Intersex Plays a Role in Microbial Homeostasis in the Brown Planthopper

Abstract

Insects harbor a wide variety of symbiotic microorganisms that are capable of regulating host health and promoting host adaptation to their environment and food sources. However, there is little knowledge concerning the mechanisms that maintain the microbial community homeostasis within insects. In this study, we found that the intersex (ix) gene played an essential role in maintaining microbial homeostasis in the brown planthopper (BPH), Nilaparvata lugens. Injection of the double-strand RNA targeting N. lugens ix (Nlix) into the newly emerged females resulted in abnormal expansion of the copulatory bursa of BPH after mating. Further observation by transmission electron microscopy (TEM) revealed that the abnormally enlarged copulatory bursa resulting from dsNlix treatment was full of microorganisms, while in contrast, the copulatory bursa of dsGFP-treated individuals stored a large number of sperm accompanied by a few bacteria. Moreover, RNA-seq analysis showed that the gene responses to bacteria were remarkably enriched in differentially expressed genes (DEGs). In addition, 16s rRNA sequencing indicated that, compared with control samples, changes in the composition of microbes presented in dsNlix-treated copulatory bursa. Together, our results revealed the immune functions of the Nlix gene in maintaining microbial homeostasis and combating infection in BPH.

Keywords: Nilaparvata lugens; RNA interference; copulation; immune deference; microbiome.

Figure A1

Shannon rarefaction curve of (A) the dsGFP-treated samples and (B) the dsNlix-treated samples. CB: copulatory bursa; FB: fat body; MG: midgut.

Figure A2

Comparison of the microbiotal structure in CB, FB, and MG of N. lugens after infection of dsNlix. The 10 species with the highest relative abundances were displayed, and the relative abundances of the remaining species were combined and grouped into Others. CB: copulatory bursa; FB: fat body; MG: midgut.

Figure 1

(A) Survival rate of BPHs at 192 h after RNAi when the newly emerged females were injected with dsRNA. dsGFP was used as control in each group, n = 100. Statistical analysis was performed by log-rank (Mantel–Cox) test. Survival rates were significantly decreased for both dsNlix-UM compared to dsGFP-UM and dsNlix-M compared to dsGFP-M. *** p < 0.001. M: samples mated with males; UM: unmated samples; (B) Transcript levels of Nlix after RNAi treatment in the newly emerged females. The RNAi efficiency of Nlix was evaluated by collecting 10 insects at 72 h after injection. Means ± SEM from three experiments. *** p < 0.001 (Student’s t-test); (C–E) Ovarian development phenotypes of unmated females after 72 h and 120 h of different treatments; (F,G) Ovarian development phenotypes of mated females after 72 h of different treatments. Yellow triangle: copulatory bursa.

Figure 2

Transmission electron microscopy observation of copulatory bursa from the 72 h females in (A) the dsGFP-injected sample, (B) the dsNlix-injected sample. A clearer view of the sperms/bacteria is shown in the top right corner of each panel. The yellow arrow marks the sperm and the red arrow marks the bacteria in the copulatory bursa.

Figure 3

GO enrichment analysis of biological processes involved in the DEGs among female adults. GO enrichment study was performed using OmicShare tools. For data regarding GO terms, see Table S1.

Figure 4

(A) qPCR verification of the 5 genes associated with the response to bacteria; (B) Number of the offspring produced from the treated females after knockdown of the 4 down-regulated genes associated with the response to bacteria, respectively. Each treatment was carried out in 10 biological replications; (C–F) Transcript levels of 4 down-regulated genes after RNAi treatment in the newly emerged females. At 72 h after injection, 10 insects were collected randomly to evaluate the RNAi efficiency. Means ± SEM from three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t-test); ns: no significant difference.

Figure 5

Phenotypes of ovary development after knockdown of 4 genes responsive to bacteria: (A–E) Ovaries dissected from the females treated with dsGFP, dsNl.chr11.425, dsNl.chr01.0531, dsNl.chr05.0253, and dsNl.chr06.0989 for 72 h, respectively. Yellow triangle: copulatory bursa.

Figure 6

(A,B) Proportional compositions of microbiome in copulatory bursa (CB), fat body (FB), and midgut (MG) of N. lugens treated with dsGFP. Composition at the phylum (A) and genus (B) levels. The 10 species with the highest relative abundances were displayed, and the relative abundances of the remaining species were combined and grouped into Others; (C) Principal coordinates analysis (PCoA) plot of the Bray–Curtis distance based on the microbial structure in CB, FB, and MG; (D) LDA Effect Size (LEfSe) analysis based on ASV abundance in CB, FB, and MG. CB: copulatory bursa; FB: fat body; MG: midgut. For microbial taxon numbers, see Table S2.

Figure 7

Microbial structural composition between dsGFP-treated group and dsNlix-treated group: (A) Relative abundances of bacteria at the genus level in CB, FB, and MG of BPH in the dsNlix-treated and control groups. The 10 species with the highest relative abundances were displayed, and the relative abundances of the remaining species were combined and grouped into Others; (B) Comparison of bacterial communities in samples treated with dsRNA via Bray–Curtis principal coordinate analysis; (C) Venn diagrams of the bacterial ASVs in CB, FB, and MG of BPH treated with dsRNA. The overlapping areas between blocks indicated the ASVs common to the corresponding groups, and the values in each block indicated the number of ASVs contained in the block; (D) LEfSe analysis based on ASV abundance in BC between dsGFP- and dsNlix-treated samples; (E) Comparative analysis of abundance in the genera of Acinetobacter, Enterococcus, candidate saccharibacteria, Arsenophous, and Ralstonia under different treatments. *, **, and *** indicate significant differences between dsGFP treatment and dsNlix treatment at p < 0.05, p < 0.01, and p < 0.001 levels, respectively. CB: copulatory bursa; FB: fat body; MG: midgut.