International Journal of Entomology and Nematology Vol. 2(1), pp , September,

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1 International Journal of Entomology and Nematology Vol. 2(1), pp , September, IJEN Research Article Behavior of Bees Associated with the Wild Blueberry Agro-ecosystem in the USA *Francis A. Drummond *School of Biology and Ecology, 305 Deering Hall, University of Maine, Orono, Maine USA Tel.: , Fax: Greenhouse and field studies were conducted between 1996 and 2014 in Maine to assess the behavior of selected bee taxa that visit wild blueberry, Vaccinium angustifolium (Ericaceae) Aiton. Some of my findings are as follows. When individual foraging efficiency was assessed on wild blueberry for four common bee pollinators, bumble bees were most efficient and honey bees were the least efficient in terms of the number of pollen grains deposited on a stigma in a single visit (P< ). However, I also found that the prior bumble bee visitation to flowers enhanced the pollination efficacy of honey bees. Field observations suggested that bumble bees recruit to plants with higher floral density and that bumble bees and andrenids forage for longer periods of time in the day than sweat bees and Osmia leaf cutting bees; honey bees showed intermediate foraging durations. Honey bees and solitary native bees were found to forage at an increasing rate with increasing air temperature, while bumble bee queens tended to forage independently of air temperature. Foraging patterns among the following bee taxa such as bumble bees, andrenids, megachilids, and honey bees also varied and the implications of these differing foraging patterns relative to pollination are discussed. Keywords: Bees, wild blueberry, Vaccinium angustifolium, pollination efficiency, flower handling time, pollen deposition, foraging pattern INTRODUCTION Wild lowbush blueberry, Vaccinium angustifolium (Ericaceae) Aiton, is the largest (in terms of land area) managed native wild fruit crop grown in North America (Jones et al.; 2014). Wild blueberry naturally occurs from the mountains of Virginia and West Virginia to the Canadian Maritimes and Quebec west to Ontario and Michigan (Vander Kloet, 1988, Jones et al., 2014). This plant species generally grows in acidic, sandy soils and is an early colonizer of disturbed areas (Hall et al., 1979; Jones et al., 2014). It is a natural occurring understory plant species in Northern Mixed Hardwood, Acadian, and Boreal Forests (Jones et al., 2014) and a valuable food resource for wildlife (Martin et al., 1951; Eaton, 1957). Management or cultivation of wild blueberry was first practiced by Native Americans (Munson, 1901; Hedrick,1919) and later introduced to European colonists (Davis, 1993). This tradition of clearing forest followed by frequent burning of the landscape to reduce plant competition with wild blueberry is still practiced by several farmers today (Rose et al., 2014). Currently, Maine is the largest producer of wild blueberry in the world with more than 24,300 hectares under cultivation (Yarborough, 2009). Insect pollination is critical for the set of wild blueberry flowers and fruit development (Lee, 1958; Vander Kloet, 1988; Bell et al., 2009; Asare, 2013). The morphology of the flower with poricidal anthers limits the species of insects that can effectively pollinate wild blueberry (Ritzinger and Lyrene, 1999; Bell et al., 2009). There are more than 120 native

2 Drummond FA. 028 bee species that are associated with wild blueberry landscapes and pollination (Boulanger et al., 1967; Finnamore and Neary, 1978; Bushmann and Drummond, 2015). Many wild blueberry growers in Maine heavily rely on honey bees for pollination (Hanes et al., 2013); however, there are still several growers that rely solely upon native bees for their pollination needs (Rose et al.; 2014). There has been a concerted effort to conserve native bees in wild blueberry habitats by identifying their floral resources, providing nests, and identifying threats from pesticide applications and pathogens (Stubbs et al., 1992; Stubbs et al., 1997; Stubbs and Drummond, 1998; 1999; Bushmann et al., 2012; Drummond 2012b; Groff et al., 2016). The importance of wild native bee pollinators in wild blueberry production reflects the development of field techniques for assessing bee populations by researchers and blueberry growers (Drummond and Stubbs, 1997b; Drummond et al., 2015). Research on the efficacy of honeybee pollination began at the University of Maine in the 1950's (Boulanger unpublished data; Boulanger et al., 1967). The importation of commercial honeybee colonies has grown from approximately 500 hives per year in the mid-1960's to approximately 60,000 hives imported for the blueberry blooming season in the year 2000 (Drummond, 2002) and more than 85,000 in 2016 (Jadczak, Maine state apiculture inspector, personal communication), an increase that reflects increasing numbers of honeybee hives per acre (Yarborough, 2013). In addition, the use of commercial bumblebees (Bombus impatiens Cresson) has become popular over the past 15 years with approximately 2,300 colonies being purchased per year for wild blueberry pollination in Maine (Stubbs and Drummond, 2001; Desjardins and Olivereira, 2006; Drummond, 2012b). In addition, several other potential commercial bees have been evaluated for wild blueberry pollination (Stubbs et al., 1994; Drummond and Stubbs, 1997a; Stubbs and Drummond, 1997a; 1997b; 1997c; 2000). Given this reliance on pollination by commercial bees, the wild blueberry production is vulnerable to disruption by lack of pollination services. However, despite the increasing reliance on commercial bee pollinators in wild blueberry, native bees are still significant in determining the fruit set and yield (Asare, 2013; Yarborough et al., 2016). Indeed, U.S. consumers are willing to pay more for blueberries pollinated by native bees (Stevens et al., 2015). However, there have been few studies on their efficacy as pollinators of wild blueberry. Whidden (1996) and