Chromatin-Remodelling ATPases ISWI and BRM Are Essential for Reproduction in the Destructive Pest Tuta absoluta

Abstract

The tomato leaf miner (Tuta absoluta) is one of the top 20 plant pests worldwide. We cloned and identified the chromatin-remodelling ATPase genes ISWI and BRM by RACE and bioinformatic analysis, respectively; used RT-qPCR to examine their expression patterns during different life cycle stages; and elucidated their roles in insect reproduction using double-stranded RNA injections. The full-length cDNA of TaISWI was 3428 bp and it encoded a 1025-aa polypeptide. The partial-length cDNA of TaBRM was 3457 bp and it encoded a 1030-aa polypeptide. TaISWI and TaBRM were upregulated at the egg stage. Injection of TaISWI or TaBRM dsRNA at the late pupa stage significantly inhibited adult ovary development and reduced fecundity, hatchability, and longevity in the adult females. To the best of our knowledge, the present study was the first to perform molecular characterisations of two chromatin-remodelling ATPase genes and clarify their roles in T. absoluta fecundity. Chromatin-remodelling ATPases are potential RNAi targets for the control of T. absoluta and other insect pests. The present study was also the first to demonstrate the feasibility of reproductive inhibitory RNAi as a putative approach for the suppression of T. absoluta and other Lepidopteran insect populations.

Keywords: RNA interference; Tuta absoluta; bromo; fecundity; imitation SWItch.

Figure 1

Phylogenetic tree based on known ISWI (A) and BRM (B) aa sequences. It was constructed in MEGA7.0 using ML method based on the Whelan Goldman (WAG) model with 1000 bootstrap replications. Lepidopterais depicted in vermilion; Hymenopterain in antique white; Coleopterain in yellow green; Diptera in sky blue; Hemipterain in cornflower blue. Table S1 lists ISWI and BRM protein sequence accession numbers.

Figure 2

Relative to RpL5, TaISWI (A) and TaBRM (B) expression levels at different life cycle stages. Relative expression levels are means ± SEM. Bars with different lowercase letters indicate significant differences between treatment means at p < 0.05.

Figure 3

Effects of double-stranded RNA (dsRNA) treatment on TaISWI (A) and TaBRM (B) mRNA expression. Controls consisted of uninjected female pupae (CK) and those injected with water or EGFP dsRNA. Expression levels are means ± SEM. Bars with different lowercase letters indicate significant differences between treatment means at p < 0.05.

Figure 4

Effects of dsISWI and dsBRM on Tuta absoluta ovary development. (A) Ovaries of uninjected 4 d females. (B) Abnormal ovaries of 4 d females injected with dsISWI. (C) Abnormal ovaries of 4 d females injected with dsBRM. The red arrow represents the mature oocytes. The ovaries were photographed under an Olympus stereomicroscope (SZX16; Olympus, Tokyo, Japan). See more pictures in Figure S5.

Figure 5

Effects of dsISWI and dsBRM on Tuta absoluta oviposition, hatching, and longevity. (A) Average number of eggs laid per female at 1–4 d, 5–8 d, and >8 d. (B) Total oviposition per female. (C) Percentage of eggs hatched at 1–4 d, 5–8 d, and >8 d. (D) Total egg hatchability. (E) Average longevity of injected F0 females. Controls consisted of uninjected female pupae (CK) and those injected with water or EGFP dsRNA. Data are means ± SEM. Bars with different lowercase letters indicate significant differences between treatment means at p < 0.05.