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    Carcass appearance does not influence scavenger avoidance of carnivore carrion

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    Phototroph-heterotroph interactions during growth and long-term starvation across Prochlorococcus and Alteromonas diversity

    All Alteromonas strains support long-term survival of Prochlorococcus under N starvationPrevious research showed that Prochlorococcus, and to some extent Synechococcus depend on co-occurring heterotrophic bacteria to survive various types of stress, including nitrogen starvation [33, 34, 42, 43]. At the first encounter between previously axenic Prochlorococcus and Alteromonas (E1), all co-cultures and axenic controls grew exponentially (Fig. 1B, C). However, all axenic cultures showed a rapid and mostly monotonic decrease in fluorescence starting shortly after the cultures stopped growing, reaching levels below the limit of detection after ~20–30 days. None of the axenic Prochlorococcus cultures were able to re-grow when transferred into fresh media after 60 days (Fig. 1C). In contrast, the decline of co-cultures rapidly slowed, and in some cases was interrupted by an extended “plateau” or second growth stage (Fig. 1B). Across multiple experiments, 92% of the co-cultures contained living Prochlorococcus cells for at least 140 days, meaning that they could be revived by transfer into fresh media. Thus, the ability of Alteromonas to support long-term N starvation in Prochlorococcus was conserved in all analyzed strains.Fig. 1: Experimental designs and overview of the dynamics of Prochlorococcus-Alteromonas co-cultures from first encounter and over multiple transfers.A Schematic illustration of the experimental design. One ml from Experiment E1 was transferred into 20 ml fresh media after 100 days, starting experiment E2. Experiment E2 was similarly transferred into fresh media after 140 days, starting experiment E3. Additional experiments replicating these transfers are described in Supplementary Fig. S1. B Overview of the growth curves of the 25 Prochlorococcus-Alteromonas co-cultures over three transfers spanning ~1.2 years (E1, E2 and E3). Results show mean + standard error from biological triplicates, colored by Prochlorococcus strain as in panel D. C Axenic Prochlorococcus grew exponentially in E1 but failed to grow when transferred into fresh media after 60, 100, or 140 days. Axenic Alteromonas cultures were counted after 60 and 100 days, as their growth cannot be monitored sensitively and non-invasively using fluorescence (optical density is low at these cell numbers). D High reproducibility and strain-specific dynamics of the initial contact between Prochlorococcus and Alteromonas strains (E1). Three biological replicates for each mono-culture and co-culture are shown. Note that the Y axis is linear in panels B, C and logarithmic in panel D. Au: arbitrary units.Full size imageIt has previously been shown that Prochlorococcus MIT9313 is initially inhibited by co-culture with Alteromonas HOT1A3, while Prochlorococcus MED4 is not [12, 32]. This “delayed growth” phenotype was observed here too, was specific to MIT9313 (not observed in other Prochlorococcus strains) and occurred with all Alteromonas strains tested (Fig. 1D). MIT9313 belongs to the low-light adapted clade IV (LLIV), which are relatively distant from other Prochlorococcus strains and differ from them in multiple physiological aspects including the structure of their cell wall [44], the use of different (and nitrogen-containing) compatible solutes [45], and the production of multiple peptide secondary metabolites (lanthipeptides, [46, 47]). LLIV cells also have larger genomes, and are predicted to take up a higher diversity of organic compounds such as sugars and amino acids [48]. It is intriguing that specifically this strain, which has higher predicted metabolic and regulatory flexibilities [49], is the only one initially inhibited in co-culture with Alteromonas.Differences in co-culture phenotype are related to Prochlorococcus and not Alteromonas strains and occur primarily during the decline stageWhile co-culture with all Alteromonas strains had a major effect on Prochlorococcus viability after long-term starvation, there was no significant effect of co-culture on traditional metrics of growth such as maximal growth rate, maximal fluorescence, and lag phase (with the exception of the previously described inhibition of MIT9313; Fig. 2A–C). However, a visual inspection of the growth curves suggested subtle yet consistent differences in the shape of the growth curve, and especially the decline phase, between the different Prochlorococcus strains in the co-cultures (Fig. 1D). To test this, we used the growth curves as input for a principal component analysis (PCA), revealing that the growth curves from each Prochlorococcus strain clustered together, regardless of which Alteromonas strain they were co-cultured with (Fig. 2D). The growth curves of all high-light adapted strains (MED4, MIT9312, and MIT0604) were relatively similar, the low-light I strain NATL2A was somewhat distinct, and the low-light IV strain MIT9313 was a clear outlier (Fig. 2D), consistent with this strain being the only one initially inhibited in all co-cultures. Random forest classification supported the observation that the growth curve shapes were affected more by the Prochlorococcus rather than Alteromonas strains, and also confirmed the visual observation that most of the features differentiating between Prochlorococcus strains occurred during culture decline (random forest is a supervised machine learning algorithm explained in more detail in Supplementary Text S2; see also Supplementary Fig. S2). Thus, co-culture with Alteromonas affects the decline stage of Prochlorococcus in co-culture in a way that differs between Prochlorococcus but not Alteromonas strains.Fig. 2: Growth analysis and principal component analysis (PCA) of the growth curves from all co-cultures during 140 days of E1.A Growth rate, B Maximum fluorescence, and C duration of lag phase during experiment E1. Box-plots represent mean and 75th percentile of co-cultures, circles represent measurements of individual cultures of the axenic controls. The only significant difference between axenic and co-cultures is in the length of the lag phase for MIT9313 (Bonferroni corrected ANOVA, p  More

