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.     

The Pine Bark Adelgid,Pineus strobi,Contains Two Novel Bacteriocyte-Associated Gammaproteobacterial Symbionts

Bacterial endosymbionts of the pine bark adelgid, Pineus strobi (Insecta: Hemiptera: Adelgidae), were investigated using transmission electron microscopy, 16S and 23S rRNA-based phylogeny, and fluorescence in situ hybridization. Two morphologically different symbionts affiliated with the Gammaproteobacteria were present in distinct bacteriocytes. One of them (“Candidatus Annandia pinicola”) is most closely related to an endosymbiont of Adelges tsugae, suggesting that they originate from a lineage already present in ancient adelgids before the hosts diversified into the two major clades, Adelges and Pineus. The other P. strobi symbiont (“Candidatus Hartigia pinicola”) represents a novel symbiont lineage in members of the Adelgidae. Our findings lend further support for a complex evolutionary history of the association of adelgids with a phylogenetically diverse set of bacterial symbionts.     

“Candidatus Accumulibacter” Population Structure in Enhanced Biological Phosphorus Removal Sludges as Revealed by Polyphosphate Kinase Genes

We investigated the fine-scale population structure of the “Candidatus Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of “Candidatus Accumulibacter” 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “Candidatus Accumulibacter” lineage. Sequences from at least five clades of “Candidatus Accumulibacter” were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using “Candidatus Accumulibacter”-specific 16S rRNA and “Candidatus Accumulibacter” clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “Candidatus Accumulibacter” lineage and the relative distributions and abundances of the five “Candidatus Accumulibacter” clades. The qPCR-based estimation of the total “Candidatus Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined “Candidatus Accumulibacter” clades. The relative distributions of “Candidatus Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “Candidatus Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.     

Assessment of Survival and Body Size Variation of Culicoides imicola (Diptera: Ceratopogonidae) as Functions of “Candidatus Cardinium” (Bacteroidetes) Infection Status

“Candidatus Cardinium hertigii” (Bacteroidetes) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of “Candidatus Cardinium” infection was documented as moderate, with no detectable sex bias. We therefore investigated whether “Candidatus Cardinium” affects important fitness parameters, such as survival and body size, in Culicoides imicola, a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account “Candidatus Cardinium” infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with “Candidatus Cardinium” infection data. The findings revealed no difference in survival of Culicoides infected with “Candidatus Cardinium” and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for “Candidatus Cardinium”-infected versus uninfected midges. In aggregate, these findings support our conclusion that “Candidatus Cardinium” does not have an overt effect on the survival and size of adult C. imicola midges. “Candidatus Cardinium” may affect immature stages or may alter adult reproductive performance.     

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.     

Diversity of Rickettsiales in the Microbiome of the Lone Star Tick,Amblyomma americanum

Ticks are important vectors for many emerging pathogens. However, they are also infected with many symbionts and commensals, often competing for the same niches. In this paper, we characterize the microbiome of Amblyomma americanum (Acari: Ixodidae), the lone star tick, in order to better understand the evolutionary relationships between pathogens and nonpathogens. Multitag pyrosequencing of prokaryotic 16S rRNA genes (16S rRNA) was performed on 20 lone star ticks (including males, females, and nymphs). Pyrosequencing of the rickettsial sca0 gene (also known as ompA or rompA) was performed on six ticks. Female ticks had less diverse microbiomes than males and nymphs, with greater population densities of Rickettsiales. The most common members of Rickettsiales were “Candidatus Rickettsia amblyommii” and “Candidatus Midichloria mitochondrii.” “Ca. Rickettsia amblyommii” was 2.6-fold more common in females than males, and there was no sequence diversity in the sca0 gene. These results are consistent with a predominantly vertical transmission pattern for “Ca. Rickettsia amblyommii.”     

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.     

Evolution and Diversity of Facultative Symbionts from the Aphid Subfamily Lachninae

