ANIMAL BEHAVIOUR, 2006, 71, 327–335 doi:10.1016/j.anbehav.2005.04.012
Geographical variation in Argentine ant aggression behaviour mediated by environmentally derived nestmate recognition cues
GRZEGORZ BUCZKOWSKI & JULES SILVERMAN Department of Entomology, North Carolina State University
(Received 21 October 2004; initial acceptance 26 January 2005; final acceptance 4 April 2005; published online 18 January 2006; MS. number: A10020R)
Social insects use a complex of recognition cues when discriminating nestmates from non-nestmate con- specifics. In the Argentine ant, Linepithema humile, recognition cues can be derived from exogenous sour- ces, and L. humile acquires prey-derived hydrocarbons that are used in nestmate discrimination. We studied Argentine ant population-level distinctions in response to external recognition cues. Ants belong- ing to a California population were strongly affected by the imposition of prey-derived hydrocarbons, with spatially isolated colony fragments that had been fed different cockroach prey (Blattella germanica or Supella longipalpa) showing high and injurious intracolony aggression when reunited. In contrast, colonies of Argentine ants from the southeastern U.S. showed only moderate and noninjurious aggression when subjected to the same treatment. Field-collected colonies of L. humile had hydrocarbons in the range of those provided by S. longipalpa, and colonies from the southeastern U.S. had significantly higher initial levels of Supella-shared hydrocarbons. When fed cockroaches, Argentine ants from both regions acquired additional amounts of Supella- and Blattella-specific hydrocarbons, with a significant increase in levels of Blattella-specific hydrocarbons. Therefore, diet partitioning produced a greater change in the proportion of prey hydrocarbons in the California than in the southeastern U.S. populations, which may be respon- sible for the altered behaviour observed in the California population. Identifying factors underlying geo- graphical variation in cue expression and/or perception may bring us closer to elucidating the selective forces driving nestmate recognition systems. Ó 2005 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
In social insects, colony integrity is maintained through (Vander Meer & Morel 1998) with recent evidence coming a well-developed system for discriminating nestmates from ants (Lahav et al. 1999; Boulay et al. 2000; Liang & from non-nestmate conspecifics, and the recognition Silverman 2000), termites (Cle`ment & Bagne`res 1998) signals are chemical and are genetically and/or environ- and wasps (Gamboa et al. 1996; Singer 1998). In social in- mentally based (Crozier & Dix 1979; Gamboa et al. 1986a; sects, cuticular hydrocarbon composition is highly diverse Howard & Blomquist 2005). Although recognition cues and may vary with age, geographical origin, sex, season, can be any aspect of an individual’s phenotype that reli- caste, rank and nest (e.g. Vander Meer et al. 1989; Ichinose ably signifies colonial membership, chemical cues are 1991; Howard 1993; Sledge et al. 2001). Such variation widely used because they are potentially information- may occur at the individual, colony or population level. rich and require little energy to produce, because they Elucidating factors responsible for variation in cuticular are metabolic by-products or passively acquired from the hydrocarbon composition and interpreting the meaning environment. In addition, quantitative and qualitative of such variation have been an active area of research for variations in the levels of chemical signals assure their re- two reasons. First, cuticular hydrocarbons are critical for quired complexity. Cuticular hydrocarbons have long nestmate recognition, and colony-level variation may been implicated in nestmate recognition in social insects provide information about a wide range of parameters, such as territoriality, breeding patterns and foraging ranges (e.g. Kaib et al. 2002; Delphia et al. 2003; Haverty Correspondence and present address: J. Silverman, Department of Ento- et al. 2003). Second, variation in cuticular hydrocarbon mology, Box 7613, North Carolina State University, Raleigh, NC patterns can be used in chemotaxonomic studies, whereby 27695-7613, U.S.A. (email: [email protected]). G. Buczkow- groups of chemical compounds are used as genetic ski is now at the Department of Entomology, Purdue University, 901 indicators in the establishment of taxonomic relation- W. State Street, West Lafayette, IN 47907-2089, U.S.A. ships (Lockey 1991). Many taxa of social insects show 327 0003–3472/05/$30.00/0 Ó 2005 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. 328 ANIMAL BEHAVIOUR, 71, 2
