Sepia Officinalis Research Articles

Graded behavioral responses and habituation to sound in the common cuttlefish Sepia officinalis.

Sound is a widely available and vital cue in aquatic environments, yet most bioacoustic research has focused on marine vertebrates, leaving sound detection in invertebrates poorly understood. Cephalopods are an ecologically key taxon that likely use sound and may be impacted by increasing anthropogenic ocean noise, but little is known regarding their behavioral responses or adaptations to sound stimuli. These experiments identify the acoustic range and levels that elicit a wide range of secondary defense behaviors such as inking, jetting and rapid coloration change. Secondarily, it was found that cuttlefish habituate to certain sound stimuli. The present study examined the behavioral responses of 22 cuttlefish (Sepia officinalis) to pure-tone pips ranging from 80 to 1000 Hz with sound pressure levels of 85-188 dB re. 1 μPa rms and particle accelerations of 0-17.1 m s(-2). Cuttlefish escape responses (inking, jetting) were observed between frequencies of 80 and 300 Hz and at sound levels above 140 dB re. 1 μPa rms and 0.01 m s(-2) (0.74 m s(-2) for inking responses). Body patterning changes and fin movements were observed at all frequencies and sound levels. Response intensity was dependent upon stimulus amplitude and frequency, suggesting that cuttlefish also possess loudness perception with a maximum sensitivity around 150 Hz. Cuttlefish habituated to repeated 200 Hz tone pips, at two sound intensities. Total response inhibition was not reached, however, and a basal response remained present in most animals. The graded responses provide a loudness sensitivity curve and suggest an ecological function for sound use in cephalopods.

Chapter One - Cephalopod Culture: Current Status of Main Biological Models and Research Priorities

A recent revival in using cephalopods as experimental animals has rekindled interest in their biology and life cycles, information with direct applications also in the rapidly growing ornamental aquarium species trade and in commercial aquaculture production for human consumption. Cephalopods have high rates of growth and food conversion, which for aquaculture translates into short culture cycles, high ratios of production to biomass and high cost-effectiveness. However, at present, only small-scale culture is possible and only for a few species: the cuttlefish Sepia officinalis, the loliginid squid Sepioteuthis lessoniana and the octopuses Octopus maya and O. vulgaris. These four species are the focus of this chapter, the aims of which are as follows: (1) to provide an overview of the culture requirements of cephalopods, (2) to highlight the physical and nutritional requirements at each phase of the life cycle regarded as essential for successful full-scale culture and (3) to identify current limitations and the topics on which further research is required. Knowledge of cephalopod culture methods is advanced, but commercialization is still constrained by the highly selective feeding habits of cephalopods and their requirement for large quantities of high-quality (preferably live) feed, particularly in the early stages of development. Future research should focus on problems related to the consistent production of viable numbers of juveniles, the resolution of which requires a better understanding of nutrition at all phases of the life cycle and better broodstock management, particularly regarding developments in genetic selection, control of reproduction and quality of eggs and offspring.

Defensive Responses of Cuttlefish to Different Teleost Predators

We evaluated cuttlefish (Sepia officinalis) responses to three teleost predators: bluefish (Pomatomus saltatrix), summer flounder (Paralichthys dentatus), and black seabass (Centropristis striata). We hypothesized that the distinct body shapes, swimming behaviors, and predation tactics exhibited by the three fishes would elicit markedly different antipredator responses by cuttlefish. Over the course of 25 predator-prey behavioral trials, 3 primary and 15 secondary defense behaviors of cuttlefish were shown to predators. In contrast, secondary defenses were not shown during control trials in which predators were absent. With seabass-a benthic, sit-and-pursue predator-cuttlefish used flight and spent more time swimming in the water column than with other predators. With bluefish-an active, pelagic searching predator-cuttlefish remained closely associated with the substrate and relied more on cryptic behaviors. Startle (deimatic) displays were the most frequent secondary defense shown to seabass and bluefish, particularly the Dark eye ring and Deimatic spot displays. We were unable to evaluate secondary defenses by cuttlefish to flounder-a lie-and-wait predator-because flounder did not pursue cuttlefish or make attacks. Nonetheless, cuttlefish used primary defense during flounder trials, alternating between cryptic still and moving behaviors. Overall, our results suggest that cuttlefish may vary their behavior in the presence of different teleost predators: cryptic behaviors may be more important in the presence of active searching predators (e.g., bluefish), while conspicuous movements such as swimming in the water column and startle displays may be more prevalent with relatively sedentary, bottom-associated predators (e.g., seabass).

