Microbial Ecology

Bacterial Community of the Digestive Tract of the European Medicinal Leech ( Hirudo verbana ) from the Danube River The original published version of this article had mistakes in figure legends. Correct figure legends are presented below.

" Candidatus Hafkinia simulans" gen. nov., sp. nov., a Novel Holospora -Like Bacterium from the Macronucleus of the Rare Brackish Water Ciliate Frontonia salmastra (Oligohymenophorea, Ciliophora): Multidisciplinary Characterization of the New Endosymbiont and Its Host Abstract We characterized a novel Holospora-like bacterium (HLB) (Alphaproteobacteria, Holosporales) living in the macronucleus of the brackish water ciliate Frontonia salmastra. This bacterium was morphologically and ultrastructurally investigated, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and performed in situ hybridization experiments with a specifically-designed probe. A new taxon, "Candidatus Hafkinia simulans", was established for this HLB. The phylogeny of the family Holosporaceae based on 16S rRNA gene sequences was inferred, adding to the already available data both the sequence of the novel bacterium and those of other Holospora and HLB species recently characterized. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and placed the new endosymbiont as the sister genus of Holospora. Additionally, the host ciliate F. salmastra, recorded in Europe for the first time, was concurrently described through a multidisciplinary study. Frontonia salmastra's phylogenetic position in the subclass Peniculia and the genus Frontonia was assessed according to 18S rRNA gene sequencing. Comments on the biodiversity of this genus were added according to past and recent literature.

Soil Properties and Multi-Pollution Affect Taxonomic and Functional Bacterial Diversity in a Range of French Soils Displaying an Anthropisation Gradient Abstract The intensive industrial activities of the twentieth century have left behind highly contaminated wasteland soils. It is well known that soil parameters and the presence of pollutants shape microbial communities. But in such industrial waste sites, the soil multi-contamination with organic (polycyclic aromatic hydrocarbons, PAH) and metallic (Zn, Pb, Cd) pollutants and long-term exposure may induce a selection pressure on microbial communities that may modify soil functioning. The aim of our study was to evaluate the impact of long-term multi-contamination and soil characteristics on bacterial taxonomic and functional diversity as related to the carbon cycle. We worked on 10 soils from northeast of France distributed into three groups (low anthropised controls, slag heaps, and settling ponds) based on their physico-chemical properties (texture, C, N) and pollution level. We assessed bacterial taxonomic diversity by 16S rDNA Illumina sequencing, and functional diversity using Biolog® and MicroResp™ microtiter plate tools. Although taxonomic diversity at the phylum level was not different among the soil groups, many operational taxonomic units were influenced by metal or PAH pollution, and by soil texture and total nitrogen content. Functional diversity was not influenced by PAH contamination while metal pollution selected microbial communities with reduced metabolic functional diversity but more tolerant to zinc. Limited microbial utilisation of carbon substrates in metal-polluted soils was mainly due to the nitrogen content. Based on these two observations, we hypothesised that reduced microbial activity and lower carbon cycle–related functional diversity may have contributed to the accumulation of organic matter in the soils that exhibited the highest levels of metal pollution.

Bacterial Community of the Digestive Tract of the European Medicinal Leech ( Hirudo verbana ) from the Danube River Abstract The digestive tract of medicinal leeches from commercial suppliers has been investigated previously and comprises of a relatively simple bacterial community. However, the microbiome of medicinal leeches collected directly from the natural habitat has not been examined. In this study, we characterized the bacterial community in the digestive tract (anterior crop, posterior crop, and intestine) of the European medicinal leech, Hirudo verbana, collected from the Danube river using culture-independent and culture-dependent approaches. Culture-independent approach confirmed that the digestive tract of H. verbana carries a relatively simple bacterial community with species richness in the individual samples ranging from 43 to164. The dominant bacterial taxon was Mucinivorans sp. (49.7% of total reads), followed by Aeromonas sp. (18.7% of total reads). Several low abundance taxa, new for H. verbana, such as Phreatobacter, Taibaiella, Fluviicola, Aquabacterium, Burkholderia, Hydrogenophaga, Wolinella, and unidentified Chitinophagia, were also detected. The aerobic culturing approach showed Aeromonas veronii (Proteobacteria), the known leech symbiont, as the most dominant taxon followed by several Pseudomonas and Acidovorax spp. No significant differences in the bacterial community composition were detected among different parts of the digestive tract of individual leeches. However, the overall composition of the bacterial community among individual specimen varied significantly and this is possibly due to differences in leech age, feeding status, and blood source. Our results showed that the core bacterial community of H. verbana collected from the natural habitat is similar to that reported from the digestive tract of commercially supplied leeches maintained in the laboratory.

