Gene Expression Detects the Factors Influencing the Reproductive Success and the Survival Rates of Paracentrotus lividus Offspring.

The increase in the demand for Paracentrotus lividus roe, a food delicacy, causes increased pressure on its wild stocks. In this scenario, aquaculture facilities will mitigate the effects of anthropogenic pressures on the wild stocks of P. lividus. Consequently, experimental studies should be conducted to enhance techniques to improve efficient aquaculture practices for these animals. Here, we for the first time performed molecular investigations on cultured sea urchins. We aimed at understanding if maternal influences may significantly impact the life of future offspring, and how the culture conditions may impact the development and growth of cultured specimens. Our findings demonstrate that the outcomes of in vitro fertilization of P. lividus are influenced by maternal influences, but these effects are largely determined by culture conditions. In fact, twenty-three genes involved in the response to stress and skeletogenesis, whose expressions were measured by Real Time qPCR, were differently expressed in sea urchins cultured in two experimental conditions, and the results were largely modified in offspring deriving from two groups of females. The findings herein reported will be critical to develop protocols for the larval culture of the most common sea urchin, both for research and industrial production purposes for mass production.

Multimodal signaling in the visuo-acoustic mismatch paradigm: similarities between dogs and children in the communicative approach.

In this review, we have analyzed the studies on the "mismatch paradigm" or "contrasting paradigm", in which the word indicates an intent that is opposite to the gesture in dogs and children. The studies on children highlighted the importance of the type of gestural messages that, when delivered in a non-ostensive manner, assume less value than the verbal indication; whereas, when more emphasis is given to the gestures, it produces opposite results. Word-trained dogs appear to rely more on words, but in the absence of such specific training, dogs rely more on gestures either in transitive or intransitive actions. Moreover, gestural communication appears easier to generalize, since dogs respond equally well to the gestural messages of familiar persons and strangers, whereas their performance lowers when a stranger provides a vocal message. Visual signals trigger faster responses than auditory signals, whereas verbal indications can at most equal the gestural latencies, but never overcome them. Female dogs appeared to be more proficient in the interpretation of gestural commands, while males performed better in the case of verbal commands. Based on a PRISMA analyses from the Web of Science database, three papers on children and four on dogs were retrieved. Our analyses revealed that gestures are more reliable reference points than words for dogs and children. Future studies should focus on choices related to objects of different values for the subjects. Moreover, the choices of dogs should be compared using known and unknown objects, which might help clarify how familiarity with the objects could differently influence their responses.

OctoPartenopin: Identification and Preliminary Characterization of a Novel Antimicrobial Peptide from the Suckers of Octopus vulgaris.

Microorganism resistance to conventional antibiotics represents one of the major global health concerns. This paper focuses on a peptide (OctoPartenopin) extracted from suckers of Octopus vulgaris; bioassay-guided chromatographic fractionation was used to identify this sequence, which holds significant antibacterial activity against Gram-positive and Gram-negative bacteria. OctoPartenopin is encrypted within the calponin sequence and was associated with the high levels of proteolytic activity already reported in octopus arm suckers. We synthesized the parent peptide and four analogues; all peptide were tested for their antibacterial and antibiofilm activities. Preliminary antibiofilm experiments showed that that one of the analogues had the best activity in both inhibition and eradication of biofilm of all three microorganisms tested. The occurrence of OctoPartenopin in arm suckers provided novel speculative information on animal behavior, as concerns maternal care of fertilized eggs. Our results highlight that suckers are a rich source of multifaceted peptides to develop alternative antimicrobial agents and food preservatives.

Oxidative stress, metabolic and histopathological alterations in mussels exposed to remediated seawater by GO-PEI after contamination with mercury.

