Prunus subgenus Cerasus (cherry) is an economically important group that distributed in temperate regions of the northern hemisphere. However, shared interspecific morphological traits and variability across taxa of Cerasus are among the impediments to taxonomic efforts to correctly delimit taxa. This is further complicated by a lack of genetic information on these taxa, with no focused genomic or phylogenetic studies being done on Cerasus. In this study, we conducted comparative analysis on the complete plastid genomes (plastomes) of 20 Cerasus species to gain a greater understanding of the attributes of the plastome of these taxa while helping resolve their phylogenetic placement in Prunus sensu lato and interspecific relationships within the subgenus. Our results displayed that (1) the plastomes of the 20 Cerasus species studied exhibited a typical quadripartite structure with conversed genome arrangement, structure, and moderate divergence. (2) The average size of complete plastomes for the Cerasus taxa studied was 157,861 bp, ranging from 157,458 to 158,024 bp. A total of 134 genes were annotated, including 86 protein-coding genes, 40 tRNAs, and 8 rRNAs across all species. In simple sequence repeat analysis, we found Cerasus had a comparable number of dispersed and tandem repeats to those identified in other angiosperm taxa, with only P. pseudocerasus found to contain trinucleotide repeats. Nucleotide diversity analysis revealed that the trnG-GCC gene and rpl32-trnL region had the highest Pi value showing potential as phylogenetic markers. (3) Two phylogenetic trees of the plastomes verified the monophyletic relationship of Cerasus and provided a more resolved species-level phylogeny. Our study provides detailed plastome information for exploring the phylogeny of subg. Cerasus taxa. We identified various types of repeats and nucleotide diversity hotspots, which can be a reference for species identification and reconstruction of phylogenetic relationships. Prunus L. subg. Cerasus (Mill.) A. Gray contains approximately 150 species that mainly distributed in temperate and subtropical regions of the northern hemisphere [Cerasus provides various edible cherries and ornamentals of economic value, and has great potential for development and application, the research on this economically important group is becoming more and more extensive. Through long-term hybridization and domestication of subg. Cerasus species, a large number of economically significant species, such as sweet cherry (P. avium), Chinese cherry (P. pseudocerasus), and variety of ornamental specie has been widely planted. In recent years, many new cultivars have been developed, indicating that the subg. Cerasus has great potential for development and utilization. However, interspecific hybridizations have complicated the taxonomy of this subgenus [Cerasus species resources. Although some studies have clarified the deep phylogenetic relationships and diversification history of Rosaceae and Prunus s.l. [Cerasus and was based on a minimal number of molecular markers [subgenus , making nus s.l. –10, only markers .The annotation and bioinformatics analysis of plastid genome play an important role in the classification and evolution of plant species, due to its unique genetic characteristics of maternal inheritance. A typical plastid genome (plastome) consists of a pair of inverted repeats (IR) regions that is separated by a large single copy (LSC) region and a small single copy (SSC) region, with standard plastome sizes ranging between 120 and 170 kb in length . CompareCerasus species from GenBank, aiming to reveal the complete structure of plastomes and hotspot regions among these 20 species while clarifying their phylogenetic placement. This information will be valuable for further evolutionary studies on subg. Cerasus and Rosaceae in general.In this study, we compared and analyzed the published complete plastomes of 20 subg. P. jamasakura and P. discoidea, since they are not endangered or protected species and were collected from the fields that are not privately owned or protected. We collected fresh young leaf samples for P. jamasakura and P. discoidea from Katano, Japan  and Huangshan, China . The leaf samples were quickly dried with silica gel in a zip lock plastic bag upon the sampling and stored at room temperature until further see.There are no specific permits required for obtaining the healthy and fresh leaves of Cerasus plants using a DNeasy Plant Mini Kit  following the manufacturer’s protocol. The extracted DNA were quantified in NanoDrop ND1000  and visualized in a 1% agarose-gel electrophoresis for the quality check. Illumina paired-end (PE) libraries (read length: 2 × 125 bp) with insert sizes of 270 to 700 bp for each of the two Cerasus species were constructed and sequenced on MiSeq platform  by Nanjing Genepioneer Biotechnologies Inc. . We removed poor quality reads (PHRED score of < 20) using the quality trim function implemented in CLC Assembly Cell package v. 4.2.1 .Total Genomic DNA were extracted from each of the two subg. P. cerasoides (GenBank accession: MF621234). The contigs obtained from the primary de novo assemblies were aligned to the reference CP genome, then the aligned contigs were assembled to each chloroplast genome in Geneious v7 (http://www.geneious.com). We annotated the CP genomes assembled using the online tool, DOGMA  [Prunus s.l., genus name Prunus was used to represent the subgenus name Cerasus. The circular plastome maps were visualized using OGDRAW v 1.3.1. The other annotated plastome sequences of 18 subg. Cerasus species we studied were all downloaded from GenBank and the accession numbers listed in We employed the low-coverage whole-genome sequence (dnaLCW) method  to assemnotator)  with a fnotator)  using thhttps://irscope.shinyapps.io/irapp/) was chosen to compare the boundaries between single copy regions (LSC and SSC) and inverted repeat (IR) regions among the 20 subg. Cerasus plastome sequences. The mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml) visualized the differences between the complete plastid sequences of 20 subg. Cerasus species in Shuffle-LAGAN mode with the annotated complete plastome of P. avium as a reference. To analyze nucleotide diversity (Pi), we applied the window size of 600bp with a 200bp step size and we extracted the shared 93 genes of 20 species in subg. Cerasus after alignment. The Pi value among the 20 subg. Cerasus species was calculated using the DnaSP v6 [The IRSCOPE  (http://pgrc.ipk-gatersleben.de/misa/) to identify SSR sequences and tandem repeats of 1–6 nucleotides were considered microsatellites. The minimum numbers of repeats were set to 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, respectively.REPuter  was selehttp://codonw.sourceforge.net/) with RSCU ratio for all protein-coding genes.We also analyzed codon usage to examine the distribution of codon usage using CodonW v1.4.2 (Cerasus in GenBank (http://tree.bio.ed.ac.uk/software/figtree/) [We selected the 20 previously sequenced plastomes from subg.  GenBank , and comigtree/) .Cerasus taxa studied have the typical quadripartite structure comprising long single copy (LSC), inverted repeat (IR) and small single copy (SSC) regions  deletion found in P. avium resulting in 77 protein coding genes for this species, with rps19 repeated twice in P. pseudocerasus (Lindl.) G. Don and P. yedoensis (Mats.) Yü et Li. The ycf1 gene also repeats twice in P. conradinae, P. discoidea, P. pseudocerasus and P. yedoensis. Compared with tRNAs in other subg. Cerasus species, P. campanulata and P. yedoensis lacked the trnfM-CAU gene. P. avium encodes the most abundant tRNA of all subg Cerasus species studied. And this species contains the unique trnS-AGA gene. At the same time, trnM-CAU & trnT-GGU are repeated twice in P. avium. Ten protein coding genes  contained one intron, two genes  contained two introns , while the protein coding gene ndhI, which is located in the LSC and SSC region, has the most nucleotide variation (0.00414). The IGS regions rpl32-trnL and trnR-atpA showed highly variable polymorphism (Pi > 0.05), with the values of nucleotide diversity 0.00822 and 0.00567, respectively  while the other 18 subg. Cerasus taxa showed codon usage bias (RSCU < 1) in 32 codons. All 20 subg. Cerasus species had 30 codons more frequently used than the expected usage at equilibrium (RSCU >1). Codons with A and/or U in the third position take up ~43% and ~47% of all codons respectively   are present across the 20 subg. ectively . Among tectively . The freSCU = 1) .Cerasus species range from 70 to 94  to 71.27% (P. leveilleana). Pentanucleotide repeats were detected in P. avium, P. kumanoensis, P. rufa, P. speciose, P. subhirtella, P. takesimensis and P. yedoensis, which accounts for the low proportion of SSRs in these seven species. Except for P. campanulata, P. subhirtella and P. yedoensis, the rest of these species all contain hexanucleotide repeats. Among all tested subg. Cerasus taxa, only one trinucleotide repeat is detected in P. pseudocerasus.SSRs, also called microsatellites, were extensively distributed in the plastome of all species studied. Using MISA software, we found the total number of SSRs in the 20 subg. 70 to 94 . Of the Cerasus species studied. Of all the subg. Cerasus species studied, P. avium contained the largest number of repeat types  analysis and Bayesian Inference (BI) analyses to perform phylogenetic analyses using the complete plastome data and CDS regions of 53 published complete plastid sequences  P. pseudocerasus is positioned at the basal node in Clade Ⅱc; c) P. kumanoensis and P. takesimensis formed a sub-clade in Clade Ⅱc; d) P. campanulata, P. spontanea and P. discoidea formed a sub-clade in Clade Ⅱc.When comparing topologies based on just plastome data or CDS regions, Clade Ⅱa and Clade Ⅱc exhibited differences . Using j regions , while tmowiczii . In bothCerasus is a challenge. The phylogenetic, diversity, and genetic relationships based on simple chloroplast markers and sequence data were previously reported in Subg. Cerasus [Analyzing the genetic background and diversity of Subg.  Cerasus ,9.Cerasus plastomes also exhibit the typical quadripartite structure and similar complete plastome sequence size of angiosperms, ranging from 157,458 bp in P. emarginata to 158,024 bp in P. discoidea  in the monophyletic subg. Cerasus clade, with P. pseudocerasus placed basal in Clade IIc [Cerasus was divided into two clades. However, the phylogenetic topology in our study based on two methods (ML and BI) found that subg. Cerasus was also divided into two major clades with high support but a short branch length. Clade Ⅰ contains P. cerasoides and P. rufa, with a central and southern Asian distribution.Plastid phylogenomics provides one possible solution for studies focused on problematic phylogenetic relationships . The rellade IIc . In theiP. emarginata + P. pensylvanica clade being placed in Clade IIa or IIc, depending on complete plastome or CDS data, respectively. CDS data also provided greater resolution on certain relationships, such as placing P. matuurae with P. speciosa in a clade that is rooted in a large polytomy in clade IIc. While the topology based on complete plastome shows no such relationship, with both species held in the large polytomy. The causes of the insignificant divergence of subg. Cerasus taxa may be due to quantum speciation or low resolution of the markers. Widespread interspecific hybridisations also have complicated the taxonomy of this subgenus [Placement of certain taxa within Clade II was also unresolved due to inconsistencies when comparing topologies based on complete plastome or CDS datasets. This includes the subgenus .P. emarginata and P. pensylvanica was notable, with these species sharing similar geographical distribution and habitat in North American. High support for the basal position of P. pseudocerasus in Clade IIc was also found in all analyses. Prunus campanulata, P. spontanea and P. discoidea formed a sub-clade in Clade Ⅱc. P. kumanoensis and P. takesimensis, these species with an eastern Asian distribution, also consistently formed a well-supported sub-clade in Clade Ⅱc in all analyses. While their relationship with P. emarginata and P. pensylvanica is still unresolved, P. itosakura, P. yedoensis and P. subhirtella showed a well-supported relationship and were consistently grouped in Clade Ⅱa.Nevertheless, certain relationships in Clade II were well-supported and consistent. Despite an unresolved placement within Clade II, the relationship between Cerasus species. We identified highly polymorphic regions of nucleotide variation that are potential molecular markers for identifying and overcoming phylogenetic problems at the species level. We found that SSRs in our plastomes might also provide intraspecific level polymorphic markers that can be used for population genetic studies.In this study, we revealed the plastome size, GC content, and gene number and order among 20 subg. P. cerasoides and P. rufa as basal in the monophyly of subg. Cerrasus, as well as a close relationship between certain groups of taxa. Undoubtedly, taxa of subg. Cerasus are a monophyletic group sister to taxa of Prunus s.l., but some infrageneric relationships within subg. Cerasus remain unresolved, as can be seen by certain low bootstrap values and changing positions of taxa depending on datasets used. Thus, it is particularly important to explore characteristics of repeat elements and in-depth information mining of plastomes in future research as well as inclusion of greater taxon sampling to help clarify these relationships.The phylogenetic trees constructed based on complete plastome and CDS regions all exhibited similar topologies, albeit with certain irregularities and discordances and a lack of resolution within certain clades. We find support for S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigCerasus species based on either (a) complete plastome data or (b) coding region (CDS) data. The support value is displayed above the branch in the order of Maximum Likelihood bootstrap support and Bayesian Inference posterior probability. “‐” indicates the branch collapse in the Bayesian tree.Combined ML and BI phylogenetic trees of 20 subg. (PDF)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.


