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Several lineages of symbiotic bacteria in insects selfishly manipulate host reproduction to spread in a population 1 , often by distorting host sex ratios. Spiroplasma poulsonii 2 , 3 is a helical and motile, Gram-positive symbiotic bacterium that resides in a wide range of Drosophila species 4 . A notable feature of S. poulsonii is male killing, whereby the sons of infected female hosts are selectively killed during development 1 , 2 . Although male killing caused by S. poulsonii has been studied since the 1950s, its underlying mechanism is unknown. Here we identify an S. poulsonii protein, designated Spaid, whose expression induces male killing. Overexpression of Spaid in D. melanogaster kills males but not females, and induces massive apoptosis and neural defects, recapitulating the pathology observed in S. poulsonii -infected male embryos 5 – 11 . Our data suggest that Spaid targets the dosage compensation machinery on the male X chromosome to mediate its effects. Spaid contains ankyrin repeats and a deubiquitinase domain, which are required for its subcellular localization and activity. Moreover, we found a laboratory mutant strain of S. poulsonii with reduced male-killing ability and a large deletion in the spaid locus. Our study has uncovered a bacterial protein that affects host cellular machinery in a sex-specific way, which is likely to be the long-searched-for factor responsible for S. poulsonii -induced male killing. The Spaid protein is identified and shown to be responsible for the male-killing effects of Spiroplasma poulsonii in Drosophila.

Abstract The endosymbiotic bacteria Spiroplasma (Mollicutes) infect diverse plants and arthropods, and some of which induce male killing, where male hosts are killed during development. Male-killing Spiroplasma strains belong to either the phylogenetically distant Citri-Poulsonii or Ixodetis groups. In Drosophila flies, Spiroplasma poulsonii induces male killing via the Spaid toxin. While Spiroplasma ixodetis infects a wide range of insects and arachnids, little is known about the genetic basis of S. ixodetis-induced male killing. Here, we analyzed the genome of S. ixodetis strains in the pea aphid Acyrthosiphon pisum (Aphididae, Hemiptera). Genome sequencing constructed a complete genome of a male-killing strain, sAp269, consisting of a 1.5 Mb circular chromosome and an 80 Kb plasmid. sAp269 encoded putative virulence factors containing either ankyrin repeat, ovarian tumor-like deubiquitinase, or ribosome inactivating protein domains, but lacked the Spaid toxin. Further comparative genomics of Spiroplasma strains in A. pisum biotypes adapted to different host plants revealed their phylogenetic associations and the diversity of putative virulence factors. Although the mechanisms of S. ixodetis-induced male killing in pea aphids remain elusive, this study underlines the dynamic genome evolution of S. ixodetis and proposes independent acquisition events of male-killing mechanisms in insects. We analyzed the genomes of Spiroplasma ixodetis in the pea aphid Acyrthosiphon pisum, highlighting their dynamic genome evolution and the diversity in the underlying mechanisms of Spiroplasma-induced male killing.

Recent studies have shown that some plants and animals harbor microbial symbionts that protect them against natural enemies. Here we demonstrate that a maternally transmitted bacterium, Spiroplasma, protects Drosophila neotestacea against the sterilizing effects of a parasitic nematode, both in the laboratory and the field. This nematode parasitizes D. neotestacea at high frequencies in natural populations, and, until recently, almost all infections resulted in complete sterility. Several lines of evidence suggest that Spiroplasma is spreading in North American populations of D. neotestacea and that a major adaptive change to a symbiont-based mode of defense is under way. These findings demonstrate the profound and potentially rapid effects of defensive symbionts, which are increasingly recognized as major players in the ecology of species interactions.

Fungus-growing ants partner with multiple microbial symbionts to obtain food and remain free from disease. Of these symbionts, those inhabiting the ant gut remain the least understood and are known only from environmental surveys. Such surveys can infer potential functions of gut symbionts, but cultures are required to experimentally validate these hypotheses. Here, we describe the first cultures of the ant gut symbionts of the fungus-growing ant Trachymyrmex septentrionalis , using comparative genomics and phenotypic experiments to describe them as two novel species: Mesoplasma whartonense sp. nov. and Spiroplasma attinicola sp. nov. This genomic analysis suggests that these species are highly specialized to T. septentrionalis and are distinct from related environmental data generated from the related ant species Acromyrmex echinatior , implying substantial host specificity. Our phenotypic experiments and genomic reconstructions highlight the highly overlapping niches and likely costs and benefits of these symbionts to their ant host, setting the stage for further experimentation.

