In vivo interference of pea aphid endosymbiont Buchnera groEL gene by synthetic peptide nucleic acids.

The unculturable nature of intracellular obligate symbionts presents a significant challenge for elucidating gene functionality, necessitating the development of gene manipulation techniques. One of the best-studied obligate symbioses is that between aphids and the bacterial endosymbiont Buchnera aphidicola. Given the extensive genome reduction observed in Buchnera, the remaining genes are crucial for understanding the host-symbiont relationship, but a lack of tools for manipulating gene function in the endosymbiont has significantly impeded the exploration of the molecular mechanisms underlying this mutualism. In this study, we introduced a novel gene manipulation technique employing synthetic single-stranded peptide nucleic acids (PNAs). We targeted the critical Buchnera groEL using specially designed antisense PNAs conjugated to an arginine-rich cell-penetrating peptide (CPP). Within 24 h of PNA administration via microinjection, we observed a significant reduction in groEL expression and Buchnera cell count. Notably, the interference of groEL led to profound morphological malformations in Buchnera, indicative of impaired cellular integrity. The gene knockdown technique developed in this study, involving the microinjection of CPP-conjugated antisense PNAs, provides a potent approach for in vivo gene manipulation of unculturable intracellular symbionts, offering valuable insights into their biology and interactions with hosts.

Host-specific co-evolution likely driven by diet in Buchnera aphidicola.

BACKGROUND: Russian wheat aphid (Diuraphis noxia Kurd.) is a severe pest to wheat, and even though resistance varieties are available to curb this pest, they are becoming obsolete with the development of new virulent aphid populations. Unlike many other aphids, D noxia only harbours a single endosymbiont, Buchnera aphidicola. Considering the importance of Buchnera, this study aimed to elucidate commonalities and dissimilarities between various hosts, to better understand its distinctiveness within its symbiotic relationship with D. noxia. To do so, the genome of the D. noxia's Buchnera was assembled and compared to those of other aphid species that feed on diverse host species. RESULTS: The overall importance of several features such as gene length and percentage GC content was found to be critical for the maintenance of Buchnera genes when compared to their closest free-living relative, Escherichia coli. Buchnera protein coding genes were found to have percentage GC contents that tended towards a mean of ~ 26% which had strong correlation to their identity to their E. coli homologs. Several SNPs were identified between different aphid populations and multiple isolates of Buchnera were confirmed in single aphids. CONCLUSIONS: Establishing the strong correlation of percentage GC content of protein coding genes and gene identity will allow for identifying which genes will be lost in the continually shrinking Buchnera genome. This is also the first report of a parthenogenically reproducing aphid that hosts multiple Buchnera strains in a single aphid, raising questions regarding the benefits of maintaining multiple strains. We also found preliminary evidence for post-transcriptional regulation of Buchnera genes in the form of polyadenylation.

Direct sequencing of insect symbionts via nanopore adaptive sampling.

Insect symbionts can alter their host phenotype and their effects can range from beneficial to pathogenic. Moreover, many insects exhibit co-infections, making their study more challenging. Less than 1% of insect species have high-quality referenced genomes available and fewer still also have their symbionts sequenced. Two methods are commonly used to sequence symbionts: whole-genome sequencing to concomitantly capture the host and bacterial genomes, or isolation of the symbiont's genome before sequencing. These methods are limited when dealing with rare or poorly characterized symbionts. Long-read technology is an important tool to generate high-quality genomes as they can overcome high levels of heterozygosity, repeat content, and transposable elements that confound short-read methods. Oxford Nanopore (ONT) adaptive sampling allows a sequencing instrument to select or reject sequences in real time. We describe a method based on ONT adaptive sampling (subtractive) approach that readily permitted the sequencing of the complete genomes of mitochondria, Buchnera and its plasmids (pLeu, pTrp), and Wolbachia genomes in two aphid species, Aphis glycines and Pentalonia nigronervosa. Adaptive sampling is able to retrieve organelles such as mitochondria and symbionts that have high representation in their hosts such as Buchnera and Wolbachia, but is less successful at retrieving symbionts in low concentrations.

Co-option of a conserved host glutamine transporter facilitates aphid/Buchnera metabolic integration.

