Comparative molecular analysis of evolutionarily distant glyceraldehyde-3-phosphate dehydrogenase from Sardina pilchardus and Octopus vulgaris

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), which is recognized as a key to central carbon metabolism in glycolysis and gluconeogenesis and as an important allozymic polymorphic biomarker, was purified from muscles of two marine species: the skeletal muscle of Sardina pilchardus Walbaum (Teleost, Clupeida) and the incompressible arm muscle of Octopus vulgaris (Mollusca, Cephalopoda). Comparative biochemical studies have revealed that they differ in their subunit molecular masses and in pI values. Partial cDNA sequences corresponding to an internal region of the GapC genes from Sardina and Octopus were obtained by polymerase chain reaction using degenerate primers designed from highly conserved protein motifs. Alignments of the deduced amino acid sequences were used to establish the 3D structures of the active site of two enzymes as well as the phylogenetic relationships of the sardine and octopus enzymes. These two enzymes are the first two GAPDHs characterized so far from teleost fish and cephalopod, respectively. Interestingly, phylogenetic analyses indicated that the sardina GAPDH is in a cluster with the archetypical enzymes from other vertebrates, while the octopus GAPDH comes together with other molluscan sequences in a distant basal assembly closer to bacterial and fungal orthologs, thus suggesting their different evolutionary scenarios.     

Early life-history processes in benthic octopus: Relationships between temperature, feeding, food conversion, and growth in juvenile Octopus pallidus

Initial growth in cephalopods is exponential, making early life-history critical in determining growth trajectories. Few captive studies have however examined the early life-history of cephalopods in fluctuating temperatures as would be encountered in the wild. This study investigates the relationship between early growth and the significant factors affecting growth, namely food intake, food conversion and fluctuating environmental temperatures. Pale octopus (Octopus pallidus) hatchlings were reared in captivity under either a warming or cooling temperature regime. Individual variations and periodicity in feeding rates (F_r), food conversion rates (C_r), growth rates (G_r), and the relationship between these variables and temperature were examined weekly. Food conversion rates were variable between individuals but also within individual octopus, which exhibited large fluctuations in C_r over time, exceeding 100% d^− 1 in one instance. Although individual F_r, C_r and G_r displayed fluctuations over time, there was no evidence of periodicity for any of the variables. Changes in temperature were not significantly correlated to changes in F_r, C_r or G_r. Feeding rate did not appear to influence growth rate or food conversion rate. Food conversion rate was negatively correlated to feeding rate in the same week, and positively correlated to growth rate. Short periods of low or no food consumption were common and the high values obtained for food conversion rate for some individuals suggest that octopus can grow substantially with little or no food intake. Individual variability observed in octopus growth may be dependent on the growth mechanism involved, specifically a fine balance between the continuous hyperplasic and hypertrophic growth found in cephalopods.     

Metallothioneins and trace elements in digestive gland,gills, kidney and gonads of Octopus vulgaris

Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (µg g^− 1, dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation.     

Distribution and selective elimination of paralytic shellfish toxins in different tissues of Octopus vulgaris

Octopus (Octopus vulgaris, Cuvier) plays a central role in the marine food web, being an important consumer with high metabolic rates and at the same time an important food item for higher predators. After harmful algal blooms, octopus can accumulate high levels of marine toxins trough trophic interrelationships. The aim of this study is to characterize the distribution of paralytic shellfish toxins (PSTs) in selected tissues of the O. vulgaris, in order to assess the translocation of toxins among organs with different physiological functions. Different retention times and selective elimination of particular toxin analogues were also investigated. Twenty three specimens of O. vulgaris were captured in Peniche (NW coast of Portugal) after PSTs have been detected in molluscan bivalves. Tissue matrices were dissected from organs with digestive function (digestive gland, stomach and salivary glands) and excretory function (kidneys and branchial hearts) and analyzed for toxin determination. Toxin analysis was carried out by high performance liquid chromatography with fluorescence detection (LC-FLD). PSTs were found in all tissues analyzed. The highest toxin concentrations were found in the digestive gland, reaching a maximum of 2980 μg STX equiv. kg⁻¹. The toxin profile was constituted by dcSTX, B1, C1 + 2, dcGTX2 + 3, dcNEO, STX and GTX2 + 3. A lower number of toxins were identified in the remaining organs, with B1 and dcSTX compromising more than 90% in molar fraction. Decarbamoyl saxitoxin was the most abundant toxin detected in digestive gland, stomach and salivary glands, while B1 was dominant in organs with excretory function. A positive correlation of concentrations of B1 and dcSTX were found in the organs analyzed. Results indicate that B1 and dcSTX are assimilated into the digestive gland in a similar proportion. Selective elimination of toxins with higher elimination of B1 and retention of dcSTX is suggested. This study contributes to better understanding of the dynamics of PSTs in O. vulgaris and the fate of PSTs in the food web.     

