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      Identification, Expression Profiling and Fluorescence-Based Binding Assays of a Chemosensory Protein Gene from the Western Flower Thrips, Frankliniella occidentalis

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          Abstract

          Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP) from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP) has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four—cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9), suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN) as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with K D values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in regulating the development of the first instar nymph and mediate F. occidentalis host recognition.

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          Odor-mediated behavior of Afrotropical malaria mosquitoes.

          The African mosquito species Anopheles gambiae sensu lato s.l. and Anopheles funestus rank among the world's most efficient vectors of human malaria. Their unique bionomics, particularly their anthropophilic, endophagic and endophilic characters, guarantee a strong mosquito-host interaction, favorable to malaria transmission. Olfactory cues govern the various behaviors of female mosquitoes and here we review the role of semiochemicals in the life history of African malaria vectors. Recent evidence points towards the existence of human-specific kairomones affecting host-seeking A. gambiae s.l., and efforts are under way to identify the volatiles mediating this behavior. Based on examples from other Culicidae spp., it is argued that there is good reason to assume that mating, sugar feeding, and oviposition behavior in Afrotropical malaria vectors may also be mediated by semiochemicals. It is foreseen that increased knowledge of odor-mediated behaviors will be applied in the development of novel sampling techniques and possibly alternative methods of intervention to control malaria.
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            A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics.

            Real-time reverse transcription (RT) PCR is currently the most sensitive method for the detection of low-abundance mRNAs. Two relative quantitative methods have been adopted: the standard curve method and the comparative C(T) method. The latter is used when the amplification efficiency of a reference gene is equal to that of the target gene; otherwise the standard curve method is applied. Based on the simulation of kinetic process of real-time PCR, we have developed a new method for quantitation and normalization of gene transcripts. In our method, the amplification efficiency for each individual reaction is calculated from the kinetic curve, and the initial amount of gene transcript is derived and normalized. Simulation demonstrated that our method is more accurate than the comparative C(T) method and would save more time than the relative standard curve method. We have used the new method to quantify gene expression levels of nine two-pore potassium channels. The relative levels of gene expression revealed by our quantitative method were broadly consistent with those estimated by routine RT-PCR, but the results also showed that amplification efficiencies varied from gene to gene and from sample to sample. Our method provides a simple and accurate approach to quantifying gene expression level with the advantages that neither construction of standard curve nor validation experiments are needed.
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              Circadian regulation of gene expression systems in the Drosophila head.

              Mechanisms composing Drosophila's clock are conserved within the animal kingdom. To learn how such clocks influence behavioral and physiological rhythms, we determined the complement of circadian transcripts in adult Drosophila heads. High-density oligonucleotide arrays were used to collect data in the form of three 12-point time course experiments spanning a total of 6 days. Analyses of 24 hr Fourier components of the expression patterns revealed significant oscillations for approximately 400 transcripts. Based on secondary filters and experimental verifications, a subset of 158 genes showed particularly robust cycling and many oscillatory phases. Circadian expression was associated with genes involved in diverse biological processes, including learning and memory/synapse function, vision, olfaction, locomotion, detoxification, and areas of metabolism. Data collected from three different clock mutants (per(0), tim(01), and Clk(Jrk)), are consistent with both known and novel regulatory mechanisms controlling circadian transcription.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                30 January 2015
                2015
                : 10
                : 1
                : e0117726
                Affiliations
                [1 ]State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
                [2 ]Institute of Plant Protection, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan, China
                Russian Academy of Sciences, Institute for Biological Instrumentation, RUSSIAN FEDERATION
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: ZKZ ZRL. Performed the experiments: ZKZ. Analyzed the data: ZKZ ZRL. Contributed reagents/materials/analysis tools: ZKZ ZRL. Wrote the paper: ZKZ ZRL. Designed the software used in analysis: ZKZ ZRL.

                Article
                PONE-D-14-40626
                10.1371/journal.pone.0117726
                4311994
                25635391
                e50819bb-f367-4273-ba72-dfb5a2cae01a
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 9 September 2014
                : 31 December 2014
                Page count
                Figures: 9, Tables: 2, Pages: 20
                Funding
                This research is partially funded by the China Agriculture Research System (CARS-25-B-07) and the National Modern Agricultural Science and Technology City Industry of Beijing (Z121100001212006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                All relevant data are within the paper and its Supporting Information files.

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