Inferring evolutionary patterns from the biogeographical distributions of mutualists and exploiters

Exploitation may lead to the breakdown of obligate species-specific mutualisms. However, the mutualism between Roridula (plants) and Pameridea (hemipterans) is often exploited by spiders. The aim of the present study was to determine when the exploiters became associated with the Roridula-Pameridea mutualism. The phylogenetic and geographical associations between Roridula and Pameridea are documented and the distribution patterns of Roridula and exploiters are overlaid to see how closely they correlate. A geographical discontinuity in Roridulas ' range divides both the host plants and associated hemipterans into two sister species so that each hemipteran species is associated with a different plant species. This suggests that Roridula was associated with Pameridea before fragmentation/vicariance events split the genus, allowing allopatric speciation. By contrast, Roridula is only associated with exploiters in parts of its current range. This suggests that exploiters are unable to traverse the disjunctions in Roridulas' distribution and that they only developed associations with the mutualism after vicariance events. It is hypothesized that Pameridea and Roridula were closely associated for a long period before the invasion of nonmutualists. The absence of associated nonmutualist species may have helped facilitate the evolution of an obligate interaction between Roridula and Pameridea .  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 541–549.     

Evolution and persistence of obligate mutualists and exploiters: competition for partners and evolutionary immunization

Mutualisms are ubiquitous in nature, as is their exploitation by both conspecific and heterospecific cheaters. Yet, evolutionary theory predicts that cheating should be favoured by natural selection. Here, we show theoretically that asymmetrical competition for partners generally determines the evolutionary fate of obligate mutualisms facing exploitation by third-species invaders. When asymmetry in partner competition is relatively weak, mutualists may either exclude exploiters or coexist with them, in which case their co-evolutionary response to exploitation is usually benign. When asymmetry is strong, the mutualists evolve towards evolutionary attractors where they become extremely vulnerable to exploiter invasion. However, exploiter invasion at an early stage of the mutualism's history can deflect mutualists' co-evolutionary trajectories towards slightly different attractors that confer long-term stability against further exploitation. Thus, coexistence of mutualists and exploiters may often involve an historical effect whereby exploiters are co-opted early in mutualism history and provide lasting 'evolutionary immunization' against further invasion.     

On mutualists and exploiters: plant-insect coevolution in pollinating seed-parasite systems

We investigate the coevolution of time of flowering and time of pollinator emergence in an obligate association between a plant and an insect that both pollinates and parasitizes flowers. Numerical analysis shows that the system in general evolves towards a time of flowering different from the time favoured by the abiotic environment. The equilibrium towards which the system evolves is a local fitness maximum (an ESS) with respect to mutational variation in flowering time but, for the insect, it can be a local fitness minimum at which selection on mutational variation in the time of insect emergence is disruptive. A consequence of evolutionary convergence to a fitness minimum is that pollinators having an earlier phenology can coexist with pollinators having a later phenology. Since late emerging insects are more likely to encounter and oviposit within previously pollinated flowers, their effect on the plant is more exploitative, leading them to function as cheaters within the system. Thus, in the long term, pollinators and exploiters are likely to be found in stable coexistence in pollinating seed-parasite systems.     

Host discrimination in modular mutualisms: a theoretical framework for meta-populations of mutualists and exploiters

Plants in multiple symbioses are exploited by symbionts that consume their resources without providing services. Discriminating hosts are thought to stabilize mutualism by preferentially allocating resources into anatomical structures (modules) where services are generated, with examples of modules including the entire inflorescences of figs and the root nodules of legumes. Modules are often colonized by multiple symbiotic partners, such that exploiters that co-occur with mutualists within mixed modules can share rewards generated by their mutualist competitors. We developed a meta-population model to answer how the population dynamics of mutualists and exploiters change when they interact with hosts with different module occupancies (number of colonists per module) and functionally different patterns of allocation into mixed modules. We find that as module occupancy increases, hosts must increase the magnitude of preferentially allocated resources in order to sustain comparable populations of mutualists. Further, we find that mixed colonization can result in the coexistence of mutualist and exploiter partners, but only when preferential allocation follows a saturating function of the number of mutualists in a module. Finally, using published data from the fig–wasp mutualism as an illustrative example, we derive model predictions that approximate the proportion of exploiter, non-pollinating wasps observed in the field.     

