Adults of Ozaena lemoulti Bänninger, G. kuntzeni, and G. parca were collected in Walker Canyon, in the Parajito Mountains just north of the US-Mexico border in Arizona (Santa Cruz County, 31°22.819'N 111°03.994'W, 1214m). This is National Forest land, where no specific permissions are required for collecting insects by hand. Walker Canyon is a riparian oak woodland, with Emory oaks (Quercus emoryi Torrey) and Mexican blue oaks (Quercus oblongifolia Torrey), as well as species of mesquite and sycamore trees. During the summer rainy season, from July to September, Goniotropis kuntzeni (Fig 1B) are common in the canyon. At night the adults actively probe holes in the bark of Emory oak trees in search of prey. They are rarely, if ever, attracted to blacklights or mercury vapor lights and seem to be strictly associated Emory oaks as they have not been found on any other tree species in the canyon. Goniotropis parca (Fig 1C) are less common than its larger bodied sister species G. kuntzeni (Fig 1B) [33]. They also appear to be strictly associated with Emory oaks, however, they are also sometimes attracted to blacklights. Ozaena lemoulti (Fig 1E) adults are far less common. Over the past two decades, only six specimens of Ozaena lemoutli have been collected by WM in Arizona, three from Emory oaks, one from a sycamore tree, and two being captured at mercury vapor lights.

Over many years of collecting in Walker Canyon, one of us (WM) has noticed that ozaenines are only found associated with the largest Emory oak trees. Those old, but living, trees contain large dead portions, especially dead branches. Many of these trees are somewhat hollow in the middle of the trunk, and they have scars with holes on the bark left by fallen branches that appear to lead into the hollow center of the trees.

The large formicine ant, Camponotus ocreatus Emery, also frequents Emory oaks, and they are always found on the same large trees with ozaenines. We noted this pattern before [25], but explore it further here as Camponotus ocreatus is the most likely host ant of O. lemoulti larvae.

Southern Arizona populations of Ozaena lemoulti and Goniotropis kuntzeni, and G. parca represent the northernmost distribution of all Western Hemisphere Ozaenini, which is primarily a tropical group. These three species are all members of a small clade of Western Hemisphere ozaenines, which also includes Goniotropis morio Klug and the genus Platycerozaena (Fig 1D) [33].

All eggs, and entire first instar of G. parca, portions of the Ozaena larva (see below) and the adult antennae were prepared for SEM. Dried specimens were first rehydrated stepwise in a graded ethanol series to distilled water and cleaned by ultrasound for 15 seconds in a soapy water solution. All specimens were then dehydrated through a series of EtOH baths of increasing concentration (70-80-90-95-100%), critical point dried (Bal-Tec CPD 030), mounted on a stub (by using self-adhesive carbon disks), sputtered with gold (Emitech k550 sputter coater), and observed with FEI Dualbeam FIB/SEM Helios Nanolab (L.I.M.E. laboratory, University ‘Roma Tre’, Rome).

Prior to dissection, the single larva specimen of O. lemoulti was photographed and drawn using a stereomicroscope Olympus SZX16 equipped with drawing tube. The right antenna, right maxilla, right mandible, right hind leg, left dorsal and lateral plates of the terminal disk and left urogomphus were removed and prepared for scanning electron microscopy. The rest of the specimen was rehydrated, cleared in 10% KOH, transferred in hot lactic acid, dehydrated through a series of EtOH baths of increasing concentration (10-20-50-70-90-95-100%), left overnight in a clove oil bath, and mounted on a slide with Canada balsam. The slide mounted specimen was studied and illustrated by using a light microscope, Olympus BX51, equipped with drawing tube. The dissected parts of the same larva were prepared according to the procedure described above and imaged by FIB/SEM.

The general terminology of larval structures follows Lawrence [34]. Notation of primary setae and pores follows the system of Bousquet and Goulet [35], modified for Metrius contractus [17]. As some of the sensilla on the abdomen and terminal disk of O. lemoulti and G. parca are homologous to those recognized by Bousquet [17] in Metrius contractus (sensilla S-I to S-V), by Di Giulio et al. [36] in Pachyteles spp. (sensilla S-I to S-VII), and by Di Giulio and Moore [20] in Arthropterus sp. (sensilla S-I to S-VIII), we adopt the same nomenclature. Notation of microsculpture follows Harris [37]. An asterisk (*) following a coded seta indicates that we question the homology between the structure on the O. lemoulti and G. parca larvae.

Total genomic DNA was extracted following the ATL protocol in the Qiagen DNeasy kit (Valenica, CA). Gene fragments from cytochrome c oxidase subunit I (COI) and 28S ribosomal DNA (28S or 28S rDNA) were amplified using the Polymerase Chain Reaction (PCR) on an Eppendorf Mastercycler Thermal Cycler with Invitrogen Platinum Taq DNA Polymerase (Carlsbad, CA).

