Genotyping of Cryptosporidium Isolates from Chamelea gallina Clams in Italy

Chamelea gallina clams collected from the mouths of rivers along the Adriatic Sea (central Italy) were found to harbor Cryptosporidium parvum (genotype 2), which is the lineage involved in zoonotic transmission. The clams were collected from the mouths of rivers near whose banks ruminants are brought to graze. This paper reports the environmental spread of C. parvum in Italy and highlights the fact that genotyping of seaborne Cryptosporidium isolates is a powerful tool with which to investigate the transmission patterns and epidemiology of this microorganism.     

Genotyping of Cryptosporidium isolates from Chamelea gallina clams in Italy

Chamelea gallina clams collected from the mouths of rivers along the Adriatic Sea (central Italy) were found to harbor Cryptosporidium parvum (genotype 2), which is the lineage involved in zoonotic transmission. The clams were collected from the mouths of rivers near whose banks ruminants are brought to graze. This paper reports the environmental spread of C. parvum in Italy and highlights the fact that genotyping of seaborne Cryptosporidium isolates is a powerful tool with which to investigate the transmission patterns and epidemiology of this microorganism.     

Mechanical disturbance affects haemocyte functionality in the Venus clam Chamelea gallina

The clam Chamelea gallina is fairly common along the western coastline of the Adriatic Sea, where it is subjected to intense fishing. To evaluate possible causes of stress in clam populations, the effects of acute mechanical disturbance on haemocyte functionality were analysed in both laboratory and field experiments. Among the various sources of stress that clams undergo during commercial fishing by hydraulic dredges, water pressure and mechanical sorting were considered, and three increasing levels of stress were applied. A reduction in immunosurveillance was highlighted, concentrations of circulating haemocytes, phagocytic and acid phosphatase activity indices decreased with increasing mechanical stress. The opposite trend shown by the beta-glucuronidase activity index is discussed. The response of clam haemocytes, detected on seasonal bases in two sites, often exhibited peculiar patterns, mostly depending on the reproductive cycle, and were probably influenced by different features of the sea bottom. Although recovery after stress was observed in laboratory experiments, some considerations are made on detrimental effects experienced in the field by under-sized clams, which are first fished and then thrown back into the sea.     

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

Morphological and cytoenzymatic characterization of haemocytes of the venus clam Chamelea gallina

A morphological and enzymatic characterization of Chamelea gallina haemocytes was carried out as a prerequisite for further studies on venus clam immunobiology. Two main types of circulating haemocytes were identified (1) hyalinocytes (79.2%), agranular cells with a central nucleus surrounded by a little cytoplasm, and (2) granulocytes (16.5%), smaller granular cells with smaller nuclei. Small cells with a strongly basophilic nucleus and a thin layer of peripheral cytoplasm, probably undifferentiated blast cells (4.3%), were also observed. Both granulocytes and hyalinocytes can assume a spreading or round morphology. The enzymatic activities of haemocytes were also investigated. Some of the granulocytes and hyalinocytes were positive for hydrolytic enzymes, suggesting a role for these cells in phagocytosis; no oxidative enzymes were detected in C. gallina haemocytes. Granulocytes and hyalinocytes can easily adhere to the substratum and exhibit a low phagocytosis activity towards foreign particles (6.3%), whereas the fraction of cells containing ingested material significantly increased after pre-incubation of test particles with cell-free haemolymph, which suggests the presence of opsonin(s) in the haemolymph.     

缺铁逆境胁迫下水稻叶片蛋白的双向电泳及其蛋白质组图谱分析

水稻幼苗经缺铁胁迫诱导分别处理1、3、5天后,用酚法和TCA/丙酮法提取叶片中的可溶性蛋白进行双向电泳分析,从而研究在缺铁条件下叶片中蛋白表达的动态变化规律.结果显示1.不同pH IPG胶条分离蛋白的效果不同.用pH3-10的IPG胶条进行双向电泳,经考马斯亮蓝染色后,可在胶面上检测到大约450个蛋白点,其中约有89%的蛋白是酸性蛋白.如果用pH4-7的IPG胶条进行双向电泳,则可检测到大约600个蛋白点,其中有29个蛋白是上调表达,1个蛋白是下调表达,5个蛋白是诱导特异表达.2.不同方法提取的可溶性蛋白质量不同.TCA法简单易操作,似乎对于碱性蛋白的抽提效果更好,在2-DE图像上,减性端显示的蛋白点多;但此方法所得蛋白的再溶性差.酚法提取的蛋白再溶性好,所抽提的蛋白量较大,纯度较高.     

