Genetic population structure of <Emphasis Type="Italic">Lefua echigonia</Emphasis> inferred from allozymic and mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> variations

We examined the genetic population structure of Lefua echigonia (Japanese name, hotoke-dojo) using polymerase chain reaction restriction-fragment length polymorphisms (PCR-RFLPs) of the mitochondrial cytochrome b gene and two allozymic loci. The phylogenetic relationships of L. echigonia and those among L. echigonia, Lefua sp. (nagare hotoke-dojo), and L. nikkonis (ezo hotoke-dojo) were also investigated based on the nucleotide sequences of the cytochrome b gene. PCR-RFLP analysis revealed 18 mitotypes in L. echigonia, 2 in Lefua sp., and 1 in L. nikkonis. Phylogenetic trees based on the cytochrome b sequences indicated that the 18 mitotypes in L. echigonia were divided into five distinct groups (South-Kanto, North-Kanto, Tohoku, Echigo, and Tokai-Kinki clades) that differed by 8.5–15.3%, reflecting region-specific geographic distributions. The distributions of alleles in two allozymic loci roughly corresponded to those of the mitotype groups. The divergence times of the five groups were estimated to be about 3.4–7.7 million years ago by applying a general rate for mitochondrial DNA, suggesting that the divergence among them might have occurred in the late Tertiary. It can be inferred that the regional differentiation of each group was mainly due to geographic isolation and that this has been maintained, because the boundaries among the groups corresponded to geological features. The trees also supported the existence of three taxa, L. echigonia, Lefua sp., and L. nikkonis. We concluded that Lefua sp. was distinguished from other species in Lefua by morphological and ecological characters and also by genetic divergences of the cytochrome b gene. Our study also demonstrated the superior efficacy and simplicity of PCR-RFLP analysis as a method for detecting genetic variation in L. echigonia.     

Evolutionary history of <Emphasis Type="Italic">Wolbachia</Emphasis> infections in the fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Background  

Wolbachia are endosymbiotic bacteria that commonly infect numerous arthropods. Despite their broad taxonomic distribution, the transmission patterns of these bacteria within and among host species are not well understood. We sequenced a portion of the wsp gene from the Wolbachia genome infecting 138 individuals from eleven geographically distributed native populations of the fire ant Solenopsis invicta. We then compared these wsp sequence data to patterns of mitochondrial DNA (mtDNA) variation of both infected and uninfected host individuals to infer the transmission patterns of Wolbachia in S. invicta.     

Negative Evidence of <Emphasis Type="Italic">Wolbachia</Emphasis> in the Predaceous Mite <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis>

The cytoplasmically inherited bacterium Wolbachia is widespread in arthropod species and has been repeatedly detected in the predaceous mite Phytoseiulus persimilis. Our original goal was to assess the prevalence of Wolbachia infection in P. persimilis and the potential fitness consequences for this host. To accomplish that goal, seven P. persimilis strains were obtained from Europe, Africa and the USA and reared on the phytophagous mite Tetranychus urticae as prey. After preliminary results showed that the T. urticae used was infected with Wolbachia, the minimum starvation time of the predators to prevent false positive results from undigested prey was determined. We tested DNA samples by PCR (polymerase chain reaction) after starving the predators or feeding them Wolbachia-free T. urticae for various periods. Those experiments showed that Wolbachia could not be detected after 16 h at 25 °C and 48 h at 20 °C. To verify the results of the PCR analyses, we furthermore conducted crossing experiments with antibiotic-treated and untreated individuals. No indications of Wolbachia effects were recorded. Additionally, we screened live eggs of four of the seven strains reared in our laboratory and alcohol samples of 10 other P. persimilis strains for the occurrence of Wolbachia by PCR, none of which tested positive. Synthesis of our study and previous reports suggests that infection of P. persimilis with Wolbachia is extremely rare and of minor importance. We discuss the significance of our findings for future studies on the presence of Wolbachia in predaceous arthropods.     

