Host-related behaviors of phorids

In order to describe host-related behaviors of phorids attacking leaf-cutting ants, the following were considered: (a) sites for host selection, (b) sites of the host body where oviposition occurs, (c) whether the fly landed on the ant or the load being carried by the ant to oviposit, and (d) the phorid's searching behavior for individual hosts. Data was collected in 12 localities in Argentina and two in Paraguay (sampling sites in Elizalde and Folgarait 2010). At each locality, and in at least three nests of each leaf-cutting ant species present, phorids were searched for in the sites where they are known to oviposit, such as foraging trails and cutting sites. In addition, phorids were searched for at external refuse piles, where some ant species dispose of their wastes (i.e., dead ants and the exhausted vegetal material used by the fungus). Once a phorid was found, its behavior was observed focusing on items (b), (c), and (d), as mentioned above. Observations of phorids from a very short distance by a trained, naked eye were more efficient for behavioral data gathering than using a video camera because video-recording in the field at the speed and distances these small phorids move was not possible. After completing the behavioral observation, phorids were captured in order to identify them to species. For completeness, unpublished data about the host-related behavior of a phorid species collected in La Selva Biological Station in Costa Rica were included. The behavioral observations were all performed during daylight hours and when ant activity was well established.

To compare differences among phorid species in time allocation to relevant aspects of their behavioral repertoire, detailed observations were performed in San Cristóbal (Santa Fe province, Argentina, 30° 12′ S, 61° 09′ W), the community with the greatest phorid richness known to date (Elizalde and Folgarait 2011). In this locality, the leaf-cutting ants present were Atta vollenweideri Forel and seven species of Acromyrmex (Ac. crassispinus Forel, Ac. fracticornis Forel, Ac. heyeri Forel, Ac. hispidus Santschi, Ac. lobicornis Emery, Ac. lundii Guerin-Meneville, and Ac. striatus Roger). Based on previous observations, and on those of Feener and Brown (1993), four host-related behaviors displayed by phorids were defined, and were the main activities performed: (1) perching: when the phorid was sitting on the side of the foraging trail, nest, or refuse pile, with the head directed towards the ants; (2) flying: when the phorid was flying to get close to an ant, came back to the perching site, went from perch to perch, or moved along the foraging trail; (3) attacking: when the phorid touched an ant for at least one second, so as to deposit an egg (as defined previously for other phorids, Tonhasca et al. 2001; Silva et al. 2008; Bragança et al. 2009) (this behavior is not referred to as oviposition because we did not confirm the presence of eggs); (4) landing on a leaf: when the phorid landed on the leaf transported by an ant, usually prior to an attack.

When a phorid female was found attacking a host, her behavior was described verbally by the observer and registered using an audiocassette recorder until at least three oviposition attempts were made on different workers. Recording observations in audiocassettes allowed quantifying the duration of each behavior. Following the observation period, the phorid was captured, and kept in a vial with alcohol, which was adequately labeled for later identification and to associate the phorid with the registered behaviors. The behavior of phorids while attacking ants was quantified for a total of 20 hours, for eight different species. Individual parasitoids were observed for nine minutes on average (SE = 0.7 min.).

Differences among phorid species in the percentage of time allocated to each of the mentioned behaviors was compared with Kruskal-Wallis analyses of variance since the data could not be normalized for most behaviors, even after transformations. The “landing on a leaf” behavior was not included, since it was performed by two species only, and just for a few seconds before ovipositing. Differences among phorid species behaviors were determined using non-parametric tests, and P-levels were adjusted using Holm's correction (Quinn and Keough 2002). Some phorid species use more than one host, but we only collected detailed behavioral data on the preferred host, because on the other hosts it was difficult to sample enough as to be used for statistical analyses. Behaviors for phorid species attacking Acromyrmex or Atta were compared separately because Atta had higher activity outside the nest than Acromyrmex (see Results), which may affect the time that phorid species allocate to each behavior, and also because they did not share hosts (Elizalde and Folgarait 2011). To compare foraging host activity among ant species, measurements of ant activity obtained in periods when no parasitoids were attacking were used, as to exclude any effect that phorids may exert on ant activity. For each ant species, at least 21 ant activity measurements were obtained for a wide range of temperatures (10–40° C) and only for adult nests. An ANOVA was performed (ant activity data was log-transformed to meet the assumptions of the test), and then a Tukey multiple comparisons test was used to find differences in foraging activity among ant species.

