Cytobiological studies on hemocyanin metabolism in the branchial heart complex of the common cuttlefish Sepia officinalis (Cephalopoda, Dibranchiata)

The present study confirms previous investigations that demonstrated a high copper content in the branchial heart and its appendage, and that gave the first indication that this organ complex might be involved in hemocyanin metabolism in Sepia officinalis L. Immunocytochemical localization of hemocyanin molecules within the endocytotic lysosomal system of the ovoid cells and tracer experiments with ¹²⁵I-labeled Sepia hemocyanin suggest its endocytotic uptake. Energy-dispersive X-ray microanalysis and histochemical methods reveal a high copper content within the ovoid cells of the branchial heart. In view of the turnover of the respiratory pigment in the branchial heart of Sepia officinalis L., we believe that the ovoid cells are a site of hemocyanin catabolism.     

Renal organs of cephalopods: a habitat for dicyemids and chromidinids

The renal organs of 32 species of cephalopods (renal appendage of all cephalopods, and renal and pancreatic appendages in decapods) were examined for parasite fauna and for histological comparison. Two phylogenetically distant organisms, dicyemid mesozoans and chromidinid ciliates, were found in 20 cephalopod species. Most benthic cephalopods (octopus and cuttlefish) were infected with dicyemids. Two pelagic cephalopod species, Sepioteuthis lessoniana and Todarodes pacificus, also harbored dicyemids. Chromidinid ciliates were found only in decapods (squid and cuttlefish). One dicyemid species was found in branchial heart appendages of Rossia pacifica. Dicyemids and chromidinids occasionally occurred simultaneously in Euprymna morsei, Sepia kobiensis, S. peterseni, and T. pacificus. The small-sized cephalopod species, Idiosepius paradoxus and Octopus parvus, harbored no parasites. Comparative histology revealed that the external surface of renal organs varies morphologically in various cephalopod species. The small-sized cephalopod species have a simple external surface. In contrast, the medium- to large-sized cephalopod species have a complex external surface. In the medium- to large-sized cephalopod species, their juveniles have a simple external surface of the renal organs. The external surface subsequently becomes complicated as they grow. Dicyemids and chromidinids attach their heads to epithelia or insert their heads into folds of renal appendages, pancreatic appendages, and branchial heart appendages. The rugged and convoluted external surface provides a foothold for dicyemids and chromidinids with a conical head. They apparently do not harm these tissues of their host cephalopods.     

Hemocyanin and the branchial heart complex of Sepia officinalis: are the hemocytes involved in hemocyanin metabolism of coleoid cephalopods?

Cytobiological experiments using isotopic- and cytochemical-labeled Sepia hemocyanin as well as immunocytochemical localization of the respiratory pigment were carried out to investigate the function of the hemocytes in hemocyanin metabolism of the common cuttlefish Sepia officinalis. For comparison, the rhogocytes (ovoid cells) of the branchial heart complex were included in this study. Hemocyanin molecules were immunocytochemically detected in the lysosomal compartment of the rhogocytes and, at lower levels, in adhesive and circulating hemocytes. (125)I-labeled Sepia hemocyanin was taken up by the rhogocytes only, whereas gold- and/or fluorescein-labeled Sepia hemocyanin was solely taken up by the adhesive and the circulating hemocytes, even though the level of uptake is different. There are also differences in the uptake of pure gold particles and/or fluorescein between rhogocytes and hemocytes. These findings give evidence that circulating and adhesive hemocytes of the branchial heart complex are not involved in hemocyanin turnover, but are a component of the cellular defense and detoxification system of adult coleoid cephalopods.     

