text:
Despite extensive study, little is known about the origins of the mutualistic bacterial endosymbionts that inhabit approximately 10% of the world's insects. In this study, we characterized a novel opportunistic human pathogen, designated “strain HS,” and found that it is a close relative of the insect endosymbiont Sodalis glossinidius. Our results indicate that ancestral relatives of strain HS have served as progenitors for the independent descent of Sodalis-allied endosymbionts found in several insect hosts. Comparative analyses indicate that the gene inventories of the insect endosymbionts were independently derived from a common ancestral template through a combination of irreversible degenerative changes. Our results provide compelling support for the notion that mutualists evolve from pathogenic progenitors. They also elucidate the role of degenerative evolutionary processes in shaping the gene inventories of symbiotic bacteria at a very early stage in these mutualistic associations.
Many insects harbor symbiotic bacteria that perform diverse functions within their hosts. However, the origins of these associations have been difficult to define. In this study we isolate a novel bacterium from a human infection and show that this bacterium is a close relative of the Sodalis-allied clade of insect symbionts. Comparative genomic analyses reveal that this organism maintains many genes that have been inactivated and lost independently in derived insect symbionts as a result of rapid genome degeneration. Our work also shows that recently derived Sodalis-allied symbionts maintain a significant population of “cryptic” pseudogenes that are assumed to have no beneficial function in the symbiosis but have not yet accumulated mutations that disrupt their translation. Taken together, our results show that genome degeneration proceeds rapidly following the onset of symbiosis. They also highlight the potential for diverse insect taxa to acquire closely related insect symbionts as a consequence of vectoring bacterial pathogens to plants and animals.
Citation: Clayton AL, Oakeson KF, Gutin M, Pontes A, Dunn DM, von Niederhausern AC, et al. (2012) A Novel Human-Infection-Derived Bacterium Provides Insights into the Evolutionary Origins of Mutualistic Insect–Bacterial Symbioses. PLoS Genet 8(11): e1002990. https://doi.org/10.1371/journal.pgen.1002990
Editor: David S. Guttman, University of Toronto, Canada
Received: June 6, 2012; Accepted: August 10, 2012; Published: November 15, 2012
Copyright: © 2012 Clayton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by National Science Foundation (www.nsf.gov) grant EF-0523818 and National Institutes of Health (www.nih.gov) grant 1R01AI095736 (to CD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Obligate host-associated bacteria often have reduced genome sizes in comparison to related bacteria that are known to engage in free-living or opportunistic lifestyles . This is exemplified by inspection of the genome sequences of mutualistic, maternally transmitted, bacterial endosymbionts of insects, many of which have been maintained in their insect hosts for long periods of evolutionary time . Often these obligate endosymbionts maintain only a small fraction of the gene inventory that is found in related free-living counterparts –, indicating that the obligate host-associated lifestyle facilitates genome degeneration and size reduction. At a simple level, the process of genome degeneration in obligate endosymbionts can be viewed as a streamlining of the gene inventory to yield a minimal gene set that is compatible with the symbiotic lifestyle. Genes that have no adaptive benefit are inactivated and deleted as a consequence of mutations that accumulate under relaxed selection at an increased rate in the asexual symbiotic lifestyle as a result of frequent population bottlenecks occurring during symbiont transmission .
Although we now have a detailed understanding of the mechanisms and evolutionary trajectory of genome degeneration in ancient obligate insect symbionts, the fundamental question of how these mutualistic associations arise remains to be answered. Studies focusing on insect-bacterial symbioses of recent origin show that closely related bacterial endosymbionts are often found in distantly related insect hosts , . This could be explained by the interspecific transmission of symbionts, mediated by parasitic wasps and mites that facilitate the transfer of symbionts between distantly related hosts , . Horizontal symbiont transmission could also be mediated by intraspecific mating, as demonstrated in the pea aphid . Another possibility is that symbionts could be acquired de novo from an environmental source.
Symbiont acquisition, at least initially, requires the symbiont to overcome or evade the insect immune response. Given that many insects are known to possess a potent immune system that repels invading microorganisms , it has been assumed that mutualistic symbionts arise from pathogenic progenitors that have evolved specialized molecular mechanisms to facilitate evasion of the immune response and invasion of insect tissues . In support of this notion, it has been shown that the genomes of recently acquired mutualistic insect endosymbionts maintain genes similar to virulence factors and toxins that are found in related plant and animal pathogens –.
In the current study we describe the discovery of a novel human-infective bacterium, designated “strain HS”, isolated from a patient who sustained a hand wound following impalement with a tree branch. Phylogenetic analyses show that strain HS is a member of the Sodalis-allied clade of insect endosymbionts. Comparative analyses of the genome sequences of strain HS and related insect symbionts suggest that close relatives of strain HS gave rise to mutualistic associates in a wide range of insect hosts.
Isolation and Culture of Strain HS
A 71-year-old male presented to his primary care physician for a routine physical examination three days after sustaining a puncture wound to the right hand. The patient fell and was impaled between the thumb and forefinger by a ∼1 cm diameter branch while removing branches from a dead crab apple tree. Upon presentation the patient denied fever or other constitutional symptoms and had a mild peripheral blood monocytosis (11.8%; reference range = 1.7–9.3%). A palpable cyst was noted in the right hand at the sight of impalement. Warm compresses were applied and cephalexin was prescribed at a dose of 500 mg four times daily for 10 days. The patient was evaluated again three days later due to continuing wound pain. The cyst was drained by aspiration and serosanguineous fluid was submitted for Gram stain and bacterial culture. The Gram stain showed scattered white blood cells, but no bacteria were visualized. A follow-up visit seven days later revealed the presence of an abscess, although the patient was afebrile and without local lymphadenopathy. The abscess was again drained by aspiration and the patient was advised to consult an orthopedic surgeon for evaluation. Subsequent surgery, approximately six weeks later, removed several foreign bodies from the wound and the patient recovered on a second course of cephalexin without incident. Two days after the original cyst aspiration, small numbers of gram negative rods resembling enteric bacteria were isolated on MacConkey agar at 35°C and 5% CO2. Colonies were wet, mucoid, variable in size, and slowly fermented lactose. The isolate could not be definitively identified by a manual phenotypic method (RapID ONE, Remel, Lenexa KS) and was misidentified as Escherichia coli at 98% confidence by an automated system (Phoenix, BD Diagnostics, Sparks, MD).
Phylogenetic Analysis of Strain HS
Phylogenetic analysis of 16S rRNA placed strain HS in a well-supported clade comprising Sodalis-allied insect endosymbionts sharing >97% sequence identity in their 16S rRNA sequences (Figure 1), which is a commonly used threshold for species-level conservation among bacteria . Aside from strain HS, the closest non-insect associated relative of this clade is Biostraticola tofi, which was isolated from a biofilm on a tufa deposit in a hard water rivulet . However, B. tofi shares only 96.5% sequence identity in 16S rRNA with its closest insect associated relative (S. glossinidius), while strain HS shares >99% sequence identity with the primary endosymbionts of the grain weevils Sitophilus oryzae and S. zeamais and with recently discovered endosymbionts from the chestnut weevil, Curculio sikkimensis and the stinkbug, Cantao occelatus –. Analysis of a protein-coding gene, groEL, corroborated these findings, confirming that strain HS is a close relative of the grain weevils, chestnut weevil and stinkbug endosymbionts (Figure 1).
Insect endosymbionts that do not have proper nomenclature are designed by the prefix “E”, followed by the name of their insect host. The numbers adjacent to nodes indicate maximum likelihood bootstrap values shown for nodes with bootstrap support >70%.
Genome Sequences of Strain HS and Related Insect Symbionts
To compare the genome sequences of strain HS and related Sodalis-allied endosymbionts, we aligned a draft sequence assembly of strain HS, comprising a total of 5.15 Mb of DNA in 271 contigs, with the complete genome sequences of the tsetse fly secondary endosymbiont, S. glossinidius (4.3 Mb) , , and the recently completed sequence of Sitophilus oryzae primary endosymbiont (SOPE; 4.5 Mb). The resulting alignments (Figure 2) reveal a remarkable level of conservation in gene content and organization between strain HS, S. glossinidius and SOPE. To determine if this high level of conservation is simply a consequence of the close evolutionary relationship between these bacteria, we also constructed a whole genome sequence alignment between strain HS and Dickeya dadantii, which represents the next most closely related free-living bacterium whose whole genome sequence is available (Figure S1). This alignment shows that strain HS and D. dadantii are substantially more divergent in terms of their gene inventories, consistent with the notion that they occupy distinct ecological niches. Considering the alignments between strain HS, S. glossinidius and SOPE, it is notable that while the genome sequences of strain HS and S. glossinidius display an increased level of co-linearity, the relationship between strain HS and SOPE is predicted to be closer based on the fact that they share a higher level of genome-wide sequence identity (Figure 2). The genome sequences of strain HS and S. glossinidius demonstrate a typical pattern of polarized nucleotide composition in each replichore (G+C skew, Figure 2), whereas the SOPE genome has numerous perturbations in G+C skew that must result from recent chromosome rearrangements. These rearrangements likely arose as a consequence of intragenomic recombination events between repetitive insertion sequence (IS)-elements, which are highly abundant in the SOPE genome (Figure S2), and have been documented as a causative agent of deletogenic rearrangements in other bacteria –.
The draft strain HS contigs are depicted in an arbitrary color scheme (outer top ring). Contigs sharing <5 kb synteny with either the SOPE or S. glossinidius genome are uncolored. The uppermost plot (colored in purple and orange) depicts G+C skew, based on a 40 kb sliding window. For upper tracks, grey bars depict genes unique to strain HS whereas green bars depict strain HS genes that share orthologs with the aligned symbiont chromosome. For lower tracks, green and red bars represent (respectively) intact and disrupted orthologs of strain HS genes in the insect symbiont genomes, whereas blue bars highlight prophage and IS-element sequences in the insect symbiont chromosomes. Plots of pairwise nucleotide sequence identity are shown in the lower alignment following in silico removal of prophage and IS-elements from the SOPE and S. glossinidius sequences. Consensus oriC and dif sequences are labeled to indicate putative origins and termini of chromosome replication.
