The Fine Structure of the Testis of a Lancelet (=Amphioxus), Branchiostoma floridae (Phylum Chordata: Subphylum Cephalochordata= Acrania)

The germinal and non-germinal cells of the ripe lancelet testis are described by transmission electron microscopy. The visceral peritoneum of the testis is composed of myoepithelial cells, and the haemal layer consists of regions of narrow sinuses and conspicuously thicker blood vessels filled with blood plasma and bounded by basal laminae. Within the germinal epithelium and the testicular lumen, the non-germinal cells, which are not abundant, contain conspicuous lysosomes and mitochondria with tubular cristae, indicating that they may be involved in steroid synthesis. In the ripe testis, the non-germinal cells do not appear to be organized into a blood-testis barrier. Ail types of spermatogenic cells may be flagellated and are joined in small groups by intercellular bridges. During differentiation of the spermatids, the Golgi complex is associated with formation of the acrosomal vesicle near the posterior pole of the cell. A remarkable feature is the dual origin of the subacrosomal material: one component originates at the posterior end of the spermatid, and the other at the anterior end. Subsequently, the two components merge into one after the acrosomal vesicle has migrated to its definitive anterior position in the mature spermatozoon.     

The Histochemistry and Fine Structure of the Nutritional Reserves in the Fin Rays of a Lancelet,Branchiostoma lanceolatum (Cephalochordata = Acrania)

Abstract Adults of the European lancelet were collected at Banyuls-sur-Mer (Mediterranean France) in mid-spring, shortly before the onset of the breeding season. The dorsal and ventral fin rays were studied by light microscopic histochemistry and by transmission electron microscopy (TEM). Each fin ray is a mass of extracellular material that accumulates beneath the mesothelium of a fin box coelom. The fin ray material is rich in lipids, proteins, and neutral mucopolysaccharides. TEM reveals no lipid droplets in this material, which consists entirely of a packed mass of 15–20 nm granules of medium electron density. It is likely that these granules consist of glycoproteins or glycolipoproteins. Our results are consistent with the proposal of Azariah (1965, Journal of the Marine Biological Association of India 7: 459–661) that lancelet fin rays are nutritional reserves supporting gametogenesis during the breeding season.     

The Wheel Organ and Hatschek's Groove in the Lancelet,Branchiostoma lanceolatum (Cephalochordata)

The wheel organ is a specialized epithelium in the roof and sides of the adult lancelet oral cavity. It borders the oral epithelium proper, separated by a thin strip of margin cells which are not ciliated but contain a few large dense-cored vesicles apically. The wheel organ cells are tall and strongly ciliated and have dark, heterochromatin-rich nuclei. Dorsally, and slightly paramedially, the organ is further specialized, forming the so-called Hatschek's groove (pit), which consists of two ciliated cell types. The first type synthesizes a dense granular material, the granules being approximately 95 nm in diameter. This is stored basally and apparently it is also released through the basal cell membrane into the blood cavities. The cells at the bottom of Hatschek's groove have peculiar rod-shaped apical cellular regions. Each cell bears one tall cilium surrounded by microvilli and it is apparently involved in the production of secretory material into the groove. It is evident that the histology, and probably also the function, of the wheel organ and its groove is much more complex than hitherto believed.     

The Oral Papilla of the Lancelet Larva (Branchiostoma lanceolatum) (Cephalochordata)

The oral papilla is a prominent larval organ in the lancelet, which develops early during ontogenesis and disappears completely at metamorphosis. Electron microscopic studies indicated that the prevailing theories of its function are unlikely. Its cells lack the cytological characteristics of sensory or mucus producing cells, and the organ is not equipped with a tuft of flagella. It was not possible to reveal the function of the papilla, but its cells seem to be involved in a pronounced substance transport through the basal cell membrane as well as in the cytoplasm. Apical specializations indicate exo- or endocytotic activity and vesicles imply some exchange also between papilla cells and the sea water.     

Serotonin-containing Cells in the Nervous System and Other Tissues During Ontogeny of a Lancelet,Branchiostoma floridae

Abstract Serotonin-containing cells are described by immunohistochemistry throughout lancelet ontogeny. Such cells are first detected in the 2-day larva: these are (1) enterochromaffin cells in the inner epithelium of the gut and (2) anterior serotonergic neurons at the rostral end of the nerve cord. In the 6-day larva, relatively low levels of serotonin appear in ventro-lateral perikarya and cell processes of intraspinal serotonergic neurons scattered along the nerve cord. In the 18-day (early metamorphic) larva, antero-lateral serotonergic neurons are detected near the rostral end of the nerve cord as two small, bilateral clusters of perikarya with axons that descend the nerve cord; at later developmental stages, these axons extend almost to the posterior end of the body. In the 21-day (mid-metamorphic) larva, serotonin can no longer be detected in the anterior serotonergic neurons, but serotonin-containing cells are found subjacent to the inner epithelium of the digestive caecum and in the peribranchial epithelium covering the primary gill bars. In the discussion, we suggest that the anterior serotonergic neurons may play a role in larval photoreception and that the antero-lateral serotonergic neurons may be homologous to vertebrate hindbrain neurons with axons descending the spinal cord to modulate undulation (if this homology is valid, the anterior limit of the lancelet hindbrain would be roughly 100 μm behind the rostral tip of the nerve cord).     

