chemical nature of phagostimulants in pollen attractive to honeybees

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Journal of Insect Behavior, Vol. 19, No. 4, July 2006 ( C 2006) DOI: 10.1007/s10905-006-9039-y Chemical Nature of Phagostimulants in Pollen Attractive to Honeybees Justin O. Schmidt 1,3 and Anita Hanna 2 Revised August 26, 2006; accepted May 19, 2006 Published online: July 13, 2006 Honey bees display a powerful ability to recognize pollen from most plants as food and non-pollen materials as not being food. We sequentially extracted a mixed species blend of pollen with a range of non polar to polar solvents and tested the extracts for attractiveness and feeding enhancement by young bees. Both non polar and polar materials were independently attractive when added in trace quantities to a plain artificial diet. The attractants have little inherent nutritional value, as addition of phagostimulants to artificial diets did not increase the life spans of bees compared to phagostimulant-free diets. These data indicate that pollen phagostimulants consist not of a single or a few specific compounds, but rather are a suite of diverse components that additively or synergistically serve to exceed a threshold level of stimulation necessary for feeding. KEY WORDS: pollen; phagostimulants; attractants; Apis; diets. INTRODUCTION Diseases, mites, insecticides, or frequent apiary movement can cause nutri- tional stresses on honeybees. Beekeepers often attempt to alleviate the ef- fects of stress by providing their bees artificial diets with or without pollen as nutritional supplement. Artificial diets are poor substitutes for natural pollen or collected pollen fed back to bees (Doull and Standifer, 1970; 1 Southwestern Biological Institute, 1961 W. Brichta Dr., Tucson, AZ 85745. 2 1721 E. Glenn St., Apt. A, Tucson, AZ 85719. 3 To whom correspondence should be addressed at; e-mail: [email protected]. 521 0892-7553/06/0700-0521/1 C 2006 Springer Science+Business Media, Inc.

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Journal of Insect Behavior, Vol. 19, No. 4, July 2006 ( C© 2006)DOI: 10.1007/s10905-006-9039-y

Chemical Nature of Phagostimulants in PollenAttractive to Honeybees

Justin O. Schmidt1,3 and Anita Hanna2

Revised August 26, 2006; accepted May 19, 2006

Published online: July 13, 2006

Honey bees display a powerful ability to recognize pollen from most plantsas food and non-pollen materials as not being food. We sequentially extracteda mixed species blend of pollen with a range of non polar to polar solventsand tested the extracts for attractiveness and feeding enhancement by youngbees. Both non polar and polar materials were independently attractive whenadded in trace quantities to a plain artificial diet. The attractants have littleinherent nutritional value, as addition of phagostimulants to artificial dietsdid not increase the life spans of bees compared to phagostimulant-free diets.These data indicate that pollen phagostimulants consist not of a single or afew specific compounds, but rather are a suite of diverse components thatadditively or synergistically serve to exceed a threshold level of stimulationnecessary for feeding.

KEY WORDS: pollen; phagostimulants; attractants; Apis; diets.

INTRODUCTION

Diseases, mites, insecticides, or frequent apiary movement can cause nutri-tional stresses on honeybees. Beekeepers often attempt to alleviate the ef-fects of stress by providing their bees artificial diets with or without pollenas nutritional supplement. Artificial diets are poor substitutes for naturalpollen or collected pollen fed back to bees (Doull and Standifer, 1970;

1 Southwestern Biological Institute, 1961 W. Brichta Dr., Tucson, AZ 85745.2 1721 E. Glenn St., Apt. A, Tucson, AZ 85719.3To whom correspondence should be addressed at; e-mail: [email protected].

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0892-7553/06/0700-0521/1 C© 2006 Springer Science+Business Media, Inc.

