The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations. Bacteroidales are abundant members of the gut microbiota of human populations across the globe. Bacteroidales use Type VI secretion systems (T6SS) to deliver toxins to neighboring cells to gain a competitive advantage. Bacteroidales T6SS are of three different genetic architectures, two of which are contained on mobile genetic elements (MGEs). Due to the phenotypes conferred by cargo genes contained on MGE, their dissemination greatly impacts the ecology and evolution of human gut microbial communities. Here, we present a comprehensive analysis of the prevalence and dissemination of these mobile T6SS loci in gut Bacteroidales species across human populations. We reveal extensive dissemination of T6SS loci to numerous Bacteroidales species in human gut microbiomes with these newly armed strains outcompeting their progenitors. Few other Bacteroidales MGE spread in as many human communities to the degree detected for the T6SS MGEs. Considering the expected cost associated with maintaining these MGEs, these observations suggest that their acquisition confers a significant benefit, increasing fitness of individual strains and possibly the Bacteroidales community as a whole. Bacteroides, Parabacteroides and Prevotella, which collectively are the most abundant Gram-negative bacteria of the healthy colonic microbiota of human populations. These bacteria secrete anti-bacterial proteins that antagonize closely related strains and species, providing a competitive advantage in the gut ecosystem . Type Vreviewed ). There reviewed . GA3 T6Sproteins –6, targeproteins . GA3 T6Scrobiota .Vibrio cholerae strains  and more rarely in other species  ] . Among tThe absence of GA1 and GA2 loci in non-gut Bacteroidales species, such as those that occupy the oral cavity or vagina , could bBacteroides and Parabacteroides species in the gut of two of these individuals, such that they have 99.997%-100% nucleotide sequence identity among them [Bacteroides and Parabacteroides strains previously isolated as part of a longitudinal study [In our previous analyses of the Bacteroidales strains from four healthy human volunteers, we showed that a GA1 ICE transferred between multiple ong them . In contong them . To deteal study  from fouB. thetaiotaomicron strain (BtCL11T00C24) containing a GA2 locus: this is the first B. thetaiotaomicron strain identified with a GA2 locus. Of the four CL06 species containing a GA2, all of which are of subtype GA2b, all loci are nearly 100% identical with only one mismatch, excluding a 445 bp duplication within the tssC gene in three strains. In contrast, a comparison of CL06 and CL11 GA2b ICE using only high scoring segment pairs of greater than 7500 bp and excluding all non-conserved regions revealed DNA identity of 97.85% with 1567 mismatches.Using the previously determined species designation of each isolate , we seleThese data clearly demonstrate that the GA2b ICEs transferred to multiple species within the gut of these two individuals, followed by fixation in the population. For the CL06 community, two species lack the GA2 locus, but each has a distinct GA1 T6SS loci, demonstrating lack of community transfer of these GA1 ICE.B. uniformis AF15-14LB and B. vulgatus AF15-6A) each contain both a GA2b and a GA2c, integrated at different chromosomal sites. This same phenomenon is observed in individual AF16, where both B. uniformis AF16-7 and B. vulgatus AF16-11 have very high sequence similarity across the whole genome with their AF15 cognates. These data indicate inter-person transmission of bacteria and that the GA2 transfer events occurred prior to passage of these bacteria to at least one of these study subjects.To further study GA1 and GA2 dissemination in a larger set of communities, we analyzed three available datasets of sequenced gut bacterial isolates from healthy adults: the BIOML  , CGR Ch. We searWe determined that GA1 and GA2 multi-species spread  are common . For comThe longitudinal BIOML dataset comprises many isolates (often more than 20) of the same species per individual community. In most cases these isolates are nearly isogenic (designated here as the same strain), but sometimes two or more distinct strains of the same species coexist at the same collection time-point . This alAcquisition of an ICE carries with it the cost of maintaining a large genetic element, but the frequent nature of ICE spread suggests that the fitness benefit of its acquisition outweighs the metabolic cost. Despite numerous examples of ICE transfers and multi-species spread, there are also examples where no transfer or single-species transfers occurred, especially for the GA2 T6SS. Therefore, fitness conferred by T6SS ICE acquisition is likely context-dependent and may be contingent upon host physiology and emergent properties of the community and its microbial composition.B. fragilis YCH46, the GA1 ICE insertion disrupted a fucosyltransferase gene (BF2787) in a polysaccharide biosynthesis locus  and B. fragilis 638R (no insertion), we verified that the duplication only spans this 7-bp sequence  and BIOML-A3 (no insertion) confirmed duplication of this recognition pattern upon ICE insertion, as it is present only once in the isogenic strain lacking the GA1 ICE. A multiple sequence alignment of the two tRNAPhe regions from species that often carry GA2a/b/c ICE insertions  indicates very little conservation in the 15-bp region downstream of the 22-bp recognition pattern. This downstream region is often AT-rich, but is also AT-rich for at least one of the tRNAPhe loci from B. thetaiotaomicron and other species that rarely acquire GA2a/b/c, indicating that this high AT region may not be a factor in species prevalence. Moreover, GA2a/b/c insertions do not strictly require an unoccupied tRNAPhe site, as we identified one instance (B. xylanisolvens CL11T00C41) where a GA2b ICE integrated in tandem downstream of a distinct ICE that integrates at the same site. This GA2b ICE integration follows the 22-bp direct repeat generated by the other ICE’s integration . The tyrosine recombinase located at the beginning of the GA2a/b/c ICE  is 81.55% identical at the protein level to the integrase of an α-mannan utilization ICE from Bacteroides thetaiotaomicron VPI-5482 [B. thetaiotaomicron and B. ovatus. The 22-bp sequence is conserved in Porphyromonas species, and while less conserved in Prevotella and Alistipes species, based on target sequence alone, a significant number of strains that do not contain GA2a/b/c ICE contain the sequence for integration.In contrast, GA2 ICE insertions are sequence-specific. For subgroups a, b and c (by far the most abundant), all integrations occurred, with roughly equal likelihood, at either of the two tRNAGGCACCAC . A compaBacteroides eggerthii AF18-1 (from the CGR collection), the GA2d T6SS region is contained on an ICE similar to GA2a/b/c ICE, with the same insertion site described above. For the other strains, the GA2d ICE is unrelated to the GA2a/b/c ICE and has one of two different integrases that dictate their site of integration. The ICE of five strains, B. vulgatus BIOML-A119, B. uniformis BIOML-A85, B. dorei BIOML-A25, B. plebeius AM31-10, and B. vulgatus VPI-4496.2, inserted into the tRNALys locus. By comparing the isogenic strains B. uniformis BIOML-A84 (no GA2d ICE insertion) and BIOML-A85 (GA2d insertion) isolated from the same person, we determined that the 20-bp recognition pattern for the integrase of this ICE  is TTCGAGCCCCAGGCGGATCA, which duplicates upon ICE insertion (B. vulgatus VPI-4496.2 (the underlined A in the pattern was substituted by a G). Interestingly, a closely related ICE harboring a porphyran utilization locus (98.57% sequence identity over 48.5% of the core ICE excluding cargo) was characterized in B. plebeius DSM 17135 [B. plebeius integrase (BACPLE_01619) is CCCCAGGCGGATCACTCA. This pattern does not exactly match the direct repeat we identified for IntGA2d  and does not have IntGA2d at the beginning of the ICE  harboring a different cargo (the B. dorei HS1_L_1_B010 ICE up to the EL88_18285 integrase). In all three cases, the ICE insertion occurred at a tRNAPro locus and is flanked by the 19-bp sequence CTGGGGGGCGTGTGGTCGC.Intriguingly, the GA2d ICE from one isolate,  the ICE . InsteadPhe, tRNALys or tRNAPro genes.In sum, these observations highlight that recombination events between related ICEs in the Bacteroidales can lead to changes in genomic target location due to integrase swaps, as well as changes in gene cargo of the ICE. In all examples identified, GA2 ICE integrate in tRNA genes, integrating at tRNAB. thetaiotaomicron, B. ovatus, B. fragilis, and B. intestinalis, while present in more than 30% of the strains of other species such as B. uniformis, B. vulgatus and B. eggerthii. Whether this bias occurs at the transfer, integration, or maintenance stage is currently unknown. It is possible that these large ICE with their T6SS loci may impose a fitness cost in some species that would outweigh any fitness advantage, precluding their selection. Alternatively, there may be species-level differences in the ability to properly regulate or selectively silence elements on the ICE or T6SS that may lead to decreased fitness. Successful integration and proper regulation of the complex T6SS machinery into different underlying cell physiologies and environmental niches is expected to be highly variable. Our data suggest that GA1, which are present in numerous species with no obvious bias, may present fewer barriers to their acquisition and maintenance.Based on these insertion sites, it is not known why GA2 T6SS loci are scarce in species such as vice-versa. In our Bacteroidales isolates genome collection (B. vulgatus AF15-6A and AF16-11). In addition, B. fragilis strains frequently harbored both a GA3 and GA1 T6SS locus (23 strains), but rarely both a GA3 and GA2 T6SS loci (1 strain). Interestingly, two of the eight strains with both a GA1 and GA2 ICE have disruptions in the T6SS loci. In B. vulgatus BIOML-A11  the tssC gene from GA1 is frameshifted by 2 bp at codon 148 of 460 , the GA1 is intact but the vgrG gene has a 5 bp frameshift at codon 346 of 603  is absent or disrupted in 11 of the 15 strains (73.3%) due to independent instances of transposase insertions. In contrast, the gene is disrupted in 31.2% of the 125 non-redundant strains with only a GA1 ICE (mhgA for (methylase-helicase GA1), encodes for a 1659-residue protein containing both an N6-adenine methylase domain and an SNF2 helicase domain. This architecture is reminiscent of type I/III restriction modification systems, although for MhgA the helicase domain does not have sequence signatures predictive of endonuclease activity [Bacillus subtilis through an unknown mechanism [mhgA gene with strains that have both a GA1 and GA2 ICE is intriguing, but it is unknown how such a product would participate in the exclusion of an element acquired in single-stranded form. Nevertheless, the observed GA2 ICE exclusion may therefore be the result of two distinct factors: ICE exclusion of GA2 by an unknown property of MhgA and T6SS exclusion. The latter could be caused by the difficulty of a cell to simultaneously maintain two complex machines that may cross-talk and are each expensive to replicate and fire. Such a combination may therefore only rarely reach fixation in a population.Additionally, we found three instances of a GA2 ICE in a strain carrying an ICE closely related to that of GA1 (99.3% identity and 95.2% coverage) but with a different cargo instead of the T6SS locus . A compa GA1 ICE  Tables  in gut Bacteroidales that undergo multi-species spread within a person’s microbiota, we scanned all four community isolate datasets  for MGEs (4 kb or larger) that are 99.99% identical in at least three different species within a community. We identified multi-species spread of at least one MGE in 40 out of 91 (43.9%) human gut communities. We identified 112 occurrences of MGE multi-species spread, which clustered into 74 MGE groups based on a threshold requiring 80% identity with at least 80% of the element shared within a group . Many ofOnly six MGEs, including GA1 and GA2b/c ICEs, were found to spread through multiple species in three or more communities with a fully conserved architecture  Tables. Bacteroides and Parabacteroides. Individuals where this ICE is absent are very rare. pMMCAT, which we only detected among Bacteroides and Parabacteroides, is ubiquitous (>70% abundance) and highly conserved (98–100% identity) in populations from USA, Japan and Thailand. It is also present at substantial frequencies (22–70%) in all the other studied populations except for Madagascar, hunter-gatherers in Tanzania and Peru. Therefore, we cannot rule out that the observed high frequencies of communities with pMMCAT shared at 99.99% identity in multiple species, could in some cases be due to the strain already carrying pMMCAT prior to colonization of the current subject.We analyzed the metagenomic dataset for the presence of the MGE with evidence of spread in more than three ecosystems  . ClusterBacteroides-dominated communities. Analyses of HGT events in Prevotella-dominated non-industrialized populations indicated less frequent HGT [One caveat to note is that our MGE spread analysis was conducted using four datasets from industrialized lifestyles, largely from uent HGT  and may Through the genomic analyses of more than 1500 Bacteroidales strains, many co-resident in the same individual, combined with analyses of nearly 1800 gut metagenomic samples of diverse global populations, we have uncovered numerous properties of the T6SS of the gut Bacteroidales. Previously thought to be rarely present in the human gut microbiota, we show that GA2 T6SS loci are the most prevalent of the three GA in the human gut metagenomes analyzed here and that T6SS of GA2 segregate into five subtypes, one of which is largely restricted to animals. GA2 subtypes in many cases demonstrate population clustering with GA2b the most abundant in Japan, but rarely present in the Mongolian population sampled where the GA2a subtype dominates. GA1 ICE acquisition and spread may be further facilitated by the low sequence selectivity necessary for integration. Most importantly, this study reveals the extensive intra-ecosystem transfer of these ICE to co-resident members of the gut microbiota, suggesting a potential community benefit by multi-species acquisition. This type of globally conserved architecture is rare for MGEs that spread to multiple species within an individual. This observation is especially interesting for GA1 and GA2, given that the T6SS variable regions harboring the effectors are frequently recombined, yet the general architecture is preserved.All genomes classified by NCBI in April 2020, as belonging to the order Bacteroidales, excluding genomes that were suppressed or considered anomalous, and excluding genomes flagged as derived from metagenomics studies or surveillance projects, were identified by an Entrez query. This initial set of 2,324 genomes was further curated to reduce clearly redundant genomic sequences , reduce longitudinal samples comprising the same strain isolated from the same individual multiple times, and to remove duplicate entries from both the GenBank and Refseq repositories, retaining the RefSeq sequence. Finally, genomic sequences that were assembled from metagenomics studies despite not being flagged as such by NCBI and those genomes not identified to at least the genus level were removed. The final set comprises 1,434 Bacteroidales genomes .Bacteroides and Parabacteroides isolates collected from four volunteers in the Comstock lab strain collection previously isolated under a protocol approved by the Partners Human Research Committee IRB and complied with all relevant federal guidelines and institutional policies [We screened 136 policies . Each st600 of ~0.8. DNA was recovered using a CTAB/NaCl DNA extraction protocol followed by sodium acetate/ethanol precipitation. SMRT sequencing was carried out at the University of Maryland’s Institute for Genome Sciences Genomics Resource Center, who performed the initial quality control, library preparation, and sequencing of the genomes using PacBio Sequel v3 SMRTcell technology. The genomes were assembled separately using Falcon/Unzip 1.2.0 [B. uniformis CL06T03C05, B. dorei CL06T03C03, B. cellulosilyticus CL06T03C01, B. xylanisolvens CL11T00C03, B. eggerthii CL11T00C20) as assessed by Bandage [B. ovatus CL03T12C18, B. vulgatus CL11T00C01 and B. xylanisolvens CL03T12C04, where multiple contigs were obtained or bubbles remained unresolved, were scaffolded using Ragout 2.3 using complete genomes from the same species as reference [The genomic sequencing of bacterial strains isolated from human fecal samples who provided formal written consent, was approved by the Partners Human Research Committee IRB and complied with all relevant federal guidelines and institutional policies. Strains  were groip 1.2.0  and Flyeip 1.2.0 , then re Bandage , the two Bandage  assemblyeference . Genes weference  and annoeference . Small ceference . The genIn addition to our own longitudinal isolates (see below), we utilized three other publicly available isolate sequence sets in our analysis of intra-community DNA transfer. Genomes from these sets were included if they were identified as belonging to the order Bacteroidales and comprised at least three different species isolated from the same individual. These included: 967 genomes from BioProject accession PRJNA544527  which are consistent as to order and identity within a GA. They also each contain variable regions encoding such things as toxin and immunity proteins, RHS proteins, and evolved TssD proteins. These variable regions would complicate analysis of the distribution of these GAs in both metagenomic datasets and in sequenced isolates. We therefore made DNA-level concatemers by removing the sequence encompassing the variable region(s) from a representative of each GA, and used these as templates for metagenomic read mapping (see above) and as queries for analysis of sequenced isolates.The T6SS loci found in species of the Bacteroidales order fall into three genetic architectures (GA). All three of these GAs contain structural genes  collection of analysis utilities were used to map reads from these sets to sequences of interest. Though the five GA2x T6SS regions are demonstrably different from one another, they do share a somewhat high level of sequence similarity that might influence short read mapping results. Thus, for these analyses, BBsplit was used, as it maps reads to multiple references simultaneously and, in the case of ambiguity (the read maps to more than one template), will determine the best match and count that read only once. Other mapping analyses were ambiguous matches were not an issue utilized BBmap.Fifteen publicly available metagenomic datasets , 48–58 wS1 FigThe bottom gene map of each pair shows the concatemers that were created after removing all genes that diverge within the same genetic architecture. These concatemers were used to query the various datasets analyzed in this study.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableA), UK(B), CGR(C) and BIOML(D) strain collections.Prevalence of GA1 and GA2 T6SS in the CL((XLSX)Click here for additional data file.S4 TableA) and GA2 subtype(B-E) T6SS regions in the CL, BIOML, CGR and UK culture collections.Genomic coordinates for the GA1((XLSX)Click here for additional data file.S5 TableA. Complete GA1 and GA2 fixation in cases where only one strain per species is present in the community (for this table we only included strains with at least 4 related isolates). B. Partial GA2 fixation in cases where only one strain per species is present in the community. C. GA1 and GA2 spread in cases where more than one unrelated strain per species is present in the community. Isolates were classified into strain groups, each designated by a different letter.(XLSX)Click here for additional data file.S6 TableA. Strains with two GA ICE integrations including at least one GA2. B. Genomic coordinates of GA ICE in strains with two integrations including at least one GA2.(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 TableDataset includes genomic coordinates for one representative from each cluster; and the list of communities where each MGE spread is observed.(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.


