Hranire Cu Polen

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studies have evaluated the quality of bee-collected pollen by direct measurement ofnutritional factors [1, 14, 21, 24, 30, 32, 43,

50, 53, 54, 56, 59, 60, 65, 66, 69, 76, 78,82, 85, 86, 90, 93, 94, 97, 101, 103], or bysuch parameters as: brood production; thegrowth, longevity, survival or protein

1. INTRODUCTION

Pollen is the only source of protein in the

diet of the honey bee,Apis mellifera L., andit also contains all of the lipids, vitaminsand minerals necessary for normal growthand development of the colony. Numerous

Original article

Pollen quality of fresh and 1-year-old single pollen dietsfor worker honey bees (Apis mellifera L.)

Stephen F. PERNAL*, Robert W. CURRIE

Department of Entomology, University of Manitoba, Winnipeg, Manitoba,Canada R3T 2N2

(Received 4 February 1999; revised 26 October 1999; accepted 4 November 1999)

Abstract Newly-emerged honey bees were placed in cages and fed sucrose syrup and one of the fol-lowing single-pollen diets:Malus domestica Borkh.,Brassica campestris L., Phacelia tanacetifoliaL.,Melilotus officinalis (L.) Pall.,Helianthus annuus L., Pinus banksiana (Lamb.), artificial supplement(Bee-Pro) or nothing. Hypopharyngeal gland protein was determined at intervals of 0, 3, 8 and14 days and ovary development was visually scored on day 14. The development of hypopharyngealglands and ovaries varied with diet and, collectively, proved to be sensitive measures of protein uti-lization and pollen quality. For workers fed 1-year-old Phacelia pollen, protein was utilized in a

differential fashion, promoting the development of ovaries over that of hypopharyngeal glands.Development of glands and ovaries was strongly correlated with the amount of protein workers con-sumed from pollen diets, and to a lesser extent, the crude protein content of diets. Storing pollen for1 year by freezing did not affect gland or ovary development.

Apis mellifera/ hypopharyngeal gland / ovary / nutrition / protein / pollen

Apidologie 31 (2000) 387409 387 INRA/DIB-AGIB/EDP Sciences

* Correspondence and reprintsE-mail: [email protected] address: Department of Biological Sciences, Simon Fraser University, Burnaby, BritishColumbia, Canada V5A 1S6


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content of workers; or the development ofhypopharyngeal glands and ovaries [6, 15,22, 23, 25, 28, 29, 31, 42, 4547, 49, 52,57, 59, 62, 7779, 81, 8789, 95, 99]. Thedirect measurement of nutritional factors,alone, may be misleading, as the importanceof the non-protein components of pollen isnot well understood [58, 91, 97].

Evaluating pollen quality by measuringcolony growth and development would pro-vide the most pertinent information aboutits potential impact on honey bee fitness.However, the collection of large quantitiesof pollen for use in studies on full-sizecolonies is not feasible at present. Measur-ing factors that are related to the workersability to utilize pollen, such as hypopha-ryngeal gland and ovary development, couldaddress any inherent differences in the effi-ciency of pollen digestion and its relativeassimilation into tissues of young workers orbrood. Because young worker bees areresponsible for feeding all castes and agecohorts within a colony [79, 11], mea-surements of hypopharyngeal gland devel-opment could also provide information

about the amount of protein potentially dis-seminated to the rest of the colony, relativeto a particular pollen source. Furthermore,by feeding similar groups of caged work-ers single pollen diets, relative consump-tion can be measured and the palatability ofpollen diets may be evaluated.

Hypopharyngeal gland development isinfluenced by the quantity and quality ofprotein ingested by workers [25, 28, 35, 47,57, 59, 8789]. The size of the hypopha-ryngeal glands, as measured by acinal diam-eter, is related to their total protein content

[5]. In nurse bees, the protein content of thehypopharyngeal glands can also be used asan indication of gland activity [37, 68].Examination of the hypopharyngeal glandsfrom caged newly-emerged workers is areliable measure of dietary protein assimi-lation, because these glands will develop inthe absence of brood and their total proteincontent is unaffected by brood quantity [10,

37]. Therefore, the protein content of thehypopharyngeal glands is an effective phys-iological parameter for evaluating the qual-ity of pollen consumed by newly-emergedworkers.

A second physiological parameter used toevaluate the quality of a pollen diet is theextent of ovary development in newly-emerged workers. Pollen that is protein-richusually promotes ovary and egg develop-ment in workers that are caged withoutqueens [42, 51, 57, 62, 63], and a lack ofpollen protein can retard or prevent ovary

development [26]. Pollen protein promotesgrowth of the fat body [57], and haemo-lymph vitellogenin titre has been linked tothe level of pollen in the diet of workers [3,12], thereby establishing the importance ofpollen protein for egg development. Hence,ovarian development is another direct mea-sure of pollen protein utilization by workers,and could indicate the potential for devel-opment of ovaries and production of eggsby queens.

In honey bee workers, the question ofwhether different physiological systems

have different nutritional requirements hasnot been adequately studied. Maurizio [57]showed similar trends in the hypopharyn-geal gland and ovary development for beesfed different pollen diets. Haydak [28], usingsimilarly designed tests, showed that thedevelopment of hypopharyngeal glands wasmore sensitive to reductions in pollen qual-ity than that of mandibular glands. If unequalpartitioning of protein between developinghypopharyngeal glands and ovaries exists, itwould affect our judgement of how theseindicators should be used in making assess-

ments of pollen quality.Ambiguity also remains over the valueof stored pollen as a protein source for honeybees. Most studies examining the nutritionalvalue of pollen after periods of storage areconfounded by the use of diets with mixed,and often unidentified, pollen species. Inaddition, poor descriptions of the techniquesused to store pollen, and incomplete


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Pollen quality for worker honey bees

drying and freezing is important, as pollenstored by freezing is less adequate for long-term brood production than pollen that isfirst dried and then frozen [15]. However,drying pollen may not adequately preventthe degradation of lipids that are nutritionallyimportant for honey bees, or prevent changesin those lipids influencing the palatabilityof pollen [73]. Freezing pollen, in combi-nation with storage in an oxygen-reducedenvironment, might prevent degradation ofpollen protein while simultaneously pre-venting the oxidation of other non-protein

constituents.

A nutritional comparison between freshand 1-year-old stored pollen, using severalidentified pollen species would provide aprecise and meaningful comparison of thequality of pollen following storage and itsutilization by different physiological sys-tems. This would provide useful informa-tion for beekeepers, who typically use theprevious years pollen to feed colonies, aswell as for researchers interested in aspectsof pollen nutrition and consumption.

