In order to precisely assess gene expression level, a suitable internal reference gene must be chosen to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data.  For greenbug, Schizaphis graminum, a suitable reference gene for assessing the level of transcriptional expression of target genes has yet to be explored.  In our study, eight reference genes, elongation fator 1 beta (Ef1β), TATA box binding protein (TBP), alpha-tubulin (α-TUB), 18S ribosomal (18S), 28S ribosomal (28S), glyceraldehyde-3-phosphate (GAPDH), actin (ACT), and ribosomal protein L18 (RPL18) were evaluated in S. graminum at different developmental stages, tissues, and insecticide treatments.  To further explore whether these genes are suitable to serve as internal control, three software-based approaches (geNorm, BestKeeper, and NormFinder), ?Ct method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes.  The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized gene expression data of three target genes, heat shock protein gene (HSP70), cytocrome P450 gene (SgraCYP18A1), and glutathione S-transferase (GST).  We found that the most suitable reference genes varied considerably under different experimental conditions.  For developmental stages, α-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACT were suitable reference genes; for insecticide treatments, 28S and α-TUB were suitable for normalizations of expression data.  In addition, 28S and α-TUB were the suitable reference gene as they had the most stable expression among different developmental stages, tissues and insecticide treatments.  This should be useful for the selection of the suitable reference genes to obtain reliable RT-qPCR data in the gene expression of S. graminum.

 

The presence of adequate nectar sources along the migratory pathway is a prerequisite for moths of oriental armyworm, Mythimna separata, to reach target habitats and lay eggs.  Identification of plant species and assessment of their suitability as wild nectar sources along the summer migration pathway of adult M. separata are critical steps in making effective forecasts of second- and third-generation armyworm outbreaks in North China.  We explored five aspects of moth-nectar source relationships: (1) Field investigations in 2014 and 2015 showed that the florescence of chaste tree, Vitex negundo var. heterophylla, a primary nectar plant in apiculture, coincided with the summer migration of oriental armyworm moths.  The flowers of chaste tree opened day and night and M. separata moths were found actively flying around and resting on tree.  (2) Identification of moth-borne pollen by scanning electronic microscopy (SEM) micrographs in 2015 and 2016 revealed that 15.4% of 369 moths trapped by blacklight traps, 17.1% of 557 moths captured by vertical-pointing searchlight traps and 24.4% of 311 moths caught by pheromone traps were found with pollen attached to their proboscis.  A total of 228 pollen-carrying individuals out of 1 237 armyworm moths were captured by the three types of traps.  The three highest percentages of pollen-bearing armyworm moths were carrying pollen of Toona sp. (59.65%), Ligustrum sp. (27.19%) and Syringa sp. (4.39%).  In total, 14 types of pollen were detected where three were identified to species, 10 to genus and one as an unknown species.  (3) Analysis of geographical distribution, flowering periods and relative abundances of candidate nectar plants suggested that Chinese toon, Toona sinensis, and privet, Ligustrum lucidum, were two important nectar plant species.  (4) Analysis on relationship between pollen and ovarian development showed that 14, 15 and 10 females carrying pollen of T. sinensis had ovaries at developmental stages I, II and III, respectively.  Many T. sinensis pollen grains were present on the proboscises of female moths.  (5) Comparison of trap effectiveness between blacklight and vertical-pointing searchlight traps indicated blacklight traps caught the highest proportion (35.2%) of female in ovarian stage V, whereas vertical-pointing searchlight traps caught the highest proportion (43.5%) of female in ovarian stage I with additional proportions of decreasing percentages for stages II, III and IV.  Integrated analysis on these five aspects suggests that T. sinensis is an important, suitable summer nectar source that potentially influences migration and reproduction of M. separata moths and thus contributes to outbreaks of this pest.  The vertical-pointing searchlight trap was the most effective tool for monitoring the migratory flight of adult M. separata.  Privet, Ligustrum lucidum, and clove, Syringa sp., are possible suitable summer nectar plants that need further study to elucidate their importance.  The reason why field investigation results on chaste tree differed from the results of moth-borne pollen analyses was discussed.