Hsp90β is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90β for proper folding and/or activity. Regulation of Hsp90β is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90β, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90β is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses  that impact Hsp90β activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90β globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90β and its secretion, through changes in the conformation of the chaperone. Heat shock protein 90 (Hsp90) is an important family of highly conserved chaperones responsible for the proper folding, the stabilization, and the targeting for degradation of many proteins ,2,3,4. IThere are two Hsp90 isoforms in the human cytosol, Hsp90α and Hsp90β ,7. The βE. coli ortholog HtpG, which lacks the CL, has a much more compact structure with little interdomain flexibility. In HtpG, the region spanning the CL insertion folds as an antiparallel β-sheet, with the CL insertion located between β-strands 8 and 9 ..94]. ThiAs shown in Our data suggests that about 95% of both sites are phosphorylated in all tested cell lines. This implies that at least 90% of Hsp90β monomers are phosphorylated on both S226 and S255 if phosphorylation is independent  and at most 95% if it is coupled . We obtained very similar values in lines of breast cancer cells (MDA-MB-468), chronic myelogenous leukemia cells (K562), pancreatic adenocarcinoma cells (AsPC-1), diffuse large B-cell lymphoma (OCI-Ly1) and untransformed, primary mammary epithelial cells (HMEC). The latter have a much slower growth rate, indicating that high occupancy on S226 and S255 is not a unique feature of rapidly proliferating cancer cells.We sought to determine the phosphorylation state of Hsp90α as well, but digestion of Hsp90α’s CL results in an even more complex mixture  and reveIn essence, our occupancy measurements indicate that phospho-occupancy at S226 and S255 on Hsp90β is constitutively high in several cultured cells under normal growth conditions.We treated K562 cells with heat shock, Hsp90 inhibitors, or stressed the cells by altering the growth conditions and compared them to untreated cells to see if CL phosphorylation correlates with Hsp90 activity. The input lysate was also analyzed to compensate for potential Hsp90 up- or downregulation.2O2 added to medium), none resulted in a significant alteration of the CL’s phosphorylation of any isoform  and with a double Hsp90βS226A/S255A alanine mutant (further on referred to as “AA”) in which both S226 and S255 are mutated to alanines, by co-immunoprecipitation (co-IP) followed by LC-MS/MS. We voluntarily limited the comparison to a double mutant only, since our data indicate that roughly 90% of Hsp90βWT should be phosphorylated on both S226 and S255. The WT and AA protein have an HA tag at the N-terminal and were expressed in Hsp90β KO cells, which allowed us to eliminate interference from WT endogenous Hsp90β that would most likely form heterodimers with mutants [We compared the proteins interacting with “wild type” Hsp90β mutants . For negThe levels of bait and input proteins were comparable between the conditions . The phoWe did not identify any protein interacting only with WT or AA. However, comparison of AA and WT co-IPs revealed substantial quantitative differences, as many proteins co-purified in greater quantity with AA . To conft-test with Benjamini-Hochberg correction highlighted 69 regulated interactors .Input lysates were comparable, but the expression of WT in the heavy cells was slightly higher than that of AA in the light cells . Correspp-value < 0.05). Even though the ratios were not very large in absolute terms, they were statistically significant. For abundant proteins like Hsp90α or Hsp70/Hsc70, even such mild changes imply a large difference in the numbers of chaperone complexes impacted. Annotation enrichment analysis on the proteins with increased binding to AA highlighted the terms typically associated with the Hsp90 chaperone machine  and detecting desthiobiotinylation by capillary Western blot analysis (CWB). Such probes have already been used in competition assays to characterize the specificity of Hsp90 inhibitors [p-value resulting from a t-test comparing WT and AA values. ATP/ADP binding was estimated for the WT Hsp90β and the AA mutant in crude cell extracts using ActivXhibitors . Table 2Both WT and AA showed greater affinity for ATP than ADP, and both appeared to be desthiobiotinylated to a similar extent. The average value for ATP binding by the AA mutant was somewhat higher than for WT, but the difference was not statistically significant, suggesting that S226 and S255 mutation to alanines does not have a dramatic effect on ATP/ADP binding.Sensitivity to proteolytic cleavage has been used to assess global changes in Hsp90 conformation . Since tCWB analysis suggests that WT is more resistant to tryptic cleavage than AA A, as theSDS-PAGE analysis and Coomassie staining revealed other protein bands near 75, 65, and 60 kDa in the samples with trypsin B. Band AIntensity analysis of peptides spanning Hsp90β from E42 to R679 in the cleavage products bands suggests that the main position where Hsp90β was cleaved is in the C domain . K607 isThe ratios of the peptides before K607 are significantly lower than those after K607 when trypsin is added. The peptides after K607 are thus less abundant in the rest of the lane compared to peptides before K607, most likely because these are part of a cleavage product smaller than 40 kDa. Taken together, the data implies that S226 and S255 mutation to alanine facilitates tryptic cleavage in the C domain and suggests that S226 and S255 phosphorylation could have the opposite effect. Since the CL region is far from K607, the differences in trypsin sensitivity are presumably caused by a change in global conformation and not by a direct inhibition of trypsin by the phosphate groups.In conclusion, limited proteolysis suggests that suppression of constitutive phosphorylation sites results in an Hsp90β dimer that has either a permanently more open conformation or increased structural dynamics, granting trypsin easier access to cleavage sites. Conversely, the structure of the WT, predominantly phosphorylated protein could be somewhat constrained by the modification on S226, S255.We next explored if nuclear Hsp90β had a different phosphorylation occupancy at S226 and S255, but occupancy in the nuclear and cytosolic fraction are in the same range  can reflect phosphorylation turnover rather than total occupancy (that we determine with our technique). Changes in phosphorylation turnover have already been reported for Hsp90 after heat stress and would not be incompatible with a high occupancy [However, other reports showed different trends. Two studies reported reduced phosphorylation of Hsp90β in cells expressing leukemogenic tyrosine kinases ,41. Kuroccupancy . TherefoWe next addressed the effect of CL modification on Hsp90 interactions. This has been addressed in specific cases but never by a large scale, unbiased approach, as presented here ,44,45,46Still, a higher amount of interactors does not necessarily imply a higher biological activity. We therefore addressed ATP binding and hydrolysis, which are key steps in the Hsp90 chaperone cycle. We showed that AA has a slightly higher binding affinity for ATP, albeit the difference was not statistically significant. Since our assay is performed in a cell lysate, a possible difference correlates with the binding of more AHSA1 (which stimulates Hsp90 ATPase activity) by the AA mutant as shown by the interactome data. Both WT and AA displayed a higher affinity for ATP than ADP, a result in agreement with one study but in conflict with others ,104,105.Conformational dynamics are critical for Hsp90β chaperone activity. We showed that S226 and S255 mutation to alanines facilitated tryptic cleavage in the C domain and possibly in the N domain, suggesting that lack of CL phosphorylation could shift Hsp90β global conformational equilibrium towards a more open state.Several studies proposed that Hsp90 phosphorylation, notably on the CL, is linked to client release at the end of the chaperone cycle ,107,108.The function of S226 and S255 phosphorylation could therefore be related to cellular or organism-related functions specific to higher eukaryotes, such as localization. Our measurements showed that nuclear-localized and cytosolic Hsp90β had virtually identical CL phosphorylation states. By contrast, we found a 10% lower phosphorylation occupancy for S255, and a 2–3% lower occupancy for S226 in eHsp90β. The difference may be greater in reality since eHsp90β can be “contaminated” by Hsp90β released by dead or necrotic cells. To explore a potential causal relationship between S226/S255 phosphorylation and secretion, we transfected cells with WT and AA and found clearly more AA in the CM than WT, suggesting a role for CL phosphosites in regulating Hsp90 secretion. This role could be direct or indirect, with dephosphorylation facilitating C-terminal cleavage events, that have been reported by Wang et al. as a hallmark of eHsp90 . CL phos213IxL215 in human Hsp90β and 205IxL207 in yeast Hsp90, dramatically impair Hsp90 secretion and chaperone activity, and alter protein-protein interactions as well as sensitivity to tryptic cleavage in the C domain [Our results on the AA mutant closely recall a previous study by Tsutsumi et al., who identified a region flanking the CL whose mutation altered Hsp90 protein protein interactions, global conformation and secretion. Specifically, alanine mutations of the conserved hydrophobic motif in β-strand 8, C domain . The autTaking these findings together with our results, we can formulate a model in which S226 and S255 phosphorylation alters the transient interactions between the C-terminal part of the CL with residual secondary structure, β-strand 9 included, and β-strand 8. Phosphate groups in the CL may form salt bridges with the numerous lysine residues in proximity and rigidify the CL, while dephosphorylation may result in increased flexibility. This mechanism could regulate contacts between β-strand 8 and the surrounding secondary structures: α-helix 8, β-strand 2 and in particular β-strand 9, as β-strands 8 and 9 are separated by the CL but do interact in the closed conformations of Hsp90, forming a β-hairpin like structure ,110,111.We provided insights on the phosphorylation occupancy of S226 and S255 in various conditions and environments and on the effects of the modifications on some key molecular properties. As it often happens with Hsp90, the answers we obtained did not provide an easily intelligible picture and several unknowns remain. Disentangling the different effects of CL phosphorylation will require further investigations at different levels. To test our structural model, it will be essential to probe, for example by hydrogen/deuterium exchange, if (de-)phosphorylation of the CL impacts the exposure of the secondary structures surrounding β-strand 8. On the functional side, it will be crucial to determine the chaperoning activity of Hsp90 in the presence/absence of the modifications. Characterizing the CL phosphorylation turnover in various conditions may also provide valuable information to understand the function of S226 and S255 in physiological states such as under stress conditions. Finally, it will be important to test the relevance of CL phosphorylation on secretion of Hsp90α, that is considered the major biologically active eHsp90 species.


