Studies on the mode of action of reutericyclin

The mode of action of reutericyclin was determined with fluorescent dyes that probed the permeability of the cytoplasmic membrane by large molecules, protons, and potassium. A comparison of reutericyclin activity with those of nisin, nigericin, and valinomycin demonstrated that reutericyclin does not form pores but selectively dissipates the transmembrane proton potential.     

Genetic Determinants of Reutericyclin Biosynthesis in Lactobacillus reuteri

Reutericyclin is a unique antimicrobial tetramic acid produced by some strains of Lactobacillus reuteri. This study aimed to identify the genetic determinants of reutericyclin biosynthesis. Comparisons of the genomes of reutericyclin-producing L. reuteri strains with those of non-reutericyclin-producing strains identified a genomic island of 14 open reading frames (ORFs) including genes coding for a nonribosomal peptide synthetase (NRPS), a polyketide synthase (PKS), homologues of PhlA, PhlB, and PhlC, and putative transport and regulatory proteins. The protein encoded by rtcN is composed of a condensation domain, an adenylation domain likely specific for d-leucine, and a thiolation domain. rtcK codes for a PKS that is composed of a ketosynthase domain, an acyl-carrier protein domain, and a thioesterase domain. The products of rtcA, rtcB, and rtcC are homologous to the diacetylphloroglucinol-biosynthetic proteins PhlABC and may acetylate the tetramic acid moiety produced by RtcN and RtcK, forming reutericyclin. Deletion of rtcN or rtcABC in L. reuteri TMW1.656 abrogated reutericyclin production but did not affect resistance to reutericyclin. Genes coding for transport and regulatory proteins could be deleted only in the reutericyclin-negative L. reuteri strain TMW1.656ΔrtcN, and these deletions eliminated reutericyclin resistance. The genomic analyses suggest that the reutericyclin genomic island was horizontally acquired from an unknown source during a unique event. The combination of PhlABC homologues with both an NRPS and a PKS has also been identified in the lactic acid bacteria Streptococcus mutans and Lactobacillus plantarum, suggesting that the genes in these organisms and those in L. reuteri share an evolutionary origin.     

Reutericyclin: biological activity,mode of action,and potential applications

Reutericyclin is an inhibitory compound produced by sourdough isolates of Lactobacillus reuteri that is structurally but not functionally related to naturally occurring tetramic acids. It is bacteriostatic or bactericidal to gram-positive bacteria based on its activity as a proton-ionophore, and a broad range of food-related spoilage organisms and pathogens is inhibited by reutericyclin. Gram-negative bacteria are resistant to reutericyclin because of the barrier properties of their outer membrane, and resistance of beer-spoiling lactobacilli towards hop bitter acids provides cross-protection to reutericyclin. Remarkably, reutericyclin-producing strains were shown to persist for a period of 10 years in an industrial sourdough fermentation, and reutericyclin was shown to be produced in concentrations active against competitors during growth of L. reuteri in sourdough. Based on the known properties of reutericyclin and L. reuteri, reutericyclin-producing strains may have applications in the biopreservation of foods. Furthermore, these strains were shown to colonize reconstituted lactobacilli-free mice at high levels. Therefore, they could serve as a suitable model system to evaluate a possible impact of antimicrobial compounds on the intestinal microflora of humans and animals.     

Characterization of Reutericyclin Produced by Lactobacillus reuteri LTH2584

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C₈ chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. Höltzel, M. G. Gänzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766–2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.     

Characterization of reutericyclin produced by Lactobacillus reuteri LTH2584

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C(8) chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. H?ltzel, M. G. G?nzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766-2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.     

In Situ Determination of Clostridium Endospore Membrane Fluidity during Pressure-Assisted Thermal Processing in Combination with Nisin or Reutericyclin

This study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat- and pressure-induced spore inactivation and membrane fluidity.     

