Linum usitatissimum) is a member of family linaceae with annual growth habit. It is included among those crops which were domesticated very early and has been used in development related studies as a model plant. In plants, Calmodulin-binding transcription activators (CAMTAs) comprise a unique set of Calmodulin-binding proteins. To elucidate the transport mechanism of secondary metabolites in flax, a genome-based study on these transporters was performed. The current investigation identified nine CAMTAs proteins, classified into three categories during phylogenetic analysis. Each group had significant evolutionary role as illustrated by the conservation of gene structures, protein domains and motif organizations over the distinctive phylogenetic classes. GO annotation suggested a link to sequence-specific DNA and protein binding, response to low temperature and transcription regulation by RNA polymerase II. The existence of different hormonal and stress responsive cis-regulatory elements in promotor region may directly correlate with the variation of their transcripts. MicroRNA target analysis revealed that various groups of miRNA families targeted the LuCAMTAs genes. Identification of CAMTA genes, miRNA studies and phylogenetic analysis may open avenues to uncover the underlying functional mechanism of this important family of genes in flax.Flax ( In addition, many eukaryotes have been identified to be equipped with CAMTA transporters including Arabidopsis thaliana [oryza sativa [Vitis vinifera [Brassica napus [Lycopersicum esculantum [Medicago truncatula [Citrus sinensis [Populus trichocarpa [Nicotiana tabacum [Musa acuminata [Phaseolus vulgaris [Zea mays [Solanum lycopersicum [Fragaria ananassa [Glycine max [Puccinia triticina and low temperature stress [The divalent ions of calcium (Caf plants . Ca2+ sistatuses . The knos (CaMs) . Among tsporters . Calmodusporters , 6–8. Amsporters . The tobthaliana , oryza sa sativa , Vitis vvinifera , Brassicca napus , Lycoperculantum , Medicaguncatula , Citrus sinensis , Populuschocarpa , Nicotia tabacum , Musa accuminata , Phaseolvulgaris , Zea mayZea mays , Solanumpersicum , Fragariananassa , and Glycine max . The CAMcine max –27. The cine max , 28. Sixcine max . For inscine max –35. AtCAe stress , 37.Linum usitatissimum), an annual plant of family Linaceae, is considered among the very first domesticated crops in the world. It has been utilized as a model species to investigate the development related processes in plants [Linum usitatissimum).Flax  database was accessed to download the sequences of six CAMTA family members from Arabidopsis thaliana [-7) search was made against the genome of flax in Phytozome database (v. 12.1) (http://www.phytozome.net/) [http://pfam.xfam.org/) [http://smart.embl-heidelberg.de/smart/batch.pl) [http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/) [http://www.expasy.org/tools/protparam.html) was employed to estimate the physical attributes of CAMTA transporters like molecular weight, isoelectric point, protein size, instability index, aliphatic index, and GRAVY [http://cello.life.nctu.edu.tw/) [http://www.genscript.com/wolf-psort.html) [The Arabidopsis information resource   using Aram.org/) , SMART (atch.pl)  and conssb.cgi/)  were usend GRAVY . Furtheredu.tw/)  and WoLFrt.html) .Arabidopsis thaliana was used as the control. The family members of LuCAMTAs were named according to the similarity of sequence and its arrangement in the phylogenetic tree.CAMTA protein sequences were aligned in Arabidopsis and flax by using the Clustal W function of MEGA 7.0. The MEGA 7.0 software was used to construct the phylogenetic tree , applyinCAMTAs were run against their corresponding genomic sequences to find out the gene structure by using Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn) [http://meme-suite.org/tools/meme) [The coding DNA sequences of flax .edu.cn) . Moreovels/meme) . The parLuCAMTA genomic DNA was retrieved from Phytozome database [http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [cis-regulatory elements with respect to stress and hormonal response.Promotor region 1kb upstream for database . The seqe/html/)  with defhttps://www.blast2go.com/) was employed for gene ontology of the LuCAMTA family members [http://mirbase.org/) [http://www.plantsrnas.org/) [http://plantgrn.noble.org/psRNATarget/analysis/) [Blast2GO software ( members . Biologise.org/) , and onlas.org/) . The taralysis/) .CAMTA gene family, flax genome was searched against identified six CAMTA genes of A. thaliana as queries in BLAST search of the Phytozome database. All non-redundant putative gene sequences were extracted from the database. SMART, CDD and PFAM databases were used for sequence analysis to confirm the existence of CAMTA-specific conserved domains i.e. IQ: calmodulin-binding IQ motifs, ANK: ankyrin repeats, IPT/TIG: Ig-like, transcription factor immunoglobulin, and CG-1: DNA-binding domain. Nine genes were finally selected and named as LuCAMTA1–9 for L. usitatissimum based on their position in relation to A. thaliana in the phylogenetic tree. The detailed physiological characteristics of the selected LuCAMTA proteins, such as isoelectric point (pI), molecular weight, length, Instability index, Aliphatic index, predicted subcellular locations and GRAVY are presented in For a comprehensive overview of the http://cello.life.nctu.edu.tw/) to revalidate the outcomes of pSORT. According to the predicted results majority of the CAMTA proteins were localized in the nucleus. However, LuCAMTA1 and LuCAMTA5 were also present in chloroplast and plasma membrane, respectively.The protein sequences were reanalyzed for subcellular localization with CELLO v. 2.5 , biological processes (BP), cellular structural components (CC). For cellular structural component (CC), LuCAMTAs were involved in the intracellular and cellular anatomical entity. The genes, specific to molecular functions (MF), were involved in sequence-specific DNA binding activities, while in biological processes (BP), the genes were responsible for response to stimuli and regulation of biological and metabolic processes , DNA binding (GO:0003677), and sequence-specific DNA binding (GO:0043565). In BP, the genes were involved in response to low temperature stress (GO:0009409) and up-regulation of transcription by RNA polymerase II (GO:0045944). Regarding CC, nucleus (GO:0005634), intracellular (GO:0005622) and cellular anatomical entity (GO:0110165) were found in GO functional annotation.Gene Ontology database was accessed to analyze the functional classification of proteins of the rocesses . Table 2LuCAMTA genes, except LuCAMTA9, were the targets of eleven different categories of miRNAs (LuCAMTA genes (LuCAMTAs and miR159 and miR164 target two LuCAMTA genes each. The predicted miRNAs were reported to involve in cleaving and inhibition of the translation of target genes.MicroRNAs (miRNAs) play a pivotal role in controlling the expression pattern of transcription factors. In the current study, potential miRNAs were searched for a set of nine identified LuCAMTA transcripts by accessing the psRNATarget . Results demonstrate that all f miRNAs . These mTA genes . For ins2+) plays a key role as a core transducer and regulator in response to environmental stimuli and developmental processes of plants [2+ signals are decoded into appropriate physiological responses and transmitted to the sink [2+-binding protein with a defined role in biochemistry, cell biology and molecular biology as a regulator that binds to a number of target proteins [CAMTA gene family. A combined N-J tree was developed to establish a phylogenetic relationship between Arabidopsis and flax. The analysis revealed an intimate association between CAMTA transporters in Arabidopsis and flax, indicating that the roles of LuCAMTAs could be like those of AtCAMTAs (LuCAMTA gene family (LuCAMTA1-3) from group- I exhibited a close association with three AtCAMTA genes (AtCAMTA1-3). Reportedly, these genes have been well-investigated for their participation in SA-regulated defense response and tolerance to cold stress [The divalent ion calcium ion , ANK (ankyrin repeats), IPT/TIG (transcription factor immunoglobulin), and IQ motifs  –27. The  species , 20, 76.cis- regulatory elements were predicted in the 1Kb upstream promotor region of LuCAMTA. The current study revealed four stress-responsive elements; low-temperature-responsive (LTR) elements [cis- regulatory elements, responsive to hormones and stress, in the promotor regions of CAMTA gene family reveals their role in corresponding environments.To elucidate the transcriptional regulation in response to stress and hormones, PlantCARE database was accessed. The elements , anaerobelements , droughtelements , and ligelements . Promoteelements  and salielements . The preLuCAMTA genes were submitted to GO database to determine the functions of these genes. According to the results, LuCAMTA genes were involved in BP, MF and CC. Regarding cellular structural component (CC), LuCAMTAs were responsible for intracellular and cellular anatomical components. The genes regarding molecular functions (MF), carried out sequence-specific DNA binding activities, while the genes in biological processes (BP) were found to regulate the responses to stimuli, biological processes and metabolic processes  are endogenous small RNA sequences of 20–24 nucleotides, performing pivotal functions in regulating growth and developmental processes of plants. They down-regulate the expression of their corresponding genes at post-transcription stage by gene silencing and target degradation or translational repression. To understand the regulatory mechanisms in plants, Identification of miRNAs is of supreme importance. Previous reports have revealed 32 conserved miRNAs in flax . Plant sCAMTA genes in flax (Linum usitatissimum). The members of CAMTA gene family in flax were identified and characterized by in silico approaches. Nine genes of CAMTA family were identified in flax genome. The analysis of these genes also suggested a potentially functional association with transporters of other plant species. The current study also identified different miRNA families targeting the identified genes of CAMTA family in flax. The present findings are of great importance to elucidate the involvement of CAMTA gene family in metabolic processes of flax and for identification of key genes in future breeding programs. The current analysis provides a deep insight into LuCAMTA gene family to enhance agronomic, ecological and economic benefits of flax.The current study reports the first systematic analysis of


Pseudomonas aeruginosa ST277 carries important antibiotic resistance determinants, highlighting the gene coding for SPM-1 carbapenemase. However, the resistance and persistence of this clone is apparently restricted to the Brazilian territory. To understand the differences between Brazilian strains from those isolated in other countries, we performed a phylogenetic analysis of 47 P. aeruginosa ST277 genomes as well as analyzed the virulence and resistance gene profiles. Furthermore, we evaluated the distribution of genomic islands and assessed in detail the characteristics of the CRISPR-Cas immunity system in these isolates.The Brazilian endemic clone P. aeruginosa ST277, confirming a strong link between sequence type and spacer acquisition. Most CRISPR spacer targets were phage sequences.The Brazilian genomes presented a typical set of resistance and virulence determinants, genomic islands and a high frequency of the CRISPR-Cas system type I-C. Even though the ST277 genomes are closely related, the phylogenetic analysis showed that the Brazilian strains share a great number of exclusively SNPs when compared to other ST277 genomes. We also observed a standard CRISPR spacers content for P. aeruginosa ST277 strains circulating in Brazil characteristically acquired In163 and PAGI-25, which can distinguish them from strains that do not accumulate resistance mechanisms and can be found on the Asian, European and North American continents. The distinctive genetic elements accumulated in Brazilian samples can contribute to the resistance, pathogenicity and transmission success that characterize the ST277 in this country.Based on our findings,  Pseudomonas aeruginosa is an important pathogen that shows a strong potential for development of multidrug resistance and is frequently implicated in healthcare-associated infections. Since the first report in 2002, SPM-1 metallo-β-lactamase is the main carbapenemase associated with P. aeruginosa in Brazil . Moreover, BLAST (blastn) was applied to check the presence of the virulence genes exoS (AY029246.1), exoU (KX641457.1), exoT , and exoY .The prediction of the resistome was made using the Comprehensive Antibiotic Resistance database (CARD) . In163 wy BLASTn  searchesFrom reordered contigs or complete genomes, CRISPR arrays and their associated proteins (Cas) were predicted using CRISPRCasFinder . The .jsacr) genes were identified based on BLAST (blastn)  [Putative anti-CRISPR ((blastn)  matches (blastn) . Eighteesoftware  [anti-I-(n = 1)] , 46, 47.n = 4634 phage files, source: ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/viral/) (downloaded on April 2018), and 2) Refseq plasmids from NCBI .When using the non-redundant spacer set as BLAT query  , followiAdditional file 1. General information about ST277 P. aeruginosa NCBI genomes.Additional file 2. P. aeruginosa ST277 pangenome.Additional file 3. Spacers from ST277 P. aeruginosa CRISPR-Cas system type I-C.Additional file 4. BLAT output from P. aeruginosa ST277 spacers against plasmids and phages.Additional file 5. Acr putative sequences.Additional file 6. Acr nucleotide sequences from plasmids.Additional file 7. Acr nucleotide sequences from phages.


