Mixed insect pest populations of Diaspididae species under control of oligonucleotide insecticides: 3'-end nucleotide matters.

Diaspididae are one of the most serious small herbivorous insects with piercing-sucking mouth parts and are major economic pests as they attack and destroy perennial ornamentals and food crops. Chemical control is the primary management approach for armored scale infestation. However, chemical insecticides do not possess selectivity in action and not always effective enough for the control of armored scale insects. Our previous work showed that green oligonucleotide insecticides (olinscides) are highly effective against armored and soft scale insects. Moreover, olinscides possess affordability, selectivity in action, fast biodegradability, and a low carbon footprint. Insect pest populations undergo microevolution and olinscides should take into account the problem of insecticide resistance. Using sequencing results, it was found that in the mixed populations of insect pests Dynaspidiotus britannicus Newstead and Aonidia lauri Bouche, predominates the population of A. lauri. Individuals of A. lauri comprised for 80% of individuals with the sequence 3'-ATC-GTT-GGC-AT-5' in the 28S rRNA site, and 20% of the population comprised D. britannicus individuals with the sequence 3'-ATC-GTC-GGT-AT-5'. We created olinscides Diasp80-11 (5'-ATG-CCA-ACG-AT-3') and Diasp20-11 (5'-ATA-CCG-ACG-AT-3') with perfect complementarity to each of the sequences. Mortality of insects on the 14th day comprised 98.19 ± 3.12% in Diasp80-11 group, 64.66 ± 0.67% in Diasp20-11 group (p < 0.05), and 3.77 ± 0.94% in the control group. Results indicate that for maximum insecticidal effect it is necessary to use an oligonucleotide insecticide that corresponds to the dominant species. Mortality in Diasp80-11 group was accompanied with significant decrease in target 28S rRNA concentration and was 8.44 ± 0.14 and 1.72 ± 0.36 times lower in comparison with control (p < 0.05) on the 10th and 14th days, respectively. We decided to make single nucleotide substitutions in Diasp20-11 olinscide to understand which nucleotide will play the most important role in insecticidal effect. We created three sequences with single nucleotide transversion substitutions at the 5'-end - Diasp20(5')-11 (A to T), 3'-end - Diasp20(3')-11 (T to A), and in the middle of the sequence - Diasp20(6)-11 (6th nitrogenous base of the sequence; G to C), respectively. As a result, mortality of mixed population of the field experiment decreased and comprised 53.89 ± 7.25% in Diasp20(5')-11 group, 40.68 ± 4.33% in Diasp20(6)-11 group, 35.74 ± 5.51% in Diasp20(3')-11 group, and 3.77 ± 0.94% in the control group on the 14th day. Thus, complementarity of the 3'-end nucleotide to target 28S rRNA was the most important for pronounced insecticidal effect (significance of complementarity of nucleotides for insecticidal effect: 5' nt < 6 nt < 3' nt). As was found in our previous research works, the most important rule to obtain maximum insecticidal effect is complete complementarity to the target rRNA sequence and maximum coverage of target sequence in insect pest populations. However, in this article we also show that the complementarity of 3'-end is a second important factor for insecticidal potential of olinscides. Also in this article we propose 2-step DNA containment mechanism of action of olinscides, recruiting RNase H. The data obtained indicate the selectivity of olinscides and at the same time provide a simple and flexible platform for the creation of effective plant protection products, based on antisense DNA oligonucleotides.

Perfect Complementarity Mechanism for Aphid Control: Oligonucleotide Insecticide Macsan-11 Selectively Causes High Mortality Rate for Macrosiphoniella sanborni Gillette.

Macrosiphoniella sanborni is a widespread pest of Chrysanthemum morifolium that causes significant damage to world floriculture. Chemical insecticides and biological methods of control have a number of disadvantages that can be improved by using oligonucleotide insecticides. In this article, we present, for the first time, the results of using oligonucleotide insecticides, for which the target sequence is an internal transcribed spacer (ITS) in a polycistronic rRNA transcript. The mortality of wingless aphid individuals after a Macsan-11 treatment was recorded at a level of 67.15 ± 3.32% 7 days after a single treatment with a solution at a concentration of 100 ng/µL and 97.38 ± 2.49% 7 days after a double treatment with a solution of the same concentration and a daily interval. The contact use of the control oligonucleotide (ACTG)2ACT-11. as well as the oligonucleotide insecticides Macsan-11(3') and Macsan-11(5') was not accompanied by insect mortality. Given the high variability in the internal transcribed spacer, which has proven to be a promising target for the action of oligonucleotide insecticides, it is possible to create selective preparations. This study showed the prospects of ribosomal insect pest genes as targets for the action of olinscides, and also demonstrated the high specificity of such insecticidal agents.

Icerya purchasi Maskell (Hemiptera: Monophlebidae) Control Using Low Carbon Footprint Oligonucleotide Insecticides.

Climate change creates favourable conditions for the growth of insect populations. Today, the world is seeing an increase in the number of insect pest infestations associated with a long-term increase in the average temperature of climatic systems. For example, local invasions of Icerya purchasi Maskell, a citrus pest recognized worldwide, have increased in size and number in recent years. Controlling this pest is complicated because not all chemical insecticides are effective, and their use is undesirable since citrus fruit is used for food and chemical agents cumulatively harm human health. In this article, we demonstrated for the first time the successful use of a short single-stranded fragment of the 28S ribosomal RNA gene called "oligoICER-11" to control cottony cushion scale, and we propose the use of green oligonucleotide insecticides with a low carbon footprint for large-scale implementation in agriculture and forestry. Using the contact oligonucleotide insecticide oligoICER-11 at a concentration of 100 ng/µL on I. purchasi larvae resulted in a mortality of 70.55 ± 0.77% within 10 days. Thus, climate change is driving the need in both agriculture and forestry for oligonucleotide insecticides (DNA insecticides, olinscides): safe, effective, affordable insecticides with a low carbon footprint and long operational life.

Oligonucleotide Insecticides for Green Agriculture: Regulatory Role of Contact DNA in Plant-Insect Interactions.

Insects vastly outnumber us in terms of species and total biomass, and are among the most efficient and voracious consumers of plants on the planet. As a result, to preserve crops, one of the primary tasks in agriculture has always been the need to control and reduce the number of insect pests. The current use of chemical insecticides leads to the accumulation of xenobiotics in ecosystems and a decreased number of species in those ecosystems, including insects. Sustainable development of human society is impossible without useful insects, so the control of insect pests must be effective and selective at the same time. In this article, we show for the first time a natural way to regulate the number of insect pests based on the use of extracellular double-stranded DNA secreted by the plant Pittosporum tobira. Using a principle similar to one found in nature, we show that the topical application of artificially synthesized short antisense oligonucleotide insecticides (olinscides, DNA insecticides) is an effective and selective way to control the insect Coccus hesperidum. Using contact oligonucleotide insecticide Coccus-11 at a concentration of 100 ng/µL on C. hesperidum larvae resulted in a mortality of 95.59 ± 1.63% within 12 days. Green oligonucleotide insecticides, created by nature and later discovered by humans, demonstrate a new method to control insect pests that is beneficial and safe for macromolecular insect pest management.