Regulating soil bacterial diversity, community structure and enzyme activity using residues from golden apple snails.

It has been shown that the golden apple snail (GAS, Pomacea canaliculata), which is a serious agricultural pest in Southeast Asia, can provide a soil amendment for the reversal of soil acidification and degradation. However, the impact of GAS residue (i.e., crushed, whole GAS) on soil bacterial diversity and community structure remains largely unknown. Here, a greenhouse pot experiment was conducted and 16S rRNA gene sequencing was used to measure bacterial abundance and community structure in soils amended with GAS residue and lime. The results suggest that adding GAS residue resulted in a significant variation in soil pH and nutrients (all P < 0.05), and resulted in a slightly alkaline (pH = 7.28-7.75) and nutrient-enriched soil, with amendment of 2.5-100 g kg-1 GAS residue. Soil nutrients (i.e., NO3-N and TN) and TOC contents were increased (by 132-912%), and some soil exocellular enzyme activities were enhanced (by 2-98%) in GAS residue amended soil, with amendment of 1.0-100 g kg-1 GAS residue. Bacterial OTU richness was 19% greater at the 2.5 g kg-1 GAS residue treatment than the control, while it was 40% and 53% lower at 100 g kg-1 of GAS residue and 50 g kg-1 of lime amended soils, respectively. Firmicutes (15-35%) was the most abundant phylum while Bacterioidetes (1-6%) was the lowest abundant one in GAS residue amended soils. RDA results suggest that the contents of soil nutrients (i.e., NO3-N and TN) and soil TOC explained much more of the variations of bacterial community than pH in GAS residue amended soil. Overuse of GAS residue would induce an anaerobic soil environment and reduce bacterial OTU richness. Soil nutrients and TOC rather than pH might be the main factors that are responsible for the changes of bacterial OTU richness and bacterial community structure in GAS residue amended soil.

Purification and Regulation of Pyruvate Kinase from the Foot Muscle of the Anoxia and Freeze Tolerant Marine Snail, Littorina littorea.

The intertidal marine snail, Littorina littorea, has evolved to survive bouts of anoxia and extracellular freezing brought about by changing tides and subsequent exposure to harsh environmental conditions. Survival in these anoxic conditions depends on the animals entering a state of metabolic rate depression in order to maintain an appropriate energy production-consumption balance during periods of limited oxygen availability. This study investigated the kinetic, physical, and regulatory properties of pyruvate kinase (PK), which catalyzes the final reaction of aerobic glycolysis, from foot muscle of L. littorea to determine if the enzyme is differentially regulated in response to anoxia and freezing exposure. PK purified from foot muscle of anoxic animals exhibited a lower affinity for its substrate phosphoenolpyruvate than PK from control and frozen animals. PK from anoxic animals was also more sensitive to a number of allosteric regulators, including alanine and aspartate, which are key anaerobic metabolites in L. littorea. Furthermore, PK purified from anoxic and frozen animals exhibited greater stability compared to the non-stressed control animals, determined through high-temperature incubation studies. Phosphorylation of threonine and tyrosine residues was also assessed and demonstrated that levels of threonine phosphorylation of PK from anoxic animals were significantly higher than those of PK from control and frozen animals, suggesting a potential mechanism for regulating PK activity. Taken together, these results suggest that PK plays a role in suppressing metabolic rate in these animals during environmental anoxia exposure.

[Effects of copper and cadmium ions on protective enzyme activity in Oncomelania hupensis].

OBJECTIVE: To evaluate the effects of Cu2+ and Cd2+ at different concentrations on superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activity in Oncomelania hupensis. METHODS: Cu2+- and Cd2+-containing solutions were prepared at 7 concentrations, and O. hupensis snails were exposed to the solutions for 24 h, of 15 snails in each concentration. Then, the snail body was collected following removal of the snail shell and homogenated, and the SOD, CAT and POS activities were detected in the supernatants. RESULTS: With the increase of the Cu2+ concentration, the SOD activity appeared a rise followed by a reduction in O. hupensis snails, and the CTA activity appeared a decline-rise-decline tendency, while the POD activity showed a tendency towards rise followed by decline. With the increase of the Cd2+ concentration, the SOD activity appeared a rise followed by a reduction in O. hupensis snails, and the CTA activity appeared a decline- rise- decline tendency, while the POD activity showed a decline-rise-decline tendency. CONCLUSIONS: Exposure to Cu2+ and Cd2+ at high concentrations results in a decline in the activity of SOD, CAT and POD in O. hupensis at the same time.

