Identifying potential candidate Culicoides spp. for the study of interactions with Candidatus Cardinium hertigii.

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors responsible for the transmission of several viruses of veterinary importance. Previous screens of Culicoides have described the presence of the endosymbiont Candidatus Cardinium hertigii (Bacteroidetes). However, any impacts of this microbe on vectorial capacity, akin to those conferred by Wolbachia in mosquitoes, are yet to be uncovered and await a suitable system to study Cardinium-midge interactions. To identify potential candidate species to investigate these interactions, accurate knowledge of the distribution of the endosymbiont within Culicoides populations is needed. We used conventional and nested PCR assays to screen Cardinium infection in 337 individuals of 25 Culicoides species from both Palearctic and Afrotropical regions. Infections were observed in several vector species including C. imicola and the Pulicaris complex (C. pulicaris, C. bysta, C. newsteadi and C. punctatus) with varying prevalence. Phylogenetic analysis based on the Gyrase B gene grouped all new isolates within 'group C' of the genus, a clade that has to date been exclusively described in Culicoides. Through a comparison of our results with previous screens, we suggest C. imicola and C. sonorensis represent good candidates for onward study of Cardinium-midge interactions.

Widespread Wolbachia infection in an insular radiation of damselflies (Odonata, Coenagrionidae).

Wolbachia is one of the most common endosymbionts found infecting arthropods. Theory predicts symbionts like Wolbachia will be more common in species radiations, as host shift events occur with greatest frequency between closely related species. Further, the presence of Wolbachia itself may engender reproductive isolation, and promote speciation of their hosts. Here we screened 178 individuals belonging to 30 species of the damselfly genera Nesobasis and Melanesobasis - species radiations endemic to the Fiji archipelago in the South Pacific - for Wolbachia, using multilocus sequence typing to characterize bacterial strains. Incidence of Wolbachia was 71% in Nesobasis and 40% in Melanesobasis, and prevalence was also high, with an average of 88% in the Nesobasis species screened. We identified a total of 25 Wolbachia strains, belonging to supergroups A, B and F, with some epidemic strains present in multiple species. The occurrence of Wolbachia in both males and females, and the similar global prevalence found in both sexes rules out any strong effect of Wolbachia on the primary sex-ratio, but are compatible with the phenotype of cytoplasmic incompatibility. Nesobasis has higher species richness than most endemic island damselfly genera, and we discuss the potential for endosymbiont-mediated speciation within this group.

A qualitative study of disengagement in disadvantaged areas of the UK: 'You come through your door and you lock that door'.

Health inequalities are a major concern in the UK. Power imbalances are associated with health inequalities and should be challenged through health promotion and empowering strategies, enabling individuals who feel powerless to take control over their own life and act on the determinants of health (Green and Tones, 2010). This study aimed to explore resident expectations of a community engagement programme that intended to empower communities to take action on pre-identified priorities. The programme targeted communities in deprived areas of a mid-sized city in the UK. A qualitative design was implemented. In-depth semi-structured interviews were undertaken with 28 adult residents at the start of the programme. Transcripts were analysed using an inductive approach to thematic analysis. Resident expectations were explored from a constructivist epistemological perspective. The qualitative inductive approach allowed a second research question to develop which led this paper to focus on exploring how disempowerment was experienced by individuals before taking part in a community engagement programme. Analysis of interviews revealed a 'process of deterioration' that provided insight into how communities might become (more) disadvantaged through disempowerment. Five master themes were identified: external abandonment at the institutional-level (master theme 1); a resulting loss of sense of community (master theme 2); this negatively affected psychological wellbeing of residents (master theme 3); who adopted coping strategies (e.g., disengagement) to aid living in such challenging areas; (master theme 4); disengagement further perpetuated the deterioration of the area (master theme 5). Distrust was identified as a major barrier to participation in community engagement programmes. Overall, our data suggested that community engagement approaches must prioritise restoration of trust and be accompanied by supportive policies to mitigate feelings of abandonment in communities.

Heritable symbionts in a world of varying temperature.

