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Aquatic organisms have to produce proteins or factors that help maintain a stable relationship with microbiota and prevent colonization by pathogenic microorganisms. In crustaceans and other aquatic invertebrates, relatively few of these host factors have been characterized. In this study, we show that the respiratory glycoprotein hemocyanin is a crucial host factor that modulates microbial composition and diversity in the hepatopancreas of penaeid shrimp. Diseased penaeid shrimp (Penaeus vannamei), had an empty gastrointestinal tract with atrophied hepatopancreas, expressed low hemocyanin, and high total bacterial abundance, with Vibrio as the dominant bacteria. Similarly, shrimp depleted of hemocyanin had mitochondrial depolarization, increased reactive oxygen species (ROS) levels, and dysregulation of several energy metabolism-related genes. Hemocyanin silencing together with ROS scavenger (N-acetylcysteine) treatment improved microbial diversity and decreased Vibrio dominance in the hepatopancreas. However, fecal microbiota transplantation after hemocyanin knockdown could not restore the microbial composition in the hepatopancreas. Collectively, our data provide, to our knowledge, new insight into the pivotal role of hemocyanin in modulating microbial composition in penaeid shrimp hepatopancreas via its effect on mitochondrial integrity, energy metabolism, and ROS production.

Hemocyanin is the oxygen transport protein of most molluscs and represents an important physiological factor that has to be well-adapted to their environments because of the strong influences of abiotic factors on its oxygen affinity. Multiple independent gene duplications and intron gains have been reported for hemocyanin genes of Tectipleura (Heterobranchia) and the caenogastropod species Pomacea canaliculata, which contrast with the uniform gene architectures of hemocyanins in Vetigastropoda. The goal of this study was to analyze hemocyanin gene evolution within the diverse group of Caenogastropoda in more detail. Our findings reveal multiple gene duplications and intron gains and imply that these represent general features of Apogastropoda hemocyanins. Whereas hemocyanin exon–intron structures are identical within different Tectipleura lineages, they differ strongly within Caenogastropoda among phylogenetic groups as well as between paralogous hemocyanin genes of the same species. Thus, intron accumulation took place more gradually within Caenogastropoda but finally led to a similar consequence, namely, a multitude of introns. Since both phenomena occurred independently within Heterobranchia and Caenogastropoda, the results support the hypothesis that introns may contribute to adaptive radiation by offering new opportunities for genetic variability (multiple paralogs that may evolve differently) and regulation (multiple introns). Our study indicates that adaptation of hemocyanin genes may be one of several factors that contributed to the evolution of the large diversity of Apogastropoda. While questions remain, this hypothesis is presented as a starting point for the further study of hemocyanin genes and possible correlations between hemocyanin diversity and adaptive radiation. Supplementary Information The online version contains supplementary material available at 10.1007/s00239-021-10036-y.

Crustacean phenoloxidase (PO) and hemocyanin (Hc) are classified as type 3 copper proteins. PO catalyzes the oxidation of mono- and di-phenol compounds, which is the rate-limiting step of melanization, while Hc generally functions as a dioxygen-transporting protein in the hemolymph of arthropods. To date, many studies have shown PO activity in Hc, which is inspired by their structural similarity. Here, the source of PO activity in crustaceans was re-examined by purifying Hc and PO exclusively from the hemolymph of kuruma prawn. The conventional procedure for the preparation of arthropod Hc, which includes precipitation of Hc by ultracentrifugation and subsequent purification by size exclusion chromatography, was not able to completely remove hemolymph-type PO from Hc. In contrast, fractionation with 50% saturation of ammonium sulfate and subsequent hydrophobic chromatography yielded sufficiently pure Hc, which contained no detectable PO protein and virtually no PO enzymatic activity. These results indicate that the main source of PO activity in the hemolymph of kuruma prawn is hemolymph-type PO and that the improved purification method of Hc is preferable for evaluating the PO activity of Hc.

In contrast to the external shells in bivalves and gastropods, most cephalopods are missing this external protection. The cuttlefish, belonging to class cephalopod, has an internal biomineralized structure made of mainly calcium carbonate for controlling buoyancy. However, the macromolecules, especially proteins that control cuttlebone mineral formation, are not sufficiently understood, limiting our understanding of the evolution of this internal shell. In this study, we extracted proteins from the cuttlebone of pharaoh cuttlefish Sepia pharaonis and performed liquid chromatography-tandem mass spectrometry to identify the shell matrix proteins (SMPs). In total, 41 SMPs were identified. Among them, hemocyanin, an oxygen-carrying protein, was the most abundant SMP. By comparison with SMPs of other marine biominerals, hemocyanin, apolipophorin, soul domain proteins, transferrin, FL-rich, and enolase were found to be unique to the cuttlebone. In contrast, typical SMPs of external shells such as carbonic anhydrase complement control protein, fibronectin type III, and G/A-rich proteins were lacking from the cuttlebone. Furthermore, the cluster analysis of biomineral SMPs suggests that the SMP repertoire of the cuttlebone does not resemble that of other species with external shells. Taken together, this study implies a potential relationship of the cuttlefish internal shell with other internal biominerals, which highlights a unique shell evolutionary pathway in invertebrates.

Current cancer treatment regimens such as chemotherapy and traditional chemical drugs have adverse side effects including the appearance of drug-resistant tumor cells. For these reasons, it is imperative to find novel therapeutic agents that overcome these factors. To this end, we explored a cationic antimicrobial peptide derived from Litopenaeus vannamei hemocyanin (designated LvHemB1) that induces cancer cell death, but sparing normal cells. LvHemB1 inhibits the proliferation of human cervical (HeLa), esophageal (EC109), hepatocellular (HepG2), and bladder (EJ) cancer cell lines, but had no significant effect on normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE-3) cells). In addition to its antiproliferative effects, LvHemB1 induced apoptosis, by permeating cells and targeting mitochondrial voltage-dependent anion channel 1 (VDAC1). Colocalization studies revealed the localization of LvHemB1 in mitochondria, while molecular docking and pull-down analyses confirmed LvHemB1-VDAC1 interaction. Moreover, LvHemB1 causes loss in mitochondrial membrane potential and increases levels of reactive oxygen species (ROS) and apoptotic proteins (caspase-9, caspase-3, and Bax (Bcl-2-associated X)), which results in mitochondrial-mediated apoptosis. Thus, peptide LvHemB1 has the potential of being used as an anticancer agent due to its antiproliferation effect and targeting to VDAC1 to cause mitochondrial dysfunction in cancer cells, as well as its ability to induce apoptosis by increasing ROS levels, and the expression of proapoptotic proteins.

Aim: Identification of full-length ORF of hemocyanin subunit-1 (Mr_HC_1) from the hepatopancreas transcriptome of freshwater prawn, Macrobrachium rosenbergii infected with Vibrio harveyi and characterization of its sequence and structure by in silico tools and softwares. Methodology: Illumina HiSeq and de novo assembled unigenes were scanned against PANM-DB to screen Mr_HC_1. FGENESH gene prediction and SMART programs were used to predict the ORF region. Subsequently, Clustal X2 and MEGA in-silico tools were used to understand the sequence relatedness and evolutionary status of Mr_HC_1. Structural prediction was performed by SWISS-MODEL and Ramachandran plot modeling programs Results: The full-length ORF was 1983 bp in length encoding a polypeptide of 661 amino acid residues. Mr_HC_1 showed a putative signal peptide of 21 amino acid residues at the N-terminus and three hemocyanin domains. Homology analysis of Mr_HC_1 amino acid sequence confirms maximum identity to M. nipponense hemocyanin subunit-1 (Mn_HC_1). Phylogenetic analysis showed that Mr_HC_1 is more closely related to the hemocyanin γ-type subunit of freshwater shrimps. Homology modeling of Mr_HC_1 showed homo-hexameric protein containing 12 copper ions. With a QMEAN score of -3.33 and model-template sequence identity of 59.15%, the predicted model of Mr_HC_1 is convincing Interpretation: This study characterizes the hemocyanin γ-type subunit protein of freshwater prawn, M. rosenbergii for future studies on host defense mechanisms.

