Transposon mutagenesis of Rickettsia felis sca1 confers a distinct phenotype during flea infection.

Since its recognition in 1994 as the causative agent of human flea-borne spotted fever, Rickettsia felis, has been detected worldwide in over 40 different arthropod species. The cat flea, Ctenocephalides felis, is a well-described biological vector of R. felis. Unique to insect-borne rickettsiae, R. felis can employ multiple routes of infection including inoculation via salivary secretions and potentially infectious flea feces into the skin of vertebrate hosts. Yet, little is known of the molecular interactions governing flea infection and subsequent transmission of R. felis. While the obligate intracellular nature of rickettsiae has hampered the function of large-scale mutagenesis strategies, studies have shown the efficiency of mariner-based transposon systems in Rickettsiales. Thus, this study aimed to assess R. felis genetic mutants in a flea transmission model to elucidate genes involved in vector infection. A Himar1 transposase was used to generate R. felis transformants, in which subsequent genome sequencing revealed a transposon insertion near the 3' end of sca1. Alterations in sca1 expression resulted in unique infection phenotypes. While the R. felis sca1::tn mutant portrayed enhanced growth kinetics compared to R. felis wild-type during in vitro culture, rickettsial loads were significantly reduced during flea infection. As a consequence of decreased rickettsial loads within infected donor fleas, R. felis sca1::tn exhibited limited transmission potential. Thus, the use of a biologically relevant model provides evidence of a defective phenotype associated with R. felis sca1::tn during flea infection.

When does the female bias arise? Insights from the sex determination cascade of a flea beetle with a strongly skewed sex ratio.

Reproduction-manipulating bacteria like Wolbachia can shift sex ratios in insects towards females, but skewed sex ratios may also arise from genetic conflicts. The flea beetle Altica lythri harbors three main mtDNA strains that are coupled to three different Wolbachia infections. Depending on the mtDNA types, the females produce either offspring with a balanced sex ratio or exclusively daughters. To obtain markers that can monitor when sex bias arises in the beetle's ontogeny, we elucidated the sex determination cascade of A. lythri. We established a RT-PCR method based on length variants of dsx (doublesex) transcripts to determine the sex of morphologically indistinguishable eggs and larvae. In females of one mtDNA type (HT1/HT1*) known to produce only daughters, male offspring were already missing at the egg stage while for females of another type (HT2), the dsx splice variants revealed a balanced sex ratio among eggs and larvae. Our data suggest that the sex determination cascade in A. lythri is initiated by maternally transmitted female-specific tra (transformer) mRNA as primary signal. This tra mRNA seems to be involved in a positive feedback loop that maintains the production of the female splice variant, as known for female offspring in Tribolium castaneum. The translation of the maternally transmitted female tra mRNA must be inhibited in male offspring, but the underlying primary genetic signal remains to be identified. We discuss which differences between the mtDNA types can influence sex determination and lead to the skewed sex ratio of HT1.

Immunogenetics, sylvatic plague and its vectors: insights from the pathogen reservoir Mastomys natalensis in Tanzania.

Yersinia pestis is a historically important vector-borne pathogen causing plague in humans and other mammals. Contemporary zoonotic infections with Y. pestis still occur in sub-Saharan Africa, including Tanzania and Madagascar, but receive relatively little attention. Thus, the role of wildlife reservoirs in maintaining sylvatic plague and spillover risks to humans is largely unknown. The multimammate rodent Mastomys natalensis is the most abundant and widespread rodent in peri-domestic areas in Tanzania, where it plays a major role as a Y. pestis reservoir in endemic foci. Yet, how M. natalensis' immunogenetics contributes to the maintenance of plague has not been investigated to date. Here, we surveyed wild M. natalensis for Y. pestis vectors, i.e., fleas, and tested for the presence of antibodies against Y. pestis using enzyme-linked immunosorbent assays (ELISA) in areas known to be endemic or without previous records of Y. pestis in Tanzania. We characterized the allelic and functional (i.e., supertype) diversity of the major histocompatibility complex (MHC class II) of M. natalensis and investigated links to Y. pestis vectors and infections. We detected antibodies against Y. pestis in rodents inhabiting both endemic areas and areas considered non-endemic. Of the 111 nucleotide MHC alleles, only DRB*016 was associated with an increased infestation with the flea Xenopsylla. Surprisingly, we found no link between MHC alleles or supertypes and antibodies of Y. pestis. Our findings hint, however, at local adaptations towards Y. pestis vectors, an observation that more exhaustive sampling could unwind in the future.

