Bartonella species are facultative intracellular bacteria and recognized worldwide as emerging zoonotic pathogens. Bartonella were isolated or identified by polymerase chain reaction (PCR) in bats and their ectoparasites worldwide, whereas the association between them was scarce, especially in Asia. In this study, a retrospective analysis with frozen samples was carried out to identify the genetic diversity of Bartonella in bats and their ectoparasites and to investigate the relationships of Bartonella carried by bats and their ectoparasites. Bats and their ectoparasites (bat flies and bat mites) were collected from caves in Hubei Province, Central China, from May 2018 to July 2020. Bartonella were screened by PCR amplification and sequencing of three genes . Bats, bat flies, and bat mites carried diverse novel Bartonella genotypes with a high prevalence. The sharing of some Bartonella genotypes between bats and bat flies or bat mites indicated a potential role of bat flies and bat mites as vectors of bartonellae, while the higher genetic diversity of Bartonella in bat flies than that in bats might be due to the vertical transmission of this bacterium in bat flies. Therefore, bat flies might also act as reservoirs of Bartonella. In addition, human‐pathogenic B. mayotimonesis was identified in both bats and their ectoparasites, which expanded our knowledge on the geographic distribution of this bacterium and suggested a potential bat origin with bat flies and bat mites playing important roles in the maintenance and transmission of Bartonella. These bats were collected with mist nets, which were settled near the entrance of karst caves at sunset when bats left roosts for night feeding, and bats were collected in the next early morning. Captured bats were put in a bag, sacrificed by inhaling of ethyl ether in the field, and then transported back to the laboratory on ice as soon as possible. Once arrived at the laboratory, bats were checked for ectoparasites with forceps, with bat flies in the fur and bat mites on the membrane wings. After collection of ectoparasites, thoracic and abdominal organs of bats were collected. All the specimen were stored at −80℃ until use. Bat species were preliminary identified by morphology, and then confirmed by polymerase chain reaction (PCR) amplification and sequencing of the cytochrome B (cytB) gene as described previously  and mitochondrial 16S rRNA genes . 2, 4 μl 2.5 mM dNTP mixture, 5 μl 10 μM each forward and reverse primer , 27.75 μl nuclease‐free water, and 5 μl sample DNA. PCR was performed with one denaturation cycle at 95˚C for 5 min followed by 40 cycles at 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 90 s, and an additional final cycle at 72˚C for 10 min. Each PCR assay included nuclease‐free water as a negative control. PCR products were analyzed by 1.2% agarose gel electrophoresis. Bands of expected size were excised from the gels, and purified using a Gel Extraction Kit . The purified amplicons were cloned into the pMD19‐T vector , and at least three positive clones were selected for sequencing with the universal primers M13‐47/ M13‐48. Chromatograms were checked with Chromas 2.5.1  to ensure sequencing accuracy. The sequences were compared with those in public databases using the Nucleotide Basic Local Alignment Search Tool (BLASTn) on the website of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi).The PCR reaction was performed in a 50 μl mixture containing 0.25 μl 5 U/μl TaKaRa Ex Taq , 5 μl 10× Ex Taq buffer, 4 μl 25 mM MgCl2.4Sequences of interest were imported into MEGA 7.0, and primers were removed after alignment with ClustalW. Phylogenetic trees were constructed based on the neighbour‐joining method using the Kimura 2‐parameter model in MEGA 7.0, and bootstrap values were calculated using 1000 replicates.2.5Bartonella prevalence by bat species, and differences were statistically significant if p‐values <.05.Since information on the age, sex, reproduction status, or ectoparasite intensity of bats was not collected in this study, statistical analyses were not performed for these factors. Chi‐squared test and Fisher's exact test were used to evaluate the 33.1Frozen bat tissue samples and bat ectoparasites used in this study were collected from bats sampled from three cities  in Hubei Province, Central China, during May 2018 to July 2020. In each of the three cities, bats were sampled once from a single karst cave  in the family Nycteribiidae. Based on the COI gene, the three bat fly species shared 98.3%, 98.3%, and 96.4% nucleotide identity with Penicillidia monoceros (GenBank accession number: MW590972), Nycteribia sp. (Korea) (GenBank accession number: MT362944), and Phthiridium hindlei (GenBank accession number: AB632568), respectively. Based on the 16S rRNA gene, the three bat fly species shared 98.2%, 95.8%, and 96.3% nucleotide identity with P. monoceros (GenBank accession number: AB632585), N. pleuralis (GenBank accession number: AB632581), and P. hindlei (GenBank accession number: AB632587), respectively. Therefore, the three bat fly species found in this study were identified as P. monoceros, Nycteribia sp. (Korea), and a potentially novel Phthiridium sp.  and Miniopterus schreibersii (3) based on the cytB gene.In September 2019, bat flies were collected from 16 bats sampled from a karst cave in Jingzhou, Hubei Province, China. Initially, these bat flies were divided into 16 pools . Based on the Bartonella analysis.Due to inability to identify bat flies and bat mites using morphological keys, molecular identification was performed to confirm their species. To meet the requirement of DNA extraction, for ectoparasites collected from Songzi in 2018, and Xianning in 2020, big bat flies were individually processed, while small bat flies and bat mites were randomly pooled for M. davidii (2) and Myotis adversus (4) based on the cytB gene. Based on the COI and 16S rRNA genes, the 15 big bat flies were identified as P. monoceros, and the one pool of small bat flies was identified as Nycteribia sp. (Korea)  were collected from six bats from a karst cave in Songzi City, Hubei Province, China. The six bats were identified as  Figures , Table 1cytB gene, the 203 bats were identified as M. davidii (105), M. adversus (29), and Rhinolophus pusillus (69). Bat flies collected from these bats included 119 big bat flies and six small bat fly pools (approximately 20 bat flies per pool). Based on the COI and 16S rRNA genes, the 119 big bat flies were all identified as P. monoceros, and the small bat fly pools consisted of Nycteribia sp. (Korea) and Phthiridium sp. , and Eyndhovenia sp. of this study shared 90.3% nucleotide identity with Eyndhovenia euryalis (GenBank accession number: EU784906). Based on the 16S rRNA gene, Spinturnix sp. of this study shared 96.7% nucleotide identity with S. myotis (GenBank accession number: FJ225969), and Eyndhovenia sp. of this study shared 94.3% nucleotide identity with E. euryalis (GenBank accession number: EU784846). Therefore, Spinturnix sp. and Eyndhovenia sp. identified in this study represented two novel species . Phylogenetic analysis based on the 3.2Bartonella positive rates for bats collected in 2018, 2019, and 2020 were 20.0% (2/10), 18.8% (3/16), and 10.3% (21/203), respectively. For individual big bat flies (P. monoceros) collected in 2018 and 2020, Bartonella infection rates were 66.7% (10/15) and 50.4% (60/119), respectively. One small bat fly pool of 2018 (Nycteribia sp.), 16 bat fly pools of 2019 , six small bat fly pools (Nycteribia sp. and Phthiridium sp.), and 11 bat mite pools (Spinturnix sp. and Eyndhovenia sp.) of 2020 were all positive for Bartonella . For individual big bat fly samples, they were firstly screened with the gltA gene. Based on the gltA gene, Bartonella identified in individual big bat flies clustered into only two groups. Therefore, representative samples were further selected for Bartonella characterization by the rpoB and ftsZ genes to reduce workload (Table For pooled samples (bat flies and bat mites) and bats, all the samples were screened for ad Table .Bartonella, clustered with previously reported bat/bat fly‐borne bartonellae. Based on the gltA gene, some Bartonella genotypes identified in this study were closely related to those identified in bats from China, as well as in bat flies from South Korea. Based on the rpoB gene, some Bartonella genotypes identified in this study were clustered with those identified in bats from other places of China as well as from other countries such as Georgia and Japan. Based on the ftsZ gene, some Bartonella genotypes identified in this study were clustered with bat‐borne Bartonella from China, Georgia, Japan, and Kenya  and the rpoB fragment (825 bp) share less than 96.0% and 95.4% sequence similarity, respectively, to those of validated species. According to this criterion, bartonellae identified in bats and their ectoparasites (bat flies and bat mites) in this study could be divided into seven and 13 Bartonella genotypes by gltA and rpoB, respectively, among which three Bartonella genotypes  defined by gltA, and 7 Bartonella genotypes  defined by rpoB represented novel genotypes of Bartonella. Based on the gltA gene, three, six, and three Bartonella genotypes were identified in bats, bat flies, and bat mites, respectively, and based on the rpoB gene, there were 4, 10, and 5 genotypes, respectively. Genotypes of Bartonella