Production of specific dsRNA against white spot syndrome virus in the yeast Yarrowia lipolytica

White spot syndrome virus (WSSV) is the most aggressive disease affecting cultured shrimp. One possibility to tackle it is by means of RNA interference (RNAi) induced by the presence of double‐stranded RNA (dsRNA). Normally, dsRNA is a product of the cellular machinery to gene regulation, but it can be produced synthetically and introduced into specific tissues or cells and thereby induce RNAi. Although in vitro production of dsRNA is possible, this is high cost. An alternative is to produce dsRNA in vivo using biological systems such as bacteria or yeasts. In this regard, Yarrowia lipolytica offers distinctive advantages for dsRNA production. The objective was to develop a Y. lipolytica strain able to produce dsRNA‐specific against WSSV and to evaluate its antiviral activity in the white leg shrimp Litopenaeus vannamei. From the 0.4 and 0.6 Kb fragments of the ORF89 gene, a dsRNA‐ORF89‐producing construct was built in the plasmid pJC410; the resulting construct (pARY410) was used to transform Y. lipolytica to drive the specific expression of dsRNA‐ORF89. Yeast colonies positive to the WSSV‐ORF89 gene were selected. The expression of dsRNA‐ORF89 and RNAse III was measured being detected at 32 and 48 hr. Subsequently, the antiviral activity of dsRNA‐ORF89 was tested in a WSSV challenge bioassay. The results showed survival in dsRNA‐ORF89 shrimp (25%) compared to control organisms treated with total RNA from the yeast P01‐AS harvested at 32 hr. In conclusion, Y. lipolytica is a convenient host to produce and deliver dsRNA‐ORF89 able to protect WSSV‐challenged shrimp.     

Demographic and competition studies on <Emphasis Type="Italic">Brachionus ibericus</Emphasis> and <Emphasis Type="Italic">Proales similis</Emphasis> in relation to salinity and algal (<Emphasis Type="Italic">Nannochloropsis oculata</Emphasis>) density

We isolated the rotifers Brachionus ibericus and Proales similis from the sediment of shrimp tanks and studied their individual demographic characters and competition between them at two food levels (0.25?×?10⁶, 1.00?×?10⁶ cells ml^?1 of Nannochloropsis oculata at 25 °C) and salinities ranging from 10 to 30‰. Our hypothesis was that growth rates would be higher with increasing food levels and salinities. Observations were taken twice a day for life table studies and daily once for population growth experiments. Using survivorship and fecundity data, we derived various life history variables. Although the average life span (7.6?±?0.4 days) and gross reproductive rate (33.8?±?2.9 neonate female^?1 day^?1) of B. ibericus were higher than those of P. similis (average life span 5.4?±?0.6 days and gross reproductive rate 13.0?±?0.6 neonate female^?1 day^?1), the population growth experiments showed that P. similis had higher r values (0.32?±?0.005 day^?1) than B. ibericus (0.23?±?0.002 day^?1) at 1.0?×?10⁶ cells ml^?1 of N. oculata. The rotifer P. similis was more adversely affected due to the presence of B. ibericus than vice versa. The data are important for developing techniques for a large-scale culture of these rotifers as food in aquaculture.     

Purification and characterization of an alpha-mannosidase from the tropical fruit babaco ( Vasconcellea x heilbornii Cv. babaco)

An alpha-mannosidase (EC 3.2.1.24) present in the lyophilized latex of babaco ( Vasconcellea heilbornii ) has been purified to apparent homogeneity by native PAGE. The purification involves a three-step procedure with successive anion exchange with Q Sepharose HP, lectin affinity chromatography using ConA Sepharose 4B, and gel filtration using Superdex 200 prep grade. The molecular mass was determined to be in the range of 260-280 kDa by Superdex 200 prep grade gel filtration, and isoelectric focusing showed a pI range between 5.85 and 6.55, suggesting different glycosylated isoforms. The optimal temperature for the alpha-mannosidase was determined to lie between 50 and 60 degrees C, and the optimal pH was 4.5 at 50 degrees C. The K(m) value for p-nitrophenyl alpha-mannopyranoside (pNPM) was found to be 1.25 mM and the V(max), 2.4 microkat mg(-1) at 50 degrees C and 1.94 microkat mg(-1) at 40 degrees C. The pure alpha-mannosidase was specific for mannose and did not display activity for any other tested synthetic substrates.     