Although the gene inventories of strain HS, S. glossinidius and SOPE share many genes in common, as expected given their close evolutionary relationship, each bacterium also maintains a fraction of unique genes. In strain HS we identified a total of 1.9 Mb of DNA encoding genes not found in either S. glossinidius or SOPE that are classified in a wide range of functional categories (Figure 3). This indicates that strain HS has many unique genetic and biochemical properties, and is consistent with the observation that strain HS, unlike the fastidious and microaerophilic S. glossinidius , grows under atmospheric conditions on minimal media. In addition, strain HS maintains a number of unique genes sharing high levels of sequence identity with virulence factors found in both animal and plant pathogens, including an Hrp-type effector protein that is characteristically utilized by plant pathogenic bacteria  (Table S1). This may be indicative of the ability of strain HS to sustain infection in plant tissues. In comparison with strain HS, the unique fractions of the S. glossinidius and SOPE chromosomes are composed almost exclusively of components of mobile genetic elements, including integrated prophage islands and IS-elements. Following excision of these mobile genetic elements in silico prior to alignment, the resulting genome sequences of S. glossinidius (3.21 Mb) and SOPE (3.15 Mb) represent near-perfect subsets of the strain HS genome (Figure 2), indicating that S. glossinidius and SOPE are abridged derivatives of a strain HS-like ancestor.
The dark shaded component of each bar refers to intact genes retained in both S. glossinidius and SOPE. The intermediate shaded component refers to intact genes retained in only S. glossinidius (upper bar) or SOPE (lower bar) and the lighter shaded component refers to genes that are either absent or disrupted in both S. glossinidius and SOPE. The COG categories are organized in five larger groups with red representing genes involved in information storage and processing, blue representing genes involved in cellular processes and signaling, black representing genes involved in metabolism, green representing genes with poorly characterized functions, and yellow representing components of phages and IS-elements.
Independent Gene Inactivation and Deletion in S. glossinidius and SOPE
To further understand genetic differences between strain HS, S. glossinidius and SOPE, we analyzed three genomic regions containing relatively high densities of pseudogenes in both S. glossinidius and SOPE (Figure 4). The most notable finding to arise from this comparison is the absence of pseudogenes in the three genomic regions of strain HS. Furthermore, our comparative analysis shows that S. glossinidius and SOPE each have a unique complement of pseudogenes. Indeed, even for orthologous genes that have been inactivated in both S. glossinidius and SOPE, mutations leading to gene inactivation in each insect symbiont genome are distinct, indicating that gene inactivation and loss took place independently in S. glossinidius and SOPE, mostly as a consequence of small frameshifting indels. However, it should also be noted that the reductions observed in the gene inventories of S. glossinidius and SOPE are very similar at the level of functional categories, indicating that the insect-associated lifestyle imposes similar constraints on the retention of genes encoding core functions such as replication, transcription, translation and energy generation (Figure 3). In order to determine the number of pseudogenes throughout the genome of strain HS, we performed a manual annotation and careful inspection of the complete draft strain HS sequence assembly. Out of a total of 4,002 intact candidate ORFs identified in the draft annotation (Table S1), only 48 (including phage and IS elements) were found to be translationally frameshifted or truncated by more than 10% of the size of their most closely related orthologs in the GenBank database (Table 1). This finding stands in stark contrast to the gene inventories of both S. glossinidius and SOPE, in which pseudogenes represent a substantial fraction of their total genomic coding capacity (Figure 2) , . Thus, for both S. glossinidius and SOPE, the predominant evolutionary trajectory following obligate insect association involved the inactivation and/or loss of a substantial component of the ancestral (strain HS-like) gene inventory.
Alignments of three regions of the S. glossinidius, strain HS, and SOPE chromosomes, corresponding to SG0948–SG0977 (A), ps_SGL0466–SG0918 (B) and ps_SGL0318–ps_SGL0330 (C) in the most recent S. glossinidius annotation . Putative ORFs and intergenic regions are drawn according to scale, oriented according to their inferred direction of transcription and color-coded according to COG functional categories. While all of the depicted strain HS genes have intact reading frames, the status of their orthologs in S. glossinidius and SOPE are shown in the outer bars (green = intact, purple = inactivated). Nonsense mutations (premature stop codons) are depicted by purple diamonds, and frameshifting indels are depicted by purple triangles. Light grey connecting bars are syntenic nucleotide alignments, while brown bars illustrate IS-element acquisitions that occur more frequently in SOPE.
Evolution of Pseudogenes in S. glossinidius and SOPE
The close evolutionary relationships between strain HS, S. glossinidius and SOPE indicate that the respective insect symbioses are recent in origin. This raises the possibility that a subset of selectively neutral genes in the S. glossinidius and SOPE genomes have not yet accumulated mutations that lead to disruption of their open reading frames. Such “cryptic” pseudogenes are assumed to have no adaptive benefit in the symbiosis and are expected to accumulate nonsense and/or frameshifting mutations in the future . To determine if the genomes of S. glossinidius and SOPE maintain cryptic pseudogenes, we compared the average size of all strain HS genes with the average sizes of strain HS orthologs that are classified either as intact, absent (lost via large deletion) or pseudogenes (visibly disrupted) in the S. glossinidius and SOPE genomes (Figure 5). First, it is important to note that the average size of the absent strain HS orthologs in S. glossinidius and SOPE is not significantly different from the average size of all strain HS ORFs, indicating that large deletion events are not significantly biased with respect to size. However, in both S. glossinidius and SOPE, genes in the pseudogene class were found to have a larger average size in comparison to all strain HS orthologs. Similarly, genes in the intact class were found to have a smaller size in comparison to all strain HS orthologs. This can be explained by the fact that larger genes have an increased likelihood of accumulating at least one disrupting mutation in a given time frame. Based on the same logic, we can infer that the intact gene class contains a subset of smaller, cryptic pseudogenes that have not yet had sufficient time to accumulate any nonsense or frameshifting mutations. Furthermore, since the difference between the average size of intact and disrupted genes is significantly larger in SOPE (192 bases) in comparison to S. glossinidius (77 bases), it follows that SOPE likely maintain a larger number of cryptic pseudogenes than S. glossinidius.
Estimating Numbers of Cryptic Pseudogenes in S. glossinidius and SOPE
In a previous study, the numbers of cryptic pseudogenes in the recently derived aphid symbiont, Serratia symbiotica, were estimated by extrapolation from a Poisson distribution of disrupting mutations found in existing pseudogenes . The expectation of a Poisson distribution is based on the assumption that the switch to an insect-associated lifestyle leads to the synchronous relaxation of selection on genes no longer required for persistence in an insect host . In the case of both SOPE and S. glossinidius, plots of the densities of disrupting mutations in pseudogenes indicate that the data is overdispersed relative to a Poisson distribution (Figure 6). This effect is exacerbated when current ORF sizes are used for the calculation of mutation densities. This results from the fact that large deletions erase any evidence of previous disrupting mutations. In order to estimate the numbers of cryptic pseudogenes in SOPE and S. glossinidius, we used a Monte Carlo simulation in which a randomly selected class of candidate pseudogenes, selected from all strain HS genes, was permitted to accumulate random disrupting mutations over time, in accordance with ORF size. In this simulation, both pseudogene counts and size differences between the strain HS orthologs of intact and disrupted S. glossinidius and SOPE genes were recorded at regular intervals. The simulation was repeated with an increasing number of neutral genes until the size difference and pseudogene count matched the empirically determined values shown in Figure 5 and Table 1. For S. glossinidius and SOPE, matches were obtained when the predicted numbers of genes evolving under relaxed selection reached 1,470 and 1,530, respectively (Figure 7). Thus, although S. glossinidius and SOPE are predicted to have almost the same numbers of genes evolving under relaxed selection, the degeneration of pseudogenes is at a more advanced stage in S. glossinidius, and SOPE has a larger proportion of neutral genes that have not yet acquired any obvious disrupting changes. Assuming that the relaxation of selection was imposed synchronously at the onset of obligate insect-association, these results suggest that the SOPE-weevil symbiosis originated more recently than the S. glossinidius-tsetse fly symbiosis. This is further supported by a comparison of the estimates of corrected mutation density derived from the simulation (Figure 7). While SOPE is estimated to maintain only 2 disrupting mutations/kb of pseudogenes, S. glossinidius is estimated to maintain more than twice that density of disrupting substitutions (4.39 disrupting mutations/kb). On a related note, we were unable to utilize dN/dS ratios to identify cryptic pseudogenes in SOPE or S. glossinidius. This is likely due to the fact that stochastic variation resulting from differences in expression level, codon bias and other factors greatly exceeds any signal resulting from a recent relaxation of selection.
The numbers of frameshifting and truncating indels and nonsense mutations were computed from alignments of strain HS, SOPE and S. glossinidius orthologs. Mutation densities were computed according to the original strain HS ORF sizes (left) or the current SOPE or S. glossinidius pseudogene sizes (right).
The simulation was repeated with an increasing number of candidate pseudogenes until estimates of pseudogene number (red) and the size difference between pseudogenes and intact genes (blue) matched empirical values shown in Figure 5 and Table 1, as highlighted by bold bars. The densities of disrupting mutations in S. glossinidius and SOPE pseudogenes (which include cryptic pseudogenes) are shown in the upper left inset, corresponding to the data points highlighted in bold.
Accelerated Sequence Evolution and Base Composition Bias in SOPE and S. glossinidius