Redescription of two Dicyemennea (Phylum: Dicyemida) from Rossia pacifica (Mollusca: Cephalopoda: Decapoda)

Two species of dicyemid mesozoan are redescribed from Rossia pacifica Berry, 1911, collected off Iwase in Toyama Bay, Honshu, Japan. Dicyemennea brevicephaloides Bogolepova-Dobrokhotova, 1962, is a large species that reaches about 4,000 microm in length. This species attaches to the surface of the branchial heart appendages. The vermiform stages are characterized as having 23 peripheral cells, a disc-shaped calotte, and an axial cell that extends to the propolar cells. An anterior abortive axial cell is absent in vermiform embryos. Infusoriform embryos consist of 35 cells; a single nucleus is present in each urn cell and the refringent bodies are absent. Dicyemennea rossiae Bogolepova-Dobrokhotova, 1962, is a medium species that reaches about 2000 microm in length. This species lives in folds of the renal appendages. The vermiform stages are characterized as having 29-34 peripheral cells, a conical calotte, and an axial cell that extends to the middle of the metapolar cells. An anterior abortive axial cell is present in vermiform embryos. Infusoriform embryos consist of 39 cells; 2 nuclei are present in each urn cell and the refringent bodies are solid.     

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.     

Cytomorphology and function of the pericardial appendages of Nautilus (Cephalopoda,Tetrabranchiata)

Summary The pericardial appendages (glands) are the principal excretory organs of the tetrabranchiate cephalopods. They were studied in Nautilus macromphalus and N. pompilius using light and electron microscopical methods. The four organs, which are homologous with the branchial heart complex in Coleoida, appear to represent a phylogenetically archaic state. Actually, they have had to take over the primary functions of secretion, and it will be seen that they are highly evolved. They are built up of numerous contractile villi which can be subdivided into three functionally important areas: the folded peripheral epithelium functioning particularly in reabsorption, the primarily secretory epithelium of the apical infoldings of the villi, and the ovoid cells with foot processes building up an ultra-filtration barrier. The filtrate seems to be expelled into the terminal secretory epithelium of the apical infoldings and not directly into the coelom.Within the brush border of the peripheral epithelia there are commensal flagellated bacteria, which may occupy an ecological niche similar to that of Dicyemida in the renal sacs of coleoids.     

The functional morphology of the branchial chambers and associated structures of Ebalia tuberosa (Crustacea: Decapoda: Leucosiidae), with special reference to ventilation of the egg-mass

Ebalia tuberosa bury themselves in sediment but do not construct permanent burrows. There are no functional Milne-Edwards' openings, the respiratory currents passing along channels formed by apposition of several interlocking buccal appendages. In adult females, the dome-like abdomen fits over the concave sternum to form a large abdominosternal chamber which completely encloses the egg-mass. The branchial chambers are connected tothe abdominosternal chamber by two branchiosternal canals plugged by modified pericardial
sacs which act as one-way valves. When the telson is extended, water is sucked into the abdominosternal chamber and then into the branchial chambers, thus ventilating the eggmass. The setation of the respiratory and associated structures serves to lock appendages
together, to form screens and gaskets and to groom the buccal appendages. Setal morphology is shown to be correlated with function.     

Cytomorphological aspects on the response of the branchial heart complex of Sepia officinalis L. (cephalopoda) to xenobiotics and bacterial infection

The ovoid cells of the branchial heart complex of Sepia officinalis L. were investigated with respect to their role in detoxification processes. The electron microscopical localization of in situ injected ferritin in the endocytotic-lysosomal system, and the fluorescence microscopical localization of protein-bound Evans blue in the ovoid cells of in vivo incubated animals, indicate that foreign materials are eliminated from the hemolymph by the branchial heart tissue. In addition to the non-circulating ovoid cells of the branchial heart, hemocytes in the circulating blood and in the wall of the branchial heart are also involved in the incorporation of allogeneic substances and bacteria, or their debris. Based on these observations, we propose that the ovoid cells, together with circulating and adhesive hemocytes in the branchial hearts, are an important component of a more comprehensive defence and detoxification system in dibranchiate cephalopods that prevents contamination of the whole organism by endocytotic removal of noxious substances from the hemolymph.     

