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    Endophytic bacterial communities are associated with leaf mimicry in the vine Boquila trifoliolata

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    Integrate geographic scales in equity, diversity and inclusion

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    Successful microbial colonization of space in a more dispersed manner

    Simulating competition for space using the “BacGo” modelTo investigate how spatial positioning of populations affects the outcome of microbial competition, we simulated two populations competing for space with a limiting size by building an individual-based model (named “BacGo”). The model was implemented in discrete grid boxes of a 20 × 20 array. As shown in Fig. 1a, our simulations were based on three basic assumptions. First, the two competing populations possess the same inherent growth rate and equal initial cell numbers, thus the only differences between them are their manners of colonizing free space. Second, the newly born daughter cell is located around its mother cell but with a random direction of spatial positioning [34], resulted in a microcolony with different spatial patterning. Lastly, if the selected box has been occupied, the newborn cell will compete for the box against the original occupants of the box and possesses a probability of 50% to survive [37].We first explored the outcome of spatial competition, which started by randomly distributing two populations on the grids with the same initial cell numbers of 10 for each. Based on our basic assumptions and the predictions of competitive exclusion theory [38], we hypothesized that only one population could win the competition and finally occupy all grids. As shown in 20,000 independent simulations with random initial distributions, we discovered that at the end of each simulation, only one population survived (Video S1 and Video S2). The Chi-square test showed no significant difference (P = 0.211) between the simulated winning times (10,177 of 20,000 simulations) and the random winning times (10,051 of 20,000 simulations) of the focus population. This result conformed with our initial assumption that cells possess a probability of 50% to survive in competing with original occupants. When we replicated simulations initiated with the same cell distribution, we found that the winning probabilities for each population changed in line with the initial distributions (Fig. S1). However, the winning probabilities never reached 100% no matter how the initial distribution changes. Together, these results suggested that unknown random factors may affect the final outcome of the competition.Next, we analyzed the dynamics of microbial colonization during our simulations. As summarized in Fig. 1b, we divided the competition process into two stages, the “occupation stage” and the “exclusion stage” (see Methods). To statistically characterize the competitive outcome at t3, we defined the winning asymmetry index, WinR, and the abundance asymmetry index, AbunR (see Methods). As shown in Fig. S2a, we found a strong positive correlation (R2 = 0.740, P  More

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    Photoheterotrophy by aerobic anoxygenic bacteria modulates carbon fluxes in a freshwater lake

