New Gall-Forming Insect Model, Smicronyx madaranus: Critical Stages for Gall Formation, Phylogeny, and Effectiveness of Gene Functional Analysis

Abstract

The molecular mechanisms underlying insect gall formation remain unclear. A major reason for the inability to identify the responsible genes is that only a few systems can be experimentally validated in the laboratory. To overcome these problems, we established a new galling insect model, Smicronyx madaranus. Our manipulation experiments using nail polish sealing and insecticide treatment revealed an age-dependent change in gall formation by S. madaranus; adult females and larvae are responsible for gall induction and enlargement, respectively. Furthermore, it has been suggested that substances released during oviposition and larval feeding are involved in each process. Phylogenetic analysis showed that gall-forming weevils, including S. madaranus, belong to two distinct lineages that utilize different host plants. This may indicate that gall-forming traits evolved independently in these Smicronyx lineages. The efficacy of RNA interference (RNAi) in S. madaranus was confirmed by targeting the multicopper oxidase 2 gene. It is expected that the mechanisms of gall formation will be elucidated by a comprehensive functional analysis of candidate genes using RNAi and the S. madaranus galling system in the near future.

Keywords: RNA interference; field dodder; insect gall; manipulation of galling insect; phylogeny of the genus Smicronyx.

Figure 1

An initial gall (A) and an egg in the initial gall (B).

Figure 2

Effects of insecticides on weevil larvae and gall growth. (A) Larval status within treated galls. White background, alive; dark grey, undetected; black, dead. Numbers in each column indicate the number of individuals in each state. Different letters (a and b) above columns indicate statistically significant differences (p < 0.05, Fisher’s exact test with Bonferroni correction). (B) Effects of insecticides on gall growth. Means ± standard deviations are shown. Sample size is given in parentheses. Different letters (a and b) indicate statistically significant differences on the same day (p < 0.05, pairwise Wilcoxon rank sum test with Bonferroni correction). ns means no significant difference.

Figure 3

Molecular phylogeny and the gall-forming trait in the genus Smicronyx. The topology and branch lengths shown were obtained using Bayesian inference methods. The scale bar indicates 0.5 substitutions per site. The Bayesian posterior probabilities/the maximum likelihood bootstrap value/the maximum parsimony bootstrap value are shown below the branches to indicate the level of support for each node (only values ≥ 50% are shown), respectively. An asterisk (*) indicates a node that was not supported by ML or MP analyses. Sequences obtained in this study are in bold type. Labels of the sequences correspond to the codes in Table S1. Filled circles in the taxa represent the gall-forming species. Sequence accession numbers are in brackets.

Figure 4

SmMCO2 expression and body color melanization. (A) Variation in SmMCO2 expression in different growth stages. Box plots show the distribution of the normalized SmMCO2 expression level. Different letters (a and b) indicate statistically significant differences on the same day (p < 0.05, pairwise Wilcoxon rank sum test with Bonferroni correction). Growth stages (P2 to A3) correspond to Figure S4. (B) Effect of RNAi on the normalized SmMCO2 expression level. The numbers in parentheses are sample sizes. The median expression level of the control group was designated as 1.0. Statistically significant differences were evaluated using Welch’s t-test. In (A,B), y axes indicate SmMCO2 gene copies/an EF1α gene copy. Each open circle indicates the value per sample. (C) Effect of RNAi for SmMCO2 on body color. Bars, 1 mm.