Hemocyanin Subunits Research Articles

A commercial seaweed extract increases growth performance, immune responses, and related gene expressions in whiteleg shrimp (Litopenaeus vannamei)

Seaweed liquid extracts are an attractive source of phytochemicals with high potential applicability in the aquafeed-additive industry. A commercial seaweed liquid extract (True Algae Max, TAM®), which has a marine seaweed odor, also dislplays significant levels of polysaccharides, phytochemicals, phenolic, and flavonoid compounds showing antioxidant activities and DPPH inhibition. This study investigates the impact of diets supplemented with TAM® as a functional additive on the growth, nutrient utilization, immune responses, and immune-related gene expressions of whiteleg shrimp (Litopenaeus vannamei). A total of 750 postlarvae (PL, with an average initial weight of 0.053±0.001 g) were divided into five experimental groups, comprising three replicates per dietary treatment. For an eight-week experimental period, all groups were fed identical diets except for the variation in TAM® inclusion levels. The basal diet (control diet) had no inclusion level of TAM® (TAM0%). Groups 2–5 each contain TAM® inclusion at levels of 1% (10 mL gk−1 diet TAM1%), 2% (20 mL gk−1 diet TAM2%), 3% (30 mL gk−1 diet TAM3%), and 4% (40 mL gk−1 diet TAM4%), respectively. The results concluded that TAM® has great potential as a feed additive for whiteleg shrimp, compared to the control group. The group TAM2% significantly achieved final weight (4.337 g), weight gain (4.287 g) specific growth rate (3.423% / day), feed conversion ratio (1.970), feed efficiency ratio (0.507), and protein efficiency ratio (1.407), compared to the control group (3.900 g, 3.850 g, 3.347%/day, 2.183, 0.458, and 1.270, respectively). Whole-body composition of protein and lipid contents were significantly improved by all TAM group including TAM2% (51.18% and 4.49%, respectively), compared to the control group (49.38% and 4.15%, respectively). As well as, shrimp in group TAM2% achieved the highest values of lysozyme (3.92 µg mL−1), superoxide dismutase (SOD, 11.92 IU mL−1), catalase (CAT, 13.04 IU g−1), lipase (31.24 IU L−1), and amylase (31.24 IU L−1), compared to the control group (3.19 µg mL−1, 8.80 IU mL−1, 10.84 IU g−1, 17.35 IU L−1, 21.65 IU L−1, respectively). For gene expressions experiment, four immune-related were performed in this study; Peroxiredoxin (Prx), Prophenoloxidase (PPO1), P53-like protein isoform delta (p53), and Hemocyanin subunit L5 (L5H). The results showed that shrimp in group TAM2% achieved the highest significant P53 gene transcription compared to the control group or the other TAM groups. Compared to other groups, the TAM1% group considerably increased PPO1 gene transcription, while TAM3% displayed the greatest Prx gene transcription. On the other hand, compared to control group, all TAM groups significantly improved L5H gene transcription. In conclusion, the current study revealed that TAM® diet supplementation (20 mL of TAM® per kg diet) represents a promising, eco-friendly, and sustainable feed additive in the shrimp aquadiet industry.

Anti-tumour Activity of Bioactive Compounds Isolated from the Hemolymph and Mucus of the Garden Snail Helix aspersa against a Panel of Human Cancer Cell Lines

Cancer remains a significant global health concern, necessitating the search for new effective and safe anti-cancer agents. Natural products derived from plants and animals, including mollusks like snails, have gained attention as potential sources of novel anti-cancer compounds. Haemocyanins (Hcs), large copper-containing glycoproteins involved in oxygen transport in mollusks, have shown promise as anti-cancer agents. This study focuses on evaluating the in vitro anti-tumour efficacy of bioactive substances obtained from the hemolymph of the garden snail Helix aspersa on various human cancer cell lines representing different types of cancer. The results demonstrate that certain hemocyanin subunits from H. aspersa exhibit cytotoxic activity comparable to cisplatin, a widely used chemotherapy drug. Additional assays confirm the cytotoxic effects of the tested substances on cancer cells. The study underscores the potential of natural compounds from H. aspersa as alternative therapeutic agents for cancer treatment, while highlighting the need for further investigation. The identification of specific proteins responsible for the observed anti-proliferative effects in the mucus of H. aspersa provides insights for the development of novel cancer therapies.

Molecular isolation and expression analysis of hemocyanin isoform 2 of Macrobrachium rosenbergii.