Prevalence of <Emphasis Type="Italic">Wolbachia</Emphasis> Infection in <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Wolbachia are obligate intracellular bacteria present in reproductive tissues of many arthropod species. It has been reported that few silverleafing populations of Bemisia tabaci were positive for Wolbachia, whereas non-silverleafing populations were more likely infected with Wolbachia and all that infect B. tabaci are Wolbachia belonging to supergroup B. However, current detection methods were shown to be not sensitive enough to uncover all infections. Herein, a protocol based on polymerase chain reaction–restriction fragment length polymorphism analysis of Wolbachia 16S ribosomal DNA is presented. A systematic survey for the prevalence of Wolbachia infection in natural populations of B. tabaci using this method revealed that (1) all populations of B. tabaci tested positive for Wolbachia and the overall infection rate reached 80.5% (293 positives in 364 tests); (2) both single infection and superinfection existed within individual whiteflies tested; and (3) silverleafing populations of B. tabaci most likely harbored A Wolbachia as single infection, whereas non-silverleafing populations tend to carry B Wolbachia as superinfection. It is clear that the Wolbachia infection pattern is closely related to the genetic races of B. tabaci, and the infection frequencies are apparently much higher than those described previously. This study shows that detection methods can significantly influence estimation of Wolbachia infection. It is supposed that Wolbachia may be acting as a biotic agent promoting rapid differentiation and speciation of B. tabaci. This is the most systematic survey of Wolbachia infection within B. tabaci.     

Effect of light and temperature on the daily rhythm of adult eclosion in the blood-sucking mosquitoes <Emphasis Type="Italic">Aedes communis</Emphasis> De Geer and <Emphasis Type="Italic">Culex pipiens</Emphasis> L. (Diptera,Culicidae)

A comparative study on the effect of light and temperature on the daily rhythm of adult eclosion was carried out on the blood-sucking mosquitoes Aedes (Ochlerotatus) communis De Geer and Culex pipiens pipiens L. Two forms of C. p. pipiens were investigated: the autogenous form molestus occurring in the underground habitats (the urban mosquito), and the anautogenous form pipiens inhabiting mostly the above-ground biotopes. The study included both field observations and laboratory experiments. Under natural conditions (southern Karelia, 62°N) at optimal temperatures A. communis and C. p. p. f. pipiens demonstrated a bimodal eclosion rhythm with morning and evening peaks, whereas in C. p. p. f. molestus only an evening maximum was observed. The fraction of adults eclosing in the middle of the day increased at lower temperatures. In all the mosquitoes studied, the daily eclosion dynamics in the nature did not differ significantly between the variants with different levels of illumination, suggesting that temperature was the main factor regulating the eclosion rhythm. However, experiments with constant temperature showed that light also could influence the timing of eclosion. The responsiveness to light was different in two studied forms of C. p. pipiens. Under a constant temperature and light-dark cycles the rhythm became weak or disappeared completely in the mosquitoes from above-ground biotopes (A. communis and C. p. p. f. pipiens) but persisted in the urban mosquito. The effect of gradual twilight transitions was more prominent than that of switching the light on or off abruptly. The observed differences in the light and temperature dependence of the eclosion rhythm are discussed in relation to the ecological conditions in different habitats.     

Polymorphism of mitochondrial DNA and infection with symbiotic cytoplasmic bacterium Wolbachia pipientis in mosquitoes of the Culex pipiens complex from Russia

A total of 208 mosquitoes of the Culex pipiens complex from 15 basement and terrestrial populations collected in different regions of the European part of Russia and Siberia were examined by genetic methods. Among these, two major mitotypes, M and P, were identified. These mitotypes differed by six substitutions in the 246-bp mitochondrial DNA cytochrome oxidase I gene fragment examined. Urban basement mosquito ecotype C. pipiens (form molestus) were characterized by the presence of mitotype M and infection with the endosymbiotic bacteria of the genus Wolbachia. Mosquitoes of the C. pipiens complex inhabiting opened biotopes harbored mitotype P, or its variety, mitotype P1, and were not infected with Wolbachia. Thus, in natural conditions marked linkage disequilibrium between cytoplasmic elements, mitochondrial DNA and Wolbachia, can be observed. Similarity of mitotypes in form molestus mosquito from different geographical localities favors the hypothesis on the common ancestry of urban mosquitoes.     

