Mycobacterium tuberculosis (M. tb) strains and co-infection with human immunodeficiency virus has escalated the need for new anti-M. tb drugs. Numerous challenges associated with the M. tb, in particular slow growth and pathogenicity level 3, discouraged use of this organism in past primary screening efforts. From current knowledge of the physiology and drug susceptibility of mycobacteria in general and M. tb specifically, it can be assumed that many potentially useful drug leads were missed by failing to screen directly against this pathogen. This review discusses recent high-throughput phenotypic screening strategies for anti-M. tb drug discovery. Emphasis is placed on prioritization of hits, including their extensive biological and chemical profiling, as well as the development status of promising drug candidates discovered with phenotypic screening.The rise of multi- and extensively drug-resistant  Tuberculosis (TB) is among the ten leading causes of death worldwide and the leading cause from a single infectious bacterium . In 2017Mycobacterium tuberculosis (M. tb) drug discovery is limited by many technical challenges. M. tb is a slow growing, most commonly respiratory, pathogen that requires handling in Biosafety Level 3 (BSL3) facilities. The lipid-rich nature of the mycobacterial cell wall contributes to its low permeability and prevents many small molecules from accessing internal molecular targets. Active multi-drug efflux pumps account for one of the many mechanisms of resistance to antibiotics [M. tb exists as a heterogeneous population in different microenvironments, which requires drugs to be active against multiple physiological states under various conditions [M. tb drug should be safe and show minimal drug–drug interactions with other antimicrobials used for treatments or major side effects over an extended treatment period of six months to 2 years. Ideally, the new regimens would decrease required treatment durations leading to increased patient compliance and improved outcomes. New leads should also be active against drug-resistant M. tb strains and be compatible with HIV therapy [The rate of progress in anti-ibiotics . Within nditions . Further therapy .M. tb drug, streptomycin [Historically, many antibiotic lead compounds were isolated from soil-derived actinomycetes during the golden era of antibiotic discovery in the mid-20th century. The original discovery platform consisted of screening for antimicrobial activity against susceptible bacteria using agar-based diffusion or cross-streak methods. These simple methods also led to the discovery of the first anti-ptomycin , and proptomycin . Severalptomycin .M. tb drug discovery efforts were principally M. bovis (BCG) or M. smegmatis, to circumvent the issues of slow growth and/or safety, while M. tb was used to a much lesser extent. Considering the abundant use of surrogate pathogens in anti-M. tb screening, it is reasonable to hypothesize that many potentially useful M. tb drug leads have been missed in the past.The bacteria used in phenotypic (whole-cell) screens of most of the early and some of the current anti-M. smegmatis and M. bovis as surrogate in vitro models [M. tb inhibitors could not be detected when screening against M. smegmatis, and 21% were missed in M. bovis screens. The genomic comparisons indicated that 30% of M. tb proteins lack conserved orthologues in M. smegmatis and 3% in M. bovis. Although, M. bovis represents a more sensitive model, M. smegmatis is often favored in HTS due to its faster growth, 4 days, compared with 14 days for M. bovis. Bedaquiline (TMC207), the first FDA approved TB drug for 40 years, was identified by HTS with M. smegmatis as surrogate [The parallel screens of ~5000 compounds from the Library of Pharmacologically Active Compounds (LOPAC), NIH Diversity Set and Spectrum Collection were performed to evaluate the efficacy of o models . The daturrogate .M. tb genome sequence in 1998 led to the identification of potential drug targets and development of new target-based screens [Phenotypic screening opens the path to discovery of compounds that inhibit new target(s) or pathway(s). However, this approach does not provide information about the drug target. The determination of the whole  screens . Neverth screens .M. bovis BCG with ~2.2 million Novartis compounds was followed by hit confirmation against M. tb H37Rv. The work from hit to lead took over 7 years and five chemical series—pyrimidineimidazoles, imidazopyridines, indolcarboxamides, pyrazolopyrimidines, and pyridones [Manjunatha and Smith reported experiences gained from TB drug discovery at the Novartis Institute for Tropical Disease (NITD) in Singapore . Both, myridones .M. tb drugs and promising candidates in clinical trials resulted from drug-to-target paths that involved HTS against whole cells  synthase and the subsequent energy supply [As previously noted, bedaquiline (TMC207), a diarylquinoline, was identified in a screen against euticals . It is hy supply . In 2012y supply . Numerouy supply .Streptomyces eurocidicus [M. tb, which brings the potential to shorten duration of the treatment. Delamanid and pretomanid appear to have a multi-target mechanism of action; acting on the inhibition of cell wall biosynthesis through inhibition of methoxy- and keto-mycolic acid synthesis and respiratory poisoning through release of nitric oxide during bacterial drug metabolism [https://www.tballiance.org/news/pretomanid-enters-FDA-review).The nitroimidazoles, delamanid (OPC-67683), and pretomanid (PA-824), are analogs of azomycin, originally isolated from ocidicus . They artabolism –22. Bothtabolism –25. Delatabolism . It has tabolism . PretomaM. tb. Among 69 hits, SQ109 was the most potent, and retained activity against isoniazid- and rifampicin-resistant M. tb strains. Activity against an ethambutol-resistant M. tb strain suggested a target, mode of action and/or activation pathways different than those of ethambutol. The SQ109 triple mode of action includes (i) the inhibition of MmpL3, a membrane transporter for trehalose monomycolate which is involved in cell wall synthesis of M. tb, (ii) the inhibition of the enzymes MenA and MenG, which are involved in menaquinone biosynthesis, and (iii) the reduction of ATP synthesis acting as an uncoupler [M. tuberculosis in the lung.The attempt to develop a new second-generation drug from the first-line drug ethambutol lead to the discovery of SQ109 , 29. A lncoupler . SQ109 eM. tb growth in mouse macrophages lead to two series of imidazopyridine amides (IPA) [1 complex which is an essential component of the electron transport chain required for ATP synthesis. The effect on energy metabolism and the potency at low doses in the chronic mouse model of TB suggest that Q203 may contribute to shortening the treatment time of TB. Q203 also appears to be synergetic with bedaquiline in the murine chronic infection model, indicating potential for new drug regimen. A phase II clinical trial by Qurient Co. Ltd is currently evaluating bactericidal activity, safety, tolerability, and pharmacokinetics of multiple oral doses of telacebec (Q203).A phenotypic HTS at the Pasteur Institute in Korea of a library of 121,156 different chemical compounds for their ability to inhibit es (IPA) . They wees (IPA) , 32. TheM. tb drugs from soil-derived actinomycetes. Emphasis is placed on the workflow for HTS phenotypic screening against virulent M. tb and on hit prioritization using assays established in the ITR laboratories. The strategies and new approaches for isolation and identification of active leads is discussed as well. An anti-M. tb drug discovery platform is presented by means of the discovery of a group of anti-mycobacterial cyclic peptides that target ClpC1 in M. tb [Natural products still appear to be a promising source of new antibiotics. Approximately, 78% of the therapeutics in the area of infectious diseases derive from natural products. In this review, we share our experience from over a decade of effort that was made in the laboratories of the Institute for Tuberculosis Research (ITR) at the University of Illinois at Chicago, in collaboration with Myongji University in Republic of Korea, to discover new anti-in M. tb –36.M. tb H37Rv using a luxABCDE reporter and the microplate alamar blue assay (MABA). This unique library, the Extract Collection of Useful Microorganisms (ECUM) (www.ecum.or.kr), was established in 1993 at Myongji University in the Republic of Korea. It includes extracts of soil microorganisms collected from sites with distinctive ecologies and unusual weather conditions, such as polar, alpine, tropical, volcanic, and desert areas. Selected actinomycete isolates were cultured in three different liquid media, i.e., glucose-soybean starch (GSS), Bennett’s (BN), and dextrin-yeast-corn steep liquor (DYC). The cell pellet was then extracted with methanol, and the supernatant with ethyl acetate. As a result, nine different extracts were generated for each actinomycete isolate , screening window coefficient (Z-factor), signal-to-noise, and signal-to-background ratios [The primary HTS of the ECUM library was performed with a whole-cell-based assay for growth inhibition of the luciferase expressing strain of d ratios . A meticM. tb lux ABCDE. The results of the single-dose primary screen were expressed as percent inhibition, calculated according to Eq.  and methanol (34%) extracts obtained from GSS (44%) and BN (35%) media. Only 20% of the hits originated from water extracts. The lower hit rate for the water extracts could be explained by the fact that larger hydrophilic compounds cannot penetrate the highly hydrophobic outer layer of utrients .M. tb under aerobic conditions, whereas the second one tests the compounds’ activity against non-replicating M. tb under hypoxic conditions. Primary hit identification was based on the assay results against replicating M. tb. Extracts with additional activity against non-replicating M. tb were prioritized due to the potential to shorten duration of TB treatment.The confirmatory screening of the hits selected after the primary HTS included two previously established in vitro assays  Supplem. The firM. tb, the identification of hits was also based on mammalian cell toxicity measurements. Primary cytotoxicity was evaluated against a green monkey kidney cell (Vero) line, and all samples with growth inhibition > 50% were deprioritized.In addition to considering the activity against replicating and non-replicating M. tb MIC values and the IC50 vs Vero cells. The MABA determines the MICs of the extracts against replicating M. tb under aerobic