Determination of Wolbachia diversity in butterflies from Western Ghats, India, by a multigene approach

Members of the genus Wolbachia are intracellular bacteria that are widespread in arthropods and establish diverse symbiotic associations with their hosts, ranging from mutualism to parasitism. Here we present the first detailed analyses of Wolbachia in butterflies from India with screening of 56 species. Twenty-nine species (52%) representing five families were positive for Wolbachia. This is the first report of Wolbachia infection in 27 of the 29 species; the other two were reported previously. This study also provides the first evidence of infection in the family Papilionidae. A striking diversity was observed among Wolbachia strains in butterfly hosts based on five multilocus sequence typing (MLST) genes, with 15 different sequence types (STs). Thirteen STs are new to the MLST database, whereas ST41 and ST125 were reported earlier. Some of the same host species from this study carried distinctly different Wolbachia strains, whereas the same or different butterfly hosts also harbored closely related Wolbachia strains. Butterfly-associated STs in the Indian sample originated by recombination and point mutation, further supporting the role of both processes in generating Wolbachia diversity. Recombination was detected only among the STs in this study and not in those from the MLST database. Most of the strains were remarkably similar in their wsp genotype, despite divergence in MLST. Only two wsp alleles were found among 25 individuals with complete hypervariable region (HVR) peptide profiles. Although both wsp and MLST show variability, MLST gives better separation between the strains. Completely different STs were characterized for the individuals sharing the same wsp alleles.     

Allele intersection analysis: a novel tool for multi locus sequence assignment in multiply infected hosts

Wolbachia are wide-spread, endogenous α-Proteobacteria of arthropods and filarial nematodes. 15-75% of all insect species are infected with these endosymbionts that alter their host's reproduction to facilitate their spread. In recent years, many insect species infected with multiple Wolbachia strains have been identified. As the endosymbionts are not cultivable outside living cells, strain typing relies on molecular methods. A Multi Locus Sequence Typing (MLST) system was established for standardizing Wolbachia strain identification. However, MLST requires hosts to harbour individual and not multiple strains of supergroups without recombination. This study revisits the applicability of the current MLST protocols and introduces Allele Intersection Analysis (AIA) as a novel approach. AIA utilizes natural variations in infection patterns and allows correct strain assignment of MLST alleles in multiply infected host species without the need of artificial strain segregation. AIA identifies pairs of multiply infected individuals that share Wolbachia and differ in only one strain. In such pairs, the shared MLST sequences can be used to assign alleles to distinct strains. Furthermore, AIA is a powerful tool to detect recombination events. The underlying principle of AIA may easily be adopted for MLST approaches in other uncultivable bacterial genera that occur as multiple strain infections and the concept may find application in metagenomic high-throughput parallel sequencing projects.     

Insight into the routes of Wolbachia invasion: high levels of horizontal transfer in the spider genus Agelenopsis revealed by Wolbachia strain and mitochondrial DNA diversity

The pandemic distribution of Wolbachia (alpha-proteobacteria) across arthropods is largely due to the ability of these maternally inherited endosymbionts to successfully shift hosts across species boundaries. Yet it remains unclear whether Wolbachia has preferential routes of transfer among species. Here, we examined populations of eight species of the North American funnel-web spider genus Agelenopsis to evaluate whether Wolbachia show evidence for host specificity and the relative contribution of horizontal vs. vertical transmission of strains within and among related host species. Wolbachia strains were characterized by multilocus sequence typing (MLST) and Wolbachia surface protein (WSP) sequences, and analysed in relation to host phylogeny, mitochondrial diversity and geographical range. Results indicate that at least three sets of divergent Wolbachia strains invaded the genus Agelenopsis. After each invasion, the Wolbachia strains preferentially shuffled across species of this host genus by horizontal transfer rather than cospeciation. Decoupling of Wolbachia and host mitochondrial haplotype (mitotypes) evolutionary histories within single species reveals an extensive contribution of horizontal transfer also in the rapid dispersal of Wolbachia among conspecific host populations. These findings provide some of the strongest evidence to support the association of related Wolbachia strains with related hosts by means of both vertical and horizontal strain transmission. Similar analyses across a broader range of invertebrate taxa are needed, using sensitive methods for strain typing such as MLST, to determine if this pattern of Wolbachia dispersal is peculiar to Agelenopsis (or spiders), or is in fact a general pattern in arthropods.     