Hemocyanins are giant oxygen transport proteins that freely float within the hemolymph of most molluscs. The basic quaternary structure of molluscan hemocyanins is a cylindrical decamer with a diameter of 35 nm which is built of 400 kDa subunits. Previously published results, however, showed that one out of two hemocyanin subunits of Rapana venosa encompasses two polypeptides, one 300 kDa and one 100 kDa polypeptide which aggregate to typical 4 MDa and 8 MDa hemocyanin (di-)decamer molecules. It was shown that the polypeptides are bound most probably by one or more cysteine disulfide bridges but it remained open if these polypeptides were coded by one or two genes. Our here presented results clearly showed that both polypeptides are coded by one gene only and that this phenomenon can also be found in the gastropod Nucella lapillus. Thus, it can be defined as clade-specific for Muricidae, a group of the very diverse Caenogastropoda. In addition, we discovered a further deviation of this hemocyanin subunit within both species, namely a region of 340 mainly hydrophilic amino acids (especially histidines and aspartic acids) which have not been identified in any other molluscan hemocyanin, yet. Our results indicate that, within the quaternary structure, these additional amino acids most probably protrude within the inner part of didecamer cylinders, forming a large extra mass of up to 800 kDa. They presumably influence the structure of the protein and may affect the functionality. Thus, these findings reveal further insights into the evolution and structures of gastropod hemocyanins. Supplementary Information The online version of this article (10.1007/s00239-020-09986-6) contains supplementary material, which is available to authorized users.

The structural stability of the hеmocyanin purified from the hеmolymph of gаrden snаils Helix lucorum (HlH) was investigated by means of far-UV circular dichroism (CD), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). For the first time, TEM analyses showed the presence of tubular polymers in hemocyanins after three-day dialysis against a stabilizing buffer containing high concentrations of Ca2+ and Mg2+ ions (100 mmol L−1). The conformational stability study of native HlH by means of CD in a wide pH range (2.5-11.5) defined the pH stability region of HlH at pH 6.5 − 8.0. DSC analyses demonstrated the thermal stability of this hemocyanin. One transition, with an apparent transition tеmperature (Tm) at 82.3 °C, was detected in the heаt capacity curve of HlH in 50 mmol L−1 Tris-HCl buffer, pH 7.2, at a heating rate of 1.0 °C min−1. The calorimetrically observed thermal transition correlates well with the unfolding transition monitored by CD measurements. The two-state kinetic model was used to analyse the process of irreversible thermal denaturation of HlH; Еa of 451 ± 4 kJ mol−1 was calculated. The obtained results on the conformational stability of HlH will facilitate the further investigation of the properties and potential biomedical applications of this respiratory protein.

Two oxygen-transporting proteins known for their immunostimulating and anticancer properties, namely hemocyanin from Helix lucorum (HlH) and hemocyanin from Rapana thomasiana (RtH), have been modified for the first time with rosmarinic acid (RA). We prepared two conjugates RA–HlH and RA–RtH containing 47 and 50 rosmarinic acid residues, respectively. The conformational analysis showed that the secondary structure of RA–HlH is less ordered than that of the native HlH, and for the modified protein we observed a decrease in α-helical structures in the favor of random coils, unordered structures, and aggregates. Calorimetric studies showed an increase in the thermal stability of RA–HlH in comparison with the native HlH which is in agreement with the observed structural changes. On the other hand, the conformation of RA–RtH was very similar to that of the native RtH although some rearrangements in the molecule were found. Interestingly, the modification of RtH with RA resulted in a decrease in its thermal stability. A 48-h in vitro experiment showed that tested in concentrations up to 0.7 mg mL−1 the two modified hemocyanins did not inhibit the cell growth of human fibroblasts.

There has been extensive research on local and systemic oxidative stress and immunosuppression in cadmium exposed crustaceans, but the underlying mechanisms remain to be elucidated. Because of multiple functions of epithelial cells, such as storing and detoxifying heavy metals, producing and secreting immune-related molecules (i.e. hemocyanin, NF-κB and CBS/H2S et al.), hepatopancreas may play an important role in immune system. In the present study, as an indication of systemic and local immune status in crayfish Procambarus clarkii, the relationship between PO activities in haemolymph and levels of CBS/H2S/NF-κBp65 in hepatopancreas was evaluated following a 96 h exposure to sub-lethal Cd2+ concentrations (1/40, 1/8 and 1/4 of the 96 h LC50). The results indicated that there was a significant increase in ROS contents accompanied by markedly decreased THC and PO levels (P

Hemocyanin, the predominant protein in the hemolymph of crustaceans, was isolated from the plasma of the giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879) and investigated, with its separated proteins MrHc1, 2, 3, and 4 (60, 114, 50, and 325 kDa, respectively) for hemagglutination (HA) activity. Hemocyanin against erythrocytes from human A, B, and O blood groups, mouse, rat sheep, water buffalo, and rabbit showed HA titer values higher than plasma. Of the four proteins isolated from hemocyanin, MrHc1 alone appeared to agglutinate the tested erythrocytes. Evaluated specific hemagglutination activity was higher for hemocyanin when compared to plasma. Sugar binding ability with N-acetylated sugars observed for plasma was restricted to N-acetyl neuraminic acid for hemocyanin and MrHc1. Inhibition of hemagglutination (HAI) by bovine submaxillary mucin (BSM) further defined the agglutination ability of hemocyanin and MrHc1. The hemocyanin protein MrHc1 on matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI–TOF/MS) and search in MASCOT database showed homology to hypothetical protein of Branchiostoma floridae Hubbs, 1922 (Cephalochordata). The results clearly indicated that the structure of the hemocyanin had evolved binding sites for sugar or sugar-linkages independently lacking homology with other hemocyanin or lectin-like proteins. The immunogenicity of this hemocyanin probably has biomedical applications as carrier adjuvants, which needs further investigation.

Prophenoloxidase is a metalloprotinase and closely associated with the copper proteins family like hemocyanin; both are extracellular proteins that are structurally similar and functionally vary in their roles. In this study, we used the mud crab Scylla serrata as a model organism to explore the mechanism of phenoloxidase (PO) and structure analysis through in silico approach also attempting to discover the chemical inhibitors for the protein molecule from different databases. We have purified metalloprotein, prophenoloxidase (proPO), exhibits the 76 kDa molecular weight in SDS-PAGE, widely distributed in hemocytes and consisted of 2 homologous polypeptide chains, proPO1 and proPO2. Enhanced PO activity was observed when using microbial pathogen-associated molecular patterns (MAMPs) and metals as an activator. In vitro and in silico approaches were employed to characterize the structural and functional properties of phenoloxidases, and also we used the virtually screened ligands to check the activity of the protein. Through virtual screening method to screen the new ligands to bind with proPO from zinc and binding databases, the binding interactions are also analyzed via docking scores. This present report paves the way to develop the new immunomodulatory ligands targeting toward the prophenoloxidase and also gives new insights about the structure of the protein.

