An Aspartic Protease With Antimicrobial Activity is Induced after Infection and Wounding in Intercellular Fluids of Potato Tubers

Aspartic proteases (APs) one of the main proteinase classes, have different physiological functions in animals, fungi and viruses. In plants, knowledge of the biological roles of APs is less well developed. An AP has been purified from potato tuber and leaves (Guevara et al., 1999, 2001). In this paper, the changes in the level of AP in response to infection by Phytophthora infestans (P. infestans) and wounding were studied in intercellular washing fluids (IWFs) from tuber disks of two potato cultivars differing in their susceptibility to P. infestans. A differential induction was observed between both cultivars: in the resistant cultivar, induction was higher and faster in infected tissues than in wounded ones. In the susceptible cultivar, a lower and later accumulation was observed than in the resistant cultivar. In addition, AP had a direct inhibitory effect on the germination of cysts of P. infestans and conidia of Fusarium solani. The pattern of accumulation and in vitro activity of AP suggest that this enzyme may have a role in the defense response of potato.     

Alternative pathways in the life history of Gracilaria gracilis (Gracilariales, Rhodophyta) from north-eastern Sicily (Italy)

The reproductive phenology of Gracilaria gracilis has been followed closely in a brackish water lake in Sicily. The life history of this red alga deviated from the typical Polysiphonia-type cycle and was characterized by the predominance of the tetrasporophytic phase over gametophytes, in situ development of germinated gametophytes and the presence of sterile pleustophytic thalli. Release of spherical agglomerates of cells produced by young tetrasporophytes was observed as well. Their ability to germinate forming new thalli appears to represent an alternative reproductive strategy of this organism. The population structure appears to reflect a predominance of the diploid phase except during the warmer summer season when only vegetative juveniles were found in the lake. Release and early stages of development of carpospores and tetraspores were critically observed in culture. A Dumontia type pattern of germination was observed for both types of spores, showing an erect thallus arising from a multicellular basal disk. Compared to tetraspores, carpospores are less readily released resulting in a subsequent delay in the development of new gametophytes.     

Osmotic adjustment,proline accumulation and cell membrane stability in leaves of Cocos nucifera submitted to drought stress

The role of drought-induced proline accumulation in coconut leaves is still unclear. With the objective of evaluating the impact of water shortage on leaf osmotic potential, proline accumulation and cell membrane stability in young plants of two Brazilian Green Dwarf coconut ecotypes from contrasting areas (Brazilian Green Dwarf from Una, Bahia, UGD, and from Jiqui, Rio Grande do Norte, JGD), a pot experiment was conducted under greenhouse conditions. Three drought cycles consisting of suspension of irrigation until the net photosynthetic rate (A) approached zero and rewatering until recovery of A to 85% of the irrigated control plants. Pre-dawn leaf water potential (Ψ_PD) reached −1.2 MPa at the point of maximum stress (PMS). Dry matter production and leaf area were severely reduced by drought treatment in the two ecotypes. Corrected values of osmotic potential were significantly reduced in stressed plants of the two ecotypes. Green dwarf coconut palm showed low osmotic adjustment (from 0.05 to 0.24 MPa) and significant accumulation of proline (from 1.5 to 2.1 times in relation to control) in leaflets in response to water deficit. Considering the growth reduction observed in both ecotypes, proline was not associated to osmoregulation. On the other hand, the absence of membrane damage, as indicated by electrolyte leakage method, suggests that the protective role of proline in this specie can be more important. The two ecotypes of Green dwarf coconut palm behaved similarly in the present experiment for most traits evaluated. Slight differences among the ecotypes were observed with respect to the response to treatments, such as higher proline accumulation in JGD.     

Transformation of Anaplasma marginale

The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, ≤1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80–90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination.     

Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method

Babesiosis and Theileriosis are tick-borne diseases widespread in tropical and sub-tropical regions with high economic impact worldwide. In Portugal there are at least 4 tick vectors known to be competent for the transmission of Babesia and Theileria sp. identified: Rhipicephalus bursa, Rhipicephalus (Boophilus) annulatus, Ixodes ricinus and Haemaphysalis punctata. All these potential Babesia and Theileria tick vectors are widely distributed in Portugal, although they are predominant in the Southern region. In this study, 1104 cattle blood samples were randomly collected from Central and Southern regions of Portugal and analyzed by PCR-reverse line blotting (RLB) for the detection of Babesia and Theileria sp. Testing indicated that 74.7% of the bovines tested were positive for either Babesia and/or Theileria sp. In addition, five different apicomplexan species, namely, Theileria buffeli, Theileria annulata, Babesia divergens, Babesia bovis, and Babesia bigemina were detected by RLB among the bovines tested. T. buffeli was the most frequently found species, being present in 69.9% of the positive samples either as single infections (52.4%), or as mixed infections (17.5%). The Babesia specie most frequently found was B. divergens, detected in 4.2% of the infected bovines. Overall, infected bovines were found in all regions tested; however the highest number of infected bovines was observed in évora district (96.2%) and in cattle from Limousin breeds (81.7%). The results indicate widespread Babesia and Theileria infections in Portuguese bovines, suggesting the need for improved control of ticks and tick-borne diseases.     

Effect of antibiotic treatment on the growth and survival of juvenile northern Chilean scallop, Argopecten purpuratus Lamarck (1819), and associated microflora in experimental cultures

Juvenile northern Chilean scallops of 937±55 μm shell height were exposed to five different concentrations of chloramphenicol (CHL) (5, 10, 25, 50 and 100 μg mL⁻¹), plus a control without antibiotics. To determine the effect of CHL on the accompanying microflora, the number of colony-forming units (CFU) that grew on TGE culture medium was counted in the seawater of containers with juveniles, and in containers with microalgae used as food. Both were exposed to the same concentrations of CHL. The growth rates of juveniles treated with CHL and the control without antibiotic showed highly significant differences ( P =0.0001). The growth rate was inversely proportional to the CHL concentration. The control sample presented the highest growth rate (84.4±14.3 μm day⁻¹), followed by the sample treated with 5 μg mL⁻¹ (64.2±14.3 μm day⁻¹). The survival in the control and in the treated samples with 5–50 μg mL⁻¹ was rather high, with a mean value of 95%. Only the sample treated with 100 μg mL⁻¹ had a low survival (36.7%). The CFU count was larger in the containers with juveniles, when compared with the ones with food. The CFU count tended to decrease with increasing CHL concentration in the juveniles.     

Dietary saponins and phytosterols do not affect growth,intestinal morphology and immune response of on‐growing European sea bass (Dicentrarchus labrax)

This study was the first to investigate the effect of soy antinutrients, saponins and phytosterols, in on‐growing sea bass. Seven diets were formulated: a control diet (fishmeal and fish oil based) and six diets containing 1 or 2 g kg^?1 levels of soya saponins, 5 or 10 g kg^?1 levels of phytosterols or a combination of 1 g kg^?1 saponins + 5 g kg^?1 phytosterols or 2 g kg^?1 saponins + 10 g kg^?1 phytosterols. After a 59 days feeding trial fish were weighed, faeces collected for digestibility studies and plasma sampled for cholesterol quantification. At day 15 and 59, distal intestine (DI) samples were collected for histological evaluation and quantification of maltase (Malt), alkaline phosphatase (AlP) and leucine aminopeptidase (LAP) activities. At day 59, DI was collected for gene expression of AlP, Malt and immune related genes. Antinutrients had no effect on fish performance, digestibility, plasma cholesterol levels and digestive enzymes activities and gene expression. Only mild morphological changes were present in groups fed high levels of saponins + phytosterols. Expression of immune related genes showed no differences between groups. Overall, on‐growing sea bass showed high tolerance to dietary antinutrients up to the levels tested.     

