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.     

Branchial vascular pathways in two species of Tetraodontiformes and the concept of secondary vessels and nutrient arteries

The vascular organisation of the branchial basket was examined in two Tetraodontiform fishes; the three-barred porcupinefish, Dicotylichthys punctulatus and the banded toadfish, Marylina pleurosticta by scanning electron microscopy of vascular casts and standard histological approaches. In D. punctulatus, interarterial anastomoses (iaas) originated at high densities from the efferent filamental and branchial arteries, subsequently re-anastomosing to form progressively larger secondary vessels. Small branches of this system entered the filament body, where it was interspersed between the intrafilamental vessels. Large-bore secondary vessels ran parallel with the efferent branchial arteries, and were found to constitute an additional arterio–arterial pathway, in that these vessels exited the branchial basket in company with the mandibular, the carotid and the afferent and efferent branchial arteries, from where they gave rise to capillary beds after exit. Secondary vessels were not found to supply filament muscle; rather these tissues were supplied by single specialised vessels running in parallel between the efferent and afferent branchial arteries in both species examined. Although the branchial vascular anatomy was generally fairly similar for the two species examined, iaas were not found to originate from any branchial component in the banded toadfish, M. pleurosticta, which instead showed a moderate frequency of iaas on other vessels in the cephalic region. It is proposed that four independent vascular pathways may be present within the teleostean gill filament, the conventional arterio–arterial pathway across the respiratory lamellae; an arterio–arterial system of secondary vessels supplying the filament and non-branchial tissues; a system of vessels supplying the filament musculature; and the intrafilamental vessels (central venous sinus). The present study demonstrates that phylogenetic differences in the arrangement of the branchial vascular system occur between species of the same taxon.     

Two different forms of gap junctions within the same organism, one with cytoskeletal attachments, in tunicates

The cells of the intestinal tract and the stigmatal cells of the branchial basket have been studied in a range of tunicates including phlebobranch, aplousobranch and stolidobranch ascidians, as well as the doliolid and pyrosomatid thaliaceans. The intercellular gap junctions between gut cells appear conventional in thin section as do those found in the lower part of adjacent stigmatal cells. However, save for the stolidobranchs, the stigmatal cells also have a second kind of gap junction which exhibit an unusual fibrous density in association with their junctional cytoplasmic surfaces; these are found in the apical region of the cells. The fibrous density is particularly well demonstrated in specimens treated with tannic acid during fixation, and subsequent en bloc uranyl acetate staining. In the branchial basket the position of these apical gap junctions is at regular intervals between adhaering junctions, which have a more substantial paramembranous fibrous mat; these two kinds of junctions alternate along deeply undulating membrane appositions. With freeze-fracture, after chemical or cryo-fixation, the gap junctions of the gut and those of the lower part of the stigmatal cells appear typical, with P-face connexons, while in the apical part of cells of the branchial basket the two faces of the gap junctions are very difficult to cleave apart. Frequently the P- and E-faces are found to adhere together in replicas, so that in these apical gap junctional regions, plaques of E-face with pits overlie the PF particles. In addition, regions of cytoplasm, into which the dense fibres project, often cleave over these adhaering E-faces of the apical gap junctions. The presence of these unusual gap junctional features in the apical region of the stigmata in the vicinity of cilia is discussed as regards their functional role.     

A comparative study of ciliary differentiations in the branchial stigmata of ascidians

