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.     

Arm regeneration in two species of cuttlefish Sepia officinalis and Sepia pharaonis

To provide quantitative information on arm regeneration in cuttlefish, the regenerating arms of two cuttlefish species, Sepia officinalis and Sepia pharaonis, were observed at regular intervals after surgical amputation. The third right arm of each individual was amputated to ~10–20 % starting length. Arm length, suction cup number, presence of chromatophores, and behavioral measures were collected every 2–3 days over a 39-day period and compared to the contralateral control arm. By day 39, the regenerating arm reached a mean 95.5 ± 0.3 % of the control for S. officinalis and 94.9 ± 1.3 % for S. pharaonis. The process of regeneration was divided into five separate stages based on macroscopic morphological events: Stage I (days 0–3 was marked by a frayed leading edge; Stage II (days 4–15) by a smooth hemispherical leading edge; Stage III (days 16–20) by the appearance of a growth bud; Stage IV (days 21–24) by the emergence of an elongated tip; and Stage V (days 25–39) by a tapering of the elongated tip matching the other intact arms. Behavioral deficiencies in swimming, body postures during social communication, and food manipulation were observed immediately after arm amputation and throughout Stages I and II, returning to normal by Stage III. New chromatophores and suction cups in the regenerating arm were observed as early as Stage II and by Stage IV suction cup number equaled that of control arms. New chromatophores were used in the generation of complex body patterns by Stage V. These results show that both species of cuttlefish are capable of fully regenerating lost arms, that the regeneration process is predictable and consistent within and across species, and provide the first quantified data on the rate of arm lengthening and suction cup addition during regeneration.     

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.     

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.     

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.     

Allometry of thermal limitation in the cephalopod Sepia officinalis

Cuttlefish (Sepia officinalis) routine metabolic rate was determined in response to acute thermal changes at a rate of 1 degrees C h(-1) for a variety of animal sizes (15-496 g wet mass, laboratory reared at 15 degrees C). In a thermal frame of 11 to 23 degrees C, oxygen consumption rates (MO(2), in mumol O(2) g(-1) min(-1)) were observed to rise with increasing temperature (T, in degrees C) and to decline with increasing body mass (m, in g), according to the formula: ln MO(2)=-3.3+0.0945T-0.215 ln m (R(2)=0.93). Outside the above thermal window, animals were not able to increase MO(2) at similar rates, indicating a beginning oxygen limitation of metabolism. Large animals (>100 g body mass) already displayed lower than expected MO(2) values at 8 and 26 degrees C, while smaller animals (15 g wet mass) were characterized by a wider thermal window (MO(2) values deviated from expected rates at 5 and 29 degrees C). Morphometric data of cuttlefish mantle skin area was obtained to discuss size - related effects of skin respiration potential on thermal tolerance. Cuttlefish growth was observed to be isometric, as constant 'Vogel numbers' of 4.2 indicated (animal body masses: 11 to 401 g). In the same mass range, specific mantle surface area declined three-fold from 10.7 (0.24) (means+/-SD) to 3.3 (0.52) cm(2) g(-1). Thus, increased thermal tolerance in smaller animals may be enabled by a higher skin respiration potential due to higher specific skin surface areas. An elevated fraction of MO(2) provided by means of skin respiration in small animals could relieve the cardiovascular system, which previously has been found a major limiting component during acute thermal stress in cuttlefish.     

Perception of visual texture and the expression of disruptive camouflage by the cuttlefish, Sepia officinalis

Juvenile cuttlefish (Sepia officinalis) camouflage themselves by changing their body pattern according to the background. This behaviour can be used to investigate visual perception in these molluscs and may also give insight into camouflage design. Edge detection is an important aspect of vision, and here we compare the body patterns that cuttlefish produced in response to checkerboard backgrounds with responses to backgrounds that have the same spatial frequency power spectrum as the checkerboards, but randomized spatial phase. For humans, phase randomization removes visual edges. To describe the cuttlefish body patterns, we scored the level of expression of 20 separate pattern 'components', and then derived principal components (PCs) from these scores. After varimax rotation, the first component (PC1) corresponded closely to the so-called disruptive body pattern, and the second (PC2) to the mottle pattern. PC1 was predominantly expressed on checkerboards, and PC2 on phase-randomized backgrounds. Thus, cuttlefish probably have edge detectors that control the expression of disruptive pattern. Although the experiments used unnatural backgrounds, it seems probable that cuttlefish display disruptive camouflage when there are edges in the visual background caused by discrete objects such as pebbles. We discuss the implications of these findings for our understanding of disruptive camouflage.     

