Rediscovery and reclassification of the dipteran taxon Nothomicrodon Wheeler, an exclusive endoparasitoid of gyne ant larvae

The myrmecophile larva of the dipteran taxon Nothomicrodon Wheeler is rediscovered, almost a century after its original description and unique report. The systematic position of this dipteran has remained enigmatic due to the absence of reared imagos to confirm indentity. We also failed to rear imagos, but we scrutinized entire nests of the Brazilian arboreal dolichoderine ant Azteca chartifex which, combined with morphological and molecular studies, enabled us to establish beyond doubt that Nothomicrodon belongs to the Phoridae (Insecta: Diptera), not the Syrphidae where it was first placed, and that the species we studied is an endoparasitoid of the larvae of A. chartifex, exclusively attacking sexual female (gyne) larvae. Northomicrodon parasitism can exert high fitness costs to a host colony. Our discovery adds one more case to the growing number of phorid taxa known to parasitize ant larvae and suggests that many others remain to be discovered. Our findings and literature review confirm that the Phoridae is the only taxon known that parasitizes both adults and the immature stages of different castes of ants, thus threatening ants on all fronts.

Almost a century ago the enigmatic taxon Nothomicrodon aztecarum (Wheeler, 1924) was raised on the basis of morphologically unusual larvae found among the brood of a carton nest ant, Azteca trigona (Dolichoderinae), from Barro Colorado Island, Panama 23 . No adult N. aztecarum were obtained and due to the similarity in larval stages, Wheeler placed Nothomicrodon in the Syrphidae as an ally of the genus Microdon whose larvae are well known predators of ant brood 24 . The true affinity of Nothomicrodon has remained unresolved because adults have not been reared and larvae have not been re-encountered since its original description. Historically, the genus has been treated as incertae sedis by syrphid experts. Cheng & Thompson 25 stated that the larva has none of the characteristics of microdontine larvae (flattened creeping sole, convex dorsal surface) and based on a suggestion from G.E. Rotheray, speculated that it might belong to the Phoridae. The most up-to-date revision of Microdontinae also treated Nothomicrodon as an unplaced taxon 26 .
In this paper we report on the rediscovery of Nothomicrodon. Scrutiny of entire nests of Azteca chartifex collected in Brazil combined with morphological and molecular studies enabled us to establish beyond doubt that these larvae belong to the Phoridae and that they were endoparasitoids of A. chartifex larvae, more specifically of the sexual female (gyne) larvae. Based on these data and a literature review of phorid parasitoids attacking social insect brood, we confirm that the Phoridae is the only insect family known with species that parasitize both the adults and immature stages of their ant hosts, thereby threatening ants on all fronts.

Results
DNA sequencing and identification. The obtained COIa fragment comprised 653 bp and the COIb 780 bp. The top 20 closest matches of the COIa sequence identification on BOLD were all Phoridae samples (except one Agromyzidae (Diptera: Opomyzoidea) sample). The highest similarity, 88.7%, was found with an unpublished Phoridae sample. Similarities of 88.5% were found with published barcodes of two phorid flies from USA and Canada (BIN BOLD: AAM9347 and BOLD: AAN8679), both from the genus Megaselia. Blasting the COIb fragment in GenBank (www.ncbi.nih.gov, on 7 March, 2016) returned a long list of Phoridae samples as closest matches. Sequence similarity of 84% was reported for a sample of the phorids Anevrina variabilis (GenBank accession number GU559934) and Dohrniphora cornuta (HM352592). Sequence similarity of 83% was reported for two samples of the phorid Apocephalus paraponerae (AF217478-9) which is a known ant parasitoid 27 . No syrphid fly species appeared in the top 20 closest matches for both COIa and COIb sequence identification. Moreover, the neighbor-joining and maximum likelihood analyses placed the Nothomicrodon sequence among all included samples of other Phoridae (Fig. 1, Table S1), herewith confirming the identification of the sample as a phorid fly, not a syrphid.
Description of Nothomicrodon third instar larva (n = 2). Overall appearance. Pear-shaped with a broad, oval-shaped abdomen and a narrow, tapering thorax; pale to brown with a coriaceous integument ( Fig. 2A); abdomen smooth except for a single pair of deep infolds across the dorsum (see Fig. S1A); head skeleton with the apex of the labium external to the fleshy pseudocephalon and comprising a pair of downwardly projecting, black, heavily sclerotized hooks, rest of the head skeleton translucent (see Fig. S2A), poorly sclerotized and lacking cibarial ridges.
