Oxygen loss compromises growth and cognition of cuttlefish newborns.

Ocean deoxygenation and standing levels of hypoxia are shrinking fundamental niches, particularly in coastal areas, yet documented repercussions on species development and behaviour are limited. Here, we tackled the impacts of deoxygenation (7 mg O2 l-1), mild hypoxia (nocturnal 5 mg O2 l-1) and severe hypoxia (nocturnal 2 mg O2 l-1) on cuttlefish (Sepia officinalis) development (hatching success, development time, mantle length), cognition (ability to learn individually and socially) and behaviour (ability to camouflage and to explore its surroundings spatially). We found that hypoxia yielded lower survival rates, smaller body sizes and inhibited predatory (increased latency to attack the prey) and anti-predator (camouflage) behaviours. Acute and chronic exposure to low oxygen produced similar effects on cognition (inability to socially learn, increased open-field activity levels, no changes in thigmotaxis). It is thus expected that, although cuttlefish can withstand oxygen limitation to a certain degree, expanding hypoxic zones will diminish current habitat suitability.

Stress produces negative judgement bias in cuttlefish.

Judgement bias tasks (JBTs) are used to assess the emotional state and welfare of animals in zoos, farms and laboratories, based on the interpretation of an ambiguous or intermediate cue. Animals in positive affective states are more likely to interpret the ambiguous cue positively, whereas animals experiencing negative affect are more likely to interpret ambiguous cues pessimistically. Here, we developed a modified JBT assay for the stumpy-spined cuttlefish, Sepia bandensis, to determine whether cuttlefish exhibit negative affective states resulting from external stressors. Positive and neutral visual cues were presented twice daily until animals learned to associate food with the reinforced visual cue. After training, one treatment group was exposed to combined exposure and handling stress produced by 6 days of impoverished housing and simulated net capture. Our control group received no stress experience. In test trials performed after the stress experience, stressed animals showed higher latencies to approach ambiguous cues, spent significantly less time in rooms with ambiguous cues once they entered, and were less likely to enter first into the ambiguous cue-paired room compared with controls. These behaviours suggest that stress induces pessimistic judgement bias in cuttlefish, the first indication of this capacity in cephalopods.

Antimicrobial and cytotoxic activities of flavonoid and phenolics extracted from Sepia pharaonis ink (Mollusca: Cephalopoda).

BACKGROUND: Several studies have been reported previously on the bioactivities of different extracts of marine molluscs. Therefore, we decided to evaluate the cytotoxic and antimicrobial activities of S. pharaonis ink as a highly populated species in the Red Sea. We extracted the flavonoids from the ink and analyzed their composition. Then we evaluated systematically the cytotoxic and antimicrobial properties of this extract. A pharmacokinetic study was also conducted using SwissADME to assess the potential of the identified flavonoids and phenolic compounds from the ink extract to be orally active drug candidates. RESULTS: Cytotoxic activity was evaluated against 5 cell lines (MCF7, Hep G2, A549, and Caco2) at different concentrations (0.4 µg/mL, 1.6 µg/mL, 6.3 µg/mL, 25 µg/mL, 100 µg/mL). The viability of examined cells was reduced by the extract in a concentration-dependent manner. The highest cytotoxic effect of the extract was recorded against A549 and Hep G2 cancer cell lines cells with IC50 = 2.873 and 7.1 µg/mL respectively. The mechanistic analysis by flow cytometry of this extract on cell cycle progression and apoptosis induction indicated that the extract arrests the cell cycle at the S phase in Hep G2 and MCF7, while in A549 cell arrest was recorded at G1 phase. However, it causes G1 and S phase arrest in Caco2 cancer cell line. Our data showed that the extract has significant antimicrobial activity against all tested human microbial pathogens. However, the best inhibitory effect was observed against Candida albicans ATCC 10,221 with a minimum inhibitory concentration (MIC) of 1.95 µg/mL. Pharmacokinetic analysis using SwissADME showed that most flavonoids and phenolics compounds have high drug similarity as they satisfy Lipinski's criteria and have WLOGP values below 5.88 and TPSA below 131.6 Å2. CONCLUSION: S. pharaonis ink ethanolic extract showed a promising cytotoxic potency against various cell lines and a remarkable antimicrobial action against different pathogenic microbial strains. S. pharaonis ink is a novel source of important flavonoids that could be used in the future in different applications as a naturally safe and feasible alternative of synthetic drugs.

