Leishmania and Bartonella DNA.In Peru, the information regarding sand fly vectors of leishmaniasis and bartonellosis in the Amazon region is limited. In this study, we carried out sand fly collections in Peruvian lowland and highland jungle areas using different trap type configurations and screened them for Leishmania spp. screening was performed by kDNA PCR and confirmed by a nested cytochrome B gene (cytB) PCR. Bartonella spp. screening was performed by ITS PCR and confirmed by citrate synthase gene (gltA). The PCR amplicons were sequenced to identify Leishmania and Bartonella species.Phlebotomine sand flies were collected in Peruvian Amazon jungle and inter Andean regions using CDC light trap, UV and color LED traps, Mosquito Magnet trap, BG Sentinel trap, and a Shannon trap placed outside the houses. Leishmania guyanensis in Lutzomyia carrerai carrerai and L. naiffi in Lu. hirsuta hirsuta were identified based on cytB sequencing. Bartonella spp. related to Bartonella bacilliformis in Lu. whitmani, Lu. nevesi, Lu. hirsuta hirsuta and Lu. sherlocki, and a Bartonella sp. related to Candidatus B. rondoniensis in Lu. nevesi and Lu. maranonensis were identified based on gltA gene sequencing.UV and Blue LED traps collected the highest average number of sand flies per hour in low jungle; UV, Mosquito Magnet and Shannon traps in high jungle; and Mosquito Magnet in inter Andean region. L. naiffi and of two genotypes of Bartonella spp. related to B. bacilliformis and Candidatus B. rondoniensis infecting sand fly species from the Amazon region in Peru.UV, Blue LED, Mosquito Magnet and Shannon traps were more efficient than the BG-Sentinel, Green, and Red LED traps. This is the first report of  Leishmania and Bartonella vectors across Andean and Amazonian regions are important steps towards the design of better control strategies and reducing the risk of transmission in endemic areas. In this paper the authors explored the effectiveness of different trap types, based in LED colors and odor attractants, for sand fly collections in endemic areas of leishmaniasis and bartonellosis. Also, we reported for the first time the detection of Bartonella DNA in sand fly species from the Peruvian Amazon jungle, and the first record in Peru of L. (V.) naiffi infecting Lu. hirsuta hirsuta. The information provided by this manuscript will serve as a baseline for future surveillance and intervention studies about sand fly species as potential vectors of Leishmania and Bartonella in highly endemic areas.Leishmaniasis and human bartonellosis, two neglected tropical diseases, are endemic in Peruvian Andean regions. However, no human bartonellosis cases has been reported in the Amazon jungle where leishmaniasis has high prevalence. The improvement of sand fly trapping methods and identification of putative  Leishmania, bacteria of the genus Bartonella, and some arboviruses [Phlebotomine sand flies are small insects of crepuscular and nocturnal activity with weak flight capabilities, distributed in tropical and sub-tropical regions around the world. Female sand flies must feed on blood to complete oogenesis and lay eggs, and obtain blood meals from a large variety of vertebrates, including humans . Due to oviruses ,3. Phleboviruses .Bartonella bacilliformis and is a severe, debilitating illness with a mortality rate of over 80% in untreated individuals [Bartonella species can cause long-lasting intra-erythrocytic infections in their mammalian reservoirs and are transmitted by other arthropod species [Lu. verrucarum and Lu. peruensis (Diptera: Psychodidae: Phlebotominae) are considered the main vectors of both diseases [The leishmaniases are a group of neglected tropical diseases endemic in 98 countries worldwide with 0.9 to 1.6 million new cases per year, between 20,000 and 30,000 deaths, and 350 million people at risk of infection [ividuals . The iniividuals ,9; no ca species –12. In P species ,14 wherediseases ,16. In cdiseases ,18. Howediseases .2, and other chemical attractants recommended for use in hematophagous insect and vector surveillance [In order to perform efficient surveillance of pathogens carried by phlebotomine sand flies, we need to understand which trapping methods are most successful for catching sand flies. Phlebotomine sand flies are generally attracted by artificial lights of different intensity and color, COeillance ,20,21. Ceillance –24. In Peillance ,22,25,26eillance ,28. Alsoeillance , which deillance ,21,30. Ieillance –35, whiceillance  such as Leishmania has been done, particularly in high and low rainforest areas where approximately 17 Lutzomyia species were found infected with L. (V.) braziliensis, L. (V.) lainsoni, L. (V.) guyanensis and L. (Viannia) spp. [Bartonella DNA in sand flies has had limited success, with most studies conducted in inter-Andean regions where Carrion’s disease is endemic, principally in Cuzco and Ancash departments where two species, Lu. peruensis and Lu. verrucarum, were found infected with B. bacilliformis [Lu. robusta and Lu. maranonensis are potential vectors of Carrion’s disease in high jungle areas in the northeastern region [Lu. maranonensis was reported recently as a new potential vector of B. bacilliformis in Cutervo, Cajamarca, where this disease is endemic [Leishmania and be transmitted by leishmaniasis vectors.Currently, more than 190 sand fly species have been identified in Peru, 80% of them in the Amazon region . Howeveria) spp. ,37. The liformis –40. Epid endemic . HoweverLeishmania and Bartonella DNA detection to identify potential vector species. Given the high sand fly diversity in selected study sites, we assumed that different Leishmania and Bartonella species are circulating in geographical regions where their presence has not been previously reported, especially in the Amazon region.In this study, we propose to evaluate for the first time in Peru different light and odor-baited trap types for catching phlebotomine sand flies, expecting to increase our understanding of the foraging behavior of sand fly species present in different ecological regions. The objective of this study was to evaluate different vector trapping methods for their efficacy in capturing sand flies and to identify potential sand fly vector species of two neglected diseases, leishmaniasis and bartonellosis, in different ecological sites in Peruvian lowland and highland jungle areas and inter-Andean regions. We hypothesized that there would be a significant variation in the response of sand flies to different trap types according to study site and sand fly fauna composition. Moreover, we screened female sand flies captured for both Bartonella and Leishmania DNA. Phylogenetic analyses were performed on DNA sequences obtained.In July 2014, and January and February 2015, multiple trap configurations were tested for their efficacy in collecting phlebotomine sand flies in four sites located in different ecological regions where leishmaniasis and/or human bartonellosis are endemic. We morphologically identified female and male sand flies, then tested non-engorged female sand flies  for molecular detection of We carried out sand fly collections in Peruvian lowland and highland jungle areas, and one inter-Andean Valley . All colFlor de Acre, Madre de Dios. A lowland rainforest area located in Iberia district, province of Tahuamanu, Madre de Dios region , near the Peru-Brazil-Bolivia triple border (southeastern Peru).Tarapoto, San Martin. A highland jungle area located in the province of San Martin, San Martin region, northeastern Peru. Sand fly collection was conducted in three rural sites located approximately 5 kilometers from Tarapoto city, along the Shilcayo River: Bocatoma de Shilcayo water station ; Centro de Rescate Urku , a protected natural area; and a small rural hostel Cordillera Escalera Lodge .San Jose de Lourdes, Cajamarca. This rural community is located in the Chinchipe Valley, highland jungle area, approximately 30 kilometers from the Peru-Ecuador border, in San José de Lourdes district, province of San Ignacio, Cajamarca region , northeastern Peru.Caraz, Ancash. A rural community (Choquechaca) located in the district of Caraz, province of Huaylas, Ancash region, along the inter Andean Santa River valley .Rainfall and high humidity are predominant in high and low jungle areas , the climate is tropical with an annual mean temperature over 26°C. The main activity of the population is agriculture and cattle breeding in Madre de Dios and San Martin, whereas in San Jose de Lourdes, Cajamarca, coffee, cacao and fruit crops are predominant. Caraz, Ancash, is a typical inter-Andean valley with dispersed housing in the rural area surrounded by crops  and livestock .2 or emit UV light. This study involved attracting sand flies using luminous traps and odor baited traps that run overnight (1800–0600 hours)  environments and at least 50 meters apart to eliminate interference. No intradomiciliary sand fly collections were performed due some traps release CO0 hours)  during tWe used the Mini CDC light traps Model 512  with a 6.3 W incandescent lamp ; Blacklight UV traps (UV) Model 1212 (John W. Hocks Co.) with a 6.3 W black light (F4T5 BLB), and LED technology with Mini CDC light trap Model 512 modified locally to replace the incandescent lamp with blue, green, or red LED color bulbs ,42. All 2, heat and water vapor, mimicking a vertebrate host and attracting biting insects [Two commercial traps were tested, BioGents Sentinel Trap  with BG Lure as the attractant; and Mosquito Magnet  Trap Model Independence MM3200B which is battery-operated and runs on R-Octenol and propane gas, which is catalytically converted to produce CO insects ,31,34. TThis trap was used as a complementary method for sand fly collection in some places and when the weather conditions allowed. Insects were collected with Shannon trap from 1800 to 2100 hours by one operator protected with a mosquito jacket acting as human bait and using a manual aspirator and head lantern to manually catch sand flies ,24. The Sand fly specimens collected were counted and sorted by location, collection method, sex and collection date, stored in 1.5 mL microcentrifuge tubes with 70% ethanol, and transported to the Entomology Department at NAMRU-6 in Bellavista, Callao region. Specimens were identified morphologically using taxonomic keys and procedures developed by Young and Duncan (1994)  with smaISA = (a + Rj)/k, where we established a rank for species abundance per site; “a” is the number of zero observations for each species in all sites multiplied by “c” which is the single largest rank in all the data set plus 1. The Rj value corresponds to the sum of ranks for a given species in all the sites whereas “k” corresponds to the number of sites. The resulting ISA values were converted into the Standardized Index of Species Abundance (SISA) using the formula SISA = (c − ISA)/(c − 1). We quantified the number of sand flies captured per hour over each day of trapping and cumulatively over all trapping days. Normality of data was assessed by the Kolmogorov-Smirnov test in Minitab 17.1.0. Since the data did not meet the normality requirement, we used a non-parametric test for data analysis. A Kruskal-Wallis test was performed to assess whether sand fly capture rates per hour varied significantly between trap types for each site on separate days. Pairwise Wilcoxon signed-rank tests corrected for multiple comparisons using a Benjamini-Hochberg procedure were used to identify the trap type that accounted for significant differences [Species abundance was calculated in Microsoft Excel 2013 using the Index of Species Abundance (ISA)  using thferences . The totach site . All anaLeishmania and Bartonella screening are provided in Pools of non-engorged phlebotomine sand flies were air-dried to remove ethanol and ground with pellet-pestles in lysis buffer. Genomic DNA was extracted according to the manufacturer’s protocol using the DNeasy Blood & Tissue kit . The final DNA was eluted in 50 μL of elution buffer, and frozen at -30°C. All PCR conditions for Leishmania was performed by PCR that targets Leishmania minicircle kinetoplast DNA  conserved among species, using primers L.MC-1S and L.MC-1R as previously described [b (cytB) [L. (V.) braziliensis and L. (Leishmania) infantum