Phylogenetics of the Spiroplasma ixodetis endosymbiont reveals past transfers between ticks and other arthropods.

The bacterium Spiroplasma ixodetis is a maternally inherited endosymbiont primarily described from ticks but also found widespread across other arthropods. While it has been identified as a male-killing agent in some insect species, the consequences of infection with S. ixodetis in ticks are entirely unknown, and it is unclear how this endosymbiont spreads across tick species. Here, we have investigated this aspect through the examination of the diversity and evolutionary history of S. ixodetis infections in 12 tick species and 12 other arthropod species. Using a multi-locus typing approach, we identified that ticks harbor a substantial diversity of divergent S. ixodetis strains. Phylogenetic investigations revealed that these S. ixodetis strains do not cluster within a tick-specific subclade but rather exhibit distinct evolutionary origins. In their past, these strains have undergone repeated horizontal transfers between ticks and other arthropods, including aphids and flies. This diversity pattern strongly suggests that maternal inheritance and horizontal transfers are key drivers of S. ixodetis spread, dictating global incidence of infections across tick communities. We do not, however, detect evidence of S. ixodetis-based male-killing since we observed that infections were widely present in both males and females across populations of the African blue tick Rhipicephalus decoloratus.

Is qualitative and quantitative metabarcoding of dung fauna biodiversity feasible?

In biodiversity assessments, especially of small-bodied organisms for which taxonomic expertise is lacking, identification by genetic barcoding may be a cost-effective and efficient alternative to traditional identification of species by morphology, ecology, and behavior. The authors tested the feasibility and accuracy of such an approach using dung insects of practical relevance in ecotoxicological assessments of veterinary pharmaceutical residues in the environment. They produced 8 known mixtures that varied in absolute and relative composition of small-bodied and large-bodied species to see whether mitochondrial cytochrome c oxidase subunit 1 barcoding picks up all species qualitatively and quantitatively. As demonstrated before in other contexts, such metabarcoding of large numbers of dung insect specimens is principally possible using next-generation sequencing. The authors recovered most species in a sample (low type I error), at minimum permitting analysis of species richness. They obtained even quantitative responses reflecting the body size of the species, although the number of specimens was not well detected. The latter is problematic when calculating diversity indices. Nevertheless, the method yielded too many closely related false positives (type II error), thus generally overestimating species diversity and richness. These errors can be reduced by refining methods and data filtering, although this requires bioinformatics expertise often unavailable where such research is carried out. Identification by barcoding foremost hinges on a good reference database, which does not yet exist for dung organisms but would be worth developing for practical applications. Environ Toxicol Chem 2016;35:1970-1977. © 2015 SETAC.

Infections with the microbe Cardinium in the Dolichopodidae and other Empidoidea.

Maternally transmitted reproductive parasites such as Wolbachia and Cardinium can drastically reshape reproduction in their hosts. Beyond skewing sex ratios towards females, these microbes can also cause cytoplasmic incompatibility. Wolbachia probably infects two thirds of insects, but far less is known about the occurrence or action of other bacteria with potentially similar effects. In contrast with the two more widespread reproductive parasites, Wolbachia and Spiroplasma, far less is known of infections with Cardinium (Bacteroidetes) and possible consequences in the Diptera. Here, in an extensive survey, 244 dipteran species from 67 genera belonging to the Dolichopodidae, Empididae, and Hybotidae were assessed for the presence of the microbe Cardinium. Although 130 of the species screened tested positive (ca. 53%), the presence of Cardinium could only be confirmed in 10 species (ca. 4%) based on analysis of sequences. Numerous additional sequences were found to be assignable to known or unknown Bacteroidetes. Considering the known issues concerning specificity of Cardinium primers and the phylogenetic uncertainties surrounding this microbe, the actual prevalence of this symbiont is worthy of further scrutiny. Potential directions for future research on Cardinium-host interactions in Diptera and in general are discussed.

Infections with Wolbachia, Spiroplasma, and Rickettsia in the Dolichopodidae and other Empidoidea.