Cuttlefish adopt disruptive camouflage under dynamic lighting.

Many animals avoid detection or recognition using camouflage tailored to the visual features of their environment.1,2,3 The appearance of those features, however, can be affected by fluctuations in local lighting conditions, making them appear different over time.4,5 Despite dynamic lighting being common in many terrestrial and aquatic environments, it is unknown whether dynamic lighting influences the camouflage patterns that animals adopt. Here, we test whether a common form of underwater dynamic lighting, consisting of moving light bands that can create local fluctuations in the intensity of light ("water caustics"), affects the camouflage of cuttlefish (Sepia officinalis). Owing to specialized pigment cells (chromatophores) in the skin,6 these cephalopod mollusks can dynamically adjust their body patterns in response to features of their visual scene.7,8,9 Although cuttlefish resting on plain or patterned backgrounds usually expressed uniform or disruptive body patterns, respectively,10,11,12 exposure to these backgrounds in dynamic lighting induced stronger disruptive patterns regardless of the background type. Dynamic lighting increased the maximum contrast levels within scenes, and these maximum contrast levels were associated with the degree of cuttlefish disruptive camouflage. This adoption of disruptive camouflage in dynamically lit scenes may be adaptive, reducing the likelihood of detection, or alternatively, it could represent a constraint on visual processing.

Water temperature effects on embryonic development and GnRH-like expression in kisslip cuttlefish (Sepia lycidas) hinting the potential role of GnRH-like peptide.

Gonadotropin-releasing hormone (GnRH)-like peptides are multifunctional neuropeptides involved in cardiac control, early ontogenesis, and reproduction in cephalopods. However, the precise role of GnRH-like peptides in embryonic development and juvenile growth in cephalopods remains unknown. In this study, we showed that GnRH-like peptides are involved in the embryonic development of kisslip cuttlefish (Sepia lycidas). We confirmed that higher water temperatures induced early hatching. Simultaneously, we found that brain GnRH-like peptide gene expression gradually increased with increasing hatching speed. However, the rise in water temperature within a suitable range had no effect on the juvenile sex ratio or early gonadal development. Our results indicate that GnRH-like peptides may play an accelerating role in embryonic development; however, they are not involved in sex determination or early gonadal development in kisslip cuttlefish.

Non-pigmented laser hair removal mediated via sepia melanin nanoparticles: in vivo study on albino mice.

OBJECTIVES: Melanin is considered the main chromophore for laser hair removal. Due to a lack of laser-absorbing chromophores, removing non-pigmented hair with laser is quite problematic with unsatisfactory outcomes. This problem could be solved by delivering more melanin to the area around the hair follicle and enhancing that area as a target for light absorption. The insolubility of Sepia melanin as an exogenous dye, in most solvents, limits its bioavailability and thus its clinical use. METHODS: In our study, to overcome the solubility problems and increase the bioavailability of melanin for biomedical and cosmetic applications, natural sepia melanin was loaded in different nano-delivery systems (spanlastics and transfersomes) to be delivered to the hair follicles. The different formulations of melanin were prepared and characterized. In vivo skin deposition and histopathological studies were conducted on albino mice. RESULTS: Transmission electron microscopy (TEM) showed the spherical shape of the prepared vesicles with an average particle size of 252 and 262 nm and zeta potential of -22.5 and -35 mV for melanin spanlastics and melanin transfersomes, respectively. Histopathological examination of hair follicles and pilosebaceous glands for the irradiated and non-irradiated albino mice skin was studied post the application of the prepared formulations topically and subcutaneously. Qualitative statistical analysis was conducted and melanin transfersomes and melanin spanlastics showed significant damage to pilosebaceous glands and hair follicles with a p-value of 0.031 and 0.009 respectively. CONCLUSION: Melanin nanovesicles as transfersomes and spanlastics could be considered a promising approach for the removal of non-pigmented hair.

Histological and scanning electron microscope observations on the developing retina of the cuttlefish (Sepia officinalis Linnaeus, 1758).

