Floral Phytochemistry: Impact of Volatile Organic Compounds and Nectar Secondary Metabolites on Pollinator Behavior and Health

Plants produce a plethora of phytochemicals including sugars, amino acids (AAs), volatile organic compounds (VOCs) and secondary metabolites (SMs) with different ecological functions. To attract pollinators and defenders and ensure reproductive success, plants mainly rely on VOCs, while to reward insects, plants synthesize nectar rich in sugars and AAs. Furthermore, plant SMs can play various roles. Some components are able to interact with the nervous system of insects by binding to neuron receptor proteins and thus manipulate pollinator behavior. Others, like alkaloids and phenolics, protect from nectar robbers and enhance memory and foraging efficiency, or, as in the case of flavonoids, exhibit high antioxidant activities supporting pollinator well-being. This review discusses the impact of VOCs and nectar SMs on insect behavior and pollinator health.     

Food Reward Chemistry Explains a Novel Pollinator Shift and Vestigialization of Long Floral Spurs in an Orchid

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The Influence of Prior Learning Experience on Pollinator Choice: An Experiment Using Bumblebees on Two Wild Floral Types of Antirrhinum majus

Understanding how pollinator behavior may influence pollen transmission across floral types is a major challenge, as pollinator decision depends on a complex range of environmental cues and prior experience. Here we report an experiment using the plant Antirrhinum majus and the bumblebee Bombus terrestris to investigate how prior learning experience may affect pollinator preferences between floral types when these are presented together. We trained naive bumblebees to forage freely on flowering individuals of either A. majus pseudomajus (magenta flowers) or A. majus striatum (yellow flowers) in a flight cage. We then used a Y-maze device to expose trained bumblebees to a dual choice between the floral types. We tested the influence of training on their choice, depending on the type of plant signals available (visual signals, olfactory signals, or both). Bumblebees had no innate preference for either subspecies. Bumblebees trained on the yellow-flowered subspecies later preferred the yellow type, even when only visual or only olfactory signals were available, and their preference was not reinforced when both signal types were available. In contrast, bumblebees trained on the magenta-flowered subspecies showed no further preference between floral types and took slightly more time to make their choice. Since pollinator constancy has been observed in wild populations of A. majus with mixed floral types, we suggest that such constancy likely relies on short-term memory rather than acquired preference through long-term memory induced by prior learning.     

Expression Divergence and Functional Redundancy of Polygalacturonases in Floral Organ Abscission

