Oryza sativa L.) is a major staple cereal worldwide, providing essential caloric requirements for more than half of the world’s population. Annual losses to rice borers are approximately 5–10%, but losses in individual fields may reach up to 50–60%. The use of traditional pest management strategies in controlling YSB is somewhat challenging due to its unique internal feeding habit. Genome sequence information of economically important crop pests is important for designing or developing pest-resistant rice varieties. In an approach to achieve this, we present our first-ever study on the draft genome sequence of YSB. The information provided from our current study might be useful in developing genome-based approaches for the management of pest species. Yellow stem borer (YSB), is the most destructive and widely occurring pest that attacks rice throughout the growing season. Rice  (Lepidoptera: Crambidae), a major monophagous insect pest of rice, causes significant yield losses. The rice–YSB interaction is very dynamic, making it difficult for management. The development of resistant lines has been unsuccessful as there are no effective resistant sources in the germplasm. Genome information is necessary for a better understanding of interaction with rice in terms of its recognition, response, and infestation mechanism. The draft genome of YSB is predicted to have 46,057 genes with an estimated size of 308 Mb, being correlated with the flow cytometry analysis. The existence of complex metabolic mechanisms and genes related to specific behavior was identified, being conditioned by a higher level of regulation. We deciphered the possible visual, olfactory, and gustatory mechanisms responsible for its evolution as a monophagous pest. Comparative genomic analysis revealed that YSB is unique in the way it has evolved. The obvious presence of high-immunity-related genes, well-developed RNAi machinery, and diverse effectors provides a means for developing genomic tools for its management. The identified 21,696 SSR markers can be utilized for diversity analysis of populations across the rice-growing regions. We present the first draft genome of YSB. The information emanated paves a way for biologists to design novel pest management strategies as well as for the industry to design new classes of safer and specific insecticide molecules.Yellow stem borer (YSB),  Oryza sativa) is the most widely grown crop in the Asian continent as about 90% of rice production and consumption takes place in this region . Results were expressed using the conversion factor 1 pg DNA = 978 Mb for genome size estimation.S. incertulas using BWA-Mem version 0.7.12 with parameters of minimum seed length 19, the penalty for mismatch 4, and gap open penalties for deletions and insertions—6. The RNA-seq reads obtained from the de novo transcriptome analysis performed at the first, third, fifth, and seventh larval developmental stages of YSB were downloaded from the NCBI SRA database (SRX733621) and mapped on the assembled genome of N. lugens, C. medinalis, and C. suppressalis. The resulting alignment file was analyzed using Mr Bayes . The phylogenetic tree was reconstructed using the Bayesian inference method implemented in the Mr Bayes program (v3.2.6). The number of substitution types was fixed to 6. The Poisson model was used for amino acid substitution, while rates variation across sites was fixed to ‘invgamma’. Four Markov chain Monte Carlo (MCMC) chains were run for 10,000 generations, sampling every 10 generations, with the first 250 sampled trees discarded as ‘burn-in’. Finally, a 50% majority-rule consensus tree was constructed.To determine how YSB is related to other insect pests, we ran multiple sequence alignment on the single-copy gene cluster generated using OrthoVenn on the proteomes of the organisms, namely, To obtain preliminary information on the YSB genome size, we deployed flow cytometry for its genome size estimation. It is a reliable method to estimate the genome size of organisms, especially when a species or an organism is being sequenced for the first time. The genome size of YSB ranged from 347.6 to 370 Mb .Drosophila melanogaster was used as a reference species. Percentage of variation within replication was 0.95–1.04% and with internal control. 1 C value is genome size of D. melanogaster—176.4 Mb; 1pg = 978Mb 1C value of YSB = 0.378 pg = 369.684.The Illumina HiSeq short reads paired-end and mate-paired raw reads were subjected to quality filtering, resulting in 24.67 and 21.12 million high-quality reads, respectively. The total data for paired-end and mate-paired reads accounted for 7.3 Gb and 6.3 Gb, respectively, totalling up to 13 Gb. The de novo assembly resulted in 310,612 scaffolds with an average scaffold size of 993 bp . The maxThe total genome size achieved upon scaffolding accounted for 308 Mb. The estimated genome size using flow cytometry (median 356 Mb) correlated with the estimated genome size (87% in terms of genome coverage) through sequencing. The average GC content of the YSB genome was observed to be 36.37%. The completeness of draft assembly validated using CEGMA (Core Eukaryotic Genes Mapping Approach) and BUSCO  respectively revealed 57.26% and 48.87% gene annotations matching with the total number of core genes in the database.biosamplehelp@ncbi.nlm.nih.gov, accessed on 30 January 2020) BioSample database under the accession SAMN12500696.The data were submitted to NCBI  genes with blast hits and the remaining 15,751 (34.19%) genes remained unannotated. The 15,751 unannotated genes did not indicate any similarity with the genes present in the existing repertoire of databases. Unannotated genes have been reported earlier in insect genomes and can be attributed to the paucity of data available for the reference genome, its completion, and assembly . Among tThe non-coding RNAs (ncRNAs) are important regulatory molecules, especially at the post-transcriptional level . SeveralTransposable elements (TEs) are fundamental to the evolution and expansion of insect genomes. The YSB genome was found to have about 1% of TEs. Among the TEs, DNA elements represented the highest number (1500), followed by SINEs, LINEs, LTR elements, non-LTR retrotransposons, retrotransposons, and other transposable elements. The transposable elements namely hAT-Charlie, TcMar-Tigger, piggyback were predominantly present in the DNA element whereas and Mobile element jockey-like, were more in non-LTR retrotransposon categories . Hox cofactors, Hox protein-regulating genes, cuticle proteins, different segments of the antenna, retinal determination network, wing development, and homeobox genes. These genes, along with the other predicted genes, might control the organ development of the YSB, as evidenced by several other studies. We identified several organ developmental genes and categorized them according to the YSB body structure . These iWe deduced three levels of gene regulation in YSB, namely, transcription factors, hormones, and epigenetic mechanisms.araucan and caupolican-like belonging to Iroquois complex; Iro-C) were also found in YSB and have been reported for controlling proneural and vein-formation in Drosophila [Several TFs such as zinc fingers, helix–turn–helix (HTH) classes containing homeodomain transcription factors, forkhead box TFs, and heat shock factor (HSF) TFs, basic DNA-binding domain TFs that include basic helix–loop–helix (bHLH), basic leucine zipper (bZIP), hormone receptors, and Myb isoforms were identified from the YSB genome, the largest being the zinc finger (391 in number) with different domains. Hox genes (homeobox-containing transcription factors) are typically found in clusters within the genome, and it has been suggested that the evolution and expansion of Hox genes have played a key role in region-specific identity during early development in the majority of insects. Other homeobox TFs (osophila .SOX transcription factors (belonging to the high-mobility group (HMG) superfamily of DNA-binding proteins) were found to be in high number (11). In addition to this, larval metamorphosis, vitellogenesis, and oocyte maturation controlling methoprene-tolerant (Met) gene that has a basic helix–loop–helix (bHLH) domain was detected in YSB as reported from other insects such as cotton bollworm (Helicoverpa armigera) [N. lugens [The rmigera) ,22,23. T. lugens .D. melanogaster that are known to be involved in the cross-talk between the signaling pathways of juvenile hormone and ecdysone were also observed in YSB [Several BTB  TFs  originally identified in d in YSB ,26. The d in YSB . Thus, tH. armigera separately from the other insect species (N. lugens for promoting vitellogenin uptake by oocytes [Amyelois transitella), a pest that belongs to the lepidopteran order (Insect molting and metamorphosis are regulated by steroid molting hormones and juvenile hormones (JHs), respectively. The key genes associated with the JH biosynthesis pathway and hormones associated with molting were principally observed under this category . The phy species . Additio oocytes  also seean order .Genomic profiling reveals the presence of several genes required for DNA methylation and histone modification . Among tThe genome data sheds light on the monophagous nature of YSB towards its host plant. It has well-equipped three-tier mechanisms related to vision, chemosensory, and digestion that assist in searching, recognizing, and successful colonization of its host.The YSB is a dynamic lepidopteran that migrates over a long distance in search of rice plants. Visual stimuli in insects are principally mediated by opsin protein, which is a member of the G protein-coupled receptor family . Opsin gIn addition to visual perception, the YSB engages strong chemosensory genes (140 genes) for olfaction that might be operated through major odorant-binding proteins (OBPs). Insect OBP genes have been identified throughout Neoptera class ,33. One Plutella xylostella)  . Serine ta class . S. furcifera and 13 were found in N. lugens [Scirpophaga (species not specified) from Indonesia through lab assay and poor field efficacy was reported [Insects have evolved specialized detoxification mechanisms to cope with plant defenses . A wide . lugens . Comparareported .takeout genes and takeout JHBP-like homologs were identified  is a phenomenon of exchanging the signal patterns between two different sex types during courtship. It was reported to be controlled by  channel ,46,47. Id in YSB , indicat insects . Along w insects .alan shepard (shep) gene. Two gene members coding for alan shepard (shep) were found in the YSB genome  is a biogenic monoamine found across most phyla of life . SerotonYSB genome has enabled us to detect the reported targets (nicotinic acetylcholine receptor (nACHR), γ-aminobutyric acid receptor (GABA), glutamate-gated chloride channel (GluCl) and voltage-gated sodium channel (VGSC), acetylcholinesterase (AchE), acetyl-CoA carboxylase (ACCase), cys-loop ligand-gated ion channel (cysLGIC) superfamily and ryanodine receptor (RyR)) for insecticides ; expressA. aegypti and Anopheles albimanus), known to inhibit the platelet aggregation [The innate immune system is the first line of defense against invading pathogens, which generally operates on a minimal scale in insects, unlike vertebrates . Howeverregation ,52, wereregation ,54,55. SWhen insects attune on the plants for their endurance, their contact on the plant surface elevates the plant defense mechanisms with the concomitant production of secondary metabolites such as alkaloids, glucosinolates, terpenoids, and phenolics . In respsid-1  involved in the regulation of gene expression , small isid-1 . We evaluated the entire genome for SSR (single sequence repeats) prediction and found 19,637 (6.3%) sequences comprised of 21,696 SSRs motifs. More than one SSR was found in 1784 scaffolds . We found that di- and trinucleotide SSRs  were significantly higher than other repeat