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SummaryEndosymbiosis – where a microbe lives and replicates within a host – is an important contributor to organismal function that has accelerated evolutionary innovations and catalysed the evolution of complex life. The evolutionary processes associated with transitions to endosymbiosis, however, are poorly understood. Here, we use comparative genomics of the genusArsenophonusto reveal the complex processes that occur on evolution of an endosymbiotic lifestyle. We compared the genomes of 38 strains spanning diverse lifestyles from environmentally acquired infections to obligate inter-dependent endosymbionts. We observed recent endosymbionts had larger genome sizes than closely related environmentally acquired strains, consistent with evolutionary innovation and rapid gain of new function. Increased genome size was a consequence of prophage and plasmid acquisition including a cargo of type III effectors, and concomitant loss of CRISPR-Cas genome defence systems enabling mobile genetic element expansion. Persistent endosymbiosis was also associated with loss of type VI secretion, likely reflecting reduced microbe-microbe competition. Thereafter, the transition to stable endosymbiosis and vertical inheritance was associated with the expected relaxation of purifying selection, pseudogenisation of genes and reduction of metabolism, leading to genome reduction. However, reduced %GC that is typically considered a progressive linear process was observed only in obligate interdependent endosymbionts. We argue that a combination of the need for rapid horizontal gene transfer-mediated evolutionary innovation together with reduced phage predation in endosymbiotic niches drives loss of genome defence systems and rapid genome expansion upon adoption of endosymbiosis. These remodelling processes precede the reductive evolution traditionally associated with adaptation to endosymbiosis.

Dicyemida is a group of small-bodied marine parasites infecting cephalopods with many unknown life history details, such as their population structure and diversity, and their relation to sexual and asexual reproductive stages. To reveal (infra)population structure of Dicyema moschatum Whitman, 1883 in its host (Eledone moschata Lamarck, 1798), we isolated microsatellite sequences from a draft genome of D. moschatum and tested the loci for amplification success and genetic diversity. Eight microsatellite loci were selected for an analysis of D. moschatum populations from several octopus individuals sampled at two Mediterranean localities. The majority of microsatellite alleles were shared across the studied range, but several private alleles were also identified. Analysis of population structure identified two to four genetic clusters, mostly concordant with the geographic origin of the samples. Allelic patterns seen in individual dicyemid genotypes revealed that although dicyemids inside one host individual show low genetic variance, they do not represent genetically identical clones. These results suggest that infection is established by several dicyemid larvae within the lifetime of the host and sexual reproduction of dicyemids occurs inside the host.

BackgroundAnimals interact with a diverse array of both beneficial and detrimental microorganisms. These interactions sometimes spark obligate symbioses where the host depends on beneficial bacteria for survival and reproduction. In insects, these obligate symbioses in many cases allow feeding on nutritionally unbalanced diets such as plant sap and vertebrate blood. It is, however, still not clear how are these obligate intracellular symbioses maintained at the cellular level for up to several hundred million years. Exact mechanisms driving host-symbiont interactions are only understood for a handful of model species and data on blood-feeding hosts with intracellular bacteria are particularly scarce.ResultsHere, we analyzed interactions at the symbiotic interfaces of an obligately blood-sucking parasite of sheep, the louse flyMelophagus ovinus. We assembled a reference transcriptome from one male and one female individual and used RNA-Seq with five biological replicates to compare expression in the midgut cells housing bacteria to the rest of the gut (foregut-hindgut). We focused on nutritional and immunity interactions between the insect host and its obligate symbiontArsenophonus melophagi, and also generated lower-coverage data for three facultative bacterial symbionts (Sodalis melophagi, Bartonella melophagi, andWolbachiasp.) and one facultative eukaryoteTrypanosoma melophagium. We found strong evidence for the importance of zinc in the system likely caused by symbionts using zinc-dependent proteases when acquiring amino acids, and for likely different immunity mechanisms controlling the symbionts than in closely related tsetse flies.ConclusionsOur results show that cellular and nutritional interactions between this blood-sucking insect and its symbionts are less intimate than what was previously found in some (but not all) plant-sap sucking insects such as aphids, psyllids, whiteflies, and mealybugs. This finding is likely interconnected to several features observed in symbionts in blood-sucking arthropods, particularly their midgut intracellular localization (as opposed to being localized in truly specialized bacteriocytes), intracytoplasmic presence (as opposed to having an outermost host-derived ‘symbiosomal’ membrane), less severe genome reduction, and relatively recent associations caused by frequent evolutionary losses and replacements.Data depositionRaw RNA-Seq data were made available through the European Nucleotide Archive (ENA) database under the study accession number PRJEB30632. All assemblies and additional large supplementary files are available on FigShare [https://doi.org/10.6084/m9.figshare.6146777.v1]. All commands used for data analyses are available on Github [https://github.com/filip-husnik/melophagus].

Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet, vertebrate blood. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, indicating that levels of B vitamins vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood and hemolymph can provide valuable informative background and understanding of general principles of insect symbiosis.In this work chromatographic separation of a mixture of eight B vitamins (B1 – thiamine, B2 – riboflavin, B3 – niacin, B5 – pantothenic acid, B6 – pyridoxine, B7 – biotin, B9 – folic acid and B12 – cyanocobalamine), four B vitamin derivatives (B3 – niacinamide, B6 – pyridoxal-5-phosphate, B6 – 4-pyridoxic acid and B9 – tetrahydrofolic acid) and 3 stable isotope labelled internal standards (B3 – niacin-13C6, B5 – pantothenic acid (di-β-alanine-13C6,15N2) and B7 – biotin-(ring-6,6-d2)) on C30 column was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Matching internal standards with analytes was done according to the results of linearity, accuracy and precision. Except for vitamin B9 (tetrahydrofolic acid) instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 μg/L, correlation coefficients from 0.9997 to 1.0000 and QC coefficients from 0.53 to 3.2 %.Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. Samples of whole blood prepared in six independent replicates were spiked at 10 μg/L and 100 μg/L level. The best results were obtained for zinc sulphate in methanol, but only nine analytes (B1 – thiamine, B2 – riboflavin, B3 – niacin, B3 – niacinamide, B5 – pantothenic acid, B6 – pyridoxine, B6 – 4-pyridoxic acid, B7 – biotin and B12 – cyanocobalamine) were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120 %, precision from 0.5 to 13 % and process efficiency from 65 to 108 %.HighlightsLC-MS/MS method for quantitation of eight B vitamins and four B vitamin derivatives was developed.Deproteinization agents trichloroacetic acid and ZnSO4/methanol were tested for protein precipitation of whole blood.Accuracy, precision and process efficiency were evaluated.Successful method validation for seven B vitamins and two B vitamin derivatives in whole blood.

Interplay between conserved host specificity and occasional host switches is an important process determining the evolution of host-parasite systems. Here, we address the dynamics of host switches at the population level in rodent-associated Eimeria. Focusing mainly on two ecologically similar host groups, Murinae and Arvicolinae, we show that the Eimeria infecting those hosts form a complex system of many genetic lineages with different host specificities. The broad geographic distribution of lineages indicates that they are well-established genetic forms which retained their host specificities while spreading across large geographic areas. We also demonstrate that genetic structure is only partially reflected by morphological traits.

We describe here a new pattern of population genetic structure in a host-parasite system that can arise after secondary contact of previously isolated populations. Due to different generation times, and therefore different tempos of molecular evolution, the host and parasite populations reach different degrees of genetic differentiation during their separation (e.g., in refugia). Consequently, upon secondary contact, the host populations are able to re-establish a single panmictic population across the area of contact, while the parasite populations stop their dispersal at the secondary contact zone and create a narrow hybrid zone. From the host's perspective, the parasite's hybrid zone functions on a microevolutionary scale as a "parasite turnover zone": while the hosts are passing from area A to area B, their parasites turn genetically from the area A genotypes to the area B genotypes. We demonstrate this novel pattern with a model composed of Apodemus mice and Polyplax lice by comparing maternally inherited markers (complete mitochondrial genomes, and complete genomes of the vertically transmitted symbiont Legionella polyplacis) with single nucleotide polymorphisms derived from louse genomic data. We discuss the circumstances that may lead to this pattern and possible reasons why it has been overlooked in studies of host-parasite population genetics.

Recent studies show that host switching is much more frequent than originally believed and constitutes an important driver in evolution of host-parasite associations. However, its frequency and ecological mechanisms at the population level have been rarely investigated. We address this issue by analyzing phylogeny and population genetics of an extensive sample, from a broad geographic area, for commonly occurring parasites of the genus Eimeria within the abundant rodent genera Apodemus, Microtus and Myodes, using two molecular markers. At the most basal level, we demonstrate polyphyletic arrangement, i.e. multiple origin, of the rodent-specific clusters within the Eimeria phylogeny, and strong genetic/phylogenetic structure within these lineages determined at least partially by specificities to different host groups. However, a novel and the most important observation is a repeated occurrence of host switches among closely related genetic lineages which may become rapidly fixed. Within the studied model, this phenomenon applies particularly to the switches between the eimerians from Apodemus flavicollis/Apodemus sylvaticus and Apodemus agrarius groups. We show that genetic differentiation and isolation between A. flavicollis/A. sylvaticus and A. agrarius faunas is a secondary recent event and does not reflect host-parasite coevolutionary history. Rather, it provides an example of rapid ecology-based differentiation in the parasite population.

A degree of host specificity, manifested by the processes of host-parasite cospeciations and host switches, is assumed to be a major determinant of parasites' evolution. To understand these patterns and formulate appropriate ecological hypotheses, we need better insight into the coevolutionary processes at the intraspecific level, including the maintenance of genetic diversity and population structure of parasites and their hosts. Here, we address these questions by analyzing large-scale molecular data on the louse Polyplax serrata and its hosts, mice of the genus Apodemus, across a broad range of European localities. Using mitochondrial DNA sequences and microsatellite data, we demonstrate the general genetic correspondence of the Apodemus/Polyplax system to the scenario of the postglacial recolonization of Europe, but we also show several striking discrepancies. Among the most interesting are the evolution of different degrees of host specificity in closely related louse lineages in sympatry, or decoupled population structures of the host and parasites in central Europe. We also find strong support for the prediction that parasites with narrower host specificity possess a lower level of genetic diversity and a deeper pattern of interpopulation structure as a result of limited dispersal and smaller effective population size.