Sequences were amplified and sequenced using the COI primers LCO1490 and HCO2198. The 28S D2/D3 region was amplified with the forward primer ls58F or ls773F (5′CACGGACCAGGGAGTCTAGCAT-3′) and the reverse primer ls1066R or ls1126R (5′TCGGAAGGAACCAGCTACTA-3′). Amplified products were cleaned, quantified, normalized and sequenced at the University of Arizona’s Genomic and Technology Core Facility using an Applied Biosystems 3730 DNA Analyzer or a 3730 XL Applied Biosystems automatic sequencer. Simultaneous contig assembly and initial base calls were performed using the Phred [38] and Phrap [39] programs as implemented in Mesquite 3.2 [40] in combination with the Chromaseq vers 1.3 package [41]. Final base calls were made after manual inspection of individual sequences in Chromaseq; universal ambiguity, IUPAC, codes were used when multiple peaks were present at individual sites. Closest published sequences to those amplified and sequenced from the food were retrieved by performing Standard Nucleotide BLAST (blastn) searches using Megablast against GenBank (NCBI).

The egg of Ozaena (Fig 2A) is 1.6 mm long and 1.1 mm wide (measured at the equatorial region), and less elliptical than the egg of Goniotropis kuntzeni (Fig 2B). The external surface of both eggs is covered with a grid pattern (Fig 2A and 2B). The hexagonal grids of the Ozaena are smaller in diameter (about 15 μm) (Fig 2C) than the more rounded grids of Goniotropis (about 30 μm in diameter) (Fig 2D). The Ozaena egg shows remnants of partially decayed follicular epithelium filling the hexagonal grids (Fig 2C). In cross section the external eggshell layer is thick (about 20 μm) and sub-oval or rhomboidal (Fig 2E) in Ozaena, but thin (about 10 μm) and rounded in Goniotropis (Fig 2F). While the internal layer of the Goniotropis eggshell (Fig 2F) is thick and spongy, containing air filled cavities (see also [25]) typical of other known ozaenine eggs (Type A sensu Kaupp et al. [32]), the internal layer of the Ozaena eggshell (Fig 2E) is thin and imperforate (similar to Type B sensu Kaupp et al. [32]).

Descriptions of the first instar larvae of Ozaena lemoulti and Goniotropis parca are provided in S1 Appendix and comparatively illustrated (Figs 3–8).

The main differences between the anatomy of the O. lemoulti larva and those of its close relatives and known burrow trappers, G. parca and G. kuntzeni are as follows.

1. The total body length, measured from tip of mandibles to apex of terminal disk, of O. lemoulti is 4.3 mm (Fig 3A and 3B), G. parca is 4.0 mm, and G. kuntzeni is 7.4 mm (Fig 3C and 3D).

2. The orientation of the head is prognathous in Ozaena but hyperprognathous in the Goniotropis species.

3. The Ozaena larva is completely blind, without stemmata (Figs 3A and 4A) whereas the larvae of Goniotropis have one stemma on each side of the parietale (Fig 3C).

4. The sensorial appendage on antennomere III is reduced, small and spherical in Ozaena (Fig 5A, arrow) but well-developed, elongate, conical, and apically pointed in Goniotropis spp. (Fig 5B, arrow).

5. The retinaculum of the mandible is slender, triangular, subapically directed in Ozaena (Fig 5E); but robust, hook-like, slightly curved inward in Goniotropis spp. (Fig 5D and 5F).

6. The lacinia is small, straight in Ozaena (Fig 5C, arrow) but well developed and hook-like in Goniotropis spp. (Fig 5D, arrow).

7. Galeomere II is short and slender in Ozaena (Fig 5C) but elongate and stout in Goniotropis spp. (Fig 5D).

8. Mesal stipital protuberance is obsolescent and positioned mesodorsally in Ozaena (Fig 5C, box), but it conspicuous, and positioned subbasally on inner side, and is directed forward in the Goniotropis spp. (Fig 5D, box).

9. The abdomen of Ozaena is extremely short and flat and is not contracted dorsally (Fig 3A and 3B); whereas the abdomen of the Goniotropis spp. is moderately short and flat, and contracted dorsally (upcurved) elevating terminal disk (Fig 3C and 3D).

10. The terminal disk of Ozaena is whitish, unsclerotized, and translucent (Fig 3A and 3B); but well sclerotized and yellowish in Goniotropis spp. (Fig 3C and 3D).

11. The terminal disk is much narrower (1.9 mm at level of lateral plates) in Ozaena (Fig 6A) than it is in the Goniotropis spp. 2.89 mm (Fig 6B).

