Lutzomyia intermedia, a vector of Leishmania braziliensis, does not protect against experimental infection. However, L. braziliensis is also transmitted by Lutzomyia whitmani, a sand fly species closely related to Lu. intermedia. Herein we describe the immune response following immunization with Lu. whitmani saliva and the outcome of this response after L. braziliensis infection.Previous works showed that immunization with saliva from Lu. whitmani saliva developed robust humoral and cellular immune responses, the latter characterized by an intense cellular infiltrate and production of IFN-γ and IL-10, by both CD4+ and CD8+ cells. Mice immunized as above and challenged with L. braziliensis plus Lu. whitmani saliva displayed significantly smaller lesions and parasite load at the challenge site. This protection was associated with a higher (p<0.05) IFN-γ production in response to SLA stimulation. Long-term persisting immunity was also detected in mice immunized with Lu. whitmani saliva. Furthermore, individuals residing in an endemic area for cutaneous leishmaniasis (CL) presented antibody responses to Lu. whitmani saliva. However CL patients, with active lesions, displayed a lower humoral response to Lu. whitmani saliva compared to individuals with subclinical Leishmania infection.BALB/c mice immunized with Lu. whitmani saliva induces protection against L. braziliensis in a murine model. We also show that Lu. whitmani salivary proteins are immunogenic in naturally exposed individuals. Our results reinforce the importance of investigating the immunomodulatory effect of saliva from different species of closely related sand flies.Pre-exposure to  Lutzomyia intermedia a vector of Leishmania braziliensis, the main species that causes cutaneous leishmaniasis (CL) in Brazil, did not protect but exacerbated CL. In the present work, we investigated if immunity to Lutzomyia whitmani, another vector of L. braziliensis, induced a similar effect or not. We observed that mice immunized with Lu. whitmani saliva develop immunity to the salivary components and that this immunity protected the mice against CL development. We further observed that people residing in areas where Lu. whitmani occurs also develop antibodies to saliva and that CL patients have lower levels of these antibodies. These evidences point to differences in the protein repertoire present in the saliva of different sand flies and highlight the concept that salivary proteins should be considered as additional components of a vaccine for leishmaniasis.The saliva from sand flies contains biologically active proteins that permit the insect to obtain a blood meal. When vertebrates are continuously exposed to these molecules, through insect biting, for example, they induce an immune response (antibody and cell-mediated immunity) in the vertebrate host. Previously, we showed that immunity to salivary proteins from  Leishmania. Sand fly saliva includes molecules that modulate the host’s hemostatic, inflammatory and immune responses creating a favorable environment that facilitate parasite infection . Expe. ExpeLeiite load –6. On thite load ,8 or immite load ,10 were (rev. in ).Lutzomyia intermedia and Lutzomyia whitmani are the main vectors responsible for Leishmania braziliensis transmission in Latin America )Lu. whitmani SGS: mice presented an increased production of anti-Lu. whitmani SGS antibodies corroborating earlier data regarding the immunogenic properties of sand fly salivary molecules [Initially we characterized the immune response to immunization with olecules . The antolecules ,25.Lu. whitmani SGS also developed a cellular response as evidenced by the presence of an intense lymphocytic infiltration, characteristic of a delayed-type hypersensitivity response (DTH) which was detected following a challenge inoculation of SGS. Moreover, we also detected production of IFN-γ, IL-10 and IL-4 by draining lymph node cells following stimulation with Lu. whitmani SGS, as well as the presence of CD4+IFN-γ+ and CD4+IL-10+ T cells. Following challenge with L. braziliensis + Lu. whitmani SGS, immune mice failed to develop cutaneous lesions and displayed a significantly reduced parasite load at the challenge site. This outcome was paralleled by a significant increase in IFN-γ production in response to stimulation with Leishmania antigens. The current hypothesis is that immunization with sand fly salivary molecules primes a CD4+ Th1 response, including IFN-γ secreting cells, and this response activates macrophages, promoting Leishmania killing [Lu. whitmani SGS may have contributed in regulating the cellular response thus inhibiting lesion development. In L. major infection, IL-10 is important to prevent cell-mediated immunopathology [L. braziliensis, we showed that Tregs secreting IL-10 are observed [Leishmania persistence [Mice immunized with  killing ,11. Our athology . The IL-observed  in accorsistence ,29.Individuals naturally exposed to sand flies in areas endemic for leishmaniasis develop a humoral immune response to salivary molecules .Lu. whitmani SGS. Moreover, anti-saliva antibody levels were higher in CL patients compared to subclinical individuals, that is those that have a positive LST test without history of CL. Herein, subclinical individuals were characterized as described by Schnorr et al. [Lu. longipalpis saliva [Leishmania responses. Our results differ from those obtained in CL areas endemic for L. tropica [L. major [P. sergenti and P. papatasi, respectively. We suggest that these discrepancies maybe related to differences in salivary components among these species [Herein, we confirmed that individuals residing in a CL endemic area display positive serology to r et al. ; howeverr et al. . We repos saliva ,33 sugge tropica  and L. mL. major ,36: in t species .Lu. whitmani also differ from those we obtained with Lu. intermedia: CL patients presented a higher IgG response to Lu. intermedia SGS compared to individuals with a positive LST [Lu. intermedia saliva increases the risk of developing CL caused by L. braziliensis [Lu. intermedia. Experimental immunization with Lu. intermedia SGS enhanced L. braziliensis infection [Lu. whitmani protected against CL development (herein). Thus, it appears that even for sand flies belonging to the same subgenus (Nyssomyia) such as Lu. intermedia and Lu. whitmani, exposure to salivary molecules leads to distinct outcomes and this is not attributed to differences in the experimental procedure as in all these works, we employed the same immunization scheme and intradermal challenge. One important aspect that has not been addressed, so far, is whether these outcomes are also obtained following a challenge with L. braziliensis-infected Lu. intermedia or Lu. whitmani sand flies. This is relevant because it has been shown that the complex scenario present in a live sand fly challenge is not fully mimicked by needle challenge [Lu. intermedia has been described [Lu. whitmani is still lacking so we cannot, presently, probe for possible differences that could explain the distinct outcomes.In terms of human exposure, the results we describe for tive LST  and expoiliensis , an effenfection ,14 wherehallenge ,39. Regaescribed  but charLu. whitmani and Lu. intermedia can be found in the same CL endemic areas we do not know if there is a cross-protective immune response to these two species of sand flies. Previous work has shown that immunization with saliva from closely related species of sand flies could result in cross-protection. Lestinova et al demonstrated that immunization with P. papatasi saliva resulted in protection upon infection with L. major plus P. papatasi or P. duboscqi saliva [Lu. longipalpis saliva were protected against L. amazonensis only against needle injection of a homologous sand fly saliva challenge; protection was not observed when mice were challenged with Phlebotomus sp. saliva [Lu. longipalpis saliva protected against a challenge with L. braziliensis plus Lu. intermedia saliva [Although i saliva . However. saliva  highligha saliva , indicata saliva , as mentLu. whitmani salivary molecules as tools to monitor human exposure in CL endemic areas [Additionally, other components present in the infected sand fly, such as the promastigote secretory gel (PSG) and parasite exosomes, are also inoculated alongside parasites and saliva into the host skin . These cic areas ,47.S1 Checklist(DOCX)Click here for additional data file.