We introduce a new pattern of population genetic structure in a host-parasite system that can arise after secondary contact (SC) of previously isolated populations. Due to different generation time and therefore different tempo of molecular evolution the host and parasite populations reach different degrees of genetic differentiation during their separation (e.g. in refugia). Consequently, during the SC the host populations are able to re-establish a single panmictic population across the whole recolonized area, while the parasite populations stop their dispersal at the SC zone and create a narrow hybrid zone (HZ). From the host’s perspective, the parasite’s HZ functions on a microevolutionary scale as a “host-cleansing filter”: while passing from area A to area B, the hosts are rid of the area A parasites and acquire the area B parasites. We demonstrate this novel pattern on a model composed ofApodemusmice andPolyplaxlice by comparing maternally inherited markers (complete mitochondrial genomes, and complete genomes of vertically transmitted symbiontLegionella polyplacis) with SNPs derived from the louse genomic data. We discuss circumstances which may lead to this pattern and possible reasons why it has been overlooked in the studies on host-parasite population genetics.

Phylogenetic diversity of symbiotic bacteria in sucking lice suggests that lice have experienced a complex history of symbiont acquisition, loss, and replacement during their evolution. By combining metagenomics and amplicon screening across several populations of two louse genera (PolyplaxandHoplopleura) we describe a novel louse symbiont lineage related toNeisseriaandSnodgrassella, and show its’ independent origin within dynamic lice microbiomes. While the genomes of these symbionts are highly similar in both lice genera, their respective distributions and status within lice microbiomes indicate that they have different functions and history. InHoplopleura acanthopus, theNeisseria-related bacterium is a dominant obligate symbiont universally present across several host’s populations, and seems to be replacing a presumably older and more degenerated obligate symbiont. In contrast, thePolyplaxmicrobiomes are dominated by the obligate symbiontLegionella polyplacis, with theNeisseria-related bacterium co-occurring only in some samples and with much lower abundance.

Population structure and biogeography of marine organisms are formed by different drivers than in terrestrial organisms. Yet, very little information is available even for common marine organisms and even less for their associated parasites. Here we report the first analysis of population structure of both a cephalopod host (Sepia officinalis) and its dicyemid parasite, based on a homologous molecular marker (cytochrome oxidase I). We show that the population of common cuttlefish in the Mediterranean area is fragmented into subpopulations, with some areas featuring restricted level of gene flow. Amongst the studied areas, Sardinia was genetically the most diverse and Cyprus the most isolated. At a larger scale, across the Mediterranean, the population structure of the parasite shows co-diversification pattern with its host, but a slower rate of diversification. Differences between the two counterparts are more obvious at a finer scale, where parasite populations show increased level of fragmentation and lower local diversities. This discrepancy can be caused by local extinctions and replacements taking place more frequently in the dicyemid populations, due to their parasitic lifestyle.

A degree of host specificity, manifested by the processes of host–parasite cospeciations and host switches, is assumed to be a major determinant of parasites’ evolution. To understand these patterns and formulate appropriate ecological hypotheses, we need better insight into the coevolutionary processes at the intraspecific level, including the maintenance of genetic diversity and population structure of parasites and their hosts. Here, we address these questions by analyzing large‐scale molecular data on the louse Polyplax serrata and its hosts, mice of the genus Apodemus, across a broad range of European localities. Using mitochondrial DNA sequences and microsatellite data, we demonstrate the general genetic correspondence of the Apodemus/Polyplax system to the scenario of the postglacial recolonization of Europe, but we also show several striking discrepancies. Among the most interesting are the evolution of different degrees of host specificity in closely related louse lineages in sympatry, or decoupled population structures of the host and parasites in central Europe. We also find strong support for the prediction that parasites with narrower host specificity possess a lower level of genetic diversity and a deeper pattern of interpopulation structure as a result of limited dispersal and smaller effective population size.

Symbiosis between insects and bacteria result in a variety of arrangements, genomic modifications, and metabolic interconnections. Here, we present genomic, phylogenetic, and morphological characteristics of a symbiotic system associated with Melophagus ovinus , a member of the blood-feeding family Hippoboscidae. The system comprises four unrelated bacteria representing different stages in symbiosis evolution, from typical obligate mutualists inhabiting bacteriomes to freely associated commensals and parasites. Interestingly, the whole system provides a remarkable analogy to the association between Glossina and its symbiotic bacteria. In both, the symbiotic systems are composed of an obligate symbiont and two facultative intracellular associates, Sodalis and Wolbachia . In addition, extracellular Bartonella resides in the gut of Melophagus . However, the phylogenetic origins of the two obligate mutualist symbionts differ. In Glossina , the mutualistic Wigglesworthia appears to be a relatively isolated symbiotic lineage, whereas in Melophagus , the obligate symbiont originated within the widely distributed Arsenophonus cluster. Although phylogenetically distant, the two obligate symbionts display several remarkably similar traits (e.g., transmission via the host's “milk glands” or similar pattern of genome reduction). To obtain better insight into the biology and possible role of the M. ovinus obligate symbiont, “ Candidatus Arsenophonus melophagi,” we performed several comparisons of its gene content based on assignments of the Cluster of Orthologous Genes (COG). Using this criterion, we show that within a set of 44 primary and secondary symbionts, “ Ca . Arsenophonus melophagi” is most similar to Wigglesworthia . On the other hand, these two bacteria also display interesting differences, such as absence of flagellar genes in Arsenophonus and their presence in Wigglesworthia . This finding implies that a flagellum is not essential for bacterial transmission via milk glands.