Many aphids harbor a variety of endosymbiotic bacteria. The functions of these symbionts can range from an obligate nutritional role to a facultative role in protecting their hosts against environmental stresses. One such symbiont is “Candidatus Serratia symbiotica,” which is involved in defense against heat and potentially also in aphid nutrition. Lachnid aphids have been the focus of several recent studies investigating the transition of this symbiont from a facultative symbiont to an obligate symbiont. In a phylogenetic analysis of Serratia symbionts from 51 lachnid hosts, we found that diversity in symbiont morphology, distribution, and function is due to multiple independent origins of symbiosis from ancestors belonging to Serratia and possibly also to evolution within distinct symbiont clades. Our results do not support cocladogenesis of “Ca. Serratia symbiotica” with Cinara subgenus Cinara species and weigh against an obligate nutritional role. Finally, we show that species belonging to the subfamily Lachninae have a high incidence of facultative symbiont infection.Many insect species harbor heritable endosymbiotic bacteria. Among the best studied of these species are aphids. Almost all aphids are infected with the obligate nutritional symbiont Buchnera aphidicola, which is generally required for the survival of aphids and provides essential amino acids that are rare in their phloem sap diet (32). Many aphids also possess additional symbionts that may be facultative from the host''s perspective and that coexist with Buchnera (20).Three lineages of facultative symbionts that are prevalent in aphids belong to the Enterobacteriaceae. Two of these lineages (“Candidatus Hamiltonella defensa” and “Candidatus Regiella insecticola”) form well-defined clades distinct from free-living bacterial species (4, 20) and confer clear advantages to their hosts by protecting them against natural enemies. “Ca. Hamiltonella defensa” prevents wasp parasitism by arresting development of wasp larvae in pea aphids, and “Ca. Regiella insecticola” provides resistance against the fungal pathogen Pandora neoaphidis (24, 31). The third lineage, “Candidatus Serratia symbiotica,” is closely related to free-living members of the genus Serratia. This symbiont is distributed sporadically among aphid species and has been proposed to have a variety of effects on hosts. In pea aphids (Acyrthosiphon pisum; Macrosiphini), “Ca. Serratia symbiotica” ameliorates the deleterious fitness effects of heat shock by protecting symbiont-harboring bacteriocyte cells (2, 19, 29). Additionally, a strain of “Ca. Serratia symbiotica” provided some resistance to parasitoid wasp attack (24). “Ca. Serratia symbiotica” has been proposed to play a role in nutrition by producing amino acids for its aphid host and by decreasing its host''s reliance on Buchnera (10, 15, 16, 26). In contrast to most Buchnera strains, Buchnera strains from Cinara cedri (Lachnini) have lost the genes for biosynthesis of the essential amino acid tryptophan, while “Ca. Serratia symbiotica” in the same host possesses at least part of the pathway, suggesting that it has a mutualistic role in the nutrition of aphids (26).In A. pisum, “Ca. Serratia symbiotica” cells are rod-shaped bacteria that are present in the sheath cells, hemolymph, and bacteriocytes of some individuals. In contrast, in C. cedri “Ca. Serratia symbiotica” occurs in all individuals, and its cells are large, round, and pleomorphic, similar to the cells of many obligate bacterial aphid endosymbionts, including Buchnera (10, 26). Furthermore, “Ca. Serratia symbiotica” has consistently been present in other Cinara species sampled (28). Both the rod-shaped and pleomorphic forms are assigned to “Ca. Serratia symbiotica” based on phylogenetic analyses of several gene sequences, but they fall into two distinct sister clades of symbiont lineages that seem to coincide with bacterial morphology (17, 20).This diversity in “Ca. Serratia symbiotica” morphology, distribution, and functions may represent evolution of different features within lineages of a single symbiont clade. If “Ca. Serratia symbiotica” is an obligate nutritional symbiont in Cinara hosts, it is expected that Cinara-associated symbionts would form a clade in which the intraclade relationships mirror those of the hosts (cocladogenesis), as observed for Buchnera and other obligate nutritional symbionts of insects (13, 21, 38). Indeed, Lamelas et al. postulated that, based on their similar phylogenies, Serratia symbionts from aphids belonging to the subgenus Cinara have had a long-term relationship with their hosts (17).In addition to the three most common facultative symbiont types found in aphids described above, several other symbiont lineages with unknown functions have been identified by amplification of bacterial 16S rRNA gene sequences from various aphid species (10, 28, 39). Here we examine the diversity of Serratia and other facultative symbionts in aphids belonging to the subfamily Lachninae. We investigated the distribution of symbionts in aphid species and geographic locations and looked for coevolutionary patterns that may correspond to the functions of facultative symbionts within their hosts.     

A Novel Bacteroidetes Symbiont Is Localized in Scaphoideus titanus, the Insect Vector of Flavescence Dorée in Vitis vinifera

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, “Candidatus Phytoplasma vitis,” which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the “Candidatus Cardinium hertigii” group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of “Ca. Cardinium hertigii.” This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of “Ca. Phytoplasma vitis” were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and “Ca. Phytoplasma vitis” have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.     

