Out of the burrow and into the nest: Functional anatomy of three life history stages of Ozaena lemoulti (Coleoptera: Carabidae) reveals an obligate life with ants

The carabid subfamily Paussinae contains many species known to be obligately associated with ants during at least one stage of their life history. Myrmecophilous larvae have been documented for members of the tribe Paussini as well as several genera in the tribes Ozaenini, including Physea and Eustra. Here we describe the first instar larva of Ozaena lemoulti, and find it to be the most highly modified ozaenine larva that we have examined to date. Many structures of the larva suggest that it is a myrmecophile. Unlike all other described ozaenine larvae, which live in burrows that they construct and seal with their terminal disk, the completely unique larval morphology suggests Ozaena has adapted to living without the protection of a burrow and therefore must have a completely different feeding strategy than the typical ambush strategy of burrow dwelling larvae. We hypothesize that Ozaena larvae live in association with ants and use their long legs for running within the nest, and modifications of the mouthparts suggest the larva feeds on soft lightly sclerotized prey, such as ant brood. Our findings support an earlier hypothesis that Ozaena is mymecophilous during the adult stage. Comparisons of the functional anatomy of the eggs, larvae and adult between Ozaena lemoulti and the closely related, non-myrmecophilous general arthropod predator, Goniotropis kuntzeni, provide complementary, yet independent, evidence suggestive of this shift in lifestyle. We also examine and molecularly identify gut contents, providing direct evidence that adult Ozaena exclusively eat Camponotus ants. We conclude that Ozaena represents an independent shift to adopting a life of myrmecophily among beetles classified within the carabid subfamily Paussinae and document the morphological changes at each life history stage associated with the shift to a nest parasite lifestyle.

Antennomere I (Figs 4A-B, 5A) with 5 additional setae: 2 short dorsolateral setae on basal half, and a crown of 3 longer setae subapically; II without additional setae; III with seta AN1 displaced anteriorly; IV with AN6 short. Mandible (Figs 4A-B, 5E) with one additional lateral seta just more distal to retinaculum; seta MN1 short; 5 additional pores on dorsal surface. Setal group gMX (Fig 5C) on stipes composed of approximately 10 elongate and thin setae distributed especially along occlusal margin; seta MX5* and another subapical additional seta at the base of maxillary palpi spine-like; seta MX6 small, subapical on lacinia; MXd* ventral at apex of galeomere I; seta MX8* subapical, close to MX9*; small sensorial area (composed of 3 short lateral and 1 longer medial basiconic sensilla) present at apex of galeomere II ( Fig 5C); maxillary palpomere IV with small dorsolateral additional seta on medial side, 1 ventral pore and 3 longitudinal subapical digitiform sensilla and apical sensorial area composed of several peglike sensilla. Prementum (Figs 4A-B) with about 26 additional spine-like setae (13 on each side) on lateral and dorsal surfaces, inserted in protuberances; seta LA1 close to midline; labial palpomere II with 2 additional setae, 1 mesodorsal and 1 small ventrolateral, 3 longitudinal subapical digitiform sensilla and apical sensorial area composed of several peg-like sensilla.
Each side of epipharynx with 3 small additional setae and 1 pore posterior to couple FR8-FR9.
Pro-, meso-and metanotum ( slightly shorter than mandibles, forwardly directed; antennomere I about 2 times longer than II and about 1.5 times longer than III; IV slightly shorter than III; III with small, globular sensorial appendage (Fig 5A, arrow). Mandibles (Figs 4A-B maxillary palpomeres gradually decreasing in length from I to III; IV digitiform, distinctly longer than others, slightly longer than II and III combined. Galeomere I short; II cylindrical, decreasing in size from base to apex, about 2 times as long as I, slightly curved inward at distal half. Lacinia short and straight, blunt tipped, basally fused with stipes ( Fig 5C). Labium (Fig 4A-B) with sclerotized prementum and 2-jointed palps; prementum distinctly transverse with setal notches present on dorsal surface and sides; ligula elongate and subrectangular, slightly shorter than labial palpomere I; labial palpomere I slightly wider than II; II digitiform, about 2 times as long as I.
Longitudinal ecdysial line well marked on pronotum, but present also on meso-and metanotum.
Spiracles.-Thoracic and abdominal spiracles (Fig 4A,C) annular with conical, protruding peritremes. Mesothoracic spiracles anterolaterally positioned on mesopleura, diameter almost 2 times wider than diameter of abdominal spiracle I. Abdominal spiracle I diameter slightly wider than diameter of abdominal spiracles II-VII, II-VII subequal, VIII extremely small, placed basally between insertion of dorsal and lateral plates of terminal disk (Fig 4C).
Coxa cylindrical, elongate, about as long as trochanter and femur combined; trochanter obliquely truncate and ventrally expanded at apex, about as long as femur and tibia combined; femur about twice as long as tibia, slightly increasing in size from base to apex; tibia cylindrical, about as long as tarsus; tarsus more slender than tibia, conical, tapered from base to apex, with 2 sharp claws; anterior claw long, regularly curved from base to apex; posterior claw shorter, thinner and almost straight.
Abdomen.-Abdominal segments I-VII very similar (Figs 2A-B, 4C-D represented by a small dorsal, subapical, setipherous protuberance; C slightly longer and more slender than B; E2 small and thin, slightly bent laterally; E1 club-shaped, extremely long and thick; lobe D obsolescent, dorsal, emerging perpendicularly to plane of other branches (Figs 6A, 8A), slightly more proximal than level of A; pygidium (Fig 6A) flattened, medioventral between urogomphal insertions.

Goniotropis parca first instar
The larva of G. parca (Figs 7B,D,F; 8B,D,F) is very similar to that of G. kuntzeni (see Moore and Di Giulio 2006), both in shape and setation. For an extensive description and illustration of the egg (Fig 2B,D,F) and of the main larval characters (Fig 3C-D, 6B) and setation of Goniotropis kuntzeni we refer to Moore & Di Giulio (2006). Here, we highlight only the main differences between G. parca and G. kuntzeni.
Measurements -Body length about 4 mm (from tip of mandibles to apex of terminal disk); cephalic capsule maximum width (at base of antennae) 1 mm, medial length (from occipital foramen to anterior margin of frontoclypeolabrum) 0.62 mm; prothorax maximum width 0.87 mm, medial length 0.5 mm; terminal disk maximum width (at level of lateral plates) 1.5 mm; dorsal plates length (from base, near articulation, to medial apex) 0.68 mm.
As compared with G. kuntzeni, G. parca is relatively smaller and more compact; the cephalic capsule is shorter, more rounded and transverse; stemmata are relatively smaller; antennae with shorter and broader antennomeres, and slightly bigger hyaline vesicle; mandibles are shorter and stouter, with retinaculum less curved inward; maxillae with stipes shorter, broader and less curved, and shorter last palpomeres; last labial palpomeres shorter; legs with segments and claws distinctly shorter; terminal disk with dorsal plates more rectangular and subparallel-sided, only slightly broadened from base to apex, and more widely separated in the middle; lateral plates more suboval and less laterally projecting; urogomphi with all branches shorter and stouter, with fewer S-I sensilla.