Co-habiting ants and silverfish display a converging feeding ecology.

BACKGROUND: Various animal taxa have specialized to living with social hosts. Depending on their level of specialization, these symbiotic animals are characterized by distinct behavioural, chemical, and morphological traits that enable close heterospecific interactions. Despite its functional importance, our understanding of the feeding ecology of animals living with social hosts remains limited. We examined how host specialization of silverfish co-habiting with ants affects several components of their feeding ecology. We combined stable isotope profiling, feeding assays, phylogenetic reconstruction, and microbial community characterization of the Neoasterolepisma silverfish genus and a wider nicoletiid and lepismatid silverfish panel where divergent myrmecophilous lifestyles are observed. RESULTS: Stable isotope profiling (δ13C and δ15N) showed that the isotopic niches of granivorous Messor ants and Messor-specialized Neoasterolepisma exhibit a remarkable overlap within an ant nest. Trophic experiments and gut dissections further supported that these specialized Neoasterolepisma silverfish transitioned to a diet that includes plant seeds. In contrast, the isotopic niches of generalist Neoasterolepisma silverfish and generalist nicoletiid silverfish were clearly different from their ant hosts within the shared nest environment. The impact of the myrmecophilous lifestyle on feeding ecology was also evident in the internal silverfish microbiome. Compared to generalists, Messor-specialists exhibited a higher bacterial density and a higher proportion of heterofermentative lactic acid bacteria. Moreover, the nest environment explained the infection profile (or the 16S rRNA genotypes) of Weissella bacteria in Messor-specialized silverfish and the ant hosts. CONCLUSIONS: Together, we show that social hosts are important determinants for the feeding ecology of symbiotic animals and can induce diet convergence.

Pinpointing the microbiota of tardigrades: What is really there?

Microbiota are considered significant in the biology of tardigrades, yet their diversity and distribution remain largely unexplored. This is partly due to the methodological challenges associated with studying the microbiota of small organisms that inhabit microbe-rich environments. In our study, we characterized the microbiota of 31 species of cultured tardigrades using 16S rRNA amplicon sequencing. We employed various sample preparation strategies and multiple types of controls and estimated the number of microbes in samples using synthetic DNA spike-ins. We also reanalysed data from previous tardigrade microbiome studies. Our findings suggest that the microbial communities of cultured tardigrades are predominantly composed of bacterial genotypes originating from food, medium, or reagents. Despite numerous experiments, we found it challenging to identify strains that were enriched in certain tardigrades, which would have indicated likely symbiotic associations. Putative tardigrade-associated microbes rarely constituted more than 20% of the datasets, although some matched symbionts identified in other studies. We also uncovered serious contamination issues in previous tardigrade microbiome studies, casting doubt on some of their conclusions. We concluded that tardigrades are not universally dependent on specialized microbes. Our work underscores the need for rigorous safeguards in studies of the microbiota of microscopic organisms and serves as a cautionary tale for studies involving samples with low microbiome abundance.

Temperature and water availability drive insect seasonality across a temperate and a tropical region.

The more insects there are, the more food there is for insectivores and the higher the likelihood for insect-associated ecosystem services. Yet, we lack insights into the drivers of insect biomass over space and seasons, for both tropical and temperate zones. We used 245 Malaise traps, managed by 191 volunteers and park guards, to characterize year-round flying insect biomass in a temperate (Sweden) and a tropical (Madagascar) country. Surprisingly, we found that local insect biomass was similar across zones. In Sweden, local insect biomass increased with accumulated heat and varied across habitats, while biomass in Madagascar was unrelated to the environmental predictors measured. Drivers behind seasonality partly converged: In both countries, the seasonality of insect biomass differed between warmer and colder sites, and wetter and drier sites. In Sweden, short-term deviations from expected season-specific biomass were explained by week-to-week fluctuations in accumulated heat, rainfall and soil moisture, whereas in Madagascar, weeks with higher soil moisture had higher insect biomass. Overall, our study identifies key drivers of the seasonal distribution of flying insect biomass in a temperate and a tropical climate. This knowledge is key to understanding the spatial and seasonal availability of insects-as well as predicting future scenarios of insect biomass change.

