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Up to now, it has been believed that invertebrates are unable to synthesize ascorbic acid (AA) in vivo. However, in the present study, the full-length CDs (Coding sequence) of L-gulonolactone oxidase (GLO) from Pacific abalone (Haliotis discus hannai Ino) were obtained through molecular cloning. The Pacific abalone GLO contained a FAD-binding domain in the N-termination, and ALO domain and conserved HWAK motif in the C-termination. The GLO gene possesses 12 exons and 11 introns. The Pacific abalone GLO was expressed in various tissues, including the kidney, digestive gland, gill, intestine, muscle and mantle. The GLO activity assay revealed that GLO activity was only detected in the kidney of Pacific abalone. After a 100-day feeding trial, dietary AA levels did not significantly affect the survival, weight gain, daily increment in shell length, and feed conversion ratio of Pacific abalone. The expression of GLO in the kidney was downregulated by dietary AA. These results implied that the ability to synthesize AA in abalone had not been lost. From the evolutionary perspective, the loss of GLO occurred independently as an independent event by matching with the genomes of various species. The positive selection analysis revealed that the GLO gene underwent purifying selective pressure during its evolution. In conclusion, the present study provided direct evidence to prove that the GLO activity and the ability to synthesize AA exist in abalone. The AA synthesis ability in vertebrates might have originated from invertebrates dating back 930.31 million years., Competing Interests: Declaration of competing interest All authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aimed to study the characteristics of prolyl endopeptidase (PEP, EC 3.4.21.26) and its possible role in the degradation of collagen, we cloned the full-length cDNA sequence of PEP from abalone (Haliotis discus hannai) (Hdh-PEP). Recombinant Hdh-PEP (rHdh-PEP) was expressed in vitro, its enzymatic properties were detected, and its secondary structure was analyzed by Circular Dichroism (CD). We for the first time determined the 1.5 Å crystal structure of rHdh-PEP. The decomposition effect of rHdh-PEP on collagen peptides was analyzed. Our data revealed that the molecular weight of rHdh-PEP is 85 kDa, consisting of a catalytic domain and a β-propeller domain. The optimal pH and temperature of rHdh-PEP were pH 6.0 and 20 °C, respectively. Using small collagen peptides as substrates, HPLC-ESI-MS analysis confirmed that rHdh-PEP specifically cleaved at the carboxyl side of proline residues, suggesting its role in the degradation of collagen peptides during autolysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bacterial infection in the marine environment is a serious problem to maintain the stability of marine ecosystems. Nevertheless, there is little report so far for the biological effects of pathogenic bacteria in coastal ecosystems. Hence, we investigated the responses of shell-less Onchidium reevesii to resist against pathogenic bacterial infection. Analysis of data here could be used as fundamental information for assessment of innate immune response of O. reevesii. The full-length OrCTL cDNA was cloned and consists of 1849 base pair (bp) encoding protein of 192 amino acids. Constructing multiple alignments suggested that OrCTL has conserved carbohydrate recognition domain (CRD) of CTLs, containing an EPS (Glu-Pro-Ser) motif that may imply the function of recognition of carbohydrates like others invertebrate. OrCTL mRNAs were mainly detected in ganglion and hepatopancreas, and expression was highly up-regulated from 2 h after Vibrio harveyi challenge, rapidly decreased at 4 h, and significantly increased at 12 h. In addition, after challenge with Vibrio parahaemolytics, OrCTL gene expression was slightly up-regulated from 2 h, peaked at 12 h. Enzyme activity (in the hepatopancreas) and cell immune (in the hemolymph) were investigated along with Superoxide dismutase (SOD) activity, alkaline phosphatase (ALP) activity and cell cycle. SOD activities were significantly higher after V. harveyi and V. parahaemolytics challenge than that in the control group, respectively. By contrast, ALP activities were significantly inhibited after challenged with bacteria than that in the control group, respectively. Enzyme activities in the hepatopancreas obviously fluctuated, and ALP activity was more sensitive to bacteria. Cell responses illustrated that there were a significant higher percentage of cells in the S and G2/M phase in hemolymph after challenged with bacteria. Our results suggested that the immune response of O. reevesii could be activated by pathogenic bacteria, and the data will provide referent for the disease prevention of systematic investigation in aquatic animal., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest to this work., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Prohormone convertases (PCs) are subtilisin-like proteases responsible for the intracellular processing of prohormones and proneuropeptides in vertebrates and invertebrates. The full-length PC2 cDNA sequence was cloned from pleuropedal ganglion of Haliotis discus hannai, consisted of 2254-bp with an open reading frame of 1989-bp and encoded a protein of 662 amino acid residues. The architecture of Hdh PC2 displayed key features of PCs, including a signal peptide, a pro-segment domain with sites for autocatalytic activation, a catalytic domain, and a pro-protein domain (P-domain). It shares the highest homology of its amino acid sequence with the PC2 from H. asinina and to lesser extent with that of Homo sapiens and Rana catesbeiana PC2. Sequence alignment analysis indicated that Hdh PC2 was highly conserved in the catalytic domain, including a catalytic triad of serine proteinases of the subtilisin family at positions Asp-195, His-236, and Ser-412. The cloned sequence contained a canonical integrin binding sequence, and four cysteine residues involved in the formation of an intramolecular disulfide link. Phylogenetic analysis revealed that the Hdh PC2 is robustly clustered with the Has PC2. Quantitative PCR assay demonstrated that the Hdh PC2 was predominantly expressed in the pleuropedal ganglion rather than in other examined tissues. Although PC2 mRNA was expressed throughout the gametogenetic cycle of male and female abalone, the expression level was significantly higher in the ripening stage of female abalone. Also, a significantly higher expression was observed in the pleuropedal ganglion and gonadal tissues at a higher effective accumulative temperature (1000°C). In situ hybridization revealed that the PC2 mRNA expressing neurosecretory cells were distributed in the cortex region of the pleuropedal ganglion. According to the results, it can be concluded that pleuropedal ganglion is the highest site of PC2 activity, and this enzyme might be involved in the abalone reproduction process., Competing Interests: All authors declare that they have no conflict of interest.

In vertebrates, the steroidogenesis enzyme 5α-reductase converts testosterone to the more potent androgen 5α-dihydrotestosterone. Homologues of 5α-reductase genes have been identified in molluscs. However, recent findings suggest that vertebrate-type steroid androgens are not utilised in molluscan reproductive development. Genomic searches have revealed that molluscs do not possess many of the steroidogenic enzymes required to make testosterone, nor a nuclear androgen receptor. Consequently, the role of 5α-reductase in molluscs presents a mystery. Here, developmental exposures of Biomphalaria glabrata to selective pharmaceutical 5α-reductase inhibitors elicited a strong, highly reproducible phenotypic response characterised by the development of elongated "banana-shaped" shell morphology. In comparison to untreated snails, the shells are open-coiled and the whorls are unattached. Dutasteride (5α-reductase inhibitor) is approximately 10-times more potent at provoking the banana-shaped shell phenotype than finasteride, paralleling the pharmaceuticals' efficacy in humans. Other enzyme inhibitors with different modes of action were tested to investigate the specificity of the phenotype. However, only the pharmaceutical 5α-reductase inhibitors provoked the response. Dutasteride elicited the same phenotype in a second gastropod, Physella acuta. In the absence of evidence for de novo androgen steroidogenesis in molluscs, these findings suggest that novel substrates for 5α-reductase exist in gastropods, lending support to the contention that molluscan endocrinology differs from the well-characterised vertebrate endocrine system.

