Evolutionary Relationships of Three New Species of Enterobacteriaceae Living as Symbionts of Aphids and Other Insects

Ecological studies on three bacterial lineages symbiotic in aphids have shown that they impose a variety of effects on their hosts, including resistance to parasitoids and tolerance to heat stress. Phylogenetic analyses of partial sequences of gyrB and recA are consistent with previous analyses limited to 16S rRNA gene sequences and yield improved confidence of the evolutionary relationships of these symbionts. All three symbionts are in the Enterobacteriaceae. One of the symbionts, here given the provisional designation “Candidatus Serratia symbiotica,” is a Serratia species that has acquired a symbiotic lifestyle. The other two symbionts, here designated “Candidatus Hamiltonella defensa” and “Candidatus Regiella insecticola,” are sister groups to one another and together show a relationship to species of Photorhabdus.     

Evolution of the secondary symbiont "Candidatus serratia symbiotica" in aphid species of the subfamily lachninae

Buchnera aphidicola BCc, the primary endosymbiont of the aphid Cinara cedri (subfamily Lachninae), is losing its symbiotic capacity and might be replaced by the coresident "Candidatus Serratia symbiotica." Phylogenetic and morphological analyses within the subfamily Lachninae indicate two different "Ca. Serratia symbiotica" lineages and support the longtime coevolution of both symbionts in C. cedri.     

Aphid-symbiotic bacteria cultured in insect cell lines

The cells and tissues of many aphids contain bacteria known as "secondary symbionts," which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names "Candidatus Consessoris aphidicola" for T type and "Candidatus Adiaceo aphidicola" for U type.     

Characterization of an obligate intracellular bacterium in the midgut epithelium of the bulrush bug Chilacis typhae (Heteroptera, Lygaeidae, Artheneinae)

Many members of the suborder Heteroptera have symbiotic bacteria, which are usually found extracellularly in specific sacs or tubular outgrowths of the midgut or intracellularly in mycetomes. In this study, we describe the second molecular characterization of a symbiotic bacterium in a monophagous, seed-sucking stink bug of the family Lygaeidae (sensu stricto). Chilacis typhae possesses at the end of the first section of the midgut a structure which is composed of circularly arranged, strongly enlarged midgut epithelial cells. It is filled with an intracellular endosymbiont. This "mycetocytic belt" might represent an evolutionarily intermediate stage of the usual symbiotic structures found in stink bugs. Phylogenetic analysis based on the 16S rRNA and the groEL genes showed that the bacterium belongs to the Gammaproteobacteria, and it revealed a phylogenetic relationship with a secondary bacterial endosymbiont of Cimex lectularius and free-living plant pathogens such as Pectobacterium and Dickeya. The distribution and ultrastructure of the rod-shaped Chilacis endosymbiont were studied in adults and nymph stages using fluorescence in situ hybridization (FISH) and electron microscopy. The detection of symbionts at the anterior poles of developing eggs indicates that endosymbionts are transmitted vertically. A new genus and species name, "Candidatus Rohrkolberia cinguli," is proposed for this newly characterized clade of symbiotic bacteria.     

Alpha-proteobacterial symbionts of marine bryozoans in the genus Watersipora

Bacterial symbionts that resembled mollicutes were discovered in the marine bryozoan Watersipora arcuata in the 1980s. In this study, we used PCR and sequencing of 16S rRNA genes, specific fluorescence in situ hybridization, and phylogenetic analysis to determine that the bacterial symbionts of "W. subtorquata" and "W. arcuata" from several locations along the California coast are actually closely related alpha-Proteobacteria, not mollicutes. We propose the names "Candidatus Endowatersipora palomitas" and "Candidatus Endowatersipora rubus" for the symbionts of "W. subtorquata" and "W. arcuata," respectively.     

Identification of sibling species of the bryozoan Bugula neritina that produce different anticancer bryostatins and harbor distinct strains of the bacterial symbiont "Candidatus Endobugula sertula".