DNA were used as positive PCR controls whereas PCR mix without DNA was used as a negative control.The initial screening for escribed  with somb (cytB) ,50. DetaBartonella DNA [gltA, the citrate synthase gene [Bartonella species but have different advantages that work well in combination. ITS is highly sensitive for detection of Bartonella DNA, but sequence alignment and phylogenetic analysis are complicated by the many insertions and deletions present. In contrast, gltA is less sensitive but is more amenable to alignment and phylogenetic analysis. Furthermore, gltA is the most commonly used marker for molecular detection of Bartonella species, creating a large database for comparison [rpoB, the β subunit of bacterial RNA polymerase gene [ftsZ, the filamenting temperature-sensitive mutant Z protein gene [gltA PCR results  [A PCR protocol that amplifies a segment between 400-600bp of the 16S–23S internal transcribed spacer (ITS) region was used for screening sand flies for ella DNA . Confirmase gene –54. Bothmparison . Additioase gene ,57, and ein gene ,59 were  results . Detailsh, Peru)  was usedLeishmania and Bartonella PCR products were analyzed by electrophoresis on 2% agarose gel stained with GelRed . The PCR amplicons were purified using the Wizard SV Gel and PCR Clean-Up System  and sequenced along both DNA strands using the BigDye Terminator v3.1 Cycle Sequencing Kit  according to the manufacturer’s instructions. Samples were sequenced at the Entomology Laboratory at NAMRU-6  using an Applied Biosystems 3130 XL Genetic Analyzer sequencer. Sequences were confirmed as being Leishmania or Bartonella DNA using the NCBI Basic Local Alignment Search Tool  prior to phylogenetic analysis.The positive cytB gene sequences obtained from sand flies was made using the ClustalW method, and sequences were analyzed with MEGA  program version X [cytB Leishmania spp. reference sequences were obtained from GenBank.The alignment of ersion X . The evoersion X . Phylogeersion X  and bootersion X . DatasetBartonella gltA sequences obtained from sand flies were aligned with reference sequences for named Bartonella species from GenBank using MAFFT v7 [MAFFT v7 . The alMAFFT v7 ,66. AligMAFFT v7  using onLutzomyia (35 species) and Brumptomyia (2 species). Abundance of species identified in all trap types used showed that Lu. davisi (SISA = 0.96), Lu. (Trichophoromyia) spp. (SISA = 0.91), Lu. yucumensis (SISA = 0.90), Lu. whitmani (SISA = 0.87) and Lu. auraensis (SISA = 0.80) were at the highest density (Lutzomyia davisi was the most abundant sand fly species in red LED trap (55.0%), Lu. auraensis was predominant in green LED and UV (70.5% and 29.6% respectively), Lu. whitmani in blue LED and CDC (34.6% and 24.6% respectively), and Lu. yucumensis was the predominant species in the sand fly collections with Shannon trap (55.7%).The CDC, blue, green and red LED, UV and Shannon traps were tested. A total of 2,328 sand fly specimens were collected edge of the forest, 913 (39.2%) females and 1,415 (60.8%) males belonging to two genera,  density . LutzomyThe UV and blue LED traps collected the highest number of phlebotomine sand flies (53.4% and 21.3% respectively), while the green LED and red LED traps collected the lowest number (1.9% and 1.7% respectively) . To evalLutzomyia (26 species) and Brumptomyia (2 species). Of them, 481 specimens were collected in the peridomicile and 854 inside the forest, but Kruskal-Wallis tests showed no significant differences in the sand fly species composition between both environments (p>0.05). Lutzomyia hirsuta hirsuta (SISA = 0.90), Lu. nevesi (SISA = 0.81) and Lu. (Trichophoromyia) spp. (SISA = 0.75) were the most abundant phlebotomine sand flies in all trap types used and all study sites. Regarding sand fly abundance by collection method, Lu. (Trichophoromyia) spp. was the predominant species in UV traps (49.3%), Lu. nevesi in blue LED (55.2%), CDC (57.6%) and MM (35.2%), Lu. hirsuta hirsuta in Shannon traps (80.2%) and MM (26.5%), and Lu. yuilli yuilli was caught principally with MM (98%)  females and 567 (42.5%) males belonging to two genera, MM (98%) .While the Shannon trap was used only on the first two days of trapping in favorable weather conditions, it captured the highest number of sand flies per hour compared to the other traps . During Lutzomyia species. Of them, 171 specimens were collected in the peridomicile and 20 in edge of the forest, but Kruskal-Wallis tests showed no significant differences in the sand fly species composition between both environments (p>0.05). Lu. maranonensis (SISA = 0.94) and Lu. robusta (SISA = 0.56) were the most abundant. Other sand fly species identified were Lu. pallidithorax, Lu. castanea and Lu. reclusa  and 29 males (15.18%) belonging to five  reclusa .The MM trap captured the greatest number of sand flies per hour compared to other trap types ; BG-S trLu. verrucarum (99.3%) and Lu. peruensis (0.7%) , followed by CDC , UV , and blue LED   and 59 males (4.8%) belonging to two species, s (0.7%) . Since ts (0.7%)  and a hi3, 5.9%) . Due to 3, 5.9%) , no signLeishmania DNA (Leishmania parasites was detected in Lu. hirsuta hirsuta (1 pool), Lu. davisi (3 pools), Lu. carrerai carrerai (2 pools), Lu. whitmani (2 pools), and Lu. (Trichophoromyia) spp. (1 pool) from Madre de Dios collected with UV, blue LED, and green LED traps; and in Lu. nevesi (7 pools) Lu. hirsuta hirsuta (2 pools), Lu. (Sciopemyia) spp. (1 pool), and Lu. (Pressatia) spp. (1 pool) from San Martin collected with UV, blue LED, CDC, and MM traps  were screened for ania DNA . DNA of MM traps . The estcytB PCR amplicons from positive pools were sequenced. One pool of Lu. carrerai carrerai collected with a UV trap from Madre de Dios was found infected with L. (V.) guyanensis; one pool of Lu. hirsuta hirsuta collected with a blue LED trap from San Martin were found infected with L. (V.) naiffi (cytB DNA sequences is shown in Nested ) naiffi . The phyBartonella DNA (Lu. whitmani (1 pool) from Madre de Dios collected with UV trap; Lu. nevesi (12 pools), Lu. hirsuta hirsuta (2 pools) and Lu. sherlocki (1 pool) from San Martin collected with CDC, blue LED, UV, and MM traps; and Lu. maranonensis (1 pool) from Cajamarca collected with blue LED were found infected with Bartonella spp. by ITS PCR, and confirmed by nested gltA PCR, and additional molecular markers rpoB and ftsZ PCR  were screened for ella DNA . Lu. whiftsZ PCR . The mingltA were sequenced and confirmed as Bartonella DNA using BLAST. ITS sequences varied in length between 461–590 bp and the closest matching Bartonella species on GenBank were B. bacilliformis (average sequence identity = 91.5%) and B. ancashensis (average sequence identity = 88.1%). The presence of insertions and deletions in the alignment of ITS sequences complicates phylogenetic analyses, so phylogenetic trees were constructed only for the gltA gene. The DNA sequences of gltA had lengths between 319–366 bp and were classified into three lineages (Lu. whitmani (1 pool) from Madre de Dios, and Lu. nevesi (11 pools) and Lu. sherlocki (1 pool) from San Martin, which were found infected with genotypes most closely related to B. bacilliformis (average sequence identity = 89.6%). Lineage 2 comprised two pools of Lu. hirsuta hirsuta from San Martin and were also related to B. bacilliformis (average sequence identity = 89.6%). The third lineage comprised of one pool of Lu. nevesi from San Martin and one pool of Lu. maranonensis from Cajamarca. These species were found infected with a genotype most closely related to Candidatus B. rondoniensis (average sequence identity = 92%).Amplicons from all positive pools by ITS and lineages . LineageLeishmania spp. and Bartonella spp. in multiple sand flies in different endemic areas outside of the Andean region. Both findings are unique in Peru and will help to better understand the behavior of phlebotomine sand fly vectors.This is the first study in Peru to evaluate the efficacy of different light and odor-baited trap types for phlebotomine sand fly collection in different ecological regions, and is the first study to determine the natural infection of Phlebotomus papatasi and results in the Brazilian rainforest [Knowledge about insect color vision suggests that there is a high sensitivity to light with short wavelengths that correspond to the ultraviolet spectrum in most insect families . Howeverinforest ,72, wher2 and other chemical attractants are very effective for collecting phlebotomine sand flies and other blood sucking insects as has been observed in Israel and Egypt [2 only successfully captured P. papatasi and other sand fly species. Most studies with MM traps have been for surveillance of mosquito vectors of malaria and arboviruses [Aedes and other mosquito species, MM trap efficacy is increased when in addition to CO2 the trap is supplemented with octenol [Anopheles species (68.13%) compared with MM (31.88%) [Except for Shannon traps and protected human bait, traps that release COnd Egypt ,32, wheroviruses ,74 and eoviruses . For Aed octenol . However(31.88%) ,77; othe2 and R-Octenol, are more selective to capture anthropophilic sand fly species, so it is recommended to use a combination of both trap types  in entomological surveillance of potential sand fly vectors of leishmaniasis and bartonellosis.Our results of the trap type evaluation conducted in different ecological regions with distinct sand fly species composition indicate that phlebotomine sand fly behavior against different colors of light or chemical attractants is variable between study sites. In low jungle regions, UV and blue LED were more efficient and collected a higher diversity of sand fly species, whereas in high jungle and inter-Andean regions, MM and UV traps were more effective. In general, in our study we have observed that traps that use short wavelength light collect a higher diversity of species , while tLutzomyia species were found naturally infected with Leishmania parasites [Lu. carrerai carrerai naturally infected with L. (V.) guyanensis in Iberia, Madre de Dios. This Leishmania species has a wide distribution in the Amazon jungle, and in Peru it has been isolated from patients with cutaneous leishmaniasis from high and low jungle regions [Lu. whitmani was found naturally infected with a genotype of Bartonella spp. (lineage 1) most closely related to B. bacilliformis based on gltA gene DNA sequences. There are no reports of autochthonous Carrion’s disease transmission in Iberia or any other sites in Madre de Dios region. Recently was reported the detection of B. bacilliformis in ticks collected from wild mammals in San Lorenzo [B. bacilliformis in other mammalian reservoirs other than humans have failed, as well as any incrimination of other arthropod vectors besides Lutzomyia species [It is known that leishmaniasis has been endemic in the Peruvian southeastern region for a long time , but thearasites ,44. In t regions ,80. At t Lorenzo , a small Lorenzo ,83. Curr species ,82–85.Lu. hirsuta hirsuta naturally infected with L. (V.) naiffi in a rural area located only 5 kilometers from Tarapoto city, along the road to Cordillera Escalera. This is the first report of L. (V.) naiffi in Peru, which increases the number of Leishmania species in our country to eight, in addition to L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, L. (L.) amazonensis, and a hybrid L. braziliensis/peruviana [Leishmania naiffi causes cutaneous leishmaniasis and is widespread in the Brazilian Amazonian region [Lu. tortura in the northern Amazonian region of Ecuador [L. (V.) naiffi has a wide distribution in the Amazon region and could be responsible for leishmaniasis cases in San Martin department, where L. (V.) guyanensis has also been detected in human patients [L. (V.) naiffi transmission in Peru. In our study sites in San Martin we also found Lu. nevesi, Lu. hirsuta hirsuta and Lu. sherlocki naturally infected with two Bartonella genotypes related to B. bacilliformis based on gltA gene sequencing (lineages 1 and 2), and a third genotype of Bartonella spp. related to Candidatus B. rondoniensis naturally infecting Lu. nevesi (lineage 3). This is the first report of detection of Bartonella DNA in Lutzomyia species from the northern Amazonian region of San Martin. Leishmaniasis cases