Till evolution do us part: The diversity of symbiotic associations across populations of Philaenus spittlebugs.

Symbiotic bacteria have played crucial roles in the evolution of sap-feeding insects and can strongly affect host function. However, their diversity and distribution within species are not well understood; we do not know to what extent environmental factors or associations with other species may affect microbial community profiles. We addressed this question in Philaenus spittlebugs by surveying both insect and bacterial marker gene amplicons across multiple host populations. Host mitochondrial sequence data confirmed morphology-based identification of six species and revealed two divergent clades of Philaenus spumarius. All of them hosted the primary symbiont Sulcia that was almost always accompanied by Sodalis. Interestingly, populations and individuals often differed in the presence of Sodalis sequence variants, suggestive of intra-genome 16S rRNA variant polymorphism combined with rapid genome evolution and/or recent additional infections or replacements of the co-primary symbiont. The prevalence of facultative endosymbionts, including Wolbachia, Rickettsia, and Spiroplasma, varied among populations. Notably, cytochrome I oxidase (COI) amplicon data also showed that nearly a quarter of P. spumarius were infected by parasitoid flies (Verralia aucta). One of the Wolbachia operational taxonomic units (OTUs) was exclusively present in Verralia-parasitized specimens, suggestive of parasitoids as their source and highlighting the utility of host gene amplicon sequencing in microbiome studies.

Microbial symbionts are shared between ants and their associated beetles.

The transmission of microbial symbionts across animal species could strongly affect their biology and evolution, but our understanding of transmission patterns and dynamics is limited. Army ants (Formicidae: Dorylinae) and their hundreds of closely associated insect guest species (myrmecophiles) can provide unique insights into interspecific microbial symbiont sharing. Here, we compared the microbiota of workers and larvae of the army ant Eciton burchellii with those of 13 myrmecophile beetle species using 16S rRNA amplicon sequencing. We found that the previously characterized specialized bacterial symbionts of army ant workers were largely absent from ant larvae and myrmecophiles, whose microbial communities were usually dominated by Rickettsia, Wolbachia, Rickettsiella and/or Weissella. Strikingly, different species of myrmecophiles and ant larvae often shared identical 16S rRNA genotypes of these common bacteria. Protein-coding gene sequences confirmed the close relationship of Weissella strains colonizing army ant larvae, some workers and several myrmecophile species. Unexpectedly, these strains were also similar to strains infecting dissimilar animals inhabiting very different habitats: trout and whales. Together, our data show that closely interacting species can share much of their microbiota, and some versatile microbial species can inhabit and possibly transmit across a diverse range of hosts and environments.

Recurrent symbiont recruitment from fungal parasites in cicadas.

Diverse insects are associated with ancient bacterial symbionts, whose genomes have often suffered drastic reduction and degeneration. In extreme cases, such symbiont genomes seem almost unable to sustain the basic cellular functioning, which comprises an open question in the evolution of symbiosis. Here, we report an insect group wherein an ancient symbiont lineage suffering massive genome erosion has experienced recurrent extinction and replacement by host-associated pathogenic microbes. Cicadas are associated with the ancient bacterial co-obligate symbionts Sulcia and Hodgkinia, whose streamlined genomes are specialized for synthesizing essential amino acids, thereby enabling the host to live on plant sap. However, our inspection of 24 Japanese cicada species revealed that while all species possessed Sulcia, only nine species retained Hodgkinia, and their genomes exhibited substantial structural instability. The remaining 15 species lacked Hodgkinia and instead harbored yeast-like fungal symbionts. Detailed phylogenetic analyses uncovered repeated Hodgkinia-fungus and fungus-fungus replacements in cicadas. The fungal symbionts were phylogenetically intermingled with cicada-parasitizing Ophiocordyceps fungi, identifying entomopathogenic origins of the fungal symbionts. Most fungal symbionts of cicadas were uncultivable, but the fungal symbiont of Meimuna opalifera was cultivable, possibly because it is at an early stage of fungal symbiont replacement. Genome sequencing of the fungal symbiont revealed its metabolic versatility, presumably capable of synthesizing almost all amino acids, vitamins, and other metabolites, which is more than sufficient to compensate for the Hodgkinia loss. These findings highlight a straightforward ecological and evolutionary connection between parasitism and symbiosis, which may provide an evolutionary trajectory to renovate deteriorated ancient symbiosis via pathogen domestication.

