The Structure of Spiroplasma Virus 4: Exploring the Capsid Diversity of the Microviridae.

Spiroplasma virus 4 (SpV4) is a bacteriophage of the Microviridae, which packages circular ssDNA within non-enveloped T = 1 icosahedral capsids. It infects spiroplasmas, which are known pathogens of honeybees. Here, the structure of the SpV4 virion is determined using cryo-electron microscopy to a resolution of 2.5 Å. A striking feature of the SpV4 capsid is the mushroom-like protrusions at the 3-fold axes, which is common among all members of the subfamily Gokushovirinae. While the function of the protrusion is currently unknown, this feature varies widely in this subfamily and is therefore possibly an adaptation for host recognition. Furthermore, on the interior of the SpV4 capsid, the location of DNA-binding protein VP8 was identified and shown to have low structural conservation to the capsids of other viruses in the family. The structural characterization of SpV4 will aid future studies analyzing the virus-host interaction, to understand disease mechanisms at a molecular level. Furthermore, the structural comparisons in this study, including a low-resolution structure of the chlamydia phage 2, provide an overview of the structural repertoire of the viruses in this family that infect various bacterial hosts, which in turn infect a wide range of animals and plants.

The endosymbiont Spiroplasma poulsonii increases Drosophila melanogaster resistance to pathogens by enhancing iron sequestration and melanization.

Facultative endosymbiotic bacteria, such as Wolbachia and Spiroplasma species, are commonly found in association with insects and can dramatically alter their host physiology. Many endosymbionts are defensive and protect their hosts against parasites or pathogens. Despite the widespread nature of defensive insect symbioses and their importance for the ecology and evolution of insects, the mechanisms of symbiont-mediated host protection remain poorly characterized. Here, we utilized the fruit fly Drosophila melanogaster and its facultative endosymbiont Spiroplasma poulsonii to characterize the mechanisms underlying symbiont-mediated host protection against bacterial and fungal pathogens. Our results indicate a variable effect of S. poulsonii on infection outcome, with endosymbiont-harboring flies being more resistant to Rhyzopus oryzae, Staphylococcus aureus, and Providencia alcalifaciens but more sensitive or as sensitive as endosymbiont-free flies to the infections with Pseudomonas species. Further focusing on the protective effect, we identified Transferrin-mediated iron sequestration induced by Spiroplasma as being crucial for the defense against R. oryzae and P. alcalifaciens. In the case of S. aureus, enhanced melanization in Spiroplasma-harboring flies plays a major role in protection. Both iron sequestration and melanization induced by Spiroplasma require the host immune sensor protease Persephone, suggesting a role of proteases secreted by the symbiont in the activation of host defense reactions. Hence, our work reveals a broader defensive range of Spiroplasma than previously appreciated and adds nutritional immunity and melanization to the defensive arsenal of symbionts. IMPORTANCE: 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.

Cloning and sequencing analysis of whole Spiroplasma genome in yeast.

Cloning and transfer of long-stranded DNA in the size of a bacterial whole genome has become possible by recent advancements in synthetic biology. For the whole genome cloning and whole genome transplantation, bacteria with small genomes have been mainly used, such as mycoplasmas and related species. The key benefits of whole genome cloning include the effective maintenance and preservation of an organism's complete genome within a yeast host, the capability to modify these genome sequences through yeast-based genetic engineering systems, and the subsequent use of these cloned genomes for further experiments. This approach provides a versatile platform for in-depth genomic studies and applications in synthetic biology. Here, we cloned an entire genome of an insect-associated bacterium, Spiroplasma chrysopicola, in yeast. The 1.12 Mbp whole genome was successfully cloned in yeast, and sequences of several clones were confirmed by Illumina sequencing. The cloning efficiency was high, and the clones contained only a few mutations, averaging 1.2 nucleotides per clone with a mutation rate of 4 × 10-6. The cloned genomes could be distributed and used for further research. This study serves as an initial step in the synthetic biology approach to Spiroplasma.

Spiroplasma infection as a cause of severe congenital keratouveitis, cataract and glaucoma.

