Heyndrickxia sporothermodurans (formerly Bacillussporothermodurans) was evaluated using whole genome sequencing. The genomes of 29 previously identified Heyndrickxiasporothermodurans and two Heyndrickxia vini strains isolated from ultra-high-temperature (UHT)-treated milk were sequenced by short-read (Illumina) sequencing. After sequence analysis, the two H. vini strains could be reclassified as H. sporothermodurans. In addition, the genomes of the H.sporothermodurans type strain (DSM 10599T) and the closest phylogenetic neighbors Heyndrickxiaoleronia (DSM 9356T) and Heyndrickxia vini (JCM 19841T) were also sequenced using both long (MinION) and short-read (Illumina) sequencing. By hybrid sequence assembly, the genome of the H. sporothermodurans type strain was enlarged by 15% relative to the short-read assembly. This noticeable increase was probably due to numerous mobile elements in the genome that are presumptively related to spore heat tolerance. Phylogenetic studies based on 16S rDNA gene sequence, core genome, single-nucleotide polymorphisms and ANI/dDDH, showed that H. vini is highly related to H. sporothermodurans. When examining the genome sequences of all H.sporothermodurans strains from this study, together with 4 H. sporothermodurans genomes available in the GenBank database, the majority of the 36 strains examined occurred in a clonal lineage with less than 100 SNPs. These data substantiate previous reports on the existence and spread of a genetically highly homogenous and heat resistant spore clone, i.e., the HRS-clone.The genetic heterogeneity of  Heyndrickxia (H.) sporothermodurans, H. oleronia and H. vini are gram-positive, rod-shaped and endospore-forming bacteria [Margalitia shackletonii and Bacillus (B.) acidicola [Bacillus species was published by Gupta et al. (2020), which proposes a reclassification and renaming of, among others, B. sporothermodurans [bacteria ,2,3. Thecidicola . Recentlmodurans . Based oHeyndrickxia sporothermodurans is a non-pathogenic bacterium whose highly-heat-resistant spores (HRS) are able to survive the food sterilization process [Heyndrickxia contamination of ultra-high-temperature (UHT)- and sterilized milk in Italy, Austria and Germany [H. sporothermodurans (Bacillus sporothermodurans sp. nov.) [H. sporothermodurans from UHT milk, feed and food [H. sporothermodurans strains and the potential spread of a specific clonal linage, the so-called HRS-clone [ process . Their p Germany  and the p. nov.) , many coand food ,14,15,16RS-clone ,18. HoweHeyndrickxia oleronia was described in 1995 by Kuhnigk et al. [Reticulitermes santonensis (Feytaud) [Heyndrickxia oleronia is phylogenetically closely related to H. sporothermodurans and was found to be difficult to distinguish from the latter on the basis of biochemical and physiological characteristics solely [H. oleronia was published 2017 by Owuso-Darko et al. [Heyndrickxia species genomes using a sequencing technique such as Illumina MiSeq sequencing that generates short-reads only. Heyndrickxia vini was isolated from an alcohol fermentation pit mud and was described in 2016 by Ma et al. [Heyndrickxia sp. showed the closest relatedness to H. sporothermodurans and H. oleronia type strains, with 98.4% and 97.2% sequence similarity of the 16S rRNA gene. Classical DNA-DNA hybridization values were 33.3% and 42.8% between H. vini vs. H. sporothermodurans and H. vini vs. H. oleronia [H. sporothermodurans and H. oleronia, the strain was then described as a new species, namely H. vini [k et al. . It was Feytaud) , but latFeytaud) ,9,13,19.s solely . The draH. vini type strain was not published and the genome sequences of only five H. sporothermodurans strains were available, i.e., those of the type strain DSM 10599T, strain BR3 , strain SAD, strain SA01 and B4102 [Campylobacter jejuni [Salmonella enterica [Listeria monocytogenes [Escherichia coli [B. cereus group [H. sporothermodurans genomic DNA sequences by sequencing an additional 29 strain genomes of H. sporothermodurans. These isolates represent problematic spoilage bacteria in the food sector and were isolated from UHT milk. In addition, the combination of short- and long-read sequencing should significantly improve the genome quality of the type strains of H. sporothermodurans, H. oleronia and H. vini. Based on the genomic sequences, this study furthermore aimed to evaluate the genetic diversity of the H. sporothermodurans group, consisting of the closely related species of H. sporothermodurans, H. oleronia and H. vini, and to determine phylogenetic relationships between H. sporothermodurans strains.Whole genome sequencing offers valuable support in understanding the genetic background and phylogenetic diversity of bacteria. It currently provides the best possibility to clarify genomic relationships with the highest discriminatory power . At the nd B4102 ,22. Their jejuni , 50 to 1enterica , 10 to 3ytogenes ,24, 80 thia coli  and 50 tus group . The aimH. sporothermodurans strains (29 isolates and the type strain DSM 10599T), three H. vini strains (two presumptively identified isolates and the type strain JCM 19841T) and the H. oleronia type strain DSM 9356T  broth  supplemented with 1 mg/L vitamin B12 (B12)  or on BHI agar (Carl Roth) + 1 mg/L B12 at 37 °C for 48 h. Default parameters were used for all protocols and software unless otherwise specified. Genomic DNA was extracted from 4 mL of culture (48 h) using the ZR fungal/bacterial DNA miniprep kit  Illumina sequencing and the Genomic Micro AX Bacteria+ kit  for Oxford Nanopore sequencing. DNA quality assurance and quantitation were performed using the Qubit fluorometer and dsDNA BR Assay kit . Additional sequence data used in this study was shown in Thirty SM 9356T  were obthttps://patricbrc.org/) [https://www.geneious.com). Contigs of less than 300 bp or with coverage <20× were removed manually and the remaining assemblies were exported as fasta files. Possible contamination of the assemblies was screened using the ContEst16S tool [The sequencing library was prepared with an Illumina TruSeq nano DNA LT library prep kit  and sequenced on the Illumina MiSeq sequencer using the Illumina MiSeq V3 reagent kit according to the manufacturer’s protocols. The fastq files were quality controlled by FastQC 0.11.7 and assembled using SPADes Version 3.10.0 on the PATRIC BRC website (rc.org/) . The ass16S tool . The gen16S tool .H. sporothermodurans type strain DSM 10599T were obtained using the same protocol, but with the following modifications, that were required due to unusual clumping of the magnetic beads during purification. For DNA clean-up of this strain, it was necessary to dilute the samples by 1:5 before adding the magnetic beads and to elute the DNA at 70 °C. Furthermore, this library was sequenced on a MinION MK1B sequencer but with a Flongle Flow Cell (FLO-FLG001). Reads were extracted from FAST5 files using the Guppy pipeline v. 2.3.1. (https://nanoporetech.com), demultiplexed by Porechop [https://www.patricbrc.org/ [® and if more than 1 contig was generated by Unicycler, the smaller contigs were mapped against the largest contig and well-mapping (redundant) sequences were removed from the assembly. Additionally, the ends of the largest contigs were checked for overlap and circularized if they matched. Circular chromosomes were rotated to dnak as starting point. Possible contamination of the assemblies was screened using the ContEst16S tool [Long sequence reads were obtained by library preparation with the MinION 1D Native DNA barcoding genomic DNA protocol (with the Oxford Nanopore Ligation Sequencing Kit (EXP-NBD104) and the Oxford Nanopore Native Barcoding Expansion Kit (SQK-LSK 109)) and by sequencing using an Oxford Nanopore MinION MK1B sequencer with a MinION Flow Cell (R9.4.1) and a Flow Cell Priming Kit (EXP-FLP002). The long sequence reads of the Porechop  and filtPorechop . The reaPorechop . Trimmedbrc.org/  with Uni16S tool . The gen16S tool .® 9.1.8. For generating the genome’s core and accessory genes, the assembled contigs were annotated with Prokka V1.14.5 [https://usegalaxy.eu/) [All assembled contigs were annotated using the NCBI Prokaryotic Genome Annotation Pipeline version 4.10 ,32 with  V1.14.5  and the  V1.14.5 . Prokka axy.eu/) . Due to http://ggdc.dsmz.de/. Phylogenies were inferred by the TYGS web server [Pairwise sequence similarities were calculated using the 16S rRNA gene sequences available via the GGDC web server  at http:b server . The folb server . Maximumb server  and TNT b server , respectb server  and subsFor further phylogenetic investigations a codon tree based on up to 1000 core genes was generated with PATRIC BRC . The defH. sporothermodurans group species. The Blast+ (ANIb) from all pairwise genome comparisons were computed at the online service JSpeciesWS [The overall genome-related indexes, i.e., average nucleotide identity (ANI) and digital DNA-DNA hybridization were used to compare the peciesWS . DigitalpeciesWS . The propeciesWS .CSI Phylogeny-based subtyping (SNP CSI Phylogeny 1.4) calls SNPs and was performed using whole genome sequence data (https://cge.cbs.dtu.dk/services/CSIPhylogeny/) [H. sporothermodurans DSM 10599T was used as reference genome and the raw reads (Illumina short-read sequence) were uploaded for each strain. Due to the unavailability of Sequence Read Archive (SRA) data (raw reads) for strain B4102 in the databases, the assembled data stored in the GenBank database were used. The analysis was submitted with the following parameters: a minimal depth at SNP positions of 25 reads, a minimal relative depth at SNP positions of 10%, a minimal distance between SNPs of 10 bp, a minimal Z-score of 1.96, a minimal SNP quality of 30, a minimal read mapping quality of 25 and ignore heterozygous SNPs activated. The pipeline and the parameters were described by Kaas et al. [The online service logeny/) . The hybs et al.  and evals et al. . BrieflyH. sporothermodurans and two presumptive H. vini strains by short-read sequencing. The two presumptive H. vini strains (identification of the strains was based on previous 16S rRNA gene Sanger sequencing) could be reclassified as H. sporothermodurans (see below). Additionally, we sequenced the type strains H. sporothermodurans DSM 10599T, H. vini JCM 19841T and H. oleronia DSM 9356T using a combination of short- and long-read sequencing technologies. Sequencing and analysis was performed according to the methodology proposed by Chun et al. [In this study, we sequenced 29 n et al. . The quaH. sporothermodurans genomes, sizes ranged from 3,372,204 bp to 3,886,000 bp with a GC content of 35.75 to 36.01 mol%. The number of gene coding sequences (CDS) ranged from 3758 to 4115. The type strain H. sporothermodurans DSM 10599T assembly yielded 302 contigs after Illumina short-read sequencing, but could be improved to 1 contig by performing a hybrid assembly of Illumina short-read and MinION long read sequences. Using this method, the H. oleronia strain DSM 9356T could be assembled into a single closed chromosome sequence, opposed to the short-read approach, which resulted in >500 contigs. When compared to the short-read assembly, the hybrid assembly noticeably increased the genome size from 3,839,826 bp to 4,417,946 bp (15.1%) for H. sporothermodurans DSM 10599T and from 5,083,966 bp to 5,198,760 bp (2.3%) for H. oleronia DSM 9356T. Similarly, the GC content increased from 35.75 to 36.34 mol% GC for H. sporothermodurans DSM 10599T and from 34.97 to 35.13 mol% GC for H. oleronia DSM 9356T. While the number of genes increased from 4032 to 4554 CDS for H. sporothermodurans DSM 10599T, a decrease from 5530 to 5397 CDS was observed for H. oleronia DSM 9356T. The massive increase in genome length, GC content and the number of genes in the hybrid assembly approach of H. sporothermodurans DSM 10599T could be explained by an elevated detection of repetitive sequences like transposons, integrases and other mobile genetic elements, which varied between 1700 and 4000 nucleotides in size. In the