826 ShortCommunications [Auk, Vol. 117

The Auk 117(3):826-830, 2000

Pollen and the Nitrogen Requirements of the LesserDouble-collared

IAN G. VAN TETS • AND SUSAN W. NICOLSON Departmentof Zoology,University of CapeTown, Rondebosch 7701, South Africa

Nectarivorousbirds typically are small, often Tets and Whelan 1997, van Tets 1998). However,the weighingless than 10 g, and havehigh basalmeta- amino acidcomposition of pollenvaries among plant bolic rates.To meet their high energyneeds, many species,and many -pollinatedplants nectarivorousspecies spend a large proportion of in North Americaproduce pollens that are deficient their time foragingon nectar.Like all ,they also in two essentialamino acids: methionine and lysine requirenutrients such as protein, vitamins, essential (Martinez del Rio 1994). fatty acids,minerals, and trace elements.Amino ac- The relative importanceof insectsand pollen in ids occur in floral nectar, and their concentrations the dietsof nectarivorousbirds varies considerably. are thoughtto be related to pollinatortype, being Anna'sHummingbirds (Calypte anna) appear to de- low in nectarof -pollinatedplants (Bakerand pendon insectsfor nitrogen.They thrive in captivity Baker 1982). However, the correlation between ami- on a diet of fruit flies and nectar (Brice 1992), but no acid concentrationand pollinatortype has been when fed pollenthey digestonly 5% of the pollen disputedby Gottsbergeret al. (1984),who suggested grains (Briceet al. 1989).In contrast,Purple-crowned that most of the variation in nectar amino acids re- Lorikeets (Glossopsittaporphyrocephala) feed exten- suitsfrom flowerdamage or contaminationwith pol- sivelyon pollenand digestup to 50%of the pollen len. In any event,the amountsare too low to satisfy grainsthey ingest (œ = 37%;Wooller et al. 1988,Rich- the protein requirementsof nectarivorousbirds ardsonand Wooller1990). New Holland Honeyeaters (Martinez del Rio 1994).Other nitrogensources are (Phylidonyrisnovaehollandiae) fall somewhere between insectsor pollen(Richardson and Wooller 1990, Brice thesetwo extremes.The role of pollen in their diet 1992),both of whichpresent birds with a numberof hasbeen studied in two siteswith stronglyconflict- potential problems. ing results (Paton 1981, 1982;Wooller et al. 1988). It In ,a large proportionof the nitrogenis in is important to note that all of thesestudies are based the formof chitin,a polymerthat may be difficultfor primarily on measures of full and empty pollen a smallnectarivorous bird to digest.Although some grains under a light microscope.Although these seabirdsare capableof producingchitinases (Jack- measurementsprovide an estimateof a bird's diges- son et al. 1992), no nectarivorous birds are known to tive capabilities,they are not accurateand can seri- do so. Accordingly,the major nitrogensources ob- ously underestimatepollen digestibility (van Tets tainedfrom insectsare likely to be the proteins,pep- and Hulbert 1999). tides, and free amino acids found in their tissues. Lesser Double-collared (Nectariniachal- However, the amino acid compositionof insects ybea)are commonin muchof SouthAfrica. They are varies greatly and often is dominatedby nonessen- importantpollinators of many plantsin the Cape tial amino acidssuch as proline (Tomlinet al. 1993). FloralKingdom of the southwesternCape, a region As a result, insectsare not always as good a source with a highfloral diversity characterized by thefam- of proteinas is widelybelieved. Furthermore, it may ilies Proteaceae, Ericaceae, and Restionaceae. In ad- be energeticallyvery expensivefor a small nectari- dition to the indigenousflora, N. chalybeafeeds ex- vorousbird to catchenough insects to meetits nitro- tensivelyon introducedspecies of Eucalyptus,which gen requirements. may haveled to increasesin the numbersand range Pollenhas long been recognized as a majorsource of this group (Fraserand Crowe1990, Parker 1994). of nitrogen for various pollinators,most no- In this study,we fed Eucalyptuspollen to LesserDou- tably honeybees(Apis mellifera; Schmidt and Buch- ble-collared Sunbirds to determine whether it was a mann 1985,Dobson and Peng1997), and it has re- viablesource of nitrogenfor this species. centlybecome clear that pollen alsois an important Methods.--Wecaptured eight sunbirds (seven elementin the diet of vertebratepollinators (Howell males and one female) in KirstenboschBotanic Gar- 1974, Richardson and Wooller 1990, Law 1992, van densin February1998 and housedthem separately in holdingcages (70 x 80 x 40 cm)in the University of Cape Town House.Except during experi- •Presentaddress: Mitrani Department of Desert ments, birds were fed a mixture of 0.7 M sucrose and Ecology,Jacob Blaustein Institute for Desert Re- Ensure© (3 g per 100 mL solution)presented in com- search,Ben-Gurion University of the Negev,Mid- mercially manufacturedfeeders and renewedtwice reshetBen-gurion 84990, Israel. daily. The sunbirdsmaintained body mass and con- E-mail: [email protected] dition on this diet. July2000] ShortCommunications 827