BACKGROUND: Only seven cases of ocular Spiroplasma infection have been reported to date, all presenting as congenital cataracts with concomitant intraocular inflammation. We describe the first case of Spiroplasma infection initially presenting as a corneal infiltrate. CASE PRESENTATION: A 1-month-old girl was referred for a corneal infiltrate in the left eye. She presented in our hospital with unilateral keratouveitis. Examination showed a stromal corneal infiltrate and dense white keratic precipitates in the left eye. Herpetic keratouveitis was suspected and intravenous acyclovir therapy was initiated. Two weeks later, the inflammation in the left eye persisted and was also noticed in the right eye. Acute angle-closure glaucoma and a cataract with dilated iris vessels extending onto the anterior lens capsule developed in the left eye. The inflammation resolved after treatment with azithromycin. Iridectomy, synechiolysis and lensectomy were performed. Bacterial metagenomic sequencing (16 S rRNA) and transmission electron microscopy revealed Spiroplasma ixodetis species in lens aspirates and biopsy. Consequently, a diagnosis of bilateral Spiroplasma uveitis was made. CONCLUSIONS: In cases of congenital cataract with concomitant intraocular inflammation, Spiroplasma infection should be considered. The purpose of this case report is to raise awareness of congenital Spiroplasma infection as a cause of severe keratouveitis, cataract and angle-closure glaucoma in newborns. Performing molecular testing on lens aspirates is essential to confirm diagnosis. Systemic macrolides are suggested as the mainstay of treatment.

Genomic insights into Spiroplasma endosymbionts that induce male-killing and protective phenotypes in the pea aphid.

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.

Comparative analysis of Spodoptera frugiperda (J. E. Smith) (Lepidoptera, Noctuidae) corn and rice strains microbiota revealed minor changes across life cycle and strain endosymbiont association.

Background: Spodoptera frugiperda (FAW) is a pest that poses a significant threat to corn production worldwide, causing millions of dollars in losses. The species has evolved into two strains (corn and rice) that differ in their genetics, reproductive isolation, and resistance to insecticides and Bacillus thuringiensis endotoxins. The microbiota plays an important role in insects' physiology, nutrient acquisition, and response to chemical and biological controls. Several studies have been carried out on FAW microbiota from larvae guts using laboratory or field samples and a couple of studies have analyzed the corn strain microbiota across its life cycle. This investigation reveals the first comparison between corn strain (CS) and rice strain (RS) of FAW during different developmental insect stages and, more importantly, endosymbiont detection in both strains, highlighting the importance of studying both FAW populations and samples from different stages. Methods: The composition of microbiota during the life cycle of the FAW corn and rice strains was analyzed through high-throughput sequencing of the bacterial 16S rRNA gene using the MiSeq system. Additionally, culture-dependent techniques were used to isolate gut bacteria and the Transcribed Internal Spacer-ITS, 16S rRNA, and gyrB genes were examined to enhance bacterial identification. Results: Richness, diversity, and bacterial composition changed significantly across the life cycle of FAW. Most diversity was observed in eggs and males. Differences in gut microbiota diversity between CS and RS were minor. However, Leuconostoc, A2, Klebsiella, Lachnoclostridium, Spiroplasma, and Mucispirilum were mainly associated with RS and Colidextribacter, Pelomonas, Weissella, and Arsenophonus to CS, suggesting that FAW strains differ in several genera according to the host plant. Firmicutes and Proteobacteria were the dominant phyla during FAW metamorphosis. Illeobacterium, Ralstonia, and Burkholderia exhibited similar abundancies in both strains. Enterococcus was identified as a conserved taxon across the entire FAW life cycle. Microbiota core communities mainly consisted of Enterococcus and Illeobacterium. A positive correlation was found between Spiroplasma with RS (sampled from eggs, larvae, pupae, and adults) and Arsenophonus (sampled from eggs, larvae, and adults) with CS. Enterococcus mundtii was predominant in all developmental stages. Previous studies have suggested its importance in FAW response to B. thuringensis. Our results are relevant for the characterization of FAW corn and rice strains microbiota to develop new strategies for their control. Detection of Arsenophonus in CS and Spiroplasma in RS are promising for the improvement of this pest management, as these bacteria induce male killing and larvae fitness reduction in other Lepidoptera species.