Vector-borne protozoan and bacterial pathogen occurrence and diversity in ectoparasites of the Egyptian Rousette bat.

Bats are known reservoir hosts for a wide variety of parasites and pathogens, including bacteria and protozoans. Some of these pathogens are vector-borne, and although their role is poorly studied, ectoparasites may contribute significantly to their transmission. The aim of this study was to molecularly detect the presence of vector-borne microorganisms in bat-associated ectoparasites to explore their diversity and distribution in these insects. We tested the presence of Bartonella spp., Polychromophilus spp., and Trypanosoma spp. in bat flies and bat fleas collected from 56 Egyptian Rousette bats (Rousettus aegyptiacus), using conventional PCR. We found a high prevalence of 43.9% (47/107) of Bartonella spp. in bat flies, but a low prevalence of 6.6% (4/61) in bat fleas. Polychromophilus and Trypanosoma DNA were absent in both bat flies and bat fleas. Furthermore, we found novel gltA Bartonella sequences, as well as genotypes that are highly similar to recently described and potentially zoonotic ones. Our results show high diversity of Bartonella in bat flies, however, their role in pathogen transmission is still unknown and should be further explored.

Genomic structural plasticity of rodent-associated Bartonella in nature.

Rodent-associated Bartonella species have shown a remarkable genetic diversity and pathogenic potential. To further explore the extent of the natural intraspecific genomic variation and its potential role as an evolutionary driver, we focused on a single genetically diverse Bartonella species, Bartonella krasnovii, which circulates among gerbils and their associated fleas. Twenty genomes from 16 different B. krasnovii genotypes were fully characterized through a genome sequencing assay (using short and long read sequencing), pulse field gel electrophoresis (PFGE), and PCR validation. Genomic analyses were performed in comparison to the B. krasnovii strain OE 1-1. While, single nucleotide polymorphism represented only a 0.3% of the genome variation, structural diversity was identified in these genomes, with an average of 51 ± 24 structural variation (SV) events per genome. Interestingly, a large proportion of the SVs (>40%) was associated with prophages. Further analyses revealed that most of the SVs, and prophage insertions were found at the chromosome replication termination site (ter), suggesting this site as a plastic zone of the B. krasnovii chromosome. Accordingly, six genomes were found to be unbalanced, and essential genes near the ter showed a shift between the leading and lagging strands, revealing the SV effect on these genomes. In summary, our findings demonstrate the extensive genomic diversity harbored by wild B. krasnovii strains and suggests that its diversification is initially promoted by structural changes, probably driven by phages. These events may constantly feed the system with novel genotypes that ultimately lead to inter- and intraspecies competition and adaptation.

Identification and response of cytochrome P450 genes in the brackish water flea Diaphanosoma celebensis after exposure to benzo[α]pyrene and heavy metals.

The cytochrome P450 (CYP) enzyme family is extensive; these enzymes participate in phase I enzyme metabolism and are involved in xenobiotic detoxification in all living organisms. Despite their significance in xenobiotic detoxification, little is known about the species-specific comparison of CYPs and their molecular responses in aquatic invertebrates. We identified 31 CYPs in the brackish water flea Diaphanosoma celebensis via thorough exploration of transcriptomic databases and measured the transcript profiles of 9 CYPs (within full sequences) in response to benzo[α]pyrene (B[α]P) and two heavy metals (cadmium [Cd] and copper [Cu]). Through phylogenetic analysis, the CYPs were separated and clustered into four clans: mitochondrial, CYP2, CYP3, and CYP4. The expression of 9 CYPs were differentially modulated (up- and/or downregulated) in response to B[α]P, Cd, and Cu. In particular, CYP370A15 was significantly upregulated in response to B[α]P, Cd, and Cu, suggesting that the identified CYPs are involved in xenobiotic detoxification and are useful as biomarkers in response to B[α]P, Cd, and Cu. This study aimed to comprehensively annotate cladoceran CYPs; our results will add to the existing knowledge on the potential roles of CYPs in xenobiotic detoxification in cladocerans.

