The best defense is a good offense: Anti-predator behavior of the common octopus (Octopus vulgaris) against conger eel attacks.

We present the description of defensive behavior in wild Octopus vulgaris against conger eel (Conger conger) attacks based on three video sequences recorded by recreational SCUBA divers in the eastern Atlantic off the coast of Galicia (NW Spain) and in the Cantabrian Sea (NW Spain). These records document common traits in defensive behavior: (1) the octopuses enveloped the conger eel's head to obscure its view; (2) they covered the eel's gills in an attempt to suffocate it; (3) they released ink; (4) the octopuses lost some appendages because of the fight. In the third video, the octopus did not exhibit the defensive behavior described in the first two videos due to an inability to utilize its arms in defense, and the conger eel's success in capturing octopuses is discussed. Additionally, both the cost that the octopus could face by losing some arms during the fight and whether the experience it acquires can be an advantage for future encounters are analyzed. The defensive behavior exhibited by octopuses in this study highlights their ability to survive in a hostile environment and serves as an example of the extensive repertoire of anti-predator strategies employed by these cephalopods.

Trophic ecology of Octopus vulgaris paralarvae along the Iberian Canary current eastern boundary upwelling system.

Our knowledge of the diet of wild octopus paralarvae, Octopus vulgaris, is restricted to the first 2 weeks of its planktonic phase when they are selective hunters found near the coastline. These small paralarvae, bearing only three suckers per arm, are transported by oceanic currents from the coast towards offshore waters, where they complete the planktonic phase over 2 months. Here, we have investigated the trophic ecology of O. vulgaris paralarvae in two contrasting upwelling sub-regions of the Iberian Canary current (ICC) eastern boundary upwelling system and have evaluated dietary change as paralarvae develop (inferred by counting the number of suckers per arm, ranging from three to 15) along the coastal-oceanic gradient during their planktonic phase. Using high-throughput amplicon sequencing, we have characterised the diet of 100 paralarvae collected along the Northwest Iberian Peninsula (n = 65, three to five suckers per arm) and off the west coast of Morocco (n = 35, three to 15 suckers per arm), identifying up to 87 different prey species. The diet of paralarvae varied along the ICC, with crabs (53.4%), siphonophores (12.2%), copepods (12.3%), cnidarians (8.4%) and pteropods (3.7%) accounting for 90% of the variability detected off NW Iberian Peninsula, whereas off W Morocco, crabs (46.2%), copepods (23.1%), cnidarians (12.9%), krill (9.3%) and fishes (4.2%) explained 95.6% of the variability observed using frequency of observance (FOO%) data. Ontogenetic changes in the diet based on groups of paralarvae with similar numbers per arm were evidenced by the decreasing contribution of coastal meroplankton and an increase in oceanic holoplankton, including siphonophores, copepods, pteropods and krill. Trophic niche breadth values ranged from 0.06 to 0.67, with averaged values ranging from 0.23 to 0.33 (generalist = 1 and specialist = 0), suggesting that O. vulgaris paralarvae are selective predators through their ontogenetic transition between coastal and oceanic environments.

Corrigendum: The significance of cephalopod beaks as a research tool: An update.

[This corrects the article DOI: 10.3389/fphys.2022.1038064.].

Population Genetic Structure of Anisakis simplex Infecting the European Hake from North East Atlantic Fishing Grounds.

The European hake, one of the most commercially valuable species in ICES fishing areas, is considered an important neglected source of zoonotic risk by nematode parasites belonging to the genus Anisakis. Merluccius merluccius is, by far, the most important host of Anisakis spp. at the European fishing grounds, in terms of demographic infection values, and carries the highest parasite burden. These high parasite population densities within an individual fish host offer a chance to explore new sources of variations for the genetic structure of Anisakis spp. populations. A total of 873 Anisakis spp. third-stage larvae, originally sampled from viscera and muscular sections of hake collected at ten fishing grounds, were primarily identified using ITS rDNA region as molecular marker. After that, we used mtDNA cox2 gene to reveal the high haplotype diversity and the lack of genetic structure for A. simplex. Dominant haplotypes were shared among the different fishing areas and fish sections analyzed. Results indicate a clear connection of A. simplex from European hake along the Northern North Sea to the Portuguese coast, constituting a single genetic population but revealing a certain level of genetic sub-structuring on the Northwest coast of Scotland. This study also provides useful information to advance the understanding of parasite speciation to different fish host tissues or microenvironments.

