Characteristics of Nectar Secretion by the Extrafloral Nectaries of Ricinus communis

The characteristics of nectar secretion by excised extrafloralnectaries of Ricinus have been examined. Secreted nectar wasfound to contain three sugars: sucrose, glucose and fructose,with glucose and fructose occurring in a 1: 1 ratio. All threesugars supported secretion when used in the culture medium andthe yield of nectar sugar was found to be concentration-dependent.Other sugar sources failed to support secretion. Experimentsusing ¹⁴C-sugars and ¹⁴CO₂ fed to intact plants allowed themovement of sugars through the nectary to be examined. Sucrosesynthesis occurs when excised glands are fed glucose and thisoccurs very early in the transport through the nectary. Themain sugar transported was sucrose, with little hydrolysis occurringuntil the final step of secretion. There was no evidence thatsucrose hydrolysis occurs either by invertase in the nectaror by a microbial flora. Inhibitors of respiration were foundto inhibit secretion as did anaerobiosis. Temperature also hada marked effect, with a temperature coefficient of 1.8. However,secretion of sucrose was not affected by anaerobic conditions,low temperatures or inhibitors of respiration as markedly asthat of glucose and fructose. Electron microscopy revealed the presence of a thickened andheavily stained wall at the inner border of the secretory epidermallayer. This wall contained numerous plasmodesmata at a frequencyof 14 per µm² and may represent an apoplastic barrier.Light microscope cytochemistry revealed that acid phosphataseis primarily located in the nectiferous tissue, while ATPaseis concentrated in the epidermis. The possibility that the nectarycontains two pathways for sucrose secretion, both apoplasticand symplastic, is discussed. Key words: Invertase, nectary, plasmodesmata, Ricinus communis, sucrose     

Extrafloral Nectaries in Cipadessa (Meliaceae)

This is the first report of an extrafloral nectary (EFN) fromAsian Meliaceae and from subfamily Melioideae. The pinnatelycompound leaf of Cipadessa baccifera has 25–35 small,ellipsoidal EFNs abaxially on the rachis, with occasional EFNson leaflets. EFNs secrete nectar until leaf maturity, then graduallywither. Each convex, ellipsoidal EFN is parenchymatous, withouta palisade epidermis, a delimiting nectary sheath, or any vascularaffiliation. This EFN differs markedly from the typical ‘Flachnektarien’EFN described earlier from neotropical Swietenia species. Cipadessa baccifera (Roth.) Miq., extrafloral nectary, Meliaceae, nectary anatomy     

Glucanases and Chitinases as Causal Agents in the Protection of Acacia Extrafloral Nectar from Infestation by Phytopathogens

Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae) contains “nectarins,” proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.Plants secrete nectar to attract mutualistic animals, which mainly function as pollinators in the case of floral nectar or as defenders against herbivores in the case of extrafloral nectar (EFN; Simpson and Neff, 1981; Heil, 2008; González-Teuber and Heil, 2009a). Because nectars usually represent aqueous solutions of monosaccharides and disaccharides together with amino acids, they are prone to infestation by microbial organisms. When present in the nectar, fungi (González-Teuber et al., 2009) and yeast (Herrera et al., 2009) in particular can alter the chemical composition of the nectar and thereby reduce its suitability for the plant''s animal mutualists (Herrera et al., 2008). Moreover, several phytopathogenic organisms may use the nectar-secreting tissues as entries to infect other plant organs (Bubán et al., 2003; Farkas et al., 2007). Therefore, being an excellent growing medium for yeast, fungi, and bacteria, nectar requires an efficient antimicrobial protection.Unfortunately, our knowledge of the means by which plants protect nectar from microorganisms is extremely limited. Although the first reports on nectar proteins date back to the 1960s and 1970s (Lüttge, 1961; Baker and Baker, 1975), most studies that considered the defensive function of nectar focused on secondary compounds such as alkaloids and phenols. These metabolites commonly protect nectar from consumption by nectar robbers (animals that feed on nectar without providing a mutualistic service to the plant [Stephenson, 1981; Johnson et al., 2006]) or limit the duration of pollinator visits (Kessler et al., 2008). Only during the last decade did a series of studies discover defensive proteins in the floral nectar of ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae; Carter et al., 1999). In this species, floral nectar contains a limited array of proteins termed “nectarins.” Nectarins serve the protection from microbial infestation through a biochemical pathway called the nectar redox cycle (Carter and Thornburg, 2004a), in which mainly three of the five nectarins are involved: NEC1, NEC3, and NEC5. NEC1 was characterized as a manganese superoxide dismutase (Carter and Thornburg, 2000), NEC3 has carbonic anhydrase and monodehydroascorbate reductase activity (Carter and Thornburg, 2004b), and NEC5 is a Glc oxidase that functions together with NEC1 in the production of high peroxide levels (Carter and Thornburg, 2004c): nectar of ornamental tobacco can accumulate up to 4 mm hydrogen peroxide, concentrations that are clearly high enough to exhibit toxicity on microorganisms. Thus, the floral nectar of ornamental tobacco is kept free of microbes mainly via the production of small reactive oxygen species.By contrast, a proteomic study on EFN of the ant-plant, Acacia cornigera, revealed the presence of several pathogenesis-related (PR) proteins (González-Teuber et al., 2009). Myrmecophytes (ant-plants) are constitutively inhabited by specialized ant species, which serve as a very efficient indirect defense against herbivores (Heil, 2008). In the most specialized cases, both the ant and the plant depend on this interaction, which thus represents an obligate mutualism. In the EFN of A. cornigera, activities of chitinase, β -1,3-glucanase, and peroxidase were detected together with proteins similar to PR-1, osmotin-like proteins, and thaumatin-like proteins (González-Teuber et al., 2009). Most of these proteins, however, were only investigated by tandem mass spectrometry (MS/MS) and characterized via MS-BLAST searches. Because no activity assays had been performed, the presence of these proteins could not be causally linked to the protection of EFN from microorganisms.This study was conducted to determine whether the antimicrobial protection of Acacia EFN can be directly and exclusively allotted to the enzymatic activity of its protein fraction, which would contrast the protective strategy of this nectar from the one that has been described by Carter, Thornburg, and colleagues (Carter et al., 1999; Carter and Thornburg, 2004a). We also aimed at investigating whether Acacia EFN inhibits the growth of phytopathogens and thus can serve in the protection from infection by pathogens that may use nectaries to enter the plant (Bubán et al., 2003). We used four sympatric Acacia species and a closely related Prosopis species, which exhibit different types of ant-plant mutualism and therefore differ in their EFN secretion schemes (Heil et al., 2004) and composition (Heil et al., 2005; González-Teuber and Heil, 2009b). The obligate myrmecophytes among Central American Acacia species secrete EFN constitutively at high rates, and the EFN of these species possesses a much higher level of proteins and of antimicrobial defense than the EFN of congeneric nonmyrmecophytes (González-Teuber et al., 2009). The nonmyrmecophytes, by contrast, secrete EFN at lower rates and only transiently in response to leaf damage; this EFN contains few proteins but high levels of Suc (Heil et al., 2005; González-Teuber et al., 2009).We studied the EFN of the obligate myrmecophytes A. cornigera, Acacia hindsii, and Acacia collinsii and of the two nonmyrmecophytes Acacia farnesiana and Prosopis juliflora. Bioassays were employed to detect inhibitory activities of the nectars against phytopathogens, and in-gel assays were used to determine the presence and functionality of basic and acidic chitinases and glucanases. Size exclusion filtration and heating of the EFN was used to investigate whether the antimicrobial activity of EFN is exclusively caused by the protein fraction. The results demonstrate that the antimicrobial protection of Acacia EFN is caused by the fraction of enzymatically active PR proteins and independent of small, soluble molecules, an observation that represents, to our knowledge, a new strategy by which plants can protect nectar from infestation by potentially deleterious microorganisms.     