Spatial and temporal variation of compositional, functional, and phylogenetic diversity in ectoparasite infracommunities harboured by small mammals.

We studied patterns of compositional, functional, and phylogenetic α- and ß-diversity in flea and gamasid mite infracommunities of small Siberian mammals, taking into account host-associated (species) and environmental (biome or sampling period) factors. We asked: (a) How do these factors and their interactions affect infracommunity diversity? (b) Does infracommunity composition, in terms of species, traits, and phylogenetic lineages, deviate from random? (c) Are species, traits, and phylogenetic lineages in infracommunities clustered or overdispersed?, and (d) Do patterns of diversity differ between the three diversity facets and/or the two ectoparasite taxa? We found that the α-diversity of infracommunities was strongly affected by host species, biome, and sampling period. The highest proportion of infracommunity diversity in both taxa was associated with the interaction between either host species and biome or host species and sampling period. Infracommunities of both taxa within, as well as between, host species, biomes, and sampling periods were characterized by the clustering of species, traits and lineages. The patterns of the effects of host species, biome, and sampling period on infracommunity diversity were congruent among the three diversity facets in both fleas and mites. We conclude that the assembly patterns in ectoparasite infracommunities mirror those characteristics of component and compound communities.

Ctenophthalmus baeticus boisseauorum (Beaucournu, 1968) and Ctenophthalmus apertus allani (Smit, 1955) (Siphonaptera: Ctenophthalmidae) as synonymous taxa: morphometric, phylogenetic, and molecular characterization.

The family Ctenophthalmidae (Order Siphonaptera) has been considered as a 'catchall' for a wide range of divergent taxa showing a paraphyletic origin. In turn, Ctenophthalmus sp. (Ctenophthalmidae) includes 300 valid described taxa. Within this genus, males are easily distinguishable basing on the size, shape, and chaetotaxy of their genitalia; however, females show slight morphological differences with each other. The main objective of this work was to carry out a comparative morphometric, phylogenetic, and molecular study of two different subspecies: Ctenophthalmus baeticus boisseauorum and Ctenophthalmus apertus allani in order to clarify and discuss its taxonomic status. From a morphological and biometrical point of view, we found clear differences between modified abdominal segments of males of both subspecies and slight differences in the margin of sternum VII of all female specimens which did not correspond with molecular and phylogenetic results based on four different molecular markers (Internal Transcribed Spacer 1 and 2 of ribosomal DNA, and the partial cytochrome c oxidase subunit 1 and cytochrome b of mitochondrial DNA). Thus, we observed a phenotypic plasticity between both subspecies, which did not correspond with a real genotypic variability nor different environmental or ecological conditions. Basing on these results, we could consider that there are no solid arguments to consider these two 'morphosubspecies' as two different taxa. We propose that C. b. boisseauorum should be considered as a junior synonym of C. a. allani.

Origin, evolution, phylogeny and taxonomy of Pulex irritans.

The human flea Pulex irritans Linnaeus, 1758 (Siphonaptera: Pulicidae) is one of the most studied species together with the cat flea Ctenocephalides felis Bouché, 1835, because they have a cosmopolitan distribution and are closely related to humans. The present study aimed to carry out a comparative morphometric and molecular study of two different populations of P. irritans (Spain and Argentina). Accordingly, internal transcribed spacer (ITS)1 and ITS2 of rDNA and the partial cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) mtDNA genes of these taxa were sequenced. Furthermore, the taxonomy, origin, evolution and phylogeny of P. irritans was assessed. The morphometric data obtained did not show significant differences between P. irritans specimens from Spain and Argentina, even when these two populations were collected from different hosts; however, there was a considerable degree of molecular divergence between both populations based on nuclear and mitochondrial markers. Thus, it is proposed that P. irritans, in contrast with other generalist fleas, maintains a certain degree of morphological similarity, at least between Western Palearctic and Neotropical areas. Furthermore, two well defined geographical genetic lineages within the P. irritans species are indicated, suggesting the existence of two cryptic species that could be discriminated by a polymerase chain reaction-linked restriction fragment length polymorphism.