The transition from hunting to herding transformed the cold, arid steppes of Mongolia and Eastern Eurasia into a key social and economic center of the ancient world, but a fragmentary archaeological record limits our understanding of the subsistence base for early pastoral societies in this key region. Organic material preserved in high mountain ice provides rare snapshots into the use of alpine and high altitude zones, which played a central role in the emergence of East Asian pastoralism. Here, we present the results of the first archaeological survey of melting ice margins in the Altai Mountains of western Mongolia, revealing a near-continuous record of more than 3500 years of human activity. Osteology, radiocarbon dating, and collagen fingerprinting analysis of wooden projectiles, animal bone, and other artifacts indicate that big-game hunting and exploitation of alpine ice played a significant role during the emergence of mobile pastoralism in the Altai, and remained a core element of pastoral adaptation into the modern era. Extensive ice melting and loss of wildlife in the study area over recent decades, driven by a warming climate, poaching, and poorly regulated hunting, presents an urgent threat to the future viability of herding lifeways and the archaeological record of hunting in montane zones. Glaciers and ice sheets serve as important travel corridors across high mountains3, and in summer, the melting ice from both glaciers and “ice patches”  provides an important source of freshwater used by animals, plants, and humans. In many areas of the world, such features function as a hunting hotspot for larger mammals, which congregate seasonally on the ice for various reasons such as thermoregulation, hydration, escape from insect pressure, and improved forage5. When organic artifacts or biological material becomes incorporated in these features, they can be preserved intact, providing a rare snapshot into cultural activity for this otherwise high-energy depositional environment that rarely preserves organic archaeological material. Due to global climate change, ancient cultural and biological material preserved in the ice for centuries or millennia is now melting out for the very first time.Artifacts emerging from melting ice provide a rare archaeological dataset to understanding the emergence of pastoral economies in the frigid steppes of interior Eurasia. Across the globe, the survey of permanent patches of snow and ice has produced new insights into the prehistoric use of high-elevation or high latitude regions which suffer from poor representation in the archaeological recordOvis ammon), Siberian ibex (Capra sibirica), and red deer (Cervus elaphus), although the area’s larger fauna are severely threatened by poaching, habitat fragmentation, and climate change6.Although the first emergence of pastoral economies in Mongolia remains poorly understood, recent decades of archaeological and ethnographic research point to a crucial role for mountain zones in this complex transition. The Altai Mountains form the western boundary of Mongolia and the eastern Eurasian steppes, stretching from Siberia in the northwest along the western edge of Gobi Desert in a southeasterly direction Fig. . Their h8, the Altai Mountains also yield the Eastern Steppe’s oldest direct evidence for domestic animal use—in the form of burials of the Afanasievo culture, apparent migrants from eastern Europe that brought with them sheep, goat, and cattle just prior to ca. 3000 BCE11. Following this initial dispersal of domestic livestock, the mountain margins of the Altai and Khangai were occupied by semi-mobile pastoral herders of the Afanasievo and later Chemurchek cultures, who raised domestic sheep, goat, and cattle across the Altai throughout the 3rd and early 2nd millennia BCE14. These early herders may have been constrained from using drier intermontane desert and steppe zones by their lack of horse transport12. However, by the late Bronze Age, the introduction of domestic horses, which appear widely in sites of the Deer Stone-Khirigsuur Culture (ca. 1250–700 BCE) across northern and western Mongolia, brought about widespread cultural and economic transformations15. Based on available evidence, the turn of the first millennium BCE saw the emergence of a highly mobile, multispecies pastoral economy characteristic of East Asian pastoralism, including ephemeral habitations and the use of domestic horses for meat and dairy16. Across the later mid-first millennium BCE, mortuary features show that western Mongolia continued to host pastoral horse cultures like the Pazyryk (ca. 400–200 BCE) and the Xiongnu (ca. 200 BCE-100 CE)17.Occupied by hunter-gatherers since at least the end of the Pleistocene16 suggest a strong dietary emphasis on sheep, goat, cattle, and horse among Bronze Age herders. However, widespread iconographic depictions of wild fauna—including elaborate deer images found on many deer stones18 imply that wild game still played a role in early eastern Eurasian pastoral culture.While some of these key events, such as initial dispersal of domestic livestock and the introduction of the horse, are apparent from research at monuments and funerary sites, almost no non-ritual faunal assemblages predating the late first millennium BCE have been identified in Mongolia. This absence of direct insights into the subsistence base of early herding societies prevents a more comprehensive assessment of the role played by wild taxa or environmental change in the transition to pastoralism. Those few faunal assemblages that have been recovered from this period12. As a result, comprehensive analysis of artifacts recovered from ice patches and their unique preservation conditions provide a promising line of inquiry into understanding the prehistoric use of mountain zones and animal economies of the region’s earliest herders. We conducted remote sensing and intensive pedestrian and horseback survey of melting ice at five locations near the mountain of Tsengel Khairkhan in western Mongolia, analyzing organic artifacts recovered from these localities using radiocarbon dating, collagen fingerprinting, and Scanning Electron Microscopy to reconstruct patterns of subsistence, tool manufacture, and cultural activities and assess the role of alpine zones and exploitation of wild fauna in the emergence of pastoral societies in East Asia.Recent applications of multimethodological archaeological science approaches have shown promise in expanding the range of information that can be garnered from organic material from early prehistoric assemblages in MongoliaAnalysis of remote sensing imagery identified several stable ice patches and glacial margins atop Tsengel Khairkhan and adjoining peaks. Comparing their present extent with imagery from the past three decades revealed a precipitous decline of more than 40% in total surface coverage of summer snow and ice extent at peak melt season since 1990 Fig. , Table 1Ovis ammon), and were typically represented only by the horns or the cranium. Given the site’s large size and our difficulty in reaching the site, which spans an elevation range of 3500–4000 m, we were unable to comprehensively assess the total number of animal bones at the site during 2019 fieldwork. Within the glacier, large horn curls can be seen protruding from the ice many tens of meters from the current melt margin, suggesting that the total number of individuals may extend into the hundreds.Our survey of five ice patches and one glacier margin revealed three localities bearing artifacts and ancient biological material, as well as one locality where faunal remains, with no direct evidence of a cultural origin, were recovered from periglacial pools frozen at the time of recovery , but some specimens dating as young as the late historic or modern era . The furthest dated downslope specimen was dated to the fifteenth-sixteenth centuries CE, and was found near the LIA moraine  exert a significant influence on taphonomic preservation –with some older objects preserved in low-lying, snow-covered microregions exhibiting better preservation than younger objects. Because of their location downslope, many artifacts may have also been exposed in prehistory, and secondarily transported downslope from an initial location higher on the glacier. Thus, at Tsengel Khairkhan, accumulated materials spanning nearly three millennia are emerging from the ice margin through recent melt activity. The rapid degradation of perishable artifacts and faunal remains after emergence from the ice reinforces the urgency of this kind of archaeological reconnaissance.To assess the impact of ice melting on artifact preservation and taphonomic processes at the site, we conducted an intensive survey of a ~ 20 m x 250 m area by walking transects spaced 5 m apart, extending from just below the actively melting ice margin downslope until no more faunal or ancient biologic material was found. Within this zone, we recorded every visible faunal specimen in situWe directly radiocarbon dated 34 archaeological and paleobiological specimens recovered from Tsengel Khairkhan Glacier . Finally, our analysis of an animal-hair rope artifact from Ice Patch 3, dated to the early Middle Ages , using Scanning Electron Microscopy (Supplementary Appendix A) suggests that the fiber artifact is likely made from hair of the Bactrian camel (Camelus bactrianus).Our taxonomic identification of bone, antler, and soft tissue from culturally-modified artifacts at Tsengel Khairkhan indicates that deer and sheep raw materials were selected for projectile production. While it is perhaps an unsurprising find that antler arrowheads were made from deer antler , the bone arrowhead appears to have been made from the bone of Salix sp., Table Hippophae sp.) and elm (Ulmus sp.).Our analysis of wooden artifacts through comparison with known materials from area wood taxa indicates that nearly all specimens identified during 2019 fieldwork were made of willow  for the willow compared to the sinew , which indicate that the arrow shaft was made of older wood, or curated/reused after its initial creation. This technology is consistent with the region’s sparse archaeological record for the early first millennium: arrowheads from the late Bronze Age Karasuk culture, in southern Siberia (ca. 1400–1000 BCE), tend to be made primarily of chipped stone or bone23, but similar tanged bronze arrowheads have been reported from the seventh century BCE burials at Arzhan 224. Recent excavations of a cenotaph from the site of Jargalantyn Am in north-central Mongolia revealed nearly identical tripartite bronze arrowheads with remnant hafting25 . The Tsengel Khairkhan arrow shaft itself shows that the area of the fletching was decorated with red pigment, a practice seen on late first millennium BCE arrows from the Russian Altai, that may be a means of indicating ownership or demarcating arrow type27. No fletching was recovered from the arrow, which may have been stripped off or lost post-depositionally. A small divot in the arrow’s tip may reflect impact damage.The arrow from Tsengel Khairkhan  through the early second millennium CE . Two antler arrowheads appear to have been hollowed out and “friction-fit” onto the end of a wooden shaft , does not retain its lower portion, and so the system of attachment or hafting could not be ascertained. Ease of manufacture and greater availability of antlers may have encouraged the use of organic points over metal counterparts for hunting.An important element of the Tsengel Khairkhan artifact assemblage is the relatively high frequency of bone and antler arrowheads . These finds outnumber metal projectiles at the site by a ratio of 2:1, showing strong technological continuity across thousands of years in the eastern Altai. Radiocarbon dating of three of these specimens demonstrates their use from at least the early first millennium BCE  and ca. 1050–927 cal. BCE , respectively. Other evidence for hunting, including nearly-intact arrows, comes from a separate ice patch along a small north-east facing terrace, where good cover may have given hunters more success with a stalking strategy. Here, hunters appear to have been originally positioned within