The effects of a diet containing the probiotic Bacillus amyloliquefaciens on the survival and growth of Haliotis discus hannai were evaluated by measuring growth and hematological parameters and the expression levels of nonspecific immune genes. In addition, the abalone's response to Vibrio parahaemolyticus infection was assessed. H. discus hannai (shell length: 29.35 ± 1.81 mm, body weight: 4.28 ± 0.23 g) were exposed to an 8-week culture experiment in indoor aquariums and a 2-week V. parahaemolyticus artificial infection experiment. In each experiment, the control group (C) was fed daily with the basal feed; the experimental groups were fed daily with the experimental feed, prepared by spraying B. amyloliquefaciens onto the basal feed at final concentrations of 10 3 (group A1), 10 5 (A2), and 10 7 (A3) cfu/g. The survival rate, body weight specific growth rate, and food conversion efficiency in A2 and A3 were significantly higher than those in A1 and C (P < 0.05). The total number of blood lymphocytes, the O 2 - and NO levels produced from respiratory burst, the activities of acid phosphatase, superoxide dismutase, and catalase, and the expression levels of catalase and thiol peroxidase in A2 were not significantly different from those in A3, but these factors were significantly higher in A2 compared to A1 and C (P < 0.05). The total antioxidant capacity and expression levels of glutathione S-transferase in A1, A2 and A3 were significantly higher than those in C (P < 0.05). At day 9 after infection with V. parahaemolyticus, all abalone in C were dead; at the end of the experiment, the cumulative mortality of abalone in A2 was significantly lower than that in any other group (P < 0.05). Thus, the experimental feed containing 10 5  cfu/g B. amyloliquefaciens not only facilitated the food intake and growth of abalone, but also effectively enhanced their non-specific immunity and resistance to V. parahaemolyticus infection. In this regard, B. amyloliquefaciens may be a useful probiotic strain for abalone aquaculture., (Copyright © 2019. Published by Elsevier Ltd.)

Desomorphine, the principal opioid in Krokodil, has an analgesic potency approximately ten-times that of morphine. Similar to other opioids, during phase II metabolism it undergoes conjugation with glucuronic acid to form desomorphine-glucuronide. Although hydrolysis of conjugated species is sometimes required prior to analysis, desomorphine-glucuronide has not been fully investigated. In this study, six hydrolysis procedures were optimized and evaluated. Deconjugation efficiencies using chemical and enzymatic hydrolysis were evaluated and stability in aqueous solution was assessed. Acid hydrolysis was compared with five β-glucuronidase sources (BGTurbo™, IMCSzyme™, Escherichia coli, Helix pomatia and Patella vulgata). At optimal conditions, each hydrolysis method produced complete hydrolysis (≥96%). However, under simulated challenging conditions, P. vulgata was the most efficient β-glucuronidase for the hydrolysis of desomorphine-glucuronide. Both BGTurbo™ and IMCSzyme™ offered fast hydrolysis with no need for sample cleanup prior to liquid chromatography-quadrupole/time of flight-mass spectrometry (LC-Q/TOF-MS) analysis. Hydrolysates using E. coli, H. pomatia and P. vulgata underwent additional sample treatment using β-Gone™ cartridges. Additionally, the stability of free and conjugated drug was evaluated at elevated temperature (60°C) in aqueous solutions between pH 4 and 10. No degradation was observed for either desomorphine or desomorphine-glucuronide under any of the conditions tested., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Benzodiazepines is a large drug group used extensively to treat sleep disorders and anxiety; these drugs are frequently associated with various crimes such as murder and drug-facilitated sexual assault. With growing use and misuse of these compounds, confirmatory assays are increasingly required in clinical laboratories. In this study, 4 β-glucuronidase enzymes were systematically evaluated for their hydrolysis efficiencies. Additionally, the matrix effects and extraction recoveries of three protein precipitation plates were systematically evaluated. The recombinant IMCSzyme β-glucuronidase enzyme showed higher hydrolysis efficiency than did β-glucuronidase from abalone, Patella vulgata and Helix pomatia. Lower ion suppression and more favorable overall recovery were observed in the Supelco protein precipitation plate than in the two other plates. Analytes were separated on a superficially porous particle column (ultra biphenyl) within 4.5 min and characterized through electrospray ionization-tandem mass spectrometry in the positive ion multiple-reaction monitoring mode. The accuracy, carryover, extraction recovery, detection limit, matrix effect, quantification limit and precision of the method were validated. Sixty opioid-positive urine samples were analyzed; a high incidence of benzodiazepines was detected in these samples. The proposed technique is robust and suitable for efficiently monitoring the numerous benzodiazepines that occur in urine samples., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from -1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme's thermal responses and foster evolutionary adaptation of function., Competing Interests: The authors declare no conflict of interest.

