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.     

Nautilus at risk--estimating population size and demography of Nautilus pompilius

The low fecundity, late maturity, long gestation and long life span of Nautilus suggest that this species is vulnerable to over-exploitation. Demand from the ornamental shell trade has contributed to their rapid decline in localized populations. More data from wild populations are needed to design management plans which ensure Nautilus persistence. We used a variety of techniques including capture-mark-recapture, baited remote underwater video systems, ultrasonic telemetry and remotely operated vehicles to estimate population size, growth rates, distribution and demographic characteristics of an unexploited Nautilus pompilius population at Osprey Reef (Coral Sea, Australia). We estimated a small and dispersed population of between 844 and 4467 individuals (14.6-77.4 km(-2)) dominated by males (83:17 male:female) and comprised of few juveniles (<10%).These results provide the first Nautilid population and density estimates which are essential elements for long-term management of populations via sustainable catch models. Results from baited remote underwater video systems provide confidence for their more widespread use to assess efficiently the size and density of exploited and unexploited Nautilus populations worldwide.     

Vertical distribution and migration patterns of Nautilus pompilius

Vertical depth migrations into shallower waters at night by the chambered cephalopod Nautilus were first hypothesized early in the early 20(th) Century. Subsequent studies have supported the hypothesis that Nautilus spend daytime hours at depth and only ascend to around 200 m at night. Here we challenge this idea of a universal Nautilus behavior. Ultrasonic telemetry techniques were employed to track eleven specimens of Nautilus pompilius for variable times ranging from one to 78 days at Osprey Reef, Coral Sea, Australia. To supplement these observations, six remotely operated vehicle (ROV) dives were conducted at the same location to provide 29 hours of observations from 100 to 800 meter depths which sighted an additional 48 individuals, including five juveniles, all deeper than 489 m. The resulting data suggest virtually continuous, nightly movement between depths of 130 to 700 m, with daytime behavior split between either virtual stasis in the relatively shallow 160-225 m depths or active foraging in depths between 489 to 700 m. The findings also extend the known habitable depth range of Nautilus to 700 m, demonstrate juvenile distribution within the same habitat as adults and document daytime feeding behavior. These data support a hypothesis that, contrary to previously observed diurnal patterns of shallower at night than day, more complex vertical movement patterns may exist in at least this, and perhaps all other Nautilus populations. These are most likely dictated by optimal feeding substrate, avoidance of daytime visual predators, requirements for resting periods at 200 m to regain neutral buoyancy, upper temperature limits of around 25°C and implosion depths of 800 m. The slope, terrain and biological community of the various geographically separated Nautilus populations may provide different permutations and combinations of the above factors resulting in preferred vertical movement strategies most suited for each population.     

Morphology and histology of the digestive tract of Nautilus pompilius and Nautilus macromphalus (Cephalopoda, Tetrabranchiata)

 This study presents histological and scanning electron microscopical findings on the structural differentiation, and the nervous and vascular supply of the digestive tracts of Nautilus pompilius and N. macromphalus, including the foregut, stomach, vestibulum, caecum, midgut and rectum. The stereoscopic reconstruction of the vestibulocaecal complex gives an idea how the digestive cycle between the stomach, vestibulum, caecum and proximal midgut could possibly proceed. All parts of the digestive tract are covered luminally by a columnar epithelium which contains numerous goblet cells. The epithelium is ciliated in the vestibulum, caecum, proximal midgut and the longitudinal groove of the rectum. On this lamina epithelialis mucosae borders the lamina propria mucosae, which consists of connective tissue and some muscle cells. In the stomach it is differentiated, forming a special bolster-like layer. The lamina propria mucosae is followed by the tunica muscularis, which consists of a stratum circulare and a stratum longitudinale in the foregut, vestibulum, caecum, midgut and rectum. In the stomach, midgut and rectum, the tunica adventitia, which consists of a thin layer of connective tissue, is located between the tunica muscularis and the cuboidal tunica serosa. Accepted: 4 August 1997     

Effects of ambient PO2 and temperature on oxygen uptake in Nautilus pompilius

This study employs closed-circuit respirometry to evaluate the effect of declining ambient oxygen partial pressure (PO₂) and temperature on mass specific rates of oxygen uptake (V˙O₂) in Nautilus pompilius. At all temperatures investigated (11, 16, and 21 °C), V˙O₂ is relatively constant at high PO₂ (oxyregulation) but declines sharply at low PO₂ (oxyconformation). The critical PO₂ below which oxyconformation begins (P _c) is temperature dependent, higher at 21 °C (49 mmHg) than at 11 °C or 16 °C (21.7 mmHg and 30.8 mmHg respectively). In resting, post-absorptive animals, steady-state resting V˙O₂ increases significantly with temperature resulting in a Q₁₀ value of approximately 2.5. The metabolic strategy of N. pompilius appears well suited to its lifestyle, providing sufficient metabolic scope for its extensive daily vertical migrations, but allowing for metabolic suppression when PO₂ falls too low. The combination of low temperatures and low PO₂ may suppress metabolic rate 16-fold (assuming negligible contributions from anaerobic metabolism and internal O₂ stores), enhancing hypoxia tolerance. Accepted: 20 January 2000     

