In-hive pollen transfer between bees enhances cross-pollination of plants

Transcription

1 LABORATORY OF ENTOMOLOGY In-hive pollen transfer between bees enhances cross-pollination of plants No.: Janneke Paalhaar May January 2007 Examinator: Marcel Dicke Wageningen UR 1

2 In-hive pollen transfer between bees enhances cross-pollination of plants Analyses of pollen on honey bees No.: Janneke Paalhaar Reg.nr.: MSc International Development Studies Thesis Entomology ENT May 2006 January 2007 Supervisors: Willem Boot, Johan Calis & Sjef van der Steen Examinator: Marcel Dicke 2

3 Preface This thesis is the outcome of research at the chair group of Entomology, Wageningen University. My direct supervisors of the study were Johan Calis and Willem Boot, working for their beekeepers firm located in Wageningen. They assisted me during the 20 weeks of preparation, experiments, analyses and writing. It was an interesting and intensive learning process, but certainly not always easy. Earlier research on in-hive transfer of pollen is difficult to interpret, because in these studies pollen from many different species with unknown age was found on the bees. In this study we chose a new experimental concept and exposed bees to pollen from three species of plants that were not yet present on the bees at the start of the experiment. Therefore, the conclusions in this report are inventive and interesting to read. I would like to thank the following people who supported me during the research. First, I would thank Johan Calis and Willem Boot, for providing me the assignment and additional, valuable information. Also for the effort they put into the research by arranging the locations for the experiments and by assisting me with collecting bees. Further, I would like to thank Sjef van der Steen and Jeroen Donders, working at the Plant Science Group, for their assistance by analysing and determining pollen on bees. I also thank Hans Smid for using his equipment and his contribution by making pictures of pollen. Further, I appreciate the help of Tibor Bukovinszky with the statistical analysis. Finally, I would like to thank Rijk Zwaan who offered me the opportunity to do the experiments in green houses in Fijnaart and De Lier. Wageningen, December 2006 Janneke Paalhaar 3

4 Summary Self-incompatible plant species need pollinators, often insects, for successful pollination. Different plant species have different strategies to attract these insects. Together with other bee species, honey bees are well known pollinators of plants. Pollen of plants can adhere to the body hairs of honey bees as they visit the flowers and can last their for a long, depending on the characteristics of the specific pollen grains (Free and Williams, 1972). Pollen from their hairs may lead to direct cross-pollination when flying from one plant to another. However, this way of cross-pollination by honey bees may not lead to the pollination of plants of the same species growing far from each other. In-hive transfer of pollen can also take place, directly, by brushing against each other, or indirectly due to eating, working and walking over the combs in the hive (Free and Williams, 1972). In-hive transfer of pollen between honey bees may enhance indirect crosspollination to an unequaled level, because honey bees forage on patches of flowers that can be more than nine kilometers from the hive (Beekman and Ratnieks, 2000). In this way bees can potentially bring pollen from one patch of flowers to another patch of flowers of the same species over huge distances. In this study in-hive transfer of pollen was researched by analysing the pollen from the bodies of different age-groups of bees from one colony, and from bees of the same colony foraging in a green house. Samples of these bees were taken every two hours during one day. The hive was located in a green house with only three plant species. Analysing and counting the amounts of pollen was done for individual bees in the laboratory. About 1 % of the pollen on foragers was found on the bodies of pre-foraging bees inside the hive. When a similar amount will be transferred in the hive to other foragers, in-hive transfer of pollen enhances cross-pollination of plants. 4

5 Table of contents Preface...3 Summary...4 Table of contents Introduction Materials and methods Experiment Analyses Results Overall comparison Activity of foragers Different pollen species Flow of pollen Discussion Different crop species Different pollen species Over one night Flow of pollen References...18 Appendix 1: Graphs of pollen flows and significant differences...20 Appendix 2: Tables of results...29 Appendix 3: Controls