In a correlated thin sectioning and freeze-fracturing study, we have examined species belonging to the orders of the ascidian class: Stolidobranchiata (Botryllus schlosseri, Botrylloides leachi, Molgula socialis, Styela plicata), Phlebobranchiata (Ascidiella aspersa, Phallusia ingeria, Ciona intestinalis) and Aplousobranchiata (Clavelina lepadiformis). Though the branchial basket varies in the complexity and filtration efficiency in the three orders, the ciliated epithelia aroand the stigmata contain a common pattern of organization; seven rows of flattened cells, each bearing a single row of long cilia flanked by a single row of microvilli. All the species examined possess ciliary specializations represented by: (a) bridges connecting doublets number 5 and 6 as well as 9.1 and 2; (b) dense material lying between the above mentioned axonemal doublets (5-6 and 1-2) and the ciliary membrane, sometimes in the shape of longitudinal strands or often as lines of dots; (c) a fuzzy coat protruding from the ciliary membrane, consisting of tufts or scattered filaments; (d) intramembrane particles (IMPs) associated with the P-face of the membrane, often arranged in clusters and orderly alignments related to the anderlying axonemal doublets; these IMPs decorate the opposite sides of each cilium facing the adjacent cilia forming the ciliary rows of adjacent cells and are absent on the lateral sides. The stigmatal cilia propel water through the stigmata and their effective strokes follow a line at right angles to the row of cilia in each cell. The usual direction of the effective stroke is toward doublets 5-6. It is possible, therefore, to refer to structure in relation to the ciliary beat cycle. The importance of these specializations is unknown, but the structures appear to vary in the different species. A correlation between the richness of the specializations and the complexity of the branchial basket was not evidenced. It was suggested that the ciliary specializations relate to the peculiar organization of the stigmatal margin and that all are involved in the regulation of the ciliary activity.     

Ultrastructure of the heart muscle cells of the cuttlefish Rossia macrosoma (Delle Chiaje) (Mollusca: Cephalopoda)

Summary The muscle cells of the ventricle, the branchial heart and the branchial heart appendages of Rossia macrosoma (Delle Chiaje) are studied. The ventricle myocardium has three muscle layers, while the other two organs exhibit a loose arrangement of muscle cells. The muscle cells of the ventricle, the branchial heart and the branchial heart appendages are similar in structure. The nuclei are surrounded by myofibrils. In the myofibrils A-, I- and discontinuous Z-bands are seen. The diameters of the thick filaments are 300–400Å, their length varies from 1.7 to 3.9 . Thin filaments have a diameter of approximately 85Å. The ratio between thick and thin filaments is roughly 1 to 11.The SR runs mostly as a longitudinal network within the myofibrils. A few short T-tubules are observed in the Z-regions. Peripheral and internal couplings exist. The latter are few in number.Intercalated discs are small and rarely observed. They have been found in all three organs. A difference in the function of these organs is not reflected in the ultrastructure of the intercalated discs. These discs are often of the interdigitating type with interfibrillar junctions and unspecialized regions. Peripheral couplings are seen at the unspecialized regions. The intercalar surfaces of the muscle cells shoulder off into the lateral surface, and the transition between the two surfaces is not a sharp one. Attachment plaques are found scattered over the whole sarcolemma.     

A Scanning Electron Microscope Study of the Branchial Sac of Benthic Filter-feeding Invertebrates (Ascidians)

Abstract With the critical point method, well preserved samples of ascidian gills have been obtained for a study by scanning electron microscopy. The electron micrographs illustrate the different stages in the structural complexity of the branchial sac in four species: Clavelina lepadiformis (Müller 1776) represents the simplest stage where the gill sheet is formed by a single screen with slits; Ciona intestinalis (Linné 1767) and Phallusia mammillata (Cuvier 1815) which exhibit intermediate stages with a secondary screen superimposed on the first, thus increasing the active surface. Maximum gill extent and, consequently, a maximum contact with the water carrying particles is observed in Styela plicata (Lesueur 1823) with the formation of gill folds. Our observations also demonstrate a morphological analogy between the zones exhibiting a positive reaction to proteolytic enzymes (chymotrypsin-like): namely the concave edge of the gill papillae of Ciona and Phallusia, the languets of the dorsal lamina of Phallusia, the folds of the longitudinal bars of Styela.     

