Science (2013) 20, 456–466, DOI 10.1111/j.1744-7917.2012.01553.x

ORIGINAL ARTICLE Effect of gland extracts on digging and residing preferences of red imported fire workers (: Formicidae)

Jian Chen and Guangmei Zhang USDA-ARS, National Biological Control Laboratory, Biological Control of Pests Research Unit, Stoneville, MS 38776, USA

Abstract There is evidence that ant-derived chemical stimuli are involved in regulat- ing the digging behavior in Solenopsis invicta Buren. However, the source gland(s) and chemistry of such stimuli have never been revealed. In this study, extracts of mandibular, Dufour’s, postpharyngeal, and poison glands were evaluated for their effect on ant digging and residing preferences of S. invicta workers from three colonies. In the intracolonial bioassays, workers showed significant digging preferences to mandibular gland extracts in 2 of 3 colonies and significant residing preferences in 1 of 3 colonies; significant digging preferences to Dufour’sgland extracts in 1 of 3 colonies and significant residing preferences in 2 of 3 colonies. No digging and residing preferences were found for postpharyngeal and poison gland extracts. In intercolonial bioassays, significant digging and residing prefer- ences were found for mandibular gland extracts in 3 of 6 colony combinations. Significant digging preferences to Dufour’s gland extracts were found in 4 of 6 colony combinations and significant residing preferences in all 6 colony combinations. For postpharyngeal gland extracts, significant digging preferences were found only in 1 of 6 colonial combinations and no significant residing preferences were found. For poison gland extracts, no signif- icant digging preferences were found; significant residing preferences were found in 1 of 6 colony combinations. However, a significant residing deterrence (negative residing preference index) was found for 2 of 6 colony combinations. Statistical analyses using data pooled from all colonies showed that mandibular and Dufour’s gland extracts caused sig- nificant digging and residing preferences in both intracolonial and intercolonial bioassays but not postpharyngeal and poison gland extracts. By analyzing the data pooled from the same three colonies used for gland extract bioassays, it was found that, in no cases, workers showed significant digging and residing preferences to 2-ethyl-3,6-dimethylpyrazine, an alarm component from mandibular gland. Key words digging preference, Dufour’s gland, mandibular gland, poison gland, postpharyngeal gland, residing preference

Introduction mass migration, in which they have to expose themselves to the open air. S. invicta construct earthen nests and for- Red imported fire , Solenopsis invicta Buren pre- aging tunnels which not only reduces the risk of an ant fer staying in a concealed environment except for a few colony being desiccated and predated, but also provides a critical events such as foraging, nuptial swarming, and unique structure for its optimum development (Tschinkel, 2006). Digging is a fundamental behavior in construction and maintenance of both nest and subterranean forag- Correspondence: Jian Chen, USDA-ARS, National Biolog- ing tunnels, which has been found to be precisely regu- ical Control Laboratory, Biological Control of Pests Research lated in ant colonies. For example, mound (aboveground Unit, Stoneville, MS 38776, USA. Tel: +1662 686 3066; fax: part of the nest) volume is directly proportional to the +1662 686 5281; email: [email protected] colony total weight in S. invicta (Tschinkel, 2006). Such a

