Overview of the bacterial community in the cotton aphids
The symbiotic bacterium was investigated based on 16S rRNA amplicon Illumina sequencing in the cotton aphid. A total number of 1,432,180 of V3-V4 rRNA raw reads were obtained by sequencing with the Illumine HiSeq 2500 (Supplyment Table 1). The average valid sequences reads in 24 samples was 37,677, and an average percentage of effective tags was 61% after trimming the barcodes and primers, and filtering low quality reads and chimaeras. Sequences analysis by Uparse software generated 170 representative sequences with assigning ≥ 97% similarity to the same OTUs. As shown in figure (Supplement Fugure 1), the rarefaction curves for all samples have almost approached the saturation plateau, indicating that the sequencing data possessed adequate abundance and depth to capture most microbial diversity information in this insect.
Based on the bacterial diversity and relative abundance, the three groups, Proteobateria, Firmicutes and Actinobacteria, were identified on phylum level in the endosymbiotic bacteria of cotton aphid (Supplement Fig. 3A, B). Of them, the Proteobateria was the most abundant bacterial phylum with an average relative abundance of over 97.9%. Buchnera and Serratia were took up the largest population of bacteria on the taxonomic distribution of genus level. Klebsiella, Bacillus, Escherichia, Bifidobacterium Staphylococcus, Pseudomonas, Erwinia and Steptococcus were also discovered, although their percentage only holded 1%.
It is worth to noticed that more kinds of symbiotic bacteria were observed In the treatment groups by the plant allelochemicals than by pesticide treatment group. We identified 224 OTUs in the treatment groups by the plant allelochemicals. However, only 84 OTUs were found in the pesticide treatment group. Actinobacreia and Firmicutes phylum in the treatment groups by the plant allelochemicals accounted for larger proportion and contain more abundant of species than in control group based on the top 10 of relative abundance species in the two main groups. In the Proteobacteria phylum, the pesticide treatment group had richer species while the treatment group by plant allelochemicals mainly reflected in Enterobacteriaceae family of Gammaproteobacteria class.
Changes of the symbiotic bacterial composition in the cotton aphid stressed by omethoate
Two doses of omethoate (1500 mg/L and 7500 mg/L, approximately equal to LC10 and LC90 respectively) were used to determine effects of the omethoate on the symbiotic bacterial composition in the cotton aphid and 0.1% Triton X-100 as the control. The overall bacteria composition was similar in these three samples, while the distribution of each group varied at 24h after treatment. Proteobacteria was the most dominant phyla, whose percentage all close to 99% in these three samples. Firmicutes was almost wiped out in the treated groups by two doses of omethoate (Fig. 1). On a genus level, Buchnera had accounted for a major proportion, and followed by the Serratia and Arsenophonus. Other types of bacteria accounts for very less percentage. The low-dose omethoate increased the relative abundance of Buchnera, while the others appeared a decreasing trend. In the meanwhile, the number of bacterium species was also less than in control and treatment group by high-dose of omethoate. The high-dose of omethoate could cause Buchnera reduced to an extent while the relative abundance of Serratia and arsenophonus exhibited increase. Buchnera is the most important bacteria in cotton aphid who have the irreplaceable positions.
Figure 1. Abundance of top 10 OTUs in omethoate treated samples. The triton is control group. LD indicates low-dose omethoate treated and HD indicates high-dose treated.
The five indexes (Observed species, Chao1, Shannon, Simpson and ACE) were used to assess the alpha diversity of bacterial community in the three samples. According to Chao1 and ACE estimator, the control group exhibited a better community richness (Table 1). Shannon and Simpson suggested that the community diversity and evenness were declined after the treatment with low-dose of omethoate. Though the high concentration of omethoate did not cause the index declined obviously, the concrete species of the bacteria had changed.
Table 1
Alpha diversity indices on different dose of omethoate effects.
