The Diversity and Dynamics of Fungi in Dryocosmus kuriphilus Community

Simple Summary Dryocosmus kuriphilus is an invasive pest species which is native to China and is widely distributed in Asia, Europe and North America. D. kuriphilus induces insect galls on chestnut trees, and fungi can cause the necrosis of chestnut trees and the death of D. kuriphilus. The aim of this research was to investigate the potential role of D. kuriphilus in the transmission of fungi. We provide the first evidence that D. kuriphilus adults shared most fungal species with associated insect galls and the galled twigs of Castanea mollissima, and were dominated by Botryosphaeria sp., Aspergillus sp. and Diaporthe sp. Furthermore, we suggest that D. kuriphilus adults may be potential vectors of plant pathogens and mediate the transmission of fungi between chestnut trees. Abstract 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.


Introduction
Galling insects are highly specialized herbivores with the ability to induce the formation of insect galls on host plants [1,2]. Insect galls are the abnormal redifferentiation and growth of infested plant tissues, providing shelter and food for the galling insects [3,4]. The major groups of gall insects include gall wasps, gall midges, gall aphids, gall moths, psyllids and thrips [5].
Dryocosmus kuriphilus (Hymenoptera: Cynipidae) is a species of gall wasp that can result in massive reductions in the yields of different chestnut trees, including Castanea henryi, Castanea mollissima and Castanea sativa [6,7]. D. kuriphilus is one of the most successful invasive pests worldwide which is native to China and is widely distributed in Asia, Europe

Library Construction and High-Throughput Sequencing
The PCR product was extracted from 2% agarose gel, following electrophoresis, and purified by using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). The PCR product was quantified by using a Quantus™ Fluorometer (Promega, Madison, Wisconsin, WI, USA). The libraries were prepared by using NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, TX, USA), and high-throughput paired-end sequencing was performed on the Illumina MiSeq (PE300) sequencing platform (Illumina, San Diego, CA, USA). Library preparation and sequencing were carried out by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw data were deposited into the NCBI Sequence Read Archive (SRA) database under Accession Number PRJNA725226.

Bioinformatics Analysis
The raw ITS1 gene sequencing reads were quality-filtered by fastp software [31] and merged, using FLASH software [32]. The sequences fulfilling the following criteria were used for the subsequent analysis: sequence length >200 bp, no ambiguous bases and mean quality score ≥20. After quality filtering, high-quality reads were clustered into Operational Taxonomic Units (OTU) at a similarity cutoff value of 97%, using UPARSE, and were screened for chimeras, using USEARCH version 7.1 [33]. The chimeric sequences were identified and then removed. The taxonomy of each OTU representative sequence was analyzed and annotated from the phylum to species level by the Ribosomal Database Project (RDP) classifier version 2.4 [34] and the UNITE database for molecular identification of fungi, using a confidence threshold of 0.7. For each sample, 54,497 sequences were randomly selected to generate an OTU table. The 54,497 represents the sequence count of the sample with the smallest acceptable number of sequences. The OTU table, which recorded the abundance and taxonomy of each OTU, was used for the subsequent statistical analysis.