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.     

Functional response of early stages of the cuttlefish Sepia officinalis preying on the mysid Mesopodopsis slabberi

The functional response of Sepia officinalis early stages, preying on mysids of the species Mesopodopsis slabberi was investigated. The effects of five prey densities (12.5, 25, 37.5, 50 and 125 mysids l^-1) and two hatchling ages (1-day-old and 7-day-old) on consumption rate and the frequency of non-feeding animals were tested. Older animals were approximately 50% heavier than newly hatched ones. Hatchlings were individually assayed under 0.25 W m^-2 natural light, 37.8 psu in salinity and 19°C. The effect of prey density on consumption rate was highly significant and no effect of age was detected within the age range tested. Maximal values recorded for consumption rate were about 0.45 mysids h^-1. The frequency of non-feeding individuals was strongly reduced at saturating prey densities. The functional response curve showed an interval of prey densities for which density-dependent prey mortality is probable.     

Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca,Cephalopoda)

Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods.     

Identification of SepCRP analogues in the cuttlefish Sepia officinalis: a novel family of ovarian regulatory peptides

In the cuttlefish, Sepia officinalis, the ovary appears to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying. Following the identification of the SepCRP-1, which is a peptide extracted from ovary and involved in egg capsule secretion, investigations were focused on the identification of related peptides. Seven related-Sepia Capsule Releasing Peptides (R-SepCRPs) were identified by means of mass spectrometry and characterized using MS/MS spectra and/or Edman degradation. Finally, primary structures were verified by the comparison of MS/MS spectra from endogenic and synthetic peptides. This new ovarian peptide family exhibits a conserved SLXKD tag involved in the biological activity. LC-MS/MS screening clearly demonstrates that R-SepCRPs are restricted to the female genital tract. Expressed during vitellogenesis, they are released by vitellogenic follicles and full-grown oocytes (FGO) in the genital coelom. Biological activities suggest that R-SepCRPs would be responsible for the storage of FGO before mating and would take part in the mechanical secretion of egg capsule products, as previously described for SepCRP-1.     

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.     

Motor and behavioural effects induced by putative neurotransmitter injection into the optic lobe of the cuttlefish, Sepia officinalis

This paper presents a technique using local injections of different drugs directly into the optic lobe of Sepia. The cholinomimetic drugs induce a general decrease of the motricity, muscle tone, frequency of respiratory movements and decoloration of the animal. Dopamine and noradrenaline produce the same complex motor effects obtained by chronic electrical stimulation. Serotonin induces complex effects, particularly on the coloration. Some serotoninergic synapses could be implicated in the triggering of colour patterns.     

Effects of stimuli shape and polarization in evoking deimatic patterns in the European cuttlefish, Sepia officinalis, under varying turbidity conditions

Cuttlefish possess the complex ability to identify approaching threats and then to selectively express the appropriate defense. We examined the visual cues used by Sepia officinalis cuttlefish during predator detection and the responses they selected. Using computer-generated stimuli, we set out to quantitate the deimatic responses to artificial looming stimuli of different shapes and contrasts. Defensive behavior gradually intensified as geometrical shapes resembled an image of a fish. Therefore, in addition to an object’s size or its sudden increase in size, cuttlefish use form recognition to identify a threat. Cuttlefish demonstrated equal performance in predator detection trough clear water when presented with intensity versus polarization contrasts. However, when the water turbidity increased, the cuttlefish still detected looming fish shapes based on polarization contrast even when intensity information alone did not suffice. These results demonstrate the interplay between intensity and polarization information transmission and processing in the spatial domain. As nectobenthic organisms, cuttlefish probably experience low visibility conditions on a regular basis. The ability to see further into turbid water and to better detect an approaching object would be beneficial for their survival.