Understanding baseline levels of physiological stress tolerance from excessive exercise in a holobenthic octopus species, Octopus pallidus.

Cephalopods receive a great deal of attention due to their socioeconomically important fisheries and aquaculture industries as well their unique biological features. However, basic information about their physiological responses under stress conditions is lacking. This study investigated the impact of a simple stressor, exercise to exhaustion, on the activity levels of antioxidant enzymes and the concentrations of molecules involved in oxidative stress response in the pale octopus (Octopus pallidus). Eight biochemical assays were measured in the humoral (plasma) and cellular (hemocyte) components of O. pallidus haemolymph, the invertebrate analogue to vertebrate blood. Overall, exercise resulted in an increase in activity of plasma catalase (CAT) and glutathione-S-transferase (GST) and the decrease in activity of plasms glutathione reductase (GR). In the hemocytes, the exercise elicited a different response, with a reduction in the activity of superoxide dismutase (SOD), GR, and glutathione peroxidase (GPX) and a reduction in nitric oxide (NO) concentration. Malondialdehyde (MDA) activity was similar in the plasma and haemocytes in control and exercised treatments, indicating that exercise did not induce lipid peroxidation. These results provide an important baseline for understanding oxidative stress in octopus, with exercise to exhaustion serving as a simple stressor which will ultimately inform our ability to detect and understand physiological responses to more complex stressors.

The Effect of Prohibitins on Mitochondrial Function during Octopus tankahkeei Spermiogenesis.

Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic expression characteristics of Ot-PHBs, observed the colocalization of Ot-PHB1 with mitochondria and polyubiquitin, and studied the effect of phb1 knockdown on mitochondrial DNA (mtDNA) content, reactive oxygen species (ROS) levels, and apoptosis-related gene expression in spermatids. Our aim was to explore the effect of Ot-PHBs on mitochondrial function during the spermiogenesis of Octopus tankahkeei (O. tankahkeei), an economically important species in China. The predicted Ot-PHB1/PHB2 proteins contained an N-terminal transmembrane, a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin domain), and a C-terminal coiled-coil domain. Ot-phb1/phb2 mRNA were widely expressed in the different tissues, with elevated expression in the testis. Further, Ot-PHB1 and Ot-PHB2 were highly colocalized, suggesting that they may function primarily as an Ot-PHB compiex in O. tankahkeei. Ot-PHB1 proteins were mainly expressed and localized in mitochondria during spermiogenesis, implying that their function may be localized to the mitochondria. In addition, Ot-PHB1 was colocalized with polyubiquitin during spermiogenesis, suggesting that it may be a polyubiquitin substrate that regulates mitochondrial ubiquitination during spermiogenesis to ensure mitochondrial quality. To further investigate the effect of Ot-PHBs on mitochondrial function, we knocked down Ot-phb1 and observed a decrease in mtDNA content, along with increases in ROS levels and the expressions of mitochondria-induced apoptosis-related genes bax, bcl2, and caspase-3 mRNA. These findings indicate that PHBs might influence mitochondrial function by maintaining mtDNA content and stabilizing ROS levels; in addition, PHBs might affect spermatocyte survival by regulating mitochondria-induced apoptosis during spermiogenesis in O. tankahkeei.

Ocean acidification increases copper accumulation and exacerbates copper toxicity in Amphioctopus fangsiao (Mollusca: Cephalopoda): A potential threat to seafood safety.