Comparative proteomic analysis of salt-responsive proteins in canola roots by 2-DE and MALDI-TOF MS

Salinity stress is a major abiotic stress that affects plant growth and limits crop production. Roots are the primary site of salinity perception, and salt sensitivity in roots limits the productivity of the entire plant. To better understand salt stress responses in canola, we performed a comparative proteomic analysis of roots from the salt-tolerant genotype Safi-7 and the salt-sensitive genotype Zafar. Plants were exposed to 0, 150, and 300 mM NaCl. Our physiological and morphological observations confirmed that Safi-7 was more salt-tolerant than Zafar. The root proteins were separated by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry was applied to identify proteins regulated in response to salt stress. We identified 36 and 25 protein spots whose abundance was significantly affected by salt stress in roots of plants from the tolerant and susceptible genotype, respectively. Functional classification analysis revealed that the differentially expressed proteins from the tolerant genotype could be assigned to 14 functional categories, while those from the susceptible genotype could be classified into 9 functional categories. The most significant differences concerned proteins involved in glycolysis (Glyceraldehyde-3-phosphate dehydrogenase, Fructose-bisphosphate aldolase, Phosphoglycerate kinase 3), stress (heat shock proteins), Redox regulation (Glutathione S-transferase DHAR1, L-ascorbate peroxidase), energy metabolism (ATP synthase subunit B), and transport (V-type proton ATPase subunit B1) which were increased only in the tolerant line under salt stress. Our results provide the basis for further elucidating the molecular mechanisms of salt-tolerance and will be helpful for breeding salt-tolerant canola cultivars.     

Glycosidases of molluscs. Purification and properties of alpha-L-fucosidase from Chamelea gallina L.

An alpha-L-fucosidase had been purified approximately 300-fold from the liver (hepatopancreas) of the marine mollusc Chamelea gallina L. (= Venus gallina L.). During the different steps of the purification procedure it was difficult to remove the contaminant N-acetylglucosaminidase activity; but, after electrofocusing, a final preparation free of this and other glycosidades present in the crude extract was obtained. The purified enzyme has a broad specificity; it hydrolyzes p-nitrophenyl alpha-L-fucoside and natural substrates such as oligosaccharides containing fucosidic residues with alpha 1--2, alpha 1--3 and alpha 1--4 linkages; also it hydrolyzes fucose-containing glycopeptides, such as thyroglobulin glycopeptide, and glycoproteins as procine submaxillary mucin (previously rendered free of sialic acid). The enzyme has a pH optimum of 5.2 +/- 0.2, with a Km of 7 X 10(-5) M using p-nitrophenyl L-fucoside as substrate. It is inhibited by Hg2+ and some sugars, and activated by CN-, Zn2+, Ca2+ and EDTA. It shows two peaks by isoelectric focusing (at 6.3 and 6.6). The molecular weight of the alpha-L-fucosidase by gel filtration was over 2000000.     

Use of shape to distinguish Chamelea gallina and Chamelea striatula (Bivalvia: Veneridae): Linear and geometric morphometric methods

The commercially fished striped venus clams Chamelea gallina and C. striatula (Bivalvia: Veneridae) are difficult to distinguish by inexperienced observers and the taxonomy of these species is still an issue of discussion. The differences in shape between C. gallina and C. striatula from Portuguese coastal waters were studied through conventional linear and geometric morphometric analysis, using both contour (elliptic Fourier analysis) and landmark-based methods. The relationships shell length vs. height, width, and total weight were significantly different between species. However, because there was a considerable overlap in the data sets, the species could not be distinguished using any combination of those linear measurements. Geometric morphometric methods provided shape variables that led to 0-6% misclassification rates between species; linear morphometric measures led to 16.8% error. Contour analysis revealed differences primarily in the shell umbo and lunular area. The umbo was more "sharp" and the lunula less pronounced in C. striatula than in C. gallina. Generalized procrustes superimposition (landmark analysis) showed that the main differences between species reside in the length of the pallial sinus. Thus, an index was developed (PI: Pallial Index = pallial sinus length/shell length), which was successfully used to separate the species (with 100% correct classification), i.e., specimens with PI lower than 0.119 belonged to C. gallina, whereas greater PI values were attributed to C. striatula. The use of these geometric morphometric methods allowed the detection of differences in shape between these two species and to develop an easy-to-use identification index. We encourage the development of analogous indices that apply the methods of geometric morphometrics to distinguish between other species whose identification is complicated.     