6 1. Introduction Pollination is necessary to evolve, for all kind of plants species in nature and under cultivation. Plant species differ in how they get pollinated. Some plant species are selfcompatible, others self-incompatible. Self-incompatible species, like apple, almond, carrot and sunflower, need to be pollinated with pollen of another individual of the same species; called cross-pollination. Besides bats, birds, flies and beetles, bees play a major role in pollination. Flying from one plant to another, pollen from their hairs may lead to direct crosspollination (Ish-Am and Eisikowitch, 1998). Bees may also shift from one patch of plants to another patch of plants of the same species which also may enhance cross-pollination (Roubik, 1999). In social bees there is another way that can lead to cross-pollination; through in-hive transfer of pollen. As different bees from one colony will forage on different locations, pollen from these different locations will be brought into the colony. Through inhive transfer of pollen, bees may bring pollen from one patch of plants to another patch of plants of the same species and enhance cross-pollination. Specifically honey bees (Apis mellifera L.) are known to forage large distances from the hive (Beekman and Ratnieks, 2000). If in-hive transfer of pollen occurs, this potentially makes cross-pollination by honey bees unequalled in nature. However, a prerequisite for long-distance cross-pollination is significant transfer of pollen from forager to forager inside the hive (Ish-Am and Eisikowitch, 1998). A lot of information is available about cross-pollination by honey bees. Many studies conclude that cross-pollination takes place according to hybride seed or fruit setting in selfincompatible plant species. For example Dag et al. (2001) introduced two-opening hives with caged flowering mango plants and did a parental analysis of the progeny. They concluded that indirect cross-pollination took place by in-hive transfer of pollen, assuming that individual bees choose the same opening every single trip. Another study analysed seed set on sunflower male-sterile plants (DeGrandi-Hoffman and Martin, 1993), but could not conclude if direct or indirect cross-pollination played a major role, because both occurred. The percentage of almond blossoms setting nuts was measured by DeGrandi-Hoffman et al. (1992). They concluded that indirect cross-pollination took place, because fruit set was homogeneously distributed throughout an orchard with different cultivars. Finally, seed set on male sterile cotton flowers was studied (Loper and DeGrandi-Hoffman, 1994). But there were insufficient numbers of bees returning to their colonies with significant amounts of cotton pollen on their bodies to ensure effective transfer of cotton pollen among nestmates in the hive. These studies were not clear about to what extend in-hive transfer of pollen contributed to cross-pollination. In other studies bees in- and outside the hive were analysed in stead of the pollinated plants. Young bees were captured and pinned upon the combs where they received a particular amount of pollen (Free and Williams, 1972). In another study hive-entrance bristles were used to show that the amount of foreign pollen adhering to honey bees departing their colony increased by these devices (Hatjina et al., 1998). However, these studies were done in open environments, accessible to many different animal species, wind and raining water, and the available pollen originating from a wide variety of plants. Further, pollen can remain on the bodies of honey bees for a long period, because they have difficulties in grooming some parts of their bodies (Free and Williams, 1972). Therefore, significance of in-hive transfer of pollen in relation to cross-pollination is still not clear. The purpose of this study was to determine to what extend in-hive transfer of pollen may contribute to cross-pollination. The flow of pollen from plants to one hive and its inhabitants during one day was studied. To limit interactions between plants and bees, clear conditions were set in this study. A green house with windows sealed with insect-gauze and 6

7 the exposure of the bees to previously inexperienced pollen from three plant species, was chosen to conduct the experiment. With these conditions the pollen on the bodies of foraging and pre-foraging honey bees were quantified to gain insight in the flow of pollen during one day. If pre-foraging bees have pollen grains on their bodies and if the density relates to the density of pollen on the bodies of foragers, in-hive transfer of pollen takes place and enhances cross-pollination. 7

8 2. Materials and methods 2.1 Experiment As a preparation for the experiment, different age-groups of one honey bee colony (about bees) were distinguished, because of the development of their different functions within the colony (Seeley, 1995). In the three consecutive weeks before the experiment three brood combs without adult bees were removed from the hive (10 combs) every Tuesday. These brood combs were put in a stove at 35 C for 24 hours. The new-born adult bees were marked on their thorax with a colour. As a result four cohorts of bees, with the age of 1-2 days, 8-9 days, days and days, got different colours respectively green, yellow, red and blue (table 1). After marking, these adult bees were put back into their original hive immediately, except the young bees of 2 days old. These new-born bees serve as a control group for the experiment and were returned to their original hive as the experiment starts the morning after marking this cohort. Table 1: Marking age-groups Age of groups (days) Colour of marking point Defining groups during the experiment blue foraging bees/foragers red foraging bees/foragers 8-9 yellow pre-foraging/young bees 1-2 green pre-foraging/young bees Half a day before the experiment started the hive was isolated from the environment to reduce the amount of strange pollen on the bees bodies. On Thursday 6 July 2006 the hive was placed in a green house with three plant species flowering; carrot, celery and endive. The windows in the green house were sealed with insect gauze to prevent bees from flying in or out the green house. The flowers of celery and endive plants both offer nectar as well as pollen to the bees. Because bees foraging on these crops always receive pollen, they are called pollen foragers. In contrast, two different types of carrot plants were planted in rows to create hybrid seeds with characteristics of both plants. The flowers of one type only produced nectar, while the flowers of the other type offered nectar and pollen. Bees foraging on the former plant are called nectar foragers and bees foraging on the latter plant are called pollen foragers. Over a period of 14 hours samples were taken every two hours; from 8:00 until 22:00. Another sampling was done at 8:00 the next day. A sample includes 10 green bees (2 days old) and 10 yellow bees (1 week old) out of the hive. Together these bees are called young or pre-foraging bees, because they were not seen outside the hive during the day. The other marked groups of bees (red and blue bees) were seen outside the hive and were therefore no representatives of in-hive bees. The sample also includes 30 pollen foragers, 10 nectar foragers and 10 water foragers. The young bees were caught on one of the combs in the middle of the colony. The pollen foragers were caught on the three different crops while they were blooming. The nectar foragers were caught on the carrot plants that only offer nectar, no pollen. Finally, the water foragers were caught at the edge of a water basket. The bees were caught by their wings with a pair of tweezers. They were put in individual Eppendorf tubes and stored in a freezer, directly after sampling. Foragers were caught from different crops. Some of the foragers just arrived at the plant while others were foraging for a longer period. As a consequence, the duration of their 8