The naturally occurring polysaccharide, β-1,3-glucans, a well-known immunostimulant, has been highly valued for many years for their health-promoting and anti-aging properties, but its mode of action is poorly understood. In this study, we first showed that oral administration of β-1,3-glucans did not affect the general condition and physiology of male mice throughout the trial period. We then showed that dietary intake of β-1,3-glucans induced a significant increase in T helper cells (CD4+) in young, middle-aged and aged male mice. We also showed that β-1,3-glucans supplementation considerably increased the delayed-type hypersensitivity (DTH) response, a T cell-mediated immune response, in young and aged mice. In addition, we found that β-1,3-glucans supplementation remarkably promoted the production of total anti-keyhole limpet hemocyanin (KLH) immunoglobulin G (IgG), anti-KLH IgG1, and anti-KLH IgG2a in young and aged mice without disturbing immune homeostasis. These data together indicate that oral administration of β-1,3-glucans enhanced the adaptive immune responses of aged mice without disturbing their general condition and physiology, supporting the idea that β-1,3-glucans are capable of counteracting the immunosenescence in mice. They also suggest that β-1,3-glucans can be clinically useful to help the elderly generate an improved response to vaccine with stronger humoral and cell-mediated immune responses.

The Neoproterozoic included changes in oceanic redox conditions, the configuration of continents and climate, extreme ice ages (Sturtian and Marinoan), and the rise of complex life forms. A much-debated topic in geobiology concerns the influence of atmospheric oxygenation on Earth and the origin and diversification of animal lineages, with the most widely popularized hypotheses relying on causal links between oxygen levels and the rise of animals. The vast majority of extant animals use aerobic metabolism for growth and homeostasis; hence, the binding and transportation of oxygen represent a vital physiological task. Considering the blood pigment hemocyanin (Hc) is present in sponges and ctenophores, and likely to be present in the common ancestor of animals, we investigated the evolution and date of Hc emergence using bioinformatics approaches on both transcriptomic and genomic data. Bayesian molecular dating suggested that the ancestral animal Hc gene arose approximately 881 Ma during the Tonian Period (1000-720 Ma), prior to the extreme glaciation events of the Cryogenian Period (720-635 Ma). This result is corroborated by a recently discovered fossil of a putative sponge ~890 Ma and modern molecular dating for the origin of metazoans of ~1,000-650 Ma (but does contradict previous inferences regarding the origin of Hc ~700-600 Ma). Our data reveal that crown-group animals already possessed hemocyanin-like blood pigments, which may have enhanced the oxygen-carrying capacity of these animals in hypoxic environments at that time or acted in the transport of hormones, detoxification of heavy metals, and immunity pathways.

Gastropods are endowed with a soluble copper containing, blue coloured respiratory protein known as hemocyanin. In the present study, for the first time, hemocyanin from Pila virens, a fresh water gastropod endemic to Indian sub-continent was studied for its conformational stability in the presence of temperature and various detergents by different spectroscopic techniques viz. UV–Vis, fluorescence and circular dichroism spectroscopy. Results from UV–Vis spectroscopy revealed that, absorbance corresponding to active site moieties were decreased with increase in temperature from 30 to 70 °C. Whereas ‘thermal reversibility’ studies showed the increase in absorbance of active site with decrease in temperature from 70 to 20 °C (is an indicative of the characteristic feature of hemocyanins—that oxygen is reversibly bound to the copper in the active site). Results from CD spectroscopy revealed that the temperature-induced conformational changes on the secondary structure of hemocyanin was reversible up to 70 °C. Beyond 80 °C, the protein was completely denatured and assumed an increased random coil conformation. Hemocyanin in the presence of high concentration of detergents lost its structure as evident from UV–Vis fluorescence (evident from the red shifts in the emission maxima accompanied by quenching in emission intensities) and CD spectroscopy. Stability of active site moieties in the presence of different detergents was also studied by UV–Vis spectroscopy. The biophysical techniques gave insights into the varied effects of each of the different denaturants on the conformational stability of hemocyanin from Pila virens.

Human norovirus is the leading cause of foodborne illness globally. Detection and quantification of norovirus commonly involves the use of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR); however, the presence of inhibitory compounds in foods limit detection and accurate quantification. Although some studies have been done on PCR inhibitors from foods, many of them are over a decade old and do not investigate inhibition in contemporary one-step RT-qPCR-based detection chemistries. The purpose of this work was to quantify the degree of inhibition that occurs from inhibitory compounds found in produce (pectin) and mollusks (hemocyanin, glycogen)—foods commonly associated with norovirus outbreaks. RT-qPCR reactions containing different amounts of genomic bacteriophage MS2 RNA, a norovirus surrogate, were spiked with different concentrations of pectin (0.0625%–0.25% w/V), glycogen (1.25%–10%), and hemocyanin (0.0625%–0.25%). Past research has implicated glycogen as an inhibitory compound in oysters; however, even high levels of glycogen (10%) had no significant effect (P > 0.05) on amplification. Conversely, both pectin and hemocyanin caused complete inhibition at 0.25%, with no significant inhibition observed at 0.0625% (P

Salinity in the oceans is changing due to climate change and global warming. Intense rainfalls and freshwater runoff decrease salinity along the coastal areas. In contrast, intense drought seasons and river damming have certainly increased salinity in lagoons and estuaries. Few studies have focused on aspects of the biology and culture of oyster Crassostrea corteziensis, but until now, physiological and immunological responses in this species have not been assessed under acute hypo– and hypersaline stress conditions. Oysters obtained from a local farm were acclimated for three weeks in laboratory conditions. To avoid closure of oyster valves during salinity induced–stress conditions, a notch was done on each organism shell not only to facilitate oyster tissue exposure to rearing water but also for sampling hemolymph. Oysters (N = 180) were abruptly exposed to three salinity treatments: (HO) hypo–, (C) control, and (HP) hypersaline stress conditions (10, 35, and 50 PSU, respectively). Four oysters per treatment were sampled at 1, 2, 3, 6, 12, 24, and 48 h after exposure. Hemolymph osmolality, water content and total protein concentration in tissues, metabolic and immune responses were assessed for each organism. Oyster survival was not different among treatments and was maintained above 96% at the end of the experimental trial. Hemolymph osmolality reached the value of rearing water at 6 and 48 h of exposure to HP and HO stress conditions, where oysters exposed to salinity increase showed less resilience than those to decrease. Higher glucose levels in plasma and lower ones of hemocyanin were assessed in the oysters exposed to HP compared to HO conditions, suggesting more stressful conditions or susceptibility of oysters during salinity increase. Total hemocyte (THC), hyalinocyte (HC), and granulocyte (GC) counts decreased in oysters exposed to HP condition, while total and differential hemocyte counts were similar among oysters exposed to HO and control conditions. Despite hemocyte phagocytosis was not different among treatments, viability decreased in those exposed to HP condition. Contrastingly, superoxide anion (SOA) production (oxidative capacity) increased in oysters exposed to both induced salinity–stress conditions, which suggest susceptibility increase in oysters, particularly during salinity increase. The results show that HP condition is particularly stressful for C. corteziensis. In turn, this condition could increase both their vulnerability to other environmental stressors, such as temperature and/or acidification or susceptibility to opportunistic pathogenic microorganisms that cause the most common oyster diseases.

