Next Article in Journal
Biological Features and Population Growth of Two Southeastern European Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) Strains
Previous Article in Journal
Ingestion of Novaluron Elicits Transovarial Activity in Stephanitis pyrioides (Hemiptera: Tingidae)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Insects
Volume 11
Issue 4
10.3390/insects11040217
insects-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Host Plants Influence the Symbiont Diversity of Eriosomatinae (Hemiptera: Aphididae)

by
Ting-Ting Xu
1,2,
Li-Yun Jiang
1,
Jing Chen
1,* and
Ge-Xia Qiao
1,2,*
1
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
2
College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
*
Authors to whom correspondence should be addressed.
Insects 2020, 11(4), 217; https://doi.org/10.3390/insects11040217
Submission received: 7 February 2020 / Revised: 16 March 2020 / Accepted: 28 March 2020 / Published: 1 April 2020
Browse Figures
Versions Notes

Abstract

:
Eriosomatinae is a particular aphid group with typically heteroecious holocyclic life cycle, exhibiting strong primary host plant specialization and inducing galls on primary host plants. Aphids are frequently associated with bacterial symbionts, which can play fundamental roles in the ecology and evolution of their host aphids. However, the bacterial communities in Eriosomatinae are poorly known. In the present study, using high-throughput sequencing of the bacterial 16S ribosomal RNA gene, we surveyed the bacterial flora of eriosomatines and explored the associations between symbiont diversity and aphid relatedness, aphid host plant and geographical distribution. The microbiota of Eriosomatinae is dominated by the heritable primary endosymbiont Buchnera and several facultative symbionts. The primary endosymbiont Buchnera is expectedly the most abundant symbiont across all species. Six facultative symbionts were identified. Regiella was the most commonly identified facultative symbiont, and multiple infections of facultative symbionts were detected in the majority of the samples. Ordination analyses and statistical tests show that the symbiont community of aphids feeding on plants from the family Ulmaceae were distinguishable from aphids feeding on other host plants. Species in Eriosomatinae feeding on different plants are likely to carry different symbiont compositions. The symbiont distributions seem to be not related to taxonomic distance and geographical distance. Our findings suggest that host plants can affect symbiont maintenance, and will improve our understanding of the interactions between aphids, their symbionts and ecological conditions.

1. Introduction

Aphids have established sophisticated symbiotic associations with bacteria that contribute to their survival and environmental suitability. Buchnera aphidicola, which is the primary endosymbiont of aphids that occupies the specialized bacteriocytes, supplies essential nutrients to its host [1,2,3,4]. B. aphidicola is strictly maternally transmitted, and exhibits a pattern of codiversification with aphid hosts during long-term evolution [1,5,6,7,8,9,10,11,12,13].
Aphids also host various facultative symbionts, which are usually distributed randomly in aphids and undergo vertical and some horizontal transmission [14,15,16,17,18,19]. Facultative symbionts are generally not required for aphid development or reproduction, but infection with facultative symbionts can have context-dependent phenotypic effects on aphid hosts that influence major ecological processes, including defence against parasitic wasps [20,21,22,23,24,25] and fungal pathogens [26,27,28], protection against heat shock [29,30], interactions with host plants [31,32,33] and modification of body colour [34,35]. In addition, except for the primary endosymbiont B. aphidicola, some Lachninae species have established co-obligate endosymbiotic associations with Erwinia haradaeae, Fukatsuia symbiotica, Hamiltonella defensa, Serratia symbiotica and Sodalis sp. [36,37,38,39,40,41,42]. Wolbachia seems to be a co-obligate symbiont in Pentalonia nigronervosa Coquerel (Aphidinae: Macrosiphini) [43] (but see Manzano-Marín [44]).
The distributions of microbial symbionts differ across aphid species or populations, which may be influenced by both internal and external factors. The strong correlations between symbiont communities and host plants were revealed in Acyrthosiphon pisum (Harris) [45,46,47], Aphis craccivora Koch [48,49], Aphis gossypii Glover [50] and Phylloxera notabilis Pergande [51]. Studies have also found that aphid geographical distribution plays an important role in shaping symbiont microbiotas [52,53,54,55,56,57]. Furthermore, some other factors are suggested to be correlated with the symbiont distribution associated with aphids, such as host aphid species, plant diversity, parasitism rate and temperature [56,58,59,60]. Henry et al. [61] investigated the bacterial communities of 133 aphid species and highlighted the important roles of ecological conditions in structuring the symbiont distributions, whereas aphid phylogeny seemed to have no effect. McLean et al. [62] surveyed the microbiota of 46 aphid species and found that the microbiota composition was influenced by aphid relatedness rather than aphid ecology.
The associations between aphids and their bacterial symbionts are very complex, and a number of studies have been carried out to understand these associations, but little research has been performed in a specific aphid group to better illustrate the interactions with symbionts. Eriosomatinae is an extraordinary aphid group exhibiting very diverse life history traits and is widely distributed in the Holarctic and Oriental regions [63]. Most eriosomatine species show a heteroecious holocyclic life history, i.e., seasonal switching between primary and secondary host plants [64,65,66]. Eriosomatinae aphids have strong primary host specificity, with different patterns of host association among tribes. The three tribes, Eriosomatini, Fordini and Pemphigini (except for Prociphilus), are primarily associated with Ulmus and Zelkova (Ulmaceae), Rhus and Pistacia (Anacardiaceae) and Populus (Salicaceae), respectively. The secondary host plants, i.e., Cypeaceae, Graminaceae, Hypnaceae, Magnoliaceae, Pinaceae are more diverse. Eriosomatinae is also typically known for inducing galls on its primary host plants, secreting a visible wax coating and producing specialized sterile soldiers [67,68,69,70,71].
Although almost all viviparous aphid species harbour B. aphidicola as their primary endosymbiont, Geopemphigus Hille Ris Lambers (Eriosomatinae: Fordini) species have lost B. aphidicola and replaced it with the Skilesia alterna symbiont (phylum Bacteroidetes) [72]. Previous studies have shown that some Eriosomatinae species harbour four facultative symbionts, Hamiltonella, Regiella, Serratia and Wolbachia [16,61,73,74,75]. However, the detailed bacterial flora of this subfamily is still unclear.
In this study, using 16S rRNA Illumina sequencing data, we aimed to define the diversity of symbiont communities across members of subfamily Eriosomatinae by an in-depth survey and determine the interaction patterns and evolutionary forces that shape their composition and complexity.

2. Materials and Methods

2.1. Sampling and DNA Extraction

Thirty-four Eriosomatinae species belonging to eleven genera were sampled in this study, including 11 species within 3 genera in Eriosomatini, 3 species within 3 genera in Fordini, 20 species within 5 genera in Pemphigini (Table S1). Twenty-nine of these species are distributed in 11 provinces in China, 2 species in Ulan Bator (Mongolia) and 3 species in Illinois (USA); 3 species feed on Anacardiaceae, 1 species on Magnoliaceae, 2 species on Oleaceae, 3 species on Rosaceae, 15 on Salicaceae (Populus) and 9 on Ulmaceae (Ulmus). There was no information of host plant for sampled species Colophina arctica Zhang & Qiao. The main morphological characteristics used to identify the Eriosomatinae species included: the shape and distribution pattern of wax plates on dorsal body, the number and distribution pattern of dorsal setae, the number of segments of antennae, length in proportion of antennal segments, the shape, distribution and number of secondary rhinaria on antennal segments III–VI, siphunculi present or absent, the shape and position of galls, the shape of claw of hind tarsal segment, the venation of fore and hind wings and so on. We used the keys of Fauna Sinica (Eriosomatinae) [69], aphids on world’s plants from Blackman and Eastop (http://www.aphidsonworldsplants.info) and the revisions on some genera. Most keys were compiled based on alate viviparous females, but some samples used in present study only were collected fundatrix or apterous viviparous females, so we have no way to identify these samples to specific species. Therefore, we used sp. 1–n to distinguish unidentified different species in the same genus. Each species analysed was represented by 3–10 individuals from the same aphid species clone, which were then mixed together. All specimens were preserved in 95% and 75% ethanol for molecular experiments and voucher specimen collections, respectively. All aphid voucher specimens and samples were deposited in the National Zoological Museum of China, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
To remove body surface contaminations, aphid specimens were immersed in 70% ethanol, washed for 5 min (with vortex and centrifugation) and rinsed four times with sterile water. Total DNA was extracted from whole aphids using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions, and two negative controls were set during DNA extraction. To verify the aphid species identification and eliminate parasitized aphids, the standard cytochrome oxidase subunit I (COI) barcode of each sample was amplified using the universal primer pair LCO1490 and HCO2198 [76].