Hemocyanin is a copper-bearing protein in the hemolymph of many arthropods and mollusks and functions as an oxygen transport and important nonspecific immune protein. In this study, complementary DNA of hemocyanin isoform 2 of the prawn Macrobrachium rosenbergii (MrHc2) was isolated by rapid amplification of cDNA ends and mRNA expression was characterized to elucidate molecular basis of its function. With a molecular mass of 77.3 kDa, MrHc2 contained three domains: hemocyanin-all-alpha, hemocyanin-copper-containing, and hemocyanin-immunoglobulin-like domains. Molecular phylogenetic analysis revealed that MrHc2 belongs to the γ-type subunit and is closely related to hemocyanin subunit 1 of the palaemonid shrimp Macrobrachium nipponense. In addition, MrHc2 resided in a different clade relative to hemocyanin (MrHc) of M. rosenbergii (α-type subunit) and in a different subclade relative to the hemocyanin proteins of penaeid shrimp. The messenger RNA transcript of MrHc2 was highly expressed in the hepatopancreas and weakly expressed in the gills, intestine, stomach, muscle, and hemocytes. Upon challenge with M. rosenbergii nodavirus (MrNV), the expression of MrHc2 was 1.96-, 2.93-, and 1.96-fold on days 3, 4, and 5, respectively, and then gradually declined to basal levels on day 7. This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV.

Proteomic Analysis Associated with the Immune Response in Hemocytes of Portunus trituberculatus Challenged with Vibrio parahaemolyticus

Vibrio parahaemolyticus belongs to an expanding group of aquatic pathogens that are widely distributed in aquatic environments. This species is a lethal pathogen for a number of economically important marine crabs. However, studies exploring host–vibrio interactions between V. parahaemolyticus and crabs are scarce, and therefore, the underlying molecular mechanisms are unclear. Herein, we performed a comprehensive proteomic analysis to investigate the immune response of Portunus trituberculatus hemocytes to V. parahaemolyticus infection. A total of 4433 proteins were identified using isobaric tags for relative and absolute quantitation (iTRAQ), and 526 differentially expressed proteins (DEPs) were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, with six DEPs further subjected to quantitative real-time PCR. Several identified DEPs were found to be mainly involved in the immune defense of the crustacean, such as a hemocyanin subunit, C-type lectin, α-2-macroglobulin, Cu/Zn-superoxide dismutase, and heat shock protein 70, playing a key role in the response to V. parahaemolyticus infection. Moreover, many immune-related KEGG pathways were markedly altered, such as cell adhesion molecules, complement and coagulation cascades, and phagosomes. Our results provide insights into how V. parahaemolyticus overcomes the innate immunity of P. trituberculatus to induce pathological alterations in affected tissues. We report the first iTRAQ-based proteomic analysis and highlight the key pathways and proteins involved in the host–vibrio interactions between P. trituberculatus and V. parahaemolyticus. These findings should enhance our understanding of the molecular mechanisms underlying such interactions.

Hemocyanins of Muricidae: New \u2018Insights\u2019 Unravel an Additional Highly Hydrophilic 800\xa0kDa Mass Within the Molecule

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.

Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia verrucosa (Crustacea, Malacostraca)

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′ and 3′ 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 γ-type of hemocyanins of other crustacean species belonging to order Decapoda, infraorder Brachyura.

PvHS9 is a novel in silico predicted antimicrobial peptide derived from hemocyanin of Penaeus vannamei

Peptides with antimicrobial potential can now be easily and efficiently predicted, designed and validated using in silico tools. Here, twelve peptides with molecular weights between 1.6 and 1.8 kDa were predicted from the sequence of the small subunit of Penaeus vannamei hemocyanin (PvHS) using three online software (AntiBP2, CAMP and APD3). Among the twelve predicted peptides, two peptides, PvHS8 and PvHS9, with the highest prediction score, were selected and their antimicrobial activities ascertained. Both peptide PvHS8 and PvHS9 had strong antimicrobial activity on Gram-negative (i.e., Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio alginolyticus, Escherichia coli, and Aeromonas hydrophila) and Gram-positive (i.e., Staphylococcus aureus, Streptococcus iniae) bacteria, with PvHS9 having the strongest activity. Structural analysis using circular dichroism (CD) and nuclear magnetic resonance (NMR) revealed that peptide PvHS9 (FWVSLKGGKTSIERK) has a β-sheet structure. Mutation of PvHS9 to distort its β-sheet structure and net positive charge attenuated its antimicrobial activity. In addition, scanning electron microscopy (SEM) analysis revealed that peptide PvHS9 destroys bacteria cells by causing membrane rupture and cell death.