Comparative mtDNA phylogeographic patterns reveal marked differences in population genetic structure between generalist and specialist ectoparasites of the African penguin (Spheniscus demersus).

To address factors affecting genetic diversity and dispersal of ectoparasites, we compared mitochondrial DNA (mtDNA) population genetic structures of the generalist soft tick Ornithodoros capensis to the more host-specific nest flea Parapsyllus humboldti. A total of 103 ticks and 92 fleas were sampled at five distinct South African island/mainland African penguin (Spheniscus demersus) colonies. With its wide host range, O. capensis showed no evidence of significant cytochrome c oxidase subunit I (COI) mtDNA population differentiation among the five sampling sites (φst = 0.00 ± 0.004; p = 0.80), as well as a higher level of genetic diversity (π = 0.8% ± 0.06%) when compared to P. humboldti. In contrast, the flea showed significant population structure among most of the same sampling sites (φst = 0.22 ± 0.11; p ≤ 0.05) and a lower level of genetic diversity (π = 0.2% ± 0.01%). Our findings suggest that despite both parasites being mostly nest bound, O. capensis have few barriers to dispersal among island and mainland colonies. However, P. humboldti are more dependent on the African penguin for dispersal and thus have more impediments to gene flow among the same colonies. These findings broadly support the SGVH (specialist generalist variation hypothesis) and provide the first evidence for this hypothesis in parasites restricted to seabird colonies.

The effects of environment, hosts and space on compositional, phylogenetic and functional beta-diversity in two taxa of arthropod ectoparasites.

We studied the effects of variation in environmental, host-associated and spatial factors on variation in compositional, phylogenetic/taxonomic and functional facets of beta-diversity in fleas and gamasid mites parasitic on small mammals and asked whether (a) the importance of these factors as drivers of beta-diversity differs among its multiple facets and (b) the effects of variation in environment, hosts and space on beta-diversity variation differ between the two ectoparasite taxa. To understand the relative effects of each group of predictors, we used a distance-based redundancy analysis and variation partitioning. The greatest portions of variation in the compositional beta-diversity of fleas were equally explained by host-associated and spatial predictors, whereas variation in host species composition contributed the most to variation in the compositional beta-diversity of mites. Variation in the phylogenetic (i.e. based on phylogenetic tree) beta-diversity of fleas was mainly due to variation in the phylogenetic composition of host communities, while the taxonomic (i.e. based on Linnean taxonomy) beta-diversity of mites was influenced by environmental variation. Unique contributions of spatial and environmental variation explained most of the variation in functional beta-diversity and its species replacement (= turnover) component (i.e. beta-diversity explained by replacement of species alone) in fleas and mites, respectively. Variation in the richness difference component (i.e. beta-diversity explained by species loss/gain alone) of functional beta-diversity was mainly affected by either variation in the functional composition of host assemblages (fleas) or its joint action with environmental variables (mites). We conclude that the pattern of the relative effects of environmental, host-associated and spatial factors on beta-diversity is context-dependent and may differ among different facets of beta-diversity, among different beta-diversity components and also among taxa dependent on biological affinities.

Gene flow and adaptive potential in a generalist ectoparasite.

BACKGROUND: In host-parasite systems, relative dispersal rates condition genetic novelty within populations and thus their adaptive potential. Knowledge of host and parasite dispersal rates can therefore help us to understand current interaction patterns in wild populations and why these patterns shift over time and space. For generalist parasites however, estimates of dispersal rates depend on both host range and the considered spatial scale. Here, we assess the relative contribution of these factors by studying the population genetic structure of a common avian ectoparasite, the hen flea Ceratophyllus gallinae, exploiting two hosts that are sympatric in our study population, the great tit Parus major and the collared flycatcher Ficedula albicollis. Previous experimental studies have indicated that the hen flea is both locally maladapted to great tit populations and composed of subpopulations specialized on the two host species, suggesting limited parasite dispersal in space and among hosts, and a potential interaction between these two structuring factors. RESULTS: C. gallinae fleas were sampled from old nests of the two passerine species in three replicate wood patches and were genotyped at microsatellite markers to assess population genetic structure at different scales (among individuals within a nest, among nests and between host species within a patch and among patches). As expected, significant structure was found at all spatial scales and between host species, supporting the hypothesis of limited dispersal in this parasite. Clustering analyses and estimates of relatedness further suggested that inbreeding regularly occurs within nests. Patterns of isolation by distance within wood patches indicated that flea dispersal likely occurs in a stepwise manner among neighboring nests. From these data, we estimated that gene flow in the hen flea is approximately half that previously described for its great tit hosts. CONCLUSION: Our results fall in line with predictions based on observed patterns of adaptation in this host-parasite system, suggesting that parasite dispersal is limited and impacts its adaptive potential with respect to its hosts. More generally, this study sheds light on the complex interaction between parasite gene flow, local adaptation and host specialization within a single host-parasite system.