The venous system of the terrestrial crab Ocypode cordimanus (Desmarest 1825) with particular reference to the vasculature of the lungs

The respiratory system of Ocypode cordimanus consists of seven pairs of gills, modified for aerial gas exchange, and a single pair of lungs. Each lung is formed from the inner surface of the branchiostegite and the thoracic wall of the branchial chamber. The branchiostegal surface is increased by a fleshy infolding, the branchiostegal shelf, whilst the surface area of the thoracic lung wall is enhanced by a large flaplike fold. The anatomy of the major sinus systems and the vascular supply to the lungs were investigated. Venous hemolymph is supplied to the lungs potentially from all the major body sinuses. The dorsal, ventral, hepatic, and infrabranchial sinuses are all connected anteriorly to the two eye sinuses which distribute hemolymph to the lungs. Each eye sinus gives off five branches to the branchiostegal lung surface and one to the thoracic lung wall. These afferent vessels are highly branched and interdigitate closely with efferent vessels. The two systems are connected by flat lacunae lying just beneath the respiratory epithelium and these are believed to be the site of gas exchange. The efferent vessels empty into two pulmonary veins on each side, one serving the branchiostegal lung wall and the other the thoracic wall. The two vessels on each side fuse before joining the pericardial cavity as a single trunk on each side.     

ESR spectra of vanadyl ion detected in branchial basket of an ascidian,ascidia ahodori

ESR spectra due to the vanadyl ion (VO²⁺, +4 oxidation state) was detected in the branchial basket of Ascidia ahodori, which is reported to contain vanadium in high amounts. The branchial basket, washed with a medium containing 1 mM EDTA, and the supernatant showed different types of vanadyl ESR spectra. On further treatment with 100 mM EDTA the branchial basket gave a characteristic ESR spectrum, indicating that the vanadyl ion binds to a high molecular weight matrix, such as proteins, which makes up the branchial basket. Judging from the relationship of the ESR parameters, g_∥ versus A_∥, the vanadyl ion is assumed to ligate with moieties such as deprotonated hydroxyl, or nitrogenous or thiolato groups from oxy- or thiolamino acid residues. The branchial basket was shown to have the ability to reduce added vanadate ion (+5 oxidation state) to the vanadyl form. On the basis of these observations, participation of the branchial basket in vanadium-accumulation by ascidians from seawater is suggested.     

Carapace movements associated with ventilation and irrigation of the branchial chambers in the semaphore crab,Heloecius cordiformis (Decapoda: Brachyura: Ocypodidae)

Summary Carapace movements in crabs are briefly reviewed. While on land and recirculating branchial water, the Australian semaphore crab Heloecius cordiformis (Decapoda: Ocypodidae), a semi-terrestrial air-breathing mangrove crab, sequentially depresses and elevates its carapace relative to its thorax (0.5–1 mm excursion) in a regular pump-like manner. In quiescent crabs each carapace-pumping cycle lasts about 4 s; carapace depression takes 3 s and elevation 1 s. Carapace movements are brought about by pressures generated within the branchial chambers by the scaphognathites, probably in combination with carapace muscles. Carapace movements are associated with bilaterally synchronised scaphognathite activity. Unilateral scaphognathite activity was not observed. During normal forward recirculation of branchial water the scaphognathites beat at about 1.5 Hz (slow-forward pumping) and the lungs (epibranchial chambers) are not ventilated. In Heloecius, the lungs are not physically separated from the gills below by an anatomical barrier. Lung ventilation is accomplished during the following sequence of events: the carapace is lowered and the scaphognathites pump in a fast-forward mode at about 2.8 Hz. This activity preferentially pumps air out of the lungs and generates suction within the branchial chambers (4–10 cm H₂O below ambient) which draws water from external body surfaces into the hypobranchial space below and around the gills. At the end of the carapace's downward travel the scaphognathites switch from fast-forward to fastreverse beating at about 4 Hz. This pumps air into the lungs and the carapace elevates. As a result, during carapace elevation the water which had previously been drawn into the branchial chambers by fast-forward pumping activity is released and flows out between the legs and into the abdominosternal cavity. When the carapace reaches its original resting or up position the scaphognathites switch from fast-reverse to slowforward beating to re-establish water recirculation through the branchial chambers. This cycle is subsequently repeated. In stationary crabs, there are 2 carapace-pumping cycles per minute, increasing to 14 per minute in active crabs (walking). When water is absent, the lungs are preferentially ventilated by slow-reverse scaphognathite pumping activity. Carapace movements do not occur in the absence of branchial water. Carapace pumping is thought to provide a mechanism which permits the scaphognathites to ventilate the lungs in the presence of recirculating branchial water, without this water interfering with lung ventilation or being lost to the environment.Abbreviations FF, FR, SF, SR fast-forward, fast-reverse, slowforward, slow-reverse scaphognathite pumping - MEA Milne Edwards aperture     

