Endosymbionts Of Insects Research Articles

Genetic Modification of Sodalis Species by DNA Transduction.

ABSTRACTBacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing, and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius. While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram-negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms.IMPORTANCE A large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherent nature of these associations, insect endosymbionts cannot be usually isolated in pure culture or genetically manipulated. Here we use a broad-host-range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad-host-range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem

Candidatus Liberibacter asiaticus (CLas), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2′-deoxy-2′-fluoro-d-arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a CLas-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a CLas essential gene. CLas relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a CLas-specific FANA ASO into infected Citrus trees significantly reduced CLas titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.

Skipping the Insect Vector: Plant Stolon Transmission of the Phytopathogen 'Ca. Phlomobacter fragariae' from the Arsenophonus Clade of Insect Endosymbionts.

Simple SummaryNumerous plant sap-feeding insects are vectors of plant-pathogenic bacteria that cause devastating crop diseases. Some of these bacteria had initially been insect endosymbionts that eventually evolved the capacity to survive in plants after being frequently transmitted to plants by their insect hosts during feeding. An example for this evolutionary transition is the bacterial symbiont ‘Candidatus Phlomobacter fragariae’ (hereafter Phlomobacter) of the planthopper Cixius wagneri. Upon transmission to strawberry plants by its insect vector, the bacterium accumulates in the plant phloem and causes Strawberry Marginal Chlorosis disease. Using quantitative PCR and transmission electron microscopy, we demonstrate an additional plant-to-plant transmission route: Phlomobacter can be transmitted from an infected plant to daughter plants through stolons, a specific type of stem from which daughter plants can develop. Our results show that Phlomobacter was abundant in stolons and was efficiently transmitted to daughter plants, which developed disease symptoms. Hence, Phlomobacter is not only able to survive in plants, but can even be transmitted to new plant generations, independently from its ancestral insect host.The genus Arsenophonus represents one of the most widespread clades of insect endosymbionts, including reproductive manipulators and bacteriocyte-associated primary endosymbionts. Two strains belonging to the Arsenophonus clade have been identified as insect-vectored plant pathogens of strawberry and sugar beet. The bacteria accumulate in the phloem of infected plants, ultimately causing leaf yellows and necrosis. These symbionts therefore represent excellent model systems to investigate the evolutionary transition from a purely insect-associated endosymbiont towards an insect-vectored phytopathogen. Using quantitative PCR and transmission electron microscopy, we demonstrate that ‘Candidatus Phlomobacter fragariae’, bacterial symbiont of the planthopper Cixius wagneri and the causative agent of Strawberry Marginal Chlorosis disease, can be transmitted from an infected strawberry plant to multiple daughter plants through stolons. Stolons are horizontally growing stems enabling the nutrient provisioning of daughter plants during their early growth phase. Our results show that Phlomobacter was abundant in the phloem sieve elements of stolons and was efficiently transmitted to daughter plants, which rapidly developed disease symptoms. From an evolutionary perspective, Phlomobacter is, therefore, not only able to survive within the plant after transmission by the insect vector, but can even be transmitted to new plant generations, independently from its ancestral insect host.

Mutational Pressure Drives Differential Genome Conservation in Two Bacterial Endosymbionts of Sap-Feeding Insects

Compared with free-living bacteria, endosymbionts of sap-feeding insects have tiny and rapidly evolving genomes. Increased genetic drift, high mutation rates, and relaxed selection associated with host control of key cellular functions all likely contribute to genome decay. Phylogenetic comparisons have revealed massive variation in endosymbiont evolutionary rate, but such methods make it difficult to partition the effects of mutation versus selection. For example, the ancestor of Auchenorrhynchan insects contained two obligate endosymbionts, Sulcia and a betaproteobacterium (BetaSymb; called Nasuia in leafhoppers) that exhibit divergent rates of sequence evolution and different propensities for loss and replacement in the ensuing ∼300 Ma. Here, we use the auchenorrhynchan leafhopper Macrosteles sp. nr. severini, which retains both of the ancestral endosymbionts, to test the hypothesis that differences in evolutionary rate are driven by differential mutagenesis. We used a high-fidelity technique known as duplex sequencing to measure and compare low-frequency variants in each endosymbiont. Our direct detection of de novode novo mutations reveals that the rapidly evolving endosymbiont (Nasuia) has a much higher frequency of single-nucleotide variants than the more stable endosymbiont (Sulcia) and a mutation spectrum that is potentially even more AT-biased than implied by the 83.1% AT content of its genome. We show that indels are common in both endosymbionts but differ substantially in length and distribution around repetitive regions. Our results suggest that differences in long-term rates of sequence evolution in Sulcia versus BetaSymb, and perhaps the contrasting degrees of stability of their relationships with the host, are driven by differences in mutagenesis.