456 Published 2012. This article is a U.S. Government work and is in the public domain in the USA. Ant digging and residing preferences 457 well-defined relationship between colony size and nest et al., 1981), and Atta laevigata (Smith)(Salzemann volume has also been found in other ant et al., 1992); trunk route markers in Pogonomyrmex bar- (Tschinkel, 1987, 1999, 2004; Mikheyev & Tschinkel, batus (Latreille), P. rugosus Emery, and P. maricopa 2004), indicating that ants are able to adjust the volume Wheeler (Holldobler¨ et al., 2004). Dufour’s gland se- of their living space through the digging and constructing cretion is also used in the establishment of hierarchies behavior. In addition to nest and tunnel construction, dig- within colonies of Leptothorax gredleri Mayr (Heinze ging is also a critical component in ant rescue behavior et al., 1998) and during nest usurpation by the slave-maker (Nowbahari et al., 2009). ant, Tinaut (Ruano et al., 2005). Most, if not all, activities of an ant colony such as for- Parallel to its great functional diversity, the Dufour’sgland aging, mating, defense, offense, immigration, nestmate has a very complex chemistry (Morgan, 2008). However, recognition, and care of brood and eggs are mediated by whether the Dufour’s gland secretion affects the digging chemical signals (Vander Meer & Alonso, 1998; Jackson behavior has not been investigated. & Ratnieks, 2006; Smith et al., 2009). In addition to re- The poison gland is the main source of ant defen- leaser such as alarm and trail pheromones, sive/offensive compounds. However, in many ant species, which cause immediate alterations in the behaviors of the it also has other functions. The venom gland was identi- recipients, queens also use primer pheromones to trig- fied as a source of trail pheromones in a number of ant ger a change of developmental events in a colony, such species (Evershed & Morgan, 1983; Morgan & Ollett, as suppression of production of sexuals (Vargo, 1998) 1987; Janssen et al., 1995; Attygalle et al., 1998; Maile and change of conspecific aggressiveness (Vander Meer et al., 2000; Leclercq et al., 2001). In S. invicta, piperidine & Alonso, 2002). There is evidence that chemical stimuli alkaloids occur in the poison gland of workers and queens are also involved in regulating digging behavior in several (Brand et al., 1972; Blum et al., 1992) and piperideine ant species such as Conomyrma pyramica (Roger) (Blum alkaloids in workers only (Chen & Fadamiro, 2009; Chen & Warter, 1966), Formica yessensis Forel (Imamura, et al., 2009a, 2010). Although they are key attractants to 1982), Lasius niger (Linnaeus) (Rasse & Deneubourg, the phorid fly, Pseudacteon tricuspis Borgmeier, an intro- 2001), Pogonomyrmex badius (Latreille) (Wilson, 1958), duced fire ant parasitoid (Chen et al., 2009b), no data have P.occidentalis (Cresson) (Spangler, 1968), and S. invicta shown they have any pheromonal functions. The queen (Hubbard, 1974). However, the chemical nature of those recognition pheromones were found in the poison gland stimuli has been identified only for a few species such of the mated queens (Rocca et al., 1983). No information as 2-heptanone in C. pyramica (Blum & Warter, 1966), is available about the effect of venom gland secretion on 4-methyl-3-heptanone in P.badius (McGurk et al., 1966; the digging behavior of S. invicta. Wilson, 1958), and carbon dioxide in S. geminata (Fabri- The chemistry of postpharyngeal glands is well stud- cius) (Hangartner, 1969). ied for some ant species (Vinson et al., 1980; Bagneres` The mandibular gland was often identified as a source & Morgan, 1991; Oldham et al., 1999; Attygalle et al., of alarm pheromones (Morgan, 2008; Holldobler¨ & 2006). Phillips and Vinson (1980) described the mor- Wilson, 1990). In P.badius, the chemical releaser of dig- phology of postpharyngeal gland of S. invicta and found ging behavior is from the mandibular gland, which also that it was most highly developed in the queen. Post- elicits alarm behavior. The presumed source of digging pharyngeal glands often contain the same compounds pheromones in F. yessensis is also the mandibular gland as those on the cuticle (Bagneres` & Morgan, 1991; (Imamura, 1982). Alkylpyrazines are commonly found in Tschinkel, 2006). For example, the postpharyngeal gland mandibular glands of many ant species (Morgan et al., of S. invicta contains long-chain hydrocarbons, which are 1999). Recently, 2-ethyl-3,6-dimethylpyrazine was iden- also found on the cuticle (Cabrera et al., 2004). Vinson tified as an alarm pheromone from mandibular gland in et al. (1980) found that the function of the postpharyngeal S. invicta (Vander Meer et al., 2010). However, whether gland also included the absorption of free fatty acids and those alkylpyrazines were involved in regulating digging triglycerides. Postpharyngeal gland secretion functions as behavior is unknown. a modifier of aggressive behavior in the myrmicine ant, The Dufour’sgland has very versatile functions. It is the Manica rubida Latreille (Hefetz et al., 1996). In S. invicta, source of both releaser and orientator of trail following the role of cuticular hydrocarbons in colony recognition behavior in S. invicta (Wilson, 1959) and Gnamptogenys was first manifested by a study on a myrmecophilus bee- striatula Mayr (Blatrix et al., 2002); territorial markers tle, Myrmecophodius excavaticollis (Blanchard). It was in Myrmica rubra (Linnaeus), M. ruginodis Nylander, M. found that the acquisition of host hydrocarbons was as- sabuleti Meinert, M. scabrinodis Nylander (Cammaerts sociated with the acceptance of this beetle into S. invicta

Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 20, 456–466 458 J. Chen & G. Zhang colonies (Vander Meer & Wojcik, 1982). The effect of postpharyngeal gland secretion on the digging behavior has never been investigated. In this study, we first evaluated the effect of extracts from mandibular, Dufour’s, postpharyngeal, and poison glands of S. invicta workers on the digging and resid- ing preferences of workers. We also evaluated the ef- fect of 2-ethyl-3,6-dimethylpyrazine, a newly identified alarm pheromone of S. invicta from the mandibular gland (Vander Meer et al., 2010), on the digging and residing preferences, aiming to see whether the alarm pheromone contributes to the observed preferences of S. invicta work- ers toward the extracts of mandibular glands.

