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     

Ocean acidification and temperature rise: effects on calcification during early development of the cuttlefish Sepia officinalis

This study investigated the effects of seawater pH (i.e., 8.10, 7.85 and 7.60) and temperature (16 and 19 °C) on (a) the abiotic conditions in the fluid surrounding the embryo (viz. the perivitelline fluid), (b) growth, development and (c) cuttlebone calcification of embryonic and juvenile stages of the cephalopod Sepia officinalis. Egg swelling increased in response to acidification or warming, leading to an increase in egg surface while the interactive effects suggested a limited plasticity of the swelling modulation. Embryos experienced elevated pCO₂ conditions in the perivitelline fluid (>3-fold higher pCO₂ than that of ambient seawater), rendering the medium under-saturated even under ambient conditions. The growth of both embryos and juveniles was unaffected by pH, whereas ⁴⁵Ca incorporation in cuttlebone increased significantly with decreasing pH at both temperatures. This phenomenon of hypercalcification is limited to only a number of animals but does not guarantee functional performance and calls for better mechanistic understanding of calcification processes.     

The effect of ration size, temperature and body weight on specific dynamic action of the common cuttlefish Sepia officinalis

The effect of meal size (shrimp Crangon crangon) [0.83–18.82% dry body weight (Dw)] on specific dynamic action (SDA) was assessed in cuttlefish Sepia officinalis (1.03–6.25 g Dw) held at 15 and 20°C. Cuttlefish <2 g significantly expended less energy in feeding and digesting their meal than cuttlefish >2 g when given the same quantity of food. Handling, eating and digesting a shrimp meal was temperature dependent with cuttlefish processing and digesting a similar sized shrimp meal faster at 20°C than at 15°C. The proportional increase in oxygen consumption (2.07 ± 0.02) was not correlated with feeding rate (FR) and was independent of temperature and cuttlefish size. The SDA peak was not correlated with FRs, and increased as cuttlefish size and temperature increased. The mean SDA coefficient was 0.87 ± 0.07% of the ingested energy; one of the lowest SDA values recorded amongst vertebrates and invertebrates. Daily energy requirements (KJ day⁻¹) for S. officinalis were calculated from laboratory estimates of energy losses due to standard (MO_{2 Standard}), routine (MO_{2 Routine}) and feeding (MO_{2 SDA}) oxygen consumption. Laboratory estimates of daily metabolic expenditures were combined with results from previous investigations to construct an energy budget for 1 and 5 g cuttlefish consuming a meal of 5 and 15% Dw at 20°C and the amount of energy available for growth was estimated to be between 35 and 80.3% of the ingested energy.     

Respiration of the eggs of the giant cuttlefish Sepia apama

On the roofs of subtidal crevices, the giant cuttlefish (Sepia apama) of southern Australia lays clutches of lemon-shaped eggs which hatch after 3 to 5 mo. Diffusion of oxygen through the capsule and chorion membrane to the perivitelline fluid and embryo was modelled using the equation V˙ _O2 = G _O2(P _O2out−P _O2in), where V˙ _O2 = rate of oxygen consumption, G _O2 = oxygen conductance of the capsule, and P _O2 values = oxygen partial pressures across the capsule. During development, V˙ _O2 rose exponentially as the embryo grew, reaching 5.5 μl h⁻¹ at hatching. Throughout development, the capsule dimensions enlarged by absorption of water into the perivitelline space, increasing G _O2 by a combination of increasing surface area, and decreasing thickness of the capsule. These processes maintained P _O2in high enough to allow unrestricted V˙ _O2 until shortly before hatching. Diffusion limitation of respiration in hatching-stage embryos was demonstrated by (1) increased embryonic V˙ _O2 when P _O2out was experimentally raised, (2) greater V˙ _O2 of resting individuals immediately after hatching, and (3) reduced V˙ _O2 of hatchlings at experimental P _O2 levels higher than P _O2in before hatching. Thus, low P _O2in may be the stimulus to hatch. Potential problems of diffusive gas-exchange are mitigated by the relatively low incubation temperature (12 °C), which may be a factor limiting the distribution of the species to cool, southern waters. Received: 14 August 1999 / Accepted: 24 January 2000     

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.     

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     

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.     

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.     