have a high prevalence in San Martin and are reported yearly by the Ministry of Health, but information about human bartonellosis in this region is very limited, with only a few cases reported during the last years but without evidence of autochthonous transmission [Bartonella and Leishmania in Madre de Dios and San Martin regions had previously been reported as potential leishmaniasis vectors in the Amazon region of Peru and other countries [In San Martin, northern Amazonian region, where leishmaniasis has higher prevalence and is an important risk area for transmission of this disease ,86, we feruviana . Leishman region  and has  Ecuador . Our respatients ,80. Furtsmission . Interes lineage . This isountries .Bartonella DNA genotype related to Candidatus B. rondoniensis (lineage 3) was also found naturally infecting Lu. maranonensis in San Jose de Lourdes, a high jungle area in Cajamarca region. This sand fly species was recently found infected with B. bacilliformis in an endemic area of Carrion’s disease in Cutervo province  and has been incriminated as a potential vector of this disease [Lutzomyia maranonensis and Lu. robusta are the most abundant phlebotomine sand flies in the Peruvian northeastern region and southern Ecuador (Zamora Chinchipe province), and it has been suggested that these sand fly species could be the vectors of Carrion’s disease and leishmaniasis in the Peru-Ecuador border regions [et al. [Lu. maranonensis is capable of harboring different Bartonella species. Similar results were reported by Villaseca et al. [Lu. peruensis naturally infected with B. bacilliformis and B. taylori in the Urubamba valley, Cuzco region, Peru, where an important outbreak of acute Carrion’s disease occurred during 1997–1998 without previous history of this disease in this region [Candidatus B. rondoniensis was first found in triatomine bugs vectors of Chagas disease, Eratyrus mucronatus from French Guiana [Bartonella species infecting triatomine bugs, but different species of Triatominae are distributed across the country, especially in the northern region  where Chagas disease is endemic [Bartonella spp.The  disease . Lutzomy regions ,89,90. O [et al.  show thaa et al.  who founs region . Candidah Guiana . In Peru endemic –96. FurtBartonella spp. in this study, the majority of them are recognized as potential vectors of leishmaniasis, including Lu. whitmani, Lu. nevesi and Lu. sherlocki [Lu. maranonensis is a suspected vector of leishmaniasis along the Peru-Ecuador border [Bartonella DNA related to B. bacilliformis in sand flies was unexpected. Due the high diversity of Bartonella species in different vertebrate hosts such as bats, rodents, cats, dogs, non-human primates and other wild vertebrates [B. bacilliformis-like lineages 1 and 2 or Candidatus B. rondoniensis-like lineage 3. While the lineages were most closely related to B. bacilliformis and Candidatus B. rondoniensis, they are still quite distant from these species and each other in absolute genetic relatedness (<95% sequence similarity) and may thus represent novel Bartonella species [gltA must be used to clarify the phylogenetic associations of these lineages with other Bartonella species [et al. [Candidatus B. rondoniensis indicate that there is an underappreciated diversity of Bartonella lineages present in South American sand flies and other insects that may give us deeper insight into the origins of B. bacilliformis. Finally, the role of these Bartonella species in human disease is unknown. Further studies are needed to determine if these Bartonella genotypes detected in sand flies from low jungle (Madre de Dios) and high jungle (San Martin and Cajamarca) regions have the ability to cause human disease, or only infect vertebrate hosts.Regarding sand fly species found naturally infected with herlocki , whereasr border . Our dettebrates ,97–101, tebrates ,102,103, species . However species ,83,104.  [et al.  with theL. (V.) naiffi in Peru and of detection of Bartonella spp. genotypes distantly related to B. bacilliformis and Candidatus B. rondoniensis infecting sand fly species from the Amazon jungle. Our results underscore the need for increased studies of the ecological aspects of Amazonian sand fly species, and to increase Carrion’s disease surveillance in non-endemic areas, especially in the Amazon jungle where the risk of transmission for this disease can be higher due the capacity of sand fly species to transmit different human and newly emerging pathogens.According to our trap type evaluation in endemic areas of leishmaniasis and bartonellosis in Peru, the effectiveness of different traps for collecting sand flies vary according to ecological characteristics, climatic conditions and sand fly fauna composition across each site. Therefore, we recommend combining different traps to efficiently survey sand fly diversity across multiple habitats and maximize the surveillance of pathogens they may carry. Regarding pathogen detection in sand flies, this is the first report of S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigComparison of sand flies per hour versus trap type in Tarapoto, San Martin, on days with good weather and permanent rain: (A) cumulative sand flies per hour; (B) sand flies per hour on separate trapping days.(TIF)Click here for additional data file.S1 Table(XLSX)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.S6 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Circular RNAs (circRNAs) are a group of noncoding RNAs that participate in gene expression regulation in various pathways. The essential roles of circRNAs have been revealed in many species. However, knowledge of circRNAs in fungi is still not comprehensive.Trichophyton rubrum (T. rubrum) is considered a model organism of human pathogenic filamentous fungi and dermatophytes. In this study, we performed a genome-wide investigation of circRNAs in T. rubrum based on high-throughput sequencing and ultimately identified 4254 circRNAs. Most of these circRNAs were specific to the conidial or mycelial stage, revealing a developmental stage-specific expression pattern. In addition, 940 circRNAs were significantly differentially expressed between the conidial and mycelial stages. PCR experiments conducted on seven randomly selected differentially expressed (DE-) circRNAs confirmed the circularized structures and relative expression levels of these circRNAs. Based on their genome locations, most circRNAs originated from intergenic regions, unlike those in plants and animals. Furthermore, we constructed circRNA-miRNA-mRNA regulatory networks that included 661 DE-circRNAs targeting 140 miRNAs and further regulating 2753 mRNAs. The relative expression levels of two randomly selected circRNA-miRNA-mRNA axes were investigated by qRT-PCR, and the competing endogenous RNA (ceRNA) network theory was validated. Functional enrichment analysis of the target genes suggested that they were significantly involved in posttranscriptional processes and protein synthesis as well as some small-molecule metabolism processes. CircRNAs are relatively more conserved in closely related dermatophytes but rarely conserved in distantly related species. Tru_circ07138_001 is a highly conserved circRNA that was conserved in all ten dermatophytes analyzed in our study and three distantly related species. Its host gene TERG_07138 was also highly conserved in two of these distantly related species Gallus gallus and Caenorhabditis elegans. The specific role of this circRNA deserves further exploration.T. rubrum as well as dermatophytes. These results could serve as valuable resources for research on circRNA regulatory mechanisms in fungi and reveal new insights for further investigation of the physical characteristics of these significant human fungal pathogens.Our study is the first to provide a global profile of circRNAs in The online version contains supplementary material available at 10.1186/s12864-021-08184-y. Circular RNAs (circRNAs) are a group of endogenous noncoding RNAs (ncRNAs) that are each composed of a single-stranded covalently bonded ring structure , 2. In 16-methyladenosine (m6A) have translational potential [Caenorhabditis elegans [Saccharomyces cerevisiae [Arabidopsis thaliana [CircRNAs are generated by back-splicing of precursor mRNA (pre-mRNA) . In contotential , 18. Theotential , mice [2otential , zebrafiotential , Caenorh elegans , Drosophrevisiae , Arabidothaliana , rice [2thaliana  and soybthaliana .Ascosphaera apis, most of which ranged in length from 200 to 600 nt, unlike circRNAs in animals and plants. These circRNAs play vital roles in metabolism, environmental stress and gene expression [Magnaporthe oryzae, which infects rice. The host genes of circRNAs in conidia were involved mainly in the synthesis of storage products for rice infection, while those of circRNAs in mycelia were involved primarily in basic metabolism for the promotion of normal fungal growth [Nosema ceranae, and circRNA-miRNA-mRNA regulatory networks showed that the target genes were engaged in various metabolic pathways, indicating that these circRNAs might play regulatory roles in N. ceranae spores [CircRNAs in fungi have been researched in only a few studies. In 2018, Guo et al. identified 551 circRNAs in pression . In 2018l growth . In addie spores . HoweverTrichophyton rubrum (T. rubrum) is the most common pathogen responsible for fungal infections worldwide and has been studied as a model organism of dermatophytes and human pathogen filamentous fungi [T. rubrum have been occasionally reported and threaten the lives of immune-deficient patients [T. rubrum continues to rise, and these infections are receiving increasing attention [T. rubrum has two major developmental stages in its life cycle: the conidial stage, in which the fungus adheres to the stratum corneum of skin, and the mycelial stage, in which the fungus penetrates and destroys the superficial layers [T. rubrum have been extensively studied at the molecular level, which has improved our understanding of this fungus [T. rubrum remain unknown.us fungi –32. In apatients , 34. Thettention . Resistattention . T. rubrl layers . The bios fungus –43. The T. rubrum based on high-throughput RNA sequencing (RNA-seq). A total of 4254 circRNAs were identified in both conidial and mycelial stages, and 940 circRNAs were considered to be significantly differentially expressed between the two stages. With further bioinformatics analysis, the genomic location, alternative splicing (AS) pattern, conservation and regulatory roles of circRNAs were also investigated. These results provide new clues for research on circRNA regulation mechanisms in fungi and could serve as a foundation for further studies on these medically important pathogenic fungi.In the current study, we performed global profiling of circRNAs in T. rubrum, we constructed cDNA libraries of conidia and mycelia with three biological replicates and sequenced them with an Illumina HiSeq platform. After the adaptors were trimmed and the low-quality reads or reads with unknown (N) bases were filtered out, averages of 125.1749 million and 124.6443 million clean reads were obtained for the conidial and mycelial stages, respectively , followed by exonic circRNAs 35.2%) and intronic circRNAs (4.3%). When the conidial and mycelial stages were compared, these three kinds of circRNAs showed similar proportions in the distribution | ≥1 and probability ≥0.8 [2 (fold change)| values of 12.64, 12.14 and 12.12, respectively, while the most significantly down-regulated circRNAs were Tru_circ05540_001, Tru_circ08130_001, and Tru_circ_sc2.6_00055, which had |log2 (fold change)| values of 8.23, 7.83 and 6.82, respectively.CircRNAs identified in the conidial and mycelial stages were compared to identify DE-circRNAs. Based on the criteria of |logity ≥0.8 , 45, 940Hierarchical cluster analysis additionally showed significantly different expression patterns between up-regulated and down-regulated circRNAs, and suggested good consistency among the three replicates  corresponded to 15 circRNAs. We also found that DE-circRNAs corresponding to identical host genes all showed consistent relative expression differences in the conidial vs. mycelial stage .The correspondence of two or more circRNAs to a single host gene may be due to