Materials and methods

Ants

Three polygyne colonies of red imported fire ants were collected from Sharkey County, Mississippi. Colonies were maintained in the same condition as described by Chen and colleagues (Chen et al., 2009a). All colonies were free of Kneallhazia solenopsae (J.D. Knell, G.E. Allen & E.I. Hazard) Sokolova & Fuxa, a microsporidian pathogen of red imported fire ants. The ant social form was determined using polymerase chain reaction on GP-9 alleles. Methods described by Valles and his colleagues were used to amplify GP-9 alleles (Valles et al., 2003).

Dissection of glands

Worker ants were frozen at −20◦C for at least 30 min. For mandibular and postpharyngeal glands, the head was excised from the body in a drop of distilled water on a glass slide. Starting from the posterior end, the exoskeleton Fig. 1 Apparatus used for evaluating digging and residing pref- of the cephalic capsule was carefully removed under a erences. Two tubes with entrance holes (A, 1) were filled with dissection microscope by a pair of dissecting forceps. moist sand: one with treated sand and the other with control To ensure intactness of the mandibular gland, it was left sand. The differences in the amount of sand removed from tubes attached to the mandible. For Dufour’s and poison glands, and in the number of ants inside tubes between the treatment and the abdomenal cuticle was first removed in distilled water control were used to calculate digging preference index (DPI) under a dissection microscope. The Dufour’s and poison and residing preference index (RPI), respectively. In this case, glands were then separated. Each gland was extracted with ants removed sand from one tube (B, 3) but not the other (B, 2). 1.0 mL of acetone and hexane (1:1) in a 2-mL centrifuge tube. (Chen, 2009). Four 2-mL centrifuge tubes were mounted under a 8.7 cm × 2.3 cm Petri dish using glue (Arrow Digging and residing preferences of gland extracts Fastener Co., Inc., Saddle Brook, NJ). Two tubes were filled with moist sand, one with sand treated with gland The bioassay apparatus is shown in Figure 1. It was a extract and the other with control sand. The other two two-choice digging bioassay arrangement which can be tubes were used to support the Petri dish. Fire ant workers used to evaluate both attractant and repellant of fire ants will dig whenever an adequate digging substrate such as

Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 20, 456–466 Ant digging and residing preferences 459 moist sand is available. The rationale for this bioassay is Data analysis that ants would dig more in tube containing a preferable substrate. The gland in 1.0 mL of acetone and hexane A paired t-test was used to compare the mean amount of was transferred into a 20 mL vial. Two milliliters of hex- removed sand or mean number of ants between the treat- ane was then added to the vial. The solution was then ment and the control. The analysis of variance followed mixed with 15 g of sand. The sand was stirred every 2 by a least significant difference mean comparison at α = min under a fume hood to facilitate the evaporation of 0.05 (SAS PROC ANOVA) was used to compare intra- the solvent. After the solvent was evaporated (5 min), colonial and intercolonial digging and residing preference 1.31 mL of distilled water was added and mixed with the indices. sand, resulting 0.06 gland equivalent per gram of moist sand. Sand in the control tube was treated only with sol- vent. Each tube was fully loaded with wet sand (approx- Results imately 3 g). There was no open space inside the tube. Twenty fire ant workers were introduced into the cen- In intracolonial bioassays where digging ants and gland ter of the Petri dish. The experiment was conducted at extracts were from the same colony, workers showed 22 ± 0.8◦C (mean ± SE). After 24 h, the sand in each significant digging preferences to sand treated with tube was collected, dried at 250◦C for at least 4 h, and mandibular gland extracts in 2 of 3 colonies (Table 1) weighed. At the same time, the number of worker ants and to sand treated Dufour’s gland extracts in 1 of 3 in each tube was recorded. Two types of bioassays were colonies (Table 2) but not with postpharyngeal and poison conducted: intracolonial and intercolonial bioassays. In gland extracts (Tables 3 and 4). Significant residing pref- the intracolonial bioassay, digging ants and gland extracts erences were found for mandibular gland extracts in 1 of were from the same colony, whereas, in the intercolo- 3 colonies (Table 1); for Dufour’s gland extracts in 2 of 3 nial bioassay, gland extracts and digging ants were from colonies (Table 2); but not with postpharyngeal and poison two different colonies. Three colonies were used. There gland extracts (Tables 3 and 4). In intercolonial bioassays were five replicates for each of three colonies in the in- where digging ants and gland extracts were from different tracolonial bioassay and for each of six colony combi- colonies, workers showed significant digging preferences nations in the intercolonial bioassay. A digging prefer- for sand treated with mandibular and Dufour’s gland ex- ence index (DPI) was calculated using the formula DPI = tracts in 3 and 4 of 6 colonial combinations, respectively (At − Ac)/(At + Ac), where Ac and At are the amounts (Tables 1 and 2); 1 of 6 colonial combinations for post- of sand removed from the control tube and treatment tube, pharyngeal gland extracts (Table 3) and none for poison respectively, 24 h after the bioassay was started. Resid- gland extracts (Table 4). Significant residing preferences ing preference index (RPI) was obtained using formula were found for mandibular gland extracts in 3 of 6 colony RPI = (Nt − Nc)/(Nt + Nc), where Nc and Nt are the combinations (Table 1) and for Dufour’s gland extracts number of ants in the control tube and treatment tube, in all 6 colony combinations (Table 2), for poison gland respectively, after each bioassay was finished. Negative extracts in 1 of 6 colonial combinations (Table 4), but DPI and RPI suggested that ants preferred control over none for postpharyngeal gland extracts (Table 3). Statis- treatment. tical analyses using data pooled from all colonies showed that S. invicta workers significantly preferred sand treated with mandibular and Dufour’s gland extracts in both in- Digging and residing preferences of alarm pheromone tracolonial and intercolonial bioassays but not postpha- ryngeal and poison gland extracts (Tables 5 and 6). For Vander Meer et al. (2010) identified 2-ethyl-3,6- the mandibular gland extracts, the difference was not dimethylpyrazine as an alarm pheromone of S. invicta. significant between intracolonial and intercolonial DPIs Its effect on digging preference was also evaluated in (F1,43 = 1.91, P = 0.17), nor was the difference be- this study using the same procedure described above. tween intracolonial and intercolonial RPIs (F1,43 = A mixture of the 2-ethyl-3,6-dimethylpyrazine and 2- 0.59, P = 0.45). For the Dufour’s gland extracts, dif- ethyl-3,5-dimethylpyrazine (Sigma–Aldrich, St Louis, ference was also not significant between intracolonial MO) was used in the bioassay. Concentrations of 25.00, and intercolonial DPIs (F1,43 = 2.91, P = 0.10) and 50.01, 100.01, 25004.00, 50008.00, and 100016.00 between intracolonial and intercolonial RPIs (F1,43 = pg/g were tested. There were 10 replicates for each 2.12, P = 0.15). In the bioassay on alarm pheromones, bioassay. For each concentration, DPI and RPI were none of tested concentrations caused significant digging calculated. and residing preferences; but in one case, the digging

Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 20, 456–466 460 J. Chen & G. Zhang

Table 1 Effect of mandibular gland extracts on digging and residing preferences of red imported fire ants.

Colony Sand removed (g) (SE)† t-test on Number of ants§ t-test on DPI (SE)‡ dug sand RPI (SE)¶ ant number Ant Extract Treatment Control t-value (P) Treatment Control t-value (P)

A A 0.91 (0.05) 0.56 (0.07) 0.25 (0.05) 7.04 (0.002) 12.20 (0.92) 4.20 (1.69) 0.52 (0.19) 3.09 (0.037) B 1.00 (0.03) 0.72 (0.06) 0.16 (0.05) 3.89 (0.018) 13.40 (1.33) 2.80 (1.63) 0.67 (0.18) 3.71 (0.021) C 0.75 (0.07) 0.61 (0.09) 0.12 (0.06) 2.45 (0.071) 12.40 (1.86) 4.80 (1.28) 0.42 (0.17) 2.49 (0.067) B A 0.77 (0.06) 0.64 (0.08) 0.10 (0.07) 1.56 (0.195) 11.80 (1.28) 2.20 (0.58) 0.68 (0.09) 6.25 (0.003) B 0.66 (0.05) 0.36 (0.05) 0.30 (0.06) 6.14 (0.004) 9.80 (2.44) 4.80 (2.08) 0.30 (0.31) 1.15 (0.313) C 0.58 (0.03) 0.54 (0.08) 0.05 (0.05) 0.62 (0.567) 4.40 (2.16) 10.00 (2.55) −0.35 (0.34) −1.19 (0.299) C A 0.81 (0.06) 0.66 (0.06) 0.10 (0.03) 3.21 (0.033) 11.00 (1.61) 3.20 (1.43) 0.56 (0.20) 2.73 (0.052) B 0.78 (0.05) 0.45 (0.06) 0.28 (0.05) 8.58 (0.001) 11.60 (1.47) 1.00 (0.45) 0.84 (0.08) 6.76 (0.003) C 0.80 (0.05) 0.75 (0.09) 0.05 (0.06) 0.80 (0.468) 9.60 (1.12) 6.40 (1.03) 0.20 (0.13) 1.559 (0.195)

†Mean (SE) weight (g) of sand removed by 20 worker ants 24 h after they were released. ‡Digging preference index. §Mean (SE) number of ants in the tubes 24 h after they were released. ¶Residing preference index.

Table 2 Effect of Dufour’s gland extracts on digging and residing preferences of red imported fire ants.

Colony Sand removed (g) (SE)† t-test on Number of ants§ t-test on DPI (SE)‡ dug sand RPI (SE)¶ ant number Ant Extract Treatment Control t-value (P) Treatment Control t-value (P)

A A 0.74 (0.06) 0.58 (0.03) 0.12 (0.04) 2.92 (0.043) 12.40 (0.25) 4.00 (1.55) 0.56 (0.12) 6.00 (0.004) B 0.88 (0.05) 0.88 (0.06) 0.00 (0.04) −0.09 (0.935) 10.20 (0.58) 5.20 (0.86) 0.33 (0.10) 3.83 (0.019) C 0.95 (0.06) 0.79 (0.05) 0.09 (0.04) 2.37 (0.077) 11.20 (0.97) 3.60 (1.12) 0.54 (0.15) 4.49 (0.011) B A 0.73 (0.05) 0.30 (0.09) 0.44 (0.15) 3.32 (0.029) 15.00 (1.05) 1.40 (0.51) 0.82 (0.06) 9.25 (0.001) B 0.74 (0.07) 0.69 (0.13) 0.05 (0.10) 0.34 (0.746) 9.20 (0.66) 5.00 (1.22) 0.32 (0.14) 2.26 (0.09) C 0.80 (0.09) 0.36 (0.11) 0.47 (0.15) 8.91 (0.001) 11.40 (1.03) 1.20 (0.49) 0.82 (0.07) 9.55 (0.001) C A 0.88 (0.04) 0.55 (0.10) 0.25 (0.08) 3.42 (0.027) 10.60 (1.47) 3.00 (1.00) 0.55 (0.13) 3.41 (0.027) B 0.90 (0.05) 0.63 (0.07) 0.19 (0.05) 5.36 (0.006) 12.00 (1.27) 3.00 (0.89) 0.59 (0.13) 4.50 (0.011) C 0.87 (0.06) 0.62(0.05) 0.17 (0.07) 2.32 (0.081) 10.00 (0.45) 2.80 (0.49) 0.57 (0.07) 8.37 (0.001)

†Mean (SE) weight (g) of sand removed by 20 worker ants 24 h after they were released. ‡Digging preference index. §Mean (SE) number of ants in the tubes 24 h after they were released. ¶Residing preference index. preference was nearly significant (100.01 pg/g, P value = in Calomyrmex sp. (Brough, 1978). This study indicated 0.06) (Table 7). that mandibular gland and/or Dufour’s gland may be the source of chemical(s) that causes the digging preference Discussion observed in Hubbard’s experiments. However, since no significant difference was found in both digging and re- Hubbard (1974) found that S. invicta workers preferred siding preference indices between intracolonial and inter- to dig in their own nest materials over either unnested colonial bioassays, chemicals from mandibular and Du- soil or nest materials from another colony. It is not un- four’s glands alone might not be enough to explain why usual that ants deposit gland substances in their nest workers preferred their own nest material over nest ma- material, such as alkaloids from the poison gland in terial from other colonies. Some other chemicals that S. invicta (Chen, 2007) and mandibular gland substances do not occur in mandibular or Dufour’s gland may be

Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 20, 456–466 Ant digging and residing preferences 461

Table 3 Effect of postpharyngeal gland extracts on digging and residing preferences of red imported fire ants.

Colony Sand removed (g) (SE)† t-test on No. of ants§ t-test on DPI (SE)‡ dug sand RPI (SE)¶ ant number Ant Extract Treatment Control t-value (P) Treatment Control t-value (P)

