Field work was conducted in the wet season between 27 June and 29 July 2007, and in the dry season between 7 and 22 February 2008 on a shaded organic coffee farm, Finca Irlanda, in the Soconusco region of Chiapas, Mexico (15° 11′ N, 92° 20′ W). The farm is located between 950-1,150 m elevation, receives approximately 4,500 mm of precipitation per year, and contains more than 200 species of shade trees. Mean temperatures during the dry season (May–August) range between 19°C and 25°C (Lin 2007). There are more than 60 arboreal ant species that occur in the farm, but A. instabilis is the most frequently encountered on the trunks of canopy trees (Philpott 2005b). Azteca instabilis builds carton nests on the tree trunks and in the lower canopy of the trees, and across the landscape, colonies of A. instabilis are distributed in patches (Vandermeer et al. 2008).
Colony Collection and Description
We collected individuals from four colonies of A. instabilis from areas of the farm under similar conditions and maintained these partial colonies in the laboratory for use in making body part extracts. We chose strong colonies with large sections of accessible carton for collection. In order to collect the nest, we cut as much of the carton as possible away from the tree and placed it into large plastic boxes, the rims of which were painted with INSECT-a-SLIP (BioQuip Products, Inc., Rancho Dominguez, 90220, CA). We collected individuals from two colonies on 27 June and the others were collected on 4 July. We collected individuals and nest material from colonies separated by a minimum of 100 m to ensure each were different colonies, and not satellite nests formed by budding. To confirm the independence of each lab colony, we placed individuals from each colony directly into tubs containing others and observed any aggressive behavior.
We provided all colonies with a coffee sapling (potted and under a meter in height) colonized by scale insects to tend, along with water, tuna, and sugar as needed. Each of the lab colonies varied in worker size, number of individuals, and in presence of reproductive individuals. Colony I was the smallest collected colony with only the smallest caste size of workers (head widths of workers are approximately 1.1 mm, 1.4 mm, and 2.1 mm for small, medium, and large workers respectively); no male or female alates were found within the collected carton. Colonies II-IV contained all worker caste sizes as well as males and winged females. At the end of the summer field season, all individuals from colony I and II and the majority of individuals from III and IV were collected in vials, the vials were then filled with hexanes and placed in the freezer. These preserved ants were then used in the winter field season for preparation of whole body extracts. Vouchers specimens of Azteca instabilis and the Pseudacteon phorid flies have been deposited at El Colegio de la Fontera Sur (ECOSUR) in Tapachula, Mexico as well as the University of Toledo in Toledo, OH.
Preparation of Extracts
To prepare head, thorax and abdomen extracts, ants were collected from each colony and frozen until dead. The ants were then trisected into head, thorax and abdomen sections with razor blades. Fifty heads, thoraxes, and abdomens were placed in separate 2-dram glass vials with 2 mL of pesticide-grade hexanes and crushed. For each colony, three extracts of heads, thoraxes and abdomens were prepared for a total of 12 extracts of each type. To prepare the dorsal and ventral abdominal extracts, 10 individuals were frozen and their abdomens were bisected under water. The dorsal and ventral segments were then placed in separate vials, each containing 2 mL of hexanes. When 10 abdomen sections were in each vial, the contents were pulverized. Colonies II, III, and IV alone were used during these trials as the workers in colony I were too small to accurately section the abdomens with available equipment. Three extracts of each body section were prepared from each colony. In preparation of whole body extracts, 50 previously frozen ants from the colonies were added to a 2-dram glass vial with 1.5 mL of hexanes and crushed.
Compounds found within the glands of A. instabilis as well as another general ant alarm compound were diluted and prepared to use for observations with phorid flies. Extracts of 2-methyl-cyclopentanone (I) were prepared using commercially available 2-methyl-cyclopentanone in a 0.17% by volume solution with hexanes (1.5 mL). The formic acid extract was prepared in a similar manner. 1-acetyl-2-methyl-cyclopentane (II) was prepared from commercially purchased 1-acetyl-2-methyl-cyclopentene by reduction of the double bond (Fig. 1b). Diphenylsilane (1.55 mol eq.), anhydrous ZnCl2 (0.40 mol eq.), and tetrakis triphenylphosphine palladium (1.8 mol%) were added to IV (60 mg) and ~2 mL of chloroform. The reaction was sealed at room temperature and allowed to stir overnight. The mixture was purified using column chromatography on silica gel give a final yield of ~30%. The identity of the final product, II, was confirmed by GC/MS and 1H NMR.
