The Variety Of Symbiotic Bacteria At Each Stage Of Musca Domestica(Diptera:Muscidae)

ObjectiveThis topic adopted the traditional isolation and culture method,PCR-DGGE and highthroughput sequencing technology based on metagenomics,through detecting the 16 S rRNA genes of bacterial to determine bacterial species,analyzed the diversity of symbiotic bacteria at each state of housefly,discusses the change rule of the symbiotic bacteria in the each state of housefly,and analyzes the pros and consof this three kinds of experimental methods in the study of insect symbiotic bacteria.Methods1Traditional isolation and culture methodLarvae were staying hungry to remove intestinal bacteria.The housefly of eggs,larvae,pupae,hungry and dietary adult flies were surface sterilized in 75% ethanol,rinsed in sterile water,then homogenized under aseptic conditions.Each sample was inoculated and isolated into the beef-protein medium and incubated at 37?.The monoclone of bacterial strains were inoculated at 37? on an orbital shaker(110 rpm)overnight.The CTAB method is used to extract DNA.The bacterial universal primer 27 F and 1527 R were used to PCR.PCR products were checked on 1% agarose gel,then sequenced by sequecing company.2Culture-independent methodLarvae were staying hungry to remove intestinal bacteria.The housefly of eggs,larvae and pupae were surface sterilized in 75% ethanol,rinsed in sterile water,then homogenized under aseptic conditions in 500?l buffer.Each sample is used to extract total DNA.2.1 PCR-DGGEEach sample of total DNA was PCR by suing the primer 27 F and 1527 R,then the PCR products were second PCR by the primer 968 GC and L1401.The PCR products were sued to DGGE.After dying by EB,the collected aim DNA band were PCR by the primer 968 and L1401.PCR products were sequenced by sequencing company.2.2 High-throughput sequencing technologyThe total DNA of the eggs,larvae and pupae were used Illumina MiSeq to sequence by sequencing company.ResultsA total of 28 species bacteria genera were isolated by traditional isolation and culture method in the each stage of the housefly,the eggs was none,including 11 from the larvae,19 from the pupae,10 from the hungry adult flies and 13 from the adult flies with dieting.A total of 7 species bacteria genera were isolated by PCR-DGGE,the majority were Staphylococcus sp.and Providencia sp..The result of high-throughput sequencing technology shows that the majority of isolates were phyla Proteobacteria and Bacte-roidetes,and genus Myroides sp.and Sphingobacterium sp.in the eggs,larvae and pupae of housefly.Due to none of symbiotic bacteria in housefly eggs,and 13 kinds of isolated bacteria from the culture medium during larvae feeding,mainly bacteria genus were Proteus sp.and Myroides sp.,9 kinds of isolated bacteria were the same with the gut living bacteria from housefly larvae,so we speculated that the intestinal symbiotic bacteria in housefly larvae mainly comes from food and environment.ConclusionThrough the adoption of the traditional isolation and culture method,PCR-DGGE and high-throughput sequencing technology based on metagenomics,analyzed the diversity of symbiotic bacteria at each state of housefly,we found that the symbiotic bacteria is complicated and various and speculated that the change of species and quantity is connected with the metamorphotic development of housefly.These three kinds of experimental methods have their own advantage.Traditional cultivation method can obtain bacteria strain thatcan be intuitive observation of morphology and physiological and biochemical characteristics of identification,for laying a foundation for a comprehensive understanding of bacteria characteristic and the function verification,but only get the culturable bacteria.The uncultivate bacteria are difficult to get any information and affected by human factors.With regard to less bacterial species diversity and separable bacteria,PCR-DGGE technique has more advantage to sort.Groups of bacteria that are too similar to separate,is hard to get ideal result,the repeatability is not high,easily influenced by artificial operation.For High-throughput sequencing method based on metagenomics,it is a new method of identification of bacteria,can get all information of bacteria.The method is simple,fast,strong repeatability,is an advanced,standardized methods for identifying mixed bacteria groups.All the information of culturable and unculturable bacteria can be available easily.Butthe same as PCR-DGGE technique,unable to obtain pure strains of bacteria,so cannot be used for functional verification of bacteria.