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.

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.

Transcriptome profiling based on protein-protein interaction networks provides a set of core genes for understanding the immune response mechanisms of the egg-protecting behavior in Octopus ocellatus.

Protection via of the immune system is indispensable to the life of organisms. Within an immune network, problems with a given link will affect the normal life activities of the organism. Octopus ocellatus is cephalopod widely distributed throughout the world's oceans. Because of its unique nervous system and locomotive organs, research on this species has gradually increased in recent years. Many immune response mechanisms associated with behaviors of O. ocellatus are still unclear. Moreover, as a factor affecting the normal growth of O. ocellatus, egg protection has rarely been considered in previous behavioral studies. In this study, we analyzed the transcriptome profile of gene expression in O. ocellatus larvae, and identified 5936 differentially expressed genes (DEGs). GO and KEGG enrichment analyses were used to search for immune-related DEGs. Protein-protein interaction networks were constructed to examine the interactions between immune-related genes. Fifteen hub genes involved in multiple KEGG signaling pathways or with multiple protein-protein interaction relationships were obtained and verified by quantitative RT-PCR. We first studied the effects of egg protection on the immunity of O. ocellatus larvae by means of protein-protein interaction networks, and the results provide valuable genetic resources for understanding the immunity of invertebrate larvae. The data serve as a foundation for further research on the egg-protecting behavior of invertebrates.

When an octopus has MS: Application of neurophysiology and immunology of octopuses for multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated disease which can cause different symptoms due to the involvement of different regions of the central nervous system (CNS). Although this disease is characterized by the demyelination process, the most important feature of the disease is its degenerative nature. This nature is clinically manifested as progressive symptoms, especially in patients' walking, which can even lead to complete debilitation. Therefore, finding a treatment to prevent the degenerative processes is one of the most important goals in MS studies. To better understand the process and the effect of drugs, scientists use animal models which mostly consisting of mouse, rat, and monkey. In evolutionary terms, octopuses belong to the invertebrates which have many substantial differences with vertebrates. One of these differences is related to the nervous system of these organisms, which is divided into central and peripheral parts. The difference lies in the fact that the main volume of this system expands in the limbs of these organisms instead of their brain. This offers a kind of freedom of action and processing strength in the octopus limbs. Also, the brain of these organisms follows a non-somatotopic model. Although the complex actions of this organism are stimulated by the brain, in contrast to the human brain, this activity is not related to a specific region of the brain; rather the entire brain area of the octopus is activated during a process. Indeed, the brain mapping or the topological perception of a particular action, such as moving the limbs, reflects itself in how that activity is distributed in the octopus brain neurons. Accordingly, various actions are known with varying degrees of activity of neurons in the brain of octopus. Another important feature of octopuses is their ability to regenerate defective tissues including the central and peripheral nervous system. These characteristics raise the question of what features can an octopus show when it is used as an organism to create experimental autoimmune encephalomyelitis (EAE). Can the immune system damage of the octopus brain cause a regeneration process? Will the autonomy of the organs reduce the severity of the symptoms? This article seeks to provide evidence to prove that use of octopuses as laboratory samples for generation of EAE may open up new approaches for researchers to better approach MS.

The bacteriolytic mechanism of an invertebrate-type lysozyme from mollusk Octopus ocellatus.

As an important economic mollusk in coastal areas, Octopus ocellatus dependents on innate immune system to resist the invasion of microorganisms. Lysozyme is a crucial effector owing to its significant lytic activity against bacterial pathogens during the immune responses. In this study, characteristic and immune function of an I-type lysozyme from O. ocellatus (OoLyz) was investigated. OoLyz shared a close relationship with the lysozymes from other bivalve mollusks. The mRNA of OoLyz exhibited a broad transcript in different tissues/organs, and with the greatest expression in hepatopancreas. The expression of OoLyz was significantly raised when O. ocellatus was infected by Vibrio anguillarum or Micrococcus luteus, suggesting OoLyz participated in innate immune response of host. Prokaryotic recombinant OoLyz (rOoLyz) exhibited obvious bacteriolysis ability towards both gram-negative bacteria V. anguillarum and Escherichia coli, and gram-positive bacteria M. luteus and Staphylococcus aureus. The bacteriolysis activities of rOoLyz towards gram-negative but not gram-positive bacteria was heat stable, indicating that OoLyz might clear gram-positive bacterium by enzyme-dependent mechanisms, but eliminate gram-negative microbe via enzymatic activity independent way. Scanning electron microscopy analysis showed that rOoLyz destroyed microbes by damaging cell wall. More importantly, the fact that rOoLyz could directly degrade the peptidoglycan, further revealed its bactericidal mechanism as a muramidase. Our results revealed the essential role of I-type lysozyme in the innate immunity of O. ocellatus, and shed new light to understand the mechanism of immune defense of mollusks.

Identification of triosephosphate isomerase as a novel allergen in Octopus fangsiao.