Heritable microbes represent an important component of the biology, ecology and evolution of many plants, animals and fungi, acting as both parasites and partners. In this review, we examine how heritable symbiont-host interactions may alter host thermal tolerance, and how the dynamics of these interactions may more generally be altered by thermal environment. Obligate symbionts, those required by their host, are considered to represent a thermally sensitive weak point for their host, associated with accumulation of deleterious mutations. As such, these symbionts may represent an important determinant of host thermal envelope and spatial distribution. We then examine the varied relationship between thermal environment and the frequency of facultative symbionts that provide ecologically contingent benefits or act as parasites. We note that some facultative symbionts directly alter host thermotolerance. We outline how thermal environment will alter the benefits/costs of infection more widely, and additionally modulate vertical transmission efficiency. Multiple patterns are observed, with symbionts being cold sensitive in some species and heat sensitive in others, with varying and non-coincident thresholds at which phenotype and transmission are ablated. Nevertheless, it is clear that studies aiming to predict ecological and evolutionary dynamics of symbiont-host interactions need to examine the interaction across a range of thermal environments. Finally, we discuss the importance of thermal sensitivity in predicting the success/failure of symbionts to spread into novel species following natural/engineered introduction.

Can maternally inherited endosymbionts adapt to a novel host? Direct costs of Spiroplasma infection, but not vertical transmission efficiency, evolve rapidly after horizontal transfer into D. melanogaster.

Maternally inherited symbionts are common in arthropods and many have important roles in host adaptation. The observation that specific symbiont lineages infect distantly related host species implies new interactions are commonly established by lateral transfer events. However, studies have shown that symbionts often perform poorly in novel hosts. We hypothesized selection on the symbiont may be sufficiently rapid that poor performance in a novel host environment is rapidly ameliorated, permitting symbiont maintenance. Here, we test this prediction for a Spiroplasma strain transinfected into the novel host Drosophila melanogaster. In the generations immediately following transinfection, the symbiont had low transmission efficiency to offspring and imposed severe fitness costs on its host. We observed that effects on host fitness evolved rapidly, being undetectable after 17 generations in the novel host, whereas vertical transmission efficiency was poorly responsive over this period. Our results suggest that long-term symbiosis may more readily be established in cases where symbionts perform poorly in just one aspect of symbiosis.

The effect of hybrid transgression on environmental tolerance in experimental yeast crosses.

Evidence is rapidly accumulating that hybridization generates adaptive variation. Transgressive segregation in hybrids could promote the colonization of new environments. Here, we use an assay to select hybrid genotypes that can proliferate in environmental conditions beyond the conditions tolerated by their parents, and we directly compete them against parental genotypes in habitats across environmental clines. We made 45 different hybrid swarms by crossing yeast strains (both Saccharomyces cerevisiae and S. paradoxus) with different genetic and phenotypic divergence. We compared the ability of hybrids and parents to colonize seven types of increasingly extreme environmental clines, representing both natural and novel challenges (mimicking pollution events). We found that a significant majority of hybrids had greater environmental ranges compared to the average of both their parents' ranges (mid-parent transgression), but only a minority of hybrids had ranges exceeding their best parent (best-parent transgression). Transgression was affected by the specific strains involved in the cross and by the test environment. Genetic and phenotypic crossing distance predicted the extent of transgression in only two of the seven environments. We isolated a set of potentially transgressive hybrids selected at the extreme ends of the clines and found that many could directly outcompete their parents across whole clines and were between 1.5- and 3-fold fitter on average. Saccharomyces yeast is a good model for quantitative and replicable experimental speciation studies, which may be useful in a world where hybridization is becoming increasingly common due to the relocation of plants and animals by humans.

First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus, important Palaearctic vectors of bluetongue virus.

Heritable bacteria have been highlighted as important components of vector biology, acting as required symbionts with an anabolic role, altering competence for disease transmission, and affecting patterns of gene flow by altering cross compatibility. In this paper, we tested eight U.K. species of Culicoides (Diptera: Ceratopogonidae) midge for the presence of five genera of endosymbiotic bacteria: Cardinium (Bacteroidales: Bacteroidaceae); Wolbachia (Rickettsiales: Rickettsiaceae); Spiroplasma (Entomoplasmatales: Spiroplasmataceae); Arsenophonus (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Cardinium spp. were detected in both sexes of Culicoides pulicaris and Culicoides punctatus, two known vectors of bluetongue virus. Cardinium spp. were not detected in any other species, including the Culicoides obsoletus group, the main vector of bluetongue and Schmallenberg viruses in northern Europe. The other endosymbionts were not detected in any Culicoides species. The Cardinium strain detected in U.K. Culicoides species is very closely related to the Candidatus Cardinium hertigii group C, previously identified in Culicoides species in Asia. Further, we infer that the symbiont is not a sex ratio distorter and shows geographic variation in prevalence within a species. Despite its detection in several species of Culicoides that vector arboviruses worldwide, the absence of Cardinium in the C. obsoletus group suggests that infections of these symbionts may not be necessary to the arboviral vector competence of biting midges.