Red color-related proteins from the shell of red swamp crayfish (Procambarus clarkii): Isolation, identification and bioinformatic analysis

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.

Protein-Level Evidence of Novel β-Type Hemocyanin and Heterogeneous Subunit Usage in the Pacific Whiteleg Shrimp, Litopenaeus vannamei

The functional diversity of crustacean hemocyanins is broad, encompassing O2 delivery, innate immune response, metabolite storage, and osmolyte balance, all in a heterogeneous protein structure. As such, the sequence diversity of this class of proteins and its subunit composition are the focus of many studies on crustacean adaptation to environmental challenges. Recent transcriptomic and genomic assemblies on the Pacific whiteleg shrimp Litopenaeus vannamei have identified unique isoforms of hemocyanin including an ancestral β-type subunit thought to be lost in penaeid shrimp. However, it is unknown the degree to which these isoforms are translated as proteins, and their abundances within the hemolymph. The present study uses proteomic approaches to characterize the protein-level abundance and organization of these hemocyanin isoforms within their native oligomeric structures. Fractions of each hemocyanin oligomeric form were purified by size-exclusion high performance liquid chromatography for identification of subunit isoforms using tandem mass spectrometry at <1% protein false discovery rate. Data are available via ProteomeXchange with identifier PXD014575. Relative abundances of hemocyanin oligomers and monomeric subunits from hemolymph and fractions were also quantified by polyacrylamide gel electrophoresis with and without denaturation for comparison of subunit heterogeneity. Hemocyanin subunits were organized primarily as hexamers (95-99% relative abundance) as opposed to dodecamers. Ten unique hemocyanins predicted by transcriptome and genome assemblies were identified by tandem mass spectrometry in both oligomer fractions including the first protein-level evidence of a β-type subunit in penaeid shrimp. Identified hemocyanins mapped to four genomic scaffolds and had two or three exons. A single small (75 kDa) subunit constituted half or more of all isoforms identified in each oligomer. The dodecameric fraction exhibited greater subunit heterogeneity utilizing a significantly lower ratio (1.04:1) of small subunit to large subunit compared to hexamers (2.05:1). One isoform, XP_027232115.1, appeared to be more dominant within dodecamers than hexamers. The ability to distinguish and quantify hemocyanin isoforms within oligomeric structures will aid future studies linking hemocyanin genes to transcripts to function and physiology as well as offer insight into the evolutionary history of crustaceans.

Broad-spectrum Antimicrobial Activity of Purified Hemocyanin Subunit IIIA Isolated from Asian Horseshoe Crab, Tachypleus gigas.

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. 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. 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. 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.

Effects of Vibro harveyi and Staphyloccocus aureus infection on hemocyanin synthesis and innate immune responses in white shrimp Litopenaeus vannamei

Hemocyanin, a multifunctional oxygen-carrying protein, has critical effects on immune defense in crustaceans. To explore the role of hemocyanin in anti-pathogen mechanism, effects of Vibrio harveyi (V. harvey) and Staphyloccocus aureus (S. aureus) on hemocyanin synthesis and innate immune responses were investigated in Litopenaeus vannamei (L. vannamei) during infection in vivo. Results showed that 105 and 106 cells mL−1V. harveyi and 106 cells mL−1S. aureus significantly affected plasma hemocyanin concentration, hepatopancreas hemocyanin mRNA and subunits expressions, plasma phenol oxidase (PO), hemocyanin-derived PO (Hd-PO), antibacterial, and bacteriolytic activities during the experiment under bacterial stress, while these parameters did not change remarkably in control group. The concentration of hemocyanin in plasma fluctuated, with a minimum at 12 h and a maximum at 24 h. Moreover, the expression of hemocyanin mRNA peaked at 12 h, while the level of hemocyanin p75 and p77 subunits reached maximum at 24 h. Besides, plasma PO and Hd-PO activities peaked at 24 h, and antimicrobial and bacteriolytic activities peaked at 12 h and 24 h, respectively. In addition, 105 cells mL−1S. aureus had no significant effect on the synthesis of hemocyanin and prophenoloxidase activating (pro-PO) system, but significantly increased antimicrobial activity at 12 h and bacteriolytic activity at 24 h. Therefore, these results suggest that the hemocyanin synthesis was initiated after invasion of pathogen, and the newly synthesized hemocyanin, acted as an immune molecule, can exerts PO activity to regulate the immune defense in L. vannamei in vivo.