types reported from the other insects . Among tB. mori genome [APN), which has a role in dietary protein digestion, was expressed at the L5 stage of YSB transcriptome [In our earlier study, we documented the transcriptome changes that occurred at four larval stages through RNA-seq. Around 88.32% of RNA-seq reads matched to the de novo assembled scaffolds , indicati genome , were excriptome . Carboxycriptome  at L7 stcriptome , was alsC. suppressalis, N. lugens, B. mori, and L. striatellus belonging to two Lepidopteran and Hemipteran orders were analyzed. The Venn diagram showed the number of shared and unique genes in YSB. The YSB shared 26,158 genes (56.79%) with C. suppressalis, 24,442 genes (53.06%) with B. mori, 15,621 genes (33.91%) with N. lugens, and 15,158 genes (32.91%) with L. striatellus. A sum of 13,348 genes was conserved among all four insects, which might be important for the Neoptera group. YSB shares 2741 genes solely with C. suppressalis, which demonstrates its closeness; 209 with N. lugens; 987 with B. mori; and only 66 genes were shared between YSB and L. striatellus. A total of 308 genes were commonly shared among C. suppressalis, N. lugens, and L. striatellus  and showed that YSB evolved closely with N. lugens followed by C. medinalis and C. suppressalis —248 Mb [Heliconius melpomene)—266 Mb [Papilio polytes)—227 Mb [Tribolium castaneum (204 Mb), and smaller than other rice pests such as Asiatic rice borer or striped rice stem borer —824 Mb [Spodoptera frugiperda)—371 Mb [N. lugens)—1140 Mb [L. striatellus Fallen)—541 Mb [N. lugens (37.59%) [L. striatellus (34.5%) [S. furcifera (31.6%) [S. frugiperda (32.97%) [Drosophila melanogaster) has a GC content of 43% [L. striatellus), white-backed planthopper (S. furcifera), and N. lugens upon flow cytometry were observed to be 587, 733, and 1110 Mb, respectively, whereas the final genome assemblies were 541, 720, and 1140 Mb, respectively [The draft genome assembly of YSB is larger than a few Lepidopteran genomes such as monarch butterfly —248 Mb , postman)—266 Mb , swallow)—824 Mb , fall ar)—371 Mb , brown p—1140 Mb , and sma)—541 Mb . The dif)—541 Mb . The GC (37.59%) , L. stri (34.5%) , S. furc (31.6%) , and S. t of 43% . Since tectively ,73, whicTribolium castaneum; 6%) [B. mori (35%) [S. frugiperda , N. lugens ) [S. furcifera  [The non-coding RNAs play a crucial role in gene regulation, and presence in YSB also suggests the same. A further level of investigation is required for deciphering regulatory functions of specific ncRNAs along with their potential relationships. Interactions among different ncRNAs will provide a clear picture of gene regulation through ncRNAs. Transposable elements in insect genomes range from as little as 1% in the Antarctic midge  to as hieum; 6%) , B. moriri (35%) , S. frugS (16%)) , S. furct class) . A lowerB. mori [D. melanogaster were reported [Apis mellifera), red flour beetle (T. castaneum), jewel wasp (Nasonia vitripennis), and fruit fly (D. melanogaster) [The transcription factors (TFs) are involved in several biological processes that work to ensure the appropriate expression of genes at a specific time at specific tissues in organisms. The zinc finger TFs were also observed in earlier-reported RNA-seq data at larval stages of YSB  and seveB. mori , implyinreported ,80,81. Hogaster) ,83,84.S. furcifera and N. lugens [Hormonal regulation is critical for insects, and specific hormones can be targeted to control the pest infestation. The silencing of Vg and VgR has been successfully demonstrated for controlling . lugens ,86. ThusN. lugens was sharply lowered after knockdown of Dnmt1 [S. furcifera were also related to DNA methylation [T. castaneum contains only two; and D. melanogaster contains seven [N. lugens genome [D. melanogaster [A. mellifera and T. castaneum [cry genes, indicating to some extent the correctness of our draft genome assembly. The Takeout family genes are another class discovered as a circadian-regulated gene found in the black-brown fly (Phormia regina) [Manduca sexta) [The occurrence of many epigenetic-associated genes in YSB confirms the existence of a typical insect epigenetic regulatory structure. The regulations of epigenetic mechanisms have not yet been exploited, except in a few studies, for instance, fecundity in of Dnmt1 . Similarhylation . The prehylation ; T. castns seven ,90, and s genome    and tobaa sexta) . These gcac), alan shepard (shep), and anoxia upregulated, which were detected initially in D. melanogaster [The behavioral genes such as cacophony , T. castaneum (11 α and 1 β subunit), and N. vitripennis (12 α and 4 β subunits) [Several insecticide targets were identified including members of the cysLGIC superfamily and nicotinic acetylcholine receptors. The cysLGIC superfamily genes are known to mediate chemical synaptic transmission in many insects and have been effectively targeted by various chemicals for crop protection strategies to date . The nicubunits) . On the N. lugens [D. melanogaster, B. mori, and H. armigera was reported as the main factor for detoxification of plant phenolics. If these genes are lost, the metabolism of plant phenolics will be impaired, and these may become toxic to the insects [Hyalophora cecropia) [B. mori [N. lugens genome. Apyrases are known to limit the plant eATP accumulation by hydrolysis of ATP, which was made evident in the case of Helicoverpa zea on tomato [Insect immune defense genes are an arsenal for host pest infestation and are critical for pest control. Several immune-related genes were present in YSB, among these, seven PGRPs were detected, whereas two PGRPs and seven βGRPs were reported in . lugens . The pol. lugens ,118. For insects . Functioecropia) , B. mori[B. mori , and a f[B. mori . Howevern tomato . ConsequH. armigera). Similarly, R-like/venom proteins from parasitoid wasps  and stink bug (Pristhesancus plagipennis) were reported to function as effectors [sid-1. Thus, the data provide strong evidence about the possibility of employing RNAi as an effective controlling strategy. Recently, our team has demonstrated the knockdown of the acetylcholinesterase (AChE) gene of YSB through RNAi as a pest control strategy [We found venom-related genes might also work as the potential effectors in YSB, along with other effectors such as oral secretion, digestive enzymes, alkaline phosphatase , and β-gffectors . The prestrategy .N. lugens might be due to the similarity in feeding behavior (monophagous rice sapsucker). The divergence with other rice pests may be admitted to the behavioral genes in addition to the effectors, immune system, etc.The evolutionary closeness of YSB with We used four representative natural populations for diversity studies using genomic SSRs. The polymorphism between YSB populations was comparatively less and might be due to the differences in the levels of insecticide exposure, external pressure, etc. Various biological traits and genetic diversity might associate with populations from different geographic localities due to geographic isolation, influenced by different climatic effects and thus different selection pressures . MicrosaC. suppressalis, N. lugens, and L. striatellus) and non-rice pests (B. mori) reveals that YSB is unique in the way it has evolved based on its visual perception, digestive enzymes, and presence of RNAi machinery. The presence of a high level of immune receptors and complete RNAi machinery gives us reasons for the non-functioning of viral suspensions on YSB. The presence of diverse effectors and their possible use by the insect provide a ray of hope to the biologist to exploit the mechanisms of these molecules for insect control. The information emanated from genome analysis matched the earlier reported transcriptome profiles and found that this information is at a much higher level than the transcriptome data. The identified SSR markers are a better genomic tool in understanding the diversity of populations/biotypes across the globe. We have added genomic SSRs to the existing repertoire in addition to our earlier EST SSRs, making them a rich source for molecular characterization of YSB populations. These SSRs can be deployed for studying the genetic diversity, evolutionary dynamics, repetitive sequence dynamics, and population dynamics of YSB populations, their related insect pests, and other insect pests with fewer genomic resources. Overall, we made a maiden effort to determine its genome size and sequence to a significant level of an agriculturally important pest, YSB, that affects the yield of rice adversely across the rice ecosystems. Our genome and transcriptome resources of YSB helps in deciphering various “omics” of this pest, leading to a better understanding of the systems biology. Our data can be mined by both the academicians and industry in developing novel management strategies and designing a new class of safer and specific insecticide molecules.The unavailability of the YSB genome sequence thus far has been hampering efforts to gain a complete hold on the YSB management strategies. The present draft genome sequence reveals that, though it has a small genome size, it uses its entire genomic information perfectly. The genome information provided a snapshot of complex metabolic mechanisms that operate in the insect to manifest and evolve in nature. We not only observed the different gene sets but also observed that YSB has a high level of gene regulation by operating a repertoire of transcription factors, hormonal control, and epigenetic mechanisms. YSB uses a strong visual and chemosensory mechanism to become monophagous. The genome analysis led to the identification of genes related to its specific behavior. Comparative genomic analysis with other rice pests (


Candidatus Microthrix is one of the most common bulking filamentous microorganisms found in activated sludge wastewater treatment plants (WWTPs) across the globe. One species, Ca. M. parvicella, is frequently observed, but global genus diversity, as well as important aspects of its ecology and physiology, are still unknown. Here, we use the MiDAS ecosystem-specific 16S rRNA gene database in combination with amplicon sequencing of Danish and global WWTPs to investigate Ca. Microthrix spp. diversity, distribution, and factors affecting their global presence. Only two species were abundant across the world confirming low diversity of the genus: the dominant Ca. M. parvicella and an unknown species typically present along with Ca. M. parvicella, although usually in lower abundances. Both species were mostly found in Europe at low-to-moderate temperatures and their growth was favored in municipal WWTPs with advanced process designs. As no isolate is available for the novel species, we propose the name “Candidatus Microthrix subdominans.” Ten high-quality metagenome-assembled genomes recovered from Danish WWTPs, including 6 representing the novel Ca. M. subdominans, demonstrated high genetic similarity between the two species with a likely preference for lipids, a putative capability to reduce nitrate and nitrite, and the potential to store lipids and poly-P. Ca. M. subdominans had a potentially more versatile metabolism including additional sugar transporters, higher oxygen tolerance, and the potential to use carbon monoxide as energy source. Newly designed fluorescence in situ hybridization probes revealed similar filamentous morphology for both species. Raman microspectroscopy was used to quantify the in situ levels of intracellular poly-P. Despite the observed similarities in their physiology (both by genomes and in situ), the two species showed different seasonal dynamics in Danish WWTPs through a 13-years survey, possibly indicating occupation of slightly different niches. The genomic information provides the basis for future research into in situ gene expression and regulation, while the new FISH probes provide a useful tool for further characterization in situ. This study is an