Effects of diets supplemented with either individual or combined probiotics,Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, on the immune response and disease resistance of the mud crab,Scylla paramamosain (Estampador)

Two trials, including firstly, diets incorporating individual or combined species of probiotics, Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, were fed to the mud crab, Scylla paramamosian, for 28 days to evaluate the growth, immunity and disease resistance, and secondly, the optimal level of B. subtilis E20 in the diet by tracking the growth, immunity and disease resistance after 28 days of feeding were analysed. No significant differences in growth, total haemocyte counts, respiratory bursts, superoxide dismutase, or glutathione peroxidase were found in the two trials. Crabs fed the B. subtilis E20‐containing diet (D20) had significantly increased phenoloxidase (PO) activity, phagocytic activity (PA) and disease resistance compared with crabs fed the control and the mixed probiotics diet (MD). The mortality of crabs fed the D20 was significantly lower than that of crabs fed the L. plantarum 7‐40‐containing diet. L. plantarum 7‐40 had a great inhibitory effect on the growth of B. subtilis E20, which may have led to the decreased probiotic effect of the MD. An analysis of the optimal level of B. subtilis E20 in the diet showed that crabs fed the B. subtilis E20‐containing diet at a level of 10^9–10 cfu kg^?1 had better immune response of PO and PA, and disease resistance in the second trial.     

Ex‐post facto analysis of diseases of the Gulf of Mexico′s white shrimp Litopenaeus setiferus

This study presents an Ex‐Post Facto analysis of diseases of wild juvenile and adults of Litopenaeus setiferus collected from a field survey at the Natural Protected Area of Terminos lagoon, southern Mexico. The objective of the present approach was to determine if sampling site and/or shrimp age were contributing risk factors for disease between juvenile and adult shrimp; if there was a determined period of time in a year cycle when diseases were more critical, and if the analysis would help to decision‐ making considering what population would pose less risk of disease‐carrying when withdrawn for experimental purposes; all under an after‐the‐fact (ex‐post facto) approach. We identified that juvenile shrimp were at more risk of contracting some diseases in the estuarine environment and June, July and August months, were found to be a critical period when colonizing and parasitic diseases maintained a significant high prevalence in the shrimp population. These assumptions may help for decision‐making when wild shrimp have to be withdrawn from their natural environment for research purposes.     

Molecular cloning and expression of kidney‐type glutaminase from common carp (Cyprinus carpio) and its up‐regulation by glutamine in primary culture enterocyte

Glutaminase (GLS) is the key enzyme of glutamine (Gln) metabolism and utilization. In this study, a cDNA encoding GLS protein was identified from common carp Cyprinus carpio intestine. The open reading frame of GLS cDNA encodes a polypeptide of 595 amino acids, which shows a high similarity with its zebrafish Danio rerio counterpart. Bioinformatic analysis showed the protein belongs to kidney‐type GLS. The putative protein has glutaminase domain and ankyrin repeats domain, which are highly conserved among vertebrate orthologues. Real‐time quantitative PCR analysis revealed that the abundance of GLS mRNA was the highest in the white muscle, followed by the brain, eyeball and pituitary. Glutaminase was ubiquitously expressed in all intestinal segments of common carp. The activity of GLS did not distribute uniformly along the entire length of the intestine. In primary culture enterocyte, and the expression of GLS mRNA is up‐regulated quickly and effectively by Gln.     

The potential of pet‐grade poultry by‐product meal to replace fish meal in the diet of the juvenile spotted rose snapper Lutjanus guttatus (Steindachner, 1869)

A 12‐week feeding trial was conducted to examine the replacement of fish meal with pet‐grade poultry by‐product meal (PBM‐PG) in the spotted rose snapper Lutjanus guttatus diet. Five experimental diets were formulated to contain graded levels of PBM‐PG at proportion of 250, 500, 75 or 900 g kg^?1. The control diet contained sardine fish meal as the main protein source. Four groups of 15 randomly assigned L. guttatus juveniles were fed to satiation 3 times day^?1. Except for the fish fed the PBM‐PG90 diet, the growth performance, survival and feed utilization efficiency of the experimental fish were not significantly lower than those of the control fish. The dietary level of PBM‐PG did significantly affect the haematological characteristics (P < 0.05). The dietary dry matter and protein apparent digestibility coefficients (ADCs) decreased with increasing dietary PBM‐PG. High values for lipid ADCs were observed in all diets, with significant differences among the dietary treatments. The fish whole‐body protein, moisture, lipid and ash contents were not affected by the inclusion of dietary PBM. These results indicate that high‐quality terrestrial PBM can successfully replace more than half of the marine fish meal protein in the L. guttatus diet.     

Dummy regression analysis for modelling the nutritionally tailored fillet fatty acid composition of turbot and sole using gilthead sea bream as a reference subgroup category

Farmed turbot and sole were sampled at different stages of the production cycle for analysis of fillet lipid content and fatty acid (FA) composition. The entire data set along with our own published data on gilthead sea bream were fitted to dummy regression equations with turbot and sole as dummy variables, gilthead sea bream as a reference subgroup category, and diet FA composition and fillet lipid content as independent variables. The relative contribution of each independent variable to the total variance was found to vary within and among FAs and fish species, but strong correlation coefficients (0.76 <  r² > 0.99) were found for almost all of the FA equations, including saturated FAs, monoenes and long‐chain polyunsaturated fatty acids (PUFA) of n‐3 and n‐6 series. Given the differences in lipogenic activities of the fish species, major interaction effects between fillet lipid content and dummy variables were found for monoenes and saturated FAs. The proposed equations (hosted at www.nutrigroup-iats.org/aquafat ) were able to fit different proportions of EPA, DPA and DHA underlying the fish species differences in FA desaturation/elongation pathways. The robustness of the model was proven with extra data from the three fish species, allowing a close linear association near to equality for the scatter plot of observed and predicted values.     

Long‐term survival and colony growth of Acropora palmata fragments transplanted by volunteers for restoration

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Comparative and collaborative studies for the validation of a nested PCR for the detection of Xanthomonas axonopodis pv. dieffenbachiae from Anthurium samples

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N‐PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N‐PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90·6, 88·7 and 47·2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92·5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N‐PCR assay. The N‐PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N‐PCR had undeniable advantages compared to the reference method (less labour‐intensive and less time‐consuming). In addition, post‐test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N‐PCR assay has since been included in a revised version of the EPPO detection protocol.     

Genetic diversity of the root‐knot nematode Meloidogyne ethiopica and development of a species‐specific SCAR marker for its diagnosis

Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species‐specific SCAR‐PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species‐specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root‐knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.