In vitro effects of glyphosate-based herbicides and related adjuvants on primary culture of hemocytes from Haliotis tuberculata.

Glyphosate-based herbicides are among the most produced and widely-used herbicides. Studies have shown that commercial formulations and adjuvants may be more toxic to non-target organisms than the active ingredients alone, but the mechanisms of action of these chemicals remain unclear. The aim of this study was to investigate the in vitro effects of glyphosate, a commercial formulation and adjuvant alone using primary culture of hemocytes from the European abalone Haliotis tuberculata, a commonly farmed shellfish. Glyphosate was found to have negligible effects on viability, phagocytic activities and lysosome stability even with very high doses (i.e. 100 mg L-1). By contrast, greater effects on viability were observed for the commercial formulation and adjuvant alone, with EC50 values of 41.42 mg L-1 and 1.85 mg L-1, respectively. These results demonstrate that the toxic sublethal effects (i.e. phagocytic activity and destabilization of lysosomal membranes) of formulated glyphosate came from adjuvants and suggest they may be related to cell and organelle membrane destabilization.

Molluscicidal activity and physiological toxicity of quaternary benzo[c]phenanthridine alkaloids (QBAs) from Macleaya cordata fruits on Oncomelania hupensis.

Schistosomiasis is a serious worldwide parasitic disease. One of the best ways to control schistosomiasis is to control the population of Oncomelania hupensis snails. We sought to identify a high-efficiency biogenic molluscicide against Oncomelania with low toxicity, to avoid chemical molluscicide contamination and toxicity in aquatic organisms. We extracted quaternary benzo[c]phenanthridine alkaloids (QBAs) from Macleaya cordata fruits. Molluscicidal activity of the QBAs against Oncomelania was determined using bioassay. Our results showed that the extracted QBAs had a strong molluscicidal effect. In treatment of O. hupensis with QBAs for 48 h and 72 h, the lethal concentration (LC50) was 2.89 mg/L and 1.29 mg/L, respectively. The molluscicidal activity of QBAs was close to that of niclosamide (ethanolamine salt), indicating that QBAs have potential development value as novel biogenic molluscicides. We also analyzed physiological toxicity mechanisms by examining the activity of several important detoxification enzymes. We measured the effect of the extracted QBAs on the activities of glutathione S-transferase (GST), carboxylesterase (CarE), acid phosphatase (ACP), and alkaline phosphatase (AKP) in the liver of O. hupensis. We found that the effects of QBAs on detoxification metabolism in O. hupensis were time and concentration dependent. The activities of GST, CarE, AKP, and ACP in the liver of snails increased significantly in the early stage of treatment (24 h), but decreased sharply in later stages (120 h), compared with these activities in controls. GST, CarE, AKP, and ACP activity in the liver of snails treated with LC50 QBAs for 120 h decreased by 62.3%, 78.1%, 59.2%, and 68.6%, respectively. Our results indicate that these enzymes were seriously inhibited by the extracted QBAs and the detoxification and metabolic functions of the liver gradually weakened, leading to poisoning, which could be the main cause of death in O. hupensis snails.

Mannan oligosaccharide increases the growth performance, immunity and resistance capability against Vibro Parahemolyticus in juvenile abalone Haliotis discus hannai Ino.

