Are ecological communities the seat of endosymbiont horizontal transfer and diversification? A case study with soil arthropod community

Maternally inherited endosymbionts of arthropods are one of the most abundant and diverse group of bacteria. These bacterial endosymbionts also show extensive horizontal transfer to taxonomically unrelated hosts and widespread recombination in their genomes. Such horizontal transfers can be enhanced when different arthropod hosts come in contact like in an ecological community. Higher rates of horizontal transfer can also increase the probability of recombination between endosymbionts, as they now share the same host cytoplasm. However, reports of community‐wide endosymbiont data are rare as most studies choose few host taxa and specific ecological interactions among the hosts. To better understand endosymbiont spread within host populations, we investigated the incidence, diversity, extent of horizontal transfer, and recombination of three endosymbionts (Wolbachia, Cardinium, and Arsenophonus) in a specific soil arthropod community. Wolbachia strains were characterized with MLST genes whereas 16S rRNA gene was used for Cardinium and Arsenophonus. Among 3,509 individual host arthropods, belonging to 390 morphospecies, 12.05% were infected with Wolbachia, 2.82% with Cardinium and 2.05% with Arsenophonus. Phylogenetic incongruence between host and endosymbiont indicated extensive horizontal transfer of endosymbionts within this community. Three cases of recombination between Wolbachia supergroups and eight incidences of within‐supergroup recombination were also found. Statistical tests of similarity indicated supergroup A Wolbachia and Cardinium show a pattern consistent with extensive horizontal transfer within the community but not for supergroup B Wolbachia and Arsenophonus. We highlight the importance of extensive community‐wide studies for a better understanding of the spread of endosymbionts across global arthropod communities.     

Bacterial endosymbiont infections in ‘living fossils’: a case study of North American vaejovid scorpions

Bacterial endosymbionts are common among arthropods, and maternally inherited forms can affect the reproductive and behavioural traits of their arthropod hosts. The prevalence of bacterial endosymbionts and their role in scorpion evolution have rarely been investigated. In this study, 61 samples from 40 species of scorpion in the family Vaejovidae were screened for the presence of the bacterial endosymbionts Cardinium, Rickettsia, Spiroplasma and Wolbachia. No samples were infected by these bacteria. However, one primer pair specifically designed to amplify Rickettsia amplified nontarget genes of other taxa. Similar off‐target amplification using another endosymbiont‐specific primer was also found during preliminary screenings. Results caution against the overreliance on previously published screening primers to detect bacterial endosymbionts in host taxa and suggest that primer specificity may be higher in primers targeting nuclear rather than mitochondrial genes.     

Cytoplasmic incompatibility involving <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Wolbachia</Emphasis> in the white-backed planthopper <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae)

Cytoplasmic incompatibility (CI) is a reproductive phenotype induced by bacterial endosymbionts in arthropods. Measured as a reduction in egg hatchability resulting from the crossing of uninfected females with bacteria-infected males, CI increases the frequency of bacteria-infected hosts by restricting the fertilization opportunities of uninfected hosts in populations. Wolbachia, a type of alpha-proteobacteria, is well known as a CI inducer in a wide range of arthropod species, while Cardinium, a member of the phylum Bacteroidetes, is known to cause CI in one wasp and three spider mite species. In this study, dual infection with Cardinium and Wolbachia induced strong CI in a single host, Sogatella furcifera (Horváth), a planthopper species that is naturally infected with both bacteria. Specifically, infection with Cardinium alone was found to cause a 76 % reduction in egg development, and dual infection with Cardinium and Wolbachia a 96 % reduction, indicating that Cardinium induces CI and the dual infection raises the CI level. This study was the first to document reproductive alteration by Cardinium in a diploid host species.     

