AbstractHaliplidae, Gyrinidae, and Dytiscidae (Coleoptera) of Cape Breton Island, Nova Scotia, Canada were surveyed during the years 2006–2007. A total of 2027 individuals from 85 species was collected from 94 different localities, which brings to 87 the number of species recorded for this locality. Among these, Heterosternutaallegheniana (Matta & Wolfe), H.wickhami (Zaitzev), Hydroporusappalachius Sherman, H.gossei Larson & Roughley, H.nigellus Mannerheim, H.puberulus LeConte, Ilybiuspicipes (Kirby), and I.wasastjernae (C.R. Sahlberg) are reported for the first time in Nova Scotia. The Nearctic component of the fauna is made up of 71 species (81.6%), the Holarctic component of 16 species (18.4%). Most species are characteristic of both the Boreal and Atlantic Maritime Ecozones and have a transcontinental distribution but 19 species (21.8%), which are generally recognized as species with eastern affinities. In an examination of the Hydradephaga of insular portions of Atlantic Canada, it was shown that the island faunas of Cape Breton Island and Prince Edward Island are very similar  despite differences in composition suggesting that more Hydradephaga species have yet to be found on Cape Breton Island.The  Lawrence. It lies within the Atlantic Maritime Ecozone along with Québec’s Gaspé Peninsula, Magdalen Islands Archipelago and portions of the south shore of the St. Lawrence River. The climate of this ecozone is strongly influenced by the Atlantic Ocean, which produces cooler summers (average 14 °C) and warmer winters (average -5 °C), with coastal areas having slightly warmer winters and cooler summers than inland. Geologically, this region is a mix of sedimentary and igneous bedrock  of the Canadian Maritimes have been sporadic and regionally variable. Recent papers .Although physically separated from the Nova Scotia peninsula by the Strait of Canso, Cape Breton is artificially connected to mainland Nova Scotia by the Canso Causeway. The island is located east-northeast of the mainland with its northern and western coasts fronting on the Gulf of Saint Lawrence; its western coast also forming the eastern limits of the Northumberland Strait. The eastern and southern coasts front the Atlantic Ocean; its eastern coast also forming the western limits of the Cabot Strait. Cape Breton Island is composed mainly of rocky shores, rolling farmland, glacial valleys, barren headlands, mountains, woods and plateaus. The boreal highlands of Cape Breton reach elevations of 300–500 m and represent true boreal forest habitat, which is rare in Nova Scotia . Geologiwww.newworldencyclopedia.org/entry/Cape_Breton_Island).Cape Breton Island’s hydrological features include the Bras d’Or Lake system, a salt-water fjord at the heart of the island, and freshwater features including Lake Ainslie, the Margaree River system, and the Mira River. Innumerable smaller rivers and streams drain into the Bras d’Or Lake estuary and onto the Gulf of St. Lawrence and Atlantic coasts , Haliplidae), Dytiscidae) and Gyrinidae: Dineutus MacLeay).Nomenclature is based on the classification in Voucher specimens are deposited in the author’s research collection .Hydradephaga were collected in this study B242F0C1-055B-5965-84E9-299B05BEC324This species is reported from 39 specimens collected in Richmond County and Inverness County .All specimens were collected on pebble substrate or in leaf litter along the margin of cold creek and streams in accordance with Heterosternutaallegheniana had only been reported from New Brunswick and southern Québec F9BFC362-3739-5770-810E-62CC0C180D6EThis species is reported from nine specimens collected in Victoria County and Inverness County .Like the previous species all specimens were collected in gravel along the margins of streams. H.wickhami in the Maritimes. Prior to this study it had only been reported from Ontario and Québec. Its presence on Cape Breton Island represents a significant extension of this species to eastern Canada .Hydroporusappalachius is usually found in habitats where there are some water movements either along the margins of small lakes or in small streams and springs .Carex along the margins of beaver ponds and roadside ponds, which is similar to the habitats where these beetles were collected in Cape Breton Island which include also eutrophic creeks.In Newfoundland and Prince Edward Island, this species has been collected from among flooded grasses and emergent Hydroporus species has generally been confused with Hydroporusrectus Fall. In the Maritime ecozone, H.gossei is also reported from the neighboring province New Brunswick and Prince Edward Island .These beetles are common in small pools with dense emergent vegetation. The two specimens collected in Cape Breton Island were from a sphagnum bog and a eutrophic pool besides a river.Prior to this study, this species had only been reported from the neighboring province New Brunswick . The NorTaxon classificationAnimaliaColeopteraDytiscidaeLeConte83D6593D-5114-5637-9DC9-2F4D6803F86CHydroporuspuberulus is reported from nine specimens form two localities in Cape Breton County and Inverness County .Carex D8761173-439C-5BF6-A53F-8FF8572DB12BIlybiuspicipes is closely similar to I.angustior  from which it can be differentiated by the relative expansion of the protarsal claw 6937479A-A336-5252-B028-370C659CA09FIlybiuswasastjernae is reported in Cape Breton Island from only one specimen collected in Victoria County (sample V27).Scirpus.These beetles are generally collected from sphagnum pools, usually in, or adjacent to forest . In CapeThis species has a Holarctic distribution more or less throughout the boreal zone. In North America it is transcontinental in the boreal region . Prior tHydradephaga species was recovered from 94 samples during a survey conducted on Cape Breton Island, Canada, between 2006–2007. According to this study and literature , H.wickhami (Zaitzev), Hydroporusappalachius Sherman, H.gossei Larson & Roughley, H.nigellus Mannerheim, H.puberulus LeConte, Ilybiuspicipes (Kirby), and I.wasastjernae (C.R. Sahlberg)  Table . Among tAgabuserythropterus (Say), Copelatusglyphicus (Say), Coptotomuslongulus LeConte, Heterosternutaallegheniana, H.pulchra (LeConte), H.wickhami, Hydroporusgossei, H.niger Say, Ilybiusconfusus Aubé, Ilybiusignarus (LeConte), I.larsoni (Fery & Nilsson), Laccornislatens , Liodessusaffinis (Say), Nebrioporusrotundatus (LeConte), Neoporuscarolinus , Neoporusclypealis (Sharp), N.spurius (LeConte), N.sulcipennis , and Platambusobtusatus (Say), which are generally recognized as species with eastern affinities (The Nearctic component of the fauna is made up of 71 species (81.6%), the Holarctic component of 16 species (17.4%). Most species are characteristic of both the Boreal and Atlantic Maritime Ecozones and have a transcontinental distribution except for finities   of the fauna, while Gyrinidae (13.8%) and Haliplidae (8.1%) are represented in lesser proportions. Forty-five species were observed at more than 5% of the sites , A.anthracinus Mannerheim, Hydroporusnotabilis LeConte, H.signatus Mannerheim, H.striola , H.tristis (Paykull), Ilybiosomaseriatum (Say), Ilybiusbiguttulus (Germar), Laccophilusm.maculosus Say, and Neoporuscarolinus  (Table The 87 a Scotia . As is tshare 78.% of the share 78.% of the Agabusleptapsis (LeConte), A.erythropterus (Say), Heterosternutaallegheniana, H.pulchra, H.wickhami, Hydroporusappalachius, Neoporusclypealis (Sharp), Neoporusdimidiatus (G. & H.), N.spurius, N.sulcipennis , and Oreodytess. scitulus (LeConte)) are worth emphasizing in that they illustrate the high abundance of lotic habitats in this portion of Cape Breton Island. In general, coexisting species may be more closely related than expected by chance if environmental features of a given habitat select for certain traits that are shared by closely related species . The extraordinary species richness of rheophilic species , Heterosternutaallegheniana, H.pulchra, H.wickhami, Hydroporusappalachius, H.badiellus Fall, H.nigellus, H.puberulus, H.rufinasus Mannerheim, Ilybiusignarus, I.picipes, I.wasastjernae, Neoporusspurius, Oreodytess. scitulus, and Platambusobtusatus) collected in Cape Breton Island have yet to be discovered in Prince Edward Island. The absence in Cape Breton Island of 12 species listed in the latter province , Dytiscusdauricus Gebler, D.harrisii Kirby, Graphoderusliberus (Say), G.perplexus Sharp, Gyrinusbifarius Fall, G.lecontei (Hope), Hygrotuscompar Fall, Peltodytestortulosus Roberts, and Sanfilippodytesplaniusculus ) is worth mentioning as it suggests that the number of Hydradephaga species on Cape Breton island may be even greater than suggested by this study.The 87 species of nditions . It is wHydradephaga in the Canadian Maritimes, with eight new records for the province of Nova Scotia. The composition of the Cape Breton fauna reflects that of the Maritime Provinces as a whole. Whereas all the species found on Cape Breton Island have also been recorded in New Brunswick  the presence on Prince Edward Island (Our study adds considerably to the previous knowledge about runswick  (except d Island  of 12 sp


