Entomophthora muscae isolate and improved long-read sequencing to obtain a largely-complete entomophthoralean genome. Our E. muscae assembly is 1.03 Gb, consists of 7,810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with other available (transcriptomic and genomic) datasets from entomophthoralean fungi, we provide new insight into the biology of these understudied pathogens. We offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex. Our findings suggest that substantial taxonomic revision is needed to define species within this group and we provide recommendations for differentiating strains and species in the context of the existing body of E. muscae scientific literature. We show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity, despite the presence of anti-transposable element defense machinery (RNAi). In addition, we find that E. muscae and its closest allies are enriched for M16A peptidases and possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We find that E. muscae has an expanded group of acid-trehalases, consistent with trehalose being the primary sugar component of fly (and insect) hemolymph. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae’s species-specific, biotrophic lifestyle. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. This is in part due to their large genome sizes and difficulty in obtaining sequenceable material. In this manuscript, we leveraged a recently-isolated, laboratory-tractable  Beauveria bassiana, Metarhizium anisopliae, etc.), many others, especially those within the fungal phylum Zoopagomycota (formerly Zygomycota), have received comparatively little attention and are poorly understood. Zoopagomycota is the earliest diverging lineage of non-flagellated fungi  was found to affect nearly the entire population  Are phylogenetic determinations made using morphological characteristics consistent with those based on molecular data? In assembling the E. muscae genome and using it to address these initial questions, we hope to provide biological insight into the Entomophthorales as well as provoke additional exploration into these understudied fungi. The answers we uncovered each reveal new avenues of research that will lead to a better understanding of the evolution of entomophthoralean insect pathogens, particularly the influence of vastly proliferated transposable elements, in support of future work and applied use of these insect pathogens.A major challenge in understanding the biology of entomophthoralean fungi is the difficulty of culturing these fungi in the laboratory. While some species can be grown in vitro  or, lessin vitro , such mein vitro . Here, wrkeley’, ). We useTrichoderma (in vitro-grown Entomophthora muscae culture inoculated from a single sporulating E. muscae-killed fruit fly. Oxford PromethION sequencing of the resultant genomic library yielded 85.71 Gb of sequenced bases (N50 = 9.54 kb) that passed quality control thresholds , proteiresholds , InterPrresholds ) and ontresholds , EggNog resholds , COG (Garesholds ) databasresholds ) and traresholds .E. muscae (this study), Entomophaga maimaiga (ARSEF 7190 v1.0) and Massospora cicadina (Pandora formicae (Strongwellsea castrans (this study) and genomes for the generalists Zoophthora radicans  to 87.4% (S. castrans), with a median value of 82.4% across this group (E. muscae genome had more double-copy (58.2%) than single-copy (23.1%) BUSCOs. The next highest proportions of double-copy BUSCOs were found in the transcriptomic datasets of P. formicae (31.1%) and S. castrans (29.7%) which includes partial transcripts and isoforms in the duplication count. Based on BUSCO scores and phylogenetic positions, we selected three other genomes aside from E. muscae to use as a core set for genomic analysis: Entomophaga maimaiga, Z. radicans and N. thromboides  using a fungal reference set (odb10) . For E. detected . For theis group . Notablyomboides .E. muscae, which was assembled from the DNA of fungal cells cultured in vitro. One possible explanation for many duplicate BUSCOs in E. muscae is that the assembled genome was functionally diploid, not haploid, which has been suggested previously for another E. muscae isolate from houseflies (Musca domestica) , this difference could not fully account for the discrepancy in gene count (E. muscae.While the high number of duplicate BUSCOs in the transcriptomes could be accounted for by contaminant host transcripts (each of these transcriptomes was assembled from fungus-killed hosts), this explanation could not account for the high number of duplicate BUSCOs in mestica) . To addrmestica) ) and plomestica) ). We obsmestica) ,B. We alne count . This suE. muscae’s genome is very large compared to the vast majority of other fungal genomes  are also extremely large, each exceeding 500 Mb. However, the impressive sizes of E. muscae and other entomophthoralean genomes do not appear to be a consequence of increased gene number (N. thromboides) to 14,701 (E. maimaiga) to 39,711 (E. muscae). Gene counts for M. cicadina are also within the low end of this range , but this number is expected to be an underestimate given the fragmented nature of the assembly. In comparison, fungi with genomes between 10-100 Mb have between ~3,000-33,000 genes. Thus, as in other fungi whose genome sizes rank within the top 1% , the predicted number of genes in entomophthoralean fungi is within the range observed for fungi with more typically-sized genomes (<100 Mb).At 1.03 Gb,  genomes . Similare number . The numConidiobolus have highly repetitive genomes ; for Z. radicans the proportion of repeated sequences genome-wide is about 20% less, at 71%. Meanwhile, N. thromboides’ genome is predominantly non-repetitive (only 13.5% sequences are repeated). The bulk of this repetitive content consists of transposable elements, which are categorized as Class I or Class II based on their mechanism of action. Class I transposons (retrotransposons) “jump” via transcription to an RNA intermediate that is then reverse transcribed to DNA and integrated in a target genomic site. Class II transposons (DNA transposons) “jump” when they are excised from donor DNA and reinserted into a target genomic site. The majority of repeat content across these four family Entomophthoraceae genomes consists of Class I retrotransposons, specifically Ty3  of Tc1 mariner DNA (Class II) transposons populating their genomes. M. cicadina is unique among the other entomophthoralean fungi in having a substantial fraction (6.3%) of L1 long, interspersed nuclear elements (LINEs) while effectively lacking any DNA transposons.Unlike gene content, repeat content across entomophthoralean genomes reveals a clear trend: fungi that shared a common ancestor after divergence from  genomes . The gened Gypsy ) long teE. muscae, E. maimaiga, M. cicadina, Z. radicans and N. thromboides genomes show trends consistent with phylogenetic relationships between these fungi (N. thromboides have expanded Ty3 and unknown LTR elements with comparable Kimura divergences (~0-50), indicating similar proliferation of these elements occurred with approximately concurrent timing. M. cicadina uniquely shows a burst of Ty3 LTR expansion around divergence time 20 and an expansion of LINE elements, occurring after Ty3 expansion. E. muscae and E. maimaiga show an increase in TcMar elements more recently than either of the Ty3 and LINE expansions. In addition, E. muscae, E. maimaiga and Z. radicans share an expansion of DNA-MULE-MuDR elements that is not observed in M. cicadina. All five genomes also possess putative repeat elements currently classified as “Unknown” beginning roughly concurrently.Kimura divergence estimates for most populous repeat elements of the se fungi . All butNeurospora crassa, an ascomycete fungus in which RIP has been extensively documented (Austropuccinia psidii (N. crassa showed high levels of RIP (17.4%) (Z. radicans occurred at an even higher rate (24.2%) than in N. crassa. We also detected RIP at low levels in the rust Austropuccinia psidii (2.6%), E. muscae (2.4%), M. cicadina (2.7%), and E. maimaga (5.7%). This analysis argues that RIP occurs in Z. radicans and could be occurring at a low level in other entomophthoralean fungi as well.Fungi are known to protect against the proliferation of TEs by mutating cytosine to thymine in repetitive regions via a process called Repeat Induced Point (RIP) mutations . This prcumented , and Ausa psidii , a basida psidii . Consist (17.4%) . SurprisNeurospora crassa, and N. crassa RID is sister to the OG0001715 clade in a tree of these proteins, albeit at low (12-14%) sequence identity  of about half the size, which is in tandem with another, complete E. muscae OG0001715 paralog (DSO57_1016267). This analysis suggests that E. muscae, E. maimaiga and Z. radicans contain multiple proteins with similar domain architecture as RID, with some direct evidence for duplication of these methyltransferases.A cytosine methyltransferase named RIP defective (RID) is required for RIP , one of identity . OG00171E. muscae and E. maimaga predicted to have several homologs of each of these genes. Thus, loss of RNAi pathway genes seems unlikely to account for transposable element proliferation in these species.Many eukaryotic organisms also employ RNA interference (RNAi) to control retrotransposon activity. RNAi occurs when double-stranded RNAs are processed into small interfering RNAs (~20-30 nucleotides long) that are used to guide degradation  of complementary RNA. In fungi, there are three proteins essential for RNAi: 1) RNA-dependent RNA polymerase (RDRP), which processes ssRNA into dsRNA, 2) Dicer, the enzyme that processes dsRNA into siRNA, and 3) Argonaute, a protein that loads siRNA to form an RNA-induced silencing complex (RISC) that identifies target RNAs by complementarity . We perfM. cicadina genome. M. cicadina was excluded because the fragmented nature of its genome assembly precluded an accurate prediction of this fungus’ proteome, a key requirement for this analysis , and more specifically at functional domains involved in metabolizing carbohydrates (using CAZy) and proteins (using MEROPS). For these analyses we used all available entomophthoralean datasets  except fanalysis .E. muscae of retroviral families , specifically Asp_protease (PF09668), Asp_protease_2 (PF13650), gag-asp_protease (PF13975), RVP_2 (PF08284) and dUTPase (PF00692) domains , which was also enriched in E. maimaiga and Z. radicans .Pfam families encompass diverse proteins with a broad range of functions . Enrichm domains . The domE. muscae (20) compared to the number of domains found to be significantly overrepresented (105) , bifunctional feruloyl and acetyl xylan esterases  and endonuclease/exonuclease/phosphatase . Alpha/beta hydrolase folds are found as catalytic domains in many different enzymes, including BD-FAE (PF20434) proteins, which specifically act on complex xylans. The BD-FAE family was significantly underrepresented in E. muscae, E. maimaiga and Z. radicans , compared to C. coronatus and N. thromboides . E. muscae, with only a single predicted BD-FAE-containing protein, was additionally significantly lower than P. formicae and S. castrans, which had 20 and 23, respectively.A much smaller number of Pfam domains were found to be significantly underrepresented in ed (105) . ProportS. castrans and P. formicae shared a unique overlap of 1,142 domains, each with 170 and 485 unique domains, respectively. E. muscae, by comparison, only