Helicobacter bizzozeronii is one of seven species in Helicobacter heilmannii sensu lato that are detected in 0.17-2.3% of the gastric biopsies of human patients with gastric symptoms. At the present, H. bizzozeronii is the only non-pylori gastric Helicobacter sp. cultivated from human patients and is therefore a good alternative model of human gastric Helicobacter disease. We recently sequenced the genome of the H. bizzozeronii human strain CIII-1, isolated in 2008 from a 47-year old Finnish woman suffering from severe dyspeptic symptoms. In this study, we performed a detailed comparative genome analysis with H. pylori, providing new insights into non-pylori Helicobacter infections and the mechanisms of transmission between the primary animal host and humans.The canine Gram-negative H. bizzozeronii possesses all the genes necessary for its specialised life in the stomach. However, H. bizzozeronii differs from H. pylori by having a wider metabolic flexibility in terms of its energy sources and electron transport chain. Moreover, H. bizzozeronii harbours a higher number of methyl-accepting chemotaxis proteins, allowing it to respond to a wider spectrum of environmental signals. In this study, H. bizzozeronii has been shown to have high level of genome plasticity. We were able to identify a total of 43 contingency genes, 5 insertion sequences (ISs), 22 mini-IS elements, 1 genomic island and a putative prophage. Although H. bizzozeronii lacks homologues of some of the major H. pylori virulence genes, other candidate virulence factors are present. In particular, we identified a polysaccharide lyase (HBZC1_15820) as a potential new virulence factor of H. bizzozeronii.Helicobacter species. In particular, we propose the hypothesis that the high metabolic versatility and the ability to react to a range of environmental signals, factors which differentiate H. bizzozeronii as well as H. felis and H. suis from H. pylori, are the molecular basis of the of the zoonotic nature of H. heilmannii sensu lato infection in humans.The comparative genome analysis performed in this study increased the knowledge of the biology of gastric  Helicobacter pylori is established as the primary cause of gastritis and peptic ulceration in humans and has been recognised as a major risk factor for mucosa-associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinoma [H. pylori gastric Helicobacter species (NHPGH), have been observed [H. pylori gastritis, severe complications of NHPGH infection have been reported, and it is important to note that MALT lymphoma is significantly more frequent in NHPGH-infected patients compared to those infected with H. pylori [arcinoma . Howeverobserved ,3. Altho. pylori .H. pylori is transmitted from human to human. In contrast, NHPGH are zoonotic agents for which animals are the natural reservoir [H. heilmannii sensu lato has been proposed to be used to refer to the whole group of NHPGH detected in the human or animal stomach when only morphology and limited taxonomical data are available [H. heilmannii s.l. comprises seven described Helicobacter species: the porcine Helicobacter suis; the feline Helicobacter felis, Helicobacter baculiformis and Helicobacter heilmannii sensu stricto (s.s.); and the canine Helicobacter bizzozeronii, Helicobacter salomonis and Helicobacter cynogastricus [Helicobacter species are very fastidious microorganisms and difficult to distinguish from each others with conventional identification procedures. Due to difficulties in the isolation and identification of H. heilmannii s.l., its epidemiology in human infections remains unclear. Although development of specific isolation methods has allowed to identify several new species considered to be uncultivable , up tpatients -8.H. bizzozeronii, described as a new species in 1996 [H. bizzozeronii in dogs remains unknown [H. bizzozeronii appears to be associated with a lower pathogenicity than H. pylori or H. felis. Even in cases of heavy bacterial loading, the only pathological finding observed was focal apoptotic loss of gastric epithelial cells [H. bizzozeronii and gastric diseases in both naturally and experimentally-infected animals, H. bizzozeronii has been associated in both human cases with severe dyspeptic symptoms [H. bizzozeronii strain CIII-1 was isolated from antrum and corpus biopsies that revealed chronic active gastritis [H. pylori-infected patients. in 1996 . Virtual unknown ,10. Expe unknown  and gerb unknown  have sugal cells . Despitesymptoms ,8. MoreoH. pylori are available, and recently, the genomes of other gastric Helicobacter species were also published [H. heilmannii s.l., the genomes of the type strain of H. felis [H. suis [H. bizzozeronii human strain CIII-1, isolated in 2008 from a 47-year-old Finnish woman suffering from severe dyspeptic symptoms [H. bizzozeronii CIII-1 is the only human H. heilmannii s.l. strain for which the genome is available and constitutes a keystone in the comprehension of non-pylori Helicobacter infections and of the mechanisms of transmission between the primary animal host and humans. Moreover, although the role of H. bizzozeronii in human gastric disease is limited compared to H. pylori, both of the species persistently colonise the same niche and possibly exploit similar mechanisms to interact with the host and induce gastritis. Thus, a comparative genome analysis between H. pylori and the human H. bizzozeronii CIII-1 would expand our knowledge of the biology of gastric Helicobacter spp. and of the pathogenesis of human gastritis.Several genome sequences of ublished -16. Amon[H. suis , both issymptoms . To our H. pylori and the human H. bizzozeronii CIII-1 strain. We advance the hypothesis that the zoonotic nature of H. bizzozeronii, and, by extension of H. heilmannii s.l., is explicable from its high metabolic versatility, which probably supports the growth of this bacterium in a wide range of niches.In this study, we provide a detailed comparative genome analysis of H. bizzozeronii CIII-1 genome were previously described [Helicobacter species [H. bizzozeronii CIII-1 genome is provided in Table The general features of the escribed . Briefly species ,16 and iH. bizzozeronii CIII-1 CDSs is shown in Table Helicobacteracae, and 95% are similar to proteobacterial proteins. H. felis is the most closely related organism, covering almost 50% of the best matches, followed by H. suis (36%) and H. pylori (2.7%). However, 33% of the CDSs have a homologue from H. pylori within the 5 best BLAST matches. Among the Campylobacter spp., C. rectus is the closest to H. bizzozeronii  even though the homology is limited to the prophage region (see below). Matches to non-ε-proteobacteria are found within the Firmicutes (1.58%), γ-proteobacteria (0.9%), Bacteroides (0.6%) and also within the Eukaryota (0.9%).The taxonomy of the best BLAST hits for the H. bizzozeronii CIII-1 with the genomes of 10 human H. pylori strains to identify proteins that are unique to H. bizzozeronii as well as proteins found in H. pylori but lacking in H. bizzozeronii. The H. pylori core genome and H. bizzozeronii CIII-1 have 1,034 groups of orthologues in common, to which 1,052 CDSs from CIII-1 belong. Moreover, 412 groups are found in H. bizzozeronii CIII-1 and in at least two H. pylori strains, and 192 groups are common in CIII-1 and in at least one H. pylori strain. The H. pylori core genome contains 151 groups of unique orthologues while a total of 562 CDSs are unique to H. bizzozeronii CIII-1. Among the 562 unique H. bizzozeronii CDSs, a putative function was predicted for only 147 (26%). Each of these 147 CDSs was searched against the NCBI nr database using BLASTp to identify homologies with other H. pylori strains. A total of 59 out of the 147 CDSs showed a homology to proteins of at least one H. pylori strain, restricting the number of unique H. bizzozeronii CDSs with a predicted function to 88. Excluding the proteins associated either with bacteriophage or with plasmid replication, the functions of the majority of the unique H. bizzozeronii genes are linked to chemotaxis and metabolism  that is essential for acid acclimation and pH homeostasis, crucial mechanisms for the gastric colonisation. The urease gene cluster showed the same syntheny as in H. pylori. Unlike the other animal gastric Helicobacter species, H. felis and H. mustelae [H. bizzozeronii CIII-1 lacks the additional urease genes (ureAB2). Other essential mechanisms for the colonisation of the gastric mucosa participate in pH homeostasis [H. bizzozeronii CIII-1 contains the periplasmic α-carbonic anhydrase orthologue HBZC1_14670, which contributes to the urease-dependent response to acidity in H. pylori [H. bizzozeronii CIII-1 carries both the asparagine and glutamine deamidase-transport systems of AnsB (HBZC1_05200 and HBZC1_12160) plus DcuA (HBZC1_00140) and γGT (HBZC1_08080) plus GltS (HBZC1_14360) that contribute to periplasmic ammonia production in H. pylori and may have a possible role in the resistance to weakly acidic conditions and in the pathogenesis of gastritis [H. bizzozeronii CIII-1 that might also contribute to acid acclimation is the presence of two copies of a putative allophanate hydrolase (HBZC1_04550 and HBZC1_09470). This enzyme catalyses the second reaction of the two-step degradation of urea to ammonia and CO2 and is usually found adjacent to a biotin-containing urea carboxylase gene , which functions as an allophanate synthase from urea [Campylobacter species and H. felis, but their role in urea degradation has been demonstrated only in the environmental α-proteobacteria species Oleomonas sagaranensis [H. bizzozeronii the orthologue could contribute to the cytoplasmic degradation of urea in combination with urease. However, other functions, such as the degradation of aromatic compounds [As described for the i genome , the H. mustelae ,16, H. beostasis : H. bizz. pylori , but lacompounds , could nHelicobacter species [H. bizzozeronii CIII-1 harbours a large group of genes distributed widely in the genome, encoded by the fla, flg, flh and fli gene families, which are involved in the regulation, secretion and assembly of flagella [Motility is fundamental for colonisation of the mucosa by gastric  species , and, siflagella .H. bizzozeronii harbours five times more MCPs than H. pylori. Twenty MCPs, one containing a PAS domain, were detected in the H. bizzozeronii CIII-1 genome compared to the four predicted in H. pylori 26695 . The repertoire of the chemotactic signal pathways of a microorganism is correlated to the environments it can transfer, the hosts it colonises and the diseases it can cause . DespiteH. felis , indicatC. jejuni is a more versatile and metabolically active pathogen than the more specialised H. pylori. The metabolic versatility of C. jejuni enables the bacterium to survive in different environments and to colonise a variety of animal