Apherusa glacialis is a common, sea ice-associated amphipod found throughout the Arctic Ocean and has long been considered permanently associated with the sea ice habitat. However, pelagic occurrences of A. glacialis have also been reported. It was recently suggested that A. glacialis overwinters at depth within the Atlantic-water inflow near Svalbard, to avoid being exported out of the Arctic Ocean through the Fram Strait. This study collated pelagic occurrence records over a 71-year period and found that A. glacialis was consistently found away from its presumed sea ice habitat on a pan-Arctic scale, in different depths and water masses. In the Svalbard region, A. glacialis was found in Atlantic Water both in winter and summer. Additionally, we analyzed A. glacialis size distributions throughout the year, collected mostly from sea ice, in order to elucidate potential life cycle strategies. The majority of young-of-the-year A. glacialis was found in the sea ice habitat during spring, supporting previous findings. Data on size distributions and sex ratios suggest a semelparous lifestyle. A synchronous seasonal vertical migration was not evident, but our data imply a more complex life history than previously assumed. We provide evidence that A. glacialis can no longer be regarded as an autochthonous sympagic species. Calanus glacialis, are able to time offspring release and also mature in the photic zone when food resources are optimal and subsequently overwinter at depth in diapause  found underneath sea ice with small individuals and juveniles also occurring within brine channels. With the exception of E. holmii, these amphipods have typically been considered permanent residents (autochthonous) of sea ice, with their entire life cycle to occur within the sea ice habitat  in deep water between 200 and 2000 m . We additionally compiled data from published and unpublished pelagic records of A. glacialis within the Arctic through literature searches and directly from individual researchers (see Acknowledgments). In total, we compiled n = 715 confirmed the presence of pelagic records  A. glacialis was found within a plankton tow to the surface, or (iii) A. glacialis was found within a depth-stratified plankton tow. In the third case, depth-stratified data were treated as individual records . A record may contain one or more individuals. Of these 715 records, 627 had corresponding vertical tow information. While some records contained both presence and absence, this was not indicative of all records. Because of the gaps in confirmed absence data, we included presence-only records in order to investigate if A. glacialis is wholly dependent on the sea ice habitat within its life. The records spanned all months of the year from 1947 through 2018, though after 5 records reported in 1947, there was a 19-year gap until 1967 and a 14-year gap from 1988 to 2001. Likely, these gaps do not represent true absences of the species in planktonic environments but rather less research efforts. Within the tow records, 15 different net types with corresponding vertical tow information were used  deployed for different research objectives, including Multinets deployed in shallower layers (deepest depths ranged from 600 to 145 m) were also checked for the presence of A. glacialis. In summary, the following nets were used: a depth-stratified zooplankton Multinet sampler  equipped with 5 nets of 0.25 m2 aperture with mesh size of 180 or 64 μm, a WP2  with 90 μm mesh, an MIK net  and a Harstad pelagic trawl with an 8 mm mesh. Sea ice was not encountered during the expedition.A new field sampling campaign targeted the Svalbard region close to the A. glacialis occurrences as individuals m−3, individuals m−2, number of individuals found, or presence only. Additionally, in some datasets, A. glacialis was originally calculated as abundances per 100 m−3, and these abundances were recalculated to abundances m−3 to compare with other datasets. We divided the pelagic presence records of A. glacialis into 3 groups. The first group consisted of n = 88 records with geographic position (latitude and longitude) and calendar date only. These records were included in the spatial mapping of the pelagic occurrence of A. glacialis on a pan-Arctic scale but were not included in additional analysis because of the lack of corresponding depth information. The second group (n = 506) contained geographic position, calendar date, and A. glacialis quantified from tows to the surface. In the literature, amphipods found within sea ice are commonly reported as individuals m−2  additionally reported A. glacialis from depth-stratified tows. The uppermost Multinet sections (i.e. the ones that terminated at the surface) were not included in the third group but instead included within the second group (tows to the surface). A. glacialis found in depth-stratified tows were only reported in the depth strata they were present, and not in the depth strata they were absent. This implies that we do not have all the information on the entire tow (or where the tow began), only sections of a depth-stratified tow where depth-specific distribution for A. glacialis was reported. Therefore, we can only report these data as abundance m−3. Given that the data were not normally distributed, a Kruskal–Wallis test was used to test for differences in A. glacialis abundances. Data analysis was conducted using R (version 3.6), and maps were created with the PlotSvalbard package  profiles  were used to investigate the relationship between profiles . Data weA. glacialis by presenting a pan-Arctic synthesis of A. glacialis body sizes and information on when young-of-the-year was present. Previously published and unpublished body length data along with information on life stage and sex were collected from a 30-year period (1979 through 2017) and from all months of the year  Surprisingly, A. glacialis was consistently found within pelagic tows during the entire year (P = 0.7). There were tows (n = 19) where abundance was not calculated, but the number of A. glacialis was reported: 1–30 individuals . These tows were taken during January, June, July, and August.Out of 506 tows to the surface, including Multinets that terminated at the surface, 85% (430 records) were located on the Canadian Beaufort Sea shelf and slope . Tows stire year  but withA. glacialis was found in a total of n = 121 depth-stratified layers [i.e. tows that did not terminate at the surface (0 m)]. Similar to the tows to the surface, A. glacialis was found on a pan-Arctic scale, but in water layers mostly shallower than 1000 m  occurred within the photic zone (0—200 m) or crossed into the photic zone from a deeper depth. Abundances of A. glacialis in these layers  were reported as: 0.004–1 m−3 and 1 to 3 individuals. One tow in the near surface layers quantified A. glacialis as 0.02 m−2. Seventy-five depth-stratified tows (62%) occurred within or crossed into the 200—1000 m depth layer, with abundances of 0.0004–1 m−3 and 1–6 individuals, respectively. A. glacialis occurred in 2 depth-stratified tows deeper than 1000 m with reported abundances of 0.002 m−3 and 2 individuals.n 1000 m . A. glaceptember . Out of Polar Night cruise, A. glacialis was present in 7 out of 20 tows to the surface between 80°N and 82°N. Tows to the surface started from 400 to 100 m, and within these 7 tows, we collected a total of 54 individuals. Within 2 Multinet layers (800—400 m), we found 2 A. glacialis. All animals found were alive, including 8 gravid females and 1 female with an empty brood pouch. Regarding pelagic occurrences coupled with hydrographic information around the Svalbard region, A. glacialis was found on the shelf, inside the Arctic Rijpfjorden, as well as off-shelf both west and north of the Svalbard Archipelago  during all months of the year, regardless of location, but was also found in other water masses  directly under sea ice using scuba equipment with a plankton hand net or electric suction sampler. The remaining individuals  were collected in depth-stratified tows and tows to the surface. The smaller juvenile size class (1–2 mm) was found between November and June and was the dominating size class during the months of March through May . Females dominated the sex ratio at all times of the year , they were also found in deep water during months when sympagic food sources were presumably available. For example, A. glacialis was found under ice and considered highly reliant on the surrounding sympagic food sources  were in good body condition and that some were gravid females. An intriguing result is that when we searched for A. glacialis in deep water during the polar night in 2017, we again found gravid females similar to the previous findings (Within our pelagic dataset, we cannot conclude whether all A. glacialis occurs during polar night, although it is unknown at what depths mating occurs. The present study supports the previous findings that juvenile offspring are likely released in late winter/early spring, evidenced by a strong increase in the number of the smallest size class found under sea ice at this time (A. glacialis breeds only once during its lifetime (A. glacialis is semelparous (Information on body size and sex ratios throughout the year can provide insights into life history strategies . Melnikohis time  and coinlifetime . Thus, oelparous .A. glacialis (A. glacialis sex ratio is in stark contrast to the other ice amphipods. G. wilkitzkii has a 1:1 sex ratio (O. nanseni and O. glacialis fluctuated between 1:1 and a dominance of either males or females depending on time of year (A. glacialis, it could be that males die soon after breeding, resulting in their particular scarcity during February to April.In all months sampled, there was a much higher proportion of females than males in the under ice habitat, although relative adult percentages were low in the spring months. Our data suggest that adult males comprise 25% of the sex ratio consistently throughout all the months but February to April, similar to previous studies of lacialis . This sa of year . Higher  of year  or malesA. glacialis life history (A. glacialis individuals anywhere at any time, it could be that adult females and males are distributed both under ice and in the water column year-round (Linking the data on body sizes, sex ratios and pelagic occurrences, we suggest an updated conceptual model of  history . We enviar-round . The secar-round . The thiA. glacialis regularly occurs within the pelagic realm, during all seasons. The scant records on body condition suggest that A. glacialis can successfully inhabit pelagic habitats. The data provide some support to the conceptual adaptive-advection model suggested by A. glacialis conducts a vertical overwintering migration and if ice drift versus water current speed makes return possible within their short life cycle. Given that A. glacialis is relatively mobile, can successfully inhabit different under ice structures and is found in the pelagic environment on a pan-Arctic scale, we suggest that A. glacialis does move in and out of the sea ice habitat and can no longer be regarded solely as an autochthonous sympagic species.We found clear evidence that A. glacialis is not as dependent on sea ice as previously assumed, sea ice habitat is still a critical part of their life history strategy, evidenced by the hatching and maturation of young A. glacialis in the under ice habitat. Due to climate change, the decline of Arctic sea ice extent basin-wide (A. glacialis. Both multiyear sea ice and pressure ridges provide a longer lasting habitat for ice-associated organisms (A. glacialis.While we have demonstrated that sin-wide  with conrganisms . IncreasA. glacialis will capitalize on this habitat when available. This study gives evidence, however, that A. glacialis does not exclusively use the sea ice habitat, allowing them to potentially adapt to future ice-free scenarios. Therefore, a more comprehensive understanding is needed of its life history and how presence away from sea ice contributes to their overall strategy. Plasticity among organisms inhabiting under ice habitat may be an adaptive trait allowing populations to sustain themselves in an ephemeral sea ice habitat. Knowing that other Arctic ice amphipods can also occur away from sea ice, future research could investigate this phenomenon in more regional or seasonal detail, especially in areas with pronounced sea ice loss.In conclusion, the combination of the occurrence of early life stages and females within the sea ice habitat, along with ice-algal food sources making up large proportions of adult diet, suggests that supplementary_table_1_records_v2_fbz072Click here for additional data file.supplementary_table_2_ctd_sources_fbz072Click here for additional data file.supplementary_table_3__records_v2_fbz072Click here for additional data file.Supp_Fig1_final_fbz072Click here for additional data file.