Coupled dinuclear copper oxygen cores (Cu2 O2 ) featured in type III copper proteins (hemocyanin, tyrosinase, catechol oxidase) are vital for O2 transport and substrate oxidation in many organisms. μ-1,2-cis peroxido dicopper cores (C P) have been proposed as key structures in the early stages of O2 binding in these proteins; their reversible isomerization to other Cu2 O2 cores are directly relevant to enzyme function. Despite the relevance of such species to type III copper proteins and the broader interest in the properties and reactivity of bimetallic C P cores in biological and synthetic systems, the properties and reactivity of C P Cu2 O2 species remain largely unexplored. Herein, we report the reversible interconversion of μ-1,2-trans peroxido (T P) and C P dicopper cores. CaII mediates this process by reversible binding at the Cu2 O2 core, highlighting the unique capability for metal-ion binding events to stabilize novel reactive fragments and control O2 activation in biomimetic systems.

BACKGROUND AND OBJECTIVES Hemocyanin Subunit IIIA is believed to possess antimicrobial properties, but its efficacy against microbial pathogens is still unclarified. Thus, this study aimed to determine antimicrobial activities of hemocyanin subunit IIIA and to identify the best activator of this protein. MATERIALS AND METHODS The hemocyanin was partially purified using spin column affinity, its fraction was applied to Hi-Prep Sephacryl Exclusion 26/60 2-200 HR column, followed by Hi-Prep 26/10 Desalting Column on fast protein liquid chromatography. The purity of hemocyanin was validated by Matrix Assisted Laser Desorption Ionization-Time of Flight/Mass Spectrometry. The antimicrobial activity was performed by Disc Diffusion Test. RESULTS Purified hemocyanin subunit IIIA was identified to have a molecular weight of 72.9 kDa. SDS was found to be the best activator of hemocyanin, as indicated by elevated level of phenoloxidase. As for antimicrobial activity, hemocyanin was minimally inhibited by all bacteria strains tested (Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae), with relatively lower Minimum Inhibitory Concentration (MIC) at 0.005 g mL-1, than recorded MIC for fungal test strains. Two fungal strains (Penicillium sp. and A. niger) show susceptible response to phenoloxidase using MgSO4 as inducer. Whereas, lysate-treated CaCl2 induced susceptibility only to A. niger. CONCLUSION Hemocyanin shows better antimicrobial activity than phenoloxidase because of its broad-spectrum activity against bacterial and fungal strains tested. Hence, the hemocyanin may potentially become a new antimicrobial candidate to be discovered for a future use in treatment of resistant bacteria.

Hemocyanin is primarily a respiratory copper-containing glycoprotein present in the hemolymph of mollusks and arthropods. Recently, hemocyanin has attracted huge research interest due to its multifunctionality and polymorphism. Most previous immune-related studies on shrimp hemocyanin have focused on the C-terminal. Moreover, we previously reported that the C-terminal domain of Litopenaeus vannamei hemocyanin possesses single nucleotide polymorphisms (SNPs), but little is known about the molecular diversity of the N-terminal domain. In the current study, diversity within the N-terminal domain of L. vannamei hemocyanin (LvHMC-N) was explored using bioinformatics and molecular biology techniques as well as immune challenge. Twenty-five LvHMC-N variants were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing, with multiple sequence alignment showing that the 25 variants shared 87%–99 % sequence homology with LvHMC (AJ250830.1). In different shrimp individuals and different shrimp tissues (i.e., hemocytes, stomach, muscle and hepatopancreas), the LvHMC-N variants were expressed differently. Pathogen challenge could modulate the molecular diversity of LvHMC-N, as three LvHMC-Nr variants (LvHMC-Nr1, LvHMC-Nr2 and LvHMC-Nr3) were identified by sequencing following Vibrio parahaemolyticus challenge. Most importantly, recombinant proteins of these three variants (rLvHMC-Nr1, rLvHMC-Nr2 and rLvHMC- Nr3) had relatively high in vitro agglutinative activities against V. parahaemolyticus, Vibrio alginolyticus and Streptoccocus iniae. Our present data indicates that the N-terminus of L. vannamei hemocyanin also possess molecular diversity, which seems to be associated with immune resistance to pathogenic infections.

Earwigs (Dermaptera) use different strategies to increase their reproductive success. Most species lay eggs; however, viviparity of the matrotrophic type has been reported in two groups: Hemimeridae and Arixeniidae. In Arixeniidae, offspring develop in two separate places: inside an ovary (the intraovarian phase) and within a uterus (the intrauterine phase). Both morphological and physiological aspects of viviparity in Arixeniidae have begun to be unraveled only recently. Here, we characterize how the first instar larvae of Arixenia esau, developing inside the mother’s reproductive system, manage respiration and gas exchange. Using modern light and electron microscopy techniques as well as immunological approach, we provide a detailed account of the maternal and larval tissue interactions during the intrauterine development. We demonstrate that respiration in the Arixenia first instar larvae relies on the extensive tracheal system of the mother as well as a respiratory pigment (hemocyanin) present within the body cavity of the larvae. Our results indicate that the larval fat body tissue is the likely place of the hemocyanin synthesis. Our study shows that characteristic cone-shaped lobes of the outgrowths located on the larval abdomen are a part of a placenta-like organ and mediate the gas exchange between the maternal and larval organisms. Based on the obtained results, we propose that Arixenia esau evolved a unique biphasic system supporting respiration of the first instar larvae during their development inside the mother’s reproductive tract.

This study investigated the effects of temperature and cooling mode on hemolymph indicators, including total protein, hemocyanin, urea nitrogen, ammonia, uric acid, carbon dioxide (CO2) and pH levels, of swimming crabs, Portunus trituberculatus, under air-exposure stress at different times. The swimming crabs were exposed to air at temperatures ranging from 0 °C to 25 °C to determine the optimal temperature. Results showed that the crabs exhibited the highest survival rate at 10 °C. After exposure to air-exposure stress at 10 °C, the gill lamella moisture content and surface humidity of the swimming crabs decreased as the air-exposure time increased. With prolonged air-exposure time, the hemolymph urea, ammonia and CO2 of the swimming crabs under air-exposure stress increased, and the hemolymph pH value of the swimming crabs slightly decreased. These results indicated that the swimming crabs had the highest survival rate under air exposure at 10 °C.

Mollusk hemocyanins have been used for decades in immunological and clinical applications as natural, nontoxic, nonpathogenic, and nonspecific immunostimulants for the treatment of superficial bladder cancer, as carriers/adjuvants of tumor-associated antigens in cancer vaccine development and as adjuvants to dendritic cell-based immunotherapy, because these glycoproteins induce a bias towards Th1 immunity. Here, we analyzed the preclinical therapeutic potential of the traditional keyhole limpet hemocyanin (KLH) and two new hemocyanins fromConcholepas concholepas(CCH) andFissurella latimarginata(FLH) in mouse models of oral squamous cell carcinoma. Due to the aggressiveness and deadly malignant potential of this cancer, the hemocyanins were applied in combination with adjuvants, such as alum, AddaVax, and QS-21, which have been shown to be safe and effective in human vaccines, to potentiate their antitumor activity. The immunogenic performance of the hemocyanins in combination with the adjuvants was compared, and the best formulation was evaluated for its antitumor effects in two murine models of oral cancer: MOC7 cells implanted in the flank (heterotopic) and bioluminescent AT-84 E7 Luc cells implanted in the floor of the mouth (orthotopic). The results demonstrated that the hemocyanins in combination with QS-21 showed the greatest immunogenicity, as reflected by a robust, specific humoral response predominantly characterized by IgG2a antibodies and a sustained cellular response manifesting as a delayed hypersensitivity reaction. The KLH- and FLH-QS-21 formulations showed reduced tumor development and greater overall survival. Hemocyanins, as opposed to QS-21, had no cytotoxic effect on either oral cancer cell line culturedin vitro, supporting the idea that the antitumor effects of hemocyanins are associated with their modulation of the immune response. Therefore, hemocyanin utilization would allow a lower QS-21 dosage to achieve therapeutic results. Overall, our study opens a new door to further investigation of the use of hemocyanins plus adjuvants for the development of immunotherapies against oral carcinoma.

