Effects Of Floral Heavy Metal Accumulation On Plant Reproduction And Bee Pollinators

Heavy metals occur in small amounts naturally in most soils,but increasing anthropogenic activities(e.g.industry,agriculture)can lead to elevating levels of these elements in soils within and surrounding urbanized and industrialized areas,causing serious soil heavy metal pollution.Researchers in University of Pittsburgh,USA,found that plants growing in nickel-rich soils could take up nickel ion and translate it into above-ground tissues,making nickel accumulate into floral organs and rewards for pollinators.They speculated that nickel accumulation in floral organs and rewards might result in fitness consequences to plants and pollinators.However,they did not conduct experiments to test the effects of floral heavy metal accumulation on plant reproduction and pollinator fitness.To clearly understand how heavy metal accumulation in flowers from soils affect plant reproduction and pollinator fitness,we conducted field and manipulative experiments to investigate metal accumulation and distribution in floral organs and floral rewards of non-accumulators by using four common soil heavy metals(zinc,copper,nickel,and lead)in Liaoning province.Meantime,we examined the effects of floral metal accumulation on plant reproductive fitness as well as the feeding behavior and survival of pollinators.The main experimental results and conclusions are listed as follows:(1)To investigate the accumulation and distribution of heavy metals from soils into flowers under natural conditions and the effect of floral metal accumulation on honeybee survival,we chose three mine areas(zinc-lead mine,copper-nickel mine and lead mine)and three nature reserves(Fenghuang mountain,Tiannv mountain and Wunv mountain)in Eastern Liaoning where metal(zinc,copper,nickel and lead)concentrations in soils around roots,floral organs(petal and nectary),floral rewards(pollen and nectar)and honey of Robinia pseudoacacia L.were determined.Meanwhile,we censused the number of dead honeybees at the entrance of each experimental hive.Our experimental results showed that the concentrations of four metals in soils around roots,floral organs and floral rewards in mine areas were higher than those in nature reserves,indicating that heavy metals in soils could be accumulated in flowers of Robinia pseudoacacia L.under natural conditions.In each sample plot,metal concentrations in floral organs were higher than those in floral rewards and metal accumulation varied depending on different floral organ/reward types.Regression analysis revealed that concentrations of main metals in flowers in each mine area were positively correlated with the metal concentrations in soils around roots,which indicated that metals accumulated in flowers were likely originated from soils.The results that the number of dead honeybees at the entrance of each hive in honey in mine areas was larger that that in nature reserves and the metal concentrations in honey from mine areas were higher than that from nature reserves indicated that metal accumulation in nectar of Robinia pseudoacacia L.might cause the high mortality of honeybees.(2)We supplemented metal(zinc,copper,nickel and lead)nitrate into soils of potted summer squash(Cucurbita pepo L.cv.Golden Apple)to investigate the accumulation and distribution of heavy metal in floral organs(pistil,anther and nectary)and floral rewards(pollen and nectar)as well as the floral metal accumulation effects on floral visitation behavior of honeybees and reproductive fitness of squash.The results showed that the four heavy metals taken up by squash did accumulate into its floral organs and rewards,although metal accumulation differed depending on various metal types and concentrations as well as floral organ/reward types,providing the first demonstration that squash was capable of accumulating metals into its flowers.Mean foraging time of honeybees to each male and female flower of squash grown in medium and high metal-treated soils was shorter relative to that of plants grown in control soils,but the visitation rate of honeybees to both male and female flowers was not affected by metal treatment in soils.Pollen viability,proportion of pollen removed,number of pollen grains deposited on stigmas,and mean mass per seed produced by metal-treated squash that received pollen from plants grown in control soils decreased at medium and high soil-supplemented metal concentrations,which indicated that floral metal accumulation of squash reduced its reproductive fitness via influencing both pollen-pistil interactions and plant-pollinator interactions.