The long-awaited first instar larva of Paussus favieri ( Coleoptera : Carabidae : Paussini )

Paussus favieri Fairmaire is one of only two species of the myrmecophilous carabid tribe Paussini known from Europe. Larvae are known from only 10 of the 580 paussine species. As in many beetles with considerably modified later instar larvae, the first instars represent a valuable source of informative characters for taxonomy and phylogenetic analyses (primary chaetotaxy, eggbursters, etc.). Therefore, the discovery of the first instar larva of P. favieri is particularly important, as it represents only the second species for which this larval stage is known. In this paper we describe the behavior and morphology of the larval first instar of P. favieri (subtribe Paussina of Paussini) and compare it with that of Arthropterus sp. (subtribe Cerapterina), which is the only other 1st instar described in the Paussini. Most surprisingly, we found that the 1st instar of P. favieri lacks a prostheca, which was previously thought to be a synapomorphy of Paussina + Platyrhopalina. Rather, P. favieri has a unique mandibular structure that seems to be functionally analogous to the protheca. It is a long, broadly lanceolate, distinctly flattened structure apparently homologous to the medial mandibular seta (MN2*), which arises from an area behind the cutting edge of mandible. We predict that the function of the protheca and this similar structure in P. favieri are involved in a specialized feeding strategy that may include soliciting trophallaxis from their host ants. We also report some observations of the first instar hatching from the egg, feeding on liquid and a behaviour we interpret as a “calling behavior,” all of which were videotaped and posted on the Tree of Life Web Project.


INTRODUCTION
Myrmecophiles (obligate symbionts of ants) are challenging to study because they are rare, they live in concealed environments (ant nests), and they have complex but little understood interactions with their hosts.While it is difficult to study their behavior and life cycle in nature, there are also challenges rearing them in laboratory conditions.Because these difficulties hamper direct observations, the nature of most interactions between myrmecophiles and ants, and the function of many structural adaptations, remain a mystery or a matter of speculation (e.g., Di Giulio & Moore, 2004).
Myrmecophiles possess specialized adaptations in order to be accepted by ants and to survive and develop within their nests.Such adaptations include: chemical and morphological mimicry; specialized behaviors to feed on and/or be fed by ants; and structural and chemical modifications to avoid ant attacks.Holometabolous myrmecophiles are unique in that they have different suites of adaptations for very different life history stages (larva, pupa, adult).For many holometabolous myrmecophiles, adults can freely enter and leave the nests, but the larvae (and pupae) must remain in the nest full-time and they are completely dependent upon the ants for survival.
Most members of the carabid beetle subfamily Paussinae are myrmecophiles and all members of this subfamily have a unique type of larva referred to as "dis-cotelic" (Di Giulio, 1999, 2008) for the presence of a wide, transverse, plate-like structure, called the terminal disk at the end of an up-curved abdomen (Bousquet, 1986;Di Giulio, 1999;Di Giulio et al., 2000).The terminal disk is composed of modified epipleurites VIII and IX, tergite VIII, and the urogomphi.In free-living, nonmyrmecophilous larvae the terminal disk is used to trap prey through an ambush feeding strategy, after the prey are attracted to it by chemical attractants (Costa et al., 1988;Di Giulio, 1999;Di Giulio & Vigna Taglianti, 2001;Moore & Di Giulio, 2006).All described species of the monophyletic tribe Paussini are thought to be parasitic myrmecophiles as both larvae and adults (Darlington, 1950;Nagel, 1979Nagel, , 1997;;Di Giulio & Moore, 2004;Geiselhardt et al., 2007).Components of the terminal disk are fused in myrmecophilous Paussini larvae such that they cannot trap prey like their free-living relatives.Substances on the fused terminal disk most likely appease ants and play important roles in establishing and maintaining of their symbiosis with ants (Bøving, 1907;Oberprieler, 1985;Bousquet, 1986;Luna de Carvalho, 1989;Di Giulio & Moore, 2004;Di Giulio, 2008).