Statistical Analysis
Statistical analysis was performed by using R version 3.6.3 (https://www.r-project.org, 26 February 2020). We counted the number of unique and common fungi in D. kuriphilus adults, associated insect galls and the galled twigs of C. mollissima at the species level.
The Sobs index and the Shannon index measures were used to evaluate the observed species richness and α-diversity, respectively, of the fungal community of D. kuriphilus adults, associated insect galls and the galled twigs at the species level. Sobs index refers to the total number of fungal species observed in D. kuriphilus adults, associated insect galls and the galled twigs. Data relating to Sobs and Shannon index were tested for normal distribution (Shapiro-Wilk test) and homogeneity of variance (Bartlett's test). The data of the Sobs index were approximately normally distributed, and the variance was homogeneous across groups. Thus, one-way analysis of variance (ANOVA) was used to evaluate whether there were overall significant differences among the Sobs index measures of different groups; if significant, the Tukey-Kramer test was then used to carry out multiple pairwise comparisons of the groups. The variance of the Shannon index was not homogeneous across groups, so the Kruskal-Wallis nonparametric test was used to evaluate whether there were overall significant differences among the Shannon index of different groups, with the Dunn test being used for multiple comparisons if the Kruskal-Wallis test was significant.
Principal coordinate analyses (PCoA) were performed to compare the fungal community structure of D. kuriphilus, associated insect galls and the galled twigs. First, the overall difference in community structure was assessed, using permutational multivariate analysis of variance (PERMANOVA). PERMANOVA was carried out by using the "adonis" function in the "vegan" package in R based on the weighted UniFrac distance with 1000 permutations [35]. Second, PCoA was carried out based on weighted UniFrac distance, using the "pcoa" function in the R package "ape" [36].
The linear discriminant analysis (LDA) Effect Size (LEfSe) (http://huttenhower.sph. harvard.edu/galaxy/, 17 March 2021) was used to reveal predominant fungi in D. kuriphilus adults, associated insect galls and the galled twigs. The Kruskal-Wallis test was used to detect those fungal taxa where the relative abundance was significantly different among D. kuriphilus adults, associated insect galls and the galled twigs from the phylum to the species level. Then, the linear discriminant analysis (LDA) was used to calculate the effect size of each taxon; the higher the LDA score, the greater the influence of taxa on the difference. For fungi with an LDA score greater than 4, the relative abundance of the fungi was showed in bubble chart and the Dunn test was used for multiple pairwise comparisons. The predominant fungi refer to the fungi with an LDA score greater than 4 and the highest abundances among D. kuriphilus adults, associated insect galls and the galled twigs.

The Fungal Community Composition of D. kuriphilus Adults, Associated Insect Galls and the Galled Twigs of C. mollissima
There was a total of four phyla, 22 classes, 56 orders, 116 families, 117 genera, 248 species and 385 OTUs in the fungal community of D. kuriphilus adults, associated insect galls and the galled twigs (Table 1). Insect galls had the most fungi, followed by the galled twigs, with D. kuriphilus having the fewest fungi from the phylum to species level ( Table 1). The fungal communities of D. kuriphilus adults, associated insect galls and the galled twigs had 176, 241 and 221 species, respectively (Table 1). At the phylum and class level, the taxa with the highest abundances were identical in the fungal communities of D. kuriphilus adults, associated insect galls and the galled twigs (Table 2). At the order, family, genus, species and OTU levels, the taxa with the highest abundances in D. kuriphilus adults were not the same as that in the insect galls and the galled twigs, whereas the taxa with the highest abundances were identical in the fungal community of the insect galls and the galled twigs ( Table 2). The fungi with the highest abundances of D. kuriphilus adults were Botryosphaeria sp. (Table 2).  A The numbers inside the parentheses represent the relative abundance expressed as the percentage of this taxon abundance in the fungal community of D. kuriphilus adults, associated insect galls or the galled twigs.

The Unique and Common Fungi of D. kuriphilus Adults, Associated Insect Galls and the Galled Twigs of C. mollissima
A total of 154 fungi were common to D. kuriphilus adults, associated insect galls and the galled twigs ( Figure 1). The relative abundance of the fungi common to D. kuriphilus adults, associated insect galls and the galled twigs was 99.36%, 98.03% and 98.71%, respectively ( Table 3). The numbers of unique fungi in D. kuriphilus adults, associated insect galls and the galled twigs were eight, two and two, respectively ( Figure 1). The relative abundance of unique fungi in D. kuriphilus adults, associated insect galls and the galled twigs was 0.14%, 0.03% and 0.04%, respectively (Table 3).