Batrachochytrium dendrobatidis (Bd) and it is possible for these community members to be used as probiotics to reduce infection levels. In the early 1990s, the emergence of Bd caused declines and disappearances of frogs in the Australian Wet Tropics; the severity of its effects varied among species and sites. Some species have since recolonized despite enzootic Bd within their populations. This variation in history among species and sites provided an opportunity to investigate the role of anti-fungal cutaneous bacteria in protecting frogs against Bd infection. We collected cutaneous swab samples from three species of frogs at two upland and two lowland sites in the Wet Tropics, and used in vitro challenge assays to identify culturable Bd-inhibitory bacterial isolates for further analysis. We sequenced DNA from cultured inhibitory isolates to identify taxa, resulting in the classification of 16 Bd-inhibitory OTUs, and determined whether inhibitory taxa were associated with frog species, site, or intensity of infection. We present preliminary results showing that the upper limit of Bd infection intensity was negatively correlated with number of inhibitory OTUs present per frog indicating that increased numbers of Bd-inhibiting taxa may play a role in reducing the intensity of Bd infections, facilitating frog coexistence with enzootic Bd. One upland site had a significantly lower prevalence of Bd infection, a significantly higher proportion of frogs with one or more culturable Bd-inhibitory OTUs, a greater number of inhibitory bacterial genera present per frog, and statistically significant clustering of individual frogs with similar Bd-inhibitory signatures when compared to all other sites. This suggests that Bd-inhibitory taxa are likely to be particularly important to frogs at this site and may have played a role in their ability to recolonize following population declines. Our findings suggest that the use of multi-taxon Bd-inhibitory probiotics to support at-risk amphibian populations may be more effective than single-taxon alternatives.Symbiotic bacterial communities resident on amphibian skin can benefit their hosts. For example, antibiotic production by community members can control the pathogen  Antibiotic-producing bacterial symbionts can protect their hosts from disease . For exaBatrachochytrium dendrobatidis  and Litoria rheocola (common mist frog), suffered either declines or local extirpation at all upland rainforest sites  appeared to suffer temporary population declines at upland sites, followed by recovery within a few years . L. nannotis are the most, and L. serrata the least, strongly associated with water, and L. serrata are the least susceptible to population fluctuations as a result of Bd infection  and the Queensland government (scientific purposes permit number WITK05922209).We collected cutaneous swab samples from the stream-dwelling rainforest frogs nfection . The uplrevalent , to maxiBd infection status.We hand-captured frogs from vegetation and rocks bordering the creeks, in the winters of 2009 and 2010. All handling was carried out with a new plastic bag and pair of vinyl gloves for each animal to prevent contact with bacteria from our skin, and to preclude the transfer of pathogens or symbionts between animals. We gently restrained each animal by hand and rinsed it twice with a stream of sterile distilled water from a wash bottle to remove transient bacteria, which can differ from the resident microbiota . We thenL. rheocola, and four were from L. nannotis. In contrast, R2A agar plates originating from L. serrata at the upland and lowland sites in Wooroonooran National Park produced very large numbers of viable bacterial colonies making isolation difficult and time-consuming; all of these plates appeared visually very similar. Because time and available effort constrained the number of these plates we could fully sample, we selected agar plates haphazardly from only four L. serrata at each of the Wooroonooran sites (Table 1). For all other combinations of site and species, all plates with bacterial colonies present were included.Inverted R2A agar plates were incubated for 48–72 h at ambient temperature (22–25°C) in the laboratory until we observed microbial culture growth. We examined plates daily for 5 days using a dissecting microscope and selected all colonies with different morphological characteristics for isolation to pure (axenic) culture using standard microbiological techniques . When inTM Ultra  and quantitative PCR  used to diagnose Bd infection status as per Bd when at least two of the three technical PCR replicates were positive. Bd intensity was calculated as the mean of the technical replicates.DNA was extracted from skin swabs with PrepManBd growth as described by Bd isolate “Gibbo River, L. Les, 06-LB-1” was incubated on TGhL agar plates  at 23°C for 3 days. Zoospores were flushed from the plate with sterile TGhL medium, filtered to remove sporangia and resuspended in sterile TGhL at 2 × 106 zoospores ml-1. We conducted challenge assays in 96-well microplates  with five replicates of each sample (50 μl cell-free supernatant and 50 μl of Bd zoospores), positive, negative, and medium only controls. Plates were incubated at 23°C for approximately 7 days until maximum growth in the positive controls was observed. Daily absorbance readings were collected at 492 nm using a spectrophotometer  and from these we calculated the proportion of inhibition observed in each sample on its maximum growth day relative to the positive control. Bacterial isolates that produced cell-free supernatants exhibiting total inhibition against Bd were selected for taxonomic identification by DNA sequencing. Due to financial constraints it was not possible to sequence other isolates.We conducted challenge assays on cell free supernatants from axenic isolates, and categorized them as totally inhibitory, partially inhibitory, or non-inhibitory according to their effects on g for 5 min to pellet cell debris. We used the supernatant directly as template in the DNA amplification reaction, but if this was unsuccessful, DNA was extracted using a Qiagen DNeasy Blood and Tissue Kit  as per the manufacturer’s protocol.We used a sterile toothpick to inoculate each axenic isolate into a 1.7 ml sterile microtube containing 400 μl molecular grade water. Samples were vortexed to create a cell suspension and subjected to three freeze-thaw cycles , and then centrifuged at 7500 ×2, 0.2 mg ml-1 BSA, 1.25 U HotStar Taq polymerase  with 1× buffer and <1 ug template DNA. The thermocycling parameters were: 95°C for 15 min followed by 35 cycles of 94°C for 1 min, 48°C for 1 min, 72°C for 1.5 min, and a final extension for 10 min at 72°C.We amplified the 16S rRNA gene from pure bacterial isolates by PCR on Bio-Rad C1000/S1000 thermal cyclers  with the bacteria-specific primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and the universal primer 1492R (5′-GGTTACCTTGTTACGACTT-3′) . The PCRBd. Although some genera could comprise multiple OTUs that each produce different antifungal compounds, it is likely that some subgeneric OTUs would have the same mechanisms. For example, multiple Pseudomonas species can produce the antifungal compound 2,4-diacetylphloroglucinol  obtained from each frog, and fitting a species accumulation curve to this relationship.PCR product purification and Sanger DNA sequencing were conducted by Macrogen, Inc. (South Korea) using both forward and reverse primers described above. We aligned forward and reverse nucleotide sequences in Geneious Pro  ggplot2 . We exclL. serrata and L. nannotis  across sites. We used Tukey’s post hoc pairwise comparisons , we used permutational multivariate analysis of variance (PERMANOVA) on the proportions of isolates from each frog (L. serrata and L. nannotis only) that were in each of two challenge assay result categories; totally inhibitory and partially inhibitory. For this, we used the function “adonis2” with marginal terms (variables were assessed in the model together to show their impact when added last) in the community ecology package vegan  with a binomial response to compare prevalence of infection in parisons  to examimultcomp , and adje method . We assehod  using the function “metaMDS.” When the nMDS failed to converge, we identified samples that were disconnected using the function “disconnected.” As a result, we removed three samples that each had just one unique Bd-inhibitory OTU from the nMDS analyses.If inhibitory bacterial assemblages have evolved in response to  measure  with 100ge vegan  with 100ge vegan  with BenBd-inhibitory cutaneous bacterial OTUs present on frogs. We used Tukey’s post hoc pairwise comparisons  on the proportion of frogs with Bd-inhibitory OTUs.We used a GLM, specifying that the response came from a Poisson distribution, to examine species and site-specific differences in the number of parisons  to examie method . For thiBd (Table 1). Our GLM on data for the two species (L. nannotis and L. serrata) that were present at all sites indicated that the prevalence of infection differed significantly between those species  and among sites ; the interaction effect was not significant . Post hoc comparisons using Benjamini and Hochberg adjusted p-values showed that the Wooroonooran upland and Kirrama upland sites differed significantly (p = 0.019). Two of the five remaining pairwise comparisons were suggestive . The remaining pairwise comparisons were not significantly different . Prevalence in L. nannotis and L. serrata combined  was 5% at Kirrama Uplands, 25% at Kirrama Lowlands, 25% at Wooroonooran Lowlands, and 55% at Wooroonooran Uplands.Thirty-two of the 110 frogs (29%) sampled were infected with L. rheocola at the Wooroonooran sites.We could not statistically compare intensity of infection among species within each site due to the low numbers of infected frogs. However, of the 29 infected frogs, 13 had infections of 10 zoospore equivalents or less indicating relatively low infection levels. Higher infection intensities occurred almost exclusively in Table 1). We obtained a total of 1005 bacterial isolates across all frog species and sites, with a mean of 11.3 cultured bacterial isolates per frog . This is broadly in line with the number of isolates reported in other studies  did not depend significantly upon infection status or species , but differed significantly among sites . Post hoc comparisons among sites using Benjamini and Hochberg adjusted p-values showed that frogs at the Kirrama upland site had a significantly greater proportion of Bd-inhibitory isolates than those at all other sites . Frogs at the Wooroonooran upland and Kirrama lowland sites also differed significantly (p = 0.0285). The remaining site-pairs were not significantly different from each other (p > 0.100 for both cases).Our PERMANOVA analysis of data for Figure 2 and Table 2). 75% of OTUs fell within the Proteobacteria phylum with small contributions from the phyla Bacteriodetes, Actinobacteria, and Firmicutes, which is in line with the findings of other studies . However, as we often sampled the majority of colonies that grew on the plates and in all cases we sampled all morphologically different isolates present, it is a limitation of culturing studies in general that it is impossible to attain adequate sampling effort. Figure 3B summarizes our analysis of the relationship between the total number of isolates we found on each individual frog and the number of unique cultured totally inhibitory OTUs included in those isolates. It provides additional information as it includes bacterial isolates that were sampled from agar plates that were not inhibitory. However, as our sampling effort is less than ideal due to the nature of culturing studies, our OTU-based analyses and results should be considered as preliminary findings.Bd-inhibitory OTUs present per frog and the upper limits of Bd infection intensity . No frogs with highly intense Bd infections and high numbers of inhibitory bacteria were found during this study.Quantile regression at the 90th percentile level demonstrated a preliminary significant negative relationship between the number of Bd-inhibitory cutaneous bacterial OTUs showed no significant interaction between species and site  and the effect of species was also not significant , however, the effect of site was significant . The multivariate dispersion of data using the Bray–Curtis distances used by the PERMANOVA was not significantly heterogeneous among sites . Inhibitory taxa present on L. nannotis and L. serrata from the Kirrama uplands clustered more tightly than those on frogs at any other site .Our PERMANOVA examining the effects of species and site on the composition of Bd-inhibitory OTUs present on frogs as a Poisson-distributed response showed a significant interaction effect, indicating that site and species had effects that were not independent . We therefore performed a post hoc