Identification of a Vibrio strain producing antimicrobial agents in the excretory organs of Nautilus pompilius (Cephalopoda: Nautiloidea)

The aim of the present study was to identify and characterize bacteria producing antimicrobial compounds in the excretory organs of Nautilus pompilius. Culture-dependent and culture-independent complementary approaches were used for bacterial identification such as: culture on selective media, Gram staining, CARD-FISH, direct DNA extraction from host tissue, PCR amplification and sequencing of the bacterial 16S rRNA gene. Results show presence of three bacterial groups: γ-Proteobacteria with three clusters (Pseudomonadales, Vibrionales, Alteromonadales), β-Proteobacteria and spirochetes. In order to screen for active strains, antimicrobial activity was tested by diffusion agar assay against Micrococcus luteus, Escherichia coli, Vibrio harveyi, and Candida albicans. Nautilus isolates showed antimicrobial activities against both Gram-positive and Gram-negative reference strains. Most of the active strains were phylogenetically related to environmental Vibrionaceae. These strains were always abundant in N. pompilius PA but were absent from Nautilus macromphalus from other geographical areas. Therefore, we suggest that antimicrobial active Vibrionaceae infect N. pompilius by environmental transmission.     

The fine structure of the branchial heart appendage of the cephalopod Octopus dofleini martini

Summary The branchial heart appendage of Octopus dofleini martini has been investigated electron microscopically. This organ is dominated by peripherally lobed blood sinuses. It contains free hemocyanin (often aligned in rows), amoebocytes, endothelial cells, and muscle cells which occur mainly in connection with neurons. The neurons are often exposed to the blood. The blood sinuses are enclosed by a basement membrane which contains collagen equivalents and fine fibrillar elements. The sinuses are covered by two different epithelia: 1) the epithelium in the caoity of the appendage consisting of irregularly shaped cells with processes, the so called ( 30 high) podocytes, and 2) the epithelium ( 40 in height) on the surface of the organ, which is composed of two parts: a) a lacuna-forming portion directly adjacent to the basement membrane, which is topped by b) a continuous tissue portion with occasional lacuna-canals. The intercellular spaces of the inner and outer epithelium are connected. The structures of these epithelial cells are discussed in relation to the formation of the pericardial fluid.Our thanks are given to Professor Dr. Georg Kümmel, Freie Universität Berlin, for suggesting the theme and his scientific guidance; to Dr. Kenneth M. Towe, Smithsonian Institution, Washington D.C., for generously allowing the use of his instruments and for his technical assistance; to Mr. Frank Denys, Medical Dental School of Georgetown University, Washington D.C., for sharing with me his technical skills and for making possible the occasional use of a dark room; and finally to Dr. Fred E. Witmer, Office of Saline Water, U.S. Dept. of Interior, Washington D.C., for his help with the translation, and for taking all the side effects of this study as patiently as he did.Supported in part by Grant number GB 17539 of the National Science Foundation. Used as part of a thesis submitted to the Freie Universität Berlin.     

Cytology of the renal appendages of Nautilus (Cephalopoda,Tetrabranchiata)

Summary The renal appendages of tetrabranchiate cephalopods, Nautilus pompilius (L.) and Nautilus macromphalus (G.B. Sow.), were studied using light- and electron-microscopical methods. The appendages, homologous to the renal appendages of dibranchiate cephalopods, possess in both species a folded transporting epithelium characterized by a secreting brush border and a high content of mitochondria and lysosome-like dense bodies as well as a basal labyrinth, and are separated from the blood lacunae by a multilayered lamina basalis.In the extracellular crypt-like infoldings of the epithelium, numerous concentric stratified concrements are found.The results strengthen the hypothesis that these concrements represent mineral-storage structures only found in calcium shell-supporting species within the class Cephalopoda.This study was supported by the Deutsche Forschungsgemeinschaft     

The ultrastructure of the lens of the cephalopod Sepiola: A scanning electron microscopic study

The ultrastructure of the eye lens of Sepiola atlantica was investigated using scanning electron microscopy. The main lens elements in both the anterior and posterior half of the Sepiola lens are plate-like configurations with fiber-like extensions at their margins. Anteriorly the plates are plano-convex, posteriorly subspherical. The central, primordial, posterior plates are spherical with no marginal extensions. The plates are mutually anchored by protrusions and invaginations and by push-button attachments. The posterior and anterior halves are separated by a septum which consists of concentric zones of radially orientated elongated cells. The marginal extensions of the plates and the septal elements are closely associated. The unique structure of the septum makes it a good candidate for the high resistance barrier between the posterior and anterior halves of the Sepiola lens (Jacob and Duncan, 1981).     