AGRAMP: machine learning models for predicting antimicrobial peptides against phytopathogenic bacteria.

Introduction: Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics for combating plant pathogenic bacteria in agriculture and the environment. However, identifying potent AMPs through laborious experimental assays is resource-intensive and time-consuming. To address these limitations, this study presents a bioinformatics approach utilizing machine learning models for predicting and selecting AMPs active against plant pathogenic bacteria. Methods: N-gram representations of peptide sequences with 3-letter and 9-letter reduced amino acid alphabets were used to capture the sequence patterns and motifs that contribute to the antimicrobial activity of AMPs. A 5-fold cross-validation technique was used to train the machine learning models and to evaluate their predictive accuracy and robustness. Results: The models were applied to predict putative AMPs encoded by intergenic regions and small open reading frames (ORFs) of the citrus genome. Approximately 7% of the 10,000-peptide dataset from the intergenic region and 7% of the 685,924-peptide dataset from the whole genome were predicted as probable AMPs. The prediction accuracy of the reported models range from 0.72 to 0.91. A subset of the predicted AMPs was selected for experimental test against Spiroplasma citri, the causative agent of citrus stubborn disease. The experimental results confirm the antimicrobial activity of the selected AMPs against the target bacterium, demonstrating the predictive capability of the machine learning models. Discussion: Hydrophobic amino acid residues and positively charged amino acid residues are among the key features in predicting AMPs by the Random Forest Algorithm. Aggregation propensity appears to be correlated with the effectiveness of the AMPs. The described models would contribute to the development of effective AMP-based strategies for plant disease management in agricultural and environmental settings. To facilitate broader accessibility, our model is publicly available on the AGRAMP (Agricultural Ngrams Antimicrobial Peptides) server.

Experimental demonstration of the transmission of Spiroplasma between different arthropod taxa.

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.

Rapid Visual Detection of Spiroplasma eriocheiris by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye.

In recent years, a new type of Spiroplasma has been found that can cause "tremor disease" of the Chinese mitten crab Eriocheir sinensis. The outbreak of epidemic tremor disease has caused a serious setback in the Chinese mitten crab farming industry, with an incidence rate of more than 30% and mortality rates of 80-100%. Therefore, finding a sensitive method to detect tremor disease in E. sinensis has become a current research focus. In this research, a loop-mediated isothermal amplification detection method coupled with hydroxynaphthol blue dye (LAMP-HNB) was developed and used to rapidly detect Spiroplasma eriocheiris. First, we designed and synthesized specific outer primers, inner primers and loop primers based on the 16S ribosomal RNA gene of S. eriocheiris. Second, the LAMP-HNB detection method for S. eriocheiris was successfully established by screening the primers, adjusting the temperature and time of the reaction, and optimizing the concentrations of Mg2+ and dNTPs. In the specific tests, only samples infected with S. eriocheiris showed positive results, and other infections caused by bacteria and parasites tested negative, proving that the test has high specificity. Moreover, the detection limit was 2.5 × 10-6 ng/µL, indicating high sensitivity. This method for detecting S. eriocheiris provides rapid visual output based on LAMP-HNB detection and is a simple, fast, sensitive, and inexpensive method that can be applied to a wide range of field investigations.

Rare Spiroplasma Bloodstream Infection in Patient after Surgery, China, 2022.

We report a case of Spiroplasma bloodstream infection in a patient in China who developed pulmonary infection, acute respiratory distress syndrome, sepsis, and septic shock after emergency surgery for type A aortic dissection. One organism closely related to Spiroplasma eriocheiris was isolated from blood culture and identified by whole-genome sequencing.

Development and application of the MIRA and MIRA-LFD detection methods of Spiroplasma eriocheiris.