2.2. Amplification and Sequencing of the 16S Ribosomal RNA Gene

Amplicons of the V3–V4 regions of the 16S ribosomal RNA (16S rRNA) gene were amplified using the primer pair 341F (5′-CCTAYGGGRBGCASCAG) and 806R (5′-GGACTACNNGGGTATCTAAT) with a barcode. All PCR amplifications were conducted in a 30 μL reaction mixture containing approximately 10 ng template DNA, 0.2 μM forward and reverse primers, 15 μL Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Massachusetts, United States) and double distilled water (ddH2O). The PCR conditions were set as follows: initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s and elongation at 72 °C for 30 s and a final extension at 72 °C for 5 min. Each species was amplified in triplicate. PCR products were mixed in the same volume with 1× loading buffer, and bright bands corresponding to lengths between 400 and 450 bp were recovered using 2% agarose gel electrophoresis. All positive PCR products were mixed at equimolar ratio and then purified with a GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Massachusetts, United States). A sequencing library was constructed using a NEBNext® UltraTM DNA Library Prep Kit for Illumina (New England Biolabs, Massachusetts, United States) following the manufacturer’s recommendations, and index codes were added. Library quality was assessed with a Qubit@ 2.0 fluorometer (Thermo Fisher Scientific, Massachusetts, United States) and an Agilent Bioanalyzer 2100 system, and then the library pool was sequenced in paired-end 250 bp format using the Illumina HiSeq 2500 platform. The raw reads were deposited in the NCBI Sequence Read Archive (SRA) database under BioProject accession number PRJNA588521.

2.3. Sequencing Data Analysis

Raw reads were assigned to each sample based on their unique barcodes. Paired-end reads were merged using FLASH (v1.2.11) [77], and low-quality tags and chimaeras were filtered by QIIME with default settings [78]. The remaining sequences were clustered into operational taxonomic units (OTUs) at 97% identity by function pick_de_novo_otus.py in QIIME. The most abundant sequences of each OTU were chosen as the representative sequences and used to obtain taxonomic annotations with the RDP Classifier based on the SILVA 132 database [79,80,81]. Rare OTUs (<0.001% relative abundance in a given sample), chloroplast OTUs and OTUs identified as resulting from sequencing or PCR errors were excluded [82]. Finally, an OTU table containing the number of sequences per sample and taxonomic information was generated.

2.4. Symbiont Community Analysis

Analyses were executed with R software (v3.6.1) [83]. To mitigate the differences in sequencing effort, the number of sequences assigned to each sample was rarefied to the value (41236 reads) corresponding to the minimum sum of sequences across all the samples. Then, the relative abundance was calculated based on these rarefied abundance data by dividing the number of sequences per OTU by the total number of sequences for a given sample. Subsequent diversity analyses were performed based on these rarefied abundance data or relative abundance data.
To evaluate the within-sample diversity (alpha diversity) of the aphid bacterial community, the Shannon index and Simpson index of each species were calculated using the phyloseq package [84] based on the OTU abundance table. A rarefaction curve was generated based on the index of observed species.
Samples of Eriosomatinae were grouped according to host plant (Table 1). All samples were divided into four groups with a sample size of ≥3 (n = 30), and samples distributing in the same geography (Gansu Province, China) were divided into two groups with a sample size of ≥3 (n = 9). We performed analysis of variance (ANOVA) followed by Tukey’s honest significant difference (HSD) post hoc test using the vegan package [85] to determine pairwise differences in the alpha-diversity indices (Shannon index and Simpson index) across all groups. Differentiation of the bacterial communities between samples (beta diversity) was quantified by calculating Bray–Curtis dissimilarities using the vegan package [85]. The bacterial communities among groups were clustered using constrained principal coordinate analysis (CPCoA) and principal coordinate analysis (PCoA) based on the relative abundance of each genus and Bray–Curtis dissimilarities in the vegan package and ape packages, respectively [85,86], and plots were created by the ggplot2 package [87]. Based on the Bray–Curtis dissimilarities, permutational multivariate analysis of variance (PERMANOVA) was performed in the vegan package [85] to assess statistically significant differences among the host plant families. To compare the relative abundance of each symbiont between samples from different host plants, we conducted a Kruskal–Wallis test and pairwise comparisons, with a false discovery rate (FDR) p-value correction for multiple tests [88].
To explore the effect of aphid geographic distribution on structuring the microbial communities, we carried out correlation analysis between geographic distance and the beta-diversity index (Bray–Curtis dissimilarity). A geographic distance matrix was constructed from geographic points (latitudes and longitudes) using the function GeoDistanceInMetresMatrix written by Peter Rosenmai. Then, the Pearson correlation coefficient (r) between the two matrices was calculated, and the significance of the statistic was evaluated by a permutation procedure using the Mantel test in the vegan package [85]. We conducted the analysis using all samples (n = 34) and samples from the same plant family Salicaceae but distributing in different locations (n = 15), respectively.
To test whether more closely related aphid species have more similar microbial community, we measured aphid phylogenetic relatedness using the proportion of COI nucleotide sites at which two aphid species differ (p-distance). Then, we used Mantel test to assess the correlation between the p-distance matrix and the Bray–Curtis matrix [85].
All beta diversity analyses were conducted using all the bacterial genera, excluding primary endosymbiont Buchnera. We also divided the community into “known facultative symbionts” and re-ran all analyses to better determine the shaping factors of facultative symbiont.
Spearman’s rank correlation coefficients (ρ) were calculated for every pair of symbionts (primary endosymbiont and facultative symbionts) associated with Eriosomatinae to determine their interactions based on their relative abundances using the Hmisc package [89].

3. Results

3.1. Sequencing Data

Sequencing of the 16S rRNA V3–V4 amplicons yielded 8989997 raw reads. After quality filtering and removal of chimaeric sequences, a total of 5922611 effective tags with an average length of 429 nt were obtained. The sequences were classified into 931 OTUs at 97% sequence identity. The rarefaction curve for each sample tended to saturate (Figure S1).

3.2. Bacterial Diversity across Eriosomatinae Aphids

After discarding rare and chloroplast sequences and contaminations, 817 OTUs were obtained and annotated as belonging to 21 phyla, 180 families and 280 genera. Overall, 50.77% of these OTUs were attributed to Proteobacteria, 21.26% to Firmicutes and 11.47% to Actinobacteria. The alpha diversity of bacteria in Eriosomatinae was low (mean Shannon index = 0.29 and mean Simpson index = 0.12; Table S2). The bacterial community was dominated by Buchnera, Regiella, Serratia, Hamiltonella, the plant pathogen Pectobacterium and several environmental bacteria, such as Gluconobacter and Acinetobacter (Figure 1a and Table S3). The total relative abundance of aphid primary endosymbiont and facultative symbiont was greater than 96.00% in most samples and that of the other bacterial genera was less than 0.50%.
The primary endosymbiont Buchnera was found in all species, with an average relative abundance of 93.21%. A total of six aphid facultative symbionts were detected in Eriosomatinae aphids (Figure 1b and Table S4). Regiella inhabited all samples (detection frequency, 34/34; average relative abundance across all samples, 2.66%) and were thus the most common, followed by Serratia (33/34; 0.41%), Hamiltonella (17/34; 1.93%), Arsenophonus (12/34; 0.40%) and Spiroplasma (12/34; 0.13%). Rickettsia (5/34) was detected in Eriosomatinae species at very low abundance (<0.01%). Every facultative symbiont was represented by a small number of OTUs. There were five OTUs for Regiella, two for Serratia and only one for the remaining four symbionts. The numbers of reads belonging to the OTUs derived from the same genus were not equal, but in each sample, a single OTU from each genus was dominant. All of the samples contained at least two facultative symbionts (Table S5). The combination of Regiella and Serratia (7/34) was the most common, followed by those of Hamiltonella, Regiella and Serratia (6/34) and Arsenophonus, Hamiltonella, Regiella and Serratia (5/34).