The draft genome sequence of Arsenophonus nasoniae, son-killer bacterium of Nasonia vitripennis, reveals genes associated with virulence and symbiosis.

Four percent of female Nasonia vitripennis carry the son-killer bacterium Arsenophonus nasoniae, a microbe with notably different biology from other inherited parasites and symbionts. In this paper, we examine a draft genome sequence of the bacterium for open reading frames (ORFs), structures and pathways involved in interactions with its insect host. The genome data suggest that A. nasoniae carries multiple type III secretion systems, and an array of toxin and virulence genes found in Photorhabdus, Yersinia and other gammaproteobacteria. Of particular note are ORFs similar to those known to affect host innate immune functioning in other bacteria, and four ORFs related to pro-apoptotic exotoxins. The genome sequences for both A. nasoniae and its Nasonia host are useful tools for examining functional genomic interactions of microbial survival in hostile immune environments, and mechanisms of passage through gut epithelia, in a whole organism context.

Characteristics of the genome of Arsenophonus nasoniae, son-killer bacterium of the wasp Nasonia.

We report the properties of a draft genome sequence of the bacterium Arsenophonus nasoniae, son-killer bacterium of Nasonia vitripennis. The genome sequence data from this study are the first for a male-killing bacterium, and represent a microorganism that is unusual compared with other sequenced symbionts, in having routine vertical and horizontal transmission, two alternating hosts, and being culturable on cell-free media. The resulting sequence totals c. 3.5 Mbp and is annotated to contain 3332 predicted open reading frames (ORFs). Therefore, Arsenophonus represents a relatively large genome for an insect symbiont. The annotated ORF set suggests that the microbe is capable of a broad array of metabolic functions, well beyond those found for reproductive parasite genomes sequenced to date and more akin to horizontally transmitted and secondary symbionts. We also find evidence of genetic transfer from Wolbachia symbionts, and phage exchange with other gammaproteobacterial symbionts. These findings reflect the complex biology of a bacterium that is able to live, invade and survive multiple host environments while resisting immune responses.

Rapid spread of male-killing Wolbachia in the butterfly Hypolimnas bolina.

Reproductive parasites such as Wolbachia can spread through uninfected host populations by increasing the relative fitness of the infected maternal lineage. However, empirical estimates of how fast this process occurs are limited. Here we use nucleotide sequences of male-killing Wolbachia bacteria and co-inherited mitochondria to address this issue in the island butterfly Hypolimnas bolina. We show that infected specimens scattered throughout the species range harbour the same Wolbachia and mitochondrial DNA as inferred from 6337 bp of the bacterial genome and 2985 bp of the mitochondrial genome, suggesting this strain of Wolbachia has spread across the South Pacific Islands at most 3000 years ago, and probably much more recently.

Hidden cytoplasmic incompatibility alters the dynamics of male-killer/host interactions.

Wolbachia manipulate the reproduction of their arthropod hosts in a variety of ways. Recent work has demonstrated that these bacteria may combine phenotypes - possessing a 'male killing' phenotype and, where males survive, induce cytoplasmic incompatibility (CI). We here develop a mathematical model to investigate the extent to which 'hidden' CI may affect the evolutionary dynamics of host genes that suppress male-killing activity. We observed that for high prevalence infections, CI drives both suppressor and bacterium to higher frequency, such that the strain appears to solely exhibit CI. In contrast, for low prevalence infections, CI impedes suppressor invasion as surviving infected males are incompatible with the majority of females in the population. Our results demonstrate that 'hidden' phenotypes as well as observable ones can impact on the dynamics of the interaction, and knowledge of these is therefore required to predict when suppressor genes will invade, and the consequences of their invasion.

