Primary co-culture as a complementary approach to explore the diversity of bacterial associations in marine invertebrates: the example of <Emphasis Type="Italic">Nautilus macromphalus</Emphasis> (Cephalopoda: Nautiloidea)

The recent application of molecular tools to address associations between bacteria and marine invertebrates has provided access to an immense diversity of unidentified microbes resistant to cultivation. However, the role of bacteria as partners in animal physiology remains unclear and in most cases difficult to investigate in the absence of adequate condition of cell growth and proliferation. In this work, we studied the reservoir of microbes associated with the excretory organs of Nautilus macromphalus as a model. Using the bacterial 16S RNA gene as a marker, we compared three complementary approaches for bacterial detection: bacterial DNA extraction from N. macromphalus tissues (“molecular approach”), strain isolation to provide a bacterial culture collection (“microbiological approach”) and finally, maintenance of N. macromphalus excretory organ cells with associated bacteria (“cellular approach”). Our results stress the potential of the “cellular approach” as a promising new tool as it promotes the detection of as yet uncultured β-proteobacteria and spirochaetes associated with N. macromphalus, and serves as a foundation for future studies describing potential roles that these bacteria may play in Nautilus.     

Laboratory rearing of Sepiolinae (Mollusca: Cephalopoda)

Five species of Sepiola and Sepictta were reared in the laboratory from egg to adult size. Spawning was achieved in 3 species of Sepiola after 5 to 7 months. The growth rate of the species reared did not depend upon temperature, which ranged from 12.5° to 20°C. A fairly constant size increase (2.5 mm mantle length/month) was observed in Sepiola during the 5 months after hatching. In Sepietta, the same growth rate was observed until the fourth month after hatching, when it increased to the rate of 5 mm mantle length/month.Laboratoire associé au C.N.R.S.     

Respiratory quotient and ammonia excretion in Tilapia mossambica

Tilapia mossambica (Peters), acclimated to and tested in fresh water at 30°C, maintained a routine respiratory quotient (R Q) of about unity and an ammonia quotient (A Q) (Vol. NH₂/Vol. O₂) of about 0.2 at high ambient oxygen concentrations. At low oxygen concentrations (below 2 ppm) R Q and A Q increased sharply to values of 8 and 1, respectively (at 0.6 ppm), indicating a close relationship of increase in anaerobic energy utilization and increase in protein metabolism at inadequate oxygen concentrations. T. mossambica (8 cm), exercised continuously with intervening sampling and flushing stops for 6 h, at a swimming speed of about 2 body lengths/sec, derived some anaerobic energy throughout the exercise (R Q: 1.2), utilizing more protein the longer the exercise. The coupling of the increased protein metabolism and anaerobic energy utilization may be of advantage in preventing acidosis and also in conserving sodium (Na⁺) in fish.Part of this work was included in a paper presented at the Centennial Meeting of The American Fisheries Society, September 13–16, 1970, New York.     

Reproduction and condition of female Octopus mimus (Mollusca: Cephalopoda)

The relationship between reproduction and condition was studied in a 15 mo sample of 919 maturing, mature and post-spawning female Octopus mimus (388 to 3714 g) caught in Iquique (North Chile). O. mimus is a semelparous species, with reproduction taking place all year round. Investment in reproductive tissues was, on average, 9.9% of mature female body weight, independent of season. However, somatic growth during maturation was dependent on season and varied between 26 and 63% of the whole-body growth in weight. The condition of females did not vary markedly until spawning, although seasonal variations were apparent, winter being the most unfavourable. Condition deteriorated dramatically after spawning, during parental care of the eggs. During this period, somatic-tissue depletion, mainly from the muscles, was >25% of the total body weight of mature females. The fecundity of O. mimus was probably limited by the costs associated with parental care of the eggs.     

Vitesse de Digestion d'Octopus cyanea (Cephalopoda: Octopoda)

In the benthic octopus Octopus cyanea Gray, acclimated to high temperature (30°C), digestion takes 12 h. Animal weight and food intake do not influence the duration of digestion. By regulating the rate of food passage in the alimentary canal, the quantity of digested food (as a percentage of ingested food) is kept constant at a particular time. A comparison with other cephalopod species and with different fish species, leads to the conclusion that the mode of life is the main factor determining the duration of digestion and its efficiency. For a given species, the duration of digestion depends on temperature. For a similar mode of life and at equal temperatures, digestion is more rapid and more efficient in cephalopods than in fishes.     

