Wolbachia effects on thermal preference of natural Drosophila melanogaster are influenced by host genetic background, Wolbachia type, and bacterial titer.

Temperature plays a fundamental role in the fitness of all organisms. In particular, it strongly affects metabolism and reproduction in ectotherms that have limited physiological capabilities to regulate their body temperature. The influence of temperature variation on the physiology and behaviour of ectotherms is well studied but we still know little about the influence of symbiotic interactions on thermal preference (Tp ) of the host. A growing number of studies focusing on the Wolbachia-Drosophila host-symbiont system found that Wolbachia can influence Tp in Drosophila laboratory strains. Here, we investigated the effect of Wolbachia on Tp in wild-type D. melanogaster flies recently collected from nature. Consistent with previous data, we found reduced Tp compared to an uninfected control in one of two fly strains infected with the wMelCS Wolbachia type. Additionally, we, for the first time, found that Wolbachia titer variation influences the thermal preference of the host fly. These data indicate that the interaction of Wolbachia and Drosophila resulting in behavioural variation is strongly influenced by the genetic background of the host and symbiont. More studies are needed to better understand the evolutionary significance of Tp variation influenced by Wolbachia in natural Drosophila populations.

Historic Museum Samples Provide Evidence for a Recent Replacement of Wolbachia Types in European Drosophila melanogaster.

Wolbachia is one of the most common bacterial endosymbionts, which is frequently found in numerous arthropods and nematode taxa. Wolbachia infections can have a strong influence on the evolutionary dynamics of their hosts since these bacteria are reproductive manipulators that affect the fitness and life history of their host species for their own benefit. Host-symbiont interactions with Wolbachia are perhaps best studied in the model organism Drosophila melanogaster, which is naturally infected with at least 5 different variants among which wMel and wMelCS are the most frequent ones. Comparisons of infection types between natural flies and long-term lab stocks have previously indicated that wMelCS represents the ancestral type, which was only very recently replaced by the nowadays dominant wMel in most natural populations. In this study, we took advantage of recently sequenced museum specimens of D. melanogaster that have been collected 90 to 200 yr ago in Northern Europe to test this hypothesis. Our comparison to contemporary Wolbachia samples provides compelling support for the replacement hypothesis. Our analyses show that sequencing data from historic museum specimens and their bycatch are an emerging and unprecedented resource to address fundamental questions about evolutionary dynamics in host-symbiont interactions. However, we also identified contamination with DNA from crickets that resulted in co-contamination with cricket-specific Wolbachia in several samples. These results underpin the need for rigorous quality assessments of museomic data sets to account for contamination as a source of error that may strongly influence biological interpretations if it remains undetected.

Wolbachia has subtle effects on thermal preference in highly inbred Drosophila melanogaster which vary with life stage and environmental conditions.

Temperature fluctuations are challenging for ectotherms which are not able to regulate body temperature by physiological means and thus have to adjust their thermal environment via behavior. However, little is yet known about whether microbial symbionts influence thermal preference (Tp) in ectotherms by modulating their physiology. Several recent studies have demonstrated substantial effects of Wolbachia infections on host Tp in different Drosophila species. These data indicate that the direction and strength of thermal preference variation is strongly dependent on host and symbiont genotypes and highly variable among studies. By employing highly controlled experiments, we investigated the impact of several environmental factors including humidity, food quality, light exposure, and experimental setup that may influence Tp measurements in adult Drosophila melanogaster flies. Additionally, we assessed the effects of Wolbachia infection on Tp of Drosophila at different developmental stages, which has not been done before. We find only subtle effects of Wolbachia on host Tp which are strongly affected by experimental variation in adult, but not during juvenile life stages. Our in-depth analyses show that environmental variation has a substantial influence on Tp which demonstrates the necessity of careful experimental design and cautious interpretations of Tp measurements together with a thorough description of the methods and equipment used to conduct behavioral studies.

Complex effects of environment and Wolbachia infections on the life history of Drosophila melanogaster hosts.