Phylogenetic heritability of geographic range size in haematophagous ectoparasites: time of divergence and variation among continents.

To understand existence, patterns and mechanisms behind phylogenetic heritability in the geographic range size (GRS) of parasites, we measured phylogenetic signal (PS) in the sizes of both regional (within a region) and continental (within a continent) geographic ranges of fleas in five regions. We asked whether (a) GRS is phylogenetically heritable and (b) the manifestation of PS varies between regions. We also asked whether geographic variation in PS reflects the effects of the environment's spatiotemporal stability (e.g. glaciation disrupting geographic ranges) or is associated with time since divergence (accumulation differences among species over time). Support for the former hypothesis would be indicated by stronger PS in southern than in northern regions, whereas support for the latter hypothesis would be shown by stronger PS in regions with a large proportion of species belonging to the derived lineages than in regions with a large proportion of species belonging to the basal lineages. We detected significant PS in both regional and continental GRSs of fleas from Canada and in continental GRS of fleas from Mongolia. No PS was found in the GRS of fleas from Australia and Southern Africa. Venezuelan fleas demonstrated significant PS in regional GRS only. Local Indicators of Phylogenetic Association detected significant local positive autocorrelations of GRS in some clades even in regions in which PS has not been detected across the entire phylogeny. This was mainly characteristic of younger taxa.

Morphological, biometrical and molecular characterization of Archaeopsylla erinacei (Bouché, 1835).

In the present work, we carried out a morphological, biometrical and molecular study of the species Archaeopsylla erinacei (Bouché, 1835) and their subspecies: Archaeopsylla erinacei erinacei (Bouché, 1835) and Archaeopsylla erinacei maura (Jordan & Rothschild, 1912) isolated from hedgehogs (Erinaceus europaeus) from different geographical regions (Seville and Corse). We have found morphological differences in females of A. erinacei from the same geographical origin that did not correspond with molecular differences. We suggest that some morphological characters traditionally used to discriminate females of both subspecies should be revised as well as we set the total length of the spermatheca as a valid criterion in order to discriminate between both subspecies. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and partial 18S rRNA gene, and partial cytochrome c-oxidase 1 (cox1) and cytochrome b (cytb) mtDNA gene sequences were determined to clarify the taxonomic status of these taxa and to assess intra-specific and intra-population similarity. In addition, a phylogenetic analysis with other species of fleas using Bayesian and Maximum Likelihood analysis was performed. All molecular markers used, except 18S, showed molecular differences between populations corresponding with geographical origins. Thus, based on the phylogenetic and molecular study of two nuclear markers (ITS1, ITS2) and two mitochondrial markers (cox1 and cytb), as well as concatenated sequences of both subspecies, we reported the existence of two geographical genetic lineages in A. erinacei corresponding with two different subspecies: A. e. erinacei (Corse, France) and A. e. maura (Seville, Spain), that could be discriminated by polymerase chain reaction-linked random-fragment-length polymorphism.

Comparative phylogeography between two generalist flea species reveal a complex interaction between parasite life history and host vicariance: parasite-host association matters.