A study of the electrical polarization of Sepia officinalis yolk envelope, a role for Na+/K+-ATPases in osmoregulation?

The cuttlefish Sepia officinalis mate and spawn in the intertidal zone where eggs are exposed during low tide to osmotic stress. Embryonic outer yolk sac is a putative site for osmoregulation of young S. officinalis embryos. By using electrophysiological recordings and immunostaining we showed, (i) that the chorion is only a passive barrier for ions, since large molecules could not pass through it, (ii) that a complex transepithelial potential difference occurs through the yolk epithelium, (iii) that ionocyte-like cells and Na+/K+-ATPases were localized in the yolk epithelium and (iv) that ouabain sensitive Na+/K+-ATPase activity could participate to this yolk polarization. These data warrant further study on the role of ion transport systems of this epithelium in the osmoregulation processes in S. officinalis embryos.

Integration of sediment contamination with multi-biomarker responses in a novel potential bioindicator (Sepia officinalis) for risk assessment in impacted estuaries

For the purpose of biomonitoring, species that combine ecological and commercial importance may provide a link between ecological and human health risk. The common cuttlefish, Sepia officinalis, holds both characteristics in south-western Europe, albeit remaining unsurveyed in ecotoxicological studies. Cuttlefish collected from an impacted estuary in SW Portugal and a reference location off the coast were analysed for a battery of biomarker responses in the digestive gland and gills. The contrast to reference animals revealed that biomarker responses, especially those related to oxidative stress, were consistent with sediment contamination by PAHs, even in a situation that combines complex toxicant mixtures, moderate levels of contamination and high ecotoxicological diversity. However, environmental parameters related to the differences between shore and estuarine habitats should not be overruled. Also, digestive gland metallothionein retained significant specificity to metals even though previous studies in the area with clams and fish failed to trigger a conclusive response. The highest net differences in biomarker responses were detected in the gills, likely indicating higher sensitivity to environmental stressors. Still, the digestive gland responses were overall the most consistent with sediment contamination and effectively differentiated between estuarine industrial- and rural-impacted sites. The results indicate that S. officinalis may be a candidate to meet the European Union's requirements for efficient biomonitoring programmes, with the additional importance of being cosmopolitan, abundant, commercially valuable and combining the molluscan biology that has been granting bivalves their high value for biomonitoring with foraging behaviour, thus better able to reflect anthropogenic stressors impacting a wider area than sedentary organisms. Nevertheless, further investigations in unpolluted sites are needed to better evaluate the background levels of biomarker responses in the species.

Optimization of Common Cuttlefish (Sepia officinalis) Protein Hydrolysate Using Pepsin by Response Surface Methodology

Protein hydrolysate was prepared from common cuttlefish (Sepia officinalis) by pepsin. Hydrolysis conditions (time, temperature, and enzyme activity) were optimized by response surface methodology (RSM) using a factorial design. The regression coefficient was close to 0.999, observed during both experimental and validation runs, and indicated the validity of the prediction model. An enzyme activity to substrate ratio (X1), 0.04 AU/g protein; time (X2), 85 min; and temperature (X3), 45°C were found to be the optimum conditions for a higher degree of hydrolysis (21%) and nitrogen recovery (90%) using pepsin. According to amino acid analysis results and chemical score, methionine and histidine are the limiting amino acids in the hydrolysates, in that order. All other amino acids are present in sufficient quantity as required.