Oxygen and carbon isotope variations in Chamelea gallina shells: Environmental influences and vital effects

Stable isotopes in mollusc shells, together with variable growth rates and other geochemical properties, can register different environmental clues, including seawater temperature, salinity and primary productivity. However, the strict biological control over the construction of biominerals exerted by many calcifying organisms can constrain the use of these organisms for paleoenvironmental reconstructions. Biologically controlled calcification is responsible for the so called vital effects that cause a departure from isotopic equilibrium during shell formation, resulting in lower shell oxygen and carbon compared to the equilibrium value. We investigated shell oxygen and carbon isotopic composition of the bivalve Chamelea gallina in six sites along with a latitudinal gradient on the Adriatic Sea (NE Mediterranean Sea). Seawater δ¹⁸O and δ¹³C_DIC varied from North to South, reflecting variations in seawater temperature, salinity, and chlorophyll concentration among sites. Shell δ¹⁸O and δ¹³C differed among sites and exhibited a wide range of values along with the ~400 km latitudinal gradient, away from isotopic equilibrium for both isotopes. These results hampered the utilization of this bivalve as a proxy for environmental reconstructions, in spite of C. gallina showing promise as a warm temperature proxy. Rigorous calibration studies with a precise insight of environment and shell growth are crucial prior to considering this bivalve as a reliable paleoclimatic archive.     

Age related properties of the Adriatic clam Chamelea gallina (L. 1758) hemocytes

The clam Chamelea gallina (L 1758) represents an important shellfish resource along Mediterranean coasts and its progressive depletion has been ascribed both to the overexploitation of stocks and to environmental or anthropic stressors. In this context, the investigation on immune parameters could represent a valid approach to measure the clam homeostasis condition and its possible influence on population dynamics. On this basis, the innate immune system, mainly represented by hemocyte phagocytosis, was investigated in organisms of different size. The results indicated a better phagocytic response in larger clams, strictly related to a greater concentration of granulocytes. A such variation in hemolymph composition appeared not dependent on environmental or endogenous factors, but rather on clam aging.     

Comparative 2-DE proteomic analysis of date palm (Phoenix dactylifera L.) somatic and zygotic embryos

Two-dimensional gel electrophoresis coupled to mass spectrometry has been used to compare the proteome of date palm (Phoenix dactylifera L. cv. Deglet Nour) zygotic and somatic embryos. Proteins were trichloroacetic acid–acetone–phenol extracted, quantified, and resolved by 2-DE in the 5 to 8 pH range. Total protein content and number of resolved spots were higher in zygotic (110 ± 14.5 mg/g DW; 349 spots) than in somatic (70.96 ± 4.8 mg/g DW; 210 spots) embryos. The 2-DE map of both systems showed qualitative (263) and quantitative (72) differences. Statistical analysis of spot intensity was performed by PCA, obtaining two accurate groupings of the samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with average linkage algorithm of the Genesis software package. Sixty-three variable spots were subjected to mass spectrometry analysis, resulting in 23 identifications. Identified proteins were classified in the following functional categories; glycolysis (8 proteins), citrate cycle (1), ATP synthesis (1), carbohydrate biosynthesis (2), amino acids metabolism (1), stress related (4), storage (3), and with no function assigned for three of them. Most of the somatic embryo specific proteins identified belonged to glycolysis pathways, whereas those of the zygotic embryo to storage and stress-related proteins. Differences are discussed in terms of metabolism and biology of both types of embryos.     

Fractionation and identification of proteins by 2-DE and MS: towards a proteomic analysis of Plasmodium falciparum

Since completion of genome sequencing of the malarial parasite Plasmodium falciparum, proteomic tools for the identification of parasite proteins have become particularly attractive as they allow a more thorough interpretation of these data. Recent advances in 2-D PAGE, MS, and bioinformatics have created great opportunities for mapping and characterization of protein populations. We employed these improvements in a proteomic approach for the analysis of proteins detected in two blood stages of P. falciparum, (i) in the schizont stage and (ii) in the merozoite stage. For the isolation of merozoites, we introduced a new protocol based on the preparation of clustered structures of merozoites upon treatment of cultures with the common cysteine proteinase inhibitor E64. Peptide mass fingerprints of excised and trypsinated protein spots, acquired by MALDI-TOF MS were generated to identify a variety of proteins. Moreover, prefractionation procedures were used to enrich and map low-abundance proteins in protein samples. The data demonstrate that classic proteomic analyses using 2-D PAGE are now feasible for P. falciparum and represent the first step in the direction of creating 2-D reference maps for this medically most relevant protozoon.     

Host‐microbiota interactions shed light on mortality events in the striped venus clam Chamelea gallina

Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate‐ and pollution‐induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA‐sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over‐expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down‐regulation of several genes involved in immune and stress response, and the over‐representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment.     

