Nestmate recognition in social wasps: the origin and acquisition of recognition odours

Anita. Behav., I986, 34, 685-695

Nestmate recognition in social wasps: the origin and acquisition of recognition odours

G E O R G E J. G A M B O A , H U D S O N K. REEVE*, I. D E A N N A F E R G U S O N & T R A C Y L. W A C K E R

Department of Biological Sciences, Oakland University, Rochester, Michigan 48063, U.S.A.

Abstract. Laboratory studies of nestmate recognition in gynes (potential queens) of the social wasp, Polistes fuscatus, indicate that recognition odours have both a heritable and an environmental component. In addition, both endogenous odours (acquired as brood) and adult-acquired odours appear to mediate nestmate recognition. Heritable and environmental, as well as endogenous and adult-acquired recognition odours, are not additive in their effect on tolerance, suggesting a 'cue similarity threshold' model of recognition rather than a model that postulates tolerance continuously increasing with increasing degree of similarity between the learned and perceived cue. Young gynes (1.7-71.8 h after emergence) clearly possess recognition odours and there is no evidence that the age of a young gyne affects its expression of recognition odours. The failure of a wasp, isolated from its natal nest at emergence, to recognize its nestmates is due to the disruption of its learning of recognition odours, not to its acquisition of recognition odours. Gynes appear not to learn recognition odours directly from themselves. Young gynes deprived of the opportunity to learn recognition odours from their natal nest treat all gynes as nestmates, regardless of relatedness. This latter result, together with additional evidence, suggests that the ontogeny of nestmate recognition ability involves the development of intolerance to unfamiliar odours rather than the development of tolerance to familiar odours.

Nestmate (kin) recognition consists of two distinct components: (1) one animal expresses a recognition cue while (2) a second animal perceives and decodes it (Beecher 1982; Sherman & Holmes 1985). A large number of recent studies of nestmate recognition in social insects have focused on the expression component of nestmate recognition, more specifi- cally, the production of the recognition cue (e.g. Greenberg 1979; Breed 1981, 1983; Getz & Smith 1983).

There is considerable evidence that social insects discriminate nestmates from non-nestmates by using odours as recognition cues. In fact, no colony or individual recognition cues other than chemical signals are known in social insects (H611dobler & Michener 1980). These recognition odours are thought to be associated with the insect integu- ment, and appear to be perceived only through contact or close approach (Jutsum et al. 1979).

Nestmate recognition odours may be categor- ized according to their ultimate origin. A recognition odour can be designated as 'environmental' if

* Present address: Section of Neurobiology and Beha- vior, Cornell University, Ithaca, New York 14850, U.S.A.

odour differences among colonies depend upon differences in inter-colony environmental factors such as food or nesting materials. A recognition odour can be designated as 'heritable' if odour differences among colonies persist even though inter-colony environmental factors are identical. Heritable odours include genetic factors and non- genetic (extrachromosomal) maternal factors.

The ultimate origin of recognition odours differs from, and cuts across, the acquisition and production of recognition odours. Environmental recognition odours may be acquired by individuals, either through ingestion or through absorption into (or adsorption to) the cuticle. Heritable recognition odours may also be acquired by individuals, either directly or indirectly, from some subset of other colony members. For example, heritable recognition odours could be externally acquired by other colony adults directly from the queen (Carlin & H611dobler 1983). Alternatively, such odours produced by the queen could be applied to the nest where other colony adults might then acquire them indirectly. Recognition odours produced by insects themselves (endogenous odours) may depend on variable environmental factors (e.g. food) or be independent of environmental factors (i.e. heri-

685

686 Animal Behaviour, 34, 3

table). Thus, acquired and endogenous recognition odours each may be heritable, environmental, or contain components of both. Our categorization of recognition odours differs from current usage, which frequently treats 'acquired' and 'environmental' and 'endogenous' and 'genetic' as syno- nyms (Crozier & Dix 1979; Jutsum et al. 1979).

Relatively few studies have rigorously investigated possible environmental recognition odours, although several studies have demonstrated that environmental factors can affect nestmate recognition. Kalmus & Ribbands (1952) showed that the manipulation of food sources can profoundly affect nestmate recognition in honey bees. Other studies providing evidence for environmental recognition odours include Wallis (1962) and Jut- sum et al. (1979) for ants, Buckle & Greenberg (1981) for sweat bees, and K6hler (1955, as dis- cussed by Ribbands 1965) for honey bees.

