Behav Ecol Sociobiol (1996) 38 : 83–88 © Springer-Verlag 1996

Sean O’Donnell

RAPD markers suggest genotypic effects on foragerspecialization in a eusocial wasp

Received: 18 July 1995/Accepted after revision: 1 October 1995

Abstract Genetic variability within insect societies mayprovide a mechanism for increasing behavioral diver-sity among workers, thereby augmenting colonyefficiency or flexibility. In order to assess the possibil-ity that division of labor has a genetic component inthe eusocial wasp Polybia aequatorialis, I asked whetherthe genotypes of workers within colonies correlatedwith behavioral specialization. Workers specialized byforaging for one of the four materials (wood pulp, insectprey, nectar, or water) gathered by their colonies. I col-lected foragers on 2 days from each of three coloniesand identified the material the foragers were carryingwhen collected. I produced random amplified poly-morphic DNA (RAPD) markers from the genomicDNA of these foragers and estimated genotypic simi-larity of foragers based on sharing of variable RAPDmarker bands. Contingency tests on 20 variable lociper colony showed statistically significant (P < 0.05)biases in RAPD marker frequencies among foragertypes in the three colonies. Patterns of association ofRAPD marker bands with specializations were con-stant in two colonies, but changed between collectiondays in one colony. RAPD marker biases suggest thatdivision of labor among workers includes a geneticcomponent in P. aequatorialis. Colony-level selectionon variation in division of labor is a possible factorfavoring the evolutionary maintenance of high geno-typic variability (low relatedness) in epiponine waspcolonies and in other eusocial insects.

Key words Division of labor · Hymenoptera · Genotypic effects · Random amplified polymorphicDNA · Polybia aequatorialis

Introduction

Kin selection theory suggests that high expected re-latedness (r = 0.75) among the daughters of single,once-mated females is an important factor in the evo-lutionary origin and maintenance of hymenopteraneusociality (Hamilton 1964, 1972). However, the con-ditions for high relatedness are often violated in insectsocieties (reviewed in Breed 1989). Intracolony relat-edness is decreased when queens mate with severalmales (polyandry) or by the presence of multiple egg-laying queens (polygyny). Many eusocial wasps(Vespidae) are typically polygynous. Excepting a fewwith small colonies, all species examined in theNeotropical swarm-founding Epiponini are polygynous(Richards and Richards 1951; Richards 1978),although queen number may fluctuate over the courseof colony development (West Eberhard 1978). Asexpected in polygynous societies, relatedness amongnest mates tends to be low in epiponine wasps (as indi-cated by protein allozyme markers; Queller et al. 1988);in other words, genetic variability within colonies ishigh.

How is social cohesion maintained in the face oflow intracolony relatedness? The diversity hypothesis(Pamilo et al. 1994) posits that high genotypic vari-ability is adaptive at the colony level due to effects ondivision of labor among workers (Crozier and Consul1976; Crozier and Page 1985). For example, if special-ization in task performance by workers has a geneticcomponent, then genotypically diverse colonies may bebetter able to respond to unpredictable environmentalcontingencies. The diversity hypothesis assumes anassociation between behavioral and genotypic similar-ity among workers within colonies. Given that geno-typic diversity is high within epiponine wasp colonies,genotypic differences may contribute to behavioralspecialization among wasp workers (O’Donnell andJeanne 1992). The purpose of this study was to deter-mine whether genotypes and behavioral specialization

S. O’DonnellDepartment of Entomology, University of California at Davis,Davis, CA 95616, USA

covary in workers of the swarm-founding wasp Polybiaaequatorialis.

I used forager specialization as measure of behav-ioral differences among workers within colonies. Inswarm-founding wasps, foraging is performed by olderworkers who typically specialize on one of the fourmaterials gathered by their colonies (Forsyth 1978;O’Donnell and Jeanne 1990). Specializations on mate-rials do not change in a predictable pattern with ageand often persist for the duration of foragers’ lives(O’Donnell and Jeanne 1992). I assessed genotypic sim-ilarity based on sharing of random amplified poly-morphic DNA (RAPD) marker bands and askedwhether genotypic differences and forager specializa-tion covary. An association between genotypic andbehavioral similarity would suggest that division oflabor among workers has a genetic component.