Improved digestibility of plant ingredient‐based diets for European seabass (Dicentrarchus labrax) with exogenous enzyme supplementation

This study evaluated the effect of supplementation of a plant ingredients‐based diet with two levels of commercially digestive enzyme products: Synergen? and Natugrain^® on the apparent digestibility coefficients (ADCs) of nutrients and on digestive enzyme activities in European seabass (Dicentrarchus labrax) juveniles. A control diet was formulated with 450 g/kg CP (200 g/kg from fish meal + 800 g/kg from plant protein) and 180 g/kg CL (fish oil), and four other diets were made similar to the control but supplemented with 0.2 g/kg or 0.4 g/kg of Synergen? and Natugrain^®. Digestive enzyme activities were not affected by dietary treatments. Nevertheless, chyme pH along the intestine of fish fed the Natugrain^®‐supplemented diets was reduced. The ADC of nutrients increased with exogenous digestive enzyme supplementation, with the highest ADC values being observed for the 0.4 g/kg Natugrain^® diet. The ADC of dry matter, protein, lipids, energy and phosphorus in this diet were 26%, 6%, 4%, 16% and 63% higher, respectively, than in the control diet. Overall, diet supplementation with exogenous enzyme complexes seems to have high potential for improving the utilization of plant feedstuff‐rich diets for seabass juveniles. This study revealed that Natugrain^® was more effective than Synergen? in improving the ADC of nutrients and energy in European seabass juveniles.     

Prebiotics effect on growth performance,hepatic intermediary metabolism,gut microbiota and digestive enzymes of white sea bream (Diplodus sargus)

This study aimed at evaluating the effects of short‐chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS) and galactooligosaccharides (GOS) on growth performance, hepatic metabolism, gut microbiota and digestive enzymes activities of white sea bream juveniles. Four diets were formulated: a control diet with fish meal (FM) and plant feedstuffs (PF) (30FM:70PF) and three test diets similar to control but supplemented with 10 g of scFOS, XOS or GOS per kilo diet, which were fed to fish during 12 weeks. Prebiotics had no effect on growth, feed efficiency or gut microbiota. Plasmatic triglycerides were lower in fish fed XOS than FOS and GOS diets. Malic enzyme activity was lower in fish fed XOS than FOS diet. Fish fed XOS diet had lower fatty acid synthetase (FAS), a key lipogenic enzyme and higher alanine aminotransferase activities. Fifteen days after the start of the trial, an enhancement of total alkaline protease, trypsin and lipase activities was observed in fish fed prebiotics, but such effect disappeared at 12 weeks. In conclusion, scFOS, XOS or GOS seem to have limited applicability in white sea bream feed.     

Stress response and lipid composition in shrimp Litopenaeus vannamei fed diets enriched with squid or scallop viscera meal

We substituted 10% of fishmeal with scallop (Argopecten ventricosus) or squid (Dosidiscus gigas) by‐product meal in a feed for shrimp (Litopenaeus vannamei) growth assay that lasted 60 days and then shrimps were submitted to handling stress. Shrimp fed the control diet had smaller weight gain and lower recovery after stress. Prostaglandins in gills were affected by diet and stress, with significant differences in shrimp fed the control diet before and after the stress. Hydroperoxides were increased after stress, while isoprostanoids were higher in shrimp fed the control diet. The gene expression of Glutathione peroxidase and Catalase was increased on the stressed shrimp fed the scallop diet, with no differences among diets for Superoxide dismutase or Cyclooxygenase. Arachidonic acid in the phospholipid fraction of muscle was the only fatty acid affected by stress and diet. Cholesterol oxidation products were significantly higher in shrimp fed the scallop diet compared to shrimp fed the control diet before applying the stress. A PCA analysis grouped in one‐factor growth variables, as well as Superoxide dismutase, esterified astaxanthin and isoprostane, while another factor was determined by stress, prostaglandin and hydroperoxide levels in gills. The partial substitution of fishmeal with scallop of squid by‐product meals affected growth and stress differently, indicating shrimp that grew up more were not necessarily more tolerant to handling stress, which can have a strong repercussion in aquaculture, particularly during density control, recording of growth and harvest.