In this study, we evaluated the quality ofseveral single pollen diets and one pollensubstitute for honey bees. The parameterswe chose to measure were physiologicalindices of pollen utilization in newly-emerged workers: i.e., the development ofthe hypopharyngeal glands and ovaries. Wealso determined whether the pattern ofhypopharyngeal gland development differedfrom that of the ovaries, to ascertain whetherone parameter was a more sensitive indica-tor of pollen protein utilization, or whether

the hypopharyngeal glands and ovaries uti-lized protein in a differential fashion. Inaddition, we examined the relationshipbetween the quality of a pollen as a foodsource for bees, and its protein content.Finally, we evaluated whether freezingpollen and storing it for 1 year in an oxy-gen-reduced environment would cause areduction in its quality.

information about the conditions maintainedduring storage are common. Thus, it is dif-ficult to assess the effects of specific storageparameters on pollen quality, and whethersuch effects are consistent among species.

The age of pollen used to feed honey beescan influence worker growth and develop-ment, or the production of brood by acolony. Workers fed dried pollen that is1-year-old or older have smaller hypopha-ryngeal glands and lower rates of weightgain than workers fed fresh pollen [28, 57].Colonies fed diets composed of 2-year-old

dried pollen rear less brood than coloniesfed freshly-dried pollen [29]. The amountand age of pollen in formulated diets alsocan affect its utilization by workers. Hage-dorn and Moeller [25] determined thatnewly-emerged workers, fed pollen sup-plement containing small quantities of driedor frozen 1-year-old pollen, have similarhypopharyngeal gland development to thosefed supplement mixed with fresh pollen.When fed supplements containing pollenstored for more than 1 year, however, work-ers have smaller glands. The rate of thoracic

weight gain in workers fed supplement con-taining dried pollen also decreases when thepollen component has been stored forextended periods.

In contrast, other studies show that theage of pollen fed to bees does not affect atleast some of the physiological measuresindicative of pollen quality. Ovarian devel-opment of queenless workers fed dried1-year-old pollen [62], and the lifespan ofcaged bees fed dried 2-year-old pollen [23],is similar to that found in bees fed freshpollen. Dietz and Stevenson [15] showed

that drying and freezing pollen can extend itsnutritional value for bees to some extent.Brood production in colonies fed freshly-collected dried pollen does not differ fromthat in colonies fed dried frozen pollen.Dried frozen pollen can support limitedbrood rearing even after 11 years; withoutfreezing, similarly-aged dried pollen is com-pletely ineffective. The combination of

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2. MATERIALS AND METHODS

2.1. Pollen collection

During the summers of 1993 and 1994,pollen was collected from blooming treesand field crops using honey bee (Apis mel-lifera) colonies fitted with OAC pollen traps[83]. Four colonies, housed in standardLangstroth hives, were placed in isolatedplots containing the following species:Malus domestica Borkh. (mixed var.)(apple), Brassica campestris L. var.

Goldrush (oilseed rape), Phacelia tanaceti-folia L. var. Angelia (phacelia),Melilotusofficinalis (L.) Pall. var. Norgold (yellowsweetclover), andHelianthus annuus L. var.Sigco 954 (sunflower). Pine pollen wasobtained by collecting male cones from pinetrees, Pinus banksiana (Lamb.) (jack pine),drying them at 35 C for 1 d, and then shak-ing the dried cones.Malus pollen was col-lected from the orchards of the Agricultureand Agri-Food Canada Research Centre inMorden, Manitoba. Pollen from field cropswas collected at the University of ManitobaGlenlea Research Station, or on co-operat-

ing producers farms in southern Manitoba.Pinus pollen was collected from the Sandi-lands Provincial Forest in eastern Manitoba.

The following protocol was observed forthe collection, handling and storage of allpollen samples to preserve the integrity ofexternal pollen lipids, and to minimize oxi-dation and desiccation of pollen. Surfacesof OAC pollen traps were rinsed withn-pentane, and the pollen collection traysof the traps were lined with tinfoil, to ensurethat pollen only came into contact with lipid-free surfaces. All laboratory tools and sur-

faces used for the handling and sorting ofpollen were also carefully cleaned so thatthey were lipid-free, and care was taken toavoid contacting pollen by hand. Pollen wasseparated from non-pollen debris manually,and corbicular loads were sorted on the basisof pollen load colour [33, 44]. Pollen fromeach colour cohort was mounted in glycer-ine jelly [19] and examined under a com-

pound microscope at 400 to confirm theidentity of the pollen species [13]. Fresh,undried pollen that was not immediatelyused for the preparation of pollen diets wasreadied for storage by placing it in glassvials (26 by 60 mm, 23 mL). After vialswere filled with pollen, any air remainingwas displaced with nitrogen. Vials wereimmediately sealed with teflon-lined screw-caps and kept frozen at 30 C.

2.2. Experimental design

The experimental design consisted of twofactors that were tested: the age of pollen(or pollen substitute) fed to bees, and itsspecies. The age of pollen was defined asbeing 1-year-old or freshly collected. One-year-old pollen (or pollen substitute) wascollected during the previous summer andstored frozen, using the previously-describedprotocol. Freshly-collected pollen was takenand used immediately, or was temporarilyfrozen until needed. This short period offreezing was necessary to preserve early-season species (Malus, Pinus) under opti-mal conditions until late-season species(Helianthus) could be collected, at whichtime all diets were evaluated simultaneously.Eight different pollen diets were tested, eachformulated from a single pollen species orpollen substitute. Individual trials consistedof the 8 pollen diets of the same pollen agefed to bees for a duration of 14 days. Trialswere performed concurrently by staggeringthe starting dates at 2-day intervals. Six indi-vidual trials were performed, enabling alltreatment combinations to be replicated threetimes.

2.3. Bioassay cages

Worker bees were confined in bioassaycages and provided a single diet. Cages(12.7 by 17.5 by 6.4 cm) were constructedof 1.3-cm thick spruce plywood, and werecovered on one face with fibreglass screen-ing (1.1 mm openings) (Fig. 1). Each cagecontained a piece of plastic comb (10.1 by

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tray for feeding bees, and a 125-mL feederbottle which provided a 2-M sucrose solu-tion. Both the pollen diets and sucrose solu-tion were fed ad libitum.

14.9 cm) (Perma Comb Systems, Wood-land Hills, CA, USA), which formed therear interior wall. Cages also contained asmall (1.6 by 1.6 by 8.9 cm) plexiglass diet

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Figure 1. Bioassay cage used for experimental treatments, constructed from 1.3-cm thick spruceplywood and fibreglass screening. Not shown is Tergal drapery lining material (200-m openings)stretched across neck of sucrose feeder bottle. Plexiglass diet tray was inserted into opening at bot-tom side wall of cage. Unit of measurement for all dimensions is centimeters.