Symbiotic interactions between insects and bacteria are ubiquitous and form a continuum from loose facultative symbiosis to greatly intimate and stable obligate symbiosis. In blood-sucking insects living exclusively on vertebrate blood, obligate endosymbionts are essential for hosts and hypothesized to supplement B-vitamins and cofactors missing from their blood diet. The role and distribution of facultative endosymbionts and their evolutionary significance as seeds of obligate symbioses are much less understood. Here, using phylogenetic approaches, we focus on the Hippoboscidae phylogeny as well as the stability and dynamics of obligate symbioses within this bloodsucking group. In particular, we demonstrate a new potentially obligate lineage of Sodalis co-evolving with the Olfersini subclade of Hippoboscidae. We also show several likely facultative Sodalis lineages closely related to Sodalis praecaptivus (HS strain) and suggest repeated acquisition of novel symbionts from the environment. Similar to Sodalis, Arsenophonus endosymbionts also form both obligate endosymbiotic lineages co-evolving with their hosts (Ornithomyini and Ornithoica groups) as well as possibly facultative infections incongruent with the Hippoboscidae phylogeny. Finally, we reveal substantial diversity of Wolbachia strains detected in Hippoboscidae samples falling into three supergroups: A, B, and the most common F. Altogether, our results prove the associations between Hippoboscoidea and their symbiotic bacteria to undergo surprisingly dynamic, yet selective, evolutionary processes strongly shaped by repeated endosymbiont replacements. Interestingly, obligate symbionts only originate from two endosymbiont genera, Arsenophonus and Sodalis, suggesting that the host is either highly selective about its future obligate symbionts or that these two lineages are the most competitive when establishing symbioses in louse flies.

Legionellaceae are intracellular bacteria known as important pathogens of man. In the environment, they are mainly found in biofilms associated with amoebas. In contrast to another gammaproteobacterial family Enterobacteriaceae which established a broad spectrum of symbioses with many insect taxa, the only instance of legionella-like symbiont has been reported from lice of the genus Polyplax. Here, we sequenced the complete genome of this symbiont and compared its main characteristics to other Legionella species and insect symbionts. Based on rigorous multigene phylogenetic analyses, we confirm this bacterium as a member of the genus Legionella and propose the name Candidatus Legionella polyplacis, sp.n. We show that the genome of Ca. Legionella polyplacis underwent massive degeneration, including considerable size reduction (529.746 bp, 484 protein coding genes) and a severe decrease in GC content (23%). We identify several possible constraints underlying the evolution of this bacterium. On one hand, Ca. Legionella polyplacis and the louse symbionts Riesia and Puchtella experienced convergent evolution, perhaps due to adaptation to similar hosts. On the other hand, some metabolic differences are likely to reflect different phylogenetic positions of the symbionts and hence availability of particular metabolic function in the ancestor. This is exemplified by different arrangements of thiamine metabolism in Ca. Legionella polyplacis and Riesia. Finally, horizontal gene transfer is shown to play a significant role in the adaptive and diversification process. Particularly, we show that Ca. L. polyplacis horizontally acquired a complete biotin operon (bioADCHFB) that likely assisted this bacterium when becoming an obligate mutualist.

The generic name Hemolivia has been used for haemogregarines characterized by morphological and biological features. The few molecular studies, focused on other haemogregarine genera but involving Hemolivia samples, indicated its close relationship to the genus Hepatozoon. Here we analyze molecular data for Hemolivia from a broad geographic area and host spectrum and provide detailed morphological documentation of the included samples. Based on molecular analyses in context of other haemogregarines, we demonstrate that several sequences deposited in GenBank from isolates described as Hepatozoon belong to the Hemolivia cluster. This illustrates the overall difficulty with recognizing Hemolivia and Hepatozoon without sufficient morphological and molecular information. The close proximity of both genera is also reflected in uncertainty about their precise phylogeny when using 18S rDNA. They cluster with almost identical likelihood either as two sister taxa or as monophyletic Hemolivia within paraphyletic Hepatozoon. However, regardless of these difficulties, the results presented here provide a reliable background for the unequivocal placement of new samples into the Hemolivia/ Hepatozoon complex.

Two morphologically similar, but phylogenetically unrelated Eimeria species from ancient mammals, African Tree Pangolin Phataginus tricuspis and Sunda Pangolin Manis javanica (Pholidota: Manidae), from two distant biogeographic realms (Afrotropical and Oriental), are characterized and compared morphologically and molecularly. Phylogenetic analyses produced an unstable topology. However, while precise position of the two Eimeria species from pangolins could not be firmly established due to the lack of related taxa, it is evident that they are not closely related and do not fall into any of the so far recognized eimerian lineages. Moreover, an eimerian found in P. tricuspis is described as a new species Eimeria nkaka n. sp., based on morphology of oocysts, endogenous developmental stages and sequence data.

Introduced species have the potential to outperform natives via the introduction of new parasites to which the native ecosystem is vulnerable. Cryptic diversity within an invasive species can obscure invasion patterns and confound proper management measures. The aim of this study is to use coalescent theory based methodology to trace recent routes of invasion in populations of Ligula intestinalis, a globally distributed fish parasite possessing both native and recently introduced populations in North Africa. Molecular analyses of mitochondrial DNA discerned a pronounced genetic divergence between introduced and native populations. Distribution of mitochondrial haplotypes demonstrated common origin of European populations with North African parasites sampled from introduced fish species in Tunisia. To test the suggested pathway of introduction, microsatellite data were examined in a model-based coalescent analysis using the software MIGRATE, where Europe to Tunisia direction of migration was favoured over alternative hypotheses of gene flow. Specificity of Tunisian populations to different host species was assessed in an epidemiologic survey confirming prevailing host-based division between introduced and native parasites in North Africa. This approach combining advanced analysis of molecular markers with host-specificity data allows revealing the evolution of host-parasite interactions following biological invasion and provides basis for devising future management measurements.