Multiple origins of interdependent endosymbiotic complexes in a genus of cicadas.

Bacterial endosymbionts that provide nutrients to hosts often have genomes that are extremely stable in structure and gene content. In contrast, the genome of the endosymbiont Hodgkinia cicadicola has fractured into multiple distinct lineages in some species of the cicada genus Tettigades To better understand the frequency, timing, and outcomes of Hodgkinia lineage splitting throughout this cicada genus, we sampled cicadas over three field seasons in Chile and performed genomics and microscopy on representative samples. We found that a single ancestral Hodgkinia lineage has split at least six independent times in Tettigades over the last 4 million years, resulting in complexes of between two and six distinct Hodgkinia lineages per host. Individual genomes in these symbiotic complexes differ dramatically in relative abundance, genome size, organization, and gene content. Each Hodgkinia lineage retains a small set of core genes involved in genetic information processing, but the high level of gene loss experienced by all genomes suggests that extensive sharing of gene products among symbiont cells must occur. In total, Hodgkinia complexes that consist of multiple lineages encode nearly complete sets of genes present on the ancestral single lineage and presumably perform the same functions as symbionts that have not undergone splitting. However, differences in the timing of the splits, along with dissimilar gene loss patterns on the resulting genomes, have led to very different outcomes of lineage splitting in extant cicadas.

One Hundred Mitochondrial Genomes of Cicadas.

Mitochondrial genomes can provide valuable information on the biology and evolutionary histories of their host organisms. Here, we present and characterize the complete coding regions of 107 mitochondrial genomes (mitogenomes) of cicadas (Insecta: Hemiptera: Auchenorrhyncha: Cicadoidea), representing 31 genera, 61 species, and 83 populations. We show that all cicada mitogenomes retain the organization and gene contents thought to be ancestral in insects, with some variability among cicada clades in the length of a region between the genes nad2 and cox1, which encodes 3 tRNAs. Phylogenetic analyses using these mitogenomes recapitulate a recent 5-gene classification of cicadas into families and subfamilies, but also identify a species that falls outside of the established taxonomic framework. While protein-coding genes are under strong purifying selection, tests of relative evolutionary rates reveal significant variation in evolutionary rates across taxa, highlighting the dynamic nature of mitochondrial genome evolution in cicadas. These data will serve as a useful reference for future research into the systematics, ecology, and evolution of the superfamily Cicadoidea.

By their own devices: invasive Argentine ants have shifted diet without clear aid from symbiotic microbes.

The functions and compositions of symbiotic bacterial communities often correlate with host ecology. Yet cause-effect relationships and the order of symbiont vs. host change remain unclear in the face of ancient symbioses and conserved host ecology. Several groups of ants exemplify this challenge, as their low-nitrogen diets and specialized symbioses appear conserved and ancient. To address whether nitrogen-provisioning symbionts might be important in the early stages of ant trophic shifts, we studied bacteria from the Argentine ant, Linepithema humile - an invasive species that has transitioned towards greater consumption of sugar-rich, nitrogen-poor foods in parts of its introduced range. Bacteria were present at low densities in most L. humile workers, and among those yielding quality 16S rRNA amplicon sequencing data, we found just three symbionts to be common and dominant. Two, a Lactobacillus and an Acetobacteraceae species, were shared between native and introduced populations. The other, a Rickettsia, was found only in two introduced supercolonies. Across an eight-year period of trophic reduction in one introduced population, we found no change in symbionts, arguing against a relationship between natural dietary change and microbiome composition. Overall, our findings thus argue against major changes in symbiotic bacteria in association with the invasion and trophic shift of L. humile. In addition, genome content from close relatives of the identified symbionts suggests that just one can synthesize most essential amino acids; this bacterium was only modestly abundant in introduced populations, providing little support for a major role of nitrogen-provisioning symbioses in Argentine ant's dietary shift.

The structured diversity of specialized gut symbionts of the New World army ants.