Chemical and biophysical properties of gelatins extracted from alkali-pretreated skin of cuttlefish (Sepia officinalis) using pepsin

Gelatins from alkali-pretreated skin of cuttlefish (Sepia officinalis) were extracted with different concentrations of pepsin at pH2.0 and characterized. The increase of enzyme concentration increased the yield of gelatin extraction. The yields of gelatins G5, G10 and G15, extracted, respectively, with 5, 10, and 15U of pepsin/g of alkali-treated skin of cuttlefish, were 6.59%, 7.69% and 9.22%, respectively, while in the absence of pepsin (G0) the yield was only 2.68%. Nevertheless, the extraction of gelatin from alkali-pretreated skin of cuttlefish with pepsin at pH2.0 affected the molecular weight, biophysical and functional properties of resulting gelatins. Gelatin extracted without pepsin showed the higher gel strength, and transition, gelling and melting temperatures, which are slightly higher than G5. All of these values decreased with increasing enzyme concentration. In addition, foam expansion (FE) was affected by enzymatic treatment, and the values decreased as pepsin increased. Nevertheless, the partially hydrolyzed gelatins showed the same emulsifying properties.

Effect of Degree of Hydrolysis and Protease Type on the Antioxidant Activity of Protein Hydrolysates From Cuttlefish (Sepia officinalis) By-Products

We have investigated the antioxidant activities of eight hydrolysates from cuttlefish by-products obtained by treatment with various gastrointestinal proteases (chymotrypsin, trypsin, and crude alkaline enzyme extracts from cuttlefish and sardinelle) and bacterial proteases (Alcalase and crude enzymes from Bacillus pumilus A1, Bacillus mojavensis A21, and Bacillus cereus BG1). The antioxidant activities of the cuttlefish by-products protein hydrolysates (CPHs) were evaluated using various in vitro antioxidant assays, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power, and total antioxidant capacity. All hydrolysates showed different degrees of hydrolysis (DH) and varying degrees of antioxidant activity. Among the different hydrolysates, cuttlefish crude enzyme hydrolysate exhibited the highest antioxidant activity, followed by sardinelle crude enzyme and Alcalase hydrolysates. Further, CPHs with different degrees of hydrolysis were prepared by treatment with proteolytic enzymes from cuttlefish, sardinelle, and B. mojavensis A21. All hydrolysates showed a greater antioxidative activity as indicated by all the methods considered. In addition, antioxidant activity in hydrolysates was positively correlated with the increase of DH. The results of this study indicated that CPHs might be a good candidate for further investigation in developing new antioxidants.

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 2-3days 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.

Sepia officinalis: A new biological model for eco-evo-devo studies

Lophotrochozoans are largely under-represented in ‘eco-evo-devo’. We aim to understand the evolution of cephalopods, a highly derived group in molluscan clade, in the light of the selective pressures that may be induced from their present mode of life. Sepia officinalis has been chosen for its particularities: a direct development, a complex nervous system allowing elaborated body patterning, and molluscan derived characters (brachial crown and funnel derived from the foot, internal calcareous shell). These characteristics have been selected because they confer adaptive advantages for its necto-benthic mode of life.We focus on the development of neuromuscular system that is implicated from environmental sensing to expression of complex behaviours. We study the expression during development of target genes identified using our EST library. We show that their role in S. officinalis is often different from those described in other species, as most genes show extended or restricted expression in the developing morphological novelties, or unexpected expression. Moreover, the nervous system – as well as the chromatophore network and the ability to perceive environment – differentiates very early and largely before the brain is constituted. These studies enlighten the particularities of molecular and physiological pathways in the cephalopod lineage. They underline the necessity to have a better evolutionary understanding of the genetic control of structure/function relationships.Genome sequencing on cephalopods – that possess the biggest genome among molluscs – appears crucial for comparative genomics in lophotrochozoan lineages. As S. officinalis is an ideally suitable eco-evo-devo model, we propose its genome sequencing as a priority.