Candidatus Arsenophonus lipopteni (Enterobacteriaceae, Gammaproteobacteria) is an obligate intracellular symbiont of the blood feeding deer ked, Lipoptena cervi (Diptera: Hippoboscidae). The bacteria reside in specialized cells derived from host gut epithelia (bacteriocytes) forming a compact symbiotic organ (bacteriome). Compared to the closely related complex symbiotic system in the sheep ked, involving four bacterial species, Lipoptena cervi appears to maintain its symbiosis exclusively with Ca. Arsenophonus lipopteni. The genome of 836,724 bp and 24.8 % GC content codes for 667 predicted functional genes and bears the common characteristics of sequence economization coupled with obligate host-dependent lifestyle, e.g. reduced number of RNA genes along with the rRNA operon split, and strongly reduced metabolic capacity. Particularly, biosynthetic capacity for B vitamins possibly supplementing the host diet is highly compromised in Ca. Arsenophonus lipopteni. The gene sets are complete only for riboflavin (B2), pyridoxine (B6) and biotin (B7) implying the content of some B vitamins, e.g. thiamin, in the deer blood might be sufficient for the insect metabolic needs. The phylogenetic position within the spectrum of known Arsenophonus genomes and fundamental genomic features of Ca. Arsenophonus lipopteni indicate the obligate character of this symbiosis and its independent origin within Hippoboscidae.

Host-parasite co-evolution belongs among the major processes governing evolution of biodiversity on the global scale. Numerous studies performed at inter-specific level revealed variety of patterns from strict co-speciation to lack of co-divergence and frequent host-switching, even in species tightly linked to their hosts. To explain these observations and formulate ecological hypotheses, we need to acquire better understanding to parasites’ population genetics and dynamics, and their main determinants. Here, we analyse the impact of co-evolutionary processes on genetic diversity and structure of parasite populations, using a model composed of the lousePolyplax serrataand its hosts, mice of the genusApodemus, collected from several dozens of localities across Europe. We use mitochondrial DNA sequences and microsatellite data to describe the level of genealogical congruence between hosts and parasites and to assess genetic diversity of the populations. We also explore links between the genetic assignment of the parasite and its host affiliation, and test the prediction that populations of the parasite possessing narrower host specificity show deeper pattern of population structure and lower level of genetic diversity as a result of limited dispersal and smaller effective population size. We demonstrate an overall complexity of the co-evolutionary processes and their variability even among closely related lineages of the parasites. In the analysis of several sympatric parasite populations, we find strong evidence for the link between the width of host specificity and genetic diversity of parasites.

Ťahyňa virus (TAHV), a mosquito-borne bunyavirus (California group), is frequently associated with inapparent or influenza-like (Valtice fever) infections in humans, rarely leading to atypical pneumonia or meningitis. Field TAHV strains exhibit a high variability in their biological properties with respect to virulence for laboratory mouse, temperature-sensitivity or character of plaques in cell culture. In consideration of the variations in the antigenic properties TAHV and its potential genetic variability, we analyzed complete nucleotide sequences of the small (S) and medium (M) genomic segments of field TAHV strains with different combinations of phenotypic markers. S segment was highly conservative in all analyzed TAHV strains. Within the M segment, the highest variability was observed in the G C gene encoding viral envelope protein and to a less extent also in the NSm gene. However, 5′ and 3′ non-coding regions of M segment, as well as in G N gene exhibited highly conservative pattern, indicating its functional importance, but minor or no role in the determination of biological properties of TAHV field strains.

Organisms with wide geographical or phenotypic diversity often constitute assemblages of genetically distinct species or lineages. Within parasites, an emergence of host-specific lineages is assumed to create such cryptic variability; however, empirical evaluation of these processes is scarce. Here, we analyse populations of a parasite with a complex life cycle, wide host spectrum and global distribution, with the aim to reveal factors underlying the evolution of host- or geography-dependent lineages. Using 15 microsatellite loci, deep genetic distances were observed between populations from distant geographical areas. On the local scale, host-mediated genetic structure was found among sympatric samples. Two lineages differing in the spectrum of infected hosts co-occurred in the Euro-Mediterranean area, and two distinct lineages were recovered from Lake Tana in Ethiopia. Although sampled across several host taxa and multiple localities, a lack of marked genetic structure was seen in the populations belonging to one of the European lineages. Only weak genetic differentiation between sympatric samples from two host species was found. Complexity of the parasite life-cycle contributed to such a stratified pattern. Differences in the immune response between fish hosts were suggested as the factor diversifying the populations locally; conversely, high mobility of the parasite due to migration with its bird (definitive) host were assessed to homogenize populations across the area of distribution. However, despite the high mobility, large bodies of salt water prevent the parasite from long-distance migrations, as was demonstrated in an example of the Mediterranean Sea which represented an effective barrier to gene flow.