identified in bats  were shared by their ectoparasites . Some genotypes of Bartonella were identified in bat flies and bat mites, but not in bats in this study   identified in this study in both bats and their ectoparasites (bat flies and bat mites) shared 95.1%−95.8% nucleotide identity with B. mayotimonesis (GenBank accession number: FJ376736), the causative agent of human endocarditis. It was close to the cutoff value (95.4%) by the gene‐sequence‐based criteria for Bartonella species definition for rpoB; therefore, Bartonella genotype (XI) could be identified as B. mayotimonesis  was statistically significant . Exactly, significant difference of the Bartonella prevalence was found between M. davidii and R. pusillus .Statistical analyses of 3.5cytB), MZ350230‐MZ350231, MZ483869, and MZ483871‐MZ483872 (bat fly COI), MZ502972‐MZ502976 (bat fly 16S rRNA), MZ483874‐MZ483876 (bat mite COI), MZ238086‐MZ238089 (bat mite 16S rRNA), MZ208681‐MZ208702 (Bartonella gltA) MZ208739‐MZ208766 (Bartonella rpoB), and MZ208703‐MZ208735 (Bartonella ftsZ).The representative sequences of this study were deposited in the GenBank database with the accession numbers: MH888178 and MW085077‐MW085079  and 12.8% . Our previous study showed that the infection rate of Bartonella in bat blood from Shandong Province in East China was 25.2% (27/107) by gltA amplification  in Peru and 45% (36/80) in Belize by gltA amplification  identified in this study in bats, bat flies, and bat mites shared 95.2%−95.7% nucleotide identify with B. mayotimonesis (GenBank accession number: FJ376736), which was close to the cutoff value (95.4%) of the gene sequence‐based criteria for Bartonella species definition proposed by La Scola et al.  shared 91.2%−92.6% nucleotide identify with B. mayotimonesis (GenBank accession number: FJ376732), which was well below the cutoff value (96.0% by gltA). The inconsistency of the gltA and rpoB gene might be explained by recombination events as observed in rodent‐borne bartonellae  . With the exception of Bartonella identified in bats from the United States that shared 99.7% nucleotide identity with B. mayotimonesis (GenBank accession number: FJ376732), Bartonella found in bats from European countries  shared 91.2%−92.6% nucleotide identity with B. mayotimonesis  in the family Nycteribiidae were identified in this study, with Phthiridium sp. as a potentially novel bat fly species.Bat flies are obligate bloodsucking ectoparasites that commonly parasitize in the fur and wing membranes of bats. Bat flies are divided into two families: the wingless, spider‐like Nycteribiidae, and the more traditionally fly‐like Streblidae  in the family Spinturnicidae were morphologically described in China, with four species in the genus Spinturnix   were identified in the big bat fly (P. monoceros), while for the small bat fly pools , Bartonella genotypes  were identified  emerging from the pupaFigure S1 Bat fly Table S4 Estimates of evolutionary divergence between Click here for additional data file.


Frontal lobe function may not universally explain all forms of attention deficit hyperactivity disorder (ADHD) but the frontal lobe hypothesis described supports an internally consistent model for integrating the numerous behaviors associated with ADHD. The paper examines the developmental trajectories of frontal and prefrontal lobe development, framing ADHD as maturational dysregulation concluding that the cognitive, motor, and behavioral abilities of the presumptive majority of ADHD children may not primarily be disordered or dysfunctional but reflect maturational dysregulation that is inconsistent with the psychomotor and cognitive expectations for the child’s chronological and mental age. ADHD children demonstrate decreased activation of the right and middle prefrontal cortex. Prefrontal and frontal lobe regions have an exuberant network of shared pathways with the diencephalic region, also having a regulatory function in arousal as well as with the ascending reticular formation which has a capacity for response suppression to task-irrelevant stimuli. Prefrontal lesions oftentimes are associated with the regulatory breakdown of goal-directed activity and impulsivity. In conclusion, a presumptive majority of childhood ADHD may result from maturational dysregulation of the frontal lobes with effects on the direct, indirect and/or, hyperdirect pathways. We think that attention deficit hyperactivity disorder (ADHD) results from differences, when compared with the normally developing child, in the trajectory of cortical maturation and well as from deviations in the trajectory of asymmetric brain development . These din utero or early in post-partum development (The beginning of the brain’s developmental interregional communication differences in ADHD as compared with neurotypical children has been thought to commence elopment . The rigelopment . Early celopment . Asymmetelopment . After 3elopment . These felopment , 2015. Telopment , 2015.This one-side-at-a-time developmental activity of the hemispheres is thought to be an important factor that is highly associated with the development and lateralization of the brain in infancy and early childhood . This asIn order to speed-up brain responsivity to external or internal voluntary action control, fronto-basal ganglia pathways must play a significant role in the control of voluntary action and in motor response inhibition. Response inhibition can be facilitated by a fast hyperdirect pathway that would connect the right inferior frontal gyrus and the pre-supplementary motor area with the subthalamic nucleus or, through the indirect pathway between the cortex and caudate. These considerations are explored further below.The brain develops from the bottom up starting in the lower brainstem and with the brainstem nuclei acting as precursors to higher levels of brain development and with the ultimate development of Brodmann areas that have both structural and functional differences . Once thThe original model by All of these circuits project from a specific area of the cortex to the basal ganglia and from there to the thalamus then returning to the cortex . Each onThere exists an additional pathway that plays a significant role in oscillating between direct and indirect pathways and is critical to this dynamic balance between these pathways and behavioral flexibility. This is termed the hyperdirect pathway and it originates from the right cerebral hemisphere alone . There aThe hyperdirect pathway suppresses unwanted movement and it will subsequently inhibit movement once an action has been completed . If therTherefore, in ADHD, we can see that many of the symptoms can be explained by overactivity of the left hemisphere’s connections to the direct pathway related to the underdevelopment and underactivity of the right hemisphere and the indirect and hyperdirect pathways  This canNeuroimaging studies have led us to theorize that the fundamental differences between rest and agency can be based on an organized level of baseline activity that is diminished during goal-oriented cognition. It has also been thought that the brain maintains a “default mode” in the absence of cognitive demands  so as toA meta-analysis  identifiDifferent groups  have disThe CEN is usually related to the appropriate functioning of the PFC and related regions such as the cingulate cortex . The CENBoth the CEN and DMN are lateralized . The CENAdditionally, not only is there a reported decrease in pain perception  as well The frontal lobes exemplify a complex neurological system. The prefrontal cortex is integrated within the frontal lobes and is thought to combine intentional responses that require intended and synchronized action sequences . FrontalThere are three classes of neuropsychological functioning associated with the prefrontal cortex: regulatory, social, and executive . The preFrontal lobe lesions in adults allows us to observe hyperactivity control mechanisms more readily . HyperacADHD as a manifestation of maturational dysregulation has been largely supported by MRI studies. Volumetric measurements of right and left hemispheres, of gray and white matter within each lobe, and cerebral and cerebellar volume have been reported to be approximately 4% smaller in ADHD individuals relative to controls . SignifiMore evidence in support of widespread volumetric reductions in ADHD subjects comes from cross-sectional studies comparing ADHD and control subjects in smaller samples than in the above studies (see reviews Cognitive and motor affect assessment in the context of the frontal lobe hypothesis of ADHD has been partly obstructed by argument about the developmental stage at which functioning of the frontal lobes matures. Earlier, The issue of developmental trajectories is singularly important as it frames the disorder of ADHD as a maturational dysfunction. The result, therefore, is that the cognitive and behavioral abilities of the ADHD child are not disordered or dysfunctional, but are rather developmentally inappropriate for the child’s chronological and mental age.Compared to neurotypical children, those with ADHD demonstrate decreased activation of the right and middle prefrontal cortex across all age groups . HoweverThe original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Both authors shared equally in preparation of the manuscript, contributed to the article, and approved the submitted version.


