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We report the draft whole-genome assembly of Microsporidia sp. MB , a symbiotic malaria-transmission-blocking microsporidian isolated from Anopheles arabiensis in Kenya. The whole-genome sequence of Microsporidia sp. MB has a length of 5,908,979 bp, 2,335 contigs, and an average GC content of 31.12%., Competing Interests: The authors declare no conflict of interest.

Importance: Microsporidia MB is a symbiont with a strong malaria transmission-blocking phenotype in Anopheles arabiensis . It spreads in mosquito populations through mother-to-offspring and sexual transmission. The ability of Microsporidia MB to block Plasmodium transmission, together with its ability to spread within Anopheles populations and its avirulence to the host, makes it a very attractive candidate for developing a key strategy to stop malaria transmissions. Here, we report tissue tropism and localization patterns of Microsporidia MB , which are relevant to its transmission. We find that Microsporidia MB accumulates in Anopheles arabiensis tissues, linked to its sexual and vertical transmission. Its prevalence and intensity in the tissues over the mosquito life cycle suggest adaptation to maximize transmission and avirulence in Anopheles arabiensis . These findings provide the foundation for understanding the factors that may affect Microsporidia MB transmission efficiency. This will contribute to the development of strategies to maximize Microsporidia MB transmission to establish and sustain a high prevalence of the symbiont in Anopheles mosquito populations for malaria transmission blocking., Competing Interests: The authors declare no conflict of interest.

Unlabelled: Insects commonly harbor facultative bacterial endosymbionts, such as Wolbachia and Spiroplasma species, that are vertically transmitted from mothers to their offspring. These endosymbiontic bacteria increase their propagation by manipulating host reproduction or by protecting their hosts against natural enemies. While an increasing number of studies have reported endosymbiont-mediated protection, little is known about the mechanisms underlying this protection. Here, we analyze the mechanisms underlying protection from parasitoid wasps in Drosophila melanogaster mediated by its facultative endosymbiont Spiroplasma poulsonii Our results indicate that S. poulsonii exerts protection against two distantly related wasp species, Leptopilina boulardi and Asobara tabida S. poulsonii-mediated protection against parasitoid wasps takes place at the pupal stage and is not associated with an increased cellular immune response. In this work, we provide three important observations that support the notion that S. poulsonii bacteria and wasp larvae compete for host lipids and that this competition underlies symbiont-mediated protection. First, lipid quantification shows that both S. poulsonii and parasitoid wasps deplete D. melanogaster hemolymph lipids. Second, the depletion of hemolymphatic lipids using the Lpp RNA interference (Lpp RNAi) construct reduces wasp success in larvae that are not infected with S. poulsonii and blocks S. poulsonii growth. Third, we show that the growth of S. poulsonii bacteria is not affected by the presence of the wasps, indicating that when S. poulsonii is present, larval wasps will develop in a lipid-depleted environment. We propose that competition for host lipids may be relevant to endosymbiont-mediated protection in other systems and could explain the broad spectrum of protection provided., Importance: Virtually all insects, including crop pests and disease vectors, harbor facultative bacterial endosymbionts. They are vertically transmitted from mothers to their offspring, and some protect their host against pathogens. Here, we studied the mechanism of protection against parasitoid wasps mediated by the Drosophila melanogaster endosymbiont Spiroplasma poulsonii Using genetic manipulation of the host, we provide strong evidence supporting the hypothesis that competition for host lipids underlies S. poulsonii-mediated protection against parasitoid wasps. We propose that lipid competition-based protection may not be restricted to Spiroplasma bacteria but could also apply other endosymbionts, notably Wolbachia bacteria, which can suppress human disease-causing viruses in insect hosts., (Copyright © 2016 Paredes et al.)