Phylogenetic characterisation of bacterial symbionts in the accessory nidamental glands of the sepioid Sepia officinalis (Cephalopoda: Decapoda)

 Female cuttlefish harbour a dense bacterial community in their accessory nidamental glands (ANG), as is also the case for the myopsid squids. Molecular approaches have been applied to explore this symbiotic association in the sepiid species Sepia officinalis. In situ localisation by Bacteria-specific probes in tissue sections of the ANG revealed the presence of a dense bacterial population in the lumina of the organ tubules. The phylogenetic identification of bacterial strains was realised by 16S rRNA gene sequencing analysis. None of the sequences obtained matched perfectly with known sequences in the database. However their similarity percentages allowed us to relate them to various bacterial groups including the taxa Agrobacterium, Roseobacter, Sporichthya, Rhodobium–Xanthobacter and Clostridium. Some bacterial species are common to both sepioids and myopsid teuthoids, others are different. Received: 28 May 1999 / Accepted: 2 November 1999     

Influence of temperature and salinity on the trace element incorporation into statoliths of the common cuttlefish (<Emphasis Type="Italic">Sepia officinalis</Emphasis>)

The use of statolith chemistry to trace migration pathways and distinguish populations of cephalopods is based on the assumption that the elemental composition of statoliths is influenced by physicochemical properties of the ambient environment. However, such influences have not been investigated experimentally up until now. This study presents the first microchemical analyses of cephalopod statoliths obtained from laboratory experiments under different controlled temperature and salinity conditions. Our results show that statolith chemical composition is strongly related to both salinity and temperature in ambient waters. The Ba/Ca ratio is negatively related to temperature and shows no relation to salinity. The I/Ca ratio is positively related to temperature and negatively to salinity. No Sr/Ca relation was found to either salinity or temperature, suggesting that the well-established proxy strontium is not as useful in cephalopod statoliths as in other biomineralized aragonites. Microanalysis of trace elements, however, shows an enormous potential for field studies on distribution, migration and stock separation of cephalopods. Furthermore, Synchrotron X-ray Fluorescence Analysis is introduced as a promising novel method for statolith analysis, providing a spatial resolution of typically 10–15 μm combined with detection limits down to 0.5 ppm.     

Respiration of Sepia officinalis during embryonic and early juvenile life

Adult Sepia officinalis L. were caught in June 1984, in the coastal waters of Wimereux (France). Deposition of the eggs took place in the seawater aquaria of the Station Marine. The oxygen consumption of S. officinalis was measured during embryonic and juvenile development. Aerobic metabolism occurs as soon as the early embryonic Stage 21. Oxygen diffuses through the initially thick egg shell; the oxygen level in the perivitelline liquid reaches a maximal value just before hatching (116.7±6.9 mm Hg). Hatchings display only a slight increase in oxygen consumption compared to embryos in the last stage of development. Respiration experiments with 40 d old juveniles showed that oxygen consumption increases with temperature, but is not affected by photoperiod. Experiments under increasing hypoxia revealed that S. officinalis juveniles are good regulators and maintain a constant oxygen consumption in the range of 4 to 7 mg O₂l^-1. Juveniles successfully recover from an hypoxic stress of 2 mg O₂l^-1 maintained for 1 h. This suggests that the respiratory pigments (pre-hemocyanins) of 40 d-old juveniles have a high oxygen affinity and/or that these juveniles have the ability to adapt to anaerobic conditions.     

Determination of copper in embryos and very young specimens of Sepia officinalis

The total amount of copper in embryos and newly hatched young individuals of Sepia officinalis L. has been determined by microtechnique, using bathocuproine-sulfonate as complexing reagent. During embryonic life, the total amount of copper does not change; it remains at a level close to 3.8 g. The copper is found in the yolk sac of very early embryos; it is subsequently transferred into the embryo proper. After hatching, the copper content diminishes quickly in starved individuals. Fed S. officinalis also usually lose copper. The reason for this may be that the inner yolk sac of newly hatched individuals contains a great deal of the total copper, which is excreted with the yolk after the latter has become superfluous. Later on, copper must be taken up from the food. The mobilization of protein and copper from the yolk into the blood may account for the early appearance of embryonic hemocyanin in the blood.     