9 presence on the flowers was not always the same and this should be taken into account. It can have effect on the amount of pollen on the body of the bee. Catching returning bees at the hive-entrance was not an option, because it would have been unclear then from which crop they returned. To determine if there is a relationship between the size of the foraging population and the amount of pollen grains on the bodies of bees inside the hive, all the bees landing in 5 min on the landing board of the hive were counted, once every period of 2 hours. 2.2 Analyses To determine the pollen density on the bees body hairs, the pollen were removed from the unfrozen bodies. For this procedure a modification of Hatjina et al. (1998) s method was used. To start, the hind legs of foraging bees were cut off to prevent that pollen in the corbiculae will be analysed. The bees were individually sonicated for 10 sec in 1.0 ml of a mixture of ethanol, water and detergent (1% vol./vol. Triton-X100 in a 70% ethanol solution) with a Sonifier B-12, micro-tip level 3. After removing the bees the tubes with the ethanol solution were centrifuged for 3.5 min on level 14 (*1000 min -1 ), the supernatant were discharged, and 50 µl distilled water was added to the remaining pollen in each tube. Tubes were vortexed for 1 min in the highest rotation to ensure a homogeneous distribution of pollen in the solution. Then 18 µl from each tube was dropped with a pipette on a haemocytometer slide; Neubauer improved and analysed with an Euromex-microscoop. When the density of pollen in the solution was low, for example in the monsters of young bees, the number of pollen grains on the total slide grid (3,6 % of the 50 µl) was counted, and the type of pollen was determined. While analysing samples with a high density of pollen, for example monsters of pollen foragers, only 1,0 % or 1,8 % of the solution was analysed. The total number of grains in the original pollen precipitate of each tube was derived under the assumption that the analysed solution was representative for the whole sample. The efficiency of the sonication removal method was tested by repeating the sonication up to four s per bee (10 bees in total). An average of 67 % (43 %-91 %) of the total pollen grains was removed from the bees by the first sonication. After a second wash treatment another 21 % percent was removed, 8 % percent was removed after the third and 3 % percent of the total pollen grains was removed after the fourth wash treatment. Only 1 % was left after 4 wash treatments, see appendix 3. One sonication was used routinely to dislodge pollen from the bodies of bees. Therefore, we should realise that the total amounts of pollen named in this report refer to an average of 67 % of the pollen on the bees bodies. The efficiency of the centrifuge method was tested by analysing the supernatant discharged from the pollen precipitate of 10 individual bees. 0 % of the total pollen grains adhering to a bee s body were still present in the supernatant. This means that all the pollen were deposit in the precipitate and stayed in the tube when supernatant was removed. Also the efficiency of vortexing the tubes with pollen and distilled water was tested to be sure that all the pollen was re-suspended. In fact 90 % of the pollen was re-suspended in the water again. With the program SPSS and the SAS System the pollen density on the bodies of 390 bees were analysed statistically. A final note has to be made about the sonication process. During the sonication process of the bees, some of them damaged by the power of this step. Therefore, the solution was spoiled with contents of the gut and further analysis was impossible. These bees are involved in the results as missing values. 9