Description. Length about 4.5 mm (1.5 mm pseudocephalon and thorax + 3 mm abdomen), abdomen 3.5 mm wide and maximum height about 0.75 mm; width of the metathorax where it joins the broader abdomen, about a quarter the maximum width of the abdomen and at the prothoracic apex, about a fifth the width of the base of the metathorax (see Fig. S1B,C); antennae on the dorso-lateral margins of the pseudocephalon, just posterior to the apex, appearing as a pair of cylindrical, tapering structures (see Fig. S2B), maxillary palpi not recognizable in the specimens examined; ventrally, apex of pseudocephalon with a pair of inwardly directed, flange-like, cuticular projections (see Fig. S1D); pseudocephalon and thorax retractile, as revealed by folds and creases along which the integument probably collapses and/or retracts, by analogy with other larvae 28 ; probable margin between the pseudocephalon and prothorax indicated by a deep infold; prothorax elongate, about twice as long as each of the pseudocephalon, mesothorax and metathorax which are all of a similar length (see Fig. S1C); towards the rear of the prothorax on the dorso-lateral margins, are the anterior respiratory processes comprising a pair of cylindrical projections inclined forward and with the spiracles across the apex (see Fig. S1C); metathorax attached to a firm infold at the apex of the first abdominal segment by a band of thin, flexible integument; lateral and posterior margins of the abdomen pinched and with a slight, continuous beading; externally segments marked only by segmental pattern of inconspicuous sensilla, each accompanied by a single hair-like seta, abdomen otherwise unmarked except for the third abdominal segment whose boundaries with adjacent segments are marked by deep infolds across the dorsum (see Fig. S1A); anal segment crescent shaped, as revealed by the pattern of sensilla round the slight, dome-shaped posterior respiratory process; this process with four pairs of short, parallel spiracles orientated dorso-ventrally, above which are a pair of cuticular scars, the paired spiracular plates separated mid-apically by a longitudinal, slit-like infold (see Fig. S2C); entire body coriaceous, locomotory organs apparently absent; head skeleton (see Fig. S2A) 0.5 mm long, form typical for a member of the Platypezoidea 28 ; sclerotization slight except for the black, sclerotized apex to the ventral, labial arm which projects externally from the apex of the fleshy pseudocephalon in the form of a pair of stout, downwardly projecting hooks; apex of labrum and mandibles tapered, inconspicuous and insignificant relative to the much larger labial hooks; ventral and dorsal cornua elongate and parallel, not diverging; ventral cornu slightly broader than dorsal cornu; cibarial ridges absent. Life cycle and developmental stages of Nothomicrodon. Parasitized A. chartifex larvae, in both early and advanced stages of parasitoid development, can be identified by the small, oval, melanized/sclerotized scar from which the posterior respiratory process of the parasitoid projects from the host cuticle (Fig. 3A). Advanced stages of development (third instar Nothomicrodon larvae) are easily observed through the host integument ( Fig. 2B,C). Breathing holes may be located on any part of the ant larva including the head. The hole is round-oval and measures 0.07 mm in diameter on average (n = 7); its rim is raised and heavily sclerotized. Upon host dissection, eggs were found firmly attached to the host cuticle ( Fig. 3B,C, n = 2 cases, Table 1). Eggs are elliptical in form with the apical portion more acute than the base. One egg was measured: length = 1.0 mm, base = 0.37 mm and apical portion = 0.19 mm. All developmental stages of Nothomicrodon remained attached by the posterior respiratory process to the host cuticle. As with other phorid species 29 , Nothomicrodon larva has three instars. The first and second are of a whitish color and the cuticle is not sclerotized (Figs 3D and 4A). First/ second instar Nothomicrodon larvae dissected from the host measured 1.34 ± 0.09 mm (mean ± SD) in width and 1.88 ± 0.11 mm in length (n = 4). Three of these larvae had the pseudocephalic region extended, length 0.56 ± 0.12 mm. Early third instar larvae are yellow in color (Fig. 4B) and the cuticle has already the leathery aspect of the fully grown, reddish-dark brown third instars (see Fig. 2A). After feeding is completed, third instar larvae cut open the host cuticle with their labial hooks (Fig. 4C). These larvae measure 3.02 ± 0.25 mm in width and 3.51 ± 0.14 mm in length (mean ± SD; n = 8).