The modern technology brought new engineering materials (e.g. nanostructured materials) with advantageous characteristics such as a high capacity to decontaminate water from pollutants (for example metal(loid)s). Among those innovative materials the synthesis of nanostructured materials (NSMs) based on graphene as graphene oxide (GO) functionalized with polyethyleneimine (GO-PEI) had a great success due to their metal removal capacity from water. However, research dedicated to environmental risks related to the application of these materials is still non-existent. To evaluate the impacts of such potential stressors, benthic species can be a good model as they are affected by several environmental constraints. Particularly, the mussel Mytilus galloprovincialis has been identified by several authors as a bioindicator that responds quickly to environmental disturbances, with a wide spatial distribution and economic relevance. Thus, the present study aimed to evaluate the impacts caused in M. galloprovincialis by seawater previously contaminated by Hg and decontaminated using GO-PEI. For this, histopathological and biochemical alterations were examined. This study demonstrated that mussels exposed to the contaminant (Hg), the decontaminant (GO-PEI) and the combination of both (Hg + GO-PEI) presented an increment of histopathological, oxidative stress and metabolic alterations if compared to organisms under remediated seawater and control conditions The present findings highlight the possibility to remediate seawater with nanoparticles for environmental safety purposes.

Effect of the algal alkaloid caulerpin on neuropeptide Y (NPY) expression in the central nervous system (CNS) of Diplodus sargus.

Recent studies have suggested that Mediterranean indigenous fish species are affected by bioactive metabolites coming from marine invasive species via food web interactions. In particular, both physiological and behavioural changes in the white sea bream Diplodus sargus were related to caulerpin (CAU), a bisindolic alkaloid particularly abundant in the invasive alga Caulerpa cylindracea, on which the fish actively feed. Dietary administration of CAU decreased aggressiveness in D. sargus, suggesting an anxiolytic-like effect of CAU possibly mediated by endogenous anxiolytic agents. This hypothesis is supported here by the finding of a significant increase of NPY transcriptional expression in the brain of fish fed with CAU enriched food, shedding more light on the neural mechanisms behind the altered behaviour of D. sargus.

Enriched Environment Increases PCNA and PARP1 Levels in Octopus vulgaris Central Nervous System: First Evidence of Adult Neurogenesis in Lophotrochozoa.

Organisms showing a complex and centralized nervous system, such as teleosts, amphibians, reptiles, birds and mammals, and among invertebrates, crustaceans and insects, can adjust their behavior according to the environmental challenges. Proliferation, differentiation, migration, and axonal and dendritic development of newborn neurons take place in brain areas where structural plasticity, involved in learning, memory, and sensory stimuli integration, occurs. Octopus vulgaris has a complex and centralized nervous system, located between the eyes, with a hierarchical organization. It is considered the most "intelligent" invertebrate for its advanced cognitive capabilities, as learning and memory, and its sophisticated behaviors. The experimental data obtained by immunohistochemistry and western blot assay using proliferating cell nuclear antigen and poli (ADP-ribose) polymerase 1 as marker of cell proliferation and synaptogenesis, respectively, reviled cell proliferation in areas of brain involved in learning, memory, and sensory stimuli integration. Furthermore, we showed how enriched environmental conditions affect adult neurogenesis.

Role of olfaction in Octopus vulgaris reproduction.

The olfactory system in any animal is the primary sensory system that responds to chemical stimuli emanating from a distant source. In aquatic animals "Odours" are molecules in solution that guide them to locate food, partners, nesting sites, and dangers to avoid. Fish, crustaceans and aquatic molluscs possess sensory systems that have anatomical similarities to the olfactory systems of land-based animals. Molluscs are a large group of aquatic and terrestrial animals that rely heavily on chemical communication with a generally dispersed sense of touch and chemical sensitivity. Cephalopods, the smallest class among extant marine molluscs, are predators with high visual capability and well developed vestibular, auditory, and tactile systems. Nevertheless they possess a well developed olfactory organ, but to date almost nothing is known about the mechanisms, functions and modulation of this chemosensory structure in octopods. Cephalopod brains are the largest of all invertebrate brains and across molluscs show the highest degree of centralization. The reproductive behaviour of Octopus vulgaris is under the control of a complex set of signal molecules such as neuropeptides, neurotransmitters and sex steroids that guide the behaviour from the level of individuals in evaluating mates, to stimulating or deterring copulation, to sperm-egg chemical signalling that promotes fertilization. These signals are intercepted by the olfactory organs and integrated in the olfactory lobes in the central nervous system. In this context we propose a model in which the olfactory organ and the olfactory lobe of O. vulgaris could represent the on-off switch between food intake and reproduction.