    SamplingWater was sampled from Cep lake in Czechia, at a regular sampling site of 10 m depth (48.944 °N, 14.877 °E). The lake originates from sand mining in the 1970–80s. It is a permanent meso-oligotrophic (chlorophyll-a concentrations ranged from 1.4–16.4 µg L−1) seepage reservoir filled with groundwater penetrating from the nearby river Lužnice. The lake area is about 1.16 km2, with the maximum depth about 11–12 m. These characteristics are representative for most of temperate and boreal lakes [16].Samples were collected every four weeks from April till October in 2018, and from April till November in 2019. Ten liters of water were collected from 0.5 m depth using a Ruttner Water Sampler (model 11.003KC Denmark AS). Temperature and oxygen profiles were taken with an EXO1 multi-parameter probe (YSI Inc., Yellow Springs, USA). Water was transported to the laboratory within 2 h from the collection in a closed container made from high-density polyethylene, rinsed three times with the sampled water and stored in a cooled box.NutrientsSamples were filtered through glass fiber filters with 0.4 µm nominal porosity (GF-5, Macherey-Nagel, Düren, Germany). Concentrations of soluble reactive phosphorus (SRP) were determined spectrophotometrically [17, 18]. Concentrations of nitrate and ammonium were measured according to Procházková [19] and Kopáčkek and Procházková [20]. Dissolved organic carbon (DOC) and dissolved nitrogen (DN) were measured with a TOC 5000 A analyzer (Shimadzu, Kyoto, Japan).PigmentsSeston from 1.43 to 3.65 L of water was collected onto GF-5 glass fiber filters (diameter 47 mm, Macherey-Nagel). The filters were dried of excess water by gently pressing in a paper towel, and flush frozen in liquid nitrogen. Pigments were extracted and analyzed by HPLC as described in Piwosz et al. [21].Net primary production (NPP) and community respiration (oxygen measurements)Oxygen concentration was measured with the Winkler method [22]. It was chosen because it allows O2 concentration to be measured directly in the water without the need to consider carbonic equilibrium, which is the case when changes in CO2 concentration is measured [23]. Samples were unfiltered to avoid the removal of particle-associated bacteria and also of free-living AAP bacteria, which tend to be larger than average freshwater bacteria [12]. Glass stoppered Winkler type oxygen bottles (115 mL nominal capacity, VTR glass, Prague, Czechia) were filled with the sampled water directly from the sampler via a rubber tube. Each bottle was first rinsed three times and then filled without the formation of air bubbles. Water was allowed to overflow the neck of the bottle for about 1 min, and the bottle was closed with a glass stopper to avoid air bubbles. The closed bottles were kept in the dark in a cooled box. On the shore, three bottles were selected as T0, and 1.2 mL of manganese (II) chloride solution (concentration 3 mol L−1) was addded, followed by the addition of 1.2 mL of a mixture containing 4 mol L−1 of sodium iodide solution and 8 mol L−1 of sodium hydroxide solution. These samples were processed in the laboratory within 3 h. The remaining bottles were incubated for 24 h at in situ temperature in the IR-box prepared from the MAKROLON IR polycarbonate sheet (4 mm thickness, Professional Plastics, Inc. Fullerton, CA, USA). These panels have a maximum transmittance of 90% in the infrared region 850–2000 nm, 50% at 780 nm, and 0% 10 °C according to our measurements (180 days, Equation 3 in Supplementary File S1). Subsequently, the differences in the Cep Lake’s carbon budget for the surface layer (down to 0.5 m depth) were calculated by multiplying the integrated values by the volume of this layer (Equation 4 and 5 in Supplementary File S1).HCO3
    − incorporationTriplicated water samples (32 mL) were incubated for 3.2–5.2 h in the IR light and the dark at in situ temperature, as described for respiration. Total activity added to each bottle was measured from 1 mL aliquot of the incubated sample that was transferred to a scintillation vial containing 20 μl of 5 mol L−1 NaOH (to prevent a loss of 14C-bicarbonate). Thirty mL of sample was filtered through 2.5 μm nitrate cellulose filters (Pragopor, Prague, Czechia, diameter 25 mm). Five mL of the filtered water was collected and subsequently filtered through a 0.17 μm nitrate cellulose filter. The resulting cell-free filtrate, which contained 14C-DOC was collected. The filtration was done at a low vacuum (0.02 MPa) to avoid cell breakage. The total CO2 assimilation rate was calculated as the sum of all these fractions.The filters were kept in an HCl-saturated atmosphere for 24 h at room temperature in a fume hood. They were placed in scintillation vials and dissolved in 1 mL of ethyl acetate (Penta, Prague, Czechia). Then, 5 mL of Ultima Golt LLT scintillation cocktail (PerkinElmer, Waltham, MA, USA) was added. Five mL of cell-free filtrates were acidified by adding 100 μL 5 mol L−1 HCl to volatilize non-incorporated H14CO3 and incubated 24 h at room temperature in a fume hood. Then, 10 mL of the scintillation cocktail was added. Finally, 5 mL of the scintillation cocktail was added to the total activity samples. Subsequently, the samples were gently mixed and left in the dark for 48 h. The radioactivity in the samples was measured using a Tri-Carb 2810 TR scintillation counter (PerkinElmer).To estimate carbon fluxes (μmol C L–1 h–1), a fraction of the added H14CO3 incorporated or released was corrected for the incubation time and multiplied by the concentration of total dissolved inorganic carbon (DIC). The DIC concentration was calculated based on temperature, pH, and alkalinity measurements (Inolab pH 720, WTW Xylem Inc. Rye Brook, NY, US) determined by Gran titration.Assimilation of organic monomersThe difference between microbial activity in the IR light and dark was also estimated based on assimilation rates of radiolabeled glucose, pyruvate, leucine and thymidine (American Radiolabeled Chemicals, St. Louis, MO, USA). Tritiated glucose (specific activity (SA): 2220 GBq mmol−1), leucine (SA: 4440 GBq mmol−1) and thymidine (SA: 2275.5 GBq mmol−1) were added to 5 mL samples to a final concentration of 5 nmol L−1. 