The significance of cephalopod beaks as a research tool: An update.

The use of cephalopod beaks in ecological and population dynamics studies has allowed major advances of our knowledge on the role of cephalopods in marine ecosystems in the last 60 years. Since the 1960's, with the pioneering research by Malcolm Clarke and colleagues, cephalopod beaks (also named jaws or mandibles) have been described to species level and their measurements have been shown to be related to cephalopod body size and mass, which permitted important information to be obtained on numerous biological and ecological aspects of cephalopods in marine ecosystems. In the last decade, a range of new techniques has been applied to cephalopod beaks, permitting new kinds of insight into cephalopod biology and ecology. The workshop on cephalopod beaks of the Cephalopod International Advisory Council Conference (Sesimbra, Portugal) in 2022 aimed to review the most recent scientific developments in this field and to identify future challenges, particularly in relation to taxonomy, age, growth, chemical composition (i.e., DNA, proteomics, stable isotopes, trace elements) and physical (i.e., structural) analyses. In terms of taxonomy, new techniques (e.g., 3D geometric morphometrics) for identifying cephalopods from their beaks are being developed with promising results, although the need for experts and reference collections of cephalopod beaks will continue. The use of beak microstructure for age and growth studies has been validated. Stable isotope analyses on beaks have proven to be an excellent technique to get valuable information on the ecology of cephalopods (namely habitat and trophic position). Trace element analyses is also possible using beaks, where concentrations are significantly lower than in other tissues (e.g., muscle, digestive gland, gills). Extracting DNA from beaks was only possible in one study so far. Protein analyses can also be made using cephalopod beaks. Future challenges in research using cephalopod beaks are also discussed.

Proteome profiling of L3 and L4 Anisakis simplex development stages by TMT-based quantitative proteomics.

Anisakis simplex is a parasitic nematode that can cause anisakiosis and/or allergic reactions in humans. The presence of invasive third-stage larvae (L3) in many different consumed fish species and the fourth-stage larvae (L4) in marine mammals, where L3 can accidentally affect to humans and develop as far as stage L4. World Health Organization and food safety authorities aim to control and prevent this emerging health problem. In the present work, using Tandem Mass Tag (TMT)-based quantitative proteomics we analyzed for the first time the global proteome of two A. simplex development stages, L3 and L4. The strategy was divided into four steps: (a) protein extraction of L3 and L4 development stages, (b) high intensity focused ultrasound (HIFU)-assisted trypsin digestion, (c) TMT-isobaric mass tag labeling following by high-pH reversed-phase fractionation, and (d) LC-MS/MS analysis in a LTQ-Orbitrap Elite mass spectrometer. A total of 2443 different proteins of A. simplex were identified. Analysis of the modulated proteins provided the specific proteomic signature of L3 (i.e. pseudocoelomic globin, endochitinase 1, paramyosin) and L4 (i.e. neprilysin-2, glutamate dehydrogenase, aminopeptidase N). To our knowledge, this is the most comprehensive dataset of proteins of A. simplex for two development stages (L3 and L4) identified to date. SIGNIFICANCE: A. simplex is a fish-borne parasite responsible for the human anisakiosis and allergic reactions around the world. The work describes for the first-time the comparison of the proteome of two A. simplex stages (L3 and L4). The strategy is based on four steps: (i) protein extraction, (ii) ultra-fast trypsin digestion under High-Intensity Focused Ultrasound (HIFU), (iii) TMT-isobaric mass tag labeling followed by high-pH reversed-phase fractionation and (iv) peptide analysis using a LTQ-Orbitrap Elite mass spectrometer. The workflow allows to select the most modulated proteins as proteomic signature of those specific development stages (L3 and L4) of A. simplex. Obtained stage-specific proteins, could be used as targets to control/eliminate this parasite and in future eradicate the anisakiosis disease.