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Standifer et al., 1978; Herbert and Shimanuki, 1980; Herbert et al., 1983;Cremonez et al., 1998; Kalev et al., 2002) and may actually harm the bees(Parker, 1926; Maurizio, 1950; Szymas and Jedruszuk, 2003). Parker (1926)observed that feeding pollen substitutes stimulates the queen to lay eggsand the nurse bees to feed the young larvae, but development was not com-pleted. We witnessed similar results in which an entire apiary, in spite ofsyrup feeding, prematurely collapsed with most colonies dying shortly af-ter feeding trials in which bees foraged on open trays of commercial diets(Schmidt unpublished). Pollen feeding usually is not a viable alternative toartificial diets for two reasons: pollen is expensive and in short supply, andpollen feeding entails significant risk of disease transmission (Hitchcock andRevel, 1963).

Artificial diets suffer serious problems including poor attractivenessand acceptability to bees, and poor nutritional value. Acceptability isgreatly enhanced by addition of pollen to the artificial diet (Standifer et al.,1970, 1978). Extracts of pollen often also dramatically increase the at-tractiveness and acceptability of artificial diets (LePage and Boch, 1968;Robinson and Nation, 1968; Doull and Standifer, 1970; Hohmann, 1970;Doull, 1974; Schmidt, 1985; Taber, 1988; Schmidt et al., 1989). The sourceof the attractants or phagostimulants in pollen was believed to be the lipidfraction, especially the lipid pollenkitt (LePage and Boch, 1968; Robinsonand Nation, 1968; Hohmann, 1970; Doull, 1974; Schmidt, 1985; Dobson,1987; Taber, 1988; Schmidt et al., 1989; Dobson et al., 1996). The chemi-cal nature of compounds that are the phagostimulants is not known. Hop-kins et al. (1969) identified E-2-Z,Z-9,12-octadecatrienoic acid that they be-lieved was a major attractant. What is known is that virtually all pollenscontain attractants (von Frisch and Rosch, 1926), even some in which thepollen itself is not consumed (Schmidt et al., 1989), yet non pollen materialslack phagostimulants. Also known is that bees exhibit preferences, oftenprofound, among pollen species (Levin and Bohart, 1955; Campana andMoeller, 1977; Schmidt, 1982; Schmidt and Johnson, 1984; Schmidt, 1984;Schmidt et al., 1987, 1995; Pernal and Currie, 2002).

Although most, or all, pollens appear to contain phagostimulants at-tractive to bees, not all pollens are equally nutritious for bees. Dandelionand sunflower, for example, are considered poorly nutritious with beesperforming poorly or declining when fed these sources (Herbert et al.,1970; Knox et al., 1971; Schmidt et al., 1995; Taber, 1996). Bees nor-mally collect a wide variety of pollen species from the environment (Mau-rizio, 1953; O’Neal and Waller, 1984; Schmidt et al., 1987), apparently,in part, to achieve a nutritional balance and avoid excess toxin overload(Schmidt, 1984; Pernal and Currie, 2001). Nevertheless, a wide variationin attractiveness and nutritional value among pollens exists (Todd and

Pollen Phagostimulants Attractive to Honey Bees 523

Bretherick, 1942; Vivino and Palmer, 1944; Maurizio, 1950; Campana andMoeller, 1977; Schmidt, 1982, 1984; Wahl and Ulm, 1983; Schmidt andJohnson, 1984; Schmidt et al., 1987). The nutritional variation can be ex-plained partly on the basis of amount of protein (Kleinschmidt and Kondos,1976; Schmidt et al., 1987; but see Pernal and Currie, 2001, 2002 for oppos-ing findings); however, much of the variation appears to be simply a matterof how readily and how much pollen is consumed (Campana and Moeller,1977). Even this correlation between amount of pollen consumed and nu-trition can be an oversimplification, as illustrated by the examples of poorperformance on dandelion and sunflower. Dandelion is low in protein anddeficient in the essential amino acids tryptophane, phenylalanine and, espe-cially, arginine (Herbert et al., 1970). Sunflower pollen is highly attractiveand avidly consumed by bees but is low in protein and the bees fail to thriveon it (Schmidt et al., 1987, 1995).

