Larval morphology of Scydmaenus tarsatus and S . hellwigii , with notes on feeding behaviour and a review of the bibliography on the preimaginal stages of ant-like stone beetles ( Coleoptera : Staphylinidae : Scydmaeninae )

Morphology of mature larvae of two Central European species of Scydmaenus Latreille is described and illustrated: S. (s. str.) tarsatus Müller & Kunze and S. (Cholerus) hellwigii (Herbst). Inaccuracies in previous descriptions of S. tarsatus are discussed and the following combination of characters is defined as diagnostic for Scydmaenus: epicranial sutures reaching posteromedian margins of antennal insertions; presence of a single pair of stemmata and epicranial supraantennal pits; anterior row of subtriangular teeth on epipharynx; mandibles falciform and without mesal teeth; antennomere 3 rudimentary; antennal sensory appendage subconical and asymmetrical; maxilla with galea and lacinia; labium strongly constricted between mentum and prementum; thoracic tergites undivided along midline; head capsule, thoracic tergites, laterotergites and abdominal segments except sternite 1 densely setose; thoracic sternites and abdominal sternite1 largely asetose; and lack of urogomphi. We also describe the feeding behaviour of immature S. tarsatus and demonstrate for the first time that Scydmaeninae larvae can feed on live springtails and not armoured mites. In the introduction we provide a summary of the literature on all hitherto known preimaginal stages of Scydmaeninae.


INTRODUCTION
Scydmaeninae is a large subfamily of Staphylinidae, comprising nearly 4900 species in about 90 genera and ten tribes (Grebennikov & Newton, 2009).Larval morphology of ant-like stone beetles, however, is very poorly known.To date, various authors have published descriptions and illustrations of larvae of only twelve genera and less than 0.5% of known species.Some published figures are highly simplified and some misleading, with morphological structures misinterpreted or taxa misidentified [e.g., a larva of the oxyteline staphylinid Syntomium Curtis assigned to Euconnus Thomson (Schmid, 1988a;Newton & Franz, 1998)].Furthermore, descriptions based on immature stages obtained ex ovo by rearing beetles are scarce and identifications based solely on the cooccurrence of larvae and adults may be unreliable.All larval stages and the pupa are described only for a single species (De Marzo, 1984) and only for one species are the details of the chaetotaxy illustrated and described using modern methods (Wheeler & Pakaluk, 1983).Although larval characters of Scydmaeninae were recently used in an extensive phylogenetic analysis (Grebennikov & Newton, 2009) and may be crucial for clarifying suprageneric relationships within this subfamily, such data is scarce, which hampers further taxonomic study.
In 1882 Tömösvary described a family Anisosphaeridae in Thysanura for a new genus, Anisosphaera, an oddlooking arthropod from North-Eastern Slovakia.Silvestri (1899) placed this family in Anisosphaeridia, a new insect order, with possible affinities to Collembola.Bergroth (1899) recognized in Anisosphaera a larva belonging to a scydmaenine genus, Cephennium Müller & Kunze, and synonymized these names; an additional discussion supporting this action was published by Dudich (1927).The larvae of Cephennium were the first to be described and are still the best known among the Scydmaeninae, with various morphological details illustrated for four species authors in identification keys, reviews and summaries (e.g., Ghilarov, 1964;Kasule, 1966;Klausnitzer, 1978Klausnitzer, , 1997;;Newton & Franz, 1998, O'Keefe, 2005).Immature stages of Plaumanniolini, Chevrolatiini and Leptoscydmini remain unknown.
Data on the biology of immature Scydmaeninae, especially their behaviour and feeding preferences, are even scarcer.Schuster (1966a, b) observed larvae of Cephennium majus Reitter and C. thoracicum Müller & Kunze feeding and carried out prey preference experiments.These species were found to be predacious and feed mostly on the armored mites (Oribatida), and only rarely on Uropodina and Gamasida; cannibalistic tendencies among the larvae were also mentioned (Schuster, 1966b).Schmid (1988) made similar observations on immature Cephennium, Stenichnus and Scydmoraphes.De Marzo (1983) reared larvae of Palaeostigus pilifer (Kraatz) and reported that the first and second instars feed exclusively on a secretion produced by the female abdominal gland, which was deposited along with the eggs.In the laboratory mature (i.e.third instar) larvae were fed on the viscera of a caterpillar of Galleria sp.De Marzo is so far the only author to provide photographic documentation of scydmaenine larvae feeding.
Recently Ja oszy ski (2012a) reported that adults of Scydmaenus tarsatus Müller & Kunze and Scydmaenus hellwigii (Herbst) can be maintained in long-term laboratory cultures by feeding them on soft-bodied arthropods.Neither of these species showed any interest in oribatid or uropodine mites, which have heavily sclerotized cuticles, whereas both readily fed on live hypogastrurid springatils (genus Ceratophysella Borner) and acaridid mites (genus Rhizoglyphus Claparède).Scydmaenus hellwigii also fed on dead springtails and S. tarsatus on dead flies, isopods and pseudoscorpions (Ja oszy ski, 2012b).During these experiments, immature scydmaenine were collected together with numerous adults of S. tarsatus and identified as conspecific with adult beetles on the basis of previous descriptions (Meinert, 1888;Brown & Crowson, 1980) and the fact that the substrate (decaying plant matter in a garden compost heap) was inhabited only by this species of ant-like stone beetles.During previous rearing of S. hellwigii adults (Ja oszy ski, 2012a) a single larva was obtained.In the present paper the previously inadequately described larva of S. tarsatus is redescribed, and the larva of S. hellwigii is described for the first time.Moreover, observations on the previously unknown feeding behaviour of larval S. tarsatus are reported.