10 3. Results 3.1 Overall comparison In table 2 the mean square and the significance are calculated for the sets of data. The dependent variable is the total amount of pollen found on the bodies of bees. To meet the normality assumption required for variance analysis, this variable was Ln transformed. The data was analysed with a General Linear Model for balanced dataset with "type of bee", "pollen species" and "" as fixed factors and with all possible interactions included. To make further comparisons of interest, post-hoc tests were carried out by using the Tukey test. All comparisons were tested at the <0.05 for significance. Data analyses was carried out using SAS The main factors "pollen species" and "type of bee" are significant, whereas the did not influence the amount of pollen collected. Furthermore, significant interaction indicated that amount of pollen depended on which type of bee collected which species of pollen. Also, different types of bees collected different amounts of pollen in the course of in the experiment. The amounts of different species of pollen collected followed a similar pattern over. Finally, there is a difference between different types of bees, with different amount of pollen of different species over the! This means that the data in general are different. Table 2: Comparison of sets of data Mean square Significance pollen species <0.001 type of bee < pollen species * type of bee <0.001 pollen species * type of bee * pollen species * type of bee * Activity of foragers The experiment was done on a sunny day on which the temperature rises to a high level inside the green house. The maximum temperature was reached between 14:00 and 18:00 o clock in the afternoon. In this period it was somes hard to catch enough bees on celery plants. After 14:00 o clock we could not find any bee on the endive plants anymore. Catching foragers on carrot plants was no problem during the whole day. The activity of foraging bees during the day was variable. The bees were released and started foraging at 8:00 o clock, and they stopped foraging at 21:00 o clock after which the hive was closed. The period with the highest activity was around 12:00 o clock (see figure 1). In total forage flights were made during the day (area under the graph in figure 1). The a forager needs for leaving the hive; collecting pollen; and returning to the hive, depends on the location of the plants in relation to the hive. After the forage flight also is needed to supply the pollen to the cells in the hive. With the estimation that the whole cycle takes 15 minutes, the maximum amount of foragers at one is about bees (around 12:00 o clock), which is a substantial part of the colony ( bees). An average amount of

11 carrot, celery and endive pollen was present on the body hairs of those bees foraging on the crops, see table 3. Figure 1: Activity of foragers activity of foragers # foragers coming back to hive :00-8:05 10:00-10:05 12:00-12:05 14:00-14:05 16:00-16:05 18:00-18:05 20:00-20:05 22:00-22:05 during day Type of pollen 3.3 Different pollen species In table 3 the total and average amounts of pollen on the different groups of bees in several periods are presented. More detailed results are showed in appendix 2. Table 3: Quantification of pollen # of pollen on the bodies of 140 pre-foraging bees inside the hive between 10:00 and 22:00 o clock 1-2 days old (70) 8-9 days old (70) average # of pollen on the bodies preforaging bees inside the hive between 10:00 and 22:00 o clock total 1-2 days old 8-9 days old # of pollen on the bodies of 210 foraging bees on crops between 10:00 and 20:00 o clock average # of pollen on the bodies of foraging bees on crops between 10:00 and 20:00 o clock total total 1-2 days old (10) # of pollen on the bodies of 20 pre-foraging bees inside the hive at 8:00 o clock after one night 8-9 days old (10) 11 average # of pollen on the bodies of preforaging bees inside the hive at 8:00 o clock after one night total 1-2 days old carrot celery endive total pollen 8-9 days old At the beginning of the experiment, at 8:00 o clock, no carrot, celery or endive pollen was found on the bodies of pre-foraging and foraging bees (see appendix 2). After starting the experiment carrot, celery as well as endive pollen was found on the body hairs of the young bees inside the hive. Some of these bees had more pollen than other bees, some had a mixture average average