The structure of some cephalopod statoliths

Summary The statoliths of Sepia officinalis, Octopus vulgaris, Alloteuthis subulata and Taonius megalops have a smooth outline, but an irregular shape. They have projections and indentations. The statoliths from a pair of statocysts are usually quite similar in size and shape, and the general pattern is probably maintained throughout the size range of the species. Statoliths from large animals are marginally larger than those from smaller ones. The statolith usually occupies only a small part of the cavity of the statocyst, and it is situated in the anterior part of the statocyst. They are joined to the macula by hairs extending from it. These hairs are very delicate and easily broken during preparation of the specimens. The hairs are much longer and narrower than the receptor cilia of the macula. The receptor cilia are enclosed within holes in the tangled hairlike anchoring fibrils.The statolith is made up of crystalline subunits, the statoconia. The crystals vary in size, they are usually elongated, hexagonal with pointed ends. The statolith consists of a closely packed mass of these crystals, sometimes they are irregularly arranged, where in others they are stacked with their long axes parallel. In Sepia officinalis and Taonius megalops, the crystals are arranged in regular shaped packets and these packets of crystals are stacked together. These larger subunits are not always arranged in a regular way, and their major axes can be organised in several different ways. The size and outline of these large subunits do vary in different parts of the statolith.The external surface of the statolith is macroscopically smooth. Over some parts there is a surface layer covering the rod-like crystals that make up the major bulk of the stone. In other regions, the surface is rough at a microscopic level, the roughness is produced by the exposed ends of the filamentous crystals. The crystals are composed of calcium carbonate in the form of aragonite.I wish to thank Professor J.Z. Young, FRS, for considerable help, advice and encouragement throughout this study. Dr. A. Boyde generously allowed me to use his scanning electron microscope and gave freely of his expertise and time. Dr. J. Fitch kindly gave me some fossil statoliths and Dr. J. Elliott examined them with his x-ray diffraction apparatus. Dr. Marion Nixon helped me to collect and prepare the specimens. Mrs. E. Bailey, Miss P. Stephens and Mr. R. Moss provided the expert technical assistance     

The fine structure of the perineural endothelium

Summary Fine strands of motor nerves were examined with the electron microscope using thin section as well as freeze-etching techniques. The specimens were taken from frog cutaneous pectoris nerve, rat sciatic nerve, mouse and shrew phrenic nerves and from human skin nerves. The perineural sheath (Henle, Ranvier, Key and Retzius) consists of one to several concentric laminae of endothelial cells; it encases nerve fascicles and eventually individual nerve fibers and terminals. The endothelial cells are extremely thin and fitted together smoothly by overlap and dove-tailing of their border zones. The cell contacts are formed by continuous zonulae occludentes, often reinforced by maculae adhaerentes, and in depth they comprise 3–15 strands with an average of 5–6 strands per junction. The membranes of endothelial cells are studded with attachment sites and stomata of plasmalemmal vesicles suggesting a high level of pinocytotic activity. This phenomenon is by no means restricted to the external laminae of the endothelial sheath. Each endothelial lamina is vested with basement membranes on both (epineural and endoneural) sides, and the spaces between laminae contain a few collagen fibers and fibroblasts. Occasionally, punctate tight junctions are seen between laminae. Cytological evidence supports the hypothesis that the perineural endothelium provides a relatively tight and highly selective barrier separating the peripheral nerves from surrounding tissue and its extracellular fluid spaces. This effect is achieved on the one hand by the sealing of pericellular spaces and on the other hand by a membrane controlled transcellular transport mechanism (pinocytosis), both of which are enhanced by their serial arrangement.Dedicated to Professor Wolfgang Bargmann, Kiel, on the occasion of his 70th birthday.The technical assistance of Dr. F. Dreyer, Mr. D. Savini, Miss H. Claassen and Miss R. Emch is gratefully acknowledged.Financial support was received by the following institutions: Swiss National Foundation for Scientific Research, grants Nrs. 3.368.0.74, 3.774.72, 3.259.74, 3.045.73. Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 38, Projekt N). The Dr. Eric Slack-Gyr Stiftung in Zürich and the Hartmann-Müller Stiftung for Medical Research in Zürich.     

