Larval Morphological Adaptations of Leiodes cinnamomea (Panzer, 1793) (Coleoptera: Leiodidae: Leiodinae)—Obligatory Feeder of Tuber Species

Simple Summary The family Leiodidae include beetles adaptated to many various habitats and foods ranging from saprophagous by obligatory mycophagists to true ectoparasites. The phylogeny of the family is complex and still requires research. Larval stages have different characters to adults and provide a lot of phylogentically valuable data. However, there are still gaps in our knowledge about immatures of the family, amongst others, in the tribe Leiodini, which is connected with endogean fungi. We provide the first detailed study of morphology of larval stages of Leiodes cinnamomea, which is an obligatory feeder and a pest of the economically valuable truffle. Our study found that larvae had unique morphological characteristics. This uniqueness, with larvae of all stages having a specific arching behaviour, is probably more common in this genus and is connected with endogean mycophagy and with other morphological characteristics. We present our hypothesis, based on our observation of the behaviour and morphology, that larvae of L. cinnamomea use a mosaic strategy for beetles living in ephemeral and in solid fungi. The study also showed that many other morphological features, such as chaetotaxy and length ratios of other parts of the body, confirmed the phylogenetic relationship that puts Leiodini close to Scotocryptini. Abstract Detailed redescription of morphology for three larval instars of truffle-feeder Leiodes cinnamomea, documented on scanning micrographs and illustrations, is provided. Systematic context, observations on biology and unique characteristics of larvae of the only well-described representative of Leiodini are discussed. Exoskeletal invaginations (pseudomycangia), granulation on the head and the median longitudinal row of sclerotized plates on tergites VII–IX—the unique structures among leiodid larvae—were confirmed, described and documented. A mosaic strategy for beetles living in ephemeral and in solid fungi is discussed.


Introduction
Leiodes cinnamomea (Panzer, 1793) is a member of one of the most species-rich genera of Leiodinae, numbering about 200 species [1][2][3]. The genus has Holarctic distribution [4][5][6][7], with some species also found in the northern part of Neotropical and in the Oriental region [3,[8][9][10][11][12], as well as some in South Africa [13,14]. It is thought that all members of the genus, as well as other genera of Leiodini, feed on subterranean fungi and, as with most Leiodinae, are obligate mycophages [15,16]. Many species are thought to develop in mycelia in the roots of grasses and rhizoids of mosses [13].

Breeding
Individuals of L. cinnamomea were bred in plastic or metallic boxes (ca. 10 cm/10 cm/ 5 cm) filled with alkali soil (a mixture of soil from the collection locality and soil from other sources with natural chalk added) and supplied with mature T. aestivum collected with the L. cinnamomea adults. The boxes were kept at a constant temperature of 10 • C and were checked frequently (every 1-2 days) in order to capture the key moments in the life cycle of the beetles. Larvae were kept together with adults and fed with the same medium. Eggs, larvae of all instars, Acarina and larvae of Diptera were collected and preserved in 75% ethanol; nematodes were preserved into 99% ethanol.

Preparation
Larvae and eggs were preserved in 75% ethanol. Two specimens of the first instar, two of the second instar and one of the third instar were macerated in warm 10% KOH and washed in distilled water. The heads of some specimens were separated before maceration and mouthparts were separated and dissected. All parts of the body were mounted in glycerol-gelatin.

Light Photography and Image Processing
Drawings, measurements and photographs of details were made at magnification up to 1000× using a Nikon ® Eclipse 80i Phase Contrast microscope (Tokyo, Japan) with a drawing tube. Photographs of morphological details were made by a Nikon Coolpix ® 4500 digital camera (Tokyo, Japan). Photographs of habitus were made under an Olympus SZ2 ® stereo microscope with an Olympus SC30 ® digital camera (Hamburg, Germany). Image stacks were processed using Combine ZP ® (Derby, UK).

SEM Preparation
One example of the third, second and first instar larva was dehydrated in a graded series of ethanol baths, dried using hexamethyldisilazane, mounted on an SEM stub with a carbon tab, coated with gold using Leica EM ACE250 (Wetzlar, Germany) and examined with a ZEISS EVO ® LS15 scanning electron microscope (Oberkochen, Germany) at magnification up to 20.000×.

Terminology and Measurements
Terminology used for the chaetotaxy follows systems proposed by Ashe and Watrous [29], Wheeler [30] and Kilian [31,32]. Table 1 provides average measurements of three larval instars.

