RNA interference-mediated knockdown of the hydroxyacid-oxoacid transhydrogenase gene decreases thiamethoxam resistance in adults of the whitefly Bemisia tabaci.

Bemisia tabaci has developed a high level of resistance to thiamethoxam, a second generation neonicotinoid insecticide that has been widely used to control this pest. In this study, we investigated whether hydroxyacid-oxoacid transhydrogenase (HOT) is involved in resistance to the neonicotinoid insecticide thiamethoxam in the whitefly. We cloned the full-length gene that encodes HOT in B. tabaci. Its cDNA contains a 1428-bp open reading frame encoding 475 amino acid residues. Then we evaluated the mRNA expression level of HOT in different developmental stages, and found HOT expression was significantly greater in thiamethoxam resistance adults than in thiamethoxam susceptible adults. Subsequently, seven field populations of B. tabaci adults were sampled, the expression of mRNA level of HOT significant positive correlated with thiamethoxam resistance level. At last, we used a modified gene silencing system to knock-down HOT expression in B. tabaci adults. The results showed that the HOT mRNA levels decreased by 57% and thiamethoxam resistance decreased significantly after 2 days of feeding on a diet containing HOT dsRNA. The results indicated that down-regulation of HOT expression decreases thiamethoxam resistance in B. tabaci adults.

[Application of FTIR and Active Ingredients Quantitative Analysis on Quality Control of Dai Medicine Alstonia scholaris (L.) R. Br.].

FTIR fingerprint of the leaves and immature stems of Alstonia scholaris (L.) R. Br. was established as a content determination method for the detection of picrinine, ursolic acid and oleanolic acid. Different medicinal parts were identified based on principal component analysis, while exploring the influence of immature stems for the leaves and the application of FTIR and HPLC in the Dai quality control in order to speed up the pace of Dai medicine modernization. Infrared spectroscopy of different batches samples were collected and the data was preprocessed as to automatic baseline correction, smooth, ordinate normalization, second order derivative, and then to PCA, all the datum in triplicate. For content determination of picrinine, mobile phase was acetonitrile (40) water (contain 0.1% ammonia water) (60) and the wavelength was set at 287 nm. For ursolic acid and oleanolic acid, the mobile phase was mixture (12∶88) of 0.1% formic acid in water (A) and methanol (B). Wavelength was 210 nm. As the results, the original spectrum difference was not obvious for leaves and stems. Pretreatment spectroscopy had a significant variation on absorption peak number and intensity in 3 000～2 800 and 1 800～500 cm(-1). The results of PCA showed that, the leaves and stems were separated; in addition the difference of different batches leaves was bigger than the stems. The mean contents of picrinine, ursolic acid and oleanolic acid in leaves were 0.79，8.47，7.51 and 0.21，1.78，1.67 mg·g(-1) in stems, respectively. The content of ursolic acid and oleanolic acid is higher than picrinine, but ursolic acid and oleanolic acid content had no obvious difference. Mean content of three ingredients in leaves is much higher than in stems. Picrinine content in leaves was 3.8 times of immature stems, ursolic acid and oleanolic acid content were 5.1 and 4.2 times of immature stems, respectively. The variety of picrinine content in different batches samples was biggest, ursolic acid and oleanolic acid content was relatively stable. The overall quality of leaves has an obvious difference compared with the immature stems, so the leaves of A. scholaris mix with immature stems could not be as Dai medicine in Dai clinic. Infrared spectroscopy combined with chromatography can quickly identify different medicinal parts and evaluate overall quality of Dai medicine, which can apply to quality control of Dai medicine.

[Distinguish and Quality Estimation of the Leaves of Alstonia scholaris (L.) R. Br. from Different Harvest Time Based on the UV-Vis·FP and HPLC·FP].

UV-Vis and HPLC fingerprint of different harvest time of the leaves of Alstonia scholaris (L.) R. Br. were establish the for identification and quality evaluation to promote the development of Dai Medicine modernization. The optimal extraction condition was used to obtain UV - vis data of different harvest time which were deducted background and eight spot smooth, were collected to make the principal component analysis in SIMCA-P(+)11.5, identifying the samples quickly with the first three principal component three-dimensional diagram. The HPLC fingerprint were obtained with Agilent ZORBAX Eclipse XDB C18 (250×4.6 mm, 5 µm) chromatographic column with the mobile phase of acetonitrile (B) - water (contain 0.1% formic acid) (A) for gradient elution (0～5 min, 5% B; 5～35 min, 5% B→26% B; 35～40 min, 26% B→56% B). The wavelength was set at 287 nm and the column temperature was maintained at 30 ℃. The flow rate was 1.0 mL·min-1 and the injection volume was 7 µL. The HPLC fingerprint of different harvest time of the leaves of Alstonia scholaris (L.) R. Br. was analysised by cluster analysis to quality evaluation. Research findings showing: (1) The UV-Vis spectrogram of different harvest time of the leaves of Alstonia scholaris (L.) R. Br. were divided into three parts according to the absorption peak position and amplitude of variation. The first was 235 to 400 nm, the second was 400 to 500 nm, and the third was 500 to 800 nm. In the first part, absorption peak were focused on 270, 287 and 325 nm, which can reflect the fingerprint character for the high absorbance and amplitude of variation. Absorption peak were distributed in 410 and 464 nm in the second part, absorbance and amplitude of variation were lower than the first part. There was a bigger absorption peak at 665 nm in the third part, but the absorbance had no difference. The UV-Vis data of different harvest time were gathered to make the principal component analysis, the result was that the samples of same month were concentrated distribution, but different month samples were dispersed distribution. (2) HPLC fingerprint were divided into three categories through hierarchical cluster analysis, 3, 4, 5 and 7 month were the first category, 6, 8, 9 month samples were second category, the others were third category. Chemical composition and content of the same category samples were similar, but the different category samples had a obvious difference, more important is that the third category samples content was the highest. Combining UV-Vis FP and HPLC FP can identify and evaluate quickly the samples of different harvest time of the leaves of Alstonia scholaris (L.) R. Br. The optimal harvest time of Alstonia scholaris (L.) R. Br. was from October to next February, which was the coldest season in the Dai calendar.