Ocean acidification (OA) and trace metal pollutants coexist to exert combined effects on the functions and services of marine ecosystems. Increasing atmospheric carbon dioxide has caused a decrease in the pH of the ocean, affecting the bioavailability and speciation of trace metals and consequently altering metal toxicity in marine organisms. As an important trace metal functioned in hemocyanin, the richness of Copper (Cu) in octopuses is remarkable. Therefore, the biomagnification and bioaccumulation capacities of Cu in octopuses may be a non-negligible risk of contamination. Here, Amphioctopus fangsiao was continuously exposed to acidified seawater (pH 7.8) and copper (50 µg/L) to investigate the combined effect of ocean acidification and Cu exposure on marine mollusks. Our results showed that A. fangsiao could adapt well to ocean acidification after 21 days of the rearing experiment. However, the accumulation of Cu in A. fangsiao intestine increased significantly in acidified seawater under high levels of Cu stress. In addition, Cu exposure can influence the physiological function of A. fangsiao, including growth and feeding. This study also demonstrated that Cu exposure disturbed glucolipid metabolism and induced oxidative damage to intestine tissue, and ocean acidification further exacerbated these toxic effects. The obvious histological damage and microbiota alterations were also caused by Cu stress and its combined effect with ocean acidification. At the transcription level, we found numerous differentially expressed genes (DEGs) and significantly enriched KEGG pathways, involving glycolipid metabolism, transmembrane transport, glucolipid metabolism, oxidative stress, mitochondrial, protein and DNA damage, all revealing the strong toxicological synergetic effect of Cu and OA exposure and the molecular adaptation mechanism of A. fangsiao. Collectively, this study demonstrated that octopuses may withstand future ocean acidification conditions, however, the complex interactions of future OA and trace metal pollution need to be emphasized. OA can influence the toxicity of trace metals, inducing a potential threat to marine organism safety.

Brain compartmentalization based on transcriptome analyses and its gene expression in Octopus minor.

Coleoid cephalopods have a high intelligence, complex structures, and large brain. The cephalopod brain is divided into supraesophageal mass, subesophageal mass and optic lobe. Although much is known about the structural organization and connections of various lobes of octopus brain, there are few studies on the brain of cephalopod at the molecular level. In this study, we demonstrated the structure of an adult Octopus minor brain by histomorphological analyses. Through visualization of neuronal and proliferation markers, we found that adult neurogenesis occurred in the vL and posterior svL. We also obtained specific 1015 genes by transcriptome of O. minor brain and selected OLFM3, NPY, GnRH, and GDF8 genes. The expression of genes in the central brain showed the possibility of using NPY and GDF8 as molecular marker of compartmentation in the central brain. This study will provide useful information for establishing a molecular atlas of cephalopod brain.

Identifying Natural Bioactive Peptides from the Common Octopus (Octopus vulgaris Cuvier, 1797) Skin Mucus By-Products Using Proteogenomic Analysis.

The common octopus is a cephalopod species subject to active fisheries, with great potential in the aquaculture and food industry, and which serves as a model species for biomedical and behavioral studies. The analysis of the skin mucus allows us to study their health in a non-invasive way, by using a hardly exploited discard of octopus in the fishing sector. A shotgun proteomics approach combined with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using an Orbitrap-Elite instrument was used to create a reference dataset from octopus skin mucus. The final proteome compilation was investigated by integrated in-silico studies, including Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, network studies, and prediction and characterization analysis of potential bioactive peptides. This work presents the first proteomic analysis of the common octopus skin mucus proteome. This library was created by merging 5937 identified spectra of 2038 different peptides. A total of 510 non-redundant proteins were identified. Obtained results show proteins closely related to the defense, which highlight the role of skin mucus as the first barrier of defense and the interaction with the environment. Finally, the potential of the bioactive peptides with antimicrobial properties, and their possible application in biomedicine, pharmaceutical, and nutraceutical industry was addressed.

Design, preparation and characterization of octopus-like self-releasing intracellular protein transporter LEB5 based on Escherichia coli heat-labile enterotoxin.