Material
Larvae together with numerous adults of Scydmaenus (s.str.) tarsatus Müller et Kunze, 1822 were collected from a large compost heap at W oc awek (Central Poland) by sifting in the last week of August 2010.No other species of ant-like stone beetles were found in this substrate.Some larvae were immediately preserved in 75% ethanol for morphological study, six others were placed alive in a 150 ml plastic container half-filled with compost and transferred to the laboratory for further observations.Numerous adults of Scydmaenus (Cholerus) hellwigii (Herbst, 1792) were collected from rotten wood and from under loose bark of a lime tree at Wroc aw-Wojnów (SW Poland) in the middle of April 2010, and transported to the laboratory in a similar manner.Scydmaenus hellwigii was reared as described previously (Ja oszy ski, 2012a): 20 females and 10 males were placed in a 30 cm high, 1 l cylindrical container 3/4 filled with moist rotten lime wood inhabited by a colony of small Isotomidae springtails of the genus Desoria Agassiz & Nicolet.The container was closed except for a 50 × 2 mm ventilation opening at the top and the substrate was dampened every two weeks.Every two months the entire substrate was sifted and searched for larvae and then put back into the same container together with all the inhabitants; this culture was terminated after six months, when a larva was found.The larva was preserved in 75% ethanol.Larvae of both of the species studied were identified as last instars based on their body length, exceeding that of adults by 20-30%.

Preparations
One larva of each species was macerated in warm 10% NaOH after separating the head; when soft tissues had dissolved specimens were washed in distilled water, slightly stained with chlorazol black and mounted in glycerol-gelatin.Drawings and measurements were made at magnifications up to 600× using a phase contrast compound microscope.

Scanning electron microscopy (SEM)
A larva of S. tarsatus was transferred from ethanol to distilled water through a series of 70/40/20% ethanol, cleaned for 1 min in 10% NaOH at ambient temperature, washed in water, transferred to absolute ethanol through a series of 20/40/75% ethanol, kept for 1 h in acetone and subsequently for 1 h in hexamethyldisilazane.The dehydrated specimen was mounted on a SEM stub with a carbon tab and sputter-coated with gold (Pirani 501, Edwards) and then examined using a LEO 435 VP scanning electron microscope.

Light photography and image processing
Habitus images were taken using an Olympus C-750UZ digital camera with a Raynox MSN-202 close-up lens.Image stacks were processed using Combine ZP (Hadley, 2010).Final image adjustments and annotations were made in Corel Photo-Paint.

Terminology and measurements
Terminology used for the chaetotaxy follows systems proposed by Wheeler & Pakaluk (1983), Wheeler (1990) and Kilian (2007).The total body length is the sum of the lengths of the head, thoracic and abdominal segments measured separately.The following abbreviations are used (abdominal segments, antennomeres and palpomeres are counted from base of abdomen, antenna and palp, respectively; i.e.An3L is the length of antennomere 3): AnL -length of antennomere; AbL -length of abdominal segment measured along midline, excluding lateral lobes projecting caudad; measurement of segment 10 does not include extruded anal membrane; AbW -width of abdominal segment; ASL -length of antennal sensory appendage; HLlength of head excluding the teeth on nasale; HW -width of head; LPL -length of labial palpomere; MPL -length of maxillary palpomere; MsL -mesothoracic length; MsW -mesothoracic width; MtL -metathoracic length; MtW -metathoracic width; PL -prothoracic length; PW -prothoracic width.