Phasing of many thousands of genotyped samples

Haplotypes are an important resource for a large number of applications in human genetics, but computationally inferred haplotypes are subject to switch errors that decrease their utility. The accuracy of computationally inferred haplotypes increases with sample size, and although ever larger genotypic data sets are being generated, the fact that existing methods require substantial computational resources limits their applicability to data sets containing tens or hundreds of thousands of samples. Here, we present HAPI-UR (haplotype inference for unrelated samples), an algorithm that is designed to handle unrelated and/or trio and duo family data, that has accuracy comparable to or greater than existing methods, and that is computationally efficient and can be applied to 100,000 samples or more. We use HAPI-UR to phase a data set with 58,207 samples and show that it achieves practical runtime and that switch errors decrease with sample size even with the use of samples from multiple ethnicities. Using a data set with 16,353 samples, we compare HAPI-UR to Beagle, MaCH, IMPUTE2, and SHAPEIT and show that HAPI-UR runs 18× faster than all methods and has a lower switch-error rate than do other methods except for Beagle; with the use of consensus phasing, running HAPI-UR three times gives a slightly lower switch-error rate than Beagle does and is more than six times faster. We demonstrate results similar to those from Beagle on another data set with a higher marker density. Lastly, we show that HAPI-UR has better runtime scaling properties than does Beagle so that for larger data sets, HAPI-UR will be practical and will have an even larger runtime advantage. HAPI-UR is available online (see Web Resources).     

Interactions of MCP1 with components of the replication machinery in mammalian cells

Eukaryotic DNA replication starts with the assembly of a pre-replication complex (pre-RC) at replication origins. We have previously demonstrated that Metaphase Chromosome Protein 1 (MCP1) is involved in the early events of DNA replication. Here we show that MCP1 associates with proteins that are required for the establishment of the pre-replication complex. Reciprocal immunoprecipitation analysis showed that MCP1 interacted with Cdc6, ORC2, ORC4, MCM2, MCM3 and MCM7, with Cdc45 and PCNA. Immunofluorescence studies demonstrated the co-localization of MCP1 with some of those proteins. Moreover, biochemical studies utilizing chromatin-immunoprecipitation (ChIP) revealed that MCP1 preferentially binds replication initiation sites in human cells. Interestingly, although members of the pre-RC are known to interact with some hallmarks of heterochromatin, our co-immunoprecipitation and immunofluorescence analyses showed that MCP1 did not interact and did not co-localize with heterochromatic proteins including HP1β and MetH3K9. These observations suggest that MCP1 is associated with replication factors required for the initiation of DNA replication and binds to the initiation sites in loci that replicate early in S-phase. In addition, immunological assays revealed the association of MCP1 forms with histone H1 variants and mass spectrometry analysis confirmed that MCP1 peptides share common sequences with H1.2 and H1.5 subtypes.     

Analysis of gain-of-function mutants of an ATP-dependent regulator of Tn7 transposition

The bacterial transposon Tn7 is distinguished by its unusual discrimination among targets, being particularly attracted to certain target DNA and actively avoiding other DNA. Tn7 transposition is mediated by the interaction of two alternative transposon-encoded target selection proteins, TnsD and TnsE, with a common core transposition machinery composed of the transposase (TnsAB) and an ATP-dependent DNA-binding protein TnsC. No transposition is observed with wild-type TnsABC. Here, we analyze the properties of two gain-of-function TnsC mutants that allow transposition in the absence of TnsD or TnsE. We find that these TnsC mutants have altered interactions with ATP and DNA that can account for their gain-of-function phenotype. We also show that TnsC is an ATPase and that it directly interacts with the TnsAB transposase. This work provides strong support to the view that TnsC and its ATP state are central to the control of Tn7 transposition.     

