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

• Main Authors: Cheng-Yuan Su, Dao-Hong Zhu, Xiao-Hui Yang
• Format: Article
• Language: English
• Published: MDPI AG 2021-08-01
• Series: Insects
• Subjects: Cynipidae; <i>Andricus hakonensis</i>; multiple infections; phage WO; <i>Wolbachia</i>
• Online Access: https://www.mdpi.com/2075-4450/12/8/713

Phage WO was first characterized in <i>Wolbachia</i>, 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 <i>Andricus hakonensis</i> and the development of highly effective primers. Based on the existing <i>Wolbachia</i> genome sequence, we designed primers (WO-TF and WO-TR) to amplify the full-length <i>orf7</i> gene of phage WO. Surprisingly, sequencing results showed a high abundance of other phage WO groups in <i>A. hakonensis</i>, in addition to the four groups previously identified. The results also showed that <i>A. hakonensis</i> contained most of the known types of <i>orf7</i> 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 <i>orf7</i> 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 <i>A. hakonensis</i> using the new primers. Importantly, all phage WO groups could be efficiently detected. Combined with the results of previous studies, our results suggest that <i>A. hakonensis</i> 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.