intraspecific variation in the types and proportions of cu- and Silverman & Liang (2001) for a single colony from ticular hydrocarbons. The potential of this variation for a single location in California cannot be universally ap- chemotaxonomic studies has been demonstrated in ants plied. Thus, we hypothesized that between-colony varia- (Oldham et al. 1994; Akino et al. 2002), wasps (Dapporto tion occurs in Argentine ant response to prey-derived et al. 2004) and most notably termites (e.g. Kaib et al. nestmate recognition cues. More specifically, from recent 1991; Haverty et al. 2000), where morphological and be- evidence of differences in L. humile population structure havioural approaches often prove unrewarding. between California and the southeastern U.S. (Buczkowski Nestmate recognition systems using cues originating et al. 2004), we predicted regional differences in prey-in- from endogenous and/or exogenous sources provide duced aggression as well. Furthermore, we hypothesized individuals with accurate information about colony mem- that any colony-level variation in prey-induced aggression bership (Gamboa et al. 1986b). The relative contribution behaviour has an underlying chemical basis, and conse- of genetic and environment-based recognition cues is of- quently, we examined qualitative and quantitative differ- ten addressed in laboratory studies, where the environ- ences in colony hydrocarbon profiles before and after mental component is kept constant and aggression rearing on Blattella germanica or Supella longipalpa. We dis- between individuals is measured. In ants, decreases cuss our results in the context of geographical differences (Obin 1986; Stuart 1987; Crosland 1989; Boulay et al. in genetic diversity within introduced L. humile popula- 2000; Chen & Nonacs 2000; Buczkowski et al. 2005) and tions (Buczkowski et al. 2004). A comparison of colonies no change or increases (Le Moli et al. 1992; Heinze et al. across populations could provide insights on how regional 1996; Holway et al. 1998; Stuart & Herbers 2000; Suarez events affect the interplay of intrinsic and extrinsic nest- et al. 2002) in aggression between colonies reared under mate recognition processes. uniform laboratory conditions have been reported. A sig- nificant decrease in aggression suggests that nestmate dis- crimination is influenced largely by environmental cues, METHODS and maintenance of aggression implies a strong genetic component. Gamboa et al. (1986b), however, cautioned Collection and Rearing of Laboratory Colonies that evidence of dominance of genetic cues in the labora- We collected ants along two 700-km transects. One tory is not evidence that genetic cues are more important transect was through an area of a supercolony in Cal- than environmental factors in field contexts. The relative ifornia (38.3–33.0 N and 122.2–117.0 W) described by importance of genetic and environmental factors ulti- Tsutsui et al. (2000), and the other spanned three states in mately depends on the organism’s genetic structure, ecol- the southeastern U.S.A.: North Carolina, South Carolina ogy and social context (Buczkowski & Silverman 2005). and Georgia (33.0–36.1 N and 72.0–84.4 W). We sampled Nestmate recognition in the Argentine ant, Linepithema seven sites in California: Berkeley, Corona, Davis, humile, integrates both genetic (Tsutsui et al. 2000; Suarez Escondido, Ojai, Pleasanton and Riverside. In the south- et al. 2002) and environmental (Chen & Nonacs 2000; eastern U.S. we sampled 11 sites: North Carolina (6: Chap- Liang & Silverman 2000) inputs. Liang & Silverman el Hill, Emerald Isle, Greenville, Jacksonville, Shallotte and (2000) examined Argentine ant nestmate recognition Winston-Salem); South Carolina (2: Greenville and Greer) and its modification by diet in a California population and Georgia (3: Barnesville, Fayetteville and Griffin). and showed that hydrocarbons acquired from cockroach For each location, we established two colony fragments prey, Supella longipalpa and Blattella germanica, were incor- each consisting of 5000 workers, several dozen queens and porated into the ant’s intrinsic cuticular hydrocarbon pro- numerous brood. Colonies were maintained in soil-free, file, and that workers from colony fragments of a single Fluon-coated trays containing nests composed of plastic colony reared in isolation on different diets displayed dishes filled with moist grooved plaster. Colonies were high aggression when reunited. Furthermore, by isolating provisioned with 25% sucrose solution and artificial diet Argentine ant colony fragments for as few as 28 days and (Bhatkar & Whitcomb 1970) ad libitum, and hard-boiled feeding them different prey (either B. germanica or S. long- egg once a week. For each location, colony fragments ipalpa), re-establishment of internest communication was were also reared on one of two prey diets: Supella longi- prevented (Silverman & Liang 2001). palpa male and female adults, or Blattella germanica female In our ongoing efforts to understand the influence of adults. The cockroaches were presented live but injured to prey-acquired hydrocarbons on nestmate