Accelerated microevolution in an outer membrane protein (OMP) of the intracellular bacteria Wolbachia

<p>Abstract</p> <p>Background</p> <p>Outer membrane proteins (OMPs) of Gram-negative bacteria are key players in the biology of bacterial-host interactions. However, while considerable attention has been given to OMPs of vertebrate pathogens, relatively little is known about the role of these proteins in bacteria that primarily infect invertebrates. One such OMP is found in the intracellular bacteria <it>Wolbachia</it>, which are widespread symbionts of arthropods and filarial nematodes. Recent experimental studies have shown that the <it>Wolbachia </it>surface protein (WSP) can trigger host immune responses and control cell death programming in humans, suggesting a key role of WSP for establishment and persistence of the symbiosis in arthropods.</p> <p>Results</p> <p>Here we performed an analysis of 515 unique alleles found in 831 <it>Wolbachia </it>isolates, to investigate WSP structure, microevolution and population genetics. WSP shows an eight-strand transmembrane β-barrel structure with four extracellular loops containing hypervariable regions (HVRs). A clustering approach based upon patterns of HVR haplotype diversity was used to group similar WSP sequences and to estimate the relative contribution of mutation and recombination during early stages of protein divergence. Results indicate that although point mutations generate most of the new protein haplotypes, recombination is a predominant force triggering diversity since the very first steps of protein evolution, causing at least 50% of the total amino acid variation observed in recently diverged proteins. Analysis of synonymous variants indicates that individual WSP protein types are subject to a very rapid turnover and that HVRs can accommodate a virtually unlimited repertoire of peptides. Overall distribution of WSP across hosts supports a non-random association of WSP with the host genus, although extensive horizontal transfer has occurred also in recent times.</p> <p>Conclusions</p> <p>In OMPs of vertebrate pathogens, large recombination impact, positive selection, reduced structural and compositional constraints, and extensive lateral gene transfer are considered hallmarks of evolution in response to the adaptive immune system. However, <it>Wolbachia </it>do not infect vertebrates. Here we predict that the rapid turnover of WSP loop motifs could aid in evading or inhibiting the invertebrate innate immune response. Overall, these features identify WSP as a strong candidate for future studies of host-<it>Wolbachia </it>interactions that affect establishment and persistence of this widespread endosymbiosis.</p

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 – for 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

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.Organismic and Evolutionary Biolog

DNA barcoding reveals diversity of Hymenoptera and the dominance of parasitoids in a sub-arctic environment

Abstract Background Insect diversity typically declines with increasing latitude, but previous studies have shown conflicting latitude-richness gradients for some hymenopteran parasitoids. However, historical estimates of insect diversity and species richness can be difficult to confirm or compare, because they may be based upon dissimilar methods. As a proxy for species identification, we used DNA barcoding to identify molecular operational taxonomic units (MOTUs) for 7870 Hymenoptera specimens collected near Churchill, Manitoba, from 2004 through 2010. Results We resolved 1630 MOTUs for this collection, of which 75% (1228) were ichneumonoids (Ichneumonidae + Braconidae) and 91% (1484) were parasitoids. We estimate the total number of Hymenoptera MOTUs in this region at 2624-2840. Conclusions The diversity of parasitoids in this sub-Arctic environment implies a high diversity of potential host species throughout the same range. We discuss these results in the contexts of resolving interspecific interactions that may include cryptic species, and developing reproducible methods to estimate and compare species richness across sites and between surveys, especially when morphological specialists are not available to identify every specimen.</p

Comparison between insect host and endosymbiotic bacteria COI for 255 specimens. (nucleotide content and variability within and between each group).

<p>Comparison between insect host and endosymbiotic bacteria COI for 255 specimens. (nucleotide content and variability within and between each group).</p

Case studies of the frequency of bacterial COI and <i>wsp</i> recovered from whole genomic DNA extracts made from 10 insect families from two orders. [46],[47].

<p>Case studies of the frequency of bacterial COI and <i>wsp</i> recovered from whole genomic DNA extracts made from 10 insect families from two orders. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036514#pone.0036514-Smith6" target="_blank">[46]</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036514#pone.0036514-Li1" target="_blank">[47]</a>.</p

Insect host geographic distribution A) Red and yellow dots indicate the collection locality for <i>Wolbachia</i> insect hosts.

<p><b>B</b>) Mantel test of pairwise Fst of <i>Wolbachia</i> COI and kilometers for the collection localities of insect hosts (r = 0.099, p = 0.92).</p

The nucleotide diversity of region of the LepR1 oligonucleotide as compared to GenBank Halictidae COI sequences (156 sequences from 93 species in black) and MLST <i>Wolbachia</i> database sequences (104 sequences in yellow) was calculated using DNASP [<b>45</b>].

<p>It is clear that the reverse primer (LepR1) is better fit to the bacterial endosymbiont than to the insect host. Specimen information for each data set is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036514#pone.0036514.s003" target="_blank">Table S2</a>.</p