Form , function and evolutionary significance of stridulatory organs in ant nest beetles ( coleoptera : carabidae : Paussini )

Stridulatory organs in the myrmecophilous carabid beetle tribe Paussini have long been recognized and used as a defining character of some genera and higher level taxa, however their morphology has only roughly been described. Here, we describe the fine morphology of Paussini stridulatory organs using scanning electron (SeM) and focused ion beam (FIB) microscopy. Within this tribe, there are three types of stridulatory organs, each with different positions of the scraper (plectrum) and file (pars stridens). type I (abdomen-femur type) is located on the abdomen (scraper) and metafemur (file) in the subtribe Paussina (sensu Geiselhardt et al., 2007, Naturwissenschaften 94: 871–894). type II (thorax-femur type) and type III (femur-thorax type) are located on the mesothorax and mesofemur in two different genera of the subtribe Platyrhopalina, however in Euplatyrhopalus the scraper is located on the mesothorax and the file is located on the mesofemur (type II), whereas in the genus Platyrhopalopsis the structures are reversed in that the file is located on the mesothorax and the scaper is located on the mesofemur (type III). the independent evolution of three types of stridula­ tory organs in three lineages of Paussini suggests that acoustical communication has played an important role in the evolution of ant nest beetles. While the roles of stridulation in this group remain speculative, we verified that all three types of stridulatory organs are present in both sexes and are similar to stridulatory organs known in their host ants which also use stridulation as a method of com­ munication. We discuss the possibility that the beetles’ stridulation could (1) facilitate their exploitation of ant colonies, and (2) be involved in mate recognition and courtship.

Beetles and ants were dissected under a stereomicroscope, kept overnight in a detergentwater solution, cleaned with ultrasound for 15 s, rinsed in water, dehydrated through a series of ethanol baths of increasing concentration (70, 80, 90, 95 and 100%), criti cal point dried (BalTec CPD 030), mounted on a stub (by using self adhesive carbon disks), and observed via FIB/SeM (Dual beam Helios -FeI, l.I.M.e.lab, university "roma tre", rome, Italy) operating with the SeM column.the FIB/SeM is equipped with two columns including one electron beam (SEM column) and one ion beam (FIB column), oriented at 52°, and focused on the same point on the sample.Specimens of Paussus favieri were sputtercoated with gold (Emitech® K550 sputter coater) before observation (FIB/SeM set at HV = 5 kV); specimens of Euplaty rhopalus vexillifer and Platyrhopalopsis picteti were observed uncoated at low voltage (FIB/SeM set at HV = 1 kV).
The spines of the scraper of P. favieri prepared for the SEM were also analyzed with the FeI Dualbeam (FIB/SeM) Helios Nanolab operated with the ion column (FIB settings: 30 KV and 0.92 nA) to investigate the internal cuticular structure of the spines (Fig. 3). the FIB was used to selectively ablate (milling process) a previously marked region of the sample using a fo cused ion current from a gallium source.The milling process was paused every few nanometers while highresolution images were taken of the cross sections with the SEM column.

