A PCR-based diagnostic assay for detecting DNA of the olive fruit fly, Bactrocera oleae, in the gut of soil-living arthropods.

Bactrocera oleae (Rossi) (Diptera: Tephritidae) is considered the most devastating pest of the olive tree worldwide. In an effort to develop management and biological control strategies against this pest, new molecular tools are urgently needed. In this study, we present the design of B. oleae-specific primers based on mitochondrial DNA sequences of cytochrome oxidase subunit I (COI) gene. Two pairs of B. oleae-specific primers were successfully designed and named as SBo1-F/SBo1-R and SBo2-F/SBo1-R, being able to amplify 108 and 214 bp COI fragments, respectively. The specificity of designed primers was tested by amplifying DNA from phylogenetically related (i.e. Diptera order) and other non-pest insects living in olive groves from the Mediterranean region. When using these primers on a PCR-based diagnostic assay, B. oleae DNA was detected in the gut content of a soil-living insect, Pterostichus globosus (Fabricius) (Coleoptera: Carabidae). The detection of B. oleae DNA in the guts of arthropods was further optimized by adding bovine serum albumin enhancer to the PCR reaction, in order to get a fast, reproducible and sensitive tool for detecting B. oleae remains in the guts of soil-living arthropods. This molecular tool could be useful for understanding pest-predator relationships and establishing future biological control strategies for this pest.

Feeding preferences and functional responses of Calathus granatensis and Pterostichus globosus (Coleoptera: Carabidae) on pupae of Bactrocera oleae (Diptera: Tephritidae).

Carabid beetles are important predators in agricultural landscapes feeding on a range of prey items. However, their role as predators of the olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), one of the most serious pests of olives, is unknown. In this context, the feeding preferences and the functional responses of two carabid beetle species, Calathus granatensis (Vuillefroy) and Pterostichus globosus (Fabricius), were studied under laboratory conditions. Feeding preference assays involved exposing carabid beetles to different ratios of B. oleae pupae and an alternative prey, the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Both species fed on B. oleae pupae however, C. granatensis always showed a significant preference for that prey whereas P. globosus switched to C. capitata pupae when the offered ratio was below 0.5. The total prey biomass consumed was significantly higher for P. globosus than for C. granatensis. Functional response curves were estimated based on different densities of B. oleae pupae and both carabid beetle species exhibited a type II functional response using Rogers' random-predator equation. P. globosus showed shorter handling time (1.223 ± 0.118 h) on B. oleae pupae than C. granatensis (3.230 ± 0.627 h). Our results suggest that both species can be important in reducing the densities of B. oleae in olive groves, although P. globosus was more efficient than C. granatensis.

Activity of Thymus capitellatus volatile extract, 1,8-cineole and borneol against Leishmania species.

In the search for new leishmanicidal agents, Thymus capitellatus Hoffmanns. & Link (family Lamiaceae) volatile extract and its major compounds, 1,8-cineole and borneol, were tested against Leishmania infantum, Leishmania tropica and Leishmania major. Plant volatile extract (essential oil) was analysed by GC and GC-MS and the activity of essential oil on Leishmania promastigotes viability was assessed using tetrazolium-dye colorimetric method (MTT). The MTT test was also used to assess the cytotoxicity of essential oil on macrophages and bovine aortic endothelial cells. Effects on parasites were also analyzed by flow cytometry in order to assess mitochondrial transmembrane electrochemical gradient (JC-1), analyze phosphatidylserine externalization (annexin V-FITC, propidium iodide) and evaluate cell cycle (DNase-free, RNase, PI). Morphological and ultrastructural studies were performed by light, scanning and transmission electron microscopy. T. capitellatus volatile extract exhibited anti-parasite activity on Leishmania species, with IC50 values ranging from 35 to 62 µg/ml. However, major compounds 1,8-cineole and borneol did not showed biological activity suggesting that these monoterpenes are not responsible for the antileishmanial activity of T. capitellatus essential oil. Appearance of aberrant-shaped cells, mitochondrial swelling and autophagosomal structures were some of the ultrastructural alterations exhibited among treated promastigote cells. T. capitellatus promoted leishmanicidal effect by triggering a programmed cell death as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. The volatile extract did not induced cytotoxic effects on mammalian cells. Taken together, these results suggest that T. capitellatus may represent a valuable source for therapeutic control of leishmaniasis in humans and animals.

Monoterpenic aldehydes as potential anti-Leishmania agents: activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major.

In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leishmania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and major compounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropica and L. major growth at IC(50) concentrations ranging from 25 to 52 µg/ml and from 34 to 42 µg/ml, respectively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastructural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures, disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citral promoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidal activity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findings lead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both may represent a valuable source for therapeutic control of leishmaniasis.

Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: effects on growth, viability, adherence and ultrastructure.

The present work evaluates the anti-Giardia activity of Syzygium aromaticum and its major compound eugenol. The effects were evaluated on parasite growth, adherence, viability and ultrastructure. S. aromaticum essential oil (IC(50)=134 µg/ml) and eugenol (IC(50)=101 µg/ml) inhibited the growth of G. lamblia. The essential oil inhibited trophozoites adherence since the first hour of incubation and was able to kill almost 50% of the parasites population in a time dependent manner. The eugenol inhibited G. lamblia trophozoites adherence since the third hour and not induce cell lyses. The main morphological alterations were modifications on the cell shape, presence of precipitates in the cytoplasm, autophagic vesicles, internalization of flagella and ventral disc, membrane blebs, and intracellular and nuclear clearing. Taken together, our findings lead us to propose that eugenol was responsible for the anti-giardial activity of the S. aromaticum essential oil and both have potential for use as therapeutic agents against giardiasis.

