Ecology

Sample collectionSamples of insects for the analyses of NAs were collected in four basins of Sierra Nevada National Park, southern Spain (Supplementary Fig. S1). Sample sites covered different spatial and temporal scales of investigation: three sampling stations across an elevational gradient and two sampling periods in spring and autumn of 2015. Given that all samples were collected from similar environments, the effect of abiotic conditions was not considered crucial for testing NAs in insects. Samples of aquatic insects were collected using a kick sampler (250 µm mesh size) by removing the substrate from at least 20 sample units (total area of 2.5 m2) taken on each station and date and distributed randomly in proportion to the occurrence of major stream habitats (i.e. rapid and slow flow, gravel, sand, zones proximal to and distant from shore and vegetated areas). All samples from each station were pooled and individuals representing six hemimetabolous and six holometabolous taxa sorted specifically for the analysis of nucleic acids (Table 1). We refer to taxa as a generic term to designate a group of one or more populations of organisms that were identified to the lowest taxonomic level possible by eye. Thus, most taxa were identified to the species (Dinocras cephalotes, and Perla marginata) or genus level (Baetis sp., Ecdyonurus sp., Epeorus sp., Rhithrogena sp., Hydropsyche sp., and Rhyacophila sp.), except for Lepidostomatidae, Limnephilidae, Brachycentridae, and Simuliidae that were identified to the family level. Although taxonomic resolution in the identification varied, taxa identified at the species and genus level represented the majority of the samples in this study. In addition, morphologically similar animals were selected for all supraspecific taxa in order to represent similar morphospecies for each taxon. When possible, up to 20 individuals per taxa that covered the full size spectrum available for each taxon were sorted into 10-mL vials containing RNAlater (Ambion Inc., Austin, Texas, USA), and transported inside a cooler to the laboratory. There, all insect samples were stored at − 80 °C until prepared for analysis. Before processing the insects, we measured body length to the nearest half millimetre under a stereoscopic microscope and verified the insect’s identity. In total, 639 individuals of 12 different taxa (six hemimetabolans and six holometabolans) were measured and analysed for NA content.Nucleic acid analysisNA analyses largely followed the methods by Wagner et al.13 with a number of recommendations by Gorokhova & Kyle25 and Bullejos et al.26. Analyses were carried out on insect legs and/or heads except for Simuliidae, where entire individuals were analysed. Preliminary analyses using legs and heads for a given individual showed that the coefficient of variation in RNA and DNA content rarely exceeded 5% (Supplementary Table S1). For the calculation of dry weight of insects where legs (one to three) were analysed, the opposite legs and the remaining body parts were separately weighed to estimate total body dry mass (total body weight = legs dry weight * 2 + remaining body parts dry weight). For the estimation of dry weight of insects where heads or the entire body were analysed, body length–weight relationships were specifically developed for each taxon in this study (Supplementary Table S2). Dry-weight was estimated by drying samples to constant weight in preweighed aluminium capsules and reweighing them with a Mettler UMT2 microbalance (± 0.1 µg; Mettler Toledo, Im Langacher, Switzerland).NAs were measured using a microplate fluorimetric high-range assay Ribo-Green assay (Initrogen, Carlsbad, California, USA) after N-laurylsarcosine extraction and RNase digestion, as described in Gorokhova & Kyle25. We used the following reagents: RiboGreenTM RNA Quantitation Kit (Invitrogen Corporation, Carlsbad, California, USA); RNase DNasefree (working solution 5 mg mL21; Q-biogen, Weston, Massachusetts, USA); N-lauroysarcosine (Sigma-Aldrich, Saint Louis, Missouri, USA); Tris-EDTA buffer (Q-biogene). Fluorescence measurements were performed using a FLUOstar Optima fluorometer (microplate reader, filters: 485 nm for excitation and 520 nm for emission; BMG Labtechnologies, Ortenberg, Germany) and black solid flat-bottom microplates (Greiner Bio-One GmbH, Frickenhausen, Germany). The plate was scanned with a 0.2-s well measurement time, making 10 measurements per well, before and after RNase digestion (30 min under dark conditions at 37 °C). Fluorescence measurements were converted into RNA and DNA concentrations (pg) by using standard curves for RNA (16S and 23S from Escherichia coli, component C of the RiboGreen Kit) and DNA (calf thymus; Sigma-Aldrich), and expressed as a percentage of body dry mass (%RNA and %DNA).Animal genome size databaseTo test the generality of our hypothesis that DNA size varied between