In vivo evaluation of oxidative stress and biochemical alteration as biomarkers in glass clover snail, Monacha cartusiana exposed to zinc oxide nanoparticles.

Oxidative stress is considered a main commonly reported mechanism of nanoparticles toxicity, so this study aimed to evaluate oxidative stress and biochemical alterations in the haemolymph and digestive gland of snail, Monacha cartusiana exposed to sublethal concentrations of zinc oxide nanoparticles (ZnONPs) for 14 days (d). The results indicated that, ZnONPs induced significant increases in lipid peroxidation (LPO) and lactate dehydrogenase (LDH) in treated animals and did not return to normal levels after recover period. A significant decline of glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities, and glutathione (GSH) content in the haemolymph and digestive gland of snails was observed when compared with control. A significant increase was observed in catalase (CAT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities of treated animals. In general, nano-materials are able to induce oxidative stress in exposed animals. The present findings indicate that, alterations of antioxidant enzyme activities, increase of LPO, LDH, and reducing of GSH content and GST, GPx activities are recognized to oxidative stress and cell damage. This species could be considered a good bioindicator to assess nano-materials exposure.

Effect of Simultaneous Snail Slime-aided Degradation and Yeast Fermentation on Terpenoid Composition of Plantain Pseudostem Waste.

BACKGROUND: In this study, local sustainable enzyme sources involving excised digestive juice of African land snail and yeast were utilized to achieve the simultaneous saccharification (SSF) and fermentation (SSF) of plantain pseudostem (PPS) waste, and afterwards their effects on terpenoids using gas chromatography coupled to a flame ionization detector (GC-FID), were examined. METHODS: The most abundant terpenoids were found in the order α-pinene > borneol > camphor > humulene > ß-caryophellene, while the least in abundance were cis ocimene (8.78x10-6 mg/100g), and cyperene (1.81x10-5 mg/100g). The application of exclusive fermentation and SSF respectively elevated azuluene by 95.46 and 99.6%, while pinene-2-ol was elevated by 83.02 and 98.57%, respectively. RESULTS: Both exclusive fermentation and SSF had no effect on myrcene, cyperene, ethyl cinnamate, germacrene b, valencene, beta selinene, aromadendrene, and taraxerol, while the degree of degradation of some of the terpenoids by both processes was respectively as follows; gama muurolene (100%), ß-caryophyllene (97.60 and 93.14%), α-terpinenyl acetate (91.95 and 83.16%), geranyl acetate (94.81 and 43.87%), and terpinen-4-ol (94.40 and 57.00%). CONCLUSION: The findings of this study encourage the imminent application of simultaneous saccharification and fermentation for the enhancement of bioactivities of terpenoids.

High-resolution crystal structures of the glycoside hydrolase family 45 endoglucanase EG27II from the snail Ampullaria crossean.

Although endogenous animal cellulases have great potential for industrial applications such as bioethanol production, few investigations have focused on these enzymes. In this study, the glycoside hydrolase family 45 (GH45) subfamily B endoglucanase EG27II from the snail Ampullaria crossean was expressed using a Pichia pastoris expression system and the crystal structure of the apo form was determined at 1.00 Šresolution; this is the highest resolution structure of an animal endoglucanase. The results showed that EG27II has a double-ψ six-stranded ß-barrel and that the structure of EG27II more closely resembles those of subfamily C enzymes than those of subfamily A enzymes. The structure of EG27II complexed with cellobiose was also determined under cryoconditions and at room temperature at three pH values, pH 4.0, 5.5 and 8.0, and no structural changes were found to be associated with the change in pH. The structural comparison and catalytic activity measurements showed that Asp137 and Asn112 function as the catalytic acid and base, respectively, and that Asp27 is also an important residue for catalysis. These high-resolution structures of EG27II provide a large amount of information for structure-based enzyme modification and cell-surface engineering, which will advance biofuel production using animal-derived cellulases.