important step toward understanding the ecology of Ca. Microthrix in WWTPs, which may eventually lead to optimization of control strategies for its growth in this ecosystem. The activated sludge process is the most common wastewater treatment technology applied worldwide. In recent years, process design has developed rapidly across the globe to meet the demands of growing urban populations. Additionally, the role of wastewater treatment plants (WWTPs) has moved toward resource recovery as societies strive toward sustainability and a circular economy. However, the unsolved problems frequently experienced at the treatment plants are bulking and foaming resulting from overgrowth of filamentous bacteria, affecting stable operation, plant hydraulic capacity, and effluent quality.Ca. Microthrix as some of the most troublesome filamentous bacteria in municipal WWTPs in addition to Gordonia, Ca. Amarolinea, and other phylotypes belonging to the phylum Chloroflexi  or long chain fatty acids (LCFAs)  and requires LCFAs  , while Ces LCFAs . Howeverrization .in situ studies using fluorescence in situ hybridization (FISH) combined with microautoradiography or nano-scale secondary-ion mass spectrometry visualized important differences compared to the isolate studies. Ca. M. parvicella has never been observed in situ to consume VFAs but only LCFAs and glycerol  granules inside Ca. M. parvicella filaments has been shown in isolates and in situ  MAGs. Both species were visualized in situ using novel FISH probes, which were further applied to investigate poly-P content using FISH-Raman microspectroscopy. Our results provide important new information about this troublesome filamentous genus in the activated sludge ecosystem, which may help to develop more targeted strategies for its control.In this study we investigated the diversity of genus Sampling of the activated sludge biomass from Danish full-scale municipal WWTPs was carried out within the MiDAS project . SamplinCa. Microthrix abundances between the two amplicon datasets. The handling of the Danish and the global dataset in regard to DNA extraction and library preparation protocol were similar, providing comparable datasets. Raw fastq files were filtered for phiX sequences using usearch -filter_phix, trimmed to 250 bp using usearch -fastx_truncate -trunclen 250, and quality filtered using usearch -fastq_filter with -fastq_maxee 1.0. Dereplication was performed using usearch -fastx_uniques with -sizeout. Amplicon sequence variants (ASVs) were generated using -unoise3 .2 Raman windows . The specific FISH probes were used to locate the target cells with a 50X dry objective (Olympus M Plan Achromat- Japan) of the in-built Olympus (model BX-41) fluorescence microscope. After bleaching, spectra from different filaments were obtained using a Horiba LabRam HR 800 Evolution (Jobin Yvon—France) equipped with a Torus MPC 3000 (United Kingdom) 532 nm 341 mW solid-state semiconductor laser. The specific settings for the spectrophotometer were: 5% neutral density (ND) filters, 600 mm/groove diffraction grating, 100 μm and 72 μm slidth width and confocal pinhole, respectively. The Raman spectra collected spanned the wavenumber region of 200 cm–1 to 3,000 cm–1. The Raman spectrometer was calibrated prior to obtaining all measurements to the first-order Raman signal of Silicon, occurring at 520.7 cm–1. Raman spectrometer operation and subsequent processing of spectra were conducted using LabSpec version 6.4 software . Absolute quantification of intracellular poly-P was carried out as described by Fresh activated sludge samples were collected from full-scale Danish WWTPs and aerated for 30 min to exhaust most intracellular carbon and refill poly-P reserves. Samples were fixed in 50% ethanol , as previously described , and stoCa. Microthrix MAGs were identified from within a set of 1083 high-quality MAGs from Danish activated sludge WWTPs (PRJNA629478). Ten MAGs were identified, and followed the MIMAG standard for high-quality draft genomes, including full-length rRNA genes, and a completeness > 90%, and contamination < 5%. GTDB-Tk v1.4.1 (Ca. M. parvicella genomes and MAGs present in NCBI but not the dereplicated GTDB-Tk Refseq release 95 database were downloaded (on 26/02/2021) and incorporated with the Danish MAGs and the GTDB-Tk set for phylogenomic analysis. The genome tree was created from the concatenated, trimmed alignment of 120 single copy proteins produced by GTDB-Tk. IQ-TREE v2.0  number , and additional checks were conducted using protein BLAST against the NCBI nr database (EnrichM v0.5.0) number  annotatelatform”  in orderlatform” . Specifidatabase .Average nucleotide identity based on blast (ANIb) was conducted using pyani v0.2.10  and the Ca. Microthrix species in the activated sludge ecosystem were analyzed using data from long-term surveys of Danish WWTPs . The two other low-abundant species were not observed as abundant in any WWTP. Ca. M. parvicella was dominant in the majority of the Danish and global plants, with the average read abundance of 1.7 ± 2.0% and 0.6 ± 1.2%, respectively. In some plants their abundance exceeded 14% likely causing severe sludge separation problems and deteriorated plant performance. Ca. M. subdominans was generally observed at lower abundances, with average and maximum read abundances in Denmark of 1.1 ± 1.3 and 13%, respectively, and average and maximum abundances globally of 0.2 ± 0.5 and 4%, respectively. Both species co-existed in the majority of the plants, indicating a similar ecological niche, with the highest global abundances recorded in a number of European countries .In the Danish nutrient removal plants nt genus , underlint genus . In somervicella , 2A, witrvicella . InteresCa. Microthrix species  and aerobic and anoxic stages , we could observe that in plants where both species were abundant, they had a similar effect on sludge settling. This was visualized by an almost identical slope representing the relationship between species read abundance and the corresponding DSVI values , which is widely used as a measure of sludge settling properties, showed a positive relationship with the relative abundance of both  species . Such re species  either fI values .Ca. Microthrix was conducted using full-length 16S rRNA gene sequences obtained from the MiDAS4 database, SILVA 138 SSURef Nr99, and from MAGs recovered from Danish WWTPs . However, only two of the species were abundant globally in WWTPs . However, it should be kept in mind that the MAGs recovered may not always represent the abundant members of the microbial community, and sequences retrieved from SILVA are clustered at 97% similarity, both of which may result in the in silico observed lack of probe coverage. All the MAG-derived sequences clustered either with Ca. M. parvicella or Ca. M. subdominans, except EsbE_MB4, which formed a separate cluster with several SILVA sequences. This particular sequence was targeted by Ca. M. parvicella-specific probe (MPA60) and the Ca. M. subdominans-specific probe (Msu181).High sequence similarity among the species in genus p values . Consequ–1), phenylalanine , lipids , amide I linkages of proteins , as well as poly-P storage   was significantly higher than similar peaks observed in other PAOs   in both dominans . No otheher PAOs , supportCa. Microthrix filaments was quantified using FISH-Raman microspectroscopy in fresh activated sludge from four full-scale EBPR plants (∗10–14 g P μm–1) and the lowest in Viby (1.12∗10–14 g P μm–1). Even though the Ca. Microthrix trichome width was relatively small, and thus also the P-content per μm of the filament, it can substantially contribute to P removal in the plants as the filament length often exceeds 100 μm, and the genus often present at high abundances , Ca. M. parvicella (4 MAGs) and Ca. M. subdominans (6 MAGs)  . One MAGMCC26207 , were foMCC26207 .Ca. Microthrix MAGs ranged between 3915 and 4743, and completeness and contamination ranged between 90.6–94.87 and 0.85–4.27%, respectively  to each other, suggesting a similar lack of diversity  regions for ectively . To datedatabase . The fouiversity . This sulibacter . Additioiversity .Ca. M. parvicella MAGs to the previously recovered genomes, metabolic reconstruction revealed similar metabolisms to the published models. The Ca. M. parvicella MAGs encoded the EMP pathway, TCA cycle, pentose phosphate pathway, as well as lipid activation and oxidation capabilities, high-affinity phosphate transporters, potential triacylglycerol (TAG) storage, and the potential to reduce nitrite and nitrate  and enoyl-CoA hydratases  were detected in every MAG, supporting lipid use as a key metabolic strategy , were detected in the KO analysis  in anaerobic respiration was also identified in Ca. M. subdominans (2O or N2 were not detected. The phosphate metabolism genes were highly similar between the two species. Genes for the high affinity phosphate transporter (pstSCAB) and transport regulator (phoU) were detected in Ca. M. subdominans . While the main metabolisms were very similar between the two species, a subset of genes comprised the species-specific core metabolisms of Ca. M. parvicella (380) and Ca. M. subdominans (364)  .Ca. M. subdominans indicated a slightly different niche to Ca. M. parvicella. Firstly, this lineage may be able to use atmospheric levels of carbon monoxide as a supplementary energy source, as it appears to encode several genes for a putative form II aerobic carbon monoxide dehydrogenase   are most closely related to those found in the ammonia oxidizing Gammaproteobacteria, Nitrosococcus, and Nitrosomonas  . This co systems . An ABC entified . The fouosomonas . These grculosis , which eella RN1 . This coella RN1 .Ca. M. parvicella prefers microaerobic conditions based on growth experiments  in one syntenic region  “Candidatus Microthrix subdominans,” . This taxon was represented by the MAG Lyne_MB9. The complete protologue can be found in Description of https://www.ncbi.nlm.nih.gov/sra) under the project number PRJNA622675.Publicly available datasets were analyzed in this study. This data can be found here: sequencing data from Danish WWTPs is available at the Sequence Read Archive (MN and PN devised the study and its main conceptual ideas. MN, CS, and MD performed data analysis with contributions from PN. FP and JP performed Raman measurements and data analysis. LT and ZK performed FISH analyses. MP and JP performed P-release lab experiments. The manuscript was drafted by MN and CS with contributions from FP, and revised by PN. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bone parameters derived from HR-pQCT have been investigated on a parameter-by-parameter basis for different clinical conditions. However, little is known regarding the interrelationships of bone parameters and the spatial distribution of these interrelationships. In this work: 1) we investigate compartmental interrelationships of bone parameters; 2) assess the spatial distribution of interrelationships of bone parameters; and 3) compare interrelationships of bone parameters between postmenopausal women with and without a recent Colles’ fracture.via spatial normalization to a common template. Mean values of the parametric maps before spatial normalization were used to assess compartmental Spearman’s rank partial correlations of bone parameters . Spearman’s rank partial correlations were also assessed for each voxel and vertex of the spatially normalized parametric maps, thus generating maps of Spearman’s rank partial correlation coefficients. Correlations were performed independently within each group, and compared between groups using the Fisher’s Z transformation.Images from the unaffected radius in fracture cases (n=84), and from the non-dominant radius of controls (n=98) were obtained using HR-pQCT. Trabecular voxel-based maps of local bone volume fraction (L.Tb.BV/TV), homogenized volumetric bone mineral density (H.Tb.BMD), homogenized μFEA-derived strain energy density (H.Tb.SED), and homogenized inter-trabecular distances (H.Tb.1/N) were generated; as well as surface-based maps of apparent cortical bone thickness (Surf.app.Ct.Th), porosity-weighted cortical bone thickness (Surf.Ct.SIT), mean cortical BMD (Surf.Ct.BMD), and mean cortical SED (Surf.Ct.SED). Anatomical correspondences across the parametric maps in the study were established All within-group global trabecular and cortical Spearman’s rank partial correlations were significant; and the correlations of H.Tb.BMD–L.Tb.BV/TV, H.Tb.BMD–H.Tb.1/N, L.Tb.BV/TV–H.Tb.1/N, Surf.Ct.BMD–Surf.Ct.SED and Surf.Ct.SIT–Surf.Ct.SED were significantly different between controls and fracture cases. The spatial analyses revealed significant heterogeneous voxel- and surface-based correlation coefficient maps across the distal radius for both groups; and the correlation maps of H.Tb.BMD–L.Tb.BV/TV, H.Tb.BMD–H.Tb.1/N, L.Tb.BV/TV–H.Tb.1/N, H.Tb.1/N–H.Tb.SED and Surf.app.Ct.Th - Surf.Ct.SIT yielded small clusters of significant correlation differences between groups.The heterogeneous spatial distribution of interrelationships of bone parameters assessing density, microstructure, geometry and biomechanics, along with their global and local differences between controls and fracture cases, may help us further understand different bone mechanisms of bone fracture. Osteoporosis is defined as a skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture . Bone stin vivo visualization of the three-dimensional (3D) bone microstructure at the distal radius and tibia with high spatial resolution and a low level of radiation exposure  enables exposure , 10. Becexposure , 12. Sinexposure .Colles’ fracture is the most common fragility fracture in the distal radius, with osteoporosis being a risk factor, especially for postmenopausal women . ExistinThis work was based on existing data from the study of Melton et al. . RecruitThis research was conducted in accordance to the regulations of the participating institutions and informed consent was obtained from all participants prior to enrollment. Analyses were performed based on de-identified data.Imaging of the distal radius for each subject was performed using an HR-pQCT system . The forearm was fixed in a carbon fiber cast, then inserted into the gantry of the scanner. The non-dominant side in controls and the unaffected side in fracture cases was scanned. The starting point of the fixed scan region was 9.5 mm proximal from a reference line set manually at the endplate in the distal radius. Scan settings were as follows: voltage 60 kVp, current 900 μA, integration time 100 ms, and effective dose 4.2 μSv. A 9.02 mm section spanned by a total of 110 slices was imaged with an isotropic voxel size of 82 μm. Subjects were rescanned if clear images were not obtained. For this study, all scans were scored for presence and severity of motion artifacts using the current artifact grading scheme of the manufacturer . Grades Bone segmentation from HR-pQCT images was performed using the standard evaluation software provided by the manufacturer as mentioned in other studies , 23. BriThe following parameters were measured for the trabecular and cortical bone compartments: trabecular BMD (Tb.BMD), trabecular bone volume fraction (Tb.BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), cortical BMD (Ct.BMD), and cortical thickness (Ct.Th) .In addition, assessment of apparent biomechanical parameters was accomplished using linear μFEA as previously described in the literature . Briefly3). SED is defined as the potential energy stored in a volume by virtue of an elastic deformation   and μFEA-derived strain energy density  .Periosteal surface-based maps encoding the local apparent cortical bone thickness (Surf.app.Ct.Th), effective cortical bone thickness taking into account porosity and partial volume effects , mean cortical BMD (Surf.Ct.BMD), and mean cortical SED (Surf.Ct.SED) at each vertex were also generated as previously described by Carballido-Gamio et al. enabling population-based vertex-wise associations of cortical bone parameters . For thiThe smooth voxel-based and the surface-based parametric maps described above were then spatially normalized to a common template of the distal radius as previously described . This spMean values of parameters of bone density, microstructure, geometry and biomechanics were compared between controls and fracture cases with linear regression models adjusting for age, height, and weight.For each compartment, within-group interrelationships of bone parameters were then evaluated using Spearman’s rank partial correlations adjusting for age, height and weight. These correlation coefficients were then compared between controls and fracture cases using the Fisher’s Z transformation.P-values in these statistical tests were considered significant if they were less than 0.05.P-value maps in the template space. Given the large number of correlations, P-value maps were corrected for multiple comparisons using the false discovery rate method  .To assess within-group spatial interrelationships of bone parameters, trabecular voxel-wise and cortical vertex-wise Spearman’s rank partial correlations adjusted for age, height, weight, and shape  were computed using the spatially-normalized parametric maps. This step generated correlation coefficient maps and their corresponding  q=0.05) . SpatialP-values in these statistical tests were considered significant if they were less than 0.05.To better understand the spatial correlations, we also performed statistical analyses of the smooth voxel-based maps of trabecular parameters and the surface-based maps of cortical parameters with no spatial normalization. In particular, we: 1) compared mean parametric values between controls and cases using linear regression models adjusting for age, height, and weight; 2) performed within-group Spearman’s rank partial correlations of bone parameters within each compartment adjusting for age, height and weight; 3) performed within-group Spearman’s rank partial correlations of bone parameters with μFEA.FL within each compartment adjusting for age, height and weight; and 4) compared the correlation coefficients between controls and fracture cases using Fisher’s Z transformations. p > 0.05).The characteristics of the subjects included in this study are shown in p < 0.01), and significantly higher Tb.Sp (p = 0.015) than controls. In addition, the fracture cases had significantly lower μFEA.FL and μFEA.Stiffness than controls (both p < 0.001).The differences between groups in BMD, bone microstructure, geometry and μFEA parameters are summarized in p < 0.001), but only the interrelationship of Tb.BMD-Tb.N was significantly different between controls and fracture cases (p < 0.05) as shown in All the interrelationships of trabecular and cortical bone parameters were significant within each group . Fracture cases also showed significantly higher H.Tb.1/N than controls (p < 0.05). However, Surf.Ct.SED did not differ between groups.Differences in homogenized and local trabecular voxel-based parameters, and in cortical surface-based parameters with no spatial normalization are summarized in p = 0.006), all within-group compartmental trabecular and cortical Spearman’s rank partial correlations were significant at p < 0.001. In addition, the correlations of H.Tb.BMD–L.Tb.BV/TV, H.Tb.BMD–H.Tb.1/N, L.Tb.BV/TV–H.Tb.1/N, Surf.Ct.BMD–Surf.Ct.SED, and Surf.Ct.SIT–Surf.Ct.SED were significantly different between groups at p < 0.05.The interrelationships of homogenized and local trabecular voxel-based parameters, and cortical surface-based parameters with no spatial normalization using Spearman’s rank partial correlations are summarized in In terms of correlations with μFEA.FL, mean values of the homogenized and local trabecular voxel-based parameters and cortical surface-based parameters manifested significant correlations in both groups, except for Ct.SED in the fracture group. There were no significant differences in the strength of these correlations between groups Table 6.In this study, we assessed the spatial distribution of interrelationships of parameters of bone density, microstructure, geometry and biomechanics in the distal radius of postmenopausal women with and without a recent Colles’ fracture using HR-pQCT. Spearman’s rank partial correlation coefficient maps showed significant heterogeneous spatial distributions of these interrelationships across the distal radius for both groups. In addition, small clusters of significant different interrelationships between controls and fracture cases were identified, particularly for trabecular bone, with stronger interrelationships in the fracture cases.As previously reported by Melton et al.  and in aρ| = 0.40–0.67). Moreover, we identified significant heterogeneous spatial distributions of interrelationships of bone parameters across the distal radius for both the trabecular (Despite global differences in trabecular and cortical bone parameters between controls and fracture cases, results showed expected global significant correlations of bone density, microstructure, geometry and biomechanics for both the trabecular and the cortical compartments within each group, for both analysis methods 5. Our rabecular , who shoabecular , 35, theabecular , comprehabecular –36 underAlthough we identified significant global and local interrelationships between all bone parameters within each group, and even similar heterogeneous spatial distributions of the correlation coefficients between groups, the results showed significant different global 5 and loIn agreement with previous studies, our results suggest that assessment of the trabecular bone in the distal radius might yield parameters that are more sensitive to prevent fragility fractures , 7, inclThis study has two main limitations. First, Colles’ fractures occurred with a median of 7 months prior to the HR-pQCT scans, i.e., this is not a prospective study, which limits to certain extent the impact of our findings. Second, the unaffected side had to be scanned for the fracture group, which might not fully represent the affected side since a previous study demonstrated side-to-side differences of cortical area and failure load at the radius of healthy women . HoweverIn conclusion, we assessed global and spatial interrelationships of bone density, microstructure, geometry, and biomechanics in postmenopausal women with and without a Colles’ fracture, as well as the global and spatial differences of these interrelationships between the control and fracture groups. We showed significant heterogeneous spatial distributions of these interrelationships across the distal radius within each group, and also found small clusters of significant differences for these interrelationships between groups, particularly in the trabecular bone compartment. Our findings indicate that local bone properties of BMD, microstructure, and geometry are heterogeneous in the distal radius, and that trabecular bone parameters may play a major role in the assessment of bone fragility at this anatomical site.The data analyzed in this study is subject to the following licenses/restrictions: The HR-pQCT images are owned by Mayo Clinic. Requests to access these datasets should be directed to principal investigator SK.KS: Analysis and interpretation of data, manuscript drafting, and revision. AB: Conception and design, interpretation of data, and manuscript revision. MO: Interpretation of data and manuscript revision. SK: Principal investigator of the original Colles’ fracture study, interpretation of data, and manuscript revision. JC-G: Conception and design, analysis and interpretation of data, manuscript drafting and revision. All authors contributed to the article and approved the submitted version.This work was supported in part by NIH R01AR068456 and NIH R01AR027065.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.