Oncomelania hupensis is the unique intermediate host of the blood fluke Schistosoma japonicum, which causes schistosomiasis. In snails, highly toxic reactive oxygen species (ROS) can be continually generated by hemocytes in response to foreign particles or pathogens, and may be involved in damaging and eliminating digenean larvae. Thioredoxin-related protein of 14 kDa (TRP14) is a member of the Trx superfamily, and plays an important role in the scavenging of ROS. This study was designed to identify and characterize TRP14 from O. hupensis (OhTRP14), and investigate the involvement of OhTRP14 in the scavenging of ROS in snail host immune response to the parasite S. japonicum. Here we expressed and purified the recombinant OhTRP14 and its mutant, and rOhTRP14 displayed oxidoreductase activity dependent on the CPDC motif. OhTRP14 protein was ubiquitously present in all the tested snail tissues, and especially immunolocalized in the cytoplasm of immune cell types (hemocytes). Both the expression of OhTRP14 and ROS level increased significantly in snails following challenge with S. japonicum. The dsRNA-mediated knockdown of OhTRP14 was successfully conducted by oral feeding, and ROS production was increased by OhTRP14 knockdown, implying that OhTRP14 was involved in the scavenging of ROS in O. hupensis circulating hemocytes. Therefore, we conclude that OhTRP14 may be involved in the scavenging of ROS in snail host immune response to the parasite S. japonicum. The results expand our understanding of the interaction between this parasite and host, and lay a foundation for the establishment of Oncomelania-schistosome infection models., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Carbamate insecticides such as carbaryl and organophosphates such as azinphos-methyl share the ability to inhibit the activity of B-esterases. This study aimed to (1) assess the inhibitory effects of carbaryl on B-esterase activity in soft tissues and hemolymph of Planorbarius corneus; (2) establish whether binary mixtures of carbaryl and azinphos-methyl depart or not from a model of concentration addition on the inhibition of cholinesterase activity; (3) determine the bioconcentration and elimination of the pesticides. The results showed that exposure of gastropods to increasing concentrations of carbaryl (0.1-5 mg L -1 ) for 48 h inhibited cholinesterase activity in a concentration-dependent manner, with an EC 50 of 1.4 ± 0.3 mg L -1 and 1.2 ± 0.1 mg L -1 for soft tissue and hemolymph, respectively. Carboxylesterase activity, measured with the substrates p-nitrophenyl butyrate and p-nitrophenyl acetate, was between 2.3 and 25 times more sensitive to carbaryl inhibition than cholinesterase activity. Binary mixtures corresponding to 0.5 EC 50 carbaryl + 0.5 EC 50 azinphos-methyl and 0.75 EC 50 carbaryl + 0.75 EC 50 azinphos-methyl produced inhibitions of cholinesterase activity similar to those of individual pesticides, following a model of concentration addition. Bioconcentration was analyzed using a one-compartment model. The absorption kinetics (k 1 ) for both pesticides alone (1.4 mg L -1 of carbaryl or 1.8 mg L -1 of azinphos-methyl) or mixed (1.4 mg L -1 of carbaryl + 1.8 mg L -1 of azinphos-methyl) were similar. The elimination kinetics ratio (k 2 ) estimated for the pesticides alone or in the mixtures showed that carbaryl was eliminated 3.5 times faster than azinphos-methyl. These results suggest that exposure of Planorbarius corneus to binary mixtures of carbaryl and azinphos-methyl for 48 h follow a concentration addition model on inhibition of cholinesterase activity and that the pesticide mixtures do not change the toxicokinetic parameters of the parent compounds., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Many invertebrates enclose their embryos within egg capsules, from which the offspring hatch. In marine gastropods that brood their egg capsules, hatching could involve radular activity by the mother or by unhatched stages, increased osmotic concentration of the intracapsular fluid, or production of hatching enzymes. The present research sought to determine whether mechanical action by the brooding female or by the encapsulated embryos was involved in the hatching for two sympatric and closely related species of calyptraeid: Crepipatella dilatata, which exhibits direct development without free-living larvae, and Crepipatella peruviana, which releases free-living veliger larvae. We also considered the role that enzymatic action or osmotic changes in the intracapsular fluid might play in hatching. Using scanning electron micrograph analyses, we found no evidence that the well-developed, pre-hatching juvenile radula of C. dilatata played any role in the hatching process and that the radula of C. peruviana did not even develop until long after hatching; so there was no evidence of radular activity involved in the hatching of either species. For C. peruviana, the intracapsular fluid osmolality was always higher than that of the surrounding seawater, suggesting that there is a strong natural water inflow during development. Moreover, when egg capsules of C. peruviana were exposed to lower ambient salinities, the substantial entry of water correlated well with high percentages of hatching, particularly for egg capsules containing advanced veligers, suggesting that an osmotic mechanism may be involved in the hatching process of this species. In contrast, hatching in C. dilatata appeared to be enzymatically mediated.

Mannitol oxidase and polyol dehydrogenases are enzymes that convert polyalcohols into sugars. Mannitol oxidase was previously investigated in terrestrial snails and slugs, being also present in a few aquatic gastropods. However, the overall distribution of this enzyme in the Gastropoda was not known. Polyol dehydrogenases are also poorly studied in gastropods and other mollusks. In this study, polyalcohol oxidase and dehydrogenase activities were assayed in the digestive gland of 26 species of gastropods, representing the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, including herbivores and carnivores were analyzed. Ultrastructural observations were undertake in species possessing mannitol oxidase, in order to investigate the correlation between this enzyme and the presence of tubular structures known to be associated with it. Mannitol oxidase activity was detected in the digestive gland of herbivores from the clades Caenogastropoda and Heterobranchia, but not in any carnivores or in herbivores from the clades Patellogastropoda, Neritimorpha and Vetigastropoda. In most of the species used in this study, dehydrogenase activities were detected using both D-mannitol and D-sorbitol as substrates. Nevertheless, in some carnivores these activities were not detected with both polyalcohols. Ultrastructural observations revealed tubular structures in digestive gland cells of some species having mannitol oxidase activity, but they were not observed in others. Based on our results, we suggest that mannitol oxidase first occurred in a herbivorous or omnivorous ancestor of Apogastropoda, the clade formed by caenogastropods and heterobranchs, being subsequently lost in those species that shifted towards a carnivorous diet.

Prolonged urinary cannabinoid excretion in chronic frequent cannabis users confounds identification of recent cannabis intake that may be important in treatment, workplace, clinical, and forensic testing programs. In addition, differentiation of synthetic Δ9-tetrahydrocannabinol (THC) intake from cannabis plant products might be an important interpretive issue. THC, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) urine concentrations were evaluated during previous controlled cannabis administration studies following tandem alkaline/E. coli β-glucuronidase hydrolysis. We optimized recombinant β-glucuronidase enzymatic urinary hydrolysis before simultaneous liquid chromatography tandem mass spectrometry (LC-MS/MS) quantification of THC, 11-OH-THC, THCCOOH, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocannabivarin (THCV) and 11-nor-9-carboxy-THCV (THCVCOOH) in urine. Enzyme amount, incubation time and temperature, buffer molarity and pH were optimized using pooled urine samples collected during a National Institute on Drug Abuse, Institutional Review Board-approved clinical study. Optimized cannabinoid hydrolysis with recombinant β-glucuronidase was achieved with 2000 IU enzyme, 2 M pH 6.8 sodium phosphate buffer, and 0.2 mL urine at 37°C for 16 h. The LC-MS/MS quantification method for hydrolyzed urinary cannabinoids was validated per the Scientific Working Group on Toxicology guidelines. Linear ranges were 1-250 μg/L for THC and CBG, 2-250 μg/L for 11-OH-THC, CBD, CBN, THCV and THCVCOOH, and 1-500 μg/L for THCCOOH. Inter-batch analytical bias was 92.4-112.4%, imprecision 4.4-9.3% CV (n = 25), extraction efficiency 44.3-97.1% and matrix effect -29.6 to 1.8% (n = 10). The method was utilized to analyze urine specimens collected during our controlled smoked, vaporized, and edible cannabis administration study to improve interpretation of urine cannabinoid test results., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

Vibrio parahemolyticus (V. parahemolyticus) is a major pathogen for abalone, an important economical shellfish in coastal area of China. There is little known about the abalone innate immune system against pathogen infection. Clip-domain serine proteases (cSPs) are increasingly recognized to play important roles in host immune defense in invertebrates. In this study, we cloned a cSP (Hdh-cSP) from abalone (Haliotis discus hannai). We found out that Hdh-cSP was widely expressed in multiple tissues of abalone, with highest level in the immune-like organ, hepatopancreas. V. parahemolyticus infection induced significantly elevated expression of Hdh-cSP in addition to better-characterized innate immune component genes including Rel/NF-κB, allograft inflammatory factor (ALInFa), macrophage expressed protein (MEP) and caspase-8. Importantly, the silencing of Hdh-cSP reduced the expression of these genes, suggesting that Hdh-cSP was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that apoptosis of hemocytes was inhibited when the transcription of Hdh-cSP was knocked down, suggesting that Hdh-cSP participated in cell apoptosis by regulation of caspase 8 expression in V. parahemolyticus infection. Therefore, our study established an important role of cSP in the innate immunity against V. parahemolyticus infection in abalone., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