Methods

Study site and organism

The study was conducted in Monteverde, Costa Rica (10°18@N,84°49@W; 1250–1550 m elevation), in November 1993 (collection ofwasps used in screening of RAPD primers) and April /May 1994.Polybia aequatorialis was among the most abundant eusocial waspsin the area. Nests typically housed populations of several thousandadult wasps. With rare exceptions, epiponine wasp colonies of thissize are permanently polygynous (Richards and Richards 1951;Jeanne 1991).

Between 10 April and 28 April 1994, three colonies’ nests wereremoved from road bank sites at night and were transported to anobservation area sheltered from rain (approximately 1300 m eleva-tion). Workers from the subject colonies foraged and repaired nestdamage beginning the day following transfer.

Quantification of forager specialization

In this part of the study I assessed whether foragers show levels ofspecialization on materials similar to other Polybia species(O’Donnell and Jeanne 1990, 1992). Workers removed from thecolonies were lightly anaesthetized with ether, individually markedon the thorax in a numerical code with paint pens, then returnedto their nests. On 6 and 7 May, I marked 185–225 workers in eachcolony. On 13–19 May, I captured and marked 20–230 additionalforagers.

I observed foraging behavior for two days at colony A (16 and17 May) and colony B (20 and 21 May) to quantify forager spe-cialization on materials. I sat near the nest entrances for two con-tinuous periods of 3 h in the morning and afternoon daily andrecorded worker identity number and material carried for all for-ager arrivals. Solid loads (prey and pulp) are carried externally andwere visible, while liquid loads (water and nectar) were distinguishedby the foragers’ posture upon transferring loads to nest mates (seeHunt et al. 1987).

To quantify the degree of specialization I used the Shannon-Wiener information variable H (Lehner 1979), which provides ameasure of how evenly observations are distributed among cate-gories (in this case, individuals’ foraging trips among materials):

Hx = []pxlog2px

where px is the proportion of a given forager’s trips devoted to mate-

rial x. This variable takes on lower values with greater specializa-tion, and with four categories (materials) the maximum value ofHx is 2. I used 2[Hx as an index of specialization so that the value of the index would increase with the degree of foragerspecialization.

Collection of foragers for genetic analysis

I collected arriving foragers from each colony on 2 days (colonyA: 18 and 24 May; colony B: 22 and 25 May; colony C: 23 and26 May) to be genotyped using RAPD markers. Upon foragerarrival, I noted the material carried, then grasped the wasp withforceps and placed it in a vial of 100% ethanol labeled for thatmaterial. Collections continued throughout the daylight hours, withoccasional breaks of 15–30 min during periods of heavy rain orwhen colonies became agitated by forager removals. Wasps werestored in ethanol at [10 °C in the field and at [20 °C in the lab-oratory until DNA extractions were performed (see Table 1 for thenumber of foragers captured).

DNA extraction and RAPD markers

DNA extractions and RAPD marker analysis were performed fol-lowing a protocol modified from Hunt and Page (1995). After blot-ting to eliminate EtOH, wasps’ wings were removed and tissueswere homogenized in 200 µl CTAB extraction buffer [1% hexade-cyltrimethyl ammonium bromide, 750 mM NaCl, 50 mM TRIS-Cl(Ph 8), 10 mM EDTA] with 1 µl of 100 ug/ml proteinase K. Sampleswere incubated at 55 °C for 2–4 h, after which 50 µl 1.5 M

NaCl/50 mM TRIS-Cl (pH 8) was added to prevent coprecipita-tion of CTAB and polysaccharide with the DNA. Samples wereextracted with 500 µl phenol/chloroform and 250 µl chloroform,and DNA was then precipitated with 25 µl 3 M sodium acetate (pH 5) and 500 µl cold ([20 °C) 100% EtOH. Following centrifu-gation for 20 min at 4000 × g, the precipitate was washed with 500 µl70% EtOH and resuspended in 50 µl of 10 mM TRIS (ph 7.6) /1 mM

EDTA. DNA was quantified with a fluorometer (Hoeffer), dilutedto 3 ng/µl in 10 mM TRIS/0.3 mM EDTA and stored frozen at[70 °C.