Bipolar Disorder (BD) and Attention Deficit and Hyperactivity Disorder (ADHD) are mental disorders with high degree of lifetime comorbidity. Both BD and ADHD are disorders with onset in childhood and early adolescence. Both disorders are often undiagnosed, misdiagnosed, and sometimes overdiagnosed, leading to high rates of morbidity and disability. The psychiatric and behavioral symptoms associated with ADHD and BD have significant overlap. Albeit the existence of a large body of literature, it is far from being clear whether comorbidity can be explained by the confounding overlap of operationally defined criteria or whether it reflects a genuine comorbidity of two biologically distinct disorders. The aim of this paper is to recognize and/or differentiate the pattern of ADHD across the course of BD from a nosological point of view, focusing on specific clinical and neurobiological dimensions. We found that some critical issues may help to fulfill the purpose of our perspective. We suggest that the relationship between ADHD and BD, based on clinical, developmental, and epidemiological commonalities, can be better clarified using four different scenarios. Bipolar Disorder (BD) and Attention-Deficit/Hyperactivity Disorder (ADHD) are often recognized as mental disorders with a high degree of comorbidity –4. BipolADHD is defined by the DSM-5 as an early-onset “neurodevelopmental disorder defined by impairing levels of inattention, disorganization and/or hyperactivity-impulsivity” . ADHD isThere is an overlap between symptoms of ADHD and BD, and in particular (hypo) manic mood episodes features, such as hyperactivity, distractibility, lack of inhibition, restlessness, racing thoughts, rapid speech, talkativeness, and irritability. The overlap of symptoms and diagnostic criteria could make the distinction between the two disorders difficult, even if ADHD is a disorder with persistent symptoms, whereas symptoms in BD are episodic , 11, 12.Many children are diagnosed with comorbid ADHD and PBD; comorbidity rates are estimated to be around 20% , 4, and In this perspective paper, we focused on specific clinical and developmental dimensions in order to recognize and/or differentiate the pattern of ADHD across the course of BD in a nosological perspective. We found that some issues may help to fulfill the purpose of our perspective; we described the results in sections according to the crucial points raised.There is a well-established psychopathological bond between ADHD and BD, even if some studies show controversial results regarding their longitudinal relationship. Rates of comorbidity are very heterogeneous; this is influenced by several aspects. Interestingly, a difference can be detected between continents. For BD, it was suggested that the prevalence may be higher in the Americas compared with Asia and European nations , 14; thiThere are genetic implications involved in ADHD and BD comorbidity. Relatives of patients with BD had a significantly higher chance of having ADHD, and among relatives of ADHD patients BD occurred more frequently ; the relShared additional risk factors are related to the prenatal, perinatal, and childhood periods. Maternal substance abuse exposes children to both conditions, and one study showed that this risk factor can be associated with the symptoms of ADHD, but not with a clinical diagnosis , 23. MatThe clinical course of ADHD to adulthood is characterized by the development of several psychiatric comorbidities during the transition from childhood/adolescence, impairment of social functioning, and deviant or rule-breaking behaviors. In adolescence, distinct cognitive profiles may emerge both reflecting continuation or potential recovery of ADHD, with or without an emotional dysregulation profile . Five diSeveral clinically informed factors such as gender, pre-mature birth, maternal education, school readiness, peer and conduct problems, cognitive and temperamental factors, early comorbidities, social context and medication could differentiate individuals following different ADHD symptom trajectories , 30. GenA large body of literature –30 suggeA 10-year follow-up study by Biederman et al.  demonstrA recent global systematic review and meta-analysis  estimateMany studies, including systematic reviews, meta-analyses, prospective and retrospective studies, have evaluated the developmental trajectory ADHD-BD , 35–38.A recent systematic meta-analytic review  estimatePredictors of BD, considering the psychopathological characteristics that forewent the disorder, could be evaluated following the model proposed by Faedda et al. . More spSeveral conditions and psychiatric comorbidities elevate the risk of progression from ADHD to BD. Among these are family history of BD, older age, Major Depressive Disorder (MDD), Anxiety Disorder, Autism Spectrum Disorder (ASD), Intelligence Disability (ID), Disruptive Behavior Disorder (DBD), Oppositional-Defiant Disorder (ODD), Conduct Disorder (CD), criminal behavior, Alcohol Use Disorder (AUD), and Cluster A or B Personality Disorder , 36, 38.There are significant clinical differences between patients diagnosed with both ADHD and BD and patients with BD without comorbid ADHD. Compared with the “pure” BD counterpart, patients with BD and ADHD have shorter period of wellness and stability , are youA recent systematic review and meta-analysis  reportedHerein, we aimed to focus on specific clinical, developmental and neurobiological dimensions to recognize and/or differentiate the pattern of ADHD across the course of BD from a nosological perspective.We are inclined to propose four scenarios that aim to clarify possible different relationships in clinical and research settings:Overestimated comorbidity of ADHD or BD due to overlap of symptoms ;ADHD can be a prodrome of BD;Comorbidity of syndromatic ADHD with strictly defined BD;ADHD-BD is a full entity.Regarding the first point, some authors , 49 haveConsidering the second scenario, misleading comorbidity may be also related to developmental sequencing, which is an example of “heterotypic continuity” in which the same developmental process has different phenotypes at different stages of life. In this case, ADHD may represent a BD precursor in a heterotypic trajectory. Unfortunately, we are not currently able to accurately predict which patients with ADHD are prone to subsequent development of BD. In fact, on one hand, a specifically increased risk of BD can be confirmed in ADHD patients compared to healthy controls, which is higher than what expected based on the general predisposing effect on other kinds of psychopathology. On the other hand, BD following ADHD may be a specific, pediatric-onset, neurodevelopmentally-based, form of BD, different from adult-onset or older-age (neurodegenerative)-onset BD, despite phenotypical similarities. Early neurodevelopmental disorders, such as co-occurring ADHD and emotional dysregulation, may be considered a precursor in the pathway to neurodevelopmental, early-onset, bipolarity .vice-versa. Lower but still rather high comorbidity rates have been reported at later ages. Therefore, in adult patients with a recent diagnosis of BD, but with an unknown history of ADHD, an accurate assessment for a previous or current diagnosis of ADHD appears mandatory (As regard to the third scenario, although the higher known rates of ADHD and BD comorbidity is in childhood , we suggandatory ; if a hiandatory .The hypothesis of a full entity ADHD-BD, different from both ADHD and BD, is supported by several familial , 54 and Apart from theoretical and nosological issues, therapeutic implications may arise from perspective paper such as the present one.Treatment of concurrent ADHD and BD, regardless of the phase of illness, remains an unresolved challenge; in a developmental perspective view, treatment may require a staged approach. By staging the introduction of treatments, one can reduce the risk of overmedicating patients and better assess the effect of each individual treatment . On the Our perspective is focused on the clinical and developmental aspects of ADHD and BD; genetic, neurobiological, neurocognitive and therapeutic aspects were not part of the aims of this perspective paper. We hope that better clinical and developmental characterization is coming and that parallel efforts through nosology can be useful.