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.

Selection of reference genes for quantitative real-time PCR in Octopus minor (Cephalopoda: Octopoda) under acute ammonia stress.

High concentration of ammonia is a common issue in the aquaculture industry and often causes detrimental effects to aquatic products. Exploring expression regulation of genes involved in acute ammonia stress can help to understand the molecular mechanisms of ammonia response. Quantitative real-time PCR (qRT-PCR) with proper reference genes is an effective way to normalize the expression of target genes. To identify the most suitable reference genes for Octopus minor (Mollusca: Cephalopoda: Octopoda) under acute ammonia stress and the normal culturing, nine candidate genes were selected for the validation: OD, RPS18b, RPL29, RPS5, EF-1α, RPL6, AA4, ACT, TUB. The results showed that the stability of candidate genes varied considerably in gill, digestive gland, brain and hemolymph. Thus, the reference genes were determined separately in different tissues. RPS5 and RPL6 showed relatively high stability in gill, while RPL6, TUB, RPL29 and OD were four suitable reference genes in digestive gland. EF-1α, TUB and RPL6 were the best combination in brain and EF-1α and RPS18b were the most appropriate reference genes in hemolymph.

Biomarkers of physiological responses of Octopus vulgaris to different coastal environments in the western Mediterranean Sea.

The increase of pollutants in coastal seawater could produce several harmful biological effects on marine organisms related to the production of reactive oxygen species (ROS) causing cellular and tissue damages through oxidative stress mechanisms. Common octopuses (Octopus vulgaris) inhabiting coastal areas under high anthropogenic activity of Mallorca (W-Mediterranean Sea) have the ability to control oxidative damage by triggering antioxidant enzyme responses. Analyzing the digestive glands, octopuses from human-altered coastal areas showed higher activity of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) compared to octopuses from non-influenced coastal waters (i.e. marine reserve area). Higher metallothionein (MT) concentrations and lack of malondialdehyde (MDA) variations also reflect adaptations of O. vulgaris to polluted areas. This is the first study assessing the levels of the oxidative stress biomarkers on O. vulgaris in the Mediterranean Sea, revealing their usefulness to assess diverse environmental pollution effects on this relevant ecological and commercial species.

Transcriptional and biochemical effects of cadmium and manganese on the defense system of Octopus vulgaris paralarvae.

Due to anthropogenic activities the relative concentrations of cadmium and manganese have increased in the marine environment. Cephalopods are able to accumulate such metals and, as inhabitant of coastal waters, Octopus vulgaris is continuously exposed to anthropogenic activities. Since no study is available on the effects of heavy metals at molecular level in developing octopuses, herein we exposed 1-day-old paralarvae for 24 h to 10, 100, and 1000 µg/L of CdCl2 or MnCl2. Cd exerted a concentration-dependent inhibition of survival and a reduction in growth rate was shown while Mn exposure did not affect the survival rate even at the highest concentrations. Gene expression profiles of hsp70, sod, cat, and gst genes were analyzed by quantitative real-time PCR and defined patterns of transcription were observed. Moreover posttranscriptional analyses were also performed suggesting the impairment of metabolic functions, under strong oxidative conditions (as occurred in paralarvae exposed to Cd) or the complete detoxification events (as occurred in paralarvae exposed to Mn).

Dose-dependent effects of the clinical anesthetic isoflurane on Octopus vulgaris: a contribution to cephalopod welfare.

Recent progress in animal welfare legislation relating to invertebrates has provoked interest in methods for the anesthesia of cephalopods, for which different approaches to anesthesia have been tried but in most cases without truly anesthetizing the animals. For example, several workers have used muscle relaxants or hypothermia as forms of "anesthesia." Several inhalational anesthetics are known to act in a dose-dependent manner on the great pond snail Lymnaea stagnalis, a pulmonate mollusk. Here we report, for the first time, on the effects of clinical doses of the well-known inhalational clinical anesthetic isoflurane on the behavioral responses of the common octopus Octopus vulgaris. In each experiment, isoflurane was equilibrated into a well-aerated seawater bath containing a single adult O. vulgaris. Using a web camera, we recorded each animal's response to touch stimuli eliciting withdrawal of the arms and siphon and observed changes in the respiratory rate and the chromatophore pattern over time (before, during, and after application of the anesthetic). We found that different animals of the same size responded with similar behavioral changes as the isoflurane concentration was gradually increased. After gradual application of 2% isoflurane for a maximum of 5 min (at which time all the responses indicated deep anesthesia), the animals recovered within 45-60 min in fresh aerated seawater. Based on previous findings in gastropods, we believe that the process of anesthesia induced by isoflurane is similar to that previously observed in Lymnaea. In this study we showed that isoflurane is a good, reversible anesthetic for O. vulgaris, and we developed a method for its use.