14C-pyruvate (SA: 2.035 GBq mmol−1) was added to a final concentration of 10 nmol L−1. Trichloroacetic acid (TCA) was added to the killed controls to a final concentration of 1%. Samples were incubated for 1 h in the dark and IR light as described for respiration. The incubations were terminated as the killed controls and kept at 4 °C in the dark until processed within 65% except for the samples from 9th May and 29th Aug 2018 (10% each), 1st Aug 2018 (21%), 25th Nov 2018 (1%), and 14th Aug 2019 (22%), Supplementary Fig. S1A). Thus, we decided to concatenate the fastaq files and analyze both fractions together as the total community. This also facilitated statistical analysis, as the activity rates were measured for the whole community without fractionations.Reads quality was evaluated using FastQC v0.11.7 (Babraham Bioinformatics, Cambridge, UK). After primer sequences trimming using Cutadapt [29] (v1.16), the number of reads per sample ranged from 49,354 to 188,942. Subsequent analyses were done in the R/Bioconductor environment using the dada2 package (version 1.14.1) [30]. Forward and reverse reads were truncated to 225 bp and low quality sequences were filtered out with the filterAndTrim function (truncLen = c(225, 225), maxN = 0, maxEE = c(2, 2), truncQ = 2), which reduced the number of reads per sample to range from 30,190 to 143,552. After merging and chimera removal using the removeBimeraDenovo function, 4,893 amplicon sequence variants (ASV) were obtained. Rare ASVs (not seen >3 times in at least 20% of the samples) were removed, which reduced the number of ASVs to 658, and the number of reads to 14,613–69,046 per sample. Taxonomic assignment was done using SILVA 138.1 database [31, 32] released on August 27, 2020. ASVs identified as Chloroplast or Cyanobacteria were excluded from the analyses, giving the final number of 546 ASVs and from 10,819 to 54,799 reads per sample. The bacterial community composition graphs were done using phyloseq [33] and ggplot2 [34] packages.AAP community compositionThe composition of AAP community was analyzed by amplicon sequencing of pufM gene encoding the M subunit of bacterial type-2 reaction centers. This gene is routinely used for diversity studies of AAP bacteria [35].PufM gene amplicons (approx. 245 bp) were prepared using pufM_UniF (5′-GGN AAY YTN TWY TAY AAY CCN TTY CA-3′) and pufM_WAW (5′-AYN GCR AAC CAC CAN GCC CA-3′) primers [36]. PCR was performed in triplicate 20 μL reactions using Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific, USA) with the following reaction conditions: 98 °C for 3 min, 27 cycles at 98 °C for 10 s, 58 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. The triplicate product reactions for each sample were pooled and gel purified using the kit Wizard SV Gel and PCR Clean-Up System (Promega, USA). The sequencing was performed on the Illumina MiSeq platform (2 × 150 bp) at Macrogen, South Korea.The fastq files were concatenated as described for bacteria communities. The Bray-Curtis similarity between two fractions for each sampling day was >70%, except for the samples from 1st Aug 2018 (47%) and 14th Aug 2019 (18%, Supplementary Fig. S1B).The samples were analyzed as described for bacterial communities. The number of reads per sample ranged from 192,360 to 239,418 after the cutadapt trimming. Forward and reverse reads were truncated to 130 bp, and the number of reads per sample after the quality filtering and denoising ranged from 189,432 to 235,311. Merging the forward and reverse reads with mergePairs function created 12,692 ASVs and reduced the number of reads to 183,136–221,281 per sample. The chimera removal lowered the number of ASVs to 1816, and the number of reads to 159,451–208,679. Rare ASVs (not seen >3 times in at least 20% of the samples) were removed, which resulted in the final 566 ASVs, and a number of reads ranging from 155,915 to 203,021 per sample. A manually curated taxonomic database was used for taxonomic assignment following the naïve Bayesian classifier method [37]. It contained 1580 unique pufM sequences, downloaded from the Fungene repository on May 16, 2019 (http://fungene.cme.msu.edu [38]), from metagenomes from the Římov Reservoir [39, 40] and from the Genome Taxonomy database accessed on September 16, 2020 [41].Statistical analysisLinear mixed-effects models were calculated in R (version 3.6.2) using lme function from the nlme package (version 3.1.143) on untransformed activity data and log10 transformed environmental variables [42]. Models’ parameters were estimated using maximum likelihood method and their significance was tested with ANOVA. Relationships between the activity measures, the environmental variables and the composition of AAP communities were investigated with distance-based linear models (DistLM) [43, 44] in Primer (version 7.0.13) with PERMANOVA + 1 add on (e-Primer, Plymouth, UK) [45]. The sequence reads were transformed with the varianceStabilizingTransformation function of the DESeq2 package [46] (version 1.14.1, blind = FALSE, fitType = “mean”).Data accessibilityThe sequences of 16S and pufM amplicons that support the findings of this study have been deposited in the EMBL database as the BioProject with the accession number PRJEB41596, together with most of the environmental metadata. The scripts and the remaining data supporting the results are included in the Supplementary Material. More