The Morphology and Adhesion Mechanism of Octopus vulgaris Suckers

The octopus sucker represents a fascinating natural system performing adhesion on different terrains and substrates. Octopuses use suckers to anchor the body to the substrate or to grasp, investigate and manipulate objects, just to mention a few of their functions. Our study focuses on the morphology and adhesion mechanism of suckers in Octopus vulgaris. We use three different techniques (MRI, ultrasonography, and histology) and a 3D reconstruction approach to contribute knowledge on both morphology and functionality of the sucker structure in O. vulgaris. The results of our investigation are two-fold. First, we observe some morphological differences with respect to the octopus species previously studied (i.e., Octopus joubini, Octopus maya, Octopus bimaculoides/bimaculatus and Eledone cirrosa). In particular, in O. vulgaris the acetabular chamber, that is a hollow spherical cavity in other octopuses, shows an ellipsoidal cavity which roof has an important protuberance with surface roughness. Second, based on our findings, we propose a hypothesis on the sucker adhesion mechanism in O. vulgaris. We hypothesize that the process of continuous adhesion is achieved by sealing the orifice between acetabulum and infundibulum portions via the acetabular protuberance. We suggest this to take place while the infundibular part achieves a completely flat shape; and, by sustaining adhesion through preservation of sucker configuration. In vivo ultrasonographic recordings support our proposed adhesion model by showing the sucker in action. Such an underlying physical mechanism offers innovative potential cues for developing bioinspired artificial adhesion systems. Furthermore, we think that it could possibly represent a useful approach in order to investigate any potential difference in the ecology and in the performance of adhesion by different species.     

Assessing the validity of stylets as ageing tools in Octopus pallidus

A method to age octopus was determined through the quantification of growth increments within the stylet structure (highly reduced internal shells). To validate the periodicity of the increments, stylets were analysed from known-age laboratory-raised Octopus pallidus. The animals, which ranged from 3 to 8 months old, were exposed either to a simulated natural or to constant temperature regime. Transverse stylet sections were embedded in thermo-plastic cement and ground until a thin section was achieved. A pre-hatch region and first post-hatch increment was identified in the stylet microstructure. The number of increments, across all ages and temperature treatments, was in remarkably close agreement to age (number of days), clearly demonstrating that stylet increments are deposited daily throughout the lifecycle of O. pallidus. Morphometric analyses of the stylet indicated that increments were laid down regularly during stylet growth and that stylets grew in concert with body size, further supporting daily periodicity. This study successfully validates daily increments in stylets and thus demonstrate a method to age octopus, therefore, potentially having critical implications for future octopus research and the effective management of stocks worldwide.     

Home occupancy by male Octopus vulgaris in a large seawater tank

Pairs of individually recognizable male Octopus vulgaris were observed in a large seawater tank containing two suitable homes (brick pots or plastic buckets). None of the animals established exclusive occupancy of one home and for much of the time both animals were associated together at the same site. Usually one of the two homes was preferred and its occupant was most likely to be the larger animal, or the earlier resident if they were of equal size. Large animals were observed to take food forcefully from smaller octopus. An arm alignment interaction is described which, it is suggested, may be a means by which two octopuses establish their relative sizes.     

The time and frequency of responses by Octopus vulgaris to an automatic food dispenser

An automatic food dispenser was designed for use with Octopus vulgaris Lamarck. One live crab was delivered each time the octopus pulled a white shape attached to the dispenser. The apparatus provided a continuous record of the time and frequency of feeding over periods of up to 15 days.     

Cardioactive peptides isolated from the brain of a Japanese octopus, Octopus minor

Octopus cardioactive peptides (Ocp-1: Gly-D-Phe-Gly-Asp, and Ocp-3: Gly-Ser-Trp-Asp) were isolated from brain extracts of the octopus, Octopus minor, using the isolated systemic heart as a bioassay. These peptides showed both positive chronotropic and inotropic effects on the heart. The stereoisomers at position 2 were also isolated, but their activities were only 1/10(3)-1/10(4) those of the corresponding isomers. The presence of the peptides in the systemic heart was confirmed by time-of-flight mass spectrometry (MS) and tandem MS analysis. The results suggested that Ocp-1 and Ocp-3 might be involved in excitatory control of the octopus cardiovascular system as neuropeptides and/or neurohormones.     

