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

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

Abstract 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 (T p) in ectotherms by modulating their physiology. Several recent studies have demonstrated substantial effects of Wolbachia infections on host T p 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 T p measurements in adult Drosophila melanogaster flies. Additionally, we assessed the effects of Wolbachia infection on T p of Drosophila at different developmental stages, which has not been done before. We find only subtle effects of Wolbachia on host T p 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 T p which demonstrates the necessity of careful experimental design and cautious interpretations of T p measurements together with a thorough description of the methods and equipment used to conduct behavioral studies

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

Abstract 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

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 ani-mals (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 mor-sitans 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

Additional file 4: of Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors

Figure S4. Kinetochore structure in different mitotic phases of S2 cells. In PM1 and PM2 cells, kinetochores (pseudo-colored in red) have an oblong appearance and do not appear to interact with MTs in an end-on fashion. Only a fraction of PM3 kinetochores show a limited end-on MT binding. Kinetochores of PM4, metaphase (M), and early anaphase (A) cells exhibit an arched structure and show end-on attached MTs. Scale bar: 0.1 Οm. (TIF 10897 kb

Additional file 3: of Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors

Figure S3. Overall MT distribution in transverse sections of S2 cells at different prometaphase stages. In the right images, the MTs and MT bundles of PM1 a, PM2 b, PM3 c, and PM4 d cells are encircled with a red line. Note that on progression through prometaphase, both the size (number of MTs) and the density (distance between MTs) of MT bundles increase. Scale bars: 1 Οm. (TIF 22315 kb

Additional file 9: of Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors

Figure S9. The QNM forms in the absence of astral microtubules. a A prometaphase cell in which the nucleation of astral MTs is completely suppressed by RNAi-mediated depletion of the centrosome component Cnn. It exhibits a QNM comparable to that observed in cells in which aster formation is not inhibited. b A prometaphase-like cell from a culture treated for 3 h with colcemid shows patches of QNM. The asterisks indicate the cell regions shown at higher magnification on the right. Scale bars: 1 Οm. (TIF 15859 kb

Additional file 6: of Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors

Figure S6. Additional examples of metaphase S2 cells. a Longitudinal section showing stacks of parallel ER membranes and kinetochores with k-fibers (arrowheads in the magnified image). b Cross section through a metaphase plate shown at different magnifications. Asterisks in the right image indicate the regions magnified in the insets. The b' inset shows a kinetochore and associated MTs (both pseudo-colored in red). The b" inset shows two MT bundles that might be either k-fibers or interpolar MT bundles (see Fig. 9 and Additional file 12: Figure S12). Scale bars: left images, 5 μm; right images, 1 μm; insets, 0.1 μm. (TIF 18615 kb