Klebsiella pneumoniae (KP) isolates from humans and other animal hosts are not well studied. Our goal was to compare the molecular epidemiology of KP strains that were isolated from urban rodents, shrews, and healthy people.The comparisons of molecular characterization and antibiotic resistance of K. pneumoniae (KP) isolates were isolated from fecal samples of rodents, shrews and healthy adults in 2015 in southern China. In total, 465 fecal samples were collected, of which 85 from rodents, 105 from shrews, and 275 from healthy adults. Antimicrobial susceptibility and production of extended-spectrum β-lactamases (ESBL) of the isolates were tested. PCR-based methods were used to detect specific genes, including ESBL genes  in ESBL-producing isolates, capsular serotypes  in hypervirulent KPs (hvKPs), and virulence genes  in hvKP isolates. Multilocus sequence type (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to exclude the homology of these isolates. The carriage rate of KP in urban rodents and shrews (78.42%) was higher than that in healthy adults (66.18%) . The prevalence rates of ESBL-producing isolates among rodents, shrews, and humans were 7.94, 12.79, and 17.03%, respectively. The positive rates of CTX-M, TEM and SHV types in ESBL-producing isolates were 29.79, 27.66, and 17.02%, respectively. Serotype K1, K5, K20, and K57 were detected in both small mammals and humans. PFGE typing revealed thirty-six clusters. PFGE cluster A was clustered by samples of shrews and healthy adult, with a similarity of 88.4%. MLST typing revealed thirty-eight types. ST23 and ST35 were detected in samples of shrews and healthy adults. ST37 was detected in samples of 2 rodents and a healthy adult.Overlapping serotypes of hvKP were observed in both the animals and humans. The same PFGE or MLST types were also found in isolates derived humans, rodents and shrews. Therefore, urban rodents and shrews might play a certain role in the transmission of drug-resistant and hypervirulent KP. Klebsiella pneumoniae (KP) is ubiquitous in humans, animals, sewage, soil, and polluted waters [d waters . KP is cd waters –4. Recend waters .Serratia marcescens strains in Europe [Efficiency of colonization and the ability to acquire resistance to antibiotics enables KP to spread rapidly in healthcare centers . With thn Europe . Since tn Europe . ESBL arn Europe . These pn Europe .In addition, a new type of virulent variant KP, known as hypervirulent KP (hvKP), which is associated with community-acquired infections, has emerged worldwide over the past three decades. HvKP infects both the healthy and non-immuno-compromised people more easily, and has more significant morbidity and mortality than the “classic” KP . InfectiEscherichia coli [Staphylococcus aureus [Salmonella spp. [Bartonella spp. [Leptospira spp. [Urban rodents and shrews have long share living environments with humans. These small mammals may play a role as reservoirs of causative agents for various bacterial, viral, and parasitic zoonoses because of their pervasiveness and their propensity toward close contact with humans . Rats hahia coli , 15, Stas aureus , and Sallla spp. . Additiolla spp.  and Leptira spp. . Thus, uira spp. . TherefoThe objectives of this study were to understand the carriage rate of KP in urban rodents and shrews, and to characterize antimicrobial resistance and hyper virulence of KP in these small mammals from a community in southern China. Meanwhile, KP isolates from healthy adults in the community were compared with those from small mammals. Based on these results, we assessed the potential transmission among urban rodents, shrews, and humans in a community.Rattus norvegicus, 3 Mus musculus, 2 Rattus flavipectus, and 105 Suncus murinus (Asian house shrews), were captured. The seasonal distribution of the capture rates was mainly in June and July  and house shrews (81.90%)   . The carriage rate of KP in the small mammals was higher than that in healthy adults , 27.65% (13/47) and 17.02% (8/47), respectively  and a healthy adult(T202–2). Whose similarity of the PFGE pattern was up to 88.4%  from the Ministry of Health, China. All surgical procedures were performed under anesthesia with diethyl ether in efforts to minimize suffering. Endangered or protected species were not involved in this study. Healthy adults participated after signing an informed consent form. After samples were collected, the animals were sent to the animal experimental center of southern medical university for conducting harmless treatment.khe gene. Microbiological tests of the purified colonies included gram staining and biochemical testing. The khe gene was identified using PCR as described previously [E. coli ATCC® 25922 were used as positive and negative controls, respectively, in each test protocol.Small mammals were captured alive around houses and buildings monthly using rat-trap cages (Yue-zong Co Ltd.) in a community in Baiyun District of Guangzhou city in southern China, between May and September 2015. Some faecal samples were collected from the animals and others from healthy adults were consecutively collected in the Physical Examination Center of the third affiliated hospital of Southern Medical University in Guangzhou from May to September 2015. All stool samples were transported to the lab in transport medium at 4 °C. Subsequently, stool samples were soaked in 5 mL of the nutrient broth  and incubated overnight at 37 ± 1 °C for bacterial enrichment. The presumptive isolates were confirmed as KP using microbiological tests combined with amplification of the species-specific eviously . KP ATCCE. coli (ATCC® 25922) were used as quality control strains.Antibiotic susceptibility testing of KP was conducted using the Kirby-Bauer disc diffusion method according to Clinical and Laboratory Standards Institute; 2018 guidelines (CLSI). A panel of 17 representative antimicrobial agents belonging to 11 different classes of antibiotics was used. Screening and phenotypic confirmatory tests for ESBL in KP were conducted according to CLSI guidelines, 2018. Results were interpreted according to CLSI. KP (ATCC® 700603) and blaTEM, blaSHV, and blaCTX-M) by PCR as described previously [magA, rmpA, uge, kfu, wcaG, and aerobactin among all hvKP isolates were screened by PCR using previously described methods [https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the CARD website (https://card.mcmaster.ca/home).PCR was used to detect the six common capsular serotype genes  , 48. Theeviously –51. Viru methods , 52–56. K. pneumoniae, placed in plugs, which were made by adding an equal volume of molten 1.0% SeaKem Gold. Slices of K. pneumoniae plugs were digested with 45 U/slice XbaI  and incubated at 37 °C for 2 h. Electrophoresis was run on the CHEF-DRIII system . PFGE patterns were analyzed using the BioNumerics software package . The similarity analysis was performed by Dice coefficient. A similarity of > 80% upon dendrogram analysis were considered to represent PFGE pattern subtypes [rpoB, gapA, mdh, pgi, phoE, infB, tonB) were detected according to the MLST official website (http://bigsdb.web.pasteur.fr/). Primer sequences, the annealing temperature, and amplified fragment sizes were shown in Additional file Genetic relatedness among the ESBL-producing KP isolates was determined by pulsed-field gel electrophoresis (PFGE). In brief, the cell suspension of subtypes . A subsep-value of < 0.05 was considered to be statistically significant. EBURST v3.0 software was used to analyze our MLST results and database data.Statistical analysis was carried out by using SPSS 20.0 software. The chi-square test was used to evaluate if the prevalence of KP was significantly different among groups. A two-sided Additional file 1: Figure S1. Tenmporal distribution of urban rodents and shrews between May and September, 2015.Additional file 2: Table S1. Primer sequences of specific gene and resistant genes for K. pneumoniae.Table S2. Primer sequences of K. pneumoniae for hypervirulent serotypes and virulent genes. Table S3. Primer sequences of seven housekeeping genes of K. peumoniae for multilocus sequence typing. Table S4. Carriage rates of KP isolates from urban rodents, house shrews, and healthy people. Table S5. Prevalence of multidrug resistance and ESBL-production among KP isolates from rodents, shrews, and healthy people (%). Table S6. Detection of hypervirulent KP isolates and virulent genes from rodents, shrews, and healthy people.