3.3. Comparison of Microbiota Associated with Eriosomatinae among Plants, Geographic Distributions and Aphid Relatedness

Measurements of within-sample diversity (alpha diversity) of all detected bacteria or secondary symbionts showed no significant differences between eriosomatine species exploiting different host plants (p > 0.05; Tukey’s HSD test).
For the beta diversity, in PCoA analysis (Figure 2a), aphids from the same plant family tended to position near each other regardless of aphid species, and the PERMANOVA revealed significant differences in bacterial communities between host plant groups (F3,26 = 0.75, R2 = 0.25 and p = 0.01; Table 2a). CPCoA analysis revealed that the bacterial communities of aphids colonizing different plants tended to form distinct clusters (20% of the total variance and p = 0.003; Figure 2b). The bacterial communities of nine aphid species feeding on Salicaceae and Ulmaceae, which distributed in the same location (Gansu Province), are significantly different (F1,7 = 5.07, R2 = 0.42 and p = 0.008; Table 2a). PCoA analysis showed distinct clusters of nine aphid species feeding on the two plant families (Figure 2c). For comparison of the facultative symbiont community, PCoA and CPCoA (21.50% of the total variance and p = 0.001) analyses showed clear clusters of aphids feeding on the same plant family (Figure 2d,e). The PERMANOVA test (Table 2a) also revealed that facultative symbiont communities of aphids differed among species feeding on four host plants (F3,26 = 0.73, R2 = 0.27 and p = 0.002) and between nine species feeding on Salicaceae and Ulmaceae (F1,7 = 9.46, R2 = 0.57 and p = 0.013). PCoA analysis showed that nine aphid species feeding on the two plant families separated from each other (Figure 2f). Among all detected symbionts, significant food plant associations were detected for Regiella (df = 3, χ2 = 15.48 and p < 0.01). The relative abundance of Regiella in aphids feeding on Ulmaceae was significantly higher than that in aphids feeding on Anacardiaceae, Rosaceae and Salicaceae.
In the Mantel test (Table 2b), we found no significant correlation between aphid geographic distances and Bray–Curtis dissimilarities of bacteria or facultative symbionts (r = −0.01–0.03 and p = 0.50–0.36). For 15 species feeding on the same plant genus (Populus), the aphid geographic distances were not significantly correlated with beta diversity of their bacteria or facultative symbionts (r = 0.14–0.14 and p = 0.19–0.20).
The genetic distance (p-distance) and Bray–Curtis dissimilarities of both bacteria and facultative symbionts were not significantly correlated in Eriosomatinae (r = 0.07–0.01 and p = 0.16–0.41; Mantel test; Table 2b). For 15 species feeding on the same plant genus (Populus), the correlations between aphid genetic distance and Bray–Curtis dissimilarities of both bacteria and facultative symbionts were also not significant (r = −0.19–0.19 and p = 0.96–0.97; Table 2b).

3.4. Correlation Test between Different Symbionts Associated with Eriosomatinae Aphids

The resulting Spearman’s correlation coefficients are shown in Table S6. Significant positive correlations were found between Arsenophonus and Hamiltonella (ρ = 0.54 and p < 0.001) and between Regiella and Serratia (ρ = 0.40 and p < 0.05). Buchnera had both negative and positive correlations with facultative symbionts, showing a significant negative correlation with Regiella (ρ = −0.52 and p < 0.01) in particular. Except for these, there were no significant correlations between other symbionts.

4. Discussion

4.1. Diversity and Composition of Eriosomatinae Aphid Symbionts

Our study revealed that the microbiota associated with Eriosomatinae was dominated by a few bacterial taxa. Of the top 10 abundant genera, 4 were derived from genera that are known to be symbionts of aphids, including Buchnera, Regiella, Serratia and Hamiltonella. Buchnera inhabited all the sampled species, with the highest relative abundance (average relative abundance: 93.21%). The ubiquity and high abundance of Buchnera in the present study seem reasonable because of its obligate nutritive role and its long-term endosymbiotic association with aphids [1,2,3,5,6,7,8,90].
Six facultative symbionts were detected in Eriosomatinae, although their relative abundances were very low. These facultative symbionts exhibited various infection patterns. Regiella was detected in all the samples in the present study, but it was only detected in one of the 22 Eriosomatinae species by Russel et al. [16] using diagnostic PCR method and not detected in the 3 Eriosomatinae species by Henry et al. [61]. Most species sampled in these two studies were not included in our study.
The defensive role of Hamiltonella, Rickettsia and Spiroplasma has been documented in many studies [21,22,23,26,27,28,91,92]. However, in the present study, only a few samples harboured these symbionts, and they did so at extremely low abundance (<0.05%); this value was lower than that detected in Hormaphidinae (<1%) [93]. Most species in Eriosomatinae induce galls on their primary host plants or live in the roots of their secondary host plants [64,67,69,94,95,96,97,98]. Galls or living underground can provide protection against parasitoids and predators to the inducer aphid and its offspring [99,100,101,102], the biological role of which is similar to that of defensive symbionts. However, carrying protective symbionts incurs a cost in some aphid species, i.e., increasing development time, and reducing longevity and fecundity, which may lead to balancing selection on symbiont maintenance [29,91,103,104,105]. Hormaphidinae species, which can induce galls on their primary host plants but are primarily collected from secondary host plants, have a higher abundance of defensive symbionts and greater proportion of infected species than Eriosomatinae [93]. Compared with only wax protection, the life history trait of living in galls or plant roots provides stronger protection to Eriosomatinae against parasites and predators. Therefore, we suppose that the special life history traits possessed by Eriosomatinae reduce the pressure of natural enemies on these aphids, which may tip the balance against the infection frequency of defensive symbionts, as observed for ant tending [61].

4.2. Structure of Symbiont Community in Relation to Ecological Conditions

Several factors have been revealed in structuring the microbial profiles of aphids. Geographical distribution has been reported to influence symbiont communities of populations of A. gossypii, whereas host plants seem to have no impact [52,53,55,56,57]. However, based on extensive sampling efforts of A. gossypii, the study revealed that host plants rather than geographical distribution structured the symbiont community [50]. Strong associations between host plants and symbiont composition have been largely documented in pea aphid A. pisum and A. craccivora [45,46,47,48,49], whereas the role of geographical distribution in structuring aphid microbial communities has not been detected in these species. In addition, the study suggested that both geographic distribution and host plant influenced the structure and composition of the bacterial community of pea aphid [54]. Studies based on comprehensive sampling within family Aphididae revealed correlations between aphid symbiont, ant attending and host plant [61] and aphid relatedness [62].
However, most studies focused on different populations of one species. In the present study, we assessed the structuring factors of symbiont community of a specific aphid group, Eriosomatinae. Our results indicated that geographical distribution did not contribute to eriosomatine microbiota composition. For species feeding on the same plant species, the symbiont diversity was also not influenced by aphid locations. Aphid relatedness has no effect on the bacterial flora of Eriosomatinae, which was in accordance with the findings of Henry et al. [61].
In contrast to geographical distribution and species relatedness, host plant is an important variable explaining the symbiont community structure associated with Eriosomatinae. Ordination analyses showed species feeding on different plants have different symbiont communities. Statistical test revealed a prominent effect of host plant on bacterial communities. These findings provide a strong evidence that aphid host plants play an important role in symbiont distribution [46,49,61].
The symbiont distribution of aphid species feeding on Ulmaceae is unique. Symbiont communities of these species were clustered separately from those in other samples in ordination analyses. The Kruskal–Wallis test also revealed that the relative abundance of Regiella in samples from Ulmaceae were significantly higher than those in samples from other plants. These findings raise the possibility that certain facultative symbionts may be involved in the adaptation of aphids feeding on certain plants. Previous studies reported a role of facultative symbionts in aphid host plant use [31,32,33], but some found mixed results for symbiont importance in interactions between aphids and host plants [106,107]. Therefore, the interactions between symbionts associated with aphids and host plants remain to be experimentally tested. Regardless, our results demonstrated that host plants are among some of the forces that drive maintenance of facultative symbionts.

