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The genetically modified cotton holding Bt proteins, is noxious to bollworms larvae but very little is known about its impact on population of non-target spiders within the field. Studies conducted with the aim to identify spiders along with their abundance, prevalence pattern, preying habit on insect pests in Bt cotton (L280, Cry2Ab4) and commonness of symbiotic bacteria in these spiders. Spiders collections were made via vial tapping/jarring, from May to end of October (2018). In total, 13,342 spiders were recorded during the entire cropping season. Least relative abundance was in May (0.28%), highest in August (30.39%) while ahead decline was observed, reached to 12% in October. The 27 species were verified, least richness was in May (3 species), high in August (19 species), later in the end, reduced to 7 species. High abundance was of Hylyphantes graminicola (68.56%) and Neoscona theisi (19.98%). Existence of H. graminicola and Pardosa astrigera observed during the whole cropping season. Presence of seven guilds among spiders had a diverse nature of hunting insect pests and on number of species existed in the crop, maximum was from direct hunting habit. Composition and community structure of microbiota varied as of spider species. Proteobacteria was the most abundant bacterial phylum and topmost genus was Wolbachia in observed spiders. Five groups of bacteria distinguished across 4 clusters of spiders depending on core community of symbiotic bacterial genera. This effort is an initial step to get knowhow about spiders in Bt cotton, which will facilitate future research studies concerning spiders.

Aphis gossypii is a globally distributed species and therefore has a highly variable life cycle. Populations of A. gossypii in northern China exhibit greater genotypic diversity and a broader host range, yet the details of life cycles of different biotypes is still unclear. In this study, the Cytb and 16S gene regions of A. gossypii collected from 5 common summer hosts and 4 primary hosts were analyzed. A total of 57 haplotypes were obtained from 1 046 individual A. gossypii sequences. The sequence included 44 variable sites, 27 of which were parsimony informative sites and 17 of which were singleton variable sites. The most frequent 3 haplotypes were found in 896 individuals, representing a total of 85.7% of all individuals and 36 haplotypes were found in 1 individual. A neighbor-joining tree was constructed using 21 haplotypes that were found in more than 2 individuals. Considering the individual host plant, 5 biotypes were identified. Type 1 corresponded exactly to the cucurbit host-race and the other 4 biotypes were found as cotton host-races. Type 3 was the most abundant biotype in cotton fields in northern China.

Odorant-binding proteins (OBPs) play a significant role in the olfactory signal transduction of insects and help them locate hosts, oviposition sites, and mating partners. The sap-sucking insect, Aphis gossypii Glover (Homoptera: Aphididae), is a destructive cosmopolitan pest and yet the molecular mechanisms by which A. gossypii perceives pheromones and host volatiles remain unknown. In this study, we identified and characterized 6 OBPs, using the A. gossypii RNA-seq transcriptome dataset previously constructed in our laboratory. Real-time PCR indicated specific expression patterns of the 6 genes, which had different levels of expression based on development stage, tissue, morph, and life cycle. In addition, binding specificities of the 6 proteins investigated using the ligand-binding assays showed that all 6 OBPs exhibited high binding affinities towards Phlorizin dehydrate while AgosOBP3 and AgosOBP4 had strong affinity to beta-ionone and AgosOBP8 displayed higher binding affinities for Nerolidol and Cis-3-hexenyl acetate compared to other OBPs.

Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L. japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L. japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L. japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT, 18S rRNA, and RPL13 during different stages; AK, RPL13, and TBP among sexes; EF1A, PPI, and RPL27 in different tissues, and EF1A, RPL13, and PPI in adults fed on different diets. Moreover, the expression profile of a target gene (odorant receptor 1, OR1) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L. japonica and will benefit subsequent genomics and functional genomics research on this natural enemy.

With increased cultivation of transgenic Bacillus thuringiensis (Bt) cotton in the saline alkaline soil of China, assessments of transgenic crop biosafety have focused on the effects of soil salinity on rhizosphere microbes and Bt protein residues. In 2013 and 2014, investigations were conducted on the rhizosphere microbial biomass, soil enzyme activities and Bt protein contents of the soil under transgenic Bt cotton (variety GK19) and its parental non-transgenic cotton (Simian 3) cultivated at various salinity levels (1.15, 6.00 and 11.46 dS m −1 ). Under soil salinity stress, trace amounts of Bt proteins were observed in the Bt cotton GK19 rhizosphere soil, although the protein content increased with cotton growth and increased soil salinity levels. The populations of slight halophilic bacteria, phosphate solubilizing bacteria, ammonifying bacteria, nitrifying bacteria and denitrifying bacteria decreased with increased soil salinity in the Bt and non-Bt cotton rhizosphere soil, and the microbial biomass carbon, microbial respiration and soil catalase, urease and alkaline phosphatase activity also decreased. Correlation analyses showed that the increased Bt protein content in the Bt cotton rhizosphere soil may have been caused by the slower decomposition of soil microorganisms, which suggests that salinity was the main factor influencing the relevant activities of the soil microorganisms and indicates that Bt proteins had no clear adverse effects on the soil microorganisms. The results of this study may provide a theoretical basis for risk assessments of genetically modified cotton in saline alkaline soil.