The tremor disease (TD) caused by Spiroplasma eriocheiris is the most destructive disease of the Chinese mitten crab, Eriocheir sinensis. This study attempts to construct Multienzyme Isothermal Rapid Amplification (MIRA), a quick and simple nucleic acid amplification method that operates at room temperature. Based on the gene sequences of S. eriocheiris, appropriate amplification primers were constructed and screened in this investigation. Both the relevant specific probe and the chosen specific amplification primers were designed and labeled. The MIRA and MIRA-LFD reaction conditions were then optimized. The result showed MIRA and MIRA-FFD could identify S. eriocheiris at 37 °C in 30 min and 15 min, respectively. To investigate the specificity of MIRA and MIRA-LFD, three Gram-negative bacteria (Bacillus subtilis, Bacillus thuringiensis, and Staphylococcus aureus), three Gram-positive bacteria (Escherichia coli, Aeromonas hydrophila, and Salmonella typhimurium) and S. eriocheiris were selected. The result showed MIRA and MIRA-LFD were highly specific to S. eriocheiris and did not react with other six pathogens. The sensitivities of PCR, MIRA, and MIRA-LFD were then evaluated. The result showed the detection limit of PCR is 1 ng/L whereas the detection limit of MIRA and MIRA-LFD is 10 pg/L. Finally, the established MIRA and MIRA-LFD detection methods had the advantages of being quick, sensitive, and specific for S. eriocheiris detection, as well as not requiring any specialized equipment.

Microbiome of Zoophytophagous Biological Control Agent Nesidiocoris tenuis.

Many insects are associated with endosymbionts that influence the feeding, reproduction, and distribution of their hosts. Although the small green mirid, Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), a zoophytophagous predator that feeds on plants as well as arthropods, is a globally important biological control agent, its microbiome has not been sufficiently studied. In the present study, we assessed the microbiome variation in 96 N. tenuis individuals from 14 locations throughout Japan, based on amplicon sequencing of the 16S ribosomal RNA gene. Nine major bacteria associated with N. tenuis were identified: Rickettsia, two strains of Wolbachia, Spiroplasma, Providencia, Serratia, Pseudochrobactrum, Lactococcus, and Stenotrophomonas. Additionally, a diagnostic PCR analysis for three typical insect reproductive manipulators, Rickettsia, Wolbachia, and Spiroplasma, was performed on a larger sample size (n = 360) of N. tenuis individuals; the most prevalent symbiont was Rickettsia (69.7%), followed by Wolbachia (39.2%) and Spiroplasma (6.1%). Although some symbionts were co-infected, their prevalence did not exhibit any specific tendency, such as a high frequency in specific infection combinations. The infection frequency of Rickettsia was significantly correlated with latitude and temperature, while that of Wolbachia and Spiroplasma was significantly correlated with host plants. The predominance of these bacteria and the absence of obligate symbionts suggested that the N. tenuis microbiome is typical for predatory arthropods rather than sap-feeding insects. Rickettsia and Wolbachia were vertically transmitted rather than horizontally transmitted from the prey. The functional validation of each symbiont would be warranted to develop N. tenuis as a biological control agent.

Self-stabilization mechanism encoded by a bacterial toxin facilitates reproductive parasitism.

A wide variety of maternally transmitted endosymbionts in insects are associated with reproductive parasitism, whereby they interfere with host reproduction to increase the ratio of infected females and spread within populations.1,2 Recent successes in identifying bacterial factors responsible for reproductive parasitism3,4,5,6,7 as well as further omics approaches8,9,10,11,12 have highlighted the common appearance of deubiquitinase domains, although their biological roles-in particular, how they link to distinct manipulative phenotypes-remain poorly defined. Spiroplasma poulsonii is a helical and motile bacterial endosymbiont of Drosophila,13,14 which selectively kills male progeny with a male-killing toxin Spaid (S. poulsonii androcidin), which encodes an ovarian tumor (OTU) deubiquitinase domain.6 Artificial expression of Spaid in flies reproduces male-killing-associated pathologies that include abnormal apoptosis and neural defects during embryogenesis6,15,16,17,18,19; moreover, it highly accumulates on the dosage-compensated male X chromosome,20 congruent with cellular defects such as the DNA damage/chromatin bridge breakage specifically induced upon that chromosome.6,21,22,23 Here, I show that without the function of OTU, Spaid is polyubiquitinated and degraded through the host ubiquitin-proteasome pathway, leading to the attenuation of male-killing activity as shown previously.6 Furthermore, I find that Spaid utilizes its OTU domain to deubiquitinate itself in an intermolecular manner. Collectively, the deubiquitinase domain of Spaid serves as a self-stabilization mechanism to facilitate male killing in flies, optimizing a molecular strategy of endosymbionts that enables the efficient manipulation of the host at a low energetic cost.