Potential dietary effects on the fatty acid composition of the common jellyfish Aurelia aurita

Fatty acid composition of the natural and aquarium-reared common jellyfish Aurelia aurita was investigated. Fatty acid composition of the aquarium-reared A. aurita clearly reflected that of the diet, brine shrimp (Artemia). In the same way, fatty acid composition of the natural A. aurita was assumed to reflect those of natural diets. Samples of natural A. aurita were collected from April 1995 to September 1995 in the Seto Inland Sea, Japan, and their fatty acids were analyzed by gas chromatography and mass spectrometry. Variation of fatty acid compositions was seasonal rather than dependent on body size. Two major seasonal groups were divided by the cluster analysis of the A. aurita fatty acid composition: the April–June and the August–September clusters. The April–June cluster was characterized by high contents of the (n − 3)-fatty acids of diatom origin, accumulated via the grazing food chain. By contrast, the August–September cluster was characterized by an increase in (n − 6)-fatty acids of macroalgal origin, probably transferred via the detritus food chain. These results suggest that the diet of natural A. aurita may shift between the diatom-based food chain and the detritus-based food chain. Received: 12 April 2000 / Accepted: 1 December 2000     

Effect of body mass,temperature and food deprivation on oxygen consumption rate of common cuttlefish <Emphasis Type="Italic">Sepia officinalis</Emphasis>

Predictions of short and long term changes in Sepia officinalis metabolism are useful, since this species is both economically important for aquaculture and also is an ideal experimental laboratory organism. In this study standard and routine oxygen consumption rates of newly hatched and juvenile laboratory raised cuttlefish S. officinalis ranging between 0.04 and 18.48 g dry body mass (Dm), were measured over a range of temperatures (10, 15, 20 and 25°C). The mass exponent (b) ranged between 0.706 and 0.992 for standard oxygen consumption and between 0.694 and 0.990 for routine oxygen consumption. Oxygen consumption scaled allometrically (b = 0.7) with body mass for cuttlefish <2 g Dm and isometrically (b = 1) thereafter. No significant differences were apparent amongst the slopes of oxygen consumption and body mass at different temperatures for standard and routine oxygen consumption. However, the intercepts differed significantly amongst the regression lines, indicating a significant effect of temperature on the magnitude of oxygen consumption. The combined effect of temperature (T) and dry body mass (Dm) are best described by the following equations: cuttlefish <2 g, MO₂ = 0.116Dm^0.7111.086 ^T and >2 g, MO₂ = 0.076Dm^0.9831.091 ^T for standard oxygen consumption; cuttlefish <2 g, MO₂ = 0.538Dm^0.7291.057 ^T and >2 g, MO₂ = 0.225Dm^0.9621.081 ^T for routine oxygen consumption. Using these equations it was estimated that a cuttlefish of 1 g Dm held at 20°C, eating 5% Dm day⁻¹ and undergoing standard and routine metabolism consumes 21.3 and 35.4%, respectively of its total daily energy intake. Juvenile cuttlefish (3.32–5.08 g Dm) held at 15°C and deprived of food for 27 days maintained a stable standard oxygen consumption rate for the first 6 days following starvation. By the 18th day without food, oxygen consumption rate had declined by 53% and further declined to 65% below the standard oxygen consumption rate on the 27th day. Upon resumption of feeding, the respiration rate returned immediately to the initial level prior to food deprivation. The present study defines the basic energy requirements and general physiological state of young cuttlefish at temperatures of 10–25°C with and without food.     

Cuttlebone calcification increases during exposure to elevated seawater pCO2 in the cephalopod Sepia officinalis

Changes in seawater carbonate chemistry that accompany ongoing ocean acidification have been found to affect calcification processes in many marine invertebrates. In contrast to the response of most invertebrates, calcification rates increase in the cephalopod Sepia officinalis during long-term exposure to elevated seawater pCO₂. The present trial investigated structural changes in the cuttlebones of S. officinalis calcified during 6 weeks of exposure to 615 Pa CO₂. Cuttlebone mass increased sevenfold over the course of the growth trail, reaching a mean value of 0.71 ± 0.15 g. Depending on cuttlefish size (mantle lengths 44–56 mm), cuttlebones of CO₂-incubated individuals accreted 22–55% more CaCO₃ compared to controls at 64 Pa CO₂. However, the height of the CO₂-exposed cuttlebones was reduced. A decrease in spacing of the cuttlebone lamellae, from 384 ± 26 to 195 ± 38 μm, accounted for the height reduction The greater CaCO₃ content of the CO₂-incubated cuttlebones can be attributed to an increase in thickness of the lamellar and pillar walls. Particularly, pillar thickness increased from 2.6 ± 0.6 to 4.9 ± 2.2 μm. Interestingly, the incorporation of non-acid-soluble organic matrix (chitin) in the cuttlebones of CO₂-exposed individuals was reduced by 30% on average. The apparent robustness of calcification processes in S. officinalis, and other powerful ion regulators such as decapod cructaceans, during exposure to elevated pCO₂ is predicated to be closely connected to the increased extracellular [HCO₃ ⁻] maintained by these organisms to compensate extracellular pH. The potential negative impact of increased calcification in the cuttlebone of S. officinalis is discussed with regard to its function as a lightweight and highly porous buoyancy regulation device. Further studies working with lower seawater pCO₂ values are necessary to evaluate if the observed phenomenon is of ecological relevance.     

