Effect of fungal infection on reproductive potential and survival time of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae)

The effect of fungal infection on the reproductive potential of two-spotted spider mite, Tetranychus urticae, was evaluated as part of the full biocontrol potential of three entomopathogenic fungi by modeling of fecundity probability. Female mites (≤2-day-old) on leaves were exposed to the sprays of Beauveria bassiana, Paecilomyces fumosoroseus and Metarhizium anisopliae at the concentrations of 1.13 × 10³, 1.55 × 10³ and 0.95 × 10³ deposited conidia mm⁻² and then individually reared at 25°C and 12:12 L:D for oviposition. Mite mortalities 10 days after spraying were 73.1, 75.4 and 67.9% in the fungal treatments versus 15.5% in control. On average, females infected by the three fungal species survived 5.8, 6.2 and 6.3 days, and laid 3.1, 4.0 and 4.0 eggs per capita, respectively. These were 3–4 fold lower than the control fecundity at 12.3. The cumulative probabilities [P(m ≤ N)] for the counts of infected and non-infected (control) females laying m eggs per capita (m ≤ N) during 10 days fit very well the equation P(m ≤ N) = 1/[1 + exp(a + bm)] (r ² ≥ 0.98), yielding a solution to the probability for the female mites to achieve a specific fecundity {P(m ≤ N)−P[m ≤ (N − 1)]}. Consequently, the infected mites had 71–78% chance to lay ≤5 eggs per capita but only 5–8% to deposit >10 eggs despite some variation among the tested fungi. In contrast, the chances for the non-infected mites to achieve the low and high fecundities were 23 and 55%. The fitted probabilities provide a full coverage of the fecundity potential of infected versus non-infected mites and are more informative than the mean fecundities.     

Application of quantitative RT-PCR to determine the distribution of <Emphasis Type="Italic">Microthrix parvicella</Emphasis> in full-scale activated sludge treatment systems

Three wastewater treatment plants in South Africa were investigated to understand the phylogeny and distribution of Microthrix parvicella using real-time polymerase chain reaction (RT-PCR). The phylogenetic analysis of the 16S rRNA of M. parvicella revealed 98% to 100% homology of South African clones to M. parvicella reported in Genbank. The standard curves for RT-PCR showed R2 values greater than 0.99, accurate for quantification. The relative occurrence of M. parvicella 16S rRNA gene copies in the three wastewater treatment plants was in the range 0% to 3.97%. M. parvicella copies increased when the environmental temperature (≤20°C) and food/microorganism (F/M) ratio was low. The M. parvicella 16S rRNA copies could be positively correlated to the sludge volume index at low temperature. At higher temperature, there was a rapid reduction in M. parvicella population irrespective of other favorable factors, indicating the strong influence of temperature on filamentous proliferation. RT-PCR has potential applications in wastewater treatment plants to monitor sudden shift in the microbial population and assessing the plants efficacy.     

Diversity and dynamics of microcystin—Producing cyanobacteria in China's third largest lake, Lake Taihu

Microcystins (MC), the most prevalent group of harmful cyanobacterial hepatotoxins, are primarily produced by strains of cyanobacteria in Microcystis, Anabaena and Planktothrix. Lake Taihu, which is the third largest freshwater lake in China, is a hypertrophic shallow lake in eastern China that has experienced lake-wide cyanobacterial blooms annually during the last few decades. In this study, PCR-DGGE was used to evaluate the diversity of potential MC-producing cyanobacteria and real-time PCR was used to analyze the dynamics of this population based on the presence of the mcy gene in samples collected during a year long study. The results revealed that all MC-producing genotypes detected belonged to the genus Microcystis. In addition, the MC-producing genotype communities were more diverse during the bloom season than the non-bloom season, and the diversity in the late bloom period was lower than the diversity in the early bloom period. Furthermore, the abundance of MC-producing genotypes increased dramatically during the bloom development period, reaching its peak in late summer (September). The results also suggested that the highest mcy gene concentration lagged behind the highest MC concentration, and the potential MC-producing cyanobacterial community shift lagged behind the development of blooms.     

Quantitative real-time PCR to determine allele number for the astringency locus by analysis of a linked marker in Diospyros kaki Thunb

Persimmon (Diospyros kaki Thunb.) is a polyploidy fruit tree species of economic importance to East Asia. Natural astringency loss is an important trait in persimmon breeding programs. Quantitative real-time PCR was used to determine the number of AST/ast alleles for fruit astringency in persimmon (D. kaki Thunb.). To this end, the cultivar Jiro was transformed with one or two copies of a gene encoding NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH), which was used as a standard for measuring the allele number of a sequenced marker tightly linked to the recessive ast locus for nonastringency. Primers for markers linked to the AST or ast allele were then used to measure the AST to ast ratio directly in the progeny of a full-sib cross. From determination of the AST to ast ratio and the results of the S6PDH copy number, the number of AST and ast alleles at the AST/ast locus was estimated. This research supported the hypothesis that D. kaki is a hexaploid with six AST and/or ast alleles. In addition to the determination of the allelic status of the AST locus, the application of real-time PCR for confirmation of the ploidy level and allelic composition of target genes in autopolyploids or allopolyploids was demonstrated.     