Drummond (2012b) both showed how bumble bees are floral constant with respect to wild blueberries and only Javorek et al. (2002) addressed the performance of native bees as pollinators and showed that the efficacy of native bees on a per bee basis is much greater than that of honey bees. Because of this lack of knowledge about native bee foraging in wild blueberry, the pollination efficacy of a range of both native and exotic bee species with regards to wild blueberry was studied in Maine. We measured and quantified: 1) selection choice of bumble bees when foraging among wild blueberry flowers with regards to flower age, size or previous visitation history, 2) the effect of flower number on stems and if this affects the bumble bee visitation choice of those stems, 3) attractiveness of five co-flowering species during wild blueberry bloom, 4) bee species-specific pollen deposition on wild blueberry flower stigmas during single visits to naive flowers, 5) bee species-specific handling time of flowers, 6) effects of sequential multiple flower visits on pollen deposition by more than one species of bee, and 7) bee-specific spatial foraging patterns in wild blueberry fields during bloom. MATERIALS AND METHODS This study was conducted over a period of twenty years ( ) in the greenhouse, laboratory, and wild blueberry fields in Maine, USA. All cage studies were conducted at the University of Maine in Orono, Maine (Penobscot county). Field studies were conducted in fifteen managed wild blueberry fields in Hancock, Lincoln, Waldo, and Washington counties, Maine. Seven experiments grouped under three studies were conducted during this period. Methods for each experiment are described below and a schematic (Fig. 1) has been included to provide an outline of the studies and experiments. Bee Preference Studies Blueberry Flower Selection Choice By Bumble Bees, Flower Size And Age During March April 2007 and then in March April 2008, the effects of wild blueberry flower age, corolla length (mm) and corolla diameter (mm) on bumble bee, Bombus impatiens (Say), choice measured by visitation frequency was studied in the greenhouse. This study was conducted in three replicate 2 m x 2 m x 3.5 m mesh cage in the University of Maine Clapp greenhouse in Orono, Maine, USA. Three bumble bee colonies (100 worker strength) were purchased from Koppert Inc. (Romulus, Michigan, USA) and placed in the flight cage for several days without flowers. The colonies derived their nutritional requirements on sugar syrup provided with the colonies upon purchase. During the previous autumn, 12 clones (genets) of Vaccinium angustifolium Aiton blueberry sods were cut and excavated from blueberry fields in Jonesboro, Maine, USA and placed in 50.8 x 40.6 cm plastic tote boxes. The boxes were kept in a walk-in refrigerator (2-4ºC) over the winter. In late February, the blueberry boxes were brought into the greenhouse (maintained at 15-22º C) and allowed to mature flowers. Upon the bloom initiation, the flowers were measured with a micrometer, dated, marked, and coded. In the

3 Int. J. Entomol. Nematol. 028 Figure 1. Outline of experiments conducted between the years 1996 and 2015, bee preference studies (A) and pollination efficacy and foraging studies (B). Cage studies are surrounded by dashed line boxes and open field studies are surrounded by solid line boxes. early morning prior to an assay, boxes of blueberry in bloom, not used for experimentation, were placed into the cages, bumble bee hives were opened and access to floral resources were allowed. Immediately prior to the start of a choice assay, all blueberry boxes were removed and two stems with a single open flower on each (standardized by either flower age (1-8 days old), corolla length, or diameter) were presented to foraging bumble bees. The stems were placed in a test tube holder such that stems were 23 cm apart. Flowers were observed until a bumble bee landed on the flower and began to extract either nectar or pollen. At this point the visited flower was recorded. Blueberry Flower Selection Choice By Bumble Bees, Previously Visited Flowers - In 2008, one choice bioassay was conducted to determine if flowers that had

4 Drummond FA. 029 not previously been visited by a bee compared to similar age and size flowers that had been visited by a bee were differentiated by foragers. Sixteen replicates of paired flowers categorized by presence or absence of previous visitation were run in the exact same manner as described above for the flower size and age assays. A non-choice bioassay using visited flowers from the previously described bioassays was used to determine if previously visited flowers (ranging from 1-6 visits) affected the flower choice by bumble bee foragers. Fiftythree replicate trials were used in this bioassay. In all bioassays conducted over both years, if no visitation occurred in 15 minutes then the trial was terminated and new pairs of stems were introduced. A total of 106 choice assays were conducted over the two-year study. Nominal logistic regression was used to test if differences among years, flower age, corolla length, and previous visitation influenced flower selection for first visit (JMP, 2015). Choice For Blueberry Flowers Compared To Five Co-Flowering Native Shrubs - This study was performed on 21 and 22 May, Flowering shrubs that bloom during wild blueberry bloom in Maine that are often found growing around the field edges of wild blueberry fields. We selected five of the most common co-flowering shrubs in Downeast Maine to test the preference of B. impatiens foragers for blueberry over the five co-flowering shrubs. The species selected for study were shadbush, Amelanchia spp.; apple, Malus pumila; rhodora, Rhododendron canadense; choke cherry, Prunus virginiana; and bunch berry, Cornus canadensis. Several flowering stems of these species were collected and each was tested against the wild blueberry stems in flower collected at the same time. A paired choice bioassay in the same manner as discussed in the former