Association of different genetic types of Francisella-like organisms with the rocky mountain wood tick (Dermacentor andersoni) and the American dog tick (Dermacentor variabilis) in localities near their northern distributional limits

Dermacentor andersoni and Dermacentor variabilis from allopatric and sympatric populations near their northern distributional limits were examined for the presence of Francisella species using molecular techniques that targeted 373 bp of the 16S rRNA gene. Although there was no evidence for the presence of Francisella tularensis in any tick, Francisella-like endosymbionts (FLEs) were common in D. andersoni and D. variabilis adults and immatures. A significantly greater proportion of female ticks contained FLEs compared to male ticks. In addition, significantly more D. variabilis adult individuals contained multiple FLE sequence types than did D. andersoni adults. Ten different types of FLEs were identified based on the sequence data, which has implications for diagnostic tests and epidemiological studies of F. tularensis in tick populations in Canada. The three most prevalent types of FLEs have been detected previously in D. andersoni or D. variabilis from other parts of their distributional ranges, whereas the other seven FLE types have not been reported previously. A comparison of the FLEs from both allopatric and sympatric populations of these two tick species provided insight into the relative host-specificity and the modes of transmission of these tick-borne bacteria. In general, each FLE type was specific for one tick species, suggesting vertical transmission of each bacterium. However, there were a few instances of potential cross-transfer of two FLE types to the other tick species at locations where D. andersoni and D. variabilis occurred in sympatry, suggesting that there may be occasional horizontal transmission of some FLEs.     

Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram-negative, rod-shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN-3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans a n d Holospora obtusa . With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus , of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN-3 as ' Candidatus Caedibacter acanthamoebae', the endosymbiont of Acanthamoeba sp. strain UWC9 as ' Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acanthamoeba sp. strain UWE39 as ' Candidatus Paracaedibacter symbiosus'. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN-3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit < 92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.     

Enhancement of in vitro cytopathogenicity by Acanthamoeba spp. following acquisition of bacterial endosymbionts

Approximately one in five isolates of Acanthamoeba spp. recovered from clinical and environmental sources are found to harbor obligate, uncultured bacterial endosymbionts of unknown clinical significance. To investigate their possible role in amoebic pathogenesis, four uninfected amoebic strains were exposed to four different endosymbionts, from which 12 stably-infected host-symbiont pairs resulted. Standardized inocula of amoebae with and without endosymbionts were placed on fibroblast monolayers to examine for cytopathic effects (CPEs). Eight to 10 days were required for monolayer effacement by endosymbiont-free amoebae; 5–8 days for amoebae containing Gram-negative rod endosymbionts; and 3 days for two amoebic isolates infected with a Chlamydia -like endosymbiont. All endosymbiont-infected amoebae produced a statistically significant enhancement in CPEs in comparison to uninfected amoebae; endosymbionts alone on monolayers produced no CPEs. This report provides evidence that obligate bacterial endosymbionts are able to enhance amoebic pathogenic potential in vitro by some as-yet unknown mechanism.     

In Situ Detection of Novel Bacterial Endosymbionts of Acanthamoeba spp. Phylogenetically Related to Members of the Order Rickettsiales

Acanthamoebae are ubiquitous soil and water bactivores which may serve as amplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectrum of uncultured endosymbionts includes gram-negative rods and gram-variable cocci, the latter recently shown to be members of the Chlamydiales. We report here the isolation from corneal scrapings of two Acanthamoeba strains that harbor gram-negative rod endosymbionts that could not be cultured by standard techniques. These bacteria were phylogenetically characterized following amplification and sequencing of the near-full-length 16S rRNA gene. We used two fluorescently labelled oligonucleotide probes targeting signature regions within the retrieved sequences to detect these organisms in situ. Phylogenetic analyses demonstrated that they displayed 99.6% sequence similarity and formed an independent and well-separated lineage within the Rickettsiales branch of the alpha subdivision of the Proteobacteria. Nearest relatives included members of the genus Rickettsia, with sequence similarities of approximately 85 to 86%, suggesting that these symbionts are representatives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the Rickettsiales. The oligonucleotide probes displayed at least three mismatches to all other available 16S rDNA sequences, and they both readily permitted the unambiguous detection of rod-shaped bacteria within intact acanthamoebae by confocal laser-scanning microscopy. Considering the long-standing relationship of most Rickettsiales with arthropods, the finding of a related lineage of endosymbionts in protozoan hosts was unexpected and may have implications for the preadaptation and/or recruitment of rickettsia-like bacteria to metazoan hosts.     