The genes of ribosomal RNA are the most popular and frequently used markers for bacterial phylogeny and reconstruction of insect-symbiont coevolution. In primary symbionts, such as Buchnera and Wigglesworthia, genome economization leads to the establishment of a single copy of these sequences. In phylogenetic studies, they provide sufficient information and yield phylogenetic trees congruent with host evolution. In contrast, other symbiotic lineages (e.g., the genus Arsenophonus) carry a higher number of rRNA copies in their genomes, which may have serious consequences for phylogenetic inference. In this study, we show that in Arsenophonus triatominarum the degree of heterogeneity can affect reconstruction of phylogenetic relationships and mask possible coevolution between the symbiont and its host. Phylogenetic arrangement of individual rRNA copies was used, together with a calculation of their divergence time, to demonstrate that the incongruent 16S rDNA trees and low nucleotide diversity in the secondary symbiont could be reconciled with the coevolutionary scenario.

Polymorphic microsatellite loci were characterised for two louse species, the anopluran Polyplax serrata Burmeister, 1839, parasitising Eurasian field mice of the genus Apodemus Kaup, and the amblyceran Myrsidea nesomimi Palma et Price, 2010, found on mocking birds endemic to the Galápagos Islands. Evolutionary histories of the two parasites show complex patterns influenced both by their geographic distribution and through coevolution with their respective hosts, which renders them prospective evolutionary models. In P. serrata, 16 polymorphic loci were characterised and screened across 72 individuals from four European populations that belong to two sympatric mitochondrial lineages differing in their breadth of host-specificity. In M. nesomimi, 66 individuals from three island populations and two host species were genotyped for 15 polymorphic loci. The observed heterozygosity varied from 0.05 to 0.9 in P. serrata and from 0.0 to 0.96 in M. nesomimi. Deviations from the Hardy-Weinberg equilibrium were frequently observed in the populations of both parasites. Fst distances between tested populations correspond with previous phylogenetic data, suggesting the microsatellite loci are an informative resource for ecological and evolutionary studies of the two parasites.

The most representative sample of molecular data, especially 16S and 12S rDNAs, is used to study the phylogeny and evolution of 57 species of three tribes, Rhodniini, Linshcosteini, and Triatomini, of the subfamily Triatominae. For the first time both New World and Old World species are brought together in a single phylogenetic analysis. Maximum-parsimony and distance estimation place both the Asiatic representatives, Linshcosteus and Triatoma rubrofasciata, as sister groups. The Linshcosteus–T. rubrofasciata clade nests firmly within Triatomini, in most analyses branching as a basalmost lineage, thus supporting a monophyletic origin of Triatominae. A paraphyly of “Triatoma” with respect to Linshcosteus, Dipetalogaster, Eratyrus, and Panstrongylus and the paraphyly of “Rhodnius” with respect to Psammolestes is observed in most of the analyses. Reinterpretation of triatomine biogeography points to the origin of Triatominae in northern areas of South America, in Central America, or in the southern region of North America. A few taxonomic changes are proposed: (1) reinclusion of Linshcosteus in Triatomini, (2) inclusion of Psammolestes in Rhodnius, (3) elevation of the “T. flavida complex” to the full genus Nesotriatoma (including N. flavida, N. bruneri, and N. obscura), (4) inclusion of the “T. spinolai complex” in Mepraia (including M. spinolai, M. gajardoi, M. eratyrusiformis, and M. breyeri), and (5) inclusion of “T.” dimidiata in Meccus (M. dimidiatus).

The phylogeny of European species of the tapeworm genus Proteocephalus was studied, based on partial 18S rDNA and morphological data. The group was found to be monophyletic. The analysis showed a low informative value of available morphological characters in comparison with molecular data. The morphological matrix resulted in a poorly resolved tree which is, however, compatible with the topology (Proteocephalus osculatus (Proteocephalus torulosus (Proteocephalus macrocephalus, Proteocephalus filicollis) (Proteocephalus tetrastomus, Proteocephalus percae, Proteocephalus longicollis))) based on the 18S rDNA data. A comparison performed by the program TreeMap showed a lack of significant congruency between parasite and host phylogenies. Therefore, the distribution of species in their hosts appears to be independent of the phylogeny and it is likely to be a result of host-switching, rather than co-speciation events.

The myzostomes are animals with five pairs of parapodia, living as commensals or (endo)parasites mostly on crinoid and ophiuroid echinoderms. They are generally considered aberrant annelids, possibly phyllodocidan polychaetes. A phylogenetic analysis of 18S and 28S ribosomal DNA sequence data of Myzostoma glabrum , together with 60 morphological, developmental, ultrastructural, and life-history characters, is presented to show that myzostomes are a sister group of the Cycliophora, closely related to the rotifer-acanthocephalan clade (= Syndermata). Myzostomes and syndermates share predominantly the highly derived spermatozoa with anteriorly directed flagella (cycliophoran sperm is insufficiently known). The myzostome-cycliophoran-syndermate clade, accommodated within the Platyzoa (including Platyhelminthes s. str. , Gastrotricha, Gnathostomulida, Syndermata, Cycliophora, and Myzostomida), is strongly supported by most analyses, regardless of alignment parameters, character combinations and weighting, species sampling, and tree-building methods. The new name Prosomastigozoa (“forward-flagellar animals”) is proposed for the group including three phyla (Cycliophora, Myzostomida, and Syndermata).

