Addicted? Reduced host resistance in populations with defensive symbionts

Heritable symbionts that protect their hosts from pathogens have been described in a wide range of insect species. By reducing the incidence or severity of infection, these symbionts have the potential to reduce the strength of selection on genes in the insect genome that increase resistance. Therefore, the presence of such symbionts may slow down the evolution of resistance. Here we investigated this idea by exposing Drosophila melanogaster populations to infection with the pathogenic Drosophila C virus (DCV) in the presence or absence of Wolbachia, a heritable symbiont of arthropods that confers protection against viruses. After nine generations of selection, we found that resistance to DCV had increased in all populations. However, in the presence of Wolbachia the resistant allele of pastrel—a gene that has a major effect on resistance to DCV—was at a lower frequency than in the symbiont-free populations. This finding suggests that defensive symbionts have the potential to hamper the evolution of insect resistance genes, potentially leading to a state of evolutionary addiction where the genetically susceptible insect host mostly relies on its symbiont to fight pathogens.     

Diversification of endosymbiosis: replacements,co-speciation and promiscuity of bacteriocyte symbionts in weevils

The processes and mechanisms underlying the diversification of host–microbe endosymbiotic associations are of evolutionary interest. Here we investigated the bacteriocyte-associated primary symbionts of weevils wherein the ancient symbiont Nardonella has experienced two independent replacement events: once by Curculioniphilus symbiont in the lineage of Curculio and allied weevils of the tribe Curculionini, and once by Sodalis-allied symbiont in the lineage of grain weevils of the genus Sitophilus. The Curculioniphilus symbiont was detected from 27 of 36 Curculionini species examined, the symbiont phylogeny was congruent with the host weevil phylogeny, and the symbiont gene sequences exhibited AT-biased nucleotide compositions and accelerated molecular evolution. These results suggest that the Curculioniphilus symbiont was acquired by an ancestor of the tribe Curculionini, replaced the original symbiont Nardonella, and has co-speciated with the host weevils over evolutionary time, but has been occasionally lost in several host lineages. By contrast, the Sodalis-allied symbiont of Sitophilus weevils exhibited no host–symbiont co-speciation, no AT-biased nucleotide compositions and only moderately accelerated molecular evolution. These results suggest that the Sodalis-allied symbiont was certainly acquired by an ancestor of the Sitophilus weevils and replaced the original Nardonella symbiont, but the symbiotic association must have experienced occasional re-associations such as new acquisitions, horizontal transfers, replacements and/or losses. We detected Sodalis-allied facultative symbionts in populations of the Curculionini weevils, which might represent potential evolutionary sources of the Sodalis-allied primary symbionts. Comparison of these newcomer bacteriocyte-associated symbiont lineages highlights potential evolutionary trajectories and consequences of novel symbionts after independent replacements of the same ancient symbiont.     

Selective and stable elimination of endosymbionts from multiple‐infected whitefly Bemisia tabaci by feeding on a cotton plant cultured in antibiotic solutions

The maternally heritable endosymbiont provides many ecosystem functions. Antibiotic elimination of a specific symbiont and establishment of experimental host lines lacking certain symbionts enable the roles of a given symbiont to be explored. The whitefly Bemisia tabaci (Gennadius) in China harbors obligate symbiont Portiera infecting each individual, as well as facultative symbionts, such as Hamiltonella, Rickettsia and Cardinium, with co‐infections occurring relatively frequently. So far no studies have evaluated the selectivity and efficacy of a specific symbiont elimination using antibiotics in whiteflies co‐infected with different symbionts. Furthermore, no success has been achieved in establishing certain symbiont‐free B. tabaci lines. In this study, we treated Hamiltonella‐infected B. tabaci line, Hamiltonella‐Rickettsia‐co‐infected line and Hamiltonella‐Cardinium co‐infected line by feeding B. tabaci adults with cotton plants cultured in water containing rifampicin, ampicillin or a mixture of them, aiming to selectively curing symbiont infections and establishing stable symbiont‐free lines. We found ampicillin selectively eliminated Cardinium without affecting Portiera, Hamiltonella and Rickettsia, although they coexisted in the same host body. Meanwhile, all of the symbionts considered in our study can be removed by rifampicin. The reduction of facultative symbionts occurred at a much quicker pace than obligate symbiont Portiera during rifampicin treatment. Also, we measured the stability of symbiont elimination in whitefly successive generations and established Rickettsia‐infected and Cardinium‐infected lines which are absent in natural populations. Our results provide new protocols for selective elimination of symbionts co‐existing in a host and establishment of different symbiont‐infected host lines.     