Although the cosmopolitan marine bryozoan Bugula neritina is recognized as a single species, natural products from this bryozoan vary among populations. B. neritina is the source of the anticancer drug candidate bryostatin 1, but it also produces other bryostatins, and different populations contain different bryostatins. We defined two chemotypes on the basis of previous studies: chemotype O contains bryostatins with an octa-2,4-dienoate substituent (including bryostatin 1), as well as other bryostatins; chemotype M lacks bryostatins with the octa-2,4-dienoate substituent. B. neritina contains a symbiotic gamma-proteobacterium "Candidatus Endobugula sertula," and it has been proposed that bryostatins may be synthesized by bacterial symbionts. In this study, B. neritina populations along the California coast were sampled for genetic variation and bryostatin content. Colonies that differ in chemotype also differ genetically by 8% in the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene; this difference is sufficient to suggest that the chemotypes represent different species. Each species contains a distinct strain of "E. sertula" that differs at four nucleotide sites in the small subunit ribosomal RNA (SSU rRNA) gene. These results indicate that the chemotypes have a genetic basis rather than an environmental cause. Gene sequences from an Atlantic sample matched sequences from the California chemotype M colonies, suggesting that this type may be cosmopolitan due to transport on boat hulls.     

Symbiotic bacteria associated with stomach discs of human lice

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation "Candidatus Riesia pediculicola" for the louse symbionts.     

Complex symbiotic systems of two treehopper species: Centrotus cornutus (Linnaeus, 1758) and Gargara genistae (Fabricius, 1775) (Hemiptera: Cicadomorpha: Membracoidea: Membracidae)

The aim of the conducted study was to describe the symbiotic systems (the types of symbionts, distribution in the body of the host insect, the transovarial transmission between generations) of two treehoppers: Centrotus cornutus and Gargara genistae by means of microscopic and molecular techniques. We found that each of them is host to four species of bacteriome-inhabiting symbionts. In C. cornutus, ancestral bacterial symbionts Sulcia and Nasuia are accompanied by an additional symbiont—the bacterium Arsenophonus. In the bacteriomes of G. genistae, apart from Sulcia and Nasuia, bacterium Serratia is present. To our knowledge, this is the first report regarding the occurrence of Serratia as a symbiont in Hemiptera: Auchenorrhyncha. Bacteria Sulcia and Nasuia are harbored in their own bacteriocytes, whereas Arsenophonus and Serratia both inhabit their own bacteriocytes and also co-reside with bacteria Nasuia. We observed that both bacteria Arsenophonus and Serratia undergo autophagic degradation. We found that in both of the species examined, in the cytoplasm and nuclei of all of the cells of the bacteriome, bacteria Rickettsia are present. Our histological and ultrastructural observations revealed that all the bacteriome-associated symbionts of C. cornutus and G. genistae are transovarially transmitted from mother to offspring.     

srDna-based taxonomic affinities of algal symbionts from a planktonic foraminifer and a solitary radiolarian

Planktonic sarcodines (acantharia, radiolaria, and planktonic foraminifera) are oceanic amoeboid protozoa that often harbor a variety of microalgae as intracellular symbionts. The identity and function of these endosymbiotic algae have intrigued and perplexed biologists for more than a century. The most conspicuous and well‐studied symbiotic algae of planktonic foraminifera and radiolaria are dinoflagellates, but a variety of nondinoflagellate taxa have also been reported. Ultrastructural features have been used to characterize some of these nondinoflagellate algae, but rarely has this led to clear taxonomic affiliations. We analyzed the nuclear small subunit ribosomal DNA (srDNA) isolated from the symbionts of the spinose planktonic foraminiferan Globigerinella siphonifera d'Orbigny (=Globigerinella aequilateralis Brady) and a solitary radiolarian (Spongodrymus sp. Haeckel) in order to determine the identity of these symbionts. The small coccoid algae isolated from G. siphonifera correspond to the Type I symbionts described by Faber et al. (1988) . Phylogenetic analysis of the srDNA sequences places these symbionts within the prymnesiophyte (haptophyte) lineage, closer to Prymnesium Conrad than to Phaeocystis Lagerheim. To our knowledge, this is the first confirmed case of a symbiotic prymnesiophyte. In addition, we were able to examine the level of sequence heterogeneity between symbionts isolated from different individuals of a single host species. The three isolates in this study had srDNA sequences that were almost identical, indicating that the three were all of the same species. Very green symbiotic algae were isolated from three solitary radiolaria identified as species of Spongodrymus. The symbiont srDNA sequences from the three individual hosts were identical to each other, again implicating a single species of algae in that symbiotic association. These symbionts are prasinophytes most closely related to the clade containing Tetaselmis convolutae Norris, Hori et Chihara. Tetraselmis convolutae is the algal symbiont of the marine flatworm, Convolutae roscoffensis Graff.     