The transition to obligate insect-association is also known to catalyze base composition bias and accelerated polypeptide sequence evolution on the part of the symbiont . The results outlined in Table 1 show that the genomic GC-contents of S. glossinidius and SOPE are lower than that of strain HS. However, to avoid any bias arising from the differential gene content of these organisms, we also performed comparative analyses focusing solely on orthologous sequences. This facilitated the comparison of 1,355 intact genes and 1,376 pseudogenes shared between strain HS and S. glossinidius, and 1,414 intact genes and 1,194 pseudogenes shared between strain HS and SOPE. Although the symbioses in the current study are anticipated to be relatively recent in origin, comparisons focusing on these shared sequences also show that both S. glossinidius and SOPE have reduced GC-contents relative to strain HS (Figure 8). This effect is most notable at 4-fold degenerate (GC4) sites in S. glossinidius, which demonstrate the highest levels of sequence divergence and AT-bias in comparison to orthologs from strain HS. Assuming that the onset of AT-bias is coincident with the origin of symbiosis, this further supports the notion that the symbiosis involving S. glossinidius is more ancient in origin. It is also notable that the number of substitutions at the 2nd codon position sites of pseudogenes (dGC2, Figure 8) is elevated by approximately the same extent (relative to intact genes) in S. glossinidius and SOPE. This implies that pseudogenes have been evolving under relaxed selection for approximately the same proportion of time since each symbiont diverged from strain HS. However, given that sequence divergence at silent sites (GC4) is greater between strain HS and S. glossinidius, this again invokes the interpretation that pseudogenes arose earlier in the S. glossinidius line of descent. It is also interesting to note that the level of divergence at GC2 sites (dGC2, Figure 8) relative to GC4 sites (dGC4, Figure 8) is greater in SOPE than in S. glossinidius. This can be explained by the fact that the pairwise comparison between strain HS and SOPE is expected to capture an increased proportion of mutations that are fixed in the insect-associated phase of life in which selection on polypeptide evolution is anticipated to be more relaxed.
The evolutionary relationships between SOPE, strain HS and S. glossinidius are depicted by bold lines drawn to scale in accordance with levels of genome-wide divergence at 4-fold degenerate (GC4) sites. Upper boxes show genome-wide GC-percentages at 2nd codon position (GC2), GC4 and intergenic (GCI) sites. Lower boxes depict the number of substitutions per site for intact genes (dGC2 and dGC4) and pseudogenes (dGC2Ψ and dGC4Ψ). The data were obtained from pairwise analysis of point mutations in 1,355 intact genes and 1,376 pseudogenes shared between strain HS and S. glossinidius, and 1,414 intact genes and 1,194 pseudogenes shared between strain HS and SOPE.
Mutational Dynamics of Gene Inactivation in SOPE and S. glossinidius
Considering only those mutations that have led to gene inactivation, we found that the relative ratios of truncating (large) indels, frameshifting (small) indels and nonsense mutations are similar in SOPE and S. glossinidius (Table 2). Inspection of the data reveals that small frameshifting deletions constitute the most abundant class of mutations leading to gene inactivation. However, it should be noted that the effects of large deletions are, for obvious reasons, not captured in our analyses. Another important point is that IS-element insertions appear to have contributed relatively little to the overall spectrum of mutations leading to gene inactivation in SOPE, representing only 10% of the total count. Indeed, the majority of IS-elements in SOPE are located either in intergenic regions or, more commonly, clustered inside other IS-elements. One potential explanation is that IS-element insertions in genic sequences might be more deleterious towards processes of transcription and/or translation in the cell, such that pseudogenes with IS-element insertions are preferentially deleted relative to pseudogenes with nonsense point mutations or small indels. However, it is conspicuous that clustering of IS-elements has also been reported for mobile DNA elements found in eukaryotes, including the MITE elements found in plants  and mosquitoes , and the Alu and L1 elements found in the human genome . The relative paucity of IS-elements in genic DNA is surprising given the fact that the SOPE genome has such a large number of pseudogenes that provide neutral space for IS-element colonization. However, the inability of IS-elements to occupy this territory can be rationalized as a consequence of an inherited adaptive bias that facilitates the avoidance of genic insertion. This makes sense when considering the perspective of an IS-element residing in a free-living bacterium that has relatively few dispensable genes. It also explains the propensity for IS-elements to insert themselves into the sequences of other IS-elements, because the safety of this approach has already been validated by natural selection. Clearly, in the case of SOPE, when the opportunity arose for expansion into novel territory (i.e. neutralized genic sequences), IS-elements were largely unable to overcome these basic evolutionary directives.
Phylogenetic analysis of strain HS indicates that it shares a close relationship with the Sodalis-allied endosymbionts that are found in a wide range of insect hosts, including tsetse flies, weevils, lice and stinkbugs. In terms of 16S rRNA sequence identity, strain HS is most closely related to endosymbionts found in the chestnut weevil, Curculio sikkimensis and the stinkbug, Cantao occelatus. Interestingly, only limited numbers of these insects maintain Sodalis-allied endosymbionts in their natural environment –, suggesting that they do not maintain persistent (maternally-transmitted) infections. Furthermore, it is notable that the sequences from strain HS, C. sikkimensis and C. occelatus are localized on very short branches in our phylogenetic trees, indicating that these particular lineages are evolving slowly in comparison to other Sodalis-allied endosymbionts. This low rate of molecular sequence evolution, along with the observation that the strain HS genome shows no sign of the characteristic degenerative changes that are known to accompany the transition to the obligate host-associated lifestyle, leads us to propose that strain HS represents an environmental progenitor of the Sodalis-allied clade of insect endosymbionts.
Closely related members of the Sodalis-allied clade of insect endosymbionts have now been identified in a wide range of distantly related insect taxa, including some that are known to feed exclusively on plants and others that are known to feed exclusively on animals . Although strain HS was isolated from the wound of a human host, it is difficult to assess the extent of its pathogenic capabilities, due to the fact that antibiotic treatment commenced three days prior to microscopic examination and culturing. In addition, the available evidence indicates that the original source of the infection was a branch from a dead crab apple tree. This implies that strain HS was present either on the bark or in the woody tissue of this tree, possibly acting as a pathogen or saprophyte. Furthermore, it is interesting to note that C. sikkimensis and C. occelatus, whose symbionts are most closely related to strain HS, are both known to feed on trees , . In addition, some wood and bark-inhabiting longhorn beetles, including Tetropium castaneum (Figure 1) have recently been found to maintain Sodalis-allied endosymbionts . Moreover, the ability of strain HS to persist in both plant and animal tissues is compatible with the observation that diverse representatives of both herbivorous and carnivorous insects have acquired Sodalis-allied symbionts.
In a comparative sense, relationships involving the Sodalis-allied endosymbionts are considered to be relatively recent in origin. Indeed, evidence of host-symbiont co-speciation only exists in the case of grain weevils, Sitophilus spp., which were estimated to have co-evolved with their Sodalis-allied endosymbionts for a period of around 20 MY, following the replacement of a more ancient lineage of endosymbionts in these insects , . The notion of a recent origin of the Sodalis-allied endosymbionts is further supported by the fact that the whole genome sequence of S. glossinidius is substantially larger than that of long-established mutualistic insect endosymbionts, and is close to the size of related free-living bacteria . However, the S. glossinidius genome does have an unusually low coding capacity resulting from the presence of a large number of pseudogenes , . This suggests that S. glossinidius is at an intermediate stage in the process of genome degeneration, in which many protein coding genes have been inactivated by indels and nonsense mutations but have not yet been deleted from the genome. In the current study we show that the genome of the grain weevil symbiont, SOPE, is at a similar stage of degeneration as evidenced by the presence of a comparable number of pseudogenes and a large number of repetitive insertion sequence elements.
In a comparative sense, it is interesting to note that SOPE and strain HS share a substantially higher level of sequence similarity, genome-wide, in comparison to S. glossinidius and strain HS (Figure 2). In the context of the progenitor hypothesis, the disparity in the relationship between strain HS, SOPE and S. glossinidius can be explained by the idea that there may be a substantial level of diversity among free-living relatives of the Sodalis-allied symbionts in the environment, and that we simply happened to characterize a representative that is more closely related to the ancestral progenitor of SOPE. While this is likely to be true to some extent, the close relationship between strain HS and SOPE can also be explained by the notion that the SOPE-grain weevil symbiosis has a more recent origin than the S. glossinidius-tsetse symbiosis. Our results provide several compelling lines of evidence in support of this idea. Most significantly, we found that the pseudogenes of S. glossinidius contain a higher average density of disrupting mutations relative to their counterparts in SOPE. This suggests that the pseudogenes of S. glossinidius have been evolving under relaxed selection for a longer period of time, consistent with the hypothesis of a more ancient origin of host association catalyzing the neutralization of these genes. In addition, the genome of SOPE is predicted to have a larger proportion of “cryptic” pseudogenes; genes evolving neutrally that have not yet had sufficient time to accumulate nonsense or frameshifting mutations that disrupt their translation. Finally, it is notable that the GC4 sites of S. glossinidius have a higher AT-content than those of strain HS and SOPE (Figure 8). Assuming that the AT-bias at GC4 sites accumulates in a clock-like manner following the onset of the symbiosis, this again supports a more ancient origin for the symbiosis involving S. glossinidius.