Effect of biodegradable polymers upon grazing activity of the sea urchin Paracentrotus lividus (Lmk) revealed by morphological, histological and molecular analyses.

In the last years biodegradable polymers (BPs) were largely used as real opportunity to solve plastic pollution. Otherwise, their wide use in commercial products, such as packaging sector, is causing a new pollution alarm, mainly because few data reported about their behaviour in the environment and toxicity on marine organisms. Our previous results showed that embryos of the sea urchin Paracentrotus lividus (Lmk) exposed to poly(ε-caprolactone) (PCL), poly(3-hydroxybutyrate) (PHB) and poly(lactic acid) (PLA) showed delay of their development and morphological malformations, also affecting at the molecular levels the expression of several genes involved in different functional responses. In the present work for the first time, we tested the effects of five microplastics (MPs) obtained from BPs such as PBS, poly(butylene succinate), PBSA, poly(butylene succinate-co-butylene adipate), PCL, PHB and PLA, upon grazing activity of the sea urchin revealed by: i. histological analysis seeing at the gonadic tissues; ii. morphological analysis of the deriving embryos; iii. molecular analyses on these embryos to detect variations of the gene expression of eighty-seven genes involved in stress response, detoxification, skeletogenesis, differentiation and development. All these results will help in understanding how MP accumulated inside various organs in the adult sea urchins, and more in general in marine invertebrates, could represent risks for the marine environment.

Exploring toxicological interactions in a changing sea: The case of the alkaloids caffeine and caulerpin.

The bisindolic alkaloid caulerpin (CAU) is a bioactive compound isolated from green algae of the genus Caulerpa that are highly invasive in the Mediterranean Sea. On the other side, the purine alkaloid caffeine (CAF) is one of the most globally consumed psychoactive substances and a widespread anthropogenic water pollutant. Both compounds display a large panel of biological properties and are well known to accumulate in the tissues of aquatic organisms and, in certain circumstances, co-occur in the human diet. On this premise, the present study aimed to investigate possible synergistic interactions between CAU and CAF by using the bivalve Mytilus galloprovincialis as a model organism. Mussels were exposed to CAF via medium while they were fed with food enriched with CAU. After treatments, biochemical analysis confirmed the toxic potential of CAF, with increased AChE activity and lipid peroxidation. Also, histopathological alterations were observed in the gills and digestive tubules. The NMR-based metabolomics analysis detected higher levels of free amino acids under CAF treatments. Conversely, the food administration of CAU did not affect the above toxicological biomarkers. In addition, we did not observe any cumulative effect between CAF and CAU toward increased cellular damage and neurotoxicity. On the other hand, a possible action of CAU in decreasing CAF toxicity could be hypothesized based on our results. This hypothesis is supported by the activity of CAU as an agonist of peroxisome proliferator-activated receptors (PPARs). PPARs mediate xenobiotic detoxification via cytochromes P450, which is involved in CAF metabolism. Overall, the results obtained not only rule out any cumulative adverse effects of CAF and CAU but also encourage further research to evaluate the possible use of CAU, a compound easily obtained through the valorization of biomass from invasive species, as a food additive to improve the clearance of xenobiotics.

Sperm-attractant peptide influences the spermatozoa swimming behavior in internal fertilization in Octopus vulgaris.

Marine invertebrates exhibit both chemokinesis and chemotaxis phenomena, induced in most cases by the release of water-borne peptides or pheromones. In mollusks, several peptides released during egg-laying improve both male attraction and mating. Unlike other cephalopods, Octopus vulgaris adopts an indirect internal fertilization strategy. We here report on the identification and characterization of a chemoattractant peptide isolated from mature eggs of octopus females. Using two-chamber and time-lapse microscopy assays, we demonstrate that this bioactive peptide is able to increase sperm motility and induce chemotaxis by changing the octopus spermatozoa swimming behavior in a dose-dependent manner. We also provide evidence that chemotaxis in the octopus requires the presence of extracellular calcium and membrane protein phophorylation at tyrosine. This study is the first report on a sperm-activating factor in a non-free-spawning marine animal.

