Function And Mechanism Of Two Kinds Of Secondary Symbiotic Bacteria In The Resistance Of Sitobion Avenae Under Fungal Infestation

Symbiotic bacteria occur widely in insects and co-evolve with host insects.There are two kinds of symbiotic bacteria in aphids,namely primary and secondary symbiotic bacteria.At present,the researches on the secondary symbiotic bacteria helping aphid resist fungal infestation mostly focus on the interaction between aphid and the obligate entomopathogenic fungus Pandora neoaphidis,and the researches on the secondary symbiotic bacteria helping aphid resist entomopathogenic fungus is still not in-depth.Sitobion avenae（Fabricius）is an important insect pest in agriculture in China,which often causes great losses in agricultural production.In this study,six aphid clones infected with one kind of secondary symbiotic bacteria were selected（infected clones,symbiotic bacteria Regiella insecticola and Rickettsia three each）by feeding antibiotics,the S.avenae lines without R.insecticola and Rickettsia of the same genotype were established.Molecular biology and ecology methods were used to explore the role and underlying mechanism of two secondary symbiotic bacteria in aphid resistance to Beauveria bassiana infestation.The mean results are as follows:1.Effects of secondary symbiont and B.bassiana infestation on the life history traits of Sitobion avenae were evaluated.Our results showed that without the infestation of B.bassiana,three S.avenae clones（i.e.,F2,M13-2,B32）infected with the secondary symbiotic R.insecticola showed higher ten-day fecundity than those corresponding clones with R.insecticola removed.The infestation of B.bassiana decreased the the total developmental time of nymphs and 1^stinstar developmental time for the clone F2 with R.insecticola,but increased its 10-d fecundity than the corresponding lines with R.insecticola removed.R.insecticola reduced the mortality rates of clone B32 after B.bassiana infestation.Rickettsia did not significantly shorten the total development time of nymphs,but reduced the mortality rates of clones Ric4,Ric7 and ZYB34 after B.bassiana infestation,and it could significantly increase the ten-day fecundity of clone Ric4.Therefore,for some particular S.avenae genotypes,the two secondary symbionts could have positive effects in the host aphid’s defense against the infestation by B.bassiana.2.The copy numbers of symbiotic bacteria R.insecticola and Rickettsia were determined by absolute fluorescence quantitative PCR at the first,third,fifth and seventh day after B.bassiana infestation.The results showed that,at day 1,3,5,and 7 after B.bassiana infestation,the copy numbers of symbiotic bacteria R.insecticola in the three S.avenae clones（F2,M13-2,B32）increased significantly（in some clones by more than ten times）compared with that of no-B.bassiana treatments.However,the copy numbers of symbiotic bacteria Rickettsia in all three clones（Ric4,Ric7,ZYB34）were significantly reduced（in some clones by more than five times reduction）from 1 to 7 days after infestation by B.bassiana.In conclusion,S.avenae infected with R.insecticola could respond to fungus infection by increasing the density of secondary symbionts.S.avenae infected with Rickettsia may respond to fungal infestation by reducing the number of secondary symbiotic copy numbers and reducing the cost of host survival.3.The fluorescence quantitative PCR has been applied to determinant the relative expression levels of four protective enzyme genes[i.e.phenoloxidase 1（PO1）,phenoloxidase 2（PO2）,catalase（CAT）,superoxide dismutase（SOD）]under treatments of B.bassiana infestation/non-B.bassiana infestation for clones infected or uninfected with secondary symbionts.Our results showed that,in a majority of the cases,the expression levels of four protective enzymes in S.avenae clones infected with the secondary symbiont were higher than those in the corresponding uninfected lines on the first day after infestation by B.bassiana.The relative expression levels of PO1,CAT and SOD of six aphid clones infected with R.insecticola and Rickettsia on the first day after infection by B.bassiana significantly increased compared with those without B.bassiana infestation.On the third day after B.bassiana infestation,only the relative expression level of PO1 in clone Ric4,B32,M13-2,the relative expression level of PO2 of clone Ric4 and ZYB34,and the relative expression level of SOD of clone Ric4 and B32 were higher than those of corresponding secondary symbiont-uninfected clones.These results indicated that two secondary symbiotic bacteria（i.e.R.insecticola and Rickettsia）could play an important role in the resistance of aphids to pathogenic fungi,such as Beauveria bassiana,through affecting the expression of protective enzyme genes.