short-read assembly (302 contigs) of H. sporothermodurans DSM 10599T there were often residues of repeat regions located at the contig ends. In the corresponding hybrid assembly, these repeat regions were distributed over the whole genome sequence and are identified with a number of 340 regions (by Geneious®). This number of repeat regions is similar to the contig number in the short-read assembly. This finding suggests a correlation between the number of contigs in short-read assembly and the number of repetitive regions in the respective genome. In the short-read assembly, there are several potential matching short-reads at the regions of repetitive sequences (>500 bp), where the assembly has to be terminated and the end of a contig arises. With hybrid assembly , these repetitive regions could be continuous sequenced and assigned. This allowed the contigs to be placed in the correct order and additional genes were therefore found within the repetitive regions.For the short-read-sequenced H. vini JCM 19841T. The sequence length, mol% GC content and gene number determined by the hybrid assembly for the H. vini JCM 19841T genome were 4,309,805 bp, 35.97 mol% GC and 4373 CDS, respectively  share 164 core genes  and thus suggests a suboptimal analysis (Roary pipeline) of the sequencing data as the numbers of core genes are expected to increase with a decreasing number of species examined. In order to reveal the underlying cause, the effects of slight changes to the parameters of the analysis pipeline were examined: When the protein identity interval was increased (sets the minimum percentage identity for protein match) to 99% (default was 95%), expectedly, less core genes were found, but the ratio of core genes between two species (44 genes) and all three species (42 genes) hinted towards an overall more reliable result (H. sporothermodurans + H. oleronia and 164 for H. sporothermodurans + H. oleronia + H. vini) was thus identified as a technical artifact in the blastp calculation (Roary pipeline). While the artifact had no significant influence on other results, it highlights the necessity of thorough manual examination of complex computational analyses.Analysis of protein-encoding genes revealed that the genomes of the three characterized re genes . Surprise result . The obsH. sporothermodurans species (n = 36 genome sequences) 1837 core genes (100% of strains harbor these genes), 490 soft core genes (95–99% of strains), 1923 shell genes (15–95% of strains), 2797 cloud genes (1–15% of strains) and 7047 different genes could be identified in total (1–100% of strains). Removing the more distant H. sporothermodurans strains SAD and B4102 (see below), the number of core genes within H. sporothermodurans genomes increased to 2187 .Within the Heyndrickxia sporothermodurans related species was done by the TYGS platform [H. sporothermodurans, H. vini and H. oleronia are related and cluster closely in a monophyletic group, which we termed the H. sporothermodurans group and in which H. sporothermodurans shows a closer relationship with H. vini than with H. oleronia.Phylogenetic tree based on 16S rRNA gene sequences of type strains of the platform  and is sH. sporothermodurans, H. vini and H. oleronia, a phylogenomic tree based on core genes was constructed. In this phylogenomic analysis the H. sporothermodurans and H. vini type strains clustered well apart from the H. oleronia type strain  shackletonii and M. camelliae form a monophyletic clade. Phylogenetically more distant to H. sporothermodurans, and without distinct clades, are W. ginsengihumi, B. acidicola, S. fordii and C. firmus. In addition, H. vini formed a clearly separated branch in the more detailed maximum-likelihood phylogenomic tree , clustered closely together with other H. sporothermodurans strains in the phylogenomic analysis, indicating that these strains were previously identified incorrectly and were in fact also H. sporothermodurans strains  or genome-genome-distance calculations (GGDCs) are considered as gold standard for species taxonomy [H. sporothermodurans  and is genetically very homogeneous. Nevertheless, when looking within the type strain group at high resolution, there was also considerable heterogeneity between strains, especially evident from the strain L1_142 and L1_141 (Germany), BR3 (Brazil) and the single isolates of Ecuador, Iran and Jordan, which formed individual lineages  between the members of a closely related clade e.g., L1_152, L1_153, L1_154 and L1_155 (red) are 3–7 SNPs . In compIn order to assess the closely related clade even more precisely, a core genome calculation within the clonally closest related isolates was performed and accessory genes were compared. The isolates L1_152, L1_153, L1_154 and L1_155 of dairy D (red) contain 3747 genes in total, of which 3418 are core genes (100% of isolates harbor these genes) and 329 are accessory (shell) genes (15–95% of isolates harbor these genes). The isolates L1_158, L1_159, L1_160 and L1_161 of dairy E (green) contain 3753 genes in total, of which 3288 are core genes and 465 are accessory genes. The isolates L1_143, L1_144 and L1_145 of dairy B (blue) contain 3969 genes in total, of which 3679 are core genes and 290 are accessory genes (shell) genes.H. sporothermodurans strains, and furthermore improved the existing H. sporothermodurans (DSM 10599T) and H. oleronia (DSM 9356T) type strain genome sequences. In addition, this study is the first to present the complete genome of H. vini (JCM 19841T).High-throughput sequencing technology development has led to a significant improvement for the discrimination of bacteria at the genomic level. This study has extended the genome sequence data from 5 so far examined to 36 H. sporothermodurans, a genome extension of 15% was achieved by long-read sequencing of the overlapping areas between the short-read contigs. At the ends of short-read contigs and in the overlapping areas, repeat regions  were frequently detected (data not shown). For species with many repetitive elements, long read sequencing is necessary to obtain the complete genome information, because repeats lead to unresolvable loops in the assembly graph that are ultimately fragmented into contigs [The here presented data show that a combination of short- and long-read sequencing data results in a considerable decrease of contig numbers from a few hundred to generally a single complete chromosome and thus represents a superior genome assembly approach. Using this approach, we frequently noticed an increase in genome length compared to the short-read assemblies. For  contigs ,48. It m contigs . As a coH. sporothermodurans has yet to be uncovered. However, it has been demonstrated that such regions can increase resistance of bacterial spores towards environmental stresses like heat exposure [Heyndrickxia species presented in this study.The reason for the high abundance of repeat regions in the genome of exposure  and we hH. sporothermodurans genome is the high degree of relationship in the respective core genes of strains, in contrast to the diversity of the respective accessory genes. Apparently, the essential core genes are much less affected by horizontal gene transfer (HGT) than the rest of the genome. In evolutionary terms, de novo mutations in Bacillus are probably less important than HGT for the variability of genomes [B. subtilis, it has been shown that after 504 generations up to 2% of the genome has been replaced by HGT [H. sporothermodurans and probably play a minor role in HGT. However, repeat regions that we found in H. sporothermodurans represent a general mechanism for horizontal gene transfer [A striking feature of the  genomes ,51. For transfer . Core geH. vini strains  could be identified as H. sporothermodurans on the basis of whole genome sequencing results. This clearly demonstrates that traditional 16S rRNA gene analysis in this case does not have sufficient resolution to discriminate between H. vini and H. sporothermodurans. The reason is probably the heterogeneity of the 16S rRNA gene copies of H. sporothermodurans, where some variants show a higher similarity to the published H. vini 16S rDNA (data not shown) than others. H. sporothermodurans and H. vini are indeed the most closely related, shown on the basis of 16S rDNA, core genes and digital whole genome comparisons (88.97% ANIb and 39.70% GGDH). Other species closely related to this H. sporothermodurans/vini clade are H. oleronia, M. shackletonii and M. camelliae. Interestingly, based on an analysis of core genes performed with 595 genes, M. shackletonii and M. camelliae form a monophyletic clade. Less related to H. sporothermodurans are Weizmannia (W.) ginsengihumi, B. acidicola, Siminovitchia (S.) fordii and Cytobacillus firmus, which deviates from previously reported work [H. sporothermodurans and related strains generally support the traditional wet-lab data  clearly shows a greater relationship between H. oleronia and H. sporothermodurans. As a result, DNA-DNA hybridization methods were not suitable for the determination of the degree of inter-species-relationships with values below 70%.Two ted work . Digitallab data  for specBacillus species was published by Gupta et al. in 2020, which proposes a comprehensive reclassification and renaming of many Bacillus species [Bacillus species clades were identified [B. oleronius and B. sporothermodurans (Oleronius Clade), for which the genus Heyndrickxia gen. nov. was proposed [B. oleronius was proposed to be renamed to Heyndrickxia oleronia and B. sporothermodurans to Heyndrickxia sporothermodurans [B. shackletonii and B. camelliae , B. ginsengihumi  and B. fordii , among others [B. firmus into Cytobacillus firmus has also been proposed [H. sporothermodurans and H. vini, the other subclade of H. oleronia. The shared core genes from the Roary pipeline (964 core genes H. sporothermodurans -H. vini vs. 158 core genes H. sporothermodurans -H. oleronia) confirm the different species subclades. It is therefore important to include genome data such as we presented here in future taxonomical Bacillus analysis . As the reclassifications were already valid (http://lpsn.dsmz.de as reference) at the time of writing, we used the new, valid species/taxon names in this study. Although the new names may initially cause some confusion, the proposed developments are justified and improve the taxonomic classification of many Bacillus species.The new classification of  species . Based oentified . A monopproposed . Accordimodurans . Furtherg others . The renproposed . Our corH. sporothermodurans genomes revealed that 34 of 36 strains belonged to the same ecotype [type strain group” accordingly in this study. The bases for these groupings were the high similarities between the strains to the type strain DSM 10599T in both GGDH (>99.5%) and ANIb (>99.75%), as well as the supporting core gene homologies and single-nucleotide polymorphisms (SNPs) analyses. The strains SAD and B4102 do not belong to this type strain group (2 of 36 strains) due to the results of whole-genome-comparisons, core gene and SNP analyses . Heyndrickx et al. [H. sporothermodurans strains based on protein levels (SDS-page subgrouping) and wet lab DNA-DNA hybridization (76–88%). There, the authors analyzed H. sporothermodurans strains from different origins , while only UHT-milk isolates were used in the present study. The limited strain variation detected here indicates that UHT-processing treatment of raw milk results in the selection of a specific ecotype.The intraspecies analysis of all available  ecotype , which wH. sporothermodurans strains in one dairy, even if they descended from a common ancestor. This coevolution of different strains can lead to the accumulation of SNPs at different speeds over time [It is difficult to provide a satisfactory answer to the question of whether and how long a strain may persist in a single dairy, or whether there are new contaminating strains being continually brought into the dairy. It can, however, be speculated that isolate