type I ("abdomen-femur" type)
The highly derived abdomenfemur mechanism of Type I is present in all genera of the derived subtribe Paussina (including the speciose genus Paussus), except for the monospecific Leleupaussus.It involves structures posi tioned symmetrically on hind legs (file) and basal abdomi nal ventrites (scraper).This organ, present in both sexes (escherich, 1898), is composed of a file comprising a slightly raised, finely ridged area positioned on the basal, inner (dorsal) face of the hind femora (Figs 1a3;, that rubs against a fixed scraper, composed of a curved row of small spines (Fig. 1a2; 2c-d) [stridulatory file sensu Dar lington, 1950, considered as pars stridens (stridulatory file) by Geiselhardt et al., 2007], positioned at both sides of ab dominal ventrite 2 (Fig. 2c; ventrites 1-3 are connate and not clearly demarked).To produce the vibratory sound, the hind legs move up and down, either singly or in tandem (Maurizi et al., 2012).
All members of the ground beetle tribe Paussini are high ly specialized myrmecophiles, integrated in the ant socie ties (mainly guests of Myrmicinae and Formicinae: Geisel hardt et al., 2007).Paussines are generally considered ant parasites since they prey on adult ants and their brood without conferring any obvious benefit to the ant colonies (Escherich, 1899;Le Masne, 1961a, b;Geiselhardt et al., 2007;Di Giulio et al., 2011;Maurizi et al., 2012).It is still unclear which type of strategy these beetles use to enter and exploit the ants' nests without being attacked.Given the prevalence of trichomes and specialized setae adorning their body, it is assumed that the beetles are able to produce rewarding/appeasing chemicals from associated special ized glands, concentrated in the antennae, pronotum, elytra and pygidium (Nagel, 1979;Geiselhardt et al., 2007;Di Giulio et al., 2009, 2012;Maurizi et al., 2012).However, chemical and acoustical mimicry (Geiselhardt et al., 2007) could also be a part of their complex and effective parasitic strategy.
The presence of three types of stridulatory organs in Paussini has long been known, however the shape of these organs has only been superficially described (Westwood, 1874;Escherich, 1898;Gahan, 1922;Luna de Carvalho, 1949, 1953;Darlington, 1950;Nagel, 1987).Nagel (1987) and Geiselhardt et al. (2007) listed a potential new type of stridulatory organ in Paussini, involving an alaryelytral structure, however its role as a stridulatory organ remains speculative.
Lack of morphological and behavioural information on the stridulatory organs in Paussini inspired us to describe and illustrate the fine morphology of the three main types of stridulatory organs in ant nest beetles using scanning electron (SeM) and focused ion beam (FIB) microscopy.We compare and contrast these independentlyevolved structures and present hypotheses for their potential func tional role(s) and impacts on ant nest beetle diversification.

MAterIAl And Methods terminology
throughout this paper we refer to the "file" as the finely ridged structure that forms one part of the stridulatory organ and the "scraper" as the peg-like stucture that forms the other part.In the literature different authors have adopted different definitions for these structures.Many follow Nichols & Schuh (1989) who defined the "scraper" as the part or the organ that is actively rubbed over the "file".However, among insects, and even within paussines, the active half of the organ has widely different shapes.therefore we refer to the "file" as the finely ridged half of the organ whether or not it is stationary or active.