Observations on feeding behaviour
Larvae of S. tarsatus were maintained under laboratory conditions as described previously for adults (Ja oszy ski, 2012a), with minor modifications.Petri dishes (diameter 3 cm) half-filled with plaster of Paris were used as arenas; they were preconditioned by filling with moist compost for 2-3 days, then the compost was removed and a fresh thin, pressed layer of compost was placed on the plaster.Six larvae were observed, three in each arena; the substrate was moistened every second day with 0.2 ml of distilled water and the Petri dishes were kept at ambient temperature (22-24°C) and in the dark.Larvae were provided with a mixture of various live mites belonging to Oribatida (mostly Phthiracaridae, Galumnidae and Carabodidae), Mesostigmata (Uropodina, Parasitina) and various springtails (mostly Hypogastruridae and Tomoceridae), all collected from the compost inhabited by S. tarsatus.Prey that were not eaten were replaced every 7 days.The larvae were kept for 4 weeks and observations were made every day for long enough to witness the entire feeding process from an attack to abandoning the remnants of the prey, which takes typically 1-2 h.
Head (Figs 4,5,(10)(11)(12)(13)(14).Prognathous, nearly as wide as long, nearly half as wide as prothorax, broadest near middle, distinctly flattened; HL 0.344 mm, HW 0.364 mm, HL/HW 0.94.Small, single stemma located dorsolaterally to each antennal insertion; stemmata distinctly darker than surrounding cuticle.Epicranial stem long, almost half as long as cranium.Epicranial sutures nearly straight, V-shaped, anteriorly nearly reaching antennal insertions; accompanied by a small epicranial supraantennal pit.Each epicranial plate with about 30 mostly asymmetrically distributed setae, and a dorsolateral cluster of five dome-shaped structures (Fig. 5); frons with about 30 mostly asymmetrically distributed setae.Anterior part of frontoclypeal region (nasale) (Fig. 11) with 8 teeth of various sizes located below distinct transverse marginal ridge but well-visible in dorsal view, two pairs of fine marginal setae, three pairs of long anterior setae and two pairs of long sub-anterior setae.Ventral side (Fig. 12): posterior tentorial pits well-visible at base of mouthparts; each lateromedian part of cranium (gena) with 9-14 asymmetrically distributed setae and two pores lateral to mouthparts, posterior part of cranium asetose.Tentorium (Figs 13,14) with broad and long anterior and posterior tentorial arms and very slender and curved dorsal tentorial arms.Tentorial bridge was not found in our preparations.
Antenna (Fig. 15).An1L 0.034 mm, An2L 0.137 mm, An3L 0.012 mm and ASL 0.032 mm.Antennomere 1 short, with one dorsal seta.Antennomere 2 almost 4 times as long as 1, with long dorsal lateral seta, two long dorsal sub-apical setae, two long apical setae, one short ventral sub-apical seta, wide and short antero-dorsal conical sensory appendage and a very small conical sensillum at its base.Antennomere 3 very small and short with one short basal seta and three apical pointed processes.
Maxilla (Fig. 18).Cardines mesally fused to submentum.Stipes broad and short, with two setae, bearing large stipital projection with galea and lacinia.Lacinia with dense and long apical trichia, its basal part and distal part of galea with dense and short trichia, base of galea with two short marginal setae; outer margin of stipital projection with one long marginal seta, its ventral surface with two sub-basal setae.Maxillary palp inserted on distinct palpifer with one seta; three-segmented, MP1L 0.037 mm, MP2L 0.050 mm and MP3L 0.081 mm; palpomere 1 slightly longer than wide, asetose; 2 elongate and slightly narrowing towards apex, with two sub-median setae; 3 strongly elongate and slender, nearly straight, with single basal seta.
medially and apically and one strikingly long subapical seta.Femur strongly elongate, on each leg with 5-6 setae.Tibiotarsus strongly elongate, as long as femur or slightly longer (metathoracic legs), on each leg with 11 setae and fine apical spines (Fig. 7).Pretarsus (Fig. 7) long and curved, pointed, with two setae.