insect metamorphosis modes across taxa and environments (terrestrial and aquatic), we incorporated the Animal Genome Size Database by Gregory16 in our analysis. The dataset covers a variety of insect groups (including 140 families and 20 orders of hemimetabolous and holometabolous insects) with a representation of most functional feeding groups, life cycles, and trait-based morphologies, comprising a total of 336 hemimetabolous and 999 holometabolous insect records.Statistical analysisTesting for differences in NAs between metamorphosis modes, we found that data were not normally distributed (Shapiro–Wilk’s W test) and could not be transformed to fit a normal distribution, so differences in NAs were tested using generalized linear mixed effects models (GLMM). Models included body length and metamorphosis mode as fixed factors, and insect taxa nested within order as random factors to account for variability within taxa subgroups. The significance of the interaction between body length and metamorphosis mode was used to test whether NA allocation to RNA differed during the ontogenetic development of animals. To examine whether insect genome size (C-value) varied between holo- and hemimetabolans using Gregory’s genome size dataset, a GLMM was also used with metamorphosis mode set as a predictor and taxa nested within order as a random variable. Before performing the models, the data were standardized (Deconstand function in R) to provide meaningful estimates of main effects in models with interaction terms27 and the best GLMM was selected according to deviance information criteria28. GLMM analyses were conducted using the ‘glmer’ function in the package ‘lme4’29. Finally, because NA data for taxon subsets were normally distributed after a log-transformation, linear-regression models were used to test the relationship between RNA and body length for each taxon. All statistical analyses were made in R30. More

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Mapping the populationThe geographic extent of the Copula sivickisi population inhabiting the east coast of Magnetic Island was mapped in the 2017 medusae season (September to November) with underwater Jellyfish Camera units (JCams; Fig. 1b;9). The photopositive C. sivickisi medusae were attracted to the light on each JCam and recorded by the adjacent camera in 30-min deployments. Following established methods for managing potential repeat counts25, the abundance of medusae was measured by counting the maximum number of medusae in any single frame of video during the deployment period (Nmax).Figure 1The Copula sivickisi population at Magnetic Island (MI) in the Townsville (TSV) region (star), Queensland, Australia. (a) Population structure analysis (green colour scale). The simulated export of C. sivickisi from MI, i.e., the tracks of the C. sivickisi medusae lost from MI reefs as adults during the 2017 season ( 33% substrate coverage by Sargassum sp. and coral) following9, and the presence of reefal habitat at the sites was later confirmed from the JCam footage. Each bay/reef, excluding Geoffrey Bay, was sampled two times over six non-consecutive nights from the 25th of September to the 30th of October. Two sites within Geoffrey Bay, randomly placed near the C. sivickisi hotspot identified in9, were sampled on each of the 6 trips to confirm the presence of C. sivickisi medusae throughout the sampling period. Geoffrey Bay was sampled at a lower spatial resolution compared to the other bays/reefs (2 sites compared to 6 sites) because we could be confident in detecting medusae in Geoffrey Bay given the information derived from the comprehensive sampling of the bay in previous medusae seasons (presented in9). Further, a higher temporal resolution (6 trips to Geoffrey Bay compared to two for the other bays/reefs) was required to verify that C. sivickisi were present during the entire sampling period, and this had associated logistical constraints such as the weather.Biophysical modellingHydrodynamic descriptionThe two-dimensional version of the Second-generation Louvain-la-Neuve Ice-ocean Model [SLIM; 26] was used to model the currents off the eastern coast of Magnetic Island and in the surrounding region. A detailed description of SLIM and the specifics of its application in this study is presented in the supplementary information. Magnetic Island lies in the central area of the Great Barrier Reef (GBR). The currents in the GBR system are largely driven by the jets of the South Equatorial Current (SEC) which flow westward across the Coral Sea and collide with the outer reefs of the GBR27. In the central GBR, the North Caledonian Jet (NCJ) from the SEC generally diverges around the Queensland Plateau before meeting the outer reefs27. In addition to these regional scale forcings, the waters within the GBR system are shallow (mostly  5 mm in diameter (half their maximum size6), are generally