Morphological and Micromorphological Description of the Larvae of Two Endemic Species of Duvalius (Coleoptera, Carabidae, Trechini)

Simple Summary The Duvalius cave beetles have a wide distribution in the Palearctic region. They have distinct adaptations to life in soil and subterranean habitats. Our present study intends to extend the knowledge on the morphology of cave Carabidae by describing two larvae belonging to different species of Duvalius and the ultrastructural details with possible implications in taxonomy and ecology. These two species are endemic for limited areas in the northern and north-western Romanian Carpathians. Our study provides knowledge on the biology and ecology of the narrow endemic cave beetles and their larvae are important in conservation and to establish management measures. Endemic species are vulnerable to extinction and, at the same time, an important target of global conservation efforts. Abstract The morphological and ultrastructural descriptions of the larvae of two cave species of Trechini—Duvalius (Hungarotrechus) subterraneus (L. Miller, 1868) and Duvalius (Biharotrechus) paroecus (J. Frivaldszkyi, 1865)—are presented in this paper. The interest in studying these larvae lays in their rarity and the limited distribution of the Duvalius species. The larvae were collected from caves in the Romanian Carpathians and were examined under a stereomicroscope and scanning electron microscopy. New important taxonomical and fine morphological characteristics are discussed together with conclusions on the larvae microhabitat as part of the measures to be taken by a proper management of caves.


Introduction
Duvalius (Delarouzée, 1859) species have distinct adaptations to life in soil and subterranean habitats, as do many species of the subtribe [1]. Duvalius has a wide distribution in the Palearctic region. Representatives of this genus were mentioned from Maghreb (Algeria), the western part of Europe in Spain (with some species in Catalonia and Mallorca) and France, central and southern Europe, Italy (especially in the Alps) but also Sicily and Sardinia, the Balkan Peninsula and the Carpathian Mountains. Towards the east, Duvalius is present from the Caucasus and the Middle East (Iran) to East China (Tien Shan Mountains) [2,3].
Duvalius consists of a complex [4] of troglomorphic genera [3], which were derived from local surface populations. Diverse ecological preferences and troglomorphism determined a speciose genus with 359 species [3,5], along with the large distribution range. Most species descriptions are based on the morphological description of the imago stage [6], and

Sampling Sites
A larva of Duvalius was collected on 21 March 2019 (legit. C. Sitar, R. Năstase-Bucur) in the entrance zone of the Izvorul Tăus , oarelor Cave (Rodna Mountains, northern Romania; Figure 1). The cave is located at 942 m a.s.l. and has a length of 8650 m, on a level difference of 329 m [27]. The habitat where the larva was collected is a descending slope with collapses and sediments, with dripping water, at about 100 m from the entrance. In the cave, adults of D. (Hungarotrechus) subterraneus (L. Miller, 1868) were also found and identified. The cave is protected under the Romanian legislation (O.U.G. nr. 57/2007) and considered as a scientific reservation with limited access.
In a passage of Varnit , a Les , ului Cave (Pădurea Craiului Mountains, north-western Romania; Figure 1) a larva was collected on 19 January 1996 (legit. O. T. Moldovan). The cave is located at 520 m a.s.l. and has a development of 124 m [28]. The larva was found at about 100 m from the entrance, in the same place as adults of D. (Biharotrechus) paroecus (Frivaldszkyi, 1865), on the muddy cave floor, covered with humid degraded wood pieces.

Microscopy Analysis
The specimens were examined under a stereomicroscope OLYMPUS SZX16 (OLYM-PUS CORPORATION, Hamburg, Germany) and for details under an OLYMPUS BX51 (OLYMPUS CORPORATION, Hamburg, Germany) microscope. All drawings were prepared using camera lucida mounted on both microscopes. Measurements were made using a micrometer. The nomenclature and the location of pores and setae used here are according to those proposed by Bousquet and Goulet [18], Makarow [19] and Grebennikov and Maddison [20]. 021, 10, x FOR PEER REVIEW 3 of 26

Microscopy Analysis
The specimens were examined under a stereomicroscope OLYMPUS SZX16 (OLYM-PUS CORPORATION, Hamburg, Germany) and for details under an OLYMPUS BX51 (OLYMPUS CORPORATION, Hamburg, Germany) microscope. All drawings were pre- The two larvae were also prepared for scanning electron microscopy (SEM) using the turbomolecular pumped coater Quorum Q150T ES (Quorum Technologies, Laughton, UK), from the Integrated Electron Microscopy Laboratory (LIME) of the National Institute for Research and Development of Isotopic and Molecular Technologies (INCDTIM), Cluj-Napoca, Romania, and examined with an SEM Hitachi SU8230 (Hitachi Group, Tokyo, Japan) (LIME-INCDTIM, Cluj-Napoca, Romania). The preparation was carried out by Dr. Lucian Barbu.