Pervasive associations between Cybaeus spiders and the bacterial symbiont Cardinium

Cardinium is a recently discovered maternally transmitted bacterial endosymbiont in the Bacteroidetes that has thus far been documented in five arthropod orders. While its effects on his hosts are largely unknown, a few strains have been shown to manipulate host reproduction in parasitic wasps and in mites, either by transforming males into females, or by causing mating incompatibilities between infected males and uninfected males. Cardinium has recently been reported to be widespread in spiders, and in this study, we document pervasive infections in Cybaeus spiders, which are some of the most abundant yet understudied spiders in the understory of moist Western North American forests. 12/20 species, as well as 96% of individuals in a local population of Cybaeus signifer were infected. Phylogenetic analysis revealed three closely related symbiont haplotypes within Cybaeus. Haplotypes clustered within geographically close species, suggesting that horizontal transmission might be quite high in this symbiont lineage.     

Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae

Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila'', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila'' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.     

Phage loss and the breakdown of a defensive symbiosis in aphids

Terrestrial arthropods are often infected with heritable bacterial symbionts, which may themselves be infected by bacteriophages. However, what role, if any, bacteriophages play in the regulation and maintenance of insect–bacteria symbioses is largely unknown. Infection of the aphid Acyrthosiphon pisum by the bacterial symbiont Hamiltonella defensa confers protection against parasitoid wasps, but only when H. defensa is itself infected by the phage A. pisum secondary endosymbiont (APSE). Here, we use a controlled genetic background and correlation-based assays to show that loss of APSE is associated with up to sevenfold increases in the intra-aphid abundance of H. defensa. APSE loss is also associated with severe deleterious effects on aphid fitness: aphids infected with H. defensa lacking APSE have a significantly delayed onset of reproduction, lower weight at adulthood and half as many total offspring as aphids infected with phage-harbouring H. defensa, indicating that phage loss can rapidly lead to the breakdown of the defensive symbiosis. Our results overall indicate that bacteriophages play critical roles in both aphid defence and the maintenance of heritable symbiosis.     

Effects of bacterial secondary symbionts on host plant use in pea aphids

Aphids possess several facultative bacterial symbionts that have important effects on their hosts'' biology. These have been most closely studied in the pea aphid (Acyrthosiphon pisum), a species that feeds on multiple host plants. Whether secondary symbionts influence host plant utilization is unclear. We report the fitness consequences of introducing different strains of the symbiont Hamiltonella defensa into three aphid clones collected on Lathyrus pratensis that naturally lack symbionts, and of removing symbionts from 20 natural aphid–bacterial associations. Infection decreased fitness on Lathyrus but not on Vicia faba, a plant on which most pea aphids readily feed. This may explain the unusually low prevalence of symbionts in aphids collected on Lathyrus. There was no effect of presence of symbiont on performance of the aphids on the host plants of the clones from which the H. defensa strains were isolated. Removing the symbiont from natural aphid–bacterial associations led to an average approximate 20 per cent reduction in fecundity, both on the natural host plant and on V. faba, suggesting general rather than plant-species-specific effects of the symbiont. Throughout, we find significant genetic variation among aphid clones. The results provide no evidence that secondary symbionts have a major direct role in facilitating aphid utilization of particular host plant species.     

Cytoplasmic incompatibility and multiple symbiont infection in the ash whitefly parasitoid, Encarsia inaron

Terrestrial arthropods are commonly infected with maternally inherited symbionts that cause reproductive incompatibilities between hosts with differing infection status. Such symbionts can have major effects on the efficacy of a biological control program if releases are comprised of mixtures of differentially infected individuals. In this study, the ash whitefly parasitoid, Encarsia inaron (Hymenoptera: Aphelinidae) from Arizona was surveyed for the presence of heritable bacterial symbionts; experiments were also performed to test for two phenotypes known to be caused by Encarsia symbionts—cytoplasmic incompatibility and changes in oviposition behavior and host use. E. inaron has successfully reduced ash whitefly to non-pest status in all three locations it has been released (California, Arizona, and North Carolina) and is also notable as one of the only Encarsia species that is not autoparasitic, with both male and female wasps developing as primary parasitoids of whiteflies. We show that E. inaron is infected with both Wolbachia and Cardinium. While there was no effect of the symbionts on oviposition behavior or host use, crosses between doubly infected male wasps and uninfected females resulted in a severe reduction in the number of female offspring; male offspring production was unaffected. This study thus serves as a further warning that ascribing a phenotype to a symbiont with confidence depends on eliminating the possibility of a mixed infection, and establishes E. inaron as a useful model for dissecting Wolbachia–Cardinium interactions.     