Myeloperoxidase of the leukocyte of normal blood. Nature of the prosthetic group of myeloperoxidase

The absorption spectra of alkaline pyridine hemochrome of myeloperoxidase in its native, acid, and modified forms were similar to those of heme a, and the molar extinction coefficient of myeloperoxidase heme was very similar to that of heme a, assuming that myeloperoxidase contains only one heme. The anaerobic titration of myeloperoxidase with dithionite showed that one electron was consumed per molecule of the enzyme for its conversion to its reduced form. The EPR spectrum of myeloperoxidase indicated that the enzyme contains both high-spin heme and non-heme iron. Carbonyl reagents, such as borohydride, hydrazine, and benzhydrazide, reacted with myeloperoxidase, causing blue shifts in its absorption spectrum. The heme was labeled with a tritium of boro[3H]hydride, suggesting that the reagents reacted with a formyl group on the porphyrin ring of the myeloperoxidase heme. When hydrazine was added to cyanide complex I of myeloperoxidase the complex was converted to the hydrazine-enzyme compound. Myeloperoxidase reacted with bisulfite to form a compound with an absorption spectrum similar to that of cyanide complex I. Borohydride-treated myeloperoxidase formed only one cyanide complex, while the native enzyme formed two different cyanide complexes, I (Kd = 0.3 muM) and II (approximate Kd = 0.1 mM). The EPR spectrum indicated that cyanide complex I of myeloperoxidase still contained high-spin heme. The results suggested that cyanide complex I and the bisulfite compound of myeloperoxidase were adducts between the nucleophilic reagents and the formyl group of myeloperoxidase heme. Based on these results, we concluded that one of the two iron atoms in a myeloperoxidase molecule exists in a formyl-heme moiety similar to heme a and the other exists as a non-heme iron.     

山羊GOLA-DQB1基因外显子2多态性与免疫性状的相关分析

利用PCR-RFLP技术, 对莱芜黑山羊、鲁波山羊和波尔山羊3个山羊种群共 175 只个体的GOLA-DQB1基因外显子2进行遗传多态性研究, 并对山羊种群的血液免疫指标的效应进行了分析。结果表明: 3个山羊种群共检测到(AA、BB、CC、AB、AC、BC、DD)7种基因型, GOLA-DQB1基因外显子2的第24、151位的碱基表现出多态性。多数指标品种效应是主要效应。莱芜黑山羊中, BC基因型的淋巴细胞百分比(W-SCR)显著高于AC 、CC基因型(P<0.05), 中性球比例(W-LCR)显著低于CC基因型(P<0.05), 大型白细胞数(W-LCC)低于AC、CC基因型, 但差异不显著(P>0.05)。波尔山羊中, BC基因型的W-LCC低于AA 、AB 、BB基因型, 但差异不显著(P>0.05)。鲁波山羊中, BC 、AC基因型的W-LCC显著低于AA基因型(P<0.05)。揭示GOLA-DQB1基因与血液免疫性状有一定的相关性。     

Interaction of the ERC family of RIM-binding proteins with the liprin-alpha family of multidomain proteins

Liprin-alpha/SYD-2 is a family of multidomain proteins with four known isoforms. One of the reported functions of liprin-alpha is to regulate the development of presynaptic active zones, but the underlying mechanism is poorly understood. Here we report that liprin-alpha directly interacts with the ERC (ELKS-Rab6-interacting protein-CAST) family of proteins, members of which are known to bind RIMs, the active zone proteins that regulate neurotransmitter release. In vitro results indicate that ERC2/CAST, an active zone-specific isoform, interacts with all of the known isoforms of liprin-alpha and that liprin-alpha1 associates with both ERC2 and ERC1b, a splice variant of ERC1 that distributes to both cytosolic and active zone regions. ERC2 colocalizes with liprin-alpha1 in cultured neurons and forms a complex with liprin-alpha1 in brain. Liprin-alpha1, when expressed alone in cultured neurons, shows a partial synaptic localization. When coexpressed with ERC2, however, liprin-alpha1 is redistributed to synaptic sites. Moreover, roughly the first half of ERC2, which contains the liprin-alpha-binding region, is sufficient for the synaptic localization of liprin-alpha1 while the second half is not. These results suggest that the interaction between ERC2 and liprin-alpha may be involved in the presynaptic localization of liprin-alpha and the molecular organization of presynaptic active zones.     

Fluorescence spectroscopy of single tryptophan mutants of apolipophorin-III in discoidal lipoproteins of dimyristoylphosphatidylcholine

The structure of the exchangeable apolipoprotein, apolipophorin-III from Locusta migratoria, apoLp-III, is described as a bundle of five amphipathic alpha-helices. To study the interaction of each of the helices of apoLp-III with a lipid surface, we designed five single-Trp mutants, each containing a Trp residue in a different alpha-helix. The Trp residues were located in the nonpolar domains of the amphipathic alpha-helices. The kinetics of the spontaneous interaction of the mutants with dimyristoylphosphatidylcholine (DMPC) indicated that all mutants behaved as typical exchangeable apolipoproteins. Circular dichroism in the far-UV indicated that all proteins have a high and similar helical content in the lipid-bound state. The interaction of the Trp residues with the lipid surface was investigated in recombinant lipoprotein particles made with DMPC. The properties of the Trp residues were investigated by fluorescence spectroscopy. These studies showed major changes in the spectroscopic properties of the Trp residues upon binding to lipid. These changes are observed with all single-Trp mutants, indicating that a major conformational change, which affects the properties of all helices, takes place upon binding to lipid. The position of the fluorescence maximum, the quenching efficiency of acrylamide as determined by steady-state and time-resolved fluorescence, and the fluorescence lifetimes of the single-Trp mutants suggest that helices 1, 4, and 5 interact with the nonpolar domains of the lipid. The properties of the Trp in helices 2 and 3 suggest that these helices adopt a different binding configuration than helices 1, 4, and 5. Helices 2 and 3 appear to be interacting with the polar headgroups of the phospholipids or constitute a different domain that does not interact with the lipid surface.     

Analysis of plasminogen-binding M proteins of Streptococcus pyogenes

Group A streptococci are common human pathogens that cause a variety of infections. They express M proteins which are important cell wall-bound type-specific virulence factors. We have found that a set of strains, associated primarily with skin infections, express M proteins that bind plasminogen and plasmin with high affinity. The binding is mediated by a 13-amino-acid internal repeated sequence located in the N-terminal surface-exposed portion of these M proteins. This sequence binds to kringle 2 in plasminogen, a domain that is not involved in the interaction with streptokinase, a potent group A streptococcal activator of plasminogen. It could be demonstrated that plasminogen, absorbed from plasma by growing group A streptococci expressing the plasminogen-binding M proteins, could be activated by exogenous and endogenous streptokinase, thereby providing the bacteria with a surface-associated enzyme that could act on the tissue barriers in the infected host.     

Effects of cold exposure on inhibition by isoproterenol of release of alanine in the rat

The effects of isoproterenol on the release of alanine during perfusion with pyruvate and valine were studied in perfused hindlimbs from rats that had been kept for 5 or 20 days at 4 degrees C. In hindlimbs perfused with Krebs bicarbonate buffer in a flow-through mode, the rate of release of alanine during perfusion with 2 mM pyruvate plus 5 mM valine was 250 nmol.min-1.leg-1, a rate that is comparable with that reported in hindlimbs perfused with complex medium. Neither the pyruvate-stimulated nor valine plus pyruvate-stimulated rates of release of alanine changed after 20 days of exposure to cold. Isoproterenol inhibited the release of alanine during perfusion with pyruvate, with valine, and with valine plus pyruvate in hindlimbs from a control group of rats. However, in hindlimbs from cold-exposed groups, isoproterenol failed to inhibit the release of alanine during perfusion with valine plus pyruvate and stimulated the release of alanine during perfusion with valine. Aminooxyacetate inhibited the effects of valine, pyruvate, and isoproterenol. The results obtained suggested that cold exposure decreases the responses to isoproterenol of the mechanism of alanine release and causes an increased supply of alanine to the liver.     

Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.     

Regulated binding of PTP1B-like phosphatase to N-cadherin: control of cadherin-mediated adhesion by dephosphorylation of beta-catenin

Cadherins are a family of cell-cell adhesion molecules which play a central role in controlling morphogenetic movements during development. Cadherin function is regulated by its association with the actin containing cytoskeleton, an association mediated by a complex of cytoplasmic proteins, the catenins: alpha, beta, and gamma. Phosphorylated tyrosine residues on beta-catenin are correlated with loss of cadherin function. Consistent with this, we find that only nontyrosine phosphorylated beta-catenin is associated with N-cadherin in E10 chick retina tissue. Moreover, we demonstrate that a PTP1B-like tyrosine phosphatase associates with N-cadherin and may function as a regulatory switch controlling cadherin function by dephosphorylating beta-catenin, thereby maintaining cells in an adhesion-competent state. The PTP1B-like phosphatase is itself tyrosine phosphorylated. Moreover, both direct binding experiments performed with phosphorylated and dephosphorylated molecules, and treatment of cells with tyrosine kinase inhibitors indicate that the interaction of the PTP1B-like phosphatase with N-cadherin depends on its tyrosine phosphorylation. Concomitant with the tyrosine kinase inhibitor-induced loss of the PTP1B-like phosphatase from its association with N-cadherin, phosphorylated tyrosine residues are retained on beta-catenin, the association of N- cadherin with the actin containing cytoskeleton is lost and N-cadherin- mediated cell adhesion is prevented. Tyrosine phosphatase inhibitors also result in the accumulation of phosphorylated tyrosine residues on beta-catenin, loss of the association of N-cadherin with the actin- containing cytoskeleton, and prevent N-cadherin mediated adhesion, presumably by directly blocking the function of the PTP1B-like phosphatase. We previously showed that the binding of two ligands to the cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase), the monoclonal antibody 1B11 and a proteoglycan with a 250-kD core protein, results in the accumulation of phosphorylated tyrosine residues on beta-catenin, uncoupling of N-cadherin from its association with the actin containing cytoskeleton, and loss of N- cadherin function. We now report that binding of these ligands to the GalNAcPTase results in the absence of the PTP1B-like phosphatase from its association with N-cadherin as well as the loss of the tyrosine kinase and tyrosine phosphatase activities that otherwise co- precipitate with N-cadherin. Control antibodies and proteoglycans have no such effect. This effect is similar to that observed with tyrosine kinase inhibitors, suggesting that the GalNAcPTase/proteoglycan interaction inhibits a tyrosine kinase, thereby preventing the phosphorylation of the PTP1B-like phosphatase, and its association with N-cadherin. Taken together these data indicate that a PTP1B-like tyrosine phosphatase can regulate N-cadherin function through its ability to dephosphorylate beta-catenin and that the association of the phosphatase with N-cadherin is regulated via the interaction of the GalNAcPTase with its proteoglycan ligand. In this manner the GalNAcPTase-proteoglycan interaction may play a major role in morphogenetic cell and tissue interactions during development.     

An invasion of cheats; the evolution of worthless nuptial gifts

Nuptial gifts are food items or inedible tokens that are transferred to females during courtship or copulation . Tokens are of no direct value to females, and it is unknown why females require such worthless gifts as a precondition of mating. One hypothesis is that token giving arose in species that gave nutritious gifts and males exploited female preferences for nutritional gifts by substituting more easily obtainable but worthless items. An invasion of such behavior would require that females accept the substitute gift and copulate for a period of time similar to that with genuine gifts. We show that both these prerequisites are met in the dance fly Rhamphomyia sulcata, in which females normally accept a nutritious gift. We removed the gift from copulating pairs and replaced it with either a large or small prey item or inedible token. We found that although pairs copulated longest with a large genuine gift, the tokens resulted in copula durations equivalent to those with a small genuine gift. We also observed that males that returned to the lek with tokens re-paired successfully. These findings suggest that female behavior in genuine gift-giving species is susceptible to the invasion of male cheating on reproductive investment.