Strong within-host selection in a maternally inherited obligate symbiont: Buchnera and aphids.

Numerous animal lineages have maternally inherited symbionts that are required for host reproduction and growth. Endosymbionts also pose a risk to their hosts because of the mutational decay of their genomes through genetic drift or to selfish mutations that favor symbiont fitness over host fitness. One model for heritable endosymbiosis is the association of aphids with their obligate bacterial symbiont, Buchnera We experimentally established heteroplasmic pea aphid matrilines containing pairs of closely related Buchnera haplotypes and used deep sequencing of diagnostic markers to measure haplotype frequencies in successive host generations. These frequencies were used to estimate the effective population size of Buchnera within hosts (i.e., the transmission bottleneck size) and the extent of within-host selection. The within-host effective population size was in the range of 10 to 20, indicating a strong potential for genetic drift and fixation of deleterious mutations. Remarkably, closely related haplotypes were subject to strong within-host selection, with selection coefficients as high as 0.5 per aphid generation. In one case, the direction of selection depended on the thermal environment and went in the same direction as between-host selection. In another, a new mutant haplotype had a strong within-host advantage under both environments but had no discernible effect on host-level fitness under laboratory conditions. Thus, within-host selection can be strong, resulting in a rapid fixation of mutations with little impact on host-level fitness. Together, these results show that within-host selection can drive evolution of an obligate symbiont, accelerating sequence evolution.

Aphid endosymbiont facilitates virus transmission by modulating the volatile profile of host plants.

BACKGROUND: Most plant viruses rely on vectors for their transmission and spread. One of the outstanding biological questions concerning the vector-pathogen-symbiont multi-trophic interactions is the potential involvement of vector symbionts in the virus transmission process. Here, we used a multi-factorial system containing a non-persistent plant virus, cucumber mosaic virus (CMV), its primary vector, green peach aphid, Myzus persicae, and the obligate endosymbiont, Buchnera aphidicola to explore this uncharted territory. RESULTS: Based on our preliminary research, we hypothesized that aphid endosymbiont B. aphidicola can facilitate CMV transmission by modulating plant volatile profiles. Gene expression analyses demonstrated that CMV infection reduced B. aphidicola abundance in M. persicae, in which lower abundance of B. aphidicola was associated with a preference shift in aphids from infected to healthy plants. Volatile profile analyses confirmed that feeding by aphids with lower B. aphidicola titers reduced the production of attractants, while increased the emission of deterrents. As a result, M. persicae changed their feeding preference from infected to healthy plants. CONCLUSIONS: We conclude that CMV infection reduces the B. aphidicola abundance in M. persicae. When viruliferous aphids feed on host plants, dynamic changes in obligate symbionts lead to a shift in plant volatiles from attraction to avoidance, thereby switching insect vector's feeding preference from infected to healthy plants.

Coordination of host and symbiont gene expression reveals a metabolic tug-of-war between aphids and Buchnera.

Symbioses between animals and microbes are often described as mutualistic, but are subject to tradeoffs that may manifest as shifts in host and symbiont metabolism, cellular processes, or symbiont density. In pea aphids, the bacterial symbiont Buchnera is confined to specialized aphid cells called bacteriocytes, where it produces essential amino acids needed by hosts. This relationship is dynamic; Buchnera titer varies within individual aphids and among different clonal aphid lineages, and is affected by environmental and host genetic factors. We examined how host genotypic variation relates to host and symbiont function among seven aphid clones differing in Buchnera titer. We found that bacteriocyte gene expression varies among individual aphids and among aphid clones, and that Buchnera gene expression changes in response. By comparing hosts with low and high Buchnera titer, we found that aphids and Buchnera oppositely regulate genes underlying amino acid biosynthesis and cell growth. In high-titer hosts, both bacteriocytes and symbionts show elevated expression of genes underlying energy metabolism. Several eukaryotic cell signaling pathways are differentially expressed in bacteriocytes of low- versus high-titer hosts: Cell-growth pathways are up-regulated in low-titer genotypes, while membrane trafficking, lysosomal processes, and mechanistic target of rapamycin (mTOR) and cytokine pathways are up-regulated in high-titer genotypes. Specific Buchnera functions are up-regulated within different bacteriocyte environments, with genes underlying flagellar body secretion and flagellar assembly overexpressed in low- and high-titer hosts, respectively. Overall, our results reveal allowances and demands made by both host and symbiont engaged in a metabolic "tug-of-war."