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.AC: conception, original draft, and revision. LP, GS, and AP: drafting and collecting data. MP: final revision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Phaseolus vulgaris, one P. lunatus and two Vigna unguiculata landraces from two different climatic backgrounds of the Andean region of South Ecuador and one P. vulgaris commercial cultivar, and we grew them under two different conditions of temperature and humidity (open field and greenhouse). Then, we recorded data for 32 characters of plant architecture, flower and fruit characteristics and yield, and 17 events in the phenology of the plants. We analysed the impact of treatment on species, climatic background, and each of the landraces, and identified both characters and landraces that are mostly affected by changes in their environmental conditions. Overall, higher temperatures were benign for all materials except for two P. vulgaris landraces from cold background, which performed better or developed faster under cold conditions. Finally, we calculated a climate resilience landrace index, which allowed us to classify the landraces by their plasticity to new environmental conditions, and found heterogeneous landrace susceptibility to warmer conditions. Two P. vulgaris landraces were highlighted as critical targets for conservation.The use and conservation of agrobiodiversity have become critical to face the actual and future challenges imposed by climate change. Collecting phytogenetic resources is a first step for their conservation; however, the genetic material must be analysed to understand their potential to improve agricultural resilience and adaptation to the new climatic conditions. We have selected nine  Climate change modifies the trends in temperature and precipitation, thus affecting the response to environmental factors of many species at different geographical levels, with drastic effects and, often, a negative impact on crops3. However, there is a consensus among different projections that indicate negative impacts of the increase in temperatures on the main crops in many agricultural regions4. Moreover, the more updated models with improved and diverse scenarios produce more pessimistic projections for yield responses for maize, rice, and soy bean, although wheat could benefit from higher CO2 concentrations5. Supporting the latter predictions, the analysis of combined published results of different analytical methods highlights the vulnerability of agriculture to climate change, suggesting a yield reduction of around 3.0 to 6.0% for each degree increase in temperature6. Besides the direct effects of temperature and rainfall on crops and plant diseases7, climate change has important economic consequences on agriculture8. However, the impact of climate change effects on crops depends on both the crop identity and its geographic location9.Improving agricultural resilience and adaptation to climate change effects is a priority to ensure crop production and food security in the upcoming years10. Therefore, prior to developing crop improvement or conservation programmes, either to face climate change challenges or to choose appropriate crop varieties that might perform well under certain local conditions, it is indispensable to gain knowledge on the plant plasticity and its potential adaptability to abiotic factors11. Moreover, the study of crop phenotypic plasticity must include the analysis of phenology, in addition to other critical traits related to plant morphology and reproduction, because phenology is highly susceptible to changes in the environmental factors12. Thus, the information gathered in this way might allow: (1) to select those varieties suitable for a particular range of environmental conditions, with important consequences for local community development, sustainable agriculture and food security; (2) it might help to identify those varieties more prone to suffer negative effects, and thus, allowing their conservation before their effective loss due to the abandoning of low yield landraces13; (3) the overall information might provide useful to develop specific indexes aimed at quantifying the resilience potential of either one species or landrace or a particular character.Plants exhibit the ability to cope with changes in their environment via phenotypic plasticity14. Beans (Phaseolus spp) originated in the New World. Common bean (P. vulgaris) had two main domestication centres at Middle and Andean South America, with four major genetic groups in Mesoamerica, Colombia, Northern Andes of Ecuador and north Peru, and the southern Andes; and exhibits both a wide morphological variability , and adaptation to a broad array of environments18. Lima beans (P. lunatus), distributed from northern Mexico to northern Argentina, have three major genetic groups, two in Mesoamerica and one in the Andes (southern Ecuador and northern Peru), which is the most likely origin of the species21. Moreover, there exists great diversity of wild bean species with potential to improve the resistance to environmental factors in common bean crops22. In addition, Latin America represents about the 50% of the bean world production, followed by Africa; per capita consumption of beans in these regions can oscillate between 12 to 60 kg per year and represent a significant source of protein23. In Ecuador, beans, which are commonly named fréjol, fríjol and poroto (P. vulgaris) and torta (P. lunatus), belong to the genus Phaseolus spp., while the cowpeas, named vaina, are Vigna spp., originated in Africa24. Both species represent the main leguminous crops with a soil surface around 32,817 ha devoted for their culture, with an overall production of 27,492 t25. Moreover, as in many other countries, most producers are smallholder farmers that grow beans mainly for self-consume, whether as crop rotation or associated to maize, and thus, beans are an important contribution to Ecuador food sovereignty26.Beans are a major food resource grown worldwide and represent the main source of protein for many societies, thus playing a vital role in the human diet of developing societies27. We lack specific predictions; however, for the impact of climate change effects on the production of legumes for the Andean region, where a negative impact on the production of cereals is expected9. The effects of climate change on the acceleration of phenological aspects of crops can be counteracted by shifting existing varieties into different regions28. Thus, it is pivotal to promote the investigation of the impact of abiotic stresses on landraces; defined by Casañas et al.29 as those “cultivated varieties that have evolved and may continue evolving, using conventional or modern breeding techniques, in traditional or new agricultural environments within a defined ecogeographical area and under the influence of the local human culture”.There are robust projections predicting a generalized decline in crops yield due to the impact of climate change, and highlighting the urgency of further research on the effects of high temperatures and other factors on crops to gain a better understanding on the uncertainties of production impactsPhaseolus spp. and Vigna spp., among other effects, and reduces yield components and biomass31; elevated CO2 concentrations have direct positive effects on stem mass, and a strong genotype × CO2 interaction for pod number, seed mass and yield on P. vulgaris32; and high temperatures negatively affect reproduction, fertilization, and post-fertilization33; Lima beans (P. lunatus); however, are more tolerant to heat than P. vulgaris14.Several studies have focused on the effects that different aspects of climate change and abiotic stress factors have on the common bean. For example, drought, the most extensively studied factor, has drastic effects on legumes because it accelerates plant maturation in P. vulgaris, P. lunatus, and Vigna unguiculata sampled from different localities at the Andes of south Ecuador, and a commercial P. vulgaris cultivar . Similarly, P. lunatus plants exhibited significant higher values in the greenhouse for composite sheet length and width and terminal leaflet width  than in the field ; but the terminal leaflet length performed better in the field compared to greenhouse (medians: 62.36 and 52.02 mm). Post hoc pairwise comparisons for treatment × climatic background highlighted that cold background landraces had higher values for branch orientation angle, composite leaf length and width, and terminal leaflet length in the greenhouse than in the field. Cold background landraces produced wider terminal leaflet widths in the greenhouse while warm background landraces did it in the field  and width (209.95 vs 169.27 mm), and terminal leaflet length (115.26 vs 93.76 mm). P. vulgaris landrace 8 failed to grow in the field. Plant architectural traits were only affected in one P. vulgaris (8) and the P. lunatus (13) landraces. P. vulgaris landrace 1, from cold background, performed better in the field than in the greenhouse. Landrace 8, from cold background as well; however, performed better in the greenhouse than in the field, suggesting a wrong identification of its real origin.Post hoc pairwise comparisons for treatment × species interaction Table , found tGreenhouse plants exhibited significant higher overall mean rank values than field plants for the total number of flowers per plant (medians: 57 vs 11 flowers), the number of pods per plant (medians: 34.5 vs 2.0 pods), the number of pods per infructescence (medians: 1.6 vs 1.4 pods), the number of grains per pod (medians: 4.2 vs 0.0 grains), the dry weight of 100 seeds (medians: 37.46 vs 0.0 g), the gross weight of seeds per plant (medians: 24.92 vs 0.0 g), the net weight of seeds per plant (medians: 24.01 vs 0.0 g), and the number of seeds (medians: 60.0 vs 0.0). On the contrary, the sheath width was larger in the field (12.48 mm) than in the greenhouse .The Kruskal–Wallis analyses of the treatment × species, treatment × climatic background (except for sheath length) and treatment × landrace interactions were significant for all the traits Table . Post hold Table  for: theld Table  for the In the treatment × climatic background, post hoc pairwise comparisons Table  found siP. vulgaris landraces 2, 4, 5, 8 and 9, and in V. unguiculata 10. Analogously, more pods per plant were produced in the greenhouse for P. vulgaris landraces 2, 4, 5 and 8, and in V. unguiculata 10. The total number of seeds produced in the greenhouse was higher than in the field for P. vulgaris landraces 2, 4, 5, 6 and 8, both V. unguiculata 10 and 11, and P. lunatus 13. P. vulgaris 3 produced higher mean rank values for the 100 seeds weight in the greenhouse than in the field. P. vulgaris landraces 5 and 6 (both from warm background), however, failed to produce enough flowers, pods, or seeds. Landrace 1, from cold background, failed to prosper in the greenhouse, and landrace 8 did not grow in the field. Otherwise, all other significant values highlighted the positive effects of warmer conditions compared to the field acting on flower, fruit, and yield characteristics  faster than P. vulgaris and P. lunatus. However, V. unguiculata