Identification and characterization of glycosylation sites on Litopenaeusvannamei hemocyanin.

The respiratory glycoprotein hemocyanin has been implicated in immune-related functions. Using lectin blotting, we show that the binding of shrimp (Litopenaeusvannamei) hemocyanin to concanavalin A decreases markedly with O-glycosidase treatment but not with PNGase F. Twelve O-glycosylation sites, three on the large hemocyanin subunit and nine on the small hemocyanin subunit (HMCs), were identified by LC-MS/MS. Importantly, when the glycosylation sites at Thr-537, Ser-539, and Thr-542 on the C terminus of HMCs were replaced with alanine, the resultant mutant hemocyanin had reduced carbohydrate content, coupled with a fourfold reduction in bacterial agglutination and 0.2-fold reduction in antibacterial activities toward Vibrioparahaemolyticus and Staphylococcusaureus. These results suggest that the glycosylation sites on shrimp hemocyanin are closely related to its immunological functions.

Physiological Effect of Eyestalk Ablation on Nutrient Utilization and Plasma Protein Expression in the Female Giant Freshwater Prawn (Macrobrachium rosenbergii) during Different Molting Cycles

Eyestalk ablation is a common technique to induce spawning in marine shrimps. Currently, little is known about how this technique affects nutritional preferences (energy sources) in response to stress induced by eyestalk ablation during reproduction and growth. Thus, this study investigated the physiological effects of eyestalk ablation on hemolymph nutrient (glucose and protein) utilization and plasma protein expression of the female giant freshwater prawn (Macrobrachium rosenbergii). Sixty female M. rosenbergii prawns weighing 15.6 ± 0.3 g were divided into unablated (no eyestalk ablation, n = 30) and ablated (unilateral eyestalk ablation, n = 30) groups. The groups were then allowed to undergo reproductive and nonreproductive molting cycles. During normal molting, hemolymph protein (1,339.90 ± 95.08 mg/mL) concentrations decreased in the postmolt stage during the reproductive cycle, indicating that protein was used to attain full ovarian maturation, as evidenced by a large rise in the Gonadosomatic index value (8.47% ± 0.52%). The ablated group was vulnerable to stress, especially during the premolt (reproductive) stage, as indicated by a significant (P < 0.05) reduction in hemolymph protein concentrations, whereas no such stress-related effect was observed during the premolt (nonreproductive) stage. Interestingly, protein profiles were unaffected by eyestalk ablation, as shown by the similarity in profiles in both the unablated and ablated groups. Using a proteomic approach, fragment hemocyanin subunit 2, an immune-related protein, was identified in the ablated group. Overall, the results suggested that protein appeared to be the main nutritional source for reproduction and growth during short-term stress associated with eyestalk ablation, which led to stimulation of hemocyanin subunit 2.

Diversity, evolution, and function of myriapod hemocyanins

BackgroundHemocyanin transports O2 in the hemolymph of many arthropod species. Such respiratory proteins have long been considered unnecessary in Myriapoda. As a result, the presence of hemocyanin in Myriapoda has long been overlooked. We analyzed transcriptome and genome sequences from all major myriapod taxa – Chilopoda, Diplopoda, Symphyla, and Pauropoda – with the aim of identifying hemocyanin-like proteins.ResultsWe investigated the genomes and transcriptomes of 56 myriapod species and identified 46 novel full-length hemocyanin subunit sequences in 20 species of Chilopoda, Diplopoda, and Symphyla, but not Pauropoda. We found in Cleidogona sp. (Diplopoda, Chordeumatida) a hemocyanin-like sequence with mutated copper-binding centers, which cannot bind O2. An RNA-seq approach showed markedly different hemocyanin mRNA levels from ~ 6 to 25,000 reads per kilobase per million reads. To evaluate the contribution of hemocyanin to O2 transport, we specifically studied the hemocyanin of the centipede Scolopendra dehaani. This species harbors two distinct hemocyanin subunits with low expression levels. We showed cooperative O2 binding in the S. dehaani hemolymph, indicating that hemocyanin supports O2 transport even at low concentration. Further, we demonstrated that hemocyanin is > 1500-fold more highly expressed in the fertilized egg than in the adult.ConclusionHemocyanin was most likely the respiratory protein in the myriapod stem-lineage, but multiple taxa may have independently lost hemocyanin and thus the ability of efficient O2 transport. In myriapods, hemocyanin is much more widespread than initially appreciated. Some myriapods express hemocyanin only at low levels, which are, nevertheless, sufficient for O2 supply. Notably, also in myriapods, a non-respiratory protein similar to insect storage hexamerins evolved from the hemocyanin.