Peroxidase and not laccase is the enzyme responsible for cell wall lignification in the secondary thickening of xylem vessels inLupinus

Summary The post-exponential growth phase of lupin (Lupinus albus cv. Multolupa) hypocotyls is characterized by a strong deposition of lignins in the primary and secondary walls of the xylem vessels. Coinciding with this phenomenon, there is a clearly peroxidatic activity in both the primary cell walls and the outer-most layers of the secondary thickening of the xylem vessels, as demonstrated by 3,3-diaminobenzidine cytochemistry. This activity was completely inhibited by KCN and the removal of H₂O₂ and was not due to laccase since this enzyme shows an almost total inability to oxidize 3,3-diaminobenzidine both in the presence and in the absence of H₂O₂. The absence of laccase-like activities in cell walls of vascular cells was supported by the fact that cell wall proteins from vascular cells were only capable of oxidizing 3,3-diaminobenzidine and coniferyl alcohol in the presence of H₂O₂. These results support the idea of an exclusive role of peroxidase (and exclude any role for laccase) in lignin formation in the secondary thickening of xylem vessels inLupinus.     

Sensory innervation of the tentacles of the polychaete,Sabella pavonina

Summary Following observation of conical groups of stiff, but motile cilia on the tentacles of the branchial crown of Sabella pavonina, these were examined with the electron microscope. The bundles consist of about 40 unenclosed standard cilia supported by one or two primary sense cells with centrally directed axons of 0.1–0.2 diameter. Axons in the distal portions of the branchial crown occur in small bundles surrounded by a basement membrane. More centrally, glial elements appear and the nerves are surrounded by a collagenous sheath. The branchial nerve trunk shows similarities in organisation to other previously investigated annelid central nervous tissue in that the whole nerve is surrounded by a fibrous sheath central to which there is a layer of glial cells with processes penetrating a central neuropile. The 0.1–0.2 axons commonly occur in glial-enveloped groups of < 40 whilst other axons of larger and mixed diameter are found together.Each tentacle has two branchial nerves on the oral side, and each nerve gives rise to two small 75-axon branches running to each pinnule. The branchial nerves fuse to form the branchial nerve trunk running to the supra-oesophageal ganglia.Sections of the branchial nerves of the branchial crown at progressively more central levels show that the branchial nerve trunk contains enough axons of 0.1–0.2 diameter to account for all the sensory cells on the tentacles. This is taken as evidence for the sensory cells having axons terminating within the central nervous system and that there is no peripheral confluence or fusion of these afferent axons.     

The isolated, perfused head of the marine teleost fish,Myoxocephalus octodecimspinosus: Hemodynamic effects of epinephrine

Summary An isolated, perfused-head preparation utilizing the marine teleost (Myoxocephalus octodecimspinosus) was developed, and the hemodynamic effects of epinephrine on this preparation were studied.The isolated head of the sculpin was found to have a relatively long-term viability as measured by stable perfusion pressures (±4.03 Torr for the first hour of perfusion) and responsiveness to epinephrine for over three hours (Fig. 1). This catecholamine induced a net decrease in the resistance of the vasculature of the head along with an increase in the dorsal aortic blood flow, and a concomitant decrease in a venous flow from the peritoneal cavity and cut muscle mass (Fig. 2, Table 1). Blocking beta adrenergic receptors during epinephrine application produced an increase in the vascular resistance and the dorsal aortic to venous flow ratio (Table 2); beta receptor stimulation was followed by a decrease in the resistance and no change in efferent flow rates (Table 3).It is concluded that alpha adrenergic receptors induce the constriction of the branchial arterio-venous anastomoses and are responsible for the efferent flow rate changes. However, beta adrenergic receptors, presumably at the level of the lamellar arterioles, appear to be the dominant factor in the control of net branchial resistance.     