In order to improve honey bee health, colony strength for pollinationand honey production, and ability to recover from stresses caused by pollenshortages, diseases, mites, and insecticides and acaricides, good nutritionis essential. The keystone to good nutrition is the availability of attractive,consumable, nutritionally balanced, affordable diets that are non toxic, andstable.

The objective of this study is to characterize the chemical nature of thephagostimulants in pollen that enhance attractiveness and feeding.

MATERIALS AND METHODS

Phagostimulant Preparation, Test Diets, and Environmental Conditions

Pollen was collected as corbicular pollen pellets from colonies locatednear Sierra Vista, Cochise County, Arizona during the spring of 2001, andstored thereafter at − 20◦C until extracted. Pollen was sequentially ex-tracted for 8 h in a soxhlet extractor using different solvents in the seriescyclopentane, ethyl acetate, ethanol, and water. All solvents except waterwere obtained from Burdick and Jackson, Muskegon, MI.

Cyclopentane was used first to remove non polar lipids, waxes, hydro-carbons and similar materials. Following extraction the liquid fraction wasevaporated by flash evaporation to near dryness, dissolved in acetone, andstored at − 20◦C until used. The residual pollen was dried and then re-extracted with ethyl acetate. Ethyl acetate was intended to collect slightlymore polar lipids than cyclopentane. After ethyl acetate extraction, thepollen residue was dried and extracted with ethanol. Ethanol extractionshould remove simple sugars, free amino acids, some vitamins and other

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semi-polar materials. Finally water was used to extract the most polar ma-terials including carbohydrates, peptides, and minerals.

Feeding Preferences

Artificial test diet: Beltsville Bee Diet (BBD), a commercial mixtureof sucrose and 2:1 torula yeast:lactalbumin (wt/wt), (BioServe, Frenchtown,NJ) was used as a base artificial diet. To BBD were added phagostimulantsderived from the various individual pollen extractions. Also tested wereBBD with the four individual extracts recombined to simulate the total ex-tractables from pollen, BBD with 5% intact pollen added, and pure pollen.To make the diets, the extract derived from pollen equivalent to 5% of theweight of diet was added. For example, for 100 g of dry diet, the extractobtained from 5 g of dry pollen was used. Extract liquid was placed in abeaker, the solvent allowed to evaporate, and powdered diet and distilledwater were added and mixed to form a moist patty. Test diets were kept at4◦C for up to a week when not fed to bees.

Fifteen grams (about 125 bees) of 1–3-days old worker bees of mainlyApis mellifera ligustica Spin. descent and taken from 4–5 different colonieswere placed in 9 cm × 6 cm × 15 cm acrylic plastic and screen cages as de-scribed by Schmidt (1984) and Schmidt et al. (1995). In each test three repli-cate cages were maintained in an environmental room designed to simulatethe conditions inside the brood chamber of a normal honey bee hive. Theroom was illuminated with red fluorescent lights and maintained at 32–34◦Cand 60–70% RH. Each cage was provided with water, a piece of beeswaxfoundation upon which the bees could rest, a 50% sucrose solution, andthe two test diets. After 48 h all diets were replaced with fresh diets in re-verse positions in each cage. Weight of consumed pollen was obtained bydetermining weight loss of the pollen remaining and subtracting from thatloss the evaporative water loss of a matched equal weight of the same dietplaced in a beeless cage in the environmental room. Consumption of thefresh diets was determined after 48 h. The tests measured feeding prefer-ences of honeybees for test diet + phagostimulant versus test diet alone.T-tests were used to indicate preferences between test diets and controls(SAS Proc., SAS Institute 1994).