BACKGROUND: In parasitic taxa, life history traits such as microhabitat preference and host specificity can result in differential evolutionary responses to similar abiotic events. The present study investigates the influence of vicariance and host association on the genetic structure of two generalist flea species, Listropsylla agrippinae, and Chiastopsylla rossi. The taxa differ in the time spent on the host (predominantly fur vs. nest) and level of host specificity. RESULTS: A total of 1056 small mammals were brushed to collect 315 fleas originating from 20 geographically distinct localities in South Africa. Phylogeographic genetic structure of L. agrippinae and C. rossi were determined by making use of 315 mitochondrial COII and 174 nuclear EF1-α sequences. Both parasites show significant genetic differentiation among the majority of the sampling sites confirming limited dispersal ability for fleas. The generalist fur flea with a narrower host range, L. agrippinae, displayed geographic mtDNA spatial genetic structure at the regional scale and this pattern is congruent with host vicariance. The dating of the divergence between the L. agrippinae geographic clades co-insides with paleoclimatic changes in the region approximately 5.27 Ma and this provides some evidence for a co-evolutionary scenario. In contrast, the more host opportunistic nest flea, C. rossi, showed a higher level of mtDNA and nDNA spatial genetic structure at the inter-populational scale, most likely attributed to comparatively higher restrictions to dispersal. CONCLUSIONS: In the present study, the evolutionary history of the flea species could best be explained by the association between parasite and host (time spent on the host). The phylogeographic pattern of the fur flea with a narrower host range correspond to host spatial genetic structures, while the pattern in the host opportunistic nest flea correspond to higher genetic divergences between sampling localities that may also be associated with higher effective population sizes. These findings suggest that genetic exchange among localities are most likely explained by differences in the dispersal abilities and life histories of the flea species.

Molecular study of Stenoponia tripectinata tripectinata (Siphonaptera: Ctenophthalmidae: Stenoponiinae) from the Canary Islands: taxonomy and phylogeny.

In the present work, we carried out a comparative molecular study of Stenoponia tripectinata tripectinata isolated from Mus musculus from the Canary Islands, Spain. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and 18S ribosomal RNA partial gene and cytochrome c-oxidase 1 (cox1) mitochondrial DNA partial gene sequences of this subspecies were determined to clarify the taxonomic status of this subspecies and to assess inter-population variation and inter-specific sequence differences. In addition, we have carried out a comparative phylogenetic study with other species of fleas using Bayesian, Maximum Parsimony, Maximum Likelihood and Neighbor-Joining analysis. A geographical signal was detected between the cox1 partial gene sequences of S. t. tripectinata isolated from M. musculus from different islands and those isolated from Apodemus sylvaticus from the Iberian Peninsula. Our results assess the monophyletic origin of Stenoponiinae and a different genetic lineage from Ctenophthalmidae. Thus, the elevation of subfamily Stenoponiinae to family level (Stenoponiidae) is suggested.

Susceptibility of Adult Cat Fleas (Siphonaptera: Pulicidae) to Insecticides and Status of Insecticide Resistance Mutations at the Rdl and Knockdown Resistance Loci.

The susceptibility of 12 field-collected isolates and 4 laboratory strains of cat fleas, Ctenocephalides felis was determined by topical application of some of the insecticides used as on-animal therapies to control them. In the tested field-collected flea isolates the LD50 values for fipronil and imidacloprid ranged from 0.09 to 0.35 ng/flea and 0.02 to 0.19 ng/flea, respectively, and were consistent with baseline figures published previously. The extent of variation in response to four pyrethroid insecticides differed between compounds with the LD50 values for deltamethrin ranging from 2.3 to 28.2 ng/flea, etofenprox ranging from 26.7 to 86.7 ng/flea, permethrin ranging from 17.5 to 85.6 ng/flea, and d-phenothrin ranging from 14.5 to 130 ng/flea. A comparison with earlier data for permethrin and deltamethrin implied a level of pyrethroid resistance in all isolates and strains. LD50 values for tetrachlorvinphos ranged from 20.0 to 420.0 ng/flea. The rdl mutation (conferring target-site resistance to cyclodiene insecticides) was present in most field-collected and laboratory strains, but had no discernible effect on responses to fipronil, which acts on the same receptor protein as cyclodienes. The kdr and skdr mutations conferring target-site resistance to pyrethroids but segregated in opposition to one another, precluding the formation of genotypes homozygous for both mutations.

[On the origin of Yersinia pestis, a causative agent of the plague: A concept of population-genetic macroevolution in transitive environment].

