Honey bee (Apis mellifera) strains differ in apple (Malus domestica ...

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tacting the anthers and stigmas of the blossom (Robinson, 1979;. Robinson & Fell, 1981; Kuhn & Ambrose, 1982; DeGrandi-Hoff- man et al., 1985; Mayer, 1987; ...
Journal of Apicultural Research 44(1): 15–20 (2005)

© IBRA 2005

ORIGINAL ARTICLE

Honey bee (Apis mellifera) strains differ in apple (Malus domestica) pollen foraging preference ARNON DAG,1* RAPHAEL A STERN2 AND SHARONI SHAFIR3 1

Institute of Horticulture, Agricultural Research Organization, Ministry of Agriculture and Rural Development, Gilat Research Station. M.P. Negev, 85280, Israel

2

Migal, Galilee Technology Center, POB. 831, Kiryat Shmona 11016, Israel

3

B. Triwaks Bee Research Center, Department of Entomology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, POB. 12, Rehovot 76100, Israel Received 8 July 2004, accepted subject to revision 5 January 2005, accepted for publication 20 January 2005

SUMMARY A large proportion of the honey bees from colonies placed in apple orchards collect pollen from competing flora. For four years we tested for a genetic component that would account for preferences for pollen from apple bloom. In the first two years we tested various genetic strains of bees and found significant differences among them in the proportion of apple pollen that they collected. In the final year we found that colonies that were progenies of colonies with high preference for apple pollen in the previous year (‘High strain’) tended to collect a higher proportion of apple pollen compared to colonies from a ‘Low strain.’ The genetic component for apple pollen preference that is evident from this study attests to the possibility of breeding a honey bee strain with high apple pollination effectiveness even under competition conditions. Keywords: Apis mellifera, Malus domestica, pollen, pollination, foraging, preference

INTRODUCTION Honey bees (Apis mellifera) collect both nectar and pollen from the flowers of apple (Malus domestica), but not necessarily at the same time (Mayer, 1984). When collecting pollen, they usually pollinate the flower effectively at each visit, but when they are collecting nectar there are gaps at the base of the stamens that enable ‘sideworking’ honey bees to obtain nectar without contacting the anthers and stigmas of the blossom (Robinson, 1979; Robinson & Fell, 1981; Kuhn & Ambrose, 1982; DeGrandi-Hoffman et al., 1985; Mayer, 1987; Thomson & Goodell, 2001; Schneider et al., 2002; Vicens & Bosch, 2000b). Furthermore, the viability of pollen carried on the body of pollen collectors is higher than that on the body of nectar collectors (Free & Williams, 1972; Kendall, 1973). This evidently arises from the fact that pollen collectors are attracted to flowers in which the pollen is fresh, whereas nectar collectors prefer older flowers in which the nectar is more readily available though viability of the pollen is low (Free, 1993). It was also found that pollen collectors have lower constancy to one cultivar compared to nectar collectors (Robinson, 1979), which improves their effectiveness in crosspollination (Kendall, 1973). Therefore, the effectiveness in apple pollination of pollen collectors is higher than that of nectar collectors (Free, 1966; Robinson & Fell, 1981; Thomson & Goodell, 2001; Vicens & Bosch, 2000b). The tendency of honey bee colonies to collect pollen has been found to have a strong genetic component (Hellmich et al., 1985; Calderone & Page, 1988; Page & Fondrk, 1995; Page et al., 2000). Honey bees may also have a genetic disposition to prefer foraging on certain plant species (Nye & Mackensen, 1970; Mackensen & Nye, 1966, 1969; Basualdo et al., 2000; Dag et al., 2003). The current work explores the genetic component for collection of apple pollen of honey bee colonies placed in blooming apple orchards. *Corresponding author: [email protected]