was the slowest species producing the flowering period , and produced more seeds per pod (9 seeds) than P. vulgaris (3.6 seeds) or P. lunatus (2.1 seeds), for other yield related traits produced lower values than the Phaseolus sp. The presence of the first shoot visible had higher mean rank values (appearance of the character delayed in time) in the field than in the greenhouse for all the species. In P. vulgaris, all significant comparisons confirmed that field conditions delayed the development of such traits, except for the fully ripe pods, which was delayed in the greenhouse for P. vulgaris and V. unguiculata . In addition, P. lunatus produced nine or more leaves unfolded later in the field than in the greenhouse (medians: 60 vs 51 days).The Kruskal–Wallis analyses of the treatment × species, treatment × climatic background and treatment × landrace were significant for all traits (Table 2). Post hoc analysis for treatment × species Table  found thPost hoc analysis for the treatment × climatic background Table , found sP. vulgaris landraces 1, 4, 6, 7 and 9, P. lunatus 13 and both V. unguiculatus 10 and 11. P. vulgaris landrace 2 finished the flowering and the fully ripe pods later in the greenhouse than in the field; and landrace 8 failed to grow under field conditions. The development of nine or more leaves for P. vulgaris landraces 3, 7 and 9 and P. lunatus, and the end of flowering for P. vulgaris landraces 1, 3, 4 and 7 were delayed in the field. Other significant differences in character expression had higher values in the field : the number of flowers per plant, the number of pods per plant, the number grains per pod, the grain length, width and thickness, the scar length, the 100 seeds weight, the gross and net weight of seeds/plant and the number of seeds. Phenologically, the most affected traits were: cotyledons completely unfolded, two full leaves unfolded, the unfolding of nine or more leaves, the emergence of the first shoot, first flower buds visible and enlarged, first petals visible, the end of flowering and the pods fully ripe.P. vulgaris landrace 8 (0.688) was highly susceptible, and landraces 1 (0.396) and 6 (0.250) and V. unguiculata landrace 11 (0.354) were very susceptible to changes in their environmental conditions. P. vulgaris landrace 12 and the commercial variety 9 were the most resilient to environmental conditions . When using this index for species, P. vulgaris was the most susceptible (0.479) and P. lunatus the most resilient (0.146) to treatment. Warm background landraces were more prone to accumulate significant differences in their traits (0.404) compared to cold background (0.298) or the commercial cultivar (0.234).The climate resilience landrace index  boosting natural soil conservation (maintenance of covered soils) and fertilisation with legume crops during fallow or crop rotation periods, either in the field or in the greenhouse; (2) in situ conservation of landraces; and (3) extra benefits for small farmers .Changing environmental conditions had a direct impact on more than half (60.4%) of the 48 analysed characters. The most informative results; however, are those related to the interaction of treatment with species, climatic background, and landraces. The treatment affected the three species differently. Moreover, some characters exhibited higher levels of variation across species and landraces than others in response to the treatment: the number of flowers and seeds per plant, the gross and net weight of seeds and the emergence of the first side shoot visible were the most plastic traits across species. In addition, we found three P. lunatus was the more tolerant species and P. vulgaris the more susceptible to changes in environmental conditions, confirming previous findings14. While warm conditions affected one architectural trait in P. vulgaris and four traits in P. lunatus, it had not effects on V. unguiculata.P. vulgaris and V. unguiculata, and most of the phenological differences were found in P. vulgaris. Cold background landraces produced larger chalice lengths, thus suggesting either a potential adaption of cold background landraces to cold environmental conditions, related to the protection of the reproductive organs or facilitating the accumulation of heat trapped within the flower structure with micro greenhouse-like effects36. Warm conditions boosted seed and yield traits in the warm background and the commercial cultivars, supporting that warm background landraces are better adapted to temperatures higher than in the field and that the commercial cultivar performs better in warmer conditions. All the phenological characters involving flower development were delayed in the field, independently of the climatic background of the landrace. The fully ripe pods happened later in the greenhouse than in the field for both cold and warm background landraces, suggesting that warmer conditions might impose higher demands of water and storage of products in the grains, thus, prolonging the time for maturation.The major effects of the treatment on characters related to reproduction and yield happened in P. vulgaris landraces that are sensible  and one landrace more resilient (12) to environmental changes. This could lead to developing specific in situ and ex situ conservation strategies. While many landraces are grown locally by the farmers, others might be present as the result of seed exchange activities  or migration of people. Flower and fruit characteristics and yield were improved in the greenhouse conditions, except for P. vulgaris landrace 1. Colder conditions delayed the emergence of many phenological traits except for the end of flowering of P. vulgaris landrace 2, from cold background, supporting its cold climatic background origin.We identified, through using the CRLI, three The first side shoot visible was the most widely affected trait by the environmental changes across landraces, highlighting the overall delay in vegetative growth in the field, and suggesting potential trade-offs in resource allocation. Warmer conditions increased stem diameter, the composite sheet length and width, and the terminal leaflet length. The latter might highlight both that some landraces had higher optimal temperatures of growth that the ones faced in the field, and that the temperatures reached in the greenhouse  are not high enough to promote plant respiration over photosynthesis. The former hypothesis seems plausible when interpreting the results after analysing the interaction between treatment and climatic background of the landraces. Moreover, that some cold background landraces performed better at higher temperatures than warm background landraces suggests that the later have reached an adaptive plateau that might not be surpassed. A potential explanation is that cold background materials could have a warm background origin, thus suggesting the later migration of the landraces by farmers through seed exchange and commercialization.37. The values found for P. vulgaris traits, are within the expected variation among previously studied landraces and cultivars38. However, differences in the timing of flowering traits among P. vulgaris landraces might alter gene flow and promote reproductive isolation; thus, affecting their evolutionary dynamics39. P. lunatus was the most productive species; moreover, P. lunatus might be considered as a potential species to grow in greenhouses and replace other crops, economically less attractive, thus, adding a value to food security and soil fertilisation.As expected, we found differences in performance and phenology among species supporting previous findingsP. vulgaris, where an increase of temperature affected negatively the number of seeds per pod, seed size and yield41. Moreover, the higher production of flowers and, thus, seeds under greenhouse conditions might be partially explained by the protection against winds that affect flower survival in the open field. Previous work; however, found that very high temperatures reduced the values for many reproductive traits33; but the latter was tested in a climate chamber a range of temperatures much higher than the ones reached in our greenhouse. The lower median values for the dry weight of the seeds in the open field; however, might be due to trade-offs between investment in reproduction and resistance against harsh abiotic or biotic conditions. Furthermore, the positive relation between temperature and seed weight was previously observed in V. unguiculata42. It is noteworthy to mention, that our results highlighted the importance of the climatic background from where the landraces come from as a fixed factor because it allowed us to better interpret the actual impact of temperature on crops and their landraces. Thus, we found that cold background plants produced the highest number of flowers and seeds and that warm background landraces performed better in the greenhouse than in the field. The behaviour of the commercial cultivar, like cold background landraces, suggests its potential cold background origin.The greenhouse conditions had a positive effect on the production of flowers and on many important components related to pods and yield. We did not expect these positive effects after previous findings in 44. Thus, global warming might be critical in several Andean crops, such as potato, which is highly sensible to high temperature45. Moreover, in addition to a negative impact on yield and quality, the rise of global temperatures will reduce the areas suitable for other crop species, such as coffee (Coffea arabica)46. In P. vulgaris, the advance of the flowering stage in the field due to warmer conditions highlighted the critical problem of flower-pollinator coupling48. Early flowering might respond to