recognition and facilitate prey handling by the ants. All colonies were colony dynamics in the Argentine ant, we attempted to maintained at 24 1 C and 50 10% RH, on a 12:12 h induce intracolony aggression within a colony of L. humile light:dark cycle. collected from Winston-Salem, North Carolina, U.S.A., by following the methodology of Liang & Silverman (2000). Despite prolonged (4-month) rearing on distinct prey di- Aggression Tests (Nestmate Discrimination ets, we were unable to induce high aggression in this col- Bioassay) ony. Former nestmates reared on the different prey displayed only moderate, noninjurious aggression (level To test whether colony partitioning alone had an effect 1 or 2 on a scale of 0–4; Suarez et al. 1999), and colony on aggression, we divided a colony (Winston-Salem, fragments merged readily when reunited. 20 000 workers) into two equal-size fragments. We then Based on this experience, we suspected that the phe- tested for aggression between the fragments 6 months nomenon described earlier by Liang & Silverman (2000) after rearing in isolation under identical environmental BUCZKOWSKI & SILVERMAN: ARGENTINE ANT BEHAVIOUR 329 and dietary conditions. We also examined aggression chromatograph equipped with a DB-1 column towards workers that had been removed from and imme- (30 m 0.25 mm 0.25 mm film thickness) and inter- diately reintroduced to their original colony. We tested for faced with a G1045A Chemstation (version A05.01). aggression at two time points: day 0 (within minutes of Oven temperature was held at 40 C for 2 min, then in- splitting the colony) and 6 months later. Sixty worker creased to 200 C at a rate of 20 C/min and finally to introductions were performed for each of two time points, 310 Cat40 C/min. Injector and flame-ionization detec- 30 introductions with workers from one colony acting as tor were at 270 C and 320 C, respectively. Helium was intruders and 30 with workers from the other colony the carrier gas, and the make-up gas was nitrogen. Quan- acting as intruders. We used an assay that measured the titative data were obtained by integrating the peaks and level of aggression in single worker introductions into calculating the percentage area under each peak. Specific a resident colony (Roulston et al. 2003). Aggression was peak identity was determined with hydrocarbon standards scored on a 0–4 scale (Suarez et al. 1999: 0: ignore, 1: and by matching diagnostic peaks with those from earlier touch, 2: avoid, 3: aggression (lunging, brief bouts of bit- studies (Jurenka et al. 1989; Liang et al. 2001). ing and/or pulling), 4: fighting (prolonged aggression, also abdomen curling to deposit defensive compounds)). Data Analysis The same assay was also used to test for changes in intra- colony aggression in colony fragments raised on distinct All behavioural and hydrocarbon data analyses were prey. Ten aggression tests per colony per interval were con- performed using SAS 8.1 statistical software (SAS 2002). ducted, five with Blattella-fed workers acting as intruders, Aggression scores were averaged in the following order: and five with Supella-fed workers acting as intruders. For (1) over replicates within a colony (N ¼ 10), (2) over colo- each test, we allowed the intruder up to 25 encounters nies within a time period (California: N ¼ 7; southeastern with resident ants. Each instance of direct physical con- U.S.: N ¼ 11) and (3) over time periods (N ¼ 9). Regional tact between the intruder and any resident was regarded differences in levels of aggression induction were com- as an encounter. Individual ants were not tested in more pared on days 12 and 162 and for an average of all time than one trial. Assays were conducted blind: the observer periods using PROC TTEST, which examines the equality who recorded worker aggression levels did not know the of variances. We report results of one of two types of t source of the interacting colony fragments and was unfa- tests, depending on the equality of variances. Results of miliar with the hypothesis being tested. Data were ana- a Student’s t test are reported when the variances were ho- lysed as the maximum score of 25 encounters (Roulston mogenous. In cases where the variances were unequal, we et al. 2003). We also report the average incidence of inju- used the Welch t test with a Satterthwaite correction (Zar rious aggression for each region. We defined injurious ag- 1999). Next, we determined the slope for aggression in- gression as aggression involving direct physical contact crease over time for each colony (PROC REG) and tested (level 3) characterized by behaviours such as biting, the hypothesis that the slope for each region (averaged pulling legs and/or antennae and spraying defensive over colonies) is not different from zero (PROC UNIVARI- chemicals. If the intruder and the resident ants engaged ATE). Subsequently, we compared regression slopes be- in a highly aggressive behaviour (level 4) for more than tween regions using PROC TTEST. We used values 10 s, a fluoned ring was placed around the fighting indi- averaged over all periods to compare the incidence of in- viduals. The trial was permitted to continue for 2 h and jurious aggression and the number of dead workers be- the physical condition of the intruder was examined every tween regions. 