Azinphos-methyl causes in Planorbarius corneus toxic effects on reproduction, offspring survival and B-esterases depending on the exposure time.

This work aimed to study in the freshwater gastropod Planorbarius corneus the effects of acute (2 days) and subchronic (14 days) exposures to an environmental concentration of the organophosphate azinphos-methyl on different reproductive parameters, offspring survival and B-esterase activities in gonads and in the whole organism soft tissue. The acute exposure inhibited only carboxylesterase activity in both tissues while the subchronic exposure also inhibited cholinesterase activity, decreased the number of hatched-eggs and increased offspring lethality (92%). On the other hand, B-esterases in gonads were more effective biomarkers than B-esterases in the whole organism due their inhibition appeared earlier in time (cholinesterase activity) and their activity remained inhibited for a longer time (carboxylesterase activity) when recovery studies were performed. We concluded that B-esterases and reproductive parameters can be used as effect biomarkers of aquatic contamination with azinphos-methyl. Our studies showed that a 14 days exposure to an environmental concentration of azinphos-methyl produced severe signs of toxicity in adult organisms, egg masses and juveniles that could cause negative effects at the population level in contaminated environments.

Connecting pattern to process: Growth of spiral shell sculpture in the gastropod Nucella ostrina (Muricidae: Ocenebrinae).

Shell morphology is a well-suited and underused system to examine the development of novel forms. The three-dimensional structure produced (the shell) is separate from the largely two-dimensional tissue that secretes it (the mantle), allowing us to disentangle the pattern from the process. Despite knowing a great deal about the mechanics of shell secretion (process), and the variety of shell shapes that exist (pattern), no effort has been made to understand how the mantle changes to produce different shell shapes. We investigated this question in the dimorphic snail Nucella ostrina, which exhibits both smooth and ribbed shells to determine how ribs are formed by the mantle. Rib thickenings are produced only in the outer calcitic shell layer and secreted by the distal Outer Mantle Epithelium (OME) with increased acid phosphatase activity. The evenly thick inner aragonitic layers are secreted by the proximal OME which expresses acid phosphatase. Here we show that locally thicker ribs in N. ostrina are produced by changing the dimensions of the distal OME: elongation in the direction of growth and increased cell height. This should increase the amount of shell material secreted, producing locally thicker shell (ribs). Preliminary evidence suggests this mechanism may be widespread in gastropods.

[Enzymology of snails under treatment of molluscicides].

Studies on the enzymology of snails are important in the study of molluscicidal mechanism. The alteration of activities of enzymes after molluscicidal treatment was reported in large numbers of papers. This paper reviews the progress of studies on the enzymology of snails under the treatment of molluscicides.

Egg-Hatching Mechanism of Human Liver Fluke, Opisthorchis viverrini: A Role For Leucine Aminopeptidases From the Snail Host, Bithynia siamensis goniomphalos.

The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24-28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.

Molluscicidal activity and physiological toxicity of Macleaya cordata alkaloids components on snail Oncomelania hupensis.