Making a master filterer: Ontogeny of specialized filtering plates in silver carp (Hypophthalmichthys molitrix)

Filter feeding fishes possess several morphological adaptations necessary to capture and concentrate small particulate matter from the water column. Filter feeding teleosts typically employ elongated and tightly packed gill rakers with secondary bony or epithelial modifications that increase filtering efficiency. The gill rakers of Hypophthalmichthys molitrix, silver carp, are anatomically distinct from and more complex than the filtering apparatus of other teleostean fishes. The silver carp filtering apparatus is composed of biserial, fused filtering plates used to capture particles ranging in size from 4 to 80 μm. Early in ontogeny, at 15–25 mm standard length (SL), silver carp gill rakers are reminiscent of other more stereotypical teleostean rakers, characterized by individual lanceolate rakers that are tightly packed along the entirety of the branchial arches. At 30 mm SL, secondary epithelial projections and concomitant dermal ossification begin to stitch together individual gill rakers. During later juvenile stages, dermal bone further modifies the individual gill rakers and creates a bony scaffold that supports the now fully fused and porous epithelium. By adulthood, the stitching of bone and complete fusion of the overlying epithelium creates rigid filtering plates with morphologically distinct faces. The inner face of the plates is organized into a net‐like matrix while the outer face has a sponge‐like appearance comprised of differently sized pores. Here, we present morphological data from an ontogenetic series of the filtering apparatus within silver carp. These data inform hypotheses regarding both how these gill raker plates may have evolved from a more basal condition, as well as how this novel architecture allows this species to feed on exceedingly small phytoplankton, particles that represent a greater filtering challenge to the typical anatomy of the gill rakers of fishes.     

Carapace movements aid air breathing in the semaphore crab,Heloecius cordiformis (Decapoda: Brachyura: Ocypodidae)

Summary While on land and recirculating branchial water the Australian semaphore crab Heloecius cordiformis (Decapoda: Ocypodidae), a semi-terrestrial airbreathing mangrove crab, sequentially depresses and elevates its carapace in a regular pump-like manner. The functional role of these carapace movements in aerial oxygen consumption is investigated. Carapace immobilisation (reversible and non-injurious) did not appear to affect branchial water circulation. In dry crabs (branchial water removed) carapace immobilisation had no effect on the rate of oxygen consumption (VO₂), heart rate or whole-body lactate (WBL) levels. In wet crabs (with branchial water) carapace immobilisation caused VO₂ to drop by 38% from 81 to 46 l O₂ · g^-1 · h^-1, heart rate to decline by 32%, from 2.5 to 1.7 Hz, and WBL levels to increase over 2.5-fold, from 0.27 to 0.67 mg · g^-1, after 3 h of carapace immobilisation. The (VO₂) of carapace-immobilised crabs with branchial water was similar to lung-occluded crabs with branchial water. Severe hypoxia induced physiological responses similar to those of carapace-immobilised crabs with branchial water. After 3 h of severe hypoxia, heart rate had declined by 80%, from 2.2 to 0.43 Hz, and the incidence of carapace pumping slowed by 85%, from 2.4 to 0.37 cycles · min^-1. It is concluded that in the absence of carapace movements branchial water in some way inteferes with lung ventilation. Under normal circumstances water circulation and lung ventilation are mutually exclusive processes (due to their singular dependence on the scaphognathites), yet in Heloecius these processes must be carried out simultaneously. Carapace movements may alleviate this conflict.Abbreviations FF, FR, SF, SR fast-forward, fast-reverse, slow-forward, slow-reverse scaphognathite pumping - MEA Milne Edwards aperture - VO₂ rate of oxygen consumption - WBL whole-body lactate     