Most recent studies of nestmate recognition in social insects have focused on possible heritable recognition odours. The design of these studies has generally involved the documentation of kin recognition under conditions of controlled environmental odo urs. Reports providing evidence of heritable recognition odours include Breed (1981, 1983), Getz et al. (1982) and Getz & Smith (1983) for honey bees, and Wallis (1962), Mintzer (1982) and Carlin & H611dobler (1983) for ants. The most conclusive evidence for a genetic recognition odour is that reported by Greenberg (1979) for sweat bees. These studies, however, do not preclude the possibility of environmental components of nestmate recognition odours, nor do they establish that heritable odours are more important than environmental odours for a given species under natural conditions. Indeed, there is convincing evidence for both environmental and heritable components of recognition odours in two extensively studied species, the sweat bee Lasioglossum zephyrum (Greenberg 1979; Buckle & Greenberg 1981) and the honey bee Apis mellifera (Kalmus & Ribbands 1952; Breed 1981).

In social wasps of the genus Polistes, nestmate recognition odours are learned from the natal nest within hours after eclosion and the memory of these odours is durable if not permanent (Pfennig et al. 1983a). Although Polistes females can acquire recognition odours from a nest and/or its adults (Pfennig et al. 1983a), it is presently unknown whether Polistes recognition odours are environmental, heritable, or made up of both components.

It is also unknown if nestmate recognition is mediated only by acquired odours. We therefore studied the primitively eusocial wasp, Polistes fuseatus, to investigate: (1) the ultimate origin of inter-colony differences in nestmate recognition odours, and (2) the possible contributions of endogenous and adult-acquired recognition odours. Our study also examined the effect of age on the expression of recognition odours.

C O N T R O L L E D E N V I R O N M E N T STUDY

Methods

Rationale The controlled environment study was designed

to reveal whether the nestmate recognition odour of P. fuscatus is heritable, environmental, or a combination of both components. We investigated the nestmate recognition ability of gynes reared in a controlled laboratory environment and compared these results with nestmate recognition studies of gynes reared in an uncontrolled field environment.

Laboratory-reared gynes Approximately 2000 gynes of P. fuscatus were

marked with a colony specific code while on their natal nest in September 1982 at the MacBride Field Campus of the University of Iowa. The following May we collected 95 marked, overwintered foundresses from 17 colonies at five sites of the field campus. These foundresses, which had not yet begun to nest, were collected while clustered in artificial nestboxes (Klahn & Gamboa 1983 des- cribe the collection site). We placed two or three foundresses that were former nestmates into each of 40 identical cardboard nestboxes (50 • 30 • 20 cm).

We provided honey, water, and construction paper from identical sources to all wasps in nestboxes maintained in a laboratory at 26~ on a natural photoperiod to stimulate nesting. A nest was constructed in each of the 40 nestboxes. As soon as a nest contained larvae, we began providing mealworms (secured from a single supplier) biweekly to the nestbox for the duration of the colony cycle. After the first workers emerged from a nest, the numbers of larvae increased rapidly and the provision of mealworms to nestboxes was

Gamboa et al.: Social wasp nestmate recognition 687

increased accordingly. By 10 September, male and female wasps were clustered on each nest, which no longer contained brood. We assumed that these females, which had extensive exposure to their natal nest and nestmates, were gynes (see Haggard & Gamboa 1980), and utilized them for our study. All of these gynes had spent their entire life cycle, i.e. from egg through to adult, in a controlled laboratory environment.

Field-reared gynes We collected 15 naturally nesting field colonies

(adults and nest) of P. fi~scatus from 7 to 19 September 1983 from nifie sites in southeastern Michigan. W e placed each colony with its nest into a separate 50;< 30 x 20-cm holding box. Nests had few, if any, brood. We provided honey and water from identical sources to all wasps and maintained them in a laboratory at 26~ on a natural photoperiod.

The females in these holding boxes, collected late in the colony cycle, were assumed to be gynes (Haggard & Gamboa 1980). These gynes, which had extensive exposure to their natal nest and nestmates, had spent their life cycle from egg through to adult in an uncontrolled field environment. Previous nestmate recognition studies of field-collected P. fuscatus females from southeastern Michigan (Pfennig et al. 1983a) and the MacBride Field Campus of the University of Iowa (Pfennig et al.~'1983b) have demonstrated that females can discriminate between nestmates and non-nestmates.

Common procedures Beginning on 22 September, we began to remove

gynes (both laboratory and field-reared) from their nestboxes and arrange them into triplets of two nestmates and a non-nestmate. The non-nestmate of a triplet was from a colony (or was the progeny ofa foundress) that was collected at least 3 km from that of the two nestmates. Thus, it was unlikely that non-nestmates were closely related (see Klahn & Gamboa 1983). We marked each wasp uniquely with the same colour of enamel for individual identification and then introduced the triplet of wasps into a cardboard observation box (15 x 15 x 15 cm) faced with clear plastic, at 1200 hours. Each triplet was observed for 45 rain at 1400, 1600 and 1900 hours on the day of the introduction and at 1100 hours the following day, for a total of 3 h. We recorded behavioural

interactions and spacing patterns (see below) of 10 triplets of laboratory and 11 triplets of field-reared gynes from 22 September to 24 October 1983. We alternated observations of laboratory and field- reared triplets.

