Ecdysone 20-monooxygenase activity in land crabs

1. Ecdysone 20-monooxygenase activity has been found in hepatopancreas, gonads, epidermis and muscle of the crab Gecarcinus lateralis. Activity was assayed by measuring the in vitro conversion of [³H]-ecdysone to [³H]-20-hydroxyecdysone. Maximal activity is obtained at 30°C and pH 8.0 in sodium phosphate buffer.2. Activity from hepatopancreas is localized in a fraction which sediments at 10,000 g, probably mitochondria.3. NADPH stimulates activity and metyrapone or oxygen deprivation inhibits it, as has been observed for cytochrome P-450-dependent monooxygenases.4. Changes in ecdysone 20-monooxygenase activity at different stages of the molt cycle are not directly correlated to changes in ecdysteroid levels in the hemolymph.     

Biodegradation of diethyl phthalate by an organic-solvent-tolerant Bacillus subtilis strain 3C3 and effect of phthalate ester coexistence

The contamination and distribution of phthalate esters - synthetic compounds widely used in plastic product production, including food and medical packaging - has raised safety concerns due to their endocrine-disrupting activity and mandated to be treated. Bacillus subtilis strain 3C3, isolated as an organic-solvent-tolerant bacterium, was capable of utilizing diethyl phthalate as a sole carbon source. Biodegradation of diethyl phthalate occurred constitutively without lag period, and its kinetics followed a first-order model. The biodegradability was significantly enhanced with the supplementation of yeast extract as a co-metabolic substrate. In the presence of Tween-80 as a solubilizing agent, cells rapidly degrade a range of short-chain phthalate esters at high concentrations (up to 1000 mg l⁻¹ for diethyl phthalate). The biodegradation of short-chain phthalates in the binary, ternary and quaternary substrate system revealed that the coexistence of other short-chain phthalates had no significant influence on the biodegradation of diethyl phthalate, and vice versa. These results substantiated that B. subtilis strain 3C3 has potential application as a bioaugmented bacterial culture for bioremediation of phthalates.     

Degradation of polychlorinated biphenyls (PCBs) by four bacterial isolates obtained from the PCB-contaminated soil and PCB-contaminated sediment

We investigated the PCB-degrading abilities of four bacterial strains isolated from long-term PCB-contaminated soil (Alcaligenes xylosoxidans and Pseudomonas stutzeri) and sediments (Ochrobactrum anthropi and Pseudomonas veronii) that were co-metabolically grown on glucose plus biphenyl which is an inducer of the PCB catabolic pathway. The aim of study was to determine the respective contribution of biomass increase and expression of degrading enzymes on the PCB degrading abilities of each isolate. Growth on 5 g l⁻¹ glucose alone resulted in the highest stimulation of the growth of bacterial strains, whereas grown on 10 mg l⁻¹, 100 mg l⁻¹, 1 g l⁻¹, or 5 g l⁻¹ biphenyl did not effected the bacterial growth. None of the strains used in this study was able to grow on PCBs as the sole carbon source. Cells grown on glucose exhibited enhanced degradation ability due to an increased biomass. Addition of biphenyl at concentrations of 1 or 5 g l⁻¹ did not increase total PCB degradation, but stimulated the degradation of highly chlorinated congeners for some of the strains. The degradation of di- and tri-chlorobiphenyls was significantly lower for cells grown on 5 g l⁻¹ biphenyl independently on glucose addition. The highest degradation of the PCBs was obtained for A. xylosoxidans grown in the presence of glucose. Thus A. xylosoxidans appears to be the most promising among the four bacterial isolates for the purpose of bioremediation.     

Effects of the complete replacement of fish oil with linseed oil on growth,fatty acid composition,and protein expression in the Chinese mitten crab (<Emphasis Type="Italic">Eriocheir sinensis</Emphasis>)

Background

The finite marine resources make it difficult for us to obtain enough fish oil (FO) used in aquatic feeds. Another sustainable ingredients should be found to substitute FO. The effects of replacing FO with vegetable oil have been studied in a variety of crustaceans, but most studies have focused on the phenotypic effects. Little is known about the mechanisms of the effects.

Methods

To understand the molecular responses during the replacement of FO in Eriocheir sinensis, we investigated the effects of feeding FO or linseed oil (LO) on growth performance, digestive enzyme activity, fatty acid composition and protein expression in E. sinensis. Twenty-four juvenile crabs were fed diets containing FO or LO for 112 days. Weight, carapace length and width were recorded. Fatty acid composition of the diets and the hepatopancreas and protein expression in the hepatopancreas were analyzed.

