Study sites and focal species

Field studies were conducted from 205 O. longinoda colonies in Gabon (Forêt des Abeilles; 0°40′S, 11°54′E; five colonies) and in different sites in Cameroon: in the city of Yaoundé (03°52′N, 11°31′E; 39 colonies) and its surroundings including Batchenga (03°51′N, 11°42′E; 21 colonies), Kala (03°50′N, 11°21′E; 46 colonies), Matomb (03°53′N, 11°14′E; 34 colonies), Mbalmayo (03°29′N, 11°30′E; 14 colonies), and Nkolbisson (03°59′N, 11°28′E; 32 colonies); and in areas close to Edea (3°47′N, 10°08′E; 11 colonies), Kribi (2°56′N, 9°54′E; seven colonies), Ebolowa (2°54′N, 11°09′E; five colonies) and Buéa (4°09′N, 9°14′E; four colonies).

Oecophylla longinoda, of the subfamily Formicinae, is a “territorially dominant arboreal ant species”1,25,26,27,28 characterized by large, polydomous (multiple nests) arboreal colonies; the workers construct the nests by manipulating larvae to glue leaves together with the silk they produce. Contrary to most formicine ants, the larvae pupate without enclosing themselves in silk cocoons. While plentiful in tree crop plantations where it is used as a biocontrol agent, in natural conditions this species occurs in relatively young or recently disturbed vegetation, whereas very old tropical rainforest trees rather shelter arboreal, carton-nesting Crematogaster spp.1,25,26,27,28.

As for most other ant species, Oecophylla workers cannot mate due to the lack of the appropriate apparatuses, but majors have fully functional ovaries and so can lay trophic eggs that are generally given to the queen. They produce unfertilized, chorioned eggs which can develop into fully functional males when the queen dies29. Because the colonies are very populous (more than 500,000 individuals1), it is difficult to conduct experiments on them. Swarms occur all throughout the year (AD, pers. obs.) which permitted us to compare the production of winged sexuals and moths.

In moths, the number of larval instars can vary intraspecifically. In the genus Eublemma (Noctuidae; Acontiinae), this number varies from eight to ten30,31 rendering it difficult to identify the different instars. Therefore, we differentiated only four “steps” in the larval life of E. albifascia (see Fig. 1 for the entire life cycle). Newly-hatched caterpillars, or first instars, develop into ≈ 4 mm-long second instars frequently found among the ant brood. The “intermediary” instars are caterpillars 8–20 mm-long able to solicit trophallaxis from workers, the same being true for last instars, up to 40 mm in length, that then pupate.

Figure 1: Representation of the different phases in the life cycle of Eublemma albifascia. (a) Egg cluster (N = 113). (b) Second instar caterpillar transported by a minor worker. (c) Left, a trophallactic exchange between a last instar caterpillar and a major worker while another worker licks the cuticle of the caterpillar; right (see arrow), a worker imbibes the anal secretion of another last instar caterpillar. (d) Last instar caterpillars in a highly parasitized colony with many males. (e) A group of agglutinated cocoons. (f) An adult moth. (g) A drawing representing the contracted gaster of an Oecophylla longinoda queen in certain parasitized colony (I) and a “normal queen” in control colonies and other parasitized colonies (II). Full size image

Voucher specimens of ants, moths (identified by Dr. M.R. Honey) and parasitoid wasps (identified by Dr. J. LaSalle) were deposited in the Museum of Natural History, London.

The search for parasitized Oecophylla longinoda colonies

To evaluate the rate of parasitism by E. albifascia caterpillars, all of the nests of 205 O. longinoda colonies (N ≥ 20 to avoid incipient colonies) on small trees (<6 m tall) to permit easy access were first inspected externally to verify if clusters of Eublemma eggs had been laid on the leaves of the nests; if so, we counted the number of eggs. The nests were then opened to verify their content, including the presence of caterpillars (the last instars were counted), dealated queen(s), winged males and females as well as their pupae (plus large last instar female larvae). Although the ants were very alarmed, this process is not destructive (except for the colonies gathered for specific studies, see below) and the workers repaired the opened nests (see the opening of the nests and the observations inside artificial nests32). In addition, ≈ 40 more colonies were inspected during preliminary and complementary studies.

Behavioral relationships between Eublemma albifascia caterpillars and Oecophylla longinoda workers

Laboratory studies were conducted on 13 O. longinoda colonies sheltering Eublemma albifascia caterpillars gathered in Yaoundé or Nkolbisson to avoid a high rate of caterpillar mortality from being transported over a long distance. Nests were collected by cutting down the supporting branches using a clipping pole, putting them into plastic bags and taking them to the laboratory. They were installed in 40 × 20 × 5 cm plastic boxes with a transparent cover (which we covered with a screen outside of observational periods to keep the workers from occulting this surface with silk) opening onto a table through holes 0.8 cm in diameter permitting the passage of emerging adult moths. The bases of the legs of the tables were placed in boxes filled with oil to prevent the workers from escaping. Extrafloral nectar-bearing potted plants (Alchornea chordifolia or Hibiscus spp.) were placed on the tables to permit the workers to forage for nectar and honeydew as they attend scale insects on these plants. The workers wove new nests in the foliage of these plants. The colonies were also provided ad libitum with water, honey and prey (mostly cricket larvae and small mealworms).