This trial was conducted to investigate the effect of mannose oligosaccharides (MOS) on the growth performance, antioxidation, immunity and disease resistance of Vibro Parahemolyticus in juvenile abalone Haliotis discus hannai Ino. Four formulated diets were produced to contain 0.00 g/kg, 0.40 g/kg, 0.80 g/kg and 1.60 g/kg Actigen®, with functional ingredients of MOS, respectively. Accordingly, the experimental diets were named as A0, A4, A8 and A16. After 120-days feeding trial, the best growth performance was observed in A8 group (P < 0.05) and there was no significant difference in A0, A4 and A16 groups. With the increase of dietary MOS, the activity of the total antioxidant capacity in hepatopancreas is increasingly elevated (P < 0.05) while no significant difference was observed on activity of glutathione S-transferase (P > 0.05). The activities of superoxide dismutase and glutathione peroxidase were firstly increased and then decreased, with the highest values in A8 group (P < 0.05). Immune-related parameters were significantly affected by dietary MOS inclusion. Specifically, the activities of alkaline phosphatase and acid phosphatase in hepatopancreas and serum of abalone fed diets containing MOS were significantly higher than those of control A0 group (P < 0.05). Moreover, the highest values of both enzymes were observed in hepatopancreas of A8 group but in serum of A16 group, respectively. The lysozyme activities in hepatopancreas and serum of A4 group were significantly higher than those of other groups (P < 0.05) and there was no significant difference in A0, A8 and A16 groups (P > 0.05). The activities of cytophagy and respiratory burst in serum of abalone were not significantly affected by dietary MOS content (P > 0.05). The mRNA levels of focal adhesion kinase and integrin-linked kinase were gradually elevated with the increase of dietary MOS, with the highest value recorded in A16 group (P < 0.05). The gene expression of caspse-3 in A8 group was dramatically higher than those of other groups (P < 0.05) and there was no significant difference in A0, A4 and A16 groups (P > 0.05). The mRNA level of nuclear factor-κB was not significantly affected by dietary MOS (P > 0.05). During 56 h of V. Parahemolyticus challenge period, the accumulated mortality rate of abalone fed diets containing MOS were significantly lower than that of control A0 group in each time point (P < 0.05). Overall, the lowest rate was happened in A8 group (P < 0.05). In conclusion, MOS inclusion in diet has obviously positive effect on growth, immunity and disease resistance capability of abalone, with the optimal level of Actigen® at 0.80 g/kg in diet.

Protection of dietary selenium-enriched seaweed Gracilaria lemaneiformis against cadmium toxicity to abalone Haliotis discus hannai.

Seaweed Gracilaria lemaneiformis is the main dietary source of the abalone mariculture industry in China. In this study, we examined the protection of selenium (Se)-enriched G. lemaneiformis against cadmium (Cd) toxicity in the abalone, Haliotis discus hannai, using various indices including metal concentration (Se and Cd), growth rate, GPx enzymatic antioxidants, and metallothionein (MT) concentration over a period of 28 days of exposure. The growth rates and Se contents increased significantly in abalones fed with Se-enriched G. lemaneiformis, while the toxicity of Cd was reduced. Seven to 12 days Cd exposure to the Se-enriched G. lemaneiformis not only affected GPx activity but the MT levels fluctuated irregularly. MT concentrations increased after 3 days exposure and then gradually decreased to the control level after Day 7. There were statistically significant positive correlations between MT levels, GPx activity and Se concentrations, and negative relationships between MT levels, GPx activity and Cd levels in abalones. These findings suggest that Se-enriched Gracilaria protects abalone against Cd toxicity. The possible mechanism is the induction of MT with a concomitant increased capacity of GPx enzymatic antioxidants.

Gastropods and Insects Prefer Different Solanum dulcamara Chemotypes.

Solanum dulcamara (Bittersweet nightshade) shows significant intraspecific variation in glycoalkaloid (GA) composition and concentration. We previously showed that constitutive differences in overall GA levels are correlated with feeding preference of the grey field slug (GFS; Deroceras reticulatum). One particularly preferred accession, ZD11, contained low GA levels, but high levels of previously unknown structurally related uronic acid conjugated compounds (UACs). Here we test whether different slug species as well as insect herbivores show similar feeding preferences among six S. dulcamara accessions with different GA chemotypes. In addition, we investigate whether slug feeding can lead to induced changes in the chemical composition and affect later arriving herbivores. A leaf disc assay using greenhouse-grown plants showed that three slug species similarly preferred accessions with low GA levels. Untargeted metabolomic analyses showed that previous slug feeding consistently increased the levels of N-caffeoyl-putrescine and a structurally related metabolite, but not the levels of GAs and UACs. Slug-induced responses only affected slug preference in one accession. A common garden experiment using the same six accessions revealed that ZD11 received the highest natural gastropod feeding damage, but suffered the lowest damage by specialist flea beetles. The latter preferred to feed on accessions with high GA levels. Our study indicates that different selection pressures imposed by generalist gastropods and specialist insects may explain part of the observed chemical diversity in S. dulcamara.

Assessing the chronic toxicity of nickel to a tropical marine gastropod and two crustaceans.