Fecundity and reproductive strategies in deep-sea incirrate octopuses (Cephalopoda: Octopoda)

Coleoid cephalopods show flexibility in their reproductive strategies or mode of spawning, which can range from simultaneous terminal spawning over a short period at the end of the animal’s life to continuous spawning over a long period of the animal’s life. Although a simultaneous terminal spawning strategy is typical of shallow water temperate octopuses, it is not known whether deep-sea octopods would have the same reproductive strategy. The reproductive strategies and fecundity were investigated in nine species of deep-sea incirrate octopuses: Bathypolypus arcticus, Bathypolypus bairdii, Bathypolypus ergasticus, Bathypolypus sponsalis, Bathypolypus valdiviae, Benthoctopus levis, Benthoctopus normani, Benthoctopus sp., and Graneledone verrucosa (total n = 85). Egg-length frequency graphs and multivariate analysis (principal components analysis) suggest that B. sponsalis has a synchronous ovulation pattern and therefore a simultaneous terminal spawning strategy. Although a simultaneous terminal spawning strategy is most likely for B. levis and B. normani, the egg-length frequency graphs and multivariate analysis also suggest a greater variation in egg-lengths which could lead to spawning over an extended period.     

Regulation of respiration during juvenile development of Sepia officinalis (Mollusca: Cephalopoda)

A culture of juvenile Sepia officinalis L. was kept during summer 1985 in the aquaria of the Station Marine, Wimereux, France. During the first four months of juvenile development, oxygen consumption under increasing hypoxia was measured with a closed respirometer. The experiments revealed a high regulatory capacity of juvenile S. officinalis. The critical oxygen concentrations were calculated and their ontogenetical evolution was studied. The critical oxygen concentration increased with increasing development. A linear relationship emerged between the critical oxygen concentration and the logarithm of the wet weight [CO_c (mg O₂ l^-1)=-0.393+0.893×log₁₀(W _w )]. The decreasing regulatory capacity of growing S. officinalis is most probably related to adaptations to a changing ecological environment during development. Another possibility is a physiological change, most probably related to the shift from embryonic to adult hemocyanin.     

Life history of Gonatus onyx (Cephalopoda: Teuthoidea): deep-sea spawning and post-spawning egg care

 A reproductive strategy consisting of deep- water spawning and egg-care was inferred for the midwater squid Gonatus onyx Young, 1972. Brooding females and associated eggs and hatchlings, captured between 1250 and 1750 m depth off southern California, are described. Brooding females appear to be senescent and lack tentacles. Large eggs of this species (3 mm) at cold temperatures (3 °C at capture depth) may require as long as 9 mo to develop. The high lipid content of the digestive gland in adult females of this species may provide fuel to support such an extended “brooding” period. Received: 22 February 1999 / Accepted: 25 May 2000     

Feeding dynamics of Octopus mimus (Mollusca: Cephalopoda) in northern Chile waters

The natural feeding of 485 Octopus mimus (164 to 3088 g) was studied in relation to the species' life cycle and environmental seasonal variations off the north of Chile from autumn 1991 to summer 1992. Analysis of digestive-tract contents revealed that O. mimus preyed upon 25 different prey items belonging to five zoological groups (Teleostei, Mollusca, Crustacea, Echinodermata and Polychaeta). Cannibalism was only occasional. The results indicate that the diet and food intake of this species are significantly affected by sex and maturation. Senescent individuals ingest a small amount of food, and their diet is mainly based on small, not very motile prey. The food intake, expressed as body weight, of non-senescent individuals is higher in females than in males. Seasonal changes in sea-water temperature seem to be followed by adjustments in food intake. Like other Octopus species, O. mimus appears to be an opportunistic predator.     