Vertically transmitted reproductive parasites are both extraordinarily widespread and diverse in their effects on their invertebrate hosts. In addition to causing skewed population sex ratios via male-killing or feminization, such bacteria can further cause cytoplasmic incompatibility or parthenogenesis. Previous surveys show that the microbes Wolbachia and Spiroplasma are common in some dipteran families, e.g. Drosophilidae or Scathophagidae, and are known to be heritable symbionts and affect reproduction in the Diptera. However, little is known of Rickettsia infections and detailed surveys targeting other Dipteran families are lacking. Here 329 samples of 247 species of Diptera belonging to the Dolichopodidae, Empididae, and Hybotidae (superfamily Empidoidea) are surveyed for the presence of the endosymbionts Wolbachia, Spiroplasma, and Rickettsia. The superfamily Empidoidea contains numerous species, which have been the targets of intense research concerning reproductive traits involved in sexual selection. 151 of the species (i.e. ca. 61%) screened here, including species from key genera such as Dolichopus, Poecilobothrus or Empis, harboured one or more symbionts. Reproductive parasites are thus also common in the Empidoidae, yet effects on hosts remain unclear. Potential endosymbiont-host interactions in this group would hence be worthy of further investigation.

Infections with Wolbachia and Spiroplasma in the Scathophagidae and other Muscoidea.

The microbes Wolbachia and Spiroplasma are common reproductive parasites of arthropods and may strongly influence reproduction of infected hosts and also impact on reproductive isolation. Such infections could hence influence results of many studies assessing reproductive behaviour and fitness of possible hosts, as well as reproductive isolation. Previous work indicates that infections with the microbes Wolbachia and Spiroplasma are common in the Drosophilidae. However, extensive and targeted surveys of other Dipteran families are lacking. Here we survey the yellow dung fly Scathophaga stercoraria and a range of other species from the Muscoidea (families Scathophagidae, Anthomyiidae, Fanniidae and Muscidae) collected in the field or obtained from museum collections for infection with the widespread reproductive parasites Wolbachia and Spiroplasma. Both have been shown to be heritable symbionts and affect reproduction in other Diptera. S. stercoraria is a very important model for the study of sexual selection, and in particular of postcopulatory processes, as it has played a major role in the history of research on sperm competition and cryptic female choice. Infections with Wolbachia were found to be widespread across the Muscoidea, whereas infections with Spiroplasma were rarer. We discuss the consequences of these findings and directions for future research on the impact of reproductive parasites on host reproduction in the Scathophagidae.

The evolutionary path to terminal differentiation and division of labor in cyanobacteria.

A common trait often associated with multicellularity is cellular differentiation, which is a spatial separation of tasks through the division of labor. In principle, the division of labor does not necessarily have to be constrained to a multicellular setting. In this study, we focus on the possible evolutionary paths leading to terminal differentiation in cyanobacteria. We develop mathematical models for two developmental strategies. First, of populations of terminally differentiated single cells surviving by the exchange of common goods. Second, of populations exhibiting terminal differentiation in a multicellular setting. After testing the two strategies against the effect of disruptive mutations (i.e. "cheater" mutants), we assess the effects of selection on the optimization of the ratio of vegetative (carbon fixing) to heterocystous (nitrogen fixing) cells, which in turn leads to the maximization of the carrying capacity for the population density. In addition, we compare the performance of differentiated populations to undifferentiated ones that temporally separate tasks in accordance to a day/night cycle. We then compare some predictions of our model with phylogenetic relationships derived from analyzing 16S rRNA sequences of different cyanobacterial strains. In line with studies indicating that group or spatial structure are ways to evolve cooperation and protect against the spread of cheaters, our work shows that compartmentalization afforded by multicellularity is required to maintain the vegetative/heterocyst division in cyanobacteria. We find that multicellularity allows for selection to optimize the carrying capacity. These results and the phylogenetic analysis indicates that terminally differentiated cyanobacteria evolved after undifferentiated species. In addition, we show that, in regimes of short daylight periods, terminally differentiated species perform worse than undifferentiated species that follow the day/night cycle; indicating that undifferentiated species have an evolutionary advantage in regimes of short daylight periods.

Real-time PCR investigation of potential vectors, reservoirs, and shedding patterns of feline hemotropic mycoplasmas.

