Efficacy of In Vivo Electroporation on the Delivery of Molecular Agents into Aphid (Hemiptera: Aphididae) Ovarioles

Abstract

While the wealth of genomic data presently available is increasing rapidly, the advancement of functional genomics technologies for the large majority of these organisms has lagged behind. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 system is an emerging gene-editing technology derived from a bacterial adaptive immune system that has proven highly effective in multiple model systems. Here, the CRISPR/Cas9 system was delivered into the ovarioles of the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphididae), with a new delivery method utilizing in vivo electroporation. To validate gene-editing, a target sequence within the marker tor pigment gene was chosen, and gene-editing was predicted to result in white pigmentation in the offspring of treated adult aphids. Adult aphids (10-d old) were injected with the tor single guide RNA and Cas9 complex and subsequently subjected to electroporation. Adult aphids were given 4 d to produce viviparous offspring. After offspring developed for 6 d, DNA was extracted and sequenced to validate if CRISPR/Cas9-directed gene editing occurred. A survival rate over 70% was found in treated adult aphids. A distinct white pigmentation was found in 2.5% of aphids; however, gene-editing within the target sequence was not found in any of the individuals screened. Presence of white aphids without gene-editing suggests other mechanisms may have influenced pigmentation. High survival rates in experimental treatments demonstrate the robustness of this new technique, and further refinement of this technique may prove it as an effective functional genomics tool for viviparous insects and/or gene editing at a somatic level.

Fig. 1.

Image depicting the electroporation station (a) and the electroporation device (b) used to introduce the CRISPR/Cas9 complex into aphid offspring in vivo.

Fig. 2.

Adult aphids were microinjected and subsequently electroporated. Survival of each treatment was monitored for 4 d, and mortality data between treatments were statistically analyzed with Kaplan–Meier mortality curves.

Fig. 3.

Bar graphs representing total aphid offspring per day for 4 d following microinjection and electroporation trials. Graphs represent aphid offspring from the Trial 1: CRISPR treatment, the Trial 2: CRISPR treatment, and the three control treatments: Carrier RNA, Carrier RNA + Cas9, and Cas9.

Fig. 4.

Image depicting the comparison between the wildtype pink phenotype and white phenotype of offspring from the Trial 1: CRISPR treatment. Sick/stressed aphids are shown in comparison with the white phenotype in trials.