alternative back-splicing events when these circRNAs share identical splicing acceptors or donors . For exaT. rubrum, we searched all the sequenced samples using the CIRI-AS algorithm to identify circ-AS events. A total of 4 types of circ-AS events, including exon skipping (ES), intron retention (IR), alternative 5′ splice site (A5SS) splicing and alternative 3′ splice site (A3SS) splicing, were identified in 101 circRNAs , followed by Tru-miR-595 (354 mRNAs) and Tru-miR-3620-5p (330 mRNAs).The up-regulated circRNA Tru_circ_sc2.6_00052 was arbitrarily selected to construct a circRNA-miRNA-mRNA network. As shown in Fig. T. rubrum development.These results illustrate the complexity of gene expression regulation. In addition, transcription factors (TFs) have been found to participate in this regulatory process . In our To further uncover the potential roles of circRNAs in the ceRNA network, 2753 target genes regulated by DE-circRNAs were subjected to GO and KEGG enrichment. Based on GO annotation of biological processes, the target genes were heavily involved in ribosome biogenesis, ribonucleoprotein complex biogenesis, rRNA processing, rRNA metabolic processes, and RNA metabolic processes, suggesting their roles in posttranscriptional regulation and protein expression-related pathways. In addition, the target genes participated in small-molecule metabolism, such as small-molecule catabolic processes, nucleobase-containing compound metabolic processes, carboxylic acid metabolic processes and monocarboxylic acid metabolic processes. In the molecular function category, the target genes were significantly involved in acetyltransferase activity and N-acyltransferase activity. In the cellular component category, target genes were significantly associated with the nucleolus, host cellular components and host cell parts  were both significantly up-regulated in conidia vs. mycelia , UBI (Gene ID: 395296) and ubq-1 (Gene ID: 175840), corresponding to three of these circRNAs, Tru_circ07138_001, circUBI and cel_circ_0000387, respectively, all belong to the short-lived polyubiquitin-related gene family, which is involved in the ATP-dependent proteolysis of impaired proteins [T. rubrum and G. gallus and 95.63% between T. rubrum and C. elegans (E < 1e−5)  and intronic regions (4.3%). This distribution pattern in  regions –69. In ccircRNAs , 28. ThecircRNAs . In addiAmong all the identified circRNAs, 1680 circRNAs mapped to 1055 host genes. In some cases, two or more circRNAs stemmed from one host gene. Some of these instances may have been due to AS events. Four types of circ-AS events corresponding to 101 circRNAs were identified in our study, including ES, IR, A5SS splicing and A3SS splicing events. Among these, A3SS is the most prevalent splicing event, and ES is the rarest. This AS pattern of circRNAs is different from that of linear mRNAs, of which IR is the most common AS type in fungi –74. AS oTrichosporon asahii (T. asahii) were compared with uninfected samples, and a differentially expressed ceRNA network was constructed [T. asahii, whereas miRNA miR-505-3p was down-regulated [It is widely accepted that circRNAs serve a regulatory role by acting as miRNA sponges and further facilitating mRNA expression, thus constructing a complex ceRNA network , 81. We structed . The relegulated . In anotegulated . The resegulated . All of egulated .Other regulatory factors, such as TFs, may also be involved in the regulation of gene expression based on a ceRNA regulating mechanism . A miRNAFunctional annotation of the target genes suggested that some of them are involved in various small-molecule metabolic processes. Moreover, the target genes were found to be heavily involved in biogenesis-, processing- and metabolism-related processes of ribosomes and RNA, suggesting that these target genes might participate in posttranscriptional processes and protein synthesis. Based on these findings, we speculate that circRNAs might interact with these target genes to affect gene expression at the posttranscriptional and translational levels in addition to acting as miRNA sponges to release mRNA, thus facilitating protein expression. Furthermore, some target genes were identified as secreted proteases and multidrug efflux transporters, suggesting that circRNAs might play a role in the virulence of this fungus.A. thaliana are orthologous to circRNAs in Oryza sativa and Solanum lycopersicum, respectively [T. rubrum exhibited rare conservation with circRNAs in some animal, plant and nematode model organisms that fewer than 1% of the circRNAs were conserved in these species. This low conservation may be due to the distant phylogenetic relationships between T. rubrum and these other species. In contrast, circRNAs in T. rubrum were relatively highly homologous to the corresponding genomic region in closely related dermatophytes, accounting for 22–42% of all identified circRNAs. In particular, the circRNA Tru_circ07138_001 was conserved in all dermatophyte species analyzed in our study. In addition, this circRNA was conserved in three distantly related species G. gallus, C. elegans and Gossypium spp., and its host gene TERG_07138 was highly homologous to the corresponding circRNA host genes in G. gallus and C. elegans. Interestingly, these three homologous host genes all belong to the polyubiquitin-related family. All of these results demonstrate high conservation of this circRNA and its host gene in these different species, suggesting that further exploration of the potential function and role of this conserved circRNA is warranted.CircRNAs are highly conserved in mammals . Approxiectively . In our T. rubrum and dermatophytes. These data could lay a foundation for further study on circRNA regulatory mechanisms and provide valuable clues for in-depth exploration of the biological characteristics of T. rubrum and closely related species. Such pursuits will facilitate the fight against these significant human fungal pathogens.Our research provides the first global profile of circRNAs in T. rubrum strain BMU 01672 was incubated on potato dextrose agar medium  at 28 °C for 15 days to induce conidia formation. Conidia were collected with distilled water at 4 °C and then filtered twice through Miracloth  to remove mycelial fragments. Conidial purity was examined with a microscope. To obtain mycelia, 1 × 106 conidia were inoculated into 100 ml YPD liquid medium (Becton Dickinson) and cultured at 28 °C under constant shaking (180 rpm) for 72 h [g for 5 min.for 72 h . The medThree biological replicates were performed for each conidial and mycelial stage, and 300 mg material was used for each replicate. Total RNA was extracted using TRIzol reagent  according to the manufacturer’s protocol. Then, RNase-free DNase  was added to eliminate potential genomic DNA contamination. The integrity and purity of total RNA were assessed with an Agilent 2100 Bioanalyzer system  and a NanoDrop ND-1000 spectrophotometer .In each sample, rRNA was removed using a Ribo-Zero rRNA Removal Kit , and linear RNA was digested with RNase R (Epicentre). The remaining RNA was fragmented, and the fragments were reverse-transcribed to construct cDNA libraries following the protocol of an NEBNext Ultra Directional RNA Library Prep Kit for Illumina . After repairing and filling the ends of double-stranded cDNA, NEBNext adaptors were ligated. cDNA fragments of 150–200 bp were selected and purified using AMPure XP Beads . Afterward, the ligated products were PCR-amplified, and the library quality and quantity were evaluated with an Agilent Bioanalyzer 2100 system and qRT-PCR. Finally, 150 bp paired-end reads were sequenced on the Illumina HiSeq™ 4000 platform.T. rubrum reference genome using BWA software to identify junction reads [T. rubrum reference genome to determine the splice sites. The junction reads with paired chiastic clipping (PCC) signals and paired-end mapping (PEM) sites were considered high-confidence back-spliced junction reads. In addition, only the back-spliced junction reads flanking the GU/AG splice signal were considered further. CircRNAs were identified using the CIRI tool based on a cutoff value of back-spliced junction reads ≥2 [Adaptors and low-quality reads that contained poly-N sequences or had low Q scores were removed to obtain clean reads, and the Q20, Q30, GC content and sequence duplication level were calculated. The clean reads were mapped to the on reads . These jreads ≥2 . We alsoreads ≥2 .2 (fold change)| ≥1 and probability ≥0.8 [The spliced reads per billion mapping (SRPBM) values were calculated to normalize the circRNA reads in each sample, and the number of junction reads per circRNA was determined with Circos software. The differential expression values of circRNAs were calculated using NOIseq, and DE-circRNAs were identified with the thresholds of |logity ≥0.8 , 93.https://www.kegg.jp/) [Gene function annotation was performed based on GO terms using the clusterProfiler R package  and KEGGegg.jp/) –97. MiRNegg.jp/) , and mRNegg.jp/) . Based oegg.jp/) .T. rubrum were blasted against circRNAs of six distantly related species, including G. gallus, D. melanogaster, Gossypium spp., A. thaliana, C. elegans, and M. musculus, using BlastN with the criteria of score > 50, identity> 50%, and E value <1e− 5. The circRNAs of these six species were deposited in the circBase database (http://www.circbase.org/) and the CircFunBase database (http://bis.zju.edu.cn/CircFunBaseBlast/). Genome sequences of ten dermatophytes, including T. equinum CBS 127.97, T. tonsurans CBS 112818, T. mentagrophytes D15P127, T. interdigitale MR816, T. benhamiae CBS 112371, T. verrucosum HKI 0517, T. violaceum CMCC (F) T31, N. gypsea CBS 118893, M. gypseum CBS 118893 and M. canis CBS 113480, were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). The genome sequences of all circRNAs identified in T. rubrum were then compared with these dermatophyte genomes using BlastN . A phylogenetic tree of circRNAs was visualized and annotated using the ggtree R package [Conservation of circRNAs was investigated in six distantly related species and ten closely related dermatophytes. CircRNAs identified in  package . Multipl package .http://www.ncbi.nlm.nih.gov/tools/primer-blast) according to the methods in previous studies [Total RNA was treated with DNase I and RNase R, and the remaining RNA was reverse-transcribed into cDNA with random primers using a SuperScript® III First-Strand Synthesis System (Invitrogen) following the manufacturer’s protocol. Genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen). Divergent primers and convergent primers of seven randomly selected DE-circRNAs were designed using Primer-BLAST ( studies , 87. All−ΔΔCt method [p < 0.05 was considered to indicate significance.The relative expression levels of the seven DE-circRNAs illustrated above were validated by qRT-PCR with divergent primers (Additional file t method . Data wet method . StatistWe randomly selected two circRNA-miRNA-mRNA axes and examined the relative expression levels of the circRNAs, miRNAs and mRNAs in these axes with respect to the conidial and mycelial stages. The primer sequences are listed in Additional file Additional file 1: Figure S1-S8. All supplementary figures.Additional file 2: Table S1. CircRNAs identified in conidial and mycelial stage of T. rubrum.Additional file 3: Table S2. Significant differentially expressed circRNAs in conidial vs. mycelial stage.Additional file 4: Table S3. GO classification of host genes for all the identified circRNAs.Additional file 5: Table S4. KEGG pathway analysis of host genes for all the identified circRNAs.Additional file 6: Table S5. GO classification of host genes for DE-circRNAs.Additional file 7: Table S6. KEGG pathways analysis of host genes for DE-circRNAs.Additional file 8: Table S7. CircRNA AS events identified in T. rubrum conidial and mycelial stages.Additional file 9: Table S8. MiRNAs targeted by DE-circRNAs.Additional file 10: Table S9. mRNAs targeted by miRNAs.Additional file 11: Table S10. GO enrichment of target genes for DE-circRNAs.Additional file 12: Table S11. KEGG pathways analysis of target genes for DE-circRNAs.Additional file 13: Table S12. The target genes related to fungal pathogenicity.Additional file 14: Table S13. Primers used in this study.