The Richness and α-Diversity at the Species Level of the Fungal Communities of D. kuriphilus Adults, Associated Insect Galls and the Galled Twigs of C. mollissima
The observed species richness (ANOVA, F2,24 = 23.36, p < 0.01) and α-diversity (Kruskal-Wallis test, H2,24 = 12.98, p < 0.01) measures at the species level differed significantly among the fungal communities of D. kuriphilus adults, associated insect galls and the galled twigs ( Figure 2). The observed species richness (Tukey-Kramer's test, p < 0.01) and α-diversity measures (Dunn test, p < 0.01) of the fungal community of D. kuriphilus adults were significantly lower than those of associated insect galls and the galled twigs at the species level (Figure 2), whereas the species richness (Tukey-Kramer's test, p = 0.27) and α-diversity (Dunn test, p = 0.115) of the fungal communities were not significantly different between associated insect galls and the galled twigs at the species level ( Figure 2). Furthermore, there was an overall significant difference among the fungal community structures of D. kuriphilus adults, associated insect galls and the galled twigs (PER-MANOVA, R 2 = 0.53, p < 0.01) (Figure 2). PCoA analysis indicated that the fungal commu-

The Richness and α-Diversity at the Species Level of the Fungal Communities of D. kuriphilus Adults, Associated Insect Galls and the Galled Twigs of C. mollissima
The observed species richness (ANOVA, F 2,24 = 23.36, p < 0.01) and α-diversity (Kruskal-Wallis test, H 2,24 = 12.98, p < 0.01) measures at the species level differed significantly among the fungal communities of D. kuriphilus adults, associated insect galls and the galled twigs ( Figure 2). The observed species richness (Tukey-Kramer's test, p < 0.01) and α-diversity measures (Dunn test, p < 0.01) of the fungal community of D. kuriphilus adults were significantly lower than those of associated insect galls and the galled twigs at the species level (Figure 2), whereas the species richness (Tukey-Kramer's test, p = 0.27) and α-diversity (Dunn test, p = 0.115) of the fungal communities were not significantly different between associated insect galls and the galled twigs at the species level ( Figure 2). Furthermore, there was an overall significant difference among the fungal community structures of D. kuriphilus adults, associated insect galls and the galled twigs (PERMANOVA, R 2 = 0.53, p < 0.01) (Figure 2). PCoA analysis indicated that the fungal community structure in D. kuriphilus adults was clearly different from that of associated insect galls and the galled twigs ( Figure 2).

The Predominant Fungal Species of D. kuriphilus Adults, Associated Insect Galls and the Galled Twigs of C. mollissima
The LEfSe analysis showed that a total of two phyla, four classes, ten orders, 14 families, 12 genera and 12 species were predominant in the fungal communities of D. kuriphilus adults, associated insect galls and the galled twigs (Figure 3). The fungal community of D. kuriphilus adults was dominated by one phylum, three orders, three families, three genera and three species. The fungal community of associated insect galls was dominated by one phylum, four classes, four orders, seven families, six genera and six species,

The Predominant Fungal Species of D. kuriphilus Adults, Associated Insect Galls and the Galled Twigs of C. mollissima
The LEfSe analysis showed that a total of two phyla, four classes, ten orders, 14 families, 12 genera and 12 species were predominant in the fungal communities of D. kuriphilus adults, associated insect galls and the galled twigs ( Figure 3). The fungal community of D. kuriphilus adults was dominated by one phylum, three orders, three families, three genera and three species. The fungal community of associated insect galls was dominated by one phylum, four classes, four orders, seven families, six genera and six species, whereas the fungal community of the galled twigs was dominated by three orders, four families, three genera and three species (Figure 3).  Notably, it was shown for the first time that Botryosphaeria sp., Aspergillus sp. and Diaporthe sp. were predominant in the fungal community of D. kuriphilus adults (Figure 3, Table 4). The relative abundances of Botryosphaeria sp., Aspergillus sp. and Diaporthe sp. in D. kuriphilus adults were 44.27%, 10.07% and 8.91%, respectively (Figure 4 and Figure S1). Furthermore, the insect galls were dominated by six fungi, namely Ascomycota species, Acremonium sp., Bullera alba, Cercospora sp., Cryptococcus aureus and Curvibasidium cygneicollum ( Figure 3, Table 4), whereas the galled twigs were dominated by three fungi, namely Didymella rosea, Cladosporium delicatulum and Capnodiales species (Figure 3 and Table 4).