1–4 min. Mortality of the intruders was recorded. Aggres- To determine whether Argentine ants acquired key prey- sion assays were performed 12, 22, 32, 42, 52, 82, 112, specific hydrocarbons, we first identified key diagnostic 132 and 162 days after the colonies were first provisioned hydrocarbons provided by each prey. For B. germanica,we with prey. selected peaks corresponding to 11- and 13- and 15-meth- ylnonacosane and 3-methylnonacosane. Both hydrocar- bons are relatively abundant in adult B. germanica, Extraction, Isolation and Chemical Analysis constituting approximately 14.5 and 10.3% of the total of Cuticular Hydrocarbons hydrocarbons, respectively (Jurenka et al. 1989). Further- more, our preliminary analysis indicated that both hydro- Ants were killed by freezing ( 20 C) before hydrocar- carbons were acquired by Argentine ants. For S. longipalpa, bon (HC) extraction. External lipids were extracted from we selected four hydrocarbons: 15,19-dimethylpentatria- the cuticle by immersing 10 whole thawed ants in 1 ml of contane, 5,9- and 5,11-dimethylpentatriacontane, 13- hexane for 10 min, followed by a brief second rinse. The and 15- and 17- and 19-methylheptatriacontane, and samples were gently shaken for the first and last 20 s of 15,19- and 17,21-dimethylheptatriacontane. These hydro- the soak period. Hexane extracts were concentrated under carbons are present in S. longipalpa at 19.0, 9.1, 8.5 and nitrogen to about 100 ml and applied to prewetted 25.7%, respectively, and each is acquired by Argentine (hexane) Pasteur pipette minicolumns filled with 500 mg ants from S. longipalpa (Liang & Silverman 2000; Liang of silica gel (63–200 mesh size, Selecto Scientific, Georgia, et al. 2001). U.S.A.). The HC fraction was eluted with 6 ml of hexane. We hypothesized that population-specific differences The extract was resuspended in 5 ml of hexane and 1 ml in response to environmental hydrocarbons may result was injected (two ant equivalents). Capillary gas chroma- from differences in the initial levels of hydrocarbons tography (GC) was carried out using an HP 5890 gas (percentage of total) that are within the range of those 330 ANIMAL BEHAVIOUR, 71, 2
provided by the prey (i.e. there would be a negative Southeastern U.S. relation between the level of aggression induced and the 4 California level of hydrocarbons similar to those provided by the prey). Therefore, we examined colonies in both regions for 3 the presence of Blattella-specific and Supella-specific hy- drocarbons and compared the initial levels of those hydro- carbons between regions (PROC TTEST). We determined 2 whether Argentine ants acquired significant amounts of prey hydrocarbons following different diet provisions by Aggression level 1 comparing their initial levels of prey-specific hydrocar- bons (field-collected colonies; day 0) to levels on day 52 0 and performing analysis of variance (ANOVA) using 12 22 32 42 52 82 112 132 162 AVE PROC GLM. We chose to examine hydrocarbon profiles Time (days) on day 52 to allow enough time for ants to acquire maxi- Figure 1. Mean SE aggression levels between L. humile colony mal levels of prey hydrocarbons. Silverman & Liang fragments from each population (southeastern U.S.: N ¼ 11 colo- (2001) reported that colony fragments fed B. germanica nies; California: N ¼ 7 colonies) that were fed different diets. X axis and S. longipalpa acquired prey hydrocarbons within 14 shows time in days since colonies were first provisioned with cock- days and aggression between colony fragments was maxi- roach prey. Initial mean SE aggression level (day 0) was mal after 28 days of isolation. We compared the relative 0.0 0.0. AVE: average across all days. amounts of prey-specific hydrocarbons acquired by Argen- tine ants in California and in the southeastern U.S. for developed their respective aggression levels within the possible regional differences in prey hydrocarbon acquisi- first 12 days of prey exposure. Aggression levels were tion using PROC GLM. All analyses were performed for maintained throughout the study despite slight fluctua- each diet in two ways: (1) by considering each hydrocar- tions between time periods. bon separately and (2) by combining diet-specific hydro- The incidence of injurious aggression ( level 3) within carbons into a single group. California colonies was relatively high (X SE instances of injurious aggression: 14.6 1.2, N ¼ 15 750 encounters (25 encounters/intruder 10 intruders 9 time intervals RESULTS 7 colonies)) compared with that of southeastern