Octopus vulgaris Exhibits Interindividual Differences in Behavioural and Problem-Solving Performance.

By presenting individual Octopus vulgaris with an extractive foraging problem with a puzzle box, we examined the possible correlation between behavioural performances (e.g., ease of adaptation to captive conditions, prevalence of neophobic and neophilic behaviours, and propensity to learn individually or by observing conspecifics), biotic (body and brain size, age, sex) and abiotic (seasonality and place of origin) factors. We found more neophilic animals showing shorter latencies to approach the puzzle box and higher probability of solving the task; also, shorter times to solve the task were correlated with better performance on the individual learning task. However, the most neophilic octopuses that approached the puzzle box more quickly did not reach the solution earlier than other individuals, suggesting that strong neophilic tendency may lead to suboptimal performance at some stages of the problem-solving process. In addition, seasonal and environmental characteristics of location of origin appear to influence the rate of expression of individual traits central to problem solving. Overall, our analysis provides new insights into the traits associated with problem solving in invertebrates and highlights the presence of adaptive mechanisms that promote population-level changes in octopuses' behavioural traits.

A chromosome-level reference genome for the common octopus, Octopus vulgaris (Cuvier, 1797).

Cephalopods are emerging animal models and include iconic species for studying the link between genomic innovations and physiological and behavioral complexities. Coleoid cephalopods possess the largest nervous system among invertebrates, both for cell counts and brain-to-body ratio. Octopus vulgaris has been at the center of a long-standing tradition of research into diverse aspects of cephalopod biology, including behavioral and neural plasticity, learning and memory recall, regeneration, and sophisticated cognition. However, no chromosome-scale genome assembly was available for O. vulgaris to aid in functional studies. To fill this gap, we sequenced and assembled a chromosome-scale genome of the common octopus, O. vulgaris. The final assembly spans 2.8 billion basepairs, 99.34% of which are in 30 chromosome-scale scaffolds. Hi-C heatmaps support a karyotype of 1n = 30 chromosomes. Comparisons with other octopus species' genomes show a conserved octopus karyotype and a pattern of local genome rearrangements between species. This new chromosome-scale genome of O. vulgaris will further facilitate research in all aspects of cephalopod biology, including various forms of plasticity and the neural machinery underlying sophisticated cognition, as well as an understanding of cephalopod evolution.

Transcriptome-wide selection and validation of a solid set of reference genes for gene expression studies in the cephalopod mollusk Octopus vulgaris.

Octopus vulgaris is a cephalopod mollusk and an active marine predator that has been at the center of a number of studies focused on the understanding of neural and biological plasticity. Studies on the machinery involved in e.g., learning and memory, regeneration, and neuromodulation are required to shed light on the conserved and/or unique mechanisms that these animals have evolved. Analysis of gene expression is one of the most essential means to expand our understanding of biological machinery, and the selection of an appropriate set of reference genes is the prerequisite for the quantitative real-time polymerase chain reaction (qRT-PCR). Here we selected 77 candidate reference genes (RGs) from a pool of stable and relatively high-expressed transcripts identified from the full-length transcriptome of O. vulgaris, and we evaluated their expression stabilities in different tissues through geNorm, NormFinder, Bestkeeper, Delta-CT method, and RefFinder. Although various algorithms provided different assemblages of the most stable reference genes for the different kinds of tissues tested here, a comprehensive ranking revealed RGs specific to the nervous system (Ov-RNF7 and Ov-RIOK2) and Ov-EIF2A and Ov-CUL1 across all considered tissues. Furthermore, we validated RGs by assessing the expression profiles of nine target genes (Ov-Naa15, Ov-Ltv1, Ov-CG9286, Ov-EIF3M, Ov-NOB1, Ov-CSDE1, Ov-Abi2, Ov-Homer2, and Ov-Snx20) in different areas of the octopus nervous system (gastric ganglion, as control). Our study allowed us to identify the most extensive set of stable reference genes currently available for the nervous system and appendages of adult O. vulgaris.

Cephalopod-omics: Emerging Fields and Technologies in Cephalopod Biology.