Tandem repeat markers as novel diagnostic tools for high resolution fingerprinting of <Emphasis Type="Italic">Wolbachia</Emphasis>

BACKGROUND: Strains of the endosymbiotic bacterium Wolbachia pipientis are extremely diverse both genotypically and in terms of their induced phenotypes in invertebrate hosts. Despite extensive molecular characterisation of Wolbachia diversity, little is known about the actual genomic diversity within or between closely related strains that group tightly on the basis of existing gene marker systems, including Multiple Locus Sequence Typing (MLST). There is an urgent need for higher resolution fingerprinting markers of Wolbachia for studies of population genetics, horizontal transmission and experimental evolution. RESULTS: The genome of the wMel Wolbachia strain that infects Drosophila melanogaster contains inter- and intragenic tandem repeats that may evolve through expansion or contraction. We identified hypervariable regions in wMel, including intergenic Variable Number Tandem Repeats (VNTRs), and genes encoding ankyrin (ANK) repeat domains. We amplified these markers from 14 related Wolbachia strains belonging to supergroup A and were successful in differentiating size polymorphic alleles. Because of their tandemly repeated structure and length polymorphism, the markers can be used in a PCR-diagnostic multilocus typing approach, analogous to the Multiple Locus VNTR Analysis (MLVA) established for many other bacteria and organisms. The isolated markers are highly specific for supergroup A and not informative for other supergroups. However, in silico analysis of completed genomes from other supergroups revealed the presence of tandem repeats that are variable and could therefore be useful for typing target strains. CONCLUSIONS: Wolbachia genomes contain inter- and intragenic tandem repeats that evolve through expansion or contraction. A selection of polymorphic tandem repeats is a novel and useful PCR diagnostic extension to the existing MLST typing system of Wolbachia, as it allows rapid and inexpensive high-throughput fingerprinting of closely related strains for which polymorphic markers were previously lacking.     

Revisiting <Emphasis Type="Italic">Wolbachia</Emphasis> Supergroup Typing Based on WSP: Spurious Lineages and Discordance with MLST

The obligate intracellular bacteria Wolbachia are taxonomically subdivided into eight supergroups (named A-H). Supergroup typing of strains has been mostly based on phylogenetic inference of the Wolbachia surface protein (wsp), a gene that recently has been shown to experience high rates of recombination. This brings into question its suitability not only for microtaxonomy, but also for supergroup classification of the genus. A Multilocus Sequence Typing (MLST) scheme for Wolbachia has recently been developed that types strains based on five conserved genes, thus providing a rigorous supergroup annotation of strains. Here we report striking discrepancies in supergroup designation between MLST and wsp inferences, and propose a revision of current methods for Wolbachia supergroup typing. Transfer of whole wsp gene sequences between supergroups A and B has occurred. Furthermore, as a result of intragenic recombination, wsp phylogeny creates spurious basal lineages that are not supported by MLST. For example, the proposed supergroup G, based upon wsp alone, likely represents only a wsp recombinant clade. Removal of supergroup G is advised until and unless the existence of this lineage is substantiated by other sequence information (e.g., MLST). We recommend a full characterization MLST for a correct strain typing, while, based on the current data set, use of a single MLST gene can be effective for supergroup designation of A and B strains. Finally, we note that the sharing of wsp sequences between A and B strains indicates a strong genetic cohesiveness of Wolbachia strains, supporting designation of these bacteria within the same species, W. pipientis.     