Neocaridina denticulate sinensis is a promising crustacean model species due to its merits in raising and breeding. However, its molecular responses to copper remains largely unknown. In the present research, RNA-seq was used to mine the alteration in transcriptome of N. denticulate sinensis hepatopancreas under copper exposure. A total of 16,423 DEGs was identified between control and Cu2+ treatment groups. GO enrichment analysis of all DEGs suggested down-regulated genes exceeded up-regulated genes in all the significantly enriched terms, except for RNA polymerase III complex (GO:0005666). KEGG analysis showed Cu exposure only induced two significantly enriched pathways, including Phagosome (ko04145) and Pathogenic Escherichia coli infection (ko05130). Besides, pattern recognition receptors as Toll, lectin B, CTL1 and SRB, AMPs as crustin type I, lysozyme, and NOS were down-regulated after Cu2+ exposure, while hemocyanin, MT, HSP70 and HSP90 were significantly up-regulated, implying these molecules may play vital role in Cu2+ detoxification of N. denticulate sinensis. Our results here provide research direction of heavy metal detoxification of N. denticulate sinensis, simultaneously enriched its genomic information.

Shellfish allergies constitute an important cause of food-induced anaphylactic reactions, which pose challenges to food safety and human health worldwide. In the present study, the specific IgE (sIgE) binding characteristics of different shrimp proteins of black tiger shrimp (Penaeus monodon) to the sera of eight shrimp-allergic patients from China were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and nanoliquid chromatography time-of-flight mass spectrometry. According to the PLGS scores (>2000) and the sequence coverage (>40%), eight proteins with sIgE binding activity were identified, including myosin heavy chain type 1 (K4Q4N8), hemocyanin (G1AP69 and Q95V28), phosphopyruvate hydratase (O96656), arginine kinase (C7E3T4), tropomyosin (A1KYZ2), sarcoplasmic calcium binding protein (H7CHW2) and glyceraldehyde-3-phosphate dehydrogenase (A0A097BQP2). Among these eight proteins, phosphopyruvate hydratase was a prevalent IgE-binding protein among these Chinese patients with binding observed in 100% of sera. Moreover, 13 peptides were predicted as epitopes of phosphopyruvate hydratase. These new details help us to understand the crustacean IgE-binding proteins especially Penaeus monodon IgE-binding proteins, that would cause allergic reaction to Chinese patients. And our findings may provide essential information to improve allergy prevention and clinical treatment to shrimp allergy in China. PRACTICAL APPLICATION: This research may have diagnostic and therapeutic value for shrimp allergies in China.

Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th1-biased cell-mediated immunity, which has beneficial effects. They are multiligand glycosylated molecules with abundant and complex mannose-rich structures. It remains unclear whether these structures influence hemocyanin-induced immunostimulatory processes in human antigen presenting cells. We have previously shown that hemocyanin glycans from Concholepas concholepas (CCH), Fissurella latimarginata (FLH) and Megathura crenulata (KLH), participate in their immune recognition and immunogenicity in mice, interacting with murine C-type lectin receptors (CLRs). Here, we studied theinteractions of these hemocyanins with two major mannose-binding CLRs on monocyte-derived human dendritic cells: MR (mannose receptor) and DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin). Diverse analyses showed that hemocyanins are internalized by a mannose-sensitive mechanism. This process was calcium dependent. Moreover, hemocyanins colocalized with MR and DC-SIGN, and were partly internalized through clathrin-mediated endocytosis. The hemocyanin-mediated proinflammatory cytokine response was impaired when using deglycosylated FLH and KLH compared to CCH. We further showed that hemocyanins bind to human MR and DC-SIGN in a carbohydrate-dependent manner with affinity constants in the physiological concentration range. Overall, we showed that these three clinically valuable hemocyanins interact with human mannose-sensitive CLRs, initiating an immune response and promoting a Th1 cell-driving potential. This article is protected by copyright. All rights reserved.

We here report the de novo transcriptome assembly and functional annotation of Eusirus cf. giganteus clade g3, providing the first database of expressed sequences from this giant Antarctic amphipod. RNA-sequencing, carried out on the whole-body of a single juvenile individual likely undergoing molting, revealed the dominant expression of hemocyanins. The mRNAs encoding these oxygen-binding proteins cumulatively accounted for about 40% of the total transcriptional effort, highlighting the key biological importance of high hemocyanin production in this Antarctic amphipod species. We speculate that this observation may mirror a strategy previously described in Antarctic cephalopods, which compensate the decreased ability to release oxygen to peripheral tissues at sub-zero temperatures by massively increasing total blood hemocyanin content compared with temperate species. These preliminary results will undoubtedly require confirmation through proteomic and biochemical analyses aimed at characterizing the oxygen-binding properties of E. cf. giganteus clade g3 hemocyanins, and at investigating whether other Antarctic arthropod species exploit similar adaptations to cope with the challenges posed by the extreme conditions of the polar environment.

Among the centipede of the chilopode Myriapoda class, the Cryptops genus is one of the most associated with accidents in humans in the metropolitan region of the state of Sao Paulo. To date, there is no study in the literature about Cryptops iheringi toxins. Thus, in this work a transcriptomic analysis of the C. iheringi venom gland was performed to obtain a profile of the toxins of this species. In addition, the crude venom was subjected to mass spectrometry analysis to establish an association between unknown sequences. These approaches for the construction of a general profile of the venom gland expression of this species led to the he identification of a Hemocyanin (Hc) subunit. Hemocyanins are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hc in the chilopode Myriapoda C. iheringi. Such respiratory proteins have long been considered unnecessary in Myriapoda, due to its tracheal systems. These respiratory proteins are potent immunogens, which induce the synthesis of large amounts of specific antibodies. Studies pointed out its interaction with polymorphonuclear monocytes and lymphocytes and in vitro tests have shown a potential anticancer activit, with in vitro significant inhibition of the growth of cancerous strains of the breast, pancreas and prostate. Currently scientific data is mostly limited to the study of native Hc of M. crenulata molluscs, therefore, the biotechnological potential of Hcs isolated from centipedes is still unexplored. Herein, the Hc sequence that was present in both proteome and transcriptome analysis have signal peptide and 76 kDa range. The Hc subunit sequence was synthesized with codon optimization for bacteria expression and the protein expressed as inclusion bodies. Refolding attempts provided soluble forms of Hc. At the moment efforts to access your biological activities are being carried out.

Hemocyanins are copper-binding proteins that play a crucial role in the physiological processes in crustaceans. In this study, the cDNA encoding hemocyanin subunit 5 from the Black sea crab Eriphia verrucosa (EvHc5) was cloned using EST analysis, RT-PCR and rapid amplification of the cDNA ends (RACE) approach. The full-length cDNA of EvHc5 was 2254 bp, consisting of a 5&prime, and 3&prime, untranslated regions and an open reading frame of 2022 bp, encoding a protein consisting of 674 amino acid residues. The protein has an N-terminal signal peptide of 14 amino acids as is expected for proteins synthesized in hepatopancreas tubule cells and secreted into the hemolymph. The 3D model showed the presence of three functional domains and six conserved histidine residues that participate in the formation of the copper active site in Domain 2. The EvHc5 is O-glycosylated and the glycan is exposed on the surface of the subunit similar to Panulirus interruptus. The phylogenetic analysis has shown its close grouping with &gamma, type of hemocyanins of other crustacean species belonging to order Decapoda, infraorder Brachyura.