Characterization of LpGPAT gene in Lilium pensylvanicum and response to cold stress.

LpGPAT was obtained from L. pensylvanicum using RT-PCR and rapid amplification of cDNA ends. The cloned full-length cDNA was 1544 bp; it encoded 410 amino acids and had a molecular size of 46 KDa. The nucleic acid sequence analysis showed that it shared high homology with other known GPATs. SMAT result suggests that there is a PlsC that exists in 176-322 amino acid sequence of LpGAPT; it means LpGPAT protein is a member of the family of acyltransferase and has acyltransferase enzymatic activity. Result of real-time quantitative PCR and semiquantitative PCR support LpGPAT gene is definitely induced by low temperature stress.

Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China.

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14-17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64).

Gene expression profiling in the thiamethoxam resistant and susceptible B-biotype sweetpotato whitefly, Bemisia tabaci.

Thiamethoxam has been used as a major insecticide to control the B-biotype sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Due to its excessive use, a high level of resistance to thiamethoxam has developed worldwide over the past several years. To better understand the molecular mechanisms underlying this resistance in B. tabaci, gene profiles between the thiamethoxam-resistant and thiamethoxam-susceptible strains were investigated using the suppression subtractive hybridization (SSH) library approach. A total of 72 and 52 upand down-regulated genes were obtained from the forward and reverse SSH libraries, respectively. These expressed sequence tags (ESTs) belong to several functional categories based on their gene ontology annotation. Some categories such as cell communication, response to abiotic stimulus, lipid particle, and nuclear envelope were identified only in the forward library of thiamethoxam-resistant strains. In contrast, categories such as behavior, cell proliferation, nutrient reservoir activity, sequence-specific DNA binding transcription factor activity, and signal transducer activity were identified solely in the reverse library. To study the validity of the SSH method, 16 differentially expressed genes from both forward and reverse SSH libraries were selected randomly for further analyses using quantitative realtime PCR (qRT-PCR). The qRT-PCR results were fairly consistent with the SSH results; however, only 50% of the genes showed significantly different expression profiles between the thiamethoxam-resistant and thiamethoxam-susceptible whiteflies. Among these genes, a putative NAD-dependent methanol dehydrogenase was substantially over-expressed in the thiamethoxamresistant adults compared to their susceptible counterparts. The distributed profiles show that it was highly expressed during the egg stage, and was most abundant in the abdomen of adult females.

Pyrosequencing the Bemisia tabaci transcriptome reveals a highly diverse bacterial community and a robust system for insecticide resistance.

BACKGROUND: Bemisia tabaci (Gennadius) is a phloem-feeding insect poised to become one of the major insect pests in open field and greenhouse production systems throughout the world. The high level of resistance to insecticides is a main factor that hinders continued use of insecticides for suppression of B. tabaci. Despite its prevalence, little is known about B. tabaci at the genome level. To fill this gap, an invasive B. tabaci B biotype was subjected to pyrosequencing-based transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes. METHODOLOGY AND PRINCIPAL FINDINGS: Using Roche 454 pyrosequencing, 857,205 reads containing approximately 340 megabases were obtained from the B. tabaci transcriptome. De novo assembly generated 178,669 unigenes including 30,980 from insects, 17,881 from bacteria, and 129,808 from the nohit. A total of 50,835 (28.45%) unigenes showed similarity to the non-redundant database in GenBank with a cut-off E-value of 10-5. Among them, 40,611 unigenes were assigned to one or more GO terms and 6,917 unigenes were assigned to 288 known pathways. De novo metatranscriptome analysis revealed highly diverse bacterial symbionts in B. tabaci, and demonstrated the host-symbiont cooperation in amino acid production. In-depth transcriptome analysis indentified putative molecular markers, and genes potentially involved in insecticide resistance and nutrient digestion. The utility of this transcriptome was validated by a thiamethoxam resistance study, in which annotated cytochrome P450 genes were significantly overexpressed in the resistant B. tabaci in comparison to its susceptible counterparts. CONCLUSIONS: This transcriptome/metatranscriptome analysis sheds light on the molecular understanding of symbiosis and insecticide resistance in an agriculturally important phloem-feeding insect pest, and lays the foundation for future functional genomics research of the B. tabaci complex. Moreover, current pyrosequencing effort greatly enriched the existing whitefly EST database, and makes RNAseq a viable option for future genomic analysis.