The fungal cytochrome P450 enzyme sterol 14α-demethylase (SDM) is a key enzyme in the ergosterol biosynthesis pathway. The binding of azoles to the active site of SDM results in a depletion of ergosterol, the accumulation of toxic intermediates and growth inhibition. The prevalence of azole-resistant strains and fungi is increasing in both agriculture and medicine. This can lead to major yield loss during food production and therapeutic failure in medical settings. Diverse mechanisms are responsible for azole resistance. They include amino acid (AA) substitutions in SDM and overexpression of SDM and/or efflux pumps. This review considers AA affecting the ligand-binding pocket of SDMs with a primary focus on substitutions that affect interactions between the active site and the substrate and inhibitory ligands. Some of these interactions are particularly important for the binding of short-tailed azoles . We highlight the occurrence throughout the fungal kingdom of some key AA substitutions. Elucidation of the role of these AAs and their substitutions may assist drug design in overcoming some common forms of innate and acquired azole resistance. The azole drug segment has dominated the global antifungal drug market since the first-generation azoles clotrimazole and miconazole were introduced into the clinic in 1960 . The triFusarium spp. and Aspergillus spp. are cross-kingdom pathogens that cause infections in plants, animals and humans [With fungal pathogens estimated to cause yield losses of up to 30%, the use of fungicides is essential for efficient food production and overall food security . Becaused humans .Aspergillus), ERG11 , or SDM , a key enzyme in the fungal-specific ergosterol biosynthesis pathway. It catalyzes the three-step conversation of lanosterol or eburicol to a 14α-demethylated product [The target of antifungal agrochemicals  and antifungal drugs is the cytochrome p450 enzyme sterol 14α-demethylase CYP51  amino acid (AA) substitutions in the SDM, affecting the active site of the enzyme, (2) overexpression of the SDM, and (3) overexpression of efflux pumps such as ATP-binding cassette (ABC) transporters and/or major facilitator superfamily (MFS) transporters ,25,27.This review will focus on key AA substitutions associated with the ligand-binding pocket of SDMs. We will summarize the known impact of these residues on resistance to short- and long-tailed azoles in pathogenic fungi of humans, animals, and plants.Fungal SDM is member of the CY51 family of the cytochrome P450 superfamily found in all eukaryotes. SDM is a bitopic endoplasmic reticulum protein, which spans the lipid bilayer once ,21,28. IIn the yeast ergosterol biosynthetic pathway, the SDM lanosterol 14α-demethylase catalyzes three reduction steps. The 14α-methyl group of the substrate lanosterol is converted first into an alcohol, an aldehyde and then released as formaldehyde together with the insertion of a 14–15 double bond yielding the reaction product 4,4 dimethylcholesta-8(9), 14, 24-trien-3beta-ol for further processing . The enzSaccharomyces cerevisiae SDM)  . These ASaccharomyces cerevisiae is a non-polar amino acid in helix A’ at the mouth of the substrate entry channel. It interacts with distinct parts of long-tailed substrates such as itraconazole (ITC) and posaconazole (PCZ) and results in resistance to these long-tailed antifungals [Glycine 73 (G73) in ifungals ,58.S. cerevisiae SDM structurally aligns with Y132 in Candida species , Y136 in Ajellomyces capsulatus (=Histoplasma capsulatum) and Uncinula necator, Y145 in Cryptococcus neoformans, Y121 in Aspergillus species , and F129 in Rhizopus arrhizus, Mucor circinelloides, and Rhizopus microsporus (Tyrosine 140 (Y140) in the B-C loop of rosporus  [49,59,6S. cerevisiae SDM structurally aligns with K143 in Candida species  (Lysine 151 (K151) in helix C of picalis) . The posS. cerevisiae SDM structurally aligns with T315 in Candida albicans and in other fluconazole-susceptible fungi  residue. Both A. fumigatus mutant strains showed lower resistance (20 µg/mL) compared to Aspergillus fumigatus strains carrying the naturally occurring I301 polymorphism (640 µg/mL) [Threonine 322 (T322) in helix I of le fungi . The A. 0 µg/mL) .S. cerevisiae SDM, the nonpolar G464 structurally aligns with G448 in A. lentulus, G464 in Sc. apiospermum, and G464 Candida albicans. It is most often substituted with serine (S). The S side chain contains a polar hydroxyl group that may cause azole resistance by modifying the heme environment.In the fungus-specific loop of S. cerevisiae SDM.In the following sections, AA substitutions in the above amino acids are discussed in the light of their impact on azole susceptibility. An overview will be provided for various pathogenic fungi of plants and animals . AA posiA. fumigatus CYP51A confer resistance to ITC and PCZ, but retain susceptibility to VCZ. This is thought to be due to long-tailed azole-specific interactions with the mouth of the SEC [S. cerevisiae mutant strains carrying the following SDM mutations: G73E, G73R, and G73W, respectively [A. fumigatus CYP51A expressed in S. cerevisiae is an important limitation that emphasizes the need to obtain a full-length structure of A. fumigatus CYP51A [Some AA substitutions have been reported at glycine 73 (G73). The structurally aligned mutations G54E/R/W in  the SEC ,62. Sagaectively . The SDMectively . Despites CYP51A .A. fumigatus CYP51A Y121F, Candida species SDM Y132F, Cryptococcus neoformans SDM Y145F, Scedosporium apiospermum SDM Y136F, Ajellomyces capsulatus SDM Y136F, and mucormycete SDM Y129F. The same modification is frequently found in fungal phytopathogens, e.g., Blumeria graminis SDM Y136F and Parastagonospora nodorum SDM Y144F  in the CYP51 promoter. Although, T289A occurs in natural isolates, but does not itself confer azole resistance [CYP51A gene and increased production of SDM [The rise of VCZ-resistant isolates of pathogen ,80. Y121sistance . Sneldern of SDM . They aln of SDM . The abun of SDM ,81.A. fumigatus sensu stricto and occurs in other species of the section Fumigati. The Fumigati contains closely related species that are morphologically indistinguishable from A. fumigatus [A. lentulus and A. felis [A. lentulus, although the exact mechanism has yet to be elucidated. Minimal inhibitory concentration (MIC) levels are generally high for ITC (0.43–16 μg/mL), VCZ (3–7.5 μg/mL), and PCZ (0.12–2.0 μg/mL) [CYP51A gene. Deletion mutants of CYP51A (Δcyp51A) had lower MICs to ITC (0.03–0.06 μg/mL), VCZ (0.25 μg/mL), and PCZ (0.015 μg/mL). A. fumigatus and S. cerevisiae mutants, carrying the A. lentulus CYP51A gene, showed higher MICs to ITC (0.5–8.0 μg/mL), VCZ (1.0–4.0 μg/mL), and PCZ (0.12–0.25 μg/mL) [A. lentulus CYP51A to suggest that the lower susceptibility to VCZ of A. lentulus is due to decreased affinity between the azole and the SDM [A. felis has also a reduced susceptibility to VCZ and ITC, but the mechanism responsible has not been determined. The possibility of a Y121F mutation was suspected, but this idea requires further research [VCZ resistance is not restricted to umigatus . Among sA. felis . Reduced0 μg/mL) ,85. Expe5 μg/mL) . Alcazar the SDM . The rarresearch .Candida species are the most frequently diagnosed fungal pathogens in humans. They can cause a variety of diseases, ranging from superficial mucosal infections to deep-seated infections such as candidemia [C. albicans, followed by C. glabrata, C. tropicalis, C. krusei, and C. parapsilosis [C. auris. This species has recently evolved as a significant human pathogen. It was first isolated in 2007 as an azole-susceptible strain from an aural sample of a female Japanese patient [C. auris [C. auris strains have spread globally and they are now recognized by the CDC to be a serious global health threat [ndidemia . The mospsilosis ,87. A sp patient . The firC. auris . Since th threat .Candida infections is fluconazole, followed by the echinocandins [Candida species is a major concern. The majority of the Candida species, including C. albicans, are naturally fully susceptible to fluconazole. This contrasts with molds, which are all intrinsically resistant to fluconazole. Resistance against FLC and VCZ in Candida species has frequently been linked to the SDM Y132F/H mutations. These SDM mutations have been reported for species including C. albicans, C. tropicalis, and C. parapsilosis and they lead to both FLC and VCZ resistance.  increases VCZ and FLC MICs in this yeast while the Y132H gives an 8-fold increase [Globally, the antifungal agent most commonly used for the treatment of ocandins . Fluconaistance. . The SDMincrease .Candida parapsilosis has emerged as the most prevalent Candida species detected in South Africa (58–63%). In other regions, including Latin America, the United States of America, Asia, and Western Pacific, the prevalence of C. parapsilosis is much lower (1%–7.7%). A 2009–2010 study showed that approximately 80% of all C. parapsilosis strains are FLC resistant. The SDM Y132F mutant was found in 68% of all FLC-resistant strains [C. parapsilosis is alarming as FLC is the antifungal drug used most often in developing countries [C. parapsilosis is a commensal of the skin and, unlike other fungi, is easily transmitted by healthcare workers, resulting in major hospital outbreaks. Isolates causing such outbreaks tend to be more virulent than isolates causing sporadic cases [C. parapsilosis with the evolution of azole resistance over time. The FLC resistance can either be acquired due to the positive selective pressure in azole-rich environments or due to horizontal transfer from other individuals. Arastehfar et al. found that SDM Y132F and the SDM Y132F K143R combination occurred in clinical outbreak strains [C. parapsilosis strains were higher than for patients infected with FLC-susceptible strains [ strains . The incountries . C. paraic cases . In Turk strains . The out strains .Candida auris isolates. In the last five years, four phylogenetically clades of C. auris have been described that correlate with different geographical origins: the South Asian clade, South American clade, African clade, and the East Asian clade [Candida auris has revealed how antifungal resistance can evolve quickly in an emerging fungal pathogen. In less than two decades, and depending on the clade, up to 100% of Candida auris strains have become FLC resistant and 41.1% VCZ resistant [C. auris SDM are Y132F and K143R. The latter mutation will be discussed in C. auris allows for such a rapid resistance development.A major concern is the multidrug resistance of an clade . Each clan clade . The SDMan clade ,95. As tesistant ,96,97. TCryptococcus includes several human pathogens. The most common of these are Cr. neoformans and Cr. gattii. Cryptococcus neoformans causes meningoencephalitis, mostly in immunocompromised patients, such as AIDS patients [Cryptococcus grows as yeast in the host and the environment. In the human host, the fungus grows as a capsulated yeast. Similar to Candida, Cryptococcus is naturally fully susceptible to fluconazole. Fluconazole as a single drug therapy or combination with 5-fluorocytosine (5-FC) is a first line treatment for cryptococcosis [The genus patients ,98. Cryppatients ,98. Crypcoccosis . Fluconacoccosis .Cryptococci are basidiomycetes that cause major disease outbreaks around the globe. As these outbreaks are clonal, the increased