Polymerase chain reactions (PCR) were made up of 12.5 µl totalvolume with 0.5 µm primer (below), 100 µm each of dATP, dGTP,dCTP, and dTTP (Pharmacia), 10 mM TRIS-HCl (ph 8.3), 50 mM

KCl, 2 mM MgCl2, 0.25 U Taq polymerase, and 3 ng genomic DNAfrom a single wasp. Reactions were topped with a drop of sterilemineral oil. PCR amplification of RAPD markers was performedin Perkin-Elmer 480 thermal cyclers with following conditions: fivecycles of 94 °C – 1 min/35 °C – 1 min/2 min ramp to 72 °C – 2 min;32 cycles of 94 °C – 10 s/35 °C – 30 s/72 °C – 1 min; one cycle of72 °C – 5 min; soak at 15 °C.

PCR reaction products (12.5 µl) were run on 20 × 25 cm gelsof 1% synergel /0.7% agarose dissolved in 250 ml 0.5 × TBE. Gelswere run at 100 V for 5 h, then stained with ethidium bromide for20 min and destained in water for 10 min. Gels were photographedon a UV transilluminator with Polaroid 667 film. Scoring of mark-ers was done from the photographs. Variable bands scored wereeither presence absence or fragment length polymorphisms. Bandswere scored blind to forager category. Approximately 10% of thePCR reactions were run twice and checked for repeatability; allscored bands showed 100% repeatability.

I screened 128 ten-nucleotide primers (Operon Technologies,Alameda, Calif.) for polymorphic markers using P. aequatorialisDNA from wasps collected at Monteverde in November 1993.Primers used in the study were those that produced some clearlyvisible, variable bands, along with several unvarying bands whichserved as internal size markers and as checks on PCR reaction qual-ity. Thirteen primers were selected which yielded from one to threevariable marker loci each; 20 variable markers were scored for each

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colony. Detailed protocols for DNA extraction, RAPD marker pro-duction, and lists of primer sequences and marker band sizes areavailable from the author upon request.

Statistical analyses

I tested whether foragers’ genotypes corresponded with the mater-ial they were carrying when collected. Genotypic similarity withincolonies was inferred from sharing of RAPD bands, and contin-gency tests of marker band frequency bias with foraged materialwere performed for each RAPD marker. Expected cell values in allcontigency tables were large enough to warrant use of likelihoodratio contingency tests (G2 statistic; Lewontin and Felsenstein 1965;Fienberg 1989). Results of Fisher’s exact tests performed on theindividual (forager category × RAPD marker band type) tables weresimilar to the G2 statistics. Assuming that all marker bands’ fre-quencies were independent, G2 statistic values were summed overall markers to obtain an overall test of bias for each colony on eachday. If marker loci were genetically linked, the degrees of freedomfor the pooled test would be overestimated by an unknown amount.This would inflate type I error only if a marker was spuriouslybiased across behavioral categories (due to sampling error) andother genetically linked markers showed the same association. Tightgenetic linkage within a set of markers generated from 20 short ran-dom primers is unlikely, especially if P. aequatorialis recombina-tion rates are high, as in other eusocial Hymenoptera (Hunt andPage 1995). G2 statistics for three-way interactions of collectionday × material × marker band frequency were calculated to test forchanges in marker frequencies between collection days (Fienberg1989).

Results

Forager specialization during observation days

Foragers did not devote equal numbers of trips to eachmaterial on observation days (for foragers making at

least four trips, colony A G2 = 862.07, df = 75,P < 0.001; colony B G2 = 266.94, df = 45, P < 0.001).Rather, an index of specialization (see Methods)showed that nearly all foragers devoted most or all tripsto a single material, and that specialization did notdecrease with an increasing number of observedforaging trips (i.e., increased sampling of behavior;Fig. 1).

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Fig. 1 Specialization on materials by Polybia aequatorialis workersduring foraging observation days. Shannon -Wiener based indexof specialization on materials (maximum value of 2) is plottedagainst foraging rate for workers from two colonies. Foraging rateswere calculated by dividing the number of observed foragingarrivals by total observation time summed over two observationdays. The maximum index value of 2 represents complete special-ization on one material; a value of 1 (horizontal line) results if equaleffort is devoted to two materials and none to the others

Material Number Material Numberforagers foragers

Colony A18 May 24 MayPrey 10 (10) Prey 42 (42)Nectar 47 (46) Nectar 22 (22)Water 8 (8) Water 7 (7)Pulp 5 (5) Pulp 19 (19)

Total 70 (69) Total 90 (90)

Colony B22 May 25 May

Prey 25 (25) Nectar 50 (47) Nectar 39 (37)Water 8 (8)Pulp 6 (6) Pulp 7 (7)

