Bettina Westermann

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.

Cytological and enzyme-histochemical investigations on the digestive organs of Nautilus pompilius (Cephalopoda, Tetrabranchiata)

Abstract The foregut, stomach, caecum, midgut, and rectum of the digestive tract of Nautilus pompilius L.were investigated with ultrastructural and enzyme-cytological methods. Three different cell types were identified within the lamina epithelialis mucosae: main cells, goblet cells, and cells with secretory granules. The main cell type is the epithelial cell with microvilli, a basal nucleus surrounded by dictyosomes, rough endoplasmic reticulum, mitochondria, and electron-dense granules identified as lysosomes in the apical part of the cell. In the caecum this cell type contains endosymbiotic bacteria. The presence of endocytotic vesicles and the storage of lipids in the caecum indicate that this organ is involved in the process of absorption. In the caecum and the longitudinal groove of the rectum the main cells are, in addition, ciliated, facilitating the transport of food particles and faeces. Two types of goblet cells are found in all organs except in the stomach, forming a gliding path for food particles and protecting the epithelium. In the foregut and rectum, cells with electron-dense granules were recognized as the third type. The conspicuous secretory cells of the rectum represent a delimited rectal gland; its possible biological function is discussed. The tunica muscularis in all organs of the digestive tract consists of obliquely striated muscle cells innervated by axons containing transparent, osmiophilic and dense-cored vesicles. Positive reactions for acid and alkaline phosphatase, monoamine oxidase, β-glucuronidase, and trypsin- and chymotrypsin-like enzymes are localized in the lamina epithelialis mucosae.

CYTOBIOLOGICAL STUDIES ON HEMOCYANIN METABOLISM IN THE BRANCHIAL HEART COMPLEX OF THE COMMON CUTTLEFISH SEPIA OFFICINALIS (CEPHALOPODA, DIBRANCHIATA)

Abstract 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.

Distribution and function of biogenic amines in the heart of Nautilus pompilius L. (Cephalopoda, Tetrabranchiata)

SummaryBiogenic amines (serotonin and catecholamines), play an important role in the control of the blood flow not only in vertebrates, but also in invertebrates such as cephalopods. In contrast to the well investigated hearts of the ȁ8modern,ȁ9 coleoid cephalopods, the innervation of the heart of the archaic Nautilus pompilius L. has not been studied in detail. In this study the distribution and effects of biogenic amines in the Nautilus heart were investigated. Serotonin and catecholamines were visualised by the glyxoylic acid induced fluorescence. High performance liquid chromatotography analysis was performed to discriminate between the catecholamines, which showed a high content of noradrenaline in the 4 auricles, the aorta and the ventricle, whereas the ventricle showed a high dopamine content. Adrenaline was found at a very low concentration in the ventricle. Serotonin and dopamine were also immunohistochemically localised to larger nerves and throughout the heart, respectively. In organ bath experiments, the auricles showed little spontaneous activity. After adding serotonin, they displayed rhythmical contractions, which were accelerated dose-dependently by noradrenaline. In summary, these data suggest an important role for biogenic amines in the control of the heart of Nautilus pompilius L., with serotonin possibly stimulating excitatory nerve fibres, whereas noradrenaline is likely to influence the muscle contraction itself.

Localization of putative neurotransmitters in the mantle and siphuncle of the mollusc Nautilus pompilius L. (Cephalopoda)

The neurotransmitter supply in the nerve endings of the mantle and the siphuncle, i.e. in organs that are responsible for the shell formation in the ectocholeate Nautilus pompilius, were investigated with electron microscopical, fluorescence-, immuno- and enzyme histochemical methods as well as with high pressure liquid chromatography (HPLC). Using antibodies against serotonin and the tetrapeptide FMRF-amide, positive reactions were demonstrated immunohistochemically within the terminal nerve fibres of the mantle and the vessels of the siphuncle. Enzyme histochemical proof of the presence of specific acetylcholinesterase yielded positive results in the muscle fibres of the mantle and siphuncle. Additionally, in the mantle, glyoxylic acid-induced fluorescence was shown within the nerve endings indicating catecholamines as neurotransmitters, whereas in the siphuncle such fluorescence did not appear. However, the HPLC-analyses showed that in the mantle and also in the siphuncle the content of dopamine is higher than that of noradrenaline whereas only traces of adrenaline occur in both organs suggesting dopamine as a putative neurotransmitter. Transmission electron microscopical examination of the nerve endings of both organs revealed that different types of vesicles were distinguished that could be considered as cholinergic, aminergic and peptidergic structures.