We here report the de novo transcriptome assembly and functional annotation of Eusirus cf. giganteus clade g3, providing the first database of expressed sequences from this giant Antarctic amphipod. RNA-sequencing, carried out on the whole body of a single juvenile individual likely undergoing molting, revealed the dominant expression of hemocyanins. The mRNAs encoding these oxygen-binding proteins cumulatively accounted for about 40% of the total transcriptional effort, highlighting the key biological importance of high hemocyanin production in this Antarctic amphipod species. We speculate that this observation may mirror a strategy previously described in Antarctic cephalopods, which compensates for the decreased ability to release oxygen to peripheral tissues at sub-zero temperatures by massively increasing total blood hemocyanin content compared with temperate species. These preliminary results will undoubtedly require confirmation through proteomic and biochemical analyses aimed at characterizing the oxygen-binding properties of E. cf. giganteus clade g3 hemocyanins and at investigating whether other Antarctic arthropod species exploit similar adaptations to cope with the challenges posed by the extremeconditions of the polar environment.

Introduction: Asthma and allergy occur frequently among seafood processing workers, with the highest prevalence seen in the crustacean processing industry. In this study we established for the first time the prevalence of allergic sensitization in the Norwegian king- and edible crab processing industry and characterized the IgE-reactive proteins.Materials and Methods: Two populations of crab processing workers participated; 119 king crab and 65 edible crab workers. The investigation included information on work tasks and health through a detailed questionnaire. Allergic sensitization was investigated by crab-specific IgE quantification and skin prick tests (SPT) to four in-house prepared crab extracts; raw meat, cooked meat, raw intestines and raw shell. Allergen-specific IgE binding patterns were analyzed by IgE immunoblotting to the four allergen extracts using worker serum samples. Total proteins in crab SPT extracts and immunoblot-based IgE binding proteins were identified by mass spectrometric analysis.Results: Positive SPTs were established in 17.5% of king- and 18.1% of edible crab workers, while elevated IgE to crab were demonstrated in 8.9% of king- and 12.2% of edible crab processing workers. There was no significant difference between the king and edible crab workers with respect to self-reported respiratory symptoms, elevated specific IgE to crab or SPT results. Individual workers exhibited differential IgE binding patterns to different crab extracts, with most frequent binding to tropomyosin and arginine kinase and two novel IgE binding proteins, hemocyanin and enolase, identified as king- and edible crab allergens.Conclusions: Occupational exposure to king- and edible crabs may frequently cause IgE mediated allergic sensitization. Future investigations addressing the diagnostic value of crab allergens including tropomyosin and arginine kinase and the less well-known IgE-binding proteins hemocyanin and enolase in a component-resolved diagnostic approach to crab allergy should be encouraged.

Tachypleus amebocyte lysate (TAL) is crucial in medical testing, but its industry in China has been restricted due to the decline of horseshoe crab population in recent years. Exploring methods of enhancing immunity and rapid hemocytes proliferation is urgent for the industrial horseshoe crab culture. In this study, β-glucan (G), peptidoglycan (P), and squalene (S) were injected to horseshoe crabs at two concentrations (5 and 10 mg/kg), in order to compare their effects on total hemocyte count (THC), reactive oxygen species (ROS), and non-specific immune enzyme activities. Results showed that the THC, superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) were significantly increased by three immunostimulants at different points of time; ROS was significantly increased except at two squalene groups; lysozyme (LZM) and alkaline phosphatase (AKP) activity were increased except at low dose (5 mg/kg) squalene group; malondialdehyde (MDA) activity was decreased in all treatments; and hemocyanin concentration (HC) changed little during the experiment. At the 48th hour, THC, ROS, SOD, CAT, T-AOC, LZM, and AKP activities were significantly higher in the two peptidoglycan groups than those in the control group; the low dose β-glucan and squalene groups showed significantly higher SOD and CAT, but their THC and AKP were not significantly different from those of the control group. In general, all three immunostimulants stimulated the hemolymph parameters of horseshoe crabs, notably, peptidoglycan could significantly increase the THC and enzyme activities, suggesting that peptidoglycan can be developed as an efficient immunostimulant for horseshoe crabs.

This study evaluated two approaches to the aquaculture of Limulus polyphemus with the ultimate goal of harvesting Limulus amebocyte lysate (LAL) at an industrial scale. To monitor Horseshoe crabs (HSCs), a combination of physical, biochemical and cellular components were examined for HSC cohorts in an indoor recirculating aquaculture system (RAS) and an outdoor on-bottom pen culture system (PCS) over a 6-month period. The metrics included body weight, hemocyanin (Hc) concentration, amebocyte density, and LAL reactivity. In addition, a simulated biomedical bleeding event (extracting 30% of the total hemolymph volume) was performed to assess the impact on physiochemical properties of the hemolymph and amebocytes. Overall, the HSCs fared better in the RAS compared to the PCS, with higher rebound kinetics with respect to Hc, amebocyte density, LAL reactivity, and with 100% survival in the RAS cohort. Further, hemolymph reinfusion (after amebocyte removal) was shown to improve HSC recovery time. In summary, outcomes of this research show that a RAS, coupled with adequate nutrition and monitoring can provide HSCs with a suitable environment for sustainable hemolymph extraction and year-round LAL production.

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

Enzymes adapted to cold temperatures are commonly characterized for having higher Michaelis-Menten constants (KM) values and lower optimum and denaturation temperature, when compared to other meso or thermophilic enzymes. Phenoloxidase (PO) enzymes are ubiquitous in nature, however, they have not been reported in spiders. It is the oxygen carrier protein hemocyanin (Hc), found at high concentrations in their hemolymph, which displays an inducible PO activity. Hence, we hypothesize that Hc-derived PO activity could show features of cold adaptation in alpine species. We analyzed the Hc from two species of Theraphosidae from different thermal environments: Euathlus condorito (2400 m a.s.l.) and Grammostola rosea (500 m a.s.l.). Hc was purified from the hemolymph of both spiders and was characterized by identifying subunit composition and measuring the sodium dodecyl sulfate (SDS)-induced PO activity. The high-altitude spider Hc showed higher PO activity under all conditions and higher apparent Michaelis-Menten constant. Moreover, the optimum temperature for PO activity was lower for E. condorito Hc. These findings suggest a potential adaptation at the level of Hc-derived PO activity in Euathlus condorito, giving insights on possible mechanisms used by this mygalomorph spider to occupy extremes and variable thermal environments.

