Drosophila melanogaster Transcriptome Response to Different Wolbachia Strains.

Wolbachia is a maternally inherited, intercellular bacterial symbiont of insects and some other invertebrates. Here, we investigated the effect of two different Wolbachia strains, differing in a large chromosomal inversion, on the differential expression of genes in D. melanogaster females. We revealed significant changes in the transcriptome of the infected flies compared to the uninfected ones, as well as in the transcriptome of flies infected with the Wolbachia strain, wMelPlus, compared to flies infected with the wMelCS112 strain. We linked differentially expressed genes (DEGs) from two pairwise comparisons, "uninfected-wMelPlus-infected" and "uninfected-wMelCS112-infected", into two gene networks, in which the following functional groups were designated: "Proteolysis", "Carbohydrate transport and metabolism", "Oxidation-reduction process", "Embryogenesis", "Transmembrane transport", "Response to stress" and "Alkaline phosphatases". Our data emphasized similarities and differences between infections by different strains under study: a wMelPlus infection results in more than double the number of upregulated DEGs and half the number of downregulated DEGs compared to a wMelCS112 infection. Thus, we demonstrated that Wolbachia made a significant contribution to differential expression of host genes and that the bacterial genotype plays a vital role in establishing the character of this contribution.

New Wolbachia pipientis Genotype Increasing Heat Stress Resistance of Drosophila melanogaster Host Is Characterized by a Large Chromosomal Inversion.

The maternally transmitted endocellular bacteria Wolbachia is a well-known symbiont of insects, demonstrating both negative and positive effects on host fitness. The previously found Wolbachia strain wMelPlus is characterized by a positive effect on the stress-resistance of its host Drosophila melanogaster, under heat stress conditions. This investigation is dedicated to studying the genomic underpinnings of such an effect. We sequenced two closely related Wolbachia strains, wMelPlus and wMelCS112, assembled their complete genomes, and performed comparative genomic analysis engaging available Wolbachia genomes from the wMel and wMelCS groups. Despite the two strains under study sharing very close gene-composition, we discovered a large (>1/6 of total genome) chromosomal inversion in wMelPlus, spanning through the region that includes the area of the inversion earlier found in the wMel group of Wolbachia genotypes. A number of genes in unique inversion blocks of wMelPlus were identified that might be involved in the induction of a stress-resistant phenotype in the host. We hypothesize that such an inversion could rearrange established genetic regulatory-networks, causing the observed effects of such a complex fly phenotype as a modulation of heat stress resistance. Based on our findings, we propose that wMelPlus be distinguished as a separate genotype of the wMelCS group, named wMelCS3.

Easy and Effective Method for Extracting and Purifying Wolbachia Genomic DNA.

A number of methods for extracting the DNA of maternally inherited obligate intracellular bacteria Wolbachia from an insect host and its subsequent purification have been described in previous scholarship. As Wolbachia is present in the hosts' organisms in rather low quantities, these techniques used to be quite labor-intensive. For this paper, we analyzed them in detail, searched for a possibility to simplify and accelerate the protocol, and proposed an easy and effective method for isolating Wolbachia DNA from Drosophila melanogaster with a purity sufficient for genomic sequencing. Our method involves the centrifugation of homogenized flies or just their ovaries, as the most Wolbachia-enriched tissue, followed by the filtration of homogenate and extraction of DNA using a modified version of the Livak buffer protocol. The proportion of Wolbachia DNA in the total DNA was quantified based on the results of sequencing with the use of the Illumina MiSeq platform and a pipeline of bioinformatic analysis. For the two analyzed D. melanogaster lines infected with two different Wolbachia strains, the proportion was at least 68 and 94%, respectively.

Fitness Analysis and Transcriptome Profiling Following Repeated Mild Heat Stress of Varying Frequency in Drosophilamelanogaster Females.

Understanding how repeated stress affects metabolic and physiological functions in the long run is of crucial importance for evaluating anthropogenic pressure on the environment. We investigated fertility, longevity and metabolism in D. melanogaster females exposed to short-term heat stress (38 °C, 1 h) repeated daily or weekly. Daily stress was shown to cause a significant decrease in both fertility and longevity, as well as in body mass and triglyceride (fat) content, but a significant increase in trehalose and glucose content. Weekly stress did not affect longevity and carbohydrate metabolism but resulted in a significant decrease in body mass and fat content. Weekly stress did not affect the total level of fertility, despite sharp fertility drops on the exact days of stressing. However, stressing insects weekly, only in the first two weeks after eclosion, caused a significant increase in the total level of fertility. The analysis of differentially expressed genes in the fat bodies and adjacent tissues of researched groups with the use of RNA-Seq profiling revealed changes in signal pathways related to proteolysis/digestion, heat shock protein 23, and in the tightly linked stress-inducible humoral factor Turandot gene network.

Drosophila female fertility and juvenile hormone metabolism depends on the type of Wolbachia infection.