Video analyses of chromatophore activity in the European cuttlefish, Sepia officinalis

There is a paucity of quantitative methods to measure chromatophore activity in cephalopods, without which, important questions regarding chromatophore function cannot be addressed. These questions include the mechanisms underlying graduated responses by chromatophores and the variations in responses by chromatophore color type. We have developed an efficient and accurate method to record and analyze the activity of individual chromatophores using a combination of video capture and computer analysis techniques. High performance video recordings were obtained from 20 to 40 chromatophores isolated from the fin of the European cuttlefish, Sepia officinalis. Captured frames were imported into MATLAB for image segmentation analyses using polygon masking to identify individual chromatophores. RGB thresholds were determined for each chromatophore analyzed and were used to follow the activity of individual chromatophores and to discriminate yellow and red-black chromatophores. Data from tests using colored paper squares and live chromatophores confirmed the utility of this novel procedure.

Microstructural and histochemical advances on the digestive gland of the common cuttlefish, Sepia officinalis L.

The cephalopod digestive gland is a complex organ that, although analogous to the vertebrate liver, has additional functions, with special (albeit not exclusive) note on its active role in digestion. Although the structure of the digestive cell and its main constituents are well known (among which “boules” and brown bodies are distinctive features), histological details of other cell types and the general structure of the digestive gland need still further research. By a thorough combination of histological and histochemical techniques, it is shown that the digestive gland diverticula of the common cuttlefish (Sepia officinalis L.) are comprised of three essential cell types: digestive, basal and excretory. Basal (“pyramidal”) cells are multi-functional, being responsible for cell replacement and detoxification, mainly through the production of calcic spherulae containing metals like copper and lead in a complex organic matrix of proteins and ribonucleins. Since copper- and lead-positive spherulae were almost absent from other cell types and lumen of the tubules, it appears that controlled bioaccumulation of these metals, rather than excretion, is the main detoxification mechanism. The results show that the organ is crossed by an intricate network of blood vessels, especially arteries and arterioles, whose contents share histochemical properties with a particular set of “boules” that are shed into the lumen of diverticula for elimination, suggesting that the organ actively removes unwanted metabolites from the haemolymph. Conversely, the rarer excretory cells appear to be specialized in the elimination of salts. Although the exact nature of many excretory and secretory products, as the metabolic pathways that originate them, remain elusive, the findings suggest an intricate interaction between the different cell types and between these and the surrounding media: haemolymph and digestive tract.

Small MPAs do not protect cuttlefish (Sepia officinalis)

Marine reserves have been widely implemented as tools for biodiversity conservation and fisheries management, amongst other goals. A large number of empirical studies have focused on their effects on reef fish populations. Yet, few studies have looked at their effectiveness on semelparous species such as cephalopods, in spite of their commercial importance in many coastal regions across the globe. In this study we combine behavioural (biotelemetry) and demographic (experimental fishing) data to understand the effects of the Luiz Saldanha Marine Park (LSMP) on local populations of cuttlefish, Sepia officinalis. We used a beyond-BACI design to analyze the possible effect of the implementation of a no-take area on the abundance and biomass of this species and acoustic telemetry data to assess its site fidelity and movements within the study area. Results indicate that there was no detectable effect of the implementation of the no-take area on the abundance or biomass of cuttlefish. We found evidence that acoustically tagged adult cuttlefish leave the reserve a few days or weeks after tagging. The fact that cuttlefish have low site fidelity inside the reserve and large movements across and beyond the study area explains why there is no increase in the population inside the MPA. These results suggest that small coastal marine reserves such as the LSMP are not effective in providing long term protection to cuttlefish populations and, probably, those of other short-lived, highly mobile cephalopods.