analysis comparing 28 pairs of combinations of species and site  and significantly greater numbers of Bd-inhibitory bacterial OTUs than L. serrata at Kirrama lowland and upland sites (p < 0.05). There was also a suggestion of greater OTU numbers in L. nannotis from the Kirrama upland site compared with Wooroonooran upland L. serrata (p = 0.061). A full set of pairwise comparisons are provided as Supplementary Table Bd-inhibitory members of the genera Acinetobacter and Duganella were unique to frogs at the Kirrama upland site .The outcome of our GLM examining the number of and site , using BBd-inhibitory bacterial OTUs on L. nannotis, the Kirrama upland site also had a significantly higher proportion of frogs with one or more Bd-inhibitory OTUs than other sites . When the Kirrama upland site was removed from the analyses, all other sites did not differ significantly from each other . The proportion of frogs with Bd-inhibitory OTUs did not differ significantly between species . This suggests that the site effect we detected was not a confounded effect of species.As well as having significantly more Bd-inhibitory cutaneous bacterial microbiota of Australian Wet Tropics frogs may have played in the reappearance of upland frog populations following population declines in the early 1990s and found that frogs from the Kirrama upland site differed from frogs at other sites in the presence and nature of their Bd-inhibitory bacteria. There was also a significant negative relationship between the upper limit of Bd infection intensity and the number of totally inhibitory bacterial genera on infected frogs. This suggests that greater inhibitory potential is linked to a reduction in the intensity of Bd infection, and that it therefore is possible that greater numbers of Bd-inhibitory bacterial genera may contribute to tolerance of Bd infection.We investigated the potential role that Figure 3A suggests that approximately 30 cultured totally inhibitory isolates per frog would be adequate. On average, our study found that 22% of the cultured isolates that we tested were totally inhibitory to Bd. Therefore, this would have required us to sample and test about 130 isolates from each frog/agar plate  in order to find 22% inhibitory. In almost all cases, there were considerably less colonies present on the agar plates than this and in many cases we sampled the majority of colonies that grew on the plate. In every case, we sampled all colony morphotypes present on the agar plates in an attempt to capture the diversity present. From our sequencing results, we often obtained identical sequences from individual frogs indicating that we probably over-sampled our agar plates, rather than under-sampled them. For these reasons, it is highly likely that we have captured the majority of cultured inhibitory OTUs present. The use of multiple culture media types may have resulted in the discovery of more OTUs, but it is likely that only a small fraction of all bacteria present on the frogs could ever be captured by culturing.While our sampling effort was not ideal, it is often impossible for studies based on cultured isolates to achieve adequate sampling effort. For example, Bd infection load. However, uncultured proteobacteria phylotype richness was negatively correlated with Bd load in E. coqui frogs in Puerto Rico  were present at all of our sites. The diverse secondary metabolites that can be produced by these principal genera might perform complementary roles. For example, one compound may act on the zoospore cell membrane while another interferes with transcription of DNA and therefore cell replication . effects . This prin hydra , and soiin hydra . Three plication . It therBd-inhibitory bacterial taxa on frog skin alters susceptibility to, or intensity of Bd infections. Similar experiments have been conducted on both plants and locusts and have demonstrated reduced pathogen density when a greater number of bacterial symbionts was present , a higher proportion of inhibitory isolates than their conspecifics at other sites, significantly more Bd-inhibitory bacterial OTUs per frog in L. nannotis , and statistically significant clustering of individual frogs with similar Bd-inhibitory taxonomic signatures . Some of the patterns we observed at the Kirrama upland site were clearly driven by L. nannotis. However, given that L. serrata only suffered temporary upland declines, it seems likely that the acquisition of greater numbers of Bd-inhibitory genera by L. nannotis at the Kirrama upland site may have facilitated their recolonization. A higher proportion of Kirrama upland frogs had Bd-inhibitory bacteria from the genera Pseudomonas and Enterobacter than at any other site, and the genera Acinetobacter and Duganella were unique to frogs from this site. Together, these differences suggest that Kirrama upland frogs, under pressure from enzootic Bd infection, have actively or passively acquired a greater number of Bd-inhibitory genera than frogs elsewhere, and that this may facilitate a reduction in the prevalence of Bd at this site.Our results from the Kirrama upland site showed that frogs at this site differed from all other sites in a number of ways, and together, provide support for protection against Bd-inhibitory isolates than frogs at the Kirrama upland site and a lower proportion of frogs with Bd-inhibitory bacteria. Given the substantial genetic separation between frogs in the two regions (Bd infection has evolved independently at the two upland sites. Therefore, factors other than symbiotic bacteria have probably played a part in aiding recolonization of the Wooroonooran upland site. These could include host immune defense, behavior, and environmental factors (All three species of frogs in this study have recolonized the Wooroonooran upland site despite having a lower proportion of  regions , it is l factors .Bd have received much attention in recent years, as it is possible that they could be cultured and used as probiotics to support at-risk populations of frogs through disease outbreaks (Bd remains essential to facilitate probiotic development, because it is not yet possible to accurately predict the functional capability of a community from 16S amplicon sequence data alone (Bd.Bacteria that can inhibit the growth of utbreaks . Therefota alone . Fortunata alone  giving cta alone  and helpBd-inhibitory genera is correlated with lower Bd infection loads in wild frogs. This highlights the necessity for the creation of appropriate multiple-isolate combinations to promote potential synergistic interactions when developing probiotic candidates for supplementation of at-risk amphibian populations.To our knowledge, we provide the first evidence that a greater number of SB and RA designed the study, conducted the data analysis, and produced the first draft of the manuscript. SB completed the fieldwork. SB and SG processed samples in the laboratory. All authors edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Membrane uncoupling protein 3 (UCP3), a member of the mitochondrial uncoupling protein family, was discovered in 1997. UCP3′s properties, such as its high homology to other mitochondrial carriers, especially to UCP2, its short lifetime and low specificity of UCP3 antibodies, have hindered progress in understanding its biological function and transport mechanism over decades. The abundance of UCP3 is highest in murine brown adipose tissue , compared to heart (2.7 pmol/mg protein) and the gastrocnemius muscle (1.7 pmol/mg protein), but it is still 400-fold lower than the abundance of UCP1, a biomarker for BAT. Investigation of UCP3 reconstituted in planar bilayer membranes revealed that it transports protons only when activated by fatty acids (FA). Although purine nucleotides (PN) inhibit UCP3-mediated transport, the molecular mechanism differs from that of UCP1. It remains a conundrum that two homologous proton-transporting proteins exist within the same tissue. Recently, we proposed that UCP3 abundance directly correlates with the degree of FA β-oxidation in cell metabolism. Further development in this field implies that UCP3 may have dual function in transporting substrates, which have yet to be identified, alongside protons. Evaluation of the literature with respect to UCP3 is a complex task because (i) UCP3 features are often extrapolated from its “twin” UCP2 without additional proof, and (ii) the specificity of antibodies against UCP3 used in studies is rarely evaluated. In this review, we primarily focus on recent findings obtained for UCP3 in biological and biomimetic systems. The role of brown adipose tissue (BAT) in obesity was suggested 40 years ago based on the ability of brown fat mitochondria to dissipate energy as heat . HoweverUncoupling protein 1  is a main player in the energy dissipation process in BAT  and BrATThe function of another member of the UCP family that was found in BAT, UCP3, has also been increasingly associated with obesity and diabetes . HoweverIn this review, we outline current knowledge on UCP3 expression and transport functions. We discuss its putative involvement in BAT functions, its interplay with UCP1 and its role in other tissues. Because several excellent reviews about different aspects of UCP1 physiology have appeared in recent years , we primThe ways how members of the mitochondrial UCP subfamily were discovered are strikingly different. UCP1 was identified due to its high protein amount in BAT, which was visible on a Coomassie stained SDS gel loaded with mitochondrial protein obtained from BAT . UCP3 s.The debates, whether UCP3, alongside with other “minor” proteins, truly belongs to the uncoupling family started shortly after they were discovered . This isUCP3 was first identified by screening a human skeletal muscle cDNA library. These initial studies revealed different patterns of UCP3 mRNA expression in human and rat . In ratsucp3 expression levels is less obvious , heart, BAT and BrAT. Increased levels of UCP3 observed in WAT of mice housed at room temperature  are thouE. coli, yeast, mammalian cells, insects, etc. for further investigation in biomimetic systems  and UCP1, all members of the SLC25 family are present at concentrations less than 20 pmol/ . Quantit systems .Analysis of UCP3 protein expression pattern in mice (BAT > > heart > muscles) implies that its presence correlates with a definite type of cellular metabolism – FA β-oxidation (FAO). BAT mitochondria have a high capacity for utilizing free long-chain FA as a substrate for β-oxidation . AlthougThe correlation between UCP3 levels and degree of FAO was investigated in murine heart . This moucp3 gene expression, which are induced by augmented lipid levels in blood plasma, e.g., during fasting/starvation, high fat diet (HFD), cold exposure  has been solved among all mitochondrial carriers . The NMRUCP3 is a typical mitochondrial carrier with a 312 amino acid peptide chain in mouse, arranged in six transmembrane domains that are connected by three long matrix loops and two shorter intermembrane (IMS) space loops . N- and m, in yeast overexpressing UCP3  and oxononenal (ONE) do not activate UCPs directly but strongly enhance the proton transport  if direcInteraction of UCP3 with HNE was investigated in SkM and heart in connection with its putative ROS-regulating role . No consm is an important factor for UCP activation. Protein activity in the presence of FA increases non-linearly at physiologically relevant potentials 130–220 mV  the β-phosphate of PN binds to R183∗ to form the loose conformation; (2) either the γ-phosphate  or β-phosphate  binds to R84∗ to generate a tight conformation, and finally, (3) α-phosphate binds to R277∗, triggering protein conformational change and inhibition. Thus, binding strengths and inhibition of di- and triphosphates were supposed to be identical, while monophosphates are unable to bind.It is generally accepted that the protonophoric activity of UCP1 and UCP3 is inhibited by PN. The mechanism of UCP1 inhibition was proposed by Garlid‘s and Klingenberg‘s groups 20 years ago . Three aRecently, we compared binding forces between different nucleotides and UCP1-UCP3 at the single molecule level using a combination of recognition imaging and force spectroscopy . We reveFor the first time, the combination of recognition and force modes of atomic force microscopy (AFM)  have allWe demonstrated that UCP3 reconstituted in bilayer membranes is completely inhibited by all PNs, regardless of phosphorylation, but IC50 increases as phosphorylation decreases. This contradicts the previous assumption that diphosphate-PNs are the most potent inhibitors of UCP3 . HoweverWe also re-evaluated the mechanism of inhibition for UCP1. We first revealed that interaction between the α-phosphate of AMP and arginine is sufficient for binding and partial inhibition of UCP1. We further proposed that each arginine-phosphate interaction contributes equally and additively to maximum inhibition, resulting in complete inhibition in the case of three interactions . The high homology between mitochondrial carriers, especially UCPs, has prompted a “take over” hypothesis . HoweverMouse overexpressing UCP3 (UCP3 Tg) in skeletal muscle was created in the same year as KO mouse . The oriImportantly, the ectopic and entopic overexpression of mitochondrial carriers also faces serious problems. The described changes may be caused by experimental artifacts. When ectopically overexpressed in HEK cells, the oxoglutarate carrier was shown to cause strong mitochondrial uncoupling due to incorrect assembly into the membrane . On the -/- mice revealed a decreased expression of UCP3 in BAT, which was even further decreased in response to cold exposure. In contrast, UCP3 expression in heart and skeletal tissues was unaffected by cold exposure. These observations give an important hint that specific (non-protonophoric) UCP3 transport function is only required in full functioning and activated BAT. Furthermore, a connection to the previously elucidated UCP1-independent thermogenic process , clearly reflect FAO type of the cell metabolism . This ob(1)It has been verified that UCP3 transports protons with a rate comparable to UCP1. However, it seems very likely that its protonophoric function may be additional to another as yet unknown transport function  similar (2)There are significant differences in inhibition by PN between UCP3 and UCP1. Maximum inhibition of UCP1 decreases with decreasing PN phosphorylation, while all PNs can fully inhibit UCP3. This is caused by different interaction mechanisms between PNs and arginine residues in UCP1 and UCP3. Increased free FA concentrations decrease the effect of all PNs on UCP1. In contrast, FAs affect only ATP-mediated inhibition in UCP3.