Description of Duvalius (Hungarotrechus) subterraneus (L. Miller, 1868) Larva
We assumed that this individual was a third instar larva based on the comparison with the data from the literature and with the larva described after.

Head
Longer than wide; flat; (length (HL): 1 mm; width (HW): 0.83 mm; ratio HL/HW: 1.20) anophthalmos; parallelized without the very slight constriction in the middle of the posterior lateral region as in other Trechinae; without postocular and epicranial grooves, any other structures (egg-bursters and teeth-like) are absent. Short epicranial suture, as long as one-fourth of the head width. Frontal sutures clearly visible, deeply curved and sinuous. Antennal ring strongly separated ( Figure 2D,E). Microsculpture of the integument of the frontal and the parietal sclerite is clearly defined ( Figure 2E,F). The frontal sclerite microsculpture is isodiametric ( Figure 2G) and the microsculpture of the parietal sclerite is transverse ( Figure 2H).

The Nasale (Anterior Margin of Epistome)
Is tri-lobed and almost symmetrical; with one median tooth and another seven teeth on each side; median lobe largest and medially protruding, projecting forwards ( Figure 2I). With four setae (NA 1-4) in the insertion area of maxilla on both sides of head and three pairs setae (NA 5-7), short and thick ( Figure 2J,K).

Labium
Labial palps with two segments, as in other representatives of the Trechini, the second one subdivided into three apparent segments ( Figure 7A,E,G). Prementum densely pubescent on the dorsal side ( Figure 7C,D) and bearing six additional setae (LA1-LA6), five on the lateral margins and one pore (LAa) on each side ( Figure 7A-D); the brushes have numerous digitiform diverticula along their entire length ( Figure 7D); seta LA6 is half the length of the first segment of the palp. The second segment with a striated structure, a sensory complex according to Makarov and Koval (2003). On first pseudo partition surface with four digitiform sensilla (Sdig 1-4) with long pores ( Figure 7E,H) and near Sdig3 one campaniform sensillum (Scam 1) ( Figure 7H,K); at the end of the first pseudo partition two campaniform sensilla (Scam 2-3) ( Figure 7G); on the second pseudo partition three campaniform sensilla (Scam 4-6) ( Figure 7G); on the third pseudo partition, in its middle length, one campaniform sensillum (Scam 7) ( Figure 7G,J) and on the apex six campaniform sensilla, and six basiconic sensilla (Sbas) ( Figure 7F-I).

Pygidium
With conical shape, 24 setae ( Figure 9D,E) and the presence of two membranous structures, retractable pseudopods in the anal tube with sclerotized teeth ( Figure 9F). The skin of both organs has many rough surfaces ( Figure 9C,E). Biology 2021, 10, x FOR PEER REVIEW 12 of 26

Description of the Duvalius (Biharotrechus) paroecus (J. Frivaldszkyi, 1865) Larva
We assumed that this individual was a second instar larva based on the comparison with the data from the literature and with the larva described previously.

Head
It is as long as it is wide; flat; (HL: 0.8 mm; HW: 0.83 mm; ratio HL/HW: 1) anophthalmos; parallelized without a very slight constriction in the middle of the posterior lateral region as in other Trechinae; without postocular and epicranial grooves, any other structures (egg-bursters and teeth-like) are absent ( Figure 10B). Short epicranial suture, as long as one-fourth of the head width. Frontal sutures clearly visible, deeply curved and sinuous. Antennal ring weakly separated ( Figure 10B-D). Microsculpture of the integument of the frontal and the parietal sclerite is clearly defined. The frontal sclerite microsculpture is isodiametric, and the microsculpture of the parietal sclerite is transverse. On the ventral side, microsculpture of the integument respects the same distribution ( Figure 10E,F).