In the current study, a comparative analysis of the genome sequences of strain HS, SOPE and S. glossinidius has provided an unprecedentedly detailed view of the nascent stages of genome degeneration in symbiosis. Taken together, our results indicate that irreversible degenerative changes, including gene inactivation and loss, in addition to base composition bias, commence rapidly following the onset of an obligate relationship. Indeed, the close relationship observed between strain HS and SOPE illustrates the potency of the degenerative evolutionary process at an early stage in the evolution of a symbiotic interaction. This is exemplified by the fact that SOPE is predicted to have lost 55% of its ancestral gene inventory (34% via gene loss and 21% via gene inactivation) in a period of time sufficient to incur a substitution frequency of only 4.3% at the highly variable GC4 sites of intact protein coding genes (Figure 8). Although estimates of genome wide synonymous clock rates vary by several orders of magnitude in bacteria , an estimate of μs = 2.2×10−7, derived recently for another insect endosymbiont, Buchnera aphidicola , places the divergence of strain HS and SOPE at only c. 28,000 years, which is much more recent than previous estimates obtained for the origin of the SOPE symbiosis , .
While the broad distribution of recently derived endosymbionts in phylogenetically distant insect hosts has previously been attributed to interspecific symbiont transfer events , , the results outlined in the current study indicate that diverse insect species can also acquire novel symbionts through the domestication of bacteria that reside in their local environment. In the case of S. glossinidius and SOPE, our comparative analyses support the notion that these symbionts were acquired independently, as evidenced by the presence of distinct mutations in shared pseudogenes. This also implies that symbionts rapidly become specialized towards a given host, likely restricting their abilities to switch hosts. Although the current study highlights the first description of a close free-living relative of the Sodalis-allied symbionts, it should be noted that environmental microbial diversity is vastly undersampled . Thus, it is conceivable that close relatives of extant insect endosymbionts, such as strain HS, are widespread in nature and provide ongoing opportunities for a wide range of insect hosts to domesticate new symbiotic associates. Furthermore, since many insects serve as vectors for plant and animal pathogens , it is conceivable that mutualistic associations arise as a consequence of the domestication of vectored pathogens. This hypothesis is compelling because such pathogens are not expected to negatively impact the fitness of their insect vectors  and under those circumstances the transition to a mutualistic lifestyle could be achieved without any need to attenuate virulence towards the insect host.
Materials and Methods
Isolation and Phylogenetic Analysis of Strain HS
Strain HS was isolated on MacConkey agar at 35°C and 5% CO2. 16S rRNA and groEL sequences were amplified from strain HS using universal primers. Following cloning of PCR products, eight clones were sequenced from each gene and consensus sequences were used in phylogenetic analyses. Sequence alignments were generated for 16S rRNA and groEL using MUSCLE . PhyML  was then used to construct phylogenetic trees using the HKY85  model of sequence evolution with 25 random starting trees and 100 bootstrap replicates.
Weevil Cultures and DNA Isolation
Synchronous cultures of Sitophilus oryzae and Sitophilus zeamais were reared on organic soft white wheat grains and corn kernels respectively, and maintained at 25°C with 70% relative humidity. Bacteriomes (containing the bacterial endosymbionts SOPE and SZPE) were isolated from 5th instar S. oryzae and S. zeamais larvae by dissection and homogenized at a sub-cellular level to release bacteria from host bacteriocyte cells; bacterial cells were then separated from host cells via centrifugation (2,000×g, 5 min). Total genomic DNA was then isolated from bacteria using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA).
SOPE and SZPE Shotgun Library Construction
Six mg of genomic DNA was hydrodynamically sheared in 5 mM Tris, 1 mM EDTA, 100 mM NaCl (pH 8) buffer to a mean fragment size of 10 kb. The sample was washed and concentrated by ultrafiltration in a Centricon-100 (Millipore, Billerica, MA) and eluted in 250 µl of 2 mM Tris (pH 8). The fragments were end-repaired by treatment with T4 DNA polymerase (New England Biolab, Beverly, MA) to generate blunt ends. The DNA was then extracted with phenol/chloroform, ethanol precipitated, and 5′ phosphorylated with T4 polynucleotide kinase (NEB). Ten mM of double-stranded, biotinylated oligonucleotide adaptors were blunt-end ligated onto the sheared genomic fragments at room temperature for 25 h using 10,000 cohesive end units of high concentration T4 DNA ligase (NEB). Unligated adaptors were removed by ultrafiltration in a Centricon-100. The adaptored fragments were bound to streptavidin-coated magnetic beads (Invitrogen), and after binding and washing, the adaptored genomic fragments were eluted in 10 mM TE (pH 8). Fragments in the 9.5–11.5 kb size range were gel purified after separation on a 0.7% 1× TAE agarose gel, and the purified DNA was electroeluted from the agarose and desalted by ultrafiltration in a Centricon-100.
SOPE and SZPE Shotgun Sequencing
pWD42 vector (GenBank: AF129072.1) was linearized by digestion with BamHI (NEB) at 37°C for 4 h, extracted with phenol/chloroform, ethanol precipitated and resuspended in 100 ml of 2 mM Tris (pH 8.0). Ten picomoles of double-stranded, biotinylated oligo adaptors were ligated onto the BamHI-digested vector at 25°C for 16 hrs using 4,000 units of T4 DNA ligase (NEB). Unligated adaptors were removed by ultrafiltration in a Centricon-100. The adaptored vector was bound to streptavidin-coated magnetic beads and the non-biotinylated adaptored vector was eluted in 10 mM TE (pH 8). One hundred ng each of adaptored vector and genomic DNA were annealed without ligase in 10 ml of T4 DNA ligase buffer (NEB) at 25°C for one hour. Two ml aliquots of the annealed vector/insert were transformed into 100 ml of XL-10 chemically competent E. coli cells (Agilent Technologies, Santa Lara, CA) and plated on LB agar plates containing 20 µg/ml ampicillin. A total of 23,808 bacterial colonies were picked into 96-well microtiter dishes containing 600 ml of terrific broth (TB)+20 µg/ml ampicillin and grown at 30°C for 16 h. Fifty ml aliquots were removed from the library cultures, mixed with 50 ml of 14% DMSO, and archived at −80°C. The 200 ml cultures were diluted 1∶4 in TB amp and runaway plasmid replication was induced at 42°C for 2.25 h. Plasmid DNA was purified by alkaline lysis, and cycle sequencing reactions were performed with forward and reverse sequencing primers using ABI BigDye v3.1 Terminator chemistry (Applied Biosystems, Foster City, CA). The reactions were ethanol precipitated, resuspended in 15 ul of dH2O, and sequence ladders were resolved on an ABI 3730 capillary instrument prepared with POP-5 capillary gel matrix.
SOPE Genome Sequence Assembly and Finishing
Following elimination of any sequences encoding contaminating plasmid vector or host insect sequences, 38,755 shotgun reads were assembled using the Phusion assembler  using the paired-end sequences as mate-pair assembly constraints. Contig assemblies were viewed and edited in Consed , and reads with high quality (Phred>20) discrepancies were disassembled. After inspection and manual assembly to extend contigs, gaps were closed by iterative primer walking (895 primer walk sequence reads) and gamma-delta transposon-mediated full-insert sequencing of plasmid clones (6,165 sequence reads across 103 transposed plasmid clones) using an established protocol . The average insert size of the plasmid library in the finished SOPE assembly was found to be 8.2 kb.
SOPE Fosmid Library Construction and Genome Sequence Validation
The SOPE fosmid library was constructed using the Epicenter EpiFOS Fosmid Library Production Kit (Epicentre Biotechnologies, Madison, WI), using SOPE total genomic DNA. 1,404 paired-end reads were generated from 702 fosmid inserts and mapped onto the assembly derived from the plasmid shotgun sequencing for validation (Figure S2).
Strain HS Sequencing
Strain HS genomic DNA was isolated from liquid culture using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). Five micrograms of total genomic DNA was used to construct a paired-end sequencing library using the Illumina paired-end sample preparation kit (Illumina, Inc. San Diego, CA) with a mean fragment size of 378 base pairs. This library was then sequenced on the Illumina GAIIx platform generating 26,891,485 paired-end reads of 55 bases in length.
Strain HS Sequence Assembly
Paired-end reads were quality filtered using Galaxy ,  and low quality paired-end reads (Phred<20) were discarded. The remaining 17,054,405 reads were then assembled using Velvet  with a k-mer value of 37, with expected coverage of 119 and a coverage cutoff value of 0.296. The resulting assembly consisted of 271 contigs with an N50 size of 231,573 and a total of 5,135,297 bases. No sequences were found to share significant sequence identity with genes encoding plasmid replication functions, suggesting that strain HS does not maintain any extrachromosomal elements.
Strain HS Annotation
The assembled draft genome sequence of strain HS was annotated by automated ORF prediction using GeneMark.hmm . The annotation was then adjusted manually in Artemis  using the published Sodalis glossinidius genome sequence  as a guide. ORFs were annotated as putatively functional only if (i) their size was ≥90% of the most closely related ORF derived from a free-living bacterium in the GenBank database, and (ii) they did not contain any frameshifting indel(s).
Genome Alignment, COG Classification, and Computational Analyses
Curation of the strain HS genome sequence was performed in Artemis . ORFs were classified into COG categories using the Cognitor software . Syntenic links shown in Figure 2 were determined by pairwise nucleotide alignments between strain HS contigs and S. glossinidius (GenBank: NC_007712.1) or the finished SOPE genome using the Smith-Waterman algorithm as implemented in the cross_match algorithm . Figure 2 was prepared from data obtained from these alignments using CIRCOS . The metrics depicted in Table 1, Table 2, and Figure 8 were computed from pairwise nucleotide sequence alignments of strain HS, S. glossinidius and SOPE ORFs using custom scripts. Candidate genes were classified as intact orthologs when their alignment spanned >99% of the HS ORF length (or 90% for ORFs <300 nucleotides in size) and did not contain frameshifting indels or premature stop codons.
Monte Carlo Pseudogene Simulation
A simple Monte Carlo approach was implemented to simulate the evolution of pseudogenes in S. glossinidius and SOPE. The simulation facilitated the progressive accumulation of random mutations in all strain HS orthologs of both intact genes and pseudogenes identified in the current S. glossinidius or SOPE gene inventories. Mutations accumulated in proportion to ORF size in a randomly selected class of neutral genes of user-defined size over a defined number of mutational cycles. At preset cycle intervals, the simulation recorded (i) the difference in size between intact and disrupted sequences, (ii) the number of neutral genes that have accumulated one or more disrupting mutations, and (iii) the density of disrupting mutations, which was calculated based on the cumulative size of all neutral genes.