12. The dorsal plates of the terminal disk are shorter in Ozaena (0.6 mm measured from base, near articulation, to medial apex) (Figs 6A and 7A); than in Goniotropis spp.(1.44 mm) (Figs 6B and 7B).

13. The shape of the dorsal plates of the terminal disk are different. In Ozaena they are thin, elongate, widely separated in the middle by a U-shaped space and apically two-lobed, the outer lobe bearing a brush of long setae (Figs 6A and 7A). In the Goniotropis spp. the dorsal plates are rectangular, slightly enlarged from base to apex and separated in middle by a thin, V-shaped space (Figs 6B and 7B).

14. The lateral plates of Ozaena area extremely thin and elongate, acutely triangular, more than 3 times wider than long (Figs 6A and 7A); in Goniotropis the lateral plates are suboval, auriform, and almost 2 times wider than long (Figs 6B and 7B).

15. The urogomphi are composed by 6 branches (Figs 6A and 8A) in Ozaena and 7 branches in Goniotropis spp. (Figs 6B and 8B). In Ozaena lobe A is the shortest, thin and highly reduced, about as long as E2, lobe B is simple, lobe C is much longer than E2 (Fig 8C), E1 is extremely long and thick, club-shaped and with a tuft of long setae at apex (Figs 6A, 8A and 8E). In Goniotropis spp. lobe A is slender and elongated, curved and tapered to tip, much longer than E2, lobe B is two-lobed at the apex (B1, B2), lobe C is as long as E2 (Fig 8D), lobe E1 and E2 are straight and subparallel sided, lobe D is small, dorsal, emerging from base of lobe E (Figs 6B, 8B and 8F).

16. The shape of sensilla S-I differs between the two genera. In Ozaena they are spiniform, and they are tightly inserted in slightly raised cuticular bases (Fig 7C and 7E); In Goniotropis spp. they are “rosette-like”, with short multilobed setae emerging from elongate, cylindrical cuticular bases (Fig 7D and 7F).

17. The shape of sensilla S-II differs between the two genera. In Ozaena they are thin and flexible, long, forming brushes at apices of dorsal and lateral plates, similar to cluster S-V on urogomphus lobe E1 (Figs 6A and 7A). In the Goniotropis spp. they are stick-like, hard, inflexible, lining the sides of dorsal and lateral plates (Figs 6B and 7B).

The moniliform antennae of Ozaena species (Fig 1E) are very different from those of all other ozaenines [30, 31], especially with respect to the shape of the last seven antennomeres (Fig 1E), which are distinctly globular and separated from one another by basal constrictions, giving this part of flagellum increased mobility. Moniliform antennae are typical of myrmecophilous beetles, including Protopaussus Gestro, and more distantly related Pselaphidae and Brentidae. The homologous antennomeres of Goniotropis species (Figs 1B, 1C and 9B) are dorsoventrally flattened and appressed. These flagellomeres are matte in Goniotropis spp. (Figs 1A,1B and 9B) and in most other ozaenines, but appear glossy in O. lemoulti (Figs 1E and 9A) due to the general loss of sensilla, especially sensilla basiconica, from most of the surfaces. In Ozaena, the apex of the last antennomere has a dense field of basiconic sensilla (Fig 9C and 9E), which are smaller than the curved and peg-like basiconic sensilla of other ozaenines. In this region, Ozaena also has some atypical coeloconic sensilla, with a relatively small medial pore, campaniform sensilla, short taste bristles and several apically fringed sensilla chaetica which surround the apical sensorial area (Fig 9C and 9E). The apical part of antennomere XI also has a distinct keel (Fig 9A, right micrograph), dividing the dorsal and ventral parts. The apical surface is covered with finely dotted microsculpture and many pores of different dimensions (Fig 9E), some of which produce filiform, paste-like material. Antennomeres V-X also have two small lateral sensorial areas, with two groups of sensilla basiconica (Fig 9A, arrows).

While the crops of Goniotropis adults were full of pieces of cuticle typical of other general arthropod predators (Fig 10A and 10B), the crops of Ozaena adults were full of a grayish pasty substance typical of other Paussinae species that feed by sucking the fluids from ant larvae, pupae and adults, including those in the myrmecophilous genus Paussus (WM and ADG personal observations).

Most of our attempts to amplify genes from the gut contents of adults, resulted in a successful amplification and clean sequence of the beetle itself or a sequence that was too messy to be meaningful. The successful results of our PCR survey of gut content are reported in Table 1. All sequences of the pasty substance in the gut of Ozaena spp. turned out to be from a Camponotus ant, including one 1116 basepair fragment of 28SrDNA from a specimen of Ozaena lemoulti from French Guiana. Individual fragments of the cuticular pieces from the guts of Goniotropis kuntzeni revealed that they feed on Camponotus as well as other arthropods in their environment (such as chrysomelid beetles).