had 14 unique Pfam domains. The trend of P. formicae and S. castrans sharing many domains was seen across all domain types. However, the proteomes for these fungi were uniquely generated from transcriptomic data, so we were concerned this pattern may reflect a methodological, not biological, signal. To test this, we recapitulated our Pfam domain analysis incorporating a proteome generated from transcriptomic data collected from E. muscae. The results of this analysis are summarized in an UpSet plot  absent in the other species. This unique pattern for S. castrans and P. formicae was consistent across all types of domains and shows a clear signal on the UpSet plot comparing domains , AA7 (glucooligosaccharide oxidases), CBM18 (chitin-binding and chitinases), CE16 (acetylesterases), CE4 (acetyl xylan esterases) and GT1 (UDP-glucuronosyltransferase)  provides insight into which carbohydrates can be metabolized. The majority (55) of CAZy domains were shared among all fungi, with  domains . E. musc domains : GH134 (sferase) . AA11 anS. castrans and P. formicae shared 448 MEROPS domains absent in the other genomes, with each having a large number of unique MEROPS domains . E. muscae by contrast had about a third to a quarter as many unique MEROPS peptidases (37). Among E. muscae MEROPS, 92 were found to be significantly different in comparisons among genomes, with 74 MEROPS domains significantly enriched and 9 domains having significantly fewer proteins  compared to S. castrans, P. formicae and N. thromboides. Considering proteins containing these 10 M16A domains, E. muscae (62 on average), E. maimaiga (19) and Z. radicans (44) were comprised of approximately 11-fold more proteins (43 vs 4) compared to S. castrans (5), P. formicae (3) and N. thromboides (2). Two metallopeptidases were uniquely enriched in E. muscae: MER0001120, a pro-collagen C-peptidase, and MER0016735, an M23 peptidase. Among serine peptidases, a lipid monooxygenase (MER0031618), two S33 prolinases (MER0036050 and MER0036051) and three kexin/kexin-like  domains were found to be significantly enriched. Additionally, E. muscae was uniquely enriched in the monoglyceride lipase MER0045883. Considering cysteine peptidases, E. muscae had a high number of metacaspases across all 10 families, though fewer than the number seen in E. maimaiga (~50 accessions for each family). Only a handful of domains were found to be significantly underrepresented in E. muscae, including aminopeptidase H11 (MER0002003), cystinyl aminopeptidases (MER0002060) and ERAP2 aminopeptidases (MER0002968)  .E. muscae is not dictated by host circadian machinery, instead suggesting that a fungal clock drives the timing (Neurospora crassa (Ascomycota) is the most highly studied and best understood circadian clock among fungi  form a heterodimeric complex . In the presence of light, WCC undergoes a conformational change and becomes a transcriptional activator of light-regulated genes, including the gene frequency (frq) that encodes an intrinsically-disordered protein. FRQ protein forms a heterodimeric complex with its partner, FRQ-interacting RNA Helicase (FRH), and this complex, called FFC (FRQ-FRH Complex), inhibits the activity of WCC. After protein synthesis, FRQ becomes progressively more phosphorylated, eventually leading to its degradation and reduced concentration of FRQ and FFC. When FRQ titers drop below a certain threshold, WCC activity can proceed unimpeded, thus closing an oscillating cycle of transcription and translation with a periodicity of about 24 hours.Beyond these protein domain and enzyme analyses, we were very interested in comparing potential circadian machinery among entomophthoralean fungi as, for many of these pathogens, infection and behavioral manipulation of the host follows a strict daily timing . How thie timing . The cirngi  and the structurally-similar domain GAF (GAF: PF01590) . For wc-2, all but E. muscae, Entomophaga maimaiga and N. thromboides also had at least one candidate gene. None of these species contained a gene encoding for a protein with the hallmark domains of frq  . All of s of frq .wc-1 homolog for all of the species analyzed, we also searched for genes encoding for known light-sensitive domains ). We did not detect any bacteriorhodopsins or phytochromes within our datasets, but we found several instances of genes containing the other light-sensitive domains, with E. muscae having far more than the rest (99 genes with rhodopsin family domains as well as 11 photolyases or FAD-binding domains thereof) (In addition to finding evidence for at least one copy of a thereof) . This anE. muscae, Entomophaga maimaga, Z. radicans and N. thromboides  contained representatives in all four species  was found for E. muscae (21%), followed by Z. radicans (6.9%), Entomophaga maimaiga (5%) and N. thromboides (2.3%). Accordingly, we observed that E. muscae genes also populate the most OGs overall, though has genes in multi-species OGs no more frequently than Entomophaga maimaiga  together with the genes that are found in species-specific OGs comprise a set of genes that are potentially unique to that species. If we consider all of the annotated genes in the E. muscae assembly, E. muscae has the highest percentage of species-specific genes among this species set at 50.9%  is trehalose  was the second-most abundant unique Pfam family (6 proteins). Among other E. muscae-specific domain families, two proteins were annotated as containing abhydrolase_8 domains (PF06259), perhaps accommodating for the apparent significant reduction in abhydrolase_3 domains in E. muscae , threonine peptidase  and cysteine peptidase  were found uniquely in E. muscae. The most abundant MEROPS family that was unique to E. muscae was a chymotrypsin family . Only three CAZy domains were uniquely found from E. muscae: GH134 , GH5 (glucanases/cellulases) and PL12 (heparin-sulfate lyase).To investigate unique characteristics of the nique to  or missinique to  E. musca. muscae , the Pfa. muscae . Nearly E. muscae genome but observed in other genomes within our core set of entomophthoralean fungi. To assess loss of key domains in E. muscae, we considered domains that were missing from E. muscae, but were significantly enriched in other entomophthoralean fungi  in E. muscae were enriched only in P. formicae and S. castrans, suggesting this signal is reflective of functionalities unique to those species, rather than functionalities lost in E. muscae.In total, 2,237 Pfam domains were absent in the an fungi . We did E. muscae. As in our previous OG analysis, we considered genes to be potentially species-specific if they either failed to cluster with an OG in E. maimaiga, Z. radicans or N. thromboides or clustered with OGs comprising only other E. muscae genes. We performed an enrichment analysis of Pfam annotations of genes that met these criteria, comparing the frequency of Pfam occurrence in this set against occurrence within all annotated genes (E. muscae-specific genes: insulinases (PF00675) M16 peptidases (PF05193) and genes that lacked any Pfam annotation. That E. muscae may have specific peptidases is consistent with our understanding of the entomopathogenic lifestyle: these enzyme families are both key in host recognition and invasion as well as host resource utilization , cell signaling , and transcriptional regulation functions . The underrepresentation of these domains in potentially E. muscae-specific genes suggests their functionalities are similar to those in other entomophthoralean species.Pfams that were underrepresented in the set of potentially E. muscae-unique genes that are predicted to encode secreted proteins  processes are underrepresented due to a both unlikelihood of being secreted and conserved function among species.We performed a similar enrichment analysis looking at the set of potentially proteins . We obseE. muscae-unique secretome included various catabolic enzymes  [PF05922], zinc carboxypeptidase [PF00246], insulinase [PF00675], lipase [PF01764], polysaccharide deacetylase [PF01522], glycosyl hydrolases ) and macromolecular recognition domains . The observation that catabolic enzymes are overrepresented within the putatively E. muscae-specific predicted secretome is consistent with our current understanding of entomopathogen host interactions  and outgroup taxa within the genus Entomophthora were mined from NCBI GenBank to determine the prevalence of each locus. Novel Sanger sequence data was also generated in support of this analysis, which has been deposited in NCBI  and three other strains from Muscidae  and Bayesian inference (BI), for the 2-gene concatenated dataset resolved the EMSC as a well-supported monophyletic group that includes tophagae . In addi Clade 2 . Clade 1Muscidae . Clade 2 grandis .Entomophthora spp. included in the study, there was considerable overlap in the ranges of size and mean number of nuclei from primary conidia  for number of nuclei and sample sizes. Nuclei data was missing for three of fourteen strains. Number of nuclei was the most commonly reported morphological character, but this measure alone did not resolve species boundaries, except for E. schizophorae.We examined the number of nuclei in, and dimensions of, primary conidia, which are potentially diagnostic morphological characters for these species . For the conidia , 6D. TheE. ferdinandii, but not E. muscae. Strains ARSEF 6716 and KVL14-17, both members of EMSC Clade 1, had overlapping spore measurements with each other and E. muscae, but not E. ferdinandii. These spore measurements further support E. scatophagae as part of the larger EMSC. They also highlight significant overlap between E. grandis and E. syrphi.Primary conidial length and width measurements provided better species-level resolution, but these measurements were only available for four of fourteen strains . ReporteE. muscae genome obtained using a long-read sequencing approach. Despite the comparative ease of obtaining high molecular weight DNA from other organisms, entomophthoralean fungi are recalcitrant to standard DNA extraction procedures. The reasons for this are not yet clear, but, along with the relatively large sizes of entomophthoralean genomes, they have stymied sequencing and assembly efforts within this fungal order. Our success came from having on-demand access to an E. muscae culture in the lab and to using a modified extraction protocol , we observed a high level of duplicate BUSCOs. Our assembly is decidedly not fully diploid ,B. Inste isolate .E. muscae genome, sourcing available transcriptomic and genomic data from species within this fungal order. The inferred phylogenetic relationship between these species, based on conserved protein sequences  in our comparative data set, was Z. radicans, which, at 655 Mb, is still >15-fold higher than the mean fungal genome size of all 6,144 genomes considered in Conidiobolus Entomophthorales: this proliferation was not recent.The second smallest genome (after One possible avenue leading to such a proliferation could be loss of defenses against repeat accumulation. Fungi use repeat induced polymorphism (RIP) and RNA interference (RNAi) as their two main strategies to combat the accumulation of repeated elements. We detected several homologues of the core RNAi pathway components in each of our fungal species: Dicer, Argonaute and RNA-dependent polymerase . This anin insecta after successfully infecting the host. Protoplast production is a necessary step for parasexual