In order to search new local plant molluscicides for the control of the vectors of schistosomiasis, leaves of Macleaya cordata (Willd) R. Br. were used to extract and separate alkaloid components by thinner acid method and column chromatography, and the molluscicidal effect of alkaloid components against snail Oncomelania hupensis was determined by bioassay. The results showed that 7 alkaloid components (AN1-7) were obtained after extracting and separating alkaloids from the leaves of M. cordata, where AN2 was found being the most toxic against snail O. hupensis with 48h LC50 and LC90 values of AN2 of 6.35mg/L and 121.23mg/L, respectively. Responses of some critical enzymes to AN2, including activities of Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate transaminase (AST), Malic dehydrogenase (MDH) and Succinate dehydrogenase (SDH) in both cephalopodium and liver, were also detected through experiments, which also explored esterase isozyme (EST) exposed to AN2 in liver tissue. The results showed that AN2 significantly inhibited the activities of SDH, MDH and esterase isozyme, as AN2 significantly stimulated the activities of ALP, ALT and AST to increase at a low concentration (e.g. 25mg/L), while significantly inhibited the activities of these enzymes at a high concentration (100mg/L). These results indicated that AN2 not only inhibited protein synthesis, and respiratory chain oxidative phosphorylation, but also caused hepatocellular injury and reduced the detoxification ability of liver.

Heat-resistant cytosolic malate dehydrogenases (cMDHs) of thermophilic intertidal snails (genus Echinolittorina): protein underpinnings of tolerance to body temperatures reaching 55°C.

Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41-44°C in summer and withstand temperatures up to at least 55°C. Here, we demonstrate that heat stability of function (indexed by the Michaelis-Menten constant of the cofactor NADH, KMNADH) and structure (indexed by rate of denaturation) of cytosolic malate dehydrogenases (cMDHs) of two congeners (E. malaccana and E. radiata) exceeds values previously found for orthologs of this protein from less thermophilic species. The ortholog of E. malaccana is more heat stable than that of E. radiata, in keeping with the congeners' thermal environments. Only two inter-congener differences in amino acid sequence in these 332 residue proteins were identified. In both cases (positions 48 and 114), a glycine in the E. malaccana ortholog is replaced by a serine in the E. radiata protein. To explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme, we used molecular dynamics simulation methods. These computational methods allow determination of thermal effects on fine-scale movements of protein components, for example, by estimating the root mean square deviation in atom position over time and the root mean square fluctuation for individual residues. The minor changes in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites. Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation.

Comparative profiling of hepatopancreas transcriptomes in satiated and starving Pomacea canaliculata.

BACKGROUND: Although Pomacea canaliculata is native to South and Central America, it has become one of the most abundant invasive species worldwide and causes extensive damage to agriculture and horticulture. Conventional physical and chemical techniques have been used to eliminate P. canaliculata, but the effects are not ideal. Therefore, it is important to devise a new method based on a greater understanding of the biology of P. canaliculata. However, the molecular mechanisms underlying digestion and absorption in P. canaliculata are not well understood due to the lack of available genomic information for this species, particularly for digestive enzyme genes. RESULTS: In the present study, hepatopancreas transcriptome sequencing produced over 223 million high-quality reads, and a global de novo assembly generated a total of 87,766 unique transcripts (unigenes), of which 19,942 (22.7%) had significant similarities to proteins in the UniProt database. In addition, 296,675 annotated sequences were associated with Gene Ontology (GO) terms. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed for the unique unigenes, and 262 pathways (p-value < 10-5) in P. canaliculata were found to be predominantly related to plant consumption and coarse fiber digestion and absorption. These transcripts were classified into four large categories: hydrolase, transferase, isomerase and cytochrome P450. The Reads Per Kilobase of transcript per Million mapped reads (RPKM) analysis showed that there were 523 down-regulated unigenes and 406 up-regulated unigenes in the starving apple snails compared with the satiated apple snails. Several important genes are associated with digestion and absorption in plants: endo-beta-1, 4-glucanase, xylanase, cellulase, cellulase EGX1, cellulase EGX3 and G-type lysozyme genes were identified. The qRT-PCR results confirmed that the expression patterns of these genes (except for the longipain gene) were consistent with the RNA-Seq results. CONCLUSIONS: Our results provide a more comprehensive understanding of the molecular genes associated with hepatopancreas functioning. Differentially expressed genes corresponding to critical metabolic pathways were detected in the transcriptome of starving apple snails compared with satiated apple snails. In addition to the cellulase gene, other genes were identified that may be important factors in plant matter metabolism in P. canaliculata, and this information has the potential to expedite the study of digestive physiology in apple snails.