The mining and processing of nickel ores from tropical regions contributes 40% of the global supply. The potential impact of these activities on tropical marine ecosystems is poorly understood. Due to the lack of ecotoxicity data for tropical marine species, there is currently no available water quality guideline value for nickel that is specific to tropical species. In this study, we investigated the toxicity of nickel to three tropical marine invertebrates, the gastropod Nassarius dorsatus, the barnacle Amphibalanus amphitrite, and the copepod Acartia sinjiensis. All toxicity tests used chronic endpoints, namely larval growth, metamorphosis (transition from nauplii to cyprid larvae) and larval development for the snail, barnacle and copepod respectively. Toxicity tests were carried out under environmentally relevant conditions (i.e. 27-30ᵒC, salinity 34-36‰, pH 8.1-8.4). Copper was also tested for quality assurance purposes and to allow for comparisons with previous studies. The copepod was the most sensitive species to nickel, with development inhibited by 10% (EC10) at 5.5 (5.0-6.0) µg Ni/L (95% confidence limits (CL)). Based on EC10 values, the gastropod and barnacle showed similar sensitivities to nickel with growth and metamorphosis inhibited by 10% at 64 (37-91) µg Ni/L and 67 (53-80) µg Ni/L, respectively. Based on existing data available in the literature, the copepod A. sinjiensis is so far the most sensitive tropical marine species to nickel. This study has provided high quality data which will contribute to the development of a water quality guideline value for nickel in tropical marine waters. A species sensitivity distribution of chronic nickel toxicity used the data generated in this paper supplemented by available literature data, comprising 12 species representing 6 taxonomic groups. A 5% hazard concentration (HC5) was determined as 8.2 µg/L Ni.

Antioxidant responses in estuarine invertebrates exposed to repeated oil spills: Effects of frequency and dosage in a field manipulative experiment.

We have experimentally investigated the effects of repeated diesel spills on the bivalve Anomalocardia brasiliana, the gastropod Neritina virginea and the polychaete Laeonereis culveri, by monitoring the responses of oxidative stress biomarkers in a subtropical estuary. Three frequencies of exposure events were compared against two dosages of oil in a factorial experiment with asymmetrical controls. Hypotheses were tested to distinguish between (i) the overall effect of oil spills, (ii) the effect of diesel dosage via different exposure regimes, and (iii) the effect of time since last spill. Antioxidant defense responses and oxidative damage in the bivalve A. brasiliana and the polychaete L. culveri were overall significantly affected by frequent oil spills compared to undisturbed controls. The main effects of diesel spills on both species were the induction of SOD and GST activities, a significant increase in LPO levels and a decrease in GSH concentration. N. virginea was particularly tolerant to oil exposure, with the exception of a significant GSH depletion. Overall, enzymatic activities and oxidative damage in A. brasiliana and L. culveri were induced by frequent low-dosage spills compared to infrequent high-dosage spills, although the opposite pattern was observed for N. virginea antioxidant responses. Antioxidant responses in A. brasiliana and L. culveri were not affected by timing of exposure events. However, our results revealed that N. virginea might have a delayed response to acute high-dosage exposure. Experimental in situ simulations of oil exposure events with varying frequencies and intensities provide a useful tool for detecting and quantifying environmental impacts. In general, antioxidant biomarkers were induced by frequent low-dosage exposures compared to infrequent high-dosage ones. The bivalve A. brasiliana and the polychaete L. culveri are more suitable sentinels due to their greater responsiveness to oil and also to their wider geographical distribution.

Fe, oxidative and nitrosative metabolism in the Antarctic limpet Nacella concinna.