Within 1-6 days after completing behavioural observations of each triplet, we periodically recorded the identity of wasps that were paired (motionless and less than 5 cm apart) in the observation box. We surveyed all observation boxes (both laboratory and field-reared gynes) from 28 September to 11 November (65 surveys, 32 different days; see below).

Recognition bioassay Observations. We recorded behavioural interac-

tions and spacing patterns among the three wasps in each observation box. Observations were blind, i.e. the observer did not know which two triplet members were nestmates. Different wasps were used for each triplet observation. Each interaction between a pair of wasps was classified as one of the 10 interaction types described by Pfennig et al. (1983b). We modified the tolerance scale developed by Pfennig et al. (1983b) by assigning a retreat index, instead of a rank, to each interaction type. The retreat index for a given interaction type is equal to

9(A - ~_ / _ 1 A - - T

where 2" is the estimated mean speed of retreats (cm/s) by wasps that were recipients (not initiators) of interactions of the given type, A is the greatest mean retreat speed recorded for any interaction type, and T is the lowest mean retreat speed recorded for any interaction type. Our modifica- tion converts the previous tolerance scale, which was based on ranks, into an interval scale of measurement. The modified tolerance scale, like that of Pfennig et al. (1983b), ranges from 1, representing the retreat index of the most intolerant interaction (chasing), to 10, representing the retreat index of the most tolerant interaction (prolonged mutual antennation). A tolerance value was obtained for each pair of wasps in a triplet by calculating the mean of the retreat indices of all interactions between the pair of wasps.

We also recorded the number of minutes two wasps were paired (motionless and less than 5 cm apart). Thus, we had two measures of spatial tolerance for each pair of wasps within a triplet, the


tolerance value and the minutes paired. For each triplet we compared the tolerance value and minutes paired for nestmates with the mean tolerance value and mean minutes paired for the two pairs of non-nestmates as our bioassay of nestmate recognition.

Surveys. Surveys were blind. Pairing ratios of nestmates were calculated for each triplet as follows.

Tn Pairing ratio =

T ,+0-5 (T~u+ T2u)

where Tn is the number of times that nestmates paired and Tl, and T2u are the number of times that the unrelated wasps (non-nestmates) paired. A pairing ratio greater than 0.50 indicates preferential nestmate pairing.

After we completed behavioural observations and pairing surveys, we measured the maximum head width of each gyne as an estimate of body size (after Haggard & Gamboa 1980).

Results

Observations Field-reared gynes were able to discriminate

nestmates from non-nestmates. Nestmates had a significantly greater mean tolerance value than non-nestmates (P < 0"01, Table I). Although nestmates spent more time paired than non-nestmates, the differences were not significant (P < 0'3, Table I). In contrast to the field-reared gynes, laboratory- reared gynes did not discriminate between nestmates and non-nestmates. Nestmates did not have a significantly greater mean tolerance value (P>0 .4 ) and did not spend significantly more

minutes paired (P>0-4) than non-nestmates (Table I).

Non-nestmate, laboratory-reared gynes had a significantly greater mean tolerance value than non-nestmate field-reared gynes (P<0.005, two- tailed Mann-Whi tney U-test; Table I). However, nestmate laboratory and nestmate field-reared gynes did not differ significantly in mean tolerance value (P>0-5 , two-tailed Mann-Whi tney U-test; Table I). There were no significant differences in minutes paired between non-nestmate laboratory and non-nestmate field gynes or between nestmate laboratory and nestmate field gynes ( P > 0.2 for both, two-tailed Mann-Whi tney U-tests; Table I), but the trends paralleled the corresponding trends in the tolerance values.

Surveys The mean pairing ratio for field-reared gynes

(0"56) was greater than that expected for random pairing (0.50) though not significantly so (P < 0.20, one-tailed Wilcoxon ranked-sum test). The mean pairing ratio for laboratory-reared gynes (0' 57) was significantly greater than 0-50, the expected mean for random pairing (P < 0.05). The mean pairing ratios for laboratory- and field-reared gynes were not significantly different (P>0.50 , two-tailed Mann-Whi tney U-test).

There was no significant difference between the size differences of nestmates and the size differences of non-nestmates for both laboratory and field- reared gynes (P>0 .20 for both; two-tailed Wil- coxon matched-pairs signed-ranks tests). Thus size differences within groups do not appear to account for the observed differences in tolerance within and between groups.

Table I. Tolerance values and minutes paired from laboratory- and field-reared gynes of Polistesfuscatus in controlled environment study

Tolerance value (mean • so) Minutes paired (mean + sD) Experimental No. of

group triplets Nestmates Non-nestmates P* Nestmates Non-nestmates P*

Field-reared 11 8.54• 7.89• <0.01 24.35• 19.18• 16.4 <0.30 gynes

Laboratory- 10 8.68• 8.60• >0-40 30.06• 19.07 33.95• >0-40 reared gynes

* One-tailed Wilcoxon matched-pairs signed-ranks tests. P values refer to comparisons between nestmates and non-nestmates. Larger mean tolerance values and mean minutes paired indicate greater tolerance.