High-resolution melting technology: a new tool for studying the Wolbachia endosymbiont diversity in the field

Stable infections by maternally transmitted symbionts are frequently found in field populations, especially in arthropods. Many questions remain regarding their contribution to host biology and ecology, and especially on environmental adaptation of their host. Wolbachia is one of the most common endosymbiont of invertebrates. This cytoplasmically inherited endocellular bacterium induces number of reproductive alterations in its arthropod hosts and various fitness effects that allow it to spread in host populations. To better understand the influence of Wolbachia on host phenotypes and consequences of the manipulation of reproduction on the host genetic differentiation, it is crucial to be able to discriminate Wolbachia strains and determine their prevalence, which requires exhaustive screening. In the present report, we proposed the use of a new tool for the population studies, based on the high resolution melting (HRM) analysis, less expensive and faster than the 'classical' methods for large-scale studies. We investigated the effectiveness of HRM to explore and characterize the diversity of Wolbachia strains. Results obtained showed that HRM is a powerful tool to identify strains and detect polymorphism in singly infected hosts. When individuals harboured a mixture of Wolbachia strains (multiple infections), there is a risk of underestimation of the diversity if the proportions of the strains are highly different. However, the same limitations exist for the other techniques commonly used. Overall, this study demonstrated that HRM analysis is a rapid and reliable technique useful for studying, without a priori, Wolbachia strains diversity in field populations.     

我国三地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响

【目的】Wolbachia 是广泛存在于节肢动物和丝状线虫体内的一类共生菌, 能够以多种方式对宿主产生影响。精卵细胞质不亲和（CI）是其引起的最普遍的表型, 即感染Wolbachia的雄性宿主与未感染或感染不同品系的雌性宿主交配后, 不能产生后代或后代极少, 而感染同品系Wolbachia的雌雄宿主交配后则能正常产生后代。我们前期研究发现, 湖北武汉、 云南六库和天津3个地区黑腹果蝇Drosophila melanogaster被Wolbachia感染。本研究旨在明确这3个地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响。【方法】利用Clustal X软件对Wolbachia的wsp基因序列进行比对, 利用MEGA软件构建系统发育树。采用多位点序列分型（MLST）的方法对Wolbachia进行分型。通过区内交配和区之间杂交的方式研究不同地区黑腹果蝇体内Wolbachia 的关系及其对果蝇生殖的影响。【结果】湖北武汉、 云南六库和天津3个地区黑腹果蝇中感染的Wolbachia都是属于A大组的Mel亚群。这3个地区果蝇感染的Wolbachia的序列类型（ST）不同, Wolbachia之间存在一定的差异。湖北武汉和天津果蝇中的Wolbachia能引起强烈的CI表型, 而云南六库果蝇中的Wolbachia引起的CI强度相对较弱。武汉果蝇中Wolbachia不能完全挽救天津果蝇中Wolbachia引起的CI表型, 而天津果蝇中Wolbachia也不能完全挽救武汉果蝇中Wolbachia引起的CI表型。【结论】武汉和天津地区黑腹果蝇中的Wolbachia可能距离较远。Wolbachia的长期共生可能对黑腹果蝇的进化产生了一定的影响, 湖北武汉与云南六库的黑腹果蝇中感染的Wolbachia属于不同的序列类型, 这2个地区的黑腹果蝇已发生了一定的分歧, 产生了一定的生殖隔离。     

Modes of acquisition of Wolbachia: horizontal transfer, hybrid introgression, and codivergence in the Nasonia species complex

Wolbachia are maternally inherited bacteria that infect a large number of insects and are responsible for different reproductive alterations of their hosts. One of the key features of Wolbachia biology is its ability to move within and between host species, which contributes to the impressive diversity and range of infected hosts. Using multiple Wolbachia genes, including five developed for Multi-Locus Sequence Typing (MLST), the diversity and modes of movement of Wolbachia within the wasp genus Nasonia were investigated. Eleven different Wolbachia were found in the four species of Nasonia , including five newly identified infections. Five infections were acquired by horizontal transmission from other insect taxa, three have been acquired by hybridization between two Nasonia species, which resulted in a mitochondrial- Wolbachia sweep from one species to the other, and at least three have codiverged during speciation of their hosts. The results show that a variety of transfer mechanisms of Wolbachia are possible even within a single host genus. Codivergence of Wolbachia and their hosts is uncommon and provides a rare opportunity to investigate long-term Wolbachia evolution within a host lineage. Using synonymous divergence among codiverging infections and host nuclear genes, we estimate Wolbachia mutation rates to be approximately one-third that of the nuclear genome.     

