Providencia rettgeri via whole genome sequencing (WGS).This study aimed to investigate the clinical infection characteristics and analyze the resistance gene carrying status of carbapenem-resistant P. rettgeri were collected from clinical patients between January 2020 and December 2021, and their susceptibility to 19 antimicrobial drugs was determined using the VITEK 2 Compact system and Kirby–Bauer (KB) disk diffusion method. The Illumina platform was used to perform WGS of the P. rettgeri isolates, and the resistance genes carried by the Carbapenem-resistant P. rettgeri strains were detected via ABRicate software. The phylogenetic tree was constructed by thirty-four strains including twenty-eight strains downloaded from NCBI database and the carbapenem-resistant six P. rettgeri strains in this study. Which based on genomic single nucleotide polymorphism (SNP) to understand the affinities of the carbapenem-resistant P. rettgeri strains.Carbapenem-resistant P. rettgeri strains were isolated from five different clinical departments using the blood, urine, sputum, and secretion specimens. These infected patients are middle-aged and elderly people with a history of severe trauma, tumors, hypertension, and various other underlying diseases, and invasive procedures. Antimicrobial sensitivity testing showed that all strains presented resistance to ampicillin-sulbactam, ceftazidime, ciprofloxacin, levofloxacin, and ertapenem, whereas they exhibited full susceptibility to cefepime and amikacin. Most strains demonstrated high resistance to β-lactams, aminoglycosides, and sulfonamides. Thirty-five resistance genes were identified by ABRicate. All carbapenem-resistant P. rettgeri strains carried aminoglycoside, fluoroquinolone, chloramphenicol, rifampicin, sulfonamide, and β-lactam resistance genes, and most importantly, all strains possessed the carbapenem resistance gene blaNDM−1. The six P. rettgeri strains in this study and the 28 carbapenem-resistant P. rettgeri strains from the NCBI database were divided into four evolutionary groups. The WF3643, WF3849, WF3822, and WF3821 strains in this study were in the same evolutionary group (clade A), while the closely related WF3099 and WF3279 strains were in different evolutionary groups (clade B and clade D), respectively. The WF3099 strain was distantly related to the other five strains.Six carbapenem-resistant P. rettgeri strains were mostly isolated from middle-aged and older patients with a history of surgery or serious underlying diseases, and they were found to cause multisystem infections. All Carbapenem-resistant P. rettgeri strains in this study carried blaNDM−1 and multiple antimicrobial drug resistance genes. Furthermore, the P. rettgeri strains in this study were closely related, suggesting the possibility of nosocomial infections. Therefore, our study highlights the need for research on P. rettgeri to control the spread of these nosocomial infections.Carbapenem-resistant The online version contains supplementary material available at 10.1186/s12866-023-03032-3. Providencia spp. are a group of urease-producing gram-negative bacteria belonging to the Enterobacteriaceae family, including P. rettgeri, P. stuartii, P. alcalifaciens, P. heimbachae, and P. rustigianii [Providencia genus is an important conditional pathogen of hospital-acquired infections [tigianii . These bfections , 3.Enterobacteriaceae poses a serious threat to human health. Carbapenems are the most potent β-lactams used for the treatment of gram-negative bacillary infections, particularly those caused by Enterobacteriaceae. NDM-1 is a class B β-lactamase encoded by the blaNDM−1 gene and confers resistance against all β-lactam antibiotics, including carbapenems [blaNDM−1, its corresponding enzyme has received widespread global attention, with outbreaks of NMD-1-producing bacteria being reported in several countries and regions, including Hong Kong and mainland China.The unreasonable and irregular application of antimicrobial drugs, coupled with the preference and increased dosage of carbapenem antimicrobial drugs in recent years has increased the severity of multi-resistant and pan-resistant bacteria. The emergence of carbapenem⁃resistant bapenems . After tP. rettgeri infections along with an increased detection rate of multi-drug resistant bacteria has been reported. In particular, carbapenem-resistant P. rettgeri has been emerging as one of the pathogens causing hospital infections, posing serious challenges to clinical disease diagnosis and treatment [P. rettgeri in our hospital using their whole genome sequencing data and elucidate their drug resistance gene carriage and homologous evolutionary relationship of the nosocomial strains. This study will provide useful data for the treatment selection of clinical antimicrobial drugs as well as for the prevention and control of hospital infections.In recent years, the occurrence of reatment . In thisP. rettgeri strains were isolated from patients visiting a 3,000-bed-capacity tertiary teaching hospital in northern China. The bacterial strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) via the VITEK MS system  and average-nucleotide identity analysis based on whole genome sequence data.Between January 2020 and December 2021, six carbapenem-resistant https://clsi.org) [Escherichia coli ATCC 25,922 and pseudomonas aeruginosa ATCC27853 were used as the quality control strain.Antimicrobials commonly used in the clinic include piperacillin-tazobactam(TZP), gentamicin(GEN), cefepime (FEP), imipenem(IPM), ceftriaxone(CRO), tobramycin(TOB), amikacin (AMK), ceftazidime (CAZ), ertapenem (ETP), levofloxacin (LVX), trimethoprim-sulfamethoxazole (SXT), ampicillin-sulbactam (SAM), and ciprofloxacin (CIP) was performed using the VITEK 2 Compact (GN13) system. Antimicrobial susceptibility testing for the drugs not included in the GN13 card, including cefoxitin(FOX), cefuroxime(CXM), aztreonam(ATM), meropenem(MEM), cefoperazone/sulbactam(CSL), and cefotaxime (CTX) was conducted using the Kirby–Bauer (KB) disk diffusion method . The susceptibility testing results were interpreted according to the 2022 Clinical and Laboratory Standards Institute guidelines (lsi.org) . Escherihttps://github.com/FelixKrueger/TrimGalore) and assembled via SPAdes (v3.13.0) (http://bioinf.spbau.ru/spades) using default parameters [Bacterial DNA was extracted using Omega Bio-Tek Bacterial DNA Kit  after overnight culture of the strains at 37 °C. DNA library preparation was performed either using the Illumina Nextera XT DNA library preparation kit  according to the manufacturer’s instructions prior to sequencing on Illumina HiSeq or MiSeq platform. Raw sequences were trimmed for quality, followed by assembly and annotation. Low-quality reads and adapters were trimmed using TrimGalore  [P. rettgeri strains required for constructing the phylogenetic tree were downloaded from the NCBI database. Consequently, the complete genomic data of 28 carbapenem-resistant P. rettgeri strains were obtained from the NCBI database , which were then identified to contain carbapenem resistance genes (blaNDM−1) by ABRicate  analysis. MEGAX 10.1.8 was used to generate unrooted maximum-likelihood phylogenetic trees with a bootstrap iteration of 1000 [The NCBI AMRFinder database was used to detect antimicrobial resistance genes using ABRicate  was in the intensive care unit (ICU) with multiple fractures. The patient was hospitalized in the ICU for 76 days and had undergone several invasive procedures, including subcutaneous tissue debridement and internal fixation of pelvic fractures. Patient 1 was discharged in good condition.P. rettgeri. Patient 2 was discharged in fair condition, with unremitting bilateral lower extremity ulceration.Patient 2 (WF3279) was in the cardiology department with severe underlying disease that resulted in bilateral lower extremity ulceration. The patient’s secretions were cultured for P. rettgeri was isolated from the patient’s urine specimen. Patient 3 was treated and discharged in good condition.Patient 3 (WF3643) had undergone cystectomy for a urologic malignancy and was hospitalized due to a urinary tract infection. P. rettgeri was cultured from the patient’s secretions. Patient 4 was hospitalized for 1 day and was discharged in poor condition with no follow-up records.Patient 4 (WF3821) was an outpatient who had skin ulceration for 4 months. P. rettgeri was isolated from the patient’s secretions. Patient 5 was discharged in good condition.Patient 5 (WF3822) had a malignant tumor of the tongue. After tongue resection, P. rettgeri was isolated from the patient’s sputum. Patient 6 eventually died.Patient 6 (WF3849) had undergone craniocerebral hematoma removal for craniocerebral trauma. Antimicrobial sensitivity characteristics of carbapenem-resistantP. rettgeri.P. rettgeri strains showed multi-antibiotic resistance  were resistant to CIP and LVX, while those possessing β-lactam resistance genes demonstrated high resistance to β-lactam/β-lactamase inhibitor combination antimicrobials. All six P. rettgeri strains harboring blaNDM−1 showed resistance to carbapenems, consistent with the resistance phenotype. Interestingly, the WF3099, WF3821, and WF3849 strains carried aminoglycoside resistance genes but did not exhibit resistance to aminoglycoside antimicrobials. Similarly, the WF3821 and WF3849 strains possessed sulforaphane resistance genes but demonstrated sensitivity to SXT.The examination of the resistance genotypes and phenotypes of P. rettgeri strains carried plasmids, with the WF3279 strain carrying two plasmids (repUS18_4_rep(pSA8589) and Col3M_1) and the remaining three strains  harboring one plasmid (Col3M_1). Plasmid repUS18_4_rep(pSA8589) is identical to ACCESSION: KC561137 in the NCBI database, with an overage of 99.78% and identity of 100%. Plasmid Col3M_1 is highly consistent with ACCESSION: JX514065, with an overage of 100% and identity of 98.09%.The PlasmidFinder prediction analysis Table  showed tP. rettgeri strains isolated in this study were used to construct a phylogenetic tree together with the 28 blaNDM−1-carrying P. rettgeri strains from the NCBI database. The results of this analysis . Most importantly, all six strains also possessed the carbapenemase resistance gene blaNDM−1, with the presence of carbapenem resistance genes being a major factor mediating carbapenem resistance. This study showed that this bacterium was resistant to both CIP and LVX, and its resistance mechanism may be related to its expression of the quinolone antimicrobial resistance genes qnrA and qnrD. All P. rettgeri strains harboring blaNDM−1 showed resistance to carbapenem antibiotics, which was consistent with the resistance phenotype. The resistance genotypes and phenotypes of P. rettgeri were the same. Three strains of P. rettgeri carried aminoglycoside resistance genes but did not show resistance to aminoglycoside antibacterial drugs. However, further investigation is required to determine whether this effect is clinically observed after in vivo administration and to understand the specific mechanism of this phenomenon. Moreover, two strains showed sensitivity to SXT although they carried the sulfonamide resistance gene. The presence of discordance between resistance phenotypes and genotypes in certain strains implies the possibility of distinct resistance mechanisms, thereby necessitating further exploration.In recent years, some strains were found to harbor carbapenem resistance genes, such as ce genes –20, wherce genes , 21, theP. vulgaris in China [The WF3279 strain carried two plasmids (repUS18_4_rep(pSA8589) and Col3M_1), while three strains  possessed one plasmid (Col3M_1). The PlasmidFinder predicted that plasmid Col3M_1 was highly similar to p3M-2 A (JX514065) found in in China . pSA8589in China . HoweverP. rettgeri were closely related, whereas only one strain isolated from the ICU ward in 2020 was distantly related. The WF3643, WF3849, WF3822, and WF3821 strains of P. rettgeri isolated in 2021 were on the same evolutionary branch. Furthermore, these four P. rettgeri strains were obtained from different departments, suggesting that their close affinities may be due to the homologous transmission of carbapenem-resistant P. rettgeri between some wards in our hospital, which should attract clinical attention. Moreover, bacteria can cross-infect inpatients through medical devices and healthcare workers’ hands, leading to nosocomial outbreaks [P. rettgeri homology in our hospital to prevent nosocomial infections.The homology analysis showed that four strains of utbreaks , 20, 24.P. rettgeri, these infections should be given sufficient attention because they involve a wide range of sites, exhibit high drug resistance, and mostly occur in immunocompromised middle-aged and older patients. Furthermore, the reasonable use of antimicrobial drugs for early treatment, combined with the identification of the infection site and antimicrobial sensitivity testing is necessary to avoid recurrent infections and actively treat the underlying disease. Our study showed that carbapenem-resistant P. rettgeri bacteria had caused nosocomial infections in some departments in a small area of our hospital. This finding should attract the attention of clinicians and microbiologists towards the need to strengthen drug resistance monitoring and avoid the generation and spread of pan-drug resistant strains.In conclusion, despite the infrequent incidence of human infection by Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Streptococcus anginosus group (SAG) pathogens  are oral flora with the potential to cause head and neck space infections including brain abscesses. Several centers noted an increase in intracranial abscesses in children in the SARS-CoV-2 era, prompting a CDC health alert in May 2022. Notably, a large proportion of such reported cases were associated with SAG and/or sinusitis. We examined the epidemiology of pediatric intracranial abscesses with a focus on SAG in the pre- and post-pandemic periods.The Cases of intracranial abscesses  of any microbiologic etiology from January 1, 2011 to December 31, 2022 were identified at Texas Children's Hospital by ICD10 codes. Subjects were cross referenced with culture results of the microbiology laboratory. Cases included were those associated with either otitis media, mastoiditis, or sinusitis. Temporal trend in these infections was investigated with linear regression.Figure 1). SAG infection was more often associated with multiple surgeries and these subjects were specifically more likely to require craniotomy or craniectomy . Among sinogenic abscesses, S. intermedius was the most common pathogen while among otogenic cases S. pyogenes predominated . There were 8 cases of craniectomies and one relapse (due to SAG) after replacement of autologous bone flap. After March 2020, 6/46 cases tested positive for SARS-CoV-2 (13%); the severity of infection was not significantly different with concomitant COVID and intracranial abscess. The proportion of SAG-associated infections was similar prior to and after the COVID pandemic.157 cases were identified and 59.9% were due to SAG. The incidence of all sinogenic/otogenic intracranial infections (p=0.006), and SAG specific infections (p=0.01), increased from 2011 to 2022 (*- p= 0.01- <0.05 ** p< 0.01Over the last decade, intracranial complications of sinusitis/otitis have been increasing, and specifically those caused by SAG. This observed trend clearly predated the COVID pandemic. SAG cases were associated with greater need for surgical intervention and specifically neurosurgery. Further work is necessary to determine the explanation for these rising infections.Sheldon L. Kaplan, MD, MeMed: Grant/Research Support|Pfizer: Grant/Research Support|Pfizer: Honoraria Jonathon C. McNeil, MD, Allergan: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support


Anopheles funestus population in various malaria-endemic countries remains relatively unknown. This study aimed to investigate the common predators that co-exist with Anopheles funestus group larvae and evaluate factors that influence their abundance in rural south-eastern Tanzania.The role of larval predators in regulating the Mosquito larvae and predators were sampled concurrently using standard dipper (350 ml) or 10 L bucket in previously identified aquatic habitats in selected villages in southern Tanzania. Predators and mosquito larvae were identified using standard identification keys. All positive habitats were geo-located and their physical features characterized. Water physicochemical parameters such as dissolved oxygen (DO), pH, electrical conductivity (EC), total dissolved solids (TDS) and temperature were also recorded.An. funestus aquatic habitats in nine villages were sampled for larvae and potential predators. A total of 8,295 predators were sampled. Of these Coenagrionidae 57.7% (n = 4785), Corixidae 12.8% , Notonectidae 9.9% (n = 822), Aeshnidae 4.9% (n = 405), Amphibian 4.5% (n = 370), Dytiscidae 3.8% (n = 313) were common. A total of 5,260 mosquito larvae were sampled, whereby Anopheles funestus group were 60.3% , Culex spp. 24.3% , An. gambie s.l. 8.3% (n = 438) and other anophelines 7.1% (n = 373). Permanent and aquatic habitats larger than 100m2 were positively associated with An. funestus group larvae (P<0.05) and predator abundance (P<0.05). Habitats with submerged vegetation were negatively associated with An. funestus group larvae (P<0.05). Only dissolved oxygen (DO) was positively and significantly affect the abundance of An. funestus group larvae (P<0.05). While predators’ abundance was not impacted by all physicochemical parameters.A total of 85 previously identified An. funestus group larvae. Additional studies are needed to demonstrate the efficacy of different predators on larval density and adult fitness traits. Interventions leveraging the interaction between mosquitoes and predators can be established to disrupt the transmission potential and survival of the An. funestus mosquitoes.Six potential predator families were common in aquatic habitats of  Such inet al., , suggestAn. funestus dynamics are not understood. The use of such predators may limit mosquito larval abundance and reduce adult densities [Many aquatic invertebrate predator species, including Aeshnidae , Notonecensities , 14–16.Anopheles species in Tanzania, Anopheles funestus is the primary vector [An. funestus larval population remains unknown.Although malaria is transmitted by several y vector –19. Thisy vector . Despitey vector –23, and y vector , the relAn. funestus aquatic habitats in south-eastern Tanzania. Specifically, this study aimed to 1) identify types of common predators co-existing with An. funestus group larvae in a rural part of Tanzania, 2) determine factors which might contributed to the abundance of these predators and 3) quantify the associations between different predator types and An. funestus group larval abundance.This study investigated the common aquatic predators and factors influencing their abundance in An. funestus is responsible for more than 85% of overall malaria transmission [An. funestus in the villages are