Symbiotic bacteria play important roles in the biology of their arthropod hosts. Yet the microbiota of many diverse and influential groups remain understudied, resulting in a paucity of information on the fidelities and histories of these associations. Motivated by prior findings from a smaller scale, 16S rRNA-based study, we conducted a broad phylogenetic and geographic survey of microbial communities in the ecologically dominant New World army ants (Formicidae: Dorylinae). Amplicon sequencing of the 16S rRNA gene across 28 species spanning the five New World genera showed that the microbial communities of army ants consist of very few common and abundant bacterial species. The two most abundant microbes, referred to as Unclassified Firmicutes and Unclassified Entomoplasmatales, appear to be specialized army ant associates that dominate microbial communities in the gut lumen of three host genera, Eciton, Labidus and Nomamyrmex. Both are present in other army ant genera, including those from the Old World, suggesting that army ant symbioses date back to the Cretaceous. Extensive sequencing of bacterial protein-coding genes revealed multiple strains of these symbionts coexisting within colonies, but seldom within the same individual ant. Bacterial strains formed multiple host species-specific lineages on phylogenies, which often grouped strains from distant geographic locations. These patterns deviate from those seen in other social insects and raise intriguing questions about the influence of army ant colony swarm-founding and within-colony genetic diversity on strain coexistence, and the effects of hosting a diverse suite of symbiont strains on colony ecology.

Patterns, causes and consequences of defensive microbiome dynamics across multiple scales.

The microbiome can significantly impact host phenotypes and serve as an additional source of heritable genetic variation. While patterns across eukaryotes are consistent with a role for symbiotic microbes in host macroevolution, few studies have examined symbiont-driven host evolution or the ecological implications of a dynamic microbiome across temporal, spatial or ecological scales. The pea aphid, Acyrthosiphon pisum, and its eight heritable bacterial endosymbionts have served as a model for studies on symbiosis and its potential contributions to host ecology and evolution. But we know little about the natural dynamics or ecological impacts of the heritable microbiome of this cosmopolitan insect pest. Here we report seasonal shifts in the frequencies of heritable defensive bacteria from natural pea aphid populations across two host races and geographic regions. Microbiome dynamics were consistent with symbiont responses to host-level selection and findings from one population suggested symbiont-driven adaptation to seasonally changing parasitoid pressures. Conversely, symbiont levels were negatively correlated with enemy-driven mortality when measured across host races, suggesting important ecological impacts of host race microbiome divergence. Rapid drops in symbiont frequencies following seasonal peaks suggest microbiome instability in several populations, with potentially large costs of 'superinfection' under certain environmental conditions. In summary, the realization of several laboratory-derived, a priori expectations suggests important natural impacts of defensive symbionts in host-enemy eco-evolutionary feedbacks. Yet negative findings and unanticipated correlations suggest complexities within this system may limit or obscure symbiont-driven contemporary evolution, a finding of broad significance given the widespread nature of defensive microbes across plants and animals.

Correlates of gut community composition across an ant species (Cephalotes varians) elucidate causes and consequences of symbiotic variability.

Insect guts are often colonized by multispecies microbial communities that play integral roles in nutrition, digestion and defence. Community composition can differ across host species with increasing dietary and genetic divergence, yet gut microbiota can also vary between conspecific hosts and across an individual's lifespan. Through exploration of such intraspecific variation and its correlates, molecular profiling of microbial communities can generate and test hypotheses on the causes and consequences of symbioses. In this study, we used 454 pyrosequencing and TRFLP to achieve these goals in an herbivorous ant, Cephalotes varians, exploring variation in bacterial communities across colonies, populations and workers reared on different diets. C. varians bacterial communities were dominated by 16 core species present in over two-thirds of the sampled colonies. Core species comprised multiple genotypes, or strains and hailed from ant-specific clades containing relatives from other Cephalotes species. Yet three were detected in environmental samples, suggesting the potential for environmental acquisition. In spite of their prevalence and long-standing relationships with Cephalotes ants, the relative abundance and genotypic composition of core species varied across colonies. Diet-induced plasticity is a likely cause, but only pollen-based diets had consistent effects, altering the abundance of two types of bacteria. Additional factors, such as host age, genetics, chance or natural selection, must therefore shape natural variation. Future studies on these possibilities and on bacterial contributions to the use of pollen, a widespread food source across Cephalotes, will be important steps in developing C. varians as a model for studying widespread social insect-bacteria symbioses.