12 of carrot, celery and endive pollen and others only had pollen of only one of these species. The amounts of pollen on the bodies of these bees were relatively low compared to the amounts of pollen on foraging bees. On the pre-foraging bees the average amount of particular pollen at one point of was not significantly different from the average at another point of. After the night young bees were sampled once again at 8:00 o clock in the morning. The amount of endive pollen was zero at that. The average amount of carrot pollen was low; 3 pollen. That means that only 30 % of the carrot pollen was left, see table 4. The percentage of celery pollen that was left on the bodies of pre-foraging bees after the night was high compared to the other pollen species; 93,5 %. Type of pollen Table 4: Pollen transport Percentage pollen on in-hive bees to foraging bees Percentage remaining pollen after one night carrot 0,72 % 30,00 % celery 1,01 % 93,75 % endive 3,08 % 0,00 % On 96 % of the pollen foragers caught at the different crops, pollen of that specific species was found on their bodies. The amount of a particular pollen on the bodies of different foraging groups was different. The range of endive pollen on endive foragers was between 0 and pollen. For celery foragers it was between 0 and celery pollen and for carrot pollen foragers the range was between 0 and carrot pollen. A lot more carrot pollen was found on the bodies of carrot pollen foragers, compared to that on carrot nectar foragers (see appendix 2). Some of the foragers also had different pollen species on their bodies at different points of, but again by none of them the average amount of pollen species at one point of was significantly different than the average at another point of. The water foragers, caught at 14:00, 16:00 and 18:00 o clock, had an average of 47 carrot pollen and 16 celery pollen on their bodies (see appendix 2). No endive pollen was present on the water foragers while the water basket was situated near the endive plants. 3.4 Flow of pollen The amounts of pollen species on specific types of bees were compared over different points of. In the graphs (see appendix 1) you can see the changes over for every group of bees. Only some cases are significant different from others, see appendix 1 for the specific cases. The pollen foragers on the carrot plants had a maximum amount of carrot pollen (7.500) on their bodies at 20:00 o clock, the last that foraging bees were caught. This average was significant different than the average at 18:00 o clock. The foragers on the endive plants had a maximum amount of endive pollen (2.500) on their bodies at 10:00 o clock; the first foraging bees were caught. After this point of average decreased significantly to 500 endive pollen. The foragers on the celery plants had a maximum average amount of celery pollen (10.000) on their bodies at 10:00 o clock. Four hours later (14:00 o clock) this average was decreased significant to celery pollen. Another 4 hours of low average followed, after which the average significantly increased to a second height at 20:00 o clock. It is interesting that there is no relation in between the highest activity of foragers, at 12:00 o clock, and the maximum amount of pollen on foragers, at 10:00 and 20:00 o clock. 12

13 Finally, also strange pollen species were found on the bodies of bees. Especially the small-sized pollen of sweet chestnut were abundantly found. The strange pollen must be 12 hours young or older. Most pre-foraging bees have strange pollen on their bodies during the day and also the morning after (range between 0 and 5723 pollen). The average amount of strange pollen on the bodies of foragers is significantly increasing during the morning and decreasing during the evening (see appendix 1). In figure 2 the different pollen species on the haemocytometer slide are visualised with an Euromex-microscoop (100x). Figure 2: Pictures of pollen species Endive pollen Carrot pollen Celery pollen Sweet chestnut pollen 13

14 4. Discussion From two hours after starting the experiment pre-foraging bees acquired about 1 % previously inexperienced, probably viable pollen, related to the amount of pollen on foragers of the same colony. This means that in-hive transfer of pollen takes place. Therefore, foraging bees of one colony can enhance cross-pollination of plants by visiting different plant patches of the same species. 4.1 Different crop species The first foragers were caught on endive plants was 10:00 o clock in the morning, when the average amount of endive pollen was maximum. The amount of endive pollen decreased significantly till 14:00 o clock, after which the endive plants stop flowering and no endive foragers were present. The average amount of celery pollen on the bodies of celery foragers was significant lower in the period between 14:00 and 18:00 o clock in the afternoon. This was also the period with the highest temperature in the green house and with the difficulty to catch foragers on celery plants. An explanation for this can be that the stock of celery pollen or nectar was running out and it became more difficult for foragers to find sufficient pollen or nectar. In this case honey bees stop foraging and stay in their hive (Free, 1963) with the result that it was more difficult for us to catch celery foragers during this period. The average amount of celery pollen on the bodies of celery foragers increased significantly between 18:00 and 20:00 o clock in the evening. This also happened with the average amount of carrot pollen on the bodies of carrot foragers in the same period. A logic explanation for this can be that the plants opened more flowers after the heat of the day, with the result that more pollen were offered to the foragers. 4.2 Different pollen species Table 3 shows the average amount of pollen on the bodies of foragers on crops outside the hive. According to Free and Williams (1972), the amount of different pollen on foragers is variable, because of differences in the size of grains, in the tendency of grains to adhere to the bee and in the amount of pollen produced by particular plants. Celery pollen are the most abundant grain species on the bodies of foraging bees. Endive pollen on foraging bees have by far the lowest contribution to the total amount. A reason for this can be that the bees quickly lose these big pollen grains. This pattern for the foraging bees is also visible when you look at the average amount of pollen on the bodies of pre-foraging bees, table 3. Celery pollen are the best dispersed among these bees, while endive pollen are present in low amounts again. The fact that there are carrot, celery and endive pollen on the bodies of pre-foraging bees, shows that transfer of pollen takes places; directly by brushing against each other or indirectly due to the combs in the hive. About 1 % of the pollen on foragers could be found back on in-hive bees of which many will probably leave the hive again to forage. In this case cross-pollination of plants by honey bees may occur by transfer of pollen in the hive. For the pollination of plants of the same species growing at different locations this will be the perfect opportunity. It is calculated which percentage of pollen on foragers is found on the body hairs of young bees, table 4. The average of 6 endive pollen on inside-hive bees is 3,08 % of the total endive pollen on foraging bees. This percentage is high, compared to that of celery and carrot percentages. This means that endive pollen are well transferred in the hive. Young bees had strange pollen on their bodies and can receive those in two possible ways. They can get the pollen directly from foragers by brushing against each other inside the hive. Or they can receive the strange pollen indirectly from non-sterile storage and brood 14