EG27I is an endogenous glucanase belonging to glycoside hydrolase family (GHF) 45 from the mollusk Ampullaria crossean. In this study, the mature EG27I peptide gene fused to the HFBII secretion signal of Trichoderma reesei was expressed under the GAP promoter of Pichia pastoris in SMD1163 strain. A bioactive EG27I with a molecular weight of 27 kDa was successfully expressed and secreted into our culture medium. The respective final OD 600 and hydrolytic activity were 333 and 1.28 U/mL when high-cell-density fermentation of the recombinant P. pastoris was performed in a 7.5 L fermenter through a fed-batch strategy for 132 h. The recombinant protein concentration of the fermentation supernatant was 47.7 mg/L. EG27I was consecutively purified from the fermentation supernatant through ultrafiltration, cation exchange, and hydrophobic interaction. The specific activity of the recombinant EG27I was 26.8 U/mg, and the optimal pH and temperature of the enzyme were 5 and 50 °C, respectively. The half-life of the enzyme activity at 100 °C could reach 40 min. The N-terminal amino acid sequence analysis of the purified recombinant protein confirmed that the amino terminal sequence was consistent with the natural structure. The high quantity and purity of the EG27I provide a basis for future structural and functional studies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone., (Copyright © 2017. Published by Elsevier B.V.)

The gastropod Nacella concinna is the most conspicuous macroinvertebrate of the intertidal zone of the Antarctic Peninsula and adjacent islands. Naturally high levels of copper and cadmium in coastal marine ecosystems are accumulated in N. concinna tissues. We aimed to study the effects of metal cations on N. concinna arginase in the context of possible adaptive microevolution. Gills and muscle had the highest argininolytic activity, which was concentrated in the cytosol in both tissues. Gills had the highest levels of arginase and may be involved in the systemic control of l-arginine levels. The relatively high argininolytic activity of the N. concinna muscular foot, with K M =25.3±3.4mmolL -1 , may be involved in the control of l-arginine levels during phosphagen breakdown. N. concinna arginases showed the following preferences for metal cations: Ni 2+ >Mn 2+ >Co 2+ >Cu 2+ in muscle and Mn 2+ >Cu 2+ in gills. Cu 2+ activation is a unique characteristic of N. concinna arginases, as copper is a potent arginase inhibitor. Cu 2+ partly neutralized N. concinna arginase inhibition by Cd 2+ , worked synergistically in muscle arginase activation by Co 2+ and neutralized muscle arginase activation by Ni 2+ . Mn 2+ was able to activate muscle arginase in the presence of Fe 3+ and Pb 2+ . The selection of arginases that are activated by Cu 2+ and resistant to inhibition by Cd 2+ in the presence of Cu 2+ over evolutionary timescales may have favored N. concinna occupation of copper- and cadmium-rich niches., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mitogen-activated protein kinase (MAPK) is involved in the regulation of cellular events by mediating signal transduction pathways. MAPK1 is a member of the extracellular-signal regulated kinases (ERKs), playing roles in cell proliferation, differentiation, and development. This is mainly in response to growth factors, mitogens, and many environmental stresses. In the current study, we have characterized the structural features of a homolog of MAPK1 from disk abalone (AbMAPK1). Further, we have unraveled its expressional kinetics against different experimental pathogenic infections or related chemical stimulants. AbMAPK1 harbors a 5' untranslated region (UTR) of 23 bps, a coding sequence of 1104 bps, and a 3' UTR of 448 bp. The putative peptide comprises a predicted molecular mass of 42.2 kDa, with a theoretical pI of 6.28. Based on the in silico analysis, AbMAPK1 possesses two N-glycosylation sites, one S&#95;TK catalytic domain, and a conserved His-Arg-Asp domain (HRD). In addition, a conservative glycine rich ATP-phosphate-binding loop and a threonine-x-tyrosine motif (TEY) important for the autophosphorylation were also identified in the protein. Homology assessment of AbMAPK1 showed several conserved regions, and ark clam (Aplysia californica) showed the highest sequence identity (87.9%). The phylogenetic analysis supported close evolutionary kinship with molluscan orthologs. Constitutive expression of AbMAPK1 was observed in six different tissues of disk abalone, with the highest expression in the digestive tract, followed by the gills and hemocytes. Highest AbMAPK1 mRNA expression level was detected at the trochophore developmental stage, suggesting its role in abalone cell differentiation and proliferation. Significant modulation of AbMAPK1 expression under pathogenic stress suggested its putative involvement in the immune defense mechanism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: In China, abalone (Haliotis discus hannai) production is growing annually. During industrial processing, the viscera, which are abundant of cellulase, are usually discarded or processed into low-value feedstuff. Thus, it is of interest to obtain cellulase from abalone viscera and investigate its application for preparation of functional oligosaccharides., Results: A cellulase was purified from the hepatopancreas of abalone by ammonium sulfate precipitation and two-steps column chromatography. The molecular weight of the cellulase was 45 kDa on SDS-PAGE. Peptide mass fingerprinting analysis yielded 103 amino acid residues, which were identical to cellulases from other species of abalone. Substrate specificity analysis indicated that the cellulase is an endo-1,4-β-glucanase. Hydrolysis of seaweed Porphyra haitanensis polysaccharides by the enzyme produced oligosaccharides with degree of polymerisation of two to four, whose monosaccharide composition was 58% galactose, 4% glucose and 38% xylose. The oligosaccharides revealed 2,2'-diphenyl-1-picrylhydrazyl free radical as well as hydrogen peroxide scavenging activity., Conclusion: It is feasible and meaningful to utilise cellulase from the viscera of abalone for preparation of functional oligosaccharides. © 2015 Society of Chemical Industry., (© 2015 Society of Chemical Industry.)

The relevance of comprehensive studies of the Rapana vital functions is determined by its considerab­le negative impact on the ecosystem of the Black Sea. The aim of the work was to find out the polymorphism and activity of the main hydrolases in the different parts of the digestive system of Rapana. Hydrolases (proteases, amylases, esterases, lipases and phosphatases) in glandular structures of the Rapana digestive system were studied by electrophoresis. It was found that different sets of hydrolytic enzymes are functioning in certain parts of the Rapana digestive tract. The gland of Leiblein and hepatopancreas played the most important role in the digestion of food components. The salivary glands had the significant influence on proteolysis.

An endo-1,4-β-glucanase from Bellamya chinensis laeta was purified to electrophoretically homogeneous state. The molecular weight of the purified enzyme was estimated 70,000 by SDS-PAGE. The enzyme was most active at pH 5.5 and 50 °C, and stable at around pH 10 and 50 °C. The enzyme exhibited the significant activity at 20 °C (30 % of the activity at optimal 50 °C). The enzyme was hydrolyzed cellohexaose into cellobiose, cellotriose, and cellotetraose as main products. Three cDNAs (BC-EG70a, BC-EG70b, and BC-EG70c) encoding the endo-1,4-β-glucanase were cloned by PCR-based method. Three endo-1,4-β-glucanases consisted of 1758 bp encoding 586 amino acids. The three genes were almost the same nucleotide sequences. The deduced proteins were consisted of a signal sequence, cellulose binding domain, linker, and catalytic domain. The amino acid sequence of BC-EG70a shares sequence identity degree with the endo-1,4-β-glucanases of Haliotis discus hannai (61 %), Ampullaria crossean (52 %), and Mizuhopecten yessoensis (51 %) which all belong to glycoside hydrolase family 9.