Wolbachia 在花蓟马中的感染及其与寄主线粒体DNA多样性的关系

【目的】Wolbachia 是一种广泛存在于节肢动物中的胞内共生细菌,影响寄主的生物学特性。花蓟马 Frankliniella intonsa (Trybom)是重要的害虫,对农作物及园林植物造成危害。本研究旨在明确 Wolbachia 在花蓟马中的感染情况,并分析其与寄主线粒体DNA多样性的关系。【方法】采集中国境内26个花蓟马自然种群,运用多位点序列分型技术（multilocus sequence typing, MLST）对其体内 Wolbachia 感染率及株系进行分析;利用线粒体 COI 分子标记研究花蓟马的遗传分化及遗传多样性;通过比较感染和未感染 Wolbachia 个体 COI 数据,探究 Wolbachia 多样性与寄主线粒体DNA多样性之间的关系。【结果】花蓟马中 Wolbachia 的感染率为0%~60%,共检测到5种 Wolbachia 株系（wFint1,wFint2,wFint3,wFint4及wFint5）,均属于B大组且形成一个单系群。Wolbachia感染情况与这些花蓟马种群（除CC, GZ, TA和TY, N<5）的线粒体DNA多样性相关,表现为不感染 Wolbachia 的种群中线粒体DNA单倍型多样性（Hd）与核苷酸多样性（Pi）均高于感染 Wolbachia 的种群,且 Wolbachia 感染率与 Hd 呈显著负相关（ P <0.05）。AMOVA分析表明花蓟马线粒体DNA遗传分化与Wolbachia 感染情况有关。【结论】 Wolbachia 可能在侵染花蓟马种群后出现遗传分化;Wolbachia 感染与寄主线粒体DNA多样性有关。     

Genetic diversity and population structure of Escherichia coli isolated from freshwater beaches

Escherichia coli is an important member of the gastrointestinal tract of humans and warm-blooded animals (primary habitat). In the external environment outside the host (secondary habitat), it is often considered to be only a transient member of the microbiota found in water and soil, although recent evidence suggests that some strains can persist in temperate soils and freshwater beaches. Here we quantified the population genetic structure of E. coli from a longitudinal collection of environmental strains isolated from six freshwater beaches along Lake Huron and the St. Clair River in Michigan. Multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) revealed extensive genetic diversity among 185 E. coli isolates with an average of 40 alleles per locus. Despite evidence for extensive recombination generating new alleles and genotypic diversity, several genotypes marked by distinct MLEE and MLST profiles were repeatedly recovered from separate sites at different times. A PCR-based phylogrouping technique showed that the persistent, naturalized E. coli belonged to the B1 group. These results support the hypothesis that persistent genotypes have an adaptive advantage in the secondary habitat outside the host.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Are Present in Southern African Scorpions and Cluster with Supergroup F

The presence and distribution of the intracellular bacteria Wolbachia in the arthropod subphylum Chelicerata (including class Arachnida) has not been extensively explored. Here we report the discovery of Wolbachia in scorpions. Five strains found in host species of the genus Opistophthalmus (Southern African burrowing scorpions) have been characterized by Multilocus Sequence Typing and by Wolbachia Surface Protein. Phylogenetic analyses indicate clustering in the supergroup F and a high genetic relatedness among all scorpion strains as a result of a potential transmission within the host genus. The F-group is an uncommon lineage compared to the A and B supergroups, although it is present in a broad range of hosts (including insects, filarial nematodes, and now arachnids) and across a large geographical area (e.g., North America, Africa, Europe, and Australia). It also shows no evidence of recombination and has a significantly higher genetic diversity than supergroup A and B. Overall, this pattern suggests an older radiation of F-strains with respect to A and B-strains, followed by limited horizontal transmission across host genera and reduced genetic flux among strains. A more extensive sampling of supergroup F-strains is required to confirm this scenario.     