Lectins are proteins that bind to the carbohydrate moieties on surface of bacteria, erythrocytes and other cells of invertebrates causing agglutination and mediate in recognition of foreign substances. In the present study, we isolated and characterized a lectin molecule present in the hemolymph of Macrobrachium rosenbergii, an important cultured freshwater prawn. Lectin in serum samples of adult prawns was assessed through hemagglutination (HA) test using rabbit RBC that showed a titre ranging from 16 to 64. This serum hemagglutinin was confirmed as a C-type lectin based on its dependency on calcium ions towards binding to rabbit RBCs. The hemagglutinin was also found to be stable at the pH range of 5.0–10.0 and temperature range of 10–40 °C. Of various sugars and glycoproteins tested in hemagglutination inhibition assay, the serum lectin was found specific only to N-acetylneuraminic acid and fetuin with respective minimum inhibitory concentrations at 50 mM and 0.31 mg/ml. Further, the lectin was purified by affinity chromatography on rabbit erythrocyte stroma, which showed hemagglutination with rabbit RBC. In electrophoretic analyses, the purified lectin showed one band with molecular weight of ~ 427 kDa in native gradient PAGE, and its two constituent polypeptide chains of ~ 81 and ~ 73 kDa in SDS-PAGE. These polypeptides were analysed in MALDI-TOF/TOF mass spectrometry and identified as hemocyanins. It was hence, concluded that hemocyanin in M. rosenbergii possesses lectin-like activity.

Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of arthropods and mollusks that attract scientific interest with their diverse biological activities and potential applications in pharmacy and medicine. The aim of the present study was to assess the in vitro antitumor activity of hemocyanins isolated from marine snail Rapana venosa (RvH) and garden snails Helix lucorum (HlH) and Helix aspersa (HaH), as well the mucus of H. aspersa snails, in the HT-29 human colorectal carcinoma cell line. The effects of the hemocyanins on the cell viability and proliferation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the alterations in the tumor cell morphology were examined by fluorescent and transmission electron microscopy. The results of the MTT assay showed that the mucus and &alpha, subunit of hemocyanin from the snail H. aspersa had the most significant antiproliferative activity of the tested samples. Cytomorphological analysis revealed that the observed antitumor effects were associated with induction of apoptosis in the tumor cells. The presented data indicate that hemocyanins and mucus from H. aspersa have an antineoplastic activity and potential for development of novel therapeutics for treatment of colorectal carcinoma.

Crab allergy is reported as a serious form of food allergy in many countries. This study was aimed to identify the major allergens of the local mud crab, Scylla tranquebarica (S. tranquebarica), and subsequently, determine the effect of vinegar treatments on the crab allergens. Crab muscles were treated with synthetic and natural vinegar. Crab proteins were then extracted from the untreated and vinegar-treated crabs. All extracts were then fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by immunoblotting; using sera from crab-allergic patients. The crab proteins were then further fractionated by two-dimensional electrophoresis (2-DE) and analyzed by mass spectrometry (MS). The untreated crab had 38 protein bands, while that was only a few bands between 18 to 73 kDa for the vinegar-treated crabs. Immunoblotting of untreated crab revealed 20 IgE-binding bands, whereas the vinegar-treated crabs could only retain a few IgE-binding bands. Five major allergens were identified with molecular weightsof38, 42, 49, 63, and 73 kDa in the untreated crab. In contrast, the vinegar-treated crabs had only a few major allergens with molecular weights of 38, 42, and 73 kDa. MS identified the 43 and 49 kDa as arginine kinase, while the 38, 63, and 73 kDa were identified as tropomyosin, actin, and hemocyanin, respectively. In conclusion, we found three common major allergens for S. tranquebarica including tropomyosin, arginine kinase, and actin, and one novel allergen known as hemocyanin. All the major allergens could retain minimal allergenic capability in vinegar-treated crabs, suggesting that vinegar treatments might be useful to reduce crab allergenicity. These data would assist the clinicians in the management of crab-allergic patients worldwide.

Two water-soluble red color-related proteins with the molecular masses of 24 and 73 kDa were purified from the shell of Procambarus clarkii. Initial color changes of these two proteins were detected at 30 °C and the large amount of red precipitate were obtained at 80 °C. PAGE analysis showed that the 24 kDa protein was the monomer, while the 73 kDa protein was the trimer. Identification revealed that these two proteins belonged to the hemocyanin subunit 2 family. With respect to the amino acid sequence similarity, the red color-related proteins shared the highest sequence identity with the hemocyanin derived from giant freshwater prawn (Macrobrachium rosenbergii). The phylogenetic tree analysis also clearly supported this finding. The shell-derived red color-related proteins show potential use as the edible thermal-sensitive indicator in food processing field.

Haemorrhagic smolt syndrome (HSS) is a disorder of unknown aetiology causing losses in the fresh water phase of Atlantic salmon farming. Normally, the mortality is limited and symptoms disappear upon seawater exposure. In this case study, classical HSS pathology with internal organ haemorrhages and nephrocalcinosis was diagnosed, and the losses were substantial. Microarray analyses of head kidney revealed association between HSS and enhanced expression of stress genes and proteins reducing bioavailability of iron, heme, and retinol. In parallel, suppression of multiple metabolic pathways was observed. Up-regulation of genes encoding acute phase proteins, complement, and lectins indicated mild inflammation but without characteristic features of viral or bacterial infections. Microarray analyses highlighted several members of tumor necrosis factor receptor superfamily that may control development of B-cell immunity. Examination of IgM at the mRNA and protein levels showed the impact of HSS on vaccine responses. In fish without HSS symptoms (non-HSS), titres of vaccine specific antibodies to A-layer of Aeromonas salmonicida subsp. salmonicida and Moritella viscosa and antibodies binding to DNP-keyhole limpet hemocyanin (DNP-KLH), which are presumably polyreactive, were respectively four- and 14-fold higher than in HSS-diseased fish. Parallel sequencing of variable regions of immunoglobulin Mrevealed a larger size of most abundant clonotypes shared by multiple individuals in the non-HSS group. The results of the current case study indicated that, in addition to direct damage, HSS suppresses humoral immune responses including the production of specific and polyreactive antibodies. Consequences of Haemorrhagic Smolt Syndrome (HSS) for the Immune Status of Atlantic salmon (Salmo salar L.) (Case Study)

Structural knowledge of gastropod hemocyanins is scarce. To better understand their evolution and diversity we studied the hemocyanin of a caenogastropod, Pomacea canaliculata (PcH). Through a proteomic and genomic approach, we identified 4 PcH subunit isoforms, in contrast with other gastropods that usually have 2 or 3. Each isoform has the typical Keyhole limpet-type hemocyanin architecture, comprising a string of eight globular functional units (FUs). Correspondingly, genes are organized in eight FUs coding regions. All FUs in the 4 genes are encoded by more than one exon, a feature not found in non- caenogastropods. Transmission electron microscopy images of PcH showed a cylindrical structure organized in di, tri and tetra-decamers with an internal collar structure, being the di and tridecameric cylinders the most abundant ones. PcH is N-glycosylated with high mannose and hybrid-type structures, and complex-type N-linked glycans, with absence of sialic acid. Terminal β-N-GlcNAc residues and nonreducing terminal α-GalNAc are also present. The molecule lacks O-linked glycosylation but presents the T-antigen (Gal-β1,3-GalNAc). Using an anti-PcH polyclonal antibody, no cross-immunoreactivity was observed against other gastropod hemocyanins, highlighting the presence of clade-specific structural differences among gastropod hemocyanins. This is, to the best of our knowledge, the first gene structure study of a Caenogastropoda hemocyanin., Instituto de Investigaciones Bioquímicas de La Plata, Facultad de Ciencias Naturales y Museo

Hepatopancreatic necrosis syndrome (HPNS) severely threatens the development of Eriocheir sinensis farming. To date, no consensus has been reached about the etiology and pathogenesis of HPNS. In this study, we investigated the effects of HPNS on the copper-containing respiratory protein (hemocyanin), the ecdysone inducible transcription factor (E75), and the zinc finger-containing transcription factor (Kruppel). We also determined the relationship between hemocyanin and the two transcription factors. Seven hemocyanin variants, four E75 isoforms, and a Kruppel-like factor were characterized in E. sinensis. The 3′ and 5′ ends of hemocyanin possessed molecular diversity, which was apparently associated with its multifunctionality. Four E75 isoforms were generated by the alternative splicing of the 5′ exons, and they originated from a single copy gene. Hemocyanins, EsE75, and EsKruppel were mainly expressed in the hepatopancreas, which is the most primary organ for the clinical characterization of HPNS. The expression of the hemocyanins and two transcriptional factors (EsE75 and EsKruppel) were significantly decreased and increased in the crabs with HPNS, respectively. Moreover, silencing of EsE75 and EsKruppel resulted in an evident increase in the synthesis of hemocyanins, indicating that EsE75 and EsKruppel acted as the transcription repressor of the hemocyanin gene. However, the detailed mechanisms by which E75 and Kruppel regulated the transcription of hemocyanin in E. sinensis need to be more thoroughly studied in the future. This study provides new information on hemocyanin, E75, and Kruppel that were influenced by HPNS in E. sinensis. The results will help to enhance the understanding of the causes and pathogenesis of HPNS.