Evolutionary relationships of "Candidatus Riesia spp.," endosymbiotic enterobacteriaceae living within hematophagous primate lice

The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body louse Pediculus humanus and provide new characterizations of the P-endosymbionts from Pediculus schaeffi from chimpanzees and Pthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium "Candidatus Arsenophonus insecticola." We propose that two additional species be added to the genus "Candidatus Riesia." The new species proposed within "Candidatus Riesia" have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated for Buchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.     

Evolutionary Relationships of “Candidatus Riesia spp.,” Endosymbiotic Enterobacteriaceae Living within Hematophagous Primate Lice

The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body louse Pediculus humanus and provide new characterizations of the P-endosymbionts from Pediculus schaeffi from chimpanzees and Pthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium “Candidatus Arsenophonus insecticola.” We propose that two additional species be added to the genus “Candidatus Riesia.” The new species proposed within “Candidatus Riesia” have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated for Buchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.     

Comparative Genomics of Serratia spp.: Two Paths towards Endosymbiotic Life

Symbiosis is a widespread phenomenon in nature, in which insects show a great number of these associations. Buchnera aphidicola, the obligate endosymbiont of aphids, coexists in some species with another intracellular bacterium, Serratia symbiotica. Of particular interest is the case of the cedar aphid Cinara cedri, where B. aphidicola BCc and S. symbiotica SCc need each other to fulfil their symbiotic role with the insect. Moreover, various features seem to indicate that S. symbiotica SCc is closer to an obligate endosymbiont than to other facultative S. symbiotica, such as the one described for the aphid Acirthosyphon pisum (S. symbiotica SAp). This work is based on the comparative genomics of five strains of Serratia, three free-living and two endosymbiotic ones (one facultative and one obligate) which should allow us to dissect the genome reduction taking place in the adaptive process to an intracellular life-style. Using a pan-genome approach, we have identified shared and strain-specific genes from both endosymbiotic strains and gained insight into the different genetic reduction both S. symbiotica have undergone. We have identified both retained and reduced functional categories in S. symbiotica compared to the Free-Living Serratia (FLS) that seem to be related with its endosymbiotic role in their specific host-symbiont systems. By means of a phylogenomic reconstruction we have solved the position of both endosymbionts with confidence, established the probable insect-pathogen origin of the symbiotic clade as well as the high amino-acid substitution rate in S. symbiotica SCc. Finally, we were able to quantify the minimal number of rearrangements suffered in the endosymbiotic lineages and reconstruct a minimal rearrangement phylogeny. All these findings provide important evidence for the existence of at least two distinctive S. symbiotica lineages that are characterized by different rearrangements, gene content, genome size and branch lengths.     

Diversity and Evolution of Paramoeba spp. and their Kinetoplastid Endosymbionts

Members of the genus Paramoeba (including Neoparamoeba) (Amoebozoa) are single‐celled eukaryotes of economic and ecological importance because of their association with disease in a variety of marine animals including fish, sea urchins, and lobster. Interestingly, they harbor a eukaryotic endosymbiont of kinetoplastid ancestry, Perkinsela sp. To investigate the complex relationship between Paramoeba spp. and Perkinsela sp., as well as the relationships between different Paramoeba species, molecular data was obtained for four novel isolates. We also acquired new data from the urchin pathogen P. invadens. Comprehensive molecular phylogenetic analyses were carried out using 33 newly obtained 18S rDNA sequences from the host amoebae and 16 new 18S rDNA sequences from their corresponding Perkinsela sp., together with all publicly available 18S molecular data. Intra‐isolate 18S rDNA nucleotide diversity was found to be surprisingly high within the various species of Paramoeba, but relatively low within their Perkinsela sp. endosymbionts. 18S rDNA phylogenies and ParaFit co‐evolution analysis revealed a high degree of congruence between the Paramoeba and Perkinsela sp. tree topologies, strongly suggesting that a single endosymbiotic event occurred in the common ancestor of known Paramoeba species, and that the endosymbionts have been inherited vertically ever since.     