U.S. Intracolony Aggression colonies (2.7 0.7, N ¼ 24 750 encounters (25 encounters/ intruder 10 intruders 9 time intervals 11 colonies); Colony partitioning did not result in aggression be- P < 0.0001). We recorded 2.2 0.3 dead workers per 10 tween colony fragments (X SE ¼ 0:0 0:0) and no ag- intruder introductions in fights involving ants from gression was observed when workers were reintroduced California, whereas no mortality was observed when ants into their own colony (0.0 0.0). Examination of geo- from the southeastern U.S. interacted (P < 0.0001). graphical variation in Argentine ant nestmate recognition behaviour revealed different responses to environmental Comparison of Hydrocarbon Profiles cues between Californian and southeastern U.S. popula- tions. Colonies from the California population were af- Field-collected colonies of L. humile already possessed fected by the imposition of prey-derived hydrocarbons hydrocarbons in the range of those provided by both in- because colony fragments raised on either B. germanica sect prey (Table 1). Colonies in both California and the or S. longipalpa displayed high aggression when reunited southeastern U.S. initially had relatively low levels of hy- (Fig. 1). In contrast, colonies from the southeastern U.S. drocarbons shared with Blattella (<1% of total hydrocar- population displayed only moderate aggression. In Cali- bons), with no difference between regions (P ¼ 0.837). fornia, the mean SE level of aggression was 3.22 0.06 Compared to Blattella hydrocarbons, colonies in both (range over time periods 2.91–3.67; range over colonies regions had higher initial levels of the diagnostic hydro- 3.02–3.42), and in the southeastern population it was carbons shared with Supella (4–9% of total hydrocarbons). 1.68 0.10 (range over time periods 1.26–2.10; range Argentine ants from California had lower initial levels of over colonies 1.22–2.37, P < 0.0001). A significant differ- hydrocarbons shared with Supella (mean SE ¼ 23.55 ence in the mean SE level of induced aggression be- 1.32%) relative to colonies from the southeastern U.S. tween regions was already evident by day 12 (California: (28.60 0.87%; P ¼ 0.016). 2.91 0.09, southeastern U.S.: 1.78 0.20, P ¼ 0.002). Argentine ants from both Californian and southeastern Over time, the difference between regions grew larger, and U.S. populations that had been fed B. germanica acquired by day 162, California colonies became highly aggressive significant levels of Blattella-derived hydrocarbons (Table 1). (3.67 0.13), while southeastern U.S. colonies remained When fed S. longipalpa, the levels of three of the four Supella relatively nonaggressive (1.60 0.17, P < 0.0001). The diagnostic hydrocarbons increased in the California colo- slopes for change in aggression across days 12–162, how- nies: 13- and 15- and 17- and 19-methylheptatriacontane ever, were not significant for either population (California: (P ¼ 0.024), 15,19-dimethylpentatriacontane (P ¼ 0.009) P ¼ 0.108, southeastern U.S.: P ¼ 0.260), and the and 5,9- and 5,11-dimethylpentatriacontane (P ¼ 0.038), difference between slopes was also not significant while the level of 15,19- and 17,21-dimethylheptatriacon- (P ¼ 0.058), indicating that colonies from both regions tane increased (P ¼ 0.0002) in the southeastern U.S. BUCZKOWSKI & SILVERMAN: ARGENTINE ANT BEHAVIOUR 331
Table 1. Initial cuticular hydrocarbon levels in California and southeastern U.S. colonies and changes in hydrocarbon levels after rearing on B. germanica and S. longipalpa prey
Geographical Cuticular Raised on Net Raised on Net region hydrocarbon* Initialy B. germanicaz changex P** S. longipalpaz change x P**
California (N¼7) BG1 0.00 0.00 4.26 0.37 4.26 <0.0001 0.16 0.16 0.16 0.356 BG2 0.26 0.26 3.97 0.30 3.71 <0.0001 0.73 0.40 0.47 0.190 BG1þBG2 0.26 0.26 8.23 0.60 7.97 <0.0001 0.89 0.38 0.63 0.096 SL1 4.17 0.14 2.80 0.24 1.37 0.0005 7.30 0.88 3.13 0.009 SL2 4.41 0.41 4.59 0.29 0.18 0.699 6.33 0.46 1.92 0.024 SL3 6.61 0.58 2.74 0.12 3.86 0.0005 4.41 0.48 2.19 0.027 SL4 8.36 0.27 4.64 0.26 3.71 <0.0001 10.47 0.75 2.11 0.038 SL1þSL2þ 23.55 1.32 14.77 0.57 8.78 0.0002 28.51 1.25 4.96 0.093 SL3þSL4 Southeastern U.S. BG1 0.32 0.18 4.83 0.61 4.51 <0.0001 0.27 0.18 0.06 0.784 (N¼11) BG2 0.00 0.00 5.64 0.69 5.64 <0.0001 0.88 0.37 0.88 0.039 BG1þBG2 0.32 0.18 10.47 1.26 10.15 <0.0001 1.15 0.43 0.83 0.100 SL1 6.78 0.22 4.60 0.39 2.18 <0.0001 7.68 0.35 0.9 0.102 SL2 6.10 0.32 3.58 0.23 2.52 <0.0001 4.60 0.16 1.50 0.003 SL3 7.06 0.26 5.62 0.59 1.43 0.053 10.74 0.60 3.68 0.0002 SL4 8.66 0.24 5.98 0.26 2.68 <0.0001 7.59 0.29 1.07 0.028 SL1þSL2þSL3þSL4 28.60 0.87 19.78 1.35 8.82 <0.0001 30.61 1.17 2.01 0.246
