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.     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

Examination of the success rate of secondary symbiont manipulation by microinjection methods in the pea aphid system

Insects harbor a wide range of microbial symbionts, but their influence on host phenotypes is described in a limited number of biological models. One experimental approach to gain knowledge on the effects of symbionts to their hosts is to create insect lines with and without symbionts and examine their phenotypes. However, the success rate of symbiont elimination and introduction methods is dependent on several parameters that are scarcely tested or described. The pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), is a model insect of symbiosis studies. It harbors a primary symbiont that supplies the host with essential amino acids, and an array of secondary symbionts whose effects have been assessed by manipulating their presence/absence in the insect. Here, we describe the influence of key parameters on the success rate of symbiont manipulation using the pea aphid–secondary symbiont system. We compared two elimination methods differing in antibiotic treatment using several aphid–symbiont combinations. We also created new aphid host–symbiont combinations by secondary symbiont introduction and examined the effects of larval stage of recipient aphids on introduction success. Our study revealed that the aphid–symbiont combination has strong influence on both symbiont introduction and elimination success rates, and that the type of antibiotics and the larval stage of recipient aphids influence the elimination and introduction success rate, respectively.     

Cover Caption

The whitefly Bemisia tabaci contains many big bacteriocytes, which house the primary symbiont “Candidatus Portiera aleyrodidarum” and a variety of secondary symbionts (see pages 194–206). The primary symbiont provides essential amino acids to hosts and the secondary symbionts can affect the life parameters of B. tabaci including fecundity, development time and sex‐ratio. In addition, the secondary symbionts of B. tabaci have been reported to confer resistance to natural enemies, heat stress and insecticides. Photo in cover shows a nymph of the whitefly and the two kidney‐like organs are the assemblage of bacteriocytes. Image by Xiao‐Li Bing.     

Diversity of bacteria associated with Hormaphidinae aphids (Hemiptera: Aphididae)

Bacteria are ubiquitous inhabitants of animals.Hormaphidinae is a particular aphid group exhibiting very diverse life history traits.However,the microbiota in this group is poorly known.In the present study,using high-throughput sequencing of bacterial 16S ribosomal RNA gene amplicons,we surveyed the bacterial flora in hormaphidine aphids and explored whether the aphid tribe,host plant and geographical distribution are associated with the distribution of secondary symbionts.The most dominant bacteria detected in hormaphidine species are heritable symbionts.As expected,the primary endosymbiont Buchnera aphidicola is the most abundant symbiont across all species and has cospeciated with its host aphids.Six secondary symbionts were detected in Hormaphidinae.Arsenophonus is widespread in Hormaphidinae species,suggesting the possibility of ancient acquisition of this symbiont.Ordination analyses and statistical tests show that the symbiont composition does not seem to relate to any of the aphid tribes,host plants or geographical distributions,which indicate that horizontal transfers might occur for these symbionts in Hormaphidinae.Correlation analysis exhibits negative interference between Buchnera and coexisting secondary symbionts,while the interactions between different secondary symbionts are complicated.These findings display a comprehensive picture of the microbiota in Hormaphidinae and may be helpful in understanding the symbiont diversity within a group of aphids.     

Parasitoid bacterial symbionts as markers of within‐host competitive outcomes: superparasitoid advantage and sex ratio bias

1. Bacterial symbionts have the potential to alter insect fitness, which could influence insect competitive ability. To investigate this possibility, within‐host competitions were staged between individuals of the parasitoid, Encarsia inaron Walker, that were differentially infected with the bacterial symbionts Cardinium and Wolbachia. 2. When parasitoids of different infection status parasitised the same whitefly host, there was no evidence that symbiont infection influenced the outcome of competition. 3. Using symbionts as markers, a significant advantage for the second (superparasitising) wasp was detected when eggs were deposited within 4 h of one another, probably as a result of ovicidal behaviour by the superparasitoid, but not when eggs were deposited 1 day apart. Additionally, the emerged sex ratio of superparasitoid offspring was male biased when eggs were deposited 4 h after the initial eggs, but had an even sex ratio when deposited 1 day later. 4. The present study demonstrates the potential utility of symbiont infection as a marking system for investigating within‐host competition among parasitoids.     