sexually mature20, and C. sivickisi medusae would take around 25 days to grow to 5 mm [unpublished data]. To generate the measure of relative connectivity, a log base 10 transformation was performed on the counts + 1 data, and the transformed data was scaled from 0 (no connections) to 1 (most connections).The potential for connectivity between the island and mainland populations, and thereby the potential for Magnetic Island to represent an isolated stock (aims 4), was assessed by tracking the positions of all adult medusae lost from habitat at/near Magnetic Island through time. The combined trajectories showed the maximum extent of the emigration plume from the Magnetic Island population.Ethics approvalThis work was supported by the ARC Centre of Excellence for Coral Reef Studies and the Australian Lions Foundation. It was conducted in accordance with James Cook University’s Animal Ethics Committee’s policies, procedures and guidelines. No specific permissions were required. More

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Nitrate (Nar) and nitrite (Nir) reductase expression in PAO1 were quantified with fluorescently tagged promoter fusions (Supplementary Fig. 1), green for the NarK subunit (NarK-GFP) and red for the NirS subunit (NirS-dsRed)35. As the first two steps in the denitrification pathway, expression of these genes indicates a metabolic switch from aerobic respiration with O2 to anaerobic respiration via denitrification. These PAO1 reporter strains were grown embedded within 3 mm diameter14 agarose particle discs held within a custom-built gastight millifluidic device (Fig. 1; Supplementary Methods), permitting continual lateral nutrient supply of rich nutrient media (with O2 and NO3–) from the bulk media via diffusion while maintaining a constant boundary condition at the particle periphery (Fig. 1a, b). This created an analog of a particle replete with dissolved organic nutrients whereby the agarose acted as an inert polymeric matrix rather than as a carbon source. Over ~24 h of growth in Luria-Bertani Broth (LB) media supplemented with NO3–, PAO1 formed densely packed stationary microcolonies (Supplementary Video 1), similar to its growth morphology in model alginate beads15,36 and agar blocks34. Seeding bacterial cells within a 1% agarose matrix allows colony expansion due only to growth (passive movement) and prevents active aerotactic and chemotactic movement driven by flagellar motility37. A subset of agarose particles contained biocompatible O2 nanosensors that could faithfully report O2 conditions varying dynamically over the scale of minutes (Supplementary Fig. 2).Fig. 1: Denitrifiers create anoxia within particles in fully aerated fluid.Agarose disc particles seeded with P. aeruginosa PAO1 were incubated with media in glass devices that permit only lateral diffusion into particles. a Topview and b sideview illustrating nutrient diffusion into particles as occurred in c, a millifluidic device with directional flow of air-saturated fluid, or a ‘domino’ device held within sealed glass bottles. Scale bar ~1.5 cm. a inset, PAO1 cells grew as dense microcolonies. d Air saturation across four particles as determined from microscopic signal of fluorescent oxygen nanosensors. Despite aerobic bulk fluid surrounding the particles, cell growth created anoxia and its onset depended on PAO1 seeding density, ranging 104–106 cells mL−1 (70–7000 cells particle−1; the 105 mL−1 cell density was tested for duplicate particles). Gray shading indicates time range shown in e and asterisks coincide with noted timepoints in e. e The air saturation for radial profiles across a single particle (105 cells mL−1 seeding) during the cellular respiration-driven transition from air-saturated to anoxic conditions. Suboxia first developed in the core (x  > 1000 μm) then spread to the particle periphery (x 103 microcolonies within each particle (e.g., Fig. 2). Both Nar and Nir expression were skewed toward the particle edge for lower bulk NO3– concentrations (Fig. 2b, c, Supplementary Fig. 4), indicative of a low NO3– flux reaching microcolonies in the particle core. In contrast, homogenous Nar expression occurred across all radial distances with 4 mM bulk NO3– (Fig. 2b), which suggests NO3– was nonlimiting for expression throughout the particle at such high bulk NO3–. Meanwhile, reduced Nir expression across the particle at 4 mM NO3– compared with lower NO3– treatments (Fig. 2c) indicates PAO1 preferentially reduced NO3– over NO2– throughout the particle under such NO3–-replete conditions. Bulk NO3– also influenced microcolony size distributions (Fig. 2b, c), with larger microcolonies manifesting near the periphery closer to the NO3– supply from the bulk fluid. Higher bulk NO3– stimulated greater microcolony growth in the particle core, i.e., at the center, the mean microcolony radius r was 6.4 ± 0.1 µm (sd) at 40 µM bulk NO3– but 12.7 ± 0.1 