The gonadotropin releasing hormone (GnRH)-like molecule in prosobranch Patella caerulea: potential biomarker of endocrine-disrupting compounds in marine environments.

It has been reported that endocrine disrupter compounds (EDCs) interfere with the endocrine system, mimicking the action of sex steroid hormones in different species of mollusks. Prosobranchs are frequently used as a reliable bioindicator to evaluate EDC exposure. In this article, we evaluate the effects of the xenoestrogen 4-n-nonylphenol (NP) in the prosobranch gastropod Patella caerulea, which exhibits protandrous hermaphroditism as its reproductive strategy. We isolated a partial sequence of a GnRH-like molecule from the gonads of Patella caerulea. The deduced amino acid sequence is highly homologous to that reported for the Lottia gigantea GnRH. Patella caerulea GnRH (pGnRH) mRNA expression is widespread in both male and female germ lines during gametogenesis. We suggest pGnRH as a novel biomarker for the early assessment of presence of EDCs and monitoring short and long-term impacts on Patella caerulea community structure.

Recording electrical activity from the brain of behaving octopus.

Octopuses, which are among the most intelligent invertebrates,1,2,3,4 have no skeleton and eight flexible arms whose sensory and motor activities are at once autonomous and coordinated by a complex central nervous system.5,6,7,8 The octopus brain contains a very large number of neurons, organized into numerous distinct lobes, the functions of which have been proposed based largely on the results of lesioning experiments.9,10,11,12,13 In other species, linking brain activity to behavior is done by implanting electrodes and directly correlating electrical activity with observed animal behavior. However, because the octopus lacks any hard structure to which recording equipment can be anchored, and because it uses its eight flexible arms to remove any foreign object attached to the outside of its body, in vivo recording of electrical activity from untethered, behaving octopuses has thus far not been possible. Here, we describe a novel technique for inserting a portable data logger into the octopus and implanting electrodes into the vertical lobe system, such that brain activity can be recorded for up to 12 h from unanesthetized, untethered octopuses and can be synchronized with simultaneous video recordings of behavior. In the brain activity, we identified several distinct patterns that appeared consistently in all animals. While some resemble activity patterns in mammalian neural tissue, others, such as episodes of 2 Hz, large amplitude oscillations, have not been reported. By providing an experimental platform for recording brain activity in behaving octopuses, our study is a critical step toward understanding how the brain controls behavior in these remarkable animals.

The Use of Isoflurane and Adjunctive Magnesium Chloride Provides Fast, Effective Anaesthetization of Octopus vulgaris.

A wide variety of substances have been used to anaesthetise invertebrates, but many are not anaesthetics and merely incapacitate animals rather than preventing pain. In essence, the role of an ideal general anaesthetic is to act as a muscle relaxant, an analgesic, an anaesthetic, and an amnesic. To achieve all these properties with a single substance is difficult, and various adjuvants usually need to be administered, resulting in a cocktail of drugs. In a clinical setting, the vast majority of patients are unaware of surgery being carried out and have no memory of it, so they can claim to have felt no pain, but this is much more difficult to demonstrate in invertebrates. Here, we show that 1% MgCl2, a muscle relaxant, is a useful adjuvant for the clinical anaesthetic isoflurane on Octopus vulgaris when applied alone in seawater for 10 min before the clinical anaesthetic. After this, full anaesthesia can be achieved in 5 min using 1% isoflurane insufflated into the saline still containing MgCl2. Full recovery takes place rapidly in about 10 to 15 min. The depth of anaesthesia was monitored using changes in respiratory rate, chromatophore pattern, and withdrawal movements of the arms and siphon. This methodology reduces stress on the animal and minimises the quantity of anaesthetic used.