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    A colonial-nesting seabird shows no heart-rate response to drone-based population surveys

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    Residual levels and dietary intake risk assessment of 11 pesticides in apricots from different ecological planting regions in China

    Chromatographic separation and mass spectrometric optimizationTo obtain the best monitoring conditions for each compound, a 0.5 mg/L mixed standard solution of 11 pesticides was mixed with the mobile phase through a syringe pump and then injected into the mass spectrometer for tuning. The precursor ion of the compound to be tested was determined by the primary mass spectrometry scan under ESI+ and ESI- modes, and then the product ion was scanned by the secondary mass spectrometry. Two groups of ion pairs with the best sensitivity were selected for detection; one group was used for quantification, and another, for qualitative analysis. The optimization results showed high sensitivity of all the 11 pesticides under the ESI+ mode. Among them, abamectin (B1a), β-cypermethrin, deltamethrin, fenpropathrin, and bifenthrin were [M + NH4]+, and other compounds were [M + H]+. MS parameters of 11 pesticides are mentioned in Table S2.Formic acid and ammonium acetate are commonly used reagents to enhance the ionization of target compounds [M + H]+ and [M + NH4]+ under the ESI+ mode, and they can effectively improve the peak pattern, making the peak sharper and more symmetrical; therefore, they need to be added during gradient elution38. To improve work efficiency, it is necessary to separate and complete the monitoring of 11 pesticides in the shortest possible time; therefore, we selected two different types of chromatographic columns (ACQUITY UPLC HSS C18 and ACQUITY UPLC HSS T3) and three different mobile phases (Ι: 0.1% formic acid aqueous solution—ACN, II: 0.05% formic acid aqueous solution—ACN, and III: 0.1% formic acid/5 mmol/L ammonium acetate aqueous solution—ACN) for optimization experiments. We observed that when using the HSS T3 chromatographic column, β-cypermethrin, deltamethrin, fenpropathrin, and bifenthrin did not show a good retention effect under the three mobile phase systems, and there was substantial tailing of the chromatographic peak. The shape of the chromatographic peak and sensitivity of the target compound were used as evaluation indicators. Compared with Ι and II, mobile phase III produced better sensitivity for all target compounds (Fig. 1), with sharper and more symmetrical peaks of β-cypermethrin, deltamethrin, fenpropathrin, and bifenthrin. This may be because the addition of 5 mmol/L ammonium acetate improved the retention performance of the HSS C18 chromatography columns without affecting the ionization efficiency of all target compounds. In summary, we selected the HSS C18 column for chromatographic separation and used 0.1% formic acid/5 mmol/L ammonium acetate aqueous solution—ACN as the mobile phase to further optimize the gradient elution procedure and effectively separate and detect all the target compounds within 8 min.Figure 1When using HSS C18, the peak areas of 11 pesticides in three different mobile phases.Full size imageOptimization of purification materialsThe flesh of apricot contains sugar, protein, calcium, phosphorus, carotene, thiamine, riboflavin, niacin, and vitamin C. Due to these diverse impurities, the analysis of the sample matrix becomes highly complex. Therefore, these impurities need to be removed from the matrix samples before analysis. Currently, PSA, C18, and MWCNTs are widely used to adsorb to the fruit substrate39. PSA has a strong adsorption capacity for metal ions, fatty acids, sugars, and fat-soluble pigments, C18 has a strong adsorption capacity for non-polar impurities (such as fat, sterol, and volatile oil), while MWCNTs have a strong adsorption capacity for pigments, which can effectively remove chlorophyll, lutein, and carotene. However, C18 and MWCNTs can also simultaneously adsorb pesticides, resulting in poor