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2.4. Diet preparation

Six of the 8 diets were prepared from thepreviously described pollen species; onewas prepared from a commercial honey beepollen substitute (Bee-Pro, Mann LakeSupply, Hackensack, MN, USA), and thefinal diet contained nothing. To simplifydescriptions, the pollen substitute Bee-Pro

will, hereafter, be referred to as a pollendiet. Honey bee-collected pollen was for-mulated into diets by mixing pollen withwater to obtain a paste-like consistency.

Each diet was prepared from a pooled sam-ple containing equal amounts of pollen fromeach collection date. For Bee-Pro andPinus pollen, sucrose (30% w/w dry mass)was added before mixing with water. Thisamount is equivalent to the total weight ofsugar normally found in bee-collectedpollen, as determined over a wide range ofplant species [60, 74, 85, 94].

2.5. Bioassay protocol

Frames of A. mellifera capped broodwere collected and incubated overnight at30 C to obtain newly-emerged adult work-ers. Cohorts of 150 newly-emerged adultworkers, collected within 12 h of emergence,were then placed in bioassay cages alongwith weighed portions of prepared diet mix-tures and were incubated at 30 1 C and70% RH. To estimate the percentage ofwater lost by diets to evaporation, equal por-tions of the same diet mixtures used in bioas-say cages were placed in an additional set ofdiet trays. These trays were placed in thesame incubator as the respective bioassay

cages. All diet trays were reweighed,cleaned, and subsequently replenished witha known mass of freshly prepared diet mix-ture on days 3 and 8. On day 14, the con-clusion of the experimental trials, diets werenot replenished. The mortality of honey beeswithin treatments was evaluated by remov-ing and counting dead bees from cages ondays 3, 8 and 14.

Hypopharyngeal glands were removedfrom randomly selected bees on days 0, 3, 8and 14. These intervals were chosen toestablish gland size. Hypopharyngeal glandsbecome large and biosynthetically active bythe time workers are 3 days old [5, 27, 36],but decrease in size and activity with age[10, 38, 48]. On day 0, 80 bees were selectedfrom the pool of newly-emerged workersto establish the extent of their gland devel-opment. On days 3, 8 and 14 of each trial, 10bees were removed from each bioassay cage,killed by crushing the thorax, and then

decapitated. Decapitated heads were imme-diately bisected with a razor blade (from theocelli to the mandibles) and both hypopha-ryngeal glands were removed by dissectionin phosphate-buffered saline (PBS), pH 7.3[18]. Glands were then placed in microcen-trifuge tubes (1.5 mL) containing 0.1 mLof PBS, and frozen until subsequent proteinanalysis.

On day 14 of the trials, ovaries were dis-sected from 25 bees, randomly selected fromeach bioassay cage. These bees were used toestablish a mean value for ovary develop-ment per treatment. Bees were killed bycrushing the thorax, pinned in a dish con-taining Apis saline [5], and dissected.Ovaries were examined using a binoculardissecting microscope, and their stage ofdevelopment was visually scored using a5-point scale, modified from Velthuis [96].A single, whole number score was assignedper ovary, after determining the stage ofdevelopment to which the majority of itsovarioles belonged. Ovaries were classifiedas: 0: undeveloped (completely restingovary, small ovarioles close to each other);1: oogenesis starting (cells swelling the top

of the ovariole and descending); 2: slightdevelopment (eggs distinguished fromtrophocytes with the nutritive follicle vol-ume not exceeding that of the egg); 3: mod-erate development (egg volume exceedingthat of the nutritive follicle); 4: highly devel-oped (eggs fully elongated sausage-shaped in appearance, with only a remnantof the trophocytes behind the eggs).

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samples were read immediately in amicroplate reader (Model 450, Bio-Rad Lab-oratories, Hercules, CA, USA) set to 595nm. Absorbances were plotted against thelinear portion of the standard curve for cal-culation of protein concentrations in theunknowns.

2.7. Protein determinationof pollen diets

Duplicate samples of fresh and 1-year-old pollen, as well as Bee-Pro, were ana-

lyzed to determine total crude protein. Sam-ples were weighed before and after drying inan oven at 70 C for 24 h, to ascertain dryweight and water content. Dried sampleswere ground with a mortar and pestle toachieve a powder-like consistency, and thenstored in a desiccator until analyzed. Totalnitrogen content of the samples was deter-mined using an elemental analyzer (ModelFP-428, Leco Instruments Ltd., Mississauga,ON, Canada), calibrated against knownnitrogen standards. To determine total crudeprotein, nitrogen values were multiplied bya conversion factor of 5.6 [65].

2.8. Calculation of diet consumption

The net weight of the pollen diet con-sumed within treatments was calculated foreach treatment and time interval by weigh-ing diet trays before and after bees con-sumed the diet, and then correcting for theamount of water lost between time inter-vals. Water loss between time intervals wasestimated using a duplicate set of dietsplaced inside incubators, which were notconsumed by bees. Sugar content of bee-collected pollen diets (sugar is added topollen during its collection by bees), wasassumed to be equal [74].

2.9. Statistical analysis

Analyses were conducted on mean valuesof hypopharyngeal gland protein, ovary

2.6. Hypopharyngeal gland protein

Hypopharyngeal gland development wasassessed using a modified Bradford dye-bind-ing assay for total protein [4]. Reagents for theBradford dye-binding assay were preparedusing Coomassie brilliant blue G-250 dye(Pharmacia LKB Inc., Uppsala, Sweden)according to the protocol outlined in Peterson[64]. A bovine serum albumin (BSA) pro-tein standard solution (0.56 mg/mL) was pre-pared from lyophilized crystals (Sigma-Aldrich Canada Ltd, Oakville, ON, Canada)

and double-distilled water. This solutionwas prepared with 1 mg/mL of sodium azide(Mallinckrodt Specialty Chemicals Canada,Inc., Mississauga, ON, Canada) and waskept frozen in 15-mL aliquots until used.

For protein determination, the frozenhypopharyngeal glands were thawed andmechanically ground in their microcen-trifuge tubes using a pestle. Twenty-five Lof 50% (w/v) aqueous n-octyl--D-glucano-pyranoside (OG) (ICN Biomedicals, Inc.,Costa Mesa, CA, USA) was added to eachsample to solubilize membrane-bound pro-

teins [20]. Individual samples were thenmixed by vortexing, and were centrifugedfor 10 min at 8 850 g. A 10-L aliquot ofthe supernatant was then diluted 10-fold inPBS (pH 7.3), or by an appropriate valueso that the concentration of the each sam-ple fell within the range of the standardcurve. An appropriate amount of OG wasadded in diluted samples to reach a finalconcentration of 0.2% (w/v) in the dyereagent [20].

