Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

Homo floresiensis: a cladistic analysis

The announcement of a new species, Homo floresiensis, a primitive hominin that survived until relatively recent times is an enormous challenge to paradigms of human evolution. Until this announcement, the dominant paradigm stipulated that: 1) only more derived hominins had emerged from Africa, and 2) H. sapiens was the only hominin since the demise of Homo erectus and Homo neanderthalensis. Resistance to H. floresiensis has been intense, and debate centers on two sets of competing hypotheses: 1) that it is a primitive hominin, and 2) that it is a modern human, either a pygmoid form or a pathological individual. Despite a range of analytical techniques having been applied to the question, no resolution has been reached. Here, we use cladistic analysis, a tool that has not, until now, been applied to the problem, to establish the phylogenetic position of the species. Our results produce two equally parsimonious phylogenetic trees. The first suggests that H. floresiensis is an early hominin that emerged after Homo rudolfensis (1.86 Ma) but before H. habilis (1.66 Ma, or after 1.9 Ma if the earlier chronology for H. habilis is retained). The second tree indicates H. floresiensis branched after Homo habilis.     

Partial protective responses induced by a recombinant cysteine proteinase from Leishmania (Leishmania) amazonensis in a murine model of cutaneous leishmaniasis

A 500 bp fragment encoding an isoform of cysteine proteinase from Leishmania (Leishmania) amazonensis was subcloned and expressed in the pHis vector, resulting in a recombinant protein of 24 kDa, rLacys24. In Western blots of L. (L.) amazonensis extracts, antibodies directed to rLacys24 recognized a cysteine proteinase isoform of 30 kDa. Analysis by fluorescence-activated cell sorter showed a significantly higher expression of CD8⁺ lymphocytes in animals immunized with rLacys24 plus CFA, whereas a low expression of CD4⁺ lymphocytes was observed in these animals. The cytotoxicity of lymphocytes isolated from mice immunized with rLacys24 plus CFA on L. (L.) amazonensis-infected macrophages was significantly higher than that observed in the presence of lymphocytes from control animals. Immunization of BALB/c mice with rLacys24 plus CFA resulted in a low but significant decrease of foot lesions after challenge with L. (L.) amazonensis compared to those exhibited by control mice.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

Nutritional value of the cryptophyte Rhodomonas lens for Artemia sp.

Juvenile or adult Artemia sp. are often used as live prey for the rearing of early life stages of some crustacean, fish and cephalopod species. The improvements of both Artemia growth and its biochemical composition are key issues for the suitable use of Artemia biomass in these rearing processes. In this study we evaluated the growth and survival rates of Artemia fed with the cryptophyte Rhodomonas lens in comparison with different microalgal species commonly used in aquaculture: the prasinophyte Tetraselmis suecica, the prymnesiophyte Isochrysis galbana Parke, and the eustigmatophyte Nannochloropsis gaditana. Microalgae were cultured semi-continuously in nutrient saturated conditions and with a daily renewal rate of 30% of the volume of cultures, to obtain biomass of controlled and optimized composition. Considerable differences in Artemia growth were observed, as well as in the survival rate. At day 8 of rearing, Artemia fed R. lens had the highest length (4.9 ±0.6 mm, P < 0.001), followed by individuals fed T. suecica (4.2 ± 0.7 mm), I. galbana (3.6 ± 0.7 mm) and finally those fed N. gaditana (1.5 ± 0.2 mm). The survival rate of Artemia fed N. gaditana (18 ± 3%) was much lower (P < 0.001) than values found for the remaining groups (69 to 88%). The growth rate of Artemia obtained with R. lens was in general much higher than with other microalgal diets previously reported in the literature. The higher protein content of R. lens could explain the higher growth obtained with this species, but differences of Artemia growth with the different diets could not be explained solely on the basis of the gross composition of microalgae. Factors such as cell size and digestibility all seem to contribute to the results observed. Another trial was carried out to investigate differences in Artemia growth and on its biochemical composition when fed the best two diets: R. lens or T. suecica. The fatty acid (FA) and total amino acid (AA) composition of both microalgal species and the composition of Artemia were assessed as well. As found in the first experiment individuals fed R. lens (group ARHO) grew faster than those fed T. suecica (group ATET), attaining 3.6 ± 0.3 mm and 3.2 ± 0.4 mm (P < 0.001), respectively, after 5 days of rearing. The much higher AA content obtained in R. lens may be on the basis of the higher growth obtained with this species. Protein and carbohydrate levels in Artemia juveniles were very similar in both groups (64-68% of dry weight, and 8-10%, respectively). Lipid was slightly lower in ARHO (12%) than in ATET (15%, P < 0.01). Regarding the FA composition, juveniles from group ARHO contained higher levels of eicosapentaenoic acid (EPA, 6.2%) than juveniles from ATET (4.1%, P < 0.01), whereas docosahexaenoic acid (DHA) was only found in juveniles from ARHO (1.1%). Taking into account that the daily productivity of R. lens culture was higher than, or at least equal, the remaining microalgal species this cryptophyte is confirmed as an excellent diet to optimize the growth of Artemia, as well as to improve its biochemical composition.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