Old and sticky-adhesive mechanisms in the living fossil Nautilus pompilius (Mollusca, Cephalopoda)

Nautiloidea is the oldest group within the cephalopoda, and modern Nautilus differs much in its outer morphology from all other recent species; its external shell and pinhole camera eye are the most prominent distinguishing characters. A further unique feature of Nautilus within the cephalopods is the lack of suckers or hooks on the tentacles. Instead, the animals use adhesive structures present on the digital tentacles. Earlier studies focused on the general tentacle morphology and put little attention on the adhesive gland system. Our results show that the epithelial parts on the oral adhesive ridge contain three secretory cell types (columnar, goblet, and cell type 1) that differ in shape and granule size. In the non-adhesive aboral epithelium, two glandular cell types (cell types 2 and 3) are present; these were not mentioned in any earlier study and differ from the cells in the adhesive area. The secretory material of all glandular cell types consists mainly of neutral mucopolysaccharide units, whereas one cell type in the non-adhesive epithelium also reacts positive for acidic mucopolysaccharides. The present data indicate that the glue in Nautilus consists mainly of neutral mucopolysaccharides. The glue seems to be a viscous carbohydrate gel, as known from another cephalopod species. De-attachment is apparently effectuated mechanically, i.e., by muscle contraction of the adhesive ridges and tentacle retraction.     

Environmental hypoxia does not constrain the diurnal depth distribution of free-swimming Nautilus pompilius

The behaviour of Nautilus pompilius swimming freely in a controlled mesocosm (tower tank, 4 m diameter x 10.5 m deep) was monitored using ultrasonic depth telemetry. Initially depths were monitored in water equilibrated with air. Then the bottom 3.5 m were rendered hypoxic (Po(2) <20 mmHg) and depths monitored again. A thermocline at 7-m depth (17 degrees C below, 20 degrees C above) prevented mixing with the top, normoxic water. Mean depth was significantly greater during the light phase (8.9 m) of the 12L : 12D photoperiod than the dark phase (5.6 m), but this was not affected by hypoxia. During the light phase animals preferred the bottom 2.5 m of the tank but showed no specific preference for any depth range during the dark phase. Hypoxia did not alter these patterns of depth preference, though one animal made regular excursions toward normoxic water during the light phase. Vertical swimming activity was almost twofold greater during the dark phase and was not affected by hypoxia. These data suggest that, at least over the short term, Nautilus are not constrained from entering areas with low dissolved oxygen. This hypoxia tolerance may be attributed to the large onboard oxygen stores and suppressed metabolism during hypoxia.     

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

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.     

Nautilus pompilius life history and demographics at the Osprey Reef Seamount, Coral Sea, Australia

Nautiloids are the subject of speculation as to their threatened status arising from the impacts of targeted fishing for the ornamental shell market. Life history knowledge is essential to understand the susceptibility of this group to overfishing and to the instigation of management frameworks. This study provides a comprehensive insight into the life of Nautilus in the wild. At Osprey Reef from 1998–2008, trapping for Nautilus was conducted on 354 occasions, with 2460 individuals of one species, Nautilus pompilius, captured and 247 individuals recaptured. Baited remote underwater video systems (BRUVS) were deployed on 15 occasions and six remotely operated vehicle (ROV) dives from 100–800 m were conducted to record Nautilus presence and behavior. Maturity, sex and size data were recorded, while measurements of recaptured individuals allowed estimation of growth rates to maturity, and longevity beyond maturity. We found sexual dimorphism in size at maturity (males: 131.9±SD = 2.6 mm; females: 118.9±7.5 mm shell diameter) in a population dominated by mature individuals (58%). Mean growth rates of 15 immature recaptured animals were 0.061±0.023 mm day⁻¹ resulting in an estimate of around 15.5 years to maturation. Recaptures of mature animals after five years provide evidence of a lifespan exceeding 20 years. Juvenile Nautilus pompilius feeding behavior was recorded for the first time within the same depth range (200–610 m) as adults. Our results provide strong evidence of a K-selected life history for Nautilus from a detailed study of a ‘closed’ wild population. In conjunction with population size and density estimates established for the Osprey Reef Nautilus, this work allows calculations for sustainable catch and provides mechanisms to extrapolate these findings to other extant nautiloid populations (Nautilus and Allonautilus spp.) throughout the Indo-Pacific.     

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

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.     

