The Molecular Mechanism Of Wing Dimorphism In English Grain Aphid, Sitobion Avenae:From Ecology To MicroRNA Levels

Aphid offers a classic model to study confluence of environmental and genetic factors on the wing dimorphism. Because of environmental variation, aphid shows two different morphs in the process of development, winged and wingless aphids. This phenotypes variation is in response to the environmental factors by choosing expression gene to achieve regulation control, which is classic epigenetic regulation. Micro RNA（mi RNA） plays a vital role in various developmental processes at the post-transcriptional level, which is an important research study in epigenetic regulation. With amount of mi RNAs identification, mi RNA regulation mechanisms on the growing development of insect have been a point of discussion for decades. While it makes excellent advances in understanding the contribution of mi RNA to wing development of insect. Studies on ecology and mi RNA mechanisms of wing dimorphism in Sitobion avenae provide insight into the future researches about wing dimorphism in aphid.In this study, the external morphology of winged and wingless in the grain aphid S. avenae were studied using stereo microscope and scanning electron microscopy; Studies on ecology of wing dimorphism were conducted by setting different external factors; Mi RNA regulation mechanisms of wing dimorphism were studied by high-throughput sequencing and quantitation real time PCR（q RT-PCR）, etc. The results are as follows:There were no obvious external differences between the 1st and 2nd instar nymphs. The antennae of both instars had five segments; the middle thorax of the 3rd instar winged nymph was greatly enlarged and the wing buds could be identified until integral wing develops in winged adult aphid. The 3rd wingless nymph was simple, without wing buds. The antennae consisted of six segments from 3rd instar stage to adult aphid; the cauda of the 1st instar nymph were tuberculiform shaped, and armed with two acicular setae. It became conical shaped in 2nd, 3rd, 4th instars stages, and then was enlarged into long taper or long tongue shape in the winged or wingless adults, respectively. Additionally, more than 6 setae were present from 2nd instar stages; there were no obvious external differences on the cornicle of 1st to 3rd instars stages, showing cylinders. The cornicles of the 4th instars nymph begin swollen at the terminal, and become slim at the proximal end. There was no obvious external difference between winged and wingless adults with polygon reticulate structure at the proximal end.We investigated wing dimorphism of S. avenae in response to the external factor, density, temperature, host plant quality, materal effects. Increasing neonatal（< 24 h old） offspring density triggered wing formation; neonatal offspring of S. avenae might tend to winged under the higher and lower temperature; Increasing materal density affected winged morph determination, linking with maternal morphs; host plant quality also correlated with wing variation in S. avenae, not only effecting the contemporary aphid, but also the second generation.We constructed two mi RNA libraries from winged and wingless adults of S. avenae. A total of 146 conserved mi RNAs and 152 species-specific candidate mi RNAs were identified using a combination of high-throughput sequencing and bioinformatics approach. More than 34% of mi RNAs was enriched in 22 nt corresponding to conventionally accepted mi RNA length. 52 differential expressed mi RNAs based on normalized deep-sequencing counts was analyzed by differential analyzation（P<0.1, 0.05 and 0.01）, including thirty up regulation mi RNAs and twenty-two down regulation mi RNAs. The targets of these mi RNAs and pathways analysis were predicted and annotated by GO（Gene Ontology） and KEGG（Kyoto Encyclopedia of Genes and Genomes）, screening out 9 mi RNAs（7 conserved mi RNAs: Let-7, mi R-1_L-1, mi R-7, mi R-277, mi R-8, m R-9a_R-2, mi R-315 and 2 novel mi RNAs: PC-5p-113190₁5, PC-3p-2743₈44）, which might be relating with wing development in S. avenae. Moreover, the expression profiling of 9 mi RNAs were assessed between winged and wingless adults of S. avenae by q RT-PCR analysis. The results showed that the expression of 9 mi RNAs showed higher expression in winged adult than that of wingless adult. Additionally, the expression of 5 conserved mi RNAs（Let-7, mi R-1_L-1, mi R-7, mi R-277, and mi R-8） were demonstrated that they had a significant difference between two wing morphs, which suggested that these mi RNAs might play a critical role in wing development in S. avenae.Subsequently, we constructed an array of mi RNA libraries from sensitive developmental period of wing dimorphism, including winged and wingless embryos, 1st and 2nd instar nymphs. More than 60% clean counts were apply to bioinformatics analysis after preliminary filtering out raw counts. There were predicted conserved pre-mi RNAs 85, unique-mi RNA 150, and new pre-mi RNAs 1050, unique-mi RNA 1016. The differential expressed mi RNAs were analyzed among two databases. The identified mi RNAs were compared with those from other insects show highly homologous to mi RNAs of A.pisum, then T.castaneum and B.mori. The expression profiling of 9 mi RNAs were assessed in different instar of both wing type in S.avenae by q RT-PCR analysis. The results showed that they were varied on the expression of different mi RNAs in different morphs and development stages, which suggested that expression of mi RNA was in spatial or temporal expression pattern.Two mi RNAs, mi R-7 and mi R-277, which had been predicted linking with wing dimorphism of S. avenae were chosed. Mi RNA agomir, double-strand small RNA, was designed artificially on based its mature sequence. It could enhance the expression of target gene by simulating mi RNAs. Concentrations of 300 n M in mi RNA agomir were applied to feed neonatal offspring for different feeding time. The acquire-to-function efficiency of mi RNA agomir on mi R-7 and mi R-277 was examined using q RT-PCR. The results showed that the expression target gene gradually increased with the increase of feeding time. Wing dimorphism on treated nymphs for different times was investigated by transferring to healthy wheat. After forth or five days, aphids, by now forth instar or early adult stage, we found that all treated nymphs could fully ensure normal growth, and the morph type was not change for feeding mi RNA agomir.