Invasive fungal infections (IFI) are adverse complications of allogeneic and autologous hematopoietic stem cell transplantation (HSCT) with significant mortality and morbidity. Randomized Controlled Trials (RCT) have addressed the optimal anti-fungal prophylaxis regimen. However, the consensus for an anti-fungal prophylaxis regimen has remained elusive. Hence, we performed a meta-analysis of currently available RCTs comparing the efficacy of fluconazole vs. other antifungal agents including voriconazole, micafungin, and itraconazole in the endpoint of preventing IFI.Randomized controlled trials were retrieved from PubMed, according to our inclusion criteria. The relative risk (RR), hazard risk (HR), and 95% confidence intervals (CI) were calculated. A random effect or fixed-effect model was used to calculate the pooled HR, based on heterogeneity. All statistical analyses were performed using RevMan software and R Core Team, and all p-values were two-tailed, and the significance level was 0.05.2=5%). However, subgroup analysis showed no statistical difference between fluconazole vs. other agents to prevent breakthrough proven IFI . Our subgroup analysis further showed that other agent’s group might have a superior role in preventing aspergillus compared with fluconazole , but no significant advantages over fluconazole for candidiasis .Ten RCTs were selected involving 2654 pts. Our results showed fluconazole is statistically inferior to other agents that include voriconazole, micafungin, and itraconazole with regards to the endpoint of a lower incidence of IFI (RR: 1.05; 95%CI: 1.02, 1.08; p=0.0002, ISuccessful Rate Without Incidence of IFIFigure 1. Successful Rate Without Incidence of IFIProven IFI vs. Suspected IFIFigure 2. Proven IFI vs. Suspected IFICandidiasis vs. AspergillusFigure 3. Candidiasis vs. AspergillusThis meta-analysis yield data that suggests fluconazole might be inferior to other agents in preventing IFI in all intent to treat patients undergoing HSCT. However, fluconazole is non-inferior in preventing proven IFI and candidiasis IFI based on our results. Thus, we continue to recommend fluconazole in selected patients who require anti-fungal prophylaxis. More RCTs are needed in the future to demonstrate the drug of choice for anti-fungal prophylaxis and address patient selection characteristics.All Authors: No reported disclosures


Naja atra bites cause severe soft tissue injury and are prone to wound infections. The pathogens of Naja atra bite-wound infections are highly variable in different geographical regions. Here, we report the first coinfection with Shewanella algae and Morganella morganii from a Naja atra bite wound with resistome analysis using whole genome sequencing. Snakebite is a major neglected tropical disease with significant mortality and morbidity. It is estimated that 4.5–5.4 million people get bitten by snakes annually. Among these patients, 1.8–2.7 million develop clinical illness, and 81,000–138,000 die from complications [Naja atra ranks third among the six most common venomous snake bites  in Taiwan [Although n Taiwan , it causn Taiwan . AlthougShewanella is derived from a limited number of case reports [Shewanella infections related to snakebites also remain scarce. To the best of our knowledge, this is the first case report of coinfection with Shewanella algae and Morganella morganii in a wound caused by N. atra bite with resistome analysis using whole genome sequencing.Our current understanding of the epidemiology and clinical manifestations of  reports . PublishN. atra) in his house. The snake was captured and identified by specialist, Dr. Yan-Chiao Mao. The bite had resulted in two puncture wounds on the lateral dorsal surface of his left foot. Local swelling, heat, and pain over his left foot extending to the medial thigh developed.A 61-year-old man with hypertension and hyperuricemia was transferred to the emergency department 20 minutes after being bitten by a cobra  with 72.0% segmented neutrophils, and hematocrit of 39.4%. His platelet count was 204 × 109/L (150–400 × 109/L). C-reactive protein was 32.98 mg/L (<0.3 mg/L). Additionally, aspartate aminotransferase was 72 U/L  and serum creatinine was 1.46 mg/dl . Computed tomography (CT) of the left leg performed with contrast material showed reticular pattern infiltration in the subcutaneous tissue over the left dorsal foot, ankle, and the anterolateral aspect of the left lower leg. There was not subcutaneous air, intramuscular abscess, nor destruction of bones.Blood tests indicated that his peripheral leukocyte count was 7.3 × 10S. algae and M. morganii were isolated from a conventional culture of aspirated bulla fluid. Intravenous piperacillin–tazobactam was administered, and intravenous antivenom was injected at 20 min, 3 h, 13 h, 37 h, and 44 h after snake bite, totaling eight vials. The local symptoms progressed rapidly over the next 2 days. He underwent surgical fasciotomy and extensive debridement of soft tissue along the dorsalis pedis and lateral side of the tibialis. Soft tissue specimens were obtained and sent for conventional culture. S. algae was designated as VGH 117, and M. morganii was designated as VGH 116. The minimum inhibitory concentrations (MICs) of M. morganii VGH116 and S. algae VGH117 are summarized in The patient subsequently underwent debridement four times after the first operation. The wound condition improved slowly after debridement, and a skin graft was performed on day 22 after sustaining the snakebite. The patient was discharged from the hospital on day 37 post-bite.The bacterial genomic DNA was extracted from an overnight culture using the QIAGEN Genomic-tip 100/G kit and Genomic DNA Buffer Set  according to the manufacturer’s instructions. A Qubit dsDNA HS Assay kit and Qubit 2.0 fluorometer  were used to measure DNA concentration. A total of 2 µg of each DNA sample was used to build indexed PCR-free libraries using a multiplexed, high-throughput sequencing TruSeq DNA Sample Preparation Kit  according to the manufacturer’s protocols with minor modifications.http://duk.sourceforge.net/, accessed on 14 November 2020). Low quality (Q-score < 10) reads were trimmed, and a read was retained if at least 50 bp was obtained by the FASTQX-toolkit . Nanopore read data were trimmed for low-quality bases via Porechop. The VGH116 genome was assembled using Velvet v. 1.2.07 and ALLPATHS v. R46652, using a 31 bp k-mer size . The VGH117 genome was reconstructed using a hybrid Illumina and Nanopore assembly pipeline called Unicyler.The genomic DNA of VGH116 and VGH117 were sequenced by Illumina MiSeq sequencer using paired-end (2 × 150 bp) sequencing, which generated 1,676,992 and 2,295,502 (69x) reads, respectively. The total read depth provided 51-fold and 69-fold coverage. VGH117 was additionally sequenced using Nanopore GridIon with 175-fold coverage (842 Mbp). Illumina read data were filtered using duk  Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP). Functional classification of the predicted genes was performed using the RPSBLAST program v. 2.2.15 in conjunction with the COGs (Clusters of Orthologous Groups of proteins) databases, using an E-value threshold <0.001. Virulence genes in the VGH116/VGH117 genomes were separately identified by running BLAST against the Virulence Factors Database (VFDB). The assembled genome was first aligned against the VFDB protein sequences of the full dataset (set B) under the following criteria: identity > 45%, aligned length > 450 bp, alignment coverage > 95%, and E-value < 1e−45. If multiple virulence factor genes overlapped at the same locus in the genome, only the best-aligned virulence factor gene was retained. Antibiotic-resistant genes were predicted via BLAST search against the Comprehensive Antibiotic Resistant Database (CARD) using the same criteria. The efflux pump genes were excluded from the analysis.We used OrthoANI to compute the Average Nucleotide Identity (ANI) among different species, as whole-genome ANI has now become the standard for species identification/confirmation by NCBI during genome submission. The OrthoANI reconstructs the phylogeny in different ways. First, the genome is chopped into pieces of 1020 bp and BLAST is used to compute the ANI of all homologous pairs. Finally, the unweighted pair group method with arithmetic mean (UPGMA) is invoked to cluster the species using the ANI as the distance metric.M. morganii VGH116 and S. algae VGH117 using the Illumina MiSeq and Oxford Nanopore GridIon at 58- to 175-fold coverage. (see Methods). Then de novo assembly was used to reconstruct a 3.8 Mbp genome for VGH116 and a 4.7 Mbp genome for VGH117 (M. morganii VGH116 was highly similar to the M. morganii L214 (>98% ANI), and S. algae VGH117 was close to S. algae MARS (>98% ANI) (Whole-genome sequencing was performed over r VGH117 . The GC 98% ANI) , confirm98% ANI) .M. morganii VGH116 was resistant to first-generation cephalosporins (cefazolin MIC ≥64 mg/L) but was susceptible to ampicillin/sulbactam (MIC ≤2 mg/L), piperacillin/tazobactam (MIC ≤2 mg/L), third and fourth generation cephalosporins , imipenem, amikacin, gentamicin, ciprofloxacin, and trimethroprim–sulfamethoxazole. Analysis of the resistome of M. morganii VGH116 revealed the presence of antibiotic resistance genes (ARGs). Several beta-lactamases were found and confirmed by multiple databases , including the NmcA beta-lactamase, cysB, and the class C beta-lactamases, AmpC (DHA-1) and AmpH.In total, 4371 and 4217 protein-coding genes and RNAs  were annotated in the VGH116 and VGH117 genomes . Phenotyalgae VGH117 was susceptible to ampicillin–sulbactam, piperacillin–tazobactam, third and fourth generation cephalosporins , imipenem, amikacin, gentamicin, ciprofloxacin, and trimethoprim–sulfamethoxazole. The resistome of this S. algae VGH 117 included the Ambler class D β-lactamases-encoding BlaOXA-55 gene.Moreover, S. N. atra harbors larger numbers of bacterial species associated with snakebite wound infections than crotaline or colubrid snake species [Envenomation resulting from snakebite frequently leads to extensive tissue destruction or an infected wound . Infecti species ,9.M. morganii, Enterococcus faecalis, and Clostridium species were the most commonly isolated pathogens in the oropharynx of N. atra. Only two of 32 oral flora of N. atra were identified as S. putrefaciens. In Taiwan [N. atra bite wound, M. morganii and Enterococcus faecalis were the most common, followed by Proteus spp. Bacteroides fragilis was an anerobic bacteria that was isolated. Mao et al. reported that only three of 112 cases were identified as S. putrefaciens [Shek et al. reported that n Taiwan ,12, amonefaciens .N. atra envenomation at Taichung and Taipei Veterans General Hospitals, 64% of cases had polymicrobial infection (≥ 2 pathogens), from which more than two organisms were isolated [Shewanella infections. Shewanella is probably an underestimated and underreported cause of wound infections following snake bites [Shewanella infection of snake bites during a twelve-year period, Po-Yu Liu et al. reported that Shewanella infection was associated with moderate to severe local envenomation [S. algae and M. morganii of wounds from the N. atra bite is rare. Furthermore, we isolated S. algae from the debrided wound and blood. Shewanella infections are increasingly being reported and have mostly been attributed to S. putrefaciens. However, recent data suggest that many S. putrefaciens isolates should be classified as the genetically distinct species S. algae rather than S. putrefaciens. This misclassification is largely due to the fact that only S. putrefaciens has been included in the databases of conventional systems. [In a previous report which reviewed all known cases of isolated . Howeverke bites . In a renomation . Coinfecsystems. .M. morganii and Shewanella putrefaciens isolated from cobra bite wounds showed that these bacteria were susceptible to amikacin, ceftazidime, ciprofloxacin, and