Hemocyanin of Litopenaeus vannamei agglutinates Vibrio parahaemolyticus AHPND (VPAHPND) and neutralizes its toxin

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.

Safety and efficacy of an oxycodone vaccine: Addressing some of the unique considerations posed by opioid abuse.

Among vaccines aimed at treating substance use disorders, those targeting opioids present several unique medication development challenges. 1) Opioid overdose is a common complication of abuse, so it is desirable for an opioid vaccine to block the toxic as well as the addictive effects of opioids. 2) It is important that an opioid vaccine not interfere with the action of opioid antagonists used to reverse opioid overdose or treat addiction. 3) Some opioids are immunosuppressive and chronic ongoing opioid use could interfere with vaccine immunogenicity. 4) Although antibody-bound oxycodone is unable to enter the brain because of its size, it might still be able to activate peripheral opioid receptors. To assess vaccine impact on opioid toxicity, rats vaccinated with oxycodone conjugated to keyhole limpet hemocyanin subunit dimer (OXY-dKLH) adsorbed to alum or controls vaccinated with dKLH were compared with regard to oxycodone-induced hotplate analgesia and oxycodone-induced respiratory depression and bradycardia. Vaccination shifted the dose-response curves to the right, representing protection, for each of these endpoints. Naloxone was equally effective in both OXY-dKLH and control groups, providing complete and rapid reversal of respiratory depression. The administration of a long-acting naltrexone formulation during vaccination did not impair vaccine immunogenicity in mice. Similarly, serum anti-oxycodone antibody titers were not altered by continuous morphine infusion during vaccination compared to opioid-naïve controls. Competitive ELISA assay showed negligible or low affinity of immune antiserum for endogenous opioids or opioid antagonists. In vitro receptor binding assays showed that antibody-bound oxycodone does not activate mu opioid receptors. These data support further study of OXY-dKLH as a potential treatment for oxycodone abuse and suggest that vaccination might also reduce the severity of oxycodone overdose.

Biomolecular changes that occur in the antennal gland of the giant freshwater prawn (Machrobrachium rosenbergii).

In decapod crustaceans, the antennal gland (AnG) is a major primary source of externally secreted biomolecules, and some may act as pheromones that play a major role in aquatic animal communication. In aquatic crustaceans, sex pheromones regulate reproductive behaviours, yet they remain largely unidentified besides the N-acetylglucosamine-1,5-lactone (NAGL) that stimulates male to female attraction. In this study, we used an AnG transcriptome of the female giant freshwater prawn (Macrobrachium rosenbergii) to predict the secretion of 226 proteins, including the most abundantly expressed transcripts encoding the Spaetzle protein, a serine protease inhibitor, and an arthropodial cuticle protein AMP 8.1. A quantitative proteome analysis of the female AnG at intermolt, premolt and postmolt, identified numerous proteins of different abundances, such as the hemocyanin subunit 1 that is most abundant at intermolt. We also show that hemocyanin subunit 1 is present within water surrounding females. Of those metabolites identified, we demonstrate that the NAGL and N-acetylglucosamine (NAG) can bind with high affinity to hemocyanin subunit 1. In summary, this study has revealed components of the female giant freshwater prawn AnG that are released and contribute to further research towards understanding crustacean conspecific signalling.

N-glycan structures of β-HlH subunit of Helix lucorum hemocyanin

The carbohydrate structures of molluscan hemocyanins have recently received particular interest due to their specific monosaccharide composition, as well as their immunostimulatory properties and application in clinical studies. For the first time, we investigated N-glycans of the structural subunit β-HlH of hemocyanin isolated from Helix lucorum. In total, 32 different glycans were enzymatically liberated and characterized by tandem mass spectrometry using a Q-Trap mass spectrometer. Our study revealed a highly heterogeneous mixture of glycans with composition Hex3-7HexNAc2-5MeHex0-4Pent0-1Fuc0-1. The oligosaccharide chains are mostly modified at the inner core by β1-2-linked xylose to β-mannose, by α1-6-fucosylation of the innermost GlcNAc residue (the Asn-bound GlcNAc), and by methylation. The glycans of β-HlH mainly contain a terminal MeHex residue; in some cases even two, three or four of these residues occur. Several carbohydrate chains in β-HlH are core-fucosylated without Xyl and also possess a high degree of methylation. This study shows the presence of mono- and bi-antennary N-glycans as well as hybrid type structures with or without core-fucosylation.