experiment in a 2 x 2 x 3 m mesh flight cage was setup in a greenhouse at the University of Maine, Orono, Maine. Commercial bumble bees, B. impatiens were purchased prior to the experiment (Koppert, Inc.) and used in all assays. All paired choice bioassays consisted of a vase of cut flowering wild blueberry stems and a vase of cut flowering stems from one of the five co-flowering shrubs.pairs of cut stems were exposed to foraging commercial worker bumble bees (B. impatiens) for five consecutive trials. For this experiment, since the age of the gathered blossoms could not be determined, the age of the blueberry blossoms was not taken into account. It was important to keep the floral displays between the blueberries and the gathered blossoms in the same volume to ensure bees would not be attracted to a larger blossom display, ensuring each flower species an equal opportunity to be chosen. For example, the apple blossoms, being rather large were paired with several blueberry stem cuttings so the overall total blossom area was similar for each flower species. Data recorded for each assay was the first floral species visited in the paired presentation and the amount of time spent on each floral species in a five-minute period of observation. The pair of stems was removed from the cage after each assay and a new pair of stems was selected. At least ten replicates for each pair of co-flowering and blueberry stems were conducted. After the trials, the flowers that had been presented in each vial were photographed and scanned into a computer to measure the amount of area associated with each presented flower blossom. Analyses of covariance (ANCOVA, covariate was area of floral display; JMP, 2015) was used to determine if the total time bumble bees spent foraging on wild blueberry was significantly greater than the time spent on the other flowering shrubs during the 5-minute assay. Nominal logistic regression (JMP, 2015) was used to assess differences in initial choice in visitation of blueberry compared to other co-flowering plant species. Recruitment of Foraging Honey Bees, Commercial Bumble Bees And Native Wild Bees To Clone Flower Density In The Field - Two studies were conducted at three locations in Maine in The first study was conducted in Union and Winterport, Maine. The fields were visited during bloom on 14 and 15 May. Ten clones (genets) in a blueberry field in Union were randomly located and flagged and 24 clones in a blueberry field in Winterport were located and flagged. In each clone 30 stems were arbitrarily selected and the total number of flowers (pre-bloom and blooming) on each stem was counted. This provided a mean floral density for each clone. A square meter quadrat was marked out with string in each clone in both fields. Bee observations were made in each clone for three minutes and the number of bumble bee queens, native wild bees other than bumble bees, and honey bees visiting each quadrat during the three minute period were recorded over the two day period. Linear general models (JMP, 2015) were used to determine if the bees recruit more heavily to clones with higher floral density. The second study was designed to assess the flowering stem selection by commercial bumble bees as a function of total and open flowers. During bloom (May) in 2008, at the University of Maine blueberry research farm, Blueberry Hill in Jonesboro, Maine a field study was conducted to determine the frequency of visitation by B. impatiens workers to wild blueberry stems with opened and non-opened flowers. Prior to bloom 20 commercial bumble bee quads (Koppert, Inc.) were placed in one of the blueberry fields (ca. 8 ha). The density of bumble bees from this stocking density of commercial bumble bees is approximately 3,500-4,000 workers. Thirty-six stems were each marked by a thread tied about the stem base. The stems were apportioned to six clones (6 marked stems in each clone). During ten observation times throughout May, each stem was observed for 2 minutes and the number of B. impatiens workers visiting flowers on each stem was recorded. A general linear model was used to determine if B. impatiens

5 Int. J. Entomol. Nematol. 030 worker visitation to stems was related to clone, total floral density, or solely open floral density (JMP, 2015). Bee Pollination Efficacy Studies During May and June of 1996 a preliminary study was conducted to assess individual bee pollination efficacy in flight cages. This study enabled us to refine methods to repeat a study with higher replication, using the modified methods in May and June of 2006, and May 2014.These studies were conducted using field cage studies in both the greenhouse and field to determine the efficacy of common bee pollinators of wild blueberry. Single Floral Visits - The bee species used in the studies were the bumble bee, Bombus impatiens (Apidae) Cresson; the leaf cutting bee, Osmia atriventris (Megachilidae) Cresson; the digging bee, Andrena carlini (Andrenidae) Cockerell, and the honey bee Apis mellifera (Apidae) (L.). Studies conducted during bloom (mid-may to mid-june) in 1997 and 2006 were aimed at determining flower handling time and the number of pollen grains placed on a new previously non-visited stigma in a wild blueberry (V. angustifolium) flower. The study was conducted in mesh flight cages either placed in a non-flower bearing field over Andrena carlini nest aggregations or in the greenhouse (see study 1 above). Bumble bee colonies, honey bee colonies, or Osmia atriventris nest blocks with active females provisioning cells, were placed in cages with 1-4 totes of flowering wild blueberries to allow foraging to take place and pollen acquisition by each of the bee species. Several of the stems in each tote (5-20) were covered with fine mesh bags to prevent bee access to the flowers. Flowers on each bagged stem were marked and the day that each flower opened was recorded. Once bees were observed actively foraging and pollinating non-bagged flowers on stems in the totes placed in the cages, the bags were