Age studies based on daily growth increments in statoliths and growth lamellae in cuttlebone of cultured Sepia officinalis

One hundred and six individuals of Sepia officinalis were cultured throughout its life cycle in two different temperature regimes: 13-15°C and 18-20°C. The number of increments in the statoliths and the number of lamellae in the cuttlebone were read at known ages in different individuals. The formation of growth increments in the statoliths was linearly related with individual age, but it was independent of temperature. By comparison between the slope of these linear relationships and the bisecting line, the hypothesis "1 increment=1 day" was validated in individuals as old as 240 days. In specimens older than 240 days, the number of increments was underestimated due to the poor resolution of the later growth increments. The maximum number of days in captivity (420) was about 80% of the life span estimated for this species in the area studied. Preliminary validation was also obtained from statoliths marked with tetracycline. Increments between tetracycline marks were not visible. The mean distance between marks in ten statoliths was 44 µm (Lj). Considering the same distance in statoliths of wild individuals, the mean number of increments counted was 37 (Lj). This result is very close to the 40 days passed between both tetracycline marks. A straight line significantly different for each culture temperature defined the relationship between number of the cuttlebone lamellae and age. The number of lamellae in the cuttlebone does not correspond to the real age. The time necessary for the formation of one lamella was 8DŽ.3 days at 13-15°C, whereas the deposition of one lamella lasted 3.1ǃ.06 days at 18-20°C. In conclusion, the periodicity for lamellar deposition can be defined only if the temperature where the animal lived is considered.     

Food composition and feeding behavior of shallow-water crinoids at Eilat (Red Sea)

The food of 3 feather-stars, Lamprometra klunzingeri Hartlaub, 1890), Heterometra savignii (J. Müller, 1841) and Capillaster multiradiatus (Linnaeus, 1758), from the shallow water of Eilat (Red Sea) was found to be composed of planktonic and semi-benthalic organisms with a strong dominance of zooplankton. Monthly analyses of stomach contents showed seasonal changes in the participation of various systematic units in the diet. Food-comprising organisms were found to be selected according to size, and those measuring up to 400 formed 90% of the diet.This research has been sponsored partly by the Oceanic Biology Branch, Office of Naval Research, U.S.A. under contract N 62558-4556 and contract F 671052 67C 0043 between the Office of N.R. and the Tel-Aviv University. It represents part of the M.Sc. thesis of the first named author (in the framework of Red Sea investigations headed by Dr. L. Fishelson).     

Age and growth of the mesopelagic squid Ancistrocheirus lesueurii (Oegopsida: Ancistrocheiridae) from the central-east Atlantic based on statolith microstructure

Statolith microstructure was studied in 56 Ancistrocheirus lesueurii (25 to 423 mm of mantle length, ML) caught in the central-east Atlantic. Statolith growth increments were grouped into three main growth zones, distinguished mainly by increment width. The second transition in the statolith microstructure (from Zone 2 to Zone 3) coincides with the life history shift from epipelagic and upper mesopelagic to a bathyal habitat. Second-order bands (mean 27.65 growth increments) and sub-bands (mean 13.6 growth increments) within statolith microstructure appeared to be related to the lunar cycle. Striking sexual dimorphism is reflected in the age and growth rates: males live ca. 1 yr, while females only start maturing at this age and obviously live >1.5 yr. A. lesueurii is a slow growing squid, attaining 25 to 30 mm ML at the age of 100 d. After ontogenetic migrations into bathypelagic waters at ML > 30 to 35 mm, growth rates gradually decrease to the minimum known values for squids. Based on back-calculated hatching dates, A. lesueurii hatches throughout the year with a peak between November and March. Received: 28 August 1996 / Accepted: 31 January 1997