Broad Distribution of Diverse Anaerobic Ammonium-Oxidizing Bacteria in Chinese Agricultural Soils

Anaerobic ammonium-oxidizing (anammox) bacteria have been detected in many marine and freshwater ecosystems. However, little is known about the distribution, diversity, and abundance of anammox bacteria in terrestrial ecosystems. In this study, anammox bacteria were found to be present in various agricultural soils collected from 32 different locations in China. Phylogenetic analysis of the 16S rRNA genes showed “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” in the collected soils, with “Candidatus Brocadia” being the dominant genus. Quantitative PCR showed that the abundance of anammox bacteria ranged from 6.38 × 10⁴ ± 0.42 × 10⁴ to 3.69 × 10⁶ ± 0.25 × 10⁶ copies per gram of dry weight. Different levels of diversity, composition, and abundance of the anammox bacterial communities were observed, and redundancy analysis indicated that the soil organic content and the distribution of anammox communities were correlated in the soils examined. Furthermore, Pearson correlation analysis showed that the diversity of the anammox bacteria was positively correlated with the soil ammonium content and the organic content, while the anammox bacterial abundance was positively correlated with the soil ammonium content. These results demonstrate the broad distribution of diverse anammox bacteria and its correlation with the soil environmental conditions within an extensive range of Chinese agricultural soils.     

Arsenophonus and Sodalis Symbionts in Louse Flies: an Analogy to the Wigglesworthia and Sodalis System in Tsetse Flies

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.     

A New Huanglongbing Species, “Candidatus Liberibacter psyllaurous,” Found To Infect Tomato and Potato, Is Vectored by the Psyllid Bactericera cockerelli (Sulc)

A new huanglongbing (HLB) “Candidatus Liberibacter” species is genetically characterized, and the bacterium is designated “Candidatus Liberibacter psyllaurous.” This bacterium infects the psyllid Bactericera cockerelli and its solanaceous host plants potato and tomato, potentially resulting in “psyllid yellowing.” Host plant-dependent HLB transmission and variation in psyllid infection frequencies are found.     

A Novel Rickettsia Species Detected in Vole Ticks (Ixodes angustus) from Western Canada

The genomic DNA of ixodid ticks from western Canada was tested by PCR for the presence of Rickettsia. No rickettsiae were detected in Ixodes sculptus, whereas 18% of the I. angustus and 42% of the Dermacentor andersoni organisms examined were PCR positive for Rickettsia. The rickettsiae from each tick species were characterized genetically using multiple genes. Rickettsiae within the D. andersoni organisms had sequences at four genes that matched those of R. peacockii. In contrast, the Rickettsia present within the larvae, nymphs, and adults of I. angustus had novel DNA sequences at four of the genes characterized compared to the sequences available from GenBank for all recognized species of Rickettsia and all other putative species within the genus. Phylogenetic analyses of the sequence data revealed that the rickettsiae in I. angustus do not belong to the spotted fever, transitional, or typhus groups of rickettsiae but are most closely related to “Candidatus Rickettsia kingi” and belong to a clade that also includes R. canadensis, “Candidatus Rickettsia tarasevichiae,” and “Candidatus Rickettsia monteiroi.”     

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 ~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.     

Genome Sequence of “Candidatus Microthrix parvicella” Bio17-1, a Long-Chain-Fatty-Acid-Accumulating Filamentous Actinobacterium from a Biological Wastewater Treatment Plant

“Candidatus Microthrix” bacteria are deeply branching filamentous actinobacteria which occur at the water-air interface of biological wastewater treatment plants, where they are often responsible for foaming and bulking. Here, we report the first draft genome sequence of a strain from this genus: “Candidatus Microthrix parvicella” strain Bio17-1.     

Diversity of “Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, “Candidatus Liberibacter africanus” in Africa and “Candidatus Liberibacter asiaticus” in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of “Ca. Liberibacter asiaticus” isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species “Ca. Liberibacter asiaticus,” even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating “Ca. Liberibacter asiaticus” isolates.     

“Candidatus Endobugula glebosa,” a Specific Bacterial Symbiont of the Marine Bryozoan Bugula simplex

The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, “Candidatus Endobugula sertula.” “Candidatus E. sertula” has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with “Candidatus E. sertula.” In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status “Candidatus Endobugula glebosa” is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.     

Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group

The phylogenetic affiliation and physiological characteristics (e.g., K_s and maximum specific growth rate [μ_max]) of an anaerobic ammonium oxidation (anammox) bacterium, “Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.