4.3. Interactions between Symbionts

The phenomenon of harbouring multiple facultative symbionts has been reported in aphids by several studies [46,54,59,108,109,110]. Similar to the findings of these studies, most eriosomatine species were superinfected with multiple facultative symbionts. Positive correlations between Arsenophonus and Hamiltonella and Regiella and Serratia were detected. Multiple-facultative symbiont infection may provide extra benefits to aphids. Coinfection of Hamiltonella–Fukatsuia and Hamiltonella–Serratia increased the resistance of A. pisum against parasites [111,112]. A. gossypii coinfected with Hamiltonella and Arsenophonus displayed enhanced fitness [113]. In contrast, Leclair et al. [114] revealed that coinfecting Hamiltonella negatively affected the beneficial phenotype provided by Rickettsiella. McLean et al. [115] revealed mixed results of multiple infections with different symbiont combinations. Furthermore, aphids hosting multiple symbionts may suffer additional costs [111,114]. These findings suggest that the interacting assemblage of facultative symbionts influences aphid fitness in different ways, which may be synergistic, additive or antagonistic.
Correlations were mostly absent between groups of symbionts, which suggested that the combinations of different symbionts were not specific. The relatively frequent superinfection with facultative symbionts in Eriosomatinae may be the result of horizontal transmission [54,108,116]. Eriosomatinae species are a typical heteroecious holocyclic aphid group [64,65]. Seasonal host alternation between primary and secondary host plants, migration between different secondary host plants and the presence of a sexual phase may greatly increase the possibility of horizontal transmission of facultative symbionts among Eriosomatinae species [116,117,118,119].

5. Conclusions

In conclusion, using Illumina sequencing of the 16S ribosomal RNA gene, we analysed the bacterial diversity in a particular aphid subfamily, Eriosomatinae. The microbiota of Eriosomatinae was dominated by heritable symbionts. The primary endosymbiont Buchnera unsurprisingly inhabited all species, in accordance with its obligate mutualist role. Regiella was the predominant facultative symbiont in the Eriosomatinae species. We found that symbiont diversity varied with host plant, suggesting an important role of the host plant in structuring the bacterial community associated with aphids. However, the aphid relatedness and geographical distribution seem to have no effect on Eriosomatinae symbiont composition. Moreover, combinations of multiple facultative symbionts were common in Eriosomatinae, but the interactions between them were very complex.

Supplementary Materials

The following are available online at https://www.mdpi.com/2075-4450/11/4/217/s1, Figure S1: rarefaction curve for each sample of Eriosomatinae based on the index of observed species, Table S1: voucher information and GenBank accession numbers of reference COI sequences, Table S2: the Shannon index and Simpson index of Eriosomatinae bacterial community, Table S3: the relative abundance of top 10 bacterial genera in Eriosomatinae, Table S4: OTUs and reads number of facultative symbionts, Table S5: infection pattern of facultative symbionts in Eriosomatinae and Table S6: spearman correlation coefficients of symbionts in Eriosomatinae.

Author Contributions

Conceptualization, G.-X.Q., L.-Y.J., J.C. and T.-T.X.; methodology, G.-X.Q., J.C. and T.-T.X.; analysis, T.-T.X.; writing–original draft preparation, T.-T.X.; writing–review and editing, G.-X.Q. and T.-T.X.; supervision, G.-X.Q.; project administration, G.-X.Q.; funding acquisition, G.-X.Q. All authors accepted the final version of the manuscript.

Funding

This research was funded by the National Key R & D Program of China (Grant number 2016YFE0203100), the National Natural Science Foundation of China (Grant numbers 31620103916 and 31772492), the Second Tibetan Plateau Scientific Expedition and Research (STEP) program (Grant number 2019QZKK0501), and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant number XDA19050303) and Youth Innovation Promotion Association of Chinese Academy of Sciences (Grant/Award number 2020087).