*Hydrocarbon designations: B. germanica specific: BG1 ¼ 11- and 13- and 15-methylnonacosane; BG2 ¼ 3-methylnonacosane; and S. longi- palpa specific: SL1 ¼ 15,19-dimethylpentatriacontane; SL2 ¼ 13- and 15- and 17- and 19-methylheptatriacontane; SL3 ¼ 15,19- and 17,21-dimethylheptatriacontane; SL4 ¼ 5,9- and 5,11-dimethylpentatriacontane. Mean SE initial levels of cuticular hydrocarbons in field- collected colonies (day 0) expressed as a percentage of total hydrocarbons. yMean SE initial levels of cuticular hydrocarbons in field-collected colonies (day 0) expressed as a percentage of total hydrocarbons. zLevels of hydrocarbons (day 62) after rearing on B. germanica or S. longipalpa. xThe change in hydrocarbon levels after rearing on cockroach prey (day 62–day 0). **Based on results of ANOVA. population. When all four Supella-derived hydrocarbons P < 0.0001) than that in our southeastern U.S. colonies were analysed as a single group, Argentine ant colonies in (5.98 versus 7.59%, P ¼ 0.0006). If 5,9- and 5,11-dimethyl- California acquired more Supella-derived hydrocarbons rel- pentatriacontane is a key nestmate recognition cue, then ative to ants from the southeastern U.S. (4.96 versus population-level differences in the net change of this hy- 2.01%), however, the net change was not significant in ei- drocarbon may underlie regional distinctions in the induc- ther population. Furthermore, L. humile raised on B. ger- tion of aggression behaviour. manica lost a significant proportion of Supella-specific When prey-specific hydrocarbons were analysed collec- hydrocarbons in both Californian (P ¼ 0.0002) and south- tively, ants fed B. germanica did not differ in either the eastern U.S. (P < 0.0001) populations. Little change in lev- amount of Blattella-specific cues that they acquired els of Blattella-specific hydrocarbons was evident in (P ¼ 0.207) or the amount of Supella-specific cues that Argentine ants raised on S. longipalpa. they lost (P ¼ 0.983), and Supella-fed ants acquired similar Interregional comparisons cannot be made by simply levels of Blattella-orSupella-specific cues (P ¼ 0.764 and evaluating changes in Supella-specific hydrocarbons in ants P ¼ 0.313, respectively; Table 2). When considered indi- fed S. longipalpa, because field-collected colonies possess Su- vidually, substantial differences between regions were pella-specific hydrocarbons and their proportion declines evident especially with respect to the amount of Supella- when fed B. germanica. Since we recorded aggression be- specific hydrocarbons acquired by colonies fed S. longi- tween colony fragments reared on both diets, we must con- palpa (Table 2). Most notably, changes in levels of three sider changes in Supella-specific hydrocarbons in colonies out of four Supella-derived hydrocarbons occurred in op- reared on S. longipalpa as well as B. germanica. The case of posite directions across populations, possibly further con- 5,9- and 5,11-dimethylpentatriacontane (SL4, Tables 1 tributing to behavioural differences between regions. and 2) is illustrative. The relative proportion of this hydro- Aggression between colony fragments raised on distinct carbon decreased in southeastern U.S. colonies reared on S. prey diets is probably caused by two factors pertaining to longipalpa ( 1.07%, P ¼ 0.028), but increased in California changes in the levels of key nestmate recognition hydro- colonies (2.11%, P ¼ 0.038; difference between regions carbons: net diet-specific changes in the levels of P ¼ 0.001). Concurrently, the relative proportion of prey-specific hydrocarbons and differences in the level of Supella-specific hydrocarbons decreased in colonies fed prey-specific hydrocarbons between diets. Ultimately, B. germanica (decline in southeastern populations: 2.68%, aggression may be induced from a combination of these P < 0.0001; decline in Californian populations: 3.71%, factors. By comparing final prey-derived hydrocarbon P < 0.0001; difference between regions: P ¼ 0.018). Conse- levels between diets for each of the two regions, differ- quently, the difference in proportions of 5,9- and 5,11- di- ences became apparent (Table 3). Liang et al. (2001) methylpentatriacontane between the two diet regimes in suggested that certain Supella-derived hydrocarbons, espe- our California colonies was larger (4.64 versus 10.47%, cially 15,19-dimethylpentatriacontane, might be critical 332 ANIMAL BEHAVIOUR, 71, 2
Table 2. Comparison of changes in cuticular hydrocarbon levels in L. humile colonies fed B. germanica and S. longipalpa
Region
Diet Cuticular hydrocarbon* California Southeastern U.S. Py
B. germanica BG1 4.26z 4.51 0.777 BG2 3.71 5.64 0.049 BG1þBG2 7.97 10.15 0.207 SL1 1.37 2.18 0.073 SL2 0.18 2.52 0.0002 SL3 3.86 1.43 0.020 SL4 3.71 2.68 0.018 SL1þSL2þSL3þSL4 8.78 8.82 0.983 S. longipalpa BG1 0.16 0.06 0.471 BG2 0.47 0.88 0.454 BG1þBG2 0.63 0.83 0.764 SL1 3.13 0.90 0.025 SL2 1.92 1.50 0.0002 SL3 2.19 3.69 <0.0001 SL4 2.11 1.07 0.001 SL1þSL2þSL3þSL4 4.96 2.01 0.313
*Hydrocarbon designations as in Table 1. yBased on results of ANOVA. zNet change in hydrocarbon levels after rearing on cockroach prey (day 62–day 0, averaged over colonies).