different environmental pressures and, for example, in oat Mediterranean landraces, it represents a potential mechanism to scape terminal drought44. However, we found that warm background landraces developed faster in the greenhouse than in the field for some characters, which might represent important adaptive characteristics  suggesting a fine-tuned genotype × environment interaction. Notwithstanding, most of the phenological traits developed later in the field for both the cold and warm background landraces. These results make us think of colder conditions imposing more costs to the plant, and then, architectural and reproductive traits develop slower in colder than in warmer conditions, where the latter facilitates fruit and seed maturation.Warmer conditions accelerated the phenological development of plants for many of the analysed traits, although the day when the pods are fully ripped, was delayed in the greenhouse. This trend has been reported in other crop species but exhibiting a negative effect on crop yieldP. vulgaris landrace 1, which seems to be very sensible to heat, or P. vulgaris landraces 6 and 8, more sensible to colder conditions than the other landraces. Moreover, this CRLI index highlighted P. vulgaris landrace 12 as the most resilient against environmental changes. The CRLI index, then, might be a powerful preliminary approach before identifying potential QTLs or traits for new breeds or genetic improvement, as well as a help for decision-making institutions or management tools23.The ecological background from where the genetic material comes from Table  plays a 49. Our work further emphasizes the importance of identifying the ecological antecedents of the landraces or crop populations because these data, being part of the characterization and later analysis, might become a robust tool for their conservation. Furthermore, the identification of appropriate standardised agromorphologic descriptors and phenological scales for each species might improve the use of plant passport data53. Finally, the characterization of the agromorphological and physiological profiles of a collection of local plant materials can generate resilience against other disasters in addition to climate change, such as the COVID-19 pandemic; since it has caused the shortage of seeds of commercial varieties in developing communities54.The hierarchical clustering of the heatmap for the 13 landraces in both treatments pointed out both that the level of expression of some characters are landrace or species specific, and that many plant morphological characters are very plastic, depending on the environmental conditions in which those traits are measured Fig. . This hiPhaseolus vulgaris, one P. lunatus and two V. unguiculata landraces collected from small farmers from the Andean region of South Ecuador during 2017 and conserved in the UTPL germplasm bank, and one commercial P. vulgaris cultivar. All UTPL landraces were collected from sites with different climatic background conditions from Loja province (Ecuador) except the UTPL-PGR-0798 landrace, collected from El Oro province , V. unguiculata as 10 and 11 (UTPL-PGR-0313 and UTPL-PGR-0317), and P. lunatus as 13 (UTPL-PGR-0230). Cold background landraces are 1, 2, 8, 12and 13; the warm background landraces are 3–7, 10 and 11. The number 9 is the commercial cultivar.We selected nine 55. Then, we first seeded an excess of seed for each landrace in a tunnel nursery at 25 °C and 45% relative humidity for 18 days. For each landrace we transplanted 72 healthy seedlings in each of the two environments (open field and greenhouse), at 50 cm between plants and one meter between rows, and drip irrigation in the greenhouse (up to twice a week to avoid hydric stress). Each environment represented one climatic condition. According to soil analysis performed in the Agrocalidad laboratory (Agencia de Regulación y Control Fito y Zoosanitario), a fertilization program was applied with mineral fertilization based on 12:36:12 (N-P2O5-K2O) as basic fertilization on both environments.We started the experiments on July 19th 2018, and lasted until the end of March 2019 at a field located in the Universidad Técnica Particular de Loja . The site has an altitude of 2160 m.a.s.l., an average minimum/maximum temperature across the year of 12.9 °C and 22.6 °C , an average annual precipitation of 780 mm and a relative humidity of 81.07%. In the same location, we settled a greenhouse with a monthly average temperature of 23.5 °C (21 °C minimum and 26 °C maximum) and relative humidity of 57.62%. Loja corresponds to the low dry montane forest (bs-MB) ecological formationhttp://www.bioversityinternational.org/publications), while integrating the particularity of each studied species: P. vulgaris50, P. lunatus51 and V. unguiculata52, and evaluated 32 morphological characters of plant architecture, inflorescence and fruit characters, and yield. Then, we used the BBCH codifications53 to register 17 phenological stages . Overall, the extracted communalities represented well our variables, with values between 0.500 and 0.966, except for Q1 (0.401), Q4 (0.431) and Q8 (0.336). Eight components had eigenvalue values above 1.000 accounting for a cumulative 80.230% of the variability of our original 48 variables. To know how many factors retain we conducted a parallel analysis58 that suggested seven factors (77.648%).We calculated an explorative correlation matrix between all pairs of the 49 variables and found that Q3 was uncorrelated with any other factor . All other 48 variables accomplished the assumptions for the PCA analysis. The Kaiser–Meyer–Olkin Measure of Sampling Adequacy (KMO) produced a meritorious value of 0.897, after Kaiser’s evaluation, the 13 landraces, the climatic background , and for the interactions between treatment × species, treatment × landrace, and treatment × climatic background using the nonparametric independent samples Kruskal–Wallis test, because the data did not meet the assumptions of the ANOVA. We further conducted post-hoc pairwise comparisons adjusted after Bonferroni. All statistical analyses were conducted in IBM® SPSS® Statistics v. 24.We analysed for differences in plant performance between both climatic conditions (open field vs greenhouse), among species p-values for each of the 13 landraces , and divide each value by the number of traits investigated . This index highlights which landraces are more likely to be affected by different aspects of the climate change effects and, thus, will provide important information about both conservation and agricultural strategies to build up resilience against climate change effects. The closer the index is to zero the lower the impact on the landrace, and the maximum impact is one. Thus, we suggest three categories to interpret these indexes: (1) weak or none effects (strong resilience) for indexes values between 0.00 and 0.33; (2) moderate effects for values between 0.33 and 0.66; and (3) strong or drastic effects  for values between 0.66 and 1.00.The information gathered up in the post-hoc pairwise comparisons about each landrace performance in both climatic conditions allowed us to create and calculate the climate resilience landrace index (CRLI). First, we quantified how many characters exhibited significant post hoc 59. During the pre-processing of the data, we applied unit variance scaling as variance normalization method because we are analysing variables of different units and intensity ranges. Afterward, we followed the Nipals  PCA method. In order to understanding the effects of the treatment (open field vs greenhouse) on agromorphological and phenological variation better, we further constructed a clustered heatmap with the 13 landraces in both environmental conditions. We produced the heatmap using Euclidean distance and complete linkage methods.We conducted a Principal Component Analysis (PCA) and clustering of the 13 landraces based on the mean values of the agromorphological characters using the ClustVis webtoolExperimental research and the field study on plants complied with Ecuadorian research normative .Supplementary Information.


Although ADHD is the most frequently diagnosed psychiatric disorder in childhood, the majority of adults with ADHD are not diagnosed and 90% of the cases remain untreated. One of the main reasons that may lead to the missed diagnosis of ADHD in adults may be the high rate of comorbid psychiatric conditions masking the main symptoms.In this study, it was aimed to present a case who was followed up with the diagnosis of ADHD since childhood and developed psychosis after a recent traumatic life event.A 19-year-old male patient was consulted because of his complaints of persecution delusions, and disorganized speech that started 2 years ago. IIt was learned that the first psychiatry application of the patient was 10 years ago with complaints of impulsivity, aggression, increased psychomotor movements, and methylphenidate treatment was started during this period. The patient, whose current clinical picture was evaluated as psychosis, was discharged after the symptoms subsided with paliperidone depot 100mg/month treatment after hospitalization. It is understood that his psychotic complaints completely regressed in the follow-ups.It is stated that approximately 80% of adult ADHD cases have at least one accompanying psychiatric disorder. However, there are limited studies in the literature on the relationship between psychotic disorders and ADHD.Recent studies indicate that beyond the fact that ADHD is a feature of the schizophrenia prodrome, ADHD diagnosis may be associated with an increased risk of psychosis in the future. Therefore, this association can be better clarified in further studies on comorbidities.No significant relationships.