Few animal groups can claim the level of wonder that cephalopods instill in the minds of researchers and the general public. Much of cephalopod biology, however, remains unexplored: the largest invertebrate brain, difficult husbandry conditions, and complex (meta-)genomes, among many other things, have hindered progress in addressing key questions. However, recent technological advancements in sequencing, imaging, and genetic manipulation have opened new avenues for exploring the biology of these extraordinary animals. The cephalopod molecular biology community is thus experiencing a large influx of researchers, emerging from different fields, accelerating the pace of research in this clade. In the first post-pandemic event at the Cephalopod International Advisory Council (CIAC) conference in April 2022, over 40 participants from all over the world met and discussed key challenges and perspectives for current cephalopod molecular biology and evolution. Our particular focus was on the fields of comparative and regulatory genomics, gene manipulation, single-cell transcriptomics, metagenomics, and microbial interactions. This article is a result of this joint effort, summarizing the latest insights from these emerging fields, their bottlenecks, and potential solutions. The article highlights the interdisciplinary nature of the cephalopod-omics community and provides an emphasis on continuous consolidation of efforts and collaboration in this rapidly evolving field.

General and species-specific recommendations for minimal requirements for the use of cephalopods in scientific research.

Here we list species-specific recommendations for housing, care and management of cephalopod molluscs employed for research purposes with the aim of contributing to the standardization of minimum requirements for establishments, care and accommodation of these animals in compliance with the principles stated in Directive 2010/63/EU. Maximizing their psychophysical welfare was our priority. General recommendations on water surface area, water depth and tank shape here reported represent the outcome of the combined action of the analysis of the available literature and an expertise-based consensus reached - under the aegis of the COST Action FA1301 - among researchers working with the most commonly used cephalopod species in Europe. Information on water supply and quality, environmental conditions, stocking density, feeding and handling are also provided. Through this work we wish to set the stage for a more fertile ground of evidence-based approaches on cephalopod laboratory maintenance, thus facilitating standardization and replicability of research outcomes across laboratories, at the same time maximizing the welfare of these animals.

Cell type diversity in a developing octopus brain.

Octopuses are mollusks that have evolved intricate neural systems comparable with vertebrates in terms of cell number, complexity and size. The brain cell types that control their sophisticated behavioral repertoire are still unknown. Here, we profile the cell diversity of the paralarval Octopus vulgaris brain to build a cell type atlas that comprises mostly neural cells, but also multiple glial subtypes, endothelial cells and fibroblasts. We spatially map cell types to the vertical, subesophageal and optic lobes. Investigation of cell type conservation reveals a shared gene signature between glial cells of mouse, fly and octopus. Genes related to learning and memory are enriched in vertical lobe cells, which show molecular similarities with Kenyon cells in Drosophila. We construct a cell type taxonomy revealing transcriptionally related cell types, which tend to appear in the same brain region. Together, our data sheds light on cell type diversity and evolution in the octopus brain.

A preliminary attempt to investigate mirror self-recognition in Octopus vulgaris.

Mirror self-recognition (MSR) is a potential indicator of self-awareness. This capability has been widely investigated among vertebrates, yet it remains largely unstudied in invertebrates. Here we report preliminary data about behavioural responses exhibited by common octopuses (Octopus vulgaris) toward reflected images of themselves and explore a procedure for marking octopus' skin in order to conduct the Mark test. Octopuses (n = 8) received four familiarization trials with a mirror and four familiarization trials with a control stimulus: a non-reflective panel (Panel group, n = 4) or the sight of a conspecific housed in an adjacent tank (Social group, n = 4). Subsequently, octopuses were marked with non-toxic nail polish in the area where the Frontal White Spots are usually expressed, and they received one test trial with the mirror and one control trial with no mirror. We found that octopuses in the Panel group tended to exhibit a stronger exploratory response toward the mirror than the non-reflective panel, but performed agonistic responses only in the presence of the mirror. In contrast, octopuses in the Social group exhibited comparable exploratory and agonistic behaviours toward the mirror and the sight of the conspecific. In the Mark test, octopuses frequently explored the mark via their arms. However, mark-directed behaviours were also observed in the absence of the mirror and in sham-marked individuals, thus suggesting that proprioceptive stimuli drove these responses. Despite the limitations associated with our marking procedure, the baseline data collected in this pilot study may facilitate the further testing of MSR in the octopus and other cephalopods.

Identification of LINE retrotransposons and long non-coding RNAs expressed in the octopus brain.