15 cells, by cleaning, eating and walking over the combs (Free and Williams, 1972). The 2 days old bees who accompanied their colony at the beginning of this experiment had already strange pollen on their bodies at this. Therefore it was fortunate to work with previously inexperienced sources of pollen. The strange pollen were probably brought in the hive by foragers in the days before the experiment started. The average amount of strange pollen on the different groups of foragers is comparable with the average amount of each particular pollen species on that group of foragers. For example the average amount of carrot pollen on the bodies of carrot pollen foragers is comparable with the average amount of strange pollen on these bees. This means that there are a lot of strange pollen on the bodies of these foragers. This phenomenon can be explained by the fact that these strange pollen are for a large part sweet chestnut pollen, which are very small compared to celery, carrot and endive pollen. Small pollen will probably disappear much slower than bigger pollen. The fact that the average amount of strange pollen on the bodies of foragers is increasing during the morning and decreasing during the evening can be explained in two ways. Foraging bees receive strange pollen by walking over the combs inside the hive while they deposit their pollen baskets. Or it is also possible that different foraging bees became active on different s during the day and groomed their bodies when they were active foragers. 4.3 Over one night Another aspect shown in table 3, is the amount of pollen on the young bees after one night (10 hours) inside a closed hive. The average amount of pollen per species on the young bees was determined at 8:00 o clock the next morning. Only the average amount of endive pollen was zero and the average amount of celery pollen was the highest compared to carrot pollen. There seems to be a species specific rate of disappearance. The size of the grains of endive pollen is much bigger than the size of carrot grains, which are again bigger than the grains of celery pollen. Size can be a reason why endive pollen on the bodies of bees disappear much quicker and celery pollen remains longer on the bodies. 4.4 Flow of pollen The amount of specific pollen on foraging bees is visualised in figure 3 by coloured arrows from the crops towards the hive. This arrow is the biggest for the flow of celery pollen, because celery was the most abundant pollen on the bees. The numbers are the average amounts on one forager. Also the remaining pollen after one night is shown with arrows in figure 3. There is no flow of endive pollen to the next morning, but there is one for celery and carrot (94 % and 30 % is remaining respectively). Finally, according to the pollen found on pre-foraging bees, arrows are drawn back from the hive to the crops. An average of 10 carrot, 16 celery and 6 endive pollen was found on one in-hive bee, see figure 3. This is 0,72 %, 1,01 % and 3,08 % related to the pollen on foragers respectively. These pollen were transferred from foragers to pre-foraging bees inside the hive, but can also represent the transfer to other foragers inside the hive. Therefore, it says something about the pollen flow from the hive to the different crops. As you can see in the appendix 1 and 2 it is determined that there are particular pollen on foragers who are not collecting on that species. For example, celery pollen is found on carrot pollen foragers, carrot pollen is found on endive foragers and endive pollen is found on carrot nectar foragers. There are two possible explanations for this. One is that these other pollen is transferred to them inside the hive by other foragers. The second explanation is that they made a mistake and went to the wrong flower patch. An average of 282 celery pollen is found on carrot pollen foragers and an average of 262 carrot pollen is found on celery 15

16 foragers. However, only 16 celery pollen and 11 carrot pollen (average) were found on the young bees inside the hive (see appendix 2). Therefore, one can assume that the carrot and celery foragers made mistakes between both crops. A reason for this can be that these two crops were next to each other in the green house. In figure 3 two black arrows are drawn between the carrot and the celery plants to show the percentage of pollen acquired by mistake. If 16 of the 277 celery pollen on carrot pollen foragers are acquired in the hive, 94 % is acquired by mistake. If 10 of the 262 carrot pollen on celery foragers are acquired in the hive, 96 % is acquired by mistake. There were less carrot pollen on the carrot nectar foragers compared to those on the carrot pollen foragers, but more compared to the carrot pollen on in-hive bees. Therefore, these nectar foragers made mistakes and are, in the same, good cross-pollinators. Figure 3: Pollen flows carrot 3 celery 15 carrot celery carrot 262 celery 282 endive 2 carrot 10 celery 16 endive 6 endive 195 Carrot plant carrot 10 Celery plant celery 77 Endive plant 16