The hypothesis of this work was that oxidative and nitrosative metabolism in the digestive gland (DG) of two limpet populations (intertidal and subtidal) of the Antarctic species Nacella concinna show different behavior when they were exposed to either intermittent (intertidal) or constant (subtidal) natural Fe. Total Fe content and labile Fe pool were higher in the DG of the subtidal compared to the intertidal population. However, no significant differences between populations were seen on the Fe atoms content of the isolated ferritin. Ascorbyl radical content was 2.0±0.4 and 6.5±0.8pmol/mg FW in the DG of the intertidal and subtidal animals, respectively. Lipid damage, assessed as content of thiobarbituric reactive substances, was different between the tissues of intertidal and subtidal samples, 491±102 and 1242±367pmol/mg FW, respectively. Catalase and superoxide dismutase activities showed no differences between the limpets. Nitric oxide (NO) content was 25±3 and 22±2pmol/mg FW in DG from intertidal and subtidal animals, respectively. NO synthase-like (NOS-like) activity was evaluated supplementing the samples with the enzyme co-factors, and the inhibitory effect of Nω-nitro-L-arginine methyl ester hydrochloride was tested. NO generation rate was 3.4±0.3 and 4.7±0.6pmol/minmg FW in DG from the intertidal and subtidal population, respectively. These results showed that the oxidative condition of the limpet population constantly covered by the Fe enriched water is more affected than the intertidal population. However, the nitrosative metabolism seems to be independent of the environmental high Fe content since similar NO steady state concentration and NOS-like activity were measured in both populations.

Dietary ascorbic acid modulates the expression profile of stress protein genes in hepatopancreas of adult Pacific abalone Haliotis discus hannai Ino.

This study was conducted to investigate the effects of dietary ascorbic acid (AA) on transcriptional expression patterns of antioxidant proteins, heat shock proteins (HSP) and nuclear factor kappa B (NF-κB) in the hepatopancreas of Pacific abalone Haliotis discus hannai Ino (initial average length: 84.36 ± 0.24 mm) using real-time quantitative PCR assays. L-ascorbyl-2-molyphosphate (LAMP) was added to the basal diet to formulate four experimental diets containing 0.0, 70.3, 829.8 and 4967.5 mg AA equivalent kg(-1) diets, respectively. Each diet was fed to triplicate groups of adult abalone in acrylic tanks (200 L) in a flow-through seawater system. Each tank was stocked with 15 abalone. Animals were fed once daily (17:00) to apparent satiation for 24 weeks. The results showed that the dietary AA (70.3 mg kg(-1)) could significantly up-regulate the expression levels of Cu/Zn superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), feritin (FT) and heat shock protein 26 (HSP26) in the hepatopancreas of abalone in this treatment compared to the controls. However, the expression levels of Mn-SOD, glutathione peroxidase (GPX), thioredoxin peroxidase (TPx), selenium-binding protein (SEBP), HSP70 and HSP90 were significantly down-regulated. Compared with those in the group with 70.3 mg kg(-1) dietary AA, the expression levels of CAT, GST and HSP26 were decreased in abalone fed with very high dietary AA (4967.5 mg kg(-1)). In addition, significant up-regulations of expression levels of Mn-SOD, GPX, TPx, SEBP, FT, HSP70, HSP90 and NF-κB were observed in abalone fed with apparently excessive dietary AA (829.8 and 4967.5 mg kg(-1)) as compared to those fed 70.3 mg kg(-1) dietary AA. These findings showed that dietary AA influenced the expression levels of antioxidant proteins, heat shock proteins and NF-κB in the hepatopancreas of abalone at transcriptional level. Levels of dietary AA that appeared adequate (70.3 mg kg(-1)) reduced the oxidative stress by influencing gene expression of antioxidant proteins, but excessive dietary AA (829.8 and 4967.5 mg kg(-1)) induced oxidative stress in Pacific abalone H. discus hannai.

Acacetin-7-rutinoside from Buddleja lindleyana, a new molluscicidal agent against Oncomelania hupensis.

Buddleja lindleyana is a medicinally important member of the family Loganiaceae distributed in Eastern China. The plant has been used in different traditional medications for the treatment of various diseases. Acacetin-7-rutinoside was isolated from the n-butanol fraction of fresh B. lindleyana leaves and found to be a new molluscicidal agent against the snail Oncomelania hupensis. The structure of acacetin-7-rutinoside was elucidated based on spectral data, including 1H NMR and 13C NMR.

Antifouling and toxic properties of the bioactive metabolites from the seagrasses Syringodium isoetifolium and Cymodocea serrulata.