Genetic diversity of isolated populations of Nautilus pompilius (Mollusca, Cephalopoda) in the Great Barrier Reef and Coral Sea

Nautilus species are the only remaining cephalopods with an external shell. Targeted heavily by the shell trade across their distribution area, these species have a poorly known population structure and genetics. Molecular techniques have been used to assess levels of inter- and intra-population genetic diversity in isolated populations of Nautilus in the northern sections of the Great Barrier Reef (GBR), Australia and in the Coral Sea. Distinct populations, physically separated by depths in excess of 1,000 m were examined. RAPD analysis of genetic differences showed limited differentiation of the “Northern GBR” populations and the “Coral Sea” populations. Discrimination between the two geographic groups was observed from these data. In addition, partial sequencing of the CoxI gene region, yielded 575 bp of sequence, which was aligned for 43 samples and phylogenetic trees constructed to examine genetic relationships. Two distinct clades were resolved in the resulting trees, representing the “Northern GBR” and “Coral Sea” population groups. Inter- and intra-population relationships are presented and discussed. The differentiation of the Nautilus populations from the Northern section of the Great Barrier Reef and those from the Coral Sea were supported by two distinctly different methodologies and the significance of this separation and the potential evolutionary divergence of these two population groups is discussed.     

The determination of sense organs in Drosophila: interaction of scute with daughterless

Summary Several genetic loci have been implicated in the formation of the peripheral nervous system during Drosophila embryogenesis. As a first step towards understanding the functional interrelationships between these genes, we have searched for dominant interactions between deficiencies for the achaete-scute complex (AS-C), daughterless (da) and six other regions necessary for peripheral neurogenesis in the embryo. We have found that adult flies doubly heterozygous for deletions of AS-C and of da, or of AS-C and a small region on the fourth chromosome, exhibit characteristic bristle defects, suggesting that these genes cooperate to form sense organs both in the embryo and in the adult.     

The fine structure of the type 2 cells in the Malpighian tubules of the stick insect,Carausius morosus

Summary Type 2 cells, or mucocytes, are present in both inferior and superior Malpighian tubules ofCarausius morosus and are concentrated towards the distal ends of the main urine-secreting parts. They are absent from the proximal few millimetres of the main part and from the distal specialised regions of the tubules. They possess numerous Golgi bodies and abundant granular E. R., which is consistent with the hypothesis that they secrete mucus. However, they possess basal infoldings and apical microvilli suggesting that they may transport substances across the tubule wall. It is suggested that they perform both functions. Reabsorption of ions or water could precipitate solid components of the urine (e.g., uric acid). Mucus may be important in nucleation of crystalline material and also prevent abrasion of the brush border.This investigation formed part of a thesis for the degree of Ph.D. in the University of Newcastle upon Tyne. It is a pleasure to thank Professor J. Shaw for his advice and encouragement and the Science Research Council for financial support.     

The fine structure of cephalopod blood vessels

Summary The fine structure of blood vessels of the retina and arms of Octopus and the lip of Sepia is described.There are two main types of vessels. The first type (type 1) has a complete basement membrane, an incomplete lining of endothelial cells formed into finger—like processes, and a complete investment of pericytes surrounding the vessel. These latter cells contain myofilaments. The second type (type 2) is smaller and contains few if any myofilaments, and has a less complexly folded endothelium. This type is subdivided into three forms depending on the number of pericytes and the form of the endothelial lining.Amoebocytes are described and these form a distinct group of cells.The fine structure of hemocyanin is observed in normally fixed material and is correlated with its previously described structure.These observations are related to their possible functional importance.