Behavioral aspects of sperm competition in cuttlefish, Sepia officinalis (Sepioidea: Cephalopoda)

Sexual selection studies in cephalopods indicate that sperm competition is a central feature of their mating systems, yet this has not been studied experimentally in any detail. In 1998 we staged 20 matings of the cuttlefish, Sepia officinalis L., in the laboratory. Males rapidly initiated mating in the “head-to-head” position, with no apparent courtship. Mating lasted an average of 10 min (range 7 to 14 min). For the first 6 min (on average 63% of the mating duration), the male flushed strong jets of water directly at the female's buccal membrane, which sometimes resulted in the expulsion of parts of spermatangia placed there in recent matings. Then, in a single discrete movement that lasted an average of only 14 s, the male's modified fourth left arm – the hectocotylus – wrapped around a single large bundle of spermatophores (ca. 150 to 300) and transferred them to the female's buccal membrane. For the remainder of the mating (average 3 min, range 1.5 to 5.0 min), the hectocotylus repeatedly broke the spermatophores open, and manipulated them, so that sperm were released and many spermatangia were attached along the ventral buccal membrane, near the paired seminal receptacles. Approximately 140 spermatangia were attached in rows 3 to 5 deep around the ventral buccal membrane in a single mating; the rest were usually discarded during mating. Histology revealed that each of the seminal receptacles consists of a series of sperm storage bulbs connected by a central duct, which leads to a single pore at the surface of the buccal membrane. Baseline data on sperm motility were obtained, but the mechanism by which sperm enter the seminal receptacle remains unknown. Females seemed to initiate termination of mating, then males guarded their mates temporarily. These results, combined with other recent laboratory experiments, provide evidence that sperm competition may be a major feature of the mating system of S. officinalis. Received: 4 January 1999 / Accepted: 18 May 1999     

Sperm motility and longevity in the giant cuttlefish, Sepia apama (Mollusca: Cephalopoda)

The sperm kinetics and fertilisation literature in marine invertebrates is heavily biased toward free-spawning species. Nonetheless, many species (e.g. cephalopods) transfer and/or fertilise gametes in confined external spaces or internally, creating very different selective pressures on sperm storage, sperm longevity and hence sperm competition. Here we report the results of an investigation into the effects of sperm age, water temperature and sperm concentration on sperm motility in the giant cuttlefish (Sepia apama). Significant positive correlations were found between percent motility and sperm concentration, and between sperm motile speed and sperm concentration. Mean percent motility of cuttlefish sperm suspension was still 9% eight hours after being released from the spermatophore and diluted into filtered seawater at 12°C (ambient field temperature during the spawning season). Sperm resuspended from spermatangia taken from (mated) females in the field were motile for up to 100 hours. When spermatophores were stored at 4°C motility was still observed in resuspended sperm after two months. Our results show that spermatangia and spermatophores can retain and release live sperm for long periods. The observed longevity of sperm in S. apama greatly increases the potential for sperm competition in this species.     

Genetic differentiation in three Sepia species (Mollusca: Cephalopoda) from Galician waters (north-west Iberian Peninsula)

The systematics of the genus Sepia is not yet clear. Morphological evidence has led to S. officinalis Linnaeus, 1758 being considered as belonging to the subgenus Sepia sensu stricto, and S. orbignyana Férussac, 1826 and S. elegans Blainville, 1827 as belonging to the subgenus Rhombosepion. Samples of 30 individuals of S. officinalis and S. orbignyana from both sides of an oceanographic boundary off the north-west Iberian Peninsula, and a sample of S. elegans from the northern side, were collected in 1993–1994. Allozyme electrophoresis for 32 presumptive loci revealed low levels of genetic variability for the three Sepia species (mean expected heterozygosity estimates were <0.052). No significant differences in allozyme frequencies were detected among populations of either S. officinalis or S. orbignyana. The genetic identities (I) of S. officinalis and S. orbignyana (I=0.12) and of S. elegans (I=0.13) were significantly different from that of S. orbignyana and S. elegans (I=0.49). The former are typical of values for confamilial genera, and a new generic status is proposed for the latter two species, which become Rhombosepion orbignyana (Férussac, 1826) and R. elegans (Blainville, 1827).     

