Effects Of Wolbachia On MtDNA Variations Of Fig Wasps Associated With Ficus Hispida

Wolbachia are widespread intracellular bacteria that are found in arthropods and nematodes. Some earlier studies showed that about 16% of the arthropods were infected with Wolbachia, however, a recent meta-analysis estimated that more than 76% of insect species harbor Wolbachia, and some hosts harbor multiple strains of Wolbachia, which probably made it the most abundant intracellular bacteria genus so far discovered.This maternally inherited endosymbiont could manipulate a lot of reproductive processes in invertebrates, including sperm-egg incompatibility (cytoplasmic incompatibility), parthenogenesis, feminization and male killing; meanwhile, it is proposed that Wolbachia also play important roles in speciation.Mitochondria possess many ideal properties: maternally inherited; smaller genome size; higher evolutionary rate and lower recombination rate than nuclear genes, and so on. mtDNA has therefore remained the marker of choice in many populations, biogeographic and phylogenetic studies.mtDNA were regarded as neutrally evolutionary markers for a long time, however, when compared with other genomes, the whole mitochondrial genome evolves under great pressures, not only from the mitochondria itself (direct) but also from other maternally inherited factors (indirect) influences. Any maternally inherited factor could influence its host mitochondrial variations, and endosymbiont Wolbachia is a splendid case, so the host mtDNA no doubt reflects their long evolutionary history with intracellular bacteria. Hurst (2005) summarized earlier relevant studies and classified into four categories: (1) symbiont-driven reduction in mtDNA diversity, (2) symbiont-driven increases in diversity, (3) symbiont-driven changes in mtDNA variation over space and (4) symbiont-associated paraphyly of mtDNA.Based on some Wolbachia prevalence investigation, fig wasps has been proved almost to be the highest infected insects, up to 59%-67%. Fig wasps live in an airtight fig fruit most of their lifetime, so the infection status and the effects of Wolbachia on different hosts attracted our great interests. In this study, we made a survey of the infection status and unveiled the effects of Wolbachia on different hosts of four fig wasps species associated with Ficus hispida.The pollinating fig wasp Ceratosolen solmsi and one of the non-pollinating fig wasps, Philotrypesis pilosa, were infected with Wolbachia, and the other two non-pollinating fig wasps, Philotrypesis sp. and Apocrypta backeri, were uninfected. Moreover, the incidences of the two infected species were different, and C. solmsi was 89.3% while P. pilosa was 100%. Based on two Wolbachia specific gene, wsp and ftsZ, all C. solmsi individuals harbored one strain of Wolbachia, while P. pilosa individuals harbored different strains of Wolbachia. Two of the P. pilosa strains were comparatively stable and one strain shared with C. solmsi except one base variation, which suggesting that Wolbachia from the two species may have horizontal transmission besides vertical transmission, although the routes were unknown.All the C. solmsi individuals were divided into two groups, according to their infection statuses, based on mtDNA neighbor-joining trees. And the two mitochondrial markers (COI and Cytb) got similar topologies. The nucleotide divergence of COI and Cytb between infected and uninfected were up to 9.2% and 15.3% respectively, meanwhile, the differences within infected and uninfected individuals were both less than 1% for either mitochondrial gene. Compared with uninfected individuals, the infected ones showed reduction in mtDNA diversity.Based on the COI and Cytb clustering trees, P. pilosa and P. sp. differed a lot in their topologies. For COI, both of the sister species owned a lot of different haplotypes and they shared a lot in nucleotide diversity; for Cytb, P. pilosa still owned many haplotypes, while P. sp. had few haplotypes, and they differed a lot from each other in nucleotide diversity.Based on three tests of neutral evolution, only the infected individuals of C. solmsi evolved departing from neutrality, and all the Cytb genes and the infected individuals of C. solmsi evolved neutrally. And the results of nuclear markers were also very interesting. For ITS2, almost the same results were got from the either species, which confirmed that all our samples were selected from the same species and excluded some demographic factors influences based on the ITS2 data. As for EF1-α, the results of sister species P. pilosa and P. sp. were in chaos, and it was hard to differentiate the two species, probably because EF1-αis conserved and it is more suitable for classifying higher taxonomic level species; on the other hand, the infected and uninfected C. solmsi differed from each other by three fixed nucleotide of EF1-α, although they didn't cause any variation at the amino acid level, and so far it has not got a satisfying answer.It was postulated that vertical and horizontal transmissions of Wolbachia co-existed in F. hispida. Probably the horizontal transmission is one of the reasons why so many fig wasps were infected with Wolbachia. wsp showed evident recombination, which is one way of bacteria speciation. Dipping into Wolbachia genomes, there was a lot of recombinant proteins, which could explain why there are so many different stains of Wolbachia.In the process of investigating the effect of Wolbachia on host mtDNA, we co-amplified many Numt (nuclear mitochondrial genes) of COI from two Philotrypesis sister species, while none were found in other two fig wasps. It may suggest that COI of the two Philotrypesis species received different pressures from other genes. The appearance of the stop codon was the most characteristic of these Numt. Many individuals possessed more than two Numt sequences. It was concluded that these transfer events happened after the divergence of the two sister species, and independent transfer from mitochondria into the nuclear and duplications of the transferred fragments were both involved in appearance of so many Numt.Based on our research, both the endosymbiont Wolbachia and Numt could influence the employment of authentic mtDNA, which make the results based on the mitochondrial marker unreliable, especially in the phylogeny reconstruction and population structure of infected arthropods. It reminded us to be more careful when using the mitochondrial markers, in case of getting false conclusions.Such a small fig fruit has brought us with so many interesting results and puzzles, more and more work needs to be done.