Mitochondrial DNA as a marker of molecular diversity: a reappraisal.

Over the last three decades, mitochondrial DNA has been the most popular marker of molecular diversity, for a combination of technical ease-of-use considerations, and supposed biological and evolutionary properties of clonality, near-neutrality and clock-like nature of its substitution rate. Reviewing recent literature on the subject, we argue that mitochondrial DNA is not always clonal, far from neutrally evolving and certainly not clock-like, questioning its relevance as a witness of recent species and population history. We critically evaluate the usage of mitochondrial DNA for species delineation and identification. Finally, we note the great potential of accumulating mtDNA data for evolutionary and functional analysis of the mitochondrial genome.

Selfish genetic elements promote polyandry in a fly.

It is unknown why females mate with multiple males when mating is frequently costly and a single copulation often provides enough sperm to fertilize all a female's eggs. One possibility is that remating increases the fitness of offspring, because fertilization success is biased toward the sperm of high-fitness males. We show that female Drosophila pseudoobscura evolved increased remating rates when exposed to the risk of mating with males carrying a deleterious sex ratio-distorting gene that also reduces sperm competitive ability. Because selfish genetic elements that reduce sperm competitive ability are generally associated with low genetic fitness, they may represent a common driver of the evolution of polyandry.

The evolution of endosymbiont density in doubly infected host species.

Multiple infection of individual hosts with several species or strains of maternally inherited endosymbionts is commonly observed in animals, especially insects. Here, we address theoretically the effect of co-infection on the optimal density of the endosymbionts in doubly infected hosts. Our analysis is based on the observation that a maternally inherited double infection is only stable if doubly infected females produce more doubly infected daughters than singly infected or uninfected females produce daughters. We consider both a general model and a model involving two endosymbionts inducing bidirectional cytoplasmic incompatibility (CI). We demonstrate that the optimal replication rate of endosymbionts in doubly infected hosts can be expected to be similar to or below the optimal replication rate in singly infected hosts. This is in contrast to some theoretical predictions for horizontally transmitted parasites and stems from the two strains of endosymbionts having coupled fitness. We discuss our results with respect to recent empirical results on endosymbiont densities, the evolution of CI-inducing bacteria and, more generally, the evolution of cooperation through direct fitness benefits.

Fitness effects of Wolbachia and Spiroplasma in Drosophila melanogaster.

Maternally inherited endosymbionts that manipulate the reproduction of their insect host are very common. Aside from the reproductive manipulation they produce, the fitness of these symbionts depends in part on the direct impact they have on the female host. Although this parameter has commonly been investigated for single infections, it has much more rarely been established in dual infections. We here establish the direct effect of infection with two different symbionts exhibiting different reproductive manipulation phenotypes, both alone and in combination, in the fruit fly Drosophila melanogaster. This species carries a cytoplasmic incompatibility inducing Wolbachia and a male-killing Spiroplasma, occurring as single or double (co-) infections in natural populations. We assessed direct fitness effects of these bacteria on their host, by comparing larval competitiveness and adult fecundity of uninfected, Wolbachia, Spiroplasma and Wolbachia-Spiroplasma co-infected females. We found no effect of infection status on the fitness of females for both estimates, that is, no evidence of any benefits or costs to either single or co-infection. This leads to the conclusion that both bacteria probably have other sources of benefits to persist in D. melanogaster populations, either by means of their reproductive manipulations (fitness compensation from male death in Spiroplasma infection and cytoplasmic incompatibility in Wolbachia infection) or by positive fitness interactions on other fitness components.

Asymmetric gene flow and constraints on adaptation caused by sex ratio distorters.

Asymmetric gene flow is generally believed to oppose natural selection and potentially impede adaptation. Whilst the cause of asymmetric gene flow has been seen largely in terms of variation in population density over space, asymmetric gene flow can also result from varying sex ratios across subpopulations with similar population sizes. We model the process of adaptation in a scenario in which two adjacent subpopulations have different sex ratios, associated with different levels of infection with maternally inherited endosymbionts that selectively kill male hosts. Two models are analyzed in detail. First, we consider one host locus with two alleles, each of which possesses a selective advantage in one of the subpopulations. We found that local adaptation can strongly be impeded in the subpopulation with the more female biased population sex ratio. Second, we analyze host alleles that provide resistance against the male-killing (MK) endosymbionts and show that asymmetric gene flow can prevent the spread of such alleles under certain conditions. These results might have important implications for the coevolution of MK bacteria and their hosts.