The Puzzle of Metabolite Exchange and Identification of Putative Octotrico Peptide Repeat Expression Regulators in the Nascent Photosynthetic Organelles of Paulinella chromatophora.

The endosymbiotic acquisition of mitochondria and plastids more than one billion years ago was central for the evolution of eukaryotic life. However, owing to their ancient origin, these organelles provide only limited insights into the initial stages of organellogenesis. The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles—termed chromatophores—that evolved from a cyanobacterium ∼100 million years ago, independently from plastids in plants and algae. Despite the more recent origin of the chromatophore, it shows tight integration into the host cell. It imports hundreds of nucleus-encoded proteins, and diverse metabolites are continuously exchanged across the two chromatophore envelope membranes. However, the limited set of chromatophore-encoded solute transporters appears insufficient for supporting metabolic connectivity or protein import. Furthermore, chromatophore-localized biosynthetic pathways as well as multiprotein complexes include proteins of dual genetic origin, suggesting that mechanisms evolved that coordinate gene expression levels between chromatophore and nucleus. These findings imply that similar to the situation in mitochondria and plastids, also in P. chromatophora nuclear factors evolved that control metabolite exchange and gene expression in the chromatophore. Here we show by mass spectrometric analyses of enriched insoluble protein fractions that, unexpectedly, nucleus-encoded transporters are not inserted into the chromatophore inner envelope membrane. Thus, despite the apparent maintenance of its barrier function, canonical metabolite transporters are missing in this membrane. Instead we identified several expanded groups of short chromatophore-targeted orphan proteins. Members of one of these groups are characterized by a single transmembrane helix, and others contain amphipathic helices. We hypothesize that these proteins are involved in modulating membrane permeability. Thus, the mechanism generating metabolic connectivity of the chromatophore fundamentally differs from the one for mitochondria and plastids, but likely rather resembles the poorly understood mechanism in various bacterial endosymbionts in plants and insects. Furthermore, our mass spectrometric analysis revealed an expanded family of chromatophore-targeted helical repeat proteins. These proteins show similar domain architectures as known organelle-targeted expression regulators of the octotrico peptide repeat type in algae and plants. Apparently these chromatophore-targeted proteins evolved convergently to plastid-targeted expression regulators and are likely involved in gene expression control in the chromatophore.

A Role for Maternal Factors in Suppressing Cytoplasmic Incompatibility.

Wolbachia are maternally transmitted bacterial endosymbionts, carried by approximately half of all insect species. Wolbachia prevalence in nature stems from manipulation of host reproduction to favor the success of infected females. The best known reproductive modification induced by Wolbachia is referred to as sperm-egg Cytoplasmic Incompatibility (CI). In CI, the sperm of Wolbachia-infected males cause embryonic lethality, attributed to paternal chromatin segregation defects during early mitotic divisions. Remarkably, the embryos of Wolbachia-infected females “rescue” CI lethality, yielding egg hatch rates equivalent to uninfected female crosses. Several models have been discussed as the basis for Rescue, and functional evidence indicates a major contribution by Wolbachia CI factors. A role for host contributions to Rescue remains largely untested. In this study, we used a chemical feeding approach to test for CI suppression capabilities by Drosophila simulans. We found that uninfected females exhibited significantly higher CI egg hatch rates in response to seven chemical treatments that affect DNA integrity, cell cycle control, and protein turnover. Three of these treatments suppressed CI induced by endogenous wRi Wolbachia, as well as an ectopic wMel Wolbachia infection. The results implicate DNA integrity as a focal aspect of CI suppression for different Wolbachia strains. The framework presented here, applied to diverse CI models, will further enrich our understanding of host reproductive manipulation by insect endosymbionts.