removed from stems with 1-5 day old flowers. Bee visits of each bee species (only a single bee species per cage) to newly un-bagged flowers were recorded. During visitation to each un-bagged flower the handling time (from initial contact of the flower until the time when the bee left the flower) was measured with a stopwatch. As soon as the visiting bee left the flower, the stem was put in an ice filled cooler for temporary storage until the end of the day s assay. All stems with marked visited flowers were taken immediately to the laboratory where the stigma was stained (Alexander stain, Alexander 1980) and inspected under the microscope for V. angustifolium tetrad pollen grains. Twenty-five replicate recordings of flower handling time and pollen deposition were made for each species in One-way analyses of variance (Welch s adjusted F-test for unequal variances, complete randomized design; JMP, 2015) were used to determine if differences due to bee species existed when considering handling time and pollen deposition per visit. Tukey posthoc multiple comparison tests were used to compare all means with one another (JMP, 2015). Sequential Floral Visits - In May 2014, a similar flight cage study to that described above was used to assess potential interactions in pollen deposition between honey bees and bumble bees when flowers were visited twice by bees. The goal of this study was to determine if pollination synergy occurs when bumble bees and honey bees both forage on blueberry flowers sequentially compared to only one of the two bee species visiting flowers sequentially. This study was conducted using two mesh flight cages in the greenhouse (see above). The bee species used in the study were the bumble bee, Bombus impatiens Cresson and the honey bee Apis mellifera (L.). Bumble bee colonies or honey bee colonies were placed in cages with 1-4 totes of flowering wild blueberries to allow foraging to take place and pollen acquisition by each of the bee species. Only a single bee was allowed to forage at a time. Stems were protected from foraging bees with mesh bags as described above and marked when flowers opened. Once bees were observed actively foraging and pollinating non-bagged flowers on stems in the totes placed in the cages, bags were removed from stems with 1-5 day old flowers and the cages were removed of all bees. Individual new bee visits of each bee species (only a single bee species per cage) to newly un-bagged flowers were recorded. As soon as a visiting bee left the first un-bagged stem (flower A), the bee was allowed to visit a second un-bagged stem (flower B). The second visited un-bagged stem (flower B) was excised and placed in a cooler until it was brought to the lab. The bee in the cage was discarded and the stem with the first visited flower (flower A) was excised and put into a test tube filled with water and transported to the flight cage with the other bee species. A new bee in this flight cage was allowed to visit the flower on the stem (flower A) that was previously visited in the other cage by the other species. After the visit to flower A in the other cage the stem (flower A) was tagged and placed in a cooler. Immediately after a bout of visitations, stems placed in the cooler were brought immediately to the laboratory where the stigma was stained and inspected under the microscope for V. angustifolium terad pollen grains as described above. Twenty replicates of each of the four treatments were performed: 1. single visit by honey bee that had previously visited a flower; 2. single visit by bumble bee that had previously visited a flower; 3. A single visit by a bumble bee that had just previously visited a flower visited by a honey bee; and 4. A single visit by a honey bee that had just previously visited a flower visited by a bumble bee. One-way analysis of variance (Welch s adjusted F-test for unequal variances, complete randomized design; JMP, 2015) was used to determine if differences in pollen deposition occurred between treatments. A Tukey post-hoc multiple

6 Drummond FA. 031 comparison test was used to compare all means with one another (JMP, 2015). Foraging Studies Diurnal Foraging Patterns Of Bees In Wild Blueberry And Response To Air Temperature - On May 21-31, 2001, observations of bee activity were made in a wild blueberry field in Winterport, Maine during peak bloom. Bees were sampled with a 30.5 cm diam. sweepnet. A total of locations in the field were sampled. At each sampling location ten 180º sweeps in the blooming crop were conducted. Sampling was conducted every 2-3 hrs starting at 6 AM, shortly after sunrise, and ending at dusk, 8 PM. Sampling was conducted each day between 21 may and 31 May. On most sampling periods, the air temperature was recorded on-site with a handheld weather data logger (Kestrel ). Bees were classified in the field into taxonomic groups and their numbers recorded. The groups were the following: honey bees, bumble bees (Bombus spp.), digger bees (Andrena spp.), sweat bees (Halictidae), and leaf cutting and mason bees (Megachilidae, almost entirely Osmia spp.). Sweepnet capture of each taxon grouping was tallied for each sampling time and the percent of relative capture by sampling time was calculated and plotted. Visual inspection of the data in a graph was used to draw conclusions about the diurnal activity of the different groups of bee foragers. Linear regression analysis was used to assess the relationship between air temperature and the percent of the honey bee, bumble bee queen, and native solitary bee (Andrena spp. + Megachilidae + Halictidae) population for each day that was foraging. Bee Spatial Foraging Patterns Within And Among Clones - During wild blueberry bloom (mid-may to mid- June) in 2003, 2004, 2005, 2006, 2007, 2008, 2009, and 2010; the spatial movement patterns of foraging honey bees and native wild bees were delineated and recorded. The blueberry fields were located in Union (Knox Co.), Winterport (Waldo Co.), and Columbia, Jonesboro, T-18, and T-19 (Washington