Organisms across the tree of life colonize novel environments by partnering with bacterial symbionts. These symbioses are characterized by intimate integration of host/endosymbiont biology at multiple levels, including metabolically. Metabolic integration is particularly important for sap-feeding insects and their symbionts, which supplement nutritionally unbalanced host diets. Many studies reveal parallel evolution of host/endosymbiont metabolic complementarity in amino acid biosynthesis, raising questions about how amino acid metabolism is regulated, how regulatory mechanisms evolve, and the extent to which similar mechanisms evolve in different systems. In the aphid/Buchnera symbiosis, the transporter ApGLNT1 (Acyrthosiphon pisum glutamine transporter 1) supplies glutamine, an amino donor in transamination reactions, to bacteriocytes (where Buchnera reside) and is competitively inhibited by Buchnera-supplied arginine-consistent with a role regulating amino acid metabolism given host demand for Buchnera-produced amino acids. We examined how ApGLNT1 evolved a regulatory role by functionally characterizing orthologs in insects with and without endosymbionts. ApGLNT1 orthologs are functionally similar, and orthology searches coupled with homology modeling revealed that GLNT1 is ancient and structurally conserved across insects. Our results indicate that the ApGLNT1 symbiotic regulatory role is derived from its ancestral role and, in aphids, is likely facilitated by loss of arginine biosynthesis through the urea cycle. Given consistent loss of host arginine biosynthesis and retention of endosymbiont arginine supply, we hypothesize that GLNT1 is a general mechanism regulating amino acid metabolism in sap-feeding insects. This work fills a gap, highlighting the broad importance of co-option of ancestral proteins to novel contexts in the evolution of host/symbiont systems.

Genome evolution of Buchnera aphidicola (Gammaproteobacteria): Insights into strand compositional asymmetry, codon usage bias, and phylogenetic implications.

Taxa of Buchnera aphidicola (hereafter "Buchnera") are mutualistic intracellular symbionts of aphids, known for their remarkable biological traits such as genome reduction, strand compositional asymmetry, and symbiont-host coevolution. With the growing availability of genomic data, we performed a comprehensive analysis of 103 genomes of Buchnera strains from 12 host subfamilies, focusing on the genomic characterizations, codon usage patterns, and phylogenetic implications. Our findings revealed consistent features among all genomes, including small genome sizes, low GC contents, and gene losses. We also identified strong strand compositional asymmetries in all strains at the genome level. Further investigation suggested that mutation pressure may have played a crucial role in shaping codon usage of Buchnera. Moreover, the genomic asymmetries were reflected in asymmetric codon usage preferences within chromosomal genes. Notably, the levels of these asymmetries were varied among strains and were significantly influenced by the degrees of genome shrinkages. Lastly, our phylogenetic analyses presented an alternative topology of Aphididae, based on the Buchnera symbionts, providing robust confirmation of the paraphylies of Eriosomatinae, and Macrosiphini. Our objectives are to further understand the strand compositional asymmetry and codon usage bias of Buchnera taxa, and provide new perspectives for phylogenetic studies of Aphididae.

Arsenophonus Interacts with Buchnera to Improve Growth Performance of Aphids under Amino Acid Stress.

Amino acids play a crucial role in the growth and development of insects. Aphids cannot ingest enough amino acids in plant phloem to meet their requirements, and therefore, they are mainly dependent on the obligate symbiont Buchnera aphidicola to synthesize essential amino acids. Besides Buchnera, aphids may harbor another facultative symbiont, Arsenophonus, which alters the requirement of the cotton-melon aphid Aphis gossypii for amino acid. However, it is unclear how Arsenophonus regulates the requirement. Here, we found that Arsenophonus ameliorated growth performance of A. gossypii on an amino acid-deficient diet. A deficiency in lysine (Lys) or methionine (Met) led to changes in the abundance of Arsenophonus. Arsenophonus suppressed the abundance of Buchnera when aphids were fed a normal amino acid diet, but this suppression was eliminated or reversed when aphids were on a Lys- or Met-deficient diet. The relative abundance of Arsenophonus was positively correlated with that of Buchnera, but neither of them was correlated with the body weight of aphids. The relative expression levels of Lys and Met synthase genes of Buchnera were affected by the interaction between Arsenophonus infections and Buchnera abundance, especially in aphids reared on a Lys- or Met-deficient diet. Arsenophonus coexisted with Buchnera in bacteriocytes, which strengthens the interaction. IMPORTANCE The obligate symbiont Buchnera can synthesize amino acids for aphids. In this study, we found that a facultative symbiont, Arsenophonus, can help improve aphids' growth performance under amino acid deficiency stress by changing the relative abundance of Buchnera and the expression levels of amino acid synthase genes. This study highlights the interaction between Arsenophonus and Buchnera to ameliorate aphid growth under amino acid stress.