Three hemotropic mycoplasmas have been identified in pet cats: Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum," and "Candidatus Mycoplasma turicensis." The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a few Ixodes sp. and Rhipicephalus sp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequently Mycoplasma coccoides PCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, "Ca. Mycoplasma turicensis" DNA was detected in saliva and feces at the early but not at the late phase of infection. M. haemofelis and "Ca. Mycoplasma haemominutum" DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.

Population genetic analysis of Pseudomonas aeruginosa using multilocus sequence typing.

To study the population genetic structure of Pseudomonas aeruginosa, we developed a multilocus sequence typing scheme. The sequences of internal fragments of seven housekeeping genes were obtained for 34 P. aeruginosa isolates from patients hospitalized in five different European cities. Twenty-six different allelic profiles were identified. The mean allelic diversity was 0.854 (range: 0.606-0.978), which was about six times greater than the results obtained with the multilocus enzyme electrophoresis method. Linkage disequilibrium was measured with the index of association. An index of 1.95+/-0.24 was calculated when all the strains were considered. This index was 1.76+/-0.27 when only one strain per sequence type was considered. Both results were different from 0, indicating linkage among loci, which means that the population structure of our set of P. aeruginosa isolates is clonal. The clonal structure of the population was also suggested by the congruence of the topology of the different trees obtained from the seven housekeeping genes. These results are in contrast to previous studies, finding a non clonal population structure. Since a small number of isolates was analyzed in this study, there might be a bias of selection which includes the possibility that they belong to widely disseminated epidemic clones. Another possibility is that recombination did not occurred homogeneously throughout the genome of P. aeruginosa, so that part of it has a clonal structure, while the remaining part of the genome is more frequently subject to recombination.

Diversity within Borrelia burgdorferi sensu lato genospecies in Switzerland by recA gene sequence.

A total of 874 Ixodes ricinus ticks were collected in Switzerland to investigate the genetic diversity of the Borrelia population. We integrated to the RT-PCR method the DNA sequence analysis of a 162-bp fragment of the recA gene. Five genospecies were detected: Borrelia afzelii, Borrelia burgdorferi s.s., Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. A heterogeneous distribution was observed within the B. burgdorferi s.l. genospecies. The most prevalent and diverse genospecies found in Switzerland was Borrelia afzelii, which might suggest a rapid evolution of this genospecies.

C-terminal region of protein kinase CK2 alpha: How the structure can affect function and stability of the catalytic subunit.

A novel mutant of the catalytic alpha subunit of human protein kinase CK2 (CK2 alpha) was designed in an attempt to clarify the role of the carboxylic-terminal segment characteristic of vertebrates, excluding fish. Starting from the sequence alignments, we constructed a phylogenetic tree of the primary structure of CK2 alpha. On this basis, we substituted two distal prolines with alanines (PA 382-384). Theoretical calculations and spectropolarimetry measurements, performed both on native and mutant subunits, indicate an increased content of alpha-helix after this double amino acidic substitution. In order to clarify the structure/function relationship of the C-terminal region, we verified if the structural change affects the catalytic activity of CK2 alpha. The mutant exhibits slightly increased phosphorylation efficiency, but reduced ability to transfer phosphate in comparison with the native subunit. At last, we compared the thermal stability of the mutant with respect to the native subunit and we tested the proteolytic degradability. The observation that the PA 382-384 mutant exhibits an increased thermal and proteolytic stability suggests that this mutant could be employed to solve the three-dimensional (3D) structure of human CK2 alpha and to overcome difficulties in crystallizing the native form.

RecA and glnA sequences separate the bacteroides fragilis population into two genetic divisions associated with the antibiotic resistance genotypes cepA and cfiA.