Movements and behaviour of European common cuttlefish Sepia officinalis in English Channel inshore waters: First results from acoustic telemetry

The increasing importance of the European common cuttlefish Sepia officinalis as a commercial fishery resource raises concerns that our limited understanding of its behaviour and movements in natural habitats may impede future management for this species. Whilst information regarding the fine-scale movements and behaviour of small marine species such as cuttlefish can be difficult to obtain, the increasing miniaturisation of electronic tags, combined with novel attachment techniques, has now enabled tracking of this cephalopod. In this study we describe the first use of electronic tags attached to adult and sub-adult S. officinalis to monitor their movements and behaviour within natural environments. Eight adults (170–205mm dorsal mantle length [DML]) were fitted with continuous acoustic transmitters and ten sub-adults (132–180mm DML) were fitted with coded acoustic transmitters. All transmitters were attached to the internal cuttlebone of the living animal. Tagged animals recovered rapidly from anaesthesia and surgical procedures and were found to retain tags for periods of up to 11weeks in this short-term study. Six of the eight adult cuttlefish released into a radio–acoustic positioning (VRAP) array left the area rapidly (≤45min) whilst two remained for multiple days (≤9days). Short-term site fidelity at a local level was observed in only two adults, whilst larger scale movements along the coastline (≤35km) occurred in three adults, indicating that a complex range of spatial behaviour patterns could occur among spawning adults. Similar patterns were observed in the space use of sub-adults monitored using a passive acoustic-receiver array on the seabed. Three sub-adults were detected within the study area over an extended period of time (≥73days), whilst the remaining sub-adults left the study area rapidly and without return. The capacity to monitor the movements of both adult and sub-adult cuttlefish in their natural environment will facilitate identification and understanding of ontogenic changes in behaviours and migratory patterns of wild populations. Our results highlight the potential of electronic tagging methods as tools to aid conservation and management of this important commercial fishery species in the future.

Hatching glands in cephalopods – A comparative study

Hatching of embryos from their eggs involves either mechanical and/or chemical support. In particular enzymes are widely used in the animal kingdom to weaken the egg layers and facilitate the embryo's escape. Although numerous morphological and biochemical studies exist on the hatching glands of invertebrates (such as sea urchins, ascidians, insects) and vertebrates (teleosts, amphibians, and mammals), little is known about the morphology of the hatching glands (Hoyle organs) in cephalopod hatchlings.In this study, the internal gland structure and the external appearance of the Hoyle organ are compared among several cephalopod species (Idiosepius pygmaeus; Euprymna scolopes; Sepia officinalis; Loligo gahi; Sepioteuthis lessoniana; Architeuthis sp.; Octopus vulgaris; Tremoctopus gracilis; Argonauta hians). In almost all cases the glandular system is restricted to the posterior part of the dorsal mantle surface. Only Octopus and Argonauta lack a specific glandular structure in this body region and the animals apparently use other mechanisms to penetrate the egg layers.In all decapod species (Idiosepius; Euprymna; Sepia; Loligo; Sepioteuthis; Architeuthis) as well as in Tremoctopus only one specific cell type is present in the Hoyle organ, which synthesizes granular material. The secretory droplets are more or less uniform in electron density in Idiosepius, Euprymna and Tremoctopus but exhibit translucent inclusions in the other decapods. The time of gland development, first synthesis of secretory products and later degeneration after hatching vary between the species.The present study contributes to our knowledge of glandular systems in cephalopods and allows comparison with hatching structures in other invertebrates and vertebrates.