Life history of Gonatus onyx (Cephalopoda: Teuthoidea): ontogenetic changes in habitat, behavior and physiology

In the present study 670 individuals of Gonatus onyx (Cephalopoda: Teuthoidea) were observed in Monterey Bay, California from a remotely operated vehicle. The vertical distribution of this species was bimodal, with peaks at 400 and 800 m depth during the day and 300 and 500 m during the night. The bimodal distribution reflects a life stage shift between younger, schooling juveniles living in shallower water and older, solitary adults which live deeper. Ontogenetic changes in behavior associated with this life stage shift are reflected in the physiology of the organisms as well. Both aerobic and anaerobic metabolism, as estimated using mitochondrial and glycolytic enzymes, decline with increasing body mass, suggesting reduced locomotory capacity in deeper-living adults. Oxygen consumption rates were also determined in relation to oxygen partial pressure. Oxygen consumption regulation was similar between juvenile and adult squids. The critical oxygen partial pressures (29 to 30 mmHg) correspond precisely to the oxygen concentrations found at the depth of maximal abundance for day and night populations of juveniles and adults, respectively. Behavioral and physiological changes with ontogeny of G. onyx are believed to result from reduced visual predator/prey interactions in the light-limited deep sea. Received: 7 February 1999 / Accepted: 26 January 2000     

Lipids in the digestive gland and the gonad of immature and mature Sepia officinalis (Mollusca: Cephalopoda)

The lipid content of the digestive gland and the gonad of cuttlefishes (Sepia officinalis L.) caught off the French coasts in 1979–1980 was determined and analysed by thinlayer chromatography. The total sterol and phospholipid levels were also determined. The results from immature and mature females and males were compared in an attempt to correlate variations of the lipids with sex or state of gonad maturation. The fluctuations in the lipid level of the digestive gland, which acts as a storage organ, seemed far better related to the diet than to sex or sexual maturity. In the gonads, the lipids were few and seemed to be mostly membrane constituents. However, the increase of phospholipids in the mature ovary could be related to yolk synthesis.     

Correlation of bioluminescence emissions with ventral photophores in the mesopelagic squidAbralia veranyi (Cephalopoda: Enoploteuthidae)

Specimens of the squidAbralia veranyi were collected off the Bahamas in 1989. The bioluminescence of the ventral photophores was recorded on videotape at 11 to 12°C and 24°C, in conjunction with measurements of the spectral emission at the same temperatures. At 11 °C, the spectrum was unimodal, peaking at 490 nm with a narrow bandwidth. At the higher temperature a shoulder at about 440 nm appeared in the emission spectrum. The short, bright flashes from the subocular photophores had a broader bandwidth and a shorter wavelength emission maximum (475 nm) than that of the ventral photophores. Video recordings at the two temperatures showed no changes in either the identities of the luminescing photophores or their relative intensities. One structurally distinct type of photophore was not illuminated. We conclude that the observed spectral changes were produced within one group of photophores, and that recruitment of the unilluminated photophores would produce an additional spectral component, previously reported from another species ofAbralia (A. trigonura).     

Phylogenetic diversity of epibiotic bacteria in the accessory nidamental glands of squids (Cephalopoda: Loliginidae and Idiosepiidae)

Bacterial communities were identified from the accessory nidamental glands (ANGs) of European and Western Pacific squids of the families Loliginidae and Idiosepiidae, as also in the egg capsules, embryo and yolk of two loliginid squid species, and in the entire egg of one idiosepiid squid species. The results of phylogenetic analyses of 16S RNA gene (rDNA) confirmed that several phylotypes of α-proteobacteria, γ-proteobacteria and Cytophaga–Flavobacteria–Bacteroides phylum were present as potential symbiotic associations within the ANGs. Several identified clones were related to reference strains, while others had no known close relatives. Gram positive strains were rare in loliginid squids. Several bacterial groups may play important roles in the function of the ANGs, such as production of the toxic compounds involved in egg protection and carotenoid pigments. Within the eggs, no bacteria were associated with embryo or yolk of Loligo vulgaris and Sepioteuthis lessoniana, but α- and γ-proteobacteria were present in the egg capsules. Most bacterial strains detected in the egg capsules were the same as those found in the ANGs. The cephalopods of temperate regions (European cuttlefishes and the squid L. vulgaris) appear to be associated with one Agrobacterium strain (Agro2) while tropical-subtropical strains (Asian and Australian loliginids) are associated with Silicibacter-related strains, suggesting a biogeographic clustering for the Agrobacterium-like strains.     