The sequences of part of the glutamine synthetase-encoding gene (glnA) and of the RecA-encoding gene (recA) were determined and aligned for 45 Bacteroides fragilis isolates from different clinical and geographical origin. The patterns of sequence divergence of glnA and recA were very similar. The sequences of a 303-bp fraction of recA showed 45 nucleotide substitutions, 40 of which allowed the separation of B. fragilis into two major divisions, which were not found when the deduced amino acid sequences were considered. The 687-bp sequences analysed for the glnA gene showed 112 nucleotide substitutions, 96 of which separated the population into the same two divisions as those described for recA. In this case, the deduced amino acid sequences showed this subdivision as well: three of the six observed amino acid substitutions were division-specific. Within the two divisions, both genes presented a high degree of sequence conservation. Each B. fragilis division was associated with the presence of a different antibiotic resistance gene: cepA encoding a serine-beta-lactamase (division I) and cfiA encoding a metallo-beta-lactamase (division II). No particular clusters associated with geographical or clinical origin, or with the production of an enterotoxin were observed. Sequencing of the cfiA gene allowed identification of two different alleles in division II. However, no association of these different cfiA alleles with the expression of imipenem resistance was observed. In conclusion, the phylogenetic patterns observed by sequencing recA and glnA are in agreement with those obtained previously by MLEE (multilocus enzyme electrophoresis). Thus, it appears that the evolution of recA and glnA genes is similar to that of the whole chromosome of B. fragilis. Horizontal gene transfer between divisions I and II seems to be low, at best. However, the results of the present study could not clarify definitively whether divisions I and II should be considered as two different B. fragilis genospecies.

Rhipicephalus ticks infected with Rickettsia and Coxiella in Southern Switzerland (Canton Ticino).

Ticks of the Rhipicephalus sanguineus species complex may be vector of various pathogens including Rickettsia conorii (the etiological agent of the Mediterranean spotted fever) and Coxiella burnetii (cause of the Query (Q) fever). R. sanguineus ticks have been imported in several parts of central and northern Europe, especially in environments such as kennels and houses providing the appropriate microclimatic conditions and the blood source necessary for their survival. Since 1940 these ticks have occasionally been recorded in Switzerland. In Ticino (the southern part of Switzerland), they have been reported since 1980 and their probable establishment in this area has been suggested in the '90s. By means of PCR and direct sequencing, we tested the identity of these ticks (using 12S rDNA gene) and the occurrence of Rickettsia spp. (using 16S rDNA, gltA and OmpA genes) as well as Coxiella sp. (using 16S rDNA). The results indicated that in Ticino, two different tick species coexist, i.e. R. sanguineus sensu stricto and Rhipicephalus turanicus. A few individuals of R. sanguineus sensu stricto are infected with Rickettsia massiliae/Bar29, which are strains of unknown pathogenicity. Coxiella sp., an endosymbiont of Rhipicephalus ticks, has also been identified in both tick species. Due to climatic changes towards global warming, imported tick species may therefore adapt to new area and might be considered as epidemiological markers for a number of infectious agents transmitted by them.

Population genetics of Helicobacter pylori in the southern part of Switzerland analysed by sequencing of four housekeeping genes (atpD, glnA, scoB and recA), and by vacA, cagA, iceA and IS605 genotyping.

The population biology of 78 Helicobacter pylori strains (71 from Swiss Italian, 4 from East Asian and 3 from South African patients) was investigated by sequence analysis of four housekeeping genes: atpD, scoB, glnA and recA. The vacA genotype, the presence of cagA and IS605, the iceA allelic type, and the resistance to metronidazole, clarithromycin and amoxycillin were determined. A high percentage of DNA polymorphic sites (19.8% for atpD, 21.3% for scoB, 23.7% for glnA and 20.3% for recA) was found. The phylogenetic trees based on the nucleotide sequences of the four gene fragments showed different topologies and were incongruent. The virulence-associated markers were distributed over the dendrograms and no association was found with phylogenetic clusters or clinical manifestations (chronic gastritis, gastric or duodenal ulcer, MALT lymphoma). Moreover, the H ratios (calculated with the homoplasy test) ranged from 0.742 to 0.799, depending on the gene fragment examined. All these observations suggest that H. pylori exists as a recombinant population. The clustering of the strains according to their geographical origin (USA/Europe, East Asia, South Africa) that has recently been demonstrated elsewhere could only be confirmed for the East Asian vacA s1c strains. In contrast, the South African strains clustered together only in the atpD tree. Presumably, recombination at the different loci has masked the evolutionary relationship among the strains.