The importance of cell separation in plant development cannot be overemphasized. The polygalacturonases (PGs) are the one of cell wall hydrolytic enzyme families that has been associated with various cell separation processes in plant development including seed germination, dehiscence, organ abscission, and fruit ripening. Both Arabidopsis and rice PG gene family have expanded in a lineage-specific fashion after the split more than 150 million years ago. Tandem duplications and large-scale duplications are the major contributors to the current PG family size in Arabidopsis. The spatial expression analysis of the 66 Arabidopsis PG family members have led us to conclude that different duplication mechanisms affect the expression divergence differently. This becomes more apparent when temporal examination of expression is conducted in five developmental stages of floral organ abscission in Arabidopsis. Nine distinct patterns of PGs are identified during floral organ abscission in Arabidopsis. Four PGs are specifically upregulated during abscission associated with cell separation process. Careful understanding of relationships among Arabidopsis PGs in a context of evolution together with expression analysis of these PGs will facilitate the functional study of PGs specifically in floral organ abscission in Arabidopsis.Key Words: cell separation, polygalacturonases, gene duplication, abscissionCell separation processes have been recognized for their involvement in plant development and more importantly, agricultural traits such as pollen dehiscence and the abscission of organs including leaves, floral organs, and fruits.^1–6 Among many cell wall hydrolytic enzymes, the PGs (polygalaturonases) have been shown to be associated with a wide range of these plant developmental programs such as seed germination, organ abscission, pod and anther dehiscence, pollen grain maturation, fruit softening and decay, xylem cell formation, and pollen tube growth.^4,7–9 Although the roles of the PG members in the various developmental programs have been hypothesized, many scientists still face challenges in developing a comprehensive understanding of the biological functions of the PG family, due to both size and possible redundancy.^10,11It has been previously reported that PGs from both tomato and Arabidopsis located in tandem clusters were derived from tandem duplications.^10,12 In addition, it has been shown by several groups that the Arabidopsis genome contains large blocks of related regions derived from whole genome duplication events.^13–15We have recently conducted a comparative analysis of PGs from Arabidopsis and rice in attempts to address following questions: (1) the patterns and extent of expansion of PG gene family in Arabidopsis and rice, (2) the mechanisms that contribute to the expansion of this gene family, (3) the degree of both spatial and temporal expression divergence amongst Arabidopsis PGs, and (4) possible mechanisms of duplicates retention and the biological roles in floral organ abscission.¹⁶In this study, we have shown that with the identification of the nodes that lead to each Arabidopsis-specific and rice-specific subfamily, there are at least 21 immediate ancestors before the split between these two organisms.¹⁶ Subsequent expansion events have been followed in a lineage specific mode in both organisms. We found that in Arabidopsis the PG family is the product of both tandem duplications and large-scale duplications, similar to other gene families such as the NBS-LRR¹⁷ and the RLK/Pelle gene family.¹⁸ As a matter of fact, more than one third of the Arabidopsis PGs (24 out of 66) were found in tandem clusters. In addition, using both AGI (Arabidopsis Genome initiative) and BHW (Blanc, Hokamp, Wolfe) blocks almost 90% (59 out of 66) of the PGs were included within large-scale duplication events.^13,15,16Interestingly, the correlation between the synonymous substitution rate (Ks) and the expression profile for the related PGs in the tandem clusters was not significant. However, the related PGs in the duplicated blocks from large-scale duplication events tend to have a similar expression pattern within the five major tissues (flowers, siliques, stems, rosette and cauline leaves, and roots). Thus, indicating tandem-duplicated PGs have higher levels of expression divergence compared with PGs found in the large-scale duplication blocks.Further examination of PG expression during specific aspects of plant growth such as the five developmental stages of floral organ abscission has led us to conclude that expression divergence between all of the PGs that showed no difference at the tissue level most likely have differences. Thus, by looking more closely at specific tissue types and time of development, the divergence in expression patterns can be revealed. This is also supported by the hypothesis that duplication mechanisms may contribute to divergence of expression differently. In addition, by identifying nine distinct patterns of PG expression during the five developmental stages of floral organ abscission, we provided candidate PGs important for studies of abscission. Considering the proposed roles of PGs in pectin modification and/or breakdown, four of the PGs (At2g41850, At2g43880, At2g43890, and At3g07970) that showed upregulated expression during floral organ abscission associated with cell separation were identified as “best candidates” for understanding floral organ abscission (Fig. 1A).Open in a separate windowFigure 1Floral organ abscission specific PGs and their T-DNA insertion lines. (A) Expression of four PGs that showed specific upregulation during floral organ abscission is demonstrated. X-axis represents the developmental stages of floral organ abscission. Pos. 1/2 and Pos. 4/5 represent pre-abscission, Pos. 7/8 represents during abscission, and Pos. 10/11 and Pos. 13/14 represent post-abscission. Y-axis represents the relative level of expression of these four PGs and the names of each gene are shown in the z-axis. Four arrows indicate the positions that are associated with cell separation and upregulation of the PGs during abscission. (B) Gene structures of abscission specific PGs and the positions of the T-DNA insertions in each mutant. Each arrow indicates the position of the T-DNA insertion for the mutant. At2g41850 (SALK_035098, red arrow head) is further examined for petal abscission zones using SEM as shown in (C). (C) Schematic diagram of flower positions in wild type in Arabidopsis and SEM of petal abscission zones from SALK_035098 and wild type. Flower position 1 denotes the flower position where white petals protrude right after anthesis and subsequent positions correspond to older stages of flowers. The days after anthesis are demonstrated with flower positions in the right. Note that at flower position 7/8 floral organs are abscised. When comparing during-and post-abscission (Pos. 7/8, Pos. 10/11 and Pos. 13/14) between the mutant and wild type, difference of a delay in floral organ abscission is not observed.In order to better understand the functions of these PGs in floral organ abscission, we also isolated homozygous T-DNA insertion lines (http://signal.salk.edu/cgi-bin/tdnaexpress) for three of these PGs (At2g41850, At2g43890, and At2g07970) associated with cell separation during abscission. We examined these mutants for a delay in floral organ abscission (Fig. 1B and C); however, nearly all of the single insertion lines examined did not display any notable phenotype regarding floral organ abscission even though in the T-DNA disruption line of At2g41850 showed slight delay positions around 7/8 (Fig. 1 and ​and1C).1C). One possible explanation is that even though each of these PGs is specifically upregulated right before or during the cell separation process of abscission, there is still redundancy in their functions. One of the approaches to resolve this problem may be the generation of multiple mutants. For example, At2g41850 and At3g07970 are derived from the same ancestor. Thus, examination of double mutants between these two diverged PGs may provide a clue as to the abscission function. In addition, At2g43880, At2g43890 and At2g41850 are also derived from the large-scale duplication events while they share different immediate ancestors. Making double mutants between the two PGs At2g43890 and At2g41850 may provide a clue as to PG function during development. Ultimately, generating triple mutants among At2g41850, At2g43890, and At3g07970 may also provide insights into the possible functions of these PGs in floral organ abscission. Studying functions of a large gene family has been always a challenge due to the size and the redundancy of the family, but these recent findings will facilitate this task.From our study, we addressed the questions as to how PG gene family has expanded and/or duplicated in the evolution, how the duplicates were retained with the possible biological functions, and how functional studies of PGs in floral organ abscission can be conducted in the future.     

