DNA insecticide developed from the Lymantria dispar 5.8S ribosomal RNA gene provides a novel biotechnology for plant protection

Abstract

Having observed how botanicals and other natural compounds are used by nature to control pests in the environment, we began investigating natural polymers, DNA and RNA, as promising tools for insect pest management. Over the last decade, unmodified short antisense DNA oligonucleotides have shown a clear potential for use as insecticides. Our research has concentrated mainly on Lymantria dispar larvae using an antisense oligoRING sequence from its inhibitor-of-apoptosis gene. In this article, we propose a novel biotechnology to protect plants from insect pests using DNA insecticide with improved insecticidal activity based on a new antisense oligoRIBO-11 sequence from the 5.8S ribosomal RNA gene. This investigational oligoRIBO-11 insecticide causes higher mortality among both L. dispar larvae grown in the lab and those collected from the forest; in addition, it is more affordable and faster acting, which makes it a prospective candidate for use in the development of a ready-to-use preparation.

Figure 1

The effect of oligoRIBO-11on the mortality of gypsy moth larvae grown under lab conditions and resistant to oligoRING insecticide: (A) 3 days after the treatment; (B) 6 days after the treatment. *Indicates results with a significant difference compared with those of the Control (p < 0.01). SE (standard errors) are given for five replicates.

Figure 2

Electrophoregram (1.8% agarose gel) representing the activity of intracellular nucleases of L. dispar (A) and Q. pubescens (B) after 0.3, 1, and 24 hours at 27 °C: 1 – control (10 μL of oligoRIBO-11 at a concentration of 150 pmol/μL); 2 – tissue homogenate (1.5 mg of biomass per 10 μL of distilled water) + 10 μL of oligoRIBO-11 at a concentration of 150 pmol/μL; 3 – control (10 μL of oligoRING at a concentration of 5 pmol/μL); 4 – tissue homogenate (1.5 mg of biomass per 10 μL of distilled water) + 10 μL of oligoRING at a concentration of 5 pmol/μL; 5 – pure tissue homogenate (1.5 mg of biomass per 10 μL of distilled water).

Figure 3

Relative concentration of 5.8S ribosomal RNA in L. dispar larvae 6 (A) and 12 (B) days after treatment with the oligoDNAs. Data represent the means and standard errors of ribosomal RNA concentrations for 3 replicates relative to the control (water-treated) group. *Indicates a significant difference between the data for the oligoRIBO-11 group and those for the control (water-treated) group when p < 0.01.

Figure 4

Images obtained via light microscopy examination of the histological slides of L. dispar larvae insect integument (I), muscle bundle (M), and midgut (G). I1, M1, G1 — Control sample (water-treated control):a — uniform distribution of heterochromatin, well expressed nucleoli; b — ordered muscle fibers with well-defined transverse striation; e — goblet cells; f — high cilia of epithelial cells; g — intercuticular space; I2, M2, G2 — oligoRIBO-11-treated larvae showing pronounced dystrophic destruction of tissues: a — condensation of heterochromatin around the periphery of the nucleus, absence of nucleolus; b — loss of transverse striation; c — defibration of muscle bundles; d — diffuse protein deposition; e — a large number of goblet cells; f — low cilia of epithelial cells.