incidence of fluconazole resistance in Cryptococcus populations is a major concern [Cryptococcus neoformans and Cr. gattii have innate heteroresistance to azoles, an adaptive mechanism involving duplication of chromosomes containing specific genes that confer azole tolerance [ concern ,101,102.olerance .Cyptococcus SDM Y145F mutation (SDM Y140F in S. cerevisiae) is one of the mutations that confers resistance to FLC and VCZ. A biochemical test of this relationship is needed for Cr. neoformans and Cr. gattii [Cr. neoformans strains has increased from 0–33% in 2001–2006 to 75–88% in 2011–2012 [Cryptococcus strains, MIC determinations should be standard practice [The . gattii . In Taiw011–2012 . In ordepractice .S. cerevisiae infections in humans are very rare. However, blood stream infections due to S. cerevisiae have been reported in COVID-19 patients [Saccharomyces cerevisiae is the fungus used most in food industries, e.g., fermented foods including wine, bread, chocolate, and beer [S. cerevisiae is also used to produce biofuel [patients  In the Wand beer . S. cere biofuel , dietary biofuel ,109, and biofuel .S. cerevisiae is one of the most widely used eukaryotic model organisms because it is easy to culture, genetically manipulate and study biochemically [S. cerevisiae has been used as a model to elucidate the impact of SDM ligand-binding site mutations in various pathogenic fungi [S. cerevisiae were from S. cerevisiae itself and then from C. albicans and C. glabrata. The high-resolution structures of wild-type and mutant SDMs in complex with substrates and inhibitors provide insight into associated phenotypes and biochemical changes and can now underpin structure-directed antifungal research and discovery [S. cerevisiae SDM has provided an effective surrogate to study mutations in the SDM of Candida species [In the life sciences, emically . S. cereic fungi . In ordeic fungi . The firiscovery . Thus fa species ,58.Scedosporium apiospermum is not well understood. To our knowledge, a single study has analyzed azole resistance patterns and correlated them with AA substitutions in the Sc. apiospermum SDM [Scedosporium species have two SDM-encoding homologs, namely, CYP51A and CYP51B. VCZ is the first line therapy for scedosporiosis [Scedosporium to azole drugs seems to be species specific. Sc. aurantiacum strains are often resistant to PCZ while the majority of strains in the Sc. apiospermum species complex are susceptible to PCZ, and with PCZ resistance more common than VCZ resistance [Sc. apiospermum strains harboring the SDM Y136F substitution in their CYP51A gene [Scedosporium species, further research is needed to elucidate its impact on resistance to short-tailed azoles.Azole resistance in rmum SDM . Similarporiosis . The sussistance . Bernhar51A gene . While tAjellomyces capsulatum, better known in medical settings as Histoplasma capsulatum, is the causative agent of histoplasmosis, the most frequent endemic mycosis in the United States of America [The dimorphic ascomycete  America . The dim America . ITC is  America . One thi America . Their F America .Rhizopus arrhizus followed by Lichtheimia corymbifera [The incidence of mucormycete infections appears to be increasing globally. Their incidence is under-reported as the disease is often misidentified because it is often hard to differentiate from other mold infections. Several mucormycetes cause severe infections in immunocompromised patients. The prevalence of individual species varies between countries. In Europe and Africa, the species reported most frequently as causing mucormycosis was ymbifera . Patientymbifera . While o: Blumeria graminis (Y136F), Mycosphaerella graminicola (Y137F), Parastagnospora nodorum (Y144F/H), Puccinia triticina (Y134F), and Uncinula necator (Y136F)  . The ass (Y136F) , so the  (Y136F) .Mycosphaerella graminicola (Zymoseptoria tritici) causes septoria leaf blotch in wheat, which causes crop losses of up to 50% [Mycosphaerella graminicola to TDM from the 1980s led to the acquisition of the SDM Y137F mutation. Its replacement with EPC and prothioconazole has resulted in the emergence of a range of alternative SDM mutations [p to 50% . SDIs arutations ,53.Blumeria graminis, the most common ascomycetous fungal pathogen infecting barley crops, causes a powdery mildew that reduces barley yields by 20–40% [Blumeria graminis, SDM Y136F has been found together with the S509T mutation [y 20–40% ,119. In mutation . Isolatemutation .Parastagonospora nodorum is an ascomycete and the causative agent of the global crop disease Stagnospora or Septoria nodorum leaf and glume blotch [Parastagonospora nodorum with a reduced susceptibility to propiconazole or TBC were found to carry either the SDM Y144F or Y144H mutations, respectively [e blotch . The ecoectively .Puccinia triticina is a basidiomycete that causes the brown rust or leaf rust. It is a common pathogen of wheat that causes up to 50% crop loss [A. fumigatus T289A may be involved.rop loss . The acqrop loss . An accoUncinula necator is an ascomycete that causes powdery mildew. It is the major fungal pathogen found in grapevines. Powdery mildew is controlled by extensive use of fungicides, including SDIs [ing SDIs . Its ecoing SDIs . SDM Y13ing SDIs , with ising SDIs .The impact of azole-resistant fungal pathogens in agriculture is an underrated problem. Most crops needed for the food supply are affected by fungal pathogens. Harvest losses caused by phytopathogens can be expected to cause food shortages for a steadily increasing human population. With extensive use of SDIs, resistance patterns that compromise their efficacy will continue to evolve. Novel antifungal agents are needed in human medicine and to guarantee efficient food production.S. cerevisiae SDM Y140F/H are, or have been, prevalent in fungal pathogens of medical or agricultural importance. We hypothesize that it represents a pan-fungal kingdom mutation that is readily induced by small azole antifungals reliant on interactions within the active site of SDM.Further research is needed to understand why the mutations structurally aligned with Candida species, including C. auris and C. tropicalis [The SDM K143R substitution is an acquired mutation in opicalis . It has opicalis . The subopicalis .C. auris has been associated with a K143R mutation that confers MIC ranging from 32 to 128 µg/mL for FLC and 0.25 to 2 µg/mL for VCZ [The high FLC and VCZ resistance of the emergent multidrug-resistant pathogen  for VCZ ,126.C. auris in hospital settings, especially in intensive care units, e.g., at Oxford University Hospital. Transmission was shown to be due to reusable axillary temperature probes. The testing of isolates according to the Sensititre YeastOne system found that 100% of the strains were resistant to FLC, 98% to VCZ, and 90% to PCZ, according to C. albicans breakpoints [There have been several outbreaks of akpoints . Other rakpoints  and Londakpoints .Escherichia coli of C. albicans SDM carrying the K143R mutation was reported to confer fluconazole resistance in vitro [Heterologous expression of in in vitro .A. fumigatus has two homologous copies of the SDM-encoding genes, namely, CYP51A and CYP51B. CYP51A is considered to be the workhorse of the ergosterol pathway. A recently discovered natural CYP51A I301 polymorphism was postulated to cause the FLC resistance of A. fumigatus. In the CYP51B homologue of A. fumigatus, T304 is structurally aligned with I301 [ith I301 . The I30ith I301 . CYP51A ith I301 .Candida species leads to fluconazole resistance [S. cerevisiae SDM mutants, they found T315A, which is equivalent to I301 in A. fumigatus CYP51A (see 2.5.1), reduces fluconazole susceptibility 4–5-fold. As this mutation is due to one single nucleotide polymorphism, it might be acquired easily in environmental and clinical settings [Lamb et al. postulated that the acquired SDM T315A mutation sistance . Using Ssettings .C. tropicalis G484D mutants [C. tropicalis SDM G464S or G464D affects the environment of the SDM heme group due to the nucleophilic serine or acidic aspartic acid side chains [C. albicans SDM G464S gave a 2- and 4-fold increase in FLC and VCZ MICs, respectively. The combination of SDM G464S and D278N further increased resistance to FLC and VCZ [The SDM G484S mutation has been linked with FLC resistance . Forasti mutants . It was e chains . C. albi and VCZ .S. cerevisiae models showed that resistance to FLC was conferred by G464H alone or in combination with other mutations. The combinations included Y132H and R467K or Y132H and H283R. The G464S Y132H combination showed a 32-fold increase in FLC resistance. As these amino acids are located at, the opposite site of the heme, crystal structures of S. cerevisiae SDM were used to show that the G464S hydroxyl replaces a water that normally creates a hydrogen bond with the heme ring D propionate [The use of opionate .Sc. apiospermum SDM G464S structurally aligns with the A. fumigatus CYP51A G448S. Mutations in G448 were reported to confer reduced susceptibility to VCZ, ITC, and PCZ, but further information on the binding of antifungal agents and its impact on azole resistance is needed [s needed .Cr. neoformans SDM G484S structurally aligns with S. cerevisiae SDM G464S. Its correlation with the development of azole resistance led to Cr. neoformans SDM G484 being suggested as a mutational hot spot [Cr. neoformans SDM G484S conferred FLC, but not to VCZ or ITC resistance [The hot spot . The Cr.sistance .Mycosphaerella graminicola SDM G460 mutations occur frequently. The M. graminicola SDM G460D mutation was first described in 1992. Strains with SDM G460 deletions (Δ460) were also found in Germany in 2004 [Mycosphaerella graminicola SDM Δ459 andΔ460 strains have been successfully engineered. These deletions and other AA substitutions in positions 459–461 are prevalent in several environmental strains with increased resistance to azoles. As G460 is part of the fungus-specific loop, early studies suggested it is involved with docking with TDM or prochloraz. The Y459 and G460 deletions were proposed to increase cavity volume and impact on resistance patterns [ in 2004 . Mycosphpatterns . It moreS. cerevisiae SDM Y140F/H are prevalent in many fungal pathogens of plants, animals, and humans. This type of modification appears to provide a pan-fungal kingdom resistance mechanism for fungi exposed to short-tailed azole drugs or agrochemicals. The mutation is found in basidiomycetes  ascomycetes  and mucormycetes . Contamination of soils and sweet- and salt-water bodies with azole antifungals, due to agricultural leaching and aquaculture makes it likely that comparable substitutions are likely to occur even in phylogenetically ancient groups of fungi such as chytridiomycetes and oomycetes. In ascomycetes and basidiomycetes, mutations equivalent to Y140F/H are acquired adaptations to azole drug exposure [Mutations or substitutions structurally aligned to exposure ,132. Theexposure . The mutAspergillus) was postulated to confer resistance to the short-tailed azole VCZ  or acquired mutation  and strains causing clinical outbreaks have been found to carry such mutations [Aspergillus, this residue does not seem to play a role in azole resistance.Other ligand-binding pocket associated substitutions detected less frequently are known to be involved in acquired or intrinsic resistance but have not been found in such a diverse range