Octopus is an important mollusk in human dietary for its nutritional value, however it also causes allergic reactions in humans. Major allergens from octopus have been identified, while the knowledge of novel allergens remains poor. In the present study, a novel allergen with molecular weight of 28kDa protein was purified from octopus (Octopus fangsiao) and identified as triosephosphate isomerase (TIM) by mass spectrometry. TIM aggregated beyond 45°C, and its IgE-binding activity was affected under extreme pH conditions due to the altered secondary structure. In simulated gastric fluid digestion, TIM can be degraded into small fragments, while retaining over 80% of the IgE-binding activity. The full-length cDNA of O. fangsiao TIM (1140bp) was cloned, which encodes 247 amino acid residues, and the entire recombinant TIM was successfully expressed in Escherichia coli BL21, which showed similar immunoreactivity to the native TIM. Different intensity of cross-reactivity among TIM from related species revealed the complexity of its epitopes. Eight linear epitopes of TIM were predicted following bioinformatic analysis. Furthermore, a conformational epitope (A71G74S69D75T73F72V67) was confirmed by the phage display technology. The results revealed the physicochemical and immunological characteristics of TIM, which is significant in the development of hyposensitivity food and allergy diagnosis.

Experimental infection of octopus vulgaris (Cuvier, 1797) with Photobacterium damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue responses.

Adult common octopus individuals were intramuscularly infected with Photobacterium damsela subsp. piscicida in order to investigate if this species is sensitive to this common and important fish pathogen. The fate of the bacterial antigens and the tissue responses of Octopus vulgaris were studied employing immunohistochemical techniques. Strong reaction at the site of injection was evident from day 2 post-infection that continued until day 14. Great numbers of hemocytes that were attracted at the site of infection were involved in phagocytosis of bacteria. Very early in the infection, a transition of cells to fibroblasts and an effort to isolate the infection was observed. During the course of the study, very large necrotic cells were seen at the site of infection, whereas during the later stages hemocytes with phagocytosed bacteria were observed in well-defined pockets inside the muscle tissue. None of the internal organs tested for the presence of the bacterium were positive with the exception of the digestive gland where antigen staining was observed which was not associated with hemocyte infiltration. The high doses of bacterial cells used in this experimental infection and the lack of disease signs from Octopus vulgaris suggest that, under normal conditions, octopus is resistant to Photobacterium damsela subsp. piscicida.

Involvement of a Serpin serine protease inhibitor (OoSerpin) from mollusc Octopus ocellatus in antibacterial response.

Serpin is an important member of serine protease inhibitors (SPIs), which is capable of regulating proteolytic events and involving in a variety of physiological processes. In present study, a Serpin homolog was identified from Octopus ocellatus (designated as OoSerpin). Full-length cDNA of OoSerpin was of 1735 bp, containing a 5' untranslated region of 214 bp, a 3' UTR of 282 bp, and an open reading frame of 1239 bp. The open reading frame encoded a polypeptide of 412 amino acids which has a predicted molecular weight of 46.5 kDa and an isoelectric point of 8.52. The OoSerpin protein shares 37% sequence identity with other Serpins from Mus musculus (NP_941373) and Ixodes scapularis (XP_002407493). The existence of a conserved SERPIN domain strongly suggested that OoSerpin was a member of the Serpin subfamily. Expression patterns of OoSerpin, both in tissues and towards bacterial stimulation, were then characterized. The mRNA of OoSerpin was constitutively expressed at different levels in all tested tissues of untreated O. ocellatus, including mantle (lowest), muscle, renal sac, gill, hemocyte, gonad, systemic heart, and hepatopancreas (highest). The transcriptional level of OoSerpin was significantly up-regulated (P<0.01) in O. ocellatus upon bacterial challenges with Vibrio anguillarum and Micrococcus luteus, indicating its involvement in the antibacterial immune response. Furthermore, rOoSerpin, the recombinant protein of OoSerpin, exhibited strong abilities to inhibit proteinase activities of trypsin and chymotrypsin as well as the growth of Escherichia coli. Our results demonstrate that OoSerpin is a potential antibacterial factor involved in the immune response of O. ocellatus against bacterial infection.

De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

BACKGROUND: Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus' well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. RESULTS: A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. CONCLUSION: The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers for octopus resistance against pathogens, which could improve octopus farming in the near future.

Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 µm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 µm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes.

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

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

Purification, cloning, and immunological characterization of arginine kinase, a novel allergen of Octopus fangsiao.