Intersexual differences of heat shock response between two amphipods (Eulimnogammarus verrucosus and Eulimnogammarus cyaneus) in Lake Baikal.

Acute temperature fluctuations are common in surface waters, and aquatic organisms may manifest physiological responses to punctuated temperature spikes long before behavioral responses. Ectotherms, especially cryophilic stenotherms such as those endemic to Lake Baikal (Siberia), may demonstrate specialized physiological responses to acute temperature increases because their proteomes have evolved to function most efficiently at lower temperatures (e.g., <10 °C). Therefore, our study questioned the nature and degree of variation in physiological response to acute thermal stress in two congenerous, endemic Baikal amphipod species, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus. We hypothesized that because interspecific and intersexual thermosensitivity varies significantly among ectotherms, there would be divergent intersexual and interspecific strategies to withstand acute thermal stress, manifested in different protein compositions and concentrations. We exposed individuals to the species’ respective LT50 for one hour followed by a three-hour recovery period. We then performed 1D-PAGE, Western blotting, 2D-PAGE, and Mass Spectrometry techniques and assessed relative intersexual and interspecific changes in proteomic composition and heat shock protein 70 level. Our results demonstrate that females tend to be more sensitive to an acute thermal stimulus than males, most likely because females allocate significant energy to reproduction and less to heat shock response, evidenced by females’ significantly lower LT50time. Lower level of Hsp70 was found in females of the thermosensitive E. verrucosus compared to males of this species. No intersexual differences were found in Hsp70 level in thermotolerant E. cyaneus. Higher levels of hemocyanin subunits and arginine kinase were found in E. cyaneus females after heat shock and recovery compared to males, which was not found for E. verrucosus, suggesting interspecific mechanisms for E. cyaneus’s higher thermotolerance. These differing responses between species and sexes of Baikal amphipods may reflect more general strategies for maintaining homeostatic conditions during acute thermal stress. As mean surface water temperatures increase worldwide, the net efficiency and efficacy of these strategies could give rise to long term changes in physiology, behavior, and interactions with other species, potentially precipitating population and community level alterations.

Uncovering Hemocyanin Subunit Heterogeneity in Penaeid Shrimp using RNA-Seq.

Aquatic crustaceans can experience low levels of O2 alone but more often in combination with high levels of CO2 both in natural estuaries and in aquaculture ponds. Hemocyanin, the respiratory pigment in many crustacean species, facilitates O2 transport and is documented to change in abundance, structure, and function in response to low O2 The impacts of high CO2 on the respiratory pigment are less clear. In this synthesis we bring together data from recently published and new RNA-Seq studies toward the aims of defining the full repertoire of hemocyanin subunits, as well as their differential expression and regulation in the Penaeoidea family in response to low O2 with or without high CO2 RNA-Seq data were collected from the hepatopancreas tissues of aquacultured Pacific whiteleg shrimp Litopenaeus vannamei, wild-caught L. vannamei, and wild-caught Atlantic brown shrimp Farfantepenaeus aztecus. De novo assembly yielded high-quality stranded transcriptomes. Manual curation of the hemocyanin subunits from all three groups of penaeid shrimp confirmed the existence of a small γ-type hemocyanin subunit (HcS), greater sequence diversity in the large γ-type hemocyanin subunit than previously identified (HcL1-3 isoforms) and expression of a β-type hemocyanin subunit (HcB) previously unidentified in Penaeid shrimp. Relative abundance of transcripts encoding these hemocyanin isoforms differed within and among the three species/strains. Exposure to low O2 induced expression of all of the subunits in aquacultured L. vannamei With concurrent exposure to high CO2, the number of γ-type hemocyanin transcripts decreased while the expression of β-type transcripts remained unchanged. Together with functional data for hemocyanins in the same shrimp species/strains, the RNA-Seq approach shows great promise to provide new insights into the connection between sequence, protein structure, and physiological function of respiratory pigments in this decapod crustacean family.