iTRAQ-based differential proteomic analysis of high- and low-virulence strains of Spiroplasma eriocheiris.

Spiroplasma eriocheiris is one of the major pathogenic bacteria in crustaceans, featuring high infectivity, rapid transmission, and an absence of effective control strategies, resulting in significant economic losses to the aquaculture industry. Research into virulence-related factors provides an important perspective to clarify how Spiroplasma eriocheiris is pathogenic to shrimps and crabs. Therefore, in this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was utilized to undertake a differential proteomic analysis of high- and low-virulence Spiroplasma eriocheiris strains at different growth phases. A total of 868 differentially expressed proteins (DEPs) were obtained, of which 31 novel proteins were identified by proteogenomic analysis. There were 62, 61, 175, and 235 DEPs between the log phase (YD) and non-log phase (YFD) of the high-virulence strain, between the log phase (CD) and non-log phase (CFD) of the low-virulence strain, between YD and CD, and between CFD and YFD, respectively. All the DEPs were compared with virulence protein databases (MvirDB and VFDB), and 68 virulence proteins of Spiroplasma eriocheiris were identified, of which 12 were involved in a total of 21 metabolic pathways, including motility, chemotaxis, growth, metabolism and virulence of the bacteria. The results of this study form the basis for further research into the molecular mechanism of virulence and physiological differences between high- and low-virulence strains of Spiroplasma eriocheiris, and provide a scientific basis for a detailed understanding of its pathogenesis.

Molecular detection of Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts in wild population of tsetse flies collected in Cameroon, Chad and Nigeria.

BACKGROUND: Tsetse flies are cyclical vectors of African trypanosomiasis (AT). The flies have established symbiotic associations with different bacteria that influence certain aspects of their physiology. Vector competence of tsetse flies for different trypanosome species is highly variable and is suggested to be affected by bacterial endosymbionts amongst other factors. Symbiotic interactions may provide an avenue for AT control. The current study provided prevalence of three tsetse symbionts in Glossina species from Cameroon, Chad and Nigeria. RESULTS: Tsetse flies were collected and dissected from five different locations. DNA was extracted and polymerase chain reaction used to detect presence of Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts, using species specific primers. A total of 848 tsetse samples were analysed: Glossina morsitans submorsitans (47.52%), Glossina palpalis palpalis (37.26%), Glossina fuscipes fuscipes (9.08%) and Glossina tachinoides (6.13%). Only 95 (11.20%) were infected with at least one of the three symbionts. Among infected flies, six (6.31%) had Wolbachia and Spiroplasma mixed infection. The overall symbiont prevalence was 0.88, 3.66 and 11.00% respectively, for Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts. Prevalence varied between countries and tsetse fly species. Neither Spiroplasma species nor S. glossinidius were detected in samples from Cameroon and Nigeria respectively. CONCLUSION: The present study revealed, for the first time, presence of Spiroplasma species infections in tsetse fly populations in Chad and Nigeria. These findings provide useful information on repertoire of bacterial flora of tsetse flies and incite more investigations to understand their implication in the vector competence of tsetse flies.

Male-killer symbiont screening reveals novel associations in Adalia ladybirds.