species, while the high specialisation of H. pylori is probably the result of a process of adaptation to the human stomach [H. pylori and H. bizzozeronii colonise similar niches, an important difference between these two species is the capability of H. bizzozeronii to move from a dog to a human host [H. bizzozeronii, which can be inferred from the genome of the human strain CIII-1, appears to be more flexible than that of H. pylori and could explain the zoonotic nature of this species.Understanding the metabolic properties of any pathogen is crucial to gain a full picture of the pathogenicity of related diseases. For example,  stomach . Althougman host . The cenH. pylori [H. bizzozeronii CIII-1 may use glucose as a source of energy through the pentose phosphate reactions and the Entner-Doudoroff pathway. However, in contrast to what was observed in H. pylori, H. bizzozeronii CIII-1 possess three genes organised in a single operon that are required for glycerol metabolism: glycerol-3-phosphate dehydrogenase , glycerol kinase  and a glycerol uptake facilitator protein . Glycerol-3-phosphate is a central intermediate in glycolysis and in phospholipid metabolism [As observed for . pylori ,29, eventabolism . The enzH. pylori [H. pylori [H. bizzozeronii CIII-1 genome contains several genes involved in the anaplerotic replenishment of oxaloacetate, succinate and α-ketoglutarate  carboxylase (HBZC1_05500) that may function in oxaloacetate synthesis. In addition, the genome harbours genes encoding enzymes potentially involved in the 2-methylcitric acid cycle  that produces succinate and pyruvate from the breakdown of propionate [gabDTP operon of E. coli but do not have orthologues in any of the H. pylori sequenced genomes. These genes are potentially involved in the production of succinic acid from the degradation of polyamines or γ-aminobutyric acid (GABA) [H. bizzozeronii trough the action of a glutamate decarboxylase (HBZC1_04360/HBZC1_04370) which does not have orthologue in H. pylori. Downstream of the putative H. bizzozeronii gabDTP operon, there are two gene homologues to the E. coli carbon starvation-induced protein  and L-2-hydroxygluturate oxidase . In E. coli, lhgO, located immediately downstream of csiD and upstream of the gabDTP operon, plays a role the recovering α-ketoglutarate, an intermediate in CAC, reduced by other enzymes [E. coli, all of these genes are regulated by the CsiR repression and σS induction acting on p csiDduring carbon starvation and the stationary phase [H. bizzozeronii CIII-1 lacks both of these transcriptional regulators but it contains a homologue of carbon-starvation regulator  that controls the H. pylori response to environmental stress [H. bizzozeronii.The absence of obvious homologues of succinyl-CoA synthetase indicates the presence of the alternative citric acid cycle (CAC), which has been described in . pylori   . The syn enzymes . In E. cry phase . H. bizzH. bizzozeronii CIII-1 harbours a cluster of 4 conserved genes (HBZC1_00410 to HBZC1_00460), annotated as acetophenone carboxylase subunits 1 to 4, which show a low homology with microbial hydantoinases. This group of genes is located near to the SCOT  and acetoacetyl-CoA thiolase  genes. A similar gene cluster, encoding a set of enzymes capable of metabolizing acetone to acetyl-CoA, has also been described in H. pylori  [H. pylori acxABC and the H. bizzozeronii acetophenone carboxylase gene cluster is very low (<30%), indicating that the H. bizzozeronii genes could be involved in an alternative pathway.Finally,  acxABC) . HoweverH. bizzozeronii appears to be highly branched and more complex than described for H. pylori , which could enable the bacteria to use formate as an electron donor, as observed in other ε-proteobacteria [H. bizzozeronii CIII-1 an extra possibility for substrate-derived electrons to be donated to the membrane-bound electron transport chain.In addition to hydrogenase HydABCDE (from HBZC1_10200 to HBZC1_10150) and other dehydrogenases in common with bacteria . In addiH. bizzozeronii requires oxygen for growth and is unable to survive under anaerobic conditions [H. pylori, H. bizzozeronii CIII-1 possesses only one terminal oxidase, ccoNOQP (from HBZC1_08510 to HBZC1_08540), which encodes a cytochrome cb-type enzyme, the major factor in aerobic respiratory metabolism [H. bizzozeronii CIII-1 could use as electron acceptors fumarate by a fumarate reductase system , as described in H. pylori [H. bizzozeronii could also utilize nitrate and nitrite as electron acceptors. This characteristic had been already shown in C. jejuni but not in H. pylori [C. jejuni system for the absence of NapL homologues and by the fact that it is not organised in a single operon  and a cytochrome-c nitrite reductase subunit c552 domain containing protein , suggesting that nitrate could be used in anaerobic respiration by this species. In addition to using nitrate and nitrite, H. bizzozeronii could also use S- or N-oxides as electron acceptors, as observed in other ε-proteobacteria [H. pylori a putative trimethylamine-N-oxide reductase is present . Although a homologue of the monoheme c type cytochrome (HBZC1_10800/Cj0265c) is absent, we cannot exclude that H. pylori may also be able to use TMAO/DMSO in respiration through a different pathway.nditions . As prevtabolism . In addi. pylori . However. pylori . Nitratebacteria . In factbacteria . In H. pH. bizzozeronii CIII-1 genome without clear homologues in H. pylori are: two copies of an NAD(P)H-dependant oxidoreductase (HBZC1_02100 and HBZC1_08240), a pyridine nucleotide-disulphide oxidoreductase (HBZC1_01010) and an aldo/keto reductase (HBZC1_08400). Although it is not possible to predict the functions of these genes, their presence increases the complexity of the electron transport chain of H. bizzozeronii and clearly differentiates this species from H. pylori.Other genes possibly involved in the electron transport chain that are present in the H. pylori [H. bizzozeronii expresses several defence mechanisms against oxidative stress. These include an iron-cofactored superoxide dismutase SodB (HBZC1_11160/11150), catalase enzyme KatA (HBZC1_12240), Cytocrome-c peroxidase (HBZC1_05800) and alkyl hydroperoxide reductase (HBZC1_01800). Moreover, because metal ions like iron and nickel favour the creation of reactive oxygen species (ROS), the detoxification of ROS needs to be coupled tightly to the control of intracellular metal concentrations, ensuring metal ion homeostasis by the regulation of metal uptake, export and storage [H. bizzozeronii contains all of the genes involved in nickel and iron uptake, storage and metabolism that have been described in other Helicobacter species [Like . pylori , becauseHBZC1_122, Cytocro species ,40.H. bizzozeronii is able to persistently colonise the stomach of both dogs [oth dogs  and humaoth dogs -8. ThereH. bizzozeronii CIII-1 genome encodes a total of 51 putative OMPs , the other eight H. bizzozeronii hof genes are organised in a single operon . This region is characterised by an anomalously low GC content and was identified as potential laterally acquired DNA by the Alien Hunter program [H. pylori proteome based on a heat-stable 50 KDa OMP [H. bizzozeronii OMPs belong to Hom Family 3, and a total of 5 putative efflux pump OMPs (Family 5) were identified. Moreover, 5 iron-regulated OMPs (Family 4) were detected in the H. bizzozeronii genome. Finally, the remaining 10 H. bizzozeronii OMPs are currently unclassified, including the putative VacA-paralog (HBZC1_11860/11850), a peptidoglycan-associated lipoprotein (HBZC1_14500), the membrane-associated lipoprotein Lpp20 (HBZC1_01250) and a putative protease (HBZC1_01500).Outer membrane proteins (OMPs) play important roles in the interaction with the host by mediating the adhesion to the mucosa and, therefore, the colonisation of the stomach and modulating the host immune response. Moreover, OMPs are essential for the general metabolism of the bacteria, preserving the selective permeability of the outer membrane to different substrates . The H. . pylori . Twenty ructures , studies gerbils ,43 and i gerbils  showed nc mucosa . Howeverc mucosa . Althoug program . Hof fam KDa OMP , but itsH. bizzozeronii CIII-1  antigens, is a primary host-interacting structure, and several lines of evidence indicate that it is involved in host-adaptation and virulence [H. pylori, H. bizzozeronii produces a predominantly low-molecular-weight LPS and does not apparently exhibit a molecular mimicry of Lewis and other blood group antigens [H. pylori LPS core antibodies reacted with the H. bizzozeronii LPS, indicating a shared epitope between these two species [H. bizzozeronii and are dispersed across the genome as they are described in H. pylori [H. bizzozeronii CIII-1 genome includes a putative α1,3-fucosyltransferase , a tandem of two putative galactosyltransferases  that are fragmented due to the presence of homopolymeric runs, a putative α1,3-sialyltransferase  and other putative glycosyltransferases that belong to GT-family 2 or 4 or are unclassified. Structural analysis of the H. bizzozeronii LPS have shown the presence of fucose [H. pylori. Although some of these genes have homologues in the H. felis and H. suis genomes [H. bizzozeronii.The communication between a microbial pathogen and its host occurs also due to the recognition of cell surface glycomolecules, including lipopolysaccharides (LPS), capsular polysaccharides and N- and O-glycoproteins, the structure of which depends of the presence and functional transcription of the genes encoding glycosyltransferases (GTs) . A totalirulence . Distincantigens . However species . Several. pylori . In addif fucose  and sial genomes ,16, the H. pylori are known to mediate pathogen-host interaction during infection [H. bizzozeronii, as a gram-negative bacterium, utilises the Sec or the twin-arginine secretion (Tat) systems to transport proteins into the periplasmic space and uses different approaches to move the proteins through the outer membrane [H. bizzozeronii CIII-1, the Sec system, which lacks a homologue to SecB, is dispersed through the genome, as it is in other Helicobacter spp. [H. pylori tatD (HBZC1_14750). Both of the systems recognise an N-terminal signal peptide of the protein that is required for translocation [H. bizzozeronii CIII-1 proteins were not identified. Several proteins that share a clear homology with excreted proteins in H. pylori were predicted to be located in the cytoplasm, indicating that the repertoire of secreted proteins in H. bizzozeronii could be considerably different from what predicted using PSORTb.The extracellular proteins of nfection . To expomembrane . In H. bter spp. ,53 and tlocation  ; the peptidyl propyl-cis,trans-isomerase (HBZC1_06110), involved in the TLR4-dependent NF-κB and in AP1 activation of macrophages [H. pylori as a carcinogenic factor [H. pylori [H. bizzozeronii CIII-1 that does not have homologues in any H. pylori is HBZC1_15820, encoding a polysaccharide lyase belonging to Family 8 , similar to other ε-proteobacteria [H. bizzozeronii. SSRs are subject to slipped-strand mutations, therefore inducing frameshift of the gene if the repeats are located in a protein-coding region or alteration of the gene expression when the repeats affect the promoter [H bizzozeronii CIII-1 genome, with 64 (the majority) located inside the coding regions. The frames of 54 genes are affected by the slipped-strand mispairing associated with SSRs. After the exclusion of essential genes which are unlikely subjected to phase variation by slipped-strand mispairing  we were able to identify a total of 43 potentially phase-variable genes  as a result of DNA recombination via natural transformation [H. bizzozeronii CIII-1 genome harbours five IS elements, with two belonging to the IS200/IS605 family and three to the IS607 family according to the semi-automatic annotation system provided by the ISsaga tool [orfA and orfB transposases. In addition, H. bizzozeronii CIII-1 contains 22 almost identical mini-IS elements of 232 bp with ~98% nucleotide identity uniformly distributed across the genome , replication A protein (HBZC1_17000) and the plasmid partitioning protein ParA (HBZC1_16910), appear originally have been plasmid-related features. These results suggest that a transposition event may have been responsible for the integration of a plasmid in the chromosome as already observed in C. jejuni [H. pylori [Large inserts of laterally acquired DNA containing functionally related genes are often referred to as genomic islands (GIs) and are typically associated with an increased virulence . The Isl. jejuni  and in H. pylori .H. bizzozeronii CIII-1 (FR871758). Among the 21 genes for which a putative function was predicted  for 4 days at 37°C in an incubator with a microaerobic atmosphere of 10% CO2 and 5% O2 . The high molecular-weight genomic DNA of H. bizzozeronii was extracted as described before [The human H. bizzozeronii CIII-1 was obtained as described previously [MluI optical map produced by OpGen Inc. . The sequences were deposited in the EMBL bank under the accession numbers FR871757 and FR871758.The genome sequence of eviously . BrieflyFor gene finding and automatic annotation, the sequences were uploaded to the RAST server . We furtH. bizzozeronii as well as the proteins found in H. pylori but missing in H. bizzozeronii, we determined the core genome of 10 H. pylori strains  using OrthoMCL with a BLAST E-value cut-off of 1.0 e-6 and an inflation parameter 1.5 [H. bizzozeronii CIII-1 genome, as previously described [H. bizzozeronii CIII-1 that did not cluster with any H. pylori proteins were considered to be specific to the species.To identify the proteins unique to eter 1.5 . We compescribed . BrieflyH. pylori [H. pylori OMPs and the phylogenetic relationships were analysed as described previously [We identified the putative outer membrane proteins using BLASTp against the OMP database  and a se. pylori  using a . pylori . For fureviously .H. bizzozeronii CIII-1 chromosome was searched for simple sequence repeats (SSRs) potentially involved in slipped-strand misparing using Msatfinder [The atfinder . The thratfinder . The lenThe regions in the genome possibly acquired by horizontal gene transfer events as well as genomic islands were predicted using Alien Hunter  and IslaACC: acetophenone carboxylase; AHD: allophanate hydrolase; NRS: nitrite reductase system; SNO: S- and N- oxidases; FDH: formate reductase system; GDH: glycerol-3-phosphate deydrogenase; PL: polysaccharide lyase; HYD: hydrogenase; NDH: NAD(P)H dehydrogenase; SDH/FR: succinate dehydrogenase/fumarate reductase; MQR: malate-quinone-reductase; LDH: lactate dehydrogenase; GLC: glycol oxidase; NAP: periplasmic nitrate reductase; MCP: methyl-accepting chemotaxis protein; CAC: citric acid cycle; PEP: phosphoenolpyruvate; TMAO: trimethylamine-n-oxide; DMSO: dimethyl sulfoxide; OMP: outer membrane protein; LPS: lipopolysaccharide; GT: glycosyltransferase; Tat: twin-arginine secretion system; MMR: mismatch repair system; SSR: simple sequence repeat; HGT: horizontal gene transfer; IS: insertion sequence; GI: genomic island; s.l.: sensu lato; s.s.: sensu stricto.TS performed the draft genome assembly and genome assembly validation and was responsible for the comparative genome analysis. PKK contributed to the data acquisition. MLH supervised the work at DFHEH. MR was responsible for the study design and coordination, contributed to the comparative genome analysis and wrote the manuscript. All authors read and approved the final manuscriptsupplementary tables. Table S1, Helicobacter bizzozeronii CIII-1 methyl-accepting chemotaxis protein; Table S2, Helicobacter bizzozeronii CIII-1 CDS involved in metabolisms and respiration; Table S3, Outer Membrane Proteins of Helicobacter bizzozeronii CIII-1; Table S4, Glycosyltransferases and genes involved in the biosynthesis of glycans in Helicobacter bizzozeronii CIII-1; Table S5, ComB system in Helicobacter bizzozeronii CIII-1; Table S6, List the CDS belonging to Helicobacter bizzozeronii CIII-1 genomic island; Table S7, List the CDS belonging to Helicobacter bizzozeronii CIII-1 plasmid pHBZ1.Click here for file


To the Editor: Scalp eschar and neck lymphadenopathy is a common clinical entity that most frequently affects women and children during spring and fall. It is usually caused by Rickettsia slovaca and R. raoultii. Typical clinical signs are a scalp lesion at the tick bite site and regional, often painful, lymphadenopathy. Acute disease can be followed by residual alopecia at the bite site (Dermacentor-borne necrosis-erythema-lymphadenopathy (both have been associated with R. slovaca); however, the most generic and all-inclusive term is scalp eschar and neck lymphadenopathy.R. massiliae belongs to the spotted fever group rickettsiae, is distributed worldwide, and is transmitted by ticks of the genus Rhipicephalus , and elevated erythrocyte sedimentation rate (43 mm/h). Ultrasonography of the neck confirmed the presence of numerous, enlarged, oval lymph nodes (maximum 17 mm) with hilar vascularity within normal limits. A scalp eschar biopsy sample and acute- and convalescent-phase (day 30) serum samples were sent to the Istituto Zooprofilattico Sperimentale della Sicilia.The patient was given doxycycline at 100 mg 2 times per day. Signs and symptoms began to improve 48–72 h later and gradually disappeared. Fever was gone after 3 days, and the other symptoms had regressed after 7 days.R. conorii was performed by microimmunofluorescence with the R. conorii/R. typhi IgG MIF Kit . Total DNA was extracted from blood and the eschar by GenElute Mammalian Genomic DNA Miniprep . To detect Rickettsia spp. DNA, we tested nucleic acids by PCR with a set of primers that amplify a 256-bp region of the gene encoding the 17-kDa antigen . DAMBE (http://dambe.bio.uottawa.ca/dambe.asp) and MEGA (www.megasoftware.net) software were used to obtain similarity percentages among analyzed sequences. To characterize Rickettsia spp., we used nucleotide sequence identity to reference strains  and ClustalW version 2.0.10 , 99% for the ompA gene (accession no. JQ480842), and 97% for the ompB gene (accession no. AF123714). Phylogenetic analysis .The presence of Rickettsia spp. Phylogenetic analysis of


Campylobacter jejuni is one of the most important zoonotic pathogens causing food-borne gastroenteritis and potentially more severe diseases such as reactive arthritis or Guillain–Barré syndrome. Crossing the intestinal epithelial barrier and host cell invasion by C. jejuni are considered to represent the primary reasons of gut tissue damage in humans and various animal model systems including monkeys, piglets, rabbits, hamsters and ferrets. C. jejuni is also able to invade underlying tissues such as the lamina propria, can enter the bloodstream, and possibly reach distinct organs such as spleen, liver or mesenteric lymph nodes. However, the molecular mechanisms as well as major bacterial and host cell factors involved in these activities are poorly understood. Various models exist by which the pathogen can trigger its own transmigration across polarized intestinal epithelial cells in vitro, the paracellular and/or transcellular mechanism. Recent studies suggest that bacterial factors such as flagellum, serine protease HtrA and lipooligosaccharide LOS may play an active role in bacterial transmigration. Here we review our knowledge on transmigration of C. jejuni as well as some other Campylobacter species, and discuss the pros and cons for the route(s) taken to travel across polarized epithelial cell monolayers. These studies provide fresh insights into the infection strategies employed by this important pathogen.Intact intercellular junctions and cellular matrix contacts are crucial structural components for the formation and maintenance of epithelial barrier functions in humans to control the commensal flora and protect against intruding microbes.  Campylobacter jejuni is a wide-spread Gram-negative bacterium living as commensal in the gut of most birds and domestic animals. However, C. jejuni is infectious for humans and consumption of contaminated food products is a major cause of human bacterial gastroenteritis, which may be responsible for as many as 400–500 million cases annually [C. jejuni infection varies from mild, non-inflammatory, self-limiting diarrhoea to severe, inflammatory, bloody diarrhoea that can continue for few weeks [C. jejuni infections can be also associated with the development of reactive arthritis and peripheral neuropathies, known as Miller–Fisher and Guillain–Barrè syndromes [C. jejuni infections, our present knowledge about the interplay between C. jejuni and its various hosts is still very limited. The availability of complete genome sequences from various C. jejuni isolates has started to improve our understanding in genetics, physiology, pathogenesis and immunity of C. jejuni infections in recent years. C. jejuni is the first bacterium reported to encode for both O- and N-linked glycosylation systems, a property that is likely influencing the host-pathogen crosstalk and disease outcome. In addition, a multitude of infection studies in various animal and in vitro cell model systems revealed the importance of C. jejuni motility and chemotaxis as critical features important for establishing successful infections [+) permits C. jejuni to effectively move to its favored colonization niche at the inner mucus layer of the human intestine. Various in vivo and in vitro studies have shown that this pathogen encodes numerous virulence determinants involved in important disease-associated processes such as bacterial adhesion to, transmigration across, invasion into and intracellular survival within infected