Host caste and developmental stage targeted. Azteca chartifex larvae are oval in form and practically hairless; the mouthparts are small, the mandibles are feebly sclerotized and, as in other dolichoderine taxa, mobility is almost lost 30 . Gyne larvae of the Dolichoderinae subfamily are much larger than worker larvae 30 . The length and width of a random sample of larvae of varying sizes were obtained (n = 212). The MDA model discriminated parasitized from unparasitized larvae according to their attributes, with parasitized larvae exclusively in the larger size class, which corresponds to gyne larvae (Fig. 5, see Fig. S3). The model explained 89 and 100% of the between group variance of the variables, and correctly assigned most of the larvae (deviance 19.8, misclassification error 0.94%). Only one parasitized and one unparasitized larvae were not correctly assigned. Parasitized larvae measured on average 3.4 ± 0.3 mm in width and 4.7 ± 0.5 mm in length (mean ± SD; n = 25); unparasitized larvae measured on average 1.5 ± 0.4 mm in width and 2.1 ± 0.6 mm in length (n = 187). Nothomicrodon was found only in the nests that contained gyne larvae: no small or fully-grown minor or major worker larvae or male larvae were parasitized. Gyne parasitism rates. Samples from three nests collected in 2012 containing a total of 1,328 adults (gynes and workers) and 1,329 larvae and pupae were examined (Table 1). All three samples contained parasitized A. chartifex gyne larvae and/or free wandering Nothomicrodon larvae (Table 1). In general only one Nothomicrodon larva develops per host and in only one occasion two parasitoid larvae were observed inside the same host larva (Fig. 4D). A single Nothomicrodon puparium was examined; it was more elliptical in body shape than the larva and seemed to have contracted. This puparium had been parasitized and the parasitoid(s) had emerged and gone as revealed by an emergence hole on its surface (see Fig. S4). Rates of Nothomicrodon parasitism for the 2012 samples were calculated as the proportion of parasitized gyne larvae with respect to the total number of examined larvae of this caste (in brackets are the corrected values that take into account wandering Nothomicrodon larvae and a puparium). Rates were as follows: sample 1: 54.2% (55.6%), sample 2: 0% (30.8%), sample 3: 100%, with an overall proportion of parasitized gyne larvae of 53.9% (57.9%). Larvae from the 2015 samples were not dissected, and estimated gyne parasitism rates were far lower (Table S2), varying from 3.5 to 75.0% with an overall proportion of parasitized gyne larvae of 8.2% (8.6%).

Discussion
In this study we resolve the long-standing enigma of the taxonomic placement of Wheeler's Nothmicrodon. Morphological and molecular data reveal that the genus belongs to the Phoridae rather than the Syrphidae where Wheeler 23 had placed it. Furthermore, our data show that these extraordinary myrmecophilous larvae develop as endoparasitoids of A. chartifex larvae, and are specific in only developing on the fully-grown gyne larvae. The larva we studied shares numerous features with that of N. aztecarum 23 and both molecular and morphological evidence support placement within the Phoridae. For example, the larval head skeleton is of a platypezoid, not a syrphoid form. Specifically, the apex of the head skeleton is the ventral labial arm which is in the form of a pair of large, sclerotized hooks projecting from the pseudocephalon which are the main food gathering structures in platypezoid larvae 28 . The DNA sequences identities and the phylogenetic analyses unambiguously show that the larva belongs to the Phoridae. With a rate of similarity of 83 to 88.5%, our sequences, however, are not closely related to any species represented by mtDNA COI sequences in the public sequence databases, and the adult stage remains to be obtained or assessed.
Species boundaries between members of the host genus Azteca are not well understood. Azteca chartifex belongs to the A. trigona group from which the type material of Nothomicrodon (N. aztecarum) was obtained. It remains possible therefore, that the same species of Nothomicrodon is associated with both A. chartifex and A. trigona.
Phorids are a group of small to minute flies comprising ca. 4,300 recognized species but the more conservative estimates consider that this figure may represent only 10% or less of the total fauna when including undescribed species 29,31 . They exhibit an array of larval feeding modes, including obligatory and facultative saprophages, predators and parasitoids 29 . Phorids are parasitic on mollusks and arthropod taxa, such as arachnids, millipedes, and insects. They are well known natural enemies of pest ants 32 and adult honey bees 33 . Most phorid flies associated with ants live either as nest commensals 34 , or as parasitoids of foraging workers 19 and occasionally alate females 35 . Apart from parasitizing ants, phorids also attack other Aculeata, including bees, stingless bees and wasps 3 . Interestingly, most dipteran parasitoid species attacking social Hymenoptera parasitize the adult stage, although scattered records exist of phorids attacking the larvae of social Hymenoptera (see Tables S3 and S4). About 40% of these cases concern species which develop as ectoparasitoids of formicid and vespid larvae (see Table S3). Larval endoparasitism by phorids is almost exclusively associated with ants (see Table S4). While only two species of the phorid genus Aenigmatias (see Table S3 and references therein) are ectoparasitoids of ant larvae, a growing bulk of records now concerns ant larval endoparasitism by phorids (see Table S4 and references therein). The discovery, in this study, of a Nothomicrodon species that is an endoparasite of ant larvae hints that other instances of ant larval parasitism exist in phorids. Our results and literature search reveal that the Phoridae is the only family with parasitoid species that attack both adult ants and their broods with, in the case of Northomicrodon, a specialization for a specific brood caste, i.e. gyne larvae. Several parasitic wasps (Hymenoptera) also attack adult ants or their brood (larvae or pupae), however, this is achieved by distinct wasp families 12 .