Coinfection of the secondary symbionts, Hamiltonella defensa and Arsenophonus sp. contribute to the performance of the major aphid pest, Aphis gossypii (Hemiptera: Aphididae).

Bacterial endosymbionts play important roles in ecological traits of aphids. In this study, we characterize the bacterial endosymbionts of A. gossypii collected in Karaj, Iran and their role in the performance of the aphid. Our results indicated that beside Buchnera aphidicola, A. gossypii, also harbors both Hamiltonella defensa and Arsenophonus sp. Quantitative PCR (qPCR) results revealed that the populations of the endosymbionts increased throughout nymphal development up to adult emergence; thereafter, populations of Buchnera and Arsenophonus were diminished while the density of H. defensa constantly increased. Buchnera reduction caused prolonged development and no progeny production. Furthermore, secondary symbiont reduction led to reduction of the total life span and intrinsic rate of natural increase as well as appearance of the deformed dead offspring in comparison with the control insects. Reduction of the secondary symbionts did not affect parasitism rate of the aphid by the parasitic wasp Aphidius matricariae. Together these findings showed that H. defensa and Arsenophonus contributed to the fitness of A. gossypii by enhancing its performance, but not through parasitoid resistance.

Obligate bacterial endosymbionts limit thermal tolerance of insect host species.

The thermal tolerance of an organism limits its ecological and geographic ranges and is potentially affected by dependence on temperature-sensitive symbiotic partners. Aphid species vary widely in heat sensitivity, but almost all aphids are dependent on the nutrient-provisioning intracellular bacterium Buchnera, which has evolved with aphids for 100 million years and which has a reduced genome potentially limiting heat tolerance. We addressed whether heat sensitivity of Buchnera underlies variation in thermal tolerance among 5 aphid species. We measured how heat exposure of juvenile aphids affects later survival, maturation time, and fecundity. At one extreme, heat exposure of Aphis gossypii enhanced fecundity and had no effect on the Buchnera titer. In contrast, heat suppressed Buchnera populations in Aphis fabae, which suffered elevated mortality, delayed development and reduced fecundity. Likewise, in Acyrthosiphon kondoi and Acyrthosiphon pisum, heat caused rapid declines in Buchnera numbers, as well as reduced survivorship, development rate, and fecundity. Fecundity following heat exposure is severely decreased by a Buchnera mutation that suppresses the transcriptional response of a gene encoding a small heat shock protein. Similarly, absence of this Buchnera heat shock gene may explain the heat sensitivity of Ap. fabae Fluorescent in situ hybridization revealed heat-induced deformation and shrinkage of bacteriocytes in heat-sensitive species but not in heat-tolerant species. Sensitive and tolerant species also differed in numbers and transcriptional responses of heat shock genes. These results show that shifts in Buchnera heat sensitivity contribute to host variation in heat tolerance.

microRNA-92a regulates the expression of aphid bacteriocyte-specific secreted protein 1.

OBJECTIVE: Aphids harbor a nutritional obligate endosymbiont in specialized cells called bacteriocytes, which aggregate to form an organ known as the bacteriome. Aphid bacteriomes display distinct gene expression profiles that facilitate the symbiotic relationship. Currently, the mechanisms that regulate these patterns of gene expression are unknown. Recently using computational pipelines, we identified miRNAs that are conserved in expression in the bacteriomes of two aphid species and proposed that they function as important regulators of bacteriocyte gene expression. Here using a dual luciferase assay in mouse NIH/3T3 cell culture, we aimed to experimentally validate the computationally predicted interaction between Myzus persicae miR-92a and the predicted target region of M. persicae bacteriocyte-specific secreted protein 1 (SP1) mRNA. RESULTS: In the dual luciferase assay, miR-92a interacted with the SP1 target region resulting in a significant downregulation of the luciferase signal. Our results demonstrate that miR-92a interacts with SP1 to alter expression in a heterologous expression system, thereby supporting our earlier assertion that miRNAs are regulators of the aphid/Buchnera symbiotic interaction.