Co-evolution and symbiont replacement shaped the symbiosis between adelgids (Hemiptera: Adelgidae) and their bacterial symbionts

The Adelgidae (Insecta: Hemiptera), a small group of insects, are known as severe pests on various conifers of the northern hemisphere. Despite of this, little is known about their bacteriocyte‐associated endosymbionts, which are generally important for the biology and ecology of plant sap‐sucking insects. Here, we investigated the adelgid species complexes Adelges laricis/tardus, Adelges abietis/viridis and Adelges cooleyi/coweni, identified based on their coI and ef1alpha genes. Each of these insect groups harboured two phylogenetically different bacteriocyte‐associated symbionts belonging to the Betaproteobacteria and the Gammaproteobacteria, respectively, as inferred from phylogenetic analyses of 16S rRNA gene sequences and demonstrated by fluorescence in situ hybridization. The betaproteobacterial symbionts of all three adelgid complexes (‘Candidatus Vallotia tarda’, ‘Candidatus Vallotia virida’ and ‘Candidatus Vallotia cooleyia’) share a common ancestor and show a phylogeny congruent with that of their respective hosts. Similarly, there is evidence for co‐evolution between the gammaproteobacterial symbionts (‘Candidatus Profftia tarda’, ‘Candidatus Profftia virida’) and A. laricis/tardus and A. abietis/viridis. In contrast, the gammaproteobacterial symbiont of A. cooleyi/coweni (‘Candidatus Gillettellia cooleyia’) is different from that of the other two adelgids but shows a moderate relationship to the symbiont ‘Candidatus Ecksteinia adelgidicola’ of A. nordmannianae/piceae. All symbionts were present in all adelgid populations and life stages analysed, suggesting vertical transmission from mother to offspring. In sharp contrast to their sister group, the aphids, adelgids do not consistently contain a single obligate (primary) symbiont but have acquired phylogenetically different bacterial symbionts during their evolution, which included multiple infections and symbiont replacement.     

Host nuclear genotype influences phenotype of a conditional mutualist symbiont

Arthropods commonly carry maternally inherited intracellular bacterial symbionts that may profoundly influence host biology and evolution. The intracellular symbiont Rickettsia sp. nr. bellii swept rapidly into populations of the sweetpotato whitefly Bemisia tabaci in the south‐western USA. Previous laboratory experiments showed female‐bias and fitness benefits were associated with Rickettsia infection, potentially explaining the high frequencies of infection observed in field populations, but the effects varied with whitefly genetic line. Here, we explored whether host extranuclear or nuclear genes influenced the variation in the Rickettsia–host phenotype in two genetic lines of the whitefly host, each with Rickettsia‐infected and uninfected sublines. Introgression between the Rickettsia‐infected subline of one genetic line and the Rickettsia‐uninfected subline of the other was used to create two new sublines, each with the maternally inherited extranuclear genetic lineages of one line (Rickettsia, two other symbionts and the mitochondria) and the nuclear genotype of the other. Performance assays comparing the original and new lines showed that in addition to Rickettsia, the interaction of Rickettsia infection with host nuclear genotype influenced development time and the sex ratio of the progeny, whereas the extranuclear genotype did not. Host nuclear genotype, but not extranuclear genotype, also influenced the titre of Rickettsia. Our results support the hypothesis that differences in host nuclear genotype alone may explain considerable within‐population variation in host–symbiont phenotype and may contribute to the observed variation in Rickettsia–whitefly interactions worldwide.     

Primary symbiont of the ancient scale insect family Coelostomidiidae exhibits strict cophylogenetic patterns