Gill receptor arrays in the horseshoe crab (Limulus polyphemus)

The branchial warts on the endopodites of the gills are covered with goblet-shaped cuticular appendages, whose internal structure shows them to be chemoreceptors. The innervated goblets have a cuticular tubule that connects an external pore through their hollow interior with the epidermal sensillum. Associated sensory neurons give rise to small axons that pass through a synaptic plexus below the epidermis. The sense organs seem specialized for sampling the exhalent water current.     

Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5.

The Dlx5 gene encodes a Distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of the mouse embryo. In later developmental stages, it appears in the branchial arches, the otic and olfactory placodes and their derivatives, in restricted brain regions, in all extending appendages and in all developing bones. We have created a null allele of the mouse Dlx5 gene by replacing exons I and II with the E. coli lacZ gene. Heterozygous mice appear normal. Beta-galactosidase activity in Dlx5+/- embryos and newborn animals reproduces the known pattern of expression of the gene. Homozygous mutants die shortly after birth with a swollen abdomen. They present a complex phenotype characterised by craniofacial abnormalities affecting derivatives of the first four branchial arches, severe malformations of the vestibular organ, a delayed ossification of the roof of the skull and abnormal osteogenesis. No obvious defect was observed in the patterning of limbs and other appendages. The defects observed in Dlx5-/- mutant animals suggest multiple and independent roles of this gene in the patterning of the branchial arches, in the morphogenesis of the vestibular organ and in osteoblast differentiation.     

Vergleichende elektronenmikroskopische Untersuchungen an den zentralen Herzorganen von Cephalopoden (Sepia officinalis)

Zusammenfassung Die vergleichenden licht- und elektronenmikroskopischen Untersuchungen am Kiemenherzen von Sepia officinalis und Loligo vulgaris verschiedener Altersstufen zeigen eine Doppelfunktion dieses Organs als Herz- und Drüsenorgan auf. — Die quantitativ wenig hervortretende quer- resp. schräggestreifte Muskulatur ist nicht geschichtet und bildet ein lockeres Maschenwerk, in dem in großer Zahl ovoide Zellformen mit größeren Einschlußkörpern eingelagert sind. — Mit dem Auftreten von A-, I- und Z-Banden und der Ausbildung eines SR- und T-Systems entspricht die Organisation der Muskelfasern der des Zentralherzens (Schipp und Schäfer, 1969 b). Im Gegensatz zum Zentralherzen wird jedoch in der Kiemenherzmuskulatur nur ein Endigungstyp der wahrscheinlich monoaminergen Nervenfasern nachgewiesen.Die Zellformen zwischen den Muskelzellen treten in 2 Typen auf, die wahrscheinlich als Ausdruckverschiedener Funktionszustände zu werten sind. Ihre Organisation (Gehalt an Einschlußkörpern, Ergastoplasma und Dictyosomen sowie Exocytosevorgänge) deutet auf eine innersekretorische Funktion hin. Die intrazellulären Vorstufen des Sekretes besitzen ein ähnlich ausgebildetes kristallines Grundmuster wie das extrazelluläre Hämocyanin. Es ist daher nicht auszuschließen, daß diesen Zellen eine blutbildende Funktion zukommt.

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Comparative electron microscopic studies of the central cardiac organs of cephalopods (Sepia officinalis)Ultrastructure and function of the branchial heart

Summary Comparative light and electron microscopic studies of the branchial heart of Sepia officinalis and Loligo vulgaris at different ages showed that the organ functions both as a heart and a glandular structure. A small number of cross- or obliquely-striated muscle cells form a loose network that contains numerous ovoid cells with large inclusion bodies. The organization of A-, I-, and Z-bands, and the formation of SR- and T-systems in the branchial heart muscle resembles those in the central heart muscle (Schipp and Schäfer, 1969 b). On the other hand, only one type of monoaminergic axon terminals is observed in the branchial heart.The cells among the branchial heart muscle fibers are of two types, that may be a representation of different functional states. The organization of the cells (in reference to cell inclusions, ergastoplasm, dictyosomes) suggests endocrine function. The cell inclusions have a crystalline inner pattern like that of extracellular hemocyanin, suggesting that the ovoid cells may have a hemopoietic function.

Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Bile pigments and bilirubin UDP-glucuronyl transferase during the metamorphosis of Rana catesbeiana tadpoles

1. Cold-acclimated (1 degree C) goldfish (Carassius auratus) branchial Na/K-ATPase activity was elevated 100% while renal Na/K-ATPase activity was not significantly affected compared with warm-acclimated (20 degrees C) goldfish. 2. Cold-acclimated goldfish branchial and renal Mg-ATPase activity was reduced 18 and 30% on a per mg protein basis, respectively. 3. Renal Na/K-ATPase activity was 4.6- and 1.6-fold greater than gill in cold- and warm-acclimated fish, respectively. 4. The elevated branchial Na/K-ATPase activity and the unchanged renal Na/K-ATPase activity are consistent with the maintenance of the reduced blood ion level in cold-acclimated goldfish.     

cDNA cloning of the Octopus dofleini hemocyanin: sequence of the carboxyl-terminal domain

A cDNA library was constructed in pUC 19, using poly(A+) RNA purified from Octopus dofleini branchial gland, which is the site of hemocyanin biosynthesis in cephalopods. The library was screened with an oligonucleotide probe derived from a portion of the partially known sequence of the C-terminal domain of Paroctopus dofleini dofleini. The clone with the longest insert--called pHC1--was sequenced and used as a probe for Northern blotting. It hybridized to a 9.5-kb RNA species, which was also visible as a band after ethidium bromide staining. The cDNA insert (approximately 1200 bp) of pHC1 contained an open reading frame of 1071 bp coding for 357 amino acids. In this insert, a region coding for 42 amino acids from the N-terminal end of the C-terminal domain is missing. These were obtained by sequencing a cloned primer extension product. By comparing our sequence with Helix pomatia beta c-hemocyanin unit D, we found 42.9% identical and 11.5% similar residues. One putative copper binding site (site B) was identified by homology to Helix hemocyanin and arthropodan hemocyanin. The location of a second possible site was identified.     

Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca,Cephalopoda)

Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods.     

Detection of salinity by the lobster, Homarus americanus.

Changes in the heart rates of lobsters (Homarus americanus) were used as an indicator that the animals were capable of sensing a reduction in the salinity of the ambient seawater. The typical response to a gradual (1 to 2 ppt/min) reduction in salinity consisted of a rapid increase in heart rate at a mean threshold of 26.6 +/- 0.7 ppt, followed by a reduction in heart rate when the salinity reached 22.1 +/- 0.5 ppt. Animals with lesioned cardioregulatory nerves did not exhibit a cardiac response to changes in salinity. A cardiac response was elicited from lobsters exposed to isotonic chloride-free salines but not to isotonic sodium-, magnesium- or calcium-free salines. There was little change in the blood osmolarity of lobsters when bradycardia occurred, suggesting that the receptors involved are external. Furthermore, lobsters without antennae, antennules, or legs showed typical cardiac responses to low salinity, indicating the receptors are not located in these areas. Lobsters exposed to reductions in the salinity of the ambient seawater while both branchial chambers were perfused with full-strength seawater did not display a cardiac response until the external salinity reached 21.6 +/- 1.8 ppt. In contrast, when their branchial chambers were exposed to reductions in salinity while the external salinity was maintained at normal levels, changes in heart rate were rapidly elicited in response to very small reductions in salinity (down to 29.5 +/- 0.9 ppt in the branchial chamber and 31.5 +/- 0.3 ppt externally). We conclude that the primary receptors responsible for detecting reductions in salinity in H. americanus are located within or near the branchial chambers and are primarily sensitive to chloride ions.     

Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca, Cephalopoda)

Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods.     