Maternally inherited intracellular bacteria Wolbachia cause both parasitic and mutualistic effects on their numerous insect hosts, including manipulating the host reproductive system in order to increase the bacteria spreading in a host population, and increasing the host fitness. Here, we demonstrate that the type of Wolbachia infection determines the effect on Drosophila melanogaster egg production as a proxy for fecundity, and metabolism of juvenile hormone (JH), which acts as gonadotropin in adult insects. For this study, we used six D. melanogaster lineages carrying the nuclear background of interbred Bi90 lineage and cytoplasmic backgrounds with or without Wolbachia of different genotype variants. The wMelCS genotype of Wolbachia decreases egg production in infected D. melanogaster females in the beginning of oviposition and increases it later (from the sixth day after eclosion), whereas the wMelPop Wolbachia strain causes the opposite effect, and the wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on these traits compared with uninfected Bi90 D. melanogaster females. The intensity of JH catabolism negatively correlates with the fecundity level in the flies carrying both wMelCS and wMelPop Wolbachia The JH catabolism in females infected with genotypes of the wMel group does not differ from that in uninfected females. The effects of wMelCS and wMelPop infection on egg production can be levelled by the modulation of JH titre (via precocene/JH treatment of the flies). Thus, at least one of the mechanisms promoting the effect of Wolbachia on D. melanogaster female fecundity is mediated by JH.

The impact of FOXO on dopamine and octopamine metabolism in Drosophila under normal and heat stress conditions.

The forkhead boxO transcription factor (FOXO) is a component of the insulin signalling pathway and plays a role in responding to adverse conditions, such as oxidative stress and starvation. In stressful conditions, FOXO moves from the cytosol to the nucleus where it activates gene expression programmes. Here, we show that FOXO in Drosophila melanogaster responds to heat stress as it does to other stressors. The catecholamine signalling pathway is another component of the stress response. In Drosophila, dopamine and octopamine levels rise steeply under heat, nutrition and mechanical stresses, which are followed by a decrease in the activity of synthesis enzymes. We demonstrate that the nearly twofold decline of FOXO expression in foxoBG01018 mutants results in dramatic changes in the metabolism of dopamine and octopamine and the overall response to stress. The absence of FOXO increases tyrosine decarboxylase activity, the first enzyme in octopamine synthesis, and decreases the enzymatic activity of enzymes in dopamine synthesis, alkaline phosphatase and tyrosine hydroxylase, in young Drosophila females. We identified the juvenile hormone as a mediator of FOXO regulation of catecholamine metabolism. Our findings suggest that FOXO is a possible trigger for endocrinological stress reactions.

Disruption of insulin signalling affects the neuroendocrine stress reaction in Drosophila females.

Juvenile hormone (JH) and dopamine are involved in the stress response in insects. The insulin/insulin-like growth factor signalling pathway has also recently been found to be involved in the regulation of various processes, including stress tolerance. However, the relationships between the JH, dopamine and insulin signalling pathways remain unclear. Here, we study the role of insulin signalling in the regulation of JH and dopamine metabolism under normal and heat stress conditions in Drosophila melanogaster females. We show that suppression of the insulin-like receptor (InR) in the corpus allatum, a specialised endocrine gland that synthesises JH, causes an increase in dopamine level and JH-hydrolysing activity and alters the activities of enzymes that produce as well as those that degrade dopamine [alkaline phosphatase (ALP), tyrosine hydroxylase (TH) and dopamine-dependent arylalkylamine N-acetyltransferase (DAT)]. We also found that InR suppression in the corpus allatum modulates dopamine, ALP, TH and JH-hydrolysing activity in response to heat stress and that it decreases the fecundity of the flies. JH application restores dopamine metabolism and fecundity in females with decreased InR expression in the corpus allatum. Our data provide evidence that the insulin/insulin-like growth factor signalling pathway regulates dopamine metabolism in females of D. melanogaster via the system of JH metabolism and that it affects the development of the neuroendocrine stress reaction and interacts with JH in the control of reproduction in this species.

B chromosomes of the Podisma sapporensis Shir. (Orthoptera, Acrididae) analysed by chromosome microdissection and FISH.

The analysis of the distribution of repetitive DNA of the B chromosomes of Podisma sapporensis in the A and B chromosomes of the natural populations and in A chromosomes of three other species of the Podismini grasshoppers were made. DNA-libraries of the B chromosome and the euchromatic segment of the A chromosome of P. sapporensis were generated by meiotic chromosome microdissection followed by degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR). Paints based on these DNA-libraries were used for FISH analysis to detect localization of homologous sequences in A and B chromosomes of P. sapporensis from different natural populations. On the basis of the FISH analysis the authors suggest that evolution of the B chromosomes in Podisma sapporensis was associated mainly with the insertions of "alien DNA sequences" into ancestral A chromosome and their further amplification. The number of initial sites of amplifications differed in the different Bs, the distance between these sites also varying. Karyotype evolution in P. sapporensis was associated partly with the insertion of "alien DNA sequences" into pericentromeric chromosomal regions. Insertion into the small short arms of the acrocentric chromosomes followed, with the DNA amplification leading to the formation of the additional C-heterochromatic arms or euchromatic-like regions of different size.