Tridacna maxima clams. We sampled five sites spanning 1,300 km  along the Red Sea's North‐South environmental gradient. We detected a diverse and structured assembly of host‐associated algae with communities demonstrating region and site‐specificity. Specimens from the Gulf of Aqaba harbored three genera of Symbiodiniaceae, Cladocopium, Durusdinium, and Symbiodinium, while at all other sites clams associated exclusively with algae from the Symbiodinium genus. Of these exclusively Symbiodinium‐associating sites, the more northern (27° and 22°) and more southern sites (20° and 18°) formed two separate groupings despite site‐specific algal genotypes being resolved at each site. These groupings were congruent with the genetic break seen across multiple marine taxa in the Red Sea at approximately 19°, and along with our documented site‐specificity of algal communities, contrasted the panmictic distribution of the T. maxima host. As such, our findings indicate flexibility in T. maxima‐Symbiodiniaceae associations that may explain its relatively high environmental plasticity and offers a mechanism for environmental niche adaptation.Giant clams (Tridacninae) are important members of Indo‐Pacific coral reefs and among the few bivalve groups that live in symbiosis with unicellular algae (Symbiodiniaceae). Despite the importance of these endosymbiotic dinoflagellates for clam ecology, the diversity and specificity of these associations remain relatively poorly studied, especially in the Red Sea. Here, we used the internal transcribed spacer 2 (ITS2) rDNA gene region to investigate Symbiodiniaceae communities associated with Red Sea  Here we demonstrate site‐ and region‐specific structuring of giant clam‐Symbiodiniaceae associations across 10° of latitude in the Red Sea. This structuring indicates a flexibility in clam‐algal associations that may explain their high environmental plasticity and offer a strategy for niche adaptation. The containers were kept in a cooling box on ice until arrival at the laboratory, where, for each of the two reefs, 2 L of seawater was immediately filtered through a 0.22 µm hydrophilic polyvinylidene fluoride (GVWP) filter  using a peristaltic pump. Filters were instantly frozen at −80°C for further analysis.2.2T. maxima mantle tissue samples were separated from the frozen seawater and homogenized using a Freezer/Mill®  by grounding up the tissues in liquid nitrogen at a rate of ten impacts per second for a total of 90 s. The Freezer/Mill® PVC tubes were cleaned thoroughly with 10% bleach between samples and the ground tissues were then dissolved in 5 ml Milli‐Q water (sterilized under UV light for 1 hr). The homogenate was transferred to 1.5 ml Eppendorf tubes and samples were frozen at −20°C until further processing.The frozen T. maxima mantle tissue samples, two water samples, and three negative controls: 1 to assess for DNA extraction kit contamination, 1 to assess for PCR reagent contamination, and 1 to assess for carryover contamination during the tissue homogenization. The carryover contamination negative sample was generated by adding Milli‐Q water to the cryotubes after they had been rinsed as part of the rinsing step carried out between each sample's processing. DNA extractions from the tissue samples were extracted with the Qiagen DNeasy 96 Blood & Tissue Kit (Qiagen) following the manufacturer's instructions with minor modifications. Briefly, 90 μl of the tissue homogenate was added to 1.5 ml Eppendorf tubes containing 90 μl of ATL buffer and 20 μl of Proteinase K and incubated in an Eppendorf ThermoMixer at 56°C and 300 rpm for 1 hr. DNA extractions were then continued according to the manufacturer's instructions. DNA concentrations were measured using a Qubit 2.0 fluorometer (ThermoFisher Scientific). Water filters were thawed and placed in 1.5 ml Eppendorf tubes, 360 μl of ATL buffer, and 40 μl Proteinase K buffer were added, and the tubes were incubated at 56°C for 20 min. DNA extractions were then continued according to the manufacturer's instructions using the Qiagen DNeasy 96 Blood & Tissue Kit (Qiagen). DNA concentrations were measured using a Qubit 2.0 fluorometer (ThermoFisher Scientific), and samples were adjusted to 10 ng/μl.In total, we extracted DNA from 50 samples, corresponding to 45 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG)GAATTGCAGAACTCCGTGAACC 3′ and SYM_VAR_REV: 5′ (GTCT CGTGGGCTCGGAGATGTGTATAAGAGACAG)CGGGTTCWCTTGTYTGACTTCATGC 3′  in any of the predicted ITS2 type profiles Figure . By cont2.3Symbiodiniaceae communities were characterized using next‐generation sequencing of the rDNA ITS2 region (ITS2). Triplicates for each sample were pooled, and samples were cleaned using ExoProStar 1‐step . Samples were then indexed using the Nextera XT Index Kit v2 . Successful addition of indexes was confirmed by comparing the length of the initial PCR product to the corresponding indexed sample on a 1% agarose gel. Samples were then cleaned and normalized using the SequalPrep Normalization Plate Kit (Invitrogen). The ITS2 libraries were pooled in an Eppendorf tube (4 μl per sample) and concentrated using a CentriVap Benchtop Vacuum Concentrator (Labconco). Following this, quality of the library was assessed using the Agilent High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). Quantification was done using Qubit . Sequencing was performed at 6 pM with 20% phiX on the Illumina MiSeq platform at 2 × 301 bp paired‐end in the KAUST Bioscience Core Laboratory (BCL). Raw sequencing data are available under NCBI BioProject accession number #657469.2.4Symbiodinium, Bray–Curtis‐derived, between‐sample dissimilarity matrix (including a square root transformation) that is output by default by SymPortal. The between‐site average dissimilarities were computed using a bespoke Python script and plotted using matplotlib's implementation of imshow. Specifically, for a given pairwise site comparison, for every sample in the first site, the distance to every sample in the second site was collected. A mean average and standard deviation were then computed from these distances. For self‐site comparisons , average within‐site distances were calculated. PCoAs were computed using Python and the scikit implementation of pcoa and plotted using matplotlib's implementation of scatter. To assess for statistical difference between sites across samples based on Symbiodinium ITS2 sequence assemblages, we ran a one‐factor PERMANOVA , and the analysis was conducted in Python using ecopy's simper function.Demultiplexed paired fastq.gz sequencing files were submitted directly to the remote instance of SymPortal for analysis  was included in the alignment (independent of any cropping). The full alignment is provided in Dryad submission https://doi.org/10.5061/dryad.k6djh9w50.Two previous giantclam‐Symbiodiniaceae characterizations exist from the Red Sea  to assess the likelihood that Symbiodiniaceae genotypes (represented in this study by ITS2 type profiles) associating with clams from the three most northern sites  may be physically available for uptake by the clams at the two most southern sites . We searched for sequences that were present in the northern site clams and the southern site seawater samples, but not present in the southern site clams.T. maxima individuals . We therefore constrained our search for sequences in the seawater samples to those that were known to be found in T. maxima individuals (from the northern sites). Finally, we were interested in finding evidence of Symbiodiniaceae diversity that the southern T. maxima individuals could potentially be associated with but had not (evidence of selectivity). As such, we further constrained our search to those sequences not found in the southern T. maxima samples.Our rationale was that while there would likely be a considerably larger richness of ITS2 sequences in the seawater samples compared to the clam samples, much of this richness would be representative of Symbiodiniaceae taxa potentially unable to form associations with Samples were distributed into three groups: clam samples from the three most northern sites, clam samples from the two most southern sites, and the two seawater samples. For each group, we generated a set of sequences that were found at least once in any of the group member samples . We then performed union, intersection, and difference operations on these sets to determine unique or shared sequence members for every combination of the groups. The set operations were performed in Python using a bespoke script, and a three‐way Venn plot was generated using venn3 from the Python package matplotlib‐venn.2.7https://doi.org/10.5061/dryad.k6djh9w50 and GitHub repository https://github.com/didillysquat/rossbach_2020. The Python scripts used in data processing and figure creation are also available at the GitHub repository.For outputs used in this study, and the results of the SIMPER analysis, please see Dryad submission 33.1T. maxima in the Red Sea, we compared the ITS2 sequences determined by Pappas et al., , while the others were either Cladocopium  or Durusdinium . Most profiles were defined by 3 or more defining intragenomic variants   where several clam samples harbored a mix of Symbiodiniaceae genera. Genera‐level profile diversity was therefore highest at this site (three genera) and absent at the four other sites . While sequences from Symbiodinium were more abundant than those from Cladocopium and Durusdinium  in the Gulf of Aqaba, differences in ITS2 array copy number between different Symbiodiniaceae taxa, specifically at the genus level, preclude the accurate inference of the relative abundance of these Symbiodiniaceae genera at this site.All non‐of Aqaba 9° where 3.4T. maxima with Symbiodinium, we did a dedicated analysis to elucidate fine‐scale differences of association. Variation of between‐sample dissimilarities within sites was highest in the two northernmost sites, the Gulf of Aqaba (29°) and Duba . However, in some cases, these dominant profiles were found at other sites at rarer abundances. Specifically, the A1‐A13a‐A13b profile dominant at Al Lith (20°) was recovered from a single sample in the Farasan Banks (19°), and the two profiles dominant at Thuwal (22°) and Duba (27°)  were recovered at the Gulf of Aqaba (29°). A reciprocal commonality was not recorded .Discounting the single‐DIV A1 profile occurrences, in general, a high site‐profile specificity was apparent, with a single specific profile dominant at each site Figure . This fi3.5Symbiodinium, Cladocopium, and Durusdinium. Eight of the 18 sequences  were DIVs from ITS2 type profiles associated with clams from either the Gulf of Aqaba or Duba.We searched for sequences that were present in the northern site clams and the southern site seawater samples, but not present in the southern site clams. We found 18 ITS2 sequences that fit these criteria  also harbored symbionts with Cladocopium and Durusdinium profiles. A similar location dependency of the specific community composition of associated Symbiodiniaceae was previously reported for giant clams from the Coral Triangle . The resolution afforded by our intragenomic diversity‐defined ITS2 profiles and the full ITS region haplotypes may be similar. Of the 9 sites sampled by Pappas et al., a site referred to as “site 1” in their study was closest to our “Thuwal” site . Three approximately equally dominant haplotypes were recovered from this “site 1.” In contrast, we recovered only a single dominant ITS2 profile. However, the single‐DIV A1 profile recovered in one of our samples is likely symptomatic of the additional extant diversity at this site. As such, differences in the number of operational taxonomic units recovered may be largely due to sampling methodology  rather than a difference in resolution. Finally, our findings of shared profiles between Thuwal (22°), Duba (27°), and the Gulf of Aqaba (29°) are also in agreement with the findings of identical ITS haplotypes recovered at Weber's sites in Egypt (North‐western Red Sea) and the Gulf of Aqaba, and Pappas et al.’s Thuwal sites in the central‐eastern Red Sea . These northern sites are characterized by the highest salinities  than in the more southerly sites. Symbiodiniaceae communities in the Gulf of Aqaba, where 4.3T. maxima host. Current evidence points toward a genetic disparity between the Red Sea T. maxima clams and other populations from the West Indian Ocean , which contrasts with the observed homogeneity of the associated Symbiodiniaceae assemblages that we identified here, especially in the more southern sites. In corals, fine‐scale resolutions of Symbiodiniaceae assemblages often strongly correlate to host genotype  at most of the Red Sea sites sampled would suggest that either one or multiple endemic panmictic populations of T. maxima are present along the latitude of the Red Sea. Given the taxonomically broad support of the 19° genetic discontinuity, it would seem exceptional that no such break is seen in T. maxima population(s). Either the host must be considered to possess an exceptional plasticity to survive in such a range of environments, or it must have some other mechanisms by which it is able to niche adapt. Given the diversity of recovered Symbiodiniaceae genotypes in this study, it would appear that T. maxima‐Symbiodiniaceae associations have a degree of flexibility and that there is a relatively high diversity of Symbiodiniaceae with which this host may associate. Our finding that T. maxima in the two most southern reefs associate with a relatively narrow diversity of Symbiodiniaceae, despite the presence of a much wider diversity that likely includes genotypes from the more northern sites, supports this notion of flexibility but also suggests a degree of selectivity by the clam hosts. Given that the high site fidelity seen in this study is most likely environmentally driven, flexibility and selectivity in these associations may offer a mechanism of adaptation for the host. In contrast to corals, where more fine‐scale resolutions increasingly reveal a relatively specific host genotype‐determined algal assemblage, such flexibility in clams represents, if confirmed, a mechanism conferring giant clams an additional resilience to warming. Indeed, although coral reefs in the Red Sea, particularly those in the southern Red Sea, have experienced intense warming‐induced bleaching in the past, such mass bleaching events have not been reported for Red Sea T. maxima populations ; Data curation ; Formal analysis ; Investigation ; Methodology ; Resources ; Visualization ; Writing‐original draft ; Writing‐review & editing . Benjamin CC Hume: Data curation ; Formal analysis ; Investigation ; Methodology ; Software ; Supervision ; Visualization ; Writing‐original draft ; Writing‐review & editing . Anny Cardenas: Data curation ; Formal analysis ; Methodology ; Resources ; Writing‐review & editing . Gabriela Perna: Data curation ; Formal analysis ; Methodology ; Resources ; Writing‐review & editing . Christian R. Voolstra: Conceptualization ; Formal analysis ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Writing‐original draft ; Writing‐review & editing . Carlos Duarte: Conceptualization ; Funding acquisition ; Project administration ; Resources ; Supervision ; Writing‐review & editing .Fig S1‐S2Click here for additional data file.