Identification of a mannose‐binding lectin‐like protein recognized by the anti‐CD25 antibody in haemocytes from Cherax quadricarinatus

The crustacean haemolymph contains three main cell populations; however, it is not clear which mechanisms participate in the regulation of cells related to innate immunity. This work aimed to identify potential interleukin‐like receptors that could regulate cellular responses in Cherax quadricarinatus. By histochemical analysis with murine anti‐CD25 staining (targeting the α‐chain of the IL‐2 receptor), we identified that this antibody recognizes cytoplasmic granules in semigranular and granular haemocytes. In haemocytes stimulated with phorbol myristate acetate (PMA), increased fluorescence was observed in these cytoplasmic granules, whereas staining with a human IL‐2 antibody after stimulation with 1–10 ng/ml PMA revealed no overexpression of the receptor or oxidative burst in haemocytes. Two‐dimensional Western blot analysis of haemocyte lysates showed that anti‐CD25 identified a 27.4‐kDa protein with an isoelectric point (pI) of 7.7 and a 46‐kDa protein with a pI of 6.9. De novo sequencing of these proteins identified that they had 32% homology with a mannose‐binding lectin (MBL) from Pacifastacus leniusculus. Our results indicate that a mannose‐binding lectin‐like protein could exert a protective effect that prevents damage from other activated immune responses.     

Measurement of pesticide residues in peppers by near‐infrared reflectance spectroscopy

BACKGROUND: Peppers are a frequent object of food safety alerts in various member states of the European Union owing to the presence in some batches of unauthorised pesticide residues. This study assessed the viability of near‐infrared reflectance spectroscopy (NIRS) for the measurement of pesticide residues in peppers. Commercially available spectrophotometers using different sample‐presentation methods were evaluated for this purpose: a diode‐array spectrometer for intact raw peppers and two scanning monochromators fitted with different sample‐presentation accessories (transport and spinning modules) for crushed peppers and for dry extract system for infrared analysis (DESIR), respectively. RESULTS: Models developed using partial least squares–discriminant analysis (PLS2‐DA) correctly classified between 62 and 68% of samples by presence/absence of pesticides, depending on the instrument used. At model validation, the highest percentage of correctly classified samples—75 and 82% for pesticide‐free and pesticide‐containing samples respectively—were obtained for intact peppers using the diode‐array spectrometer. CONCLUSION: The results obtained confirmed that NIRS technology may be used to provide swift, non‐destructive preliminary screening for pesticide residues; suspect samples may then be analysed by other confirmatory analytical methods. Copyright © 2010 Society of Chemical Industry     

Phytochemical and antiprotozoal activity of Ocotea lancifolia

Thirteen known isoquinoline alkaloids were isolated from Ocotea lancifolia, popularly known as < canela pilosa > in Brasil and < laurel né > by the Guarani people which means smell laurel. Their activities against the promastigote forms of three Leishmania strains and the bloodstream form of Trypanosoma cruzi were evaluated, as well as their hepatocytotoxicity. Among them, the noraporphine alkaloid (-) caaverine has shown the most interesting antiprotozoal activity against Leishmania and T. cruzi parasites.     

Apoptosis inhibition of Atlantic salmon (Salmo salar) peritoneal macrophages by Piscirickettsia salmonis

To improve the understanding of the piscirickettsiosis pathogenesis, the in vivo apoptosis modulation of peritoneal macrophages and lymphocytes was studied in juvenile Salmo salar intraperitoneally injected with Piscirickettsia salmonis. Five fish were sampled at post‐exposure days 1, 5, 8 (preclinical), 20 (clinical) and 40 (post‐clinical period of the disease), and the leucocytes of their coelomic washings were analysed by flow cytometry (using the JC‐1 cationic dye), TUNEL and cytology to detect apoptotic cells. A selective and temporal pattern of apoptosis modulation by P. salmonis infection was observed. Apoptosis in lymphocytes was not affected, whereas it was inhibited in macrophages but only during the preclinical stage of the induced piscirickettsiosis. Hence, it is postulated that P. salmonis inhibits macrophage apoptosis at the beginning of the disease development to survive, multiply and probably be transported inside these phagocytes; once this process is complete, macrophage apoptosis is no longer inhibited, thus facilitating the exit of the bacteria from the infected cells for continuing their life cycle.     