The attack rate for each parasitoid species in the high-diversity site was estimated as the number of attacking bouts that phorids performed per minute. Because the attack rate could depend on ant activity in foraging trails, a regression analysis was carried out between the number of ants per minute and attack rate. Ants per minute were estimated by averaging the number of ants returning to the nest during one minute counted before and after recording the observations of phorid behavior. Attack rates and ant activity were log-transformed to meet the assumptions of the analysis. Then, the residuals of the regression were used in a one-way ANOVA with parasitoid species as factor. In addition, to test for an effect of ant activity on phorid attack rates within species, non-parametric correlations between attack rates and average ant activity recorded during the same observation period were carried out.

The activity of ants outside the nest can be regarded as a feature at the species level, since some species of leaf-cutting ants had more foragers outside the nest than others (Hölldobler and Wilson 1990; Elizalde and Folgarait 2010; see Results), and therefore may affect the rate of parasitoid attack. To assess this hypothesis, and test the effect of variation of ant activity among phorid species, a correlation was carried out between the mean attack rate of parasitoid species and the mean ant activity at species level. The activity of each ant species was the average activity recorded for all sampling periods when no parasitoids were attacking, as to exclude any effect that phorids may exert on ant activity.

Ant behaviors in response to phorids

To evaluate the association between the presence of parasitoids and the ants’ responses typically exhibited against phorids (i.e., hitchhiking and body postures), their incidence was recorded in three nests of each ant species present in the same 14 localities mentioned above. This sampling yielded information for more ant species than sampling at only one locality would have. The presence of ant body postures and hitchhikers was recorded while crawling slowly along the main part of a foraging trail for 30 minutes. All phorids observed during these 30-minute periods were collected in order to later identify them to species. Thirty-minute samplings for ant defense behaviors and phorid presence were also carried out at refuse piles, external to the nest, when present.

The relationships between the dependent variable (presence of hitchhikers or presence of defensive postures) and the independent variables (presence of phorids and ant species) were established using two logistic models (also called logit models in this case because dependent variables were categorical, Agresti 2002). These models give better estimations of ant defenses against phorids as an overall feature of the interaction than the proportions obtained directly from the sampled data because these proportions are smoothed by using the information from all the observations, not just the information involved in the proportion being considered (Agresti 2002). Log-likelihood ratio tests were used to evaluate the importance of the independent variables in the model. For these analyses, ant species in which defensive behaviors were not observed (Acromyrmex balzani Emery, Ac. fracticornis, and Ac. rugosus Smith) and Ac. striatus, which showed defensive behaviors at a very low frequency, were not included. In addition, Ac. hispidus was not included in the model accounting for hitchhiker presence because almost all their phorids were attacking in refuse piles, where no hitchhikers were registered (see Results). Data for each ant species were pooled across localities.

While recording focal observations of phorid behaviors in the high-diversity site, ants’ responses to phorids were registered in order to describe them and to evaluate whether ant species differed in their defenses according to phorid species attacking. Based on the repertoire of responses reported for Atta ants in reaction to phorids (Eibl-Eibesfeldt and Eibl-Eibesfeldt 1967; Feener and Moss 1990; Feener and Brown 1993; Tonhasca 1996; Bragança et al. 2002; Bragança et al. 2009), the behaviors described for Solenopsis ants in response to their phorid parasitoids (Wuellner et al. 2002), and our observations, several behaviors of ants in response to phorids were defined. The percentage that each behavior was performed by each ant species was calculated, discriminated by the phorid species attacking. Since phorids, the focus of the observations, flew very fast, it was not possible to record the reactions of all ants.

The responses of ants against phorid attacks included: (1) not reacting, (2) outrunning the pursuing phorid (the change in velocity was very obvious, although it was not measured), or (3) thrashing legs and antennae trying to dislodge the phorid. Additionally, the responses included (4) adopting one of the following body postures, for which ants generally stopped walking and sometimes dropped the load: (a) ball: the ant curled on itself, forming a small ball, with the head down and the gaster between the legs; (b) biting: the head was directed upwards, antennae and legs were stretched out, and the mandibles were open upwards; (c) C posture: the gaster was located between the legs, forming a “C” with the body (see Figure 1c in Eibl-Eibesfeldt and Eibl-Eibesfeldt 1967) (the biting and C postures were very similar, and although they were recorded separately, in some instances an ant would switch from one to the other); (d) gaster down: the gaster was directed towards the ground in a way that the tip was not accessible (the gaster was not located between the legs, and the ant was able to walk while adopting this posture); (e) head against thorax: the ant put its head against the thorax, impeding the access to the neck area; (f) laying: the ant lay down on its side or crouched against the ground.