Experimental Testing of Dispersal Limitation in Soil Bacterial Communities with a Propagule Addition Approach Abstract The role of dispersal in the assembly of microbial communities remains contentious. This study tested the importance of dispersal limitation for the structuring of local soil bacterial communities using an experimental approach of propagule addition. Microbes extracted from soil pooled from samples collected at 20 localities across ~ 400 km in a temperate steppe were added to microcosms of local soils at three sites; the microcosms were then incubated in situ for 3 months. We then assessed the composition and diversity of bacterial taxa in the soils using 16S rRNA gene amplicon sequencing. The addition of the regional microbial pool did not cause significant changes in the overall composition or diversity of the total bacterial community, although a very small number of individual taxa may have been affected by the addition treatment. Our results suggest a negligible role of dispersal limitation in structuring soil bacterial communities in our study area.

Early Changes in Nutritional Conditions Affect Formation of Synthetic Mutualism Between Chlorella sorokiniana and the Bacterium Azospirillum brasilense Abstract The effect of three different nutritional conditions during the initial 12 h of interaction between the microalgae Chlorella sorokiniana UTEX 2714 and the plant growth–promoting bacterium Azospirillum brasilense Cd on formation of synthetic mutualism was assessed by changes in population growth, production of signal molecules tryptophan and indole-3-acetic acid, starch accumulation, and patterns of cell aggregation. When the interaction was supported by a nutrient-rich medium, production of both signal molecules was detected, but not when this interaction began with nitrogen-free (N-free) or carbon-free (C-free) media. Overall, populations of bacteria and microalgae were larger when co-immobilized. However, the highest starch production was measured in C. sorokiniana immobilized alone and growing continuously in a C-free mineral medium. In this interaction, the initial nutritional condition influenced the time at which the highest accumulation of starch occurred in Chlorella, where the N-free medium induced faster starch production and the richer medium delayed its accumulation. Formation of aggregates made of microalgae and bacteria occurred in all nutritional conditions, with maximum at 83 h in mineral medium, and coincided with declining starch content. This study demonstrates that synthetic mutualism between C. sorokiniana and A. brasilense can be modulated by the initial nutritional condition, mainly by the presence or absence of nitrogen and carbon in the medium in which they are interacting.

Genetic and Genome Analyses Reveal Genetically Distinct Populations of the Bee Pathogen Nosema ceranae from Thailand Abstract The recent global decline in Western honeybee (Apis mellifera) populations is of great concern for pollination and honey production worldwide. Declining honeybee populations are frequently infected by the microsporidian pathogen Nosema ceranae. This species was originally described in the Asiatic honeybee (Apis cerana), and its identification in global A. mellifera hives could result from a recent host transfer. Recent genome studies have found that global populations of this parasite are polyploid and that humans may have fueled their global expansion. To better understand N. ceranae biology, we investigated its genetic diversity within part of their native range (Thailand) and among different hosts (A. mellifera, A. cerana) using both PCR and genome-based methods. We find that Thai N. ceranae populations share many SNPs with other global populations and appear to be clonal. However, in stark contrast with previous studies, we found that these populations also carry many SNPs not found elsewhere, indicating that these populations have evolved in their current geographic location for some time. Our genome analyses also indicate the potential presence of diploidy within Thai populations of N. ceranae.