Competition between insects and their endosymbiotic bacteria for environmentally limited nutrients can compromise the fitness of both organisms. Tsetse flies, the vectors of pathogenic African trypanosomes, harbor a species and population-specific consortium of vertically transmitted endosymbiotic bacteria that range on the functional spectrum from mutualistic to parasitic. Tsetse's indigenous microbiota can include a member of the genus Spiroplasma, and infection with this bacterium causes fecundity-reducing phenotypes in the fly that include a prolonged gonotrophic cycle and a reduction in the motility of stored spermatozoa post-copulation. Herein we demonstrate that Spiroplasma and tsetse spermatozoa compete for fly-derived acylcarnitines, which in other bacteria and animals are used to maintain cell membranes and produce energy. The fat body of mated female flies increases acylcarnitine production in response to infection with Spiroplasma. Additionally, their spermathecae (sperm storage organs), and likely the sperm within, up-regulate expression of carnitine O-palmitoyltransferase-1, which is indicative of increased acylcarnitine metabolism and thus increased energy demand and energy production in this organ. These compensatory measures are insufficient to rescue the motility defect of spermatozoa stored in the spermathecae of Spiroplasma-infected females and thus results in reduced fly fecundity. Tsetse's taxonomically simple and highly tractable indigenous microbiota make the fly an efficient model system for studying the biological processes that facilitate the maintenance of bacterial endosymbioses, and how these relationships impact conserved mechanisms (mammalian spermatozoa also use acylcarnitines as an energy source) that regulated animal host fecundity. In the case of insect pests and vectors, a better understanding of the metabolic mechanisms that underlie these associations can lead to the development of novel control strategies.

Endosymbiotic bacteria associated with eukaryotic hosts are omnipresent in nature, particularly in insects. Studying the bacterial side of host-symbiont interactions is, however, often limited by the unculturability and genetic intractability of the symbionts. Spiroplasma poulsonii is a maternally transmitted bacterial endosymbiont that is naturally associated with several Drosophila species. S. poulsonii strongly affects its host’s physiology, for example by causing male killing or by protecting it against various parasites. Despite intense work on this model since the 1950s, attempts to cultivate endosymbiotic Spiroplasma in vitro have failed so far. Here, we developed a method to sustain the in vitro culture of S. poulsonii by optimizing a commercially accessible medium. We also provide a complete genome assembly, including the first sequence of a natural plasmid of an endosymbiotic Spiroplasma species. Last, by comparing the transcriptome of the in vitro culture to the transcriptome of bacteria extracted from the host, we identified genes putatively involved in host-symbiont interactions. This work provides new opportunities to study the physiology of endosymbiotic Spiroplasma and paves the way to dissect insect-endosymbiont interactions with two genetically tractable partners. IMPORTANCE The discovery of insect bacterial endosymbionts (maternally transmitted bacteria) has revolutionized the study of insects, suggesting novel strategies for their control. Most endosymbionts are strongly dependent on their host to survive, making them uncultivable in artificial systems and genetically intractable. Spiroplasma poulsonii is an endosymbiont of Drosophila that affects host metabolism, reproduction, and defense against parasites. By providing the first reliable culture medium that allows a long-lasting in vitro culture of Spiroplasma and by elucidating its complete genome, this work lays the foundation for the development of genetic engineering tools to dissect endosymbiosis with two partners amenable to molecular study. Furthermore, the optimization method that we describe can be used on other yet uncultivable symbionts, opening new technical opportunities in the field of host-microbes interactions. The discovery of insect bacterial endosymbionts (maternally transmitted bacteria) has revolutionized the study of insects, suggesting novel strategies for their control. Most endosymbionts are strongly dependent on their host to survive, making them uncultivable in artificial systems and genetically intractable. Spiroplasma poulsonii is an endosymbiont of Drosophila that affects host metabolism, reproduction, and defense against parasites. By providing the first reliable culture medium that allows a long-lasting in vitro culture of Spiroplasma and by elucidating its complete genome, this work lays the foundation for the development of genetic engineering tools to dissect endosymbiosis with two partners amenable to molecular study. Furthermore, the optimization method that we describe can be used on other yet uncultivable symbionts, opening new technical opportunities in the field of host-microbes interactions.

Spiroplasma (Mycoplasmatales: Spiroplasmataceae) is one of the most widely distributed symbionts of arthropods. Spiroplasma species can infect their hosts via vertical or horizontal transmission. However, the mode of transmission of Spiroplasma between different arthropod taxa has not been elucidated. In this study, we investigated the potential for the transmission of Spiroplasma to non-native arthropod species, using 2 Spiroplasma spp. isolated from ticks, namely Spiroplasma ixodetis and Spiroplasma mirum, and 3 species of mosquito laboratory colonies, namely Aedes albopictus, Aedes aegypti, and Culex pipiens pallens (Diptera: Culicidae).After feeding the adult mosquitoes with Spiroplasma-containing artificial meals, they were kept at 25 °C for 10 days. Homogenates prepared from Spiroplasma-fed mosquitoes were used to re-isolate Spiroplasma using the in vitro culture method. Nine weeks after culture initiation, the presence of Spiroplasma was tested using the polymerase chain reaction (PCR).The results revealed that only S. ixodetis was detected from all 3 species of mosquitoes and re-isolated from 2 of them.The differences in the infection ability of different Spirolasma species could be attributed to several factors, including environmental effects. Nevertheless, this is the first experimental demonstration of Spiroplasma transmission among different arthropod taxa. Further studies are needed to elucidate the evolutionary mechanism that supports the survival of Spiroplasma in nature. Graphical Abstract