These postures were generally adopted by the ants at the moment the phorids touched them, and were called “post-attack responses”. But in some instances, an ant could adopt the posture when the phorid was near, and was thus a “pre-attack response”. Colony level responses of ants to phorids (i.e., ants showing a reaction although they were not being pursued by phorids) included the biting or C postures. Usually these ants were near the attacked ant or detected the phorid that was flying past them. Another colony level response, called “tending,“ occurred when 5– 15 nest-mates approached an attacked ant in a defensive posture and touched the attacked ant with their mandibles and antennae (Feener and Moss 1990; Tonhasca et al. 2001).

Ant species were identified in the field and corroborated later in the laboratory using the available keys (Bonetto 1959; Gonçalves 1961). Phorids could not be identified in the field, and were collected and identified in the laboratory using keys (Brown 1997; Disney et al. 2006; Disney et al. 2008; Disney et al. 2009; Brown et al. 2010). Voucher specimens of phorids and ants were deposited in Museo Bernardino Rivadavia (Buenos Aires, Argentina). Statistical analyses were performed in the R environment (R Development Core Team 2010).

Host-related behaviors of phorids

The generalities of host-related behaviors of phorids attacking Acromyrmex and Atta ants were described by collecting data from ant nests at 14 localities. In addition, from a high-species richness locality, the existence of inter-specific differences in host attack rate, and the time allocated to perform host-related behaviors, were evaluated, as well as the possible effect of the number of ant workers outside the nest on attack rates within and among phorid species.

Phorids belonging to the same species showed the same host-related behaviors, such as site of egg insertion, landing on host body or load to oviposit, and strategy to select individual hosts even when attacking different host species, in all localities sampled. Most phorid species searched for their leaf-cutting ant hosts in foraging trails (Table 1), and several phorid species were reported attacking ants when taking waste out of the nest and working on the external refuse piles (Table 1). One of these parasitoids, Myrmosicarius longipalpis Disney, was exclusively found attacking ants at the refuse piles of its host Ac. hispidus, whereas Myrmosicarius crudelis Borgmeier and Myrmosicarius gracilipes Borgmeier were found at higher abundance at refuse piles, since only 28% (out of a total 88 individuals) and 39% (of 18) of the individuals were found in foraging trails of Ac. crassispinus, respectively. On the other hand, Apocephalus neivai Borgmeier and Myrmosicarius cristobalensis Disney were found mostly on foraging trails, with only 4% of the individuals (of 141) and 11% (of 97) attacking ants at refuse piles of Ac. crassispinus and Ac. lobicornis, respectively. In addition, Myrmosicarius catharinensis Borgmeier occasionally (4% of 115 individuals) attacked ants that were performing nest maintenance tasks (i.e., arranging sticks). Since phorids from the same locality were attacking the same host species on trails and refuse piles, or while they were maintaining the nest, this behavioral variation was not related to having pooled data across different host species or sampling localities.

Table 1.

Summary of main host-related phorid parasitoid behaviors (for more details see Appendix). Phorids that attack Acromyrmex host species are underlined. Abbreviations: ? = no information gathered; for “Site for host selection” column: C = cutting site, R = refuse pile, T = foraging trail; for “Oviposition site” column: C = clypeus area, G = tip of gaster, M = mandible or maxillae insertion, O = occiput or near; for “Landing site” column: G = gaster, H = head, Lc = leaf being cut, Lt = leaf transported, N = does not land; for “Search for ants” column: F = while flying, P = from a perch.

The site of oviposition on the host's body varied across, but not within, phorid species (Table 1). Most species used different areas of the ants’ heads to oviposit (in the front, back, mouth area; see Appendix), and Apocephalus species oviposited exclusively in the head (Table 1). However, some species belonging to Eibesfeldtphora and Myrmosicarius also oviposited in the gaster.

Phorid species could be classified according to how they searched for hosts. Some species searched for their hosts mostly from a perch (Table 1). These phorids started to fly, following a particular host, and then usually returned to the perch. These species, such as Ap. neivai among the species that attack Acromyrmex, and Eibesfeldtphora trilobata Disney among the species that attack Atta, employed the ambush strategy (see Barrows 2001). Other species used an actively searching strategy (see Barrows 2001), mainly choosing their hosts from the air (Table 1). Examples using this strategy included Myrmosicarius brandaoi Disney and M. gonzalezae Disney among species that attack Atta, and M. catharinensis Borgmeier and M. longipalpis among species that use Acromyrmex as hosts (Table 1). All species in the genera Apocephalus known to date are mainly ambush parasitoids, while all the Myrmosicarius species use the actively searching strategy (Table 1, Appendix). In the case of Eibesfeldtphora species, E. inferna behaved as an actively searching parasitoid, while E. trilobata used an ambush behavior; however other species of this genus reported in the literature (E. curvinervis and E. tonhascai; Feener and Brown 1993; Tonhasca 1996) have shown to be ambush parasitoids.