Uptake, transfer and elimination kinetics of paralytic shellfish toxins in common octopus (Octopus vulgaris).

Marine phycotoxins derived from harmful algal blooms are known to be associated with mass mortalities in the higher trophic levels of marine food webs. Bivalve mollusks and planktivorous fish are the most studied vectors of marine phycotoxins. However, field surveys recently showed that cephalopod mollusks also constitute potential vectors of toxins. Thus, here we determine, for the first time, the time course of accumulation and depuration of paralytic shellfish toxins (PSTs) in the common octopus (Octopus vulgaris). Concomitantly, the underlying kinetics of toxin transfer between tissue compartments was also calculated. Naturally contaminated clams were used to orally expose the octopus to PSTs during 6 days. Afterwards, octopus specimens were fed with non-contaminated shellfish during 10 days of depuration period. Toxins reached the highest concentrations in the digestive gland surpassing the levels in the kidney by three orders of magnitude. PSTs were not detected in any other tissue analyzed. Net accumulation efficiencies of 42% for GTX5, 36% for dcSTX and 23% for C1+2 were calculated for the digestive gland. These compounds were the most abundant toxins in both digestive gland and the contaminated shellfish diet. The small differences in relative abundance of each toxin observed between the prey and the cephalopod predator indicates low conversion rates of these toxins. The depuration period was better described using an exponential decay model comprising a single compartment - the entire viscera. It is worth noting that since octopuses' excretion and depuration rates are low, the digestive gland is able to accumulate very high toxin concentrations for long periods of time. Therefore, the present study clearly shows that O. vulgaris is a high-potential vector of PSTs during and even after the occurrence of these toxic algal blooms.

Behavioural and immunological responses to an immune challenge in Octopus vulgaris.

Behavioural and immunological changes consequent to stress and infection are largely unexplored in cephalopods, despite the wide employment of species such as Octopus vulgaris in studies that require their manipulation and prolonged maintenance in captivity. Here we explore O. vulgaris behavioural and immunological (i.e. haemocyte number and serum lysozyme activity) responses to an in vivo immune challenge with Escherichia coli lipopolysaccharides (LPS). Behavioural changes of immune-treated and sham-injected animals were observed in both sight-allowed and isolated conditions, i.e. visually interacting or not with a conspecific. Immune stimulation primarily caused a significant increase in the number of circulating haemocytes 4h after the treatment, while serum lysozyme activity showed a less clear response. However, the effect of LPS on the circulating haemocytes begins to vanish 24h after injection. Our observations indicate a significant change in behaviour consequent to LPS administration, with treated octopuses exhibiting a decrease of general activity pattern when kept in the isolated condition. A similar decrease was not observed in the sight-allowed condition, where we noticed a specific significant reduction only in the time spent to visually interact with the conspecific. Overall, significant, but lower, behavioural and immunological effects of injection were detected also in sham-injected animals, suggesting a non-trivial susceptibility to manipulation and haemolymph sampling. Our results gain importance in light of changes of the regulations for the use of cephalopods in scientific procedures that call for the prompt development of guidelines, covering many aspects of cephalopod provision, maintenance and welfare.

Differential proteins of the optic ganglion in octopus vulgaris under methanol stress revealed using proteomics.

An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 µg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.

DNA damage and metal accumulation in four tissues of feral Octopus vulgaris from two coastal areas in Portugal.