The present study documents the antifouling and toxic properties of seagrasses Syringodium isoetifolium and Cymodocea serrulata. For that, the seagrasses S. isoetifolium and C. serrulata were extracted individually using organic solvents viz. dichloromethane, acetone and methanol. Amongst the extracts, the maximum antimicrofouling and antimacrofouling activities were exhibited by methanol extracts of both the seagrasses. The Minimal Inhibitory Concentration (MIC) of methanolic extracts of seagrasses was ranged from 1.0 to 10µg/ml against test biofilm bacteria and microalgal strains. Similarly, 100% fouling inhibition of limpet Patella vulgata was found at 6.0mg/ml of methanolic extracts of seagrasses. The mussel Perna indica showed 50% of byssal production and attachment inhibition at 21.51±2.03, 17.82±1.07µg/ml and the anticrustaecean activity for 50% mortality of Artemia salina was recorded at 732.14±9.21 and 394.16±5.16µg/ml respectively for methanolic extracts of S. isoetifolium and C. serrulata. The minimal inhibitory and higher lethal concentrations of active methanol extracts shows it׳s less toxic nature. Based on the prolific results, methanol extracts of S. isoetifolium and C. serrulata were subjected to purification using silica gel column and thin layer chromatography. Then the active compounds of the bioassay guided fractions were partially characterized using gas chromatography coupled with mass spectroscopy (GC-MS) and keyed out that fatty acids (C16 to C24) were the major components which responsible for the antifouling properties of the candidate seagrasses.

Comparative effects of petrol and diesel on enzyme activity in Tympanotonus fuscatus after sublethal exposure.

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.

Hydrogen-bonded complexes and blends of poly(acrylic acid) and methylcellulose: nanoparticles and mucoadhesive films for ocular delivery of riboflavin.

Poly(acrylic acid) (PAA) and methylcellulose (MC) are able to form hydrogen-bonded interpolymer complexes (IPCs) in aqueous solutions. In this study, the complexation between PAA and MC is explored in dilute aqueous solutions under acidic conditions. The formation of stable nanoparticles is established, whose size and colloidal stability are greatly dependent on solution pH and polymers ratio in the mixture. Poly(acrylic acid) and methylcellulose are also used to prepare polymeric films by casting from aqueous solutions. It is established that uniform films can be prepared by casting from polymer mixture solutions at pH 3.4-4.5. At lower pHs (pH < 3.0) the films have inhomogeneous morphology resulting from strong interpolymer complexation and precipitation of polycomplexes, whereas at higher pHs (pH 8.3) the polymers form fully immiscible blends because of the lack of interpolymer hydrogen-bonding. The PAA/MC films cast at pH 4 are shown to be non-irritant to mucosal surfaces. These films provide a platform for ocular formulation of riboflavin, a drug used for corneal cross-linking in the treatment of keratoconus. An in vitro release of riboflavin as well as an in vivo retention of the films on corneal surfaces can be controlled by adjusting PAA/MC ratio in the formulations.

Histopathological changes in tissues of Bithynia siamensis goniomphalos incubated in crude extracts of camellia seed and mangosteen pericarp.

The present study was performed to observe histopathological changes in tissues of Bithynia siamensis goniomphalos (Gastropoda, Bithyniidae) incubated in crude extract solutions of camellia (Camellia oleifera) seed and mangosteen (Garcinia mangostana) pericarp, and furthermore to estimate the molluscicidal effects of 2 plant substances. Substantial numbers of bithyniid snails were incubated in various concentrations of 2 plant solution for 24 hr. As the positive control, snails incubated in various concentrations of niclosamide, a chemical molluscicide, were used. The histopathological findings were observed in sectioned snail specimens of each experimental and control groups. The results showed that both camellia and mangosteen extracts had molluscicidal effects at 24 hr with 50% lethal concentration (LC50) at concentrations of 0.003 and 0.002 g/ml, respectively, while niclosamide had LC50 at concentrations 0.599 ppm. B. siamensis goniomphalos snail tissues (foot, gill, and digestive system) showed disruption of columnar muscle fibers of the foot, reduction of the length and number of gill cilia, numerous mucous vacuoles, and irregularly shaped of epithelial cells. Irregular apical and calciferous cells, dilatation of the digestive gland tubule, and large hemolymphatic spaces, and irregular apical surfaces, detachment of cilia, and enlargement of lysosomal vacuoles of epidermis were also shown in all groups. By the present study, it is confirmed that 2 plants, camellia and mangosteen, are keeping some substance having molluscicidal effects, and histopathological findings obtained in this study will provide some clues in further studies on their action mechanisms to use them as natural molluscicides.