In this work we present a detailed study of the major events during retinal histogenesis of the cuttlefish Sepia officinalis from early embryos to newly hatched animals and juveniles. For this purpose, we carried out morphometric and histological analyses using light and scanning electron microscopy. From St19, the first embryonic stage analysed, to St23/24 the embryonic retina is composed of a pseudostratified epithelium showing abundant mitotic figures in the more internal surface. At St24 the first photoreceptor nuclei appear in the presumptive inner segment layer, while an incipient layer of apical processes of the future rhabdomeric layer become visible at St25. From this stage onwards, both the rhabdomeric layer and the inner segment layer increase in size until postnatal ages. In contrast, the width of the supporting cell layer progressively decreases from St25/26 until postnatal ages. S. officinalis embryos hatched in a morphologically advanced state, showing a differentiated retina even in the last stages of the embryonic period. However, features of immaturity are still observable in the retinal tissue during the first postnatal weeks of life, such as the existence of mitotic figures in the apical region of the supporting cell layer and migrating nuclei of differentiating photoreceptors crossing the basal membrane to reach their final location in the inner segment layer. Therefore, postnatal retinal neurogenesis is present in juvenile specimens of S. officinalis.

Natural melanin nanoparticles (MNPs) extracted from Sepia officinalis: A cost-effective, chemo-photothermal, synergistic nanoplatform for osteosarcoma treatment.

Osteosarcoma conventional chemotherapeutics are known for their side effects, limited options, and induction of drug resistance. This creates the need to develop new therapeutics capable of effectively destroying cancer cells with low toxicity, improving patient survival rate and their life quality. This work reports a novel drug delivery nanoplataform made of Natural Melanin Nanoparticles (MNPs), obtained from Sepia officinalis ink, with 99% incorporation efficiency of doxorubicin (Dox) without the use of non-toxic solvents. A significant photothermal effect was shown by a 36ºC increment after 10 min of laser irradiation, surpassing reported values for synthetic melanin. A sustained drug release of ca. 23% with photothermal stimuli was observed, compared to 15% without stimuli, after 48 h. This nanoplatform is obtained as a food industry side product, which makes it a natural cost-effective biomedical material. Natural MPs were applied in an osteosarcoma cell line (SaOs-2), and internalized by the cells in less than 2 h, showing cytocompatibility up to 1000 µg/mL after 72 h of contact with cells. On the contrary, when natural MNPs loaded with Dox (Dox-MNPs) were placed in contact with the SaOs-2 cells and were simultaneously receiving NIR light it was observed a 93% reduction in cancer cells in 48 h, revealing a synergistic effect between chemotherapy and phototherapy. To our knowledge this is the first time that natural MNPs extracted from Sepia officinalis were tested on an osteosarcoma cell line as chemo-photothermal agent, showing these NPs are an effective, cost-effective, reproducible, non-toxic nanoplatform for osteosarcoma treatment using combined effects.

Comparative transcriptomic analysis of the different developmental stages of ovary in the cuttlefish Sepia pharaonis.

The cuttlefish, Sepia pharaonis, is characterized by rapid growth and strong disease resistance, making it an important commercially farmed cephalopod species in the southeastern coastal regions of China. However, in the reproductive process of S. pharaonis, there are challenges such as a low output of eggs, poor quality, and low survival rates of newly hatched juveniles. Therefore, there is an urgent need to study the molecular mechanisms underlying ovarian development in this species. In this study, we conducted the first transcriptomic analysis of the ovary at four developmental stages: the undeveloped stage, developing stage, nearly-ripe stage, and ripe stage, and compared the transcriptomics among these four stages using Illumina sequencing technology. The total numbers of clean reads of the four stages ranged from 40,890,772 to 52,055,714 reads. A total of 136,829 DEGs were obtained, GC base ratios of raw data were between 38.44 and 44.59%, and the number of uniquely mapped reads spanned from 88.08 to 95.90%. The Pearson correlation coefficient demonstrated a strong correlation among different samples within the same group, PCA and Anosim analysis also revealed that the grouping of these four stages was feasible, and each stage could be distinguished from the others. GO enrichment analysis demonstrated that ovarian follicle growth, sex differentiation, and transforming growth factor beta receptor, played a foreshadowing role at the early ovarian development stage, and the terms of small molecule metabolic process, peptide metabolic process, and catalytic activity were prominent at the mature stage. Meanwhile, KEGG analysis showed that the early ovarian development of S. pharaonis was mainly associated with the cell cycle, DNA replication, and carbon metabolism, while the mid-late ovarian development was involved with the signal transduction, endocrine system, and reproduction pathway. RT-qPCR further confirmed the consistent expression patterns of genes such as 17ß-HSD, GH, VGS, NFR, and NYR in the ovaries of S. pharaonis, exhibiting elevated levels of expression during the maturation stage. Conversely, ER and OM exhibited high expression levels during the early stages of ovarian development. These transcriptomic data provide insights into the molecular mechanisms of S. pharaonis ovarian development. The findings of this study will contribute to improving the reproduction and development of cuttlefish and enriching the bioinformatics knowledge of cephalopods.