The first Paussini larva was discovered more than hundred years ago (Bøving, 1907;Di Giulio, 2008).Since then our knowledge of the group has grown very slowly.To date, we know larvae of only 10 of approximately 580 described species (for a list, see Di Giulio & Moore, 2004;Di Giulio, 2008;Nagel, 2009), representative of 3 of the 7 subtribes (Cerapterina, Platyrhopalina, Paussina) and 4 of the 22 genera (Arthropterus W.S. MacLeay, 1838, Platyrhopalopsis Desneux, 1905, Paussus Linnaeus, 1775, Granulopaussus Kolbe, 1938).Two other larval descriptions (Wasmann, 1918;Brauns, 1914;van Emden, 1922) are too vague and superficial to be useful, the identifications having been based solely upon speculations, and the deposition of material is unknown (Di Giulio et al., 2003;Di Giulio & Moore, 2004;Di Giulio, 2008).While first instar larvae are widely acknowledged to be an important source of diagnostic characters (i.e., primary chaetotaxy and egg burster shape) (Bousquet & Goulet, 1984), most of the described Paussini larvae are second or third instars.To date the only first instar larva described in this tribe is that of Arthropterus sp., classified in the subtribe Cerapterina (Di Giulio & Moore, 2004).
The tribe Paussini is mainly tropical and subtropical with very few species known in the Palearctic Region (see Nagel, 2003 for an updated list).Only two species are distributed in Europe, Paussus turcicus I. Frivaldszky von Frivald, 1835 and P. favieri Fairmaire, 1851.Paussus turcicus occurs in West-Balcan, Turkey, S-Caucasus and Turkmenistan (Nagel, 1987(Nagel, , 2003) ) and P. favieri is an Atlanto-Mediterranean species (Nagel, 1987(Nagel, , 2003) ) present in southwestern France, Spain, Portugal, Morocco, Algeria and Tunisia (Casale et al., 1982;Nagel, 1987).There are some museum specimens of P. favieri from Sicily and Sardinia (Casale et al., 1982), and one from Corsica (Zerche, 1990), however, no specimens have been collected on these islands during the past 100 years.The closest relatives of P. favieri, are P. biflagellatus Luna de Carvalho, 1973and P. krelli Kaupp & Rödel, 1997(Luna de Carvalho, 1980;Kaupp & Rödel, 1997) known only from the sub-Saharan countries of Ghana and the Ivory Coast.For over 150 years P. favieri has attracted the intense interest of researchers and collectors, due to its rarity, its bizarre structural adaptations to a myrmecophilous lifestyle, and the fact that it is one of the few paussine species known from the Mediterranean Region.Through the years, many researchers have tried to learn about the life cycle and preimaginal stages of this species but to no avail (an apodous, physogastric larva was erroneously attributed to P. favieri by Xambeu, 1892).
Recently, on a collecting expedition to the High Atlas Mountains of Morocco we collected a gravid female of P. favieri and in our laboratory in Rome we were able to rear the first instar larva of this remarkable species.These specimens represent the first record ever of first instar specimens of the subtribe Paussina.In this paper we describe P. favieri first instar larval morphology, provide notes on collecting adults, rearing conditions and behavior of the larva in captivity.We also compare this larva to the first instar of Arthropterus, the only other first instar described for the tribe Paussini.

