Applied and Environmental Microbiology

ABSTRACT Bemisia tabaci (Hemiptera: Aleyrodidae) is a species complex containing >28 cryptic species, some of which are important crop pests worldwide. Like many other sap-sucking insects, whiteflies harbor an obligatory symbiont, “ Candidatus Portiera aleyrodidarum,” and a number of secondary symbionts. So far, six genera of secondary symbionts have been identified in B. tabaci . In this study, we report and describe the finding of an additional bacterium in the indigenous B. tabaci cryptic species China 1 (formerly known as B. tabaci biotype ZHJ3). Phylogenetic analysis based on the 16S rRNA and gltA genes showed that the bacterium belongs to the Alphaproteobacteria subdivision of the Proteobacteria and has a close relationship with human pathogens of the genus Orientia . Consequently, we temporarily named it Orientia -like organism (OLO). OLO was found in six of eight wild populations of B. tabaci China 1, with the infection rate ranging from 46.2% to 76.8%. Fluorescence in situ hybridization (FISH) of B. tabaci China 1 in nymphs and adults revealed that OLOs are confined to the bacteriome and co-occur with “ Ca . Portiera aleyrodidarum.” The vertical transmission of OLO was demonstrated by detection of OLO at the anterior pole end of the oocytes through FISH. Quantitative PCR analysis of population dynamics suggested a complex interaction between “ Ca . Portiera aleyrodidarum” and OLO. Based on these results, we propose “ Candidatus Hemipteriphilus asiaticus” for the classification of this symbiont from B. tabaci .

ABSTRACT The recently described bacterium “ Candidatus Methylomirabilis oxyfera” couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of “ Ca . Methylomirabilis oxyfera” is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of “ Ca . Methylomirabilis oxyfera” to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple “ Ca . Methylomirabilis oxyfera” enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC 16:0 ). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the Δ7 position (10MeC 16:1Δ7 ), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC 16:0 and 10MeC 16:1Δ7 are key and characteristic components of the lipid profile of “ Ca . Methylomirabilis oxyfera.” The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.

ABSTRACT Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus Nysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont Schneideria nysicola within a pair of bacteriomes in the abdomen. Our survey of Nysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of Wolbachia . Diagnostic PCR survey of 343 individuals representing 24 populations of four Nysius species revealed overall detection rates of the alphaproteobacteria at 77.6% in Nysius plebeius , 87.7% in Nysius sp. 1, 81.0% in Nysius sp. 2, and 100% in Nysius expressus . Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the order Rickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, and Midichloria spp., the mitochondrion-associated endosymbionts of ticks. In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of N. plebeius , including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation “ Candidatus Lariskella arthropodarum” is proposed for the endosymbiont clade.

ABSTRACT Here we present comparative data on the localization and identity of intracellular symbionts among the superfamily Lygaeoidea (Insecta: Hemiptera: Heteroptera: Pentatomomorpha). Five different lygaeoid species from the families Blissidae and Lygaeidae (sensu stricto; including the subfamilies Lygaeinae and Orsillinae) were analyzed. Fluorescence in situ hybridization (FISH) revealed that all the bugs studied possess paired bacteriomes that are differently shaped in the abdomen and harbor specific endosymbionts therein. The endosymbionts were also detected in female gonads and at the anterior poles of developing eggs, indicating vertical transmission of the endosymbionts via ovarial passage, in contrast to the posthatch symbiont transmission commonly found among pentatomoid bugs (Pentatomomorpha: Pentatomoidea). Phylogenetic analysis based on 16S rRNA and groEL genes showed that the endosymbionts of Ischnodemus sabuleti , Arocatus longiceps , Belonochilus numenius , Orsillus depressus , and Ortholomus punctipennis constitute at least four distinct clades in the Gammaproteobacteria . The endosymbiont phylogeny did not agree with the host phylogeny based on the mitochondrial cytochrome oxidase I ( COI ) gene, but there was a local cospeciating pattern within the subfamily Orsillinae. Meanwhile, the endosymbiont of Belonochilus numenius (Lygaeidae: Orsillinae), although harbored in paired bacteriomes as in other lygaeoid bugs of the related genera Nysius , Ortholomus , and Orsillus , was phylogenetically close to “ Candidatus Rohrkolberia cinguli,” the endosymbiont of Chilacis typhae (Lygaeoidea: Artheneidae), suggesting an endosymbiont replacement in this lineage. The diverse endosymbionts and the differently shaped bacteriomes may reflect independent evolutionary origins of the endosymbiotic systems among lygaeoid bugs.