Montivipera are among the paramount examples of Mediterranean neo-endemism, with restricted ranges in the mountains of Anatolia, the Levant, Caucasus, Alborz, and Zagros. Here we explore the phylogenetic and ecological diversification of Montivipera to reconstruct its ecological niche evolution and biogeographic history. Using 177 sequences of three mitochondrial genes, a dated molecular phylogeny of mountain vipers was reconstructed. Based on 320 occurrence points within the entire range of the genus and six climatic variables, ecological niches were modelled and used to infer ancestral niche occupancy. In addition, the biogeographic history and ancestral states of the species were reconstructed across climate gradients.The orogeny of the eastern Mediterranean region has substantially affected ecological speciation patterns, particularly of mountain-dwelling species. Mountain vipers of the genus Montivipera colonised coastal regions from a mountain-dwelling ancestor. We detected a highly complex ecological niche evolution of mountain vipers to temperature seasonality, a variable that also showed a strong phylogenetic signal and high contribution in niche occupation.Dated phylogenetic reconstruction revealed that the ancestor of mountain vipers split into two major clades at around 12.18 Mya followed by multiple vicariance events due to rapid orogeny. Raising mountain belts in the Eastern Mediterranean region and subsequent remarkable changes in temperature seasonality have led to the formation of important centres of diversification and endemism in this biodiversity hotspot. High rates of niche conservatism, low genetic diversity, and segregation of ranges into the endemic distribution negatively influenced the adaptive capacity of mountain vipers. We suggest that these species should be considered as evolutionary significant units and priority species for conservation in Mediterranean mountain ecosystems.The online version contains supplementary material available at 10.1186/s12862-021-01863-0. The recent decline in biodiversity necessitates a deeper understanding of current species distribution patterns and their evolution through time. This knowledge is particularly important in ecology, evolution, and conservation planning , 2. In tEcological divergence has been emphasised as one of the main drivers of speciation , 13. DurMountains are one of the main cradles of diversification, as half of the world’s biodiversity hotspots are located in mountainous landscapes , 22. A tMontivipera, which have experienced considerable speciation in mountainous landscapes [M. xanthina, M. wagneri, and M. bornmuelleri, and a clade of two closely-related sister species M. albizona and M. bulgardaghica, although M. albizona has also been considered as a subspecies of M. bulgardaghica based on the low genetic divergence between them [M. raddei, M. latifii, M. albicornuta and, M. kuhrangica. While in the Xanthina complex M. xanthina ranges from the semi-elevated uplands of southern Turkey to low-elevated plains and islands of the Mediterranean Sea, other species of both complexes disperse in mountain-top isolated populations . The Raddei complex consists of four species, M. raddei, M. albicornuta, M. latifii, and M. kuhrangica, showing a strongly supported monophyly that has split around 1.97 Mya (HDP = 0.79–3.15). The species inside this complex have diverged between 1.38 Mya (95% HDP = 0.64–2.12) and 1.01 Mya (95% HDP = 0.62–1.39). A strong cladogenic divergence is visible in the Xanthina complex, with the complex having split into the Xanthina and Bornmuelleri clades around 6.65 Mya (95% HDP = 5–8.3). A comparably early split in the Xanthina clade was the divergence of the coastal groups  from their mountain-dwelling sister group (M. xanthina Taurus) at 4.49 Mya (95% HDP = 3.47–5.51). In the Bornmuelleri clade, consisting of M. bornmuelleri, M. wagneri, M. albizona and M. bulgardaghica, the divergence from the most recent common ancestor (MRCA) was estimated at 3.16 Mya (95% HDP = 2.18–4.14), 2.54 Mya (95% HDP = 1.73–3.35), and 1.62 Mya (95% HDP 1.42–1.82), subsequently. The complete unpruned phylogenetic tree of the species is shown in the Additional file The dated phylogenetic tree of mountain vipers Fig.  allowed M. xanthina to 0.95 and 0.94 in M. bornmuelleri, respectively , which have evolved in different directions compared to their close sister taxa. Notwithstanding, they showed between-group convergent evolution, particularly with respect to the precipitation variables.The ancestral niche occupancy profiles Fig.  providedd) of 0 and a high cladogenetic dispersal rate (j) of 0.407. Reconstruction of the biogeographic history of mountain vipers using a BEAST chronogram in BIOGEOBEARS is illustrated in Fig. M. xanthina clade, where coastal lineages  dispersed toward Mediterranean coastal habitats in southwest and western Turkey from a mountain-dwelling ancestor. Reconstructions within the Bornmuelleri clade indicated two subsequent dispersal events from the ancestor to the Levant, i.e. M. bornmuelleri, and to the northeastern Turkey, i.e. M. wagneri, followed by a vicariance in Anatolian Diagonal between M. bulgardaghica and M. albizona. Supporting our results, Stümpel et al. [Ancestral range estimation analyses revealed that based on the likelihood-ratio test comparing six biogeographic models, the DIVALIKE model is supported best  and Blomberg’s K (P < 0.05) for four of the six climatic variables: temperature seasonality, maximum temperature of the warmest month, minimum temperature of the coldest month, and precipitation of the driest month  have highly likely diverged from a mountain-dwelling population. Relatively firm phylogenetic conservatism was seen in species of the Raddei complex. However, in this group, M. latifii in comparison to its ancestor has evolved toward extreme environmental conditions with a colder climate and higher temperature seasonality. Similarly, in the Bornmuelleri clade, M. bornmuelleri and M. wagneri have evolved divergently toward opposite climatic conditions compared to their most recent common ancestor.We found phylogenetic signals under both Pagel’s λ P < 0.0 and Blomth Table  based onMontivipera has long been the subject of debate among many herpetologists and taxonomists. All species in this group experienced a complex taxonomic history. For example, Mertens et al. [M. bornmuelleri as Vipera lebetina bornmuelleri, a subspecies of the later Macrovipera genus. Sigg et al. [Vipera xanthina when the Montivipera genus had not yet been introduced, and many species of this genus along Macrovipera and Daboia were considered to be monophyletic groups under the genus Vipera. Nilson and Andrén [Montivipera and Macrovipera as subgenera of Vipera, and Joger [Montivipera. Since our phylogenetic inference is based on only mitochondrial data we acknowledge that it might raise levels of uncertainty in species tree reconstruction as reliance on mitochondrial data can have ramifications for both evolutionary inference and taxonomy [The taxonomic status of mountain vipers of the genus s et al.  resolvedg et al.  suggested Andrén  first sund Joger  later retaxonomy . Indeed,taxonomy  or even taxonomy , 38. AltMontivipera species. Generally, our estimated divergence times in the Montivipera genus are consistent with those of previous studies, for example see Stümpel et al. [The results of this study highlight the role of mountain ranges in producing and maintaining diversity in the eastern Mediterranean region. Particularly during times of orogeny, mountains facilitate geographic isolation and formation of allopatric speciation , a pattel et al.  and Behrl et al. .Macrovipera lebetina in the Mediterranean coastal ranges [Montivipera that occupies lower-elevated habitats in co-existing ranges. Overall, in the Bornmuelleri and Raddei clades, all the divergence events have occurred during Pliocene and Pleistocene climatic oscillations. Avise et al. [M. latifii, M. kuhrangica, M. xanthina Taurus, M. albizona, and M. bulgardaghica, as supported by niche evolution and ancestral state estimation, emphasises the classic model of niche conservatism: conservation of niches after allopatric speciation in sister taxa [A considerable divergence in mountain vipers is the split of coastal vipers from their mountain-dwelling ancestor around 4.6 Mya, confirmed by niche evolution and ancestral range estimations. A possible reason for this divergence could be the absence of l ranges , a domine et al.  reportede et al.  and Plöte et al.  argued tter taxa , 20. Herter taxa .M. wagneri and M. latifii have convergently evolved toward areas with the highest temperature seasonality and negative extremes of temperature, M. bornmuelleri has adapted to humid ranges with the lowest temperature seasonality. Consistently, temperature seasonality showed the strongest phylogenetic signal and was ranked among the most important variables contributing to the current distribution of mountain vipers , and also obtained from other herpetologists’ field surveys (n = 62), existing databases , and scientific publications (n = 143). Particularly, the study by Mebert et al.  has notiIn order to reconstruct a dated molecular tree, we downloaded 177 mitochondrial sequences from NCBI GenBank generated by previous studies. Specifically, our dataset comprised 82 sequences of cytochrome b  and ND4 (640 bp) generated by Behrooz et al.  for the M. bornmuelleri to 53 sequences for M. albicornuta  the split between species of M. xanthina and M. raddei complexes using a normal distribution with a mean of 12.6 Mya and a standard deviation of 1.2 Mya. Accordingly, additional sequences of Cytb for two species of Macrovipera, including Levantine viper Macrovopera lebetina and Razi’s viper Macrovipera razii, were obtained from GenBank and incorporated to our dataset. Two independent runs were performed at 200 million generations and sampled every 10,000 generations, and omitting 25% as burn-in. Tracer 1.5 [Divergence dates among 12 main lineages of l et al. , were cal et al. . Molecull et al.  includinacer 1.5  was usedMontivipera BEAST tree using the drop.tip function in the R package ape [We used the ancestral niche occupancy reconstruction approach proposed by Evans et al.  to reconkage ape .Species’ potential distribution was modelled by using the maximum entropy (MaxEnt) method  and six To reconstruct ancestral niche occupancy profiles, we combined each species’ potential distribution at a given grid cell in the study area with the corresponding scores of the six original climatic variables to create a vector of probabilities per binned climatic values . To generate profiles of predicted niche occupancy (PNO), the map of each climatic variable was converted to a histogram of 100 equal-interval bins, and the total MaxEnt raw probabilities in each bin were calculated for each species. By using this method, instead of extracting climatic data from species localities to reconstruct ancestral climatic tolerances, we linked habitat suitability with bins of climatic variables . We thenWe inferred ancestral ranges and the biogeographic history of mountain vipers based on the pruned divergence time tree and three models of biogeographical range expansion, including Dispersal-Extinction-Cladogenesis (DEC), Dispersal-Vicariance (DIVA-like), and Bayesian inference (BAYAREA-like) models, all using the BioGeoBEARS package  in the Rphylosig function of package phytools [fitContinuous function of package geiger [contMap function of package phytools [We illustrated the ancestral state estimations of six climatic variables to assess phylogenetic niche conservatism (PNC) in mountain vipers . First, phytools . We thene geiger . Selectie geiger  using thphytools .Additional file 1: Table S1. Number of occurrence points and mitochondrial sequences used to reconstruct phylogenetic time tree and perform ecological niche modelling of mountain vipers. Table S2. Average percent contribution of six climatic variables, area under the receiver operating characteristic curve (AUC) and true statistics skill (TSS) of the ecological niche models performed for mountain vipers of the genus Montivipera in the eastern Mediterranean region. Table S3. Results of assessing the best evolutionary models of bioclimatic variables affecting Montivipera based on AICc statistics, using the BEAST chronogram and the fitContinuous function of geiger package. Bold values show the best model. AICc: Akaike Information Criterion for small sample-sized data, Ll: Log-likelihood. Table S4. List of samples, geographical origin, id and accession numbers for genes of the genus Montivipera and out-groups used in the phylogeographic and molecular dating analyses. Fig. S1. The Bayesian phylogeny tree reconstructed from the 177 sequences of mountain vipers (Montivipera). The tree was reconstructed based on a partitioned analysis with three Mitochondrial genes . Values above and below the branches are the posterior probabilities and mean age estimates, respectively. Colors of the branches corresponds to the geographic origin of the species as shown in Fig. 1.