Insect–Plant Interactions: A Multilayered Relationship

AbstractDuring herbivory, insects recognize their host plant based on specific chemical cues, whereas the plants induce various chemical and morphological defense responses to resist this attack. However, the seemingly bidirectional insect–plant interaction involves various confounding aspects that influence the performance and fitness of the two participants. These interactions are often mediated by associated microbiota, competitors, predators, and parasitoids that interact in either obligate or facultative manner. Insect endosymbionts play a crucial role in the perception, nutrition, metabolism as well as reproduction of their host, which together determine its survival and fitness on the plant. Endosymbionts also help their host to overcome plant defenses by detoxifying plant metabolites. On the contrary, plant-associated microbes contribute in induced systemic plant resistance by enhancing chemical and morphological defense. These interactions determine the association of insect and plant, not only with the high trophic levels but also with the ecosystem as a whole. Thus, insect–plant interaction is a multilayered relationship extending to various micro- and macro-organisms associated either temporally or spatially. All these relationships may be considered to obtain a wholesome perspective of the natural environment.

Food Resource Sharing of Alder Leaf Beetle Specialists (Coleoptera: Chrysomelidae) as Potential Insect-Plant Interface for Horizontal Transmission of Endosymbionts.

Recent studies suggest that endosymbionts of herbivore insects can be horizontally transferred to other herbivores feeding on the same host plants, whereby the plant acts as an intermediate stage in the chain of transmission. If this mechanism operates, it is also expected that insect communities sharing the same host plant will have higher chances to share their endosymbionts. In this study, we use a high-throughput 16S rRNA metabarcoding approach to investigate the presence, diversity, and potential sharing of endosymbionts in several species of leaf beetles (Coleoptera: Chrysomelidae) of a local community specialized on an alder diet in North America. Rickettsia and Wolbachia were predominant in the sample, with strong evidence for each species having their own dominant infection, of either or both types of bacteria. However, all species shared a much lower proportion of a particular Wolbachia type, compatible with the same strain dominant in one of the species of leaf beetles. Crucially, the same 16S rRNA haplotype of Wolbachia was found on alder leaf extracts. The combined evidence and the absence of this strain in a syntopic species of leaf beetle feeding on a different host plant support the hypothesis that at least the initial stages of the mechanism that would allow horizontal transmission of endosymbionts across species feeding on the same plant is possible. The accessibility and characteristics of endosymbiont associations of this system make it suitable for deeper analyses of their diversity and transmission in natural conditions.

Modelling lifestyle changes in Insect endosymbionts, from insect mutualist to plant pathogen

Several insect endosymbionts have evolved to become plant pathogens, but the causes of this transition are currently unknown. In this paper, we use adaptive dynamics to develop hypotheses to explain why an insect endosymbiont would evolve to become a plant pathogen. We develop a model of facultative insect endosymbionts, capable of both vertical transmission within the insect population and horizontal transmission between insect and plant populations. We assume that an evolutionary trade-off between vertical and horizontal transmission exists. The transmission method of an endosymbiont is correlated with the nature of the symbiotic relationship between host and symbiont. We assume that vertical transmission represents an insect endosymbiont lifestyle and horizontal transmission represents a plant pathogen lifestyle. Our results suggest that temperature increases, increased agricultural intensification, disease dynamics within the plant host, insect mating system and change in the host plant of the insect may influence an evolutionary transition from an insect endosymbiont to a plant pathogen.