An ecological scenario is proposed for the origin of causative agent of the plague (the bacterium Yersenia pestis) from the clone of pseudotuberculous microbe of the first serotype Y. pseudotuberculosis O:1b. Disclosed are the conditions of gradual intrusion of psychrophile saprozoonosis ancestor into the blood of the primary host, Mongolian tarbagan marmot Marmota sibirica. As an inductor of speciation acted the Sartan cooling that occurred in the end of late Pleistocene under conditions of arid ultra-continental climate in Central Asia. Soil freezing down to the level of hibernating chambers in marmot burrows initiated the transition of marmot flea, Oropsylla silantiewi, larvae to optional hemophagy on the mucous coat inside the mouth cavity of sleeping marmots. In its turn, this promoted the conditions of mass traumatic intrusion of Y pseudotuberculosis into marmots bloodstream from faecal particles getting in their mouth cavity in course of building up a plug in a burrow for hibernating. In marmot populations, the selection of bacteria underwent under conditions of heterothermy with repeated changes of hibernating marmots body temperature within the range of 5-37 degrees C (torpor-euthermy). During the warm season, when pseudotuberculous microbes are totally eliminated from the bloodstream of healthy marmots with body temperature about 37 degrees C, bacteria could survive in fleas' digestive tract in the form of biofilm developing in proventriculus as a so called blockage. Final isolation between ancestral and daughter species was helped by the development of intrapopulation antagonism related with the beginning of full-scale synthesis of bacteriocin pesticin. Population-genetic processes in the "marmot-flea" system have led to a macroevolutionary event, that is, to passage of bacteria in a new ecological niche and adaptive zone that are principally different from those of the ancestor. All the present intraspecies forms of Y. pestis that appeared due to microevolution, have originated with the subspecies Y. pestis tarbagani that has formed in Central Asia during the Sartan cooling.

Comparative phylogeography of the plateau zokor (Eospalax baileyi) and its host-associated flea (Neopsylla paranoma) in the Qinghai-Tibet Plateau.

BACKGROUND: Specific host-parasite systems often embody a particular co-distribution phenomenon, in which the parasite's phylogeographic pattern is dependent on its host. In practice, however, both congruent and incongruent phylogeographic patterns between the host and the parasite have been reported. Here, we compared the population genetics of the plateau zokor (Eospalax baileyi), a subterranean rodent, and its host-associated flea species, Neopsylla paranoma, with an aim to determine whether the two animals share a similar phylogeographic pattern. RESULTS: We sampled 130 host-parasite pairs from 17 localities in the Qinghai-Tibet Plateau (QTP), China, and sequenced a mitochondrial DNA (mtDNA) segment (~2,500 bp), including the complete COI and COII genes. We also detected 55 zokor and 75 flea haplotypes. AMOVA showed that the percentage of variation among the populations of zokors constituted 97.10%, while the within population variation was only 2.90%; for fleas, the values were 85.68% and 14.32%, respectively. Moreover, the flea Fst (fixation index) values were significantly smaller than in zokor. Although the Fst values between zokors and fleas were significantly and positively correlated (N=105, R=0.439, p=0.000), only a small amount (R2=0.19) of the flea Fst variations could be explained by the zokor Fst variations. The two animals showed very distinct haplotype network structures from each other while co-phylogenetic analyses were unable to reject the hypothesis of an independence of speciation events. CONCLUSIONS: Zokors and fleas have very distinct population genetic patterns from each other, likely due to the influence of other sympatrically-distributed vertebrates on the transmission of fleas.

Phylogenetic structure of host spectra in Palaearctic fleas: stability versus spatial variation in widespread, generalist species.

We investigated spatial variation in the phylogenetic structure of host spectra in fleas parasitic on small mammals. Measures of phylogenetic host specificity ((phylogenetic species clustering (PSC) and phylogenetic species variability (PSV)) varied significantly more between than within flea species, but the proportion of variation which accounted for among-species differences was low. In 13 of 18 common flea species, at least one of the indices of the phylogenetic structure of regional host spectra revealed a significantly positive association with the phylogenetic structure of regional host assemblage, while relationships between PSC or PSV of the regional host spectrum and the distance from either the region of a flea's maximal abundance or latitude were not supported. Overall, results of this study demonstrated that although the degree of phylogenetic host specificity in fleas can be considered as a true attribute of a flea species, it is highly spatially variable, with phylogenetic structure of the surrounding host pool being the main reason behind this variation.