Bacterial symbionts play a critical role in the physiology, ecology and evolution of a diverse range of insects. Such symbionts with unknown roles in the ecology and evolution of their hosts have been reported from archaeococcoid scale insects of family Coelostomidiidae. We examine in detail the bacterial community associated with the remaining species of this family, and calculate the cophylogenetic relationship between the hosts and their symbionts. The 28S ribosomal RNA (rRNA) and mitochondrial cytochrome oxidase I genes were used to reconstruct the host phylogeny while the 16S rRNA gene was used for the bacterial phylogeny. Three well-supported clades were detected within the phylogeny of the monophyletic family Coelostomidiidae. Besides the known symbionts, a novel Sodalis-like symbiont was detected from three of the species. The primary bacteriome inhabiting B-symbiont (Bacteroidetes; ‘Candidatus Hoataupuhia coelostomidicola’) was widespread across the host family. Cophylogenetic comparison using Jungles-based reconciliation analysis and ParaFit statistical test revealed a strongly congruent phylogeny of this symbiont with the host family, with no host-switches and few losses and duplications. A similar pattern was observed across a relatively unrelated neococcoid family that exhibits a different physiology and symbiont community, besides a related Bacteroidetes symbiont. We reconfirm that the B-symbiont is a primary symbiont, owing to its strongly congruent evolution with the host and its bacteriome-inhabiting nature. Our analysis affirms recent suggestions that the Bacteroidetes-affiliated symbionts may have driven the hyper-diversification of scale insects worldwide.     

Cohabitation and roommate bias of symbiotic bacteria in insect hosts

Symbiotic interactions between insects and bacteria have long fascinated ecologists. Aphids have emerged as the model system on which to study the effect of endosymbiotic bacteria on their hosts. Aphid‐symbiont interactions are ecologically interesting as aphids host multiple secondary symbionts that can provide broad benefits, such as protection against heat stress or specialist natural enemies (parasitic wasps and entomopathogenic fungi). There are nine common aphid secondary symbionts and individual aphids host on average 1–2 symbionts. A cost‐benefit trade‐off for hosting symbionts is thought to explain why not all aphids host every possible symbiont in a population. Both positive and negative associations between various symbionts occur, and this could happen due to increased costs when cohosting certain combinations or as a consequence of competitive interactions between the symbionts within a host. In this issue of Molecular Ecology, Mathé‐Hubert, Kaech, Hertaeg, Jaenike, and Vorburger (2019) use data on the symbiont status of field‐collected aphids to inform a model on the evolution of symbiont co‐occurrence. They vary the effective female population size as well as the rate of horizontal and maternal transmission to infer the relative impact of symbiont‐symbiont interactions versus random drift. Additional data analysis revisits an association between two symbionts in a fruit fly species using a long‐term data set to highlight that such interactions are not limited to aphids.     

Selective elimination of aphid endosymbionts: effects of antibiotic dose and host genotype, and fitness consequences

Multiple endosymbionts commonly coexist in the same host insects. In order to gain an understanding of the biological roles of the individual symbionts in such complex systems, experimental techniques for enabling the selective removal of a specific symbiont from the host are of great importance. By using the pea aphid-Buchnera-Serratia endosymbiotic system as a model, the efficacy, generality, and fitness consequences of selective elimination techniques at various antibiotic doses and under a variety of host genotypes were investigated. In all the disymbiotic aphid strains examined, the facultative symbiont Serratia was selectively eliminated by ampicillin treatment in a dose-dependent manner, suggesting a generality of the elimination technique irrespective of host genotype. However, fitness consequences of the Serratia elimination differed between the aphid strains, indicating substantial effects of host genotype. In all the disymbiotic aphid strains, the obligate symbiont Buchnera was selectively eliminated by rifampicin treatment irrespective of the antibiotic dose. However, the survival and reproduction of the Buchnera-free aphids varied in a dose-dependent manner, and the dose dependence was strikingly different between the aphid genotypes. These results provide a basis for the development of new protocols for manipulating insect endosymbiotic microbiota.     

Coexistence of novel gammaproteobacterial and Arsenophonus symbionts in the scale insect Greenisca brachypodii (Hemiptera,Coccomorpha: Eriococcidae)