Antimicrobial Activities of Cysteine-rich Peptides Specific to Bacteriocytes of the Pea Aphid Acyrthosiphon pisum.

Aphids have a mutualistic relationship with the bacterial endosymbiont Buchnera aphidicola. We previously reported seven cysteine-rich peptides in the pea aphid Acyrthosiphon pisum and named them Bacteriocyte-specific Cysteine-Rich (BCR) peptides; these peptides are exclusively expressed in bacteriocytes, special aphid cells that harbor symbionts. Similar symbiotic organ-specific cysteine-rich peptides identified in the root nodules of leguminous plants are named Nodule-specific Cysteine-Rich (NCR) peptides. NCR peptides target rhizobia in the nodules and are essential for symbiotic nitrogen fixation. A BacA (membrane protein) mutant of Sinorhizobium is sensitive to NCR peptides and is unable to establish symbiosis. Based on the structural and expressional similarities between BCR peptides and NCR peptides, we hypothesized that aphid BCR peptides exhibit antimicrobial activity, similar to some NCR peptides. We herein synthesized BCR peptides and investigated their antimicrobial activities and effects on the bacterial membrane of Escherichia coli. The peptides BCR1, BCR3, BCR5, and BCR8 exhibited antimicrobial activities with increased membrane permeability. An sbmA mutant of E. coli, a homolog of bacA of S. meliloti, was more sensitive to BCR peptides than the wild type. Our results suggest that BCR peptides have properties that may be required to control the endosymbiont, similar to NCR peptides in legumes.

A Freeloader? The Highly Eroded Yet Large Genome of the Serratia symbiotica Symbiont of Cinara strobi.

Genome reduction is pervasive among maternally inherited bacterial endosymbionts. This genome reduction can eventually lead to serious deterioration of essential metabolic pathways, thus rendering an obligate endosymbiont unable to provide essential nutrients to its host. This loss of essential pathways can lead to either symbiont complementation (sharing of the nutrient production with a novel co-obligate symbiont) or symbiont replacement (complete takeover of nutrient production by the novel symbiont). However, the process by which these two evolutionary events happen remains somewhat enigmatic by the lack of examples of intermediate stages of this process. Cinara aphids (Hemiptera: Aphididae) typically harbor two obligate bacterial symbionts: Buchnera and Serratia symbiotica. However, the latter has been replaced by different bacterial taxa in specific lineages, and thus species within this aphid lineage could provide important clues into the process of symbiont replacement. In the present study, using 16S rRNA high-throughput amplicon sequencing, we determined that the aphid Cinara strobi harbors not two, but three fixed bacterial symbionts: Buchnera aphidicola, a Sodalis sp., and S. symbiotica. Through genome assembly and genome-based metabolic inference, we have found that only the first two symbionts (Buchnera and Sodalis) actually contribute to the hosts' supply of essential nutrients while S. symbiotica has become unable to contribute towards this task. We found that S. symbiotica has a rather large and highly eroded genome which codes only for a few proteins and displays extensive pseudogenization. Thus, we propose an ongoing symbiont replacement within C. strobi, in which a once "competent" S. symbiotica does no longer contribute towards the beneficial association. These results suggest that in dual symbiotic systems, when a substitute cosymbiont is available, genome deterioration can precede genome reduction and a symbiont can be maintained despite the apparent lack of benefit to its host.

mTOR Complex 1 Implicated in Aphid/Buchnera Host/Symbiont Integration.