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

Abstract. 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. 125I-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.

Immunohistochemical Localization of Cardio-Active Neuropeptides in the Heart of a Living Fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata)

Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neuro-secretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).

Tracer studies of food absorption in the digestive tract of Nautilus pompilius (Cephalopoda, Tetrabranchiata)

Abstract. In Nautilus pompilius, tracer experiments with 14C-labelled food show that the midgut gland, caecum and crop are involved in absorption of nutrients. According to liquid scintillation and light- and electron-microscopic autoradiography, the midgut gland exhibits the highest activity, followed by the caecum and crop. The density of silver precipitates is highest in the terminal alveoli of the midgut gland. Precipitates are also seen in the main cells of the caecal epithelium. Few precipitates are found in the lamina epithelialis mucosae of the crop, indicating that, in addition to food storage, digestive processes begin in this organ. These results have been confirmed by injection of the protein ferritin into the buccal cavity. The largest amount of ferritin is seen in the dense bodies of the main cells of the midgut gland, whereas those of the main cells of the caecum and crop contain less ferritin.

Hemocyanin re-uptake in the renal and branchial heart appendages of the coleoid cephalopod Sepia officinalis.

Abstract. The renal and branchial heart appendages of Sepia officinalis L. were investigated in order to elucidate a possible involvement of their excretory epithelia in hemocyanin metabolism. Immunocytochemical findings and tracer experiments indicate that after passing the barrier of ultrafiltration the hemocyanin molecules are taken up by the epithelial cells of the renal and branchial heart appendages and are subsequently carried back to the circulatory system, suggesting a mechanism of hemocyanin recycling. Apart from a function in maintaining constant hemocyanin levels, the present study indicates that the renal and branchial heart appendages are also sites of temporary hemocyanin storage.

Morphological and histological organization of the pyriform appendage of the tetrabranchiate Nautilus pompilius (Cephalopoda, Mollusca)

The pyriform appendage, an organ only found in nautiloid cephalopods was investigated with histological, histochemical and ultrastructural methods in order to characterize the anatomical and the cytological structure of this organ. The pyriform appendage is situated within the genital septum and lies in close contact with the ventricle of the heart. The proximal side ends blindly near the gonad whereas the distal side is developed into a duct. The duct was observed to open into the mantle cavity in juvenile and adult Nautilus pompilius of both sexes. Injections of India ink in the heart demonstrate that the organ is supplied with hemolymph from an artery that extends from the heart. The pyriform appendage is a hollow organ consisting mainly of glandular tissue. The lumen is covered with a columnar epithelium, the tunica mucosa, consisting of only one cell type containing vacuoles with different inclusions. Underneath the tunica mucosa is the tunica muscularis, which is embedded in connective tissue and folded, enlarging the internal surface. A cuboidal tunica serosa surrounds this organ. The vacuoles and the secretory products contain neutral mucopolysaccharides, glycoproteins and glycolipids. Acid phosphatase and serotonin were localized in the tunica mucosa. Acetylcholinesterase, catecholamines and the tetrapeptide FMRF‐amide were demonstrated within the nerve endings of the tunica muscularis indicating a dual “cholinergic‐aminergic” neuroregulation, possibly modulated by FMRF‐amide. These findings suggest that the pyriform appendage is not a rudimentary organ but instead has distinct biological functions in nautiloid cephalopods, possibly in intraspecific communication. J. Morphol. 2009.