The myzostomes are animals with five pairs of parapodia, living as commensals or (endo)parasites mostly on crinoid and ophiuroid echinoderms. They are generally considered aberrant annelids, possibly phyllodocidan polychaetes. A phylogenetic analysis of 18S and 28S ribosomal DNA sequence data of Myzostoma glabrum, together with 60 morphological, developmental, ultrastructural, and life-history characters, is presented to show that myzostomes are a sister group of the Cycliophora, closely related to the rotifer-acanthocephalan clade (=Syndermata). Myzostomes and syndermates share predominantly the highly derived spermatozoa with anteriorly directed flagella (cycliophoran sperm is insufficiently known). The myzostome-cycliophoran-syndermate clade, accommodated within the Platyzoa (including Platyhelminthes s. str., Gastrotricha, Gnathostomulida, Syndermata, Cycliophora, and Myzostomida), is strongly supported by most analyses, regardless of alignment parameters, character combinations and weighting, species sampling, and tree-building methods. The new name Prosomastigozoa ("forward-flagellar animals") is proposed for the group including three phyla (Cycliophora, Myzostomida, and Syndermata).

A phylogenetic analysis of tapeworms (Eucestoda) based on complete sequences of the 18S rRNA genes of 43 taxa (including new sequences of 12 species) was carried out, with the emphasis on the groups parasitising teleost fish and reptiles. Spathebothriidea and Trypanorhyncha (the latter group being paraphyletic) appeared as basal groups of the Eucestoda but their position was not stable. The tetrafossate orders (Litobothriidea, Lecanicephalidea, Tetraphyllidea, Proteocephalidea, Nippotaeniidea, Tetrabothriidea and Cyclophyllidea) were well separated from the remaining groups. Results supported polyphyly of the Pseudophyllidea formed by two distinct clades: one with diphyllobothriids (Diphyllobothrium, Schistocephalus, Spirometra and Duthiersia) and another including Abothrium, Probothriocephalus, Eubothrium and Bothriocephalus. The former pseudophyllidean clade formed a separate branch with the Caryophyllidea (Khawia and Hunterella) and Haplobothriidea (Haplobothrium), the latter taxon being closely related to either caryophyllideans or diphyllobothriids in different analyses. Proteocephalideans formed a monophyletic group in all analyses and constituted a clade within the Tetraphyllidea thus rendered paraphyletic. Within the Proteocephalidea, the Acanthotaeniinae (Acanthotaenia from reptiles in Africa) and Gangesiinae (Gangesia and Silurotaenia from silurid fish in the Palearctic Region) were separated from parasites of freshwater fish and mammals. The family Proteocephalidae was found to be paraphyletic due to the placement of a monticelliid species, Monticellia sp., in a clade within the former family. The genus Proteocephalus appeared as an artificial assemblage of unrelated taxa which is congruent with previous molecular analyses.

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.

Ligula intestinalis is a species with a complex biology. Several strains, distinct in the host specificity and biogeography, are supposed to exist; however, their morphological or molecular identification remains unresolved. We describe the isolation of 16 polymorphic microsatellite markers. The number of alleles ranged from four to 14 in a population of 21 L. intestinalis individuals. Loci showed amplification success across samples of worldwide distribution. A cross-amplification of 14 markers in Digramma interrupta (Cestoda: Pseudophyllidea) was observed.

The review is devoted to the gut lectins/hemagglutinins of the following representatives of important disease vectors: ticks, kissing-bugs, mosquitoes, sandflies and tsetse flies. The paper surveys the recent knowledge on these carbohydrate binding factors with respect to their structural and functional properties, and their significance for pathogen/parasite transmission by the blood-sucking arthropods. Recent results suggest that in most vectors the gut lectin activities are blood-meal enhanced, might participate in blood-meal processing and digestion and could serve as antibacterial and antiparasitic agents.

A hemagglutinin was identified and partially characterized in the crop of the bug Triatoma infestans. It agglutinated mouse and rabbit erythrocytes, and was strongly inhibited by bacterial lipopolysaccharides. SDS-PAGE and immunoblotting with specific antibodies identified a dominant protein with molecular mass of 20 kDa under reducing conditions. The protein seems to be synthesized in the crop epithelial cells and deposited on the inner crop surface. Its possible biological role is discussed. © 1995 Wiley-Liss, Inc.

Bacteria of the genus Sodalis live in symbiosis with various groups of insects. The best known member of this group, a secondary symbiont of tsetse flies Sodalis glossinidius, has become one of the most important models in investigating establishment and evolution of insect-bacteria symbiosis. It represents a bacterium in the early/intermediate state of the transition towards symbiosis, which allows for exploring such interesting topics as: usage of secretory systems for entering the host cell, tempo of the genome modification, and metabolic interaction with a coexisting primary symbiont. In this study, we describe a new Sodalis species which could provide a useful comparative model to the tsetse symbiont. It lives in association with Melophagus ovinus, an insect related to tsetse flies, and resembles S. glossinidius in several important traits. Similar to S. glossinidius, it cohabits the host with another symbiotic bacterium, the bacteriome-harbored primary symbiont of the genus Arsenophonus. As a typical secondary symbiont, Candidatus Sodalis melophagi infects various host tissues, including bacteriome. We provide basic morphological and molecular characteristics of the symbiont and show that these traits also correspond to the early/intermediate state of the evolution towards symbiosis. Particularly, we demonstrate the ability of the bacterium to live in insect cell culture as well as in cell-free medium. We also provide basic characteristics of type three secretion system and using three reference sequences (16 S rDNA, groEL and spaPQR region) we show that the bacterium branched within the genus Sodalis, but originated independently of the two previously described symbionts of hippoboscoids. We propose the name Candidatus Sodalis melophagi for this new bacterium.