Ice XIX representsthe latest discovery of ice polymorphs and existsin the medium pressure range near 1–2 GPa. Ice XIX is a partiallyhydrogen-ordered phase, by contrast to its disordered mother phaseice VI, which shares the same oxygen-atom network with ice XIX. IceXIX differs in terms of the ordering of the hydrogen-atom sublattice,and hence the space group, from its hydrogen-ordered sibling ice XV,which also features the same type of oxygen network. Together, iceVI, XV, and XIX form the only known trio of ice polymorphs, wherepolymorphic transformations from order to order, order to disorder,and disorder to order are possible, which also compete with each otherdepending on the thermodynamic path taken and the cooling/heatingrates employed. These transitions in the H-sublattice have barelybeen investigated, so we study here the unique triangular relationin the ice VI/XV/XIX trio based on calorimetry experiments. We revealthe following key features for H-sublattice transitions: (i) uponcooling ice VI, domains of ice XV and XIX develop simultaneously,where pure ice XV forms at ≤0.85 GPa and pure ice XIX formsat ≥1.60 GPa, (ii) ice XIX transforms into ice XV via a transientdisordered state, (iii) ice XV recooled at ambient pressure featuresa complex domain structure, possibly containing an unknown H-orderedpolymorph, (iv) recooled ice XV partly transforms back into ice XIXat 1.80 GPa, and (v) partial deuteration slows down domain reorderingstrongly. These findings not only are of interest in understandingpossible hydrogen-ordering and -disordering processes in the interiorof icy moons and planets but, more importantly, also provide a challengingbenchmark for our understanding and parameterizing many-body interactionsin H-bonded networks. The structuralmodel in the tetragonal space group 2 and afterrecovery to ambient pressure.1 Ice XIXis antiferroelectric, just like its triclinic P1̅sibling ice XV.3 This makes the uniquecase for a “trio” of ice polymorphs sharing the sametopological network of oxygen (O) atoms. Other ice polymorphs appearas “order–disorder duos” or “singles”,yet awaiting the discovery of their counterpart. For this reason,ice VI/XV/XIX represents a unique case that allows for the study ofthe triangular relationship between one disordered parental phaseand two ordered children. In particular, the order-to-order transitionfrom ice XIX to XV at ambient pressure is unprecedented in ice chemistry.4 Please note that both ice XIX and XV are partly ordered, where thephase transition between them results in a change of space group symmetry.Our notion of order follows the tradition in ice chemistry that hasbegun in the 1970s with the report of the first (partly) ordered icephase, ice XI.6 That is, the order-to-order transitiondescribed here reflects a transition from one H-ordered space groupto another, where the order is partial both before and after the transition.The criterion for the transition from one type of H-order to anothertype of H-order is the change of symmetry elements within the unitcell induced through H-atom rearrangement. Already years before thediscovery of ice XIX, Shephard and Salzmann had noticed that the disorderingtransition from ice XV to VI shows features7 unknown for any other ordered ice polymorph. Rosu-Finsen et al.attributed the complexity to a disordered, deep glassy H-atom networkbelow 129 K.9 By contrast, our group has advocatedthe existence of a novel H-ordered ice polymorph,10 nowknown as ice XIX. In a very recent work, Salzmann et al. claimed that“ice XIX” is H-disordered and originates from ice VIthrough lattice distortion.11 As pointedout by Hansen in an accompanying comment, it is unlikely though, “weare really looking at the same ice phase” because their preparationprocedures are different.12Thefamily of ice polymorphs has recently grown through the discoveryof ice XIX.14 The ice XIX unit cell has double the volume of the ice VI and XVunit cells, making supercells a necessity to be considered in theorywork. Nanda and Beran have asked about the governing factors for theorder in ice VI.15 Concerning the ice VIunit cell, Komatsu et al. found that all ordered configurations arealmost degenerate.16 Because of the near-degeneracy,it seems conceivable that ice VI has a multitude of reaction pathsupon cooling, possibly leading to a range of differently ordered domainswithin the ice VI matrix. For instance, a Cc phasehas been considered in theory work but not discovered in the experiment.13 In this experimental work, we take a close lookat pressure as a governing factor for the type of H-order that developsupon cooling ice VI. Furthermore, we examine whether it is possibleto directly switch between the two ordered low-temperature phasesice XV and XIX, without taking the detour via the high-temperatureform ice VI. As our main analytical tool, we use low-temperature differentialscanning calorimetry, where subtle changes in heat flow indicate (endothermic)disordering and (exothermic) ordering transitions in the H-sublatticeof the ice.Not onlyin experiments but also from a theoretical viewpoint,the ordering of ice VI represents a challenge.10 and XIX.1 In the following, we brieflydescribe the specific details such as sample volume, isotopic composition,dopant, and the thermodynamic path. Cooling rates represent a kineticaspect and determine the time provided in the experiment for the H-atomsublattice to rearrange. For the data shown in 2O and 5% D2O and containing 0.01 MHCl as a dopant, were pipetted into an indium container and placedinside the 8 mm diameter bore of the cylinder. The role of the indiumcontainer is to reduce friction between the sample and the piston–cylinderat cryogenic temperatures, as originally proposed by Mishima et al.17 The sample was then (i) cooled to 77 K, (ii)compressed to one of the pressures specified above, (iii) heated to255 K, (iv) cooled to 77 K, (v) decompressed, and (vi) recovered underliquid nitrogen. In step (iv), the samples were cooled from 160 to90 K at a rate of 3 K min–1. Above 160 K and below90 K, cooling rates of up to 20 K min–1 were applied.As a reference, 0.01 M HCl in H2O samples, without anyadded D2O, were prepared at pressures of 0.85, 1.00, 1.20,1.45, 1.60, and 1.80 GPa.In general, thesample preparationfollows the procedures outlined in our previous work on ice β-XV2O) cooled at 1.80 GPa as mentioned above for the data in –1 to 77 K. For the data in 2O) rapidly quenched at 1.00 GPa. After releasing the pressure, thesample was heated to 130 K at 4 K min–1 and cooledto 77 K at 1 K min–1 to obtain ice XVrec(130 K).For the data in Supporting Information.All of the samples were characterizedusing both X-ray diffraction and differential scanning calorimetry.A cylindrical sample batch recovered from the piston–cylinderequipment was 600 mg. For calorimetry and diffraction experiments,small chunks were cut from the cylinder and transferred under liquidnitrogen to the instrument. More details about the instruments anddata acquisition and handling are provided in the 19 Up to ≈148 K, ice VI itself merely produces a baseline featuringa subtle glass transition20 so that calorimetrydoes not allow for the quantification of the ice VI fraction. IceXV features a single endotherm near 130 K, indicating the disorderingtransition to ice VI. Ice XIX, by contrast, shows a very complex thermalsignature featuring two endotherms (near 100 and 130 K), and an exothermin between. The first endotherm has been assigned to the H-disorderingof ice XIX and the second one to the H-disordering of ice XV.4 The exotherm near 120 K signifies an H-ordering process: nascentice VI (from ice XIX) orders to ice XV, which is thermodynamicallymore stable than ice VI at 120 K. The observation of the exothermprecludes ice XV at 77 K. This is also refuted based on our neutrondiffraction data: the ice XIX sample prepared at 1.80 GPa revealsno evidence for any initial ice XV at 80 K, but ice XV slowly appearsabove 120 K at ambient pressure.1 The transientcharacter of ice VI emerging from ice XIX leads us to mark it as atransition state, ice VI‡.10In the first set of experiments, wecooled ice VI from 250 to 77 K at pressures in the range of 0.85–2.00GPa  through cooling ice VI at high pressure and (ii) through heatingice XIX at ambient pressure. Consistent with this, also the area ofthe ice VI‡ exotherm is the smallest at 1.70 GPa.This good correlation between the area of the exotherm and the areaof the second endotherm  from nascent ice VI‡ at 118 K and (ii) fromstable ice VI at 140 K. At 1 bar, ice VI orders upon cooling to formice XV,10 whereas ice XIX formation kinetics is by far tooslow to be relevant. ‡ endotherm is missing. Upon heating to 118 K(or beyond), ice XIX disappears, where ice XV forms upon recoolingat ambient pressure. The area of the ice XV → VI endothermsignificantly grows from 45 to 65 J mol–1 for case(i) and to 150 J mol–1 for case (ii). That is, someice XV is gained upon recooling ice VI‡, and largeamounts are produced from stable ice VI. The tripling in size comparedto the pressure-cooled ice XV sample23 suggests a better ordering and faster kinetics at ambient pressure.Moreover, the shape of the endotherm in recooled ice XV ” and “ice XVrec(140 K)” as suggested earlier in ref. (rec(140 K) is much more ordered than all other variants of ice XV producedthrough different pathways.The second set of experimentsis devoted to a deeper understanding of the complex transformationsequence at ambient pressure. To this end, we recooled the 1.80 GPaice XIX sample 10 (circled d in rec(130 K) to 1.80 GPa and kept it isothermally at 80 K for 4 h  (circled d in rec(130 K) (wine red trace in rec(140 K) (black trace in ‡ (orange diamonds) are likely found in the sample.While ice VI cannot be directly assessed from the calorimetry trace,the relatively small latent heat for the ice XV endotherm speaks infavor of unconverted ice VI. Ice VI‡ domains arepresent, e.g., at the interface between the ice XIX and ice XV domains.These domains are ill-defined and may be closer to either ice XV orice XIX. The domain structure is significantly different in recooledice XV . Also, the peak frontingand the width are reduced from about 45 to 35 K. This suggests thatsome of the less stable domains of recooled ice XV tend to disorderat 1.80 GPa, converting to ice VI‡. No ice XIX reappearsat 80 or 100 K and 1.80 GPa. –1. The XV → VI enthalpy decreases to 105 J mol–1, and the peak width decreases to less than 20 K. That is, about1/3 of the ice XV domains in ice XVrec(130 K) haveconverted to ice VI‡ and some ice XIX. While stillfar from being a complete back-transformation XV → XIX, thisexperiment demonstrates that the order–order transition inthe triangular relation can in principle take place in both directions.Yet, about two-thirds of the ice XV domains remain unaffected. Itseems that the fully developed, most stable ice XV domains are unaffectedat 1.80 GPa. Only partly developed, less stable ice XV domains canbe converted back to ice VI‡ or ice XIX. It needsto be investigated in future experiments whether all ice XV domains,including the stable ones, can be converted to ice XIX, maybe at higherpressure or after longer waiting. Higher temperatures cannot be probedbecause at 125 K and 1.80 GPa, the stable phase is already ice VIaccording to Yamane et al.2 so that iceXV would convert to stable ice VI, not ice XIX.Let usfinally move to thelast set of calorimetric experiments. The observation of the ice XIX→ ice VIdice XV 3d.As al2O samples of ice VI topology. Theice VI topology is of high interest because it is the only one thatin addition to the H-disordered parent phase features two types ofH-ordered polymorphs, namely, ice XV and ice XIX. Analogously to otherice polymorphs, both of them only originate from their parent phasein the presence of point defects in their lattice, as