Involvement of bacteria in the development of fungal infections in the Colorado potato beetle.

Entomopathogenic fungi may interact with insects' symbiotic bacteria during infection. We hypothesized that topical infection with Beauveria bassiana may alter the microbiota of the Colorado potato beetle (CPB) and that these modifications may alter the course of mycoses. We used a model with two concentrations of conidia: (1) high concentration that causes rapid (acute) pathogenesis with fast mortality followed by bacterial decomposition of insects; (2) lower concentration that leads to prolonged pathogenesis ending in conidiation on cadavers. The fungal infections increased loads of enterobacteria and bacilli on the cuticle surface and in hemolymph and midgut, and the greatest increase was detected during the acute mycosis. By contrast, stronger activation of IMD and JAK-STAT signaling pathways in integuments and fat body was observed during the prolonged mycosis. Relatively stable (nonpathogenic) conditions remained in the midgut during both scenarios of mycosis with slight changes in bacterial communities, the absence of mesh and stat expression, a decrease in reactive oxygen species production, and slight induction of Toll and IMD pathways. Oral administration of antibiotic and predominant CPB bacteria (Enterobacteriaceae, Lactococcus, Pseudomonas) led to minor and mainly antagonistic effects in survival of larvae infected with B. bassiana. We believe that prolonged mycosis is necessary for successful development of the fungus because such pathogenesis allows the host to activate antibacterial reactions. Conversely, after infection with high concentrations of the fungus, the host's resources are insufficient to fully activate antibacterial defenses, and this situation makes successful development of the fungus impossible.

Links between Soil Bacteriobiomes and Fungistasis toward Fungi Infecting the Colorado Potato Beetle.

Entomopathogenic fungi can be inhibited by different soil microorganisms, but the effect of a soil microbiota on fungal growth, survival, and infectivity toward insects is insufficiently understood. We investigated the level of fungistasis toward Metarhizium robertsii and Beauveria bassiana in soils of conventional potato fields and kitchen potato gardens. Agar diffusion methods, 16S rDNA metabarcoding, bacterial DNA quantification, and assays of Leptinotarsa decemlineata survival in soils inoculated with fungal conidia were used. Soils of kitchen gardens showed stronger fungistasis toward M. robertsii and B. bassiana and at the same time the highest density of the fungi compared to soils of conventional fields. The fungistasis level depended on the quantity of bacterial DNA and relative abundance of Bacillus, Streptomyces, and some Proteobacteria, whose abundance levels were the highest in kitchen garden soils. Cultivable isolates of bacilli exhibited antagonism to both fungi in vitro. Assays involving inoculation of nonsterile soils with B. bassiana conidia showed trends toward elevated mortality of L. decemlineata in highly fungistatic soils compared to low-fungistasis ones. Introduction of antagonistic bacilli into sterile soil did not significantly change infectivity of B. bassiana toward the insect. The results support the idea that entomopathogenic fungi can infect insects within a hypogean habitat despite high abundance and diversity of soil antagonistic bacteria.

Faint gray bands in Drosophila melanogaster polytene chromosomes are formed by coding sequences of housekeeping genes.

In Drosophila melanogaster, the chromatin of interphase polytene chromosomes appears as alternating decondensed interbands and dense black or thin gray bands. Recently, we uncovered four principle chromatin states (4НММ model) in the fruit fly, and these were matched to the structures observed in polytene chromosomes. Ruby/malachite chromatin states form black bands containing developmental genes, whereas aquamarine chromatin corresponds to interbands enriched with 5' regions of ubiquitously expressed genes. Lazurite chromatin supposedly forms faint gray bands and encompasses the bodies of housekeeping genes. In this report, we test this idea using the X chromosome as the model and MSL1 as a protein marker of the lazurite chromatin. Our bioinformatic analysis indicates that in the X chromosome, it is only the lazurite chromatin that is simultaneously enriched for the proteins and histone marks associated with exons, transcription elongation, and dosage compensation. As a result of FISH and EM mapping of a dosage compensation complex subunit, MSL1, we for the first time provide direct evidence that lazurite chromatin forms faint gray bands. Our analysis proves that overall most of housekeeping genes typically span from the interbands (5' region of the gene) to the gray band (gene body). More rarely, active lazurite chromatin and inactive malachite/ruby chromatin may be found within a common band, where both the housekeeping and the developmental genes reside together.

Structural and Functional Dissection of the 5' Region of the Notch Gene in Drosophila melanogaster.

Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. Here, we provide the results of targeted 5'-end editing of the Drosophila Notch gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, we obtained a founder fly stock in which a 4-kb fragment, including the 5' nontranscribed region, the first exon, and a part of the first intron of Notch, was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5'untranscribed region of the gene were obtained by ΦC31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level we defined a DNA segment (~250 bp) in the Notch5'-nontranscribed region which when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.

Molecular and genetic organization of bands and interbands in the dot chromosome of Drosophila melanogaster.

The fourth chromosome smallest in the genome of Drosophila melanogaster differs from other chromosomes in many ways. It has high repeat density in conditions of a large number of active genes. Gray bands represent a significant part of this polytene chromosome. Specific proteins including HP1a, POF, and dSETDB1 establish the epigenetic state of this unique chromatin domain. In order to compare maps of localization of genes, bands, and chromatin types of the fourth chromosome, we performed FISH analysis of 38 probes chosen according to the model of four chromatin types. It allowed clarifying the dot chromosome cytological map consisting of 16 loose gray bands, 11 dense black bands, and 26 interbands. We described the relation between chromatin states and bands. Open aquamarine chromatin mostly corresponds to interbands and it contains 5'UTRs of housekeeping genes. Their coding parts are embedded in gray bands substantially composed of lazurite chromatin of intermediate compaction. Polygenic black bands contain most of dense ruby chromatin, and also some malachite and lazurite. Having an accurate map of the fourth chromosome bands and its correspondence to physical map, we found that DNase I hypersensitivity sites, ORC2 protein, and P-elements are mainly located in open aquamarine chromatin, while element 1360, characteristic of the fourth chromosome, occupies band chromatin types. POF and HP1a proteins providing special organization of this chromosome are mostly located in aquamarine and lazurite chromatin. In general, band organization of the fourth chromosome shares the features of the whole Drosophila genome.