Following the classification of luna de Carvalho (1953), we describe three basic types of stridulatory organs in the tribe Paussini, hereafter called type I, type II and type III, based on the position of the scraper (also known as plectrum) and the file (also known as pars stridens).oriented longitudinally along the leg axis, composed of 38-45 subparallel ridges; ridges 1.57 ± 0.14 μm thick and approximately 1.62 ± 0.43 μm apart; most ridges complete and parallel, others incomplete and convergent.scraper (Fig. 2c-d).The scraper comprises an outward ly curved row (length 415 μm) of cuticular spines (25-30 elements) on abdominal ventrite 2, posterolateral to hind coxae (Fig. 2c).each spine (Figs 2d) flattened and sub triangular, 32.56 ± 3.62 μm long, obliquely emerging from an elliptical socket (length ~ 15 μm, max width ~ 6 μm), apically bearing a trichoid projection that is directed up ward (Figs 2d;3).Distance between spines approximately 9.46 ± 1.50 μm. the spines are not parallel to one another but radially oriented along the curved row, their apices pro jecting laterally.Their orientation is parallel to the ridges of the file.
FIB/seM analysis (Fig. 3a-d).the FIB/SeM dissec tions reveal that the triggerlike spines of the scraper may not be modified setae, but new complex cuticular struc tures.Each spine is a cuticular outgrowth of the sternal plate, deeply invaginated in the thickness of the cuticle, not showing a thin cuticular membrane like the typical trichoid sensilla.The spines are solid, rather than hollow, making them strong and the tips are tapered giving them flexibility.
The origin of the triggerlike spines comprising the scraper is unclear, but additional investigation may reveal whether the spines are highly modified setae, or completely novel structures.
comments.There is a wide range of variation in certain aspects of the type I stridulatory organ.the scraper varies in the number and shape of the spines, their spacing, and the degree of curvature of the row of spines (e.g., see Es cherich, 1898; Nagel, 1987).
stridulatory organ of the host ant (Fig. 4a-f).The stucture of the stridulatory organ of Pheidole pallidula is very similar to most organs described in ants (see Höll dobler & Wilson, 1990).It is positioned on the abdominal tergites III-IV (Fig. 4b).the file is located mid-dorsally on the pretergite of the fourth abdominal segment (Fig. 4c-d), while the scraper is located on the posterior margin of the third abdominal tergite (postpetiole) (Fig. 4e-f).
Stridulatory file (Fig. 4c-d).the file is a rounded tegu mental area with a very regular organization, characterized by parallel transversally arranged cuticular ridges (Fig. 4c).This area extends longitudinally for the whole length of the pretergite.the file is easily distinguishable from the surrounding cuticle which is more irregular (Fig. 4d).
scraper (Fig. 4e-f).The scraper is located on the medial portion of the posterior margin of the third abdominal ter gite, bent downward, lying over the file.