MATERIAL AND METHODS
Several adult specimens of Paussus favieri, collected during an expedition to High Atlas Mountains in Morocco (2009) from nests of Pheidole pallidula (Nylander, 1849), were kept in captivity for about one month under controlled conditions.During this time, their behavior and interactions with their host ants were studied (Di Giulio et al., unpubl.).One of the females laid two eggs on the moist filter paper.Eggs were held at room temperature (about 25°C), inside a vial closed by a moist cotton plug.Two larvae hatched eleven days after isolation.Larvae were observed under Olympus SZX16 stereomicroscope, and interactions among the larvae, adults and host ant brood were recorded by a camera (ColorView II, AnalySIS software Olympus SIS, Cell *D, 2006).Two days after hatching, the small larvae were submerged in boiling distilled water for a few seconds and then transferred to 70% EtOH.Larvae were drawn using Olympus SZX16 equipped with drawing tube (Figs 1-5).Then, one specimen was rehydrated, cleared in 10% KOH, transferred to hot lactic acid, dehydrated through a series of EtOH baths of increasing concentration (10, 20, 50, 70, 90, 95 and 100%), left overnight in a clove oil bath, and mounted on a slide with Canada balsam.This specimen was illustrated by using a light microscope Olympus BX51 equipped with drawing tube.The second specimen was dehydrated through a series of EtOH baths of increasing concentration (70, 80, 90, 95 and 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 Philips XL30 scanning electron microscope and FEI Dualbeam FIB/SEM Helios Nanolab (L.I.M.E.laboratory, University "Roma Tre", Rome).In this paper, the general terminology of larval structures follows Lawrence (1991).The term "frontoclypeolabrale" is used here instead of "frontale" or "frontal plate" (more often used in larval taxonomy of Carabidae) or "frons" or "frontoclypeal region" (see Lawrence, 1991), following the recent taxonomic descriptions of Paussinae (Di Giulio & Moore, 2004, 2009;Moore & Di Giulio, 2006) and the available larval key (http://tolweb.org/notes/?note_id=3430).Notation of primary setae and pores follows the system of Bousquet & Goulet (1984), modified for Metrius contractus Eschscholtz, 1829 (Bousquet, 1986).As some of the sensilla of P. favieri larva present on the abdomen and terminal disk are homologous to those recognized by Bousquet (1986) in Metrius contractus (sensilla S-I to S-V), by Di Giulio (1999) and Di Giulio et al. (2000) in several species of Pachyteles Perty, 1830 (sensilla S-I to S-VII), and by Di Giulio & Moore (2004) in Arthropterus sp.(sensilla S-I to S-VIII), we adopted the same nomenclature used by these authors.Notation of microsculpture follows Harris (1979).An asterisk (*) following a coded seta indicates that the homology between the structure in the P. favieri larva and the corresponding code is questionable.

Behavioral observations on Paussus favieri first instar larva
Immediately after hatching the terminal disk is closed.That is, the dorsal and lateral plates are stuck to the ventral plates (http://tolweb.org/media/44075).The larva actively moves, twisting its head and thorax up, down and side to side for about one hour.After this period of hyperactivity, it remains still, stretching and inflating its body until the terminal disk is completely opened and sclerotized.Once the body becomes white-yellow and head and claws more sclerotized, the larva becomes active again, opening and closing its mandibles (which it can move independently of one another) with abdomen and terminal disk bent dorsally.It is unable to use its short legs for walking, but rather for support.On several occasions we observed the larva grasping the filter paper that lined the observation chamber with its mandibles, thereby creating an anchoring point.The larva would then flex its body vertically, bringing the terminal disk up and over its head (http://tolweb.org/media/44071).We also observed what we interpret to be a "calling behavior" in which the larva balances on its hind legs and urogomphi as it lifts its head and thorax into the air, moving from side to side while opening and closing its mandibles, and waving its mid and fore legs (http://tolweb.org/media/44074).
Most often the larva did not show interest toward the host ant brood, but on one occasion we observed an unsuccessful attempt to pierce the brood with its mandibles (http://tolweb.org/media/44076).However, when offered damaged brood the larva began to actively suck the host's hemolymph, demonstrating an aptitude for liquid feeding (http://tolweb.org/media/44077).