Biology
Eggs ( Figure 1A) were laid separately, directly in the ground or on the surface of truffle, at random locations. Different larval stages were simultaneously found on, inside and around the truffle body and in the soil. Larvae moved very quickly and used pygopods for moving and for defense ( Figure 1B-F). They bent up their abdomen, straightened their head and moved large mandibles. Mature larvae (Figure 2A,B) made cells for diapause and pupation [33] in the fungi using corridors in the hymenium ( Figure 2C), which were closed by a cup formed from the hymenium chewed by the mandibles ( Figure 2D) or in the soil ( Figure 2E,F). A complete life cycle from an egg to adult, in the laboratory, at 10 • C, is not known, as we did not observe pupation.
We also observed other invertebrates: numerous representatives of Acarina Zerconopsis remiger (Kramer, 1876) ( Figure 3A), some dipteran larvae Bradysia smithae Menzel & Heller, 2005 [34] and numerous nematodes ( Figure 3B) on the truffle body. However, we did not observe interactions between L. cinnamomea and these organisms, except one incidence of nematodes in the cranium of a dead larva of L. cinnamomea.
Total body length: Instar I: 4.5 mm. Instar II: 6.15 mm. Instar III: 10 mm. White, with thin smooth cuticle, with large head (proportionally to the body). Head with very small antennae and without stemmata and characteristic granulation on frontal part and paired sclerotized invaginations below antennal. Large asymmetric mandibles with reddishbrown apices (Figure 2A,B). Larvae in all instars densely setose with small setae on ventral side of abdomen. Setae simple, brownish. Each segment of abdomen divided into tergite (mediotergite) and laterotergite with annular spiracle. Tergites (mediotergites) undivided medially, without carina, with paired reservoirs present and with two pairs of long posterior setae. Sternites with three pairs of long, posterior setae and numerous minute setae on mediosternites (only in instar III) and group of minute setae on laterosternites, which are less isolated than laterotergites. Asperities arranged into transverse short rows on mediotergites and mediosternites. Abdominal mediotergites VII-IX with medial row of sclerotized plates. Very small two-segmented urogomphi on abdominal segment IX. Brownish tarsunguli of legs.
Anal membrane. Short and wide. Instar I. Chaetotaxy: dorsal side with two long setae (D1); ventral side with two long setae and three medium setae. Instar II ( Figure 13F). Instar III. (Figures 15A,B and 16A