Biology of the deep-sea octopus Bathypolypus sponsalis (Cephalopoda: Octopodidae) from the western Mediterranean Sea

Several aspects of the biology of Bathypolypus sponsalis were studied from 297 individuals (115 males, 180 females and 2 indeterminates) caught in a depth range of 200–800 m depth in the western Mediterranean Sea. The paper presents data on sizes (length-weight relationships, size-frequency distributions) and reproduction (sex ratio, maturation, condition), and also analyses of the diet of B. sponsalis from samples taken throughout the year. Length-weight relationships showed that females are heavier than males at the same mantle length. Although mature individuals were found all year round, the maximum number occurred in spring and summer. Sexual maturation data revealed that males mature at smaller sizes than females. The gonadosomatic index increased with maturity in both sexes; the increase was gradual in males, but abrupt in females. The digestive gland index was used as a condition index and showed a differential behaviour with maturity; it increased gradually in females, but decreased in males. Like other octopus species, B. sponsalis appears to be an opportunistic predator, feeding on a great variety of preys. Stomach content analysis yielded a total of 19 different prey items belonging to four major groups (Crustacea, Mollusca, Ophiuroidea and Osteichthya). The first three groups were the more frequent preys, since crustaceans, molluscs and ophiuroids appeared in 76%, 49% and 30% of the stomachs, respectively. Decapoda Reptantia (among crustaceans) and cephalopods and bivalves (among molluscs) constituted the more abundant prey items. While the Decapoda Reptantia group was significantly more abundant in stomach contents of females, gastropods were taken more frequently by males. These differences in diet could reveal females as a more active predators than males. Received: 5 March 2000 / Accepted: 7 November 2000     

Sodium- and potassium-activated adenosine triphosphatase in the excretory organs of Sepia officinalis (Cephalopoda)

Sodium- and potassium-activated ATPase (Na⁺–K⁺-ATPase) has been demonstrated in excretory organs of Sepia officinalis, using a cytochemical procedure. In the renal appendages, both epithelia of the pancreatic appendages, the folded epithelium of the branchial heart appendage and the transport-active epithelium of the gill, the enzyme is localized exclusively in the basolateral cell membranes, i.e., the membranes of the basal labyrinth and the lateral plasma membranes. In addition, Na⁺–K⁺-ATPase is also located in the sarcolemma of the muscle fibres of the branchial heart. Distribution and localization of the enzyme is further substantiated by [³H]-ouabain autoradiography. The possible involvement of Na⁺–K⁺-ATPase in the excretion of ammonia and in ionic regulation in dibranchiate cephalopods is discussed.This study was supported by the Deutsche Forschungsgemeinschaft and is part of a doctoral dissertation     

Surface oceanography of the inferred hatching grounds of Illex argentinus (Cephalopoda: Ommastrephidae) and influences on recruitment variability

Interannual variability in the mesoscale oceanography of the inferred hatching area of winter-spawned Illex argentinus (i.e. those belonging to the Southern Patagonic Stock) was examined during the hatching months of June and July. The hatching area was defined as the region 32°-39°S, 49°-61° W, and indicators of the surface oceanography in this area were derived from remotely sensed advanced very high resolution radiometer (AVHRR) sea-surface temperature (SST) data. Interannual variability in the mesoscale oceanic circulation in the inferred hatching area was examined in terms of (1) the area occupied by "frontal" waters (defined as areas with SST gradients of ́°C over an area of 15 km by 15 km), and (2) the area occupied by waters of "favourable" SST (defined as those with temperatures in the range 16-18°C). Oceanographic indicators were compared with an index of I. argentinus recruitment to the Falkland Islands (Malvinas) fishery (catch per licensed vessel) for the period 1987 to 1999. The two oceanographic indicators were seen to co-vary, with a decrease in the proportion of the inferred hatching area occupied by frontal waters corresponding to an increase in the proportion of water of favourable SST. High squid abundance was found to be associated with a lower proportion of frontal waters or a higher proportion of favourable-SST waters within the inferred hatching area in the year preceding the fishery. Interannual variability in the interaction of the Brazil and Falkland Currents in the inferred hatching area during the early life stages is shown to be important in influencing the population size of I, argentinus, and mechanisms influencing squid recruitment in this highly dynamic region are discussed.