Gonadal Transcriptome Sequencing Analysis Reveals the Candidate Sex-Related Genes and Signaling Pathways in the East Asian Common Octopus, Octopus sinensis.

The East Asian common octopus (Octopus sinensis) is an economically important species among cephalopods. This species exhibits a strict dioecious and allogamous reproductive strategy, along with a phenotypic sexual dimorphism, where the third right arm differentiates into hectocotylus in males. However, our understanding of the molecular mechanisms that underlie sex determination and differentiation in this species remains limited. In the present study, we surveyed gene-expression profiles in the immature male and female gonads of O. sinensis based on the RNA-seq, and a total of 47.83 Gb of high-quality data were generated. Compared with the testis, we identified 8302 differentially expressed genes (DEGs) in the ovary, of which 4459 genes were up-regulated and 3843 genes were down-regulated. Based on the GO enrichment, many GO terms related to sex differentiation were identified, such as sex differentiation (GO: 0007548), sexual reproduction (GO: 0019953) and male sex differentiation (GO: 0046661). A KEGG classification analysis identified three conserved signaling pathways that related to sex differentiation, including the Wnt signaling pathway, TGF-ß signaling pathway and Notch signaling pathway. Additionally, 21 sex-related DEGs were selected, of which 13 DEGs were male-biased, including Dmrt1, Foxn5, Foxj1, Sox30, etc., and 8 DEGs were female-biased, including Sox14, Nanos3, ß-tubulin, Suh, etc. Ten DEGs were used to verify the expression patterns in the testis and ovary using the RT-qPCR method, and the results showed that the expression level shown by RT-qPCR was consistent with that from the RNA-seq, which confirmed the reliability of the transcriptome data. The results presented in this study will not only contribute to our understanding of sex-formation mechanisms in O. sinensis but also provide the foundational information for further investigating the molecular mechanisms that underline its gonadal development and facilitate the sustainable development of octopus artificial breeding.

Passive elasticity properties of Octopus rubescens arms.

In this report, passive elasticity properties of Octopus rubescens arm tissue are investigated using a multidisciplinary approach encompassing biomechanical experiments, computational modeling, and analyses. Tensile tests are conducted to obtain stress-strain relationships of the arm under axial stretch. Rheological tests are also performed to probe the dynamic shear response of the arm tissue. Based on these tests, comparisons against three different viscoelasticity models are reported.

Developing isotopic proxies to reconstruct the metabolic rates and thermal histories of octopus.

Understanding an animal's metabolic rate and thermal history is pivotal for ecological research. Recent studies have proposed the use of stable carbon and oxygen isotopes (δ13C and δ18O) in biogenic carbonates as proxies of metabolic rate and experienced temperature, respectively, to overcome the challenges of directly measuring these data in the field. Our study represents the first experimental investigation to develop δ13C and δ18O proxies in octopus. Octopus berrima hatchlings were raised in captivity, at varying water temperatures, for up to 110 days. O. berrima statoliths were then subsequently analysed for δ13C and δ18O values. The proportion of metabolically derived carbon, or respired carbon (Cresp), increased as the octopus grew (slope = 0.076, R2 = 0.72), suggesting an influence of somatic growth rate and body mass on δ13C values. Additionally, we identified an inverse correlation between δ18O values and environmental temperature (slope = -0.163, R2 = 0.91), which was subsequently used to develop a thermal reconstruction model. Our experiment aids in interpreting stable isotopic values in statoliths and their application as temperature and metabolic proxies in wild-caught octopus. Such proxies will increase our monitoring capabilities of these ecologically and commercially significant cephalopods and contribute to their conservation and effective management.

Expression profile and immunomodulatory roles of methionine-enkephalin and delta opioid receptor in Octopus ocellatus.