Intracellular lifestyle of Brucella spp. Common genes with other animal pathogens, plant pathogens, and endosymbionts

Brucella spp. are intracellular pathogens that belong, like Agrobacterium, Rhizobium and Rickettsia, to the alpha-2-subgroup of proteobacteria. The genome organization of most Brucella spp. is characterized by the presence of two chromosomes. The intracellular lifestyle of Brucella, as well as the possible genes involved in pathogenesis and host cell signaling, are discussed, including the presence of genes with high similarity to those from other animal pathogens, plant pathogens and endosymbionts.     

Neoparamoeba spp. and their eukaryotic endosymbionts similar to Perkinsela amoebae (Hollande, 1980): coevolution demonstrated by SSU rRNA gene phylogenies

The molecular phylogeny of Neoparamoeba spp. based on SSU rDNA was updated by including new sequences of strains isolated from an invertebrate and an alga. In total, 59 sequences of strains representating N. pemaquidensis, N. branchiphila, N. aestuarina and N. perurans were analysed. Sequences of SSU rDNA of eukaryotic endosymbionts (Perkinsela amoebae-like organisms) were prepared from 34 samples of genomic DNA of strain-representatives of N. pemaquidensis, N. branchiphila and N. aestuarina. Comparison of phylograms reconstructed from corresponding SSU rDNA sequences of host amoebae and their symbionts revealed a high level of congruence, which argues very strongly for coevolution of these two eukaryotic organisms.     

Limited Multiplication of Symbiotic Cyanobacteria of Azolla spp. on Artificial Media

We examined various media and conditions to isolate symbiotic cyanobacteria from the leaf cavities of Azolla spp. Cyanobacteria survived and multiplied to a limited extent on a medium with fructose, Casamino Acids, yeast extract, and NaNO₃ under 1% O₂. These cyanobacteria were antigenically identical to the endosymbionts.     

The genus Caedibacter comprises endosymbionts of Paramecium spp. related to the Rickettsiales (Alphaproteobacteria) and to Francisella tularensis (Gammaproteobacteria)

Obligate bacterial endosymbionts of paramecia able to form refractile inclusion bodies (R bodies), thereby conferring a killer trait upon their ciliate hosts, have traditionally been grouped into the genus CAEDIBACTER: Of the six species described to date, only the Paramecium caudatum symbiont Caedibacter caryophilus has been phylogenetically characterized by its 16S rRNA gene sequence, and it was found to be a member of the Alphaproteobacteria related to the RICKETTSIALES: In this study, the Caedibacter taeniospiralis type strain, an R-body-producing cytoplasmatic symbiont of Paramecium tetraurelia strain 51k, was investigated by comparative 16S rRNA sequence analysis and fluorescence in situ hybridization with specific oligonucleotide probes. C. taeniospiralis is not closely related to C. caryophilus (80% 16S rRNA sequence similarity) but forms a novel evolutionary lineage within the Gammaproteobacteria with the family Francisellaceae as a sister group (87% 16S rRNA sequence similarity). These findings demonstrate that the genus Caedibacter is polyphyletic and comprises at least two phylogenetically different bacterial species belonging to two different classes of the PROTEOBACTERIA: Comparative phylogenetic analysis of C. caryophilus, five closely related Acanthamoeba endosymbionts (including one previously uncharacterized amoebal symbiont identified in this study), and their hosts suggests that the progenitor of the alphaproteobacterial C. caryophilus lived within acanthamoebae prior to the infection of paramecia.     