(3)i is a new inhibitor of UCP1 and UCP3 that causes partial inhibition while utilizing a mechanism distinct from that of PNs.P(4)UCP3 tissue distribution shows a clear dependence on cell metabolism, directly correlating with a preference for FAO. Expression of this protein is adaptive in nature, emerging short-term during metabolic necessity. Hence, drawing conclusions about UCP3 function based solely on mRNA data is not feasible. KO models have limited potential for the exploration of UCP3 function because mice may adapt their metabolism to the existing nutrition supply.EP and AR contributed to the conceptualization and wrote the original draft. KH, AR, and especially GM contributed to the visualization. EP acquired funding. All authors reviewed and edited the draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Production of drinking water is accompanied by a nearly equivalent volume of brine discharge with a salinity of ~80 that is twice the normal, which can potentially impact marine ecosystems. The goal of this study was to examine whether benthic foraminifera, a known sensitive marine bio-indicator, are affected by this brine-discharge. For that, we investigated the seasonal and cumulative effect of brine discharges of three operating desalination facilities along the Israeli coast. Those facilities are located in Ashkelon, Hadera, and Sorek. The brine-discharge in the first two desalination plants is associated with thermal pollution, while the Sorek facility entails increased salinity but no thermal pollution. In four seasonal cruises during one year, we collected surface sediment samples in triplicates by grabs from the outfall (near the discharge site), and from a non-impacted control station adjacent to each study site. Our results highlight that the most robust responses were observed at two out of three desalination shallow sites (Ashkelon and Hadera), where the brine was discharged directly from a coastal outfall and was accompanied with thermal pollution from the nearby power plants. The total foraminiferal abundance and diversity were, generally, lower near the outfalls, and increased towards the control stations. Moreover, changes in the relative abundances of selected species indicate their sensitivity to the brine discharge. The most noticeable response to exclusively elevated salinity was detected at Sorek discharge site, where we observed a sharp decline in organic-cemented agglutinated benthic foraminifera, suggesting that these are particularly sensitive to elevated salinity. The herein study contribute new insights into the effect of brine discharge from desalination plants, on benthic foraminifera, and propose a scientifically-based ecological monitoring tool that can help stakeholders.Desalination plants along the Mediterranean Israeli coastline currently provide ~587 million m Temperature records were similar between the outfall and the control stations and varied between ~29°C and ~17°C during summer and winter, respectively . The salA well-distinguished difference in surface grain size mode (μm) between the three sampling sites was correlated with water depth, as reported for the Israeli shelf ,48. An aValues recorded at the surface sediments of the three sites were very low and homogeneous for all sampling sites and stations (<0.1%). These values are typical of shallow siliciclastic sandy sediments in an oligotrophic sea ,49, whilMost of the measured trace elements showed very low values and did not vary between the sampling stations. The only exceptions were chromium (Cr) and manganese (Mn), which presented slightly higher concentrations at the Sorek outfall station compared to the control station, while an opposite trend was observed at both the Ashkelon and Hadera sites .2O5 and SO3 are consistently low at all sites and stations . The only exception was the concentration of Fe2O3, which exhibited higher values at the outfall station at the Sorek site and the control station at the Ashkelon site. The concentration of Fe2O3 at the Hadera site was low at both stations compared to that at the two other facilities . The results indicate that there is a difference between the sites and the stations .An ANOSIM test was performed to evaluate the differences between all three sites and six stations, regarding all environmental parameters  of the total assemblage at shallow water depths from 3 to 9 m at Ashkelon and Hadera. Other common species at these water depths were Ammonia beccarii (avg. 11.5% ±12%), Ammonia sp.1 (10% ±7%), Ammonia tepida (11% ±11%), Pararotalia calcariformata (17% ±29% mainly in Hadera), Buccella granulata (5.5% ±5%), and Spiroplectammina sp.1 (10% ±14% only in Sorek), each constituting up to 20% of the total assemblage. . On the other hand, the relatively high abundance of stained organic-cemented agglutinated species (arenaceous foraminifera) such as Eggerelloides advenus and Spiroplectammina sp. 1 at the control station of the Sorek site suggests that these species inhabit shallower depths than previously reported [P. calcariformata that was mostly found in the Hadera site seemed to be restricted to the northern and central coast of Israel, where it was abundant in both hard-bottom and soft-sediment habitats and frequently accompanied with high water temperatures [A clear bathymetric zonation based on foraminiferal species was observed in this study, as reported in previous studies ,52. For reported ,53,54. Teratures ,55.Ashkelon, Hadera: R = 0.685, p = 0.001%, Ashkelon, Sorek: R = 0.557, p = 0.001%, Hadera, Sorek: R = 0.662, p = 0.001%). This finding means that the foraminiferal assemblages of the three sites are incomparable, potentially to their geographical, physical, chemical, or sedimentological differences (substrate type).In order to illustrate the visual statistical differences between all sampling sites and stations, we performed an nMDS plot diagram using foraminiferal relative abundance. This diagram showed a clear separation between the three sampling sites  and stations  . ANOSIM  = 21.998, p<0.001;  = 9.75, p<0.001; and Sorek: F = 5.84, p = 0.002; 2O3. Desalination outputs  were effective in opposite directions at Ashkelon and Hadera and the same direction at the Sorek site  sandy sediments originating from the Nile River ,54. PrevThe low foraminiferal abundances in the naturally unstable and diluted sandy habitats of the Israeli coastline (SE Mediterranean Sea) therefore, present a challenge for the detection of the anthropogenic footprint of elevated salinity and temperature in that area. Species richness should be independent and provides a reliable parameter for assessing the diversity of species, even when low, in soft-sediment habitats. Consequently, it is clear that this comparison between the foraminiferal assemblages of the control stations vs. outfall stations should be focused on changes in their relative abundances and species richness, rather than on numerical abundances.Considering the results of the statistical analyses  = 4.88, p = 0.03; Figs Ammonia parkinsoniana and Porosononion granosum at the control station. Ammonia beccarii showed an opposite trend, being highly abundant at the outfall station, possibly because it prefers the coarser sediments characteristic of the energetic shallower water depths  = 30.54 p<0.001, Figs Pararotalia calcariformata at the control station  and Quinqueloculina seminula did not display any noticeable differences in their relative abundances between the stations, indicating that they are less sensitive to salinity and temperature anomalies  = 19.98, p<0.001, Figs Spiroplectammina sp. 1 and Eggerelloides advenus observed at the control station  on benthic foraminifera, with minimum interference from other natural or anthropogenic factors. Moreover, as the deepest site with finer sediments and lower water energy, this site exhibited the highest species richness (up to 28 species/sample) and therefore provided a better representation of the soft-sediment benthic foraminiferal community . nMDS pl on benthion Figs . Typicalion Figs . It is, Seasonal changes in benthic foraminiferal assemblages were monitored at three desalination facility sites  along the Mediterranean coast of Israel during 2016–2017. Two of the study sites, Ashkelon and Hadera, are very shallow, and mixing with the cooling water of adjacent power plants occurs at these sites operating since 2005 and 2009, respectively. The outcome is the local elevation of water temperatures in addition to the brine discharged. On the other hand, a diffuser system without cooling water operates at the Sorek site since 2013, allowing us to assess the effect of brine alone on the shallow-water ecosystem, without the added parameter of elevated temperatures.All sites exhibited significant differences between the outfall and the control stations, indicating a robust synergic effect of elevated brine levels and temperature  as well as an independent influence of brine (Sorek).The most noticeable effects of the brine and temperatures were the decreases in abundance and species richness observed between most of the samples at the outfall and the control stations from the same water depth.The Sorek site facilitated an isolated evaluation of the impact of elevated salinity on the foraminiferal assemblages. The most noticeable negative response to brine discharge was observed among different lineages of organic-cemented agglutinated species. Their similar responses indicated mutual sensitivity of this shell-type strategy to an increase in salinity above ambient levels.This study demonstrates that an increase of 2–3 salinity units caused by desalination plants has a minor, local effect on calcareous benthic foraminiferal assemblages and a more prominent impact on organic-cemented agglutinated species. Therefore, to ensure a minor effect on the local benthic ecosystem, it is pertinent to maintain low levels of brine while the industry continues to evolve.S1 TableNumerical abundances , the total number of specimens counted, and the number of species, of each site, station, season, and replicate.(XLSX)Click here for additional data file.S2 TablesSTATISTICA 10 software output.Table A in S2 Tables. Two-way ANOVA is comparing salinity measurements of the three studied sites , and the stations  for each site.Table B in S2 Tables. Tukey HSD post-hoc test demonstrating the salinity differences between the outfall to the control of the three sites. Stars indicate homogenous groups.Table C in S2 Tables. One-way ANOVA is comparing salinity measurements of the outfall and control stations at Ashkelon.Table D in S2 Tables. A non-parametric, Mann-Whitney U test comparing salinity measurements of the outfall and control stations at Hadera. This test was performed because the ANOVA Assumption was violated.Table E in S2 Tables. One-way ANOVA is comparing salinity measurements of the outfall and control stations at Sorek.(PDF)Click here for additional data file.S1 FigDemonstrating the differences of Foraminifera total abundances (top) and species richness (bottom), between the outfall to the control of each site.(PDF)Click here for additional data file.