In this study, the expressions and distributions of methionine-enkephalin (Met-enk) and δ opioid receptor in the nervous system of Octopus ocellatus, and the immune regulatory mechanisms of Met-enk on O. ocellatus were explored. The distributions and expressions of Met-enk and δ opioid receptor were assessed by immunohistochemistry and enzyme-linked immunosorbent assay. UV-spectrophotometer, microplate reader, and flow cytometer were used to examine the effects of different concentrations of Met-enk on phagocytosis, antioxidant effects, and body surface mucus immunity of O. ocellatus hemocytes. The data were used to study the mechanisms of Met-enk immunity regulation in O. ocellatus. According to the results, the expression levels of Met-enk and δ opioid receptor in O. ocellatus lymphocytes were higher than those in hemocytes. The expression levels of Met-enk in the ganglia of O. ocellatus decreased in the following order: pedal ganglia > cerebral ganglia > visceral ganglia > optic ganglia > stellate ganglia. Moreover, the phagocytic activity of O. ocellatus hemocytes was enhanced with increasing Met-enk concentration. With increasing Met-enk concentration, the expressions of nitric oxide, total nitric oxide synthase, inducible nitric oxide synthase, catalase, hydrogen peroxide, myeloperoxidase, reduced glutathione, α-naphthy acetate esterase, and methionine aminopeptidases decreased in serums of O. ocellatus in the experimental group compared to the blank group. Similarly, the content of reduced glutathione in the hemocytes of O. ocellatus was also lower in the experimental group than in the blank group; however, the expressions of other substances were higher compared to the blank group. Furthermore, α-naphthy acetate esterase, myeloperoxidase, and hydrogen peroxide expressions in mucus immunity trials of the body surface were lower in the experimental group compared to the blank group. These results indicate that the distributions and expressions of Met-enk and δ opioid receptor in the nervous system of O. ocellatus were related to axoplasmic transport and immune regulation mechanisms. Met-enk participates in cellular immunity, humoral immunity, and mucus immunity in the form of neurotransmitters, thereby regulating the immune response of O. ocellatus.

Climate change consequences on the systemic heart of female Octopus maya: oxidative phosphorylation assessment and the antioxidant system.

There is evidence that indicates that temperature modulates the reproduction of the tropical species Octopus maya, through the over- or under-expression of many genes in the brain. If the oxygen supply to the brain depends on the circulatory system, how temperature affects different tissues will begin in the heart, responsible for pumping the oxygen to tissues. The present study examines the impact of heat stress on the mitochondrial function of the systemic heart of adult O. maya. The mitochondrial metabolism and antioxidant defense system were measured in the systemic heart tissue of female organisms acclimated to different temperatures (24, 26, and 30°C). The results show that acclimation temperature affects respiratory State 3 and State 4o (oligomycin-induced) with higher values observed in females acclimated at 26°C. The antioxidant defense system is also affected by acclimation temperature with significant differences observed in superoxide dismutase, glutathione S-transferase activities, and glutathione levels. The results suggest that high temperatures (30°C) could exert physical limitations on the circulatory system through the heart pumping, affecting nutrient and oxygen transport to other tissues, including the brain, which exerts control over the reproductive system. The role of the cardiovascular system in supporting aerobic metabolism in octopus females is discussed.

3D octopus kinematics of complex postures: Translation to long, thin, soft devices and their potential for clinical use.

INTRO/BACKGROUND: Octopuses are capable of complex arm movements. Unfortunately, experimental barriers and lack of a robust analysis method made it difficult to quantify the three-dimensional (3D) kinematics of soft, flexible bodies, such as the octopus arm. This information is not only crucial for understanding the posture of the animal's arm but also for the development of similarly designed soft, flexible devices. OBJ/GOAL: The primary goal of this work was to create a method to comprehensively quantify complex, 3D postures of octopus (Octopus Bimaculoides) arms in a manner that is conducive and translatable to octopus arm-inspired devices for health monitoring and rehabilitation. METHODS: In this study, 3D underwater motion capture was used to collect kinematic data on both live octopuses and disembodied arms that still had neural activity. A new method was developed to define how arm curvature and regional segments were oriented relative to each other in 3D. This included identification of the bend within a segment along with the computation of the relative orientation between segments, thus permitting the complete quantification of complex arm motions. RESULTS: By comparing vector-based and radius of curvature-based approaches to magnitude of curvature, it was clear that the vector-based approach was less dependent on the length of a segment and that its reported ranges of motion were translatable for outcome measures associated with clinical use. The new approach for the relative orientation of each segment of the octopus arm resulted in the capability of describing the octopus arm in many unique postures, such as straight, simple bending, and complex bending as it utilized the three rotational angles. OUTCOME/IMPACT: This method and its application to octopus arms will yield new information that can be used to better communicate and track not only octopus arm movements but in the development of complex, segmented, soft-bodied devices that can be used in health monitoring and rehabilitation.