The GenBank accession numbers for sequences used in Figure 1 are as follows: Endosymbiont of Circulio sikkimensis 16S rRNA, (AB559929.1), groEL, (AB507719); Vibrio cholerae 16S rRNA, (NC_002506.1), groEL, (NC_002506.1); Dickeya dadantii 16S rRNA (CP002038.1), groEL, (CP002038.1); Escherichia coli 16S rRNA, (NC_000913.2), groEL, (NC_000913.2); Candidatus Moranella endobia 16S rRNA, (NC_015735), groEL, (NC_015735); Sodalis glossinidius 16S rRNA, (NC_007712.1), groEL, (NC_007712.1); Yersinia pestis 16S rRNA, (NC_008150.1), groEL, (NC_008150.1); Wigglesworthia glossinidia 16S rRNA, (NC_004344.2), groEL, (NC_004344.2); Candidatus Blochmannia pennsylvanicus 16S rRNA, (NC_007292), groEL, (NC_007292); Endosymbiont of Cantao ocellatus 16S rRNA, (AB541010), groEL, (BAJ08314); Endosymbiont of Columbicola columbae 16S rRNA, (AB303387), groEL, (JQ063388); Sitophilus zeamais primary endosymbiont 16S rRNA, (AF548142), groEL (JX444567); Sitophilus oryzae primary endosymbiont 16S rRNA, (AF548137), groEL (AF005236); Strain HS 16S rRNA, (JX444565), groEL (JX444566). The GenBank accession numbers for sequences used in Figure 4 are as follows: Strain HS Figure 4A (JX444569), Figure 4B (JX444571), Figure 4C (JX444572); Sitophilus oryzae primary endosymbiont Figure 4A (JX444568), Figure 4B (JX444570), Figure 4C (JX444573).
Alignment between strain HS contigs (top) and the chromosome of Dickeya dadantii. The draft strain HS contigs are depicted in an arbitrary color scheme (outer top ring). On the upper track, grey bars depict genes unique to strain HS whereas green bars depict strain HS genes that share orthologs with the aligned D. dadantii chromosome. On the lower track, green and red bars represent intact and disrupted genes (respectively) in the D. dadantii chromosome, and blue bars indicate prophage and IS-element ORFs.
Genome assembly validation of the 4.5 Mb SOPE genome using plasmid and fosmid mate-pair coverage. The finished SOPE chromosome sequence assembly was obtained by paired-end plasmid shotgun sequencing, primer walking and directed transposon-based full-insert plasmid sequencing. The physical map of the plasmid paired-ends are represented in blue. Clones from underrepresented regions and IS-element clusters were completely sequenced by transposon-mediated sequencing (red; see Materials and Methods). The resulting finished assembly (circular chromosome: 4,513,139 bp) was validated by fosmid paired-end sequencing (orange). Depth of plasmid clone physical coverage is depicted in the histogram (yellow). The locations of the four most abundant families of IS-elements are depicted by the inner bars (red, IS903; blue, IS256; green, IS21; purple, ISL3).
List of complete strain HS gene products and status of orthologs in S. glossinidius and SOPE. Candidate virulence genes are highlighted in the column labeled “V”, according to the presence of orthologs in animal (A) or plant (P) pathogens.
Conceived and designed the experiments: ALC KFO RBW MF CD. Performed the experiments: ALC KFO MG AP DMD ACvN RBW MF CD. Analyzed the data: ALC KFO MG AP DMD ACvN RBW MF CD. Contributed reagents/materials/analysis tools: RBW MF CD. Wrote the paper: ALC KFO RBW MF CD.
- 1. Andersson SG, Kurland CG (1998) Reductive evolution of resident genomes. Trends Microbiol 6: 263–268.
- 2. Dale C, Moran NA (2006) Molecular interactions between bacterial symbionts and their hosts. Cell 126: 453–465.
- 3. Pérez-Brocal V, Gil R, Ramos S, Lamelas A, Postigo M, et al. (2006) A small microbial genome: the end of a long symbiotic relationship? Science 314: 312–313.
- 4. McCutcheon JP, Moran NA (2007) Parallel genomic evolution and metabolic interdependence in an ancient symbiosis. Proc Natl Acad Sci U S A 104: 19392–19397.
- 5. Nakabachi A, Yamashita A, Toh H, Ishikawa H, Dunbar HE, et al. (2006) The 160-kilobase genome of the bacterial endosymbiont Carsonella. Science 314: 267.
- 6. Moran NA, McCutcheon JP, Nakabachi A (2008) Genomics and evolution of heritable bacterial symbionts. Annu Rev Genet 42: 165–190.
- 7. Nováková E, Hypsa V, Moran NA (2009) Arsenophonus, an emerging clade of intracellular symbionts with a broad host distribution. BMC Microbiol 9: 143.
- 8. Snyder AK, McMillen CM, Wallenhorst P, Rio RV (2011) The phylogeny of Sodalis-like symbionts as reconstructed using surface-encoding loci. FEMS Microbiol Lett 317: 143–151.
- 9. Huigens ME, de Almeida RP, Boons PA, Luck RF, Stouthamer (2004) Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia in Trichogramma wasps. Proc Biol Sci 271: 509–515.
- 10. Jaenike J, Polak M, Fiskin A, Helou M, Minhas M (2007) Interspecific transmission of endosymbiotic Spiroplasma by mites. Biol Lett 3: 23–25.
- 11. Moran NA, Dunbar HE (2006) Sexual acquisition of beneficial symbionts in aphids. Proc Natl Acad Sci U S A 103: 12803–12806.
- 12. Hoffmann JA (1995) Innate immunity of insects. Curr Opin Immunol 7: 4–10.
- 13. Pontes MH, Smith KL, De Vooght L, Van Den Abbeele J, Dale C (2011) Attenuation of the sensing capabilities of PhoQ in transition to obligate insect-bacterial association. PLoS Genet 7: e1002349
- 14. Dale C, Young S, Haydon DT, Welburn SC (2001) The insect endosymbiont Sodalis glossinidius utilizes a type-III secretion system for cell invasion. Proc Natl Acad Sci U S A 98: 1883–1888.
- 15. Dale C, Plague GR, Wang B, Ochman H, Moran NA (2002) Type III secretion systems and the evolution of mutualistic endosymbiosis. Proc Natl Acad Sci U S A 99: 12397–12402.
- 16. Degnan PH, Yu Y, Sisneros N, Wing RA, Moran NA (2009) Hamiltonella defensa, genome evolution of protective bacterial endosymbiont from pathogenic ancestors. Proc Natl Acad Sci U S A 106: 9063–9068.
- 17. Nováková E, Hypsa V (2007) A new Sodalis lineage from bloodsucking fly Craterina melbae (Diptera, Hippoboscoidea) originated independently of the tsetse flies symbiont Sodalis glossinidius. FEMS Microbiol Lett 269: 131–135.
- 18. Darby AC, Choi J-H, Wilkes T, Hughes MA, Werren JH, et al. (2009) Characteristics of the genome of Arsenophonus nasoniae, son-killer bacterium of the wasp Nasonia. Insect Mol Biol Suppl 1: 75–89.
- 19. Stackebrand E, Goebel BM (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44: 846–849.
- 20. Verbarg S, Frühling A, Cousin S, Brambilla E, Gronow S, et al. (2008) Biostraticola tofi gen. nov., spec. nov., a novel member of the family Enterobacteriaceae. Curr Microbiol 56: 603–608.
- 21. Kaiwa N, Hosokawa T, Kikuchi Y, Nikoh N, Meng XY, et al. (2010) Primary gut symbiont and secondary, Sodalis-allied symbiont of the Scutellerid stinkbug Cantao ocellatus. Appl Environ Microbiol 76: 3486–3494.
- 22. Toju H, Hosokawa T, Koga R, Nikoh N, Meng XY, et al. (2009) “Candidatus Curculioniphilus buchneri,” a novel clade of bacterial endocellular symbionts from weevils of the genus Curculio. Appl Environ Microbiol 76: 275–282.
- 23. Toju H, Fukatsu T (2011) Diversity and infection prevalence of endosymbionts in natural populations of the chestnut weevil: relevance of local climate and host plants. Mol Ecol 20: 853–868.
- 24. Toh H, Weiss BL, Perkin SA, Yamashita A, Oshima K, et al. (2006) Massive genome erosion and functional adaptations provide insights into the symbiotic lifestyle of Sodalis glossinidius in the tsetse host. Genome Res 16: 149–156.
- 25. Belda E, Moya A, Bentley S, Silva FJ (2010) Mobile genetic element proliferation and gene inactivation impact over the genome structure and metabolic capabilities of Sodalis glossinidius, the secondary endosymbiont of tsetse flies. BMC Genomics 11: 449.
- 26. Nevers P, Saedler H (1977) Transposable genetic elements as agents of gene instability and chromosomal rearrangements. Nature 268: 109–115.
- 27. Bickhart DM, Gogarten JP, Lapierre P, Tisa LS, Normand P, et al. (2009) Insertion sequence content reflects genome plasticity in strains of the root nodule actinobacterium Frankia. BMC Genomics 10: 468.
- 28. Parkhill J, Berry C (2003) Genomics: Relative pathogenic values. Nature 423: 23–25.
- 29. Tampakaki AP, Skandalis N, Gazi AD, Bastaki MN, Sarris PF, et al. (2010) Playing the “Harp”: evolution of our understanding of hrp/hrc genes. Annu Rev Phytopathol 48: 347–370.
- 30. Burke GR, Moran NA (2011) Massive genomic decay in Serratia symbiotica, a recently evolved symbiont of aphids. Genome Biol Evol 3: 195–208.
- 31. McCutcheon JP, Moran NA (2011) Extreme genome reduction in symbiotic bacteria. Nat Rev Microbiol 10: 13–26.
- 32. Tarchini R, Biddle P, Wineland R, Tingey S, Rafalski A (2000) The complete sequence of 340 kb of DNA around the rice Adh1–adh2 region reveals interrupted colinearity with maize chromosome 4. Plant Cell 12: 381–391.
- 33. Feschotte C, Mouchès C (2000) Recent amplification of miniature inverted-repeat transposable elements in the vector mosquito Culex pipiens: characterization of the Mimo family. Gene 250: 109–116.
- 34. Jurka J, Kohany O, Pavlicek A, Kapitonov VV, Jurka MV (2004) Duplication, coclustering, and selection of human Alu retrotransposons. Proc Natl Acad Sci U S A 101: 1268–1272.
- 35. Toju H, Fukatsu T (2011) Diversity and infection prevalence of endosymbionts in natural populations of the chestnut weevil: relevance of local climate and host plants. Mol Ecol 20: 853–868.
- 36. Kaiwa N, Hosokawa T, Kikuchi Y, Nikoh N, Meng XY, et al. (2010) Primary gut symbiont and secondary, Sodalis-allied symbiont of the Scutellerid stinkbug Cantao ocellatus. Appl Environ Microbiol 76: 3486–3494.
- 37. Grünwald S, Pilhofer M, Höll W (2010) Microbial associations in gut systems of wood- and bark-inhabiting longhorned beetles [Coleoptera: Cerambycidae]. Syst Appl Microbiol 33: 25–34.
- 38. Lefèvre C, Charles H, Vallier A, Delobel B, Farrell B, et al. (2004) Endosymbiont phylogenesis in the dryophthoridae weevils: evidence for bacterial replacement. Mol Biol Evol 21: 965–973.
- 39. Conord C, Despres L, Vallier A, Balmand S, Miquel C, et al. (2008) Long-term evolutionary stability of bacterial endosymbiosis in curculionoidea: additional evidence of symbiont replacement in the dryophthoridae family. Mol Biol Evol 25: 859–868.
- 40. Morelli G, Didelot X, Kusecek B, Schwarz S, Bahlawane C, et al. (2010) Microevolution of Helicobacter pylori during prolonged infection of single hosts and within families. PLoS Genet 6: e1001036
- 41. Moran NA, McLaughlin H, Sorek R (2009) The Dynamics and Time Scale of Ongoing Genomic Erosion in Symbiotic Bacteria. Science 323: 379–382.
- 42. Green J, Bohannan BJ (2006) Spatial scaling of microbial diversity. Trends Ecol Evol 21: 501–507.
- 43. Nadarasah G, Stavrinides J (2011) Insects as alternative hosts for phytopathogenic bacteria. FEMS Microbiol Rev 35: 555–575.
- 44. Stavrinides J, No A, Ochman H (2010) A single genetic locus in the phytopathogen Pantoea stewartii enables gut colonization and pathogenicity in an insect host. Environ Microbiol 12: 147–155.
- 45. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797.
- 46. Guindon S, Gascuel O (2001) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696–704.
- 47. Hasegawa M, Kishino H, Yamo T (1985) Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol 22: 160–174.
- 48. Mullikin JC, Ning Z (2003) The phusion assembler. Genome Res 13: 81–90.
- 49. Gordon D, Abajian C, Green P (1998) Consed: a graphical tool for sequence finishing. Genome Res 8: 195–202.
- 50. Robb FT, Maeder DL, Brown JR, DiRuggiero J, Stump MD, et al. (2001) Genomic sequence of hyperthermophile, Pyrococcus furiosus: implications for physiology and enzymology. Methods Enzymol 330: 134–157.
- 51. Blankenberg D, Von Kuster G, Coraor N, Ananda G, Lazarus R, et al. (2010) Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol 19: Unit 19.10.1–21.
- 52. Goecks J, Nekrutenko A, Taylor J (2010) Galaxy Team (2010) Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 11: R86.
- 53. Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18: 821–829.
- 54. Lukashin AV, Borodovsky M (1998) GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res 26: 1107–1115.
- 55. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, et al. (2000) Artemis: sequence visualization and annotation. Bioinformatics 16: 944–945.
- 56. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, et al. (2003) The COG database: and updated version includes eukaryotes. BMC Bioinformatics 4: 41.
- 57. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, et al. (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19: 1639–1645.