The alkaline comet assay has been employed for the first time to estimate the basal DNA damage in the digestive gland, gills, kidney and gonads of Octopus vulgaris. Octopuses were captured in two coastal areas adjacent to the cities of Matosinhos (N) and Olhão (S), Portugal. The area of Matosinhos is influenced by discharges of the Douro River, city of Porto, industries and intensive agriculture, while Olhão is an important fisheries port. Previous works point to contrasting metal availability in the two coastal areas. Among the analysed tissues digestive gland presented the highest levels of Zn, Cu, Cd and Pb. Tissues of specimens from Matosinhos exhibited high levels of Cd and from Olhão enhanced Pb concentrations. The DNA damages in digestive gland, gills and kidney were more accentuated in specimens from Matosinhos than from Olhão, suggesting a stronger effect of contaminants. Elevated strand breakages were registered in digestive gland, recognised for its ability to store and detoxify accumulated metals. The DNA damages in kidney, gills and gonads were lower, reflecting reduced metal accumulation or efficient detoxification. The broad variability of damages in the three tissues may also mirror tissue function, specific defences to genotoxicants and cell-cycle turnover.

Acrosome reaction in Octopus tankahkeei induced by calcium ionophore A23187 and a possible role of the acrosomal screw.

The octopod sperm is unique especially in two aspects: the screw-shaped acrosome and its inner layered substructure (striation). The present study aims to investigate morphological changes of Octopus tankahkeei spermatozoa during the acrosome reaction (AR) and to pursue functions of the internal substructures revealed by inducing AR with the calcium ionophore A23187. Gradual changes of the spermatozoa were traced using fluorescence and electron microscopy. The AR process included the bulging, vesiculation, and dehiscence of the plasma membrane around the acrosome and the nucleus, as well as the vesiculation of the mitochondrial sheath. Membrane vesiculation outside the nucleus has never been reported in the order Octopoda. The rigid screw and the inner striation of the acrosome remained intact surmounting the nucleus, suggesting that these two structures have potential functions during fertilization. In addition, the detachment of the sperm head and the tail was commonly observed in this study, both in intact and acrosome-reacted sperm. Fluorescence microscopy revealed that the detached mitochondrial sheath usually gave weaker and more dispersive signals than the joint ones. This phenomenon implied that the intense energy release might promote the detachment of the mitochondrial sheath.

Neuropeptidergic control of Octopus oviducal gland.

The oviducal gland of the female of Octopus vulgaris lies about halfway along the oviduct. Progesterone and 17beta-estradiol receptors have been immunolocalized in the nuclei of the cells of the glandular compartment of previtellogenic glands. We also have evidence of FMRFamide-like and cGnRH-I-like immunoreactivity in the nerve endings that reach the oviducal gland. Moreover, we have recently shown APGWamide immunoreactivity in the glandular cells of the inner part of the oviducal gland. Here we report a review on these findings as well as our latest studies on the effect that neuropeptides may exert on the secretory activity of the oviducal gland. cAMP seems to be a possible second messenger involved in such a process. We discuss the findings of a neuropeptidergic action on the glandular cells of oviducal gland in a more complex frame of molecules, such as steroids, biogenic amines and neuromodulators, controlling the activity of the gland.

Cholinergic modulation of L-type calcium current in isolated sensory hair cells of the statocyst of octopus, Eledone cirrhosa.

Whole-cell voltage-clamp recordings from dissociated hair cells of the statocyst of octopus, Eledone cirrhosa, demonstrated that application of ACh, carbachol or muscarine (10 microM) reversibly decreased the amplitude of L-type calcium current (I(Ca,L)), while nicotine (10-100 microM) did not have any effect. Furthermore, atropine blocked the effect of ACh and agonists suggesting that ACh reduces I(Ca,L) through activation of muscarinic receptors. Internal dialysis of these cells with guanosine 5'-O-3-thiotriphosphate (GTPgammaS), a non-hydrolysable GTP analogue, mimicked the ACh-induced inhibition of I(Ca,L) and occluded any further ACh-induced inhibition. Internal dialysis of these hair cells with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) reduced the ACh-induced inhibition I(Ca,L). The inhibitory effects of ACh were abolished by pre-incubation of these cells with pretussis toxin (PTX) suggesting that ACh-induced inhibition of I(Ca,L) involves a PTX-sensitive G protein pathway.

Effects of nitric oxide donors on the afferent resting activity in the cephalopod statocyst.