HOST SWITCHING IN DICYEMIDS (PHYLUM DICYEMIDA).

Dicyemids (phylum Dicyemida) are the most common and most characteristic endosymbionts in the renal sacs of benthic cephalopod molluscs: octopuses and cuttlefishes. Typically, 2 or 3 dicyemid species are found in a single specimen of the host, and most dicyemids have high host specificity. Host-specific parasites are restricted to a limited range of host species by ecological barriers that impede dispersal and successful establishment; therefore, phylogenies of interacting groups are often congruent due to repeated co-speciation. Most frequently, however, host and parasite phylogenies are not congruent, which can be explained by processes such as host switching and other macro-evolutionary events. Here, the history of dicyemids and their host cephalopod associations were studied by comparing their phylogenies. Dicyemid species were collected from 8 decapodiform species and 12 octopodiform species in Japanese waters. Using whole mitochondrial cytochrome c oxidase subunit 1 (COI) sequences, a phylogeny of 37 dicyemid species, including 4 genera representing the family Dicyemidae, was reconstructed. Phylogenetic trees derived from analyses of COI genes consistently suggested that dicyemid species should be separated into 3 major clades and that the most common genera, Dicyema and Dicyemennea, are not monophyletic. Thus, morphological classification does not reflect the phylogenetic relationships of these 2 genera. Divergence (speciation) of dicyemid species seems to have occurred within a single host species. Possible host-switching events may have occurred between the Octopodiformes and Decapodiformes or within the Octopodiformes or the Decapodiformes. Therefore, the mechanism of dicyemid speciation may be a mixture of host switching and intra-host speciation. This is the first study in which the process of dicyemid diversification involving cephalopod hosts has been evaluated with a large number of dicyemid species and genera.

Projected ocean temperatures impair key proteins used in vision of octopus hatchlings.

Global warming is one of the most significant and widespread effects of climate change. While early life stages are particularly vulnerable to increasing temperatures, little is known about the molecular processes that underpin their capacity to adapt to temperature change during early development. Using a quantitative proteomics approach, we investigated the effects of thermal stress on octopus embryos. We exposed Octopus berrima embryos to different temperature treatments (control 19°C, current summer temperature 22°C, or future projected summer temperature 25°C) until hatching. By comparing their protein expression levels, we found that future projected temperatures significantly reduced levels of key eye proteins such as S-crystallin and retinol dehydrogenase 12, suggesting the embryonic octopuses had impaired vision at elevated temperature. We also found that this was coupled with a cellular stress response that included a significant elevation of proteins involved in molecular chaperoning and redox regulation. Energy resources were also redirected away from non-essential processes such as growth and digestion. These findings, taken together with the high embryonic mortality observed under the highest temperature, identify critical physiological functions of embryonic octopuses that may be impaired under future warming conditions. Our findings demonstrate the severity of the thermal impacts on the early life stages of octopuses as demonstrated by quantitative proteome changes that affect vision, protein chaperoning, redox regulation and energy metabolism as critical physiological functions that underlie the responses to thermal stress.

Genome-wide identification of toll-like receptors in Octopus sinensis and expression analysis in response to different PAMPs stimulation.

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation.

Soft octopus-inspired suction cups using dielectric elastomer actuators with sensing capabilities.

Bioinspired and biomimetic soft grippers are rapidly growing fields. They represent an advancement in soft robotics as they emulate the adaptability and flexibility of biological end effectors. A prominent example of a gripping mechanism found in nature is the octopus tentacle, enabling the animal to attach to rough and irregular surfaces. Inspired by the structure and morphology of the tentacles, this study introduces a novel design, fabrication, and characterization method of dielectric elastomer suction cups. To grasp objects, the developed suction cups perform out-of-plane deflections as the suction mechanism. Their attachment mechanism resembles that of their biological counterparts, as they do not require a pre-stretch over a rigid frame or any external hydraulic or pneumatic support to form and hold the dome structure of the suction cups. The realized artificial suction cups demonstrate the capability of generating a negative pressure up to 1.3 kPa in air and grasping and lifting objects with a maximum 58 g weight under an actuation voltage of 6 kV. They also have sensing capabilities to determine whether the grasping was successful without the need of lifting the objects.

