First Record of an Encyrtid Wasp (Hymenoptera: Chalcidoidea) as a True Primary Parasitoid of Ants (Hymenoptera: Formicidae)

ABSTRACT 
 Numerous cases of associations of encyrtid wasps with ants have already been reported. In the majority of these cases, however, wasps are associated only indirectly with ants (interference associations) through primary parasitism of the trophobionts (Coccoidea), which are exploited and protected by ants. Suspected direct parasitism cases are unusual and no direct attack of encyrtids on ants has ever been demonstrated. Here we provide both a revised list of all known cases of associations between encyrtid wasps and ants, and a report of the first record of a true primary encyrtid parasitoid of ants. Of two colonies of the arboreal ponerine ant, Pachycondyla goeldii (Forel), examined from French Guiana, one had 3 pupae parasitized by males and females of a gregarious, minute encyrtid wasp species, identified as Blanchardiscus pollux Noyes, and 2 other cocoons presented evidence of pararasitism. This first host record for the genus Blanchardiscus, which has always been placed near other genera that parasitize scale-insects, supports a hypothesis of a shift from a myrmecophilous host to an ant host. Our findings increase to 9 the number of parasitoid wasp families known to attack ants as primary hosts. A closer examination of other arboreal ants, particularly those involved in ant-garden building and nest weaving, will certainly yield new ant-parasitoid associations.

Here we describe the first known case of true primary parasitism of a species of ant by an encyrtid wasp (Chalcidoidea: Encyrtidae). Our results are based on ant specimens collected in 2002 in the course of investigations on the eucharitid fauna associated with formicids in French Guiana. An exhaustive recent review of all the material preserved in alcohol, focusing on eucharitid wasps associated with poneromorph ants , fortuitously allowed us to detect new examples of hymenopteran parasitoids of ants, other than eucharitids. Moreover, a survey of the literature over the last 100 years allowed us to list both all the recorded cases of associations between encyrtid wasps and ants and the exact nature of their known relationships.

MATERIALS AND METHODS
Two nests of the arboreal ant-garden species, Pachycondyla goeldii (Forel) (Formicidae: Ponerinae), were collected on 12 Nov 2002 in French Guiana, at Km 13 along the road leading to the Hydroelectric complex at Petit Saut, Sinnamary (N 05° 07' 25.3" W 52° 57' 16.7"). Pachycondyla goeldii is a monogynous, polydomous ponerine species (Denis et al. 2006) that colonizes pioneer vegetal formations where plants are characterized by a rapid, continuous growth and a high production of leaves and soft wood (Corbara & Dejean 1996;Dejean et al. 2000b). Founding queens and first generation workers initiate their own ant garden by building a cardboard-like structure into which epiphyte seeds are integrated. Following the growth of the epiphyte, the colony establishes its nest within the root system (Corbara & Dejean 1996;Orivel et al. 1998). Ant gardens were transported to the laboratory, and their content preserved in alcohol for later examination.
The presence and number of ant dealate females, alate females, males, workers, cocoons, and larvae, as well as the presence of any adult myrmecophile (especially eucharitid parasitoids), were recorded. At first, the contents of cocoons preserved in alcohol were superficially examined while backlit to check for the presence of any ectoparasitoid. In particular, we looked for wasp remains (exuvia) within empty ant cocoons denoting previous parasitoid emergence and for the presence of scars upon ant larvae resulting from unsuccessful attacks of eucharitid first instar larvae. During June 2011, preserved material was revised again: all of the cocoons were dissected under a stereomicroscope and ant pupae were this time thoroughly checked for the presence of both ecto-and endoparasitoids or for any evidence of parasitism. We discovered some encyrtids, some of these were slide-mounted and identified by direct comparison with type material of the relevant species. Voucher specimens were deposited in the Natural History Museum, London, England and in the Arthropod collection of El Colegio de la Frontera Sur-Chetumal, Quintana Roo, Mexico.