text:
Kunkletown nursing home missing tools
The Pennsylvania State Police reported that multiple power tools and standard tools were stolen from a nursing home in Kunkletown on Thursday.
A Dewalt hammer drill, three Sawzall tools and a variety of other tools were taken from Getz Personal Care Home on Scenic Drive sometime between 11 a.m. and noon.
If anyone has more information about this incident, contact Trooper Flynn in Lehighton at 610-377-4270.
We reserve the right to remove any content at any time from this Community, including without limitation if it violates the Community Rules. We ask that you report content that you in good faith believe violates the above rules by clicking the Flag link next to the offending comment or fill out this form. New comments are only accepted for two weeks from the date of publication.

domain: law


The Judicial Service Commission (JSC) has unveiled a list of shortlisted candidates for jobs they advertised in October 2021.
In a statement released by Chief Justice Martha Koome on March 10, it was revealed that the 500 vacant slots for the position of Court Assistant II had attracted over 50,000 applications from Kenyans.
At the time, it was revealed that the monthly gross salary for the position ranged from Ksh61,400 to Ksh79,300.
“JSC would like to notify applicants and the members of the public in general that the shortlisting process for the positions that were advertised on October 8, 2021 has been completed after a thorough evaluation of the applications.
“Among positions that attracted a high number of applicants include that of Court Assistant II which has 57,245 applications,” read the statement by the CJ.
How to Check Shortlisting and Interview Requirements
Kenyans who applied for the position have been advised to visit the JSC website using the link, to check if they were shortlisted for the job.
Upon opening the page, applicants are advised to check for their names by carrying out a search using their National Identification (ID) numbers.
“You can search for an applicant by ID number or name on the webpage. A yellow highlight indicates a match. To find a word or phrase quickly, use the keyboard shortcuts Ctrl + f (Windows, Linux, and Chrome OS) or + ⌘ f (Mac OS) (Mac).
“Yellow markers appear on the right scrollbar, indicating the location of all of the matches on the page,” read the statement.
Additionally, JSC announced that interviews will begin from March 15 to April 14 at the Kenya School of Monetary Studies located along Thika road.
Apart from getting clearance from the Credit Reference Bureau (CRB), shortlisted candidates were also advised to carry with them clearance certificates from the Kenya Revenue Authority (KRA), Higher Education Loans Board (HELB), Directorate of Criminal Investigations (DCI), and the Ethics and Anti-Corruption Commission (EACC).
“Shortlisted candidates are requested to read the schedule carefully and adhere to the timelines. Specific venues will be notified to candidates on arrival at the venue.
Candidates are asked to bring their original certificates and must remember to take them back after the interview,” read the statement in part.


Cats have been companions of mankind for thousands of years. However, misunderstandings persist between the two species. A new study published on May 3, 2023 in the specialized journal animals provides a boost for a better understanding.
A still incomplete understanding
Pets often spend more time with a human than with their fellow animals. It then becomes necessary to understand each other better in order to live in good harmony. And like dogs, cats have developed a special sensitivity to communication signals emitted by humans. “For example, cats can read human pointing gestures to locate hidden food and follow human gaze to get reference information.“, details the new study. They are also able to distinguish our expressions.
As for us, we are still a long way from being able to read them like an open book. Thus, the study of communication signals between cats and humans remains modest. Do they prefer voice-only communication? Are they more comfortable with a fleeting gaze or with a frank gaze? Charlotte de Mouzon and Gérard Leboucher, two researchers from the Ethology Cognition Development Laboratory (Paris Nanterre University), conducted the survey on an ideal field of study: cat cafes located in Bordeaux and Toulouse.
Read alsoCats attach themselves to us like babies
Uncomfortable cats facing a curious situation
The two scientists examined three types of communication: vocal (the cat is called by its first name, but eye contact is avoided), visual (the experimenter presents his hands to the animal and alternates gazes turned towards him and towards the floor) or bimodal, i.e. both visual and vocal. For this, they sifted through the videos of 12 felines (6 males and 6 females) living in cafes and filmed when the establishments were not yet open to the public. During a control condition, the experimenter emitted no communication signal to the animal.
Read also“Despite our fascination with cats, we still have a lot to learn about them”
Depending on the method of communication used, the cat took more or less time to approach. Thus, the results show that these felines interacted more quickly when the communication was visual or bimodal, but were more reserved when the person did not seek to communicate with them or when he only used his voice for that. This last observation perhaps reveals the hesitation of cats in the face of a curious situation: very often, a human who calls them also applies to look at them.
“Knowing the importance of vocal communication between cats and their humans, we expected a greater importance of vocal signals, remark to Science and Future Dr. Charlotte de Mouzon. Our results highlight, once again, the great adaptive capacity of cats who could adjust their use of different communication channels depending on the human they are interacting with.”
Read alsoTraces of an elusive species of feline discovered on Everest
In addition, the researchers noticed that the cats wagged their tails significantly more when the experimenter ignored them, compared to the “visual” and “bimodal” conditions. They also waggled their tail more during vocal communication compared to bimodal one. A behavior adopted when felines are faced with a frustrating situation.
Still a long way to fully understand cats
This study therefore highlights that cats prefer either visual or visual and vocal communication when in contact with strangers. The two scientists therefore encourage people “use appropriate visual communication signals when interacting with cats, especially if they are unfamiliar“Indeed, different results might have been obtained with a human familiar to them.”We had seen in a previous study that cats did not react the same way to the voice of their human as to that of a stranger.“, specifies the researcher. Thus, future research on the cat-owner couple could reveal another dynamic and allow us to improve our knowledge even further.
Read alsoThe intriguing “cat-fox” is indeed a specific feline of Corsica
“Our understanding of cats is improving as humans are increasingly attentive to animal welfare, rejoices Dr. de Mouzon. But there is still a little way to go, so it is important to be able to give the floor to cat behavior specialists, so that they can make the general public aware of their needs and behaviors.“. And to add: “However, with the growing attachment of humans to their cats – and this is a very good thing – we must remain vigilant against anthropomorphic excesses.“, she warns. Understanding the cat also involves respecting its nature.


Latest Tenders for Musical Instruments & Accessories
Music is the essence of life. It speaks what can not be expressed. Musical Instrument industries cater to wide range of consumer base. Analysts are predicting several prime area of growth in retail sales of musical instruments. Get all the latest information on Indian tenders for Musical Instruments & Accessories at Indiamart.


text:
Title: Diagnosis and Classification of 17 Diseases from 1404 Subjects via Pattern Analysis of Exhaled Molecules
Authors: Hossam Haick et al.
Publication Info: ACS Nano, 2017, 11 (1), pp 112–125 DOI: 10.1021/acsnano.6b04930
This work reports on the development of a portable, inexpensive, and non-invasive breath analyzer that couples the use basic fundamental chemical principles with pattern recognition algorithms to simultaneously detect and discriminate between 17 different diseases including various forms of cancer and Parkinson’s disease.
Since antiquity breath odor has been employed to assess human health. Through normal metabolic activity endogenous volatile organic compounds are released into the bloodstream and subsequently excreted through exhalation. As breath analysis has evolved over the years, specific relative combinations of certain exhaled organic compounds have been identified as biomarkers for various health conditions.
While breath analysis has become a valid health diagnostic tool, its ubiquity is currently hindered by a few aspects that this work seeks to overcome. It typically requires patients to visit clinics staffed with trained technicians to perform the diagnostic and maintain, calibrate, and operate costly and sensitive instrumentation – typically a mass spectrometer coupled to a gas chromatography implement. The inexpensive and disposable breath sensors proposed in this work will enable health assessment in the convenience of one’s own home – like a home pregnancy test or blood insulin level test.
The breath analyzers for this work were fabricated on inexpensive silicon wafers and the entire footprint of a sensor is about the size of a fingernail. The actual sensing component is an array of 3 millimeter diameter discs made conductive through drop-casting a small quantity of gold nanoparticles or carbon nanotubes from solution. Each disc in the array is made unique through molecular modification with different small molecules such as dodecanethiol, chlorobenzene, or methanethiol.
Various electrical responses (such as resistance, impedance, and time-resolved resistance and impedance) measured across each modified pad provide the sensing outputs of the breath analyzer. When a sample of breath passes over the conductive pads, the various organic compounds present can induce measurable changes in the electrical properties of the conductive pads. In response to the types, extent, and timescales of basic intermolecular forces (i.e. dispersion, dipole-dipole, and hydrogen bonding interactions) that occur between the endogenous organic compounds exhaled in the breath and the molecular modifiers on the conductive pads, the electrical characteristics of the pads can be altered and readily measured. For example, significant sorption of a ketone in the breath may occur at a pad functionalized with an amide group. Inclusion of this ketone analyte may cause swelling of the molecular layers which function to bridge the individual gold nanoparticles, and thus changing the electrical properties (e.g. the conductivity, dielectric constant, impedance, etc.) of that specific pad over a period of time until the analyte desorbs back into the flowing air stream.
The analysis component of this sensor is an interesting attempt at biomimicry of the human olfactory system. The human olfactory system is composed of ~350 different olfactory receptors and and each of these receptor types exhibit subtle differences in composition and structure. None of these receptors are exclusively selective for any single odorant (e.g. there is not a specific receptor for cinnamaldehyde). Each receptor type actually exhibits relatively poor selectivity and have evolved compositional and structural features that enable a wide range of intermolecular interactions to occur between it and the odorant. An electrical response based on the strength and dynamics of the receptor-odorant interactions is conducted to the brain from the array of olfactory neurons. It is the simultaneous combination of electrical responses of many neurons, not a single neuron, that allow the brain to amazingly identify a specific odorant. Smell does not occur in the nose, but in the brain!
In this sensor platform, the array of olfactory neurons composing the human olfactory system are synthesized by the uniquely functionalized discs, and the brain functionality is synthesized by a software artificial neural network that is trained with breath samples of patients with previously known health status. While this report emphasis the pattern recognition algorithms and information processing employed to detect disease biomarkers in complex breath samples, the root of this work is in the fundamental basic chemistry that enables it all – intermolecular interactions between the functionalized pads and various organic compounds in the breath.