An alien metabolite vs. a synthetic chemical hazard: An ecotoxicological comparison in the Mediterranean blue mussel.

Bioactive natural products from marine invasive species may dramatically impact native communities, while many synthetic pharmaceutical drugs are released into the marine environment and have long-lasting harmful effects on aquatic life. Sometimes, metabolites from alien species and synthetic compounds share similar mechanisms of action, suggesting comparable ecotoxicological impacts. This applies to the alkaloid caulerpin (CAU) from the green algae Caulerpa cylindracea, highly invasive in the Mediterranean Sea, and to the synthetic lipid-lowering drug fenofibrate (FFB), both acting as agonists of peroxisome proliferator-activated receptors (PPARs). Analogies with FFB, which is widely considered hazardous to the aquatic environment, have led to concerns about the ecotoxicological potential of CAU. The problem has implications for public health as CAU is well known to enter the food web accumulating in fish of commercial importance. Here, we compared the effects of FFB and CAU through biochemical and histopathological analysis on a relevant bioindicator molluscan species, the mussel Mytilus galloprovincialis. Under laboratory conditions, mussels were fed with food enriched with CAU or FFB. After treatment, biochemical markers were analyzed revealing metabolic capacity impairments, cellular damage, and changes in acetylcholinesterase activity in mussels fed with FFB-enriched food. NMR-based metabolomic studies also showed significant alterations in the metabolic profiles of FFB-treated mussels. In addition, dietary administration of FFB produced morphological alterations in the mussels' gills and digestive tubules. Obtained results confirm that FFB is harmful to aquatic life and that its release into the environment should be avoided. Conversely, dietary treatment with CAU did not produce any significant alterations in the mussels. Overall, our results pave the way for the possible valorization of the huge biomass from one of the world's worst invasive species to obtain CAU, a natural product of interest in drug discovery.

Neuroecology: Forces that shape the octopus brain.

Octopuses inhabit almost all seas in the world. A new study on tropical species suggests that, as in vertebrates, folding in the brain and visual system might be linked to habitat and lifestyle.

In an octopus's garden in the shade: Underwater image analysis of litter use by benthic octopuses.

Benthic octopuses have been widely documented in artificial shelters for decades, and this use is apparently increasing. Despite any possible positive effects, the use of litter as shelter could have negative implications. In this work, we aimed to elucidate the interactions of octopuses with marine litter, identifying types of interactions and affected species and regions. To achieve this, we obtained 261 underwater images from 'citizen science' records, and identified 8 genera and 24 species of benthic octopuses interacting with litter. Glass objects were present in 41.6% of interactions, and plastic in 24.7%. Asia presented the highest number of images, and most records were from 2018 to 2021. Citizen science provided important evidence on octopus/marine litter interactions, highlighting its value and the need for more investigations on the subject. This information is fundamental to help prevent and mitigate the impacts of litter on octopuses, and identify knowledge gaps that require attention.

The differential role of Leydig cells in the skin and gills of Lissotriton italicus larvae.

Larval urodeles are provided with external gills involved, along with the skin, in gas exchange and osmoregulation. Gills and skin epithelia are different, each showing a peculiar set of specialized cells but both provided with Leydig cells (LCs). Information on LCs in the gills is lacking as the literature has focused primarily on the epidermis. Contradictory and fragmentary results highlight that LCs origin, fate, and functions remain not fully understood. Here, we investigated the morpho-functional differences of LCs in the skin and gills of Lissotriton italicus larvae for the first time. LCs showed the same morphological and ultrastructural features in both tissues, even if LCs were significantly larger in the epidermis. Despite the uniform morphology within the LCs population, the proliferative ability was different. The putative diversity in the mucus composition was evaluated using a panel of 4 lectins as markers of specific carbohydrate moieties, revealing that sites of specific glycoconjugates were comparable in two tissues. To disclose the involvement of LCs in water storage and transport, immunofluorescence assay for aquaporin-3 has also been performed, demonstrating the expression of this protein only in gills epithelium. By demonstrating that LCs can multiply by cell division in gills, our results will also contribute to the discussion about their proliferative ability. Finally, we found that the LCs cytoplasm is rich in glycoconjugates, which are involved in many diverse and essential functions in vertebrates. RESEARCH HIGHLIGHTS: In gills LCs can multiply by cell division and express aquaporin-3 demonstrating a tissue-specific role of LCs. LCs cytoplasm is rich in glycoconjugates. LCs population show a uniform morphology in both gills and skin.