Survival Studies

Longevity and survival tests were conducted in the environmentalroom. Sixty 1-day-old worker bees were placed in acrylic plastic and screen

Pollen Phagostimulants Attractive to Honey Bees 525

Table I. Dry Weight and Percentage of Extracts De-rived from 1000 g of Pollen

Solvent Weight of extract (g) % of Pollen

Cyclopentane 21.57 2.16Ethyl Acetate 9.89 0.99Ethanol 282.1 28.21Water 533.4 53.34Total 846.96 84.7

cages (9 cm × 6 cm × 15 cm) and provided with a single diet, plus wa-ter, 50% sucrose solution, and a piece of beeswax foundation to rest upon.Seven diets were tested: four single extract diets containing BBD plusphagostimulants from each of the single solvent pollen extracts, BBD plusthe four solvent extracts recombined, BBD plus 5% intact pollen, and con-trol BBD diet. Diet consumption was measured and the dead bees werecounted and removed every 2 or 3 d until the last bee died (Schmidt et al.,1987, 1995). Three replicates of each diet were simultaneously conducted.Survival profiles were analyzed by the SURVIVE procedure followed byWilcoxon and Log-Rank tests (SAS Institute, 1994) and diet consumptionwas analyzed by analysis of variance (SPSS, 1990).

RESULTS

The relative quantities of materials extractable from pollen are shownin Table I. About 2% of the pollen was non polar and extracted by theleast polar solvent, cyclopentane. Very little further non polar or slightlypolar material was extractable with ethyl acetate. The bulk of the solublematerial in pollen was moderately polar and extracted into ethanol, or wasvery polar and water soluble. Overall, the lipophilic extractables accountedfor about 2.2% of pollen and the polar extractables comprised about 81.5%of the total.

When given a choice, bees generally preferred diets containingBBD + phagostimulants over BBD alone (p < 0.05, t-test) (Fig. 1). The oneexception, however, was the choice between BBD and BBD + phagostim-ulants from ethyl acetate in which the diets were consumed about equally(p > 0.05). Honey bees also responded positively to diet + 5% pollen com-pared to diet of BBD only (p < 0.001). Bees fed BBD + phagostimulantsfrom mixed solvents also consumed significantly more diet than bees fed onBBD alone (p < 0.001).

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Fig. 1. Feeding preferences of bees given a choice of diet + phagostimulant versus untreateddiet (∗p<0.05, ∗∗p<0.001; t-test)

Survival rates of bees fed diets with and without phagostimulants areshown in Fig. 2. Bees fed pure pollen have the longest survival rates (themean 24.4 ± 0.7 days) and their survival was significantly greater than onall other diets (p < 0.05). When fed BBD + cyclopentane extract ofpollen, mean survival was 14.2 ± 0.4 days (Fig. 2a). Mean survival rates forthe other treatments were: ethyl acetate extract 14.6 ± 0.4 days (Fig. 2b);ethanol extract 14.2 ± 0.4 days (Fig. 2c); water extract 12.4 ± 0.4 days (Fig.2d); 5% pollen 9.6 ± 0.4 days (Fig. 2e); and the recombined four solvents13.2 ± 0.4 days (Fig. 2f).

DISCUSSION

In nature honey bees collect and feed almost exclusively on pollen forall their needs except energy (Schmidt and Johnson, 1984; Herbert, 1992).

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Fig. 2. Survival curves of bees fed only one diet. In all pan-els the curves are compared to bees fed pure pollen: (a) diet+ cyclopentane extract of pollen; (b) diet + ethyl acetateextract of pollen; (c) diet + ethanol extract of pollen; (d)diet + water extract of pollen; (e) diet + 5% whole intactpollen; (f) diet + combined cyclopentane, ethyl acetate,ethanol, and water extracts of pollen.

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Pollen Phagostimulants Attractive to Honey Bees 529

Pollen is produced by thousands of different plant species in hundreds ofdifferent genera. Yet, despite numerous examples of foragers collectingodd materials including road and coal dust, sawdust, plant spores, and evencheese mites (Spencer-Booth, 1960) during periods of extreme deficiency ofpollen or protein starvation, under normal conditions bees have a remark-able ability to recognize and generalize a material as being either pollen ornot pollen. This is particularly true in the case of the nurse bees that con-sume the pollen, unlike the foragers that collect pollen but consume little ornone of it. The overall generalization by bees of pollens as a category indi-cates that pollens share common properties that are recognizable by bees.Size and protein content of pollen are unlikely to serve for recognition cuesbecause acceptable pollens range over an order in magnitude in diametersand by a factor of at least three in protein content (Schmidt and Johnson,1984; Schmidt et al., 1987). Moreover, many other materials have size andprotein properties within the range of pollen.

