Cryptic community structure and metabolic interactions among the heritable facultative symbionts of the pea aphid.

Most insects harbour influential, yet non-essential heritable microbes in their hemocoel. Communities of these symbionts exhibit low diversity. But their frequent multi-species nature raises intriguing questions on roles for symbiont-symbiont synergies in host adaptation, and on the stability of the symbiont communities, themselves. In this study, we build on knowledge of species-defined symbiont community structure across US populations of the pea aphid, Acyrthosiphon pisum. Through extensive symbiont genotyping, we show that pea aphids' microbiomes can be more precisely defined at the symbiont strain level, with strain variability shaping five out of nine previously reported co-infection trends. Field data provide a mixture of evidence for synergistic fitness effects and symbiont hitchhiking, revealing causes and consequences of these co-infection trends. To test whether within-host metabolic interactions predict common versus rare strain-defined communities, we leveraged the high relatedness of our dominant, community-defined symbiont strains vs. 12 pea aphid-derived Gammaproteobacteria with sequenced genomes. Genomic inference, using metabolic complementarity indices, revealed high potential for cooperation among one pair of symbionts-Serratia symbiotica and Rickettsiella viridis. Applying the expansion network algorithm, through additional use of pea aphid and obligate Buchnera symbiont genomes, Serratia and Rickettsiella emerged as the only symbiont community requiring both parties to expand holobiont metabolism. Through their joint expansion of the biotin biosynthesis pathway, these symbionts may span missing gaps, creating a multi-party mutualism within their nutrient-limited, phloem-feeding hosts. Recent, complementary gene inactivation, within the biotin pathways of Serratia and Rickettsiella, raises further questions on the origins of mutualisms and host-symbiont interdependencies.

Food provisioning to Pardosa spiders decreases the levels of tissue-resident endosymbiotic bacteria.

The diversity, host specificity, and physiological effects of endosymbiotic bacteria in spiders (Araneae) are poorly characterized. We used 16S rDNA sequencing to evaluate endosymbionts in the cephalothorax and legs of a wolf spider Pardosa agrestis. We tested the effects of feeding once or twice daily with fruit flies, aphids, or starved and compared them to those of syntopically occurring Pardosa palustris. The feeding increased traveled distance up to five times in some of the groups provisioned with food relative to the starved control. The Shannon diversity t-test revealed significant differences between these component communities of the two spider species. The increased frequency of feeding with fruit flies, but not aphids, increased the dominance and decreased the alpha diversity of OTUs. The obligate or facultative endosymbionts were present in all analyzed spider individuals and were represented mostly by Rickettsiella, Rhabdochlamydia, Spiroplasma, and the facultative intracellular parasite Legionella. Vertically transmitted endosymbionts were less common, represented by Wolbachia pipientis and Rickettsia sp. H820. The relative abundance of Mycoplasma spp. was negatively correlated with provisioned or killed aphids. In conclusion, the tissues of Pardosa spiders host tremendously diverse assemblages of bacteria, including obligate or facultative endosymbionts, with yet unknown phenotypic effects.

A rapidly spreading deleterious aphid endosymbiont that uses horizontal as well as vertical transmission.

Endosymbiotic bacteria that live inside the cells of insects are typically only transmitted maternally and can spread by increasing host fitness and/or modifying reproduction in sexual hosts. Transinfections of Wolbachia endosymbionts are now being used to introduce useful phenotypes into sexual host populations, but there has been limited progress on applications using other endosymbionts and in asexual populations. Here, we develop a unique pathway to application in aphids by transferring the endosymbiont Rickettsiella viridis to the major crop pest Myzus persicae. Rickettsiella infection greatly reduced aphid fecundity, decreased heat tolerance, and modified aphid body color, from light to dark green. Despite inducing host fitness costs, Rickettsiella spread rapidly through caged aphid populations via plant-mediated horizontal transmission. The phenotypic effects of Rickettsiella were sensitive to temperature, with spread only occurring at 19 °C and not 25 °C. Body color modification was also lost at high temperatures despite Rickettsiella maintaining a high density. Rickettsiella shows the potential to spread through natural M. persicae populations by horizontal transmission and subsequent vertical transmission. Establishment of Rickettsiella in natural populations could reduce crop damage by modifying population age structure, reducing population growth and providing context-dependent effects on host fitness. Our results highlight the importance of plant-mediated horizontal transmission and interactions with temperature as drivers of endosymbiont spread in asexual insect populations.

The inside scoop: Comparative genomics of two intranuclear bacteria, "Candidatus Berkiella cookevillensis" and "Candidatus Berkiella aquae".

"Candidatus Berkiella cookevillensis" (strain CC99) and "Candidatus Berkiella aquae" (strain HT99), belonging to the Coxiellaceae family, are gram-negative bacteria isolated from amoebae in biofilms present in human-constructed water systems. Both bacteria are obligately intracellular, requiring host cells for growth and replication. The intracellular bacteria-containing vacuoles of both bacteria closely associate with or enter the nuclei of their host cells. In this study, we analyzed the genome sequences of CC99 and HT99 to better understand their biology and intracellular lifestyles. The CC99 genome has a size of 2.9Mb (37.9% GC) and contains 2,651 protein-encoding genes (PEGs) while the HT99 genome has a size of 3.6Mb (39.4% GC) and contains 3,238 PEGs. Both bacteria encode high proportions of hypothetical proteins (CC99: 46.5%; HT99: 51.3%). The central metabolic pathways of both bacteria appear largely intact. Genes for enzymes involved in the glycolytic pathway, the non-oxidative branch of the phosphate pathway, the tricarboxylic acid pathway, and the respiratory chain were present. Both bacteria, however, are missing genes for the synthesis of several amino acids, suggesting reliance on their host for amino acids and intermediates. Genes for type I and type IV (dot/icm) secretion systems as well as type IV pili were identified in both bacteria. Moreover, both bacteria contain genes encoding large numbers of putative effector proteins, including several with eukaryotic-like domains such as, ankyrin repeats, tetratricopeptide repeats, and leucine-rich repeats, characteristic of other intracellular bacteria.

The Genetic Diversity of Rickettsiella Symbionts in Ixodes ricinus Throughout Europe.

Rickettsiella species are bacterial symbionts that are present in a great variety of arthropod species, including ixodid ticks. However, little is known about their genetic diversity and distribution in Ixodes ricinus, as well as their relationship with other tick-associated bacteria. In this study, we investigated the occurrence and the genetic diversity of Rickettsiella spp. in I. ricinus throughout Europe and evaluated any preferential and antagonistic associations with Candidatus Midichloria mitochondrii and the pathogens Borrelia burgdorferi sensu lato and Borrelia miyamotoi. Rickettsiella spp. were detected in most I. ricinus populations investigated, encompassing a wide array of climate types and environments. The infection prevalence significantly differed between geographic locations and was significantly higher in adults than in immature life stages. Phylogenetic investigations and protein characterization disclosed four Rickettsiella clades (I-IV). Close phylogenetic relations were observed between Rickettsiella strains of I. ricinus and other arthropod species. Isolation patterns were detected for Clades II and IV, which were restricted to specific geographic areas. Lastly, although coinfections occurred, we did not detect significant associations between Rickettsiella spp. and the other tick-associated bacteria investigated. Our results suggest that Rickettsiella spp. are a genetically and biologically diverse facultative symbiont of I. ricinus and that their distribution among tick populations could be influenced by environmental components.

Molecular Detection of Tick-borne Pathogens in Ticks Collected from Hainan Island, China.

Pathogens like bacteria and protozoa, which affect human and animal health worldwide, can be transmitted by vectors like ticks. To investigate the epidemiology and genetic diversity of bacteria and protozoans carried by ticks in Chengmai county of Hainan province, China, 285 adult hard ticks belonging to two species [ Rhipicephalus sanguineus ( sensu lato): 183, 64.21% and Rhipicephalus microplus: 102, 35.79%] from dogs, cattle, and goats were collected. Microbial families were identified in these ticks by amplifying the 18S rRNA, 16S rRNA ( rrs), citrate synthase ( gltA), and heat shock protein ( groEL) genes. Our data revealed the presence of four recognized species and two Candidatus spp. of Anaplasmataceae and Coxiellaceae. In sum, these data reveal an extensive diversity of Anaplasmataceae bacteria, Coxiellaceae bacteria, Babesiidae, and Hepatozoidae in ticks from Hainan Island, highlighting the need to understand the tick-borne pathogen infection in local animals and humans.

A robust phylogenetic framework for members of the order Legionellales and its main genera (Legionella, Aquicella, Coxiella and Rickettsiella) based on phylogenomic analyses and identification of molecular markers demarcating different clades.

The order Legionellales contains several clinically important microorganisms. Although members of this order are well-studied for their pathogenesis, there is a paucity of reliable characteristics distinguishing members of this order and its constituent genera. Genome sequences are now available for 73 Legionellales species encompassing ≈90% of known members from different genera. With the aim of understanding evolutionary relationships and identifying reliable molecular characteristics that are specific for this order and its constituent genera, detailed phylogenetic and comparative analyses were conducted on the protein sequences from these genomes. A phylogenomic tree was constructed based on 393 single copy proteins that are commonly shared by the members of this order to delineate the evolutionary relationships among its members. In parallel, comparative analyses were performed on protein sequences from Legionellales genomes to identify novel molecular markers consisting of conserved signature indels (CSIs) that are specific for different clades and genera. In the phylogenomic tree and in an amino acid identity matrix based on core proteins, members of the genera Aquicella, Coxiella, Legionella and Rickettsiella formed distinct clades confirming their monophyly. In these studies, Diplorickettsia massiliensis exhibited a close relationship to members of the genus Rickettsiella. The results of our comparative genomic analyses have identified 59 highly specific molecular markers consisting of CSIs in diverse proteins that are uniquely shared by different members of this order. Four of these CSIs are specific for all Legionellales species, except the two deeper-branching "Candidatus Berkiella" species, providing means for identifying members of this order in molecular terms. Twenty four, 7 and 6 CSIs are uniquely shared by members of the genera Legionella, Coxiella and Aquicella, respectively, identifying these groups in molecular terms. The descriptions of these three genera are emended to include information for their novel molecular characteristics. We also describe 12 CSIs that are uniquely shared by D. massiliensis and different members of the genus Rickettsiella. Based on these results, we are proposing an integration of the genus Diplorickettsia with Rickettsiella. Three other CSIs suggest that members of the genera Coxiella and Rickettsiella shared a common ancestor exclusive of other Legionellales. The described molecular markers, due to their exclusivity for the indicated taxa/genera, provide important means for the identification of these clinically important microorganisms and for discovering novel properties unique to them.

Coxiellaceae in Ticks from Human, Domestic and Wild Hosts from Sardinia, Italy: High Diversity of Coxiella-like Endosymbionts.

PURPOSE: Coxiella burnetii is known for its potential as veterinary and human bacterial pathogen. The bacteria have been described in ticks, but their role in transmission of Q fever in humans is considered low. Coxiella endosymbionts closely related to C. burnetii have been also isolated from an extensive range of tick species and evidence is growing that these endosymbionts could be linked to human bacteremia. The aim of this study was to get new information on the presence of Coxiella species in ticks infesting wild and domestic hosts in Sardinia, Italy. METHODS: Here, 138 ticks collected from the study area were analyzed for the presence of C. burnetii and Coxiella-like bacteria by polymerase chain reaction (PCR), sequencing and philogenetic analyses using a set of primers targeting the 16S rRNA gene. RESULTS: DNA of Coxiella species was detected in 69% of the total ticks examined. Based on phylogenetic analysis, the 16S rRNA Coxiella genotypes identified in this study grouped in strongly supported monophyletic clades with identified reference sequences of CLEs detected from Rhipicephalus, Dermacentor, Haemaphysalis and Ornithodoros species and with Coxiella burnetii strains isolated worldwide. CONCLUSION: This study reports the molecular detection of a high diversity of Coxiella-like bacteria in Sardinian ticks and confirms also the presence of C. burnetii in tick species previously identified in the island. The role that Coxiella-like endosymbionts play in Sardinian ticks and in their vertebrate hosts needs to be explored further.

Molecular Detection of Tick-borne Pathogens in Ticks Collected from Hainan Island, China / 生物医学与环境科学（英文）

Pathogens like bacteria and protozoa, which affect human and animal health worldwide, can be transmitted by vectors like ticks. To investigate the epidemiology and genetic diversity of bacteria and protozoans carried by ticks in Chengmai county of Hainan province, China, 285 adult hard ticks belonging to two species [

Rickettsia hoogstraalii and a Rickettsiella from the Bat Tick Argas transgariepinus, in Namibia.

Ectoparasites were collected from Eptesicus hottentotus, the long-tailed serotine bat, caught in Namibia as part of an ecological study. Larvae of Argas transgariepinus, a blood-feeding ectoparasite of bats in Africa, were removed from 3 of 18 bats. We present scanning electron microscope images of unengorged larvae. As with other ectoparasites, this bat tick might transmit pathogens such as Borrelia and Rickettsia to their hosts as has been reported for bat ticks in Europe and North America. We screened 3 pools (25 total) of larvae of A. transgariepinus removed from the long-tailed serotine bat Eptesicus hottentotus caught in Namibia. Two microbes of unknown pathogenicity, including Rickettsia hoogstraalii, a spotted fever group pathogen, and a Rickettsiella sp. were detected by molecular techniques.

Infection With a Novel Rickettsiella Species in Emperor Scorpions (Pandinus imperator).

Rickettsiella infection was diagnosed in 4 adult emperor scorpions (Pandinus imperator) from 2 different collections over a 3-year period. One case had a 2-day history of weakness, failure to lift the tail, or respond to stimulation, with rapid progression to death. The other 3 cases were found dead. There were no gross lesions, but histologically the hemolymphatic vasculature and sinuses, presumed hematopoietic organ, heart, midgut and midgut diverticula, nerves, and skeletal muscle were infiltrated with phagocytic and granular hemocytes with necrosis. Phagocytic hemocytes contained abundant intracellular microorganisms that were Fite's acid-fast-positive, Macchiavello-positive, variably gram-positive or gram-negative, and Grocott's methenamine silver-negative. By transmission electron microscopy, hemocytes contained numerous phagocytic vacuoles with small dense bacterial forms (mean 0.603 × 0.163 µm) interspersed with large bacterial forms (mean 1.265 × 0.505 µm) and few intermediary forms with electron-dense nucleoids and membrane-bound crystalline arrays (average 4.72 µm). Transmission electron microscopy findings were consistent with bacteria of the family Coxiellaceae. Based on sequencing the 16S ribosomal RNA gene, the identity was confirmed as Rickettsiella, and phylogenetic analysis of protein-coding genes gidA, rspA, and sucB genes suggested the emperor scorpion pathogen as a new species. This study identifies a novel Rickettsiella causing infection in emperor scorpions and characterizes the unique pathological findings of this disease. We suggest this organism be provisionally named Rickettsiella scorpionisepticum.

Endosymbiotic Rickettsiella causes cytoplasmic incompatibility in a spider host.

Many arthropod hosts are infected with bacterial endosymbionts that manipulate host reproduction, but few bacterial taxa have been shown to cause such manipulations. Here, we show that a bacterial strain in the genus Rickettsiella causes cytoplasmic incompatibility (CI) between infected and uninfected hosts. We first surveyed the bacterial community of the agricultural spider Mermessus fradeorum (Linyphiidae) using high throughput sequencing and found that individual spiders can be infected with up to five different strains of maternally inherited symbiont from the genera Wolbachia, Rickettsia, and Rickettsiella. The Rickettsiella strain was pervasive, found in all 23 tested spider matrilines. We used antibiotic curing to generate uninfected matrilines that we reciprocally crossed with individuals infected only with Rickettsiella. We found that only 13% of eggs hatched when uninfected females were mated with Rickettsiella-infected males; in contrast, at least 83% of eggs hatched in the other cross types. This is the first documentation of Rickettsiella, or any Gammaproteobacteria, causing CI. We speculate that induction of CI may be much more widespread among maternally inherited bacteria than previously appreciated. Further, our results reinforce the importance of thoroughly characterizing and assessing the inherited microbiome before attributing observed host phenotypes to well-characterized symbionts such as Wolbachia.

Isolation of Rickettsia, Rickettsiella, and Spiroplasma from Questing Ticks in Japan Using Arthropod Cells.

Ticks are blood-sucking ectoparasites that transmit zoonotic pathogens to humans and animals. Ticks harbor not only pathogenic microorganisms but also endosymbionts. Although some tick endosymbionts are known to be essential for the survival of ticks, their roles in ticks remain poorly understood. The main aim of this study was to isolate and characterize tick-borne microorganisms from field-collected ticks using two arthropod cell lines derived from Ixodes scapularis embryos (ISE6) and Aedes albopictus larvae (C6/36). A total of 170 tick homogenates originating from 15 different tick species collected in Japan were inoculated into each cell line. Bacterial growth was confirmed by PCR amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8-week observation period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA PCR products indicated that they were previously known tick-borne pathogens/endosymbionts in three different genera: Rickettsia, Rickettsiella, and Spiroplasma. These included four previously validated rickettsial species namely Rickettsia asiatica (n = 2), Rickettsia helvetica (n = 3), Rickettsia monacensis (n = 2), and Rickettsia tamurae (n = 3) and one uncharacterized genotype Rickettsia sp. LON (n = 2). Four isolates of Spiroplasma had the highest similarity with previously reported Spiroplasma isolates: Spiroplasma ixodetis obtained from ticks in North America and Spiroplasma sp. Bratislava 1 obtained from Ixodes ricinus in Europe, while two isolates of Rickettsiella showed 100% identity with Rickettsiella sp. detected from Ixodes uriae at Grimsey Island in Iceland. To the best of our knowledge, this is the first report on successful isolation of Rickettsiella from ticks. The isolates obtained in this study can be further analyzed to evaluate their pathogenic potential in animals and their roles as symbionts in ticks.

'Candidatus Aquirickettsiella gammari' (Gammaproteobacteria: Legionellales: Coxiellaceae): A bacterial pathogen of the freshwater crustacean Gammarus fossarum (Malacostraca: Amphipoda).

Invasive and non-native species can pose risks to vulnerable ecosystems by co-introducing bacterial pathogens. Alternatively, co-introduced bacterial pathogens may regulate invasive population size and invasive traits. We describe a novel candidate genus and species of bacteria ('Candidatus Aquirickettsiella gammari') found to infect Gammarus fossarum, from its native range in Poland. The bacterium develops intracellularly within the haemocytes and cells of the musculature, hepatopancreas, connective tissues, nervous system and gonad of the host. The developmental cycle of 'Candidatus Aquirickettsiella gammari' includes an elementary body (496.73 nm ±â€¯37.56 nm in length, and 176.89 nm ±â€¯36.29 nm in width), an elliptical, condensed spherical stage (737.61 nm ±â€¯44.51 nm in length and 300.07 nm ±â€¯44.02 nm in width), a divisional stage, and a spherical initial body (1397.59 nm ±â€¯21.26 nm in diameter). We provide a partial genome for 'Candidatus Aquirickettsiella gammari', which clades phylogenetically alongside environmental 16S rRNA sequences from aquatic habitats, and bacterial symbionts from aquatic isopods (Asellus aquaticus), grouping separately from the Rickettsiella, a genus that includes bacterial pathogens of terrestrial insects and isopods. Increased understanding of the diversity of symbionts carried by G. fossarum identifies those that might regulate host population size, or those that could pose a risk to native species in the invasive range. Identification of 'Candidatus Aquirickettsiella gammari' and its potential for adaptation as a biological control agent is explored.

Genomic Insight into Symbiosis-Induced Insect Color Change by a Facultative Bacterial Endosymbiont, "Candidatus Rickettsiella viridis".

Members of the genus Rickettsiella are bacterial pathogens of insects and other arthropods. Recently, a novel facultative endosymbiont, "Candidatus Rickettsiella viridis," was described in the pea aphid Acyrthosiphon pisum, whose infection causes a striking host phenotype: red and green genetic color morphs exist in aphid populations, and upon infection with the symbiont, red aphids become green due to increased production of green polycyclic quinone pigments. Here we determined the complete genome sequence of the symbiont. The 1.6-Mb circular genome, harboring some 1,400 protein-coding genes, was similar to the genome of entomopathogenic Rickettsiella grylli (1.6 Mb) but was smaller than the genomes of phylogenetically allied human pathogens Coxiella burnetii (2.0 Mb) and Legionella pneumophila (3.4 Mb). The symbiont's metabolic pathways exhibited little complementarity to those of the coexisting primary symbiont Buchnera aphidicola, reflecting the facultative nature of the symbiont. The symbiont genome harbored neither polyketide synthase genes nor the evolutionarily allied fatty acid synthase genes that are suspected to catalyze the polycyclic quinone synthesis, indicating that the green pigments are produced not by the symbiont but by the host aphid. The symbiont genome retained many type IV secretion system genes and presumable effector protein genes, whose homologues in L. pneumophila were reported to modulate a variety of the host's cellular processes for facilitating infection and virulence. These results suggest the possibility that the symbiont is involved in the green pigment production by affecting the host's metabolism using the secretion machineries for delivering the effector molecules into the host cells.IMPORTANCE Insect body color is relevant to a variety of biological aspects such as species recognition, sexual selection, mimicry, aposematism, and crypsis. Hence, the bacterial endosymbiont "Candidatus Rickettsiella viridis," which alters aphid body color from red to green, is of ecological interest, given that different predators preferentially exploit either red- or green-colored aphids. Here we determined the complete 1.6-Mb genome of the symbiont and uncovered that, although the red-green color transition was ascribed to upregulated production of green polycyclic quinone pigments, the symbiont genome harbored few genes involved in the polycyclic quinone biosynthesis. Meanwhile, the symbiont genome contained type IV secretion system genes and presumable effector protein genes, whose homologues modulate eukaryotic cellular processes for facilitating infection and virulence in the pathogen Legionella pneumophila We propose the hypothesis that the symbiont may upregulate the host's production of polycyclic quinone pigments via cooption of secretion machineries and effector molecules for pathogenicity.

Sex ratios of the tick Ixodes arboricola are strongly female-biased, but there are no indications of sex-distorting bacteria.

Studies on sex ratio are of fundamental importance for understanding the biology of populations and biological control of pests and pathogens. In most Ixodes tick species, only females feed in the adult stage and, hence, contribute to pathogen transmission. The tree-hole tick Ixodes arboricola infests cavity-nesting birds and has limited dispersal possibilities. It plays an important role in the maintenance of zoonotic disease cycles. Here, we quantified the sex ratio of 718 adult I. arboricola ticks obtained from a laboratory stock at nine distinct periods (cohorts) from 2008 to 2015. In addition, we screened 93 specimens, collected from four study sites in 2011 and 2012, for the presence of six maternally inherited bacterial parasites known to manipulate arthropod sex ratios. We found significantly female-biased sex ratios in seven out of nine cohorts. There were no infections with members of the Wolbachia, Arsenophonus or Cardinium bacterial genera, whereas 96.8% of the screened ticks were infected with Rickettsia vini, 22.6% with Rickettsiella sp., and 14.0% with Spiroplasma ixodetis. Male and female I. arboricola were found equally infected. Our results suggest skewed sex ratios in I. arboricola are not caused by these bacterial infections, although there may be other, untested candidates driving sex ratios. Alternatively, female-biased sex ratios may be an adaptation in females to high local densities and low dispersal, where the production of daughters has a selective advantage because a few sons can fertilise all daughters.

Context-dependent vertical transmission shapes strong endosymbiont community structure in the pea aphid, Acyrthosiphon pisum.

Animal-associated microbiomes are often comprised of structured, multispecies communities, with particular microbes showing trends of co-occurrence or exclusion. Such structure suggests variable community stability, or variable costs and benefits-possibilities with implications for symbiont-driven host adaptation. In this study, we performed systematic screening for maternally transmitted, facultative endosymbionts of the pea aphid, Acyrthosiphon pisum. Sampling across six locales, with up to 5 years of collection in each, netted significant and consistent trends of community structure. Co-infections between Serratia symbiotica and Rickettsiella viridis were more common than expected, while Rickettsia and X-type symbionts colonized aphids with Hamiltonella defensa more often than expected. Spiroplasma co-infected with other endosymbionts quite rarely, showing tendencies to colonize as a single species monoculture. Field estimates of maternal transmission rates help to explain our findings: while Serratia and Rickettsiella improved each other's transmission, Spiroplasma reduced transmission rates of co-infecting endosymbionts. In summary, our findings show that North American pea aphids harbour recurring combinations of facultative endosymbionts. Common symbiont partners play distinct roles in pea aphid biology, suggesting the creation of "generalist" aphids receiving symbiont-based defence against multiple ecological stressors. Multimodal selection, at the host level, may thus partially explain our results. But more conclusively, our findings show that within-host microbe interactions, and their resulting impacts on transmission rates, are an important determinant of community structure. Widespread distributions of heritable symbionts across plants and invertebrates hint at the far-reaching implications for these findings, and our work further shows the benefits of symbiosis research within a natural context.

'Candidatus Cochliophilus cryoturris' (Coxiellaceae), a symbiont of the testate amoeba Cochliopodium minus.

Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as 'Candidatus Cochliophilus cryoturris'. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.

House spider genome uncovers evolutionary shifts in the diversity and expression of black widow venom proteins associated with extreme toxicity.

BACKGROUND: Black widow spiders are infamous for their neurotoxic venom, which can cause extreme and long-lasting pain. This unusual venom is dominated by latrotoxins and latrodectins, two protein families virtually unknown outside of the black widow genus Latrodectus, that are difficult to study given the paucity of spider genomes. Using tissue-, sex- and stage-specific expression data, we analyzed the recently sequenced genome of the house spider (Parasteatoda tepidariorum), a close relative of black widows, to investigate latrotoxin and latrodectin diversity, expression and evolution. RESULTS: We discovered at least 47 latrotoxin genes in the house spider genome, many of which are tandem-arrayed. Latrotoxins vary extensively in predicted structural domains and expression, implying their significant functional diversification. Phylogenetic analyses show latrotoxins have substantially duplicated after the Latrodectus/Parasteatoda split and that they are also related to proteins found in endosymbiotic bacteria. Latrodectin genes are less numerous than latrotoxins, but analyses show their recruitment for venom function from neuropeptide hormone genes following duplication, inversion and domain truncation. While latrodectins and other peptides are highly expressed in house spider and black widow venom glands, latrotoxins account for a far smaller percentage of house spider venom gland expression. CONCLUSIONS: The house spider genome sequence provides novel insights into the evolution of venom toxins once considered unique to black widows. Our results greatly expand the size of the latrotoxin gene family, reinforce its narrow phylogenetic distribution, and provide additional evidence for the lateral transfer of latrotoxins between spiders and bacterial endosymbionts. Moreover, we strengthen the evidence for the evolution of latrodectin venom genes from the ecdysozoan Ion Transport Peptide (ITP)/Crustacean Hyperglycemic Hormone (CHH) neuropeptide superfamily. The lower expression of latrotoxins in house spiders relative to black widows, along with the absence of a vertebrate-targeting α-latrotoxin gene in the house spider genome, may account for the extreme potency of black widow venom.

Consequences of coinfection with protective symbionts on the host phenotype and symbiont titres in the pea aphid system.

Symbiotic associations between microbes and insects are widespread, and it is frequent that several symbionts share the same host individual. Hence, interactions can occur between these symbionts, influencing their respective abundance within the host with consequences on its phenotype. Here, we investigate the effects of multiple infections in the pea aphid, Acyrthosiphon pisum, which is the host of an obligatory and several facultative symbionts. In particular, we study the influence of a coinfection with 2 protective symbionts: Hamiltonella defensa, which confers protection against parasitoids, and Rickettsiella viridis, which provides protection against fungal pathogens and predators. The effects of Hamiltonella-Rickettsiella coinfection on the respective abundance of the symbionts, host fitness and efficacy of enemy protection were studied. Asymmetrical interactions between the 2 protective symbionts have been found: when they coinfect the same aphid individuals, the Rickettsiella infection affected Hamiltonella abundance within hosts but not the Hamiltonella-mediated protective phenotype while the Hamiltonella infection negatively influences the Rickettsiella-mediated protective phenotype but not its abundance. Harboring the 2 protective symbionts also reduced the survival and fecundity of host individuals. Overall, this work highlights the effects of multiple infections on symbiont abundances and host traits that are likely to impact the maintenance of the symbiotic associations in natural habitats.