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.

A sRNA in a reduced mutualistic symbiont genome regulates its own gene expression.

Similar to other nutritional endosymbionts that are obligate for host survival, the mutualistic aphid endosymbiont, Buchnera, has a highly reduced genome with few regulatory elements. Until recently, it was thought that aphid hosts were primarily responsible for regulating their symbiotic relationship. However, we recently revealed that Buchnera displays differential protein regulation, but not mRNA expression. We also identified a number of conserved small RNAs (sRNAs) that are expressed among Buchnera taxa. In this study, we investigate whether differential protein regulation in Buchnera is the result of post-transcriptional gene regulation via sRNAs. We characterize the sRNA profile of two Buchnera life stages: (i) when Buchnera is transitioning from an extracellular proliferating state in aphid embryos and (ii) when Buchnera is in an intracellular nonproliferating state in aphid bacteriocytes (specialized symbiont cells). Overall, we identified 90 differentially expressed sRNAs, 97% of which were upregulated in aphid embryos. Of these sRNAs, the majority were predicted to be involved in the regulation of various metabolic processes, including arginine biosynthesis. Using a heterologous dual expression vector, we reveal for the first time that a Buchnera antisense sRNA can post-transcriptionally interact with its cognate Buchnera coding sequence, carB, a gene involved in arginine biosynthesis. These results corroborate our in vivo RNAseq and proteomic data, where the candidate antisense sRNA carB and the protein CarB are significantly upregulated in aphid embryos. Overall, we demonstrate that Buchnera may regulate gene expression independently from its host by utilizing sRNAs.

Efficacy of In Vivo Electroporation on the Delivery of Molecular Agents into Aphid (Hemiptera: Aphididae) Ovarioles.

While the wealth of genomic data presently available is increasing rapidly, the advancement of functional genomics technologies for the large majority of these organisms has lagged behind. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 system is an emerging gene-editing technology derived from a bacterial adaptive immune system that has proven highly effective in multiple model systems. Here, the CRISPR/Cas9 system was delivered into the ovarioles of the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphididae), with a new delivery method utilizing in vivo electroporation. To validate gene-editing, a target sequence within the marker tor pigment gene was chosen, and gene-editing was predicted to result in white pigmentation in the offspring of treated adult aphids. Adult aphids (10-d old) were injected with the tor single guide RNA and Cas9 complex and subsequently subjected to electroporation. Adult aphids were given 4 d to produce viviparous offspring. After offspring developed for 6 d, DNA was extracted and sequenced to validate if CRISPR/Cas9-directed gene editing occurred. A survival rate over 70% was found in treated adult aphids. A distinct white pigmentation was found in 2.5% of aphids; however, gene-editing within the target sequence was not found in any of the individuals screened. Presence of white aphids without gene-editing suggests other mechanisms may have influenced pigmentation. High survival rates in experimental treatments demonstrate the robustness of this new technique, and further refinement of this technique may prove it as an effective functional genomics tool for viviparous insects and/or gene editing at a somatic level.

Band-aids for Buchnera and B vitamins for all.

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

Midgut fungal and bacterial microbiota of Aedes triseriatus and Aedes japonicus shift in response to La Crosse virus infection.

Understanding how midgut microbial communities of field-collected mosquitoes interact with pathogens is critical for controlling vector infection and disease. We used 16S rRNA and internal transcribed spacer sequencing to characterize the midgut bacterial and fungal communities of adult females of Aedes triseriatus and Aedes japonicus collected as pupae in tree holes, plastic bins and waste tires and their response to La Crosse virus (LACV) infection. For both mosquito species and across all habitat and virus treatments, a total of 62 bacterial operational taxonomic units (OTUs) from six phyla and 21 fungal OTUs from two phyla were identified. The majority of bacterial (92%) and fungal (71%) OTUs were shared between the mosquito species; however, several OTUs were unique to each species. Bacterial and fungal communities of individuals that took either infectious or noninfectious bloodmeals were less diverse and more homogeneous compared to those of newly emerged adults. Interestingly, LACV-infected A. triseriatus and A. japonicus had higher bacterial richness and lower fungal richness compared to individuals that took a noninfectious bloodmeal, suggesting that viral infection was associated with an increase in bacterial OTUs and a decrease in fungal OTUs. For both mosquito species, several OTUs were identified that had both high fidelity and specificity to mosquito midguts that were infected with LACV. Overall, these findings demonstrate that bacterial and fungal communities that reside in mosquito midguts respond to host diet and viral infection and could play a role in modulating vector susceptibility to LACV.