New approach to the mitotype classification in black honeybee <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> and Iberian honeybee <Emphasis Type="Italic">Apis mellifera iberiensis</Emphasis>

The black honeybee Apis mellifera mellifera L. is today the only subspecies of honeybee which is suitable for commercial breeding in the climatic conditions of Northern Europe with long cold winters. The main problem of the black honeybee in Russia and European countries is the preservation of the indigenous gene pool purity, which is lost as a result of hybridization with subspecies, A. m. caucasica, A. m. carnica, A. m. carpatica, and A. m. armeniaca, introduced from southern regions. Genetic identification of the subspecies will reduce the extent of hybridization and provide the gene pool conservation of the black honeybee. Modern classification of the honeybee mitotypes is mainly based on the combined use of the DraI restriction endonuclease recognition site polymorphism and sequence polymorphism of the mtDNA COI–COII region. We performed a comparative analysis of the mtDNA COI–COII region sequence polymorphism in the honeybees of the evolutionary lineage M from Ural and West European populations of black honeybee A. m. mellifera and Spanish bee A. m. iberiensis. A new approach to the classification of the honeybee M mitotypes was suggested. Using this approach and on the basis of the seven most informative SNPs of the mtDNA COI–COII region, eight honeybee mitotype groups were identified. In addition, it is suggested that this approach will simplify the previously proposed complicated mitotype classification and will make it possible to assess the level of the mitotype diversity and to identify the mitotypes that are the most valuable for the honeybee breeding and rearing.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Infections of the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

We report the first systematic survey for the presence of Wolbachia endosymbionts in aphids and whiteflies, particularly different populations and biotypes of Bemisia tabaci. Additional agriculturally important species included were predator species, leafhoppers, and lepidopterans. We used a polymerase chain reaction (PCR)-based detection assay with ribosomal 16S rDNA and Wolbachia cell surface protein (wsp) gene primers. Wolbachia were detected in a number of whitefly populations and species, whitefly predators, and one leafhopper species; however, none of the aphid species tested were found infected. Single, double, and triple infections were detected in some of the B. tabaci populations. PCR and phylogenetic analysis of wsp gene sequences indicated that all Wolbachia strains found belong to group B. Topologies of the optimal tree derived by maximum likelihood (ML) and a ML tree in which Wolbachia sequences from B. tabaci are constrained to be monophyletic are significantly different. Our results indicate that there have been at least four independent Wolbachia infection events in B. tabaci. The importance of the presence of Wolbachia infections in B. tabaci is discussed. RID= ID= <E5>Correspondence to: </E5>K. Bourtzis; <E5>email:</E5> kbourtz&commat;cc.uoi.gr Received: 9 September 2002 / Accepted: 25 September 2002     

<Emphasis Type="Italic">Wolbachia</Emphasis>-induced cytoplasmic incompatibility in Japanese populations of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae)

Intracellular bacteria of the genus Wolbachia (alpha Proteobacteria) induce cytoplasmic incompatibility (CI) in many arthropod species, including spider mites, but not all Wolbachia cause CI. In spider mites CI becomes apparent by a reduced egg hatchability and a lower daughter:son ratio: CI in haplodiploid organisms in general was expected to produce all-male offspring or a male-biased sex ratio without any death of eggs. In a previous study of Japanese populations of Tetranychus urticae, two out of three green-form populations tested were infected with non-CI Wolbachia strains, whereas none of six red-form populations harbored Wolbachia. As the survey of Wolbachia infection in T. urticae is still fragmentary in Japan, we checked Wolbachia infection in thirty green-form populations and 29 red-form populations collected from a wide range of Japanese islands. For Wolbachia-infected populations, we tested the effects of Wolbachia on the reproductive traits and determined the phylogenetic relationships of the different strains of Wolbachia. All but one green-form populations were infected with Wolbachia and all strains belonged to the subgroup Ori when the wsp gene was used to determine the phylogenetic relationships of different strains of Wolbachia. Six out of 29 red-form populations harbored Wolbachia and the infected strains belonged to the subgroups Ori and Bugs. Twenty-four of 29 infected green-form populations and five of six infected red-form populations induced CI among the hosts. Thus, CI-Wolbachia strains are widespread in Japan, and no geographical trend was observed in the CI-Wolbachia. Although three red-form populations harbored other intracellular bacteria Cardinium, they did not affect host reproduction.     

