In vivo and ex vivo studies support the immunostimulant and immunoprotective effect of Damiana (Turnera diffusa Willd) in Almaco Jack (Seriola rivoliana).

Damiana (Turnera diffusa Willd) was evaluated in vitro for antioxidant and antibacterial activities against Staphylococcus aureus and Streptococcus pyogenes (as a preliminary screening assessment) by high-performance thin-layer chromatography (HPTLC)-Direct bioautography. A study was performed in vivo to evaluate the effects of Damiana enriched diets at 0.5 % on immune parameters in mucus and serum and gene expression in Almaco Jack (Seriola rivoliana) intestine after two and four weeks; an infection with Aeromonas hydrophila at 1x107 colony forming units (CFU) followed and an ex vivo study was carried out using head-kidney leukocytes. Ferric reducing ability of plasma (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays showed high antioxidant activities in Damiana leaves; even in the ABTS assay, Damiana at 300 µg/mL showed similar activity to ascorbic acid - the standard control. Damiana exhibited strong in vitro antimicrobial activity against S. aureus and S. pyogenes. In vivo studies showed a strong enhancement of myeloperoxidase, nitric oxide, superoxide dismutase, and catalase activities in mucus and serum of S. rivoliana supplemented with Damiana; their immunological response enhanced after infection with A. hydrophila. IL-1ß, TNF-α, and IL-10 gene expressions upregulated in the fish intestine challenged with the bacterium. Piscidin and macrophage (MARCO) receptor gene expression up-regulated at week 4 and down-regulated after infection. Intestinal histology results confirm that Damiana not cause inflammation or damage. Finally, the ex vivo study confirmed the immunostimulant and protective effects of Damiana through increased phagocytic, respiratory burst, myeloperoxidase activities and nitric oxide generation before and upon the bacterial encounter. These results support the idea that Damiana has the potential as an immunostimulant additive for diets in aquaculture by enhancing immune parameters and protecting Almaco Jack against A. hydrophila infections upon four weeks of supplementation.

Genetic architecture of multiple mutualisms and mating system in Turnera ulmifolia.

Plants often associate with multiple arthropod mutualists. These partners provide important services to their hosts, but multiple interactions can constrain a plant's ability to respond to complex, multivariate selection. Here, we quantified patterns of genetic variance and covariance among rewards for pollination, biotic defence and seed dispersal mutualisms in multiple populations of Turnera ulmifolia to better understand how the genetic architecture of multiple mutualisms might influence their evolution. We phenotyped plants cultivated from 17 Jamaican populations for several mutualism and mating system-related traits. We then fit genetic variance-covariance (G) matrices for the island metapopulation and the five largest individual populations. At the metapopulation level, we observed significant positive genetic correlations among stigma-anther separation, floral nectar production and extrafloral nectar production. These correlations have the potential to significantly constrain or facilitate the evolution of multiple mutualisms in T. ulmifolia and suggest that pollination, seed dispersal and defence mutualisms do not evolve independently. In particular, we found that positive genetic correlations between floral and extrafloral nectar production may help explain their stable coexistence in the face of physiological trade-offs and negative interactions between pollinators and ant bodyguards. Locally, we found only small differences in G among our T. ulmifolia populations, suggesting that geographic variation in G may not shape the evolution of multiple mutualisms.

Hepatodamianol as hepatoprotective constituent of Turnera diffusa.

It is well known that liver diseases are a major health problem and that there is a lack of hepatoprotective agents. Turnera diffusa (damiana) is a plant with a widespread distribution in México, which has many traditional uses, including the treatment of hepatic illnesses. Based on the bioassay-guided fractionation of a methanolic extract obtained from the aerial part of T. diffusa, we purified and identified a compound called hepatodamianol (1). This C-glycoside exhibited a four times greater hepatoprotective effect than the widely used hepatoprotective agent silibinin against carbon tetrachloride damage in an in vitro model using HepG2 cells. Hepatodamianol produced no cytotoxic effects and it exhibited a high antioxidant capacity. Therefore, hepatodamianol is a good candidate compound for testing as a hepatoprotective agent in a preclinical trial.

Two Ways to Achieve the Same Goal-Two Validated Quantitative NMR Strategies for a Low-Abundance Natural Product in Standardized Extracts: The Case of Hepatodamianol in Turnera diffusa.

