Bacterial Diversities Associated With The Fig And Four Fig Wasp Species Of Ficus Hispida

Insects, as the most successful and largest population in the terrestrial, have high fitnessfor lots of inhabits, that partially benefits from their microbial symbionts. Host-associatedmicrobial symbionts take part in many life process of macroorganism, including nutrition,immunization, fitness, and reproduction, and have played significant roles on the cospeciationand coevolution with their host insects. Fig-pollinating wasp mutualism is widely regarded asone of the most classical models to explain mutualisms and coevolution between insect andplant host. Hundreds of species of pollinating fig wasps (PFW), keeping extreme “one to one”pattern, distinguish one responsible fig species especially from others to finish pollination andoviposition in the past millions of years.Ecologists have increasingly pay attention to a third-party, Bacterial or fungi which havebeen known to play very important roles in more and more mutualism interaction, to interpretand reveal these previously unexplored components of even the most classic two-partnerassociations. To date, we have known little knowledge about the microbial communityassociated with fig and fig wasps, except the inheritable endosymbiont, Wolbachia, which hashigh prevalence across fig wasp species. Janzen have reviewed “fig pseudolocule sterility”and indicated that microbial or fungal clones were never found growing in the pseudoloculeof undamaged developing figs and female wasps remain relatively intact for many weeks afterdying in syconium. Miller et al. cultured a single bacterial species, Serratia plymuthica(Lehmann and Neumann) Bergey et al., and a single yeast species, Candida guilliermondii(Castellani), from healthy fig and associated PFW Blastophaga psenes. In contrast, a largefungal microflora was revealed from six species of developing and healthy figs in Panama,using culture-free methods. We supposed that there should also be a specific bacteriacommunity in the fig-fig wasp symbiosis system, and the bacterial microflora may even playsome important roles in uncovering lots of unexplored components of the classic model. Inthe other word, the genes or composition of microbial community associated with fig and figwasps should evolve faster than fig wasps under the constraint of syconia. And the mutualismof fig and fig wasp would be an ideal model to verify the hypothesis “microbe are everywhereand environment select” and the theory of hologenome.In the present study, we have screened the microbial communities of the four species offig wasps (PFW: Ceratosolen solmsi; NPFWs:(Apocrypta bakeri, Philotrypesis pilosa andPhilotrypesis sp.) and the fig ovaries associated with Ficus hispida using culture-free methodsincluding16S rDNA clone libraries and high throughput database from genome. Herein, we asked five questions:(1) whether the bacterial communities associated with the fig-fig waspsymbiosis system are as simple as the “sterile” syconium, consistent with previous studies?(2)Is the bacterial community of the system, different from the other habit?(3) Where does thebacteria community of fig wasps come from, fig fruit, mother wasp or some other uncertainvariables?(4) Whether the bacterial communities of the four species of wasps varied, due totheir different ecological niches in the small house?(5) Is there any other endosymbiontexisting within fig wasps, response to the regulation of wasps’ reproduction, in addition toWolbachia?We have reached to the following conclusions with our research: Firstly, the bacterialcommunities associated with the four fig species of Ficus hispida present three characters:1)Fig wasps contain large bacterial communities much more than previous estimation.2) Figwasps and other insects living outside fig share similar bacterial diversity on the level ofphylum, however, they distinguish with each other on lower level with taxa.3) Different figspecies and their fig wasp probable contain different bacterial diversity.Secondly, the four fig wasp species with different taxonomic status and life habitspresent different bacterial compositions:1) There are small differences with intraspecies andlarge differences with interspecies.2) There are three distinct bacterial compositions, one forC. solmsi, the second for P. pilosa, and the third for A. bakeri and P. sp.3) The bacterialdiversity of C. solmsi shows the typical characters of the bacterial communities withphytophagous insects, which are rich in family Enterobacteriaceae.4) A. bakeri and P. sp.which belong to two different genera within the same family displays almost same bacterialcompositions corresponding to their similar life habits, and they are especially rich in genusTepidimonas.5) The bacterial composition with P. pilosa is different with those of the otherthree fig wasp species, and implies its different feeding habit.6) The three different bacterialcompositions with four wasp species are corresponding to their different ecological niches butnot their genetic relationship.Thirdly, through the thoroughly DNA extraction of bacteria within fig by cell disruptionand chemical extraction and targeted detection using specific primers which could not bind tothe ctDNA, we have succeeded to detect the bacterial diversity of fig ovaries with Ficushispida. Though simpler than the complicated bacterial diversity of rhizosphere, there are alsokinds of bacteria existing in fig ovaries other than sterile. The dominant consortium whichtypically includes genera Delftia and Acinetobacter and so on, are not infected apparently bythe behavior of oviposition. However, Accs strain emerged from ovaries after oviposition, andthen we got four evidences to verify the vertical transmission for the Accs strain.1) Accs strain existed in every clone libraries of C. solmsi with average relative richness2.2%whichsimilar to that of Wolbachia.2) The relative richness of Accs within ovaries is about12%,3times more than Acfh.3) Accs strains cluster into one branch on the NJ tree, with97%similarity with the most similar sequence in NCBI.4) There are18ribonucleotides divergencebetween Accs and Acfh.The genome database of C. solmsi is equal to the hologemoe of C. solmsi, which includealmost all the genome messeges of microbe. Through statistial analysis with these sequencesof microbe within the genome database, we found that:1) The real diversity of microbeswithin the holobiont of C. solmsi is much more than the detected result though clone libraries.2) the assembled genome of C. solmsi only contain little microbial sequences whichcorrespond to those dominant bacteria within clone libraries.3) There are some potentiallyimportant fungi and viruses sequences exsiting in the database which provide new chance toexplore the extensive interaction between fig wasps and their microbes.The interesting concordence between bacterial compositions and ecological niches of figwasp species within the same inclosed syconia would play an important role on the furtherresearch on interaction between hosts and its symbionts. Moreover, this system provides anideal model for hologenome theory. Great amounts of microbes, including bacteria, fungi andviruses, were uncovered in this study, which would provide more pathways to mine fig and figwasp system.