Comparative transcriptomics of aphid species that diverged > 22 MYA reveals genes that are important for the maintenance of their symbiosis.

Most plant-sap feeding insects have obligate relationships with maternally transmitted bacteria. Aphids require their nutritional endosymbiont, Buchnera aphidicola, for the production of essential amino acids. Such endosymbionts are harbored inside of specialized insect cells called bacteriocytes. Here, we use comparative transcriptomics of bacteriocytes between two recently diverged aphid species, Myzus persicae and Acyrthosiphon pisum, to identify key genes that are important for the maintenance of their nutritional mutualism. The majority of genes with conserved expression profiles in M. persicae and A. pisum are for orthologs previously identified in A. pisum to be important for the symbiosis. However, asparaginase which produces aspartate from asparagine was significantly up-regulated only in A. pisum bacteriocytes, potentially because Buchnera of M. persicae encodes its own asparaginase enzyme unlike Buchnera of A. pisum resulting in Buchnera of A. pisum to be dependent on its aphid host for aspartate. One-to-one orthologs that explained the most amount of variation for bacteriocyte specific mRNA expression for both species includes a collaborative gene for methionine biosynthesis, multiple transporters, a horizontally transmitted gene, and secreted proteins. Finally, we highlight species-specific gene clusters which may contribute to host adaptations and/or accommodations in gene regulation to changes in the symbiont or the symbiosis.

Strong Linkage Between Symbiotic Bacterial Community and Host Age and Morph in a Hemipteran Social Insect.

The relationships between symbionts and insects are complex, and symbionts usually have diverse ecological and evolutionary effects on their hosts. The phloem sap-sucking aphids are good models to study the interactions between insects and symbiotic microorganisms. Although aphids usually exhibit remarkable life cycle complexity, most previous studies on symbiotic diversity sampled only apterous viviparous adult females or very few morphs. In this study, high-throughput 16S rDNA amplicon sequencing was used to assess the symbiotic bacterial communities of eleven morphs or developmental stages of the social aphid Pseudoregma bambucicola. We found there were significant differences in bacterial composition in response to different morphs and developmental stages, and for the first time, we revealed male aphids hosted very different symbiotic composition featured with low abundance of dominant symbionts but high diversity of total symbionts. The relative abundance of Pectobacterium showed relatively stable across different types of samples, while that of Wolbachia fluctuated greatly, indicating the former may have a consistent function in this species and the latter may provide specific function for certain morphs or developmental stages. Our study presents new evidence of complexity of symbiotic associations and indicates strong linkage between symbiotic bacterial community and host age and morph.

Compartmentalized into Bacteriocytes but Highly Invasive: the Puzzling Case of the Co-Obligate Symbiont Serratia symbiotica in the Aphid Periphyllus lyropictus.