With a little help from my friends: the roles of microbial symbionts in insect populations and communities.

To understand insect abundance, distribution and dynamics, we need to understand the relevant drivers of their populations and communities. While microbial symbionts are known to strongly affect many aspects of insect biology, we lack data on their effects on populations or community processes, or on insects' evolutionary responses at different timescales. How these effects change as the anthropogenic effects on ecosystems intensify is an area of intense research. Recent developments in sequencing and bioinformatics permit cost-effective microbial diversity surveys, tracking symbiont transmission, and identification of functions across insect populations and multi-species communities. In this review, we explore how different functional categories of symbionts can influence insect life-history traits, how these effects could affect insect populations and their interactions with other species, and how they may affect processes and patterns at the level of entire communities. We argue that insect-associated microbes should be considered important drivers of insect response and adaptation to environmental challenges and opportunities. We also outline the emerging approaches for surveying and characterizing insect-associated microbiota at population and community scales. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Facultatively intrabacterial localization of a planthopper endosymbiont as an adaptation to its vertical transmission.

Transovarial transmission is the most reliable way of passing on essential nutrient-providing endosymbionts from mothers to offspring. However, not all endosymbiotic microbes follow the complex path through the female host tissues to oocytes on their own. Here, we demonstrate an unusual transmission strategy adopted by one of the endosymbionts of the planthopper Trypetimorpha occidentalis (Hemiptera: Tropiduchidae) from Bulgaria. In this species, an Acetobacteraceae endosymbiont is transmitted transovarially within deep invaginations of cellular membranes of an ancient endosymbiont Sulcia-strikingly resembling recently described plant virus transmission. However, in males, Acetobacteraceae colonizes the same bacteriocytes as Sulcia but remains unenveloped. Then, the unusual endobacterial localization of Acetobacteraceae observed in females appears to be a unique adaptation to maternal transmission. Further, the symbiont's genomic features, including encoding essential amino acid biosynthetic pathways and its similarity to a recently described psyllid symbiont, suggest a unique combination of the ability to horizontally transmit among species and confer nutritional benefits. The close association with Acetobacteraceae symbiont correlates with the so-far-unreported level of genomic erosion of ancient nutritional symbionts of this planthopper. In Sulcia, this is reflected in substantial changes in genomic organization, reported for the first time in the symbiont renowned for its genomic stability. In Vidania, substantial gene loss resulted in one of the smallest genomes known, at 108.6 kb. Thus, the symbionts of T. occidentalis display a combination of unusual adaptations and genomic features that expand our understanding of how insect-microbe symbioses may transmit and evolve.IMPORTANCEReliable transmission across host generations is a major challenge for bacteria that associate with insects, and independently established symbionts have addressed this challenge in different ways. The facultatively endobacterial localization of Acetobacteraceae symbiont, enveloped by cells of ancient nutritional endosymbiont Sulcia in females but not males of the planthopper Trypetimorpha occidentalis, appears to be a unique adaptation to maternal transmission. Acetobacteraceae's genomic features indicate its unusual evolutionary history, and the genomic erosion experienced by ancient nutritional symbionts demonstrates the apparent consequences of such close association. Combined, this multi-partite symbiosis expands our understanding of the diversity of strategies that insect symbioses form and some of their evolutionary consequences.

Limited variation in microbial communities across populations of Macrosteles leafhoppers (Hemiptera: Cicadellidae).