Purification and Characterization of Lactate Dehydrogenase in the Foot Muscle and Hepatopancreas of Otala lactea.

Lactate dehydrogenase (LDH) has a crucial role in maintaining ATP production as the terminal enzyme in anaerobic glycolysis. This study will determine the effect of posttranslational modifications (PTMs) on the activity of LDH in the foot muscle and hepatopancreas of an estivating snail, Otala lactea. LDH in foot muscle of O. lactea was purified to homogeneity and partially purified in hepatopancreas in a two-step and three-step process, respectively. The kinetic properties and stability of these isoforms were determined where there was a significant difference in Km and I50 values with pyruvate and urea separately in foot muscle; however, hepatopancreas exhibited significant differences in Km and I50 in salt between control and stress. Interestingly, hepatopancreas has a higher affinity for pyruvate in the control state whereas foot muscle has a higher affinity for its substrate in the estivated state. PTMs of each isoform were identified using immunoblotting and dot blots, which prove to be significantly higher in the control state. Overall, foot muscle LDH enters a low phosphorylation state during estivation allowing more efficiency in consuming pyruvate with higher thermal stability but less structural stability. Hepatopancreas LDH becomes dephosphorylated in the estivating snail that decreases the efficiency of the enzyme in the forward direction; however, the snail has an increased tolerance to the presence of salt when water becomes scarce. Such tissue-specific regulations indicate the organism's ability to reduce energy consumption when undergoing metabolic depression.

In vitro and in vivo studies of cholinesterases and carboxylesterases in Planorbarius corneus exposed to a phosphorodithioate insecticide: Finding the most sensitive combination of enzymes, substrates, tissues and recovery capacity.

Organophosphate insecticides (OPs) continue to be an important class of agrochemicals used in modern agriculture worldwide. Even though these pesticides persist in the environment for a relatively short time, they show a high acute toxicity that may represent a serious hazard for wildlife. Sub-lethal effects on non-target species are a focus in pest management programs and should be used as biomarkers. Cholinesterases (ChEs) are the most used biomarker of OP exposure in vertebrate and invertebrate species. However, the combined monitoring of ChE and carboxylesterase (CE) activities may provide a more useful indication of exposure and effect of the organisms. The objective of the present work was to find the most sensitive combination of enzyme, substrate, tissue and capacity to recovery of B-esterases in the freshwater gastropod Planorbarius corneus exposed to the OP azinphos-methyl. For this purpose, ChE and CE activities in different tissues of P. corneus (head-foot, pulmonary region, digestive gland, gonads and whole organism soft tissue) were studied. Measurements of ChE activity were performed using three substrates: acetylthiocholine, propionylthiocholine and butyrylthiocholine and CE activity using four different substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1-naphthyl acetate, and 2-naphthyl acetate in control and exposed organisms. Finally, the recovery rates of ChE and CE activities following 48h exposure to azinphos-methyl were analyzed. Our results show a preference for acetylthiocholine as substrate, a high inhibition with eserine (a selective ChE inhibitor) and inhibition with excess of substrate in all the analyzed tissues. The highest ChE and CE activity was found in the pulmonary region and in the digestive gland, respectively. The highest CE Vmax was obtained with 1 and 2-naphthyl acetate in all the tissues. CEs were more sensitive than ChE to azinphos-methyl exposure. The highest sensitivity was found using p-nitrophenyl acetate and butyrate as substrates. On the other hand, CEs of the digestive gland and the pulmonary region were more sensitive than CEs of the whole organism soft tissue. Regarding the recovery of enzyme activities after 48h exposure, ChE and CEs with p-nitrophenyl butyrate reached control values after 14days in the digestive gland and after 21days in the pulmonary region. Our results show marked differences in P. corneus basal ChE and CE activities depending on substrates and the tissue. Also, both tissue-dependent and substrate-dependent variations in sensitivity to azinphos-methyl exposure and recovery were obtained. CEs measured with p-nitrophenyl butyrate in the pulmonary region were the best combination to be used as biomarker of exposure to azinphos-methyl due to their sensitivity and low recovery capacity. Environmental concentrations of azinphos-methyl inhibited CE activity so they could be used as effective biomarkers of aquatic contamination.