intestinal epithelial cells [C. jejuni research on how this bacterium can breach the gut epithelial barrier and transmigrate across polarised cell layers. In particular, we focus on the two major known routes that could be taken, the transcellular and paracellular ways of C. jejuni transmigration. Better molecular understanding of these pathways and the identification of involved bacterial and host factors is crucial for the future development of effective treatment regimes.annually . The cliew weeks -5. In soyndromes ,7. Despifections ,8-10. Inal cells . In the The intestinal mucosal epithelium in humans is an important cell layer that controls not only digestive, absorptive and secretory functions, but also forms the first barrier against pathogenic microbes . The intC. jejuni have adapted mechanisms during evolution to exploit TJs, AJs, FAs and/or HDs in infected cells in order to proliferate, survive and sometimes persist within the host [TJs are based on junction adhesion molecules (JAMs), claudins, occludin and other proteins, which represent important structural elements in establishing epithelial cell polarity. They are crucial for the tight sealing of the cellular sheets, thus controlling paracellular ion flux and therefore maintaining tissue homeostasis. The tight apposition of the membranes at TJs, which are localized at the apical end of the lateral membrane, also blocks lateral mobility of membrane proteins and lipids allowing the segregation of membrane components in an apical and basolateral compartment Figures B and 2A.the host ,19-21.C. jejuni research is to define the exact role of bacterial adhesion, invasion and transmigration across enterocytes for the induction or absence of pathogenesis in different hosts. Several in vivo studies of human biopsies and infected animal models reported on observations of C. jejuni entering gut epithelial cells and underlying subepithelial tissues during infection , invade into (Inv+) and survive inside a defined intracellular compartment (Surv+), called the Campylobacter-containing vacuole. These phenotypes, have been reported for both C. jejuni infection of non-polarised and polarised epithelial cells , gain access to deeper tissues, trigger cell damage and cause disease in humans. There are two general mechanisms how bacterial pathogens can overcome the epithelial barrier, described as the paracellular and the transcellular migration routes [C. jejuni across an intestinal epithelium layer in vitro have been performed with multiple strains and polarized cell lines grown in transwell chambers  obtained from the lower chamber, GPA and other functional assays. Application of chloramphenicol, a well-known inhibitor of bacterial protein biosynthesis, reduced the transmigration potential of C. jejuni significantly [de novo[C. jejuni adherence, penetration and transmigration activities were also inhibited at lower temperatures when investigated at 20°C and 4°C as compared to 37°C [C. jejuni require active bacterial and host cell processes at optimal temperature. The current, common opinion is that C. jejuni can effectively transmigrate in vivo and in vitro, but the involved mechanisms (paracellular and/or transcellular) are controversial in the literature . The majority of utilised C. jejuni strains can markedly interact with these cells and actively transmigrate in large quantities within 1-6 h, while a negative control (non-pathogenic Escherichia coli) did not  would be limited to the paracellular pathway. Bras and Ketley [C. jejuni isolates exhibiting various phenotypes in the Caco-2 infection model including Inv+/Trans+, Inv+/Trans- and Inv-/Trans+ strains, respectively. This study demonstrates that at least some Inv-/Trans+C. jejuni strains are to abound, which should travel exclusively by the paracellular route. In addition, the study shows that Inv+/Trans- strains exist. This means that invasive C. jejuni strains do not necessarily exit polarised epithelial cells at the basal membrane to complete the transmigration step, but obviously stay within the intracellular environment. In another study, transcytosis across polarized Caco-2 monolayers was seen in 18 of the 21 colitis-associated C. jejuni strains, compared with only 11 of the 23 isolates from non-inflammatory diarrhoea [-/Trans+ phenotype; again in such cases transcytosis can be unambiguously attributed to a paracellular passage [C. jejuni wild-type isolates and showed that they differ by at least 10-fold in invasiveness and transmigration across Caco-2 cells. C. jejuni transmigration did not quantitatively correlate with the intracellular invasiveness of these isolates and a similar repertoire of strains including Inv+/Trans+, Inv+/Trans- and Inv-/Trans+ isolates were found as described above. Taken together, these data suggest that different phenotypic wild-type C. jejuni isolates exist in nature and that bacterial transmigration capabilities may correlate with colitis disease outcome. However, more studies are certainly necessary to substantiate this hypothesis.Most of the polarised cell lines applied to study d Ketley  describeiarrhoea . Interes passage . Harvey Listeria, Shigella, Neisseria and Salmonella reduced TER substantially over time, infection with the C. jejuni Inv+/Trans+ strains 81–176, F38011 und NCTC11168 did not influence TER significantly [C. jejuni infection [C. jejuni traversed the polarised cell monolayers without any apparent damage to the host cells. Commonly, within 4–8 h almost no difference was observed in the morphologic appearance of host cells and TER values of C. jejuni-infected versus non-infected monolayers [C. jejuni Inv+/Trans+ and Inv-/Trans+ strains traversed the Caco-2 cell monolayer without causing apparent cell damage as judged by TER [C. jejuni cannot pass the epithelial barrier by the paracellular route, at least not at early times of infection [C. jejuni[in vitro indicates that opening of TJs and AJs by C. jejuni associated with massive cell monolayer disruption can occur at late times of infection. Immunofluorescence microscopy of polarized T84 monolayers infected for 24 h with C. jejuni revealed a redistribution of the TJ transmembrane protein occludin from an intercellular to an intracellular location associated with a change in phosphorylation [in vivo  with electrodes . For thificantly ,45. Manynfection ,50,84,85nolayers ,50,84,85d by TER . This innfection ,49,84,85C. jejuni. The drorylation . Host ceC. jejuni with polarized Caco-2 cells. The efficiency of C. jejuni invasion of Caco-2 cells was 2- to 3-fold less as compared to non-polarised INT-407 cells [C. jejuni, and even smaller percentage of Caco-2 cells contained 5–20 internalized bacteria per cell after 2 h of infection [C. jejuni were present extracellularly between two neighboring Caco-2 cells as well as intracellularly [C. jejuni, which remain tightly surrounded by a host-derived membrane (the Campylobacter vacuole) after invasion into epithelial cells in vitro and in vivo[C. jejuni could translocate across polarized cell monolayers by passing through single cells  or transmission electron microscopy (TEM), which also illustrated the presence of microvilli and well-defined brush borders at the apical cell surface ,40. In a07 cells . Interesnfection . Furtherllularly ,40. Simid in vivo,50,86. IC. jejuni transmigration across polarised intestinal cells is not fully understood because only a handful putative bacterial and host factors have been reported yet. The application of pharmacological inhibitors has indicated that the activity of phosphoinositid-3-kinase is necessary for C. jejuni transcytosis [C. jejuni transmigration was enhanced by adding interferon-gamma, probably because of its TER-reducing capabilities during inflammation [C. jejuni resulted in a colonization-negative phenotype in various animal models [C. jejuni motility or the flagellin gene products or both appear to be essential for translocation across the polarized monolayer in vitro[C. jejuni mutants GRK5 and GRK7 (FlaA FlaB Mot-) and GRK17 (FlaA FlaB+ Mot-) were unable to cross the Caco-2 cellular barrier as compared to Mot+ control strains [flaA/B) and ΔflgF mutants with Mot- phenotype were also diminished in passing polarised T84 or MKN-28 cells, respectively [C. jejuni variant that has reduced motility (Mot−/+) despite retaining intact flagella, was unable to colonise the intestinal tract of infant mice [C. jejuni-positive blood culture [C. jejuni.The process of scytosis . The rolscytosis . In addiammation . Many otl models ,8,9. Ear in vitro. For exa strains . Anotherectively ,44,85. Iant mice  and elic culture . It was ciaB mutant was able to transmigrate across polarised T84 cells like wild-type bacteria suggesting that apical cell invasion is not necessary for C. jejuni transmigration, thus favoring the paracellular route [cadF mutant (deficient in fibronectin-binding and invasion) also transmigrated as effectively as wild-type C. jejuni strain F38011 [cstII mutant in C. jejuni strain GB11 exhibited a strong deficiency of invasion and transmigration as determined by GPA, immunofluorescence microscopy and transwell assays, thus favoring a transcellular route [The flagellum does not only have a distinct function in bacterial motility and cell binding, but also acts as a type III secretion system (T3SS) for the delivery of Cia (Campylobacter invasion antigens) proteins into the extracellular space or into the host cell -93. The ar route . Furthern F38011 . This rear route . UnfortuC. jejuni, the serine protease HtrA (high temperature resistant protein A) [htrA gene in two strains resulted in strong deficiency of C. jejuni to travel across polarised MKN-28 cells [per se is not sufficient for C. jejuni transmigration [htrA-mediated cell binding maybe involved [C. jejuni, although this class of proteases has well-known functions as chaperone and protein quality controllers in the periplasm of E. coli[C. jejuni was enhanced during host cell contact or in the presence of calf serum, but was independent of the flagellar T3SS [in vitro[Shigella, Helicobacter and EPEC, but not for the urogenital pathogen Neisseria[H. pylori or C. jejuni to enter the intercellular space. In further studies, the HtrA ortholog in H. pylori has also been shown to cleave another protein, fibronectin, but C. jejuni HtrA has obviously lost this activity over fibronectin during evolution [C. jejuni binding to integrins and host cell invasion, at least during infection of non-polarised cells [Recently, another factor was identified to be a novel virulence determinant in otein A) . Deletio28 cells ,45. Thisigration , but htrinvolved . Anotherf E. coli,45. Secrlar T3SS . Infecti[in vitro,45. Intevolution ,96. The ed cells ,72,97,98htrA or substitution of htrA with a protease-deficient S197A point mutant in the bacteria resulted in severe defects for E-cadherin cleavage and