Figure 1.
The time course of behavioral phase change in fourth-instar nymphs of Locusta migratoria.
(A) Isolation of gregarious locusts (IG). (B) Crowding of solitarious locusts (CS). Pgreg, probabilistic metric of gregariousness. Arrows indicate median Pgreg values.
Table 1.
Variables retained in binary logistic model.
Figure 2.
Candidate genes involved in locust phase change.
(A) The numbers of up- and down-regulated genes during the time course of IG or CS. Numbers following IG or CS indicate hours of treatment. (B) Venn diagram indicating 453 mutually differentially expressed genes during the time courses of IG and CS. These 453 genes were categorized into 8 clusters by hierarchical clustering using a complete linkage algorithm. Cluster A and cluster H exhibit consistent changes over the time course of treatments and display a negative correlation of expression trends (Pearson correlation, r = −0.896, p<0.0001). CSP and takeout genes are highlighted in these two categories (red arrows for CSP and blue arrows for takeout). Horizontal stripes represent genes, and columns represent experimental treatments. Logarithmic fold change of treatment vs. control (gregarious during IG or solitarious during CS) is shown in the heat map using red and green colors to indicate up- and down-regulation, respectively.
Table 2.
Top ten gene categories enriched in InterPro annotations during the time course of IG or CS.
Figure 3.
The tissue-specific and time course expression of LmigCSP3 and LmigTO1 genes in fourth-instar nymphs.
(A) RNA relative expression levels of LmigCSP3 (Left) and LmigTO1 (Right) in six selected tissues. A, antenna; L, labial palp; B, brain; W, wing; HL, hind leg; FB, fat body. Means labeled with the same letter within each treatment are not significantly different and error bars represent SEM. *, p<0.05; **, p<0.01. (B) RNA relative expression levels of LmigCSP3 and LmigTO1 in antennal tissues during the time course of IG (Left) or CS (Right).
Figure 4.
The effects of RNAi knockdown of LmigCSP3 and LmigTO1 genes on their expression levels.
(A) The expression levels of LmigCSP3 in gregarious (Student's t-test, t = 5.308, 5.229, p = 0.006, 0.006) or solitarious (Student's t-test, t = 10.880, 12.731, p<0.0001, 0.0001) fourth-instar nymphs after dsLmigCSP3 or dsLmigCSP3&TO1 injection, respectively (B) The expression levels of LmigTO1 in gregarious (Student's t-test, t = 4.825, 6.852, p = 0.005, 0.002) or solitarious (Student's t-test, t = 10.401, 13.366, p<0.0001, 0.0001) fourth-instar nymphs after dsLmigTO1 or dsLmigCSP3&TO1 injection, respectively (C) Effects of dsLmigCSP3 injection on RNA relative expression levels of five other CSP genes.(Student's t-test, t = 4.353, 3.496, 4.347, 6.315, 3.271, p = 0.012, 0.025, 0.007, 0.001, 0.017, respectively). In all groups, treatment (inj-dsGFP, inj-dsLmigCSP3, inj-dsLmigTO1 or inj-dsLmigCSP3&TO1) compared with the corresponding non-injected controls. *, p<0.05; **, p<0.01; n.s., not significant.
Figure 5.
Behavioral phenotype of locust nymphs in arena after RNAi knockdown of LmigCSP3/LmigTO1.
(A) Changes of attraction behavior induced by LmigCSP3, LmigTO1 or LmigCSP3&TO1 knockdown in gregarious nymphs. (B) Effects of LmigCSP3, LmigTO1 or LmigCSP3&TO1 knockdown on total distance moved in gregarious nymphs. (C) Effects of LmigCSP3, LmigTO1 or LmigCSP3&TO1 knockdown on total duration of movement in gregarious nymphs. (D) Change of repulsion behavior induced by LmigCSP3, LmigTO1 or LmigCSP3&TO1 knockdown in solitarious nymphs. (E) Effects of LmigCSP3, LmigTO1 or LmigCSP3&TO1 knockdown on total distance moved in solitarious nymphs. (F) Effects of LmigCSP3, LmigTO1 or LmigCSP3&TO1 knockdown on total duration of movement in solitarious nymphs. In all groups, treatment (inj-dsGFP, inj-dsLmigCSP3, inj-dsLmigTO1 or inj-LmigCSP3&TO1) compared with non-injected controls. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant.
Figure 6.
Dual-choice of locust nymphs in Y-tube olfactometer after RNAi knockdown of LmigCSP3 or LmigTO1.
(A) Choice behavior of gregarious locusts injected by dsLmigCSP3 or dsLmigTO1. (B) Choice behavior of solitarious locusts injected by dsLmigCSP3 or dsLmigTO1. Volatiles represent volatiles from 30 gregarious fourth-instar nymphs. Marks (*, ** and n.s.) inside white stripe indicate the significance of Chi square test for comparing individual numbers in each arm. Marks (** and n.s.) outside the stripe indicate the significance of treatment (inj-dsGFP, inj-dsLmigCSP3 or inj-dsLmigTO1) compared with non-injected controls. n, individual numbers used in significance test. N.C., no choice, excluded in significance test. *, p<0.05; **, p<0.01; n.s., not significant.