Two 10-L aliquots of each diluted sam-ple were pipetted into separate wells of a

microassay plate. Standards, ranking in con-centration from 0.05 to 0.5 g/L of BSA,were prepared and added to the microassayplates, along with reagent blanks. DiluteCoomassie dye reagent (200 L) was thenadded to all wells containing samples andstandards. The microassay plate was thenagitated on an orbital plate shaker, and incu-bated at 20 C for 5 min. Optical densities of

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score and diet consumption for each bioas-say cage rather than for each individual bee,to provide comparisons based on the cor-rect experimental unit. Differences inhypopharyngeal gland protein and diet con-sumption among treatments were examinedusing a split-plot design ANOVA withpollen age, replicate and diet as main fac-tors, and time as a repeated measure [70].Honey bee mortality was analyzed using thesame design, but counts of dead bees pertime interval were converted to cumulativeproportions of the initial population. Pro-

portions were then transformed using anangular transformation [84] prior to analy-sis. Ovary development was analyzed as afactorial ANOVA, with pollen age, repli-cate and diet as factors. For all analyses,comparisons between means were madeeither with single degree of freedom con-trasts or least-significant difference tests(LSD) [70].

3. RESULTS

Consumption of experimental diets wasindependent of any interaction between diettype and age (F= 1.74; df = 6, 12; P = NS)(Fig. 2). For all diets, consumption differedwith the age of pollen used (F= 9.20; df = 1,12; P < 0.05), with workers consumingslightly more fresh than 1-year-old pollen.This preference continued to remain evi-dent after Bee-Pro (a pollen substitute)was excluded from the previous analysis(F= 5.41; df = 1, 10; P < 0.05). The quan-

tity of diet consumed also varied with thetype of pollen fed (F= 62.44; df = 6, 12;P < 0.0001), as workers ate substantiallymoreMalus,Melilotus, Phacelia,BrassicaandHelianthus pollen than that of Bee-Pro

or Pinus.

Temporal patterns of pollen consump-tion were influenced by the type of pollen

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Figure 2. Pollen consumption for worker bees fed freshly collected and 1-year-old pollen. Valuesrepresent the mean amount of pollen consumed within treatments per worker bee per day. Mean num-bers of bees in treatments were calculated from the populations in cages at the midpoint of each exper-imental time interval, corrected for sampling loss and mortality, and weighted by the duration of theinterval. Significant differences between diets are indicated by different letters (LSD, = 0.05).


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relationship between the crude protein con-tent of a pollen diet and its consumption byworker bees (r2 = 0.054; P = NS). Estimatesof the actual amount of protein consumedby bees in treatments were also calculated byconverting consumption values to dry massquantities and multiplying by the appropri-ate crude protein conversion. The con-sumption of dietary protein was influencedby the interaction between diet type and age(F= 4.96; df = 6, 12; P < 0.01) (Fig. 4).Workers consumed greater quantities ofprotein from fresh Phacelia (F= 29.79;

df = 1, 12; P < 0.0001) or Bee-Pro

(F= 7.14;df = 1, 12; P < 0.05) diets than those thatwere 1 year old. However, protein con-sumption fromMalus,Melilotus,Brassica,Helianthus and Pinus diets did not differbetween ages of pollen (F= 3.08; df = 1,10; P = NS).

Temporal patterns of protein consump-tion varied with the type of pollen diet fed toworkers (F=23.51; df = 12, 24; P < 0.0001),but not its age (F= 2.68; df = 2, 24; P = NS)

diet fed to workers (F= 26.51; df = 12, 36;P < 0.0001), but not its age (F= 2.58; df = 2,36; P = NS) (Fig. 3). Over both ages ofpollen, the consumption ofMalus,Melilotus,Phacelia, Brassica , Helianthus andBee-Pro differed among time intervals(F= 679.61; df = 2, 30; P < 0.0001). Forthese diets, maximum consumption occurredduring days 03, exceeding values for theremaining time intervals (F= 1237.99;df = 1, 30; P < 0.0001). Consumption duringthe 38-day interval substantially declinedrelative to the 03-day interval (F= 623.30;

df = 1, 30; P < 0.0001), but was greater thanthat for the 814-day interval (F= 121.23;df = 1, 30; P < 0.0001). The consumption ofPinus pollen did not vary with time (F= 1.81;df = 2, 36; P = NS). Within each time inter-val, Bee-Pro and Pinus were consumedless than other pollen diets, over both ages ofpollen.

Table I shows the total crude protein con-tent of each pollen diet and its accompany-ing water content. There was no linear

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Figure 3. Pollen consumption during each experimental time interval for worker bees fed freshly col-lected and 1-year-old pollen. Values represent mean consumption per bee per day, based on populationsin cages at the midpoint of the time interval, corrected for sampling loss and mortality.


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S.F. Pernal, R.W. Currie396

Table I. Protein and water content of pollen diets.

Pollen diet Age of pollen % Proteina, % Water,g/100 g dry g/100 g

mass wet massSpecies Common name

Malus domestica Apple Fresh 25.12 10.71Melilotus officinalis Yellow sweetclover Fresh 24.15 23.48Phacelia tanacetifolia Phacelia Fresh 30.10 14.56Brassica campestris Oilseed rape Fresh 25.72 29.53Bee-Pro Bee-Pro Fresh 29.92 5.89Helianthus annuus Sunflower Fresh 14.86 17.66Pinus banksiana jack pine Fresh 14.03 7.50Malus domestica Apple 1-year-old 24.29 11.50Melilotus officinalis Yellow sweetclover 1-year-old 23.92 13.88Phacelia tanacetifolia Phacelia 1-year-old 26.02 18.43Brassica campestris Oilseed rape 1-year-old 24.67 19.99Bee-Pro Bee-Pro 1-year-old 29.89 5.80Helianthus annuus Sunflower 1-year-old 15.00 23.94Phacelia banksiana jack pine 1-year-old 14.00 7.31

a Protein determination for Bee-Pro and jack pine was performed after the addition of 30% (w/w dry mass)sucrose.

Figure 4. Protein consumption for worker bees fed freshly collected and 1-year-old pollen. Valuesfor consumption were calculated using the protein content of each diet. Mean numbers of bees in treat-ments were calculated from populations in cages at the midpoint of each experimental time interval,corrected for sampling loss and mortality, and weighted by the duration of the interval. Significantdifferences between diets are indicated by different letters (LSD, = 0.05).