The Possibility of Fungal Horizontal Transmission among D. kuriphilus Adults, Associated Insect Galls and the Galled Twigs of C. mollissima
To our knowledge, this study provided the first evidence that D. kuriphilus adults, their associated insect galls and the galled twigs share most of the species in the fungal community. Previous studies have shown that the insect galls of D. kuriphilus and host plants shared C. aureus [24], C. cygneicollum [37], Cercospora spp. [38], Cladosporium spp. [21] and D. rosea [25,39]. Furthermore, D. kuriphilus and associated insect galls shared C. parasitica [26], Fusarium spp. [29] and G. castaneae [27]. Therefore, the sharing of fungi among D. kuriphilus adults, associated insect galls and the galled twigs may be common.
We speculated that the fungi might horizontally transmit among D. kuriphilus adults, associated insect galls and the galled twigs. We suggest that structural (vascular) connections, transport of substances, contact and feeding relationships play an essential role in the potential horizontal transmission among D. kuriphilus adults, associated insect galls and the galled twigs.
The insect galls of D. kuriphilus are structurally connected with the galled twigs [40]. This structural connection provides a physical route for the horizontal transmission of fungi between D. kuriphilus-induced insect galls and the galled twigs. For example, endophytic fungi can grow into insect galls from the neighboring leaf in the form of mycelia or by directly penetration of the gall via spores [16]. Moreover, the supply of water and most nutrients to insect galls are obtained from the host plants via xylem vessels and phloem sieve tubes, respectively [41,42]. The spores of Ceratocystis fagacearum could spread from the primary infection site to other parts of the host plant through the xylem vessels and the phloem sieve tubes [43,44]. Thus, the transport of water and nutrients may provide favorable conditions for the horizontal transmission of fungi between D. kuriphilus-induced galls and the galled twigs. Furthermore, D. kuriphilus lives in the gall chambers of insect galls, making constant contact with the insect galls before eclosion [45]. During this contact process, the fungi associated with the insect galls may adhere to the exoskeleton surface of D. kuriphilus adults or be collected and transported within the body of D. kuriphilus. The fungi associated with insect galls may enter the digestive system of D. kuriphilus when the latter feeds on the insect galls. Therefore, such contact and feeding relationships are conducive to the horizontal transmission of fungi between D. kuriphilus adults and the insect galls.

The Differences in Fungal Community Structure among D. kuriphilus Adults, Associated Insect Galls and the Galled Twigs of C. mollissima
The differences in fungal community structure between the insect galls and the galled twigs may be associated with the differences in chemical composition and content between the insect galls and the galled twigs. Previous studies have confirmed differences in chemical composition and concentration between the host plant and both aphid galls [46] and midge galls [47]. The fungal community structures of aphid galls [48] and midge galls [49] also differed from those associated with the corresponding host plants. For the insect galls of D. kuriphilus and other gall wasps, the chemical composition, as well as the concentrations of amino acids, carbohydrates, lipids, lignin and secondary metabolites were markedly different from those of the host plants [42,[50][51][52][53][54]. We propose that the chemical components and concentrations of insect galls induced by D. kuriphilus may affect the fungal community structure and provide a particular habitat for the fungi associated with the insect galls. For example, Cornell has shown that the high tannin concentration in the insect galls of galls wasps prevents the colonization of some fungi [55].
Furthermore, the ability of fungi to utilize particular plant chemicals may also associate with the differences in fungal community structure between the insect galls and the galled twigs. The lignification degree of cynipid galls is higher than that of the host plants [56]. Lignin is a complex, polyphenolic macromolecule, which is refractory to degradation and assimilation [57]. However, some fungi, such as the white-rot fungi, can break down and use lignin by producing diverse extracellular oxidases, including phenol oxidases, lignin peroxidase and manganese peroxidase [58]. These fungi, which can utilize the substances making up the insect galls of D. kuriphilus, may be better adapted to the environment of insect galls.
The fungal community structure of D. kuriphilus adults was obviously different from that of insect galls. We suggest that the inter-kingdom barriers between D. kuriphilus and insect galls may prevent the colonization of some fungi and hence contribute to fungal community structure differences between D. kuriphilus adults and associated insect galls. Fungal colonization at the cross-kingdom level is not as well-known as that within the animal or plant kingdoms. The fungi must come into close and frequent contact with potential hosts and overcome the host defense of another kingdom [59,60].

