Knockdown of Methoprene-Tolerant Arrests Ovarian Development in the Sogatella furcifera (Hemiptera: Delphacidae)

Abstract

Juvenile hormone (JH) is responsible for repressing larval metamorphosis and inducing vitellogenesis and egg production in insects. Methoprene-tolerant (Met) is known to be an intracellular receptor and transducer of JH. We examined the role of Met in ovarian development in the rice pest Sogatella furcifera (Horváth). We first cloned and sequenced S. furcifera Met (SfMet). The SfMet protein belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) family with a bHLH domain and two PAS domains (PAS-A and PAS-B). SfMet was expressed in all developmental stages and tissues but was most highly expressed in the ovaries of adult females. Furthermore, RNA interference (RNAi) mediated silencing of SfMet substantially reduced the expression of SfVg, decreased yolk protein deposition and blocked oocyte maturation and ovarian development. These results demonstrate that SfMet plays a key role in female reproduction in S. furcifera and suggest that targeting this gene could be an effective way of controlling this pest.

Keywords: Sogatella furcifera; juvenile hormone; methoprene-tolerant; vitellogenin.

Fig. 1.

Identification and sequence analysis of Sogatella furcifera Methoprene-tolerant (SfMet). (A) Gene structure of SfMet predicted by InterPro tool. (B) Alignment of amino acid sequences of SfMet with those of other hemipteran species; Nilaparvata lugens (NlMet, ALT45968.1), Myzus persicae (MpMet, AYI50057.1), Planococcus kraunhiae (PkMet, BAU79435.1), and Aphis gossypii (AgoMet, ANZ54966.1). The bHLH, PAS-A and PAS-B domains are indicated by the black line.

Fig. 2.

Neighbor-joining phylogenetic tree of Sogatella furcifera SfMet and those in other insects, using Portunus trituberculatus (PtMet) as the outgroup. Numbers at the nodes of branches indicate bootstrap values (%) based on 1,000 replicates. The number in parenthesis are the GenBank accession numbers for each species’ gene. The blue triangle indicates the protein sequence of SfMet. DmMetA and DmMetB, Drosophila melanogaster; GmmMet, Glossina morsitans morsitans; SmMet, Sitodiplosis mosellana; Cppmet, Culex pipiens pipiens; AsMet, Anopheles sinensis; BmMet, Bombyx mori; DsMet, Dendrolimus spectabilis; PiMet, Plodia interpunctella; HaMet, Helicoverpa armigera; MseMet, Mythimna separate; TcMet, Tribolium castaneum; CbMet, Colaphellus bowringi; LdMet, Leptinotarsa decemlineata; MsMet, Monochamus saltuarius; MaMet and MalMet, Monochamus alternatus; DpMet, Diploptera punctate; ZnMet, Zootermopsis nevadensis; NlMet, Nilaparvata lugens; PkMet, Planococcus kraunhiae; MpMet, Myzus persicae; AgoMet, Aphis gossypii.

Fig. 3.

Tissues and temporal expression profiles of SfMet. (A) Relative expression levels of SfMet in the first to fifth nymphal instars (N1, N2, N3, N4, N5), female (F) and male adults (M). (B) Relative expression levels of SfMet in females at different times after emergence. HAE, hours after emergence. (C) Relative expression levels of SfMet in various tissues of females. Mg, midgut; Ov, ovary; Hd, head; Th, thorax; Fb, fat body. Bars indicate the mean (±SE) of three biological replicates. Different letters above bars represent significant differences (ANOVA followed by Tukey’s test, P < 0.05).

Fig. 4.

Functional analysis of SfMet in the ovarian development of female Sogatella furcifera. Expression levels of SfMet (A), SfKr-h1 (B), and SfVg (C) in whole bodies of females 48, 72, and 96 h after injected with dsMet and dsEGFP. (D) Effect of SfMet RNAi on ovarian development with dsEGFP as a control. Ovaries were dissected 132 h post-injection. Scale bar, 0.5 mm. Asterisks represent values statistically different from the EGFP dsRNA group (t-test: *P < 0.05, **P < 0.01).