Genomic basis of endosymbiont-conferred protection against an insect parasitoid.

Bacterial endosymbionts exert a variety of beneficial effects on insect hosts. In pea aphids (Acyrthosiphon pisum), several inherited endosymbiont species protect their hosts against parasitoid wasps, which are major natural enemies. However, strains of these symbiont species vary in their ability to confer protection against parasitoids, with some conferring almost complete protection and others conferring almost none. In this study, two strains of the endosymbiont Regiella insecticola (R. insecticola 5.15 and R. insecticola LSR1) were found to differ in ability to protect pea aphids attacked by the parasitoid Aphidius ervi. Parasitism trials reveal that R. insecticola 5.15, but not R. insecticola LSR1, significantly reduced parasitoid success and increased aphid survivorship. To address the potential genetic basis of protection conferred by R. insecticola 5.15 we sequenced the genome of this symbiont strain, and then compared its gene repertoire with that of the already sequenced nonprotective strain R. insecticola LSR1. We identified striking differences in gene sets related to eukaryote pathogenicity. The protective strain R. insecticola 5.15 encoded five categories of pathogenicity factors that were missing or inactivated in R. insecticola LSR1. These included genes encoding the O-antigen biosynthetic pathway, an intact Type 1 Secretion System and its secreted RTX toxins, an intact SPI-1 Type 3 Secretion System and its effectors, hemin transport, and the two-component system PhoPQ. These five pathogenicity factors and translocation systems are hypothesized to collectively play key roles in the endosymbiont's virulence against parasitoids, resulting in aphid protection. Mechanisms through which these factors may target parasitoids are discussed.

The impact of microbial symbionts on host plant utilization by herbivorous insects.

Herbivory, defined as feeding on live plant tissues, is characteristic of highly successful and diverse groups of insects and represents an evolutionarily derived mode of feeding. Plants present various nutritional and defensive barriers against herbivory; nevertheless, insects have evolved a diverse array of mechanisms that enable them to feed and develop on live plant tissues. For decades, it has been suggested that insect-associated microbes may facilitate host plant use, and new molecular methodologies offer the possibility to elucidate such roles. Based on genomic data, specialized feeding on phloem and xylem sap is highly dependent on nutrient provisioning by intracellular symbionts, as exemplified by Buchnera in aphids, although it is unclear whether such symbionts play a substantive role in host plant specificity of their hosts. Microorganisms present in the gut or outside the insect body could provide more functions including digestion of plant polymers and detoxification of plant-produced toxins. However, the extent of such contributions to insect herbivory remains unclear. We propose that the potential functions of microbial symbionts in facilitating or restricting the use of host plants are constrained by their location (intracellular, gut or environmental), and by the fidelity of their associations with insect host lineages. Studies in the next decade, using molecular methods from environmental microbiology and genomics, will provide a more comprehensive picture of the role of microbial symbionts in insect herbivory.

Evaluating insect-microbiomes at the plant-insect interface.

Plants recognize biotic challengers and respond with the appropriate defense by utilizing phytohormone signaling and crosstalk. Despite this, microbes and insects have evolved mechanisms that compromise the plant surveillance system and specific defenses, thus ensuring successful colonization. In nature, plants do not experience insect herbivores and microbes in isolation, but in combination. Over time, relationships have developed between insects and microbes, varying on a continuum from no-relationship to obligate relationships that are required for both organisms to survive. While many reviews have examined plant-insect and plant-microbe interactions and the mechanisms of plant defense, few have considered the interface where microbes and insects may overlap, and synergies may develop. In this review, we critically evaluate the requirements for insect-associated microbes to develop synergistic relationships with their hosts, and we mechanistically discuss how some of these insect-associated microbes can target or modify host plant defenses. Finally, by using bioinformatics and the recent literature, we review evidence for synergies in insect-microbe relationships at the interface of plant-insect defenses. Insect-associated microbes can influence host-plant detection and/or signaling through phytohormone synthesis, conserved microbial patterns, and effectors, however, microbes associated with insects must be maintained in the environment and located in opportunistic positions.

