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.     

Molecular analysis of a novel FMRFamide-related peptide gene (SOFaRP(2)) and its expression pattern in the brain of the European cuttlefish Sepia officinalis

FMRFamide-related peptides (FaRPs) are among several neurotransmitters known to regulate the chromatophore function in the European cuttlefish Sepia officinalis. Here we report the cloning and sequencing of a novel S. officinalis FaRP gene (SOFaRP(2)). The complete 835-base pair cDNA sequence of the SOFaRP(2) gene contains an open reading frame of 567 base pairs encoding 188 amino acids and four putative FaRPs, NSLFRFamide, GNLFRFamide, TIFRFamide and PHTPFRFamide. All except TIFRFamide cause chromatophore expansion when assayed in an in vitro chromatophore bioassay. To investigate the expression pattern of SOFaRP(2) gene in the cuttlefish brain, in situ hybridization was performed using a full length RNA probe. The SOFaRP(2) gene was expressed primarily in the posterior chromatophore, anterior chromatophore, lateral basal and optic lobes among other brain locations. The SOFaRP(2) gene appears to be expressed in all brain regions involved in chromatophore regulation. These data suggests that some or all of the four FaRPs encoded by SOFaRP(2) might be involved in controlling chromatophore activity in cuttlefish.     

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.     

Peripheral and central nervous responses evoked by small water movements in a cephalopod

Potentials were recorded from the epidermal head lines and from the CNS of young cuttlefish, Sepia officinalis, in response to weak water movements. 1. Within the test range 0.5-400 Hz a sinusoidal water movement elicits up to 4 components of response if the electrode is placed on a headline: (i) a positive phasic ON response; (ii) a tonic frequency-following microphonic response; (iii) a slow negative OFF response; and (iv) compound nerve impulses. 2. The amplitude of both the ON wave and the microphonic potential depends on stimulus frequency, stimulus amplitude and stimulus rise time. Frequencies around 100 Hz and short rise times are most effective in eliciting strong potentials. The minimal threshold was 0.06 microns peak-to-peak water displacement at 100 Hz (18.8 microns/s as velocity). 3. Change of direction of tangential sphere movement (parallel vs. across the head lines) has only a small effect on the microphonic and the summed nerve potentials. 4. Frequency and/or amplitude modulations of a carrier stimulus elicit responses at the onset and offset of the modulation and marked changes in the tonic microphonic response. 5. Evoked potentials can be recorded from the brain while stimulating the epidermal lines with weak water movements. The brain potentials differ in several aspects from the potentials of the head lines and show little or no onset or offset wave at the transitions of a frequency and amplitude modulation.     

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.     

FaRP cell distribution in the developing CNS suggests the involvement of FaRPs in all parts of the chromatophore control pathway in Sepia officinalis (Cephalopoda)

The FMRFamide-related peptide (FaRP) family includes a wide range of neuropeptides that have a role in many biological functions. In cephalopods, these peptides intervene in the peculiar body patterning system used for communication and camouflage. This system is particularly well developed in the cuttlefish and is functional immediately after hatching (stage 30). In this study, we investigate when and how the neural structures involved in the control of body patterning emerge and combine during Sepia embryogenesis, by studying the expression or the production of FaRPs. We detected FaRP expression and production in the nervous system of embryos from the beginning of organogenesis (stage 16). The wider FaRP expression was observed concomitantly with brain differentiation (around stage 22). Until hatching, FaRP-positive cells were located in specific areas of the central and peripheral nervous system (CNS and PNS). Most of these areas were implicated in the control of body patterns, suggesting that FaRPs are involved in all parts of the neural body pattern control system, from the ‘receptive areas’ via the CNS to the chromatophore effectors.     

Quantification of cuttlefish (Sepia officinalis) camouflage: a study of color and luminance using in situ spectrometry