Bartonella infections in small rodents in the Shangdang Basin, China. Small rodents were captured using snap traps, and their liver, spleen, and kidney tissues were harvested for Bartonella detection and identification using a combination of real-time PCR of the ssrA gene (296 bp) and conventional PCR and sequencing of the gltA gene (379 bp). Results showed that 55 of 147 small rodents to be positive for Bartonella, with a positivity rate of 37.41%, and 95% confidence interval of 29.50%- 45.33%. While the positivity rate across genders  and tissues  of small rodents was not statistically different, that in different habitats  was statistically different. There were 42 Bartonella sequences identified in six species, including 30 B. grahamii, three B. phoceensis, two B. japonica, two B. queenslandensis, one B. fuyuanensis and four unknown Bartonella species from Niviventer confucianus, Apodemus agrarius and Tscherskia triton. In addition to habitat, Bartonella species infection could be affected by the rodent species as well. Among the Bartonella species detected in this area, B. grahamii was the dominant epidemic species (accounting for 71.43%). B. grahamii exhibited four distinct clusters, and showed a certain host specificity. In addition, 11 haplotypes of B. grahamii were observed using DNASP 6.12.03, among which nine haplotypes were novel. Overall, high occurrence and genetic diversity of Bartonella were observed among small rodents in the Shangdang Basin; this information could potentially help the prevention and control of rodent-Bartonella species in this area.The aim of this study was to investigate the occurrence and molecular characteristics of  Bartonella species are recognized as emerging zoonotic pathogens that are mainly transmitted by blood-sucking arthropods, some being infectious to humans. Rodents are important natural reservoirs of Bartonella species. Bartonella infection has been detected in various countries worldwide, and their prevalence in rodents varies greatly across countries and regions. This is the first report of Bartonella prevalence and genetic diversity in Shangdang Basin of China. It revealed a high Bartonella-positivity rate in Niviventer confucianus and Apodemus agrarius in this area. Relatively higher occurrence of Bartonella species was detected in forests and farmlands. Six Bartonella species were detected, B. grahamii, B. phoceensis, B. japonica, B. queenslandensis, B. fuyuanensis and one unknown Bartonella species, of which B. grahamii was the dominant species, with high genetic diversity, and potentially pathogenicity in humans. Our study provided a better understanding of the occurrence and genetic diversity of Bartonella species in the small rodents in Shangdang Basin. Bartonella species are newly discovered ancient gram-negative hemotrophic bacteria that are mainly transmitted by blood-sucking arthropods [Bartonella have been described till date, and they have a wide range of reservoirs, including cats, dogs, rodents, bats, carnivores and ruminants [Bartonella to be 90.4% in Onychomys torridus in the United States, 69% in Apodemus in Japan, 17% in Micromys minutus in Russia, 78% in the rodents in Thailand, and 8.38%, 14.9%, 26.08%, and 57.7% in the rodents from eastern, southeastern, northwestern and northeastern of China respectively [thropods . Over 40uminants , among wBartonella species have been recognized as human pathogens, such as B. bacilliformis [B. quintana [B. henselae [B. elizabethae [B. clarridgeiae [B. koehlerae [B. vinsonii subsp. arupensis [B. vinsonii subsp. berkhoffii [B. grahamii [B. rochalimae [B. tamiae [B. ancashensis [B. washoensis [Bartonella infection can cause human diseases with different clinical manifestations, being especially fatal in immunocompromised patients [Humans can be infected via close contact with rodents, cats and dogs, or upon being bitten by blood-sucking arthropods, such as ticks, lice, fleas and mites , resultiliformis , B. quinquintana , B. henshenselae , B. elizzabethae , B. clarridgeiae , B. koehoehlerae , B. vinsrupensis , B. vinsrkhoffii , B. grahgrahamii ,20, B. rchalimae , B. tami. tamiae , B. ancaashensis , and B. shoensis . Differepatients .Sciurotamias davidianus, Tamiops swinhoei, Apodemus agrarius, Apodemus peninsulae, Tamias sibiricus, Myospalax fontanieri, Tscherskia triton, Rattus norvegicus, Mus musculus, Cricetulus barabensis [Bartonella species. However, the Bartonella species in small rodents in this area have not yet been explored. This study aimed to investigate the occurrence and genetic diversity of Bartonella species in small rodents in Shangdang Basin. Our findings provided the insights into the distribution and genetic diversity of Bartonella in small rodents and presented a scientific basis for the control and prevention of Bartonella infection in humans in this area.The Shangdang Basin is located southeast of Shanxi Province, China, between the Taihang and Taiyue Mountains, with an average elevation of 900–1000 m. It has a complex natural environment, with plains, hills, and mountains, and more than 10 species of rodents have been reported to inhabit this area since the 1990s, including but not limited to rabensis . IncreasThis study was approved by the Ethics Committee of Changzhi Medical College (No: DW2021052). All animals were treated according to the Guidelines of Regulations for the Administration of Laboratory Animals  and the Guidelines for Treating Animals Kindly from Ministry of Science and Technology of the People’s Republic of China. All efforts were made to minimize discomfort to the animals.Small rodents in Shangdang Basin  of Shanxi Province were captured using snap traps in July 2020. Six rodent sampling sites from Luzhou District, Shangdang District, Huguan County, Xiangyuan County, and Tunliu District were randomly selected, and their geographical distribution is shown in Bartonella by targeting a fragment of 296 bp of the transfer-mRNA (ssrA) gene according to a previous study [2O, 0.8 μL (10 μmol/L) of each primer and 0.4 μL (10 μmol/L) probe  [DNA was extracted from approximately 10 mg of each liver, spleen and kidney tissue according to the manufacture’s protocols of TIANamp Micro DNA Kit (TIANGEN Biotech (Beijing) Co., Ltd., China). Real-time PCR was performed to detect the us study . BrieflyCG-BHQ1) , and 3 μssrA-positive samples, the Bartonella citrate synthase (gltA) gene amplification was further performed according to the manufacture’s protocols of TaKaRa PCR Amplification Kit , with 20 μL reaction mixtures containing 2 μL of 10 × PCR buffer, 1.6 μL of dNTP mix, 0.1 μL of Taq, 13.5 μL of double-distilled H2O, 0.4 μL (10 μmol/L) of each primer  [gltA amplification was performed under the following conditions: one cycle for 5 min at 94°C; 35 cycles for 30 s at 94°C, 30 s at 55°C, and 60 s at 72°C; and a final extension for 10 min at 72°C. Next, PCR products with 379 bp were identified by 1.5% agarose gel electrophoresis, and then sent to Shanghai BioGerm Medical Technology Co., Ltd  for sequencing. Briefly, the purified PCR products were sequenced using an ABI Prism dye terminator cycle sequencing ready reaction kit and an ABI PRISM 3730XL DNA sequencer .For GTAAACA) , and 2 μBartonella species sequences in the GenBank using the BLAST program at the National Center for Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic tree was created using the maximum-likelihood method with MEGA version 7.0, and bootstrap values were calculated with 1000 replicates [Brucella abortus was used as the outgroup.The sequences generated in this study were submitted to the GenBank . The nucleotide sequence homology was blasted against reported plicates ,31. BrucBartonella-positive sequences were analyzed for polymorphism based on the number of polymorphic sites (S), the number of haplotypes (H), nucleotide diversity (π), average number of nucleotide differences (κ) and haplotype diversity (Hd) using DNASP 6.12.03. Then, the sequences were analyzed based on a median-joining network using the Population Analysis with Reticulate Trees (PopART) software version 1.7 (http://popart.otago.ac.nz/index.shtml) with the default setting (epsilon = 0). Bartonella strains were downloaded from the GenBank, and their accession numbers are listed in Bartonella in different tissues, habitats and genders of small rodents were analyzed using the Chi-square test. Multivariate logistic regression analysis was performed to explore whether risk factors were associated with the occurrence of Bartonella species in rodents (Bartonella DNA positive vs Bartonella DNA negative). Odds ratios (ORs) and 95% confidence intervals were calculated to determine the strength of the associations. All data were analyzed using SPSS 22.0 . Statistical significance was set at P < 0.05.The positivity rates of Mus musculus (66), Niviventer confucianus (39), Apodemus agrarius (22), Rattus tanezumi (16), Tscherskia triton (2), and Apodemus draco (2). Thereafter, rodents from different classifications were selected and their kidney tissues used for CO I gene sequencing. Since M. musculus, N. confucianus, and A. agrarius are easy to identify by morphology, we selected some of the samples for CO I gene sequencing. On the other hand, all rodents from R. tanezumi, T. triton, and A. draco species were confirmed by CO I gene sequencing. Sequencing results were consistent with the morphological classification. Geographical distribution of the trapped rodents was shown in In total, 147 trapped small rodents were identified into six species by morphology, including Bartonella detection by real-time PCR amplification of the ssrA gene and cPCR of the gltA gene, both of them being positive in at least one tissue was considered positive for Bartonella. In total, 55 small rodents were positive for Bartonella, with a positivity rate of 37.41% , and were classified into four species (N. confucianus (34/39), A. agrarius (18/22), T. triton (2/2), and A. draco (1/2)). After deleting the missing specimens, 147 liver, 128 spleen, and 136 kidney specimens were used for further detection. The positivity rates in liver, spleen, and kidney were 28.57% , 33.59% , and 36.76% , respectively, and the difference in positivity rate in different tissues was not statistically significant   .