Bacterial Diversity Of Bacteriomes And Reproductive Organs From Five Cicada Species

Both nymphs and adults of cicadas feed on xylem sap which is an extremely imbalanced food source that limited in carbohydrates,essential amino acids and vitamins.There are mounts of bacteria located in bacteriomes,intestinal tract,reproductive organs,and so on,and they supply nutrient for cicadas in nutrition metabolism,growth and development.At present,studies on bacterial composition and diversity associated with bacteriomes and reproductive organs are rare.In the present study the bacterial composition and diversity in female bacteriomes from two cicada species(i.e.,Platypleura kaempferi and Meimuna mongolica)was initially investigated with 16 S rRNA polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)analysis,then,the distribution of dominant bacteria in various organs from P.kaempferi and M.mongolica was confirmed by diagnose PCR.Further,the bacterial composition and diversity in bacteriomes(female and male)and reproductive organs from P.kaempferi,M.mongolica,Karenia caelatata,Tanna sinensis and Hyalessa maculaticollis were researched with Illumina High-throughput sequencing technology.Main results are as follows:1.PCR-RFLP analysis showed that bacteria in female bacteriomes from P.kaempferi were mainly distributed in Proteobacteria and Deinococcus-Thermus.Thereinto,Hodgkinia sp.and a Rickettsia sp.(previously detected from the green rice leafhopper Nephotettix cincticeps)were the dominant.Bacteria in female bacteriomes from M.mongolica were mainly distributed in Bacteroidetes,Tenericutes,Deinococcus-Thermus,Actinobacteria and Firmicute.Thereinto,Sulcia muelleri and Spiroplasma sp.were the dominant bacteria.Additionally,Bacterial diversity of female bacteriomes in M.mongolica was greater than in P.kaempferi.2.The High-throughput Sequencing analysis of high quality sequences,OTUs and composition of bacteria associate with bacteriomes and reproductive organs from five cicada species revealed that,(1)A total of 3,011,673 high quality sequences and 9,042 OTUs were obtained from the bacteriomes and reproductive organs from five cicada species,and the sequencing depth of each sample was reliable to reflect the bacterial diversity.(2)The bacteria located in P.kaempferi were distributed across 12 assigned phyla.Thereinto,Bacteroidetes was dominant in bacteriomes(female and male)and ovaries,andProteobacteria was dominant in testes.At the genus(species)level,S.muelleri was dominant in bacteriomes(female and male)and ovaries.Bacterial communities associated with testes were complex,Rickettsia was dominant in Pt 1,Rhodococcus,S.muelleri and Rickettsia were dominant in Pt 2,and Meiothermus was dominant in Pt 3.(3)The bacteria identified in M.mongolica were classified into 11 phyla.Thereinto,Bacteroidetes was dominant in bacteriomes(female and male)and ovaries,and Tenericutes was dominant in testes.At the genus(species)level,S.muelleri was dominant in bacteriomes(female and male)and ovaries,and Spiroplasma sp.was dominant in testes.(4)The bacteria resided in K.caelatata were distributed across 9 assigned phyla.Thereinto,Proteobacteria was dominant in bacteriomes(female and male)and testes,and Bacteroidetes and Proteobacteria were dominant in ovaries.At the genus level,Dietzia was dominant in bacteriomes(female and male),and there were many bacteria species located in testes and ovaries,and their richness were similar,so the dominant bacteria was not obvious.(5)The bacteria identified in T.sinensis were classified into 8 phyla.Thereinto,Proteobacteria was dominant in bacteriomes(female and male)and ovaries,and Proteobacteria and Verrucomicrobia were dominant in testes.At the genus level,Rhodoplanes was dominant in bacteriomes(female and male)and ovaries,and Corynebacterium was dominant in testes.(6)The bacteria resided in H.maculaticollis were distributed across 8 assigned phyla.Thereinto,Bacteroidetes was dominant in bacteriomes(female and male),and Proteobacteria was dominant in testes and ovaries.At the genus(species)level,S.muelleri was dominant in bacteriomes(female and male),and Rickettsia was dominant in testes and ovaries.3.The Illumina High-throughput sequencing analysis of bacterial alpha diversity associate with bacteriomes and reproductive organs from five cicada species revealed that:(1)Within cicada species,the comparision analysis of bacterial alpha diversity revealed that,in P.kaempferi,bacterial richness associated with different organs were: testes >ovaries > male bacteriomes > female bacteriomes;bacterial diversity were: ovaries > testes >female bacteriomes > male bacteriomes.In M.mongolica,bacterial richness associated with different organs were: female bacteriomes > testes > ovaries > male bacteriomes;bacterial diversity were: ovaries > testes > male bacterioms > female bacteriomes.In K.caelatata,bacterial richness associated with different organs were: testes > male bacteriomes > ovaries >female bacteriomes;bacterial diversity were: testes > ovaries > female bacterioms > male bacteriomes.In T.sinensis,bacterial richness associated with different organs were: testes >ovaries > male bacteriomes > female bacteriomes;bacterial diversity were: testes > ovaries >female bacteriomes > male bacteriomes.In H.maculaticollis,bacterial richness associatedwith different organs were: testes > ovaries > male bacteriomes > female bacteriomes;bacterial diversity were: ovaries > female bacteriomes > male bacteriomes > testes.(2)Compared the alpha diversity associated with the corresponding organs among the five cicada species,it showed that bacterial richness associated with bacteriomes(female and male),ovaries and testes from K.caelatata were the highest,and H.maculaticollis was the lowest.Bacterial diversity in bacteriomes(female and male)and ovaries from K.caelatata were the highest,and bacterial diversity in bacteriomes(female and male),testes from M.mongolica and ovaries from T.sinensis were the lowest.4.Diagnostic PCR performed on different tissues from P.kaempferi and M.mongolica showed that,for different tissues from P.kaempferi,S.muelleri was distributed in bacteriomes(female and male),ovaries,testes,foreguts;Hodgkinia sp.was distributed in bacteriomes(female and male)and ovaries;Rickettisia sp.(previously detected from Nephotettix cincticeps)was distributed in salivary glands,midguts,malpighian tubules and testes;Spiroplasma sp.wasn't distributed in any dissected tissues.For different tissues from M.mongolica,S.muelleri was detected from bacteriomes(female and male),foreguts,ovaries and testes;Hodgkinia sp.wasn't distributed in any dissected tissues;Rickettisia sp.(previously detected from Nephotettix cincticeps)was distributed in midguts and individual salivary glands;Spiroplasma sp.was distributed in bacteriomes(female and male),midguts and hindguts.5.Bacterial composition and diversity were significantly different in bacteriomes and reproductive organs from different cicada species,the discrepancy could be derived from the cicada species,individual difference,living conditions and the interaction of bacteria within the host.Additionally,S.muelleri and Hodgkinia wasn't present in some ciacada species,it probably that the two bacteria were replaced or disappeard in the evolution of host insects.