ABSTRACT “ Candidatus Liberibacter asiaticus” is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of “ Ca . Liberibacter” associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes ( hyv I and hyv II ) were identified in the prophage regions of the Psy62 “ Ca. Liberibacter asiaticus” genome. Sequence analyses of the hyv I and hyv II genes in 35 “ Ca. Liberibacter asiaticus” DNA isolates collected globally revealed that the hyv I gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyv II contains up to 2 NITRs and 4 partial repeats and shares homology with hyv I . Frequent deletions or insertions of these repeats within the hyv I and hyv II genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of “ Ca. Liberibacter asiaticus” bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single “ Ca. Liberibacter asiaticus”-infected sample. This is the first evidence of different “ Ca. Liberibacter asiaticus” populations coexisting in a single HLB-affected sample. The Florida “ Ca. Liberibacter asiaticus” isolates contain both hyv I and hyv II , while all other global “ Ca. Liberibacter asiaticus” isolates contain either one or the other. Interclade assignments of the putative Hyv I and Hyv II proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple “ Ca. Liberibacter asiaticus” populations in the world and a multisource introduction of the “ Ca. Liberibacter asiaticus” bacterium into Florida.

ABSTRACT 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.

ABSTRACTOne emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by “CandidatusPhytoplasma solani” and spread in vineyards by the planthopperHyalesthes 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 “CandidatusSulcia muelleri” cluster in theBacteroidetesand in the “CandidatusPurcelliella pentastirinorum” group in theGammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to theBetaproteobacteria, 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 tickHaemaphysalis longicornis, we propose the new name “CandidatusVidania fulgoroideae.” Other bacterial endosymbionts identified inH. obsoletuswere related to the intracellular bacteriaWolbachia pipientis,Rickettsiasp., and “CandidatusCardinium hertigii.” Fluorescentin situhybridization 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 inH. obsoletus, it may occur in a bipartite or even tripartite relationship between “Ca.Sulcia” and “Ca.Purcelliella,” “Ca.Vidania,” or both.

ABSTRACT Mealybugs (Coccoidea: Pseudococcidae) are sap-sucking plant parasites that harbor bacterial endosymbionts within specialized organs. Previous studies have identified two subfamilies, Pseudococcinae and Phenacoccinae, within mealybugs and determined the primary endosymbionts (P-endosymbionts) of the Pseudococcinae to be Betaproteobacteria (“ Candidatus Tremblaya princeps”) containing Gammaproteobacteria secondary symbionts. Here, the P-endosymbionts of phenacoccine mealybugs are characterized based on 16S rRNA from the bacteria of 20 species of phenacoccine mealybugs and four outgroup Puto species (Coccoidea: Putoidae) and aligned to more than 100 published 16S rRNA sequences from symbiotic and free-living bacteria. Phylogenetic analyses recovered three separate lineages of bacteria from the Phenacoccinae, and these are considered to be the P-endosymbionts of their respective mealybug hosts, with those from (i) the mealybug genus Rastrococcus belonging to the Bacteroidetes , (ii) the subterranean mealybugs, tribe Rhizoecini, also within Bacteroidetes , in a clade sister to cockroach endosymbionts ( Blattabacterium ), and (iii) the remaining Phenacoccinae within the Betaproteobacteria , forming a well-supported sister group to “ Candidatus Tremblaya princeps.” Names are proposed for two strongly supported lineages: “ Candidatus Brownia rhizoecola” for P-endosymbionts of Rhizoecini and “ Candidatus Tremblaya phenacola” for P-endosymbionts of Phenacoccinae excluding Rastrococcus and Rhizoecini. Rates of nucleotide substitution among lineages of Tremblaya were inferred to be significantly faster than those of free-living Betaproteobacteria . Analyses also recovered a clade of Gammaproteobacteria , sister to the P-endosymbiont lineage of aphids (“ Candidatus Buchnera aphidicola”), containing the endosymbionts of Putoidae, the secondary endosymbionts of pseudococcine mealybugs, and the endosymbionts of several other insect groups.

