Larval RNAi in Drosophila?

RNA interference (RNAi) has become a common method of gene knockdown in many model systems. To trigger an RNAi response, double-stranded RNA (dsRNA) must enter the cell. In some organisms such as Caenorhabditis elegans, cells can take up dsRNA from the extracellular environment via a cellular uptake mechanism termed systemic RNAi. However, in the fruit fly Drosophila melanogaster, it is widely believed that cells are unable to take up dsRNA, although there is little published data to support this claim. In this study, we set out to determine whether this perception has a factual basis. We took advantage of traditional Gal4/upstream activation sequence (UAS) transgenic flies as well as the mosaic analysis with a repressible cell marker (MARCM) system to show that extracellular injection of dsRNA into Drosophila larvae cannot trigger RNAi in most Drosophila tissues (with the exception of hemocytes). Our results show that this is not due to a lack of RNAi machinery in these tissues as overexpression of dsRNA inside the cells using hairpin RNAs efficiently induces an RNAi response in the same tissues. These results suggest that, while most Drosophila tissues indeed lack the ability to uptake dsRNA from the surrounding environment, hemocytes can initiate RNAi in response to extracellular dsRNA. We also examined another insect, the red flour beetle Tribolium castaneum, which has been shown to exhibit a robust systemic RNAi response. We show that virtually all Tribolium tissues can respond to extracellular dsRNA, which is strikingly different from the situation in Drosophila. Our data provide specific information about the tissues amenable to RNAi in two different insects, which may help us understand the molecular basis of systemic RNAi.     

How does Drosophila melanogaster overwinter?

In temperate regions low temperatures seem to be the most restrictive factor for survival of Drosophila natural populations, which depends on the capacity of one or more developmental stages to resist unfavourable winter conditions. In this study we have attempted to answer the question of how D. melanogaster overwinters under natural temperature conditions. Only adults overwintered and no diapause was observed in any developmental stage. Thus, developmental duration becomes a decisive component with respect to overwintering potential and, therefore, the preadult stages are unlikely to overwinter. Possible evolutionary steps in adaptation to cold regions are discussed.     

中国特有果蝇 Drosophila curviceps 种亚组在Drosophila immigrans种组中的种系发生地位

果蝇immigrans种组中的curviceps种亚组是1992年新建立的中国特有果蝇类群。该种亚组中的物种主要分布在中国大陆和台湾。目前除了形态学水平的研究外,还没有其他证据支持建立该种亚组的合理性及其起源和种系发生地位。为了在DNA分子水平上探讨果蝇curviceps种亚组在果蝇immigrans种组中的种系发生地位,从而为今后更深入地研究中国特有果蝇,甚至为果蝇亚属的进化遗传学提供理论依据,测定了immigrans种组5个种亚组（nasuta、immigrans、hypocausta、quadrilineata、curviceps)中12个代表物种的rDNA的ITS1和部分Adh基因的序列。其中ITS1序列的长度为513－587bp,共有191个信息位点;Adh基因片段的长度在714－747bp之间,共99个信息位点。考虑到单个分子提供的信息较少,将两个分子的序列综合起来,组成一个较长的复合序列。分别根据ITS1,Adh和两个分子的复合序列排比（Alignment)结果,和最大简约法和邻接法构建分子系统树,其中根据复合序列构建的系统树与形态学研究结果最为一致。分子树显示curviceps种亚组的特种确定单独形成一个分枝,为种亚组级的分类阶元,支持了形态学将其建立为一个新种亚组。根据Kimura距离,估算了复合分子的替换速率约为每百万年1．48％,进而计算出5个种亚组的分 歧年代。结合各物种的地理分布,推测了immigrans种组的进化历史：curviceps种亚组与quadrilineata种亚组的亲缘关系最近,主要分布在中国南部的温带地区。它们之间的分歧时间大约为3．4百万年,是最年轻的两个种亚组。主要分布在苏门答腊及附近的热带地区的hypocausta种亚组的物种是最早分化出来的,与其他种亚组的分歧时间约为9．2百万年。该结果与形态学和生物地理学研究相吻合。值得一提是的,目前归属仍存在争议的物种D.neohypocausta,在分子系统树中与hypocausta种亚组的物种相距较远,而与immiagrasn种亚组的关系较近,但分枝置信度较低（＜50％）。由于还缺乏其他方面的证据,因此D.neohypocausta的归属有待今后的研究来作定论。     

Drosophila Mechanotransduction—Linking Proteins and Functions

The sensation of touch, gravity, and sound all rely on dedicated ion channels that transduce mechanical stimulus forces into electrical response signals. The functional workings and molecular identities of these mechanotransducer channels are little understood. Recent work shows that the mechanotransducers for fly and vertebrate hearing share equivalent gating mechanisms, whereby this mechanism can be probed non-invasively in the mechanics of the Drosophila ear. Here, we describe how this mechanics can be used to evaluate the roles of identified proteins in the process of mechanosensation and, specifically, their contributions to mechanotransduction.     