Wolbachia bacteria are common endosymbionts of many arthropods found in gonads and various somatic tissues. They manipulate host reproduction to enhance their transmission and confer complex effects on fitness-related traits. Some of these effects can serve to increase the survival and transmission efficiency of Wolbachia in the host population. The Wolbachia-Drosophila melanogaster system represents a powerful model to study the evolutionary dynamics of host-microbe interactions and infections. Over the past decades, there has been a replacement of the ancestral wMelCS Wolbachia variant by the more recent wMel variant in worldwide D. melanogaster populations, but the reasons remain unknown. To investigate how environmental change and genetic variation of the symbiont affect host developmental and adult life-history traits, we compared effects of both Wolbachia variants and uninfected controls in wild-caught D. melanogaster strains at three developmental temperatures. While Wolbachia did not influence any developmental life-history traits, we found that both lifespan and fecundity of host females were increased without apparent fitness trade-offs. Interestingly, wMelCS-infected flies were more fecund than uninfected and wMel-infected flies. By contrast, males infected with wMel died sooner, indicating sex-specific effects of infection that are specific to the Wolbachia variant. Our study uncovered complex temperature-specific effects of Wolbachia infections, which suggests that symbiont-host interactions in nature are strongly dependent on the genotypes of both partners and the thermal environment.

TEM Imaging of Membrane Choreography During Mitosis of Drosophila Tissue Culture Cells.

Schneider 2 (S2) cells are one of the most widely used Drosophila cell lines, and are specifically suitable for genetic dissection of biological processes by RNA interference. We have recently developed a method that allows an easy preparation of samples for transmission electron microscopy (TEM) analysis of S2 cells. This method is based on the collection and pelleting of the cells in test tubes, followed by fixation and staining of pellets in the same tubes. Pellets are then embedded in resin and used to prepare ultrathin sections for TEM observation. Our Method allows clear visualization of the complex membrane transformations that characterize mitosis in S2 cells. It also allows precise analysis of microtubule behavior during the different mitotic phases. Although the method was specifically developed for S2 cells, our preliminary results indicate that it can be successfully applied to other types of Drosophila tissue culture cells.

Restriction of Wolbachia Bacteria in Early Embryogenesis of Neotropical Drosophila Species via Endoplasmic Reticulum-Mediated Autophagy.

Wolbachia are maternally transmitted intracellular bacteria that are not only restricted to the reproductive organs but also found in various somatic tissues of their native hosts. The abundance of the endosymbiont in the soma, usually a dead end for vertically transmitted bacteria, causes a multitude of effects on life history traits of their hosts, which are still not well understood. Thus, deciphering the host-symbiont interactions on a cellular level throughout a host's life cycle is of great importance to understand their homeostatic nature, persistence, and spreading success. Using fluorescent and transmission electron microscopy, we conducted a comprehensive analysis of Wolbachia tropism in soma and germ line of six Drosophila species at the intracellular level during host development. Our data uncovered diagnostic patterns of infections to embryonic primordial germ cells and to particular cells of the soma in three different neotropical Drosophila species that have apparently evolved independently. We further found that restricted patterns of Wolbachia tropism are determined in early embryogenesis via selective autophagy, and their spatially restricted infection patterns are preserved in adult flies. We observed tight interactions of Wolbachia with membranes of the endoplasmic reticulum, which might play a scaffolding role for autophagosome formation and subsequent elimination of the endosymbiont. Finally, by analyzing D. simulans lines transinfected with nonnative Wolbachia, we uncovered that the host genetic background regulates tissue tropism of infection. Our data demonstrate a novel and peculiar mechanism to limit and spatially restrict bacterial infection in the soma during a very early stage of host development. IMPORTANCE All organisms are living in close and intimate interactions with microbes that cause conflicts but also cooperation between both unequal genetic partners due to their different innate interests of primarily enhancing their own fitness. However, stable symbioses often result in homeostatic interaction, named mutualism, by balancing costs and benefits, where both partners profit. Mechanisms that have evolved to balance and stably maintain homeostasis in mutualistic relationships are still quite understudied; one strategy is to "domesticate" potentially beneficial symbionts by actively controlling their replication rate below a critical and, hence, costly threshold, and/or to spatially and temporally restrict their localization in the host organism, which, in the latter case, in its most extreme form, is the formation of a specialized housing organ for the microbe (bacteriome). However, questions remain: how do these mutualistic associations become established in their first place, and what are the mechanisms for symbiont control and restriction in their early stages? Here, we have uncovered an unprecedented symbiont control mechanism in neotropical Drosophila species during early embryogenesis. The fruit fly evolved selective autophagy to restrict and control the proliferation of its intracellular endosymbiont Wolbachia in a defined subset of the stem cells as soon as the host's zygotic genome is activated.

Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies.

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host's brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

Transport Oligonucleotides-A Novel System for Intracellular Delivery of Antisense Therapeutics.

Biological activity of antisense oligonucleotides (asON), especially those with a neutral backbone, is often attenuated by poor cellular accumulation. In the present proof-of-concept study, we propose a novel delivery system for asONs which implies the delivery of modified antisense oligonucleotides by so-called transport oligonucleotides (tON), which are oligodeoxyribonucleotides complementary to asON conjugated with hydrophobic dodecyl moieties. Two types of tONs, bearing at the 5'-end up to three dodecyl residues attached through non-nucleotide inserts (TD series) or anchored directly to internucleotidic phosphate (TP series), were synthesized. tONs with three dodecyl residues efficiently delivered asON to cells without any signs of cytotoxicity and provided a transfection efficacy comparable to that achieved using Lipofectamine 2000. We found that, in the case of tON with three dodecyl residues, some tON/asON duplexes were excreted from the cells within extracellular vesicles at late stages of transfection. We confirmed the high efficacy of the novel and demonstrated that MDR1 mRNA targeted asON delivered by tON with three dodecyl residues significantly reduced the level of P-glycoprotein and increased the sensitivity of KB-8-5 human carcinoma cells to vinblastine. The obtained results demonstrate the efficacy of lipophilic oligonucleotide carriers and shows they are potentially capable of intracellular delivery of any kind of antisense oligonucleotides.

3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes' chromatin.

How chromosomes are folded, spatially organized and regulated in three dimensions inside the cell nucleus are among the longest standing questions in cell biology. Genome-wide chromosome conformation capture (Hi-C) technique allowed identifying and characterizing spatial chromatin compartments in several mammalian species. Here, we present the first genome-wide analysis of chromatin interactions in chicken embryonic fibroblasts (CEF) and adult erythrocytes. We showed that genome of CEF is partitioned into topologically associated domains (TADs), distributed in accordance with gene density, transcriptional activity and CTCF-binding sites. In contrast to mammals, where all examined somatic cell types display relatively similar spatial organization of genome, chicken erythrocytes strongly differ from fibroblasts, showing pronounced A- and B- compartments, absence of typical TADs and formation of long-range chromatin interactions previously observed on mitotic chromosomes. Comparing mammalian and chicken genome architectures, we provide evidence highlighting evolutionary role of chicken TADs and their significance in genome activity and regulation.

Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors.

BACKGROUND: S2 cells are one of the most widely used Drosophila melanogaster cell lines. A series of studies has shown that they are particularly suitable for RNAi-based screens aimed at the dissection of cellular pathways, including those controlling cell shape and motility, cell metabolism, and host-pathogen interactions. In addition, RNAi in S2 cells has been successfully used to identify many new mitotic genes that are conserved in the higher eukaryotes, and for the analysis of several aspects of the mitotic process. However, no detailed and complete description of S2 cell mitosis at the ultrastructural level has been done. Here, we provide a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM). RESULTS: We analyzed by TEM a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behaviors. This unbiased approach provided a comprehensive ultrastructural view of the dividing cells, and allowed us to discover that S2 cells exhibit a previously uncharacterized behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. CONCLUSIONS: We discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells.

A New Look at Etiological Factors of Idiopathic Scoliosis: Neural Crest Cells.