Acknowledgments

We are grateful to all collectors for their collecting specimens and Fen-Di Yang for making voucher specimens. We thank Peter Rosenmai for the R code of calculating a distance matrix for geographic points (function GeoDistanceInMetresMatrix) published in his blog (https://eurekastatistics.com/calculating-a-distance-matrix-for-geographic-points-using-r/). We also thank the anonymous reviewer from a professional language editing service for the language revision of this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Buchner, P. Endosymbiosis of Animals with Plant. Microorganisms; Interscience Publishers: New York, NY, USA, 1965. [Google Scholar]
  2. Douglas, A.E.; Prosser, W.A. Synthesis of the essential amino-acid tryptophan in the pea aphid (Acyrthosiphon pisum) symbiosis. J. Insect. Physiol. 1992, 38, 565–568. [Google Scholar] [CrossRef]
  3. Douglas, A.E. Nutritional interactions in insect-microbial symbioses: Aphids and their symbiotic bacteria Buchnera. Annu. Rev. Entomol. 1998, 43, 17–37. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Shigenobu, S.; Watanabe, H.; Hattori, M.; Sakaki, Y.; Ishikawa, H. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. Aps. Nature 2000, 407, 81–86. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Munson, M.A.; Baumann, P.; Clark, M.A.; Baumann, L.; Moran, N.A.; Voegtlin, D.J.; Campbell, B.C. Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families. J. Bacteriol. 1991, 173, 6321–6324. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Moran, N.A.; Munson, M.A.; Baumann, P.; Ishikawa, H. A molecular clock in endosymbiotic bacteria is calibrated using the insect hosts. Proc. R. Soc. B 1993, 253, 167–171. [Google Scholar] [CrossRef]
  7. Baumann, P.; Baumann, L.; Lai, C.Y.; Rouhbakhsh, D.; Moran, N.A.; Clark, M.A. Genetics, physiology, and evolutionary relationships of the genus Buchnera: Intracellular symbionts of aphids. Annu. Rev. Microbiol. 1995, 49, 55–94. [Google Scholar] [CrossRef]
  8. Baumann, P.; Moran, N.A.; Baumann, L. The evolution and genetics of aphid endosymbionts. Bioscience 1997, 47, 12–20. [Google Scholar] [CrossRef] [Green Version]
  9. Clark, M.A.; Moran, N.A.; Baumann, P.; Wernegreen, J.J. Cospeciation between bacterial endosymbionts (Buchnera) and a recent radiation of aphids (Uroleucon) and pitfalls of testing for phylogenetic congruence. Evolution 2000, 54, 517–525. [Google Scholar] [CrossRef]
  10. Jousselin, E.; Desdevises, Y.; Coeur d’acier, A. Fine-scale cospeciation between Brachycaudus and Buchnera aphidicola: Bacterial genome helps define species and evolutionary relationships in aphids. Proc. R. Soc. B 2009, 276, 187–196. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  11. Liu, L.; Huang, X.L.; Zhang, R.L.; Jiang, L.Y.; Qiao, G.X. Phylogenetic congruence between Mollitrichosiphum (Aphididae: Greenideinae) and Buchnera indicates insect-bacteria parallel evolution. Syst. Entomol. 2013, 38, 81–92. [Google Scholar] [CrossRef]
  12. Liu, L.; Li, X.Y.; Huang, X.L.; Qiao, G.X. Evolutionary relationships of Pemphigus and allied genera (Hemiptera: Aphididae: Eriosomatinae) and their primary endosymbiont, Buchnera aphidicola. Insect Sci. 2014, 21, 301–312. [Google Scholar] [CrossRef]
  13. Xu, T.T.; Chen, J.; Jiang, L.Y.; Qiao, G.X. Historical and cospeciating associations between Cerataphidini aphids (Hemiptera: Aphididae: Hormaphidinae) and their primary endosymbiont Buchnera aphidicola. Zool. J. Linn. Soc. 2018, 183, 604–613. [Google Scholar] [CrossRef]
  14. Chen, D.Q.; Purcell, A.H. Occurrence and transmission of facultative endosymbionts in aphids. Curr. Microbiol. 1997, 34, 220–225. [Google Scholar] [CrossRef] [PubMed]
  15. Sandström, J.P.; Russell, J.A.; White, J.P.; Moran, N.A. Independent origins and horizontal transfer of bacterial symbionts of aphids. Mol. Ecol. 2001, 10, 217–228. [Google Scholar] [CrossRef] [PubMed]
  16. Russell, J.A.; Latorre, A.; Sabater-Munoz, B.; Moya, A.; Moran, N.A. Side-stepping secondary symbionts: Widespread horizontal transfer across and beyond the Aphidoidea. Mol. Ecol. 2003, 12, 1061–1075. [Google Scholar] [CrossRef]
  17. Russell, J.A.; Moran, N.A. Horizontal transfer of bacterial symbionts: Heritability and fitness effects in a novel aphid host. Appl. Environ. Microbiol. 2005, 71, 7987–7994. [Google Scholar] [CrossRef] [Green Version]
  18. Vorburger, C.; Siegrist, G.; Rhyner, N. Faithful vertical transmission but ineffective horizontal transmission of bacterial endosymbionts during sexual reproduction of the black bean aphid, Aphis fabae. Ecol. Entomol. 2017, 42, 202–209. [Google Scholar] [CrossRef] [Green Version]
  19. Rock, D.I.; Smith, A.H.; Joffe, J.; Albertus, A.; Wong, N.; O’Connor, M.; Oliver, K.M.; Russell, J.A. Context-dependent vertical transmission shapes strong endosymbiont community structure in the pea aphid, Acyrthosiphon pisum. Mol. Ecol. 2018, 27, 2039–2056. [Google Scholar] [CrossRef]
  20. Oliver, K.M.; Russell, J.A.; Moran, N.A.; Hunter, M.S. Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proc. Natl. Acad. Sci. USA 2003, 100, 1803–1807. [Google Scholar] [CrossRef] [Green Version]
  21. Oliver, K.M.; Moran, N.A.; Hunter, M.S. Variation in resistance to parasitism in aphids is due to symbionts not host genotype. Proc. Natl. Acad. Sci. USA 2005, 102, 12795–12800. [Google Scholar] [CrossRef] [Green Version]
  22. Vorburger, C.; Gehrer, L.; Rodriguez, P. A strain of the bacterial symbiont Regiella insecticola protects aphids against parasitoids. Biol. Lett. 2010, 6, 109–111. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Hansen, A.K.; Vorburger, C.; Moran, N.A. Genomic basis of endosymbiont-conferred protection against an insect parasitoid. Genome. Res. 2012, 22, 106–114. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Brandt, J.W.; Chevignon, G.; Oliver, K.M.; Strand, M.R. Culture of an aphid heritable symbiont demonstrates its direct role in defence against parasitoids. Proc. R. Soc. B 2017, 284, 20171925. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Frago, E.; Mala, M.; Weldegergis, B.T.; Yang, C.; McLean, A.; Godfray, H.C.J.; Gols, R.; Dicke, M. Symbionts protect aphids from parasitic wasps by attenuating herbivore-induced plant volatiles. Nat. Commun. 2017, 8, 1860. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Ferrari, J.; Darby, A.C.; Daniell, T.J.; Godfray, H.C.J.; Douglas, A.E. Linking the bacterial community in pea aphids with host-plant use and natural enemy resistance. Ecol. Entomol. 2004, 29, 60–65. [Google Scholar] [CrossRef]
  27. Scarborough, C.L.; Ferrari, J.; Godfray, H.C. Aphid protected from pathogen by endosymbiont. Science 2005, 310, 1781. [Google Scholar] [CrossRef]
  28. Łukasik, P.; van Asch, M.; Guo, H.; Ferrari, J.; Godfray, H.C. Unrelated facultative endosymbionts protect aphids against a fungal pathogen. Ecol. Lett. 2013, 16, 214–218. [Google Scholar] [CrossRef]
  29. Chen, D.Q.; Montllor, C.B.; Purcell, A.H. Fitness effects of two facultative endosymbiotic bacteria on the pea aphid, Acyrthosiphon pisum, and the blue alfalfa aphid, A. Kondoi. Entomol. Exp. Appl. 2000, 95, 315–323. [Google Scholar] [CrossRef]
  30. Montllor, C.B.; Maxmen, A.; Purcell, A.H. Facultative bacterial endosymbionts benefit pea aphids Acyrthosiphon pisum under heat stress. Ecol. Entomol. 2002, 27, 189–195. [Google Scholar] [CrossRef]
  31. Leonardo, T.E.; Muiru, G.T. Facultative symbionts are associated with host plant specialization in pea aphid populations. Proc. R. Soc. Lond. B 2003, 270, S209–S212. [Google Scholar] [CrossRef] [Green Version]
  32. Tsuchida, T.; Koga, R.; Fukatsu, T. Host plant specialization governed by facultative symbiont. Science 2004, 303, 1989. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Wagner, S.M.; Martinez, A.J.; Ruan, Y.M.; Kim, K.L.; Lenhart, P.A.; Dehnel, A.C.; Oliver, K.M.; White, J.A. Facultative endosymbionts mediate dietary breadth in a polyphagous herbivore. Funct. Ecol. 2015, 29, 1402–1410. [Google Scholar] [CrossRef]
  34. Tsuchida, T.; Koga, R.; Horikawa, M.; Tsunoda, T.; Maoka, T.; Matsumoto, S.; Simon, J.C.; Fukatsu, T. Symbiotic bacterium modifies aphid body color. Science 2010, 330, 1102–1104. [Google Scholar] [CrossRef] [PubMed]