Bacterial reverse mutation (BRMT) and acute oral toxicity tests were employed to assess the PFS for their possible mutagenicity and toxicity. Results indicated that the PFS were devoid of mutagenic effects in BRMT and showed higher safety profile in rodent acute oral toxicity test.Herbal products are being used and trusted globally for thousands of years for their health benefits and limited side effects. Globally, a general belief amongst the consumers is that herbal supplements are always safe because they are “natural.” But later, research reveals that they may not be safe. This raises concern on their safety and implications for their use as feed supplement or medicine. Toxicity testing can reveal some of the risks that may be associated with use of herbs, therefore avoiding potential harmful effects. The present study was designed to investigate five poultry feed supplements (PFS), EGMAX® , FEED-X Poultry industry is one of the most fertile areas to ease out the pressure of population on crop cultivation. Amongst the livestock based vocations, poultry farming occupies a pivotal position due to its enormous potential to bring about rapid economic growth with low input investment , 2. ThouProducts that are effective, environmentally, and user friendly will provide safe, economical, and long-term success to poultry health programs . In this C. STODI prevents and controls wet litter in poultry during high risk periods, for example, disease outbreak, susceptible season or age, changes in environmental conditions, peak production/growth, and dietary errors. It helps to prevent/treat the noninfectious diarrhoea. It is also supportive to treat infectious diarrhoea along with antibiotic treatment.EGMAX minimizes the gap between standard and actual egg production. It minimizes hairline cracks of eggs and also improves fertility in female breeders. FEED-X optimizes the feed efficiency in poultry. It improves the gastrointestinal function for better absorption of nutrients and also improves carcass characteristics. KOLIN PLUS is a natural replacer of synthetic choline chloride. It reduces the abdominal and liver fat levels. It maintains the healthy liver and supports the growth of poultry birds. PHYTOCEE enhances and maintains the immune competence. It protects the bird from the ill effects of production stress and other stresses. It compensates the stress-induced depletion of Vitamin in vitro BRMT and in vivo acute oral toxicity test.Even though proofs of efficacy on these PFS are available , 6, it iIn the current study, BRMT was performed using AMES MPF fluctuation procedure in microplate format (MPF). The AMES MPF test is a fluctuation method which is different from traditional AMES preincubation and plate incorporation method. AMES MPF uses liquid media and 384-well microplates with readout based on a colour change . Several in vivo. Acute toxicity is involved in estimation of LD50  to 50% of the animals tested) and also in examining the adverse effects that occurs on first exposure to single oral dose administration of test substance [Acute oral toxicity test is usually an initial screening step in the assessment and evaluation of the toxic characteristics of test substances inubstance , 11.EGMAX, FEED-X, KOLIN PLUS, PHYTOCEE, and STODI are herbal PFS developed and manufactured by Natural Remedies Pvt. Ltd., Bangalore, India.EGMAX, FEED-X, KOLIN PLUS, PHYTOCEE, and STODI are standardized botanical powder found to contain not less than 0.1% aloin A and aloin B, 0.5% andrographolide, 8.0% polyphenols, 0.5% of gallic acid, and 0.20% punicalagin, respectively.4-aminocytidine, 9-aminoacridine, and 4-nitroquinoline-n-oxide were procured from Sigma-Aldrich. Dimethyl sulphoxide (DMSO) and carboxymethylcellulose sodium salt (CMC) were procured from Himedia Laboratories Pvt. Ltd.2-Aminoanthracene, 2-nitrofluorene, N in vitro BRMT. PFS as such were subjected for in vivo acute oral toxicity evaluation.PFS were individually extracted as per  Salmonella typhimurium (S. typhimurium)  and Escherichia coli (E. coli) (WP2 uvrA) strains, Aroclor 1254 induced rat liver microsomal fraction (S9), buffer salts, G-6-P, NADP, growth media, exposure media, and reversion indicator media. Genotyping of tester strains was performed by Xenometrix and verified with the help of certificate of analysis. Tester strains were analyzed for their spontaneous mutation rate and genotypic characteristics such as mutations at his, bio loci (histidine and biotin dependency), rfa mutation (defective lipopolysaccharide (LPS) layer that coats the cell surface), and uvrB deletion (eliminating the accurate excision repair mechanism) and for the presence of plasmids pKM101  by Xenometrix and confirmed their genetic integrity.AMES MPF™ Penta I test kit was procured from Xenometrix. The kit containsFemale albino Wistar rats that were bred and reared at Natural Remedies Pvt. Ltd., Bangalore, India, were used in acute oral toxicity studies. S. typhimurium strains like TA 98, TA 100, TA 1535, and TA 1537 and tryptophan loci in E. coli (WP2 uvrA). BRMT with fluctuation method (AMES MPF Penta I-Micro Plate Format mutagenicity assay) was followed to determine the possible genotoxic potential of the four PFS. Based on solubility and precipitation test, DMSO (4%) was used as vehicle control. Cytotoxicity test was conducted using TA 98 strain in both absence and presence of S9 (4.5%) to select the test concentrations for mutagenicity test. For cytotoxicity test, percentage of reduction was determined and the concentrations which showed cell reduction >50% were expelled for mutagenicity testing [ S. typhimurium and E. coli strains were treated with PFS/vehicle control/appropriate positive controls in absence and presence of S9 (4.5%) in triplicate along with indicator media and kept for 48 hours of incubation at 37 ± 2°C. Revertant colonies were counted and positive response was determined [BRMT was used to identify the ability of test substance to induce reverse mutation at histidine loci in testing . In the termined , 14.The animal experiment was conducted according to the CPCSEA  guidelines and after approval by the Institutional Animal Ethics Committee (IAEC). Acute oral toxicity study was performed as per the OECD test guideline for testing of chemicals  of three replicates. Statistical significance was evaluated using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons using GraphPad Prism 5.0 . Statistical significance level was set at Cytotoxicity test on PFS was conducted by using TA 98 strain in both presence and absence of S9 to select appropriate concentrations for the main study. Cytotoxicity is determined by calculating the percentage of reduction on cell viability with the following formula: Based on the cytotoxicity results, the concentrations of test substances which showed less than 50% reduction in cell viability  were conP < 0.05) and also showed fold increase more than two with respect to the vehicle control and thus demonstrated the validity of test  and E. coli (WP2 uvrA) both with and without metabolic activation. This indicates that our formulations cause neither any frame shift mutations in the tester stains TA98 and TA1537 nor base pair mutations in the tester strains TA100, TA1535, and E. coli WP2 uvrA.The BRMT and acute oral toxicity test are recommended by several regulatory agencies  for substance evaluation to determine the safety. Due to its simplicity and relatively low cost, BRMT is commonly employed as an initial screening method for genotoxic activity , 19. EGM Aloe barbadensis (A. barbadensis) and Solanum xanthocarpum (S. xanthocarpum), found to be nonmutagenic in both presence and absence of metabolic activation in BRMT. Similarly, water and methanolic extract of A. barbadensis were reported to be nonmutagenic to S. typhimurium strains TA 98 and TA 100 [μg/plate [50 was found to be more than 5000 mg/kg. Similar to the above finding, Solanum xanthocarpum was reported to be safe with an LD50 value of >2000 mg/kg [EGMAX containsd TA 100 . Aloin, μg/plate . EGMAX w00 mg/kg . Andrographis paniculata (A. paniculata), P. granatum, and E. officinalis was found to be nonmutagenic. Several reports demonstrated that extracts of A. paniculata, P. granatum, and E. officinalis were found to be safe in in vitro, in vivo, and clinical trials. A. paniculata standardized to andrographolide was reported to be nongenotoxic in AMES, chromosomal aberration, and micronucleus tests [50 of A. paniculata extract was found to be greater than 5 g/kg in rodents at the single oral administration [ A. paniculata was reported to be devoid of reproductive toxicity [FEED-X composed ofus tests . Aqueousus tests . Aqueousus tests , 25. LD5stration . Also A.toxicity , 27. In  Acacia nilotica (A. nilotica) and Curcuma longa (C. longa), was found to be nonmutagenic. Similarly, acetone extract of A. nilotica was reported to exhibit antimutagenic activity against 2-amino fluorine [ C. longa was reported to be nongenotoxic to rodents [ C. longa was found to be nonmutagenic to S. typhimurium strains like TA98 and TAMix. Also it was found to be safe up to 5000 mg/kg to rodents after a single oral administration [50 of Acacia arabica was found to be greater than 2000 mg/kg [KOLIN PLUS, an herbal amalgamation offluorine . C. long rodents . Polysacstration . LD50 of00 mg/kg . Safety  E. officinalis, Ocimum sanctum (O. sanctum), and Withania somnifera (W. somnifera) were found to be safe. Antigenotoxic activity of W. somnifera against malathion-induced DNA damage in mice leucocytes was reported [ W. somnifera extract was reported to protect the albino mice against toxicity induced by lead nitrate [ In vivo study on W. somnifera root extract in mice reported nongenotoxic effect as evident from no significant changes of chromosome morphology [ O. sanctum at a dose 5 g/kg body weight did not cause micronucleus induction in bone marrow cells of rats. Ethanol extract of O. sanctum was reported to be antimutagenic to cyclophosphamide-induced micronucleus formation at a dose of 5 g/kg body weight. The antimutagenicity of O. sanctum was attributed to the enhanced levels of liver detoxification enzymes. O. sanctum has been reported to have anticlastogenic effect against Mitomycin C- and chromium-induced genotoxicity in human peripheral blood lymphocytes [ in vivo chromosomal aberration study, leaves of O. sanctum exhibited protective effect on chromium- and mercury-induced structural chromosomal aberrations [PHYTOCEE used in poultry feeds was reported to be nonmutagenic to TA98, and TAMix strains in both the absence and presence of metabolic activation . The revreported . W. somn nitrate . In vivorphology . Adminisphocytes . In an irrations . In this P. granatum, A. arabica, H. antidysenterica, A. paniculata, and Terminalia bellerica (T. bellerica). In this study, STODI was found to be nonmutagenic to S. typhimurium and E. coli strains in presence and absence of metabolic activation. The antimutagenic effect of H.antidysenterica was