recovery. Nano-ZrO2 has a large specific surface area and good adsorption stability and has recently been used to purify substrates. It can selectively remove fats and pigments from samples compared to conventional C18 fillers.In the current study, different purification materials were combined for the analysis of 11 pesticide residues and to propose the best purification strategy in the pretreatment of apricot samples. As displayed in Fig. 2, the average recovery of 11 pesticides in the apricot was higher using the C18/nano-ZrO2/MWCNTs than other combinations. Nano-ZrO2 showed better adsorption than PSA in purifying fatty acids, organic acids, polar pigments, and sugars in apricot, owing to its larger specific surface area, better adsorption capacity, and stability. To conclude, the combination of 10 mg C18, 30 mg nano-ZrO2, and 5 mg MWCNTs demonstrated the best recovery for 11 pesticides, with recovery in the range of 72% to 114%, at a pesticide spiking level of 0.01 mg/kg. In summary, we finally determined that among the tested combinations, C18/nano-ZrO2/MWCNTs (10 mg/ 30 mg/5 mg) is the best purification combination for the pre-treatment of apricot samples.Figure 2The recoveries of 11 pesticides in apricot matrix under different scavenger combinations (2–1 C18/nano-ZrO2/MWCNTs, 2–2 PSA/C18/MWCNTs, 2–3 nano-ZrO2/PSA/MWCNTs; 0.01 mg/kg, n = 3).Full size imageLinearity, matrix effects, limit of detection and limit of quantificationThe standard curve obtained from the standard working solutions of 11 pesticides and the calibration curve from blank apricot matrix spiked with 11 pesticides showed good linearity (0.001, 0.005, 0.01, 0.05, 0.1, and 0.5 mg/L), with R2 ≥ 0.9959 for all tested samples (Table 1).Table 1 The standard curves, R2 and MEs of 11 pesticides in apricot.Full size tableTo evaluate MEs, the slopes of matching 11 pesticide standards with solvent and apricot matrix were calculated at the same concentration. According to the derived slope of the matrix-matched calibration curve, MEs of 11 pesticides in apricot were between 89 and 113% (Table 1), well within the range of 80% to 120%, indicating that the MEs could be ignored. It also suggests that the current pre-treatment method has a good purification effect and eliminates the matrix effect very well, laying a robust foundation for the subsequent step of quantitative analysis of samples. We next used the standard solution curve to quantify the 11 pesticide residues in apricot.The LOD refers to the minimum concentration or minimum amount of a component to be tested that can be detected from a test sample under a given confidence level by an analytical method. Its physical meaning is the amount of the measured component when the signal is 3 times the standard deviation (S = 3σ) of the reagent blank signal (background signal). Sometimes it also refers to the amount of the measured component corresponding to when the signal is three times the background signal generated by the reagent blank (S = 3 N). The LOQ refers to the minimum amount of the analyte in the sample that can be quantitatively determined, and the determination result should have a certain accuracy40. The LOQ reflects whether the analytical method has the sensitive quantitative detection ability. The LOQ is the lowest validated level with sufficient recovery and precision, which was estimated to be 0.001 mg/L, while the LOD is the lowest calibration level, which was 2 µg/kg, according to SANTE/12,682/2020.Accuracy and precisionIn the matrix, 11 pesticides were spiked at four levels (0.002, 0.02, 0.1, and 1 mg/kg), and for each spiked sample, there were six replicates. The recoveries of 11 pesticides in apricot at all levels ranged between 72 and 119%. The inter- and intra-level relative standard deviations (RSDs, %) of 11 pesticides in apricot were  More

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    Longitudinal analysis of the Five Sisters hot springs in Yellowstone National Park reveals a dynamic thermoalkaline environment

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