Dependence on multiple nutritional symbionts that form a metabolic unit has evolved many times in insects. Although it has been postulated that host dependence on these metabolically interconnected symbionts is sustained by their high degree of anatomical integration (these symbionts are often housed in distinct symbiotic cells, the bacteriocytes, assembled into a common symbiotic organ, the bacteriome), the developmental aspects of such multipartner systems have received little attention. Aphids of the subfamilies Chaitophorinae and Lachninae typically harbor disymbiotic systems in which the metabolic capabilities of the ancient obligate symbiont Buchnera aphidicola are complemented by those of a more recently acquired nutritional symbiont, often belonging to the species Serratia symbiotica. Here, we used microscopy approaches to finely characterize the tissue tropism and infection dynamics of the disymbiotic system formed by B. aphidicola and S. symbiotica in the Norway maple aphid Periphyllus lyropictus (Chaitophorinae). Our observations show that, in this aphid, the co-obligate symbiont S. symbiotica exhibits a dual lifestyle: intracellular by being housed in large syncytial bacteriocytes embedded between B. aphidicola-containing bacteriocytes in a well-organized compartmentalization pattern, and extracellular by massively invading the digestive tract and other tissues during embryogenesis. This is the first reported case of an obligate aphid symbiont that is internalized in bacteriocytes but simultaneously adopts an extracellular lifestyle. This unusual infection pattern for an obligate insect symbiont suggests that some bacteriocyte-associated obligate symbionts, despite their integration into a cooperative partnership, still exhibit invasive behavior and escape strict compartmentalization in bacteriocytes. IMPORTANCE Multipartner nutritional endosymbioses have evolved many times in insects. In Chaitophorinae aphids, the eroded metabolic capabilities of the ancient obligate symbiont B. aphidicola are complemented by those of more recently acquired symbionts. Here, we report the atypical case of the co-obligate S. symbiotica symbiont associated with P. lyropictus. This bacterium is compartmentalized into bacteriocytes nested into the ones harboring the more ancient symbiont B. aphidicola, reflecting metabolic convergences between the two symbionts. At the same time, S. symbiotica exhibits highly invasive behavior by colonizing various host tissues, including the digestive tract during embryogenesis. The discovery of this unusual phenotype for a co-obligate symbiont reveals a new face of multipartner nutritional endosymbiosis in insects. In particular, it shows that co-obligate symbionts can retain highly invasive traits and suggests that host dependence on these bacterial partners may evolve prior to their strict compartmentalization into specialized host structures.

Ploidy dynamics in aphid host cells harboring bacterial symbionts.

Aphids have evolved bacteriocytes or symbiotic host cells that harbor the obligate mutualistic bacterium Buchnera aphidicola. Because of the large cell size (approximately 100 µm in diameter) of bacteriocytes and their pivotal role in nutritional symbiosis, researchers have considered that these cells are highly polyploid and assumed that bacteriocyte polyploidy may be essential for the symbiotic relationship between the aphid and the bacterium. However, little is known about the ploidy levels and dynamics of aphid bacteriocytes. Here, we quantitatively analyzed the ploidy levels in the bacteriocytes of the pea-aphid Acyrthosiphon pisum. Image-based fluorometry revealed the hyper polyploidy of the bacteriocytes ranging from 16- to 256-ploidy throughout the lifecycle. Bacteriocytes of adult parthenogenetic viviparous females were ranged between 64 and 128C DNA levels, while those of sexual morphs (oviparous females and males) were comprised of 64C, and 32-64C cells, respectively. During post-embryonic development of viviparous females, the ploidy level of bacteriocytes increased substantially, from 16 to 32C at birth to 128-256C in actively reproducing adults. These results suggest that the ploidy levels are dynamically regulated among phenotypes and during development. Our comprehensive and quantitative data provides a foundation for future studies to understand the functional roles and biological significance of the polyploidy of insect bacteriocytes.

Elucidation of host and symbiont contributions to peptidoglycan metabolism based on comparative genomics of eight aphid subfamilies and their Buchnera.

Pea aphids (Acyrthosiphon pisum) are insects containing genes of bacterial origin with putative functions in peptidoglycan (PGN) metabolism. Of these, rlpA1-5, amiD, and ldcA are highly expressed in bacteriocytes, specialized aphid cells that harbor the obligate bacterial symbiont Buchnera aphidicola, required for amino acid supplementation of the host's nutrient-poor diet. Despite genome reduction associated with endosymbiosis, pea aphid Buchnera retains genes for the synthesis of PGN while Buchnera of many other aphid species partially or completely lack these genes. To explore the evolution of aphid horizontally-transferred genes (HTGs) and to elucidate how host and symbiont genes contribute to PGN production, we sequenced genomes from four deeply branching lineages, such that paired aphid and Buchnera genomes are now available for 17 species representing eight subfamilies. We identified all host and symbiont genes putatively involved in PGN metabolism. Phylogenetic analyses indicate that each HTG family was present in the aphid shared ancestor, but that each underwent a unique pattern of gene loss or duplication in descendant lineages. While four aphid rlpA gene subfamilies show no relation to symbiont PGN gene repertoire, the loss of aphid amiD and ldcA HTGs coincides with the loss of symbiont PGN metabolism genes. In particular, the coincident loss of host amiD and symbiont murCEF in tribe Aphidini, in contrast to tribe Macrosiphini, suggests either 1) functional linkage between these host and symbiont genes, or 2) Aphidini has lost functional PGN synthesis and other retained PGN pathway genes are non-functional. To test these hypotheses experimentally, we used cell-wall labeling methods involving a d-alanine probe and found that both Macrosiphini and Aphidini retain Buchnera PGN synthesis. Our results imply that compensatory adaptations can preserve PGN synthesis despite the loss of some genes considered essential for this pathway, highlighting the importance of the cell wall in these symbioses.

