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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.

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.

Background Endosymbiotic bacteria of the genus Spiroplasma (Mollicutes) are widespread among arthropods and plants with some lineages inducing male killing in several insect hosts. In the pea aphid, Acyrthosiphon pisum , three divergent clades of Spiroplasma ixodetis have been described, but male killing has been so far reported in only one clade. Here, we aimed to assess the distribution of male-killing phenotype among the three clades and investigated links between genomic variation and male-killing phenotypes in S. ixodetis infecting pea aphids. Results Field collections in eastern France revealed heterogeneous infection patterns across host-plant-associated biotypes, with 17% of established aphid lines carrying S. ixodetis strains spanning three clades. Sexual reproduction of these lines was induced under laboratory conditions to assess male production as a proxy for male killing, revealing a wide range of variation from complete absence of males to normal male production. Comparative genomic analyses of Spiroplasma strains associated with reduced or normal male production and from the different clades uncovered striking divergence in genome structure, including marked differences in synteny and gene content. Considerable variation was found in the copy number of putative virulence-associated genes amongst the clades, including those encoding ribosome-inactivating proteins (RIPs), ankyrin repeat domains, and ovarian tumour (OTU) domains. Notably, none of the strains encoded Spaid, the effector responsible for male killing in S. poulsonii from Drosophila . Strains within the same clade exhibited highly similar gene repertoires yet differed in putative male killing phenotype, and no unique gene nor genomic feature could be linked to reduced male production. Conclusions This study investigated the evolutionary dynamics and effects of Spiroplasma ixodetis infections on male production in the pea aphid Acyrthosiphon pisum . Our findings provide insights into the diversity and potential evolutionary trajectories of Spiroplasma –aphid associations, laying the groundwork for elucidating the genetic basis of male killing in this system. Moreover, our results highlight the challenges of pinpointing the genetic determinants of reproductive manipulation across diverse symbiont–host systems.

Tsetse ( Glossina spp.) are vectors of African trypanosomes, the causative agents of Human and African Animal trypanosomiases, diseases that remain significant medical and socioeconomic challenges in sub-Saharan Africa. In addition to trypanosomes, tsetse harbor both obligate and facultative symbiotic bacteria that can influence vector competence and reproductive biology. One such facultative symbiont, Spiroplasma glossinidia , infects several tsetse species within the Palpalis subgroup. In Glossina fuscipes fuscipes ( Gff ), the Spiroplasma glossinidia strain s Gff induces a trypanosome-refractory phenotype and negatively impacts reproductive fitness by reducing female fecundity. However, the mechanisms behind these Spiroplasma -derived phenotypes remain poorly understood. Here, we report successful in vitro cultivation of s Gff and present complete genomes from three sources: in vitro cultured s Gff and s Gff isolated from both laboratory-maintained and wild-caught (Uganda) Gff flies. Comparative genomic analyses revealed a high degree of similarity in gene content and synteny among these s Gff samples, confirming that they represent isolates of the same strain. Phylogenomic analyses placed s Gff within the Spiroplasma poulsonii clade. We found the s Gff genome to be highly dynamic, containing numerous mobile genetic elements. Additionally, in silico annotations indicated that s Gff relies on its host for both lipids and carbohydrates and can produce several toxins, all of which could be implicated in the observed trypanosome refractory phenotype. Finally, comparative transcriptomic analysis of s Gff from host hemolymph versus in vitro culture provided insights into potential factors relevant to host-symbiont interactions. Our findings provide a foundation for understanding the nutritional dialogue between s Gff and its host and identify symbiotic products that may contribute to trypanosome resistance. Furthermore, the establishment of an in vitro culture system for s Gff represents a significant resource for future functional studies with potential implications for vector control.

Profiling of wild and laboratory tsetse populations using 16S rRNA gene amplicon sequencing allowed us to examine whether the “ Wigglesworthia - Sodalis - Wolbachia dogma” operates across species and populations. The most abundant taxa, in wild and laboratory populations, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. The species richness of the microbiota was greater in wild than laboratory populations. Spiroplasma was identified as a new symbiont exclusively in Glossina fuscipes fuscipes and G . tachinoides , members of the palpalis sub-group, and the infection prevalence in several laboratory and natural populations was surveyed. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse-associated Spiroplasma , present in G . f . fuscipes and G . tachinoides . Spiroplasma density in G . f . fuscipes larva guts was significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density was higher in testes than ovaries, and was significantly higher density in live versus prematurely deceased females indicating a potentially mutualistic association. Higher Spiroplasma density in testes than in ovaries was also detected by fluorescent in situ hybridization in G . f . fuscipes .

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.

Defensive endosymbiotic bacteria conferring protection to their hosts against parasites and pathogens are widespread in insect populations. However, the mechanisms by which most symbionts confer protection are not fully understood. Here, we studied the mechanisms of protection against bacterial and fungal pathogens mediated by the Drosophila melanogaster endosymbiont Spiroplasma poulsonii . We demonstrate that besides the previously described protection against wasps and nematodes, Spiroplasma also confers increased resistance to pathogenic bacteria and fungi. We identified Spiroplasma -induced iron sequestration and melanization as key defense mechanisms. Our work broadens the known defense spectrum of Spiroplasma and reveals a previously unappreciated role of melanization and iron sequestration in endosymbiont-mediated host protection. We propose that the mechanisms we have identified here may be of broader significance and could apply to other endosymbionts, particularly to Wolbachia , and potentially explain their protective properties.

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.

Bacteria commonly deploy toxic proteins that act with specificity on target molecules to support invasion and improve survival in competitive environments. Many toxin-encoding bacteria have evolved into host-associated defensive partnerships, in which they use toxins to improve host survival during infection. The stability of these relationships requires that symbiont toxins target diverse parasites while minimizing damage to the host. We investigate the specificity of a group of ribosome-targeting toxins (ribosome-inactivating proteins) encoded by heritable Spiroplasma symbionts that contribute to defense against parasite infection in fruit fly hosts. Using Escherichia coli to express five divergent copies of this toxin, we show that distantly related members of the family all retain the ability to inactivate ribosomes by adenine cleavage at the α-sarcin/ricin loop, the enzymatic hallmark of RIPs. However, when exposed to live insect and fungal cells, ribosome inactivation varies across the five toxins, suggesting cellular recognition or localization play a role in target specificity. To identify toxin domains required for specificity, we removed rapidly evolving “accessory” domains from two toxins. Both truncated toxins exhibit significantly increased activity on purified ribosomes in vitro, suggesting one role of accessory domains is to reduce toxicity, which may help protect hosts from collateral damage. One of the truncated toxins also showed significantly reduced inactivation of cellular ribosomes in vivo, indicating a role for accessory domains in cell specificity. Together, these data reveal a mechanism for symbiont discrimination between hosts and parasites and highlight how dynamic toxin evolution can contribute to stability and novelty in defensive symbiosis.