The effects of bath applications of the nitric oxide (NO) donors sodium nitroprusside (SNP), diethylamine sodium (DEA), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-penicillamine (SNAP) on the resting activity (RA) of afferent crista fibers were studied in isolated statocysts of the cuttlefish Sepia officinalis. The NO donors had three different effects: inhibition, excitation, and excitation followed by an inhibition. The SNAP analog N-acetyl-DL-penicillamine (xSNAP; with no NO moiety) had no effect. When the preparation was pre-treated with the NO synthase inhibitor N(G)-nitric-L-arginine methyl ester HCl (L-NAME), the NO donors were still effective. When the preparation was pre-treated with the guanylate cyclase inhibitors methylene blue (M-BLU) or cystamine (CYS), NO donors had only excitatory effects, whereas their effects were inhibitory only when pre-treatment was with the adenylate cyclase inhibitors nicotinic acid (NIC-A), 2',3'-dideoxyadenosine (DDA), or MDL-12330A. When pre-treatment was with a guanylate and an adenylate cyclase inhibitor combined, NO donors had no effect; in that situation, the RA of the afferent fibers remained and the preparation still responded to bath applications of GABA. Selective experiments with statocysts from the squid Sepioteuthis lessoniana and the octopod Octopus vulgaris gave comparable results. These data indicate that in cephalopod statocysts an inhibitory NO-cGMP and an excitatory NO-cAMP signal transduction pathway exist, that these two pathways are the key pathways for the action of NO, and that they have only modulatory effects on, and are not essential for the generation of, the RA.

Scopolamine impairs memory recall in Octopus vulgaris.

The involvement of the central cholinergic system in predatory performance, and on the recall of individual and observational memory in Octopus vulgaris was studied by treating the animals with the muscarinic antagonist scopolamine (2 mg/kg). The absence of the effects of the injection of scopolamine on blood circulation was also checked. Scopolamine did not affect the ability of octopuses to prey on live crabs. However, it interfered significantly with memory recall. In fact, the ability to solve the jar problem was impaired within the first hour after injection (short-term effects) and was only partially recovered after 24 h (long-term). Moreover, both individual and observational learning of a visual discrimination were significantly reduced at the short- and long-term testing. These results support a role of the cholinergic system in the processes of memory recall of O. vulgaris.

The effect of L-glutamate on the afferent resting activity in the cephalopod statocyst.

The effects of bath application of L-glutamate and of excitatory amino acid agonists and antagonists on the resting activity of afferent crista fibers were studied in isolated preparations of the statocyst of the cuttlefish, Sepia officinalis. L-Glutamate (threshold 10(-5) M) and its agonists quisqualate and kainate (thresholds 10(-6) M) increased the resting activity in a dose-dependent manner. Glutamine (threshold 10(-5) M) was also excitatory, while D-glutamate had no effect. Also, no obvious excitatory effects were seen for NMDA and L-aspartate, nor was any antagonistic effect seen for the selective NMDA-receptor antagonist D-2-amino-5-phosphonovaleric acid (D-AP-5). The spider toxin Argiotoxin636 (threshold 10(-11) M), 2-amino-4-phosphonobutyric acid (AP-4), glutamic acid diethyl ester (GDEE), gamma-D-glutamylaminomethyl-sulfonic acid (GAMS), and kynurenic acid decreased the resting activity and effectively blocked or reversed the effect of L-glutamate and its non-NMDA agonists. Preliminary experiments with statocysts from the squid Sepioteuthis lessoniana and the octopod Octopus bimaculoides gave comparable results. All data show that in cephalopod statocysts L-glutamate, via non-NMDA receptors, has an excitatory effect on the activity of afferent fibers, an effect consistent with its possible function as a hair cell transmitter.

Chronotropic and inotropic effects of atrial peptides on the isolated systemic heart of Octopus vulgaris.

The chronotropic and inotropic effects of four atrial peptides (cardiodilatin 1-16, atrial natriuretic factor 8-33 and atriopeptin I and III) on the isolated systemic heart of Octopus vulgaris were studied. Using a preparation that produces a physiological stroke volume at physiological input pressures, it was found that ANF, atriopeptin I and atriopeptin III exerted both negative chronotropic and inotropic effects. In contrast, cardiodilatin produced a positive inotropic effect. A dose-response curve of ANF is reported, showing a threshold concentration of about 10(-12) M. The pharmacological and physiological implications of these results are discussed in relation to some characteristics of the cephalopod systemic heart.