type II ("thorax-femur" type)
Only known for the genus Euplatyrhopalus (7 species, subtribe Platyrhopalina), this structure was first described by Westwood (1874) but without functional interpreta tion.Later Fowler (1912) recognized it as a stridulatory organ and hypothesized a function in mating.This organ, present in both sexes, consists of a file composed of a fan of several short ridges, strongly raised and widely sepa rated from one another, located on the internal side of both meso femora (Figs 1b3; 5a), and a scraper located on each side of metasternum (Fig. 1b2), composed of pointed seti ferous protuberances (Fig. 5c-d).
host ant: unknown.(c-d) and the solid nature of the spine (d).Note that the gold layer, sputtered on the cuticular surface for conductive purposes, was removed by the ions during focusing operations, except under the spines that worked as shelters (triangular whitish "shadows" under the tip of the spines).

type III ("femur-thorax" type)
the type III stridulatory organ is currently known only in the nominotypical subgenus (2 described species) of the small genus Platyrhopalopsis (Luna de Carvalho, 1953); it is absent in P. (Platy rhopalides) badgleyi (see Gahan, 1922).In type III, the scraper and file are inverted as com pared with types I and II.this organ, present in both sexes, is composed of a wide fan of many fine costae (file), pre sent on each side of metasternum (Figs 1c2; 6a-b), that is rubbed against a scraper positioned basally on the internal side of each mesofemur (Figs 1c3;. Stridulatory file (Figs 1c2;.the type III file com prises a wide cuticular surface (about 1 mm 2 ) at anterior corners of metasternum furrowed by a fan of about 100 fine subparallel, elongate ridges (Fig. 6a-b), radially directed; each ridge, 5.25 ± 1.1 μm thick, distant from the adjacent ridge by approximately 5.04 ± 0.54 μm; most ridges are complete and parallel, others are incomplete and conver gent (Fig. 6b) as in type I. scraper (Figs 1c3;.The scraper is composed of a row of 4 small setiferous keels (each about 50 μm long, seta excluded), positioned on mesofemur on top of a con vex region near the base (Figs 1c3; 6c).Each keel is pro duced apically and directed longitudinally along the femur axis, thereby redirecting the orientation of the associated seta (Fig. 6d).Setae associated with the keels are well de veloped and apically branched, like the other femoral se tae, but are oriented tangentially to the surface rather than perpendicular to it (Fig. 6c-d).Distances between spines vary between 30-90 μm.
The host ant of Platyrhopalopsis picteti is unknown, but the host ant of P. melleii is Pheidologeton diversus (Myrmicinae) (Luna de Carvalho, 1989;Geiselhardt et al., 2007).dIscussIon the file and scraper of the three types of stridulatory or gans in Paussini are located on different body parts, and therefore they are not homologous, but rather they evolved independently, once in Paussina (type I) (Fig. 1a) and twice in Platyrhopalina (type II and III) (Fig. 1b-c).that acoustical communication is important to the biology of these beetles is supported by the fact that stridulatory or gans evolved three times independently in this group and that each type of organ is present and morphologically similar in all species of these clades (Fig. 7) .The absence of the type I stridulatory organ in the monotypic Leleu paussus is interesting.However the phylogenetic position of this enigmatic genus is uncertain and we suspect that it lies outside of Paussina, as suggested by Luna de Car valho (1989).the type III stridulatory organ occurs only within the subgenus Platyrhopalopsis; it does not occur in the monotypic subgenus Platyrhopalides, containing only Platyrhopalopsis badgleyi.In the absence of a detailed phylogenetic framework for Platyrhopalina, we can only assume that the type III stridulatory organ evolved in the most recent common ancestor of the nominotypical subge nus rather than the genus.
Paussines can perceive vibratory sound by means of mechanoreceptors.While this may not seem surprising, Darlington (1950) questioned the ability of paussines to perceive mechanical stimuli since their antennal pedicel is greatly reduced and therefore he suspected that they may lack a Johnston's organ, the widely acknowledged seat of the insect acoustical reception (McIver, 1985;Bailey, 1991;Fedorova & zhantiev, 2009).However, a recent study revealed (1) the presence of a well developed, albeit very small, Johnston's organ in P. favieri and (2) that the antennal trichomes serve as mechanoreceptors (in addition to their role facilitating the spread of glandular secretions) and are potentially involved in the reception of vibratory signals (Di Giulio et al., 2012).
Due to the rarity of Paussus, Platyrhopalopsis and Eu platyrhopalus species and the challenges in rearing them, sounds emitted from their stridulatory organs are still poor ly known (yet they are actively being investigated in ADG laboratory at roma tre), and the biological significance of these sounds remains a matter of speculation (Fowler, 1912;Nagel, 1987;Geiselhardt et al., 2007).Based on the observations of Nagel (1987), stridulation was reported and considered as a response to molestation (Geiselhardt ).However, based on the combined indirect evi dence described below, we predict that stridulations are involved in both intra and interspecific communication in these myrmecophilous species.

Intraspecific communication
In many insect species stridulatory organs are well known to function in intraspecific communication.Most commonly males stridulate to attract females and in these species the males have stridulatory organs and the females generally do not (but see Wessel, 2006 for a few excep tions).Observations of P. favieri reared in captivity re vealed that stridulations are also performed during the premating behavior, especially by females (Maurizi et al., 2012).Luna de Carvalho (1953) also speculated that the function of these organs could be related to mate recog nition inside crowded ant nests where acoustical signals could be more effective than visual or chemical signals.

Interspecific communication
Generally, when stridulatory organs are present in both sexes of a species, as in the case of paussines, they are as sumed to also be involved in interspecific communication (but see Wessel, 2006).For a variety of reasons presented below we suspect that the type I stridulatory organ, present in all members of the diverse genus Paussus, facilitates acoustical communication between these beetles and their ant hosts, similar to the situation demonstrated in larvae and pupae of myrmecophilous butterflies (see De Vries et al., 1993;Barbero et al., 2009a, b;Casacci et al., 2013).