(3)We experimentally manipulated four heavy metals(zinc,copper,nickel and lead)in a native ornamental plant,Hosta ensata F.Maekawa,to investigate the effects of heavy metals in nectar on the visitation behavior of pollinators and nectar robbers as well as the reproductive fitness of Hosta.The results showed that pollinating honeybees and bumblebees spent shorter time foraging metal-treated flowers,but their visitation rate to metal-treated flowers was significantly higher than that to control flowers.Moreover,pollinators removed less nectar from flowers treated with metals,demonstrating that pollinating honeybees and bumblebees responded negatively to heavy metals in nectar.We also found that nectar-robbing bumblebees responded negatively to the presence of metals in nectar,robbing metal-treated flowers less frequently.Although metals in nectar had no significant direct effects on reproduction measured as fruit set,mean mass per seed and seed germination rate via hand-pollination,we detected their positive indirect effects on components(number of pollen grains deposited on stigma,fruit set,mean mass per seed and seed germination rate)of female fitness,which indicated that nectar metal accumulation enhanced female reproductive fitness of Hosta via modification of mutualistic interactions between plant and pollinators(more frequent visitation of pollinating honeybees and bumblebees)and antagonistic interactions between plant and nectar robbers(lower robbing rate of nectar-robbing bumblebees).(4)Effects of heavy metals(zinc,copper,nickel and lead)on feeding behavior and survival of bumblebees(Bombus koreanus Skorikov)were investigated in laboratory.The experimental results showed that proportion of individual bumblebees extending their proboscis(%PER)reduced when their antenna were stimulated by higher concentrations of zinc,copper and nickel,but lead stimulation on antennas imposed no significant effects on the proportion of individual bumblebees extending their proboscis(%PER).However,when proboscises of bumblebees were stimulated by metals,we found that metals(zinc,copper and nickel)at any experimental concentrations had no effect on the proportion of individual bumblebees completely consuming the testing droplet,but lead at lower concentrations decreased the proportion of individual bumblebees completely consuming the testing droplet.These results revealed that the response of bumblebees to heavy metals varied depending on different metal types and concentrations and also the sensory system stimulated during the assay.Although the capacity of bumblebees to discriminate different concentrations of sucrose was not affected by pretreatment with nickel,pretreatment with zinc,copper and lead reduced the capacity of bumblebees to discriminate different concentrations of sucrose,indicating that the ability of bumblebees to evaluate the energy values of foods might decrease if they consumed foods with heavy metals(zinc,copper and lead).The results of acute toxicity and chronic toxicity of heavy metals to bumblebees showed that mortality of bumblebees increased with elevated metal concentrations.Moreover,chronic toxicity of heavy metals to bumblebees was slightly stronger that acute toxicity,which probably demonstrated that long-term exposure of bumblebees to heavy metals with lower concentrations might lead to high mortality relative to short-term exposure to metals with higher concentrations.(5)Small nucleus colonies of honeybees were exclusively fed on sugar syrup and pollen patty spiked with heavy metals(zinc,copper,nickel and lead)to test effects of heavy metals on the whole-colony fitness of honeybees.The results showed that nickel decreased the brood surface area and total worker weight,indicating that nickel retarded the growth of brood and worker in the colony.Although zinc and copper increased the number of total capped broods,more dead pupae were found in the colony,which may demonstrate that pupae were more sensitive to heavy metals(zinc and copper).The results that the accumulation concentrations of all heavy metals(zinc,copper,nickel and lead)in workers were higher than other survived members may indicate that one possible cause for bee decline in areas where heavy metal pollution occurs is the toxicity resulting from metal accumulation in bees.We detected higher concentrations of zinc and copper in honey relative to control,signifying that workers might detoxify metals(zinc and copper)via producing honey.The concentrations of copper and nickel in queens were not significantly different from controls,which demonstrated that queens might detoxify metals by laying eggs.Overall,when small nucleus colonies of honeybees were fed on foods with heavy metals,either growth of broods and workers was slowed or mortality of pupae was higher.Therefore,heavy metal accumulation reduced the whole-colony fitness of honeybees.In conclusion,floral heavy metal accumulation caused by soil heavy metal pollution may impose either negative or beneficial effects on plant reproductive fitness,which likely depended on different plant-floral visitor systems according to this study.However,heavy metal accumulation in floral rewards definitely had negative effects on feeding behavior and survival of pollinators.