Thrips palmi is a widely distributed major agricultural pest in the tropics and subtropics, causing significant losses in cucurbit and solanaceous crops through feeding damage and transmission of tospoviruses. Thrips palmi is a vector of capsicum chlorosis virus (CaCV) in Australia. The present understanding of transmission biology and potential effects of CaCV on T. palmi is limited. To gain insights into molecular responses to CaCV infection, we performed RNA-Seq to identify thrips transcripts that are differentially-abundant during virus infection of adults. De-novo assembly of the transcriptome generated from whole bodies of T. palmi adults generated 166,445 contigs, of which ~24% contained a predicted open reading frame. We identified 1,389 differentially-expressed (DE) transcripts, with comparable numbers up- (708) and down-regulated (681) in virus-exposed thrips compared to non-exposed thrips. Approximately 59% of these DE transcripts had significant matches to NCBI non-redundant proteins (Blastx) and Blast2GO identified provisional functional categories among the up-regulated transcripts in virus-exposed thrips including innate immune response-related genes, salivary gland and/or gut-associated genes and vitellogenin genes. The majority of the immune-related proteins are known to serve functions in lysosome activity and melanisation in insects. Most of the up-regulated oral and extra-oral digestion-associated genes appear to be involved in digestion of proteins, lipids and plant cell wall components which may indirectly enhance the likelihood or frequency of virus transmission or may be involved in the regulation of host defence responses. Most of the down-regulated transcripts fell into the gene ontology functional category of ‘structural constituent of cuticle’. Comparison to DE genes responsive to tomato spotted wilt virus in Frankliniella occidentalis indicates conservation of some thrips molecular responses to infection by different tospoviruses. This study assembled the first transcriptome in the genus Thrips and provides important data to broaden our understanding of networks of molecular interactions between thrips and tospoviruses. Frankliniella occidentalis is world-wide the most devastating invasive species, with a broad host range, transmitting multiple tospoviruses  including the economically important tomato spotted wilt virus (TSWV)  are multi-subunit storage proteins with phenol oxidase activity that are involved in insect innate immunity, humoral immune response to pathogens and disease resistance [2 fold change 1.75  vs 13.31 ] was shown to have phenol oxidase activity in melanogenesis [2 fold change 2.08  vs 2.88 ] has a physiological role in pH and ion regulation pathways. Endoglucanase [log2 fold change 1.2–1.3  vs 1.95 ] is a salivary gland-associated cell wall digestive enzyme [T. palmi transcriptome. Deoxyribonuclease I [log2 fold change 1.31  vs 2.62 ] is an endonuclease that cleaves DNA and has been suggested to play a role in apoptosis [2 fold change of 2.82 associated with vector response to TSWV infection in F. fusca adult transcriptome [The up-regulated genes common to both virus-vector interactions are associated with innate immunity and salivary glands. Hexamerins [logsistance . Similarogenesis . Carbonipoptosis . This ge2 fold change 5.0–7.1  vs 4.5 ], which is a structural constituent of cuticle and alpha-tocopherol transfer-like [log2 fold change 7.0  vs 4.9 ] which has been shown to be involved in antiviral immunity in Drosophila melanogaster [F. occidentalis larvae [T. palmi adults.When down-regulated genes in adult transcriptomes were compared, none of the most highly down-regulated TP transcripts were shared between the two systems. However, genes of endocuticle structural glycoprotein [lognogaster  were dows larvae  and T. pThrips. We analysed T. palmi transcriptome in response to CaCV infection to identify DE transcripts and to classify these genes by sequence homologies (ontologies) to known proteins to begin to dissect the global response of this thrips species to tospovirus infection. Here, we present hypotheses about the effect of virus infection on three dynamic processes in vector biology: innate immunity, growth and development, and fitness.Quantifying changes in global gene expression is one means of inferring cellular and physiological responses of insects to infection by insect-pathogenic viruses or insect-vectored plant-viruses . This isT. palmi. Cathepsins are known to be involved in various biological processes including protein degradation, apoptosis and signalling activated in late endosome and lysosome and implicated in virus entry and replication [Based on Blastx descriptions and GO terms for DE transcripts, we identified 86 up-regulated transcripts putatively associated with innate immune response that may have been triggered by CaCV infection . The majlication , 106. Thlication .T. palmi. Besides pigmentation, melanin deposits and encapsulates foreign targets such as nematodes, parasitoids and virus-infected tissues and inhibits progression of infection [T. palmi transcriptome, we identified several PO genes and a CLIP domain-containing SP that were up-regulated in virus-exposed thrips. Previous studies have shown that PO cascade is activated in mosquitoes in response to Semliki Forest virus infection resulting in inhibition of virus spread in cell culture [Genes associated with melanisation seem to play a significant role in virus-exposed nfection , 107. Ennfection , serine nfection  and laccnfection  are the nfection . In T. p culture , 110. CL culture , 60. In  culture –65.Genes involved in other innate immune system pathways such as Toll, antigen processing and presentation, and complement and coagulation cascades  were also found up-regulated. Among the up-regulated transcripts in CaCV-exposed thrips, 63 encoded proteolytic enzymes, including 30 serine protease and trypsin genes. According to GO assignment all serine proteases and trypsins in this thrips transcriptome were predicted to exhibit serine-type endopeptidase activity. The role that these proteases may play in virus-exposed thrips is not known. Previously it was shown that insect serine proteases and trypsin-like serine proteases are generally involved in hemolymph coagulation, activation of antimicrobial peptide synthesis, and melanin synthesis , 112. ThT. palmi identified activation of different immunity-related pathways than the results reported by Zhang and collaborators [F. occidentalis-TSWV, such as Toll, JAK-STAT and RNA interference. Either these pathways have no significant role in adult T. palmi against CaCV or they were active only in larvae at the time of virus acquisition and initial replication and intercellular movement, which was not captured by our adult transcriptome. In another study, Schneweis and colleagues [F. occidentalis exposed to TSWV, 75% of innate immune response related transcripts were annotated as insect storage proteins, hexamerins which are also associated with humoral immunity. We did not identify hexamerins among the DE transcripts of T. palmi suggestive of activation of different set of genes in T. palmi’s innate immunity. When compared with F. fusca-TSWV adult DE genes, we identified up-regulation of several similar immune system-related genes such as cathepsin B, aminopeptidase N, serine proteases and heat shock protein 70. Therefore, it is likely that more generally in adult thrips infected with tospoviruses, immune reactions like apoptosis, phagocytosis and proteolysis are activated.Our study of borators  for F. olleagues  showed tin silico. All lipase 1 sequences lacked a signal peptide, hence were considered unlikely to be components of saliva and more likely involved in innate immunity. Of the 15 lipase 3 genes, nine sequences contained a signal peptide and a predicted cleavage site at their N-terminus, suggestive of secreted proteins. Lipases have previously been identified to be associated with salivary glands of other insects, including the potato leafhopper (Empoasca fabae) [Bemisia tabaci) [F. occidentalis [T. palmi, contained predicted signal peptides and cleavage sites. Similar proteins were identified in salivary gland transcriptomes of E. fabae and F. occidentalis [Salivation is an essential process in insect feeding , 114 anda fabae) , whitefl tabaci)  and F. odentalis . Other ldentalis , 88. Onedentalis  and the dentalis , 119. UpF. occidentalis [E. fabae [Lygus hesperus [T. palmi transcriptome is consistent with findings in other insects. Based on signal peptide predictions, some of these enzymes may be secreted, a requisite for extra-oral digestion.Plant-feeding insects secrete a range of cell wall-degrading enzymes to release cell contents and to facilitate subsequent ingestion . Pectin dentalis , E. fabaE. fabae  and the hesperus . Other chesperus , 123–125Apis melifera) [Ades aegypti) [Bombyx mori) [Tribolium castaneum) [F. occidentalis [E. fabae, [F. occidentalis salivary gland transcriptome [T. palmi transcriptome.A α-amylase that may be involved in sugar metabolism was identified among up-regulated transcripts in virus-exposed thrips. This sequence lacked an obvious signal peptide, but its GO term ‘extracellular exosome’ suggests extracellular localization and a