cFLIP is critical for oligodendrocyte protection from inflammation

Neuroinflammation associated with degenerative central nervous system disease and injury frequently results in oligodendrocyte death. While promoting oligodendrocyte viability is a major therapeutic goal, little is known about protective signaling strategies. We report that in highly purified rat oligodendrocytes, interferon gamma (IFNγ) activates a signaling pathway that protects these cells from tumor necrosis factor alpha (TNFα)-induced cytotoxicity. IFNγ protection requires Jak (Janus kinase) activation, components of the integrated stress response and NF-κB activation. Although NF-κB activation also occurred transiently in the absence of IFNγ and presence of TNFα, this activation was not sufficient to prevent induction of the TNFα-responsive cell death pathway. Genetic inhibition of NF-κB translocation to the nucleus abrogated IFNγ-mediated protection and did not change the cell death induced by TNFα, suggesting that NF-κB activation via IFNγ induces a different set of responses than activation of NF-κB via TNFα. A promising candidate is the NF-κB target cFLIP (cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein), which is protease-deficient caspase homolog that inhibits caspase-3 activation. We show that IFNγ-mediated protection led to upregulation of cFLIP. Overexpression of cFLIP was sufficient for oligodendrocyte protection from TNFα and short hairpin RNA knockdown of cFLIP-abrogated IFNγ -mediated protection. To determine the relevance of our in vitro finding to the more complex in vivo situation, we determined the impact on oligodendrocyte death of regional cFLIP loss of function in a murine model of neuroinflammation. Our data show that downregulation of cFLIP during inflammation leads to death of oligodendrocytes and decrease of myelin in vivo. Taken together, we show that IFNγ-mediated induction of cFLIP expression provides a new mechanism by which this cytokine can protect oligodendrocytes from TNFα-induced cell death.Interferon gamma (IFN-γ), the only type-II class IFN, has a paradoxical role in modulating cell function. It is critical for innate and adaptive immunity, but has multiple other functions. In the central nervous system (CNS), IFNγ has contrasting effects on the oligodendrocyte progenitor cells (O-2A/OPCs) that generate myelin-producing oligodendrocytes. O-2A/OPCs show suppressed division when exposed to IFNγ.^{1, 2, 3} However, when O-2A/OPCs differentiate into oligodendrocytes, IFNγ becomes pro-apoptotic.^{4, 5, 6, 7} Although IFNγ has a critical role in the pathogenesis of immune-mediated demyelinating disease;^{8, 9} the response of committed oligodendrocytes to IFNγ is more complex. For example, tumor necrosis factor alpha (TNFα) can show enhanced cytotoxicity in oligodendrocytes and transformed human neural cell lines when co-exposed with IFNγ.^{3, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19}In contrast with reported toxic effects of IFNγ on oligodendrocytes, other studies did not see negative effects on mature oligodendrocytes^{5, 9, 20} or saw protection of glial lineage cells. IFNγ protects the Oli-neu oligodendrocyte-like cell line from reactive oxygen and nitrogen species,²¹ and overexpression of IFNγ before the induction of experimental autoimmune encephalomyelitis (EAE) protected oligodendrocytes from immune-mediated damage.⁹ The mechanism of such protection remains elusive.We now report that IFNγ protects purified, committed oligodendrocytes from TNFα-mediated apoptosis via Janus kinase (Jak)-mediated activation of the stress kinase PKR (double-stranded RNA-dependent protein kinase) and NF-κB-induced expression of cFLIP (cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein), which inhibits caspase activation. Moreover, gain-of-function and loss-of-function experiments show that cFLIP is necessary and sufficient for oligodendrocyte protection from TNFα. These results demonstrate induction of cFLIP in a stress response and NF-κB-dependent manner, leading to inhibition of caspase-mediated apoptosis, and reveal an important role for cFLIP in oligodendrocyte protection in vivo.     