to nestmate recognition in L. humile. The difference in the the levels of hydrocarbons shared with certain prey, (2) level of 15,19-dimethylpentatriacontane between colonies an environmental factor (exposure to prey hydrocarbons) reared on B. germanica and S. longipalpa was 62% (2.80 ver- differentially affected the two populations, and conse- sus 7.30) in the California population, but only 40% (4.60 quently, (3) different levels of aggression developed in versus 7.68) in the southeastern U.S. population (P ¼ 0.01 the two populations, producing mortality in one but not between regions). the other. Geographical variation in response to extrinsic environ- mental cues can result from proximate environmental DISCUSSION effects, from underlying genetic factors, as well as from the interaction between the two (Bradshaw 1965; Via & We found profound geographical variation in Argentine ant response to extrinsic nestmate recognition cues; ants Table 3. Final levels of prey-specific hydrocarbons in L. humile from a California population were strongly affected by the colonies fed B. germanica and S. longipalpa imposition of prey-derived hydrocarbons, and colonies from a population in the southeastern U.S. were signifi- Diet cantly less affected. In other hymenopteran taxa, various environmental factors affect the expression of social Cuticular B. S. behaviour, including latitude (Packer 1990; Miyanaga Region hydrocarbon* germanica longipalpa Py et al. 1999; Richards 2000), altitude (Eickwort et al. 1996), food and nest site availability (Banschbach & Herb- California BG1 4.26 0.16 <0.0001 ers 1996; Herbers & Banschbach 1999) and season (Yanega BG2 3.97 0.73 <0.0001 < 1993; Richards & Packer 1995). We have also highlighted BG1þBG2 8.23 0.89 0.0001 SL1 2.80 7.30 0.002 the potential importance of environmental factors in the SL2 4.59 6.33 0.009 evolution and persistence of kin and/or nestmate recogni- SL3 2.74 4.41 0.012 tion behaviour and emphasize the importance of laboratory SL4 4.64 10.47 <0.0001 manipulations in revealing phenotypic variation that SL1þSL2þ 14.77 28.51 0.0002 SL3þSL4 might otherwise remain undiscovered in studies under natural conditions. Southeastern BG1 4.83 0.27 <0.0001 U.S. BG2 5.64 0.88 <0.0001 Earlier studies have reported either local variation in the BG1þBG2 10.47 1.15 <0.0001 expression of nestmate recognition behaviour (Breed et al. SL1 4.60 7.68 <0.0001 1999) or variation in the types and proportions of social SL2 3.58 4.60 0.002 insect cuticular hydrocarbons for chemotaxonomic pur- SL3 5.62 10.74 <0.0001 poses (e.g. Brown et al. 1996; Akino et al. 2002; Dapporto SL4 5.98 7.59 0.0006 SL1þSL2þ 19.78 30.61 <0.0001 et al. 2004). We provide new evidence for a sequence of SL3þSL4 events that ties intraspecific variation in hydrocarbon lev- els with colony-level consequences: (1) Argentine ants *Hydrocarbon designations as in Table 1. from the two populations showed intrinsic differences in yBased on results of ANOVA. BUCZKOWSKI & SILVERMAN: ARGENTINE ANT BEHAVIOUR 333
Lande 1985; West-Eberhard 1989). Although natural selec- than does the California population that we sampled tion has been traditionally considered to be very influen- (Buczkowski et al. 2004; but see Ingram & Gordon tial in producing phenotypic variants adapted to 2003), showing that not all introduced populations with- conditions present in a certain geographical region, evi- in the U.S. are genetically and behaviourally uniform. dence for the influence of environmental factors inducing Regional differences in genetic diversity may also be phenotypic plasticity is less common (e.g. Greene 1989; manifest in differential response to exogenous cues Arnqvist & Johansson 1998). The pattern of geographical through modification of the structure and operation of variation in nestmate recognition behaviour in L. humile the nestmate recognition system. This system consists of may result from diversity in colony-level experiences re- three distinct components: (1) expression, (2) perception sulting from ecological differences and/or from differen- and (3) action (Gamboa et al. 1986b; Waldman 1987; tial rates of evolution of genotypic traits affecting Reeve 1989). In the Argentine ant, the origins of the ex- agonistic and/or sensory behaviour. Evidence for the con- pression component (cue ontogeny) are both genetic tribution of environmental factors comes from our exam- (Tsutsui et al. 2000; Giraud et al. 2002; Suarez et al. ination of hydrocarbon profiles in L. humile from both 2002) and environmental (Chen & Nonacs 2000; Liang ranges. Regional environmental differences may especially & Silverman 2000). Although field-collected L. humile re- promote a differential response to prey-derived hydrocar- veal regional differences in levels of certain hydrocarbons bons. For example, regional distinctions in diet and nest- shared with Supella, we do not know whether these are ing material diversity might