putative digestive role. α-amylases have been identified in the saliva of many insects including honeybee (elifera) , mosquitaegypti) , silkworyx mori)  and red staneum) , and alsdentalis  and E. f. fabae, . Genes ecriptome  but wereT. palmi ACE may have similar functions.Among the other salivary gland-associated genes identified in this study were angiotensin-converting enzyme-like (ACE) genes which may have a role in inhibition of plant defence , 91. Wan2 fold change >9) in adult and pupal transcriptome of F. fusca infected with TSWV [2-fold change of 2.0. Abundance of Vg transcripts and their receptors in virus-exposed T. palmi implies transovarial transport of egg yolk protein components to the growing oocytes had been promoted in the presence of CaCV infection. Enhanced vitellin and vitellogenin levels in response to a plant virus infection was first reported from whitefly, MEAM1 (B biotype) B. tabaci that fed on tomato yellow leaf curl China virus-infected plants [F. occidentalis [Recilia dorsalis that fed on rice gall dwarf virus (RGDV)-infected plants showed significantly reduced longevity and fecundity associated with reduced levels of Vg compared to non-viruliferous individuals [Among the up-regulated genes in CaCV-exposed thrips, 54 transcripts represented vitellogenin (Vg) homologues . Vg contith TSWV . The priith TSWV . In femaith TSWV . Accumulith TSWV . Among td plants . These adentalis . In contividuals .Dialeurodes citri) [A. aegypti [Apis mellifera) [T. palmi may play a role in innate immunity to CaCV infection. In support, Vg was found to be 9-fold up-regulated in pupae of F. fusca exposed to TSWV [Laodelphax striatellus) [T. palmi, Vg was the most abundant transcript in virus-exposed small brown planthopper transcriptome [T. palmi cannot be excluded due to a current lack of knowledge in tissue tropism and virus localization in the body of T. palmi. Furthermore, Vg was not among the most abundant unigenes and DE genes in F. occidentalis transcriptome infected with TSWV [T. palmi interaction, but the exact role that Vg may play needs to be investigated further.There are other reports that have shown involvement of Vg in immune responses of insects including citrus whitefly (s citri) , A. aegy aegypti  and honellifera) . Based o to TSWV . Anotheratellus) . Similarcriptome . Previoucriptome . Furtherith TSWV . TherefoT. palmi and estimated differential abundance of transcripts when exposed to CaCV infection. The majority of DE transcripts did not contain Blastx annotations or conserved functional domains, but their differential abundance reflects unique roles in T. palmi-virus interaction and T. palmi biology in general. Although we cannot directly compare our data with studies of F. occidentalis [F. fusca [T. palmi appeared to be species-specific. Aside from the inherent differences across thrips species and tospoviruses species, differences in the experimental methods and computational protocols and parameters used in data analyses could explain the weak overlap in transcripts responsive to virus across the three studies. For example, DE analyses for F. fusca [T. palmi were performed using de novo transcriptome assemblies, whereas Schneweis and colleagues [F. occidentalis.In summary, we have assembled a whole-body transcriptome of adult dentalis  and F. fF. fusca  exposed F. fusca  and T. plleagues  used a dWe identified virus-responsive up-regulated DE transcripts that have putative roles in thrips innate immunity, feeding and fecundity. Down-regulated transcripts that are likely associated with structural component of cuticle and integral components of membrane may be responsible for delaying thrips development and increased longevity to ensure virus persistence and transmission. However, functions of those genes need to be experimentally validated in future. For example, transcripts associated with thrips feeding and fecundity could be candidates for potential targets in thrips and tospovirus management. In addition, transcriptomic data generated in this study will enrich genomic information of thrips and will allow functional studies on other economically important thrips and insects more broadly.S1 Table(DOCX)Click here for additional data file.


Brassica napus (oilseed rape) is an economically important oil crop worldwide. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a threat to oilseed rape production. Because the flower petals play pivotal roles in the SSR disease cycle, it is useful to express the resistance-related genes specifically in flowers to hinder further infection with S. sclerotiorum. To screen flower-specific promoters, we first analyzed the transcriptome data from 12 different tissues of the B. napus line ZS11. In total, 249 flower-specific candidate genes with high expression in petals were identified, and the expression patterns of 30 candidate genes were verified by quantitative real-time transcription-PCR (qRT-PCR) analysis. Furthermore, two novel flower-specific promoters (FSP046 and FSP061 promoter) were identified, and the tissue specificity and continuous expression in petals were determined in transgenic Arabidopsis thaliana with fusing the promoters to β-glucuronidase (GUS)-reporter gene. GUS staining, transcript expression pattern, and GUS activity analysis indicated that FSP046 and FSP061 promoter were strictly flower-specific promoters, and FSP046 promoter had a stronger activity. The two promoters were further confirmed to be able to direct GUS expression in B. napus flowers using transient expression system. The transcriptome data and the flower-specific promoters screened in the present study will benefit fundamental research for improving the agronomic traits as well as disease and pest control in a tissue-specific manner. Brassica napus L. is an evolutionarily young allotetraploid species formed about 7500 years ago by natural hybridization between Brassica oleracea and Brassica rapa, followed by chromosome doubling [B. napus is an economically important oil crop all over the world. It not only provides vegetable oil for people, but also provides high quality fodder for animals. In addition, owing to its favorable agronomic properties, such as cultivation under different seasons and rotation with cereals, B. napus is preferred by farmers worldwide.doubling ,2. B. naSclerotinia sclerotiorum (Lib.) de Bary, a cosmopolitan pathogen of many economically important crops. As a necrotrophic pathogen, it infects more than 600 plant species, including important oil crops such as oilseed rape, soybean, and sunflower [However, Sclerotinia stem rot (SSR) is destructive to oilseed rape production. SSR is caused by unflower ,4,5,6. Sunflower .S. sclerotirum causes severe yield losses in oilseed rape in the field [Numerous studies have shown that flower petals play vital roles across the whole infection cycle of SSR, by supplying nutrition for ascospore germination and hyphae development ,9,10. AsS. sclerotirum infection and extension will likely be effective.Genetic engineering of crops, where resistant genes are introduced to improve crops resistance against disease, is a rapid breeding approach . The cauArabidopsis thaliana PISTILLATA (PI) and APETALA3 (AP3) are B-class organ identity genes in the ABC model, which are required for petal and stamen development [PI promoter driven GUS expression only occurs in the petals and stamens in A. thaliana [B. napus homolog gene PISTILLATA-1 showed low flower specificity, under which GUS could also express in leaf and silique [BnQRT3 promoter drives GUS expressing in branch connective tissue between the pedicels and stem, besides in flowers [A. thaliana and B. napus are different, even though they all belong to Brassicacea. Consequently, it is necessary to identify new flower-specific promoters in B. napus per se. In this study, flower-specific promoters in B. napus were identified based on transcriptome data from 12 different tissues including root, stem, leaf, flower bud, and six dissected flower parts. Two novel strictly flower-specific promoters were cloned and experimentally confirmed by a GUS reporter gene expression system. The transcriptome data and the flower-specific promoters screened in the present study will benefit fundamental research for improving the agronomic traits as well as disease and pest control in a tissue-specific manner.To date, several flower-specific promoters have been reported and applied to research in the cut-flower industry. Most of these promoters are flavonoid synthesis-related gene promoters, and are used for altering corolla colors to create novel varieties in ornamental flowers, such as lily, petunia, and rose ,20,21,22elopment ,24,25,26thaliana . However silique . BnQRT3  flowers . The expB. napus line ZS11 were collected and used to construct libraries for RNA sequencing. After quality filtering, we obtained 18 million to 51 million clean reads for each tissue library (SRA accession: PRJNA474576). Through mapping to the B. napus genome database, 67.85 to 87.88% of the clean reads could be mapped. Over 12 million to 26 million unique reads were obtained, which occupied 51.02 to 75.54% of the mapped data  exhibited specific and high expression in flower buds (FSP102) were highly expressed in BP or WP, while only FSP046 and FSP061 