Rapid development of 36 polymorphic microsatellite markers for Tetranychus truncatus by transferring from Tetranychus urticae

Tetranychus truncatus Ehara is a phytophagous spider mite that is now one of the most important pests of agricultural and economic crops in East and Southeast Asia. However, population genetics and other studies of T. truncatus have been impeded by the lack of microsatellite markers, which are expensive and time-consuming to identify. Previous studies indicated a high potential of cross-amplification of microsatellites in Tetranychus species, meaning that the microsatellite flanking sequences are sufficiently homologous among Tetranychus species that the primers for one species may work in another species. Here, we tested 205 primer pairs designed from the whole genome sequence of Tetranychus urticae Koch, a sister species of T. truncatus, for microsatellite markers in three populations of T. truncatus in China (N = 94). About half (102) of these primer pairs yielded the desired PCR products, 36 of which revealed polymorphism in T. truncatus. Each of the 36 markers harbored between 2 and 23 alleles, with a mean polymorphic information content of 0.589 (0.119–0.922 range). The mean observed and expected heterozygosity across loci and the three populations were 0.468 and 0.628, respectively. Of the 36 primer pairs, 22 also worked in Tetranychus piercei, but only a few of them worked in T. ludeni and T. phaselus. Cross-amplification is thus a cost-effective way to develop microsatellite markers, which can be of great value in population genetics studies.     

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0?×?10⁹ to 3.2?×?10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8?×?10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.     

Sex steroid changes during temperature‐induced gonadal differentiation in Paralichthys olivaceus (Temminck & Schegel, 1846)

Sex steroid changes during temperature‐induced gonadal differentiation were evaluated in the olive flounder, Paralichthys olivaceus. Larvae were reared at 21 ± 0.5°C, 24 ± 0.5°C and 28 ± 0.5°C from day 40 post‐hatching (dph) to 90 dph. The proportion of males was 61.1, 76.7, 87.8 and 47.8% in 21°C, 24°C, 28°C and in control groups, respectively. Gonadal differentiation was circa 65 dph, when fishes were a mean 39 mm total length (TL). The gonads developed faster when fishes were reared in higher temperatures. Radioimmunoassay (RIA) analyses indicated that the level of estradiol‐17β (E2) changed during the period of gonadal differentiation and peaked at an onset of ovarian differentiation in all groups. Compared with fish in control groups, the levels of E2 were lower in thermal‐treated groups, especially in the highest temperature groups. The present results indicate that E2 plays a major role in the process of ovarian differentiation, and suggest that temperature‐induced masculinization in P. olivaceus is mainly due to a decrease in the E2 level during the period of ovarian differentiation.     

Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology

Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.     

Different utilizable substrates have different effects on cometabolic fate of imidacloprid in Stenotrophomonas maltophilia

Imidacloprid, the largest selling insecticide in the world, is more stable in soil, and its environmental residue and effects are attracting people's close attention. One of imidacloprid metabolism pathways was degraded to CO₂ through olefin imidacloprid pathway. Here, we report that sucrose as a utilizable substrate enhanced the cometabolism of imidacloprid by Stenotrophomonas maltophilia CGMCC 1.1788 to produce 5-hydroxy imidacloprid, whereas when succinate was used as a utilizable substrate, 5-hydroxy imidacloprid from imidacloprid was transformed to olefin imidacloprid, and the latter was further degraded. The hydroxylation of imidacloprid required NAD(P)H, whereas the dehydration of 5-hydroxy imidacloprid to form olefin imidacloprid required succinate rather than NAD(P)H. NADPH greatly favored the hydroxylation of imidacloprid more than NADH, and NADPH inhibited the dehydration of 5-hydroxy imidacloprid to olefin imidacloprid, but NADH did not. Therefore, sucrose may be metabolized through hexose monophosphate pathway to produce mainly NADPH which participated in the hydroxylation of imidacloprid to 5-hydroxy imidacloprid and meanwhile inhibited the dehydration of 5-hydroxy imidacloprid to olefin imidacloprid, whereas succinate may be metabolized mainly through the tricarboxylic acid cycle to produce NADH which was involved in hydroxylation of imidacloprid to 5-hydroxy imidacloprid but did not inhibit the dehydration of 5-hydroxy imidacloprid to olefin imidacloprid. Our results have a significant meaning in further understanding the influence of different utilizable substrates on the cometabolic pathways and the fate of environmental imidacloprid.     

Genetic structure of silver pomfret (Pampus argenteus (Euphrasen, 1788)) in the Arabian Sea,Bay of Bengal,and South China Sea as indicated by mitochondrial COI gene sequences

The silver pomfret (Pampus argenteus) is a commercially important species native to the Indo‐western Pacific area. In the present study, the genetic structure of five P. argenteus populations from the Arabian Sea, Bay of Bengal, and South China Sea were investigated using sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. All populations showed high levels of haplotype diversity (h) and low levels of nucleotide diversity (π). Phylogenetic trees indicated that the haplotypes could be divided into three clusters, indicating the geographic distribution of the five populations. Analysis of molecular variance (amova ) indicated the intra‐region variation among these populations to be highly significant. The average pairwise differences and F_st values among the three regions were also found to be significant. These results suggest that P. argenteus in these regions has strong geographic genetic structure. The present results provide new information for the genetic assessment, fishery management, and conservation of this species.     

Development of thermodynamic optimum searching (TOS) to improve the prediction accuracy of flux balance analysis

Flux balance analysis (FBA) has been widely used in calculating steady‐state flux distributions that provide important information for metabolic engineering. Several thermodynamics‐based methods, for example, quantitative assignment of reaction directionality and energy balance analysis have been developed to improve the prediction accuracy of FBA. However, these methods can only generate a thermodynamically feasible range, rather than the most thermodynamically favorable solution. We therefore developed a novel optimization method termed as thermodynamic optimum searching (TOS) to calculate the thermodynamically optimal solution, based on the second law of thermodynamics, the minimum magnitude of the Gibbs free energy change and the maximum entropy production principle (MEPP). Then, TOS was applied to five physiological conditions of Escherichia coli to evaluate its effectiveness. The resulting prediction accuracy was found significantly improved (10.7–48.5%) by comparing with the ¹³C‐fluxome data, indicating that TOS can be considered an advanced calculation and prediction tool in metabolic engineering. Biotechnol. Bioeng. 2013; 110: 914–923. © 2012 Wiley Periodicals, Inc.