Diversity and recombination in <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> from <Emphasis Type="Italic">Bryobia</Emphasis>spider mites

BACKGROUND: Wolbachia and Cardinium are endosymbiotic bacteria infecting many arthropods and manipulating host reproduction. Although these bacteria are maternally transmitted, incongruencies between phylogenies of host and parasite suggest an additional role for occasional horizontal transmission. Consistent with this view is the strong evidence for recombination in Wolbachia, although it is less clear to what extent recombination drives diversification within single host species and genera. Furthermore, little is known concerning the population structures of other insect endosymbionts which co-infect with Wolbachia, such as Cardinium. Here, we explore Wolbachia and Cardinium strain diversity within nine spider mite species (Tetranychidae) from 38 populations, and quantify the contribution of recombination compared to point mutation in generating Wolbachia diversity. RESULTS: We found a high level of genetic diversity for Wolbachia, with 36 unique strains detected (64 investigated mite individuals). Sequence data from four Wolbachia genes suggest that new alleles are 7.5 to 11 times more likely to be generated by recombination than point mutation. Consistent with previous reports on more diverse host samples, our data did not reveal evidence for co-evolution of Wolbachia with its host. Cardinium was less frequently found in the mites, but also showed a high level of diversity, with eight unique strains detected in 15 individuals on the basis of only two genes. A lack of congruence among host and Cardinium phylogenies was observed. CONCLUSIONS: We found a high rate of recombination for Wolbachia strains obtained from host species of the spider mite family Tetranychidae, comparable to rates found for horizontally transmitted bacteria. This suggests frequent horizontal transmission of Wolbachia and/or frequent horizontal transfer of single genes. Our findings strengthens earlier reports of recombination for Wolbachia, and shows that high recombination rates are also present on strains from a restrictive host range. Cardinium was found co-infecting several spider mite species, and phylogenetic comparisons suggest also horizontal transmission of Cardinium among hosts.     

Multilocus sequence typing scheme for bacteria of the Bacillus cereus group

In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.     

节肢动物内共生菌Wolbachia的研究进展

沃尔巴克氏体Wolbachia为母系传播的胞内共生菌,可通过对宿主产生多种调控方式扩大其自身在宿主种群的传播。据推测,有40%~60%的节肢动物都感染有Wolbachia,并可根据不同株系间的系统发育关系将其分为多个超群。为了有助于深入研究Wolbachia对其宿主的调控方式及其调控机制及提出更为有效的害虫生物防治策略,本文综述了节肢动物内共生菌Wolbachia的研究现状。1924年Wolbachia被报道首次发现于尖音库蚊Culex pipiens的生殖组织中,1971年确认其与宿主的胞质不亲和现象有关。Wolbachia可以通过胞质不亲和、杀雄、雌性化、孤雌生殖等作用方式调控宿主的生殖。除生殖调控之外,Wolbachia对宿主的调控方式还包括调控宿主新陈代谢、抵制病原菌、影响宿主生殖力等。Wolbachia调控的胞质不亲和现象可用“修饰-营救”(modification-rescue)模型解释,且已有与Wolbachia诱导宿主胞质不亲和相关的功能基因被报道。wMel株系是首个公布全基因组序列的Wolbachia株系,随后又有数十种不同株系的Wolbachia基因组陆续被破译。wMel株系Wolbachia可起到抑制登革热病毒传播的作用;同时,Wolbachia和昆虫不育技术的结合对白纹伊蚊Aedes albopictus野外种群起到良好的控制效果。鉴于目前节肢动物内共生菌Wolbachia的研究现状,我们认为未来应开展以下研究：(1)Wolbachia基因组及生殖调控作用关键功能基因的研究;(2)Wolbachia与宿主间互作机制的研究;(3)Wolbachia在生物防治方面的应用。     

Supergroup C Wolbachia,mutualist symbionts of filarial nematodes,have a distinct genome structure