they enhancethe mobility.4 Weintroduce these point defects through the use of a dilute solutionof HCl, producing a combination of ionic H3O+ defects in combination with Bjerrum-L-defects. This combinationof point defects turns out to be most efficient for the ice VI →XV transformation.24 Approximately every millionthH2O molecule is replaced by a HCl molecule substitutionallyin the ice lattice. As has been shown previously in the examples ofice V/XIII, VI/XV, and XII/XIV, this does not affect the global structure,phase behavior, or thermodynamics of the ice phase, yet enhances themobility of the H-atoms in the ice lattice by 3–4 orders ofmagnitude. The case of doped ice XIX is unique, showing the most complexthermodynamics involving three stable ice phases. We here aim at resolvingkinetic aspects governing the phase behavior of ice XIX based on themethod of calorimetry that has helped to create our understandingof H-atom dynamics in ice phases for many decades.25 The ordering process also shows a huge isotopeeffect in deuterated samples, where deuteration slows down the sublatticedynamics tremendously. The sublattice dynamics in deuterated ice VIcan be massively accelerated by adding small amounts of H2O.1 The reason for the much faster dynamicsin such isotopically mixed samples is the subject of our ongoing research.In the present work, we reveal pressure as an excellent parameterfor governing the kinetics of ice XV and XIX formation upon coolingprotiated ice VI. The rate of ice XIX formation increases with increasingpressure, but the rate of ice XV formation decreases with increasingpressure. Below 1.00 GPa, ice XIX formation is too slow to be significant,whereas above 1.60 GPa, ice XV formation is too slow. Between 1.00and 1.60 GPa, there is a strong competition, with the formation ofboth ice XV and XIX domains. Furthermore, some of the initial iceVI remains disordered throughout. This competition is sketched in ‡, starting above 100K. At 100–129 K, nascent ice VI‡ slowly convertsto ice XV, which itself transforms into stable ice VI above 129 K.The fraction of ice VI‡ that was lacking time totransform into ice XV remains ice VI, whereas the fraction that hadconverted to ice XV turns back into ice VI. This rich behavior uponheating also impacts the recooling process at 1 bar, yielding recooledice XV. While pressure-cooled ice XV at 0.85 GPa shows a relativelysharp endotherm, the peak width of recooled ice XV is 3 times larger.This implies an inhomogeneous domain structure of recooled ice XV,where each differently ordered ice XV domain shows a unique onsettemperature of disordering. Notably, a prominent shoulder just above120 K, visible in 9 thus, turns out to be a mixture of severaldifferently ordered domains, where domain walls separate them. Twoof these domain structures are known today, ice XV and XIX, and otherssuch as the Cc phase13 might find experimental corroboration in future crystallographicwork. As pointed out by the reviewers of our work, the Cc phase might actually be a Pc phase consistent withstructures #25 and #38 in Supporting Information Table S3 in ref. , the ordered pendants show quite low permittivities (ε< 3).32 Also the plastic deformationand creep behavior differ very much, depending on the type of H-bondnetwork.34 For this reason, it is of crucial importanceto know how and how fast the H-sublattice (dis)orders as a functionof temperature and pressure—the key question addressed in thepresent work. For Ganymede and other icy Jupiter and Saturn moonsin our solar system, it seems that the temperatures in the interiorare probably too high to allow for H-ordering of ice VI.Theseresults reveal the subtle interplay of thermodynamics andkinetics that governs the type of H-ordering and its rate of formationas a function of pressure. As such, this represents a great benchmarkfor any kind of potential describing H-bonded networks, especiallycondensed water. There are currently no potentials that accuratelydescribe this competition between different types of H-orders to thebest of our knowledge. In addition to the importance of describingthe H-bond interactions in this many-body system exactly, the resultsare also of interest in our understanding of ice in the interior ofplanets and moons. Ice VI is thought to exist in the interior of manyouter planets and icy moons,


We infer that cellular carbon accumulation in the weathering crust exceeds fluvial export, promoting biomass sequestration, enhanced carbon cycling, and biological albedo reduction. We estimate that up to 37 kg km−2 of cellular carbon is flushed from the weathering crust environment of the western Greenland Ice Sheet each summer, providing an appreciable flux to support heterotrophs and methanogenesis at the bed.The Greenland Ice Sheet harbours a wealth of microbial life, yet the total biomass stored or exported from its surface to downstream environments is unconstrained. Here, we quantify microbial abundance and cellular biomass flux within the near-surface weathering crust photic zone of the western sector of the ice sheet. Using groundwater techniques, we demonstrate that interstitial water flow is slow (~10 Microbes that colonise ice sheet surfaces are important to the carbon cycle, but their biomass and transport remains unquantified. Here, the authors reveal substantial microbial carbon fluxes across Greenland’s ice surface, in quantities that may sustain subglacial heterotrophs and fuel methanogenesis. One important carbon source is the ice sheet surface6, which is host to diverse and active microbial assemblages10. Recent work has highlighted the global significance of these supraglacial microbial communities: they hold key roles in amplifying ice melt by lowering bare-ice albedo14, and driving biogeochemical cycling of carbon16, nitrogen17 and anthropogenic contaminants19.The Greenland Ice Sheet sequesters and exports organic carbon3 km2 bare-ice ablation area20, microbes, organic and inorganic debris and associated nutrients are washed downslope by the supraglacial meltwater drainage networks that develop21. These networks commonly terminate in moulins that provide meltwater pathways to the subglacial environment22. Such seasonal meltwater transfer affects the labile, bioavailable carbon exported from the ice sheet23 and ultimately influences the microbial community composition and nutrient supply to downstream aquatic and marine ecosystems25. However, supraglacial drainage networks comprising streams, rivers and lakes account for a small fraction of the total ablating area of the ice sheet28 and the majority of summer-season runoff is generated over extensive bare-ice areas between supraglacial meltwater channels29. The hydrological functioning of this wide-spread interfluvial area is unconstrained30 and its role in modulating the delivery of microbes, organic and mineral dusts and debris, and associated nutrients to the subglacial environment remains unquantified.During seasonal melt, across Greenland’s 220 × 1031) and has an assumed meltwater throughflow velocity of ~1–10 m d−1 (ref. 28). Smith et al.32 demonstrate that the weathering crust has an important hydrological function and delays surface meltwater runoff, challenging the commonly held assumptions of efficient and rapid supraglacial drainage. Furthermore, the weathering crust photic zone is increasingly recognised as an important microbial habitat35; yet little is known about how, where and what quantities of microbial biomass are stored and produced within, or exported from, the weathering crust to downstream environments and ecosystems.The bare-ice interfluve area is characterised by the presence of an up to ~1 m thick, porous ice weathering crust, that forms due to subsurface shortwave radiation penetration, melting and percolation. In west Greenland, this weathered near-surface ice exhibits an effective porosity of up to 47% with a specific water storage potential of up to 0.18 m  derived from unmanned aerial vehicle (UAV) surveys  to 10.9 (± 5.4) cm below the ice surface, respectively. A total of 47 successful recharge experiments were conducted, yielding a mean hydraulic conductivity (K) of 0.28 (± 0.34) m d−1 . Comparison of K against melt rate  it is not associated with the instantaneous melt rate .The study period of July 23 to 29, 2014 was characterised by predominantly clear-sky conditions with a mean two-metre air temperature of 1.9 °C  , but at the 95% confidence level suggests a slight negative relationship with time since daily peak melt , where both melt variables provide proxies for diurnal energy receipt and weathering crust development .The 73 water samples recovered from both fully and partially recharged auger holes in bare-ice show a mean microbial abundance of 2.28 × 10on) Fig. . Abundanr2 = 0.50, p < 0.001: Fig. 37. Examination of the association between K and abundance independently for these nominal bacteria and algae classes highlights similar non-linear, inverse relationships  of 22% (ref. 31), the cross-sectional water flow area can be determined. Considering saturated transport through the weathering crust under Darcian flow36, the throughflow velocity (vt) is given by:h is the average local surface slope (0.022 m m−1). With a mean value for hydraulic conductivity (K) of 0.28 m d−1, we determine a spatially averaged weathering crust throughflow velocity of 0.028 m d−1 and a specific meltwater discharge of 0.0018 m3 d−1. Typically, at our study site located within the western sector of the Greenland Ice Sheet, ablating bare-ice and, by inference, its hydraulically active weathering crust is exposed, on average, for around 70 days (or 76%) of the summer melt season38. Accordingly, using our derived mean and maximum vt values, respectively, only 22 to 66% of the catchment’s weathering crust area delivers interstitial meltwater directly to the stream network seasonally  into the stream network. Coupling this stream bank area with our estimates of mean throughflow velocity and mean microbial abundance we derive a daily microbial cell flux of 2.80 × 1012, which equates to 5.73 × 1012 cells km−2 d−1 when scaled across the entire catchment. Using our uppermost observed K-value, the specific discharge, total and specific cell fluxes increase to 0.008 m3 d−1, 1.27 × 1013 cells d−1 and 2.61 × 1013 cells km−2 d−1.From aerial photogrammetry of our study catchment, we calculate a bare-ice weathering crust area of 6.84 × 102) to the supraglacial stream network lies between 8.36 × 10−5 and 0.017 kg C km−2 d−1; on inclusion of the contribution from the larger (>15 μm) algal class, these values upwardly adjust to 1.36 × 10−4 and 0.055 kg C km−2 d−1 (Table To convert the daily cell fluxes to biomass estimates (see Methods), we apply a constant carbon content to derive a minimum, and a biovolume ratio to determine a maximum. Employing these approaches, for a conservative throughflow velocity, we propose the carbon efflux from the study catchment’s interfluvial area  stream-defining contributing area threshold aggregates the two hydrological functions of the slow interstitial near-surface matrix, and fast rills and micro-channels43, yielding more rapid transit times. The hydrological function of the weathering crust is further compounded by macro-spatial variations in hydraulics, the saturated and unsaturated zones, near-surface fracturing processes and ice structure and crystal sizes44. Given these intrinsic environmental controls, the derivation of a spatially and/or temporally consistent estimate of the hydraulic conductivity from coarse resolution topographic metrics without any ground measurements is problematic.Our analysis demonstrates that an extensive saturated porous layer, with a water table extending from ~0.1 m below the surface, is hydraulically active in a study catchment located on the ablating western margin of the