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Zusammenfassung Die Ultrastruktur von Blutgefäßen der Retina und der Arme von Octopus und der Lippe von Sepia wird beschrieben.Es bestehen vorwiegend zwei Gefäßtypen. Der erste Typ (Typ 1) zeigt eine geschlossene Basalmembran, eine unvollständige Begrenzung durch Endothelzellen, die fingerförmige Fortsätze bilden und eine vollständige Pericytenhülle um das Gefäß. Die letztere enthält Myofilamente. Der zweite Typ (Typ 2) ist kleiner und enthält wenig oder keine Myofilamente. Er besitzt ein weniger komplex gefaltetes Endothel. Dieser Typ wird gemäß der Zahl der Pericyten und der Form der endothelialen Begrenzung in drei Gruppen unterteilt.Deutlich verschiedene Amoebozyten werden beschrieben.Die Ultrastruktur von Haemocyanin ist an normal fixiertem Material zu beobachten. Sie wird zu ihrer in früheren Arbeiten beschriebenen Struktur in Beziehung gebracht.Die Beobachtungen werden in Hinblick auf ihre mögliche funktionelle Bedeutung diskutiert.

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Acknowledgements. We would like to acknowledge the encouragement and advice of Professor J. Z. Young and Dr. E. G. Gray. Mrs. J. I. Astafiev did the drawings and Mr. A. Aldrich and Mr. S. Waterman helped with the photography.     

Sperm nucleomorphogenesis in the cephalopod Sepia officinalis

Sperm nucleomorphogenesis in the cephalopod Sepia officinalis is the product of the interaction between perinuclear microtubules and condensing chromatin. This interaction occurs during spermiogenesis and is established through the nuclear membrane. As in other cephalopod species, the perinuclear microtubules are transient structures. In the case of S. officinalis, they begin to appear in the basal area of the early spermatid and progress from there, establishing contact with the external nuclear membrane and follow a defined, but not symmetric, geometry. Thus, the microtubules accumulate preferentially in one area of the nuclear membrane which we refer to here as the "dorsal zone". Later, the microtubules will be eliminated before the mature spermatid migrates to the epidydimis. The chromatin is condensed within the nucleus following a complex pattern, beginning as fibro-granular structures until forming fibres of approximately 45 nm diameter (patterning phases). From this stage on, an increase in the chemical basicity of DNA-interacting proteins is produced, and chromatin fibres coalesce together, being recruited to the dorsal zone of the membrane, where there is a higher density of microtubules. This last step (condensation phases) allows the chromatin fibres to be arranged parallel to the axis of the elongating nucleus, and more importantly, is deduced to cause a lateral compression of the nucleus. This lateral compression is in fact a recruitment of the ventral zone toward the dorsal zone, which brings about an important reduction in nuclear volume. The detailed observations which comprise this work complement previous studies of spermiogenesis of Sepia and other cephalopods, and will help to better understand the process of cellular morphology implicated in the evolution of sperm nuclear shape in this taxonomic group.     

The fine structure and photosynthetic cost of structural leaf variegation

The leaves of some plants display an optical patchiness on their upper side, displaying light- and dark-green areas with high and low reflectance, respectively. In this investigation, we studied the fine structure of the corresponding sectors and we asked whether the lost reflected light entails a photosynthetic cost to these leaves. Four species, i.e. Arum italicum, Ranunculus ficaria, Cyclamen hederifolium and Cyclamen persicum were investigated. Scanning electron microscope examination revealed that epidermal cells of light-green sectors of all species are more bulgy than corresponding cells of neighboring dark-green leaf sectors. The comparative anatomical study revealed that (i) epidermis thickness of the light-green areas and the number of mesophyll cell layers does not differ from those of the adjacent dark-green leaf sectors and (ii) palisade cells of light-green sectors are slightly larger and more loosely arranged, allowing a much higher percentage of intercellular air spaces. The latter histological feature seems to provide the structural basis for the different optical properties between the two leaf sectors. Contrary to expectations, net photosynthetic rates (expressed on a leaf area basis) were similar in the light-green and the dark-green areas of the two cyclamen species. Yet, in C. persicum net photosynthesis was higher in the light-green areas, if expressed on a dry mass basis. The small size of the light-green spots in the rest of the test plants precluded CO₂ assimilation measurements, yet maximum linear photosynthetic electron transport rates displayed no differences between the two sectors in all plants. Thus, the assumption of a photosynthetic cost in the light-green areas was not confirmed. On the contrary, a higher construction cost was evident in the dark-green areas of three species, displaying a significantly higher specific leaf mass, without any photosynthetic benefit. The results on net photosynthesis were compatible with leaf optical properties and pigment levels. Thus, in spite of the considerably higher reflectance of the light-green areas and their lower (yet normal for a green leaf) chlorophyll levels, corresponding differences in absorptance were slight. In addition, dry mass-based pigment contents in dark-green areas were higher, while chlorophyll a/b (in two species) and carotenoid/chlorophyll ratios (in three species) were lower, pointing to a shade adaptation in these sectors. We conclude that in variegated leaves of this kind, dark-green areas are more costly to build and probably less photosynthetically active. We argue that the high pigment contents of dark-green areas establish steep light gradients in the corresponding mesophyll, rendering deeper chloroplast layers more shade adapted.     