<Emphasis Type="Italic">Wolbachia</Emphasis> and bacteriophage WO-B density of <Emphasis Type="Italic">Wolbachia</Emphasis> a-infected <Emphasis Type="Italic">Aedes albopictus</Emphasis> mosquito

Wolbachia are maternally inherited symbiotic bacteria capable of inducing an extensive range of reproductive abnormalities in their hosts, including cytoplasmic incompatibility (CI). Its density (concentration) is likely to influence the penetrance of CI in incompatible crosses. The variations of Wolbachia density could also be linked with phage WO density. We determined the relative density (relative concentration) of prophage WO orf7 and Wolbachia (phage-to-bacteria ratio) during early developmental and adult stages of singly infected Aedes albopictus mosquito (Wolbachia A-infected) by using real-time quantitative PCR. Phage WO and Wolbachia did not develop at the same rate. Relative Wolbachia density (bacteria-to-host ratio) was high later in development (adult stages) whilst relative prophage WO density (phage-to-bacteria ratio) was low in the adult stages. Furthermore, 12-d-old adults of singly infected female mosquito had the highest Wolbachia density. In contrast, the larval stage 4 (L4) contained the highest prophage WO-B orf7 density. The association of hosts-Wolbachia-phage among diverse species is different. Thus, if phage and Wolbachia are involved in CI mechanism, the information of this association should be acquired for each specific type of organism for future use of population replacement or gene drive system.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Strains Typing in Different Geographic Population Spider, <Emphasis Type="Italic">Hylyphantes Graminicola</Emphasis> (Linyphiidae)

The Wolbachia endosymbiont of spiders has not been extensively examined. In order to investigate the distribution, evolutionary history, and reproductive phenotype of Wolbachia in spiders in China, we tested 11 geographic populations of Hylyphantes graminicola. Wolbachia infection has been detected in each population. 10 Wolbachia strains have been characterized by multilocus sequence typing (MLST). Phylogenetic analyses indicated that eight Wolbachia strains in H. graminicola belonged to supergroup B, and two belonged to supergroup A. No correlation existed between Wolbachia diversity and host’s geographic distance. The significant correlation was observed between pairwise distance of H. graminicola COI and genetic divergence of associated Wolbachia strains. We also found that Wolbachia infection frequencies in hosts varied over geographic space.     

Molecular phylogeography of two sibling species of <Emphasis Type="Italic">Eurema</Emphasis> butterflies

The common yellow butterfly Eurema hecabe is widely distributed in East Asia, and is one of the most burdensome species for taxonomists due to the numerous geographic and seasonal wing colour patterns. Moreover, within this species, individuals with a yellow wing fringe that occur in temperate regions of Japan (Y type) proved to be biologically different from others that occur widely in subtropical regions of Japan and all over East Asia (B type). To unveil the genetic variation within and between the two types, a total of 50 butterflies collected at 18 geographic localities in East Asia were examined for nucleotide sequence variation of three mitochondrial regions: cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit III (COIII) and NADH dehydrogenase subunit 5 (ND5). In addition, they were also examined for infection status with the endosymbiotic bacteria Wolbachia. The three mitochondrial sequences consistently showed that (i) Y type and B type were highly divergent, (ii) nucleotide variation within B type was very small although sampled from a geographically wide range, and (iii) a weak association existed between mitochondrial DNA haplotypes and Wolbachia infection status.     