Cotton (Gossypium hirsutum Linn.) is widely cultivated in China. The polyphagous insect Adelphocoris suturalis (Jakovlev) is a serious insect pest in cotton growing regions. Plants have evolved sophisticated systems to cope with herbivore attacks. However, the cotton defense mechanisms induced by A. suturalis feeding have lagged behind. We carried out untargeted proteomic analysis using the iTARQ technique and metabolomics based on LC-MS/MS analysis of cotton leaves fed upon by A. suturalis. Proteomic analysis identified 775 upregulated proteins and 477 downregulated proteins in plants that were infested by A. sututralis compared to the controls. Metabolomic analysis identified 50 differentially expressed metabolites in the positive ion mode and 14 in the negative ion mode compared to the controls. The tryptophan metabolism pathway was significantly changed in both the positive and negative ion mode in the metabolomics analysis. The alpha-linolenic acid pathway was significantly changed in both the proteomic and metabolomics analyses. Furthermore, the result was validated by RT-qPCR analysis of 5 related genes involved in alpha-linolenic acid pathway. These results indicate that tryptophan metabolism and the alpha-linolenic acid pathway may be important in cotton defense against herbivores and would enhance our understanding of plant defenses induced by A. sututrali feeding.

The ecological fitness of transgenic cotton and its effects on the insect communities in cotton fields is one of the key aspects of the evaluation of the environmental safety of transgenic cotton. New transgenic GAFP (Gastrodia anti-fungal protein) cotton and its parental varieties were used in this study to explore their ecological fitness and their effects on insect community infield in Anyang, Henan Province in 2013 and 2014. The results showed that there was no significant difference in dry mass for transgenic cotton leaves compared to that of parental cotton. Specific leaf areas of transgenic cotton were lowered obviously at seedling stage, while enhanced significantly at budding, flowering and bolling stages relative to parental cotton. The plant height of transgenic cotton was lowered only at seedling stage, and no significant difference was showed between the two cultivars at budding, flowering and bolling stages. No significant differences were discovered on plant branch numbers, bud numbers and falling numbers between the transgenic cotton and control material in any of the four key stages during the cotton growth. However, the number of bolls per plant for transgenic cotton was lower than that of the control cotton at the bolling stage. In the 2nd, 3rd, and 4th generation of cotton bollworm (Helicoverpa armigera), the mortality rate of cotton bollworm and beet armyworm (Spodoptera exigua) of transgenic cotton had no significant difference with parental cotton. Compared to parental cotton, total individuals of insect community, pest sub-communities and enemy sub-communities in transgenic cotton field didn't show any significant difference. The above results showed that after the GAFP gene was imported into cotton, the cotton growth was enhanced significantly, while the whole yield component traits and the insect community in the field were not significantly changed. Our study on the competition of new transgenic cotton and survival of transgenic cotton insect communities in cotton field would provide the theoretical basis for the evaluation of new transgenic cotton and environmental safety, and accumulate scientific data for environmental safety evaluation of the transgenic cotton.新型转基因棉花生态适合度及其对棉田昆虫群落的影响是转基因棉花环境安全评价的重点内容.本文选用新型转