Comparison of Cd, Cu, Se, and Zn Concentration in the Muscle and Hepatopancreas of Sepia pharaonis and Uroteuthis duvauceli in the North of Persian Gulf (Iran).

The objective of this research was to examine and contrast the levels of cadmium (Cd), copper (Cu), zinc (Zn), and selenium (Se) in the muscle and hepatopancreas tissues of two species, namely pharaoh cuttlefish (Sepia pharaonis) and Indian squid (Uroteuthis duvauceli), from the Persian Gulf. A total of thirty individuals of each species were gathered in January 2009 from the northern waters of the Persian Gulf. The metal concentrations were significantly higher in muscle tissue (p < 0.05) than in other tissues. S. pharaonis had higher metal concentrations than U. duvauceli. In the muscle and hepatopancreas samples of S. pharaonis, the highest mean concentrations were found to be for Zn (58.45 ± 0.96 µg/g dw) and Cu (1541.47 ± 192.15 µg/g dw), respectively. In U. duvauceli, the highest concentration of measured elements was seen for Zn in both muscle (36.52 ± 0.56 µg/g dw) and hepatopancreas (60.94 ± 2.65 µg/g dw). Se had the lowest concentration among the elements measured in both species. There was a negative and significant correlation between Cu and biometrical factors (total body length and weight) in both muscle and hepatopancreas samples of S. pharaonic and only in the muscle samples of U. duvauceli (p < 0.01, R2 = - 052; p < 0.01, R2 = - 0.055). However, there was a strong correlation between Zn and biometrical factors in hepatopancreas samples of both species. The comparison of metal concentrations with standards revealed that only Cd levels in S. pharaonis exceeded the ESFA and WHO standards, whereas other metals were below the standards.

Comparative proteomic analysis of the protein profile in the cuttlefish Sepia pharaonis associated with skin ulceration syndrome.

Skin ulceration syndrome (SUS) is becoming a severe problem in the breeding and culturing process of the cuttlefish Sepia pharaonis. However, limited knowledge is available about the occurrence of this devastating disease. In this study, proteomic analysis was used to identify the differentially expressed proteins (DEPs) and the biological pathways enriched in SUS-diseased S. pharaonis. Both the healthy group and diseased group were analyzed in triplicate, with 4 cuttlefish in each replicate. The results showed that 85 DEPs were identified between the two groups, including 36 upregulated proteins and 49 downregulated proteins in the diseased group compared to the healthy group. GO enrichment analysis revealed that the DEPs were mainly enriched in cellular component organization or biogenesis, nucleus and ion binding processes. The results of the KEGG pathway analysis indicated that extracellular matrix (ECM)-receptor interaction was the most enriched upregulated pathway. Real-time reverse transcriptase PCR was used to identify the expression of two differentially expressed matrix metalloproteinases (MMPs), and the results showed that the mRNA expression of MMP14 and MMP19 was significantly upregulated in the skin tissue of the diseased group. Furthermore, the protease activity of the diseased group was higher than that of the healthy group. Our results offer basic knowledge on the changes in protein profiles during the occurrence of SUS in the cuttlefish S. pharaonis.

Gene network analyses of Sepia esculenta larvae exposed to copper and cadmium: A comprehensive investigation of oxidative stress, immune response, and toxicological mechanisms.

Copper (Cu) and Cadmium (Cd), prevalent heavy metals in marine environments, have known implications in oxidative stress, immune response, and toxicity in marine organisms. Sepia esculenta, a cephalopod of significant economic value along China's eastern coastline, experiences alterations in growth, mobility, and reproduction when subjected to these heavy metals. However, the specific mechanisms resulting from heavy metal exposure in S. esculenta remain largely uncharted. In this study, we utilized transcriptome and four oxidative, immunity, and toxicity indicators to assess the toxicological mechanism in S. esculenta larvae exposed to Cu and Cd. The measurements of Superoxide Dismutase (SOD), Malondialdehyde (MDA), Glutathione S-Transferase (GST), and Metallothioneins (MTs) revealed that Cu and Cd trigger substantial oxidative stress, immune response, and metal toxicity. Further, we performed an analysis on the transcriptome data through Weighted Gene Co-expression Network Analysis (WGCNA) and Protein-Protein Interaction (PPI) network analysis. Our findings indicate that exposure methods and duration influence the type and the extent of toxicity and oxidative stress within the S. esculenta larvae. We took an innovative approach in this research by integrating WGCNA and PPI network analysis with four significant physiological indicators to closely examine the toxicity and oxidative stress profiles of S. esculenta upon exposure to Cu and Cd. This investigation is vital in decoding the toxicological, immunological, and oxidative stress mechanisms within S. esculenta when subjected to heavy metals. It provides foundational insights capable of advancing invertebrate environmental toxicology and informs S. esculenta artificial breeding practices.