Living in a dynamic environment: The effects of multi-ways temperature variation on embryo and newborn juveniles of a shallow-water octopus (Amphioctopus fangsiao).

Shallow waters are characterized by fluctuating environmental conditions, modulating marine life cycles and biological phenomena. Multiple variations in water temperature could affect eggs and embryos during spawning events of many marine invertebrate species, yet most of the findings on embryonic development in invertebrates come from experiments based on the constant temperature. In this study, to examine the effects of temperature variation on octopus embryos, Amphioctopus fangsiao, a common shallow-water octopus along the coast of China, was exposed to the constant temperature (18 °C, in situ temperature of the seawater in Lianyungang), ramping temperatures (from 18 to 24 °C), diel oscillating temperatures (18 °C and 20 °C for 12 h each day), and acute increasing temperatures (the temperature increased sharply from 18 °C to 24 °C at embryonic development stage XIX) for 47 days (from embryogenesis to settlement). The results demonstrated that the temperature variations accelerated the development time of A. fangsiao embryos. Temperature fluctuations could cause embryonic oxidative damage and disorder of glycolipid metabolism, thereby affecting the growth performance of embryos and the survival rate of hatchings. Through transcriptome sequencing, the mechanistic adaption of the embryo to environmental temperature variations was revealed. The pathways involved in the TCA cycle, DNA replication and repair, protein synthesis, cell signaling, and nervous system damage repair were significantly enriched, indicating that the embryo could improve heat tolerance to thermal stress by regulating gene expression. Moreover, acute warming temperatures posed the most detrimental effects on A. fangsiao embryos, which could cause embryos to hatch prematurely from the vegetal pole, further reducing the survival of hatchings. Meanwhile, the diel oscillating temperature was observed to affect the normal morphology of the embryo, resulting in embryo deformities. Thus, the constant temperature is critical for balanced growth and defense status in octopuses by maintaining metabolism homeostasis. For the first time, this study evaluates the effects of multiple temperature fluctuations on embryos of A. fangsiao, providing new insights into the physiological changes and molecular responses of cephalopod embryos following dynamic temperature stress.

Understanding baseline levels of physiological stress tolerance from excessive exercise in a holobenthic octopus species, Octopus pallidus.

Cephalopods receive a great deal of attention due to their socioeconomically important fisheries and aquaculture industries as well their unique biological features. However, basic information about their physiological responses under stress conditions is lacking. This study investigated the impact of a simple stressor, exercise to exhaustion, on the activity levels of antioxidant enzymes and the concentrations of molecules involved in oxidative stress response in the pale octopus (Octopus pallidus). Eight biochemical assays were measured in the humoral (plasma) and cellular (hemocyte) components of O. pallidus haemolymph, the invertebrate analogue to vertebrate blood. Overall, exercise resulted in an increase in activity of plasma catalase (CAT) and glutathione-S-transferase (GST) and the decrease in activity of plasms glutathione reductase (GR). In the hemocytes, the exercise elicited a different response, with a reduction in the activity of superoxide dismutase (SOD), GR, and glutathione peroxidase (GPX) and a reduction in nitric oxide (NO) concentration. Malondialdehyde (MDA) activity was similar in the plasma and haemocytes in control and exercised treatments, indicating that exercise did not induce lipid peroxidation. These results provide an important baseline for understanding oxidative stress in octopus, with exercise to exhaustion serving as a simple stressor which will ultimately inform our ability to detect and understand physiological responses to more complex stressors.

Carboxymethyl cellulose-based hydrogel with high-density crack microstructures inspired from the multi-tentacles of octopus for ultrasensitive flexible sensing microsystem.