Candida auris, azole-resistant Aspergillus species, or Lomentospora prolificans [In recent decades, the prevalence of resistant fungal isolates has been steadily increasing both in veterinary and human medicine as well as in agriculture ,2. This lificans ,2,3,4. NCandida species. A total of nine papers were published in this Special Issue, including one review and eight original articles. The review article presents our current knowledge about C. glabrata, including its resistance, adaptation, and survival strategies [Copaifera leaf extracts, especially Copaifera paupera and Copaifera reticulata leaf extracts, inhibited C. glabrata biofilm formation and increased the cell vacuolization and cell membrane damage of this species [Ruta graveolens showed high activity against clinically relevant Candida species; moreover, it demonstrated a remarkable synergizing effect in combination with traditional antifungal drugs [C. auris isolates, where the caffeic acid phenethyl ester reduced the biomass and the metabolic activity of C. auris biofilm and decreased its adhesion to cultured human epithelial cells, indicating a potent antifungal and antivirulence effect [Candida species. In addition, they demonstrated favorable cytotoxicity properties as well [Candida cells, supporting the usage of this compound for the treatment of oral fungal infections [3 and EDTA [3 or EDTA can decrease the viability of the biofilms formed by C. albicans [C. albicans by using in silico, in vitro, and in vivo approaches [Candida cells [This Special Issue addresses current state-of-the-art investigations with regard to alternative therapeutic approaches against different potentially resistant rategies . Andrade species . Regardial drugs . In anote effect . It is n as well . Araujo  as well . They obfections . Gómez-Cand EDTA . They foalbicans . A compralbicans . Furtheralbicans . Drug realbicans . Santos proaches . Based oda cells .In summary, this Special Issue is a great resource that highlights novel and innovative work on alternative therapeutic approaches. Hopefully, this Special Issue will be able to stimulate further exciting research in the future.


Ganoderma species in Polyporales are ecologically and economically relevant wood decayers used in traditional medicine, but their genomic traits are still poorly documented. In the present study, we carried out a phylogenomic and comparative genomic analyses to better understand the genetic blueprint of this fungal lineage. We investigated seven Ganoderma genomes, including three new genomes, G. australe, G. leucocontextum, and G. lingzhi. The size of the newly sequenced genomes ranged from 60.34 to 84.27 Mb and they encoded 15,007 to 20,460 genes. A total of 58 species, including 40 white-rot fungi, 11 brown-rot fungi, four ectomycorrhizal fungi, one endophyte fungus, and two pathogens in Basidiomycota, were used for phylogenomic analyses based on 143 single-copy genes. It confirmed that Ganoderma species belong to the core polyporoid clade. Comparing to the other selected species, the genomes of the Ganoderma species encoded a larger set of genes involved in terpene metabolism and coding for secreted proteins . Of note, G. australe has the largest genome size with no obvious genome wide duplication, but showed transposable elements (TEs) expansion and the largest set of terpene gene clusters, suggesting a high ability to produce terpenoids for medicinal treatment. G. australe also encoded the largest set of proteins containing domains for cytochrome P450s, heterokaryon incompatibility and major facilitator families. Besides, the size of G. australe secretome is the largest, including CAZymes , proteases G01, and lipases GGGX, which may enhance the catabolism of cell wall carbohydrates, proteins, and fats during hosts colonization. The current genomic resource will be used to develop further biotechnology and medicinal applications, together with ecological studies of the Ganoderma species.The  Ganoderma species  are both ecologically and economically relevant fungi found in forest ecosystems. As wood-decay fungi, Ganoderma species efficiently decompose the components of plant cell walls, including lignin, cellulose and hemicellulose [G. boninense Pat. causes a basal stem rot (BSR) on oil palm trees [G. lucidum and G. australe have been used for degradation of environmental pollutants [Ganoderma fruiting bodies have been used over the last 2000 years to produce drugs used for improving immunity, and in anti-aging and anti-cancer treatments in humans [G. lingzhi Sheng H. Wu, Y. Cao, and Y.C. Dai and G. tsugae Murrill are used in Asian traditional medicine to improve health. On the other hand, polysaccharides and glycans extracted from G. sinense J.D. Zhao, L.W. Hsu and X.Q. Zhang have been used as clinical drugs [ellulose . G. bonilm trees ,3, whilellutants ,5. As men humans ,7,8. Foral drugs .Ganoderma is the largest genus in Ganodermataceae including 461 taxa recorded in Index Fungorum  as of 15 September 2021. However, only four complete genomes of Ganoderma species have been reported at this date. The genome of the well-known medicinal polypore, G. lucidum, was published by Chen et al. [Ganoderma species to unravel phylogenetic relationships within the Polyporales. The sequencing and analyses of G. sinense, another well-known medicinal fungus, provided new highlights on the genome methylation patterns, small RNA transcriptome, SM and defense processes [G. boninense isolated from an oil palm tree with severe symptoms of BSR disease. Additional genomes are required to explore the full diversity of Ganoderma gene sets involved secondary metabolism and wood white-rot decay and identify the genetic traits specific to the different species. Therefore, we sequenced and analyzed three additional genomes of Ganoderma species and compared them to other previously sequenced taxa, i.e., G. australe, G. leucocontextum and G. lingzhi.n et al. . This stn et al.  used therocesses . Utomo erocesses  sequenceGanoderma australe is widely distributed on deciduous trees in central, eastern and southern China, and G. lingzhi is widely cultivated thanks to its high medicinal value, whereas G. leucocontextum was recently described from southwestern China and is now cultivated in Yunnan and Tibet for medicinal production. In addition to these three new genomes, we included in our analysis seven publicly available genomes of Ganoderma species. Finally, we compared the Ganoderma gene sets to those obtained on 51 Basidiomycota species, including white-rot and brown-rot wood decayers, and biotrophic fungi. Here, we provided new insights on the evolutionary relationships between Ganoderma species and other Polyporales, and we identified idiosyncrasies in the gene repertoire involved in SM and secreted enzymes.Ganoderma leucocontextum Dai 12418 was collected on Quercus, and the strain was cultured on Potato Dextrose Agar ; G. australe Cui 17254 and G. lingzhi Cui 9166 were collected on other angiosperm logs., and the strains were cultured on Malt Extract Agar . The strains were cultured for 7–14 days in the dark at 25 °C. The taxonomic affiliation of the three strains was confirmed by phylogenetic analyses based on ITS sequences. Isolates were deposited in the culture collection of the Institute of Microbiology, Beijing Forestry University and are available upon request.Fruiting bodies of Ganoderma australe was extracted by using the sodium dodecylsulfate (SDS) method [G. leucocontextum and G. lingzhi. For genomic DNA extraction, fresh 7-day-old vegetative agar mycelial cultures grown on cellophane sheets were harvested, snap frozen in liquid nitrogen and stored at −80 °C. High molecular weight genomic DNA of ) method , whereas) method  was usedGanoderma leucocontextum was sequenced using the Pacific Biosciences platform ; 1.5 μg of genomic DNA was sheared to 10 kb using Covaris g-Tube or Diagenode megaruptor tube. The sheared DNA was treated with DNA prep to remove single-stranded ends and DNA damage repair mix followed by end repair/A Tail and ligation of barcoded overhang adapters using SMRTbell Express Template Prep 2.0 Kit . The library was purified with AMPure PB beads and libraries with different barcodes were pooled at equimolar for up to four maximum fungal genomes with a total sum of genome sizes of 200 Mb. A BluePippin size selection  was then performed on the pool to remove the shorter peaks physically. PacBio Sequencing primer was then annealed to the SMRTbell template library and sequencing polymerase was bound to them using Sequel II Binding kit 2.0. The prepared SMRTbell template libraries were then sequenced on a Pacific Biosystems’ Sequel II sequencer using 8 M v1 SMRT cells and Version 2.0 sequencing chemistry with 1 × 1800 sequencing movie run times. Filtered subread data was processed to remove artifacts. Mitochondria was assembled separately with the CCS reads using an in-house tool (assemblemito.py), used to filter the CCS reads, and polished with gcpp—algorithm arrow version SMRTLink v8.0.0.80529 . The mitochondria-filtered CCS reads were then assembled with Flye version 2.7.1-b1590  (—g 40 M—asm-coverage 50—pacbio-corr) and polished with gcpp—algorithm arrow version SMRTLINK v8.0.0.80529. Contigs less than 1000 bp were excluded.The genome of the strain Dai 12418 from Ganoderma australe Cui 17254 and G. lingzhi Cui 9166 genomes were sequenced using PacBio and Illumina platform. Sequencing libraries for Illumina were generated using NEBNext® Ultra™ DNA Library Prep Kit . One μg of genomic DNA was fragmented by sonication to 350 bp (400 bp for G. lingzhi DNA) using Covaris g-Tube. The DNA fragments were end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification. The libraries were purified by AMPure XP system  and were analyzed for size distribution by Agilent2100 Bioanalyzer and quantified using real-time PCR. The libraries for single-molecule real-time (SMRT) on PacBio platform was constructed with an insert size of 20 kb using the SMRT bell TM Template kit . The DNA fragments were repaired DNA damage and ends and prepared blunt ligation reaction. The library was purified with 0.45X AMPure PB beads and size-selection using the BluePippin System . The libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer . The whole genome of G. australe was sequenced using PacBio Sequel and Illumina NovaSeq PE150 at Beijing Novogene Bioinformatics Technology Co., Ltd. . The whole genome of G. lingzhi was sequenced using PacBio RS II platform and Illumina MiSeq platform at Shanghai Personalbio Technology Co., Ltd. . The low-quality reads were filtered (less than 500 bp) to obtain clean data. Preliminary assembly was conducted with SMRTLink v5.0.1 , and long reads (more than 6000 bp) were selected. By the variant Caller module of the SMRT Link software, the arrow algorithm was used to correct and count the variant sites in the preliminary assembly results.The Ganoderma leucocontextum was annotated using the JGI Annotation Pipeline with the support of their corresponding Trinity transcriptomes. Both assembly and annotations are available from JGI genome portal in MycoCosm  [G. australe and G. lingzhi were conducted by Augustus v3.03 [The genome of ry 2022) . The de us v3.03 , Genewisus v3.03  and Evidhttps://busco-data.ezlab.org/v4/data/lineages/basidiomycota_odb10.2020-09-10.tar.gz, accessed on 16 January 