C. jejuni transcytosis through MKN-28 cells [C. jejuni may transmigrate intercellularly between neighboring cells using the paracellular route. Interestingly, in a time course of C. jejuni infection the total amount of cell-associated E-cadherin dropped to some extent, but did not lead to a complete cleavage, not even in 8h infections [C. jejuni and suggests that these bacteria could somehow open and close the “door” between two neighboring cells [C. jejuni across polarised host epithelial cells.Deletion of 28 cells ,45. Thusfections . It was fections . As the ng cells . Such a ng cells . If our Campylobacters other than C. jejuni are rare in the literature, with only a few reports on C. fetus, C. rectus and C. coli. In a first study, the ability to translocate across epithelial barriers has been investigated during infection with C. fetus, a recognized pathogen of cattle, sheep and humans [C. fetus was found to translocate efficiently within 24h without altering TER, similar to C. jejuni as discussed above. C. fetus was also observed to invade and subsequently egress from Caco-2 cells as shown in a modified GPA procedure and this occurred independently of C. fetus S layer expression [C. fetus both at apical and basal surfaces as well as in intracellular locations, but not in the paracellular space. Pharmacological inhibitor studies demonstrated the requirement of a functional tubulin cytoskeleton, and together with the TEM data support a transcellular mechanism for C. fetus transmigration across Caco-2 monolayers [C. fetus infections in various hosts and can explain bacterial recovery from extraintestinal sites [C. rectus, a periodontal pathogen associated with human fetal exposure and adverse pregnancy outcomes including preterm delivery, was investigated [C. rectus can translocate from a distant site of infection to the placenta where it can induce fetal growth restriction and impairs placental development [C. rectus was detected in 63% of placentas after two weeks and significantly decreased fetoplacental weight. In invasion assays, C. rectus was able to effectively invade human trophoblasts in vitro (but not trophoblasts of murine origin), and showed a trend for higher invasiveness as compared to C. jejuni. Interestingly, C. rectus infection significantly upregulated IL-6 and TNF-α levels in a dose-dependent manner in human trophoblasts, but not in murine cells, suggesting a correlation between invasion and cytokine activation [C. rectus in human trophoblasts may play a role in facilitating bacterial translocation and placental inflammation during early gestation [C. coli, which is the dominant Campylobacter species commonly found in pigs [C. coli strain 5981 in weaned pigs over 28 days. Excretion of C. coli 5981 was seen for all piglets 7 days after inoculation and highest counts were detectable on day 10 [C. coli was observed for gut tissues of the small intestine and for the gut associated lymphatic tissues, such as jejunal mesenteric lymph nodes and tonsils as well as for spleen and gall bladder. In conclusion, this trial indicates that C. coli exhibit translocation and invasion capabilities in pigs making it a useful model system to study colonisation and pathogenicity of this pathogen [Studies on the epithelial transmigration of d humans . Using cpression . SEM andnolayers . Thus, tal sites . In a sestigated . Infectielopment . Infectitivation . It was estation . A third in pigs . An expen day 10 . Post-mopathogen .C. jejuni is a predominant zoonotic pathogen causing enterocolitis in humans worldwide. However, despite the high prevalence of C. jejuni induced disease and research progress made in recent years, our knowledge is still relatively limited as compared to other invasive pathogens such as Salmonella, Listeria or Shigella. A series of studies on human biopsies and animal infection experiments have demonstrated that C. jejuni is able to cross the intestinal epithelial barrier and enter underlying tissues, bloodstream and even other organs  in multiple non-polarised cell types [C. jejuni strains invading polarised cells from apical or basal membranes are currently not available. Alternative possibilities include the involvement of ganglioside-like LOS in apical invasion, thus favoring a transcellular route [C. jejuni can open the TJs after longer co-incubation times is yet unclear. Several studies exist that could support the apical invasion model, but can C. jejuni also enter host cells from basal surfaces? Basal engulfment and entry of C. jejuni into non-polarised Chang or polarised Caco-2 cells has been demonstrated by TEM and immunofluorescence microscopy, and this process has been called subvasion [C. jejuni strains to travel across polarised intestinal epithelial cells, either by a transcellular or paracellular pathway or a mix of both. It should be also considered that individual C. jejuni strains might switch from one to the other mode under specific culturing or infection conditions. Finally, besides the commonly applied transwell system, a vertical diffusion chamber model system has been recently described, which creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured intestinal epithelial cells, thus producing an in vitro system that probably closely mimics in vivo conditions of the human intestine [C. jejuni with intestinal epithelial cells demonstrated the importance of performing such experiments under conditions that converge to the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms [in vitro studies are already transformed because they originate from cancer patients. A possible alternative option came from recent studies demonstrating that single Lgr5 (the receptor for the Wnt-agonistic R-spondins)-positive stem cells isolated from the intestine can grow in culture into complex epithelial organoid structures, called miniguts, which retain their original organ identity [Campylobacter. It therefore appears that transmigration of C. jejuni and that of many other Campylobacter species will continue to be a fascinating and rewarding research subject in the future.Different mechanisms are used by various enteropathogens to transmigrate across the host intestinal epithelium, including transcytosis through specialized M cells, phagocytosis by interepithelial leukocytes or transcytosis of enterocytes ,81,82. C rabbits . From ininvasion . In continvasion ,98,104. ll types ,97,98, bar route , but thiar route ,45. How ubvasion ,105. Howntestine . The usechanisms . In addiidentity -109. ThiThe authors declare that they have no competing interests.SB performed the data collection and wrote the text. MB designed Table 1 and SW designed Table 2. NT was drawing the figures and wrote the figure legends. All authors discussed, read and approved the text including the final manuscript.


Candida albicans has been much studied, little is known about the life cycles of non-pathogen species of the CTG clade. Taking advantage of the recently obtained sequence of the biotechnological Millerozyma (Pichiasorbitophila) farinosa strain CBS 7064, we used MLST to better define phylogenic relationships between most of the Millerozyma farinosa strains available in public collections. This led to the constitution of four phylogenetic clades diverging from 8% to 15% at the DNA level and possibly constituting a species complex (M. farinosa) and to the proposal of two new species:Millerozyma miso sp. nov. CBS 2004T ( = CLIB 1230T) and Candida pseudofarinosa sp. nov.NCYC 386T( = CLIB 1231T).Further analysis showed that M. farinosa isolates exist as haploid and inter-clade hybrids. Despite the sequence divergence between the clades, secondary contacts after reproductive isolation were evidenced, as revealed by both introgression and mitochondria transfer between clades. We also showed that the inter-clade hybrids do sporulate to generate mainly viable vegetative diploid spores that are not the result of meiosis, and very rarely aneuploid spores possibly through the loss of heterozygosity during sporulation. Taken together, these results show that in this part of the CTG clade, non-Mendelian genetic exchanges occur at high rates through hybridization between divergent strainsfrom distinct clades and subsequent massive loss of heterozygosity. This combination of mechanisms could constitute an alternative sexuality leading to an unsuspected biodiversity.Among ascomycetous yeasts, the CTG clade is so-called because its constituent species translate CTG as serine instead of leucine. Though the biology of certain pathogenic species such as  Saccharomyces species is well known. They propagate as haploid or diploids according to the environment, mate, sporulate and undergo meiosis to yield haploid spores Saccharomyces species are also known for forming inter-specific hybrids under the pressure of industrial fermentation conditions Candida sp. pathogens belonging to the CTG clade were shown to have various life cycles. Candida albicans is a diploid that does mate and shows parasexuality, but does not perform sporulation or meiosis Clavispora lusitaniae, on the other hand, can mate and sporulate. Its progeny is complex and comprises haploids resulting from meiosis, diploids and aneuploids Candida tropicalis was also recently shown to display parasexuality The life cycle of Debaryomyces hansenii. This species is thought to have a rather clonal reproduction through fusion between mother and daughter cells followed by meiosis, whilemating between individual cells israrely observed Pichia farinosa (Lindner) E.C. Hansen var. farinosa (teleomorph of Candida cacaoi) is a ubiquitous, halotolerant yeast found mainly in food  but alsoon various substrates  P. farinosa to Millerozyma farinosaM. farinosa and other yeasts, mainly Candida albicans, were found in patients undergoing radiotherapy for cancer of the mouth M. farinosa strain was found as an oral cavity colonizer M. farinosa were also described However, little is known about non-pathogenic species of this clade like the most studied Pichia sorbitophila, was described M. farinosa and P. sorbitophila can produce high yields of glycerol and xylitol, which makes them valuable in biotechnology. The placementof some strains in the M. farinosa species is a matter of debate M. farinosaand P. sorbitophila. On the basis of DNA relatedness, Debaryomyces halotolerans, P. sorbitophila, and Pichia petrophilum were considered to be conspecific with M. farinosaM. farinosa CBS 7064 carries two divergent URA3 genes, which were also different from that of the M. farinosa type strain [20]. The presence of the two divergent URA3 gene copies and the higher number of chromosomes in pulse field gel electrophoresis was in agreement with CBS 7064 being diploid.In 1980, a related species, M. farinosa type strain CBS 185T. CBS 7064 contains sevenpairs of chromosome. In three ofthese both parents contribute one chromosome; in