Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae).

Liriomyza trifolii is a highly-invasive leafmining insect that causes significant damage to vegetables and horticultural crops worldwide. Relatively few studies have quantified gene expression in L. trifolii using real-time quantitative PCR (RT-qPCR), which is a reliable and sensitive technique for measuring gene expression. RT-qPCR requires the selection of reference genes to normalize gene expression data and control for internal differences between samples. In this study, nine housekeeping genes from L. trifolii were selected for their suitability in normalizing gene expression using geNorm, Normfinder, BestKeeper, the ΔCt method and RefFinder. HSP21.7, which encodes heat shock protein 21.7, was used as a target gene to validate the expression of candidate reference genes. Results indicated that ACTIN and 18S were optimal for developmental stage and low temperature, TUB and 18S showed the most stable expression for sex, and GAPDH and ACTIN were the best reference genes for monitoring gene expression at high temperature. Selection and validation of appropriate reference genes are critical steps in normalizing gene expression levels, which improve the accuracy and quality of expression data. Results of this study provide vital information on reference genes and is valuable in developing a standardized RT-qPCR protocol for functional genomics research in L. trifolii.

Cloning and expression of genes encoding heat shock proteins in Liriomyza trifolii and comparison with two congener leafminer species.

The polyphagous agromyzid fly, Liriomyza trifolii, is a significant and important insect pest of ornamental and vegetable crops worldwide. The adaptation of insects to different environments is facilitated by heat shock proteins (HSPs), which play an important role in acclimation to thermal stress. In this study, we cloned and characterized five HSP-encoding genes of L. trifolii (Lthsp20, Lthsp40, Lthsp60, Lthsp70, and Lthsp90) and monitored their expression under different thermal stresses using real-time quantitative PCR. Pupae of L. trifolii were exposed to 19 different temperatures ranging from -20 to 45°C. The results revealed that Lthsp20, Lthsp40, Lthsp70 and Lthsp90 were significantly upregulated in response to both heat and cold stress, while Lthsp60 was induced only by heat temperatures. The temperatures of the onset (Ton) and maximal (Tmax) expression of the five Lthsps were also determined and compared with published Ton and Tmax values of homologous genes in L. sativae and L. huidobrensis. Although L. trifolii occurs primarily in southern China, it has cold tolerance comparable with the other two Liriomyza species. Based on the heat shock proteins expression patterns, L. trifolii has the capacity to tolerate extreme temperatures and the potential to disseminate to northern regions of China.

[Probabilistic evaluation of dietary exposure to lead and age-related exposure centile for residents in Jiangsu].

OBJECTIVE: To establish a probabilistic model for evaluation of dietary exposure to lead and construct age-related exposure centiles for the residents in Jiangsu. METHODS: Lead contamination data were obtained from the national food contamination monitoring program during 2001 - 2006 and 2791 samples from 232 food products in Jiangsu were included. Food consumption data were from the national diet and nutrition survey conducted in 2002, including 3938 subjects in Jiangsu. A non-parametric probabilistic model using Monte Carlo simulation was applied to derive the intake distribution. The intake data was then analyzed using the LMS method, which constructs exposure percentiles adjusted for the median (M), the coefficient of variation (S) and the skewness (L) of the intake distribution. RESULTS: The median and P(99) of the lead exposure for the residents in Jiangsu were 1.02 µg×kg(-1)×d(-) and 9.29 µg×kg(-1)×d(-1), respectively.6.38% of the total population showed to have a lead intake exceeding the tolerable limit, which for the urban and rural population were 4.31% and 7.06%, respectively. The exceeding rate for children of 2 - 10 years old from the urban and rural areas were 13.17% and 17.70%, respectively. CONCLUSION: There was a large variation in the lead exposure level of the population in Jiangsu; People in rural areas are in greater risk for higher lead exposure than urban people; The dietary exposure to lead for children and the high-end population was serious.

[Development and verification of Chinese dietary exposure evaluation model software].

OBJECTIVE: To develop the dietary exposure evaluation model software accredited of Chinese intellectual property rights and to verify the rationality and accuracy of the results from the probabilistic model in Chinese dietary exposure evaluation model software according to international standards. METHODS: The software of SAS was used to build various evaluation model based on the data from Chinese dietary survey and the chemical compound in food surveillance and to design an operation interface. The results from probabilistic dietary exposure model for children 2 - 7 years old were compared with that from duplicate portion study of 2-7 years children dietary exposure in Jinhu, Jiangsu province in order to analyze the rationality of model. The results from probabilistic model of dietary exposure were compared with the results from @Risk software to verify the correction of the probabilistic model by using the same data of randomly selected 10 000 study subjects from national dietary survey. While, the mean drift was used as an internal index to illustrate the accuracy of the computation. RESULTS: Chinese dietary exposure evaluation software was developed successfully. On the rationality, the results from probabilistic model were lower than that from the point estimation (e.g., cucumber: the result of point estimation of acephate was 4.78 microg x kg(-1) x d(-1), while the results of probabilistic model which was 0.39 microg x kg(-1) x d(-1)). Meanwhile the results from probabilistic model were higher than the results of duplicate portion study (on the P95, the result of probabilistic model of Pb exposure in children was 11.08 microg x kg(-1) x d(-1), while the results of duplicate portion study was 5.75 microg x kg(-1) x d(-1)). On accuracy, the results from @Risk and the probabilistic model were highly consistent (on the P95, the result of probabilistic assessment of acephate diet exposure was 4.27 microg x kg(-1) x d(-1), while the results of duplicate portion study was 4.24 microg x kg(-1) x d(-1)), and the mean drift was of random distribution, the drift region varied from 0.05% to 11.9%. CONCLUSION: The results computed by the software of Chinese dietary exposure evaluation model are reliable and reasonable, which is a meaningful step to improve the dietary exposure evaluation technique in China.