Drosophila Memory: Dopamine Signals Punishment?

Dopamine-containing neurons are widespread in the fly brain and have been implicated in negatively reinforced memory. Current technology allows the investigator to watch dopaminergic neurons in action in the brain of a learning fly.     

Komplementäre DNA-Replikationsmuster bei Drosophila melanogaster

In D. melanogaster salivary gland chromosomes continuous and discontinuous labeling patterns have been described. Arcos-Terán (1972) has devided those of the X-chromosome in into a 6 class sequence beginning with continuous labeling and ending with labeling restricted to a few heavy bands. Labeling patterns reverse to the described ones have not been reported. — In the present communication new types of discontinuous labeling patterns are described for the X of melanogaster females. One type shows unlabeling of the majority of heavy bands and of the chromocenter whereas interbands and faint bands are labeled. In other types the number of unlabeled heavy bands is decreased and the chromosome is nearly totally labeled. These new labeling patterns are the reverse of Arcos-Terán's patterns II, IV, and V. It must therefore be assumed that the continuous pattern of labeling corresponds to the (early) middle of the replication cycle and that replication in melanogaster females starts with the labeling patterns described here.     

An Adaptive Immune Response in Drosophila?

The ability to mount an adaptive immune response is thought to be an attribute restricted to vertebrates. A new study conducted in Drosophila demonstrates that invertebrate immunity can adapt to an immune challenge and mount a specific immune response.     

Heterosis × nutrition interaction in Drosophila melanogaster

Summary The relationship between heterozygosity and the expression of heterosis at two different nutrition levels was investigated using Drosophila melanogaster. Average daily egg production and egg hatchability were measured in two parental strains and in F₁, F₂ and reciprocal backcross generations. Heterosis was more pronounced in the poor nutritional conditions. Two electrophoretic markers used to estimate the level of heterozygosity in F₂ and backcrosses revealed an excess of heterozygous genotypes. Quantitative genetic effects (an additive line effect and individual and maternal heterosis) were estimated for both traits in the two environments. Although this model gave a reasonable fit in most cases, some epistatic interaction would have to be invoked in order to explain fully the results.     

Thermal stability of α-glycerophosphate dehydrogenase in Drosophila

Thermal stability of -glycerophosphate dehydrogenase-1 (-Gpdh-1) in nine Drosophila species was studied at pH's ranging from 6.4 to 8.5. This was done by measuring the changes in the activity of enzymes during the heat denaturation process. In addition to temperature, the rate of denaturation is highly dependent on the pH of the incubation buffer. The results of this study show that the thermal stability of enzyme molecules is different in different species. This holds true also in the species in which the enzymes have been found to be identical by other means. The differences between species of the Drosophila virilis group are discussed.This study was supported by funds from the National Research Council of Sciences of Finland.     

Die optischen Übertragungseigenschaften der Komplexaugen von Drosophila

Summary The transfer properties of the optical system in the arthropod compound eye are determined by the interommatidial angle , influencing the resolving power, and by the width of the visual fields of single ommatidia , influencing the response at high spatial frequencies of brightness distributions in the object space. The energy transfer/ receptor is proportional to ( )² and decreases with in-inreasing approximation of the perfect-imaging condition: gD 0; 0. However, a value > 0 has to be maintained in order to overcome the threshold of nervous excitation at a certain minimum-brightness level. Theoretical treatment yields /=0.62 to 0.88 as the corresponding optimum-imaging relation. The actual ratio can be derived from measurements of the optomotor reactions to the movement of periodic brightness patterns. The approximate value 0.76 is obtained from the fruitfly Drosophila with normal and mutant eye pigmentation. As a result, the parameters of this imaging system are found to be established in a way that enables optimum performance at sufficient illumination. An dieser Stelle möchte ich Dr. W. Reichardt für sein eingehendes Interesse und manche anregende Diskussion über die Sehvorgänge im Komplexauge meinen Dank sagen. Dr. K. Kirschfeld verdanke ich ebenfalls wertvolle Hinweise. Herrn E. Freiberg bin ich für die Anfertigung der Abbildungen sehr verbunden.     