Can maternally transmitted endosymbionts facilitate the evolution of haplodiploidy?

Whilst many invertebrate taxa are haplodiploid, the factors underlying the evolution of haplodiploidy remain unresolved. We investigate theoretically whether haplodiploidy might evolve as an outcome of the co-evolution between maternally inherited endosymbionts and their hosts. First, we substantially extend a recently developed model that involves maternally inherited endosymbionts that kill male offspring by eliminating the paternal genome. We also put forward a new hypothesis and develop a model that involves bacteria that induce cytoplasmic incompatibility (CI). Based on these models, we explore the co-evolutionary events that might occur between hosts and symbionts. We find that both with male-killers and CI-inducing endosymbionts, the hosts are likely to develop increased viability of haploid males, which can be considered a preadaptation to haplodiploidy. In addition, populations with haploidizing male-killers can in some cases evolve directly towards a genetic system of paternal genome elimination, a special form of haplodiploidy. These results are combined with consideration of mechanism and ecology to appraise the likelihood of male-killers and CI inducing bacteria being involved in the evolution of haplodiploidy.

Voice recognition for radiology reporting: is it good enough?

AIM: To compare the efficiency and accuracy of radiology reports generated by voice recognition (VR) against the traditional tape dictation-transcription (DT) method. MATERIALS AND METHODS: Two hundred and twenty previously reported computed radiography (CR) and cross-sectional imaging (CSI) examinations were separately entered into the Radiology Information System (RIS) using both VR and DT. The times taken and errors found in the reports were compared using univariate analyses based upon the sign-test, and a general linear model constructed to examine the mean differences between the two methods. RESULTS: There were significant reductions (p<0.001) in the mean difference in the reporting times using VR compared with DT for the two reporting methods assessed (CR, +67.4; CSI, +122.1s). There was a significant increase in the mean difference in the actual radiologist times using VR compared with DT in the CSI reports; -14.3s, p=0.037 (more experienced user); -13.7s, p=0.014 (less experienced user). There were significantly more total and major errors when using VR compared with DT for CR reports (-0.25 and -0.26, respectively), and in total errors for CSI (-0.75, p<0.001), but no difference in major errors (-0.16, p=0.168). Although there were significantly more errors with VR in the less experienced group of users (mean difference in total errors -0.90, and major errors -0.40, p<0.001), there was no significant difference in the more experienced (p=0.419 and p=0.814, respectively). CONCLUSIONS: VR is a viable reporting method for experienced users, with a quicker overall report production time (despite an increase in the radiologists' time) and a tendency to more errors for inexperienced users.

Male-killing Spiroplasma naturally infecting Drosophila melanogaster.

Elucidation of the mechanism of action of selfish genetic elements is difficult outside species with well-defined genetics. Male-killing, the phenomenon whereby inherited bacteria kill male hosts during embryogenesis, is thus uncharacterized in mechanistic terms despite being common and important in insects. We characterized the prevalence, identity and source of the male-killing infection recently discovered in Drosophila melanogaster in Brazil. Male-killing was found to be present in 2.3% of flies from Recife, Brazil, and was uniquely associated with the presence of Spiroplasma infection. The identity of sequences across part of the 16S and across the 16S-23S ITS region indicated that the male-killing infection of D. melanogaster was very closely related to S. poulsonii, the source of the male-killing infection in willistoni group flies also found in South America. The sequences of two further protein-coding genes indicated the D. melanogaster infection to be most closely related to that found in D. nebulosa, from the willistoni group. Our data suggest that the establishment of D. melanogaster in South America was associated with the movement of male-killing bacteria between species.

Analysis of ligand-receptor cross-linked fragments by mass spectrometry.

G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable alpha-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to cross-linked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an alpha-factor analog with the photoaffinity group p-benzoyl-l-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of alpha-factor and a region of Ste2p covering residues 251-294.