Several morphological features appear to adapt the Nothomicrodon larva to a parasitic feeding mode. The labial hooks facilitate piercing, tearing and loosening fragments of host tissue which are then sucked up by the pump in the head skeleton, and guided towards it by the relatively immobile labrum and at either side of it, the tapered mandibles. The long, parallel dorsal and ventral cornua of the head skeleton suggest that it is covered in short, wide muscles. Such a characteristic delivers a shallow but strong pumping action 36 , which is typical of many zoophagous larvae 37 . Perhaps the most distinctive feature of the Nothomicrodon larva is its pear shape with an extremely broad, smooth and apparently inflexible abdomen, contrasting with a highly narrowed, tapered, flexible and retractile thorax. Such a shape is also known in larvae of another taxon of cyclorrhaphan endoparasitoids, the Conopidae, whose larvae attack principally the adults of aculeate Hymenoptera 38 . The pear shape in conopid larvae is adaptive in that the broad abdomen resides in the abdomen of the host while the narrow thorax reaches through the petiole into the thorax to feed on the high density of muscle tissue. The pear shape of the Nothomicrodon larva appears to be similarly adapted. Flexibility in the thorax probably facilitates reaching in and around the host body in order to gather food, and might also help egression from the host after completion of larval growth. The Nothomicrodon larva might well eat the host remains surrounding its body, as occurs in other parasitoids, such as the braconid wasp Toxoneuron bicolor (= Toxoneuron nigriceps). In this endoparasitoid, post-egression feeding enhances growth and survival 39 . However, on the basis of mouthpart structure, it would likely be difficult for the Northomicrodon larva to fragment the host remains, unlike the braconid which has chewing mandibles. In the Nothomicrodon larva, the absence of locomotory structures on the ventrum of the abdomen is possibly explained by the position of the larva inside a host where locomotion is not required. The relative size of the anterior respiratory processes is surprising given the position of the thorax inside the host. In  Aztc 017  0  0  1295  1170  96  1266  52  54.2  55.6  2 eggs, 13 L 1-2 , 12  early L 3 , 25 L 3  3   Aztc 032  0  0  0  0  9  9  0  0  30.8  3  1 parasitized  puparium   Aztc 033  7  44  26  0  10  10  10  100  100  10 L 3  4   Total  7  44  1321  1170  115  1285  62 53.9 57.9 62 10 1 Table 1. Azteca chartifex material examined for this study, gyne parasitism rate and number and developmental stage of Nothomicrodon. a Corrected to take into account the free wandering Nothomicrodon larvae and a puparium. contrast, the posterior respiratory process projects only slightly which is probably an adaptive shape in that it is less likely to become caught in host tissue. The flat ventral and slightly convex dorsal surfaces of the larva, together with their hard, leather-like cuticle, and the possibility of retraction of the pseudocephalon and thorax (the most fragile parts of the body) seem to be adaptations to live on the very hard and concave carton walls of the host nest chambers, and might well provide protection from aggressive worker ants.
Whether the Nothomicrodon female places its eggs near the host (or host habitat) and the fly larva actively seeks for its host, or lays eggs directly on an Azteca ant larva within the host nest remains to be assessed. Whatever, our results show that only the fully-grown gyne larvae of the ant host are targeted, and suggest that host size may be a limiting factor to Nothomicrodon larval development. Ant parasitoids impose variable fitness costs on both individuals and colonies 13,33,40,41 . For high rates of parasitism, parasitoids may significantly reduce resource intake, colony size, and colony fitness. By exclusively parasitizing gyne larvae, Nothomicrodon parasitism directly affects the reproductive success of the colony and thereby imposes a high fitness cost to A. chartifex. Other parasites and parasitoids impose high reproductive costs on their hosts as for example, in Nosema infections of bumble bees 42 . However, fitness costs are not inevitable; not all A. chartifex colonies we studied suffered high rates of Nothomicrodon parasitism.