µm (sd) at 4 mM. Notably, under 4 mM bulk NO3–, size was skewed toward the particle periphery even while Nar expression was not, suggesting maximum cell-specific Nar expression rates occurred across all radial locations despite biomass production in the particle core remaining NO3– limited.Fig. 2: Denitrification gene expression and microcolony size within particles in fully anoxic fluid.a Example images showing PAO1 microcolonies expressing NarK-GFP (nitrate reductase) after 40 h of growth in bulk anoxic media amended with 3 nitrate concentrations. Scale bar = 700 μm. b Mean relative expression of NarK-GFP for microcolonies, and mean radii of those microcolonies, shown in relationship to the radial distance to the nearest particle edge. The mean NarK-GFP expression for microcolonies in each particle (3–4 replicates) is indicated by a thin green line and the mean for all particles by a thick line. Similarly, an exponential model fit of microcolony radii for each particle is indicated by a thin black line and the mean for all particles by a thick line inset, Results for one example particle in a, illustrating the data for n microcolonies relative to the respective fits for those data. c Same as b but for reporter strain NirS-dsRed (nitrite reductase) expression in separate particles incubated in parallel.Full size imageNot only did particle denitrification readily occur in anoxic bulk fluid as expected, it was also prevalent among particles in oxygenated bulk fluid. Moreover, the spatiotemporal expression of Nar and Nir coincided with the development and microscale distribution of suboxic conditions. In these experiments, agarose particles were embedded with either NarK-GFP or NirS-dsRed PAO1 (106 cells mL−1) as previously, then incubated in partially oxygenated LB media (50% air saturation) containing NO3– (40 µM). A subset of particles was co-embedded with oxygen nanosensors to enable imaging of the O2 landscape. Minimal Nar and Nir expression were detected for the first 14 h of incubation. Strikingly, Nar and Nir activation then occurred as a wave that traced the initiation and expansion of suboxia (Fig. 3a–c). At the earliest stage of the wave (following 16–18 h of growth), expression increased substantially in the vicinity of the particle core (Fig. 3d), crafting a transition zone of only 390 ± 50 µm (sd) width that differentiated low- from high-expression microcolonies (Fig. 3e, Supplementary Fig. 5, Supplementary Fig. 6). This upregulation of expression coincided spatially with the development of anoxia in the core (Fig. 4, Supplementary Fig. 7; (x) > 1000 µm). During the next stage of the wave (t = 20–22 h), expression near the core elevated further while also initiating farther afield, widening the transition zone and reflecting expansion of suboxia toward the particle periphery. As the wave progressed (t = 24–28 h), expression diminished near the core, and consequently, the transition zone width contracted (200 ± 15 µm (sd)) even while its outermost edge neared to within 200 µm of the particle edge, where O2 conditions had become anoxic (Fig. 4). Finally, in the final stage of the wave (exemplified at t = 40 h), high expression was confined to a fine band distantly from the core, yet not directly at the edge, as evidenced by a very narrow transition zone (57 ± 13 µm (sd) width). Notably, at the last stage, O2 throughout the particle was elevated above the minimal observed levels, in contrast to previous stages (t = 24–28 h; Fig. 4, Supplementary Fig. 7). This may reflect anoxic acclimation by PAO1 to preferentially perform denitrification over oxidative respiration thereby permitting O2 to diffuse more readily through the particle matrix.Fig. 3: Radial migration of denitrification expression within particles in partially aerated fluid.Particles seeded with cells were incubated in LB media saturated with 50% air and supplemented with 40 μM NO3–, then stopped at various timepoints. a Example images showing PAO1 microcolonies expressing NarK-GFP (nitrate reductase). Scale bar = 700 μm and applies to all images. Separate particles with the reporter strain for NirS-dsRed (nitrite reductase) were incubated in parallel. b Relative expression of NarK-GFP and c NirS-dsRed in particles. All microcolonies from 3–4 replicate particles per timepoint are represented. Nar expression initiates at the particle core while Nir expression initiates just proximal to it. For both Nar and Nir, maximal expression migrates outward creating a wave over subsequent timepoints. d Relative microcolony expression (as in b and c) shown as a microcolony’s radial location within the particle. An expression intensity fit was calculated as mean fits of each strain at each timepoint (Supplementary Note). Here the maximum fit value for each reporter strain at t = 24 h was set equal to 1, and shown are the expression values for all microcolonies relative to 1. e The