Co.), Maine. Using a digital audio recorder, we assessed the distances between stems that were visited by foraging bees, distances between visited flowers within a stem, the number of flowers visited per stem by bees, and the cardinal direction bees flew when moving from one stem to the next. The bees observed and mapped in this study were honey bees, (Apis mellifera), Bombus spp. (mostly B. ternarius), Andrena spp., and Osmia spp. Data was recorded on individual bees until the bee was lost among the blueberry foliage or it left on a long distance flight where it was not possible to follow it any longer. At this point the foraging bout was marked as terminated and another bee was selected for observation. Care was taken not to disturb the bee by observers maintaining a one-meter distance from the foraging bee, minimizing casting shadows on the foragers, and moving slowly while observing foragers. Frequency distributions of distances among visited stems, cardinal direction between stems, flowers, number of flowers visited per stem, and stems visited per bout were constructed and fit to theoretical probability distributions for each bee taxon group observed (JMP, 2015). RESULTS Study Bee Preference Studies Blueberry Flower Selection Choice By Bumble Bees, Flower Size And Age - Over both years (2007 and 2008) wild blueberry flower corolla length and diameter were highly correlated (r = , n=90, P< ). Flower age was not correlated with either corolla length or diameter (P> 0.05). Because of this only year, corolla length and flower age were modeled to determine if bumble bees select flowers to visit in choice bioassays. Bumble bees appear to demonstrate preference for longer wild blueberry flowers (x 2 (1) = 7.221, P = 0.007, Figure 2), but they did not discriminate for flower age (P> 0.05) when the choice was made between flowers that differed in age up to 6 days. However, a non-significant trend was found, which suggests a tendency to select younger flowers (P = 0.106). Year and the interaction between corolla length and flower age was also not significant. The odds ratio for the significant corolla length effect suggests that for every difference in 1.0 mm length between flowers that it is 2.3 times more likely that the larger flower will be first selected by a bumble bee forager. Blueberry Flower Selection Choice By Bumble Bees, Previously Visited Flowers - The trials that focused on previously visited flowers compared to flowers that had never been by a bumble bee showed no evidence of choice (P = 0.715) and the trials assessing visitation to flowers that had been previously visited from 1 to 6 times showed no evidence of an effect on bumble bee foragers (P = 0.363). Recruitment Of Foraging Honey Bees, Commercial Bumble Bees And Native Wild bees To Clone Flower Density In The Field - I found that floral density per stem (both open and non-opened flowers) in clones did determine the recruitment of honey bees, bumble bee queens, and native bees other than bumble bees, but this effect was dependent upon site (Table 1).With regards to the entire foraging bee community (all bee taxa), bees recruited to clones with higher total flower density. There was an effect of bee density due to site which is not unexpected as bee densities, especially native bees, vary by an order of magnitude from field to field and geographic region to region. In the field,the frequency at which stems were selected by foraging bumble bees in Jonesboro, Maine was not influenced by clone (genet; P = 0.178), nor the

7 Int. J. Entomol. Nematol. 032 Figure 2. Modeled probability of a bumble bee forager selecting a flower when given a choice of two flowers. As number become more negative on x-axis the flower chosen was smaller compared to larger flower (mm), as number become more positive, the flower chosen was larger compared to smaller flower (mm). Table 1. Recruitment of foraging bees to flower density / stem in a clone. Bee taxa group Significant Factors F-test Proportion variance explained, r 2 Total bee community site F(1,30) = 5.407, P= * (0.418)** flower density F(1,30) = , P=0.001 Bumble bee queens site F(1,30) = 5.707, P= (0.224) flower density F(1,30) = 6.221, P=0.020 Native bees (other flower density F(1,30) = 6.101, P= than bumble bees) Honey bees site flower density site x flower density F(1,30) = , P= F(1,30) = , P= F(1,30) = , P= * proportion variance explained by entire model ** proportion variance explained by flower density, based upon sequential sums of squares number of open flowers (P = 0.417) on the stem. The initial choice to land on the stem was significantly determined by the number of total flowers (open and nonopen flowers: F(1,28) = 4.292, P = ), but only 12% of the variance in the frequency of visitation to stems was explained by the total number of flowers per stem. Choice For Blueberry Flowers Compared To Five Co-Flowering Native Shrubs - Bumble bee foragers do appear to prefer wild blueberry flowers to most of the coflowering species both in % first visits (x 2 (4) = , P = (0.350) ) and time spent foraging on flowers (F(4,111) = 2.568, P = 0.042; Figure 3). Comparing the ratio of initial visitation to blueberry vs. the other species (t-test comparing percent choice of blueberry to 50%, no difference in species choice) and relative time on flowers of blueberry compared to the other species (t-test comparing relative time to 1.0, no difference in relative time on two species), it can be seen that blueberry is highly attractive to bumble bee foragers compared to the other five species (Figure 3). The only co-flowering