Detailed behavioral observations of a group of phorid species in the high-diversity site supported the above dichotomy found in searching strategies. Phorids that searched their host from a perch, like E. trilobata and Ap. neivai, spent more time perching than other phorids (65 and 95% of the time, respectively, Table 2; H = 15.9, df = 2, p < 0.01 for perching behavior in Atta's phorids; H = 15.7, df = 4, p < 0.01 for Acromyrmex's phorids). Meanwhile, Myrmosicarius species, using an actively searching strategy, were flying for a higher percentage of time compared to species using the ambush strategy (Table 2; H = 6.1, df = 2, p = 0.05 for flying behavior in phorid parasitoids of Atta; H = 20.6, df = 4, p < 0.01 for phorid parasitoids of Acromyrmex). Two Myrmosicarius species (M. cristobalensis and M. crudelis) spent, however, higher percentages of their time compared to species using the ambush strategy (Table 2; H = 6.1, df = 2, p = 0.05 for flying behavior in phorid parasitoids of Atta; H = 20.6, df = 4, p < 0.01 for phorid parasitoids of Acromyrmex). Two Myrmosicarius species (M. cristobalensis and M. crudelis) spent, however, higher percentages of their time perching than flying (Table 2). These results were possibly due to these phorids resting more than the other species. On the other hand, some species showed differences in the time spent attacking ants, with M. gonzalezae being the species that spent a greater proportion of the time attacking (almost 15% of total time, Table 2; H = 14.9, df = 2, p < 0.01 for attacking behavior in Atta phorids), whereas the other species only spent 1–3% of the time performing this behavior (Table 2). Phorid parasitoids of Acromyrmex differed in the time spent attacking as well (H = 12.0, d.f = 4, p = 0.02), with M. longipalpis spending less time attacking than Ap. neivai and M. crudelis (Table 2). Because At. vollenweideri had higher activity outside the nest than most Acromyrmex species (F7,274 = 24.5, p < 0.001; Figure 1), and that difference may affect the time that phorids attacking each host allocate to different behaviors as well as attack rates, phorid species attacking each genera were compared separately.

Table 2.

Percentage of the time (median, first, and third quartiles between parentheses) that each phorid species spent performing host-related behaviors: attacking, perching, and flying; and mean attack rates per minute (standard deviation between parentheses). a) Phorid species attacking foragers of Atta vollenweideri. b) Phorid species that attack Acromyrmex hosts, first indicating the host species on which data was collected, and the task that ants were performing between parentheses (f = foraging, w = waste removal).

There were no differences in attack rates among phorid species attacking Atta (F_2,54 = 2.14,p = 0.13; Table 2) or Acromyrmex (F_4,39 = 1.45, p = 0.23; Table 2). Only for M. gonzalezae and M. crudelis was there a significant and positive correlation between phorid attack rate and ant activity (rho = 0.48, p = 0.03, N = 16; rho = 0.60,p = 0.01, N = 13, respectively), and no correlation was found in any of the other species tested (M. brandaoi: rho = 0.32, p = 0.20, N = 9; M. catharinensis: rho = -0.16, p = 0.63, N = 7; M. cristobalensis: rho = -0.18, p = 0.72, N = 13; E. trilobata: rho = 0.08, p = 0.32, N = 32). However, mean ant activity measured as a species trait (Figure 1) showed a positive correlation with mean attack rates per parasitoid species (rho = 0.84,p = 0.009, N = 7).

Figure 1.

Box-whisker plot for ant foraging activity from eight leaf-cutting ant species sampled in a high-richness locality. Ant names are abbreviated with the first three letters of the specific epithet: Acromyrmex fracticornis, Acromyrmex hispidus, Acromyrmex lundii, Acromyrmex crassispinus, Acromyrmex heyeri, Acromyrmex striatus, Acromyrmex lobicornis, and Atta vollenweideri). Lines group ant species with similar values of activity (P > 0.05, Tukey multiple comparison test). High quality figures are available online.

A complete description of the behavior of each phorid species and the defensive behaviors of the ants is given in the Appendix.

Ant behaviors in response to phorids

The association of hitchhiking and defensive body postures of 11 ant species with the presence/absence of phorids was tested using logistic models. Furthermore, differences among host species in their behavioral defenses displayed against phorid species were described and compared in the high-richness locality.

Hitchhikers were recorded in 36% of the 30-minute samplings and defensive body postures in 37% of them (577 total sampling periods of 30 minutes, where Atta saltensis was the species less sampled (with 34 sampling periods), and Ac. lundii the species most sampled (with 145 periods)). However, since phorids were not frequently found in most ant nests (only 26% of the nests sampled for 30 minutes had phorids), these percentages increased to 51% in periods with hitchhikers and to 69% in periods with defense postures of the ants when only considering the sampling periods with phorids’ presence.