stridulatory organ of type I -a key innovation
The acquisition of a key innovation often precedes an adaptive radiation (Yoder et al., 2010).One of the most apparent key innovations in Paussus is the acquisition of the type I stridulatory organs (Fig. 7).We suspect that the sophisticated stucture of the scraper (Figs 2d; 3a-d) led to more effective acoustical communication between these beetles and their hosts resulting in an increase in the rate of species diversification in the large genus Paussus (390 species) (Moore & robertson, in prep.)(see Fig 7).un like the scraper found in type II (Fig. 5c-d) and type III (Fig. 6c-d), the scraper portion of type I may represent a completely new cuticular structure, rather than modifica tions and fortifications of setae.the apical projections of the type I scraper may be the first part of this stucture to come into contact with the fine ridges of the stridulatory file and we predict they are mainly responsible for the fine scraping; however it is also possible that the whole distal edge of the spines can be rubbed aganist the file; such that eventually both parts, the trichoid apex and the distal edge, could be used to create different patterns of vibrations.
Most Paussus species, all of which have the type I strid ulatory organ, have been found in association with Myr micinae (Geiselhardt et al., 2007), especially Pheidole, whose members are able to stridulate (Moore, 2006).In both ants (Fig. 4a-f) and paussines (Fig. 2a-d), the stridu latory organs are composed of a finely ridged suboval file and some distinct prominences that function as the scraper.
But our most compelling evidence comes from direct ob servations of the interactions between Paussus favieri and its host ant, Pheidole pallidula, reared in captivity (Mau rizi et al., 2012).Males and females of Paussus favieri fre quently stridulate (exemplified by the rhythmic movement of the beetle hind legs singly or in tandem) during their interactions with the host ants.Movements of the beetle's hind legs, presumably associated with stridulations, elic ited benevolent responses from their host ants in which the minor and major worker ants antennated and actively licked the beetle.
While our knowledge of paussine host ant association is still very limited, there are interesting exceptions to this general pattern in both directions.There are myrme cophilous paussines without stridulatory organs that are known to live with stridulating host ants and some pauss ine species with stridulatory organs are known to live with non stridulating ants.Acoustic and chemical communi cation strategies most certainly vary within this diverse group.Future research aimed at furthering our knowledge of paussinehost associations as well as behavioral studies on the interactions between paussines and their hosts will certainly reveal new insight into the range and relative

Fig. 3 .
Fig. 3. Cuticular spine of the scraper of Paussus favieri (stridulatory organ type I), dissected by using FIB/SeM microscope.the micrographs show different stages (a-d) of the milling process operated by the focused ion beam.Note the thickness of the cuticle at the base of the spine (a-d), the extent of the invaginated part of the spine (b-d), the basal space allowing moderate spine deflection(c-d) and the solid nature of the spine (d).Note that the gold layer, sputtered on the cuticular surface for conductive purposes, was removed by the ions during focusing operations, except under the spines that worked as shelters (triangular whitish "shadows" under the tip of the spines).

Fig. 4 .
Fig. 4. Pheidole pallidula, exemplifying the Paussus favieri host ant's stridulatory organ.a -part of the colony showing the queen (in the middle), surrounded by workers and one soldier (bottom); b-f, SEM micrographs: b -worker specimen in left lateral view, arrows indicate the position of the stridulatory organ; c -stridulatory file of soldier specimen on meso-dorsal part of the first gastral segment (i.e., the fourth abdominal segment) showing the elliptical ridged area; d -close-up of left peripheral part of stridulatory file, showing the distinct change of the microsculpture; e -anterior part of the abdomen of soldier specimen, dissected from the gaster, showing the position of the scraper (arrow) on the posterior edge of the postpetiole (i.e., the third abdominal tergite); f -closeup of the scraper, consisting of a medial cuticular prominence represented by a downward folding of the postpetiole edge.

Fig. 5 .
Fig. 5. Euplatyrhopalus vexillifer male, exemplifying type II. a -stridulatory file in sub-basal position on left mesofemur (posterior side); b -close-up of medial ridges of stridulatory file; c -left part of metasternum showing the curved rows of pointed setiferous protuberances (scraper); d -left part of metasternum in obliquely posterior view showing the length of the cuticular setiferous protu berances of the scraper.

Fig. 6 .
Fig. 6.Platyrhopalopsis picteti male, exemplifying type III. a -stridulatory file on left part of metasterum showing the length and the fan-like arrangement of the ridges; b -close-up of medial ridges of left stridulatory file; c -strongly convex basal part of left femur showing the arrangement of the pointed setiferous keels (scraper); d -closeup of the setiferous keels of the scraper.

Fig. 7 .
Fig. 7. Summary of phylogenetic relationships of Paussini af ter Darlington (1950).Terminal clades are drawn proportional to speciesrichness.The three types of stridulatory organs in Pauss ini are mapped on the phylogeny (gray hash marks), illustrating the inferred evolutionary independence and significance of each type.