Head.Strongly transverse (Figs 2a-b, 7a), two times as wide as long, prognathous, subparallel-sided, basally nar-rowed, with maximum width at base of antennae; cephalic capsule steeply sloped dorsally from base to apex, not retracted into prothorax (Fig. 7a); base of head capsule and occipital foramen lined with a sclerotized band.Frontoclypeolabrale sub-hexagonal (Fig. 2a); anterior margin not sclerotized, distinctly concave, medially very thin and slightly emarginated; surface distinctly convex posteriorly and anterolaterally, deeply concave anteromedially; transverse frontal keel absent; eggbursters well developed, consisting of two strongly sclerotized longitudinal keels, posteriorly slightly convergent, ending anteriorly in a sharply pointed spine directed anteriorly (Figs 2a, 7a-c); coronal suture absent; frontoclypeolabrale almost fused with parietalia, frontal sutures very fine but still visible in light microscope.Parietalia (Figs 2a-c, 7a-b) subparallel-sided, without stemmata, protruding at base of antennae; ventral walls of parietalia medially fused into a short gular suture.Antennae (Figs 2a,7a,d) 4-jointed, directed forward and slightly convergent anteriorly, not reaching apices of the mandibles, inserted in extensive membranous, soft elevations; antennomeres I-III wide; I asymmetrical, shorter on ental side, slightly longer than broad, more than three times as long as II; II short, three times wider than long; III about six times as long as II; II slightly shorter than IV; IV very small compared to others; sensorial appendage ovoid, positioned ventrolaterally on antennomere III (Fig. 7d), about as long as IV.Mandibles (Figs 3c, 7e) subtriangular, slightly falcate apically, 1.5 times as long as wide at base, with single, ventral cutting edge; dorsal surface deeply excavate along occlusal margin, particularly at base of modified mandibular seta; retinaculum slender, triangular, sharp and pointed, displaced and directed apically, forming, together with the pointed apex, a bidentate mandible; prostheca and penicillus absent.Maxilla (Figs 3f-g) with small, subtriangular cardo and subquadrate stipes; maxillary palpus 4-jointed: I partially fused with stipes, about twice wider than long; II slightly longer than I; III subquadrate, twice as long as II; IV conical and subulate; galea 1-jointed (corresponding to galeomere II of Ozaenini, galeomere I being fused with stipes), digitiform, almost straight, distinctly tapered from base to apex; lacinia small, vestigial; lateral margin of stipes distinctly curved, occlusal margin straight without basal tooth.Labium composed of a membranous mentum, ventrally sclerotised prementum and 2-jointed palps.Prementum (Figs 3d-e) slightly enlarged from base to apex, particularly in ventral view; basal half of prementum dorsally bulging, distal half with vestigial subapical ligula, represented by prominent bases of strong setae LA6.Labial palpomeres subequal in legth: I subquadrate, II conical, subulate (Figs 3d-e).Hypopharynx densely covered by transverse parallel rows of pointed papillae (Fig. 7f), closely fitting in the vault of the oral cavity.
Legs.Short and highly modified, all similar in type and length (Figs 4a,6d), composed of 2 joints subequal in length: basal joint representing coxa, basally embedded into soft membranous areas, dorsally articulated with medially subdivided pleural sclerite; distal part of coxa obliquely truncate to allow folding of second joint; latter cylindrical, digitiform, slightly tapered to apex, composed of completely fused trochanter, femur, tibia and tarsus; sutures between segments hardly visible.Procoxae more slender and shorter than meso-and metacoxae.Second joint bearing single conspicuous claw (Figs 4a, 6e), apically curved and pointed.
Abdomen.Subparallel-sided, gradually enlarged toward teminal disk, scarcely or not sclerotised, distinctly curved in an upturned position (Figs 1, 6a, c); narrowest at segments III-V; terga flattened or slightly convex, pleura and sterna swelling.Terminal disk (Figs 5, 8a-b) wider than segment VII, regularly round, with perimeter strongly raised, corrugated and markedly sclerotized; disk composed of 6 symmetrical subtriangular plates joined by thin membranous lines; dorsal plates wider than others, medially convex and bearing abundant sensilla S-I; lateral plates small; ventral plates corresponding to modified urogomphi, similar in shape to dorsal plates, with surface flattened or slightly concave.Pygidium cylindrical, dorso-ventrally flattened, ventral to urogomphi.