Scale insects are commonly associated with obligate, intracellular microorganisms which play important roles in complementing their hosts with essential nutrients. Here we characterized the symbiotic system of Greenisca brachypodii, a member of the family Eriococcidae. Histological and ultrastructural analyses have indicated that G. brachypodii is stably associated with coccoid and rod‐shaped bacteria. Phylogenetic analyses have revealed that the coccoid bacteria represent a sister group to the secondary symbiont of the mealybug Melanococcus albizziae, whereas the rod‐shaped symbionts are close relatives of Arsenophonus symbionts in insects – to our knowledge, this is the first report of the presence of Arsenophonus bacterium in scale insects. As a comparison of 16S and 23S rRNA genes sequences of the G. brachypodii coccoid symbiont with other gammaprotebacterial sequences showed only low similarity (～90%), we propose the name ‘Candidatus Kotejella greeniscae’ for its tentative classification. Both symbionts are transovarially transmitted from one generation to the next. The infection takes place in the neck region of the ovariole. The bacteria migrate between follicular cells, as well as through the cytoplasm of those cells to the perivitelline space, where they form a characteristic ‘symbiont ball’. Our findings provide evidence for a polyphyletic origin of symbionts of Eriococcidae.     

Effects of antibiotics on fitness of the B biotype and a non-B biotype of the whitefly Bemisia tabaci

The whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae), harbors primary and secondary endosymbionts. Previous research showed that the invasive B biotype and an indigenous non‐B biotype (named non‐B ZHJ‐1 population) of B. tabaci from Zhejiang, China, harbored different endosymbionts. To investigate the function of these endosymbionts in the two biotypes of B. tabaci, we fed adult whiteflies with three antibiotics, tetracycline, ampicillin trihydrate, and rifampicin, and evaluated the fitness of their offspring on cotton plants. These three antibiotics did not remove the primary endosymbiont Portiera aleyrodidarum but were capable of eliminating the secondary endosymbionts. In the B biotype, treatments of adults with tetracycline or ampicillin trihydrate accelerated development and increased the survival of their offspring, while treatment of adults with rifampicin significantly retarded the development of their offspring but did not affect their survival. In the non‐B ZHJ‐1 population, treatments of adults with tetracycline or ampicillin trihydrate also accelerated the development of their offspring but did not significantly affect their survival, while treatment of adults with rifampicin significantly retarded development and reduced the survival of their offspring. These results suggest that removal of some secondary endosymbionts and/or reduction of the primary endosymbiont from B. tabaci may produce both favorable and unfavorable effects on the fitness of the host insects.     

Band‐aids for Buchnera and B vitamins for all

Evolution lacks foresight, and hence, key adaptations may produce major challenges over the long run. The natural world is rife with examples of long‐term ‘side effects’ associated with quick‐fix tinkering, including blind spots in vertebrate eyes. An important question is how nature compensates for imperfections once evolution has set a course. The symbioses associated with sap‐feeding insects present a fascinating opportunity to address this issue. On one hand, the substantial diversity and biomass of sap‐feeding insects are largely due to ancient acquisitions of nutrient‐provisioning bacterial symbionts. Yet, the insularity and small population sizes enforced by intracellular life and strict maternal transfer inevitably result in the degradation of symbiont genomes and, often, the beneficial services that symbionts provide. Stabilization through lateral transfer of bacterial genes into the host nucleus (often from exogenous sources) or replacement of the long‐standing symbiont with a new partner are potential solutions to this evolutionary dilemma (Bennett & Moran 2015 ). A third solution is adoption of a cosymbiont that compensates for specific losses in the original resident. Ancient ‘co‐obligate’ symbiont pairs in mealybugs, leafhoppers, cicadas and spittlebugs show colocalization, codiversification, metabolite exchange and generally nonredundant nutrient biosynthesis (Bennett & Moran 2015 ). But in this issue, Meseguer et al. ( 2017 ) report on a different flavour of cosymbiosis among conifer‐feeding Cinara aphids.     

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.     

Cellular and potential molecular mechanisms underlying transovarial transmission of the obligate symbiont Sulcia in cicadas

Vertical transmission of symbionts in insects is critical to persistence of symbioses across host generations. The key time point and related cellular/molecular mechanisms underlying the transmission in most insects remain unclear. Here, we reveal that in the bacteriome–endosymbiont system of the cicada Meimuna mongolica, the obligate symbiont Candidatus Sulcia muelleri (hereafter Sulcia) proliferates and migrates to the ovaries mainly after the adult emergence of cicadas. Sulcia cells swell to approximately twice their previous size with the outer membrane changed to be more irregular during this process. Almost all the Sulcia genes involved in biosynthesis of essential amino acids, heat shock protein, energy metabolism, DNA replication and repair and protein export were highly expressed in all life stages of cicadas. Among which, genes involved in DNA replication and synthesis of leucine and arginine were upregulated in the newly emerged adults relative to fifth-instar nymphs. Signal transduction is the pronounced function exhibited in both Sulcia and the cicada bacteriomes in newly emerged adults. The results suggest host sensing of arginine and leucine integrate Sulcia's output of host-EAAs into mTORC1 signalling. This study highlights the importance of signalling pathways in regulating the host/symbiont interaction and symbiont transmission in sap-feeding auchenorrhynchous insects.     

