Wolbachia transfer from Rhagoletis cerasi to Drosophila simulans: Investigating the outcomes of host-symbiont coevolution

Wolbachia is an endosymbiont of diverse arthropod lineages that can induce various alterations of host reproduction for its own benefice. Cytoplasmic incompatibility (CI) is the most common phenomenon, which results in embryonic lethality when males that bear Wolbachia are mated with females that do not. In the cherry fruit fly, Rhagoletis cerasi, Wolbachia seems to be responsible for previously reported patterns of incompatibility between populations. Here we report on the artificial transfer of two Wolbachia variants (wCer1 and wCer2) from R. cerasi into Drosophila simulans, which was performed with two major goals in mind: first, to isolate wCer1 from wCer2 in order to individually test their respective abilities to induce Cl in the new host; and, second, to test the theoretical prediction that recent Wolbachia-host associations should be characterized by high levels of CI, fitness costs to the new host, and inefficient transmission from mothers to offspring. wCer1 was unable to develop in the new host, resulting in its rapid loss after successful injection, while wCer2 was established in the new host. Transmission rates of wCer2 were low, and the infection showed negative fitness effects, consistent with our prediction, but CI levels were unexpectedly lower in the new host. Based on these parameter estimates, neither wCer1 nor wCer2 could be naturally maintained in D. simulans. The experiment thus suggests that natural Wolbachia transfer between species might be restricted by many factors, should the ecological barriers be bypassed.

Contrasting mutual sexual selection on homologous signal traits in Drosophila serrata

The nature of male mating preferences, and how they differ from female mating preferences in species with conventional sex roles, has received little attention in sexual selection studies. We estimated the form and strength of sexual selection as a consequence of male and female mating preferences in a laboratory-based population of Drosophila serrata. The differences between sexual selection on male and female signal traits (cuticular hydrocarbons [CHCs]) were evaluated within a formal framework of linear and nonlinear selection gradients. Females tended to exert linear sexual selection on male CHCs, whereas males preferred intermediate female CHC phenotypes leading to convex (stabilizing) selection gradients. Possible mechanisms determining the nonlinear nature of sexual selection on female CHCs are proposed.

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS-GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR(-) stopphenotype and induces specific swellings in heterochromatin.

Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L(-/-) germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L(-/-) meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.

Distinct types of glial cells populate the Drosophila antenna

Background: The development of nervous systems involves reciprocal interactions between neurons and glia. In the Drosophila olfactory system, peripheral glial cells arise from sensory lineages specified by the basic helix- loop- helix transcription factor, Atonal. These glia wrap around the developing olfactory axons early during development and pattern the three distinct fascicles as they exit the antenna. In the moth Manduca sexta, an additional set of central glia migrate to the base of the antennal nerve where axons sort to their glomerular targets. In this work, we have investigated whether similar types of cells exist in the Drosophila antenna. Results: We have used different P( Gal4) lines to drive Green Fluorescent Protein ( GFP) in distinct populations of cells within the Drosophila antenna. Mz317:: GFP, a marker for cell body and perineural glia, labels the majority of peripheral glia. An additional similar to 30 glial cells detected by GH146:: GFP do not derive from any of the sensory lineages and appear to migrate into the antenna from the brain. Their appearance in the third antennal segment is regulated by normal function of the Epidermal Growth Factor receptor and small GTPases. We denote these distinct populations of cells as Mz317- glia and GH146- glia respectively. In the adult, processes of GH146- glial cells ensheath the olfactory receptor neurons directly, while those of the Mz317- glia form a peripheral layer. Ablation of GH146- glia does not result in any significant effects on the patterning of the olfactory receptor axons. Conclusion: We have demonstrated the presence of at least two distinct populations of glial cells within the Drosophila antenna. GH146- glial cells originate in the brain and migrate to the antenna along the newly formed olfactory axons. The number of cells populating the third segment of the antenna is regulated by signaling through the Epidermal Growth Factor receptor. These glia share several features of the sorting zone cells described in Manduca.

Age determination in individual wild-caught Drosophila serrata using pteridine concentration

Fluorescence spectrophotometry can reliably detect levels of the pteridine 6-biopterin in the heads of individual Drosophila serrata Malloch 1927. Pteridine content in both laboratory and field captured flies is typically a level of magnitude higher than the minimally detectable level (mean(lab)=0.54 units, mean(field)=0.44 units, minimum detectable level=0.01 units) and can be used to predict individual age in laboratory populations with high certainty (r(2)=57%). Laboratory studies of individuals of known age ( from 1 to 48 days old) indicate that while pteridine level increases linearly with age, they also increase in a linear manner with rearing temperature and ambient light levels, but are independent of sex. As expected, the longevity of laboratory-reared males ( at least 48 days) is higher than the range of predicted ages of wild-caught males based on individual pteridine levels (40 days). However, the predictive equation based on pteridine level alone suggested that a number of wild-caught males were less than 0 days old, and the 95% confidence for these predictions based on the inverse regression broad. The age of the oldest wild-caught male is to fall within the range of 2 to 50 days. The effects of temperature and light intensity determined in laboratory study (effect sizes omega(2)=14.3 and respectively) suggests that the calibration of the prediction equation for field populations would significantly improved when combined with fine scaled studies of habitat temperature and light conditions. ability to determine relative age in individual wild-caught D. serrata presents great opportunities for a variety evolutionary studies on the dynamics of populations.