Obligate nutritional endosymbioses are arguably the most intimate of all interspecific associations. While many insect nutritional endosymbioses are well studied, a full picture of how two disparate organisms, a bacterial endosymbiont and a eukaryotic host, are integrated is still lacking. The mTOR pathway is known to integrate nutritional conditions with cell growth and survival in eukaryotes. Characterization and localization of amino acid transporters in aphids suggest the mTOR pathway as a point of integration between an aphid host and its amino acid-provisioning endosymbiont Buchnera aphidicola The mTOR pathway is unannotated in aphids and unstudied in any nutritional endosymbiosis. We annotated mTOR pathway genes in two aphid species, Acyrthosiphon pisum and Myzus persicae, using both BLASTp searches and Hidden Markov Models. Using previously collected RNAseq data we constructed new reference transcriptomes for bacteriocyte, gut, and whole insect tissue for three lines of M. persicae Annotation of the mTOR pathway identified homologs of all known invertebrate mTOR genes in both aphid species with some duplications. Differential expression analysis showed that genes specific to the amino acid-sensitive mTOR Complex 1 were more highly expressed in bacteriocytes than genes specific to the amino acid-insensitive mTOR Complex 2. Almost all mTOR genes involved in sensing amino acids showed higher expression in bacteriocytes than in whole insect tissue. When compared to gut, the putative glutamine/arginine sensing transporter ACYPI000333, an ortholog of SLC38A9, showed 6.5 times higher expression in bacteriocytes. Our results suggest that the mTOR pathway may be functionally important in mediating integration of Buchnera into aphid growth and reproduction.

Targeting symbiosis-related insect genes by RNAi in the pea aphid-Buchnera symbiosis.

The growth and reproduction of phloem sap-feeding insects requires the sustained function of intracellular bacteria localized in specialized cells known as bacteriocytes, giving the potential to target the bacterial symbiosis as a novel strategy for controlling sap-feeding insect pests. We focused on two genes in the pea aphid Acyrthosiphon pisum, amiD and ldcA1, which were acquired horizontally from bacteria and have the annotated function to degrade immunogenic bacterial peptidoglycan. We hypothesized that AmiD and LdcA1 function to eliminate peptidoglycan fragments released by the bacterial symbiont Buchnera inhabiting the bacteriocytes, thereby protecting the Buchnera from host attack. Consistent with this hypothesis, expression of amiD and ldcA1 was enriched in bacteriocytes and varied significantly with aphid age, conforming to an inverse curvilinear relationship for amiD and negative linear relationship for ldcA1. RNAi against amiD and ldcA1 administered orally to larval pea aphids caused a significant reduction in Buchnera abundance and activity, accompanied by depressed aphid growth rates. For RNAi experiments, the aphids were co-administered with dsRNA against an aphid nuclease nuc1, protecting the dsRNA against non-specific degradation. These experiments demonstrate that selective suppression of insect symbiosis-related gene function can reduce the performance of an insect pest. Phylogenetic analysis identified amiD and ldcA1 in sequenced genomes of other aphid species, and amiD in related groups of phloem-feeding insects, offering the opportunity for specific controls against a range of insect pests.

Differential accumulation of leucine and methionine in red and green pea aphids leads to different fecundity in response to nitrogen fertilization.

BACKGROUND: Nitrogen fertilization affects plants directly and herbivorous insects indirectly. Although insect species and even genotypes are known to differ in their responses to nitrogen fertilization, the physiological and molecular mechanisms remain unclear. This study assessed the fecundity and related regulatory signaling pathways in the green and red morphs of pea aphid (Acyrthosiphon pisum) feeding on Medicago truncatula with and without nitrogen fertilization. RESULTS: Nitrogen fertilization significantly increased foliar amino acid concentrations and consequently increased the concentrations of several individual essential amino acids in body tissue of the green morph. The increased concentration of Leu, Ile, Met and Val was consistent with enhanced biosynthesis of these amino acids in the endosymbiont Buchnera. Under nitrogen fertilization, Leu and Met accumulated in the green morph enhanced the target of rapamycin (TOR) signaling pathway, which consequently increased fecundity by promoting vitellogenin synthesis. In the red morph, however, nitrogen fertilization did not change the concentration of essential amino acids, TOR signaling or fecundity. CONCLUSION: Specific amino acids accumulation and the nutrient transduction pathway in pea aphids are responsible for genotype-specific fecundity in response to nitrogen fertilization, which could be used as potential target for pest control. © 2018 Society of Chemical Industry.

Bacteriocyte cell death in the pea aphid/Buchnera symbiotic system.