of fungal pathogens. SDM K143R/Q mutations have been described as single substitutions or occurring with Y132F. Thus far, this mutation has only been described in  species ,124. In utations . In moldA. fumigatus appears to be mediated via SDM 301I [Candida albicans carries the structurally aligned polar AA T315. This residue is too distant to affect the binding of FLC directly and it sidechain hydrogen bond is instead thought to increase the flexibility of helix I. The non-polar I301 in A. fumigatus SDM cannot form this hydrogen bond and will therefore expected to stiffened helix I. Whether this AA substitution mediates FLC resistance in other intrinsically FLC-resistant molds remains to be determined.Intrinsic fluconazole resistance in SDM 301I . This fiS. cerevisiae SDM G73 and G464 are infrequent. A more comprehensive picture of the relevance of all the ligand binding site substitutions and their impact on long- and short-tailed azole resistance requires further structural and functional analysis.Mutations structurally aligned with S. cerevisiae SDM Y140, K151, and V311 we have described represent the tip of an iceberg across the fungal kingdom. Fungi occur in almost all known ecological niches. Most of these niches can be contaminated with azoles, but their impact is poorly understood. While it is clear that there is positive selection pressure for azole-resistant strains and species, how the complex composition of fungal communities in different niches is affected is not known. Current reports of SDM binding site mutations are limited to a few fungal pathogens of plants and animals. The effects of azole contamination that does not directly affect commercial production or human health are not known, e.g., how is fungal biodiversity affected in azole contaminated water and soils?We are confident that AA mutations/substitutions structurally aligned with CYP51A mutations may encroach into clinical settings [Candida species or isolates with acquired azole-resistance in patients’ mycobiomes. The use of cosmetics and personal hygiene products containing azole antifungals may aid colonization of hair and skin with azole-resistant Candida species [Fungi with acquired azole resistance are likely to have a fitness advantage in azole-contaminated environments ,136,137.settings ,77. Azol species . While l species .Candida and Aspergillus, respectively, has created azole-rich niches in patient cohorts. Because mucormycetes are intrinsically resistant to FLC, echinocandins and VCZ, high-risk patients receiving antifungal prophylaxis with any of these three agents can provide a suitable niche for these fungi. The presence of SDIs in agricultural soils and on food might contribute to significant exposure of these patient cohorts. For example, contamination of flour and bakeries with azole-resistant aspergilli and mucormycetes has recently been found and the associated risk of exposure to bakers of these azole-resistant fungi discussed [High prevalence of mucormycetes in homes and workspaces  has been confirmed . Selectiiscussed . Professiscussed . Furtheriscussed ,77.We need to limit the use of azole drugs and agrochemicals by applying them more judiciously. A key aim should be to reduce azole contamination of the environment and in food. Ideally, azoles should be used for very specific applications and a strict structural separation of azole inhibitors used in agriculture and medicine is required. The wide availability of azoles favors the emergence of azole-resistant fungi. Within a few decades, azole-resistance may become so wide spread that the utility of this valuable substance class will be lost to both agriculture and medicine. By understanding the molecular basis of azole binding to its target, it should now be possible to design azole inhibitors that meet these challenges.New antifungal compounds are urgently needed due to the emergence of azole- and multidrug-resistant fungi. Understanding the mechanism of acquired and intrinsic azole resistance in a wide range of fungal pathogens is therefore highly relevant to the field of antifungal drug discovery.The linkage of azole resistance in agriculture and medicine is a major problem that needs to be addressed. Two major concerns are: (1) the exposure of people to azole drugs via azole-contaminated food products and subsequently the selection pressure on the human mycobiome, and (2) the selection for and evolution of azole-resistant fungal pathogens in the environment. Azole resistance in the medical setting is particularly troublesome since only three major substances classes are available for the treatment of systemic fungal infections . Some fungal pathogens of humans have intrinsic resistance against one or multiple drug classes, narrowing treatment options. The addition of acquired azole resistance can often make combination therapies the only treatment option.We suggest that drugs used in the clinical setting need to differ structurally from those used in agriculture, if they act via the same cellular mechanism. For example, should long-tailed azoles be reserved for medical applications and not developed as agrochemicals? The alternative is to focus on the development of separate drugs and agrochemicals that attack different cellular targets in order to overcome the cross-habitat resistance found throughout the fungal kingdom.The knowledge gained from understanding how key AA changes in the ligand-binding pocket of SDMs affect substrate and inhibitor binding can now be used to design a new generation of azole drugs that are more potent and resistant to fungal adaptation via the known mechanisms. Stronger binding could be achieved by modulating key interactions of these ligands with the active site and the substrate entry channel of SDMs.


Leishmania donovani with limited molecular validation. In order to molecularly characterize the causative parasite of the cutaneous disease, parasite specific Internal-Transcribed Spacer 1 (ITS1) PCR RFLP and sequence analysis was performed on skin lesional biopsies from cutaneous leishmaniasis patients. Interestingly, we found the presence of Leptomonas seymouri in 38.5% (22/57) of the patients along with L. donovani detected in all the samples. L. seymouri is a monoxenous insect trypanosomatid, generally incapable of infecting humans. In recent years, the parasite is also reported to co-infect humans with L. donovani in visceral and post kala-azar dermal leishmaniasis (PKDL) cases prevalent in northeastern India. The finding of L. seymouri-L. donovani co-infection in unusual cutaneous cases from Himachal Pradesh is the first ever to our knowledge and imply a newer disease paradigm. There is an urgent need to understand the biology of Leptomonas co-infection with L. donovani and its possible role in visceral and/or dermotropic disease outcome. Importantly, L. seymouri co-infection in cutaneous cases and previously reported visceral and PKDL cases needs to be recognized as a newer phenomenon by the leishmaniasis surveillance program in India.Himachal Pradesh in India is a newer endemic state with co-existence of cutaneous and visceral leishmaniasis. The cutaneous leishmaniasis cases are on an increase in the region and reported to be unusually caused by  Leishmania parasite with a digenetic life cycle in the sandfly vector and the mammalian host. In India, visceral leishmaniasis (VL) caused by L. donovani predominates in the northeast belt in the state of Bihar, West Bengal, Uttar Pradesh, and Jharkhand with fewer cutaneous leishmaniasis (CL) cases by L. tropica in the hot arid western region of the Thar Desert in Rajasthan  caused by L. donovani variants (unpublished data). Classically, Leptomonas spp. comprise insect parasite with a monoxenous life cycle and are considered non-pathogenic to humans. Lately, adaption of the parasite to dixenous life cycle has been reported under specific conditions such that L. seymouri can exist as a co-infectant with other pathogens in immunocompromised subjects , Shimla and Mahatma Gandhi Medical Services Complex (MGMSC) Khaneri, Rampur, Shimla at the time of diagnosis. Written informed consent was obtained from all the patients. The study design was approved by the Institutional Ethics Committee IGMC, Shimla, Himachal Pradesh, Approval no. HFW(MS)G-5(Ethics)/2014-10886 and Central University of Punjab, Approval no. CUPB/IEC/2016/034.Lesional biopsy samples were processed for parasite detection using Giemsa stained touch smears, Hematoxylin and Eosin (H & E) stained paraffin-embedded tissue sections as per standard protocol  isolation from patient samples and laboratory-grown L. donovani  promastigote culture was done using standard protocol  culture used as a positive control. HaeIII restriction enzyme digestion of the amplified ITS1 region between the small subunit (ssu) rRNA and 5.8S rRNA DNA provides a Leishmania spp. specific RFLP pattern  using BLAST analysis while the sequences corresponding to ~400 bp band, showed maximum identity with the standard L. seymouri ITS1 sequences with accession numbers KP717899, EU623433, and JN84880 . All the sequences corresponding to ~320 bp band and ~400 bp band were deposited with their accession numbers in Genbank . ITS1 se JN84880  . Nine out of 11 CL test samples also clustered closely with the standard L. seymouri isolates from India with respect to the ~400 bp sequence represented by HPCL_L. seymouri series  from the CL samples and the ITS1 sequences representing standard d method . Phylogeectively . All thei series , Table 1i series , Table 1L. donovani, ~320 bp and HP L. seymouri, ~400 bp ITS1 sequences  along with the ~320 bp ITS1 sequences of standard L. donovani isolates from India (AJ634376), Sri Lanka (AM901447), Bhutan (JQ730001) and ~400 bp ITS1 sequences of standard L. seymouri isolates from India  exhibited distinct differences in ITS1 sequences specific to L. donovani and L. seymouri. Additional nucleotide patches unique to L. seymouri ITS1 sequence in comparison to L. donovani ITS1 region were apparent in the sequence alignment shown in ITS1 DNA region is highly conserved among different trypanosomatid parasite species  of the CL cases caused by L. donovani. This is the first-ever report of L. seymouri co-infection in CL patients, typed with unusual cutaneous manifestation by L. donovani with no visceral features, detailed in a recent communication from our laboratory (unpublished data).Detection of the ITS1 sequences corresponding to the two bands in clinical specimens clearly exhibited the presence of L. seymouri as a L. donovani co-infectant in leishmaniasis patients from HP, a newer endemic region in India in line with earlier reports of L. seymouri co-infection in VL and PKDL patients from the northeastern VL zone  can be found in the article/The studies involving human participants were reviewed and approved by Institutional Ethics Committee IGMC, Shimla, Himachal Pradesh, Approval no. HFW(MS)G-5(Ethics)/2014-10886 and Central University of Punjab, Approval no. CUPB/IEC/2016/034. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.LT collected the samples, performed the experiments, and analyzed the data. HK constructed the phylogenetic tree. AN provided the patient samples. AJ and MJ contributed in concept and design of study, data analysis, drafting of article, critical revision, and final approval of manuscript. 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.