Despite the great potential of protein drugs as intracellular therapeutic agents, the unmet challenge in breaking through the cell membrane barrier and delivering them to intracellular targets remains. Therefore, developing safe and effective delivery vehicles is critical for fundamental biomedical research and clinical applications. In this study, we designed an octopus-like self-releasing intracellular protein transporter, the LEB5, based on the heat-labile enterotoxin. This carrier comprises five identical units, each of which has three main components: a linker, a self-releasing enzyme sensitivity loop, and the LTB transport domain. The LEB5 comprises five purified monomers that self-assemble to create a pentamer with ganglioside GM1 binding capacity. The fluorescent protein EGFP was used as a reporter system to identify the LEB5 features. The high-purity fusion protein ELEB monomer was produced from modified bacteria carrying pET24a(+)-eleb recombinant plasmids. EGFP protein could effectively detach from LEB5 by low dosage trypsin, according to electrophoresis analysis. The transmission electron microscopy results indicate that both LEB5 and ELEB5 pentamers exhibit a relatively regularly spherical shape, and the differential scanning calorimetry measurements further suggest that these proteins possess excellent thermal stability. Fluorescence microscopy revealed that LEB5 translocated EGFP into different cell types. Flow cytometry showed cellular differences in the transport capacity of LEB5. According to the confocal microscopy, fluorescence analysis and western blotting data, EGFP was transferred to the endoplasmic reticulum by the LEB5 carrier, detached from LEB5 by cleavage of the enzyme-sensitive loop, and released into the cytoplasm. Within the dosage range of LEB5 10-80 µg/mL, cell counting kit-8 assay revealed no significant changes in cell viability. These results demonstrated that LEB5 is a safe and effective intracellular self-releasing delivery vehicle capable of transporting and releasing protein medicines into cells.

Non-sulfated steroidal glycosides cistoindosides from marine 'old woman octopus' Cistopus indicus attenuate pro-inflammatory lipoxygenase.

Two non-sulfated steroidal glycosides, cistoindosides A-B were isolated from organic extract of the marine 'old woman octopus' Cistopus indicus (family Octopodidae). Their structures were characterized as 3ß-acteoxy-23ß-hydroxy-cholesta-9-ene-ß-D-xylopyranoside (cistoindoside A) and 22,23-epoxy-3ß-hydroxy-cholesta-5-ene-ß-D-4'-O-acetoxy-xylopyranoside (cistoindoside B). Cistoindoside B, glycosylated with ß-D-4'-O-acetoxy-xylopyranoside in conjunction with epoxy moieties displayed superior anti-inflammatory properties as acknowledged by its promising 5-lipoxygenase attenuation potential (IC50 2.11 µM) than the 5-lipoxygenase inhibitor drug zileuton (IC50 3.76 µM). The anti-inflammatory properties were corroborated by the promising antioxidant activities (IC50 ∼ 1.0-1.5 mM) of these steroid glycosides. Sizeably greater electronic properties, balanced hydrophobic-lipophilic properties (log POW ∼ 4.0) and comparatively lower steric factors were directly proportional to their bioactivities. Molecular simulation studies in the active sites of 5-lipoxygenase displaying lesser binding energies and inhibition constant (Ki) of cistoindoside B could be correlated with anti-inflammatory properties. Cistoindosides could be projected for their utilization as potential bioactive leads in functional food and pharmaceutical applications.

Oxaspiro Indiculides from Old Woman Octopus Cistopus indicus as Dual Inhibitors of Inducible Cyclooxygenase and Lipoxygenase.

The organic extract of the old woman octopus Cistopus indicus (Octopodidae), ubiquitous in the Central and South Indo-Pacific to the tropical Indian Ocean, was chromatographically fractionated over a reverse-phase adsorbent to yield two oxygenated spiro heterocyclic compounds, named indiculides A and B. Their structures were elucidated by using comprehensive spectroscopic methods. The radical scavenging potential displayed by indiculide A (IC50 ∼1.2 mM) besides attenuating the cyclooxygenase isoforms (COX-1/COX-2; IC50 3.36/3.02 µM) showed considerably superior activities when equated to those showed by indiculide B (IC50 3.45/3.22 µM). The inhibition property of indiculide A against 5-LOX (IC50 2.57 µM) was significantly greater than that of the standard 5-LOX inhibitor zileuton (IC50 3.70 µM, p<0.05). A greater selectivity index (anti-COX-1/anti-COX-2, 1.11) was perceived for indiculide A than that demonstrated by indiculide B (1.07) and anti-inflammatory drug diclofenac (0.96). Structure bio-activity relation study of indiculide A disclosed proportionality to the electronic properties besides permissible hydrophobicity-lipophilicity equilibrium, which could result in its efficient interface with the active site of inflammatory enzyme causing promising anti-inflammatory potential. Larger hydrogen bond networks of indiculide A on account of the more electronic-rich centers in conjunction with reduced docking factors reinforced its noteworthy attenuation potential against 5-LOX. The in vitro bioactivity assessment and in silico docking results were further validated by the superior drug-like characteristics of indiculide A (drug-likeness score, 0.21) than B analog, and therefore, the former metabolite could be a potential anti-inflammatory lead.