SUMMARYCoccidian oocysts from feces of 46 individuals of the garden dormouse, Eliomys quercinus (Rodentia: Gliridae), were morphologically and molecularly characterized. Both morphological and sequence data (18S rDNA and ORF 470) showed low variability, indicating that all samples represent a single species. By comparison with published morphological descriptions of coccidia from glirid rodents, we determined that the samples represent Eimeria myoxi. Molecular data suggest that this species does not fall within the 2 known rodent-specific groups but branches as a third independent lineage. However, its exact position in respect to other eimerian clusters could not be established due to the lack of phylogenetic information at this taxonomic level for the 18S rRNA and ORF 470 genes. Based on these results, we provide a re-description of Eimeria myoxi, which contains morphological and molecular characteristics sufficient for its further unequivocal identification.

Endocytobionts from Triatoma infestans are described (from salivary glands, ganglia, visceral muscles, hemocytes, pericardial cells, nephrocytes, ovaries, testes, and dorsal vessel). They are determined as gram-negative bacteria and they resemble other intracellular symbionts found in insects. Tissue distribution was similar in all specimens. The glial cells in CNS ganglia and salivary glands are the most heavily infected tissues. Even massive infections do not cause any apparent pathogenic effect. The nurse cells and oocytes are without endocytobionts. Nonetheless, transovarial transmission of endocytobionts is deduced from their presence in embryonic gut lumen. In the embryo and first larval stage the organs are without endocytobionts. Their dispersal begins after the first blood meal and molting. Virus-like particles were observed in both the endocytobionts and the host cells. The possible influence of endocytobionts on the host is discussed.

Polymorphisms in the isotype I beta-tubulin gene are important genetic determinants of benzimidazole (BZ) resistance in a number of parasitic nematode species including Teladorsagia circumcincta, a major gastrointestinal nematode of sheep. This study investigates the genetic diversity at this locus in a BZ-resistant isolate of T. circumcincta (MTci5) derived from a sheep farm in the United Kingdom (UK) that was open to animal, and therefore parasite, migration. Pyrosequencing was used to determine the frequency of single nucleotide polymorphisms (SNPs) known to be associated with BZ resistance. This was followed by a combination of single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing to sample allelic diversity in a 276bp fragment immediately surrounding the isotype I beta-tubulin F200Y mutation. The genetic diversity at this locus was extremely high, with seven different haplotypes found to contain the resistant F200Y polymorphism in this single resistant isolate. Genotyping by SSCP interfaced with pyrosequencing demonstrated that the P200(Y) mutation is also present on multiple haplotypes in two other BZ-resistant T. circumcincta isolates from the UK. This contrasts with much lower levels of haplotype diversity in BZ-resistant alleles present in T. circumcincta isolates from French goat farms that are closed to parasite migration. Taken together with our knowledge of T. circumcincta population genetic structure, these results are most consistent with multiple independent origins of resistance and mixing of alleles due to the large amount of livestock movement in the UK.

Background The genus Arsenophonus is a group of symbiotic, mainly insect-associated bacteria with rapidly increasing number of records. It is known from a broad spectrum of hosts and symbiotic relationships varying from parasitic son-killers to coevolving mutualists. The present study extends the currently known diversity with 34 samples retrieved mainly from hippoboscid (Diptera: Hippoboscidae) and nycteribiid (Diptera: Nycteribiidae) hosts, and investigates phylogenetic relationships within the genus. Results The analysis of 110 Arsenophonus sequences (incl. Riesia and Phlomobacter), provides a robust monophyletic clade, characterized by unique molecular synapomorphies. On the other hand, unstable inner topology indicates that complete understanding of Arsenophonus evolution cannot be achieved with 16S rDNA. Moreover, taxonomically restricted Sampling matrices prove sensitivity of the phylogenetic signal to sampling; in some cases, Arsenophonus monophyly is disrupted by other symbiotic bacteria. Two contrasting coevolutionary patterns occur throughout the tree: parallel host-symbiont evolution and the haphazard association of the symbionts with distant hosts. A further conspicuous feature of the topology is the occurrence of monophyletic symbiont lineages associated with monophyletic groups of hosts without a co-speciation pattern. We suggest that part of this incongruence could be caused by methodological artifacts, such as intragenomic variability. Conclusion The sample of currently available molecular data presents the genus Arsenophonus as one of the richest and most widespread clusters of insect symbiotic bacteria. The analysis of its phylogenetic lineages indicates a complex evolution and apparent ecological versatility with switches between entirely different life styles. Due to these properties, the genus should play an important role in the studies of evolutionary trends in insect intracellular symbionts. However, under the current practice, relying exclusively on 16S rRNA sequences, the phylogenetic analyses are sensitive to various methodological artifacts that may even lead to description of new Arsenophonus lineages as independent genera (e.g. Riesia and Phlomobacter). The resolution of the evolutionary questions encountered within the Arsenophonus clade will thus require identification of new molecular markers suitable for the low-level phylogenetics.

SUMMARYMonophyly of all 11 valid Eimeria species from rabbits (Oryctolagus cuniculus Linnaeus, 1758) was revealed based on nuclear 18S rDNA sequence data. This finding implies that these species, which vary considerably in terms of their morphology and biology, diversified on a single host or several closely related species. Phylogenetic analysis divided rabbit Eimeria species into 2 sister lineages, corresponding to the presence/absence of the oocyst residuum. Other morphological or biological traits (oocyst shape and size, presence/absence of oocyst inner structures, pathogenicity, infection site, pre-patent and patent periods, sporulation time, and number of asexual generations) do not explicitly correlate with the phylogeny of rabbit coccidia.