-I and UEA-I) gave a weak or moderate reaction, and Con A was also observed in the perinuclear region. In vitellogenic oocytes, these 4 lectins were found to also bind to the nuclear envelope, karyoplasm and nucleolus, and, with the exception of Con A, could also be found in the nuclei of more mature stages. The cytoplasmic yolk granules and Golgi vesicles of the vitellogenic oocyte, were moderately positive for Con A, HPA, RCA-I and UEA-I, but HPA, RCA-I and UEA-I were only weakly bound at the oocyte surface. In mature oocytes, all 5 lectins bound moderately or strongly to yolk granules and cell surface. HPA, RCA-I and UEA-I bound moderately or strongly to various nuclear compartments. Thus, carbohydrate content varied with the development and maturation of the oocytes, and the PAS results were in agreement with the lectin-binding results. Charged carbohydrate residues were observed in the egg envelope and Golgi bodies.These results suggest that the appearence of Con A-, HPA-, RCA-I- and UEA-I-binding glycoconjugates in the nuclei of developing oocytes show a varying pattern indicating different phases of nuclear activity which correlate with different carbohydrate synthetic activities of the oocyte.     

BfCBR: a cannabinoid receptor ortholog in the cephalochordate Branchiostoma floridae (Amphioxus)

A gene encoding an ortholog of vertebrate CB(1)/CB(2) cannabinoid receptors was recently identified in the urochordate Ciona intestinalis (CiCBR; [Elphick, M.R., Satou, Y., Satoh, N., 2003. The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis. Gene 302, 95-101.]). Here a cannabinoid receptor ortholog (BfCBR) has been identified in the cephalochordate Branchiostoma floridae. BfCBR is encoded by a single exon and is 410 amino acid residue protein that shares 28% sequence identity with CiCBR and 23% sequence identity with human CB(1) and human CB(2). The discovery of BfCBR and CiCBR and the absence of cannabinoid receptor orthologs in non-chordate invertebrates indicate that CB(1)/CB(2)-like cannabinoid receptors originated in an invertebrate chordate ancestor of urochordates, cephalochordates and vertebrates. Furthermore, analysis of the relationship of BfCBR and CiCBR with vertebrate CB(1) and CB(2) receptors indicates that the gene/genome duplication that gave rise to CB(1) and CB(2) receptors occurred in the vertebrate lineage. Identification of BfCBR, in addition to CiCBR, paves the way for comparative analysis of the expression and functions of these proteins in Branchiostoma and Ciona, respectively, providing an insight into the ancestral functions of cannabinoid receptors in invertebrate chordates prior to the emergence of CB(1) and CB(2) receptors in vertebrates.     

Carbohydrates in the epidermal mucous cells of a fresh-water fish Mastacembelus pancalus (mastacembelidae,Pisces) as studied by electron-microscopic cytochemical methods

Carbohydrates in the mucous cells of the epidermis of the fish Mastacembelus pancalus were studied by means of electron-microscopic cytochemical methods using physical development procedures. Three types of mucous cells (types A-C) were differentiated on the basis of the reactivities of the secretory products elaborated by them. The carbohydrate contents of mucous globules predominantly comprised sulfate esters and traces of oxidizable vicinal diols in type-A cells, oxidizable vicinal diols in type-B cells, and moderate amounts of both sulfate esters and oxidizable vicinal diols in type-C cells. Glycogen particles were also found to occur in the cytoplasm of these cells, and glycoproteins containing oxidizable vicinal diols were visualized in Golgi cisternae, rough endoplasmic reticulum, nuclear envelopes, and plasma membranes. In the type-A and type-B cells situated in the superficial layers of the epidermis, extensive cisternae of the Golgi apparatus and copious rough endoplasmic reticulum suggested the active syntheses of secretory contents, in contrast to the type-C mucous cells, which displayed poor development of these organelles, in the deeper layers.     