Transfection of Feminizing <Emphasis Type="Italic">Wolbachia</Emphasis> Endosymbionts of the Butterfly, <Emphasis Type="Italic">Eurema hecabe</Emphasis>, into the Cell Culture and Various Immature Stages of the Silkmoth, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Wolbachia are maternally inherited endosymbiotic bacteria of invertebrates that can manipulate the reproductive systems of their arthropod hosts in a variety of ways. To establish a useful model system for investigating the mechanism of Wolbachia-induced host feminization, we conducted the following series of experiments: (1) feminizing Wolbachia of the butterfly, Eurema hecabe, were transferred into cell cultures of the silkmoth, Bombyx mori, and (2) the transfected Wolbachia in cell cultures were inoculated into B. mori at four immature stages. Wolbachia were successfully transfected into the cell cultures and stably maintained for more than 1 year (>30 passages). However, none of the inoculated insects produced mature oocytes that were Wolbachia-positive. This finding was consistent with the fact that Wolbachia was not detected in individuals in subsequent generations. In contrast, Wolbachia were detected at relatively high frequencies (60–80% of individuals) in the somatic tissues of inoculated insects. Real-time quantitative polymerase chain reaction revealed that the Wolbachia densities in the cultured cells were approximately tenfold higher than those in the native host E. hecabe. Among B. mori individuals inoculated at various developmental stages, those inoculated at early stages exhibited higher Wolbachia densities at the adult stage. The Wolbachia densities in individuals inoculated at the second-instar stage were comparable to those in intact E. hecabe. These results suggest that infection and/or proliferation of Wolbachia in germline cells are actively hindered by regulation in B. mori but feasible in somatic cells and that the Wolbachia densities in somatic tissues are regulated by the living host insects.     

Establishment of a new continuous cell line of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> strain infected by the intracellular endosymbiotic bacterium <Emphasis Type="Italic">Wolbachia pipientis</Emphasis> under natural conditions

Wolbachia pipientis is an obligately intracellular bacterium infecting a number of arthropod and nematode species. At the body level, Wolbachia infection may cause parthenogenesis, feminization of genetic males, male killing, or cytoplasmic incompatibility; it may also be asymptomatic. Of special interest is DNA transfer from Wolbachia to the host insect genome, which was discovered recently. At the cellular level, the effects caused by Wolbachia have been studied more poorly. Only one of the known insect cell lines has been obtained from an insect species (the mosquito Aedes albopictus) infected by Wolbachia. In this study, a continuous cell line Dm2008Wb1 has been obtained from embryos of Drosophila melanogaster infected under natural conditions. Wolbachia both persists in a primary cell culture and is retained upon its transformation into a continuous culture. The presence of this bacterium in cells in a free form is evidenced by the fact that tetracycline treatment can cure the cells of Wolbachia and by successful transfer of Wolbachia to another cell line (S2), where it has not been detected before.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Replication and Host Cell Division in <Emphasis Type="Italic">Aedes albopictus</Emphasis>

Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species. The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing. Like other vertically transmitted intracellular symbionts, Wolbachias replication rate must not outpace that of its host cells if it is to remain benign. The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis. During diapause in mosquito eggs, no host cell division is believed to occur. Further development is triggered only by subsequent exposure of the egg to water. This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate. We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.     

Incidence of the endosymbionts <Emphasis Type="Italic">Wolbachia</Emphasis>, <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Spiroplasma</Emphasis> in phytoseiid mites and associated prey

Endosymbiotic bacteria that potentially influence reproduction and other fitness-related traits of their hosts are widespread in insects and mites and their appeal to researchers’ interest is still increasing. We screened 20 strains of 12 agriculturally relevant herbivorous and predatory mite species for infection with Wolbachia, Cardinium and Spiroplasma by the use of PCR. The majority of specimens originated from Austria and were field collected or mass-reared. Eight out of 20 strains (40%) tested, representing seven of 12 mite species (58%), carried at least one of the three bacteria. We found Wolbachia in the herbivorous spider mites Tetranychus urticae and Bryobia rubrioculus, with the former also carrying Spiroplasma and the latter also carrying Cardinium. Cardinium was furthermore found in two populations of the predatory mite Euseius finlandicus and the spider mite Eotetranychus uncatus. Spiroplasma was detected in the predatory mite Neoseiulus californicus. All bacteria positive PCR products were sequenced, submitted to GenBank and analyzed in BLAST queries. We found high similarities to complete identity with bacteria found in the same and different mite species but also with bacteria found in insect species like ladybirds, butterflies and minute pirate bugs, Orius. We discuss the significance of potential (multiple) infections with the investigated bacteria for biological control.     