Altered tRNA characteristics and 3' maturation in bacterial symbionts with reduced genomes.

Translational efficiency is controlled by tRNAs and other genome-encoded mechanisms. In organelles, translational processes are dramatically altered because of genome shrinkage and horizontal acquisition of gene products. The influence of genome reduction on translation in endosymbionts is largely unknown. Here, we investigate whether divergent lineages of Buchnera aphidicola, the reduced-genome bacterial endosymbiont of aphids, possess altered translational features compared with their free-living relative, Escherichia coli. Our RNAseq data support the hypothesis that translation is less optimal in Buchnera than in E. coli. We observed a specific, convergent, pattern of tRNA loss in Buchnera and other endosymbionts that have undergone genome shrinkage. Furthermore, many modified nucleoside pathways that are important for E. coli translation are lost in Buchnera. Additionally, Buchnera's A + T compositional bias has resulted in reduced tRNA thermostability, and may have altered aminoacyl-tRNA synthetase recognition sites. Buchnera tRNA genes are shorter than those of E. coli, as the majority no longer has a genome-encoded 3' CCA; however, all the expressed, shortened tRNAs undergo 3' CCA maturation. Moreover, expression of tRNA isoacceptors was not correlated with the usage of corresponding codons. Overall, our data suggest that endosymbiont genome evolution alters tRNA characteristics that are known to influence translational efficiency in their free-living relative.

Aphid genome expression reveals host-symbiont cooperation in the production of amino acids.

The evolution of intimate symbiosis requires the coordination of gene expression and content between the distinct partner genomes; this coordination allows the fusion of capabilities of each organism into a single integrated metabolism. In aphids, the 10 essential amino acids are scarce in the phloem sap diet and are supplied by the obligate bacterial endosymbiont (Buchnera), which lives inside specialized cells called bacteriocytes. Although Buchnera's genome encodes most genes for essential amino acid biosynthesis, several genes in essential amino acid pathways are missing, as are most genes for production of nonessential amino acids. Additionally, it is unresolved whether the supply of nitrogen for amino acid biosynthesis is supplemented by recycling of waste ammonia. We compared pea aphid gene expression between bacteriocytes and other body tissues using RNA sequencing and pathway analysis and exploiting the genome sequences available for both partners. We found that 26 genes underlying amino acid biosynthesis were up-regulated in bacteriocytes. Seven of these up-regulated genes fill the gaps of Buchnera's essential amino acid pathways. In addition, genes underlying five nonessential amino acid pathways lost from Buchnera are up-regulated in bacteriocytes. Finally, our results reveal that two genes, glutamine synthetase and glutamate synthase, which potentially work together in the incorporation of ammonium nitrogen into glutamate (GOGAT) cycle to assimilate ammonia into glutamate, are up-regulated in bacteriocytes. Thus, host gene expression and symbiont capabilities are closely integrated within bacteriocytes, which function as specialized organs of amino acid production. Furthermore, the GOGAT cycle may be a key source of nitrogen fueling the integrated amino acid metabolism of the aphid-Buchnera partnership.

Unveiling metabolic integration in psyllids and their nutritional endosymbionts through comparative transcriptomics analysis.

Psyllids, a group of insects that feed on plant sap, have a symbiotic relationship with an endosymbiont called Carsonella. Carsonella synthesizes essential amino acids and vitamins for its psyllid host, but lacks certain genes required for this process, suggesting a compensatory role of psyllid host genes. To investigate this, gene expression was compared between two psyllid species, Bactericera cockerelli and Diaphorina citri, in specialized cells where Carsonella resides (bacteriomes). Collaborative psyllid genes, including horizontally transferred genes, showed patterns of conserved gene expression; however, species-specific patterns were also observed, suggesting differences in the nutritional metabolism between psyllid species. Also, the recycling of nitrogen in bacteriomes may primarily rely on glutamate dehydrogenase (GDH). Additionally, lineage-specific gene clusters were differentially expressed in B. cockerelli and D. citri bacteriomes and are highlighted here. These findings shed light on potential host adaptations for the regulation of this symbiosis due to host, microbiome, and environmental differences.

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.