Discussion
Compared to the other leiodid larvae, which are mostly campodeiform [2,32], L. cinnamomea has very soft bodied, grub-like, large larvae (achieving a length of 1 cm in the final instar). They have a c-inverse shaped body, with a large cranium and large, strongly sclerotized mandibles, short legs and urogomphi: all these characteristics have an adaptive role for subterranean mycophagy [35].
Head morphology of L. cinnamomea is worth mentioning since it possesses two unique structures for leiodid larvae: granulation ( Figure 7E,F) and paired invaginations (Figures 7C,D and 8D-F) on the dorsal side of the cranium. Granulation, regular, present frontally, and in all instars, seem to have a stridulatory function, discussed below. Invaginations also presented in all instars and were prolonged dorsal mandibular articulations placed symmetrically near the basement of antennae. Such cuticular pockets on the head, documented here for the first time, among leiodid larvae, are known in the adult beetle of Curculionidae (Scolytinae and Platypodinae) for transporting of symbiotic fungi [48]. Exoskelatal cavities are also distributed on other parts of the body, mainly in adult beetles, also in the leiodid Camiarinae [1,48]. They also occur on the abdominal tergites of the camiarini larvae of Inocatops Broun, 1893 [49]. It is believed that L. cinnamomea help in dispersion of spores and mycelium propagation of Tuber species [18,28], but functioning of these invaginations as mycangia seems to be impossible because the pockets are smaller in size than the spores (pers. obs. AK). Grebennikov and Leschen [48] suggested that such structures called mycangia-like or pseudomycangia can be reservoirs of secretions or for the thermal sensing. The last hypothesis could be an interesting explanation as to how larvae of L. cinnamomea can react to light without stemmata.
Mouthparts of the larvae of L. cinnamomea are characterized by large asymmetrical mandibles, with a brown-reddish apex and transverse ledges on mola ( Figure 10A-D,F). They are similar to illustrations of the mandibles of larvae of C. spinaculeus [37]. Another leioidid larvae-Colenisia Fauvel, 1902 and Nargomorphus filitarsis Szymczakowski, 1973 [51]-have molae with ledges but are not as similar to L. cinnamomea as the molae of larvae of the tenebrionid bark beetle Synchroa punctata Newman, 1838 (Figures 36 and 64 in [52]), and the agyrtid larva of Necrophilus hydrophiloides Guérin-Méneville, 1835 [51]. This convergence of morphology of mandibular mola should point to its adaptative role to feeding on the same substrate, but, in Staphylinoidea, changing in type of food is very often not reflected in the morphology of mouthparts [51,53]. Agyrtid Necrophilus immatures are not sporophagous [51] but are found in small decaying carcasses and feces [36]. Leioidid Nargomorphus and Creagrophorus larvae feed on spores inside puffballs (Gasteromycetes) [36,37]. Leioidid Colenisia is a facultative spore feeder of Basidiomycetes [15] and guts of tenebrionid Synchroa larvae included woody and fungali material; fungi seem to play a leading role as nutrients [52]. Most of the spores in the alimentary canals of larvae of L. cinnamomea are not crushed and have visible walls and ornamentation. This poses the question 'if undamaged spores are found in the gut, how can the beetle take nutrients from undamaged spores'?
Larvae of Zearagytodes maculifer (Broun, 1880) are obligate spore-feeders specialising on the bracket fungi Ganoderma, but ingested spores are broken in less than 10% of cases [54]. However, these ingested spores have thinner outer and inner walls and a loss of cytoplasm [55]. Larvae of L. cinnamomea probably cut hard material (peridium and hymenium of the fungus before it softens) using the strong apices of their mandibles; however, ledges of the mola crush spores only partially and uptaking of nutrients likely occurs in Z. maculifer. Although insect-fungus relationships and evolution have been investigated for decades, many questions are still unanswered and need further research.
Other parts of mouthparts-the undivided mala of the maxilla, without brushes, the protruding anterior part of labrum and the slender labium-resemble those in Scotocryptini (S. meliponae and C. spinaculeus) [37,38] and Pseudoliodini (Colenisia) [51]. However, there are a pair of unique sensilla on the ligula of maxilla ( Figure 11F), where other leiodid larvae only have setae.
Soft-bodied larvae can have abdominal segments transversely or longitudinaly divided into folds [56]. Larvae of all instars of L. cinnamomea have each segment of the abdomen divided into a main tergite (mediotergite) and laterotergite, which is the area around the spiracle. Additionally, the sternites in mature larvae also look divided, forming additional lateral folds named here as laterosternites (LS) and visible on SEM photos, although they are not really separated sclerites. Chaetotaxy of the thorax and abdomen are very simple, excluding smaller setae: two pairs of long posterior setae on tergite and three pairs of long posterior setae on sternite, long seta on laterotergit near annular spiracle; this pattern is present in all instars. Only the number of smaller setae on the head, antennae, sternites and legs is different. Setae are simple-pointed apically, similar to some of Agathidiini but different to Scotocryptini, where the setae have an expanded top and are short and robust. Chaetotaxy of the abdominal sternite and the head have the same pattern as S. meliponae [38]. Tergites of the last three segments possess a median row of large, blunt-ended, strongly sclerotized rectangular plates (RP) on the last three tergites of the abdomen ( Figure 15A,C,D): in addition to the two unique structures on the head, this is the third unique character of L. cinnamomea among larvae of Leiodidae. These structures were illustrated by Arzone [19] and were called serie di scagliette rettangolari rossicce; such structures were also described by Lyszkowski [25] for L. rufipennis, although he did not illustrate them. Along with granulation on the head, they seemed to be