BACKGROUND: Transposable elements (TEs) widely contribute to the evolution of genomes allowing genomic innovations, generating germinal and somatic heterogeneity, and giving birth to long non-coding RNAs (lncRNAs). These features have been associated to the evolution, functioning, and complexity of the nervous system at such a level that somatic retrotransposition of long interspersed element (LINE) L1 has been proposed to be associated to human cognition. Among invertebrates, octopuses are fascinating animals whose nervous system reaches a high level of complexity achieving sophisticated cognitive abilities. The sequencing of the genome of the Octopus bimaculoides revealed a striking expansion of TEs which were proposed to have contributed to the evolution of its complex nervous system. We recently found a similar expansion also in the genome of Octopus vulgaris. However, a specific search for the existence and the transcription of full-length transpositionally competent TEs has not been performed in this genus. RESULTS: Here, we report the identification of LINE elements competent for retrotransposition in Octopus vulgaris and Octopus bimaculoides and show evidence suggesting that they might be transcribed and determine germline and somatic polymorphisms especially in the brain. Transcription and translation measured for one of these elements resulted in specific signals in neurons belonging to areas associated with behavioral plasticity. We also report the transcription of thousands of lncRNAs and the pervasive inclusion of TE fragments in the transcriptomes of both Octopus species, further testifying the crucial activity of TEs in the evolution of the octopus genomes. CONCLUSIONS: The neural transcriptome of the octopus shows the transcription of thousands of putative lncRNAs and of a full-length LINE element belonging to the RTE class. We speculate that a convergent evolutionary process involving retrotransposons activity in the brain has been important for the evolution of sophisticated cognitive abilities in this genus.

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.

Cephalopod Behavior: From Neural Plasticity to Consciousness.

It is only in recent decades that subjective experience - or consciousness - has become a legitimate object of scientific inquiry. As such, it represents perhaps the greatest challenge facing neuroscience today. Subsumed within this challenge is the study of subjective experience in non-human animals: a particularly difficult endeavor that becomes even more so, as one crosses the great evolutionary divide between vertebrate and invertebrate phyla. Here, we explore the possibility of consciousness in one group of invertebrates: cephalopod molluscs. We believe such a review is timely, particularly considering cephalopods' impressive learning and memory abilities, rich behavioral repertoire, and the relative complexity of their nervous systems and sensory capabilities. Indeed, in some cephalopods, these abilities are so sophisticated that they are comparable to those of some higher vertebrates. Following the criteria and framework outlined for the identification of hallmarks of consciousness in non-mammalian species, here we propose that cephalopods - particularly the octopus - provide a unique test case among invertebrates for examining the properties and conditions that, at the very least, afford a basal faculty of consciousness. These include, among others: (i) discriminatory and anticipatory behaviors indicating a strong link between perception and memory recall; (ii) the presence of neural substrates representing functional analogs of thalamus and cortex; (iii) the neurophysiological dynamics resembling the functional signatures of conscious states in mammals. We highlight the current lack of evidence as well as potentially informative areas that warrant further investigation to support the view expressed here. Finally, we identify future research directions for the study of consciousness in these tantalizing animals.

Metacestodes of Elasmobranch Tapeworms in Octopus vulgaris (Mollusca, Cephalopoda) from Central Mediterranean-SEM and Molecular Data.

Cephalopods are intermediate/paratenic hosts in the life cycle of elasmobranch tapeworms, nevertheless most records of infection in this group of mollusks are outdated and fragmentary. The present work aimed to investigate the cestode fauna of the common octopus Octopus vulgaris from the Tyrrhenian Sea (Central Mediterranean). The parasitic stages were characterized by light and Scanning Electron Microscopy (SEM) and sequencing of 28S rDNA. Three cestode taxa were identified to the genus level: the onchoproteocephalidean Acanthobothrium sp. (prevalence 28%), the "tetraphyllidean" Anthobothrium sp. (prevalence 13%) and the trypanorhynch Nybelinia sp. (prevalence 3%). The remarkable prevalence observed for gastrointestinal cestodes highlight a possible important role of O. vulgaris in the transmission of elasmobranch tapeworms, particularly Onchoproteocephalideans. Furthermore, the present work provides, for the first time, detailed morphological (SEM) and molecular support to confirm the occurrence of Anthobothrium sp. in cephalopod hosts. In order to gain higher taxonomic resolution for the identified taxa, we stress the need to collect further morphological and molecular data of adult cestodes infecting their elasmobranch definitive hosts.

E Pluribus Octo - Building Consensus on Standards of Care and Experimentation in Cephalopod Research; a Historical Outlook.