Wolbachia pipientis is possibly the most widespread endosymbiont of arthropods and nematodes. While all Wolbachia strains have historically been defined as a single species, 16 monophyletic clusters of diversity (called supergroups) have been described. Different supergroups have distinct host ranges and symbiotic relationships, ranging from mutualism to reproductive manipulation. In filarial nematodes, which include parasites responsible for major diseases of humans (such as Onchocerca volvulus, agent of river blindness) and companion animals (Dirofilaria immitis, the dog heartworm), Wolbachia has an obligate mutualist role and is the target of new treatment regimens. Here, we compare the genomes of eight Wolbachia strains, spanning the diversity of the major supergroups (A–F), analysing synteny, transposable element content, GC skew and gene loss or gain. We detected genomic features that differ between Wolbachia supergroups, most notably in the C and D clades from filarial nematodes. In particular, strains from supergroup C (symbionts of O. volvulus and D. immitis) present a pattern of GC skew, conserved synteny and lack of transposable elements, unique in the Wolbachia genus. These features could be the consequence of a distinct symbiotic relationship between C Wolbachia strains and their hosts, highlighting underappreciated differences between the mutualistic supergroups found within filarial nematodes.     

Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest Streptococcus pneumoniae Clonal Complex in the MLST Database

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.     

SPECIALIZATION AND GEOGRAPHIC ISOLATION AMONG WOLBACHIA SYMBIONTS FROM ANTS AND LYCAENID BUTTERFLIES

Wolbachia are the most prevalent and influential bacteria described among the insects to date. But despite their significance, we lack an understanding of their evolutionary histories. To describe the evolution of symbioses between Wolbachia and their hosts, we surveyed global collections of two diverse families of insects, the ants and lycaenid butterflies. In total, 54 Wolbachia isolates were typed using a Multi Locus Sequence Typing (MLST) approach, in which five unlinked loci were sequenced and analyzed to decipher evolutionary patterns. AMOVA and phylogenetic analyses demonstrated that related Wolbachia commonly infect related hosts, revealing a pattern of host association that was strongest among strains from the ants. A review of the literature indicated that horizontal transfer is most successful when Wolbachia move between related hosts, suggesting that patterns of host association are driven by specialization on a common physiological background. Aside from providing the broadest and strongest evidence to date for Wolbachia specialization, our findings also reveal that strains from New World ants differ markedly from those in ants from other locations. We, therefore, conclude that both geographic and phylogenetic barriers have promoted evolutionary divergence among these influential symbionts.     

Multi-virulence-locus sequence typing of Listeria monocytogenes

A multi-virulence-locus sequence typing (MVLST) scheme was developed for subtyping Listeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST). A set of 28 strains (eight different serotypes and three known genetic lineages) of L. monocytogenes was selected from a strain collection (n > 1,000 strains) to represent the genetic diversity of this species. Internal fragments (ca. 418 to 469 bp) of three virulence genes (prfA, inlB, and inlC) and three virulence-associated genes (dal, lisR, and clpP) were sequenced and analyzed. Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types. Comparison of MVLST with automated EcoRI-RT and PFGE with ApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970). Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype 1/2a and 4b strains than MLST. Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology of L. monocytogenes.     

Wolbachia and DNA barcoding insects: patterns, potential, and problems

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein--wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor--which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region.     

Recent changes in phenotype and patterns of host specialization in Wolbachia bacteria

Wolbachia are a genus of bacterial symbionts that are known to manipulate the reproduction of their arthropod hosts, both by distorting the host sex ratio and by inducing cytoplasmic incompatibility. Previous work has suggested that some Wolbachia clades specialize in particular host taxa, but others are diverse. Furthermore, the frequency with which related strains change in phenotype is unknown. We have examined these issues for Wolbachia bacteria from Acraea butterflies, where different interactions are known in different host species. We found that bacteria from Acraea butterflies mostly cluster together in several different clades on the bacterial phylogeny, implying specialization of particular strains on these host taxa. We also observed that bacterial strains with different phenotypic effects on their hosts commonly shared identical gene sequences at two different loci. This suggests both that the phenotypes of the strains have changed recently between sex ratio distortion and cytoplasmic incompatibility, and that host specialization is not related to the bacterial phenotype, as suggested from previous data. We also analysed published data from other arthropod taxa, and found that the Wolbachia infections of the majority of arthropod genera tend to cluster together on the bacterial phylogeny. Therefore, we conclude that Wolbachia is most likely to move horizontally between closely related hosts, perhaps because of a combination of shared vectors for transmission and physiological specialization of the bacteria on those hosts.