Materials and Methods
Insect sampling and preparation. Azteca chartifex adults and brood, as well as Nothomicrodon larvae, were collected in the state of Bahia in Brazil in 2012 and 2015 (SI Text: Material and Methods). Azteca workers, larvae and pupae were examined under a stereomicroscope (Nikon SMZ745T) and dissected if parasitized (Table 1). Late instar Nothomicrodon larvae found in the nest galleries along with workers and ant brood were collected and examined. Ant larvae collected in 2015 were checked only externally, without dissection, and used essentially for estimating gyne parasitism rates (see Table S4). Nothomicrodon larvae and a subsample of A. chartifex larvae (including both parasitized and unparasitized larvae) were measured to the nearest 0.1 mm (width × length) using a stereomicroscope provided with an ocular micrometer. A Mixture Discriminant Analysis (MDA) model was fitted to the A. azteca data set to test for differences in size between parasitized and unparasitized larvae. MDA is effective for selecting the suitable subclass division of a data set (Gaussian mixture of subclasses) 43 . The statistical analysis was performed using the MDA package in R 44 . An overall gyne parasitism rate was calculated taking into account all of the potential host larvae examined, with a correction for parasitoid larvae/pupae found freely in the nest chambers. Nothomicrodon larvae preserved in alcohol were prepared for description by rehydration and then fixation in Kahle's solution 38 . To examine the larval head skeleton, the apex of the thorax of a preserved larva was cut off and soaked in hot KOH for about 5 minutes. Excess tissue was removed from the head skeleton and it was washed in acetic acid and stored in glycerol. It was examined using a Wild 5 stereo-microscope in a solid watch glass containing 70% ethanol. The drawing was made with a drawing tube attached to the microscope. Terminology generally follows Rotheray & Gilbert 28 . Stacked images were obtained using Helicon Focus© (Helicon Soft Ltd). Specimens were also critical point dried and sputter coated before observation with a SM-51 TOPCON Scanning Electron Microscope.
DNA sequencing, identification and clustering. Three second instar larvae of Nothomicrodon, obtained by dissection of the hosts, were used for molecular work (labelled Aztc 017B-I, Aztc 017B-II and Aztc 017B-III).
DNA was extracted from a small piece of tissue (0.5-1.0 mm sample) of the larvae using the Phire ™ Tissue Direct PCR master Mix #F-170S kit (Thermo Scientific Baltics UAB, Vilnius, Lithuania) following the Dilution & Storage protocol with some modifications (SI Text: Material and Methods).
The obtained COIa and COIb sequence fragments of our species of Nothomicrodon, referred as incertae sedis in Table S1, were individually blasted against the BOLD systems v3 (boldsystems.org, accessed 7 March, 2016) and the NCBI GenBank databases, respectively, using BLASTn for the sequence comparisons and identifications. Sequences produced in this study were deposited in the European Nucleotide Archive (http://www.ebi.ac.uk/ ena), accession numbers LT592267 (COIa) and LT592268 (COIb).
We additionally used a dataset of COIb sequences retrieved from GenBank with the aim to test the placement of our species of Nothomicrodon among samples of the closely related cyclorrhaphan fly families using sequence clustering. The dataset comprised eight COIb sequences of Phoridae species, nine of Syrphidae, five of Platypezidae, four of Pipunculidae, one of Lonchopteridae, and used Sciadoceridae as outgroup (Table S1). The COIb sequence dataset comprised 764 nucleotides and was analyzed using Neighbor-Joining and Maximum Likelihood in software MEGA v.6 45 using the K2P and General Time Reversible 46 models of evolution, respectively.
Third instar larvae of our species were further compared to the description and figures of N. aztecarum in Wheeler 23 and to the images of the paratype in the Syrphidae Community Website http://syrphidae.myspecies. info/taxonomy/term/75. Voucher material of ants and parasitoids was deposited in the following collections: Centro de Pesquisa do Cacau at Ilhéus, Brazil (CPDC collection, CEPEC/CEPLAC) (10 host workers, five third instar Nothomicrodon larvae); El Colegio de la Frontera Sur at Chetumal, Mexico (Colección de Formicidae and Colección de Artrópodos) (10 host workers, three third instar Nothomicrodon larvae); the National Museums at Edinbugh, Scotland (three third and one second instar Nothomicrodon larvae); and the Finnish Museum of Natural History at Helsinki (Finland) (three second instar Nothomicrodon larvae, 2 host workers).