Shell growth and chamber formation of aquarium-reared Nautilus pompilius (Mollusca, Cephalopoda) by X-ray analysis

Observations on the growth rate of aquarium maintained Nautilus pompilius in different developmental stages, i.e. juveniles (shell length about 8.75 cm), late juveniles (approximately 10 cm), and early adolescent (approximately 13.5 cm), indicate that this species is fully grown at an age of 7.3-8 years. The age calculations are based on two different computations: (1) the measurement of the increase of the shell length per day and (2) the formation of new septa in time intervals of 150+/-5 days, as demonstrated by X-ray analyses. After N. pompilius hatches, its shell grows about 139 mm to reach full growth and approximately 28 septa are formed. With an increase of the shell length of 0.052 mm per day, it takes about 2,673 days (7.3 years) to reach maturity. Provided that the process of chamber formation follows an exponential function, these computations result in approximately 2,925 days (8 years) to reach full maturity. Supposing that N. pompilius may live for several years after onset of maturity like Nautilus belauensis, the total life span for this species may exceed 11-12 years.     

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.     

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 Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (M_r = ∼3.5 million), assembled from 10 identical copies of an ∼350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of ∼50 kDa each (FUs a–g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5′ UTR of 58 bp, a 3′ UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (M_r = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin “OdH” from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 ± 24 million years ago, in close agreement with fossil records from the early Devonian. [Reviewing Editor: Dr. Axel Meyer] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ619741.     

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 neurosecretory 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).     

Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a “pearl-chain” of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O₂ molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 Å cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 Å resolution (FSC_1/2-bit criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 Å structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.     

Arginine kinase from Nautilus pompilius, a living fossil. Site-directed mutagenesis studies on the role of amino acid residues in the Guanidino specificity region

Arginine kinases were isolated from the cephalopods Nautilus pompilius, Octopus vulgaris, and Sepioteuthis lessoniana, and the cDNA-derived amino acid sequences have been determined. Although the origin and evolution of cephalopods have long been obscure, this work provides the first molecular evidence for the phylogenetic position of Cephalopoda in molluscan evolution. A crystal structure for Limulus arginine kinase showed that four amino acid residues (Ser(63), Gly(64), Val(65), and Tyr(68)) are hydrogen-bonded with the substrate arginine. We introduced three independent mutations, Ser(63) --> Gly, Ser(63) --> Thr, and Tyr(68) --> Ser, in Nautilus arginine kinase. One of the mutants had a considerably reduced substrate affinity, accompanied by a decreased V(max). In other mutants, the activity was lost almost completely. It is known that substantial conformational changes take place upon substrate binding in arginine kinase. We hypothesize that the hydrogen bond between Asp(62) and Arg(193) stabilizes the closed, substrate-bound state. Site-directed mutagenesis studies strongly support this hypothesis. The mutant (Asp(62) --> Gly or Arg(193) --> Gly), which destabilizes the maintenance of the closed state and/or perhaps disrupts the unique topology of the catalytic pocket, showed only a very weak activity (0.6-1.5% to the wild-type).     

The siphuncle in Georginidae and other Ordovician actinoceroid cephalopods

The lower Middle Ordovician carbonate sediments of the Georgina Basin in Australia contain many and varied Actinoceratida, including an endemic family, Georginidae Wade (1977), with siphuncular calcification consisting of radial lamellae separated by spaces (now sediment-filled) enclosed within calcified annulus walls. In each segment a distinct series of more massive calcareous engrafts grows inward from the inside of the connecting ring and is engrafted into adjacent annuli across the interannulus; this divides the perispatium into longitudinal perispatial sinuses. At each extremity each perispatial sinus is connected with passages leading through the segments to the axial space and, nearer to, or at the interannulus, with radial canals. Axial canals are few, but more than one is normal. Good preservation of Armenoceras and Actinoceras allows recognition of similar structures in the annuli of normal Actinoceratida.     

A molecular and karyological approach to the taxonomy of Nautilus

Nautiloids, the externally shelled cephalopods of Cambrian origin, are the most ancient lineage among extant cephalopods. Their ancestral characters are explored based on morphological and molecular data (18S rDNA sequence) to investigate the evolution of present cephalopod lineages. Among molluscs, nautilus 18S rDNA gene is the longest reported so far, due to large nucleotidic insertions. By comparison with other 18S sequences, the complete gene of N. macromphalus helps to clarify the taxonomic status of the three universally recognised Nautilus species. The range of interspecific molecular differences supports separation of the present species into two surviving ectocochleate genera, Nautilus and Allonautilus. Nautiloid 18S is considered as corresponding to the ancestral form of 18S as is the number of chromosomes in Nautilus (52), the lowest among cephalopods. Comparison of karyological characteristics amongst cephalopods in a phylogenetic context suggests a possible correlation between duplication events and lineage divergence.     

Redundancy in response to DNA damage

Comment on: Ciznadija D, et al. Cell Cycle 2011; 10:2714-23.