Diversity analysis of intestinal bacterial flora of Culex pipiens pallens at different developmental stages

doi:10.12140/j.issn.1000-7423.2022.04.007

Abstract

Abstract:

Objective To determine the composition and diversity of intestinal microflora of Culex pipiens pallens at different developmental stages. Methods The intestinal tracts of 20 Cx. pipiens pallens Ⅳ-instar larvae, pupae and female mosquitoes were dissected to collect intestinal samples for extraction of genomic DNA of intestinal bacteria, with triplicate for each developmental stage. The 16S rRNA highly variable regions of intestinal bacteria was amplified by PCR and sequenced using the Illumina NovaSeq6000 sequencing platform. The number of sequences and operational taxonomic units (OTU) were compiled and statistically analyzed using structural histogram and Veen diagram to reveal the flora composition and abundance of intestinal bacteria at different developmental stages of Cx. pipiens pallens. The abundance and diversity of microbial flora were analyzed by the Alpha diversity index. Principal coordinate analysis (PCoA) was used to examine the variance in the flora structure at different developmental stages. Linear discriminant analysis (LEFSe) was used to differentiate the species with significant differences in abundance between groups. SPSS 22.0 statistical software was used to conduct one-way ANOVA for genus-based analysis of the core bacterial abundance of Cx. pipiens pallens at different stages. Results The intestinal microbiota of Cx. pipiens pallens larvae, pupae and female mosquitoes were 1 972 OTUs, belonging to 22 phyla, 45 classes, 112 orders, 178 families and 329 genera. At the genus level, the fourth instar larvae, pupae and female mosquitoes were composed of 230, 233 and 228 genera, respectively. The dominant genera were Bacteroides with the abundance of (16.83 ± 3.5) %, (26.98 ± 0.94) % and (20.82 ± 2.63) %, respectively; Muribaculaceae, the abundance was (12.36 ± 1.94) %, (19.98 ± 1.24) %, (14.90 ± 1.52) %, respectively; the abundance of Alistipes was (4.13 ± 0.62)%, (7.29 ± 1.28)%, (5.36 ± 1.00)%, respectively; Lachnospiraceae_NK4A136_group were (3.39 ± 0.39)%, (5.77 ± 0.21) % and (3.93 ± 0.40)%, respectively. At the level of OTU, Veen diagram analysis showed that the number of OTU shared by the fourth instar larvae, pupae and female adult was 687, and the unique OTU was 250, 284 and 309, respectively. Alpha diversity showed that the composition and diversity of Cx. pipiens pallens were different at different developmental stages (F = 36.83, P < 0.01; F = 28.91, P < 0.01), the species abundance and diversity of pupae were the highest (6.58 ± 0.04). The results of PCoA showed that the microflora was presented in three groups, with each stage falling into one group, respectively. There were statistically significant differences in the composition of bacteria among different stages (Df = 2, R² = 0.707 44, P < 0.01). The results of LEFSe showed that 17 taxa were different at developmental stages. There were statistically significant differences in the abundance of Leifsonia, Bacteroides, Muribaculaceae and Wolbachia in different developmental stages of Cx. pipiens pallens (F = 48.331, 10.826, 17.759, 95.139, P < 0.01). Leifsonia mainly existed in the fourth instar larval stage (0.887 ± 0.022), Bacteroidetes (0.270 ± 0.009) and Muribaculaceae (0.200 ± 0.012) mainly existed in the pupa stage. Wolbachia mainly existed in the female mosquito stage (0.172 ± 0.025). Conclusion The dominant intestinal flora of Cx. pipiens pallens at different developmental stages are Bacteroides and Muribaculaceae, and the pupa shows the highest species abundance and species diversity.

Key words: Culex pipiens pallens, Intestinal bacteria, Diversity, High throughput sequencing

CLC Number:

R384.112

LV Wen-xiang, CHENG Peng, PENG Hui, WANG Hai-yang, LIU Hong-mei, WANG Hai-fang, GUO Xiu-xia, GONG Mao-qing, LIU Li-juan. Diversity analysis of intestinal bacterial flora of Culex pipiens pallens at different developmental stages[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(4): 460-467.

Figures/Tables 6

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样品Sample	原始标签数No. raw tag	有效标签数 No. effective tag	平均片段长度/bp Average length/bp	不同分类水平物种数量 Statistics of the number of classify distribution
样品Sample	原始标签数No. raw tag	有效标签数 No. effective tag	平均片段长度/bp Average length/bp	门Phyla	纲Class	目Order	科Family	属Genus
Ⅳ龄幼虫The fourth instar larva
L1	67 114	58 096	415.40	11	19	55	90	162
L2	72 466	64 056	415.25	12	21	58	93	171
L3	71 951	62 972	414.63	15	25	72	105	187
蛹Pupa
P1	73 525	63 673	415.01	12	23	61	95	164
P2	69 258	60 714	415.03	14	23	58	89	160
P3	69 857	60 684	414.74	16	27	64	102	179
雌成蚊 Female mosquito
A1	67 119	59 037	412.94	11	19	55	81	141
A2	74 434	66 009	414.13	12	23	60	93	162
A3	70 069	62 294	414.62	13	25	60	94	166
总计Total				22	45	112	178	329

样品 Sample	Chao1指数 Chao1 index	香农指数Shannon index	辛普森指数Simpson index	覆盖度Good’s coverage
Ⅳ龄幼虫The fourth instar larva	1 035.17 ± 41.71^a	6.05 ± 0.16^a	0.95 ± 0.00^a	0.99
蛹Pupa	1 038.76 ± 31.56^a	6.58 ± 0.04^a	0.95 ± 0.00^a	0.99
雌成蚊Female mosquito	963.24 ± 71.00^a	5.93 ± 0.05^b	0.93 ± 0.01^b	0.99