17 However, endive plants grew separately and mistakes were not usual. It is calculated that an average of 10 carrot pollen was found on the body of a forager from an endive plant. This amount is comparable with the pollen density on the bodies of the young bees. Also celery pollen were found on endive foragers, both visualised by arrows in figure 3. Further, the average amount of endive pollen on nectar foragers (2) was comparable with the average amount of endive pollen on pre-foraging bees (6). Also the average amount of carrot (47) and celery pollen (16) on water foragers is comparable with the pollen density on the pre-foraging bees. Therefore, it is likely that these pollen are transferred inside the hive. The fact that there were no endive pollen on the water foragers, while the basket was situated near the endive plants strengthen this conclusion. Finally, the following conclusions can be drawn. Different crop species have different strategies to attract insects for pollination, and their pollen have different shapes and characteristics. Some pollen species remain longer on the bodies of honey bees than other species, which has consequences for the transfer of pollen in the hive. Probably, the duration of viability will vary for different pollen, which is important to realise. But at least, in this study, we know the age of the different pollen grains on the bees. An answer can also be given on the question if in-hive transfer of pollen takes place. Besides honey bees make mistakes in the location they were foraging on, honey bees transfer pollen inside the hive. Pre-foraging as well as foraging bees have pollen on their bodies acquired from other foragers, directly or indirectly. These conclusion can be made because the age of the bees could be determined, the supply of pollen was limited and the offered pollen species were not available outside the green house at that. Because in-hive transfer of pollen takes place at a significant level, honey bees enhance cross-pollination. Foraging honey bees can cover a great distance from their hive and can lay genetic contacts between plants of one species growing far from each other, by transferring pollen from one plant, through in-hive transfer of pollen, to another plant. 17

18 5. References Beekman M. and Ratnieks F.L.W. (2000) Long-range foraging by the honey-bee, Apis mellifera L. In: Functional Ecology, Vol. 14, Is. 4, p Dag A., Degani C. & Gazit S. (2001) In-hive pollen transfer in Mango. In: Acta Horticulture, Vol. 561, p DeGrandi-Hoffman G., Thorp R., Loper G. & Eisikowitch D. (1992) Identification and distribution of cross-pollinating honey-bees on almonds. In: Journal of Applied Ecology, Vol. 29, p DeGrandi-Hoffman G. & Martin J.H. (1993) The size and distribution of the honey bee (Apis mellifera L.) cross-pollinating population on male sterile sunflowers (Helianthus annuus L.). In: Journal of Apicultural Research, Vol. 32, Is. 3-4, p Free J.B. (1963) The flower constancy of honeybees. In: Journal of Animal Ecology, Vol. 32, Is. 1, p Free G.B. & Williams I.H. (1972) The transport of pollen on the body hairs of honeybees (Apis mellifera L.) and bumblebees (Bombus spp.). In: Journal of Apicultural Research, Vol. 9, p Hatjina F., Free J.B. & Paxton R.J. (1998) Hive-entrance pollen transfer devices to increase the cross-pollination potential of honey bees. I. Examination of six materials. In: Journal of Apicultural Research, Vol. 37, Is. 4, p Ish-Am G. & Eisikowitch D. (1998) Mobility of honey bees (Apidae, Apis mellifera L.) during foraging in avodaco orchards. In: Apidologie, Vol. 29, p Loper G.M. and DeGrandi-Hoffman G. (1994) Does in-hive pollen transfer by honey bees contribute to cross-pollination and seed set in hybrid cotton? In: Apidologie, Vol. 25, Is. 1, p Roubik, D.W. (1999) The foraging and potential outcrossing pollination ranges of African honey bees (Apiformes : Apidae; Apini) in Congo Forest. In: Journal of the Kansas Entomological Society, Vol. 72, Is. 4, p Seeley T.D. (1995) The wisdom of the hive. The social physiology of honey bee colonies. Harvard University Press, London. 18