Increased Mutation Rate Is Linked to Genome Reduction in Prokaryotes

The evolutionary processes that drive variation in genome size across the tree of life remain unresolved. Effective population size (Ne) is thought to play an important role in shaping genome size [1-3]-a key example being the reduced genomes of insect endosymbionts, which undergo population bottlenecks during transmission [4]. However, the existence of reduced genomes in marine and terrestrial prokaryote species with large Ne indicate that genome reduction is influenced by multiple processes [3]. One candidate process is enhanced mutation rate, which can increase adaptive capacity but can also promote gene loss. To investigate evolutionary forces associated with prokaryotic genome reduction, we performed molecular evolutionary and phylogenomic analyses of nine lineages from five bacterial and archaeal phyla. We found that gene-loss rate strongly correlated with synonymous substitution rate (a proxy for mutation rate) in seven of the nine lineages. However, gene-loss rate showed weak or no correlation with the ratio of nonsynonymous/synonymous substitution rate (dN/dS). These results indicate that genome reduction is largely associated with increased mutation rate, while the association between gene loss and changes in Ne is less well defined. Lineages with relatively high dS and dN, as well as smaller genomes, lacked multiple DNA repair genes, providing a proximate cause for increased mutation rates. Our findings suggest that similar mechanisms drive genome reduction in both intracellular and free-living prokaryotes, with implications for developing a comprehensive theory of prokaryote genome size evolution.

P finder: genomic and metagenomic annotation of RNase P RNA gene (rnpB)

BackgroundThe rnpB gene encodes for an essential catalytic RNA (RNase P). Like other essential RNAs, RNase P’s sequence is highly variable. However, unlike other essential RNAs (i.e. tRNA, 16 S, 6 S,...) its structure is also variable with at least 5 distinct structure types observed in prokaryotes. This structural variability makes it labor intensive and challenging to create and maintain covariance models for the detection of RNase P RNA in genomic and metagenomic sequences. The lack of a facile and rapid annotation algorithm has led to the rnpB gene being the most grossly under annotated essential gene in completed prokaryotic genomes with only a 24% annotation rate. Here we describe the coupling of the largest RNase P RNA database with the local alignment scoring algorithm to create the most sensitive and rapid prokaryote rnpB gene identification and annotation algorithm to date.ResultsOf the 2772 completed microbial genomes downloaded from GenBank only 665 genomes had an annotated rnpB gene. We applied P Finder to these genomes and were able to identify 2733 or nearly 99% of the 2772 microbial genomes examined. From these results four new rnpB genes that encode the minimal T-type P RNase P RNAs were identified computationally for the first time. In addition, only the second C-type RNase P RNA was identified in Sphaerobacter thermophilus. Of special note, no RNase P RNAs were detected in several obligate endosymbionts of sap sucking insects suggesting a novel evolutionary adaptation.ConclusionsThe coupling of the largest RNase P RNA database and associated structure class identification with the P Finder algorithm is both sensitive and rapid, yielding high quality results to aid researchers annotating either genomic or metagenomic data. It is the only algorithm to date that can identify challenging RNAse P classes such as C-type and the minimal T-type RNase P RNAs. P Finder is written in C# and has a user-friendly GUI that can run on multiple 64-bit windows platforms (Windows Vista/7/8/10). P Finder is free available for download at https://github.com/JChristopherEllis/P-Finder as well as a small sample RNase P RNA file for testing.

Effect of host fruit, temperature and Wolbachia infection on survival and development of Ceratitis capitata immature stages.

The Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), holds an impressive record of successful invasions promoted by the growth and development of international fruit trade. Hence, survival of immatures within infested fruit that are subjected to various conditions during transportation seems to be a crucial feature that promotes invasion success. Wolbachia pipientis is a common endosymbiont of insects and other arthropods generating several biological effects on its hosts. Existing information report the influence of Wolbachia on the fitness traits of insect host species, including the Mediterranean fruit fly. However, little is known regarding effects of Wolbachia infection on immature development in different host fruits and temperatures. This study was conducted to determine the development and survival of immature stages of four different Mediterranean fruit fly populations, either infected or uninfected with Wolbachia, in two hosts (apples, bitter oranges) under three constant temperatures (15, 25 and 30°C), constant relative humidity (45-55 ± 5%), and a photoperiod of 14L:10D. Our findings demonstrate both differential response of two fruit fly lines to Wolbachia infection and differential effects of the two Wolbachia strains on the same Mediterranean fruit fly line. Larva-to-pupa and larva-to-adult survival followed similar patterns and varied a lot among the four medfly populations, the two host fruits and the different temperatures. Pupation rates and larval developmental time were higher for larvae implanted in apples compared to bitter oranges. The survival rates of wildish medflies were higher than those of the laboratory adapted ones, particularly in bitter oranges. The Wolbachia infected medflies, expressed lower survival rates and higher developmental times, especially the wCer4 infected line. High temperatures constrained immature development and were lethal for the Wolbachia infected wCer4 medfly line. Lower temperatures inferred longer developmental times to immature stages of all medfly populations tested, in both host fruits. Implications on the ecology and survival of the fly in nature are discussed.