Pollution effects were assessed by means of biochemical biomarkers (catalase, glutathione S-transferase and acetylcholinesterase activities, and metallothioneins content) in five species at selected coastal sites across the Eastern Mediterranean and the Black Sea. The mussel Mytilus galloprovincialis, a well-established sentinel species, was investigated in the Adriatic Sea, Aegean Sea, and Black Sea. The mussel Brachidontes pharaonis and the striped red mullet Mullus surmuletus were used in the Levantine Sea where M. galloprovincialis is not present. The white seabream Diplodus sargus sargus and the gastropod Rapana venosa were additionally sampled in the Adriatic and the Black Sea, respectively. Mussels showed catalase, glutathione S-transferase, and acetylcholinesterase responses to pollution in most geographical areas while the response of metallothioneins was restricted to a few sites. R. venosa showed marked responses of catalase and metallothioneins whereas both fish species did not generally exhibit variations in biomarker values among sites. The approach based on the reference deviation concept using the "Integrated Biological Responses version 2" index was useful for the interpretation of overall biomarker responses.

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca(2+)/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.

The small abalone Haliotis diversicolor is an economically important mollusk that is widely cultivated in Southern China. Gonad precocity may affect the aquaculture of small abalone. Polyamines, which are small cationic molecules essential for cellular proliferation, may affect gonadal development. Ornithine decarboxylase (ODC) and antizyme (AZ) are essential elements of a feedback circuit that regulates cellular polyamines. This paper presents the molecular cloning and characterization of AZ from small abalone. Sequence analysis showed that the cDNA sequence of H. diversicolor AZ (HdiODCAZ) consisted of two overlapping open reading frames (ORFs) and conformed to the +1 frameshift property of the frame. Thin Layer chromatography (TLC) analysis suggested that the expressed protein encoded by +1 ORF2 was the functional AZ that targets ODC to 26S proteasome degradation. The result demonstrated that the expression level of AZ was higher than that of ODC in the ovary of small abalone. In addition, the expression profiles of ODC and AZ at the different development stages of the ovary indicated that these two genes might be involved in the gonadal development of small abalone.

The intertidal and subtidal environments in the Antarctic Peninsula are vulnerable to pollutants, such as diesel oil, a commonly used fuel. Nacella concinna is capable of accumulating polycyclic aromatic hydrocarbons and is a potential biomonitor of diesel oil contamination. This work investigates the interaction of diesel oil, temperature and salinity on the activity of antioxidants enzymes defense of the gills, foot muscle and digestive glands. Upregulation of superoxide dismutase occurred in the three tissues by warming. The foot muscle catalase and the gill glutathione reductase were upregulated only by diesel. The inability to upregulate catalase and glutathione S-transferase in the digestive gland, as well as the increase of lipoperoxidation, suggested that this gland is more susceptible to the deleterious effects from oxidative stress., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Detecting impacts of organophosphorus biocides (OP) is facilitated by analysing "biomarkers" - biological responses to environmental insults. Understanding is hampered by studying biomarkers in isolation at different levels of biological response and limited work on ecologically-important species. We tested the relevance of esterases as biomarkers of OP-exposure in limpets (Patella vulgata), abundant prosobranchs that structure the assemblages on rocky shores through their grazing. We characterized esterases in haemolymph and tissue, and quantified their dose-dependent inhibition by chlorfenvinphos (0.1-3.0 mM) in vitro. To determine whether esterases are useful biomarkers we exposed limpets to chlorfenvinphos (0-10 μg L(-1)). Despite reduced tenacity (ability to stick to a surface) and haemocyte-viability, esterases remained unaffected. Tenacity was reduced by >50% at 5 μg L(-1) and by 95% at 10 μg L(-1), whilst haemocyte-viability was more sensitive with >40% reductions at concentrations of 0.5 μg L(-1) and above. We discuss results in relation to linking sub-lethal and ecological impacts at contaminated sites., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Increasing temperatures can be a significant stressor for aquatic organisms. Abalones, a type of large marine gastropods, are the most commercially important species in aquaculture for Asia. To evaluate the potential ecological risk posed by temperature stress, we measured biological responses such as survival rate, adhesion ability (falling rate), and foot abnormalities in the abalone Haliotis discus hannai. Additionally, biochemical and molecular responses were evaluated in H. discus hannai exposed to various temperature gradients. The survival rate was reduced in abalones exposed to relative high temperatures (more than 26 °C). Increased temperature stress induced a higher falling rate and abnormal foot structure. Furthermore, increased antioxidant enzyme activities were observed in abalones exposed to relative high temperatures (26 and 28 °C). The activities of superoxide dismutase were induced in a time-dependent manner after high temperature stress. Generally, heat shock protein 90 also increased significantly in H. discus hannai exposed to temperature gradients (more than 24 °C) for 12 h. These results provide valuable information regarding stress responses to increased temperatures, in H. discus hannai: adverse biological and molecular outcomes could be utilized as risk assessments and stress monitoring of marine ecosystems under increased water temperatures., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The level of cellulolytic activity in different areas of the gut of the terrestrial slug Arion ater was assayed at different temperatures and pH values. To do this, crude gut proteins were isolated and assayed using modified dinitrosalicylic acid reducing sugar assay. Crude protein samples were also separated and cellulolytic activity identified using in gel CMC zymography and esculin hydrate activity gel assays. pH and temperature profiling revealed optimum cellulolytic activity between pH5.0 and 6.0 for different gut regions and retention of up to 90% of activity at temperatures up to 50°C. Zymograms and activity gels revealed multiple endoglucanase and β-glucosidase enzymes. To further investigate the source of this cellulolytic activity bacterial isolates from the gut were tested for endoglucanase and β-glucosidase activity using growth plate assays. 12 cellulolytic microbes were identified using 16S rDNA gene sequencing. These include members of the genera Buttiauxella, Enterobacter, Citrobacter, Serratia and Klebsiella. Gut metagenomic DNA was then subjected to PCR, targeting a 400bp region of the 16SrDNA gene which was subsequently separated and individuals identified using DGGE. This identified members of the genera Citrobacter, Serratia, Pectobacterium, Acinetobacter, Mycoplasma, Pantoea and Erwinia. In summary, multiple glycoside hydrolase enzymes active over a broad range of temperature and pH values in a relatively under studied organism were detected, indicating that the gut of A. ater is a viable target for intensive study to identify novel carbohydrate active enzymes that may be used in the biofuel industry., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Pharmaceutical compounds like antidepressants found in surface waters raise concerns due to their potential toxicity on non-target aquatic organisms. This study aimed at investigating the in vitro cytotoxicity and immunomodulatory properties of four common antidepressants, namely Amitriptyline, Clomipramine, Citalopram and Paroxetine, on primary cultures of abalone hemocytes (Haliotis tuberculata), after 48 h-exposure. Effects on immunocompetence (phagocytosis, levels of reactive oxygen species, esterase activity and lysosomal membrane destabilization) were assessed. Results obtained by MTT assays revealed that acute toxicity is unlikely to occur in the environment since the LC50s of the four antidepressants are at the mg/L level. The different immunological endpoints displayed a biphasic response, with an increase at the lowest concentration (i.e. 1 μg/L) followed by a decrease at higher concentrations. Overall, Amitriptyline and Clomipramine, the two tricyclic antidepressants, had higher immunomodulatory capacities than the two selective serotonin reuptake inhibitors Citalopram and Paroxetine. Amitriptyline was the most potent and Citalopram the least potent drug in altering immune function in H. tuberculata., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study examined the potential of abalone β-glucuronidase as a viable and cost effective alternative to current hydrolysis procedures using acid, Helix pomatia β-glucuronidase and Escherichia coli β-glucuronidase. Abalone β-glucuronidase successfully hydrolyzed oxazepam-glucuronide and lorazepam-glucuronide within 5% of the spiked control concentration. Benzodiazepines present in authentic urine specimens were within 20% of the concentrations obtained with the current hydrolysis procedure using H. pomatia β-glucuronidase. JWH 018 N-(5-hydroxypentyl) β-d-glucuronide was hydrolyzed within 10% of the control concentration. Authentic urine specimens showed improved glucuronide cleavage using abalone β-glucuronidase with up to an 85% increase of drug concentration, compared with the results obtained using E. coli β-glucuronidase. The JWH 018 and JWH 073 carboxylic acid metabolites also showed increased drug concentrations of up to 24%. Abalone β-glucuronidase was able to completely hydrolyze a morphine-3-glucuronide control, but only 82% of total morphine was hydrolyzed in authentic urine specimens compared with acid hydrolysis results. Hydrolysis of codeine and hydromorphone varied between specimens, suggesting that abalone β-glucuronidase may not be as efficient in hydrolyzing the glucuronide linkages in opioid compounds compared with acid hydrolysis. Abalone β-glucuronidase demonstrates effectiveness as a low cost option for enzyme hydrolysis of benzodiazepines and synthetic cannabinoids.