Arginine kinase (AK) is an important enzyme participating in energy metabolism in invertebrates, but, to date, there have been no reports that AK from octopus is an allergen. In this study, octopus AK was purified, and its molecular biological, immunological, and physicochemical characterizations were analyzed. The results showed that octopus AK was purified and confirmed by mass spectrometry for the first time, and its molecular mass was 38 kDa. The full-length gene sequence of octopus AK encompassed 1209 bp and was predicted to encode a protein with 348 amino acid residues. The homology of octopus AK and crustacean AK was about 54%, but the similarity between their three-dimensional structures was high. Octopus AK could react with mouse anti-shrimp AK and rabbit anti-crab AK polyclonal antibody singly. Octopus AK could also react with specific IgE of the sera from octopus-allergic patients effectively, whereas crab AK could inhibit the reaction between them. Finally, the IgE-binding activity of octopus AK could be reduced in the processes of thermal or acid-alkali treatment. In summary, AK was identified as a novel allergen in octopus, which had a sensitizing ability similar to that of crustacean AK. This is significant in allergy diagnosis and the treatment of octopus-allergic disorders.

The prevalence of food hypersensitivity in young adults.

rising prevalence of food hypersensitivity (FHS) and severe allergic reactions to foods have been reported in the last decade. However, little is known on the prevalence in young adults. This study estimated the prevalence of FHS to the most common allergenic foods in an unselected population of young adults. We investigated a cohort of 1272 young adults 22 years of age by questionnaire, skin prick test (SPT) and histamin release (HR) followed by oral challenge to the most common allergenic foods. FHS was divided into primary and secondary FHS. Primary FHS was defined as being independent of pollen sensitization, whereas secondary FHS was defined as reactions to pollen related fruits and vegetables in pollen allergic patients. The questionnaire was returned by 77.1%. Primary FHS was reported by 19.6% and secondary FHS by 16.7% of the participants. Confirmed primary FHS by oral challenge was 1.7% [1.1% - 2.95%]. In primary FHS, the most common allergenic food was peanut (0.6%) followed by additives (0.5%), shrimp (0.2%), codfish (0.1%), cow's milk (0.1%), octopus (0.1%) and soy (0.1%). In secondary FHS, kiwi allergy was reported by 7.8% of the participants followed by hazelnut (6.6%), pineapple (4.4%), apple (4.3%), orange (4.2%), tomato (3.8%), peach (3.0%) and brazil nut (2.7%). This study found a 1.7% [1.1% - 2.95%] prevalence of primary FHS confirmed by oral challenge to the most common allergenic foods in an unselected population of young adults.

Lysozyme and antiprotease activity in the lesser octopus Eledone cirrhosa (Lam.) (Cephalopoda).

Antiprotease and lysozyme activities were detected in various tissue samples including the haemocytes and haemolymph of Eledone cirrhosa. Injection of live Vibrio anguillarum caused an increase in lysozyme activity in the branchial heart over 48 hours and a decrease in the lysozyme activity of haemocytes over 24 hours. Haemocytes from control PBS injected animals demonstrated increased lysozyme levels 4 hours after injection whereas it decreased after the injection of live bacteria in PBS. The lysozyme activity of the haemolymph was not affected by these procedures. Bacteria injections had no effect on the antiprotease activity of the organ samples but increased the antiprotease activity of the haemocytes compared to controls in the 4 h samples. Haemolymph antiprotease activity decreased at a greater rate following bacteria injection than in control PBS injected animals. Haemocyte numbers/ml increased for both the control and bacteria injected animals with a greater increase demonstrated for the bacteria injected animals in the 4 h sample. Concomittant with the increase in the numbers of circulating haemocytes live V. anguillarum were cleared from the circulation of E. cirrhosa in less than 4 hours.

Isolation and characterization of a lectin from the hemolymph of the cephalopod Octopus vulgaris (Lam.) inhibited by alpha-D-lactose and N-acetyl-lactosamine.

The hemolymph of Octopus vulgaris is known to contain a lactose-specific agglutinin. This has been isolated by a two-step affinity-chromatographical procedure using, firstly immobilized asialofetuin followed by its binding to and elution from Con A-Sepharose. The lectin is a glycoprotein with a carbohydrate content of about 8%, and has a molecular weight (M.W.) of 260 kDa. Disulfide bridges connect subunits of molecular weights ranging between 30-32 kDa and of differing isoelectric points (pH 6.4, 6.6, 7.0, 7.3). Ca++-ions are required for ligand binding of the lectin; the pH-optimum for binding reactions is between pH 8.2-8.5 as determined by precipitation experiments with asialofetuin. Heating to 60-80 degrees C reduces agglutinating activity which is completely destroyed at 90 degrees C.

[Immunogenic activity of hemocyanin from the hemolymph of Helix pomatia and Octopus vulgaris]. / Attività immunogena di emocianine da emolinfa di Helix pomatia e di Octopus vulgaris.

The immunogenic activity of Helix Pomatia Haemocyanin (HPH) and Octopus vulgaris Haemocyanin (OVH) has been tested in rabbits. Primary and secondary antibody response has been evaluated in sera by agar double immuno-diffusion test (Ouchterlony), counter immunoelectrophoresis and complement fixation. Bath haemocyanins proved highly antigenic: antibody titers rising up to 1/3.200 after the second antigenic challenge. The Ouchterlony test showed a partial identity between H.P.H. and O.V.H. No adverse effect was evident in injected animals.