Feeding behaviour of immature Scydmaenus tarsatus
The only prey observed to be attacked and eaten by larvae of S. tarsatus were live springtails: Ceratophysella denticulata (Bagnall, 1941)  Although after 7 days some mites were found dead, their bodies were apparently intact and undamaged.Larvae did not show any interest in mites present in the arena, but readily attacked springtails.All Collembola attacked and devoured were smaller than the Scydmaenus larvae, typically about 1-1.5 mm in length.Beetle larvae actively searched for prey by patrolling the arena and the springtails they encountered were usually attacked from behind or from the side.During four weeks, the entire feeding from attack to abandoning the remains of the prey was observed 22 times.In each case the first contact with maxillary palps was immediately followed by a quick grip with mandibles; the feeding time depended on the size of prey captured and was from about 20 to 50 min.During this time the prey showed irregular movements for several minutes.The Scydmaenus larvae frequently changed their grip on their prey during feeding (often turning the springtail upside down and consuming it from the ventral surface) and chewing intensively with their mandibles.The prey was devoured nearly completely; the abandoned remains were not recognizable as a springtail.

Description of mature larva of Scydmaenus hellwigii
Body (Fig. 56).Total body length 2.31 mm.Strongly elongate, sub-cylindrical and only slightly flattened, more convex dorsally than ventrally, nearly parallel-sided up to abdominal segment 2, remaining part of abdomen gradually narrowing; pigmentation creamy-white; integument weakly sclerotized, densely setose, all setae simple (i.e., non-modified, slender and pointed at apex) and with simple (i.e., not papillate) insertions.Tergal and sternal plates indistinct and undivided along midline.Microsculpture absent except for fine isodiametric granulation on bases of abdominal segments 9 and 10 and most of the surface of abdominal sternite 9.There are no microtrichia on the body segments.Head .Prognathous, nearly as wide as long, only slightly narrower than prothorax, broadest slightly posterior to middle, distinctly flattened; HL 0.264 mm, HW 0.269 mm and HL/HW 0.981.Small, single stemma located dorso-laterally and posteriorly to each antennal insertion; stemmata distinctly darker than surrounding cuticle.Epicranial stem long, about half as long as cranium.Epicranial sutures nearly straight, v-shaped, anteriorly nearly reaching antennal insertion, accompanied by a small epicranial supra-antennal pit.Each epicranial plate with 40-50 mostly asymmetrically distributed setae; frons with ca. 25 setae.Anterior part of frontoclypeal region (nasale) (Fig. 58) with five large teeth located below distinct transverse marginal ridge and not visible in dorsal view, one pair of fine marginal setae, three pairs of long anterior setae and two pairs of long sub-anterior setae.Ventral side (Fig. 59): tentorial pits not visible; each latero-median part of cranium (gena) with ca. 10 asymmetrically distributed setae and one pore lateral to mouthparts; posterior part of cranium asetose.Tentorium similar to that in S. tarsatus (not shown).
Mandibles (Fig. 62).Slightly darker than body, symmetrical, each falciform, with broad base and slender Maxilla (Fig. 63).Cardines mesally fused to submentum, each with one seta.Stipes with two setae and large projection bearing lacinia and galea.Lacinia with dense, long apical trichia, its basal part and nearly entire surface of galea with sparser and shorter trichia; base of galea with two short ventral setae; outer margin of stipital projection with one marginal seta, ventral surface of stipital projection with one sub-basal seta.Maxillary palp threesegmented, inserted on distinct palpifer with one seta; MP1L 0.031 mm, MP2L 0.051 mm and MP3L 0.055 mm; palpomere 1 short and nearly as long as broad, asetose; 2 strongly elongate and narrowing towards apex, with two sub-median setae; 3 strongly elongate, slender, slightly curved, asetose.