An Octopus-Derived Peptide with Antidiuretic Activity in Rats.

Discovering new drug candidates with high efficacy and few side effects is a major challenge in new drug development. The two evolutionarily related peptides oxytocin (OXT) and arginine vasopressin (AVP) are known to be associated with a variety of physiological and psychological processes via the association of OXT with three types of AVP receptors. Over decades, many synthetic analogs of these peptides have been designed and tested for therapeutic applications; however, only a few studies of their natural analogs have been performed. In this study, we investigated the bioactivity and usefulness of two natural OXT/AVP analogs that originate from the marine invertebrate Octopus vulgaris, named octopressin (OTP) and cephalotocin (CPT). By measuring the intracellular Ca2+ or cyclic AMP increase in each OXT/AVP receptor subtype-overexpressing cell, we found that CPT, but not OTP, acts as a selective agonist of human AVP type 1b and 2 receptors. This behavior is reminiscent of desmopressin, the most widely prescribed antidiuretic drug in the world. Similar to the case for desmopressin, a single intravenous tail injection of CPT into Sprague-Dawley rats reduced urine output and increased urinary osmolality. In conclusion, we suggest that CPT has a significant antidiuretic effect and that CPT might be beneficial for treating urological conditions such as nocturia, enuresis, and diabetes insipidus.

B-esterases characterisation in the digestive tract of the common octopus and the European cuttlefish and their in vitro responses to contaminants of environmental concern.

Cephalopods are a group of marine invertebrates that have received little attention as sentinel species in comparison to other molluscs, such as bivalves. Consequently, their physiological and biochemical xenobiotic metabolism responses are poorly understood. Here we undertake a comparative analysis of the enzymatic activities involved in detoxification reactions and neural transmission in the digestive tract of two commercial cephalopods: the Common octopus, Octopus vulgaris, and the European cuttlefish, Sepia officinalis. For methodological purposes, several common B-esterases (five carboxylesterase (CE) substrates and three cholinesterase (ChE) determinations) were assayed as a proxy of metabolic and neuronal activities, respectively. Four components of the digestive tract in each species were considered: salivary glands, the stomach, the digestive gland and the caecum. The in vitro responses of digestive gland homogenates to model chemicals and contaminants of environmental concern were contrasted between both cephalopod species. The baseline biochemical activities in the four digestive tract components were also determined. Moreover, in order to validate the protocol, purified proteins, recombinant human CE (CE1 and CE2) and purified eel acetylcholinesterase (AChE) were included in the analysis. Overall, carboxylesterase activities were higher in octopus than in cuttlefish, with the activity quantified in the digestive tract components in the following order: digestive gland ≈ caecum > stomach ≈ salivary glands, with higher hydrolysis rates reached with naphthyl-derived substrates. In contrast, cuttlefish hydrolysis rates with ChE substrates were higher than in octopus. This trend was also reflected in a higher sensitivity to CE inhibitors in octopus and to AChE inhibitors in cuttlefish. Given the detoxification character of CEs and its protective role preventing AChE inhibition, octopus could be regarded as more efficiently protected than cuttlefish from neurotoxic exposures. A full characterisation of B-esterases in the digestive tract of the two common cephalopods is also provided.

Impact of Short- and Long-Term Exposure to Elevated Seawater Pco2 on Metabolic Rate and Hypoxia Tolerance in Octopus rubescens.