Sample | species | chao1 | ACE | shannon | simpson |
Triton.1 | 38 | 40.5 | 40.867 | 1.021 | 0.293 |
Triton.2 | 45 | 47.5 | 48.254 | 1.006 | 0.285 |
Triton.3 | 40 | 47.857 | 53.999 | 0.883 | 0.269 |
LD1 | 26 | 27 | 27.551 | 0.657 | 0.21 |
LD2 | 22 | 22.143 | 22.906 | 0.668 | 0.221 |
LD3 | 24 | 38 | 32.628 | 0.482 | 0.143 |
HD1 | 45 | 47.154 | 49.134 | 0.881 | 0.298 |
HD2 | 27 | 40.75 | 42.284 | 0.831 | 0.297 |
HD3 | 30 | 30.545 | 31.953 | 0.999 | 0.335 |
Table 1. Alpha diversity indices on different dose of omethoate effects.
Changes in the bacterial 16s rRNA genes under the stress of plant allelochemicals
The five indexes were employed to determine the community richness and diversity, after normalizing the different samples (cutoff = 28,325)(Figure 2). The 2-tridecannone and gossypol possessed more influence on the bacteria community, which brought about richer specie. However, the gossypol resulted in the community evenness slightly decreased (Simpson index). Tannin acid and quercetin possessed smaller influence on the symbiotic bacteria compared to 2-tridecanone and gossypol.
Figure 2. Within-habitat diversity in cotton aphids after four plant allelochemicals treated.
The heat map was drawn according the genus annotation and abundance in the species level (Fig. 3). More bacterial species were observed in the treatment groups by 2-tridecanone and gossypol. However, the stress of gossypol resulted in Buchnera abundance decrease. In the meanwhile, tannin acid and quercetin had little influence on Buchnera.
Figure 3. The top 35 bacteria abundance changed after four plant allelochemicals treated. CK indicates control group. SD, SH, SF and ST indicates tannic acid, quercetin, gossypol and 2-tridecanone treated groups respectively.
Variation of Buchnera and Serrtia after the treatment by xenobiotic chemicals
The Principal Coordinate Analysis (PcoA) was performed to determine the inter-group difference using the maximum variation factor based on a distance matrix of weighted unifrac (Fig. 4A). The significant changes in the community were observed after the treatment by omethoate. Three groups of samples were separated based on the top two characteristics which showed the independence among treatment groups. The LDA in LefSe was used to calculate the distinct species among different groups (Fig. 5A). In the treatment group by LC10 of omethoate, the major feature was the significant increasing of species in Buchnera. In the group by LC90, Marcescens notably increased in Serratia genus. Gossypol is a typical secondary metabolite existed in cotton. Based on the Principal Coordinate Analysis (PcoA) (Fig. 4B), the treated group by gossypol was separated from the other groups and the three repeat gather in a same quadrant (Fig. 5C). It suggests that the community composition in the treated group by gossypol was distinctive with other groups. In the previous analysis, more species were observed in the group treated by gossypol and numbers of the original species have greatly changed in abundance.
Figure 4. Difference between experimental and control groups by principal coordinates analysis. The greatest two eigenvalue in distance matrix were chose as ordinate and abscissa.
Figure 5. Species with significant differences between groups by Linear discriminant analysis Effect Size (LefSe) analysis. LefSe determines the features most likely to explain differences between classes by coupling standard tests for statistical significance with additional tests encoding biological consistency and effect relevance.
In order to find the intergroup different species, we used Kruskal Wallis and Wilcoxon to test the difference and liner discriminant analysis to obtain the LDA score [36]. The different spices all distributed in Enterobacteriaceae family, Enterobacteriales order, Gammaproteobacteria class. Buchnera when setting the LDA score as Fig. 5B, D. Buchnera turned into sharp decline after gossypol stressing in the meanwhile the Marcescens, Serratia and Klebsiella appeared a significant increase. The t-test also displayed the same variation tendency in Buchnera and Serratia. This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.