Effect of tidal regime on the thermal tolerance of the marine gastropod Lunella smaragda (Gmelin 1791).

The tidal cycle around New Zealand results in spring low tides consistently occurring during the hottest part of the day (mid-afternoon) in north-eastern New Zealand, and during the cooler dawn/dusk periods in the north-west of the country. We hypothesised that due to mid-afternoon spring low tides, intertidal populations residing at north-eastern sites would show greater thermotolerance than their north-west conspecifics. To test this we used the marine gastropod, Lunella smaragda, which were collected from sites on both the East and West coasts of the Auckland region and exposed to an acute heat shock. Thermotolerance was measured as survivorship (LT50), drop down time (time to heat coma) and thermal stability of the anaerobic energy producing enzyme Tauropine dehydrogenase. Furthermore, temperature loggers were deployed at each site so as to record and compare thermal regimes among sites. A strong temperature spike associated with spring low tide was found at all sites, and maximal temperatures of all East coast sites were higher than West coast sites (in some case by up to 10°C). In terms of thermotolerance, mortality of L. smaragda occurred at 42°C leading to 100% mortality at 45°C. However, comparison of LT50 showed snails were equally thermotolerant regardless of site of collection. Similar results were found in TDH thermal stability with animals from all sites showing an approximately 80% decrease in enzyme activity after 10min exposure to 42°C. Whilst drop down times were different among sites these were correlated with animal size as opposed to site of collection. Thus, East coast populations of L. smaragda appear no more thermotolerant than their West coast counterparts. Such a result is concerning as maximal temperatures at East coast sites already exceed the LT50 values of L. smaragda recorded in the lab suggesting these populations have less of a thermal safety margin.

Morphological and enzymatical observations in Oncomelania hupensis after molluscicide treatment: implication for future molluscicide development.

A preparation of niclosamide named 50 % wettable powder of niclosamide ethanolamine salt (WPN), the only chemical molluscicide available in China, has been widely used for Oncomelania hupensis control over the past 20 years, but its molluscicidal mechanism has not been elucidated yet. Recently, a derivative of niclosamide, the salt of quinoid-2',5-dichloro-4'-nitro-salicylanilide (Liu Dai Shui Yang An, LDS), has been proven to have equivalent molluscicidal effects as WPN but with lower cost and significantly lower toxicity to fish than WPN. In our previous study, gene expression profiling of O. hupensis showed significantly effects after these two molluscicides had been applied. This study was designed to use morphological and enzymological analyses to further elucidate the mechanism by which these molluscicides cause snail death. After WPN or LDS treatment, the number of mitochondria of O. hupensis was reduced and their cristae appeared unclear, heterochromatin gathered to be polarized, ribosome numbers of the rough endoplasmic reticulums (rERs) decreased, myofilaments in muscle cells became disordered and loose, and cytoplasm in some liver cells was concentrated. Damage of cell structures and organelles suggested inhibited movement ability and effects on liver and energy metabolism following treatment. In parallel, activities of enzymes related with carbohydrate metabolism were inhibited except lactate dehydrogenase (LDH) increased in muscle tissue, and activities of enzymes related with stress response increased followed by decreasing to lower levels than those of the H2O-treated group. This shift of carbohydrate metabolism patterns led to insufficient energy supply and lactic acid accumulation, and variations of nitric oxide synthase (NOS), alanine aminotransferase (ALT), and superoxide dismutase (SOD) during process of molluscicide treatment suggested a stress response of snail to the molluscicides at early stages and later fatal damage in liver and nervous system. In general, effects of WPN and LDS were similar although LDS-treated snails showed more serious damage in the liver and a stronger inhibition of enzymes related with aerobic respiration and stress response. This was consistent with the transcriptome profile obtained previously. However, considering enzyme activities at post-transcriptional and protein levels, comprehensive identification and annotation of potential enzyme-related genes and regulation pattern would be necessary to provide great benefit for understanding of potential mechanism of these molluscicides and even for future molluscicide development.