Gill microcirculation of the air-breathing climbing perch, Anabas testudineus (Bloch):relationships with the accessory respiratory organs and systemic circulation

The general macrocirculation and branchial microcirculation of the air-breathing climbing perch, Anabas testudineus, was examined by light and scanning electron microscopy of vascular corrosion replicas. The ventral aorta arises from the heart as a short vessel that immediately bifurcates into a dorsal and a ventral branch. The ventral branch distributes blood to gill arches 1 and 2, the dorsal branch to arches 3 and 4. The vascular organization of arches 1 and 2 is similar to that described for aquatic breathing teleosts. The respiratory lamellae are well developed but lack a continuous inner marginal channel. The filaments contain an extensive nutritive and interlamellar network; the latter traverses the filament between, but in register with, the inner lamellar margins. Numerous small, tortuous vessels arise from the efferent filamental and branchial arteries and anastomose with each other to form the nutrient supply for the filament, adductor muscles, and arch supportive tissues. The efferent branchial arteries of arches 1 and 2 supply the accessory air-breathing organs. Arches 3 and 4 are modified to serve primarily as large-bore shunts between the dorsal branch of the ventral aorta and the dorsal aorta. In many filaments from arches 3 and 4, the respiratory lamellae are condensed and have only 1-3 large channels. In some instances in arch 4, shunt vessels arise from the afferent branchial artery and connect directly with the efferent filamental artery. The filamental nutrient and interlamellar systems are poorly developed or absent. The respiratory and systemic pathways in Anabas are arranged in parallel. Blood flows from the ventral branch of the ventral aorta, through gill arches 1 and 2, into the accessory respiratory organs, and then returns to the heart. Blood, after entering the dorsal branch of the ventral aorta, passes through gill arches 3 and 4 and proceeds to the systemic circulation. This arrangement optimizes oxygen delivery to the tissues and minimizes intravascular pressure in the branchial and air-breathing organs. The efficiency of this system is limited by the mixing of respiratory and systemic venous blood at the heart.     

Oxygen uptake and the potentiating effects of increased hemolymph lactate on oxygen transport during exercise in the blue crab,Callinectes sapidus

Summary At the onset of moderate swimming activity,Callinectes sapidus rapidly increased branchial ventilation, heart rate, and oxygen uptake, reaching steady state values in 2–3 min, with a half-time of 30 sec. Although O₂ extraction efficiency decreased slightly (50% to 43%) upon reaching steady state, O₂ uptake was increased 2.6 fold over resting (routine) levels. HemolymphP _{O 2} did not change during sustained (30–60 min) exercise, but a marked decrease in pH (7.60 to 7.10), associated with a 14-fold increase in hemolymph lactate concentration, caused decreases in both pre-and postbranchial O₂ content due to a large hemocyanin Bohr shift. The effect of the Bohr shift on O₂ binding, however, was minimized by an increase in hemocyanin O₂ affinity induced by lactate ions; the influence of lactate on hemocyaninP ₅₀ was shown to be the same in vivo and in vitro. As a result of the interaction between the Bohr and lactate effects, only slight increases were observed in the a-v O₂ difference (13%) and the quantitative role of hemocyanin in oxygen transport (11%) during exercise. The increase in O₂ delivery was therefore attributed primarily to a 2.3 fold increase in cardiac output (Fick estimate), resulting from increases in both heart rate (1.61 X) and stroke volume (1.42X). During exercise hemocyanin remained 21% oxygenated upon leaving the tissues, thus maintaining a substantial venous O₂ reserve which could be utilized to fuel more strenuous levels of exercise at least partly by aerobic pathways. The high hemolymph lactate levels, however, indicate that anaerobic metabolism makes a significant contribution to energy production even during moderate exercise. These results are similar to the respiratory and circulatory responses reported for other decapod crustaceans and fish during mild exercise.C. sapidus, however, appears to be highly resistant to fatigue, which correlates with its welldeveloped locomotor capabilities.