Identification and expression of two oxytocin/vasopressin-related peptides in the cuttlefish Sepia officinalis

Two novel members of the oxytocin/vasopressin superfamily have been identified in the cephalopod Sepia officinalis. Oxytocin/vasopressin gene sequences were cloned by Race PCR. The two precursors we identified exhibit the classical organization of OT/VP superfamily precursors: a signal peptide followed by a nonapeptide and a neurophysin domain. The neurophysin domain is entirely conserved for the cuttlefish precursors, but the nonapeptides and the signal peptides differ. The first nonapeptide, called sepiatocin, is highly homologous to Octopus vulgaris octopressin. The second nonapeptide, called pro-sepiatocin, shows sequence homologies with a Crustacean oxytocin/vasopressin-like peptide identified in Daphnia culex and with a novel form of oxytocin described in New World monkeys. The expression of pro-sepiatocin is restricted to the supraesophageal and subesophageal masses of the brain whereas sepiatocin is expressed in the entire central nervous system. Sepiatocin, as described for octopressin, modulates the contractile activity of several muscles such as penis, oviduct and vena cava muscles; this suggests its involvement in reproduction and blood circulation. Pro-sepiatocin is released in the hemolymph; it is a neurohormone able to target numerous peripheral organs.     

Symmetrical crypsis and asymmetrical signalling in the cuttlefish Sepia officinalis

The salience of bilateral symmetry to humans has led to the suggestion that camouflage may be enhanced in asymmetrical patterns. However, the importance of bilateral symmetry in visual signals (and overall morphology) may constrain the evolution of asymmetrical camouflage, resulting in the bilaterally symmetrical cryptic patterns that we see throughout the animal kingdom. This study investigates the cuttlefish (Sepia officinalis), which can control the degree of symmetry in its coloration. Ten juvenile S. officinalis were filmed in two behavioural contexts (cryptic and threatened) to test the prediction that cryptic patterns will be expressed more asymmetrically than an anti-predator signal known as the 'deimatic display'. Cryptic body patterns, particularly those with a disruptive function, were found to exhibit a high degree of bilateral symmetry. By contrast, the components of the deimatic display were often expressed asymmetrically. These results are contrary to the predicted use of symmetry in defensive coloration, indicating that the role of symmetry in both crypsis and visual signalling is not as straightforward as previously suggested.     

A preliminary analysis of sleep-like states in the cuttlefish Sepia officinalis

Sleep has been observed in several invertebrate species, but its presence in marine invertebrates is relatively unexplored. Rapid-eye-movement (REM) sleep has only been observed in vertebrates. We investigated whether the cuttlefish Sepia officinalis displays sleep-like states. We find that cuttlefish exhibit frequent quiescent periods that are homeostatically regulated, satisfying two criteria for sleep. In addition, cuttlefish transiently display a quiescent state with rapid eye movements, changes in body coloration and twitching of the arms, that is possibly analogous to REM sleep. Our findings thus suggest that at least two different sleep-like states may exist in Sepia officinalis.     

Food imprinting, new evidence from the cuttlefish Sepia officinalis

Imprinting provides precocial offspring with an efficient means to optimize their subsequent behaviours. We discovered food imprinting using a sophisticated invertebrate model: the cuttlefish. We showed that early juveniles preferred the prey to which they have been visually familiarized, when the amount of information was sufficient and only if such familiarization occurred during a short sensitive period. We also demonstrated that the effects of visual food imprinting overcame those of the first food ingested. Our study shows that visual imprinting is a critical process in animals, surpassing more direct reward experiences that occur outside the critical exposure period.     

Effects of early visual experience on the background preference in juvenile cuttlefish Sepia pharaonis

Although cuttlefish are capable of showing diverse camouflage body patterns against a variety of background substrates, whether they show background preference when given a choice of substrates is not well known. In this study, we characterized the background choice of post-embryonic cuttlefish (Sepia pharaonis) and examined the effects of rearing visual environments on their background preferences. Different rearing backgrounds (enriched, uniformly grey and checkerboard) were used to raise cuttlefish from eggs or hatchlings, and four sets of two-background-choice experiments (differences in contrast, shape, size and side) were conducted at day 1 and weeks 4, 8 and 12 post-hatch. Cuttlefish reared in the enriched environment preferred high-contrast backgrounds at all post-embryonic stages. In comparison, those reared in the impoverished environments (uniformly grey and checkerboard) had either reversed or delayed high-contrast background preference. In addition, cuttlefish raised on the uniformly grey background, exposed to a checkerboard briefly (0.5 or 3 h) at week 4 and tested at week 8 showed increased high-contrast background preference. Interestingly, cuttlefish in the enriched group preferred an object size similar to their body size at day 1 and week 4, but changed this preference to smaller objects at week 12. These results suggest that high-contrast backgrounds may be more adaptive for juvenile cuttlefish, and visually enriched environments are important for the development of these background preference behaviours.     