well known and have been previously characterized [A cross-sectional survey was conducted, between March and May 2022, in nine villages in south-eastern Tanzania, namely Chikuti , Mzelezi , Chirombola , Ebuyu , Mwaya  and Tulizamoyo  in Ulanga district and Ikwambi , Kisawasawa  and Mpofu  in Kilombero district . In thiscterized . Eighty-Mosquito larvae and predators were sampled using standard dippers (350 ml) or 10 L buckets, as previously described , 15, 24.An. funestus s.s. whilst 28% were An. rivulorum and 12% were An. leesoni [An. funestus group, An. gambiae s.l. or Anopheles sp. following the identification key by Gilles and Coetzee [In a previous study, mosquito larvae from the same villages were taken to the laboratory in Ifakara, allowed to emerge and eventually identified to species by PCR. Of those identified 53% were  leesoni . All thr Coetzee , 26. Cul Coetzee  and GerbAn. funestus group larvae were sampled for mosquito larvae and predators. Their overall physical characteristics were recorded and physicochemical parameters of the water , total dissolved solids (TDS) were measured using a portable water quality meter . A Trans Instruments Dissolved Oxygen Meter (HD3030) was used to measure dissolved oxygen (DO), using standard recording procedures. Habitats were classified as being either: swamp, stream, river, rice-field, stream-pool, ground-pool, ditch, spring-fed pool, puddle, hoof-print, man-made wells, brick or sand pit. Water colour was categorized as being clear (transparent and odourless) or coloured .Only positive aquatic habitats for 2 or more than 100 m2. Vegetation quantity and vegetation type were also classified as  and  respectively. Water bodies known to have existed for three months or more were considered to be permanent whilst other collections of water were considered to be ‘temporary’. Water depth was classified as being less than 50 cm or more than 50 cm deep. The distance from aquatic habitats to the nearest houses were estimated visually and classified as being less than 100 m or more than 100 m.The source of water was also classified as rainwater or others (non-rainwater). Algal quantities in the habitats were classified as none, moderate, or abundant. Algal type was classified as filamentous, green, blue-green or brown. Water was also classified as being stagnant, slow or fast moving. The land use surrounding the aquatic habitats was classified as scrub, cattle grazing or cultivated field. Shade over the habitats was classified as none, partial or heavy. Habitat size was measured using tape and classified as being less than 100 mglmmTMB package [An. funestus group larvae (iii) assess which habitat characteristics contributed to the abundance of predators and An. funestus group larvae and (iv) assess the impact of each predator family on the abundance of An. funestus group larvae. All variables (i-iv) were assessed individually and later combined in the final model.Analysis was done using open source software R version 4.2.1. . General package  were useDue to a large number of dips with zero larvae the negative binomial with zero inflated models were used. In all models, the study villages in which the aquatic habitats were identified and habitat ID were used as random terms to capture unexplained variations between villages and habitats. The best fitting models were selected using Akaike Information Criterion (AIC) and results presented as risk ratios (RR) at 95% CI and statistical significance was considered when the P-value < 0.05.Research proposal was presented to the Nelson Mandela Institute of Science and Technology and approval for this study was obtained from the institutional review board of Ifakara Health Institute (Ref: IHI/IRB/No: 13–2022) and from the Medical Research Coordinating Committee (MRCC) at the National Institute for Medical Research (NIMR) (Ref: NIMR/HQ/R.8a/Vol. IX/3353). The consent of publication this manuscript was obtained from the National Institute for Medical Research (NIMR) (Ref. No: NIMR/HQ/P.12 VOL XXXV/61). Written permission to conduct study was obtained from local leaders in each village whereby the purpose, procedure and benefits of the study were clearly explained. Verbal and written informed consents were obtained from community members who assisted to sample aquatic predators.Anopheles funestus group larvae were identified and characterized. In these habitats, a total of 8,295 predators were sampled. Among all sampled predators, only 7,906 predators were identified belonging to eight families. Of these, Coenagrionidae accounted for 57.7% (n = 4785), Corixidae 12.8% , Notonectidae 9.9% (n = 822), Aeshnidae 4.9% (n = 405), Amphibian 4.5% (n = 370), Dytiscidae 3.8% (n = 313), Belostomatidae 1.2% (n = 103) and Nepidae 0.6% (n = 48)  of the total, Culex spp 24.3 , An. gambiae s.l. 8.3% (n = 438), and other anopheline larvae 7.1% (n = 373)  . Three h(n = 48) . A totaln = 373) .An. funestus group larvae and predators were samples from different aquatic habitats both man made and natural habitats: includes grounded pool, Brick/ sand pit, man-made wells, river stream, swamp and spring fed pool   and predator abundance P>0.05, . Tempera P<0.05, .An. funestus group larval abundance (P<0.05) whilst Notonectidae and Corixidae were negatively associated with An. funestus abundance (P<0.05). No strong association between abundance of An. funestus group larval abundance and some predator families including Aeshnidae and Belostomatidae were found (P>0.05) (Coenagrionidae and Dytiscidae were positively associated with (P>0.05) .An. funestus habitats, a total of 46 were co-inhabited by An. gambiae s.l, 23 habitats had other anopheline larvae and 60 habitats had Culex spp  [In aquatic habitats, higher total dissolved solids have harmful impacts on the aquatic organisms . It chanet al., , but con,214.31) .An. funestus group larvae coexist with various mosquito species and other organisms in their aquatic habitats, which is consistent with previous studies conducted by Nambunga et al., [et al., [This study revealed that various predator families share similar aquatic habitats. Furthermore, it found that  et al.,  and Dida[et al., .An. funestus group larvae and predators was varying. In particular, this study noted some predator families namely; Notonectidae and Corixidae were bounded in area with low abundance of An. funestus group larvae. However, the highest number of predators and low number of mosquito larvae could also reflect the direct predation in the aquatic habitats. Whilst Coenagrionidae and Dytiscidae were bounded in the area with higher abundance of An. funestus group larvae, showing the positive association between these predators and An. funestus group larvae. These could be due to variation in feeding preferences among each predator family. More important, further studies should be done to confirm this because another study has shown that Coenagrionidae are not only significant predators for Anopheles larvae but also Aedes aegypti larvae [An. funestus group larvae.The association of i larvae . HoweverThis study suggests that, for effective malaria vector control, intervention strategies should focus on both, permanent aquatic habitats and temporary/seasonal as well as micro-habitats such as ditches and some man-made wells. The evidence by this study also suggests that these temporary and micro habitats can significantly produce higher numbers of disease transmitting mosquitoes though they limit predator’s colonization abilities. One benefit of utilising biological control is that it may target mosquito species at low densities, it has no impact on non-target organisms and it is simple to use in the field .One of the limitations of this study was that it did not focus on understanding anthropogenic factors and how they might influence the abundance of predators. Though it is very important to assess how both natural and human activities influence abundance of predators, the current study focused on water physicochemical parameters and other physical characteristics of the aquatic habitats. Further studies should also, morphologically identify the aquatic predators to species level using an appropriate identification key. This should help to understand how the aquatic predators are distributed in different aquatic habitats. Although this does not affect our interpretation of results, a cross sectional study doesn’t represent the variation among habitat characteristics over time including the changes in temperature and water physicochemical parameters. A longitudinal study would help capture seasonal variations between predator and prey abundance. Such study may help in the design of novel interventions focussed on this relationship.An. funestus group larvae and factors influencing their abundance. Six predator families were commonly identified; Coenagrionidae, Corixidae, Notonectidae, Amphibians, Aeshndae, and Dytiscidae. The abundance of predator families with An. funestus group larvae varied. The only physicochemical parameter influencing An. funestus group larvae abundance was dissolved oxygen. Additional studies are needed to demonstrate the efficacy of predators on mosquito larval densities and adult fitness traits. Interventions leveraging the interaction between mosquitoes and predators can be established to disrupt the transmission potential and survival of the An. funestus mosquitoes.This study demonstrated the existence of common predators in aquatic habitats colonized by S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Dataset(CSV)Click here for additional data file.