Constructing high-density contact-separation sites on conductive materials highly determines the sensitivity of flexible resistance-type sensors relying on the crack microstructures. Herein, inspired from the multiple-tentacle structures on octopus, we demonstrated a sort of novel carbonized ZIF-8@loofah (CZL) as conductive material to develop ultrasensitivity flexible sensor, in which the carbonized ZIF-8 nanoparticles (~100 nm) served as tentacles. Originating from the formation of high-density contact-separation sites, the fabricated CZL-based strain sensor delivered ultrahigh sensitivity of GFmax = 15,901, short response time of 22 ms and excellent durability over 10,000 cycles. These features enable the sensor with efficient monitoring capacity for complex human activities, such as pulse rate and phonation. Moreover, when CZL was assembled into triboelectric nanogenerator (TENG), CZL-based TENG can effectively convert the irregular biomechanical energy into electric energy, providing sustainable power supply for the continuous operation of the sensing micro-system. Our findings established a novel platform to develop high-performance self-powered sensing systems of physiological parameter of human inspired from the nature.

Permeable Skin Patch with Miniaturized Octopus-Like Suckers for Biosignal Monitoring.

Wearable electronics demand high adhesion properties through various skin conditions. Here, 3D-printed porous skin patches with octopus-like suckers of different geometries are presented. Experimental and theoretical studies are investigated to show an enhanced, low-cost 3D-printed bioinspired patches that successfully obtain biosignals comparable to commercial electrodes.Clinical Relevance- This work establishes low-cost, highly-adhesive skin patches that are irritation- and contamination-free with effortless peel-off technique for biosignal measurement.

The complete mitochondrial genome of Octopus vulgaris.

BACKGROUND: The Octopus vulgaris species complex consists of numerous morphologically similar but genetically distinct species. The current publicly available mitogenome of this species has been generated from a specimen collected from Tsukiji Fish Market, Tokyo, Japan. Octopus from the northwestern Pacific Ocean are now considered to be a separate species, Octopus sinensis. For this reason, we hypothesised that the current record of O. vulgaris was sequenced from a specimen of O. sinensis. Here, we sequenced the first complete mitogenome of a specimen of Octopus vulgaris sensu stricto that was collected from the species' confirmed distribution areas in northeastern Atlantic. METHODS AND RESULTS: The complete mitogenome was assembled de novo and annotated using 250 bp paired-end sequences. A single circular contig 15,655 bp in length with a mean read coverage of 1089 reads was reconstructed. The annotation pipeline identified 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNA) and two ribosomal RNAs. A maximum likelihood phylogenetic tree recovered the assembled mitogenome as the sister taxon of a monophyletic group comprising O. sinensis and the previously published mitogenome of "O. vulgaris" from Japan. This confirms that the latter was a Japanese specimen of O. sinensis. CONCLUSION: The mitogenome sequenced here is the first to be published for Octopus vulgaris sensu stricto. It represents an important first step in genetics-informed research on the evolution, conservation, and management of this commercially important species.

Genomic evidence for West Antarctic Ice Sheet collapse during the Last Interglacial.

The marine-based West Antarctic Ice Sheet (WAIS) is considered vulnerable to irreversible collapse under future climate trajectories, and its tipping point may lie within the mitigated warming scenarios of 1.5° to 2°C of the United Nations Paris Agreement. Knowledge of ice loss during similarly warm past climates could resolve this uncertainty, including the Last Interglacial when global sea levels were 5 to 10 meters higher than today and global average temperatures were 0.5° to 1.5°C warmer than preindustrial levels. Using a panel of genome-wide, single-nucleotide polymorphisms of a circum-Antarctic octopus, we show persistent, historic signals of gene flow only possible with complete WAIS collapse. Our results provide the first empirical evidence that the tipping point of WAIS loss could be reached even under stringent climate mitigation scenarios.

Cephalopod chemotactile sensation.

Allard et al. describe the remarkable 'taste by touch' abilities of cephalopods, in particular octopuses.

Cephalopod behaviour.

Underlying all animal behaviors, from the simplest reflexive reactions to the more complex cognitive reasoning and social interaction, are nervous systems uniquely adapted to bodies, environments, and challenges of different animal species. Coleoid cephalopods - octopuses, squid, and cuttlefish - are widely recognized as the most behaviorally complex invertebrates and provide exciting opportunities for studying the neural control of behaviour. These unusual molluscs evolved over 400 million years ago from slow-moving armored forms to active predators of coastal and open ocean ecosystems. In this primer we will discuss how, during cephalopod evolution, the relatively simple ganglion-based molluscan nervous system has been extensively transformed to control the complex bodies and process extensive visual, tactile, and chemical sensory inputs, and summarize some recent findings about their fascinating behaviors.