  35. Nikoh, N.; Tsuchida, T.; Maeda, T.; Yamaguchi, K.; Shigenobu, S.; Koga, R.; Fukatsu, T. Genomic insight into symbiosis-induced insect color change by a facultative bacterial endosymbiont, Candidatus Rickettsiella viridis. mBio 2018, 9, e00890-18. [Google Scholar] [CrossRef] [Green Version]
  36. Pérez-Brocal, V.; Gil, R.; Ramos, S.; Lamelas, A.; Postigo, M.; Michelena, J.M.; Silva, F.J.; Moya, A.; Latorre, A. A small microbial genome: The end of a long symbiotic relationship? Science 2006, 314, 312–313. [Google Scholar] [CrossRef] [Green Version]
  37. Lamelas, A.; Gosalbes, M.J.; Manzano-Marín, A.; Pereto, J.; Moya, A.; Latorre, A. Serratia symbiotica from the aphid Cinara cedri: A missing link from facultative to obligate insect endosymbiont. PLoS Genet. 2011, 7, e1002357. [Google Scholar] [CrossRef] [Green Version]
  38. Manzano-Marín, A.; Latorre, A. Settling down: The genome of Serratia symbiotica from the aphid Cinara tujafilina zooms in on the process of accommodation to a cooperative intracellular life. Genome Biol. Evol. 2014, 6, 1683–1698. [Google Scholar] [CrossRef]
  39. Manzano-Marín, A.; Simon, J.C.; Latorre, A. Reinventing the wheel and making it round again: Evolutionary convergence in Buchnera-Serratia symbiotic consortia between the distantly related Lachninae aphids Tuberolachnus salignus and Cinara cedri. Genome Biol. Evol. 2016, 8, 1440–1458. [Google Scholar] [CrossRef] [Green Version]
  40. Manzano-Marín, A.; Szabo, G.; Simon, J.C.; Horn, M.; Latorre, A. Happens in the best of subfamilies: Establishment and repeated replacements of co-obligate secondary endosymbionts within Lachninae aphids. Environ. Microbiol. 2017, 19, 393–408. [Google Scholar] [CrossRef]
  41. Meseguer, A.S.; Manzano-Marín, A.; Coeur d’acier, A.; Clamens, A.L.; Godefroid, M.; Jousselin, E. Buchnera has changed flatmate but the repeated replacement of co-obligate symbionts is not associated with the ecological expansions of their aphid hosts. Mol. Ecol. 2017, 26, 2363–2378. [Google Scholar] [CrossRef]
  42. Manzano-Marín, A.; Coeur d’acier, A.; Clamens, A.L.; Orvain, C.; Cruaud, C.; Barbe, V.; Jousselin, E. A freeloader? The highly eroded yet large genome of the Serratia symbiotica symbiont of Cinara strobi. Genome Biol. Evol. 2018, 10, 2178–2189. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. De Clerck, C.; Fujiwara, A.; Joncour, P.; Leonard, S.; Felix, M.L.; Francis, F.; Jijakli, M.H.; Tsuchida, T.; Massart, S. A metagenomic approach from aphid’s hemolymph sheds light on the potential roles of co-existing endosymbionts. Microbiome 2015, 3, 63. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Manzano-Marín, A. No evidence for Wolbachia as a nutritional co-obligate endosymbiont in the aphid Pentalonia nigronervosa. bioRxiv 2019, 609511. [Google Scholar] [CrossRef] [Green Version]
  45. Tsuchida, T.; Koga, R.; Shibao, H.; Matsumoto, T.; Fukatsu, T. Diversity and geographic distribution of secondary endosymbiotic bacteria in natural populations of the pea aphid, Acyrthosiphon pisum. Mol. Ecol. 2002, 11, 2123–2135. [Google Scholar] [CrossRef] [PubMed]
  46. Ferrari, J.; West, J.A.; Via, S.; Godfray, H.C. Population genetic structure and secondary symbionts in host-associated populations of the pea aphid complex. Evolution 2012, 66, 375–390. [Google Scholar] [CrossRef] [PubMed]
  47. Gauthier, J.P.; Outreman, Y.; Mieuzet, L.; Simon, J.C. Bacterial communities associated with host-adapted populations of pea aphids revealed by deep sequencing of 16S ribosomal DNA. PLoS ONE 2015, 10, e0120664. [Google Scholar] [CrossRef]
  48. Brady, C.M.; White, J.A. Cowpea aphid (Aphis craccivora) associated with different host plants has different facultative endosymbionts. Ecol. Entomol. 2013, 38, 433–437. [Google Scholar] [CrossRef]
  49. Brady, C.M.; Asplen, M.K.; Desneux, N.; Heimpel, G.E.; Hopper, K.R.; Linnen, C.R.; Oliver, K.M.; Wulff, J.A.; White, J.A. Worldwide populations of the aphid Aphis craccivora are infected with diverse facultative bacterial symbionts. Microb. Ecol. 2014, 67, 195–204. [Google Scholar] [CrossRef]
  50. Xu, S.F.; Jiang, L.Y.; Qiao, G.X.; Chen, J. The bacterial flora associated with the polyphagous aphid Aphis gossypii Glover (Hemiptera: Aphididae) is strongly affected by host plants. Microb. Ecol. 2019. [Google Scholar] [CrossRef] [Green Version]
  51. Medina, R.F.; Nachappa, P.; Tamborindeguy, C. Differences in bacterial diversity of host-associated populations of Phylloxera notabilis Pergande (Hemiptera: Phylloxeridae) in pecan and water hickory. J. Evol. Biol. 2011, 24, 761–771. [Google Scholar] [CrossRef]
  52. Najar-Rodriguez, A.J.; McGraw, E.A.; Mensah, R.K.; Pittman, G.W.; Walter, G.H. The microbial flora of Aphis gossypii: Patterns across host plants and geographical space. J. Invertebr. Pathol. 2009, 100, 123–126. [Google Scholar] [CrossRef]
  53. Jones, R.T.; Bressan, A.; Greenwell, A.M.; Fierer, N. Bacterial communities of two parthenogenetic aphid species cocolonizing two host plants across the Hawaiian islands. Appl. Environ. Microbiol. 2011, 77, 8345–8349. [Google Scholar] [CrossRef] [Green Version]
  54. Russell, J.A.; Weldon, S.; Smith, A.H.; Kim, K.L.; Hu, Y.; Lukasik, P.; Doll, S.; Anastopoulos, I.; Novin, M.; Oliver, K.M. Uncovering symbiont-driven genetic diversity across North American pea aphids. Mol. Ecol. 2013, 22, 2045–2059. [Google Scholar] [CrossRef]
  55. Zhao, Y.; Zhang, S.; Luo, J.Y.; Wang, C.Y.; Lv, L.M.; Cui, J.J. Bacterial communities of the cotton aphid Aphis gossypii associated with Bt cotton in northern China. Sci. Rep. 2016, 6, 22958. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Gallo-Franco, J.J.; Duque-Gamboa, D.N.; Toro-Perea, N. Bacterial communities of Aphis gossypii and Myzus persicae (Hemiptera: Aphididae) from pepper crops (Capsicum sp.). Sci. Rep. 2019, 9, 5766. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Guo, J.; Liu, X.; Poncelet, N.; He, K.; Francis, F.; Wang, Z. Detection and geographic distribution of seven facultative endosymbionts in two Rhopalosiphum aphid species. Microbiologyopen 2019, 8, e00817. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Zytynska, S.E.; Weisser, W.W. The natural occurrence of secondary bacterial symbionts in aphids. Ecol. Entomol. 2016, 41, 13–26. [Google Scholar] [CrossRef] [Green Version]
  59. Zytynska, S.E.; Meyer, S.T.; Sturm, S.; Ullmann, W.; Mehrparvar, M.; Weisser, W.W. Secondary bacterial symbiont community in aphids responds to plant diversity. Oecologia 2016, 180, 735–747. [Google Scholar] [CrossRef]
  60. Fakhour, S.; Ambroise, J.; Renoz, F.; Foray, V.; Gala, J.L.; Hance, T. A large-scale field study of bacterial communities in cereal aphid populations across Morocco. FEMS Microbiol. Ecol. 2018, 94, fiy003. [Google Scholar] [CrossRef] [Green Version]
  61. Henry, L.M.; Maiden, M.C.; Ferrari, J.; Godfray, H.C. Insect life history and the evolution of bacterial mutualism. Ecol. Lett. 2015, 18, 516–525. [Google Scholar] [CrossRef]
  62. McLean, A.H.C.; Godfray, H.C.J.; Ellers, J.; Henry, L.M. Host relatedness influences the composition of aphid microbiomes. Environ. Microbiol. Rep. 2019, 11, 808–816. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Remaudière, G.; Remaudière, M. Catalogue of the World’s Aphididae: Homoptera Aphidoidea; Institut National de la Recherche Agronomique: Paris, Franch, 1997. [Google Scholar]
  64. Smith, C.F. Keys to and descriptions of the genera of Pemphigini in North America (Homoptera: Aphididae: Pemphiginae). North Carolina Agri. Exp. Station Tech. Bull. 1974, 226, 1–61. [Google Scholar]
  65. Moran, N.A. The evolution of host-plant alternation in aphids: Evidence for specialization as a dead end. Am. Nat. 1988, 132, 681–706. [Google Scholar] [CrossRef]
  66. Moran, N.A.; Whitham, T.G. Evolutionary reduction of complex life cycles: Loss of host alternation in Pemphigus (Homoptera: Aphididae). Evolution 1988, 42, 717–728. [Google Scholar] [CrossRef] [PubMed]