Pollution of the aquatic environment by petroleum and its products is common the world over. This study is aimed at examining sublethal effects of petrol and diesel on enzymes in Tympanotonus fuscatus namely: Aspartate Transaminase (AST) (E.C. 2.6.1.1), Alanine Transaminase (ALT) (E.C. 2.6.2.2) and Alkaline Phosphatase (ALP) (E.C. 3.1.3.1) activity after exposure. The periwinkles were exposed to 10.40, 15.60, 21.00, 26.00 ml L(-1) and a control. The organs were removed on the sixth day and were prepared for enzymatic analysis. Enzyme activities were compared to the control value and between the toxicants. The effects of the toxicants on AST activity in the muscle and viscera were significantly different (p > 0.05) from the control value (137.50 ± 15.10 IU L(-1)). AST activity were raised more in petrol concentrations than the diesel concentrations in the muscle. The reverse was the case in the viscera at 15.60 ml L(-1) (227.50 ± 24.75 IU L(-1)). ALT activity in the muscle were not significant (p > 0.05) between the toxicant media. In the viscera, significant differences (p > 0.05) were observed in some of the concentrations with petrol showing higher activity. ALP activity in the muscle were not significant (p > 0.05) in both media, but were more elicited in the diesel concentrations. In the viscera, the activities of ALP were more pronounced in the petrol concentrations and were significant (p > 0.05) at the higher diesel concentrations. The exposure of Tympanotonus fuscatus to petrol and diesel concentrations caused changes in the enzymatic activities in the organism with those of petrol more pronounced than those of the diesel.

Our objective was to determine if the Lake Baikal endemic gastropod Benedictia limnaeoides ongurensis, which inhabits in stable cold waters expresses a thermal stress response. We hypothesized that the evolution of this species in the stable cold waters of Lake Baikal resulted in a reduction of its thermal stress-response mechanisms at the biochemical and cellular levels. Contrary to our hypothesis, our results show that exposure to a thermal challenge activates the cellular and biochemical mechanisms of thermal resistance, such as heat shock proteins and antioxidative enzymes, and alters energetic metabolism in B. limnaeoides ongurensis. Thermal stress caused the elevation of heat shock protein 70 and the products of anaerobic glycolysis together with the depletion of glucose and phosphagens in the studied species. Thus, a temperature increase activates the complex biochemical system of stress response and alters the energetic metabolism in this endemic Baikal gastropod. It is concluded that the deepwater Lake Baikal endemic gastropod B. limnaeoides ongurensis retains the ability to activate well-developed biochemical stress-response mechanisms when exposed to a thermal challenge., (© 2013.)

In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 μg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 μg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 μg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 μg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 μg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hexokinase from the hepatopancreas and foot muscle of Littorina littorea undergoes stable modification of its kinetic and structural properties in response to prolonged oxygen deprivation. In the hepatopancreas, a reduction in the Km glucose for hexokinase from the anoxic animal suggests a more active enzyme form during anoxia. Conversely, in the foot muscle, an increase in Km ATP and a decrease in Vmax for anoxic snail hexokinase were consistent with a less active enzyme form during anoxia. In either case, the molecular basis for the stable modification of hexokinase kinetics is reversible phosphorylation. The activation of endogenous PKC and AMPK increased the Km glucose for anoxic hepatopancreas hexokinase to a value that was similar to the control Km glucose. Alternatively, stimulation of endogenous PKA, PKG, and CamK for control foot muscle hexokinase increased the Km ATP to a value similar to that seen for the anoxic enzyme form. In both tissues, activation of endogenous phosphatases reversed the effects of protein kinases. Dephosphorylation and activation of hepatopancreas hexokinase during anoxia may allow for increased shunting of glucose-6-phosphate into the pentose phosphate pathway, thereby producing reducing equivalents of NADPH needed for antioxidant defense upon tissue re-oxygenation. Conversely, phosphorylation and inhibition of foot muscle hexokinase during anoxia may reflect the decreased need for glucose oxidation during hypometabolism., (© 2013 Elsevier Inc. All rights reserved.)

