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

SIMPLE SUMMARY: Andricus hakonensis is thought to contain the most complex and diverse phage types known and should be an ideal model material for studying interactions among bacteriophages, bacteria and eukaryotes. As shown in previous studies, existing primers are not effective enough to amplify all virus groups in A. hakonensis. Based on a comprehensive analysis of all virus groups reported to date, we designed a relatively conservative primer for virus detection. This primer can accurately and efficiently detect the presence of phage WO in arthropod hosts. Using gene alignment, clear evidence was provided for the existence of hitherto unreported base deletions, which are an important cause of diversity in phage WO associated with A. 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.