fusing in the laboratory setting. However, the existence of parasexuality has been questioned in this group of fungi, as cell-to-cell fusion has only been observed to occur between genetically-identical hyphal bodies prior to resting spore formation  revealed several functions enriched in the predicted E. muscae proteome  on our core set of entomophthoralean genomes  and the blue-light sensitive white-collar 1 (wc-1). While we found many genes encoding light-sensitive protein domains, including several wc-1 homologs, we did not find homologs of frq within our entomophthoralean fungi. However, absence of frq does not preclude a functional clock in these fungi; many fungi that lack a frq homolog have clear circadian phenotypes under free-running conditions , indicating that there are frq-independent mechanisms for keeping time within fungi that have yet to be discovered (Verticillium dahliae has a homolog of frq that does not cycle over circadian time (frq genes have only been observed in Ascomycota (except for the subphylum Saccharomycotina), so it is unsurprising that entomophthoralean fungi would lack homologs of this gene  into putative orthologous groups , followed by E. muscae, Entomophaga maimaiga and Z. radicans (7.5%). Each species also possessed species-specific OGs, with E. muscae possessing the most. An enrichment analysis of Pfam annotations for E. muscae genes that were assigned to core OGs revealed that many processes that may be host-specific  are underrepresented within the core set , and wi species .Massospora experimentally detected several secondary metabolites that, based on current bioinformatic models of biosynthesis, should not exist in this species  in early infection , a constituent of subtilisins  that is expressed immediately after invasion of the host hemolymph helps fungal cells avoid detection by the host immune system (E. muscae’s unique collagen triple helix domain may play a role in evading the host immune system.An enrichment for the collagen triple helix repeat domain (PF01391) caught our attention. In the generalist fungal pathogen e system . In a siE. muscae-specific OGs. Berberine-like proteins are involved in the synthesis of isoquinoline alkaloids, a class of compounds that includes the analgesics morphine and codeine, and usually require FAD as a cofactor  among secreted cofactor . Intriguuinoline . Isoquinuinoline , and somEntomophthora spp. were identified using a combination of morphology, host species, and/or location  overlapped with measurements for E. ferdinandii and outside the reported measurements for E. muscae despite occupying the same well-supported clade as strains whose spore measurements agreed with E. muscae.To explore the utility of DNA sequence data (ITS + 28S) for identification purposes in the EMSC, we assessed the congruence of available sequence and morphology data for EMSC isolates. We recovered two well-supported clades (both in the single gene trees and the concatenated tree) that contain strains previously identified as tophagae -C. Usingtophagae . Furtherzophorae . InteresTaken together, these results indicate that the morphological characters examined here, which are traditionally used to identify Entomophthorales, are incongruent with molecular data especially within the EMSC. However, this disagreement between morphology and sequence data could reflect 1) the paucity of character data, 2) insufficient taxon sampling that failed to capture the full range of morphological and genetic variation, and/or 3) the presence of misidentified specimens in collections and studies that are potentially masking the presence of morphologically cryptic species . While ITS and 28S loci may have insufficient phylogenetic signal to fully resolve these species boundaries especially within the EMSC, resolution may be achievable with the addition of other loci. In addition, the evolutionary history of this group may include a combination of introgression and rare sexual recombination events that have softened species boundaries within the EMSC, though data supporting this are currently limited . OverallMany strains of EMSC exist in fungal collections (ARSEF in particular) that have no associated sequence or morphology data. We propose that for each individual specimen, whether novel or from a collection, researchers minimally generate ITS and 28S sequence data and collect morphological data , so that future research can assess these features in combination to define the species boundaries of this group. We encourage researchers to further deposit voucher specimens, live cultures and/or DNA with fungal collections  so that future researchers can return to the original sample to examine traits or generate more sequence data if needed. Likewise, historic studies based solely on morphology from herbarium specimens may need to be resampled for both DNA sequencing and morphology.Entomophthora muscae has been under scientific scrutiny since its description in 1855, yet, even under the new light of our genomic investigation, this fungus remains “one of the strangest and most interesting apparitions” . Cultures were grown without shaking, with flasks laid on the long edge to maximize surface oxygen exchange, in the dark at room temperature (19-21C). DNA was isolated from a log-phase culture via methods of  and sequenced sequentially on the Oxford Nanopore PromethION platform by the The Bauer Core Facility at Harvard University  to 82x coverage. Reads are accessible through the NCBI Sequence Read Archive (SRA) via accession number SRR18312934. Nanopore reads that passed filtering were assembled using Flye v2.8.3 . The resE. muscae (SRR18312935. This was accomplished by demultiplexing with longranger v2.2.2 (10x genomics), reads mapped with bwa-mem2  caught in a cabbage field . The fly contained a clearly visible large hole on the side of the abdomen characteristic of S. castrans infection and was kept alive for a few hours until being snap-frozen in liquid nitrogen. The infected fly was ground to a fine powder in liquid nitrogen before extracting total RNA using a Plant RNEasy Kit (QIAGEN #74904) following manufacturer’s instructions. Total RNA was sequenced using Illumina HiSeq 2000 technology and TRUseq library building by Beijing Genomics Institute , and transcriptome sequences were assembled in de novo mode using Trinity v2.11.0. Raw reads are deposited at the European Nucleotide Archive (ENA) with accession number: ERR12023556. Assembled transcripts are available in zenodo archive under Supporting_data folder  using the PHYling v1.1 (Phylogenetic relationships of species were determined by identifying conserved proteins from the protein translation of predicted genes or transdecoder ORFs from the transcriptome-only sampled species (ing v1.1  BUSCO / ing v1.1  searchesing v1.1  using thing v1.1 ) which dGenome completeness was computed with BUSCO v5.2.2  using thData used to generate de novo library of elements and classify their likely lineage using default parameters and LTR finding with LTRStruct option. The elements were classified by similarity to RepBase v20170127  was limited to MEROPS, CAZy and Pfam annotations. These annotations were completed according to a pipeline identifying domains by sequence similarity and motif searches https://github.com/stajichlab/Comparative_pipeline. The identified domain counts per species aggregated as total counts or total number of unique genes with a domain were compiled into a single table for comparison between species.Domain analysis for seven entomophthoralean proteomes  generated from the same strain (NCBI GEO GSE111046).Count tables were used to determine lists of domains present within each genome for set analysis including UpSet plots (ComplexUpset and UpSetR) and analysis of unique/missing genes. A second set analysis was conducted with Pfam domains including an additional Putative circadian genes were surveyed using Pfam domain annotations. The following domains were used for this survey: 7tm_1 (PF00001), FRQ (PF09421), GATA (PF00320), PAS_3 (PF08447), PAS_9 (PF13426), PAS (PF00989), Bac_rhodopsin (PF01036), GpcrRhopsn4 (PF10192), DNA_photolyase (PF00875), FAD_binding_7 (PF03441), PHY (PF00360) and GAF (PF01590). These were used to filter single-domain circadian candidates from each genome for our domain analysis. Additionally, we considered the expected domain pattern of known circadian genes to identify curated candidates for FRQ (one FRQ domain), WC1 (one GATA and one PAS_3 and one PAS_9), WC2 (one GATA and one PAS_3 and no PAS_9) and 7tm_1 .RNAi pathway candidates were similarly surveyed using Pfam domain annotations. Domains included in this survey were: RdRP (PF05183), Dicer_dimer (PF03368), PAZ (PF02170), Piwi (PF02171), Ribonuclease_3 (PF00636) and DEAD (PF00270). Identified candidates containing surveyed domains, but we further employed an expected domain pattern to curate candidates for RNAi pathway proteins: RDRP (containing RdRP domain), Dicer (containing Dicer_dimer), Ago (containing both PAZ and Piwi), Dicer_Alt (containing Ribonuclease_3 and either DEAD or PAZ).Orthologous genes were identified between the compared entomophthoralean species by first taking the longest peptide for each gene to avoid including alternative spliced isoforms in the analyses. Proteins were clustered with OrthoFinder v2.5.2  using Diin vivo samples .Gene expression was estimated by aligning a pooled set of 27 SE111046  to the aN. crassa. This alignment was used to produce a protein tree using RAxML . This tree was manually rooted to separate orthogroups, maintaining the clade containing RID and OG0001715. The basal E. muscae protein DSO57_1016266-T1 is apparently partial (140 aa) compared to other E. muscae proteins in OG0001715 (both are >1200 aa).Orthogroups were used to identify methyltransferases that share RID domain architecture. RID is annotated with multiple Pfam DNA_methylase (PF00145) domains. RID candidate proteins, containing more than one DNA_methylase, were identified with the same approach we used to identify circadian protein candidates. Where necessary, homologs that corresponded with genomes used in orthogroup analysis were identified via BLAST. All proteins within orthogroups that contained RID candidates (OG0001715 and OG0003300) were grouped in Geneious Prime (2021.2.2) and aligned using MAFFT  with the sequence of RID (AAM27408.1) from E. maimaiga (EMA), Z. radicans (ZRA), N. thromboides (CTH), C. coronatus (Conco1), and Basidiobolus meristosporus (Basme2finSC).Protein sequences containing extracellular trehalase enzymes (PF01204) were retrieved from the data and aligned using MAFFT, which was used to calculate a maximum likelihood phylogenetic tree with RAxML using default parameters. In addition, total number of genes and number of genes encoding the PFAM domains Lipases (Lipase_3), Subtilisin-like serine peptidases (Peptidase_S8), Trehalases , Trypsins (Trypsin), and Chitinases (Glycohydro_18) in representative fungal species of Zoopagomycota and Ascomycota were retrieved from JGI Mycocosm and compared to our data of E. muscae genome and its relatives, fungiSMASH v6.0.0 of antiSMASH . DNA was extracted from individual EMSC-killed cadaver drosophilids collected across California as per . All resulting trees & alignments are available in zenodo archive , and in.ac.il/; . Alignme.ac.il/; ) and a B.ac.il/; ), for a .ac.il/; . Trees w.ac.il/;  and Inks archive .Supplement 1Supplement 2Supplement 3Supplement 4