Mangifera indica (M. indica) and their antibacterial efficacy against Aeromonas hydrophila (A. hydrophila) in Cirrhinus mrigala . The prepared M. indica mediated silver nanoparticles (Mi-AgNPs) were found to be polycrystalline in nature, spherical in shapes with average size of 62 ± 13 nm. C. mrigala (n = ±15/group) were divided into six groups i.e., G1: control, G2: A. hydrophila challenged, G3: A. hydrophila challenged + Mi-AgNPs (0.01 mg/L), G4: A. hydrophila challenged + Mi-AgNPs (0.05 mg/L), G5: A. hydrophila challenged + Mi-AgNPs (0.1 mg/L) and G6: A. hydrophila challenged + M. indica extract (0.1 mg/L). Serum biochemical, hematological, histological and oxidative biomarkers were evaluated after 15 days of treatment. The liver enzyme activities, serum proteins, hematological parameters and oxidative stress markers were found to be altered in the challenged fish but showed retrieval effects with Mi-AgNPs treatment. The histological analysis of liver, gills and kidney of the challenged fish also showed regaining effects following Mi-AgNPs treatment. A CFU assay from muscle tissue provided quantitative data that Mi-AgNPs can hinder the bacterial proliferation in challenged fish. The findings of this work suggest that M. indica based silver nanoparticles can be promising candidates for the control and treatment of microbial infections in aquaculture.The present study reports the green synthesis of silver nanoparticles from leaves’ extract of  Aquaculture is playing a key role in supplying world food and nutrition needs as about 17% of the animal source protein comes from aquaculture. Moreover, fish meat is rich in essential minerals and omega-3 fatty acids which have great health benefits .Aeromonas, Edwardsiella, Flavobacterium, Citrobacter, Mycobacterium, Shewanella, Streptococcus, and Pseudomonas [In order to meet the growing needs of fish, it’s important to address the issues and challenges faced by fisheries sector, especially diseases caused by fish pathogens. The parasitism and disease outbreaks not only reduce the fish production but also cause serious economic losses to aquaculture globally ,4. Diffeudomonas .Aeromonas hydrophila (A. hydrophila), one of the Aeromonas species, is a motile, rod-shaped, opportunistic Gram-negative, facultative anaerobic, nonspore forming ubiquitous bacteria. The most common fish disease caused by this bacterium is called ‘Motile Aeromonas Septicemia’ which is also known by different names including ‘Hemorrhagic Septicemia, Ulcer Disease, or Red-Sore Disease. The list of symptoms in fish infected by A. hydrophila includes lack of appetite, skin ulcerations, movement abnormalities, pale gills, and swollen appearance. The fish organs such as gills, liver, kidneys, spleen, pancreas, and skeletal muscle are commonly affected by A. hydrophila. Furthermore, A. hydrophila is an afflictive increase in the list of multidrug resistant bacteria and fish infection caused by A. hydrophila is a ‘zoonotic disease’ and may be transferred to the human by infected fish consumption [sumption ,9,10,11.A. hydrophila challenge. Nevertheless, growing multidrug resistance against bacteria and undesirable residual product gathering in aquatic environment can cause huge outbreaks of epidemics leading to serious losses to aquaculture in particular and to the environment in general. Regarding vaccination, different challenges have to be faced, such as, specific vaccines have to be administered against particular pathogen, limited types of vaccines are available, higher prices of commercial vaccines and laborious vaccine administration methods [Antibiotics and vaccination are the most common prophylactic strategies being used against  methods ,12. Thercandida and Acaricides) and Photocatalytic Activities [In the green synthesis, nanomaterials are made by green chemistry-based methods using the natural products such as plants and microorganisms as reducing and stabilizing agents. This method is cost-effective, and eco-friendly as it reduces the use of noxious chemicals. Moreover, in this synthesis, surfaces of nanoparticles can be functionalized with extract biomolecules such as polyatomic alcohols, proteins, organic ligands, and polysaccharides leading to the enhanced antimicrobial efficacy against different microbes ,14. Nanotivities ,18.The use of silver in different forms as a medicinal remedy to prevent infection transmission, to treat various maladies including burns, ulcerations, and infected wounds can be found in literature since the ancient civilization. The antibacterial properties of silver are also well documented ,20. SilvMangifera indica (M. indica) commonly known as Mango, has been used for various medical applications since centuries owing to biological properties including antibacterial, antioxidant, antiviral, anti-inflammatory, antidiabetic and antiallergic effects. Leaves of M. indica are considered important because they have a variety of important biomolecules such as phenolic acid, phenolic esters, flavonoids, triterpenoids, thiamine, mangiferin and chinonin [chinonin ,25.Cirrhinus mrigala  is an important fish of the Carp family and is a preferred food source in South Asian countries. In the present study, we prepared AgNPs using the leaves’ extract of M. indicia and investigated their anti-bacterial efficacy against A. hydrophila in C. mrigala. Our hypothesis was that M. indicia mediated AgNPs can help fight A. hydrophila challenge in C. mrigala and thereby, can be employed as promising, novel anit-bacterial measures in aquaculture.M. indica were collected from Botanical Garden of the University of the Punjab, Lahore, Pakistan. In the first step, leaves were washed thrice with running tap water followed by thorough rinsing with deionized (DI) water. The cleansed leaves were shade dried for 10 days before grinding into fine powder by electric grinder. 6 g of this powder was mixed in 50 mL of DI water and was heated up to 70 °C under constant stirring (200 rpm). After 45 min of heating and stirring, solution was taken off from the hot plate and let it to cool down to room temperature in order to sediment the residue. The solution was then centrifuged for 15 min at 4000 rpm and supernatant was separated as extract. Finally, extract was collected by filtering it through Whatmann filter paper (Fresh leaves of er paper .3) precursor solution was heated up to 70 °C under constant magnetic stirring (200 rpm). Thereafter, 3.0 mL of the M. indica leaves’ extract was added into the precursor solution dropwise. The optimized pH value of the solution was maintained to 7.6. The color of the solution changed with time from transparent to light brown to dark brown at the end. The changing of the solution color is an indication of reduction reaction and formation of stable AgNPs. The solution was then cooled down to room temperature. In M. indica leaves’ extract mediated AgNPs are shown.For the biosynthesis of Mi-AgNPs, procedure mentioned by Philip et al. ,27,28,292) clean piece of soda lime glass slide and dried to obtain sufficient amount Mi-AgNPs for SEM and EDX measurements. Prior to conduct SEM, a thin coating of gold was applied to avoid any possible charging effects.The prepared Mi-AgNPs were characterized using Ultraviolet-visible (UV-Vis) spectroscopy , X-ray diffractometer (XRD) , Fourier transform infrared spectroscopy (FTIR) , Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) . For SEM analysis, few drops of Mi-AgNPs were cast on a small . The obtained bacterial culture was centrifuged at 3000 rpm for 15 min. A bacterial pellet was formed that was rinsed several times with sterilized phosphate bufered saline (PBS), having a pH 7.1. The final bacterial pellet was suspended in PBS. A dilution of bacteria corresponding to 1.8 × 105 CFU/mL was prepared and 0.5 mL of this dilution was intramuscularly injected to the juveniles for challenge assays. Afterwards, juveniles were randomly distributed into following six groups in triplicates (n = ±15/group):G1: control ,A. hydrophila challenged,G2: A. hydrophila challenged + Mi-AgNPs-administered in aquaria water on a daily basis at a concentration of 0.01 mg/L),G3: A. hydrophila challenged + Mi-AgNPs-administered in aquaria water on a daily basis at a concentration of 0.05 mg/L),G4: A. hydrophila challenged + Mi-AgNPs-administered in aquaria water on a daily basis at a concentration of (0.1 mg/L),G5: A. hydrophila challenged + M. indica extract-administered in aquaria water at a concentration of 0.1mg/L),G6: G7: Mi-AgNPs treated- Mi-AgNPs-administered in aquaria water on a daily basis at a concentration of (0.1mg/L).For challenge studies, hypervirulent Mi-AgNPs and Mi-Extract were freshly prepared each day, sonicated and administrated to the aquaria water at the above-mentioned concentrations. The concentrations of the AgNPs were chosen based on previous literature regarding nanoparticles administration to fish. Furthermore, optimization was also performed through pilot experiments.At the 15th day of treatment fish were anesthetized by using clove oil (100 µg/L). Blood was collected from the caudal vein with disposable sterile syringe. For serum preparation blood was allowed to stand at room temperature for one hour and afterwards was centrifuged for 15 min at 3500 rpm. The activities of Alanine aminotransferase (ALT) Aspartate