Small copepods could channel missing carbon through metazoan predation.

Global ecosystem models are essential tools for predicting climate change impacts on marine systems. Modeled biogenic carbon fluxes in the ocean often match measured data poorly and part of this could be because small copepods (<2 mm) are modeled as unicellular feeders grazing on phytoplankton and microzooplankton. The most abundant copepods from a seasonal upwelling region of the Eastern North Atlantic were sorted, and a molecular method was applied to copepod gut contents to evaluate the extent of metazoan predation under two oceanographic conditions, a trophic pathway not accounted for in global models. Scaling up the results obtained herein, based on published field and laboratory estimates, suggests that small copepods could ingest 1.79-27.20 gigatons C/year globally. This ignored metazoan-copepod link could increase current estimates of biogeochemical fluxes (remineralization, respiration, and the biological pump) and export to higher trophic levels by 15.6%-24.4%. It could also account for global discrepancies between measured daily ingestion and copepod metabolic demand/growth. The inclusion of metazoan predation into global models could provide a more realistic role of the copepods in the ocean and if these preliminary data hold true at larger sample sizes and scales, the implications would be substantial at the global scale.

Feeding Relationship between Octopus vulgaris (Cuvier, 1797) Early Life-Cycle Stages and Their Prey in the Western Iberian Upwelling System: Correlation of Reciprocal Lipid and Fatty Acid Contents.

Under the influence of the Western Iberian upwelling system, the Iberian Atlantic coast holds important hatcheries and recruitment areas for Octopus vulgaris. Recently identified as an octopus hatchery, the Ría de Vigo harbors an important mesozooplankton community that supports O. vulgaris paralarvae during the first days of their planktonic stage. This study represents a preliminary approach to determine the nutritional link between wild O. vulgaris hatchlings, paralarvae and their zooplankton prey in the Ría de Vigo, by analyzing their lipid class content and fatty acid profiles. The results show that octopus hatchlings are richer in structural lipids as phospholipids and cholesterol, while the zooplankton is richer in reserve lipids like triacylglycerol and waxes. Zooplankton samples are also particularly rich in C18:1n9 and 22:6n3 (DHA), that seem to be successfully incorporated by O. vulgaris paralarvae thus resulting in a distinct fatty acid profile to that of the hatchlings. On the other hand, content in C20:4n6 (ARA) is maintained high through development, even though the zooplankton is apparently poorer in this essential fatty acid, confirming its importance for the development of O. vulgaris paralarvae. The content in monounsaturated fatty acids, particularly C18:1n7, and the DHA: EPA ratio are suggested as trophic markers of the diet of O. vulgaris paralarvae.

Diet Composition and Variability of Wild Octopus vulgaris and Alloteuthis media (Cephalopoda) Paralarvae: a Metagenomic Approach.

The high mortality of cephalopod early stages is the main bottleneck to grow them from paralarvae to adults in culture conditions, probably because the inadequacy of the diet that results in malnutrition. Since visual analysis of digestive tract contents of paralarvae provides little evidence of diet composition, the use of molecular tools, particularly next generation sequencing (NGS) platforms, offers an alternative to understand prey preferences and nutrient requirements of wild paralarvae. In this work, we aimed to determine the diet of paralarvae of the loliginid squid Alloteuthis media and to enhance the knowledge of the diet of recently hatched Octopus vulgaris paralarvae collected in different areas and seasons in an upwelling area (NW Spain). DNA from the dissected digestive glands of 32 A. media and 64 O. vulgaris paralarvae was amplified with universal primers for the mitochondrial gene COI, and specific primers targeting the mitochondrial gene 16S gene of arthropods and the mitochondrial gene 16S of Chordata. Following high-throughput DNA sequencing with the MiSeq run (Illumina), up to 4,124,464 reads were obtained and 234,090 reads of prey were successfully identified in 96.87 and 81.25% of octopus and squid paralarvae, respectively. Overall, we identified 122 Molecular Taxonomic Units (MOTUs) belonging to several taxa of decapods, copepods, euphausiids, amphipods, echinoderms, molluscs, and hydroids. Redundancy analysis (RDA) showed seasonal and spatial variability in the diet of O. vulgaris and spatial variability in A. media diet. General Additive Models (GAM) of the most frequently detected prey families of O. vulgaris revealed seasonal variability of the presence of copepods (family Paracalanidae) and ophiuroids (family Euryalidae), spatial variability in presence of crabs (family Pilumnidae) and preference in small individual octopus paralarvae for cladocerans (family Sididae) and ophiuroids. No statistically significant variation in the occurrences of the most frequently identified families was revealed in A. media. Overall, these results provide new clues about dietary preferences of wild cephalopod paralarvae, thus opening up new scenarios for research on trophic ecology and digestive physiology under controlled conditions.