conditions, while the low oxygen recovery assay (LORA) determines their MICs against non-replicating M. tb under hypoxic conditions. All calculations were based on the assumption that the concentration of the DMSO-reconstituted extracts was 1 mg/mL, and the MIC and IC50 values were then used to calculate the selectivity index (SI) as IC50/MIC against both replicating and non-replicating M. tb. The SI can be considered as a preliminary estimation of the extract’s therapeutic window. To prioritize highly promising extracts, a SI > 50 was used as a threshold.The subsequent prioritization step considered both the anti-M. tb isolates with resistance to known antibiotics. This included mono-resistant strains to isoniazid (INH), rifampin (RIF), streptomycin (SM), kanamycin (KM), cycloserine (CS), bedaquiline (TMC207), and the in-house generated M. tb strains with mono-resistance to clofazimine (CLF), moxifloxacin (MOX), and capreomycin (CAP)  . This apStaphylococcus aureus, Gram-negative bacterium—Escherichia coli, yeast—Candida albicans, and non-tuberculous mycobacterium—M. smegmatis (Supplementary data), all of which are widely used in HTS campaigns. Extracts with activity against one of the tested species have a higher chance of containing a known compound. Moreover, some antimicrobial compounds share similar structural cores or related targets or exhibit universal antimicrobial activity. Considering the long duration of TB treatment, it is preferable that new TB drugs possess a narrow SOA. This decreases the risk of bacterial resistance emerging in other microbial species during the long course of TB antimicrobial therapy.Additional information for the biological dereplication was obtained from the evaluation of the spectrum of activity (SOA) against a microbial panel consisting of a Gram-positive bacterium—M. smegmatis were prioritized. Activity against M. smegmatis was not deprioritized due to the common molecular targets observed between M. tb and other mycobacteria. Therefore, activity against M. tb and other mycobacteria is commonly seen and does not diminish a compound’s potential use in TB treatment.The assessment of SOA reveals the specificity of the antimicrobial compounds according to the Gram reaction, taxonomy, and pathogenicity of the bacteria. Only extracts with low activity against yeasts and bacteria other than M. tb strain, mc27000 luxABCDE [M. tb activity results of the samples could be determined after only 24 h, versus 8–10 days for common M. tb in vitro assays. The combination of TLC with MS and/or NMR allowed the chemical characterization of the separated compounds, and by using bioautography, their chemical profile could be matched with their biological activity.To correlate biological dereplication with chemical identification/characterization of the hits , the HPTluxABCDE , 45, whiM. tb strains. The hits in the second group had (a) lower activity against replicating M. tb, (b) activity against at least one of the non-tuberculous species, and (c) in comparison to the first group, some toxicity against Vero cells. The hits in the third group had (d) even lower activity against replicating M. tb, (e) high activity against at least one non-tuberculous species, (f) toxicity against Vero cells, and (g) cross-resistance with mono-resistant M. tb strains. The fourth group contained the hits with the least favorable biological profile.All hits were divided into four priority groups based on their activity profiles. The first group contained hits with highly selective potency against replicating, non-replicating, and mono-resistant M. tb. All extracts that failed to reproduce anti-M. tb activity were removed from the hit prioritization list.The source organisms of the prioritized hits of the four groups were refermented at medium scale, and extracted as before. The stock solutions of the newly fermented extracts were prepared at an accurate concentration in DMSO, and their activity was reassessed against replicating and non-replicating M. tb, mono-drug-resistant M. tb strains, E. coli, C. albicans, M. smegmatis, and ESKAPE  panels, as well as Vero cells  using reverse phase silica (C18) gel with a gradient elution 20–100% methanol in 20% increments and a final chloroform wash. The resulting six fractions were prepared in DMSO, and their activity was evaluated against replicating and non-replicating ro cells . Priorities Fig. .Fig. 3Th50, MABA, and LORA values. The fractionation scheme included an initial separation via VLC (C18) to crudely separate the compounds of different polarities, followed by fractionation with a Sephadex LH-20 column to separate compounds of different polarity and molecular size. High-speed counter current chromatography (HSCCC) and (semi)preparative high-performance liquid chromatography (HPLC) were used in the final steps of purification.After establishing the priority list of the strains, the source organisms of hits from the first group were refermented in larger volumes, e.g., 60 L. A bioactivity-guided fractionation (BGF) strategy was applied to isolate the anti-TB active compounds, i.e., all fractionation steps were done while monitoring ICM. tb leads using counter current separation (CCS) methods [The Bio-GUESS concept has been introduced recently to facilitate a more targeted isolation procedure of potential anti- methods . For thi methods  was comb methods .The molecular weight of isolated compounds was determined via high-resolution mass spectrometry (HR-MS). Molecular formula prediction software was used to determine the elemental composition, and various public databases of mass spectral data were utilized for rapid dereplication. A more recent approach includes the use of MS/MS-based Molecular Networks algorithm which associated MS/MS spectra based on similar fragmentation patterns, as per the assumption that structurally related molecules will fragment in a similar way to give analogous patterns . New mol1H and various 2D  experiments. The 1H NMR iterative Full Spin Analysis (HiFSA) was used for unambiguous assignment, confirmation of the proposed structure and validation of the newly identified natural product lead [δH, in ppm), and 1H,1H spin–spin coupling constants  present in a molecule from high-resolution 1H NMR spectra. It also enables resolution of highly complex 1H NMR signal patterns, yielding information about a molecular structure that otherwise is inaccessible and/or ignored.Further structural elucidation was based primarily on NMR data collected with 400–900 MHz NMR instruments and including 1D uct lead . The metM. tb leads.Table Nonomuraea sp. MJM5123, the producing strain of the new macrocyclic tridecapeptide family, ecumicin and its analogs [M. tb in vitro, including non-replicating cultures, clinical isolates representing six major global clades , MDR and XDR strains. Ecumicin also shows in vivo activity; the complete inhibition of M. tb growth in mouse lungs was achieved after 12 doses at 20 or 32 mg/kg [M. tb [The first HTS campaign of 65k actinomycetes led to the prioritization of 22 extracts for further studies. Among them was a mycelial methanolic extract of  analogs –35. Ecum32 mg/kg . The absStreptomyces atratus, MJM3502 revealed that the active components were rufomycins [Streptomyces [M. tb and M. abscessus.The third HTS campaign of 20k actinomycete extracts led to the prioritization of three strains. The rapid dereplication of the ethyl acetate extract of fomycins . These cptomyces –56, and ptomyces –59. OngoM. tb drugs of both ecumicin and rufomycin suggested a novel mechanism of action. The target has recently been identified as the ClpC1 ATPase a protein complex essential for the growth of M. tb [M. tb natural products, lassomycin [M. tb lead compound (Fig. The lack of cross-resistance with existing anti-of M. tb , 61. Thessomycin  and cyclssomycin , 64, havM. tb, most of the screens were performed against the rapidly growing, M. smegmatis or BCG. However, the use of surrogate strains for screening risks missing compounds that are highly selective for M. tb. The experience with a screening and prioritization workflow, shared in this review, may facilitate the discovery of new active compounds with narrow spectrum activity and good safety profiles. So far, the phenotypic screening showed bigger potential than the target-based approach. All recently introduced anti-M. tb drugs or the promising candidates in clinical trials come from phenotypic screening. The new anti-M. tb drugs bring hope to more effective TB treatment.The selection of an adequate screening assay has always been an important part of drug discovery programs. Due to the slow growth and safety restrictions of Supplementary Data


Mycobacterium tuberculosis. In this review, we outline the most abundant of over 170 NTM species and inadequacies of diagnostics and treatments and weigh the advantages and disadvantages of currently available in vivo animal models of NTM. In order to effectively combat this group of mycobacteria, more research focused on appropriate animal models of infection, screening of chemotherapeutic compounds, and development of anti-NTM vaccines and diagnostics is urgently needed.Seemingly innocuous nontuberculous mycobacteria (NTM) species, classified by their slow or rapid growth rates, can cause a wide range of illnesses, from skin ulceration to severe pulmonary and disseminated disease. Despite their worldwide prevalence and significant disease burden, NTM do not garner the same financial or research focus as  Pulmonary diseases due to NTM are an increasing global health concern.Prevalence of NTM is increasing in the US and has surpassed pulmonary tuberculosis (TB).Successful treatment for NTM infection is complicated by the large number of NTM species (>170), difficulties with diagnosis, and few therapeutic options once diagnosed.Mycobacterium avium complex (MAC) lung disease treatment is costly, lengthy, and often results in toxic side effects.New therapeutic strategies are urgently needed, including more efficacious drugs with fewer adverse side effects and novel treatment options that could reduce treatment time and toxicity.Prioritizing funding and research using NTM animal models to dissect NTM pathogenesis and host-directed responses and test novel therapeutic regimens against these infections may help combat these widespread pathogens.Am J Respir Crit Care Med. 2007;175(4):367–416.Griffith DE, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Thorax. 2017;72(Suppl 2):ii1-ii64.Haworth CS, et al. British Thoracic Society guidelines for the management of non-tuberculous mycobacterial pulmonary disease (NTM-PD). Mycobacterium avium complex infection and evaluation of drug combinations. Antimicrobial agents and chemotherapy. 