were highly expressed in BP and WP at the same time. In addition, the expression levels of the four candidates  in the stamen were relatively high.Thirty of 249 flower-specific candidates, most of which specifically expressed in BP or WP, were selected for quantitative real-time transcription-PCR (qRT-PCR) analysis. In the first round of detection, we defined flower-specific genes as those with expression values in the flower buds that were at least three times higher than that in other tissues . Six genwer buds a. Then, wer buds b. To scrFSP046 and FSP061) from the ZS11 cultivar were cloned and sequenced. The cis elements in these two promoter regions were analyzed using the PlantCARE online tool; and the results are illustrated in FSP061 promoter sequence, but none are identified in the FSP046 promoter sequence. There are several MYB, and MYC elements located in both promoter regions. Many cis elements related to light responsiveness, such as Box 4, I-box, TCT-motif, and GT1-motif, are found in both FSP046 and FSP061 promoters.To detect the flower-specificity, about 3.5 Kb of upstream sequences of two flower-specific candidate genes (FSP046 and FSP061 gene were fused to the GUS reporter gene (FSP046::GUS and FSP061::GUS) and transformed into A. thaliana. Histochemical staining was detected during the two-leaf, six-leaf, and flowering stages of T2 generation plants by histochemical assay  according to the P-box consensus sequence [B. napus. In future studies, GUS activity of different deletions of the two promoters will be implemented to find novel flower-specific motifs, and this method will also be used to identify the core regions of FSP046 and FSP061 promoters.In present study, both vulgaris , chsA ofa hybrid  and chs s hybrid . Howeversequence . TherefoAtPI and AtAP3 promoters also drive GUS expressing in petal and stamen [XY355::OvPAP2 transgenic B. napus plants exhibited red petals and stamen [InMYB1 promoter) driving GUS protein expression in petals distinctly and uniquely in A. thaliana [S. sclerotiorum for apetalous winter oilseed rape [FSP046 and FSP061 promoters are suitable to apply in SSR disease control in a flower-specific manner.From d stamen ,27. Oilsd stamen , while Xd stamen . It was thaliana . Young aeed rape . ConsequA. thaliana, both of the GUS gene expression level and protein activity in FSP046::GUS transgenic plants were higher than that of FSP061::GUS in flowers  could be stained by GUS reaction buffer  was a major pest at the inflorescence stage [The multiple functions of oilseed rape  are heavily researched. Many regions in China have constructed large-scale oilseed flower sightseeing tourism, which has become an important part of the local economy . To add ce stage . In recece stage ,46,47. Hce stage . Our floB. napus line ZS11 were obtained from Key Laboratory of Biology and Genetic Improvement of Oil Crops at OCRI and sown in October in the field located in Wuhan , Hubei province, China. Hubei province is the major production area for winter rapeseed planting in the middle and lower Yangtze River. The trial management followed standard breeding field protocols. Tissue samples were collected at the full-bloom stage in the following spring. Root, stem, leaf, flower bud, little siliques (about 4 cm long), and dissected flower parts including blossomy petal , wilting petal , pistil, stamen, sepal, ovule, and pericarp were collected from 15 plants in the same developmental stage in the same test plot.Seeds of Total RNA was extracted from different tissues using TRIzol reagent . Poly-A-containing mRNA was isolated from the total RNA using Oligotex mRNA Mini Kit  according to the manufacturers’ instructions. RNA libraries with insert sizes of 250 bp were constructed for each tissue sample and sequenced on an Illumina HiSeq 2000 platform  at BGI Co. Ltd. The libraries were sequenced for paired-end reads of 150 bp.B. napus line Darmor-bzh  [The raw reads were filtered to remove adapter sequences, low-quality reads, and reads containing poly-N using the tools from the NGS QC Toolkit (v2.3) . The deranapus/)  using Toanapus/) . The mapanapus/) .S. sclerotiorum tending to infect the plant petals primarily in the SSR disease cycle, the genes with FPKM values ≥15 in BP or WP were finally selected as flower-specific candidates. GO functional annotation and classification of the flower-specific candidate genes were conducted in the PlantTFDB 4.0 online tool (http://planttfdb.cbi.pku.edu.cn/) with the threshold of p-value ≤ 0.01 [http://www.ehbio.com/ImageGP/).Tissue-specific genes were screened and calculated using the threshold of FPKM value >2. We defined flower-specific genes as genes with threshold FPKM values ≥15 in either flower tissues , while the FPKM values were <1 in all of the other tissues . Furthermore, according to the trait of e ≤ 0.01 ,52. The B. napus β-actin gene (AF111812) was used as a reference standard. The relative expression was calculated using the 2Ct−∆∆ method [To verify the tissue specificity of flower-specific candidate genes, qRT-PCR primers of flower-specific candidate genes were designed . Primer t method .B. napus line ZS11 genome [http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [About 3.5 Kb sequences upstream of the start codon ATG were selected, and the primers  were des1 genome . To anale/html/) .CaMV 35S promoter using the ClonExpress II One Step Cloning Kit . The primer pairs are listed in Agraobecterium tumefaciens GV3101 competent cell . A. thaliana transformation was performed using the floral dipping method [B. napus, a syringe-press method was used for Agro-infiltration [Agrobacterium cells were harvested and adjusted to OD600 2.0. The resuspension solutions were put into a 50 mL syringe, and then, 10–15 flowers from B. napus line Westar were added into the solution. Positive pressure was used for 30 s, and the inoculated flowers were placed into wet box for preserving moisture. Forty-eight hours later, the flower tissues were collected for GUS staining. Three independent biological repeats were performed.To construct the promoter::GUS reporter system, flower-specific gene promoters were cloned into pBI121 at HindIII and BamHI sites to replace the g method . For traltration . BrieflyA. thaliana seedlings, organs, and tissues from different T2 generation lines and flowers of B. napus were infiltrated in GUS reaction solution  for 12 h at 37 °C in the dark. Several washes of 70% ethanol were performed to stop the GUS reaction and remove chlorophyll at room temperature. Then the stained tissues were observed under an anatomical lens .Histochemical detection of GUS staining was performed as described by Jefferson et al. . TransgeA. thaliana tissues was measured according to Jefferson et al. [® Explorer Multimode Microplate Reader . The GUS enzyme activity was calculated as picomoles of 4-MU produced per milligrams of protein per minute. The GUS gene expression in different tissues was examined by qRT-PCR analysis. The A. thaliana actin gene (AT3G18780) was used as a reference gene. The used primers were listed in GUS activity from different transgenic positive n et al.  by measun et al. . FluoresB. napus, which provide a foundation for the expression patterns of gene family studies. Moreover, based on the RNA sequencing data, other tissue-specific promoters (such as in the root and stem) could also be screened using similar procedures, which could be used for disease and pest management in a tissue-specific manner, for example with clubroot disease and aphids. Therefore, the transcriptome data present in this study will provide practical guidance for other studies. In conclusion, the transcriptome data and the flower-specific promoters screened in the present study will benefit fundamental research and disease and pest control in a tissue-specific manner.In the present study, we sequenced the transcriptomes of 12 different tissues from