While male-killing bacteria are known to infect across arthropods, ladybird beetles represent a hotspot for these symbioses. In some host species, there are multiple different symbionts that vary in presence and frequency between populations. To further our understanding of spatial and frequency variation, we tested for the presence of three male-killing bacteria: Wolbachia , Rickettsia and Spiroplasma , in two Adalia ladybird species from a previously unexplored UK population. The two-spot ladybird, A. bipunctata, is known to harbour all three male-killers, and we identified Spiroplasma infection in the Merseyside population for the first time. However, in contrast to previous studies on two-spot ladybirds from continental Europe, evidence from egg-hatch rates indicates the Spiroplasma strain present in the Merseyside population does not cause embryonic male-killing. In the related ten-spot ladybird, A. decempunctata, there is only one previous record of a male-killing symbiont, a Rickettsia , which we did not detect in the Merseyside sample. However, PCR assays indicated the presence of a Spiroplasma in a single A. decempunctata specimen. Marker sequence indicated that this Spiroplasma was divergent from that found in sympatric A. bipunctata. Genome sequencing of the Spiroplasma -infected A. decempunctata additionally revealed the presence of cobionts in the form of a Centistes parasitoid wasp and the parasitic fungi Beauveria. Further study of A. decempunctata from this population is needed to resolve whether it is the ladybird or wasp cobiont that harbours Spiroplasma , and to establish the phenotype of this strain. These data indicate first that microbial symbiont phenotype should not be assumed from past studies conducted in different locations, and second that cobiont presence may confound screening studies aimed to detect the frequency of a symbiont in field collected material from a focal host species.

The toxins of vertically transmitted Spiroplasma.

Vertically transmitted (VT) microbial symbionts play a vital role in the evolution of their insect hosts. A longstanding question in symbiont research is what genes help promote long-term stability of vertically transmitted lifestyles. Symbiont success in insect hosts is due in part to expression of beneficial or manipulative phenotypes that favor symbiont persistence in host populations. In Spiroplasma, these phenotypes have been linked to toxin and virulence domains among a few related strains. However, these domains also appear frequently in phylogenetically distant Spiroplasma, and little is known about their distribution across the Spiroplasma genus. In this study, we present the complete genome sequence of the Spiroplasma symbiont of Drosophila atripex, a non-manipulating member of the Ixodetis clade of Spiroplasma, for which genomic data are still limited. We perform a genus-wide comparative analysis of toxin domains implicated in defensive and reproductive phenotypes. From 12 VT and 31 non-VT Spiroplasma genomes, ribosome-inactivating proteins (RIPs), OTU-like cysteine proteases (OTUs), ankyrins, and ETX/MTX2 domains show high propensity for VT Spiroplasma compared to non-VT Spiroplasma. Specifically, OTU and ankyrin domains can be found only in VT-Spiroplasma, and RIP domains are found in all VT Spiroplasma and three non-VT Spiroplasma. These domains are frequently associated with Spiroplasma plasmids, suggesting a possible mechanism for dispersal and maintenance among heritable strains. Searching insect genome assemblies available on public databases uncovered uncharacterized Spiroplasma genomes from which we identified several spaid-like genes encoding RIP, OTU, and ankyrin domains, suggesting functional interactions among those domain types. Our results suggest a conserved core of symbiont domains play an important role in the evolution and persistence of VT Spiroplasma in insects.

Screening and functional analysis of three Spiroplasma eriocheiris glycosylated protein interactions with Macrobrachium nipponense C-type lectins.