Increased Biodiversity in the Environment Improves the Humoral Response of Rats

Previous studies have compared the immune systems of wild and of laboratory rodents in an effort to determine how laboratory rodents differ from their naturally occurring relatives. This comparison serves as an indicator of what sorts of changes might exist between modern humans living in Western culture compared to our hunter-gatherer ancestors. However, immunological experiments on wild-caught animals are difficult and potentially confounded by increased levels of stress in the captive animals. In this study, the humoral immune responses of laboratory rats in a traditional laboratory environment and in an environment with enriched biodiversity were examined following immunization with a panel of antigens. Biodiversity enrichment included colonization of the laboratory animals with helminths and co-housing the laboratory animals with wild-caught rats. Increased biodiversity did not apparently affect the IgE response to peanut antigens following immunization with those antigens. However, animals housed in the enriched biodiversity setting demonstrated an increased mean humoral response to T-independent and T-dependent antigens and increased levels of “natural” antibodies directed at a xenogeneic protein and at an autologous tissue extract that were not used as immunogens.     

Investment into Defensive Traits by Anuran Prey (Lithobates pipiens) Is Mediated by the Starvation-Predation Risk Trade-Off

Prey can invest in a variety of defensive traits when balancing risk of predation against that of starvation. What remains unknown is the relative costs of different defensive traits and how prey reconcile investment into these traits when energetically limited. We tested the simple allocation model of prey defense, which predicts an additive effect of increasing predation risk and resource availability, resulting in the full deployment of defensive traits under conditions of high risk and resource saturation. We collected morphometric, developmental, and behavioural data in an experiment using dragonfly larvae (predator) and Northern leopard frog tadpoles (prey) subject to variable levels of food availability and predation risk. Larvae exposed to food restriction showed limited response to predation risk; larvae at food saturation altered behaviour, development, and growth in response to predation risk. Responses to risk varied through time, suggesting ontogeny may affect the deployment of particular defensive traits. The observed negative correlation between body size and activity level for food-restricted prey – and the absence of a similar response among adequately-fed prey – suggests that a trade-off exists between behavioural and growth responses when energy budgets are limited. Our research is the first to demonstrate how investment into these defensive traits is mediated along gradients of both predation risk and resource availability over time. The interactions we demonstrate between resource availability and risk level on deployment of inducible defenses provide evidence that both internal condition and extrinsic risk factors play a critical role in the production of inducible defenses over time.     

Nectar spur evolution in the Mexican lobelias (Campanulaceae: Lobelioideae)

Phylogenetic studies are often hampered by the independent evolution of characters that may potentially obscure relationships. The adaptive significance of the nectar spur and its evolution within the Mexican lobeliads (Campanulaceae) is considered here. The taxonomic delimitations of Heterotoma from the Mexican species within the genera Lobelia and Calcaratolobelia were tested. Independent molecular data were gathered to determine whether the Mexican spurred lobeliads should be treated as distinct genera. The internal transcribed spacer (ITS) region from 18-26S nuclear rDNA and chloroplast DNA from the 3' trnK intron were sequenced from 14 representative species. Our data suggest that Heterotoma, as originally conceived, is a good evolutionary unit within Lobelia and that the presence of a nectar spur is an important morphological character that can be used in defining phylogenetic position. This study also suggests that morphological changes associated with hummingbird pollination have evolved more than once in the Mexican lobeliads, from small blue-flowered, insect-pollinated relatives.     

Nectar Sucking Pollinators in Hybrid Derivatives of Epimedium (Berberidaceae)

Abstract The followings are detected in the plants derived from hybridization between Epimedium diphyllum and E. sempervirens. Bombus ardens queen sucked nectar from flowers with spurs of which lengths correspond to those of the bees' proboscides. Queens of B. ardens and males and females of Tetralonia nipponensis sucked nectar indiscriminately from reddish purple and white flowers. B. hypocrita queens, which were not pollinators, perforated spurs and thieved nectar.     

Mutualistic degradation of the lignin model compound veratrylglycerol-beta-(o-methoxyphenyl) ether by bacteria.

Complete degradation of the lignin model compound veratrylglycerol-beta-(o-methoxyphenyl) ether is accomplished mutualistically by a two-membered bacterial culture. Bacterial isolate E1, which has been tentatively identified as an Acinetobacter, grows on veratrylglycerol-beta-(o-methoxyphenyl) ether producing guaiacol (o-methoxyphenol) as a non-metabolizable, bacteriocidal by-product. When Nocardia corallina (strain A81) is also present in media containing veratrylglycerol-beta-(o-methoxyphenyl) ether as the only carbon/energy source, it is able to grow on the guaiacol produced from veratrylglycerol-beta-(o-methoxyphenyl) ether by isolate E1. Strain A81 alone does not grow on veratrylglycerol-beta-(o-methoxyphenyl) ether. In the absence of strain A81, isolate E1 is rapidly killed by accumulated guaiacol. In the presence of the Nocardia, isolate E1 maintains its viability.     

Latitudinal Variation of a Defensive Symbiosis in the Bugula neritina (Bryozoa) Sibling Species Complex

Mutualistic relationships are beneficial for both partners and are often studied within a single environment. However, when the range of the partners is large, geographical differences in selective pressure may shift the relationship outcome from positive to negative. The marine bryozoan Bugula neritina is a colonial invertebrate common in temperate waters worldwide. It is the source of bioactive polyketide metabolites, the bryostatins. Evidence suggests that an uncultured vertically transmitted symbiont, “Candidatus Endobugula sertula”, hosted by B. neritina produces the bryostatins, which protect the vulnerable larvae from predation. Studies of B. neritina along the North American Atlantic coast revealed a complex of two morphologically similar sibling species separated by an apparent biogeographic barrier: the Type S sibling species was found below Cape Hatteras, North Carolina, while Type N was found above. Interestingly, the Type N colonies lack “Ca. Endobugula sertula” and, subsequently, defensive bryostatins; their documented northern distribution was consistent with traditional biogeographical paradigms of latitudinal variation in predation pressure. Upon further sampling of B. neritina populations, we found that both host types occur in wider distribution, with Type N colonies living south of Cape Hatteras, and Type S to the north. Distribution of the symbiont, however, was not restricted to Type S hosts. Genetic and microscopic evidence demonstrates the presence of the symbiont in some Type N colonies, and larvae from these colonies are endowed with defensive bryostatins and contain “Ca. Endobugula sertula”. Molecular analysis of the symbiont from Type N colonies suggests an evolutionarily recent acquisition, which is remarkable for a symbiont thought to be transmitted only vertically. Furthermore, most Type S colonies found at higher latitudes lack the symbiont, suggesting that this host-symbiont relationship is more flexible than previously thought. Our data suggest that the symbiont, but not the host, is restricted by biogeographical boundaries.     

Time-Varying Wing-Twist Improves Aerodynamic Efficiency of Forward Flight in Butterflies

Insect wings can undergo significant chordwise (camber) as well as spanwise (twist) deformation during flapping flight but the effect of these deformations is not well understood. The shape and size of butterfly wings leads to particularly large wing deformations, making them an ideal test case for investigation of these effects. Here we use computational models derived from experiments on free-flying butterflies to understand the effect of time-varying twist and camber on the aerodynamic performance of these insects. High-speed videogrammetry is used to capture the wing kinematics, including deformation, of a Painted Lady butterfly (Vanessa cardui) in untethered, forward flight. These experimental results are then analyzed computationally using a high-fidelity, three-dimensional, unsteady Navier-Stokes flow solver. For comparison to this case, a set of non-deforming, flat-plate wing (FPW) models of wing motion are synthesized and subjected to the same analysis along with a wing model that matches the time-varying wing-twist observed for the butterfly, but has no deformation in camber. The simulations show that the observed butterfly wing (OBW) outperforms all the flat-plate wings in terms of usable force production as well as the ratio of lift to power by at least 29% and 46%, respectively. This increase in efficiency of lift production is at least three-fold greater than reported for other insects. Interestingly, we also find that the twist-only-wing (TOW) model recovers much of the performance of the OBW, demonstrating that wing-twist, and not camber is key to forward flight in these insects. The implications of this on the design of flapping wing micro-aerial vehicles are discussed.     

Extrafloral Nectaries in Aspen (Populus tremuloides): Heritable Genetic Variation and Herbivore-induced Expression

Annals of Botany 100:     

Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

New viral strains can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. However, such mutants generally harbor amino acid changes that affect viral binding to a single class of carbohydrate receptors. We describe the rational design and synthesis of novel, chimeric adeno-associated virus (AAV) strains that exploit an orthogonal glycan receptor for transduction. A dual glycan-binding AAV strain was first engineered as proof of concept by grafting a galactose (Gal)-binding footprint from AAV serotype 9 onto the heparan sulfate-binding AAV serotype 2. The resulting chimera, AAV2G9, continues to bind heparin affinity columns but interchangeably exploits Gal and heparan sulfate receptors for infection, as evidenced by competitive inhibition assays with lectins, glycans, and parental AAV strains. Although remaining hepatotropic like AAV2, the AAV2G9 chimera mediates rapid onset and higher transgene expression in mice. Similarly, engraftment of the Gal footprint onto the laboratory-derived strain AAV2i8 yielded an enhanced AAV2i8G9 chimera. This new strain remains liver-detargeted like AAV2i8 while selectively transducing muscle tissues at high efficiency, comparable with AAV9. The AAV2i8G9 chimera is a promising vector candidate for targeted gene therapy of cardiac and musculoskeletal diseases. In addition to demonstrating the modularity of glycan receptor footprints on viral capsids, our approach provides design strategies to expand the AAV vector toolkit.     

Nectar Sugar Composition in Relation to Pollination Syndromes in Sinningieae (Gesneriaceae)

A putative correlation between nectar sugar composition andpollination syndrome was evaluated in the tribe Sinningieae(Neotropical Gesneriaceae). Sucrose, fructose and glucose werequantified in the nectar of 45 species using high performanceanion-exchange chromatography. Representative species of thehummingbird, bee, bat and moth pollination syndromes were sampledin relation to their numeric importance in the tribe. In hummingbirdand bee flowers, which represent 95% of the species in Sinningieae,nectar was sucrose-dominant (ratio [sucrose]/[hexose] > 1).Sugar ratios below one were only found in the nectar of threespecies with moth and bat syndromes. Sugar concentration averaged23.9 ± 10.6% (wt/total wt) in hummingbird flowers and28.7 ± 10.6% in bee flowers, whereas diluted nectar (7.1± 3.4%) was restricted to bat flowers. Similarities inthe nectar of hummingbird and bee flowers contrast with thepresence of specific morphological traits associated with thesetwo syndromes, indicating that plant-pollinator relationshipsrely on flower display rather than on nectar characteristics.By contrast, distinct nectar chemistry is correlated with thebat syndrome in which a particularly low sucrose productionis responsible for hexose dominance. Copyright 2001 Annals ofBotany Company Nectar sugar composition, pollination syndrome, Sinningia, Gesneriaceae, Brazil     

Phylogenetic and Experimental Tests of Interactions among Mutualistic Plant Defense Traits in Viburnum (Adoxaceae)

Abstract Plant traits that mediate mutualistic interactions are widespread, yet few studies have linked their macroevolutionary patterns with the ecological interactions they mediate. Here we merged phylogenetic and experimental approaches to investigate the evolution of two common mutualistic plant traits, extrafloral nectaries (EFNs) and leaf domatia. By using the flowering plant clade Viburnum, we tested whether macroevolutionary patterns support adaptive hypotheses and conducted field surveys and manipulative experiments to examine whether ecological interactions are concordant with evolutionary predictions. Phylogenetic reconstructions suggested that EFN-bearing species are monophyletic, whereas the evolution of domatia correlated with leaf production strategy (deciduous or evergreen) and climate. Domatia were also more common in the EFN clade, suggesting that the two traits may jointly mediate ecological interactions. This result was further investigated in a common-garden survey, where plants with domatia and EFNs on the leaf blade had more mutualistic mites than plants with other trait combinations, and in manipulative field experiments, where the traits additively increased mutualist abundance. Taken together, our results suggest that mutualistic traits in Viburnum are not ecologically independent, as they work in concert to attract and retain mutualists, and their long-term evolution may be influenced by complex interactions among multiple traits, mutualists, and geography.     

Pollination Ecology and Effect of Nectar Removal in Macleania bullata (Ericaceae)1

The floral syndrome of Macleania bullataYeo (Ericaceae) reflects its adaptation to hummingbird pollination. Its flowers, however, are subject to high levels of nectar robbing. I examined the floral visitor assemblage of M. bullata in a tropical montane wet forest in southwestern Colombia, focusing on the behavior of the visitors. I also tested for the presence of nocturnal pollination and the effects of nectar removal on new nectar production. The principal floral visitors were the nectar robbing hummingbirds Ocreatus underwoodii (19.1% of visits) and Chlorostilbon mellisugus (18.9%). Only two species of long–billed hummingbirds visited the flowers of M. bullata as “legitimate” pollinators: Coeligena torquata (14.7% of visits) and Doryfera ludoviciae (14.3%). The remaining visits constituted nectar robbing by bees, butterflies, and other species of hummingbirds. Nocturnal pollination took place, although fruit set levels were 2.4 times higher when only diurnal pollination was allowed as opposed to exclusively nocturnal pollination. Nectar robbers removed floral nectar without pollinating the flower. Treatments of experimental nectar removal were carried out to examine if flowers synthesize more nectar after nectar removal. Nectar removal increased the total volume of nectar produced by each flower without affecting sugar concentration. Thus, nectar robbing can impose a high cost to the plants by forcing them to replace lost nectar.     

Densovirus Is a Mutualistic Symbiont of a Global Crop Pest (Helicoverpa armigera) and Protects against a Baculovirus and Bt Biopesticide

Mutualistic associations between symbiotic bacteria and their hosts are common within insect systems. However, viruses are often considered as pathogens even though some have been reported to be beneficial to their hosts. Herein, we report a novel densovirus, Helicoverpa armigera densovirus-1 (HaDNV-1) that appears to be beneficial to its host. HaDNV-1 was found to be widespread in wild populations of H. armigera adults (>67% prevalence between 2008 and 2012). In wild larval populations, there was a clear negative interaction between HaDNV-1 and H. armigera nucleopolyhedrovirus (HaNPV), a baculovirus that is widely used as a biopesticide. Laboratory bioassays revealed that larvae hosting HaDNV-1 had significantly enhanced resistance to HaNPV (and lower viral loads), and that resistance to Bacillus thuringiensis (Bt) toxin was also higher at low doses. Laboratory assays indicated that the virus was mainly distributed in the fat body, and could be both horizontally- and vertically-transmitted, though the former occurred only at large challenge doses. Densovirus-positive individuals developed more quickly and had higher fecundity than uninfected insects. We found no evidence for a negative effect of HaDNV-1 infection on H. armigera fitness-related traits, strongly suggesting a mutualistic interaction between the cotton bollworm and its densovirus.