MATERIALS AND METHODS Site selection, honey bee colonies and pollen collection The experiments were conducted in three sites in northern Israel (33°N, 35°E, c. 600 m above sea level), during four years: 1998, 1999, 2001 and 2002. The selected sites were in large apple orchards in which the main variety was ‘Red Delicious’ with its pollenizers ‘Golden Delicious’ and ‘Granny Smith.’ The experimental colonies were assessed for their strength before introduction into the orchards. Only colonies populated by more than 10 frames, at least seven of which with brood (in compliance with local standards for beehives rented for pollination), were used in the study. The beehives were introduced into the orchards at the beginning of blooming (Delaplane & Mayer, 2000) and fitted with entrance pollen traps. The blooming period in Israel usually starts between the beginning of April and the beginning of May and lasts 1–3 weeks depending on the weather. Pollen was collected from traps every day or two in the afternoon after the most active period of bee flight, except on rainy and cold days without bee foraging activity. We collected pollen until the end of the apple bloom. The collected pollen pellets were kept in a refrigerator at 4 °C. A few weeks after collection, a sample of c. 100 pellets was taken for each hive for each collection date. This sample was examined for the percentage of apple pellets. Apple pollen was distinguished by its white-green color. When in doubt, the discrimination was done by observing pollen grains under a light microscope with pollen pellets collected from a bee captured on an apple bloom serving as reference for apple pollen (Stern et al., 2001). The decision on the plant origin for each pellet was made under the assumption that each pellet contains just one type of pollen (Free, 1963).

16 Assessing preference of different strains, 1998 The goals of the first year study were to establish a working protocol for comparing the levels of apple pollen collected by different colonies over the season, to assess whether large variability exists between colonies, and whether such variability has a genetic component. The experiment was conducted in Site 1 (Kibbutz Malkia). We compared a local Italian-based strain from the Zriffin breeding programme (n = 31 colonies) with an imported Italian-based strain from Hawaii (n = 8). To avoid placing a large number of colonies together in one location, we haphazardly split the beehives from the two genetic lines between two locations in the orchard; the two locations were 500 m apart. In the first location we placed 20 colonies from the Zriffin line and five from Hawaii, and in the second location we placed 11 colonies from Zriffin and three from Hawaii. The Zriffin queens were daughters of four different queens that had been artificially inseminated with drones from the Zriffin stock. The tested queens had freely mated with drones from the same breeding programme. The Hawaii queens arrived mated from the Kona Farms Apiary. Assessing preference of different strains, 1999 The goals of the second year were to expand the search for a genetic component for apple pollen collection by testing the performance of selected strains and of a wider pool of genotypes. The experiment was conducted in Site 2 (Kibbutz Yiftach). We compared four different strains. One strain (n = 14) consisted of a selected line. We raised a daughter queen (F1) from each of two colonies (P) that had the highest proportion of daily mean apple pollen in the 1998 season and instrumentally inseminated each queen with drones from the other colony. We then raised daughter queens (F2) from these colonies and allowed them to mate freely. The other strains consisted of free-mated queens from three other breeding programmes: Buckfast bees in Israel (n = 5), Italian-based bees from Hawaii (same source as in 1998) (n = 5), and Italian-based bees from Australia (n = 7). We haphazardly mixed and distributed the colonies from the different strains in the orchard in the recommended way for distribution of beehives in pollination, in five locations, with 300 m between locations. Assessing preference of selected strains Strain selection, 2001

Dag, Stern, Shafir the top 1, 2, and 8 in terms of mean apple pollen percent) with drones from these colonies (but crossing between colonies to avoid inbreeding). We set up five low-strain lines by different permutations of instrumentally inseminating daughter queens from four low-performance colonies (ranked the bottom 1, 2, 3, and 6) with drones from three of these colonies. Of the three NWC colonies tested, one was included in the high-performance group and one in the low-performance group. The virgin queens were instrumentally inseminated with 9 µl of semen from 10–20 drones from one colony. The queens were then transferred to small mating nuclei and after they had sealed brood they were transferred to queenless colonies (Laidlaw & Page, 1996). We tested 19 colonies from the high strain and 15 colonies from the low strain. Statistical analyses We analysed each year separately by MANOVA with repeated measures (sampling date) for each colony using JMP 5 (SAS Institute). Several multivariate tests are possible, and in some cases they yield the identical F value. In cases where they differ, we present the approximate F value and corresponding degrees of freedom based on the Pillai’s Trace test which generally is preferred in terms of power. As appropriate, we tested the effects of genotype, location within the orchard, and time (repeated measures), and their interaction, on the proportion of apple pollen collected by each colony. Data were transformed by the arcsin square root of the proportion.