Increased Mutation Rate is Linked to Genome Reduction in Prokaryotes

The evolutionary processes that drive variation in genome size across the tree of life remain unresolved. Effective population size (Ne) is thought to play an important role in shaping genome size, a key example being the reduced genomes of insect endosymbionts, which undergo population bottlenecks during transmission. However, the existence of reduced genomes in marine and terrestrial prokaryote species with large Ne demand an alternative explanation. One such alternative is enhanced mutation rate, which might increase an organism’s ability to adapt to novel environments, but might also promote gene loss. We employed a novel approach involving molecular evolutionary and phylogenomic analyses of nine lineages of prokaryotes that display various levels of genome reduction: Blattabacterium and Buchnera insect endosymbionts, marine Prochlorococcus and Synechococcus, the thermophilic archaean Thermococcus, and five lineages of free-living bacteria belonging to the Proteobacteria, Actinobacteria, and Bacteroidetes. We found that rates of gene loss strongly correlate with mutation rates in all of these groups, with the exception of two lineages of free-living bacteria, and show weak or no correlation with the ratio of nonsynonymous/synonymous substitution rates (dN/dS). This indicates that genome reduction is largely associated with increased mutation rate, while the association between gene loss and changes in Ne is less well defined. Lineages with relatively high dS and dN, as well as smaller genomes, lacked multiple DNA repair genes, providing a proximate cause for increased mutation rates. Our findings suggest that similar underlying mechanisms drive genome reduction in both intracellular and free-living prokaryotes, with implications for developing a comprehensive theory of prokaryote genome size evolution.

Evolution from Free-Living Bacteria to Endosymbionts of Insects: Genomic Changes and the Importance of the Chaperonin GroEL.

Major insect lineages have independently acquired bacterial species, mainly from Gamma-proteobacteria and Bacteroidetes class, which could be nutritional mutualistic factories, facultative mutualists that protect against biotic and abiotic stresses, or reproductive manipulators (which alter the fertility of the host species in its benefit). Some of them are enclosed in bacteriocytes to assure their maternal transmission over generations. All of them show an increased level of genetic drift due to the small population size and the continuous population bottlenecking at each generation, processes that have shaped their genome, proteome, and morphology. Depending on the nature of the relationship, the degree of genome plasticity varies, i.e., obligate nutritional mutualistic symbionts have extremely small genomes lacking mobile elements, bacteriophages, or recombination machinery. Under these conditions, endosymbionts face high mutational pressures that may drive to extinction or symbiont replacement. How do then they survive for such long evolutionary time, and why do they show a genome stasis? In this chapter, after a brief introduction to the problem, we will focus on the genome changes suffered by these endosymbionts, and on the mutational robustness mechanisms, including the moonlighting chaperone GroEL that could explain their long prevalence from an evolutionary perspective by comparing them with free-living bacteria.

Genetic variability on worldwide populations of the scale insect Pulvinariella mesembryanthemi

