Biodiversity Data Journal :
Research Article
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Corresponding author: Hao Yuan (2022020001@xiyi.edu.cn)
Academic editor: Anna Sandionigi
Received: 29 Nov 2022 | Accepted: 28 Feb 2023 | Published: 15 Mar 2023
© 2023 Yantong Liu, Lina Zhao, Zhongying Qiu, Hao Yuan
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Liu Y, Zhao L, Qiu Z, Yuan H (2023) The gut microbiota diversity of five Orthoptera (Insecta, Polyneoptera) insects determined by DNA metabarcoding. Biodiversity Data Journal 11: e98162. https://doi.org/10.3897/BDJ.11.e98162
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Most orthopteran insects are phytophagous and some are important pests in agriculture and forests. Many intestinal microflora of Orthoptera insects have been reported, primarily from Acridoidea and there have been few reports of other taxa. In this study, we collected 15 individuals representing five species (Ruspolia lineosa, Tetrix japonica, Erianthus versicolor, Gryllotalpa orientalis and Teleogryllus emma) belonging to five orthopteran superfamilies (Tettigonioidea, Tetrigoidea, Eumastacoidea, Gryllotalpoidea and Grylloidea) to characterise and compare the gut microbiota with greater taxonomic width by performing sequencing analysis of the 16S rRNA V4 region in gut material. A total of 606,053 high-quality sequences and 3,105 OTUs were acquired from 15 gut samples representing 24 phyla, 48 classes, 69 orders, 133 families and 219 genera. Firmicutes and bacteria were the most abundant phyla, followed by Bacteroidetes, Cyanobacteria, Actinobacteria and Acidobacteria. At the genus level, Serratia, Citrobacter, Wolbachia, Lactobacillus and Parabacteroides were the most predominant genera in R. lineosa, T. japonica, E. versicolor, G. orientalis and T. emma, respectively. Both Principal Coordinates Analysis (PCoA) and heatmap results revealed significant differences in bacterial community composition across species. Additionally, alpha diversity analysis indicated the bacterial richness was significantly different amongst the five species.
gut microbiota, DNA metabarcoding, Orthoptera, biodiversity
Large numbers of microorganisms colonise the insect gut and form complex symbiotic relationships with their host. Insect-gut symbiotic microorganisms play important roles in parasitifer mating preference (
DNA metabarcoding, a high-throughput DNA barcoding technique, is a fast and efficient method to assess biodiversity (
Most orthopterans are phytophagous and some are important pests in agriculture and forests. Most reports of intestinal microflora in Orthoptera have primarily concentrated on Acridoidea (
A total of 15 orthopteran specimens across five species (Ruspolia lineosa belonging to Tettigonoidea, Gryllotalpa orientalis belonging to Gryllotalpoidea, Teleogryllus emma belonging to Grylloidea, Erianthus versicolor belonging to Eumastacoidea and Tetrix japonica belonging to Tetrigoidea) were collected, with three specimens per species collected in the same region (see Table
Superfamily |
Species |
SampleID |
Location |
Date |
|
Tetrigoidea |
Tetrix japonica |
Z |
Z1 |
Shaanxi, Xi’an |
21/08/2016 |
Z2 |
Shaanxi, Xi’an |
21/08/2016 |
|||
Z3 |
Shaanxi, Xi’an |