Idiopathic scoliosis is one of the most common disabling pathologies of children and adolescents. Etiology and pathogenesis of idiopathic scoliosis remain unknown. To study the etiology of this disease we identified the cells' phenotypes in the vertebral body growth plates in patients with idiopathic scoliosis. Materials and methods: The cells were isolated from vertebral body growth plates of the convex and concave sides of the deformity harvested intraoperatively in 50 patients with scoliosis. Cells were cultured and identified by methods of common morphology, neuromorphology, electron microscopy, immunohistochemistry and PCR analysis. Results: Cultured cells of convex side of deformation were identified as chondroblasts. Cells isolated from the growth plates of the concave side of the deformation showed numerous features of neuro- and glioblasts. These cells formed synapses, contain neurofilaments, and expressed neural and glial proteins. Conclusion: For the first time we demonstrated the presence of cells with neural/glial phenotype in the concave side of the vertebral body growth plate in scoliotic deformity. We hypothesized that neural and glial cells observed in the growth plates of the vertebral bodies represent derivatives of neural crest cells deposited in somites due to alterations in their migratory pathway during embryogenesis. We also propose that ectopic localization of cells derived from neural crest in the growth plate of the vertebral bodies is the main etiological factor of the scoliotic disease.

The Drosophila orthologue of the INT6 onco-protein regulates mitotic microtubule growth and kinetochore structure.

INT6/eIF3e is a highly conserved component of the translation initiation complex that interacts with both the 26S proteasome and the COP9 signalosome, two complexes implicated in ubiquitin-mediated protein degradation. The INT6 gene was originally identified as the insertion site of the mouse mammary tumor virus (MMTV), and later shown to be involved in human tumorigenesis. Here we show that depletion of the Drosophila orthologue of INT6 (Int6) results in short mitotic spindles and deformed centromeres and kinetochores with low intra-kinetochore distance. Poleward flux of microtubule subunits during metaphase is reduced, although fluorescence recovery after photobleaching (FRAP) demonstrates that microtubules remain dynamic both near the kinetochores and at spindle poles. Mitotic progression is delayed during metaphase due to the activity of the spindle assembly checkpoint (SAC). Interestingly, a deubiquitinated form of the kinesin Klp67A (a putative orthologue of human Kif18A) accumulates near the kinetochores in Int6-depleted cells. Consistent with this finding, Klp67A overexpression mimics the Int6 RNAi phenotype. Furthermore, simultaneous depletion of Int6 and Klp67A results in a phenotype identical to RNAi of just Klp67A, which indicates that Klp67A deficiency is epistatic over Int6 deficiency. We propose that Int6-mediated ubiquitination is required to control the activity of Klp67A. In the absence of this control, excess of Klp67A at the kinetochore suppresses microtubule plus-end polymerization, which in turn results in reduced microtubule flux, spindle shortening, and centromere/kinetochore deformation.

Restricted distribution and lateralization of mutualistic Wolbachia in the Drosophila brain.

Microbial symbionts are universal entities of all living organisms that can significantly affect host fitness traits in manifold ways but, even more fascinating, also their behaviour. Although better known from parasitic symbionts, we currently lack any cases where 'neurotrophic' symbionts have co-evolved mutualistic behavioural interactions from which both partners profit. By theory, most mutualistic associations have originated from ancestral parasitic ones during their long-term co-evolution towards a cost-benefit equilibrium. To manipulate host behaviour in a way where both partners benefit in a reciprocal manner, the symbiont has to target and remain restricted to defined host brain regions to minimize unnecessary fitness costs. By using the classic Drosophila paulistorum model system we demonstrate that (i) mutualistic Wolbachia are restricted to various Drosophila brain areas, (ii) form bacteriocyte-like structures within the brain, (iii) exhibit strictly lateral tropism, and (iv) finally propose that their selective neuronal infection affects host sexual behaviour adaptively.

A simple and effective method for ultrastructural analysis of mitosis in Drosophila S2 cells.