Greenland Ice Sheet. Calculations of the weathering crust’s hydraulic conductivity of 10−1 for the weathering crust are markedly lower than supraglacial stream and river velocities of 104–106 m d−1 (ref. 46), but are similar to those reported for low-gradient ablating sea ice surfaces47. Though slow, the throughflow velocities could increase if the water table within the weather crust rose into the higher porosity uppermost unsaturated ice layer48. Our calculated meltwater efflux from the weathering crust to the stream network represents a relatively small proportion of the total discharge in the supraglacial channels, emphasising the potential importance of flow contributions from within the unsaturated layer and micro-channels, and which merits further investigation. Nonetheless, the slow transit of water through the saturated weathering crust hydraulically acts to delay surface meltwater runoff32 and impedes any associated microbe and nutrient transport.Unsurprisingly, our throughflow velocities of <0.13 m d49 as well as alternative methods51. We derive a microbial load of 1.46 × 109 cells m−2 for the hydraulically active weathering crust, with a minimum of 1.46 × 108 cells m−2 , the mean abundance is increased to 6.12 × 104 cells mL−1, and our load rises to 3.9 × 109 cells m−2 or 0.05 to 34.1 kg C km−2.Our estimates for microbial abundance in the recharge water compare well to other supraglacial enumerations using flow cytometry53; extrusion of exopolysaccharides (EPS) or other critical compounds56; or hydrological elution (flushing) with increased melt, analogous to the ‘first flush effect’57 at diurnal, synoptic and/or seasonal timescales as alluded to by the weak correlation between abundance and time since daily peak melt  using an allometric best-estimate of biomass. If we consider our study catchment to be representative of all the moulin terminating supraglacial catchments across the ice sheet’s western ablation zone22, upscaling our catchment data suggests that between 1.8 × 1018 and 8.3 × 1018 microbial cells were delivered to the subglacial drainage system over the 2014 ablation season. Accounting for the different biomass estimation methods, this equates to a seasonal carbon delivery of 25 kg C to 3.0 × 105 kg C from western Greenland’s ablation zone under a mean throughflow velocity . These biomass assessments increase to 102 to 105 kg C under the maximum observed throughflow velocity.At our study catchment, we estimate that between 1.4 × 10−2 of microbial cellular carbon was delivered from the supraglacial drainage network to the subglacial environment in western Greenland. However, weathering crust development and biomass transport are defined by the cumulative shortwave radiation receipt and the length of the bare-ice melt season. Therefore, to contextualise our observations in 2014, a typical or average melt year, we recalculate our biomass fluxes for 2006 and 2012: anomalously low and high melt years. Using the mean throughflow velocity and all the biomass estimates, we derive a flux of between 7.2 × 10−4 kg C km−2 and 0.5 kg C km−2 for the 2006 low melt year. For 2012, which sustained high melt, these estimates increase fourfold to 3.1 × 10−3 kg C km−2 and 2.1 kg C km−2. Refining these biomass export estimates on a by-catchment basis, we find a mean of 9.8 kg C km−2 and a maximum of 21.8 kg C km−2 in 2014, with 4.1 kg C km−2 and 16.6 kg C km−2 in 2006, and 15.9 kg C km−2 and 23.6 kg C km−2 in 2012; inclusion of large algae more than doubles these seasonal biomass flux values.Using the mean hydraulic conductivity and the allometric biomass conversion, we calculate that over the entire 2014 ablation season 0.3 to 1.1 kg C km48 and, hence we report a cellular carbon liberation to serve as a benchmark that invites future refinement through further investigation of the hydrological configuration, dynamics and functioning of the supraglacial interfluve environment.The wide range of values in our assessment of cellular carbon delivery from the weathering crust emphasises its dependence on the melt season duration and intensity, the near-surface hydraulic conductivity of glacier ice, the representative microbial abundance in transport, the biomass conversion utilised, and the treatment of larger algal size fractions. Moreover, the definition of the supraglacial stream network that transports the cells released from the weathering crust also influences these estimates; a simple linear relationship exists between the seasonal mass of cellular carbon entrained and the proportion of a catchment that is defined as stream bank, with a halving of stream-adjacent area leading to a 50% reduction in the carbon biomass transported to downstream environments. We focus our attention on biomass efflux from the saturated weathering crust, which does not account for meltwater and microbe transport through the unsaturated portion of the near-surface ice and, therefore, represents a baseline. Our calculations also assume that the weathering crust is not responding dynamically over synoptic time-scales−1 coupled with a mean distance-to-stream index across our catchment of 7.24 ± 6.6 m  as a conservative estimate of antecedent near-surface microbial load, we calculate microbial abundance increases of 1.5 × 102 to 2.3 × 103 cells mL−1 d−1 using published doubling times. This translates to between 1.4 × 10−4 and 1.4 kg km−2 d−1 of accumulated organic carbon  in the weathering crust: the minimum estimate assumes 11 fg per cell, and an 11 d doubling time , while the maximum includes >15 µm algal cells, and assumes a 1 d doubling time with constant biovolume ratios  assuming that optimal conditions persist throughout the average melt-season (Table −2 d−1)58 and mineral-rich cryoconite that typically covers up to 8% of the ice surface (8.7 kg C km−2 d−1)16. It is important to note that these accumulation rates do not include the antecedent concentrations, long-residence time, or emergent populations. Hence, with equivalent allometric biomass efflux projections of only 0.01 kg C km−2 d−1 , our analysis demonstrates the potential for cellular biomass accumulation within the near-surface weathering crust. This hypothesis is further emphasised by coupling our weathering crust data with published algal and cryoconite productivity estimates for the location58 to account for the three constituent supraglacial interfluve environments. We assume that 35% of the bare-ice surface is characterised by active supraglacial streams, as found in our study catchment, with the remaining interfluvial area occupied by the weathering crust and algae, and exhibiting a cryoconite coverage of 8%. This yields near-surface biomass accumulation estimates ranging from 0.59 to 0.82 kg C km−2 d−1, which far exceed the projected maximum efflux of 0.25 kg C km−2 d−1 for the site. Consequently, the weathering crust theoretically undergoes a substantive gain in cellular carbon over the melt-season.Numerous factors affect supraglacial microbial growth, such as exposure to sunlight, nutrient and water availability50 confirming that the ice sheet’s weathering crust is a biologically-active habitat. Our study catchment suggests that the near-surface weathering crust in western Greenland exhibits an in situ storage of 1.5 × 1015 cells km−2, equivalent to between 0.02 and 14.0 kg C km−2, depending on the biomass conversion applied. This near-surface habitat is hydrologically active and characterised by protracted residence times owing to slow transport of meltwater through the spatially extensive porous interfluve area. To improve models of near-surface microbial transport, it is essential to explore the microbe entrainment and transport-controlling environmental characteristics  and biophysical responses56. For example, the crystal size and dust content of Greenland’s ablation area is known to vary, in part due to the era when the emergent ice was formed61; such properties will influence spatial patterns in surface hydraulic conductivity and microbe entrainment. Nonetheless, the mean near-surface weathering crust hydraulic conductivity measured across the study catchment, during a typical Greenland melt season62, compares well to other supraglacial evaluations36, and yields a specific daily microbe flux of only 4.1 × 107 cells. Here, we expand on three core implications of this low rate of near-surface cellular biomass transport: the fluvial export of biomass from the ice sheet surface, the accumulation of biomass in the weathering crust, and the associated supraglacial carbon cycling.Our observations of microbial abundance at the S6 site resonates with those previously reported elsewhere for supraglacial meltwaters3 km2 in western Greenland during 2014, our allometric best-estimate of the ablation season biomass export from the surface weathering crust to moulins descending into the ice sheet interior is 3.8 to 15.5 tonnes of cellular carbon. This highlights the importance of the flushing of organic matter from the ice sheet’s surface to the subglacial environment. In low-melt years, these biomass estimates can decrease by an order of magnitude, while in high-melt years the values can double. However, current understanding of the nature of meltwater routing, transit times and the redox conditions in Greenland’s subglacial environment is incomplete. Therefore, we propose that the transport and potential subglacial deposition and storage of cellular organic carbon and associated compounds by inefficient basal drainage networks enhances microbe-water-rock interaction times and ratios, and provides a viable and contributory source of carbon for the support of heterotrophs23 and for methanogenesis63 at the ice sheet bed. Moreover, with knowledge that at least a portion of the weathering crust communities are active58, through their export from the supraglacial environment, they may deliver functionality to, and inoculate downstream sub- and pro-glacial aquatic systems. It is also established that the weathering crust evolves and decays, respectively, under clear-sky and cloudy or rainfall-dominated conditions48. Consequently, the synoptic and seasonal patterns, and future of supraglacial cellular biomass export warrant further investigation given the increasing clear-sky ablation season phases64, expanding bare-ice area20 and/or more frequent rainfall events65 forecast for Greenland under future atmospheric warming.Across an area of ~14 × 1014. Our findings highlight how cell export from the ice surface is also conditioned by these environmental variables indicating that, during high melt years, two competing amplification mechanisms interact at seasonal time-scales: in situ microbial biomass accumulation and hydrological export both increase. Nonetheless, our assertion that cellular biomass export lies below the equivalent cellular load or estimated accumulation is of particular significance given the biological-darkening of the ice sheet , where microbial abundance strongly influences the bare-ice reflectivity, darkens surface ice and enhances surface melt cycles58. Studies suggest that the darkening phenomenon on the ice sheet is driven by predominantly phototrophic algal growth in the top few centimetres of the ice sheet58. Our results indicate the potential for autochthonous microbial carbon accumulation in this shallow near-surface weathering crust; such accumulation also contributes to this biological darkening of the ice. Variability in the accumulation and export of cellular biomass, respectively, within and from the weathering crust can contribute to the strong inter- and intra-annual variation in observed ice sheet albedo66.The abundance and proliferation of microbial cells across the bare-ice ablation zone is dependent on the duration of the melt season and cumulative shortwave irradiance48 and hydraulically linked44 to the porous