Wolbachia Strengthens Cardinium-Induced Cytoplasmic Incompatibility in the Spider Mite Tetranychus piercei McGregor

Wolbachia and Cardinium are maternally inherited intracellular bacteria that can manipulate the reproduction of their arthropod hosts, such as by inducing cytoplasmic incompatibility (CI). Although the reproductive alteration induced by Wolbachia or Cardinium have been well investigated, the effects of these two endosymbionts co-infecting the same host are poorly understood. We found that Tetranychus piercei McGregor is naturally infected with Wolbachia and Cardinium. We performed all possible crossing combinations using naturally infected and cured strains, and the results show that Wolbachia induced a weak level of CI, while Cardinium-infected and doubly infected males caused severe CI. Wolbachia and Cardinium could not rescue CI each other; however, Wolbachia boosted the expression of Cardinium-induced CI. Quantitative PCR results demonstrated that CI was associated with the infection density of Wolbachia and Cardinium.     

Endosymbionts moderate constrained sex allocation in a haplodiploid thrips species in a temperature-sensitive way

Maternally inherited bacterial endosymbionts that affect host fitness are common in nature. Some endosymbionts colonise host populations by reproductive manipulations (such as cytoplasmic incompatibility; CI) that increase the reproductive fitness of infected over uninfected females. Theory predicts that CI-inducing endosymbionts in haplodiploid hosts may also influence sex allocation, including in compatible crosses, however, empirical evidence for this is scarce. We examined the role of two common CI-inducing endosymbionts, Cardinium and Wolbachia, in the sex allocation of Pezothrips kellyanus, a haplodiploid thrips species with a split sex ratio. In this species, irrespective of infection status, some mated females are constrained to produce extremely male-biased broods, whereas other females produce extremely female-biased broods. We analysed brood sex ratio of females mated with males of the same infection status at two temperatures. We found that at 20 °C the frequency of constrained sex allocation in coinfected pairs was reduced by 27% when compared to uninfected pairs. However, at 25 °C the constrained sex allocation frequency increased and became similar between coinfected and uninfected pairs, resulting in more male-biased population sex ratios at the higher temperature. This temperature-dependent pattern occurred without changes in endosymbiont densities and compatibility. Our findings indicate that endosymbionts affect sex ratios of haplodiploid hosts beyond the commonly recognised reproductive manipulations by causing female-biased sex allocation in a temperature-dependent fashion. This may contribute to a higher transmission efficiency of CI-inducing endosymbionts and is consistent with previous models that predict that CI by itself is less efficient in driving endosymbiont invasions in haplodiploid hosts.Subject terms: Evolutionary genetics, Evolutionary ecology, Parasitology     

Antagonistic interaction between male-killing and cytoplasmic incompatibility induced by Cardinium and Wolbachia in the whitefly,Bemisia tabaci

Cardinium and Wolbachia are maternally inherited bacterial symbionts of arthropods that can manipulate host reproduction by increasing the fitness of infected females.Here,we report that Cardinium and Wolbachia coinfection induced male-killing and cytoplasmic incompatibility(CI)when they coexisted in a cryptic species of whitefly,Bemisia tabaci Asia II7.Cardinium and Wolbachia symbionts were either singly or simul-taneously localized in the bacteriocytes placed in the abdomen of B.tabaci nymphs and adults.Cardinium-Wolbachia coinfection induced male-killing and resulted in a higher female sex ratio in the intraspecific amphigenetic progeny of Asia II7 IcwH and IcwL lines;interestingly,male-illing induction was enhanced with increased Cardinium titer.Moreover,single infection of Wolbachia induced partial CI in the Asia II7 Iw line and resulted in reduced fecundity,higher embryonic mortality,and lower female sex ratio.The uninfected Asia II7 Iu line had significantly higher fecundity,lower embryonic and nymphal mortalities,and a lower level of CI than both the Wolbachia infected Asia II7 Iw line and the Cardinium--Wolbachia-coinfected Asia II7 IcwH line.Our findings indicate that Cardinium-Wolbachia coinfection induced male-killing,which may have had antag-onistic effects on Wolbachia-induced CI in the Asia II7 whiteflies.For the first time,our study revealed that B.tabaci Asia II7 reproduction is co-manipulated by Cardinium and Wolbachia endosymbionts.     