piperacillin/tazobactam [M. morganii VGH116 genome were AmpC and AmpH. AmpC encodes a chromosomal class C beta-lactamase. AmpH bound strongly to penicillin G, cefoxitin, and cephalosporin C; was temperature sensitive; and disappeared from cells after overnight incubation in the stationary phase [BlaOXA-55 were found in the S. algae VGH117 genome. BlaOXA-55 has a narrow spectrum of hydrolysis on penicillins and narrow-spectrum cephalosporins and imipenem [M. morganiii harboring ampC and ampH, which conferred resistance to cephamycin and the first-generation cephalosporin. S. algae strains harbored the quinolone-resistance-associated genes  regardless its resistance to ciprofloxacin. S. algae VGH117 also contained qnrA, gyrA, gyrB but had no resistance to quinolones [Previous reports on zobactam . In our ry phase . Antibioimipenem . In thisinolones .S. algae VGH117 genome showed prominent GC-skews, which have been proven to be useful as an indicator of the DNA leading strand, lagging strand, replication origin (ori), and replication terminal (ter) [The al (ter) . The twoN. atra harbors a wide range of pathogenic bacteria; however, bacterial coinfection with snakebites has not been common in previous studies. Snake venom toxins contributed to the development of wound infection, and the pathogenicity of microorganisms could further worsening the wound condition [M. morganii and S. algae following snakebites. Further studies are required to clarify such an issue.The oral cavity of ondition . The bacN. atra bite wound infection caused by M. morganii carrying AmpC and AmpH and S. algae carrying BlaOXA-55. Empiric antibiotics for snake bite wound infection need to consider the reported pathogen of N. atra wound infection and associated resistance genes. With the rising of antimicrobial resistance [In this study, we demonstrated sistance , the app


Picea mongolica. This tree species is endemic to semiarid habitats of Inner Mongolia in China. To extend what is known about the molecular events underpinning its zygotic embryogenesis, comparative transcriptomic analyses of gene expression in zygotic embryos were performed by RNA sequencing in P. mongolica. Our results showed that most changes in transcript levels occurred in the early embryonic pattering determination and formation of mature embryos. Transcripts related to embryogenic competence, cell division pattern, hormones, and stress response genes were identified during embryogenesis. Auxin is essential for early embryo patterning and pre-cotyledon embryonic formation. However, ABA is a major regulator of embryo maturation. Moreover, we found that methylation-related gene expression is associated with activation of early-stage embryos, late embryogenesis abundant proteins, and storage/energy-related genes with late and mature embryos. Furthermore, network analysis revealed stage-specific and multistage gene expression clusters during embryogenesis. WOX, MYB, AP2, and HLH transcription factors seem more relevant to embryogenesis in different stages. Our results provide large-scale and comprehensive transcriptome data for embryo development in P. mongolica. These data will lay a foundation for the protection and utilization of P. mongolica resources.Zygotic embryogenesis is a critical process during seed development in gymnosperms. However, knowledge on the genome-wide transcriptional activation that guides this process in conifers is limited, especially in  Arabidopsis thaliana  reproduction system, the best pathway to enhance P. mongolica yield (id areas . The conca yield . We founPinus sylvestris), and Pinus pinaster  were identified during seed development. Many genes related to transcriptional regulation, signal transduction, and metabolic pathways were identified. Additionally, weighted gene co-expression network analysis (WGCNA) was performed to reveal key genes for specific time points in seed development. Our results provide data on molecular and biochemical events associated with ZE development, a foundation for improving the vegetative propagation of conifers via somatic embryogenesis in P. mongolica. Moreover, this work expands our understanding of the development of ZEs in conifers.In the present study, to gain insight into the regulatory pathways associated with zygotic embryo development and reveal the transcriptomic activation mechanism during embryogenesis, we sequenced the transcriptome of zygotic embryo (ZE) over several developmental stages covering most embryogenesis stages in P. mongolica were obtained every 10 days after pollination between June and September 2019, from adult trees in the Baiyinaobao Nature Reserve of Inner Mongolia, China. Cones were periodically collected every week for all desired developmental stages of ZEs. The megagametophytes containing different developmental stages of ZEs were divided for RNA sequencing based on stereomicroscope observations  sequencing.For transcriptome sequencing, total RNA was obtained using the Plant RNA kit  following the manufacturer’s instructions. RNA degradation and contamination were detected in 1% agarose gels. RNA quantity and integrity were assessed using the NanoPhotometer spectrophotometer  and RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . Total RNA (1 µg) was used to generate sequencing libraries using the NEBNextp-value < 0.05 were used to determine DEGs. Further analysis will be based on the fold-change value of EEGs using Expander 7 software  (1.16.1) . Pairwissoftware  with thesoftware .http://systemsbiology.cau.edu.cn/agriGOv2/) was used in Gene Ontology (GO) enrichment analysis of obtained DEGs, which was corrected by a p value less than 0.05. The cluster Profiler R package was used to test the enrichment of DEGs in KEGG pathways. Venn diagrams were constructed using online software (http://bioinformatics.psb.ugent.be/webtools/Venn/). Hierarchical clustering heat maps were built, and cluster analyses were conducted using MultiExperiment Viewer (http://www2.heatmapper.ca/expression/).The agriGO (version 2.0) , and cDNA was obtained using the PrimeScript™ RT Reagent Kit . PCRs were performed using AceQ qPCR SYBR Green Master Mix with the following thermal cycling conditions: 95°C for 10  min, 40 cycles of 95°C for 10 s, and 60°C for 60  s . RelativP. mongolica, we isolated ZEs from June to September after pollination  and false discovery rate of 0.05 or less were used as thresholds. Each pairwise comparison identified a large proportion of DEGs, including 12,473 in proembryogenic versus cleavage polyembryony stages, 8,868 in cleavage polyembryony versus dominant embryo stages, 5,389 in dominant embryos versus pre-cotyledonary embryos, 9,077 in pre-cotyledonary embryos versus cotyledonary embryos, 15,878 in cotyledonary embryos versus mature embryos, and 16,987 in mature embryos versus proembryo embryos . Next, wP. mongolica , response TFs  were highly abundant. More upregulated transcripts were highly enriched in response to stimulus, such as salt, drought, cold, and wound stress. Moreover, histone H2A, lysine-specific demethylase JMJ706 and JMJ25, and histone RNA stem-loop-binding two genes were up-expressed in early proembryos. On the contrary, histone acetylation-related genes (HAC1 and histone deacetylase 6) and chromatin formation or remodeling genes  were highly expressed at the mature phases. ABA response, dehydration, photosynthesis initiation, and storage/energy-related genes were associated with mature stages of embryo development. In addition, many DEGs were annotated as containing “terminal inverse repeats (TIR),” “leucine rich repeats,” or “NB-ARC domains,” which are molecular switches implicated in immune signal transduction mechanisms . These gp value < 0.001). Next, we analyzed the functions of these genes in each model and found that the translation initiation factor, CCAAT-box binding factor, and mRNA splicing factor were significantly associated with early embryogenic stage, suggesting that this period may be the transcriptional activation stage. Heat shock protein was significantly correlated with dominant embryonic developmental stage. Auxin-responsive protein was mainly enriched in the pre-cotyledon stage. Several late-embryogenesis abundant-like proteins were found during late embryogenesis and embryonic maturation stages. Therefore, LEA are likely key genes for embryo maturation in P. mongolica. In addition, the top 50 genes associated with MEdarkred were of unknown function. This may be due to incomplete genome annotation in P. mongolica.Because all DEGs represent different functions, a WGCNA assay was used to define clusters and obtain gene modules with specific patterns of expression during the six-embryo development stages . Based oP. mongolica, the MGFR tool  plays a critical role in maintaining embryogenesis by regulating tissue differentiation and determining the physiological characteristics of late embryos . MoreoveP. pinaster  play an important role during the ZE development. WOX2 is expressed in the apical daughter cell, and WOX8 is specifically expressed in the basal daughter cells of the zygote . The YDAP. mongolica.Several genes are sufficient for embryogenesis when ectopically expressed. For example, when LEC1 and LEC2 are expressed in seedlings, SE is induced . SERK1 eP. mongolica. Our datasets and approach are expected to facilitate the discovery of molecular mechanisms underlying patterns of embryonic development. Moreover, functional understanding of these shared and distinct expression patterns of signaling, transcriptional, and epigenetic factors will help to address how embryonic development shapes the divergence of seed development in P. mongolica. Furthermore, our data lay a foundation for the protection and utilization of P. mongolica resources.In summary, our results establish a set of stringently defined temporal markers for identifying the key players involved in embryogenesis of


Mikania micrantha is a notorious invasive weed that causes substantial economic losses and negative ecological consequences in southern China. However, the contributions of gene flow, environmental variables, and functional genes, all generally recognized as important factors driving invasive success, to its successful invasion of southern China are not fully understood. Here, we utilized a genotyping-by-sequencing approach to sequence 306 M. micrantha individuals from 21 invasive populations. Based on the obtained genome-wide single nucleotide polymorphism (SNP) data, we observed that all the populations possessed similar high levels of genetic diversity that were not constrained by longitude and latitude. Mikania micrantha was introduced multiple times and subsequently experienced rapid-range expansion with recurrent high gene flow. Using FST outliers, a latent factor mixed model, and the Bayesian method, we identified 38 outlier SNPs associated with environmental variables. The analysis of these outlier SNPs revealed that soil composition, temperature, precipitation, and ecological variables were important determinants affecting the invasive adaptation of M. micrantha. Candidate genes with outlier signatures were related to abiotic stress response. Gene family clustering analysis revealed 683 gene families unique to M. micrantha which may have significant implications for the growth, metabolism, and defense responses of M. micrantha. Forty-one genes showing significant positive selection signatures were identified. These genes mainly function in binding, DNA replication and repair, signature transduction, transcription, and cellular components. Collectively, these findings highlight the contribution of gene flow to the invasion and spread of M. micrantha and indicate the roles of adaptive loci and functional genes in invasive adaptation.A long-standing and unresolved issue in invasion biology concerns the rapid adaptation of invaders to nonindigenous environments.  