weathering crust, it is unknown whether these features represent biomass sinks where a proportion of the entrained microbes within the near-surface become bound to cryoconite granules55 and are removed from the water column. Carbon cycling within the weathering crust itself is also promoted through cell mortality, from grazing by protists, viral lysis and photolysis34. Indeed, the cycling of organic carbon and exudation or release of dissolved organic carbon (DOC) by microbes is commonly reported in supraglacial meltwaters68 and is evidenced by elevated DOC in near-surface ice69. There will likely be a preference for the cycling of new, labile, rather than older, recalcitrant, ‘fossil’ carbon70. Moreover, the extended residence time of microbes within the weathering crust can also account for DOC consumption56. Combined, these processes will modulate the organic carbon and associated compounds supplied to subglacial and downstream environments. Given geophysical evidence of saturated sediments at least 1 m thick beneath the western Greenland Ice Sheet72, coupled with indications of subglacial methane cycling63 and the emergence of methane-saturated proglacial waters4, our findings confirm a considerable organic carbon flux enters and is likely sequestered and/or transformed within the subglacial environment. Consequently, there remains a need to better constrain microbial carbon cycling pathways and their controls across supraglacial, subglacial and proglacial environments in Greenland.The disparity between the accumulation and export of cellular biomass across the western sector of the ice sheet also imparts a number of biogeochemical cycling processes. For example, with cryoconite holes punctuating73. Observations over the last two decades suggest an annual ablation at the site of −1.96 m w.e.62. Situated on the western edge of the Dark Zone, a combination of enhanced dust content, black carbon and microbial community loading reduce the local bare-ice albedo at the site75. Neighbouring ice temperature observations76 show that at ~1000 m a.s.l. only the uppermost 1–2 m of the western sector of the ice sheet’s ablation zone reaches 0 °C during the summer ablation season. This shallow, seasonally transient, temperate ice layer is likely to develop as a porous and hydrologically active weathering crust during summer31.Our study site was located proximate to the S6 automatic weather station (AWS) on the Kangerlussuaq Transect in western Greenland , 38 km from the ice margin at an elevation of approximately 1020 m a.s.l. within the well-reported Dark Zone Fig. . The loc73 were used as input to a point-based energy balance model77 to estimate local melt conditions across the low elevation range supraglacial catchment during the observation period. We estimated ice surface albedo for the catchment simplistically using a 12-hour running mean of the ratio of incoming and outgoing shortwave radiation at the S6 AWS, filtered for erroneous values, and which ranged between 0.37 and 0.50. The surface aerodynamic roughness parameter (z0) was kept constant, and taken as an average derived from a set of ten random 10 m transects78 oriented perpendicular to the dominant south-easterly wind (mean orientation: 116°) during the field campaign with elevation data extracted from the 0.25 m resolution DEM  within the supraglacial catchment of interest: z0 = 1.142 mm compared well to the 10−3–10−4 m range quoted for bare-ice in the locality79. The 2014 summer season was unexceptional in surface mass balance terms, suggesting our study period provides a representative baseline73.Hourly meteorological data from the S6 AWS36: briefly, shallow (0.26 or 0.36 m) holes were made using a 50 mm diameter Kovacs ice auger, and bespoke capacitance piezometers were used to record bail-recharge experiments, yielding water level recovery records at 2 s time intervals. Hydraulic conductivity (K) was assessed from these recharge curves following standard groundwater techniques36 (see Supplementary Method S2). A total of 9 experimental auger hole sites, separated by distances of ~9 m, were located in a quasi-random grid aligned perpendicular to the primary channel flowing to the northwest  of all the recharge experiments undertaken bore incomplete recharge, which precluded confident estimation of K-values36.Observations of the weathering crust and its hydrological functioning were made following Stevens et al.est Fig. . Auger hFollowing auger hole recharge experiments, using a polyethylene syringe and 30 cm polypropylene tube rinsed three times with supraglacial stream water prior to sampling, a 15 mL depth-integrated sample of recharge water was abstracted. Of this, 10 mL was decanted into a 15 mL sterile polyethylene centrifuge tube, and fixed using 50 µL glutaraldehyde . The preserved samples were kept dark and cool (~4 °C) for up to 8 days while in the field and in transit, and subsequently fast frozen and stored at −80 °C until analysis.80 to enumerate the microbes in the recharge water samples, employing the following protocol: samples were thawed at ambient laboratory room temperature, gently agitated, and stained with SYBR Gold  at a final concentration of 1× and stored in the dark at 20 °C for a maximum of 240 minutes prior to analysis. Enumeration was conducted with a Sony SH-800EC Cell-Sorter , using gates set within the Sony Cell Sorter v.2.1.3 software package to describe stained and non-stained particles as optimised for the sample water type15  following the manufacturer’s instructions (see Supplementary Method S3). Our size assessment was constrained by the instrumental ~0.5 μm analytical limit, with a threshold applied to eliminate detection noise.We used flow cytometry5 cells mL−1 were treated as outliers. These ten samples, we found, were proximate to highly localised microbial blooms and/or anomalously high particle concentrations, as reported by other authors58. We justify our approach here based on cell abundances of <105 mL−1 being distinctive of supraglacial meltwaters50, weathering crust water35, and glacier ice82. Further, eighteen ancillary water samples taken from exploratory auger and cryoconite holes in bare-ice areas at the S6 site demonstrated no cell concentrations >105 mL−1.To remain conservative in our assessment of the weathering crust hydrology, recharge water samples yielding microbial abundance of >1.0 × 1037. The microbes classed as algae, typically accounted for <10 % of the observed abundance. Given the filamentous nature of many cyanobacteria in glacial environments55, it is likely that a proportion of the cyanobacteria were included in our algal category. Because viruses, filamentous cyanobacteria and ice algae were not specifically identified, differentiated, or excluded by gating in our approach, uncertainties lie in the classification (and therefore biomass) of microbes particularly in the lower- and upper-most size fractions reported here.We employed a 10 µm size threshold to nominally classify microbes as bacteria or algae52: (i) constant carbon content, (ii) allometric, or (iii) constant carbon ratio approximations. Previous work has suggested constant values for bacteria and archaea  fg C cell−1 (ref. 83)) or equivalent estimates for cyanobacteria and algal cells ). However, these approaches may not fully reflect variations in microbe size. Consequently, biomass can also be approximated using either a size-dependent allometric model85 or a constant biovolume ratio86.Microbial abundance can be converted to carbon biomass using three established techniques85; algae were described as simple 10 µm diameter cylinders87. For each of these three geometries, the cell length was defined by the midpoint of the size fraction reported by the cytometric analysis. Allometric methods ascribe the mass of carbon  as a function of V (in µm−3) and scaling factors (c and a):c = 162, a = 0.91 (ref. 85); and for algae, c = 109, and a = 0.991 (ref. 84). Norland52 argued that the allometric biomass method provides the most robust calculation of biomass, and is therefore referred to as our best estimate. Derived constant biovolume ratios assessed for bacteria and algae indicate, respectively, 560 fg C µm−3 (ref. 86) and 110 fg C µm−3 for algal plasma volume88.Here, owing to the wide variety of unknown microbe geometries, biovolumes (V) were estimated by assuming bacteria <1 µm are spherical, with larger bacteria as rods  exhibiting length:width ratios defined by typical bacterial geometries89. However, due to the uncertainty over the size of algal cells or filaments in the >15 µm category owing to the limitations of the FCM analysis, these were ascribed a nominal mean length of 27.5 μm, which equates to that typical of an Ancylonema nordenskiöldii alga, common to the locality14. These algal contributions are included as accompanying data in our carbon calculations given their capacity to exaggerate the biomass estimates.Despite the potential for including large viruses in the <1 μm cell counts, the size fraction was included in the biomass estimates based on the existing evidence of such small bacteria within Greenland’s iceTo contextualise our biomass results, and in recognition of the extent of the Dark Zone and presence of elevated abundances associated with emergent dusts and algal blooms across the region, we re-ran our calculations using the mean cell concentration and size distributions derived from our entire sample set. The inclusion of outlying samples increased the mean abundance by less than a factor of 3, while the difference in proportion of cells in each size fraction was ≤1%.11. Briefly, 740 overlapping RAW format images were acquired at 350 m above the ice surface using a Sony NEX-5N camera. These images were georeferenced with on-board GPS and UAV altitude data and processed into orthomosaics and DEMs using Agisoft’s PhotoScan Pro. The resulting orthomosaic and DEM products yielded a horizontal ground resolution of ~0.1 m 91 using 75 control points manually located in both images. A thin-planted spline warping was applied to the UAV-derived DEM. To correct the hydrological inconsistencies the two DEMs were resampled to a resolution of 0.25 m and detrended using 31 × 31 plane fitting filter in the RSGISLib software92. The detrending process yielded two raster surfaces, the first high frequency signal  and the second, the low frequency signal . To generate the final DEM the high frequency signal from the UAV was combined with the low frequency signal from the ArcticDEM, providing a hydrologically consistent DEM, and an 11 × 11 kernel, two-standard-deviation Gaussian smoothing filter was applied. The combined, tuned elevation model was then resampled to a 1 m horizontal resolution using a cubic spline.Due to the absence of constrained ground control points on the ice surface to geolocate the processed DEM, and uncertainty associated with the onboard GPS and limited area-of-observation relating to the flightpath, the DEM contained well-known photogrammetric artefacts in its 3D geometry93. Analyses of the DEM and ortho-imagery within ESRI’s ArcGIS v.10.5 Spatial Analyst indicated that abundant, large cryoconite holes were associated with local topographic depressions or sinks. Therefore, sinks within the DEM were filled using a threshold depth of 0.51 m based on the maximum depth recorded in a survey of 90 random cryoconite holes with diameters ranging from 0.01 to 1 m at the study site. Sinks >0.51 m in depth were retained, to account for any crevasse or fracture features, despite their relative paucity in the local catchment area.To estimate supraglacial drainage, a surface flow routing method that accounts for features causing hydrological fragmentation is a viable protocol2 to define active supraglacial channels. This threshold returned a ~0.5 km2 supraglacial catchment containing our experimental