DISCUSSION
The larva of Scydmaenus tarsatus was described by Meinert (1888) and his illustrations were redrawn several times (e.g., Ganglbauer, 1899;Larsson, 1941;Kühnelt, 1961;Ghilarov, 1964;Klausnitzer, 1978), in some cases losing or even gaining some details.Meinert's description (in Danish) is rather brief, but the genus can be determined using the figures, which are fairly accurate except that showing the stipital projection with a single apical lobe, instead of the two distinct lobes reported in the present study.Some additional details provided later by Brown & Crowson (1980) disagree with the previous description.These authors state that the ocelli are absent, the stipital projection is illustrated with a simple subtriangular apex and the pygopod (i.e., the abdominal segment 10) with a distinct pair of lateral projections, apparently inserted on the extruded anal membrane.The present study confirms Meinert's observations concerning the presence of a pair of small stemmata (Fig. 10) and the absence of any lateral lobes or projections on the abdominal apex (Figs 48,49).Moreover, the nasale illustrated by Brown & Crowson is devoid of teeth, while in a slide preparation and in SEM images a row of small subtriangular teeth can be seen (Figs 4,10,11).
The larva of Scydmaenus tarsatus is unique among known Scydmaeninae in having distinctly separated and strongly expanded lateral parts on thoracic and abdominal segments, the protergite with a large anterior part separated from the tergal plate, the nasale with a row of teeth 598 visible in dorsal view, a very large number of setae that are mostly asymmetrically distributed and inserted on distinct papillae.In general body form this larva highly resembles immature Scydmaenus tachyoryctidis (Jeannel & Paulian, 1944) and S. longicollis (Böving & Craighead, 1953).However, the newly described larva of Scydmaenus hellwigii (Fig. 56) strikingly differs from the nearly onisciform S. tarsatus in having a more cylindrical body, without expanded and demarcated lateral parts on the segments, its setae have simple (i.e., not papillate) insertions and abdominal segment 9 is strongly elongate, not transverse (major differences are listed in Table 1).The subcylindrical body shape resembles that of a cyrtoscydmine species, Stenichnus turbatus Casey (Wheeler & Pakaluk, 1983), but Scydmaenus hellwigii clearly differs in a number structures, e.g., medially undivided thoracic tergites (divided in Stenichnus), much denser and asymmetrically distributed setae, anterior parts of epicranial sutures adjacent to posteromedian (and not anteromesal) margins of antennal insertions, the antennomere 3 rudimentary (and not nearly half as long as 2) and the two lobes of maxilla with dense microtrichia (and not a single apical lobe bearing only several robust setae).The larva of Scydmaenus hellwigii shares many more characters with S. tarsatus than with any of the known immatures of species of Eutheiini, Cephenniini, Cyrtoscydmini, Mastigini, Leptomastacini or Clidicini.Larvae of S. tarsatus and S. hellwigii can be distinguished from all remaining Scydmaeninae by the following set of characters, diagnostic for Scydmaenus: the epicranial sutures reach posteromedian margins of antennal insertions; the presence of epicranial supra-antennal pits and a single pair of stemmata; the anterior margin of epipharynx with a row of subtriangular teeth; the mandibles falciform, without additional teeth on the mesal margin; the antennomere 3 rudimentary; the antennal sensory appendage sub-conical and asymmetrical; the maxillae with two lobes; the labium strongly constricted between the mentum and prementum; thoracic tergites undivided along midline; the head, thoracic tergites, laterotergites and abdominal segments except sternite 1 densely setose; thoracic sternites and abdominal sternite 1 largely asetose; and lack of urogomphi.
Scydmaenus is a highly diverse, speciose and cosmopolitan genus divided into about 30 subgenera (Newton & Franz, 1998); it is even more heterogeneous than Euconnus, the largest and taxonomically most challenging scydmaenine genus.Ja oszy ski et al. ( 2012) recently demonstrated that adults of S. tarsatus and S. hellwigii, although externally similar, differ strikingly in the architecture of their cephalic central nervous system; also structures on the metaventrite are clearly different between the nominotypical subgenus of Scydmaenus and Cholerus (Ja oszy ski, unpubl.observ.).It is therefore not surprising that the larvae of these species differ significantly.
This study, for the first time, demonstrates that scydmaenine larvae can feed on Collembola.Under laboratory conditions, the larvae of S. tarsatus showed no interest in mites and fed only on springtails.Ja oszy ski (2012a, b) reports that adults of this species ignore armoured oribatid and uropodine mites, but feed readily on softbodied acaridids and springtails (including Ceratophysella and Tomocerus, accepted also by the larvae) and scavenge various dead arthropods.Although their behaviour or food preferences may have been affected by laboratory conditions and in nature they may prefer other prey, it is clear that both adults and larvae of S. tarsatus are capable of capturing and feeding on springtails.Since the mouthparts of adult and immature Scydmaenus show a number of differences, this observation is important for understanding the evolution of morphological adaptations for feeding on a particular kind of prey, characteristic of some Scydmaeninae [e.g., Cephenniini (Schuster, 1966a, b;Schmid, 1988a, b)].

TABLE 1 .
Major morphological differences between larvae of S. tarsatus and S. hellwigii.