Microbial symbionts play crucial roles in insect biology, yet their diversity, distribution, and temporal dynamics across host populations remain poorly understood. In this study, we investigated the spatio-temporal distribution of bacterial symbionts within the widely distributed and economically significant leafhopper genus Macrosteles, with a focus on Macrosteles laevis. Using host and symbiont marker gene amplicon sequencing, we explored the intricate relationships between these insects and their microbial partners. Our analysis of the cytochrome oxidase subunit I (COI) gene data revealed several intriguing findings. First, there was no strong genetic differentiation across M. laevis populations, suggesting gene flow among them. Second, we observed significant levels of heteroplasmy, indicating the presence of multiple mitochondrial haplotypes within individuals. Third, parasitoid infections were prevalent, highlighting the complex ecological interactions involving leafhoppers. The 16S rRNA data confirmed the universal presence of ancient nutritional endosymbionts-Sulcia and Nasuia-in M. laevis. Additionally, we found a high prevalence of Arsenophonus, another common symbiont. Interestingly, unlike most previously studied species, M. laevis exhibited only occasional cases of infection with known facultative endosymbionts and other bacteria. Notably, there was no significant variation in symbiont prevalence across different populations or among sampling years within the same population. Comparatively, facultative endosymbionts such as Rickettsia, Wolbachia, Cardinium and Lariskella were more common in other Macrosteles species. These findings underscore the importance of considering both host and symbiont dynamics when studying microbial associations. By simultaneously characterizing host and symbiont marker gene amplicons in large insect collections, we gain valuable insights into the intricate interplay between insects and their microbial partners. Understanding these dynamics contributes to our broader comprehension of host-microbe interactions in natural ecosystems.

Implementing high-throughput insect barcoding in microbiome studies: impact of non-destructive DNA extraction on microbiome reconstruction.

Background: Symbiotic relationships with diverse microorganisms are crucial for many aspects of insect biology. However, while our understanding of insect taxonomic diversity and the distribution of insect species in natural communities is limited, we know much less about their microbiota. In the era of rapid biodiversity declines, as researchers increasingly turn towards DNA-based monitoring, developing and broadly implementing approaches for high-throughput and cost-effective characterization of both insect and insect-associated microbial diversity is essential. We need to verify whether approaches such as high-throughput barcoding, a powerful tool for identifying wild insects, would permit subsequent microbiota reconstruction in these specimens. Methods: High-throughput barcoding ("megabarcoding") methods often rely on non-destructive approaches for obtaining template DNA for PCR amplification by leaching DNA out of insect specimens using alkaline buffers such as HotSHOT. This study investigated the impact of HotSHOT on microbial abundance estimates and the reconstructed bacterial community profiles. We addressed this question by comparing quantitative 16S rRNA amplicon sequencing data for HotSHOT-treated or untreated specimens of 16 insect species representing six orders and selected based on the expectation of limited variation among individuals. Results: We find that in 13 species, the treatment significantly reduced microbial abundance estimates, corresponding to an estimated 15-fold decrease in amplifiable 16S rRNA template on average. On the other hand, HotSHOT pre-treatment had a limited effect on microbial community composition. The reconstructed presence of abundant bacteria with known significant effects was not affected. On the other hand, we observed changes in the presence of low-abundance microbes, those close to the reliable detection threshold. Alpha and beta diversity analyses showed compositional differences in only a few species. Conclusion: Our results indicate that HotSHOT pre-treated specimens remain suitable for microbial community composition reconstruction, even if abundance may be hard to estimate. These results indicate that we can cost-effectively combine barcoding with the study of microbiota across wild insect communities. Thus, the voucher specimens obtained using megabarcoding studies targeted at characterizing insect communities can be used for microbiome characterizations. This can substantially aid in speeding up the accumulation of knowledge on the microbiomes of abundant and hyperdiverse insect species.

Unrelated facultative endosymbionts protect aphids against a fungal pathogen.

The importance of microbial facultative endosymbionts to insects is increasingly being recognized, but our understanding of how the fitness effects of infection are distributed across symbiont taxa is limited. In the pea aphid, some of the seven known species of facultative symbionts influence their host's resistance to natural enemies, including parasitoid wasps and a pathogenic fungus. Here we show that protection against this entomopathogen, Pandora neoaphidis, can be conferred by strains of four distantly related symbionts (in the genera Regiella, Rickettsia, Rickettsiella and Spiroplasma). They reduce mortality and also decrease fungal sporulation on dead aphids which may help protect nearby genetically identical insects. Pea aphids thus obtain protection from natural enemies through association with a wider range of microbial associates than has previously been thought. Providing resistance against natural enemies appears to be a particularly common way for facultative endosymbionts to increase in frequency within host populations.

Uncovering symbiont-driven genetic diversity across North American pea aphids.