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P = NS) and between different time peri-ods (F= 2.38; df = 2, 94; P = NS). Themean cumulative proportion of dead beesat the end of the experiment was 3.6 0.5%,with values ranging from 016%.

The overall analysis of samples from1 600 newly-emerged workers clearly showsthat the type of pollen consumed by beeshad a pronounced effect on their hypopha-ryngeal gland development (F = 23.22;df = 7, 14; P < 0.0001) (Fig. 6). Further-more, these effects were consistent between

freshly-collected and 1-year-old pollen diets(F= 0.54; df = 7, 14; P = NS). The age ofpollen used to feed bees did not affect thedegree of hypopharyngeal gland develop-ment (F= 0.28; df = 1, 14; P = NS). Forboth ages of pollen, diets composed ofMalus,Melilotus, Phacelia andBrassicapollen promoted greater hypopharyngealgland development than diets prepared fromPinus or no pollen, with Bee-Pro andHelianthus diets being intermediate.

(Fig. 5). Over both ages of pollen, the con-sumption ofMalus ,Melilotus, Phacelia,Brassica,Helianthus and Bee-Pro differedamong time intervals (F= 531.10; df = 2,20; P < 0.0001). For these diets, maximumconsumption occurred during days 03, sig-nificantly exceeding values for the remain-ing time intervals (F= 964.46; df = 1, 20;P < 0.0001). Consumption during the 38-day interval declined relative to the 03-dayinterval (F= 481.88; df = 1, 20; P < 0.0001),but was greater than that for the 814-dayinterval (F= 97.74 ; df = 1, 20; P < 0.0001).

Protein consumption from Pinus pollen dietsdid not vary with time (F= 0.64; df = 2, 24;P = NS). Within each time interval, less pro-tein was consumed fromHelianthus, Bee-Pro or Pinus pollen than from all otherdiets, over both ages of pollen.

Overall worker bee mortality, analyzed asthe cumulative proportion of bees dying percage, was similar in bees fed different agesof pollen (F< 0.01; df = 1, 14; P = NS),different pollen diets (F= 2.27; df = 7, 14;

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Figure 5. Protein consumption during each experimental time interval, for worker bees fed freshlycollected and 1-year-old pollen. Values for consumption were calculated using the protein content ofeach diet. Mean bee numbers per cage are based on populations at the midpoint of the time interval,corrected for sampling loss and mortality.


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Temporal patterns of hypopharyngealgland protein content changed with the ageof pollen fed to workers (F = 18.27; df = 2,74; P < 0.0001) and were also dependenton the type of diet consumed (F = 2.64;df = 14, 74; P < 0.01) (Fig. 7). For workersfed freshly-collected pollen, the hypopha-ryngeal glands contained an average of44.5 3.2 protein on day 0, with valuesranging from 14.280.5 . Workers fed freshMalus,Melilotus, Phacelia,Brassica andHelianthus pollen developed glands that

varied in protein content among time inter-vals (F= 35.86; df = 2, 16; P < 0.0001).Workers consuming Malus pollen dietsdeveloped glands that were largest duringthe 03-day interval (F= 8.94; df = 1, 28;P < 0.001). However, those fedMelilotus,Phacelia,Brassica andHelianthus pollendeveloped glands that were smallest duringdays 03 (F= 59.37; df = 1, 12; P < 0.0001),

peaking in size during the 38-day interval(F= 68.64; df = 1, 12; P < 0.0001). Forworkers fed Bee-Pro, Pinus or no pollen,gland protein did not vary significantly overtime (F= 3.71; df = 2, 12; P = NS). Withinindividual time intervals, workers fedMalus,Melilotus, Phacelia and Brassica pollenconsistently developed larger glands thanbees fed other diets; workers fed Pinus orno pollen showed the least development.

For workers fed 1-year-old pollen,hypopharyngeal gland development showed

slightly different temporal patterns (Fig. 7).The hypopharyngeal glands from these beescontained an average of 38.9 2.9 pro-tein on day 0, with values ranging from14.167.3 . For all diets, the mean valuesfor gland protein were greatest during the03-day interval; however, only those forMalus,Melilotus, Bee-Pro andHelianthusshowed significant changes over time

398

Figure 6. Hypopharyngeal gland development for worker bees fed freshly collected and 1-year-oldpollen. Values for hypopharyngeal gland protein represent mean development, averaged over thetime intervals ending on days 3, 8 and 14. Significant differences between diets are indicated bydifferent letters (LSD, = 0.05).


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The analyses of pooled ovary scores from1 200 dissections showed similar patternsto those indicated by hypopharyngeal glanddevelopment. The overall analysis showedthat ovary development was greatly affectedby the type of pollen fed to workers(F= 10.39; df = 7, 47; P < 0.0001) (Fig. 8).The age of pollen used to feed newly-emerged workers had no effect on the extentof their ovarian development (F= 0.72; df=1,47; P =NS), and no interaction betweenpollen age and diet type existed (F= 0.34;df = 7, 30; P = NS). For both ages of pollen,diets composed of Malus, Mel ilotus ,Phacelia and Brassica pollen promotedgreater ovary development than diets pre-pared from Pinus or no pollen. Bee-Pro

andHelianthus diets produced intermediateovary development.

The linear relationship between ovaryscore and hypopharyngeal gland protein isshown in Figure 9. These two measures ofprotein utilization are well correlated(r2 = 0.904; P < 0.0001). The crude proteincontent of diets was also found to be

(F= 20.28; df = 2, 12; P < 0.0001). For thesediets, hypopharyngeal gland developmentwas greatest during the 03-day interval(F= 27.54; df = 2, 12; P < 0.001), anddeclined as the experiment progressed. Pro-tein levels from bees fed the remainingpollen diets showed little variation over time,or possessed greater variability per timeinterval. The relative ranking of 1-year-olddiets within time intervals showed some sim-ilarity to fresh pollen diets, but fewer con-sistent patterns among groups of diets wereseen across time periods. Workers fedMalusandMelilotus diets during the 03-day inter-val developed larger hypopharyngeal glandsthan bees fed all other diets, and continuedto develop larger glands than with most

remaining diets during the 38-day inter-val. Workers fed Pinus or no pollen had lowhypopharyngeal gland protein during the03-day interval, and during the 38-dayand 814-day time intervals, produced thelowest hypopharyngeal protein of all diets.All other 1-year-old diets were similar forhypopharyngeal gland development, withinall time periods.

399

Figure 7. Hypopharyngeal gland development during each experimental time interval, for worker beesfed freshly collected and 1-year-old pollen.