The Nasale (Anterior Margin of Epistome)
Is tri-lobed and almost symmetrical; median lobe largest and medially protruding, strongly projecting forwards, with two median teeth and another 11 teeth on each side; the teeth are arranged in layers ( Figure 10G

Mandible
Narrow, regularly and moderately curved, without additional teeth, only with a small excision in front of retinaculum ( Figure 13A); with two setae (MN1-MN2) and one pore (MNa; Figure 13B); MN1 is long, MN2 short and conical sensillum chaeticum as in the previous species ( Figure 13C); penicillum moderately developed.

Maxilla
Cardo very small with seta (MX1) not evident ( Figure 14A). Stipes with six setae (MX2-MX7) and a variable number of short setae (gMX). Lacinia absent. Galea with two segments; second larger than the first; on first segment of galea one pore (MXa); second segment with two sensilla chaetica (MX8, MX9) and three apical membranous sensilla, one campaniform sensillum (Scam), one styloconic sensillum (Ssty) and one basiconic sensillum (Sbas) ( Figure 14G). Maxillary palps with five segments as in others representatives of the tribe Trechini; it is composed by four palpomeres inserted on the basal segmentpalpiger PG. On the third segment three sensilla chaetica (Sct 1-3) and one pore (MXc) ( Figure 14B,C); fourth segment with six campaniform sensilla (Scam 1-6) located at the distal end of the segment ( Figure 14B,E); on the fifth segment one sensillum chaeticum, three sensilla digitiformia (Sdig) with long pores, and on the apex and on the apex six campaniform sensilla, and six basiconic sensilla ( Figure 14D,F) incompletely formatted.

Mandible
Narrow, regularly and moderately curved, without additional teeth, only with a small excision in front of retinaculum ( Figure 13A); with two setae (MN1-MN2) and one pore (MNa; Figure 13B); MN1 is long, MN2 short and conical sensillum chaeticum as in the previous species ( Figure 13C); penicillum moderately developed.

Labium
Labial palps with two segments, as in other representatives of the tribe Trechini. The second segments subdivided into three apparent segments ( Figure 15A,D). Prementum densely pubescent on the dorsal side and bearing four additional setae, three on the lateral margins (LA1-3; Figure 15A,B) on each side; the brushes have numerous digitiform diverticula as well as the species described above ( Figure 15C). On first pseudo partition surface with two sensilla digitiformia with long pores (Figure 15D,E) and three campaniform sensilla ( Figure 15D); on the second pseudo partition three campaniform sensilla and one sensillum chaeticum ( Figure 15F,G); on the third pseudo partition, in the middle of the length one campaniform sensillum ( Figure 15D) and on the apex six campaniform sensilla (Scam 1-6), and nine basiconic sensilla (Sbas1-9; Figure 15H).

Maxilla
Cardo very small with seta (MX1) not evident ( Figure 14A). Stipes with six setae (MX2-MX7) and a variable number of short setae (gMX). Lacinia absent. Galea with two segments; second larger than the first; on first segment of galea one pore (MXa); second segment with two sensilla chaetica (MX8, MX9) and three apical membranous sensilla, one campaniform sensillum (Scam), one styloconic sensillum (Ssty) and one basiconic sensillum (Sbas) ( Figure 14G). Maxillary palps with five segments as in others representatives of the tribe Trechini; it is composed by four palpomeres inserted on the basal segmentpalpiger PG. On the third segment three sensilla chaetica (Sct 1-3) and one pore (MXc) ( Figure 14B,C); fourth segment with six campaniform sensilla (Scam 1-6) located at the distal end of the segment ( Figure 14B,E); on the fifth segment one sensillum chaeticum, three sensilla digitiformia (Sdig) with long pores, and on the apex and on the apex six campaniform sensilla, and six basiconic sensilla ( Figure 14D,F) incompletely formatted.

Labium
Labial palps with two segments, as in other representatives of the tribe Trechini. The second segments subdivided into three apparent segments ( Figure 15A,D). Prementum densely pubescent on the dorsal side and bearing four additional setae, three on the lateral margins (LA1-3; Figure 15A,B) on each side; the brushes have numerous digitiform diverticula as well as the species described above ( Figure 15C). On first pseudo partition surface with two sensilla digitiformia with long pores (Figure 15D,E) and three campaniform sensilla ( Figure 15D); on the second pseudo partition three campaniform sensilla and

Leg
With one claw ( Figure 16A,B), similar to that of the previous species.