Impact of FXIII-A Val34Leu polymorphism on coronary artery disease in Croatian patients

Arterial thrombosis is the major reason for severe complications of coronary artery disease (CAD). Recently it has been suggested that the FXIII-A Val34Leu polymorphism, affecting clot stability, provides protection against thrombosis. Results published up to date implicate that there is a significant correlation between geographical area and the Leu34 allele prevalence and that its contribution to arterial thrombosis is different in different populations. The purpose of this study was to determine frequency of Leu34 allele in Croatian subjects as well as to estimate its association with a CAD. FXIII-A Val34Leu genotyping was carried out by real-time PCR method on the LightCycler using melting curve analysis with forward 5′-AACTTCCAGGACCGGCTTT-3′ and reverse 5′-ACCCAGAGTGGTGGGGAA-3′ primers. The Leu34 allele frequency in studied Croatian subjects was 24.3%. No significant differences were found in the prevalence of FXIII-A Val34Leu genotype or Leu34 allele distribution between studied subjects (P > 0.05). Carriage of the Leu34 allele was not significantly associated with CAD or MI risk reduction (P > 0.05). This is the first report that studies the prevalence of the Leu34 allele frequency in Croatian subjects and our results suggest that possession of the Leu 34 alele does not provide protection against MI.     

Influence of Temperature on Virulence of Fungal Isolates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to the Two-Spotted Spider Mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Twenty-three isolates of Metarhizium anisopliae (Metschnikoff) Sokorin and three isolates of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales: Clavicipitaceae) were assessed for their virulence against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Based on the screening results, nine isolates of M. anisopliae and two isolates of B. bassiana were tested for their virulence against young adult (1- to 2-day-old) female T. urticae at constant temperatures of 20, 25, 30 and 35°C. At all temperatures tested, all the fungal isolates were pathogenic to T. urticae but mortality varied with isolates and temperatures. Fungal isolates were more virulent at 25, 30 and 35°C than at 20°C. The lethal time to 50% mortality (LT₅₀) and lethal time to 90% mortality (LT₉₀) values decreased with increased temperature. There were no significant differences in virulence between fungal isolates at 30 and 35°C; however, significant differences were observed at 20 and 25°C.     

An improved ultrafast 2D NMR experiment: Towards atom-resolved real-time studies of protein kinetics at multi-Hz rates

Multidimensional NMR spectroscopy is a well-established technique for the characterization of structure and fast-time-scale dynamics of highly populated ground states of biological macromolecules. The investigation of short-lived excited states that are important for molecular folding, misfolding and function, however, remains a challenge for modern biomolecular NMR techniques. Off-equilibrium real-time kinetic NMR methods allow direct observation of conformational or chemical changes by following peak positions and intensities in a series of spectra recorded during a kinetic event. Because standard multidimensional NMR methods required to yield sufficient atom-resolution are intrinsically time-consuming, many interesting phenomena are excluded from real-time NMR analysis. Recently, spatially encoded ultrafast 2D NMR techniques have been proposed that allow one to acquire a 2D NMR experiment within a single transient. In addition, when combined with the SOFAST technique, such ultrafast experiments can be repeated at high rates. One of the problems detected for such ultrafast protein NMR experiments is related to the heteronuclear decoupling during detection with interferences between the pulses and the oscillatory magnetic field gradients arising in this scheme. Here we present a method for improved ultrafast data acquisition yielding higher signal to noise and sharper lines in single-scan 2D NMR spectra. In combination with a fast-mixing device, the recording of ¹H–¹⁵N correlation spectra with repetition rates of up to a few Hertz becomes feasible, enabling real-time studies of protein kinetics occurring on time scales down to a few seconds.     

Influence of testosterone and a novel SARM on gene expression in whole blood of Macaca fascicularis

Anabolic hormones, including testosterone, have been suggested as a therapy for aging-related conditions, such as osteoporosis and sarcopenia. These therapies are sometimes associated with severe androgenic side effects. A promising alternative to testosterone replacement therapy are selective androgen receptor modulators (SARMs). SARMs have the potential to mimic the desirable central and peripheral androgenic anabolic effects of testosterone without having its side effects.In this study we evaluated the effects of LGD2941, in comparison to testosterone, on mRNA expression of selected target genes in whole blood in an non-human model. The regulated genes can act as potential blood biomarker candidates in future studies with AR ligands.Cynomolgus monkeys (Macaca fascicularis) were treated either with testosterone or LGD2941 for 90 days in order to compare their effects on mRNA expression in blood. Blood samples were taken before SARM application, on day 16 and on day 90 of treatment.Gene expression of 37 candidate genes was measured using quantitative real-time RT-PCR (qRT-PCR) technology.Our study shows that both testosterone and LGD2941 influence mRNA expression of 6 selected genes out of 37 in whole blood. The apoptosis regulators CD30L, Fas, TNFR1 and TNFR2 and the interleukins IL-12B and IL-15 showed significant changes in gene expression between control and the treatment groups and represent potential biomarkers for androgen receptor ligands in whole blood.     

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

The generation of airborne microorganisms from concentrated animal-feeding operations (CAFOs) is a concern from a human and animal health perspective. To better understand the airborne microorganisms found in these environments, a number of collection and analytical techniques have been utilized and will be discussed in this review. The most commonly used bioaerosol collection method is the liquid impingement format, which is suitable with a number of culture-based and non-culture molecular-based approaches, such as polymerase chain reaction. However, the vast majority of airborne microorganism studies conducted at CAFOs utilize culture-based analyses. Because of the limitations often associated with culture-based analyses, we focused our discussion on the application of molecular-based techniques to identify and/or quantify microorganisms, as they have promising application in bioaerosol research. The ability to rapidly characterize airborne microorganisms will help to ensure protection of public and environmental health. The use or mention of any commercial products does not imply any endorsement of that product by either the authors or the US Department of Agriculture.