Hitchhikers were found in several ant species without previous records of this behavior: At. saltensis Forel, At. vollenweideri, Acromyrmex ambiguus Emery, Ac. crassispinus, Ac. heyeri, Ac. hispidus, Ac. lobicornis, Ac. lundii, and Ac. striatus. Only three Acromyrmex species had neither hitchhikers nor body postures: Ac. fracticornis, Ac. rugosus, and Ac. balzani. In fact, no phorids were collected attacking the last species, and phorids over Ac. rugosus were recorded in one opportunity only. No hitchhiking was recorded in waste remover workers, although they did display defensive postures. No more than three hitchhikers were recorded per leaf, the most frequent situation being only one.

Figure 2.

Estimated proportions (dots) and 95% confidence intervals (lines) from the logistic model for: a) hitchhiker presence, and b) ant body postures presence. Probabilities are discriminated by ant host species (x-axis, ant names abbreviated with the first three letters of the specific epithet: Atta vollenweideri, Atta saltensis, Acromyrmex lundii, Acromyrmex lobicornis, Acromyrmex hispidus, Acromyrmex heyeri, Acromyrmex crassispinus, Acromyrmex ambiguus); and phorid presence (full dots and bolded lines indicate phorid presence, absence shown with white dots). Asterisks next to species labels indicate the species that differed significantly according to Fisher's exact tests (see Results). High quality figures are available online.

Both the presence of phorids and the host species were important determinants of the probability for the presence of hitchhikers (phorid presence: deviance = 17.9, df = 1,p < 0.001; host species: deviance = 87.2, df = 6,p < 0.001; deviance of the logistic model = 2.08, df = 6,p = 0.91 indicates a good fit to the data (Agresti 2002); Figure 2a). Phorid presence (estimated coefficient from the model b = 0.95, SE = 0.24) increased the chances of finding hitchhikers by e^0.95. It was thus 2.58 times more likely to find them during a 30-minute sampling period compared to periods without phorids (the adjusted mean probability of hitchhiker presence for each ant species was higher when phorids were present in comparison to absent, Figure 2a). Since confidence intervals overlapped considerably in some ant species, the proportion of samplings with hitchhikers when phorids were present or absent for each ant species were compared using Fisher's exact tests. Only At. vollenweideri and Ac. lundii showed a significant association between the presence of phorids and hitchhikers (p < 0.05). Meanwhile, for Ac. ambiguus, the association between these variables was nearly significant (p = 0.07).

The presence of hitchhikers was recorded when phorids that land on leaves were the only species of parasitoid ovipositing, and when other phorid species were attacking alone. Thus, to evaluate whether phorids that do not land on the leaves also triggered the hitchhiker defense, another logistic model was used, but excluding from the dataset sampling periods with phorid species landing on the leaf to oviposit (Ap. neivai and Apocephalus vicosae Disney; see Table 1). Similar results to those with the complete data set were obtained, with both phorid presence and ant species as important variables to account for hitchhiker presence (phorid presence: deviance = 10.8, df = 1,p < 0.01; host species: deviance = 82.7, df = 6,p < 0.01; deviance of the logistic model = 4.01, df = 6, p = 0.71), suggesting that hitchhikers may also have a defense function against phorids that do not land on the leaves.

Both phorid presence and ant species were important when accounting for occurrence of ant body postures according to a logistic model (phorid presence: deviance = 61.4, df = 1, p < 0.001; host species: deviance = 70.8, df = 7, p < 0.001; deviance of the logistic model = 8.87, df = 7 and p = 0.26 indicates a good fit to the data; Figure 2b). Phorid presence (estimated coefficient b= 1.65, SE = 0.24) increased the chances to find defensive postures by 5.2 times (e^1.65). In addition, ant species showed different probabilities of displaying postures (Figure 2b).

Defensive behaviors of ants against phorids were similar for most ant species, irrespective of the phorid species attacking, although the proportion in which they were displayed differed (Table 3). Atta and Acromyrmex species showed the same body postures in response to phorids (Table 3, pooling species of each genus), except for the “thorax against head” and “gaster down” behaviors found only in Atta and in some Acromyrmex, respectively (but see Appendix for the last behavior elicited by an Atta parasitoid, Eibesfeldtphora inferna Brown). However, the percentages of display for each behavior differed according to which phorid species was attacking (Table 3). Most ants reacted against the phorids with the ball posture when they were touched by the parasitoids (Table 3). At. vollenweideri reacted to attacks by E. trilobata and M. gonzalezae, phorid species with different oviposition strategies, with similar postures, although varying in the percentage that each one was displayed (Table 3). Similar defensive reactions by the ants were found when comparing Acromyrmex species; six behaviors were shared by Ac. lobicornis foragers reacting against M. cristobalensis and by Ac. crassispinus waste removers against M. crudelis (pooling pre and post-attack behaviors; Table 3). Both phorid species attacked the gaster, and ants reacted against them by lowering it, even when the focal ants were foragers or waste removers.