The South African scale insect Pulvinariella mesembryanthemi was introduced worldwide in several coastal areas with Mediterranean climate, probably through infested plants of Carpobrotus sp. Its high host specificity and its capacity to produce severe damages in the invasive Carpobrotus sp. plants makes this insect a potential biocontrol agent. To test the efficiency and host range of insects used for biocontrol, population genetic studies can help to unravel cryptic complexes and intraspecific diversity. In this study we performed a genetic analysis including native and exotic populations of P. mesembryanthemi, through Sanger sequencing of mitochondrial (cytochrome c oxidase I, COI) and ribosomal (D2–D3 expansion segments of the large subunit ribosomal RNA gene 28S) gene fragments. Accidentally, an endosymbiont was sequenced with one of the pair of primers used. The exotic populations of the insect did not show any variability among populations for both studied genes, which suggest a common origin of all studied introduced populations. Contrastingly, native populations showed high variability and seemed to be a cryptic species complex. Moreover, the Gauteng populations (from NE South Africa) were phylogenetically the closest to the exotic ones, suggesting that the exotic populations could be original from somewhere near this area. An endosymbiont of P. mesembryanthemi was detected, and the sequenced coxA gene was similar to that of the Rickettsiaceae family from the α-Proteobacteria, and close to other insect endosymbionts. To the best of our knowledge, this was the first mention of this endosymbiont in P. mesembryanthemi, although α-Proteobacteria endosymbionts have been reported for other sap-sucking insects.

Quantitative methods for assessing local and bodywide contributions to Wolbachia titer in maternal germline cells of Drosophila

BackgroundLittle is known about how bacterial endosymbionts colonize host tissues. Because many insect endosymbionts are maternally transmitted, egg colonization is critical for endosymbiont success. Wolbachia bacteria, carried by approximately half of all insect species, provide an excellent model for characterizing endosymbiont infection dynamics. To date, technical limitations have precluded stepwise analysis of germline colonization by Wolbachia. It is not clear to what extent titer-altering effects are primarily mediated by growth rates of Wolbachia within cell lineages or migration of Wolbachia between cells.ResultsThe objective of this work is to inform mechanisms of germline colonization through use of optimized methodology. The approaches are framed in terms of nutritional impacts on Wolbachia. Yeast-rich diets in particular have been shown to suppress Wolbachia titer in the Drosophila melanogaster germline. To determine the extent of Wolbachia sensitivity to diet, we optimized 3-dimensional, multi-stage quantification of Wolbachia titer in maternal germline cells. Technical and statistical validation confirmed the identity of Wolbachia in vivo, the reproducibility of Wolbachia quantification and the statistical power to detect these effects. The data from adult feeding experiments demonstrated that germline Wolbachia titer is distinctly sensitive to yeast-rich host diets in late oogenesis. To investigate the physiological basis for these nutritional impacts, we optimized methodology for absolute Wolbachia quantification by real-time qPCR. We found that yeast-rich diets exerted no significant effect on bodywide Wolbachia titer, although ovarian titers were significantly reduced. This suggests that host diets affects Wolbachia distribution between the soma and late stage germline cells. Notably, relative qPCR methods distorted apparent wsp abundance, due to altered host DNA copy number in yeast-rich conditions. This highlights the importance of absolute quantification data for testing mechanistic hypotheses.ConclusionsWe demonstrate that absolute quantification of Wolbachia, using well-controlled cytological and qPCR-based methods, creates new opportunities to determine how bacterial abundance within the germline relates to bacterial distribution within the body. This methodology can be applied to further test germline infection dynamics in response to chemical treatments, genetic conditions, new host/endosymbiont combinations, or potentially adapted to analyze other cell and tissue types.

Liberibacter crescens Is a Cultured Surrogate for Functional Genomics of Uncultured Pathogenic 'Candidatus Liberibacter' spp. and Is Naturally Competent for Transformation.

'Candidatus Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. 'Ca. L. asiaticus', causal agent of Huanglongbing or citrus "greening," and 'Ca. L. solanacearum', causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Cicada Endosymbionts Have tRNAs That Are Correctly Processed Despite Having Genomes That Do Not Encode All of the tRNA Processing Machinery.