RECIPROCAL NEST EXPOSURE S T U D Y

Methods

Rationale

Pfennig et al. (1983a) showed that adult P. fuscatus gynes can acquire recognition odours from a nest and/or its adults. We designed the reciprocal nest exposure experiment to determine whether endogenous odours, odours acquired as adults, or both, mediate nestmate recognition in gynes of P. fuscatus.

Preliminary procedures We collected 33 naturally-nesting field colonies

of P.fuscatus from 20 August to 5 September 1983 from eight distinct sites (more than 10 km apart) in southeastern Michigan. These pupae-laden nests had few, if any, larvae or eggs. After being cleared of adults, each nest was cut into fragments of 10-15 cmL Each nest fragment was placed into a separate, labelled, 0.4-1itre uncovered plastic cup and maintained at 25-27~ in a laboratory. We monitored nest fragments for adult emergences from 26 August to 8 September 1983 and assumed that emerging females were gynes (Haggard & Gamboa 1980).

Controls Control gynes were temporarily removed from

their natal nest fragment after they had removed their pupal caps. These gynes were given a unique, red paint-mark and then returned to their natal nest fragment. After control gynes became inactive on their natal nest fragment, they were provided with honey and water from a single source and the plastic cup was covered with a perforated, trans- parent lid. Nest fragments were used only once as an exposure substrate for a gyne.

After a variable period of exposure (2?=45 h-/-SD 18 h; r ange=15-70 h) to their natal nest fragment and subsequently eclosing nestmates, control gynes ( N = 36) were grouped into 12 triplets, each consisting of two nestmates and a non- nestmate matched as closely as possible for duration of exposure to their natal nest fragment (Fig. 1). At 1100 hours, we introduced each triplet of control gynes into a 30 x 30 x 30-cm cardboard box faced with clear plastic. Each triplet was observed for 30 min at 1300, 1500, and 1700 hours on the day of introduction and at 1100 hours the

U ~ N 2 U N2 Control Treatment

Figure l. Diagrams depicting the two experimental conditions of the reciprocal nest exposure study. Control: nestmates (N~ and N2) learn common recognition odours from different fragments of their natal nest (N). Treatment: nestmates (N~ and N2) learn common recognition odours from different fragments of the non-nestmate's nest (U) while the non-nestmate (U) learns recognition odours from the nestmates' nest fragment (N).

following day, for a total of 2 h. We recorded behavioural interactions and spacing patterns (see below) of 12 control triplets from 28 August to 9 September 1983.

Treatment Treatment gynes were treated like control gynes,

except that after emergence and marking, each gyne was placed upon a different fragment of a foreign nest (one collected from a distant site) instead of being returned to its natal nest fragment. Nest fragments were used only once as an exposure substrate for a gyne.

After a variable period of exposure 0 ? = 3 9 h + s o 14 h; r ange= 19.4-63 h) to the foreign nest fragment and subsequently emerging unrelated wasps, treatment gynes ( N = 36) were grouped into 12 triplets, each consisting of two nestmates and a non-nestmate matched as closely as possible for duration o f exposure to a foreign nest fragment. The two nestmates in a treatment triplet had been exposed to the nest (and nestmates) of the non- nestmate in the triplet; conversely, the non-nestmate had been exposed to the nest (and nestmatcs) of the nestmates in the triplet (see Fig. 1). Observa- tions of treatment triplets were made at approximately the same times and between the same dates as observations of control triplets.

Recognition bioassay We used the same recognition bioassay that we

described for the observations of the controlled environment study.


Predicted experimental results All of our predictions of experimental results

include the assumption that gynes learn recognition odours only fi-om the natal nest and/or its brood and that this learning occurs entirely when they are adults. These assumptions are consistent with the results of previous studies (Shellman & Gamboa 1982; Pfennig et al. 1983a,b) as well as with those of our variable age study.

In the control observations, the nestmates should be the most tolerant pair of wasps in a triplet. This result is expected based on previous studies (e.g. Shellman & Gamboa 1982; Pfennig et al. 1983a) in which control triplet members also had extensive exposure to both their natal nest and nestmates. Control nestmates have the opportunity to learn, from the natal nest, the common odour that they also express (Pfennig et al. 1983a).