Small-scale rearing of cuttlefish (Sepia officinalis) for research purposes

We report a methodology refined over 20 years to culture and maintain a small colony of Sepia officinalis for research year-round. Wild-caught eggs were obtained annually from England and reared in semi-closed natural seawater systems of large volume. Constant temperature (15 °C) and day length (12L/12D) delayed sexual maturation and reproductive behavior. This extended life cycle by roughly 50%, thus providing small research animals for about 18 months from every annual batch of eggs. A novel live food – gammarid crustaceans collected from washed up seagrass– was provided to hatchlings and juveniles. Juveniles were then trained to take thawed shrimp thereafter, thus reducing the expense of live foods. Typical survival to one year was >65%. With these methods, healthy sexually immature cuttlefish were available year-round for behavioral and physiological studies without the confounding influences of hormonally-induced fighting, mating, and egg laying that typically occur within six months.     

Effects of temperature on the growth and microstructure formation of cuttlebone from cuttlefish Sepia pharaonis

ABSTRACT

This study investigated the effects of three temperatures (20°C, 27°C, and 31°C) on the physiological performance (survival and growth) and cuttlebone micromorphological features (chamber number, chamber height and lamellae number) of cuttlefish Sepia pharaonis. We examined gross morphological characteristics of the internal calcareous cuttlebone to determine whether lamellar matrix was impacted by temperature. Juvenile survival was significantly affected by temperature (χ²?=?54.580, df?=?2, P?<?0.001). Cuttlebone weight, length and width were also positively correlated with temperature. Scanning electron microscopy revealed that a single chamber structure consists of septa, lamellae and pillars. At 20°C the chamber number of 89.0?±?10.8 was significantly higher than at 27°C with 44.8?±?3.6 or 31°C with 47.5?±?4.3 (Kruskal–Wallis [KW], χ^2?=?26.391, df?=?2, P?=?0.0001), whilst chamber height was lower at 20°C (KW χ^2?=?27.842, df?=?2, P?=?0.0001). Moreover, lamellae number varied among the treatments (KW χ^2?=?22.411, df?=?2, P?=?0.0002). Lamellae numbers at 20°C, 27°C and 31°C were 3–6, 6–9 and 5–8, respectively. The results indicated that the intrinsic lamellar matrix structure was significantly affected by temperature and that this effect may be used in cuttlebone growth studies.     

Role of blood-oxygen transport in thermal tolerance of the cuttlefish, Sepia officinalis

Mechanisms that affect thermal tolerance of ectothermic organismshave recently received much interest, mainly due to global warmingand climate-change debates in both the public and in the scientificcommunity. In physiological terms, thermal tolerance of severalmarine ectothermic taxa can be linked to oxygen availability,with capacity limitations in ventilatory and circulatory systemscontributing to oxygen limitation at extreme temperatures. Thepresent review briefly summarizes the processes that definethermal tolerance in a model cephalopod organism, the cuttlefishSepia officinalis, with a focus on the contribution of the cephalopodoxygen-carrying blood pigment, hemocyanin. When acutely exposedto either extremely high or low temperatures, cuttlefish displaya gradual transition to an anaerobic mode of energy productionin key muscle tissues once critical temperatures (T_crit) arereached. At high temperatures, stagnating metabolic rates anda developing hypoxemia can be correlated with a progressivefailure of the circulatory system, well before T_crit is reached.However, at low temperatures, declining metabolic rates cannotbe related to ventilatory or circulatory failure. Rather, wepropose a role for hemocyanin functional characteristics asa major limiting factor preventing proper tissue oxygenation.Using information on the oxygen binding characteristics of cephalopodhemocyanins, we argue that high oxygen affinities (= low P₅₀values), as found at low temperatures, allow efficient oxygenshuttling only at very low venous oxygen partial pressures.Low venous PO₂s limit rates of oxygen diffusion into cells,thus eventually causing the observed transition to anaerobicmetabolism. On the basis of existing blood physiological, molecular,and crystallographical data, the potential to resolve the roleof hemocyanin isoforms in thermal adaptation by an integratedmolecular physiological approach is discussed.     