2015;59(4):2129–35.Andrejak C, et al. Characterization of mouse models of Science. 2016;354:751.Bryant J, et al. Emergence and spread of a human transmissible multidrug-resistant nontuberculous mycobacterium. https://doi.org/10.1016/j.meegid.2018.10.003.Huh HJ, et al. Recent advances in molecular diagnostics and understanding mechanisms of drug resistance in nontuberculous mycobacterial diseases. Infection, Genetics and Evolution, Mycobacterium tuberculosis (Mtb) or M. leprae, include over 170 different species varying in their ability to cause disease [M. avium complex [MAC], M. abscessus), others  are regionally significant ) and rough (loss of GPL) phenotypes have divergent effects on infected macrophages that induce either protective granulomas and chronic infection or abscess formation and acute infection, respectively . S. SM. absbscessus  for rapiM. abscessus [Mtb. For example, our group has engineered a vaccine against Mtb, ID93  + glucopyranosyl lipid adjuvant (GLA) formulated in an oil-in-water stable nanoemulsion (SE), which is currently being tested in a Phase 2a trial in South Africa for safety, immunogenicity, and dose selection in 60 TB patients administered post-treatment [Despite the significant global impact of opportunistic NTM and the need to stop subsequent reinfection from the environment in susceptible hosts , 106, thbscessus , 114. Foreatment . Escalatreatment –122, andreatment , 122. Threatment . Numeroureatment , 124–141reatment , providireatment . As the M. avium and M. kansasii [M. intracellulare and M. simiae, perhaps because BCG lacks cross-reactive antigens against these NTM [M. ulcerans, including increased CD4+ TH1 responses, following vaccination with BCG [Mtb-specific chemokine (C-X-C motif)  memory T cells in non-TB–exposed healthy control (HC) donors has been observed [H1-inducing adjuvant GLA-SE [M. ulcerans infection, as shown by delayed progression of infection and reduced bacterial burden in wild-type compared to GKO mice [M. ulcerans Ag85A in the Buruli ulcer mouse model [Mtb experience for possible immune interference or boosting effects and should be a major priority for the research community.Variable host factors and mycobacterial experience are particularly important considerations for rational anti-NTM vaccine design. It has long been hypothesized that NTM-based preexisting immunity could lead to variability and/or lack of Bacillus Calmette–Guérin (BCG) vaccine efficacy . Converskansasii . BCG washese NTM . Fraga awith BCG . Recentlobserved . The post GLA-SE . For exaGKO mice . Other dse model . ProgresChallenges facing the prevention and treatment of NTM disease include inadequate research—in part no doubt due to insufficient funding—the lack of knowledge of factors leading to susceptibility in mouse strains with different genetic backgrounds, and a poor understanding of the mechanisms utilized by NTM that allow for the evasion of host immunity resulting in bacterial persistence. By addressing these challenges, more effective prophylactic and therapeutic approaches for the prevention and treatment of NTM will likely emerge.Use of the current minimum inhibitory and minimum bactericidal concentration (MIC/MBC) method in the clinic may need to be updated and/or complemented with newer techniques, and there is continued debate over an optimal animal model for compound screening ongoing in the research community . Neverth


AbstractYponomeutamontanatus is sequenced and compared with other published yponomeutoid mitogenomes. The mitogenome is circular, 15,349 bp long, and includes the typical metazoan mitochondrial genes  and an A + T-rich region. All 13 protein-coding genes use a typical start codon ATN, the one exception being cox1, which uses CGA across yponomeutoid mitogenomes. Comparative analyses further show that the secondary structures of tRNAs are conserved, including loss of the Dihydorouidine (DHU) arm in trnS1 (AGN), but remarkable nucleotide variation has occurred mainly in the DHU arms and pseudouridine (TψC) loops. A + T-rich regions exhibit substantial length variation among yponomeutoid mitogenomes, and conserved sequence blocks are recognized but some of them are not present in all species. Multiple phylogenetic analyses confirm the position of Y.montanatus in Yponomeutoidea. However, the superfamily-level relationships in the Macroheterocera clade in Lepidoptera recovered herein show considerable difference with that recovered in previous mitogenomic studies, raising the necessity of extensive phylogenetic investigation when more mitogenomes become available for this clade.The complete mitochondrial genome (mitogenome) of  PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs)), and an A + T-rich region  and T.yunnanensis   is a circular and double-stranded molecule that usually encodes 37 genes  .Lepidoptera is the second largest insect order after Coleoptera, with more than 157,000 extant species in 43 superfamilies . Relative to other species-rich orders, however, the current number of sequenced mitogenomes is still limited. Moreover, deep-level lepidopteran phylogeny is still poorly understood despite previous investigations based on various data including mitogenome sequences  (Praydidae)  (Plutellidae)  (Lyonetiidae)  , Plutellellidae) , and Leuetiidae) . Thus, tYponomeutoidea lineages would play an important role for better understanding the evolution of the superfamily or even Lepidoptera as a whole. In the present study, we sequenced the complete mitogenome of Yponomeutamontanatus Moriuti, 1977, the first mitogenome from the family Yponomeutidae. Moreover, detailed comparative analyses were conducted based on this and all other published yponomeutoid mitogenomes. In addition, extensive phylogenetic analyses using three different datasets and three different tree-constructed methods were performed to test phylogenetic implications of the Y.montanatus mitogenome in Lepidoptera phylogeny. This study contributes to further understanding the mitogenome evolution and phylogeny of the Yponomeutoidea and Lepidoptera.Mitogenomic data of major Y.montanatus specimens were sampled by light trap at Mountain Jigongshan, Henan, China in May 2018. Fresh specimens were stored in 95–100% ethanol in the field and then maintained at –80 °C until used for DNA extraction. Dry specimens were identified based on the morphological description and illustrations provided by cox1 barcode sequence in GenBank. Thorax muscle tissues were used to extract genomic DNA with the DNeasy tissue kit  according to the manufacturer’s instructions. Voucher specimens are deposited in the Biology Laboratory of Zhoukou Normal University, China.Adult Y.montanatus. Briefly, total genomic DNA was firstly quantified and fragmented to an average size of 400 bases using Covaris M220 system with the Whole Genome Shotgun method . Then, a library was constructed using the TruSeq DNA PCR-Free Sample Preparation Kit . Lastly, Illumina HiSeq 2500 was used for sequencing with the strategy of 251 paired-ends.Next-generation sequencing methods were used to obtain the complete mitogenome sequence of Y.montanatus. FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used for quality control . After processing with AdapterRemoval v. 2  and MEGA v. 6.06 (http://tandem.bu.edu/trf/trf.html) (RSCU) were calculated using MEGA v. 6.06 (The MITOS webserver was employed to annotate the complete mitogenome sequence with the invertebrate genetic code . The tRN v. 6.06 . To ensu v. 6.06  with invrf.html) . All oth v. 6.06 . Strand  v. 6.06 . The nuc v. 6.06 .Y.montanatus mitogenome in Lepidoptera phylogeny, a total of 33 mitogenomes representing 15 lepidopteran superfamilies with mitogenome available , PCG123R (PCG123 dataset plus two rRNAs and 22 tRNAs), and PCGAA (amino acid sequences translated from 13 PCGs). Nucleotide sequence substitution model was selected using PartitionFinder v. 1.1.1 (BIC) algorithm under a greedy search. The best partition scheme and corresponding models are shown in Suppl. material To investigate phylogenetic implications of the platform . MEGA v.platform  was usedplatform  was alsov. 1.1.1 , with thML) analyses were conducted using two methods. The raxmlGUI version 1.539 interface  analysis was performed using MrBayes v. 3.1.2 . For theY.montanatus (GenBank accession number: MK256747) is circular, double-stranded, and 15,349 bp long  and an A + T-rich region are included. Among them, 23 (nine PCGs and 14 tRNAs) are encoded on majority strand (J-strand), and the remaining 14 are located on minority strand (N-strand). As in most ditrysian members of Lepidoptera, the trnM-trnI-trnQ can be recognized in Yponomeutoidea, in contrast to the trnI-trnQ-trnM in most non-ditrysian lineage such as the Hepialoidea (Drosophilayakuba)  .Yponomeutoidea mitogenomes, which ranges from 81% in P.lutella (KM023645) to 82.5% in L.malifoliella  were calculated and drawn for all five yponomeutoid mitogenomes. As shown in Figure Y.montanatus mitogenome, a number of 3,727 amino acids are translated, of which 1,787 (47.9%) are encoded by the five frequently used codons above. However, the codons absent in yponomeutoid mitogenomes are different, but most of them are rich in C/G nucleotides. In general, the high A/T content in frequently used codons effectively contributes to the high A + T composition in PCGs and the whole mitogenome.The total length of the 13 PCGs in yponomeutoid mitogenomes use the conventional ATN start codon  to 71 bp (trnK)  to 1,488 in L.malifoliella  lacks the DHU arm, a feature generally present in all Lepidoptera insects as well as in other metazoan mitogenomes  Fig. , Table 1ogenomes . In tRNAogenomes .trnS1 (AGN). However, substantial nucleotide variation exists, most of which occurred in the DHU arm and TψC loops  were rarely mutated relative to other yponomeutoid species. For trnS1 (AGN), the TCT is used instead of routinely used codon GCT. This phenomenon has been recognized in previous reports such as two species of Hepialoidea and three species of Noctuoidea , respectively.Similar to other yponomeutoid mitogenomes, two rRNA genes, ome Fig. , Table 1Y.montanatus mitogenome, 36 gene overlapping sites were recognized across 13 gene junctions from one to eight bp in length   .Y.montanatus mitogenome n sequence but with different repeat numbers. P.xylostella is an important agricultural pest, and this microsatellite (TA)n sequence may be used as a candidate marker to test the population structure for pest management. The one between the trnQ and nad2 genes exhibits substantial sequence variation (except two sequences for P.xylostella) among reported yponomeutoid mitogenomes. This intergenic region is also widely present in other lepidopterans such as those in the Lasiocampidae n element, respectively. Also, insect A + T-rich region is generally characterized by the presence of multiple tandem repeat elements ), which is consistent with that of Y.montanatus is nested within Yponomeutoidea, confirming its phylogenetic position using mitogenomic data. In previous studies, the Yponomeutoidea is recovered either sister to Gracillarioidea  were recovered by studies based on various data, such as mitogenome sequences (Bombycoidea + (Geometroidea + Noctuoidea), which was identical to that of Kawahara and Breinholt (Regarding the phylogenetic pattern of other superfamilies, mostly identical results were obtained by different analyses, which are also similar to other mitogenome-based studies . We noti studies  as well  studies  where thequences , multi-gequences  and 741 equences . Interesreinholt . This re


Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae). In contrast to expectations, we find that the genome and transcriptome sizes of this parasitoid wasp are comparable to other members of the Chalcidoidea superfamily. Moreover, compared to other chalcid wasps the gene content of M. amalphitanum is remarkably conserved. Intriguingly, we observed significant changes in M. amalphitanum transposable element dynamics over time, in which an initial burst was followed by suppression of activity, possibly due to a recent reinforcement of the genome defense machinery. Overall, while the M. amalphitanum genomic data reveal certain features that may be linked to the unusual biological properties of this organism, miniaturization is not associated with a large decrease in genome complexity.Body size reduction, also known as miniaturization, is an important evolutionary process that affects a number of physiological and phenotypic traits and helps animals conquer new ecological niches. However, this process is poorly understood at the molecular level. Here, we report genomic and transcriptomic features of arguably the smallest known insect–the parasitoid wasp,  Megaphragma amalphitanum  is one of the smallest known insects, whose size (250 μm adult length) is comparable with unicellular eukaryotes and even some bacteria  developing on the shrubs Viburnum tinus (Adoxaceae) and Myrtus communis (Myrtaceae) [Hercinothrips femoralis (Thysanoptera: Thripidae) [M. amalphitanum belongs to chalcid wasps, which represent one of the largest insect superfamilies [Miniaturization in animals is an evolutionary process that is frequently accompanied by structural simplification and size reduction of organs, tissues and cells , 2. The bacteria . Parasitrtaceae) , and posripidae) . The wasripidae) , 4. M. a species)–10 that M. amalphitanum, this species has only ~4,600 neurons in its brain, which is substantially fewer than in the brains of other wasps, e.g., the parasitoid chalcid wasp Trichogramma pretiosum (Trichogrammatidae: Trichogrammatinae) , Hemiptarsenus sp.  , and the honey bee Apis mellifera (Apidae) . Moreover, by the final stage of M. amalphitanum development, up to 95 percent of the neurons of the central nervous system have lost their nuclei [Amongst notable anatomical features of r nuclei , 13. Nevr nuclei .M. amalphitanum partial genome assembly and the adult transcriptome, and compare these with several parasitoid wasp species of different body sizes from the Chalcidoidea and Ichneumonoidea hymenopteran superfamilies. We performed general gene ontology and pathway analyses as well as specific gene categories of interest, such as chemosensory receptors and venom components. Additionally, we investigated transposable element (TE) content and dynamics across M. amalphitanum and other parasitoid wasp species and analyzed the major components of the genome defense machinery. As body size reduction and loss of physiological or phenotypic traits is often correlated with genome size diminution [M. amalphitanum genome would be greatly simplified.In this study, we present a minution , 16 and/minution , includiminution , 19, we M. amalphitanum individuals were reared in the laboratory conditions from eggs of Heliothrips haemorrhoidalis (Thysanoptera: Thripidae) collected in Santa Margherita, Northern Italy . Unfortunately, we could collect only a dozen M. amalphitanum individuals because their habitats are difficult to detect (culture in the laboratory is currently impossible), the imago life span is short (5 days), and the animal is extremely small. With several insects we could cleanly recover, we were therefore able to obtain only around 1–5 ng of genomic DNA for the each paired-end DNA and cDNA libraries. DNA was extracted from ten individuals  using NucleoSpin Tissue XS kit  for each DNA-library. Three DNA libraries  were constructed using Ovation Ultralow Systems V2 kit . Limited amount of biological material and low quantity of starting material (1–3 ng) did not permit construction of mate-paired libraries. Genome libraries were sequenced using Illumina HiSeq 1500  with 150 bp paired-end reads. RNA was extracted from ten M. amalphitanum individuals  using the Trizol reagent  by a standard protocol, and cDNA libraries were constructed using Ovation RNA-Seq System V2 kit  with poly(A) enrichment.de novo genome assembly. To assemble the genome of M. amalphitanum, we used 102,188,833 paired-end reads. Genome assemblies were constructed using different assembly algorithms, and their performance was compared to each other  of M. amalphitanum (SRR5982987) and from head (DNA-library3) of M. amalphitanum (SRR5982986) were prepared. In total, 79,317,970 (paired-end sequencing: 2×100 bp) and 85,409,775 (single-end sequencing: 50 bp) DNA reads were sequenced and used for M. amalphitanum coverage increase and as additional evidence during the search for potentially missing genes  [The output from Illumina sequencing of the genomic DNA library (source format *.fastq) was used for ch other . Then, Mng genes . Then, tv.3.6.1) .de novo assembly was conducted using the default k-mer size in the Trinity software package (v. 2.4.0) [M. amalphitanum transcriptome assembly was performed using the Trinotate pipeline [Illumina RNA sequencing generated a total of 59,790,973 paired-end reads. Transcriptome . 2.4.0) , which cpipeline .Transposable element (TE) de novo identification and analysis. For de novo TE library construction, we used the REPET package [www.repeatmasker.org), which was also used to build the corresponding TE landscape divergence plots. package  which coM. amalphitanum from other Hymenoptera, we performed whole-genome shotgun sequencing of DNA (DNA-library1) isolated from ten adult individuals , using the Illumina platform . The resulting partial genome assembly (PRJNA344956) has a cumulative length of 346 megabases (Mb), with a scaffold N50 of 10,296 bp. The total genome coverage is 88.6-fold. Thus, the genome of M. amalphitanum is comparable in size with other Chalcidoidea wasps, such as Copidosoma floridanum, T. pretiosum or Nasonia vitripennis [To gain insight into the genomic signatures of miniaturization that would distinguish platform . The resripennis , 25. TheM. amalphitanum genome assemblies were evaluated with the BUSCO v3  Hymenoptera gene set [M. amalphitanum assembly as putatively missing . Transcriptome de novo assembly (PRJNA344956) was performed using the Trinity software [). The Illumina paired-end RNA-Seq data from M. amalphitanum were mapped to the previously assembled genome using Bowtie2 [We also performed whole-body transcriptome analysis using RNA extracted from ten software . A total Bowtie2 . InspectThe BUSCO analysis shows the low completeness of the present partial genome and transcriptome assemblies, with 28–29% of BUSCO genes listed as fragmented. This may be caused by inability to use mate-paired DNA-libraries or single-molecule sequencing ; possible high heterozygosity and/or significant structural variation between different parasitoid wasp individuals that were used for genome and transcriptome assemblies; BUSCO database incompleteness; and other factors. An additional factor in poor transcriptome completeness could be a high number of short and chimeric isotigs: while nearly 80% of transcriptome reads map to the genome, only 24% of assembled contigs are represented in the complete BUSCO set.M. amalphitanum gene products in three independent categories: biological processes (We used Gene Ontology (GO) analysis terms to describe characteristics of rocesses , moleculrocesses , and celrocesses . BLASTX rocesses .M. amalphitanum transcripts were matched to the Clusters of Orthologous Groups (COG) database to predict and classify their functions. In total, 8,810 genes were assigned to 25 COG functional categories. One of the largest groups is represented by the cluster for post-translational modification, protein turnover, and chaperones , followed by intracellular trafficking, secretion, and vesicular transport , DNA replication, recombination and repair , signal transduction mechanisms  and transcription   .To better understand incorporation of genes into diverse pathways, all annotated transcripts were mapped against the KEGG database for pathway-based analysis. As a result, 6,130 transcripts out of a total of 46,841 were assigned to a KEGG pathway, and were present in 328 different KEGG pathways. The KEGG pathway distribution is summarized in M. amalphitanum and the available transcriptome assemblies of other parasitoid wasps from the families Trichogrammatidae  and Braconidae including Cotesia vestalis (a diamondback moth parasitoid), Diachasma alloeum (an apple maggot parasitoid) and Fopius arisanus (tephritid fruit fly parasitoid) were used for comparative analysis of the most represented gene functions in parasitoids. We also used transcriptome assemblies from the Agaonidae fig wasp, Ceratosolen solmsi. We found significant similarities between M. amalphitanum, T. pretiosum and C. vestalis major GO enrichment categories  using OrthoMCL [krueppel-1, knirps or short gastrulation [Given the incomplete nature of the OrthoMCL . The corOrthoMCL . Our finrulation ).M. amalphitanum is associated with significant gene loss that could be detected even in a partial genome assembly, we used genomic data of six larger hymenopteran species , as well as the well-annotated genome of the honeybee (A. mellifera) as reference (body sizes are presented in S5 Table). We mapped the M. amalphitanum (DNA-library1), T. pretiosum, C. vestalis, C. floridanum, F. arisanus, N. vitripennis, N. giraulti DNA reads on the A. mellifera genome sequence    in TBLASTN searches reduced the number of putatively missing genes to just five: centrosomin, phosphoglycerate mutase 5, phosphoglycerate mutase 5–2, 26S proteasome complex subunit DSS1, and mucin-1/nucleoporin NSP1-like. We detected short M. amalphitanum genome sequences encoding protein fragments (~8–23 amino acid residues) with some similarity to four of them, suggesting that they may be in the process of degeneration in this species. Despite a thorough search, we were unable to find any homologous sequence related to centrosomin (cnn) gene either in the partially assembled genome or in our cDNA libraries. Although cnn is regarded as rapidly evolving [T. pretiosum, suggesting that this gene is specifically absent in M. amalphitanum. In Drosophila melanogaster, Cnn has important roles at the centrosome in mitotic spindle formation, cytoskeleton organization and neuronal morphogenesis [cnn remains tentative. Globally, however, the analysis of the available genome assembly argues for relatively little gene loss in M. amalphitanum. Confident identification of true gene losses in this species will require additional DNA sequencing and improved genome assembly.To determine whether miniaturization in NA10625) , and detibrary3)  and obseibrary3) . Howeverevolving , sequencogenesis , 32, altogenesis . Since tM. amalphitanum, whose central and peripheral nervous systems are highly reduced [M. amalphitanum precluded accurate annotation of full-length sequences in this species for the majority of loci. Nevertheless, comparison with chemosensory receptor repertoires of other insects allowed us to define probable orthologous relationships with receptors of known function in other species and obtain initial estimates of the size of each family.Chemosensory receptors are encoded by some of the largest gene families in insect genomes, reflecting their important and wide-ranging roles in detection of environmental odors and tastants. We asked how these gene families have evolved in  reduced , 14. TheIr8a, Ir25a , Ir93a and Ir76b), as well as four genes encoding tuning IRs related to acid-sensing receptors in other species. We also identified orthologs of IR68a, which functions in hygrosensation [M. amalphitanum is therefore very similar in size and content to that of N. vitripennis [The most deeply conserved family of chemosensory receptors in insects are the Ionotropic Receptors (IRs), which are distantly related to ionotropic glutamate receptors , 38. IRsensation  and IR21ensation , 41. OveM. amalphitanum ortholog of Orco and 83 additional Or-related sequences. We caution that many of these Or sequences are small fragments (often located near the end of the assembled contigs), so it is currently difficult to determine whether these are intact genes or pseudogenes. Within the GR repertoire, we identified genes encoding proteins related to GR43a, a sensor of both external and internal fructose [Gr gene fragments. The sizes of these repertoires are smaller than in N. vitripennis (300 Ors (including 76 pseudogenes) and 58 Grs (including 11 pseudogenes) [Meteorus pulchricornis and Macrocentrus cingulum [Insects possess a second superfamily of chemosensory ion channels–distinguished by a heptahelical protein structure–comprising Odorant Receptor (OR) and Gustatory Receptor (GR) subfamilies, which generally function in detection of volatile and non-volatile stimuli, respectively –45. Simifructose , two othfructose , and 25 dogenes) ), but sicingulum , 50. HowM. amalphitanum has retained the conserved chemosensory receptors of larger wasps (and other insects), and appears to have numerous additional order- or species-specific receptors to allow detection of environmental chemical cues.In sum, these analyses reveal that despite drastic nervous system reduction, N. vitripennis toxin constituents in M. amalphitanum and other parasitoid wasps , using previously published venom data [). We identified 28 transcripts encoding putative venom proteins  constitute a measurable fraction of virtually all eukaryotic genomes, and can play important roles in their function and evolution. In insects, TE activity has been implicated in evolution of eusociality, based on comparison of ten bee genomes with increasing degrees of social complexity . We perfdominula , 25, 57.de novo TE identification . The N. vitripennis genome was 32.5% repetitive, in close agreement with the published estimate [D. alloeum was highly repetitive at 52.8% , and Reestimate , and D. opterans , 59 . We note, however, a recent comprehensive study [Wolbachia infection or other representatives of the Rickettsiaceae family were found in M. amalphitanum individuals [T. pretiosum carries the Wolbachia symbiont [Nasonia strain was maintained on antibiotics to cure it of infection.While TE dynamics may be affected by different factors, the observed drop in active TE content in families , 60, 61.families , howeverve study  describeividuals , while tsymbiont ; the seqM. amalphitanum, including Dicer (Dcr)-like and Argonaute (Ago)/Piwi-like protein-coding genes. In insects, Ago-1 and Dcr-1 homologs represent the key components of the miRNA pathway; Ago-2 and Dcr-2 mediate antiviral RNA interference; and Piwi and Ago-3/Aub suppress TE activity in the germline [M. amalphitanum and T. pretiosum possess equal numbers of Dcr-1 and Dcr-2 homologs, as well as Ago-2 and Ago-3 homologs , weM. amalphitanum integrase-related TE transcripts showed that high-copy hits represent MITEs  will be essential to confidently assess specific genetic adaptations that may be linked with body miniaturization.Our study provides a first view of the genomic content of one of the smallest insects currently known, the parasitoid wasp M. amalphitanum displaying a relatively recent decline in TE activity preceded by a burst, a pattern not observed in most other parasitoid wasps. The decline in TE activity may have been associated with evolution of additional Ago and Piwi copies, not present in T. pretiosum, which could have reinforced the genome defense machinery to prevent uncontrolled TE expansion. This hypothesis is strengthened by identifying duplications of Piwi-like proteins accompanied by a decline in TE activity over time in two additional species of Hymenoptera; by contrast, most other hymenopterans show no such decline.Interestingly, transposable element dynamics over time were found to differ substantially between the analyzed wasp species, with The relationship between body size and genome size has been discussed for a long time. Significant correlations of these values have been described for flatworms and copepods ; by contS1 FigK-mer sizes were matched for ABySS, SPAdes and Velvet. Note: CLC Genomics Workbench does not use k-mer size; CLC assembly was performed with default settings, and the statistics are given in (PNG)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIFF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 FigY-axis: number of genes; X-axis: number of hymenopteran genomes analysed.(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 FigMultiple alignments of CDS sequences were performed using Muscle v3.8 with default settings. Phylogenetic trees were generated under the maximum likelihood criterion using PhyML 3.0 . All manipulations of phylogenetic trees were performed using FigTree. Scale bar, nucleotide substitutions per site.(PNG)Click here for additional data file.S13 Fig(PNG)Click here for additional data file.S14 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


Rhagoletis fly (Diptera: Tephritidae) pests, apple maggot fly, Rhagoletis pomonella , and western cherry fruit fly, Rhagoletis indifferens Curran, in Washington State (WA), U.S.A. using biologically relevant environmental variables. We tested the hypothesis that abundance of the two species is influenced by different environmental variables, based on the fact that these two species evolved in different environments, have different host plants, and that R. pomonella is an introduced pest in WA while R. indifferens is native. We collected data on fly and host plant abundance at 61 randomly selected sites across WA in 2015 and 2016. We obtained land-cover, climate, and elevation data from online sources and used these data to derive relevant landscape variables and modeled fly abundance using generalized linear models. For R. pomonella, relatively high winter mean minimum temperature, low elevation, and developed land-cover were the top variables positively related to fly abundance. In contrast, for R. indifferens, the top variables related to greater fly abundance were high Hargreaves climatic moisture and annual heat-moisture deficits (indication of drier habitats), high host plant abundance, and developed land-cover. Our results identify key environmental variables driving Rhagoletis fly abundance in WA and can be used for understanding adaptation of insects to non-native and native habitats and for assisting fly quarantine and management decisions.Well-adapted and abundant insect pests can negatively affect agricultural production. We modeled the abundance of two  The threat of invasive insects including those that threaten agriculture is a function of how well they adapt to their non-native habitats and ultimately their abundance in or near agroecosystems –4. Pest Habitat heterogeneity and complexity as governed by vegetation type, structure, topography, and host abundance may contribute to abundance of some insect species . InsectsRhagoletis pomonella  (Diptera: Tephritidae), is a major quarantine pest of commercial apple , which is the top fruit commodity in the region. In Washington State (WA) alone, apple production in 2016 was valued at U.S. $2.4 billion  Eaton)  Eaton) . Howeverid-1800s ,15. The stern WA , indicatR. pomonella and R. indifferens are not the same due to differences in the flies’ environmental niches ) in western North America [The climatic conditions of the study sites were represented using climate variables obtained from the ClimateWNA program , 35. Cli, U.S.A. , and the America .R. pomonella and R. indifferens abundance at the 61 locations were modeled using host abundance data, landscape metrics, and topographic and climate variables. We used linear models for preliminary analysis and investigated the magnitude and direction of relationships among the response and predictor variables (each variable was tested separately). Abundance data were log-transformed (log10+1) prior to linear modeling. This preliminary analysis helped us identify the important