Cephalopods are renowned for their ability to adaptively camouflage on diverse backgrounds. Sepia officinalis camouflage body patterns have been characterized spectrally in the laboratory but not in the field due to the challenges of dynamic natural light fields and the difficulty of using spectrophotometric instruments underwater. To assess cuttlefish color match in their natural habitats, we studied the spectral properties of S. officinalis and their backgrounds on the Aegean coast of Turkey using point-by-point in situ spectrometry. Fifteen spectrometry datasets were collected from seven cuttlefish; radiance spectra from animal body components and surrounding substrates were measured at depths shallower than 5 m. We quantified luminance and color contrast of cuttlefish components and background substrates in the eyes of hypothetical di- and trichromatic fish predators. Additionally, we converted radiance spectra to sRGB color space to simulate their in situ appearance to a human observer. Within the range of natural colors at our study site, cuttlefish closely matched the substrate spectra in a variety of body patterns. Theoretical calculations showed that this effect might be more pronounced at greater depths. We also showed that a non-biological method (“Spectral Angle Mapper”), commonly used for spectral shape similarity assessment in the field of remote sensing, shows moderate correlation to biological measures of color contrast. This performance is comparable to that of a traditional measure of spectral shape similarity, hue and chroma. This study is among the first to quantify color matching of camouflaged cuttlefish in the wild.     

Visual interpolation for contour completion by the European cuttlefish (Sepia officinalis) and its use in dynamic camouflage

Cuttlefish rapidly change their appearance in order to camouflage on a given background in response to visual parameters, giving us access to their visual perception. Recently, it was shown that isolated edge information is sufficient to elicit a body pattern very similar to that used when a whole object is present. Here, we examined contour completion in cuttlefish by assaying body pattern responses to artificial backgrounds of 'objects' formed from fragmented circles, these same fragments rotated on their axis, and with the fragments scattered over the background, as well as positive (full circles) and negative (homogenous background) controls. The animals displayed similar responses to the full and fragmented circles, but used a different body pattern in response to the rotated and scattered fragments. This suggests that they completed the broken circles and recognized them as whole objects, whereas rotated and scattered fragments were instead interpreted as small, individual objects in their own right. We discuss our findings in the context of achieving accurate camouflage in the benthic shallow-water environment.     

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.     

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.     

Food of the cuttlefish Sepia officinalis and S. elegans in the Ria de Vigo (NW Spain) (Mollusca: Cephalopoda)

A study of the diet of Sepia officinalis and S. elegans in the Ria de Vigo has shown that crustaceans are the most abundant prey in both species, followed by fish. Changes in the food composition of both species occur with growth. The type of prey eaten by the two sexes of these species is very similar. The possibility of trophic competition between juveniles of S. qficinalis and S. elegans is discussed.     

Cytobiological studies on hemocyanin metabolism in the branchial heart complex of the common cuttlefish Sepia officinalis (Cephalopoda, Dibranchiata)

The present study confirms previous investigations that demonstrated a high copper content in the branchial heart and its appendage, and that gave the first indication that this organ complex might be involved in hemocyanin metabolism in Sepia officinalis L. Immunocytochemical localization of hemocyanin molecules within the endocytotic lysosomal system of the ovoid cells and tracer experiments with ¹²⁵I-labeled Sepia hemocyanin suggest its endocytotic uptake. Energy-dispersive X-ray microanalysis and histochemical methods reveal a high copper content within the ovoid cells of the branchial heart. In view of the turnover of the respiratory pigment in the branchial heart of Sepia officinalis L., we believe that the ovoid cells are a site of hemocyanin catabolism.     

Biochemical composition of cuttlefish (Sepia esculenta) eggs during embryonic development

Changes in egg volume, water content, proteins, carbohydrates, lipids, amino acids and fatty acids in cuttlefish (Sepia esculenta) were determined during embryogenesis to understand the nutritional requirements in the early life phase. The egg volume and the water content decreased significantly (P<0.05) during early embryonic development, and then increased abruptly after the beginning of organ differentiation. During embryonic development, protein was the major content in the yolk and the per cent composition varied to a large extent (55.19–78.45%) with carbohydrates (9.86–15.56%) and lipids (1.62–2.97%). Proteins and lipids decreased 23.3% and 0.4%, respectively, in dry weight, while carbohydrates increased 0.46%. Total amino acids and essential amino acids (EAAs) were stable during the early embryonic developmental stage, but decreased significantly until the eggs hatched (P<0.05). The largest utilisation of the yolk protein possibly occurred with respect to EAAs (25.7%) because of a decrease in methionine (70.3%), valine (63.0%) and phenylalanine (58.5%). The most important fatty acids were saturated fatty acid (SFA) C16:0 and polyunsaturated fatty acids (PUFAs) C22:6 and C20:5. Unsaturated fatty acids (UFAs) and SFA decreased at a similar rate during embryonic development (5.69% and 6.15%, respectively). For UFAs, monounsaturated fatty acids were consumed at a greater rate than PUFAs (29.6% and 0.05%, respectively).     

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.     

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.