Systematic investigation of Amphioxus (Branchiostoma floridae) microRNAs

Ascaris lumbricoides and Ascaris suum are parasitic nematodes living in the small intestine of humans and pigs, and can cause the disease ascariasis. For long, there has been controversy as to whether the two ascaridoid taxa represent the same species due to their significant resemblances in morphology. However, the complete mitochondrial (mt) genome data have been lacking for A. lumbricoides in spite of human and animal health significance and socio-economic impact globally of these parasites. In the present study, we sequenced the complete mt genomes of A. lumbricoides and A. suum (China isolate), which was 14,303 bp and 14,311 bp in size, respectively. The identity of the mt genomes was 98.1% between A. lumbricoides and A. suum (China isolate), and 98.5% between A. suum (China isolate) and A. suum (USA isolate). Both genomes are circular, and consist of 36 genes, including 12 genes for proteins, 2 genes for rRNA and 22 genes for tRNA, which are consistent with that of all other species of ascaridoid studied to date. All genes are transcribed in the same direction and have a nucleotide composition high in A and T (71.7% for A. lumbricoides and 71.8% for A. suum). The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. Phylogenetic analyses of A. lumbricoides and A. suum using concatenated amino acid sequences of 12 protein-coding genes, with three different computational algorithms (Bayesian analysis, maximum likelihood and maximum parsimony) all clustered in a clade with high statistical support, indicating that A. lumbricoides and A. suum was very closely related. These mt genome data and the results provide some additional genetic evidence that A. lumbricoides and A. suum may represent the same species. The mt genome data presented in this study are also useful novel markers for studying the molecular epidemiology and population genetics of Ascaris.     

Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae)

Glycoproteins (GPs) were visualised histochemically in the secretory cells – the mucous goblet cells (the type A and the type B), the serous goblet cells, the club cells and the epithelial cells in the gill epithelium of Rita rita. The type A mucous goblet cells, the type B mucous goblet cells and the epithelial cells elaborate GPs with oxidizable vicinal diols and GPs with sialic acid residue without O-acyl substitution. In addition, GPs with O-sulphate esters are elaborated by the type A and GPs with O-acyl sugars by the type B mucous goblet cells. GPs are absent in the serous goblet cells and are with oxidizable vicinal diols in low moieties in the club cells. The analysis of the results elucidates interesting differences in the composition and concentration of GPs in the mucus elaborated by the epithelium of the gill arches and the gill rakers; and the gill filaments and the secondary lamellae indicating the potential importance of the glycoproteins at these locations. GPs elaborated on the surfaces of the gill arches and the gill rakers could be associated to assist in feeding activities and on the surfaces of the gill filaments and the secondary lamellae in the respiratory activity.     

文昌鱼秦皇岛、青岛和厦门地理种群形态特征的分化

把分别采自秦皇岛、青岛和厦门(各25尾)的文昌鱼种群的形态特征分为计数性状8个和计量性状10个,然后对18个形态特征进行ANOVA、聚类和主成分分析。结果表明:3地种群全长、体高和背腹鳍条数等18个形态特征的平均值均存在显著差异(P<0·05),且种群间的变异大于种群内。进一步比较秦皇岛和青岛种群,除在口笠触须、背鳍条数、腹鳍条数和肌节总数4个特征较为相似外,其余14个特征均存在显著差异(P<0·05);而青岛和厦门种群在全长、体高、腹孔肛门间肌节数、生殖腺数、吻鳍高、背鳍高、上尾鳍高等7个特征上更为相似(P>0·05);此外,秦皇岛和厦门种群在腹鳍高、上尾鳍长和高、下尾鳍长和高等5个特征上存在相似性(P>0·05)。聚类分析结果表明,75个样本明显分成两个类群:第二类群主要由厦门样本构成,而第一类群则主要由秦皇岛和青岛样本构成。由前3个主成分分析结果表明,所有样本可大致分为3个类群:秦皇岛、青岛和厦门类群。其中来自厦门的个体聚成一个明显的独立类群,而来自秦皇岛和青岛的样本在主成分分析图上则存在一定的交叉和重叠。总之,厦门地理种群分化最为明显,而秦皇岛和青岛种群在总体分化的趋势下,个体间形态特征仍存在相似性。     