variables and reduced the variables from more than one-hundred to just 20. In addition, cross-correlation tests conducted in R statistical software [R. pomonella and R. indifferens, choosing one representative variable out of each set of highly correlated variables (Pearson’s correlation r ≥ ± 0.7) from the models. Despite a slightly higher correlation with the climate variables, we kept elevation in one of the models (the R. pomonella model), because elevation improved model results when either used separately or in combination with other variables.Relationships with host availability, climate variability, and landscape heterogeneity have been modeled using different approaches including linear models and their extensions , 39. In software  indicateglm function and R packages including stats, MASS, and Hmisc [For the final analyses, generalized linear models (GLM) were selected based on the assumption that our non-transformed fly count data have a Poisson distribution. GLM fits less-complex models, and its results are relatively easy to interpret. The statistical analyses were conducted in R statistical software environment using the nd Hmisc . We ran nd Hmisc , 42.R. pomonella and R. indifferens abundance across western, central, and eastern WA, the mean numbers of flies caught on traps per county in each region were calculated. The numbers of counties included in the study within each region were replicates. Western WA comprised seven counties, central WA four counties, and eastern WA eight counties . Within each county, there were one to nine sites (= traps). Means for each county were calculated based on data from the two seasons. Because the data did not meet the assumption of normality, a Kruskal-Wallis test was conducted on rankings of abundance within each species across regions.To test for differences in R. pomonella abundance in the final GLM model included winter mean minimum temperature (Tmin_wt), elevation, and four different land-cover types . The mean numbers of R. pomonella caught ± SE in western, central, and eastern WA were 12.6 ± 9.0, 0.1 ± 0.1, and 0.6 ± 0.5, respectively. There was no difference in numbers of flies caught in central and eastern WA.Our results indicate that western WA counties had greater counties . StatistR. indifferens abundance in the GLM model included winter Hargreaves climatic moisture deficit (CMD_wt), annual heat-moisture index (AHM), host plant abundance, and three different land-cover types , the abundance of R. indifferens also increased . The mean numbers of R. indifferens caught ± SE in central, western, and eastern WA were 50.4 ± 21.6, 7.1 ± 3.9, and 17.5 ± 12.6, respectively.In contrast to omonella , R. indistern WA . The numR. pomonella and R. indifferens is influenced by different environmental variables. Results show how two members within a genus of frugivorous insects can have different habitat requirements to maximize population growth, as measured by abundance, even though they are found in broadly overlapping habitats. This may not be surprising given the relatively distant phylogenetic positions of the two species within Rhagoletis [R. pomonella is more abundant in the relatively wet region of western WA than drier central WA while the reverse is true of R. indifferens.The results of our study support the hypothesis that abundance of agoletis  and theiR. pomonella in WA is most abundant and therefore survives optimally in areas with warmer winters. These areas are the lower-lying regions west of the Cascade Mountain Range. The lower abundance and apparent poorer survival of R. pomonella in colder regions may be related to where the fly evolved in eastern North America. Winter temperatures where R. pomonella evolved in eastern North America are generally warmer than those at higher elevations in WA [Our analyses show that ns in WA –48. HoweR. pomonella abundance, probably because higher elevation is directly related to lower temperatures. Ultimately, R. pomonella may have poorer survival in the colder, drier upland forests or foothills of the Cascade Mountains than in the warmer, lower elevations west of the Cascades. Fly abundance in relation to three measures of temperature  were not among the top predictors of eriments , 21. HowP = 0.017) suggests that lack of human intervention by way of irrigation or planting of host trees slightly reduces the chances of large R. pomonella populations establishing.In addition to the developed category, shrub-scrub, cultivated crops, and pasture-hay categories were also positively significant, even though fly abundance in the latter was low . For shrR. pomonella, for R. indifferens most of the climatic variables had a positive relationship with abundance. The top variables most positively related to R. indifferens abundance in WA in the linear model were related to evapotranspiration and moisture deficit, i.e., drier conditions. These results suggest dry conditions are more conducive to high R. indifferens abundance than wetter conditions. Evapotranspiration is the sum of evaporation and plant transpiration from the Earth’s land and ocean surface to the atmosphere, called the reference evapotranspiration (ET0) [R. indifferens [R. indifferens abundance across WA (In contrast to on (ET0) –54. Althon (ET0) , air vapon (ET0) . Consistifferens . Furthercross WA  indicateR. indifferens abundance. However, host abundance was not among the top variables related to R. indifferens abundance in the linear model, suggesting that there are differences in fly abundance given equal tree abundance between western and central WA. Cherry trees in western WA seem abundant enough to support larger fly populations, yet fly populations there are lower than in central WA. Drier climate positively affecting host fruit loads and other ecological differences may be reasons. Under the relatively wet, humid, and warm winter climate in western WA, cherry set may be lower than in central WA; trees may be subject to more fungal growth, e.g., brown rot (Monilinia spp.) [Cherry host abundance was one of the significant variables in the GLM model, suggesting areas that are hot and dry with the most unmanaged cherry trees will have the greatest ia spp.) ,57, and R. pomonella above, is associated with residences, lawn grasses, and thus irrigated habitat. Furthermore, as for R. pomonella, the cultivated crops category was significant as well, probably because cultivated sweet cherry trees are common in this land-cover class. This suggests the abundance of R. indifferens is dependent on the availability of sweet cherries rather than irrigation per se. For example, if urban areas were planted with less preferred host plants than sweet cherry, then perhaps the developed land-cover class would not be significant due to lower host quality. If true, then the significance of the developed land-cover class is more likely due to host plant quality than irrigation. The categories grassland-herbaceous and wetlands were highly significant but negatively so, likely because most host trees are unable to survive in these habitats.The developed land-cover category was significant in the GLM model, as this variable, also discussed in relation to R. indifferens to survive in drier habitats with high sweet cherry abundance has increased the range and abundance of the fly since pre-human settlement. There may be more R. indifferens now in WA than before colonists from the eastern U.S. arrived with their cultivated cherry trees and began irrigating the region in the mid-1800s [Results indicate that planting of cultivated cherry trees in dry, hot irrigated habitat had unanticipated consequences for pest management. The ability of id-1800s .R. pomonella is to keep it out of commercial apple orchards to maintain export markets [R. pomonella larva in commercially-packed apples from WA [R. pomonella for apple export purposes include risk assessments for apple orchards and establishment of Areas of Low Pest Prevalence or Pest Free Areas (ALPP or PFAs) [R. pomonella in those areas. In our study, apparently due in large part to low winter temperatures, no R. pomonella were detected in Okanogan, Stevens, Pend Oreille, Asotin, Garfield, Chelan and Walla Walla Counties. Washington State Department of Agriculture did detect R. pomonella in Okanogan County in 2017 [Our results can be used in fly management efforts. The goal for managing  markets , 58. The from WA . Implicaor PFAs) , which w in 2017  after ouRhagoletis indifferens is occasionally found infesting commercial cherries at low levels [R. pomonella, ALPP and PFAs do not seem possible for R. indifferens in the dry commercial cherry-growing areas in central WA as it is abundant there. To reduce the threat of R. indifferens invading and then infesting cherries in orchards, unmanaged cherry trees within 0.29 km of cherry orchards  need to Wallace . Our finRhagoletis pests in WA. Furthermore, we explained the observed abundance and distribution patterns of these pests in relation to climatic factors, host abundance, land-cover, and landscape metrics. Our results identify pest abundant sites and can be used for understanding adaptation of insects to non-native and native habitats and for aiding fly quarantine and management decisions. Experimental work on proximate and ultimate causes of the relationships we established in this study will be needed to more fully understand the intricacies of factors affecting fly population abundance.In summary, we used primary survey data, biologically relevant environmental variables, and modeling approaches to assess the abundance of two S1 FigR. pomonella abundance while (B) winter mean minimum temperature (Tmin_wt), (C) winter degree days below 5°C (DD5_wt), and (D) frost-free period (FFP) had positive relationships with R. pomonella abundance.(A) elevation had a negative relationship with (TIF)Click here for additional data file.S2 FigR. indifferens abundance, suggesting that moisture deficit plays a significant role in explaining the distribution of the pest in the state. (A) CMD = Hargreaves climatic moisture deficit, (B) CMD_wt = winter Hargreaves climatic moisture deficit, (C) SHM = summer heat-moisture index, (D) Eref = Hargreaves reference evaporation.Most of the climatic variables used in the analysis had a positive relationship with (TIF)Click here for additional data file.S1 AppendixThe following land-cover categories were found within 125 m radius of the trap locations.(DOCX)Click here for additional data file.S2 AppendixThe following annual and seasonal climate variables were obtained using ClimateWNA program—V.5.30.(DOCX)Click here for additional data file.