RESULTS
The first nest collected was composed of 1 queen, 77 workers, 4 pupae, 2 larvae, and several eggs. The second nest contained no queen, 5 alate females, 208 workers, 67 pupae (of which 16 large pupae, presumably winged sexuals), and neither larvae nor eggs. Initial cursory inspection of the brood, in 2002, showed no signs of external attack.
During the later examination, in 2011, it was noted that the color of the gaster of some ant pupae looked slightly different and, in one instance, very small dark points were visible through the cuticle. In this last case, dissection showed the presence of already pigmented developing individuals of a gregarious, very minute (body length < 1 mm) endoparasitoid wasp. Five ant pupae from nest no. 2 were found to be parasitized. One worker pupa contained 15 almost fully developed and pigmented adult wasps (8 females, 7 males). A second worker pupa contained 11 wasp pupae (at that developmental stage sex could not be determined, see Fig. 1), and a female (queen) pupa contained 4 white wasp larvae-prepupae. Two other queen pupae had a hole in their gaster, from which parasitoids had previously emerged (Fig.  1).
The wasp was identified as Blanchardiscus pollux Noyes (Chalcidoidea: Encyrtidae: Encyrtinae). Only 2 species of the genus Blanchardiscus de Santis are currently known: B. scutellaris De Santis and B. pollux. Blanchardiscus scutellaris was described from a female collected with sweep net upon vegetation in Tucuman, Argentina (De Santis 1964), and has been reported from Brazil (Noyes 1980), whereas B. pollux is known from both the male and the female and was collected from Costa Rica and Belize (Noyes 2004). The biology of both species was heretofore unknown and no host data were available for any of the two species.
According to our observations, B. pollux is a gregarious endoparasitoid of P. goeldii pupae. Wasp larvae pupate inside the ant host pupae, which are protected by a cocoon. Developing wasps were found in the dorsal anterior portion of the gaster of the ant host, near the petiole. They were grouped in a compact mass, and for the 15 nearly adult specimens whose sexual gender could be assessed, the wings were somewhat distorted. Only a small portion of the ant's gaster was occupied by the parasitoids: B. pollux adults measure less than 1 mm, whereas P. goeldii workers measure 9-12 mm. At emergence, adult wasps chew a hole (1 mm in diam), in the host cuticle and through the host cocoon. A single emergence hole was observed in the apical, ventral portion of the gaster, near the meconium (Fig. 1), in the 2 queen pupae from which wasps had already emerged. Both worker and queen pupae of P. goeldii were parasitized and both male and female wasps emerged from the same host. It is unknown whether more than a single generation occurs inside the ant nest or whether adults leave the host nest to mate and disperse, but the latter seems more likely because both males and females were collected outside ant nests (Noyes 2004). However, even if no free adult wasp was found within both P. goeldii nests, they might have passed unnoticed when nests were collected.