Results

Growth performance and molting interval were unchanged by diet. Crabs fed FO and LO had same activity of lipase and amylase, but comparing with crabs fed LO, crabs fed FO had higher trypsin activity and lower pepsin activity. Hepatopancreas fatty acid composition changed to reflect the fatty acid composition of the diets. In total, 194 proteins were differentially expressed in the hepatopancreas between the diets. Expression of heat shock proteins was higher in crabs fed LO. Expression of fatty acid synthase, long-chain fatty acid transport protein 4, acyl-CoA delta-9 desaturase, and fatty acid-binding protein 1, was higher in crabs fed FO.

Conclusions

The substitution of FO with LO didn’t have any effects on the growth and molting of mitten crab, but could significantly decrease the ability of mitten crab to cope with stress. The high content of HUFAs in the hepatopancreas of mitten crab fed FO is due to the high abundance of the proteins relative to the transport of the HUFAs. These findings provide a reason of the high content of EPA and DHA in crabs fed with FO, and provide new information for the replacement of FO in diets of mitten crab.

     

Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda,Grapsidae)

This work was aimed at evaluating the gill carbonic anhydrase (CA) activity of the estuarine crab Chasmagnathus granulata exposed in vivo to cadmium, at different salinities. The in vivo effect of the specific inhibitor acetazolamide (AZ) was also assayed. Besides, the inhibition of CA activity by different heavy metals (cadmium, copper, zinc) and AZ were evaluated under in vitro conditions. For the in vivo assays, adult males were acclimated to salinities of 2.5 or 30‰. The corresponding 96-h LC₅₀ of cadmium was 2.69 mg l⁻¹ at 2.5‰, and >50 mg l⁻¹ at 30‰. Cadmium only caused a significant lower CA activity than control at 2.5‰. EC₅₀ for CA inhibition was estimated to be 1.59 mg l⁻¹ at 2.5‰. Statistical differences in Na⁺ hemolymphatic levels (P<0.05) were only detected at 2.5‰, between 0 and 1.25 mg l⁻¹ of cadmium, but no statistical differences were observed for Cl⁻ levels at any assayed salinity. As CA inhibition registered at 2.5‰ was followed by only changes in Na⁺ concentration, it is likely that cadmium exposure could differentially affect ions permeability, among others factors. The concentrations that inhibited in vitro 50% of enzymatic activity (IC₅₀) were 2.15×10⁻⁵, 1.62×10⁻⁵, 3.75×10⁻⁶ and 4.4×10⁻¹⁰ M for cadmium, copper, zinc and AZ, respectively. The comparison with IC₅₀ values of other aquatic species, indicates a higher CA sensitivity for C. granulata to pollutants.     

CYP330A1 and CYP4C39 enzymes in the shore crab Carcinus maenas: sequence and expression regulation by ecdysteroids and xenobiotics

Cytochrome P450 enzymes (CYP enzymes) catalyse important metabolic reactions of exogenous and endogenous substrates, including steroid hormones. Here, we report the first two CYP sequences from the shore crab, Carcinus maenas. Two complete cDNAs isolated from crab hepatopancreas encode CYP enzymes named CYP330A1, the first member of a new family, and CYP4C39. CYP330A1 is closest related to members of the CYP2 family (37.3% identical to mouse CYP2J6) and CYP4C39 is most identical to crayfish CYP4C15 (59.5%). CYP330A1 gene expression was induced in hepatopancreas of male green intermoult crabs by ecdysone and ponasterone A, but also by benzo(a)pyrene and phenobarbital. CYP330A1 induction was not observed in red crabs. The present results indicate that the CYP330A1 enzyme may be involved in ecdysteroid metabolism, presumably catabolism, and in the detoxification of environmental pollutants. Ecdysteroids or xenobiotics did not affect CYP4C39 gene expression. The fact that both ecdysteroids and xenobiotics affect CYP330A1 gene expression indicates that mutual interactions between chemical exposures and endocrine functions may exist in the shore crab.     