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correlated with hypopharyngeal gland pro-tein (r2 = 0.471; P < 0.01) and ovary score(r2 = 0.601; P < 0.001). However, thestrongest linear relationships were foundbetween protein consumption and hypopha-ryngeal gland development (r2 = 0.869;P < 0.0001) (Fig. 10a), and between proteinconsumption and ovary score (r2 = 0.905;P < 0.0001.) (Fig. 10b).

4. DISCUSSION

The development of the hypopharyngealglands and ovaries in newly-emerged workerbees both appear to be reliable and sensi-tive measures of protein utilization, andwhen used together provide a good indica-tion of the quality of the pollen that is beingconsumed. This conclusion is supported bya strong correlation between the amount ofprotein consumed from pollen diets and the

extent of hypopharyngeal gland or ovarydevelopment.

Hypopharyngeal gland and ovary devel-opment were chosen for analysis in thisstudy because they were considered the bestindicators of how pollen diets affect colony-level fitness. Hypopharyngeal gland devel-opment in nurse bees is positively corre-lated with pollen consumption [35], andprotein synthesis from these glands utilizesprotein derived from pollen [8]. In a colony,nurse bees actively consume and digest thelargest quantities of stored pollen [11] and

secrete it as brood food from their hypopha-ryngeal and mandibular glands [80]. Thequantity and quality of brood food producedby nurse bees have important ramificationsfor the fitness of the colony as a whole. Mostbrood food is fed to the developing larvaewithin the colony; however, a significantproportion is also fed to the adult membersof each caste [9]. The quality of food

400

Figure 8. Ovary development for worker bees fed freshly collected and 1-year-old pollen. Values forovary score represent the mean extent of ovary development for bees on day 14 of the experimentalperiod. Significant differences between diets are indicated by different letters (LSD, = 0.05).


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received by the brood and the queen, espe-cially, has the potential to influence the over-all rate of colony growth. In addition, thesurvival of workers is directly affected bythe total amount of pollen protein consumed[45, 46, 77]. Therefore, diets that enhancehypopharyngeal gland development innurses potentially influence colony popu-lation size, a variable that is related to suchcolony-level fitness components as increasedcolony survival and reproductive perfor-mance [71]. In contrast, worker ovariandevelopment provides a direct measure of

the ability of bees to convert pollen proteinsinto vitellogenin [3, 12], a lipoprotein whichis required for egg-laying. Greater or moreefficient production of vitellogenin in queenscould increase fecundity, colony populationsize, and therefore colony-level fitness.

Although the utilization of pollen pro-tein by the hypopharyngeal glands andovaries closely paralleled each other, ourresults showed that there are importantdifferences in the way that the ovaries andhypopharyngeal glands assimilate proteinfrom some pollen sources. Workers fed

401

Figure 9. Relationship between mean ovary scoreand hypopharyngeal gland protein for worker beesfed freshly collected and 1-year-old pollen. Valuesfor hypopharyngeal gland protein represent meandevelopment, averaged over the intervals endingon days 3, 8 and 14. Values for ovary score rep-resent the mean extent of ovary development forbees on day 14 of the experimental period.

Figure 10. Correlations between protein consumption per worker (from Fig. 4) and indices of workerdevelopment, for freshly collected and 1-year-old pollen diets. a) Hypopharyngeal gland protein.Values represent mean development, averaged over the intervals ending on days 3, 8 and 14.b) Ovary development. Values are the mean extent of development on day 14 of the experimental period.


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1-year-old Phacelia pollen developedsmaller hypopharyngeal glands than work-ers fed fresh Phacelia pollen, while beesfed other diets had no change in gland sizebetween both ages of pollen. In contrast,ovary development for workers fed fresh or1-year-old Phacelia pollen was similar, eventhough bees consumed less 1-year-oldpollen. It is likely that decreased consump-tion of 1-year-old pollen limited the proteinavailable for metabolic processes in theseworkers. As a result, protein may have beenutilized according to a physiological prior-

ity, or sink, which favoured the develop-ment of the ovaries over that of the hypopha-ryngeal glands. If this is true, suchdifferential utilization of protein would notoccur under queenright conditions, whereovary development is naturally suppressed,but hypopharyngeal gland development isnot [40, 41]. Our results suggest, that underqueenless conditions, more than one physi-ological index of protein utilization shouldbe used to evaluate honey bee nutrition.Such measures should include ovariandevelopment.

The significant positive correlationbetween the crude protein content of pollendiets and hypopharyngeal gland or ovarydevelopment in workers indicates that, inthe absence of conducting a nutritionalbioassay, crude protein content could beused as a general guideline for evaluatingpollen quality. Although most species ofpollen that have been quantitatively ana-lyzed exhibit similar amino-acid profilesand contain the minimum levels of essen-tial amino acids [23] necessary for normalhoney bee growth and development [14, 21,23, 59, 66, 85, 102], protein content is

important. The developmental rate of thehypopharyngeal glands in workers is notrelated to the essential amino-acid compo-sition of the pollen consumed [59], but iscorrelated with the level of protein in thediet [59, 88] and the amount of protein thatis ingested [59, 89]. Furthermore, the addi-tion of essential amino acids has oftenproven to be unsuccessful at improving the

nutritional status of specific pollen diets [2,23, 52]. Even for a species such as dandelion(Taraxacum officinale Weber ex Wigg.),which does not support brood rearingbecause of amino acid deficiencies [31, 52],crude protein content is characteristicallylow (9.9%) [53]. Therefore, our results sup-port the use of crude protein content as aparameter for evaluating the quality of apollen diet.

Freezing pollen and storing it in an oxy-gen-reduced environment for up to 1 yeardid not degrade its nitrogen content, or

change its nutritional value for honey beeworkers. Although workers consumedslightly more fresh than 1-year-old frozenpollen, overall hypopharyngeal gland andovary development did not differ betweenfresh or stored pollen diets. Although thecrude protein content among species ofpollen used in this study differed by up to16%, protein levels for fresh and 1-year-oldconspecific pollen differed by less than 1%(except for Phacelia at 4.1%). These resultsare in agreement with studies that havereported little change in the content of pro-teins, minerals, carbohydrates, and lipids inpollen after storage by freezing [14, 103].However, this is in contrast with reports ofreductions in digestible proteins [93], dete-rioration or lowered availability of proteins[29], decreased vitamin content [24], orincreased mortality and loss of brood-rearingcapacity [15] of stored pollen. Our 1-year-oldpollen samples may have benefitted from alower storage temperature (30 C) than thatused in other studies, and also the presenceof an oxygen-reduced atmosphere. Thepollen species chosen for this study mayalso have inherently influenced the nature

of our results. Lack of proper taxonomicidentification of pollen species confoundsthe interpretation of results from many otherstudies, as mixed or unidentified pollensources have often been utilized. Our find-ings with pure pollen samples of known ori-gin indicate that the use of 1-year-old frozenpollen for use as feeding supplements incolonies could be nutritionally equivalent