Extraintestinal gamogony of Aggregata octopiana in the reared common octopus (Octopus vulgaris) (Cephalopoda: Octopodidae)

Aggregata octopiana (Apicomplexa, Aggregatidae) is the most prevalent coccidian in the wild common octopus (Octopus vulgaris), whose heteroxenous life cycle includes gamogony and sporogony undergoing in the octopus digestive tract. In the infected reared octopi, we observed an unusual extraintestinal distribution of the coccidian, with both gamogony and sporogony ongoing in dermal and gill tissue. Oocysts and macrogamonts were embedded in the dermal connective tissue of octopian arms, demarcated by a thin cyst wall or multilayered dark membrane. In gill connective and epithelial tissue all developmental stages were observed, eliciting hemocytic infiltration. Sometimes a complete substitution of the tissue by cysts and developmental stages occurred, resulting in necrosis of gill tissue.     

pH evaluation in the digestive tract of the pygmy octopus,Paractopus digueti

This study is considered the first report on the digestive tract pH of the pygmy octopus (Paroctopus digueti). Adult octopuses obtained from the wild (mean ± SD) (42.1 ± 15.1 g), and those acclimated to captivity in a fed (25.4 ± 9.0 g, n = 15) or fasted (23.1 ± 6.1 g, n = 15) state, were studied. The digestive tract regions of buccal mass (BMA), anterior salivary glands (ASG), posterior salivary glands (PSG), crop (CRO), stomach (STO), caecum (CAE), digestive gland (DGL) and intestine (INT) were dissected. The pH of the internal part of the digestive tract regions was measured. Food intake (dry weight) per octopus was 53.8 ± 35.1 mg to 214.9 ± 157.6 mg at 15 min and 8 h, respectively. The apparent food transit time was approximately 8 h for the appearance of feces in the posterior intestine. In all cases, the pH of the digestive tract regions was lower than pH 7.0. No statistical difference was found when comparing the pH by digestive tract regions between wild octopuses and octopuses in captivity (fasting and feeding). In acclimatized octopuses, the average pH was 6.41 ± 0.22 and 6.41 ± 0.23 for fasting and fed octopuses, respectively. Although DGL had the lowest pH values relative to other digestive tract tissues (p < 0.05), pH was always >5.0 (6.04 ± 0.12 in the wild and 5.97 ± 0.17 in feeding octopuses). In conclusion, the pygmy octopus has an acidic pH in its digestive tract under fasting and feeding conditions.     

Profiles of digestive enzymes of two competing planktivores,silver carp and gizzard shad,differ

Typically, studies in digestive physiology in fish focus on a few enzymes and provide insight into the specific processes of the enzyme in a targeted species. Comparative studies assessing a wide number of digestive enzymes on fishes that compete for food resources are lacking, especially in the context of an introduced species. It is generally thought that the invasive silver carp (SVC; Hypophthalmichthys molitrix) directly compete for food resources with the native gizzard shad (GZS; Dorosoma cepedianum) in waters where they coexist. We compared 19 digestive enzymes between SVC and GZS throughout a year and in two rivers in the Midwestern United States: Illinois River and Wabash River. All digestive enzymes analyzed were detected in both SVC and GZS in both rivers. However, the profiles of the digestive enzymes varied by species. Alkaline phosphatase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase were all much higher in SVC than in GZS. Differences between digestive enzyme profiles were also observed between rivers and months. This study demonstrates the utility of using an ecological approach to compare physiological features in fishes.     

Protection of the hermit crab Pagurus pollicaris say from predators by hydroid-colonized shells

Only one study has shown that a hydroid-colonized gastropod shell was a deterrent to predation on hermit crabs. In the present study, the hydroid-colonized shell protected the hermit crab Paguruspollicaris Say from the shell-crushing stone crab Menippe mercenaria (Say) and the non-shell-crushing octopus Octopus joubini Robson. The shell-crushing calico crab Hepatus epheliticus (Johansson) was not deterred, however, by a hydroid-colonized shell.     