Candida albicans is likely the most studied fungus in the context of IBD, being a near universal gut commensal in humans and also a major barrier-invasive pathogen. There is emerging evidence that intra-strain variation in C. albicans virulence factors exerts a critical influence on IBD pathophysiology. In this review, we describe the immunological impacts of variations in C. lbicans colonisation, morphology, genetics, and proteomics in IBD, as well as the clinical and therapeutic implications.The gut microbiome is a diverse microbial community composed of bacteria, viruses, and fungi that plays a major role in human health and disease. Dysregulation of these gut organisms in a genetically susceptible host is fundamental to the pathogenesis of inflammatory bowel disease (IBD). While bacterial dysbiosis has been a predominant focus of research for many years, there is growing recognition that fungal interactions with the host immune system are an important driver of gut inflammation.  Inflammatory bowel disease (IBD) is a group of chronic inflammatory conditions affecting the intestine that are subclassified into Crohn’s disease (CD) and ulcerative colitis (UC). CD causes patchy transmural inflammation that can affect any part of the gastrointestinal tract, whereas UC is defined by continuous mucosal inflammation of the colon. IBD pathogenesis is thought to arise from dysregulation of the immune system in genetically predisposed individuals exposed to environmental triggers. There is a rising global burden from chronic inflammatory and autoimmune diseases, including IBD, which is increasing too quickly to be due to genetics alone ,2. The hThe gastrointestinal tract is a sophisticated immune organ that has evolved to maintain a diverse microbial community through a range of tolerance mechanisms whilst restricting the growth of pathogens. We now appreciate that the microbiota plays a major role in several symbiotic relationships with the human host. Metagenomic data have demonstrated how a diverse microbiome is important to sustain the mucosal barrier integrity, immune tolerance of commensal organisms, and host metabolism . Host–miCandida spp. are now known to colonise the gut in most humans, they are not natural commensals in the rodent intestine [Candida albicans appears to play a pivotal role in shaping human host immunity, a concept that was first identified in IBD through research into anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-glycan antibodies.Our understanding of the bacterial microbiome in human health and disease has increased significantly in recent years, with evidence of dysbiosis being reported in a wide range of disorders. Intestinal dysbiosis can broadly be defined as an alteration in the gut microbial community that functionally contributes to intestinal or systemic pathology . The keyntestine . Among tC. albicans and other yeasts can induce the production of ASCA as well as other anti-glycan antibodies associated with CD, namely anti-mannobioside (AMCA), anti-laminaribioside (ALCA), and anti-chitobioside (ACCA), although their clinical use is inferior to that of ASCA [An association between intestinal fungi and IBD has long been suspected, given the early research linking ASCA and CD. ASCA is an anti-glycan antibody against mannan, a conserved component of fungal cell walls, that serves as a serological biomarker for disease onset and clinical course in IBD . C. albi of ASCA ,15,16,17The ASCA status varies depending on the IBD type and disease location. Approximately 50 to 60% of CD patients are ASCA positive, compared to only 10–15% of UC patients and 5% of the general population. Patients with small bowel Crohn’s disease also exhibit higher rates of ASCA positivity compared to those with isolated colonic disease ,19. ASCAC. albicans, the ASCA status, and CD has produced mixed results. Although C. albicans is an immunogen for ASCA, a study into familial CD found that ASCA levels were stable and high in CD patients independent of colonisation, despite C. albicans being more likely to colonise patients with CD and their first-degree relatives [Candida tropicalis was the only fungus that positively correlated with the ASCA level. This study, however, reported a surprisingly low C. albicans abundance in both CD and healthy controls compared to other similar studies [C. albicans was not investigated [Research into the associations among elatives ,31. In a studies . Lastly,stigated . While cstigated .Candida spp. diversity that correlates with disease activity [Candida spp. [Much like the bacterial microbiome, the mycobiome is imbalanced in IBD with differing patterns reported in CD and UC . There iactivity ,33. Howeactivity . These factivity . Moreoveactivity . In contida spp. .C. albicans and a decrease in S. cerevisiae, with these variations being amplified in patients with active disease [C. albicans; however, the authors also detected a marked expansion of Saccharomyces species [C. albicans in disease [There are distinct mycobiome shifts in IBD compared to the general population. Numerous studies have observed an increased proportion of  disease ,34,36,37 species . The geo disease .Faecalibacterium prausnitzii was shown to inhibit the growth of C. albicans, virulence factor expression, and transition to pathogenic behaviour in vitro [F. prausnitzii and C. albicans had less severe colitis with a significantly reduced mucosal Candida burden compared to mice infected with C. albicans alone [Escherichia coli can inhibit Candida spp. biofilm and hyphal development [C. albicans bloom, which is thought to be promoted by the restoration of mucosal hypoxia and gut re-colonisation by Clostridia [Candida tropicalis levels appear to be positively correlated with several pathogenic bacteria within the guts of CD patients, and their co-existence within biofilms is associated with more filamentous growth [Inter- and intra-kingdom interactions that are not yet fully understood occur within the gut. in vitro . In a dens alone . A propons alone . A separelopment . A recenostridia . Separats growth . The intCandida species, among which C. albicans is the most prevalent human pathogen. This polymorphic fungus is a commensal coloniser of the human skin and mucosal surfaces where it can exist in yeast and filamentous hyphal forms. Previous reports suggest that the human GI tract is colonised by Candida species in up to 60% of healthy individuals, typically in the yeast form that establishes symbiotic commensalism with the host [C. albicans gut isolates which impacts the colonising potential of different strains. Certain transcriptional regulators, such as Efg1, control commensalism by repression of the hyphal growth gene EFH1 [C. albicans hyphae have pathogenic potential through the invasion of the epithelial cell layer and the release of cytolytic enzymes [2/CO2 tension, and interactions with other gut organisms all affect yeast-to-hyphal morphological switching, and understanding how these environmental factors induce C. albicans pathogenicity could be key when considering possible therapeutic targets [There are several the host ,44, alththe host . There iene EFH1 . C. albi enzymes ,47. Hyph enzymes . Tempera targets .Candida and Saccharomyces species, and luminal fungi that tend to be transient in nature [C. albicans can lead to the promotion of the gut barrier integrity through JAK/STAT signalling, Th17 cell responses, and IL22 secretion [C. albicans, however, is a well-recognised opportunistic pathogen in immunocompromised and intensive care patients, with the gut microbiome being the main reservoir for infection and dissemination [C. albicans was recently included on the WHO fungal priority pathogen lists [There is a distinction between fungi that exist in close proximity to the intestinal mucosa, typically immunogenic species such as n nature . Host–fuecretion . C. albimination . Furthermination . Given ten lists , emphasiC. albicans expresses several major virulence factors, with adhesion to hosts’ epithelial surfaces being a critical first step in establishing infection. Adhesion factors are crucial for enabling C. albicans to bind to and remain associated with the gut epithelium [ALS, HWP, and IFF/HYR gene families. Members of the agglutinin-like sequence (Als) family act as both epithelial adhesins and invasins. Als3, in particular, is a hyphae-specific surface protein that binds to host epithelial cell receptors such as E- and N-cadherin, thereby triggering endocytosis [C. albicans adhesion to epithelial surfaces is conceptually an attractive therapeutic target, and several novel small molecules and monoclonal antibody drugs have shown promising results [ithelium . Adhesinocytosis . Inhibit results . Als3 isC. albicans to exist as a commensal organism. For example, Ume6 is a filamentation regulator that inhibits commensalism via the downstream activation of proteases and adhesion factors [C. albicans did not correlate with the degree of histological inflammation [C. albicans compared to the yeast form, and Als adhesins were shown to be crucial in orchestrating this [C. albicans being linked to filamentation [C. albicans isolates, which blunted the ability of ‘high damage’ filamenting strains to inflict host cell injury [C. albicans strains from the same host, was independent of the disease status [A central factor in both the development of epithelial damage and immune evasion is the yeast-to-hyphal transition. Several transcription factors  control the yeast–hyphae transformation . The exp factors . Dependiammation . In conting this . An in ventation . They exl injury . Importae status . These rC. albicans, the ECE1 gene encodes a novel cytolytic peptide known as candidalysin that mediates epithelial cell damage [ECE1 gene expression between C. albicans strains were not found to influence keratinocyte damage levels in an in vitro model, although there was a strong correlation between cell injury and IL-1α release in these same cultures [ECE1 allele identified in a vaginal C. albicans strain was associated with reduced secretion of the candidalysin peptide, leading to diminished virulence [Candida spp., such as C. dubliniensis and C. tropicalis, produce candidalysin of variable potency levels to induce cellular damage and cytokine responses [C. albicans pathogenicity.In l damage . Candidal damage . Epithell damage ,62,63. Tl damage ,65. The l damage ,67. In al damage . Interescultures . Conversirulence . In addiesponses . Intra-sC. albicans intra-species diversity is a critical determinant of pathogenicity in human hosts. There is significant genetic diversity in C. albicans gut isolates between IBD patients that is independent of the disease status and geographic location [C. albicans isolates, with these outcomes being inversely correlated [C. albicans triggered an increased neutrophil response and IL-17 pathway activation through alarmin release, resulting in significant mucosal inflammation that, in turn, led to rapid fungal clearance. Interestingly, the filamentation ability did not significantly vary between the pro- and anti-inflammatory strains, indicating an alternative cause for the differences in host persistence and cell damage [location . Genomiclocation ,71,72. Urrelated . Proinfll damage .C. albicans strains were categorised into low- and high-damage groups based on their ability to injure murine macrophages, referenced against a known highly virulent strain, SC5314 [C. albicans strains, rather than the