Aeromonas veronii is a gram-negative species abundant in aquatic environments that causes disease in humans as well as terrestrial and aquatic animals. In the current study, 41 publicly available A. veronii genomes were compared to investigate distribution of putative virulence genes, global dissemination of pathotypes, and potential mechanisms of virulence. The complete genome of A. veronii strain ML09-123 from an outbreak of motile aeromonas septicemia in farm-raised catfish in the southeastern United States was included. Dissemination of A. veronii strain types was discovered in dispersed geographical locations. Isolate ML09-123 is highly similar to Chinese isolate TH0426, suggesting the two strains have a common origin and may represent a pathotype impacting aquaculture in both countries. Virulence of strain ML09-123 in catfish in a dose-dependent manner was confirmed experimentally. Subsystem category disposition showed the majority of genomes exhibit similar distribution of genomic elements. The type I secretion system (T1SS), type II secretion system (T2SS), type 4 pilus (T4P), and flagellum core elements are conserved in all A. veronii genomes, whereas the type III secretion system (T3SS), type V secretion system (T5SS), type VI secretion system (T6SS), and tight adherence (TAD) system demonstrate variable dispersal. Distribution of mobile elements is dependent on host and geographic origin, suggesting this species has undergone considerable genetic exchange. The data presented here lends insight into the genomic variation of A. veronii and identifies a pathotype impacting aquaculture globally. Aeromonas veronii is a Gram-negative, rod-shaped, mesophilic, motile bacterium in the Aeromonadaceae family. It is widespread in aquatic environments across the globe . Alo. AloAero animals , and a n animals , tilapia animals , sea bas animals , channel animals , and rai animals . Clinica animals , 10. It  animals –13.A. hydrophila is associated with significant economic losses in the Chinese cyprinid fish industry [A. hydrophila has also been attributed to catastrophic losses in the catfish industry in the southeastern United States since 2009 [A. hydrophila pathotype has a Chinese origin [A clonal pathotype of industry . An emernce 2009 . Phylogee origin .A. veronii causes disease in fish worldwide. A. veronii has been reported from disease outbreaks in cultured channel catfish in China [A. veronii might contribute to ulcerative syndrome in Chinese longsnout catfish (Leiocassis longirostris) [Aeromonas strains from different species are sometimes more virulent than either strain alone [A. veronii is often reported as a co-infection with other Aeromonas species. An A. veronii-A. sobria complex was identified from soft shell disease in turtles [A. veronii-A. jandai complex was reported from Nile tilapia [Similarly, in China  and tilain China . It is hrostris) . Interesin alone . In agre turtles , while a tilapia .A. veronii genomes, revealing considerable genetic exchange within the species and geographic dissemination of strain types. We also report the genome sequence of A. veronii strain ML09-123 isolated from diseased channel catfish in a commercial aquaculture pond in the southeast U.S., and we identified that it shares high identity with an isolate from Chinese aquaculture. Comparative virulence of A. hydrophila ML09-119 [A. veronii ML09-123 in catfish confirmed that this clonal A. veronii pathotype has potential to cause disease in aquaculture. This work provides novel insights into the pathogenicity and epidemic potential of A. veronii in fish as well as genome variability within the species.Here we report a comparison of 41 publicly available ML09-119  and A. vAeromonas veronii ML09-123 and A. hydrophila ML09-119 isolates were recovered from disease outbreaks from commercial catfish farms in Alabama and maintained in the archival collection of the Mississippi State University College of Veterinary Medicine. All A. veronii genomes , was sequenced using barcoded Illumina libraries prepared using a Nextera DNA Sample Prep Kit . Paired sequences were obtained from an Illumina GAIIx sequencer using 150 bp read length  . Reads were screened using the “trim sequences” option of CLC Workbench version 6.5.1. (CLC Bio). Adaptors were removed, low quality sequences were removed, and contig creation and de novo assembly were conducted using CLC Workbench and Sequencher version 5.4 (Gene Codes Corporation). For annotation, the draft genome was submitted to RAST [ to RAST  and NCBICore- and pan-genome analyses were performed using EDGAR 2.0  employing a generic orthology criterion based on BLAST score ratio values (SRV). BLAST scores were normalized in relation to the best hit possible. Based on the distribution of SRVs in the dataset, cutoffs were estimated as described . The numA. veronii genomes, a phylogenetic tree was constructed based on the complete core genome of 41 A. veronii genomes  for subsystem categorization and comparison [The genomes of ML09-123 and 40 other mparison . For annRAST annotated protein files were submitted to MacSyFinder , 31, whiA. veronii genomes were identified through searches of the Virulence Factors Database (VFDB) [http://www.mgc.ac.cn/VFs/download.htm, and BLAST searches were conducted with all predicted protein files using CLC Genomic Workbench (version 6.5.1). Only E-values <1*10−50 were deemed significant. Results were processed with a custom Python script to extract matches with virulence genes. Hits present in almost all (N-2 or more) or almost none (2 or fewer) strains were excluded from analysis, and a binary strain 207 virulence gene matrix was constructed with 1 indicating presence and 0 indicating absence of a match with a virulence gene within a strain. The R library pheatmap [A. veronii virulence factors in metabolic pathways were analyzed by Blast2GO [Putative virulence factors encoded in e (VFDB) . Full dapheatmap  was usedBlast2GO .A. veronii genome to ISsaga [Insertion elements were determined by submitting nucleotide files of each o ISsaga  using thA. veronii genomes was determined using PHASTER (PHAge Search Tool Enhanced Release) [The presence of prophages in Release) . NucleotRelease)  to identIdentification of integrons and their components in the genomes was performed by IntegronFinder .A. veronii genomes were determined by CRISPRfinder [mandatory, accessory, forbidden) defined as follows: mandatory elements are identifiable and ubiquitous; accessory elements could be essential but not identifiable. If the identified element is partly homologous, it is considered a forbidden element.CRISPR-Cas systems and their distribution in PRfinder  and MacSPRfinder . The fol−10, and duplicates were removed.Because the majority of the genomes are not closed, contig files were concatenated, and concatenated nucleotide files were submitted to CARD (Comprehensive Antibiotic Resistance Database)  to perfoAll fish disease challenges were conducted in compliance with protocol #17–288 approved by the Mississippi State University Institutional Animal Care and Use Committee (IACUC). The approved protocol included humane endpoints, and when morbid fish met established criteria, they were immediately euthanized by immersion in tricaine methane sulfonate (MS-222). Criteria for euthanasia were loss of balance, hanging at the water surface, or non-responsiveness to external stimuli. Some of the fish died during the study as a result of the experimentally induced systemic infection due to its rapid progression. All personnel on this experiment received IACUC-approved trained in animal care and welfare by the University Laboratory Animal Veterinarian.A. hydrophila ML09-119 [A. veronii ML09-123 were tested using a channel catfish challenge model. Briefly, 12 month-old channel catfish  reared indoors for disease research at Mississippi State University were stocked into twenty-seven 40-liter flow-through tanks (10 fish/tank) and acclimated for four days. Tanks were assigned randomly to two treatment groups (A. veronii and A. hydrophila) and four doses  with three replicate tanks for each dose. A negative control group was also included. Water temperature was maintained at 32°C (±2) during the experiments. Fish were fed to satiation twice daily and monitored three times daily for morbidity and mortality. On the day of challenge, fish were anesthetized by immersion in tricaine methane sulfonate (MS-222), and each pre-determined dose was administered intraperitoneally (IP) in 0.1 ml volume. Fish were monitored twice daily, and mortalities were recorded for a total of seven days. The experiment was terminated when no fish mortality was observed for three consecutive days. Mean percent mortalities for each dose and treatment group were calculated and arcsine transformed. The mean transformed percent mortality for fish challenged with four doses for A. hydrophila and A. veronii was compared against sham challenged fish using the Student's t-test (p < 0.05).The relative virulence of ML09-119  and A. vA. veronii strains used in this study and their genome features are listed in A. veronii genomes and 2855 genes in the core genome. The extrapolation of the core genome size  U.S. dairy cattle isolates and Greece surface sediment isolates (strain pamvotica), 2) U.S. strain ML09-123 and China strain TH0426 (catfish isolates), 3) U.S. human isolates  and China pond sediment isolate B565, and 4) U.S. surface water and Germany surface water isolates. The remaining A. veronii isolates did not fit into these or other subgroups. These findings were confirmed and supported by Average Nucleotide Identity (ANI); ANI values in conserved branches (genetic subgroups) were above 99.91% , and unknown source isolate LMG 13067 from the U.S. , entF, rmlC, farB, mprA, phoQ, and mshD are the second most prevalent (shared by 38 A. veronii genomes), and regX3 and wbfY genes are shared by 37 A. veronii genomes. The zot toxin gene is present in only six A. veronii genomes, including strains ML09-123 and TH0426. Some of the T6SS elements such as AHA_1837  are present only in two strains (ML09-123 and TH0426).Two hundred seven putative virulence genes were identified, representing 29 categories. Secretion systems were the most common category (68 genes), followed by adherence (56 genes), immune evasion (23 genes), antiphagocytosis (11 genes), iron uptake (7 genes), and toxins (4 genes). The represented categories are shown in A. veronii genomes carry insertion elements. The number of insertion elements per genome varies from 16 to 163. Strain AVNIH1 has the highest number of insertion elements, whereas strain A29 carried the least