bowshot of the patch, where arrows were apparently lost in the ice after being released.The spatial concentration of projectiles near the ridge that forms the glacier’s northern margin, along with the presence of other modified wooden artifacts in this area, could be a reflection of downslope transport—but it appears likely that ancient hunters utilized this natural landform to drive and ambush big game, primarily argali sheep. Almost all of the projectiles recovered by our project originated from the runnel formed by the intersection of the ice margin with the ridge’s southern slope Fig. . AdditioCervus elaphus) and Siberian ibex (Capra sibirica). At present, all three taxa inhabit high elevation zones in the study area31. Modern argali occupy a variety of ecozones, including steppe, semi-desert, desert, and mountain environments, across elevations ranging from 300 m to 5750 m30. Modern argali distribution is also shaped by competition from livestock and other wild ungulates, predation, and availability of critical resources such as water and forage36. The high frequency and visibility of argali remains at Tsengel Khairkhan imply that argali were relatively abundant at the study site in antiquity, and more detailed future study may help understand the relationship between prehistoric argali populations and climate dynamics at high altitudes in the Altai.Surface survey strongly suggests that the primary animal taxon hunted at Tsengel Khairkhan was the argali sheep, comprising the overwhelming majority of identifiable visible finds; however, we also identified the remains of red deer (5 where bighorn sheep (Ovis canadensis) skulls and horn cores are the predominate faunal element at ice patch sites. Future zooarchaeological analysis will be necessary to assess evidence for butchery on argali remains at Tsengel Khairkhan, but it is possible the presence of large and unwieldly horns and antlers and the difficulty of mountain transit factored into the choice of leaving behind cranial elements. Our isolated finds of sinew may support the idea that animals were at least partially butchered near the glacial margin after a successful hunt.While we did not conduct a comprehensive zooarchaeological study, the predominance of cranial elements and horns visible at the surface suggest that decapitation at the kill site was a standard practice at Tsengel Khairkhan from the first millennium BCE. Interestingly, cranial or mandibular elements also comprised the vast majority of identified materials at ice patches in the Greater Yellowstone area of the United Statesovoo, or stone cairn37. During the late Bronze Age, the ritual placement of the head, neck, and hoof bones of deceased horses was standard practices at burials and monument sites of the DSK complex (ca. 1200–700 BCE)39. We radiocarbon dated one argali skull pile at Tsengel Khairkhan, consisting of the horns and crania of at least five individuals, to ca. 250 CE . Based on their size and the pattern of horn sheath growth rings, all of the skulls in this arrangement seem to be large, older males, which might have been preferentially hunted for their larger body size and/or cultural significance. While we cannot rule out the possibility that the piles we identified at Tsengel Khairkhan were created by later visitors using ancient skulls, our findings raise the possibility that following a successful hunt, early pastoralists afforded similar respect to their prey, leaving the skull of the animal atop the mountain intentionally out of respect or spiritual practice, as observed in ice patches at the Greater Yellowstone Area, USA, with bighorn sheep5.Our results also reveal that hunting activities played an important role in the worldview and spiritual beliefs of early pastoralists, as revealed by the treatment of argali sheep skulls at Tsengel Khairkhan Fig. . Nearly Ovis through collagen fingerprinting) indicates that this specimen is of modern origin but at least several decades old . Other materials from near this patch, including a horseshoe and nails, appear to indicate horse-riding activity in recent centuries. At the slightly higher elevation Ice Patch 3 (~ 3250 m), we also identified a rope fragment made of animal hair, identified as Bactrian camel through SEM, from the fifth/sixth centuries CE. This artifact could represent a component of a bridle/halter, as similar artifacts have been recovered from Xiongnu tombs40, or could have been used for securing pack bags during transport or hunting.Archaeological finds from lower-elevation patches at the nearby location of Khultsuut also show the use of mountain zones for other activities, including transport and herding. Present-day herders use the area around Ice Patches 1 and 3 for pasturing sheep and goats during the summer months. It is likely that these high mountain patches—fed by melting snow and ice—provided a seasonally-productive summer pasture for pastoral purposes in the past as well. Radiocarbon dating of a single sheep tooth from Ice Patch 1 . Hunting of argali was also not a purely pragmatic act—piles of skulls and horn sheaths, formed intentionally along the glacial margin, appear to represent intentional post-hunt offerings or spiritual behavior. Together, this work shows that big game hunting was an integral aspect of early herding life.This research presents the first direct evidence for Bronze Age organic weapon manufacture and hunting strategies in the Mongolian Steppe. Only stone and metal arrowheads are typically recovered from surface surveys, but our glacier finds show that antler and bone arrowheads—made from deer antler and bone of 38. Monumental standing stones of this culture are often decorated with large deer, and deer (probably Cervus elaphus) and wild game form an important component of Bronze and Iron Age assemblages in adjoining regions of Central and Northeast Asia42. To date, however, no archaeological projects have identified evidence for wild game consumption in DSK or other early Mongolian pastoral cultures. However, our inference of a major cultural and economic role for Ovis ammon across the hunting/herding transition in the Altai and other areas of eastern Eurasia finds some support in the petroglyph record, where animals such as argali and ibex are frequently depicted43  criteria, and populations are highly fragmented across the country47. The situation is exacerbated by poorly regulated ‘hunting tourism’ and an opaque permitting system49. In 2019, Donald Trump, Jr. was widely criticized for a trip to the study region in which two argali sheep were hunted and exported using a permit that was not issued until after the hunt was completed50. This intense hunting pressure, along with a warming climate and changes in livestock density have fragmented the distribution of argali in Mongolia, creating small, isolated populations that are more prone to extinction.While these impacts of summer warming on Altai ice are pernicious, the present study also indicates that diminishing wildlife populations portend serious deleterious impacts on the future of pastoral herding in the Altai. Ice patch research in other areas of the world has shown that mountain hunting served as a bulwark against periods of low productivity or stressOur new findings demonstrate that the decline of wildlife populations is not only a conservation concern for species like argali, but also a threat to the viability of pastoral lifeways. Archaeological materials from Tsengel Khairkhan show that since the Bronze Age, herding societies have relied upon big-game hunting in mountain zones to provide additional subsistence resources, and that these species have also played a central role in ceremony, identity and belief systems. Argali hunting has therefore remained a key aspect of lifeways in the Altai for at least 3000 years. The modern loss of big game populations, and accompanying restrictions on legal hunting for residents, may compound issues related to climate change and ice loss and negatively impact the viability and resilience of pastoral lifeways. As populations decline further, the loss of big game will likely continue to reduce the region’s ecosystem functioning and viability for eco-tourism.While challenges in preservation have to this point prevented a clear assessment of the role of montane zones in the emergence of pastoral societies in eastern Eurasia, results from ice patch archaeological survey show that alpine hunting played an important role in pastoral subsistence strategies since at least the final Bronze Age. Our results provide the first direct insights into projectile construction and hunting strategies of the late second and early first millennium BCE, indicating that perishable antler and bone points were often used for hunting . Projectile artifacts show that mountain glaciers and ice patches were used for ambush hunting with arrows, darts or spears, which were in use during the late Iron Age. The impact of hunting was not limited to subsistence—raw material selection choices and the placement of argali sheep skulls and horn sheaths suggest a post-hunt ritual treatment, underscoring the impact of big game hunting in cultural or spiritual beliefs. As the climate warms and big game populations continue to decline from overhunting and illegal wildlife trade, the loss of mountain ice and wildlife will likely undercut the resilience of traditional herding lifeways in a rapidly changing world.Using publicly available satellite (LANDSAT 7) imagery taken during the late summer (August and early September), we analyzed the area for ice patches that appeared to be permanent during the period 2010–2018 and exhibited a flat forefield potentially conducive to artifact preservation. From this dataset, we identified five ice patches of interest. Through discussions with local collaborators, we also identified a sixth locality along the margin of the Tsengel Khairkhan glacier itself, where climbers reported finding wood and antler projectiles during prior summers. For pedestrian survey, we surveyed the margin of maximum melt extent  using a handheld GPS. At all ice patch locations, we conducted systematic surveys downslope from the ice margin until reaching the edge of lichen-free areas (which provide a proxy for regularly ice-covered zones) at 10 m transect intervals. At the Tsengel Khairkhan glacier, due to logistical constraints we were only able to conduct two 10 m transects downslope from the ice margin, and one area of full coverage survey using 5 m transects Fig. . In the 19. All wooden artifacts and a selection of animal bones were sampled for radiocarbon dating at the AMS Laboratory at the University of Oxford, United Kingdom and the University of Arizona AMS Laboratory in Tucson, Arizona. Each radiocarbon date was calibrated using OxCal  using the INTCAL20 calibration curve, and duplicated dates were combined in OxCal using the ‘R_Combine’ function. We sampled each artifact made of bone, antler, or sinew for taxonomic identification through collagen fingerprinting at the ZooMS Laboratory at the Max Planck Institute for the Science of Human History in Jena, Germany, following protocols outlined in Taylor et al.12.Next, we conducted osteological and biomolecular analyses of recovered artifacts to assess their age, preservation, and material composition. For bone objects, we made taxonomic identifications using reference images, and assessed the state of weathering for each recorded specimen based on criteria from Behrensmeyer52. Glacier outlines were derived from Landsat 5 Thematic Mapper (TM), Landat 8—OLI, and Sentinel-2A imagery classified using a Near Infrared/Shortwave Infrared band ratio. For these glacier outlines, all glaciers identified in the band ratio needed to meet a minimum size threshold of 0.01 km2. Remote sensing analysis and figures were performed using QGIS 3.10 (http://www.qgis.org).Finally, we analyzed the impact of contemporary ice loss on the study area using remote sensing. To calculate ice loss in the region over the last three decades, we used glacier outlines from Tsengel Khairkhan for 1990, 2000, 2010, and 2016 available from the Global Land Ice Measurements from Space (GLIMS) databaseSupplementary Information.