relative abundance, resulted in candidalysin-dependent macrophage IL-1β production which, in turn, correlated clinically with endoscopic disease severity, as assessed by Mayo scores [C. albicans strain variation has also been studied in other gut disorders, including irritable bowel syndrome, where the importance of the genetic and phenotypic diversity was highlighted at the strain level [C. albicans variants with high expression of virulence factors in an attempt to prevent tissue invasion and barrier damage  is an antibody involved in intestinal immunity and homeostasis that coats microbes and controls the host immune response to commensal and pathogenic bacteria . It is sidalysin ,75. sIgAensalism . InteresAntifungal immunity is dependent on signalling cascades triggered by pattern recognition receptors (PRRs) that sense fungal pathogen-associated molecular patterns (PAMPs) such as β-glucans. C-lectin receptors (CLRs) are a key family of PRRs that mediate antifungal signalling through caspase recruitment domain 9 (CARD9)-dependent pathways . GWAS stC. albicans yeasts is dependent on Dectin-1, which results in phagocytosis by binding β-1,3-glucans and triggering Toll-like receptor (TLR) pathways [C. tropicalis and a decrease in non-pathogenic Saccharomyces spp. in their microbiome. Interestingly, concomitant fluconazole treatment reduced the colitis severity [Dectin-1 was the first C-lectin receptor to be identified and was initially described in myeloid lineage cells. High expression of Dectin-1 in monocytes, macrophages, dendritic cells, and neutrophils facilitates the innate detection of β-glucan ligands, leading to the rapid production of reactive oxygen species and the upregulation of cytokines including IL-1β, IL-6, IL-23, and TNF-α . Macrophpathways . CLRs arpathways . Dectin-pathways ,82,83,84pathways . In the severity , whereasseverity . The facThe caspase recruitment domain 9 (CARD9) gene encodes an adaptor protein that mediates PRR signalling and the downstream induction of antifungal immunity . DiffereC. albicans, induces granulopoiesis through IL-6 and candidalysin signalling, resulting in the sustained expansion of neutrophil populations [Neutrophils instigate and orchestrate mucosal defence against microbes, but can also induce pathological inflammation through excessive recruitment and activation . An impoulations . Severalulations ,96,97. Tulations . Viral-iulations .C. albicans. Neutrophils are capable of sensing the size of microbes, including C. albicans hyphae, to selectively release NETs [C. albicans [Once resident in mucosal tissue, neutrophils contribute to intestinal inflammation through several mechanisms, including antifungal immune responses. Neutrophil extracellular traps (NETs) are antimicrobial structures composed of chromatin and microbicidal molecules such as cathelicidin and calprotectin, peptides that are both associated with IBD ,100. Thease NETs . An addiase NETs . It is pase NETs . NETosisalbicans ,100,104.albicans . TNFα stalbicans ,106. Neualbicans ,108. A fC. albicans challenge [Mononuclear phagocytes (MNP) include monocytes, macrophages, and dendritic cells. Their role within mucosal immunity is to encourage the immune tolerance of commensal organisms while eliminating pathogens in order to maintain mucosal homeostasis. The MNP phenotype and function vary between tissue compartments. In the gut, the fractalkine receptor CX3CR1 is of special interest in the context of IBD and antifungal immunity . Fractalhallenge . There aC. albicans, and promote the activation of the inflammasome in DSS colitis [C. albicans is the primary inducer of antifungal IgG in both mice and humans, which is thought to be achieved through CARD9 signalling in CX3CR1+ macrophages [C. albicans virulence factor candidalysin induces the activation of the NLRP3 inflammasome, leading to the cytolysis of MNPs, a potential de facto state of CX3CR1 deficiency [An important feature of CX3CR1+ mononuclear phagocytes is their high expression of CLRs that trigger downstream signalling via the spleen tyrosine kinase (Syk) pathway ,115. CX3 colitis ,115. C. rophages . As prevficiency . Indeed,ficiency ,117. In C. albicans mucocutaneous infections, demonstrating a crucial role for this cytokine family in mediating antifungal immunity [C. albicans is the major inducer of the antifungal Th17 pathway in humans, resulting in the production of IL-17A and IL-22 [Human antifungal immunity depends on Th17 cells, which defend mucosal barriers, and Th1 cells, which prevent fungal dissemination, with both axes being strongly linked to IBD pathology ,119. Th1immunity ,121,122.nd IL-22 ,124,125.nd IL-22 ,126.C. albicans is not a natural commensal in rodents and therefore requires specific measures to induce replication in the murine GI tract [C. albicans, with murine blood being unable to reduce the fungal burden or filamentation [C. albicans. We know that IL-17 knockout mice develop more severe DSS colitis than wild-type animals, but the IL-17 pathway is crucial for preventing fungal mucosal overgrowth; hence, there is clearly a fine balance to be struck between the under- and overexpression of IL-17 [C. albicans do not develop spontaneous inflammation; however, colonisation is promoted in the context of DSS-induced colitis, with associated increases in inflammation and immune responses directed against fungal antigens [C. albicans immunity, while also highlighting gaps in our understanding. These gaps are exemplified by the failure of anti-IL17 therapy in IBD trials that is discussed later in the article.The links among Th17, antifungal immunity, and IBD pathogenesis are complex and challenging to model. DSS colitis in mice has been a valuable tool for enhancing our understanding of intestinal inflammation, but nonetheless, this model has major shortcomings . The comGI tract . Indeed,entation . Regardlof IL-17 ,130. Micantigens ,131. TheC. albicans [C. albicans [Commensal-specific T cells regulate gut inflammation and homeostasis, promoting tolerance and epithelial barrier function . They aralbicans . There ialbicans . More realbicans , suggestalbicans . CD161+ albicans . These palbicans .Tissue-resident immune cells, including various types of unconventional T cells that are strongly enriched at epithelial barrier sites, are increasingly being recognised as important and distinct components of mucosal immunity. Among these are the γδ T cells, which play important roles in antimicrobial defence but have also been implicated in IBD pathogenesis . In miceDebaryomyces hansenii  was found to colonise inflamed tissue in CD and impair mucosal healing through macrophage induction of the type 1 interferon–CCL5 axis [Aberrant type 1 immunity is heavily linked with IBD and may also increase the susceptibility to mucosal fungal infections. The importance of Th1 responses in IBD is unequivocal, with numerous studies demonstrating that mucosal inflammation is associated with increased levels of Th1 cytokines including IFN-γ, TNFα, and IL-12 . Indeed,CL5 axis . The cliC. albicans overgrowth through CD4+ and CD8+ T cell-mediated IFNγ release [C. albicans epithelial invasion, despite the presence of autoantibodies that neutralised IL-17A, IL-17F, and IL-22. However, the results of this study proved controversial, with several commentaries contesting the evidence that interferonopathy, rather than IL-17 autoantibodies, underpins CMC in APS-1 patients [Given the number of inflammatory mechanisms involved in mucosal fungal immunity, it is perhaps unsurprising that there have been varying signals about the pathologic predominance of individual pathways. Chronic mucocutaneous candidiasis (CMC) is pathognomonic of autoimmune polyendocrine syndrome type 1 (APS-1), a monogenic deficiency in an autoimmune regulator gene. A recent paper by Break et al. reported that aberrant Th1 cell responses cause inflammation and breakdown of the mucosal integrity, leading to  release . Th17 repatients ,151. Varpatients ,153.Clostridium difficile infection [p < 0.0001) [Over the last 20 years, there has been a flurry of new immunological therapies for IBD, but there are still no licensed treatments that target microbial dysregulation. Despite this, there have been promising efforts to develop faecal microbial transplantation (FMT) ,155,156,nfection . Given tnfection ,159,160. 0.0001) . The eviC. albicans burden [C. albicans levels in the gut prior to FMT were associated with an improved clinical response, while a decrease in the C. albicans burden post-FMT was linked with an improved bacterial diversity and superior clinical outcomes [C. albicans as a biomarker in predicting the FMT efficacy.Efforts have been made to understand the effects of FMT on the microbiomes of recipient patients, with bacterial diversity appearing to increase in individuals who respond compared with those who do not ,162. A fs burden . Elevateoutcomes . These fC. albicans as a potential therapeutic target, a small number of studies have investigated the effects of oral antifungal medications in CD and UC patients. A recent pilot study was performed to assess whether 6 months of oral fluconazole could prevent post-operative recurrence in CD patients [C. albicans colonisation [C. albicans, which published studies rarely factor into their analyses. Despite some positive signs, oral fluconazole is unlikely to be the golden bullet for antimicrobial treatment in IBD, especially when factoring in the risks of drug-associated toxicity and fungal resistance. More targeted therapeutic approaches are needed that can preferentially deplete pathogenic over commensal strains.With the growing interest in patients . Intrigupatients ,166. HowC. albicans filamentation and adhesion to epithelial surfaces are core virulence factors and potential therapeutic targets. The induction or provision of antibodies capable of neutralising key pathogenic factors could therefore prove to be effective treatments for different subtypes of IBD. For example, the NDV-3A vaccine induces IgG and IgA responses to the adhesin Als3, which plays major roles in filamentation and binding to the epithelia. NDV-3A has already proven to be safe and effective for inducing host lymphocyte responses and reducing symptoms in a phase II RCT for recurrent vaginal candidiasis [C. albicans-induced damage in DSS colitis [didiasis . Followi colitis . Another colitis ,169. A sCandida immunity, J.F. Colombel and colleagues advanced the hypothesis that the failure of the IL-17 blockade in CD was due to the uncontrolled growth of intestinal C. albicans [C. albicans infections but also with the new onset and exacerbation of IBD [C. albicans and IBD pathogenesis.Given the link between IL-17 and IBD, much promise was held for the IL-17A monoclonal antibody Secukinumab. This proved to be a disappointment, as the phase II clinical trial had to be halted early after futility criteria were met . The evialbicans . IL-17 in of IBD ,174,175.C. albicans is an important component of IBD-associated dysbiosis. The variable expression of virulence factors between C. albicans strains and their subsequent impacts on the mucosal immune system have changed the way that we view their roles as commensals and pathogens. As we further our understanding of the immune mechanisms involved, there is growing evidence that pathogenic C. albicans strains should be future targets for IBD therapies and that the organisms themselves may be suitable predictive markers for treatments such as FMT.


