In vitro and in vivo effects of chemotherapeutants on the oyster parasite, Perkinsus marinus

To investigate the potential of chemotherapeutants to control the oyster pathogen Perkinsus marinus, anticoccidial and antifungal compounds were tested in vitro on infected hemolymph and cultured P. marinus cells and in vivo on infected oysters. In addition, acute toxicity to oysters was determined for six anticoccidials. In vitro experiments with infected hemolymph consisted of 24 h exposure of 0.2 mL hemolymph aliquots to concentrations ranging from 100 mg/L to 0.01 mg/L of amphotericin-B, amprolium, arprinocid, cycloheximide, lasalocid, malachite green, monensin, sulfadimethoxine, and a potentiated sulfadimethoxine, followed by incubation in fluid thioglycollate medium (FTM) to determine prezoosporangia abundance. Lasalocid, malachite green, and amphotericin-B were the most effective compounds reducing prezoosporangia abundance, relative to the untreated control group, at concentrations as low as 10 mg/L. Cycloheximide, monensin, and to a lesser extent sulfadimethoxine, were also effective but only at the highest concentration tested (100 mg/L). In vitro experiments with cultured P. marinus consisted of 24 h exposure of 10&\sp5& cells to 100 mg/L, 10 mg/L, and 1 mg/L of amphotericin-B, and 100 mg/L of cimetidine, cycloheximide, fumagillin, 5-fluorocytosine, ketoconazole, lasalocid, and monensin, followed either by incubation in FTM to determine abundance and size of prezoosporangia, or by addition of Neutral Red to determine cell viability. Amphotericin-B, lasalocid, and monensin were effective in reducing prezoosporangia abundance, size, and/or cell viability. No effects of cycloheximide on cultured cells were apparent. Lasalocid, monensin, and malachite green, were toxic to oysters at concentrations below 10 mg/L. The 96-hr. LC50 for lasalocid was 0.59 mg/L. No median lethal dose was determined for monensin or malachite green, but oyster mortality resulted from exposures ranging from 1 mg/L to 10 mg/L of either compound. In three in vivo experiments, infected oysters were exposed to amprolium, arprinocid, cycloheximide, lasalocid, monensin, malachite green, potentiated sulfadimethoxine, and sulfadimethoxine at various concentrations. Only cycloheximide was effective in reducing P. marinus infections. After 15 days of exposure to 10 mg/L of cycloheximide, weighted prevalence significantly declined from 3.78 in untreated controls to 2.10 in treated oysters. Infections progressed after treatment was discontinued as indicated by an increase in weighted prevalence from 0.71 at the end of treatment to 1.31 one month later. (Abstract shortened by UMI.)

In-Vitro And In-Vivo Effects Of 8 Chemotherapeutants On The Oyster Parasite Perkinsus-Marinus (Mackin, Owen, And Collier)

Eight therapeutants were tested for in vitro inhibition of Perkinsus marinus (Mackin, Owen, and Collier) enlargement and in vivo control of established infections. In addition, acute toxicity of six anticoccidials to oysters was determined. For in vitro experiments 0.2 ml aliquots of infected hemolymph were exposed to 5 concentrations (100 mg/l, 10 mg/l, 1 mg/l, 0.1 mg/l and 0.01 mg/1) of amprolium, arprinocid, cycloheximide, lasalocid, malachite green, monensin, sulfadimethoxine, and a potentiated sulfadimethoxine. Exposure lasted 1 day and was followed by incubation in fluid thioglycollate medium. Lasalocid and malachite green were the most effective compounds, showing significant anti-P. marinus activity at concentrations as low as 10 mg/l. Cycloheximide, monensin, and to a lesser extent sulfadimethoxine, were also effective but only at the highest concentration tested (100 mg/1). At concentrations lower than 10 mg/l, no compound tested had a significant effect on P. marinus. Lasalocid, monensin, and malachite green, were toxic to oysters at concentrations below 10 mg/l. The 96-hr LC50 for lasalocid was 0.59 mg/l. No median lethal dose was obtained for monensin or malachite green, but oyster mortality resulted from exposures ranging from 1 mg/l to 10 mg/l of either compound. In two in vivo experiments, infected oysters were exposed to amprolium, cycloheximide, malachite green, and sulfadimethoxine at various concentrations. Only cycloheximide was effective in reducing P. marinus infections. After 15 days of exposure to 10 mg/l of cycloheximide, weighted prevalence significantly declined from 3.78 in untreated controls to 2. 10 in treated oysters. In addition, infections as measured by repeated hemolymph samples from individual oysters, significantly decreased after treatment. Extension of cycloheximide exposure to 30 days similarly reduced disease prevalence and weighted prevalence. Infections, however, were not completely eliminated even after 30 days of exposure to 10 mg/l of cycloheximide. Furthermore, infections progressed after treatment was discontinued as indicated by an increase in weighted prevalence from 0.71 at the end of treatment to 1.31 one month later