Table 3.

Percentages for the behavioral responses of ant species towards attacking phorid species, depending on whether the attack was displayed previously (Pre) or after (Post) the ant was touched by the phorid. In addition, colony responses by ants not pursued by phorids (but from the trail where an ant was attacked) are included. The number of ants for which behaviors were recorded is included for pre and post attack behaviors; for colony level responses ‘n’ represents the number of times that the behavior was recorded.

Detailed description of phorid behavior

Apocephalus neivai (observations made over Ac. crassispinus, Ac. heyeri, Ac. lobicornis, and Ac. lundii, mostly in San Cristóbal, Santa Fe province, and Noetinger, Córdoba province, Argentina). This phorid was usually perched at the side of the foraging trail, with its head directed towards the foraging trail, occasionally flying from perch to perch. When an ant carrying a leaf passed under the perch, the phorid flew to the leaf and walked on it towards the ant's head. Then, it touched the back of the head with its fore legs and then turned around to insert the ovipositor, near the insertion of the mandible in the right or left side, using the leaf as a platform. However, in several instances the female left the ant immediately after landing on the leaf or after touching the ant's head with its fore legs, without probing it with its ovipositor. This species went flying from perch to load, and in few cases from load to load, selecting ants to oviposit. After attacking, the phorid walked back to the tip of the leaf, flying from there to the perch.

The attacked ant as well as those near it adopted, in general, ball or biting postures. Two phorids were captured by the ants and then dismembered.

Apocephalus setitarsus (observations made over At. saltensis in La María, INTA, Santiago del Estero, and At. vollenweideri at San Cristóobal, Santa Fe province; and Mercedes, Corrientes province, Argentina). The behavior of this species was observed on few occasions, due to its low abundance. Most of the time, phorids of this species were ovipositing on Atta workers while they were cutting leaves at cutting sites. In a few instances, this species was collected on the foraging trail; however, no attacks were recorded there. This species approached the host by walking. Once over the ant, the female touched with its forelegs the back of the head and the area of mandible insertion. Then, the ovipositing female turned around and positioned its ovipositor in the area of insertion of the mandibles, and remained there for 30–50 seconds.

If the ant detected the phorid, she moved her legs and antennae touching the area where the phorid was located. These ants stopped cutting and abandoned the area. In these cases, the phorid left the ant. In one case, a phorid was captured by the ants.

Apocephalus vicosae (observations made over At. vollenweideri in San Cristóbal, Santa Fe, Argentina). This phorid was attacking ants in the foraging trails using the transported leaf as a platform. The ovipositing female went from perch to perch in short flying bouts, and was very difficult to follow when flying from the perch to the ant's load. Ap. vicosae reached the ant's load by walking or by a short flight towards the distal tip of the leaves carried by the ants. Once there, the phorid walked towards the side of the ant head, positioning its body perpendicular to the main axis of the ant body. Then, it extended its ovipositor to reach the area near the clypeus.

The ants kept walking with the phorids over them but some stopped when the phorids landed on the leaf. To capture this phorid while ovipositing on an ant, it was necessary to collect the ant, as the phorid seemed to get stuck by the ovipositor inside the ant's body. It was not possible to collect it with the aspirator. In one case, a phorid was captured by the ants.

Eibesfeldtphora inferna (observations made over At. cephalotes in La Selva Biological Station, Costa Rica). This species was found most often on At. cephalotes trails, but was also collected near nest entrances. To attack, the fly approached the ant from its front and then positioned herself behind the ant. The fly followed the ant closely for as long as 100 cm. The phorid oriented its head towards the head of the ant and put the ovipositor in the direction of the ant anus, touching it while flying or after landing for approximately a second on the gaster. The exact place of oviposition could not be determined, but it was near the end of the gaster, most likely the anus itself.

E. inferna usually attacked ants that were carrying leaves, but it also attacked ants without leaves. Two flies of this species were riding on leaves carried by ants. After attacking a host, or trying to attack several ants, a phorid would either land on the sides of the trail, using a dry leaf or stick as a perch, or continue flying, searching for another host. If the phorid landed on a perch, it began flying again after some time, apparently unrelated to the number or size of the ants passing by the perch. This species did not use a perch for detecting appropriate hosts, because it was always observed flying for a while before attacking ants.