Organized microtubule arrays in γ-tubulin-depleted Drosophila spermatocytes

To assess the role of γ-tubulin in spindle assembly in vivo, we have followed meiosis progression by immunofluorescence and time-lapse video microscopy in γTub23C^PI mutant spermatocytes. We have found that centrosomes associate with large numbers of astral microtubules even though γ-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells. Several lines of evidence suggest that these cones may be related to wild-type central spindles. First, they are assembled midway through meiosis and elongate during anaphase. Second, they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone. Third, Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones. Finally, ectopic cytokinesis furrows are often formed at the distal end of the cone. Our results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a γ-tubulin-independent manner in Drosophila spermatocytes. However, γ-tubulin seems to be essential for spindle assembly in these cells. Finally, our results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles.     

Pervasive Natural Selection in the Drosophila Genome?

Over the past four decades, the predominant view of molecular evolution saw little connection between natural selection and genome evolution, assuming that the functionally constrained fraction of the genome is relatively small and that adaptation is sufficiently infrequent to play little role in shaping patterns of variation within and even between species. Recent evidence from Drosophila, reviewed here, suggests that this view may be invalid. Analyses of genetic variation within and between species reveal that much of the Drosophila genome is under purifying selection, and thus of functional importance, and that a large fraction of coding and noncoding differences between species are adaptive. The findings further indicate that, in Drosophila, adaptations may be both common and strong enough that the fate of neutral mutations depends on their chance linkage to adaptive mutations as much as on the vagaries of genetic drift. The emerging evidence has implications for a wide variety of fields, from conservation genetics to bioinformatics, and presents challenges to modelers and experimentalists alike.     

Drosophila olfaction: the end of stereotypy?

Recent work has demonstrated substantial wiring and functional stereotypy in the fly olfactory system. In this issue of Neuron, Murthy et al. demonstrate that in the mushroom body, a site of olfactory associative learning, this initial peripheral stereotypy gives way to functionally nonstereotyped circuits.     

Axon–glial interactions at the Drosophila CNS midline

The glia that reside at the midline of the Drosophila CNS are an important embryonic signaling center and also wrap the axons that cross the CNS. The development of the midline glia (MG) is characterized by migration, ensheathment, subdivision of axon commissures, apoptosis, and the extension of glial processes. All of these events are characterized by cell-cell contact between MG and adjacent neurons. Cell adhesion and signaling proteins that mediate different aspects of MG development and MG–neuron interactions have been identified. This provides a foundation for ultimately obtaining an integrated picture of how the MG assemble into a characteristic axonal support structure in the CNS.     

