Dryocosmus kuriphilus (Hymenoptera: Cynipidae) is a gall wasp that induces insect galls on chestnut trees and results in massive yield losses worldwide. Fungi can cause the necrosis of chestnut trees and the death of gall wasps. The aim of this research was to investigate the potential role of D. kuriphilus in the transmission of fungi. We sequenced the ribosomal RNA internal transcribed spacer region 1 of fungi in D. kuriphilus adults, associated insect galls and the galled twigs of Castanea mollissima, using high-throughput sequencing. We compared the species richness, α-diversity and community structure of fungi in D. kuriphilus adults, insect galls and the galled twigs. We provide the first evidence that D. kuriphilus adults shared most fungal species with associated insect galls and the galled twigs, and were dominated by Botryosphaeria sp., Aspergillus sp. and Diaporthe sp. We suggest D. kuriphilus adults may be potential vectors of plant pathogens and may facilitate the transmission of fungi between chestnut trees. Furthermore, the fungi may horizontally transmit among D. kuriphilus adults, associated insect galls and the galled twigs.

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.

Dryocosmus kuriphilus (Hymenoptera: Cynipidae) induces galls on chestnut trees, which results in massive yield losses worldwide. Torymus sinensis (Hymenoptera: Torymidae) is a host-specific parasitoid that phenologically synchronizes with D. kuriphilus. Bacteria play important roles in the life cycle of galling insects. The aim of this research is to investigate the bacterial communities and predominant bacteria of D. kuriphilus, T. sinensis, D. kuriphilus galls and the galled twigs of Castanea mollissima. We sequenced the V5–V7 region of the bacterial 16S ribosomal RNA in D. kuriphilus, T. sinensis, D. kuriphilus galls and galled twigs using high-throughput sequencing for the first time. We provide the first evidence that D. kuriphilus shares most bacterial species with T. sinensis, D. kuriphilus galls and galled twigs. The predominant bacteria of D. kuriphilus are Serratia sp. and Pseudomonas sp. Furthermore, the bacterial community structures of D. kuriphilus and T. sinensis clearly differ from those of the other groups. Many species of the Serratia and Pseudomonas genera are plant pathogenic bacteria, and we suggest that D. kuriphilus may be a potential vector of plant pathogens. Furthermore, a total of 111 bacteria are common to D. kuriphilus adults, T. sinensis, D. kuriphilus galls and galled twigs, and we suggest that the bacteria may transmit horizontally among D. kuriphilus, T. sinensis, D. kuriphilus galls and galled twigs on the basis of their ecological associations.

Insect galls are the abnormal growth of plant tissues induced by a wide variety of galling insects and characterized by high concentrations of auxins and cytokinins. It remains unclear whether the auxins and cytokinins affect the bacterial community structure of insect galls. We determined the concentrations of indoleacetic acid (IAA) as an example of auxin, trans-zeatin riboside (tZR) and isopentenyladenine (iP) as cytokinins in Lithosaphonecrus arcoverticus (Hymenoptera: Cynipidae) galls and the galled twigs of Lithocarpus glaber (Fagaceae) using liquid chromatography–tandem mass spectrometry. Moreover, for the first time, we compared the bacterial community structure of L. arcoverticus galls and galled twigs by high-throughput sequencing, and calculated the Spearman correlation and associated degree of significance between the IAA, tZR and iP concentrations and the bacterial community structure. Our results indicated the concentrations of IAA, tZR and iP were higher in L. arcoverticus galls than in galled twigs, and positively correlated with the bacterial community structure of L. arcoverticus galls. We suggest the high concentrations of IAA, tZR and iP may affect the bacterial community structure of L. arcoverticus galls.

This study aimed to characterize the sources and spatio-temporal distribution patterns of cytokinins in Dryocosmus kuriphilus and its induced galls, providing observational data on possible gall-inducing mechanisms. Specifically, the contents of six cytokinins (TZR, DZR, cis-Zeatin, trans-Zeatin, IP, and IPR) in the epidermal layer (IPR: 3.92 ± 0.15 ng/g), outer protective layer, inner protective layer, and nutritive layer of galls, as well as in D. kuriphilus at different developmental stages, were quantified by UPLC-MS/MS. The results showed that IPR (63.12 ± 2.55 ng/g) was relatively high in larvae. This observational finding is consistent with the hypothesis that cytokinins in D. kuriphilus may originate from the insect itself or from plant sources. Elevated cytokinin levels were found in plant galls compared to normal tissues.