Leg
With one claw ( Figure 16A,B), similar to that of the previous species.

Urogomphi
Is similar to that of the previous species ( Figure 16C). In dorsal view, with eight long setae (UR1-UR8) on each side ( Figure 16D); on the surface numerous sensilla chaetica (Sct) ( Figure 16F).

Pygidium
Is similar to that of the previous species ( Figure 16C). With conical shape, 24 long setae, numerous short setae and two membranous retractable pseudopods in the anal tube with sclerotized teeth. The skin of both organs has many rough areas ( Figure 16E-G).

Discussion and Conclusions
The larvae of these specialized troglobiotic (cave-adapted) carabid are difficult to find, even in the caves where adult individuals are abundant [10,30] (O.T.M., personal observations). Yet, another difficulty is to determine the presence or absence of sets or pores under an optical microscope for comparison with the classical description [2,15,18]. From our observations, the breaking of the setae at the time of collection and preparation can lead to confusion in identifying the pores and determining the number of setae under the optical microscope. Despite their importance, there are a few studies related to the ultrastructural organization of the head and appendages, as well as the sensilla present on these structures [22][23][24][25][26].
The larval morphology of the Duvalius larvae described in this paper conforms to the diagnosis of the Trechini by the absence of lacinia, presence of one claw, division of the apical palpal segments into apparent segments [7,13,18,[31][32][33][34] and chaetotaxy [35]. The Duvalius larvae also have a set of apical and subapical sensilla on the antennae that are characteristic of the tribe [7], and the presence of secondary setae on the frontal part of the cephalic capsule [20]. The main morphological differences between the Duvalius larvae were considered to be the length/width ratio of the cephalic capsule, the shape of the nasale and slight differences in the development of setae [10]. These differences were also observed in the two described larvae, although it is not certain that they are individual variations and do not reflect inter-species variations. Therefore, we analyzed microstructures of Duvalius larvae, frequently overlooked due to the lack of SEM analysis, and that can provide more reliable taxonomic characters. The two larvae described here, D. subterraneus and D. paroecus, have a different chaetotaxy of the cephalic capsule (Figures 9 and 11). These fine structures, such as nasale, number and arrangement of setae on the mandible or sensilla complex, represent structural elements of taxonomic importance and show the specialization of an organ such as the mandible, antenna and labium to subterranean microhabitats. The larvae are often found under rocks, in wet conditions, even inside the caves. The pubescence on the head and body, and the densely pubescent labium, shows possible adaptation to life on muddy soil and water film on a periodically flooded cave floor [14] or in "terrestrial-phreatic habitats" according to Jeannel [17]. The role of pubescence is to retain air bubbles in immersion conditions and to ensure floating [36]. The labial brushes observed in the two described larvae have multiple digitiform branches that increase the retention capacity of the organic particles from water [37], as also described in cave Leptodirini [38].
The nasale (2 I-K, 10 G-I) of the two species presents structural differences. The nasale of D. subterraneus is more prominent than of D. paroecus. D. subterraneus has seven setae and D. paroecus has eight. The nasale of the two species is tri-lobed (a central lobe and two lateral lobes). Each lobe has several small lobes at a fine structural level. These small lobes differ in number and shape between the two species. The central lobe of the two species is a formation consisting of two layers. The D. subterraneus first layer has four small lobes on each side. In the anterior part, between the two NA7 setae, the nasale has a slightly more prominent small lobe. The small lobes have a rounded apical part. The D. paroecus first layer has three small lobes on each side. The small lobes have a sharp apical part. The anterior small lobe (between the two NA8) is rounded and slightly contoured. The D. subterraneus second layer is an irregular formation in the anterior part. Laterally, there are three small, slightly contoured lobes on each side. The D. paroecus second layer is well contoured with five small sharp lobes on each side. In the anterior part, the second layer has six small sharp lobes.