DISCUSSION
Within the subfamily Paussinae, larvae are known from 4 of the 5 tribes (Metriini, Mystropomini, Ozaenini, Paussini), the larvae of Protopaussini being still unknown.Within these tribes, only one or few species of 11 genera (out of 46; Lorenz, 2005) are known as larvae, often described based on a single specimen of second and/or third instar.Though our knowledge of Paussinae larvae is very limited, the taxon sampling is broad enough to provide information about relationships among major clades.However since larvae are rarely collected in the field and difficult to rear in the laboratory, phylogenetic analyses based on larval morphology compared different instars of the taxa included in the analyses (Bousquet, 1986;Beutel, 1992;Vigna Taglianti et al., 1998;Di Giulio et al., 2003;Di Giulio & Moore, 2004).For this reason, only morphological characters related to shape of the head capsule, head appendages, legs and terminal disk have been used.It has not been possible to include first instar larval characters, such as those related to eggbursters or primary chaetotaxy (Bousquet & Goulet, 1984), which are widely acknowledged to be phylogenetically informative (Bousquet & Goulet, 1984;Bousquet, 1986;Arndt, 1998;Meier & Lim, 2009).In fact, until now first instar larvae in Paussinae were known for only four species: Metrius contractus (Metriini), Pachyteles vignai Deuve, 2000 (Ozaenini), Goniotropis kuntzeni (Bänninger, 1927) (Ozaenini), and Arthropterus sp.(Paussini).
The discovery, description and observations of the 1st instar larva of Paussus favieri presented herein are significant in many respects.First the paper provides information about the larval stage, behavior, and life cycle of a rare species that is increasingly endangered due to the destruction of suitable habitats (P.favieri is endemic to the Mediterranean Region, and is one of only two species of Paussini occurring in Europe).It also increases the number of described myrmecophilous larvae, thereby broadening our understanding of evolutionary adaptations to a myrmecophilous life in Paussini.In the following discussion, the morphological characters observed in P. favieri 1st instar larva are analyzed and discussed in comparison with those of Arthropterus sp., the only other known species of the Paussini.
All the synapomorphic characters of Paussini (Di Giulio & Moore, 2004) are found in the larva of Paussus favieri, including: Neck not constricted, head prognathous, coronal suture absent, antenna short and broadly inserted in bulging membranous base, large antennal sensorial appendage, mandibles short and pointed with some long setae, stipes without basal tooth, setal group gMX extremely reduced, prementum bulging, one tarsal claw, urogomphi plate-like, terminal disk perfectly round, pygidium ventral to urogomphi.
Most Paussini larvae have an elongate digitiform prostheca, a soft structure with an anterior ridge that almost reaches the apex and arises near the base of the ental surface of the mandible.Surprisingly, the prostheca is not present in the 1st instar of P. favieri, although it has been considered a synapomorphy of the Paussina + Platyrhopalina.While P. favieri does not have a prostheca, it does have a similar structure that is apparently homologous to the medial mandibular seta (MN2*).It is a long, broadly lanceolate, distinctly flattened structure (Figs 2a, 3c, 7a,e-f), arising from an area behind the cutting edge of mandible.The functions of the prostheca and of this similar structure in P. favieri are unknown, but possibly these larvae have a specialized feeding strategy that may include soliciting trophallaxis from the host ants.
The reduction of the base of the cephalic capsule (frontale + parietalia), with loss of coronal suture and basal part of frontal sutures up to the base of the egg bursters, is shared by the larvae of P. favieri (Figs 2a,7a) and other known larvae of the Paussini subtribes Paussina and Platyrhopalina, being possibly their synapomorphy (Di Giulio & Moore, 2004).This reduction from the ancestral state found in other Paussinae tribes (e.g., Metriini, Ozaenini) may be related to the secondary acquisition of a prognathous position of mouthparts and head derived from a hyperprognathous ancestral state (Di Giulio et al., 2003).Such a basal degeneration of the head (including the widening of the occipital foramen and a disappearance of the neck) could be an adaptation for living with ants.