AbstractMuch of the CO2 released by human activity into the atmosphere is dissolving into the oceans, making them more acidic. In this study we provide the first data on the short- and long-term impacts of ocean acidification on octopuses. We measured routine metabolic rate (RMR) of Octopus rubescens at elevated CO2 pressure (Pco2) with no prior acclimation and 1 or 5 wk of acclimation and critical oxygen pressure (Pcrit) after 5 wk of acclimation. Our results show that with no prior acclimation, octopuses had significantly higher RMRs in 1,500-µatm Pco2 environments than octopuses in 700- or 360-µatm environments. However, after both 1 and 5 wk of acclimation there was no significant difference in RMRs between octopuses at differing Pco2, indicating that octopuses acclimated rapidly to elevated Pco2. In octopuses acclimated for 5 wk at 1,500 µatm Pco2, we observed impaired hypoxia tolerance, as demonstrated by a significantly higher Pcrit than those acclimated to 700 µatm Pco2. Our findings suggest that O. rubescens experiences short-term stress in elevated Pco2 but is able to acclimate over time. However, while this species may be able to acclimate to near-term ocean acidification, compounding environmental effects of acidification and hypoxia may present a physiological challenge for this species.

Development of a 3D scanning method to discriminate blocks of Octopus minor with surplus water gain.

A 3D scanning method was developed to differentiate Octopus minor blocks which had surplus water to increase weight of O. minor. Effects of soaking time (0.5, 1 and 3 h) and apparent density of O. minor were determined using the number of O. minor in a block (4, 5, 6, and 7). A 0.5, 1, and 3 h soaking time increased O. minor weight by 11.85, 16.02, and 24.53%, respectively. Apparent density of non-weight gained O. minor blocks was significantly higher than those of 3 h soaked samples (p < 0.05). A 3D scanning method had limited ability to differentiate 1 h soaked and non-soaked samples, whereas it had high potential to discriminate 3 h soaked samples. Blind test using 25 blocks of O. minor showed that 3D scanning method evaluated 88% of prediction percentage. The total time of 3D scanning took <30 min for one block with a relatively high precision.

Synapsins are expressed at neuronal and non-neuronal locations in Octopus vulgaris.

Synapsins are a family of phosphoproteins fundamental to the regulation of neurotransmitter release. They are typically neuron-specific, although recent evidence pointed to their expression in non-neuronal cells where they play a role in exocytosis and vesicle trafficking. In this work, we characterized synapsin transcripts in the invertebrate mollusk Octopus vulgaris and present evidence of their expression not only in the brain but also in male and female reproductive organs. We identified three synapsin isoforms phylogenetically correlated to that of other invertebrates and with a modular structure characteristic of mammalian synapsins with a central, highly conserved C domain, important for the protein functions, and less conserved A, B and E domains. Our molecular modeling analysis further provided a solid background for predicting synapsin functional binding to ATP, actin filaments and secretory vesicles. Interestingly, we found that synapsin expression in ovary and testis increased during sexual maturation in cells with a known secretory role, potentially matching the occurrence of a secretion process. This might indicate that its secretory role has evolved across animals according to cell activity in spite of cell identity. We believe that this study may yield insights into the convergent evolution of ubiquitously expressed proteins between vertebrates and invertebrates.

Fish-Parasitic Gnathiid Isopods Metamorphose Following Invertebrate-Derived Meal.

Organisms with a parasitic lifestyle comprise a high proportion of biodiversity in aquatic and terrestrial environments. However, there is considerable variation in the ways in which they acquire nutrients. Hematophagy is a common consumption strategy utilized by some terrestrial, aquatic, and marine organisms whereby the parasite removes and digests blood from a host. Gnathiid isopods are marine hematophagous parasites that live in benthic substrates from the intertidal to the abyss. Although ecologically similar to ticks and mosquitoes, they feed only during each of 3 juvenile stages and adults do not feed. They have long been considered as generalist fish parasites and to date, there have been no reports of their successfully feeding on invertebrates. Based on observations of gnathiids attached to soft-bodied invertebrates collected from light traps, we conducted a laboratory experiment in which we collected and individually housed various common Caribbean invertebrates and placed them in containers with gnathiids to see if the gnathiids would feed on them. All fed gnathiids were subsequently removed from containers and given the opportunity to metamorphose to the next developmental stage. In total, 10 out of the 260 gnathiids that were presented with 1 of 4 species of potential invertebrate hosts had fed by the next morning. Specifically, 9 of a possible 120 gnathiids fed on lettuce sea slugs (Elysia crispata), and 1 of a possible 20 fed on a bearded fireworm (Hermodice carunculata). Eight of these 10 fed gnathiids metamorphosed to the next stage (5 to adult male, 2 to adult female, and 1 to third-stage juvenile). Even though feeding rates on invertebrates were considerably lower than observed for laboratory studies on fishes, this study provides the first documented case of gnathiids' feeding on and metamorphosing from invertebrate meals. These findings suggest that when fish hosts are not readily available, gnathiids could switch to soft-bodied invertebrates. They further provide insights into the evolution of feeding on fluids from live hosts in members of this family.

