Studies On Symbiotic Bacterium In Pandora Neoaphidis And Their Interactions

Pandora neoaphidis（Entomophthorales）,an obligated aphid-pathogenic fungi,can cause a high level epizootic to make aphid population collapse.It has increasing scientific attention on this fungi.Some evidences on the presence of symbiotic bacteria in P.neoaphidis have been proved in our previous studies.Here we would explore the biological characteristic of symbiotic bacteria in fungi and their interactions,using P.neoaphidis isolation F98028 and Acinetobacter calcoaceticus ATCC 23055 as the main study object.Results are following:1.Design and application of the outdoor isolation unit for Entomophthoralean fungi.In order to better verify the existence of symbiotic bacteria in P.neoaphidis,an outdoor simple isolation unit for collecting the asexual spores,or conidia was designed and developed.Its conidial collection ability and contamination rate were evaluated for his application advantage.Compared statistically the new isolation unit with the traditional cryotube method,it found that the number of spores collected in the isolation unit was significantly greater than that of cryotubes from the third collection day.The cumulative number of spores collected by the isolation unit was larger than the cryopreservation tube method in the fifth day.The percentage of successful isolation attempts was not significantly different between the conventional method（78±17%）and the isolation unit（76±18%）（t=0.43,P=0.67）.The novel isolation unit was as effective as the ‘descending conidia’ method,much easier to use,especially under field conditions,and did not require laminar flow conditions.The isolation unit can thus be used under relatively primitive field conditions without the need to return to the laboratory.Therefore,use of the isolation unit could improve the isolation efficiency greatly in remote areas where access to laminar flow facilities are limited.2.Construction of genetic transformation system of A.calcoaceticusNew plasmid p MU125 was constructed from E.coli-A.calcoaceticus Shuttle plasmid p WH1266 which intergrated with Green fluorescent protein（GFP）gene.Subsequently,the electrocompetent cells,prepared by Na OH-ethanol,were used for electroporation transformation.Using an orthogonal experimental design,optimal parameters for high-efficiency transformation of Acinetobacter calcoaceticus were defined: highest transformation efficiency（7.51×103 transformants/μg DNA）was achieved at a growth phase of OD600=0.5;700ng plasmid DNA;200Ohms resistance.This provides a useful tool for genetic manipulation of A.calcoaceticus.3.Verification of symbiotic bacteria in P.neoaphidis based on Koch’s postulatesFirstly,the groove was used to form a physical isolation environment for co-cultivating P.neoaphidis and symbiotic bacteria.The unique biological characteristics of P.neoaphidis,spore-discharging capbility are used to exclude the bacteria from attaching to the fungal spore surface based on the negative results on bacterial liquid culture of the fungal mycelia outside the groove（formed by the spore discharged from groove inside）.Secondly,extraction of the total mycelium DNA of P.neoaphids outside the grooves and subsequent PCR using specific primers for A.calcoaceticus were carried out to prove A.calcoaceticus can enter the fungus body.Thirdly,it found that there was A.calcoaceticus in the fungal mycelia outside the groove using transmission electron microscopy.Furthermore,the co-culturing A.calcoaceticus can be isolated from the mechanically disrupted mycelium which were collected from the groove outside.All above were the verification process of symbiotic bacteria in P.neoaphidis based on Koch’s postulates.4.Preliminary study on the origin of the symbiotic bacteria of P.neoaphidis.Firstly,aphid honeydew of various feeding period was collected and coated on LB solid medium.The number of colonies illustrated the dynamic change of intestinal microorganism from peach aphid.The results showed that the rifampicin and tetracycline had achieved a certain degree of aposymbiosis of the intestinal microorganism from peach aphid.A.calcoaceticus fed to the peach aphid can be detected in honeydew after 3 days.It suggested that A.calcoaceticus could survive in aphids.Moreover,the aphids was living well during the whole feeding cycle.Secondly,the distribution of the intestinal tract of symbiotic bacteria through the intestinal tract of peach aphid was investigated.Aphids were fed with GFP-Labeled Acinetobacter calcoaceticus for 3 days.After aphids were frozen and sectioned,both the treatment group and the control group were observed using confocal laser scanning microscopy.The sections were stained with Sudan III to determine the fat distribution located around the internal organs,body cavity walls,and the head of aphid.Subsequently,aphid was dissected and its fat-containing tissue fluid was sucked with a capillary tube then were spread on ampicillin-resistant LB media to observe existing A.calcoaceticus or not.Results showed that the GFP fluorescence spots on the peach aphid slices gradually increased and diffusely distributed as the feeding days rising.It suggested that the target bacteria A.calcoaceticus could be spread through the intestine to the outside of the tissue,especially near the fat tissue.Moreover,symbiotic bacteria might enter into the mycelium of P.neoaphidis by attaching to the fat droplets which P.neoaphidis could engulf normally.5.RNA-seq on P.neoaphidis containing the symbiotic bacteria A.calcoaceticus.The transcriptome information was obtained by RNA-seq in P.neoaphidis containing the symbiotic bacteria A.calcoaceticus.The transcriptome data of the interaction between fungal and bacteria were analyzed from the bacterial perspective.The results showed that two samples（Pn-Ac₀1and Pn-Ac₀2）have obtained 3,365 genes in total by mapping to genome guided or reference sequence.Pn-Ac₀1 and Pn-Ac₀2 have 527 and 3,365 genes respectively.22 genes was found to related to the Type VI secretory system（T6SS）which played an important role in the symbiotic relationship between fungi and bacteria.The expression level FPKM values of T6 SS system pipeline structural protein Hcp gene Pn-Ac₀1 and Pn-Ac₀2 were 3856.48 and 6607.49 respectively.As for Pn-Ac₀2,four types of Vgr G pin structure proteins genes were found,and the expression levels of FPKM were 91.77,77.94,138.41 and 65.07 respectively.As for the Imp series protein genes,the expression level FPKM values of Imp B,Imp C and Imp H in the two samples were 1928.24 and 687.82,936.57 and 58.2,623.63 and 703.95 respectively.T6 SS energy supply related ATP binding protein subunit Clp B,Clp X,Clp P and Clp A expression level FPKM values were 7797.11 and 6467.52,3528.19 and 2325.44,1582.88 and 1140.18,402.34 and 677.78 respectively.Validation of the symbiotic bacteria in P.neoaphidis and their interactions was explored and verified in this study.Furthermore,a useful support was provided for future study on the immunological correlation betweenthe host aphids and P.neoaphidis.