Cryptogein and capsicein promote defence responses in Quercus suber against Phytophthora cinnamomi infection

The decline of cork oak (Quercus suber) stands in Iberian Peninsula is associated with infection by Phytophthora cinnamomi. Most Phytophthora species secrete elicitins, which can enhance defence reactions against some pathogens. Here cytological and physiological effects of the elicitins cryptogein and capsicein on cork oak root infection by P. cinnamomi were evaluated. The progression of the pathogen in root tissue and its effects on total fatty acid (TFA) composition of roots and leaves were analysed in seedlings. Net photosynthesis (P _n ), stomatal conductance (g _s ), chlorophyll a fluorescence measurements (quantum yield of linear electron transport ? _e , photochemical quenching q _P, non-photochemical quenching NPQ) and carotenoid determinations were carried out in well established (4?months) plants. In elicitin-treated roots, 2?days after inoculation, the pathogen which presented loss of viability and membrane degradation was mainly restricted to the intercellular spaces of the cortical parenchyma, and did not reach the vascular cylinder. Electron dense materials accumulated in the intercellular spaces of the cortex next to disorganized hyphae, suggested to be related with defence reactions. Cryptogein (or its interaction with P. cinnamomi) induced enhanced lipid synthesis in leaves, which may contribute to preserve membrane stability. P. cinnamomi decreased P _n , g _s , ? _e , and q _P, whereas elicitin-treated plants displayed values similar to controls. Overall, the results indicated a resistance response of cork oak against this oomycete, induced by the elicitins.     

Isolation of a Protein Bound to Canker-forming Factor from Citrus Plant

Interaction analysis using affinity analysis (Affinity Sensors, Cambridge, UK) indicated the presence of proteins bound to Apl1, a virulence factor of Xanthomonas campestris pv. citri required for the formation of canker symptom on citrus, in the fraction 25–50% ammonium sulfate of citrus crude extracts. Three proteins of 25, 50 and 110-kD were eluted from an Apl1-affinity column. Western analysis revealed that Apl1 binds specifically to the 25-kD and 110-kD but not to the 50-kD protein. When crude extracts of soybean and of tobacco were applied to the Apl1-affinity column, only faint bands were detected. This result suggests that Apl1 targets exist specifically in citrus plants. The amino acid sequence of the N-terminal of the 25-kD protein was determined, and homology search analysis revealed that this sequence was almost identical to those commonly present in S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT) from several plants. This enzyme is specific to the substrate trans -caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA for lignin formation. Received 4 November 1999/ Accepted in revised form 26 November 1999     

Study of peripheral blood cell populations involved in the immune response of goats naturally infected with Mycobacterium caprae

Tuberculosis in goats caused by Mycobacterium bovis and Mycobacterium caprae has noteworthy sanitary and economic implications. Current diagnostic assays are based on cellular immunity and although they have demonstrated a high sensitivity, some animals remain undetected. In the present study, flow cytometry has been used to determine changes in CD4+, CD8+ and CD25+ T cell populations in peripheral blood from naturally infected goats. Proportion of lymphocytes producing PPD-specific interferon-gamma (IFN-γ) was calculated and an ELISA for detection of PPD-specific IFN-γ was performed to measure the cytokine in plasma. The infected goats showed percentages of CD4+ T cells between 27.31% and 47.23% and there were not significant differences (p=0.113) with the non-infected control goats although the mean percentage was lower in this group. Regarding CD8+ T cells, a higher percentage was observed in healthy goats compared to controls (p=0.081). The mean percentage of lymphocytes expressing CD25 without antigen stimulation (30.65±3.91) was higher in lesion and/or culture-positive animals than in the controls (21.84±1.21; p=0.053). The percentage of CD4+/IFN+ T cell population stimulated with bovine PPD was a reliable marker of infection, since the mean percentage in the infected goats was significantly higher than in the controls (p<0.05). Tuberculosis in goats caused by M. caprae induced changes in cellular populations similar to those described for M. bovis in cattle.     

Alpha‐Linolenic Acid Supplementation in Tris Extender Can Improve Frozen–Thawed Bull Semen Quality

