Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 Plays an Important Role in the Development and Reproduction of Nilaparvata lugens

Abstract

The brown planthopper, Nilaparvata lugens, is a difficult-to-control insect pest affecting rice yields in Asia. As a structural component of the inter-alpha-trypsin inhibitor (ITI), the inter-alpha-trypsin inhibitor heavy chain (ITIH) has been reported to be involved in various inflammatory or malignant disorders, ovarian development, and ovulation. To reveal the function of ITIH4 in N. lugens, the gene encoding N. lugens ITIH4 (NlITIH4) was cloned and characterized. NlITIH4 contains a signal peptide, a vault protein inter-alpha-trypsin domain, and a von Willebrand factor type A domain. qPCR analysis showed that NlITIH4 was expressed at all developmental stages and in all tissues (fat body, ovary, and gut), with the highest expression in the fat body. Double stranded NlITIH4 (dsNlITIH4) injection clearly led to an RNAi-mediated inhibition of the expression of NlITIH4 and resulted in reduced survival, delayed ovarian development, and reduced egg production and egg hatching. These results indicate that NlITIH4 plays an important role in the development and reproduction of N. lugens.

Keywords: Nilaparvata lugens; RNAi; development; inter-alpha-trypsin inhibitor heavy chain 4; reproduction.

Figure 1

Nucleotide and encoded amino acid sequences of NlITIH4 ORF. The signal peptide is indicated by a box. The vault protein inter-alpha-trypsin domain is underlined with a dotted line, and the von Willebrand factor type A domain is underlined with a solid line.

Figure 1

Nucleotide and encoded amino acid sequences of NlITIH4 ORF. The signal peptide is indicated by a box. The vault protein inter-alpha-trypsin domain is underlined with a dotted line, and the von Willebrand factor type A domain is underlined with a solid line.

Figure 2

A phylogenetic analysis of ITIH4 based on amino acid sequence. Sequences were retrieved from the NCBI protein database. These sequences included the mammal ITIH4s from Homo sapiens (NP_001159921.1), Sus scrofa (XP_020924178.1), Bos taurus (XP_024838404.1), Mus pahari (XP_029397816.1), and all available insect ITIH4s from Aricia agestis (XP_041974567.1), Bombyx mandarina (XP_028042060.1), Manduca sexta (XP_030027688.2), B. mori (XP_021202615.2), Sitophilus oryzae (XP_030759847.1), Asbolus verrucosus (RZB41582.1), Tribolium madens (XP_044254677.1), Leptinotarsa decemlineata (XP_023015426.1), Photinus pyralis (XP_031349540.1), Homalodisca vitripennis (XP_046679415.1), Rhopalosiphum maidis (XP_026822307.1), Aphis craccivora (KAF0720343.1, Melanaphis sacchari (XP_025205475.1), Myzus persicae (XP_022172078.1), Nilaparvata lugens (QCI55997.1), Cryptotermes secundus (XP_023714342.1), Zootermopsis nevadensis (XP_021930042.1), Blattella germanica (PSN48673.1), and Thrips palmi (XP_034239235.1). The phylogenetic tree was constructed with the neighbor-joining method (bootstrap = 1000) using MEGA 10.0.

Figure 3

Expression patterns of NlITIH4 in different developmental stages and tissues. (A) Expression of NlITIH4 in different developmental stages, including 1st to 5th instar nymphs, one- to five-days old brachypterous adult females (B1D to B5D) and macropterous adult females (M1D to M5D). (B) Expression of NlITIH4 in ovary, gut, and fat body of female adults. Data are expressed as mean ± SE. Significant differences: * p < 0.05, ** p < 0.01. Different lower-case letters above the bars indicate significant differences for which p < 0.05.

Figure 4

Effects of dsNlITIH4 injection on the growth and reproduction of N. lugens: (A) the efficiency of RNAi by dsNlITIH4 injection at 1 to 5 days post injection of dsRNA (d.p.i.); (B) effect of dsNlITIH4 injection on the survival of N. lugens at 1 to 4 d.p.i.; (C) effect of dsNlITIH4 injection on the number and hatching rate of eggs; and (D) effect of dsNlITIH4 injection on the vitellogenesis of ovarioles. EV (early vitellogenesis prophase), MV (middle vitellogenesis), LV (late vitellogenesis), and TV (terminal vitellogenesis) at 1 to 4 d.p.i. Data are expressed as mean ± SE. Significant differences: * p < 0.05, ** p < 0.01.

Figure 5

Effects of dsRNA injection on ovarian development at 1 to 5 days post injection of dsRNA (d.p.i.). At 1 to 2 d.p.i., there was no visible difference between the ovaries from dsNlITIH4-treated and dsGFP-treated groups, but notable differences are present at 3 to 5 d.p.i. Scale bar: 400 μm.

Figure 6

Effects of RNAi on the transcription levels of Vg (A), VgR (B), MeT (C) at 1 to 4 days post injection of dsRNA (d.p.i.). Data are expressed as mean ± SE. Significant difference: different lower-case letters above the bars indicate significant differences for which p < 0.05.