Chemicals that may have little or no nutritional value are obvious can-didates to serve as pollen recognition cues. Numerous studies have pro-vided evidence for a chemical source for pollen recognition cues (LePageand Boch, 1968; Robinson and Nation, 1968; Doull and Standifer, 1969;Hohmann, 1970; Doull, 1974; Schmidt, 1985; Taber, 1988; Schmidt et al.,1989; Hanna and Schmidt, 2004), but none have successfully identified anycompounds in pollen that serve this role. Indeed, given the diversity of tax-onomic sources of pollen that honey bees consume, we would be surprisedif any one “magic bullet” phagostimulory compound exists. Rather, we ex-pect that a variety of compounds, both individually and combined, servethat role. In all likelihood bees recognize pollen on the basis of a summa-tion of chemical phagostimulants that reach an acceptance threshold leveland that concurrent threshold levels of deterrent chemicals are not present.An important, little recognized factor in the natural product chemistry ofpollen is the relative infrequency of deterrent chemicals.

A selection procedure by bees for food that is based upon a summationor a synergism of phagostimulatory chemicals in pollen is consistent withthe results of this investigation. Three of the four sequential solvent extractsof a multi-species pollen blend, and the recombination of all four extractsenhanced feeding preference for an artificial diet. This suggests that phagos-timulants fall into both non polar (lipophilic) and polar (hydrophilic) cat-egories. The apparently anomalous result of lack of attraction of the ethylacetate extract appears likely to be an artifact of two factors: the minutequantity of material extracted by that solvent, and a possible removal ofmost of the lipophilic phagostimulatory material in the earlier cyclopen-tane extract. In support of this idea are the results from earlier studies inwhich ethyl acetate extracts of pollen from the same source (without prior

530 Schmidt and Hanna

extraction with cyclopentane) were highly attractive to bees (Hanna andSchmidt, 2004). Although the design of tests in this report does not allowcomparisons of the extracts among themselves, it is worth noting that whenall four extracts were combined, the attractiveness of diet with extract com-pared to plain diet was greater than with any of the individuals extracts.Whether this was caused by additive or synergistic factors cannot be deter-mined.

The greatly reduced longevity of bees fed any of the diet preparationsrelative to natural pollen indicates that the artificial diet was not adequatefor good bee nutrition. In the longevity tests, all six diets produced similarlyshortened life span outcomes. This is not surprising: all diets were identi-cal except for the addition of traces of phagostimulants. Phagostimulantsenhance feeding but likely provide little of nutritional value other than pos-sibly vitamins. The diet itself appeared to be nutritionally deficient and thesource of the problem – a problem that appears to have plagued the bee-keeping industry with artificial diets in general.

The results of these studies suggest two problems in designing futureartificial diets for honey bees. First is the problem of getting the bees toeat the diet; the second is the problem of making the diet nutritious – thatis – digestible, utilizable, and complete. The results of this study indicatethat phagostimulants in pollen can be used to enhance the consumption ofthe artificial diet. The larger problem appears to be designing a basic dietitself. No currently available artificial diet approximates perfection; never-theless, if we can induce better feeding via addition of phagostimulants tocurrent diets, our efforts can be beneficial to provide temporary nutritionalassistance to bees during brief times of pollen dearth, and for pollinatingcrops lacking adequate pollen, or for seasons requiring colony populationincreases.

ACKNOWLEDGMENTS

We thank Leonard Hines for providing the pollen, Jose Villa and BillRubink for reviews, and two anonymous reviewers.

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