Previously, we isolated two α-amylase isozymes, HdAmy58 and HdAmy82, from the digestive fluid of the Pacific abalone Haliotis discus hannai (Kumagai et al., 2013, Comp. Biochem. Physiol., B 164, 80-88). These enzymes degraded starch producing maltooligosaccharides but not glucose. However, the digestive fluid itself could produce glucose from starch, indicating that the digestive fluid contains α-glucosidase-like enzymes together with the α-amylases. Thus, in the present study, we isolated this α-glucosidase-like enzyme from the digestive fluid and characterized it to some extent. Isolation of this enzyme was carried out by ammonium sulfate fractionation followed by conventional column chromatographies and FPLC. The purified enzyme showed an apparent molecular mass of 97kDa on SDS-PAGE, and optimal temperature and pH of 45°C and 3.8-5.5, respectively. This enzyme could degrade various sizes of maltooligosaccharides into glucose and released glucose from starch producing no appreciable intermediate oligosaccharides. We concluded that this enzyme is an α-glucosidase (EC 3.2.1.20) exolitically acting on polymer substrate and named HdAgl. HdAgl efficiently degraded maltose but hardly degraded p-nitrophenyl α-D-glucopyranoside (α-pNPG) and isomaltose. This enzyme was regarded as a maltase-like α-glucosidase that preferably degrades maltose but scarcely aryl glucosides. When starch was used as a substrate, HdAgl converted approximately 40% (w/w) of the starch to glucose. If an abalone α-amlylase HdAmy58 was added to the reaction mixture, the glucose yield increased to 60% (w/w). These results suggested that both HdAgl and HdAmy58 play important roles for the production of glucose from dietary starch in the digestive fluid. The amino-acid sequence of 887 residues for HdAgl was deduced by the cDNA method. This sequence showed 41-46% amino-acid identities to those of mammalian and avian α-glucosidases belonging to glycoside-hydrolase-family31., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chlorpyrifos is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. In recent years, there has been an important decrease in the number of organisms of Planorbarius corneus. Since the presence of pesticides in the water can be one of the reasons for this decrease, it is very important to study the effect of subchronic exposure to environmental concentrations of pesticides on these organisms. The aim of the present work was to investigate different effects of the subchronic exposure to low concentrations of the organophosphate chlorpyrifos in P. corneus and the possibility to use these as biomarkers. To this end, we have exposed the organisms to 0.4 and 5 μg L(-1) of chlorpyrifos for 14 days and recorded the number of egg masses, the number of eggs per mass, the number of eggs without embryo, the time for hatching, and the % of hatching and survival. We have also determined the activities of cholinesterases, carboxylesterases and glutathione S-transferase in whole organism soft tissue and in the gonads. A 14 days exposure to 0.4 μg L(-1) caused an increase in the number of egg masses without eggs and a decrease in carboxylesterases measured with p-nitrophenyl butyrate. However the exposure to 5 μg L(-1) also caused an increase in the time for hatching, a decrease in the % of hatching and survival and also inhibition of cholinesterases and carboxylesterases with p-nitrophenyl acetate and butyrate. In contrast, the glutathione S-transferase has not been modified with the tested concentrations. We concluded that when P. corneus exposed to chlorpyrifos for 14 days, the CES determined with p-nitrophenyl butyrate proved to be the most sensitive biomarker. However, exposure to environmental concentrations showed a decrease in the reproduction ability which could cause a decrease in the number of organisms of this species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

HSD17B12 is a member of the hydroxysteroid dehydrogenase superfamily, a multifunctional group of enzymes involved in the metabolism of steroids, retinoids, bile and fatty acids. Whether the main role of HSD17B12 in mammals is in steroid or fatty acid metabolism is a subject of intense debate. In mollusks it has been shown that an HSD17B12 orthologue can convert estrone into estradiol in vitro, although its primary in vivo function remains unknown. To gain insight into its role in gastropods, we provide here the first cloning of Hsd17b12 in Nucella lapillus and its detailed tissue distribution through quantitative PCR. Furthermore, given that the endocrine disruptor tributyltin (TBT) has been reported to unbalance steroid and lipid levels in gastropods, we tested its impact in on NlHsd17b12 transcript expression. Our results show that NlHsd17b12 is ubiquitously expressed in all tissues analyzed, with higher levels in organs with high metabolic rates, such as kidney and digestive gland, a pattern consistent with an involvement in lipid metabolism. Exposure to TBT chloride at 100 ng Sn/L caused a decrease in NlHsd17b12 mRNA levels in digestive gland, after one and two months, while no effect was observed in gonads. Overall, these results suggest that in mollusks, as in mammals, this enzyme is likely to be involved in lipid metabolism, and emphasize the need to perform more detailed studies on its in vivo function, in order to understand its physiological role and the biological impact of its disruption by pollutants such as TBT., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Two α-amylase (EC 3.2.1.1) isozymes, HdAmy58 and HdAmy82, with approximate molecular masses of 58 kDa and 82 kDa, respectively, were isolated from the digestive fluid of the Pacific abalone Haliotis discus hannai. Optimal temperatures and pHs for HdAmy58 and HdAmy82 were at 30 °C and 6.7, and 30 °C and 6.1, respectively. Both enzymes similarly degraded starch, glycogen, and maltooligosaccharides larger than maltotriose producing maltose and maltotriose as the major degradation products. However, the activity toward maltotetraose was appreciably higher in HdAmy82 than HdAmy58. cDNAs encoding HdAmy58 and HdAmy82 were cloned and the amino-acid sequences of 511 and 694 residues for HdAmy58 and HdAmy82, respectively, were deduced. The putative catalytic domains of HdAmy58 and HdAmy82 were located in the 17-511th and 19-500th amino-acid regions, respectively, and they showed approximately 50% amino-acid identity to each other. These sequences also showed 62-99% amino-acid identity to the catalytic domains of known α-amylases that belong to glycoside-hydrolase-family 13. The difference in the molecular masses between HdAmy58 and HdAmy82 was ascribed to the extension of approximately 190 residues in the C-terminus of HdAmy82. This extended region showed 41-63% amino-acid identity with the ancillary domains of several α-amylases previously reported., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Marine invertebrates have evolved multiple responses to naturally variable environmental oxygen, all aimed at either maintaining cellular oxygen homeostasis or limiting cellular damage during or after hypoxic or hyperoxic events. We assessed organismal (rates of oxygen consumption and ammonia excretion) and cellular (heat shock protein expression, anti-oxidant enzymes) responses of juvenile and adult abalone exposed to low (~83% of saturation), intermediate (~95% of saturation) and high (~115% of saturation) oxygen levels for one month. Using the Comet assay, we measured DNA damage to determine whether the observed trends in the protective responses were sufficient to prevent oxidative damage to cells. Juveniles were unaffected by moderately hypoxic and hyperoxic conditions. Elevated basal rates of superoxide dismutase, glutathione peroxidase and catalase were sufficient to prevent DNA fragmentation and protein damage. Adults, with their lower basal rate of anti-oxidant enzymes, had increased DNA damage under hypoxic and hyperoxic conditions, indicating that the antioxidant enzymes were unable to prevent oxidative damage under hypoxic and hyperoxic conditions. The apparent insensitivity of juvenile abalone to decreased and increased oxygen might be related to their life history and development in algal and diatom biofilms where they are exposed to extreme diurnal fluctuations in dissolved oxygen levels., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Strombus gigas and Strombus pugilis are threatened species and aquaculture represents a good alternative solution to the fishing. In this study, we highlighted the intracellular digestion process in the digestive gland of two Strombidae species, S. gigas and Strombuspugilis, by the cytochemical characterization of two lysosomal enzymes: acid phosphatase and arylsulfatase. In order to check the efficiency of artificial food digestion, we conducted the characterization on freshly collected, starved and artificially fed individuals of S. pugilis. TEM observations of digestive gland sections from freshly collected individuals of both species revealed the presence of acid phosphatase and arylsulfatase activity mostly located in the apical third of digestive cells. Both enzymes were also detected in artificially fed individuals. In response to the starvation, acid phosphatase is not produced anymore by digestive cells, while arylsulfatase is still present. To our knowledge, this is the first cytochemical validation of intracellular digestion of artificial food in Strombidae. This study highlights the intracellular digestion of artificial food developed for Strombidae aquaculture. Moreover, we have shown that the lysosomal activity could be used as a feed index., (Copyright © 2012 Wiley Periodicals, Inc.)