NF-��B in the Immune Response of Drosophila

The nuclear factor κB (NF-κB) pathways play a major role in Drosophila host defense. Two recognition and signaling cascades control this immune response. The Toll pathway is activated by Gram-positive bacteria and by fungi, whereas the immune deficiency (Imd) pathway responds to Gram-negative bacterial infection. The basic mechanisms of recognition of these various types of microbial infections by the adult fly are now globally understood. Even though some elements are missing in the intracellular pathways, numerous proteins and interactions have been identified. In this article, we present a general picture of the immune functions of NF-κB in Drosophila with all the partners involved in recognition and in the signaling cascades.The paramount roles of NF-κB family members in Drosophila development and host defense are now relatively well established and have been the subject of several in-depth reviews in recent years, including some from this laboratory (e.g., Hoffmann 2003; Minakhina and Steward 2006; Ferrandon et al. 2007; Lemaitre and Hoffmann 2007; Aggarwal and Silverman 2008). To avoid excessive duplication, we limit this text to the general picture that has evolved over nearly two decades—since the initial demonstration that the dorsal gene plays a role in dorsoventral patterning in embryogenesis of Drosophila and that it encodes a member of the NF-κB family of inducible transactivators (Nüsslein-Volhard et al. 1980; Steward 1987; Roth et al. 1989). In the early nineties, it became apparent that NF-κB also plays a role in the antimicrobial host defense of Drosophila (Engström et al. 1993; Ip et al. 1993; Kappler et al. 1993; Reichhart et al. 1993). We focus in this article on the immune functions of NF-κB and refer the reader to recent reviews for the roles of NF-κB in development (Roth 2003; Brennan and Anderson 2004; Moussian and Roth 2005; Minakhina and Steward 2006).The Drosophila genome codes for three NF-κB family members (Fig. 1). Dorsal and DIF (for dorsal-related immunity factor) are 70 kDa proteins, with a typical Rel homology domain, which is 45% identical to that of the mammalian counterparts c-Rel, Rel A, and Rel B. Dorsal and DIF lie some 10 kbp apart on the second chromosome and probably arose from a recent duplication (Meng et al. 1999). Both proteins are retained in the cytoplasm by binding to the same 54-kDa inhibitor protein Cactus, which is homologous to mammalian IκBs (Schüpbach and Wieshaus 1989; Geisler et al. 1992). The single Drosophila Cactus gene is closest to mammalian IκBα (Huguet et al. 1997). The third member of the family in Drosophila, Relish, is a 100-kDa protein with an amino-terminal Rel domain and a carboxy-terminal extension with typical ankyrin repeats, as found in Cactus and mammalian IκBs. Relish is similar to mammalian p100 and p105 and its activation requires proteolytic cleavage as in the case for these mammalian counterparts (reviewed in Hultmark 2003).Open in a separate windowFigure 1.The NF-κB and IκB proteins in Drosophila. The length in amino acids is indicated by numbers. REL, Rel-homology domain; NLS, nuclear localization sequence; PEST, proline, glutamic acid, serine, and threonine-rich segment; Ac, acidic domain.Put in simple terms, NF-κB family members function in the host defense of Drosophila to control the expression of genes encoding immune-responsive peptides and proteins. Prominent among the induced genes are those encoding peptides with direct antimicrobial activity. To exert this function, Dorsal and DIF are translocated to the nucleus following stimulus-induced degradation of the inhibitor Cactus, whereas Relish requires stimulus-induced proteolytic cleavage for nuclear translocation of its amino-terminal Rel domain. This paradigm is similar to that observed in mammalian immunity. Again, for the sake of simplicity, we may say that the stimulus-induced degradation of Cactus, and the concomitant release of Dorsal or DIF, is primarily observed during Gram-positive bacterial and fungal infections and mediated by the Toll signaling pathway. In contrast, stimulus-induced proteolytic cleavage of Relish, and concomitant nuclear translocation of its amino-terminal Rel domain, is the hallmark of the response to Gram-negative bacterial infection and mediated by the Imd signaling pathway. Whether these pathways are also involved in the multifaceted defense against viruses remains an open question (Zambon et al. 2005). The Toll pathway was further shown to be involved in hematopoiesis of flies (Qiu et al. 1998). Of note, the Cactus-NF-κB module also plays a central role in the elimination of Plasmodium parasites in infected mosquitoes (Frolet et al. 2006). In the following, we review our information of the two established signaling pathways, Toll and Imd, which lead to gene reprogramming through NF-κB in response to bacterial and fungal infections. We first consider the upstream mechanisms that mediate the recognition of infection and allow for a certain level of discrimination between invading microorganisms. Gene reprogramming in this context is best illustrated by the induction of the antimicrobial peptide genes, which serve as the most convenient readouts of the antimicrobial defense of Drosophila (see Samakovlis et al. 1990; Reichhart et al. 1992; Ferrandon et al. 1998). Flies produce at least seven families of mostly cationic, small-sized, membrane-active peptides, with spectra variously directed against Gram-positive (defensins) and Gram-negative (diptericins, attacins, and drosocin) bacteria, and against fungi (drosomycins and metchnikowins), or with overlapping spectra (cecropins) (reviewed in Bulet et al. 1999; Hetru et al. 2003). The primary site of biosynthesis of these peptides is the fat body, a functional equivalent of the mammalian liver. Blood cells also participate in the production of antimicrobial peptides. As a rule, these molecules are secreted into the hemolymph where they reach remarkably high concentrations to oppose invading microorganisms (Hetru et al. 2003). This facet of the antimicrobial host defense is generally referred to as systemic immune response. Of note, the gut and the tracheae also produce antimicrobial peptides in response to microbes (see Tzou et al. 2000; Onfelt Tingvall et al. 2001; Liehl et al. 2006; Nehme et al. 2007).During infection, the Toll and Imd pathways control the expression of hundreds of genes. In addition to the antimicrobial peptides, these genes encode proteases, putative cytokines, cytoskeletal proteins, and many peptides and proteins whose function in the host defense are still not understood (De Gregorio et al. 2001; Irving et al. 2001).