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protein [66]. Low protein values among var-ious species of Pinus (1.912.1%) have alsobeen reported for bee-collected and hand-collected samples [14, 23, 45, 66, 85, 93,94]. Published values for the crude proteincontent of Phacelia tanacetifolia pollen werenot found; in our study it contained the high-est level of crude protein (28.1%) of all nat-ural pollen diets. Although variation in pro-tein content occurs between geographiclocations and with different collection tech-niques [53], our reported values comparefavourably with other studies. The polylec-

tic nature of honey bee foraging may be anadaptation to prevent colonies from becom-ing dependent on a small number of pollensources lacking in protein, and lessen theimpact of vitamin, mineral or lipid defi-ciencies, or toxin overloads [72], associatedwith individual species.

An interesting finding in this study con-cerns the lack of relationship between thecrude protein content of the diets tested andtheir relative consumption by newly-emerged workers. This lack of relationshipsuggests that young worker bees, particu-larly nurses, may have no inherent mecha-nism through which they can discriminatethe protein content of the diet that they areconsuming. If workers could discriminatethis aspect of pollen quality, it would beexpected that larger quantities of somepollen species, such as Helianthus andPinus, would have been consumed to com-pensate for their low protein content [100].In colonies, large increases in pollen con-sumption occur when the level of pollenprotein decreases by 10%, in an apparentattempt to meet protein requirements [46]. Inour study, which used caged queenless

workers, consumption did not increase witha decrease in pollen protein.Helianthuspollen, which is low in protein, was con-sumed in the same proportions as higher-protein pollens, and Pinus pollen was con-sumed in small quantities. In contrast withour findings, Schmidt and Johnson [75]showed a weak positive correlation betweenworker feeding preference and the protein

to that of fresh pollen if improved storageprotocols such as those used in this studywere followed.

Reduced palatability of stored pollen forworker bees may be responsible for theslight reduction in consumption of 1-year-old pollen relative to freshly collected pollenobserved in this study. The attractivenessand palatability of pollen is affected by itslipid composition [16, 17, 34, 39, 55, 73, 78,92]. Although the lipid composition changeswhen pollen is stored in the hive [54, 98], ithas not been demonstrated whether suchchanges occur under other storage condi-tions. Changes in pollen lipids could explainthe slight reductions in pollen consumptionin studies such as ours, where protein contenthas been shown to remain stable in storage.Reduced palatability of stored pollen occurswhen pollen has been dried and stored con-tinuously at room temperature [15, 95].

The species of pollen we examined inthis study, with the exception of Pinus, areusually collected in large quantities by honeybees. The relative differences in protein con-tent among these species show that foraginghoney bees collect pollen that varies greatlyin quality. Our mean crude protein valuesfor Malus domestica (24.7%),Melilotusofficinalis (24.0%), Hel ianthus annuus(14.9%),Brassica campestris (25.2%) andPinus banksiana (14.0%) are comparableto other studies that have analyzed bee-col-lected pollen, after standardizing their datafor the protein conversion factor of 5.6.From these studies, it is apparent that proteinmay vary within a particular species, or aclosely related group of species. Reportedprotein values for apple (Malus pumila

Mill.) range between 22.724.6%, whilethose for Trifolium species vary between17.6 18.4% [59, 87]. Oilseed rape pollen isreported to contain between 19.422.7%protein forBrassica campestris [87, 94],24.3% for Brassica napus L. [66], and21.6% for an unidentified mixture of bothspecies [86]. In addition,Helianthus annuuspollen has been described to contain 15.8%

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content of pollen diets. Their results are,however, consistent with our findings inshowing that workers do not increase pollenconsumption to compensate for reductions indietary protein. Schmidt and Johnsons workalso suggests that consumption may be influ-enced by physical or chemical cues that areunrelated to pollen quality.

Factors other than protein may be impor-tant in determining the amount of pollenconsumed by individual workers. Theamount of pollen consumed is, in part, alsoa measure of how attractive and palatable

a diet is. Bee-Pro, a pollen substitute con-taining no natural pollen, is as high in pro-tein content asMalus,Melilotus,Brassica orPhacelia pollen. However, Bee-Pro con-sumption was low relative to natural pollen.This may have occurred because Bee-Pro

lacks phagostimulants normally associatedwith the lipid components of pollen [67,73]. The lack of phagostimulants or the pres-ence of repellents in Pinus pollen, ananemophilous species, may also explain itslow level of consumption. Many anemophi-lous species of pollen are readily collectedby bees [61], but others remain unpreferredor contain toxic compounds [77]. Our studydemonstrates that Pinus pollen is not read-ily consumed by workers, and confirms thepoor value of Pinus pollen for honey beedevelopment and longevity [23, 93]. Its lackof nutritional value does not appear to bethe result of deficient amino-acid composi-tion as the supplementation of amino acidsthat are absent, or present in low concen-trations in Pinus pollen, do not improve itsquality [23]. Therefore, the existence of com-pounds that reduce the palatability of Pinuspollen likely influence its utilization by

honey bees.Modern agricultural systems entail the

use of large monocultures, severely limit-ing floral diversity for bees. Our findingssuggest that the colony performance ofhoney bees is not likely to be adverselyaffected if bees are limited to foraging onmonocultures ofMalus domestica,Melilo-tus officinalis, Brassica campestr is or

Phacelia tanacetifolia. However, honey beecolonies restricted to foraging onHelianthusannuus during its bloom period may suffera slight loss of fitness. Pollen supplementsmade from Bee-Pro might benefit fromthe addition of pollen, or phagostimulantsfrom pollen, that bees normally prefer toconsume. Pinus banksiana pollen is nutri-tionally deficient, and is not palatable tobees. Although sometimes marketed as apollen substitute, it should not be used as afeed supplement for honey bees.

In conclusion, the development of

hypopharyngeal glands and ovaries innewly-emerged honey bee workers isstrongly correlated with the amount of pollenprotein consumed from diets, and to a lesserextent, with the crude protein content of thediets themselves. For certain pollen diets,workers physiologically allocate protein ina differential fashion, promoting greaterdevelopment of the ovaries over that of thehypopharyngeal glands. Over the specieswe have tested, the consumption of pollenby workers appears to be unrelated to itsnutritional content, and may be more influ-enced by the presence or absence of phagos-timulants or repellants. We also ascertainedthat pollen quality remains unaffected afterstorage for 1 year, following our protocolof freezing in an oxygen-reduced environ-ment. Although pollen quality is best eval-uated in single-pollen nutritional bioassays,the crude protein content of pollen can beused as a general guideline for evaluatingthe quality of pollen collected by foragers.