Convergent and parallel evolution in a voltage-gated sodium channel underlies TTX-resistance in the Greater Blue-ringed Octopus: Hapalochlaena lunulata.

The natural history and pharmacology of tetrodotoxin (TTX) has long intrigued biologists. This toxin has a remarkable distribution that spans two domains of life (Bacteria and Eukarya). Within Eukaryotes, TTX has only been identified in animals but is known to be present in over five-dozen species of phylogenetically distant Metazoans. Despite decades of work, the origin and biosynthetic pathways of TTX remain unresolved. Investigations in puffer fishes and salamanders have provided insights into the acquisition of auto-resistance to TTX through the evolution of voltage-gated sodium ion channels (VGSCs) that have reduced binding affinity for TTX. To date there have been no studies of these proteins in tetrodotoxic Blue-Ringed Octopuses. Here we report data demonstrating that the Greater Blue-ringed Octopus (Hapalochlaena lunulata) expresses a VGSC (HlNaV1) gene with mutations that reduce the channel's TTX-binding affinity and likely render the organism TTX resistant. We identified three amino-acid substitutions in the TTX-binding site of HlNaV1 that likely confer TTX-resistance to both the channel and the organism. These substitutions are associated with organismal TTX-resistance in other TTX-bearing taxa and are convergent with substitutions that have evolved in fish, salamanders, and some TTX-resistant invertebrates.

Methyl-end desaturases with ∆12 and ω3 regioselectivities enable the de novo PUFA biosynthesis in the cephalopod Octopus vulgaris.

The interest in understanding the capacity of aquatic invertebrates to biosynthesise omega-3 (ω3) long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) has increased in recent years. Using the common octopus Octopus vulgaris as a model species, we previously characterised a ∆5 desaturase and two elongases (i.e. Elovl2/5 and Elovl4) involved in the biosynthesis of LC-PUFA in molluscs. The aim of this study was to characterise both molecularly and functionally, two methyl-end (or ωx) desaturases that have been long regarded to be absent in most animals. O. vulgaris possess two ωx desaturase genes encoding enzymes with ∆12 and ω3 regioselectivities enabling the de novo biosynthesis of the C18 PUFA 18:2ω6 (LA, linoleic acid) and 18:3ω3 (ALA, α-linolenic acid), generally regarded as dietary essential for animals. The O. vulgaris ∆12 desaturase ("ωx2") mediates the conversion of 18:1ω9 (oleic acid) into LA, and subsequently, the ω3 desaturase ("ωx1") catalyses the ∆15 desaturation from LA to ALA. Additionally, the O. vulgaris ω3 desaturase has ∆17 capacity towards a variety of C20 ω6 PUFA that are converted to their ω3 PUFA products. Particularly relevant was the affinity of the ω3 desaturase towards 20:4ω6 (ARA, arachidonic acid) to produce 20:5ω3 (EPA, eicosapentaenoic acid), as supported by yeast heterologous expression, and enzymatic activity exhibited in vivo when paralarvae were incubated in the presence of [1-14C]20:4ω6. These results confirmed that several routes enabling EPA biosynthesis are operative in O. vulgaris whereas ARA and docosahexaenoic acid (DHA, 22:6ω3) should be considered essential fatty acids since endogenous production appears to be limited.

Fatty acids and elemental composition as biomarkers of Octopus vulgaris populations: Does origin matter?