plot area, with elevations increasing up-glacier by ~20 m over a 2.5 km distance and a mean pixel surface slope of 2.23 (±1.70)%. Following the GIS-based identification of the primary stream network based on the flow accumulation threshold, the streams were removed or ‘burned’28 from the DEM and the hydrological assessment to calculate supraglacial interfluve flowpath lengths was rerun  algorithms were used on the filled DEM to derive flow accumulation and stream network maps. Cross-examination of these with the high-resolution UAV ortho-imagery indicated a high degree of fit, and facilitated identification of a flow accumulation threshold of 165 mrun Fig. . SensitiK36, to reveal a regional picture we upscaled our observed data across 795 moulin-terminating supraglacial catchments in western Greenland defined by Yang and Smith22, employing the bare-ice duration maps from Ryan et al.20 for 2006, 2012, and the study year 2014. The mean summer season (June to August: JJA) bare-ice duration (bi) for each catchment was used to account for latitudinal and elevational gradients, and annual variability. We presume that the depth of the hydraulically active weathering crust evolved over time: deepening from the onset of bare-ice, reaching a maximum depth (≤0.29 m), and declining thereafter. We used a modified cosine function centred on July 15 (day of year (DOY) 196) and defined by our observation period to describe this growth and decay of the weathering crust: for short bare-ice durations (bi <38 days), the hydraulically active layer did not reach the 0.29 m maximum but reaches its greatest depth of 0.0076·bi, while for longer bare-ice durations, the depth increases to 0.29 m and plateaus at that maximum prior to decaying (see Supplementary Method S5). From this simple evolving depth model and our mean microbial abundance, for each catchment within the 1.38 × 103 km2 region, we calculated a cell flux to supraglacial streams assuming a constant proportion (14%) for stream bank area over each of the internally drainage catchments, as found in our study catchment. We assume that rapid (<1 d) in-stream transit time46 from source to moulins results in minimal change in microbial abundance and biomass during transport.Given the absence of a clear relationship between melt rate and Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Streptomyces are characterized by complex morphological differentiation. Mostly, industrial production of NPs is done by submerged fermentation, where streptomycetes grow as a vegetative mycelium forming pellets. Often, suboptimal growth peculiarities are the major bottleneck for industrial exploitation. In this work, we employed genetic engineering approaches to improve the production of moenomycins (Mm) in Streptomyces ghanaensis, the only known natural direct inhibitors of bacterial peptidoglycan glycosyltransferses. We showed that in vivo elimination of binding sites for the pleiotropic regulator AdpA in the oriC region strongly influences growth and positively correlates with Mm accumulation. Additionally, a marker- and “scar”-less deletion of moeH5, encoding an amidotransferase from the Mm gene cluster, significantly narrows down the Mm production spectrum. Strikingly, antibiotic titers were strongly enhanced by the elimination of the pleiotropic regulatory gene wblA, involved in the late steps of morphogenesis. Altogether, we generated Mm overproducers with optimized growth parameters, which are useful for further genome engineering and chemoenzymatic generation of novel Mm derivatives. Analogously, such a scheme can be applied to other Streptomyces spp.Streptomycetes are soil-dwelling multicellular microorganisms famous for their unprecedented ability to synthesize numerous bioactive natural products (NPs). In addition to their rich arsenal of secondary metabolites,  Streptomyces are well known as one of the richest sources of biologically active secondary metabolites of microbial origin [Streptomyces biology.The members of the genus l origin . Becausel origin . These uIn contrast to most bacteria, streptomycetes are multicellular microorganisms, which are also notable for their complex morphological differentiation . NormallEscherichia coli and Bacillus spp., the Streptomyces growth type is less attractive from a biotechnological point of view, for several reasons. Particularly, such growth results in mass- and heat-transfer problems, and in oxidative stress [cslA/glxA operon and the mat cluster [In a submerged environment streptomycetes typically grow in a form of vegetative mycelium, predominantly forming dense particles or clumps . In conte stress ,7. Thesee stress . Large me stress . Formati cluster ,11,12, w cluster .Streptomyces ghanaensis ATCC14672 is famous for the production of Mm, a mixture of similar phosphoglycolipid antibiotics with a unique mechanism of action. Acting as the only known direct inhibitors of bacterial peptidoglycan glycosyltransferases—the enzymes involved in the penultimate step of cell wall biosynthesis—Mm have been considered to serve as the blueprint for the development of a novel class of antibacterials [S. ghanaensis, the strain also forms large mycelial particles growing in submerged cultures [moe cluster); however, the level of Mm biosynthesis was still lower than in the wild type [moe cluster and numerous global regulators were shown to influence Mm production [gh, which was shown to directly activate the transcription of moe genes [Streptomyces and control the expression of numerous genes involved in morphogenesis and both primary and secondary metabolism [Streptomyces coelicolor also demonstrated a capacity of AdpA to inhibit the chromosome replication at the initial stage by binding to the replication origin (oriC), thus decreasing the access of the initiator protein (DnaA) [wblA, a gene encoding a small Fe–S-containing cluster regulatory protein, drastically affects the antibiotic biosynthesis in many streptomycetes, including Mm production [cterials . In addicultures . This foild type ,17. Seveoduction ,24,25,26oe genes . Ortholotabolism ,28. Inten (DnaA) . Furtheroduction ,32,33,34S. ghanaensis strains where the beneficial changes were sequentially combined, enabling the generation of a triple mutant strain with significantly improved growth particularities and Mm production titers. We showed that AdpAgh plays an important role in the regulation of chromosome replication, thus crucially influencing the growth type of S. ghanaensis. Based on this observation, we created a mutant strain with deletion of amidotransferase moeH5, leading to accumulation of a significantly narrowed spectrum of Mm. Finally, we eliminated the negative regulator of Mm biosynthesis wblA to increasing antibiotic production levels. Overall, our findings set up a putative strategy for optimizing the natural products biosynthesis in other strains.In this work, we applied genetic engineering approaches to construct a series of Escherichia coli strains were grown in Luria-Bertani (LB) medium at 37 °C. Soya mannitol agar (SFM), oatmeal agar (OAT) and tryptic soy broth (TSB) liquid or agar (TSA) media were used to grow S. ghanaensis. Where necessary, the media were supplemented with appropriate antibiotics.E. coli XL1-Blue according to standard procedures [Routine cloning manipulations were made in ocedures . Polymerocedures . VerificE. coli ET12567 (pUZ8002) was used as a donor for conjugal transfer of plasmids into S. ghanaensis strains. Fresh recipient spore suspension was harvested from 7-day-old S. ghanaensis strains grown on OAT at 37 °C. After a 10 min heat shock at 50 °C, spores (4 × 107 cfu) were mixed with E. coli cells (1 × 108 cfu) carrying the different vectors and then plated on SFM supplemented with 60 mM CaCl2. Next, after 15 h of incubation at 28 or 37 °C (depending on a vector), plates were overlaid with 1 mL of water containing 1.6 mg of apramycin and 5 mg of phosphomycin. Usually, exconjugants appeared after 3 to 5 days of incubation. Nonsporulating mutant strains were first pre-grown in TSB for 24 h and then 1 mL of the resulting culture (~3 × 107 cfu) was directly mixed with donor cells using the aforementioned procedure.S. ghanaensis in liquid-grown cultures was monitored using a Carl Zeiss Axiolab 1 microscope. For scanning electron microscopy (SEM), slices of S. ghanaensis lawns were cut off SFM agar plates and directly analyzed on a Quanta 250 environmental scanning electron microscope (ThermoFisher).The mycelial morphology of ghbs in the oriC region of the S. ghanaensis chromosome were substituted to a nonsense sequence, the following scheme was used. Firstly, a 3-kb-long DNA fragment, consisting of AdpAghbs plus two 1.5-kb-long regions of homology, was amplified from the S. ghanaensis chromosomal DNA by PCR using primers oriC_for/oriC_rev. The obtained amplicon was digested with XbaI and EcoRV and cloned into XbaI-EcoRV-cleaved pBluescriptKS+ to give pBloriC. In order to mutate AdpAghbs, PCR mutagenesis was applied to amplify a 6 kb DNA fragment from pBloriC with primers oriC_mut_for/oriC_mut_rev. The obtained PCR fragment was first treated with T4 polynucleotide kinase and then self-ligated giving pBloriC-mut. Next, pBloriC-mut was cut with XbaI and EcoRV, and the resulting 3 kb DNA fragment was ligated into the respective sites of pKG1139, a vector based on a thermosensitive pSG5 replicon carrying a reporter gusA gene [S. ghanaensis. To promote an integration of pKGoriC-mut into the S. ghanaensis chromosome via homologous recombination, one exconjugant was inoculated in 15 mL of TSB containing apramycin. Five passages were conducted, where after each 24 h of growth at 40 °C (nonpermissive temperature for the pSG5 replication), 10 μL of the culture was inoculated into a fresh medium with apramycin. Next, 1 mL of the resulting culture was plated on oatmeal agar. After seven days of incubation at 37 °C, serial dilutions of spore suspension were plated on SFM supplemented with 5-bromo-4-chloro-3-indolyl-D-glucuronide (X-Gluc) solution (50 μg mL−1). White colonies (indication of vector lost) were checked for the sensitivity to apramycin and then subjected to the PCR analysis. Substitution of AdpAghbs was confirmed by sequencing, leading to the creation of S. ghanaensis O1.To construct a mutant strain, where AdpAusA gene . The finmoeH5 from the S. ghanaensis O1 chromosome, a 6.4 kb DNA fragment comprising moeH5 with two 2.4-kb-long regions of homology was PCR synthesized from the chromosome using primers moeH5_for/moeH5_rev. The resulting amplicon was ligated into pBluescriptKS+ cleaved with EcoRV to give pBlmoeH5. Next, PCR mutagenesis was used to remove the moeH5 coding sequence. To do this, a pair of primers, moeH5_mut_f/moeH5_mut_r, was used to amplify a 7.8 kb DNA fragment comprising no moeH5 gene from pBlmoeH5. Next, the PCR product was treated with T4 polynucleotide kinase and self-ligated, yielding pBlΔmoeH5. A 4.8 kb DNA fragment was cut out of pBlΔmoeH5 using XbaI-EcoRV and ligated into the respective sites of pKG1139, creating pKGΔmoeH5. Then, pKGΔmoeH5 was transferred to S. ghanaensis O1 by intergeneric conjugation with E. coli. Screening for double crossover mutants was done as described above, giving a mutant strain S. ghanaensis O1ΔH5.To delete wblA deletion, the analogous scheme was used. First, a 4.3 kb DNA fragment, consisting of wblA with two 2 kb flanking regions of homology, was amplified from the S. ghanaensis chromosome by PCR using the primers wblA_for/wblA_rev. The amplicon was ligated into pBluescriptKS+ cut with EcoRV to give pBlwblA. Then, PCR mutagenesis was done to generate a 6.9 kb fragment (containing no wblA) from pBlwblA with the primers wblA_mut_f/wblA_mut_r. It was subsequently treated with T4 polynucleotide kinase and self-ligated to create pBlΔwblA. A 4 kb XbaI-EcoRV cut DNA fragment was retrieved