Rsum Qualit de diffrents pollensmonofloraux, frais et dun an, comme

nourriture pour les ouvrires dabeillesdomestiques (Apis mell ifera L.). Desgroupes de 150 abeilles naissantes ont tplaces dans des cages en bois, nourriesavec une solution de saccharose 2 M etavec lun des six pollens suivants :Malusdomestica Borkh.,Brassica campestris L.,Phacelia tanacetifolia L.,Melilotus offici-nalis (L.) Pall., Helianthus annuus L. et

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von jeweils 150 frisch geschlpften Honig-bienen (Apis mellifera L.) wurden in hl-zernen Testkfigen mit 2 M Zuckerlsungversorgt und mit jeweils einer der folgen-den Pollennahrungen gefttert:Malus dome-stica Borkh.,Brassica campestris L., Pha-celia tanacetifolia L.,Meliotus officinalis(L.) Pall.,Helianthus annuus L. und Pinusbanksiana (Lamb.). Weiterhin wurdenzustzlich die kommerzielle Pollenersatz-nahrung Bee-Pro und eine pollenfreieErnhrung untersucht. Den gekfigten Bie-nen wurde entweder frisch gesammelter Pol-

len verfttert oder Pollen, der ein Jahr langbei 30 C in Glasgefen in einer sauer-stoffreduzierten Atmosphre gelagert wordenwar. Zu Beginn des Experiments, nach 3,8 und 14 Tagen (dem Versuchsende) wur-den jeweils 10 Arbeiterinnen aus jedemKfig entnommen und die Hypopharynx-drsen herausprpariert. Zu jedem dieserZeitpunkte wurde die Nahrungsaufnahmegemessen, der Roheiweigehalt jeder Nah-rung wurde mit einer Elementaranalysebestimmt. Anhand des Untersuchungsver-fahrens von Bradford wurde der Gesamt-proteingehalt der Drsen bestimmt und alsMa fr den Entwicklungsgrad und dieGre der Drsen benutzt. Weiterhin wur-den am 14. Tag des Experiments 25 Bienenprpariert und die Entwicklung der Ovarienvisuell in 5 Stufen eingeteilt. Der Entwick-lungszustand der Hypopharynxdrsen undder Ovarien war bei den verschiedenenErnhrungen unterschiedlich und erwiessich als zuverlssige und empfindliche Mess-gre fr die Eiweinutzung und die Pol-lenqualitt. Bei der Verftterung von einJahr lang gelagertem Phacelia Pollen wurdedas Eiwei allerdings abweichend genutzt

und frderte die Entwicklung der Ovarienstrker als die der Hypopharynxdrsen. DieEntwicklung der Drsen und der Ovarienwar stark mit der Menge von Eiwei kor-reliert, die die Arbeiterinnen mit der Pol-lennahrung aufnahmen, zu einem geringerenGrad hing sie mit dem Roheiweigehalt derNahrung zusammen. Die tiefgekhlte Lage-rung des Pollens ber ein Jahr hatte auf die

Pinus banksiana (Lamb.). Un succdancommercial de pollen, Bee-Pro, et unrgime sans pollen ont galement t tes-ts. Le pollen fourni aux abeilles encagestait soit du pollen frachement rcolt, soitdu pollen congel 30 C pendant un an etconserv dans des rcipients en verre sousatmosphre rduite en oxygne. Au dbutde lexprience, puis aux jours J3, J8 et J14,dix abeilles ont t prleves dans chaquecage et leurs glandes hypopharyngiennesextraites. La teneur totale en protines desglandes a t dtermine par le test de Brad-ford et utilise comme mesure du dvelop-pement et de la taille des glandes. A la fin delexprience (J14), 25 abeilles de chaquecage ont t dissques et leur dveloppe-ment ovarien class visuellement de 0 4.Le dveloppement des glandes hypopha-ryngiennes et des ovaires a vari en fonc-tion du rgime et sest comport, danslensemble, comme une mesure fiable et sen-sible de lutilisation des protines et de laqualit du pollen. Pour les ouvrires nourriesavec du pollen de phaclie dun an, les pro-tines ont t utilises diffremment : ellesont plus favoris le dveloppement des

ovaires que celui des glandes hypopharyn-giennes. Le dveloppement des ovaires etdes glandes tait fortement corrl avec laquantit de protines consommes issuesdu pollen et, un moindre degr, avec lateneur brute en protines des pollens. Laconservation du pollen par conglationdurant un an na eu aucun effet sur le dve-loppement des glandes ni des ovaires. Lepollen de tournesol et le succdan Bee-Pro devraient tre complts par dautresespces de pollen lorsquils sont donns ennourrissement ; le pollen de pin devrait trevit.

Apis mellifera/ glande hypopharyngienne /ovaire / nourrissement / protine / pollen

Zusammenfassung Qualitt vonfrischem und ein Jahr altem unifloralenPollen als Nahrung von Honigbienenar-beiterinnen (Apis mellifera L.). Gruppen

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Entwicklung der Ovarien und Drsen keinenEinfluss. Helianthus Pollen und die Bee-Pro Pollenersatznahrung sollten zur Ftte-rung von Bienenvlkern mit anderen Pol-lenarten angereichert werden; der Pollenvon Pinus sollte vermieden werden.

Apis mellifera / Hypopharynxdrse /Ovarien / Ernhrung / Protein / Pollen

ACKNOWLEDGMENTS

We would like to acknowledge the assistanceprovided by Christine Abraham, WendyGraham and Mike Patterson for dissecting thehypopharyngeal glands and ovaries. Thanks arealso owed to Douglas Olafson for his assistancein the collection of pollen. Cam Davidson pro-vided access to the orchards at the Agricultureand Agri-Food Canada Research Centre in Mor-den, Manitoba. Ren Mabon of Brett YoungSeeds Ltd. provided phacelia seed, and HaroldPauls, Clayton Mannes, Bernard Mariash wereco-operating producers, who allowed access totheir crops. The elemental analysis of pollen wasprovided by the Department of Plant Science,University of Manitoba. We also acknowledge

the efforts of Earl Thompson, Manitoba Hydro,for producing the bioassay cage drawing. Thisresearch was financially supported by ManitobaAgriculture, a University of Manitoba Fellow-ship to SFP and a Research Development FundGrant to RWC.

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