Preparation of 3-D Model of Swarming Ants
To have the capacity to experimentally isolate chemical from visual cues as phorid attractants, we prepared a 3-D model of swarming ants to simulate ant movement without chemical signals. This model consisted of magnets (10 × 4 mm) placed on a platform itself sitting atop a magnetic conveyor belt made from bike chain and powered by a servo motor charged by a 6 V battery (Fig. 2). When in motion, the magnets move in a pattern that gives the effect of ants swarming in many directions. To examine visual cues necessary for host location and oviposition, we prepared two different types of model ants to place on the magnets and magnetic platform to attract phorids. We used freshly killed A. instabilis and ant-shaped clay models on the platform of our model. Ants used in visual cue observations were collected fresh from A. instabilis nests and frozen until dead. To examine whether size of host matters for Pseudacteon parasites of A. instabilis, we collected individuals from the largest (5 mm), middle (3-4 mm) and smallest (<3 mm) caste size for our experiments. We then washed these ants with hexanes to remove cuticular hydrocarbons or any other chemical cues that may be of further use to the phorids in host location. Once dry, we attached the ants to rectangular magnets using gorilla glue (The Gorilla Glue Co., 45227, OH). To examine whether color is an important visual cue for Pseudacteon parasites of A. instabilis, we molded red, yellow and brown ant shapes (approximately 5 mm in size) out of Fimo dough (Eberhard Faber, 91311, CA) and glued them to small square magnets to make ant models for color choice tests. Azteca instabilis ants most closely match the red color used in clay models. We prepared fresh versions of these models every day this type of observation was performed.
During the summer field season, we placed extracts made from different A. instabilis body parts near A. instabilis nests to record phorid attraction. Twelve strong A. instabilis colonies, defined as those with between 5–30 workers foraging at the base of the nest, each separated by at least 30 m were used as trial sites. To deliver pheromone into the air surrounding the nest, we placed open vials containing the prepared extract, with a filter paper wick, near the base of the nest. We presented each of the five extract types (head, thorax, abdomen, ventral abdominal section, dorsal abdominal section) on different days to prevent any potential contamination or any possible synergistic effects due to the presence of multiple compounds. On a sixth day, wicks doused in solvent controls were presented at A. instabilis colonies. To prevent differences between ant activities at each observation site from affecting the outcome, each field colony was visited each day at approximately the same time, and in the same order. The source of the extracts (i.e. lab colony) presented to each colony was randomized to account for any possible differences in extracts (concentration, composition) produced by individual lab colonies.
After presenting extracts to the air, the area surrounding the vials (approximately 30 cm2) was observed for 10 min and any phorid attack sessions were recorded. The phorid flies tend to attack by hovering over a moving ant, then diving at the ant between approximately 1 and 30 times before leaving the trial site. We considered independent phorid attack sessions to be any time in which the phorid fly approached the vial, then hovered over a single ant and dove toward the ant at least once. Multiple attacks on the same ant directly after one another were counted as the same attack session.
During the winter field season, we performed 15 min observations with extracts of whole body A. instabilis, along with formic acid, compound I, and compound II (each 0.17% by volume in 1.5 mL hexanes). Due to relative availability of the extract types, sample size differed for each of the extracts. We observed eleven replicates with compound I, nine with formic acid, six using compound II, six with and whole body extract. Otherwise, observation methods were the same as described above.
To examine visual cues used by phorids, we placed the 3-D swarming ant model within one meter of a strong colony of A. instabilis. We then added to the center of the platform, 6 model ants (either two of each color (yellow, brown, and red) or two of each size (small, medium, and large), along with a vial containing extract prepared from whole bodies of A. instabilis (Fig. 2). Extract preparation and set up of wires required several minutes during which the model ants remained stationary on the platform and vials with extracts remained closed. It is important to note that no phorid flies appeared until the extract of 50 crushed workers was opened and no flies attacked a model until the motor was turned on. To begin a 15 min observation period, we turned on the motor after opening the extract vial and inserting the filter paper wick. During observations, we recorded the number of phorid attack sessions on each ant type. As a control, we placed 6 magnets without ant models on the platform, along with the whole body extract and observed the number of phorid attack sessions on moving magnets for 15 min. Additional control observations were performed with each model type where the models remained stationary throughout the 15 min observation period. We performed 12 replicates each of type of visual cue choice (size, color, and control observations as well as a duplicate set of no movement control observations).
The five extract types as well as the abdominal extracts were compared using a Chi-square test of proportions. Additionally, the number of phorid attack sessions for each chemical and visual cue was compared using univariate ANOVAs and Tukey post hoc tests. We used ANOVA to compare the number of phorid attack sessions depending on a) chemical cues presented to phorids, b) size of visual cues, c) color of visual cues, and d) type of visual cue (dead ant, clay, control). In order to meet conditions of normality, all data were log-transformed (ln (1+ number of phorid attack sessions)).