Superantigenic activity of toxic shock syndrome toxin-1 is resistant to heating and digestive enzymes

Aims: To elucidate the stability of superantigenic activity and pathogenesis of toxic shock syndrome toxin 1 (TSST‐1) and staphylococcal enterotoxin A (SEA) against heating and digestive enzymes. Methods and Results: Purified TSST‐1 and SEA were treated with heating, pepsin and trypsin that are related to food cooking, stomach and intestine conditions. The integrity, superantigenic activity and toxicity of treated TSST‐1 and SEA were analysed by Western blotting, spleen cell culture, cytokine assay and toxic shock models. Both TSST‐1 and SEA showed strong resistance to heating, pepsin and trypsin digestion. Furthermore, the treated TSST‐1 showed significant higher induction of interferon‐γ and toxic shock compared with that of SEA. Pepsin‐ or trypsin‐digested TSST‐1 fragments still showed significant superantigenic and lethal shock toxicities. Conclusions: The superantigenic activity of TSST‐1 was stable to heating and digestive enzymes. Pepsin‐ and trypsin‐digested TSST‐1 fragments still showed superantigenic and lethal shock activities, indicating that digested TSST‐1 could cross epithelial cells and induce systemic toxicity. Significance and Impact of the Study: This study found, for the first time, that pepsin‐ or trypsin‐digested smaller TSST‐1 retained significant superantigenic and lethal shock activities. The different resistance of TSST‐1 and SEA participates in the different pathogenic activities during food poisoning and toxic shock syndrome.     

Quantification of the age-pigment lipofuscin in known-age octopus (Octopus pallidus): A potential tool for age determination

Stylet increment analysis (SIA) is the key method to age octopus, however, currently it is not reliable for all species. The suitability of the age-pigment lipofuscin as an alternative ageing method for octopus was examined. To determine the relationship between age and lipofuscin known-age octopus (Octopus pallidus) were reared in the laboratory from hatching to eight months old. Twenty-eight individuals at three different ages (3, 6 and 8 months old) were collected for lipofuscin analysis. The first two age groups (n = 5 each) were reared under ambient temperatures, while the oldest group (n = 18) was reared under three different controlled temperature regimes (n = 6 per treatment). For comparison, five wild O. pallidus were also collected for lipofuscin analysis and aged using SIA. Lipofuscin was analysed in the brain tissue and quantified at a commercial ageing centre using standard histological methods. Lipofuscin granules were clearly discernable in the brain tissue, and there was a strong exponential relationship between age and lipofuscin (R² = 0.86). Lipofuscin concentration was not related to sex, temperature or body weight in same-age individuals. Except for one individual, the predicted age of the wild animals, based on the relationship between lipofuscin and age, was close to the age determined using SIA. This study is the first to report lipofuscin in an octopus species and shows that lipofuscin has excellent potential as an alternative ageing method for octopus. This research will have important applications for species which cannot be reliably aged using current ageing methods.     

Receptor-mediated inhibition of octopamine-stimulated adenylate cyclase in the optic lobe of Octopus vulgaris

1. Inhibition of octopamine-stimulated adenylate cyclase was studied in the optic lobe of Octopus vulgaris.2. The octopamine antagonist, mianserin, and the dopamine D₂ agonists, PPHT and metergoline, induced dose-dependent inhibition of octopamine-stimulated adenylate cyclase activity.3. The binding of the tritiated benzazepine neuroleptic YM-09151-2 to octopus membranes and the displacement of [³H]YM-09151-2 by PPHT, metergoline and spiperone were consistent with the presence of a D₂-like dopamine receptor in the octopus optic lobe.4. The conclusion is drawn that octopamine-stimulated adenylate cyclase in the octopus is negatively regulated by a dopamine D₂-like receptor.     

Bipedal locomotion in Octopus vulgaris: A complementary observation and some preliminary considerations

Lacking an external shell and a rigid endoskeleton, octopuses exhibit a remarkable flexibility in their movements. Bipedal locomotion is perhaps the most iconic example in this regard. Until recently, this peculiar mode of locomotion had been observed only in two species of tropical octopuses: Amphioctopus marginatus and Abdopus aculeatus. Yet, recent evidence indicates that bipedal walking is also part of the behavioral repertoire of the common octopus, Octopus vulgaris. Here we report a further observation of a defense behavior that encompasses both postural and locomotory elements of bipedal locomotion in this cephalopod. By highlighting differences and similarities with the other recently published report, we provide preliminary considerations with regard to bipedal locomotion in the common octopus.     

Factors affecting the spatial distribution of natural populations of Octopus joubini robson

Observations were made on the density and distribution of Octopus joubini in its natural environment. The average density of octopuses was 1/33 m² but their distribution was clumped. This distribution correlated significantly with the distribution of molluscan shells in which they hid. In addition, octopuses were attracted to sites that had been enriched with extra empty gastropod shells. Since site stability was not observed and they were not repelled from one another when placed at a high density, octopuses were probably not spaced by social pressure. Small crab species, the normal prey of O. joubini, were at a very high density of 30/m², and food abundance probably did not limit octopus number. The main factor affecting distribution of this population of octopuses may be predator pressure acting through the availability of places to hide.