Biological invasion has been reported at increasing rates and on expanding scales since the 20th century, gaining the attention of ecologists and evolutionary biologists . A long-Ambrosia artemisiifolia individuals from 37 locations and found that SNPs active in geographic differentiation occurred more often within pathways related to growth and defense responses than in other pathways. Van Boheemen et al.  [A. artemisiifolia individuals from North America, Europe, and Australia, and found multiple introductions and adaptive signatures in response to environmental selection pressure. These studies have shown that the GBS approach can provide insight into the genetic basis underlying population invasion and contribute to the exploration of the environmental variables and genes involved in population adaptation.Early studies using anonymous markers  and amplified fragment length polymorphisms (AFLPs)) may have included bias in their assessment of genetic variation, while the utility of these markers to detect adaptive loci associated with environmental variables, especially when using few molecular markers, is limited. Furthermore, studies using anonymous markers cannot be used to determine the gene function of candidate loci. However, with the development of sequencing technology, reduced-representation genomic sequencing has provided an opportunity to obtain genome-wide sequence information across populations, greatly promoting research into population expansion and adaptive evolution in invasive plants . Genotyp7, 2020) ,15 analyMikania micrantha H.B.K. (Asteraceae), a tropical American perennial creeping vine, is one of the top 10 most problematic noxious weeds worldwide [M. micrantha covers a relatively wide array of biome types, including tropical and subtropical moist and dry broadleaf forests, tropical and subtropical coniferous forests, tropical and subtropical grasslands, deserts and xeric shrublands, savannas, and temperate broadleaf and mixed forest [M. micrantha plants can grow in a variety of soil types, in both dry and cold areas, in aquatic habitats, and in broad light niches [M. micrantha indicates its strong adaptability to different environmental conditions. The population of M. micrantha can not only adapt well to climate conditions similar to those of its native range, but also invade novel climatic niches [M. micrantha into different climatic niches demonstrate its wide ecological amplitude and strong environmental adaptability. In addition, M. micrantha can propagate sexually via thousands of lightweight seeds [M. micrantha populations. Considering its high dispersal potential and strong adaptability, M. micrantha is an ideal candidate for investigating the contribution of gene flow to population expansion, the response of genetic loci to environmental variables, and the role of functional genes in invasive adaptation.orldwide ,20. The orldwide ,22. The d forest . M. micrt niches ,25,26. Tc niches . Lineagec niches . The expht seeds . Long-diht seeds . FrequenM. micrantha spread rapidly throughout southern China following its introduction to the Hong Kong Zoological and Botanical Gardens in 1884 [M. micrantha in southern China has been investigated in several genetic studies, all of which used a small number of anonymous genomic markers [M. micrantha populations. Some key issues have not been resolved in these genetic studies. First, different molecular markers detected obviously different levels of genetic differentiation across populations distributed in similar geographic ranges , hindering the understanding of how gene flow contributes to the invasive success of M. micrantha in southern China. Secondly, genetic loci that respond to environmental variables have not been detected; consequently, the effect of environmental variables on genetic variation and population adaptation remains unresolved. Third, candidate functional genes related to colonization and population adaptation have not yet been identified. Population genomic data for natural populations of M. micrantha are needed to address these issues. in 1884 . The rol markers ,33,34,35M. micrantha individuals from 21 populations in six regions, covering the main part of its invasive range in southern China. Based on the SNP data, we detected the effect of gene flow on the genetic variation in the introduced populations of M. micrantha. We also identified outlier SNPs associated with environmental variables. Furthermore, the gene family clustering analysis of the genomes of M. micrantha and 12 other plants revealed unique gene families in M. micrantha that are important for adaptation. We also detected candidate genes that showed a significant positive selection signature in M. micrantha and explored their functions. The current study provides new insights into the population expansion and adaptation of M. micrantha.In the current study, we used the GBS approach to generate a large number of SNPs to analyze 306 M. micrantha individuals were sampled from 21 invasive populations distributed in six regions in southern China, including Hong Kong, Macao, Shenzhen, Neilingding Island, Dongguan, and Zhuhai  method [Genomic DNA from 306 ) method . DNA quaM. micrantha DNA samples using MseΙ  and EcoRΙ restriction enzymes. Briefly, the DNA samples were incubated with MseΙ at 37°C. The digested DNA samples were ligated to MseΙ Y adapter N containing individual-specific barcodes with T4 DNA ligase. To further reduce the complexity and increase genome coverage, the MseΙ-digested samples were redigested with EcoRΙ. EcoRΙ-digested DNA fragments were purified using Agencourt AMPure XP (Beckman) and PCR-amplified using Phusion Master Mix (NEB), universal primer, and index primer. Amplified DNA from 306 M. micrantha individuals was purified and screened for size. Size-selected fragments (insert sizes of 265–315 bp) were purified and diluted for sequencing. Samples for all M. micrantha individuals were divided into 12 pooled libraries, which were sequenced using the Illumina NovaSeq platform  to generate 150 bp paired-end reads.GBS libraries were constructed for 306 M. micrantha individual were sorted based on the individual-specific barcode within the raw reads. Raw reads for each individual were quality-controlled with in-house Perl scripts. Clean reads were obtained by removing reads that contained more than 10% ambiguous bases (N), more than 50% low-quality  bases, and bases that aligned to adapters exceeding 10 nt.Sequences from each M. micrantha reference genome (unpublished genome data from our laboratory) using the Burrows-Wheeler Aligner (BWA) v0.7.17 [M. micrantha genome, ANNOVAR [Clean reads from 306 individuals were aligned to the  v0.7.17  with the v0.7.17 . mpileup v0.7.17  was also ANNOVAR  was usedHO), gene diversity (HS), allelic richness (AR), and inbreeding coefficients (FIS), were calculated for each population and region using the R package hierfstat [p < 0.05).The genetic diversity indices, including the observed heterozygosity /4FST.To determine the level of genetic differentiation in the study areas, pairwise Weir and Cockerham’s age in R . To testage in R . Hierarcage in R  to identM. micrantha, PLINK v1.9 [2) = 0.8. Filtered SNPs (5966 SNPs) were used for genetic structure analysis. ADMIXTURE v1.23 [K) was set from 2 to 22. Cross-validation (CV) error was used to select the most likely number of clusters. The K value with the lowest CV error was determined to be the optimal number of clusters. GCTA v1.26 [M. micrantha. Based on the pairwise genetic distances among individuals, the unweight pair group method with the arithmetic mean (UPGMA) method implemented in MEGA v5.10 [To investigate the genetic structure of INK v1.9  was usedRE v1.23  was usedTA v1.26  was usedGA v5.10  was usedGA v5.10 .http://www.worldclim.org (accessed on 5 March 2019)) at a 2.5-arc-minute resolution. The soil factors, including fresh water content; air-dried water content; pH; electrical conductivity; organic matter content; total N, C, K, Ca, Na, Mg, Al, P, S, Si, Fe, Mn, Zn, Cu, Pb, Cr, As, Se, Ni, and Cd were measured following the method of Shen et al. (2021) [http://lpdaac.usgs.gov (accessed on 8 March 2019), recorded in 2006–2016). The yearly mean value for each ecological variable derived from the MODIS dataset was computed using the maximum value composites function [The environmental data comprised 19 bioclimatic variables, 25 soil factors, and 6 ecological variables. The bioclimatic variables corresponding to historic conditions (from 1960 to 1990) for each population were obtained from WorldClim v1.4 (. (2021) . The ecofunction . To redufunction . EnvironFST-based outlier approach was used with the BayeScan software [FST coefficient (α) shared by all populations and a population-specific FST coefficient (β) shared by all loci [10 (PO) > 2 were considered outlier SNPs under selection. Given that purifying or balancing selection is more prone to elevated false-positive rates when identifying outlier loci in BayeScan under the context of range expansion [To determine whether there were SNP loci potentially under selection in the 21 populations, an software . It has all loci . A positxpansion , only ou−5.The genes containing outlier SNPs identified by BayeScan were functionally annotated. Annotation based on the Pfam protein database was performed using Hmmscan implemented in HMMER v3.1 . Based oK) was selected using the snmf function, which uses a Bayesian clustering algorithm to estimate the admixture coefficients of individuals. Based on the results of the snmf analysis obtained with 10 repetitions and 20,000 iterations for each K value from 1 to 22, the K value with the minimum cross-entropy was identified. This K value was used to perform LFMM analysis with the lfmm function. The lfmm function was implemented using 50,000 burn-in, 100,000 iterations, and 10 replicates. The p-values were used to test the significance of the correlation, with a p-value < 0.005 representing a significant association.The latent factor mixed model (LFMM) was used to detect the associations of outlier SNPs with environmental variables in the R package LEA . LFMM haFST matrix using a Mantel test in the R package ade4 [The Bayesian method implemented in the BAYENV v2.0 package  was alsoage ade4  with 100M. micrantha and 12 other plants were identified using the OrthoMCL program [M. micrantha proteins, protein sets from 12 other plants were obtained from public sources  enrichment analysis was performed using KOBAS [p-values were used to determine the significance of the KEGG pathway enrichment, with adjusted p-values < 0.05 considered significant. In addition, ANNOVAR [M. micrantha have developed nonsynonymous amino acid mutations at the population level, overlaps between genes containing nonsynonymous SNPs  were annotated and used for KEGG enrichment analysis. Furthermore, the overlaps between genes containing nonsynonymous SNPs and PSGs in M. micrantha were detected.Based on the gene family clustering data for g MUSCLE . To iden of PAML  was usedM. micrantha individuals from 21 invasive populations was performed. The analysis yielded a total of 3320.63 million raw reads, with a mean of 10.85 million reads per individual  varying from 1.743 (HK3) to 1.886 (HK1), observed heterozygosity (HO) ranging from 0.280 (HK4) to 0.387 (DG4), and gene diversity (HS) ranging from 0.363 (MA1) to 0.458 (SZ1) (FIS) values were positive in all 21 populations. The distribution of genetic diversity across 21 populations was not limited by longitude and latitude (p > 0.05), except for a weak correlation between AR and latitude   . The inblatitude . In addi > 0.05) .FST value was highest between HK3 and NLD2 (0.133) and lowest (0.004) between NLD2 and NLD6  to 0.043 (SZ versus NLD)  between populations ranged from 1.63 to 62.25 and from 5.564 to 31 between regions  . These r regions .M. micrantha, genetic clustering of the 306 individuals was conducted using ADMIXTURE, which also provided evidence of an admixed structure at the optimal clustering