Comparative Genomics Suggests an Independent Origin of Cytoplasmic Incompatibility in Cardinium hertigii

Terrestrial arthropods are commonly infected with maternally inherited bacterial symbionts that cause cytoplasmic incompatibility (CI). In CI, the outcome of crosses between symbiont-infected males and uninfected females is reproductive failure, increasing the relative fitness of infected females and leading to spread of the symbiont in the host population. CI symbionts have profound impacts on host genetic structure and ecology and may lead to speciation and the rapid evolution of sex determination systems. Cardinium hertigii, a member of the Bacteroidetes and symbiont of the parasitic wasp Encarsia pergandiella, is the only known bacterium other than the Alphaproteobacteria Wolbachia to cause CI. Here we report the genome sequence of Cardinium hertigii cEper1. Comparison with the genomes of CI–inducing Wolbachia pipientis strains wMel, wRi, and wPip provides a unique opportunity to pinpoint shared proteins mediating host cell interaction, including some candidate proteins for CI that have not previously been investigated. The genome of Cardinium lacks all major biosynthetic pathways but harbors a complete biotin biosynthesis pathway, suggesting a potential role for Cardinium in host nutrition. Cardinium lacks known protein secretion systems but encodes a putative phage-derived secretion system distantly related to the antifeeding prophage of the entomopathogen Serratia entomophila. Lastly, while Cardinium and Wolbachia genomes show only a functional overlap of proteins, they show no evidence of laterally transferred elements that would suggest common ancestry of CI in both lineages. Instead, comparative genomics suggests an independent evolution of CI in Cardinium and Wolbachia and provides a novel context for understanding the mechanistic basis of CI.     

An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host–symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host–symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host–symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed.     

Lateral transfers of insertion sequences between Wolbachia,Cardinium and Rickettsia bacterial endosymbionts

Various bacteria live exclusively within arthropod cells and collectively act as an important driver of arthropod evolutionary ecology. Whereas rampant intra-generic DNA transfers were recently shown to have a pivotal role in the evolution of the most common of these endosymbionts, Wolbachia, the present study show that inter-generic DNA transfers also commonly take place, constituting a potent source of rapid genomic change. Bioinformatic, molecular and phylogenetic data provide evidence that a selfish genetic element, the insertion sequence ISRpe1, is widespread in the Wolbachia, Cardinium and Rickettsia endosymbionts and experiences recent (and likely ongoing) transfers over long evolutionary distances. Although many ISRpe1 copies were clearly expanding and leading to rapid endosymbiont diversification, degraded copies are also frequently found, constituting an unusual genomic fossil record suggestive of ancient ISRpe1 expansions. Overall, the present data highlight how ecological connections within the arthropod intracellular environment facilitate lateral DNA transfers between distantly related bacterial lineages.     

A divergent Cardinium found in daddy long-legs (Arachnida: Opiliones)

Recent studies indicate that a newly described bacterial endosymbiont, Cardinium, is widespread in arthropods and induces different reproductive manipulations in hosts. In this study, we used a portion of the 16S rRNA gene of the Cardinium to screen 16 Opilionid species from the suborder Palptores. We found the incidence of Cardinium in these Opiliones was significantly higher than in other pooled arthropods (31.2% versus 7.2%, P = 0.007). Phylogenetic analyses using maximum parsimony (MP) and Bayesian analysis revealed two distinct clades in Opiliones. One is a divergent monophyletic clade with strong support that has so far not been found in other arthropods, and a second one contains Cardinium both from Opiliones and other arthropods. There is not complete concordance of the Cardinium strains with host phylogeny, suggesting some horizontal movement of the bacteria among Opiliones. Although the divergence in the sequenced 16S rRNA region between the Cardinium infecting Opiliones and Cardinium from other arthropods is greater than among Cardinium found in other arthropods, all are monophyletic with respect to the outgroup bacteria (endosymbionts of Acanthamoeba). Based on high pairwise genetic distances, deep branch, and a distinct phylogenetic grouping, we conclude that some Opiliones harbor a newly discovered Cardinium clade.     