A novel red color-related pigment-binding protein named LvPBP75 isolated from the shell of Litopenaeus vannamei has recently been identified as hemocyanin. However, information on the functional and structural properties of LvPBP75 is insufficient. This study aimed to elucidate the thermal properties and pigment-binding ability of LvPBP75.; Results: LvPBP75 showed significant red color change after heat treatment with high concentrations of NaCl (>0.1 mol L-1 ), acidic ( 9) pH values and alcohols. LvPBP75 mRNA expression analysis revealed that expression level was highest in hepatopancreas and weakest in muscle. Reconstruction and structural analysis revealed that astaxanthin could bind to hemocyanin derived from the shell of L. vannamei but not to hemocyanins derived from the hepatopancreas or hemolymph of other invertebrates. Three-dimensional models of hemocyanin monomer displayed significant structural differences between native LvPBP75 and hemocyanin derived from shrimp hepatopancreas.; Conclusion: The results suggest a novel function of hemocyanin as binding with pigment and its involvement in L. vannamei shell color change. The pigment-binding ability of hemocyanins has species and tissue specificity, and their unique structural features play an important role in binding ability. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry.

Phenoloxidases (POs) play a crucial role in melanization of crustaceans. There are at least two types of POs characterized in crustaceans: the conventional type (POα here) that is expressed in hemocytes and POβ, a secreted protein synthesized in the hepatopancreas. We investigated the source of PO activity in the hemolymph of a lobster and determined the kinetic parameters of mono- and di-PO activities. In the lobster hemolymph, POα, which formed a hexamer similar to both POβ and hemocyanin, contributed to PO activity, whereas the amount of POβ was low. Kinetic analyses using purified prophenoloxidase of crustaceans showed that lobster POα has a higher rate constant, while shrimp POβ has higher specificity in both mono- and di-PO reactions, when tyramine and dopamine were employed as substrates. There should be at least two types of PO molecules in crustacean hemolymph, but the dominant PO molecule type varies among species.

Type-3 copper proteins (T3CPs) are complex proteins which share similar active sites. Two copper atoms (CuA and CuB) bind dioxygen as a peroxide in a side on coordination. This protein family comprises the enzymes tyrosinase and catechol oxidase as well as the oxygen transporter hemocyanin. T3CPs occur in almost all organisms and exhibit a number of essential functions. In particular, they are involved in all kinds of enzymatic browning reactions and immune defense. The chemical basis of the two catalytic processes, i.e., the o-hydroxylation of monophenols and the two-electron oxidation to o-quinones, is still discussed. Investigations on natural enzymes with known crystal structures, along with site directed mutagenesis experiments, provide deeper insight into mechanistic details of these reactions. In particular, they shed light on the deprotonation of monophenolic substrates which appears to be required for the enzymatic conversion to o-quinones. Moreover, they allow drawing definite conclusions regarding the role of particular amino acids (glutamate and asparagine/aspartate) in these processes, which in turn provides a structure-based argument for the functional difference between tyrosinases and catechol oxidases.

Hemocyanin is a multifunctional respiratory glycoprotein, which has also been implicated in other biological functions in shrimp. Moreover, recent studies have revealed that hemocyanin is also involved in a broad range of immune-related activities in shrimp. However, in spite of the considerable interest in unraveling the reasons behind the multiple immune-related functions of hemocyanin, little is known about its transcriptional regulation. Here, DNA pull-down and Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) analyses were used to isolate and identify the putative transcription factor(s) that are involved in the transcriptional regulation of the small subunit hemocyanin gene of Penaeus vannamei (PvHMCs). Kruppel-like factor (designated PvKruppel), a zinc finger transcription factor homolog in P. vannamei, was identified among the putative transcription factors, while bioinformatics analysis revealed the presence of Kruppel-like factor binding site (KLF motif) on the core promoter region of PvHMCs. Mutational analysis and electrophoretic mobility shift assay (EMSA) confirmed that PvKruppel could bind to the KLF motif on the core promoter region of PvHMCs. Moreover, in response to lipopolysaccharide (LPS), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenge, transcript levels of PvKruppel and PvHMCs were negatively correlated. Furthermore, overexpression of PvKruppel significantly reduced the promoter activity of PvHMCs, while PvKruppel knockdown by RNA interference or lipopolysaccharides (LPS) stimulation resulted in a significant increase in the transcript level of PvHMCs. Taken together, our present study provides mechanistic insights into the transcriptional regulation of PvHMCs by PvKruppel during shrimp immune response to pathogens.

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific strain of Vibrio parahaemolyticus (VPAHPND), results in great loss of global shrimp production. Despite this, studies on shrimp defense mechanisms protecting against AHPND are few. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes from white shrimp Litopenaeus vannamei hepatopancreas upon VPAHPND infection at the early stages: 3 and 6 h post challenge and in the late stage at 48 h post challenge. Hemocyanin (HMC) is the most abundant gene identified as the up-regulated gene in the SSH library. Various hemocyanin subunits such as hemocyanin (HMC), hemocyanin subunit L1 (HMCL1), L2 (HMCL2), L3 (HMCL3), and L4 (HMCL4) were analyzed for their expression levels upon VPAHPND infection and in response to challenge with partially purified toxin of VPAHPND by qRT-PCR. Only HMC was highly up-regulated at 3 and 6 h post challenge in response to VPAHPND challenge. Two HMC subunits, HMCL3 and HMCL4, were up-regulated in the early phase of VPAHPND toxin injection. Furthermore, all subunits were down-regulated in the late phase of VPAHPND and toxin challenges. The native hemocyanin protein purified from shrimp hemolymph, identified as mixture of HMC and HMCL1, exhibited agglutination activity on VPAHPND. Injecting the purified native hemocyanin along with VPAHPND into shrimp decreased the number of bacteria in the hemolymph as compared to the VPAHPND challenged control. Moreover, pre-incubation of the purified native hemocyanin and VPAHPND toxin prior to injection into shrimp resulted in the decrease of cumulative mortality of shrimp when compared to the control. In addition, protein-protein interaction analysis carried out by ELISA technique indicated that hemocyanin exhibited VPAHPND toxin-neutralizing activity through direct interaction with PirA subunit with a dissociation constant of 6.83 × 10−6 M. Our results indicated that upon VPAHPND infection the expression of hemocyanin was induced and hemocyanin functions might involve agglutination of invading VPAHPND and also neutralization of VPAHPND secreted toxin via direct interacting with the PirA protein.