Sex biases in the genome-wide distribution of DNA methylation and gene expression levels are some of the manifestations of sexual dimorphism in mammals. To advance our understanding of the mechanisms that contribute to sex biases in DNA methylation and gene expression, we conducted whole genome bisulfite sequencing (WGBS) as well as RNA-seq on liver samples from mice with different combinations of sex phenotype and sex-chromosome complement. We compared groups of animals with different sex phenotypes, but the same genetic sexes, and vice versa, same sex phenotypes, but different sex-chromosome complements. We also compared sex-biased DNA methylation in mouse and human livers. Our data show that sex phenotype, X-chromosome dosage, and the presence of Y chromosome shape the differences in DNA methylation between males and females. We also demonstrate that sex bias in autosomal methylation is associated with sex bias in gene expression, whereas X-chromosome dosage-dependent methylation differences are not, as expected for a dosage-compensation mechanism. Furthermore, we find partial conservation between the repertoires of mouse and human genes that are associated with sex-biased methylation, an indication that gene function is likely to be an important factor in this phenomenon. Mammalian males and females carry different sex-chromosome complements ; produce different levels of sex hormones; have different anatomy and physiology; and, in humans, have different risks for developing certain diseases. Gene expression is a major player in defining cell phenotype and is regulated by epigenetic factors, such as DNA methylation and chromatin organization. Hence, it is logical to hypothesize that the difference between the male and female cells results from differences in gene expression, which are accompanied by differences between the male and female cell epigenomes.In both mice and humans, the expression levels of a subset of genes vary between males and females in somatic cells ,2,3,4,5.DNA methylation or chromatin modifications are essential regulatory layers involved in the orchestration of gene expression. They also contribute to the sex-biased gene expression and, thereby, sexual dimorphism in phenotypes. Indeed, in humans, global levels of DNA methylation and methylation of repetitive elements, in particular, are higher in males ,9,10. InTo better understand how sex-biased DNA methylation patterns arise and whether they are associated with sex-biased expression, we undertook a survey of DNA methylation and gene expression in the liver of adult mice with different combinations of sex-chromosome complement and phenotypic sex: XY males (XY.M), XX females (XX.F), females with monosomy X (XO.F), and sex reversed XY females (XY.F) . We genePaf mice (referred to as Paf from this point on) were generated by backcrossing C3H/HeSn-Paf/J carriers of the patchy fur (Paf) mutation purchased from the Jackson Laboratory to C57BL/6J mice . Males that carry the Paf mutation were identified based on their hair loss phenotype and crossed to C57BL/6J females. Female offspring from these crosses were genotyped using reverse transcription followed by PCR (RT-PCR) for the Xist gene, which is expressed in XX females, but not in XO females  were considered and only tiles with at least one CpG site were used. Differentially methylated tiles were detected with ) method , and witOne thousand nanograms of DNA per sample was treated with sodium bisulfite using EpiTect Bisulfite Kit . Primers for sex-associated differentially methylated regions (sDMRs) (50–70 bp) were designed using the PyroMark Assay Design 2.0 Software . The list of primers is provided in https://www.encodeproject.org/files/ENCFF580WIS) that had predicted 15 chromatin states of liver genome of male mice (strain C57BL/6J) with chromHMM [Basic annotations, including CpG island (CGI) annotation, genic annotation, and chromatin state estimation, of sDMR or CpG sites were performed for sDMRs from the four comparisons and one background group containing all CpG sites  with the R package annotatr (v1.12.1) . The CpGchromHMM . The 15 p < 5 × 10−3).sDMRs were annotated with UCSC track RepeatMasker ,45,46 foThe genomic coordinates of all sDMRs were then used to detect enrichments of previously known transcription factor binding sites with Homer (v.4.9.1) , applyinTotal RNA was isolated using TRIzol Reagent  according to the manufacturer’s instructions and followed by purification using the RNeasy MinElute Cleanup Kit . Library preparation and RNA-sequencing (RNA-seq) were performed at the McGill University and Genome Quebec Innovation Centre. Paired-end sequencing using an Illumina NovaSeq 6000S4 sequencer was distributed across two lanes in two sequencing runs. Each lane had a mix of sequencing libraries to avoid batch effects. Once demultiplexed, each sample had a minimum of 100 million raw paired reads of 100 bp each.RNA-seq” pipeline [Differential gene expression analysis was performed using the GenPipes “pipeline . Brieflypipeline . The abupipeline . PC analpipeline  and Edgepipeline  packagesp-value below 0.05) was used to calculate the distance to the closest sDMR, using BEDtools (v. 2.26.0) [Additionally, differential transcript expression was estimated using the Kallisto–Sleuth pipeline ,54. Brie 2.26.0) .The proximal genes of genomic loci/regions (sDMC or sDMR) were identified by annotatr (v1.12.1) , where tAssociated genes for intergenic sDMR were not included because the confidence level of identifying true sDMR-associated genes was low and inconsistent with that used for identifying sDMC-proximal or sDMR-proximal genes for sDMC or sDMR overlapping with genic regions.phyper function in the R package stats (v.3.6.1) [sDMR-proximal genes were identified for each sDMR using a distance cutoff of up to 5 Kb upstream of TSS (genic region + 5 kb upstream of TSS). For each comparison (XX.F vs. XY.M and XY.F vs. XY.M), we evaluated the enrichment of DEG in the sDMR-proximal genes using a hypergeometric test with v.3.6.1) ,57.For the overlaps between sDMR-proximal genes and DEG, we also tested the association between the methylation level of sDMR  and expression levels of DEG  for both XX.F versus XY.M and XY.F versus XY.M comparisons. Chi-square test was applied in testing the significance of higher/lower expression levels of DEG being associated with enrichments of sDMRs with lower/higher methylation.http://www.informatics.jax.org).The list of 15,779 orthologous genes  of human and mouse was retrieved from the vertebrate homology section on the Mouse Genome Informatics (MGI)  (q < 0.05), 2582 on the X chromosome and 4192 on autosomes, were used for our analysis of sDMC-proximal genes.The human high-density lipoprotein (HDL) dataset  was geneThe Turner dataset  focused phyper function in the R package stats (v.3.6.1) [We identified sDMR-proximal genes for sDMR identified in the XX.F versus XY.M comparison of our mouse dataset and sDMC-proximal genes for sDMC from the human HDL dataset. For the Turner dataset, we extracted genes associated with sDMC. We focused on the orthologous genes between humans and mice for all three datasets. We inspected the overlaps of genes between human and mouse datasets. The significance of overlaps was tested with the hypergeometric test with v.3.6.1) ,57.Paf) (Methods). TIR mice carry a sex determining region of Chr Y (Sry) gene variant, which is, on the C57BL/6J genetic background, inefficient to upregulate its target Sry-box transcription factor 9 (Sox9) gene that is necessary for initiating testicular differentiation [TIR mice developing bilateral ovaries with female phenotype, and the rest developing bilateral testes with male phenotype or unilateral ovary and contralateral testis with intersex external genitalia (true hermaphrodites) [Paf mice carry a Paf mutation on the X chromosome. The mutation is located within the boundary of the pseudoautosomal region and interferes with segregation of the X and Y chromosomes [PafY males are XO females .To dissect the impacts of sex phenotype and sex-chromosome complement on DNA methylation, we used crosses from two strains of mice that show high rates of sex reversal (TIR) or X chromosome monosomy  ,61. The omosomes ,63,64. T females . In our Paf females (XXPaf.F) from the Paf cross , XY males (XY.M), and sex-reversed XY females (XY.F) from the TIR cross and XO females (XO.F) and XXaf cross , Methodsckground . To minickground . Extremeckground .Paf cross (XO.F and XXPaf.F), (ii) females from the TIR cross (XX.F and XY.F), and (iii) males (XY.M)  and mice with one X . It is worth noting that males (XY.M) tend to cluster separately from females with one X . When plotting the top two principal components (PCs) for autosomes, explaining 76.3% of the variance, samples clustered into three main groups: (i) females from the s (XY.M) a. The feth one X c. The efth one X c,d.Paf.F versus XO.F. To minimize the impact of the genetic background on our ability to detect sex-biased DNA methylation, we used the three experimental groups from the TIR cross as the main discovery set and conducted comparisons between XXPaf.F and XO.F separately. For each comparison, we detected sDMC using the DSS tool  [Paf.F versus XO.F, identified fewer sDMC on autosomes, but more on the X chromosome . The highest number of sDMC was identified in the comparison between groups with both different sex phenotypes and sex-chromosome complements, XX.F versus XY.M .To identify the CpGs with methylation levels that depended on sex phenotype and/or sex-chromosome complement ), we compared CpG methylation levels in four comparisons: XX.F versus XY.M, XX.F versus XY.F, XY.F versus XY.M, and XXMethods) . The comPaf.F vs. XO.F) are controlled by the number of X chromosomes. The comparison XX.F versus XY.M had 7595 unique sDMC  not found in other comparisons. Further, 2737 of the XX.F versus XY.M sDMC  were also found in the XY.F versus XY.M comparison (different sex phenotypes), whereas 6756  of the sDMC from the XX.F versus XY.M were found in the XX.F versus XY.F comparison (different sex-chromosome complement). A total of 816 sDMC (15 on autosomes and 801 on the X chromosome) overlapped between the XX.F versus XY.M and XXPaf.F versus XO.F comparisons. A total of 2984 sDMC (2 on autosomes and 2982 on the X chromosome) were shared across the three comparisons with different X-chromosome dosage .To isolate the effects of sex phenotype and sex-chromosome complement on sex-biased DNA methylation levels, we identified overlaps between sDMC across comparisons for all chromosomes as well as for autosomes only b. The exX-chromosome dosage is associated with X-inactivation and different methylation levels on the active and inactive X chromosomes. To separate the effect of X-inactivation on X-linked loci from other mechanisms, we analyzed autosomal and X-linked sDMC separately  (Aldh3b3) (Caprin1 and Ch6qA1) (Snrpn) was used as a control, where 50% methylation independent of the sex or sex-chromosome complement was expected (n = 4–8 per group). We observed the expected hypermethylation of the Xist and hypomethylation of the Pgk1 promoters in mice with a single X, and the opposite patterns in mice with two X chromosomes. Interestingly, several autosomal sDMRs identified by one of the packages (Comt and Bcl6) or only one of the comparisons (Ergic1 and Esr1) were confirmed by pyrosequencing as belonging to the sex-phenotype associated