Chromosomal-level assembly of Bactericera cockerelli reveals rampant gene family expansions impacting genome structure, function and insect-microbe-plant-interactions.

Lineage specific expansions and gene duplications are some of the most important sources of evolutionary novelty in eukaryotes. Although not as prevalent in eukaryotes compared to bacteria, horizontal gene transfer events can also result in key adaptations for insects, especially for those involved in insect-microbe interactions. In this study we assemble the first chromosomal assembly of the psyllid Bactericera cockerelli and reveal that the B. cockerelli genome has experienced significantly more gene expansion events compared to other Hemipteran representatives with fully sequenced genomes. We also reveal that B. cockerelli's genome is the largest psyllid genome (567 Mb) sequenced to date and is ~15% larger than the other two psyllid species genomes sequenced (Pachypsylla venusta and Diaphorina citri). Structurally, B. cockerelli appears to have an additional chromosome compared to the distantly related psyllid species P. venusta due to a previous chromosomal fission or fusion event. The increase in genome size and dynamic nature of the B. cockerelli genome may largely be contributed to the widespread expansion of type I and II repeat elements that are rampant across all of B. cockerelli's. chromosomes. These repeat elements are distributed near equally in both euchromatic and heterochromatic regions. Furthermore, significant gene family expansions and gene duplications were uncovered for genes that are expected to be important in its adaptation to insect-plant and microbe interactions, which include transcription factors, proteases, odorant receptors, and horizontally transferred genes that are involved in the nutritional symbioses with their long-term nutritional endosymbiont Carsonella.

Divergent Host-Microbe Interaction and Pathogenesis Proteins Detected in Recently Identified Liberibacter Species.

Candidatus (Ca.) Liberibacter taxa are economically important bacterial plant pathogens that are not culturable; however, genome-enabled insights can help us develop a deeper understanding of their host-microbe interactions and evolution. The draft genome of a recently identified Liberibacter taxa, Ca. Liberibacter capsica, was curated and annotated here with a total draft genome size of 1.1 MB with 1,036 proteins, which is comparable to other Liberibacter species with complete genomes. A total of 459 orthologous clusters were identified among Ca. L. capsica, Ca. L. asiaticus, Ca. L. psyllaurous, Ca. L. americanus, Ca. L. africanus, and L. crescens, and these genes within these clusters consisted of housekeeping and environmental response functions. We estimated the rates of molecular evolution for each of the 443 one-to-one ortholog clusters and found that all Ca. L. capsica orthologous pairs were under purifying selection when the synonymous substitutions per synonymous site (dS) were not saturated. These results suggest that these genes are largely maintaining their conserved functions. We also identified the most divergent single-copy orthologous proteins in Ca. L. capsica by analyzing the ortholog pairs that represented the highest nonsynonymous substitutions per nonsynonymous site (dN) values for each pairwise comparison. From these analyses, we found that 21 proteins which are known to be involved in pathogenesis and host-microbe interactions, including the Tad pilus complex, were consistently divergent between Ca. L. capsica and the majority of other Liberibacter species. These results further our understanding of the evolutionary genetics of Ca. L. capsica and, more broadly, the evolution of Liberibacter. IMPORTANCE "Candidatus" (Ca.) Liberibacter taxa are economically important plant pathogens vectored by insects; however, these host-dependent bacterial taxa are extremely difficult to study because they are unculturable. Recently, we identified a new Ca. Liberibacter lineage (Ca. Liberibacter capsica) from a rare insect metagenomic sample. In this current study, we report that the draft genome of Ca. Liberibacter capsica is similar in genome size and protein content compared to the other Ca. Liberibacter taxa. We provide evidence that many of their shared genes, which encode housekeeping and environmental response functions, are evolving under purifying selection, suggesting that these genes are maintaining similar functions. Our study also identifies 21 proteins that are rapidly evolving amino acid changes in Ca. Liberibacter capsica compared to the majority of other Liberibacter taxa. Many of these proteins represent key genes involved in Liberibacter-host interactions and pathogenesis and are valuable candidate genes for future studies.

Origin and examination of a leafhopper facultative endosymbiont.