[Progress of researches on lysozyme and its expression in Oncomelania hupensis].

Lysozyme generally exists in animals, plants and microorganisms, and it is used as a natural anti-infection material and one of the important non-specific immune factors in organisms. This paper reviews the progress of researches on its classification, gene structure and function, and expression regulation in Oncomelania hupensis, and on the factors affecting its activities in recent years, in order to further discuss its distribution in O. hupensis.

Exposure of composite tannery effluent on snail, Pila globosa: A comparative assessment of toxic impacts of the untreated and membrane treated effluents.

Effluent from tannery industries can significantly affect the aquatic environment due to the presence of a variety of recalcitrant components. The present study focuses on a comparative assessment of the toxic impacts of an untreated tannery effluent and membrane treated effluents using snail, Pila globosa as an aquatic model. Composite tannery effluent collected from a common effluent treatment plant was selected as the untreated effluent. To investigate the effect of treated effluents on the aquatic organism the effluent was treated by two ways, viz. a single stage microfiltration (MF) using ceramic membrane and a two-step process involving MF followed by reverse osmosis (RO). The whole body tissue, gonad and mantle of P. globosa were subjected to enzyme assays like superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GSH-GPx), glutathione S- transferase (GST), etc. for assessing toxic impact. Changes in the biochemical parameters like protein, carbohydrate and amino acid were observed including histological studies of gonad and mantle tissue upon treatment with tannery effluents. To examine potential DNA damage due to the exposure of the effluent, comet assay was conducted. The study revealed that with an exposure to the untreated effluent, activity of the antioxidant enzymes increased significantly while the protein and carbohydrate content reduced largely in the whole body tissue, gonad as well as mantle tissues of P. globosa. Histological study indicated considerable damage in the gonad and mantle tissues following exposure to the untreated effluent. Comet assay using hemolymph of P. globosa following exposure to tannery effluent, showed significant genotoxicity. Interestingly, compared to the untreated effluent, damaging effect was reduced in molluscs tissues when exposed to MF treated effluent and even lesser when exposed to MF+RO treated effluent. Apart from the reduced activities of oxidative stress enzymes, the protein, amino acid and carbohydrate content of molluscs exposed to both of the treated effluent were found close to that of control. Comet assay revealed no damage in the DNA for MF and MF+RO treated effluent indicating that the membrane based treatment procedure restores environmental condition to control level.

[Enzyme kinetic analysis of Oncomelania hupensis exposed to active ingredient of Buddleja lindleyana (AIBL)].

OBJECTIVE: To analyze the enzyme kinetics of active ingredient of Buddleja lindleyana (AIBL) against Oncomelania hupensis, the intermediate host of Schistosoma japonicum. METHODS: O. hupensis snails were placed in 1 000 ml of 3.55 mg/L AIBL solution for 24, 48 h and 72 h, respectively, and the enzyme kinetics of alanine aminotransferase (GPT) was determined by Reitman-Frankel assay, lactate dehydrogenase (LDH) by the chemical inhibition lactic acid substrate method, alkaline phosphatase (AKP) by the disodium phenyl phosphate colorimetric method, acetylcholine esterase (AChE) and malate dehydrogenas (MDH) by ELISA, and succinate dehydrogenase (SDH) by the phenazine methyl sulfate reaction method (PMS) in the soft tissues of O. hupensis before and after AIBL treatment. RESULTS: Following exposure to 3.55 mg/L AIBL solution for 24 h, the GPT, LDH, and AKP activities significantly improved in the soft tissues of O. hupensis, while the SDH and MDH activities were significantly lowered in the head-foot and liver. However, AIBL treatment did not cause significant effect on AChE activity in O. hupensis. CONCLUSIONS: AIBL causes significant damages to O. hupensis liver and can efficiently act on anaerobic and aerobic respiration loci, which will hinder energy metabolism, and cause inadequate energy supply in cells used for normal secretion, eventually leading to O. hupensis death.