As in all known larvae of Paussina, the basal maxillary palpomere and the stipes of P. favieri are partially fused (Figs 3f-g); in some other Paussus species this fusion is complete.A small remnant lacinia is present in P. favieri (Fig. 3f), whereas it is completely absent in other Paussina and Platyrhopalina.Arthropterus has a highly derived lacinia with a row of strong hook-like setae.
In contrast to the larva of Arthropterus, which has distinct and functional leg articles, the trochanter, femur, tibia, and tarsus of P. favieri are fused (Fig. 4a) and remnant sutures are only visible with SEM (Fig. 6d).Such fusion is a synapomorphy of Paussina and Platyrhopalina, though in Platyrhopalopsis traces of oblique sutures between the fused articles are clearly visible.The reduction from two (Metriini + Mystropomini + Ozaenini) to one claw seems to be a defining character of Paussini.Most Paussina and Platyrhopalina larvae have a very small and thin tarsal claws, whereas Arthropterus and P. favieri larvae have conspicuous, robust tarsal claws.It is possible that this is a characteristic of all first instar larvae as compared with latter instars.
As in the other Paussus larvae (Paussina) and in Platyrhopalopsis (Platyrhopalina), an anterior medial emargination of frontoclypeolabrale is present in P. favieri (Figs 2a,c,7a), while this modification is absent in the Arthropterus larva.For this reason we consider this character to be synapomorphic of the subtribes Paussina and Platyrhopalina.In P. favieri, we observed that the medial anterior margin moves during liquid feeding and we predict that this movement may increase the efficiency of suction.
There are several differences between the terminal disk of the first instar Arthropterus and P. favieri larvae (Figs 5, 8c, e): (1) The marginal setae of P. favieri are lanceolate, thin, with a pointed tip, while in Arthropterus they are clavate (sensilla S-VIII of Di Giulio & Moore, 2004, possibly homologous to sensilla S-II).Such a feature has not been observed in any other larva of Paussini and could be a feature only present in the first instars.In P. favieri a dense substance emerges from the bases of these setae and runs onto the surface of the disk.A possible glandular function was also reported for the homologous setae of Arthropterus sp.(Di Giulio & Moore, 2004).The different shapes of these setae could be functionally related to different ways of supplying the substance to the ants, from the apex of the club-shaped sensilla in Arthropterus, or from the surface of the cup-shaped terminal disk in P. favieri.
(2) The disk of P. favieri is cup-shaped, as in all species of the subtribes Paussina and Platyrhopalina, with upcurved margin, while in Arthropterus the disk is biconvex with a simple margin.
(3) The dorsal plates of P. favieri are slightly wider than ventral plates, and the lateral plates are small, while in Arthropterus the dorsal plates are almost twice as large as the ventral, and the lateral plates are wide.
(4) The sensilla S-I located on the terminal disk are complex with multispinulate, dome-like bases and short fringed setae in P. favieri, whereas the homologous structures in Arthropterus are short, simple and coniform, bearing an elongate, simple, blunt-tipped seta.
Nothing is known about the way of life of Paussini larvae except for several field observations which confirm that they are myrmecophilous.In most cases larvae have been found inside ant nests or carried about by the host ants (Luna de Carvalho, 1959, 1992;Geiselhardt et al., 2007).No data on oviposition, feeding, development or behavior are available, not even for the first instar larvae of Arthropterus, hatched in captivity from eggs laid by an unidentified female (Di Giulio & Moore, 2004).Various authors have speculated that Paussini larvae feed on host brood and that the round cup-shaped terminal disk is probably adapted for supplying attractive substance to worker ants (Oberprieler, 1985;Luna de Carvalho, 1992).Based on the observation of several characters, unusual for a predaceous carabid larva (i.e., shortened and somewhat degenerated head capsule, reduced mouthparts, unique presence of a prostheca, partial atrophy of legs), Di Giulio (2008) advanced the possibility that they could at least partially be fed by the ants through trophallaxis.The behavioral observations reported here do not exclude this hypothesis.In fact, the "calling behavior" described above (first paragraph of Results section) is similar to that reported for ant larvae soliciting trophallaxis (Hölldobler & Wilson, 1990).