Piotr Łukasik

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.

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 individuals 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.

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 ants 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.

Herbivorous turtle ants obtain essential nutrients from a conserved nitrogen-recycling gut microbiome

Nitrogen acquisition is a major challenge for herbivorous animals, and the repeated origins of herbivory across the ants have raised expectations that nutritional symbionts have shaped their diversification. Direct evidence for N provisioning by internally housed symbionts is rare in animals; among the ants, it has been documented for just one lineage. In this study we dissect functional contributions by bacteria from a conserved, multi-partite gut symbiosis in herbivorous Cephalotes ants through in vivo experiments, metagenomics, and in vitro assays. Gut bacteria recycle urea, and likely uric acid, using recycled N to synthesize essential amino acids that are acquired by hosts in substantial quantities. Specialized core symbionts of 17 studied Cephalotes species encode the pathways directing these activities, and several recycle N in vitro. These findings point to a highly efficient N economy, and a nutritional mutualism preserved for millions of years through the derived behaviors and gut anatomy of Cephalotes ants.Gut bacteria are prevalent across insects including ants, but their precise roles are often unclear. Here, Hu et al. show that microbes aid ants by recycling nitrogen into bio-available amino acids. This function is conserved across the turtle ants, suggesting an ancient nutritional mutualism.

Multiple origins of interdependent endosymbiotic complexes in a genus of cicadas

Significance Highly reduced genomes from bacteria that are long-term beneficial endosymbionts of insects often show remarkable structural stability. Endosymbionts in insects diverged by tens or hundreds of millions of years often have genomes almost completely conserved in gene order and content. Here, we show that an endosymbiont in some cicadas has repeatedly and independently fractured into complexes of distinct genomic and cellular lineages present in the same host. Individual endosymbiont lineages, having lost many of the essential ancestral genes, rely on each other for basic function and together seem to provide the same nutritional benefits as the ancestral single symbiont. These cicada endosymbionts show genomic parallels to mitochondria and provide another example of how normally stable genomes can lose structural stability. 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.

Dramatic differences in gut bacterial densities help to explain the relationship between diet and habitat in rainforest ants

Abundance is a key parameter in microbial ecology, and important to estimates of potential metabolite flux, impacts of dispersal, and sensitivity of samples to technical biases such as laboratory contamination. However, modern amplicon-based sequencing techniques by themselves typically provide no information about the absolute abundance of microbes. Here, we use fluorescence microscopy and quantitative PCR as independent estimates of microbial abundance to test the hypothesis that microbial symbionts have enabled ants to dominate tropical rainforest canopies by facilitating herbivorous diets, and compare these methods to microbial diversity profiles from 16S rRNA amplicon sequencing. Through a systematic survey of ants from a lowland tropical forest, we show that the density of gut microbiota varies across several orders of magnitude among ant lineages, with median individuals from many genera only marginally above detection limits. Supporting the hypothesis that microbial symbiosis is important to dominance in the canopy, we find that the abundance of gut bacteria is positively correlated with stable isotope proxies of herbivory among canopy-dwelling ants, but not among ground-dwelling ants. Notably, these broad findings are much more evident in the quantitative data than in the 16S rRNA sequencing data. Our results help to resolve a longstanding question in tropical rainforest ecology, and have broad implications for the interpretation of sequence-based surveys of microbial diversity.

Genome Evolution of Bartonellaceae Symbionts of Ants at the Opposite Ends of the Trophic Scale

Abstract Many insects rely on bacterial symbionts to supply essential amino acids and vitamins that are deficient in their diets, but metabolic comparisons of closely related gut bacteria in insects with different dietary preferences have not been performed. Here, we demonstrate that herbivorous ants of the genus Dolichoderus from the Peruvian Amazon host bacteria of the family Bartonellaceae, known for establishing chronic or pathogenic infections in mammals. We detected these bacteria in all studied Dolichoderus species, and found that they reside in the midgut wall, that is, the same location as many previously described nutritional endosymbionts of insects. The genomic analysis of four divergent strains infecting different Dolichoderus species revealed genes encoding pathways for nitrogen recycling and biosynthesis of several vitamins and all essential amino acids. In contrast, several biosynthetic pathways have been lost, whereas genes for the import and conversion of histidine and arginine to glutamine have been retained in the genome of a closely related gut bacterium of the carnivorous ant Harpegnathos saltator. The broad biosynthetic repertoire in Bartonellaceae of herbivorous ants resembled that of gut bacteria of honeybees that likewise feed on carbohydrate-rich diets. Taken together, the broad distribution of Bartonellaceae across Dolichoderus ants, their small genome sizes, the specific location within hosts, and the broad biosynthetic capability suggest that these bacteria are nutritional symbionts in herbivorous ants. The results highlight the important role of the host nutritional biology for the genomic evolution of the gut microbiota—and conversely, the importance of the microbiota for the nutrition of hosts.

Changes in endosymbiont complexity drive host-level compensatory adaptations in cicadas

For insects that depend on one or more bacterial endosymbionts for survival, it is critical that these bacteria are faithfully transmitted between insect generations. Cicadas harbor two essential bacterial endosymbionts, Sulcia muelleri and Hodgkinia cicadicola. In some cicada species, Hodgkinia has fragmented into multiple distinct cellular and genomic lineages that can differ in abundance by more than two orders of magnitude. This complexity presents a potential problem for the host cicada, because low-abundance-but-essential Hodgkinia lineages risk being lost during the symbiont transmission bottleneck from mother to egg. Here we show that all cicada eggs seem to receive the full complement of Hodgkinia lineages, and that in cicadas with more complex Hodgkinia this outcome is achieved by increasing the number of Hodgkinia cells transmitted by up to six-fold. We further show that cicada species with varying Hodgkinia complexity do not visibly alter their transmission mechanism at the resolution of cell biological structures. Together these data suggest that a major cicada adaptation to changes in endosymbiont complexity is an increase in the number of Hodgkinia cells transmitted to each egg. We hypothesize that the requirement to increase the symbiont titer is one of the costs associated with Hodgkinia fragmentation.