Floating ice shelves are the Achilles’ heel of the Antarctic Ice Sheet. They limit Antarctica’s contribution to global sea level rise, yet they can be rapidly melted from beneath by a warming ocean. At Filchner-Ronne Ice Shelf, a decline in sea ice formation may increase basal melt rates and accelerate marine ice sheet mass loss within this century. However, the understanding of this tipping-point behavior largely relies on numerical models. Our new multi-annual observations from five hot-water drilled boreholes through Filchner-Ronne Ice Shelf show that since 2015 there has been an intensification of the density-driven ice shelf cavity-wide circulation in response to reinforced wind-driven sea ice formation in the Ronne polynya. Enhanced southerly winds over Ronne Ice Shelf coincide with westward displacements of the Amundsen Sea Low position, connecting the cavity circulation with changes in large-scale atmospheric circulation patterns as a new aspect of the atmosphere-ocean-ice shelf system. New data from five hot-water drilled boreholes show how atmospheric anomalies affect the circulation beneath Filchner-Ronne Ice Shelf on multi-year time scales. The apparent link of the dense water formation to remote teleconnections is an important step for better predicting contributions to future sea level rise from this sector of Antarctica. Over recent decades ongoing ocean-induced ice shelf thinning has driven increased mass loss from the Antarctic ice sheet2. Hence, understanding the fate of oceanic heat that flows toward the cavities beneath Antarctic ice shelves is one of the greatest challenges for determining future sea level. Located in the southern Weddell Sea, Filchner-Ronne Ice Shelf  that drives a thermohaline circulation in the southward-deepening ice shelf cavity5. As a result of the pressure dependence of the melting point of ice, HSSW cooled to surface freezing temperatures can drive basal melting over the deepest parts of the ice shelf13, creating a colder, but fresher, and therefore slightly less dense14 water mass known as Ice Shelf Water (ISW). ISW is primarily exported from beneath FRIS through the Filchner Trough16 that connects the ice shelf cavity with the deep ocean. At present, these outflows are dense enough to block the inflow of Warm Deep Water that circulates along the Weddell Sea continental shelf break17. For a warmer and wetter future climate, model simulations suggest that HSSW production is reduced far enough that the ISW density barrier will be eroded and warmer waters will be able to flush the FRIS cavity18. However, the uncertainties in these projections are large19 and the likelihood of such a regime shift due to ongoing climate change remains largely unknown.Increased mass loss from FRIS can be triggered by a drop in the production of dense water on the continental shelf that currently prevents warmer offshore waters from entering the FRIS cavity20 beneath FRIS from five hot-water drilled boreholes (Methods). Together with ship-based observations from the ice shelf front22, the data show that since 2015 a strengthening in wind-driven sea ice formation in the Ronne polynya caused an intensification of the density-driven circulation in the FRIS cavity. We observe dense water pulses propagating from Ronne Ice Shelf and driving a mode shift of the circulation under the northern Filchner Ice Shelf that affects the water mass properties in the Filchner Trough on interannual time scales. Combining our observations with analysis of satellite-based sea ice concentration24 and atmospheric reanalysis products25 further suggests that the sustained dense water production is related to recent atmospheric anomalies, in particular the position, and possibly the strength, of the Amundsen Sea Low (ASL) that occurred during an increasingly positive Southern Annual Mode (SAM). These findings further our understanding of how this sector of the marine-based West Antarctic Ice Sheet may respond to changes in large-scale climate and the implications for future sea level rise.This study employs new observations of water masses, currents, and basal melt ratesThe first 9–12 months of the ocean time series (starting in December 2016) from the northern drill sites on Filchner Ice Shelf  and 34.87 g kg−1 (34.7 psu) at FNE1 and FNE2   transects in front of Filchner Ice Shelf  in the southern Weddell Sea (Methods) reveal that extended periods of open water in front of western Ronne Ice Shelf have fostered anomalously high SFRs since 2015 Fig.  before R 33 Fig. . In part 33 Fig. , followe 33 Fig. , stands  33 Fig. .−1 at the upper and lower current meter deployed at Site 5  show southward velocities at the lowermost instrument that prevail during the observational period regardless of the mode shift Fig. . During FSE Fig. , howeverode Fig. . A recenMore subtle changes in flow velocities are seen at the mid-depth and upper current meters at FNE1 Fig. , where n12, and an increase again from 2015. Some clusters of enhanced SFR events  per decade between 1978 and 2019 (2015). Although correlations between SAM and SFRs are hardly significant on monthly time scales .To assess which atmospheric conditions may promote the Ronne mode beneath FRIS, monthly anomalies of surface pressure (SP), wind speed, and direction derived from ECMWF ERA-Interim38. Variability in the depth and location of the ASL strongly affects climate and sea ice conditions in West Antarctica by influencing the meridional wind fields40. The position of the ASL is related to the existence (or non-existence) of an anticyclone in the EBS and thus the direction of the general flow across the ice shelf edge , indicating westward ASL movements as a factor promoting enhanced sea ice formation and hence dense water production in the southern Weddell Sea. Indeed, the recent transition from a phase of decline in polynya activity12 to enhanced SFRs that reinforced the Ronne mode beneath FRIS coincides with a shift from a more eastward to a more westward ASL position , a climatological low pressure area in the Pacific sector of the Southern Oceandge Fig.  a. Monthion Fig. . Negativion Fig.  togetherion Fig. .r = −0.69). The increased ice transport northward from the Ronne Ice Front is supported by an increased sea ice export from the northern Weddell Sea due to recently stronger westerly winds under a more positive SAM. Note that the ice drift deviates slightly to the left of the main wind direction41, which is of minor importance for the clockwise transport around cyclones, but in the case of strong westerly winds leads to a northward displacement of the ice (edge), which releases pressure from the ice pack further south. In particular, this was the case in austral spring 2016/2017, which exhibited an extraordinarily early start of the melt season, and with positive anomalies in sea ice extent in the Weddell Sea related to atmospheric forcing43. This mechanism would be consistent with the apparent delayed increase of SFRs one season after a more positive SAM. Also, the Maud Rise polynya, which has earlier been linked to wind-driven upwelling was more pronounced during that period44, as well as exceptionally warm and prolonged inflows of Warm Deep Water that were observed on the eastern continental shelf in the southern Weddell Sea45. Although the exact mechanisms controlling each of these anomalies remain to be resolved, their coincidence may point at a common origin, such as general intensification of the circulation associated with a strong positive SAM that persisted over this period.While positive SP anomalies in the EBS during enhanced SFR events appear largely independent of the long-term trend, the frequent occurrence of extreme low pressure events in the EWS since 2015 aligns with an overall decreasing SP anomaly as part of the more positive SAM  that maintains the dense water outflow in the Filchner Trough. This transition from a more localized overturning circulation under the northern Filchner Ice Shelf (Berkner mode) was caused by pulses of Ronne HSSW that propagated through the cavity along different pathways. Herein, the ice shelf acts as a delayed low-pass filter, such that the variability of HSSW inflows from the Ronne polynya affects water mass properties in the Filchner Trough on interannual time scales.11, further work is required to find out whether the reinforcement of the Ronne mode is a persistent trend or whether it stems from the internal variability of the system.Our analysis further suggests a predominantly wind-driven control on SFRs in front of Ronne Ice Shelf in the present climatic regime. The anticyclonic atmospheric circulation that intensifies sea ice formation in the southern Weddell Sea is driven by synoptic-scale pressure anomalies that are linked to variability of the Amundsen Sea Low and the Southern Annual Mode. While this differs from the suggested gradual decline in dense water production that would make FRIS more susceptible to warm inflows in response to future climate change46. A sustained Berkner mode would eventually decrease the ISW density on the eastern part of the continental shelf, causing a potential feedback on the Berkner HSSW production and weakening the density barrier11 that prevents modified WDW from entering the ice shelf cavity18. Although varying amounts of remotely sourced Ronne ISW and more locally produced Berkner HSSW have been observed in front of Filchner Ice Shelf . However, most of the meltwater from FRIS is currently exported through the Filchner Trough and does not affect the western continental shelf regions where the densest HSSW is produced that drives the Ronne mode. This indicates that the polynya activity in front of Ronne Ice Shelf really is the main regulator of the circulation of the southern Weddell Sea continental shelf.HSSW formation is also influenced by glacial meltwater inflow49. It depends, however, on the exact response, whether this should support stronger southerly winds over the Ronne Ice Shelf and an increase in the area of open water that facilitates increased dense water production at the ice shelf front. Changes in ASL location and strength were previously employed to explain the interannually varying warm water inflows toward ice shelves in the Amundsen Sea51. Our results imply a potential see-saw behavior between basal melt rates in that region and dense water production in the southern Weddell Sea on interannual time scales. However, the complex relationship between the ASL and hemispheric modes of climate variability  paired with the extremely high interannual variability of SP in the Amundsen-Bellingshausen and Ross Seas regions, hampers accurate predictions of the longitudinal position and strength of the ASL in a changing climate. In particular, the Antarctic Dipole56 connects tropical signals to Antarctic sea ice, where the Atlantic and Pacific centers of the dipole react in opposite ways to temperature anomalies in the tropics. Warm ENSO events are related to higher (lower) temperatures and less (more) sea ice than on average in the Pacific (Atlantic) and a persisting high-pressure center in the Bellingshausen Sea, the response usually lagging the tropical signal by several seasons. A very strong warm ENSO event in 2015/16 (defined using the ONI (NOAA)) might be connected to the increase in SFR after 2016 found in this study. Those ENSO-induced anomalies in Antarctica are modulated by SAM depending on their phase relationship58. McKee et al. found54 statistically significant relationships between Weddell Sea Bottom Water temperature and ENSO/SAM. They suggest that dense shelf water production might be influenced by anomalous winds via modulating the fraction of open water over the shelf. Our study proves this hypothesis and similar mechanisms were found for the Ross Sea in the Pacific sector of the Southern Ocean59, which is under more direct influence of ENSO.Our study emphasizes the link between the atmospheric forcing and the oceanic circulation, which is shown by our measurements on an interannual-scale. Long-term implications of the described physical mechanisms depend on changes in the mean atmospheric conditions above the Southern Ocean. The majority of climate models exhibit an increasing SAM index in the 21st century, associated with a deepening and change in location of the ASL61 that deserves further attention. Reduced sea ice formation rates in a warmer, wetter climate have been suggested as the future paradigm for the southern Weddell Sea11. The mechanisms revealed by our study imply potentially wide ranging teleconnections and lend atmospheric projections predictive power over ocean conditions beneath an ice shelf. That is, if we know how the remote atmospheric patterns are likely to change (through coupled climate models), we can improve predictions about the future sea ice formation rates that will dominate the long-term response at the Filchner-Ronne Ice Shelf.Although the statistical evidence from short available time series is weak, our results highlight a new aspect of the atmosphere-ocean-ice shelf system33; in 2015/16 at a southeastern site (FSE) and a southwestern site (FSW) beneath the Filchner Ice Shelf; and in 2016/17 at two northern sites (FNE1 & FNE2) beneath the Filchner Ice Shelf  and six (FSE) calibration profiles through the sub-ice shelf water column at each site prior to the mooring deployment. To minimize icing in the conductivity cell, the instrument was stored in a heated bath at the surface of the ice shelf, but sometimes needed to flush for a few minutes below the ice shelf base until conductivity readings had stabilized. CTD profiles were corrected after post-calibration of the instrument and the raw data were smoothed, corrected for thermal inertia of the cell and averaged into 1 m vertical bins using the