Acorus calamus (AC), commonly known as Sweet Flag, has been widely used in India to treat neurological, metabolic, and respiratory disorders, indicating its potential therapeutic value. This study aimed to determine the antidepressant and antioxidative effects of AC on rats subjected to long-term, social isolation-induced stress.Social isolation stress (SIS) and individual housing have been shown to cause abnormal cognitive insufficiencies, altered anxiety levels, and signs of psychiatric diseases. This study involved 2-month-old male rats (24) weighing approximately 180–200 g bred in-house. The rats were divided into four groups (n = 6): Group 1 received saline, Group 2 received SIS, Group 3 received only 50 mg/kg AC, and Group 4 received 50 mg/kg AC and SIS for 6 weeks. After this, behavioral, biochemical, and neuronal assays were conducted.Behavioral experiments showed significantly higher activity levels (p < 0.001) in AC-treated rats than in the SIS group. In addition, rats subjected to SIS with AC treatment exhibited enhanced total antioxidants, superoxide dismutase, and neuronal assays compared to rats subjected to SIS alone.Acorus calamus treatment improved the antidepressant and antioxidant potential against SIS in rat brain tissue. Moreover, we proved that AC can effectively reverse the neurotoxicity induced by SIS in animal models. As we battle against the coronavirus disease 2019 pandemic and social isolation, AC could be considered a supplementary treatment to alleviate depressive-like symptoms in our present-day lifestyle. It iet al.  found th [et al.  reportedEarly childhood SIS, whether acute or chronic, has been linked to abnormal cognitive functions , 14, altet al. [The antioxidant defense mechanism of the central nervous system is sufficient to counteract continuous oxidative damage despite being sensitive to OS. Oxidative stress has been extensively studied in neurological diseases using various stressors. Stress-induced oxidative damage in the brain can alter cellular homeostasis. Rai et al.  demonstret al. .Early SIS has been linked to impaired mitochondrial function, leading to increased nitric oxide (NO) and ROS production and a significant decrease in glutathione and ATP levels , 31. Reset al. [Social isolation stress has been found to significantly affect the prefrontal cortex . Tang etet al.  found thet al. , 39. In et al. . The atret al. . Social et al. . Clinicaet al.  and postet al. . Hence, Acorus calamus (AC) Linn. (Acoraceae), known as Vacha in Sanskrit and Sweet Flag in English, is an annual, semiaquatic, aromatic herb widely used in Ayurveda  and traditional Chinese medicine [Acorus calamus has numerous health benefits, including neuroprotection, antioxidant properties, antidepressant effects, anticonvulsant activity, antihypertensive properties, anti-inflammatory effects, immunomodulatory activity, cardioprotective effects, and potential benefits for obesity [medicine . This plmedicine . The rhimedicine  and is amedicine . Acorus  obesity , 49. At Acorus calamus has also shown effectiveness against noise stress-induced memory impairment [Acorus calamus rhizome powder has demonstrated a beneficial effect on restraint stress-induced modulatory changes in Na-K-ATPase activity, antioxidants, and cognitive functions [Research has shown that AC exhibits a neuroprotective effect in autism-induced Wistar rats . In addipairment  and has pairment . Acorus unctions . Furtherunctions . Forouzaunctions  argued tThis study aimed to evaluate the following: (1) behavioral changes (motor and cognitive functions) in rats subjected to SIS and AC-treated rat brains; (2) the level of antioxidants in rat brains subjected to SIS and AC-treated rats; (3) histomorphological changes in the neurons of the mPFC in rats subjected to SIS AC treatment; and (4) the effect of AC on oxidative damage, as well as behavioral and histomorphological changes in rat brains subjected to SIS.The study was approved by Institutional Animal Ethics Committee of Kasturba Medical College, Mangalore (KMC/MNG/IAEC/10-2019).This study was conducted from July 2020 to December 2020 at Central Animal House, Kasturba Medical College, Mangalore.ad libitum. They were retained in controlled environments with respect to humidity, temperature, and light and dark cycles. Paddy husk was used as bedding and rats were housed in polypropylene cages. The rules set forth by the Indian Government for the use of laboratory animals were followed in this investigation [This study used 2-month-old male rats (weighing 180 g ± 20) bred in-house. Food and water were available to animals tigation .Acorus calamus powder was purchased from Natural Remedies, Bangalore, India. The experiment included the following animal groups, each consisting of six rats (n = 6). The initial weight of all rats was recorded before the experiment commenced. The experimental rats received AC orally through an oral gavage tube. The rats were allowed to acclimatize for a week before being randomly divided.Group 1: Six socially grouped (SG) rats received saline (3 rats/cage)Group 2: Six rats subjected to SIS for 6 weeks (1 rat/cage)Group 3: Six rats received only 50 mg/kg of AC daily for 6 weeks (3 rats/cage)Group 4: Six rats received AC at 50 mg/kg daily + SIS for 6 weeks (1 rat/cage).Acorus calamus powder was administered at 50 mg/kg daily [Acorus calamus powder was purchased from Natural Remedies, Bangalore, India. All other chemicals and reagents were high-performance liquid chromatography or analytical grade  purchased from Sri Durga Laboratories, Mangalore, Karnataka, India.kg daily . This adThe open-field test is the most commonly employed method for analyzing rats’ emotional, exploratory, and motor activities . A rectaet al. [The TST was performed as defined by Nandi et al. . Using aet al. [The memory recall of the rats in all groups was tested using a passive avoidance device, as described and applied in a study by Rai et al. . This test day of testing, the rats were positioned for exploration in the center of the bright compartment, facing away from the entry to the small, dark compartment. The door separating both compartments was kept open, and three test trials lasting 5 min each were conducted after the rats had 5 min to explore each chamber. The time spent in each compartment was noted during every trial, and the rats were placed in the home cage after each trial.Exploration test: On the 1Aversive stimulation and learning phase (passive avoidance acquisition): After the third test trial, a foot shock  was applied across the grid floor immediately after the rats entered the dark compartment. The rats were given an additional 10 s to establish a link between the characteristics of the chamber and the foot shock. The rats were then placed back into the home cage.st time was recorded. The rats were given a maximum of 300 s to explore. Normal rats do not explore the dark compartment, where they received a shock the preceding day, restricting their natural behavior. A decreased latency to enter the dark section suggests inadequate memory retention.Retention test: The memory retention test was conducted 24 h after the foot shock. The rats were placed in the bright compartment, and the time taken (latency) to enter the dark compartment for the 1g for 20 min at 4°C, a portion of the supernatant was removed for biochemical analysis.After 24 h, biochemical tests were conducted. The rats were sacrificed through cervical decapitation, and the entire brain was quickly separated and cleaned with 0.1 M/L saline phosphate buffer (pH 7.4). The tissue was then weighed and homogenized (1:10 w/v) in 0.1 M/L saline phosphate buffer. After centrifuging the homogenate at 10,000× et al. [The assay principle involves a Fenton-type reaction, in which a standardized solution of the Fe-Ethylenediaminetetraacetic acid complex combines with hydrogen peroxide to produce hydroxyl radicals. These ROS break down benzoate, generating compounds that react with thiobarbituric acid. Antioxidants in the brain homogenate inhibit the production of thiobarbituric acid–reactive substances. The reaction was quantified using spectrophotometry, and the inhibition rate of color development was proportional to the concentration of antioxidant activity. The TAO level was calculated following the method described by Koracevic et al. .et al. [−5 M nitroblue tetrazolium, 1.17 × 10−6 M riboflavin, and 1 × 10−2 M methionine in 0.05 M potassium phosphate buffer (pH 7.8), along with appropriately diluted tissue homogenate in a volume of 3 mL. The solution was placed in a beaker and illuminated for 10 min using a 15-volt fluorescent bulb inside an aluminum-lined foil box. The control sample was prepared without the enzyme. The absorbance was determined at 560 nm using a Systronic-117 ultraviolet-visible spectrophotometer, and SOD activity was quantified as the specific activity of the enzyme (U/mg protein). The technique developed by Lowry et al. [The procedure developed by Marklund and Marklund  and Rao et al.  was usedy et al.  was used2 area of the mPFC were counted . Afterward, 25 sections were serially mounted on air-dried gelatinized slides from each animal. Cresyl violet stain was used to stain the sections . The Nis counted . Six secThe data were expressed as mean ± SE, and a one-way analysis of variance was performed to assess the significance of the differences among the groups. A significance level of p < 0.05 was considered significant.Acorus calamus-treated rats also showed a significant increase (p < 0.001) in these activities when compared to SIS rats  in socially isolated rats compared to SG rats. However, the rats subjected to AC treatment with SIS showed an increase in these emotional activities compared to the SIS group. SIS rats .Acorus calamus treatment in SIS rats was unable to completely reverse the anxiety-like behavior (p > 0.01) in the open-field test compared to SG rats , indicating anxiety-like behavior.  