Task 1: Candidate gene selection

Realization: We have completed all the activities in this task and used the results in Task 2. The genome of the Colorado potato beetle, essential for gene selection, was published in a scientific journal (https://doi.org/10.1038/s41598-018-20154-1).

Task 2: Selection of target genes based on expression

Realization: We have completed all the activities in this task and used the results in Task 3.

Task 3: dsRNA production and delivery method establishment

Realization: We have completed all the activities in this task and used the delivery method of dsRNA in Task 4.

Task 4: Evaluation of dsRNAs’ insecticidal potential

Realization: We have completed activities related to the planned laboratory testing, collection, and data processing. The manuscript preprint was uploaded to bioRxiv and published in a scientific journal (https://doi.org/10.3389/fpls.2020.01250).

Hypothesis 1 (suppression of selected candidate genes will result in increased mortality, abnormal development or decreased growth of CPB larvae) was confirmed only for the selected target gene mesh and rejected for the JHBP gene. For the third selected target gene, the hypothesis could not be confirmed or rejected, since we did not succeed in effectively silencing the expression of the alas gene using the designed dsRNA.

Task 5: dsRNA-induced effects on gut tissue transcriptome using RNA-Seq

Realization: We have completed activities related to the planned laboratory testing, preparation of bioinformatic works, generation, and data processing. The manuscript is in preparation.

Hypothesis 2 (suppression of genes whose biological function has not yet been confirmed will lead to changes at the level of transcriptome and phenotype associated with the function of the gene) has been changed. We have decided to study the effect of non-specific dsRNA on the CPB gut transcriptome instead of the influence of insecticidal dsRNA. The results of this analysis show that the gut responds with a change in expression of about 100 genes. dsRNA thus does not cause major reprogramming of the gut transcriptome. Comparison with the response of Tribolium beetle and honey bee to non-specific dsRNA response does not suggest that insects would have a general and preserved response to non-specific dsRNA at the level of gene expression.

Task 6: dsRNA-induced effects on gut metagenome

Realization: We have completed activities related to the planned laboratory testing, preparation of bioinformatic works, generation, and data processing. The time delay from the original plan was due to difficulties in establishing effective isolation of metagenomic DNA from the content of the beetle’s gastrointestinal tract.

Hypothesis 3 (RNAi insecticide will change the composition of intestinal bacterial clades and accelerate or reduce the abundance of particular clades) has been confirmed by metagenomic analysis. These results will have to be confirmed by qPCR and expanded to samples from the remaining experiments.