TABLE1. Percentageof intactand emptypollen grains in Eucalyptuscalophylla pollen (n = 8), in the exper- imentaldiet (n = 15 batches,5 eachwith low,medium, and high pollendensity), and in the cloacalfluid of Nectariniachalybea (n = 16). Valuesare œ_ SE.

Pollen E. calophylla Experimental Cloacal state pollen diet fluid Intact 88 + 1.5 85.2 _+_1.0 68.6 -+ 2.9 Empty 12 --_1.5 10.9 +_0.9 29.8 + 2.7 Partially empty 0 0.3 + 0.1 1.7 + 0.3 Germinating 0 3.6 + 0.9 0

Duringthe feeding trials, birds were kept in cylin- empty, partially empty, and germinating pollen dricalcages (diameter 36 cm,height 50 cm) in a con- grainsin pollenthat had beenin the diet solutions stant-environment room at 20øC, 50% relative hu- for 24 h. The delayensured that any effectson the midity, and a 12L:12D light cycle.Birds were fed pollen of immersionin sucrosesolution, such as in- from glass feeders constructedfrom modified bu- ducedgermination, were takeninto account.Similar retteswith the bottomend curvedupward to pre- measurementswere alsomade using fresh pollen. ventspillage and the openingenlarged .to a diameter We conductedthree trials,with a 72-h restperiod of 3 to 4 mm. We recordedbody massof eachbird at between each trail, so that each bird was tested on the beginningand end of eachfeeding trial. eachdiet. The diet givento an individualduring a For the first 48 h of each trial, birds were fed ad trial wasdetermined randomly, with the soleproviso libitum a maintenancediet of 1.2 M sucrosesupple- that no bird was giventhe samediet twice.When all mentedwith 3 g of Ensureper 100 mL solution.For three trials were completed,the samplesof cloacal the next48 h, theywere fed oneof threediets: 1 g, 3 fluid were thawed and diluted to 50 mL with dis- g, or 5 g of bee-collectedpollen suspendedin 100mL tilled water.The level of organicnitrogen in eachwas of 1.2 M sucrosesolution. We useda relativelyhigh thendetermined using Kjeldahl analysis. The nitro- concentrationto minimize settlingof the pollen;N. gen contentof all threeexperimental diets was de- chalybeareadily feeds on sugarsolutions of thiscon- terminedat thesame time. Using these values, we de- centration(Lotz and Nicolson1999). The pollen was termined nitrogen intake and nitrogen output of obtained commercially(Ridley Bee Products,West- eachbird. By regressingnitrogen balance (the dif- ern Australia)and was collectedby beeskept in an ferencebetween intake and output) againstnitrogen Eucalyptuscalophylla plantation. The pollen con- intake,we determinedthe dietary maintenanceni- tained95% Eucalyptus pollen (measured using light trogen requirements of N. chalybeaon E. calophylla microscopy),almost all of whichwas from a single pollen.We alsocalculated the standarderror of the species,presumably E. calophylla.A singledrop of a dietary maintenancenitrogen requirement. commercialmultivitamin supplement(Abbott Lab- Results.--Alleight birds maintainedbody mass oratories)was added to each 100 mL of sucroseso- and conditionduring the threefeeding trials. At the lution. The mixture was kept at 4øCand a freshso- start of the three trials, meanbody masswas 8.0 -+ lution madeevery 24 h. SE of 0.2 g, 8.0 -+ 0.2 g, and 8.2 _%0.1g, respectively; To minimizethe amountof pollenthat settledout, at the endof the trials,body mass averaged 8.1 + 0.2 the experimentaldiet availableto eachbird did not g, 7.9 ___0.1 g, and 8.0 -+ 0.2 g. exceed2 mL. Small volumesof fresh food were pro- In the pollen-sucrosemixtures fed to the birds, videdat intervalsof 2 to 3 h throughoutthe day,and 10.9%of the pollengrains were empty and 3.6%had the added volumes recorded. At the end of each trial, begunto germinateafter 24 h (Table1). In thecloacal the remainingsolution was filtered and the dry mass fluid, 29.8% of the pollen grainswere empty and of pollen determined. none was germinating(Table 1). This suggeststhat During the second48 h of the trial, traysof liquid 18.9%of the pollengrains had beenemptied in tran- paraffinwere placed beneath the cagesto collectthe sit, aswell as all thosethat had begunto germinate. cloacal fluid. Cloacal fluid was then removed from Becausethe latter were less than 4% of the total, and the traysusing Pasteur pipettes. During the second becausemost pollen was eaten in lessthan 24 h, 19% and third feedingtrials, three subsamples,each of is a reasonableestimate of the ability of N. chalybea less than 0.1 mL, were removed from the cloacalfluid to digestE. calophyllapollen grains. of eachbird and placedon microscopeslides. From Valuesfor nitrogen balanceversus dietary nitro- thesesubsamples we measuredthe meanpercentage gen intake are shownin Figure 1. The maintenance of full, empty,germinating, and partially empty pol- nitrogen requirement of a bird is its nitrogen intake len grains.The remainderof the cloacalfluid was when nitrogenbalance equals zero. In this case,the frozen. We also measuredthe percentageof full, dietary maintenancenitrogen requirement of N. chal- 828 ShortCommunications [Auk, Vol. 117