Total 89 (86) Total 46 (44)

Colony C23 May 26 May

Prey 19 (19) Prey 7 (7)Nectar 44 (44) Nectar 49 (49)Water 4 (4) Water 2 (2)Pulp 3 (3) Pulp 5 (5)

Total 63 (63) Total 63 (63)

Table 1 Numbers of foragersdevoted to gathering each offour materials on collectiondays (numbers of foragers onwhich RAPDs were run inparenthesis)

Forager collections

More foragers were devoted to food materials than tonest materials on all collection days; nectar foragerswere typically the most abundant category (Table 1).Foraging rates for each material decreased over the dayon collection days and foraging eventually ceased(excepting nectar, which continued arriving at low ratesuntil nightfall). Materials gathered by fewer individu-als generally ceased arriving earliest in the day(personal observations). Most collected foragers wereunmarked in all samples. Marked foragers typicallydevoted most of their trips to carrying the same ma-terial during behavioral observations as they were

gathering when collected several days later (Fig. 2).Therefore, foraging specializations did not change formost individuals; furthermore, no regular sequence ofchange in specializations with age was evident amongthose foragers that switched (switching occurred amongall materials; Fig. 2).

RAPD marker frequency biases across materials

A subset of the RAPD markers (1– 4 markers) showedstatistically significant frequency biases with foragedmaterial on each collection day. There was an overallbias (G2 summed across markers, P < 0.05) in markerfrequencies with foraged material for one of the twocollection days in each colony; frequencies showed anon-significant trend toward bias (0.05 < P < 0.10) ontwo other collection days (Table 2). Pooling over col-lection days, marker band frequencies were significantlybiased with forager specializations in each colony(Table 2). Colony A showed significant changes in pat-terns of allele frequency bias with materials betweenthe first and second collection days, while colonies Band C did not (Table 2).

Discussion

Over several days time, P. aequatorialis foragers exhibited high levels of specialization on materials sim-ilar to those used by P. occidentalis (O’Donnell andJeanne 1990). RAPD marker band frequencies showedbiases associated with forager specialization in eachcolony, suggesting that behavioral differences among P. aequatorialis workers are based in part on geneticvariability.

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Fig. 2 Consistency of P. aequatorialis forager specialization fromobservation days to collection days. The x-axis lists the materialforagers were carrying when collected for genetic analysis. Stackedshaded bars represent numbers of foragers devoting all ( filled bars)or most (hatched bars) trips during observations to the same mate-rial they were carrying when collected; open bars represent foragersdevoting most trips to another material

Colony A Colony B Colony C

Day 1 Day 2 Day 1 Day 2 Day 1 Day 2

Overall within-daytesta G2 = 77.2 G2 = 133.5 G2 = 84.0 G2 = 70.2 G2 = 77.0 G2 = 91.7

df = 60 df = 60 df = 60 df = 60 df = 63 df = 63NS P < 0.005 P < 0.05 NS NS P < 0.05(P < 0.10) (P < 0.10)

Overall test pooledacross daysb G2 = 80.2 G2 = 113.7 G2 = 91.3

df = 57 df = 60 df = 60P < 0.05 P < 0.005 P < 0.01

Overall three-wayinteractionc G2 = 119.9 G2 = 40.8 G2 = 74.4

df = 60 df = 60 df = 63P < 0.005 NS NS

a Likelihood ratio tests for allele frequency bias with forager specialization summed across allmarkers within collection daysb Excluding markers with significant 3-way interaction (below)c 3-way interaction of (collection day × forager specialization × marker band frequency), summed overall markers

Table 2 Results of contingencytests for biases in RAPDmarker band frequenciesamong forager types in Polybia aequatorialis

For the foragers used in genetic analysis, identifi-cation of specialization categories was imperfectbecause it was based on a single foraging trip. Due tooccasional switching among materials by foragers, it islikely that I assigned some individuals to materialsother than their specialty. Furthermore, the ability todetect biases statistically was limited by the small numbers of foragers gathering building materials.Significant biases of RAPD marker band frequencieswith forager specialization, occurring even in the faceof these sampling uncertainties, provide strong evidencethat genotypic variation corresponded to differences inforager behavior.