Population and Culture Age Influence the Microbiome Profiles of House Dust Mites Abstract Interactions with microorganisms might enable house dust mites (HDMs) to derive nutrients from difficult-to-digest structural proteins and to flourish in human houses. We tested this hypothesis by investigating the effects of changes in the mite culture growth and population of two HDM species on HDM microbiome composition and fitness. Growing cultures of laboratory and industrial allergen-producing populations of Dermatophagoides farinae (DFL and DFT, respectively) and Dermatophagoides pteronyssinus (DPL and DPT, respectively) were sampled at four time points. The symbiotic microorganisms of the mites were characterized by DNA barcode sequencing and quantified by qPCR using universal/specific primers. The population growth of mites and nutrient contents of mite bodies were measured and correlated with the changes in bacteria in the HDM microbiome. The results showed that both the population and culture age significantly influenced the microbiome profiles. Cardinium formed 93% and 32% of the total sequences of the DFL and DFT bacterial microbiomes, respectively, but this bacterial species was less abundant in the DPL and DPT microbiomes. Staphylococcus abundance was positively correlated with increased glycogen contents in the bodies of mites, and increased abundances of Aspergillus, Candida, and Kocuria were correlated with increased lipid contents in the bodies of mites. The xerophilic fungus Wallemia accounted for 39% of the fungal sequences in the DPL microbiome, but its abundance was low in the DPT, DFL, and DFT microbiomes. With respect to the mite culture age, we made three important observations: the mite population growth from young cultures was 5–8-fold higher than that from old cultures; specimens from old cultures had greater abundances of fungi and bacteria in their bodies; and yeasts predominated in the gut contents of specimens from young cultures, whereas filamentous mycelium prevailed in specimens from old cultures. Our results are consistent with the hypothesis that mites derive nutrients through associations with microorganisms.

Geographic and Temporal Variation of Distinct Intracellular Endosymbiont Strains of Wolbachia sp. in the Grasshopper Chorthippus parallelus : a Frequency-Dependent Mechanism? Abstract Wolbachia is an intracellular endosymbiont that can produce a range of effects on host fitness, but the temporal dynamics of Wolbachia strains have rarely been experimentally evaluated. We compare interannual strain frequencies along a geographical region for understanding the forces that shape Wolbachia strain frequency in natural populations of its host, Chorthippus parallelus (Orthoptera, Acrididae). General linear models show that strain frequency changes significantly across geographical and temporal scales. Computer simulation allows to reject the compatibility of the observed patterns with either genetic drift or sampling errors. We use consecutive years to estimate total Wolbachia strain fitness. Our estimation of Wolbachia fitness is significant in most cases, within locality and between consecutive years, following a negatively frequency-dependent trend. Wolbachia spp. B and F strains show a temporal pattern of variation that is compatible with a negative frequency-dependent natural selection mechanism. Our results suggest that such a mechanism should be at least considered in future experimental and theoretical research strategies that attempt to understand Wolbachia biodiversity.

Titanium Ions Inhibit the Bacteria in Vase Solutions of Freshly Cut Gerbera jamesonii and Extend the Flower Longevity Abstract Titanium ions significantly promote plant growth, but the mechanism is still unclear. Cut flowers are ideal materials for the study of plant growth and senescence. In this study, freshly cut Gerbera jamesonii were used to study the effects of titanium ions (8 mg/L) on the flower longevity. Flowering observation showed that the gerbera vase life was significantly prolonged in the presence of titanium ions. Plate colony counts showed that the amounts of bacteria in the vase solution of the control group were approximately 1700 times more than that of titanium ion treatment group. High-throughput sequencing was used to determine the sequences of 16S rRNA gene V3-V4 variable regions of the vase solutions to analyze bacterial species, their average proportions, and absolute abundance. The results showed that the titanium ions reduced the entire bacterial counts as well as altered the absolute abundance of different bacterial species in the vase solution. The most prevalent bacteria were mainly Enterobacteriaceae, Pseudomonas veronii, Pseudomonas sp., Delftia sp., Agrobacterium sp., Sphingobacterium multivorum, Acinetobacter johnsonii, and Clostridiaceae. In combination with plate colony counts, we demonstrated that all the bacterial growths were significantly inhibited by titanium ions, regardless of their average proportions increased or decreased. These results showed that titanium ions could extend effectively the longevity of gerberas and possess the broad-spectrum antibacterial properties. This study provides a basis for further mechanism exploration of titanium ions action and its applications in cut flower preservation and agricultural production.