Eukaryotes engage in intimate interactions with microbes that range in age and type of association. Although many conspicuous examples of ancient insect associates are studied (e.g., Buchnera aphidicola), fewer examples of younger associations are known. Here, we further characterize a recently evolved bacterial endosymbiont of the leafhopper Euscelidius variegatus (Hemiptera, Cicadellidae), called BEV. We found that BEV, continuously maintained in E. variegatus hosts at UC Berkeley since 1984, is vertically transmitted with high fidelity. Unlike many vertically transmitted, ancient endosymbioses, the BEV-E. variegatus association is not obligate for either partner, and BEV can be cultivated axenically. Sufficient BEV colonies were grown and harvested to estimate its genome size and provide a partial survey of the genome sequence. The BEV chromosome is about 3.8 Mbp, and there is evidence for an extrachromosomal element roughly 53 kb in size (e.g., prophage or plasmid). We sequenced 438 kb of unique short-insert clones, representing about 12% of the BEV genome. Nearly half of the gene fragments were similar to mobile DNA, including 15 distinct types of insertion sequences (IS). Analyses revealed that BEV not only shares virulence genes with plant pathogens, but also is closely related to the plant pathogenic genera Dickeya, Pectobacterium, and Brenneria. However, the slightly reduced genome size, abundance of mobile DNA, fastidious growth in culture, and efficient vertical transmission suggest that symbiosis with E. variegatus has had a significant impact on genome evolution in BEV.

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.

Potato leafroll virus reduces Buchnera aphidocola titer and alters vector transcriptome responses.

Viruses in the Luteoviridae family, such as Potato leafroll virus (PLRV), are transmitted by aphids in a circulative and nonpropagative mode. This means the virions enter the aphid body through the gut when they feed from infected plants and then the virions circulate through the hemolymph to enter the salivary glands before being released into the saliva. Although these viruses do not replicate in their insect vectors, previous studies have demonstrated viruliferous aphid behavior is altered and the obligate symbiont of aphids, Buchnera aphidocola, may be involved in transmission. Here we provide the transcriptome of green peach aphids (Myzus persicae) carrying PLRV and virus-free control aphids using Illumina sequencing. Over 150 million paired-end reads were obtained through Illumina sequencing, with an average of 19 million reads per library. The comparative analysis identified 134 differentially expressed genes (DEGs) between the M. persicae transcriptomes, including 64 and 70 genes that were up- and down-regulated in aphids carrying PLRV, respectively. Using functional classification in the GO databases, 80 of the DEGs were assigned to 391 functional subcategories at category level 2. The most highly up-regulated genes in aphids carrying PLRV were cytochrome p450s, genes related to cuticle production, and genes related to development, while genes related to heat shock proteins, histones, and histone modification were the most down-regulated. PLRV aphids had reduced Buchnera titer and lower abundance of several Buchnera transcripts related to stress responses and metabolism. These results suggest carrying PLRV may reduce both aphid and Buchnera genes in response to stress. This work provides valuable basis for further investigation into the complicated mechanisms of circulative and nonpropagative transmission.

Uncovering Symbionts Across the Psyllid Tree of Life and the Discovery of a New Liberibacter Species, "Candidatus" Liberibacter capsica.

Sap-feeding insects in the order Hemiptera associate with obligate endosymbionts that are required for survival and facultative endosymbionts that can potentially modify resistance to stress, enemies, development, and reproduction. In the superfamily Psylloidea, the jumping plant lice (psyllids), less is known about the diversity and prevalence of their endosymbionts compared to other sap-feeding pests such as aphids (Aphididae). To address this knowledge gap, using 16S rRNA sequencing we identify symbionts across divergent psyllid host lineages from around the world. Taking advantage of a new comprehensive phylogenomic analyses of Psylloidea, we included psyllid samples from 44 species of 35 genera of five families, collected from 11 international locations for this study. Across psyllid lineages, a total of 91 OTUs were recovered, predominantly of the Enterobacteriaceae (68%). The diversity of endosymbionts harbored by each psyllid species was low with an average of approximately 3 OTUs. Two clades of endosymbionts (clade 1 and 2), belonging to Enterobacteriaceae, were identified that appear to be long term endosymbionts of the psyllid families Triozidae and Psyllidae, respectively. We also conducted high throughput metagenomic sequencing on three Ca. Liberibacter infected psyllid species (Russelliana capsici, Trichochermes walkeri, and Macrohomotoma gladiata), initially identified from 16S rRNA sequencing, to obtain more genomic information on these putative Liberibacter plant pathogens. The phylogenomic analyses from these data identified a new Ca. Liberibacter species, Candidatus Liberibacter capsica, that is a potential pathogen of solanaceous crops. This new species shares a distant ancestor with Ca. L. americanus, which occurs in the same range as R. capsici in South America. We also detected the first association between a psyllid specializing on woody hosts and the Liberibacter species Ca. L. psyllaurous, which is a globally distributed pathogen of herbaceous crop hosts in the Solanaceae. Finally, we detected a potential association between a psyllid pest of figs (M. gladiata) and a Ca. Liberibacter related to Ca. L. asiaticus, which causes severe disease in citrus. Our findings reveal a wider diversity of associations between facultative symbionts and psyllids than previously reported and suggest numerous avenues for future work to clarify novel associations of ecological, evolutionary, and pathogenic interest.