Levels of ochratoxin A in serum from urban and rural Portuguese populations and estimation of exposure degree.

Urban and rural population exposure to ochratoxin A (OTA) in central zone of Portugal was investigated in three places: Coimbra, Verride and Ereira. The analytical method proposed for the determination of ochratoxin A involved extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column (IAC), high performance liquid chromatography (HPLC) with spectrofluorimetric detection (FD) for separation and identification of ochratoxin A, and confirmation with HPLC-FD after OTA methylation in serum. The limit of quantification of the proposed method was 0.1 microg/L for serum and 0.05 microg/L for blood. OTA recoveries in serum ranged from 70.3% to 115.3% for levels at 0.25 microg/L and 0.5 microg/L, respectively, with a within-day RSD between 8.0% and 16.2%. Ochratoxin A serum levels were evaluated in an hundred and four donors from Coimbra city, Verride, and Ereira. The study revealed a frequency of detection of 100%. The ratio of ochratoxin A level in serum to whole blood was 2.0+/-0.7. The overall concentrations range from 0.25 to 2.49 microg/L, 0.14 to 1.91 microg/L, and 0.19 to 0.96 microg/L, for samples of Verride, Ereira, and Coimbra, respectively. The mean concentration and standard deviation were 0.78+/-0.53 microg/L, 0.44+/-0.31 microg/L, and 0.42+/-0.18 microg/L for the same samples. A significant difference was found in Verride population (P-value=0.000). Levels of OTA are clearly higher in males from rural areas than in females. For all samples, a significant difference was found in Verride male population (P-value=0.014).

Ochratoxin A in nephropathic patients from two cities of central zone in Portugal.

Ochratoxin A (OTA) produced by Aspergillus and Penicillium genera contaminates several foods. OTA is nephrotoxic to all animal species studied so far, and most likely to humans, who show the longest half-life for elimination of this toxin among all examined species. OTA has other toxic effects such as teratogenicity, immunotoxicity, genotoxicity, and is also mutagenic and carcinogenic, all of which lead to life-threatening pathologies through several molecular pathways. A sensitive, specific and rapid method applying high performance liquid chromatography coupled to a spectrofluorimeter for the determination of ochratoxin A in human serum was validated. Serum samples were extracted with chloroform-orthophosphoric acid, and cleaned-up through immunoaffinity column (IAC). The separation and identification was performed by HPLC coupled to a spectrofluorimeter, and, after OTA methylation, the confirmation was achieved. Chromatographic separation of the analyte was performed on a reverse phase column with a mobile phase of water:acetonitrile:glacial acetic acid (49.5:49.5:1.0). Linearity was established between the range of 1 and 10 ng/ml. Under the optimized conditions, the recoveries were higher than 83.0% for all fortification levels. The intra-day precision oscillated between 8.0 and 5.0% at levels of 0.25 and 0.5 microg/l, while the inter-day precision was in the range of 10.7-16.0%. The limit of quantification of the method was 0.05 microg/l. The method is appropriate for quantitative determination of OTA in human serum and has been successfully applied to the analysis of OTA in haemodialysis patients from two principal cities of Portugal, in order to evaluate its exposure degree. Levels of OTA in Coimbra were higher than in Aveiro, 0.50 microg/l versus 0.49 microg/l. In respect to gender, levels of OTA were higher in males from Aveiro than in females, 0.52 microg/l versus 0.44 microg/l, and in Coimbra were similar, 0.50 microg/l versus 0.51 microg/l. However, in none of the cases, significant statistical differences were found.

Ultrastructure of the mature pollen of Michelia figo (Lour.) Spreng. (Magnoliaceae).

The structural organization of the Michelia figo mature pollen was investigated. The pollen wall consisted of an outer exine and an inner intine, the former being coated by a thin polysaccharide pellicle. The intine comprised three structurally distinct layers that were equally thick throughout the pollen surface. The generative cell (GC) was closely associated with the vegetative cell (VC) nucleus and its periplasm was found to maintain communication with the sporoderm through a complex plasmalemmic cord. In the freeze-fixed pollen a fluffy coat was detected on the cytoplasmic face of the VC plasmalemma bordering the GC. The plastids were present in only the VC and usually contained abundant small starch grains. In a few pollen grains, however, little or no starch existed and in this case one or more electron dense inclusions appeared in the plastids. Microbodies were found in both the VC and GC. In the VC they presumably have a glyoxysomal function as indicated by the numerous lipid droplets in the cytoplasm and the spatial relationship of microbodies with lipid droplets and/or mitochondria. In the GC the function of the microbodies is unclear once this cell had no abundant lipid reserves and the microbodies did not show any preferential relationship with other organelles. The most conspicuous feature of the VC cytoplasm was the high amount of storage vacuoles which displayed a striking different appearance after one and the other of the fixation techniques used. In contrast to the chemically fixed pollen they were quite polymorphic in the freeze-fixed pollen, and appeared uniformly filled with fibrillar material. Enzymatic digestion with protease has revealed most of this material to be proteinaceous in nature. The existence of phytin reserves is, however, also probable. These protein storage vacuoles closely resemble those in storage tissues of seeds and fruits.