The Drosophila S2 tissue culture cells are a widely used system for studies on mitosis. S2 cells are particularly sensitive to gene silencing by RNA interference (RNAi), allowing targeted inactivation of mitotic genes. S2 cells are also well suited for high-resolution light microscopy analysis of mitosis in fixed cells, and can be easily immunostained to detect mitotic components. In addition, S2 cells are amenable to transformation with plasmid encoding fluorescently tagged mitotic proteins, allowing in vivo analysis of their behavior throughout cell division. However, S2 cells have not been widely used for transmission electron microscopy (TEM) analysis, which provides ultrastructural details on the morphology of the mitotic apparatus that cannot be obtained with high-resolution confocal microscopy. Here, we describe a simple method for the ultrastructural analysis of mitosis in Drosophila S2 cells. •Our method, which involves fixation and sectioning of a cell pellet, provides excellent preservation of mitotic structures and allows analysis of a higher number of mitotic divisions per sample, compared to correlative light-electron microscopy.•Dividing cells are randomly oriented within the pellet and are sectioned along different planes, providing all-around information on the structure of the mitotic apparatus.

Drosophila melanogaster brain invasion: pathogenic Wolbachia in central nervous system of the fly.

The pathogenic Wolbachia strain wMelPop rapidly over-replicates in the brain, muscles, and retina of Drosophila melanogaster, causing severe tissue degeneration and premature death of the host. The unique features of this endosymbiont make it an excellent tool to be used for biological control of insects, pests, and vectors of human diseases. To follow the dynamics of bacterial morphology and titer in the nerve cells we used transmission electron microscopy of 3-d-old female brains. The neurons and glial cells from central brain of the fly had different Wolbachia titers ranging from single bacteria to large accumulations, tearing cell apart and invading extracellular space. The neuropile regions of the brain were free of wMelPop. Wolbachia tightly interacted with host cell organelles and underwent several morphological changes in nerve cells. Based on different morphological types of bacteria described we propose for the first time a scheme of wMelPop dynamics within the somatic tissue of the host.

Imaging yeast NPCs: from classical electron microscopy to Immuno-SEM.

Electron microscopy (EM) has been used extensively for the study of nuclear transport as well as the structure of the nuclear pore complex (NPC) and nuclear envelope. However, there are specific challenges faced when carrying out EM in one of the main model organisms used: the yeast, Saccharomyces cerevisiae. These are due to the presence of a cell wall, vacuoles, and a densely packed cytoplasm which, for transmission EM (TEM), make fixation, embedding, and imaging difficult. These also present problems for scanning EM (SEM) because cell wall removal and isolation of nuclei can easily damage the relatively fragile NPCs. We present some of the protocols we use to prepare samples for TEM and SEM to provide information about yeast NPC ultrastructure and the location of nucleoporins and transport factors by immunogold labeling within that ultrastructure.

Spatial and temporal distribution of pathogenic Wolbachia strain wMelPop in Drosophila melanogaster central nervous system under different temperature conditions.

The pathogenic Wolbachia strain, wMelPop, of Drosophila melanogaster is propagated in the fly's brain and muscles. To determine how wMelPop spreads in the host's central nervous system (CNS) during its life cycle, we used whole-mount fluorescent in situ hybridization to demonstrate the spatial distribution of wMelPop in D.melanogaster larvae and adults. To assess the effect of temperature on the pattern of wMelPop spread, we performed this analysis under moderate (25°C) and high (29°C) temperature conditions. Wolbachia distribution pattern in the third instar larva and adult brain was similar at both temperatures. wMelPop was generally localized to the subesophageal ganglion and the central brain of the host, whereas optic lobe anlagen cells of third instar larvae and cells of the optic lobe, lamina and retina of adult flies were mostly free of bacteria. Interestingly, high temperature had no significant effect on wMelPop titer or localization in the brain during larval development, but considerably altered it in adults immediately after eclosion. At both temperatures and within all tested stages of the life cycle, the bacterial titer varied only slightly between individuals. The observed differences in wMelPop titers in the central brain, subesophageal ganglion and optic lobe anlagen cells of third instar larva's CNS, together with the observation that these patterns are conserved in the adult brain, suggest that Wolbachia distribution is determined during fly embryogenesis.