20 suchpollen has begun to germinate,it wouldbe eas- ier for the birds to digest. The nitrogenrequirement of N. chalybea,6.8 mg N day% is exceptionallylow. Costa'sHummingbird (Calyptecostae) has lower requirementswhen main- tainedon an artificialdiet (4.5mg N day% Briceand (• lO Grau 1991)but is only half the size of N. chalybea. When allometric effects are taken into account, the two valuesare very similar (using a massexponent of 0.75, and with body massesof 8 g and 4 g respec- tively, N. chalybeahas a requirementof 254 mg N kg -ø'75day -• and C. costae283 mg N kg -ø75day-•). Be- causethe low nitrogen requirementtakes into ac- countthe low digestibilityof E. calophyllapollen to N. chalybea,it is clear that the nitrogenthat is ab- sorbedfrom pollenis usedefficiently. This is almost 0 5 10 15 2o 25 certainlydue to the amino acid compositionof the Dietary Nitrogen Intake pollen.Levels of methionineand lysine (1.7% and 5.5% of total amino acid composition,respectively; (mgN day'l) van Tets and Hulbert 1999) are typical of thoseof FIG. 1. Relationshipbetween nitrogen balance good protein sources(Moir 1994), and the birds (NB, the differencebetween dietary nitrogenintake shouldretain the aminoacids they absorbedduring and nitrogen output) and dietary nitrogen intake digestion. (NI) for Nectariniachalybea on a diet of Eucalyptuscal- It is possiblethat avian nectarivoresas a group ophyllapollen. NB = 0.62 NI - 4.2 (R2 = 0.94). have unusuallylow nitrogenrequirements. Anna's Hummingbirdscan survive for 10 dayson a zero ni- trogen diet of 22% sucrose(Brice 1992). Recent work on Orange-tufted Sunbirds (Nectariniaosea) in Israel showsthat this species(mass = 6.9 g) requiresonly ybeawhen fed on E. calophyllapollen was 6.8 + 0.45 2.8 mg N day-• to maintain nitrogen balance on a mg N day% whichis equivalentto 57 + 4 mg of pol- diet of Drosophila(L. Roxburghpers. comm.). Given len per day.A very stronglinear relationship existed the potentialdifficulty for small nectarivoresto si- betweennitrogen balance and intake (R2 = 0.94). multaneouslygather nitrogen and sufficientnectar Discussion.--Theproportion of empty pollen to meettheir energyneeds, it is easyto imaginethat grains in the excretaand the low dietary mainte- strongselective pressure exists in favorof birdswith nancenitrogen requirements suggest that N. chalybea low nitrogen needs.Unfortunately, the data set on is capableof meeting its nitrogen requirementssole- aviannitrogen requirements is too small to make de- ly from pollen.However, the extentof polleninges- tailed comparisons. tion in the field is unknown.Some mammalian pol- It is particularlyinteresting to note that the re- linators are capableof gatheringsimilar or larger gressionof nitrogenbalance against nitrogen intake quantities of pollen (Law 1992, van Tets 1998), but for N. chalybeawas stronglylinear (Fig. 1), indicating the systemsare so differentthat it is impossibleto that as more nitrogenwas consumed,more was re- make valid comparisons. tained.In theory,this shouldnot occur,because the The percentageof empty pollen grains(19%) fell birds were neither growing nor reproducing(Brice betweenthe earlier valuesfor lorikeets(37%; Wooller and Grau 1991).In practice,however, it is a very com- et al. 1988)and (5%; Brice et al. 1989). mon result that for somereason is consistentlyig- Interestingly,Cockatiels (Nymphicus hollandicus) fed nored (e.g. Smith and Green 1987, Brice and Grau pollenfrom the samespecies of Eucalyptusachieved 1991, Witmer 1998, van Tets and Hulbert 1999). Al- a nearly identical result (18%; Brice et al. 1989). thoughit is possiblethat someform of unmeasured These values are much lower than those for mam- nitrogenexcretion is taking place,it seemsmore like- mals that feed on flowers(50 to 70%; van Tets1997, ly that vertebratesare capableof