Transposable elements (TEs) increase the frequency of spontaneous mutations in the genome and are capable of altering the gene structure and expression. TE activities and genomic positions are therefore important to study. A combination of two sequencing methods proved advantageous in searching for TE insertions and chromosomal rearrangements, i.e., full-genome nanopore sequencing allowed detection of TE insertions, and transcriptome sequencing on the Illumina platform evaluated their effects on gene expression. Genome sequencing data were obtained for Drosophila melanogaster strains with the SS (w1, flamenco mutant) and MS (w1, flamenco mutant, active gypsy copy) flamenco phenotypes. The wild-type laboratory strain D32 was used as a control. TE insertions and deletions in euchromatin genome regions and gene introns were found in the mutant and wild-type strains as compared with a reference genome (NCBI GCF_000001215.4). The genomes under study were searched for insertions and deletions in RNA interference system genes and genes differentially expressed in the SS and MS strains. TE insertions were detected in various regions of the AGO3, CG17147, Su(var)3-3, Gasz, CG43348, moody, and CG17752 genes. A change in TE position did not correlate with a decrease or an increase in gene transcription in most genes. A chromosomal rearrangement affecting the 3'-untranslated region was observed in the vig gene. A de novo genome assembly was conducted for the MS strain based on the long-read sequencing data. Higher expression of CR45822 and pst in the SS and MS strains was found to be due to a triplication rather than to changes in regulatory sequences or a TE insertion.

This study investigated the effect of knockout of six Hsp70 genes (orthologues of the mammalian genes Hspa1a, Hspa1b, Hspa2, and Hspa8) on age-related changes in gene expression in the legs of Drosophila melanogaster, which contain predominantly skeletal muscle bundles. For this, the leg transcriptomic profile was examined in males of the w(1118) control strain and the Hsp70(-) strain on the 7th, 23rd and 47th days of life. In w(1118) flies, an age-related decrease in the locomotion (climbing) speed (a marker of functional state and endurance) was accompanied by a pronounced change in the transcriptomic profile of the leg skeletal muscles, which is conservative in nature. In Hsp70(-) flies, the median lifespan was shorter and the locomotion speed was significantly lower compared to the control; at the same time, complex changes in the age-related dynamics of the skeletal muscle transcriptome were observed. Mass spectrometry-based quantitative proteomics showed that 47-day-old Hsp70(-) flies, compared with w(1118) flies, demonstrated multidirectional changes in the contents of key enzymes of glucose metabolism and fat oxidation (glycolysis, pentose phosphate pathway, Krebs cycle, beta-oxidation, and oxidative phosphorylation). Such dysregulation may be associated with a compensatory increase in the expression of other genes encoding chaperones (small Hsp, Hsp40, 60, and 70), which regulate specific sets of target proteins. Taken together, our data show that knockout of six Hsp70 genes slightly reduced the median lifespan of flies, but significantly reduced the locomotion speed, which may be associated with complex changes in the transcriptome of the leg skeletal muscles and with multidirectional changes in the contents of key enzymes of energy metabolism.

Drosophila melanogaster is a common genetic object for research of RNA interference pathways and regulation of mobile elements. At present, taking part in control of retrotransposon expression, the system of piRNA interference is well studied in ovary tissues. It is strongly believed that, in D. melanogaster, the system of piRNA interference is used for retrotransposon suppression only in the gonads, and two distinct pathways of piRNA biogenesis exist. Both mechanisms use transcripts of piRNA clusters (accumulations of truncated and defective copies of mobile elements): from uni-strand clusters in the first case and from dual-strand clusters in the second case, transcribed from one or both DNA chains, respectively. It is well known that proper function of dual-strand clusters depends on the gene rhino, while uni-strand clusters are transcribed and then spliced independently of rhino. In this paper, we show that rhino participates in splicing of uni-strand cluster flamenco transcripts. Moreover, the system of piRNA interference is necessary for regulation of several retrotransposons not only in the gonads but also in other organs.

To study the causes of impaired control of the activity of mobile genetic elements in the strains with the flamenco phenotype SS (w, flamenco mutant) and MS (w, flamenco mutant, active copy of gypsy), sequencing of these transcriptomes was performed. The D32 strain was used as control (laboratory wild type strain). An algorithm was developed for the search for amino acid substitutions in high-throughput RNA sequencing data using a triplet code for analysis. With the help of this algorithm, seven nonsense mutations were detected. The allele-specific PCR method confirmed the presence of five of the seven nonsense mutations found in silico. However, the detected nonsense mutations are not associated with the flamenco phenotype. A search for mutations in 89 genes of the RNA interference system in the SS and MS strains relative to the reference BDGP6 genome and the wild type D32 strain was carried out. No deletions, insertions, nonsense codons, and other disorders that can unambiguously lead to a change in the function of the gene are detected. To identify genes with specific expression for the strains with the flamenco phenotype, the transcriptomes of the SS and MS strains were compared with the control strains D-32, OregonR, and w1118. A set of 25 genes with differential expression was identified, among which two genes, sosie and CR45822, significantly changed the expression in the SS and MS strains. Both genes, directly or indirectly, are involved in oogenesis. Thus, the expression of the sosie and CR45822 genes can be used as a marker of the flamenco phenotype in the SS and MS strains.

The flamenco locus is one of the main components of the piRNA pathway of regulation of mobile genetic elements (MGEs) in Drosophila melanogaster. Mutations at this locus lead to an increase in the transposition activity of MGEs and, as a result, to genetic instability. In this paper, the fertility of a genetically unstable MS strain obtained more than 25 years ago and characterized by a mutation in the flamenco locus and the presence of a functionally active copy of gypsy retrotransposon was investigated. Complex violations of the ovarian morphology were revealed in the MS strain in females: defects in the follicular layer and ring channels, as well as degradation of trophocytes, which in turn led to a decrease in reproductive abilities. Analysis of the MS strain transcriptome showed a decrease in the expression level of 40 genes encoding chorionic proteins and expression specificity at different stages of follicle development. In the F1 and F2 hybrid females from the crosses of MS females with wild type males, restoration of reproductive abilities was observed, despite the fact that half of the F2 females had the flamenco genotype and genetic instability caused by transposition of gypsy (according to the ovo^(D) test). Moreover, the frequency of gypsy transposition in the hybrid F2 females with the flamenco genotype doubled in comparison with the MS strain females. Thus, the MS strain had acquired partial suppression of the flamenco phenotype and accumulated several recessive mutations in the genes that control oogenesis after cultivation for over 25 years.