level (K = 15)  revealed an admixed distribution pattern in which individuals from different populations clustered randomly . To furt(K = 15) . Consiste branch .p < 0.001, FST = 0.042; p < 0.001). The Mantel test showed that the genetic differentiation between populations included a weak genetic relatedness caused by isolation by distance (IBD) .The analysis of molecular variance (AMOVA) revealed that 95.84% of the total variation occurred within populations, while only 4.16% occurred among populations (10 (PO) > 2  showed an FST < 0.1. Outlier SNPs also exhibited low FST values, with a mean value of 0.151, implying that the 21 populations did not show significant adaptive differentiation at the outlier loci. The outlier SNPs were located in different regions of the M. micrantha genome, with 11 outlier SNPs residing in exon regions of 11 genes  > 2 . These 211 genes . The 11 11 genes . In addi11 genes . Under tM. micrantha populations. These two approaches have great power for the identification of outlier loci associated with environmental variables. We identified 31 associations between 18 outlier SNPs and six environmental variables in the LFMM analysis , “biosynthesis of unsaturated fatty acids” (ko01040), “photosynthesis” (ko00195), “other glycan degradation” (ko00511), and “ether lipid metabolism” (ko00565)—all of which are important for the growth, metabolism, and defense response of M. micrantha  and “integral to membrane” (GO: 0016021), respectively.Further, we found that eight genes unique to ectively . These cM. micrantha and the 12 other plants. Following the likelihood ratio test, 41 genes were considered candidate genes that had undergone positive selection in M. micrantha  were significantly enriched , and lower than the interpopulation genetic differentiation level reported by previous genetic studies based on a small number of molecular markers (8–483 markers) [M. micrantha. Low genetic differentiation levels may occur because M. micrantha plants can produce enormous numbers of lightweight seeds that are dispersed over long distances by the wind [M. micrantha mainly depends on clonal propagation, which may also be an important factor leading to low genetic differentiation. Human activities can result in seeds or cloned fragments being carried to different locations, increasing genetic exchange between individuals from different populations and weakening population divergence. In addition, the homogenization effect of frequent gene flow would reduce adaptive differentiation between populations. Consistent with this, we detected a high rate of gene flow (1.63 ≤ Nm ≤ 62.25) among M. micrantha populations. Similarly, a high rate of gene flow has been found among populations within the Asian invasion range [M. micrantha population invasion is accompanied by a high rate of gene flow.The genetic differentiation levels in markers) ,31,34. Ithe wind , promotiM. micrantha populations [M. micrantha exhibits patterns of population genetics that tend to enhance its invasion potential and that gene flow and multiple introductions (see the following paragraph) may mitigate the negative influence of the founder effect, genetic bottleneck, or inbreeding on genetic diversity and mediate genetic diversity distribution in its invasive range.Generally, the range limit linked to species invasions affects the genetic diversity of invasive populations . The genulations ,34,75, hulations , and notM. micrantha was also confirmed by the weak correlation between genetic differentiation and geographic distance. Geographical isolation can explain genetic differentiation during the early period of population invasion, when populations are geographically dispersed. The effect of geographical isolation on population differentiation becomes weaker with a high rate of gene flow during population expansion. The continued invasion of M. micrantha in southern China (more than 130 years) and high rate of gene flow among populations can explain the weak correlation between genetic differentiation and geographic distance. Overall, M. micrantha was introduced multiple times and subsequently experienced a rapid range expansion with recurrent high rates of gene flow.ADMIXTURE and PCA analysis revealed multiple genetic clusters and an admixed population structure in the study area, which was consistent with the genetic structure previously detected in populations that invaded Asia ,69. MultM. micrantha invasion from tropical America to southern China, the new environments encountered likely induced candidate-selected loci that respond to environmental conditions. The LFMM and BAYENV analysis revealed significant associations between 38 outlier SNPs and 13 environmental variables, including temperature, precipitation, soil, and ecological variables, all of which may play roles in the invasive adaptation of M. micrantha. Specifically, (i) rapid adaptation to climate is conducive to range expansion of invasive plants. Temperature and precipitation are important determinants of the growth, adaptation, and potential distribution of M. micrantha [M. micrantha genome responds to temperature and precipitation fluctuations. (ii) The significant effect of soil composition on the invasion of M. micrantha has been confirmed [M. micrantha depends on accelerated activation of soil K [M. micrantha. We found significant associations between outlier SNPs, the percentage of non-vegetation cover, and the percentage of tree cover. M. micrantha often forms a dense thicket above nearby plants [M. micrantha genome may respond positively to tree or vegetation cover. Taken together, these results reveal that the M. micrantha genome already has selective signatures that respond to the changing environment to promote its invasive adaptation.The detection of association between SNPs and environmental variables helps us to recognize the bioclimatic, soil, and ecological variables that contribute to population adaptation. During the process of icrantha . Consisticrantha ,78 and ionfirmed . The soionfirmed . C depleonfirmed . Ca playonfirmed . Furtheronfirmed . Indeed,onfirmed  and it hf soil K . Finallyf soil K . In addif soil K . (iii) Ey plants  and escay plants . These cM. micrantha. Unique gene families were also observed in the genomes of Artemisia annua [Chrysanthemum nankingense [M. micrantha were enriched in functional pathways related to important physiological processes and stress response, such as oxidative phosphorylation, the biosynthesis of unsaturated fatty acids, and photosynthesis. Oxidative phosphorylation is an important metabolic pathway that generates ATP required for plant growth and metabolism. There is evidence that oxidative phosphorylation plays a crucial role in abiotic stress responses. For instance, oxidative phosphorylation is enriched by genes associated with the cold tolerance in Anthurium andraeanum [M. micrantha, providing the energy supply needed for rapid vegetative growth [M. micrantha. Similarly, the upregulation of genes related to photosynthesis promotes the adaptation of Solidago canadensis to a shaded habitat, greatly improving the plant’s ability to adapt to different light conditions [M. micrantha with functional importance will provide a valuable resource for further research on the adaptive mechanisms of this species.Based on gene family clustering analysis, we identified gene families unique to ia annua  and Chrykingense . Gene fadraeanum . Unsaturdraeanum . High phe growth . The enrnditions . The ideM. micrantha to drought and biotic stress. We also found unique genes related to ‘integral to membrane’. This functional category is related to the defense response of apple root to pathogen infection [Unique genes that harbor mutations leading to nonsynonymous amino acid substitutions among populations are worthy of attention. These unique genes encode functional proteins and are involved in important function categories. For example, GSTT3 is a member of GST superfamily and is related to antioxidants that respond to drought and biotic stress ,95. We dnfection . AlthougM. micrantha would enable us to better understand the molecular associations underpinning its invasive adaptation. Accordingly, we identified 41 genes showing a significant positive selection signature in M. micrantha. These PSGs were found to be involved in (i) binding, (ii) DNA replication and repair, (iii) signature transduction, and (iv) transcription and cellular components  [M. micrantha to cold stimulus. Finally, key cellular components  and transcriptional regulation are essential for plant growth and environmental adaptation. The positive selection of genes related to the nucleus and transcription suggests their potential roles in M. micrantha. In addition, two functionally important PSGs harbored mutations that resulted in nonsynonymous amino acid substitutions among 21 populations, which implies that they may contribute to the population adaptation of M. micrantha.Genes under evolutionary pressure (such as positive selection) may be important for the survival and spread of various species . The idemponents . Functionis seed ,99. DNA nis seed . Plants nis seed . Exonuclnis seed . The posl change . For exavuglare) . This paM. micrantha, providing insights into M. micrantha adaptation. We detected 11 outlier SNPs distributed in 11 genes as candidate targets of adaptive importance. The GO annotation analysis of the 11 genes revealed that they were related to stress response, which should be considered in future population genomic research. Under the biological process category, “metabolic process”, “oxidation–reduction process”, and “response to stimulus” were highly representative GO terms important for the environmental adaptation of invasive and non-invasive plants. The metabolic process may contribute to the cold tolerance in the northward invasion of Alternanthera philoxeroides [Amorpha fruticosa seedlings [Kandelia obovata [Phragmites karka [Tamarix ramosissima in response to water deficiency [During rapid population invasion, genes that regulate important biological processes are expected to function in xeroides . The oxieedlings  and in t obovata . Effecti obovata . In the  obovata  and the es karka . In termficiency .M. micrantha in southern China. Genome-wide SNP analysis revealed, for the first time, the effects of gene flow, environmental variables, and functional genes on the population variation and adaptation of M. micrantha. The presented findings demonstrate the importance of a high rate of gene flow coupled with genetic admixture for the invasion success of M. micrantha. We identified 38 outlier SNPs associated with environmental variables. Soil composition, temperature, precipitation, and ecological variables were important determinants affecting the invasive adaptation of M. micrantha. Candidate genes with outlier signatures were related to abiotic stress responses. Gene family clustering analysis revealed 683 gene families unique to M. micrantha that may have significant implications for the growth, metabolism, and defense response in M. micrantha. Forty-one genes with significant positive selection signatures were identified. These PSGs mainly function in binding, DNA replication and repair, signature transduction, transcription, and cellular components. In addition, eight genes unique to M. micrantha and two PSGs harbored mutations leading to nonsynonymous amino acid substitutions across 21 populations; these may play crucial roles in the population expansion and adaptation of M. micrantha. Overall, the presented population genomic analysis indicates that the sustained high rate of gene flow, response of adaptive loci, and regulation of functional genes have enabled the invasion and adaptation of M. micrantha in southern China. These findings can be used to guide prevention and monitoring efforts for this invasive species.In the present study, we used population genomics to characterize the range expansion and invasive adaptation of