The Directive 2010/63/EU "on the protection of animals used for scientific purposes" originally induced some concern among cephalopod researchers, because of the inclusion of cephalopod mollusks as the only invertebrates among the protected species. Here we reflect on the challenges and issues raised by the Directive on cephalopod science, and discuss some of the arguments that elicited discussion within the scientific community, to facilitate the implementation of the Directive 2010/63/EU in the scientific research context. A short overview of the aims of the COST Action FA1301 "CephsInAction," serves as a paradigmatic instance of a pragmatic and progressive approach adopted to respond to novel legislative concerns through community-building and expansion of the historical horizon. Between 2013 and 2017, the COST Action FA1301 has functioned as a hub for consolidation of the cephalopod research community, including about 200 representatives from 21 countries (19 European). Among its aims, CephsInAction promoted the collection, rationalization, and diffusion of knowledge relevant to cephalopods. In the Supplementary Material to this work, we present the translation of the first-published systematic set of guidelines on the care, management and maintenance of cephalopods in captivity (Grimpe, 1928), as an example of the potential advantages deriving from the confluence of pressing scientific concerns and historical interests.

Global patterns of parasite diversity in cephalopods.

We compiled an updated global catalogue of parasites in cephalopods. Data were used to assess changes in taxonomic distinctness of parasites over two centuries and across the world's oceans, to quantify turnover and nestedness components of parasite ß-diversity, and to attempt estimating their γ-diversity at a global scale. A total of 309 parasites infecting 164 cephalopods were found. We hypothesize that this diversity counts for less than half the potential parasite richness in this molluscan taxon. Taxonomic breadth of parasites was significantly above expectations from null models for Mediterranean Sea and NE Atlantic Ocean, whereas the opposite occurred for NW Pacific Ocean, where a few closely related genera characterized the parasite pool. ß-diversity of parasites was very high and dominated by turnover, except for the Atlantic Ocean where a nested pattern among sub-basins emerged. Taxonomic relatedness of parasite species remained substantially unchanged through time, but species replacements largely occurred over the last two centuries. Our findings highlighted potential hotspots of taxonomic distinctness in cephalopod parasites, geographic regions deserving future research, and the need for a deeper understanding of the magnitude of marine parasite diversity, their biogeography, and their role in marine ecosystems. Our global overview may represent a baseline step for future advances in this direction.

Locomotory behaviour of the intertidal marble crab (Pachygrapsus marmoratus) supports the underwater spring-loaded inverted pendulum as a fundamental model for punting in animals.

In aquatic pedestrian locomotion the dynamics of terrestrial and aquatic environments are coupled. Here we study terrestrial running and aquatic punting locomotion of the marine-living crab Pachygrapsus marmoratus. We detected both active and passive phases of running and punting through the observation of crab locomotory behaviour in standardized settings and by three-dimensional kinematic analysis of its dynamic gaits using high-speed video cameras. Variations in different stride parameters were studied and compared. The comparison was done based on the dimensionless parameter the Froude number (Fr) to account for the effect of buoyancy and size variability among the crabs. The underwater spring-loaded inverted pendulum (USLIP) model better fitted the dynamics of aquatic punting. USLIP takes account of the damping effect of the aquatic environment, a variable not considered by the spring-loaded inverted pendulum (SLIP) model in reduced gravity. Our results highlight the underlying principles of aquatic terrestrial locomotion by comparing it with terrestrial locomotion. Comparing punting with running, we show and increased stride period, decreased duty cycle and orientation of the carapace more inclined with the horizontal plane, indicating the significance of fluid forces on the dynamics due to the aquatic environment. Moreover, we discovered periodicity in punting locomotion of crabs and two different gaits, namely, long-flight punting and short-flight punting, distinguished by both footfall patterns and kinematic parameters. The generic fundamental model which belongs to all animals performing both terrestrial and aquatic legged locomotion has implications for control strategies, evolution and translation to robotic artefacts.

A standardized battery of tests to measure Octopus vulgaris' behavioural performance.

Here we introduce a series of behavioural tasks to assess inter-individual variability in behaviours exhibited by the cephalopod mollusc Octopus vulgaris. We propose that, by using octopus' predatory behavioural response, it is possible to measure: (1) the ability to adapt to the captive condition (acclimatization), (2) the response towards novel stimuli (neophobia), (3) the capability of social learning, (4) the ability of solving problems (problem solving), and (5) the response to artificial stimuli (preferences, individual learning). To assure comparability and reproducibility of results, this battery of tests is here applied to a large sample of individuals in standardized experimental conditions. Such battery of tests serves as an in vivo screening that should be adopted not only to investigate cognitive abilities in specific behavioural domains, but also to monitor the welfare status of animals under captivity, thus to check sensory functions as well as motor abilities in other investigations within the fields of biology and neuroscience. Our aim was to provide a reliable tool to exploit this animal species for research in different fields.