RESULTS Assessing preference of different strains, 1998 Colonies of two genotypes (Zriffin and Hawaii) were located in two locations in Site 1. The Genotype × Location interaction was not significant (F = 0.08, df = 1,35, NS) and removed from the model. Apple pollen proportions changed significantly over the season (Time: F = 20.1, df = 4,33, P < 0.0001). While the mean performance in the two locations was similar (Location: F = 0.16, df = 1,36, NS), the proportions of apple pollen changed differently at the two locations (Time × Location: F = 4.34, df = 4,33, P = 0.006). The Zriffin colonies collected significantly higher levels of apple pollen than the Hawaii colonies (fig. 1; Genotype: F = 6.90, df = 1,36, P = 0.013), and the performance of the two genotypes changed differently along the season (Time × Genotype: F = 2.76, df = 4,33, P = 0.044).

The goal of the 2001 study was to assess the performance of a large number of hives (n = 33) that were all located in one orchard, in order to select outstanding colonies for instrumental insemination the following year. The experiment was conducted in site 3 (Kibbutz Baraam). We used 30 free-mated Italian-based queens from the Zriffin breeding programme, 1–3 daughter queens of each of 11 different queens. We also tested three queens of the New World Carniolan (NWC) breeding programme (Cobey, 1999), which arrived from Kona Farms Apiary in Hawaii. To avoid placing a large number of colonies together in one location, we haphazardly split the beehives from the different genetic lines between the northern (n = 16) and southern (n = 17) parts of the orchard; the two locations were 500 m apart. We kept the queens from the low and high colonies in terms of daily mean percent apple pollen, from which to raise virgin queens and drones for instrumental insemination the following year.

Colonies of four genotypes were located in five locations in Site 2. Apple pollen proportions changed significantly over the season (Time: F = 8.50, df = 8,6, P = 0.009). There were significant differences between locations (Location: F = 5.18, df = 4,13, P = 0.01), and the temporal pattern varied among locations (Time × Location: F = 1.89, df = 32,36, P = 0.03). The temporal pattern was similar for all genotypes (Time × Genotype: F = 0.89, df = 24,24, NS), but the mean performance differed between genotypes (fig. 2; Genotype: F = 3.48, df = 3,13, P = 0.047). A post-hoc contrast revealed that the Italian-based bees from Australia collected a significantly higher proportion of apple pollen than all the other genotypes (F = 9.09, df = 1,13, P = 0.01). The ‘Selected strain’ did not show any significant tendency to collect apple pollen compared to the other strains.

Testing selected strains, 2002

Strain selection, 2001

The goal of the 2002 study was to compare the performance of the strains established from the previous year’s colonies which were either high or low in the proportion of apple pollen that they collected, in the same orchard in which the colonies had been tested the previous year. For instrumental insemination we used only queens from 2001 that had survived to the next year. We set up three high-strain lines by instrumentally inseminating daughter queens from three high-performance colonies (ranked

All the colonies were located in one orchard, but to test for possible microhabitat differences we compared the colonies in the northern part of the orchard with those in the southern; performance did not differ between the two locations (F = 0.78, df = 1,31, NS). The apple blooming season was relatively short, and the percentage of apple pollen in the hives was relatively stable over the season (time: F = 2.3, df = 4,28, P = 0.08). There were, however, large differences in performance among colonies. Figure

Assessing preference of different strains, 1999

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Differences in apple-pollen foraging 80% Italian Hawaii Italian Zriffin

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Date FIG. 2. Effect of honey bee strain on percentage of apple pollen pellets collected by colonies placed in a blooming apple orchard, Site 2 (Kibbutz Yiftach), 1999. 3 shows the performance of the top eight colonies (in terms of mean apple pollen percent) from which three queens parented the high strain colonies the following year, as well as the bottom six colonies from which four queens parented the low strain the following year.

Testing selected strains, 2002 Two days after the beehives were introduced, a storm approached and the temperature dropped to less than 12 °C (daily maximum). These climatic conditions did not allow bee flight activity (Vicens & Bosch, 2000a), except for one warmer day on 31 March when temperature reached 15 °C. Weather conditions improved again on 5 April, and foraging resumed. The

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Dag, Stern, Shafir H1

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FIG. 3. Percentage of apple pollen pellets collected by colonies placed in a blooming apple orchard, Site 3 (Kibbutz Baraam), 2001. Colonies with the highest (H) and lowest (L) apple pollen percentages are shown. The colonies that parented the selected strains in 2002 are shown with solid lines. 100% High Low

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FIG. 4. Effect of honey bee strain (‘High’ or ‘Low’) on percentage of apple pollen pellets collected by colonies placed in a blooming apple orchard, Site 3 (Kibbutz Baraam), 2002. percentage of apple pollen initially increased and then showed a monotonic decline (time: F = 142, df = 9,18, P < 0.0001), with both high and low strains showing a similar pattern (fig. 4; Time × Genotype: F = 1.23, df = 9,18, NS). The high strain colonies maintained a higher apple pollen percentage throughout the season, approaching statistical significance (Genotype: F = 3.57, df = 1,26, P = 0.07).