8 Drummond FA. 033 Figure 3. Preference of Bombus impatiens workers (stipled bar) for species other than blueberry (measured in paired bioassays as the percentage of first landings on blueberry vs the other species). In addition, the relative time spent on blueberry flowers relative to the co-flower (hatched bar). The red dashed line indicates a region where foragers spent the same time on blueberry and the coflowering plant and the blue dotted line indicates the region of no-preference in initial floral choice between blueberry and a coflowering plant species. Asterisks indicate significant differences in 1) time spent on the co-flower relative to blueberry and 2) preference for choice of initial flower selection. species that appeared to be as attractive to bumble bees as blueberry in initial visitation were apple and choke cherry, but even these two species had 3-4 times the foraging effort on blueberry once the initial flower was selected. There were no visits to bunch berry for any of the assays. Rhodora was almost ignored when first initial visits were considered, but the amount of time that bumble bees spent foraging on this species was equivalent to blueberry. Bee Pollination Efficacy Studies Single Floral Visits - Efficacy in pollinating wild blueberry, on a per bee basis, varied by species. This was the case for pollen deposition on floral stigmas after single visits (F(3,43.8) = , P< ) and for handling time of flowers for each visit (F(3,50.2) = 9.377, P< ). Figure 4 shows that pollen deposition on the blueberry flower stigma was significantly greater after a single visit by B. impatiens and O. atriventris, and least by the honey bee, A. mellifera. The amount of time spent handling the flower was greatest for the honey bee and least for the bumble bee and leaf cutting bee. When one considers both pollen deposition / visit and handling time, the bumble bee and Osmia leaf cutting bee were the most efficient pollinators tested on wild blueberry. Javorek et al. (2002) showed similar results of bee pollination efficiency with several of the same taxa associated with wild blueberry. Sequential Floral Visits - When sequential visits were assessed by honey bees, bumble bees, or a mixture of both bee species, we found that honey bees visiting a flower previously visited by a bumble bee significantly (F(3,33.8) = , P< ), increased the amount of pollen deposited on the stigma of the subsequent flower visited. This pattern was not seen for bumble bees visiting previously visited flowers by bumble bees or honey bees (Figure 5). Foraging Studies Diurnal Foraging Patterns Of Bees In Wild Blueberry - Figure 6 shows that during the peak bloom, honey bees do not start foraging until mid-morning and they terminate foraging by early evening. The megachilids (mostly Osmia spp. during this study) were similar to honey bees in their foraging periodicity. The halictids had a shorter foraging period, starting in the midmorning and ending by mid-afternoon. The bumble bees started the earliest of the foraging bee community and continued until dark. The digger bees (Andrena spp.) also started foraging around dawn, but they finished foraging by early evening. Response To Air Temperature - Air temperature was often found to be a major determinant of bee foraging, however, light intensity, precipitation and wind can also contribute. We found that honey bees and native solitary bees as groups responded in their foraging activity to increasing air temperatures (F (1,26) = , P<0.0001, and F (1,26) = , P< ; solitary native bees and honey bees, respectively). The proportion of the variance in foraging activity for solitary native bees

9 Int. J. Entomol. Nematol. 034 Figure 4. Average pollen deposition on floral stigmas after a single visit and flower handling time by common wild blueberry pollinators: bumble bees, B. impatiens, digger bees, Andrena carlini, leaf cutting bees, O. atriventris, and honey bees, A. mellifera. Bars with the same letters (uppercase handling time, lowercase pollen deposition) are not significantly different, Tukey s test, experiment-wise error rate at P< Figure 5. Pollen deposition on a floral stigma by a bumble bee visiting a flower previously visited by a bumble bee and then visiting a new flower (BB/BB), or by a bumble bee visiting a flower previously visited by a honey bee and then visiting a new flower (HB/BB), or by a honey bee visiting a flower previously visited by a honey bee and then visiting a new flower, or by a honey bee visiting a flower previously visited by a bumble bee and then visiting a new flower (BB/HB). Bars with the same letters are not significantly different, Tukey s test, experiment-wise error rate at P< and honey bees was 44.1 and 79.1%, respectively. It can be seen in Figure 7 that the threshold for foraging of native solitary bees was approximately 8ºC and that for honey bees was approximately 11ºC. Bumble bee queen foraging did not appear to respond to increases in air temperature (P> 0.05). This can be expected due to the thermoregulation exhibited by bumble bees, especially queens (Heinrich, 1972a; 1972b; 1972c). Bee Spatial Foraging Patterns Within And Among Clones - The foraging bee taxa that we observed had

10 Drummond FA. 035 Figure 6. Diurnal foraging periodicity of honey bees and native bees, May, 2001 in Winterport, Maine. Figure 7. Relationship between air temperature at time of sampling and the percent of a specific taxon group s population (native solitary bees, bumble bee queens, and honey bees) for a given day. very different spatial patterns (Table 2). For instance, the cardinal direction that a forager took upon leaving a stem to move to the subsequent stem was fairly random and non-directional for bumble bees and honey bees, but almost unidirectional as if bees were following a trap line for Andrena spp. The number of flowers per stem visited were low for honey bees (mean = 1.6 flowers / stem, range 1-16 flowers visited per stem) compared to bumble bees (mean = 2.5 flowers / stem, ra nge = 1-27 flowers visited per stem), Osmia spp. (mean = 2.2 flowers / stem, range = 1-7 flowers visited / stem), and Andrena spp. (mean = 2.7 flowers / stem, range = 1-10 flowers visited / stem). The average distance that honey bees moved between stems for foraging was 0.1 m, while Bombus spp. was 0.3 m, Osmia spp. was 0.4 m, and Andrena spp. was 1.7 m. An example of the distribution of flowers visited per stem between honey bees and Andrena spp. is shown in Figure 8.