21/08/2016 |
|||
Tettigoniidae |
Ruspolia lineosa |
ZS |
ZS1 |
Shaanxi, Xi’an |
22/08/2016 |
ZS2 |
Shaanxi, Xi’an |
22/08/2016 |
|||
ZS3 |
Shaanxi, Xi’an |
22/08/2016 |
|||
Eumastacoidea |
Erianthus versicolor |
M |
M1 |
Guangdong, Ruyuan |
15/09/2016 |
M2 |
Guangdong, Ruyuan |
15/09/2016 |
|||
M3 |
Guangdong, Ruyuan |
15/09/2016 |
|||
Gryllotalpidae |
Gryllotalpa orientalis |
LG |
LG1 |
Henan, Nanyang |
29/08/2016 |
LG2 |
Henan, Nanyang |
29/08/2016 |
|||
LG3 |
Henan, Nanyang |
29/08/2016 |
|||
Gryllidae |
Teleogryllus emma |
HLYHL |
HLYHL1 |
Shaanxi, Xi’an |
21/08/2016 |
HLYHL2 |
Shaanxi, Xi’an |
21/08/2016 |
|||
HLYHL3 |
Shaanxi, Xi’an |
21/08/2016 |
Microbial genomic DNA was extracted from the gut samples using the phenol-chloroform method as previously described (
To integrate raw paired-end sequences, we quality-screened for paired-end sequences in FASTQ format using Trimmomatic (v.0.36, http://www.usadellab.org/cms/index.php?page=trimmomatic) (
Operational taxonomic units (OTUs) were generated with sequence similarity greater than 97% using the uclust function (
Chao1, ACE, Shannon and Simpson indices for each sample were calculated using the summary.single command in the MOTHUR software package (http://www.mothur.org/) (
We utilised the V4 region of the 16S rRNA amplicon to assess the gut microbiota composition of five orthopterans using Illumina MiSeq DNA metabarcode sequencing. A total of 778,780 paired-end reads were acquired from all intestinal samples, with an average read length of 450 bp. After quality control, 606,053 high-quality reads were acquired. Based on 97% species similarity and chloroplast and mitochondrial sequences and OTUs with < 0.001% abundance in all samples being removed, a total of 3,105 OTUs were obtained from all intestinal samples. The fifteen insect samples were divided into five groups, each with three samples. The number of OTUs in each group (ZS, M, HLYHL, Z and LG) was 978, 818, 951, 952 and 1,417, respectively. Amongst these, 43 OTUs present in all groups were defined as core OTUs and 94, 81, 648, 90 and 1,039 OTUs were uniquely identified in ZS, M, HLYHL, Z and LG, respectively (Fig.
The Dendrogram and heatmap revealed the differences of the top 100 OTUs amongst the 15 samples (Fig.
Dendrogram and heatmap of bacterial distributions of the top 100 abundant OTUs present in the microbial community of the fifteen samples. The numbers indicate the actual reads number of the OTU. The heatmap plot depicted the relative abundance of each sample and the relative values for OTUs are indicated by colour intensity. Z: Tetrix japonica; ZS: Ruspolia lineosa; M: Erianthus versicolor; LG: Gryllotalpa orientalis; HLYHL: Teleogryllus emma.
PCoA plot based on an unweighted UniFrac distance matrix depicting differences in the composition of the gut microbiota of the five groups. In the unweighted UniFrac analysis of the gut samples, the first principal coordinate, explained 40.11% of sample variation and separated groups of LG and HLYHL from others. The third principal coordinate (7.49% of sample variation) separated groups (M) from others. Z: Tetrix japonica; ZS: Ruspolia lineosa; M: Erianthus versicolor; LG: Gryllotalpa orientalis; HLYHL: Teleogryllus emma.
