Infection of army ant pupae by two new parasitoid mites (Mesostigmata: Uropodina)

A great variety of parasites and parasitoids exploit ant societies. Among them are the Mesostigmata mites, a particularly common and diverse group of ant-associated arthropods. While parasitism is ubiquitous in Mesostigmata, parasitoidism has only been described in the genus Macrodinychus. Yet information about the basic biology of most Macrodinychus species is lacking. Out of 24 formally described species, information about basic life-history traits is only available for three species. Here we formally describe two new Macrodinychus species, i.e. Macrodinychus hilpertae and Macrodinychus derbyensis. In both species, immature stages developed as ecto-parasitoids on ant pupae of the South-East Asian army ant Leptogenys distinguenda. By piercing the developing ant with their chelicera, the mites apparently suck ant hemolymph, ultimately killing host individuals. We compare infection rates among all studied Macrodinychus species and discuss possible host countermeasures against parasitoidism. The cryptic lifestyle of living inside ant nests has certainly hampered the scientific discovery of Macrodinychus mites and we expect that many more macrodinychid species await scientific discovery and description.


BACKGROUND
In 1982 David H. Kistner published an influential book chapter with the title ''The Social Insects' Bestiary'' (Kistner, 1982), a metaphor referring to the many thousand arthropod species exploiting social insect societies (Kistner, 1979;Kistner, 1982;Hölldobler & Wilson, 1990). Among them are such diverse groups as beetles, flies, wasps, ants, millipedes, silverfish, and mites (Donisthorpe, 1927;Rettenmeyer, 1961;Kistner, 1979;Kistner, 1982;Hölldobler & Wilson, 1990;Buschinger, 2009;Parker, 2016). The latter are particularly abundant guests of social insect colonies (Kistner, 1982;Eickwort, 1990;Gotwald Jr, 1996). The mite order Mesostigmata is notable in this respect because 20 out of 109 of its families are considered to have some kind of relationship with ants (Walter & Proctor, 1999; When Hirschmann wrote these lines, his hypothesis was speculative and lacked solid evidence. For most Macrodinychus species we still lack information about their basic biology including possible symbiosis with ants. Today it is known that about one third of the Macrodinychus species are indeed associated with ants, with three definite examples of parasitoidism (Lachaud, Klompen & Pérez-Lachaud, 2016). In the present study, we provide further support for Hirschmann's hypothesis by adding two additional species to the list of Macrodinychus parasitoids. We formally describe and provide life history information for two hitherto undescribed Macrodinychus species, Macrodinychus hilpertae Brückner, Klompen & von Beeren sp. nov. and Macrodinychus derbyensis Brückner, Klompen & von Beeren sp. nov. Both species were collected from colonies of the South-East Asian army ant Leptogenys distinguenda. Like other Macrodinychus parasitoids, the entire juvenile development of the new species took place as ecto-parasitoids on host pupae, ultimately killing the host individuals.