11 Int. J. Entomol. Nematol. 036 Table 2. Probability density function fit to the frequency distributions of the number of stems per bout, distance between stems (m), cardinal direction to the next stem, and flowers visited per stem for four taxa of common bee pollinators of wild blueberry in Maine. bee taxon group Osmia spp. Andrena spp. Bombus spp. Honey bee, A. mellifera sample size of measure measure distribution parameters goodness of fit* 47 stems / bout Gamma (P) Poisson 45 distance between stems Gamma (m) 38 cardinal direction Gamma (P) Poisson 87 flowers visited / stem Poisson (P) (CM) 15 stems / bout Exponential (K) 25 distance between stems (m) Weibull 24 cardinal direction Gamma Poisson (LR) (K) 81 flowers visited / stem Exponential (LR) 173 stems / bout Log Normal u = (D) = distance between stems Weibull (m) 2508 cardinal direction uniform na** na 2536 flowers visited / stem Beta 610 stems / bout Beta 4509 distance between stems (m) (LR) (K) (K) Exponential (LR) 4903 cardinal direction uniform na na 5333 flowers visited / stem Poisson (P) * probability value for goodness of fit to theoretical distribution, (test statistic) = P: Pearson chisquare, CM: Cramer von Mises, K: Kolmogorov s D, LR: adequacy LR test. ** na: not applicable. DISCUSSION Over 120 species of bees have been found to be associated with wild blueberry in Maine (Bushmann and Drummond, 2015). Little is known, however, about their behavior and activity during the blooming season. The collection of studies, reported here, begin to shed light on how individual bee species or groups of species play a role in wild blueberry pollination. There are several major areas that I would like to address in this discussion. First of all, it is apparent that bees, as shown by our work with bumble bees, make choices both at the blueberry flower level, but also at the stem floral density level and they make choices between competing flowering shrub species and wild blueberry flowers.bumble bees (B. impatiens) apparently choose flowers according to size, the longer (or wider, as the two traits are highly correlated)were preferred. Blueberry flowers reflect UV light (Schaefer et al., 2004) and is attractive to several species of bees (Kevin and Baker, 1983); thus, it is not surprising that such a preference takes place during foraging. It was surprising to see that flower age did not affect the choice in foraging bumble bees. Despite intact floral resources and most likely not lower than younger flowers, it has been demonstrated that wild blueberry flowers lose their receptivity to pollen over a 8-10 day period with maximal receptivity only

12 Drummond FA. 037 Figure 8. Frequency distribution for distance between stems moved by honey bees (a) and Andrena spp. (b). lasting for 4-5 days (Drummond, unpublished data). It has been shown that as many flowers age and start to senesce they change in color and become less attractive to bees (Gori, 1989). This could be interpreted as a plant signaling phenomenon, beneficial to the plant and the bees (Casper and La Pine, 1984). I found a nonsignificant trend (P = 0.106), which suggests that rejection of older resource depleted flowers by bees might be occurring among a background of relatively rich nectar and pollen resources. In most bee pollinated crops the choice of visitation may not operate in the field since most crops are monocultures of a single genotype, unlike wild blueberries (Bell et al., 2009). Wild blueberry is a mass flowering crop that produces up to 8,000 flowers / m 2 (Jones et al., 2014). Flower production per stem can vary greatly from clone (genet) to clone (Bell, 2009). Therefore, despite a managed field being a continuous carpet of plants or clones, flower density varies spatially across fields. We found that bumble bees, and we hypothesize that this is the case with other bee taxa, recruit preferentially to clones with a higher density of flowers. The proportion of variance in bumble bee foragers recruiting to clones was high (r 2 = 0.418), which suggests that there may be a selective advantage to individual plants or genotypes to produce as many flowers per stem as possible as has been suggested in other mass flowering plant species (Augspurger, 1980). One would also presume that this is an advantage to foraging bees as high floral density should minimize the energy output per unit energy acquired from flowers per unit of time (Waddington et al., 1979). The last of my findings relative to flower choice by bumble bees was contrary to the findings of previous studies (Goulson et al., 1998; Stout et al., 1998). It has been observed in other studies that bumble bees tend not to visit flowers that have previously been visited as frequently. It is often suggested that several species of bumble bees mark flowers when visiting them and this chemical signal is thought to communicate a lower level of floral resources to subsequent bees potentially decreasing the chance that they will visit a flower previously visited and marked (Cameron, 1981). In two different experiments we did not find any evidence that suggests this is happening with bumble bees visiting wild blueberry flowers multiple times. Wild blueberry flowers each have on average ovules (Bell et al., 2012a). Flowers may need to be visited several times by bees in order to result in enough ovules fertilized by deposited pollen grains for fruit to be retained by the plant. Thus mechanisms that would lower the chance of multiple visits might reduce fruit production. However, this is probably not the case with bumble bee queens, probably the most efficient pollinator of wild blueberry (see below). Bushmann and Drummond (2015) demonstrated that there is great floral constancy in native bees in wild

13 Int. J. Entomol. Nematol. 038 blueberry fields. We had hypothesized that this might be due to the ratio of wild blueberry flowers in a location compared to other flowering plants that might compete for bees adjacent to where blueberries grow. However, our cage study suggests that at least with five species of woody shrubs that overlap with blueberry bloom there is a moderate degree of preference for visiting and foraging on wild blueberry (3 of 5 species preferred and 4 of 5 species foraged on more intensely). The flowering shrubs that are less preferred relative to blueberry might be good candidate species for pollinator plantings used to enhance densities of native bees for wild blueberry pollination (Venturini, 2015). However, these species would be important during the every other non-flowering year of blueberry production. Under typical wild blueberry management, flowers only occur every other year in the two year production cycle, the other year being the prune year where plants are in a vegetative growth phase (Yarborough, 2009). Not all bees appear to be equal as pollinators of wild blueberry. Our field cage studies suggest that when measuring both pollen deposition per visit and flower handling time, queen B. impatiens and O. atriventris are more efficient that A. carlini and A. mellifera. The nonnative honey bee (A. mellifera) is the least efficient, but this bee is often placed in fields during bloom providing a foraging force of hundreds of thousands per hectare (up to 10 colonies per hectare, Drummond, 2012a). Therefore, in this case, the inefficiency of the species as a pollinator of wild blueberry can be countered by its high density, a factor often overlooked by pollination ecologists. It is most likely the poricidal anthers (Bell et al., 2009) that make wild blueberry a difficult flower for honey bees to extract and then deposit pollen. Our observations suggest that honey bees