Symbiotic associations play a pivotal role in multicellular life by facilitating acquisition of new traits and expanding the ecological capabilities of organisms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells (bacteriocytes) or organs (bacteriomes) have evolved for harboring beneficial microbial partners. The processes regulating the cellular life cycle of these endosymbiont-bearing cells, such as the cell-death mechanisms controlling their fate and elimination in response to host physiology, are fundamental questions in the biology of symbiosis. Here we report the discovery of a cell-death process involved in the degeneration of bacteriocytes in the hemipteran insect Acyrthosiphon pisum This process is activated progressively throughout aphid adulthood and exhibits morphological features distinct from known cell-death pathways. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of nonautophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, activation of reactive oxygen species, and Buchnera endosymbiont degradation by the lysosomal system. We showed that this multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization spread throughout the entire cytoplasm and surrounding Buchnera aphidicola endosymbionts. Our findings provide insights into the cellular and molecular processes that coordinate eukaryotic host and endosymbiont homeostasis and death in a symbiotic system and shed light on previously unknown aspects of bacteriocyte biological functioning.

Probing behavior of aposymbiotic green peach aphid (Myzus persicae) on susceptible Solanum tuberosum and resistant Solanum stoloniferum plants.

The green peach aphid, Myzus persicae Sulzer (Hemiptera: Aphididae) is one of the potato important pests; it is the most efficient vector of potato viruses. Myzus persicae harbors the endosymbiotic bacteria Buchnera aphidicola which supplements their diet. There is increasing evidence that B. aphidicola is involved in plant-aphid interactions and we previously demonstrated that B. aphidicola disruption (aposymbiosis) affected the probing behavior of M. persicae on radish plants, delaying host plant acceptance. In this work, we evaluated the effect of aposymbiosis on the probing behavior of M. persicae on 2 Solanum species with different compatibility with M. persicae, Solanum tuberosum (susceptible) and Solanum stoloniferum (resistant) with the electrical penetration graph technique (EPG). To disrupt B. aphidicola, rifampicin was administered to aphids through artificial diets. Aposymbiotic aphids, on both plant species, showed increased pathway activities, mechanical problems with the stylets, and delayed salivation in the phloem. The extended time in derailed stylet mechanics affected the occurrence of most other probing activities; it delayed the time to the first phloem phase and prevented ingestion from the phloem. The effect of aposymbiosis was more evident in the compatible interaction of M. persicae-S. tuberosum, than in the incompatible interaction with S. stoloniferum, which generated the M. persicae-S. tuberosum interaction to become incompatible. These results confirm that B. aphidicola is involved in the plant-aphid interaction in relation to plant acceptance, presumably through a role in stylets penetration in the plant.

Genetic Structure of the Bacterial Endosymbiont Buchnera aphidicola from Its Host Aphid Schlechtendalia chinensis and Evolutionary Implications.

Buchnera aphidicola is a primary symbiotic bacterium which provides essential amino acids to aphids. In this study, we sequenced nuclear 16s rDNA and atpAGD genes for 156 individuals of B. aphidicola from eight geographically distant populations to investigate the genetic diversity and structure of B. aphidicola associated to the sumac gall aphid Schlechtendalia chinensis in central and southern China. Our analyses of the combined sequences showed that B. aphidicola from S. chinensis had high haplotype and nucleotide diversity (h = 0.893; π = 0.00164). One of the 16 haplotypes detected had a wide geographic distribution across the central and southern China and was probably the ancestral haplotype of B. aphidicola from S. chinensis. A network and phylogenetic analysis revealed a geographic structure in which the 16 haplotypes of B. aphidicola were divided into the northern and southern clades separated by the Yangtze River. The two clades diverged from each other at 22.1 ± 3.7 Mya according to our divergence time estimations. Therefore, the modern genetic structure in B. aphidicola from S. chinensis has been probably impacted by historical geological events. Combined with the data from GenBank, we also reconstructed the phylogenetic relationships of three aphid subfamilies and their symbiont bacteria. The results indicated significant topological correlations between the aphid and bacterial phylogenies at interspecific levels.

Happens in the best of subfamilies: establishment and repeated replacements of co-obligate secondary endosymbionts within Lachninae aphids.