Impact of heat stress on the fitness outcomes of symbiotic infection in aphids: a meta-analysis.

Beneficial microorganisms shape the evolutionary trajectories of their hosts, facilitating or constraining the colonization of new ecological niches. One convincing example entails the responses of insect-microbe associations to rising temperatures. Indeed, insect resilience to stressful high temperatures depends on the genetic identity of the obligate symbiont and the presence of heat-protective facultative symbionts. As extensively studied organisms, aphids and their endosymbiotic bacteria represent valuable models to address eco-evolutionary questions about the thermal ecology of insect-microbe partnerships, with broad relevance to various biological systems and insect models. This meta-analysis aims to quantify the context-dependent impacts of symbionts on host phenotype in benign or stressful heat conditions, across fitness traits, types of heat stress and symbiont species. We found that warming lowered the benefits (resistance to parasitoids) and costs (development, fecundity) of infection by facultative symbionts, which was overall mostly beneficial to the hosts under short-term heat stress (heat shock) rather than extended warming. Heat-tolerant genotypes of the obligate symbiont Buchnera aphidicola and some facultative symbionts (Rickettsia sp., Serratia symbiotica) improved or maintained aphid fitness under heat stress. We discuss the implications of these findings for the general understanding of the cost-benefit balance of insect-microbe associations across multiple traits and their eco-evolutionary dynamics faced with climate change.

Aphid hologenomics: current status and future challenges.

Aphids are important model organisms in ecological, developmental, and evolutionary studies of, for example, symbiosis, insect-plant interactions, pest management, and developmental polyphenism. Here, we review the recent progress made in the genomics of aphids and their symbionts: hologenomics. The reference genome of Acyrthosiphon pisum has been greatly improved, and chromosome-level assembly is now available. The genomes of over 20 aphid species have been sequenced, and comparative genomic analyses have revealed pervasive gene duplication and dynamic chromosomal rearrangements. Over 120 symbiont genomes (both obligate and facultative) have been sequenced, and modern deep-sequencing technologies have identified novel symbionts. The advances in hologenomics have helped to elucidate the dynamic evolution of facultative and co-obligate symbionts with the ancient obligate symbiont Buchnera.

Phylosymbiotic Structures of the Microbiota in Mollitrichosiphum tenuicorpus (Hemiptera: Aphididae: Greenideinae).

Aphids harbor an array of symbionts that provide hosts with ecological benefits. Microbial community assembly generally varies with respect to aphid species, geography, and host plants. However, the influence of host genetics and ecological factors on shaping intraspecific microbial community structures has not been fully understood. In the present study, using Illumina sequencing of the V3 - V4 hypervariable region of the 16S rRNA gene, we characterized the microbial compositions associated with Mollitrichosiphum tenuicorpus from different regions and plants in China. The primary symbiont Buchnera aphidicola and the secondary symbiont Arsenophonus dominated the microbial flora in M. tenuicorpus. Ordination analyses and statistical tests suggested that geography and aphid genetics primarily contributed to the variation in the microbiota of M. tenuicorpus. We further confirmed the combined effect of aphid genetics and geography on shaping the structures of symbiont and secondary symbiont communities. Moreover, the significant correlation between aphid genetic divergence and symbiont community dissimilarity provides evidence for intraspecific phylosymbiosis in natural systems. Our study helped to elucidate the eco-evolutionary relationship between symbiont communities and aphids within one given species.

Genome sequence of the English grain aphid, Sitobion avenae and its endosymbiont Buchnera aphidicola.