Context: The Boraginaceae family comprises plants that have important therapeutic and cosmetic applications. Their pharmacological effect is related to the presence of naphthaquinones, flavonoids, terpenoids, phenols, or purine derivative – allantoin.Objective: In the present study, comparison of some secondary metabolite content and phytochemical relationship between 17 species of the Boraginaceae family were analyzed.Materials and methods: High performance capillary electrophoresis (HPCE) was used to perform a chemometric analysis in the following Boraginaceae species: Anchusa azurea Mill., Anchusa undulata L., Borago officinalis L., Buglossoides purpurocaerulea (L.) I.M. Johnst., Cerinthe minor L., Cynoglossum creticum Mill, Echium italicum L., Echium russicum J.F. Gmel., Echium vulgare L., Lindelofia macrostyla (Bunge) Popov (syn. Lindelofia anchusoides (Lindl.) Lehm.), Lithospermum officinale L., Nonea lutea (Desr.) DC., Omphalodes verna Moench , Pulmonaria mollis Wulfen ex Hornem., Pulmonaria obscura Dumort., Symphytum cordatum Waldst. & Kit ex Willd., and Symphytum officinale L.Results: Six active compounds in shoot extracts  and four compounds in root extracts  were identified. The presence and abundance of these compounds were used for the characterization of the species and for revealing their phytochemical similarity and differentiation.Discussion and conclusion: The present study provides the first comprehensive report of the extraction and quantification of several compounds in Boraginaceae species (some of them for the first time). Among the 17 species studied, species with potentially high pharmacological activity were recognized. Borago officinalis L. or Echium vulgare L., have been recognized as a good source of unique fatty acids γ-linolenic acid  and stearidonic acid   I.M. Johnst., Cerinthe minor L., Cynoglossum creticum Mill, Echium italicum L., Echium russicum J.F. Gmel., Echium vulgare L., Lindelofia macrostyla (Bunge) Popov (syn. Lindelofia anchusoides (Lindl.) Lehm.), Lithospermum officinale L., Nonea lutea (Desr.) DC., Omphalodes verna Moench , Pulmonaria mollis Wulfen ex Hornem., Pulmonaria obscura Dumort., Symphytum cordatum Waldst. & Kit ex Willd., and Symphytum officinale L.) were analyzed. The phytochemical analysis of these species was conducted in two steps. First, a traditional strategy based on qualitative and quantitative analysis of several compounds in the plant extracts by high-performance capillary electrophoresis (HPCE) was employed. In the second strategy, multivariate chemometric techniques were applied to reveal similarities and differences between the Boraginaceae species studied.In the present study, some secondary metabolites content and phytochemical relationships between 17 Boraginaceae species  was extracted with 4 mL hexane in an ultrasonic bath for 30 min (extraction for shikonin derivatives). Afterwards, the extract was centrifuged at 10,000 g for 5 min and the supernatant was evaporated; next, the residue was dissolved in 2 mL of methanol and filtered through a 0.22 μm membrane filter.Fifty-one individuals of 17 species of the Boraginaceae family   (190–600 nm) . The separation was performed according to the modified method described previously  method with the max shift parameter set at 10  and four compounds in the roots . Rosmarinic acid and allantoin were present in all the species studied , whereas the other compounds were present in a detectable quantity only in some species  to chemical reference standards, we identified and quantified several biologically active compounds: six compounds in the shoots  has been used for centuries as a poultice for treating wounds or burns and in treatment of rheumatism  were found in the roots of E. italicum. As a heterocyclic nitrogen compound, allantoin plays a role in nitrogen metabolism in plants . Moreover, its role in the chemical interactions between plants and other organisms, including insects and microorganisms, has been shown  of this compound was found in the roots of A. undulata. Moreover, the shoots of C. minor, O. verna, P. obscura and S. cordatum could also be a good source of rosmarinic acid.Some earlier reports indicated presence of rosmarinic acid in Boraginaceae plants  in several Boraginaceae species. Among these compounds, rutin exhibits significant biological activities such as anti-oxidant, anti-inflammatory, anti-diabetic and anti-adipogenic effects  contained hydrocaffeic acid, and chlorogenic acid was detected in the shoots of seven Boraginaceae species. However, the content of these compounds was in general relatively low – below 0.1%.Although some authors reported the presence of rutin in the cts Chua . Of the Lithospermum erythrorhizon Siebold & Zucc, Arnebia euchroma (Royle) I.M. Johnst, Arnebia guttata Bunge, Onosma paniculatum Bureau & Franch, Onosma hookeri C.B. Clarke  and from L. officinale. It should be noted that the presented values represent the sum of shikonin derivatives since shikonin in the Boraginaceae plants usually occurs in many derivative forms  generally accumulating high concentrations of allantoin and rosmarinic acid, while the other subcluster grouped six species .Based on the phytochemical data, chemometric analyses were performed separately for the shoots and roots. Generally, two peaks of the two main compounds, allantoin and rosmarinic acid, together with some unidentified compounds eluting at 9–10 min had the greatest influence on the grouping process. The hierarchical cluster analysis (HCA) of the shoot data revealed species grouping in two main clusters . One cluoot data . The firp-hydroxybenzoic acid, rutin, hydrocaffeic acid, rosmarinic acid, chlorogenic acid, and shikonin) in Boraginaceae plants. Among the studied Boraginaceae species, some with potential high pharmaceutic activity have been distinguished. Besides S. officinale, which is commonly known as a source of allantoin, E. italicum appears to be another species that is rich in this compound (approx. 3.5% in the roots). Moreover, some species, e.g. B. purpurocaerulea, P. molis, A. undulata and A. azurea, could be good sources of rosmarinic acid for pharmacological purposes.Several biologically active compounds were identified and compared between 17 Boraginaceae species. The use of two modes of data acquisition  showed the phytochemical profile of each species and allowed recognition of plants that could be a potential source of important biologically active compounds. For the first time, the report has shown extraction and quantification of several compounds (allantoin,


Ensifera ensifera and its guild of long-flowered plant species. We combined occurrence data and environmental variables to predict E. ensifera distribution, in addition to range polygons available for plant species in order to evaluate the geographical variation in bill length and plant species richness. A positive relationship between bill length and plant species richness within the E. ensifera range suggests a geographical structuring of the interaction networks. At mid-latitude locations of E. ensifera range, where hummingbirds attained the longest bills, richness of long-flowered plant species was higher than at low latitude locations. These locations likely represent coevolutionary vortices where long-lasting reciprocal selection probably drove the evolution of long traits, consequently drawing new plant species into the coevolutionary network. Conversely, areas where the sword-billed hummingbird was absent or had shorter bills probably represent coevolutionary coldspots. Our results provide a first insight into this phenotypically specialized plant-pollinator network across the landscape and show candidate areas to test the predictions of the coevolutionary hypothesis, such as reciprocal selection.The coevolutionary process among free-living mutualists with extremely long matching traits may favor the formation of mutualistic interaction networks through coevolutionary escalation, complementarity and convergence. These networks may be geographically structured; the links among the species of a local network are shaped by the biotic composition of the community, thus creating selection mosaics at broader geographical scales. Therefore, to fully understand a coevolutionary process, it is crucial to visualize the geographical structure of the interaction network across the landscape. In this study we focused on the poorly known interaction system between  Ensifera ensifera Bois.), which bears by far the longest bill among pollinating birds [For nearly every pollinator guild, species bearing extremely long reward-collecting appendages have evolved. These include a fly ,2 and a ng birds . The appng birds  was inspng birds . Only inThese relationships among free-living mutualists often do not consist of pairwise interactions, but rather of species that locally interact with a guild of several partner species. Consequently, the coevolutionary process among free-living mutualists favors the formation of mutualistic networks through coevolutionary complementarity and convergence –14. SeveTo our knowledge, the effect of coevolutionary escalation has been focused on free-living antagonists, rather than on free-living mutualists, as a coevolutionary process concurrent with complementarity and convergence. In antagonistic interactions, coevolutionary escalation is expected to drive away species from interaction networks, promoting a higher ecological specialization and eventually pairwise coevolution ,18. ContE. ensifera and its guild of plants, whose flowers are exclusively pollinated by the sword-billed hummingbird [Passiflora species has similar divergence time to that of E. ensifera. Although specific literature on the interaction ecology of this hummingbird and the guild of plants that it pollinates is surprisingly scarce, the sword-billed hummingbird is known to visit a set of long-flowered species [E. ensifera is known to vary geographically [Studies on plant-hummingbird networks have shown that hummingbirds that best match flower length improve nectar intake and that plants whose flowers that best match hummingbird bill lengths benefit from stronger interaction ,20. Hencmingbird ,21,22. Smingbird , who sho species ,24–26. Sphically , long-flphically ).E. ensifera acquired the longest bills, i.e., where coevolutionary escalation has been strongest or most persistent in time, thus representing possible coevolutionary hotspots. Conversely, since short-billed hummingbirds would not be the most efficient pollinators for the long-flowered guild of plants [E. ensifera either is absent or did not acquire long bills. Species distribution models (SDMs) can be used to project species range by combining occurrence data and environmental variables [E. ensifera and used available range polygons of the guild of long-flowered species, to estimate overlapping ranges. Community studies suggest that very long matching traits are shaped by diffuse coevolution, thus pollinator with long appendages interacts more frequently with long-tubed plants [We hypothesized that reciprocal selection would have occurred or may be occurring at sites where f plants ,20, coevariables . In thisd plants . TherefoE. ensifera, is distributed in the high Andean forests  in Venezuela, Colombia, Ecuador, Peru and Bolivia. It exhibits the longest bill among hummingbirds, ranging between 81 and 120 mm in length , nearly as long as the body [The sword-billed hummingbird, the body ,27.E. ensifera, since no other hummingbird pollinator in its Neotropical communities would be able to pollinate the deepest flowers  to differentiate them from similarly long hawkmoth-pollinated species. The threshold of 80 mm was chosen as a conservative limit of inclusion, since this is the minimum bill length of E. ensifera. We also searched for long-flowered species among the specimens deposited in different herbaria . These procedures resulted in a list of 24 species with mean corolla lengths from 95.59 to 146 mm potentially pollinated by the sword-billed hummingbird . We commingbird , S1 Fig.mingbird . Followiensifera .E. ensifera was obtained from 51 georeferenced museum records of male individuals published by Sánchez Osés [E. ensifera individuals by fitting a generalized linear model (GLM) with gamma error distribution and log link function in R [We measured corolla tube length and flower operative length (i.e. distance from anthers and stigma to nectary) of the 24 species. Measurements were taken from 2 to 44 individuals per plant species, either in the field or from voucher specimens deposited in different herbaria . Bill lehez Osés . Mean vaion in R . The modE. ensiferawere obtained from the Global Biodiversity Information Facility (http://www.gbif.org/). If information source was ambiguous, occurrence points were discarded for greater reliability.To reduce potential sampling bias, initial georeferenced points were spatially thinned; to this end, clusters were eliminated by keeping 4 km as the minimum distance between points . OccurreE. ensifera using one topographic  and 19 bioclimatic variables obtained from WorldClim database (http://www.worldclim.org/bioclim) with a spatial resolution of 2.5 arc-min. SDM was performed from presence-only records via maximum entropy method using the program MAXENT v.3.3.3k [Species distribution was modeled for v.3.3.3k . Before v.3.3.3k . Optimalv.3.3.3k . Model pv.3.3.3k . In addiv.3.3.3k . Backgrov.3.3.3k . The finv.3.3.3k . The 10 S. dombeyi, A. mutisii, P. bracteosa, P. leptomischa, and P. tenerifensis. Thus, these plant species were not included in the following analyses.Range polygons were obtained for 19 of the 24 plant species of the guild from the BIEN package of R . Range pE. ensifera was converted into presence-absence maps considering the 10 percentile threshold. For the guild of plants, a map was generated by adding all the plant species range polygons (“richness map”). To quantify the areas of overlap between E. ensifera and its plant guild and reciprocally, i.e. between the plant guild and E. ensifera, the number of overlapped pixels on the resulting maps was counted and converted to km2. A visual representation of the number of plant species overlapping with E. ensifera along its range was generated by trimming the richness map to fit the SDM of E. ensifera.The SDM of To determine the relationship between bill length and plant richness we fitted a GLM with a gamma error distribution using the 51 bill length records of male hummingbird. We visualized the regressions using the ggplot2 package in R .P. parvifolia up to 146 ± 2.70 mm in P. loxensis  and long-flowered species of Passiflora supersection Tacsonia (11.6 ma) [E. ensifera. A similar pattern was also evidenced in the pollination mutualism between the extremely curved-billed Eutoxeres hummingbirds and their matching plant species [The high geographical overlap between 11.6 ma) ,52. In a species .E. ensifera guild, mean operative flower length was greater than bill length, as expected for morphological complementarity in coadapted plant-pollinator relationships .Xantopham morganii praedicta and the long-spurred orchid A. sesquipedale, other long-spurred species have been proposed as possible food plants [Ensifera ensifera bill length varied across the geographical range, possibly creating differences in the degree of asymmetry of interactions across the landscape, as expected under a geographical mosaic of coevolution [Extreme one-to-one specialization is rare in plant-pollinator systems, since the flower resources exploited by the nectar-feeding visitors are ephemeral, rendering the dependence on a single plant species unreliable. Even in the highly specialized interaction between the long-tongued hawkmoth d plants . Plant-pd plants , with lod plants . Our resvolution .E. ensifera distribution. There, E. ensifera exhibited the longest bills, probably acquired by escalated reciprocal selection, which presumably drew several species to converge matching traits. We cannot ascertain whether escalation, if present, was driven by pairwise interaction and later increased in the number of interacting species, or if the joint action of the plant guild has driven coevolution in a diffuse manner. Both possibilities are suggested by the high richness of the plant community in these areas. Despite a non-significant relationship, probably due to the small sample size, we observed a positive trend between bill length and species plant richness. Accordingly, a study on the relationship between proboscides length of a long-tongued fly and the long-flowered guild it pollinates showed that morphological traits of pollinators were more exaggerated at sites where the local plant community consisted of several species with even longer tubes than the proboscides [We detected an area where species are predicted to participate in a “coevolutionary vortex” at mid-latitudes of boscides . HoweverE. ensifera are much shorter than floral tubes, unilateral selection of bills is the most likely scenario, since the hummingbirds with the longest bills will be favored. We expect this to happen in the south of the E. ensifera range, where bills are short (mean = 96.91 mm) and plant richness is low. In these areas, P. tripartita, P. mixta and B. sanguinea overlapped with E. ensifera, and their flower tubes are much longer  than the bill length. Based on these facts, we postulate that these areas represent coevolutionary coldspots. When both hummingbird and flower match at low trait magnitudes, we expect an incipient hotspot since we assume that escalated reciprocal selection will likely shape long complementary traits over time. Other coldspots are represented by areas where E. ensifera was absent within the range of the plant guild. Plants might colonize other areas outside the range of this hummingbird, where abiotic conditions are favorable, via alternative modes of reproduction .In a geographical context, the coevolutionary scenario in some communities may be intermingled with other communities in which selection is one-sided or absent . For insE. ensifera range, where the whole plant guild overlapped with the hummingbird and where its bill was longest. On the other hand, we detected presumptive coevolutionary coldspots where the hummingbird either has short bill or is absent. Our study provides an approach to the geographical dimension of coevolution in this phenotypically specialized plant-pollinator system. It is important to note that other selective forces than those exerted by flower-operative length could be determining the longest bills attained in the mid-latitudes of hummingbird distribution. For instance, long bills could reflect a selection to reduce interspecific competition for food resource [Our results using SDMs suggest a geographical structuring of the relationship between the sword-billed hummingbird and its guild of long-flowered plants. We observed a pattern expected under a geographical mosaic scenario where the overlapping ranges of interacting species varied across the landscape. On the one hand, we observed a possible coevolutionary vortex area at mid-latitudes within resource . Meanwhiresource . TherefoS1 FigEnsifera ensifera: (a) Aetanthus dichotomus (Lorantaceae), (b) Brugmansia sanguinea (Solanaceae), (c) Salvia dombeyi (Lamiaceae), (d) Passiflora mixta (Passifloraceae), (e) P. tripartita var. mollisima (Passifloraceae), (f) P. cumbalensis (Passifloraceae), and (g) Salpichroa didierana (Solanaceae).Representative plant species pollinated by the sword-billed hummingbird Photo credits: (a) A. Kay*, (b) and (e) S. Leiva González, (c) and (g) A.A. Cocucci, d) L. Agudelo*, and (f) R. Culbert*. Scale bars equal to 1 cm. * Photo shared by Flickr.com.(EPS)Click here for additional data file.S2 Fignaturalearthdata.com.Probability of occurrence averaged after 10 cross-validation runs at the potential suitable habitat based on the SDMs is indicated with yellow to red colors. Background vector maps of was obtained from the public domain dataset Natural Earth @(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Myxomas are the most common type of cardiac benign tumors and most of them are located in the left atrium, followed by the right atrium. The majority of Myxomas is located in the left atrium and has a variable clinical presentation. Myxomas affect patients within a wide age range (15–80 years), and the average age is approximately 50 years. There is a female predominance in the sporadic form. Myxomas are usually pedunculated, solitary, and sporadic but may be associated with familial autosomal dominant syndromes.We report a 38-year-old female presented with large myxoma in the right atrium and atypical presentation and successfully underwent surgical excision of right atrial myxoma. The patient was asymptomatic on 6 months follow up.Cardiac myxomas are the most frequent finding among primary cardiac tumors. Clinical manifestations depend on the involvement in valvular obstruction, distant arterial embolisms, or nonspecific, constitutional symptoms. Transoesophageal Echocardiography is the cornerstone for diagnosis of atrial myxoma. Cardiac myxomas should be managed with surgical resection. •Myxomas are the most common type of cardiac benign tumors and most of them are located in the left atrium, followed by the right atrium.•We report a case of 40-year-old female patient, presented with chest pain, epigastric abdominal pain, vomiting for 3 days, easy fatigability since 1 yr, intermittent episodes of presyncope since 1 year. No constitutional disturbances such as fever, weight loss, skin rash, myalgia or arthralgia. Cardiovascular system examination was normal with no clinically detectable murmurs.•The patient underwent open surgical resection of the RA mass with no pre-operative or post-operative complications Gross macroscopic examination showed a nearly spherical gelatinous mass of the size of a cricket ball measuring around 4 cm in diameter with a smooth surface and elastic consistency.•Histo-pathological examination confirmed the diagnosis of cardiac myxoma. Microscopic showing tumor displaying myxoid change and stellate cells having bland nuclei arranged in reticular meshwork. The patient was asymptomatic on 6 months follow up. Our case is important because of the atypical size and location of cardiac myxoma in the right atrium.•RA myxoma is considered as differential diagnosis of right atrial thrombus. Early diagnosis and treatment prevent the complications of thromboembolic events. Primary cardiac tumors are very rare, with an incidence of 0.001%–0.03% . More th83 years . With a 83 years . Most my1.1We report a case of 40-year-old female patient, presented with chest pain, epigastric abdominal pain, vomiting for 3 days, easy fatigability since 1 yr, intermittent episodes of presyncope since 1 year. No constitutional disturbances such as fever, weight loss, skin rash, myalgia or arthralgia. Cardiovascular system examination was normal with no clinically detectable murmurs. ECG showed sinus rhythm, incomplete RBBB with T inversion in leads III and avF. Chest X-Ray revealed normal. Transthoracic Echocardiography revealed a large mass in the right atrium, attached to the interatrial septum. Trans-Esophageal Echocardiography was done to clearly delineate the mass, which confirmed the attachment of RA mass to the interatrial septum in the region of fossa ovalis, with no extension into inferior vena cava, measuring 5.0 × 4.0 cm A–B. UltrOur case is important because of the atypical size and location of cardiac myxoma in the right atrium.2Right atrial myxoma accounts for only 15%–20% of all cardiac myxomas. It is usually found in the interatrial septum , but in 3RA myxoma is considered as differential diagnosis of right atrial thrombus. Early diagnosis and treatment prevent the complications of thromboembolic events. We report a 38-year-old female presented with large myxoma in the right atrium and atypical presentation and successfully underwent surgical excision of right atrial myxoma.Ethical commitee approved.None.Dr. H.S Natraj Setty: Writing the paper.Dr. Yeriswamy M.C: Data analysis.Dr. Santhosh Jadav: Data analysis.Dr. Jayashree Kharge: Reference Collection.Dr. T.R Raghu: Study Design.Dr. Rahul Patil: Study Design.Dr. B.K Geetha: Reference Collection.Dr. Veeresh Patil: Data analysis.Dr. Sathwik Raj: Data analysis.Dr. C.N Manjunath: Final Approval.Consent obtained.N/A.Dr. Natraj Setty H.S.Not commissioned, externally peer reviewed.None.