N-glycosylation, one of the main protein posttranslational modifications (PTMs), plays an important role in the pathogenic process of pathogens through binding and invasion of host cells or regulating the internal environment of host cells to benefit their survival. However, N-glycosylation has remained mostly unexplored in Spiroplasma eriocheiris, a novel type of pathogen which has serious adverse effects on aquaculture. In most cases, N-glycoproteins can be detected and analyzed by lectins dependent on sugar recognition domains. In this study, three Macrobrachium nipponense C-type lectins, namely, MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3, were used to screen S. eriocheiris glycosylated proteins. First, qRT-PCR results showed that the expression levels of the three kinds of lectins were all significantly up-regulated in prawn hearts when the host was against S. eriocheiris infection. A bacterial binding assay showed that purified recombinant MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3 could directly bind to S. eriocheiris in vitro. Second, three S. eriocheiris glycosylated proteins, ATP synthase subunit beta (ATP beta), molecular chaperone Dnak (Dnak) and fructose bisphosphate aldolase (FBPA), were screened and identified using the three kinds of full-length C-type lectins. Far-Western blot and coimmunoprecipitation (CO-IP) further demonstrated that there were interactions between the three lectins with ATP beta, Dnak and FBPA. Furthermore, antibody neutralization assay results showed that pretreatment of S. eriocheiris with ATP beta, Dnak and FBPA antibodies could significantly block this pathogen infection. All the above studies showed that the glycosylated protein played a vital role in the process of S. eriocheiris infection.

Diverse Molecular Mechanisms Underlying Microbe-Inducing Male Killing in the Moth Homona magnanima.

Male killing (MK) is a type of reproductive manipulation induced by microbes, where sons of infected mothers are killed during development. MK is a strategy that enhances the fitness of the microbes, and the underlying mechanisms and the process of their evolution have attracted substantial attention. Homona magnanima, a moth, harbors two embryonic MK bacteria, namely, Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), and a larval MK virus, Osugoroshi virus (OGV; Partitiviridae). However, whether the three distantly related male killers employ similar or different mechanisms to accomplish MK remains unknown. Here, we clarified the differential effects of the three male killers on the sex-determination cascades and development of H. magnanima males. Reverse transcription-PCR demonstrated that Wolbachia and Spiroplasma, but not OGVs, disrupted the sex-determination cascade of males by inducing female-type splice variants of doublesex (dsx), a downstream regulator of the sex-determining gene cascade. We also found that MK microbes altered host transcriptomes in different manners; Wolbachia impaired the host dosage compensation system, whereas Spiroplasma and OGVs did not. Moreover, Wolbachia and Spiroplasma, but not OGVs, triggered abnormal apoptosis in male embryos. These findings suggest that distantly related microbes employ distinct machineries to kill males of the identical host species, which would be the outcome of the convergent evolution. IMPORTANCE Many microbes induce male killing (MK) in various insect species. However, it is not well understood whether microbes adopt similar or different MK mechanisms. This gap in our knowledge is partly because different insect models have been examined for each MK microbe. Here, we compared three taxonomically distinct male killers (i.e., Wolbachia, Spiroplasma, and a partiti-like virus) that infect the same host. We provided evidence that microbes can cause MK through distinct mechanisms that differ in the expression of genes involved in sex determination, dosage compensation, and apoptosis. These results imply independent evolutionary scenarios for the acquisition of their MK ability.

African Queens find mates when males are rare.

In butterflies and moths, male-killing endosymbionts are transmitted from infected females via their eggs, and the male progeny then perish. This means that successful transmission of the parasite relies on the successful mating of the host. Paradoxically, at the population level, parasite transmission also reduces the number of adult males present in the final population for infected females to mate with. Here we investigate if successful female mating when males are rare is indeed a likely rate-limiting step in the transmission of male-killing Spiroplasma in the African Monarch, Danaus chrysippus. In Lepidoptera, successful pairings are hallmarked by the transfer of a sperm-containing spermatophore from the male to the female during copulation. Conveniently, this spermatophore remains detectable within the female upon dissection, and thus, spermatophore counts can be used to assess the frequency of successful mating in the field. We used such spermatophore counts to examine if altered sex ratios in the D. chrysippus do indeed affect female mating success. We examined two different field sites in East Africa where males were often rare. Surprisingly, mated females carried an average of 1.5 spermatophores each, regardless of male frequency, and importantly, only 10-20% remained unmated. This suggests that infected females will still be able to mate in the face of either Spiroplasma-mediated male killing and/or fluctuations in adult sex ratio over the wet-dry season cycle. These observations may begin to explain how the male-killing mollicute can still be successfully transmitted in a population where males are rare.