The quantification of low-abundance secondary metabolites in plant extracts is an analytical problem that can be addressed by different analytical platforms, the most common being those based on chromatographic methods coupled to a high-sensitivity detection system. However, in recent years nuclear magnetic resonance (NMR) has become an analytical tool of primary choice for this type of problem because of its reliability, inherent simplicity in sample preparation, reduced analysis time, and low solvent consumption. The versatility of strategies based on quantitative NMR (qNMR), such as internal and external standards and electronic references, among others, and the need to develop validated analytical methods make it essential to compare procedures that must rigorously satisfy the analytical well-established acceptance criteria for method validation. In this work, two qNMR methods were developed for the quantification of hepatodamianol, a bioactive component of T. diffusa. The first method was based on a conventional external standard calibration, and the second one was based on the pulse length-based concentration determination (PULCON) method using the ERETIC2 module as a quantitation tool available in TopSpin software. The results show that both procedures allow the content of the analyte of interest in a complex matrix to be determined in a satisfactory way, under strict analytical criteria. In addition, ERETIC2 offers additional advantages such as a reduction in experimental time, reagent consumption, and waste generated.

Chemical Characterization of Flowers and Leaf Extracts Obtained from Turnera subulata and Their Immunomodulatory Effect on LPS-Activated RAW 264.7 Macrophages.

The anti-inflammatory properties of Turnera subulata have been evaluated as an alternative drug approach to treating several inflammatory processes. Accordingly, in this study, aqueous and hydroalcoholic extracts of T. subulata flowers and leaves were analyzed regarding their phytocomposition by ultrafast liquid chromatography coupled to mass spectrometry, and their anti-inflammatory properties were assessed by an in vitro inflammation model, using LPS-stimulated RAW-264.7 macrophages. The phytochemical profile indicated vitexin-2-O-rhamnoside as an important constituent in both extracts, while methoxyisoflavones, some bulky amino acids (e.g., tryptophan, tyrosine, phenylalanine), pheophorbides, and octadecatrienoic, stearidonic, and ferulic acids were detected in hydroalcoholic extracts. The extracts displayed the ability to modulate the in vitro inflammatory response by altering the secretion of proinflammatory (TNF-α, IL-1ß, and IL-6) and anti-inflammatory (IL-10) cytokines and inhibiting the PGE-2 and NO production. Overall, for the first time, putative compounds from T. subulata flowers and leaves were characterized, which can modulate the inflammatory process. Therefore, the data highlight this plant as an option to obtain extracts for phytotherapic formulations to treat and/or prevent chronic diseases.

Turnera diffusa extract attenuates profibrotic, extracellular matrix and mitochondrial markers in activated human hepatic stellate cells (HSC).

INTRODUCTION AND OBJECTIVES: Hepatic fibrosis is characterized by the accumulation of extracellular matrix which includes the accumulation of α-smooth muscle actin (α-SMA), collagen type I (COL1α1), as well as remodeling induced by metalloproteinases and tissue inhibitor of metalloproteinase (TIMPs), where hepatic stellate cells (HSCs) play a central role. In addition, the transcription factor SNAI1 (which participates in epithelial-mesenchymal transition, EMT) and mitofusin 2 (MFN2, a mitochondrial marker) plays an important role in chronic liver disease. Turnera diffusa (TD), a Mexican endemic plant, has been shown to possess antioxidant and hepatoprotective activity in vitro. We treated human HSC (LX2 cells) with a methanolic extract of Turnera diffusa (METD) to evaluate the mechanism involved in its hepatoprotective effect measured as fibrosis modulation, EMT, and mitochondrial markers. MATERIALS AND METHODS: HSC LX-2 cells were treated with METD (100 and 200ng/mL) alone or combined with TGF-ß (10ng/mL) at different time points (24, 48, and 72h). α-SMA, COL1α1, MMP2, TIMP1, SNAI1, and MFN2 mRNAs and protein levels were determined by real-time quantitative PCR and Western Blot analysis. RESULTS: We found that METD decreases COL1α1-mRNA, α-SMA, and TIMP1 protein expression in LX2 cells treated with and TGF-ß. This treatment also decreases MFN2 and TIMP1 protein expression and induces overexpression of MMP2-mRNA. CONCLUSIONS: Our results suggest that a methanolic extract of Turnera diffusa is associated with an antifibrotic effect by decreasing profibrotic and mitochondrial markers together with the possible induction of apoptosis through SNAI1 expression in activated HSC cells.

Species diversification in the lowlands of mid-latitude South America: Turnera sidoides subsp. carnea as a case study.