SeaBird processing software. Absolute salinity and conservative temperature were computed using the TEOS-10 Gibbs thermodynamic potential for seawater. For comparability with earlier studies, source water salinities are also stated in units of practical salinity, following the Unesco 1983 algorithms for computation of fundamental properties of seawaterAll CTD data were interpolated on a common 2-hourly time vector and then smoothed with a 27-day Hanning window moving-average filter using the jlab MATLAB-toolbox. Time series from temperature sensors of all five instrument-depths at FNE1 are shown in Fig. 21 and in January 2017 during cruise JR16004 with RRS James Clark Ross22, are used to provide context for the sub-ice shelf measurements  space. Neglecting minor corrections due to heat conduction at the ice–ocean interface, the slope of these lines is given as −1K−1 and latent heat of fusion −1 and salinity 26 to discriminate between Berkner HSSW and Ronne HSSW source water types for FRIS ISW. Assuming that ISW being observed at the mooring sites originated from HSSW on the continental shelf, the “source water salinity” of this HSSW is found as the intersection of the ISW’s Gade-line with the surface freezing point temperature. Based on this, time series of source water salinity was computed from the filtered in-situ temperature and salinity data from the lowermost CTD instrument at each ice shelf mooring  and the difference between the 2 m air temperature (T) and the ocean surface freezing temperature, computed as T. Time series of 0.125 degree spherical grid resolution atmospheric fields were interpolated onto the 25 km x 25 km polarstereographic SIC grid. To avoid edge effects near the time-evolving ice front, only SIC grid points that are masked as ocean points during the entire time series are being used. Despite their relatively low spatial resolution, these products provide long time series, and comparison of the obtained SFRs with estimates from shorter higher resolving sea ice concentration products, as well as sea ice assimilating model simulations21 yield similar results for the overlapping time periods, confirming the robustness of the method.To assess the evolution of HSSW production on the southern Weddell Sea continental shelf, daily averages of open-ocean heat loss were calculated as a proxy for sea ice formation rates (SFRs), using passive microwave sea ice concentration (SIC) from the Special Sensor Microwave Imager/Sounder (SSMIS)U were computed as the arithmetic average of all grid points within the polygon at the Ronne Ice Shelf front shown in Fig. Spatially varying fields of absolute SFR anomalies averaged from 2015 to 2018, relative to the period from 2002 to 2018, are shown in Fig. U are shown in Supplementary Table Correlations between monthly anomalies in sea ice formation, SIC, entclass1pt{minimaentclass1pt{minima36 downloaded from https://legacy.bas.ac.uk/met/gjma/sam.html  as well as a reanalysis product of Amundsen Sea low (ASL) indices49, version 2 downloaded from https://scotthosking.com/asl_index . Atmospheric composites and time series of seasonally corrected pressure anomalies were computed using monthly averaged 0.125 degree ERA-Interim surface pressure (SP) and 10-m wind velocity vectors and speed fields. Time series of local seasonally corrected SP anomalies in the eastern Weddell Sea (EWS) and the eastern Bellingshausen Sea (EBS) were calculated from spatial averages of all grid points within a radius of 50 km of the locations indicated in Fig. Large-scale climate variability is assessed using monthly time series of the observation-based Southern Hemisphere Annular Mode Index (SAM index)p = 0.01. Covariance was computed for monthly and 36-month low-pass filtered time series of SFRs and SAM index with lag between 0 and ±18 months , one of which was deployed approximately 6.3 km west of FNE1, the other being deployed at FNE1 itself , non-melt-induced variations in thickness are difficult to extract. This is important for tidally induced vertical strain, which is expected to make a strong contribution in this region. For the purposes of this study, in which longer-term variability is of interest, this difficulty can be simply overcome by filtering the signal to remove variability at time scales of the dominant tidal frequencies.−1.Here, 5-day bin averages were computed from the hourly data (and including data from both instruments in the overlapping period in 2017) to obtain basal melt rate time series Fig.  uncontamSupplementary InformationPeer Review File


Both ERK1/2 and Akt kinases activate mTORC1, but only the former is bidirectionally regulated by the status of serine S1364 on TSC2 that confers input-selective mTORC1 amplification or attenuation. Tuberous sclerosis complex-2 (TSC2) negatively regulates mammalian target of rapamycin complex 1 (mTORC1), and its activity is reduced by protein kinase B (Akt) and extracellular response kinase (ERK1/2) phosphorylation to activate mTORC1. Serine 1364 (human) on TSC2 bidirectionally modifies mTORC1 activation by pathological growth factors or hemodynamic stress but has no impact on resting activity. We now show this modification biases to ERK1/2 but not Akt-dependent TSC2-mTORC1 activation. Endothelin-1–stimulated mTORC1 requires ERK1/2 activation and is bidirectionally modified by phospho-mimetic (S1364E) or phospho-silenced (S1364A) mutations. However, mTORC1 activation by Akt-dependent stimuli (insulin or PDGF) is unaltered by S1364 modification. Thrombin stimulates both pathways, yet only the ERK1/2 component is modulated by S1364. S1364 also has negligible impact on mTORC1 regulation by energy or nutrient status. In vivo, diet-induced obesity, diabetes, and fatty liver couple to Akt activation and are also unaltered by TSC2 S1364 mutations. This contrasts to prior reports showing a marked impact of both on pathological pressure-stress. Thus, S1364 provides ERK1/2-selective mTORC1 control and a genetic means to modify pathological versus physiological mTOR stimuli. The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth, synthetic activity, metabolism, and protein homeostasis . MTORC1 Given the multiplicity of signaling kinase inputs that rarely occur in isolation, their interactions and hierarchy is important to understand, yet this remains under studied. A dominant mechanism of TSC1/TSC2 regulation involves its intracellular localization to or away from the lysosomal membrane where Rheb and mTORC1 reside . Nutrienq,11-GPCR agonist such as endothelin-1 (ET-1), or cardiac pressure-overload, mTORC1 activity and associated pathological growth and disease are markedly amplified when a S1364A mutation is expressed and equally markedly attenuated with a S1364E mutation  or mTORC1 (rapamycin) inhibition (Myocytes expressing the phospho-silenced mutation TSC2hibition . Our prihibition  showed aS1364A (C42S) that enhances its localization to the plasma membrane and in turn regulation of S1364. One way to explain these results is that unlike PDE5 inhibition, the two other modes of PKG activation also suppress ERK1/2 activation proximal to TSC2. If so, then our current data indicate this should circumvent a S1364A mutation. We tested this hypothesis in NRVMs expressing both TSC2S1364A and PKG1αC42S . Insulin-activated Akt and S6K in MEFs, co-activating ERK1/2 as well  but to aC2S1364E . This coC2S1364E . Cells eC2S1364E . We foun mutants  and S2B.S1364E had reduced p/t S6K overall (P = 0.02 for genotype-ERK1/2 inhibition interaction). A small but significant increase in p/t S6K from thrombin in MEFs with TSC2WT or TSC2S1364E and co-ERK1/2 inhibition was statistically similar and likely reflected unaltered effects from Akt activation  inhibitors or vehicle control. S1364 phosphorylation increased with ET-1 and was unaltered by co-ERK1/2 inhibition yet blocked by co-PKG inhibition  potently altered mTORC1 activation and the corresponding pathophysiology  is considered central to the pathophysiology, contributing to fat deposition in the liver , type-2 Most of the experiments in this study stimulated pathways using receptor ligands as opposed to over-expressing a constitutively active kinase. We chose this approach to generate acute changes rather than have prolonged signaling that could trigger other compensations. Although these ligands do not trigger only one pathway , their balance  were sufficiently different to address the hypothesis. We also used selective kinase inhibitors rather than genetic silencing methods, as the latter may alter other signaling as well. We have not yet identified the intra-molecular mechanism by which TSC2 S1364 selectively modulates ERK1/2 but not Akt signaling. However, recent structural data and ongoing efforts should pave the way for such insights in the future . The finTo our knowledge, S1364 is the first TSC2 regulatory site revealed that leaves basal mTORC1 signaling intact despite mutagenesis to a phospho-silenced or mimetic form yet confers selective modulation of mTORC1 activity upon co-stimulation with bias to ERK1/2 but not Akt activation. From a translational perspective, the results open opportunities to modulate TSC2-mTORC1 regulation in a more selective manner, with potential applications to cell therapies for cancer and autoimmune disease. From a general perspective, they reveal how phosphorylation of TSC2 can confer selective amplification or attenuation of other kinase inputs via the same protein to control net mTORC1 activity.WT, TSC2S1364A, or TSC2S1364E  protein using either adenovirus or plasmid expression vectors applied at reported MOI  were freshly isolated as previously described  and cultrted MOI  for 3–4 rted MOI . PlasmidThe following pharmaceuticals were used: recombinant endothelin-1 , thrombin , PDGF-BB , ERK1/2 inhibitors , Akt inhibitor , insulin , Torkinib , Rapamycin , PDE9 inhibitor , PDE5 inhibitor , and soluble guanylate cyclase activator .Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) , and p-TSC2 S1365 (mouse) #120718 . Total protein was determined with Revert Total Protein Stain . TSC2 S1364A and S1364E mutants (human TSC2) both plasmid and adenovirus were generated as previously described . PKG1α CS1364A, or TSC2S136E mutants, at 10 MOI. After 48 h in culture, cells were placed in serum-free media (DMEM) overnight and stimulated with activators  for 15  or 30  minutes.In studies using NRVMs, cells were stimulated with endothelin-1 (ET-1 100 nM for 15 min). MEFs were stimulated with either thrombin (100 nM thrombin for 15 min), insulin (0.5 μM for 30 min), or platelet-derived growth factor , all dissolved in distilled water. In these studies, cells were further pretreated with ERK1/2 inhibition (SCH772984 or U0126 [both 10 μM]) and Akt inhibition  each provided 1 h before stimulation. These doses and incubation times were based on screening assays for inducing Akt, ERK1/2, and S6K activation. In another experiment, NRVMs were pre-incubated with phosphodiesterase type 9 , type 5 , or guanylate cyclase activator . In many of the studies, cells were pre-transfected (plasmid) or infected (adenovirus) to express TSC2 WT, TSC23, 1.2 mM KH2PO4, 1.2 mM CaCl2, 1.25 mM MgSO4, 5 mM sodium lactate, 8 mM KCl, 20 mM Hepes, and 20 mM 2-deoxyglucose (pH adjusted to 6.6) as described , Akt , PKG , or vehicle for 1 h before ET-1 (100 nM), insulin (10 μg/ml), or vehicle for 15 min. Cells were then lysed and assayed for TSC2 S1364 phosphorylation.oC overnight. A total protein stain  was used at 5–10 ml per membrane. Antibody binding was visualized with an infrared imaging system  and bands were quantified with Odyssey Application Software 3.1.Cells were lysed in lysis buffer  and protein concentrations determined by BCA assay (Pierce). Samples were prepared in Licor protein sample loading buffer (LI-COR) or SDS Tris-glycine buffer  and run on TGX 7.5% or 4–20% Tris-glycine gels  and blotted onto nitrocellulose membranes. The membranes were blocked in blocking buffer , and incubated with primary antibodies diluted in antibody buffer  at 4Age matched male mice harboring homozygous KI mutations for SA (n = 8), SE (n = 14) or littermate WT controls (n = 14) were fed 60% high fat diet  for 18 wk, starting at age 10–12 wk. An additional group of n = 3–5 mice of each genotype were placed on a standard chow diet for the same time. Serial weights were obtained every other week. After 18 wk resting plasma glucose and glucose tolerance tests were obtained after overnight fasting (18 h). For the glucose tolerance test, the mice were injected intraperitoneally with glucose (1 g/kg) and a baseline glucose measurement was taken from a tail-clip blood droplet using a glucometer. Blood glucose was then measured serially at 15, 30, 60, and 120 min. At the end of the protocol, livers were harvested for histology and fixed in SafeFix Fixative. Paraffin-embedded, dewaxed, 5-μm sections were stained using hematoxylin and eosin. The study was approved by the Johns Hopkins Animal and Care Use Committee in accordance with NIH guidelines on the humane care and use of animals.Data are mostly analyzed by one- or two-way ANOVA or by nonparametric Kruskal–Wallis or Mann–Whitney U tests, as appropriate. Each test is identified in the figure legends. Multiple-comparisons tests  were used to obtain within group comparisons, and results are also provided in the legends. Statistical analysis was performed using Prism Version 9.0.The study does not report any large-scale data bases, sequences, computation models, or atomic coordinates. We have provided original gels for all data displayed in the manuscript, as well as source data for all summary figures. Each figure is accompanied by a PDF file with the original gels, and an Excel spreadsheet providing the numerical data shown in the summary graphs.