2022.The quality of the predicted proteomes was evaluated by using the tool Benchmarking Universal Single-Copy Orthologs (BUSCO v.4.1.3)  with theGanoderma genomes. Pair-wise comparisons and identification of syntenic blocks were performed by using the R package DECIPHER [The synteny analyses were conducted on the 10 largest scaffolds of each of the selected DECIPHER  with defDECIPHER . Data maDECIPHER .Ganoderma species were conducted by Maximum Likelihood (ML) with 23 ITS sequences. Sanguinoderma sp. Cui 17238 was used as the outgroup. The ML analyses were performed in RAxML-HPC v. 8.2.3 [http://tree.bio.ed.ac.uk/software/figtree/, accessed on 16 January 2022). The ML bootstrap ≥ 50% were presented on topologies from ML analyses.The phylogenetic analyses of 12 v. 8.2.3  involvinMelampsora larici-populina Kleb. and Ustilago maydis (DC.) Corda were used as outgroup species  using tBLASTx [http://www.repeatmasker.org, accessed on 16 January 2022).Transposable elements (TEs) were identified as described in Payen et al. . Briefly tBLASTx . The covhttps://itol.embl.de/, accessed on 16 January 2022) [http://www.cazy.org, accessed on 16 January 2022). The annotation of secreted proteases and lipases was performed by BLASTP search  against MEROPS  and Lipase Engineering Database .The gene clusters related to SM biosynthetic pathways were predicted by antiSMASH 4.2.0  and visury 2022) . PFAM dory 2022) . The mosry 2022) , and proGanoderma species were newly sequenced in this study: Ganoderma australe strain Cui 17254, G. leucocontextum strain Dai 12418 and G. lingzhi strain Cui 9166. The size of the G. australe assembly was 84.27 Mb and 20,460 protein-coding genes were predicted  were found in genome assemblies, indicating that assembled genomes captured most of the coding gene space, although the gene annotation for G. lingzhi appeared to be more fragmented  in the average genome size were found between white-rot fungi, brown-rot fungi and ectomycorrhizal fungi. The number of duplicated BUSCO genes was higher (23.4%) in G. australe. The synteny analyses showed no evidence for whole-genome or segmental duplications  retrotransposons. Besides, the proportion of unknown TEs in Ganoderma species was large, especially in G. australe  and G. lingzhi , indicating they likely played a key role in genome rearrangements. G. leucocontextum displayed the largest TE coverage , and the most diverse TE distribution, including simple repeats (Repetitive), IS3EU and Dada of DNA transposon, RTEX and L1 of non-LTR retrotransposon which are unique repeat elements in Ganoderma species  to 41.69% (Fomitiporia mediterranea) (F. mediterranea (p < 0.01), among which Ganoderma species have relatively higher TE coverage (p < 0.05). Ectomycorrhizal fungi showed the larger repeat element coverage, in which unknown TE elements were the most abundant (>12%).TE coverage in the 58 analyzed genomes ranged from 0.37% (erranea) . CompariRhizopogon vinicolor, the pathogens, ectomycorrhizal fungi and endophytes generally had a lower number of genes involved in SM than saprotrophic species. This difference was mainly driven by a lower content in t1pks, terpenes related genes and miscellaneous genes tagged as “putative” and “other” by the antiSMASH software. The antrodia clade composed of brown-rot species was segregated from other Polyporales clades by an enrichment in t1pks, indole, and fatty acid associated genes, and a depletion in NRPS. Among white-rot fungi in Polyporales, the phlebioid clade displayed a higher content in t3pks and a lower content in terpenes associated genes. The core polyporoid clade contained the species with diverse sets of biosynthetic genes clusters. Gene clusters involved in terpene synthesis and unknown metabolites  were enriched in Ganoderma species, especially in G. australe which contained 31 terpene associated gene clusters and 80 putative biosynthetic gene clusters. Noteworthy, G. lingzhi and G. lucidum as the main medically relevant species showed the lowest number of terpenes associated gene clusters, whereas G. australe and the pathogenic G. boninense had the highest content. G. lucidum, G. leucocontextum, Trametes versicolor, and T. pubescens genomes encoded a cluster related to lantipeptide production, and the first three were identified as associated with a t1pks.We identified a total of 16 types of SM biosynthetic clusters . Except Ganoderma australe showed the higher number of Pfam protein domains, i.e., five-fold larger than Ustilago maydis (with 864 protein domains). In G. australe, 13 protein domains were prominent, including cytochrome P450s (PF00067: p450) involved in SM, heterokaryon incompatibility protein (PF06985: HET) often related to vegetative incompatibility (VI) and membrane transporters of the major facilitator superfamily (PF07690: MFS_1). Besides, other protein domains putatively playing a role in epigenetic regulation  and protein–protein interactions (PF12937: F-box-like) were also enriched in G. australe. Comparing to other Ganoderma species, G. boninense and G. leucocontextum encoded additional protein domains , which were only found in parasitic and symbiotic fungi. Protein kinases domains  and protein–protein interactions domains  were especially enriched in ectomycorrhizal fungi.More than 5000 Pfam protein domains were identified in the 58 selected genomes. A total of 32 Pfam categories were sorted according to their gene copy number (>100) . GanoderGanoderma species have the largest secretome among the 58 selected fungal species  in the other species of the core polyporoid clade. Compared to other species, G. australe had the largest set of secreted CAZymes, in which GH18 and EXPN families were highly enriched in this species. It also contained the largest repertoire of PCWDEs, MCWDEs, and enzymes acting on pectin, peptidoglycans, and chitin , but five protease families  were only found in white-rot fungi, although with low gene copy numbers. Among these families, the M57 protease family including four proteins, was found in Ganoderma australe, G. leucocontextum, G. lingzhi, and G. lucidum. In addition, Ganoderma species have more G01, M28E, and M35 proteases.A total of 3880 genes encoding for secreted proteases (in 73 MEROPS families) were identified in 58 fungal genomes . Eight pp < 0.001) and among the 58 analyzed fungal secretomes, Ganoderma australe contained the highest number of secreted lipases, the most abundant being GGGX lipases  [G. tsugae CCMJ4178 [G. leucocontextum DH-8 [G. leucocontextum was newly sequenced and its genome was 50.05 Mb with 58 scaffolds [G. leucocontextum sequenced in our study was 60.34 Mb with 843 scaffolds. In our study, 21 terpene gene clusters were predicted in strain Dai 12418 against the 10 terpene gene clusters predicted in strain DH-8. The two-fold enrichment in strain Dai 12418 compared to strain DH-8 was unexpected, despite taking into differences in annotation tools and the potential bias of assembly quality. It may suggest a substantial intraspecific genome polymorphism. Direct comparisons of other functional gene categories were hampered by major differences in annotation methodologies. For example, Liu et al. [eptides) ,69. The CCMJ4178  and G. ltum DH-8 . The strcaffolds , the genu et al.  predicteGanoderma species. Compared to other species, the terpene genes of Ganoderma species were indeed expanded, however, substantial differences were observed among this genus. G. lingzhi and G. lucidum as the main medically species have the lowest number of terpenes associated clusters whereas the widespread G. australe and the pathogenic G. boninense have the highest content. Terpene related gene expansion in G. australe and G. boninense might explain their ecological ability to develop on a broader set of substrates. On the other hand, a shift of the terpenome composition in G. lingzhi, G. tsugae, and G. leucocontextum might have led to the production of terpenes with beneficial properties for human health. This study also allowed us to identify a polyketide synthase complex involved in the synthesis of the antibacterial lantipeptide [G. lucidum, G. leucocontextum, Trametes versicolor, and T. pubescens, likely explaining the use of formulations based on these fungi as antibiotics [G. australe, suggested an outstanding ability to synthesize a large set of secondary metabolites of yet unknown function. This confirmed that the economically and medically relevant secondary metabolites of these fungi represent an untapped resource.One of the main targets for medically-relevant products are the secondary metabolites associated genes, especially the terpenes in ipeptide  in G. luibiotics ,72,73,74As Polyporales is an important group of wood decayers, we investigated the decay ability of these species by analyzing their secretome. The current evolution was posited that early Agaricomycetes were saprotrophic and different lifestyles were derived from it ,76. It aGanoderma species deepened their lignocellulose degradation ability with further expansion of the ligninolytic PODs (AA1), hemicellulose , BCW- (GH25), and FCW-  degrading enzymes. Aminopeptidase Ap1 (M28E), deuterolysin (M35) and scytalidoglutamic peptidase (G01) were also enriched in Ganoderma species, but further functional analyses are needed to clarify their role(s). Noteworthy, G. australe showed an even wider degrading gene repertoire including higher PCW-degrading CAZyme, lipase and protease content. Similarly, numerous cytochrome P450 monooxygenases (PF00067: p450) were found in G. australe. These enzymes are known for their role in lignin and xenobiotic degradation [G. australe to more efficiently decompose lignocellulose than other Ganoderma species. An increased CAZyme repertoire in BCW- and FCW-degrading enzymes was also found in G. australe, which could perform the higher competitiveness than other microorganism for the substrate during colonization on the hosts. Associated with its enriched number of biosynthetic genes and clusters, such as lytic transglycolase (PF03330) and cerato-platanins (PF07249), this repertoire could be used to combat with miscellaneous biotic threats.The various white-rot Polyporales clades also displayed divergent wood degrading abilities. The potential ligninolytic ability and more generally PCW degradation ability of the core polyporoid clade was the highest among Polyporales due to the expansion of ligninolytic PODs (AA1) and hemicellulose degrading enzyme . The difference among white-rot clades also showed in the enzyme arsenal to compete with other microbes for substrates and copy with biotic threats. The specific expansion in BCW-degrading enzyme (GH25) and FCW-degrading enzyme  was also observed in the core polyporoid clade. radation . This paGanoderma australe indicated that these mechanisms play a key role in the stable reproduction and evolution of this wood decayer [Ganoderma species , MYB transcription factors were also abundant in Ganoderma species. These transcription factors are the largest transcription factor families in eukaryotic organisms and play key role in variable development and physiological activities [Ganoderma species after manually curation, and of the gene copy number found in each species was lower than the detected results in our study. This is likely due to the differences between strains and technical methods used here. Further exploration of MYB genes can help to clarify its potential function during the growth and development of Ganoderma fungi.The abundance of HET genes related to heterokaryon incompatibility, MFS transporters, and epigenetic regulation in  decayer ,80,81. Wtivities ,83. WangGanoderma genomes revealed a series of genetic features specific to this lineage of wood decayers. This study provided foundational information to characterize further ecological traits of this important group of decomposers. In addition, this information will be used to characterize the regulation of genes involved in SM biosynthesis pathways at the transcriptomic level, including antimicrobial compounds and medicinal drugs.The present comparative analysis of the publicly available