A A 0.97 (0.05) 1.09 (0.07) −0.06 (0.05) −1.11 (0.329) 2.80 (1.02) 3.60 (2.62) 0.29 (0.34) −0.24 (0.822) B 1.21 (0.05) 1.38 (0.10) −0.07 (0.07) −1.05 (0.351) 6.60 (1.89) 2.40 (1.75) 0.58 (0.32) 1.35 (0.250) C 0.99 (0.06) 0.99 (0.06) 0.00 (0.03) −0.07 (0.949) 4.80 (1.36) 7.00 (1.87) −0.17 (0.22) −1.03 (0.360) B A 1.14 (0.07) 1.18 (0.04) −0.02 (0.02) −0.98 (0.380) 6.40 (1.44) 6.20 (2.27) 0.09 (0.29) 0.06 (0.958) B 1.07 (0.08) 1.18 (0.04) −0.02 (0.10) −1.59 (0.187) 2.80 (0.86) 11.00 (1.30) −0.31 (0.25) 1.15 (0.313) C 1.22 (0.04) 1.05 (0.06) 0.08 (0.02) 4.30 (0.013) 4.60 (1.63) 8.80 (1.77) −0.31 (0.25) −1.26 (0.280) C A 0.95 (0.07) 1.09 (0.06) −0.07 (0.04) −1.64 (0.176) 8.80 (1.83) 6.40 (2.29) 0.20 (0.27) 0.61 (0.578) B 1.30 (0.05) 1.30 (0.09) 0.00 (0.02) 0.00 (1.000) 7.00 (1.27) 7.80 (1.69) −0.04 (0.12) −0.35 (0.740) C 1.07 (0.10) 1.04 (0.06) 0.01 (0.03) 0.45 (0.676) 8.60 (1.21) 5.40 (1.33) 0.24 (0.17) 1.31 (0.260)

†Mean (SE) weight (g) of sand removed by 20 worker ants 24 h after they were released. ‡Digging preference index. §Mean (SE) number of ants in the tubes 24 h after they were released. ¶Residing preference index.

Table 4 Effect of poison gland extracts on the digging and residing preferences of red imported fire ants.

Colony Sand removed (g) (SE)† t-test on Number of ants§ t-test on DPI (SE)‡ dug sand RPI (SE)¶ ant number Ant Extract Treatment Control t-value (P) Treatment Control t-value (P)

A A 0.89 (0.04) 0.85 (0.06) 0.030 (0.04) 0.70 (0.523) 9.60 (1.17) 7.60 (1.81) 0.15 (0.18) 0.69 (0.526) B 0.90 (0.07) 0.95 (0.06) −0.030 (0.015) −2.26 (0.087) 8.80 (1.24) 6.40 (1.91) 0.20 (0.21) 0.78 (0.478) C 0.65 (0.04) 0.63 (0.04) 0.020 (0.02) 0.63 (0.563) 7.80 (1.46) 9.00 (2.07) −0.04 (0.19) −0.35 (0.744) B A 0.53 (0.07) 0.59 (0.03) −0.060 (0.08) −0.63 (0.562) 3.60 (1.50) 14.80 (2.31) −0.61 (0.15) −3.48 (0.025) B 0.60 (0.08) 0.61 (0.07) −0.017 (0.07) −0.17 (0.871) 5.00 (0.78) 10.00 (2.10) −0.25 (0.19) −2.08 (0.107) C 0.69 (0.04) 0.62 (0.14) 0.120 (0.16) 0.53 (0.622) 12.20 (1.32) 4.20 (1.20) 0.48 (0.15) 3.19 (0.033) C A 0.67 (0.06) 0.70 (0.07) −0.020 (0.06) 0.45 (0.686) 4.00 (1.52) 12.80 (1.46) −0.54 (0.16) −3.23 (0.032) B 0.76 (0.08) 0.76 (0.04) −0.010 (0.06) 0.00 (1.000) 11.20 (1.46) 5.60 (1.29) 0.33 (0.14) 2.38 (0.076) C 0.68 (0.05) 0.61 (0.04) 0.050 (0.04) 1.60 (0.184) 9.00 (1.14) 6.80 (1.11) 0.14 (0.13) 1.06 (0.351)

†Mean (SE) weight (g) of sand removed by 20 worker ants 24 h after they were released. ‡Digging preference index. §Mean (SE) number of ants in the tubes 24 h after they were released. ¶Residing preference index. involved in determining such colony specificity. Hubbard A mere attractant or an arrestant may cause such digging (1974) also found that ants made no distinction between preferences because they may increase the chance for ants unnested soil and soil in which another colony had nested. to find and/or stay longer on the treated sand. If this is the In contrast, in this study, ants preferred sand treated with case, the chemical may not be associated with digging be- mandibular or Dufour’sgland extracts from other colonies havior itself. If a chemical changes the digging readiness over control sand. It indicated that colony-specific chem- and digging dynamics such as digging speed and volume, ical signals may be able to completely mask the effect it may be proper to label it as a digging pheromone. How- of non-colony-specific chemicals that causes digging and ever, the data in this study do not provide any insights to residing preferences. those aspects. Ant digging behavior is sophisticated and so is the The mandibular gland often functions as a source of mechanism of digging preferences observed in this study. alarm pheromones (Morgan, 2008). However, in some ant

Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 20, 456–466 462 J. Chen & G. Zhang

Table 5 Intra- and intercolonial effect of gland extracts on the digging preferences of red imported fire ants.