Clostridium baratii strains are rare opportunistic pathogens associated with botulism intoxication. They have been isolated from foods, soil and be carried asymptomatically or cause botulism outbreaks. Is not taxonomically related to Clostridium botulinum, but some strains are equipped with BoNT/F7 cluster. Despite their relationship with diseases, our knowledge regarding the genomic features and phylogenetic characteristics is limited. We analyzed the pangenome of C. baratii to understand the diversity and genomic features of this species. We compared existing genomes in public databases, metagenomes, and one newly sequenced strain isolated from an asymptomatic subject. The pangenome was open, indicating it comprises genetically diverse organisms. The core genome contained 28.49% of the total genes of the pangenome. Profiling virulence factors confirmed the presence of phospholipase C in some strains, a toxin capable of disrupting eukaryotic cell membranes. Furthermore, the genomic analysis indicated significant horizontal gene transfer (HGT) events as defined by the presence of prophage genomes. Seven strains were equipped with BoNT/F7 cluster. The active site was conserved in all strains, identifying a missing 7-aa region upstream of the active site in C. baratii genomes. This analysis could be important to advance our knowledge regarding opportunistic clostridia and better understand their contribution to disease. Clostridium species among the most characterized and studied of its members [Clostridium is an important genus of Gram-positive, obligate anaerobes, bar-shaped spore-forming rods. Clostridium species could be found in soil and residing in several animals’ intestines, showing their widespread distribution [C. tetani), gastroenteritis [C. perfringens), Clostridioides difficile infection, and botulism [C. botulinum), among several others [Clostridium species are well studied for their industrial properties in producing important bioproducts such as ethanol, organic acids, and therapeutic proteins [Species belonging to the Firmicutes phylum are the most numerous in the gut microbiota, with several  members . Clostriribution . This taribution  . These outbreaks are described as sporadic and rare episodes [C. baratii strains have been found in liver pastes, meat, soil, and other foods. The species is considered an opportunistic pathogen that can cause disease in subjects with altered gut microbiomes or undergoing antibiotherapy, both infants and adults.episodes ,13,14,15C. baratii isolates responsible for the outbreaks have been shown to carry the botulinum neurotoxin (BoNT) and its gene cluster responsible for the production of the Type F botulinum toxin [C. botulinum strains and some strains of C. baratii. There are nine subtypes of the botulinum toxin F serotype [Botulism intoxication is characterized by flaccid paralysis due to the blocking of neurotransmitter release  at the neuromuscular junction. There are eight serotypes of BoNT toxins  , which dum toxin ,21,22,23serotype . These sserotype . The BoNserotype . The nonserotype . The Uviserotype .Clostridium species play a pivotal and intricate role in the gut microbiota. Under normal circumstances, they promote gut homeostasis and contribute to the barrier effect and balance in mucosal immunity [C. baratii genomes to better understand their diversity and their contribution to disease. To do so, we compared and studied all available C. baratii genomes, including one derived from a new isolate obtained by us, focusing on the identification of genes associated with the pathogenicity of this species.immunity ,27. HoweClostridium baratii C3 strain was isolated from fecal samples from a healthy subject in a previous study [Clostridium Medium under anaerobic conditions in an anaerobic jar (Anaerocult), and genomic DNA was extracted using a modified phenol-chloroform protocol. DNA was submitted to the sequencing service MicrobesNG  for sequencing using Illumina MiSeq with a minimum coverage of 30×, and 250 bp pair-end reads.us study . The strus study  after thReads were trimmed with Trimmomatic . TrimmedC. baratii strain was used to perform comparative genomics with the existent genomes available in the Reference sequence (RefSeq) database at NCBI available on September 2021 [The genome of our isolated ber 2021 . The datber 2021 .C. baratii strain, we included in our analysis all Clostridium pathogenic strains used in Kiu et al. [To obtain a better resolution of the phylogenetic relationships of our isolated u et al. . Then peu et al.  with theu et al. . We usedu et al.  to searcu et al. , a tool u et al. .Homologous groups of protein-coding genes from multiple bacterial genomes were built using Roary . Roary wWe employed the reference pangenome created by Roary as an input to EggNOG 4.5.1 eggNOG-mapper v2 genome-wide functional annotation tool , ran defWe evaluate the Clusters of Orthologous Groups (COGs)  found inhttp://www.cazy.org/ (accessed on 5 January 2021) to identify genes of carbohydrate metabolism. CaZy hits were considered positive if they had a 60% identity and coverage in blast result.We used a blastp search against the Carbohydrate-Active Enzymes (CaZy)  DatabaseGenes related to the GH (glycoside hydrolases) CaZy category in the pangenome were extracted from each strain using in-house Perl and bash scripts. Bar plots were generated using the R ggplot2 package .Clostridium strains from the Virulence Factor Database  [To identify the virulence potentials, we performed an in silico analysis of 29 identified toxins and virulent enzyme sequence data found in different ly 2020) ,42. To ily 2020) ,57 (PHAgly 2020)  (CARD), C. baratii strains, we performed an in silico analysis using the Comprehensive Database of Plasmid Sequences [C. baratii strains with BLAST+ [To identify the plasmid in equences . With thh BLAST+ .−5 were applied. Heat maps were generated using the R [BLAST+  was emplng the R  pheatmapJX847735 and used to build a BLAST database. BLAST+ was employed for sequence similarity search (blastP) using thresholds for percentage identity and coverage of 60% and e value of 1 × 10−5. Heat maps were generated using the R pheatmap package. To understand the phylogenetic relationship between the BoNT genes in C. baratii and C. botulinum strains, we used RAxML [BoNT gene clusters were downloaded from the NCBI database from the code ed RAxML  to calcued RAxML .C. baratii genomes, we used the BoNT/F7 gene from AGR53840.1 and performed a tertiary structure prediction with Phyre2 [. We compared this gene to the known three-dimensional structure of BoNT/A1 (PDB: 3BTA) in VMD [To evaluate the possible effect on the three-dimensional structure of the loss of seven amino acids upstream of the active site in BoNT toxin in h Phyre2 . We comp) in VMD  using MuC. baratii C3 was routinely cultured in Reinforced Clostridium Medium  supplemented with 0.5 g/L of L-cysteine . All incubations were performed at 37 °C for 24–48 h in an anaerobic jar  with anaerobic packs . Two overnight cultures of C. baratii were made on Blood Agar and Starch Agar plates . The bacteria were also inoculated in API-20A strips  following the manufacturer’s instructions. Finally, antibiotic resistance patterns were evaluated using antibiotic discs in RCM agar plates incubated for 48 h  for 48 h. Cefazolin (30 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), erythromycin (15 µg), penicillin G (10 µg), piperacill/tazobactam (110 µg), trimethoprim-sulfamethoxazole (25 µg), and vancomycin (30 µg) were used.C. baratii available from IMG [C. baratii C3. In publicly accessible metagenomes studies from the NCBI [C. baratii genomes across human gut metagenomes was very low: from 2588 human gut meta-genome bioprojects only four contained C. baratii genomes  to undeu et al. . The pat genomes . C. barad 771-14 .C. baratii, we constructed the pangenome of this species from all fifteen available genomes. The pangenome of C. baratii comprised 6122 genes, 1744 core genes, and 4378 accessory genes  database [C. baratii genomes have variations in their repair mechanisms and adaptations to stress. Similarly, genes associated with cell wall/membrane/envelope biogenesis (“M” COG code) were found more often in the accessory pangenome (233 genes) than in the core genes (68 genes). Finally, the number of genes associated with carbohydrate transport and metabolism (“G” COG code) was higher among accessory genes than in core genes (accessory: 288 genes vs. core: 127 genes). This suggests that carbohydrate utilization functions are conserved among strains of C. baratii.We then analyzed the functions of the core and accessory genes of the database . Genes wC. baratii genomes  was present in all C. baratii genomes , theta-toxin/perfringolysin O (pfoA), mu-toxin , and microbial collagenase (colA).We later examined the presence of virulence factors among all analyzed ological . The BoN genomes . C. perfClostridium genomes (C. baratii strains had a rpoB gene (CARD code ARO:3004563) with point mutations conferring putative resistance to rifampin and rifampicin. The genome of C. sordelli contains the most diverse repertoire of AMR genes, including EF-Tu mutations (CARD code ARO:3003357) conferring resistance to elfamycin, gyrA (fluoroquinolones), rpoC (rifampicin), tetA, and tetB (tetracycline resistance). C. baratii strains 2789STDY5834956, C3, and MGYG-HGUT-00064 had a tetA gene. Strain C3 carried point mutations in gyrB (CARD code ARO:3004562) with point mutations conferring resistance to fluoroquinolone antibiotics. Strain XCM carried tetA and tetB genes  and phage content among these  genomes . All buttB genes . These rC. perfringens [C. baratii strains 771-14, XCM, Sullivan 2789STDY5834956, and 2789STDY5834907. The PHASTER Putative prophages analysis showed that all C. baratii genomes except strain 695-15 contain several prophage proteins, ranging from eight to more than a hundred . Only a 7-aa region upstream of the active site appeared to be missing across C. baratii genomes compared with C. botulinum strains  events, as suggested by the high number of genes associated with transposases, integrases, and phages (“L” COG category). This high unusual number could explain a high potential for gene gain or loss events  were more represented within the core pangenome, indicating that these functions are conserved among C. baratii species.The pangenomic variation of the s events . InteresC. baratii and other pathogen clostridia described in Kiu et al. [C. perfringens, C. baratii strains. The phospholipase C (plc) is a hydrolase involved in signal transduction processes [C. baratii genomes. And we found only two C. baratii strains with complete plasmids (plasmid pNPD11_1 in CDC51267 and plasmid pCBJ in Sullivan strains). Additionally, we only found proteins from a plasmid (plasmid pNPD11_1) in strains that have the BoNT cluster . This is relevant because these are the only genomes that are closed, the other strains have a draft genome. Knowing how they were acquired is a big challenge because is necessary to have more information on these strains and ideally have closed the genomes instead of what is available today. This bioinformatics prediction correlates with the β-hemolytic activity of C. baratii C3. Finally, predicted AMR results should be taken with caution, and should be validated experimentally. For example, CARD predicted fluoroquinolone resistance in C. baratii C3 through gyrB mutations, however, this microorganism was sensitive to ciprofloxacin. Apparently, C3 is resistant to tetracycline and rifampin, but we were not able to confirm this estimation. Clostridium species appear to be resistant in great numbers to sulfonamides.To understand the pathogenicity of u et al. , we evalrocesses . Bacterirocesses , and undrocesses  and gas rocesses . We founC. baratii 693-15, 694-15, 695-15, 771-14, 796-15, Sullivan, and CDC51267 strains). Considering the clinical importance of this microorganism and that most studies reporting genomic information about this taxa are related to botulinum outbreaks, the proportion of C. baratii genomes with BoNT production potential is unclear and could be overlooked. The phylogeny of BoNT genes (C. baratii and C. botulinum strains showed a monophyletic lineage, indicating that the amino acid sequence is well conserved between these species. These findings agree with the high degree of conservation of the active site of the BoNT protein. In addition, BoNT sequences within C. baratii strains analyzed in this study, have a lack of genetic variation among BoNT sequences when compared to other Clostridium species and its presence in seven of fifteen genomes suggest a recent horizontal gene transfer event that is also supported by a large number of prophage proteins. That C. baratii strains acquired the BoNT cluster by HGT was proposed before based on the evidence of the presence of the same toxins in different groups of bacterial species [The BoNT/F gene cluster was present in seven out of fifteen strains (NT genes  in C. ba species  and that species .C. botulinum and C. baratii strains was on average 72%, and the active site HEXXH was conserved between proteins of both species (C. baratii genomes. However, this gap is not likely to impact the toxicity of BoNT because these amino acids do not interact with the molecular target of BoNT, the VAMP2 protein [C. baratii needs to recognize a longer peptide of the VAMP sequence to achieve cleavage [C. botulinum.The percentage of identity between the BoNT type F of  species . We foun protein . It is rcleavage  when ideC. baratii is an understudied clostridial species that could pose a potential threat to human health considering some isolates could produce the botulinum toxin. Our comparative genomic analysis of fifteen genomes of C. baratii indicated a heterogeneous open pangenome and diversity in adaptation to stress processes. Certain C. baratii species seem to be resistant to tetracycline, the majority of C. baratii strains were equipped with phospholipase C. Seven out of fifteen genomes carried a complete BoNT/F7 cluster that is highly conserved among C. baratii genomes and displayed distinct differences compared to C. botulinum BoNT. The analysis we report helps to understand the properties of rare clostridial taxa and their understudied clinical relevance, highlighting their toxin-producing capabilities and likely resistance and adaptation mechanisms.


Murraya exotica L. (Rutaceae) has important horticultural and medicinal values. Here, we reported the complete chloroplast (cp) genome of M. exotica using the next-generation sequencing method. The cp genome is 160,179 bp in length, including a large single-copy region , a small single-copy region , and a pair of inverted repeats (IR) regions 26,994 bp. A maximum-likelihood phylogenomic analysis showed that M. exotica was sister to Murraya paniculate. These findings will provide useful information for further investigation of cp genome evolution in Murraya. Murraya exotica (Rutaceae) is a perennial tree widely cultivated in tropical and subtropical regions  genome can be used as a tool for phylogenetic analysis, and it can be used for other usages such as species identification , Yunnan Province, China. The voucher specimen was deposited in the herbarium of Kunming Institute of Botany (KUN), Chinese Academy of Sciences . The genomic DNA was extracted following the modified CTAB method from leaf tissue (Doyle and Doyle M. exotica was deposited in GenBank (Genbank accession number: MW722359).The fresh leaves of M. exotica was 160,179 bp in length, including a large single-copy region , a small single-copy region , and a pair of inverted repeats (IR) regions of 26,994 bp. The overall GC content was 38.6%, with the LSC, SSC, and IR regions being 37.10%, 34.80%, and 43.00%, respectively.The cp genome of M. exotica and other species classified in the family Rutaceae and two species of Meliaceae designated as outgroups. The alignment was conducted using MAFFT (Katoh and Standley Murraya exotica was sister to Murraya paniculata, especially, these two species and Atalantia kwangtungensis belonged to one clade (Murraya is not a monophyletic taxon, the result support previous systematic findings (Samuel et al. Atalantia, Murraya, Glycosmis, and Clausena in Aurantioideae formed monophyly (Wang et al. M. exotica.The phylogenetic analysis was performed using the complete cp genome of ne clade . Therefo


WOX) gene family is a class of plant-specific transcriptional factors and plays a crucial role in forming the shoot apical meristem and embryonic development, stem cell maintenance, and various other developmental processes. However, systematic identification and characterization of the kiwifruit WOX gene family have not been studied. This study identified 17 and 10 WOX genes in A. chinensis (Ac) and A. eriantha (Ae) genomes, respectively. Phylogenetic analysis classified kiwifruit WOX genes from two species into three clades. Analysis of phylogenetics, synteny patterns, and selection pressure inferred that WOX gene families in Ac and Ae had undergone different evolutionary patterns after whole-genome duplication (WGD) events, causing differences in WOX gene number and distribution. Ten conserved motifs were identified in the kiwifruit WOX genes, and motif architectures of WOXs belonging to different clades highly diverged. The cis-element analysis and expression profiles investigation indicated the functional differentiation of WOX genes and identified the potential WOXs in response to stresses. Our results provided insight into general characters, evolutionary patterns, and functional diversity of kiwifruit WOXs.The WUSCHEL (WUS)-related homeobox ( Drosophila, and they belong to a large transcriptional factor (TF) family harboring a short stretch of amino acids (60–66 residues) conserved DNA-binding domain (referred to as homeodomain)   proteins were primarily identified in odomain) . HB protodomain) . The homodomain) . The HB odomain) . Previouem (SAM) . The WOXn plants . Genome-d poplar .WOX gene family has often been classified into three clades which include the ancient clade, the intermediate clade, and the modern/WUS clade   in Arabidopsis   . The AtWity axis . Howeverelopment . The AtWus roots . The modbidopsis . Besidesression) . WOX generistems . In summntiation .Actinidia genus, including 54 species and 75 taxa (A. chinensis (Ac) and A. eriantha (Ae) has been reported  were obtained from the Kiwifruit Genome Database (http://kiwifruitgenome.org/). The WOX protein sequences of Arabidopsis and rice were collected from the PlantTFDB v4.0 (http://planttfdb.gao-lab.org/). The local BLAST tool was used to construct the protein database of the two kiwifruit species. The combined protein sequences of Arabidopsis and rice WOX protein were used to query the kiwifruit protein database by the BLASTp. The candidate WOX proteins in kiwifruit were identified by BLASTp search scores of ≥ 100 and an e-value of ≤ 1 × e−10. The Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and the simple modular architecture research tool (SMART) (http://smart.embl.de/) were used to confirm the homeodomain of the candidate WOX protein, and the candidate WOX protein harboring the homeodomain were obtained and used for further analysis.The whole-genome sequences and protein sequences of the two kiwifruit species (WOX gene family in the two kiwifruit species were computed using the ProtParam on ExPASy server (http://web.expasy.org/protparam/). The subcellular localization of kiwifruit WOX proteins was predicted using the online web software CELLO .The protein length, theoretical isoelectric point (pI), grand average of hydropathicity (GRAVY), and molecular weight (MW) of the WOX genes of the two kiwifruit species were extracted. The gene structures of WOX genes were investigated using the Gene Structure Display Server . The conserved motifs of WOX proteins were identified using MEME (http://meme-suite.org/tools/meme) with a maximum of 10 motifs   .Arabidopsis thaliana, Oryza sativa, A. chinensis, and A. eriantha were performed using ClustalX with default parameters . The duplication patterns of kiwifruit WOXs were identified using the MCScanX software with default parameters  from NCBI (https://www.ncbi.nlm.nih.gov/). We further re-analyzed these transcriptome data using genomes of the Actinidia chinensis ‘Red5’ cultivar and A. eriantha white cultivar as reference genome  to 354 a.a (AcWOX3a) and from 169 a.a (AeWOX4c) to 371 a.a (AeWOX10)  . The preAeWOX10) . MoreoveAeWOX10) . The subAeWOX10) .WOX genes in kiwifruit, the neighbor-joining (NJ) tree was constructed using the full-length protein sequences of the identified 17 AcWOXs, 10 AeWOXs, and previously published 15 AtWOXs and 10 OsWOXs (WOX genes from Arabidopsis and rice). Consistent with previous reports in Arabidopsis, rice, and other species    . Eleven  AeWOXs) . The inte AeWOXs . AcWOXs and rice .AcWOX genes were randomly distributed on 12 chromosomes of Ac (WOX genes (three AcWOX genes), followed by three chromosomes  containing two genes and the other seven chromosomes  containing one AcWOX gene  possessed one AeWOX gene  and ancient clade WOXs (motif 4), respectively  to the rate of synonymous substitution (Ks)  . Generalion (Ks) . In thisof 0.259 . These rcis-element plays a crucial control in transcriptional regulation and significantly affects gene function  to 11 (AcWOX13b)   and 7B.  species . The cisergently , suggestunctions .AcWOX genes in three tissues  based on transcriptome data  or decreased (AcWOX4a/4b/13b) their expressions along with fruit development  which was the pathogen of kiwifruit bacterial canker disease (AcWOX genes significantly responded to ethylene treatments (AcWOX3b/4b/5/13a) were upregulated by ethylene treatment and gradually reduced their expressions after the treatment, and the AcWOX13a had the highest expression level  comparative transcriptomes of the resistance cultivar and the susceptible cultivar  than in the susceptible variety (HY)  twocultivar ; (c) witcultivar . In the on times . AcWOX13ety (HY) , indicatmparison . The thireatment .WOX gene family is the core regulator for forming the shoot apical meristem and embryonic development, stem cell maintenance, and various other developmental processes in plants . Further, we compared the characters and evolutionary patterns of kiwifruit WOXs of the two species. In addition, we analyzed and compared cis-element organizations for promoter regions of kiwifruit WOXs identified in the two species. We also investigated the expression profiles of AcWOXs in different tissues and different fruit developmental stages and the influence of hormonal treatment and biotic stresses on AcWOXs expression.The n plants . Genome-l plants . HoweverWOX genes were identified in Ac and Ae, respectively  has been reported in rice  number, deceased AR length or increased AR roughtnees  and A. eriantha (Ae) and conducted a detailed investigation of their evolutionary relationship, genome organization, duplication events and expression profiles. In total, 17 and 10 WOX genes have been identified from Ac and Ae genomes. They were classified into three clades  based on the sequence alignment and phylogenetic analysis, which was corresponding to previous report of Arabidopsis. Gene structures and motif patterns showed that WOX members in the same clade displayed more similarly. Gene duplications and selective pressure analysis indicated that WOX genes in two kiwifruit species have undergone different evolution patterns after the shared genome duplication events. Subcellular localization revealed that all WOX genes were located inside the nucleus. Furthermore, expression patterns detected tissue-specific expressed genes and hormonal or abiotic-stress responding genes. Overall, WOX genes identified in Ac and Ae genomes provided insight into general characters, evolutionary patterns, and functional diversity of kiwifruit WOXs.In conclusion, we performed genome-wide identification and characterization of the 10.7717/peerj.12348/supp-1Supplemental Information 1WOX gene family.Sequence logos for the ten conserved motifs identified in the kiwifruit Click here for additional data file.10.7717/peerj.12348/supp-2Supplemental Information 2Sequence alignment of the homeodomain in the kiwifruit WOX proteins.Click here for additional data file.10.7717/peerj.12348/supp-3Supplemental Information 3WOX genes. AcWOXs with high expression levels in three tissues and three fruit developmental stages were shown in (a) and (b). DAP, days after pollination. AcWOXs with high expression levels in samples treated with ethylene was shown in (c). DAFB, days after full bloom of fruit; DAT, day after treated with ethylene. (d) showed AcWOXs with high expression levels in two kiwifruit cultivars infected with Psa. HT and HY represented resistance and susceptible cultivar, respectively. The number in cultivar names showed hours post the Psa invasion. (e) shows expression profiles of AcWOXs with high expression levels in two susceptible cultivars to the invasion of Psa. DPI, days post-infection. (f) was expression profiles of AcWOXs with high expression levels in samples with or without Acibenzolar-S-methyl (ASM) treatments during the Psa infection. Healtycontrol represents samples without ASM treatment and Psa infection. The number presented in sample names indicated hours post the Psa infection.Expression profiles of Click here for additional data file.10.7717/peerj.12348/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.12348/supp-5Supplemental Information 5Click here for additional data file.


Primulina tenuituba is a species in the Gesneriaceae family that is widely distributed in China. It is a karst-dwelling species with an enormous tolerance for extreme drought and high temperatures. The species is also used in traditional Chinese medicine. In this study, the complete chloroplast genome of P. tenuituba was assembled and characterized for the first time. The complete chloroplast genome exhibited a typical quadripartite cycle of 153,236 bp in length, including a pair of inverted repeats (IRs) of 25,494 bp, which were separated by a large single-copy (LSC) region of 84,364 bp and a small single copy (SSC) region of 17,884 bp. The GC content was 37.6%. The complete chloroplast genome of P. tenuituba contains 114 genes, including 80 protein-coding genes, 30 tRNAs genes, and four rRNAs. The phylogenetic analysis showed that P. tenuituba is closely related to P. eburnea. The newly reported chloroplast genome of P. tenuituba would provide valuable data for further studies on its evolution and adaptation mechanism. Primulina tenuituba (W.T. Wang) Yin Z. Wang (Gesneriaceae), a karst-dwelling species, is widely distributed in Guizhou, Hunan, Sichuan, and Hubei Provinces in China, and appears to have a strong tolerance for extreme drought and high temperatures. Its rhizome is also used in traditional Chinese medicine in treating rheumatism , and a voucher specimen (WF200808-12) was deposited at the herbarium of Guizhou Normal University . Total genomic DNA was extracted from the fresh leaves of P. tenuituba using a modified cetyltrimethylammonium bromide (CTAB) method  for next-generation sequencing. Short-insert (350 bp) paired-end read libraries preparation and 2 × 150 bp sequencing were performed on an Illumina (HiSeq4000) genome analyzer platform. A total of 13,101,144 raw reads were obtained, and 98.27% of which were declared as clean reads. The clean reads were mapped to P. ophiopogoides (D. Fang & W.T. Wang) Yin Z. Wang  in Geneious Primer  was built. The IR region was detected by the Repeat Finder option in Geneious Primer and was reverse copied to obtain the complete plastid genome. The annotation approach was performed using CPGAVAS2 and PGA , containing one large single-copy (LSC) region of 84,364 bp and one small single-copy (SSC) region of 17,884 bp, which are separated by two inverted repeat (IR) regions of 25,494 bp. The GC content was 37.6%. A total of 114 unique genes were annotated in the complete chloroplast genome, including 80 protein-coding genes, four ribosomal RNA genes, and 30 transfer RNA genes.The complete chloroplast genome of Aphelandra Knappiae Wassh. representing Acanthaceae and Premna microphylla Turcz. and Stachys sylvatica L. representing Labiatae were employed as outgroups. The result showed that Gesneriaceae is monophyletic. Primulina tenuituba fell into a monophyletic group Primulina and was closely related to P. eburnea (Hance) Yin Z. Wang. This relationship was congruent with their morphological similarity, such as the narrowly long leaves. The newly reported complete chloroplast genome of P. tenuituba would provide valuable data for further studies on its evolution and adaptation mechanism.To investigate the phylogenetic relationships with the closely related species, the maximum likelihood (ML) phylogenetic tree was constructed by RAxML (Stamatakis


Ureaplasma parvum is usually part of the normal genital flora. Rarely can it cause invasive infections such as genitourinary infections, septic arthritis, or meningitis.U. parvum infection. U. parvum was detected in synovial fluid and in a urine sample. Treatment consisted of double-J stenting and targeted antibiotic therapy. Evolution showed resolution of urinary symptoms and clinical improvement of arthritis despite functional sequelae.Here we present the first description of chronic ureterocystitis in a 56-year-old immunocompromised patient, complicated first by reactive arthritis and secondarily by contralateral septic arthritis due to U. parvum colonisation, this diagnosis should remain a diagnosis of exclusion. However, because of the difficulty in detecting this microorganism, it should be considered in unexplained subacute urethritis or arthritis, including reactive arthritis, especially in immunosuppressed patients. Real-time PCR positivity in the absence of a differential diagnosis should not be overlooked.Given the high prevalence of The online version contains supplementary material available at 10.1186/s12879-021-06733-0. Ureaplasma, part of the Mycoplasmataceae family together with Mycoplasma, were pathogens causing non-gonococcal urethritis [Ureaplasma urealyticum and Ureaplasma parvum are the main species identified as pathogenic and were identified in 1999 [In 1954, Shepard et al., discovered that species from the genera  in 1999  .U. urealyticum is a common pathogen in genitourinary infections, reactive arthritis [U. parvum is usually considered part of the normal genital flora and therefore non-pathogenic. Nevertheless, it can occasionally be an opportunistic or iatrogenic [U. parvum positive screening on multiplex real-time PCR for sexually transmitted infection (STI) is usually not reported because of lack of clinical significance. However, a few reports of U. parvum infections, including post-surgical meningitis [rthritis , and occrthritis . In conttrogenic  pathogentrogenic  or respitrogenic . Given ttrogenic , U. parvningitis  and septU. parvum septic arthritis following reactive arthritis as a complication of genitourinary infection in an immunosuppressed woman.Here, we report a case of A 56-year-old woman was admitted in March 2019 for diagnostic work up. She had received treatment for follicular lymphoma with R-CHOP from October 2017 to April 2018 and was still on rituximab. She had no other significant medical history.She first presented in April 2018 with arthritis of the right knee and received ceftriaxone, then cefpodoxime-proxetil as a probabilistic antibiotic therapy, with favourable outcome. Arthrocentesis was not performed.3 and red cell count > 1000/mm3, but culture remained sterile. Bladder ultrasonography revealed vesical thickening and intravesical clot deposits, evocative of haemorrhagic cystitis. The patient declined cystoscopy and received empirical treatment with furadantine 50 mg target dose for 5 days.In October 2018, the patient reported dysuria and haematuria. Urine tests showed leukocyte count > 1000/mm3) with no crystals and negative Gram stain. Cultures remained sterile; broad-range 16 S ribosomal RNA PCR of synovial fluid was negative. Reactive arthritis was considered, but urine samples were negative for Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium. STI multiplex real-time PCR of a urine sample was positive for U. parvum on retrospective analysis (not done at that time). No antibiotics were administered, and the evolution was favourable without sequelae.In November, she presented left-knee arthritis, recurrence of haemorrhagic cystitis and bilateral conjunctivitis. The right knee was clinically normal. Arthrocentesis showed an inflammatory liquid  knee arthritis and acute renal failure. The left-knee examination was unremarkable at that time. CT revealed cystitis and bilateral ureteral stenosis responsible for dilatation of the urinary tract . Then, hypotension developed: piperacilline-tazobactam was started, surgical knee drainage was performed, and intracellular arthritis was suspected. Multiplex STI real-time PCR , was strongly positive for U. parvum on synovial fluid and on a urine sample. Specific culture media was inoculated , and A7 agar culture was positive for U. parvum in urine sample, but antibiotic susceptibility testing was negative. Bladder biopsy showed aspecific inflammation; PCR was negative for U. parvum although after 5 days of doxycycline treatment. 18 F-fluoro-2-deoxy-D-glucose positron emission tomography showed right-knee and right latero-uterine hypermetabolism  and subsequently by obstructive renal failure and septic arthritis. Antibiotic therapy with doxycycline 100 mg twice a day was started for 3 months. The fever receded, but local inflammation persisted. A control arthrocentesis after 15 days without antibiotics showed persistent arthritis but negative PCR results for U. parvum. Clarithromycin was introduced for 6 weeks, with marked clinical improvement: signs of erythema, swelling and pain disappeared, despite functional sequelae (flexion limitation 90°). Antibiotic treatment was well tolerated, and no clinical or biological event appeared during treatment.The diagnosis retained was U. parvum infection.We report a case of right-knee reactive arthritis that was spontaneously regressive without sequelae. The right-knee arthritis preceded left-knee septic osteoarthritis associated with bilateral ureteral stenosis secondary to chronic ureterocystitis due to U. parvum colonisation of the urogenital tract has been reported, and a high load of U. parvum has been found associated with chronic non-gonococcal urethritis in men, although whether it was a cause or a marker of non-gonococcal urethritis was unclear [U. parvum infection suggests opportunistic pathogenicity. Indeed, most infections have been reported in immunosuppressed or post-surgical patients. Ureaplasma are a common observation in patient with B-cell specific dysfunction such as patients with Common Variable Immune Deficiency [U. parvum prosthetic joint infection [U. parvum and Mycoplasma hominis [U. parvum orchitis complicated by gleno-humeral arthritis and endocarditis in a 56-year-old man with lymphoma history [U. parvum has never been reported. unclear . Currentficiency . For exanfection  was repo hominis  as well  history . HoweverU. parvum. However, given the frequency of genital colonization [U. parvum, this result was not considered significant, and only C. trachomatis, N. gonorrhoeae and M. genitalium results were reported. In the absence of other causal pathogens, clinical presentation and evolution pointed towards a diagnosis of U. parvum cystoureteritis at that time. Hence, we suggest that testing for U. parvum in subacute cystitis/ureteritis should be considered in immunosuppressed patients after excluding usual (including intracellular) pathogens and mycobacterial sampling.In the present case, retrospective assessment of the November 2018 STI multiplex real-time PCR of a urine sample was positive for Ureasplama sp. are not observed on Gram staining, and cultures are only contributory for high bacterial load and require specific media. Moreover, classical identification techniques may require 3 to 7 days, whereas real-time PCR is faster and more sensitive for Ureaplasma detection [U. parvum are resistant to doxycycline by carrying the resistance gene tet(M) [etection . Use of etection  and someetection . Therefoe tet(M) .U. parvum as a possible etiology for the reactive arthritis delayed the diagnosis and allowed septic complications to develop. Indeed, in November, the patient presented left-knee arthritis associated with cystitis and conjunctivitis . Direct examination, culture and 16 S real-time PCR of arthrocentesis fluid gave negative results. This episode receded in the absence of antibiotic treatment. Together with spontaneous healing, the association of conjunctivitis and ureterocystitis with a sterile arthritis suggested reactive arthritis to U. parvum ureterocystitis.In our case, overlooking C. trachomatis, but Salmonella spp., Shigella spp., Campylobacter jejuni and Yersinia spp. are also frequently involved. Chlamydophila pneumoniae, Clostridium difficile, HIV and U. urealyticum [Reactive arthritis is a rare immune-mediated arthritis following a genitourinary or gastrointestinal infection in predisposed patients and corresponds to the classic clinical triad: urethritis, eye disorders and arthritis. The principal microorganism involved in genitourinary infections responsible for reactive arthritis is alyticum  have beeU. parvum ureterocystitis in an immunosuppressed patient.To our knowledge, we report the first case of oculo-urethro-synovial syndrome due to U. parvum are not relevant, and PCR analysis is the best way to diagnosis the infection. PCR analysis of synovial liquid should be performed for immunosuppressed patients, especially in the presence of gynaecologic or urinary symptoms. U. parvum is also likely responsible for reactive arthritis.In immunosuppressed people, arthritis can be caused by mycoplasma species. Cultures of It did not need formal ethics approval as this complies with national guidelines  2016/679 du Parlement européen et du Conseil du 27 avril 2016, relatif à la protection des personnes physiques à l’égard du traitement des données à caractère personnel et à la libre circulation de ces données ») .Additional file 1. Timeline.


Portogloboviridae is a family of viruses with circular, double-stranded DNA genomes of about 20 kbp. Their icosahedral virions have a diameter of 87 nm, and consist of an outer protein shell, an inner lipid layer and a nucleoprotein core wound up into a spherical coil. Portogloboviruses infect hyperthermophilic archaea of the genus Saccharolobus, order Sulfolobales and are presumably nonlytic. Portogloboviruses encode mini-CRISPR arrays which they use to compete against other co-infecting viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Portogloboviridae, which is available at ictv.global/report/portogloboviridae. T=43 icosahedral shell. The fivefold vertices of the capsid are decorated with the vertex complexes extending above the capsid surface and potentially involved in receptor binding [Adnaviria [Virions are icosahedral, with a diameter of about 87 nm from vertex to vertex and 83 nm from facet to facet , Fig. 1a binding . The int binding . Lipids  binding . The nuc binding . The vir binding , as prevdnaviria .The circular dsDNA genome of Sulfolobus polyhedral virus 1 is 20 222 bp long and contains 45 open reading frames (ORFs) . ORFs arViral replication occurs by chronic infection without an apparent lytic cycle . No infowww.ictv.global/taxonomy. The family Portogloboviridae comprises a single genus, Alphaportoglobovirus, with two species. Portogloboviruses infect members of the hyperthermophilic archaeal genus Saccharolobus, order Sulfolobales.Current taxonomy: Portogloboviridae: www.ictv.global/report/portogloboviridae.Full ICTV Report on the family


Treponema pallidum is on the rise. In this narrative literature review, we show that the incidence of rare manifestations of syphilis, such as intracerebral gummata, is increasing and should be considered in the differential diagnosis of intracerebral lesions. With the exemplary case that we present here, we aim to raise awareness of the resurgence of this disease, which should be considered in the differential diagnosis of intracerebral lesions, especially for patients who have a risk profile for syphilis, and serological testing for T. pallidum prior to surgery should be discussed in order to avoid an unnecessary operation.Infection with  Treponema pallidum is a Gram-negative, spiral-shaped bacterium (spirochete) of the family Spirochaetaceae that causes syphilis in humans [n humans ,2. The cn humans ,4. Howevn humans ,5. Neuron humans ,7. On thToxoplasma species and T. pallidum were negative. Staining with antibodies against the glial fibrillary acidic protein (GFAP), p53, and IDH1-R132H were not suggestive of a brain tumor. Therefore, the histopathological diagnosis was necrotizing meningoencephalitis. Lumbar puncture for cerebrospinal fluid analysis was performed but did not show evidence of an infection. Although human immunodeficiency virus (HIV) infection was not detected, serum testing was positive for several treponemal tests (chemiluminescence assay (CLIA); T. pallidum hemagglutination assay (TPHA): 160,000 ; rapid plasma regain (RPR) test for anti-cardiolipin antibodies: 8 ), as was cerebrospinal fluid testing (TPHA: 5000 (reference < 20 titer); RPR: 1 ). IgM antibodies for T. pallidum were positive (Treponema IgM: 51 IE/mL (reference < 20 IE/mL)). Prior to discharge on day 8 after surgery, the patient was referred to the dermatology department for a 21-day course of intravenous penicillin G .A 51-year-old man with leucocytic colitis presented in August 2016 with a one-month history of vertigo and blurry vision. He reported two episodes of tinnitus in February and April and a history of having sex with men. He was in a long-time monogamous relationship and his partner had no known previous infections. The patient had never been diagnosed with a sexually transmitted disease, nor had he received any serological testing for sexually transmitted diseases in the past. Active medications included antiandrogenic therapy for alopecia. A vestibular examination with Frenzel goggles showed spontaneous nystagmus. The remainder of his clinical examination was unremarkable. No skin or mucosal lesions were observed. Because of the inconspicuous clinical examination, no laboratory or instrumental testing was conducted, except imaging testing. Magnetic resonance imaging of the neurocranium showed a small right frontolateral dural-based contrast-enhancing lesion measuring approximately 1.5 cm in diameter with perifocal oedema . After cT. pallidum serology parameters improved as well . Four months later, the patient had recovered completely. Serological parameters for T. pallidum were: CLIA: > 70; TPHA: 2560; RPR: < 1; IgM: 11 IE/mL.The T. pallidum were not always positive. White blood cells were often present and increased or reactivated VDRL or a positive T. pallidum antibody index. Increased protein levels and pleocytosis were also often present. On imaging, syphilitic gummata presented mostly solid and contrasted medium, with perifocal edema and signs of inflammatory processes  . In totaAlthough reports of intracerebral gummata are rare , the inc


Aspergillus infection, typing methods of Aspergillus species are increasingly being used. Accordingly, studying the spread and population dynamicsof strains isolating from clinical and environment, from a single host to large-scale ecosystems is definitely needed. In the current study, we carried out a genetic analysis of nine microsatelliteloci in isolates from different regions of Iran to compare and explore the genetic diversity between environmental and clinical A. fumigatus strains.  Because of the growing incidence of A. fumigatus, have collected from six cities of Iran. All A. fumigatus isolates identified based on macroscopic andmicroscopic characters, the ability to grow at above 45°C, and confirmed using DNA sequencing of the partial b-tubulin gene. Sixty-six A. fumigatus isolates were subjected by microsatellitetyping using three separate multiplex PCRs with a panel of nine short tandem repeats (STR) to evaluate the genetic relatedness.  Sixty-six clinical (n=43) and environmental (n= 23) isolates of A. fumigatus isolates revealed 38 distinct genotypes distributed among environmental and clinical isolates. We identified 12 clones including 40 different isolatesrepresenting 60% of all isolates tested, which each clone included 2–7 isolates. The STR typing of 66 A. fumigatus isolates.These findings show that the high genetic diversity observed of Iranian A. fumigatus isolates with those outside Iran and formed a separate cluster. The STR typing is considered as a valuable tool with excellent discriminatory power to study the molecular epidemiology and genotypic diversity of  Aspergillus fumigatus is an everywhere saprophytic mold with a global distribution frequently occurred in human fungal infections ranging from slight allergic reactions, colonization to serioussystemic infections. Invasive aspergillosis (IA) is a main reason of infections among immunocompromised individuals with over 200,000 life-threatening fungal infections yearly[ A. fumigatus is the predominant species to be found from cultures of respiratorysamples in cystic fibrosis patients with frequency approximately from 6% to 60% [ A. fumigatus infections in susceptible patients has dramatically increased in recent years due to construction, renovation, demolition and excavation activities in hospitalsand clinical centers [ A. fumigatus isolates have been increasingly reported, recently.[ Aspergillus flavus is another species of Aspergillus that is more common in Iran due to tropical and subtropical climatic conditions. Various studies in Iran from clinical andenvironmental isolates of A. flavus have identified using molecular and genotyping techniques [ A. fumigatus azole resistance designed a global project to realize how resistance is created in the environment. This project conductsvarious studies with the aim of maintaining the use of azoles for the production of food and human medicines [ Aspergillus infection, typing methods of Aspergillus species are extremely being used. Accordingly, studying the spread and populationdynamics of strains isolating from clinical and environment, from a single host to large-scale ecosystems is definitely needed[ A. fumigatus methods such as (AFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE),random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), RNA-induced silencing complex (RISC), repetitive element sequence-based PCR (rep-PCR),single-stranded DNA binding protein (SSDP), and variable number tandem repeat (VNTR) is lack of discriminatory power and poor interlaboratory reproducibility.However, microsatellites were used to determine and analyzed the genetic distances and have demonstrated to be powerful instruments for molecular typing[ A. fumigatus from different sources in Iran are underestimated. Hence, the main object of this study was to investigate the molecular epidemiology of clinical andenvironmental A. fumigatus isolates from Iran using microsatellite typing. yearly  - 4 . Re to 60%   , 6 .Thcenters   , 8 . Inecently.  - 11 .Ahniques   - 14 .Ahniques   - 17 .Tdicines   .Regard needed  , 20 . N typing  . Micros typing  , 23 . NSixty-six clinical (n=43) and environmental (n= 23) isolates were obtained from the culture collection of invasive fungi research centre (IFRC), Mazandaran University of Medical Sciences,Sari, Iran. This study was approved by the ethics committee of Mazandaran University of Medical Sciences under the ethics committee code 92-181(2013.4.12).Stock cultures were maintained on malt-extract agar  at 24 °C for one week prior to use. All isolates were collected from six cities of Iran comprising of Mashhad, Tehran , Sari , Shiraz , Hamadan , Babol . Clinical isolates comprising bronchoalveolar lavage ,endotracheal n = 5; 11.6%), sputum , sinus discharge , lung biopsy  and ear swabs , but environmental isolates collected from soilsamples surrounding from hospital gardens , air . All isolates were initially screened by macro- and microscopic features, ability to grow at>45°C and were confirmed to the species level by DNA sequencing of the ß-tubulin as previously described [ .6%, sputTaq DNA polymerase (Roche Diagnostics), 0.2 mM deoxynucleoside triphosphates and 2µl of target DNA in 1× reaction buffer. Thermocycling was done in a thermocycler(Westburg- Biometra USA ) as described before [ A. fumigatus strains was determined by comparing the profiles with BioNumerics v6.6 software .To generate the dendrogram, or the option to generate a minimum spanning tree directly from the categorical data, un-weighted paired group method was applied using arithmetic average(UPGMA) algorithm was applied. The Simpson index diversity (D) was used to measure the genetic distinction or diversity between A. fumigatus isolates. A ‘D’ valueof 1 indicates that all isolates are different whereas a ‘D’ value of 0 indicates that all isolates are identical[ To perform microsatellite typing, three distinct multiplex PCRs with a panel of nine short tandem repeats (STR) were designed to evaluate the genetic relationshipbetween the isolates as previously described   . To dif before   . PCR prentical  , 27 .A. fumigatus isolates from different sources were genotyped using the full panel of 9 short tandem repeats markers. The Simpson’s index of diversity was less than0.9 for all nine markers combined. According to the STR typing, 38 distinct genotypes were distributed among environmental and clinical isolates.Remarkably, A. fumigatus isolates.Eight genotype clusters included only clinical isolates, two cluster only environmental isolates and two clusters contained clinical and environmental isolates.A. fumigatus isolates. The STR typing depicted no genotypic correlation of Iranian A. fumigatus with isolates from other countries.In addition, the high genetic diversity observed of Iranian A. fumigatus isolates with those outside Iran and formed a separate cluster .The size of the circle is correlated with the number of isolates possessing the corresponding Gt. Dark, dashed and thin connecting bars corresponds to one,2 or >2 different markers observed between linked Gt. Gts with a shaded background contain a minimum of 2 isolates that differ maximum in 1 microsatellitemarker as the possible result of microevolutionary events and are likely to be clonally related. A. fumigatus isolates from different cities were shared in a cluster. Among different genotypes, 6 clusters comprised isolates of different cities.The image shows that some A. fumigatus isolates with those outside Iran and formed a separate cluster.The image shows that the high genetic diversity observed of Iranian A. fumigatus is globally found in the hospital environments and most frequently isolated from patients with IA or aspergilloma[ Aspergillus conidia concentrations dueto the construction inside or surroundings area of the hospitals as well as problems in the ventilation or conditioner systems have proposed the environment to bethe potential source of infection [ Aspergillus genotyping, but [ et al, newly described a novel panel of 9 STRs for genotypingof A. fumigatus with highly discriminatory power, clear assignment, inter-laboratory exchangeability of the results [ et al. [ et al.[ Af asa main genotyping tool. Unlike other Aspergillus species, there is little information about the genotypic diversity of A. fumigatus from different sources in Iran[ A. fumigatus isolates.Hence, 38 distinct genotypes were identified within 66 Iranian A. fumigatus isolates using a panel of nine microsatellite markers. The large number of genotypeswas in concordances with the results of other studies [ et al., we were able to two cluster isolates by their clinical or environmental origin [ et al. reported that majority of the patients were affected with only one genotype [ A. fumigatus strains with the TR34/L98H mutation, isolated from compost in13 different Iranian cities, shared the same allele across all nine examined microsatellite loci [ A. fumigatus isolates from different parts of India.This strains shared the same genotype not found in any other evaluated samples within or outside of India [ gilloma  - 34 . tfection   , 36 .Dfection   .Severafection   . In sevng, but   , they lng, but   , 41 . dresults   .Howeve et al.    and Bal et al.   designein Iran  . Here, studies   , 46 .Uenotype   .This ote loci   .Like of India   .A. fumigatus isolates.Generally, these findings show that the high genetic diversity observed of Iranian A. fumigatus isolates with those outside Iran and formed a separate cluster.The STR typing is considered as a valuable tool with excellent discriminatory power to study the molecular epidemiology and genotypic diversity of H. B., T. SH and M. N., conceived the study. M. N., M. M, and M. F prepared sampling and performed molecular identification and analyzed the data. S. Kh performed Microsatellite assayand H.B, M.N interpreted the data. H. B., T. SH, S. Kh and M. N prepared the manuscript. All authors approved the final version of the manuscript.No financial interests related to the material of this manuscript have been declared.


Aprostocetus brevipedicellus Yang and Cao (Eulophidae: Tetrastichinae) is one of the most promising biocontrol agents for forest pest control. Mass rearing of A. brevipedicellus is critical for large-scale field release programs, but the optimal rearing hosts are currently not documented. In this study, the parasitism of A. brevipedicellus and suitability of their offspring on Antheraea pernyi eggs with five different treatments were tested under laboratory conditions to determine the performance and suitability of A. brevipedicellus. Among the host egg treatments, A. brevipedicellus exhibited optimal parasitism on manually-extracted, unfertilized, and washed (MUW) eggs of A. pernyi, and A. brevipedicellus offspring emerging from MUW eggs had high egg load. The results indicate that MUW eggs are optimal for the mass production of A. brevipedicellus.The egg parasitoid Aprostocetus brevipedicellus, a eulophid gregarious egg parasitoid of lepidopterous pests, is a potential biological control agent for the control of many forest pests. A dominant factitious host, Antheraea pernyi, has been widely used for mass rearing several parasitoids in China. However, whether A. pernyi eggs are suitable for A. brevipedicellus rearing remains unclear. Here we evaluated A. brevipedicellus parasitism and fitness of their offspring on A. pernyi eggs with five different treatments, including manually-extracted, unfertilized and washed eggs (MUW), naturally-laid, unfertilized and washed eggs (NUW), naturally-laid, unfertilized, and unwashed (NUUW) eggs, naturally-laid, fertilized and washed eggs (NFW), and naturally-laid, fertilized and unwashed eggs (NFUW). The results showed that A. brevipedicellus could parasitize host eggs in all treatments but significantly preferred MUW eggs to other treatments. Moreover, A. brevipedicellus preferred unfertilized eggs to fertilized eggs and parasitized more washed eggs than unwashed. The pre-emergence time of parasitoid offspring emerging from fertilized eggs was shorter than that from unfertilized eggs. More parasitoid offspring emerged from unwashed eggs than that from washed eggs. The offspring emergence rate was high (>95%) and also female-biased (>85%) among all egg treatments. The egg load of female parasitoid offspring emerging from MUW and NUW eggs was 30–60% higher than the remaining treatments. Overall, MUW eggs of A. pernyi are the most suitable for the mass production of A. brevipedicellus. In China, forest insect pests have posed a serious threat to forest trees and agricultural crops. There are 5055 pest species accounting for damage in 11.4282 million ha of forestry in China . ManagemBiological control is a key method for pest management in forestry ,13,14. AAprostocetus brevipedicellus Yang and Cao (Eulophidae: Tetrastichinae) is an egg parasitoid first found in 2005 [Caligula japonica Moore, Dendrolimu spp., and Lebeda nobilis Walker and is one of the most promising biocontrol agents for forest pest control [Aprostocetus parasitoids have successfully been used in forestry to control midge, beetle, and weevil pests [ in 2005 . It can  control . Many Apil pests ,26,27,28Aprostocetus parasitoids relies on natural hosts for field release programs, such as mass rearing of Aprostocetus hagenowii Ratzeburg on the American cockroach Periplaneta americana L. for control [A. brevipedicellus has been documented. The factitious eggs of Chinese oak silkworm Antheraea pernyi Guérin-Méneville are widely used for mass production of egg parasitoids such as Trichogramma and Anastatus because they are simple and cheap to mass-produce, and easy to transport [A. pernyi from unfertilized females have been reported to use mass rearing of egg parasitoids [Trichogramma parasitoids prefer parasitizing manually-extracted unfertilized washed A. pernyi eggs. Trichogramma offspring had increased fitness when reared on this factious host compared to natural hosts [A. brevipedicellus mass production remains unclear.Currently, mass rearing of  control . To our ransport ,31,32,33ransport ,37,38,39ransport ,36,37,38asitoids . A recenal hosts . HoweverA. pernyi eggs can be used as a rearing host for A. brevipedicellus. We also evaluate the parasitism preference and fitness of A. brevipedicellus offspring on A. pernyi eggs under different host statuses. The objective of the current study was to determine the optimal A. pernyi egg treatment for mass production of A. brevipedicellus.Additionally, host egg washing has been proved to influence parasitism preference and host orientation ,41,42,43Aprostocetus brevipedicellus was initially collected from parasitized eggs of Japanese giant silkworm Caligula japonica in walnut orchards in Kangxian , Gansu Province, China, in 2017. The species were identified based on the morphological characteristics as described by Yao [d by Yao  and furtA. pernyi, were collected in Yongji City, Jilin Province, China, and then stored at 4 °C in the Institute of Biological Control, Jilin Agricultural University. After storage for 2–3 months, cocoons were transferred to an emergence room at 25 °C to allow adult emergence. Newly emerged adults (<6 h) were collected for the experiments.Cocoons of Chinese oak silkworm, A. pernyi were conducted to determine the optimal condition for the rearing of Aprostocetus brevipedicellus. The five treatments including (1) manually-extracted, unfertilized and washed eggs (MUW), (2) naturally-laid, unfertilized and washed eggs (NUW), (3) naturally-laid, unfertilized and unwashed eggs (NUUW), (4) naturally-laid, fertilized and washed eggs (NFW), and (5) naturally-laid, fertilized and unwashed eggs (NFUW). The preparation of the five egg treatments was similar to our previous study [Egg treatments of us study . The MUWA. brevipedicellus on treated A. pernyi eggs was conducted under laboratory conditions . Newly emerged A. brevipedicellus females (<1 h old) were collected and introduced with males in glass tubes provided with 10% honey-water solution as food  to allow mating for 3 days before use in experiments. Based on our observations, one A. brevipedicellus female can parasitize up to 20 A. pernyi eggs within 24 h. Therefore, mated female parasitoids were individually exposed to 40 host eggs in a no-choice test for 24 h to allow a surplus supply of hosts. For each treatment, forty host eggs were glued onto a paper card (0.5 cm × 5.0 cm) using nontoxic glue and placed inside a glass tube . One three-day-old, mated female A. brevipedicellus was also introduced into the glass tube containing an egg card. Our previous study found that a 10% honey-water solution was suitable for A. brevipedicellus. Therefore, a 10% honey-water solution was provided as food during the test. After 24 h, A. brevipedicellus was removed from the glass tube. The host eggs on the card were then cut out from the card and held individually in a glass tube, and maintained in an incubator under the conditions as described above. All parasitized eggs were checked daily until the emergence of all adults. After there was no further adult emergence, host eggs were individually dissected under a stereomicroscope to check the number of dead wasps left inside the chorion. For each egg treatment, the number of parasitized eggs (number of the host eggs with emergence hole + number of host eggs without emergence hole but containing parasitoids), pre-emergence time (d) (the number of days from exposure of host eggs to the parasitoid to the adult offspring emerging from host egg), the number of emerged adults per host egg (dead adults inside host eggs were excluded), the emergence rate , percentage of female progeny , the number of dead parasitoids left inside the eggs, female offspring body size , and the egg load per female offspring were recorded. For HTL, thirty female adults from each treatment were selected randomly, and HTL was measured using an ultramicroscope (Keyence VHX-2000). To record the egg load per female offspring, thirty newly-emerged females were randomly selected from each treatment and then dissected under a stereomicroscope to examine the ovary to document the number of eggs. For each egg treatment, 30 female A. brevipedicellus were tested.A no-choice experiment of A. brevipedicellus for A. pernyi eggs among the five treatments previously listed. One three-day-old and mated female A. brevipedicellus, as described above, was introduced into a glass tube  containing an egg card. The egg card carried forty A. pernyi eggs from all egg treatments, with eight eggs per treatment, and was randomly placed on the paper card (0.5 cm × 5.0 cm). 10% honey-water solution was also provided as food. After 24 h, parasitoids were removed, and host egg cards were cut out individually and kept in an incubator as described above for posttreatment observation. After 6 days, the eggs were examined under a stereomicroscope to document the number of parasitized eggs of each treatment. This experiment was replicated 30 times.A choice experiment was tested under laboratory conditions  to determine the host preference of p < 0.05. All data were subject to a normality test (Shapiro–Wilk test) prior to ANOVA. All percentage data were arcsine square-root-transformed prior to the Shapiro–Wilk test. The analysis was performed on the transformed data, and untransformed means ± SE were presented. For the choice test, data were analyzed using the Friedman non-parametric analysis to determine the effect of treatment on the number of parasitized eggs. All data analyses were performed using SPSS 23.0 .For the no-choice bioassay, a one-way analysis of variance (ANOVA) was conducted to determine the effect of treatment on the number of parasitized eggs, pre-emergence time, number of emerged adults per egg, emergence rate, number of dead wasps left inside per egg, percentage of female progeny, hind tibia length and egg load of per female offspring. Tukey’s honestly significant difference (HSD) test was used to compare means at F1,116 = 8.10, p = 0.0052) regardless of washing or not. However, the interaction between fertilization and washing treatments did not affect the number of parasitized eggs . Aprostocetus brevipedicellus successfully parasitized A. pernyi eggs in all egg treatments, and there was a significant difference in the number of parasitized eggs among treatments  (Fertilization significantly affected the number of eggs parasitized ( 0.0001) A. The maA. pernyi resulted in the maximum number of parasitized eggs, which was 259–2533% higher than the remaining treatments  , and parasitized 633% more NFW eggs than NFUW eggs . The number of parasitized eggs was equivalent between NUUW and NFUW treatments .In the choice test, MUW eggs of  0.0001) B. AprostA. brevipedicellus offspring was 6–10% shorter on NFW and NFUW treatments compared with MUW, NUW, and NUUW treatments  (The pre-emergence time of  0.0001) A. A. brevipedicellus adults per A. pernyi egg was 10–31% higher in the NFUW treatment compared with the remaining egg treatments  . Aprostocetus brevipedicellus offspring emerging from MUW egg treatment was 3% more female-biased than the NUW egg treatment , and ranged from 95% to 97%. Similarly, there were no significant differences in the number of dead parasitoids per host egg between all egg treatments . The number of dead parasitoids per egg was the highest in those from NFUW host eggs (0.35), followed by NUUW (0.23), NFW (0.22), MUW (0.17), and NUW host eggs (0.17). The number of emerged  0.0001) B. MUW anreatment C. EmergeA. brevipedicellus female offspring ranged from 410.50 μm to 428.10 μm, and no significant differences were found in the hind tibia length among all egg treatments . Female offspring emerging from MUW egg treatment resulted in the longest hind tibia length, followed by NUW, NFW, NUUW, and NFUW. Aprostocetus brevipedicellus female offspring emerging from MUW egg treatment resulted in maximum egg load, which was 2–12% higher than the remaining treatments  (Hind tibia length of  0.0001) . A. pernyi, have been demonstrated as an excellent substitute host for mass-production for several egg parasitoids, especially Trichogramma and Anastatus [A. pernyi egg treatments by A. brevipedicellus, and the fitness of resulting A. brevipedicellus offspring. The results showed that A. brevipedicellus successfully parasitized A. pernyi eggs in all egg treatments with varied parasitism and host suitability performance. Our study indicated that the MUW A. pernyi eggs showed great potential for mass production of A. brevipedicellus, since the results showed that this treatment resulted in the maximum number of parasitized host eggs in both the choice and no-choice experiments and high egg load in offspring from this treatment.The large eggs of the Chinese oak silkworm, nastatus ,30,33,34A. brevipedicellus could recognize both fertilized and unfertilized host eggs and preferred parasitizing unfertilized eggs. Similar findings were also reported for T. japonicus and T. dendrolimi, which prefer unfertilized H. halys, and A. peryni eggs for mass rearing [A. brevipedicellus parasitized significantly more washed eggs than unwashed. Wang et al. found the same results for T. dendrolimi parasitizing A. pernyi eggs [A. pernyi eggs after washing treatments.Previous studies have demonstrated that the host quality ,44,45 an rearing ,39. In anyi eggs . This maA. pernyi eggs, A. brevipedicellus parasitized significantly more manually-extracted eggs than naturally-laid eggs. The difference in preference may be due to the immature chorion of eggs, which is easier for A. brevipedicellus oviposition. The early manual extraction of eggs may have terminated the tanning process related to hardening the egg chorion [A. pernyi chorion is a limiting factor for some parasitoids [Furthermore, the washing treatment may soften and thin the egg chorion due to the friction between the eggs during washing. However, further studies are required to verify this phenomenon. Among the washed  chorion . Previouasitoids ,34. ResuA. brevipedicellus offspring was also affected by the fertilization of host eggs [A. brevipedicellus developing in fertilized eggs of A. pernyi was significantly shorter than that in unfertilized eggs. Similarly, Yang et al. reported that three Trichogramma species had shorter developmental time in fertilized eggs of Chilo suppressalis Walker than those in nonfertilized [A. brevipedicellus offspring on fertilized eggs than unfertilized eggs. Other factors may also be involved, e.g., egg deposition period [The performance of ost eggs ,35,37,38rtilized . The difn period , but theA. brevipedicellus adults emerged per host egg from fertilized eggs than from unfertilized eggs. Similarly, egg parasitoids Gonatocerus morrilli Howard and Telenomus coloradensis Crawford parasitizing on Homalodisca vitripennis Germar eggs had higher mortality on unfertilized host eggs [A. pernyi eggs. We suspect that embryonic development of fertilized A. pernyi egg improves accessibility to nutrients in the host egg for the parasitoid. Therefore, when parasitizing fertilized eggs, the parasitoids might lay the larger number of eggs per host egg. A recent study on Trichogramma parasitizing A. pernyi eggs also indicated that the number of emerged adults per fertilized host egg was significantly higher than per unfertilized host egg [Many female parasitoids can assess host quality and quantity to determine suitability for offspring development ,47,48,49ost eggs . This ishost egg . There whost egg ,51. A. brevipedicellus offspring was strongly female-biased  in all treatments. The percentage of female progeny under the natural field conditions was 77% [Ooencyrtus kuvanae Howard positively affects its fecundity [Oomyzus sokolowskii Kurdjumov adults per host was negatively correlated with egg load [A. pernyi eggs are the most optimal host for the mass rearing of A. brevipedicellus.Results showed that biased >8% females was 77% ,52. Ther was 77% ,53,54,55 was 77% ,57. For ecundity . Host quecundity ,59. Li eegg load . AlthougA. pernyi were most suitable for oviposition based on host preference, parasitism, and parameters of offspring fitness. Our results provide evidence to support that MUW eggs of A. pernyi are desirable for mass production of A. brevipedicellus. They also provide the possibility for large-scale application of A. brevipedicellus to control the forest pests effectively. However, further studies are needed to optimize the rearing ratio of A. brevipedicellus to MUW eggs and their biocontrol efficiency against target pests in field release programs.In conclusion, MUW eggs of