Gut microbiota alpha diversity was estimated using alpha diversity curves (rarefaction curves and Shannon–Wiener curves) and alpha diversity indices (Chao1, ACE, Simpson and Shannon indices). The rarefaction curves (
The diversity indices for each sample are shown in Table
SampleID |
Chao1 |
ACE |
Simpson |
Shannon |
Z1 |
371 |
478.18 |
0.78 |
3.36 |
Z2 |
522 |
648.34 |
0.89 |
4.82 |
Z3 |
446 |
577.26 |
0.83 |
4.11 |
ZS1 |
498 |
582.61 |
0.86 |
5.11 |
ZS2 |
665 |
788.12 |
0.97 |
6.43 |
ZS3 |
594 |
694.67 |
0.97 |
6.43 |
M1 |
339 |
395.71 |
0.89 |
4.83 |
M2 |
306 |
396.69 |
0.77 |
3.56 |
M3 |
579 |
629.24 |
0.98 |
7.19 |
LG1 |
865 |
865.00 |
0.99 |
7.87 |
LG2 |
898 |
969.45 |
0.92 |
6.36 |
LG3 |
932 |
971.13 |
0.98 |
7.62 |
HLYHL1 |
436 |
468.90 |
0.96 |
6.18 |
HLYHL2 |
582 |
621.68 |
0.97 |
6.76 |
HLYHL3 |
602 |
621.76 |
0.98 |
6.87 |
p-value |
0.001 |
0.002 |
0.100 |
0.027 |
Amongst the identified sequences, a total of 219, 133, 69, 48 and 24 microbes at the genus, family, order, class and phylum taxonomic levels, respectively, were identified across all samples. Table
SampleID |
Phylum |
Class |
Order |
Family |
Genus |
OTUs |
Z1 |
13 |
25 |
37 |
84 |
111 |
581 |
Z2 |
18 |
33 |
41 |
93 |
131 |
694 |
Z3 |
16 |
29 |
41 |
92 |
125 |
616 |
ZS1 |
15 |
26 |
36 |
83 |
114 |
624 |
ZS2 |
16 |
28 |
42 |
95 |
136 |
811 |
ZS3 |
16 |
31 |
43 |
91 |
124 |
726 |
M1 |
15 |
29 |
40 |
90 |
113 |
515 |
M2 |
12 |
25 |
35 |
83 |
105 |
455 |
M3 |
16 |
31 |
38 |
95 |
151 |
656 |
LG1 |
16 |
28 |
39 |
63 |
83 |
1049 |
LG2 |
14 |
27 |
39 |
66 |
78 |
1104 |
LG3 |
15 |
25 |
35 |
55 |
65 |
1080 |
HLYHL1 |
5 |
13 |
20 |
40 |
41 |
512 |
HLYHL2 |
7 |
15 |
25 |
42 |
50 |
725 |
HLYHL3 |
7 |
14 |
24 |
34 |
36 |
680 |
Z |
18 |
35 |
48 |
105 |
160 |
955 |
ZS |
18 |
36 |
53 |
109 |
155 |
980 |
M |
17 |
35 |
46 |
107 |
165 |
827 |
LG |
18 |
34 |
45 |
75 |
101 |
1417 |
HLYHL |
8 |
18 |
29 |
51 |
65 |
951 |
Total |
24 |
48 |
69 |
133 |
219 |
3105 |
Amongst 24 phyla, Firmicutes (45.0%), bacteria (31.4%), Bacteroidetes (17.8%), Actinobacteria (2.1%) and Acidobacteria (2.0%) were present in all samples and abundant in the majority of samples, representing more than 98% of total sequences (Fig.
Amongst 219 genera, Lactococcus (9.95%), Lactobacillus (9.00%), Citrobacter (7.87%), Parabacteroides (7.67%), Sediminibacterium (6.77%), Serratia (6.65%), Bacteroides (5.18%), Streptococcus (4.37%), Wolbachia (4.27%), Geobacillus (3.14%), Bacillus (2.72%), Rhodanobacter (1.89%), Pseudomonas (1.69%), Ralstonia (1.63%), Ochrobactrum (1.58%), Burkholderia (1.49%), Ruminococcus (1.48%), Sphingomonas (1.42%), Rhodococcus (1.41%) and Oscillospira (1.07%) were the most abundant genera, accounting for more than 81% of total sequences (Fig.