Epidemiology of asexuality induced by the endosymbiotic Wolbachia across phytophagous wasp species: host plant specialization matters

Among eukaryotes, sexual reproduction is by far the most predominant mode of reproduction. However, some systems maintaining sexuality appear particularly labile and raise intriguing questions on the evolutionary routes to asexuality. Thelytokous parthenogenesis is a form of spontaneous loss of sexuality leading to strong distortion of sex ratio towards females and resulting from mutation, hybridization or infection by bacterial endosymbionts. We investigated whether ecological specialization is a likely mechanism of spread of thelytoky within insect communities. Focusing on the highly specialized genus Megastigmus (Hymenoptera: Torymidae), we first performed a large literature survey to examine the distribution of thelytoky in these wasps across their respective obligate host plant families. Second, we tested for thelytoky caused by endosymbionts by screening in 15 arrhenotokous and 10 thelytokous species for Wolbachia, Cardinium, Arsenophonus and Rickettsia endosymbionts and by performing antibiotic treatments. Finally, we performed phylogenetic reconstructions using multilocus sequence typing (MLST) to examine the evolution of endosymbiont‐mediated thelytoky in Megastigmus and its possible connections to host plant specialization. We demonstrate that thelytoky evolved from ancestral arrhenotoky through the horizontal transmission and the fixation of the parthenogenesis‐inducing Wolbachia. We find that ecological specialization in Wolbachia's hosts was probably a critical driving force for Wolbachia infection and spread of thelytoky, but also a constraint. Our work further reinforces the hypothesis that community structure of insects is a major driver of the epidemiology of endosymbionts and that competitive interactions among closely related species may facilitate their horizontal transmission.     

Endosymbiont costs and benefits in a parasitoid infected with both Wolbachia and Cardinium

Theory suggests that maternally inherited endosymbionts can promote their spread and persistence in host populations by enhancing the production of daughters by infected hosts, either by improving overall host fitness, or through reproductive manipulation. In the doubly infected parasitoid wasp Encarsia inaron, Wolbachia manipulates host reproduction through cytoplasmic incompatibility (CI), but Cardinium does not. We investigated the fitness costs and/or benefits of infection by each bacterium in differentially cured E. inaron as a potential explanation for persistence of Cardinium in this population. We introgressed lines infected with Wolbachia, Cardinium or both with the cured line to create a similar genetic background, and evaluated several parasitoid fitness parameters. We found that symbiont infection resulted in both fitness costs and benefits for E. inaron. The cost was lower initial egg load for all infected wasps. The benefit was increased survivorship, which in turn increased male production for wasps infected with only Cardinium. Female production was unaffected by symbiont infection; we therefore have not yet identified a causal fitness effect that can explain the persistence of Cardinium in the population. Interestingly, the Cardinium survivorship benefit was not evident when Wolbachia was also present in the host, and the reproduction of doubly infected individuals did not differ significantly from uninfected wasps. Therefore, the results of our study show that even when multiple infections seem to have no effect on a host, there may be a complex interaction of costs and benefits among symbionts.     

Diversity of secondary endosymbionts among different putative species of the whitefly Bemisia tabaci