Lygus pratensis (L.) is an important cotton pest in China, especially in the northwest region. Nymphs and adults cause serious quality and yield losses. However, the genetic structure and geographic distribution of L. pratensis is not well known. We analyzed genetic diversity, geographical structure, gene flow, and population dynamics of L. pratensis in northwest China using mitochondrial and nuclear sequence datasets to study phylogeographical patterns and demographic history. L. pratensis (n = 286) were collected at sites across an area spanning 2,180,000 km2, including the Xinjiang and Gansu-Ningxia regions. Populations in the two regions could be distinguished based on mitochondrial criteria but the overall genetic structure was weak. The nuclear dataset revealed a lack of diagnostic genetic structure across sample areas. Phylogenetic analysis indicated a lack of population level monophyly that may have been caused by incomplete lineage sorting. The Mantel test showed a significant correlation between genetic and geographic distances among the populations based on the mtDNA data. However the nuclear dataset did not show significant correlation. A high level of gene flow among populations was indicated by migration analysis; human activities may have also facilitated insect movement. The availability of irrigation water and ample cotton hosts makes the Xinjiang region well suited for L. pratensis reproduction. Bayesian skyline plot analysis, star-shaped network, and neutrality tests all indicated that L. pratensis has experienced recent population expansion. Climatic changes and extensive areas occupied by host plants have led to population expansion of L. pratensis. In conclusion, the present distribution and phylogeographic pattern of L. pratensis was influenced by climate, human activities, and availability of plant hosts.

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels.

In this study, we detected and clarified the roles of transgenic Cry1Ac+Cry2Ab cotton "639020" in controlling cotton bollworm (Helicoverpa armigera) during critical periods of bud stage (second generation of bollworm), flowering stage (third generation of bollworm) and bolling stage (fourth generation of bollworm) as well as the influences of 639020 cotton on functional response of the main predators (Chrysopa sinica larvae, Propylaea japonica, Orius and Erigonidium graminicola ) on whitefly using transgenic Cry1Ac cotton "CCRI41" and conventional cotton "CCRI49" as the control. Our results showed that the 639020 cotton well controlled the second and third generation of bollworm, and the level of insect resistance increased by 52.85% and 16.22% separately compared with that of CCRI41, with a significant effect on the second generation of bollworm. Moreover, the number of bollworm eggs in 639020 cotton field was lower than that in CCRI41 and CCRI49 cotton fields (except the second generation of bollworm) during the cotton bud, flowering and bolling stages. Although the number of bollworm larvae in 639020 cotton field was significantly lower than that in CCRI49 field, and both under the controlling index, it has no significant difference compared with that in CCRI41 cotton field. There were also no obvious changes in predator functions of Chrysopa sinica, Propylaea japonica, Orius and Erigonidium graminicola on bemisia tabaci between 639020, CCRI41 and CCRI49 cotton filed. This study evaluated the safety of new transgenic cotton on environment, anti-insect activity of exogenous gene and the safety of production and application prospect.

Reference genes have been utilized in estimating gene expression levels using quantitative reverse transcriptase-quantitative polymerase chain reaction (qRT-PCR) analysis. Aphidius gifuensis Ashmaed is one of the most widely used biological control agents for aphids. The biological properties of this species have been studied in detail, and current investigations are focused on elucidating the regulatory mechanisms in its host However, the appropriate reference genes for target gene expression studies have not been identified. In this study, the expression profiles of 12 candidate reference genes were evaluated under different experimental conditions(development stage, sex, tissue type, diet) by using dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt. In addition, RefFinder was used to rank the overall stability of the candidate genes. Finally, we recommend three optimal reference genes for the normalization of qRT-PCR data in the presence of specific variables, which include ACTB, RPL13, and PPI for different developmental stages; RPS18, ACTB, and RPL13 for sexes; RPL13, PRII3, and RPS18 in different tissue types; and RPL13, RPL27, and ACTB in diverse diets. The present study has identified optimal reference genes that could be used in estimating the expression levels of specific genes under these conditions following the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines, which would facilitate in advancements in functional genomics research on A. gifuensis.

To explore the efficacy and safety of tandospirone for adolescents with anxiety disorder.In this multicenter open-label 12-week study, a total of 169 adolescents met the criteria of DSM-IV for anxiety disorder were treated with flexible dose of tandospirone. The effects and safety of treatment were assessed by the Hamilton anxiety scale (HAMA), state-trait anxiety inventory (STAI), clinical global impression (CGI) and side effects at baseline and Weeks 2, 4, 8 and 12.In 151 cases completing the 12-week treatment, the scores of HAMA decreased by 18% ± 12%, 38% ± 16%, 54% ± 20% and 63% ± 17% at Weeks 2, 4, 8 and 12. Significant reductions in HAMA scores were seen at Weeks 2-12 (P0.01) and marked improvement appeared in 81.5% cases at Week 12 (reduction rate of HAMA: 50%). At Week 12, the HAMD(17) scores were markedly lower versus baseline (7 ± 2 vs 13 ± 3, P0.01). Adverse effect of tandospirone was scarcely found (9.9%).Tandospirone is an effective and safe anti-anxiety drug for adolescents.