The present study describes the novel use of fatty acids (FAs) and element profiles of Octopus vulgaris inhabiting three coastal areas in the W-Mediterranean Sea. These populations are exposed to different anthropogenic activities, and were compared at different geographical scales. The FA composition in the mantle of O. vulgaris exhibited significant differences in 22:6 n-3 (DHA) and 22:5 n-3 (EPA) among the sampled populations. The essential microelements Fe, Cu, Zn and Ni, and the non-essential microelements As, Sr, Al and Cd were the main contributors of variability among sampled octopus populations, with some notable differences among tissues. The variations in the FAs and elemental composition in octopus tissues were detected with other populations throughout the species distribution range, which might reflect differences in natural habitats and foraging strategies. Therefore, these may be considered biomarkers as a proxy to distinguish the origin of octopus specimens at different scales.

Characterization and bioactive potentials of secondary metabolites from mollusks Crassostrea madrasensis and Amphioctopus marginatus.

Chemical investigations of the ethyl acetate-methanol (EtOAc-MeOH) extract of the selected mollusks from the south-west coast of Arabian Sea led to the isolation of methyl-3-(26-phenylacetyloxy)-icosahydro-19-hydroxy-4,4,20-trimethylpicene-23-carboxylate (1) and methyl-3-(26-phenylacetyloxy)-icosahydro-1,19-dihydroxy-4,4,20-trimethylpicene-23-carboxylate (2) from Crassostrea madrasensis, whereas cholesta-5-en-3ß-yl-(32-methyl-(30-((E)-34-amino-36-ethyl-39-oxohept-36-enyl)-pentanedioate (3) and 7-ethyl-9-vinyl-octahydroazuleno [1,8-bc]pyran-3,12-dione (4) were isolated from Amphioctopus marginatus. Their structures were assigned by detailed spectroscopic experiments. The studied compounds were checked for antioxidant and anti-inflammatory activities by various in vitro experiments. The radical quenching potential of 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis-3-ethylbenzothiozoline-6-sulfonic acid diammonium salt were greater for 2 (IC50 0.85 and 1.10 mg/mL, respectively) than other studied compounds. The pro-inflammatory cyclooxygenase-2 inhibitory activity of 2 was greater (IC50 0.95 mg/mL) than those displayed by other studied compounds (>1.0 mg/mL). The 5-lipoxygenase inhibitory potential of 1 and 2 (~1.36 mg/mL) were greater than those displayed by 3 (1.64 mg/mL) and 4 (1.45 mg/mL).

The evolution and origin of tetrodotoxin acquisition in the blue-ringed octopus (genus Hapalochlaena).

Tetrodotoxin is a potent non-proteinaceous neurotoxin, which is commonly found in the marine environment. Synthesised by bacteria, tetrodotoxin has been isolated from the tissues of several genera including pufferfish, salamanders and octopus. Believed to provide a defensive function, the independent evolution of tetrodotoxin sequestration is poorly understood in most species. Two mechanisms of tetrodotoxin resistance have been identified to date, tetrodotoxin binding proteins in the circulatory system and mutations to voltage gated sodium channels, the binding target of tetrodotoxin with the former potentially succeeding the latter in evolutionary time. This review focuses on the evolution of tetrodotoxin acquisition, in particular how it may have occurred within the blue-ringed octopus genus (Hapalochlaena) and the subsequent impact on venom evolution.

Shotgun Proteomics Analysis of Saliva and Salivary Gland Tissue from the Common Octopus Octopus vulgaris.

The salivary apparatus of the common octopus ( Octopus vulgaris) has been the subject of biochemical study for over a century. A combination of bioassays, behavioral studies and molecular analysis on O. vulgaris and related species suggests that its proteome should contain a mixture of highly potent neurotoxins and degradative proteins. However, a lack of genomic and transcriptomic data has meant that the amino acid sequences of these proteins remain almost entirely unknown. To address this, we assembled the posterior salivary gland transcriptome of O. vulgaris and combined it with high resolution mass spectrometry data from the posterior and anterior salivary glands of two adults, the posterior salivary glands of six paralarvae and the saliva from a single adult. We identified a total of 2810 protein groups from across this range of salivary tissues and age classes, including 84 with homology to known venom protein families. Additionally, we found 21 short secreted cysteine rich protein groups of which 12 were specific to cephalopods. By combining protein expression data with phylogenetic analysis we demonstrate that serine proteases expanded dramatically within the cephalopod lineage and that cephalopod specific proteins are strongly associated with the salivary apparatus.