Histopathological Changes in Tissues of Bithynia siamensis goniomphalos Incubated in Crude Extracts of Camellia Seed and Mangosteen Pericarp

The present study was performed to observe histopathological changes in tissues of Bithynia siamensis goniomphalos (Gastropoda, Bithyniidae) incubated in crude extract solutions of camellia (Camellia oleifera) seed and mangosteen (Garcinia mangostana) pericarp, and furthermore to estimate the molluscicidal effects of 2 plant substances. Substantial numbers of bithyniid snails were incubated in various concentrations of 2 plant solution for 24 hr. As the positive control, snails incubated in various concentrations of niclosamide, a chemical molluscicide, were used. The histopathological findings were observed in sectioned snail specimens of each experimental and control groups. The results showed that both camellia and mangosteen extracts had molluscicidal effects at 24 hr with 50% lethal concentration (LC50) at concentrations of 0.003 and 0.002 g/ml, respectively, while niclosamide had LC50 at concentrations 0.599 ppm. B. siamensis goniomphalos snail tissues (foot, gill, and digestive system) showed disruption of columnar muscle fibers of the foot, reduction of the length and number of gill cilia, numerous mucous vacuoles, and irregularly shaped of epithelial cells. Irregular apical and calciferous cells, dilatation of the digestive gland tubule, and large hemolymphatic spaces, and irregular apical surfaces, detachment of cilia, and enlargement of lysosomal vacuoles of epidermis were also shown in all groups. By the present study, it is confirmed that 2 plants, camellia and mangosteen, are keeping some substance having molluscicidal effects, and histopathological findings obtained in this study will provide some clues in further studies on their action mechanisms to use them as natural molluscicides.

Two new triterpenoids from the bark of Eucalyptus exserta and their molluscicidal and cytotoxic activities.

Two new triterpenoids, 2α-hydroxyurs-12-en-28-oic acid-3ß-trans- isoferulate (1) and 2α, 3α, 24-trihydroxyolean-18-en-28-oic acid (2), together with three known triterpenoids (3-5) were isolated from the methanol extract of the bark of Eucalyptus exserta. Their structures were determined by spectroscopic means. Compounds 1, 2, and 5 exhibited molluscicidal activity against Pomacea canaliculata. All isolated compounds showed significant cytotoxic activity against Spodoptera litura (SL) cells.

Molecular cloning, characterization and mRNA expression of selenium-dependent glutathione peroxidase from abalone Haliotis discus hannai Ino in response to dietary selenium, zinc and iron.

A novel selenium-dependent glutathione peroxidase (Se-GPX) was cloned from abalone Haliotis discus hannai Ino (HdhGPx) by homology cloning with degenerate primers and RACE techniques. The full length of HdhGPx cDNA was 963bp with a 669bp open reading frame (ORF) encoding 222 amino acids and a 101bp eukaryotic selenocysteine insertion sequence (SECIS) in 3' untranslated region (UTR). It was showed that HdhGPx has a characteristic codon at (235)TGA(237) that corresponds to selenocysteine (SeC) as U(72). Sequence characterization revealed that HdhGPx contains a characteristic GPx signature motif 2 ((96)LGLPCNQF(103)), an active site motif ((179)WNFEKF(184)). In addition, two potential N-glycosylation sites ((112)NGTE(115) and (132)NLTQ(135)) were identified in HdhGPx. 3D modeling analysis showed that the overall structure of HdhGPx monomer had more similarity to human GPx3 than human GPx1. Relatively higher-level mRNA expression was detected in hepatopancreas, mantle and gonad by real-time PCR assays. The relative expression levels of HdhGPx mRNA in hepatopancreas and haemocytes were detected by real-time PCR in abalone fed with nine different diets containing graded levels of selenium (0.15, 1.32 and 48.7mgkg(-1)), zinc (6.69, 33.85 and 710.63mgkg(-1)) and iron (29.17, 65.7 and 1267.2mgkg(-1)) for 20weeks, respectively. The results showed that the expressions of HdhGPx mRNA were statistically higher at adequate dietary selenium (1.32mgkg(-1)), zinc (33.85mgkg(-1)) and iron (65.7mgkg(-1)) than those in low dietary minerals, respectively. But HdhGPx mRNA expression levels were down-regulated by high contents of dietary selenium (48.7mgkg(-1)), zinc (710.63mgkg(-1)) and iron (1267.2mgkg(-1)), respectively. These results indicated that adequate dietary minerals could increase the mRNA expression of HdhGPx, and then to increase the total antioxidant capacities in abalone.