Oxidant Trade-Offs in Immunity: An Experimental Test in a Lizard

Immune system functioning and maintenance entails costs which may limit investment into other processes such as reproduction. Yet, the proximate mechanisms and ‘currencies’ mediating the costs of immune responses remain elusive. In vertebrates, up-regulation of the innate immune system is associated with rapid phagocytic production of pro-oxidant molecules (so-called ‘oxidative burst’ responses). Oxidative burst responses are intended to eliminate pathogens but may also constitute an immunopathological risk as they may induce oxidative damage to self cells. To minimize the risk of infection and, at the same time, damage to self, oxidative burst activity must be carefully balanced. The current levels of pro- and antioxidants (i.e. the individual oxidative state) is likely to be a critical factor affecting this balance, but this has not yet been evaluated. Here, we perform an experiment on wild-caught painted dragon lizards (Ctenophorus pictus) to examine how the strength of immune-stimulated oxidative burst responses of phagocytes in whole blood relates to individual oxidative status under control conditions and during an in vivo immune challenge with Escherichia coli lipopolysaccharide (LPS). Under control conditions, oxidative burst responses were not predicted by the oxidative status of the lizards. LPS-injected individuals showed a strong increase in pro-oxidant levels and a strong decrease in antioxidant levels compared to control individuals demonstrating a shift in the pro-/antioxidant balance. Oxidative burst responses in LPS-injected lizards were positively related to post-challenge extracellular pro-oxidants (reflecting the level of cell activation) and negatively related to pre-challenge levels of mitochondrial superoxide (suggesting an immunoregulatory effect of this pro-oxidant). LPS-challenged males had higher oxidative burst responses than females, and in females oxidative burst responses seemed to depend more strongly on antioxidant status than in males. Our results confirm the idea that oxidative state may constrain the activity of the innate immune system. These constraints may have important consequences for the way selection acts on pro-oxidant generating processes.     

An Experimental Test of Competition among Mice,Chipmunks, and Squirrels in Deciduous Forest Fragments

Mixed hardwood forests of the northeast United States support a guild of granivorous/omnivorous rodents including gray squirrels (Sciurus carolinensis), eastern chipmunks (Tamias striatus), and white-footed mice (Peromyscus leucopus). These species coincide geographically, co-occur locally, and consume similar food resources. Despite their idiosyncratic responses to landscape and patch variables, patch occupancy models suggest that competition may influence their respective distributions and abundances, and accordingly their influence on the rest of the forest community. Experimental studies, however, are wanting. We present the result of a large-scale experiment in which we removed white-footed mice or gray squirrels from small, isolated forest fragments in Dutchess County, New York, and added these mammals to other fragments in order to alter the abundance of these two species. We then used mark–recapture analyses to quantify the population-level and individual-level effects on resident mice, squirrels, and chipmunks. Overall, we found little evidence of competition. There were essentially no within-season numerical responses to changes in the abundance of putative competitors. Moreover, while individual-level responses (apparent survival and capture probability) did vary with competitor densities in some models, these effects were often better explained by site-specific parameters and were restricted to few of the 19 sites we studied. With only weak or nonexistent competition among these three common rodent species, we expect their patterns of habitat occupancy and population dynamics to be largely independent of one another.     

Diversity and Distribution of Floral Resources Influence the Restoration of Plant–Pollinator Networks on a Reclaimed Strip Mine

Plant–pollinator mutualisms are one of the several functional relationships that must be reinstated to ensure the long‐term success of habitat restoration projects. These mutualisms are unlikely to reinstate themselves until all of the resource requirements of pollinators have been met. By meeting these requirements, projects can improve their long‐term success. We hypothesized that pollinator assemblage and structure and stability of plant–pollinator networks depend both on aspects of the surrounding landscape and of the restoration effort itself. We predicted that pollinator species diversity and network stability would be negatively associated with distance from remnant habitat, but that local floral diversity might rescue pollinator diversity and network stability in locations distant from the remnant. We created plots of native prairie on a reclaimed strip mine in central Ohio, U.S.A. that ranged in floral diversity and isolation from the remnant habitat. We found that the pollinator diversity declined with distance from the remnant habitat. Furthermore, reduced pollinator diversity in low floral diversity plots far from the remnant habitat was associated with loss of network stability. High floral diversity, however, compensated for losses in pollinator diversity in plots far from the remnant habitat through the attraction of generalist pollinators. Generalist pollinators increased network connectance and plant‐niche overlap. As a result, network robustness of high floral diversity plots was independent of isolation. We conclude that the aspects of the restoration effort itself, such as floral community composition, can be successfully tailored to incorporate the restoration of pollinators and improve success given a particular landscape context.     

Pollinator functional response and plant population dynamics: Pollinators as a limiting resource

Summary During recent years, much work has focused on which factors limit the reproductive success in plants. Several studies show a strong influence of either resource limitation, pollen limitation or a combined effect of both. The theoretical arguments for resource limitation are abundant, but there has been very little work done concerning the effect of pollinator availability. In this paper we construct a model to study how the reproductive success in plants is influenced by the foraging behaviour of the pollinators. The pollinator population is assumed to have a constant population density. A functional response function for the pollinators is derived. It is similar to a Holling type II functional response. It is shown that, since the pollinators are regulated by factors not included in the model and their capability to pollinate is limited by the functional response, this is sufficient for regulating the plant population. There also exists a threshold condition for the persistence of the plant population that depended on the search rate of the pollinators and the demographic parameters of the plant population. If this threshold condition is not satisfied the plant population cannot persist and will become extinct. If the condition is satisfied the plant population grows until it is limited at the equilibrium mentioned above.     

Pollinator directionality as a response to nectar gradient: promoting outcrossing while avoiding geitonogamy

Plants with multiple flowers could be prone to autonomous self‐pollination and insect‐mediated geitonogamy, but physiological and ecological features have evolved preventing costs related to autogamy. We studied the rare perennial herb Dictamnus albus as a model plant, with the aim of describing the plant–pollinator system from both plant and pollinator perspectives and analysing features that promote outcrossing in an entomophilous species. The breeding system and reproductive success of D. albus were investigated in experimental and natural conditions, showing that it is potentially self‐compatible, but only intra‐inflorescence insect‐mediated selfing is possible. Nectar analysis showed gender‐biased production towards the female phase, which follows the male phase, and during flowering, full blooming is found in flowers at the bottom of the raceme. Among a wide spectrum of insect visitors, three genera (Bombus, Apis, Megachile) were found to be principal pollinators. A study of insect behaviour showed a tendency towards bottom‐to‐top flights for the most important pollinators Bombus spp. and Apis mellifera: upward movements on the racemes could be explained by foraging behaviour, from more to less rewarding flowers. In accordance with the ‘declining reward hypothesis’, bumblebees and honeybees leave the plant when gain of reward is low, after which few flowers are visited, reducing the chance of self‐pollen transfer among flowers. Intra‐flower self‐pollination is prevented in D. albus by protandry and herkogamy, while the nectar‐induced sequential pattern of pollinator visits avoids geitonogamy and tends to maximise pollen export, promoting outcrossing. All these features for preventing selfing benefit plant fitness and population genetic structure.     

Pollinator rarity as a threat to a plant with a specialized pollination system

An increasing diversity of highly specialized pollination systems are being discovered, many of which are likely to be vulnerable to anthropogenic landscape modification. Here, we investigate if a specialized pollination system limits the persistence of Caladenia huegelii (Orchidaceae), an endangered species pollinated by sexual deception of thynnine wasps. Once locally common in part of its geographical range, C. huegelii is now largely restricted to small habitat remnants in urban areas. Pollinator surveys coupled with DNA barcoding detected a single pollinator taxon, a small form of Macrothynnus insignis. Phylogenetic analysis revealed that small M. insignis from within the range of C. huegelii are strongly divergent from other wasp populations, suggesting that some reproductive isolation may exist. Although common in intact landscapes outside the range of C. huegelli, small M. insignis individuals were recorded at only 4% of sites in suitable C. huegelii habitat. Accordingly, reproductive success in C. huegelii was low compared with related Caladenia spp., with 33–60% of populations failing to set fruit in any given year. As such, populations are likely to now persist primarily through individual plant longevity rather than reproduction. Due to the low reproductive success of C. huegelii, ongoing human intervention will almost certainly be needed to sustain the species. Future research will need to focus on optimizing hand pollination to maintain reproduction and high seed fitness. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 511–525.     

Change of Floral Orientation within an Inflorescence Affects Pollinator Behavior and Pollination Efficiency in a Bee-Pollinated Plant,Corydalis sheareri

Vertical raceme or spike inflorescences that are bee-pollinated tend to present their flowers horizontally. Horizontal presentation of flowers is hypothesized to enhance pollinator recognition and pollination precision, and it may also ensure greater consistency of pollinator movement on inflorescences. We tested the hypotheses using bee-pollinated Corydalis sheareri which has erect inflorescences consisting of flowers with horizontal orientation. We altered the orientation of individual flowers and prepared three types of inflorescences: (i) unmanipulated inflorescences with horizontal-facing flowers, (ii) inflorescences with flowers turned upward, and (iii) inflorescences with flowers turned downward. We compared number of inflorescences approached and visited, number of successive probes within an inflorescence, the direction percentage of vertical movement on inflorescences, efficiency of pollen removal and seed production per inflorescence. Deviation from horizontal orientation decreased both approaches and visits by leafcutter bees and bumble bees to inflorescences. Changes in floral orientation increased the proportion of downward movements by leafcutter bees and decreased the consistency of pollinator movement on inflorescences. In addition, pollen removal per visit and seed production per inflorescence also declined with changes of floral orientation. In conclusion, floral orientation seems more or less optimal as regards bee behavior and pollen transfer for Corydalis sheareri. A horizontal orientation may be under selection of pollinators and co-adapt with other aspects of the inflorescence and floral traits.     

Segment-Specific Adhesion as a Driver of Convergent Extension

Convergent extension, the simultaneous extension and narrowing of tissues, is a crucial event in the formation of the main body axis during embryonic development. It involves processes on multiple scales: the sub-cellular, cellular and tissue level, which interact via explicit or intrinsic feedback mechanisms. Computational modelling studies play an important role in unravelling the multiscale feedbacks underlying convergent extension. Convergent extension usually operates in tissue which has been patterned or is currently being patterned into distinct domains of gene expression. How such tissue patterns are maintained during the large scale tissue movements of convergent extension has thus far not been investigated. Intriguingly, experimental data indicate that in certain cases these tissue patterns may drive convergent extension rather than requiring safeguarding against convergent extension. Here we use a 2D Cellular Potts Model (CPM) of a tissue prepatterned into segments, to show that convergent extension tends to disrupt this pre-existing segmental pattern. However, when cells preferentially adhere to cells of the same segment type, segment integrity is maintained without any reduction in tissue extension. Strikingly, we demonstrate that this segment-specific adhesion is by itself sufficient to drive convergent extension. Convergent extension is enhanced when we endow our in silico cells with persistence of motion, which in vivo would naturally follow from cytoskeletal dynamics. Finally, we extend our model to confirm the generality of our results. We demonstrate a similar effect of differential adhesion on convergent extension in tissues that can only extend in a single direction (as often occurs due to the inertia of the head region of the embryo), and in tissues prepatterned into a sequence of domains resulting in two opposing adhesive gradients, rather than alternating segments.     

Quantifying Poverty as a Driver of Ebola Transmission

Background

Poverty has been implicated as a challenge in the control of the current Ebola outbreak in West Africa. Although disparities between affected countries have been appreciated, disparities within West African countries have not been investigated as drivers of Ebola transmission. To quantify the role that poverty plays in the transmission of Ebola, we analyzed heterogeneity of Ebola incidence and transmission factors among over 300 communities, categorized by socioeconomic status (SES), within Montserrado County, Liberia.

Methodology/Principal Findings

We evaluated 4,437 Ebola cases reported between February 28, 2014 and December 1, 2014 for Montserrado County to determine SES-stratified temporal trends and drivers of Ebola transmission. A dataset including dates of symptom onset, hospitalization, and death, and specified community of residence was used to stratify cases into high, middle and low SES. Additionally, information about 9,129 contacts was provided for a subset of 1,585 traced individuals. To evaluate transmission within and across socioeconomic subpopulations, as well as over the trajectory of the outbreak, we analyzed these data with a time-dependent stochastic model. Cases in the most impoverished communities reported three more contacts on average than cases in high SES communities (p<0.001). Our transmission model shows that infected individuals from middle and low SES communities were associated with 1.5 (95% CI: 1.4–1.6) and 3.5 (95% CI: 3.1–3.9) times as many secondary cases as those from high SES communities, respectively. Furthermore, most of the spread of Ebola across Montserrado County originated from areas of lower SES.

Conclusions/Significance

Individuals from areas of poverty were associated with high rates of transmission and spread of Ebola to other regions. Thus, Ebola could most effectively be prevented or contained if disease interventions were targeted to areas of extreme poverty and funding was dedicated to development projects that meet basic needs.     

An Experimental Study of Competition Between Seed-eating Desert Rodents and Ants

Reciprocal increases in rodent and ant densities on 0.1 ha plotsfrom which the other taxon had been excluded demonstrate thatthese distantly related desert granivores compete for seeds.Relative to unmanipulated control plots, numbers of ant coloniesincreased 71% on plots where rodents were excluded; rodentsincreased 20% in numbers of individuals and 29% in biomass inthe absence of ants. Comparisons of seed levels in the soiland of annual plant densities on experimental and control plotsprovide evidence that the rodent and ant populations are limitedby and compete for food. Greater numbers of seeds and annualsoccurred on plots where rodents and ants had been excluded thanon plots where both taxa were present. Particular species ofannuals were reduced in density by foraging of rodents. Antsincreased species diversity by differentially harvesting seedsof the most common species. Results of these and other recentstudies suggest that competition among distantly related organismsplays a major role in the organization of ecological communities.     

Pollinator availability as a determinant of flowering time in ocotillo (Fouquieria splendens)

Summary Ocotillo, a perennial shrub of Sonoran and Chihuahuan Deserts, produces its red tubular flowers in spring. This timing coincides with northward migration of hummingbirds through desert areas. Observations of visitors, pollen collections, and seed set reductions following exclusion of different flower visitors indicate that both hummingbirds and solitary bees pollinate ocotillo in southern Arizona. Seed sets of flowers on marked plants varied considerably within and between years, and this variation was related to the temporal match between flowering and hummingbird migration. This suggests that selection acts on plants to synchronize flowering with periods of pollinator abundance.