χ2 = 1.208, P = 0.272). Seventy eight small rodents of four species were captured in villages, with a Bartonella positivity rate of 5.13% . Forty five small rodents of five species were captured in forests, with a positivity rate of 84.44% . Twenty four small rodents of four species were captured in farmlands, with a positivity rate of 54.17% . Thus, the positive rates in different habitats were significantly different   in males and 33.72%  in females; the difference was not statistically significant (< 0.001) . The mulgltA sequences were obtained from 42 Bartonella-positive small rodents. DNA sequence homology analysis and phylogenetic analysis of the gltA gene indicated that six Bartonella species were detected in the liver, spleen, and kidney of small rodents, and that the Bartonella species detected in different tissues of each small rodent were consistent. Thirty sequences were shown to be B. grahamii with 96.05–100% identity, including 23 from N. confucianus (with highest identity of 97.30% with Myodes rutilus in China (KJ175044)), 6 from A. agrarius (including 4 sequences with highest identity of 99.08% with A. agrarius in northern China (KJ175032) and two sequences with highest identity of 100% with A. agrarius in southern China (EU179229)), and one from T. triton (with highest identity of 98.68% with T. triton in China (MH748115)); three sequences from A. agrarius were B. phoceensis with 96.86–99.74% identity (with highest identity of 99.74% with A. agrarius in China (KX549997)); two sequences from A. agrarius were B. japonica with 99.70% identity (with Apodemus argenteus in Japan (AB242289)); two sequences from N. confucianus were B. queenslandensis with 99.47–99.48% identity (with highest identity of 99.48% with N. confucianus in China (MH748120)); one sequence from A. agrarius was B. fuyuanensis with highest identity of 98.93% with A. agrarius in China (MH748123); three sequences of AA87SXHG, AA126SXTL and AA130SXTL from A. agrarius and one sequence of NC56SXHG from N. confucianus were unknown Bartonella species, which shared 94.23–95.26% nucleotide sequence similarity in their gltA fragment with the nearest species of Bartonella, B. krasnovii (with highest identity of 95.26% with Flea in Israel (CP031844)), B. gabonensis (with highest identity of 95.25% with Lophuromys sp. in Gabon (MT274297)) and B. elizabethae (with highest identity of 95.24% with Meriones libycus in Georgia (KT327032)), respectively  showed 17 haplotypes in Bartonella species in this area , among which B. grahamii exhibited 11 haplotypes , unknown Bartonella had two haplotypes , and B. phoceensis, B. japonica, B. queenslandensis had one haplotype respectively  for Hap 1, and four sequences  for Hap 4, 47, 48, and 49 respectively), which might be associated with the strains isolated from Apodemus spp. from China, Japan, South Korea, and the Russian Far East and from Cricetulus longicaudatus in China. Twenty three B. grahamii sequences from N. confucianus contained five haplotypes , which clustered separately, and might be associated with the strains isolated from Myodes rutilus and Microtus fortis in China and from Apodemus agrarius in the Russian Far East. One B. grahamii sequence (TT79SXHG) from T. triton might be associated with a strain isolated from T. triton in China , A. agrarius (81.82%), T. triton (100%) and A. draco (50%). Considering that only two A. draco and two T. triton were captured, the positivity rates of 50% and 100% might not be the real Bartonella prevalence rates in these rodents, and further studies might be required for validation. However, no Bartonella was detected in M. musculus and R. tanezumi in our study, in contrast to the reports of some previous studies, such as no infection in M. musculus but 15.51% infection in R. tanezumi in Fujian, China [M. musculus in Lithuania [R. tanezumi in Vietnam [Bartonella in the rodents inhabit the farmland and forest was higher, which was similar to that reported in a previous study [Bartonella in the farmland and forest were obviously higher than that in the village, indicating that the risk of Bartonella infection is significantly increased when people are engaged in wild activities; therefore, improvement in risk awareness and taking corresponding preventive measures would be necessary.Generally, spleen tissue is used for of China . Bartonen, China , 8.3% inA. draco %. Considus study . The infgltA gene sequencing, 60 sequences were obtained from 42 small rodents, and the Bartonella species detected in different tissues of each small rodent were found to be consistent. Therefore, we selected 42 sequences from 42 small rodents for further analyses. DNA sequence homology and phylogenetic analyses. indicated that six Bartonella species were detected in the small rodents, including B. grahamii, B. phoceensis, B. japonica, B. queenslandensis, B. fuyuanensis and unknown Bartonella species. In addition, three species of Bartonella  were detected in N. confucianus and five species of Bartonella  were detected in A. agrarius. The Bartonella species detected in N. confucianus and A. agrarius in the same habitat were not completely the same, B. queenslandensis was only detected in N. confucianus, B. phoceensis and B. japonica were only detected in A. agrarius, suggesting that in addition to habitat, Bartonella species infection could be affected by the rodent species as well, and had a certain host specificity, which was similar to that reported in previous studies [Based on  studies ,33,36,52B. grahamii has been reported in many hosts, including A. agrarius (AB529500), Apodemus flavicollis (EU014266), T. triton (MH748118), Microtus pennsylvanicus (MK984788), Microtus ochrogaster (AB426656), etc. Moreover, we had previously identified B. grahamii in Ochotona curzoniae (KT445929), Cricetulus longicaudatus (MT815312), and Microtus oeconomus (MT815315). Here, it indicated that the sequences of B. grahamii from A. agrarius were clustered with the strains from A. agrarius, the sequence from T. triton were similar to the strain from T. triton, and the sequences from N. confucianus gathered in one unique cluster, which showed a certain host specificity in this area. To the best of our knowledge, B. grahamii was the first detected in N. confucianus in China. Additionally, B. grahamii was identified from three sampling sites, accounting for 71.43% (30/42) of all Bartonella species, suggesting that it was the dominant Bartonella species in Shangdang Basin. B. grahamii has been defined as a human pathogen [B. grahamii and cause human disease in this area. Whether the pathogenicity of B. grahamii is different in different rodents would require further study. A previous study had suggested that one Bartonella species could be identified to validated Bartonella species when they shared ≥ 96% nucleotide sequence similarity of gltA sequences [B. heixiaziensis and B. fuyuanensis have been considered as new species identified in China [unknown Bartonella species, which shared ≤ 96% nucleotide sequence similarity in their gltA fragment with that of the reported Bartonella species, might be considered potential novel Bartonella species.pathogen ,20, implatus MT8112, and Mrarius AB29500, ApgltA and rpoB when similar parameters were calculated for sequences of known Bartonella species [gltA sequences for B. grahamii. Polymorphic level (Hd = 0.789) and nucleotide diversity (π = 0.01853) of B. grahamii (30 sequences) in our study were slightly greater than that of B. grahamii isolates  from the northeast China [B. grahamii in Shangdang Basin, which could be the result of accelerated evolution of Bartonella species in rodents [B. grahamii, we constructed a haplotype network by comparing the sequences in our study with those of other B. grahamii strains isolated from different rodents and regions. Currently, 55 haplotypes of B. grahamii were observed among approximately 15 genera of rodents, which exhibited a complex network, suggesting the evolution of B. grahamii to be complex. Generally, sequences from A. agrarius and T. triton are mainly related to the Asian strains isolated from Apodemus and Cricetulus, whereas the sequences from N. confucianus are mainly related to the Chinese strains isolated from Myodes and Microtus and the Russian Far East strains isolated from Apodemus. Additionally, among the 11 haplotypes detected in this study, only the sequences of two haplotypes were the same as that of the known B. grahamii, the remaining nine haplotypes were novel, suggesting that B. grahamii evolved rapidly in this area.A previous study revealed that the highest polymorphism was within  species . Our stu Hd = 0.79 and nuc rodents . To furtT. triton and A. draco were too small; thus, sample size would need to be increased in future to understand the real prevalence of Bartonella species. Secondly, some tissue samples did not contain a high quantity of bacteria, and hence not all the PCR-positive samples were sequenced successfully. The sequencing success rate was 76.37% (42/55), was higher than 14.68% (16/109) and 17.61% (56/318) in Thailand and Lithuania, respectively, similar to 78.95% (45/57) in Tanzania [unknown Bartonella species based on gltA gene analysis, which did not cluster with any reported Bartonella species. However, we only sequenced 326 bp segments within the gltA gene, and whether these unclassified Bartonella species are indeed novel would require further validation.Our study has some limitations. First, the sample sizes of Tanzania ,55,56, wBartonella, including B. grahamii, B. phoceensis, B. japonica, B. queenslandensis, B. fuyuanensis and unknown Bartonella were detected in three species of rodents, N. confucianus, A. agrarius and T. triton in Shangdang Basin. Wild rodents were found to be more susceptible to Bartonella than domestic rodents. B. grahamii was the dominant species and had high genetic diversity in this area; its pathogenicity would require further investigation. Our study investigated the occurrence and molecular characteristics of Bartonella species among small rodents in Shangdang Basin; the information could potentially benefit the prevention and control of rodent-Bartonella species in this area.In conclusion, six species of S1 Table(PDF)Click here for additional data file.