At the phylum level, we analysed the differences in Firmicutes, bacteria, Bacteroidetes, Actinobacteria and Acidobacteria in different groups. Amongst these, Acidobacteria (P < 0.01) and bacteria (P < 0.001) demonstrated significant differences and Actinobacteria, Bacteroidetes and Firmicutes showed no differences. We further calculated multiple comparisons to show differences between each two groups of Acidobacteria and bacteria, the relative abundance of the phylum bacteria in Z was mostly significantly higher than others and the relative abundance of the phylum Acidobacteria in ZS and M were significantly higher than LG and HLYHL (Fig.
The relative abundance (% of individual taxonomic group) of Acidobacteria and bacteria present in the microbial community of the different groups. Differences were analysed by employing ANOVA analysis and Tukey Post Hoc HSD Significance Test (* P < 0.05, ** P < 0.01, *** P < 0.001). Z: Tetrix japonica; ZS: Ruspolia lineosa; M: Erianthus versicolor; LG: Gryllotalpa orientalis; HLYHL: Teleogryllus emma.
Amongst the 20 most abundant genera, ANOVA indicated significant differences for Lactococcus (P < 0.05), Citrobacter (P < 0.001), Parabacteroides (P < 0.01), Sediminibacterium (P < 0.01), Wolbachia (P < 0.001), Geobacillus (P < 0.01), Bacillus (P < 0.05), Rhodanobacter (P < 0.05), Pseudomonas (P < 0.05), Ralstonia (P < 0.01), Ochrobactrum (P < 0.05), Burkholderia (P < 0.01) and Rhodococcus (P < 0.01) (Suppl. material
Based on the results obtained for 15 samples across five orthopteran species using DNA metabarcoding, the predominant phyla in the insect gut were Firmicutes and bacteria, representing 70.1% of total sequences. This result is quite similar to those obtained in previous studies. Yun et al. studied gut samples from 305 individuals belonging to 218 species in 21 taxonomic orders and found the predominant phyla to be Firmicutes and bacteria, representing 82.8% of total sequences (
According to our study, the predominant genera in the gut were Lactococcus and Lactobacillus, belonging to the order Lactobacillales and the class Bacilli. Bacilli species reportedly exert beneficial effects in terms of preventing intestinal disorders and reducing inflammation (
When comparing gut bacteria amongst samples, we identified differences in diversity and abundance. Stanley et al. analysed samples from 207 chicken caecal microbiota across three similar trials and demonstrated the ability of host genes and environmental factors to alter the composition of the intestinal microflora (
To evaluate the relationships between the gut microbiota and host in five species, we collected 15 samples and classified them into five groups. Amongst the six most abundant phyla, ANOVA analysis revealed that Acidobacteria and bacteria differed significantly. bacteria abundance was highest in Z, followed by ZS, M, LG and HLYHL. Acidobacteria abundance was highest in ZS, followed by M and Z and low abundance in LG and HLYHL. Bacteroidetes, Cyanobacteria and Firmicutes did not differ significantly. Amongst the 20 most abundant genera, 13 to 20 were significantly different. Of these, all were low in LG and HLYHL with the exception of Parabacteroides. According to our PCoA and heatmap analysis, different individuals in the same group had relatively close relationships and, thus, bacterial community composition similarity was higher in same-group individuals than in different-group individuals. Alpha diversity analysis showed significant differences for Chao1, ACE and Shannon, illustrating higher bacterial community richness and diversity in the different groups.
In summary, our study revealed the composition and diversity of the gut microbiota of 15 individuals belonging to five orthopteran species using DNA metabarcode sequencing. The results revealed a bacterial community composition comprising 24 phyla and 219 genera. The most abundant phyla were Firmicutes and bacteria and the most abundant genera were Lactococcus and Lactobacillus. We also compared differences in bacterial composition of distinct species at the phylum and genus levels. The results suggested the gut bacteria composition differed significantly across the five species.
The raw data are available at the National Center for Biotechnology Information (NCBI) SRA (https://www.ncbi.nlm.nih.gov/sra/): SRR20722952 - SRR20722966.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This study was supported by the Key Research and Development Project of Shaanxi Province (2019SF-172).