demonstrated against NaN3(sodium azide) and MMS (methyl methane sulfonate) in TA 97a, TA 100, TA 104, and TA 102 tester strains [50 of STODI more than 5000 mg/kg body weight in rats. Aqueous, ethanol, and hydroalcoholic extracts of H. antidysenterica seeds are reported to be safe up to 2000 mg/kg body weight in rats [ H. antidysenterica leaves was reported to be safe up to 2000 mg/kg after single oral dose in rats [ T. bellerica was reported to be antimutagenic against 4-O-nitrophenylenediamine (NPD) and NaN3 in AMES Salmonella/microsome assay [ T. bellerica was reported to possess LD50 value higher than 2000 mg/kg body weight of Wistar rats [STODI is a herbal blend of strains . The cur in rats , 38. Eth in rats . Acetoneme assay . T. belltar rats . Based o S. typhimurium and E. coli strains in presence and absence of metabolic activation. In acute oral toxicity studies, no major gross pathological changes were observed in any of the PFS treated animals. PFS fed rats exhibited comparable weight gain. This suggested that the PFS did not exert any deleterious effects on the general health status and metabolic growth of the rats. Under our test conditions, the oral administration of EGMAX, FEED-X, KOLIN PLUS, PHYTOCEE, and STODI was found to be safe up to 5000 mg/kg in female albino Wistar rats. Based on the results, all the PFS were declared as unclassified in the hazard category according to Globally Harmonized System.The current study reckoned that PFS, EGMAX, FEED-X, KOLIN PLUS, PHYTOCEE, and STODI were nonmutagenic in in vitro BRMT. Also it was concluded that the five poultry feed supplements EGMAX, FEED-X, KOLIN PLUS, PHYTOCEE, and STODI were nontoxic to rats and LD50 was found to be more than 5000 mg/kg rat body weight.In conclusion, the results of this investigation revealed that the four poultry feed supplements EGMAX, FEED-X, KOLIN PLUS, and STODI were nonmutagenic in


Due to low primer universality (matK) and/or the existence of multiple copies (ITS), the commonly used barcodes matK and ITS were not appropriate for Adiantum. The PCR amplification rate was extremely low in all nuclear genes except for IBR3_2. rbcL had the highest PCR amplification rate (94.33%) and sequencing success rate (90.78%), while trnH-psbA had the highest species identification rate (75%). With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. The nuclear IBR3_2 showed 100% PCR amplification success rate in Adiantum, however, it seemed that only diploid species could acquire clean sequences without cloning. With cloning, IBR3_2 can successfully distinguish cryptic species and hybrid species from their related species. Because hybridization and allopolyploidy are common in ferns, we argue for including a selected group of nuclear loci as barcodes, especially via the next-generation sequencing, as it is much more efficient to obtain single-copy nuclear loci without the cloning procedure.DNA barcoding is a fast-developing technique to identify species by using short and standard DNA sequences. Universal selection of DNA barcodes in ferns remains unresolved. In this study, five plastid regions ( Purified PCR products were sequenced in both directions with the PCR primers on an ABI 3730xl DNA Sequencer .Total DNAs were extracted from silica-gel dried-leaf material and herbarium specimens using the CTAB procedure  and DnearbcL, matK, trnH-psbA, trnL-F and rps4-trnS) and eight candidate nuclear regions  [Adiantum. The primer information and thermocycling conditions used in this study were listed in Five candidate plastid regions ( SQD1_1) –48 were IBR3_2, but this resulted in chromatograms with multiple peaks for most species. We then cloned IBR3_2 from all samples, using the ZeroBack Fast Ligation Kit  and following standard protocols for cloning, colony selection, and post-cloning re-amplification with pZeroBack/Blunt Vector primers. At least six and up to 20 colonies were picked for sequencing for each individual. Some taxa ultimately yielded fewer than six sequences despite multiple cloning attempts because of technical difficulties in generating sequences.A subset of samples were sequenced via direct sequencing without cloning using the amplification primers of P-distance model [P-distance model, and used to evaluate whether individual samples of a species clustered in species-specific monophyletic clades. Robustness of inference was assessed by running 5, 000 bootstrap replicates [Sequences were aligned with Clustal X  and thence model . Intra- ce model , and “bace model . The neiplicates .matK showed the lowest PCR amplification success rate (33.33%), while rbcL, trnH-psbA, trnL-F, and rps4-trnS showed much higher PCR amplification and sequencing success rates. PCR amplification success rate of the four DNA regions was 94.33% (rbcL), 93.20% (trnH-psbA), 85.82% (trnL-F), and 94.20% (rps4-trnS), and sequencing success rate of the four markers was 90.78%, 84.16%, 77.01%, and 83.57%, for rbcL, trnH-psbA, trnL-F, and rps4-trnS, respectively  were low (<50%) or did not obtain clean sequences by direct sequencing without cloning. IBR3_2 had 100% PCR amplification success (A. ailaoshanense) were obtained by sequencing the clones  were downloaded from GenBank. The data analyses included 133 rbcL sequences, 122 trnH-psbA sequences, 126 rps4-trnS sequences, and 116 trnL-F sequences.The newly acquired DNA sequences have been deposited in GenBank and their accession numbers were provided in e.g., poly-A/G/C) within trnH-psbA was detected in taxa in ser. Reniformia, ser. Gravesiana, ser. Caudata, and ser. Venusta. The mononucleotide repeats near the end of sequences led to unclean reverse sequences in some cases. So only forward sequences of psbA in some individuals were used for the analyses. There are some poly structures in trnL-F and rps4-trnS, so the sequences of the “f” end of trnL-F and those of the trnS-F end of rps4-trnS of some samples were difficult to obtain. The forward reads alone were added to the analyses in these samples that the mononucleotide repeats exists.Homopolymer  and rbcL (73.33%), and trnL-F (65.52%) (A tree-based method (NJ) was used for the species identification of (65.52%) .A. subpedatum and A. myriosorum, A. formosanum and A. refractum, A. juxtapositum and A. chienii, A. meishanianum and A. malesianum were not identified by the present data. Adiantum formosanum (Kuo430) strongly clustered with A. refractum. Samples of A. juxtapositum  and A. chienii (LuJM568) grouped together with a high BP value. The samples of two Vietnamese individuals of “A. juxtapositum” (CP050 and CP076) did not cluster with the Chinese A. juxtapositum clade  strongly clustered with A. roborowskii var. roborowskii in the NJ trees . With the lack of sampling of the close relative A. formosanum, A. refractum was successfully identified in the trnH-psbA tree.Twenty-one of the 28 species were each supported to be monophyletic in the sbA tree . Three srbcL and rps4-trnS, 22 of the 30 species each formed a clade with high bootstrap values .Based on the ic group . Five grL-F tree . AdiantuThe combination of DNA barcodes can slightly improve the ability of species identification .IBR3_2 was successful in identifying the parental species of the presumed hybrid taxa. The maternal parent of A. meishanianum was shown to be A. malesianum, and the maternal parent of the hybrid species A. ailaoshanense was identified to be A. sinicum in the present analysis .analysis . HoweverAn ideal DNA barcode should be routinely retrievable with a single primer pair with little requirement for manual editing of sequence traces, can provide maximal discrimination among species ,54, and matK is one of the most variable coding regions within cpDNA [trnK exons [et al. [matK as a barcode for ferns, matK showed low universality in Adiantum using the same primers FWPtmatKF1 and FWPtmatK rAGK designed by Li et al. [Cheilanthes of the same family (Pteridaceae), and primers FWPtmatK fEDR and FWPtmatK rAGK designed by Kuo et al. [matK primer development in ferns to ensure efficient PCR and sequencing, and at present this marker seems to be a difficult barcode locus for ferns.Although in cpDNA –56, it inK exons . Taxon snK exons ,57. Even [et al.  endorsedi et al.  based onrbcL, trnH-psbA, trnL-F, and rps4-trnS) were shown in this study .Relatively well-defined gaps between intraspecific and interspecific divergences of the four selected barcodes , except that the four species groups (eight species) cannot be differentiated by all four barcode markers. trnH-psbA was ranked the second for PCR amplification success in Adiantum, however, its sequencing success rate was lower because of the existence of poly-A/G/C repeats [trnH-psbA can be used as an effective barcode in Adiantum with its short length and relatively high variability, and may be used to identify specimens or traditional medicinal materials. barcode –4,58. Pr species ,59. The  repeats ,59–60. ttrnL-F is shown here to have the highest sequence divergence, even at the intraspecific level in Adiantum. The amplification of trnL-F was, however, relatively difficult. PCR amplification success rate was 82.8% when total DNAs were extracted using the CTAB procedure. Higher-quality DNA can improve this rate, and it can reach about 90% when Dneasy extraction kits were used to extract the total DNAs. The sequencing of this marker was also relatively difficult (sequencing success rate at 77.01%) because of the universal presence of mononucleotide repeats in the intergenic spacer. The presence of mononucleotide repeat in trnL-F affected sequence quality, and reduced the universality of this marker.Reported as the most variable locus in ferns ,61, trnLtrnL-F, rps4-trnS ranked the third for PCR amplification rate, but the lowest sequencing success rate. Only a single forward sequence was obtained from 26% samples because of the presence of the mononucleotide repeat structure in trnL-F IGS and rps4-trnS IGS.With the rates of sequence divergence just lower than A. subpedatum and A. myriosorum, A. formosanum and A. refractum, A. juxtapositum and A. chienii, A. meishanianum and A. malesianum, A. gravesii and A. mariesii cannot be identified in the trnL-F tree because the species pairs had identical trnL-F sequences. Adiantum refractum was successfully identified in the rbcL tree, and A. myriosorum was successfully identified in rps4-trnS tree, wih the absence of A. formosanum and A. subpedatum in the individual datasets. In fact, the identified species were the same by rbcL, trnL-F,and rps4-trnS, with all five groups of species not identified while trnH-psbA could identify A. mariesii from A. gravesii.Five species groups including Adiantum davidii fell into two respective clades  [pgiC [gapCp) -gapCpSh (gapCp “short”) [gapCpLg (gapCp “long”) [Chase et al.  suggesteet al. –63, the FY (LFY) ,64–66, tY) [pgiC ,63,66–68“short”) ,63,68–71et al. [DET1, gapCpSh, IBR3, pgiC, SQD1, TPLATE, and transducin gene which were readily amplified and sequenced from 15 diploid Polypodiales species. The