Predators and mutualists influence the exclusion of aphid species from natural communities

We investigated why two species of aphids (Aphis jacobaeae and Brachycaudus cardui) were very rare in a study site despite their abundance in the surrounding area. The study site contained many common species of aphid and we tested the hypothesis that the community of aphid predators in the field excluded the missing species. Colonies of the two aphid species were artificially initiated in the experimental site and allocated to one of four treatments: control; ground predator exclusion; total predator exclusion, and provision of mutualist ants. Two measures of colony performance were analysed: longevity and cumulative aphid density. Colonies decline naturally in late summer but control colonies disappeared very quickly while colonies protected from all predators survived the longest. The performance of colonies protected from just ground predators was intermediate. We failed to persuade ants to tend A. jacobaeae. Colonies of B. cardui attended by ants performed better than controls and those with ground predators excluded, but not as well as those with all predators excluded. We conclude that the absence of the two species of aphid in the study site is influenced by the resident predator community, and by the availability of mutualists. Received: 27 April 1998 / Accepted: 30 November 1998     

Proximate Costs of Competition for Nectar

Food competition among coexisting nectarivorous birds is conspicuousand often intense, affecting patterns of flower choice, dailybehavior budgets, and timing of successful reproduction. Exploitativecompetition involves loss of accumulated nectar to other individualsthat visited a flower first. Preliminary data support the useof Poisson models of the frequencies of point-source visitationand overlap for determining the probabilities of actual competitiveevents. Nectar losses from monitored flowers can be estimatedin terms of time intervals between visits weighted by flower-specificnectar production and bird-specific nectar removal capabilities.Foraging time budgets then provide a meaningful common denominatorfor assessing the impacts of competitive nectar losses, becausecompensatory increases in foraging time are required to maintaina balanced energy budget. Flexibility in foraging time budgetsmade possible by high efficiency foraging and predictably lowcompetitive losses may be an important determinant of reproductivetiming and success in nectar feeding birds. Aggressive displacement of competitors and territorial defenseof flowers are common forms of interference competition in nectar-feedingbirds. Aggression has definable caloric costs that ultimatelymust relate to caloric gains. Defense of flowers increases theaggressor's exclusive use of nectar, increases the predictabilityof a nectar supply, and increases the average amount of nectarobtained per flower. Simple cost-benefit models of territorialitydefine conditions when net benefits of territoriality are greaterthan those of alternatives.     

The adsorption of biomolecules to multi-walled carbon nanotubes is influenced by both pulmonary surfactant lipids and surface chemistry

Background

During production and processing of multi-walled carbon nanotubes (MWCNTs), they may be inhaled and may enter the pulmonary circulation. It is essential that interactions with involved body fluids like the pulmonary surfactant, the blood and others are investigated, particularly as these interactions could lead to coating of the tubes and may affect their chemical and physical characteristics. The aim of this study was to characterize the possible coatings of different functionalized MWCNTs in a cell free environment.

Results

To simulate the first contact in the lung, the tubes were coated with pulmonary surfactant and subsequently bound lipids were characterized. The further coating in the blood circulation was simulated by incubating the tubes in blood plasma. MWCNTs were amino (NH₂)- and carboxyl (-COOH)-modified, in order to investigate the influence on the bound lipid and protein patterns. It was shown that surfactant lipids bind unspecifically to different functionalized MWCNTs, in contrast to the blood plasma proteins which showed characteristic binding patterns. Patterns of bound surfactant lipids were altered after a subsequent incubation in blood plasma. In addition, it was found that bound plasma protein patterns were altered when MWCNTs were previously coated with pulmonary surfactant.

Conclusions

A pulmonary surfactant coating and the functionalization of MWCNTs have both the potential to alter the MWCNTs blood plasma protein coating and to determine their properties and behaviour in biological systems.     

Evolutionary origins and diversification of proteobacterial mutualists

Mutualistic bacteria infect most eukaryotic species in nearly every biome. Nonetheless, two dilemmas remain unresolved about bacterial–eukaryote mutualisms: how do mutualist phenotypes originate in bacterial lineages and to what degree do mutualists traits drive or hinder bacterial diversification? Here, we reconstructed the phylogeny of the hyperdiverse phylum Proteobacteria to investigate the origins and evolutionary diversification of mutualistic bacterial phenotypes. Our ancestral state reconstructions (ASRs) inferred a range of 34–39 independent origins of mutualist phenotypes in Proteobacteria, revealing the surprising frequency with which host-beneficial traits have evolved in this phylum. We found proteobacterial mutualists to be more often derived from parasitic than from free-living ancestors, consistent with the untested paradigm that bacterial mutualists most often evolve from pathogens. Strikingly, we inferred that mutualists exhibit a negative net diversification rate (speciation minus extinction), which suggests that mutualism evolves primarily via transitions from other states rather than diversification within mutualist taxa. Moreover, our ASRs infer that proteobacterial mutualist lineages exhibit a paucity of reversals to parasitism or to free-living status. This evolutionary conservatism of mutualism is contrary to long-standing theory, which predicts that selection should often favour mutants in microbial mutualist populations that exploit or abandon more slowly evolving eukaryotic hosts.     

Competition Between Hummingbirds and Insects for Nectar

Preliminary data are provided suggesting that hummingbirds competewith bees and hawkmoths for flower nectar while on southwardmigration through the Sierra Nevada of California during thesummer. Evidence is given that removal of hummingbirds enhancesforaging in bees and hawkmoths on plants shared with the birds.The birds were aggressive against moths but not usually againstbees. Competition between these taxa leads to temporal and spatialpartitioning of the resource that is apparently behaviorallymediated. It is speculated that for hummingbirds competitionwith insects is less predictable and more difficult to contendwith than is competition with other hummingbirds.     

Nectar from willow catkins as a food source for Blue Tits

Bird-plant interactions are not so well known as they ought to be. Nectar is a potential food source to birds, while the plants could benefit also from having the birds act as pollinators.     

Epiregulin is not essential for development of intestinal tumors but is required for protection from intestinal damage

Epiregulin, an epidermal growth factor family member, acts as a local signal mediator and shows dual biological activity, stimulating the proliferation of fibroblasts, hepatocytes, smooth muscle cells, and keratinocytes while inhibiting the growth of several tumor-derived epithelial cell lines. The epiregulin gene (Ereg) is located on mouse chromosome 5 adjacent to three other epidermal growth factor family members, epigen, amphiregulin, and betacellulin. Gene targeting was used to insert a lacZ reporter into the mouse Ereg locus and to ablate its function. Although epiregulin is broadly expressed and regulated both spatially and temporally, Ereg null mice show no overt developmental defects, reproductive abnormalities, or altered liver regeneration. Additionally, in contrast to previous hypotheses, Ereg deficiency does not alter intestinal cancer susceptibility, as assayed in the ApcMin model, despite showing robust expression in developing tumors. However, Ereg null mice are highly susceptible to cancer-predisposing intestinal damage caused by oral administration of dextran sulfate sodium.     

Ethylene signaling is critical for synergid cell functional specification and pollen tube attraction

ETHYLENE INSENSITIVE 3 (EIN3) is a key regulator of ethylene signaling, and EIN3‐BINDING F‐BOX1 (EBF1) and EBF2 are responsible for EIN3 degradation. Previous reports have shown that the ebf1 ebf2 double homozygous mutant cannot be identified. In this study, the genetic analysis revealed that the ebf1 ebf2 female gametophyte is defective. The pollination experiment showed that ebf1 ebf2 ovules failed to attract pollen tubes. In female gametophyte/ovule, the synergid cell is responsible for pollen tube attraction. Observation of the pEIN3::EIN3‐GFP transgenic lines showed that EIN3 signal was over‐accumulated at the micropylar end of ebf1 ebf2 female gametophyte. The overexpression of stabilized EIN3 in synergid cell led to the defect of pollen tube guidance. These results suggested that the over‐accumulated EIN3 in ebf1 ebf2 synergid cell blocks its pollen tube attraction which leads to the failure of ebf1 ebf2 homozygous plant. We identified that EIN3 directly activated the expression of a sugar transporter, SENESCENCE‐ASSOCIATED GENE29 (SAG29/SWEET15). Overexpression of SAG29 in synergid cells blocked pollen tube attraction, suggesting that SAG29 might play a role in ethylene signaling to repel pollen tube entry. Taken together, our study reveals that strict control of ethylene signaling is critical for the synergid cell function during plant reproduction.     

Scent chemistry and pollinator attraction in the deceptive trap flowers of Ceropegia dolichophylla

Ceropegia species (Apocynaceae, Asclepiadoideae) have pitfall flowers and are pollinated by small flies through deception. It has been suggested that these flies are attracted by floral scent. However, the scent that is emitted from Ceropegia flowers has not been studied using headspace and gas chromatography mass spectrometry methods. It has also been unclear whether or not the flowers are mimics of particular models that attract flies. In the present study, we determined the composition as well as the spatial and temporal patterns of floral scent emitted by C. dolichophylla. Furthermore, we determined the pollinators in the native (China) and non-native (Germany) range of this species, and tested the capability of the floral scent to attract flies in the non-native range. Our data demonstrate that the floral scent, which is emitted from morning until evening, primarily from the tips of the corolla lobes, consists mainly of spiroacetals and aliphatic compounds. Milichiid flies were common visitors/pollinators in the native as well as non-native range, and were attracted by floral scent in bioassays performed in the non-native range. The compounds emitted by C. dolichophylla are unusual for flowers, but are well known from insect pheromones and occur in the glandular secretions of insects. The milichiid flies that visit and pollinate the flowers are kleptoparasites that feed on the prey (haemolymph or other secretions) of predatory arthropods, e.g. spiders, to which they are attracted by scent. Our data thus suggest that the floral scent of C. dolichophylla mimics the feeding sites of kleptoparasitic flies.     

FolX from Pseudomonas aeruginosa is octameric in both crystal and solution

FolX encodes an epimerase that forms one step of the tetrahydrofolate biosynthetic pathway, which is of interest as it is an established target for important drugs. Here we report the crystal structure of FolX from the bacterial opportunistic pathogen Pseudomonas aeruginosa, as well as a detailed analysis of the protein in solution, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). In combination, these techniques confirm that the protein is an octamer both in the crystal structure, and in solution.     

Silencing NaTPI Expression Increases Nectar Germin,Nectarins, and Hydrogen Peroxide Levels and Inhibits Nectar Removal from Plants in Nature

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellant, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H₂O₂) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H₂O₂ compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H₂O₂ and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H₂O₂ levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H₂O₂ to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.Floral nectar is an innovative feature of plants that is thought to have evolved as a reward for pollen-transporting floral visitors. Sugars (e.g. Glc, Fru, and Suc), amino acids, and lipids (Baker and Baker, 1982, 1986) provide nutritional rewards that are essential for many pollinators. But nectar is also known to contain other compounds, such as volatile organic compounds (VOCs), alkaloids, phenolics, and nonprotein amino acids (Baker, 1977, 1978; Raguso, 2004; Kessler and Baldwin, 2007), which do not increase the nutritional value of nectar. Nectar is also exploited as a food source by nectar robbers and nectar-infesting microorganisms, which do not provide mutualistic services to the plant and are known to directly reduce a plant''s fitness either by competing with pollinators or by infesting reproductive organs (Traveset et al., 1998; Irwin and Brody, 1999; Maloof and Inouye, 2000; Farkas et al., 2007). Therefore, flowers must solve the dilemma of repelling nectar thieves or florivores that provide no pollination services while simultaneously attracting fitness-enhancing pollinators.Most of the defensive compounds in nectar have been reported to act selectively (i.e. only on antagonists). For example, the floral nectar of Catalpa speciosa contains iridoid glycosides that fend off nectar robbers but not the plant''s specific pollinators (Stephenson, 1981). Similarly, the presence of phenols in the floral nectar of Aloe vryheidensis lowers its palatability to generalist floral visitors like sunbirds or honey bees while not affecting the attractiveness of the nectar to a specialist bird, the dark-capped bulbul (Johnson et al., 2006). In its native habitat, Nicotiana attenuata (Solanaceae) maximizes its maternal and paternal reproductive success while repelling herbivores, florivores, and nectar robbers by producing a sophisticated blend of both repellants (nicotine) and attractants (benzylacetone) in its nectar and floral head space (Kessler et al., 2008) as well as by changing its floral phenology in response to herbivore attack, so as to switch from the use of night-active hawkmoth pollinators, which oviposit herbivores on the plants they pollinate, to day-active hummingbird pollinators, which do not (Kessler et al., 2010). While this sophisticated use of chemical attractants and repellants is likely a common solution to the dilemma, very little is known about the function of most secondary metabolites found in nectar (Thornburg, 2007).Similar chemically mediated strategies are used to solve a similar problem when plants use a combination of direct and indirect defenses to protect their leaves from herbivore attack (Halitschke et al., 2008). In N. attenuata, attack by the specialist herbivore Manduca sexta elicits a remarkable array of direct and indirect defenses, most of which are elicited by the jasmonate signaling pathway in response to herbivore-specific elicitors (Baldwin, 2001; Kessler and Baldwin, 2002; Wu and Baldwin, 2009). These herbivory-elicited responses include the accumulation of toxins and digestibility reducers, which function as direct defenses, as well as the release of a complicated blend of VOCs (Gaquerel et al., 2009), which repel further oviposition by M. sexta moths and attract predacious bugs that feed on M. sexta eggs or larvae, thereby functioning as an indirect defense (Kessler and Baldwin, 2001).Once herbivores start feeding on N. attenuata leaves, they are frequently repelled by a suite of locally and systemically elicited direct defenses (Steppuhn et al., 2008). Trypsin protease inhibitors (TPIs) are an effective component of this inducible defensive system that reduces the performance of folivores by targeting their main proteolytic digestive enzymes and is strongly induced by herbivore attack (van Dam et al., 2000; Glawe et al., 2003; Zavala et al., 2004b, 2008; Horn et al., 2005). However, in N. attenuata, the biosynthesis of TPIs incurs substantial fitness costs (Zavala et al., 2004a); silencing the TPI gene in N. attenuata abolishes the plant''s capacity to produce TPIs and allows it to grow faster, flower earlier, and produce more seed capsules compared with TPI-producing genotypes (Zavala et al., 2004a). Similarly, restoring TPI production by transforming an ecotype of N. attenuata naturally deficient in TPI production (Wu et al., 2007) reduces lifetime seed production (Zavala et al., 2004a). TPIs are not only restricted to leaves but accumulates in reproductive organs, where they may protect these fitness-valuable tissues against attack from florivores and microbes. Atkinson et al. (1993) and Johnson et al. (2007) elegantly demonstrated that TPIs dramatically accumulate in Nicotiana alata stigmas to become the most abundant protein in these tissues. PIs have been reported to accumulate in Solanum americanum seeds, where they were shown to play an important role in seed development (Suk-Fong et al., 2006). These studies highlight that while it is clear that TPIs occur at high levels in reproductive organs, their role in floral function has not been thoroughly explored.As part of a research program to study the defensive functions of nicotine and TPIs against folivores, we planted N. attenuata plants that had been transformed with RNA interference constructs to silence their nicotine (ir-pmt), TPI (ir-pi), or both (ir-pmt/pi) in the plant''s native habitats in Utah during four field seasons. Serendipitously, we noticed that the amount of nectar removed by the native community of floral visitors from ir-pmt/pi plants did not differ from that removed from wild-type plants, although we had recently discovered that silencing nicotine alone (ir-pmt) consistently increased nectar removal (Kessler and Baldwin, 2007). These observations suggested that silencing TPIs alone might impede nectar removal by the native community of floral visitors. During two field seasons (2007 and 2009), we compared the amount of nectar removed from wild-type plants and from TPI-silenced plants (ir-pi) and found that, indeed, less nectar was consistently removed from ir-pi plants. To understand these observations, we compared the floral chemistry of wild-type and ir-pi plants, including floral volatiles, nectar sugar, nicotine, and proteomes. We found that silencing TPIs increased the accumulation of nectar proteins, especially the nectar germin-like proteins (GLPs) and nectarins, which are known to participate in the nectar redox cycle and generate hydrogen peroxide (H₂O₂; Carter and Thornburg, 2000). Consistent with data on nectar proteins, we also found significantly higher levels of H₂O₂ in the nectar of ir-pi plants compared with those of wild-type plants. To test whether the differences in the accumulation of H₂O₂ in the nectar of ir-pi and wild-type plants could explain the nectar removal observations in the field, we experimentally increased H₂O₂ in the nectar of wild-type plants to the levels found in ir-pi nectar using a mixture of Glc oxidase (GOX) and Glc and compared nectar removal by the native community of floral visitors.