SG rats .Acorus calamus treatment in SIS rats significantly enhanced (p < 0.001) the number of crossings in peripheral squares compared to SIS rats, suggesting enhanced locomotor and exploratory activities. Acorus calamus treatment in SIS rats also resulted in a significant decrease (p < 0.001) in peripheral square crossings compared to the AC treatment group  in SIS rats compared to SG rats, indicating an impact on general locomotor activity. nt group .The immobility time was significantly higher (p < 0.001) in SIS rats than SG rats, indicating depressive-like behavior. However, the SIS rats treated with AC showed a reduced immobility time compared to the SIS rats, indicating a reversal of depressive-like behavior .The time taken for SIS rats to enter the dark compartment was significantly shorter (p < 0.001) than SG rats, suggesting poor retrieval of learning behavior. Treatment with AC alone enhanced the time of entry into the dark compartment, indicating improved learning abilities. However, AC treatment in SIS rats did not increase the duration to enter the dark compartment, as the potential to enter the dark compartment was significantly lower (p < 0.001) compared to SG rats. This result showed that SIS rats treated with AC had poor recovery of learning behavior but had an enhanced latency to enter the dark compartment compared to rats subjected to isolated stress alone .The TAO levels in the brain homogenate were significantly reduced (p < 0.001) in isolated rats compared to SG rats. However, AC treatment in SIS rats resulted in a significant enhancement (p < 0.001) in the level of TAO in the brain tissue compared to SIS rats. Rats subjected to AC treatment alone also showed a highly significant (p < 0.001) increase in brain TAO levels when compared to SIS rats .Acorus calamus treatment alone showed a significant (p < 0.001) increase in brain TAO levels compared to SG and SIS rats  in the level of SOD in the brain tissue compared to SG rats. However, AC-treated SIS rats revealed a significant increase (p < 0.05) in the level of SOD in the brain homogenate compared to SIS and SG rats (p < 0.001). SIS rats .Acorus calamus treatment in SIS rats resulted in a significant increase in normal neurons compared to SIS rats (p < 0.01) and AC-treated rats (p < 0.001)  and AC treatment groups (p < 0.05), suggesting degenerating neurons. < 0.001) .et al. [et al. [et al. [In this study, we observed that SIS rats spent less time in the center squares, displaying increased anxiety-like behavior than SG rats, which was not reversed by AC treatment. However, the locomotor and exploratory activities of the SIS rats were lower in the peripheral squares compared to the SG rats, and this impairment was significantly improved by AC treatment. These findings are consistent with Grigoryan et al. , who evaet al. . Chronicet al. , is know [et al. . In an a [et al. . In addi [et al. . However [et al. , 72.Social isolation stress rats exhibit symptoms similar to human anxiety, depression, schizophrenia, and memory loss , 18, 73.et al. [Social isolation stress-induced for 8 weeks caused an antioxidant imbalance, inducing reduced production of ROS and decreased levels of SOD, catalase, and GPx in the rat brain , 74. Ouret al.  also demThe mPFC is crucial in integrating cognitively and emotionally significant information , 40. SocAcorus calamus, the promising drug in this study, exhibited antidepressant and antioxidant potential against SIS in brain tissue. The results indicate that AC effectively combats SIS-induced neurotoxicity in animal models. Considering the importance of social therapy during the COVID-19 pandemic, AC could serve as a valuable supplement in combating SIS-induced psychiatric symptoms and in addressing the challenges of our present-day lifestyle.The rat model of SIS used in this study effectively mimics human stress conditions. The long-term effect of stress on neuronal loss, including cognitive decline and accelerated aging, is well known. Thus, we focused on the role of antioxidants, cognitive behavior, and neuronal assays in the mPFC. ARR and TJ: Planned and conducted the study and drafted the manuscript. MP and AM: Conducted the animal experiment and stained the slides. MMP and BVM: Statistical analysis, prepared graphs, and drafted and revised the manuscript All authors have read, reviewed, and approved the final manuscript.


Subtercola sp. PAMC28395, a strain isolated from cryoconite in Uganda. This strain possesses several active carbohydrate-active enzyme (CAZyme) genes involved in glycogen and trehalose metabolism. Additionally, two specific genes associated with α-galactosidase (GH36) and bacterial alpha-1,2-mannosidase (GH92) were identified in this strain. The presence of these genes indicates the likelihood that they can be expressed, enabling the strain to break down specific polysaccharides derived from plants or the shells of nearby crabs. The authors performed a comparative analysis of CAZyme patterns and biosynthetic gene clusters (BGCs) in several Subtercola strains and provided annotations describing the unique characteristics of these strains. The comparative analysis of BGCs revealed that four strains, including PAMC28395, have oligosaccharide BGCs, and we confirmed that the pentose phosphate pathway was configured perfectly in the genome of PAMC28395, which may be associated with adaptation to low temperatures. Additionally, all strains contained antibiotic resistance genes, indicating a complex self-resistance system. These results suggest that PAMC28395 can adapt quickly to the cold environment and produce energy autonomously. This study provides valuable information on novel functional enzymes, particularly CAZymes, that operate at low temperatures and can be used for biotechnological applications and fundamental research purposes.This study reports the complete genome sequence of  Although glaciers are generally thought to only appear in cold environments, glaciers also exist in equatorial Africa, such as on Mt. Kilimanjaro in Tanzania, Mt. Kenya in Kenya, and the Rwenzori Mountains in Uganda ,2. AmongAlthough glacier regions have sub-zero temperatures and harsh environments, they are home to several microbes that are significant sources of cold-adapted microorganisms . MicroorSubtercola genus, a member of the family Microbacteriaceae, belongs to the phylum Actinomycetota. The use of Subtercola boreus strain DSM13056T as a new psychrophilic actinobacterium isolated from groundwater was first mentioned in 2000 [Subtercola species isolated from cold environments such as high-altitude extreme cold environmental niches, including snowy mountains [Subtercola genus in cold environments, such as the cold habitats of Antarctic and Arctic Oceans. These results show indication of a species of Psychrophiles [Subtercola genus is still considered new. So far, few studies have performed comparative genomic analysis of this genus. Subtercola species are a relatively established genus within the family Microbacteriaceae. It currently contains five recognized members: S. boreus, S. frigoramans, S. lobariae, S. vilae, and S. endophyticus. Genome sequences of 16 strains belonging to the Subtercola species have been registered in the National Center for Biotechnology Information (NCBI) database. Moreover, Jiang et al. [Subtercola genus. Strain PAMC28395 was obtained from a glacier located in the Rwenzori Mountains, and this study presents the complete genome sequence of this strain. A comparative genomic analysis was conducted using complete genome data in the NCBI database to gain insights into its characteristics within the Subtercola genus. This analysis helped predict gene functions and provided a deeper understanding of the mechanisms by which PAMC28395 adapts to its natural environment, uncovering emerging evolutionary characteristics. This study’s comprehensive examination of complete genomes offers valuable insights into the evolution and genetic composition of various strains and species inhabiting cold environments.A comparative genomic analysis based on genome data of available bacteria strains could reveal common species characteristics of large populations and the physiological characteristics of bacteria. Moreover, some research studies on the characteristics of related bacteria have proven that unique functional genes might be acquired from each natural environment and enhance their potential applications, such as in bioremediation, biomedical, and healthcare contexts . The Sub in 2000 . Subtercountains , cold voountains , glacierountains , have berophiles ,16,17. Crophiles . The Subg et al.  recentlySubtercola sp. PAMC28395 strain was isolated from glaciers of Mt. Rwenzori, Uganda, using 0.001 × TSB Gellan gum. This strain was isolated at 10 °C. It grew at temperatures of up to 15 °C using a pure Tryptic Soy Broth  and produced enough cells for extraction of genomic DNA. The genomic DNA of PAMC28395 was extracted using a QIAamp DNA Mini Kit  according to the manufacturer’s protocol. Purified genomic DNA was quantified with a spectrophotometer . In addition, the quality of the extracted DNA was determined based on A260/A280 and agarose gel e We have checked and provided sharper figurelectrophoresis before performing further research.The https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). In addition, coding DNA sequences (CDSs) were predicted and annotation was performed with a Rapid Annotation using Subsystem Technology (RAST) server (https://rast.nmpdr.org/) [https://proksee.ca/) [Before the sequencing process, quantity and purity were determined using an Agilent 2100 Bioanalyzer  to obtain complete genome sequencing. Genome sequencing was performed using PacBio RS II single-molecule real-time (SMRT) sequencing technology . SMRTbell library inserts 20 kb) were sequenced using SMRT cells. Raw sequence data were generated and de novo assembled using the hierarchical genome assembly process (HGAP) protocol  and RS Hdr.org/) . A circu kb were www.ezbiocloud.com). Finally, 16S rRNA sequences were aligned using MUSCLE [The PAMC28395 strain was compared with diverse gena in Microbacteriaceae using 16S rRNA phylogenetic analysis. Alignments were performed using the NCBI database’s Basic Local Alignment Search Tool and analyzed using EzBioCloud . We then determined the relationship of PAMC28395 with other strains from the same species and confirmed their similarity by comparing values of ANI calculated using an OrthoANI in OAT (the Orthologous Average Nucleotide Identity Tool) [https://ggdc.dsmz.de/ggdc_background.php) with recommended parameters and/or default settings [We downloaded whole genome sequences of ty Tool) . In addisettings .https://bcb.unl.edu/dbCAN2/) [https://arts.ziemertlab.com/).We used diverse annotation tools to survey the genomic features of PAMC28395. First, the genome of strain PAMC28395 was annotated using the RAST server. Second, predicted gene sequences were translated and searched in the NCBI non-redundant database. Next, we obtained functional annotation genes, including Clusters of Orthologous Genes (COGs) from EGGNOG 5.0  and funcdbCAN2/)  with an dbCAN2/)    to improdbCAN2/)  with defdbCAN2/)  web servAs shown in The circular genome map provided a comprehensive characterization of PAMC28395. In addition, we provided classification in 20 functional COG (Cluster of Orthologous Groups) categories . The