Collection and specimen depository
Two Macrodinychus (Mesostigmata: Uropodina) species were discovered during a project aiming to uncover the interactions of the army ant Leptogenys distinguenda and its diverse myrmecophile fauna (Witte et al., 1999;Witte et al., 2002;Kistner, Witte & Maschwitz, 2003;Witte et al., 2008;Maruyama, von Beeren & Hashim, 2010;Maruyama, von Beeren & Witte, 2010;Mendes, von Beeren & Witte, 2011;Ott et al., 2015). The mites were initially hidden, enclosed in ant pupal cocoons, and collection took place incidentally by collecting ant pupae (Fig. 1). The latter were collected during army ant colony emigrations using aspirators and forceps (for more information see von Beeren et al., 2011b). Collection took place in Malaysia, primarily at the Ulu Gombak Field Studies Centre of the University Malaya (latitude: 3.325, longitude: 101.750, elevation: 260) and additionally at the Biodiversity Institute Bukit Rengit (latitude: 3.596,longitude: 102.182,elevation: 72), between April and May 2008, August and September 2008, February and March 2009, August and September 2009, February and March 2010, and March and April 2011. The specimens were stored in absolute ethanol and deposited at the Ohio State University Acarology Collection, Columbus, Ohio, USA (OSAL). Macrodinychid mites are vouchered together with their respective ant pupa. Further specimens are deposited at the Adam Mickiewicz University in Poznań (three specimens of M. hilpertae labeled as ''Tank mite'' and two specimens of M. derbyensis labeled as ' 'Smooth shell''). Borrowing the latter specimens for morphological analysis was not possible in a reasonable time frame, because of an entire re-organisation of the department's mite collection. All other specimens have been lost during several institutional moves of one of the authors (CvB).
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:84ADDB13-56F3-431D-9244-E19C3A2F7E04. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Prevalence of mites
To evaluate mite prevalence and screen for different development stages we gently opened a total of 2,360 L. distinguenda pupal cocoons from a total of six different colonies. Since many adult specimens found during these dissections have been lost, we were not able to reliably identify all Macrodinychus specimens to the species level. As a consequence of this, we could not determine the prevalence for each of the two species separately, but instead evaluated the overall parasitism rate among Macrodinychus spp., i.e., the total number of pupal infections by Macrodinychus mites. In addition, after an initial screening of 1,391 pupal cocoons for adult Macrodinychus mites in 2009 and 2010, all L. distinguenda pupae from different colonies were combined in 2012 for storage at the LMU Munich. Therefore, we only have limited data about the colony of origin of Macrodinychus mites. For three additional L. distinguenda colonies we estimated the total number of pupae allowing us to estimate the number of pupal infections per colony. For this, Leptogenys bivouac sites were marked with tape and checked every 30 min for ongoing colony emigrations. Upon the start of an emigration, defined as workers carrying larvae or pupae to the new nest site, the number of Leptogenys workers carrying pupae and heading toward the new bivouac site was repeatedly counted for 30 s, followed by a 150 s break till the emigration was finished. We did not collect pupae for dissections from these colonies.

Morphological protocol and imaging
Specimens were dissected and slide mounted in Hoyer's medium or lactophenol (Walter & Krantz, 2009) and studied with bright-field, differential interference contrast and phase contrast microcopy. Morphological structures were drawn based on images taken during the phase contrast microscopy. In addition, focus-stacked images were taken with a Keyence VHX-5000 digital microscope (Keyence Deutschland GmbH, Neu-Isenburg, Germany) using the VH-Z50L lens. All measurements were taken using internal scale function as implemented in the Keyence system software (version 1.5.1.1; system version 1.0.4). A total of 37 images were uploaded to the Barcode of Life Database. Images can be accessed using the sample ID (provided in results) as search term. Images of all immature stages can be found on BOLD (search using the sample ID). Holotype label information is listed verbatim, with the different labels separated by forward slashes.

Observations in laboratory nests
Interactions between host ants and adult Macrodinychus specimens were studied in laboratory nests containing 110-170 ant workers, 44-55 ant pupae, 22-30 callows (freshly hatched workers) and three to six clusters of ant larvae. Behavioral tests were carried out with workers of the myrmecophile's colony of origin. Details about the nest set-ups were described previously (von Beeren et al., 2011a). Myrmecophiles were tested individually. Frequently, myrmecophiles behaved excitedly for a short period after transferring them to laboratory nests, which sometimes initiated ant aggression. To avoid biases caused by the specimen transfer we gave myrmecophiles two minutes settling time before recording the ant behaviors. We then observed the interactions of the myrmecophile in the first 50 consecutive encounters with host ant workers (for definition of behavioral categories see Table S1). At the study time, we did not realize that there are two different Macrodinychus species and therefore the data presented here cannot be assigned to the species level. Nonetheless, we consider the behavioral data as valuable because behavioral interactions with host ants have not been studied systematically for any Macrodinychus species. To compare the host-symbiont interactions of Macrodinychus spp. with those of other L. distinguenda myrmecophiles, we additionally tested the following associates: the silverfish Malayatelura ponerophila (Mendes, von Beeren & Witte 2011) the spider Sicariomorpha maschwitzi (Wunderlich, 1994), the snail Allopeas myrmecophilos (Janssen & Witte, 2002), and the rove beetles Maschwitzia ulrichi (Kistner, 1989), Witteia dentrilabrum (Maruyama, von Beeren & Hashim, 2010), and Togpelenys gigantea (Kistner, 1989). Data on rove beetles were published previously (von Beeren et al., 2011a).