Recent technical developments have resulted in robotic-assisted pedicle screw placement techniques. However, the use of robotic-assisted navigational techniques is still subject to controversy. This study aims to assess the accuracy and safety of a self-developed navigation system, the point spine navigation system (PSNS), for robotic-assisted pedicle screw placement surgery. Fifty-nine pedicle screws were implanted in three porcine vertebrae at the T6–T10 and L1–L5 levels, with the assistance of the PSNS. The navigation and planning system provides virtual surgical guide images, including sagittal, coronal, axial, oblique planes, and customized three-dimensional reconstructions for each vertebra to establish accurate pedicle screw trajectories and placement tracts. After pedicle screw placement, post-operative spiral computer tomographic scans were performed and screws were evaluated using the Gertzbein–Robbins classification. Differences between the actual pedicle screw position and pre-operative planning paths, including the angle, shortest distance, and entry trajectory were recorded. The 59 pedicle screw placements were all within a safe zone, and there was no spinal canal perforation or any other damage under postoperative computed tomography image data. Fifty-one screws were categorized as group A, seven screws were noted as group B, and one screw was identified as group E under the Gertzbein–Robbins classification. The mean entry point deviation was 2.71 ± 1.72°, mean trajectory distance was 1.56 ± 0.66 mm, and average shortest distance between two paths was 0.96 ± 0.73 mm. Pedicle placement remains a challenging procedure with high reported incidences of nerve and vascular injuries. The implementation of a robotic-assisted navigational system yields an acceptable level of accuracy and safety for the pedicle screw placement surgery. Free-handed pedicle screw placement remains a high-risk procedure owing to the many important structures near the pedicle, such as the spinal cord, nerve root, and associated vessels, despite the advancements in fluoroscopy . ComplicThe PSNS consists of a navigation workstation ; handpiece robot , and navigational instruments . Each part of the system was designed with the workspace availability and necessity for operation in mind .The navigation and planning system provides virtual surgical guide images, including sagittal, coronal, axial, oblique planes, and customized three-dimensional reconstructions for each vertebra to establish and transfer the pedicle screw trajectories and directions to the computer monitor. Using an optical tracking system, we can match the porcine anatomy with pre-operative planning images. After completing the planned surgical path, this information was transferred to the handpiece robot by the handpiece robot control unit. Intraoperatively, the navigation system was also able to integrate the tracking information and display it on a monitor in real time. Further, the admission path on the spine and the real-time positions of the instruments were also displayed on the screen.The infrared optical tracking system comprised a camera , passive markers, and probe. The markers were attached to the robot and vertebra to be navigated, and the probe was used to define the point coordinates in space. With these markers, the tracker can monitor the positions and movements of the robot end-effector in real-time. Calibrations must be performed before commencing operation. We used the handpiece robot to create a variety of gestures and to record the kinematics of the robot through the optical tracking system to create a precise set of kinematic parameters. For the registration in the PSNS system, we selected fiducial points (reference pins) on the image displayed and selected the corresponding positions of the points using the probe in real-world coordinates. Through repeated selections and measurements, the coordinate frame of the image and that of the real porcine body were synchronized. We fixed eight markers on the PSNS handpiece robot, which were tracked by the tracking device. After registration, the positions and orientations of the markers were converted to the positions and orientations of the handpiece robot.The handpiece robot was composed of a 6-DOF Stewart platform, and the end of the robot was equipped with an operation tool used for drilling the screw path. Intraoperatively, the movement of the surgical target is tracked in real-time, and the robot automatically compensates for the correct target.The pedicles of the experimental porcine model were evaluated to ensure that they were intact through preoperative computed tomography (CT) scans. The pedicle screws selected were 4.5 mm in diameter and 30 mm in length, which is a feasible size. Incomplete and narrow pedicles were excluded.As shown in A total of three porcine vertebrae at the T6–T10 and L1–L5 levels were used, and the porcine spines used in our study was procured from National Taiwan University and approved by the Certified Agricultural Standards (CAS) of Taiwan. The porcine spines were immobilized on the operating table in the prone position, and the procedure was performed as described above .After the operation, postoperative CT scans weres performed. Standard measurements based on postoperative CT scans and acknowledged criteria, the Gertzbein–Robbins classification , were adA total of 60 screws were placed in 30 vertebrae, and one of the screw placements was excluded from analysis owing to robotic guidance abortion for unknown reason. This study demonstrated a low overall screw malposition rate of 1.7% for robotic-assisted screw placements, and the deviations were assessed by postoperative CT scans. For the remaining 59 pedicle screws, surgical procedures were smoothly performed using the PSNS. According to the Gertzbein–Robbins classification, 51 screws (86.4%) fell into group A, 7 screws (11.9%) fell into group B, and 1 screw (1.7%) fell into group E . All pedDuring the surgery, the optical tracking system was used to record the tip position data at a frequency of 60 Hz, and a linear regression curve was calculated. Differences between the actual pedicle screw position (green line) and preoperative planning path (red line), including the angle, shortest distance, and entry point, were also recorded . The meaSurgical robotics applications have developed rapidly since the 1990s to optimize the available robotic technologies, and they are designed to enhance and complement the surgeon’s abilities . SurgeonThere have been several debates regarding the safety and accuracy of robotic-assisted pedicle screw placements , 12. In Differences between the actual pedicle screw position  improves accuracy of pedicle screw placements and ensures optimal safety of pedicle screw placement surgery.