Abstract Endosymbionts are important components of arthropod biology. The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex composed of ≥ 28 putative species. In addition to the primary endosymbiont Portiera aleyrodidarum, six secondary endosymbionts (S‐endosymbionts), Hamiltonella, Rickettsia, Wolbachia, Cardinium, Arsenophonus and Fritschea, have been identified in B. tabaci thus far. Here, we tested five of the six S‐endosymbiont lineages (excluding Fritschea) from 340 whitely individuals representing six putative species from China. Hamiltonella was detected only in the two exotic invaders, Middle East‐Asia Minor 1 (MEAM1) and Mediterranean (MED). Rickettsia was absent in Asia II 1 and MED, scarce in Asia II 3 (13%), but abundant in Asia II 7 (63.2%), China 1 (84.7%) and MEAM1 (100%). Wolbachia, Cardinium and Arsenophonus were absent in the invasive MEAM1 and MED but mostly abundant in the native putative species. Furthermore, phylogenetic analyses revealed that some S‐endosymbionts have several clades and different B. tabaci putative species can harbor different clades of a given S‐endosymbiont, demonstrating further the complexity of S‐endosymbionts in B. tabaci. All together, our results demonstrate the variation and diversity of S‐endosymbionts in different putative species of B. tabaci, especially between invasive and native whiteflies.     

Effects of different temperature regimes on survival of Diaphorina citri and its endosymbiotic bacterial communities

The Asian citrus psyllid, Diaphorina citri, is a major pest of citrus and vector of citrus greening (huanglongbing) in Asian. In our field‐collected psyllid samples, we discovered that Fuzhou (China) and Faisalabad (Pakistan), populations harbored an obligate primary endosymbiont Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.) and a secondary endosymbiont, Wolbachia surface proteins (WSP) which are intracellular endosymbionts residing in the bacteriomes. Responses of these symbionts to different temperatures were examined and their host survival assessed. Diagnostic PCR assays showed that the endosymbionts infection rates were not significantly reduced in both D. citri populations after 24 h exposure to cold or heat treatments. Although quantitative PCR assays showed significant reduction of WSP relative densities at 40°C for 24 h, a substantial decrease occurred as the exposure duration increased beyond 3 days. Under the same temperature regimes, Ca. C. ruddii density was initially less affected during the first exposure day, but rapidly reduced at 3–5 days compared to WSP. However, the mortality of the psyllids increased rapidly as exposure time to heat treatment increased. The responses of the two symbionts to unfavorable temperature regimes highlight the complex host‐symbionts interactions between D. citri and its associated endosymbionts.     

Transinfection of the olive fruit fly Bactrocera oleae with Wolbachia: towards a symbiont‐based population control strategy

The olive fruit fly Bactrocera oleae is responsible for worldwide economic damage. In this report, we describe the first B. oleae lines transinfected with the Wolbachia strain wCer2, an endosymbiont of the cherry fruit fly Rhagoletis cerasi. Immunostaining followed by confocal microscopy, detects high numbers of Wolbachia in embryos as well as in ovarioles and sperm from individuals of both transinfected lines. wCer2 was uniformly distributed in B. oleae egg chambers and the cortex of preblastoderm embryos. Wolbachia is known to manipulate host reproduction with several strategies, one of which is cytoplasmic incompatibility (CI), resulting in embryonic mortality in incompatible crosses. Wolbachia was found to induce complete CI in the novel host, suggesting that symbiont‐based approaches can be used as novel environmentally friendly tools for the control of natural olive fruit fly populations.     

Evidence for Horizontal Transmission of Secondary Endosymbionts in the Bemisia tabaci Cryptic Species Complex

Bemisia tabaci (Hemiptera: Aleyrodidae) is a globally distributed pest composed of at least 34 morphologically indistinguishable cryptic species. At least seven species of endosymbiont have been found infecting some or all members of the complex. The origin(s) of the associations between specific endosymbionts and their whitefly hosts is unknown. Infection is normally vertical, but horizontal transmission does occur and is one way for new infections to be introduced into individuals. The relationships between the different members of the cryptic species complex and the endosymbionts have not been well explored. In this study, the phylogenies of different cryptic species of the host with those of their endosymbionts were compared. Of particular interest was whether there was evidence for both coevolution and horizontal transmission. Congruence was observed for the primary endosymbiont, Portiera aleyrodidarum, and partial incongruence in the case of two secondary endosymbionts, Arsenophonus and Cardinium and incongruence for a third, Wolbachia. The patterns observed for the primary endosymbiont supported cospeciation with the host while the patterns for the secondary endosymbionts, and especially Wolbachia showed evidence of host shifts and extinctions through horizontal transmission rather than cospeciation. Of particular note is the observation of several very recent host shift events in China between exotic invader and indigenous members of the complex. These shifts were from indigenous members of the complex to the invader as well as from the invader to indigenous relatives.     