Peripheral and central nervous responses evoked by small water movements in a cephalopod

Potentials were recorded from the epidermal head lines and from the CNS of young cuttlefish, Sepia officinalis, in response to weak water movements. 1. Within the test range 0.5-400 Hz a sinusoidal water movement elicits up to 4 components of response if the electrode is placed on a headline: (i) a positive phasic ON response; (ii) a tonic frequency-following microphonic response; (iii) a slow negative OFF response; and (iv) compound nerve impulses. 2. The amplitude of both the ON wave and the microphonic potential depends on stimulus frequency, stimulus amplitude and stimulus rise time. Frequencies around 100 Hz and short rise times are most effective in eliciting strong potentials. The minimal threshold was 0.06 microns peak-to-peak water displacement at 100 Hz (18.8 microns/s as velocity). 3. Change of direction of tangential sphere movement (parallel vs. across the head lines) has only a small effect on the microphonic and the summed nerve potentials. 4. Frequency and/or amplitude modulations of a carrier stimulus elicit responses at the onset and offset of the modulation and marked changes in the tonic microphonic response. 5. Evoked potentials can be recorded from the brain while stimulating the epidermal lines with weak water movements. The brain potentials differ in several aspects from the potentials of the head lines and show little or no onset or offset wave at the transitions of a frequency and amplitude modulation.     

The fine structure of the compound sense organs on the cirri of Nereis diversicolor

Summary A study of the fine structure of the sense organs on the prostomial cirri and palps of Nereis diversicolor shows them to consist of two types of cell. There are between 7 and 15 sensory cells and a similar number of associated cells which contain many osmiophilic granules. The cell bodies of both are sub-epidermal, having a long distal process which reaches the surface in a raised sensory hillock. The sensory cells carry a cilium, which passes through the cuticle and emerges surrounded by a sheath formed from the outer layers of the epicuticle. Scanning electron micrographs show the surface of the cirrus to be covered by hair-like epicuticular microvilli, through which the sheathed cilia protrude. There is also a second type of sensory cell which occurs singly between the epithelial cells. The distal membrane of this cell is formed into a tuft of approximately 55 large microvilli which open through a pore in the epicuticle. It is suggested by their position and structure, that both these receptors resemble chemoreceptors.We should like to acknowledge the advice and technical help of Dr. J. A. Nott of the N.E.R.C. unit of Electron Microscopy, Menai Bridge, and Dr. P. E. Secker of the School of Electronic Engineering for use of the Cambridge Stereoscan. The work is supported by a grant from the Science Research Council to D.A.D.     

The development of the lymphoid organs of flounder, Paralichthys olivaceus, from hatching to 13 months

The growth of the lymphoid organs, such as head kidney, spleen and thymus were studied in flounder, Paralichthys olivaceus Temminck & Schlegel, from hatching to 13 months of age. Except for the thymus, all organs grew as the fish grew. By 2 months of age the lymphoid organs attained their maximum relative weight. The organ weight showed a closer correlation to body weight than they did to age. The total number of leucocytes in the lymphoid organs increased with age, but the number per milligram of lymphoid organ remained constant. A micro and ultrastructural study of the lymphoid organs showed that the full development of the lymphoid organs was not achieved until the juvenile stage. The spleen and head kidney had mixed populations of "red" and "white" cells. The head kidney was more lymphoid than the spleen. The thymus involuted quickly during the first 6 months. The blood components had no obvious relationship with age or season during the period studied.     