Carbonic anhydrases (CAs) represent a diversified family of metalloenzymes that reversibly catalyze the hydration of carbon dioxide. They are involved in a wide range of functions, among which is the formation of CaCO(3) skeletons in metazoans. In the shell-forming mantle tissues of mollusks, the location of the CA catalytic activity is elusive and gives birth to contradicting views. In the present paper, using the European abalone Haliotis tuberculata, a key model gastropod in biomineralization studies, we identified and characterized two CAs (htCA1 and htCA2) that are specific of the shell-forming mantle tissue. We analyzed them in a phylogenetic context. Combining various approaches, including proteomics, activity tests, and in silico analyses, we showed that htCA1 is secreted but is not incorporated in the organic matrix of the abalone shell and that htCA2 is transmembrane. Together with previous studies dealing with molluskan CAs, our findings suggest two possible modes of action for shell mineralization: the first mode applies to, for example, the bivalves Unio pictorum and Pinctada fucata, and involves a true CA activity in their shell matrix; the second mode corresponds to, for example, the European abalone, and does not include CA activity in the shell matrix. Our work provides new insight on the diversity of the extracellular macromolecular tools used for shell biomineralization study in mollusks., (© 2012 WILEY PERIODICALS, INC.)

L-amino acid oxidase is widely found in diverse organisms and has different properties. It is thought to contribute to antimicrobial activity, amino acid catabolism, and so forth. The purpose of this communication is to summarize the advances in non-snake venom L-amino acid oxidase, including its enzymatic and structural properties, gene cloning and expression, and biological function. In addition, the mechanism of its biological function as well as its application is also discussed.

The 17-beta-hydroxysteroid dehydrogenases (17β-HSDs) are key enzymes for sex steroid biosynthesis. To date, relatively little is known about the presence and function of 17β-HSDs in marine gastropods. In the present study, a cDNA sequence encoding putative 17β-HSD type 11 (17β-HSD-11) was identified in marine abalone (Haliotis diversicolor supertexta). The full-length cDNA contains 1058bp, including an open reading frame (ORF) of 900bp that encodes a protein of 299 amino acids. Comparative structural analysis revealed that abalone 17β-HSD-11 shares relatively high homology with other 17b-HSD-11 hormologues, and a lesser degree of amino acid identity with other forms of 17b-HSD, especially in the functional domains, including the cofactor binding domain (TGxxxGxG) and catalytic site (YxxSK). Phylogenetic analysis showed that abalone 17β-HSD-11 belongs to the short-chain dehydrogenase/reductase (SDR) family. Functional analysis following transient transfection of the ORF into human embryonic kidney-293 (HEK-293) cells indicated that abalone 17β-HSD-11 has the ability to convert 5α-androstane-3α,17β-diol (3α-diol) to androsterone (A) and testosterone (T) to androstenedione (4A). Expression analysis in vivo demonstrated that abalone 17β-HSD-11 is differentially expressed during three stages (non-reproductive, reproductive, and post-reproductive). Taken together, these results indicate that ab-17β-HSD-11 is an SDR family member with a potential role in steroid regulation during the reproductive stage., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Among metals, cadmium, a non-essential element, is an important pollutant that is released into aquatic environments. Due to its persistence and bioaccumulation, this metal has been shown to exert immunological effects on organisms. The objective of the present study was to investigate the in vitro effects of cadmium chloride using a haemocyte primary culture from the European abalone, Haliotis tuberculata. Most studies have maintained viable haemocytes in vitro for periods ranging from several hours to several days during acute exposures. Few investigations have reported the effects of metals using longer in vitro exposures, which are more realistic with regard to mimicking environmental conditions. In this study, we exposed abalone haemocytes to concentrations from 0.5 to 50,000 μgL(-1) of CdCl2 for 10 days. The effects of cadmium chloride were reflected in a significant decrease in the number of viable cells and morphological modifications in a concentration-dependent manner beginning at a concentration of 500 μgL(-1) as well as in some physiological processes, such as phagocytotic activity and the number of lysosome-positive cells. In contrast, phenoloxidase (PO) activity and reactive oxygen species (ROS) production were increased beginning at a concentration of 5 μgL(-1), which is consistent with environmental concentrations in polluted sites. For PO activity and ROS production, maximally 9-fold and 130% inductions, respectively, were recorded under the highest dose. These results thus indicate that cadmium chloride alters immune parameters of abalone haemocytes and that the long-term (10 days) primary culture system used here represents a suitable, sensitive in vitro model for assessing cytotoxic responses., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nitric oxide synthase (NOS) has been characterized in several opistobranchs and pulmonates but it was much less investigated in prosobranchs, which include more than 20,000 species and account for most of the gastropod diversity. Therefore, new data from this large group are needed for a better knowledge of the molecular evolution of NOS enzymes in molluscs. This study focused on NOS expressed in the nervous system of the prosobranch neogastropod Stramonita haemastoma. In this study we report compelling evidence on the expression of a constitutive Ca(2+) /CaM-dependent neuronal NOS in the central and peripheral nervous system. The prevailing neuronal localization of NADPHd activity was demonstrated by NADPHd histochemistry in both central and peripheral nervous system structures. L-arginine/citrulline assays suggested that Stramonita NOS is a constitutive enzyme which is both cytosolic and membrane-bound. Molecular cloning of the full-length Stramonita NOS (Sh-NOS) by reverse-transcription polymerase chain reaction (RT-PCR) followed by 5' and 3' RACE showed that Sh-NOS is a protein of 1,517 amino acids, containing a PDZ domain at the N-terminus and sharing similar regulatory domains to the mammalian neuronal NOS (nNOS). Regional expression of the Sh-NOS gene was evaluated by RT-PCR. This analysis showed different expression levels in the nerve ring, the osphradium, the cephalic tentacles, the buccal tissues, and the foot, whereas NOS expression was not found in the salivary glands and the gland of Leiblein. The present data provide a solid background for further studies addressing the specific functions of NO in neogastropods., (Copyright © 2011 Wiley-Liss, Inc.)

Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The esophagus and intestine form the longest regions of the digestive tract in the giant keyhole limpet and are lined by epithelial cells sharing a common morphology and releasing materials into the gut lumen by apocrine secretion. The purpose of this study was to determine if these morphologically similar regions release similar digestive enzymes and compare their contributions to digestive enzymes released from other regions of the gut. Principal component analysis of enzymes detected by the API ZYM system for 19 enzymes plus EnzChek assays for protease, α-amylase, lipase, cellulase, and lysozyme identify four distinct regions of the gut: 1) crystalline style and style sac, 2) digestive gland, 3) salivary glands, and 4) esophagus and intestine. Heterogeneity in enzymatic activity was observed in regions of the gut with similar cell morphology (middle and posterior esophagus and intestine) as well as regions with different cell morphology (salivary glands, digestive gland and crystalline style). Enzyme activity in each of these regions is compared to other gastropods, in particular the abalone. Although much of the length of the digestive tract is lined by a morphologically similar epithelium, different regions of the alimentary tract produce a different suite of enzymes which may contribute to the digestive process. These data will help enhance our limited understanding of the digestive physiology of Megathura crenulata and lead to improvement of its culture for clinical research., (Copyright © 2011 Elsevier Inc. All rights reserved.)