Molecular Detection Of Wolbachia And Its Effect On Reproduction In Hylyphantes Graminicola

Hylyphantes graminicola is one of the most prevalent spiders in the cotton field. In this study, the effect of Wolbachia on host’s reproduction and fittness were analysed, and the routine for Wolbachia horizontal transmission were also conducted. We firstly detected that CI(cytoplasmis incompatibility) was not happened in H. graminicola, and Wolbachia can be transmitted by mating of H. graminicola in this study. All these results will provide useful theoretical and empirical foundation for later studys. The followings are the main results of this study.1 Wolbachia infection and genetic divergence analyses of Wolbachia wsp gene in different Hylyphantes graminicola populationsThe result of Wolbachia detecting in 14 geogrophical populations (XX, HZ, WZS, LY, GL, LS, BN, WM, SJZ, TG, NJ, FD, XS, WH) of Hylyphantes graminicola indicated that Wolbachia infection existed in all 14 populations of H. graminicola. The infection frequency of XX (Henan Province) popuation was the highest (52.94%), then the HZ (Shandong Province) population (45.45%). The lower infection frequency of Wolbachia existed in FD population (8.62%), XS population (8.1%) and WH population (7.69%).The result for DNA polymorphic sites analyses of Wolbachia wsp gene (69 sequences from 7 populations) by DnaSP4.10 indicated that total number of sites was 560. Invariable (monomorphic) sites was 380, and variable (polymorphic) sites was 185. The result of genetic distance analyses showed that genetic distances within a same population of H, graminicola were not always near or equal to 0, and genetic distances of Wolbachia wsp gene between different populations will be equal to 0 sometimes. The result of wsp gene haplotype showed that the haplotype diversity of WZS population was the highest (0.90000). The mean number of pairwise differences of WZS population was the highest (83.70000), and the the nucleotide diversity of WZS population was the highest (0.45243). Through AMOVA analyses,84.96% genetic variation happened within population, and the genetic differentiation within popultion was so significant (Fst=0.15042, P<0.01).2 Wolbachia MLST typing of Hylyphantes graminicola A total of 10 STs, new to the MLST database, were found in 11 H. graminicola geographic populations, and the STs from LY and HZ are identical. The 10 STs were assigned to supergroup A and supergroup B respectively based on the phylogenies of concatenated MLST data. Based on the ML, MP and BI analyses, eight STs (ST-177, ST-178, ST-179, ST-180, ST-181, ST-182, ST-183 and ST-184) were assigned to supergroup B, and two STs (ST185 and ST 186) to supergroup A.The result of Mantel test indicated the correlation was significant between pairwise distance of H. graminicola COI and associated Wolbachia MLST concatenated data (r2=0.394,p=0.013,p<0.05). We also checked the correlation between Wolbachia MLST concatenated data and host’s geographic distance, and the result showed that no correlation was found between Wolbachia divergence and host’s geographic distance (r2=0.027,p=0.665, p>0.05). No correlation was found between pairwise distance of H. graminicola COI and its geographic distance (r2=0.027,p=0.135, p>0.05).3 The effect of Wolbachia on the reproductive and fittness of H. graminicolaThe result shows that 4 types of mating pairs in H. graminicola((?)+×(?)-、(?)-×(?)+(?)+×(?)+�'�(?)-×(?)-) all have normal mating behavior and can produce valid egg sacs, and CI was not found within mating pair(?)-×(?)+. Through analysing the number of egg sacs, developmental duration of eggs and decendents of each egg sacs between Wolbachia positive H. graminicola and Wolbachia negative H. graminicola, the significance was found in developmental duration of the first eggs sacs(P<0.01).The result of Wolbachia removing from its host showed that 40% Wolbachia positive H. graminicola lost Wolbachia through feeding on l0mg/ml antibiotics solution for 50 days, and 50% Wolbachia positive H. graminicola also lost Wolbachia through feeding on 3.75mg/ml antibiotics artificial feed (the density of antibiotics in artificial feed) for 50 days.4 Wolbachia horizontal transmission Through Wolbachia negative H. graminicola predating Wolbachia positive Drosophila melangastor, the result of Wolbachia detecting showed that no Wolbachia infection was detected in H. graminicola (who have predated Wolbachia positive Drosophila melangastor).Two mating groups between Wolbachia positive individuals ((?)+or (?)+) and negative individuals (()?-or(?)-) were assigned in this study, and the molecular evidence was detected by sequencing wsp (Wolbachia surface protein) gene fragments. The results shows that through mating, positive spiders ((?)+or(?)+) can transmit Wolbachia to negative spiders ((?) or(?)-), and this is the first report about Wolbachia horizontal transfer through mating behavior in intraspecific level.