Virtually all aphids maintain an obligate mutualistic symbiosis with bacteria from the Buchnera genus, which produce essential nutrients for their aphid hosts. Most aphids from the Lachninae subfamily have been consistently found to house additional endosymbionts, mainly Serratia symbiotica. This apparent dependence on secondary endosymbionts was proposed to have been triggered by the loss of the riboflavin biosynthetic capability by Buchnera in the Lachninae last common ancestor. However, an integral large-scale analysis of secondary endosymbionts in the Lachninae is still missing, hampering the interpretation of the evolutionary and genomic analyses of these endosymbionts. Here, we analysed the endosymbionts of selected representatives from seven different Lachninae genera and nineteen species, spanning four tribes, both by FISH (exploring the symbionts' morphology and tissue tropism) and 16S rRNA gene sequencing. We demonstrate that all analysed aphids possess dual symbiotic systems, and while most harbour S. symbiotica, some have undergone symbiont replacement by other phylogenetically-distinct bacterial taxa. We found that these secondary associates display contrasting cell shapes and tissue tropism, and some appear to be lineage-specific. We propose a scenario for symbiont establishment in the Lachninae, followed by changes in the symbiont's tissue tropism and symbiont replacement events, thereby highlighting the extraordinary versatility of host-symbiont interactions.

Intraspecific genetic variation in hosts affects regulation of obligate heritable symbionts.

Symbiotic relationships promote biological diversification by unlocking new ecological niches. Over evolutionary time, hosts and symbionts often enter intimate and permanent relationships, which must be maintained and regulated for both lineages to persist. Many insect species harbor obligate, heritable symbiotic bacteria that provision essential nutrients and enable hosts to exploit niches that would otherwise be unavailable. Hosts must regulate symbiont population sizes, but optimal regulation may be affected by the need to respond to the ongoing evolution of symbionts, which experience high levels of genetic drift and potential selection for selfish traits. We address the extent of intraspecific variation in the regulation of a mutually obligate symbiosis, between the pea aphid (Acyrthosiphon pisum) and its maternally transmitted symbiont, Buchnera aphidicola Using experimental crosses to identify effects of host genotypes, we measured symbiont titer, as the ratio of genomic copy numbers of symbiont and host, as well as developmental time and fecundity of hosts. We find a large (>10-fold) range in symbiont titer among genetically distinct aphid lines harboring the same Buchnera haplotype. Aphid clones also vary in fitness, measured as developmental time and fecundity, and genetically based variation in titer is correlated with host fitness, with higher titers corresponding to lower reproductive rates of hosts. Our work shows that obligate symbiosis is not static but instead is subject to short-term evolutionary dynamics, potentially reflecting coevolutionary interactions between host and symbiont.

Seasonal Changes in the Endosymbiotic Consortia of Aphids from the Genus Cinara.

Buchnera aphidicola is the primary endosymbiont of aphids with which it maintains an obligate mutualistic symbiotic relationship. Insects also maintain facultative symbiotic relationships with secondary symbionts, and Serratia symbiotica is the most common in aphids. The presence of both symbionts in aphids of the subfamily Lachninae has been widely studied by our group. We examined two closely related aphids, Cinara tujafilina and C. cedri in the present study. Even though both B. aphidicola strains have similar genome sizes and gene contents, the genomes of the two S. symbiotica strains were markedly different. The SCc strain has the smallest genome known for this species, while SCt possesses a larger genome in an intermediate stage between the facultative S. symbiotica of Acyrthosiphon pisum (SAp) and the co-obligate S. symbiotica SCc.Aphids are vulnerable to high temperatures. Previous studies indicated that S. symbiotica SAp confers resistance to heat-shock stress. In order to clarify whether S. symbiotica strains from genus Cinara also play a role in heat stress protection, we performed a quantitative determination of the consortium Buchnera/Serratia from two geographically close populations, each of which belonged to the Cinara species examined, over two years in natural environments. We found no variation in the consortium from our C. cedri population, but a positive correlation between both endosymbiont densities and average daily temperatures in the C. tujafilina population. Even though S. symbiotica SCt may retain some protective role against heat stress, this does not appear to be due to the release of protective metabolites by cell lysis.

Direct flow cytometry measurements reveal a fine-tuning of symbiotic cell dynamics according to the host developmental needs in aphid symbiosis.