Behavioral, physiological and biochemical responses and differential gene expression in Mytilus galloprovincialis exposed to 17 alpha-ethinylestradiol and sodium lauryl sulfate.

Mixture of contaminants often determine biological responses of marine species, making difficult the interpretation of toxicological data. The pharmaceutical 17 alpha-ethinylestradiol (EE2) and the surfactant Sodium Lauryl Sulfate (SLS) commonly co-occur in the marine environment. This study evaluated the effects of EE2 (125.0 ng/L) and SLS (4 mg/L), acting individually and combined, in the mussel Mytilus galloprovincialis. Contaminated mussels closed their valves for longer periods than control ones, especially in the presence of both contaminants, with longer closure periods immediately after spiking compared to values obtained one day after spiking. Nevertheless, males and females increased their metabolism when in the presence of both contaminants (males) and SLS (females), and independently on the treatment males and females were able to activate their antioxidant and biotransformation defences. Although enhancing defences mussels still presented cellular damage and loss of redox balance, especially noticed in the presence of EE2 for males and SLS for females. Histopathological damage was found at mussel's gills in single and mixture exposure, and qPCR analysis revealed a clear estrogen receptor expression with no additive effect due to combined stressors. The results obtained highlight the harmful capacity of both contaminants but further research on this matter is needed, namely considering different climate change scenarios.

MicroRNAs are deeply linked to the emergence of the complex octopus brain.

Soft-bodied cephalopods such as octopuses are exceptionally intelligent invertebrates with a highly complex nervous system that evolved independently from vertebrates. Because of elevated RNA editing in their nervous tissues, we hypothesized that RNA regulation may play a major role in the cognitive success of this group. We thus profiled messenger RNAs and small RNAs in three cephalopod species including 18 tissues of the Octopus vulgaris. We show that the major RNA innovation of soft-bodied cephalopods is an expansion of the microRNA (miRNA) gene repertoire. These evolutionarily novel miRNAs were primarily expressed in adult neuronal tissues and during the development and had conserved and thus likely functional target sites. The only comparable miRNA expansions happened, notably, in vertebrates. Thus, we propose that miRNAs are intimately linked to the evolution of complex animal brains.

Research Trends in Octopus Biological Studies.

Octopuses represent interesting model studies for different fields of scientific inquiry. The present study provides a bibliometric analysis on research trends in octopuses biological studies. The analysis was executed from January 1985 to December 2020 including scientific products reported in the Web of Science database. The period of study was split into two blocks ("earlier period" (EP): 1985-2010; "recent period" (RP): 2011-2020) to analyze the evolution of the research topics over time. All publications of interest were identified by using the following query: ((AK = octopus) OR (AB = octopus) OR (TI = octopus)). Data information was converted into an R-data frame using bibliometrix. Octopuses studies appeared in 360 different sources in EP, while they increased to 408 in RP. Sixty countries contributed to the octopuses studies in the EP, while they were 78 in the RP. The number of affiliations also increased between EP and RP, with 835 research centers involved in the EP and 1399 in the RP. In the EP 5 clusters (i.e., "growth and nutrition", "pollution impact", "morphology", "neurobiology", "biochemistry") were represented in a thematic map, according to their centrality and density ranking. In the RP the analysis identified 4 clusters (i.e., "growth and nutrition", "ecology", "pollution impact", "genes, behavior, and brain evolution"). The UK with Ireland, and the USA with Canada shared the highest number of publications in the EP, while in the RP, Spain and Portugal were the leading countries. The current data provide significant insight into the evolving trends in octopuses studies.