Data analysis
Behavioral counts were expressed as compositional data (%) by standardizing for the total number of interactions per specimen (approx. 50 interactions per specimen: mean ± SD = 50.83 ± 3.20 interactions, N = 97). These multivariate data were analyzed with a permutational analysis of variance (PERMANOVA) with 9,999 permutations based on Bray-Curtis similarities. Due to the rareness of certain associates, some specimens were tested multiple times (Table S2). This was considered in the PERMANOVA design (Myrmecophile species = fixed factor; Specimen ID = random factor). In addition to the multivariate analysis of behavioral interactions, we calculated an aggression index (AI in (%)) to measure the total aggression of ants towards a focal myrmecophile. For this, the sum of aggressive behaviors (chased, snapped, stung) was divided through the total number of interactions. We applied PERMANOVA for univariate cases based on Euclidean distances with the same design as described above. PERMANOVAs were run with the software Primer 7 (Primer-E Ltd., Ivybridge, UK, vers. 7.0.12) with the add-on PERMANOVA+1 (Anderson, Gorley & Clarke, 2008).

Macrodinychus (Monomacrodinychus) derbyensis Brückner, Klompen & von
Descriptions of immature morphology of Macrodinychus are hitherto really limited. Comparing the two species described here, the deutonymphs of M. derbyensis differs from those of M. hilpertae in relative length of dorsal and ventral setae (especially relative lengths of St4 vs. St1-3), lack of ornamentation of the dorsum, and presence (vs. absence) of seta pd1 on tibia IV.

RESULTS-LIFE HISTORY SECTION Host infection rate
Out of 2,360 inspected L. distinguenda pupae 40 were infected with one of the two Macrodinychus species, i.e., the pupal infection rate at Ulu Gombak was 1.69%. Each pupa was only infected by a single Macrodinychus specimen. The inspection of host pupae from a single colony in 2009 demonstrated that Macrodinychus species can co-occur in the same colony (M. hilpertae = 2 infected pupae; M. derbyensis = 4 infected pupae).
The pupal number per colony was estimated for three different L. distinguenda colonies: 6,456 pupae, 5,845 pupae, and 9,846 pupae. With an infection rate of 1.69%, the total number of pupal infection per colony was estimated to be 109, 99, and 166, respectively.

Life-history of M. hilpertae and M. derbyensis
The dissection of 2,360 ant pupae recovered 20 immature and 20 adult mite stages. The following two immature development stages were found: three protonymphs (M. derbyensis, N = 2; M. hilpertae, N = 1) and 17 deutonymphs (M. derbyensis, N = 7; M. hilpertae, N = 10). All parasitized ant pupae had small, brownish scars (Fig. 4), which were not present in unparasitized ant pupae. Adult mites and deutonymphs that were detached from ant pupae left behind a conspicuous abnormal cavity in pupal bodies (Fig. 4). We did not find Macrodinychus larvae. However, we detected larval exuviae of three M. hilpertae individuals (Fig. 4). Exuviae of proto-and deutonymph were frequently detected inside pupal cocoons, often still attached to the mite or to the ant specimen (e.g., see BOLD image of sample 'cvb757macro002').

Life-history of macrodinychid mites
Adopting Kistner's metaphor of a social insect bestiary, the two herein described Macrodinychus parasitoids are extraordinary examples of specialized beasts invading ant colonies. Both species fulfill their immature development inside army ant colonies, which constitutes a stable and predator-free space with sufficient supply of food (Kistner, 1979;Hölldobler & Wilson, 1990;Hughes, Pierce & Boomsma, 2008). More specifically, Macrodinychus immatures were attached to and most likely fed on defenseless ant pupae.
While we do not provide direct evidence here that M. hilpertae and M. derbyensis were feeding on the host's hemolymph or tissue, this seems to be the most parsimonious explanation to us. First, parasitized pupae possessed scars which can be interpreted as feedings marks. We consider it most likely that scars represent areas where mites used their chelicera to pinch the ant's cuticle in order to feed on host tissue and/or drink from the excreting hemolymph. Second, we found exuviae of different development stages inside individual pupal cocoons, indicating that the mites grew by feeding on the ant pupae. Consumption of host tissue/hemolymph is also indicated by the fact that detached mites left behind physical impressions constituting substantial parts of the ants' gasters.
In ant-associated Mesostigmata, parasitoidism has only been described in the genus Macrodinychus (Lachaud, Klompen & Pérez-Lachaud, 2016). The five more extensively studied Marodinychus species (including M. hilpertae and M. derbyensis) seem to share the following key life-history traits (González, Gómez & Mesa, 2004;Breton, Takaku & Tsuji, 2006;Krantz, Gómez & González, 2007;Lachaud, Klompen & Pérez-Lachaud, 2016): all species seem to fulfill their entire immature development, including larval, proto-and deutonymphal stage, by feeding on individual ant pupa. For this, they seem to pierce the pupal cuticle with their chelicera to consume host tissue and/or to suck host hemolymph leaving behind small, brownish feeding marks. The larvae have well-developed legs and hence seem to be the mobile instar to find suitable hosts, while proto-and deutonymphs are more likely immobile feeding instars. Macrodinychus adults finally occupy a substantial part of the pupa's body. Once removed from the ant, they leave behind a conspicuous cavity, providing visual evidence for a lethal feeding strategy.