The English grain aphid, Sitobion avenae, is a major agricultural pest of wheat, barley and oats, and one of the principal vectors of barley yellow dwarf virus leading to significant reductions in grain yield, annually. Emerging resistance to and increasing regulation of insecticides has resulted in limited options for their control. Using PacBio HiFi data, we have produced a high-quality draft assembly of the S. avenae genome; generating a primary assembly with a total assembly size of 475.7 Mb, and an alternate assembly with a total assembly size of 430.8 Mb. Our primary assembly was highly contiguous with only 326 contigs and a contig N50 of 15.95 Mb. Assembly completeness was estimated at 97.7% using BUSCO analysis and 31,007 and 29,037 protein-coding genes were predicted from the primary and alternate assemblies, respectively. This assembly, which is to our knowledge the first for an insecticide resistant clonal lineage of English grain aphid, will provide novel insight into the molecular and mechanistic determinants of resistance and will facilitate future research into mechanisms of viral transmission and aphid behavior.

Microbe Profile: Buchnera aphidicola: ancient aphid accomplice and endosymbiont exemplar.

Buchnera aphidicola is an obligate endosymbiont of aphids that cannot be cultured outside of hosts. It exists as diverse strains in different aphid species, and phylogenetic reconstructions show that it has been maternally transmitted in aphids for >100 million years. B. aphidicola genomes are highly reduced and show conserved gene order and no gene acquisition, but encoded proteins undergo rapid evolution. Aphids depend on B. aphidicola for biosynthesis of essential amino acids and as an integral part of embryonic development. How B. aphidicola populations are regulated within hosts remains little known.

Horizontal-Acquisition of a Promiscuous Peptidoglycan-Recycling Enzyme Enables Aphids To Influence Symbiont Cell Wall Metabolism.

During evolution, enzymes can undergo shifts in preferred substrates or in catalytic activities. An intriguing question is how enzyme function changes following horizontal gene transfer, especially for bacterial genes that have moved to animal genomes. Some insects have acquired genes that encode enzymes for the biosynthesis of bacterial cell wall components and that appear to function to support or control their obligate endosymbiotic bacteria. In aphids, the bacterial endosymbiont Buchnera aphidicola provides essential amino acids for aphid hosts but lacks most genes for remodeling of the bacterial cell wall. The aphid genome has acquired seven genes with putative functions in cell wall metabolism that are primarily expressed in the aphid cells harboring Buchnera. In analyses of aphid homogenates, we detected peptidoglycan (PGN) muropeptides indicative of the reactions of PGN hydrolases encoded by horizontally acquired aphid genes but not by Buchnera genes. We produced one such host enzyme, ApLdcA, and characterized its activity with both cell wall derived and synthetic PGN. Both ApLdcA and the homologous enzyme in Escherichia coli, which functions as an l,d-carboxypeptidase in the cytoplasmic PGN recycling pathway, exhibit turnover of PGN substrates containing stem pentapeptides and cross-linkages via l,d-endopeptidase activity, consistent with a potential role in cell wall remodeling. Our results suggest that ApLdcA derives its functions from the promiscuous activities of an ancestral LdcA enzyme, whose acquisition by the aphid genome may have enabled hosts to influence Buchnera cell wall metabolism as a means to control symbiont growth and division. IMPORTANCE Most enzymes are capable of performing biologically irrelevant side reactions. During evolution, promiscuous enzyme activities may acquire new biological roles, especially after horizontal gene transfer to new organisms. Pea aphids harbor obligate bacterial symbionts called Buchnera and encode horizontally acquired bacterial genes with putative roles in cell wall metabolism. Though Buchnera lacks cell wall endopeptidase genes, we found evidence of endopeptidase activity among peptidoglycan muropeptides purified from aphids. We characterized a multifunctional, aphid-encoded enzyme, ApLdcA, which displays l,d-endopeptidase activities considered promiscuous for the Escherichia coli homolog, for which these activities do not contribute to its native role in peptidoglycan recycling. These results exemplify the roles of enzyme promiscuity and horizontal gene transfer in enzyme evolution and demonstrate how aphids influence symbiont cell wall metabolism.

Geography-dependent symbiont communities in two oligophagous aphid species.