Endosymbiotic associations constitute a driving force in the ecological and evolutionary diversification of metazoan organisms. Little is known about whether and how symbiotic cells are coordinated according to host physiology. Here, we use the nutritional symbiosis between the insect pest, Acyrthosiphon pisum, and its obligate symbiont, Buchnera aphidicola, as a model system. We have developed a novel approach for unculturable bacteria, based on flow cytometry, and used this method to estimate the absolute numbers of symbionts at key stages of aphid life. The endosymbiont population increases exponentially throughout nymphal development, showing a growing rate which has never been characterized by indirect molecular techniques. Using histology and imaging techniques, we have shown that the endosymbiont-bearing cells (bacteriocytes) increase significantly in number and size during the nymphal development, and clustering in the insect abdomen. Once adulthood is reached and the laying period has begun, the dynamics of symbiont and host cells is reversed: the number of endosymbionts decreases progressively and the bacteriocyte structure degenerates during insect aging. In summary, these results show a coordination of the cellular dynamics between bacteriocytes and primary symbionts and reveal a fine-tuning of aphid symbiotic cells to the nutritional demand imposed by the host physiology throughout development.

A metagenomic approach from aphid's hemolymph sheds light on the potential roles of co-existing endosymbionts.

BACKGROUND: Aphids are known to live in symbiosis with specific bacteria, called endosymbionts which can be classified as obligate or accessory. Buchnera aphidicola is generally the only obligatory symbiont present in aphids, supplying essential nutrients that are missing in the plants phloem to its host. Pentalonia nigronervosa is the main vector of the banana bunchy top virus, one of the most damageable viruses in banana. This aphid is carrying two symbionts: B. aphidicola (BPn) and Wolbachia sp. (wPn). The high occurrence of Wolbachia in the banana aphid raises questions about the role it plays in this insect. The goal of this study was to go further in the understanding of the role played by the two symbionts in P. nigronervosa. To do so, microinjection tests were made to see the effect of wPn elimination on the host, and then, high-throughput sequencing of the haemolymph was used to analyze the gene content of the symbionts. RESULTS: We observed that the elimination of wPn systematically led to the death of aphids, suggesting that the bacterium could play a mutualistic role. In addition, we identify and annotate 587 and 250 genes for wPn and BPn, respectively, through high-throughput sequencing. Analysis of these genes suggests that the two bacteria are working together for the production of several essential nutrients. The most striking cases are for lysin and riboflavin which are usually provided by B. aphidicola alone to the host. In the banana aphid, the genes involved in the production pathways of these metabolites are shared between the two bacteria making them both essential for the survival of the aphid host. CONCLUSIONS: Our results suggest that a co-obligatory symbiosis between B. aphidicola and Wolbachia occurs in the banana aphid, the two bacteria acting together to supply essential nutrients to the host. This is, to our knowledge, the first time Wolbachia is reported to play an essential role in aphids.

Heritable symbiosis: The advantages and perils of an evolutionary rabbit hole.

Many eukaryotes have obligate associations with microorganisms that are transmitted directly between generations. A model for heritable symbiosis is the association of aphids, a clade of sap-feeding insects, and Buchnera aphidicola, a gammaproteobacterium that colonized an aphid ancestor 150 million years ago and persists in almost all 5,000 aphid species. Symbiont acquisition enables evolutionary and ecological expansion; aphids are one of many insect groups that would not exist without heritable symbiosis. Receiving less attention are potential negative ramifications of symbiotic alliances. In the short run, symbionts impose metabolic costs. Over evolutionary time, hosts evolve dependence beyond the original benefits of the symbiosis. Symbiotic partners enter into an evolutionary spiral that leads to irreversible codependence and associated risks. Host adaptations to symbiosis (e.g., immune-system modification) may impose vulnerabilities. Symbiont genomes also continuously accumulate deleterious mutations, limiting their beneficial contributions and environmental tolerance. Finally, the fitness interests of obligate heritable symbionts are distinct from those of their hosts, leading to selfish tendencies. Thus, genes underlying the host-symbiont interface are predicted to follow a coevolutionary arms race, as observed for genes governing host-pathogen interactions. On the macroevolutionary scale, the rapid evolution of interacting symbiont and host genes is predicted to accelerate host speciation rates by generating genetic incompatibilities. However, degeneration of symbiont genomes may ultimately limit the ecological range of host species, potentially increasing extinction risk. Recent results for the aphid-Buchnera symbiosis and related systems illustrate that, whereas heritable symbiosis can expand ecological range and spur diversification, it also presents potential perils.