Puffins reveal contrasting relationships between forage fish and ocean climate in the North Pacific

Long‐term studies of predator food habits (i.e., ‘predator‐based sampling’) are useful for identifying patterns of spatial and temporal variability of forage nekton in marine ecosystems. We investigated temporal changes in forage fish availability and relationships to ocean climate by analyzing diet composition of three puffin species (horned puffin Fratercula corniculata, tufted puffin Fratercula cirrhata, and rhinoceros auklet Cerorhinca monocerata) from five sites in the North Pacific from 1978–2012. Dominant forage species included squids and hexagrammids in the western Aleutians, gadids and Pacific sand lance (Ammodytes personatus) in the eastern Aleutians and western Gulf of Alaska (GoA), and sand lance and capelin (Mallotus villosus) in the northern and eastern GoA. Interannual fluctuations in forage availability dominated variability in the western Aleutians, whereas lower‐frequency shifts in forage fish availability dominated elsewhere. We produced regional multivariate indicators of sand lance, capelin, and age‐0 gadid availability by combining data across species and sites using Principal Component Analysis, and related these indices to environmental factors including sea level pressure (SPL), winds, and sea surface temperature (SST). There was coherence in the availability of sand lance and capelin across the study area. Sand lance availability increased linearly with environmental conditions leading to warmer ocean temperatures, whereas capelin availability increased in a non‐linear manner when environmental changes led to lower ocean temperatures. Long‐term studies of puffin diet composition appear to be a promising tool for understanding the availability of these difficult‐to‐survey forage nekton in remote regions of the North Pacific.     

The Algal Meroterpene 11-Hydroxy-1′-O-Methylamentadione Ameloriates Dextran Sulfate Sodium-Induced Colitis in Mice

Inflammatory bowel disease (IBD) is a complex class of immune disorders. Unfortunately, a treatment for total remission has not yet been found, while the use of natural product-based therapies has emerged as a promising intervention. The present study was aimed to investigate the anti-inflammatory effects of the algal meroterpene 11-hydroxy-1′-O-methylamentadione (AMT-E) in a murine model of dextran sodium sulphate (DSS)-induced colitis. AMT-E was orally administered daily (1, 10, and 20 mg/kg animal) to DSS treated mice (3% w/v) for 7 days. AMT-E prevented body weight loss and colon shortening and effectively attenuated the extent of the colonic damage. Similarly, AMT-E increased mucus production and reduced myeloperoxidase activity (marker for anti-inflammatory activity). Moreover, the algal meroterpene decreased the tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10 levels, and caused a significant reduction of the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Our results demonstrate the protective effects of AMT-E on experimental colitis, provide an insight of the underlying mechanisms of this compound, and suggest that this class of marine natural products might be an interesting candidate for further studies on the prevention/treatment of IBD.     

Dissipation and movement of flumioxazin in soil at four field sites in Chile

BACKGROUND: Flumioxazin is a soil-applied herbicide recommended for broadleaf weed control in soybeans and peanuts, and was recently introduced for vineyard weed management. Considering the limited information available in relation to flumioxazin field soil behaviour, the main objectives of this study were to determine the persistence, adsorption and movement of flumioxazin in soil in four Chilean vineyard production areas. RESULTS: DT(50) values ranged from 10.6 +/- 1.0 to 32.1 +/- 3.1 days between localities, being correlated with rain events, time between herbicide application and first heavy rain event, and soil pH. Flumioxazin soil residue found at 90 days after application (DAA) varied from 9.6 to 24.9% of the initial amount applied, and depended on the total rainfall amount that occurred during the first 90 DAA. Herbicide leaching below 15 cm was approximately 45% of the flumioxazin detected at 90 DAA in the whole soil profile. Flumioxazin maximum leaching soil depth was 45 cm at all locations. K(d) values varied from 2.54 to 6.51 mg L(-1), depending on localities and soil profile depth, and correlated positively with organic carbon and clay content. CONCLUSIONS: These results indicate that flumioxazin is a herbicide with low environmental risk owing to its short DT(50), reduced soil residues 3 months after application and low effective dose.