It seems that the ants detected the fly when being pursued. Ants reacted in two ways to E. inferna: walking faster to outrun the phorid, or lowering their abdomens against the ground, so that the tips were not accessible to the phorid. Both of these reactions were effective in dissuading the fly from ovipositing. Some ants even dropped the leaf and started walking faster.

When the ants were attacked, they kept walking or stopped, stunned, generally holding the leaf between their mandibles. In the case of being stunned, 5–15 ants of the same size or smaller approached the ant attacked and touched her with their antennae. This tending behavior towards the attacked ant could last for several minutes. Then, the attacked ant continued with the journey back to the nest. Usually, some workers stayed on the leaf carried by the attacked ant as hitchhikers. One E. inferna was captured by a hitchhiker. If a non-attacked ant nearby detected a fly, she tried to catch it with her mandibles. These ants opened their mandibles and raised their heads, adopting a biting posture once they detected the fly.

Eibesfeldtphora trilobata (observations made over At. vollenweideri in San Cristóbal, Santa Fe province; Mercedes, Corrientes province; Argentina). These phorids perched at the side of the foraging trail on a leaf or stick, or even on the ground. They began flying after an ant walked under or near their perch. They followed the ant from behind for 5–10 sec (c. 2 m of the foraging trail), keeping a short distance. If by that time they did not reach the ant, they generally returned to the same perch or one near it, or, less frequently, they searched at flight for another close ant. They attacked on the neck, between the head and the thorax, probably on the foramen magnum, positioning their body in the same direction as the ant's body in a way that both heads were in the same direction. They attacked ants with or without loads, walking in both directions on the foraging trails.

The attacked ant stopped walking at the moment that it perceived the phorid, and when the phorid finished the attack, the ant adopted a C or ball posture, and maintained it for almost a minute before resuming its march. Some phorids of this species re-attacked the same individual ants after the ants abandoned the posture adopted in the previous attack. Some ants kicked the phorid flying near them using their hind legs. Others tried to dislodge the phorid by touching it with their legs and antennae. Some ants lay down on their side on the ground, and stayed there for some seconds; others started to run or did not show any reaction to the phorids. Around the attacked ant, many small ants adopted the biting or C postures. Several ants in biting postures were frequently observed near the area where the phorid perched. These flies seemed to be quite territorial with their perching site, as they kept the same perch or site in the foraging trail during the whole observation period.

Lucianaphora folgaraitae Disney (observations made over Ac. crassispinus, Ac. heyeri, and Ac. lundii in Noetinger, Córdoba province, Argentina). This phorid went from perch to perch with short flying bouts, like jumping, and walked between the ants on the foraging trails, seemingly undetected by the ants. Phorids landed or walked on the leaves transported by the ants, and then moved to the ventral part of the head to put the ovipositor near the maxillae. It took these phorids 8–11 sec to oviposit.

If the ants detected the phorid, they stayed still or put their heads down towards the thorax, touching with their forelegs the ventral part of the head. In general, these reactions were effective to dissuade the phorid.

Myrmosicarius brandaoi (observations made over At. saltensis in La María-INTA, Santiago del Estero, and At. vollenweideri in San Cristóbal, Santa Fe; P.N. Estero Poí, Formosa; R.N. Formosa, Formosa; Argentina). Most of the time, this phorid was flying along the foraging trail without following a particular ant. To attack, it touched the right side (14 times vs. 1 in left side) of the ant's head very fast (< 2 sec). The attacked ants could be carrying a leaf piece or not, and going in or out of the nest. This species was also collected flying inside the nest entrance hole.

The attacked ants adopted the ball posture, the C posture, or stayed in biting posture. Some ants adopted the biting or C postures without being touched by the phorid passing by. In several occasions, colony level response was evident by several small ants with the biting or C postures around the attacked ant. Those that were closer to the attacked ant could touch it with their antennae. One phorid of this species was captured by the ants.

Myrmosicarius catharinensis (observations made over Ac. ambiguus, Ac. heyeri and Ac. lundii mostly in Magdalena, Buenos Aires province; San Cristóbal, Santa Fe province and Noetinger, Córdoba province, Argentina). This species flew along the foraging trail for great distances and speed without stopping. For example, a female covered 2 m in 30 seconds, and another 2 m in 25 seconds. In some instances, this species attacked ants performing nest maintenance, i.e., arranging sticks in the nest mound. Phorids of this species were able to fly sideways and backwards, maybe to locate or visually evaluate the ant. To attack, M. catharinensis went to the front of the ant and then darted to the ant's head, near the clypeus or the insertion of the antenna. It was frequent for this species to attack ants with their mandibles busy, carrying a load or clearing out sticks or soil particles from the trail, although it also attacked ants without loads.