Influences of two coexisting endosymbionts,CI‐inducing Wolbachia and male‐killing Spiroplasma,on the performance of their host Laodelphax striatellus (Hemiptera: Delphacidae)

The small brown planthopper Laodelphax striatellus (Hemiptera: Delphacidae) is reported to have the endosymbiont Wolbachia, which shows a strong cytoplasmic incompatibility (CI) between infected males and uninfected females. In the 2000s, female‐biased L. striatellus populations were found in Taiwan, and this sex ratio distortion was the result of male‐killing induced by the infection of another endosymbiont, Spiroplasma. Spiroplasma infection is considered to negatively affect both L. striatellus and Wolbachia because the male‐killing halves the offspring of L. striatellus and hinders the spread of Wolbachia infection via CI. Spiroplasma could have traits that increase the fitness of infected L. striatellus and/or coexisting organisms because the coinfection rates of Wolbachia and Spiroplasma were rather high in some areas. In this study, we investigated the influences of the infection of these two endosymbionts on the development, reproduction, and insecticide resistance of L. striatellus in the laboratory. Our results show that the single‐infection state of Spiroplasma had a negative influence on the fertility of L. striatellus, while the double‐infection state had no significant influence. At late nymphal and adult stages, the abundance of Spiroplasma was lower in the double‐infection state than in the single‐infection state. In the double‐infection state, the reduction of Spiroplasma density may be caused by competition between the two endosymbionts, and the negative influence of Spiroplasma on the fertility of host may be relieved. The resistance of L. striatellus to four insecticides was compared among different infection states of endosymbionts, but Spiroplasma infection did not contribute to increase insecticide resistance. Because positive influences of Spiroplasma infection were not found in terms of the development, reproduction, and insecticide resistance of L. striatellus, other factors improving the fitness of Spiroplasma‐infected L. striatellus may be related to the high frequency of double infection in some L. striatellus populations.     

Cryptic diversity,reproductive isolation and cytoplasmic incompatibility in a classic biological control success story

Molecular genetics and symbiont diagnostics have revolutionized our understanding of insect species diversity, and the transformative effects of bacterial symbionts on host life history. Encarsia inaron is a parasitoid wasp that has been shown to harbour two bacterial endosymbionts, Wolbachia and Cardinium. Known then as E. partenopea, it was introduced to the USA in the late 1980s from populations collected in Italy and Israel for the biological control of an ornamental tree pest, the ash whitefly, Siphoninus phillyreae. We studied natural populations from sites in the USA, the Mediterranean and the Middle East as well as from a Cardinium‐infected laboratory culture established from Italy, with the aims of characterizing these populations genetically, testing reproductive isolation, determining symbiont infection status in their native and introduced range, and determining symbiont role. The results showed that the two Encarsia populations introduced to the USA are genetically distinct, reproductively isolated, have different symbionts and different host–symbiont interactions, and can be considered different biological species. One (‘E. inaron’) is doubly infected by Wolbachia and Cardinium, while only Cardinium is present in the other (‘E. partenopea’). The Cardinium strains in the two species are distinct, although closely related, and crossing tests indicate that the Cardinium infecting ‘E. partenopea’ induces cytoplasmic incompatibility. The frequency of symbiont infection found in the native and introduced range of these wasps was similar, unlike the pattern seen in some other systems. These results also lead to a retelling of a successful biological control story, with several more characters than had been initially described.     