The hydrodynamics of jet propulsion swimming in hatchling and juvenile European common cuttlefish, Sepia officinalis.

Cuttlefish swim using jet propulsion, taking a small volume of fluid into the mantle cavity before it is expelled through the siphon to generate thrust. Jet propulsion swimming has been shown to be more metabolically expensive than undulatory swimming, which has been suggested to be due to the lower efficiency of jet propulsion. The whole-cycle propulsive efficiency of cephalopod molluscs ranges from 38 to 76%, indicating that in some instances jet propulsion can be relatively efficient. Here, we determined the hydrodynamics of hatchling and juvenile cuttlefish during jet propulsion swimming to understand the characteristics of their jets, and whether their whole-cycle propulsive efficiency changes during development. Cuttlefish were found to utilise two jet types: isolated jet vortices (termed jet mode I) and elongated jets (leading edge vortex ring followed by a trailing jet; termed jet mode II). The use of these jet modes differed between the age classes, with newly hatched animals nearly exclusively utilising mode I jets, while juveniles showed no strong preferences. Whole-cycle propulsive efficiency was found to be high, ranging from 72 to 80%, and did not differ between age classes. During development, Strouhal number decreased as Reynolds number increased, which is consistent with animals adjusting their jetting behaviour in order to maximise whole-cycle propulsive efficiency and locomotor performance. Although jet propulsion swimming can have a relatively high energetic cost, in cuttlefish and nautilus, both neutrally buoyant species, the whole-cycle propulsive efficiency is actually relatively high.

Preliminary study on toxicological mechanism of golden cuttlefish (Sepia esculenta) larvae exposed to cd.

BACKGROUND: Cadmium (Cd) flows into the ocean with industrial and agricultural pollution and significantly affects the growth and development of economic cephalopods such as Sepia esculenta, Amphioctopus fangsiao, and Loligo japonica. As of now, the reasons why Cd affects the growth and development of S. esculenta are not yet clear. RESULTS: In this study, transcriptome and four oxidation and toxicity indicators are used to analyze the toxicological mechanism of Cd-exposed S. esculenta larvae. Indicator results indicate that Cd induces oxidative stress and metal toxicity. Functional enrichment analysis results suggest that larval ion transport, cell adhesion, and some digestion and absorption processes are inhibited, and the cell function is damaged. Comprehensive analysis of protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to explore S. esculenta larval toxicological mechanisms, and we find that among the 20 identified key genes, 14 genes are associated with neurotoxicity. Most of them are down-regulated and enriched to the neuroactive ligand-receptor interaction signaling pathway, suggesting that larval nervous system might be destroyed, and the growth, development, and movement process are significantly affected after Cd exposure. CONCLUSIONS: S. esculenta larvae suffered severe oxidative damage after Cd exposure, which may inhibit digestion and absorption functions, and disrupt the stability of the nervous system. Our results lay a function for understanding larval toxicological mechanisms exposed to heavy metals, promoting the development of invertebrate environmental toxicology, and providing theoretical support for S. esculenta artificial culture.

Biological light-weight materials: The endoskeletons of cephalopod mollusks.