from pBlΔwblA and then cloned into pKG1139 digested with the respective enzymes to give pKGΔwblA. The final construct was conjugally transferred to S. ghanaensis O1ΔH5 and screening for the mutants was conducted as mentioned above. This resulted in construction of S. ghanaensis O1ΔH5ΔwblA.To generate a triple mutant with a S. ghanaensis strains, Mm extraction and analysis of Mm production were performed according to the previously established protocols [S. ghanaensis WT extracts: MmA , nosokomycin B , moenomycin G  and desmethylated NoB , whereas nosokomycin A  and desmethylated NoA  were monitored in the strains carrying moeH5 deletion.Cultivation of rotocols . Routinerotocols . Four maS. ghanaensis, a reversed-phased InfinityLab Poroshell 120 EC-C18 column  was employed with the following mobile system: phase A—water and mobile phase B—MeCN, both with 0.5% acetic acid (vol/vol) as a solvent modifier. The solvents were delivered at 0.5 mL/min under a gradient elution program: 0 min 95% A, 0.5 min 95% A, 23 min 5% A, 26.5 min 5% A, 27 min 95% A, and 30 min 95% A. The mass spectrometer was operated in negative ESI mode using the parameters described earlier [For qualitative analysis of extracts from  earlier .S. ghanaensis biosynthesis of Mm is strictly depended on the AdpAgh activity, where it directly activates the transcription of key moe genes [S. coelicolor AdpA binds to the oriC region, thus inhibiting chromosome replication [ghbs in oriC influences growth and Mm production in S. ghanaensis. For this purpose, we first compared the oriC regions between S. ghanaensis and S. coelicolor for the presence of AdpAbs. As depicted in S. ghanaensis DNA region, responsible for AdpA and DnaA binding, shares a high degree of similarity to that of S. coelicolor [ghbs , indicating their involvement in the regulation of chromosome replication. It was shown that AdpA binding led to the extended protection of the DNA region and thus reduce accessibility of DnaAbs to the DnaA initiator protein [Streptomyces lividans 66 oriC region suggested that replication of its chromosome depends on all DnaAbs, including those that surround AdpAbs [In oe genes . Moreovelication . Therefoelicolor . Particuor [ghbs . Analogo protein . Previoud AdpAbs   to eliminate the overlapping AdpAghbs 1 and 2 in vivo. We substituted them for a nonsense sequence in a way that the distance between two adjacent DnaAbs remained intact  were used to initiate the fermentation and then the strains were cultivated in parallel in TSB for four days. Aliquots were collected from the growing cultures at certain time points and were subjected to further analysis. Biomass accumulation was used to monitor the growth dynamic. As shown in S. ghanaensis O1 accumulated two-fold more biomass during the first 20 h of growth compared to the wild type. Moreover, the mutant strain entered the stationary phase already after 14 h of growth, whereas it took 22–24 h to reach it for the wild type. After 30 h of cultivation, both strains displayed a similar growth pattern, indicating that mutation of AdpAghbs strongly affects the type of growth, especially at early stages.Next, we assessed changes in both Mm and biomass accumulation by S. ghanaensis strains. As depicted in S. ghanaensis [S. ghanaensis O1 produced a higher amount of Mm over the first three days of growth and reached its maximum after 72 h. After four days of cultivation, a concentration of Mm in biomass pellet slightly dropped down, most likely due to the cell lysis. In contrast, the control strain gradually accumulated the antibiotic over the entire fermentation time course and gained its highest titer after 96 h  was inoculated into TSB. Sample aliquots were collected from growing cultures and then mycelial morphology was directly analyzed. The beginning of aggregation of the germinated spores was already noticeable after 2 h of growth in the wild type, whereas there was a 2 h delay in S. ghanaensis O1 (data not shown). After 6 h of growth both strains formed mycelial structures; however, they were bigger and denser in the parental strain , MmA (m/z = 789.8), dNoB (m/z = 734.8) and MmG (m/z = 770.8). A contribution of other Mm to the total antibiotic production in S. ghanaensis O1 was relatively low. In contrast to the parental strain, deletion of moeH5 greatly narrowed down Mm spectrum in S. ghanaensis O1ΔH5  in the chromosome of the producing strain, making it easily susceptible for the next genetic manipulations.In summary, we constructed the mutant S. ghanaensis, Mm biosynthesis includes no cluster-situated regulators involved in the regulation of metabolic pathway [wblA—a gene encoding a Fe–S cluster-containing transcription factor, involved in the late stages of morphological differentiation, was shown to negatively operate Mm synthesis in S. ghanaensis [S. ghanaensis O1ΔH5, we created a triple mutant where ghwblA was deleted from the chromosome via homologous recombination. The gene knockout was performed in the same way as described above, leading to the construction of a marker- and “scar”-less mutant S. ghanaensis O1ΔH5ΔwblA.Unlike the majority of antibiotic biosynthetic gene clusters, in  pathway ,17,19. Tanaensis ,24. To iS. ghanaensis O1ΔH5ΔwblA and O1ΔH5 were grown in TSB for four days. Extracts from their biomass were subjected to analysis via LC-MS. As depicted in ghwblA significantly improved antibiotic production in O1ΔH5ΔwblA compared to the parental strain. On average, the ghwblA-deficient mutant accumulated 2.6-fold more NoA than the O1ΔH5 strain  [S. ghanaensis O1ΔH5 accumulated the growth benefits of the parental S. ghanaensis O1 strain and produced a significantly narrowed spectrum of Mm accumulating almost exclusively NoA. The strain may be used as a background for the chemoenzymatic synthesis of Mm analogues with altered biological activities.Common for NP biosynthetic pathways, there are few final compounds accumulated during the fermentation course ,66,67. Ttructure . The thrtructure . In thisre, Flp) ,69, leavre, Flp) , it permS. ghanaensis synthesizes Mm in milligram quantities, which has forced numerous investigations aimed to improving production levels. Because of the absence of cluster-situated regulators associated with moe genes, improvement of Mm biosynthesis is challenging. Some global regulators were shown to be involved in governing of Mm production in streptomycetes. In this study, we deleted the regulatory gene ghwblA from the chromosome of S. ghanaensis O1ΔH5 using the aforementioned marker- and “scar”-less technique. The wblA orthologs are conserved across the Streptomyces genus and many studies, including that for Mm synthesis, reported them as negative regulators of antibiotic production [ghwblA strongly increased NoA accumulation by the resulting triple mutant strain S. ghanaensis O1ΔH5ΔwblA, while completely blocking the morphogenesis at the aerial mycelium level.Biosynthesis of secondary metabolites in streptomycetes undergoes a fine-tune regulation at multiple levels. Usually, natural producers accumulate a relatively low amount of desired compound and antibiotic titer improvement is the main requirement for the industry. oduction ,32,33. DE. coli and S. ghanaensis. Interestingly, we observed a profound impact of substitution of AdpAghbs in the oriC region on the conjugal efficiency. In comparison to the wild-type strain, both pSET152 and pKC1139 were transferred with 2.5–3-fold higher frequency using S. ghanaensis O1 as a recipient. Likely, the way that the mutant grows is the most plausible explanation of the increased conjugal ability. In contrast to the parental strain, which forms dense and large mycelial particles that would limit the physical contacts with donor cells, S. ghanaensis O1 produces almost no mycelial pellets, establishing an efficient and close contact between interacting cells. As anticipated, deletion of amidotransferase moeH5 did not influence the morphological progression and resulted in no changes in the exconjugants occurrence compared to S. ghanaensis O1. Lastly, the frequency of conjugation was greatly reduced when S. ghanaensis O1ΔH5ΔwblA was used as a recipient strain. This perfectly correlates with the inability of the ghwblA-defficient mutant to produce spores. Even though we used an equal amount of recipient cells (~107 cfu), the exconjugants occurrence was decreased more than 6-fold when compared to S. ghanaensis O1ΔH5. In many tested cases, the conjugal efficiency was shown to be much higher when a spore suspension rather than mycelium was used [S. ghanaensis O1ΔH5ΔwblA, its further utilization might be limited in experiments demanding a high efficiency of conjugation.Successful genome engineering relies on an efficient and relatively easy way of DNA transfer into streptomycetes. Therefore, we evaluated the influence of applied genetic manipulations on the efficacy of DNA transfer in intergeneric conjugation between was used ,72. TherS. ghanaensis strains with improved growth and antibiotic production characteristics. First, we in vivo substituted AdpAghbs in the oriC region in the S. ghanaensis chromosome. Strikingly, in contrast to the wild type, the mutant S. ghanaensis O1 strain grew in a dispersed way, forming no mycelial pellets and displaying an accelerated growth rate. Moreover, the maximum Mm biosynthesis was observed 24 h in advance in the mutant compared to the parental strain. Next, a marker- and “scar”-less elimination of the amidotransferase gene moeH5 greatly reduced the spectrum of Mm produced by S. ghanaensis O1ΔH5, leading to the accumulation of almost exclusively NoA. Additionally, knockout of the negative regulator of antibiotic production ghwblA strongly improved the NoA biosynthetic titers but resulted in a strain deficient in sporulation. Finally, we compared the efficiency of DNA transfer using intergeneric conjugation with E. coli. We observed the strong beneficial effect of the AdpAghbs substitution on the frequency of exconjugants occurrence, whereas deletion of ghwblA drastically reduced the conjugal efficacy. Overall, we constructed a triple mutant carrying no extra foreign DNA, whose type and accelerated growth, narrowed spectrum and increased level of antibiotic synthesis are clearly supreme over the original strain. The technology described in this paper can be useful background information for further projects focused on production titers optimization in other strains.In this work, we employed genome engineering approaches to construct