Listeria prophages on their host genome organizations, this work sequenced two L. monocytogenes strains (134LM and 036LM), previously identified as lysogens by mitomycin C induction. Draft genomes were generated with assembly sizes of 2,953,877 bp and 3,000,399 bp. One intact prophage  was inserted into the comK gene of the 134LM genome. Two intact prophages  were inserted in tRNA-Lys and elongation-factor genes of the 036LM genome. The findings confirmed the presence of three corresponding induced phages previously obtained by mitomycin C induction. Comparative genomic analysis of three prophages obtained in the newly sequenced lysogens with 61 prophages found in L. monocytogenes genomes, available in public databases, identified six major clusters using whole genome-based phylogenetic analysis. The results of the comparative genomic analysis of the prophage sequences provides knowledge about the diversity of Listeria prophages and their distribution among Listeria genomes in diverse environments, including different sources or geographical regions. In addition, the prophage sequences and their insertion sites contribute to the genomic diversity of L. monocytogenes genomes. These data of prophage sequences, prophage insertion sites, and prophage sequence comparisons, together with ANIb confirmation, could be useful for L. monocytogenes classification by prophages. One potential development could be refinement of prophage typing tools for monitoring or surveillance of L. monocytogenes contamination and transmission.A prophage is a phage-related sequence that is integrated into a bacterial chromosome. Prophages play an important role in bacterial evolution, survival, and persistence. To understand the impact of  L. monocytogenes is a foodborne pathogen and the causative agent of listeriosis, a potentially fatal infection with a mortality rate up to 30% [L. monocytogenes in a variety of foods [L. monocytogenes is well adapted to survive in stressful environments [p to 30% ,2. A numof foods , includironments ,6,7, whironments .L. monocytogenes isolates from food environments previously showed that prophages play an important role in the evolution of L. monocytogenes [Listeria lysogens (prophage-carrying Listeria) [L. monocytogenes. Multiple prophages, or monocins (an incomplete phage particle derived from a cryptic prophage), were observed in multiple strains, such as F6854, L99, HCC23, J0161 [L. monocytogenes [Comparative genomic analysis of ytogenes , facilitytogenes ,11,12. Pisteria) ,14. They3, J0161 . In addiytogenes . TherefoListeria phages have been sequenced [Listeria phages induced from the lysogens found in food and food-related environment were sequenced in a previous study [Listeria phages.Several temperate equenced ,18,19. Tequenced , and phaequenced . In addius study . The seqL. monocytogenes lysogens isolated from seafood and a seafood-related environment were sequenced to explore the prophage regions or any potential reorganization, compared to the genome sequences of corresponding induced phages. Potential insertion sites and attachment sites were predicted to confirm the prophage border in the host Listeria genomes. Analysis of the prophage sequences was performed with sequences of their corresponding induced phages and those publicly available. Overall, information obtained here allows us to better understand (i) the relationship between prophages and prophage-related regions in the genomes of L. monocytogenes and (ii) the genomic diversity of temperate Listeria phages from different environments or various geographical regions.Specifically, genomes of two selected L. monocytogenes lysogens, 2 isolates  were selected for sequencing. These two lysogens were isolated from different sources, including a seafood product (fish stick) by Vongkamjan et al. (2016) [L. monocytogenes lysogen 134LM harbors one inducible prophage and the L. monocytogenes lysogen 036LM harbors two inducible prophages [To understand the prophage content and their organization in the . (2016)  and a se. (2016) . The L. rophages .Listeria isolate was inoculated in 10 mL of Brain Heart Infusion  broth and incubated at 30 °C with shaking (220 rpm) for 16 h, followed by a centrifugation at 6000 rpm for one minute before cell harvesting. Then, bacterial DNA was extracted using GF-1 bacterial DNA extraction kit  following the manufacturer’s instruction. DNA quality and concentration were measured with a Maestro Nano Drop Spectrophotometer . The extracted DNA was subsequently used for sequencing library preparation by random fragmentation of the DNA sample using Illumina TruSeq DNA PCR-free kit, followed by 5′ and 3′ adapter ligation. Then, the high-quality sequencing fragmentation libraries were sequenced using the Illumina HiSeq2500 platform with 100-bp paired-end reads at Macrogen Inc. .A single colony of each L. monocytogenes F2365 (GenBank accession no. NC-002973.6) was used as a reference genome for alignment and ordering contigs using MAUVE genome alignment version 2.4.0 [http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html, accessed date 13 July 2018) before receiving the GenBank accession numbers. tRNAscan-SE [L. monocytogenes lysogens were submitted to the Center for Genomic Epidemiology website  MLST tool for MLST sequence typing [After sequencing, raw reads was filtered to remove the low-quality reads using Trimmomatic , then deon 2.4.0 . SubsequAscan-SE  was usedAscan-SE  was usede typing .L. monocytogenes strains used below without the following information, the corresponding clonal complex (CC) and Lineage were defined in BIGSdb via links to the PubMLST website  [L. monocytogenes newly sequenced here and those previously sequenced, the average nucleotide identity based on BLAST+ (ANIb) was calculated using the JSpeciesWS webserver  [For these two sequences, and any ne 2021) ,34. Serone 2021) . Moreovene 2021) .Listeria isolate was submitted to PHASTER for identification of prophages and prophage-related regions [int) that was adjacent to a potential bacterial gene, which could be a potential insertion site. To identify a complete sequence of each identified intact prophage, full gene clusters of prophages [The draft genome of each  regions . This torophages  were conattL and attR).Each complete sequence of the intact prophage was extracted for comparative analysis starting from 3′ end of the corresponding phage integrase gene through the 5′ end of the last phage genes detected by PHASTER. Generally, this site was several genes after a phage endolysin gene. PHASTER also detects a prophage attachment (att) site by searching for a sequence region with short nucleotide repeats (12–80 nucleotides) in both left and right junction fragments  [Then, the extracted intact prophage sequence was submitted for annotation using RAST . Annotatne 2021) . In addine 2021) .L. monocytogenes strains with complete genomes available on the National Center for Biotechnology Information (NCBI) database were subjected to prophage screening by PHASTER  with default settings. “Intact prophage” were manually confirmed as the prophages by the presence of the attachment site (att), integrase gene, terminases, and structural viral proteins. All 163 L. monocytogenes strains were subjected to MLST sequence typing (ST) [L. monocytogenes strains for allow up analysis  and prophage complement (having complete prophage sequence(s) and identified attachment site(s)) were used in parallel for the selection of L. monocytogenes strains to be included in our study.A total of 163 ing (ST) . ST and analysis . The 14 ribution ,42,43. Forldwide . The infL. monocytogenes genomes were submitted to PHASTER [comK-prophages, ten tRNA-Lys prophages, tRNA-Arg, tRNA-Ser, tRNA-Thr, elongation factor gene, and/or ribosomal protein S9.All of these detected intact prophages obtained from 35  PHASTER  for propL. monocytogenes lysogens newly sequenced and the closely related prophages [For comparative genomic analysis of prophages, the sequences of three obtained prophages in the two newly sequenced lysogens and this database set were included. Overall comparison of 64 prophage sequences was conducted using intergenomic distances inferred with the Genome-Blast Distance Phylogeny (GBDP) tool  using serophages .L. monocytogenes lysogens sequenced in this study and three Listeria phages  that were sequenced previously [Listeria phage sequences were extracted for nucleotide sequence alignment using Clustal Omega [Comparative genomic analysis was performed using sequences of the three prophages  found in the genomes of the two eviously . Inductieviously . Linear eviously . The regal Omega .Listeria phages available on the NCBI database were obtained  database at NCBI with SRA accession number SRP162378 and SRP163030 under BioProject PRJNA484544 and PRJNA480937, respectively.Sequences of L. monocytogenes 134LM and 036LM yielded 21 and 19 million reads with an estimated coverage of 715× and 643×, respectively . Assembly size of L. monocytogenes 036LM was 3,000,399 bp with 3027 detected CDSs (11 contigs). The GC content of both L. monocytogenes lysogens was 37.8%. A total of 54 tRNAs and four complete rRNA operons (16S-23S-5S) were found in 134LM genome, while 52 tRNAs and three complete rRNA operons were found in 036LM genome. The genomes of these two lysogens showed 99.94% sequence similarity as observed by ANIb , lineage I, and belonged to L. monocytogenes serogroup IVb .The draft genomes of ectively . Genome  by ANIb , and shacomK-prophage insertion resulted in a separation of the comK gene. However, prophage pp-036LM-EF was inserted in a translation elongation factor gene (EF) and prophage pp-036LM-tRNA-Lys was located at the 3′-end of tRNA-Lys of the 036LM genome. Various repeat nucleotide sequences as phage attachment sites were identified: (i) “ATGATTATAATAAT” for prophage pp-134LM-comK, (ii) “TGATAACAAAGC” for prophage pp-036LM-EF and (iii) “ACTCTTAATCAGCGGGTCGGGGGTTCGAAACCCTCACAACCCATA” for prophage pp-036LM-tRNA-Lys.PHASTER program identified one intact prophage in the 134LM genome  and two intact prophages in the 036LM genome  . In addiL. monocytogenes genomes, has been previously reported as a monocin [lmaDCBA operon identified as a virulence factor of L. monocytogenes.The GC content of prophage pp-134LM-comK and pp-036LM-tRNA-Lys was 37.8%, while GC content of prophage pp-036LM-EF was 37.1%. Genome annotation of three prophages  revealed monocin ,65. ThesOverall, sequence comparison of prophages and the corresponding induced phages showed similar genes and gene functions . HoweverListeria phage vB_LmoS_293. A 55-bp-fragment