Structural biological hard tissues fulfill diverse tasks: protection, defence, locomotion, structural support, reinforcement, buoyancy. The cephalopod mollusk Spirula spirula has a planspiral, endogastrically coiled, chambered, endoskeleton consisting of the main elements: shell-wall, septum, adapical-ridge, siphuncular-tube. The cephalopod mollusk Sepia officinalis has an oval, flattened, layered-cellular endoskeleton, formed of the main elements: dorsal-shield, wall/pillar, septum, siphuncular-zone. Both endoskeletons are light-weight buoyancy devices that enable transit through marine environments: vertical (S. spirula), horizontal (S. officinalis). Each skeletal element of the phragmocones has a specific morphology, component structure and organization. The conjunction of the different structural and compositional characteristics renders the evolved nature of the endoskeletons and facilitates for Spirula frequent migration from deep to shallow water and for Sepia coverage over large horizontal distances, without damage of the buoyancy device. Based on Electron-Backscatter-Diffraction (EBSD) measurements and TEM, FE-SEM, laser-confocal-microscopy imaging we highlight for each skeletal element of the endoskeleton its specific mineral/biopolymer hybrid nature and constituent arrangement. We demonstrate that a variety of crystal morphologies and biopolymer assemblies are needed for enabling the endoskeleton to act as a buoyancy device. We show that all organic components of the endoskeletons have the structure of cholesteric-liquid-crystals and indicate which feature of the skeletal element yields the necessary mechanical property to enable the endoskeleton to fulfill its function. We juxtapose structural, microstructural, texture characteristics and benefits of coiled and planar endoskeletons and discuss how morphometry tunes structural biomaterial function. Both mollusks use their endoskeleton for buoyancy regulation, live and move, however, in distinct marine environments.

The dynamics of pattern matching in camouflaging cuttlefish.

Many cephalopods escape detection using camouflage1. This behaviour relies on a visual assessment of the surroundings, on an interpretation of visual-texture statistics2-4 and on matching these statistics using millions of skin chromatophores that are controlled by motoneurons located in the brain5-7. Analysis of cuttlefish images proposed that camouflage patterns are low dimensional and categorizable into three pattern classes, built from a small repertoire of components8-11. Behavioural experiments also indicated that, although camouflage requires vision, its execution does not require feedback5,12,13, suggesting that motion within skin-pattern space is stereotyped and lacks the possibility of correction. Here, using quantitative methods14, we studied camouflage in the cuttlefish Sepia officinalis as behavioural motion towards background matching in skin-pattern space. An analysis of hundreds of thousands of images over natural and artificial backgrounds revealed that the space of skin patterns is high-dimensional and that pattern matching is not stereotyped-each search meanders through skin-pattern space, decelerating and accelerating repeatedly before stabilizing. Chromatophores could be grouped into pattern components on the basis of their covariation during camouflaging. These components varied in shapes and sizes, and overlay one another. However, their identities varied even across transitions between identical skin-pattern pairs, indicating flexibility of implementation and absence of stereotypy. Components could also be differentiated by their sensitivity to spatial frequency. Finally, we compared camouflage to blanching, a skin-lightening reaction to threatening stimuli. Pattern motion during blanching was direct and fast, consistent with open-loop motion in low-dimensional pattern space, in contrast to that observed during camouflage.

Fungi as a source of eumelanin: current understanding and prospects.

Melanins represent a diverse collection of pigments with a variety of structures and functions. One class of melanin, eumelanin, is recognizable to most as the source of the dark black color found in cephalopod ink. Sepia officinalis is the most well-known and sought-after source of non-synthetic eumelanin, but its harvest is limited by the availability of cuttlefish, and its extraction from an animal source brings rise to ethical concerns. In recent years, these limitations have become more pressing as more applications for eumelanin are developed-particularly in medicine and electronics. This surge in interest in the applications of eumelanin has also fueled a rise in the interest of alternative, bio-catalyzed production methods. Many culinarily-utilized fungi are ideal candidates in this production scheme, as examples exist which have been shown to produce eumelanin, their growth at large scales is well understood, and they can be cultivated on recaptured waste streams. However, much of the current research on the fungal production of eumelanin focuses on pathogenic fungi and eumelanin's role in virulence. In this paper, we will review the potential for culinary fungi to produce eumelanin and provide suggestions for new research areas that would be most impactful in the search for improved fungal eumelanin producers.

A brain atlas for the camouflaging dwarf cuttlefish, Sepia bandensis.

