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 <i>Wolbachia</i> 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

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

Prevalence and genetic diversity of Wolbachia endosymbiont and mtDNA in Palearctic populations of Drosophila melanogaster

Abstract Background Maternally inherited Wolbachia symbionts infect D. melanogaster populations worldwide. Infection rates vary greatly. Genetic diversity of Wolbachia in D. melanogaster can be subdivided into several closely related genotypes coinherited with certain mtDNA lineages. mtDNA haplotypes have the following global distribution pattern: mtDNA clade I is mostly found in North America, II and IV in Africa, III in Europe and Africa, V in Eurasia, VI is global but very rare, and VIII is found in Asia. The wMel Wolbachia genotype is predominant in D. melanogaster populations. However, according to the hypothesis of global Wolbachia replacement, the wMelCS genotype was predominant before the XX century when it was replaced by the wMel genotype. Here we analyse over 1500 fly isolates from the Palearctic region to evaluate the prevalence, genetic diversity and distribution pattrern of the Wolbachia symbiont, occurrence of mtDNA variants, and finally to discuss the Wolbachia genotype global replacement hypothesis. Results All studied Palearctic populations of D. melanogaster were infected with Wolbachia at a rate of 33–100%. We did not observe any significant correlation between infection rate and longitude or latitude. Five previously reported Wolbachia genotypes were found in Palearctic populations with a predominance of the wMel variant. The mtDNA haplotypes of the I_II_III clade and V clade were prevalent in Palearctic populations. To test the recent Wolbachia genotype replacement hypothesis, we examined three genomic regions of CS-like genotypes. Low genetic diversity was observed, only two haplotypes of the CS genotypes with a ‘CCG’ variant predominance were found. Conclusion The results of our survey of Wolbachia infection prevalence and genotype diversity in Palearctic D. melanogaster populations confirm previous studies. Wolbachia is ubiquitous in the Palearctic region. The wMel genotype is dominant with local occurrence of rare genotypes. Together with variants of the V mtDNA clade, the variants of the ‘III+’ clade are dominant in both infected and uninfected flies of Palearctic populations. Based on our data on Wolbachia and mtDNA in different years in some Palearctic localities, we can conclude that flies that survive the winter make the predominant symbiont contribution to the subsequent generation. A comprehensive overview of mtDNA and Wolbachia infection of D. melanogaster populations worldwide does not support the recent global Wolbachia genotype replacement hypothesis. However, we cannot exclude wMelCS genotype rate fluctuations in the past

Classification of Fruit Flies by Gender in Images Using Smartphones and the YOLOv4-Tiny Neural Network

The fruit fly Drosophila melanogaster is a classic research object in genetics and systems biology. In the genetic analysis of flies, a routine task is to determine the offspring size and gender ratio in their populations. Currently, these estimates are made manually, which is a very time-consuming process. The counting and gender determination of flies can be automated by using image analysis with deep learning neural networks on mobile devices. We proposed an algorithm based on the YOLOv4-tiny network to identify Drosophila flies and determine their gender based on the protocol of taking pictures of insects on a white sheet of paper with a cell phone camera. Three strategies with different types of augmentation were used to train the network. The best performance (F1 = 0.838) was achieved using synthetic images with mosaic generation. Females gender determination is worse than that one of males. Among the factors that most strongly influencing the accuracy of fly gender recognition, the fly’s position on the paper was the most important. Increased light intensity and higher quality of the device cameras have a positive effect on the recognition accuracy. We implement our method in the FlyCounter Android app for mobile devices, which performs all the image processing steps using the device processors only. The time that the YOLOv4-tiny algorithm takes to process one image is less than 4 s

Classification of Fruit Flies by Gender in Images Using Smartphones and the YOLOv4-Tiny Neural Network

The fruit fly Drosophila melanogaster is a classic research object in genetics and systems biology. In the genetic analysis of flies, a routine task is to determine the offspring size and gender ratio in their populations. Currently, these estimates are made manually, which is a very time-consuming process. The counting and gender determination of flies can be automated by using image analysis with deep learning neural networks on mobile devices. We proposed an algorithm based on the YOLOv4-tiny network to identify Drosophila flies and determine their gender based on the protocol of taking pictures of insects on a white sheet of paper with a cell phone camera. Three strategies with different types of augmentation were used to train the network. The best performance (F1 = 0.838) was achieved using synthetic images with mosaic generation. Females gender determination is worse than that one of males. Among the factors that most strongly influencing the accuracy of fly gender recognition, the fly&rsquo;s position on the paper was the most important. Increased light intensity and higher quality of the device cameras have a positive effect on the recognition accuracy. We implement our method in the FlyCounter Android app for mobile devices, which performs all the image processing steps using the device processors only. The time that the YOLOv4-tiny algorithm takes to process one image is less than 4 s

Fitness Analysis and Transcriptome Profiling Following Repeated Mild Heat Stress of Varying Frequency in Drosophila melanogaster 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 &deg;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