Air exposure and functionality of Chamelea gallina haemocytes: effects on haematocrit, adhesion, phagocytosis and enzyme contents

The Venus clam Chamelea gallina is fairly common along the western coasts of the Adriatic and is subjected to intense fishing. Since over the last 20 years extensive hypoxic and anoxic conditions have repeatedly damaged this natural resource, we decided to study the effects of anoxic stress on the functionality of clam haemocytes and the consequences on immune responses. Clams, exposed to air, close their valves and tissues become anoxic and metabolism processes switch to anaerobiosis. In these conditions, a significant decrease in the haematocrit value and in the percentage of acid phosphatase-positive haemocytes was observed, while the number of cells with beta-glucuronidase significantly increased after day 1. The above indices generally returned to control values when clams were re-immersed in seawater after 1 day of treatment. Clams exposed to air for 2 days and then re-immersed, attempted to recover in the subsequent 3 days. Animals had fully recovered on day 4. Three-day-exposed clams did not recover. Phagocytic and adhesion indices decreased significantly after the first day of air exposure. The change in frequency of three types of circulating cells (spreading, round, apoptotic) was also monitored.     

Biochemical and energy dynamics throughout the reproductive cycle of the striped venus Chamelea gallina (Mollusca,Bivalvia)

The striped venus Chamelea gallina is an important commercial bivalve species in Europe. However, large inter-annual fluctuations in stock abundance and periodic recruitment failure threaten the biological and economic sustainability of this fishery. This study aimed to improve the knowledge of the reproductive cycle and reproductive strategies of this species from the Algarve coast (southern Portugal) in order to contribute to the establishment of management measures and to assess its potential for aquaculture. The reproductive cycle of C. gallina followed a seasonal cycle, significantly influenced by sea surface temperature and food availability. Gametogenesis took place in winter, coinciding with the phytoplankton bloom. Spawning occurred during summer, followed by a short period of sexual inactivity in autumn. Condition index did not reflect the reproductive cycle of C. gallina and generally, followed the same trend of chlorophyll a. Glycogen was positively correlated with gonadal index and chlorophyll a. High total lipid values were recorded throughout gonad ripeness and spawning, but decreased at the end of the spawning and in the rest period. The extended spawning period of C. gallina will allow larvae to be obtained for much of the year by artificial spawning of wild broodstock.     

Comparative 2-DE proteomic analysis of date palm (Phoenix dactylifera L.) somatic and zygotic embryos

Two-dimensional gel electrophoresis coupled to mass spectrometry has been used to compare the proteome of date palm (Phoenix dactylifera L. cv. Deglet Nour) zygotic and somatic embryos. Proteins were trichloroacetic acid–acetone–phenol extracted, quantified, and resolved by 2-DE in the 5 to 8 pH range. Total protein content and number of resolved spots were higher in zygotic (110 ± 14.5 mg/g DW; 349 spots) than in somatic (70.96 ± 4.8 mg/g DW; 210 spots) embryos. The 2-DE map of both systems showed qualitative (263) and quantitative (72) differences. Statistical analysis of spot intensity was performed by PCA, obtaining two accurate groupings of the samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with average linkage algorithm of the Genesis software package. Sixty-three variable spots were subjected to mass spectrometry analysis, resulting in 23 identifications. Identified proteins were classified in the following functional categories; glycolysis (8 proteins), citrate cycle (1), ATP synthesis (1), carbohydrate biosynthesis (2), amino acids metabolism (1), stress related (4), storage (3), and with no function assigned for three of them. Most of the somatic embryo specific proteins identified belonged to glycolysis pathways, whereas those of the zygotic embryo to storage and stress-related proteins. Differences are discussed in terms of metabolism and biology of both types of embryos.     

Polymorphic analysis of the three MHC-linked HSP70 genes

Three genes encoding members of the M _r 70 000 heat shock protein family (HSP70) are known to lie in the class III region of the human major histocompatibility complex. IN order to determine whether these genes or their protein products exhibit any polymorphism the three genes have been specifically amplified from genomic DNA and sequenced. The HSP70-1 and HSP70-2 genes encode the major heat-inducible HSP70. A comparison of the nucleotide sequences of these genes from B8, SC01, DR3, B18, F1C30, DR3, and B7, SC30, DR2 haplotypes has revelad only very limited sequence variation which is not associated with any amino acid polymorphism. The HSP70-Hom gene encodes a protein that is highly related to HSP70-1, but which is not heat-inducible. Nucleotide sequence analysis of this gene from different haplotypes has revealed a Met Thr amino acid substitution at residue 493 in a number of the haplotypes tested. This variable amino acid lies in the proposed peptide-binding site of the HSP70-Hom protein. Address correspondence and offprint requests to: R. D. Campbell.     

产热凝胶土壤杆菌ATCC31749蛋白质组双向电泳图谱的构建

建立了热凝胶生产茵土壤杆茵茵体总蛋白的蛋白质提取方法和双向电泳方案,确定了使用蛋白质裂解液(7 mol/L尿素,2 mol/L硫脲,1%ASB-14去垢剂,40 mmol/L Tris,0.001%溴酚蓝,1 mmol/L EDTA,1%TBP和1%两性电解质)结合超声破碎法来提取茵体总蛋白的方案为最佳,选择17 cm...