The coleoid cephalopods (cuttlefish, octopus, and squid) are a group of soft-bodied marine mollusks that exhibit an array of interesting biological phenomena, including dynamic camouflage, complex social behaviors, prehensile regenerating arms, and large brains capable of learning, memory, and problem-solving.1,2,3,4,5,6,7,8,9,10 The dwarf cuttlefish, Sepia bandensis, is a promising model cephalopod species due to its small size, substantial egg production, short generation time, and dynamic social and camouflage behaviors.11 Cuttlefish dynamically camouflage to their surroundings by changing the color, pattern, and texture of their skin. Camouflage is optically driven and is achieved by expanding and contracting hundreds of thousands of pigment-filled saccules (chromatophores) in the skin, which are controlled by motor neurons emanating from the brain. We generated a dwarf cuttlefish brain atlas using magnetic resonance imaging (MRI), deep learning, and histology, and we built an interactive web tool (https://www.cuttlebase.org/) to host the data. Guided by observations in other cephalopods,12,13,14,15,16,17,18,19,20 we identified 32 brain lobes, including two large optic lobes (75% the total volume of the brain), chromatophore lobes whose motor neurons directly innervate the chromatophores of the color-changing skin, and a vertical lobe that has been implicated in learning and memory. The brain largely conforms to the anatomy observed in other Sepia species and provides a valuable tool for exploring the neural basis of behavior in the experimentally facile dwarf cuttlefish.

Embryonic exposure to environmentally relevant levels of tributyltin affects embryonic tributyltin bioaccumulation and the physiological responses of juveniles in cuttlefish (Sepia pharaonis).

Tributyltin (TBT) is a typical organic pollutant that persists in aquatic sediments due to its wide usage as an antifouling fungicide during the past few decades. Despite increased awareness of the serious negative consequences of TBT on aquatic species, studies on the effects of TBT exposure on cephalopod embryonic development and juvenile physiological performance are scarce. To investigate the lasting effects of TBT toxicity on Sepia pharaonis from embryo to hatchling, embryos (gastrula stage, 3-5 h post fertilization) were exposed to four levels of TBT until hatching: 0 (control), 30 (environmental level), 60, and 120 ng/L. Subsequently, juvenile growth performance endpoints and behavioral alterations were assessed over 15 days post-hatching. Egg hatchability was significantly reduced and embryonic development (i.e., premature hatching) was accelerated in response to 30 ng/L TBT exposure. Meanwhile, TBT-induced alterations in embryonic morphology primarily included yolk-sac lysis, embryonic malformations, and uneven pigment distributions. During the pre-middle stage of embryonic development, the eggshell serves as an effective barrier to safeguard the embryo from exposure to 30-60 ng/L TBT, according to patterns of TBT accumulation and distribution in the egg compartment. However, even environmental relevant levels of TBT (30 ng/L) exposure during embryonic development had a negative impact on juvenile behavior and growth, including slowing growth, shortening eating times, causing more irregular movements, and increasing inking times. These findings indicate that after TBT exposure, negative long-lasting effects on S. pharaonis development from embryo to hatchling persist, suggesting that long-lasting toxic effects endure from S. pharaonis embryos to hatchlings.

Effect of polycyclic aromatic hydrocarbons on homeobox gene expression during embryonic development of cuttlefish, Sepia officinalis.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) commonly found in marine environments. Their bioaccumulation can cause harm to aquatic organisms, including invertebrates, particularly during the early stages of embryonic development. In this study, we evaluated, for the first time, the patterns of PAH accumulation in both capsule and embryo of common cuttlefish (Sepia officinalis). In addition, we explored the effects of PAHs by analysing the expression profiles of seven homeobox genes [i.e., gastrulation brain homeobox (GBX), paralogy group labial/Hox1 (HOX1), paralogy group Hox3 (HOX3), dorsal root ganglia homeobox (DRGX), visual system homeobox (VSX), aristaless-like homeobox (ARX) and LIM-homeodomain transcription factor (LHX3/4)]. We found that PAH levels in egg capsules were higher than those observed in chorion membranes (35.1 ± 13.3 ng/g vs 16.4 ± 5.9 ng/g). Furthermore, PAHs were also found in perivitellin fluid (11.5 ± 5.0 ng/ml). Naphthalene and acenaphthene were the congeners present at highest concentrations in each analysed egg component suggesting higher bioaccumulation rates. Embryos with high concentrations of PAHs also showed a significant increase in mRNA expression for each of the analysed homeobox genes. In particular, we observed a 15-fold increase in the ARX expression levels. Additionally, the statistically significant variation in homeobox gene expression patterns was accompanied by a concomitant increase in mRNA levels of both aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). These findings suggest that bioaccumulation of PAHs may modulate developmental processes of cuttlefish embryos by targeting homeobox gene-mediated transcriptional outcomes. Mechanisms underlying the upregulation of homeobox genes could be related to the ability of PAHs to directly activate AhR- or ER-related signaling pathways.