Cloning, characterization and TBT exposure response of CuZn superoxide dismutase from Haliotis diversicolor supertexta.

The full-length cDNA and genomic DNA of a cytoplasmic copper, zinc superoxide dismutase (CuZn-sod) were cloned from the hepatopancreas of small abalone Haliotis diversicolor supertexta by RT-PCR, RACE and TAIL PCR. The full-length cytoplasmic CuZn-sod cDNA (designated sasod) comprises 984 bp. Its ORF encodes a polypeptide of 154 amino acids with a predicted molecular mass of 15.7 kDa and theoretical isoelectric point of 6.30. The deduced amino acid (designated saSOD) shares a common consensus pattern with the SODs of vertebrate and invertebrate animals. The full-length sasod genomic DNA comprises 5,574 bp, containing five exons and four introns. The splice donor and acceptor sequence of the four introns is 5'GT-AG3'. Real time quantitative PCR analysis revealed that sasod expression level in hepatopancreas of small abalone was no significant difference at 2, 6, 48 and 192 h post TBT exposure (P > 0.05). However, the sasod expression level at 12 and 24 h post TBT exposure was decreased significantly (P < 0.05).

Factors promoting marine invasions: a chemoecological approach.

The Mediterranean Sea is losing its biological distinctiveness, and the same phenomenon is occurring in other seas. It gives urgency to a better understanding of the factors that affect marine biological invasions. A chemoecological approach is proposed here to define biotic conditions that promote biological invasions in terms of enemy escape and resource opportunities. Research has focused on the secondary metabolite composition of three exotic sea slugs found in Greece that have most probably entered the Mediterranean basin by Lessepsian migration, an exchange that contributes significantly to Mediterranean biodiversity. We have found toxic compounds with significant activity as feeding deterrents both in the cephalaspidean Haminoea cyanomarginata and in the nudibranch Melibe viridis. These findings led us to propose aposematism in the former and dietary autonomy in producing defensive metabolites in the latter case, as predisposing factors to the migration. In the third mollusk investigated, the anaspidean Syphonota geographica, the topic of marine invasions has been approached through a study of its feeding biology. The identification of the same compounds from both the viscera of each individual, separately analyzed, and their food, the seagrass Halophila stipulacea, implies a dietary dependency. The survival of S. geographica in the Mediterranean seems to be related to the presence of H. stipulacea. The initial invasion of this exotic pest would seem to have paved the way for the subsequent invasion of a trophic specialist that takes advantage of niche opportunities.

Molecular characterization and mRNA expression during metal exposure and thermal stress of copper/zinc- and manganese-superoxide dismutases in disk abalone, Haliotis discus discus.

Complementary DNAs encoding copper/zinc superoxide dismutase (Cu/Zn-SOD; SOD1) and manganese superoxide dismutase (Mn-SOD; SOD2) were isolated from disk abalone, Haliotis discus discus. The open reading frame sequences of Cu/Zn- and Mn-SODs encoded 154 and 226 amino acids, respectively. Multiple sequence alignments using the deduced amino acid sequences revealed that both abalone SODs showed considerable sequence similarities with their orthologues from diverse aerobic organisms, in which the amino acid residues forming metal ligands were highly conserved. All phylogenetic trees for both SOD genes inferred from maximum likelihood and Bayesian inference analyses presented the monophyletic status of Teleostei and Aves/Tetrapoda clades, and recovered relatively close genetic affiliation of H. discus discus with some molluscan species. Expression of both SODs at mRNA levels were highly modulated in various tissues (gill, muscle and hepatopancreas from juveniles, and haemocytes from adults) by experimental exposures to heavy metals (copper, zinc and cadmium) and also by thermal treatments (elevation of temperature). The mRNA levels of both SODs were increased in general during the metal or thermal treatments; however, the transcriptional responses of SOD genes were quite variable depending upon isoforms and tissues based on semi-quantitative and/or real-time RT-PCR assays.