Morphological differences and molecular phylogeny of freshwater blooming species, Peridiniopsis spp. (Dinophyceae) from China

In 2008–2010, several freshwater dinoflagellate blooms caused by Peridiniopsis spp. were observed in China. P. penardii and P. niei sampled from various geographical localities were examined by means of light and scanning electron microscopy. After comparing morphological and molecular differences, the new freshwater variety Peridiniopsis penardii var. robusta var. nov. (Peridiniales, Dinophyceae) found in Manwan Reservoir, Yunnan Province was described. The new variety differed from P. penardii since it possessed numerous robust antapical spines and a conspicuous apical spine. Molecular phylogenetic analyses based on SSU, LSU and ITS indicated P. niei, P. penardii and P. penardii var. robusta were closely related with P. kevei, and clustered into a monophyletic clade. The new variety possessed an endosymbiotic diatom which was similar to P. penardii and P. kevei, whereas the endosymbiont was not present in cells of P. niei. The endosymbiont SSU and ITS phylogenies showed that the endosymbionts of these three dinoflagellates were closely related to members of Thalassiosirales. Furthermore it was concluded that the endosymbionts might originate from Discostella-like species.     

Assessment of bacterial endosymbiont diversity in <Emphasis Type="Italic">Otiorhynchus</Emphasis>spp. (Coleoptera: Curculionidae) larvae using a multitag 454 pyrosequencing approach

BACKGROUND: Weevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. So far, little is known on the presence of endosymbionts in Otiorhynchus spp.. Investigation of endosymbiosis in this genus may help to understand the evolution of different reproductive strategies in these weevils (parthenogenesis or sexual reproduction), host-symbiont interactions, and may provide a future basis for novel pest management strategy development. Here, we used a multitag 454 pyrosequencing approach to assess the bacterial endosymbiont diversity in larvae of four economically important Otiorhynchus species. RESULTS: High-throughput tag-encoded FLX amplicon pyrosequencing of a bacterial 16S rDNA fragment was used to characterise bacterial communities associated with different Otiorhynchus spp. larvae. By sequencing a total of ~48,000 PCR amplicons, we identified 49 different operational taxonomic units (OTUs) as bacterial endosymbionts in the four studied Otiorhynchus species. More than 90% of all sequence reads belonged either to the genus Rickettsia or showed homology to the phylogenetic group of "Candidatus Blochmannia" and to endosymbionts of the lice Pedicinus obtusus and P. badii. By using specific primers for the genera Rickettsia and "Candidatus Blochmannia", we identified a new phylogenetic clade of Rickettsia as well as "Candidatus Nardonella" endosymbionts in Otiorhynchus spp. which are closely related to "Candidatus Blochmannia" bacteria. CONCLUSIONS: Here, we used multitag 454 pyrosequencing for assessment of insect endosymbiotic communities in weevils. As 454 pyrosequencing generates only quite short sequences, results of such studies can be regarded as a first step towards identifying respective endosymbiotic species in insects. In the second step of our study, we analysed sequences of specific gene regions for a more detailed phylogeny of selected endosymbiont genera. As a result we identified the presence of Rickettsia and "Candidatus Nardonella" endosymbionts in Otiorhynchus spp.. This knowledge is an important step in exploring bacteria-insect associations for potential use in insect pest control.     

Survey of Heritable Endosymbionts in Southern Mexico Populations of the Fruit Fly Species Anastrepha striata and A. ludens

Heritable endosymbiotic bacteria associated with insects are ubiquitous and taxonomically diverse. Many of these endosymbionts influence the fitness of their hosts and/or manipulate their host reproduction. Exploiting the effects of endosymbionts on hosts for pest control is a growing research area, but requires knowledge of endosymbionts associated with the target pest population. In this study, we used molecular methods to screen southern Mexico populations of two species of tephritid fruit fly pests, Anastrepha ludens and A. striata, for heritable bacteria. The only heritable endosymbiont found was Wolbachia in A. striata. Based on multilocus sequence typing and phylogenetic analyses, this Wolbachia strain is new and belongs to the Wolbachia supergroup B. Wolbachia strains previously reported in members of the genus Anastrepha in South America belong to supergroup A. We discuss the potential implications for pest control of the presence of a different Wolbachia strain in southern Mexico.     