radial location and range of the transition zone for each timepoint, approximated as the sloped region closest to the particle edge in the expression profile for NarK-GFP (green) and NirS-dsRed (red) (see also Supplementary Fig. 6). The midpoint of the slope (white diamonds) and the inflection point of the slope (circles) are indicated. NarK-GFP shows significantly higher expression between timepoints for midpoints (one-way ANOVA; F 1,35 = 27.9, p = 4.0 × 10−11) and for inflection points (one-way ANOVA; F 1,35 = 17.2, p = 9.5 × 10−9). NirS-dsRed also showed significantly higher expression between timepoints for midpoints (one-way ANOVA; F 1,35 = 14.9, p = 4.5 × 10−8) and for inflection points (one-way ANOVA; F 1,35 = 21.0, p = 1.0 × 10−9).Full size imageFig. 4: Evolution of anoxia within particles in partially aerated fluid.Two-dimensional profiles of air saturation were generated from particles co-seeded with the NarK-GFP reporter strain (nitrate reductase) and oxygen nanosensors. Scale bar = 700 μm. Separate analogous particles with the NirS-dsRed reporter strain (nitrite reductase) were incubated in parallel (Supplementary Fig. 7). For both, suboxic conditions develop in the particle core and then migrate outward toward the particle periphery over time.Full size imageThroughout the evolution of particle suboxia, Nar and Nir activity corresponded with the estimated spatiotemporal availability of O2 and NO3– in particles. Curve fits for microcolony fluorescence signal data were generated by assuming that O2 and NO3– concentrations at each radial location were the only controlling factors on expression, wherein O2 inhibits expression exponentially and NO3– has a directly proportional relationship, i.e., (Epropto {e}^{-k[{{mathrm{O}}}_{2}]}times [{mathrm{N}}{mathrm{{O}}}_{3}^{-}]). Approximating the distributions of O2 and NO3– in the particle as simple logistic functions, denitrification gene expression, E, is governed by the following relationship:$$E=alpha times {e}^{-beta left[2{[{{mathrm{O}}}_{2}]}_{{mathrm{bulk}}}left(1-frac{1}{1+{e}^{-gamma x}}right)right]}times {[{mathrm{N}}{{mathrm{O}}}_{3}^{-}]}_{{mathrm{bulk}}}left(1-frac{1}{1+{e}^{-delta (x-varepsilon )}}right)$$whereby α, β, γ, δ, and ε are fitting parameters and x is the distance to the particle edge. (Supplementary Note). The shape of this fit prediction matches the observed empirical data remarkably well (Fig. 3d, Supplementary Fig. 5), and the midpoints and inflection points from one timepoint to the next were significantly different (one-way ANOVA, F 1,8 = 27.9, p = 4.0 (times) 10−11 for Nar midpoints, F 1,8 = 14.9, p = 4.5 (times) 10−8 for Nir midpoints, F 1,8 = 17.2, p = 9.5 (times) 10−9 for Nar inflection points, and F 1,8 = 21.0, p = 1.0 (times) 10−9 for Nir inflection points). These fits illustrate how the response of PAO1 nar and nir gene expression reflects the balance between bacterial consumption and diffusion of O2 and NO3– from bulk surrounding fluid. In this manner, the fluorescence signal diminishes after achieving its peak and microcolonies remain small behind the advancing fluorescence wave while colonies continue to expand ahead of it (Supplementary Fig. 5, Supplementary Fig. 8). Nar was downregulated in the wake of the wave, causing microcolony expansion to slow or cease in the absence of respiration. Putatively, continued O2 and then NO3– uptake by large microcolonies at the periphery created growth limitation for microcolonies in their shadows farther from the bulk fluid source. Nir expression also advanced as a wave but interestingly created an annulus of maximal expression bounded by lower expression toward both the periphery and center of the particle. This expression pattern likely reflects localized production and utilization of NO2– within the particle interior. Exterior to the ring, ample NO3– from the bulk favored nitrate reductase, but interior to the ring, NO3– and NO2– were diffusion-limited.Importantly, the heterogeneous distribution of Nar and Nir expression across the particle also manifested among PAO1 cells at the scale of individual microcolonies. High magnification colony-scale images near the particle periphery and in the transition zone revealed a common phenotype reflecting the overall expression across the particle whereby the core of a single colony is expressive but the outer margin is not. As quantified for >103 microcolonies per particle, the subregion expressing Nar or Nir (i.e., “on”) varied with distance from the particle edge (Fig. 5). A thin radial zone within the particle (distance from the particle edge, x ~90–240 µm) harbored high heterogeneity with colonies ranging from 0–100% as shown (Fig. 5b, c). This narrow transition zone aligned with that quantified at lower magnification (Figs. 3, 4), indicative of a sharp transition from O2 to nitrate- and nitrite-driven respiration. In the flanking region exterior to this zone (x  240 µm), Nar was predominately on (median colony fraction expression = 0.78 ± 0.40 interquartile range; Fig. 5b) and Nir was almost completely on (median colony fraction expression = 0.95 ± 0.25 interquartile range; Fig. 5c). These expression characteristics resulted in a significantly stronger population bimodality for Nir than for Nar (Fig. 5d); Kolmogorov–Smirnov nonparametric test for probability distribution similarity, n1 = 1554, n2 = 1354, p = 1.2 (times) 10−40, Dn = 0.25. Akin to the annular feature observed at lower magnification (Fig. 3), this binary Nir expression likely reflects localized endogenous production and utilization of NO2–. Since NO2– is not continuously supplied from bulk media via lateral external diffusion like NO3–, microcolonies in the interior use Nar to produce NO2–, which is then preferentially consumed via Nir within each microcolony as the next most available oxidant for generating energy.Fig. 5: Heterogenous denitrification gene expression within individual microcolonies.a PAO1 NarK-GFP (top) or NirS-dsRed (bottom) were grown in separate particles in LB media saturated with 50% air and supplemented with 40 μM NO3–. Arrows indicate the particle edge; scale bars = 100 μm. For hundreds of microcolonies, the fraction expressing either NarK or NirS was quantified, and example microcolonies (right) illustrate ‘on’ fractions ranging from 0 to 0.90, with ‘on’ subregions noted by blue dotted lines. b Microcolony fraction expression for NarK-GFP and c NirS-dsRed, as a function of distance to the nearest particle edge. Shown are the median (blue crosses) and quartiles binned over 25 μm of radial particle space. Colonies were primarily ‘off’ in the aerated zone nearest bulk fluid (x  250 μm). In the aerobic zone, the median expression fraction of NarK-GFP is 3.1 × 10−5 (interquartile range 5.5 × 10−5) whereas the NirS-dsRed expression fraction is 2.7 × 10−5 (interquartile range 1.5 × 10−5). Expression of both genes are not significantly different from each other (Wilcoxon rank sum; p = 0.1). In the anaerobic zone, the median expression fraction of NarK-GFP and NirS-dsRed are 0.70 (interquartile 0.39) and 0.94, (interquartile 0.24), respectively. These anoxic fractional expressions are significantly different from each other (n1 = 1160, n2 = 1078, Wilcoxon rank sum; p = 2.2 × 10−32, w = 2724). The occurrence of heterogenous partially-on microcolonies reflects a sharp transition zone between presumptive aerobic and anoxic conditions (x ~ 100–250 μm). d Probability density functions for each reporter strain indicate stronger bimodality and higher binary expression for NirS-dsRed than for NarK-GFP. The distribution of NarK v. NirS expression are significantly different from each other (two sample Kolmogorov–Smirnov test; n1 = 1554, n2 = 1354 p = 1.2 × 10−40, Dn = 0.25) with significantly different medians (n1 = 1554, n2 = 1354, Wilcoxon rank sum; p = 1.9 × 10−33, w = 1.9 × 106).Full size imageRespiratory shading by exterior colonies and cells was a key emergent feature among microcolonies within the agarose particles, occurring in a fractal-like geometry. At the particle-scale ((R) ~1500 µm), nutrient consumption by microcolonies at the periphery prevented uptake by those at the interior; similarly, at the microcolony-scale (r ~25 µm), cells at the margin prevented uptake by those at the center, with consumption generating gradients of uptake flux at both scales. Shading did not occur for microcolonies in the particle core through the early stages of suboxia, as Nar and Nir expression were absent and microcolony size distribution was uniform over the first 14 h (Fig. 3, Supplementary Fig. 8). Since microcolonies were small over this stage, the O2 flux to the center outpaced aerobic respiration, and cell biomass at the particle-scale had not yet substantially diminished diffusive O2 availability. Then, owing to cell growth and the onset of shading, the O2 concentrations decreased rapidly over ~2 h (Fig. 4). As such, respiratory shading should be diminished when the density of microcolonies is lower. We tested this hypothesis with particles seeded at very low density (~102 cells mL−1) resulting in 1–6 microcolonies particle−1. Indeed, after 40 h of growth, the resultant microcolonies were quite large (r = 62 ± 11 µm (sd)); Supplementary Fig. 9) regardless of radial location within the particle, reflecting low intercolony competition for oxidants that readily diffused throughout the particle. While here respiratory shading across scales occurred for a clonal population, natural multispecies communities may additionally distribute functional roles among diverse taxa, e.g., anammox aggregates spatially differentiate such that aerotolerant species encase the outer perimeter to respire O2, shielding Planctomycetes to perform oxygen-inhibited anammox in the interior10. More

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