exclusively nectar feed on wild blueberry flowers and lack a behavioral repertoire for manipulating the anthers; thus, they only extract pollen inadvertently while probing nectaries. The native bees we studied had behavioral mechanisms for extracting copious amounts of pollen. Bumble bees and andrenids vibrated (buzz pollinated the flowers; Buchmann et al., 1983) wild blueberry flowers and Osmia leaf cutting bees crawled into the flowers and drummed the anthers with their forelegs displacing pollen. I found that the efficiency of the honey bee as a pollen vector can be enhanced if the honey bee visits a flower previously visited by a bumble bee and then subsequently visits a new flower. We think that this might be due to the following phenomenon. When a bumble visits a flower it either vibrates the flower releasing a large amount of pollen that coats the interior of the flower or a bumble bee that has previously vibrated a flower itself gets coated with pollen on its head and abdomen, then when nectar-feeding, the bumble bee will coat the interior of the flower. Now when a honey bee visits a flower to nectar feed it might transfer much of the pollen residue from the inside of a flower to the stigma. This is a conjecture that will need to be tested. Another aspect of pollination efficiency might be the spatial pattern of bee movement between stems and within a stem between flowers. Wild blueberry flowers, for the most part are obligate out-crossers, except for % of clones in a field that have the capacity to self fertilize (Bell et al., 2010; Bell et al., 2012b). The implications of this is that bees that forage with larger intra-stem movements and fewer visits of flowers per stem, and with directional movement in a blueberry field (i.e., not randomly moving independent of cardinal direction) should be a better pollinator from the plant s perspective. My data suggests that honey bees might not be the best pollinator relative to its random movement and short distance moved between stems. The bumble bee might not be the best pollinator relative to its random directional movement. The andrenids might not be the best pollinator relative to the number of flowers that they visit on a stem, but this might be the best in terms of their long flights made between stems. These attributes along with the efficiency of pollen deposition and flower handling time will have to be tested with simulation in order to determine which bee species might be the best pollinators in the wild blueberry ecosystem. The last aspect of bee behavior that I looked at was diurnal foraging and how temperature affects bee activity. Certainly, different taxa of bees forage at different times of day, but all taxa that we observed foraged during the middle of the day during what would be suspected of being the warmest time of the day. Early morning after dawn and early and late evening had a lower percentage of each taxon actively foraging and some bees such as honey bees and megachilids were not seen at these times. This is most likely due to air temperatures (Corbet et al., 1993), but also possibly due to light levels, although this is best documented for crepuscular bees (Kelber et al., 2006). We observed that the solitary bees and honey bees that we sampled increased in activity in relation to increasing temperature. Bumble bee queens appear to be active, for the most part, independently of air temperature. This is most likely due to their ability to thermoregulate and increase their internal body temperature metabolically (Heinrich, 1972a). A question might be asked how climate change might affect bee foraging in wild blueberry. The first response might be that warmer temperatures during bloom would increase the length of time during the days that bees forage on flowers, increasing pollination. However, during our study we did not have exceptionally high temperatures during the day. From personal experience during the summer when temperatures can occasionally reach > 30ºC, bumble bees will stop foraging at mid-day and then commence foraging in the evening. If air temperatures during bloom (mid-may to mid-june) in

14 Drummond FA. 039 Maine increase dramatically, then, at least for some bee taxa, foraging activity might decrease. This could be detrimental for the range of some species of bumble bees as has been documented by (Kerr et al., 2015). CONCLUSION An understanding of crop pollination requires an understanding of the interactions between: bees and crop plant reproductive biology, as well as among morphology, bee species, the abiotic environment and bee behavior and plant behavior and physiology. This work reports on several isolated studies that begin to increase our understanding of several of these interactions. The contribution of these studies is in providing quantitative relationships that can be used to construct a computer simulation model of bee pollination of wild blueberry. Specifically, I have shown that bumble bees discriminate and select larger flowers, but that flower age or whether flowers were previously visited did not affect flower selection. Flower density does affect long-distance recruitment to clones, with clones characterized by higher flower density receiving higher bee visitation. I also found that blueberry flowers are much more attractive than five other native co-flowering shrubs. This is important because highly attractive shrubs that flower at the same time as blueberry might pull bees away from the blueberry crop during pollination. Another important finding is that native bees are much more efficient, on a per bee basis, than the introduced honey bee at pollinating blueberry, in terms of flower handling time and pollen deposition on the stigma of the flower. However, the presence of bumble bees visiting flowers prior to honey bees appears to increase the pollination efficacy of honey bees. The last important finding in this study that has direct implications to modeling pollination is that I found differential lengths of time during the day that different taxa foraged on blueberry and that this was partly explained by air temperature. Bee movement in blueberry fields was found to be dependent upon the taxon. This aspect of bee foraging has not been investigated in simulation models constructed to investigate the dynamics of crop pollination. I believe that all of my findings will allow the construction of a preliminary model. ACKNOWLEDGEMENTS I would like to thank the assistance of Constance Stubbs, Stephanie L. Allard, Lisa Campbell, Judith Collins, and several undergraduate research assistants that helped on data collection for these studies over the past 20 years. This is Maine Agricultural and Forest Experiment Station Publication3500.Financial support for some of the objectives in this project ( ) was provided by the U.S. Department of Agriculture National Institute of Food and Agriculture - Specialty Crops Research Initiative Contract/Grant/Agreement No REFERENCES Alexander MP (1980). A versatile stain for pollen, fungi, yeast, and bacteria. Stain Technol. 55 (1): Asare E (2013). The Economic Impacts of Bee Pollination on the Profitability of the Lowbush Blueberry Industry in Maine. Electronic Theses and Dissertations. Paper etd/2038 Augspurger CK (1980). Mass-flowering of a tropical shrub (Hybanthusprunifolius): influence on pollinator attraction and movement. Evol.: Bell DJ (2009). Spatial and genetic factors influencing yield in lowbush blueberry (Vaccinium angustifolium Ait.) in Maine. PhD dissertation, University of Maine, Orono, Maine, 144 pp. Bell DJ, Rowland LJ, Smagula J, Drummond F (2009). 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