Design and Testing of Effective Primers for Amplification of the orf7 Gene of Phage WO Associated with Andricus hakonensis

Abstract

Phage WO was first characterized in Wolbachia, an obligate intracellular Rickettsiales known for its ability to regulate the reproduction of arthropod hosts. In this paper, we focus on the study of virus diversity in Andricus hakonensis and the development of highly effective primers. Based on the existing Wolbachia genome sequence, we designed primers (WO-TF and WO-TR) to amplify the full-length orf7 gene of phage WO. Surprisingly, sequencing results showed a high abundance of other phage WO groups in A. hakonensis, in addition to the four groups previously identified. The results also showed that A. hakonensis contained most of the known types of orf7 genes (I, III, IV, V and VI) and the level of diversity of harbored phage WO was very high. Therefore, we speculated that existing primers were not specific enough and that new primers for the detection of phage WO were needed. Based on the existing orf7 gene sequence, we designed specific detection primers (WO-SUF and WO-SUR). Sequencing results showed that the primers effectively amplified all known types of phage WO. In addition to amplifying most of the known sequences, we also detected some new genotypes in A. hakonensis using the new primers. Importantly, all phage WO groups could be efficiently detected. Combined with the results of previous studies, our results suggest that A. hakonensis contains the largest number of phage types (up to 36 types). This study is novel in that it provides practical molecular evidence supporting base deletions, in addition to gene mutations and genetic recombination, as an important cause of phage WO diversity.

High Levels of Multiple Phage WO Infections and Its Evolutionary Dynamics Associated With Wolbachia-Infected Butterflies

“…The Wolbachia strain w Ri, infecting Drosophila simulans , showed a single WO haplotype according to the initial PCR detection using phage WO-specific gene markers, but the genome sequence confirmed four protophage copies in the genome, three of which were unique ( Gavotte et al, 2007 ; Klasson et al, 2009 ). In fact, primers used to screen for the presence or absence of the orf7 gene were not degraded enough to detect all orf7 variants ( Kent and Bordenstein, 2010 ; Su et al, 2021 ). Su et al (2021) designed specific detection primers, and may have significantly improved the detection efficiency of orf7 variants.…”

“…In fact, primers used to screen for the presence or absence of the orf7 gene were not degraded enough to detect all orf7 variants ( Kent and Bordenstein, 2010 ; Su et al, 2021 ). Su et al (2021) designed specific detection primers, and may have significantly improved the detection efficiency of orf7 variants. Thus, the diversity of phage WO in Wolbachia -infected arthropods might be greater than current estimates.…”

“… Zhu et al (2021) provided empirical molecular evidence of orf7 gene recombination, suggesting that intragenic recombination was the important evolutionary force, which effectively promoted the diversity of phage WO associated with gall wasps which harbor multiple phage WO types. Furthermore, base deletions during replication also significantly promoted the evolution of the phage genome, resulting in the diversity of phage WO associated with A. hakonensis ( Su et al, 2021 ).…”