Because colonies ceased gathering materials roughlyin ascending order of the number of workers devotedto each material, recruitment of new foragers withindays appeared to be low. I assumed that within collec-tion days, the samples represented the genotypic diver-sity of foragers active on that day and were not stronglyaffected by recruitment of new foragers. Presumablyforager collections on the second day sampled workersrecruited in response to destructive sampling (foragerremovals) on the first collection day. In colony A thedistribution of marker bands among specializationschanged significantly between collection days, while inthe other colonies the associations of RAPD markerbands with specialization were constant. In colony A,the foragers recruited on the second collection day wereapparently drawn from a different pool of genotypesthan those on the first day.

The relative importance of different sources ofgenetic variation within P. aequatorialis colonies isunknown. It is likely that the presence of multiplequeens (polygyny) contributed to genotypic variabilityin the worker force. Genetic recombination amongheterozygous loci in queens and multiple mating(polyandry) may have also contributed to genotypicvariability; multiple mating has not been documentedin epiponines, but is possible. Anecdotal field observa-tions suggest that female Polybia reproductives visitmating aggregations of males (R.L. Jeanne, personalcommunication), which may promote outcrossing andincrease genetic variation within colonies. Protein allo-zyme markers did not produce evidence of inbreedingin three species of epiponine wasps (Queller et al. 1988,1993).

One plausible mechanism for the observed cor-respondence of genetic and behavioral variation isthat marker loci were linked to genes with strong be-havioral effects. Genes with major effects on behav-ioral variation have been demonstrated for colonyhygiene and foraging specialization in honey bees(Rothenbuhler 1964; Page et al. in press). No markerbands appeared to be associated with particular behav-ioral roles across all colonies in this study, althoughtwo colonies showed consistent associations of RAPDmarker bands with behavior over time, even afterdestructive sampling of foragers. Genotypic effects on

behavior may result from complex developmentalprocesses, and the behavioral phenotype associatedwith a given genotype may vary depending on the socialand external environmental conditions under which aworker develops.

Evidence for genotypic effects on worker behaviorhas now been found in a diversity of insect societies,including polygynous ants (Stuart and Page 1991;Snyder 1993), polyandrous honey bees (Dreller et al.1995; Oldroyd et al. 1994; Page et al. in press), andpolygynous wasps (present study). Taken by itself, anassociation between behavioral specialization andgenetic variability is necessary but not sufficient todemonstrate that low intracolony relatedness is adap-tive due to increased colony fitness. It is, however, animportant first step in determining the bases for theevolutionary maintenance of within-colony genotypicdiversity. Polygyny and other factors that decreaseintracolony relatedness may have arisen for other reasons, however, genotypic diversity might still bemaintained by selection for increased colony fitness viamore efficient or plastic division of labor. A thoroughtest of the diversity hypothesis in eusocial wasps wouldinclude measures of the effects of differing degreesof genetic variability on colony performance. Severalrecent studies support the notion of increased per-formance and/or homeostatic ability with increasedgenetic diversity in honey bee colonies (Oldroyd et al.1992; Fuchs and Schade 1994; Page et al. 1995). Thisrelationship may be possible to test in epiponine waspsin the field: variation in queen number has beendocumented among species, among colonies (Richardsand Richards 1951), and over the course of colonydevelopment (Queller et al. 1993). If the diversityhypothesis is correct, then variation in queen numberwill be found to translate into measurable differencesin worker genotypic diversity, worker behavior, and incolony performance.

Acknowledgements Rob Page was most generous in providing sup-port and laboratory space, as well as discussions on design andanalysis. Dave Nielsen and Greg Hunt gave valuable advice ondeveloping RAPD markers. Frank Joyce and Katie van Deusensmoothed many logistical bumps in the field, and David McDonaldand Susan Bulova assisted ably with field work. Susan Bulova, HughDingle, Benjamin Oldroyd, Robert Page, and three anonymousreviewers made helpful comments on the manuscript. Jim Carpenteridentified the wasps; vouchers of Polybia aequatorialis from the sub-ject colonies are deposited in the Bohart Entomological Museumat the University of California at Davis. The Organization forTropical Studies assisted in obtaining research permits from theCosta Rican Ministry of Natural Resources (permit numbers00106590 and 00108567). Thanks to Paul Hanson and HumbertoLezama of the University of Costa Rica for loans of specimens.Financial support was provided by postdoctoral fellowships fromthe University of California at Davis U.S. National ScienceFoundation-funded Animal Behavior Research Training Grantand from the NSF-Division of Environmental Biology (DEB-9303244).

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