Protein biomarker discovery and fast monitoring for the identification and detection of Anisakids by parallel reaction monitoring (PRM) mass spectrometry.

UNLABELLED: Anisakids are fish-borne parasites that are responsible for a large number of human infections and allergic reactions around the world. World health organizations and food safety authorities aim to control and prevent this emerging health problem. In the present work, a new method for the fast monitoring of these parasites is described. The strategy is divided in three steps: (i) purification of thermostable proteins from fish-borne parasites (Anisakids), (ii) in-solution HIFU trypsin digestion and (iii) monitoring of several peptide markers by parallel reaction monitoring (PRM) mass spectrometry. This methodology allows the fast detection of Anisakids in <2h. An affordable assay utilizing this methodology will facilitate testing for regulatory and safety applications. SIGNIFICANCE: The work describes for the first time, the Protein Biomarker Discovery and the Fast Monitoring for the identification and detection of Anisakids in fishery products. The strategy is based on the purification of thermostable proteins, the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by High-Intensity Focused Ultrasound (HIFU) and the monitoring of several peptide biomarkers by Parallel Reaction Monitoring (PRM) Mass Spectrometry in a linear ion trap mass spectrometer. The workflow allows the unequivocal detection of Anisakids, in <2h. The present strategy constitutes the fastest method for Anisakids detection, whose application in the food quality control area, could provide to the authorities an effective and rapid method to guarantee the safety to the consumers.

Cannibalistic behavior of octopus (Octopus vulgaris) in the wild.

The first description of cannibalism in wild adult Octopus vulgaris is presented from 3 observations made in the Ría de Vigo (NW Spain), which were filmed by scuba divers. These records document common traits in cannibalistic behavior: (a) it was intercohort cannibalism; (b) attacks were made by both males and females; (c) in 2 of the records, the prey were transported to the den, which was covered with stones of different sizes; (d) the predator started to eat the tip of the arms of its prey; (e) predation on conspecifics occurred even if there were other abundant prey available (i.e., mussels); and (f) the prey/predator weight ratio in the 3 cases ranged from 20% to 25% body weight. The relationships between this behavior and sex, defense of territory, energy balance, food shortage, competition and predation, as well as how the attacker kills its victim are discussed.

An assessment of contaminant concentrations in toothed whale species of the NW Iberian Peninsula: part II. Trace element concentrations.

Concentrations of Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V and Zn were investigated in the liver and kidney of the five most common toothed whales off the Northwest Iberian Peninsula (NWIP), specifically common dolphin, long-finned pilot whale, harbour porpoise, striped dolphin and bottlenose dolphin. Differences were observed in the bioaccumulation of the above elements between the five species. The differences are probably related to biological factors such as age and sex and/or to ecological factors specific to each species such as feeding habits or bioavailability of the various elements. However, no significant relationship was observed between element accumulation and sex. Pilot whale and striped dolphin showed the highest concentrations of renal Cd and the highest concentrations of hepatic Hg and Se, while bottlenose dolphin showed the highest concentrations of Hg in kidneys. An analysis of inter-elemental relationships showed strong positive correlations between Hg and Se in the five species, however most individuals have Hg:Se molar ratio less than 1:1 indicating an excess of Se compare to Hg. This result, probably reflect the high proportion of young animals in the sample available for this study and/or that these animals had a good health status. We also observed a positive correlation in striped dolphins between Cd and Cu and between Cd and Zn in kidneys. In addition, comparing with other studies world-wide, the element concentrations (Hg and Cd) found in Iberian toothed whales indicate that these populations are not specially threatened by Hg and Cd exposure in the area.