D. kuriphilus Adults as Potential Vectors of PLANT Pathogens
Many species of the Botryosphaeria, Aspergillus and Diaporthe genera are plant pathogens [61][62][63]. Previous studies had shown that C. mollissima is attacked by a range of fungi, including plant pathogens such as Botryosphaeria dothidea [63], Aspergillus sp. [64,65] and Diaporthe nobilis [66]. Furthermore, many fungi have been isolated from the insect galls induced by D. kuriphilus, including B. dothidea [24,67,68], D. nobilis [24] and Aspergillus spp. [28]. We noticed that plant pathogens B. dothidea was isolated from five phytophagous insects and the dispersal and propagule pressure of Botryosphaeria spp. in oak trees were affected by insect vectors [69]. Thus, the predominant fungi in D. kuriphilus adults, such as Botryosphaeria sp., Aspergillus sp. and Diaporthe sp., may be plant pathogens.
The ovipositor of D. kuriphilus is a needle-like apparatus used to introduce wasp eggs into buds of the host plants and may result in fresh wounds in buds [30,49]. The injuries provide entry points for fungi and a potential approach for the fungal transmission between D. kuriphilus and the host tree, C. mollissima. For example, spores of the parasitic C. parasitica infected host plants through fresh wounds [27,67]. Panzavolta et al. indicated that the galling insects associated with the transport of plant pathogens to oak trees [70]. Here, we suggest that D. kuriphilus adults may be potential vectors of plant pathogens and can mediate the transmission of fungi between chestnut trees, and the pathogen pervasiveness of chestnut trees may be enhanced by their association with D. kuriphilus.

The Predominant Fungi in D. kuriphilus
The fungi associated with galling insects can be saprotrophs, symbionts and insect pathogens [71]. The available literature suggests that the death of gall wasps was associated with several fungi, including Cladosporium sp. [72], D. quercina [16], G. castaneae [28], Gnomoniopsis smithogilvyi, Fusarium oxysporum and Fusarium avenaceum [73]. Previous studies have confirmed that some species of the Aspergillus genus contribute to the death of members of the insect orders Hymenoptera [74], Lepidoptera [75], Coleoptera [76] and Diptera [77]. Furthermore, some species of the Diaporthe genus are pathogens of dipteran [78] and lepidopteran species [20,79] and can result in the death of insects [61]. However, there is no firm evidence indicating that Botryosphaeria sp., Aspergillus sp. or Diaporthe sp. can result in the death of D. kuriphilus adults.
In future studies, we will focus on the isolation and cultivation of predominant fungi in D. kuriphilus adults and plan to evaluate the role of these fungi.

Conclusions
In conclusion, this study indicated that D. kuriphilus adults, associated insect galls and the galled twigs of C. mollissima shared most of the species in the fungal community for the first time. This study also provided the first evidence that Botryosphaeria sp., Aspergillus sp. and Diaporthe sp. were predominant in the fungal community of D. kuriphilus.
We suggest that structural (vascular) connections, the transport of substances, contact, feeding and oviposition relationships play an important role in the potential horizontal transmission of fungal species among D. kuriphilus adults, the associated insect galls and the galled twigs. Furthermore, differences in fungal community structure among D. kuriphilus adults, the insect galls and the galled twigs may be associated with differences in the chemical composition and concentrations between insect galls and galled twigs, differences in the ability of fungi to use key chemicals and cross-kingdom barriers between D. kuriphilus and the plant tissue forming the insect galls. In addition, Botryosphaeria sp., Aspergillus sp. and Diaporthe sp. may be plant pathogens. We suggest that D. kuriphilus adults may be potential vectors of plant pathogens.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/insects12050426/s1. Figure S1: The relative abundance of fungi predominant in the Dryocosmus kuriphilus adults, associated insect galls and the twigs of Castanea mollissima at the species level.