DISCUSSION
Encyrtid wasps are widespread and common throughout the world. The family currently includes more than 480 genera and 4500 species and is one of the most important chalcidoid families for biological control of insect pests (Noyes & Hayat 1994;Noyes 2000Noyes , 2012Trjapitzin 2008). Though a few species are egg predators (e.g. in Microterys), most encyrtids are endoparasitoids of insects and arachnids, including other hymenopteran parasitoids. About half of all encyrtids whose host is known are parasitic upon Coccoidea (Noyes 2012). Two subfamilies are recognized: the Tetracneminae with 111 genera and 848 species, and the Encyrtinae with 370 genera and 3700 species (Trjapitzin 1973a(Trjapitzin , 1973bNoyes 2012).
Numerous encyrtids have been recorded associated with ants belonging to 3 subfamilies (Dolichoderinae, Formicinae, and Myrmicinae). However, almost all these records referred to indirect associations with ants (i.e. interference, see Table 1) through primary parasitism of the trophobionts that ants tend (e.g. Bartlett 1961;Cudjoe et al. 1993;Hübner & Völkl 1996;González-Hernández et al. 1999;Barzman & Daane 2001;Mgocheki & Addison 2009). These associations are more numerous in Encyrtinae, with 27 species from 17 genera involved in 45 associations     with ants, than in Tetracneminae, with 15 species from 8 genera involved in 27 associations. However, such a difference in the number of reported associations likely reflects species richness within each subfamily, because there are more encyrtine than tetracmine species. Various encyrtid species reported in interference association with ants (Table 1) have a very wide primary host range, and some are even hyperparasitoids: e.g. Prionomitus mitratus is parasitic on a fairly wide range of psyllids, Prochiloneurus pulchellus is a hyperparasitoid of many species of mealybugs, and Syrphophagus aphidivorus is a hyperparasitoid of virtually any aphid that feeds on grasses or herbaceous vegetation. Likewise, in numerous genera (Aenasius, Anagyrus, Anicetus, Coccidoxenoides, Comperiella, Leptomastix, Ooencyrtus, Prochiloneurus, Syrphophagus, see Table 1), the indirect association with the ant is not specific and can even involve species from different ant subfamilies. However, in some genera, e.g. Ananusia, Encyrtus, Holcencyrtus, Metaphycus, Microterys, Prionomitus, and Tetracnemoidea (Table 1), more specific interferences with ants-at species or, at least, at subfamily level-can occur and could suggest some level of selection of the ant associates by the parasitoids.
A few cases of assumed direct associations involving encyrtids and ants have been reported (Table 1), but true primary parasitism has never been demonstrated before. An unidentified species of encyrtid was recorded from a refuse heap of Eciton burchellii (Westwood) (Rettenmeyer et al. 2011), but direct interaction with this ant host has not been observed and the encyrtid may have only been a prey. The 2 females of Taftia prodeniae Ashmead found by Roepke (1919) clanging to the antennae of Dolichoderus thoracicus (Fr. Smith) (referred to as D. bituberculatus Mayr) may have been phoretic rather than parasitic. Only Holcencyrtus wheeleri (Ashmead) (referred to as Pheidoloxenus wheeleri), found in nests of the myrmicine ants Pheidole tepicana Pergande (referred to as P. instabilis) (Wheeler 1907) and P. ceres Wheeler (referred to as P. ceres var. tepaneca Wheeler) (Mann 1914), seems to have symphilic relationships with its hosts and has been suspected of being "probably also entoparasitic on these ants or their progeny during its larval stages" (Wheeler 1910). Wheeler (1907) stated that this "exquisite little Chalcidid . . . runs about in the dense throng of Pheidole workers like one of their number. It is not easily detected, as it resembles the workers in its small size (1 mm) and in being subapterous or practically wingless". In some occasions as many as 6 or 8 H. wheeleri have been observed in a single nest of P. tepicana (Wheeler 1907 Noyes & Hayat 1994;Noyes 2012) that are often associated with ants, and therefore association with ants might be fortuitous. Nevertheless, in the case of H. wheeleri, considering that it seems to be a regular myrmecophile in P. tepicana nests, that adults present subapterism, and that ant hosts are not associated with Aphididae or Coccidae (Wheeler 1907), such an association is unlikely to be only circumstancial; however, a primary parasitic relationship was never proved and the wasp developmental stages remained unknown. Therefore, our record of B. pollux from French Guiana reared from pupae of the neotropical ant P. goeldii constitutes both the first record of primary parasitism of ants for the Encyrtidae and the first case associating an encyrtid species with a ponerine ant. Our finding increases to 9 the number of parasitic wasp families that attack ants. As hypothesized by Huggert and Masner (1983) and Hanson et al. (1995), a possible evolutionary path to the parasitism of ants by hymenopterous parasitoids could have been through the occurence of a shift from the initial primary host-an ant symbiont-to the ant host through a gradual process of association and integration with the ant hosts. Such a hypothesis seems plausible for numerous families , and a supporting example has recently been proposed among eulophids for 2 Horismenus species associated with the weaver ant Camponotus sp. near textor (Hansson et al. 2011). Phylogenetically, Blanchardiscus is very probably near genera that include species known to parasitize scale-insects. Our record of B. pollux parasitizing ants may also support this hypothesis.
Little is known about the diversity of parasitoids of ants in general, though knowledge on this topic has significantly increased in the last two decades . Likewise, the hosts of most parasitic hymenopteran species already described are still unknown, especially those of rare or rarely collected species. Several recent reports (Hansson et al. 2011;Gates & Pérez-Lachaud 2012) have called attention to the diversity of parasitoids that attack ants, particularly in the case of arboreal ant species. For example, hymenopterous myrmecophiles associated with the neotropical weaver ant Camponotus sp. near textor, another arboreal ant, include 2 species of Eucharitidae, 2 of Eulophidae, and 1 of Eurytomidae (Hansson et al. 2011;Gates & Pérez-Lachaud 2012;Pérez-Lachaud & Lachaud, unpublished data). Those findings and the present record of a new family of parasitic wasps attacking ants inhabiting ant-gardens strongly suggest that arboreal ant nests may constitute a hot spot of diversity that has little been studied. As highlighted by Schmid-Hempel (1998) and , parasitic wasps associated with ants as the primary host are diverse, but most associations still await discovery.