Are we underestimating the diversity and incidence of insect bacterial symbionts? A case study in ladybird beetles

Vertically transmitted bacterial symbionts are common in arthropods. However, estimates of their incidence and diversity are based on studies that test for a single bacterial genus and often only include small samples of each host species. Focussing on ladybird beetles, we collected large samples from 21 species and tested them for four different bacterial symbionts. Over half the species were infected, and there were often multiple symbionts in the same population. In most cases, more females than males were infected, suggesting that the symbionts may be sex ratio distorters. Many of these infections would have been missed in previous studies as they only infect a small proportion of the population. Furthermore, 11 out of the 17 symbionts discovered by us were either in the genus Rickettsia or Spiroplasma, which are rarely sampled. Our results suggest that the true incidence and diversity of bacterial symbionts in insects may be far greater than previously thought.     

No sex in fungus-farming ants or their crops

Asexual reproduction imposes evolutionary handicaps on asexual species, rendering them prone to extinction, because asexual reproduction generates novel genotypes and purges deleterious mutations at lower rates than sexual reproduction. Here, we report the first case of complete asexuality in ants, the fungus-growing ant Mycocepurus smithii, where queens reproduce asexually but workers are sterile, which is doubly enigmatic because the clonal colonies of M. smithii also depend on clonal fungi for food. Degenerate female mating anatomy, extensive field and laboratory surveys, and DNA fingerprinting implicate complete asexuality in this widespread ant species. Maternally inherited bacteria (e.g. Wolbachia, Cardinium) and the fungal cultivars can be ruled out as agents inducing asexuality. M. smithii societies of clonal females provide a unique system to test theories of parent–offspring conflict and reproductive policing in social insects. Asexuality of both ant farmer and fungal crop challenges traditional views proposing that sexual farmer ants outpace coevolving sexual crop pathogens, and thus compensate for vulnerabilities of their asexual crops. Either the double asexuality of both farmer and crop may permit the host to fully exploit advantages of asexuality for unknown reasons or frequent switching between crops (symbiont reassociation) generates novel ant–fungus combinations, which may compensate for any evolutionary handicaps of asexuality in M. smithii.     

Macronutrients mediate the functional relationship between Drosophila and Wolbachia

Wolbachia are maternally inherited bacterial endosymbionts that naturally infect a diverse array of arthropods. They are primarily known for their manipulation of host reproductive biology, and recently, infections with Wolbachia have been proposed as a new strategy for controlling insect vectors and subsequent human-transmissible diseases. Yet, Wolbachia abundance has been shown to vary greatly between individuals and the magnitude of the effects of infection on host life-history traits and protection against infection is correlated to within-host Wolbachia abundance. It is therefore essential to better understand the factors that modulate Wolbachia abundance and effects on host fitness. Nutrition is known to be one of the most important mediators of host–symbiont interactions. Here, we used nutritional geometry to quantify the role of macronutrients on insect–Wolbachia relationships in Drosophila melanogaster. Our results show fundamental interactions between diet composition, host diet selection, Wolbachia abundance and effects on host lifespan and fecundity. The results and methods described here open a new avenue in the study of insect–Wolbachia relationships and are of general interest to numerous research disciplines, ranging from nutrition and life-history theory to public health.     

Molecular mechanisms of cell–cell spread of intracellular bacterial pathogens

Several bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and Rickettsia spp., have evolved mechanisms to actively spread within human tissues. Spreading is initiated by the pathogen-induced recruitment of host filamentous (F)-actin. F-actin forms a tail behind the microbe, propelling it through the cytoplasm. The motile pathogen then encounters the host plasma membrane, forming a bacterium-containing protrusion that is engulfed by an adjacent cell. Over the past two decades, much progress has been made in elucidating mechanisms of F-actin tail formation. Listeria and Shigella produce tails of branched actin filaments by subverting the host Arp2/3 complex. By contrast, Rickettsia forms tails with linear actin filaments through a bacterial mimic of eukaryotic formins. Compared with F-actin tail formation, mechanisms controlling bacterial protrusions are less well understood. However, recent findings have highlighted the importance of pathogen manipulation of host cell–cell junctions in spread. Listeria produces a soluble protein that enhances bacterial protrusions by perturbing tight junctions. Shigella protrusions are engulfed through a clathrin-mediated pathway at ‘tricellular junctions’—specialized membrane regions at the intersection of three epithelial cells. This review summarizes key past findings in pathogen spread, and focuses on recent developments in actin-based motility and the formation and internalization of bacterial protrusions.     