domain: chemistry


BILCO Size BR-2 Sloped Wall Steel Cellar Door - Primer Finish
BR-2 Sloped Wall Cellar Doors:
- Door Opening Size W40" x L60-1/4" | Overall Door Size: W 47-3/16" x L67-5/8"
- Heavy Gauge Steel with Flanged Construction
- Lift Assistance w/ Supplemental Coil Springs
- Sturdy Concealed Hinges
- Baked-on Sherwin Williams Primer
The BR Sloped Wall Cellar Door is the ideal solution for any basement opening. This heavy gauge steel door is built with a flanged construction for maximum strength and comes complete with gas spring operators and supplemental coil springs for lift assistance. The sturdy concealed hinges and baked-on Sherwin Williams primer provide an extra layer of strength and protection. Installation is quick and easy, making the BR-2 the perfect choice for any home or business. With its heavy-duty design and superior construction, the BR Sloped Wall Basement Door is sure to last for years to come.
BILCO Basement Door Features
- BR Sloped Wall Basement Doors provide an inside opening width of 40 inches and an inside opening length of 60.25 inches, allowing for easy access.
- Gas Spring Operators and Supplemental Coil Springs provide lift assistance, allowing for easy opening and closing.
- Flanged construction and heavy gauge steel provide a sturdy, durable design.
- Sturdy concealed hinges and a baked-on Sherwin Williams Primer ensure maximum protection from the elements.
- Installation is quick and easy, thanks to pre-drilled holes and simple instructions.


text:
Google removes underlined links. My teenage spirit wails.
Google has gone ahead and removed the underlined links from their search results. It’s a knife into the gut of my teenage years. Such links! So Geocities. Fuck, man. Time waits for no one, especially the Old Internet. Where format was madness, everyone had a website with their own noodlings, and I could find a fucking link by looking for the underlined text.
Google started life as a research project in 1996 to crawl the internet and create a search engine. 18 years later, Google is now removing the last of the design left over from that era. Starting today, the ’90s-style underlined links are being removed from Google search results. It truly marks the end of an era of the web, with underlined links a familiar method used by web developers to highlight links on sites like Geocities, Altavista, and pets.com. It’s the biggest visual change today, but Google is also tweaking other parts of its desktop search results.
“We’ve increased the size of result titles, removed the underlines, and evened out all the line heights,” says Google lead designer Jon Wiley. “This improves readability and creates an overall cleaner look.” Google is also bringing over the new ad labels from its mobile site, and the whole search interface looks a little more consistent with its mobile and app variants. The changes might seem minor, but for Google it’s a significant change to a search engine that rarely changes its design. It also matches more modern touch-friendly sites that have opted for increased page elements as more consumers move towards tablets and smartphones. It’s not the drastic homepage redesign that Google trialed for less than a day back in 2010, but today signals the end of Google’s 1990’s web era just as the World Wide Web celebrates its 25th anniversary. [The Verge]

domain: news


text:
Market Optimism Boosting Sterling
The Pound continued to make headway yesterday as better than expected inflation data and speculation that the Bank of England would take a less dovish (low interest rates) stance provided support. Also, vaccine rollout optimism and global risk appetite continued to reinforce support. The Euro fell to below 1.1300, 8 month lows, and Sterling re-tested the 1.3700 mark against the Dollar but failure helped trigger a significant correction.
Bank of England Governor, Andrew Bailey, opined that economic fallout would not be severe as after the 1980’s recession and noted that inflation was still very low and there’s uncertainty over what people will do with increased savings. Finally, he added that no decision had been made on negative interest rates. The Pound consolidated near 1.3650 on the Dollar and the Euro recovered to 1.1273.
Global risk appetite remains strong and Sterling tests the 1.3700 mark again this morning and the Euro again weakening to near 1.1300.
The US NAHB housing index retreated to 83 for January from 86 the previous month and compared with expectations of an unchanged reading. Higher yields and fresh coronavirus restrictions may have had an impact, although confidence remained very high in historic terms.
US equity futures held gains into yesterday’s New York open amid positive risk appetite. Wall Street equities posted fresh record highs amid expectations of further strong fiscal support and a very accommodative Federal Reserve (Fed) policy. Although equities moved higher, there was no increase in bond yields which curbed potential dollar support.
There was relief that President Biden’s inauguration passed with no significant incident. As expected, Biden issued a series of executive orders to reverse many of Trump’s policies with the mandating of mask wearing in federal buildings.
The Euro’s rally against the Dollar has stalled over the last two weeks, with the potential for the pair to suffer deeper losses if the ECB expresses displeasure over the single currency’s strength at Policy meeting.
Currently, the currency pair is trading near 1.2125, having pulled back from multi-month highs around the 1.2350 earlier this month. The central bank is widely expected to keep the interest rate and the asset purchase program unchanged, having partly boosted the level of policy accommodation until early 2022 in December. However, the possibility of the central bank jawboning the currency cannot be ruled out.
Data to watch
07:45 – EUR – Monetary Policy Statement
07:45 – EUR – Main Refinancing Rate
08:30 – EUR – ECB Press Conference
08:30 – USD – Philly Fed Manufacturing Index
08:30 – USD – Unemployment Claims


HYATTSVILLE – Record turnout for Hyattsville’s May 2 election led to little surprises as three incumbents won their races. However, two new faces will be joining the council from Wards 3 and 5. Approximately 1,575 Hyattsville residents took to the polls last Tuesday to elect the councilmember of their choosing and voice their opinion on […]
HYATTSVILLE – Record turnout for Hyattsville’s May 2 election led to little surprises as three incumbents won their races. However, two new faces will be joining the council from Wards 3 and 5.
Approximately 1,575 Hyattsville residents took to the polls last Tuesday to elect the councilmember of their choosing and voice their opinion on the city’s non-binding referendum.
Among the myriad of decisions the public made, Wards 3 and 5 had to pick between three brand new candidates for the open seats.
Ward 5 Councilwoman Ruth Ann Frazier and Ward 3 Councilman Patrick Paschall both stepped away from the council through not seeking reelection. That gave a path for two new faces to join the seasoned Hyattsville City Council.
“It’s surreal,” said Erica Spell, Ward 5’s new councilwoman. “I’m a bit speechless, but I’m just so thrilled.”
For the first few minutes after the election results were announced, Spell did not quite know what happened; she was not aware she had won the Ward 5 Hyattsville City Council seat.
“It just took me a while to process it all, because I’ve been working day-in and day-out doing everything I could possibly do. So, it took me a while to register,” she said.
Spell won the ward’s seat with 78 total votes – just 27 more votes than second place contender Ben Zeitler. Derrika Durant, the third resident vying for the seat, pulled in four votes.
Both Spell and Carrianna Suiter, the newcomer who won the open Ward 3 seat, were filled with emotions after the election results were announced.
Suiter hugged her husband and accepted congratulations while holding back tears.
“I am so excited and I, honestly, just finishing eight years of federal service, I couldn’t think of a better way to spend the next four years of my life than giving it back to this community,” she said.
Suiter raked in 138 votes in the election, surpassing both Vinni Anandham, who received 19 votes, and Ayanna Shivers, who brought home 85.
Suiter spent the last decade working for former President Barack Obama. She said she decided to run for the council after the most recent national election, which she said made it “even more important to engage in a local way.”
That was a common theme among the 12 candidates on the May 2 ballot. This year’s Hyattsville election saw six women throw their hats into the ring, which both Spell and Suiter said was inspiring.
“I feel incredibly empowered by the strong slate of candidates that Ward 3 brought forth and I certainly am looking forward to bringing that work to the council,” Suiter said.
The May 2 election also saw the reelection of three sitting council members, Ward 1’s Bart Lawrence, Ward 2’s Robert Croslin and Ward 4’s Edouard Haba.
Ward 1, though loud and contested, shaped up to be quite the run-away victory for Lawrence.
“I am surprised. I had no idea. I just had no idea,” Lawrence said. “I had no idea that that many people would come out to vote for me.”
Twenty-four percent of the ward showed up and showed out for the election. Contenders for the seat included Lawrence, who won with 466 votes, Talib Karim, who gathered 168, and Ian Herron with 50 total.
The Ward 1 race was perhaps the most contentious of the five races, with numerous councilmembers backing Lawrence and Mayor Candace Hollingsworth herself getting involved in campaigning for the incumbent.
“I ran the first time and it was pretty much just me. It was a totally different race. This time I had great friends, incredible support. It was just amazing,” he said. “I think this will make me a better council member and things will be better moving forward.”
Croslin ran unopposed in Ward 2, though there were 20 write-in votes in the race that included multiple votes for Ray Caron and Emily Strab. One voter even said “not Croslin, anyone but Croslin.”
Still, Croslin brought in 352 votes from the 15 percent of his ward that voted.
“I feel good,” he said. “It was good to stand out there and talk to all my neighbors, and feeling the support from my community was great. It was wonderful.”
Haba said he felt similarly, though his ward only saw an 8 percent turnout. Haba won his reelection with 106 votes to his opponent, Shirley Ann Bender’s, 16 total votes.
“I’m feeling happy, excited and encouraged because this is a statement that what we have been doing is working and residents are listening to what we do and they’re watching what we do,” he said. “It says we’re on the right track, but we need to keep working.”
Haba also called the election a “double-win” for him because of the overwhelming response to the city’s referendum question.
With 15 percent of the city’s 10,000 residents taking to the polls, most races were handily decided. However one part of the multi-part referendum question was decided by just one vote.
The referendum question focused on the size and makeup of the council and whether the city’s residents wanted to see the size or composition change in the coming years.
Residents came out in force against any reduction in size of the council, voting 947 to 609 against question one, which asked if the council should consider reducing the number.
For Part A of the second question, which asked if there should be a reduction to one councilmember for each of the five wards, 581 voted in favor, 768 voted against. Similarly, in Part B, which suggested reducing the number of members per ward and reducing the number of wards, residents voted 121 for and 1,186 against.
Part C suggested reducing the number to one councilmember per ward, but increasing the number of wards. That question had 403 in favor, but 942 opposed. Part D asked if there should be a reduction in wards, but allow for two councilmembers per ward. That question was also opposed with 986 against and 358 for.
“I was opposed to the questions from the beginning,” Haba said. “The way it’s written, I didn’t think it was fair and I said that to residents.”
Haba said the questions were one-sided and leaned only toward a reduction, when he believes the council’s issues can be solved by agreeing to “work together better as a team.”
The last question on the ballot asked if the city should have “one or more councilmembers elected at large by all the city voters.” The “ayes” for this ballot question won out by just one vote, 723-722.