Aphids and their diverse symbionts have become a good model to study bacteria-arthropod symbiosis. The feeding habits of aphids are usually influenced by a variety of symbionts. Most studies on symbiont diversity have focused on polyphagous aphids, while symbiont community patterns for oligophagous aphids remain unclear. Here, we surveyed the bacterial communities in natural populations of two oligophagous aphids, Melanaphis sacchari and Neophyllaphis podocarpi, in natural populations. Seven common symbionts were detected, among which Buchnera aphidicola and Wolbachia were the most prevalent. In addition, an uncommon Sodalis-like symbiont was also detected in these two aphids, and Gilliamella was found in some samples of M. sacchari. We further assessed the significant variation in symbiont communities within the two aphid species, geographical regions and host specialization using statistical and ordination analyses. Geography was an important factor in shaping the symbiont community structure in these oligophagous aphids. Furthermore, the strong geographical influence may be related to specific environmental factors, especially temperature, among different regions. These findings extend our knowledge of the significance of geography and its associated environmental conditions in the symbiont community structure associated with oligophagous aphids.

Natural Selection Shapes Maintenance of Orthologous sRNAs in Divergent Host-Restricted Bacterial Genomes.

Historically it has been difficult to study the evolution of bacterial small RNAs (sRNAs) across distantly related species. For example, identifying homologs of sRNAs is often difficult in genomes that have undergone multiple structural rearrangements. Also, some types of regulatory sRNAs evolve at rapid rates. The high degree of genomic synteny among divergent host-restricted bacterial lineages, including intracellular symbionts, is conducive to sRNA maintenance and homolog identification. In turn, symbiont genomes can provide us with novel insights into sRNA evolution. Here, we examine the sRNA expression profile of the obligate symbiont of psyllids, Carsonella ruddii, which has one of the smallest cellular genomes described. Using RNA-seq, we identified 36 and 32 antisense sRNAs (asRNAs) expressed by Carsonella from the psyllids Bactericera cockerelli (Carsonella-BC) and Diaphorina citri (Carsonella-DC), respectively. The majority of these asRNAs were associated with genes that are involved in essential amino acid biosynthetic pathways. Eleven of the asRNAs were conserved in both Carsonella lineages and the majority were maintained by selection. Notably, five of the corresponding coding sequences are also the targets of conserved asRNAs in a distantly related insect symbiont, Buchnera. We detected differential expression of two asRNAs for genes involved in arginine and leucine biosynthesis occurring between two distinct Carsonella-BC life stages. Using asRNAs identified in Carsonella, Buchnera, and Profftella which are all endosymbionts, and Escherichia coli, we determined that regions upstream of these asRNAs encode unique conserved patterns of AT/GC richness, GC skew, and sequence motifs which may be involved in asRNA regulation.

The boom and bust of the aphid's essential amino acid metabolism across nymphal development.

Within long-term symbioses, animals integrate their physiology and development with their symbiont. In a model nutritional mutualism, aphids harbor the endosymbiont, Buchnera, within specialized bacteriocyte cells. Buchnera synthesizes essential amino acids (EAAs) and vitamins for their host, which are lacking from the aphid's plant sap diet. It is unclear if the aphid host differentially expresses aphid EAA metabolism pathways and genes that collaborate with Buchnera for the production of EAA and vitamins throughout nymphal development when feeding on plants. It is also unclear if aphid bacteriocytes are differentially methylated throughout aphid development as DNA methylation may play a role in gene regulation. By analyzing aphid gene expression, we determined that the bacteriocyte is metabolically more active in metabolizing Buchnera's EAAs and vitamins early in nymphal development compared to intermediate or later immature and adult lifestages. The largest changes in aphid bacteriocyte gene expression, especially for aphid genes that collaborate with Buchnera, occurred during the 3rd to 4th instar transition. During this transition, there is a huge shift in the bacteriocyte from a high energy "nutrient-consuming state" to a "recovery and growth state" where patterning and signaling genes and pathways are upregulated and differentially methylated, and de novo methylation is reduced as evidenced by homogenous DNA methylation profiles after the 2nd instar. Moreover, bacteriocyte number increased and Buchnera's titer decreased throughout aphid nymphal development. These data suggest in combination that bacteriocytes of older nymphal and adult lifestages depend less on the nutritional symbiosis compared to early nymphal lifestages.