Unlabelled: Spiroplasmas are helical and motile members of a cell wall-less eubacterial group called Mollicutes. Although all spiroplasmas are associated with arthropods, they exhibit great diversity with respect to both their modes of transmission and their effects on their hosts; ranging from horizontally transmitted pathogens and commensals to endosymbionts that are transmitted transovarially (i.e., from mother to offspring). Here we provide the first genome sequence, along with proteomic validation, of an endosymbiotic inherited Spiroplasma bacterium, the Spiroplasma poulsonii MSRO strain harbored by Drosophila melanogaster. Comparison of the genome content of S. poulsonii with that of horizontally transmitted spiroplasmas indicates that S. poulsonii has lost many metabolic pathways and transporters, demonstrating a high level of interdependence with its insect host. Consistent with genome analysis, experimental studies showed that S. poulsonii metabolizes glucose but not trehalose. Notably, trehalose is more abundant than glucose in Drosophila hemolymph, and the inability to metabolize trehalose may prevent S. poulsonii from overproliferating. Our study identifies putative virulence genes, notably, those for a chitinase, the H2O2-producing glycerol-3-phosphate oxidase, and enzymes involved in the synthesis of the eukaryote-toxic lipid cardiolipin. S. poulsonii also expresses on the cell membrane one functional adhesion-related protein and two divergent spiralin proteins that have been implicated in insect cell invasion in other spiroplasmas. These lipoproteins may be involved in the colonization of the Drosophila germ line, ensuring S. poulsonii vertical transmission. The S. poulsonii genome is a valuable resource to explore the mechanisms of male killing and symbiont-mediated protection, two cardinal features of many facultative endosymbionts., Importance: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. These endosymbionts play key roles in their hosts' fitness, including protecting them against natural enemies and manipulating their reproduction in ways that increase the frequency of symbiont infection. Little is known about the molecular mechanisms that underlie these processes. Here, we provide the first genome draft of a vertically transmitted male-killing Spiroplasma bacterium, the S. poulsonii MSRO strain harbored by D. melanogaster. Analysis of the S. poulsonii genome was complemented by proteomics and ex vivo metabolic experiments. Our results indicate that S. poulsonii has reduced metabolic capabilities and expresses divergent membrane lipoproteins and potential virulence factors that likely participate in Spiroplasma-host interactions. This work fills a gap in our knowledge of insect endosymbionts and provides tools with which to decipher the interaction between Spiroplasma bacteria and their well-characterized host D. melanogaster, which is emerging as a model of endosymbiosis., (Copyright © 2015 Paredes et al.)

Unlabelled: Spiroplasma is a diverse bacterial clade that includes many vertically transmitted insect endosymbionts, including Spiroplasma poulsonii, a natural endosymbiont of Drosophila melanogaster. These bacteria persist in the hemolymph of their adult host and exhibit efficient vertical transmission from mother to offspring. In this study, we analyzed the mechanism that underlies their vertical transmission, and here we provide strong evidence that these bacteria use the yolk uptake machinery to colonize the germ line. We show that Spiroplasma reaches the oocyte by passing through the intercellular space surrounding the ovarian follicle cells and is then endocytosed into oocytes within yolk granules during the vitellogenic stages of oogenesis. Mutations that disrupt yolk uptake by oocytes inhibit vertical Spiroplasma transmission and lead to an accumulation of these bacteria outside the oocyte. Impairment of yolk secretion by the fat body results in Spiroplasma not reaching the oocyte and a severe reduction of vertical transmission. We propose a model in which Spiroplasma first interacts with yolk in the hemolymph to gain access to the oocyte and then uses the yolk receptor, Yolkless, to be endocytosed into the oocyte. Cooption of the yolk uptake machinery is a powerful strategy for endosymbionts to target the germ line and achieve vertical transmission. This mechanism may apply to other endosymbionts and provides a possible explanation for endosymbiont host specificity., Importance: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. Studies have shown that many facultative endosymbionts, including Spiroplasma, confer protection against different classes of parasites on their hosts and therefore are attractive tools for the control of vector-borne diseases. The ability to be efficiently transmitted from females to their offspring is the key feature shaping associations between insects and their inherited endosymbionts, but to date, little is known about the mechanisms involved. In oviparous animals, yolk accumulates in developing eggs and serves to meet the nutritional demands of embryonic development. Here we show that Spiroplasma coopts the yolk transport and uptake machinery to colonize the germ line and ensure efficient vertical transmission. The uptake of yolk is a female germ line-specific feature and therefore an attractive target for cooption by endosymbionts that need to maintain high-fidelity maternal transmission.