Nestmates in treatment triplets should be the most tolerant pair of wasps if recognition odours are entirely acquired in the adult stage. This result is expected since nestmates (but not non-nestmates) have the opportunity to learn, from different fragments of the same nest, a common odour that they both share via acquisition (see Fig. 1). In contrast, nestmates in treatment triplets should be the least tolerant pair of wasps if recognition odours are individually produced (endogenous) or acquired before emergence. This result is expected since nestmates will learn different odours from those they express. Each non-nestmate, however, will learn the same odour that the other non- nestmate expresses and thus non-nestmates should treat each other tolerantly (see Fig. 1). I f both endogenous and adult-acquired recognition odours mediate the ontogeny of nestmate recognition, then results intermediate between the two

hypothesized results (as outlined above) are expected.

Results

Control gynes previously exposed to their natal nest and nestmates discriminated nestmates from non-nestmates. Nestmates had a significantly greater mean tolerance value (P < 0.02) and spent significantly more minutes paired (P<0"02) than non-nestmates (Table II). Gynes previously exposed to the natal nest and nestmates of the unrelated triplet member (treatment) were not more tolerant of nestmates than non-nestmates. Nestmates did not differ significantly from non- nestmates in mean tolerance value (P>0.10) or mean minutes paired ( P > 0.10, Table II).

Treatment non-nestmates were more tolerant of each other than control non-nestmates. Treatment non-nestmates had a significantly higher mean tolerance value than control non-nestmates (P < 0'01) and also spent more minutes paired than control non-nestmates, although the difference was not significant (P < 0"20, Table II). There were no significant differences in either tolerance values or minutes paired between control nestmates and treatment nestmates or between control nestmates and treatment non-nestmates (P>0 .40 for both, two-tailed Mann-Whi tney U-tests; Table II).

Control non-nestmate tolerance values and minutes paired (Table II) were significantly nega- tively correlated with the duration of nest exposure ( r = - 0 . 6 9 , P < 0 ' 0 1 and r = - 0 . 5 3 , P<0 .05 , respectively). In other words, control non-nestmates were increasingly less tolerant of each other the longer they had been exposed to their natal nest and nestmates (or as they became older), In

Table II. Tolerance values and minutes paired for nestmates and non-nestmates in control and treatment gynes Of P. fuscatus in reciprocal nest exposure study

Tolerance value (mean _+ sD) Minutes paired (mean _+ sD) Experimental No. of

group triplets Nestmates Non-nestmates P* Nestmates Non-nestmates P*

Control 12 7.55_+ 1.19 6-78_+0-65 <0.02 50-3+34-0 36.9+29.2 <0-02

Treatment 12 7.75_+1-05 7-84_+0.78 >0.10 48.7_+36.9 50.0+25.2 >0.t0

* Values for treatments and controls are based on two-tailed and one-tailed Wilcoxon matched-pairs signed-ranks tests, respectively. P values refer to comparisons between nestmates and non- nestmates. Larger tolerance values indicate greater tolerance.


contrast, the control nestmate tolerance values and minutes paired (Table II) were not significantly correlated with the duration of nest exposure (r = 0'25, P > 0.20 and r = - 0' 34, P > 0"20, respect-

ively). Treatment non-nestmate tolerance values (but

not minutes paired; r=0 .0 , P > 0'5) were significantly positively correlated with the duration of nest exposure (r = 0'59, P < 0"05; Table II). Thus, treatment gynes were increasingly more tolerant of non-nestmates the longer they had been exposed to the natal nest and nestmates of the unrelated triplet member (or as they became older). Treatment nestmate tolerance values and minutes paired (Table II) were not significantly correlated with the duration of nest exposure (r=0.22, P > 0 ' 2 0 and r = 0, P > 0"5, respectively).

There were no significant differences between the mean ages of nestmates and non-nestmates in either the controls or treatments (P > 0.10 for both, two-tailed Wilcoxon matched-pairs signed-ranks tests). In addition, the mean age differences of nestmates and non-nestmates in treatments and controls did not differ significantly (P > 0" 10, two- tailed Mann-Whi tney U-test). Thus age differences do not appear to explain the observed differences in tolerance within and between groups.

V A R I A B L E AGE S T U D Y

Methods

Rationale The results of the reciprocal nest exposure study

were consistent with the hypothesis that the age of a female may affect its expression of recognition odours. Thus, we designed the variable age study to determine whether the age of a female affected its tolerance by nestmate or non-nestmate females that had extensive prior exposure to their own natal nest and nestmates.

Procedures We collected 15 naturally nesting field colonies

(adults and nest) of P. fuscatus on 7 and 22 July 1984 from three sites (more than 10 km apart) in southeastern Michigan. Each nest, which contained eggs, larvae and pupae, was cleared of adults and then placed into a separate 15 • 15 x 15-cm cardboard box faced with clear plastic. The adults cleared from each nest were placed into separate, similar boxes. Honey and water were provided to

boxes containing adult wasps while only water was provided to boxes containing a brood-filled nest.