Genetic variability,community structure,and horizontal transfer of endosymbionts among three Asia II‐Bemisia tabaci mitotypes in Pakistan

Endosymbionts associated with the whitefly Bemisia tabaci cryptic species are known to contribute to host fitness and environmental adaptation. The genetic diversity and population complexity were investigated for endosymbiont communities of B. tabaci occupying different micro‐environments in Pakistan. Mitotypes of B. tabaci were identified by comparative sequence analysis of the mitochondria cytochrome oxidase I (mtCOI) gene sequence. Whitefly mitotypes belonged to the Asia II‐1, ‐5, and ‐7 mitotypes of the Asia II major clade. The whitefly–endosymbiont communities were characterized based on 16S ribosomal RNA operational taxonomic unit (OTU) assignments, resulting in 43 OTUs. Most of the OTUs occurred in the Asia II‐1 and II‐7 mitotypes (r² = .9, p < .005), while the Asia II‐5 microbiome was less complex. The microbiome OTU groups were mitotype‐specific, clustering with a basis in phylogeographical distribution and the corresponding ecological niche of their whitefly host, suggesting mitotype‐microbiome co‐adaptation. The primary endosymbiont Portiera was represented by a single, highly homologous OTU (0%–0.67% divergence). Two of six Arsenophonus OTUs were uniquely associated with Asia II‐5 and ‐7, and one occurred exclusively in Asia II‐1, two only in Asia II‐5, and one in both Asia II‐1 and ‐7. Four other secondary endosymbionts, Cardinium, Hemipteriphilus, Rickettsia, and Wolbachia OTUs, were found at ≤29% frequencies. The most prevalent Arsenophonus OTU was found in all three Asia II mitotypes (55% frequency), whereas the same strain of Cardinium and Wolbachia was found in both Asia II‐1 and ‐5, and a single Hemipteriphilus OTU occurred in Asia II‐1 and ‐7. This pattern is indicative of horizontal transfer, suggestive of a proximity between mitotypes sufficient for gene flow at overlapping mitotype ecological niches.     

Detection of symbionts and virus in the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae), vector of the <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> in Central India

Legume crops in Central India, the main soybean production area of the country, may suffer from yellow mosaic disease caused by the Mungbean yellow mosaic India virus (MYMIV). MYMIV is transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), which is a species complex composed of various genetic groups. This vector species harbors different endosymbionts among regional strains and among individuals. To elucidate fundamental aspects of this virus vector in the state of Madhya Pradesh, the infection status of the symbionts and the virus in whiteflies was studied. A polymerase chain reaction (PCR) survey of the whiteflies collected in Madhya Pradesh found four secondary endosymbionts, Arsenophonus, Hemipteriphilus, Wolbachia, and Cardinium, in addition to the primary endosymbiont Portiera. Arsenophonus and Hemipteriphilus were highly infected but the infection rates of Wolbachia and Cardinium were low. MYMIV was detected in whitefly populations collected from various host plants in Madhya Pradesh. The whitefly populations belonged to the Asia I and II genetic groups; several different Asia II populations were also distributed. Specific relations were not observed among symbiont infection status, virus infection, and the whitefly genetic groups in the populations of Madhya Pradesh, though Cardinium was highly detected in the Asia II-1 group. New primers, which can be used for PCR template validation and for discriminating two phylogenetically close endosymbionts, were designed.     