Embryos and Larvae of a Lancelet,Branchiostoma floridae,from Hatching through Metamorphosis: Growth in the Laboratory and External Morphology

Florida lancelets were raised in laboratory cultures from the egg to the juvenile stage. At frequent intervals during development, elongation of the embryonic and larval body was measured at room temperature (22.5°C) and at the approximate temperature of the natural environment (30°C). Development was slower at the lower temperature, with metamorphosis commencing during the fifth week as compared to the third week at the higher temperature. Scanning electron microscopy (SEM) was used to describe a frequently sampled series of hatched embryos, pre-metamorphic larvae, metamorphic larvae, and juveniles. The advent (and sometimes subsequent disappearance) of the following structures was determined from the SEM data: general epidermal ciliation, peroral pit, mouth, primary gill slits, ciliary tuft, external opening of the club-shaped gland, sense cells, anus, metapleural folds, and preoral cirri. Our SEM did not substantiate the claims of van Wijhe for a transitory larval mouth near the anteriovental end of the larvae. The general epidermal cilation, which is uniformly distributed in the embryos, becomes somewhat reduced in the pre-metamorphic larvae and then disappears almost entirely during metamorphosis. The epidermis includes two distinct sense cell types (I and II) and possibly a third type (the ventral pit cells, to which an adhesive role has alternatively been attributed). The anus first opens on the right-hand side and only later migrates across the mid-ventral line to assume a position on the left-hand side of the larva; this is contrary to the established view that the anus of the larval lancelets opens on the left-hand side and remains there.     

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.     

Acrosome reaction in spermatozoa from the amphioxus Branchiostoma belcheri (Cephalochordata, Chordata)

The formation of an acrosomal process at acrosomal exocytosis in spermatozoa of the amphioxus was described in the present report for the first time. A non-reacted acrosome was located in front of the nucleus, where a cup-shaped acrosomal vesicle covered a conical accumulation of subacrosomal material. When naturally spawned spermatozoa were treated with a calcium ionophore, ionomycin, the acrosomal vesicle opened at the apex and an acrosomal process was projected. The process exhibited a filamentous structure. The reaction followed the mode typically seen in marine invertebrates. These observations suggest that the features and function of the acrosome of amphioxus, whose position is on the border between invertebrates and vertebrates, reflect their ecological adaptation and phylogenic position.     

Age Structure,Growth, and Weight of Branchiostoma senegalense (Acrania,Branchiostomidae). off North-West Africa

Branchiostoma senegalense from a population off North-West Africa reach an age of 4–5 years. Relative growth decreases logarithmically with age. B. senegalense reaches 50% of its maximum length during the first year. Differences in the average length of these lancelets in samples taken in close proximity of each other are ascribed to the absence of individual year classes. The immigration of larvae into suitable sediments depends on the current and follows the pattern of the larval swarms in the plankton. Colonized areas are not normally left. The functions relating wet and dry weights (WW and DW respectively). to length L are lg WW = 2.71 × lg L – 2.16, and lg DW = 2.96 × lg L – 3.45. The dry weight is 11.85% of the wet weight.     

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.     

The ultrastructure of the blood vessels of Branchiostoma lanceolatum (Pallas) (Cephalochordata)

Summary The ultrastructure of the blood vessels in the caudal region of Branchiostoma is described in specimens injected with indian ink. None of the vessels have endothelial cells delimiting the luminal surface. The vessels are delimited either by dense connective tissue or by the characteristic basement lamella underneath the basal lamina of the myocoelic epithelium. It is proposed that the main blood flow in the caudal region follows different pathways depending on the activity of the animal. During swimming the muscle activity of the caudal muscles may have the effect that more blood flows from the aorta to the myoseptal plexi and is drained to the caudal vessel. In the resting animal it is possible that the blood flow through the myosepta is insignificant, and that the caudal blood flow is more or less restricted to the direct connections between the aorta and the caudal vessel: the dorsoventral anastomosis and the segmental connecting vessels.Supported by a grant from the Danish Natural Science Research Council     