results showed that IBR3_2, DET1, and SQD1_1 were amplified well, and clean sequences were obtained via direct Sanger sequencing from most taxa of Polypodiales examined.Rothfels et al.  presentepgiC, gapC, LEAFY, IBR3_2, DET1, and SQD1_1) screened in this study, ITS exhibited high PCR amplification success, but it did not produce a single band for PCR amplification due to additive banding and incomplete concerted evolution. ITS is thus difficult to be used as a barcode in ferns. The PCR amplification rates in six candidate nuclear regions  were extremely low and/or clean sequences were not generated via direct sequencing without cloning.Of the eight nuclear candidate markers  and the pgiC gene is possibly of value for phylogenetic reconstruction at the specific and generic levels. The primers 14F/15R and 15F/16R were developed and applied to study mating systems and other population genetic traits [pgiC gene was also used to detect the origins of polyploids and hybrids in Dryopteris [pgiC gene are lacking [Ishikawa et al.  developec traits . The pgiyopteris ,45,68. B lacking .et al. [gapCp, and pgiC, to explore patterns of reticulate evolution in Asplenium. The results showed that all three nuclear markers amplified well and several copies were recovered by cloning PCR products in Asplenium. Rothfels et al. [pgiC situated in exons 14 and 16, to amplify introns 14, 15, and exon 15 (about 600–700 bp); however, samples of Adiantum failed to be amplified and directly sequenced in their study.Schneider et al.  employeds et al.  designedpgiC using the primers of 14F/16R and 15F/16R, and obtained relatively weak bands in Adiantum when the 14F/16R primers were used. However, the sequences of pgiC are only about 300 bp in Adiantum, and showed multiple peaks in the sequencing signals. The primer pair 15F/16R failed to amplify taxa of Adiantum.We amplified et al. [gapCp gene that encodes the glyceraldehyde-3-phosphate dehydrogenase. Their survey across ferns demonstrated that these primers are nearly universal for ferns, and holds considerable potential for addressing species-level questions across the tree of life in ferns. Rothfels et al. [gapCpSh, which overlaps with the gapCp region amplified with the primers of Schuettpelz et al. [gapCpSh amplified and sequenced well in fern taxa, however, they did not obtain clean sequences for Adiantum  and a few other genera (cloning not attempted). We amplified gapCp using the primers of Schuettpelz et al. [Adiantum.Schuettpelz et al.  designeds et al.  designedz et al. , and ranz et al. , and obtIBR3_2 had a high PCR amplification rate even though it is difficult to obtain sequences via direct sequencing. It seems that clean sequences may be obtained from autoploid species whereas allopolyploid or hybrid ones failed. IBR3_2 successfully distinguished two presumed hybrid species—A. meishanianum from its maternal parent A. malesianum [A. ailaoshanense [A. sinicum by the degenerate base. The hybrid A. meishanianum [A. malesianum had the same plastid sequences. Nevertheless, they can be distinguished in morphology and the IBR3_2 sequences. Therefore, IBR3_2 has the potential to be further explored as a nuclear barcode locus in some fern groups such as Adiantum and its close relatives.lesianum , and A. shanense  from itshanianum  and its Adiantum, and the result is nearly congruent with morphology-based taxonomy except for a few species. Ultimately the systematics community relies on morphology for species delimitation [A. subpedatum and A. myriosorum, A. formosanum and A. refractum, A. juxtapositum and A. chienii, A. meishanianum and A. malesianum cannot be identified by the plastid barcodes. We herein discuss these species groups in light of a morphological framework.DNA barcoding is generally successful for species identification in mitation –75. FourAdiantum subpedatum was recorded only from the Longtang Mountain in Zhejiang province [A. myriosorum [A. subpedatum and A. myriosorum lie in plant size and sori number [A. subpedatum is about 24–28 cm, whereas A. myriosorum is taller, about 40–60 cm. Adiantum subpedatum was described to have 1–2 sori per pinnule [A. myriosorum, while most samples from the two populations of the Longtang Mountain  of A. subpedatum have 3 sori per pinnule. Plant size and sori number may vary with the habitats. Adiantum subpedatum and A. myriosorum fell into the same clade in nuclear IBR3-2 tree . Both species are epilithic plants, and have 2–4 sori on the thin papery and fan-shaped pinnules. The primary differences between A. formosanum and A. refractum are plant height and pinnule size. Although A. formosanum differ with A. refractum in a few nucleotides, it still fell into A. refractum clade in nuclear IBR3-2 tree  [A. juxtapositum. The veins on lower surface of the samples from Vietnam are more visible than those of the Chinese samples. The fronds of the former are rather leathery while the latter are sub-leathery. The Chinese A. juxtapositum thus may be best treated as a synonym of A. chienii while the samples from Vietnam probably represent a new species.Lu et al.  proposedlue Figs  Figs. Ho A. sp.)  did not Adiantum meishanianum was validated in 2009 [A. philippense was suggested as its paternal species, and A. malesianum as its maternal parent [A. meishanianum and its maternal A. malesianum had identical plastid sequences. They can be distinguished by morphology . Adiantum meishanianum can also be distinguished from the maternal parent A. malesianum using IBR3_2. in 2009 . A cryptl parent . The preet al. [et al. [A. × ailaoshanense, which probably originated from A. sinicum x A. menglianense [Adiantum in our present study. Our IBR3_2 data also support the parents of the hybrid species A. ailaoshanense (Yan 12413 and Yan 12410) to be A. sinicum and A. menglianense  and the shape of pinnule margin (entire or undulate-crenate vs. bluntly serrate) [undulate-crenate pinnule margins clustered with A. roborowskii var. taiwanianum whereas another sample was nested into another subclade in the A. roborowskii var. roborowskii clade. Our DNA barcoding results and morphology thus support the treatment of Mao et al. [Mao et al.  placed Aserrate) . The difo et al. .Adiantum menglianense was published in 1992 [Flora Yunnanica treatment [Adiantum menglianense formed a highly-supported clade, and was sister to the closely related species A. philippense with strong support (BP≥82) in all trees ; the false indusia are short and straight in A. menglianense (vs. long and cupped in A. philippense); and pinnules are palmate and thin in A. menglianense (vs. semilunar and thick in A. philippense). Adiantum menglianense was described to have 6–10 sori per pinnule [A. philippense has 2–6 sori [et al. [A. philippense is the paternal parent of A. meishanianum. The present analyses confirmed the paternal species of A. meishanianum as A. menglianense. in 1992 , and wasreatment . Adiantuees Figs –S13 Figs pinnule  while A.2–6 sori . Zhang e [et al.  pointed Adiantum gravesii and A. mariesii failed to be identified except by trnH-psbA and IBR3_2. Adiantum gravesii can be distinguished from A. mariesii using morphological characters with the former being a bigger plant with reniform or lunate indusia vs. the latter a smaller plant with circular indusia.matK and the existence of multiple copies of ITS, these two commonly used barcodes were not appropriate for Adiantum. With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. Coupling DNA barcoding with morphology provides important insights into species delimitations for several taxa in our case study. Overall DNA barcoding provides additional DNA diagnostic characters to discriminate the individuals lacking diagnostic features because of rapid diversification, morphological stasis, and phenotypic variations [Due to low primer universality of riations . Becauseriations , 83–84. riations ).S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S13 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Acyrthosiphon pisum), the parasitoid Aphidius ervi competitively displaced other parasitoids, except for the native Praon pequodorum. The pea aphid exhibits tremendous clonal variation in resistance to A. ervi, primarily through infection with the heritable bacterial symbiont Hamiltonella defensa, although some symbiont-free aphid genotypes encode endogenous resistance. Interestingly, H. defensa strains and aphid genotypes that protect against A. ervi, provide no protection against the closely related, P. pequodorum. Given the specificity of aphid defenses, we hypothesized that aphid resistance traits may contribute to the continued persistence of P. pequodorum. We conducted multiparasitism assays to determine whether aphid resistance traits mediate internal competition between these two solitary parasitoid species, but found this was not the case; P. pequodorum was the successful internal competitor across lines varying in susceptibility to A. ervi. Next, to determine whether resistance traits influence competitive interactions resulting in the stable persistence of P. pequodorum, we established replicated cages varying in the proportion of resistant aphids and recorded successful parasitism for each wasp species over time. As expected, A. ervi outcompeted P. pequodorum in cages containing only susceptible aphids. However, P. pequodorum not only persisted, but was the superior competitor in populations containing any proportion (20–100%) of resistant aphids (20–100%). Smaller scale, better replicated competition cage studies corroborated this finding, and no-competition and behavioral assays provide insight into the processes mediating competition. Genetic variation, including that acquired via infection with protective symbionts, may provide a supply of hosts susceptible only to particular enemies, mediating competition with effects on community richness and stability.Insects use endogenous mechanisms and infection with protective symbionts to thwart attacks from natural enemies. Defenses that target specific enemies, however, potentially mediate competition between rivals and thereby impact community composition. Following its introduction to North America to control pea aphids ( Diverse eukaryotic taxa show substantial variation in susceptibility to attack by specific natural enemies –5. Most Acyrthosiphon pisum, is a leading model of protective symbiosis. In addition to the obligate, nutrient-provisioning symbiont, Buchnera aphidicola allowing exploitation of plant phloem [Regiella insecticola, provide protection against the entomopathogenic fungi Pandora neoaphidis [Hamiltonella defensa defends against attacks by the hymenopteran parasitoid Aphidius ervi [H. defensa confers varying levels of protection against attack by A. ervi depending on symbiont strain and associated bacteriophages called APSEs that infect the bacterial symbiont [H. defensa and phage variant APSE3 receive high levels of protection, while those infected with strains lacking APSE receive no protection [H. defensa