produce qualitative and due to genetic and/or environmental factors and whether quantitative differences in cuticular hydrocarbon compo- colonies in either region show temporal variation in cutic- sition, which might affect worker response to novel nest- ular hydrocarbon patterns. In some ants, recognition cues mate recognition cues, especially if the new cues overlap are dynamic and may change throughout the life of the with similar preexisting cues. Workers may not recognize colony (e.g. Vander Meer et al. 1989) and show seasonal newly acquired hydrocarbons as foreign if these are al- variation (Ichinose 1991; Nielsen et al. 1999). Differences ready part of their cuticular constituents. These effects in cue perception may also reflect regional distinctions in might be especially pronounced with threshold levels of genetic diversity. Perception involves the development of hydrocarbons, above which increases in hydrocarbon lev- a recognition template and processing of perceived cues. els do not cause a change in behaviour. Relative to Argen- The template is a collection of recognition cues that an tine ants from California, ants from the southeastern U.S. individual considers acceptable (Waldman et al. 1988) had higher initial levels of hydrocarbons shared with with the cues based on a referent. The referent can be any- Supella, particularly 15,19-dimethylpentatriacontane and thing that the individual contacts, including objects in 13- and 15- and 17- and 19-methylheptatriacontane. Lev- the nesting environment, its diet, and most importantly, els of these hydrocarbons may be partly responsible for other individuals living in the same group (nest or colo- the lack of within-colony aggression induced in ants ny). Argentine ant colonies are highly polygynous from this region. Liang et al. (2001) suggested that certain (Markin 1968, 1970), with each reproductive female possi- Supella-derived hydrocarbons (especially 15,19-dimethyl- bly contributing unique recognition labels. As a result, pentatriacontane) might be critical to nestmate recogni- workers learn the colony’s collective odour cues, thereby tion in L. humile, because their addition alters creating a uniform template (Crozier & Dix 1979; Breed hydrocarbon ratios within a spectrum that is sensitive to & Bennett 1987). When the diversity of the referents modification. In Camponotus spp., the ratios of certain hy- that form the recognition system is wide, a broader tem- drocarbons affect behaviour between worker castes (Bona- plate presumably is formed and more labels are deemed vita-Cougourdan et al. 1993) as well as between individual acceptable (Vander Meer & Morel 1998). The breadth of workers (Boulay et al. 2000). We found that Argentine ants the template ultimately depends on the genetic diversity in the southeastern U.S. not only have higher initial levels between individuals that contribute to the collective colo- of Supella-shared hydrocarbons, but they also incorporate ny odour. Within the area of California that we sampled, lower amounts of some of those hydrocarbons relative to genetic diversity across colonies was lower (23 alleles at ants from California. Supella longipalpa hydrocarbons alter 7 microsatellite loci) than that of colonies in the south- nestmate recognition in the Argentine ant more pro- eastern U.S. (47 alleles at 7 microsatellite loci; Buczkowski foundly than do those from a number of other insects, in- et al. 2004). As a result, the template in the California col- cluding B. germanica (Liang et al. 2001). Therefore, onies may be relatively narrow, such that addition of regional comparisons of Supella-specific hydrocarbons S. longipalpa and/or B. germanica hydrocarbons may trigger might be especially important in explaining population- changes in perception and provoke aggression between level behavioural differences. We found that the propor- former nestmates. In contrast, in southeastern U.S. colo- tions of three of four Supella-shared hydrocarbons nies, where genetic diversity is higher, L. humile may increased in L. humile from California, but they either de- have a wider template and therefore perceive certain exog- creased or increased only slightly in the southeastern U.S. enous hydrocarbons within this template, such that colonies. behaviour is not altered. Besides its being derived from environmental sources, Whether variation in expression and/or perception can geographical variation in the induction of aggressive explain regional responses to exogenous cues, it appears behaviour may result from regional differences in genetic that similar selection pressures, such as perturbation to an diversity. The southeastern U.S. L. humile population has ant colony’s physical and biotic environment that both higher genotypic diversity and intercolony aggression fragments the colony and provides potentially novel 334 ANIMAL BEHAVIOUR, 71, 2
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