involved with very interesting behaviour, i.e., arching up of the abdomen over the heads and moving the mandibles. This most characteristic behaviour of larvae of L. cinnamomea was observed by Laboulbéne [26], Arzone [19] and Lyszkowski [25] for L. rufipennis. Lyszkowski hypothesized that these larvae could stridulate using these structures. Strong, regular, dense granulation of the head could be a pars stridens; the row of plates on abdomen could be a plectra [57]. Stridulation can be used both for calling and mating songs, but also as a defense sound, which, together with the higher posture achieved by arching and moving large mandibles, may serve as a warning signal. Unfortunately, we did not observe or record any act of stridulation. Arzone [19] stated that larvae arched their abdomen dependent on the intensity of stimulation (vibration, light), which was observed by AK, and MK; however, we did not observe that they could arch the body and bring the head close enough to the abdomen to produce sounds. However, there were no other explanations of the function of these unique structures.
During this behaviour, larvae have an inverse c-shape curvature of the body. Such positioning of the body was described for living subterranean Leiodinae by Newton [15], Wheeler [38], Baranowski [13] and, generally, as behaviour of subterranean mycophagists among beetles by Lawrence [35]. Most authors have interpreted it as a defensive behaviour, together with moving mandibles. Larvae probably protect themselves from other inhabitants of truffles, namely nematodes, acari and dipteran larvae [19,24,26,34,58] (pers. obs. AK, MK), but there are no reports of behaviour to suggest whether they are enemies of these beetles or incidental cohabitants of fungi. We observed Acarina Zerconopsis remiger ( Figure 3A) on the truffle body but no interactions between them and the larvae. Nematodes on ( Figure 3B) and inside truffle bodies were also very numerous (also recorded by Arzone [19]) as cohabitants of L. cinnamomea, but only one specimen was found inside the cranium of the dead larva (pers. obs. AK). Thus, it is most likely that larvae of L. cinnamomea defend against other individuals and their strong mandibles. However, neither Arzone [18,19] nor we observed aggregations of larvae. On the other hand, Baranowski [13] mentioned groups of three to eight larvae of Leiodes obesa (Schmidt, 1841) in small chambers about 4-20 cm underground. However, neither Arzone [18,19] nor we observed such aggregations of larvae. Our knowledge of the biology of subterranean fungi feeders and factors to which they are exposed is still scarce. Mycophages very often feed on ephemeral, unpredictable (spatially and temporally) sources of food [15,54,59]. Therefore, reproduction of mycophagists is often fast and is connected with a high population density (adults and immatures). Although we did not observe a high density of immatures of L. cinnamomea in the laboratory, we expect that, since this species is known to cause a lot of damage in economically valuable truffles, in some circumstances, larvae can be numerous and such defensive behaviour can be very important. Until now, the observations of Arzone [18,19] and ours (AK) have shown that adults lay only a few single eggs on different occasions (gradual egg laying), so larvae of different stages coexist.
It is also known that the duration and texture of fungi influence the fauna that inhabit them, influencing their biology, behaviour [59] and morphology, for both adult and larval [35,51,53]. Antipredator behaviours correlate with structure, durability and the place on the fungi, where beetles live, whether inside or on the surface [59]. Tough and persistent fungal structures correlate with the presence of egg clumping, egg insertion, aposematism, gregariousness, pupation within fungi and pupal aggregation [59]. Beetles living on, or in, ephemeral and soft fungi do not show aposematism, gregariousness, egg insertion, pupation within fungi nor pupal aggregation [59]. The most conspicuous antipredator behaviour among Leiodidae is present in the camiarinae larvae of Z. maculifer, which live in dense populations on wood-decaying bracket fungi (tough and persistent) Ganoderma, where larvae have aposematic pigmentation of the body and extremely long urogomphi [54,60].
Larval morphology of L. cinnamomea is characteristic of those living within fungi: its larvae are white and soft, grub-like in the last instar, C-inverse shape, with shortened legs and urogomphi, and enlarged head and mandibles. They drill corridors inside truffles and feed hymenium with spores. These features were identified by Lawrence [35] for internal feeders. However, enlarged mandibles have a reddish-brown apex, which, along with the white head, gives aposematic effect. Aposematism is a strategy for surface, persistent and tough fungi. Pupation of this species shows a mixed strategy for tough and ephemerid fungi; it can proceed inside fungi, near their surface ( Figure 2D) (pers. Obs. AK, MK) or in soil, but always in cocoons ( Figure 2E,F) [19] (pers. obs. AK).
To summarise, it seems that L. cinnamomea uses a mosaic strategy for beetles living in ephemeral and in solid fungi. Truffles, similar to other hypogean fungi, have characteristics of both of them. These fungi are quite solid and tough until maturation, when the hymenium undergoes autodigestion [35]. Are they persistent or ephemeral? Certainly, they are temporally and spatially unpredictable.

3.
Granulation and sclerotized plates seem to be correlated with arching up behaviour as organ of stridulation, although the act was not observed.

5.
Morphology of larvae, mostly head, mouthparts, tergites and ratios of antennae and urogomphi, similar to those in Agathidiini and Scotocryptini, roughly confirmed the phylogenetic relationship in the family because we do not know larval morphology of other tribes of Leiodinae. 6.
Small differences of chaetotaxy among instars but tendency toward grub-like form of body with tergites and sternites divided into folds. 7.
Mosaic strategy-for beetles living in ephemeral and in solid fungi.