  67. Ghosh, A.K. Fauna of India and the Adjacent Countries: Homoptera: Aphidoidea. Part. 3, Subfamilies: Pemphiginae; Zoological Survey of India: Calcutta, India, 1984. [Google Scholar]
  68. Smith, R.G. Wax glands, wax production and the functional significance of wax use in three aphid species (Homoptera: Aphididae). J. Nat. Hist. 1999, 33, 513–530. [Google Scholar] [CrossRef]
  69. Zhang, G.X.; Qiao, G.X.; Zhong, T.S.; Zhang, W.Y. Fauna Sinica Insecta, Vol. 14. Homoptera: Mindaridae and Pemphigidae; Science Press: Beijing, China, 1999. [Google Scholar]
  70. Pike, N.; Foster, W. Fortress repair in the social aphid species Pemphigus spyrothecae. Anim. Behav. 2004, 67, 909–914. [Google Scholar] [CrossRef]
  71. Pike, N.; Whitfield, J.A.; Foster, W.A. Ecological correlates of sociality in Pemphigus aphids, with a partial phylogeny of the genus. BMC Evol. Biol. 2007, 7, 185. [Google Scholar] [CrossRef] [Green Version]
  72. Chong, R.A.; Moran, N.A. Evolutionary loss and replacement of Buchnera, the obligate endosymbiont of aphids. ISME J. 2018, 12, 898–908. [Google Scholar] [CrossRef] [Green Version]
  73. Burke, G.R.; Normark, B.B.; Favret, C.; Moran, N.A. Evolution and diversity of facultative symbionts from the aphid subfamily Lachninae. Appl. Environ. Microbiol. 2009, 75, 5328–5335. [Google Scholar] [CrossRef] [Green Version]
  74. Jousselin, E.; Coeur d’Acier, A.; Vanlerberghe-Masutti, F.; Duron, O. Evolution and diversity of Arsenophonus endosymbionts in aphids. Mol. Ecol. 2013, 22, 260–270. [Google Scholar] [CrossRef]
  75. Wang, Z.; Su, X.M.; Wen, J.; Jiang, L.Y.; Qiao, G.X. Widespread infection and diverse infection patterns of Wolbachia in Chinese aphids. Insect Sci. 2014, 21, 313–325. [Google Scholar] [CrossRef]
  76. Folmer, O.; Black, M.; Hoeh, W.; Lutz, R.; Vrijenhoek, R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 1994, 3, 294–299. [Google Scholar] [PubMed]
  77. Magoc, T.; Salzberg, S.L. Flash: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011, 27, 2957–2963. [Google Scholar] [CrossRef] [PubMed]
  78. Caporaso, J.G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F.D.; Costello, E.K.; Fierer, N.; Pena, A.G.; Goodrich, J.K.; Gordon, J.I.; et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 2010, 7, 335–336. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Wang, Q.; Garrity, G.M.; Tiedje, J.M.; Cole, J.R. Naïve bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 2007, 73, 5261–5267. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  80. Quast, C.; Pruesse, E.; Yilmaz, P.; Gerken, J.; Schweer, T.; Yarza, P.; Peplies, J.; Glockner, F.O. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013, 41, D590–D596. [Google Scholar] [CrossRef] [PubMed]
  81. Yilmaz, P.; Parfrey, L.W.; Yarza, P.; Gerken, J.; Pruesse, E.; Quast, C.; Schweer, T.; Peplies, J.; Ludwig, W.; Glockner, F.O. The SILVA and “All-Species Living Tree Project (LTP)” taxonomic frameworks. Nucleic Acids Res. 2014, 42, D643–D648. [Google Scholar] [CrossRef] [Green Version]
  82. Navas-Molina, J.A.; Peralta-Sanchez, J.M.; Gonzalez, A.; McMurdie, P.J.; Vazquez-Baeza, Y.; Xu, Z.; Ursell, L.K.; Lauber, C.; Zhou, H.; Song, S.J.; et al. Advancing our understanding of the human microbiome using QIIME. Methods Enzymol. 2013, 531, 371–444. [Google Scholar] [CrossRef] [Green Version]
  83. R Core Team. R: A language and environment for statistical computing; R Foundation for Statistical Computing: Vienna, Austria, 2019; Available online: https://www.R-project.org/ (accessed on 15 January 2020).
  84. McMurdie, P.J.; Holmes, S. Phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 2013, 8, e61217. [Google Scholar] [CrossRef] [Green Version]
  85. Oksanen, F.J.; Blanchet, F.G.; Friendly, M.; Kindt, R.; Legendre, P.; McGlinn, D.; Minchin, P.; O’Hara, R.B.; Simpson, G.; Solymos, P.; et al. Vegan: Community ecology package. R Package Version 2.5-4. 2018. Available online: https://CRAN.R-project.org/package=vegan (accessed on 15 January 2020).
  86. Paradis, E.; Claude, J.; Strimmer, K. Ape: Analyses of phylogenetics and evolution in R language. Bioinformatics 2004, 20, 289–290. [Google Scholar] [CrossRef] [Green Version]
  87. Wickham, H. ggplot2: Elegant Graphics for Data Analysis; Springer: New York, NY, USA, 2016. [Google Scholar]
  88. Benjamini, Y.; Hochberg, Y. Controlling the false discovery rate-a practical and powerful approach to multiple testing. J. R. Statist. Soc. B 1995, 57, 289–300. [Google Scholar] [CrossRef]
  89. Harrell, F.E., Jr. With contributions from Charles Dupont and many others. Hmisc: Harrell Misc. R Package Version 4.2-0. 2019. Available online: https://CRAN.R-project.org/package=Hmisc (accessed on 15 January 2020).
  90. Tamas, I.; Klasson, L.; Canback, B.; Naslund, A.K.; Eriksson, A.S.; Wernegreen, J.J.; Sandstrom, J.P.; Moran, N.A.; Andersson, S.G. 50 million years of genomic stasis in endosymbiotic bacteria. Science 2002, 296, 2376–2379. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  91. Oliver, K.M.; Smith, A.H.; Russell, J.A. Defensive symbiosis in the real world-advancing ecological studies of heritable, protective bacteria in aphids and beyond. Funct. Ecol. 2014, 28, 341–355. [Google Scholar] [CrossRef] [Green Version]
  92. Vorburger, C.; Rouchet, R. Are aphid parasitoids locally adapted to the prevalence of defensive symbionts in their hosts? BMC Evol. Biol. 2016, 16, 271. [Google Scholar] [CrossRef] [Green Version]
  93. Xu, T.T.; Chen, J.; Jiang, L.Y.; Qiao, G.X. Diversity of bacteria associated with Hormaphidinae aphids (Hemiptera: Aphididae). Insect Sci. 2019. [Google Scholar] [CrossRef] [Green Version]
  94. Smith, C.F. Pemphiginae associated with the roots of conifers in north America (Homoptera: Aphididae). Ann. Entomol. Soc. Am. 1969, 62, 1128–1152. [Google Scholar] [CrossRef]
  95. Stroyan, H.L.G. The life cycle and generic position of Aphis tremulae L., 1761 (Aphidoidea: Pemphiginae), with a description of the viviparous morphs and a discussion of spruce root aphids in the British Isles. Biol. J. Linn. Soc. 1975, 7, 45–72. [Google Scholar] [CrossRef]
  96. Danielsson, R. The root-feeding generations of gall-making aphids of the genera Pachypappa Koch, Pachypappella Baker, and Gootiella Tullgren (Homoptera. Aphidoidea. Pemphigidae). Acta Phytopathol. Entomol. Hung. 1990, 25, 77–88. [Google Scholar]
  97. Zhang, H.C.; Qiao, G.X. Systematic status of the genus Formosaphis Takahashi and the evolution of galls based on the molecular phylogeny of Pemphigini (Hemiptera: Aphididae: Eriosomatinae). Syst. Entomol. 2007, 32, 690–699. [Google Scholar] [CrossRef]
  98. Pike, K.S.; Graf, G.; Foottit, R.G.; Maw, H.E.L.; von Dohlen, C.; Harpel, J.; Pantoja, A.; Emmert, S.; Hagerty, A.M. Eriosomatine aphids (Hemiptera: Aphididae: Eriosomatinae) associated with moss and roots of conifer and willow in forests of the Pacific Northwest of North America. Can. Entomol. 2012, 144, 555–576. [Google Scholar] [CrossRef] [Green Version]
  99. Cornell, H.V. The secondary chemistry and complex morphology of galls formed by the Cynipinae (Hymenoptera): Why and how? Am. Midl. Nat. 1983, 110, 225–234. [Google Scholar] [CrossRef]
  100. Price, P.W.; Waring, G.L.; Fernandes, G.W. Hypotheses on the adaptive nature of galls. Proc. Entomol. Soc. Wash. 1986, 88, 361–363. [Google Scholar]
  101. Stone, G.N.; Schönrogge, K. The adaptive significance of insect gall morphology. Trends Ecol. Evol. 2003, 18, 512–522. [Google Scholar] [CrossRef]
  102. Miller, D.G.; Ivey, C.T.; Shedd, J.D. Support for the microenvironment hypothesis for adaptive value of gall induction in the California gall wasp, Andricus quercuscalifornicus. Entomol. Exp. Appl. 2009, 132, 126–133. [Google Scholar] [CrossRef]
  103. Vorburger, C.; Gouskov, A. Only helpful when required: A longevity cost of harbouring defensive symbionts. J. Evol. Biol. 2011, 24, 1611–1617. [Google Scholar] [CrossRef] [Green Version]
  104. Polin, S.; Simon, J.C.; Outreman, Y. An ecological cost associated with protective symbionts of aphids. Ecol. Evol. 2014, 4, 826–830. [Google Scholar] [CrossRef]