English

The present study aims to evaluate the antitumor, antioxidant and anti-inflammatory activities of two molluscan extracts, Sepia officinalis, ink extract (IE) and Coelatura aegyptiaca extract (CE). The antioxidant activities of both extracts were evaluated using 1,1- diphenyl-2-picrylhydrazyl (DPPH) scavenging and lipid peroxidation assays. The analgesic effects were evaluated using the writhing, hot plate and formalin tests. Cytotoxicity assay was performed using sulphorhodamine B (SRB) method on hepatocellular carcinoma (HepG2) cell lines. The IE extract exhibited dose dependent radical scavenging activity. The two tested extracts showed inhibition of thiobarbituric acid–reactive substances (TBARAS) at all concentrations, with an IC50 value of 176.77 and 177.23 (µg/ml), respectively. IE and CE extracts showed analgesic action by inhibiting the acetic acid-induced writhing. IE exhibited significant anti-nociceptive actions in mice by increasing the latency period in the hot-plate test. Both extracts significantly decreased the time of paw lickings in both early and late phases. Furthermore, IE and CE showed cytotoxic activities HepG2 cell lines with IC50 value of 67 and 49.24 µg/ml, respectively. In conclusion, IE and CE extracts have antioxidant, anti-inflammatory and cytotoxic properties and can be considered as promising anticancer drugs. Key words: Sepia ink, Coelatura aegyptiaca, antioxidant, anti-inflammatory, cytotoxicity.

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.

Variability of cadmium accumulation in cephalopods (Octopus vulgaris, Sepia officinalis, Loligo vulgaris and Todarodes sagittatus) collected in Sardinia in 2008-2012

Cephalopods are known to accumulate cadmium and play an important role in its biomagnification. They are an essential link in marine trophic chain and represent an important case in studies on cadmium transfer to man through the food chain. Since cadmium concentration widely varies in different tissues of the cephalopods – mainly accumulating in the hepatopancreas – evisceration represents a recommended preliminary step to reduce cadmium intake in view of cephalopods consumption; yet, the residual concentration in the edible part may still be a risk for public health. This study is intended to assess cadmium levels variability in the muscles of Cephalopoda, considering the different feeding habitats and marine trophic webs. In compliance with EU regulation, a survey on cadmium and other heavy metal levels in various sea food, including cephalopods, was conducted by the Istituto Zooprofilattico of Sardinia in co-operation with local health authorities. During a five-year survey (2008- 2012), 90 samples were collected from the following species: commons octopus (Octopus vulgaris), cuttlefish (Sepia officinalis), European squid (Loligo vulgaris), and flying squid (Todarodes sagittatus), located in different coastal areas and representatives of either benthic or nektonic habitats. Determination of cadmium levels was carried out according to Regulations (EC) No. 882/2004, No. 1881/2006 and No. 333/2007. Analysis of the edible portion (muscle) of fresh homogenised samples was carried out by inductively coupled plasma mass spectrometry (ICP-MS). The results showed a highly skewed distribution of data. No statistically significant differences were observed among four distributions of the natural logs of cadmium levels in the species considered.

Complete mitochondrial genome of the common cuttlefish Sepia pharaonis (Sepioidea, Sepiidae)

In this study, the complete mitochondrial genome of the common cuttlefish Sepia pharaonis was determined first. The genome was 16,208 bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 main non-coding regions [both are control regions (CR)], the gene composition and order of which were similar to most other invertebrates. The overall base composition of S. pharaonis is T: 36.3%, C: 14.7%, A: 40.9% and G: 8.1%, with a hightly A + T bias of 77.2%. Two control regions all contain termination-associated sequences and conserved sequence blocks. This mitogenome sequence data would play an important role in the investigations of the phylogenetic relationships, taxonomic resolution and phylogeography of the Sepiidae.

Pump–Probe Microscopic Imaging of Jurassic-Aged Eumelanin

Melanins are biological pigments found throughout the animal kingdom that have many diverse functions. Pump-probe imaging can differentiate the two kinds of melanins found in human skin, eumelanin and pheomelanin, the distributions of which are relevant to the diagnosis of melanoma. The long-term stability of the melanin pump-probe signal is central to using this technology to analyze melanin distributions in archived tissue samples to improve diagnostic procedures. This report shows that most of the pump-probe signal from eumelanin derived from a Jurassic cephalopod is essentially identical to that of eumelanin extracted from its modern counterpart, Sepia officinalis. However, additional classes of eumelanin signals found in the fossil reveal that the pump-probe signature is sensitive to iron content, which could be a valuable tool for pathologists who cannot otherwise know the microscopic distributions of iron in melanins.