Although the workers became quickly used to being observed, most of the inside nest observations were conducted at night using red lighting. Other observations were conducted between 8:00 and 12:00 to record the number of cases when a major worker furnished trophic eggs to the queen in control colonies and to four last instar caterpillars in parasitized colonies.

To verify if E. albifascia caterpillars acquired the colony odor inside their host nests or, rather, have an intrinsic, appeasing odor for their host ants, we conducted two series of confrontation experiments based on the notion of colony mate recognition11. A control experiment was conducted using 40 O. longinoda workers transferred from one nest to another from the same colony and 40 others between two nests from two different colonies. We then transferred 20 caterpillars from one nest to another belonging to a neighboring, alien colony. Then, 20 other caterpillars were transferred from one nest to a distant one belonging to the same colony (two colonies used, one of them reared in artificial nests).

The impact of caterpillar pressure on the sex ratio in Oecophylla longinoda colonies

All of the nests from each colony were gathered, put into large plastic bags and transported to the laboratory where they were placed in a refrigerator. Then, we counted the caterpillars in parasitized colonies as well as the pupae and winged males and females in both types of colonies. While doing so, we noticed that parasitized colonies can have a queen morphologically similar to those in control colonies (thereafter “normal queens”) or a queen with a “contracted gaster” (Fig. 1).

We first used a general linear model (GLM) (R v. 2.14.2 software; R Development Core Team used for all statistical comparisons) to test the hypothesis that highly parasitized colonies (N = 8; they sheltered 131, 139, 144, 151, 177, 184, 201 and 214 last instar caterpillars, respectively; Table 1) contained more males than did control colonies (N = 12; no caterpillar found whatever the instar; queens with a normal gaster). Because our response variable (i.e., the sex of the ants) was binary, we modeled it with a binomial error distribution and tested the effect of the presence of parasites on the probability of being a male. These two lots of colonies were composed of a similar number of nests (mean ± SE: 28.75 ± 2.00 ‘nests’ per control colony vs. 29.50 ± 2.65 ‘nests’ per parasitized colony; t = 0.23; 18 df; P = 0.82).

Table 1 Composition of the colonies studied. Full size table

Second, in the four parasitized colonies having a queen with a contracted gaster (i.e., containing 206, 241, 260 and 274 last instar caterpillars, respectively; Table 1), the male pupae and adults were too numerous to be counted. So, we counted the pupae and winged females in the field and verified if worker pupae were present.

Third, the same process was applied to six parasitized colonies having lost their queen (i.e., containing 55, 64, 69, 288, 235 and 359 last instar caterpillars, respectively); some nests belonging to the first three colonies were full of agglutinated cocoons (Fig. 1 and Table 1). The numbers of pupae and winged males and females, which correspond to discrete, positive variables, were compared using a Poisson error distribution in a GLM.

After opening the nests of both the control colonies and the parasitized colonies, we noted the number of eggs surrounding the queen in each colony on an ordinal scale or gathered them using a fine paintbrush, and put them into a box to be counted in the laboratory. To test the effect of parasites on the number of eggs, we used the non-parametric Wilcoxon rank test to account for the ordinal nature of the response variable (Table 1).

Starvation of Oecophylla longinoda queens in parasitized colonies

For two O. longinoda control colonies and four parasitized colonies bred in the laboratory, we conducted 54 series of 10-minute observations (9 h) during which we noted each time it occurred the duration of worker-queen trophallaxis in the former case and worker-caterpillar trophallaxis in the latter case (one caterpillar targeted in each colony during each observation session). The durations of the trophallaxis, an over-dispersed positive variable, were compared using a GLM with a quasi Poisson error distribution. The numbers of trophallactic events, a discrete and positive but not over-dispersed variable, were compared using a GLM with a Poisson error distribution.

For the same colonies, we also compared using a GLM with a Poisson error distribution the numbers of trophic eggs provided by major workers to the queens in control colonies and to caterpillars in parasitized colonies (20 series of 10-minute observations per queen and per caterpillar).

Using a microscale (Mettler® AE 260), we weighed minor, media and major workers, males, winged queens and mated queens (N = 10 in each case) taken from control colonies, and last instar caterpillars (N = 28) as well as “normal” queens (N = 5) and queens with a contracted gaster (N = 4) taken from parasitized colonies. The weight of 10 queens from control colonies and nine from parasitized colonies were compared with a classical GLM with a normal error distribution.