somedegree of ni- van Tetsand Whelan 1997),but 19%is high enough trogen retention. Determining whether and how to suggestthat pollenis a potentiallyuseful nitrogen they do so shouldbe an interestingavenue of future sourcefor thesebirds. Although in our study the research. number of germinating pollen grains was not Althoughour resultsshow that pollenis a viable enoughto havea majoreffect on digestion,it wasin- food sourcefor N. chalybea,the resultsdo not pre- terestingthat germinationoccurred in the sucrose cludethe use of insects.Cloacal fluid producedby solution.Nectarivorous birds often ingest pollen that freshlycaught N. chalybeacontains pollen and insect has fallen into the nectar.If a large proportionof fragments.The relativeimportance of the two nitro- July2000] ShortCommunications 829 gen sourcesmay vary in different times and places. mountain fynbos in the Cape of Good Hope. The Cape Floral Kingdom includesa large number South African Journalof Zoology 25:97-108. of plants that flower in winter, many of which have GOTTSBERGER,G., J. SCHRAUWEN, AND H. E LIN- large inflorescencesthat enableefficient pollen har- SKENS.1984. Amino acids and sugars in nectar, vesting.Because insect availability is likely to be low and their putative evolutionary significance. duringthe coldwinters typical of the area,whereas Plant Systematicsand Evolution 145:55-77. pollen availabilityis likely to be high, pollenmay be HOWELL,D. J.1974. Bats and pollen:Physiological as- an important seasonalsource of nitrogenfor N. chal- pectsof the syndromeof chiropterophily.Com- ybea.The same may apply to the honeyeatersof Aus- parativeBiochemistry and Physiology A 48:263- tralia'scoastal temperate regions for similar reasons. 276. In tropical areas such as Central America, it is less JACKSON,S., A. R. PLACE, AND L. J. SEIDERER.1992. likely that insectactivity dropsduring winter. Chitin digestion and assimilationby seabirds. The amino acid compositionof pollen and insects Auk 109:758-770. will play a majorrole in determiningthe extentto LAW, B. S. 1992. Physiologicalfactors affecting pol- whicheither food type servesas a nitrogensource for len use by Queenslandblossom bats, Syconycteris nectarivorousbirds. If insectsor pollen are deficient australis.Functional Ecology 65:634-648. in one or more amino acids, then birds will require LOTZ,C. N., AND S. W. NICOLSON.1999. Energy and access to those amino acids from another source. Be- water balance in an African sunbird, Nectarinia causefeeding on pollen versusinsects involves very chalybea,feeding on different nectar concentra- differentstrategies, it is importantthat the relative tions.Journal of ComparativePhysiology B 169: merits of the two nitrogen sourcesare taken into ac- 200-206. count in studies of the foraging behavior of small MARTINEZDEL RIO, C. 1994. Nutritional ecologyof nectarivorous birds. fruit-eating and flower-visiting birds and bats. Acknowledgments.--Wethank Cape Nature Conser- Pages103-127 in The digestive systemin mam- vation and KirstenboschBotanic Gardens for per- mals: Food,form and function (D. J. Chiversand mission to capture sunbirds, and C. Lotz and C. P. Langers, Eds.). Cambridge University Press, Spottiswoodefor their assistancein capturing and Cambridge,United Kingdom. maintaining the birds. ProfessorW. Stockallowed us More, R. J. 1994.The 'carnivorous'herbivores. Pages to use his Kjeldahl system. Financial support was providedby the SouthAfrican Foundationfor Re- 87-102 in The digestivesystem in mammals: Food, form and function (D. J. Chivers and P. search Development and the University of Cape Town. Langers, Eds.). Cambridge University Press, Cambridge,United Kingdom. PARKER, V. 1994. Swaziland bird atlas, 1985-1991. LITERATURE CITED Websters, Mbabane, Swaziland.

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