Development of RNAi Methods for the Mormon Cricket, Anabrus simplex (Orthoptera: Tettigoniidae)

Abstract

Mormon crickets are a major rangeland pest in the western United States and are currently managed by targeted applications of non-specific chemical insecticides, which can potentially have negative effects on the environment. In this study, we took the first steps toward developing RNAi methods for Mormon crickets as a potential alternative to traditional broad-spectrum insecticides. To design an effective RNAi-based insecticide, we first generated a de novo transcriptome for the Mormon cricket and developed dsRNAs that could silence the expression of seven housekeeping genes. We then characterized the RNAi efficiencies and time-course of knockdown using these dsRNAs, and assessed their ability to induce mortality. We have demonstrated that it is possible to elicit RNAi responses in the Mormon cricket by injection, but knockdown efficiencies and the time course of RNAi response varied according to target genes and tissue types. We also show that one of the reasons for the poor knockdown efficiencies could be the presence of dsRNA-degrading enzymes in the hemolymph. RNAi silencing is possible in Mormon cricket, but more work needs to be done before it can be effectively used as a population management method.

Keywords: Mormon cricket; Orthoptera; RNAi; dsRNA.

Figure 1

(A) Mormon crickets cannibalizing each other during a population outbreak in Idaho in 2020; (B) A population outbreak of Mormon crickets along a major highway in Idaho in 2019. (Photo credits: (A): H.S.; (B): D.A.W.).

Figure 2

Knockdown efficiency assay in different tissues. (A) Head tissue, (B) Thorax tissue. Each gene tested had a sample size of at least 5. Error bars indicate the standard error of the mean for each control (GFP) and target gene. Data were analyzed using the ΔΔCt method. The statistical significance of differences was analyzed with the Student’s t-test or Wilcoxon test. Significant differences between the control (GFP) and target gene are indicated by asterisks (*: p < 0.05; **: p < 0.005; ****: p < 0.0001).

Figure 3

Time course assay for EF1, GAPDH, Hsp70, and RpL5 target genes. Each gene per day had at least a sample size of 3. Error bars indicate the standard error of the mean for each control (GFP) and target gene. Data were analyzed using the ΔΔCt method. The arrow indicates the booster injection. The statistical significance of differences was analyzed with the Student’s t-test or Wilcoxon test. Significant differences between the control (GFP) and target gene are indicated by asterisks (*: p < 0.05; **: p < 0.005; ***: p < 0.0001; ****: p < 0.00001).

Figure 4

dsRNA degradation assay for hemolymph. dsRNA completely degrades in undiluted hemolymph, strongly suggestive of high dsRNase activity.