aminotransferase (AST) and Alkaline phosphatase (ALP) were assessed by Kinetic enzyme assays. Total protein (TP) was estimated through Photometric Colorimetric-Biuret method by using total protein kit . Albumin kit  was used to calculate the amount of Albumin (Alb). Globulin (Glo) was calculated by subtracting Alb from TP. Albumin/Globulin Ratio (A/G) was also calculated.Ethylenediaminetetra cetic acid (EDTA) was added to the fresh blood to stop clotting. For Total leukocyte count (TLC) and Total erythrocyte count (TEC) blood was diluted in Turk’s solution and Toisson’s solution respectively. Cells were counted on hemocytometer. Hemoglobin (Hb) was measured by Cyanmethemoglobin method using Drabkin’s fluid, and absorbance was recorded on the spectrophotometer at 540 nm. Hematocrit (Hct %) was perceived by using microhematocrit tubes. Blood smears from fresh blood were prepared immediately after blood collection for Differential Leukocyte count (DLC). Blood smears were air dried and later were dipped in 95% methanol for cell fixation. Giemsa stain was used for staining smears. The stained smears were studied under light microscope. Number of Lymphocytes (Lym), Monocytes (Mon), Basophils (Bas), Eosinophils (Eos) and Neutrophils (Neu) were documented.Kidney, liver and gill tissues were immediately fixed in 10% buffered formalin to maintain cell structure and shape. Following the fixation tissues were dehydrated by passing through different grades of ethanol, for 15 min each, in ascending order. Finally, they were kept in absolute ethanol. Tissues were then put in parafin wax and sections of 6–7 µm were cut by using a Microtome machine (ERM-2301). Later the sections were washed successively with xylene and ethanol to remove any extra material. The sections were shifted onto glass slides, stained with hematoxylin and eosin (H&E) and were imaged through optical microscope .Liver and kidney tissues were snap-frozen on liquid nitrogen, later they were rinsed with chilled PBS. Tissues were then homogenized with a Tenbroek glass homogenizer and were centrifuged at 4 °C for 15 min at 10,000 rpm. The supernatant was used to measure malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) through spectrophotometer.A.hydrophila challenged, A. hydrophila + Mi-AgNPs (0.1 mg/L) and A. hydrophila + Mi-Extract (0.1 mg/L) groups to compare the bacterial load. The tissues were suspended in sterile PBS (0.5 mL). Equal volume (100 µL) of suspensions was poured and spread on LB agar plates and placed in the incubator overnight at 37 °C. The growth of bacteria was checked on the next day.Equal amounts (0.5 mg) of muscle tissues were collected from Data was analyzed by GraphPad prism software . Values in the tables and graphs represent averages ± standard error (SE) of three independent experiments. ANOVA was applied to find significant differences between various groups.3, Mi-extract and Mi-AgNPs are shown. In case of DI and AgNO3 samples no absorption peaks were observed in the entire wavelength range of 300–900 nm confirming the absence of AgNPs. A series of narrow peaks can be noticed at lower wavelength values below 400 nm in the case of Mi-extract. Similar absorption properties of M. indica leaves extract has also been reported in literature [In terature ,27,28,29nsitions ,27. A siThe XRD pattern of Mi-AgNPs showed four noticeable diffraction peaks at 2θ values of 38.25° (111), 45.85° (200), 64.75° (220), and 77.3° (311) b. These FTIR is prominent techniques for the identification of different functional groups and bonds. It can be utilized to study the potential biomolecules of Mi-extract involved in the bioreduction and stabilization of Mi-AgNPs .−1  starts as soon as precursor salt, AgNO3 dissolves in DI water. These Ag-ions require some reducing agent to convert onto free Ag-atoms (Ag0), in this case metabolites of Mi-leaves extract serve as bio-reducing agent to convert Ag+ to Ag0. The heating and construal stirring helped in enhancing the reaction rate and developing the suitable reaction condition for the generation of nuclei from the free Ag-atoms under the effect of Brownian motion and van der Waals interactions. As the reaction proceeds, nucleation and growth processes go side by side. Next, Mi-extract biomolecules came into play and start capping the Mi-AgNPs by behaving as stabilizing agents.A. hydrophila challenged fish. Infection caused the enzyme concentrations to rise significantly in the infected fish , that help them to cope with the microbial challenges. The oxidative burst results in various enzymatic and biochemical changes in the host to control the pathogenic spread. Results of the biochemical parameters indicated that the liver enzyme ALT, AST and ALP were increased in the ted fish . HoweverL. rohita against A. hydrophila challenge [Azadirachta indica have been reported in C. mrigala fingerlings challenged with A. hydrophila. Similar to our finding the effects of AgNPs were reported to be more promising than the extract [Moringa oleifera leaves resulted in low amounts of SOD, CAT and POD activities in rice plants with biotic stress. AgNPs reduced the biotic stress by adjusting the production of proteins, enzymatic and nonenzymatic compounds and controlled the proliferation of Aspergillus flavus [Mango kernel has been shown to stimulate immunity in hallenge . Moreove extract . The effs flavus .A. hydrophila infection caused decline in TLC, TEC, Hb and Hct. Mi-AgNPs showed improvements by recovering these parameters  denaturation in the Carrageenan-induced rat hind paw edema model [Lindera strychnifolia exhibited wound healing property [For histopathological analysis, 0.1 mg/L concentration of Mi-AgNPs was employed, being the most effective concentration in the previous experiments. Same concentration of the extract was also used for this assay for a comparable analysis. In the control group normal tissue morphology of liver, gills and kidney was observed a,f,k. A.uolation b,g,l. A.ol group c,h,m. Thol group d,i,n. Fiol group e,j,o. Mima model . In anotproperty . Some poproperty .A. hydrophila challenged group. It was noted that Mi-AgNPs restored the levels of these markers in a dose dependent manner. In the group treated with only Mi-AgNPs, no significant differences were found than the control group showing that the prepared Mi-AgNPs did not cause any oxidative burden of their own on the fish. It has been reported that AgNPs prepared from M. indica seed aqueous extract perform dose dependent antioxidant activity [Catharanthus roseus flower extracts also showed stronger antioxidant potential in comparison to its flower extract [MDA levels were increased significantly both in the gills and liver of the challenged fish . Howeveractivity . Moreove extract . Plants  extract . The hig extract . Thus, tM. indica extract were seen to perform strong antibacterial performance against Gram-negative bacteria viz, P. aeruginosa K. pneumoniae E. coli and S. typhimurium [Moringa oleifera leaf extract and showed antimicrobial activity against clinical isolates of S. aureus, C. tropicalis, C. krusei and K. pneumonia [S. aureus and E. coli [Lavandula angustofolia, L. Otostegia persica, Catharanthus roseus, Lantana camara and Albizia procera have also been exhibited significant antibacterial effects against S. aureus, E. coli and Pseudomonas aeruginosa [Bacterial colonies were counted from the agar plates which showed that Mi-AgNPs (0.1 mg/L) notably decreased the proliferation of bacteria . Bacterihimurium . In anotneumonia . AgNPs p E. coli . AgNPs pruginosa ,57,58,59ruginosa ,61.M. indica plant extract which served as reducing and stabilizing agent. The biosynthesized Mi-AgNPs exhibited significant biochemical, histological and antioxidant potential against A. hydrophila in C. mrigala. The bacterial proliferation was low in the challenged fish treated with Mi-AgNPs. Overall, AgNPs showed stronger antibacterial potential than the M. indica extract. We observed that the highest concentration of the Mi-AgNPs (0.1 mg/L) was most effective in the disease recovery and therefore, it is recommended that for future studies the reported highest concentration or even higher doses should be tested. Based on these findings it is concluded that M. indica mediated AgNPs can be promising candidates for antibacterial applications against pathogens in aquaculture. Our study is novel in presenting a first in vivo challenge assay for the anti-bacterial effect of M. indica leaves’ extract mediated AgNPs in C. mrigala and provides innovative insights of biogenic remedies for disease treatment in aquaculture. This will help in minimizing the use of pernicious drugs and antibiotics in fisheries which by means of bio-magnification in animal tissues can also be a health hazard to humans.Nanotechnology offers numerous benefits for biological applications but the synthesis methods bring forth some concerns. The synthesis of nanoparticles through the utilization of green resources as a substitute for the synthesizing chemicals has started to revolutionize this approach. This study presents an eco-friendly and biocompatible method for the synthesis of AgNPs from