To determine whether an SRC homology 2 domain-containing phosphotyrosine phosphatase 2 (SHP2) inhibitor would increase muscle-specific kinase (MuSK) phosphorylation and override the inhibitory effect of MuSK-antibodies (Abs).The effect of the SHP2 inhibitor NSC-87877 on MuSK phosphorylation and AChR clustering was tested in C2C12 myotubes with 31 MuSK-myasthenia gravis (MG) sera and purified MuSK-MG IgG4 preparations.In the absence of MuSK-MG Abs, NSC-87877 increased MuSK phosphorylation and the number of AChR clusters in C2C12 myotubes in vitro and in DOK7-overexpressing C2C12 myotubes that form spontaneous AChR clusters. In the presence of MuSK-MG sera, the AChR clusters were reduced, as expected, but NSC-87877 was able to protect or restore the clusters. Two purified MuSK-MG IgG4 preparations inhibited both MuSK phosphorylation and AChR cluster formation, and in both, clusters were restored with NSC-87877.Stimulating the agrin-LRP4-MuSK-DOK7 AChR clustering pathway with NSC-87877, or other drugs, could represent a novel therapeutic approach for MuSK-MG and could potentially improve other NMJ disorders with reduced AChR numbers or disrupted NMJs. In a variable percentage of patients, muscle-specific kinase antibodies (MuSK-Abs) are present.3 The subsequent recruitment of intracellular downstream of kinase 7 (DOK7) further stimulates MuSK phosphorylation, causing activation of a phosphorylation cascade that ultimately leads to clusters of acetylcholine receptor (AChR) anchored by intracellular rapsyn on the postsynaptic membrane of the NMJ.4MuSK is normally activated following binding of agrin, secreted from the motor nerve terminal, to low-density lipoprotein receptor-related protein 4 (LRP4). LRP4 then binds to MuSK leading to its dimerization and to auto- and trans-phosphorylation in the MuSK juxtamembrane region.6 Considering this mechanism, increasing MuSK phosphorylation could represent a potential therapeutic strategy for the development of specific treatments.MuSK-Abs are predominantly of the IgG4 subclass and inhibit the interaction between LRP4 and MuSK, preventing MuSK phosphorylation and AChR clustering.7 We tested NSC-87877 for its ability to increase MuSK phosphorylation and to reverse or prevent the effects of MuSK-Abs in 2 in vitro models.Among several regulators of the AChR clustering pathway, the SRC (Rous sarcoma gene) homology 2 domain-containing phosphotyrosine phosphatase 2 (SHP2) is a phosphatase that reduces MuSK phosphorylation. Importantly, for the purposes of this study, NSC-87877, an SHP2 inhibitor, has been shown to enhance agrin-induced and agrin-independent AChR clustering in vitro.9 Sera were heated, dialyzed, and sterile filtered before use. IgG fractions were purified from plasmapheresis of 2 additional patients with MuSK-MG using protein G sepharose and an IgG4 affinity matrix.8 Effectiveness of IgG subclass purification was tested with CBA. Briefly, MuSK-transfected human embryonic kidney cells were incubated with the different IgG subclasses (1:20) and probed with anti-human-IgG1, -IgG2, -IgG3, and -IgG4 monoclonal mouse antibodies (1:50) . After fixing with 3% formaldehyde, cells were stained with Alexa Fluor 488 goat anti-mouse IgG (1:200)  and images captured using the Olympus IX71 fluorescence microscope with Simple PCI . The SHP2 inhibitor NSC-87877 (#2613) was obtained from Tocris Bioscience, Bristol, United Kingdom.Serum samples were collected, with informed consent, from 31 patients with typical MuSK-MG symptoms and immunotherapy responses. MuSK-Ab titers of patients' sera and purified immunoglobulin G (IgG) fractions were determined by radioimmunoassay and cell-based assay (CBA).9 MuSK-MG sera with a broad range of MuSK-Abs (nM) were chosen according to availability. MuSK-MG sera  or purified MuSK-Ab subclasses (0.5 nM) were applied to myotubes for 30 minutes and then incubated overnight with agrin 1:800  in the presence and absence of NSC-87877 100 μM. AChR clusters were then labeled with Alexa Fluor 594 α-bungarotoxin   and fixed in 3% formaldehyde. Twenty fields selected with bright field were analyzed for number and cluster length (>3 μm) using ImageJ software.8–11C2C12 mouse myoblasts  were maintained and differentiated into myotubes after 5–6 days in differentiation medium as previously reported .Musk “knockout” (KO) C2C12s were generated with the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system using standard methods.12 Briefly, Musk guide oligonucleotides (Integrated DNA Technologies) were designed and cloned into a modified plasmid pX335-U6-Chimeric_BB-CBh-hSpCas9n-D10A . Guide A oligonucleotide sequences were A-forward: 5′-CACCGCATTCTCCCGGATGCTGTAG-3′ and A-reverse: 5′-AAACCTACAGCATCCGGGAGAATGC-3′. Guide B oligonucleotide sequences were B-forward: 5′-CACCGCTCCTCACCATTCTGAGCG-3′ and B-reverse: 5′-AAACCGCTCAGAATGGTGAGGAGC-3′. Myoblasts were electroporated with 10 μg of each plasmid using the Neon Transfection System (Life Technologies), selected with Geneticin Antibiotic , and cloned in 96-well plates using fluorescence-activated cell sorting. Clones were screened by Sanger sequencing after PCR of genomic DNA using the primers 5′-TGGTGCTTTGGTTATGGAGCC-3′ and 5′-GAGGAGGGGTCTAAGGCTTG-3′. Musk KO generation was confirmed by Western blot.The 11 The myotubes were exposed to MuSK-Ab or control sera or 0.5 nM MuSK-Ab of each purified preparation (IgG or IgG4) as illustrated in the figures.DOK7-overexpressing myoblasts were prepared as previously described.MuSK phosphorylation was assessed by Western blotting on immunoprecipitates from the C2C12 myotubes. They were starved of fetal calf or horse serum for 3 hours, incubated in medium alone or medium plus NSC-87877  for 40 minutes at 37°C, lysed in cold lysis buffer , and centrifuged. The supernatant was immunoprecipitated with anti-MuSK-Ab  using Protein G Dynabeads  or directly with patients' MuSK-Abs for experiments with purified IgG and IgG4. Immunoprecipitated proteins were eluted into sodium dodecyl sulphate sample buffer, and the Western blots probed with anti-phosphotyrosine antibody 1:1,000 . The nitrocellulose was then stripped and reprobed with AF562. Band densitometry was performed using ImageJ software. MuSK phosphorylation levels were normalized to levels of immunoprecipitated MuSK.p values <0.05 were considered statistically significant.Statistical tests were performed with GraphPad Prism 6 as indicated in the figure legends. Error bars represent the standard error of the mean unless otherwise stated. Differences with The study of archived patient samples was approved by the Oxfordshire Research Ethics Committee A (07 Q160X/28).The authors confirm that all the data supporting the findings of this study will be made available on reasonable request to the corresponding author.7 NSC-87877 at 100 µM, without agrin, increased the numbers of AChR clusters similar to those achieved with agrin alone  for 40 minutes. MuSK was immunoprecipitated by commercial anti-MuSK AF562. Phosphorylation was identified in the immunoprecipitates by Western blotting with an anti-tyrosine phosphorylation antibody and MuSK expression by incubating the stripped membranes with anti-MuSK AF562. AChR clusters were measured on parallel myotube cultures by labeling with fluorescent alpha-bungarotoxin after 12 hours. The ratio of phosphorylated MuSK to total MuSK increased substantially  with maximal effect at 100 µM; at higher concentrations, phosphorylation decreased . C2C12 mp < 0.05 at 40′, p < 0.01 at 90′)  , and the11 MuSK phosphorylation was clearly present in the DOK7-overexpressing cells without added agrin and was further increased by NSC-87877  were studied. All patients except 3 were on immunotherapies, mainly prednisolone and/or azathioprine, but all had predominantly bulbar disease (as indicated by b grades), and 8 still had severe involvement  of 21 MuSK-Abs and 2 control sera were incubated with the myotubes for 30 minutes before adding agrin with or without NSC-87877; 12 hours later, the numbers of AChR clusters were measured  of control-treated myotubes. Nevertheless, NSC-87877 increased the number of clusters  of control, whereas on DOK7-overexpressing myotubes, the number of clusters was 69.6 ± 10.9%, n = 10; clusters  at 1:10 Finally, to demonstrate that the effects were due (at least mainly) to IgG4 MuSK-Abs, purified MuSK-Ab IgGs and IgG4 subfractions were prepared from plasmapheresis material of 2 additional patients with typical MuSK-MG. For myotubes not exposed to patient antibodies, MuSK was immunoprecipitated by the anti-MuSK AF562 antibody from the cell lysates . For myotubes exposed to patient antibodies, to ensure that only phosphorylation of the antibody-bound MuSK was assessed, MuSK was immunoprecipitated by the patient IgG. To adjust for this difference in the precipitation technique, the phosphorylation signals were always normalized to the MuSK protein signal. An example immunoblot is shown in 8 Moreover, in each case, NSC-87877 protected the myotube clusters from the inhibitory effects.MuSK-MG can be a life-threatening disease that responds poorly to symptomatic drugs such as cholinesterase inhibitors and often requires high doses of steroids and other immunosuppressive drugs for long-term treatment. Patients may have myasthenic crises with respiratory and bulbar involvement that often require urgent treatment in intensive care. The development of a therapy that acts specifically on the mechanisms of the disease could achieve quick control of symptoms and in the longer term might help reduce the potential side effects from immunosuppressive therapies. Thus, targeting the intracellular regulation of the AChR clustering pathway by inhibition of the specific phosphatase, SHP2, as we show here, could represent a novel and specific therapeutic strategy for MuSK-MG. The results also draw attention to the potential relevance of targeting intracellular pathways in diseases caused by extracellular antibodies or other mechanisms.14 play an important role in cell growth. By contrast, Src homology 2 domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a potentially safer target. Under physiologic conditions, through a negative-feedback loop, SHP2 reduces MuSK phosphorylation, providing homeostatic control of AChR clusters at the NMJ. SHP2 also enhances cell replication inducing different pathways such as Ras/Erk, PI3K, serine/threonine kinase 1, and janus kinase 2-signal transducer and activator of transcription15–17; thus, SHP2 inhibition should not increase the risk of tumorigenesis. Indeed, pharmacologic inhibition of SHP2 has already demonstrated efficacy in the treatment of a mouse glioma xenograft model.18There are several proteins downstream of MuSK that could be potential pharmacologic targets, including Abl tyrosine kinases and guanosine triphosphatases of the Rho family. However, increasing the activity of these regulators to enhance AChR clustering could lead to tumor development as many of them, such as ErbB2 and mitogen-activated protein kinase/extracellular-signal-regulated-kinase,7; here, we confirm that this works through increasing phosphorylation, which was clearly seen irrespective of the presence of agrin or MuSK-Abs. Although NSC-87877 is known to act on both SHP1 and SHP2, only SHP2 has been detected in C2C12 myotubes,19 and, reassuringly, conditional knockout of SHP2 in skeletal muscle did not compromise the formation and maintenance of the NMJ, suggesting that pharmacologic inhibition of SHP2 should be well tolerated in skeletal muscle,20 although other SHP2-specific inhibitors should also be studied.The selective inhibition of the SHP2 phosphatase that controls MuSK phosphorylation by NSC-87877 was shown previously to increase AChR clustering in C2C12 myotubes8 The inhibitory effects of MuSK-Abs on AChR clustering were present to varying degrees in all sera studied, correlating broadly with MuSK-Ab titers. NSC-87877 improved phosphorylation and AChR clustering at each concentration of the sera tested. Moreover, when the phosphorylation and AChR clustering experiments were conducted comparing total IgG and the IgG4 subfraction from 2 patients, both reduced MuSK phosphorylation and AChR clustering, and SHP2 inhibition by NSC-87877 was able to reverse their pathologic effects. Of interest, the inhibitory effect of IgG4 on AChR clustering does not depend on its monovalency and divalent monoclonal antibodies to MuSK also inhibit clustering.22 These recent findings suggest that MuSK antibodies could have additional pathogenic mechanisms, other than the direct inhibition of agrin/MuSK interaction, which contribute to the disruption of the AChR clustering pathway. Nevertheless, the inhibition of MuSK phosphorylation still remains the main effect of MuSK antibodies, as confirmed by our results with purified total IgG and by the effectiveness of the inhibition of SHP2.MuSK-Abs are mainly IgG4 subclass and are known to inhibit MuSK activation and phosphorylation by blocking the interaction between LRP4 and MuSK.There were some limitations to the experimental procedure. For the phosphorylation experiments, the patient antibody-precipitated MuSK was less than the MuSK precipitated by commercial antibody from the control cultures. There were 2 reasons for this. First, we chose a concentration of patient MuSK antibody (0.5 nM) that was submaximal for inhibition of clustering, making sure that any effects on clustering were clearly evident, but we needed to ensure that we only measured the phosphorylation in those MuSK molecules that were bound by patient IgG. Second, the commercial antibody was applied to the cell lysate from the myotubes rather than to the live cells and thus could also precipitate MuSK that was not on the surface. To adjust for these differences in MuSK immunoprecipitation, the phosphorylation was normalized to the MuSK protein signal for each lane. Further in vitro studies examining the intracellular molecular mechanisms need to be performed, and the protective effects of SHP2 inhibition against MuSK antibodies demonstrated in vivo.23 Using new or repurposed drugs to enhance MuSK phosphorylation, or other downstream components of the signaling pathway, could be beneficial in MuSK-Ab myasthenia and perhaps in other disorders of neuromuscular transmission where loss of AChRs or disruption of the NMJ structure underlies the muscle weakness.Most novel therapies in MG depend on monoclonal antibodies targeting the immune system, but given the understanding of the mechanisms involved in the MuSK pathway, faster pharmacologic treatments should be explored. Recently, a monoclonal MuSK antibody was shown to stimulate MuSK phosphorylation and to preserve NMJs in a motor neuron disease model.