We monitored nests for adult emergences from approximately 0600 to 2400 hours from 22 July to 6 August 1984. Immediately upon eclosion, each female was isolated into a 0.23-1itre paper cup (provided with water and covered with an opaque perforated lid). Beginning 25 July, we began to remove female wasps of various ages (1.7 71.8 h after eclosion) from their isolation cups in prep- aration for behavioural observations. These females had no prior exposure to either their natal nest or nestmates as adults. Since we wanted to focus on those behavioural interactions directed at these previously isolated wasps (i.e. introduced wasps), we cooled them to minimize their activity (see Reeve & Gamboa 1983 for a similar procedure) as follows. We placed a previously isolated wasp into a 0"13-1itre glass jar, immersed this jar in an equilibrated ice-water mixture for 10 rain, opened the jar to mark the wasp's thorax with yellow enamel, and then returned the jar containing the wasp to the ice-water bath for an additional 5 min. At the end of this cooling period, we removed the wasp from its jar, waited a few minutes for leg movement, and then placed the wasp onto the surface of a nest lying (open cells facing upward) on the bot tom of a 15 x 15 x 15-cm cardboard box faced with clear plastic. The box contained unmarked adult females (residents), some of which were on the nest. The nest had been placed together with its respective field-collected colony adults 1 h before the introduction of the marked wasp. The field-collected unmarked females (residents) probably had extensive previous exposure to both their natal nest and nestmates.

The marked, introduced wasp was either placed into a box containing its female nestmates and natal nest (control) or into a box containing the females and nest of an unrelated colony (treatment). We recorded behavioural interactions (see below) between the introduced wasp and the resident wasps for 30 min. Observations were made from 1109 to 1452 hours from 25 July to 9 August 1984.

Each colony (nest plus resident females) was used for both control observations and corresponding treatment observations. The corresponding control and treatment observations for each of 15 colonies were matched as closely as possible for age of the introduced wasp (the mean age difference was 1.94_+ SD 2.92 h). The order of observations (control or treatment) was randomized.


Recognition bioassay Observations were blind, i.e. the observer did not

know the colony affiliation or age of the introduced wasp. We recorded the identity of the initiator and recipient of an interaction. After we completed our observations, we examined the interactions initiated by resident wasps and by the introduced wasp separately. We used the modified tolerance scale as described in the observations of the controlled environment study for calculating the corresponding tolerance values of nestmates and non-nestmates for both resident-initiated interactions and interactions initiated by the introduced female.

Results

Interactions initiated by resident females Resident females clearly have the ability to

discriminate between introduced nestmates and introduced non-nestmates. Resident (R) females were significantly more tolerant of introduced (I) nestmates than they were of introduced non- nestmates ( P < 0.01, Table III). In addition, there was no significant correlation between the age of an I wasp and the corresponding difference in tolerance value between the matched control and treatment observation (r=0"05, P>0"50; two- tailed Spearman rank correlation). There was also no correlation between the age of an I nestmate or the age of an I non-nestmate and its tolerance by R females (P>0.20 , r = - 0 . 2 8 and -0 .02 , respectively; two-tailed Spearman rank correlation). Thus, there is no evidence that the age of a female

affects its tolerance by R females. There was no significant difference in the mean age of I nestmates (30"78_+SD23.42h) and I non-nestmates (29.77_+ SD 22-46 h, P > 0'20; two-tailed Wilcoxon matched-pairs signed-ranks test). Therefore, age differences do not explain the differential tolerance of I nestmates and I non-nestmates by R females.

Interactions initiated by introduced females Unlike R females, which were more tolerant of I

nestmates than I non-nestmates, I females were equally tolerant of nestmates and non-nestmates. The mean tolerance values for interactions initiated by I wasps toward R nestmates and R non- nestmates were not significantly different (P > 0"50, Table III). In fact, the mean tolerance values for controls of the R initiated interactions were not significantly different from the mean tolerance values for either control or treatment 1 initiated interactions ( P > 0.50 for both, two-tailed M a n n - Whitney U-tests; Table III). In other words, 1 wasps treated all R wasps as nestmates, regardless of their relatedness to the R wasps. It might be argued that the cooling of the introduced gyne, by reducing its activity level, increased its tolerance of other gynes. Thus, tolerance values derived from the interactions initiated by the introduced gyne may simply be artefacts of the reduction of its activity level. However, we found no significant correlation between the interaction rate or activity level (see Reeve & Gamboa 1983) of the introduced wasp and its tolerance of other gynes (r = 0"05 and 0.28, respectively; P>0-20 for both, two-tailed Spearman rank correlation).

Table IlL Tolerance values for 15 matched control and treatment observations of interactions between an introduced female and resident females of P. fuscatus in variable age study

R initiated interactions I initiated interactions

Nestmate Non-nestmate Nestmate Non-nestmate

7.96_+0.80 5.86+1.8 8.10+ 1-00 8.14+_0.50

P* < 0.01 P* > 0.50

Figures give mean tolerance value__+ SD. Values for the R and I columns are for those interactions initiated by resident and introduced females respectively. Larger values indicate greater tolerance. * One-tailed Wilcoxon matched-pairs signed-ranks tests.