Background:Candida spp can cause a variety of infections known as candidiasis, ranging from severe invasive infections to superficial mucosal infections of the mouth and vagina. Fluconazole, a triazole antifungal, is commonly prescribed to treat candidiasis but increasing fluconazole resistance is a growing concern for several Candida spp. Although C. albicans has historically been the most common cause of candidiasis, other species are increasingly common and antifungal resistance is more prevalent in these non-albicans species, including C. glabrata, C. parapsilosis, and C. tropicalis, which were the focus of this analysis. Methods: We used the PINC AI healthcare data (PHD) database to examine fluconazole resistance for inpatient isolates between 2012 and 2021 from 187 US acute-care hospitals with at least 1 Candida spp culture with a fluconazole susceptibility result over the entire period. We calculated annual percentage fluconazole resistance for C. glabrata, C. tropicalis, and C. parapsilosis isolates using the clinical laboratory interpretation for resistance. Results: We identified 4,264 C. glabrata, 2,482 C. parapsilosis, and 2,283 C. tropicalis isolates between 2012 and 2021 with susceptibility results. The percentage of C. glabrata isolates resistant to fluconazole doubled between 2020 and 2021 (14.6% vs 29.3%) . The percentage of C. parapsilosis isolates resistant to fluconazole steadily increased since 2017 , with an 82% increase in 2021 compared with 2020 (3.8% in 2020 vs 6.9% in 2021). Fluconazole resistance among C. tropicalis isolates varied over the years, with a 0.3% decrease in 2021 from 2020 . Of hospitals reporting at least 1 result each year 2020–2021, 44% observed an increase in the proportion of C. glabrata isolates resistant to fluconazole in 2021 compared to 2020. Conclusions: Our analysis highlights a concerning increase in fluconazole resistance among C. glabrata and C. parapsilosis isolates in 2021 compared with previous years. Further investigation of the observed increases in fluconazole resistance among these Candida spp could provide further insight on potential drivers of resistance or limitations in reported results from large databases. More analyses are needed to understand rates, sites of Candida infections, and risk factors  associated with resistance.Disclosures: None


Dioecy, a sexual system of single-sexual (gynoecious/androecious) individuals, is rare in flowering plants. This rarity may be a result of the frequent transition from dioecy into systems with co-sexual individuals.Diospyros species, was identified in the diploid D. oleifeara historically. We characterized potential genetic mechanisms that underlie the dissolution of dioecy to monoecy and andro(gyno)monoecy, based on multiscale genome-wide investigations of 150 accessions of Diospyros oleifera. We found all co-sexual plants, including monoecious and andro(gyno)monoecious individuals, possessed the male determinant gene OGI, implying the presence of genetic factors controlling gynoecia development in genetically male D. oleifera. Importantly, discrepancies in the OGI/MeGI module were found in diploid monoecious D. oleifera compared with polyploid monoecious D. kaki, including no Kali insertion on the promoter of OGI, no different abundance of smRNAs targeting MeGI (a counterpart of OGI), and no different expression of MeGI between female and male floral buds. On the contrary, in both single- and co-sexual plants, female function was expressed in the presence of a genome-wide decrease in methylation levels, along with sexually distinct regulatory networks of smRNAs and their targets. Furthermore, a genome-wide association study (GWAS) identified a genomic region and a DUF247 gene cluster strongly associated with the monoecious phenotype and several regions that may contribute to andromonoecy.In this study, co-sexual expression (monoecy and hermaphroditic development), previously thought to be polyploid-specific in D. oleifera, presumably also a result of genomic features of the Y-linked region.Collectively, our findings demonstrate stable breakdown of the dioecious system in The online version contains supplementary material available at 10.1186/s12870-023-04610-3. Dioecy—male and female reproductive organs in separate individuals—is found in only approximately 6% of angiosperm species, but in diverse plant lineages –3. This Diospyros is diverse, and several key genetic components controlling sexual expression have been identified [D. lotus is dioecious, exhibiting separate male and female plants; polyploid D. kaki encompasses gynoecious , androecious , monoecious , androgynomonoecious , and andromonoecious  individuals [D. oleifera -coding gene OGI determines the formation of male trees via repression of the feminising gene MeGI [MeGI promoter may contribute to monoecy in hexaploid D. kaki [Sex expression in the genus entified . Diploidividuals –12. Simiera Fig. . In Dioss system . In diplene MeGI . In cont D. kaki .Fig. 1DiD. spp. have been identified, the fundamental molecular mechanisms that underlie the sexual diversity of some D. spp.  are unclear [D. oleifera population. The diploidy of D. oleifera with all types of sex expressions was confirmed   was conducted to determine candidate genomic regions and genes that contribute to the sexual diversity.In mid-April 2019 and 2021, we surveyed the sex phenotype of E) Fig. S. Two hunE) Fig. S. The genOGI gene in 150 D. oleifera individuals . In contrast, all 45 androecious individuals contained the MSR and OGI . All co-sexual plants , except for one monoecious individual, contained the MSR and were OGI+; these findings strongly suggested that OGI is required for male tissue production in general sex determination of D. oleifera, as in monoecious D. kaki [OGI were considered female-biased monoecious plants. We did not include plants with inconsistent phenotypes (four gynoecious with OGI) in subsequent analyses.We analysed the presence of the MSR  and the ls Table . We foun D. kaki . Among tOGI, form two- or three-flower cymes with a female  flower in the middle and one or two abnormal small female flowers at the sides  on the OGI promoter [OGI+D. oleifera individuals harboured Kali on the promoter of OGI  [OGI gene were used to represent the OGI expression levels here. The abundance of smRNAs on OGI gene was significantly higher in male floral buds obtained from all the androecious, monoecious and andromonoecious D. oleifera trees  than that in the female floral buds obtained from gynoecious trees   . Thus, tOGI and MeGI regions (smMeGI) in D. oleifera  than in floral buds from gynoecious plants   Fig. A, similaril Fig. F, which  D. kaki , 10.smMeGI patterns in the co-sexual types were distinct from the findings in previous reports. In the monoecious D. oleifera, where individuals exhibited separate male and female flowers, smMeGI accumulation was not significantly different between the female  and male floral buds    Fig. C. Simila_M) Fig. F.smMeGI. Among andromonoecious plants, the level of smMeGI accumulation was significantly lower in hermaphroditic floral buds  than in male floral buds    Fig. D. In con_H) Fig. A and D. M_H Fig. F.OGI― pseudo-monoecious plants, low smMeGI accumulation was observed in both solitary female floral buds  and lateral floral buds of the three flower cymes   Fig. E. MeGI eent Fig. F.D. kaki, a lower methylation level of the MeGI promoter is associated with female flower formation in genetically male plants [D. oleifera, where sex expression does not match with the component of OGI/MeGI system. The DNA methylation levels of floral buds and stems were analysed by whole-genome bisulphite sequencing  than in male tissues (M_M) in almost all genomic regions  [D. oleifera.As a potential regulator of DNA methylation, we investigated the expression levels of DNA methyltransferase/demethylase genes by RNA-Seq. A demethylase gene (evm.model.Chr7.1501), homologous to REPRESSOR OF SILENCING1 (ROS1) , was dowTo characterise the functional overlap of gynoecia/androecia development in different sexual expression systems, transcriptome analyses were conducted using 26 samples of floral buds and stems of flowering shoots Text S-S26. We i.e., low-level miRNA and high-level mRNA targets in females), were identified in single- and co-sexual systems , auxin response factor 18 family members, cinnamoyl-CoA reductase 2, myosin-11, and evm.model.Chr12.1832.1. Specifically, NOZZLE and GAM1 were detected in all three networks. In contrast, at least two of the three female-active networks  , SPL9, SPL16, SPL17, lysine-specific demethylase (JMJ25), BHLH25, PCS1, MAR-binding filament-like protein 1–1 (MFP1-1), origin of replication complex subunit 1A (ORCS-1A), Fanconi anaemia group M protein homolog, and cucumisin, growth-regulating factor 6 (GRF6), ORCS-1A, ATP sulphurylase 1-chloroplastic, and SOBIR1.Based on the interaction network analysis and functional annotation, highly expressed miRNAs in female (or hermaphroditic) and their mRNA targets downregulated in female (or hermaphroditic) floral buds, and vice versa  and male tissues revealed that numerous Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were commonly enriched in both single- and co-sexual systems, including the female-active pathways phenylpropanoid biosynthesis, biosynthesis of secondary metabolites, and plant hormone signal transduction, etc.Fig. G, as welTo evaluate the regulatory paths of sex differentiation in female and male floral buds in co- and single-sexual systems, coexpression patterns were visualised by weighted correlation network analysis using all female and male floral buds that had been subjected to RNA-Seq analysis Table S. A scaleP < 0.05) enriched in five GO terms: regulation of transcription DNA-templated, regulation of primary metabolic process, regulation of gene expression, regulation of cellular macromolecule biosynthetic process, and copper ion binding  in the midnight-blue module and B3 domain-containing transcription repressor (VAL1) in the yellow module were shown to be potential key genes for male development , Myb-related protein B (myb12) and bHLH96 (BHLH96) in the green module were shown to be potential key genes for female development  and 318,863 indels were obtained for further analysis. The mean PI_HAT value was 0.056, indicating a low level of familial relationship in the population. PCA showed that the sampled individuals could be divided into three clusters  .We performed whole-genome sequencing of 150 To identify the genomic factors that confer the monoecious phenotype, we performed GWAS for the proportion of female shoots in genetically male plants. Ninety individuals with male flower production were selected; and 3,502,197 SNPs and 311,512 indels were retained for further analysis after LD pruning Table S.DUF247 family were identified in this block  (Table SD. lotus [OGI−) were homozygous (XX type), whereas most of the 89 male-functional individuals were heterozygous (XY type)  Table S of 150 p) Table S. This reD. lotus  Fig. 9BBOGI+ (Tape) Fig. . Genotyppe) Fig. . The occOGI  usually fully overlapped. This finding may be attributed to the low viability of the YY genotype . Genetic modelling has shown that under such conditions, stable coexistence of single- and co-sexual plants in a population (subdioecy) can be achieved, because it is likely that Y chromosome has not yet evolved to fully suppress female functions [GLO and ARR9  and androecious  plants  and increased expression of putative miRNA regulators were identified in female (or hermaphroditic) floral buds compared with male floral buds. GAMYB is a trans-activator of GA signalling [GAMYB in male floral buds implies that GA signalling promotes the development of androecious tissues, consistent with our previous findings that GA promotes the male function in monoecious [D. kaki. Furthermore, SPL family genes , JMJ25, and GRFs were commonly activated in female tissues, presumably through miRNA regulation. SPL family genes have diverse functions in plant development [LEAFY, FRUITFULL, and APETALA1 to control the timing of flower formation [JMJ25 is a histone H3K9 demethylase gene that reportedly affects DNA methylation [GRFs are plant-specific transcription factors, and the miR396/GRF regulatory network is required for the proper development of the pistil in Arabidopsis [D. oleifera were consistent with the working hypothesis regarding sexual expression in D. oleifera.We also identified several putative key relationships between miRNA expression and target expression. Decreased expression of gnalling , 30 and gnalling , 32. Thenoecious , 34 and noecious  D. kaki.elopment ; one of ormation . JMJ25 ihylation . GRFs arbidopsis , 39. TheHSFB3) was reported to be firmly correlated with abiotic and biotic stress [HSFB3 was sharply higher expressed in male floral buds than that in female, suggesting that when plants were suffering from abiotic or biotic stress, they tended to bear male flowers. Thus, protection against abiotic or biotic stress contributes to the feminising scenario in D. oleifera.Expression of heat stress TF B-3 , implying functional divergence. The monoecious determinant, as well as its molecular genetic control, must be identified in subsequent studies. Although a very strong signal was obtained for chromosome 7, it could not explain all monoecious phenotypes  [D. oleifera plants bearing hermaphroditic flowers, whereas Masuda et al. regarded the hermaphrodite mechanism in Diospyros as polyploid species-specific. Our results indicate that the evolution of hermaphroditic flower development in Diospyros is not ploidy-dependent; however, considering the similarity in sexual expression between D. kaki and D. oleifera, as well as their close phylogenetic relationship [D. kaki and D. oleifera are needed.Masuda et al. (2022b) reported that  D. kaki . This maes Fig. S, as in D (2022b)  is that tionship , a commoD. oleifera, female shoots mainly develop from mixed dormant buds that developed on the tips of the flowering mother branches in monoecious plants which are genetically male with OGI. In contrast, male shoots mainly develop on the basal parts of the mother branches monoecy, based on multiscale genome-wide investigations of 150 accessions of Diospyros oleifera. We found all co-sexual plants, including monoecious and andro(gyno)monoecious individuals, possessed the male determinant gene OGI, implying the presence of genetic factors controlling gynoecia development in genetically male D. oleifera. In both single- and co-sexual plants, female function was expressed in the presence of a genome-wide decrease in methylation levels, along with sexually distinct regulatory networks of smRNAs and their targets. Furthermore, a genomic region and a DUF247 gene cluster strongly associated with the monoecious phenotype and several regions that may contribute to andromonoecy were identified. Collectively, our findings demonstrate stable breakdown of the dioecious system in D. oleifera, presumably also a result of genomic features of the Y-linked region.Co-sexual expression (monoecy and hermaphroditic development), previously thought to be polyploid-specific in D. oleifera collection from Guilin, Guangxi Zhuang Autonomous Region, China, was evaluated for sexual expression in the 2019 and 2021 seasons. Two hundred eight D. oleifera trees were found in the natural population. In D. oleifera, the flower sex on a single shoot is uniform. Thus, for simplicity and accuracy, large flowering mother branches (approximately 1.5 m in height × 1.5 m in width) containing ≥ 20 flowering shoots  were used to calculate the proportions of female, male, and hermaphroditic shoots in monoecious, androgynomonoecious, and andromonoecious trees. At least five large flowering mother branches of each tree were selected; the highest female and hermaphroditic shoot proportions of the large flowering mother branches were used for GWAS as an indication of feminising ability on the co-sexual tree.A D. oleifera 1’ was used to optimise the published version of the D. oleifera genome [D. oleifera main genome. This genome was deposited in figshare (https://doi.org/10.6084/m9.figshare.20101664.v3) [D. oleifera individuals (detail information of resequencing reads was introduced in the following text) were mapped to the D. oleifera main genome with the Burrows-Wheeler Aligner (BWA) mem option and the paired-end model [https://doi.org/10.6084/m9.figshare.20407386.v1) [D. oleifera main genome [D. oleifera chloroplast genome [The female plant ‘a genome  using a 1664.v3) . The malnd model . The unmnd model , and thend model  to const7386.v1) . The D. n genome , male-unn genome , and thet genome  were comD. oleifera genome. Details of library construction, sequencing, and analysis are provided in Methods SFloral bud and immature stem tissues of flowering shoots were sampled in mid-April (April 15–17th), 2019, which is a key period for the differentiation of flower sex types Method S. Those sOGI sequence from the D. lotus genome [MeGI sequence from the D. oleifera reference genome, and the ‘Kali’ sequence reported by [. Here, the smRNAs-Seq reads mapped onto the MeGI gene body were referred to as smMeGI. The accumulation levels of smMeGI and each fragment were recorded as reads per million reads. Two independent-samples T test  was used to determine the significant difference between female (or hermaphroditic) and male floral buds (or stems) in each sexual systems.The smRNAs-Seq reads were also mapped to the s genome , MeGI seorted by , using torted by . Here, tD. oleifera  were used to label SNPs and insertion-deletions (indels). Polymorphisms that matched the following four criteria were filtered out: > 2 alleles, variants beyond the read depth between half and twice the genome-wide average, missing rates ≥ 0.25, and minor allele frequency < 0.05. The linkage disequilibrium (LD) was evaluated using the pairwise squared Pearson’s correlation coefficient (r2) calculated by PLINK version 1.9 [A set of 150 ra Table  was selesequence  using thsion 1.9 . LD prunUsing the filtered variant sets, the following analyses were conducted. First, the familial relationships and sample uniqueness were evaluated based on the PI_HAT value computed by PLINK. Then the population structure of the 150 trees was estimated using principal component analysis (PCA) in EIGENSTRAT software . The maxP < 0.05) was used to determine the genome-wide significance thresholds. The LD patterns surrounding GWAS peaks were visualised using the R package LDheatmap [http://www.broadinstitute.org/haploview) for chromosomes 4, 2, 11, and 14. The regions with pairwise r2 > 0.5 were regarded as candidate LD blocks.GWAS was performed using the linear mixed model in the R package rrBLUP . A kinshDheatmap  for chroAdditional file 1.Additional file 2.Additional file 3.Additional file 4.


Sium L. (Apiaceae) is a small genus distributed primarily in Eurasia, with one species also occurring in North America. Recently, its circumscription has been revised to include 10 species, however, the phylogenetic relationships within its two inclusive clades were poorly supported or collapsed in previous studies based on nuclear ribosomal DNA ITS or cpDNA sequences. To identify molecular markers suitable for future intraspecific phylogeographic and population genetic studies, and to evaluate the efficacy of plastome in resolving the phylogenetic relationships of the genus, the complete chloroplast (cp) genomes of six Sium species were sequenced.Sium plastomes exhibited typical quadripartite structures of Apiaceae and most other higher plant plastid DNAs, and were relatively conserved in their size , gene arrangement and content (with 114 unique genes). A total of 61–67 SSRs, along with 12 highly divergent regions  were discovered in the plastomes. No significant IR length variation was detected showing that plastome evolution was conserved within this genus. Phylogenomic analysis based on whole chloroplast genome sequences produced a highly resolved phylogenetic tree, in which the monophyly of Sium, as well as the sister relationship of its two inclusive clades were strongly supported.The The plastome sequences could greatly improve phylogenetic resolution, and will provide genomic resources and potential markers useful for future studies of the genus.The online version contains supplementary material available at 10.1186/s12870-023-04376-8. Sium L. is a small genus belonging to the tribe Oenantheae of Apiaceae subfamily Apioideae, which included 12 species in previous delimitation [S. ventricosum (H.de Boissieu) L.S.Wang & M.F.Watson, S. latifolium L., S. medium Fisch. & C.A.Mey., S. ninsi Thunb., S. serra (Franch. & Sav.) Kitag., S. sisaroideum DC., S. sisarum L., S. suave Walter-it is also distributed in North America, and S. tenue Kom.). In sub-Saharan Africa, the genus has one species (S. repandum Welw. ex Hiern), while Sium burchellii (Hook. f.) Hemsl. and S. bracteatum (Roxb.) Cronk are endemic to the island of Saint Helena. The genus compassed species that often live in moist to aquatic habitats, and characterized by glabrous throughout, fascicled, fusiform or fibrous roots, simple pinnate leaves with sessile pinnae, long and reflexed styles, fruits with prominent and corky-thickened ribs [mitation . The majned ribs .Sium are used as herbal medicines in Chinese folk remedies [Sium sisarum are rich in carbohydrates, and served as food before the potato was introduced [Sium suave is often sold in medicinal markets as an adulterant of “Gao-ben” [Some species of remedies . They arremedies , and canremedies . The thitroduced , 8. FurtGao-ben” .Sium and its infrageneric phylogenetic relationships have been controversial. For example, members of the traditionally delimited Berula and Sium have long been considered closely related and sometimes even synonymous [Sium is unclear [Sium, and the taxonomic status of some species were resolved. Among them, Pimpinella crispulifolia H.de Boissieu was transferred into the Sium based on morphological and molecular analysis [S. serra and S. ventricosum were recognized as belonging to the genus Sium [Sium and related genera [Sium, as previously circumscribed (with 12 species), was shown to be polyphyletic. Its three species  outside of Eurasia (from sub-Saharan African/Saint Helena), together with Berula  and Afrocarum (Africa), formed a strongly supported clade (Berula s.l.). Berula s.l. has been recognized at the generic level and these African/Saint Helena species were thus transferred into the genus Berula. The remaining species constituted the Sium s.s., including the northern Holarctic clade  and the southern Palearctic clade . However, the phylogenetic relationships between the two inclusive clades were poorly supported in the ITS trees [rps16-trnK) analysis [The circumscription of nonymous . Moreove unclear . In receanalysis , and S. nus Sium . Furthernus Sium , 13, alod genera , showed TS trees , or theyanalysis . TherefoPlastomes can provide valuable information for taxonomy and phylogeny, and have proven to be a powerful tool for exploring phylogenetic relationships , 16. It Sium species were sequenced, characterized and compared. We aimed to: (1) analyze the global features and structural patterns of Sium cp genomes; (2) identify simple sequence repeats (SSRs) and hotspot regions within Sium; (3) evaluate the efficacy of the whole cp genome in resolving the relationships within the genus, and compared with the results of ITS analysis. This is the first comprehensive analysis on cp genomes of the genus Sium, the results of which will provide genetic resources and molecular markers for future studies of this genus.In this study, the complete chloroplast (cp) genomes of six Sium range in length from 153,029 bp (S. ventricosum) to 155,006 bp (S. ninsi), with the GC content identically 37.40% , 7 tRNA genes , and four rRNA genes  are duplicated in the IR regions  having one intron and two genes (ycf3 and clpP) having two introns. The two individuals of S. suave present minor difference in length, with 154,642 bp (S. suave 1) and 154,676 bp (S. suave 2). In general, the plastomes of Sium are highly identical in their structural organization, gene order and gene content.The seven complete cp genomes of Sium cp genomes  to 67 (S. suave) . In which, 2630 variable sites and 1245 parsimony informative sites are included. The divergence levels of the noncoding and single-copy regions (LSC and SSC) are higher than that of the coding and IR regions, respectively  show the sequence divergence of 0.0060, and the largest sequence divergence value is observed between S. crispulifolium and S. medium (0.0080).The aligned cp genomes of six Pi) range from 0.0000 to 0.0327  are recognized as hotspot regions with nucleotide diversity > 0.015. Of those, six regions are located in the LSC, and six were in the SSC region. The NJ trees show that all these regions could identify Sium species separately or jointly  duplicated in the IRa. Similarly, the SSC/IRa junction are located in the ycf1 gene across all species, resulting in the presence of ψycf1 in the IRb. There are two copies of the rpl2 genes located in the IR regions (IRa and IRb), which is 117–139 bp away from the LSC/IR junction, while the trnH gene is just located at the junction of LSC/IRa region in all plastomes. The ndhF gene of S. ninsi, S. tenue and S. ventricosum are completely located in the SSC region, which extends into the IRb region by about 7–39 bp in other species. These variations at boundary regions lead to the length variation in the cp genomes of Sium.The expansion and contraction of the junction regions are analyzed for the six ted Fig. . The genSium species are inferred. The phylogenies estimated using ML and BI analyses are well-resolved and fully consistent with one another . Within Sium, two monophyletic sister clades are recognized with high support . Clade I includes two species distributed in the northern Holarctic [S. medium is sister group to the clade of eastern Asiatic-North American S. suave. The remaining species of the genus constitute the Clade II, in which the two species endemic to China (S. ventricosum and S. crispulifolium) ally together, and comprise a sister group relationship to the group of S. ninsi and S. tenue. The ITS and plastome trees produce consistent topologies for Sium species , along with the monotypic African Afrocarum, were nested within Berula forming the Berula s.l. clade. These four African/Saint Helena species have been transferred into the genus Berula [Sium constituted the Sium s.s., and consisted of two clades: the northern Holarctic clade  and the southern Palearctic clade . In all analyses, the monophyly of each of the two clades is strongly supported, however, their sister group relationship was poorly supported or collapsed in previous studies [Sium crispulifolium was a poorly known species endemic to China, and it was initially described under Pimpinella as P. crispulifolia H.de Boissieu [Sium , it was transferred into the Sium [Sium was expanded to accommodate 10 species. Sium ventricosum is an aquatic and dwarf plant endemic to high montane regions in southwest of China [Apium, Chamaesium, Sium, and Sinocarum [Sium ventricosum and S. crispulifolium constituted a well-supported sister group, and allied strongly with two other eastern Asia representatives (S. tenue and S. ninsi) within Clade II. Therefore, phylogenomic analysis further confirmed that they belong to the genus Sium. The phylogenetic positions of other Sium species were generally consistent with those inferred by previous studies [In recent years, several molecular studies on tribe Oenantheae and the genus ried out , 12–14.  studies , 14. WitBoissieu . Howeverthe Sium . Thus, tinocarum –43. In t studies , 14, butSium species were analyzed and compared. The results revealed that the Sium plastomes are conserved in structural organization, gene order and content. The SSRs and hotspot regions identified can be used as molecular markers in the future intraspecific diversity study of Sium. Furthermore, our study showed that plastome sequences could greatly improve phylogenetic resolution. Overall, the complete plastome sequences reported herein enriched the genomic information available for Sium.In this study, the cp genomes of six Sium species were sampled for analysis . DNA extractions were conducted using the Plant Genomic DNA Kit from Tiangen Biotech (Beijing) Co., Ltd., China. According to the manufacturer’s instructions, the total DNA was fragmented ultrasonically and used for 350-bp insert libraries construction. All of the genomic data were sequenced using the Illumina platform at Novogene , with the paired-end reads 2 × 150 bp.A total of seven accessions, representing six is Table . The colTiedemannia filiformis subsp. greenmannii (Mathias & Constance) M.A. Feist & S.R. Downie [Sium cp genomes and ITS sequences were submitted to GenBank .Raw sequence reads were quality trimmed using Trimmomatic v.0.36 . Remaini. Downie . The tRN. Downie . The cir. Downie . The sevSium plastomes were identified with MISA [The simple sequence repeats (SSRs) of ith MISA . ThreshoS. crispulifolium as a reference. To determine the nucleotide diversity (Pi) among the cp genomes of Sium, the sliding window analysis was conducted using DnaSP v.6.10 [Sium species.The mVISTA program was used to evaluate the variability of the seven complete cp genome sequences under Shuffle-LAGAN mode , using SP v.6.10 , with a P v.6.10  to detecChamaesium taxa were selected as outgroup because the genus holds a sister-group relationship to all other apioid genera, excluding those of its most early-diverging branches [In total, forty-one cp genomes, representing the taxa from major clades of Apioideae and seven newly obtained taxa, were used in the phylogenetic analysis. Two branches , 57. Foubranches , with thbranches , and manbranches . Maximumbranches  using thbranches . Four MaSium species, we performed phylogenetic analysis on these regions separately or jointly using MEGA 7.0. Neighbor-Joining (NJ) trees were calculated according to the Kimura 2-parameter (K2P) model using a bootstrap test with 1000 replicates.To verify the ability of highly divergent regions to identify Additional file 1: Figure S1. Neighbor-Joining trees constructed for the 12 variable regions. A. ndhE-ndhG; B. ndhF-rpl32; C. rpl16; D. rpl32-trnL; E. rps4-trnT; F. trnE-trnT; G. trnG-atpA; H. trnQ; I. ycf1a; J: ycf1b; K. ycf1-ndhF; L. accD-psbI.Additional file 2: Figure S2. Neighbor-Joining tree constructed for the combined 12 variable regions.Additional file 3: Figure S3. Phylogenetic relationships of Sium species inferred from Bayesian Inference (BI) and Maximum Likelihood (ML) analyses of the ITS sequences. Bootstrap values (BS) and posterior probabilities (PP) are shown next to the branches. – indicated that the branch has no support value from BI analysis.Additional file 4: Table S1. List of genes found in the Sium chloroplast genome.Additional file 5: Table S2. More detail of SSRs in Sium species.Additional file 6: Table S3. Primers for PCR amplification of the 12 variable regions among the seven Sium taxa.