category  a and oneAldh3b3) b. We als Ch6qA1) c and two Ch6qA1) d. Finallexpected e. We percategory a. In the females c.Paf.F vs. XO.F), fewer sDMR were detected on autosomes in total  than on the X chromosome . The comparison XX.F versus XY.M, with both different sex phenotypes and sex-chromosome complements, identified multiple sDMRs on both autosomes and X chromosome . Similar to the trends observed in sDMC, males showed lower methylation levels in the majority of autosomal sDMRs in comparisons between groups with different sex phenotypes (2521/2853 in XX.F vs. XY.M and 3414/3847 XY.F vs. XY.M), while nearly half of the autosomal sDMR showed lower methylation in mice with a single X in comparisons between groups with the same sex phenotype, but different sex-chromosome complements (784/1563 in XX.F vs. XY.F and 379/650 in XXPaf.F vs. XO.F).The overall distribution of sDMRs across chromosomes  resemblePaf.F versus XO.F comparison showed higher percentages of autosomal sDMR in CpG islands, but lower percentage in CpG shelves compared with all other groups. While on the X chromosome, all four comparisons showed higher percentages of sDMR in CpG islands and CpG shelves compared with the background group, with the XXPaf.F versus XO.F and XY.F versus XY.M comparisons having the highest and lowest percentage, respectively  the distributions of CpG islands (CGI), genic regions, and chromatin states; (2) repetitive elements; and (3) DNA motifs (Methods). To measure enrichment in any category, we contrasted our findings to a set of random regions extracted from all CpG sites referred to as the background group. More than 70% of autosomal sDMRs were found to overlap with intergenic (inter-CpG) regions in all four comparisons, as well as in the background group . The XXPectively . When inectively . To estiound set . On the ound set .Paf.F vs. XO.F) (Paf.F versus XO.F comparisons. One specific subfamily (IAPEz-int) of ERVK showed over-representation in all comparisons between groups with different sex-chromosome complements. In contrast to autosomal sDMRs, sDMRs on the X chromosome showed less repeat enrichment  with a clear separate cluster of phenotypic males  in the comparison XX.F versus XY.M, 207 DEGs in XY.F versus XY.M, 14 DEGs in XX.F versus XY.F, and 2 DEGs in XXPaf.F versus XO.F (Paf.F vs. XO.F and XX.F vs. XY.F), which is consistent with the relative numbers of sDMR detected. Larger portions of DEGs showed lower expression levels on chromosome 5, 7, 10, 12, 15, 19, and X in males in the XX.F versus XY.M and XY.F versus XY.M comparisons, while DEGs on Y chromosomes all were detected only in Y chromosome carriers in both XX.F versus XY.M and XX.F versus XY.F comparisons . For the XY.F versus XY.M comparison, 51 (out of 207) DEGs overlapped with sDMR-proximal genes  (p < 2.511 × 10−11). In XY.M of the XY.F versus XY.M comparison, 37 genes showed lower methylation and higher expression, 9 genes showed higher methylation and lower expressions, 2 genes showed lower methylation and lower expression, and no gene showed higher methylation and higher expression . While intergenic and distant regulatory DNA elements have a significant impact on transcriptional regulation, the confidence of identifying proximal genes for intergenic sDMR was low. Therefore, the association between intergenic sDMR and sex-biased transcription was not investigated using the hypergeometric test or the Chi-square test.For the two comparisons that had a sufficient number of DEGs (XX.F vs. XY.M and XY.F vs. XY.M), we checked the cumulative distribution of distances between their TSS and the nearest sDMR (Methods). We found that sDMRs were particularly enriched around the TSS of transcripts that were significantly over-expressed in phenotypic males (XY.M) . The XX.× 10−16) . SpecifiPaf.F versus XO.F comparison  as biologically the most relevant for comparing to the human HDL and Turner datasets, respectively.To determine the functionality of sex-biased DNA methylation found in our crosses, we compared our mouse liver dataset with two publicly available human DNA methylation datasets. The expectation was that functionally important sex bias in DNA methylation would be conserved in humans and associated with orthologous genic regions in a tissue-specific fashion. Therefore, we used genes as an anchor point in our analyses. The human HDL dataset explored sex differences in the liver methylome . The humPaf.F versus XO.F genes were compared to the Turner dataset  (p < 5.18 × 10−5)  (p < 5.74 × 10−7)  a. Among  × 10−5) a. Among  < 0.21) b. Among  × 10−7) b. Taken In this study, we used two tools to identify regions with different methylation levels: DSS for identifying sDMC and both methylkit and DSS for identifying sDMR. Here, we discuss some caveats that require caution in interpreting the sDMC and sDMR results. To estimate DMC, statistical significances of millions of CpG sites having different methylation levels between groups were assessed independently. In contrast to the large number of statistical tests needed, the sequencing coverage and numbers of samples sequenced for each group is limited by the cost of WGBS. One common practice in response to such a challenge is to leverage the strong spatial correlation of methylation levels among CpG sites nearby. By default, DSS applied a 500 bp smoothing window to allow CpG sites with insufficient sequencing coverage (depth), borrowing information from nearby CpG sites with higher coverages. By nature, the choice of the smoothing window size is one source of selection bias. Estimation of sDMR is a better compromise by reducing the degrees of freedom . DSS and methylKit have distinct approaches in defining the boundaries of DMR. DSS uses CpG sites as candidate boundaries and merges nearby sDMC/sDMR, while methylKit uses pre-defined tiles for grouping spatially close CpG. The selection of a sweet spot for merging nearby sDMC/sDMR in DSS and the size of tiles applied in methylKit are hence also sources of potential selection bias for sDMR identification. For instance, the selection of the size of pre-defined tiles can dramatically influence the number of resulting sDMR. The threshold of methylation differences is another parameter that can be tweaked in detecting sDMR. Therefore, we used a 20% threshold for methylation differences, which has been used by other groups in their studies of sex-biased methylation in mice ,71.In our study, validation experiments conducted on a small subset of sDMR found no false positive results. However, several results were false negative. Indeed, a number of sDMRs identified in one of the comparisons were validated by pyrosequencing in other comparisons despite the negative result of the WGBS analysis . This haSexual dimorphism in mammalian phenotypes and gene expression levels are often attributed to the action of gonadal sex hormones . Howeversex-phenotype dependent sDMRs promote sex differences in gene expression for autosomal genes represented by equal number of alleles in both sexes, whereas most X-linked sDMR reflect dosage compensation for X-linked genes that attenuates rather than amplifies sex differences in expression, an observation consistent with current understanding of the function of X-inactivation [We find thousands of autosomal and only 146 X-linked sDMR whose methylation depends on the sex phenotype of the mouse. These findings are consistent with other studies of sex-biased DNA methylation in mouse liver implicating testosterone as the strongest influence on autosomal DNA methylation and X-chromosome inactivation as the major influence on the methylation patterns of X-linked loci ,71,76. Ftivation ,78.The Y-chromosome dependent DMC group consisted of 6643 X-linked, 113 autosomal, and 2 Y-linked sDMC. The caveat here is that the tools used to detect DMC have a number of limitations, as discussed above, and further analyses and validation are necessary to better understand the underlying mechanism. Indeed, the mouse Y chromosome harbors several interesting gene candidates encoding proteins involved in transcriptional regulation or chromatin remodeling that are also expressed in the mouse liver.Thus, the ensemble of our data and data from other studies shows that, in liver, the sex phenotype is the strongest influence on sex differences in autosomal gene expression and DNA methylation levels with the largest repertoire of target genes on autosomes. Moreover, our data for mouse liver provide little support for the role of epigenetic memory of the embryonic states as a major contributor to the sex bias in autosomal DNA methylation . In contPaf cross, portions of the C3H genome that were present on a largely C57BL/6J background led to increase in inter-individual variation in DNA methylation that was most pronounced in large regions of chromosomes 1, 5, and 10. Moreover, filtering of SNPs has not completely alleviated genetic influences on methylation, which suggests, as expected, that there is more to genetic variation than SNPs  and methylcytosine dioxygenase ten-eleven translocation (TET) proteins are critical for the establishment, maintenance, or erasure of DNA methylation in different cell types and different developmental stages ,87. Highth males ,89. MoreTranscription factor motif analysis of sex-phenotype dependent autosomal sDMRs found enrichment of binding sites for transcription factors CUX2 and HNF6 that have sex-biased expression in the mouse liver, in agreement with findings reported by other groups . HoweverMotif analysis of X-dosage dependent X-linked sDMR shows little motif enrichment. The one comparison with same genotype, but different sex phenotypes and no X-inactivation, XY.F versus XY.M, shows enrichment for the same factors that are detected in sex-phenotype dependent autosomal sDMR. The caveat here is that only 146 sex-phenotype dependent sDMRs were identified on the X compared with ~1500 X-dosage dependent sDMRs. Therefore, the striking difference between XY.F versus XY.M and the three other comparisons should be taken with caution.Sex bias in gene expression or DNA methylation in somatic cells has been reported in different vertebrate species, including fish, reptiles, birds, and mammals. In mammals, with most data coming from mouse and human studies, sex bias in methylation is usually associated with sex-biased gene expression ,15, and Paf.F versus XO.F comparison may be the result of cell-type specificity of sex-biased methylation as different cell types were analyzed in humans and mice.Here, we compared sex-biased DNA methylation in mouse and human livers and found a significant overlap between sDMR-proximal orthologous genes. This suggests that the mechanisms underlying sex-biased DNA methylation are likely associated with gene function and cell type and are at least partially conserved between humans and mice. The enrichment of X-linked sDMR-proximal genes overlapping in mice and humans is an expected outcome as both mammalian species have X-chromosome inactivation. The lack of significant overlap between autosomal genes identified in the Turner dataset and those identified in the mouse XXThe relationship between sex-biased methylation, sex chromosome evolution, sex-biased expression, and sexually dimorphic phenotypes becomes more complex in species that are evolutionarily more distant or where sex determination is modulated by environmental conditions, such as certain species of fish and reptiles ,99,100.