Continent-Scale Sampling Reveals Fine-Scale Turnover in a Beneficial Bug Symbiont.

Many members of animal-associated microbial communities, including the gut flora, are acquired from their host's environment. While many of these communities are species rich, some true bugs (Hemiptera) in the superfamilies Lygaeoidea and Coreidae allow only ingested Burkholderia to colonize and reproduce in a large portion of the midgut. We studied the spatial structuring of Burkholderia associated with a widespread omnivorous bug genus, Jalysus (Berytidae). We sampled Wickham's stilt bug, Jalysus wickhami, across the United States and performed limited sampling of its sister species, the spined stilt bug Jalysus spinosus. We asked: (1) What Burkholderia strains are hosted by Jalysus at different locations? (2) Does host insect species, host plant species, or location influence the strain these insects acquire? (3) How does Burkholderia affect the development and reproductive fitness of J. wickhami? We found: (1) Sixty-one Burkholderia strains were present across a sample of 352 individuals, but one strain dominated, accounting for almost half of all symbiont reads. Most strains were closely related to other hemipteran Burkholderia symbionts. (2) Many individuals hosted more than one strain of Burkholderia. (3) J. wickhami and J. spinosus did not differ in the strains they hosted. (4) Insects that fed on different plant species tended to host different Burkholderia, but this accounted for only 4% of the variation in strains hosted. In contrast, the location at which an insect was collected explained 27% of the variation in symbiont strains. (5) Burkholderia confers important fitness benefits to J. wickhami. In laboratory experiments, aposymbiotic (Burkholderia-free) insects developed more slowly and laid fewer eggs than symbiotic (Burkholderia-colonized) insects. (6) In the lab, nymphs sometimes acquired Burkholderia via indirect exposure to adults, indicating that horizontal symbiont transmission can occur via adult insect-mediated enrichment of Burkholderia in the local environment - a phenomenon not previously reported in bug-Burkholderia relationships. Taken together, the results suggest that for these bugs, critical nutritional requirements are outsourced to a highly diverse and spatially structured collection of Burkholderia strains acquired from the environment and, occasionally, from conspecific adults.

The Effects of Different Diets and Transgenerational Stress on Acyrthosiphon pisum Development.

Despite the fact that sap-feeding hemipterans are major agricultural pests, little is known about the pea aphid's (Acyrthosiphon pisum) nymphal development, compared to other insect models. Given our limited understanding of A. pisum nymphal development and variability in the naming/timing of its developmental events between different environmental conditions and studies, here, we address developmental knowledge gaps by elucidating how diet impacts A. pisum nymphal development for the LSR1 strain when it develops on its universal host plant (Vicia faba), isolated leaves, and artificial diet. Moreover, we test how plant age and transgenerational stressors, such as overcrowding and low plant vigor, can affect nymphal development. We also validate a morphological method to quickly confirm the life stage of each nymphal instar within a mixed population. Overall, we found extremely high variation in the timing of developmental events and a significant delay in nymphal (~5-25-h/instar) and pre-reproductive adult (~40-h) development when reared on isolated leaves and artificial diets, compared to intact host plants. Also, delays in development were observed when reared on older host plants (~9-17-h/event, post 2nd instar) or when previous generations were exposed to overcrowding on host plants (~20-h delay in nymph laying) compared to controls.