L1_151 persisted in dairy D and developed into the clonal related strains L1_152, L1_153, L1_154, L1_155 (3–7 SNPs) within the 11 months these strains were isolated. The SNPs between isolate L1_151 (origin) and L1_152, L1_153, L1_154, L1_155 were 18, 16, 21, 22 SNPs, respectively. However, this does not correspond with the times of sampling, if accumulation of SNPs during time would be assumed. Additionally, the clonal isolates L1_152, L1_153, L1_154 and L1_155 harbor 3418 core and 329 accessory genes. This was quite a surprising high rate of accessory genes for strains with such low numbers of 3–7 SNPs in their core genomes. One explanation would be the coexistence and coevolution of different ver time . Furtherver time .The strains from Germany also showed individual clusters in which strains showed clonal relationships, but different clusters of German strains also showed that these probably presented individual lineages . A remarListeria (L.) monocytogenes outbreak analysis, the distances are set at 4 to 25 (up to 63) SNPs for a clonal line, depending on the species and outbreak period  [L. monocytogenes strains with under 10 SNPs can occur in different retail deli environments without any links of known transmission [In general, there is no strict rule for a separation between the terms clone and strain. For r years) . Other asmission . Today, smission .Only when horizontal gene transfer and the core genome are evaluated together, is it possible to distinguish between strain and clonal line . Here, wHowever, this very close relationship indicates, that due to the high thermotolerance of their spores, bacteria persist within a milk processing environment for long time periods of up to several months. Moreover, the occurrence of phylogenetic clusters often correlated with their respective geographic origin, indicating that these heat-resistant bacteria not only co-exist for extended periods of time in specific milk processing environments, but also display a similar dissemination behavior between closely located processing environments. Supporting this assumption, we determined one monophyletic cluster containing highly related strains, which originated from proximal geographic sites, i.e., Italy, Albania and Czech Republic.H. vini spores has not been estimated so far, this should also be done in future investigations. Thermal preservation is of great importance for the food industry. If the thermal processes are not sufficient for spore-inactivation, this can lead to premature food spoilage. Our investigations show the persistence of H. sporothermodurans for months in food processing plants. For this reason, molecular detection systems should also be developed for selective and sensitive detection of H. sporothermodurans and other relevant Bacillus species contamination to prevent financial losses for companies from loss of quality of the processed milk products.It is unclear which physiological adaptations  were triggered by the genetic changes. For this, further studies should be carried out, if possible with a greater heterogeneity of strains. Determining the heat resistance of the spores is one of the important points to clarify if some clusters are more heat resistant than others. It is possible that isolates from one dairy may become more resistant over time. These results on heat resistance should then be compared with the genomic and genomic change  data, similar to those presented in this study. Also, the heat resistance of H. sporothermodurans DSM 10599T, H. oleronia DSM 9356T and H. vini JCM 19841T as well as the genomes of 31 H. sporothermodurans strains isolated from UHT-treated milk from different continents. This study is the first to present the complete genome of H. vini (JCM 19841T). The type strains  were clustered into one phylogenetic clade, whereby H. sporothermodurans was more closely related to H. vini than to H. oleronia. Moreover, this clade was found to be distinct from the M. shackletonii and M. camelliae cluster.This study presents the complete genomes of the type strains Heyndrickxia species genomes using a short-read sequencing approach [H. sporothermodurans DSM 10599T, which mostly yielded a single coherent chromosome sequence instead of a few hundred contigs. Furthermore, genome size and G + C value differed significantly between both assembly approaches. We could detect a major increase in genome length and a minor increase in the mol% G + C content. The high amount of contigs obtained after short-read sequencing is probably caused by the occurrence of mobile elements which are overrepresented in H. sporothermodurans. As the assembly of genomes containing multiple repetitive genetic elements can be challenging, this species can be considered a model organism for improving and evaluating sequencing methods and assemblies.Previous work reported difficulties in assembling approach ,18,20. TH. sporothermodurans strains, which were classified as type strain group accordingly. The remaining two strains (SAD and B4102) could be identified as H. sporothermodurans as well, based on digital whole genome comparison. Nevertheless, both strains probably belong to distinct phylogenetic clades due to their clear genetic distance from the type strain group.Furthermore, a high clonal relationship of core genes was shown in 34 of 36 H. sporothermodurans.In conclusion, our results indicate that clonally related isolates with <10 SNPs occur in the course of few months in industrial dairy environments and can persist and (co)evolve quickly. Therefore, this study represents the basis for the development of detection systems and avoidance strategies to prevent contamination of food with highly heat-resistant spore formers like


The Chengjiang biota is one of the most species-rich Cambrian Konservat-Lagerstätten, and preserves a community dominated by non-biomineralized euarthropods. However, several Chengjiang euarthropods have an unfamiliar morphology, are extremely rare, or incompletely preserved.Jianshania furcatus. We reveal new morphological details, and demonstrate that the specimens assigned to this species represent two different taxa. The holotype of J. furcatus features a head shield with paired anterolateral notches, stalked lateral eyes, and an articulated tailspine with a bifurcate termination. The other specimen is formally redescribed as Xiaocaris luoi gen. et sp. nov., and is characterized by stalked eyes connected to an anterior sclerite, a subtrapezoidal head shield covering three small segments with reduced tergites, a trunk with 15 overlapping tergites with a well-developed dorsal keel, and paired tail flukes.We employed micro-computed tomography to restudy the enigmatic euarthropod X. luoi nov. as a representative of Fuxianhuiida, an early branching group of stem-group euarthropods endemic to the early Cambrian of Southwest China. X. luoi nov. represents the fifth fuxianhuiid species described from the Chengjiang biota, and its functional morphology illuminates the ecological diversity of this important clade for understanding the early evolutionary history of euarthropods.The presence of antennae, biramous appendages with endopods composed of 15 articles, and multiple appendage pairs associated with the trunk tergites identify  Jianshania furcatus )Additional file 1: Video S1. Dorsal side of Xiaocaris luoi nov. as shown in Fig. Additional file 2: Video S2. Ventral side of Xiaocaris luoi nov. as shown in Fig. Additional file 3: Video S3. Head of Xiaocaris luoi nov. as shown in Fig. Additional file 4: Video S4. Head of Xiaocaris luoi nov. as shown in Fig.


We present here an updated checklist for the butterflies of Matheran, Maharashtra, India, an eco-sensitive zone, with identification remarks for locally rare or very rare butterflies. This is the first dedicated checklist for butterflies of Matheran after 125 years. A total of 140 species of butterflies were recorded belonging to six families. Amongst them, 15 species were either listed under Schedule I, II or IV of the Indian Wildlife (Protection) Act, 1972. We also list the habitats of the species along with the data for their activity at the time of recording the observation. We propose a uniform colour code system for representing season and activity for the Indian butterflies. Examples of colour barcodes are provided with the images of rare and very rare butterflies. The lack of abundance data is a limitation of the study for which we propose long term monitoring with dedicated efforts. Butterflies are an ideal taxonomic group for ecological studies of landscapes  and theiHere, we provide a checklist for butterflies of Matheran surveyed between the years 2011 and 2019. Ours is the first dedicated checklist for the butterflies of Matheran after 18.9866°N 73.2679°E, 772 m a.s.l., WGS 84) is a small hill station located in Karjat Tehsil of Raigad District in the Indian State of Maharashtra  which are available as a data table on the India Biodiversity Portal , monsoon (Jun-Sept) and winter (Oct-Jan) throughout the year from September 2011 to March 2019. Intermittent observations were taken between 06.00 hrs and 17.00 hrs for around three days a month. The butterflies were observed in all possible habitats at six localities and on two trails in and around Matheran  was prepared in R  using thhttps://colordesigner.io/color-mixer). Grey colour represents the occurrence of the species in all seasons. All other colours were selected from the RGB scheme for it provides a wider range of colours. These colours were selected in such a way that they represent the correponding activity, for example, brown for mud puddling, honey colour  for nectaring, amber colour for tree sap feeding etc., except basking which is represented by magenta.The colour codes Table  were preLycaenidae with 46 species (32.86%), followed by Nymphalidae with 43 species (31.43%), were amongst the most species-rich families in the area. Species belonging to the family Hesperiidae (25 species), Pieridae (14 species) and Papilionidae (10 species) were amongst other common species found in the area. The range of Cheritrafreja  which was earlier recorded from Amboli, Sindhudurga, Maharashtra  . The family Riodinidae was represented by only one species namely, Abisarabifasciata (Double Banded Judy).The SAC gained a plateau and standard deviation for species richness declined from 97.75 ± 17.07 to 141.0 ± 0.0 as the number of sights increased from one to eight, predicting sufficient efforts to record all the species found in the area    is extenHesperiidae for which the maximum numbers of species (N = 23) were recorded during the monsoon  and 34.40% (N = 43) of total species of butterflies recorded during respective seasons  were recorded during winter, while minimum numbers of species (N = 80) were recorded during the monsoon Fig. . Maximum were recNymphalidae and Lycaenidae dominated the species diversity at all the sites studied in and around Matheran. Members of the family Lycaenidae were particularly present in higher numbers at Charlotte Lake while those of Hesperiidae were particularly present in higher numbers at Garbett Point  with 78.22% of relativised strict nestedness (nestedness without considering the effect of species replacement) and lowest relativised beta diversity of 29.42%. Relativised strict nestedness was the highest (85.67%) for the family Hesperiidae with a similarity of 65.91% and beta diversity of 34.10%, while relativised nestedness (nestedness considering the effect of species replacement) was the highest (93.56%) for the family Pieridae. Similarity of species composition between the sites was the lowest (49.10%) for the family Lycaenidae with the highest relativised richness difference (31.99%) indicating more site specific species composition for the members of the family Lycaenidae, unlike the members of other families.Members of the family int Fig. . The Simint Fig. . SimilarNo seasonal activity pattern could be observed Table , Table 4Our list contains 15 such species which are scheduled under the Wildlife (Protection) Act, 1972 of India Table , 12, 13Abbreviations: FW-Forewing, HW-Hindwing, UN-Underside, UNF-Underside of Forewing, UNH-Underside of Hindwing, UP- Upperside, UPF-Upperside of Forewing, UPH-Upperside of HindwingBibasis Moore, 1881Genus Bibasissena   Fig. a.Common name: Orange-tailed awlet.Identification remarks: Bright orange fringe on HW and on the tip of the abdomen. Broad, pure white, outwardly diffused, central band on UN. Wingspan 42–50 mm.Season: Monsoon.Habitat and activity: The species was observed in forested patches while nectaring.Burara Swinhoe, 1893Genus Burarajaina   Fig. b.Common name: Orange awlet.Identification remarks: UN pale brown. UNH with orange stripes along veins and has orange fringe. UNF purplish. Wingspan 60–70 mm.Season: Monsoon.Habitat and activity: The species was observed in forested patches while nectaring.Celaenorrhinus Hübner, 1819Genus Celaenorrhinusruficornis Hampson, 1889   Fig. d.Common name: Plain banded awl.Identification remarks: Outwardly diffused broad white or bluish-white band on UNH. Female has an additional spot on UPF. UN paler, inner half has greenish gloss. Wingspan 45–55 mm.Season: Monsoon.Habitat and activity: The species was observed in forested patches while nectaring.Matapa Moore, 1881Genus Matapaaria   Fig. e.Common name: Common Redeye.Identification remarks: Dark buff-brown with no markings on UP. HW has greyish fringe tinged with pale yellow. UN more yellowish orange-brown. Indistinct black brand on UPF of male. Wingspan 40–55 mm.Season: Monsoon and winter.Habitat and activity: The species was observed in forested patches while nectaring.Arhopala Boisduval, 1832Genus Arhopalaamantes   Fig. f.Common name: Large oakblue.Identification remarks: Tailed with lobe. UNH has central squarish spots in spaces 4 and 5 at right angles. Metallic scales at UNH lower tip. Wingspan 45–57 mm.Season: Winter.Habitat and activity: The species was observed in forested patches while mud puddling, basking or feeding on rotten fruits.Arhopalacentaurus   Fig. a.Common name: Centaur oakblue.Identification remarks: HW tailed. No HW lobe. Metallic scaling on UNH faint or absent. UNF band continuous and curved. UNF cell spots outlined by silver lines. Male UP brilliant violet-blue, narrow dark borders. Females UP paler blue, broad wing borders. Wingspan 53–62 mm.Season: Winter.Habitat and activity: The species was observed in forested patches while mud puddling, basking or feeding on rotten fruits.Cheritra Moore, 1881Genus Cheritrafreja (Fabricius 1793)  Fig. b.Common name: Common Imperial.Identification remarks: Two tails. UN of both sexes white to pale brown; faint bars at cell-ends. Narrow dark outer central line on UNF. UNH with outer central and marginal lines and black spots crowned with metallic scales at lower tip. Wingspan 38–42 mm.Season: Monsoon and winter.Habitat and activity: The species was observed in forested patches while basking or nectaring.Chliaria Moore, 1884Genus Chliariaothona   Fig. c.Common name: Orchid Tit.Identification remarks: Two tails. UN white, faint cell-end bars, black-edged brown markings. UNF band upper part wider than the lower part. UNH central band broken twice; prominent black spot near base. Wingspan 24–27 mm.Season: Winter.Habitat and activity: The species was observed in forested patches while mud puddling, basking or nectaring.Spalgis Moore, 1879Genus Spalgisepius .Common name: Apefly Fig. d.Identification remarks: HW Tailless. UN with several fine wavy vertical lines. Male FW has acute apex and straight outer edge. Female has rounded outer edge. Caterpillars feed on mealy bugs. Wingspan 20–30 mm.Season: Winter.Habitat and activity: The species was observed in forested patches while feeding on bird droppings.Spindasis Donzel, 1847Genus Spindasisvulcanus   Fig. e.Common name: Common silverline.Identification remarks: Two tails, one lobe on HW. UN light yellow, black or brown bordered brilliant reddish bands with central silver lines. Separate spots at base of UNH and outer basal band of spots does not extend downwards to first costal vein. Orange-crowned black spot on UNH lobe. Female larger than male and with more rounded FW. Wingspan 26–34 mm.Season: Summer.Habitat and activity: The species was observed in plains and undulating terrains while either mud puddling, basking, nectaring or feeding on carcass.Tarucus Moore, 1881Genus Tarucusananda   Fig. f.Common name: Dark Pierrot.Identification remarks: HW Tailed. Resembles Assam Pierrot, differs in having the central spot in space 5 joined to the band of spots near margin on UN. Wingspan 22–28 mm.Season: Winter.Habitat and activity: The species was observed in forested patches while mud puddling.Athyma Westwood, 1850Genus Athymainara Westwood, 1850   Fig. bCommon name: Common sergeant.Identification remarks: A prominent row of black spots always towards the inner edge of the white band on both sides of HW. UPF white cell streak divided into four parts. Wingspan 60–70 mm.Season: Winter.Habitat and activity: The species was observed in forested patches while mud puddling or basking.Charaxes Ochsenheimer, 1816Genus Charaxespsaphon Westwood, 1847   Fig. d.Common name: Rustic.Identification remarks: Basal area of UPF reddish-brown, a broad yellow or white central band and broad black apex. Two darker marginal lines of crescents on UPH. Sexes similar. Wingspan 50–60 mm.Season: Monsoon and winter.Habitat and activity: The species was observed in forested patches while mud puddling, basking or nectaring.Euploea Fabricius, 1807Genus Euploeaklugii Moore, 1858   Fig. f.Common name: Double branded crow.Identification remarks: Similar to Common Crow, but male has two parallel brands on UPF; female has two similar faint streaks near inner edge on UPF. Wingspan 95–105 mm.Season: Summer.Habitat: The species was observed in forested patches while mud puddling, basking or nectaring.PolyuraGenus Polyurabharata Drury, 1773.Common name: Cryptic Nawab Fig. a.Identification remarks: Pale greenish-yellow, wide central band on both sides. Large pale green spot near FW apex on both sides. Wingspan 60–75 mm.Season: Winter.Habitat: The species was observed in forested patches while mud puddling or basking, feeding on tree sap, animal waste or carcasses.Tanaecia Butler, 1869Genus Tanaecialepidea   Fig. b.Common name: Grey Count.Identification remarks: UP dark brown with pale grey border. Border broad on HW and narrow on FW, ending before apex. FW apex produced and outer edge incurved. Female, larger and duller coloured than male, with extra pale brown markings. Wingspan 65–85 mm.Season: Monsoon and winter.Habitat and activity: The species was found at forest edges while mud puddling or basking or feeding on tree sap, carcasses, animal waste, bird droppings or rotten fruits.Tirumala Moore, 1880Genus Tirumalaseptentrionis   Fig. c.Common name: Dark Blue Tiger.Identification remarks: Similar to Blue Tiger, but markings narrower and darker. UNH has a long V-shaped pale blue marking in the cell. UN darker than Blue Tiger. Male UNH has scent scales pouch. Wingspan 75–95 mm.Season: Summer and winter.Habitat and activity: The species was observed in forested patches while mud puddling, basking or nectaring.Pachliopta Reakirt, 1865Genus Pachlioptaaristolochiae   Fig. a.Common name: Common Rose.Identification remarks: HW tailed. UNF black with pale greyish stripes between veins. UNH has large white patch of five elongate spots around end-cell, series of bright red or brownish-red spots on outer edge. Body red. Wingspan 80–110 mm.Season: Winter.Habitat and activity: The species was observed at forests edges, scrubs and in grasslands while nectaring.Pachlioptahector   Fig. b.Common name: Crimson rose.Identification remarks: HW tailed. Markings on both sides similar. Body bright crimson. Female duller, with larger crimson crescents and spots on HW. Wingspan 90–110 mm.Season: Winter.Habitat and activity: The species was observed at forests edges, scrubs and in grasslands while nectaring.Papilio Linnaeus, 1758Genus Papiliohelenus Linnaeus, 1758   Fig. d.Common name: Common Albatross.Identification remarks: Male UPF with dark dusting in apical area and along outer edge, but may be absent. No dark spot on UPF. Pale dull yellow UNH unmarked. Seasonal variation seen in both sexes. In female, UPF apex, leading edge and outer edge bordered with black with four to five white spots near apex. No cell spot. UPH has toothed black border. Wingspan 60–75 mm.Season: Monsoon and winter.Habitat and activity: The species was observed in forested patches while nectaring.Appiasindra Moore, 1857  edges black with two to five apical white spots. Males of northern population have complete row of four or five apical spots on UPF. UPF has black area along outer edge which extends inwards. In female, UPF black, with central white patch and two white spots at apex. UPH with black outer half and dusky grey or white basal half. UNF with broad dark band from leading edge to outer edge. UNH variable. Wingspan 60–70 mm.Season: Winter.Habitat and activity: The species was observed in forested patches while nectaring.Appiaslibythea Fabricius, 1775  and Celaenorrhinus, Taractrocera and Spialia (Family Hesperiidae). These butterflies could be observed in open areas on hill-tops and hill-slopes during monsoon and winter months, but their number becomes less in these areas during the months of summer when they could be observed in dark, shady habitats. We were, however, unable to determine the cause of the high number of hesperiid observations during the monsoon and this needs a detailed behavioural study.The butterfly diversity and distribution is known to be affected by seasons . This isThe patterns for the diversity of butterflies of Matheran are very similar to those of the California Channel Island Birds and Vanuatu Birds, mentioned by https://www.inaturalist.org/) for conveying information regarding the seasons and activities of butterflies.This novel approach is expected to improve the representation of the data for seasons and activities of the Indian butterflies. We encourage adding more activities and unique colour codes to make this system more universal, uniform and reader friendly. We also recommend its use while uploading records on open databases, such as Butterflies of India  and iNatA total of 140 species of butterflies belonging to six families were recorded from Matheran, India. This list includes 77 new records for Matheran. We observed a strong seasonal variation in butterfly diversity. The maximum diversity (N = 125) of butterflies was recorded during winter, while the least (N = 80) during monsoon. A high similarity of butterfly species composition was observed between the pairs of sites studied, tending towards perfect nestedness. This also emphasises the fact that the butterfly diversity in the region is quite stable and chances of emigration to, or immigration from, surrounding regions are very low. A strong seasonal gradient for activity patterns was not observed; however, we did observe a 'pocket effect' of dry season on butterflies. Butterflies during the dry season tend to aggregate near damp and shady places. Further, we introduce a novel barcode system for denoting seasons and activities of Indian butterflies and hope that this will help butterfly biologists to concisely and effectively present the data.B7F5EC55-BD5D-5C37-8899-426DCA2A73CD10.3897/BDJ.8.e54333.suppl1Supplementary material 1Percentage matrix fill and percentage contributions from the SDR-simplex analyses of family-wise and overall species richness.Data typeTableFile: oo_429763.docxhttps://binary.pensoft.net/file/429763Sawant, M., Sarang, S., Modak, N.E51D2D46-C59D-525F-B592-5880AD01F3F310.3897/BDJ.8.e54333.suppl2Supplementary material 2Similarity-Richness difference-Species replacement simplex plot for overall butterfly diversity of Matheran showing high similarity. Points denote pair of sites (N = 28)Data typeImageFile: oo_429764.docxhttps://binary.pensoft.net/file/429764Sawant, M., Sarang, S., Modak, N.


Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis are the most important causative agents of leishmaniases in the New World. These two parasite species may co-circulate in a given endemic area but their interactions in the vector have not been studied yet. We conducted experimental infections using both single infections and co-infections to compare the development of L. (L.) infantum (OGVL/mCherry) and L. (V.) braziliensis (XB29/GFP) in Lutzomyia longipalpis and Lutzomyia migonei. Parasite labelling by different fluorescein proteins enabled studying interspecific competition and localization of different parasite species during co-infections. Both Leishmania species completed their life cycle, producing infective forms in both sand fly species studied. The same happens in the co infections, demonstrating that the two parasites conclude their development and do not compete with each other. However, infections produced by L. (L.) infantum reached higher rates and grew more vigorously, as compared to L. (V.) braziliensis. In late-stage infections, L. (L.) infantum was present in all midgut regions, showing typical suprapylarian type of development, whereas L. (V.) braziliensis was concentrated in the hindgut and the abdominal midgut (peripylarian development). We concluded that both Lu. migonei and Lu. longipalpis are equally susceptible vectors for L. (L.) infantum, in laboratory colonies. In relation to L. (V.) braziliensis, Lu. migonei appears to be more susceptible to this parasite than Lu. longipalpis.Leishmaniases are neglected tropical diseases and  The causative agents, flagellates of the genus Leishmania (Kinetoplastida: Trypanosomatidae), subgenera Leishmania and Viannia, are transmitted by phlebotomine sand flies (Diptera: Psychodidae)2.Leishmaniases are important parasitic diseases, causing serious medical problems in many countries, as they rank in the top-three list of neglected tropical diseases caused by protistsLeishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis are the most important causative agents of leishmaniases in humans3. L. (V.) braziliensis causes a typical cutaneous leishmaniasis (CL), which may progress to mucosal disease, whereas L. (L.) infantum infections is responsible for a life-threatening form of the disease – visceral leishmaniasis (VL). These two parasite species also differ in their development in the sand fly vector; both colonize the sand fly midgut, but only L. (V.) braziliensis was documented to colonize the hindgut (peripylarian development)4.In Brazil, 5, the significance of co-infections by different Leishmania in sand flies is poorly understood. So far, rather limited number of investigations has been conducted to study the simultaneous development of different Leishmania species in the same sand fly7. In particular, it would be interesting and important to study simultaneous development of a suprapylarian Leishmania and a peripylarian Viannia.As different parasite species may co-circulate in a given endemic areaL. (L.) infantum and L. (V.) braziliensis in Lutzomyia longipalpis, a known permissive vector9, and Lutzomyia migonei, which is susceptible to the development of L. (V.) braziliensis10 and to different strains of L. (L.) infantum11. Parasite labelling by different fluorescein proteins enabled us to study interspecific competition during co-infections.The present study was designed to fill this gap in knowledge and conducted experimental co-infections to compare the development of Lu. migonei females were dissected. On day 2 post infection (PI), L. (L.) infantum infection rate was 95% and parasites grew very vigorously, whereas L. (V.) braziliensis infection rate was 70% and parasites grew slower .In total, 322 wer Fig. . Both pawer Fig. . There wL. (L.) infantum infection rate was still high  braziliensis infection rate was lower (50%) and parasites colonized only the midgut and the hindgut. Differences in infection rates were non-significant , whereas differences in the location of the parasites were significant . Similar differences between L. (L.) infantum and L. (V.) braziliensis were also observed in parasite loads and their localization during coinfections  infantum developed very successfully (infection rate 90%), whereas L. (V.) braziliensis grew less successfully (infection rate 68%), but there was no statistical difference . L (L.) infantum colonized the cardia and the stomodeal valve significantly more frequently , whereas L. (V.) braziliensis did not colonize the stomodeal valve at all  infantum produced high infection rates  braziliensis infections were limited to the midgut and hindgut and some co-infected females became negative for L. (V.) braziliensis   Fig. , with pa3%) Fig. .L. (L.) infantum developed more successfully than L. (V.) braziliensis .On day 5 PI, Lu. longipalpis infected with L. (L.) infantum had a heavy infection rate (75%), whereas in L. (V.) braziliensis parasites were less numerous and the infection rate (66%) was significantly lower   infantum parasites frequently colonized the stomodeal valve while L. (V.) braziliensis infections were mostly limited to in the midgut and pylorus region of the hindgut  Fig. . L. (L.)gut Fig. . The samCo-infection did not alter the dynamics of infection of the individual parasite species and did not favour or harm any of the parasite species and the same differences in parasite localization were observed in coinfections.L. (L.) infantum, elongated nectomonads were the prevailing forms in both vectors  . On the other hand, significant differences were found between morphological forms of L. (V.) braziliensis  in both vectors: elongated nectomonads  but short nectomonads . The metacyclic  Fig. , and difads Fig.  prevaileads Fig.  were thelic Fig.  forms weLeishmania species were more pronounced: in Lu. migonei and Lu. longipalpis infected by L. (L.) infantum the majority of parasites were present as elongated nectomonads, whereas in Lu. migonei and Lu. longipalpis infected by L. (V.) braziliensis the prevailing forms were short nectomonads  braziliensis  infantum and L. (V.) braziliensis grew similarly until the third day of cultivation . From day 4 PI, L. (V.) braziliensis grew slower compared to L. (L.) infantum. The differences were statistically significant .To investigate the ion Fig. , there wLeishmania spp. in two New World phlebotomine sand fly species, using both single infections and co-infections. Differences found between L. (L.) infantum and L. (V.) braziliensis were more pronounced than the differences between Lutzomyia species. In particular, L. (L.) infantum produced higher infection rates and grew more vigorously, as compared to L. (V.) braziliensis, in both Lu. longipalpis and Lu. migonei. Both sand fly species tested were similarly susceptible to infection by L. (L.) infantum, which confirms previous findings11.We evaluated the developmental patterns of two Leishmania species9. Specific vectors, like Phlebotomus papatasi support development of a single parasite species or two closely related parasite species12 In contrast, permissive vectors support experimental infections by a broad range of Leishmania spp. The most important example of a permissive vector is Lu. longipalpis, which was involved in the establishment of L. (L.) infantum in Latin America9 and is proven vector of this parasite in many countries, from Costa Rica to northern Argentina13. Here we confirmed that Lu. migonei should be also considered as the permissive vector, as females of this species support the development of all Leishmania species tested so far, namely Leishmania (Leishmania) amazonensis, L. (V.) braziliensis and L. (L.) infantum11.Sand fly vectors have been classified into two categories, specific vectors and permissive vectors, based on their ability to support late-stage development of different Lu. migonei was more susceptible to L. (V.) braziliensis than Lu. longipalpis. Previously, several sand fly species were described as vectors of L. (V.) braziliensis14, but the role of Lu. longipalpis and Lu. migonei in the circulation of this parasite was unknown5. Natural infections of Lu. migonei by L. (V.) braziliensis were repeatedly found in the Rio de Janeiro State15. Nieves and Pimenta10 conducted a laboratory study on susceptibility of Lu. migonei to L. (V.) braziliensis resulting in 77% positivity of the dissected females. Lu. longipalpis carrying DNA of L. (V.) braziliensis has been described in the South-Eastern region of Brazil16 and in laboratory conditions 70% of Lu. longipalpis females developed late stage infections of L. (V.) braziliensis17.Nevertheless, our detailed comparative study revealed that L. (L.) infantum developed more rapidly in both sand fly species than L. braziliensis. This correlates with growth curves we observed in vitro: during initial three days both species grew similarly, but then, from day 3 onwards, L. (L.) infantum grew significantly faster. These results suggest that development in the sand fly is affected not only by the susceptibility of the vectors, but also by the growth characteristics of the studied strains. In late-stage infections, L. (L.) infantum was present in all midgut regions, the cardia and stomodeal valve in both sand fly species from day 5 PI, showing typical suprapylarian type of development. In contrast, L. (V.) braziliensis was concentrated in the hindgut and the abdominal midgut (peripylarian development). The same happens in the co infections, demonstrating that the two parasites conclude their development and do not compete with each other. Previous study with Lu. migonei and two Leishmania species was done using single infections only, but observations by Nieves and Pimenta10 were similar to our results: suprapylarian L. (L.) amazonensis grew faster than peripylarian L. (V.) braziliensis.This interspecific difference appears to be strain-independent as almost identical patterns of development were described for the L. (V.) braziliensis strain m290310 and for two different L. (L.) infantum strains M4192 and CUK3 in Lu. migonei11.In single infections and co-infections, 18. Both Leishmania species completed the life cycle, producing infective forms in both sand fly species studied. We conclude that both Lu. migonei and Lu. longipalpis are equally susceptible vectors for L. (L.) infantum, in laboratory colonies. In relation to L. (V.) braziliensis, Lu. migonei appears to be more susceptible to this parasite than Lu. longipalpis.From an epidemiological point of view, it is very important to carry out studies, assessing the development of two species of parasites that cause different forms of leishmaniasis, especially when they coexist in endemic regionsLu. longipalpis  and Lu. migonei  were used and maintained under standard conditions, as previously described19. A fluorescent strains of L. (V.) braziliensis (XB29 marked with GFP) and L. (L.) infantum (OGVL marked with mCherry) by stable integration of mCherry into the 18 S rRNA locus as previously described20. We have used parasites within less than 10 passages in vitro and were maintained at 23 °C on Medium 199  supplemented with 10% fetal calf serum , 1% BME vitamins (Sigma-Aldrich), 2% human urine and 250 μg/ml amikin .Established laboratory colonies of in-vitro, two initial doses were used: 104 promastigotes/ml and 105 promastigotes/ml. The concentration of parasites was analyzed daily for 7 days, using counting in Burker chamber. The experiments were repeated twice.For study of growth curves 6 promastigotes/ml for single infections and 5 × 105 of each species for co-infections . Three groups of females of Lu. migonei were studied, the first infected with L. (V.) braziliensis (XB29 marked with GFP), the second with L. (L.) infantum (OGVL marked with mCherry) and the third co-infected with both Leishmania species. The same procedure was performed with L. longipalpis. Engorged females were separated and maintained in the same conditions as the colony and dissected on days 2, 5 and 8 post-infection (PI). Individual guts were placed into a drop of saline and analyzed by fluorescence microscopy for the localization, infection intensity and morphology of Leishmania infections. Parasite loads were graded according to Myskova et al.21 as light (<100 parasites per gut), moderate (100 to 1000 parasites per gut) and heavy (>1000 parasites per gut). The experiments were repeated four times.Female sand flies (3–6 days old) of both species were fed through a chick-skin membrane on heat-inactivated rabbit blood containing 10Lu. migonei and Lu. longipalpis infected with L. (L.) infantum and L. (V.) braziliensis, alone or in combination  were prepared on days 5 and 8 PI, being fixed with methanol and stained with Giemsa. Stained smears were then examined under a light microscope with an oil-immersion objective and photographed with an Olympus D70 camera . Body length, body width and flagellar length of 200 randomly selected promastigotes from four females/smears were measured for each sand fly species and time intervals using Image-J 1.x software. Promastigote forms were distinguished according to Walters et al.22 and Sadlova et al.23. L. (L.) infantum, developmental forms were identified as follows: (i) elongated nectomonads (body length ≥14 μm) (ii) short nectomonads (body length <14 μm and flagellar length ≤2 times body length); (iii) metacyclic promastigotes (body length <14 μm and flagellar length ≥2 times body length); and (iv) haptomonads . L. (V.) braziliensis, developmental forms were identified as follows: (i) elongated nectomonads (body length ≥13 μm); (ii) short nectomonads (body length <13 μm and flagellar length  ≤ 2 times body length); (iii) metacyclic promastigotes (body length <13 μm and flagellar length ≥2 times body length); and (iv) haptomonads .Smears from midguts of Animals used for blood-feeding of sand fly colonies were maintained and handled in the animal facility of Charles University in Prague, in accordance with institutional guidelines and Czech legislation , which complies with all relevant European Union guidelines for experimental animals. All experiments were approved by the Committee on the Ethics of Laboratory Experiments of the Charles University in Prague and were performed under the Certificate of Competency (Registration Number: CZ 03069).


Candida auris is associated with invasive infections in critically ill patients and has been isolated in different countries worldwide. Ease of spread, prolonged persistence in the environment and antifungal drug resistance pose a significant concern for the prevention of transmission and management of patients with C. auris infections. Early and correct identification of patients colonized with C. auris is critical in containing its spread. However, this may be complicated by C. auris strains being misidentified as other phylogenetically related pathogens. In this review, we offer a brief overview highlighting some of the critical aspects of sample collection, laboratory culture-dependent and independent identification and the susceptibility profile of C. auris.The multidrug-resistant yeast  Candida auris, the yeast pathogen firstly isolated from a Japanese patient’s external ear canal in 2009, has been involved in invasive healthcare-associated outbreaks and sporadic cases reported in various countries worldwide ..C. aurisC. auris may be misidentified as Candida haemulonii, Candida sake, Rhodotorula glutinis or other Candida species by these conventional biochemical systems because it is not always present in databases or because of the overlapping biochemical profiles [profiles . In the profiles .C. auris with high reliability. However, Ambaraghassi and colleagues showed that this software precisely identifies isolates from the South American clade, but had limited power to correctly identify C. auris from the African and East Asian clades [C. auris, C. famata, and species in the C. haemulonii complex should be confirmed by MALDI-TOF or DNA sequencing. It has been reported that VITEX 2 XL (bioMérieux version 8.01) is capable of detecting n clades . Hence, Therefore, the methods listed above are currently not precise enough to identify this microorganism. Moreover, the slow turnaround time of enrichment cultures could be a significant limiting factor .C. auris may be used [Both culture- and non-culture-based molecular methods for the detection of  be used ,42,43,44C. auris over other Candida species [Culture-based molecular methods rely on the yeast growth conditions (10% NaCl and 40 °C) and on its use of dulcitol as a carbon source to enrich  species ,42. Then species . It must species . Culture species ,33,41,42Moreover, they were confirmed by testing on large yeast panels and are time-saving as they reduce the overall turnaround time from days to working hours, allowing the rapid identification of colonized patients ,40,41,46C. albicans/C. tropicalis, C. parapsilosis and C. glabrata/C. krusei) directly from blood specimens [C. auris panel, it detects this microorganism with a limit of detection < 5 CFU/mL in less than 5 h [Currently, a T2 magnetic resonance assay  is available for the rapid (<3 h) and accurate and sensitive (1–3 CFU/mL) detection of specific Candida species  detection of amplicon and melting-point analysis—distinguishes between C. auris, C. duobushaemulonii, C. haemulonii, and C. lusitaniae [C. auris cells; in the case of a positive result from a nonsterile specimen , it could indicate past or present colonization with C. auris.Kordalewska and colleagues developed two rapid, accurate, easy-to-perform molecular diagnostic assays based on real-time PCR to discriminate sitaniae . Both PCsitaniae . Of noteC. auris from colonies recovered in the enrichment broth, distinguishing them from other Candida spp. only if C. auris spectrum is included in the reference database [C. auris is currently more likely with the FDA-approved MALDI-TOF Biotyper , using their updated research use only (RUO) library (Versions 2014 5627 and more recent) or CA System library (Version Claim 4), or with the VITEK (MALDI-TOF) MS (bioMérieux) FDA-approved IVD v3.2 or the RUO Version 4.14 with the Saccharomycetaceae update [MALDI-TOF mass spectrometry (MS) can reliably differentiate database . Precisee update ,29.C. auris isolates, even with an upgraded RUO [Some authors reported higher accuracy and efficiency for the Bruker Biotyper over the Vitek 2 MS in the identification of aded RUO .C. auris to the species level (https://www.cdc.gov/microbenet/index.html) [C. auris isolates, compared to conventional identification methods. However, it represents a time-consuming method when applied to all colonies recovered from colonized patients and requires a substantial upfront investment [In addition to these options, the CDC, in collaboration with Bruker, provides an online tool for the accurate classification of ex.html) . Three dex.html) . In spitex.html) . Therefovestment .C. auris isolates [RPB1 and RPB2, have been reported to be highly discriminatory for strain differentiation between C. haemulonii complex and C. auris [Multilocus sequence typing (MLST), a proteomic analysis performed with MALDI-TOF MS, amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS), can be used to evaluate the genetic relatedness of isolates . For MLSC. auris .C. auris isolates clonality as sequence variation between individual strains within a clonal lineage is very small  [For AFLP analysis, genomic DNA was subjected to a combined restriction–ligation procedure containing EcoRI and MseI restriction enzymes  and complementary adaptors . However genome) ,53,54,55C. auris. In concordance with WGS analysis, the authors identified five major different C. auris clusters  and most isolates differing by >30 SNPs were determined [Recently, de Groot and colleagues developed a short tandem repeat (STR) typing assay for termined .C. auris [C. auris outbreak investigations because only this technique grouped the isolates into four main clusters, in accordance with WGS data. The other typing tools showed poor performances with the highest agreement between microsatellite typing and ITS sequencing with 45% similarity, followed by microsatellite typing and FTIR with 33% similarity. The lowest agreement was observed between FTIR spectroscopy, MALDI-TOF MS and ITS sequencing.In a recent study, Vatanshenassan and colleagues compared different typing techniques  to evaluate their application in typing C. auris . ResultsC. auris is its ability to develop resistance to all three of the main classes of antifungal drugs , with severely limiting clinical and therapeutic management options [C. auris isolates, with a small proportion (4%) exhibiting resistance to all classes of antifungals. Therefore, when in the presence of an unidentified yeast resistant to more than one antifungal drug, further testing for C. auris identification should be performed [One of the most concerning aspects of  options ,59,60,61erformed ,16,55,62C. auris in New York, three were found to have pan-resistant C. auris that had developed after treatment with antifungal medications, including echinocandins. All three patients had multiple comorbidities, but no recent domestic or foreign travel [Recently, among 801 patients identified with n travel .C. auris with distinct drug susceptibility profiles with a C. albicans reference strain using a multiomics approach. Their results highlighted that, despite the distinctive drug resistance profile, C. auris isolates were very similar. In contrast, their profile was different from that of C. albicans, both in terms of carbon utilization and in lipid and protein content, supporting a multifactorial mechanism of drug resistance [Zamith−Miranda and colleagues compared two clinical isolates of sistance .C. auris, especially among isolates from India, where a study of 350 isolates showed around 90% minimal inhibitory concentrations (MICs) of fluconazole greater than 16 μg/mL, and South Africa [C. auris can also, to a lesser extent, exhibit reduced susceptibility to other triazole antifungals  [C. auris commonly exhibits susceptibility to the polyene amphotericin B; however, geographical differences are found. For example, resistance to amphotericin B was detected in 30% of C. auris U.S. isolates [C. auris. Still, some authors related it to a reduction in ergosterol content in the cellular membrane [C. auris isolates from multiple geographic areas in patients initially treated with an echinocandin [In general, high levels of resistance to fluconazole are observed in h Africa ,68,69,70isolates . The priC. auris can be performed using CLSI and EUCAST broth microdilution methods, as well as the E-test gradient diffusion method (bioMérieux), Sensititre YeastOne (Thermo Fisher Scientific) and Vitek-2 Yeast susceptibility system (bioMérieux). Nonetheless, these in vitro methodologies may present limitations, such as slow turnaround time (24 h after isolation) and, not lastly, the need of specific know-how to readout C. auris MIC values [Antifungal susceptibility testing (AFST) for C values . In TablC. auris. Some epidemiologic cut-off values (ECVs) have been suggested by Arendrup and colleagues comparing the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) reference microdilution MICs, which appeared to have minor differences [C. auris isolates from different clades [C. auris MIC interpretation, based on information gathered for Candida spp. and several expert opinions .Currently, no established susceptibility breakpoints are available for ferences . Howevert clades ,72. MoreC. auris MIC values, we must mention the possibility of running into the “eagle” effect or paradoxical growth effect of echinocandins, which is the reduced antifungal behavior at higher doses of the drug in vitro. It is important to underline that the eagle effect does not reflect the potential in vivo response of C. auris to echinocandins, as indicated in an invasive murine candidiasis model [Talking about the need of specific know-how to readout is model .As regards automated systems for routine AFST, some authors observed a good (96.7%) agreement between VITEK 2 and CLSI methods in fluconazole AFST, although others have reported suboptimal performance for amphotericin B ,75.C. albicans and then in anidulafungin-resistant C. glabrata with known FKS2 mutations, where they obtained less satisfactory results [C. albicans and C. glabrata and of C. auris [C. auris isolates nonsusceptible against echinocandin antifungals within 6 h, making it a promising candidate for AFST in clinical laboratories in the future [MALDI-TOF MS has also been increasingly used for AFST. Vella and colleagues have developed a rapid AFST assay based on MALDI-TOF MS analyzing changes in the MS profile spectra induced by antifungals after 3–6 h of incubation, firstly in caspofungin-resistant  results ,77. ReceC. auris ,79. The C. auris screening, a duplex ERG11 assay enabling detection of mutations at positions Y132 and K143, and a simplex FKS1 HS1 assay enabling detection of mutations at position S639 were developed to identify known mutations related to resistance to azoles and echinocandins, respectively [m). These assays are not only very reproducible but may also be updated each time a new mutation is detected [In place of traditional methods, rapid molecular methods can be used to identify resistance-conferring mutations . These mectively . Resultsectively . Moreovedetected .C. auris has two characteristics that make it an epidemiologically important microorganism: the ability to spread rapidly and a multidrug resistance phenotype. For these reasons, its detection requires the implementation of basic infection control measures [C. auris is detected the ECDC recommends activating the screening of close contact patients, possibly extending contact tracing based on a case-by-case risk evaluation  [C. auris are also crucial. Meanwhile, point prevalence surveys should be run to identify colonized patients in hospital units where the index patient is or was present. It is also suggested to review patient records to determine any prior healthcare exposures, mainly overnight stays in healthcare facilities in the month prior to culture positivity [As previously said, measures . To contization) . Prompt sitivity .C. auris colonization/infection status is recommended [C. auris infection/colonization at the moment of hospital admission is of utmost importance [When patients are moved to other healthcare facilities, notification of ommended . For theportance ,80.C. auris has proven susceptible to chlorhexidine in vitro in recent works; however, in a cohort of UK patients, despite daily chlorhexidine bathing, colonization with C. auris was not eradicated [Infection control should include single room isolation or patient cohorting and dedicated nursing staff. Currently, there is no established decolonization protocol, so the above-described measures should be applied promptly. adicated ,81. Furtadicated .C. auris. It is therefore crucial to have a cleaning protocol in place and strengthen the regular decontamination of the equipment and environment, especially high-touch areas [However, it is known that reusable equipment may be a source of transmission of infection of ch areas .C. auris has been associated with nosocomial outbreaks on five continents in the last ten years. Prevalence of infection is unpredictable due to misidentification and unreported cases. Proper microbiological identification, rigorous epidemiological surveillance, adequate treatment and prevention and containment strategies, combined with higher awareness on the side of physicians, microbiologists and healthcare workers, are indispensable to limit further spreading of this pathogen. Further research should investigate rapid and accurate laboratory identification methods and evaluate the clinical role of new therapeutic options to counteract C. auris antifungal resistance.The emerging pathogen


Snodgrassella alvi affects honeybee immune gene expression. We show that both live and heat-killed S. alvi protect honeybees from the opportunistic pathogen Serratia marcescens and lead to the expression of host antimicrobial peptides. Honeybee immune genes respond differently to live S. alvi compared to heat-killed S. alvi, the latter causing a more extensive immune expression response. We show a preference for Toll pathway upregulation over the Imd pathway in the presence of both live and heat-killed S. alvi. Finally, we find that live S. alvi aids in clearance of S. marcescens from the honeybee gut, supporting a potential role for the symbiont in colonization resistance. Our results show that colonization by the beneficial symbiont S. alvi triggers a replicable honeybee immune response. These responses may benefit the host and the symbiont, by helping to regulate gut microbial members and preventing overgrowth or invasion by opportunists.The gut microbiome plays a critical role in the health of many animals. Honeybees are no exception, as they host a core microbiome that affects their nutrition and immune function. However, the relationship between the honeybee immune system and its gut symbionts is poorly understood. Here, we explore how the beneficial symbiont  Thesl motifs . For Toll motifs . The Imdl motifs . Dorsal l motifs . Honeybeoptaecin –22.apidaecin in response to S. alvi colonization [S. alvi in honeybee immune priming. However, neither study measured transcriptional responses of fat bodies—the centre of the insect immune response [Honeybees upregulate expression of nization ,24. Combresponse . AdditioS. alvi plays in activating the honeybee immune system and protecting hosts from pathogens. Using both live and heat-killed bacteria, we show that colonization is not required for pathogen protection, but aids in pathogen clearance, and that heat-killed S. alvi causes a more extensive immune expression response than the live symbiont. These results suggest that S. alvi is not only priming the honeybee immune system but also potentially modulating it.In this study, we investigated the role 2.(a)Escherichia coli strain MG1655 was grown at 37°C on LB agar. S. alvi strain wkB2 and S. marcescens strain N10 were grown as previously described, on Columbia agar supplemented with sterile sheep blood at 35°C and 5% CO2 [S. alvi and S. marcescens isolates were previously derived from honeybee guts.(b)E. coli and S. alvi on agar plates were pooled individually and suspended in 500 μl PBS. The OD600 of bacterial cultures was measured, and the amount of S. alvi and E. coli used for heat-killing was diluted to an OD representative of 5 × 108 CFUs. Heat-killed cells were prepared by then suspending S. alvi or E. coli in 1 ml of PBS and heating to 80°C. Cells were left at 80°C for 30 min for S. alvi and 10 min for E. coli. Direct plating of heat-killed bacterial suspensions confirmed that no live cells were present.Overnight growths of (c)Apis mellifera samples were obtained from hives at the University of Texas at Austin. To obtain microbiota-free bees, dark-eyed pupae were removed from capped brood cells using sterilized forceps and placed in sterile plastic cages as previously described [All escribed . Bees obescribed . Newly e(d)−1 of input RNA. Relative gene expression was determined using quantitative PCR. The source of primers used in qPCR assays can be found in electronic supplementary material, table S1. Absolute quantification of the housekeeping gene RPS18 was accomplished by cloning RPS18 into the Promega pGEM-T vector . Primer efficiency was determined using 10-fold serial dilutions of primer standards. Standards for target genes not cloned into a pGEM-T vector were created by PCR amplification of the control group cDNA with target gene qPCR primers. Reactions were run in an Eppendorf MasterCycler Realplex machine using technical replicates for each sample and Bio-Rad iTaq Universal SYBR Green Supermix  for fluorescence. Relative gene expression for the genes, abaecin, apidaecin, hymenoptaecin, cactus-1, cactus-2, dorsal, relish, dredd, pgrp-lc, pirk and toll, were determined using a 2-ΔΔCt method and log2 transformed [Bees were sampled on their respective days post bacterial inoculation and moved into 15 ml centrifuge tubes to be frozen at −80°C. Abdomens were removed from each bee and placed into 600 μl of RNA lysis buffer and pestle homogenized. RNA was then isolated using a Zymo Research Quick-RNA Tissue/Insect Microprep Kit . RNA was eluted in 50 μl of RNase-free water and stored at −80°C. Concentrations were quantified using a Thermo Fisher Scientific NanoDrop Lite Spectrophotometer. RNA was reverse-transcribed using a QuantaBio qScript cDNA Synthesis Kit  with all reactions normalized to 500 ng μlnsformed .(e)S. marcescens diluted to an OD600 of 1 for survival assays and 0.5 for clearance assays. In survival assays, bees were then returned to their respective cup cages and the number of dead bees was recorded daily for 10 days. Survival data were pooled from three independent trials. A Cox proportional hazard model was used to test any effect independent trials might have on bee survivorship . In the clearance assay, whole guts were extracted from bees 1 day and 3 days post-inoculation with S. marcescens. These guts were immediately homogenized into 200 μl PBS. We prepared serial dilutions and spot-plated 10 μl of each dilution onto LB agar plates. S. marcescens colonies were counted after 1 day at 37°C in aerobic conditions and total CFUs per gut calculated. Less than 10 discernible CFUs were found in bees given no bacterial exposure.Microbiota-free bees were fed their respective bacterial, heat-killed bacterial or PBS treatment. Five days post-inoculation bees were chilled and placed in 0.5 ml microcentrifuge tubes, each with a hole cut in the bottom. Each bee was then hand-fed 5 μl of a mixture of 20% sucrose and 80% PBS or this same mixture plus a 24 h growth of (f)https://rstudio.com/) [S. marcescens clearance assays was accomplished using the Tukey honest significant difference method [Survival curves were created using the survminer and survival packages in RStudio (io.com/) ,29. Curvio.com/) . The Forio.com/) . Statistio.com/) ,33. Geneio.com/) . Statiste method .3.(a)S. marcescens [S. alvi, heat-killed S. alvi or sterile sugar syrup. Five days post-treatment, each bee was hand-fed S. marcescens or sterile PBS. Across treatments, bees receiving no pathogen had higher survival than bees challenged with the pathogen S. marcescens by heat-killed S. alvi supports immune priming as a factor in pathogen defence. To determine if live and dead S. alvi trigger a host immune response, we treated microbiota-free bees with live S. alvi, heat-killed S. alvi and heat-killed E. coli and assessed expression of immune genes 5 days later. While S. alvi is a co-evolved symbiont of honeybees, E. coli is not a typical bee gut microbiome member. However, both bacteria are Gram-negative. Therefore, by including heat-killed E. coli we could assess whether gene expression responses were general to Gram-negative bacterial components or if they were symbiont-specific. In all treatments, we found significant upregulation of the AMPs abaecin, apidaecin and hymenoptaecin relative to uninoculated microbiota-free bees (a). In a replicated experiment, sampling bees 1, 2 and 5 days post-inoculation, we saw similar upregulation, suggesting that this response spans sampling dates . These results show that both live and dead S. alvi trigger an immune response and that this response is not symbiont-specific, as heat-killed E. coli treatments show similar upregulation of AMPs.Protection against ree bees a. In a r(c)Drosophila, Dorsal and Relish play critical roles as transcription factors in the expression of AMPs [S. alvi, we measured relative gene expression via qPCR of dorsal, relish and cactus after inoculation with live S. alvi, heat-killed S. alvi or heat-killed E. coli. Surprisingly, bees inoculated with live S. alvi did not have as high upregulation compared to both heat-killed treatments (b). In a replicated experiment, sampling bees 1, 2 and 5 days post-inoculation, we saw similar patterns . There appears to be no correlation between transcriptional regulator gene expression and AMP gene expression, as all treatment groups had similarly elevated AMP expression regardless of dorsal or relish expression . Interestingly, while S. alvi contains components sufficient for immune regulator upregulation, live cells do not trigger the same magnitude of response found in heat-killed treated groups.In  of AMPs ,17. To featments b. In a rpression a,b. Inte(d)S. alvi on bee immune gene expression, we expanded our gene targets to include the receptor toll and pgrp-lc, as well as the Imd regulatory genes pirk and dredd. In Drosophila sp. Pirk is known to inhibit the function of the Imd pathway while Dredd modifies Relish, triggering its translocation to the nucleus [S. alvi. If live S. alvi alone fails to upregulate immune genes, we would expect signals generated by heat-killed S. alvi to dominate. However, if live S. alvi actively suppresses host immune gene upregulation, we would expect a gene expression pattern more comparable to live S. alvi alone. While live and heat-killed S. alvi treatments on their own confirmed previous results, our mixed treatment did not favour one or the other . For the Toll pathway genes toll, cactus and dorsal, the mixed treatment group favoured expression similar to treatment with heat-killed S. alvi alone (b). While we found no change in pirk levels, the expression of the Imd pathway genes dredd, pgrp-lc, and relish in bees treated with both live and heat-killed S. alvi was reduced compared to live or heat-killed treatments alone (c). These results show a context-dependent loss of Imd pathway gene expression when bees were treated with a mixture of S. alvi and heat-killed S. alvi. Additionally, these results suggest that live S. alvi can reduce the expression of Imd pathway genes activated by heat-killed S. alvi.We saw considerable upregulation of AMPs nt group a. For thvi alone b. While ts alone c. These (e)S. alvi increase host survival following subsequent challenge with S. marcescens, and both trigger differential immune gene expression. To determine if these treatments could aid in S. marcescens clearance, microbiota-free bees were inoculated with live S. alvi, heat-killed S. alvi or a mixture of the two, then challenged with S. marcescens. Treatment with live S. alvi reduced S. marcescens CFUs both 1 day and 3 days after pathogen challenge .Binding of the inhibitor Cactus prevents the transcription factor Dorsal from translocating to the nucleus . Activat figures b and 3b.Drosophila Imd pathway, Relish activation requires both cleavage and phosphorylation, carried out by other enzymes. Cleavage and phosphorylation are required for translocation to the nucleus and recruitment of RNA polymerase II [relish may not lead to a heightened host immune response if these other regulatory processes are hindered. Future studies should investigate how post-transcriptional processes affect expression and should quantify immune gene protein levels in response to these treatments.In the erase II ,39. TherS. alvi both enhance survival following S. marcescens exposure and upregulate immune genes, but only live S. alvi or a mixed treatment aids in pathogen clearance (figures S. alvi plays a role in resistance beyond immune gene upregulation, potentially by direct antagonism or by out-competing invading pathogens for space and resources. However, a protective host response does not imply a fitness benefit.Both resistance and tolerance play roles in pathogen defence in animals –42. Resi figures  and 4. TDrosophila, exposure to Salmonella typhimurium or Listeria monocytogenes triggers anorexia and altered immune responses, increasing tolerance to the former while decreasing resistance to the latter [Mycobacterium marinum activates the Drosophila immune system, causing the dysregulation of metabolic pathways leading to host wasting and mortality [S. alvi could reflect reduced investment in costly immune responses made superfluous by the presence of this defensive symbiont.In e latter . Similarortality . In beesSodalis glossinidius requires the outer membrane protein OmpA to form a biofilm and colonize the fly gut [OmpA causes the clearance of Sodalis by the host immune system [S. alvi requires outer membrane proteins for its colonization of the honeybee, forming a biofilm directly on the gut epithelia of the ileum [Sodalis, this biofilm may prevent targeting of S. alvi by immune effectors.Bacterial biofilms can prevent the immune system from recognizing bacterial components and clearing an infection . However fly gut . The knoe system . Similarhe ileum ,47. LikeS. alvi lyses during colonization and growth, however, the dynamics of S. alvi and phage interactions remains unclear. Regardless, the biofilm produced by S. alvi may prevent lysed bacterial components and host immune receptors from coming into contact. Heat-killed cells cannot form a biofilm, and their components likely spread throughout the gut, interacting with membrane receptors in regions not typically associated with S. alvi. These features of heat-killed cells could explain the higher induction by heat-killed S. alvi and the mixed treatment for Toll pathway genes.The honeybee gut harbours a diverse community of lytic phages ,49. It iS. alvi can form a biofilm or in places where S. alvi does not colonize. Future experiments should compare priority effects between live and heat-killed treatments to determine the impact of S. alvi colonization prior to feeding with heat-killed cells and vice versa. Additionally, using S. alvi mutants deficient for biofilm production would help to study the biofilm's significance in immune activation [In the mixed treatment group, bees were fed both live and heat-killed cells simultaneously. Under this simultaneous exposure, heat-killed bacterial components may interact with immune receptors before tivation .Drosophila, Gram-negative bacteria typically trigger the Imd pathway, while Gram-positive bacteria trigger the Toll pathway [S. alvi show similar expression patterns for Imd pathway genes, while live S. alvi treated bees have lower expression of Toll pathway genes (S. alvi is mixed with heat-killed cells, Toll pathway gene expression favours that of bees given heat-killed S. alvi alone. However, we find a sharp drop in Imd pathway gene expression (figures c and S. alvi appears capable of reducing expression of Imd pathway genes (In  pathway ,52. Beesay genes . Once li figures c and 5. ay genes . KnockdoS. alvi over an approximately 80 million-year period has likely resulted in highly specific host–symbiont interaction networks [S. alvi is highly resistant to apidaecin, which is present in the gut lumen after inoculation with gut microbes [S. alvi by the host immune system. We propose that the gut symbiont S. alvi can possibly modulate the host immune system.Coevolution of bees and networks . For examicrobes . This an


Phrynocephalus vlangalii from the subfamily Agaminae. In most previous studies of these sex chromosomes, the focus has been on Australian species from the subfamily Amphibolurinae, but only the sex chromosomes of the Australian central bearded dragon (Pogona vitticeps) are well-characterized cytogenetically. To determine the level of synteny of the sex chromosomes of P. vitticeps across agamid subfamilies, we performed cross-species two-colour FISH using two bacterial artificial chromosome (BAC) clones from the pseudo-autosomal regions of P. vitticeps. We mapped these two BACs across representative species from all six subfamilies as well as two species of chameleons, the sister group to agamids. We found that one of these BAC sequences is conserved in macrochromosomes and the other in microchromosomes across the agamid lineages. However, within the Amphibolurinae, there is evidence of multiple chromosomal rearrangements with one of the BACs mapping to the second-largest chromosome pair and to the microchromosomes in multiple species including the sex chromosomes of P. vitticeps. Intriguingly, no hybridization signal was observed in chameleons for either of these BACs, suggesting a likely agamid origin of these sequences. Our study shows lineage-specific evolution of sequences/syntenic blocks and successive rearrangements and reveals a complex history of sequences leading to their association with important biological processes such as the evolution of sex chromosomes and sex determination.Dragon lizards (Squamata: Agamidae) comprise about 520 species in six subfamilies distributed across Asia, Australasia and Africa. Only five species are known to have sex chromosomes. All of them possess ZZ/ZW sex chromosomes, which are microchromosomes in four species from the subfamily Amphibolurinae, but much larger in  Reptiles are well known for their diverse modes of sex determination and sex chromosomes ,2. They Agamid lizards (Squamata: Agamidae), commonly known as dragons in Australasia, are notorious for their variability in forms of sex determination ,21,22,23Phrynocephalus vlangalii from the subfamily Agaminae are macrochromosomes [Pogona vitticeps, P. barbata, Diporiphora nobbi and Ctenophorus fordi, have micro sex chromosomes [Only about one fifth 91 species) of Agamid species have been karyotyped, with diploid chromosome numbers ranging from 2n = 32 to 2n = 54 ,50. Agam species omosomes , whereasomosomes ,20. The omosomes . Neverthomosomes ,52 with omosomes ,30,53.Pogona vitticeps, has a well-annotated genome with well-characterized ZZ/ZW sex microchromosomes, homologous to chicken chromosomes 17 and 23 [P. vitticeps ZW sex chromosomes as probes and hybridized them to the metaphase chromosomes of 14 acrodont taxa , comprising species that span the spectrum of sex determination, including TSD, GSD and obligatory parthenogenesis.Molecular cytogenetics is a powerful tool for discovering homology and evolutionary trends in reptile sex chromosomes ,55 and h7 and 23 ,58,59,607 and 23 ,62, inclP. vitticeps, Tympanocryptis lineata and Rankinia diemensis) were performed following the Animal Ethics Guidelines of the University of Canberra , with permits issued by the ACT Government (license number LT2017960). Fieldwork conducted for Agama picticauda was under Miami-Dade County Parks and Recreation Scientific Research Permit number 263-2016 and Marquette University IACUC AR-288. Calotes versicolor and Leiolepis reevesii rubritaeniata specimen collection, animal care and procedures were approved by the Animal Experiment Committee, Kasetsart University, Thailand . Phrynocephalus cf. guttatus, Bronchocela cristatella, Leiolepis cf. ngovantrii, Saara loricata and Chamaeleo calyptratus were sampled in collaboration with breeders in Czech Republic. Samples of Hydrosaurus weberi and Trioceros johnstoni were provided by Czech zoological gardens . All experimental procedures in Czech Republic were approved by the Committee for Animal Welfare of the Ministry of Agriculture of the Czech Republic, permissions No. 29555/2006-30 and 8604/2019-7.In total, 22 individuals of 14 species of acrodont lizards—12 agamids (from six subfamilies) and 2 chamaeleonid species—were chosen for the study . Animal P. vitticeps, T. lineata, R. diemensis and A. picticauda for the cytogenetic analyses. Cells were cultured, and metaphase chromosomes were harvested following the procedures as described by Ezaz et al. [C. versicolor and L. reevesii rubritaeniata cells were also cultured from tail tissues. Cell culture and chromosome harvesting followed the procedures as described by Chaiprasertsri et al. [P. cf. guttatus, B. cristatella, L. cf. ngovantrii, S. loricata, C. calyptratus, H. weberi and T. johnstoni were obtained by cultivation of leukocytes and the preparation of the cell cultures and chromosome harvesting followed a detailed protocol described in Mazzoleni et al. [Fibroblast cells were cultured from the tail tissues of z et al. . C. versi et al. . Mitotici et al. .P. vitticeps ZW sex chromosome BAC clones (Pv03_L07 and Pv150_H19) from the P. vitticeps Bacterial Artificial Chromosome (BAC) library  [P. vitticeps have been found to be highly repetitive in nature [ZNF135-like and a fragment of ORPRD1 [NR5A1 gene [P. vitticeps. In addition, BAC Pv03_L07 hybridizes onto the telomeric region of the second-largest chromosome (chromosome 2) of P. vitticeps [Two WA, USA)  were mapn nature . The BACf ORPRD1 . BAC Pv15A1 gene , which i5A1 gene . These t5A1 gene . The twoitticeps ,57,59. Titticeps .All slides were observed, and images of metaphases were captured using a Zeiss Axio Scope A1 epifluorescence microscope fitted with a high-resolution microscopy camera AxioCam MRm Rev. 3 . Images were analyzed using Metasystems Isis FISH Imaging System V 5.5.10 software . P. vitticeps , it is possible that the microchromosome pair with Pv150_H19 could be a sex chromosome in the GSD species. If so, those same homologous chromosomes could be autosomes in the TSD species while still contributing to the sex-differentiation cascade or pathways. Further investigation is required on this aspect.The second BAC clone, Pv150_H19, was derived from Pv03_L07 a. This pPv03_L07 . This sustycinae . The lacelements ,55, whicelements ,26,62. T systems .) Pv150_Pogona vitticeps derived BAC sequences among agamids indicate that there has been conservation of chromosome segments across agamid lineages. The BAC sequence contained in the BAC Pv150_H19 is largely conserved in microchromosomes across the agamid phylogeny, while the other (BAC Pv03_L07) in macrochromosomes appears to have been only translocated to microchromosomes in the ancestor of the studied members of the subfamily Amphibolurinae. It was then likely later duplicated to a microchromosome containing the BAC sequence Pv150_H19, while the original microchromosome copy of the BAC Pv03_L07 has been lost in the ancestor of P. vitticeps (P. vitticeps is thus likely a result of a rather complex history of rearrangements [P. vitticeps and its relatives.The ancestral vertebrate karyotype has remained relatively stable over the last ~370 million years as large segments of ancestral chromosomes are still retained among all lineages . These sitticeps . Since nngements . Future