Comparative Field Study Of Crassostrea gigas (Thunberg, 1793) And Crassostrea virginica (Gmelin, 1791) In Relation To Salinity In Virginia

To evaluate and compare the performance of triploid juvenile C. gigas (mean shell height = 19.2 mm) and triploid juvenile Crassostrea virginica (mean shell height = 31.7 mm), 600 oysters of each species were deployed for 1 year in floating mesh cages at three replicate sites within low, medium, and high salinity regimes (respectively,25%) in the Chesapeake Bay and the Atlantic Coast of Virginia. The comparative performance of the two oyster species varied with salinity. At low salinity sites, cumulative mortality of C. virginica (10%) was significantly (P \u3c .05) lower than that of C. gigas (63%), and over-all mean growth rate of C. virginica (2.9 mm mo(-1)) was significantly (P \u3c .05) higher than that of C. gigas (1.6 mm mo(-1)). At medium salinity sites, survival and growth rate of C. virginica and C. gigas were nor significantly (P \u3e .05) different. Both species experienced moderately high cumulative mortality at the medium salinity sites-35% for C. virginica and 53% for C. gigas-but considerable variation among sires was observed. Ar high salinity sites, mean cumulative mortality was similarly low

A Comparative Field Study of Crassostrea ariakensis and Crassostrea virginica in Relation to Salinity in Virginia

In accordance with the Rational Plan for Testing Application of Non-Native Oyster Species (VIMS 1996) we conducted a field experiment to examine survival, growth and disease susceptibility of Crassostrea ariakensis (=rivularis) in relation to salinity in Virginia. The performance of triploid C. ariakensis in comparison with that of diploid C. virginica, (n = 250, age = 2 years, mean shell height = 60- 64 mm) was evaluated at replicate sites within low, medium, and high salinity regimes (respectively, \u3c 15‰, 15-25‰, \u3e 25‰) in Chesapeake Bay and the Atlantic Coast. During the course of this study, from June 1998 to September 1999, there was a severe oyster disease epizootic prevailing in Chesapeake Bay. At the end of the study C. ariakensis exhibited lower disease prevalence and intensity and superior survival and growth than C. virginica. At low salinity sites cumulative mortality in C. ariakensis (14%) was significantly lower than that in C. virginica (81%). At medium and high salinity sites, cumulative mortality in C. ariakensis was less than 16% whereas all C. virginica were dead by the end of the experiment. After one year of deployment, mean shell height of C. ariakensis at low, moderate, and high salinity sites, was respectively 96 mm, 125 mm, and 140 mm. In comparison, mean shell height of C. virginica was respectively 72 mm, 85 mm, and 75 mm. Prevalence and intensity of Perkinsus marinus infections were significantly lower in C. ariakensis than in C. virginica. During the second summer of disease exposure, prevalence in C. ariakensis ranged form 0-28% whereas prevalence in C. virginica was 100% at all sites. Only light infections were present in C. ariakensis whereas heavy infections were found in C. virginica. MSX was absent in C. ariakensis and present in C. virginica. Mud worms were present in both oyster species but infestations were low and did not appear to affect condition or growth. In summary, wide salinity tolerance and low disease susceptibility were associated with high survival and growth of C. ariakensis in Chesapeake Bay and the Atlantic Coast of Virginia

Sequencing of \u3ci\u3eAspergillus nidulans\u3c/i\u3e and comparative analysis with \u3ci\u3eA. fumigatus\u3c/i\u3e and \u3ci\u3eA. oryzae\u3c/i\u3e

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso, and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation. Document includes all supplementary information (820 pages). Supplementary files are also attached below as Related files. THERE IS NO SUPPLEMENTARY FILE #7. PDF file size (with supplementary files included) is 10 Mbytes. An optimized version of the ARTICLE ONLY is attached as a Related File and is 1.9 Mbytes

Sequencing of \u3ci\u3eAspergillus nidulans\u3c/i\u3e and comparative analysis with \u3ci\u3eA. fumigatus\u3c/i\u3e and \u3ci\u3eA. oryzae\u3c/i\u3e

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso, and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation. Document includes all supplementary information (820 pages). Supplementary files are also attached below as Related files. THERE IS NO SUPPLEMENTARY FILE #7. PDF file size (with supplementary files included) is 10 Mbytes. An optimized version of the ARTICLE ONLY is attached as a Related File and is 1.9 Mbytes

Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans

Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the DeltagprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the DeltagprD has a much lower PKA activity upon starvation. Transcriptomics and (1)H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the DeltagprB and DeltagprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in DeltagprB, while in the DeltagprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the DeltagprD strain. The (1)H NMR analysis revealed significant expression of essential amino acids with elevated levels in the DeltagprD strain, compared to the wild-type and DeltagprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development

Sub-Telomere Directed Gene Expression during Initiation of Invasive Aspergillosis

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio