Some insects are infected with maternally inherited bacterial endosymbionts that protect them against pathogens or parasitoids. The weevil Sitona obsoletus (=Sitona lepidus) is invasive in New Zealand, and suspected to contain such defensive symbionts, because it is particularly resistant to a Moroccan strain of the parasitoid Microctonus aethiopoides (which successfully attacks many other weevil species), and shows geographic variation in susceptibility to an Irish strain of the same parasitoid. Using 454 pyrosequencing, we investigated the bacterial community associated with S. obsoletus, two other exotic weevils (Sitona discoideus and Listronotus bonariensis) and two endemic New Zealand weevils (Irenimus aequalis and Steriphus variabilis). We found that S. obsoletus was infected by one strain of Wolbachia and two strains of Rickettsia, none of which were found in any other weevil species examined. Using diagnostic PCR, we found that S. obsoletus in the Northland region, where parasitism is highly variable, were primarily infected with Wolbachia and Rickettsia strain 2, indicating that these two symbionts should be investigated for potential defensive properties. In comparison, S. discoideus lacked any apparent maternally inherited bacterial endosymbionts. In the other weevil species, we found a different strain of Wolbachia and two different strains of Spiroplasma. Two weevil species (St. variabilis and L. bonariensis) were infected with distinct strains of Nardonella, the ancestral endosymbiont of weevils, whereas three weevil species (S. obsoletus, S. discoideus, and I. aequalis) lacked evidence for Nardonella infection. However, I. aequalis was consistently infected with a novel Enterobacteriaceae strain, suggesting that a symbiont replacement may have taken place, similar to that described for other weevil clades.

In many arthropods, maternally inherited endosymbiotic bacteria can increase infection frequency by manipulating host reproduction. Multiple infections of different bacteria in a single host population are common, yet few studies have documented concurrent endosymbiont phenotypes or explored their potential interactions. We hypothesized that spiders might be a particularly useful taxon for investigating endosymbiont interactions, because they are host to a plethora of endosymbiotic bacteria and frequently exhibit multiple infections. We established two matrilines from the same population of the linyphiid spider Mermessus fradeorum and then used antibiotic curing and controlled mating assays to demonstrate that each matriline was subject to a distinct endosymbiotic reproductive manipulation. One matriline was co-infected with Rickettsia and Wolbachia and produced offspring with a radical female bias. Antibiotic treatment eliminated both endosymbionts and restored an even sex ratio to subsequent generations. Chromosomal and fecundity observations suggest a feminization mechanism. In the other matriline, a separate factorial mating assay of cured and infected spiders demonstrated strong cytoplasmic incompatibility (CI) induced by a different strain of Wolbachia. However, males with this Wolbachia induced only mild CI when mated with the Rickettsia-Wolbachia females. In a subsequent survey of a field population of M. fradeorum, we detected these same three endosymbionts infecting 55% of the spiders in almost all possible combinations, with nearly half of the infected spiders exhibiting multiple infection. Our results suggest that a dynamic network of endosymbionts may interact both within multiply infected hosts and within a population subject to multiple strong reproductive manipulations.

Maternally inherited bacterial endosymbionts are common in arthropods, but their distribution and prevalence is poorly characterized in many host taxa. For example, spiders (Araneae) have received little attention, but initial surveys suggest that vertically transmitted symbionts may be common. Here, we characterized endosymbiont infection in a community of agricultural spiders.Using a combination of diagnostic PCR and high-throughput sequencing of the bacterial microbiome, we evaluated symbiont infection in 267 individual spiders representing 14 species in 3 families. We found 27 Operational Taxonomic Units (OTUs) that are likely endosymbiotic, including several strains of Wolbachia, Rickettsia and Cardinium, all of which are vertically transmitted and frequently associated with reproductive manipulation of arthropod hosts.Seventy percent of spider species had individuals that tested positive for one or more endosymbiotic OTUs, and specimens frequently contained multiple symbiotic strain types. The most symbiont-rich species, Idionella rugosa, had eight endosymbiotic OTUs, with as many as five present in the same specimen. Individual specimens within infected spider species had a variety of symbiotypes, differing from one another in the presence or absence of symbiotic strains. Our sample included both starved and unstarved specimens, and dominant bacterial OTUs were consistent per host species, regardless of feeding status. We conclude that spiders contain a remarkably diverse symbiotic microbiota. Spiders would be an informative group for investigating endosymbiont population dynamics in time and space, and unstarved specimens collected for other purposes (e.g., food web studies) could be used, with caution, for such investigations.

Acknowledgements:We thank J. Harwood and J. Dryer for providing and collecting 22 specimens.Abstract 31 Maternally inherited bacterial endosymbionts are common in arthropods, but their distribution 32 and prevalence is poorly characterized in many host taxa. For example, spiders (Araneae) have 33 received little attention, but initial surveys suggest that vertically transmitted symbionts may be 34 common. Here, we characterized endosymbiont infection in a community of agricultural spiders. 35Using a combination of diagnostic PCR and high-throughput sequencing of the bacterial 36 microbiome, we evaluated symbiont infection in 267 individual spiders representing 14 species 37 in 3 families. We found 27 Operational Taxonomic Units (OTUs) that are likely endosymbiotic, 38 including several strains of Wolbachia, Rickettsia and Cardinium, all of which are vertically 39 transmitted and frequently associated with reproductive manipulation of arthropod hosts. 40Seventy percent of spider species had individuals that tested positive for one or more 41 endosymbiotic OTUs, and specimens frequently contained multiple symbiotic strain types. The 42 most symbiont-rich species, Idionella rugosa, had eight endosymbiotic OTUs, with as many as 43 five present in the same specimen. Individual specimens within infected spider species had a 44 variety of symbiotypes, differing from one another in the presence or absence of symbiotic 45 strains. Our sample included both starved and unstarved specimens, and dominant bacterial 46OTUs were consistent per host species, regardless of feeding status. We conclude that spiders 47 contain a remarkably diverse symbiotic microbiota. Spiders would be an informative group for 48 investigating endosymbiont population dynamics in time and space, and unstarved specimens 49 collected for other purposes (e.g., food web studies) could be used, with caution, for such 50 investigations. 51 52 53The majority of terrestrial arthropod species are infected by inherited bacterial symbionts. 54The most common of such bacteria, Wolbachia, is estimated to infect approximately half of 55 arthropod species, albeit often at a low frequency of infected individuals within a given species 56 [1-3]. These bacteria are primarily transmitted vertically, but horizontal transfers among host 57 taxa occasionally occur through a variety of ecological mechanisms such as shared host 58 substrates or shared enemies [4-8]. Following successful establishment in a new host lineage, the 59 endosymbiont then promotes the continued existence of that infected lineage, either by providing 60 some sort of fitness benefit to the host [9,10], or by manipulating host reproduction such that 61 infected mothers produce more daughters than uninfected mothers [11]. Over generations, the 62 result is an infection that can spread through the host population via selection, rather than 63 contagion. 64 It is inevitable that this process of infection and spread sometimes occurs in the context 65 of other vertically-transmitted endosymbionts, ...

We investigated the potential for gene flow in a dependent lineage (DL) system of the harvester ant Pogonomyrmex. Each DL system is composed of 2 reproductively isolated lineages that are locked in an obligate mutualism. The genetic components that produce the worker phenotype are acquired by hybridizing with the partner lineage. In the mating flight, queens of both lineages mate with multiple males from each lineage. During colony growth and reproduction, eggs fertilized by partner-lineage sperm produce F(1) hybrid workers with interlineage genomes, whereas eggs fertilized by same-lineage sperm result in the development of new queens with intralineage genomes. New males are typically produced from unfertilized eggs laid by the pure-lineage queen but in her absence may be produced by interlineage F(1) workers. We investigated the potential for interlineage gene flow in this system using 2 classes of lineage-specific nuclear markers to identify hybrid genome combinations. We confirmed the production of viable interlineage F(1) reproductive females in field colonies, the occurrence of which is associated with the relative frequencies of each lineage in the population: interlineage F(1) queens occurred only in the rare lineage of the population with dramatically skewed lineage frequencies. In laboratory colonies, we detected fair meiosis in interlineage F(1) workers leading to the production of viable and haploid interlineage F(2) males. We conclude that the genomes of each lineage recombine freely, suggesting that extrinsic postzygotic selection maintains the integrity of each lineage genome. We compare our findings with those of the H1/H2 DL system.