The study was conducted to evaluate the effects of α ‐linolenic acid (ALA) on frozen–thawed quality and fatty acid composition of bull sperm. For that, twenty‐four ejaculates obtained from three bulls were diluted in a Tris extender containing 0 (control), 3, 5, 10 and 15 ng/ml of ALA. Extended semen was incubated at 37°C for 15 min, to allow absorption of ALA by sperm cell membrane. The sample was chilled for 2 h, packed into 0.25‐ml straws and frozen in liquid nitrogen for 24 h. Subsequently, straws were thawed and evaluated for total sperm motility (computer‐assisted semen analysis), membrane functional integrity (hypo‐osmotic swelling test), viability (eosin‐nigrosin), fatty acid composition (gas chromatography) and lipid peroxidation (thiobarbituric acid‐reactive substances (TBARS)). A higher (p < 0.05) percentage of total sperm motility was observed in ALA groups 5 ng/ml (47.74 ± 07) and 10 ng/ml (44.90 ± 0.7) in comparison with control (34.53 ± 3.0), 3 ng/ml (34.40 ± 2.6) and 15 ng/ml (34.60 ± 2.9). Still, the 5 ng/ml ALA group presented a higher (p < 0.05) percentage of viable sperms (74.13 ± 0.8) and sperms with intact membrane (74.46 ± 09) than all other experimental groups. ALA concentration and lipid peroxidation in post‐thawed sperm was higher in all treated groups when compared to the control group. As such, the addition of 5 ng/ml of ALA to Tris extender improved quality of frozen–thawed bull spermatozoa.     

Contamination of the Coastal Waters of Gijón (North West Spain) by Butyltin Compounds

An innovative method for the determination of butyltin compounds in seawater samples based on the use of isotope dilution analysis has been applied to study the contamination of butyltin compounds in the Bay of Gijón (Spain) where no previous data existed. The sampling in this bay allowed the evaluation of mono-, di-and tributyltin (MBT, DBT and TBT, respectively) contamination in very different environmental compartments (harbour, marina, shipyards and enclosed and open beaches). The results showed clearly a strong influence of the type of location, the water renewal and the proximity to contaminated areas on the levels of butyltin compounds. It was found that in more than 75% of the samples the TBT values were found to be higher than the US-EPA water quality criteria of 7.4 ng L^?1 and that in 95% of the samples the concentration of TBT exceeded 1 ng L^?1 which has been reported to be a toxic level for many marine species.     

Toxocara canis infection induces antigen-specific IL-10 and IFNgamma production in pregnant dogs and their puppies

Toxocara canis (T. canis) is originally a parasite of canine bitches and their pups. The pathogenicity of T. canis infection is enhanced during pregnancy and puppyhood. The aim of this study was to investigate if modification of IFNgamma and IL-10 secretion occurs during infection in pregnant dogs and puppies. Analysis of cytokines secreted could let us hypothesize a role for IL-10 and/or IFNgamma in T. canis infection. We tested T. canis-specific production of IFNgamma and IL-10 by lymphocytes of pregnant dogs and their puppies after in vitro re-exposure to purified excretory/secretory antigen (ESAg) from T. canis. Blood mononuclear cells (BMC) isolated from pregnant dogs and their puppies were cultured in the presence of ESAg. Cultures' supernatants were tested for cytokine levels by ELISA. Results obtained showed that IL-10 concentrations increased during pregnancy in infected animals and in the meantime IFNgamma production decreased. In puppyhood, we observed that, IL-10 concentration decreased with the age of puppies mainly in infected animals while IFNgamma increased. In conclusion, our data suggests that BMC of infected dogs have a particular modification of IL-10 and IFNgamma synthesis. These data could be the basis to design immunotherapeutic approaches.     

Growth, Survival, Yield, and Size Distributions of Freshwater Prawn Macrobrachium rosenbergii and Tilapia Oreochromis niloticus in Polyculture and Monoculture Systems in Puerto Rico

Abstract.— Tilapia and freshwater prawn production in Puerto Rico for monoculture and polyculture systems were compared. The experiment consisted of three treatments with three replicates each. The stocking rates for the prawn monoculture, fish monoculture and polyculture treatments were respectively: 7 prawns/m², 1 tilapia/M², and 7 prawns with 1 tilapia/ m². The mean stocking size for tilapia and prawn were respectively, 7–8 g and 1–1.3 g. After 145 d of culture, yields and mean weight of tilapia in monoculture and polyculture system were not significantly different. Total yields were 2,942 and 2,769 kg/ha, respectively. Mean weights were 348 g in monoculture and 331 g in polyculture. Yields and mean weight of prawns in monoculture and polyculture were significantly different. Total yields were 1,367 and 951 kg/ha, respectively. Mean weights were 55 g in monoculture and 31 g in polyculture. Total yield in polyculture was 3,720 kg/ha showing an increase over the production separately obtained in prawn and fish monoculture.     