The lowlands of mid-latitude South America comprise complex temperate ecoregions characterized by a unique biodiversity. However, the processes responsible for shaping its species diversity are still largely unknown. Turnera sidoides subsp. carnea is a variable subspecies occurring in the lowlands of northeastern Argentina and Uruguay, extending to southern Paraguay and Brazil. It constitutes a good model to perform evolutionary studies. Here we used an integrative approach to understand the process of diversification within this subspecies and to increase the knowledge concerning patterns and processes responsible for shaping the species diversity in the temperate lowlands of South America. The results provided strong evidences that this subspecies is an autopolyploid complex per se, being in an active process of intrasubspecific diversification. Morphological and genetic data show that the diversity of T. sidoides subsp. carnea is in congruence with the great past and present abiotic and biotic variability of the mid-latitude South American lowlands. The evolutionary history of this subspecies is consistent with past fragmentation and allopatric differentiation at diploid level. Geographic isolation and local adaptation would have promoted strong morphological, ecological, and genetic differentiation, resulting in two morphotypes and different genetic groups indicative of incipient speciation.

Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species.

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.

Assessment of chemical, biological and immunological properties of "Damiana de California" Turnera diffusa Willd extracts in Longfin yellowtail (Seriola rivoliana) leukocytes.

In Mexican herbal medicines or natural remedies, Turnera diffusa (Turneraceae) known as "Damiana de California", has ethnopharmacological relevance, including aphrodisiac, diuretic, and antimicrobial activities. To explore the immunological effect of infusion and methanolic extracts from Damiana de California, this study investigated its chemical, biological, antimicrobial and immunological properties in Longfin yellowtail Seriola rivoliana leukocytes. The analysis of chemical compounds revealed a considerable level of total phenolic and flavonoid contents in the infusion compared with methanolic extract. Furthermore, the antioxidant activity showed high hydroxyl radical scavenging activity in infusion extract compared with BHT positive control. Superoxide radical scavenging activity and ion chelation were higher in methanolic extract followed by infusion treatment. Interestingly, notable antimicrobial activity was observed in both extracts of T. diffusa against Vibrio parahaemolyticus. An in vitro study was performed using leukocytes of S. rivoliana treated with infusion or methanolic extracts at 12.5, 25 and 50 µg/mL for 24 h. Remarkably, infusion extract induced proliferation at any concentration but not the methanolic extract, which was diminished in a dose-dependent fashion. The immunostimulation study demonstrated that the phagocytosis activity increased in those leukocytes stimulated with methanolic extract but diminished the respiratory burst activity, in contrast to the activity observed in those leukocytes stimulated with infusion treatment. Finally, leukocytes incubated with the extracts and confronted with V.parahaemolyticus up-regulated the transcription of proinflammatory cytokine IL-1ß gene in a dose response relationship. These findings suggest that the infusion treatment has potential therapeutic properties, promoting the antioxidant capacity and enhancing immune parameters in Longfin yellowtail S. rivoliana.

The long and short of the S-locus in Turnera (Passifloraceae).

Distyly is an intriguing floral adaptation that increases pollen transfer precision and restricts inbreeding. It has been a model system in evolutionary biology since Darwin. Although the S-locus determines the long- and short-styled morphs, the genes were unknown in Turnera. We have now identified these genes. We used deletion mapping to identify, and then sequence, BAC clones and genome scaffolds to construct S/s haplotypes. We investigated candidate gene expression, hemizygosity, and used mutants, to explore gene function. The s-haplotype possessed 21 genes collinear with a region of chromosome 7 of grape. The S-haplotype possessed three additional genes and two inversions. TsSPH1 was expressed in filaments and anthers, TsYUC6 in anthers and TsBAHD in pistils. Long-homostyle mutants did not possess TsBAHD and a short-homostyle mutant did not express TsSPH1. Three hemizygous genes appear to determine S-morph characteristics in T. subulata. Hemizygosity is common to all distylous species investigated, yet the genes differ. The pistil candidate gene, TsBAHD, differs from that of Primula, but both may inactivate brassinosteroids causing short styles. TsYUC6 is involved in auxin synthesis and likely determines pollen characteristics. TsSPH1 is likely involved in filament elongation. We propose an incompatibility mechanism involving TsYUC6 and TsBAHD.

A brainstorm on the systematics of Turnera (Turneraceae, Malpighiales) caused by insights from molecular phylogenetics and morphological evolution.