Danio rerio) are ideal sentinels for detecting environmental changes due to their biomedical tool kit, widespread geographic distribution, and well-characterized phenotypic responses to environmental disturbances. Here, we demonstrate the utility of zebrafish sentinels by characterizing phenotypic differences in wild zebrafish between two field sites in India. Site 1 was a rural environment with flowing water, low-hypoxic conditions, minimal human-made debris, and high iron and lead concentrations. Site 2 was an urban environment with still water, hypoxic conditions, plastic pollution, and high arsenic, iron, and chromium concentrations. We found that zebrafish from Site 2 were smaller, more cohesive, and less active than Site 1 fish. We also found sexually dimorphic body shapes within the Site 2, but not the Site 1, population. Advancing zebrafish sentinel research and development will enable rapid detection, evaluation, and response to emerging global health threats.Environmental change poses a devastating risk to human and environmental health. Rapid assessment of water conditions is necessary for monitoring, evaluating, and addressing this global health danger. Sentinels or biological monitors can be deployed in the field using minimal resources to detect water quality changes in real time, quickly and cheaply. Zebrafish ( Danio rerio) can serve as effective biological monitors for site differences because of their widespread distribution [Environmental change is rapidly accelerating, producing profound changes in natural environments ,2,3,4,5.ribution  and techribution .Ecosystems are differentially impacted by environmental change due to the diversity of environmental conditions among regions ,25,26. CMany aspects of environmental change including temperature, oxygenation, water flow, and pollution alter morphology and behavior in multiple taxa ,44,45,46Zebrafish are powerful model organisms utilized in multiple disciplines due to their vast biomedical toolkit and well-characterised phenotypic and genotypic changes in response to environmental change ,54,55,56Here, we present simple morphometric and behavioral measures that can be conducted at various field sites by both specialists and non-specialists to detect local site differences using zebrafish. All analyses can be performed rapidly in real-time using free and open-source software, allowing for expeditious assessment and minimal financial burden on investigators. Our goal is that these assays be used for community scientists living where zebrafish are found to facilitate the evaluation of environments and to guide decisions to improve health outcomes. To highlight the utility of wild zebrafish as sentinels using these proposed metrics, we characterized phenotypic differences in zebrafish sampled from two Indian populations that differed in environmental conditions. One population (Site 1) was obtained from a rural environment with flowing water with low-hypoxic conditions that had minimal human-made debris and iron and lead concentrations that exceeded acceptable limits, and the other population (Site 2) was from an urban environment with still water with hypoxic conditions, plastic pollution, and arsenic, iron, and chromium concentrations that exceeded acceptable WHO and BIS arsenic limits ,67,68,69We conducted a field study at two field sites in India during the post-monsoon season in November 2017. Wild zebrafish were previously studied at these field sites ,70,71,72We sampled zebrafish from a rural site (Site 1), a fast-moving river in West Bengal, northeast India (a tributary of the Torsa River in Cooch Behar) with low normoxic conditions. Site 1 had arsenic concentrations below (0.01 mg/L), iron (0.3 mg/L) and lead (10 µg/L) concentrations above the WHO and BIS acceptable limits, and was situated in a location with a low human population density (~700 persons/km²) ,73. The All procedures involving animals were approved by Arizona State University’s Institutional Animal Care Use Committees under protocol 17-1596R on 22 June 2017, and complied with the existing rules and guidelines outlined by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, the Institutional Animal Ethics Committee (IAEC), and the Indian Institute of Science Education and Research (IISER) Kolkata.Following a post-capture acclimation period of at least 1 h, we divided zebrafish into equal groups of 6 fish, creating 12 groups per field site. Before behavioral testing, we photographed all fish from above. All photos were taken at the same height and with a scale within the frame to allow for morphological analysis. We then sequentially placed zebrafish into a white plastic tank (dimensions 30.8 cm × 34 cm) filled to a height of 5 cm with habitat water, where they acclimated for an additional 5 min before behavioral assessments, giving a total acclimation time of 65 min. The tanks were recorded from above using a GoPro Hero 5 Session camera. The acclimation period appeared sufficient as the fish did not freeze and displayed exploratory behavior consistent with an absence of stress responses ,81.We extracted behavioral data from video recordings using ToxTrac video tracking software in a 3 min sample period ,83. We it-tests between populations.We categorized video data as either Social measures or Activity measures. Social measures analyzed were inter-individual distance , time in proximity (number of seconds spent within two body-lengths of a group member), and nearest-neighbour distance (average distance to the closest group member). As the threshold for size detection for video tracking was most reliable between 400–500 pixels for both groups, we standardized the distance used for the two-body-length metric as 1000 pixels. Pixel data were converted to millimeters for statistical analysis. Activity measures analyzed were distance traveled , swim speed (the number of mm traversed divided by the sample duration), tank exploration (mm² of the tank visited), and number of seconds in motion. We collected data for each individual and collapsed data into tank means for analysis between levels of Population to account for the independence of observations, leaving a total of 12 observations per field site. As residuals were normally distributed we analyzed behavioral data with Welch’s two-sample t-tests. Finally, we calculated standard length as the distance between landmarks 1 and 4 (length from the anterior to posterior points of the body) and analyzed length data with a Kruskal Wallis test. All morphometric analyses were conducted using the geomorph [We collected morphometric and geomorphometric data using the TPS series software . We placgeomorph  and lme4geomorph  packagesF(1140) = 3.44, p = 0.001), as well as a non-significant trend for an interaction between Population and Sex (F(1140) = 1.137, p = 0.07). Consensus shape differed between sexes, but did not reach statistical significance, F(1140) = 0.872, p = 0.19. To characterize these differences in morphology between populations, we then analyzed body size ratio and standard length.We found distinct fish shape differences between the two populations. We analyzed partial warps of fish using PCA and found that the first two axes explained 74.69% of the total variance in body shape . Morphomp < 0.0001). Further analysis showed that the ratio between posterior and anterior body regions of Site 2 male fish  was 22% smaller than Site 1 female fish , 21% smaller than Site 1 male fish , and 17% smaller than Site 2 female fish   = 7.79, p = 0.01. When analyzed with Tukey’s post-hoc test, we found that all fish had relatively larger posterior regions than Site 2 male fish . This shows that Site 1 fish and Site 2 female fish have a significantly more torpedo shape with elongated posterior ends than male fish from Site 2 [We compared the relative area of the posterior body region  to the anterior body region  . The pos = 0.03) . This dim Site 2 .H(78) = 101.15, p = 0.040 , measuring more than twice as long as both male  and female  fish from Site 2, and nearly twice as long as female fish from Site 1 . These differences in standard length led to a significant main effect of Population on standard length,  = 0.040 .t(22) = −2.69, p = 0.014  (t(22) = 2.384, p = 0.028  (t(22) = 1.42, p = 0.175   . Fish fr.49 SEM) . We foun.09 SEM) .t(22) = 2.42, p = 0.027  (t(22) = 3.40, p = 0.003   = −0.82, p = 0.423, Site 1 M = 159.3 s ± 4.74 SEM, Site 2 M = 164.5 s ± 4.29 SEM) (t(22) = −0.79, p = 0.44, Site 2 = 69.7% explored ± 3.96 SEM, Site 1 = 65.9% explored ± 2.59 SEM)  . Similar.56 SEM) . We foun.29 SEM) , with no.59 SEM) .F = 7.06, p = 0.02). Mixed- and single-sex groups did not significantly differ across other metrics. However, the Site 2 sample had 11 mixed-sex tanks and 1 single-sex tank while the Site 1 sample had 4 mixed-sex tanks and 8 single-sex tanks. Thus, the difference in distance travelled may reflect population, but not group composition-dependent activity.As there were disproportionately more male than female fish sampled, we investigated whether behavior across these levels differed between single-sex and mixed-sex groups. We found that fish in mixed-sex groups from both populations swam nearly half the distance as single-sex groups  research practices are required to effectively implement biological sentinels ,130,131.Startups are engines of ingenuity that can help introduce zebrafish sentinel devices into the market as commercial tools . EntreprEnvironmental change poses a considerable threat to natural environments and global health ,2,3,4,5.