There were between two to seven colonies in each of the three lines of the high strain and five of the low strain. We calculated the mean percent apple pollen of each of the eight lines and ranked them. The three high-strain lines ranked 1–3, and the five low-strain lines ranked 4–8. By Monte Carlo simulation we calculated that the probability of achieving such ranking by chance is less than 0.02.

Differences in apple-pollen foraging

DISCUSSION Pollen collectors are considered to be more efficient than nectar collectors in apple pollination in general, and in ‘Red Delicious’ in particular. However, Mayer et al. (1985) estimated that only 20% of the foragers in apple orchards are pollen collectors. Vicens & Bosch (2000b) found in Spain that in an apple orchard, with cultivars similar to those in the current study, only 3% of the foragers collected apple pollen. They concluded that ‘Delicious’ apple is not a preferred pollen source for bees. Competition was also found to be a major constraint for pollination of apple in Israel (Stern et al., 2001), as well as in other countries were apple is grown (Free, 1968; Severson & Parry, 1981; Mayer & Lunden, 1991). Several attempts to reduce the effect of competition in apple orchards with attractants did not result in a significant increase in bee foraging activity (Mayer, 1984; Mayer et al., 1989; Currie et al., 1992). More evidence for the importance of pollen collection was shown by Stern et al. (2001). They found strong correlations between the percent of apple pollen of the total pollen collected and bee activity in the orchard, and between bee activity and fruit set and yield in apple orchards. These findings emphasize the importance of the pollen sources of beehives located in apple orchards in affecting the dynamics of apple pollination. Analysis of pollen sources contributes to the understanding of conditions when pollination is a yield-limiting factor. Furthermore, any genetic component that may exist in a honey bee colony, which would enhance its preference for apple pollen, would result in better pollination and higher yields. The variation in the tendency to collect apple pollen between different years found in the current study may be due to different levels of competition or weather conditions. In most of the experimental years, a general trend of decline in percent of apple pollen was observed (figs 1, 2 and 4). This general trend may be explained by the tendency of foragers to widen their forage area gradually, which results in abandonment of the target crop (Free et al., 1960). The high level of apple pollen in 2002 in the third sampling date (fig. 4) may be explained by the stormy weather that prevented bee activity during the previous days. The bees that emerged after a few days in the hive were naïve to the environment and the first flowers that they encountered was the apple bloom. Similar behaviour may be found in sequential introduction of beehives into apple orchards; bees from beehives introduced at the peak of the bloom tend to visit apple bloom more than bees from hives located in the orchard from the beginning of the bloom (Stern et al., 2001). The only year in which the level of apple pollen was stable along the blooming period was 2001 (fig. 3). In that year bees maintained a relatively high activity level throughout the blooming period; it seems that since the bloom was short and intensive it was highly attractive to the bees, and there was insufficient time for them to switch to competing flora. Much variation was also found between colonies in the same year. The highest level of apple pollen during the blooming period was 97.8% in one of the colonies in 2001, and the lowest value was 11% in one of the colonies in 1998. Large variations between colonies placed in the same site in their preferences for different pollen sources was also reported by Shimanuki et al. (1967) in cranberry. Severson & Parry (1981) claim that pre-conditioning of honey bee colonies, i.e. their food source allocation before introducing them into the apple orchard, plays a major role in their preference for apple versus competing flora pollen while they are in the orchard. In our study, all the honey bee colonies from the different genetic stocks were placed in the same location before being introduced into the apple orchard, so different regimes of pre-conditioning may be excluded. Pollen selection between various plant species may be mediated by specific volatiles emitted that are attractive to honey bees (Standifer, 1966; Lepage & Bosch, 1968; Hopkins et al., 1969; Dobson & Bergstorm, 2000). Pollen selection can also be mediated by deterrent compounds (Schmidt, 1982). Honey bee genetic strains may differ in their sensitivity to particular pollen odours.