Cytochrome P450 expression is moult stage specific and regulated by ecdysteroids and xenobiotics in the crab Carcinus maenas

The shore crab Carcinus maenas has a high capacity for metabolizing polycyclic aromatic hydrocarbons (PAHs). Cytochrome P450 (CYP) enzymes are involved in this metabolism and also have a role in development and reproduction. This investigation is a systematic gene expression analysis of six CYPs in C. maenas. Expression of CYP2 and CYP3-like genes was predominant in hepatopancreas, while expression of CYP4-like genes was predominant in gills and epidermis. Expression of all six CYP genes fluctuated over the moult cycle in the hepatopancreas and structurally related genes were regulated coordinately. The study suggests that hepatopancreas is a major site of CYP gene expression in C. maenas confirming previous biochemical studies showing that this tissue is the major compartment for CYP mediated xenobiotic metabolism in crustaceans. In addition, the data show that CYP2 and CYP3 related genes respond to ecdysteroid and xenobiotic treatment, while those related to CYP4 genes do not and likely are involved in a more general physiological function such as fatty acid metabolism. The developmental variations of CYP expression suggest a molecular mechanism for the stage specific susceptibility of crabs exposed to environmental pollutants.     

The effects of copper, iron and zinc on digestive enzyme activity in the hybrid tilapia Oreochromis niloticus (L.) ×Oreochromis aureus (Steindachner)

The present experiment was conducted to study effects of Cu, Fe and Zn on activities of digestive enzymes of the hybrid tilapia Oreochromis niloticus×Oreochromis aureus. The acidic protease activities increased 65·5 and 55·1% by addition of homogenates of digesta‐containing stomach with copper (75 mg l⁻¹) and zinc (50 mg l⁻¹) respectively. Addition of Cu and Zn increased the activities of protease in the hepatopancreas homogenates by 132·7 and 38·1% respectively, and reduced the activity of protease in the digesta‐containing intestine homogenates by 11·0 and 13·8% respectively. Addition of Fe (50 mg l⁻¹) increased the acidic protease activity by 96·7% but did not alter the activities of protease in the intestine and hepatopancreas. Addition of Cu markedly inhibited activities of amylase in intestine and hepatopancreas homogenates, while Zn addition showed no effects. Addition of Fe reduced activities of amylase in the intestine homogenates by 47·9% but had no effect on amylase activities in the hepatopancreas. When Cu (75 mg kg⁻¹), Fe (50 mg kg⁻¹) and Zn (50 mg kg⁻¹) were supplemented to basal diet for 3 weeks, the activities of amylase in hepatopancreas homogenates increased 125·3, 215·6 and 70·0%, respectively, the activities of amylase in intestine increased 79·8, 74·6 and 48·5%, respectively, and the activities of lipase in intestine increased 90·5, 149·8 and 84·0%, respectively. Supplementation of Cu, Fe or Zn into diet had no effects on activity of protease in all digestive organs. Therefore, the results suggest that effects of Cu, Fe and Zn on activity of digestive enzymes in vitro were different from those seen in vivo, and that the positive effects of Cu, Fe and Zn supplemented to fish diet would be valuable information for formulating fish feed.     

Effect of cadmium exposure on lactate dehydrogenase activity in the hepatopancreas and abdominal muscle of the fiddler crab,Uca pugilator

1. Lactate dehydrogenase (LDH) activity in the hepatopancreas and abdominal muscle of fiddler crabs, Uca pugilator, was determined after 24 and 48 hr of exposure to 2 ppm cadmium chloride.2. For the cadmium exposed crabs, LDH activity in the hepatopancreas decreased, whereas that in the abdominal muscle increased.3. The increased LDH activity in the abdominal muscle may reflect increased dependence on anaerobic carbohydrate metabolism in fiddler crabs exposed to cadmium in their environment.     

In vitro and in vivo effects of copper on haemocyanin-o2 binding in the shore crab,Carcinus maenas

1. When added in vitro to crab haemolymph at concentrations of 50 or 100 mg.1⁻¹, copper decreased both haemocyanin-O₂ affinity and the cooperativity of O₂ binding.2. In crabs contaminated by a lethal dose of waterborne copper (2mg.l⁻¹), haemolymph total concentration of the metal never reached levels that could affect O₂ binding properties directly.3. Exogenous copper added in vitro or entering the animal in contaminated water was found for the most part non filterable and thus probably bound to haemolymph proteins.     