Gene loss and genome reduction are defining characteristics of endosymbiotic bacteria. The most highly reduced endosymbiont genomes have lost numerous essential genes related to core cellular processes such as replication, transcription, and translation. Computational gene predictions performed for the genomes of the two bacterial symbionts of the cicada Diceroprocta semicincta, "Candidatus Hodgkinia cicadicola" (Alphaproteobacteria) and "Ca Sulcia muelleri" (Bacteroidetes), have found only 26 and 16 tRNA genes and 15 and 10 aminoacyl tRNA synthetase genes, respectively. Furthermore, the original "Ca Hodgkinia cicadicola" genome annotation was missing several essential genes involved in tRNA processing, such as those encoding RNase P and CCA tRNA nucleotidyltransferase as well as several RNA editing enzymes required for tRNA maturation. How these cicada endosymbionts perform basic translation-related processes remains unknown. Here, by sequencing eukaryotic mRNAs and total small RNAs, we show that the limited tRNA set predicted by computational annotation of "Ca Sulcia muelleri" and "Ca Hodgkinia cicadicola" is likely correct. Furthermore, we show that despite the absence of genes encoding tRNA processing activities in the symbiont genomes, symbiont tRNAs have correctly processed 5' and 3' ends and seem to undergo nucleotide modification. Surprisingly, we found that most "Ca Hodgkinia cicadicola" and "Ca Sulcia muelleri" tRNAs exist as tRNA halves. We hypothesize that "Ca Sulcia muelleri" and "Ca Hodgkinia cicadicola" tRNAs function in bacterial translation but require host-encoded enzymes to do so.IMPORTANCE The smallest bacterial genomes, in the range of about 0.1 to 0.5 million base pairs, are commonly found in the nutritional endosymbionts of insects. These tiny genomes are missing genes that encode proteins and RNAs required for the translation of mRNAs, one of the most highly conserved and important cellular processes. In this study, we found that the bacterial endosymbionts of cicadas have genomes which encode incomplete tRNA sets and lack genes required for tRNA processing. Nevertheless, we found that endosymbiont tRNAs are correctly processed at their 5' and 3' ends and, surprisingly, that mostly exist as tRNA halves. We hypothesize that the cicada host must supply its symbionts with these missing tRNA processing activities.

Illumina-based 16S metagenomic analysis of the indigenous gut microbiota of cavity-nesting bee Megachile centuncularis: a comparison with the cavity-nesting wasp Ancistrocerus antilope

Insect guts are colonized by different species of microorganisms, most of which are benign or beneficial to their host. However, little is known about how the microbial gut communities vary among different host species. In this study, using 16S metagenomic sequencing, we compared the indigenous gut microbiota in adult of two solitary cavity-nesting Hymenoptera, a bee (Megachile centuncularis) and a wasp (Ancistrocerus antilope). The richness of operational taxonomic units (OTUs) was estimated in the guts of freshly eclosed imagoes, which had had no contact with the environment. We obtained 123,237 total reads from M. centuncularis and 125,942 from A. antilope. Three phyla, Proteobacteria, Actinobacteria, and Firmicutes, were dominant. Nine OTUs in M. centuncularis and five OTUs in A. antilope represented ≥1% of all reads. Wolbachia, a well-known insect endosymbiont, dominated in both species, representing 27% of reads in M. centuncularis and 48% in A. antilope. The most abundant in the gut of M. centuncularis was Arthrobacter (38%), while the dominants in the gut of A. antilope were Microbacterium (18%) and Erwinia (14%). These differences in microbiota richness and composition between bee and wasp are likely to be caused by their disparate trophic specialization.

Chaperonin overproduction and metabolic erosion caused by mutation accumulation in Escherichia coli.

Bacterial cells adapting to a constant environment tend to accumulate mutations in portions of their genome that are not maintained by selection. This process has been observed in bacteria evolving under strong genetic drift, and especially in bacterial endosymbionts of insects. Here, we study this process in hypermutable Escherichia coli populations evolved through 250 single-cell bottlenecks on solid rich medium in a mutation accumulation experiment that emulates the evolution of bacterial endosymbionts. Using phenotype microarrays monitoring metabolic activity in 95 environments distinguished by their carbon sources, we observe how mutation accumulation has decreased the ability of cells to metabolize most carbon sources. We study if the chaperonin GroEL, which is naturally overproduced in bacterial endosymbionts, can ameliorate the process of metabolic erosion, because of its known ability to buffer destabilizing mutations in metabolic enzymes. Our results indicate that GroEL can slow down the negative phenotypic consequences of genome decay in some environments.