This paper significantly advances this field of research by investigating for the first time the fungal abundance and diversity associated with algal blooms causing the darkening of the GrIS. There is a strong association between the abundance and diversity of fungal species and the blooming of algae on the surface ice of the Greenland Ice Sheet.Recent studies have highlighted the importance of ice-algal blooms in driving darkening and therefore surface melt of the Greenland Ice Sheet (GrIS). However, the contribution of fungal and bacterial communities to this microbially driven albedo reduction remains unconstrained. To address this significant knowledge gap, fungi were isolated from key GrIS surface habitats , and a combination of cultivation and sequencing methods utilized to characterize the algal-associated fungal and bacterial diversity and abundance. Six hundred and ninety-seven taxa of fungi were obtained by amplicon sequencing and more than 200 fungal cultures belonging to 46 different species were isolated through cultivation approaches. Basidiomycota dominated in surface ice and water samples, and Ascomycota in snow samples. Amplicon sequencing revealed that bacteria were characterized by a higher diversity (883 taxa detected). Results from cultivation as well as ergosterol analyses suggested that surface ice dominated by ice algae and cryoconite holes supported the highest fungal biomass (10 Recentlyosphere , the GrIyosphere . Signifiyosphere . These ayosphere  and can yosphere . Such alrggrenii  and, duegal taxa , impart gal taxa . Togethegal taxa  and consgal taxa . IncreasTo-date, the majority of microbial studies conducted on GrIS supraglacial habitats have focused on ice algal blooms , and theThe aim of this study was therefore to provide the first comprehensive analysis of the fungal diversity from the surface of the GrIS. To this end, a combination of culturing and ITS amplicon sequencing was applied to five different GrIS surface habitats: snow, supraglacial water, sediment and water from cryoconite holes, and surface ice containing a low and high abundance of ice algae. Bacterial diversity was determined in parallel by cultivation and amplicon sequencing techniques. Special consideration was given to the comparison between the dark ice and other habitats.Whirl-Pak® plastic bags. Samples were collected from supraglacial water, sediment and water from cryoconite holes (“slurry”), dispersed cryoconite (only in 2016), fresh snow (only in 2017), surface ice with high abundance of ice algae (104 cells ml-1) – hereafter referred to as dark ice, and with low algal abundance (101 cells ml-1), referred to as clear ice.Two sampling campaigns were conducted during the 2016 (July–August) and 2017 (June–July) melt seasons in the south-western ablation zone of the GrIS, covering in 2 years an entire ablation season: from snow to the end of melt. The sampling site was located ∼60 km  east of Kangerlussuaq, and was situated within the ‘dark zone,’ a region running along the western edge of the GrIS that is characterized by particularly low albedo and extensive ice algal blooms during summer melt seasons  Figure . The samin situ at site temperature and due to the unknown fungal abundances and in order to obtain single colony forming units, aliquots of 10 ml and 100 ml were filtered through Milli-pore membrane filters (0.45 μm pore size) in duplicate. Filters were placed on two enumeration and four different selective agar media with either low nutrient content (used only in 2017 sampling) or with low water activity (aw). 100 μl of the original sample from all environments were directly applied on all media and dispersed with a sterile Drigalski spatula. Media used included DRBC – a general-purpose enumeration medium (aw = 1) (w = 0.946) (w = 0.880) (w = 1), (w = 1) used in 2017 sampling  ; DG-18 –= 0.946) ; MY10-12= 0.880) ; R2A – a(w = 1), ; and SNAsampling . For the2 of mycelium according to Taq DNA Polymerase according to manufacturer’s protocol. Reactions were run in a PCR Mastercycler Ep Gradient (Eppendorf) with an initial denaturation of 2 min at 95°C, followed by 30 cycles of denaturation at 95°C for 45 s, annealing at 54°C for 30 s, and elongation at 72°C for 2 min, with a final elongation at 72°C for 4 min. For identification of Penicillium strains, the partial β-tubulin gene (benA) was amplified and sequenced with Ben2f and Bt2b primers  sequences, amplified with ACT-512F and ACT-783R primers  and R2A media, respectively. DNA from filamentous fungi was extracted by mechanical lysis of approximately cm primers . Initial primers . Initial primers . Initial primers  and a to primers  implemen primers  and exam primers . Maximum primers  were use primers   and immediately frozen at -20°C until further analysis in the laboratory. Replicates were treated as independent, with a total of 15 samples labeled with corresponding habitat type followed by the collection year and the number of replicate: C-ice16-1; C-ice16-2; C-ice17-1; C-ice17-2; Cry17-1; Cry17-2; D-ice16-1; D-ice16-2; D-ice17-1; D-ice17-2; S-wtr16-1; S-wtr17-1; S-wtr17-2; Snow17-1; Snow17-2. DNA was extracted from filters and cryoconite sediment using the PowerWater DNA Isolation Kit  and then from the same filters using the PowerLyzer PowerSoil DNA Isolation Kit , according to the manufacturer’s instructions with a slight modification to increase the DNA yield and quality. To increase efficiency of fungal cells lysis an additional heating incubation at 65°C for 10 min was used after adding PW1 solution. DNA from both isolation methods was pooled together and stored at -80°C until PCRs were performed.For 2016 samples, DNA was isolated from filtered biomass  of clear ice (C-ice), dark ice (D-ice) and supraglacial water (S-wtr). From 2017 samples, DNA was isolated from 700 ml of dark ice, 1 L of clear ice, 2 L of melted snow (Snow), and 1 L of supraglacial water. With the exception of supraglacial water from 2016, two biological replicates of each sample were assessed. All filters and 1 g of cryoconite sediment (Cry) were placed in 1.5 ml microcentrifuge tubes containing RNAlaterFor the analysis of fungal diversity, Illumina Miseq V3 (300 bp paired-end) sequencing was carried out on the ITS2 region of the ITS rDNA gene amplified using the primers ITS4-Fun (5′ AGCCTCCGCTTATTGATATGCTTAART-3′) and 5.8S-Fun (5′-AACTTTYRRCAAYGGATCWCT-3′) . Amplifi® High-Fidelity DNA Polymerase according to manufacturer’s protocol. Reactions were run in a PCR Mastercycler Ep Gradient (Eppendorf) with initial denaturation of 3 min at 95°C, followed by 20 cycles of 20 s at 95°C, 30 s at 56°C and of 30 s at 72°C, with a final elongation of 5 min at 72°C.For analysis of bacterial diversity, Illumina Miseq V2 (250 bp paired-end) sequencing was carried out on the hypervariable V3 and V4 regions of the 16S rRNA gene by using the 341F_ill (5′-CCTACGGGNGGCWGCAG-3′) and 802R_ill (5′-GACTACHVGGGTATCTAATCC-3′) universal bacterial primers . Due to Single-end reads were quality checked and trimmed  and analyzed with QIIME2 2018.8 software package  . The forin situ until analysis. In the laboratory (Ljubljana), filters and corresponding methanol were transferred to 5 ml sterile centrifuge tubes, and 3 ml of 100% chloroform was added and vortexed for 15 min at room temperature. The methanol phase was transferred to a sterile 1.5 ml microcentrifuge tube and dried in a biosafety cabinet overnight. The residues were re-dissolved in 1.5 ml 90% methanol and vortexed for 5 min at room temperature. Samples were then filtered through 0.22 μm pore size filters and transferred to HPLC tubes. HPLC analyses were performed using Waters HPLC system 2965 , equipped with a degasser, a quaternary pump, an auto-sampler and 2998 PDA detector set at 280 nm. A Kinetex XB-C18  column  was used. 50 μl of each sample was injected. Ergosterol was eluted from the column using an isocratic mobile phase of 100% methanol and a flow rate of 1.0 ml/min. Before HPLC analysis, standard ergosterol  was purified according to Quantification of ergosterol as a biomarker molecule for the determination of fungal presence was performed for 2017 environmental samples. Filtration of 500 ml of melted snow and 100 ml of supraglacial water, dark ice, clear ice and cryoconite was performed through Milli-pore membrane filters (0.45 μm pore size) in biological triplicates (in duplicate for snow). Filters were then stored in sterile microcentrifuge tubes containing 1.5 ml of 90% methanol and frozen w media: on MY10-12 up to 2.5 × 10 CFU/100 ml and on DG-18 up to 3 × 105 CFU/100 ml. DRBC counts ranged from 0 to 2.3 × 102 CFU/100 ml. Significantly higher counts were obtained on low nutrient media: on MM up to 7 × 104 CFU/100 ml and on SNA up to 3.6 × 105 CFU/100 ml.Abundances of cultivable fungi from samples collected in 2017 are presented in 4 CFU/100 ml (DG-18 and MM) to 3.6 × 105 CFU/100 ml on SNA. Fungal abundance in dark ice spanned 2–8 × 10 CFU/100 ml on the two low aw media (MY10-12 and DG-18), but increased considerably on media with low nutrient concentrations , where CFU ranged from 2 to 7 × 104 CFU/100 ml. Clear ice contained considerably lower counts than cryoconite and dark ice, ranging from 0 on DG-18 and MY10-12 to 6 × 10 CFU/100 ml on MM and SNA. CFU in supraglacial water were considerably higher relative to clear ice and snow and ranged from no isolates (DRBC and MY10-12) to 1 × 104 CFU/100 ml.The overall lowest counts were detected in fresh snow, with fungal counts, ranging from 0 (DRBC) to 2 × 10 CFU/100 ml (MM and DG-18). In contrast, samples from cryoconites holes had the highest fungal abundance with counts ranging from 5 × 10Quantification of ergosterol in samples collected during 2017 fieldwork is shown in Ergosterol levels were highest in cryoconite slur and dark ice compared to the other sampled habitats, with concentrations up to 162 ng/ml and 177 ng/ml, respectively. Interestingly, biological replicates of samples with the highest levels showed high variability in ergosterol concentrations, with values ranging between 21.6 and 162 ng/ml in cryoconite slur, and 30 and 177 ng/ml in dark ice. However, even the smallest ergosterol quantities in the abovementioned samples were 10 times higher than any of the samples of fresh snow, clear ice and supraglacial water. Concentrations of ergosterol in supraglacial water and clear ice were similar, with values from 3.6 to 3.9 ng/ml, and 2.4 to 3.6 ng/ml, respectively. The lowest levels of ergosterol, from undetectable to 0.66 ng/ml, were found in fresh snow.Oleoguttula mirabilis and two basidiomycetous