Characterization of candidate intermediates in the Black Box of the ecdysone biosynthetic pathway in Drosophila melanogaster: Evaluation of molting activities on ecdysteroid-defective larvae

Early steps of the biosynthetic pathway of the insect steroid hormone ecdysone remains the “Black Box” wherein the characteristic ecdysteroid skeleton is built. 7-Dehydrocholesterol (7dC) is the precursor of uncharacterized intermediates in the Black Box. The oxidation step at C-3 has been hypothesized during conversion from 7dC to 3-oxo-2,22,25-trideoxyecdysone, yet 3-dehydroecdysone is undetectable in some insect species. Therefore, we first confirmed that the oxidation at C-3 occurs in the fruitfly, Drosophila melanogaster using deuterium-labeled cholesterol. We next investigated the molting activities of candidate intermediates, including oxidative products of 7dC, by feeding-rescue experiments for Drosophila larvae in which an expression level of a biosynthetic enzyme was knocked down by the RNAi technique. We found that the administration of cholesta-4,7-dien-3-one (3-oxo-Δ^4,7C) could overcome the molting arrest of ecdysteroid-defective larvae in which the expression level of neverland was reduced. However, feeding 3-oxo-Δ^4,7C to larvae in which the expression levels of shroud and Cyp6t3 were reduced inhibited molting at the first instar stage, suggesting that this steroid could be converted into an ecdysteroid-antagonist in loss of function studies of these biosynthetic enzymes. Administration of the highly conjugated cholesta-4,6,8(14)-trien-3-one, oxidized from 3-oxo-Δ^4,7C, did not trigger molting of ecdysteroid-defective larvae. These results suggest that an oxidative product derived from 7dC is converted into ecdysteroids without the formation of this stable conjugated compound. We further found that the 14α-hydroxyl moiety of Δ⁴-steroids is required to overcome the molting arrest of larvae in loss of function studies of Neverland, Shroud, CYP6T3 or Spookier, suggesting that oxidation at C-14 is indispensable for conversion of these Δ⁴-steroids into ecdysteroids via 5β-reduction.     

Silver nanoparticle pollutants activate oxidative stress responses and rosmarinic acid accumulation in sage

In this study, physiological and molecular responses of sage (Salvia officinalis) to silver nanoparticles (SNPs) were studied. It is supposed that sage oxidative responses can be activated to overcome the negative effects of SNPs. Results showed the penetration of SNPs via leaf epidermis into the parenchyma cells after foliar application. A significant decrease of photosynthetic pigments and increase of cell injury indicators, the activity of enzymatic antioxidants and also the content of non-enzymatic antioxidants were observed after exposure of sage plants to 50 and 1000 mg l⁻¹ SNPs compared to control plants. Phenolic compounds generally increased, but not in linear response to the dose level. The most abundant phenolic acid, rosmarinic acid (RA), increased more than eightfold at 100 mg l⁻¹ SNPs. Furthermore, the content of RA, salvianolic acid A and B was positively correlated with the activity of phenylalanine ammonia-lyase and RA synthase, but not with tyrosine aminotransferase. It could be concluded that the content of phenolic compounds increased in response to lower SNPs concentrations (50 and 100 mg l⁻¹). However, the oxidative stress responses continued above these concentrations.     

三疣梭子蟹蜕壳周期肝胰腺、外壳和鳃中钙含量的变化

研究测定了三疣梭子蟹蜕壳周期肝胰腺、鳃和外壳中的钙含量。结果显示,在不同时间,鳃中钙含量变化较小,各时间点差异不显著;蜕壳前期至蜕壳刚完成阶段三疣梭子蟹(软壳蟹)肝胰腺中钙含量最高,蜕壳后第4 d肝胰腺中钙含量最低。三疣梭子蟹外壳中钙含量随着外壳的硬化(钙化)逐渐增加,完全硬化后外壳中钙含量达到最高。研究表明,蜕壳后三疣梭子蟹肝胰腺中钙含量的降低与外壳的钙化之间具有关联性,肝胰腺在三疣梭子蟹蜕壳后钙的储存中起着重要作用,而鳃在钙离子储存中所起的作用可能小于钙转移的作用。     

The ecdysteroid receptor

Summary

The steroid molting hormone of insects and other arthropods regulates gene activity in target tissues through its association with a specific, high affinity receptor protein. In this review we summarize recent advances in several areas of ecdysteroid receptor research, including efforts to characterize and purify the receptor protein, cytochemical studies of its tissue distribution and subcellular localization during development, and current molecular genetic studies ecdysteroid action.     