Cytoplasmic incompatibility and multiple symbiont infection in the ash whitefly parasitoid,Encarsia inaron

Terrestrial arthropods are commonly infected with maternally inherited symbionts that cause reproductive incompatibilities between hosts with differing infection status. Such symbionts can have major effects on the efficacy of a biological control program if releases are comprised of mixtures of differentially infected individuals. In this study, the ash whitefly parasitoid, Encarsia inaron (Hymenoptera: Aphelinidae) from Arizona was surveyed for the presence of heritable bacterial symbionts; experiments were also performed to test for two phenotypes known to be caused by Encarsia symbionts—cytoplasmic incompatibility and changes in oviposition behavior and host use. E. inaron has successfully reduced ash whitefly to non-pest status in all three locations it has been released (California, Arizona, and North Carolina) and is also notable as one of the only Encarsia species that is not autoparasitic, with both male and female wasps developing as primary parasitoids of whiteflies. We show that E. inaron is infected with both Wolbachia and Cardinium. While there was no effect of the symbionts on oviposition behavior or host use, crosses between doubly infected male wasps and uninfected females resulted in a severe reduction in the number of female offspring; male offspring production was unaffected. This study thus serves as a further warning that ascribing a phenotype to a symbiont with confidence depends on eliminating the possibility of a mixed infection, and establishes E. inaron as a useful model for dissecting Wolbachia–Cardinium interactions.     

Conjugation genes are common throughout the genus Rickettsia and are transmitted horizontally

Rickettsia are endosymbionts of arthropods, some of which are vectored to vertebrates where they cause disease. Recently, it has been found that some Rickettsia strains harbour conjugative plasmids and others encode some conjugative machinery within the bacterial genome. We investigated the distribution of these conjugation genes in a phylogenetically diverse collection of Rickettsia isolated from arthropods. We found that these genes are common throughout the genus and, in stark contrast to other genes in the genome, conjugation genes are frequently horizontally transmitted between strains. There is no evidence to suggest that these genes are preferentially transferred between phylogenetically related strains, which is surprising given that closely related strains infect similar host species. In addition to detecting patterns of horizontal transmission between diverse Rickettsia species, these findings have implications for the evolution of pathogenicity, the evolution of Rickettsia genomes and the genetic manipulation of intracellular bacteria.     

Blood parasites of livestock in certain Regions in Saudi Arabia

Blood samples from camels, sheep, goats and cattle from six Regions in Saudi Arabia were examined for blood parasites. Asir Region camels were disinfected while those of the Eastern, Jazan, Northern Frontiers, Riyadh and Tabouk Regions were infected with Trypanosoma evansi (5–40%), those of Riyadh and the Eastern Regions were infected with Dipetalonema evansi (1–6%) and those of the Eastern, Jazan and Riyadh Regions were infected with Eperythrozoon species (8–20%). Sheep and goats of all tested regions were infected with Theilaria hirci (4–20% and 6–14%, respectively), Theilaria ovis (5–19% and 6–24%, respectively) and Eperthrozoon ovis (2–9% and 2–8%, respectively). Sheep of the Eastern and Northern Frontiers Regions were also infected with Anaplasma ovis (2%) and also those of the Eastern Region were infected with Babesia motasi (4%) as well. Cattle of Asir and Eastern Regions were infected with Anaplasma marginale (1–3.4%) and those of the Eastern, Jazan and Riyadh Regions were infected with Theileria annulata (11.3–25%) and Eperthrozoon wenyoni (1–4%). Moreover, Jazan cattle were infected with Babesia bigemina (6%) and a benign Theileria species (27%). Some of these parasites are recorded in new localities indicating that they are spreading in the country. Also, this is the first report in Saudi Arabia of D. evansi in camels, A. ovis and B. motasi in sheep and A. marginale and B. bigemina in cattle. These parasites may be introduced into the country with infected livestock infested with the vectors of these parasites. The suspected vectors of the detected parasites in Saudi Arabia is discussed. Follow up surveys of blood parasites are recommended to assess their distribution and infection rates in the livestock of all Regions of Saudi Arabia, to make plans for control measures against their vectors.