19 Janneke Paalhaar Wageningen UR December

20 # carrot pollen (*500) # carrot pollen (*500) Appendix 1: Graphs of pollen flows and significant differences Carrot pollen green bees Error Bars show Mean +/- 1.0 SE 0.20 Dot/Lines show Means yellow bees Error Bars show Mean +/- 1.0 SE 0.08 Dot/Lines show Means

21 # carrot pollen (*500) # carrot pollen (*500) carrot pollen foragers Error Bars show Mean +/- 1.0 SE 20 Dot/Lines show Means carrot nectar foragers Error Bars show Mean +/- 1.0 SE 3 Dot/Lines show Means

22 # carrot pollen (*500) Foragers endive foragers celery foragers

23 # celery pollen (*500) Celery pollen green bees Error Bars show Mean +/- 1.0 SE 0.08 Dot/Lines show Means yellow bees 0.20 Error Bars show Mean +/- 1.0 SE Dot/Lines show Means # celery pollen (*500)

24 # celery pollen (*500) # celery pollen (*500) # celery pollen (*500) celery foragers 30 Error Bars show Mean +/- 1.0 SE Dot/Lines show Means Foragers 2.5 carrot nectar foragers carrot pollen foragers endive foragers

25 Endive pollen green bees 0.10 Error Bars show Mean +/- 1.0 SE Dot/Lines show Means # endive pollen (*500) yellow bees 0.25 Error Bars show Mean +/- 1.0 SE Dot/Lines show Means # endive pollen (*500)

26 # endive pollen (*500) endive foragers 6 Error Bars show Mean +/- 1.0 SE Dot/Lines show Means carrot nectar foragers Error Bars show Mean +/- 1.0 SE Dot/Lines show Means # endive pollen (*500)

27 # strange pollen (*500) # strange pollen (*500) Strange pollen green bees 3 Error Bars show Mean +/- 1.0 SE Dot/Lines show Means yellow bees Error Bars show Mean +/- 1.0 SE 1.0 Dot/Lines show Means

28 # strange pollen (*500) # strange pollen (*500) Foragers 30 carrot nectar foragers carrot pollen foragers endive foragers celery foragers Significant differences type of pollen group of bees increase/decrease significance 18:00-20:00 carrot carrot pollen foragers increase 0, :00-14:00 celery celery foragers decrease <0, :00-20:00 celery celery foragers increase 0,01 10:00-12:00 endive endive foragers decrease 0, :00-12:00 strange carrot nectar foragers increase 0, :00-16:00 strange carrot nectar foragers increase 0,001 16:00-18:00 strange carrot nectar foragers decrease <0, :00-12:00 strange carrot pollen foragers increase <0, :00-16:00 strange carrot pollen foragers decrease 0,04 18:00-20:00 strange carrot pollen foragers decrease <0, :00-14:00 strange celery foragers increase 0,003 14:00-18:00 strange water foragers increase 0,

29 Appendix 2: Tables of results 8:00 10:00 12:00 14:00 2 days old bees average stdev min max days old bees average stdev min max carrot nectar foragers average stdev min max carrot pollen foragers average stdev min max endive foragers average stdev min max celery foragers average stdev min max water foragers average stdev min max

30 16:00 18:00 20:00 22:00 2 days old bees average stdev min max days old bees average stdev min max carrot nectar foragers average stdev min max carrot pollen foragers average stdev min max celery foragers average stdev min max water foragers average stdev min max

31 8:00 next day 2 days old bees average stdev min max days old bees average stdev min max

32 Appendix 3: Controls bee 1e washtr.m. percent 2e washtr.m. percent 3e washtr.m. percent 4e washtr.m. percent total percent supernatr contr. percent pellet contr. percent 1a b total 6 55% 3 27% 1 9% 1 9% % 0 0% 8 73% 2a b total % 95 7% 27 2% 9 1% % 0 0% 9 0,65% 3a b total 8 80% 2 20% 0 0% 0 0% % 0 0% 1 10% 4a b total 98 71% 22 16% 14 10% 4 3% % 0 0% 4 3% 5a b total 22 43% 21 41% 7 14% 1 2% % 0 0% 1 2% 6a b total % 87 36% 27 11% 16 7% % 0 0% 1 0,41% 7a b total 50 67% 3 4% 16 21% 6 8% % 0 0% 2 3% 8a b total % 36 21% 10 6% 3 2% % 1 0,58% 2 1% 9a b total 85 73% 27 23% 4 3% 0 0% % 1 0,86% 1 0,86% 10a b total % % 48 7% 4 1% % 0 0% 17 2,48% percent 67% 21% 8% 3% 100% 0% 10% 32