ABSTRACT “ Candidatus Accumulibacter” and total bacterial community dynamics were studied in two lab-scale enhanced biological phosphorus removal (EBPR) reactors by using a community fingerprint technique, automated ribosomal intergenic spacer analysis (ARISA). We first evaluated the quantitative capability of ARISA compared to quantitative real-time PCR (qPCR). ARISA and qPCR provided comparable relative quantification of the two dominant “ Ca . Accumulibacter” clades (IA and IIA) detected in our reactors. The quantification of total “ Ca . Accumulibacter” 16S rRNA genes relative to that from the total bacterial community was highly correlated, with ARISA systematically underestimating “ Ca . Accumulibacter” abundance, probably due to the different normalization techniques applied. During 6 months of normal (undisturbed) operation, the distribution of the two clades within the total “ Ca . Accumulibacter” population was quite stable in one reactor while comparatively dynamic in the other reactor. However, the variance in the clade distribution did not appear to affect reactor performance. Instead, good EBPR activity was positively associated with the abundance of total “ Ca . Accumulibacter.” Therefore, we concluded that the different clades in the system provided functional redundancy. We disturbed the reactor operation by adding nitrate together with acetate feeding in the anaerobic phase to reach initial reactor concentrations of 10 mg/liter NO 3 -N for 35 days. The reactor performance deteriorated with a concomitant decrease in the total “ Ca . Accumulibacter” population, suggesting that a population shift was the cause of performance upset after a long exposure to nitrate in the anaerobic phase.

ABSTRACT Previously isolated dissimilatory perchlorate-reducing bacteria (DPRB) have been primarily affiliated with the Betaproteobacteria . Enrichments from the cathodic chamber of a bioelectrical reactor (BER) inoculated from creek water in Berkeley, CA, yielded a novel organism most closely related to a previously described strain, WD (99% 16S rRNA gene identity). Strain VDY T has 96% 16S rRNA gene identity to both Magnetospirillum gryphiswaldense and Magnetospirillum magnetotacticum , and along with strain WD, distinguishes a clade of perchlorate-reducing Magnetospirillum species in the Alphaproteobacteria . In spite of the phylogenetic location of VDY T , attempted PCR for the key magnetosome formation genes mamI and mamL was negative. Strain VDY T was motile, non-spore forming, and, in addition to perchlorate, could use oxygen, chlorate, nitrate, nitrite, and nitrous oxide as alternative electron acceptors with acetate as the electron donor. Transient chlorate accumulation occurred during respiration of perchlorate. The organism made use of fermentation end products, such as acetate and ethanol, as carbon sources and electron donors for heterotrophic growth, and in addition, strain VDY T could grow chemolithotrophically with hydrogen serving as the electron donor. VDY T contains a copy of the RuBisCo cbbM gene, which was expressed under autotrophic but not heterotrophic conditions. DNA-DNA hybridization with strain WD confirmed VDY T as a separate species (46.2% identity), and the name Magnetospirillum bellicus sp. nov. (DSM 21662, ATCC BAA-1730) is proposed.