can be costly, and balancing selection likely contributes to the maintenance of intermediate frequencies observed in field populations [H. defensa in lab studies have been found to be highly susceptible to attack by A. ervi, some aphid clones lacking HFS do have endogenous resistance [Aphids (Hemiptera: Sternorrhyncha), a diverse group of phloem-feeding herbivores, often show tremendous variation in resistance to parasitoids that can arise from endogenous sources and infection with maternally transmitted bacterial symbionts , 19–27. t phloem , individt phloem  but see t phloem ). For exoaphidis –32 and Hius ervi . Infectisymbiont , 34, 35.otection . Infectiulations , 37. Whisistance .th century, and numerous native and introduced parasitoids successfully attacked this aphid until the introduction of A. ervi in 1959 [A. ervi (Braconidae: Aphidiinae) emerged as the dominant parasitoid of pea aphids, outcompeting and displacing other native and introduced parasitoid species. The native wasp, Praon pequodorum, also an aphidiine braconid, was the only parasitoid not displaced by A. ervi in field populations, although the proportion of pea aphids successfully parasitized by this wasp dropped sharply (ca. 40% to < 10%). Prior studies have identified processes that may contribute to the continued persistence of P. pequodorum. First, P. pequodorum may be a superior internal competitor (i.e. within host) in instances of multiparasitism with A. ervi and related aphidiine braconids, where individuals of both species compete within a single aphid host, but only one can survive [A. ervi is the superior external (i.e. among hosts) competitor, exhibiting better foraging skills and faster attack rates relative to P. pequodorum [A. ervi’s foraging efficiency is more negatively impacted by non-target aphids [H. defensa strains and HFS-free innately resistant clones provide protection that is specific to A. ervi and has no significant effect on P. pequodorum development [P. pequodorum. Resistance traits specific to A. ervi may result in a reservoir of hosts susceptible only to P. pequodorum, or directly impact internal competition during instances of multiparasitism. A recent study using a European pea aphid line found that H. defensa influenced internal competition between A. ervi and the distantly related parasitoid, Aphelinus abdominalis  [The pea aphid was accidentally introduced to North America in the 19 in 1959 . After i survive –41. Secoquodorum , althougt aphids . A recenelopment . Hence, linidae) .H. defensa or aphid-based defenses mediate internal competition between these two wasps during instances of multiparasitism. Second, we conducted population cage studies, at two scales, to determine whether resistance traits influence competitive interactions resulting in the stable persistence of P. pequodorum. Replicated cage treatments varied in the proportion of aphids carrying highly protective H. defensa (treatments ranged from 0% to 100%) or with 100% aphid-based resistance . We also conducted assays without competition, as well as behavioral assays, aimed at better understanding processes mediating competition.Here we examine whether the specificity of pea aphid resistance traits identified in our earlier study  influencMedicago sativa, in Utah, USA 2007) is infected with an APSE3 containing H. defensa strain that confers high levels of resistance to A. ervi. Line AS30-Hd- resulted from the antibiotic curing [A. ervi [A. ervi [Vicia faba) in cup cages (Georgia (GA) experiments) or 60x60x60cm Bug Dorm 6610 cages (North Dakota (ND) experiments) reared at 20±1°C with a 16L: 8D photoperiod. Aphid lines were screened for expected symbionts as well as all other pea aphid HFS using the PCR primers and reaction conditions as in [H. defensa was detected, and this symbiont was only present in line AS3-Hd+, as expected. We further confirmed the presence of phage APSE3 using diagnostic PCR [Three experimental pea aphid lines were used in this study. Line AS3-Hd+  and field-collected mummies from North Dakota and Wisconsin, while the ND culture contained only ND collected wasps. The A. ervi colony was maintained under similar conditions and on the same aphid lines as P. pequodorum.The P. pequodorum and A. ervi, we parasitized cohorts of 20 (X 10 replicates) third instar aphids that were A) highly susceptible (line AS30-Hd-), B) have symbiont-based resistance (AS3-Hd+), or C) have aphid-based resistance (CJ1130-R). Half the replicates (N = 5) for each treatment were parasitized first by P. pequodorum then again <6 hrs later with A. ervi, and half vice-versa; ensuring that females of each species lay only one egg in each aphid. There was no difference in multiparasitism outcome due to wasp parasitism order, , so we Vicia faba plants) in each cage (Bug Dorm_1 DP1000 30x30x30cm). For Experiment 2A, Treatments were A) uninfected, susceptible aphids (line AS30-Hd-), B) aphids infected with APSE3-H. defensa which is highly resistant to A. ervi (line AS3-Hd+), and C) uninfected aphids with a resistant aphid genotype (line CJ1130-R). For Experiment 2B, treatments were A) 50% with symbiont-based resistance (AS3-Hd+) and 50% highly susceptible aphids (AS30-Hd-), B) 80% with symbiont-based resistance (AS3-Hd+) and 20% with highly susceptible aphids (AS30-Hd-), and C) 20% with symbiont-based resistance (AS3-Hd+) and 80% highly susceptible aphids (AS30-Hd-). Four replicate cages of each treatment (A- C) were created for a total of 12 cages. The 60 adult aphids used to seed each cage were allowed to reproduce for four days producing a cohort of approximately 800–1100 2nd and 3rd instar nymphs, the instars preferred for both A. ervi and P. pequodurum suitable for parasitism [A. ervi and P. pequodorum to each cage and allowed them to parasitize aphids for 24 hours before they were aspirated from cages. After ~10 days, we removed all mummies and recorded the total number of each parasitoid species per cage. Mummies are easily distinguished as P. pequodorum pupates underneath the aphid’s mummified exoskeletons and A. ervi within it. Mummies from each species were placed in separate cages to eclose as adults and mate before being used in the next round of parasitism. Population cages were reset with 20 aphid adults per plant and given 3–4 days to reproduce as above to maintain a static resistance target. This is to ensure similar number of aphids among treatments as susceptible aphids suffered high mortality due to parasitism. After such time, all wasps that emerged from a specific replicate cage were placed back into the same cage and again allowed to parasitize aphids for 24 hours. The cages were repeated until either a) one wasp species lost, b) enemy frequencies stabilize, or c) four bouts of parasitism occurred.Population cages were established by placing 60 adult aphids of a particular treatment . To determine rates of successful parasitism, we then counted the number of mummies produced by each parasitoid species after 10 days. Parasitoids were provided with honey and water prior to experiments, and had never before encountered aphids. The number of aphids and exposure time were chosen to reduce the likelihood of superparasitism and larval competition and thus make the parasitism assay more reflective of wasp foraging behavior. Each treatment was replicated 24 times, although four replicates (2 from 100% AS3-Hd+ and 2 from 50% of each) were not included in our analysis because one or both of the parasitoids were not found at the end of the oviposition period.We also conducted smaller scale competition assays to complement our larger cage studies but with much greater replication, and to determine if the presence of a competitor affected the pattern of parasitism of the parasitoids relative to our no competition experiment (below). Treatments varied by cohort composition, and included either 100% AS3-Hd+ aphids, 100% AS30-Hd- aphids, or 50% of each. Each 40x10cm cylindrical replicate cage contained a cohort of 30 aphids on a 10-day-old fava seedling. Next, we simultaneously introduced single 2–3d-old, mated A. ervi or P. pequodorum, for 4 hrs (10:00 to 14:00), with mummies counted after 10 days. This created a 3x2 treatment factorial, with the first factor being the three aphid treatments and the second the two parasitoid species. The experiment was replicated over four experimental blocks, each spaced one week apart. The first two experimental blocks included 8 replicates of each treatment, while the latter two experimental blocks included 12 replicates of each treatment. In a small number of replicates, the parasitoid could not be found at the end of the 4 hr oviposition period to be removed. Because the fate of these parasitoids is unknown, and they may have had the opportunity to continue ovipositing after the 4 hr oviposition period, these replications were excluded from analysis. Similarly, replicates in which no mummies formed were excluded from analysis to use consistent methodology with [We also performed parasitism assays in the absence of competition to determine patterns of parasitism of each wasp species in response to different proportions of resistant and susceptible aphids. Treatments were the same as above: either 100% AS3-Hd+ aphids, 100% AS30-Hd- aphids, or 50% of each. In this assay, each cohort of 20 aphids was exposed to single, mated female of either species, ogy with .A. ervi changes in response to both aphid resistance traits and the presence of P. pequodorum, we conducted behavioral observations to see if the foraging behavior of A. ervi changes with these factors. We set up a 3x2 factorial arrangement of treatments. The first factor was the composition of the aphid cohort exposed to the parasitoids: 100% AS3-Hd+, 100% AS30-Hd-, or 50% of each. The second factor consisted of whether the A. ervi foraged alone or foraged alongside a P. pequodorum. The arenas consisted of 10-day-old fava seedlings infested with a total of 30 second instar aphids. We used 2–3 day old female parasitoids that were provisioned with honey and water and that had never encountered aphids prior to the experiments.Because our parasitism assays indicated that the parasitism success of P. pequodorum approaching A. ervi. Upon making contact, the P. pequodorum would behave evasively (e.g. orient away from A. ervi or fly away), behave aggressively , or ignore the A. ervi . In case these interactions affected parasitism, they were also analyzed for differences by aphid cohort.We recorded the amount of time parasitoids spent walking, resting, grooming, and feeding on honeydew or from extrafloral nectaries, in addition to encounters with aphids and encounters with other parasitoids. For each aphid/parasitoid encounter, we recorded whether the aphid was parasitized, the aphid escaped , or the aphid was rejected . These observations focused on whether or not the parasitoid probed the aphid with her ovipositor, but we did not confirm whether or not eggs were laid. However, the decision to lay eggs and the