The ants were able to detect the phorid while it was flying, and responded by either running or halting their march, disrupting ant traffic. While being attacked, ants adopted a ball or C posture. After the phorid attack, nestmates sometimes touched with their legs and antennae the area where the ant was attacked. In a few occasions, the ants showed no reaction to the phorid.

Myrmosicarius cristobalensis (observations made over Ac. fracticornis and Ac. lobicornis mostly in San Cristóbal, Santa Fe province, and Noetinger, Córdoba province, Argentina). This species flew along the foraging trails, following ants that were (23 times) or not (8 times) carrying a leaf fragment, and following them very close to the gaster. For instance, one phorid followed an ant for 10 seconds at a 3-4 mm distance from the gaster. They were able to follow ants for 30 cm (circa 8 seconds), flying sideways or backwards. They attacked at the tip of the gaster, landing there for some seconds, with their head positioned in the same direction of the ant's head. During summer time, when ant foraging activity was reduced to a short time-window during the day, this species was attacking ants at refuse piles on a few occasions (11%), coincident with times when foragers were not active.

The attacked ants adopted C, ball, or biting postures; they halted for a few seconds or ran away. They also lowered the gaster against the ground, and some continued walking with the gaster between the hind legs. In one case, a phorid was chasing an ant from behind, and a nearby ant captured the phorid with its mandibles.

Myrmosicarius crudelis (observations made over Ac. crassispinus and Ac. lundii, in San Cristóbal, Santa Fe and Noetinger, Córdoba, Argentina). These phorids showed several peculiarities. First, they were ovipositing in ants mainly at refuse piles, although they also searched for ants in foraging trails, always near the nest. They flew in a distinctive way, selecting one ant in which to oviposit while hovering in the air. They were also able to fly sideways and backwards, and go for a short distance into the entrance holes of the nest to attack ants. They attacked ants on the tip of the gaster, positioning their body in the same line as the ant's body, but with the ovipositor towards the left side of the ant. They attack ants loaded or not with the exhausted fungal substrate.

The attacked ant adopted a ball posture or lowered down the gaster while running back to the nest. In addition, the ants showed the biting posture when the phorids flew over them.

Myrmosicairus gonzalezae (observations made of At. vollenweideri, in San Cristóbal, Santa Fe; and Mercedes, Corrientes, Argentina). This phorid flew fast along the foraging trail (for example, in one case a female moved 15 m in 30 seconds). This species oviposited between the head and thorax, in the right side of the host, with its body perpendicular to that of the ant. However, due to the posture of its body and the curved shape of its ovipositor (Disney et al. 2006), it is highly likely that the ovipositor tip reached the ventral side of the neck. M. gonzalezae mainly attacked ants not carrying a leaf (31 times) that were going out of the nest to forage, but it could also attack ants carrying a leaf (5 times). When flying, it followed the ants from behind.

The attacked ants stopped and touched with their legs and antennae the part of their bodies where the phorid oviposited. Other ants came and touched the attacked ant with their mouthparts and antennae. These ants adopted a C or biting posture as a colony response. After circa 15 seconds, the attacked ant did one of several things: started walking with the phorid still attacking her, turned themselves to one side against the ground, adopted a ball posture, or did not show any reaction at all. One phorid was captured by the ants.

Myrmosicarius gracilipes (observations made over Ac. crassispinus in Noetinger, Córdoba province, Argentina). This phorid attacked ants at refuse piles, although it was also collected on foraging trails. The attacked ants were carrying a piece of exhausted fungal substrate or not, and the phorid attacked them on the right side, where the head articulates with the thorax, positioning its body perpendicular to the ant's body. The attacked ants adopted a biting posture when phorids flew over them.

Myrmosicarius longipalpis (observations made over Ac. hispidus in San Cristóóbal, Santa Fe province; P.N. Chaco, Chaco province; P.N. Estero Poí, Formosa province, Argentina). This species searched for ants at the refuse pile. They followed ants very close to the host's front, flying backwards. They very quickly attacked the head of ants with refuse loads. In addition, they were able to search for an ant in which to oviposit from a fixed point in the air, from where they darted to the selected ant, in a similar way as M. crudelis. Usually, several females were attacking ants at a refuse pile, where it was frequently observed that two phorids touched in the air, and then separated to opposite sides of the refuse pile.

The attacked ant adopted biting or ball postures, and other ants approached the attacked ant, touching it with their mouth parts and antennae. When M. longipalpis phorids were flying, several ants adopted the biting posture.