Presence of Nosema ceranae in honeybees (Apis mellifera) in Uruguay

The microsporidium Nosema ceranae is an emergent pathogen of European honeybees Apis mellifera. Using a PCR-RFLP diagnosis, 29 samples of infected honeybees obtained in 2007-2008 (N = 26), 2004 (N = 2) and before 1990 (N = 1) were analyzed for the presence of Nosema apis and N. ceranae. Only N. ceranae was found in all samples, indicating that this species dispersed to Uruguay (and likely the region) at some time before 1990. The presence of N. ceranae in Uruguay is not associated with an increase of Nosemosis, and its role in colony loss seems to be irrelevant.     

Nematicidal activity of Haplophyllum tuberculatum and Plectranthus cylindraceus oils against Meloidogyne javanica

The potentials of Haplophyllum tuberculatum and Plectranthus cylindraceus oils to control Meloidogyne javanica were investigated in vitro and in a greenhouse. A mixture of Haplophyllum and Plectranthus oils (1:1) was highly toxic to M. javanica in vitro, as it killed all nematode juveniles and inhibited hatching of eggs at 12.5 μg/ml concentration after 24 h exposure time, as did carbofuran at the same concentration. In the green-house, tomatoes grown in soil treated with a combination (1:1) of the two oils developed fewer root galls than those grown in soil treated with higher doses of either oil. The oil mixture, at 2.5 and 5.0 μg/ml of soil, was not phytotoxic to tomato plants as evident from the appearance and height of plants after 12 weeks exposure time, compared to treatment over the same period at lower effective doses. The nematicidal activity of the combined essential oils was suggested by the presence of C₁₀ dienes, C₁₀ trienes and C₁₀ phenol.     

Acylated flavonol tetraglycosides from Delphinium gracile

An ethanol extract of the aerial parts of Delphinium gracile DC. yielded five flavonol glycosides quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-caffeoyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (1), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (2), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(Z-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (3), kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (4) kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (5) in addition to 4-(β-d-glucopyranosyloxy)-6-methyl-2H-pyran-2-one (6) and rutin. Structures were elucidated by spectroscopic methods.     

Decolorization of malachite green by cytochrome c in the mitochondria of the fungus Cunninghamella elegans

We studied the decolorization of malachite green (MG) by the fungus Cunninghamella elegans. The mitochondrial activity for MG reduction was increased with a simultaneous increase of a 9-kDa protein, called CeCyt. The presence of cytochrome c in CeCyt protein was determined by optical absorbance spectroscopy with an extinction coefficient (E_550-535) of 19.7 ± 6.3 mM⁻¹ cm⁻¹ and reduction potential of + 261 mV. When purified CeCyt was added into the mitochondria, the specific activity of CeCyt reached 440 ± 122 μmol min⁻¹ mg⁻¹ protein. The inhibition of MG reduction by stigmatellin, but not by antimycin A, indicated a possible linkage of CeCyt activity to the Qo site of the bc1 complex. The RT-PCR results showed tight regulation of the cecyt gene expression by reactive oxygen species. We suggest that CeCyt acts as a protein reductant for MG under oxidative stress in a stationary or secondary growth stage of this fungus.     