Sand removed (g) (SE)† Relationship between digging Gland DPI (SE)‡ t-value (P) ants and extracts Treatment Control

Intracolonial Mandibular 0.79 (0.04) 0.56 (0.06) 0.20 (0.04) 5.12 (0.0002) Dufour’s 0.78 (0.04) 0.63 (0.05) 0.11 (0.04) 2.57 (0.0219) Postpharyngeal 1.04 (0.04) 1.11 (0.04) −0.03 (0.02) −1.43 (0.1757) Poison 0.72 (0.04) 0.69 (0.04) 0.02 (0.03) 0.98 (0.3442) Intercolonial Mandibular 0.78 (0.03) 0.62 (0.03) 0.13 (0.03) 5.17 (<0.0001) Dufour’s 0.86 (0.03) 0.59 (0.05) 0.24 (0.05) 6.42 (<0.0001) Postpharyngeal 1.14 (0.04) 1.16 (0.04) −0.01 (0.02) −0.78 (0.4416) Poison 0.70 (0.03) 0.71 (0.04) 0.002 (0.03) −0.27 (0.7868)

†Mean (SE) weight (g) of sand removed by 20 worker ants 24 h after they were released. ‡Digging preference index.

Table 6 Intra- and intercolonial effect of gland extracts on the residing preferences of red imported fire ants.

Number of ants† Relationship between digging Gland RPI (SE)‡ t-value (P) ants and extracts Treatment Control

Intracolonial Mandibular 10.53 (0.93) 5.13 (0.92) 0.34 (0.12) 3.06 (0.0085) Dufour’s 10.53 (0.45) 3.93 (0.67) 0.48 (0.07) 7.34 (<0.0001) Postpharyngeal 4.73 (0.92) 6.67 (1.30) −0.02 (0.16) −1.01 (0.3276) Poison 7.87 (0.78) 8.13 (0.99) 0.01 (0.10) −0.17 (0.8699) Intercolonial Mandibular 10.77 (0.82) 4.00 (0.77) 0.47 (0.10) 4.45 (0.0001) Dufour’s 11.73 (0.50) 2.90 (0.40) 0.61 (0.05) 11.13 (<0.0001) Postpharyngeal 6.36 (0.64) 6.43 (0.82) 0.06 (0.11) −0.05 (0.9587) Poison 7.93 (0.80) 8.80 (0.97) −0.03 (0.10) −0.51 (0.6145)

†Mean (SE) number of ants in the tubes 24 h after they were released. ‡Residing preference index. species, alarm pheromones also serve as releasers of dig- veloped in S. invicta (Phillips & Vinson, 1980; Billen, ging behavior. For example, 4-methyl-3-heptanone from 1990), with workers containing only about 300 pg/ant of the mandibular gland of P. badius (Latreille) released 2-ethyl-3,6-dimethylpyrazine (Vander Meer et al., 2010). digging behavior (Wilson, 1958; McGurk et al., 1966). If 2-ethyl-3,6-dimethylpyrazine was responsible for the 4-Methyl-3-heptanone has been found in the mandibu- digging preferences observed in the bioassay for mandibu- lar glands of many other ant species, including Cera- lar gland extracts, similar preferences would be observed pachys sp (Morgan et al., 2008), Atta sexdens (Linnaeus), in the bioassays for 2-ethyl-3,6-dimethylpyrazine at A. opaciceps (Borgmeier) (Francelino et al., 2006), A. 300 pg in 15 g of sand (20 pg/g). That is equivalent to texana (Buckley) (Moser et al., 1968), Manica bradleyi the 750 pg of the mixture of 2-ethyl-3,6-dimethylpyrazine (Wheeler), M. mutica Emery (Fales et al., 1972), Pachy- (40%) and 2-ethyl-3,5-dimethylpyrazine (60%) in 15 g of condyla villosa (Fabricius) (Duffield & Blum, 1973), sand (50 pg/g). However, no significant digging prefer- Pogonomyrmex californicus (Buckley), P. desertorum ence was observed at any tested concentrations of the Wheeler, P.occidentalis (Cresson), and P.rugosus Emery mixture including 50.00 pg/g, although it was close to be- (McGurk et al., 1966). Unfortunately, whether 4-methyl- ing significant at 100.01 pg/g. Vander Meer et al. (2010) 3-heptanone releases digging behavior of those species is found that 2-ethyl-3,6-dimethylpyrazine had a lower ac- still unknown. tive alarm response threshold than a mixture of 2-ethyl- 2-Ethyl-3,6-dimethylpyrazine is an alarm pheromone 3,6-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine. in S. invicta. Because mandibular glands are not well de- However, since no digging preference was observed at

Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 20, 456–466 Ant digging and residing preferences 463

any concentrations above 25.00 pg/g, it could be con- cluded that 2-ethyl-3,6-dimethylpyrazine might not con- ) 11) 90) 86) 33) 17) 26)

P tribute to the digging preferences observed in bioas- ...... says with mandibular gland extracts or, 2-ethyl-3,6- 67 (0 13 (0 18 (0 99 (0 36 (0 15 (0 ...... dimethylpyrazine might not be a major contributor. -value ( 1 0 ants. t − − Mixtures of farnesene compounds have been found in -test on ant number

t Dufour’s glands in S. invicta workers. Dufour’s gland is a source of recruitment pheromones in S. invicta.The ¶ 10) 12) 12) 0 10) 0 14) 1 11) 1 recruitment process has been reduced into three subcate- ...... gories of behaviors: attraction (Vander Meer et al., 1988), 18 (0 06 (0 02 (0 12 (0 11 (0 07 (0 orientation induction (Vander Meer et al., 1990), and ori- ...... 0 RPI (SE)

− entation (Vander Meer et al., 1981). The attraction and orientation induction require multiple chemical compo- nents; however, Z,E-α-farnesene accounts for 100% of

§ the orientation. It will be very interesting to see how each individual component and their various combinations af- fect the digging behavior of S. invicta. 86) 9.33 (0.87) 61) 5.77 (0.81) 0 86) 5.83 (0.77) 0 85) 7.17 (0.86) 0 98) 4.60 (0.74) 0 98) 6.90 (0.76) 0 ......

Number of ants Acknowledgments 80(0 . 70 (0 60 (0 07 (0 70 (0 77 (0 . . . . . We thank Dr. Walker A. Jones and Dr. Yucheng Zhu, USDA-ARS, Stoneville, Mississippi and Dr. David Oi, USDA-ARS, Gainesville, Florida for critical reviews of ) Treatment Control 16) 6 41) 5 67) 6 86) 8 06) 6 29) 8 P ...... an early version of the manuscript. We thank Dr. David Oi and Mr. Euripides V. Mena for their assistance in check- 45 (0 83 (0 44 (0 18 (0 00 (0 09 (0 ...... value (

1 ing for the presence of Kneallhazia solenopsae in red im- t- −

-test on dug sand ported fire ants. We also thank Ms. Sharla Burke for her t technical assistance. Mention of trade names or commer-

‡ cial products in this publication is solely for the purpose 05) 10) 0 08) 0 05) 0 08) 2 07) 1 ...... of providing specific information and does not imply rec- ommendation or endorsement by the U.S. Department of 11 (0 07 (0 03 (0 11 (0 05 (0 08 (0 ...... 0 DPI (SE) Agriculture. −

† Disclosure 027) 040) 0 040) 0 048) 0 050) 0 035) 0 ...... The authors declare that no competing interests exist. 57 (0 29 (0 30 (0 52 (0 29 (0 46 (0 ......

References 038) 0 040) 0 040) 0 037) 0 050) 0 038) 0 ...... Attygalle, A.B., Jham, G.N. and Morgan, E.D. (2006) Contents Sand removed (g) (SE) 32 (0 32 (0 53 (0 37 (0 51 (0 50 (0 ...... Treatment Control of the hypertrophied postpharyngeal gland of workers and soldiers of the fire ant Solenopsis geminata (Hymenoptera: Myrmicinae). Sociobiology, 47, 471–482. Attygalle, A.B., Kern, F., Huang, Q. and Meinwald, J. (1998) Trail pheromone of the myrmicine ant Aphaenogaster 00 0 01 0 00 0 01 0 00 0 00 0 ...... rudis (Hymenoptera: Formicidae). Naturwissenschaften, 85, 25 50

100 38–41. 50008 25004 Effect of mixture of 2-ethyl-3,6-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine on the digging and residing preferences of red imported fire Bagneres,` A.G. and Morgan, E.D. (1991) The postpharyn- 100016 geal glands and the cuticle of Formicidae contain the same characteristics hydrocarbons. Experientia (Basel), 47, Residing preference index. Mean (SE) weight (g) ofDigging sand preference removed by index. 20 worker antsMean 24 h (SE) after number they of were ants released. in the tubes 24 h after they were released. Concentration in sand (pg/g) Table 7 † ‡ § ¶ 106–111.

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