Removal of a specialization-associated symbiont does not affect aphid fitness

Symbioses are ubiquitous in nature. However, these interactions, in particular those which are facultative from the perspective of the host, are still poorly understood. One major question is whether facultative symbionts affect host fitness, and in particular whether they can benefit the host. In pea aphid, host plant specialization was found to be associated with the presence of Pea Aphid U‐type Symbiont [Proc. R. Soc. Lond. B 270 (2003) S209]. Host and symbiont genomes are normally co‐transmitted, making it difficult to determine which is responsible for causing this variation in fitness. Here antibiotic treatment is used to selectively remove the symbiont, thus partitioning symbiont and host genomes. I find that PAUS is not responsible for causing host plant specialization. Examination of aphids in which PAUS presence has been naturally manipulated also supports this conclusion. Additional work will be necessary to determine the forces maintaining this symbiont in host populations, and whether it benefits the host insect under different conditions.     

Protection against a fungal pathogen conferred by the aphid facultative endosymbionts Rickettsia and Spiroplasma is expressed in multiple host genotypes and species and is not influenced by co‐infection with another symbiont

Many insects harbour facultative endosymbiotic bacteria, often more than one type at a time. These symbionts can have major effects on their hosts' biology, which may be modulated by the presence of other symbiont species and by the host's genetic background. We investigated these effects by transferring two sets of facultative endosymbionts (one Hamiltonella and Rickettsia, the other Hamiltonella and Spiroplasma) from naturally double‐infected pea aphid hosts into five novel host genotypes of two aphid species. The symbionts were transferred either together or separately. We then measured aphid fecundity and susceptibility to an entomopathogenic fungus. The pathogen‐protective phenotype conferred by the symbionts Rickettsia and Spiroplasma varied among host genotypes, but was not influenced by co‐infection with Hamiltonella. Fecundity varied across single and double infections and between symbiont types, aphid genotypes and species. Some host genotypes benefit from harbouring more than one symbiont type.     

Differential Responses of the Whitefly Bemisia tabaci Symbionts to Unfavorable Low and High Temperatures

The whitefly Bemisia tabaci complex contains many cryptic species, of which the Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) are notorious invasive pests. In our field-collected whitefly samples, MEAM1 harbors an obligate primary symbiont “Candidatus Portiera aleyrodidarum” and two secondary symbionts, “Candidatus Hamiltonella defensa” and Rickettsia sp., whereas MED has only “Ca. Portiera aleyrodidarum” and “Ca. Hamiltonella defensa.” Both “Ca. Portiera aleyrodidarum” and “Ca. Hamiltonella defensa” are intracellular endosymbionts residing in the bacteriomes, whereas Rickettsia sp. has a scattered distribution throughout the host body cavity. We examined responses of these symbionts to adverse temperatures as well as survival of the host insects. After cold treatment at 5 or 10 °C or heat treatment at 35 or 40 °C for 24 h, respectively, the infection rates of all symbionts were not significantly decreased based on diagnosis PCR. However, quantitative PCR assays indicated significant reduction of “Ca. Hamiltonella defensa” at 40 °C, and the reduction became greater as the duration increased. Compared with “Ca. Hamiltonella defensa,” “Ca. Portiera aleyrodidarum” was initially less affected in the first day but then showed more rapid reduction at days 3–5. The density of Rickettsia sp. fluctuated but was not reduced significantly at 40 °C. Meanwhile, the mortality rates of the host whiteflies elevated rapidly as the duration of exposure to heat treatment increased. The differential responses of various symbionts to adverse temperatures imply complex interactions among the symbionts inside the same host insect and highlight the importance of taking the whole bacterial community into account in studies of symbioses.