number of insertions. Insertion families and subgroups IS1595-subtype-ISPna2, IS51, IS3, IS2, IS481, IS4, IS903, and ISL3 are encoded by a majority of the evaluated A. veronii genomes  do not carry any type of phage regions, whereas the remaining genomes have at least one type of identified phage element. The general G+C content for phage elements identified within elements .A. veronii genomes carry questionable CRISPR regions. Cas systems are divided into three categories , with these three categories further subdivided into 10 different subcategories . Only a handful of A. veronii genomes encode these elements , while the remaining genomes do not carry Cas elements  or none of the integron regions (Only two strains (AVNIH1 and Ae52) encode complete integron regions. The other  regions .A. veronii genomes had relatively similar resistome profiles. The AVNIH1 genome possessed the largest number of antimicrobial resistance elements. Antimicrobial resistance elements encoded by channel catfish isolate ML09-123 and Chinese channel catfish isolate TH0426 were consistent with the profiles from most of the evaluated A. veronii genomes. Antimicrobial inactivation genes imiH, imiS, and cphA derivates were conserved across the 41 A. veronii genomes , 105 CFU/fish (36.67%), 106 CFU/fish (86.67%), and 107 CFU/fish (93.33%). Virulent A. hydrophila strain ML09-119 resulted in no mortalities at 104 CFU/fish and 105 CFU/fish, but it caused 90% and 100% mortalities at 106 CFU/fish and 107 CFU/fish, respectively. Results are shown in A. veronii strain ML09-123 from diseased catfish in the U.S. aquaculture industry. Comparative genomics analysis suggests that ML09-123 and Chinese isolate TH0426 have a common origin. By the injection route of exposure, A. veronii strain ML09-123 is virulent in catfish and is a potential pathogen that could impact U.S. aquaculture. However, experimental infection by injection bypasses the normal mucosal barriers, so this trial does not address the potential necessity for A. veronii to require predisposing conditions to cause infection in catfish under standard aquaculture conditions.In the current study, we sequenced the genome of A. veronii ML09-123 genome falls with respect to the publicly available A. veronii genomes. Four discrete groups were identified with a high degree of genomic homology (>99%). Interestingly, members within the groups have disparate geographic origins. Core-genome based phylogenetic analysis was consistent with and supported genetic groupings based on ANI calculations.ANI calculations can be used for an accurate digital investigation of bacterial systematics . As suchA. veronii clades and strains. However, the core genome size (2272 genes) relative to the average number of protein coding genes for A. veronii (3927 genes) is 70.6%. Thus, the species has a relatively large set of core functions with highly variable sections of genome that potentially enable different environmental adaptations.Pan/core genome analysis revealed a relatively low proportion of core-genome genes relative to pan-genome genes (30.9%), indicating a substantial amount of gene acquisition in Based on SEED subsystem analysis, the Chinese catfish isolate TH0426 and U.S. catfish isolate ML09-123 carry the most abundant elements in the category “phages, prophages, transposable elements and plasmids” . Thus, bA. veronii genetic subgroups based on ANI and core genome comparison ..A. veronA. veronii genomes encode the T2SS system, which is known for exporting hydrolytic enzymes that are important virulence factors of A. hdyrophila [A. veronii colonize the gut of leeches; in particular, export of hemolysin by T2SS is an important step for initial colonization of the leech gut [A. veronii, suggesting A. veronii strains rely heavily on the T2SS. T2SS components are similar to Tad pilus and T4P, especially in their structural components [A. veronii genomes.Similarly, all 41 yrophila , 50. T2Seech gut  Fig 5)..A. veronmponents , 53. T4Pmponents . All theA. veronii genomes encode the mandatory genes for flagellum. Interestingly, eleven of the A. veronii genomes do not encode T3SS  , and it A. veronii genomes encode the T6SSi system, which is well-known for contributing to virulence and the ability to interact with environment, host, and competitor bacteria [A. hydrophila, T6SS contributes to virulence and translocates the effector protein Hcp into eukaryotic cells, which can modulate activation of host immune cells [tssH, also known as clpV) is conserved across all the evaluated A. veronii genomes (except strain X11). TssH is an ATPase implicated in the recycling of other T6SS components, TssB/TssC, which are part of the tubular sheath [tssH is present in A. veronii genomes that lack genes encoding core T6SS components, TssH may have another important role. In our analysis, we could not identify a pattern for presence of this system based on genetic subgroups, geographic location, or host. However, presence of T6SS in A. hydrophila genomes varies based on the geographic location [There are three type of T6SS: T6SSi, T6SSii, and T6SSiii. The majority of the bacteria . In A. hne cells , 66. Intr sheath , 68. It r sheath . Howeverlocation .Actinobacillus actinomycetemcomitans [A. veronii genomes (30 out of 41) do not encode this system. Interestingly, all the genomes carry at least two, some as many as four, copies of tadZ, which encodes one of the core components of TAD system. While functionality of this component remains unclear, it is thought to be peripherally linked with the inner membrane [The Tad system is essential for colonization, biofilm formation, and virulence of some species . For exacomitans . The majmembrane .Aeromonas spp. are important pathogens in humans and other organisms. Surface polysaccharides, iron binding systems, exotoxins and other extracellular enzymes, secretion systems, adhesins, and flagella are some of the main factors contributing to Aeromonas virulence [A. hydrophila and A. veronii-Aeromonas sobria groups contain more virulence genes than the Aeromonas caviae-Aeromonas media group, suggesting that A. hydrophila and A. veronii-A. sobria may be more adapted to a pathogenic lifestyle [A. veronii isolates from diseased Gibel carp have some variation in the virulence genes they carry, but both encode and express several secreted enzymes [A. veronii genomes, followed by adherence and immune evasion . U.S. catfish isolate ML09-123 and China catfish isolate TH0426 have different numbers of bacteriophage elements, but they share one intact phage region. We did not identify a conserved phage element present in all 41 A. veronii genomes , which are separated from each other by spacers . They pr regions .intI) and its promoter region (PintI) and 2) attachment site of the integron (attI) and constitutive promoter (Pc) that integrates gene cassettes at attI. For a functional integron, only these core elements (intI gene and attI integration site) are required [attC genes, which mediate integration into integrons. Complete integron elements include integrase and at least one attC region. Incomplete integrons include ln0 elements that consist of integrase without attC sites and clusters of attC lacking integron-integrase (CALIN) [A. veronii genomes either carry only one, two, or none of the CALIN elements in their genome.Integrons are a major type of genetic element resposible for spread of antibiotic resistance genes. They consist of two main components: 1) integron-integrase (required . Gene ca (CALIN) , 81–83. A. veronii genomes we analyzed, which could be the result of encoding three integrons. Our antimicrobial resistance analysis showed a majority of the putative antimicrobial resistance elements are shared by all the A. veronii genomes with some exceptions  is an important cause of MAS outbreaks in catfish aquaculture in the southeast U.S. By the experimental injection route of exposure, A. veronii strain ML09-123 had similar virulence as vAh strain ML09-119. One important difference is that ML09-119 did not cause any mortality at doses of 104 and 105 CFU/fish, while ML09-123 caused a dose response that correlated with mortalities at the intermediate doses  larvae is also controlled by quorum sensing [Virulent te doses . This suhila SSU . Virulen sensing .A. veronii genomes analyses shows that A. veronii strain ML09-123 is highly similar to China strain TH0426. This observation was confirmed by ANI, core genome comparison, and subsystem category distribution . Our results confirm that A. veronii is a potential pathogen in Ictalurus catfish and that its virulence by the experimental injection route is similar to vAh. Thus, strains ML09-123 and TH0426 appear to be inherited from a common ancestor strain and affect aquaculture in both China and the U.S. We also found that pan genome is still open and the core genome may reduce as further strains are added to the repertoire. However, approximately 30% of A. veronii genomes show considerable variation, particularly in putative virulence genes, which contributes to a relatively large pan-genome for the species.In conclusion, our comparative S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)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 Fig(DOCX)Click here for additional data file.