Substitution of fishmeal with soybean meal in practical diets for juvenile white shrimp Litopenaeus schmitti (Pérez‐Farfante & Kensley 1997)

The feasibility of substituting soybean meal for fishmeal diets for juvenile white shrimp Litopenaeus schmitti (0.35±0.01 g) was evaluated, and an adequate substitution level was determined. Five diets were evaluated using 46%, 59%, 75%, 88% and 100% substitution levels. Pellet water stability was significantly affected by dietary soybean content (P<0.05). Increased soybean content produced lower pellet stability, ranging from a dry matter loss of 14–22% after a 2‐h immersion, and 20–33% after an 8‐h immersion. After 52 days, significant differences (P<0.05) were found in shrimp weight, feed conversion ratio and protein efficiency ratio. The values were 0.64–1.06 g, 2.8–7.9 and 0.45–1.21, respectively, for the three measurements. Overall, better results were obtained with diets where soybean meal was substituted for fishmeal up to 75%. The 100% soybean meal diet resulted in poor growth performance of shrimp. Survival rates were acceptable for all treatments (90% or higher) and no significant differences were found in survival between treatments. Regression analysis using the broken‐line methodology indicated that 76.5±2% is an optimum soybean substitution level in diets that contained fishmeal and soybean as the major protein sources for grow‐out of juvenile white shrimp.     

An Integration of Water Physicochemistry,Algal Bioassays,Phytoplankton, and Zooplankton for Ecotoxicological Assessment in a Highly Polluted Lowland River

Results of different approaches potentially useful for the evaluation of water ambient quality were analysed and compared in a small temperate lowland river with mixed diffuse and multiple-point source pollution. The Reconquista River (Buenos Aires Province, Argentina), one of the most polluted watercourses of Latin America, receives agrochemicals as well as domestic and industrial (mostly untreated) effluents. Physical and chemical water variables were determined; unispecies algal bioassays (with Chlorella pyrenoidosa and Scenedesmus acutus) were carried out in laboratory; and density and structure of phyto- and zooplankton were analysed at three sites in four dates (representing a range of likely conditions in the river). A general scheme of association among plankton, bioassays and physical/chemical variables was elaborated, that helped to infer possible control factors in this multi-stressed system. Some empirical methods, but mainly mathematical ones including multivariate techniques (as PCA, cluster analysis), were applied for evaluation of samples. A preliminary selection of indices and attributes as potential indicators of the water river quality was made, and then applied for assay a tentative integrative ordination of samples. The relative best water quality was recorded when-where dissolved oxygen concentration, algal diversity and planktonic crustacean density were higher. The worst water quality corresponded to the lack of cladocerans and lowest crustacean density, and higher: organic and industrial pollution, major nutrients (ammonium and orthophosphates), BOD, hardness, conductivity, algal biomass in bioassays, phytoplankton density (>10 000 ind. mL^-1), dominance of a single algal species (>90%), and rotifer proportion in zooplankton (>85%).     

Microbial community structure and function in a soil contaminated by heavy metals: effects of plant growth and different amendments

We studied the effects of in situ remediation of a heavy metal (HM) contaminated soil on some soil chemical properties, microbial function and microbial structural diversity after 18 months. The experiment was carried out at semifield scale in containers filled with HM contaminated soil from the Aznalcóllar mine accident (Southern Spain, 1998). The remediation measures consisted of the application of different amendments and/or establishment of a plant cover (Agrostis stolonifera L.). Seven treatments were established: four organic treatments (municipal waste compost (MWC), biosolid compost (BC), leonardite (LEO) and litter (LIT)), one inorganic treatment (sugar beet lime (SL)) and two controls (control with plant cover (CTRP) and control without plant cover (CTR)). Several soil chemical (pH, soluble HM, total organic C (TOC), water-soluble C (WSC) and available-P) and biochemical properties (microbial biomass C (MBC), MBC/TOC ratio and enzyme activities) were determined. Microbial community structure was studied by means of ARDRA (amplified ribosomal DNA restriction analysis). The SL, MWC and BC treatments were the most efficient to raise soil pH and decrease soluble HM concentrations. Total organic C was increased in the organic treatments by 2 to 4-fold, whereas water-soluble C was statistically similar in the CTRP, SL and the organic treatments, probably due to the presence of a root system in all these treatments. Available-P was also increased in the BC, SL and MWC treatments due to the higher P content of the amendments applied in these treatments. Soil microbial function was generally enhanced in the amended and CTRP treatments. The MWC, BC and SL treatments were particularly efficient to increase microbial biomass C, the MBC/TOC ratio and the dehydrogenase and aryl-sulphatase enzyme activities. These results could be attributed to the amelioration of some of the soil chemical properties: increase in soil pH and water-soluble C and decrease of HM soluble concentrations. ARDRA analyses showed changes in structural diversity in both the bacterial and fungal community under the different treatments. Fingerprinting patterns of the 16S rDNA obtained with Hinf-I and of the 18S rDNA with Hpa-II revealed higher similarity percentages among samples from the same treatment compared with samples from the other treatments. In addition, a higher similarity was found between samples from all treatments under the Agrostis influence. The use of certain amendments and/or a plant cover is important for in situ remediation of HM contaminated soils, since these practices can affect soil chemical properties, as well as the microbial community function and structure.