An assessment of contaminant concentrations in toothed whale species of the NW Iberian Peninsula: part I. Persistent organic pollutants.

Concentrations and patterns of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in the blubber of the five most common toothed whales off the Northwest Iberian Peninsula (NWIP), specifically common dolphin, long-finned pilot whale, harbour porpoise, striped dolphin and bottlenose dolphin, were investigated. The study revealed that differences in PCB and PBDE concentrations among the species are highly dependent on age and sex but also on ecological factors such as trophic level, prey type and habitat. Of the five species studied, bottlenose dolphin and harbour porpoise showed the greatest concentrations of PCBs. Both species exceed the toxic threshold of 17µgg(-1) lipid weight (PCB Aroclor equivalent) for health effects on marine mammals, for 100% and 75% of the individuals tested, respectively. Overall, the PCB and PBDE levels observed in the NWIP toothed whales were of the same order of magnitude or lower than those reported by previous studies in areas of the NE Atlantic. However, they are often higher than those for toothed whales from the southern Atlantic and Pacific Ocean.

Optimization of the pepsin digestion method for anisakids inspection in the fishing industry.

During the last 50 years human anisakiasis has been rising while parasites have increased their prevalence at determined fisheries becoming an emergent major public health problem. Although artificial enzymatic digestion procedure by CODEX (STAN 244-2004: standard for salted Atlantic herring and salted sprat) is the recommended protocol for anisakids inspection, no international agreement has been achieved in veterinary and scientific digestion protocols to regulate this growing source of biological hazard in fish products. The aim of this work was to optimize the current artificial digestion protocol by CODEX with the purpose of offering a faster, more useful and safer procedure for factories workers, than the current one for anisakids detection. To achieve these objectives, the existing pepsin chemicals and the conditions of the digestion method were evaluated and assayed in fresh and frozen samples, both in lean and fatty fish species. Results showed that the new digestion procedure considerably reduces the assay time, and it is more handy and efficient (the quantity of the resulting residue was considerably lower after less time) than the widely used CODEX procedure. In conclusion, the new digestion method herein proposed based on liquid pepsin format is an accurate reproducible and user-friendly off-site tool, that can be useful in the implementation of screening programs for the prevention of human anisakiasis (and associated gastroallergic disorders) due to the consumption of raw or undercooked contaminated seafood products.

Sperm ultrastructure in Bathypolypus bairdii and B. sponsalis (Cephalopoda: Octopoda).

A morphological comparison of the spermatozoa of the octopods Bathypolypus bairdii and B. sponsalis has been carried out by electron microscopy. Although the mature spermatozoon of B. bairdii is longer and thinner than that of B. sponsalis, its general ultrastructure is similar except for some minor differences. Their characteristic acrosomes, described here for the first time, consist of a periodically banded cone surrounded by a double helix whose arrangement has been defined by a numeric expression. The plasma membrane of sponsalis that surrounds the acrosome has many projections filled with abundant granular cytoplasm, which appears as a star-shaped acrosome in cross-section. The mitochondrial sheath contains 9-11 mitochondria in bairdii but only 9 in sponsalis. A comparison with other Octopodidae shows that the nucleus of both species is the largest ever seen. Our results support the capability of sperm morphology to discriminate between species and could even discern at higher taxonomic levels.

Parasite recruitment and oceanographic regime: evidence suggesting a relationship on a global scale.

We here investigate the relationship between oceanographic processes and variability in parasite recruitment to host populations using existing data from host-parasite systems encountering differing hydrographic conditions. Combined epidemiological data obtained from both exploited fish and cephalopod populations indicate that variability in recruitment of parasite infracommunities tends to be associated with major current systems of the World's oceans. It appears that instability in water masses caused by physical perturbations (e.g. water mass convergence and turbulent mixing in upwelling systems) is associated with instability of trophic interactions over time, which in turn leads to a paucity of parasite communities in that area. The likely relationship between parasite recruitment and oceanographic regime should be extremely useful to the fishing industry and also as an indicator of ecosystem health.