Different functions of tight junctions in the ascidian branchial basket

The stigmatal cells in the branchial basket of ascidians from a number of genera have been examined as to the nature and distribution of their intercellular junctions. The branchial wall consists of ciliated and parietal cells; the ciliated cells are arranged in seven rows and are associated by junctions with other cells in the same row as well as with those in adjacent rows. They are also associated by junctions with peripheral parietal cells. Junctions between adjacent ciliated cells in all cases exhibit tight junctions or zonulae occludentes. However, these cell borders also possess fasciae or zonulae adhaerentes if they are in the same row and the ciliary rootlets insert-into these junctions. If the cells are in adjacent rows they exhibit adhaerentes junctions only in species belonging to the orders Phlebobranchiata and Aplousobranchiata. In contrast, if the cells in adjacent rows belong to the order Stolidobranchiata. they never exhibit any adhaerentes junctions and the ciliary rootlets of the basal bodies from the cilia insert instead into the tight junctions and the non-junctional membrane below them. At the homologous junctional borders between adjacent parietal cells and also at heterologous junctional borders between parietal and ciliated cells, tight junctions alone occur, with no co-existing adhaerentes junctions along their lateral borders. Again, fibrils from ciliary rootlets insert into zonulae occludentes. This shows that tight junctions are capable both of forming permeability barriers, in that they can be seen to prevent the entry of exogenous tracers such as lanthanum, and of acting as adhesive devices.     

The Rhinal Bone and Its Evolutionary Significance

Bjerring, H. C. (Section of Palaeozoology, Swedish Museum of Natural History, Stockholm, Sweden.) The rhinal bone and its evolutionary significance. Zool. Scripta 1 (5): 193–201, 1972.– On the basis of serially sectioned embryos of Amia calva, the ethmoidal region of the endocranium and certain adjacent exo-skeletal elements are analysed. The results include evidence of the existence of branchial moieties pertaining to the first or premandibular metamere. Each of these moieties comprises infrapharyngobranchial (the ethmobasal), suprapharyngobranchial (the orbitonasal lamina), and epibranchial (the palatoquadrate pterygoid process) endoskeletal components as well as horizontal infrapharyngeal (the vomer), ascending infrapharyngeal (the rhinal), and epal (the dermopalatines and ectopterygoid) dental plates. The anterior cerebral arteries may represent the efferent blood vessels of the first-metamere branchial moieties. A hypothesis on the origin of the nasal sacs is offered.      

Observations of valve-like structures and evidence for rectification of flow within the gill lamellae of the crab Carcinus maenas (Crustacea,Decapoda)

Summary The isolated gills of Carcinus maenas, perfused at pressure drops of 1–10 cm of water, exhibited flow rectification, the resistance to perfusion via the afferent vessel being many times lower than that for efferent perfusion. The asymmetry was greater at the lower end of this pressure range.The overall afferent branchial resistance for Carcinus of weight 65 g, and with no ventilatory component in the transmural pressure difference, was estimated to be 0.05 cm of water. l^–1 · sec. The corresponding overall reverse (efferent) branchial resistance was 0.36 cm of water · l^–1 · sec.LM, TEM and SEM examination of the gills indicated that haemolymph leaves each gill lamella via several discrete parallel efferent channels which drain different regions of the lamella, and that each efferent channel is nearly closed, at its junction with the efferent branchial vessel, by a diaphragm of loosely interwoven and very elongated cells. It is concluded that these cells may constitute efferent valves and that narrow apertures between them may contribute a major component to the branchial resistance and be primarily responsible for the rectification of flow. Relatively wide apertures lead directly from the afferent vessel into the lamellae and are not asociated with valves of any kind.The valves may be important in enabling changes in transmural pressure associated with ventilatory reversals to pump haemolymph unidirectionally through the lamellae. Similarly valves may allow the oscillating venous pressures associated with locomotor activity to improve gill perfusion during exercise.The elongated tails of the cells of the efferent valve contain numerous microtubules. The wider cell bodies contain the nucleus and many mitochondria. Unusual organelles composed of many short (about 0.25 m long) microtubules and often lying close to the nuclear membrane may be microtubule organising centres. It is speculated that, in addition to their simple mechanical function, the valve cells may play a more dynamic role in regulating flow of haemolymph through different lamellar routes, or that they may monitor composition, pressure or flow of the efferent lamellar circulation.