two the chromosomes contain regions that are identical and regions that are diverged;two consist of identical chromosomes, indicative of massive loss of heterozygosity (LOH)From therecently sequenced CBS 7064 genome M. farinosa species and to gain insight into the life cycle of CTG species, we took advantage of the available complete genome sequence of the CBS 7064 strain M. farinosa strains available in public collections. In this paperwe use MLST to constitute phylogenetic groups. We demonstrate reproductive isolation of these groups. We show that M. farinosa is a species complex made of distinct taxa, and in which isolates exist as haploid organisms and as hybrids resulting from crosses between strains from these taxa. We also show that the hybrids can sporulate to generate viable diploid spores that are not the result of meiosis. In this species complex, an alternative sexuality allows the formation of hybrids subjected to LOH and of rare aneuploids.In order to define the parental contributors of CBS 7064, to resolve the taxonomy of the Debaryomyces hansenii, a closely related species to M. farinoseD. hansenii. The most informative intron sequences were those of the ACT1 and RPL33 genes. In order to analyze the M. farinosa species, we therefore amplified and sequenced the introns of these two genes in 41 M. farinosa strains  T. An example in T and CBS 2001. These observations strongly suggest that the strains displaying heterozygosity result from crosses between strains belonging to different clades, as already observed for D. hanseniiIn a previous study, we have observed that  strains . In a nuACT1 alleles of 10 strains including the type strain CBS 185T. As mentioned above, the results from the analysis of the RPL33 alleles gave essentially the same clades and are not further discussed here. Clade 2 contained 18sequencesincluding one of theACT1 alleles of CBS 7064. Sequence divergence in the ACT1 intron between these two clades was 8.5%  from that of clade 1  was defined by the sequence of the was 8.5% . We noteas 11.5% . The fou clade 1 . Finallyd clades .ACT1 gene intron tree in RPL33 gene intron tree  was different from all sequenced RPL33 alleles; the other allele belongs to clade 2 as does the unique ACT1 allele of this hybrid. Since CBS 7064 was homozygous for clade 2 RPL33 allele .Finally, two strains, CECT 10348 and NCYC 386ACT1 and RPL33, we were able to define five distinct clades in the M. farinosa species (see above). This MLST analysisshowed unambiguously that strains belonging to clade 2 and clade 4 contributed to the hybrid CBS 7064. In the description of the genome of CBS 7064, a distinction was made between the two parental contributing genomes (designated Pγ and Pε?) on the basis of the unequal distribution of NUMT insertions and on the G+C percentage of each subgenome T, 2001, 2007 and 2004T from clades 1, 2, 3 and 5, respectively), using 16 markers located at the extremities of each of the chromosomes of CBS 7064 to define the origin of each of the chromosomes of CBS 7064 and to better define each of M. farinosa clades.Using two housekeeping genes T for clade 1, CBS 2007 for clade 3 and CBS 2004T for clade 5 displayed 81 to 96% identity with that of CBS 7064. These results there fore confirmed what we had observed with the ACT1 and the RPL33 markers, i.e.a clade 2 strain is a parental contributor of CBS 7064,whileneither clade 1, clade 3 nor clade 5 strains contributed toCBS 7064.Interestingly, none of the strains tested gave 100% identity with the right end markers of chromosomes A and B of CBS 7064 . A total of 70 spores were analyzedfurther. As the NUMT content of CBS 2006 is not known, we used SNP in genomic markers to analyze the progeny. We amplified and sequenced the AB-r and CD-l markers and it was interesting to examine potential genetic exchanges in haploid strains. Mitochondria constitute good markers to follow genetic exchanges.Within M.farinosa inter-clade hybrids, an MLST analysis of the COX2 and COX3 genes was first performed using sequence of the part of these two genes in the typical strains of each clade and in all the inter-clade hybrids. A schematic representation of the relationship between the mtDNA sequences in relation with the different clades is shown in Tas expectedcarrieddivergentmtDNA alleles, while surprisinglystrains from clade 1 and clade 2 carried the same mtDNA alleles. All the tested hybrids,except CBS 7064 (clade 2/clade 4) and CBS 2167 (clade 3/clade 5), carried either the mtDNA alleles of clade 2 or that of clade 3, consistent with the idea that the hybridswere derived from crosses betweenthe clades. Our results show that, like in SaccharomycesMetschnikowia inter-specific hybrids M. farinosa species complex. This is in contrast with several reportson Saccharomycesfermentative hybrids involving S. cerevisiaeshowingthat mtDNA from the non-cerevisiaeparental contributor wasspecifically favored To address thequestion of the inheritance of mtDNA in the T and CBS 7064 had already shown that they differed Interestingly, the mtDNA carried CBS 7064 did not correspond to any of the alleles of clades 1, 2, 3 and 5. Comparison of the mitochondrial genomes of CBS 185Tand clade 2 CBS 2001 typical strains carried the same mtDNA alleles prompted us to analyze the distal COX2 and COX3 geneson the mitochondrial genome of all haploid strains from clade 1 and 2 in order to try to identify the missing mtDNA type. As represented in COX2(0.5%) and five SNP in COX3(0.8%), demonstratingthat CBS 2020 carried another mtDNA type. These results suggest that mating events between clade 1 and clade 2 ancestors have occurred associated with unidirectional transfer of mtDNA. Unlike in MetschnikowiaThe fact that both clade 1 CBS 185M. farinosa strains and confirm the existence of genetic exchanges between clades 1 and 2. By monitoring the presence /absence of NUMT in PCR products, we analyzed nine heterozygous and two homozygous NUMT loci present in the CBS 7064 genome in the three haploid strains CBS 185T, (clade 1), CBS 2001 (clade 2) and CBS 2007 (clade 3) as well as and two hybrids CBS 5730 (clade 2/clade 3) and CBS 8045 (clade 2/clade 4). Most insertions were absent in the typical strains from clades 1, 2 and 3, consistent with their presence on chromosomes E, J and M that are characteristic of the clade 4 parent of CBS 7064(T.NUMTinsertions can be found in most yeast genomes  CBS 7064. ConversT and displayed 9% divergence with the sequence of the parentCBS 2001(clade 2), which contributed the rest of the chromosome. Moreover,the clade 2/clade 4 hybrid CBS 8045 (see above) also displayed the clade 1 sequence for this locus. These observations suggest that some genetic exchange has occurred between clades 1 and 2 after reproductive isolation, in agreement with the presence of an identical mtDNA in most strains of clade 1 and clade 2.During the analysis of a NUMT locus on chromosome E (NUMT s9) M. farinosaspecies was shown to be made of related, but distant clades, and it was therefore necessary to establish a phylogenetic analysis of the species and clades constituting the genus Millerozyma based on a reliable multigenic analysis. Kurtzman et al Pichia, which now contains the type strain of the genus, Pichia membranifaciens, and various species from other previously described genera such as Issatchenkia. Kurtzman and Suzuki P. farinosa belongs as Millerozyma. We generated a phylogeny with the typical strain of each of the four clades, the clade 4 (the Pε subgenome) parent of CBS 7064, NCYC 386T,and the type strains of the previously decribed Millerozymaspecies using the RPB1, RPB2 and ACT1 exon2 sequences  previously used for CBS 2004T by Mogi in 1942 (but which had not been described) under the name Millerozyma miso and we would like to define a new species Candida pseudofarinosa (type strain NCYC 386T = CLIB 1231T).The equences . All of In agaro YPD post dies 2 ad 28°C cellulae vegetativae ovoidae aut oblongae, per gemmationem multipolarem reproducentes. Cultura est granula, albida vel cremea, margo glabro vel undulato.In agaro Zea mayidis pseudomycelia formatur.Asci non conjugate et non rumpuntur. Habentes 3–4 ascosporae globosae.Glucosum, galactosum fermentatur. Maltosum, sucrosum, trehalosum, melibiosum, lactosum, cellobiosum, melezitosum et raffinosum non fermentatur.d-ribosum, l-rhamnosum, trehalosum, erythritolum, gluconatum et glycerolum assimilantur. Xylosum, sucrosum, maltosum, lactosum, cellobiosum, α-methylum-d-glucosidum, raffinosum,melezitosum, inositolum,acidum lacticum, sorbosum, d-glucosaminum, l-arabinosum, melibiosum, et sodium glucuronatum non assimilantur.Ethylaminum, lysinum et cadaverinum, assimilantur at non nitratum, nitritum creatinum etcreatininum.Vitamina externa crescentia non sunt necessaria. Crescere potest in 10% NaCl et 50% glucosum. 0.01% cycloheximidi non crescit.Maxima temperature crescentiae: 42°C. Materia amyloidea iodophila, acidum aceticum non formantur. Diazonium caeruleum B non respondens. Ureum non hydrolysatur.Glucosum, galactosum, Typus stirps CBS 2004T( = CLIB 1230T)After 48 h at 28°C on YPD ovoid or oblong vegetative cells are formed . Cell diFermentation:d-glucose andd-galactose were fermented. d-maltose, saccharose, trehalose, melibiose, lactose, cellobiose, melezitose and raffinose are not fermented.Assimilation reaction and other growth tests: d-glucose, d-galactose, d-ribose, l-rhamnose, trehalose, erythritol, 2-keto-d-gluconate, d-gluconate and glycerol are assimilated. d-xylose, sucrose, d-maltose, d-lactose, d-cellobiose, α-methyl-d-glucoside, d-raffinose, d-melezitose, inositol, dl-lactate, l-sorbose, d-glucosamine, l-arabinose, d-melibiose, sodium glucuronate, are not assimilated. Ethylamine, l-lysine and cadaverine are assimilated; potassium nitrate, sodium nitrite, creatine and creatinine are not assimilated.Growth in vitamin-free medium, 10% NaCl and 50% glucose are positive. 0.01% cycloheximide is negative. Maximum growth temperature is 42°C. Starch-like compounds and acetic acid are not produced. Diazonium blue B reaction is negative. Urease activity is negative.Zygopichia miso Mogi, J. Agric. Chem. Soc. Japan 18∶543-554, 629–641, 733–741, 940–944, 1942Synonym: T( = CLIB 1230T)Type: CBS 2004In agaro YPD post dies 6 ad 28°C cellulae vel elongatae pseudohyphae formantes, per gemmationem multipolarem reproducentes. Cultura est butyrosa, sulcuma, centrum sublatum est, albida. In agaro Zea mayidis pseudomycelia formatur.Ascosporae nullae.Glucosum, galactosum, maltosum, sucrosum, trehalosum, melibiosum, lactosum, cellobiosum, melezitosum et raffinosum non fermentatur.Glucosum, gluconatum et galactosum assimilantur. D-ribosum, xylosum, L-rhamnosum, sucrosum, maltosum, lactosum, trehalosum, cellobiosum, alpha-methylum-D-glucosidum, raffinosum, melezitosum, glycerolum, erythritolum, inositolum, sorbitolum, D-gluconatum, acidum lacticum, sorbosum, D-glucosaminum, L-arabinosum, melibiosum, sodium glucuronatum et N-acetyl-glucosaminum non assimilantur.Nitratum, ethylaminum, lysinum, cadaverinum, creatinum et creatininum assimilantur at non nitritum et glucosaminum.Vitamina externa crescentia non sunt necessaria. Crescere potest in 10% NaCl et 50% glucosum. 