Background Tsetse flies, the biological vectors of African trypanosomes, have established symbiotic associations with different bacteria. Their vector competence is suggested to be affected by bacterial endosymbionts. The current study provided the prevalence of three tsetse symbiotic bacteria and trypanosomes in Glossina species from Burkina Faso. Results A total of 430 tsetse flies were captured using biconical traps in four different collection sites around Bobo-Dioulasso (Bama, Bana, Nasso, and Peni), and their guts were removed. Two hundred tsetse were randomly selected and their guts were screened by PCR for the presence of Sodalis glossinidius , Spiroplasma sp., Wolbachia and trypanosomes. Of the 200 tsetse, 196 (98.0%) were Glossina palpalis gambiensis and 4 (2.0%) Glossina tachinoides . The overall symbiont prevalence was 49.0%, 96.5%, and 45.0%, respectively for S. glossinidius , Spiroplasma and Wolbachia . Prevalence varied between sampling locations: S. glossinidius (54.7%, 38.5%, 31.6%, 70.8%); Spiroplasma (100%, 100%, 87.7%, 100%); and Wolbachia (43.4%, 38.5%, 38.6%, 70.8%), respectively in Bama, Bana, Nasso and Peni. Noteworthy, no G. tachnoides was infected by S. glossinidius and Wolbachia , but they were all infected by Spiroplasma sp . A total of 196 (98.0%) harbored at least one endosymbionts. Fifty-five (27.5%) carried single endosymbiont. Trypanosomes were found only in G. p. gambiensis , but not G. tachinoides . Trypanosomes were present in flies from all study locations with an overall prevalence of 29.5%. In Bama, Bana, Nasso, and Peni, the trypanosome infection rate was respectively 39.6%, 23.1%, 8.8%, and 37.5%. Remarkably, only Trypanosoma grayi was present. Of all trypanosome-infected flies, 55.9%, 98.3%, and 33.9% hosted S. glossinidius , Spiroplasma sp and Wolbachia , respectively. There was no association between Sodalis , Spiroplasma and trypanosome presence, but there was a negative association with Wolbachia presence. We reported 1.9 times likelihood of trypanosome absence when Wolbachia was present. Conclusion This is the first survey reporting the presence of Trypanosoma grayi in tsetse from Burkina Faso. Tsetse from these localities were highly positive for symbiotic bacteria, more predominantly with Spiroplasma sp . Modifications of symbiotic interactions may pave way for disease control.

Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense.

Tsetse flies (Glossina spp.) are vectors of parasitic trypanosomes, which cause human (HAT) and animal African trypanosomiasis (AAT) in sub-Saharan Africa. In Uganda, Glossina fuscipes fuscipes (Gff) is the main vector of HAT, where it transmits Gambiense disease in the northwest and Rhodesiense disease in central, southeast and western regions. Endosymbionts can influence transmission efficiency of parasites through their insect vectors via conferring a protective effect against the parasite. It is known that the bacterium Spiroplasma is capable of protecting its Drosophila host from infection with a parasitic nematode. This endosymbiont can also impact its host's population structure via altering host reproductive traits. Here, we used field collections across 26 different Gff sampling sites in northern and western Uganda to investigate the association of Spiroplasma with geographic origin, seasonal conditions, Gff genetic background and sex, and trypanosome infection status. We also investigated the influence of Spiroplasma on Gff vector competence to trypanosome infections under laboratory conditions. Generalized linear models (GLM) showed that Spiroplasma probability was correlated with the geographic origin of Gff host and with the season of collection, with higher prevalence found in flies within the Albert Nile (0.42 vs 0.16) and Achwa River (0.36 vs 0.08) watersheds and with higher prevalence detected in flies collected in the intermediate than wet season. In contrast, there was no significant correlation of Spiroplasma prevalence with Gff host genetic background or sex once geographic origin was accounted for in generalized linear models. Additionally, we found a potential negative correlation of Spiroplasma with trypanosome infection, with only 2% of Spiroplasma infected flies harboring trypanosome co-infections. We also found that in a laboratory line of Gff, parasitic trypanosomes are less likely to colonize the midgut in individuals that harbor Spiroplasma infection. These results indicate that Spiroplasma infections in tsetse may be maintained by not only maternal but also via horizontal transmission routes, and Spiroplasma infections may also have important effects on trypanosome transmission efficiency of the host tsetse. Potential functional effects of Spiroplasma infection in Gff could have impacts on vector control approaches to reduce trypanosome infections.