mosMicrobacteriaceae  showed that the PAMC28395 strain was most closely related to Subtercola species. This strain was phylogenetically placed with S. frigoramans and S. vilae. Results from phylogenetic analysis, the Basic Local Alignment Search Tool, and EzBioCloud revealed that it was closely related to S. frigoramans K265T (99.44%), S. vilae DB165T (97.83%), and S. lobariae 9583bT (96.87%). These results confirm that the PAMC28395 strain belongs to the family Microbacteriaceae, phylum Actinobacteria. However, strain S. frigoramans K265T was reported based only on 16S rRNA sequence comparison. Since there were little chemical taxonomic and phenotypic data to support this, it was reported that strain K265 should be included in a genus as a new species, for which the name S. frigoramans gen. nov. was suggested [frigoramans K265T (1437 bp obtained through PCR). EZBioCloud calculated the similarity based on the completeness of the reference sequences. For instance, when the completeness of S. frigoramans K265T is 99.3%, the calculated similarity with strain PAMC28395 is 99.44%. Similarly, when considering S. vilae DB165T with a completeness of 100%, the calculated similarity is 97.83%. These results suggest that strain PAMC28395 likely belongs to the Subtercola genus and exhibits similarity to S. frigoramans. However, it is important to conduct further confirmatory experiments to provide more concrete evidence. Based on the observed differences and similarities, there is a possibility that PAMC28395 could be classified as a new species within the Subtercola genus, potentially positioned between S. vilae and S. frigoramans.The identification of PAMC28395 was verified using 16S rRNA sequence analysis A. Furtheuggested . Indeed,uggested . Based oSubtercola genus in the NCBI database. Not all strains mentioned above have whole genomes. Among registered strains in the NCBI database, only the two strains of PAMC28395 and AK-R2A1-2 have complete genome sequences among Subtercola species. However, strain AK-R2A1-2 has recently been identified as S. endophyticus by Jiang et al. [frigoramans DSM13057 (60.7%) and S. vilae DB165 (18.7%). This conclusion is supported by the significantly lower values observed for PAMC28395, which fall below the 70% threshold. Therefore, both ANI and dDDH values support that PAMC28395 should be identified as a novel species between S. vilae and S. frigoramans in the Subtercola genus. Further clarification of the new species within the Subtercola genus would require additional experiments. By conducting comprehensive experiments such as phenotypic characterization, biochemical assays, growth conditions analysis, and additional genetic analysis, the potential new species’ unique characteristics and distinct traits can be elucidated, leading to more accurate classification and characterization. While phylogenetic analysis can provide valuable insights and comparing ANI and dDDH calculates the value, experimental validation is crucial for definitively establishing a new species. Thus, we have mentioned the strain as Subtercola sp. PAMC28395.There is little genomic information on the g et al. . These sg et al. ,39. The g et al. . HoweverCAZymes are carbohydrate-degrading enzymes that break down complex polysaccharides such as cellulose into simple sugars. This is crucial for the survival of bacteria. Through complete genome analysis of various strains and cold-adapted species, evolutionary insights can be revealed. For example, it has been reported that Bacillus sp. TK-2 exhibits adaptability to cold environments through the expression of CAZyme genes involved in the degradation of polysaccharides such as cellulose and hemicellulose . HoweverSince genes related to carbohydrate metabolism are expected to be highly conserved, we conducted a detailed analysis of CAZymes. Among a total of 2849 identified protein-encoding genes in strain PAMC28395, 92 were annotated and classified into the group of CAZymes  using dbCAN3. Results provided insight into the carbohydrate utilization mechanisms of strain PAMC28395. Using the Signal P tool, we predicted the retention of 12 genes encoding signal peptides in the CAZymes of strain PAMC28395. In addition, we anticipated that enzymes involved in carbohydrate metabolism would be distributed as follows: 31 GHs, 47 GTs, 7 CEs, 4 AAs, 3PLs, and 2 CBMs A.Shigella sp. strain PAMC28760, Arthrobacter sp. strain PAMC25564, and Nakamurella sp. strain PAMC28650 isolated from cold environments have been reported to be able to adapt and survive in cold environments through glycogen metabolism [GH gene annotation revealed that the PAMC28395 genome sequence contained genes involved in glycogen and trehalose metabolism pathways, such as β-glucosidase , 1,4-alpha-glucan (glycogen) branching enzyme , malto-oligosyltrehalose synthase , limit dextrin alpha-1,6-maltotetraose-hydrolase , trehalose phosphorylase , and malto-oligosyltrehalose trehalohydrolase  . Glycogetabolism ,45,46.agaSK. When expressed in vivo, it might contribute to α-galactosidase activity. Enzyme AgaSK isolated from Ruminococcus gnavus E1 (RG E1) has recently been characterized as a bifunctional enzyme composed of a GH36 α-galactosidase and a kinase domain that can phosphorylate sucrose provided by raffinose hydrolysis, highlighting a putative novel glycolytic pathway in bacteria for sucrose assimilation. It shows partial plant oligosaccharide-degrading abilities. It plays an essential role in metabolizing complex oligosaccharides, working in conjunction with other GH enzymes and contributing to the degradation of mixed fibers [Moreover, we found interesting CAZyme-related genes in genome information. Microbial α-galactosidases (GH36) play a crucial role in the breakdown of plant-derived α-galactosidases by human gut microbiota. Here, we identified another GH36 gene, similar to d fibers . We alsod fibers . Recentld fibers ,50.Subtercola species, this study offers valuable insights into the evolutionary strategies of these microorganisms. It presents exciting opportunities for developing novel biotechnological tools with diverse industrial applications. Considering the accessibility of available genome data, complete genomes of five strains, including our strain, were chosen for comparative analysis of CAZymes. Our results showed that the number of total CAZymes in each genome ranged from a minimum of 95 (PAMC28395) to a maximum of 157 (Subtercola sp. DB165). Our genome sequence analysis of the five strains confirmed our predictions, revealing a consistent pattern of 23 CAZyme genes across all strains, including AA3, AA7, CE4, CE14, CE9, GH1, GH3, GH13, GH15, GH23, GH63, GT1, GT2, GT4, GT20, GT28, GT35, GT39, GT51, GT76, GT83, CBM32, and CBM48  and bacterial alpha-1,2-mannosidase (GH92) had the highest probability of expression in this strain. In conclusion, our genomic comparison demonstrates the potential of these strains to degrade complex polysaccharides and store energy.By revealing key CAZymes involved in the cold adaptation of nd CBM48 B. Of parWe investigated the glycogen/trehalose and pentose phosphate metabolic pathway to confirm related cold adaptation to a cold environment in PAMC28395 . This anAs a result, this strain probably produces polysaccharides by themselves and from an external source using phosphoenolpyruvate rather than consuming energy because they have a related recycling gene for degradation or production. To adapt and survive in a cold environment, organisms must have well-developed functional energy storage systems. Glycogen is one of the carbohydrates stored in animal cells. It is used as an energy reserve to adapt and survive in a cold environment. It has a three-dimensional molecular structure comprising the most representative α-1,4-glucoside linkage and α-1,6-glucosidase linkage among carbohydrates . EnzymesMoreover, the pentose phosphate pathway (PPP) is a major pathway for glucose catabolism, along with glycolysis. However, the extent of its contribution to bacterial metabolic adaptation, particularly in the context of bacteria, remains largely unexplored. The PPP comprises two branches: an oxidative branch and a non-oxidative branch. Glucose flux through the oxidative branch generates NADPH, which is an essential reductant in anabolic processes. Meanwhile, the non-oxidative branch produces ribose-5P (R-5P) from glucose, which can be converted reversibly into glycolytic intermediates such as glyceraldehyde 3P (GA-3P) and fructose-6P (F-6P) ,53. AlthSubtercola genus evolutionarily, explicitly targeting those associated with cold environment adaptation to shed light on factors contributing to the bacterium’s diverse range of secondary metabolites. As shown in Subtercola sp. PAMC28395, Subtercola sp. AK-R2A1-2, Subtercola sp. Z020, S. frigoramans DSM13057, and S. vilae DB165 suggested that four clusters  were conserved with few differences , which contain genes encoding core biosynthetic enzymes, transporters, and regulators found physically co-located. This knowledge drove us to investigate the genetic traits of the ferences . Except ferences A, which clusters B. ConsidSubtercola sp. PAMC28395, Subtercola sp. AK-R2A1-2, Subtercola sp. Z020, S. frigoramans DSM13057, and S. vilae DB165 revealed that strain PAMC28395 contained more core genes in BGCs than other strains  and bacterial alpha-1,2-mannosidase (GH92) had the highest probability of expression in this strain. Moreover, a comparative analysis of BGCs in several Subtercola strains, including PAMC28395, revealed they had oligosaccharide BGCs which might be associated with their adaptation to low temperatures. To the best of our knowledge, this is the first report of antibiotic resistance gene analysis in the Subtercola genus. Results of ARTS analysis revealed that multiple antibiotic resistance genes were present in all strains analyzed, which might suggest that they provide a complex self-resistance system for these strains to survive. Additional studies are needed to characterize these BGCs and determine their products and biological roles. In summary, genome sequence analysis can provide valuable information on novel functional enzymes, particularly CAZymes, that operate at low temperatures. The CAZymes can be used for biotechnological applications and fundamental research purposes. Moreover, this study forms a basis for understanding how the PAMC28395 strain produces energy in cold environments.The complete genome sequence of PAMC28395 was elucidated in this study, and a comparative genome analysis with other species was conducted to investigate CAZyme patterns. This isolate was obtained from cryoconite under laboratory conditions. It was confirmed as a