Antheraea frithi mitogenome was sequenced to understand its phylogenetic and evolutionary relationship. It comprises 22 tRNAs, 13 protein-coding genes, two rRNAs and a AT rich region. The arrangement of mitogenome gene follows the pattern of dytrysian lepidoptera. rrnL gene consists of two unique long consensus repeats copy. tRNAs like structure of trnL and trnP are present in rrnL gene and AT rich region, respectively. The phylogenetic analysis by maximum-likelihood method showed that A. frithi was clustered with A. pernyi and A. yamamai, the commercialized wild silkworms. Antheraea frithi, the oak tasar silkworm, is an unexplored wild sericigenous insect that showed commercial potentiality in Manipur. Proper biological and genetic characterizations of A. frithi are essential for its successful conservation and commercialization. The complete mitochondrial genome of A. frithi was determined to examine its comprehensive evolutionary relationship and maternal inheritance among sericigenous groups.A. frithi were collected from Phaibung (1200 m ASL), Senapati district , Manipur, India. Taxonomically identified A. frithi was stored in the IBP, IBSD under the number WS/IBSD/008. The silk gland was used for extraction of mitochondrial DNA (mtDNA). mtDNA was extracted following the protocol of Clark and Nicklas (http://lowelab.ucsc.edu/tRNAscan-SE/). Phylogenetic trees based on maximum likelihood analysis were constructed on MEGA 5.1 Live samples of  Nicklas  with sliA. frithi (GenBank KJ740437) was 15,338 bp in length. It contains typical composition of 37 genes found almost in mitochondrial genomes of insects, having 13 protein coding genes (PCGs), 22 tRNA genes, two subunit of rrnS and rrnL, and AT-rich region. The mitogenome follows orientation and gene order of ditrysian species of Lepidoptera   and minimum in the cox1 gene (72.15%). Antheraea frithi has high AT% (89.19%) in the control region as similar with Artogeia melete (89.17%)  stretches at the 18 bp downstream of rrnS gene. trnP-like structure having proper anticodon but partial DHU loop is also detected.Mitogenome of A. frithi, built from the 13 PCGs of mitogenomes shows clustering of A. pernyi and A. yamamai in one distinct clade. Within Bombycoidea superfamily, Bombycidae, Saturniidae and Sphingoidae designated a cluster following the traditional morphology and molecular based classification (Liu et al. The phylogenetic tree of


Rickettsia and their arthropod hosts from entomological surveillance. Methods: During 2015–2016, we screened 1825 samples of rodents and ectoparasites collected from rodents and domestic mammals  across Thailand for Rickettsia. The citrate synthase gene was amplified to identify Rickettsia to species, while the Cytochrome Oxidase subunit I (COI) and subunit II (COII) genes were used as target genes for ectoparasite identification. All target gene amplicons were pooled for library preparation and sequenced with Illumina MiSeq platform. Result: The highest percentage of Rickettsia DNA was observed in fleas collected from domestic animals (56%) predominantly dogs. Only a few samples of ticks from domestic animals, rodent fleas, and rodent tissue were positive for Rickettisia DNA. NGS based characterization of Rickettsia by host identified Rickettsia asembonensis as the most common bacteria in positive fleas collected from dogs (83.2%) while “Candidatus Rickettsia senegalensis” was detected in only 16.8% of Rickettsia positive dog fleas. Sequence analysis of COI and COII revealed that almost all fleas collected from dogs were Ctenocephalides felis orientis. Other Rickettsia species were detected by NGS including Rickettsia heilongjiangensis from two Haemaphysalis hystricis ticks, and Rickettsia typhi in two rodent tissue samples. Conclusion: This study demonstrates the utility of NGS for high-throughput sequencing in the species characterization/identification of bacteria and ectoparasite for entomological surveillance of rickettsiae. A high percentage of C. f. orientis are positive for R. asembonensis. In addition, our findings indicate there is a risk of tick-borne Spotted Fever Group rickettsiosis, and flea-borne murine typhus transmission in Tak and Phangnga provinces of Thailand.Background: Next generation sequencing (NGS) technology has been used for a wide range of epidemiological and surveillance studies. Here, we used amplicon-based NGS to species identify  Rickettsia is comprised of obligate intracellular Gram-negative organisms that cause diseases in people throughout the world. Rickettsioses are zoonotic diseases that can be transmitted to human by ectoparasites including fleas, ticks, mites, and lice. Mammals including rodents, domestic animals, and wildlife species have been recognized as reservoirs of rickettsioses. Currently there are 30 identified Rickettsia species [Rickettsia are classically characterized into two groups, the spotted fever group  which cause the tick-borne spotted fever group (SFG) rickettsioses, and typhus group  which cause louse-borne epidemic typhus, and flea-borne murine or endemic typhus, respectively. There are also some pathogenic rickettsial species classified into the transitional group, as they share features between SFG and typhus group. The transitional rickettsiae includes Rickettsia akari, Rickettsia australis, Rickettsia felis which are causative agents of mite-borne rickettsialpox, Queensland tick typhus, and flea-borne spotted fever, respectively [The genus  species , and som species . Rickettectively .Rickettsia spp. worldwide is defined by a combination of climate, the geographical distribution of the primary vector and distribution of the natural animal hosts required to amplify and maintain its presence in the environment. Most SFG Rickettsia are distributed in the limited geographic regions where their specific tick host species is found [R. rickettsii, is distributed throughout the Americas, and Dermacentor ticks serve as the primary vector for RMSF in southeast and south central United States [Amblyomma cajennense sensu lato species complex transmit RMSF in central and South America [Rhipicephalus sanguineus sensu lato, which commonly infests dogs, has been implicated in an RMSF outbreak in the tribal lands of Arizona and in Sonora, Mexico [Rickettsia conorii, is distributed across southern Europe, northern Africa, with occasional cases reported in Asia, with the potential vector R. sanguineus s.l. (brown dog tick) [Rickettsia africae causes African tick bite fever in sub-Saharan Africa which correlates with the distribution of its known vectors Amblyomma hebraeum and Amblyomma variegatum [The epidemiology of is found ,3,4. Ford States , while A America . However, Mexico ,8. Meditog tick) ,9. Rickeriegatum .Rickettsia in Thailand, SFG Rickettsia are considered to be an under recognized cause of febrile illness in Thailand [Rickettsia vectors and animal reservoirs to better understand transmission and epidemiology is lacking. Briefly, there is serologic and molecular evidence that Rickettsia honei, and Rickettsia japonica cause humans infections in Thailand, [R. honei has been identified from Ixodes spp. and Rhipicephalus spp. larval ticks, and detection of R. japonica from Haemaphysalis hystricis suggesting these tick species as potential vectors [R. typhi, and flea-borne SFG disease caused by R. felis are distributed worldwide. Murine typhus is endemic in Thailand, as shown to be the second leading cause of acute undifferentiated febrile illness in Bangkok [Rickettsia typhi is transmitted by rat flea (Xenopsylla cheopis), and Rattus spp. serve as the primary reservoir [R. typhi can also be transmitted by cat flea  [R. felis and R. felis-like Rickettsia spp. DNA in dog, flea, and febrile patients suggests these bacteria can infect in human and domestic animals in Thailand [Although, there are a number of published reports of Thailand  and broahailand, ,13,14. I vectors ,16. Flea Bangkok . Ricketteservoir . In somes felis) ,20. The Thailand ,22,23.Rickettsia in wildlife and domestic animals, as well as their ectoparasites. Most studies perform PCR and sequence analysis of target genes to screen and characterize Rickettsia species and other interested pathogens [Rickettsia DNA by real-time PCR assay. Here, we develop the amplicon-based next generation sequencing (NGS) to characterize Rickettsia and their arthropod hosts into species. Amplicons of the target genes for Rickettsia and ectoparasites species identification were sequenced simultaneously using Miseq platform. This alternative approach facilitates identification of Rickettsia spp. from a large number of positive samples acquired from our multi-province surveillance. The identification of bacteria, vector, and animal reservoirs would aid our understanding of the risk of rickettsial disease transmission and epidemiology.To better understand the epidemiology of rickettsiosis and assess the risk for emerging rickettsial zoonoses in human, we performed surveillance for athogens ,23,24,25athogens ,27,28,29Rickettsia DNA from a total of 1825 samples including rodents, and ectoparasites collected from rodents and domestic animals, dog, cat, and cattle, (2015–2016). Rickettsia DNA by sample type and province where samples were collected. There was a high percentage of Rickettsia DNA in fleas were collected from domestic animals (97/173 = 56%), all positive fleas were collected from dogs. Of other sample types, only a few tick pools from domestic animals (3/157 = 1.9%), rodent flea pools (1/52 = 1.9%), and rodent tissue samples (2/1384 = 0.1%) were Rickettsia positive. No Rickettsia were found in lice collected from domestic animals or ticks collected from rodents. The data of domestic animals infested with ectoparasites were provided as One hundred and three samples were PCR-positive for Rickettsia-positive samples, 82 samples were selected for amplicon-based NGS species identification for Rickettsia species and Sanger sequencing was used for the remaining pools. Rickettsia gltA sequences were obtained from 101 samples. Phylogenetic analysis of gltA sequences for Rickettsia species characterization is shown in Rickettsia species were identified based on their highest DNA sequence identity to the reference sequences retrieved from Genbank database. There were four Rickettsia species identified from our samples, R. asembonensis, “Candidatus Rickettsia senegalensis”, R. helongjiangensis and R. typhi with 99.2–100% identity to references sequences. The result of Rickettsia species characterization by gltA gene is shown in Rickettsia species found were identified as R. asembonensis  followed by “Ca. R. senegalensis” (16.8%), R. helongjiangensis (2%) and R. typhi (2%). All Rickettsia from rodents (n = 2) and rodent fleas (n = 1) were R. typhi and R. asembonensis, respectively. The majority of Rickettsia identified from fleas collected from dogs (n = 95) were R. asembonensis , while “Ca. R. senegalensis” was detected in 16.8% (16/95) of fleas from dogs. Of three Rickettsia-positive ticks collected from dogs, two from Phangnga province were R. helongjiangensis and one from Si Sa Ket province was “Ca. R. senegalensis”.From a total of 103 COI and COII sequences by NGS and Sanger sequencing were available for 92 Rickettsia-positive pools (91 flea pools from dogs and 1 flea pool from rodent), and phylogenetic analysis was shown in COI and COII characterization for almost all flea samples (Rickettsia-positive fleas from dogs (89/91 = 97.8%) were identified as Ctenocephalides felis orientis (C. f. orientis) with 98–100% identity to the reference sequence, only 2 of them (2.2%) were Echidnophaga gallinacea. (95–97% identity). Interestingly, two flea pools from dogs were unidentified in which their COI sequences have only 91 and 82% identity to C. f. orientis sequence reference, but their COII sequences are 100% identical to C. f. orientis. Thus, they are classified as C. f. orientis. One positive rat flea was classified as an unidentified group by both COI and COII gene sequences.Results of flea species identification using  samples . The majCOI sequence was used to characterize three Rickettsia-positive tick pools by Sanger sequencing. Two tick pools were characterized as H. hystricis with 99% identity to the reference sequence (data not shown), while one tick pool that had been previously identified as R. sanguineus s.l. by morphology failed in sequencing. When the distribution of Rickettsia species among ectoparasite species was examined, R. asembonensis and “Ca. R. senegalensis” were found in various species of fleas and Rhipicephalus ticks from dogs. Among C. f. orientis population (n = 89), 83% (74/89) carried R. asembonensis and 17% (15/89) had “Ca. R. senegalensis”. However, it was noted that R. heilongjiangensis was found in only H. hystricis ticks.For tick species identification, only Rickettsia and ectoparasite species in parallel. In our previous surveillance during 2015–2016 in Thailand, high percentage of flea samples were PCR-positive for Rickettsia DNA. Instead of using Sanger sequencing to characterize Rickettsia and flea species using their specific target genes which would require large amounts of time to assemble and analyze each individual sequence, we applied the amplicon-based NGS technology. The Citrate synthase (gltA) gene, Cytochrome Oxidase subunit I (COI) and subunit II (COII) amplicons were sequenced simultaneously using Miseq platform. The advantage of NGS over traditional Sanger sequencing is that amplicons of several target genes could be sequenced simultaneously and up to 384 samples could be analyzed in one experimental run, if dual indexes were used in library preparation (Illumina). NGS sequencing strategies are used widely and provide a range of applications for epidemiological and surveillance analysis. For example, whole genome sequencing and genome-wide screening for single nucleotide polymorphisms have been used for microbial typing for outbreak analysis [Rickettsia and flea species, to enable rapid analysis of a large number of samples collected from a surveillance study.Here, we demonstrated the utility of next generation sequencing (NGS) for high-throughput sequencing to identify analysis . Other ranalysis ,27,28,29Rickettsia species and most were identified later as R. asembonensis. Molecular identification of flea species indicates all flea from dogs are Ctenocephalides felis orientis, except for two Echidnophaga gallinacea (sticktight flea) that may occasionally be found in dogs [C. f. orientis positive for R. asembonensis correspond to the study in India, suggesting a specific host-bacteria relationship [R. asembonensis DNA were also detected among fleas from cats in Khon Kaen province, northeastern Thailand [C. felis by morphology rather than molecular identification. Rickettsia asembonensis belongs to a group of R. felis-like organisms which are ubiquitously distributes. It was firstly detected as Rickettsia sp. RF2125 in Ctenocephalides canis in western Thailand near the Myanmar border [Pulicidae, Ceratophyllidae, and Coptopsyllidae [Rickettsia asembonensis could be a potential human pathogen as it has been reported from patients with fever of unknown origin in Thailand, Malaysia, and Peru [Rickettsia asembonensis was also detected in healthy dogs in South Africa [More than a half of all fleas from dogs during our 2015–2016 surveillance were PCR-positive for  in dogs . The evitionship ,33. SimiThailand , althougr border . Its hosand Peru ,35,36,37h Africa , sick doh Africa , and heah Africa . NonetheRickettsia DNA (<2%) was observed in dog tick, rodent flea, and rodent samples. Interestingly, R. heilongjiangensis was found in only H. hystricis ticks from dog in Phangnga province, while R. typhi was found in rodent samples from Tak province. R. heilongjiangensis belonging to Spotted Fever group (SFG), cause Far-Eastern spotted fever which is endemic in northeastern China [R. japonica, later confirmed to be R. heilongjiangensis [R. heilongjiangensis, causative agent of SFG rickettsiosesis, in H. hystricis ticks in Thailand. Spotted Fever Group Rickettsia apparently adapts itself to a specific tick species, and the relationship between SFG Rickettsia, tick, and vertebrate host have been documented [A low percentage of rn China , Siberiarn China . In Thaiangensis ,16. Howecumented .Rickettsia based on the Citrate synthase (gltA) gene  gene  and fleatA) gene . The gltR. asembonensis DNA (>50%) was observed in fleas from dogs (C. f. orientis). Rickettsia asembonensis belongs to a group of R. felis-like organisms which distribute ubiquitously, but its definite pathogenicity to human remains unknown. We also identified R. heilongjiangensis, causative agent of SFG rickettsioses, in H. hystricis ticks from dog in Phangnga province and R. typhi in rodents from Tak province. Our findings demonstrate the circulation of tick- and flea-borne SFG rickettsioses, and flea-borne murine typhus, and thus risk of transmission from potential vectors and animal reservoir in Tak and Phangnga provinces of Thailand. Moreover, our study illustrates another application of NGS for simultaneous characterization of Rickettsia and flea species, to enable rapid analysis of a large number of samples collected from a surveillance study.In conclusion, a high percentage of Rickettsia species (2015–2016), rodents and ectoparasites collected from rodents and domestic mammals  in Phangnga, Ranong, Si Sa Ket, Tak, and Udon Thani provinces of Thailand were screened for Rickettsia spp. Rodents were captured from plantations, cultivated rice-fields, and grassland areas using live traps, as previously described [During field surveillance for escribed . Captureescribed . Ectoparescribed ,44,45 an® SP instrument  with QIAsymphony® DNA Mini Kit . For ectoparasites, a modified protocol of QIAamp DNA Mini Kit (Qiagen) was used to extract genomic DNA from each pool as previously described [Genomic DNA was extracted from rodent tissue, and ectoparasite pools, as previously described . Brieflyescribed . DNA wasRickettsia using TaqMan real-time PCR (qPCR) targeting 17 kDa protein gene and some of the positive signals from qPCR were confirmed by conventional PCR of Rickettsia citrate synthase (gltA) gene. Details of primers, probes and PCR conditions were previously published by Takhampunya et al. 2019 [Rodent samples and ectoparasite pools were screened for al. 2019 .Rickettsia-positive ectoparasite pools were selected for NGS characterization of Rickettsia (82/103) and flea species (92/103), and the remaining pools was characterized by Sanger sequencing, while there was not enough DNA to do sequencing for two samples. Citrate synthase (gltA) gene was amplified for Rickettsia species characterization, while Cytochrome Oxidase subunit I (COI) and subunit II (COII) genes were used as target genes for flea species identification. The gltA amplification was done following the protocol for the Rickettsia confirmation assay as mention earlier [COI and COII and PCR conditions were described in the previous study [gltA, COI, and COII amplicons of each sample were pooled and subjected to NGS. Since the length of these amplicons is more than 600 bp, direct amplicon sequencing on Illumina system could not provide sequence information across the entire length. So, the Nextera XT DNA library prep kit was used to prepare amplicon libraries, according to the manufacturer’s protocol (Illumina). Briefly, amplicons were fragmented and tagged with adapter sequences by Nextera transposome. Then, library was amplified using a limit-cycle PCR to tag DNA fragments with dual indices and Illumina sequencing adapters. The final products were purified using Agencourt AMPure XP beads to remove excess primers, nucleotides, salts, and enzymes. The purity of the libraries was verified using the QIAxcel Advanced System (Qiagen) with a QIAxcel DNA High Resolution Cartridge. The purified Index library concentration was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen) and DNA concentration was normalized to 4.0 nM for each library. Five microliters of DNA from each library at 4.0 nM were pooled for one NGS run. Pooled libraries were denatured with NaOH, according to the manufacturer’s protocol (Illumina). Denatured libraries were diluted to a final concentration of 4 pM. A 10% Phix control adapter-ligated library (Illumina) was included to serve as internal control of each run for low-diversity library. Sequencing was performed on the Illumina MiSeq System using the MiSeq Reagent Kit v2 (300-cycles), following manufacturer’s instructions. High throughput sequencing (HTS) datasets was deposited in the NCBI’s Sequence Read Archive under BioProject # PRJNA691717, accession number SRR13425361-SRR13425442.Most  earlier . Primer us study ,47. The http://www.ncbi.nlm.nih.gov/Genbank/) under accession number MW492078-MW492178, MW492179-MW492261, and MW492262-MW492354. The full list of sequences and their database identifiers are given as For Sanger sequencing, the PCR product of each gene was sequenced. Excess primers and nucleotides were removed from PCR amplicons using ExoSAP-IT™ (Applied Biosystem) according to the manufacturer’s instructions. The purified amplicons were sequenced using an ABI BigDye™ Terminator v3.1 Cycle Sequencing Kit, ethanol precipitated, and run on a SeqStudio Genetic Analyzer . Sequences were assembled using Sequencher™ ver. 5.1 . Nucleic acid sequences generated during this study (Sanger sequences and consensus sequences from NGS) were deposited in the GenBank database . Consensus sequences were extracted from read mapping, according to default settings, and used for phylogenetic tree analysis. Sequences from NGS and Sanger sequencing were aligned with reference sequences retrieved from the GenBank database using the MUSCLE codon alignment algorithm [The sequence reads generated on MiSeq sequencers were processed on the CLC Genomics workbench software v 20.0.3 . High-throughput sequences were imported into CLC Genomics Workbench according to quality scores of Illumina pipeline 1.8 . The sequences were trimmed to remove adaptor sequences, low quality sequence (limit = 0.05), ambiguous nucleotides , and read lengths shorter than 100 bp. Trimmed reads were mapped to reference sequences of lgorithm . Maximumlgorithm .