Influence of the concentrations of d-xylose and yeast extract on ethanol production by Pachysolen tannophilus

To determine the most favorable conditions for the production of ethanol by Pachysolen tannophilus, this yeast was grown in batch cultures with various initial concentrations of two of the constituents of the culture medium: d-xylose (s_o), ranging from 1 g·l⁻¹ to 200 g·l⁻¹, and yeast extract (l_o), ranging from 0 g·l⁻¹ to 8 g·l⁻¹. The most favorable conditions proved to be initial concentrations of S_o=25 g·l⁻¹ and l_o=4 g·l⁻¹, which gave a maximum specific growth rate of 0.26 h⁻¹, biomass productivity of 0.023 g·l⁻¹·h⁻¹, overall biomass yield of 0.094 g·g⁻¹, specific xylose-uptake rate (q_s) of 0.3 g·g⁻¹·h⁻¹ (for t=50 h), specific ethanol-production rate (q_E) of 0.065 g·g⁻¹·h⁻¹ and overall ethanol yield of 0.34 g·g⁻¹; q_s values decreased after the exponential growth phase while q_E remained practically constant.     

Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis

A feather-degrading bacterium was isolated from the gut of the tarantula Chilobrachys guangxiensis, and was classified as Bacillus subtilis (named Bacillus subtilis CH-1) according to both the phenotypic characteristics and 16S rRNA profile. The improved culture conditions for feather-degrading were 10.0 g l⁻¹ mannitol, 10.0 g l⁻¹ tryptone, 0.1 g l⁻¹ MgCl₂, 0.4 g l⁻¹ KH₂PO₄, 0.3 g l⁻¹ K₂HPO₄, 0.5 g l⁻¹ NaCl, and 2.0 g l⁻¹ intact feather, with pH 8.5 and 37 °C. In the optimized medium, the intact black feather was completely degraded by Bacillus subtilis CH-1 in 24 h. Furthermore, four kinds of enzymes which include extracellular protease Vpr, peptidase T, γ-glutamyl transpeptidase and glyoxalmethylglyoxal reductase were identified as having principal roles. Simultaneously, the relationship between the disulfide bond reducing activity (DRT) and the keratinase activity (KT) in B. subtilis CH-1 fermentation system was discussed. This is the first report for a feather-degrading enteric bacterium from tarantula. The identification of the enzymes shines a light on further understanding the molecular mechanism of feather-degrading by microbes.     

Effect of heavy metals (Cu,Cd and Pb) on aspartate and alanine aminotransferase in Ruditapes philippinarum (Mollusca: Bivalvia)

The accumulation of cadmium, copper and lead and their effects on aspartate and alanine aminotransferases in digestive gland, gills, foot and soft body in the clam Ruditapes philippinarum were examined. The animals were exposed to different concentrations: Cd (200–600 μg·l⁻¹), Pb (350–700 μg·l⁻¹) and Cu (10–20 μg·l⁻¹) for 7 days. The highest concentrations were found in digestive gland for cadmium and copper, and in gills for lead, and the lowest values were observed in the foot. Aspartate aminotransferase activity (AST), in general, was not inhibited by cadmium, lead or copper during the exposure. Only in clams exposed to cadmium (600 μg·l⁻¹, 7 days) and copper (20 μg·l⁻¹, 5 days) were observed significant differences (P<0.05) in foot and gills, respectively, with respect to control. In the case of alanine aminotransferase activity (ALT), significant differences were observed for cadmium and lead in treated animals with respect to control. With regard to copper, a decrease in ALT was observed in gills and foot exposed to 20 μg·l⁻¹. A significant correlation (P<0.05) was observed between ALT and metal accumulation for cadmium, copper and lead in gills. In the case of soft body, only cadmium and lead showed a significant correlation. In summary, R. philippinarum can be considered a bioindicator species for cadmium and lead accumulation and ALT could be useful as biomarker of sublethal stress for these metals in soft tissues and gills. Only gills can be considered an adequate target tissue for copper.     

Ethanol fermentation by an efficient strain,NRRL B-4286, of Zymomonas mobilis

We studied the effect of the initial substrate concentration over the range of 100–250 g·l⁻¹ on the fermentation kinetics in batch cultures of Zymomonas mobilis NRRL B-4286 on glucose, fructose, and sucrose, using an adapted initial inoculum. With increasing concentrations of substrate, parameters related to growth were more rapidly and strongly affected than those related to ethanol production. This strain produced 94.0 g·l⁻¹, 76.9 g·l⁻¹, and 66.5 g·l⁻¹ of ethanol at glucose, fructose, and sucrose concentrations of 200 g·l⁻¹, respectively, more than the amount produced by the efficient strain ZM4 (NRRL B-14023).