19 Our results support the hypothesis that there is a genetic component to honey bee preference for apple pollen. The Zriffin strain was found to collect a significantly higher proportion of apple pollen than the Hawaii strain (fig. 1); the Australian strain collected a higher proportion of apple pollen compared to the other strains tested (fig. 2); and the ‘High’ colonies, which were daughters of colonies with high preference for apple pollen in the previous year (fig. 3), tended to collect a higher proportion of apple pollen compared to the ‘Low’ colonies (fig. 4). We do not know the mechanism responsible for differences between colonies in preference for apple pollen. Such a mechanism may be specific to apple pollen or may be more general. Specific preference for apple pollen has been reported also for Osmia cornuta (Vicens & Bosch, 2000b). A more general trait which could lead to greater apple pollen collection would be a reduction in flight distance, as reported for the ‘Hy-Queen’ strain and which explained the apparent preference of these bees for alfalfa (Gary et al., 1978). The ‘Selected strain’ tested in 1999 that originated from outstanding colonies in 1998 did not show outstanding levels of apple pollen (fig. 2). These queens originated from a ‘High’ genotype, but mated freely; thus, selection was only on the maternal side, probably weakening the strength of selection. Furthermore, the colonies in 1999 were tested at a site different from where the selected colonies had been in 1998. A potential genotype × environment interaction might have obscured the response to selection, as reported for the ‘Hy-Queen’ bees in alfalfa pollen collection (Gary et al., 1978). In 1998 and 1999, the differences in proportions of apple pollen between strains seemed consistent throughout the season. A similar pattern was reported by Basualdo et al. (2000) for pollen foraging on sunflowers, with consistent differences between colonies from the beginning to the end of the bloom. Such a pattern suggests that mean performance can be used as a reliable index for selection purposes. This is the index that we used in selecting the ‘High’ and ‘Low’ colonies from the 2001 season for testing in 2002 (fig. 3). The different dynamics of apple pollen collection seen in the 2002 season (fig. 4) between the ‘High’ and ‘Low’ strains attests to a potential mechanism by which the differences may arise. At the beginning of the bloom, colonies from both strains showed a similar pattern of apple pollen collection. Only later in the season did the two strains diverge. Foragers from the ‘High’ strain maintained a greater fidelity to apple flowers, while those from the ‘Low’ strain had a greater tendency to abandon the apple in favour of competing flora. Such a behavioural trait might be of little commercial advantage due to the fact that the early, king bloom flowers produce the best fruit and they are the important target for pollination (Delaplane & Mayer, 2000). However, where there is strong competition and low bee activity and fruit set, all the flowers count for potential fruits (Stern et al., 2001). Switching from one alternative to another (apple flower to competing flora) resembles the cognitive process during a reversal learning paradigm, in which after several conditioning trials a rewarding option becomes unrewarding and a previously unrewarding option becomes rewarding. Chandra et al. (2000) demonstrated a genetic component to the speed at which bees learn to switch between the alternatives in a reversal-learning task using the proboscis-extension response. Reversal-learning performance is a relatively simple bioassay to employ, and the consequences of selection are evident after one generation (Chandra et al., 2000). This raises the possibility of using this bioassay to select ‘High’ strains for apple pollination (i.e. slow reversal learning), if there is in fact a common underlying mechanism to reversal-learning performance and the speed at which bees abandon the apple bloom. Similarly to our findings, a genetic component for preference for collecting pollen from the ‘target’ crop was found for alfalfa (Nye & Mackensen, 1970; Mackensen & Nye, 1966, 1969) and sun-

20 flower (Basualdo et al., 2000). Lately, a genetic component was also found for collecting nectar from avocado flowers (Dag et al., 2003). A genetic component for foraging on a specific plant may be exploited in a selection programme for bee strains with a preference for the target crop (Jay, 1986). The selection effort for bees with a preference for apple pollen could be combined with selection for pollen hoarding (Hellmich et al., 1985; Calderone & Page, 1988; Page & Fondrk, 1995; Page et al., 2000), and possibly with selection for slow reversal-learning. The combined effects of a high level of pollen collectors with a high level of apple pollen gatherers and a high fidelity for apple should provide a strain of honey bee with enhanced performance as apple pollinators. Acknowledgements We thank Gil Menda for performing the instrumental inseminations and Dan Apiary for providing the beehives for the experiments. This work was funded in part by the Israel Fruit Board and by grant no. 1998232 from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel.

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