On Branchiostoma californiense (Cephalochordata) from the Gulf of Nicoya estuary, Costa Rica

The cephalochordates are represented by the lancelets, of which species of the genus Branchiostoma are the best known. In recent years, these organisms have been the center of activity of studies focusing on the phylogenetic relationships of the chordates. In 1980, a survey of the benthos at 48 stations in the Gulf of Nicoya estuary, Pacific coast of Costa Rica, yielded 265 specimens of the lancelet Branchiostoma californiense. A total of 48 specimens was also collected at an intertidal flat in the mid upper estuary. Of the 48 subtidal stations, only eight had B. californiense, and these sites all had a sand fraction above 72%. The remaining stations ranged in their sand content from as low as 1% to as high as 92%, with an average of 25.9%, with 29 stations having a sand content lower than 72%. Lower salinities and muddy sediments may limit the distribution of the lancelet further upstream. This information is useful when changes over decades in the ecology of the estuary need to be evaluated against the background of local, regional, and global dynamics.     

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     

The myosepta in Branchiostoma lanceolatum (Cephalochordata): 3D reconstruction and microanatomy

Zoomorphology - Myosepta have been subject to comparative and evolutionary studies in aquatic groups of the Craniata, because they are likely to play a role in transmission of muscular forces to...     

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.     

A Neurosecretory System in the Brain of the Lancelet, Branchiostoma lanceolatum

Ultrastructural and histochemical studies indicate a neurosecretory system exists in the lancelet brain with basal properties resembling a primitive hypothalamic system. A nucleus of secreting neurons, containing peptide granules (115 nm), is prominent in the dorsal walls of the brain. The axons establish contacts with the ventral brain surface, probably releasing their secretory product out of the brain. The neurons are innervated by dopaminergic "boutons en passant" often very active with a high number of electron translucent vesicles as well as dense-core vesicles (90 nm). Ventrally located cellbodies containing what are probably secretory peptide granules (110 nm) establish contacts with their basal processes on the ventral brain surface.     

Fine Structure of Tentacles,Arms and Associated Coelomic Structures of Cephalodiscus gracilis (Pterobranchia,Hemichordata)

The tentacles of the pterobranch Cephalodiscus, a hemisessile ciliary feeder, originate from the lateral aspects of the arms and are covered by an innervated epithelium, the majority of its cells bearing microvilli. Each side of a tentacle has two rows of ciliated cells and additional glandular cells. The coelomic spaces in the tentacles are lined by cross-striated myoepithelial cells, allowing rapid movements of the tentacles. One, possibly two, blood vessels accompany the coelomic canal. On their outer sides the arms are covered by a simple ciliated epithelium with intra-epithelial nerve fibres; the inner side is covered by vacuolar cells. On both sides different types of exocrine cells occur. The collar canals of the mesocoel are of complicated structure. Ventrally their epithelium is pseudostratified and ciliated; dorsally it is lower and forms a fold with specialized cross-striated myoepithelial cells of the coelomic lining. Arms, tentacles, associated coelomic spaces and the collar canal of the mesocoel are considered to be functionally interrelated. It is assumed that rapid regulation of the pore width is possible and even necessary when the tentacular apparatus is retracted, which presumably leads to an increase of hydrostatic pressure in the coelom.     

Epidermal receptor development and sensory pathways in vitally stained amphioxus (Branchiostoma floridae)

Chloromethyl (CM) DiI was applied to the exterior of living embryos, larvae, and metamorphic juveniles of amphioxus. This fluorescent dye is taken up preferentially (but not highly selectively) by epidermal receptors and often stains sensory axons to their full extent. Type I primary receptors in the epidermis first become morphologically detectable along the rostrocaudal axis of the 2.5 day larva when their epidermal perikarya extend unbranched axons to the nerve cord. These axons run posteriorly or anteriorly within the nerve cord, depending on whether their perikarya are located, respectively, rostral or caudal to the most posterior pharyngeal slit. In later larvae, axons of type I receptors are organized into a dorsal and a subdorsal sensory tract on either side of the nerve cord. In the epidermis of metamorphic juveniles, CM-DiI also stains type II receptors (which are axonless, secondary receptors) and ventral pit cells (which may not be receptors). It is probable, but not yet conclusively demonstrated, that peripheral neurites from Retzius bipolar cells (primary intramedullary sensory neurones) synapse with type II secondary epidermal receptors or ramify freely among the other epidermal cells. The discussion considers homologies among epidermal sensory receptors in chordates.     

Genomic organization and evolution of actin genes in the amphioxus Branchiostoma belcheri and Branchiostoma floridae

We previously described the cDNA cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203–215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon–intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages and that gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates.