Blicca bjoerkna and Abramis brama and cross-breeds (the F1 generation of hybrids) under the condition of similar frequencies of pure and hybrid genotypes was investigated. We also examined the potential effect of the maternal origin of hybrids  on monogenean abundance.Host specificity is one of the outputs of the coevolution between parasites and their associated hosts. Several scenarios have been proposed to explain the pattern of parasite distribution in parental and hybrid genotypes ranging from hybrid resistance to hybrid susceptibility. We hypothesized that host-parasite co-adaptation limits the infection of host-specific parasites in hybrid genotypes even under the condition of the high frequency of hybrids. The experimental monogenean infection in pure breeds of B. bjoerkna, the next with A. brama in the maternal positions) were exposed to infection by monogeneans naturally occurring in B. bjoerkna and A. brama. The experiment was run under similar frequencies of the four breed lines.Pure breeds of two cyprinids and two cross-breeds (one with B. bjoerkna and A. brama. However, each species harboured specific monogenean fauna. Hybrids harboured all monogenean species specifically infecting one or the other species. Monogenean infection levels, especially those of Dactylogyrus specific to A. brama, were lower in hybrids. For the majority of host-specific parasites, there was no effect of the maternal origin of hybrids on monogenean abundance. Asymmetry was found in the distribution of specific parasites in favour of specialists of B. bjoerkna in the monogenean communities of hybrids.We showed similar levels of monogenean infection in B. bjoerkna. Our results revealed a difference between the degree of host-parasite coadaptation in specific parasites of A. brama and the degree of host-parasite coadaptation in specific parasites of B. bjoerkna and their associated hosts.Our results indicate that the maternal mtDNA of hybrids is not an important predictor of host-specific monogenean infection, which may suggest that mitochondrial genes are not strongly involved in the coadaptation between monogeneans and their associated hosts. The asymmetry of species-specific parasites suggests similarity between the molecular components of the immune mechanisms in hybrids and  Mus musculus and M. domesticus) and their natural hybrids [Abramis brama) and roach (Rutilus rutilus) [The evolution of host-parasite relationships is associated with reciprocal genetic co-adaptations between the two interacting partners , 2. Such hybrids , 5. Host hybrids , 7. Howe hybrids , 9. WoliFish hybridization is a frequent event in nature , 11. SucFrom the evolutionary point of view, hybridization generates novel morphological and genetic variants of organisms. However, novel features of hybrid organisms may affect the compatibilities between hosts and specific parasites. Generally, host-parasite incompatibility (predicted by the resistance hypothesis) or, alternatively, host-parasite compatibility  result from a wide range of molecular determinants . MoreoveA. brama and R. rutilus. This may indicate the limited inheritance of defense mechanisms from one parental species and suggests different degrees of host-parasite coadaptation between different host-specific parasites and their associated hosts [Differences in monogenean species richness and abundance between hybrids and their parental species were previously shown in fish , 16, 29.ed hosts , 19.Blicca bjoerkna) and common bream (A. brama). With respect to these species, the intermediate survival rate of the F1 generation, hybrid fertility, and the capacity to produce high quality gametes have previously been documented [Blicca bjoerkna and A. brama also exhibit similar ecology. Each of these cyprinid species harbours specific monogenean parasites [In the present study, two widely distributed cyprinid species in central European waters exhibiting low evolutionary divergence and high morphological similarities were studied: silver bream  in order to investigate the susceptibility of the F1 hybrids of two phylogenetically closely-related cyprinid species to the specific monogenean parasites of the parental species and to test whether the maternal ancestry of F1 hybrids (mtDNA) has an effect on the level of monogenean parasite infection.The aim of this study was to use breed lines of B. bjoerkna  and A. brama  were caught by electrofishing from the River Dyje near the city of Břeclav  and transported alive to the facility of the Institute of Vertebrate Biology, Czech Academy of Sciences. Fish separated according to sex into two well-aerated tanks were stimulated for ovulation/spermiation by carp pituitary  and by subsequently increasing the water temperature to 22 °C. Oocytes of ovulating females were obtained by the dry method and sperm was sampled according to Linhart et al. [B. bjoerkna; (ii) female and male A. brama; (iii) female B. bjoerkna and male A. brama; and (iv) female A. brama and male B. bjoerkna. As a result of artificial spawning, four lines of offspring were obtained: pure breed B. bjoerkna; pure breed A. brama; and two crossbreed lines, F1 hybrids with A. brama maternal origin (mtDNA) and F1 hybrids with B. bjoerkna maternal origin (mtDNA). Four lines of offspring were reared subsequently up to 1-year-old.Specimens of t et al. . HatcherA. brama and 7 of B. bjoerkna captured from nature (the River Dyje near the city of Břeclav) were placed in the tank with aerated water. To initiate infection, the batches of the A. brama and B. bjoerkna breed lines and the crossbreed lines were kept in the same tank with the infected specimens of A. brama and B. bjoerkna caught in nature.The experimental infection was conducted in a well aerated and filtered tank. A total of 15 specimens: 8 of Three weeks after the initiation of the experiment, all fish specimens were sacrificed by severing the spinal cord in accordance with Law 246/1992 of the Czech Republic and subsequently dissected following Ergens & Lom . All parA. brama, B. bjoerkna, F1 hybrids with maternal origin of A. brama and F1 hybrids with maternal origin of B. bjoerkna). In accordance with Bush et al. [Prevalence and intensity of infection were calculated for each parasite species in each of the host lines  between A. brama and the two hybrid lines, i.e. F1 hybrids with A. brama maternal origin and F1 hybrids with B. bjoerkna maternal origin, and (ii) between B. bjoerkna and the two abovementioned hybrid lines. Asymmetrical infection by host-specific parasites in F1 hybrids with different maternal origins  was tested using the Wilcoxon signed-rank test. The Mann-Whitney U-test was used to test the effect of the maternal origin of hybrids on the asymmetrical infection by host-specific parasites in F1 hybrids; i.e. the difference between B. bjoerkna-specific parasite species richness (or abundance) and A. brama-specific parasite species richness (or abundance) was used as a dependent variable in the Mann-Whitney U-test. Statistical analyses were performed using Statistica 13.3 for Windows .Kruskal-Wallis H-test followed by multiple comparison tests was applied to test the differences in monogenean abundance among and between fish breeds. As host-specific parasites were not shared between A. brama and 4 monogenean species in B. bjoerkna, all monogeneans were found on the gills (Table U(15) = 16.5, Z = − 1.27, P = 0.203). Using fish from artificial breeding, significant differences in total monogenean abundance among A. brama, B. bjoerkna, and F1 hybrids were found  = 6.04, P = 0.049). Even when monogenean abundance tended to reach higher values in common bream and silver bream when compared to hybrids, multiple comparisons revealed only a significant difference between F1 hybrids and B. bjoerkna (P = 0.043) . The differences in Dactylogyrus abundance among A. brama, B. bjoerkna, and hybrids were not significant  = 2.90, P = 0.235, Fig. Dactylogyrus zandti and D. falcatus were the parasite species with the highest infection levels in the experimentally infected breed line of A. brama. Concerning host-specific parasites of A. brama present in hybrids, D. zandti achieved the highest infection level  in the F1 hybrids of both maternal origins. Monogenean communities of the A. brama breed line consisted of 5 monogenean species; 4 of them host-specific Dactylogyrus species, which were also identified in the source specimens of this species  = 17.09, P = 0.002, multiple comparisons between A. brama and F1 hybrids with A. brama maternal origin: P < 0.001 and between A. brama and F1 hybrids with B. bjoerkna material origin: P = 0.002) was revealed for total Dactylogyrus abundance. The same difference was found for the abundance of 3 Dactylogyrus species, i.e. D. falcatus  = 18.82, P < 0.001, multiple comparisons: P < 0.001 and P = 0.006, respectively), D. wunderi  = 24.38, P < 0.001, multiple comparisons: P = 0.005 and P = 0.008, respectively), and D. zandti  = 9.84, P = 0.007, multiple comparisons: P = 0.033 and P = 0.042, respectively). However, no differences in total Dactylogyrus abundance or the abundance of individual Dactylogyrus species between the 2 lines of hybrids (P > 0.05) were found. No difference in the abundance of least numerous D. auriculatus between A. brama and hybrids  = 0.51, P = 0.775) was found.Concerning Dactylogyrus specific to B. bjoerkna, the Kruskal-Wallis H-test followed by multiple comparisons revealed a weakly significant difference in the abundance of only D. sphyrna between B. bjoerkna and hybrids with B. bjoerkna maternal origin  = 6.99, P = 0.03, multiple comparison: P = 0.046). Concerning P. bliccae, a significant difference was found between B. bjoerkna and hybrids with A. brama maternal origin  = 21.58, P < 0.001, multiple comparison: P = 0.009). The difference between B. bjoerkna and hybrids with B. bjoerkna maternal origin was not significant (multiple comparison: P = 0.052).Concerning Gyrodactylus vimbi was the only monogenean species infecting both pure breed lines and both lines of hybrids  = 4.33, P = 0.228).ds Table . No signB. bjoerkna  and the hybrid line with A. brama maternal origin . The species richness of B. bjoerkna-specific parasites was higher than the species richness of A. brama-specific parasites in the hybrid line with B. bjoerkna maternal origin (P = 0.049) and the hybrid line with A. brama maternal origin (P = 0.003)  = 462.00, Z = 1.20, P = 0.230) or abundance (U(67) = 552.00, Z = 0.07, P = 0.945) between A. brama-specific and B. bjoerkna-specific parasites.The Wilcoxon signed-rank test revealed asymmetry in the abundance of host-specific monogeneans in favor of parasites specific to kna Fig. a. The abA. brama and B. bjoerkna. Although there is phylogenetic proximity between these cyprinid species [Dactylogyrus. The majority of host-specific Dactylogyrus in European cyprinid hosts evolved by intra-host speciation (speciation within host species) [D. zandti and D. wunderi specific to A. brama evolved by intra-host speciation within A. brama, and D. distinguendus, D. cornu and D. cornoides specific to B. bjoerkna evolved by intra-host speciation within B. bjoerkna. Both A. brama and B. bjoerkna are also parasitized by Dactylogyrus species, representatives of other phylogenetic lineages unrelated to those originating from intra-host speciation . Despite the phylogenetic proximity of A. brama and B. bjoerkna, their Dactylogyrus species (or lineages) evolved independently  in the F1 hybrids of cyprinid species with low evolutionary divergence,  species , they haspecies) , i.e. D. related ).The present work is the first experimental study investigating the distribution of host-specific monogeneans in F1 hybrids of cyprinids under the following conditions: (i) the experiment was run using similar proportions of parental and hybrid genotypes; and (ii) the immunity of breed and crossbreed lines was not affected by any previous infection prior to experimental infection by monogeneans  or failed parasite reproduction under our experimental conditions (for D. paradoxum). In contrast, P. bliccae, host-specific parasite of B. bjoerkna, was not identified in our low sample size of dissected wild fish specimens, but this diplozoid species was evidently formerly present in cohabited wild fish specimens and was successfully reproducing in experimental B. bjoerkna and both lines of hybrids.We applied co-habitation design of experiment when specimens of breed and crossbreed lines were placed together with wild Dactylogyrus) tended to be higher in parental species when compared to hybrids . However, for the majority of host-specific parasites , we found higher abundance in a corresponding pure species when compared to parasite abundance in hybrids. Fritz et al. [We found that the total abundance of monogeneans  than parental species, this pattern of infection was revealed in a different time and space. The same finding was demonstrated by Šimková et al. [Cyprinus carpio and Carassius gibelio, that share some Dactylogyrus species exhibiting so-called phylogenetic specificity, i.e. parasite species restricted to phylogenetically closely-related host species  does not play a significant role in host-specific parasite selection.Wolinska et al.  proposedá et al.  using twá et al. ). Krasnoá et al.  suggesteá et al.  is relatá et al. , 7 invesá et al.  reporteda et al. , that thMus musculus and M. domesticus) and their natural hybrids, and suggested that a broken system of host-parasite genetic coadaptation might explain their finding. In the present study, each parental species harbored unique monogenean fauna , and interspecific hybrids resulting from artificial breeding harbored all parasites specific to A. brama and B. bjoerkna, mostly at lower intensities of infection when compared to parental species. This pattern of distribution of parental species-specific parasite in hybrids likely results from a lack of genetic co-adaptation between the host-specific parasite and associated host genome (here the genome of A. brama or genome of B. bjoerkna) as suggested by Krasnovyd et al. [Dactylogyrus species: D. distinguendus; D. auriculatus; D. cornu; and the generalist G. vimbi. However, the last three species were found in very low intensities in parental species and hybrids. To test the hybrid bridge hypothesis, other experiments with hybridizing systems of A. brama and B. bjoerkna (or other fish species with host-specific parasites) will need to be performed in the future, these focusing on the different rate of genetic introgression between two species  and the potential to transfer host-specific parasites. However, the hybridization of A. brama and B. bjoerkna does not represent a serious threat to the genetic integrity of these species (because of the low frequency of hybrids in nature) and presents only a minimal risk with respect to transferring host-specific parasites (because of host-parasite co-adaptation).The hybrid bridge hypothesis , 45 suggd et al. . Some moParadiplozoon bliccae, a strict specialist of B. bjoerkna, was the only parasite species exhibiting a significant difference in abundance between silver bream and hybrids with common bream maternal origin. Due to the low parasite intensity of infection, this finding cannot clearly support the effect of mtDNA on the presence and/or level of parasite infection. However, even with no statistical support, our data indicate a higher intensity of infection by B. bjoerkna-specific parasites in hybrid specimens with B. bjoerkna maternal position.In the present study, we also examined the potential effect of mtDNA on the presence of host-specific parasites. Cyto-nuclear incompatibility, i.e. incompatibilities between the alleles of mitochondrial and nuclear genomes resulting from hybridization, may cause hybrid breakdown even in the F1 generation of hybrids . Chou & Dactylogyrus parasites in pure and hybrid specimens across hybrid zones. In a study focusing on the temporal and spatial variation of parasite infection in a hybridizing system of cyprinid species with high evolutionary divergence, Krasnovyd et al. [R. rutilus, which was the species achieving higher monogenean diversity but lower parasite  abundance when compared to A. brama. In our study, we reported asymmetry in the monogenean communities of hybrids toward a higher abundance of B. bjoerkna-specific parasites. However, source fish caught in nature as well as breed lines of A. brama and B. bjoerkna exhibited no differences in monogenean intensity of infection, and A. brama even exhibited slightly higher total monogenean species richness.Šimková et al.  suggested et al.  demonstrDactylogyrus specific to A. brama. Our findings contradict Wollinska et al. [B. bjoerkna potentially resulting from different inheritances of immune protective mechanisms from both parental species. The maternal origin of hybrids has no principal role in determining the presence of host-specific parasites, which seems to suggest that mitochondrial genes are not primarily involved in co-evolutionary associations between cyprinid hosts and specific monogenean parasites. We highlight the need for genomic studies to identify the genes involved in reciprocal genetic co-adaptations of host-specific parasites and their associated hosts.Our results confirm that the presence of host-specific parasites is affected by hybridization. We showed that host-specific monogeneans reached higher levels of infection in pure species when compared to their reciprocal intergeneric hybrids under the condition of there being similar proportions of pure species genotypes and hybrid genotypes, which again supports the static scenario of hybrid resistance. This was more strongly evidenced for the abundant a et al. , who hyp


Apis nigrocincta, a native bee species from Sulawesi and the Philippines, plays a vital role in pollinating flowering plants in local ecosystem and agriculture. In this study, we assessed the intraspecific genetic variation of A. nigrocincta using the sequence of cytochrome c oxidase subunit I (COI). Molecular phylogenetic analysis showed that there were three main clades in A. nigrocincta specimens from Sulawesi based on their respective locations . Genetic distance analysis using the Kimura 2-parameter (K2P) model showed that the intraspecific genetic distance in Sulawesi specimens ranged from 0.000 to 0.055. There are 26 nucleotide polymorphic sites within Sulawesi A. nigrocincta. The variation was dominated by transition T ↔ C. The molecular identification result was supported by morphological identification. The results of the two methods agree that the specimen under study was A. nigrocincta. The result of genetic distance calculation indicated that although the tested specimens were derived from remote locations, the genetic variation was still within the range of intraspecific variation.Asian cavity-nesting honey bee  Apis. These insects play a significant role in the pollination of important crops. Currently, there are nine species of honey bees known to inhabit the world: Apis dorsata, A. laboriosa, A. mellifera, A. florea, A. andreniformis, A. cerana, A. koschevnikovi, A. nigrocincta , and A. nuluensis , with approximately 44 subspecies [There are about 30,000 bee species distributed worldwide. About 17,000 of them have been described . Apidae,A. nigrocincta has been poorly studied. In recent years, molecular methods have been applied to study intraspecific genetic variation, species identification, and phylogenetic relationship among close-related taxa [A. nigrocincta to facilitate species identification and analyze their genetic distances.The honey bees are prone to extinction due to their haplodiploid sex-determining mechanism, limited population because of anthropogenic activities , and lowted taxa . One tected taxa . For aniSphagneticola trilobata (L.) Pruski, Tachytarpheta jamaicensis (L.) Vahl., and Mikania micrantha Kunth.The adult worker bees were captured using a swingnet from an abandoned local garden in Mahakeret village, city of Manado, North Sulawesi province, Indonesia (1°28'53”N and 124°50'21”E). The location was situated at an altitude of about 40 meters above sea level. The average temperature at sample collection place (March 2019) was between 29°C and 32°C with humidity of 85–89%. The percentage of vegetation cover was 80%. The vegetation consisted mainly of bushes, grasses, and wild flowers such as The observed morphological characters included length of following parts: head (HD), antenna (AT), proboscis (PB), thorax (TO), abdomen (AB), fore-wing (FW), hind-wing (FL), midleg (ML), and hind-leg (HL) using a caliper .Total DNA was extracted from coxa connected to the abdomen using ZR Tissue & Insect DNA MiniPrep™ (Zymo Research). The genomic DNA was cleaned with DNA Clean & Concentrator™-5 (DCC™-5) (Zymo Research) for the generation of high-quality DNA for PCR. The DNA barcode of COI region was amplified using Toyobo KOD FX Neo PCR Master Mix with primer pairs LCO1490 and HCO2198 . The conhttp://www.ncbi.nlm.nih.gov). Identification was performed using BLAST identity search provided by the same platform. The clean sequences were aligned using Clustal O (1.2.1) multiple sequence alignment  with other allied honey bee COI sequences from different parts in Sulawesi retrieved from GenBank. The evolutionary history was inferred by the Maximum Likelihood (ML) method based on the Kimura 2-parameter (K2P) model [Chromatograms were subjected to the procedure as was done previously . The cleP) model . EvolutiP) model .A. nigrocincta provided by Hadisoesilo et al. [The photograph of honey bee specimen obtained from Manado Mahakeret is shown in o et al. . The moro et al. .A. nigrocincta CAL01 has been deposited in GenBank with accession number MK880239. The complete BLAST search is presented in The cytochrome oxidase I (COI) sequence of A. nigrocincta specimens. Estimation of genetic distance amongst A. nigrocincta is shown in A. nigrocincta. Variation of intraspecific COI gene showed that transition T ↔ C dominated the polymorphic pattern.The molecular phylogenetic analysis by the ML method based on the K2P model is shown in v was equal to 100. The number of transition substitution was 86.89, while the number of substitution was 13.11. Therefore, the transition/transversion ratio (ti/tv) was 6.63. However, sampling size strongly influences the ti/tv. The estimated maximum likelihood ti/tv bias (R) was 9.05 under the K2P model [Estimation of substitution using the maximum composite likelihood method can be seen in Apis nigrocincta . This phenomenon was supported by studies conducted by Damus and Otis [A. cerana and A. cerana japonica were confirmed as being distinct from the rest of A. cerana. This indicates that Apis nigrocincta found in Pakistan was most likely the part of the group A. cerana japonica. Another finding showed that A. nigrocincta from Sulawesi mainland and Sangihe Island were embedded in the A. cerana group [A. nigrocincta had similarities with A. cerana [The specimen KY834222 from Islamabad Pakistan was observed to be out of the Sulawesi group. In the phylogenetic tree created by involving its sister species, and Otis  which stna group . The phy. cerana , 22.A. nigrocincta maintained a high specific genetic diversity on Sulawesi Island.The closest genetic distance was 0.000, between haplotype CAL01 and DQ020233 from Bogani Nani Wartabone National Park, Gorontalo (Indonesia). The farthest genetic distance was 0.082, between haplotype CAL01 and KY834222 from Islamabad (Pakistan), and 0.052 with DQ020223 from Bantimurung, South Sulawesi (Indonesia). The genetic distance was 0.055 between NC038114 from Sangihe Island North Sulawesi (Indonesia) and DQ020223. With this value, Takahasi et al. [A. mellifera in 10 locations across China and Pakistan was 0.039 [A. cerana was detected between Flores' and Taiwan's haplotypes [The result of genetic distance calculation indicated that although the tested specimens were derived from remote locations, it was still within the range of intraspecific variation, except with specimen from Pakistan. Maximum pairwise distance of as 0.039 . The resplotypes . The cloplotypes . Differeplotypes , 27. Theplotypes , 29. Theplotypes , 31.A. nigrocincta. However, six polymorphic sites were detected in Bombus koreanus in China [Apis mellifera in United States [A. cerana in India [No information was provided earlier on the polymorphic sites of in China  and Apisd States . The train India . Due to in India .A. nigcrocinta group from different locations in Sulawesi has been demonstrated in this study. This molecular marker was also able to identify A. nigrocincta successfully based on the similarity sequence with other A. nigrocincta specimens. This finding provides valuable information on the effective use of COI as molecular marker in research on phylogenetic studies.The ability of COI gene to differentiate between A. nigrocincta using COI gene sequences. The results showed that there were three main clades of Sulawesi specimen, namely, North, Central, and South Sulawesi. This finding suggested that genetic variation in A. nigrocincta in Sulawesi is still in the category of intraspecific variation.The current study assessed the intraspecific genetic variation in


Haementeria acuecueyetzin (Hirudinida: Glossiphoniidae) based on specimens collected in Tabasco, Mexico. The circular genome is 14,985 bp in length, and consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and an AT-rich control region. Phylogenetic analysis based on the 13 protein-coding genes and two rRNA genes places H. acuecueyetzin sister to H. officinalis within the family Glossiphoniidae. Mitochondrial gene order in H. acuecueyetzin is consistent with other members of Clitellata with no evidence of gene gain/loss, duplication, or rearrangement.Here, we present the mitogenome of the blood feeding leech  Haementeria acuecueyetzin Oceguera-Figueroa . For a parallel project regarding the leech bacterial endosymbionts, total DNA of a mixed pool of 120 bacteria bearing organs or bacteriomes from 30 leeches  was extracted using a commercial extraction kit . DNA libraries were constructed using the NGS Nextera FLEX DNA library preparation kit  according to the manufacturer’s protocol and total DNA was multiplexed together with 11 other samples and sequenced on a single lane on the HiSeqX platform (150 bp paired-end) at SickKids, Toronto, Canada. Adapters were trimmed with the FastX-Toolkit , Helobdella robusta (AF178680) and Haementeria officinalis (LT159848) as reference for bacterial endosymbiont, host (leech), and leech mitochondrial sequences, respectively. We recovered a linear contig of ∼15 Kbp with strong similarity in both nucleotide identity and length when compared to Haementeria officinalis, the only previously sequenced mitogenome from a congener. Online BLASTn and BLASTx searches confirmed the leech affinity of the contig and its components (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Shorter sequencing reads were mapped to the single contig using Bowtie 2.3.5.1 .On 18 March 2018, a total of 30 specimens of H. acuecueyetzin within Hirudinea using all available mitogenomes from GenBank. A maximum-likelihood tree was obtained using RaxML v.8.2.4 (Stamatakis H. acuecueyetzin as the sister taxon to H. officinalis, within a clade exclusively formed by other members of Glossiphoniidae. The glossiphoniid clade, in turn, is the sister to the rest of hirudineans (H. acuecueyetzin is consistent with that of other members of Clitellata with no evidence of gene gain/loss, duplication, or rearrangement.Sequences of the 13 protein coding genes and two ribosomal RNAs were used to infer the phylogenetic position of udineans . Mitocho


Oryza sativa L. ssp. Indica) were cultured with control (10 mM NO3-) and nitrate deficient solution (0 mM NO3-) for four weeks. Nitrogen (N) deficiency significantly decreased the content of N and P, dry weight (DW) of the shoots and roots, but increased the ratio of root to shoot in O. sativa. N deficiency decreased the photosynthesis rate and the maximum quantum yield of primary photochemistry (Fv/Fm), however, increased the intercellular CO2 concentration and primary fluorescence (Fo). N deficiency significantly increased the production of H2O2 and membrane lipid peroxidation revealed as increased MDA content in O. sativa leaves. N deficiency significantly increased the contents of starch, sucrose, fructose, and malate, but did not change that of glucose and total soluble protein in O. sativa leaves. The accumulated carbohydrates and H2O2 might further accelerate biosynthesis of lignin in O. sativa leaves under N limitation. A total of 1635 genes showed differential expression in response to N deficiency revealed by Illumina sequencing. Gene Ontology (GO) analysis showed that 195 DEGs were found to highly enrich in nine GO terms. Most of DEGs involved in photosynthesis, biosynthesis of ethylene and gibberellins were downregulated, whereas most of DEGs involved in cellular transport, lignin biosynthesis and flavonoid metabolism were upregulated by N deficiency in O. sativa leaves. Results of real-time quantitative PCR (RT-qPCR) further verified the RNA-Seq data. For the first time, DEGs involved oxygen-evolving complex, phosphorus response and lignin biosynthesis were identified in rice leaves. Our RNA-Seq data provided a global view of transcriptomic profile of principal processes implicated in the adaptation of N deficiency in O. sativa and shed light on the candidate direction in rice breeding for green and sustainable agriculture.Rice cultivar “Weiyou916” ( Nitrogen (N) is one of essential macronutrients required for plant growth and development. It is a constituent of amino acids, nucleic acids, vitamins, chlorophyll, alkaloids, some plant hormones and secondary compounds in plants. N is the most abundant mineral element in plants, which constitutes about 2% - 4% of plant dry weight (DW) . AlthougArabidopsis . Si. SiPIPs onoplast , which miculture . Interesmitation . Our RNA2 assimilation and Fv/Fm in N deficient O. sativa leaves  signaling and likely to act as both local and systemic signals coordinating N demand and acquisition . CytokinO. sativa leaves , chalcone-flavonone isomerase (CHFI), flavanone 3-dioxygenase 2-like, etc., which implicated in flavonoids biosynthesis were apparently upregulated by N deficiency in O. sativa leaves , which have antioxidant activity, are the largest class of phenolics. They are resulted from the addition of malonyl CoA to the phenylpropanoid molecule coumaroyl CoA and are widely distributed plant secondary metabolites . Many gea leaves . Huang emitation . More remitation , 69. TheO. sativa. N deficiency decreased the photosynthesis and Fv/Fm, however, increased the intercellular CO2 concentration and Fo. N deficiency significantly increased the production of H2O2, the contents of MDA, lignin, starch, sucrose and fructose, but did not change that of glucose in O. sativa leaves. A total of 1635 genes showed differential expression in response to N deficiency revealed by RNA-Seq and the transcriptomic data were further validated by RT-qPCR. Of which, 999 DEGs and 636 DEGs were downregulated and upregulated by N deficiency in O. sativa leaves, respectively. GO analysis showed that 195 DEGs were found to highly enrich in nine GO terms. Most of DEGs involved in photosynthesis, biosynthesis of ethylene and gibberellins were downregulated, whereas most of DEGs involved in cellular transport, lignin biosynthesis and flavonoid metabolism were upregulated by N deficiency in O. sativa leaves Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Medicago truncatula. To investigate the potential role of ethylene, a M. truncatula ethylene- insensitive mutant, sickle, was analysed. The sickle mutant occurs in the accession A17 that has moderate resistance to Acyrthosiphon kondoi, A. pisum and Therioaphis trifolii. The sickle mutant resulted in increased antibiosis-mediated resistance against A. kondoi and T. trifolii but had no effect on A. pisum. When sickle was introduced into a genetic background carrying resistance genes, AKR (A. kondoi resistance), APR (A. pisum resistance) and TTR (T. trifolii resistance), it had no effect on the strong aphid resistance mediated by these genes, suggesting that ethylene signaling is not essential for their function. Interestingly, for the moderate aphid resistant accession, the sickle mutant delayed leaf senescence following aphid infestation and reduced the plant biomass losses caused by both A. kondoi and T. trifolii. These results suggest manipulation of the ethylene signaling pathway could provide aphid resistance and enhance plant tolerance against aphid feeding.Ethylene is important for plant responses to environmental factors. However, little is known about its role in aphid resistance. Several types of genetic resistance against multiple aphid species, including both moderate and strong resistance mediated by R genes, have been identified in  R) genes, that modulate plant defenses against aphids have been identified in a range of plant species against various aphid species ( × 100, in which A: average of the non-infested plant dry weight; B: dry weight of individual aphid-infested plant. To assess the aphid performance and plant tolerance on whole plants, non-choice experiments were conducted under glasshouse conditions. Plants of With each experiment, the aphid weight, the number of necrotic flecks or plant biomass reduction were analyzed by one-way ANOVA and compared by the LSD test at a 5% significance level using GenStat .


One unmet challenge in lung cancer diagnosis is to accurately differentiate lung cancer from other lung diseases with similar clinical symptoms and radiological features, such as pulmonary tuberculosis (TB). To identify reliable biomarkers for lung cancer screening, we leverage the recently discovered non-canonical small non-coding RNAs  in human peripheral blood mononuclear cells and develop a molecular signature composed of distinct ts/rs/ysRNAs (TRY-RNA). Our TRY-RNA signature precisely discriminates between control, lung cancer, and pulmonary TB subjects in both the discovery and validation cohorts and outperforms microRNA-based biomarkers, which bears the diagnostic potential for lung cancer screening.The online version contains supplementary material available at 10.1186/s12943-020-01280-9. One unmet challenge in current lung cancer diagnosis is to accurately differentiate lung cancer from other lung diseases with similar clinical symptoms and radiological features. Imaging-based screening methods, such as low-dose computed topography (LDCT), could sometimes be false positives, as indeterminate pulmonary nodules may also be caused by other lung diseases such as pulmonary tuberculosis (TB) , which iRecent sncRNA sequencing (sncRNA-seq) attempts have ubiquitously detected several non-canonical sncRNA types, which are fragments derived from canonically transcribed parent large RNAs, including tRNA-derived small RNAs (tsRNAs), rRNA-derived small RNAs (rsRNAs), and YRNA-derived small RNAs (ysRNAs) . ts/rs/ySPORTS1.0, which was designed to optimize the annotation and quantification of non-canonical sncRNAs  in addition to miRNAs [Spearman’s rank correlation test: ρ > 0.700 and P < 10− 9)     . In total, nine tsRNA species, eight rsRNA species, and eight ysRNA species  signature. Both principal component analysis   was 1.000 between the cancer and non-cancer subjects and 0.994 between the TB and non-TB subjects  (Fig. AUC was 0.930 between the cancer and non-cancer subjects and 1.000 between the TB and non-TB subjects (Fig. We further assessed the TRY-RNA signature in the validation cohort with 35 human PBMC samples collected from 12 healthy controls, 15 lung cancer patients, and 8 pulmonary TB patients (Additional file We also compared the expression profile of miRNAs among the control, lung cancer, and pulmonary TB subjects in the discovery cohort and identified a signature with 43 miRNA species, referred to as the MIR signature (Additional file Our TRY-RNA signature derived from the repertoire of PBMC non-canonical sncRNAs makes it possible for the early diagnosis of lung cancer and pulmonary TB, which may reflect the host responses to different antigens and would represent an improvement over the previous studies focusing solely on tsRNAs in cancer tissues , 5, 8. IAdditional file 1: Supplementary Figures. Fig. S1. The workflow of the study. Fig. S2. The landscape of non-canonical sncRNAs in human PBMCs. Fig. S3. The mapping profile of tsRNAs. Fig. S4. Comparison of the expression of the prioritized sncRNA subcategories between lung cancer stages. Fig. S5. Comparison of the expression of the prioritized sncRNA subcategories between lung cancer histological types. Fig. S6. Comparison of the expression of the prioritized sncRNA subcategories between the lung cancer patients with and without lymph node involvement. Fig. S7. Comparison of the expression of the prioritized sncRNA subcategories between the lung cancer patients with and without distant metastasis. Fig. S8. Comparison of the expression of the prioritized sncRNA subcategories between the lung cancer patients with and without smoking history. Fig. S9. The TRY-RNA signature. Fig. S10. The TRY-RNA index in the discovery cohort. Fig. S11. Comparison of the expression of the sncRNA species within the TRY-RNA signature between lung cancer stages. Fig. S12. Comparison of the expression of the sncRNA species within the TRY-RNA signature between lung cancer histological types. Fig. S13. Comparison of the expression of the sncRNA species within the TRY-RNA signature between the lung cancer patients with and without lymph node involvement. Fig. S14. Comparison of the expression of the sncRNA species within the TRY-RNA signature between the lung cancer patients with and without distant metastasis. Fig. S15. Comparison of the expression of the sncRNA species within the TRY-RNA signature between the lung cancer patients with and without smoking history. Fig. S16. Expression heatmap of the MIR signature in the discovery cohort. Fig. S17. The MIR index in the discovery cohort. Fig. S18. Comparison between the TRY-RNA and MIR signatures.Additional file 2: Supplementary Tables. Table S1. The human subjects of the discovery cohort. Table S2. The TRY-RNA signature. Table S3. The human subjects of the validation cohort. Table S4. The MIR signature.Additional file 3. Methods.


Oncorhynchusmasou, Oncorhynchusketa, Parahuchoperryi, Salvelinusleucomaenisleucomaenis and Salvelinusmalmakrascheninnikova). The invasive alien rainbow trout Oncorhynchusmykiss was detected at all 16 sites and it was the most commonly detected salmonid. Although we found no obvious competitive exclusion of native salmonids by rainbow trout in the study area, the invasive species occurred more often and at more sites than any of the natives. We also determined the occurrence and seasonal changes in the fish community, classified as native salmonids, invasive rainbow trout, Cypriniformes and other benthic fishes. There were fewer species overall in winter, but the sites with higher species richness in winter were on the lower reaches of the river. In addition, we detected domestic invaders, such as the topmouth gudgeon, Pseudorasboraparva, although they were less prevalent than rainbow trout. These results show the effectiveness of eDNA metabarcoding, which can be used for surveying species richness at an ecosystem scale. In particular, the detection of the early stages of establishment and spread of invasive species can be achieved by eDNA monitoring.In freshwater ecosystems, invasive salmonid fishes can have a significant impact on native fish species. Detecting the invasion and its negative effects is critical for the conservation of native fish communities. We examined the species composition and seasonal changes in the freshwater fish community, including salmonids, on the Kamikawa Plain, Hokkaido Island, Japan, using environmental DNA (eDNA) metabarcoding. We detected 23 fish species in 176 samples collected from 16 sites over 12 months (October 2018 – August 2019). Between 11 and 20 species were detected at each site, including five native salmonids ( Parahuchoperryi (Brevoort 1856) , Salvelinusleucomaenisleucomaenis  (white-spotted char), Salvelinusmalmakrascheninnikova Taranetz 1933 (Dolly Varden char), Salvelinusmalmamiyabei Oshima 1938, Oncorhynchusmasou (Brevoort 1856) , Oncorhynchusketa  , Oncorhynchusgorbuscha  , Oncorhynchusnerka   and Oncorhynchustshawytscha  . In Japan, the translocation of freshwater fishes, including salmonids, both within the country and from overseas, began in the 1800s  (rainbow trout) and Salmotrutta Linnaeus 1758 (brown trout) have had negative effects on native species, including through antagonistic relationships (i.e. predation) and competitive exclusion (There is a high species richness (seven species and two subspecies) of native salmonid fish on Hokkaido Island in the Japanese Archipelago, as he 1800s . Since the 1800s . In Hokkxclusion . For exaSalmonid species, such as salmon, trout and charr, generally need different habitats, based on the season of the year and stage of their life cycle . As manySilrusasotus Linnaeus 1758 (Far Eastern catfish) was found in the middle reaches of the Ishikari River in Hokkaido (Channaargus (Cantor 1842) (northern snakehead) also has been found in parts of Hokkaido, including the capital, Sapporo City (Pseudorasboraparva (Temminck & Schlegel 1846) (topmouth gudgeon), an endemic Japanese gobionid fish, is known as the “Asian killer fish” in Europe. It has been established in Europe and its parasitic pathogens  have caused a drastic decrease in native European cyprinid species  metabarcoding allows sequences found in eDNA to be associated with a taxonomic name. This technique is a powerful molecular tool for surveying species richness non-invasively, which works in many ecosystems . NakagawThe Kamikawa Plain, Hokkaido, contains Daisetsuzan National Park, the largest national park in Japan; the Ishikari River, the largest river in Hokkaido, originates in the Daisetsu Mountains. Thus, conservation of the fish community in this area is important for freshwater ecosystems in Hokkaido. We investigated the species composition and seasonal changes in the freshwater fish community, including salmonids, on the Kamikawa Plain using eDNA metabarcoding. We executed monthly water sampling, including heavy snow conditions in midwinter, using methods developed in our previous studies  to assesWe selected 16 sites along the Ishikari River, its tributary including the Chubetsu River, the Masutori-gawa River and the Antaroma River, in the Kamikawa Plain, Central Hokkaido  with the E-Gel Precast Agarose Electrophoresis System (Thermo Fisher Scientific). The size of the library sample was confirmed using an Agilent 2100 Bioanalyzer . This process was performed at Environmental Research and Solutions Co. Ltd.  for the samples collected in October and November 2018 and at Kobe University  for the rest of the samples. The library sample was sequenced using an Illumina MiSeq or iSeq with 2 × 150 bp pair-end kits .Raw reads from a high-throughput sequencer were preprocessed and analysed using USEARCH v10.0.240 . The criTo scrutinise the results obtained, we performed the following three steps. First, the number of species reads detected in the negative controls (i.e. filtration blanks or PCR blanks) was subtracted from the corresponding samples. Second, pure marine fish species were excluded as contamination because there were no marine fish in the studied area. Finally, every species detected in only one sample was regarded as a potential false positive and so these species were excluded from the subsequent analysis. We converted the read counts to presence/absence data and used these in further analyses.All statistical analyses, including mapping, were conducted using R 3.5.2 (R Core Team 2018 https://www.R-project.org). Geographical information about the sites was collected using Google Maps (geodetic system: WGS84). The great-circle distance between the sites was calculated using the function spDistsN1 of the “sp” package. The “maptools” and “rgdals” packages were used to draw the results on the map. We used the “tidyverse” package to tidy up the data and we used the “vegan” package to examine the fish species composition and its spatial autocorrelation. The spatial autocorrelation was calculated using the mantel function of “vegan” with the distance matrix with the Jaccard Index because we collected presence/absence data. Mapping was conducted with geographical data from the Ministry of Land, Infrastructure, Transport and Tourism of Japan (http://nlftp.mlit.go.jp/ksj/index.html). The “ggplot2” and “gplot” packages were used to draw graphs, including a heatmap.The factors affecting the fish community at each site on each sampling date were analysed with permutational multivariate analysis of variance (PerMANOVA) using the adonis function of “vegan”, with site, sampling date and presence of rainbow trout as explanatory variables. In PerMANOVA, sites were classified into three categories: mainstream , mid-sized stream  and small tributaries . The sampling dates were categorised for four seasons, according to the Japanese seasonal categories and the degree of snowfall in Hokkaido, as follows: autumn (October and November 2018), winter , spring (April and May 2019) and summer .O.mykiss, a generalised linear model (GLM), using a binomial distribution (link function was 'logit') with the explanatory variables as site, sampling date and presence of rainbow trout, was run. Model selection was performed using Akaike's Information Criterion involving the function stepAIC of package 'MASS'.Finally, to determine whether there was a competitive exclusion relationship between each major fish species and Cyprinidae and Salmonidae   (stone loach) of Nemacheilidae, Misgurnusanguillicaudatus (Cantor 1842) (weather loach) of Cobitidae and Cottusnozawae Snyder 1911 (wrinklehead sculpin) of Cottidae were detected at all 16 sites, as was the DNA of the invasive rainbow trout.The number of species per site ranged from 11 to 20 , Salvelinusleucomaenisleucomaenis  (white-spotted charr), Salvelinusmalmakrascheninnikova (Dolly Varden char), Oncorhynchusmasou  and O.keta . The ASVs of these salmonids, except P.perryi, did not identify the fish to the species level though eDNA metabarcoding because there are closely-related subspecies or other species with which they can be misidentified. For example, there are a few char subspecies in Japan’s Honshu Island that are very similar to S.leucomaenisleucomaenis (white-spotted charr). Other species that have similar DNA are Oncorhynchusmasou Brevoort 1856 (satsukimasu trout) and O.masou ; Salvelinusfontinalis (Mitchill 1814) (brook trout) and S.malmakrascheninnikova (Dolly Varden charr); and humpback salmon and chum salmon. Author AI directly identified the species present here through specimen capture, as shown in Suppl. material We detected native salmonids s Pallas 814 , Carassiuscuvieri Temminck & Schlegel 1846 (Japanese crucian carp), Pseudorasboraparva (topmouth gudgeon), Gnathopogoncaerulescens (Sauvage 1883) (hon-moroko), Tanakialanceolata (Temminck & Schlegel 1846) (slender bitterling), Rhynchocyprislagowskii (Dybowski 1869) (Amur minnow), Pseudaspiushakonensis (Günther 1877) (Japanese dace) [the former genus Tribolodon Sauvage 1883 is currently considered a synonym of Pseudaspius Pallas 1776, according to Cypriniformes species Rhynchocyprispercnurus  (swamp minnow) was also detected  of Cobitidae and domestic invader Silurusasotus Linnaeus 1758 (Far Eastern catfish) of Siluridae, from some sites on a few sampling dates.We also detected native sand lamprey Barbatulabarbatula (Stone loach) DNA was detected at all 16 sites throughout the study (165 of 176 samples), making it the most common ASV. Cottusnozawae (wrinklehead sculpin) DNA was also detected at all sites throughout the year (113 samples), although there was a lower rate of detection at near-reservoir sites. DNA from Pseudaspiussachalinensis (Ezo-ugui), Pseudaspiushakonensis (Japanese dace) and Misgurnusanguillicaudatus (weather loach) was detected in 82, 48,and 66 samples, respectively, with higher rates at the lower-reach sites and lower rates at the upper reaches and smaller tributaries.The number of samples of each salmonid and other frequently-detected species  was detected at all sites throughout the research (105 samples), making it the most abundant salmonid and it was detected repeatedly at the near-reservoir sites. Although O.masou  DNA was detected in almost all (15 of 16) sites, there was a total of just 72 samples, with fewer samples from the near-reservoir sites, which is a pattern different from that of the O.mykiss  DNA was detected at 12 sites, in 32 total samples. S.malmakrascheninnikova (Dolly Varden charr) DNA was detected at 11 sites and in more samples in the upper sites and around-dam sites than elsewhere . DNA of chum salmon was detected at 11 sites and in 18 samples in total, although the detection from the above-reservoir site was not reliable because no stock has been reported there. DNA of critically-endangered species Sakhalin taimen, which has not been captured around our research sites since the 1970s , native salmonids, Cypriniformes and others, drawing species-count graphs for each site by research date  in winter, although the winter samples from other sites tended to contain fewer species (2–5). These sites are at the lower reaches of our research rivers Fig. . Higher We drew a heatmap based on the Jaccard Similarity Index of the species composition pooled for yearly data for each site Fig. . The hea2 = 0.036, Suppl. material We analysed the effects of rainbow trout on the fish community using PerMANOVA and GLM. The PerMANOVA showed that all variables of site, sampling date and rainbow trout had significant effects (RO.mykiss (rainbow trout), have been established and is considered to negatively affect native salmonids and other fish. As O.mykiss DNA was detected at all sites and showed the highest frequency (105 samples) of all the salmonids, it seems that the species could be the dominant salmonid around the Kamikawa Plain. Around the Chubetsu Reservoir, salmonids, other than the rainbow trout, were detected at fewer sites and lower frequency than that of O.mykiss. This is because the species is thought to have been artificially stocked into the Reservoir for recreational fishing.We successfully detected the fish community composition using eDNA metabarcoding. Our research targeted the freshwater ecosystems in Hokkaido, Japan, where the invasive salmonid, O.masou  DNA was detected at 15 of the 16 sites. It was observed that at Site A, O.masou up-migrated into the Masutori-gawa River to spawn in autumn, although larger rainbow trout individuals inhabited this area sympatrically along with the masu salmon. This species is not considered to be endangered, although continuous and long-term assessments are needed, as earlier studies have found that the rainbow trout is a potential threat to native salmonids  was detected at just 11 sites, this species tending to coexist with rainbow trout in the upper reaches, however, as previously reported by S.malmakrascheninnikova-positive samples. This result and our GLM, support the report of S.malmakrascheninnikova and O.mykiss.Salvelinusleucomaenisleucomaenis (white-spotted charr) was detected in 32 samples. Although the species has not been denoted in the Japanese Red List, we believe there is a future threat for this species on the Kamikawa Plain. Oncorhynchusmykiss DNA was detected in 25 of these 32 samples; however, S.leucomaenisleucomaenis and O.mykiss. Since S.leucomaenisleucomaenis tends to migrate seasonally (i.e. sea-run and up-migration), they are the native salmonid most negatively affected by artificial structures, such as dams  was detected 18 times across 11 sites. It was detected four times near the Chubetsu Reservoir, at one below-reservoir site (Site J in December 2018) and three above-reservoir sites . While the species has not been reported to up-migrate to Site J, author AI observed them spawning at the lower site (Site G). The other sites are above the Reservoir and the species cannot pass the dam structure. It is possible that the DNA found at the above-reservoir sites was derived from the eggs of the salmon, which are commonly used for recreational fishing. Since the Chubetsu Reservoir is a large barrier of more than 80 m height, there is no possibility of the transportation of the carcass by wild animals.The DNA of P.perryi , which is denoted as critically endangered (CR) in the Japanese Red List, in two samples from two sites. We detected DNA from In all, we detected the DNA of 25 fish species by metabarcoding eDNA in water samples. We examined the differences in species composition amongst the research sites and the seasonal changes in this composition. The low spatial autocorrelation of the species composition and the heatmap analysis revealed that the composition at each site was determined by multiple factors. We were able to detect DNA from samples taken in midwinter, which is the heavy snow season. The number of species tended to be lower in winter (December to March) than in other seasons. However, Sites A, C, D, F and G contained more species in winter than in summer, where they were located on the lower reaches of our sample rivers and could contain deeper pools, compared with the smaller, fast flowing tributaries found at sites in the upper reaches. This result suggests that they formed a winter refuge for the fish community. Although further studies are required on the quantitative effect and mechanisms of eDNA flow in lotic water , HandleyB.barbatula (stone loach) and/or M.anguillicaudatus (weather loach) DNA was detected at those sites year-round. It is possible that the loach species do not migrate and thus overwinter around the Reservoir. At present, however, since we do not have detailed environmental data for the sites, we cannot fully explain the multiple factors influencing species composition.The reservoir sites had lower numbers of species, probably not because of competition with rainbow trout, but due to other unfavourable conditions or overwintering migration. While very little salmonid DNA was detected at reservoir sites in winter, C.carpio (common carp), Carassius sp. (crucian carp), T.lanceolata (slender bitterling), P.parva (topmouth gudgeon), G.caerulescens (hon-moroko) and S.asotus (Far Eastern catfish), that are not native to Hokkaido Island. Their detailed distribution in Hokkaido was not identified in this study. Repeated detection of these species indicates that they should indeed be present in this area, if sampled using traditional methods. These species are thought to be domestic invaders from inside the Japanese Archipelago (T.lanceolata (slender bitterling) and G.caerulescens (hon-moroko), were detected repeatedly in this study, although they were not previously reported to be present on Hokkaido Island. They are native to Japan’s Honshu Island and are thought to have been artificially introduced to Hokkaido for fisheries or recreational angling.We found the DNA of species, such as hipelago  and so mPseudorasboraparva (topmouth gudgeon) is the most notable of the domestic and non-defined invaders found in this study and should receive the most attention. This species was detected at six sites , specifically four of the lower sites and two of the near-reservoir sites. This species has invaded many European water bodies and is colloquially called the "Asian killer fish" because its parasitic pathogen kills native Cypriniformes and might kill native salmonid fishes (P.parva in a future study.d fishes . It is pC.carpio (common carp), C.cuvieri (Japanese crucian carp) and S.asotus (Far Eastern catfish), which have established populations in Hokkaido Island , C.nozawae (wrinklehead sculpin) and M.anguillicaudatus (weather loach), were detected at most sites and in most water samples. It is not clear whether M.anguillicaudatus (weather loach) is truly native to these sites or is a domestic invader. However, these benthic species are thought to be important elements of the fish community in Hokkaido, although they have not received much attention from fishery or commercial viewpoints.As for other domestic invaders, we detected o Island . For exaO.mykiss, Cypriniformes and other benthic fish, such as B.barbatula, C.nozawae and M.anguillicaudatus. We also detected less-common domestic invaders. These findings used the technique of eDNA metabarcoding, which is capable of surveying species composition at the ecosystem scale. In particular, the detection of the early stages of establishment and spread of the invaders is best achieved by eDNA monitoring rather than by traditional surveys, although the traditional suveys should be combined to verify the results. We revealed that there is high species richness of Cypriniformes in the lower reaches and benthic species are important across the river system. This is important information about fish communities along the Ishikari River system on the Kamikawa Plain and the conservation of the aquatic ecosystems of Hokkaido Island.In conclusion, we detected the distribution and seasonal changes of the fish community, including the native salmonids, the invasive B7CB6857-9B56-53F1-8FE8-2291AE4EA2FD10.3897/BDJ.8.e56876.suppl1Supplementary material 1Table S1 Results of eDNA metabarcoding for 16 sites from October 2018 to August 2019.Data typexlsx spreadsheetxlsx spreadsheetBrief descriptionResults of eDNA metabarcoding for 16 sites from October 2018 to August 2019. Total number of species per site is shown numerically and the presence/absence is indicated by 1/0, respectively. Site abbreviations correspond to those in Figures.File: oo_458807.xlsxhttps://binary.pensoft.net/file/458807A. Imamura, K. Hayami, M.K. Sakata, T. MinamotoA. Imamura, K. Hayami, M.K. Sakata, T. Minamoto39663944-8E81-5784-AA2B-F7A7B2C515D010.3897/BDJ.8.e56876.suppl2Supplementary material 2Raw data of eDNA metabarcoding.Data typeSpread sheetBrief descriptionMarine fish species are omitted. Species identification is tentative.File: oo_458806.xlsxhttps://binary.pensoft.net/file/458806A. Imamura, K. Hayami, M. K. Sakata, T Minamoto2C8A8F90-5B40-582A-BFB6-1CD960339BD810.3897/BDJ.8.e56876.suppl3Supplementary material 3Table S2Data typexlsx spreadsheetsBrief descriptionResults of fish species detected.File: oo_458724.xlsxhttps://binary.pensoft.net/file/458724A. Imamura, K. Hayami, M. K. Sakata, T Minamoto05D4E492-E57C-5721-BAFA-6D8BF768128B10.3897/BDJ.8.e56876.suppl4Supplementary material 4Supplementary figures of the environmental DNA surveyData typejpeg imagesBrief descriptionLethenteron sp.; SI_b: Tanakialanceolata (slender bitterling); SI_c: Rhynchocyprislagowskii (Amur minnow); SI_d: Gnathopogoncaerulescens (hon-moroko); SI_e: Silurusasotus (Far Eastern catfish); SI_f: Pungitius sp.; SI_g: Oryzias sp.; SI_i: Rhinogobius sp.Fig. S1 Results of the environmental DNA survey for each fish species. Circle size indicates the total number of samples in which that species was detected out of the 176 samples taken throughout the year (October 2018–August 2019). The explanatory circle with “10” indicates that the species was detected in ten samples. SI_a: File: oo_458729.pdfhttps://binary.pensoft.net/file/458729A. imamura, K. Hayami, M. K. Sakata, T. Minamoto8EAB09A3-8A1A-504D-BBC4-4E0BC40CE90110.3897/BDJ.8.e56876.suppl5Supplementary material 5Table S3 Result of Permutational Multivariate Analysis of Variance (PerMANOVA) for the fish community detected with eDNA metabarcoding. The sites were categorised as mainstream, mid-size stream or small tributaryData typexlsx spreadsheetFile: oo_445872.xlsxhttps://binary.pensoft.net/file/445872A. Imamura, K. Hayami, M.K. Sakata, T. Minamoto7C507588-3915-5CA1-A9DE-41055161E40D10.3897/BDJ.8.e56876.suppl6Supplementary material 6Supplementary Tables S4-S10Data typexlsx spreadsheetsBrief descriptionResult of Generalised Linear Model (GLM) for the detection of major fish species.File: oo_458725.xlsxhttps://binary.pensoft.net/file/458725A. Imamura, K. Hayami, M.K. Sakata, T. Minamoto


Cytokine studies of ME/CFS as published. We have added clarifying or corrective details to several papers listed in the table, and we have added text to the figure legend to clarify its organization. We have corrected instances in which Blundell et al. (104) reported some studies' cytokine findings as “no significant difference,” when the original paper had actually reported those cytokines as below detection level. We have corrected instances in which cited papers made typographical or counting errors in their reported findings. We have added Stringer et al. (142), which was not included in the original table but should have been.In the original article, there were some minor mistakes in Cytokine studies of ME/CFS appears below.The corrected Table A1. J Trans Med. (2015) 13:299. doi: 10.1186/s12967-015-0653-3In the original article, the reference for Hardcastle et al. (2015) was incorrectly written as Hardcastle SL, Brenu EW, Johnston S, Nguyen T, Huth T, Ramos S, et al. Longitudinal analysis of immune abnormalities in varying severities of chronic fatigue syndrome/myalgic encephalomyelitis patients. Int J Med Sci. (2015) 12:764–72. doi: 10.7150/ijms.12399That citation should have instead been: Hardcastle SL, Brenu EW, Johnston S, Nguyen T, Huth T, Ramos S, et al. Serum immune proteins in moderate and severe chronic fatigue syndrome/myalgic encephalomyelitis patients. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Shewanella algae is an emerging marine zoonotic pathogen and accounts for considerable mortality and morbidity in compromised hosts. However, there is scarce literature related to the understanding of the genetic background of virulence determinants in S. algae. In this study, we aim to determine the occurrence of common virulence genes in S. algae using whole-genome sequence and comparative genomic analysis. Comparative genomics reveals putative-virulence genes related to bile resistance, chemotaxis, hemolysis, and motility. We detected the existence of hlyA, hlyD, and hlyIII involved in hemolysis. We also found chemotaxis gene cluster cheYZA operon and cheW gene. The results provide insights into the genetic basis underlying pathogenicity in S. algae. Shewanella algae is an emerging marine zoonotic pathogen. The organism was first classified in 1990 by Simidu et al. [S. algae is found in marine environments throughout the world and has been linked with both human and marine animal infections [Shewanella species found in clinical specimens and S. algae accounts for the majority of isolates from humans [S. algae has also been reported to cause diseases in marine animal, both wild and cultured [S. algae.u et al. , and desfections , 4. Currm humans , 6. S. acultured –9. HowevS. algae isolates could provide clues for pathogenic or fitness determinants [Marine ecosystem consists of a large variety of organisms that impact human health . The advrminants .S. algae isolates from clinical setting using whole-genome sequence and comparative genomic analysis and to explore the relationship among the tested genomes.The aims of the study were to determine the occurrence of common virulence genes found in S. algae strains ACCC, YHL, and CHL were obtained from various clinical sources . Raw sequence files were artifact-filtered and trimmed with DUK (http://duk.sourceforge.net/) and FASTX-toolkit fastx_trimmer (https://github.com/agordon/fastx_toolkit), respectively. Assembly was performed with a hybrid approach by ALLPATHS, version R46652 and Velvet version 1.2.07.Nucleic acids were extracted from overnight culture using the QIAGEN Genomic-tip 100/G kit and the Genomic DNA Buffer Set  according to the manufacturer's protocol. The DNA concentrations were measured by Qubit dsDNA HS Assay kit using Qubit 2.0 fluorometer . The DNA sample was sheared, in a microTUBE using Covaris S2 , into the desired size fragment of the library. The indexed PCR-free library preparation was performed using multiplexed high-throughput sequencing TruSeq DNA Sample Preparation Kit (Illumina) with 2 S. algae MARS 14 was retrieved from the NCBI Genome website (https://www.ncbi.nlm.nih.gov/assembly/GCF_000947195.1/).Genome sequence of human isolated Genome-based phylogenic analysis was performed using pairwise comparison of average nucleotide identity. The whole-genome average nucleotide identity (ANI) was calculated with the use of a modified algorithm . Phylogehttp://edgar.computational.bio) [S. algae genomes were generated for Prokka-annotated genomes using EDGAR . We compared the S. algae genomes using the MUMmer software package [The annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP)  and the nal.bio) . The cor package  together package .The genomic sequencing consisted of 250 bp paired-end reads, yielding approximately 0.88 Gbp to 1.24 Gbp for each isolate. The de novo assembly of genome sequence data revealed that the number of contigs (>200 bp) varied from 27 to 74 for each genome. The maximum contig size among the genomes was 976,090 bp aligned to YHL. The GC content ranged from 52.96% for CHL to 53.08% for ACCC. S. algae strains were identical in terms of nucleotide sequences, as shown in The average nucleotide identity (ANI) was calculated and revealed that tested S. algae strains. S. algae genes. The numbers of orthologous and strain-specific unique genes are shown in the Venn diagram. Core genome for the S. algae strains consists of 1354 coding sequences (S. algae YHL to 466 for S. algae CHL. Following genome map construction, we conducted genome mapping among the S. algae strains in the study. In this comparison, colored arcs indicate regions of high similarity as revealed by the NUCmer script from the MUMmer software package. As shown in We constructed a pan-genome dataset using whole-genome sequence of sequenced equences . The setexbBD, galU, and htpB are shared with S. algae genomes. Heat shock protein gene clpP and hemolysis homologous genes, hlyA, hlyD, hlyIII, and tolC, were found in each S. algae genome. Gene cluster cheYZA operon and cheW involved in chemotaxis were detected in all tested S. algae. Flagellar gene operons are present in all tested S. algae genome.As illustrated in S. algae has become an emerging marine zoonotic pathogen world-wide [S. algae infection is broad with considerable morbidity and mortality in compromised hosts [S. algae is important for determining molecular epidemiology, understanding its pathogenesis, identifying specific biomarkers, tracing evolution of these strains, and developing control strategy of these pathogens in host reservoirs. In this study, we investigated the core genetic structure underlying S. algae virulence. The pathogenicity and distribution patterns of the S. algae strains extended our understanding of their pathogenic potential.rld-wide . The speed hosts , 32. ThuS. algae from various sources [S. algae detected the presence of chemotaxis gene cluster cheYZA operon that is conserved in the chemotactic bacteria [cheA was demonstrated to be essential for chemotaxis using a two-component pathway [cheY and then is dephosphorylated by the phosphatase cheZ [S. algae habitats, the drivers of its chemotaxis could be very diverse. Previous studies have demonstrated that pathogenic bacteria use chemotaxis to localize reservoirs. Further study would be needed to identify the microenvironments suit for S. algae and the trigger of its chemotaxis.Previous attempts have been made to report the basic features of the genome of  sources , 34. In bacteria . Chemota pathway . In briease cheZ . Previouase cheZ . CheW isase cheZ . Owing tS. algae infection, and bile resistance has been noted in pathogenic strains [exbBD gene encodes Ton energy transduction system implicated in the response to bile [galU, htpB, and wecA involved in bile resistance [Shewanella.Biliary tract infection is main manifestation of  strains . In the  to bile , 40. We sistance –43. The S. algae [S. algae genomes. These flagellar systems are unique and require more study regarding the evolution and organization. Hemolysis is a main pathogenic feature in S. algae [hlyA encodes RTX pore-forming toxin α-hemolysin, which alters membrane permeability and causes cell lysis in a variety of human and animal hosts [Motility is one characteristic of S. algae . We idenS. algae . The genal hosts .S. algae. Although the number of strains was limited, we highlight the unique characteristics of core virulence determinants in these strains, as a high level of genomic conservation.In conclusion, this is one of the few studies tracking genetic background of putative virulence-related genes in


Torreya nucifera (L.) Siebold & Zucc. (Taxaceae) is a tertiary relict tree species with a distribution that is limited to South Korea and Japan. In the present study, the complete chloroplast (cp) genome of T. nucifera was sequenced and analyzed. The genome was 136,985 bp in length and contained 118 genes, including 82 protein-coding genes, 33 tRNA genes, and 4 rRNA genes. Fifteen of the genes contained a single intron, whereas ycf3 contained two introns and there were no inverted repeat sequences in the genome. Phylogenetic analysis supported the monophyly of Torreya species within the Taxaceae and T. nucifera was closely related to its congener T. grandis. Torreya nucifera (L.) Siebold & Zucc., which is a member of the tertiary relict genus Torreya Arn. (Taxaceae), is a long-lived, dioecious, and evergreen gymnosperm tree species that can grow up to 25 m in height  genome of T. nucifera was sequenced using high-throughput sequencing technology with the aim of generating informatics data that could be used to elucidate the phylogeny of the genus and to facilitate the conservation of T. nucifera. oil Lee . In SoutT. nucifera Forest  in Pyungdae-ri, Jeju-do Island . Total genomic DNA was extracted using the DNeasy Plant Mini Kit  and subsequently stored at the Plant DNA Bank of the National Institute of Forest Science . Whole genome sequencing was conducted using the Ion Torrent sequencing platform  and the cp genome sequence of T. nucifera was assembled using Geneious R10  as a reference sequence. Annotations were assigned using NCBI BLAST searches and the tRNAscan-SE program .Fresh leaves were collected from an individual tree of T. nucifera was 136,985 bp in length with a circular structure. The overall GC content was 35.4%. The genome contained 118 genes, including 82 protein-coding genes, 33 tRNA genes, and 4 rRNA genes. Fifteen genes contained a single intron, whereas ycf3 had two introns. The cp DNA of T. nucifera did not contain a pair of IRs separated by LSC and SSC regions, which is consistent with the cp genomes of other conifers  and Taxodium distichum (L.) Rich. (LC177556) as outgroup taxa. The dataset was analyzed using the RAxML BlackBox web-server (Stamatakis et al. T. nucifera was closely related to T. grandis (NC_034806; T. nucifera.A maximum-likelihood phylogenetic tree was constructed using 81 protein-coding gene sequences from 13 gymnosperm taxa, with


Streptomyces produce a great diversity of specialized metabolites, including highly volatile compounds with potential biological activities. Volatile organic compounds (VOCs) produced by nine Streptomyces spp., some of which are of industrial importance, were collected and identified using gas chromatography–mass spectrometry (GC-MS). Biosynthetic gene clusters (BGCs) present in the genomes of the respective Streptomyces spp. were also predicted to match them with the VOCs detected. Overall, 33 specific VOCs were identified, of which the production of 16 has not been previously reported in the Streptomyces. Among chemical classes, the most abundant VOCs were terpenes, which is consistent with predicted biosynthetic capabilities. In addition, 27 of the identified VOCs were plant-associated, demonstrating that some Streptomyces spp. can also produce such molecules. It is possible that some of the VOCs detected in the current study have roles in the interaction of Streptomyces with plants and other higher organisms, which might provide opportunities for their application in agriculture or industry.The  Volatile organic compounds (VOCs) are small odorous molecules up to C20) with low molecular mass , high vapor pressure, low boiling point and a lipophilic moiety 0 with lo, humans Streptomyces are ubiquitous soil dwellers, but are also found in the rhizosphere or as symbionts of plants and invertebrates [Streptomyces are recognized as industrially relevant because of their ability to produce a variety of specialized metabolites, which are widely used in medicine and agriculture [Non-motile sporulating Gram-positive bacteria from the genus tebrates . The Striculture . Many meiculture . In compiculture . Specialiculture , leadingStreptomyces spp. are also known to produce a variety of VOCs, some of which are classified as specialized metabolites [Streptomyces life cycle called exploration is also mediated by the VOC trimethylamine (TMA) [Streptomyces venezuelae, which leads to decreased iron solubility due to pH changes, thereby reducing its availability for other competing microorganisms [S. venezuelae explorative growth, which enables it to rapidly move over solid surfaces [Streptomyces intraspecies signalling involves two common VOCs, namely geosmin and 2-methylisoborneol (2-MIB), which are also known for their strong, earthy odor. The two metabolites attract Folsomia candida (springtails), which feed on Streptomyces colonies, where spores attach to their hydrophobic cuticles and are dispersed by the soil-dwelling hexapods to new and relatively distant locations [Solenopsis invicta (red imported fire ant) are attracted by geosmin and 2-MIB to Streptomyces-rich locations for nesting, which have reduced levels of pathogenic fungi [Drosophila melanogaster (common fruit fly) for oviposition, where other Streptomyces specialized metabolites, such as cosmomycin D and avermectin, kill the larvae, providing a potential nutrient source for the Streptomyces [Streptomyces use different VOCs for eliciting trans-kingdom interactions and for influencing biological outcomes, demonstrating the importance of such molecules in the environment.abolites ,31 and habolites , symbiosabolites . A recenne (TMA) ,35. Whenrganisms . In addiocations . A recenic fungi . In addiptomyces . TherefoStreptomyces spp., making them good candidates for the production of such compounds [Streptomyces spp. extensively used as model organisms to study different biological phenomena or in the production of important human and veterinary drugs were chosen for analysis in the current study. The selected microorganisms were cultured under different nutritional conditions and VOC production was analyzed using gas chromatography–mass spectrometry (GC-MS). Using this strategy, we obtained a snapshot of the volatile production capabilities of the respective Streptomyces spp., and some of our major findings are discussed below.In addition to some of the known examples described above, genome mining has revealed an abundance of potential VOC-associated BGCs of unknown function in ompounds ,39. Impoompounds . TherefoS. aureofaciens ATCC 12551 was purchased from Cedarlane Laboratories ; S. coelicolor M145 [S. avermitilis, S. clavuligerus, S. exfoliatus SMF19, S. griseofuscus, S. hygroscopicus NRRL 15879, S. lividans TK24 and S. parvulus B1682) were gifts from Dr. Susan E. Jensen . All media/reagents were purchased from Fisher Scientific  or VWR International . All Streptomyces cultures were grown at 28 °C and liquid cultures were agitated by shaking at 200 rpm. Spore stocks were prepared using International Streptomyces Project (ISP)-4 medium; seed cultures were grown using 5 mL of BD™ Tryptic Soy Broth (TSB) for 48 h. For inoculation, 1% v/v of seed culture was transferred to 25 mL of YMS [Bacteria used for VOC production are listed in lor M145  was provL of YMS , SFM [43L of YMS  medium iFor VOC analyses, cultures of each strain grown in the three growth media  were mixed together, and 25 mL of this mixture was transferred into one 8 oz Mason/Canning jar . As a control, volatiles were also collected from uninoculated media processed similarly. Volatile collection and extraction was performed according to the method described by Light et al.  with somStreptomyces spp. Volatiles collected using the PVAS22 system were eluted in 200 μL of hexane  under a flow of nitrogen. Extracts were concentrated under ultra-high purity nitrogen to 75 μL and transferred to a 250 μL glass insert. Samples were analyzed using a Scion 456 Gas Chromatograph–Single Quad Mass Spectrometer . A non-polar capillary column Rxi®-5silms  linked to a Bruker mass spectrometer  was used for analysis. Samples underwent splitless injection. The oven temperature was programmed for 1 min at 40 °C then increased to 100 °C at 6 °C/min and held for 1 min, then increased to 250 °C at 10 °C/min, and temperature held for 5 min. Both the injector and transfer line were maintained at 250 °C. Helium was used as a carrier gas. We used a detection threshold of 100 MCPS (megaCounts per second) to assemble a list of the high-abundance volatiles in each sample. These compounds were identified using analytical standards and NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library Version 2.0 g build in 2011 . Matches suggested by NMSS were evaluated based on spectral similarity, as well as comparison of their Kovats retention indices (RI) to published values  to determine the composition of VOCs being released from control (sterile medium) and media inoculated with different d values . IdentifatpD, gyrB, recA, rpoB and trpB, were downloaded and aligned with MAFFT . The evolutionary history was inferred by using the maximum likelihood method and the general time reversible model, and the tree with the highest log likelihood (−39385.69) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. This analysis involved ten nucleotide sequences and there were 10,954 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [Mycobacterium tuberculosis H37Rv (NCBI accession number NC_000962.3) served as an out-group.Strains for which genomic data were used for phylogenetic and BGC analysis are listed in n MEGA X  using a BGC prediction was performed using the bacterial version of antiSMASH 5.0 .Streptomyces spp. used as model organisms to study various aspects of general biology (S. coelicolor and S. lividans), protein and specialized metabolite production , or large scale industrial production of pharmaceutically important compounds , were chosen for analysis in the current study. Prior to culturing the respective species for VOC detection, their publicly available genome sequences were examined to obtain a better understanding of their relative phylogeny and genetic capabilities for specialized metabolism.Nine Streptomyces spp. chosen in this study were obtained from the National Center for Biotechnology Information (NCBI) database. The sequences were selected based on quality and completeness, and the genomes ranged in size from 6.51 to 10.38 Mbp  clustered together and contained the highest numbers of BGCs on their chromosomes  and S. hygroscopicus 5008 are also known to contain plasmids. In addition, plasmids from three species contain predicted BGCs, with the highest number being 30 in the case of S. clavuligerus ATCC 27064 (S. coelicolor A3(2) also contains two plasmids, one of which harbors three predicted BGCs. However, because the S. coelicolor strain (M145) used in the current study lacks plasmids [The genome sequences of the nine 0.38 Mbp . The phyand trpB ,50,51,52ens DM‑1 A. In addomosomes . Of the CC 27064 . S. coelplasmids , we did Streptomyces spp. The three most abundant categories were those containing terpene synthases (60/16.3%), non-ribosomal peptide synthetases  and type I polyketide synthases  (Streptomyces [Streptomyces genome contained between 25 and 65 BGCs (S. hygroscopicus contained the highest number (n = 65), which is consistent with it having the largest genome (10.38 Mbp) in the current study. Despite its relatively small chromosome, S. clavuligerus contained the second highest number of BGCs (n = 63), due to the 30 that are located on the 1.8 Mbp plasmid present in this species. In comparison, S. exfoliatus had the lowest number of BGCs (n = 25) amongst the species analyzed in the current study  . This isptomyces ,54,55. E 65 BGCs , where Snt study . Overallnt study . This isces spp. . The higressures . TherefoStreptomyces cultures grown under three different nutritional conditions  were from S. hygroscopicus (n = 10)  when subjected to GC-MS analysis [Streptomyces spp.The largest number of scopicus , which a(n = 10) . Howevert origin , containur study , even thur study . Previou acetate . In a seanalysis . It is pStreptomyces-specific VOCs were identified in the current study, of which 17 (including nine terpenoids) have been reported to be produced by members of the genus previously (Drosophila spp. [Streptomyces [Fusarium moniliforme (Family: Nectriaceae), which is known to cause cutaneous disease in humans [Sclerotinia sclerotiorum (plant pathogenic fungus) [Thirty-three eviously , Table 1ila spp. , geosminila spp. , whereasptomyces . Moreoven humans . Addition humans ,74, whicn humans  and Scle fungus) .S. globisporus [Argemone ochroleuca (Mexican poppy) [Carum montanum (Family: Apiaceae) [S. griseus DSM40236 cultures [S. albus [Streptomyces spp. [S. albus [S. alboflavus [S. globisporus [Other terpenoids detected in the current study include α-muurolene , which ibisporus . In addin poppy)  and Carupiaceae) , respectcultures . DihydroS. albus  and in mS. albus . Cholestces spp.  and was S. albus , 1H-indeboflavus  and 1H-ibisporus , but no S. albidoflavus [Scolytus schevyrewi (banded elm bark beetles) are attracted by 1-hexanol production in apricot trees, indicating a role for it in host tree selection [Streptomyces spp. [Rhodnius prolixus (Family: Reduviidae) and Triatoma infestans (kissing bug), both of which are vectors of the Chagas disease parasite [Ceratocystis fimbriata (Family: Ceratocystidaceae) [Streptomyces [Streptomyces yanglinensis [Chanmemelum nobil (Roman chamomile) [Malus domestica (apple) [Streptomyces spp. However, molecules with similar functional groups and structures have been described from the genus, suggesting that the Streptomyces are capable of producing such metabolites [Organic alcohols were another major class of VOCs detected in the current study , Table 1doflavus . It consdoflavus ,61,62, adoflavus . On the election . Anotherces spp. ,64,125 aces spp. . It has parasite . Phenyleidaceae) . Exampleptomyces ,126,127.linensis , Chanmemamomile)  and Malu (apple) . Other dabolites .Streptomyces, 13 are also produced by plants (Brickellia cavanillesii (Family: Asteraceacae) [Niponia nodulosa (Family: Cryptodesmidae) [Streptomyces spp. are known plant symbionts, and they have been identified in various arthropod species found to produce specialized metabolites [Streptomyces spp. and their possible contribution to specialized metabolite/VOC production in plants and arthropods should be taken into consideration while studying such phenomena in the future.It is worth noting that among the 17 known VOCs previously reported from y plants , Table 1aceacae) . In addiaceacae)  and in tesmidae) ,129. Strabolites . TherefoStreptomyces, and seven of them are terpenoids  with applications in phytotherapy for skin inflammation, but it has been shown to have some in vitro cellular toxicity [Phyllotreta and Aphthona flea beetles [Atractylodes lancea (Family: Asteraceae) [Zingiber zerumbet (bitter ginger) [+, K+-ATPase activity [Juniperus virginiana (eastern red cedar) essential oil [Valeriana jatamansi  [Parthenium hysterophorus (Santa Maria feverfew) [Pamburus missionis (Family: Rutaceae) essential oil [Eryngium carlinae (toad’s herb) and displays potential antioxidant activity [Sixteen VOCs identified in the current study have not been previously reported from rpenoids , Table 1rpenoids . α-Elemotoxicity . α-Himac beetles ,131, but beetles , antimic beetles  and anti beetles  activiti beetles . Other deraceae)  and Zing ginger) , and has ginger) , inhibitactivity  and stimactivity . β-Cedretial oil  and the tial oil . β-Vatiranaceae)  and Parteverfew) . Aromadetial oil , which itial oil ,136, wheactivity .Streptomyces-specific VOCs found in this study were non-terpenoid molecules , a traditional Chinese herbal medicine [Tinospora cordifolia (heart-leaved moonseed) [Lathyrus sativus (grass pea) [Aphis craccivora (cowpea aphids) are attracted by it in Lathyrus sativus [Cyrtocarpa procera (Chupandia plant) and has been shown to be toxic to Artemia salina (brine shrimp) [Aedes aegypti (yellow fever mosquito) [Spirodela polyrrhiza (HZ1) and Lemna minor (WX3), and has also been shown to stimulate nitrogen utilization by bacteria for remediation purposes [Streptomyces, but we were unable to find any information on their biosynthesis in other organisms or their biological activities, respectively.Nine olecules . p-Cresomedicine . Cetene oonseed)  and Lathass pea) , where f sativus . Moreove shrimp) . Another shrimp) ,144,145, shrimp) , human e shrimp)  and duri shrimp) . In addi shrimp) , mushroo shrimp)  and leaf shrimp) . It mimi shrimp) . Methyl osquito) . It is ppurposes . CycloheStreptomyces spp. are known to produce VOCs that are normally associated with higher organisms [Streptomyces spp. can naturally synthesize certain plant-associated VOCs further highlights the possibility of using these bacteria for the large-scale production of such molecules. As prokaryotes, Streptomyces are relatively easy to manipulate and culture, allowing for the improved and sustainable production of certain chemicals [Streptomyces reveromyceticus and the titer of botryococcene (a triterpene) was increased by manipulating biosynthetic genes and promoters [Certain rganisms . Many VOrganisms , but therganisms . In addirganisms . The finhemicals . For exaromoters . It has romoters ,156,157,Streptomyces, also have similar functions [S. coelicolor, promotes the growth of Arabidopsis thaliana by altering auxin/cytokinin homeostasis [Streptomyces albulus inhibit the growth of the fungal pathogens Sclerotinia sclerotiorum (Family: Sclerotiniaceae) and Fusarium oxysporum (Family: Nectriaceae), which are responsible for cucumber Fusarium wilt and sclerotinia stem rot of oilseed rape, respectively [D. melanogaster by geosmin produced by the Streptomyces has already been discussed in detail [Streptomyces has been previously reported [Streptomyces spp. needs to be verified. Understanding the ecological roles of such VOCs can allow for their exploitation in different fields, particularly in agriculture, where microbial products can provide greener alternatives for managing plant diseases or to promote growth [Streptomyces VOCs also have applications in the food, cosmetic, chemical and pharmaceutical industries. Therefore, with the increasing number of new metabolites being routinely identified from different Streptomyces spp., members from the genus can also serve as an important resource for the industrial production of both endogenous and exogenous VOCs for future use.The ecological function of many plant VOCs, such as those produced by flowering varieties to attract pollinators, is well-documented . As a deunctions . For exaeostasis . Other Vectively . In addin detail . Althougreported , we usede growth . Further


Rickettsia aeschlimannii, R. africae, and R. raoultii bacteria, all of which can cause disease in humans. Bird migrations might facilitate the spread of these pathogens into new areas.Migratory birds can transport infected ticks across continents. We evaluated pathogens in ticks collected from migratory birds in Italy. We found DNA from  Borrelia burgdorferi sensu lato, spotted fever group (SFG) rickettsiae, and Crimean-Congo hemorrhagic fever virus (CCHFV) in Ixodes ricinus and Hyalomma marginatum ticks collected from birds that migrate annually from Africa to Europe , the dunnock (Prunella modularis), the Eurasian blackcap (Sylvia atricapilla), the European robin (Erithacus rubecula), the song thrush (Turdus philomenos), and the subalpine warbler (Sylvia cantillas).We conducted fieldwork activities at the Ponza Ringing Station on the island of Ponza  during spring (March–May) 2016 and 2017. We captured 744 migratory birds belonging to 20 different species  during rhttps://diagnostics.roche.com) and DNA  extraction. We used the RealStar CCHFV RT-PCR Kit 1.0  for CCHFV detection; we used conventional PCR with protocols described elsewhere , Anaplasma phagocytophilum , Ehrlichia canis (clone), Rickettsia raoultii (clone) and B. burgdorferi (clone) as positive controls for each amplification.We collected 231 engorged ticks and identified them using standard morphologic keys , I. frontalis (3 nymphs), I. ventalloi (3 nymphs), and Amblyomma marmoreum (1 nymph). Amplification of the ITS region failed in the remaining ticks, identifying only the genus; these ticks were mostly Hyalomma spp.  or Ixodes spp. .Using PCR amplification of the ITS region, we identified 94 ticks at the species level: ompA gene in all the SFG rickettsiae–positive ticks (https://www.lgcgroup.com) and compared with sequences deposited in GenBank. Results revealed R. aeschlimannii DNA in 47 (94.0% [95% CI 83.8%–97.9%]) of 50 ticks (R. aeschlimanni strain documented from Egypt (GenBank accession no. HQ335157), Turkey (GenBank accession no. MF379299), and Italy (GenBank accession no. MH532239) and 1 sequence identical to R. aeschlimanni strain RH (GenBank accession no. HM050286) from Senegal; the latter strain differed from the others by 1 nt (T instead of C at nt 425). Two (4.0% [95% CI 1.1%–13.5%]) sequences were identical to R. africae (GenBank accession no. HQ335132), and 1 (2.0% [95% CI 0.4%–10.5%]) sequence was identical to R. raoultii (GenBank accession no. MF166732). We also screened a subset of positive ticks using primers targeting a fragment of the gltA gene . To determine the species, we amplified a fragment of the 50 ticks . We idenH. marginatum species complex  are the most widespread ticks in Africa, they also have been found in some countries in Europe, such as the United Kingdom (Hyalomma ticks are vectors and reservoirs of this virus; birds, which are the primary hosts for the immature stages of these ticks, can maintain and spread the virus into new areas through migration (Although ticks of the R. aeschlimannii and R. africae, which are zoonotic bacterial species endemic to Africa, are transmitted by ticks belonging to the genera Hyalomma and Amblyomma. However, these bacteria have also been detected in ticks from other regions, such as Oceania, the Caribbean islands, and Europe (R. aeschlimannii have been recently described in Greece (R. africae in H. rufipes and R. raoultii in Hyalomma sp., which are not known vectors for these pathogens. Because we did not test the birds for Rickettsia spp. and the ticks were engorged, we cannot exclude the possibility that the ticks acquired these microorganisms by feeding on positive birds. Nevertheless, our results agree with those observed in a study in Italy (Rickettsia species into areas to which they are not endemic. They also highlight role of migratory birds in the passive transportation of infected ticks.H. marginatum complex ticks attached to birds migrating from Africa to Europe (Hyalomma ticks in areas previously free of these vectors. Finally, RNA of another relevant human pathogen, the recently discovered Alkhurma hemorrhagic virus (H. marginatum complex.Although no ticks tested positive for CCHFV in our study, some studies report this virus in In summary, we found zoonotic bacteria in ticks carried by birds across their migratory routes and assessed the risk for pathogen introduction in Italy. However, further studies are needed to clarify the role of these ticks in the epidemiology of zoonotic pathogens.