  105. Zytynska, S.E.; Thighiouart, K.; Frago, E. A meta-analysis on the benefits and costs of hosting secondary endosymbionts in sap-sucking insects. bioRxiv 2019, 563031. [Google Scholar] [CrossRef]
  106. Ferrari, J.; Scarborough, C.L.; Godfray, H.C. Genetic variation in the effect of a facultative symbiont on host-plant use by pea aphids. Oecologia 2007, 153, 323–329. [Google Scholar] [CrossRef]
  107. McLean, A.H.; van Asch, M.; Ferrari, J.; Godfray, H.C. Effects of bacterial secondary symbionts on host plant use in pea aphids. Proc. R. Soc. B. 2011, 278, 760–766. [Google Scholar] [CrossRef]
  108. Henry, L.M.; Peccoud, J.; Simon, J.C.; Hadfield, J.D.; Maiden, M.J.; Ferrari, J.; Godfray, H.C. Horizontally transmitted symbionts and host colonization of ecological niches. Curr. Biol. 2013, 23, 1713–1717. [Google Scholar] [CrossRef] [Green Version]
  109. Smith, A.H.; Lukasik, P.; O’Connor, M.P.; Lee, A.; Mayo, G.; Drott, M.T.; Doll, S.; Tuttle, R.; Disciullo, R.A.; Messina, A.; et al. Patterns, causes and consequences of defensive microbiome dynamics across multiple scales. Mol. Ecol. 2015, 24, 1135–1149. [Google Scholar] [CrossRef] [PubMed]
  110. Zhang, S.; Luo, J.; Wang, L.; Zhang, L.; Zhu, X.; Jiang, W.; Cui, J. Bacterial communities in natural versus pesticide-treated Aphis gossypii populations in north China. Microbiologyopen 2019, 8, e00652. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  111. Oliver, K.M.; Moran, N.A.; Hunter, M.S. Costs and benefits of a superinfection of facultative symbionts in aphids. Proc. R. Soc. B. 2006, 273, 1273–1280. [Google Scholar] [CrossRef] [PubMed]
  112. Guay, J.F.; Boudreault, S.; Michaud, D.; Cloutier, C. Impact of environmental stress on aphid clonal resistance to parasitoids: Role of Hamiltonella defensa bacterial symbiosis in association with a new facultative symbiont of the pea aphid. J. Insect Physiol. 2009, 55, 919–926. [Google Scholar] [CrossRef] [PubMed]
  113. Ayoubi, A.; Talebi, A.A.; Fathipour, Y.; Mehrabadi, M. Coinfection of the secondary symbionts, Hamiltonella defensa and Arsenophonus sp. contribute to the performance of the major aphid pest, Aphis gossypii (Hemiptera: Aphididae). Insect Sci. 2020, 27, 86–98. [Google Scholar] [CrossRef]
  114. Leclair, M.; Polin, S.; Jousseaume, T.; Simon, J.C.; Sugio, A.; Morliere, S.; Fukatsu, T.; Tsuchida, T.; Outreman, Y. Consequences of coinfection with protective symbionts on the host phenotype and symbiont titres in the pea aphid system. Insect Sci. 2017, 24, 798–808. [Google Scholar] [CrossRef]
  115. McLean, A.H.C.; Parker, B.J.; Hrcek, J.; Kavanagh, J.C.; Wellham, P.A.D.; Godfray, H.C.J. Consequences of symbiont co-infections for insect host phenotypes. J. Anim. Ecol. 2018, 87, 478–488. [Google Scholar] [CrossRef]
  116. Moran, N.A.; Dunbar, H.E. Sexual acquisition of beneficial symbionts in aphids. Proc. Natl. Acad. Sci. USA 2006, 103, 12803–12806. [Google Scholar] [CrossRef] [Green Version]
  117. Gehrer, L.; Vorburger, C. Parasitoids as vectors of facultative bacterial endosymbionts in aphids. Biol. Lett. 2012, 8, 613–615. [Google Scholar] [CrossRef]
  118. Chrostek, E.; Pelz-Stelinski, K.; Hurst, G.D.D.; Hughes, G.L. Horizontal transmission of intracellular insect symbionts via plants. Front. Microbiol. 2017, 8, 2237. [Google Scholar] [CrossRef] [Green Version]
  119. Pons, I.; Renoz, F.; Noel, C.; Hance, T. Circulation of the cultivable symbiont Serratia symbiotica in aphids is mediated by plants. Front. Microbiol. 2019, 10, 764. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Insects 11 00217 g001 550
Figure 1. Bar plots of all bacteria (a) and facultative symbionts (b) associated with Eriosomatinae.
Figure 1. Bar plots of all bacteria (a) and facultative symbionts (b) associated with Eriosomatinae.
Insects 11 00217 g001
Insects 11 00217 g002 550
Figure 2. PCoA plot illustrating the separation of all samples from four host plant groups (a and d) and species distributing in the same location from two host plant groups (c and f) based on differences in bacterial community structure (a and c) and facultative symbiont community structure (d and f); CPCoA plot illustrating the separation of samples from four host plant groups based on differences in bacterial community structure (20.0% of the total variance and p = 0.003) (b) and facultative symbiont community structure (21.5% of the total variance and p = 0.001) (e). Colours correspond to different plant families, as shown in the legend. Ellipses cover 95% of the data for each plant families.
Figure 2. PCoA plot illustrating the separation of all samples from four host plant groups (a and d) and species distributing in the same location from two host plant groups (c and f) based on differences in bacterial community structure (a and c) and facultative symbiont community structure (d and f); CPCoA plot illustrating the separation of samples from four host plant groups based on differences in bacterial community structure (20.0% of the total variance and p = 0.003) (b) and facultative symbiont community structure (21.5% of the total variance and p = 0.001) (e). Colours correspond to different plant families, as shown in the legend. Ellipses cover 95% of the data for each plant families.
Insects 11 00217 g002
Table
Table 1. Grouping information for host plant of Eriosomatinae species.
Table 1. Grouping information for host plant of Eriosomatinae species.
Host PlantSample NumberSample Name
Species across Eriosomatinae
Anacardiaceae3Chaetogeoica sp., Kaburagia rhusicola, Schlechtendalia chinensis
Magnoliaceae1Formosaphis micheliae
Oleaceae2Ligustrum lucidum, Prociphilus sp.
Rosaceae3Eriosoma lanigerum, Prociphilus kuwanai, Prociphilus pini
Salicaceae15Epipemphigus imaicus, Epipemphigus niisimae, Epipemphigus yunanensis, Pemphigus borealis, Pemphigus bursarius, Pemphigus matsumurai, Pemphigus populitransversus, Pemphigus sinobursarius, Pemphigus sp. 1, Pemphigus sp. 2, Pemphigus tibetensis, Thecabius beijingensis, Thecabius sp. 1, Thecabius sp. 2, Thecabius sp. 3
Ulmaceae9Eriosoma sp. 1, Eriosoma sp. 2, Eriosoma sp. 3, Eriosoma sp. 4, Tetraneura sp. 1, Tetraneura sp. 2, Tetraneura sp. 3, Tetraneura sp. 4, Tetraneura sp. 5
Species from Gansu Province
Salicaceae4Pemphigus sinobursarius, Thecabius sp. 1, Thecabius sp. 2, Thecabius sp. 3
Ulmaceae5Tetraneura sp. 3, Tetraneura sp. 4, Tetraneura sp. 5, Eriosoma sp. 2, Eriosoma sp. 3
Table
Table 2. Statistical test results for bacterial and facultative symbiont communities in relation to different factors.
Table 2. Statistical test results for bacterial and facultative symbiont communities in relation to different factors.
(a)
Bacterial CommunityFacultative Symbiont Community
Host plantAll 4 groups2 groupsAll 4 groups2 groups
0.25, 0.0100.42, 0.0080.27, 0.0020.57, 0.013
(b)
Bacterial CommunityFacultative Symbiont Community
All species15 speciesAll species15 species
Geographic distribution−0.01, 0.500.14, 0.190.03, 0.360.14, 0.20
Aphid relatedness0.07, 0.16−0.19, 0.960.01, 0.41−0.19, 0.97
(a): Permutational multivariate analysis of variance (PERMANOVA) for the effects of host plant on bacterial and facultative symbiont diversity. The values in each cell represent R2 and p. (b): Mantel test between aphid geographical distance and Bray–Curtis dissimilarity and between p-distance of aphid species and Bray–Curtis dissimilarity. The values in each cell represent r and p. Significant p values (p < 0.05) are in bold.

Share and Cite

MDPI and ACS Style

Xu, T.-T.; Jiang, L.-Y.; Chen, J.; Qiao, G.-X. Host Plants Influence the Symbiont Diversity of Eriosomatinae (Hemiptera: Aphididae). Insects 2020, 11, 217. https://doi.org/10.3390/insects11040217

AMA Style

Xu T-T, Jiang L-Y, Chen J, Qiao G-X. Host Plants Influence the Symbiont Diversity of Eriosomatinae (Hemiptera: Aphididae). Insects. 2020; 11(4):217. https://doi.org/10.3390/insects11040217

Chicago/Turabian Style

Xu, Ting-Ting, Li-Yun Jiang, Jing Chen, and Ge-Xia Qiao. 2020. "Host Plants Influence the Symbiont Diversity of Eriosomatinae (Hemiptera: Aphididae)" Insects 11, no. 4: 217. https://doi.org/10.3390/insects11040217

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop