Abstract: In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca2+ binding EF-hand motifs, possessed Ca2+-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca2+-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca2+-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca2+. Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca2+-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

Abstract: Bumblebees and other pollinators provide a vital ecosystem service for the agricultural sector. Recent studies however have suggested that exposure to systemic neonicotinoid insecticides in flowering crops has sub-lethal effects on the bumblebee workforce, and hence in reducing queen production. The mechanism behind reduced nest performance, however, remains unclear. Here we use Radio Frequency Identification (RFID) technology to test whether exposure to a low, field realistic dose (0.7 ppb in sugar water and 6 ppb in pollen) of the neonicotinoid imidacloprid, reduces worker foraging efficiency. Whilst the nectar foraging efficiency of bees treated with imidacloprid was not significantly different than that of control bees, treated bees brought back pollen less often than control bees (40 % of trips vs 63 % trips, respectively) and, where pollen was collected, treated bees brought back 31 % less pollen per hour than controls. This study demonstrates that field-realistic doses of these pesticides substantially impacts on foraging ability of bumblebee workers when collecting pollen, and we suggest that this provides a causal mechanism behind reduced queen production in imidacloprid exposed colonies.

Abstract: Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning.

Abstract: Honey bee hives are filled with stored pollen, honey, plant resins and wax, all antimicrobial to differing degrees. Stored pollen is the nutritionally rich currency used for colony growth and consists of 40–50% simple sugars. Many studies speculate that prior to consumption by bees, stored pollen undergoes long-term nutrient conversion, becoming more nutritious ‘bee bread’ as microbes predigest the pollen. We quantified both structural and functional aspects associated with this hypothesis using behavioural assays, bacterial plate counts, microscopy and 454 amplicon sequencing of the 16S rRNA gene from both newly collected and hive-stored pollen. We found that bees preferentially consume fresh pollen stored for 96 h. The estimated microbe to pollen grain surface area ratio was 1:1 000 000 indicating a negligible effect of microbial metabolism on hive-stored pollen. Consistent with these findings, hive-stored pollen grains did not appear compromised according to microscopy. Based on year round 454 amplicon sequencing, bacterial communities of newly collected and hive-stored pollen did not differ, indicating the lack of an emergent microbial community co-evolved to digest stored pollen. In accord with previous culturing and 16S cloning, acid resistant and osmotolerant bacteria like Lactobacillus kunkeei were found in greatest abundance in stored pollen, consistent with the harsh character of this microenvironment. We conclude that stored pollen is not evolved for microbially mediated nutrient conversion, but is a preservative environment due primarily to added honey, nectar, bee secretions and properties of pollen itself.

Abstract: Summary Reactive oxygen species (ROS) produced by NAD(P)H oxidases play a central role in plant stress responses and development. To better understand the function of NAD(P)H oxidases in plant development, we characterized the Arabidopsis thaliana NAD(P)H oxidases RBOHH and RBOHJ. Both proteins were specifically expressed in pollen and dynamically targeted to distinct and overlapping plasma membrane domains at the pollen tube tip. Functional loss of RBOHH and RBOHJ in homozygous double mutants resulted in reduced fertility. Analyses of pollen tube growth revealed remarkable differences in growth dynamics between Col–0 and rbohh–1 rbohj–2 pollen tubes. Growth rate oscillations of rbohh–1 rbohj–2 pollen tubes showed strong fluctuations in amplitude and frequency, ultimately leading to pollen tube collapse. Prior to disintegration, rbohh–1 rbohj–2 pollen tubes exhibit high-frequency growth oscillations, with significantly elevated growth rates, suggesting that an increase in the rate of cell-wall exocytosis precedes pollen tube collapse. Time-lapse imaging of exocytic dynamics revealed that NAD(P)H oxidases slow down pollen tube growth to coordinate the rate of cell expansion with the rate of exocytosis, thereby dampening the amplitude of intrinsic growth oscillations. Using the Ca2+ reporter Yellow Cameleon 3.6, we demonstrate that high-amplitude growth rate oscillations in rbohh–1 rbohj–2 pollen tubes are correlated with growth-dependent Ca2+ bursts. Electrophysiological experiments involving double mutant pollen tubes and pharmacological treatments also showed that ROS influence K+ homeostasis. Our results indicate that, by limiting pollen tube growth, ROS produced by NAD(P)H oxidases modulate the amplitude and frequency of pollen tube growth rate oscillations.

Abstract: Anther cuticle and pollen exine act as protective envelopes for the male gametophyte or pollen grain, but the mechanism underlying the synthesis of these lipidic polymers remains unclear. Previously, a tapetum-expressed CYP703A3, a putative cytochrome P450 fatty acid hydroxylase, was shown to be essential for male fertility in rice (Oryza sativa L.). However, the biochemical and biological roles of CYP703A3 has not been characterized. Here, we observed that cyp703a3-2 caused by one base insertion in CYP703A3 displays defective pollen exine and anther epicuticular layer, which differs from Arabidopsis cyp703a2 in which only defective pollen exine occurs. Consistently, chemical composition assay showed that levels of cutin monomers and wax components were dramatically reduced in cyp703a3-2 anthers. Unlike the wide range of substrates of Arabidopsis CYP703A2, CYP703A3 functions as an in-chain hydroxylase only for a specific substrate, lauric acid, preferably generating 7-hydroxylated lauric acid. Moreover, chromatin immunoprecipitation and expression analyses revealed that the expression of CYP703A3 is directly regulated by Tapetum Degeneration Retardation, a known regulator of tapetum PCD and pollen exine formation. Collectively, our results suggest that CYP703A3 represents a conserved and diversified biochemical pathway for in-chain hydroxylation of lauric acid required for the development of male organ in higher plants.

Abstract: Summary 1. Generalist herbivorous insects, which feed on plant tissue that is nutritionally heterogeneous or varies in its content of secondary metabolites, often benefit from dietary mixing through more balanced nutrient intake or reduced exposure to harmful secondary metabolites. Pollen is similarly heterogeneous as other plant tissue in its content of primary and secondary metabolites, suggesting that providing their offspring with mixed pollen diets might be a promising strategy for pollen generalist bees to complement nutrient imbalances or to mitigate harmful secondary metabolites of unfavourable pollen. 2. In the present study, we compared larval performance of the pollen generalist solitary bee species Osmia cornuta (Megachilidae) on five experimental pollen diets that consisted of different proportions of unfavourable pollen diet of Ranunculus acris (Ranunculaceae) and favourable pollen diet of Sinapis arvensis (Brassicaceae). In addition, we microscopically analysed the pollen contained in the scopal brushes of field-collected females of O. cornuta and three closely related species to elucidate to what degree these pollen generalist bees mix pollen of different hosts in their brood cells. 3. In striking contrast to a pure Ranunculus pollen diet, which had a lethal effect on most developing larvae of O. cornuta, larval survival, larval development time and adult body mass of both males and females remained nearly unaffected by the admixture of up to 50% of Ranunculus pollen diet to the larval food. 4. Between 42% and 66% of all female scopal pollen loads analysed contained mixtures of pollen from two to six plant families, indicating that pollen mixing is a common behaviour in O. cornuta and the three related bee species. 5. The present study provides the first evidence that the larvae of pollen generalist bees can benefit from the nutrient content of unfavourable pollen without being negatively affected by its unfavourable chemical properties if such pollen is mixed with favourable pollen. We conclude that the widespread pollen mixing by females of pollen generalist bees should also be considered as a possible strategy to exploit flowers with unfavourable pollen and to optimize larval food quality.

Abstract: Research was done during 2012 to evaluate the potential exposure of pollinators to neonicotinoid insecticides used as seed treatments on corn, cotton, and soybean. Samples were collected from small plot evaluations of seed treatments and from commercial fields in agricultural production areas in Arkansas, Mississippi, and Tennessee. In total, 560 samples were analyzed for concentrations of clothianidin, imidacloprid, thiamethoxam, and their metabolites. These included pollen from corn and cotton, nectar from cotton, flowers from soybean, honey bees, Apis mellifera L., and pollen carried by foragers returning to hives, preplanting and in-season soil samples, and wild flowers adjacent to recently planted fields. Neonicotinoid insecticides were detected at a level of 1 ng/g or above in 23% of wild flower samples around recently planted fields, with an average detection level of about 10 ng/g. We detected neonicotinoid insecticides in the soil of production fields prior to planting at an average concentration of about 10 ng/g, and over 80% of the samples having some insecticide present. Only 5% of foraging honey bees tested positive for the presence of neonicotinoid insecticides, and there was only one trace detection (< 1 ng/g) in pollen being carried by those bees. Soybean flowers, cotton pollen, and cotton nectar contained little or no neonicotinoids resulting from insecticide seed treatments. Average levels of neonicotinoid insecticides in corn pollen ranged from less than 1 to 6 ng/g. The highest neonicotinoid concentrations were found in soil collected during early flowering from insecticide seed treatment trials. However, these levels were generally not well correlated with neonicotinoid concentrations in flowers, pollen, or nectar. Concentrations in flowering structures were well below defined levels of concern thought to cause acute mortality in honey bees. The potential implications of our findings are discussed.

Abstract: One expected effect of climate change on human health is increasing allergic and asthmatic symptoms through changes in pollen biology. Allergic diseases have a large impact on human health globally, with 10–30% of the population affected by allergic rhinitis and more than 300 million affected by asthma. Pollen from grass species, which are highly allergenic and occur worldwide, elicits allergic responses in 20% of the general population and 40% of atopic individuals. Here we examine the effects of elevated levels of two greenhouse gases, carbon dioxide (CO2), a growth and reproductive stimulator of plants, and ozone (O3), a repressor, on pollen and allergen production in Timothy grass (Phleum pratense L.). We conducted a fully factorial experiment in which plants were grown at ambient and/or elevated levels of O3 and CO2, to simulate present and projected levels of both gases and their potential interactive effects. We captured and counted pollen from flowers in each treatment and assayed for concentrations of the allergen protein, Phl p 5. We found that elevated levels of CO2 increased the amount of grass pollen produced by ,50% per flower, regardless of O3 levels. Elevated O3 significantly reduced the Phl p 5 content of the pollen but the net effect of rising pollen numbers with elevated CO2 indicate increased allergen exposure under elevated levels of both greenhouse gases. Using quantitative estimates of increased pollen production and number of flowering plants per treatment, we estimated that airborne grass pollen concentrations will increase in the future up to ,200%. Due to the widespread existence of grasses and the particular importance of P. pratense in eliciting allergic responses, our findings provide evidence for significant impacts on human health worldwide as a result of future climate change.

Abstract: Summary 1. Production of many flowering crops often benefits from elevated pollinator diversity and abundance. Nevertheless, the opposite relationship may arise if bees impair fruit or seed production and/or quality by damaging flowers during visitation, despite transferring pollen. 2. We assessed pollination and drupelet set (i.e. the number of drupelets per fruit) in 16 raspberry Rubus idaeus fields along a gradient of bee abundance in north-west Patagonia, Argentina. Using pollen supplementation, we also tested whether drupelet set was pollen limited in a subset of six fields. 3. Managed Apis mellifera and the invasive bumblebee Bombus terrestris accounted for 50% and 45% of all bee visits, respectively, to raspberry flowers. Pollen loads on stigmas increased with visit frequency of all bees combined and particularly with visitation by A. mellifera, but not by B. terrestris. Drupelet set was not pollen limited along the gradient of bee abundance. 4. Instead, drupelet set decreased with the proportion of damaged styles, which varied more strongly with the frequency of visits by B. terrestris than by A. mellifera. In fields with the highest bee frequency of visits ( � 300 visits flower � 1 day � 1 ), � 80% of styles were damaged in flowers and these developed into fruits with � 30% fewer drupelets compared to flowers in fields with the lowest bee visitation rates ( � 4 visits flower � 1 day � 1 ). 5. Synthesis and applications. Extreme bee visitation, particularly by Bombus terrestris, damaged the styles of raspberry flowers, precluding ovule fertilization by deposited pollen and limiting crop production by reducing drupelet set. Only a few bee visits are required to maximize fruit production in raspberry plants, therefore, pollinator management in north-west Patagonia should focus principally on reducing the abundance of the invasive bumblebee B. terrestris and secondarily controlling the number of honeybee hives in nearby cultivated fields. Although mainstream pollinator management relies on the assumption that more visits enhance fruit set, high bee visitation rates can be detrimental for fruit development and, consequently, for crop yield.

Abstract: Male fertility in flowering plants depends on proper cellular differentiation in anthers. Meiosis and tapetum development are particularly important processes in pollen production. In this study, we showed that the tomato male sterile (ms10(35)) mutant of cultivated tomato (Solanum lycopersicum) exhibited dysfunctional meiosis and an abnormal tapetum during anther development, resulting in no pollen production. We demonstrated that Ms10(35) encodes a basic helix-loop-helix transcription factor that is specifically expressed in meiocyte and tapetal tissue from pre-meiotic to tetrad stages. Transgenic expression of the Ms10(35) gene from its native promoter complemented the male sterility of the ms10(35) mutant. In addition, RNA-sequencing-based transcriptome analysis revealed that Ms10(35) regulates 246 genes involved in anther development processes such as meiosis, tapetum development, cell-wall degradation, pollen wall formation, transport, and lipid metabolism. Our results indicate that Ms10(35) plays key roles in regulating both meiosis and programmed cell death of the tapetum during microsporogenesis.

Abstract: Pollen tubes are among the fastest tip-growing plant cells and represent an excellent experimental system for studying the dynamics and spatiotemporal control of polarized cell growth. However, investigating pollen tube tip growth in the model plant Arabidopsis remains difficult because in vitro pollen germination and pollen tube growth rates are highly variable and largely different from those observed in pistils, most likely due to growth-promoting properties of the female reproductive tract. We found that in vitro grown Arabidopsis pollen respond to brassinosteroid (BR) in a dose-dependent manner. Pollen germination and pollen tube growth increased nine- and fivefold, respectively, when media were supplemented with 10 µM epibrassinolide (epiBL), resulting in growth kinetics more similar to growth in vivo. Expression analyses show that the promoter of one of the key enzymes in BR biosynthesis, CYP90A1/CPD, is highly active in the cells of the reproductive tract that form the pathway for pollen tubes from the stigma to the ovules. Pollen tubes grew significantly shorter through the reproductive tract of a cyp90a1 mutant compared to the wild type, or to a BR perception mutant. Our results show that epiBL promotes pollen germination and tube growth in vitro and suggest that the cells of the reproductive tract provide BR compounds to stimulate pollen tube growth.

Abstract: Total and individual carotenoids, fatty acid composition of total lipids, and main lipid classes of 16 fresh bee-collected pollen samples from Romania were determined by high-performance liquid chromatography with photodiode array detection and capillary gas chromatography with mass detection. Analyzed samples were found rich in lutein, whereas β-criptoxanthin and β-carotene were present in a wide range of amounts correlated with predominant botanical origin of the samples. High amounts of lutein were correlated with the presence of Callendula officinalis, Taraxacum officinale and Anthylis sp. The highest amount of total lipids was found in samples where pollen from Brassica sp. was predominant. Lipid classes were dominated by polyunsaturated fatty acids. Saturated fatty acids were determined in variable amounts. Lipid and carotenoid contents present great variability, explained by the various botanical species present in the samples.

Abstract: Seventeen phenolic compounds related to the botanical origins of 13 Anzer pollens from Turkey have been analyzed by RP-HPLC. LOD values of standards were found to be between 0.0192 and 0.1313 mg/L with good linearity (r > 0.9977). The mean content of identified total phenolics ranges from 0.5 mg/100 g pollen to 2.6 mg/100 g pollen. While a common profile of phenolic compounds comprising p-OH benzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, rutin, trans-cinnamic acid, and cis,trans-abscisic acid was detected in all Anzer pollen, catechin was not determined in any of the pollen samples. Furthermore, antioxidant properties of pollens were determined using total phenolic content, FRAP, CUPRAC and DPPH● (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity tests. The antioxidant activities showed a marked correlation with total phenolics. Practical Applications Bee-collected pollen is one of the richest and the purest natural food supplements packed by honeybees into granules and subsequently harvested from hives by humans. Bee pollen is composed of nutritionally essential substances such as carbohydrates, proteins, amino acids, lipids, vitamins, mineral substances and trace elements. It also contains considerable amounts of polyphenolic substances, mainly flavonoids, which are regarded as potent antioxidants. Flavonoids can be used to evaluate botanical origin and to set up quality standards for the assessment of nutritional and physiological properties of bee pollen. This paper provides an applicable RP-HPLC method for the determination of 17 phenolic compounds in Anzer pollens and for the investigation of their total phenolic contents and antioxidant capacities.

Abstract: Crop production is highly sensitive to elevated temperatures. A rise of a few degrees above the optimum growing temperature can lead to a dramatic yield loss. A predicted increase of 1–3 degrees in the twenty first century urges breeders to develop thermo-tolerant crops which are tolerant to high temperatures. Breeding for thermo-tolerance is a challenge due to the low heritability of this trait. A better understanding of heat stress tolerance and the development of reliable methods to phenotype thermo-tolerance are key factors for a successful breeding approach. Plant reproduction is the most temperature-sensitive process in the plant life cycle. More precisely, pollen quality is strongly affected by heat stress conditions. High temperature leads to a decrease of pollen viability which is directly correlated with a loss of fruit production. The reduction in pollen viability is associated with changes in the level and composition of several (groups of) metabolites, which play an important role in pollen development, for example by contributing to pollen nutrition or by providing protection to environmental stresses. This review aims to underline the importance of maintaining metabolite homeostasis during pollen development, in order to produce mature and fertile pollen under high temperature. The review will give an overview of the current state of the art on the role of various pollen metabolites in pollen homeostasis and thermo-tolerance. Their possible use as metabolic markers to assist breeding programs for plant thermo-tolerance will be discussed.

Abstract: This review focuses on the energy metabolism during pollen maturation and tube growth and updates current knowledge. Pollen tube growth is essential for male reproductive success and extremely fast. Therefore, pollen development and tube growth are high energy-demanding processes. During the last years, various publications (including research papers and reviews) emphasize the importance of mitochondrial respiration and fermentation during male gametogenesis and pollen tube elongation. These pathways obviously contribute to satisfy the high energy demand, and there are many studies which suggest that respiration and fermentation are the only pathways to generate the needed energy. Here, we review data which show for the first time that in addition plastidial glycolysis and the balancing of the ATP/NAD(P)H ratio (by malate valves and NAD(+) biosynthesis) contribute to satisfy the energy demand during pollen development. Although the importance of energy generation by plastids was discounted during the last years (possibly due to the controversial opinion about their existence in pollen grains and pollen tubes), the available data underline their prime role during pollen maturation and tube growth.

Abstract: Deformed wing virus (DWV) is a honeybee pathogen whose presence is generally associated with infestation of the colony by the mite Varroa destructor, leading to the onset of infections responsible for the collapse of the bee colony. DWV contaminates bee products such as royal jelly, bee-bread and honey stored within the infected hive. Outside the hive, DWV has been found in pollen loads collected directly from infected as well as uninfected forager bees. It has been shown that the introduction of virus-contaminated pollen into a DWV-free hive results in the production of virus-contaminated food, whose role in the development of infected bees from virus-free eggs has been experimentally demonstrated. The aim of this study was twofold: (i) to ascertain the presence of DWV on pollen collected directly from flowers visited by honeybees and then quantify the viral load and (ii) determine whether the virus associated with pollen is infective. The results of our investigation provide evidence that DWV is present on pollen sampled directly from visited flowers and that, following injection in individuals belonging to the pollinator species Apis mellifera, it is able to establish an active infection, as indicated by the presence of replicating virus in the head of the injected bees. We also provide the first indication that the pollinator species Osmia cornuta is susceptible to DWV infection.

Abstract: . Recent studies have suggested that the ice-nucleating ability of some types of pollen is derived from non-proteinaceous macromolecules. These macromolecules may become dispersed by the rupturing of the pollen grain during wetting and drying cycles in the atmosphere. If true, this mechanism might prove to be a significant source of ice nuclei (IN) concentrations when pollen is present. Here we test this hypothesis by measuring ambient IN concentrations from the beginning to the end of the 2013 pollen season in Raleigh, North Carolina, USA. Air samples were collected using a swirling aerosol collector twice per week and the solutions were analysed for ice nuclei activity using a droplet freezing assay. Rainwater samples were collected at times when pollen grain number concentrations were near their maximum value and analysed with the drop-freezing assay to compare the potentially enhanced IN concentrations measured near the ground with IN concentrations found aloft. Ambient ice nuclei spectra, defined as the number of ice nuclei per volume of air as a function of temperature, are inferred from the aerosol collector solutions. No general trend was observed between ambient pollen grain counts and observed IN concentrations, suggesting that ice nuclei multiplication via pollen grain rupturing and subsequent release of macromolecules was not prevalent for the pollen types and meteorological conditions typically encountered in the southeastern US. A serendipitously sampled collection after a downpour provided evidence for a rain-induced IN burst with an observed IN concentration of approximately 30 per litre, a 30-fold increase over background concentrations at −20 °C. The onset temperature of freezing for these particles was approximately −12 °C, suggesting that the ice-nucleating particles were biological in origin.

Abstract: Pollen quality is important for growers and breeders. This study was carried out to determine in vitro pollen viability and pollen germination in seven genotypes of cherry laurel (Prunus laurocerasus L.). Two pollen viability tests, TTC (2,3,5-triphenyl tetrazolium chloride) and IKI (iodine potassium iodide), were used. Pollen traits of genotypes were studied using an in vitro medium containing 0%, 5%, 10%, 15%, and 20% sucrose to determine the best sucrose concentrations for germination. In the second step, the germinated pollen was counted 1, 4, 6, 10, 12, 24, and 48 hours later until there was no further germination. The viability rates were different according to genotypes and tests used. The IKI and TTC staining tests and pollen germination had low correlation (r(2) = 0.0614 and r(2) = 0.0015, resp.). Painted pollen rate was higher and pollen was well-stained with IKI test and pollen viability estimated with TTC staining test was better than that estimated with the IKI staining test. 15% sucrose gave the best germination rates in most of the genotypes. Pollen germination rates were recorded periodically from one hour to 48 hours in 15% sucrose and the results showed that pollen germination rates increased after 6 hours of being placed in culture media.

Abstract: Bumblebees (Bombus spp.) are efficient pollinators of many flowering plants, yet the pollen deposition performance of individual bees has not been investigated. Worker bumblebees exhibit large intraspecific and intra-nest size variation, in contrast with other eusocial bees; and their size influences collection and deposition of pollen grains. 2. Laboratory studies with B. terrestris workers and Vinca minor flowers showed that pollination effectiveness PE, as measured from pollen grains deposited on stigmas in single visits (SVD), was significantly positively related to bee size; larger bees deposited more grains, while the smallest individuals, with proportionally shorter tongues, were unable to collect or deposit pollen in these flowers. Individuals did not increase their pollen deposition over time, so handling experience does not influence SVD in Vinca minor. 3. Field studies using Geranium sanguineum and Echium vulgare, and multiple visiting species, confirmed that individual size affects SVD. All bumblebee species showed positive SVD/size effects, though even the smallest individuals did deposit pollen. Apis with its limited size variation showed no such detectable effect when visiting Geranium flowers. Two abundant hoverfly species also showed size effects, particularly when feeding for nectar on Echium. 4. Mean size of foragers also varied diurnally, with larger individuals active earlier and later, so that pollination effectiveness varies through a day; flowers routinely pollinated by bees may best be served by early morning dehiscence and visits from larger individuals. 5. Thus, while there are well-documented species-level variations in pollination effectiveness, the fine-scale individual differences between foragers should also be taken into account when assessing the reproductive outputs of biotically-pollinated plants.

Abstract: (p. 288) New palynological information from Greece is presented. The information of Holocene lake sediments is used to compare the representation of pollen types with those from modern surface samples and with the pollen types derived from the herb and tree species listed in plant-sociological relevees from (semi-)natural forest. Differences in the qualitative composition of pollen assemblages from these sources are used to trace types that indicate human activity and influence on the vegetation in prehistoric and historical times. The concept of diversity in vegetation is compared with that in pollen assemblages. Some pollen types from trees, herbs or weeds connected with the activity of man are dealt with in particular. It is investigated in how far such pollen types could mark the first farming activities in Greece. Attention is paid to the various demographic events that are thought to have happened in Greece and their (possible) impact upon the environment.

Abstract: Almost all bees collect nectar and pollen from flowers. Female bees collect pollen to provision their nest cells, whereas they use nectar for individual energy supply and nest cell provisioning. Bees fine-tune nectar foraging to the amount and to the concentration of nectar, but the individual bees' response to variability of amount and concentration of pollen reward has not yet been studied thoroughly in laboratory settings. We developed an experimental set-up in which bumblebees simultaneously collected sugar solution and pollen from artificial flowers; natural pollen was mixed with cellulose powder or glass powder as a pollen surrogate. Here we show that bumblebee (Bombus terrestris) workers do not specialise in nectar or pollen collection, but regularly collect both rewards on the same day. When offered a fixed pollen reward and varied amounts and concentrations of sugar solution, the bumblebees fine-tuned sugar solution foraging dependent on both the volume and concentration, with strong preferences for the highest concentration and the greatest volume. In the reciprocal tests, when offered a fixed sugar reward and varied amounts and concentrations of pollen mixed with a nutrient-free pollen surrogate, the bumblebees follow more an all-or-none rule for pollen, accepting all amounts and concentrations except pure surrogate. It is discussed how the bumblebees' ability to sense sugar, and their apparent inability to sense the pollen protein content, shaped their foraging behaviour. It is argued that the rarity of nectar mimicry and the frequency of pollen mimicry in natural flowers might be interpreted in the context of divergent abilities of nectar and pollen recognition in bees.

Abstract: A palynological and sedimentological record from the Mahwaqa Mountain in KwaZulu-Natal, South Africa, provides evidence of the vegetation dynamics in this part of the Grassland Biome during the last c. 18,000 years. The wetland is located at 1,850 m on an isolated outlier of the Ukhahlamba–Drakensberg Mountain range on an ecotone along a climatic gradient. The vegetation responded to humidity and temperature changes during the late Pleistocene and Holocene. The period c. 18,000–13,500 cal. bp is characterized by high Ericaceae and Restionaceae percentages and decreasing values of charred particles, indicating cool conditions. Around 13,500–8,500 cal. bp, Ericaceae were gradually replaced by Poaceae, signaling climate warming. Growing environmental wetness during the same time period is inferred from Phragmites-type and Cliffortia pollen percentages. Since c. 8,500 cal. bp, Cliffortia, Restionaceae, and Phragmites-type percentages have maintained low levels. Ericaceae were almost completely replaced by grasses and Asteraceae by c. 7,500 cal. bp. All indications are that warm and fluctuating moisture conditions followed until 4,600 cal. bp but they became driest between c. 4,600 and 3,500 cal. bp, when high Asteraceae, Pentzia-type and Scabiosa percentages were prominent. From c. 3,500–800 cal. bp, the increase of sedges, Aponogeton and grass pollen (including Phragmites-type) at the expense of Asteraceae pollen suggests the return of slightly more humid conditions. Since c. 1,000 cal. bp an increase of water demanding Podocarpus and Cliffortia occurred. Pine pollen indicates the recent introduction of alien plants in the 19th and 20th centuries.

Abstract: Cucumber green mottle mosaic virus (CGMMV) is transmitted mechanically in cucurbits, but whether it is transmittedvia pollen to healthy plants and onto the subsequent generation of seedlings is unknown. Greenhouse experiments wereconducted to investigate the importance of this route of infection. Cucumber seedlings at the 3-true-leaf stage weremechanically inoculated with CGMMV. At anthesis, scanning electron microscopy (SEM) and reverse transcriptionpolymerase chain reaction (RT-PCR) were used to verify the presence of CGMMV. Pollen was collected from the flow-ers of the infected plants and used to fertilize non-inoculated plants. The rate of CGMMV transmission to the resultingfruits ranged from 17 1to51 2% compared with 33 3–100% for mechanically inoculated plants. Seeds were harvestedfrom the cucumber fruits of both treatments and tested for the presence of CGMMV by RT-PCR. The CGMMV-posi-tive seeds harvested from the two treatments were sown separately. The seed transmission rates for the inoculated andnon-inoculated plants were 16 7–100% and 12 8–76 7%, respectively. It was concluded that CGMMV can be trans-mitted both horizontally via cucumber pollen and vertically, to the next generation, in infected seeds. In addition therate of seed transmission was much higher than previous reports. These findings have important implications for thedisease management of CGMMV.Keywords: Cucumber green mottle mosaic virus, pollen transmission, pollination, seed transmission, tobamovirus,transmission rate

Abstract: Herbivory defence chemicals in plants can affect higher trophic levels such as predators and parasitoids, but the impact on pollinators has been overlooked. We show that defensive plant chemicals can damage pollinator fitness when expressed in pollen. Crop lupins (Lupinus species from Europe and South America) accumulate toxic quinolizidine alkaloids in vegetative tissues, conferring resistance to herbivorous pests such as aphids. We identified the alkaloid lupanine and its derivatives in lupin pollen, and then provided this compound at ecologically-relevant concentrations to queenless microcolonies of bumblebees (Bombus terrestris) in their pollen to determine how foraging on these crops may impact bee colony health and fitness. Fewer males were produced by microcolonies provided with lupanine-treated pollen and they were significantly smaller than controls. This impact on males was not linked to preference as workers willingly fed lupanine-treated pollen to larvae, even though it was deleterious to colony health. Agricultural systems comprising large monocultures of crops bred for herbivore resistance can expose generalist pollinators to deleterious levels of plant compounds, and the broader environmental impacts of crop resistance must thus be considered.