H. pylori infection and eradication cause perturbations of the gut microbiome. The gut microbiota has been identified as a potential contributor to metabolic diseases. We evaluate whether these alterations in intestinal microbiota composition produced by H. pylori infection and its posterior eradication with antibiotic treatment could be associated with glucose homeostasis in metabolically healthy subjects.H. pylori and 20 control subjects were recruited. The infected subjects were evaluated before and two months after eradication treatment . The microbiota composition in fecal samples was determined by 16S rRNA gene (V3-V4) sequencing using Illumina Miseq.Forty adult patients infected with H. pylori eradication) showed a decreased bacterial richness and diversity with respect to controls. There was an improvement in glucose homeostasis in subjects two months after H. pylori eradication treatment. Changes in the amount of Rikenellaceae, Butyricimonas, E. biforme, B. fragilis, and Megamonas were inversely associated with changes in the glucose level or related parameters (Hb1ac) in H. pylori eradication subjects.Patients (pre- and post-H. pylori infection and eradication with antibiotic treatment causes alteration of the human gut microbiome. The increase in SCFA-producing bacteria and glucose-removing bacteria, specifically members of Megamonas, Rikenellaceae and Butyricimonas, has been related with an improvement in glucose homeostasis after H. pylori eradication with antibiotic treatment. Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa of humans and non-human primates  -3’ and 5'GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[GACTACHVGGGTATCTAATCC]-3’. Each 25 μL of polymerase chain reaction (PCR) reaction holds 12.5ng of fecal genomic DNA as template, 12.5 μL of Master Mix (2x KAPA HiFiHotStartReady Mix) and 5 μL of 1μM of each primer.The PCR products were checked using electrophoresis in 2% (w/v) agarose gels. A bioanalyzer  was used to verify the size of the PCR product.The fecal bacterial microbiota composition was determined using tag-encoded16S rRNA gene Miseq-based  high-throughput sequencing. The 16S rRNA V3-V4 amplicon (amplicon size ~460bp) was amplified by polymerase chain reaction (PCR)  using the universal primers reported by Klindworth et al.  fused wiAMPure XP beads  were used to purify the free primers and primer dimer species in the amplicon product. Dual indices and Illumina sequencing adapters were attached to sequence the amplicons, using the Nextera XT Index Kit  and purified the amplicon again using AMPure XP beads . Before sequencing, DNA concentration of each PCR product was determined using a Qubit Fluorometric double-stranded DNA assay (Thermo Fisher Scientific) and Bioanalyzer DNA 1000 chip to verify the size . The amplicons from each reaction mixture were pooled in equimolar ratios based on their concentration. The sample pool (4nM) was denatured and diluted following Illumina guidelines. Paired-end sequencing of amplicons was conducted on the Illumina MiSeq platform using the v3 kit generating 2 × 301 nucleotide reads .The merged paired-end reads were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) tool  . Operati1c levels. For regression and correlation analysis, the variables AUC and HbA1c were expressed as % of change, as well as the OTU differential. Statistical significance was set at P<0.05, reported by the conservative false discovery rate (FDR)-corrected p-value for multiple comparisons or p-value, as appropriate.The statistical analysis was performed with SPSS 22.0  and QIIME .The data were expressed as mean ± standard deviation. In order to check changes in the relative abundance (%) of operational taxonomic units (OTUs) and in clinical, biochemical and anthropometric variables between groups, the Wilcoxon’s signed-rank test (for paired samples) and the Mann-Whitney U test (for independent samples) were used, whereas to check qualitative changes in OTUs (presence/absence) between groups, the G-test of independence (g_test) was used. Alpha diversity between different groups of the samples was assessed by non-parametric two-sample t-test . The analysis of similarity (ANOSIM) statistical test was performed via QIIME (compare_categories.py—method anosim) to test the statistical significance between groups. Spearman’s correlation coefficient was also used to examine the relationship between the OTUs and biochemical variables. A multivariate regression analysis was performed to identify individual changes in OTUs as independent predictors for changes in the AUC and HbAH. pylori eradication treatment, as well as those of the control subjects, are depicted in The anthropometric and biochemical variables of the patients before and after H. pylori patients and control subjects were balanced according to age and sex. No differences were found in anthropometric parameters such as BMI and waist circumference or biochemical parameters such as glucose, insulin, HOMA.IR, HOMA-β, triglycerides, and cholesterol levels. However, the HDL-cholesterol level significantly increased after H. pylori eradication therapy, while the LDL-cholesterol level was lower in controls than in patients before H. pylori eradication treatment.Regarding the dietary assessment, no statistically significant differences were observed in the comparisons of micronutrients and macronutrients between patients and controls (p> 0.05) (data not shown).1c are shown in H.pylori eradication patients are depicted in H. pylori eradication patients (p = 0.01) .There were no statistically significant differences in AUC and HbA1c between patients with infection and controls .Fasting plasma glucose concentrations and HbA = 0.01) . There wA total of 13,747,554 high-quality sequences and 59,614 OTUs were obtained from the total samples, although samples were rarefied to 34,208 sequences per sample, which corresponded to 85% of the lowest number of quality reads obtained from any individual sample in the dataset. Moreover, in order to increase the statistical power, OTUs that were not found in at least five different samples, were excluded from the analysis. These reads/OTUs were assigned to 12 phyla, 49 families, 75 genera and 42 different species.H. pylori patients differed significantly . Taking into account the abundance of the bacteria, a better explanation was observed as the percentage of variance explained was higher , indicating a clear effect of this factor on the ecological diversity of the groups. Delving further into the results, no differences were observed between the control and pre-treatment groups and pre- and post-treatment patients , while a significant difference was found between the control and post-treatment fecal communities , indicating a clear influence of the antibiotic treatment for H. pylori on the fecal ecology of the patients . Within the H. pylori patients, the eradication treatment affected richness (p = 0.041), indicating a decrease in the number of registered OTUs, and evenness was almost statistically significant (p = 0.051)  and Shannon (Diversity).As expected, control subjects showed the greatest diversity and richness, showing statistical differences with respect to the = 0.051) .H. pylori eradication treatment and associated significantly with the variables of our study (AUC and HbA1c) are highlighted. In this manner, we observed a lower presence of the Rikenellaceae family, the Butyricimonas genus and E. biforme and a greater presence of B. Fragilis and Megamonas genus in patients after H. pylori eradication treatment.According to the qualitative assessment of the OTU discovered, a different occurrence (presence/absence) is depicted . Due to Firmicutes and Bacteroidetes. Actinobacteria, Proteobacteria and Verrucomicrobia contributed smaller proportions, between 1–5%  . Paraprevotella, Lachospira, Oscillospira, Dialister, Phascolartobacterium, Ruminonococcus, Sutterella, and Akkermansia contributed lower proportions, between 1–5% .Regarding the relative abundance of each OTU in the fecal samples collected, the dominant bacterial phyla were, as expected, een 1–5% . Bacteroae (>5%) , while teen 1–5% .With resH. pylori eradication treatment were found in our study , while the Bacteroidetes and Firmicutes phyla remained at similar levels in the control subjects  (Bacteroidetes/Firmicutes ratio did not significantly differ between patients before and after H. pylori eradication treatment (p<0.05), with greater values than control subjects in both cases . Moreover, we found a decrease in the relative abundance of Actinobacteria post-H. pylori eradication compared with pre-H. pylori eradication  and controls .Significant changes in relative abundance comparing controls and patients before and after ur study . Bactero = 0.82) . In thisActinobacteria phylum, we found that the H. pylori eradication treatment led to a significant decrease in the relative abundance of the family Bifidobacteriaceae, and especially in the genus Bifidobacterium and B. longum and B. adolescentis species. Firmicutes and Proteobacteria phyla also experimented a decrease after the H. pylori eradication treatment, specifically a decrease in the relative abundance of the family Streptococcaceae and the genus Streptococcus. Moreover, after the eradication treatment a decrease in the abundance of Turicibacteraceae and the genera Turicibacter, Ruminococcaceae and Oscillospira, as well as the family Oxalobacteriaceae and the genus Oxalobacter and O. Formigenes species, and the family Enterobacteriaceae were reported with respect to the control group , as well as with the proportion of HbA1c changes  in patients after H. pylori eradication treatment.In order to establish a possible relationship between the glucose metabolism status of the studied groups and their gut microbiota profiles, correlation studies were performed. Significant univariate correlations were found between changes in the amount of specific bacteria and the proportion of changes in the glucose AUC  and Butyricimonas  predicted the proportion of changes in the glucose AUC in patients after the eradication treatment. These results remained statistically significant even after correcting for age, sex and BMI.Multivariate regression analyses, for all the bacterial groups analyzed, were assessed. Only the changes in H. pylori eradication with antibiotic treatment produces specific bacterial changes associated with an improvement in glucose homeostasis and HbA1c levels in patients with normal blood glucose concentrations. HbA1c is an index of long-term glycemic control and a risk predictor used in the monitoring of diabetes. However, HbA1c levels are acquiring a big relevance also in apparently healthy subjects : Pre- vs. Post-H. pylori eradication; [b]: control vs. Pre-H. Pylori eradication; [c]: Control vs. Post-H. pylori eradication.(TIFF)Click here for additional data file.