Molecular Identification of Algal Endosymbionts in Large Miliolid Foraminifera: 2. Dinoflagellates

Large miliolid foraminifers of the subfamily Soritinae bear symbiotic dinoflagellates morphologically similar to the species of the "Symbiodinium" complex, commonly found in corals and other marine invertebrates. Soritid foraminifers are abundant in coral reefs and it has been proposed that they share their symbionts with other dinoflagellate-bearing reef dwellers. In order to test this hypothesis, we have analysed partial large subunit ribosomal DNA sequences from dinoflagellates symbionts obtained from 28 foraminiferal specimens, and compared them to the corresponding sequences of Symbiodinium-like endosymbionts from various groups of invertebrates. Phylogenetic analysis of our data shows that all soritid symbionts belong to the "Symbiodinium" species complex, within which they form seven different molecular types (Frl-Fr7). Only one of these types (Fr1) branches within a group of invertebrate symbionts, previously described as type C. The remaining six types form sister groups to coral symbionts previously designed as types B, C, and D. Our data indicate a high genetic diversity and specificity of Symbiodinium-like symbionts in soritids. Except for type C, we have found no evidence for the transmission of symbionts between foraminifers and other symbiont-bearing invertebrates from the same localities. However, exchanges must have occurred frequently between the different species of Soritinae, as suggested by the lack of host specificity and some biogeographical patterns observed in symbiont distribution. Our data suggest that members of the subfamily Soritinae acquired their symbionts at least three times during their history, each acquisition being followed by a rapid diversification and independent radiation of symbionts within the foraminiferal hosts.     

The motility symbiont of the termite gut flagellate Caduceia versatilis is a member of the "Synergistes" group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the "Synergistes" phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the "Synergistes" phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The "Synergistes" phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, "Candidatus Tammella caduceiae," for this unique motility ectosymbiont of Caducaia versatilis.     

Molecular evidence for the broad distribution of anaerobic ammonium-oxidizing bacteria in freshwater and marine sediments

Previously available primer sets for detecting anaerobic ammonium-oxidizing (anammox) bacteria are inefficient, resulting in a very limited database of such sequences, which limits knowledge of their ecology. To overcome this limitation, we designed a new primer set that was 100% specific in the recovery of approximately 700-bp 16S rRNA gene sequences with >96% homology to the "Candidatus Scalindua" group of anammox bacteria, and we detected this group at all sites studied, including a variety of freshwater and marine sediments and permafrost soil. A second primer set was designed that exhibited greater efficiency than previous primers in recovering full-length (1,380-bp) sequences related to "Ca. Scalindua," "Candidatus Brocadia," and "Candidatus Kuenenia." This study provides evidence for the widespread distribution of anammox bacteria in that it detected closely related anammox 16S rRNA gene sequences in 11 geographically and biogeochemically diverse freshwater and marine sediments.     

Betaproteobacterial symbionts of the ciliate Euplotes: origin and tangled evolutionary path of an obligate microbial association

The Polynucleobacter-Euplotes association is an obligatory symbiotic system between a monophyletic group of ciliate species belonging to the genus Euplotes and bacteria of the species Polynucleobacter necessarius (Betaproteobacteria). Both organisms are unable to survive independently. Several studies revealed the existence of free-living populations of Polynucleobacter bacteria which are phylogenetically closely related to the endosymbiotic ones, but never share associations with Euplotes in the natural environment. Hence, following the most parsimonious explanation on the origin of the association, this symbiosis should represent a synapomorphic character for the hosts' clade. Nevertheless, phylogenetic analyses performed on an increased number of strains here presented suggest that Euplotes species, during their evolution, recruited Polynucleobacter bacteria as symbionts more than once. Moreover, in three cases, we observed different bacteria as obligate symbionts. These symbionts are the first characterized representatives of a phylogenetic lineage branching in a basal position with respect to the genus Polynucleobacter. The hypothesis that the original obligate symbionts belonged to this newly discovered clade and that, only subsequently, in most cases they have been replaced by Polynucleobacter bacteria recruited from the environment is proposed and discussed. The evolutionary path of this association seems anyway to have been more complex than so far supposed.     