D I S C U S S I O N

Controlled Environment Study

The ultimate origin of recognition cues The results of our observations indicate that the

recognition odour of P. fuseatus has a significant environmental component. Non-nestmates reared in a controlled (homogeneous) laboratory environment were significantly more tolerant of each other than non-nestmates reared in uncontrolled (hetero- geneous) field environments ( P < 0 ' 0 0 5 for tolerance values, Table I).

The results of our later surveys indicate that the recognition odour of P. fuscatus also has a heritable component. In the surveys, laboratory gynes,


which spent their entire life cycle from egg through adult in a controlled laboratory, preferentially paired with nestmates (P<0.05, pairing ratios). Nevertheless, one could argue that these gynes might have learned non-chemical (e.g. visual) fea- tures of their nestmates while they were together on the natal nest. However, prior exposure to nestmates is neither necessary (Pfennig et al. 1983a,b) nor sufficient (Shellman & Gamboa 1982) for the development of nestmate recognition ability. In fact, even after extensive exposure (up to 115 days) to adult nestmates, gynes fail to recognize their familiar nestmates (Shellman & Gamboa 1982). Therefore, based on the results of our observations and surveys, we conclude that the nestmate recognition odour of gynes of P.fuscatus is composed of both environmental and heritable components.

Nestmate and non-nestmate laboratory gynes, and nestmate field gynes did not differ significantly in tolerance values (P> 0.10, Table I). Therefore, laboratory nestmates, which shared both heritable and environmental recognition odours, were not more tolerant than laboratory non-nestmates, which shared only environmental recognition odours. This indicates that environmental and heritable recognition odours are not additive in their effects on tolerance. These data also show that environmental recognition odours can override heritable recognition odours since laboratory non- nestmates, which share environmental but not heritable recognition odours, treated each other as nestmates.

In observations, which were made during a period of 2 days, laboratory gynes failed to discriminate between nestmates and non-nestmates. However, in later surveys, which spanned a period of approximately 30 days, these same gynes discriminated between nestmates and non-nestmates. In the observations, gynes had recently been exposed to their natal nest while in the surveys they had not (see Methods, controlled environment study). It may be that in the observations, environmental odours recently acquired from the natal nest temporarily masked the heritable odour differences of unrelated gynes, but later in the surveys the acquired environmental odours decayed, thereby unmasking the heritable odour differences of unrelated gynes. Our results do not support stochastic factors (e.g. odours arising from fungi, bacteria, etc.) as an explanation for the preferential pairing of nestmates in surveys. If such factors caused each laboratory colony to have a unique odour, we

would expect these environmental odours to exert their greatest influence on preferential nestmate pairing immediately after nestmates had been exposed to these common odours.

Reciprocal Nest Exposure Study The contribution o f endogenous and adult-acquired recognition odours

Since the treatment results are intermediate between our two predicted results, both endogenous (or brood-acquired) and adult-acquired odours mediate recognition in P. fuscatus. Treat- ment wasps that could have used only adult- acquired odours to mediate recognition (i.e. the nestmates) and treatment wasps that could have used only endogenous odours to mediate recognition (i.e. the non-nestmates) both treated each other as nestmates. Nestmates in the treatments were as tolerant of each other as nestmates in the controls, indicating that adult-acquired cues were used in nestmate recognition. Non-nestmates in the treatments were also as tolerant of each other as nestmates in the controls, indicating that endogenous odours were used in nestmate recognition. In addition, treatment non-nestmates were significantly more tolerant of each other than control non-nestmates, which further supports the hypothesis that endogenous odours mediate nestmate recognition.

Although our assays of recognition are sensitive to continuous differences in tolerance, our results show that endogenous and adult-acquired odours are not additive in their effects on tolerance; otherwise, control nestmate tolerance should exceed treatment nestmate and non-nestmate toler- ances (Table II). This result (and the non-additi- vity of heritable and environmental odour effects on tolerance) is more consistent with a 'cue similarity threshold' model of recognition than with a model that postulates tolerance continuously increasing with increasing degree of similarity between the learned and perceived cue. A wasp probably matches the odour of an encountered wasp with the odour learned previously from the natal nest as a young adult. Depending on the similarity (match) between the learned and perceived odour, a wasp may treat another wasp, in terms of mean tolerance, either as a nestmate or as a non-nestmate.