With 145 species, Turnera is the largest genus of Turneraceae (Malpighiales). Despite several morphotaxonomic and cytogenetic studies, our knowledge about the phylogenetic relationships in Turnera remains mainly based on morphological data. Here, we reconstruct the most comprehensive phylogeny of Turnera with molecular data to understand the morphological evolution within this group and to assess its circumscription and infrageneric classification. We analyzed two nuclear and six plastid markers and 112 taxa, including species and infraspecific taxa, 97 from Turnera, covering the 11 series of the genus. Bayesian inference, maximum parsimony and maximum likelihood analyses show that Turnera, as traditionally circumscribed, is not monophyletic. The genus is divided into two well-supported independent clades; one of them is sister to the genus Piriqueta and is here segregated as the new genus Oxossia. According to our reconstructions, Turnera probably evolved from an ancestor without extrafloral nectaries and with solitary, homostylous flowers with yellow petals. The emergences of extrafloral nectaries and distyly, both common in extant taxa, played an important role in the diversification of the genus. An updated infrageneric classification reflecting the relationships within Turnera is now possible based on morphological synapomorphies and is here designed for further studies.

Modulatory effect of Turnera diffusa against testicular toxicity induced by fenitrothion and/or hexavalent chromium in rats.

Oxidative stress and increased production of reactive oxygen species have been implicated in pesticides and heavy metals toxicity. The objective of this study was to investigate the efficacy of Turnera diffusa Willd (damiana) on counteracting fenitrothion (FNT) and/or potassium dichromate (CrVI)-induced testicular toxicity and oxidative injury in rats. FNT and/or CrVI intoxicated animals revealed a significant increase in thiobarbituric acid reactive substances and hydrogen peroxide levels. While, reduced glutathione and protein content, as well as antioxidant enzymes, phosphatases, and aminotransferases activities, were significantly decreased. In addition, significant changes in testosterone and follicle-stimulating hormone levels were detected. Furthermore, histological and immunohistochemical alterations were observed in rat testes and this supported the observed biochemical changes. On the other hand, rats treated with damiana alone decreased lipid peroxidation and increased most of the examined parameters. Moreover, damiana pretreatment to FNT and/or CrVI-intoxicated rats showed significant improvement in lipid peroxidation, enzyme activities, and hormones as compared with their respective treated groups. Conclusively, rats treated with both FNT and/or CrVI showed pronounced hazardous effect especially in their combination group in addition, Turnera diffusa had a potential protective role against FNT and/or CrVI induced testicular toxicity.

Selective Inhibition of Human Monoamine Oxidase B by Acacetin 7-Methyl Ether Isolated from Turnera diffusa (Damiana).

The investigation of the constituents that were isolated from Turnera diffusa (damiana) for their inhibitory activities against recombinant human monoamine oxidases (MAO-A and MAO-B) in vitro identified acacetin 7-methyl ether as a potent selective inhibitor of MAO-B (IC50 = 198 nM). Acacetin 7-methyl ether (also known as 5-hydroxy-4', 7-dimethoxyflavone) is a naturally occurring flavone that is present in many plants and vegetables. Acacetin 7-methyl ether was four-fold less potent as an inhibitor of MAO-B when compared to acacetin (IC50 = 50 nM). However, acacetin 7-methyl ether was >500-fold selective against MAO-B over MAO-A as compared to only two-fold selectivity shown by acacetin. Even though the IC50 for inhibition of MAO-B by acacetin 7-methyl ether was ~four-fold higher than that of the standard drug deprenyl (i.e., SelegilineTM or ZelaparTM, a selective MAO-B inhibitor), acacetin 7-methyl ether's selectivity for MAO-B over MAO-A inhibition was greater than that of deprenyl (>500- vs. 450-fold). The binding of acacetin 7-methyl ether to MAO-B was reversible and time-independent, as revealed by enzyme-inhibitor complex equilibrium dialysis assays. The investigation on the enzyme inhibition-kinetics analysis with varying concentrations of acacetin 7-methyl ether and the substrate (kynuramine) suggested a competitive mechanism of inhibition of MAO-B by acacetin 7-methyl ether with Ki value of 45 nM. The docking scores and binding-free energies of acacetin 7-methyl ether to the X-ray crystal structures of MAO-A and MAO-B confirmed the selectivity of binding of this molecule to MAO-B over MAO-A. In addition, molecular dynamics results also revealed that acacetin 7-methyl ether formed a stable and strong complex with MAO-B. The selective inhibition of MAO-B suggests further investigations on acacetin 7-methyl as a potential new drug lead for the treatment of neurodegenerative disorders, including Parkinson's disease.

Cytotoxic Properties of Damiana (Turnera diffusa) Extracts and Constituents and A Validated Quantitative UHPLC-DAD Assay.

In our continuing search for new cytotoxic agents, we assayed extracts, fractions, and pure compounds from damiana (Turnera diffusa) against multiple myeloma (NCI-H929, U266, and MM1S) cell lines. After a first liquid-liquid solvent extraction, the ethyl acetate layer of an acetone (70%) crude extract was identified as the most active fraction. Further separation of the active fraction led to the isolation of naringenin (1), three apigenin coumaroyl glucosides 2⁻4, and five flavone aglycones 5⁻9. Naringenin (1) and apigenin 7-O-(4″-O-p-E-coumaroyl)-glucoside (4) showed significant cytotoxic effects against the tested myeloma cell lines. Additionally, we established a validated ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) method for the quantification of the isolated components in the herb and in traditional preparations of T. diffusa.

The role of triploids in the origin and evolution of polyploids of Turnera sidoides complex (Passifloraceae, Turneroideae).

Triploids can play an important role in polyploid evolution. However, their frequent sterility is an obstacle for the origin and establishment of neotetraploids. Here we analyzed the microsporogenesis of triploids (x = 7) and the crossability among cytotypes of Turnera sidoides, aiming to test the impact of triploids on the origin and demographic establishment of tetraploids in natural populations. Triploids of T. sidoides exhibit irregular meiotic behavior. The high frequency of monovalents and of trivalents with non-convergent orientations results in unbalanced and/or non-viable male gametes. In spite of abnormalities in chromosome pairing and unbalanced chromosome segregation, triploids are not completely sterile and yielded up to 67% of viable pollen. Triploids that originated by the fusion of 2n × n gametes of the same taxon showed more regular meiotic behavior and higher fertility than triploids from the contact zone of diploids and tetraploids or triploids of hybrid origin. The reproductive isolation of T. sidoides cytotypes of different ploidy level is not strict and the 'triploid block' may be overcome occasionally. Triploids of T. sidoides produce diploid and triploid progeny suggesting that new generations of polyploids could originate from crosses between triploids or from backcrosses with diploids. The capability of T. sidoides to multiply asexually by rhizomes, would enhance the likelihood that a low frequency of neopolyploids can be originated and maintained in natural populations of T. sidoides.

Acute Hypoglycemic and Antidiabetic Effect of Teuhetenone A Isolated from Turnera diffusa.

Diabetes mellitus is a chronic degenerative disease that causes long-term complications and represents a serious public health problem. Turnera diffusa (damiana) is a shrub that grows throughout Mexico and is traditionally used for many illnesses including diabetes. Although a large number of plant metabolites are known, there are no reports indicating which of these are responsible for this activity, and this identification was the objective of the present work. Through bioassay-guided fractionation of a methanolic extract obtained from the aerial part of T. diffusa, teuhetenone A was isolated and identified as the main metabolite responsible for the plant's hypoglycemic activity. Alpha-glucosidase inhibitory activity and cytotoxicity of this metabolite were determined. Hypoglycemic and antidiabetic activities were evaluated in a murine model of diabetes in vivo, by monitoring glucose levels for six hours and comparing them with levels after administering various controls. Teuhetenone A was not cytotoxic at the tested concentrations, and did not show inhibitory activity in the glucosidase test, and the in vivo assays showed a gradual reduction in glucose levels in normoglycemic and diabetic mice. Considering these results, we suggest that teuhetenone A has potential as an antidiabetic compound, which could be further submitted to preclinical assays.

Three's a Crowd: Trade-Offs between Attracting Pollinators and Ant Bodyguards with Nectar Rewards in Turnera.

Many plants attract insect pollinators with floral nectar (FN) and ant "bodyguards" with extrafloral nectar (EFN). If nectar production is costly or physiologically linked across glands, investment in one mutualism may trade off with investment in the other. We confirmed that changes in FN and EFN availability alter pollination and ant defense mutualisms in a field population of Turnera ulmifolia. Plants with additional FN tended to produce more seeds, while plants with reduced EFN production experienced less florivory. We then mimicked the consumptive effects of mutualists by removing FN or EFN daily for 50 days in a full factorial design using three Turnera species (T. joelii, T. subulata, and T. ulmifolia) in a glasshouse experiment. For T. ulmifolia and T. subulata, but not T. joelii, removing either nectar reduced production of the other, showing for the first time that EFN and FN production can trade off. In T. subulata, increased investment in FN decreased seed set, suggesting that nectar production can have direct fitness costs. Through the linked expression of EFN and FN, floral visitors may negatively affect biotic defense, and extrafloral nectary visitors may negatively affect pollination.

Evidence of the occurrence of structural chromosome changes at the initial diploid diversification of the autopolyploid Turnera sidoides L. (Passifloraceae) complex.

Turnera sidoides is an autopolyploid complex of obligate outcrossing perennial herbs. It includes five subspecies and five morphotypes in which diploid to octoploid cytotypes were found. Based on phenetic analyses of the complex and karyotype data of polyploid cytotypes, it has been hypothesized that morphological and chromosome differentiation of T. sidoides occurred at the diploid level. To test this hypothesis, we present the first detailed chromosome analysis of diploid populations of three subspecies and four morphotypes. CMA(+)/DAPI(-) bands were restricted to secondary constrictions (except in the andino morphotype) and varied in number and position among taxa. By contrast, DAPI staining was uniform in all the materials investigated. The number and position of 45S rDNA loci were coincident with the CMA(+)/DAPI(-) bands associated with secondary constrictions. Only one pair of 5S rDNA loci was detected in all the taxa (except in subsp. holosericea), but its position was variable. The identified chromosome markers varied among the three subspecies analyzed, but they were more conserved among the morphotypes of subsp. pinnatifida. Cluster analysis of these chromosome markers supports the current taxonomic arrangement of diploids and demonstrates that structural chromosome changes would have led or accompanied the initial differentiation of T. sidoides at the diploid level.

Plant defence as a complex and changing phenotype throughout ontogeny.

BACKGROUND AND AIMS: Ontogenetic changes in anti-herbivore defences are common and result from variation in resource availability and herbivore damage throughout plant development. However, little is known about the simultaneous changes of multiple defences across the entire development of plants, and how such changes affect plant damage in the field. The aim of this study was to assess if changes in the major types of plant resistance and tolerance can explain natural herbivore damage throughout plant ontogeny. METHODS: An assessment was made of how six defensive traits, including physical, chemical and biotic resistance, simultaneously change across the major transitions of plant development, from seedlings to reproductive stages of Turnera velutina growing in the greenhouse. In addition, an experiment was performed to assess how plant tolerance to artificial damage to leaves changed throughout ontogeny. Finally, leaf damage by herbivores was evaluated in a natural population. KEY RESULTS: The observed ontogenetic trajectories of all defences were significantly different, sometimes showing opposite directions of change. Whereas trichome density, leaf toughness, extrafloral nectary abundance and nectar production increased, hydrogen cyanide and compensatory responses decreased throughout plant development, from seedlings to reproductive plants. Only water content was higher at the intermediate juvenile ontogenetic stages. Surveys in a natural population over 3 years showed that herbivores consumed more tissue from juvenile plants than from younger seedlings or older reproductive plants. This is consistent with the fact that juvenile plants were the least defended stage. CONCLUSIONS: The results suggest that defensive trajectories are a mixed result of predictions by the Optimal Defence Theory and the Growth-Differentiation Balance Hypothesis. The study emphasizes the importance of incorporating multiple defences and plant ontogeny into further studies for a more comprehensive understanding of plant defence evolution.

Acute effects of a herb extract formulation and inulin fibre on appetite, energy intake and food choice.

The impact of two commercially available products, a patented herb extract Yerbe Maté, Guarana and Damiana (YGD) formulation and an inulin-based soluble fermentable fibre (SFF), alone or in combination, on appetite and food intake were studied for the first time in a double blind, placebo-controlled, cross-over design. 58 normal to slightly overweight women consumed a fixed-load breakfast followed 4h later by an ad libitum lunch. They were administered YGD (3 tablets) and SFF (5g in 100ml water), YGD and water (100ml), SFF and placebo (3 tablets) or water and placebo 15min before meals. Appetite was assessed using visual analogue scales, and energy intake was measured at lunch. Significant reductions in food intake and energy intake were observed when YGD was present (59.5g, 16.3%; 112.4kcal, 17.3%) and when SFF was present (31.9g, 9.1%; 80kcal, 11.7%) compared with conditions were products were absent. The lowest intake (gram and kcal) was in the YGD+SFF condition. Significant reductions in AUC hunger and AUC desire to eat were also observed after YGD+SFF combination. The data demonstrate that YGD produces a robust short-term effect on caloric intake, an effect augmented by SFF. Caloric compensation for SFF indicates independent effects on appetite regulation.