<Emphasis Type="Italic">Wolbachia pipientis</Emphasis> in Australian Spiders

Wolbachia pipientis is an endosymbiotic bacterium common to arthropods and filarial nematodes. This study presents the first survey and characterization of Wolbachia pipientis that infect spiders. All spiders were collected from Queensland, Australia during 2002–2003 and screened for Wolbachia infection using PCR approaches. The Wolbachia strains present in the spiders are diverse, paraphyletic, and for the most part closely related to strains that infect insects. We have also identified several spider Wolbachia strains that form a lineage outside the currently recognized six main Wolbachia supergroups (A–F). Incongruence between spider and Wolbachia phylogenies indicates a history of horizontal transmission of the bacterium in these host taxa. Like other arthropods, spiders are capable of harboring multiple Wolbachia strains.     

Iranian <Emphasis Type="Italic">Trichogramma</Emphasis>: ITS2 DNA characterization and natural <Emphasis Type="Italic">Wolbachia</Emphasis> infection

Egg parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae) are important natural enemies of many lepidopterous pests in agricultural and forest areas. Because the use of indigenous Trichogramma populations/species can significantly increase the biological control efficiency, the characterization of endemic populations and precise species identification is important. In this study, Trichogramma wasps were collected from parasitized eggs of several lepidopteran pests in Northern and Central Iran. Seven Trichogramma species were identified based on the morphology and the nucleotide sequence, size and restriction profile (PCR-RFLP) of the internal transcribed spacer 2 (ITS2) region of the rDNA of T. brassicae Bezdenko, T. cacoeciae Marchal, T. embryophagum Hartig, T. evanescens Westwood, T. euproctidis Girault, T. pintoi Voegelé and T. tshumakovae Sorokina. This is the first report of T. euproctidis in Iran. Additionally, natural populations were surveyed for the prevalence of Wolbachia. Two populations of T. brassicae were found to be infected by a single Wolbachia strain which was identified by using a multi-locus sequencing approach.     

Male rescue maintains low frequency parthenogenesis-inducing <Emphasis Type="Italic">Wolbachia</Emphasis> infection in <Emphasis Type="Italic">Trichogramma</Emphasis> populations

Parthenogenesis-inducing (PI) Wolbachia bacteria are reproductive parasites that cause infected (W ⁺) female haplodiploid parasitoids to produce daughters without fertilization by males. Theoretically, PI Wolbachia infection should spread to fixation within Trichogramma populations as males are no longer required to produce female offspring. Infections in some naturally occurring Trichogramma populations are, however, maintained at frequencies ranging from 4 to 26%. Here we describe discrete equation models to examine if the PI Wolbachia infection in Trichogramma populations can be maintained at relatively low frequencies by mating regularity. Model outcomes suggest the probability of W ⁺ females mating could stabilize Wolbachia infection frequency at low levels in Trichogramma populations. The primary mechanism maintaining low-level PI Wolbachia infection in Trichogramma populations is reducing the survivorship from egg to adult in infected relative to uninfected females. The model successfully demonstrates that the relatively low PI Wolbachia infection frequency in host populations can be maintained by fertilization, or male rescue, of infected eggs, which avoids potentially hazardous gamete duplication that occurs during Wolbachia-induced parthenogenesis.     

<Emphasis Type="Italic">Haematospirillum</Emphasis> and insect <Emphasis Type="Italic">Wolbachia</Emphasis> DNA in avian blood

In this study, blood samples of 259 Acrocephalus sp. warblers were molecularly analysed for Anaplasmataceae and Rhodospirillaceae based on PCR amplification of 16S rRNA gene fragments. One bird blood sample (from Reed Warbler, Acrocephalus scirpaceus) yielded a sequence with 99.8% identity to Haematospirillum jordaniae. This is the first molecular evidence for the occurrence of this species in the blood of any vertebrate other than human. Another bird blood sample (from Marsh Warbler: Acrocephalus palustris) yielded a Wolbachia sequence, closely related to a moth endosymbiont with 99.8% identity. A nematode origin of Wolbachia DNA detected here in avian blood can be excluded, because results of phylogenetic analysis showed its closest alignment with insect wolbachiae. This is the first finding of insect Wolbachia DNA in the circulatory system of birds, which can be explained either by the inoculation of wolbachiae by blood-sucking vectors, or passing of Wolbachia DNA from the gut into the blood of this insectivorous bird species.