Mandibles of the two larvae have a different number of setae (three for D. subterraneus ( Figure 5A-C) and two for D. paroecus ( Figure 13A-C). We noticed the presence of a chaetic sensillum on the inner side of the terebra at its distal third of each mandible ( Figure 5A-C and Figure 13A-C). Until now, the presence of these sensilla has not been reported in the literature. The presence of this pair of sensilla in both species suggests that this character might be present in other Duvalius species, but was not observed under classical microscopes. The arrangement of the pairs of chaetic sensilla, both on the inner and on the outer curvature, highlights the possible mechanoreceptor role of these sensilla. Thus, in addition to the role in food intake, the mandibles provide information on the mandibles' position relative to their food sources, and possible obstacles in the vicinity of the larva. Several authors have investigated the sensilla equipment of the larval head in Carabids: Altner and Bauer [22], Bauer and Kredler [23], Moore and Di Giulio [24], Li et al. [25] and Giglio et al. [26].
Attention was given especially to the ultrastructure and function of a sense organ located on the second segment. In carabid beetles, this structure is called the hyaline vesicle, and it was described by van Emden [31] and Jeannel [4]. It is a modified or complex sensilla basiconicum, very developed, and with an increased surface of the receptor membrane. This organ, with a presumed olfactory role, can receive highly diluted chemical cues [26,39]. On the last segment of the antenna there is a grooved peg sensillum, also mentioned by Altner and Prillinger [40], Keil [41], Steinbrecht [42] and Zacharuk [43], with an olfactory receptor or receptors with a combined olfactory/thermoreceptive function [40,44,45]. Two sensilla styloconica with possible thermo-hygroreceptive function were present in our larvae, as also described by Altner and Loftus [46], Steinbrecht and Kittmann [47] and Zacharuk [45]. In both Duvalius larvae described here, the hyaline vesicles and grooved peg sensillum were well represented, which indicated sensitivity to substances dissolved in the humid cave air or water.
The description of Duvalius larvae at an ultrastructural level highlights the presence of particular sensory ultrastructures with a fundamental role in the reception and transmission of information from the environment [48]. As with most beetles, carabid larvae have a large number of sensory structures located on the labial and maxillary palps, as these appendages are directly involved in the detection and recognition of food or prey by means of both tactile and chemical receptors [26]. The distal segments of both maxillary and labial palps possess very similar sensory structural units, which are the sensilla campaniformia, sensilla chaetica, sensilla basiconica and sensilla digitiformia [26]. Sensilla digitiformia are very interesting receptors with roles in thermoreception, hygroreception and CO 2 reception [49]. Although initially thought to be vibration receptors, used by larvae to determine terrain conformation [50], their number, shape and size may differ from species to species, which can be an adaptation to the used microhabitats, soil, microcaverns, caves, etc. Sensilla campaniformia and sensilla chaetica are mechanoreceptors. Sensilla basiconica have the role of contact chemoreceptors [26]. Trichoid sensilla present on galea and maxillary palps have an olfactory role [51]. Bland et al. [52] suggested that these sensilla are involved in the chemoreception of pheromones.
Our study should be completed by comprehensive phylogenetic analysis on Duvalius. It will, thus, provide valuable information on the specificity of vulnerable cave microhabitats, knowledge needed to establish proper management of caves. Knowledge on the biology and ecology of the narrowly endemic cave beetles and their larvae are important in conservation and to establish management measures. Endemic species are vulnerable to extinction and, at the same time, an important target of global conservation efforts [53,54] because they have specific climatic and environmental requirements and limited dispersal capacity [55][56][57][58][59]. Cave endemics are of particular note, for their very narrow distribution and low or no resilience to human impact, similarly to island faunas [60]. For the two caves where the new larvae were sampled, the main conservation measures are the restricted and controlled access of tourists, especially in the context of a changing climate. Increasing temperatures on the surface can have an impact on the distribution of subterranean Trechini, a group with sensitive representatives, by a shift in their preferred habitats, from more superficial to deeper habitats (bigger voids inside the karstic massifs, such as the caves) [61]. Therefore, the delimitation of paths inside caves with Duvalius or other cave species is absolutely obligatory, to avoid the microhabitats of their adults and larvae. On the surface, conservation measures must aim at conserving the forest habitats above the caves, ensuring a humid and cooler environment for the more superficial possible habitats of cave beetles and other cave species.