Based on previous research of nestmate recognition in Polistes (Pfennig et al. 1983a), it is not obvious to us why tolerance of control non-


nestmates decreases and tolerance of treatment non-nestmates increases with duration of nest exposure (or increasing age of wasps). Three explanations appear consistent with these results. (1) Increased exposure to the nest may allow more complete learning of recognition odours. However, this explanation does not appear to be entirely consistent with available evidence. Pfennig et al. (1983a) suggested that the learning of recognition odours in P.fuscatus may be completed within 4-16 h after eclosion since increasing a newly emerged gyne's exposure to its natal nest from 4.16 to 13"5 h had no demonstrable effect on its subsequent tolerance of nestmates and non-nestmates. Breed (1983) also presented evidence that learning of recognition cues in honey bees reaches an asymp- tote 5-6 h after the initial exposure to recognition cues. (2) Discrimination ability may increase with age, i.e. improve with maturation. (3) The expression of a recognition odour may increase with age. Qualitative evidence from studies of social wasps (Mischocyttarus) indicates that colony adults tolerate female non-nestmates 1 2 days old, but not female non-nestmates 2-4 days old, suggesting that young adults may accumulate odour with age (Litte 1976). We discuss the results of our test of this hypothesis in the next section.

Variable Age Study

The effect o f age on the expression of recognition odours

Our results conclusively show that young adult females (1.7 71.8 h after emergence) possess recognition odours. Experienced resident females discriminated young unfamiliar nestmates from young unfamiliar non-nestmates 'and their tolerance of these young females was independent of the age of the young female. Thus, we conclude that the correlations of tolerance with age as documented in the reciprocal nest exposure study are unlikely to be explained by the effect of age on the expression of recognition odours. The most likely explanation for this correlation is that discrimination ability increases with age. The results of the variable age study do not reveal whether the recognition odours of young adult females are acquired before emergence, endogenous, or a composite of both. There is, however, no significant correlation between the age of a wasp and its tolerance by resident females (P > 0.20). This indicates that if recognition odours are acquired as brood, there is no evidence that they

decay over the time interval of 1'7-71-8 h after eclosion.

Shellman & Gamboa (1982) and Pfennig et al. (1983a) documented that the isolation of a Polistes gyne from its natal nest at emergence disrupts its nestmate recognition. However, it was unknown whether the isolation of a gyne disrupted its acquisition of recognition odours, its learning of recognition odours, or both. Since our results conclusively demonstrate that young gynes isolated at emergence possess recognition odours, and since young gynes are sufficiently mature to exhibit recognition ability (see controls of the reciprocal nest exposure study), their failure to exhibit nestmate recognition after isolation from the natal nest must be due to the disruption of the learning of recognition odours. These results thus eliminate the possibility that the learning of recognition cues occurs in the larval and/or pupal stage; otherwise, young gynes isolated at emergence should be able to recognize their nestmates. Since young isolated gynes have the opportunity to learn their own odours but show no evidence that they do, they appear not to learn recognition odours directly from themselves. However, a single individual may have only a fraction of the odour components of an entire colony and, as a result, may learn its own odour yet fail to recognize many of its nestmates. We consider this hypothesis unlikely since three independent studies of both young and mature isolated Polistes gynes (Shellman & Gamboa 1982; Pfennig et al. 1983a; this study) have been unable to detect even a trend toward preferential nestmate tolerance in previously isolated gynes.

Gynes that were isolated from their natal nest at emergence showed no evidence of nestmate recognition ability (Table III), a result that corroborates earlier findings by Shellman & Gamboa (1982) and Pfennig et al. (1983a) that prior exposure to the natal nest (and/or its contents) is necessary for the ontogeny of nestmate recognition ability. One might argue that the social naivety of isolated wasps dramatically affects their behaviour towards (hence recognition of) newly encountered individuals. However, this argument appears to be untenable since female P. fuseatus with no prior exposure as adults to adult conspecifics (butwith a short 1 or 4.16-13.5 h exposure to only the natal nest) later recognize nestmates (Pfennig et al. 1983a).

Young gynes that were deprived of the opportunity to learn recognition odours from the natal nest

Gamboa et al.. Social wasp nestmate recognition 695

treated all resident females as nestmates, regardless of their relatedness to the resident females. This result ( together with the negative corre la t ion of age and non-nes tmate tolerance in controls of the reciprocal nest exposure study) suggests tha t the ontogeny of nes tmate recogni t ion ability in Polistes involves the development of intolerance to unfamiliar odours ra ther than the deve lopment of tolerance to familiar odours.

A C K N O W L E D G M E N T S

This s tudy was suppor ted by N S F BNS 81-15932; G. J. G. was principal investigator. We thank K. A. Berven, W. M. Getz, W. G. Holmes, D. W. Pfennig, and J. Shel lman Reeve for critically reviewing our manuscr ip t and J. E. K lahn for mark ing gynes and assisting us in collecting foundresses.

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(Received 29 October 1984; revised 12 April 1985; MS. number: A4410)

Nestmate recognition in social wasps: the origin and acquisition of recognition odours

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