Association between osteoporosis and polymorphisms of the bone Gla protein, estrogen receptor 1, collagen 1-A1 and calcitonin receptor genes in Turkish postmenopausal women

In this study, we have investigated the association between osteoporosis and osteocalcin (BGLAP) − 298 C>T, estrogen receptor 1 (ER1) 397 T>C, collagen type1 alpha 1 (Col1A1) 2046 G>T and calcitonin receptor (CALCR) 1340 T>C polymorphisms. Genomic DNA was obtained from 266 persons (158 osteoporotic and 108 healthy controls). Genomic DNA was extracted from EDTA-preserved peripheral venous blood of patients and controls by a salting-out method and analyzed by PCR-RFLP. As a result, there was no statistically significant difference in the genotype and allele frequencies of patients and controls for BGLAP − 298 C>T, Col1A1 2046 G>T, ER1 397 T>C and CALCR 1340 T>C polymorphisms. However, ER1 CC genotype compared with TT + TC genotypes was found to increase the two fold the risk of osteoporosis [p = 0.039, OR = 2.156, 95% CI (1.083–4.293)] and CALCR CC genotype compared with TT + TC genotypes was found to have protective effect against osteoporosis [p = 0.045, OR = 0.471, 95% CI (0.237–0.9372)]. In the combined genotype analysis, ER1/CALCR TCCC combined genotype was estimated to have protective effect against osteoporosis [p = 0.0125, OR = 0.323, 95% CI (0.1383–0.755)] whereas BGLAP/Col1A1 CCTT and ER1/CALCR CCTT combined genotypes were estimated as risk factors for osteoporosis in Turkish population (p = 0.027, p = 0.009 respectively).     

Protective role of Mangifera indica, Cucumis melo and Citrullus vulgaris peel extracts in chemically induced hypothyroidism

An investigation was made to evaluate the pharmacological importance of fruit peel extracts of Mangifera indica (MI), Citrullus vulgaris (CV) and Cucumis melo (CM) with respect to the possible regulation of tissue lipid peroxidation (LPO), thyroid dysfunctions, lipid and glucose metabolism. Pre-standardized doses (200 mg/kg of MI and 100 mg/kg both of CV and CM), based on the maximum inhibition in hepatic LPO, were administered to Wistar albino male rats for 10 consecutive days and the changes in tissue (heart, liver and kidney) LPO and in the concentrations of serum triiodothyronine (T₃), thyroxin (T₄), insulin, glucose, α-amylase and different lipids were examined. Administration of three test peel extracts significantly increased both the thyroid hormones (T₃ and T₄) with a concomitant decrease in tissue LPO, suggesting their thyroid stimulatory and antiperoxidative role. This thyroid stimulatory nature was also exhibited in propylthiouracil (PTU) induced hypothyroid animals. However, only minor influence was observed in serum lipid profile in which CM reduced the concentrations of total cholesterol and low-density lipoprotein-cholesterol (LDL-C), while CV decreased triglycerides and very low-density lipoprotein-cholesterol (VLDL-C). When the combined effects of either two (MI + CV) or three (MI + CV + CM) peel extracts were evaluated in euthyroid animals, serum T₃ concentration was increased in response to MI + CV and MI + CV + CM treatments, while T₄ level was elevated by the combinations of first two peels only. Interestingly, both the categories of combinations increased T₄ levels, but not T₃ in PTU treated hypothyroid animals. Moreover, a parallel increase in hepatic and renal LPO was observed in these animals, suggesting their unsafe nature in combination. In conclusion the three test peel extracts appear to be stimulatory to thyroid functions and inhibitory to tissue LPO but only when treated individually.     

Structural characterization and protective effect against murine sepsis of fucogalactans from Agaricus bisporus and Lactarius rufus

Fucogalactans from edible Agaricus bisporus (RFP-Ab) and wild Lactarius rufus (RFP-Lr) mushrooms were obtained on aqueous extraction followed by purification. RFP-Ab had M_w 43.8 × 10⁴ g mol⁻¹ and RFP-Lr M_w 1.4 × 10⁴ g mol⁻¹. RFP-Lr had a (1 → 6)-linked α-d-Galp main-chain partially substituted at O-2 by nonreducing end-units of α-l-Fucp (29%). While RFP-Ab had a similar main chain, it was partially substituted at O-2 by nonreducing end-units of α-l-Fucp (2.8%) and β-d-Galp (14.5%), and partially methylated at HO-3. Both RFP-Lr and RFP-Ab were tested in mice against polymicrobial sepsis. Lethality rate, myeloperoxidase (MPO) activity and cytokine levels were determined. It was observed a reduction in late mortality rate by 62.5% and 50%, respectively, prevention of neutrophil accumulation in ileum and decreasing in TNF-α and IL-1β serum levels.