number of eggs laid may vary according to host type . On seveP. pequodorum treatment, which was replicated 11 times.To reduce the number of inactive parasitoids, we initially tested each parasitoid in a 10 cm Petri dish with 20 susceptible second instar aphids. Parasitoids that did not attack an aphid in the Petri dish within five minutes were discarded and not used in experiments. Wasps that did parasitize an aphid were introduced to the cage containing the aphids and fava seedling and their behaviors were recorded for one hour. Observations were terminated early if no parasitoids made contact with an aphid for 15 minutes. Host plants were small enough that even when two parasitoids were included, both could be simultaneously observed. Each treatment was replicated 10 times except for the 50% of each aphid line without P. pequodorum success among lines and thereby determine whether aphid resistance mediated internal competition. A Fisher’s Exact Test was also used with the multiparasitism assays to determine whether competition outcome differed from the null expected . For the smaller scale competitive and non-competitive parasitism assays, we used ANOVA to test for differences in the number of mummies produced by each wasp species separately to test for block effects in the non-competitive parasitism assays and to confirm that parasitism differed between the two pure aphid cohort treatments. For the smaller scale competitive and non-competitive parasitism assays, we used ANOVA to analyze differences in mummy production between the different aphid cohort compositions. Both wasp species were included in each ANOVA. To conduct statistical analysis, the number of mummies was square-root transformed to achieve normally distributed data. For the parasitoid behaviors, we used MANOVA to compare the time each parasitoid allocated to walking, grooming, resting, and feeding. We also used MANOVA to compare the number of aphids parasitoids encountered and the numbers of those encounters that included stings, aphid escapes, or rejections of aphids. We used MANOVA to compare the numbers of interactions between A. ervi and P. pequodorum in which A. ervi fled from, ignored, or attacked P. pequodorum. Finally, we compared the numbers of mummies that formed after each behavioral observation using ANOVA. All significant results in ANOVA were further resolved using Tukey’s HSD post-hoc tests. Statistical analyses were conducted in R v. 3.2.0.In the population cages, each mummy count from each cage at each new time point was compared against the values in the T0 time point through a Fisher’s Exact Test to determine whether the wasp proportions significantly differed from starting wasp proportions. For the multiparasitism assay, a logistic regression analysis was used to compare P. pequodorum and A. ervi. We found no difference in outcome whether A. ervi or P. pequodorum attacked the aphid first thus results were combined (P = 0.3853). We find that P. pequodorum produces significantly more mummies than A. ervi in all three aphid lines  was conducted to determine whether symbiont- and aphid-based resistance mediates internal competition between id lines  indicatiwo wasps .A. ervi outcompeted its rival P. pequodorum wasps and competitively excluded this wasp after 4 bouts of parasitism (Experiment 2A: P. pequodorum outcompetes A. ervi in the majority of cases (In the population cage containing only susceptible aphids (AS30-Hd-), of cases  Cages wiP. pequodorum outcompetes A. ervi in all cages, even when the percentage of resistant aphids is only 20%, suggesting that even low levels of resistance specific to A. ervi in pea aphid populations can result in the persistence of P. pequodorum (We conducted a second population cage to determine what percentage of symbiont-based resistance in a population could affect the competition among wasps (Experiment 2B). Surprisingly, we find that quodorum .F2,154 = 9.84, P < 0.0001) (A. ervi produced significantly more mummies when attacking 100% AS30-Hd- aphids than 100% AS3 Hd+ aphids (Tukey’s HSD: P<0.0001) or the 50:50 mix (Tukey’s HSD: P = 0.0011) , we found that the number of mummies produced varied according to the resistance composition of the aphid population and parasitoid species ( 0.0001) . A. ervi 0.0011) . The num cohorts . While tA. ervi mummy counts varied significantly according to aphid treatment  (P = 0.00015) and the 50:50 mix of the two aphid lines (Tukey’s HSD: P = 0.0029). P. pequodorum mummy counts did not differ among aphid lines  , we found that 0.00010)  with a sA. ervi behavioral responses based on the presence of competitors, nor aphid lines varying in resistance (Experiment 5). The amount of time A. ervi spent walking, resting, grooming, or feeding did not differ between treatments , nor the number of aphids that were parasitized, escaped, or rejected did not differ between treatments . The number of interactions between A. ervi and P. pequodorum in which P. pequodorum was aggressive toward, retreated from, or ignored A. ervi was also unaffected by treatment . Finally, we found no differences in the number of A. ervi mummies that formed in response to each aphid cohort composition , which may be an artifact of the generally low and highly variable number of mummies produced with only one hour of foraging.We found no differences in A. ervi is the dominant parasitoid, having competitively displaced numerous native and introduced parasitoids other than P. pequodorum, which remains at low levels in most sampled fields [P. pequodorum. In cages containing only aphids susceptible to attack by A. ervi, this wasp outcompetes and displaces P. pequodorum. However, in cages containing any proportion of aphids infected with the protective symbiont H. defensa (from 20 to 100%), or those with 100% endogenous resistance, P. pequodorum maintained viable populations for multiple generations and even displaces A. ervi in most instances  may have biased our experiments in favor of P. pequodorum. Larger cages with greater aphid abundances and lower overall parasitism rates may favor A. ervi relative to P. pequodorum, because the superior host-finding ability and faster attack rates of A. ervi gives it a greater intrinsic rate of increase than P. pequodorum [A. ervi foraging behavior due to competitors or aphid resistance traits (Experiment 5). In addition to aphid resistance traits and foraging abilities, other factors may directly or indirectly impact P. pequodorum abundance in field populations, including the availability of alternative aphid hosts [It was surprising that even in cages (Experiment 2) with the lowest proportion (20%) of symbiont-resistant aphids,  defensa  indicatiquodorum , 48. We id hosts  or diffeid hosts , 49.A. ervi’s parasitism potential  were largely consistent with our larger cages, showing that defensive symbionts can alter the outcome of competitive interactions between rival wasps as host resistance traits and the presence of a competitor decreased otential . In bothrum Figs . HoweverP. pequodorum consistently outperformed A. ervi in the presence of aphid resistance traits specific to A. ervi, we note an interesting anomaly in one cage replicate (Experiment 2). In the final sampling point (T4) of the four replicate cages containing 100% aphids with symbiont resistance . This result is perplexing because at the penultimate sampling point (T3) P. pequodorum produced 89% of the mummies indicating a rapid and complete turnaround following just a single bout of parasitism.While H. defensa or APSE can lead to the instant loss of resistance to A. ervi [H. defensa and APSE. Of course, it is unlikely that symbiont or APSE loss among a subset of the population would have resulted in such a rapid and total change in competition outcomes. Another possibility is that the rapid evolution of counter-resistance specific to symbiont-based defense contributed to A. ervi’s resurgence, which could occur given that the same individual wasps emerging from prior parasitism bouts were reintroduced into the same replicate cage each round. While parasitoids, including A. ervi, have been shown to rapidly evolve counter-resistance specific to symbiont-based defenses [P. pequodorum’s performance in this single cage. Follow up studies are needed to determine whether A. ervi wasps can rapidly evolve the ability to overcome symbiont-based resistance, and if so, examining of mechanisms underlying increased wasp counter-resistance.Loss of  A. ervi  so we vedefenses , 50, 51,P. pequodorum is a superior internal competitor in cases of multiparasitism with A. smithi [P. pequodorum is a superior internal competitor in cases of multiparasitism with A. ervi [P. pequodorum outperformed A. ervi in population cages containing resistant aphids. Instead, we found that P. pequodorum successfully outcompetes A. ervi in the multiparasitism assay , we did not find that P. pequodorum produced more mummies when attacking H. defensa infected aphids (Vicia faba) of host plant, thus indirect effects may be stronger or weaker in field settings with other host plants or other genotypes. Nonetheless, symbiont-based resistance traits targeting one enemy may have both direct and indirect effects on non-targets.Pea aphid resistance traits specific to  defensa , 54, whid aphids  comparedP. pequodorum in field populations after the establishment of A. ervi, which displaced other parasitoids [Here we show that aphid resistance traits from both endogenous and symbiont-based sources can influence competition between rival parasitoids, even when found at low percentages (20%) within populations. This result likely contributes to the persistence of asitoids . Thus, basitoids ), and thComplex aphid genotypes, comprising both aphid- and symbiont-encoded resistance to specific primary parasitoids, may influence the diversity and richness of the primary parasitoid community with effects that extend to other members within the food web, including other mutualists and other natural enemies. In turn, changing compositions of higher-order enemies and primary parasitoids can exert pressure leading to the maintenance of target specific resistance traits, allowing for more diversity in the food web.S1 TableA. ervi; Pq = P. pequodorum.Successful parasitism as indicated by the number of mummies at each time point  for each replicate among population cages varying in modes (symbiont vs endogenous) and levels of resistance. * indicates that mummy production at that time differs significantly from that at time zero (T0) as determined by Fisher’s Exact Test. Ae = (PDF)Click here for additional data file.S2 TableA. ervi; Pq = P. pequodorum.Successful parasitism as indicated by the number of mummies at each time point  for each replicate among population cages varying in proportion of aphids with symbiont resistance. * indicates that mummy production at that time differs significantly from that at time zero (T0) as determined by Fisher’s Exact Test. Ae = (PDF)Click here for additional data file.


Gentiana contains 362 species, but there are only two valuable complete cp genomes. The purpose of this study is to report the characterization of complete cp genome of G. lawrencei var. farreri, which is endemic to the Qinghai-Tibetan Plateau (QTP).The chloroplast (cp) genome is useful in plant systematics, genetic diversity analysis, molecular identification and divergence dating. The genus G. lawrencei var. farreri cp genome. The comparison analysis including genome difference and gene divergence was performed with its congeneric species G. straminea. The simple sequence repeats (SSRs) and phylogenetics were studied as well.Using high throughput sequencing technology, we got the complete nucleotide sequence of the G. lawrencei var. farreri is a circular molecule of 138,750 bp, containing a pair of 24,653 bp inverted repeats which are separated by small and large single-copy regions of 11,365 and 78,082 bp, respectively. The cp genome contains 130 known genes, including 85 protein coding genes (PCGs), eight ribosomal RNA genes and 37 tRNA genes. Comparative analyses indicated that G. lawrencei var. farreri is 10,241 bp shorter than its congeneric species G. straminea. Four large gaps were detected that are responsible for 85% of the total sequence loss. Further detailed analyses revealed that 10 PCGs were included in the four gaps that encode nine NADH dehydrogenase subunits. The cp gene content, order and orientation are similar to those of its congeneric species, but with some variation among the PCGs. Three genes, ndhB, ndhF and clpP, have high nonsynonymous to synonymous values. There are 34 SSRs in the G. lawrencei var. farreri cp genome, of which 25 are mononucleotide repeats: no dinucleotide repeats were detected. Comparison with the G. straminea cp genome indicated that five SSRs have length polymorphisms and 23 SSRs are species-specific. The phylogenetic analysis of 48 PCGs from 12 Gentianales taxa cp genomes clearly identified three clades, which indicated the potential of cp genomes in phylogenetics.The cp genome of G. lawrencei var. farreri mainly consistent of ndh genes which could be dispensable under chilling-stressed conditions in the QTP. The complete cp genome sequence of G. lawrencei var. farreri provides intragenic information that will contribute to genetic and phylogenetic research in the Gentianaceae.The “missing” sequence of  The chloroplast (cp) is the photosynthetic organelle that provides essential energy for plants, and is hypothesized to have arisen from ancient endosymbiotic cyanobacteria . In angiRecently, the number of completely sequenced cp genomes from higher plants has increased significantly. The cp genome is useful in plant systematics research because of its maternal inheritance, haploid nature and highly conserved structures. It is widely used in the study of genetic diversity, molecular identification, phylogenetic classification and divergence dating . The comGentiana is the largest genus in the Gentianaceae, containing 15 sections and about 362 species  Satake & Toyokuni ex Toyokuni. It has very beautiful flowers and has been used in traditional Chinese and Tibetan medicine  from a single plant. Total genomic DNA was isolated from young leaves using a Dzup plant genomic DNA extraction kit  following the manufacturer’s instructions. After DNA isolation, the procedure was performed in accordance with the standard Illumina protocol, including sample preparation and sequencing. Approximately 5–10 μg of genomic DNA was fragmented using ultrasound, which was purified using the CASpure PCR Purification Kit , followed end repair with poly-A on the 3′ ends. The DNA were then linked to adapters, extracted at specific size after agarose gel electrophoresis and amplified by PCR to yield a sequencing library. Then, a quarter of one flow-cell lane containing the fragmented genomic DNA of G. lawrencei var. farreri was sequenced using the Illumina HiSeq 4000 platform , yielding 36.08 million 150-bp paired-end reads from a library of approximately 350-bp DNA fragments. Reads corresponding to plastid DNA were identified using a BLASTN  search against the plastome sequences of two Gentiana taxa: G. straminea (GenBank accession no. NC_027441) and G. crassicaulis (NC_027442). A total of 2,517,802 reads (6.97%) were recovered and assembled using Velvet 1.2.10 (G. lawrencei var. farreri plastome sequence was deposited in GenBank (accession no. KX096882).t 1.2.10 . Eight chttps://code.google.com/archive/p/msdb/downloads) with minimal repeat numbers of 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, respectively.The protein coding genes (PCGs), tRNAs and rRNAs in the cp genome were predicted and annotated using Dual Organellar GenoMe Annotator (DOGMA) using default parameters . The posG. straminea (NC_027441) was obtained from the National Center for Biotechnology Information (NCBI). Genome comparison to identify the differences between G. lawrencei var. farreri and G. straminea was performed using mVISTA  found in all of the species were extracted from the selected cp genomes. The amino acid sequences of each of the 48 cp PCGs were aligned using MSWAT (http://mswat.ccbb.utexas.edu/) with default settings, and back translated to nucleotide sequences. Phylogenetic analyses were performed using the concatenated nucleotide sequences and PhyML3.1 software , as determined using the Akaike information criterion implemented in jModelTest 2.1.7  and two PCGs (clpP and ndhF) and 1 ycf (ycf3) have two introns. Like most other land plants, rps12 is trans-spliced, with its two 3′end residues separated by an intron in the IR region, and the 5′end exon is in the LSC region , GapB and GapD contain exon 2 of ndhB and GapC contains five PCGs . A comparative analysis between G. lawrencei var. farreri and G. straminea cp genomes revealed that the sequence similarities between the trnH-GUG-psbA, trnK-UUU-italictrnQ-UUG, trnS-GCU-trnG-GCC, atpH-atpI, rpoB-trnC-GCA, psbC-trnS-UGA, trnT-UGU-trnL-UAA, atpB-rbcL, ycf1-ndhF, rpl32-trnL-UAG and trnL-CAA-ycf15 intergenic regions are very low.A comparative analysis between the cp genomes in ectively . Four biectively . CompareG. lawrencei var. farreri and G. straminea were compared using the mVISTA program to determine the level of sequence divergence. The comparison showed that the coding regions of both cp genomes are highly conserved compared with the noncoding regions. In particular, the intergenic regions showed the greatest divergence between the two cp genomes. More divergence was found in the sequences of clpP, ndhB, ndhD, ndhE, ndhF and ndhH, which are distributed mainly in the SSC regions, compared with other PCGs. The nucleotide and amino acid sequences of the PCGs of G. lawrencei var. farreri and G. straminea are highly similar, with average sequence similarities of 95.0 and 93.0%, respectively. Between the two species, the nucleotide sequence identities of the LSC, SSC, and IR are 88.7, 61.0, and 92.9%, respectively. The most conserved genes include all the rRNA genes, the genes from photosystem I, the cytochrome b/f complex genes and the ATP synthesis genes  bootstrap support.An ML phylogenetic tree constructed using 48 PCGs from 12 Gentianales taxa clearly identified the three families  in the analysis as being monophyletic with high bootstrap value . The treG. lawrencei var. farreri is 10,241 bp shorter than G. straminea. Although the cp genome size is variable, ranging from 120 kb to 160 kb, huge genome losses in congeneric taxa are rarely reported. In general, most of the size changes in angiosperm cp genomes can be accounted for by rare deletions and duplications leading to massive changes in the size of the IR region  and LSC  regions rather than the two IR regions . More than half (50.33%) of the sequence length in the SSC region was lost. Therefore, the cp genome size variation in the two Gentiana taxa was not caused by deletions in the IR regions, but by deletions in the SSC and LSC regions. Although the IR region can vary from 10 to 76 kb among angiosperms, in the great majority of species it is a rather constant 22–26 kb in size  value than the LSC (0.0551) and IR (0.0243) regions. Two possible explanations about variation in the SSC region were proposed in previous studies. Firstly, the higher rate of molecular evolution in the SSC than other regions was also observed in vs. noncoding regions in the sequence. However, this does not appear to be true in our study. Secondly, the SSC region is a “hotspot” for recombination  subunits. The cp DNA of most of the higher plants contains 11 ndh genes, which encode protein subunits of the thylakoid NDH complex. The complex is analogous to mitochondrial complex I (EC 1.6.5.3), which catalyzes the transfer of electrons from NADH to plastoquinone , indicating that they had evolved under positive selection. The clpP gene also showed a high Ka/Ks ratio in Fagopyrum tataricum (ndhB and ndhF genes experienced positive selection pressure. In the absence of nine ndh genes in G. lawrencei var. farreri, the remaining ndhB and ndhF genes might play an important role in cyclic photophosphorylation, although the functions of ndhB and ndhF genes are unknown. The ndhB and ndhF genes are probably transcribed independently as monocistronic mRNAs (ndhF gene requires the nuclear-encoded sigma4 factor; the ndhF product in turn would stimulate the transcription of the other plastid ndh genes. Therefore, the selection pressure on the ndhF gene may play an important role in evolution of ndh genes.Variation in the divergence of the coding region was observed between the two  regions . Our datataricum . Interesic mRNAs . Favory Gentiana, several phylogeny studies have been carried out (Gentiana being controversial due to inconsonant sectional classification and the low support for relationships (Chondrophyllae, which has 10 series and 163 species, derived within a very short period of time followed by subsequent rapid radiation (Cruciata but failed to find corresponding morphological circumscriptions to support them (Gentiana; therefore, to realize the full potential of the cp genome in phylogenetic analysis, more taxa of different secttions should be included in the cp genome comparison analysis.The ML phylogenetic tree of Gentianales constructed using 48 PCGs clearly grouped the taxa from the three families into three clades. The phylogenetic relationships were consistent with previous studies that classified the three families as three monophyletic clades and identified the Rubiaceae as the base group in the Gentianales . The cp ried out . Howeverionships . For exaadiation , making ort them . Our anaG. lawrencei var. farreri and G. straminea, which belong to different sections. SSRs are more polymorphic than cp loci that are amplified by universal primers; therefore, the polymorphic SSRs could offer higher resolution for phylogenetic tree construction in Gentiana.Chloroplast SSRs are good tools for studies in plant ecology and evolution . MicrosaG. lawrencei var. farreri and describe its evolutionary characteristics in comparison with G. straminea. About 10kb sequence which mainly consistent of nine ndh genes were lost in G. lawrencei var. farreri. The divergence hotspots and SSRs clarified here could be used as molecular markers and will be useful for further studies on population genetics, phylogenetics and evolution of the genus Gentiana.We present the first report of the complete cp genome sequence of 10.7717/peerj.2540/supp-1Table S1Click here for additional data file.10.7717/peerj.2540/supp-2Table S2Click here for additional data file.10.7717/peerj.2540/supp-3Table S3Click here for additional data file.10.7717/peerj.2540/supp-4Table S4Click here for additional data file.10.7717/peerj.2540/supp-5Table S5Click here for additional data file.


Sarcopenia has been associated with reduced physical activity (PA). We aimed to determine if sarcopenia, and specific components of muscle size, function, and physical performance, are associated with high impacts achieved during habitual PA, as these are related to bone strength in community-dwelling older women.Participants were older women from the Cohort of Skeletal Health in Bristol and Avon. We defined sarcopenia using the EWGSOP criteria. Lower limb peak muscle power and force were assessed using Jumping Mechanography (JM). High vertical impacts were assessed by tri-axial accelerometry (at least 1.5g above gravity). Cross-sectional associations were analyzed by linear regression, adjusting for age, height and weight (or fat mass for models including appendicular lean mass index), comorbidities, smoking, alcohol, and Index of Multiple Deprivation.SD 3.0) years; 242 (64%) also completed JM. In age-adjusted analysis, a negative relationship was observed between severity of sarcopenia and high, but not medium or low, impacts (p = .03 for trend). Regarding components of sarcopenia underlying this relationship, multivariable analyses revealed that gait speed  and peak force  were independently associated with high impacts.Our analyses included 380 participants, with mean age 76.7 (Older women with sarcopenia experienced fewer bone-strengthening high impacts than those with presarcopenia or without sarcopenia. To increase bone strengthening activity in older women, interventions need to improve both lower limb muscle force and walking speed. Sarcopenia, a common disorder of older age, is associated with increased mortality  and consSarcopenia has been associated with reduced PA; sarcopenic adults (defined using EWGSOP criteria) participated in less moderate-vigorous PA (MVPA) than nonsarcopenic adults in a recent large population-based analysis . LongituInterest is growing regarding the influence of vertical impacts achieved during habitual PA, particularly on Bone Mineral Density (BMD), since historic animal studies first highlighted the relationship between mechanical strain and bone formation . A meta-Although sarcopenia has been associated with lower generalized PA , the extParticipants were recruited from the Cohort of Skeletal Health in Bristol and Avon (COSHIBA), comprising women recruited during 2007–2009 through general practitioner registries within Southwest England, born between January 1, 1927 and December 31, 1942 . A detaiMuscle power and force were assessed using a Leonardo Mechanography Ground Reaction Force platform. The platform consists of two plates with corner sensors which detect voltage proportional to applied force . Sensor SPPB comprises tests of balance , chair rise time (five timed chair rises as fast as possible [without using arms]), and gait speed , each test being scored out of four points giving a maximal total score of 12 .2.All consenting participants underwent DXA scanning of the total body to measure lean mass (LM) and fat mass (FM); the methodology has previously been described  see Sup. Before 3 minus total bone content, applied filters derived smoothed total mCSA.The methodology for pQCT acquisition is described elsewhere  see Sup. Muscle Grip strength was assessed using a JAMAR digital handgrip dynamometer , with the handle set at the second point. Three tests were performed on each hand (with 30 seconds rest between each test) with the participant standing upright (unless unable to stand) with their arm fully extended next to their body and a stiff wrist. The participant was instructed to squeeze as hard as they can for as long as they can, using standard instructions. Measurements alternated between each hand with a total of three attempts for each hand and the maximal value was taken. Measurements were recorded to the nearest 0.1 kg.x time for swimming). Data was uploaded and imported into Stata  and processed using custom-designed code which cleaned data to remove artifacts  and nonwear time. A valid day was defined as at least 10 hours of wear time. A peak was identified as any vertical acceleration greater than the previous or subsequent recorded acceleration. Peaks were classified into three impact bands: low (0.5≤g<1.0), medium (1.0≤g<1.5), and high (≥1.5 g), over and above Earth’s gravity. The number of counts in each impact band were normalized to 7 days of 14 hours.The protocol for PA monitoring, and derivation of impact variables, has been described elsewhere . Briefly2), sarcopenia as low muscle mass and low grip strength (<20 kg) or gait speed (<0.8 m/s), and severe sarcopenia as low muscle mass, grip strength, and gait speed (Presarcopenia was defined as low muscle mass (ALMI ≤ 5.45 kg/mit speed . Due to SD]) for continuous variables, except for accelerometry counts which were positively skewed and are presented as median (interquartile range [IQR]), and log-transformed.Descriptive statistics are presented as counts and percentages for categorical variables, and mean . All continuous exposure variables were standardized. As outcome variables were log-transformed, coefficients represent the percentage change in outcome per SD change in exposure, and percentage difference in geometric mean for categorical variables. Data analysis was performed using Stata version 13 . Sensitivity analyses are described in the Supplementary Material.Relationships between sarcopenia/individual components of muscle size and function, and accelerometry counts  in each PA impact band, were examined using linear regression. We adjusted for the following a priori confounders: age at clinic session (model 1); model 1 plus height and weight  (model 2); model two plus Index of Multiple Deprivation (IMD), smoking, alcohol consumption , and comorbidities  (model 3). Methods regarding acquisition of covariates are described in full in the Supplementary Material. To identify independent determinants of accelerometry counts, muscle function variables were included in the same multivariable model, and model fit subsequently evaluated (One thousand sixty-four members of the COSHIBA population were invited to reattend of whom 380 (36%) provided complete data for SPPB, sarcopenia exposures, covariates, and PA outcomes and formed the study population . Compare2  and the mean BMI 27.0 (4.6) kg/m2 . 6.3% wep for trend = .03; p < .01).Sarcopenia status was unrelated to low or medium impacts; however, sarcopenia status was inversely associated with high impacts in our age-adjusted analyses, with lower levels seen in sarcopenia, whereas little difference was observed in presarcopenia (p < .05), including our fully-adjusted model (model 3) .In analyses examining differences in impact counts between those above and below the grip strength, gait speed and ALMI thresholds for defining sarcopenia, grip strength below 20 kg and gait speed slower than 0.8 m/s were associated with fewer low, medium, and high impacts over 1 week in all models  . ALMI ≤5When gait speed was included as a continuous variable, similar associations were observed, with positive associations for all three impact categories and models . Chair rr2 = 0.18).Multivariable linear regression analyses were performed with all predictor variables, based on model 3, to determine which measures of muscle size and function were independently associated with impacts. Gait speed was the only measure associated with low and medium impacts  , whereasWe investigated relationships between sarcopenia and habitual levels of PA, categorized according to level of vertical impact, in a community-based population of older women. We found that sarcopenia, defined using EWGSOP criteria, was associated with fewer high, but not low or medium, vertical impacts. In contrast, physical capability, as reflected by SPPB score, was related to low, medium, and high impacts. To clarify these relationships, we examined associations between individual components of sarcopenia and vertical impacts, with a focus on lower limb muscle function. Gait speed was independently associated with low, medium, and high impacts; peak lower limb muscle force was independently associated with high impacts only. Our results are broadly consistent with previous reports that sarcopenic individuals participated in less MVPA . HoweverThere is significant overlap in the definitions of sarcopenia and physical frailty—gait speed and grip strength assessment can be used to identify both conditions ,23. Our Our finding that grip strength and gait speed were associated with reduced vertical impacts in all categories is consistent with previous studies. A longitudinal analysis identified grip strength to be associated with MVPA in men and women aged 60+ . In oldeHigh impacts have previously been associated with bone strength in our study population . Hence, The strengths of this study include the objective measurement of high impact PA by accelerometers. In addition, data were available for a wide range of variables allowing adjustment for a priori confounders. The availability of JM-derived variables as additional measures of lower limb muscle function is another strength.The study populations were younger and more highly educated than the source population, which limits generalizability of our results to all community-dwelling older women. The population is female only; the relationship between sarcopenia and high impact PA has yet to be investigated in older men. Restricting JM analysis to those participants scoring ≥6 on SPPB meant we were unable to analyze the relationship between muscle function measures and high impact activity in participants with low physical function. This study is cross-sectional and therefore a causal relationship between low muscle function and high impact counts cannot be inferred; it is plausible that lack of high impact PA led to declines in muscle mass and function. We used the EWGSOP criteria to define sarcopenia as this is the most widely used criteria. However, a recent study of the performance of sarcopenia definitions found that the EWGSOP definition was worse at predicting physical and health-related outcomes  compared to a simple strength test . The EWGWe investigated relationships between sarcopenia and habitual levels of PA categorized according to level of vertical impact, in a community-based population of older women. We found that gait speed and lower limb peak muscle force were independently associated with the number of high, potentially osteogenic, impacts during everyday activity. These findings underlie the importance of including exercises to increase gait speed and lower limb muscle force (such as resistance exercises) in PA interventions, intended to prevent osteoporosis through greater participation in high impact activities.The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online.Supplementary data is available at Supplementary InformationClick here for additional data file.The VIBE study is funded by the Medical Research Council (grant ref MR/KO24973/1). A.H. is funded by the Wellcome Trust (grant ref 20378/Z/16/Z). C.L.G. is funded by a Clinician Scientist Fellowship from Arthritis Research UK (grant ref 20000). The COSHIBA cohort was originally funded through a Clinician Scientist Fellowship for EMC from Arthritis Research UK (grant ref 17823).None Reported.Study Design: K.H., E.M.C., J.H.T. Study Conduct: K.H., K.C.D., A.H., J.H.T. Data Collection: K.H., K.C.D., J.H.T. Data Analysis: A.H., C.L.G., J.H.T. Data Interpretation: A.H., C.L.G., J.H.T. Drafting manuscript: A.H., C.L.G., J.H.T. Revising manuscript and approving final version: A.H., C.L.G., K.H., K.C.D., E.M.C., J.H.T. A.H. takes responsibility for the integrity of data analysis.