Central distribution and three-dimensional arrangement of fin chromatophore motoneurons in the cuttlefish Sepia officinalis

Cephalopod body patterning is a most complex invertebrate behavior. Generated primarily by pigment-containing chromatophore organs, this behavior enables rapid alteration of body coloration as a result of direct innervation of chromatophores by motoneurons. This study focuses on location and arrangement of fin chromatophore motoneurons in the cuttlefish Sepia and investigates the possibility of central topography. Retrograde labeling of topographically arranged fin nerve branches in the periphery revealed the posterior subesophageal mass (PSEM) of the brain as the primary location of fin chromatophore motoneurons; within this region, most cells were located in the posterior chromatophore and fin lobes. Additionally, a small percentage of labeled motoneurons occurred in the anterior subesophageal mass and the stellate ganglia. Data from three-dimensional reconstructions of PSEMs showed the arrangement of labeled motoneurons within individual lobes; these data suggest no obvious topographic arrangement. Further, electrical stimulation of the PSEM generated chromatophore activity on the fin and mantle. These stimulation results, coupled with the retrograde labeling, suggest that chromatophore motoneurons are located across multiple PSEM lobes.     

Behavioural responses of juvenile cuttlefish (Sepia officinalis) to local water movements

Physiological studies have shown that the epidermal head and arm lines in cephalopods are a mechanoreceptive system that is similar to the fish and amphibian lateral lines (Budelmann BU, Bleckmann H. 1988. A lateral line analogue in cephalopods: Water waves generate microphonic potentials in the epidermal head lines of Sepia officinalis and Lolliguncula brevis. J. Comp. Physiol. A 164:1-5.); however, the biological significance of the epidermal lines remains unclear. To test whether cuttlefish show behavioural responses to local water movements, juvenile Sepia officinalis were exposed to local sinusoidal water movements of different frequencies (0.01-1000 Hz) produced by a vibrating sphere. Five behavioural responses were recorded: body pattern changing, moving, burrowing, orienting, and swimming. Cuttlefish responded to a wide range of frequencies (20-600 Hz), but not to all of the frequencies tested within that range. No habituation to repeated stimuli was seen. Results indicate that cuttlefish can detect local water movements (most likely with the epidermal head and arm lines) and are able to integrate that information into behavioural responses.     

Acetylcholine release and choline uptake by cuttlefish (Sepia officinalis) optic lobe synaptosomes

Acetylcholine (ACh), which is synthesized from choline (Ch), is believed to hold a central place in signaling mechanisms within the central nervous system (CNS) of cuttlefish (Sepia officinalis) and other coleoid cephalopods. Although the main elements required for cholinergic function have been identified in cephalopods, the transmembrane translocation events promoting the release of ACh and the uptake of Ch remain largely unsolved. The ACh release and Ch uptake were quantitatively studied through the use of in vitro chemiluminescence and isotopic methods on a subcellular fraction enriched in synaptic nerve endings (synaptosomes) isolated from cuttlefish optic lobe. The ACh release evoked by K+ depolarization was found to be very high (0.04 pmol ACh.s(-1).mg(-1) protein). In response to stimulation by veratridine, a secretagogue (a substance that induces secretion) that targets voltage-gated Na+ channels, the release rate and the total amount of ACh released were significantly lower, by 10-fold, than the response induced by KCl. The high-affinity uptake of choline was also very high (31 pmol Ch.min(-1).mg(-1) protein). The observed ACh release and Ch uptake patterns are in good agreement with published data on preparations characterized by high levels of ACh metabolism, adding further evidence that ACh acts as a neurotransmitter in cuttlefish optic lobe.     

Perception of edges and visual texture in the camouflage of the common cuttlefish, Sepia officinalis

The cuttlefish, Sepia officinalis, provides a fascinating opportunity to investigate the mechanisms of camouflage as it rapidly changes its body patterns in response to the visual environment. We investigated how edge information determines camouflage responses through the use of spatially high-pass filtered 'objects' and of isolated edges. We then investigated how the body pattern responds to objects defined by texture (second-order information) compared with those defined by luminance. We found that (i) edge information alone is sufficient to elicit the body pattern known as Disruptive, which is the camouflage response given when a whole object is present, and furthermore, isolated edges cause the same response; and (ii) cuttlefish can distinguish and respond to objects of the same mean luminance as the background. These observations emphasize the importance of discrete objects (bounded by edges) in the cuttlefish's choice of camouflage, and more generally imply that figure-ground segregation by cuttlefish is similar to that in vertebrates, as might be predicted by their need to produce effective camouflage against vertebrate predators.