Evolutionary dynamics of wAu-Like Wolbachia variants in neotropical Drosophila spp.

Wolbachia bacteria are common intracellular symbionts of arthropods and have been extensively studied in Drosophila. Most research focuses on two Old Word hosts, Drosophila melanogaster and Drosophila simulans, and does not take into account that some of the Wolbachia associations in these species may have evolved only after their fast global expansion and after the exposure to Wolbachia of previously isolated habitats. Here we looked at Wolbachia of Neotropical Drosophila species. Seventy-one lines of 16 Neotropical Drosophild species sampled in different regions and at different time points were analyzed. Wolbachia is absent in lines of Drosophild willistoni collected before the 1970s, but more recent samples are infected with a strain designated wWiL Wolbachia is absent in all other species of the willistoni group. Polymorphic wWil-related strains were detected in some saltans group species, with D. septentriosaltans being coinfected with at least four variants. Based on wsp and ftsZ sequence data, wWil of D. willistoni is identical to wAu, a strain isolated from D. simulans, but can be discriminated when using a polymorphic minisatellite marker. In contrast to wAu, which infects both germ line and somatic tissues of D. simulans, wWil is found exclusively in the primordial germ line cells of D. willistoni embryos. We report on a pool of closely related Wolbachia strains in Neotropical Drosophila species as a potential source for the wAu strain in D. simulans. Possible evolutionary scenarios reconstructing the infection history of wAu-like Wolbachia in Neotropical Drosophild species and the Old World species D. simulans are discussed.

Measuring natural and sexual selection on breeding values of male display traits in Drosophila serrata

Fundamental to many theories of sexual selection is the expectation that sexual traits, which males use in an attempt to increase mating success, confer costs as well as benefits to individual males. Although evolution of exaggerated male traits is predicted to be halted, by costs applied by natural selection, there is a lack of empirical work devoted to quantitatively establishing whether natural selection opposes sexual selection generated by the preferences of females. In this study, we quantified natural and sexual selection gradients on breeding values for cuticular hydrocarbon (CHC) components of male contact pheromones in Drosophila serrata. As male sexual traits may often be environmentally condition dependent, breeding values were used in the selection analysis to remove the possibility of environmental correlations between the measured trait and fitness biasing estimates of selection. The direction of natural selection was found to oppose sexual selection on a subset of CHCs examined. Opposing natural and sexual selection suggests that further evolution of the male pheromone may in part be limited by costs associated with attractive male CHC blends.

AP-2 complex-mediated endocytosis of Drosophila Crumbs regulates polarity via antagonizing Stardust

Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. Here, we show that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The adaptor protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutations in AP-2 causes expansion of the Crumbs-positive plasma membrane and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking-down both AP-2 and Stardust retains Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity.

Alternative exon usage in the single CPT1 gene of Drosophila generates functional diversity in the kinetic properties of the enzyme:differential expression of alternatively spliced variants in drosophila tissues

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A.Weperformed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1mRNAwere found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K 0.5) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

NOTCH1 Gain of Function in Germ Cells Causes Failure of Spermatogenesis in Male Mice

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icretransgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63genes' expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation.

Drosophila Flight Stabilization Depends on Apparent Distances in Optic Flow Field

To perform daily flight tasks, insects rely heavily on their visual perception of a dynamic environment. They must process visual signals quickly and accurately and update their behavior. Flies are vulnerable to environmental disturbances, such as gusts of wind blowing them off course, but they may use the altered visual field to compensate and regain their original course. In studies using Drosophila melanogaster, it has been shown that their corrective responses can be analyzed by measuring changes in their wing beats. By enclosing a tethered fly in a cuboidal visual arena displaying a computerized optic flow field, it is possible to calculate the change in wing beat amplitudes from an infrared shadow of its wings using photodiodes and a custom wing beat analyzer. In this experiment, manipulations ofthe optic flow field are used to create a field where points have varying relative forward speed, to study how the insect performs corrective maneuvers. The results show that Drosophila have a stronger corrective response to the quickly moving, apparently near points compared to the slower moving, apparently distant points. This implies the flies are distinguishing points based on their relative speeds, inferring distance, and adjusting their corrective actions with this information.

EFFECTS OF A VISUAL HORIZON ON DROSOPHILA MELANOGASTER OBJECT-TRACKING RESPONSES

To navigate effectively in three-dimensional space, flying insects must approximate distances to nearby objects. Humans are able to use an array of cues to guide depth perception in the visual world. However, some of these cues are not available to insects that are constrained by their rigid eyes and relatively small body size. Flying fruit flies can use motion parallax to gauge the distance of nearby objects, but using this cue becomes a less effective strategy as objects become more remote. Humans are able to infer depth across far distances by comparing the angular distance of an object to the horizon. This study tested if flying fruit flies, like humans, use the relative position of the horizon as a depth cue. Fruit flies in tethered flight were stimulated with a virtual environment that displayed vertical bars of varying elevation relative to a horizon, and their tracking responses were recorded. This study showed that tracking responses of the flies were strongly increased by reducing the apparent elevation of the bar against the horizon, indicating that fruit flies may be able to assess the distance of far off objects in the natural world by comparing them against a visual horizon.