0.01% cycloheximidi non crescit.Maxima temperature crescentiae: 40°C. Materia amyloidea iodophila, acidum aceticum non formantur. Diazonium caeruleum B non respondens. Ureum non hydrolysatur.Typus stirps NCYC 386T( = CLIB 1231T)After 6 days at 28°C on YPD, cells are elongated and mycelial pseudohyphae are formed . BuddingFermentation: D-glucose, D-galactose, D-maltose, saccharose, trehalose, melibiose, lactose, cellobiose, melezitose and raffinose are not fermented.Assimilation reaction and other growth tests: D-glucose, D-gluconate and D-galactose are assimilated. D-ribose, D-xylose, L-rhamnose, sucrose, D-maltose, D-lactose, trehalose, D-cellobiose, alpha-methyl-D-glucoside, D-raffinose, D-melezitose, glycerol, erythritol, inositol, D-sorbitol, 2-keto-D-gluconate, D L-lactate, L-sorbose, D-glucosamine, L-arabinose, D-melibiose, sodium glucuronate, N-acetyl-glucosamine are not assimilated. Potassium nitrate, ethylamine, L-lysine, cadaverine, creatine and creatinine are assimilated; sodium nitrite and glucosamine are not assimilated. Growth in vitamin-free medium, 10% NaCl and 50% glucose are positive. 0.01% cycloheximide is negative. Maximum growth temperature is 40°C. Starch-like compounds and acetic acid are not produced. Diazonium blue B reaction is negative. Urease activity is negative.T ( = CLIB 1231T), was isolated in Japan and was deposited at National Collection Yeast Culture by IFO collection during December 1953.The type strain, NCYC 386M. farinosa was composed of at least four diverged taxa. In addition, two taxa that presented high sequence divergence were considered as new species: Candida pseudofarinosa and Millerozyma miso. We have previously shown that another CTG species, D. hansenii, whose taxonomy has been discussed over the years Candida parapsilosis was also shown to be a complex made of three species Species whose taxonomy is debatable may reflect the possible existence of a species complex containing cryptic species. By evaluating the sequence divergence in regions little subjected to selective pressure, we have shown that S. pastorianus strains Saccharomyces, in which hybrids provide new technological potentialitiessuch as specific fermentations M. farinosa isolates (data not shown), in contrast to what was described by M. farinosa remains to be found.Since the discovery of the inter-specific hybrid lager yeast M. farinosa hybrids, sequence divergence in the ACT1 intron varied between 8.5 and 14.5%. The overall average genomic divergence between the contributors of CBS 7064 is 15.3% ACT1 and RPL33. Introns are therefore good indicators of overall sequence divergence in yeast genomes, and are superior to sequences like the rDNA D1/D2 ACT1coding gene In the M. farinosahybrids, the overall divergence may be reduced by LOH. Indeed, massive LOH was observed in M. farinosa CBS 7064 to the point where some of the chromosome pairs became entirely homozygous M. farinosa hybrids.The distribution of markers in the diploid spore G6showed that the genetic information from one of the parents became predominant. This processseems to vary according to the strain analyzed, as seen from the comparison betweenCBS 7064and spore G6 of CBS 2006. In our study of LOH in a large number of diploids inD. hansenii,we observed a tendency, which favored only one parental contributor M. farinosa shares common featureswith other described CTG species such as C. albicansCandidadubliniensisD. hansenii, for the existence of hybrids and LOH. In addition, natural populations of both haploids and hybrids exist in M. farinosa and D. hansenii.In the S. cerevisiaeis greatly diminished. Sequence divergence(as low as 3.6% in populations of S. paradoxus) was shown to drastically lower spore viability M. farinosa clades (from 8.5 to 14%) is well above these thresholds and would almost certainly prevent recombination at meiosis if meiosis occurred. Consistent with this observation, our study has shown that there is no shuffling of alleles between analyzed strains using two markers. This suggests that M. farinosa has mainly a clonal propagation. Hence, it is possible that the concept of species defined by fecundity as described by Liti et al Saccharomyces does not apply to yeasts of the CTG clade.In the case of hybrids, LOH would certainly alleviate the heterozygosity-driven inhibition of meiotic recombination. Liti et al M. farinosa hybrids did sporulate, one can predict that they do not undergo meiosis. In Zygosaccharomyces bailii, a species that is closer to the Saccharomyces and does not belong to the CTG clade, a lack of meiotic spores was observed. This was explained by the absence of nuclear fusion after mating C. lusitaniae, whose progeny from crosses was complexand contained 70% haploid spores, 21% diploid spores with the rest being aneuploid M. farinosa may be due to the important sequence divergence between the parents, which was not the case in C. lusitaniae. On the other hand, all the spores from D. hanseniiinter-clade hybrids are also diploid (our unpublished observation), though in this case the sequence divergence was lower  than that of M. farinosa clades. This observationsuggests that sequence divergence may not be the only reason for the lack of meiosis in these species. Alternatively, in these species, a 3% sequence divergence may still be too high to allow meiosis to occur. It has been proposed that errors in meiosis such as meiosis I non-disjunction or precocious sister segregationor a possibleabsence of synaptonemal complex could lead to aneuploid and diploid sporesin C. lusitaniaeSaccharomycesM. farinosa.Although most of the M. farinosa could undergo LOH leading to a reduced heterozygosity allowing meiosis in the resulting organism. The study of the spore G6 confirmed that LOH could be massive in M. farinosa, as seen in M. farinosa CBS 7064. As in C. albicansin which stress affects LOH M. farinosa. C. parapsilosisM. farinosaclade 1 and 2.After sporulation, M. farinosa is a mix of haploid clones belonging to different clades, hybrids resulting from mating between these clades and subjected to LOH and aneuploids having variable DNA content resulting from the sporulation of the hybrids through abortive meiosis or possible other mechanisms. The haploid or close to haploid spores resulting from the hybrids therefore display a non-Mendelian marker inheritance. For instance, these haploids may be the ancestor of strains of M. farinosa clade 1 or clade 2, which have exchanged material, as deduced from the transmission of mtDNA and from the introgression of unknown extent that we have evidenced from the NUMT analysis. Together, these results indicate that hybridization followedbyLOH is an alternative sexuality for these very divergent strains, although arare occurrence of meiosis isnot excluded. In conclusion, M. farinosa constitutes a species complex made of diverged cryptic species, whose genomes are affected by secondary contacts and LOH. We hypothesizethat this situation could be the case for a number of species of the CTG clade. This ensemble of observations invites reflection on thespecies concept in this clade.The progeny of some of the CTG yeast species can therefore be complex, contributing to the diversity in this clade. http://www.CBS.knaw.nl), CECT (http://www.cect.org/), CIRM-Levures (http://www.inra.fr/cirmlevures), DSMZ (http://www.dsmz.de/), MUCL (http://bccm.belspo.be/about/mucl.php), NBRC (http://www.nbrc.nite.go.jp/e/), NCYC (http://www.ncyc.co.uk/), PYCC (http://www.crem.fct.unl.pt/) and VTT (http://culturecollection.vtt.fi/). They are listed in The yeast strains used in this study are from CBS (http://fokker.wi.mit.edu/primer3) on the alignments of genes from complete sequence genomes (http://www.genolevures.org/). Some of the primers were already described The sequences of the nucleotide primersused in this study are listed in P. sorbitophila) farinosa CBS7064 around each NUMT locus. For the nine heterozygous loci , we chose primer sequences that were identical on both alleles. Two pairs of primers were designed for each of the two homozygous NUMT loci to increase the chance to amplify these regions in all strains.Primers were designed on the complete sequence of M.  or the primers used for the PCR amplification.PCR products were purified using Nucleospin extract II purification kit . They were cloned intoa pGemT Easy  using T4 DNA ligase (Promega). DH10B http://www.ncbi.nlm.nih.gov). Sequence alignments were generated by using ClustalX http://www.psc.edu/biomed/genedoc). Phylogenetic trees were reconstructed with Neighbor-Joining program implemented in ClustalX and in MAFFT or with Phyml PCR fragments were sequenced on both strands by Cogenics  and Eurofins MWG Operon . Sequences were assembled with the phred/phrap/consed package. Sequences were analysed with various programs including BLAST implemented at NCBI (Flow cytometry was performed as described in http://www.mycobank.org) accession numbers for Millerozyma miso sp. nov. and Candida pseudofarinosasp. nov. are MB 563405 and MB 563406, respectively.The GenBank/EMBL/DDBJ accession numbers for sequences described in this article are listed in Figure S1RPL33 gene intron of M. farinosa isolates. Bootstrap values (%) based on 1000 replicates are indicated at the nodes for main groups and clades. All positions containing gaps and missing data were eliminated from the dataset. Clades are indicated.Typical strains are in bold characters. Heterozygous alleles in hybrids were arbitrarily given the prefix A or B. Bar, 0.02 substitutions per site.Neighbor-joining phylogram of the (TIF)Click here for additional data file.Figure S2M. farinosa isolates after propidium iodide staining.Fluorescence histograms of various (TIF)Click here for additional data file.Figure S3Schematic representation of the localization of the studied markers on the CBS 7064 chromosomes. The representation of the CBS 7064 chromosomes is taken from (TIF)Click here for additional data file.Figure S4Micrograph of sporulating cells after two weeks on Malt agar at room temperature of (A) CBS 7064 and (B) CBS 2006.Bar,5 µm.(TIF)Click here for additional data file.Table S1Primers used in this study.(DOCX)Click here for additional data file.Table S2Accession numbers.(XLSX)Click here for additional data file.Table S3CBS 7064 markers used in this study.(XLSX)Click here for additional data file.Table S4M. farinosa typical strains  and M. miso (CBS 2004T) with M. (P. sorbitophila) farinosa CBS 7064 and with each other.Sequence identity(%) of various markers of the (XLSX)Click here for additional data file.


Adelphocoris suturalis (Jakovlev), Adelphocoris lineolatus (Goeze) and Adelphocoris fasciaticollis (Reuter) (Hemiptera: Miridae) are common pests of several agricultural crops. These three species have vastly different geographical distributions, phenologies and abundances, all of which are linked to their reliance on local plants. Previous work has shown notable differences in Adelphocoris spp. host use for overwintering. In this study, we assessed the extent to which each of the Adelphocoris spp. relies on some of its major overwinter hosts for spring development. Over the course of four consecutive years (2009–2012), we conducted population surveys on 77 different plant species from 39 families. During the spring, A. fasciaticollis used the broadest range of hosts, as it was found on 35 plant species, followed by A. suturalis (15 species) and A. lineolatus (7 species). Abundances of the species greatly differed between host plants, with A. fasciaticollis reaching the highest abundance on Chinese date (Ziziphus jujuba Mill.), whereas both A. suturalis and A. lineolatus preferred alfalfa (Medicago sativa L.). The host breadths of the three Adelphocoris spp. differed greatly between subsequent spring and winter seasons. The generalist species exhibited the least host fidelity, with A. suturalis and A. lineolatus using 8 of 22 and 4 of 12 overwinter host species for spring development, respectively. By contrast, the comparative specialist A. fasciaticollis relied on 9 of its 11 overwinter plants as early-season hosts. We highlight important seasonal changes in host breadth and interspecific differences in the extent of host switching behavior between the winter and spring seasons. These findings benefit our understanding of the evolutionary interactions between mirid bugs and their host plants and can be used to guide early-season population management.The mirid bugs  Host plant use forms the basis of niche breadth and the evolutionary success of herbivores Adelphocoris suturalis (Jakovlev), A. lineolatus (Goeze) and A. fasciaticollis (Reuter) are three common pest species on cotton, alfalfa and many other crops Bacillus thuringiensis) cotton and the subsequent reduction in insecticide use in this crop have increased Adelphocoris spp. infestation levels In China, Adelphocoris spp. have different geographical distributions, seasonal occurrences and infestation levels. A. suturalis is mainly found in temperate areas, such as the Yangtze River Region and the southern part of the Yellow River Region, whereas A. lineolatus and A. fasciaticollis are usually confined to colder regions, namely certain parts of the Yellow River Region Adelphocoris spp. population abundances in each of these regions Adelphocoris species has a specific range of overwintering host plants that it uses, largely consistent with each species’ distribution and phenology The three Adelphocoris species overwinter as eggs. Some insect species rely on a wide range of plant species for overwintering, whereas others have a far more restricted host range. The eggs of A. suturalis have successfully eclosed from 115 plant species, whereas A. lineolatus and A. fasciaticollis have overwintered on 40 and 35 plant species, respectively Adelphocoris spp. overwintering sites is particularly important given the limited dispersal capacities of the newly emerged nymphs Adelphocoris spp. rely on overwinter hosts for spring development and whether early-season host use relates to the dietary breadth of a given species.On these winter hosts, the different Adelphocoris species with previously reported winter host use patterns. The results may help explain interspecific differences in the distributions and phenologies of Adelphocoris spp. Additionally, a sound understanding of early-season host switching and population buildup could ultimately help predict Adelphocoris spp. infestation levels in summer crops such as cotton and alfalfa.In this study, we contrasted the early-season host plant range of the three No specific permits were required for the described field studies.Adelphocoris spp. have similarly low population levels Field surveys were conducted from mid-April to mid-June of the year 2009–2012 at the natural areas and agricultural fields near the Langfang Experiment Station, Chinese Academy of Agricultural Sciences (CAAS) , in Hebei Province, China. Here, all three 2 (in 2009), 67 species covering 11,769 m2 (2010), 43 species covering 8,417 m2 (2011) and 56 species covering 4,345 m2 (2012)  that are common and widely distributed in the agroecosystems of northern China based on information from local plant guides. A total of 77 plant species from 39 families were sampled, including 53 weeds, 20 trees, 2 pasture crops and 2 agricultural crops. We sampled 65 plant species covering 10,790 m2 (2012)  and 2.Adelphocoris spp. abundance on different plants was assessed using a standard white pan beating method. For herbaceous plants, we examined the entire plant; whereas for tree crops, we sampled the young branches. Sampling was performed every 3–5 days, the plant material was shaken over a 40 cm×26 cm×11 cm white pan, and the dislodged Adelphocoris individuals (adults and nymphs) were counted Adelphocoris species was based on morphological features 2, whereas for uncommon species, all of the plants at a given site were sampled. At each event, we determined the exact area covered by each plant species  and recorded the plant growth stage. To correctly identify the associations of a particular Adelphocoris sp. with a given plant species, we only selected uniform patches or carefully chose single stems of a given plant species for sampling. Plant species were identified using regional weed guides Adelphocoris sp. were found were defined as ‘host plants’ of the respective species Adelphocoris sp. were regarded as the species’ ‘dominant hosts’ The sampling protocol was adapted from an existing one Adelphocoris sp., the average abundance on each plant species was computed on a yearly basis, i.e., by dividing the total number of captured individuals on one plant species by the total area covered by this respective plant throughout the entire sampling period Adelphocoris spp. The abundance of each Adelphocoris sp. was compared between different dominant plant species using a two-way un-replicated ANOVA with a Friedman's test, with years and plant species as fixed factors. A Chi-square test was performed to compare the rate at which overwinter host plants were also used as spring hosts between the three Adelphocoris species. All of the statistical analyses were performed using SAS software For each A. suturalis, 15 species of host plants were found in the spring . From the analyses of plant distribution and A. suturalis abundance on these 15 plant species, alfalfa Medicago sativa L. , and four weeds Cnidium monnieri (L.) Cuss. (0.17), Kochia scoparia (L.) Schrad (0.07), Humulus scandens (Lour.) Merr. (0.02), and Chenopodium album L. (0.02) were regarded as the major spring host plants. From a total of 22 A. suturalis overwinter hosts, 8 species were confirmed as spring host plants, including C. album, H. scandens, K. scoparia, M. sativa, Prunus armeniaca L., Prunus persica (L.) Batsch, Salsola collina Pall., and Vitis vinifera L. . The second highest abundance was 0.34 individuals per m2 on another pasture crop Melilotus suaveolens Ledeb, and those on all of the other host species were less than 0.01. From a total of 12 overwinter host plants, 4 species  were found to be A. lineolatus’s spring host plants . Chinese date, Z. jujuba, was considered a key spring host plant because of its large growing area and the high abundance of A. fasciaticollis , and the population abundance on 4 host species, including Morus alba L., P. armeniaca, Crataegus pinnatifida Bge., and Pyrus bretschneideri Rehd, was less than 0.01. Among 11 A. fasciaticollis winter hosts, 9 species were regarded as its spring hosts, including Artemisia argyi Levl. et Vant., Artemisia scoparia Waldst. et Kit., H. scandens, K. scoparia, P. armeniaca, P. bretschneideri, S. collina, V. vinifera, and Z. jujuba  and 33.3% (4/12) of their overwinter plants. However, for A. fasciaticollis, 81.8% (9/11) of overwinter plants were also used as early-season hosts. The extent of using overwinter plants as early-season hosts significantly differed between the three Adelphocoris spp. . Additionally, 5 plant species, including Cirsium setosum (Willd.) MB., H. scandens, Lepidium sativum L., M. sativa, and S. collina, were shared as early-season host plants by all three mirid bug species , Lygus hesperus Knight, Apolygus lucorum (Meyer-Dür)) in the spring have been determined A. suturalis, 7 species for A. lineolatus, and 35 species for A. fasciaticollis in northern China. Several early-season host plants had been previously identified for these Adelphocoris spp. in China Adelphocoris spp. and plants, their results cannot be used to explore between-species differences in distribution and seasonal occurrence. Our present study effectively complements previous work because all three Adelphocoris spp. coexist at similar population levels at the study site For A. suturalis, A. lineolatus and A. fasciaticollis, respectively A. suturalis, A. lineolatus and A. fasciaticollis to 270, 245 and 127 species, maintaining the previous interspecific differences in host breadth , carrot (Daucus carota L.), garland chrysanthemum (Chrysanthemum coronarium L.), celery (Apium graveolens L.), alfalfa and hairy vetch (Vicia villosa Roth), are cultivated to a large extent Stark differences were found between the host breadth of the three ectively . In the A. lineolatus, alfalfa was the principal early-season host plant and is also an important overwinter host for this species A. lineolatus in one of China’s key cattle growing areas  A. lineolatus adults greatly prefer alfalfa to other host plants A. lineolatus adults gradually migrate back to the alfalfa fields A. lineolatus, which greatly affects its distribution and phenology.For A. fasciaticollis, the early-season host range was similarly as broad as the overwinter host range, with 35 plant species reported as overwinter hosts A. fasciaticollis, but because no individuals were found on other fruit trees, such as P. persica and Malus domestica Borkh., the A. fasciaticollis life cycle may be mainly restricted to Chinese date A. fasciaticollis is mainly confined to this part of the country For Adelphocoris species exhibited varying levels of fidelity to their overwinter host plants, with the (comparative) specialist A. fasciaticollis exhibiting the greatest extent of host fidelity. This finding supports the above general viewpoint on host fidelity of phytophagous insects.During the host-plant selection process of phytophagous insects, the successful colonization of suitable host plants is pivotal for their individual survival and population build-up. For specialist insect species, it may be more difficult and dangerous to change food plants and seek a new host than for generalists A. suturalis and A. lineolatus, that use an entirely new set of plants for early-season development, host switching could constitute an additional mortality factor Adelphocoris spp. on different hosts can thus help explain between-population differences in many life-history traits.Host fidelity does not necessarily imply increased survival because host switching can cause additional mortality. Even for species that use overwinter host plants for spring development, survival rates can be as low as 30% A. suturalis and A. lineolatus, a relatively small set of host plants was recorded during the spring compared to their overall host range, which comprises 270 and 245 species, respectively  nymphal development versus physical attributes that provide shelter for winter eggs. Nevertheless, the large differences in host plant ranges of both A. suturalis and A. lineolatus between subsequent seasons must be further analyzed. More precisely, the relationship between (autumn) adult oviposition preference and offspring performance merits further study Our work shows large seasonal variability in host usage patterns. For ectively . A. fascAdelphocoris spp. complete their first generation on early-season host plants, these plants act as important sources for subsequent infestation of cotton and other summer agricultural crops. Hence, strategic management of early-season host plants could lead to important reductions of those summer populations. For instance, in the United State, broadleaf weeds are the main early-season host plants of the tarnished plant bug L. lineolaris before its movement into cotton fields L. lineolaris numbers in cotton fields Adelphocoris spp. in cotton agroecosystems in China.Because