<Emphasis Type="Italic">Wolbachia</Emphasis>-induced cytoplasmic incompatibility in Japanese populations of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae)

Intracellular bacteria of the genus Wolbachia (alpha Proteobacteria) induce cytoplasmic incompatibility (CI) in many arthropod species, including spider mites, but not all Wolbachia cause CI. In spider mites CI becomes apparent by a reduced egg hatchability and a lower daughter:son ratio: CI in haplodiploid organisms in general was expected to produce all-male offspring or a male-biased sex ratio without any death of eggs. In a previous study of Japanese populations of Tetranychus urticae, two out of three green-form populations tested were infected with non-CI Wolbachia strains, whereas none of six red-form populations harbored Wolbachia. As the survey of Wolbachia infection in T. urticae is still fragmentary in Japan, we checked Wolbachia infection in thirty green-form populations and 29 red-form populations collected from a wide range of Japanese islands. For Wolbachia-infected populations, we tested the effects of Wolbachia on the reproductive traits and determined the phylogenetic relationships of the different strains of Wolbachia. All but one green-form populations were infected with Wolbachia and all strains belonged to the subgroup Ori when the wsp gene was used to determine the phylogenetic relationships of different strains of Wolbachia. Six out of 29 red-form populations harbored Wolbachia and the infected strains belonged to the subgroups Ori and Bugs. Twenty-four of 29 infected green-form populations and five of six infected red-form populations induced CI among the hosts. Thus, CI-Wolbachia strains are widespread in Japan, and no geographical trend was observed in the CI-Wolbachia. Although three red-form populations harbored other intracellular bacteria Cardinium, they did not affect host reproduction.     

Multiple endosymbiont infections and reproductive manipulations in a linyphiid spider population

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.     

Host genotype–endosymbiont associations and their relationship with aphid parasitism at the field level

1. The relationship between endosymbionts and insects represent complex eco‐evolutionary interactions. Vertically transmitted endosymbionts can be a source of evolutionary novelty by conferring ecologically important traits to their insect hosts, such as protection against natural enemies. Host–endosymbiont associations could constitute an adaptive complex (holobiont) on which selective pressures present in the environment can act, being transferred to the next generation. 2. Although several laboratory‐based studies have confirmed host genotype × symbiont interactions, few studies have been directed at those associations in the natural populations and their ability to protect themselves from parasitism pressure at the field level. 3. A field‐based approach to study the aphid genotype–endosymbiont associations and its relationship with the total parasitism in the grain aphid Sitobion avenae was conducted. From the field study, experiments were carried out to study the defensive effect of the two most common facultative endosymbionts (Regiella insecticola and Hamiltonella defensa) present in S. avenae against one of the most important parasitoid species, Aphidius ervi. 4. Evidence is presented here of a high specificity of the aphid clone–endosymbiont associations in the field; however, the field and experimental results here do not support a relationship between the aphid clone–endosymbiont associations and a proxy of total parasitism in S. avenae. These findings highlight the importance of particular host clone–endosymbiont couplings as a key factor in gaining an understanding of the coevolutionary dynamics of endosymbionts in nature and their effect on the invasive potential of pest insects.     

Age,tissue, genotype and virus infection regulate Wolbachia levels in Drosophila

The bacterial symbiont Wolbachia can protect insects against viral pathogens, and the varying levels of antiviral protection are correlated with the endosymbiont load within the insects. To understand why Wolbachia strains differ in their antiviral effects, we investigated the factors controlling Wolbachia density in five closely related strains in their natural Drosophila hosts. We found that Wolbachia density varied greatly across different tissues and between flies of different ages, and these effects depended on the host–symbiont association. Some endosymbionts maintained largely stable densities as flies aged while others increased, and these effects in turn depended on the tissue being examined. Measuring Wolbachia rRNA levels in response to viral infection, we found that viral infection itself also altered Wolbachia levels, with Flock House virus causing substantial reductions in symbiont loads late in the infection. This effect, however, was virus‐specific as Drosophila C virus had little impact on Wolbachia in all of the five host systems. Because viruses have strong tissue tropisms and antiviral protection is thought to be cell‐autonomous, these effects are likely to affect the virus‐blocking phenomenon. However, we were unable to find any evidence of a correlation between Wolbachia and viral titres within the same tissues. We conclude that Wolbachia levels within flies are regulated in a complex host–symbiont–virus‐dependent manner and this trinity is likely to influence the antiviral effects of Wolbachia.