text:
Every single wall art are designed by their unique model and identity. It's fine to use a classy accent to your decor by incorporating wall art into your style. You are able to enhance the wall art in your house that suitable with your personal taste that designed by their beautiful and attractive look.
Consider the room you want to put the wall art. Have you got a large space that will require outdoor wall art decors for this you need to find the right for your space, or are you experiencing a tiny space? That you don't wish to buy wall art that won't proper, therefore make sure you calculate your room to check out free area just before buy it. When you've finished of the designated room, you can begin your search.
Each wall art are exciting that may be used in any space or room of the house. These are one of cheap option to give a old room a mini-renovation to get a fresh look and nuance in your room. Decorating with outdoor wall art decors is ideal for each people, of every age, as well as for any interior design which you want along with change the design of the room.
All of the outdoor wall art decors can allow you to set up exclusive style and create different nuance for the home. Using wall art for your room will let you to set up the perfect ambiance for each room. Analyzing the style of your home before shopping wall art may also enable you to find the correct model, size, measurement, and shape for the room.
- Fashionable Large Outdoor Wall Art Metal House Outside Design Pictures Extra With Outdoor Wall Art Decors View Photo 1 of 20
- Most Popular Outdoor Wall Art Decors Within Wall Decor For Outdoor Patios Enthralling Patio At Elegant Art Ideas View Photo 2 of 20
- 2018 Outdoor Wall Art Decors With Regard To Sofa Ideas View Photo 3 of 20
- Outdoor Wall Art Decors Pertaining To Trendy How To Hang Decor On Vinyl Siding Our Storied Home Shel Silverstein View Photo 4 of 20
- Outdoor Wall Art Decors Within Well Known Metal Wall Art Outdoor Perth – Outdoor Designs View Photo 5 of 20
- Well Known Exterior Metal Wall Art Cushty Panels Outdoor Uk Home Fullsize Of Within Outdoor Wall Art Decors View Photo 6 of 20
- Well Liked Garden Wall Plaques Outdoor Outside Wall Art Decor Large Wall With Regarding Outdoor Wall Art Decors View Photo 7 of 20
- Widely Used Outdoor Wall Art Decors Pertaining To Outdoor Wall Art Sun View Photo 8 of 20
- Best Of Wall Art Ideas For Garden View Photo 9 of 20
- Amazing Outdoor Patio Wall Decor Ideas U Pic For Decorations And Pertaining To Favorite Outdoor Wall Art Decors View Photo 10 of 20
- Popular Wrought Iron Wall Decor Large Metal Wall Art Decor Large Metal Wall Intended For Outdoor Wall Art Decors View Photo 11 of 20
- Fancy Outdoor Coastal Wall Decor Gift Wall Art Ideas Dochistafo In Preferred Outdoor Wall Art Decors View Photo 12 of 20
- Outdoor Wall Art Decors Throughout Most Up To Date Amazon: Deco 79 63533 Metal Fish Wall Decor: Home & Kitchen View Photo 13 of 20
- Well Known Outdoor Wall Art Decors With 34 Best Porch Wall Decor Ideas And Designs For View Photo 14 of 20
- Reflect The Home Owner's Creative Personality With These 12 Outdoor With Newest Outdoor Wall Art Decors View Photo 15 of 20
- Outdoor Wall Art Decors For Newest Wrought Iron Garden Decor Wrought Iron Garden Accessories Cape Town View Photo 16 of 20
- Outdoor Wall Art Decors With Regard To Newest Metal Butterfly Wall Decor – Talentneeds – View Photo 17 of 20
- Outdoor Metal Wall Art Design Ideas — Indoor & Outdoor Decor In Most Up To Date Outdoor Wall Art Decors View Photo 18 of 20
- Most Recent Outdoor Wall Art Decors Throughout Outside Wall Decoration – Jscollectionofficial View Photo 19 of 20
- Outdoor Wall Art Decors In Newest Metal Wall Art – Walmart View Photo 20 of 20
Are you currently looking for out where to buy wall art for your home? The good news is here offers anything you covered with numerous outdoor wall art decors for your home! Decorating with wall art is really a fun way to give a different appearance to a space, or bring in a unique style. Also you will discover wall art in numerous dimensions and in a variety of patterns, colors, shapes and variations.
So there is likely outdoor wall art decors can be a preferred choice and come in equally large and small shapes. Apply wall art as accent could certainly creates you to be unique element for your home and makes you more satisfied with your space!
Using outdoor wall art decors to your interior may allow you to change the area in your house and focus on numerous appearance that you have on display. Most wall art are ideal for setting the nuance in specific rooms as well as applying for others purposes. In advance of ordering wall art, be sure you match their design and texture with the existing model in your home. It is possible to search wide selection of wall art to find the good for your house.
Each and every outdoor wall art decors are available various size, dimensions, patterns, models, variations, and designs, that is helping to make them a perfect alternative to makeover your previous design. Feel free to use ideas from the current interior decoration to get wall art for your home. Each wall art comes with an iconic styles and shapes that every homeowner may get pleasure from it! They are also great makes the environment of an area very much more wonderful.

domain: artistic


text:
Find Perfect Pairs Game
Do you guys want to test how careful you are and how quickly you can remember the location of objects? With today's game, you will have such an opportunity! We have prepared for you an exciting game, where you will look for perfect pairs, or rather identical pictures. Well, are you ready? Then let's try! On the first level in front of you there will be four cards and hint with which you need to remember the meaning of the game. But in the next level the number of cards will be increased, which means that to remember the location of similar ones, you will have more difficulties. Once the level starts, all pictures and their location you will see for a few seconds, after that they will close and you will have to find all pairs from memory. If you suddenly forgot, don't worry, click on all the pictures, then try to remember what is on it and keep looking for the similar one. Let's test your attention together and have fun. Good luck!

domain: hobby


text:
Massachusetts Man Sentenced to Almost 4 Years
on Federal Firearm Charge
Contact: Jonathan R. Chapman
Assistant United States Attorney
Tel: (207) 780-3257
Portland, Maine: United States Attorney Thomas E. Delahanty II announced that Jamie
Figueroa, 22, of Lawrence, Massachusetts, was sentenced today in U.S. District Court by Judge
D. Brock Hornby to 46 months of imprisonment to be followed by three years of supervised
release for possession of a firearm by a felon. Figueroa pled guilty to the charge on November
According to court records, on September 2, 2013, Figueroa, a convicted felon since
2012, was arrested by Windham, Maine police officers. The police officers had responded to a
call regarding a residential disturbance and had obtained a description of the vehicle used by the
suspects. Officers located the vehicle, which had been reported stolen in New Hampshire,
stopped in the driveway of another residence in Windham. At gunpoint, officers ordered
Figueroa, who was the driver, out of the vehicle and removed a loaded revolver from his coat
The investigation was conducted by the Bureau of Alcohol, Tobacco, Firearms and
Explosives and the Windham, Maine Police Department.
Training and seminars for Federal, State, and Local Law Enforcement Agencies.
Help us combat the proliferation of sexual exploitation crimes against children.
Our nation-wide commitment to reducing gun crime in America.
Making sure that victims of federal crimes are treated with compassion, fairness and respect.


text:
How do you find
Wuhan Biet Co., Ltd.
WUHAN BIET CO.,LTD was set up in July 2010.It specializes in theproduction,research,development and
Rech Chemical Co., Ltd.
Twenties years ago, Rech Chemical Co.,Ltd were mainly a solid manganese sulphate supplier. Today, b
Giga Fine Chem.
Giga Fine Chem. is over 40 years history and headquarters in Taipei. Main business is to do the fun
Alabiochem company locates at a beautiful chinese city-SuZhou. We are a professional biochemical co
Autech Industry Co.,Lim
Autech Industry Co., Ltd was established in 2006, the head office is in Jinan City, Shandong Provin
Nanjing Shunxiang Pharm
Shunxiang is a comprehensive company specializing in the development, production and marketing of n


text:
view from near Dublin flea market,Dublin 8
The two above are from photos taken at Experience Farmleigh in Phoenix Park in 2011.
This is a statue in the gardens of the Imma.
This is a statue in the Iveagh Gardens.
This was a robot at Kings of concrete which does be at wood Quay a few times a year.
This is a sand sculpture by a group of irish sculptors. They sometimes do sand sculptures in the ground of Dublin Castle in August. Duthain Dealbh is the irish phrase meaning fleeting sculptures. I really like some of the things they've made in the past. If you want to see more of their work, i think that their website is www.duthaindealbh.com.
The above picture was done in the cafe of Hugh Lane Gallery.
The above two pictures are both of statues. The first statue was in the gardens of the IMMA, the second is from the National Art Gallery.


text:
When asked by an audience member to reprise a performance of the song "Confrontation" from Les Miserables, the two did not miss a beat. Harris is a very convincing Javert as Segel is a Jean Valjean.
|comments powered by Disqus|
(Shane Keyser/Kansas City Star)
A feral cat who came to stay
Reader recounts her experience with a stray cat that showed up and made herself at home 13 years ago. Advice from an expert has helped her keep the cat healthy all these years.
Cindy Crawford viral 'real' photo a fake
Photographer from 2013 Marie Clare photo shoot says picture was stolen and altered.

domain: entertainment


text:
Unlike the rest of us, Arizona Congressman Ben Quayle can rest easy -- Texas tough guy Chuck Norris doesn't want to kick his ass. In fact, the played-out punchline of endless jokes is diggin' him some Ben Quayle.
The man who will "never have a heart attack because his heart isn't foolish enough to attack him" apparently considers himself an authority on border violence, and recently penned an article for the website Townhall.com, in which he touts the freshman Arizona congressman's assessment of Homeland Security Director Janet Napolitano's take on border violence.
Check it out below:
I agree with Rep. Ben Quayle, R-Ariz., who said that for cattle ranchers, the daily reality of drug and human smugglers traversing their property is "far more impacting" than Napolitano conveys. Quayle went on to say, "Statistics and averages might mean something to government bureaucrats and analysts in Washington, but try telling the people who deal with these realities every day that the violence along the border has subsided.
The Delta Force star is referring to an op-ed Quayle wrote that appeared in the Arizona Republic earlier this month.
Check it out here.
Quayle's piece points out nothing new. It's a fairly obvious critique of Napolitano's take on border violence that's been echoed by Republicans from Phoenix to Philadelphia -- and that's about as critical as we feel comfortable being now that we know Chuck Norris has Quayle's back.