Background: Motor symptom disorders in patients with Parkinson disease (PD) are closely related to reduced inhibitory ability. Although exercise has been shown to improve this ability in patients with PD, its effects on proactive and reactive inhibition have not been determined. Most previous studies of inhibitory control disorder in people with PD have been behavioral, and little attention has been paid to functional cortical connectivity. Wu Qin Xi, a low–medium-intensity qigong exercise that is safe and easy to do for elderly individuals, can support physical well-being and help prevent and alleviate disease. In this study, our aims were to explore the effects of a long-term Wu Qin Xi intervention on response inhibition and to examine how improved inhibition control relates to cortical connectivity using dual-site paired-pulse transcranial magnetic stimulation (ppTMS), in patients with mild–moderate PD.Methods: A single-blind randomized controlled trial will be conducted. A total of 90 elderly subjects will be recruited and allocated randomly to Wu Qin Xi, balance exercise, and healthy control groups. The exercise interventions will be implemented in three 90-min sessions per week for 24 weeks; the healthy control group will receive no intervention. The primary assessments will be response inhibition metrics and task-based ppTMS. The secondary outcomes will include motor symptom severity, mobility, balance, emotional state, and quality of life. Assessments will be conducted at baseline, at the conclusion of the intervention period (week 24), and a few months after the intervention (week 36 follow-up).Discussion: This study is designed to provide insights into the effects of practicing Wu Qin Xi on response inhibition function in people with PD. The results will provide evidence on the value of traditional Chinese exercise as a therapeutic rehabilitation option for these patients. They will also provide data addressing how brain function–related cortical connectivity is related to reactive vs. proactive inhibition in people with PD participating in an exercise intervention.Clinical Trial Registration: This study has been registered prospectively in the Chinese Clinical Trial Registry . A central part of motor control is response inhibition, defined as the ability to cancel planned movements effectively . InhibitThe frontal–subcortical network may be important for inhibitory control , 7; chanThe motor symptoms of PD have been related to decreased inhibitory ability . PharmacWu Qin Xi is an integrated qigong exercise system that consists of movements that imitate the technical actions and breathing of five animals  while attending to unification of the regulation of one's body, respiration, and mind  is used as a metric of one's ability to respond to stop orders and considered to be an indicator of reactive inhibition efficiency . MeanwhiWu Qin Xi training on the primary outcomes of response inhibition ability and cortical connectivity using task-based ppTMS and on the secondary outcomes of balance, emotional status, and quality of life in patients with mild–moderate PD. We hypothesize that Wu Qin Xi training will improve response inhibition ability and increase brain network connectivity and response inhibition coherence in the motor network in this population. We further hypothesize that observed network connectivity changes will occur in association with improvements in response performance.We propose a prospective randomized clinical trial designed to examine the effects of Wu Qin Xi and balance groups at a 1:1 ratio. Both the Wu Qin Xi group and the balance training group will receive group-based exercise intervention at the sports laboratory of Shanghai University of Sport. Participants who meet the criteria will undergo evaluations at baseline, immediately after the intervention, and at 36-week follow-up intervals , overt signs of cognitive impairment [Montreal Cognitive Assessment (MoCA) score <26], symptoms of impulse control disorder (which accelerate the stopping of initiated motor actions), history of epilepsy, and participation in any other behavioral or instructor-led exercise program during the study period.Wu Qin Xi exercise intervention, we estimated the required sample size based on the number of subjects included in a previous behavioral experimental study (*power software indicated that 63 participants (21/group) will be required to demonstrate effects on primary and secondary outcomes with repeated-measures analyses of variance (ANOVAs) at a 0.05 significance level and 80% power. The observation of significant interhemispheric inhibition of motor cortex changes in a sample of elderly individuals (10/group) obtained with a similar TMS paradigm , and a 10-min cooldown consisting of limb range-of-motion movements, sustained stretching, and relaxing . The balance training will consist of: (1) static balancing on unstable surfaces with the maintenance of postural control and progression to weight shifting; (2) dynamic balancing with postural control while standing and performing upper-limb and trunk movements; (3) balance strategy development with a focus on the hips while maintaining ankle balance, including stepping in different directions under interference; (4) and adaptation to varying bases of support and standing in a narrow space and on an uneven surface  muscle with 9-mm-diameter Ag-AgCl surface electrodes. The active and reference electrodes will be placed over the stomach muscle and metacarpophalangeal joint, respectively. The signal will be amplified , bandpass filtered , digitized at 5 kHz by an analog–digital interface , and saved for off-line analysis.1mV will be defined as the lowest TMS intensity required to generate MEPs of 1 mV in the relaxed FDI muscle in at least five of 10 trials. The resting motor threshold was defined as the lowest TMS intensity required to generate MEPs of more than 50 μV in at least five out of 10 trials with the target muscle completely relaxed  connected to two single-pulse monophasic stimulators . The M1 hotspot will be defined as the scalp location inducing the largest peak–peak motor-evoked potential (MEP) amplitude in the contralateral FDI muscle. The handle of the test stimulus (TS) coil will be positioned posteriorly, 30°–45° off the midsagittal line. TS relaxed .To investigate changes in connectivity between the left M1 and right hemispheric response inhibition after long-term exercise training, we will apply ppTMS with two high-power Magstim 200 devices and two figure-of-eight coil sites . Coil plTMS assessments will be performed before and after the intervention and at a follow-up appointment 12 weeks after completion of the 24-week intervention period, with the subjects sitting relaxed in a chair  in front of a computer monitor placed 75 cm in front of their eyes. Before the assessment, the subjects will receive instructions and practice the behavioral task for familiarization. They will be instructed to respond as quickly and accurately as possible to the arrow and not to delay their responses in anticipation of the stop signal. The zero time point will be set at the onset of the stimulus, and the TS will appear for 50, 100, 150, 200, and 250 ms [10 times per interstimulus interval (ISI) and 10 times for TS-alone trials; The task will consist of randomly interleaved no-go and stop-signal trials, enabling us to examine both types of inhibition . A totalThe ppTMS will be used to assess changes in cortical connectivity in specific brain regions associated with inhibitory control before and after the intervention and at the follow-up appointment.The subjects' mobility, balance, emotional state, motor symptoms, daily living ability, and sleep quality will be assessed using the following instruments: Timed up-and-go test (TUG), Hospital Anxiety and Depression Scale (HADS), Unified Parkinson's Disease Rating Scale Part III (UPDRS-III), Parkinson Disease Questionnaire (PDQ-39), and Parkinson Disease Sleep Scale (PDSS).The TUG is a simple measure of mobility and motor ability in older adults. The total time, in s, needed for the subject to get up from a chair and walk 3 m at their usual walking pace, then turn around and walk back to the chair, and sit down (with his or her back against the seat) will be measured . The parThe HADS is a 14-item clinical and research instrument widely used to screen patients with chronic illness for depression and anxiety symptoms in the past week and recommended for use in people with PD; due to potentially overlapping somatic symptoms, parkinsonian manifestations are not assessed with this scale , 37. It The UPDRS-III is currently recognized as the standard tool for the clinical evaluation of PD motor symptoms. Higher scores indicate greater PD motor sign severity.The 39-item PDQ was used to assess health-related quality of life  in the previous month in people with PD. Each item is rated on a 5-point (0–4) ordinal scale, with higher scores indicating worse quality of life .The 15-item PDSS was used to rate the severity of sleep disturbances in people with PD. Items inquire about daytime sleepiness, nocturia, and nocturnal sleep quality, restlessness, and psychiatric and movement symptoms. Patients rate the frequency or intensity of symptoms on a scale ranging from 0 (poor or frequent) to 10 (good or never), with a total possible score of 150 and higher scores indicating better sleep quality .Wu Qin Xi practice on inhibition of responses, between- and within-group differences in behavioral outcomes over time  will be examined using mixed-model ANOVA. Peak–peak MEP amplitudes from the conditioned (CS and TS) TMS trials will be expressed as percentages of the mean peak–peak MEP amplitude from the unconditioned (TS only) test pulse. Values <100% and >100% will be taken to indicate inhibition and facilitation, respectively. Values will be reported as means ± standard errors of the mean. MEP values will be interpreted as representations of the influence of the DLPFC, pre-SMA, and rIFG on M1 under the given condition. Three-factor mixed-design repeated-measures ANOVA (three groups × three times × five ISIs) will be used to analyze the effect of Wu Qin Xi practice on the inhibitory control of brain network connections in people with PD. The MEP values will serve as the dependent variable, while time, group, and ISI will be treated as independent variables. Repeated-measures ANOVAs will be used to determine the effects of Wu Qin Xi practice on the secondary outcomes, with group and time as factors.To explore the effects of Wu Qin Xi practice on different inhibitory control modes in people with PD will improve our understanding of inhibitory disorders in this population and support the development of diversified rehabilitation training programs.Exercise experience has been shown to improve inhibitory control , and aerWu Qin Xi exercise intervention relative to baseline. We will explore the effects of long-term Wu Qin Xi exercise on functional changes coincident with cortico-cortical interaction connectivity changes with behavioral assessments of dyskinesia and executive ability for response inhibition control. To further clarify the effects of the long-term Wu Qin Xi intervention, the relationship between the two modes of response inhibition will be explored in relation to changes in prefrontal network connectivity. This neurological evidence will improve our understanding of the physiological basis of impulse control disorder in people with PD. If the present results provide evidence of beneficial effects of Wu Qin Xi on response inhibition ability in people with PD, then this study will provide neurological evidence favoring the use of a supplemental exercise-based clinical therapy.Long-term specialized exercise generates exercise experience-related changes in neural mechanisms –23 and eThis study will have several limitations. First, because we are recruiting only subjects with mild–moderate PD, our findings will not be generalizable to patients with advanced PD. Second, we did not plan to match subtypes of the disease, and the cohort may include patients with and without SWEDD (scans without evidence of a dopaminergic deficit). Studies have suggested that the inhibitory ability associated with abnormal activity in cerebello-thalamo-cortical circuits during movement in SWEDD cases may differ from those of patients with imaging evidence of a dopaminergic deficit. In future studies, we intend to consider patients with different PD subtypes comprehensively and to conduct experiments designed to delineate neural mechanisms . FinallyThe studies involving human participants were reviewed and approved by Shanghai University of Sport research ethics committee. The patients/participants provided their written informed consent to participate in this study.ZW contributed to the conceptualization, methodology, investigation, and writing the original draft. L-LZ contributed to the visualization, software, formal analysis, and validation. YW contributed to data curation. JZ contributed to the writing, review, and editing and project administration. KL contributed to the conceptualization, supervision, and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.