Bacterial endosymbiont localization in Hyalesthes obsoletus, the insect vector of Bois noir in Vitis vinifera

One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by "Candidatus Phytoplasma solani" and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the "Candidatus Sulcia muelleri" cluster in the Bacteroidetes and in the "Candidatus Purcelliella pentastirinorum" group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name "Candidatus Vidania fulgoroideae." Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and "Candidatus Cardinium hertigii." Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As "Ca. Sulcia" is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between "Ca. Sulcia" and "Ca. Purcelliella," "Ca. Vidania," or both.     

Refining the roots of the beewolf-Streptomyces symbiosis: antennal symbionts in the rare genus Philanthinus (Hymenoptera, Crabronidae)

Insects engage in symbiotic associations with a large diversity of beneficial microorganisms. While the majority of well-studied symbioses have a nutritional basis, several cases are known in which bacteria protect their host from pathogen infestation. Solitary wasps of the genera Philanthus and Trachypus (beewolves; Hymenoptera, Crabronidae) cultivate the actinomycete "Candidatus Streptomyces philanthi" in specialized antennal gland reservoirs. The symbionts are transferred to the larval cocoon, where they provide protection against pathogenic fungi by producing at least nine different antibiotics. Here we investigated the closest relatives of Philanthus and Trachypus, the rare genus Philanthinus, for the presence of antennal gland reservoirs and symbiotic streptomycetes. Molecular analyses identified "Ca. Streptomyces philanthi" in reservoirs of Philanthinus quattuordecimpunctatus. Phylogenies based on the 16S rRNA gene suggest that P. quattuordecimpunctatus may have acquired "Ca. Streptomyces philanthi" by horizontal transfer from other beewolf species. In histological sections and three-dimensional reconstructions, the antennal gland reservoirs were found to occupy six antennal segments (as opposed to only five in Philanthus and Trachypus) and to be structurally less complex than those of the evolutionarily more derived genera of beewolves. The presence of "Ca. Streptomyces philanthi" in antennal glands of Philanthinus indicates that the symbiosis between beewolves and Streptomyces bacteria is much older than previously thought. It probably evolved along the branch leading to the monophyletic tribe Philanthini, as it seems to be confined to the genera Philanthus, Trachypus, and Philanthinus, which together comprise 172 described species of solitary wasps.     

16S rRNA phylogeny of sponge-associated cyanobacteria

Phylogenetic analyses of 16S rRNA sequences of sponge-associated cyanobacteria showed them to be polyphyletic, implying that they derived from multiple independent symbiotic events. Most of the symbiont sequences were affiliated to a group of Synechococcus and Prochlorococcus species. However, other symbionts were related to different groups, such as the Oscillatoriales.     

Lucinidae/sulfur-oxidizing bacteria: ancestral heritage or opportunistic association? Further insights from the Bohol Sea (the Philippines)

The first studies of the 16S rRNA gene diversity of the bacterial symbionts found in lucinid clams did not clarify how symbiotic associations had evolved in this group. Indeed, although species-specific associations deriving from a putative ancestral symbiotic association have been described (coevolution scenario), associations between the same bacterial species and various host species (opportunistic scenario) have also been described. Here, we carried out a comparative molecular analysis of hosts, based on 18S and 28S rRNA gene sequences, and of symbionts, based on 16S rRNA gene sequences, to determine as to which evolutionary scenario led to modern lucinid/symbiont associations. For all sequences analyzed, we found only three bacterial symbiont species, two of which are harbored by lucinids colonizing mangrove swamps. The last symbiont is the most common and was found to be independent of biotope or depth. Another interesting feature is the similarity of ctenidial organization of lucinids from the Philippines to those described previously, with the exception that two bacterial morphotypes were observed in two different species (Gloverina rectangularis and Myrtea flabelliformis). Thus, there is apparently no specific association between Lucinidae and their symbionts, the association taking place according to which bacterial species is present in the environment.     

Multiple origins of the symbioses in Paramecium bursaria

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.     

Evolutionary relationships of "Candidatus endobugula" bacterial symbionts and their Bugula bryozoan hosts

Ribosomal gene sequences were obtained from bryozoans in the genus Bugula and their bacterial symbionts; analyses of host and symbiont phylogenetic trees did not support a history of strict cospeciation. Symbiont-derived compounds known to defend host larvae from predation were only detected in two out of four symbiotic Bugula species.