Host selection by ovipositing cowpea weevils: patterning of input from separate sense organs

Ablations were performed to identify the sense organs used in host selection by ovipositing cowpea weevils, Callosobruchus maculatus. Antennae, foretarsi and palpi (maxillary + labial) were removed singly or in combination, and females were offered pairwise choices of four host species. Removal of the palpi consistently had the greatest effect on host choice, whereas the relative importance of other organs depended on the pair of hosts tested. Different organs may provide conflicting input as to the preferred host; certain ablations led to a complete reversal in preference (as opposed to a simple loss of discrimination). Input from separate organs appeared to be received in a dominance hierarchy.

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Sélection de l'hôte lors de la ponte de Callosobruchus maculatus: modélisation des influx provenant séparément des divers organes sensoriels

Résumé Les ablations ont servi d'étape initiale lors de la détermination des organes sensoriels impliqués dans la sélection du lieu de ponte par C. maculatus. Les antennes, les tarses antérieurs, les palpes (maxillaires et labiaux) ont été retirés seuls ou en combinaison. Les femelles ont eu des choix binaires entre les graines de 4 légumineuses: Cicer arietinum, Glycine max, Phaseolus vulgaris, Vigna unguiculata. Chaque combinaison d'ablations était accompagnée d'un témoin chez lequel les mêmes ablations étaient unilatérales. C'est l'ablation des palpes qui a le plus modifié le choix de l'hôte; l'influence relative des autres organes dépendait des paires d'hôtes offerts. Différents organes peuvent fournir des influx opposés quant à l'hôte préféré certaines ablations ont conduit à une inversion complète des préférences. Il semble que les influx sensoriels formaient une gamma avec hiérarchie dominante. Dans les choix binaires, les préférences initiales des femelles intactes ont été supprimées avec l'ablation des palpes seuls, cependant elles n'avaient pas été modifiées par l'ablation conjointe des palpes et des tarses. Dans ces expériences, l'influx antennaire ne devenait dominant qu'après ablation des deux autres organes sensoriels. Les préférences des femelles avec ablations unilatérales ne différaient pas de celles des femelles intactes.

     

The larval eye of the aeolid nudibranch Trinchesia aurantia (Alder and Hancock)

Summary The larval eye of the aeolid nudibranch Trinchesia aurantia has been investigated at three different stages; in all, the eyes remain closely attached to, and in cellular contact with, the central ganglia. The larval eye is a simplified version of the adult eye in that, the eye and the constituent cells, nuclei, lens, microvilli and pigment granules are all smaller, and the interdigitation between the retinal cells is not developed. The absence of the small cells of the cornea and of the spherical vesicles in the cytoplasm of the sensory cells, is further evidence of the incomplete formation of the eye. The possible origin of the eye of Trinchesia is discussed and compared with that of other gastropods.I am very grateful for the help and guidance of my supervisor Dr. D. A. Dorsett throughout the preparation of this paper. I was sponsored by a grant from the N.E.R.C.     

The fine structure of ascospore shape and development inCeratocystis fimbriata

Ascospore development inCeratocystis fimbriata Ell. & Halst. commenced in an eight-nucleate ascus. A single vesicle formed along the periphery of the ascus from fragments of ascospore delimiting membranes, surrounded all eight nuclei and eventually invaginated, first forming pouches with open ends, then finally enclosing each of the eight nuclei in a separate sac, thus delimiting ascospores. Pairing of the ascospores followed and brim formation occurred at the contact area between two ascospores. Osmiophilic bodies contributed to the formation of brim-like appendages by fusing to the ascospore walls. Additional brims were observed at opposite ends of the ascospores giving them a double-brimmed appearance.Abbreviations AV ascus vesicle - DM delimiting membrane - EV electron translucent bodies - G granules - M mitochondria - N nucleus - OB osmiophilic bodies - PMV plasmamembrane vesicles - PW primary wall - SW secondary wall