Parasitism rates of macrodinychid mites-native versus invasive host ants
Besides similarities among species, we also detected a notable difference between the Macrodinychus species studied previously and those studied here. The prevalence of infection, measured as the percentage of infected to non-infected host pupae, was markedly lower in M. hilpertae and M. derbyensis (approx. 2% vs. 15%-90% in other Macrodinychus species; see González, Gómez & Mesa, 2004;Breton, Takaku & Tsuji, 2006;Krantz, Gómez & González, 2007;Lachaud, Klompen & Pérez-Lachaud, 2016). Various explanations could be responsible for the vastly different parasitism rates among studied macrodinychid mites. For example, the particular sampling methods or seasonal and spatial differences in parasitoid prevalence could conceivably cause such variation. Another possible cause is that Macrodinychus hilpertae and M. derbyensis have been studied in a native host-parasitoid system, while other Macrodinychus spp. have exclusively been studied in association with invasive ant species. Parasitoids are often a major source of host mortality and intense selection on the host to evolve counter-defenses against parasitoid attacks can be expected (Godfray, 1994). In species drifts across continents, however, local and alien interaction partners have no coevolutionary history (Thompson, 2005;Simberloff, 2013).

Possible counter-adaptations against macrodinychid mites
Hidden inside the pupal silk cocoons, the immature mites studied here are practically invisible to adult host workers. In contrast, once eclosed from the pupal cocoon, adult mites are exposed and thus are accessible for host inspection. Similar to socially integrated species such as the spider Sicariomorpha maschwitzi (Witte et al., 2009;von Beeren, Hashim & Witte, 2012) and the silverfish Malayatelura ponerophila (Witte et al., 2009;von Beeren et al., 2011b), adult Macrodinychus spp. were mostly ignored or unnoticed by host ants. Nonetheless, host workers regularly antennated adult parasitoids and ultimately attacked them, although at a relatively low level. Low levels of aggression towards myrmecophiles are still biologically relevant. For instance, soft bodied myrmecophiles such as the silverfish M. ponerophila were occasionally killed in behavioral assays (Witte et al., 2008;von Beeren et al., 2011b). We interpret the occasional attacks towards Macrodinychus mites as a behavior to fight off the adult parasitoid before host brood become infected with parasitoid larvae. However, Macrodinychus specimens survived these attacks unscathed owing to their protective morphology, embodied by a well-sclerotized cuticle and the possibility to retract all extremities into cuticular cavities (pedofossae) (see Figs. 2 and 3). A more efficient host defense might be the ants' behavior following the initial attacks. Macrodinychus spp. were often picked up by workers in laboratory nests and dumped at the ants' refuse site, outside the inner nest part where the parasitoid target, i.e., ant brood, is located. The adult mites were mobile and regularly re-entered the brood chambers in laboratory nests, only to be picked up and dumped at the refuse site again. In addition to this, the frequent emigrations of army ants might represent another counter-measurement to reduce a colony's total fitness loss imposed by parasites and parasitoids (Witte et al., 2008;von Beeren et al., 2011a) because parasites/parasitoids can be left behind at the abandoned nest site (Witte, 2001). Support for this hypothesis comes from an observation during a nest emigration of Leptogenys distinguenda initiated in the laboratory at the field site. We collected three Macrodinychus spp. adults at the abandoned nest site (a 1 m ×1 m ×1 m plastic box filled with leaf litter), in other words, the emigrated colony shed off these parasitoids.