Cerebrotypes in Cephalopods: Brain Diversity and Its Correlation With Species Habits, Life History, and Physiological Adaptations.

Here we analyze existing quantitative data available for cephalopod brains based on classical contributions by J.Z. Young and colleagues, to cite some. We relate the relative brain size of selected regions (area and/or lobe), with behavior, life history, ecology and distribution of several cephalopod species here considered. After hierarchical clustering we identify and describe ten clusters grouping 52 cephalopod species. This allows us to describe cerebrotypes, i.e., differences of brain composition in different species, as a sign of their adaptation to specific niches and/or clades in cephalopod molluscs for the first time. Similarity reflecting niche type has been found in vertebrates, and it is reasonable to assume that it could also occur in Cephalopoda. We also attempted a phylogenetic PCA using data by Lindgren et al. (2012) as input tree. However, due to the limited overlap in species considered, the final analysis was carried out on <30 species, thus reducing the impact of this approach. Nevertheless, our analysis suggests that the phylogenetic signal alone cannot be a justification for the grouping of species, although biased by the limited set of data available to us. Based on these preliminary findings, we can only hypothesize that brains evolved in cephalopods on the basis of different factors including phylogeny, possible development, and the third factor, i.e., life-style adaptations. Our results support the working hypothesis that the taxon evolved different sensorial and computational strategies to cope with the various environments (niches) occupied in the oceans. This study is novel for invertebrates, to the best of our knowledge.

Prospective severity classification of scientific procedures in cephalopods: Report of a COST FA1301 Working Group survey.

Cephalopods are the first invertebrate class regulated by the European Union (EU) under Directive 2010/63/EU on the protection of animals used for scientific purposes, which requires prospective assessment of severity of procedures. To assist the scientific community in establishing severity classification for cephalopods, we undertook a web-based survey of the EU cephalopod research community as represented by the participants in the European COoperation on Science and Technology (COST) Action FA1301, CephsInAction'. The survey consisted of 50 scenarios covering a range of procedures involving several cephalopod species at different life stages. Respondents (59 people from 15 countries) either allocated a severity classification to each scenario or indicated that they were unable to decide (UTD). Analyses evaluated score distributions and clustering. Overall, the UTD scores were low (7.0 ± 0.6%) and did not affect the severity classification. Procedures involving paralarvae and killing methods (not specified in Annexe IV) had the highest UTD scores. Consensus on non-recovery procedures was reached consistently, although occasionally non-recovery appeared to be confused with killing methods. Scenarios describing procedures above the lower threshold for regulation, including those describing behavioural studies, were also identified and allocated throughout the full range of severity classifications. Severity classification for scenarios based on different species (e.g. cuttlefish vs. octopus) was consistent, comparable and dependent on potentially more harmful interventions. We found no marked or statistically significant differences in the overall scoring of scenarios between the demographic subgroups (age, sex, PhD and cephalopod experience). The COST Action FA1301 survey data provide a basis for a prospective severity classification for cephalopods to serve as guide for researchers, project assessors and regulators.

From injury to full repair: nerve regeneration and functional recovery in the common octopus, Octopus vulgaris.

Spontaneous nerve regeneration in cephalopod molluscs occurs in a relative short time after injury, achieving functional recovery of lost capacity. In particular, transection of the pallial nerve in the common octopus (Octopus vulgaris) determines the loss and subsequent restoration of two functions fundamental for survival, i.e. breathing and skin patterning, the latter involved in communication between animals and concealment. The phenomena occurring after lesion have been investigated in a series of previous studies, but a complete analysis of the changes taking place at the level of the axons and the effects on the animals' appearance during the whole regenerative process is still missing. Our goal was to determine the course of events following injury, from impairment to full recovery. Through imaging of the traced damaged nerves, we were able to characterize the pathways followed by fibres during regeneration and end-target re-innervation, while electrophysiology and behavioural observations highlighted the regaining of functional connections between the central brain and periphery, using the contralateral nerve in the same animal as an internal control. The final architecture of a fully regenerated pallial nerve does not exactly mirror the original structure; however, functionality returns to match the phenotype of an intact octopus with no observable impact on the behaviour of the animal. Our findings provide new important scenarios for the study of regeneration in cephalopods and highlight the octopus pallial nerve as a valuable 'model' among invertebrates.