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    Cooperative herbivory between two important pests of rice

    Plants and insectsRice (Oryza sativa) cultivar Minghui63 was used in this study. Rice plants were grown in a greenhouse at 27 ± 3 °C with 75 ± 10% RH (relative humidity) and a photoperiod of 16:8 h L:D (light:dark). The cultivation of rice plants followed the same procedure as described previously27. Plants were used for experiments when they were at the tillering stage, which occurred about 44–49 days after sowing.C. suppressalis larvae were reared on an artificial diet as described70. Ten percent honey water solution was provided to supply nutrition for the adults. N. lugens were maintained on a BPH-susceptible rice variety Taichung Native 1 (TN1)38. T. japonicum were obtained from Keyun Industry Co., Ltd (Jiyuan, China). Newly emerged adult wasps were maintained in glass tubes (3.5 cm diameter, 20 cm height) and supplied with 10% honey water solution as a food source and were maintained for at least 6 h to ensure free mating, before females were used for the following experiments. All three species were maintained in climatic chambers at 27 ± 1 °C, 75 ± 5% RH, and a photoperiod of 16:8 h L:D.Performance of caterpillars on insect-infested rice plantsMultiple types of rice plants were prepared: (i) uninfested plants, meaning that potted rice plants remained intact without insect infestation; (ii) SSB-infested plants, each potted rice plant was artificially infested with one 3rd instar SSB larva that had been starved for >3 h for 48 h; (iii) BPH-infested plants, each potted rice plant was artificially infested with a mix of fifteen 3rd and 4th instars BPH nymphs for 48 h; (iv) SSB/BPH-infested plants, each potted rice plant was simultaneously infested with one SSB larva and 15 BPH nymphs for 48 h; (v) SSB → BPH-infested plants, each potted rice plant was artificially infested with one SSB larvae alone for the first 24 h, then 15 BPH nymphs were additionally introduced for another 24 h; (vi) BPH → SSB-infested plants, namely each potted rice plant was artificially infested with 15 BPH nymphs for the first 24 h, then one SSB larvae were additionally introduced for another 24 h. Plant treatments were conducted as described in detail in our previous study27. During herbivory treatment, the uninfested plants were placed in a separate room to avoid possible volatile-mediated interference. During the subsequent bioassays, both SSB caterpillar and BPH nymphs remained in or on the rice plants.Two bioassays were conducted to test the performance of C. suppressalis larvae feeding on differently treated rice plants. The first bioassay included the plant treatments i, ii, iii, and vi, and the second bioassay included the plant treatments i, ii, v, and vi. Three 2-day-old larvae of C. suppressalis were gently introduced onto the middle stem of each rice plant using a soft brush. The infested rice plants were then placed in climatic chambers at 27 ± 1 °C, 75 ± 5% relative humidity, and a photoperiod of 16:8 h L:D. The C. suppressalis larvae were retrieved from the rice plants after 7 days feeding, and they were weighed on a precision balance (CPA2250, Sartorius AG, Germany; readability = 0.01 mg). The mean weight of the three caterpillars on each plant was considered as one biological replicate. The experiment was repeated four times using different batches of plants and herbivores, resulting in a total of 30–46 biological replicates for each treatment.Oviposition-preferences of C. suppressalis females choosing among differently infested rice plantsGreenhouse experimentIn the greenhouse, seven choice tests were conducted with C. suppressalis females including (i) SSB-infested plants versus uninfested plants; (ii) BPH-infested plants versus uninfested plants; (iii) SSB/BPH-infested plants versus uninfested plants; (iv) SSB-infested plants versus BPH-infested plants; (v) SSB-infested plants versus SSB/BPH-infested plants; (vi) BPH-infested plants versus SSB/BPH-infested plants; and (vii) the test in which C. suppressalis females were exposed to all four types of rice plants. The experiments were performed as described in detail by Jiao et al.30. In brief, four potted plants were positioned in the four corners of a cage (80 × 80 × 100 cm) made of 80-mesh nylon nets for each test. For paired comparisons, two potted plants belonging to the same treatment were placed in opposite corners of each age, and in the test with four types of rice plants, each type of plant was positioned in one of the four corners of each cage. Five pairs of freshly emerged moths (less than 1 day) were released in each cage, and a clean Petri dish (9 cm diameter) containing a cotton ball soaked with a 10% honey solution was placed in the center of the cage as food source. After 72 h, the number of individual eggs on each plant were determined. The experiment was conducted in a greenhouse at 27 ± 3 °C, 65 ± 10% RH, and a photoperiod of 16:8 h L:D. Each choice test was repeated with 9–11 times (replicates).Field cage experimentThe oviposition preference of SSB females was further assessed in a field near Langfang City (39.58° N, 116.48° E), China. Four choice tests were conducted: (i) SSB-infested plants versus uninfested plants; (ii) BPH-infested plants versus uninfested plants; (iii) SSB/BPH-infested plants versus uninfested plants; and (iv) SSB/BPH-infested plants versus SSB-infested plants. The treated rice plants were prepared as described above and were transplanted into experimental plots (1.5 × 1.5 m). For each pairwise comparison, six plots of rice plants were covered with a screened cage (8 × 5 × 2.5 m) made of 80-mesh nylon net to prevent moths from entering or escaping. Each of the six plots contained nine rice plants of a particular treatment, with three plots per cage representing the same treatment. Plots were separated by a 1 m buffer and they were alternately distributed in a 3 × 2 grid arrangement in each cage (Supplementary Fig. 4). Approximately 50 mating pairs of newly emerged C. suppressalis adults ( More