烟粉虱复合种内共生菌多样性及其生物学意义

烟粉虱复合种Bemisia tabaci（Gennadius） complex内共生菌的种类可分为5类：变形菌门（Proteobacteria）γ变形菌纲的初级内共生菌Candidatus Portiera aleyrodidarum和次级内共生菌,变形菌门α变形菌纲Wolbachia属细菌,衣原体门（Chlamydiae）衣原体纲细菌CandidatusFritschea bemisiae以及拟杆菌门（Bacteroidetes）类噬胞细菌（Cytophaga-like-organism, CLO）。系统发育分析表明,烟粉虱初级内共生菌分为2支,一些初级内共生菌和宿主系统发育并不严格一致; 次级内共生菌分为3支,一些次级内共生菌可能存在水平传播或多次侵入的现象。上述5类内共生菌在烟粉虱不同生物型/地理种群内的分布存在差异。B型烟粉虱可能具有独特的初级、次级内共生菌,并且不含有或很少含有Wolbachia属细菌、衣原体纲细菌Candidatus Fritschea bemisiae以及类噬胞细菌（CLO）。最后探讨了不同内共生菌对烟粉虱宿主的生物学意义及其研究方向。     

昆虫内共生菌及其功能研究进展

昆虫内共生菌与宿主之间的互作关系已逐渐成为昆虫学的研究热点之一。昆虫内共生菌具有协助宿主营养代谢、 逃避天敌攻击和增强抗药性等功能： 通过协助宿主营养代谢, 提供食物中缺乏的营养物质来弥补食物中营养物质的不足; 分泌抗菌肽、 毒素等物质以增强对外源寄生物等的防御能力, 抑制对宿主的不利影响; 同时, 也可以增强宿主抗逆性, 调控植物生理反应, 抑制植物对宿主的不利影响; 利用对抗逆性基因精确的表达调控来增强宿主抗药性等。因此, 内共生菌介导的宿主生物学性状的改变, 扩大了宿主昆虫的生态位, 成为昆虫生长发育过程中的重要调控因子。目前, 昆虫内共生菌的功能往往是通过研究宿主感染共生菌前后性状的变化而证实。近几年, 转录组学、 蛋白质组学、 基因组学等技术的进步, 促进了内共生菌与宿主昆虫共生机制研究的发展。通过研究内共生菌及其功能基因在昆虫种群动态中的作用, 特别是内共生菌感染对宿主生殖、 存活、 适应环境能力的影响, 将有利于揭示内共生菌与宿主的共生机制, 并最终为开发新的防控技术提供理论依据。本文针对昆虫内共生菌的功能进行了综述, 并对日后的研究方向进行了展望, 提供了研究昆虫内共生菌与宿主互作关系的方法及建议。     

Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families.

Aphids (superfamily Aphidoidea) contain eubacterial endosymbionts localized within specialized cells (mycetocytes). The endosymbionts are essential for the survival of the aphid hosts. Sequence analyses of the 16S rRNAs from endosymbionts of 11 aphid species from seven tribes and four families have indicated that the endosymbionts are monophyletic. Furthermore, phylogenetic relationships within the symbiont clade parallel the relationships of the corresponding aphid hosts. Our findings suggest that this endocytobiotic association was established in a common ancestor of the four aphid families with subsequent diversification into the present species of aphids and their endosymbionts.     

Genetic Variation among Endosymbionts of Widely Distributed Vestimentiferan Tubeworms

Vestimentiferan tubeworms thriving in sulfidic deep-sea hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts. In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent and seep habitats form two distinct clades with little variation within each clade. In the present study, we used two different approaches to assess the genetic variation among biogeographically distinct vestimentiferan symbionts. DNA sequences were obtained for the noncoding, internal transcribed spacer (ITS) regions of the rRNA operons of symbionts associated with six different genera of vestimentiferan tubeworms. ITS sequences from endosymbionts of host genera collected from different habitats and widely distributed vent sites were surprisingly conserved. Because the ITS region was not sufficient for distinguishing endosymbionts from different habitats or locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the bacterial endosymbionts. Most of the endosymbionts displayed unique REP-PCR patterns. A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors, including host genera, geographic location, and bottom type.