with some nucleotide modifications was found in the sequence of prophage pp-036LM-tRNA-Lys, thus differentiating this from the sequence of corresponding induced phage LP040. This could be a single/multiple nucleotide polymorphisms changed between the two sequences (induced phage and integrated prophage).Several sequence variations were observed regions , tRNA-Lys (n = 15) and tRNA-Arg (n = 14), followed by tRNA-Ser (n = 7), EF-Ts (n = 5), Ribosomal protein S9 (n = 3), and tRNA-Thr (n = 2).A total of 61 intact prophages detected by PHASTER within the genomes of 35 selected  genomes . ProphagL. monocytogenes genomes with sequence similarity show a high degree of prophage conservation despite these strains being isolated from different sources, distinct geographical locations, and different times. For example, the strain J1776 (from human), J1926 (from food), and J1817 (from environment), shared 99.99–100% sequence similarity by ANIb , HCC23 , and M7  shared 99.99–100% sequence similarity, and they contained the same set of prophages , with 85–100% nucleotide sequence similarity as determined by BLASTn. Among 12 strains, L. monocytogenes ST1 included for the analysis, L. monocytogenes lysogens 134LM (food source), and PNUSAL000474 (unknown source) shared 99.94% sequence similarity by ANIb; they also harbored a highly conserved comK-prophage with 90% nucleotide sequence similarity.The results reveal that many  by ANIb . They alL. monocytogenes genomes with high sequence similarity contained different prophages. In other words, the prophage sequence and their insertion sites also contribute to the genomic diversity of L. monocytogenes genomes. For instance, although the genome of the lysogenic isolate 134LM  showed high sequence similarity (99.97%) to the genomes of three L. monocytogenes strains 02-1792, 02-1289, and 02-1103  as identified by ANIb, prophage pp-134LM-comK was found in a different cluster than prophages of the 3 strains 02-1792, 02-1289 and 02-1103 whose insertion sites were tRNA-Lys gene. Similarly, the L. monocytogenes 036LM  and five L. monocytogenes strains J1-108, 81-0592, 81-0558, 81-0861 and 10-0809  shared 99.71% sequence similarity by ANIb. These strains contained a highly similar tRNA-Lys prophage by 99% nucleotide sequence similarity. The differences were from an extra 48,684 bp of the EF-TS-prophage inserted in the genome of the L. monocytogenes 036LM. Another example, an additional tRNA-Arg prophage, increased the distance of L. monocytogenes Scott A sequence  to three sequences of L. monocytogenes 02-1792, 02-1289, 02-1103 .However, many comK, tRNA-Lys, tRNA-Thr, and ribosomal protein S9. In addition, cluster VI contained 13 prophages with four insertion sites: comK, tRNA-Lys, tRNA-Arg, and tRNA-Thr.Full genome comparison of 64 prophage sequences by VICTOR classified those into six distinct clusters, I to VI , supportLinear genome visualizations of prophages were generated using Easyfig 2.1 . Result L. monocytogenes strains from various sources. For example, the pp-036LM-EF prophage showed high similarity to prophage of L. monocytogenes strains N1-011A, FDA00011238, and FDA00006905  and F4244  by 96% nucleotide sequence similarity. The prophage pp-036LM-tRNA-Lys showed high similarity to prophage of L. monocytogenes strains J1-108, 81-0592, 81-0558, 81-0861, and 10-0809  by 99% nucleotide sequence similarity. The comK-prophage of 134LM was the most closely related to a comK-prophage of L. monocytogenes strains PNUSAL000474  and F4244  by 90% nucleotide sequence similarity.Three prophages found in the two lysogens sequenced in this study were classified into clusters II, V, and VI. Interestingly, these prophage sequences were closely related to prophages of Listeria phage vB_LmoS_188, which was a temperate phage previously isolated from wild mushroom in Ireland. Phage LP040 was closely related to phage vB_LmoS_293, which was also a temperate phage previously isolated from mushroom compost in Ireland. Phage LP041 was closely related to a unique Listeria phage B054, which was previously induced from L. innocua WSLC 2054 in the U.S.Genomes of phages sequenced here were closely related to several temperate phages . Phage LListeria phage and the closely related phages indicates some regions with high nucleotide sequence similarity (>90%), especially the late genes, which encoded for DNA packaging, head, tail, and base plate structure , GC content (~38%) as reported previously [Listeria genomes as commonly reported, which typically showed 13–18 repeat rRNA regions across the chromosome [L. monocytogenes lysogens sequence genomes [Overall, the genomes of these lysogens displayed typical genome features of eviously ,66. Howeromosome . This co genomes .L. monocytogenes newly sequenced here shared 99.94% sequence similarity by ANIb. The two intact prophages in the lysogenic genome of 036LM could account for the difference, compared to the genome of 134LM. In addition, genome sequences of both lysogens showed high similarity to the genome of L. monocytogenes reference strain F2365, but prophage was not detected in F2365 and no insertion site was found. Furthermore, the extra EF-Ts prophage in 036LM or the tRNA-Arg prophage in L. monocytogenes Scott A made the difference between these strains and their closely related strains. These findings suggest that prophage sequences could contribute to evolutionary changes of L. monocytogenes genomes. Similarly, Orsi et al. (2008) reported that the differences in a 42-kb-prophage sequence differentiate four similar genome backbone L. monocytogenes isolates of a strain which persisted over 12 years, indicating the importance of prophages in differentiation of closely related L. monocytogenes [The two genomes of lysogenic L. monocytogenes genomes as indicated by various insertion sites detected. The comK gene and tRNA-Lys gene were reported as the most common insertion site for Listeria prophages. Previous studies have shown that multiple L. monocytogenes strains such as J0161, J2818, R479a, HCC23 and J1-108 containing comK and/or tRNA-Lys prophage(s) [comK-prophages have been previously reported to facilitate the survival over 10 years in foods and food processing environments [comK-prophage could also help the Listeria lysogen escape from activated macrophages, facilitating the virulence of L. monocytogenes when this bacteria infects a human host [Listeria lysogen. In addition, the EF gene was another insertion site for Listeria prophages detected in this study. This is considered as a rare insertion site, which has also previously been found in a few strains of L. monocytogenes such as SLCC2755 [L. innocua strain Clip11262 [L. monocytogenes. Overall, prophage insertion loci could be influenced by the integrase gene or the bacterial attachment sites [Prophages were found in different loci of phage(s) . Lysogenronments . The comman host . This prSLCC2755  and L. ilip11262 . The EF nt sites .L. monocytogenes 134LM newly sequenced here and L. monocytogenes strains on the database such as 02-1792, 02-1289, and 02-1103. However, the prophage in the genome of L. monocytogenes 134LM showed a different insertion site and was found in a different phylogenetic cluster than those closely related strains. These findings suggest that prophage sequences and the insertion sites could provide additional information for identifying the variations in the genomes of Listeria beyond the analysis by ANIb. Similarly, previous studies have revealed that the genetic diversity of L. monocytogenes was affected by gain or loss of genetic islands, especially the presence of prophage sequences [L. monocytogenes strains showing prophage with the same genome size), insertion site, and classified into the same cluster, could have relatedness confirmed by ANIb analysis. Therefore, data from the detection of prophages, differentiation of insertion sites in specific loci of Listeria, and prophage sequence comparison, together with the ANIb confirmation, could be used to further classify L. monocytogenes strains.Full genome comparison showed high sequence similarity by ANIb analysis between equences . In addiL. monocytogenes genomes, which contributes to the genomic diversity of L. monocytogenes genomes. Moreover, variation of prophage sequences was found in the genomes of L. monocytogenes, suggesting diverse types of prophages were found in L. monocytogenes genomes, which play an important role in the host genome diversity. Previous studies have reported the role of prophages, as a contributor to the Listeria diversity [Listeria lysogens could contribute to the variation of host genomes as mentioned in a previous study [We found that the prophage insertion sites could be diverse in iversity . Diverseus study ,25.Listeria phages [Listeria genome during prophage induction or the acquisition of prophage genes is a main source of L. monocytogenes genome diversity [Prophages showed genome features with typical six gene clusters of temperate a phages ,18,19,20a phages , suggesta phages ,18. Furta phages ,71 or raa phages . These fiversity .Listeria species. However, low sequence similarity was observed in the region of cell lysis and lysogeny control between the induced phages and the closely related temperate phages. In the cell lysis cluster, neither holin nor endolysin genes were conserved among the examined phages in this study. Similar to report of previous study by Dorscht et al. (2009), genes encoding for holin and endolysin showed a low similarity among the Listeria phages included in the analysis. These temperate and induced phages were not only from different sources, but also from distinct regions in the world, e.g., Thailand, Ireland, and the U.S.Genomes of the induced phages from seafood and a seafood-related environment are closely related to the temperate phages from natural environments  or induced phages from lysogens of other The lysogeny control cluster of phages LP040 and LP041 showed high percentage (%) similarity to their closely related temperate phages. This suggests the possibility that this phage may insert into the same genomic locus or have the same phage insertion site. This could be from the phage attachment site and the bacterial attachment site recombining between two DNA recognition sequences, resulting in the phage genome integrated into the bacterial chromosome by site-specific recombination ,72. ThisL. monocytogenes (134LM and 036LM) obtained from seafood and a seafood-related environment. Intact prophages were found in the genomes of these two lysogens. A comparative prophage sequence analysis suggests that various prophage insertion sites, as well as diverse prophage sequences, could contribute to the diversity of Listeria in food processing environments. Overall, the study here provides a model for comparative analysis among prophages in diverse genomes of L. monocytogenes lysogens. Data from the detection of prophages, differentiation of insertion sites in specific loci of Listeria, and prophage sequence comparison together with the ANIb analysis, could further classify closely related L. monocytogenes strains/isolates. This study also suggests the potential development for using a prophage as a typing tool for monitoring and surveillance of foodborne pathogen contamination and transmission.Here, we reported the draft genome sequences of two lysogenic isolates of