Heritable genetic variation is required for evolution, and while typically encoded within nuclear and organellar genomes, several groups of invertebrates harbour heritable microbes serving as additional sources of genetic variation. Hailing from the symbiont-rich insect order Hemiptera, pea aphids (Acyrthosiphon pisum) possess several heritable symbionts with roles in host plant utilization, thermotolerance and protection against natural enemies. As pea aphids vary in the numbers and types of harboured symbionts, these bacteria provide heritable and functionally important variation within field populations. In this study, we quantified the cytoplasmically inherited genetic variation contributed by symbionts within North American pea aphids. Through the use of Denaturing Gradient Gel Electrophoresis (DGGE) and 454 amplicon pyrosequencing of 16S rRNA genes, we explored the diversity of bacteria harboured by pea aphids from five populations, spanning three locations and three host plants. We also characterized strain variation by analysing 16S rRNA, housekeeping and symbiont-associated bacteriophage genes. Our results identified eight species of facultative symbionts, which often varied in frequency between locations and host plants. We detected 28 cytoplasmic genotypes across 318 surveyed aphids, considering only the various combinations of secondary symbiont species infecting single hosts. Yet the detection of multiple Regiella insecticola, Hamiltonella defensa and Rickettsia strains, and diverse bacteriophage genotypes from H. defensa, suggest even greater diversity. Combined, these findings reveal that heritable bacteria contribute substantially to genetic variation in A. pisum. Given the costs and benefits of these symbionts, it is likely that fluctuating selective forces play a role in the maintenance of this diversity.

The diversity and fitness effects of infection with facultative endosymbionts in the grain aphid, Sitobion avenae.

Mutualisms with facultative, non-essential heritable microorganisms influence the biology of many insects, and they can have major effects on insect host fitness in certain situations. One of the best-known examples is found in aphids where the facultative endosymbiotic bacterium Hamiltonella defensa confers protection against hymenopterous parasitoids. This symbiont is widely distributed in aphids and related insects, yet its defensive properties have only been tested in two aphid species. In a wild population of the grain aphid, Sitobion avenae, we identified several distinct strains of endosymbiotic bacteria, including Hamiltonella. The symbiont had no consistent effect on grain aphid fecundity, though we did find a significant interaction between aphid genotype by symbiont status. In contrast to findings in other aphid species, Hamiltonella did not reduce aphid susceptibility to two species of parasitoids (Aphidius ervi and Ephedrus plagiator), nor did it affect the fitness of wasps that successfully completed development. Despite this, experienced females of both parasitoid species preferentially oviposited into uninfected hosts when given a choice between genetically identical individuals with or without Hamiltonella. Thus, although Hamiltonella does not always increase resistance to parasitism, it may reduce the risk of parasitism in its aphid hosts by making them less attractive to searching parasitoids.

No Transcriptional Compensation for Extreme Gene Dosage Imbalance in Fragmented Bacterial Endosymbionts of Cicadas.

Bacteria that form long-term intracellular associations with host cells lose many genes, a process that often results in tiny, gene-dense, and stable genomes. Paradoxically, the some of the same evolutionary processes that drive genome reduction and simplification may also cause genome expansion and complexification. A bacterial endosymbiont of cicadas, Hodgkinia cicadicola, exemplifies this paradox. In many cicada species, a single Hodgkinia lineage with a tiny, gene-dense genome has split into several interdependent cell and genome lineages. Each new Hodgkinia lineage encodes a unique subset of the ancestral unsplit genome in a complementary way, such that the collective gene contents of all lineages match the total found in the ancestral single genome. This splitting creates genetically distinct Hodgkinia cells that must function together to carry out basic cellular processes. It also creates a gene dosage problem where some genes are encoded by only a small fraction of cells while others are much more abundant. Here, by sequencing DNA and RNA of Hodgkinia from different cicada species with different amounts of splitting-along with its structurally stable, unsplit partner endosymbiont Sulcia muelleri-we show that Hodgkinia does not transcriptionally compensate to rescue the wildly unbalanced gene and genome ratios that result from lineage splitting. We also find that Hodgkinia has a reduced capacity for basic transcriptional control independent of the splitting process. Our findings reveal another layer of degeneration further pushing the limits of canonical molecular and cell biology in Hodgkinia and may partially explain its propensity to go extinct through symbiont replacement.