yeasts, Glaciozyma antarctica-like and Rhodotorula “svalbardensis”. Other yeasts that were isolated sporadically were Vishniacozyma victoriae, Dothiora sp., Mrakia sp., Phenoliferia glacialis, Sporobolomyces ruberrimus, Tilletiopsis washingtonensis and Basidiomycota sp. 2, 4, 6 . In comparison to dark ice, a much lower recovery of cultivable species was obtained from clear ice. However, Oleoguttula mirabilis, Glaciozyma antarctica-like and Rhodotorula “svalbardensis” remained the most common taxa, with Baeospora myosura, Basidiomycota sp. 2  of the cultivable fungal diversity showed dark ice and snow as the habitats with the most diverse fungal communities compared to cryoconite, supraglacial water and clear ice . Filamentous fungi were represented by lear ice . Furtherlear ice , with soArticulospora sp., present to a lesser extent also in plated ice algae. As in clear and dark ice, Rhodotorula “svalbardensis” was the dominant species also in cryoconites. Other sporadic fungal species were Cladosporium sp., Vishniacozyma victoriae, Dothiora europaea, Mrakia sp., Glaciozyma antarctica-like, and Preussia sp.Cryoconite holes differed from all other communities by a high presence of PRJNA507743.In total 4,357,151 single-end reads were obtained from 15 samples, corresponding to 697 different operational taxonomic units (OTUs). The number of reads per sample ranged from 109,100 (Cry17-1) to 434,630 (S-wtr17-1), with the exception of sample S-wtr16, where the number of reads was much lower , therefore this sample was excluded from analyses of alpha and beta diversity. Sequence data generated and analyzed during the current study are available in GenBank under the BioProject with the accession code p = 0.001; the number of permutations = 999). However, no statistically significant differences were found in the pairwise PERMANOVA comparisons of sample types, possibly due to the small numbers of samples of each sample type.Shannon indices  varied oMicrobotryomycetes (Basidiomycota) , followed by Chytridiomycota phylum (22.8–26.3%). The phylum Chytridiomycota was also present in all the other samples, except for snow, with lower frequencies . Snow samples were dominated by 45.2–75.6% Dothideomycetes (Ascomycota) and by 45.7–5.5% Agaricomycetes (Basidiomycota). Although black yeast genus Aureobasidium  represented 0.7–3% of the Dothideomycetes, the majority of this class (40.5–66.7%) belonged to the Pleosporales, with the two most abundant families being Pleosporaceae (16.2–32.6%) and Sporormiaceae (14.8–23.7%) and the most abundant genus Comoclathris (9.4–31.4%). Snow samples and clear ice 2017 samples were characterized by the presence of Rozellomycota phylum . Dark ice from 2016 and 2017, clear ice from 2017 and snow contained Oleoguttula mirabilis sequences with a relative abundance <1%. The class Malasseziomycetes was present in clear ice, supraglacial water and cryoconite samples with a frequency <1%.Clear ice, dark ice and supraglacial water samples from both years (2016 and 2017) were dominated by omycota) , with reThe PCA of ordination patterns revealed four separate sample clusters based on Bray-Curtis dissimilarity index . The thr5 CFU/100 ml, respectively. Clear ice followed with 1.3 × 102 CFU/100 ml, while fresh snow had the lowest bacterial counts – 2 CFU/100 ml.Abundances of bacteria on R2A medium are summarized in Pseudomonas sp., followed by Rugamonas rubra, Janthinobacterium sp. and Undibacterium sp. Amongst sporadically isolated species were unidentified Oxalobacteraceae, and Sphingomonas sp.Forty-six bacterial isolates were obtained from all five environments sampled in 2017. Cryoconites had the most diverse bacterial community. The most isolated species was Bacillus sp., Curtobacterium sp., Frigoribacterium sp., uncultured Oxalobacteraceae similar to isolates from Arctic sea ice , corresponding to 883 different features OTUs. Number of reads per sample ranged from 2,067 (D-ice17-1) to 108,084 (S-wtr17-1). Sequence data generated and analyzed during the current study are available in GenBank under the BioProject with the accession code p = 0.001, and the number of permutations = 999). Pairwise PERMANOVA identified statistically significant differences between dark ice and supraglacial water (p < 0.005), but no other significant differences were found .Shannon indices  for bactMicrococcus , which was represented by few sequences in other samples (<1%), while the genus Hymenobacter (Bacteroidetes) and the phylum Acidobacteria were present in all other samples except snow. Phylum Armatimonadetes was present in ice and supraglacial water from 2017 in high abundance (10.8–60.8%). Its presence was lower in cryoconites (4.17–5.6%), while it was absent from snow. Cryoconite samples were characterized by the classes Chloroflexi (mainly class Ktedonobacteria) and WPS-2. Genus Polaromonas was found in ice from 2016 and supraglacial water from both sampling years.The common bacterial phyla across all 15 samples were Actinobacteria, Bacteroidetes, Proteobacteria, and Cyanobacteria, with broad ranges of relative abundances  . HoweverThe PCA of ordination patterns revealed five separate sample clusters based on Bray–Curtis dissimilarity index . The thrThis is the first study to characterize the fungal diversity and abundance across different habitats of the surface of the GrIS using both cultivation and sequencing approaches. Particular emphasis was given to algal dominated ice in comparison to other habitats due to its impact on surface albedo and potential to exacerbate surface melt. In this study, we use the information about fungal diversity in the different supraglacial habitats to speculate potential roles of supraglacial fungal community in association with ice algae, such as a saprotrophic role in degradation of the algal biomass, but also possibly pathogenic or symbiotic relationship, either as endophytes relieving stress conditions or akin to those of lichens helping the ice algae in stabilizing themselves on the ice. Interactions between fungi and ice algae may thus influence GrIS ice algal bloom dynamics, and consequently hold potential to indirectly impact ice surface albedo and melt.p = 0.011). Ergosterol on Canadian soil samples measured with HPLC procedure by Fungal abundance was estimated across five different surface habitats of the GrIS via cultivation and ergosterol measurements. The relative amounts of ergosterol confirmed the fungal abundance determined by cultivation in cryoconite and dark ice. MM, due to its low nutrient concentration, limits the growth of highly sporulating fungi and thus reduces the overestimation of their number. This may be the explanation of why in our study, MM is the medium that better reflected the real abundance of fungi and that correlated with the ergosterol quantification with a correlation coefficient of 0.726 , as previously observed for Icelandic  are two genera known for their stress tolerance, and are consistently isolated from extreme environments . Oleoguttula mirabilis, reported for the first time in the Arctic, is a new species from a recently described genus of black meristematic fungi . Its presence was observed in close association with clumps of dead and alive ice algae, kept together by algal EPS, suggesting a potential role in the degradation of the ice algae. Articulospora has been previously described in cryoconite holes in Svalbard  that furthermore showed the presence of the plant-associated black yeast genus Cladophialophora (9.2–12.6%). Other species obtained by cultivation were mainly basidiomycetous yeasts, such as Mrakia sp., Rh. “svalbardensis”, and Glaciozyma antarctica-like typical for polar habitats (see above).Ascomycetous genera, such as m nivale , which cm nivale , and basurements . High prurements  that mayurements . Ancestrar areas . TherefoSvalbard  and its Penicillium bialowiezense-like (data not shown), dominating in all the environments, while in 2017 the isolates were much more diverse and belonged to 33 different genera. Two possible reasons for the observed differences could be the different snow conditions on the GrIS between 2016 (after the snow melting) and 2017 (before the snow melting), and the use of a wider selection of media in 2017. The results of amplicon sequencing, however, did not show such stark dissimilarity between the seasons, possibly pointing to insufficient sampling of the total fungal community with cultivation methods or their greater bias.Finally, we observed a substantial difference in cultivable fungal diversity between the sampling seasons of 2016 and 2017. The majority of isolates cultivated from 2016 sampling belonged to Heterotrophic bacteria are common within the various supraglacial habitats where they play an important role in nutrient cycling of decaying organic matter and remineralization of the limited nutrients available. Bacterial populations of the GrIS supraglacial environments have been studied for years , facilitIn line with previous studies  ProteobaThis is the first report of fungal communities in GrIS water-based ecosystems and one of the few culture independent studies of microbial diversity in ice. In our amplicon sequencing based study of the microbial variety of GrIS supraglacial habitats, we described more diverse bacterial communities than fungal communities. Combining different approaches was thus beneficial in these extreme environments with low biodiversity. Dark ice fungal communities were characterized by plant pathogenic fungi and endophytes and a much higher fungal and bacterial abundance than in other supraglacial habitats on the GrIS, underlining its distinctiveness and suggesting that ice algal clumps probably influence the associated microbial diversity and abundance. In particular, fungal roles in the prospering and degradation of ice algal blooms should be investigated further. Since the darkening of ice  has the potential to significantly increase GrIS surface melt, interactions between all microbial communities in this specific habitat should be studied in more detail.The datasets generated for this study can be found in GenBank, MK460310–MK460422, MK454775–MK454866, MK453054–MK453127, PRJNA507743, and PRJNA517071.LP collected the samples with the support of AA and CW. LP performed the wetlab analyses. CG performed the bioinformatic analyses of NGS data. LP and CG analyzed the data. LP, NG-C, and CG interpreted the data with contributions from AA and CW. LP drafted the manuscript. All authors contributed to design of the experiments, revised the manuscript draft, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.