Fine structure of the excretory system of Amphioxus (Branchiostoma floridae) and its response to osmotic stress

Summary The excretory organs of Amphioxus occur as segmentally arranged structures throughout the pharyngeal region and may be divided into three components: the solenocytes, the renal tubule, and the renal glomerulus.The solenocytes possess foot processes that rest upon the coelomic surface of the ligamentum denticulatum. The tubular apparatus of the solenocytes consists of ten triangular rods surrounding a central flagellum. The distal end of the tubular apparatus enters branches of the renal tubule. The renal tubule eventually opens into the atrial cavity of Amphioxus. The renal glomerulus is a sinus within the connective tissue of the ligamentum dentieculatum where it connects elements of the branchial circulation with the dorsal aorta. The renal glomerulus, like other blood vessels of Amphioxus, lacks an endothelial lining.If Amphioxus is adapted to artificial sea water at different concentrations there is no change in kidney morphology suggesting that Amphioxus is either is osmotic with its environment or is osmoregulating with other organs.This work was supported by U.S. Public Health Service Grant 5-T01-GM 00582.     

Special secretory cells in the soybean seed coat

Summary The seed coat of soybean (Glycine max L. Merr.) is of physiological interest for synthesis and transport of amino acids and photosynthates during embryo development. A transmission and scanning electron microscopic study to elucidate the structure of the seed coat disclosed a specialized convex area (antipit) appressed to a concave pit in the center of the abaxial surface of the cotyledon. The antipit, which lies on the inner surface of the seed coat at a medial point in the anterior to posterior direction of the seed, contained specialized secretory cells bounded by loose multi-layered cell walls. These cells were rectangular in the developing seed, varied in length, and contributed directly to the convex morphology of the antipit seen on the ventral surface of the seed coat. At maturity these cells assumed the shape of a cone, extending from the aleurone layer in a perpendicular array. The aleurone and cone cells contained numerous Golgi apparatus, laminated rough endoplasmic reticulum, secretory vesicles, and amyloplasts. Secretory vesicles arose directly from tubules of fenestrated trans cisternae of the Golgi apparatus. Mitochondria were clustered with the amyloplasts; stacks of lamellar cisternae of rough endoplasmic reticulum were associated with the nucleus and Golgi apparatus. The cellular contents, the interconnections by plasmodesmata, and the close physical association with the cotyledon suggested that the aleurone and cone cells may be involved in symplastic transport of nutrients for use by the developing embryo.This paper is dedicated to the memory of my parents, Joseph and Theresa Yaklich, who by their example taught me the value of work and the enjoyment of simple things.     

Ultrastructural studies on the secretory cavities ofCitrus deliciosa ten. I. Early stages of the gland cells differentiation

Summary Secretory cavities ofCitrus deliciosa seem to originate from a pair of meristematic cells (an epidermal cell and a second one placed under it). These cells undergo successive divisions resulting in the formation of a globular/oval gland situated in the parenchyma, and a conical stalk, which joins the gland with the epidermis. The young gland consists of a central group of polyhedral cells ensheathed by layers of radially flattened cells.During the early differentiation stages of the gland cells a close association of cytoplasmic microtubules with various organelles is observed. Plastids increase progressively in number and size and their matrix locally contains tubular networks accompanied by small oil droplets. In peripheral cytoplasm numerous myelin-like lomasomes have been observed. Peripheral cells of the gland are gradually modified from the inner cells following a different developmental pattern. Their walls become thicker and plastids do not contain tubular complexes, but only a few thylakoids partly surrounding the newly formed starch grains.