Morphological description of the last instar larva of Tipula (Pterelachisus) cinereocincta mesacantha Alexander 1934 (Diptera: Tipuloidea: Tipulidae), a newly recorded crane fly from China

Tipuloidea (crane flies) is one of the most species-rich groups of Diptera, including over 15,000 species worldwide, with a wide variety of habitats. However, relatively less attention has been paid to the larvae of crane flies in contrast to the better-known adults. In this study, Tipula (Pterelachisus) cinereocincta mesacantha Alexander, 1934 is added to the fauna of China, from larvae found beneath a piece of moss in a forest of eastern Liaoning and reared and associated with the adult stage. The external structures of the larva are described thoroughly accompanied by light microscopy photographs. In addition, with scanning electron microscopy, more detailed structures of the larval head capsule, the antenna and the mouthparts are presented for the first time for the larvae of Tipula (Pterelachisus). Some microscopic structures, including the paired peg-like sensilla on the central teeth of epipharynx, and a pair of cushiony lobes anterior to the teeth, are newly recorded for the larva of Tipuloidea. On the basis of the comparison of larval characters, the described larvae of Pterelachisus are roughly categorized into three groups. The variations of larval heads between T. (P.) cinereocincta mesacantha and other related species are also discussed. This discovery will provide new data and insights for further research on the taxonomy and phylogeny of Tipuloidea, as well as some evidence on the biology and adaptions of crane fly larvae.


Introduction
The superfamily Tipuloidea, commonly known as crane flies, is one of the most species-rich groups of the order Diptera, including over 15,000 species worldwide (Oosterbroek 2022). The larval stage of a crane fly, composed of four instars, occupies most of the life cycle just like most other dipteran insects. Crane fly larvae, also known as leatherjackets, occupy a wide variety of habitats, from strictly aquatic to entirely terrestrial (Alexander 1920;Alexander and Byers 1981;Pritchard 1983;Rogers 1942).
Pterelachisus Rondani, 1842 is one of the largest subgenera of the vast crane fly genus Tipula, composed of 205 described species worldwide, among which forty-five had been recorded from China before (Oosterbroek 2022). The larvae of Pterelachisus are terrestrial, usually found in four types of habitats, i.e., humus, rotten wood, moss on rotten wood and moss on rocks (Brindle 1960;Alexander 2002;Polevoi and Pilipenko 2016;Kramer and Langlois 2019;Gorban and Podeniene 2022). To date, only eleven species of Pterelachisus have had their larvae described and illustrated, none of which occur in China (Table 1). Most of these descriptions are focused on the anal and spiracular regions, as well as the marcoseta arrangements on the thoracic and abdominal segments.
All the described Pterelachisus species share the following features: the three pairs of spiracular lobes are relatively short, among which the lateral lobes are somewhat longer than dorsal lobes, and the ventral lobes are extremely shorter and broader; the dorsal lobe is conical with a reduced 1 3 sclerotized area at the base or below (absent in T. (P.) luridorostris), which never extended to the tip; the lateral lobe has a sclerotized area as a thin vertical line near the base, or completely disappeared, relatively dilated in T. (P.) apicispina and T. (P.) irrorata; the ventral lobe always has extensively sclerotized area on the upper surface (inner surface); anal field possesses two pairs of short and stout anal papillae; abdominal macrosetae D2 is very close to D3 and far away from D1, D4 and D6 are both much shorter than D1, while D5 is vestigial, almost invisible. Since most of these features were not unique to the larvae of Pterelachisus, while similar situations could be, respectively, observed in some other Tipula species, especially in the subgenera Lunatipula, Vestiplex, Beringotipula and Serratipula, it is still not easy to recognize the larvae of Pterelachisus by any single character (Gelhaus 1986). In addition, only a few features of the larval head capsule of Pterelachisus have been described so far (Podeniene 2003), so it remains a mystery how the head capsule varies between different species or subgenera.
In the present study, we found a brood of crane fly larvae beneath a piece of moss in a forest of Liaoning, Northeast China. After being reared to adults, they were identified as Tipula (Pterelachisus) cinereocincta mesacantha Alexander 1934, which had not been recorded from China before.
Herein, detailed morphological data on the last instar (fourth instar) larvae of T. (P.) cinereocincta mesacantha is provided using the combination of scanning electron microscopy (SEM) and light microscopy (LM). The morphological differences and variations of the larval structures between T. (P.) cinereocincta mesacantha and its related species or genera are discussed.

Insect collection and rearing
The last instar larvae of T. (P.) cinereocincta mesacantha were collected from Hemu National Forest Park, Fushun, Liaoning, China, by the author Wan-Xin Cai, on July 23rd, 2021. A total of 72 larvae were found under a group of moss (Brachythecium sp., recognized based on images) on a rock near a stream in the forest ( Fig. 1A-C).
After being brought back to the laboratory, twenty of the larvae were preserved for examination, while the other 52 were placed with the moss from the collecting site in a plastic box for rearing. Nearly a month later, 11 male and 12 female adults emerged successively. The species was then identified from the adult features, especially those of the male hypopygium, using Alexander (1934) and Savchenko (1964).  Savchenko (1954); Theowald (1957Theowald ( , 1967; Podeniene (2003) Westpalaearctic (Europe); Eastpalaearctic (West Siberia of Russia) Under moss on live or decaying wood or stone Tipula truncorum Meigen, 1830 Savchenko (1954); Theowald (1957Theowald ( , 1967; Podeniene (2003) Westpalaearctic (Europe); Eastpalaearctic (West Siberia of Russia) Under moss in low lying woody swamps Tipula varipennis Meigen, 1818 Savchenko (1954); Chiswell (1956); Theowald (1957Theowald ( , 1967 All the immature and adult specimens studied in this work were deposited in the insect specimen room of the College of Plant Protection, Shenyang Agricultural University (SYAU), Shenyang, China.

Morphological study
The twenty last instar larvae were fixed in hot Dietrich's solution (formalin: 95% ethanol: glacial acetic acid: distilled water = 6:15:1:80, v/v), standing for 24 h under a fume hood at room temperature before being preserved in 75% ethanol, following the method of Jiang and Hua (2015).
Larvae were studied using a Leica EZ4HD stereoscopic microscope (Leica, Wetzlar, German). Color photographs of the larval habitus were taken by Olympus OM-D E-M5 II digital camera (Olympus, Tokyo, Japan). Other color photographs were taken by Nikon SMZ 25 stereoscopic microscope (Nikon, Tokyo, Japan) or Keyence VHX-7000 digital microscope (Keyence, Osaka, Japan).

Head
The head capsule (Fig. 3A, B) is hemicephalic, oval in dorsal view, with length 1.59-1.62 mm, width 1.01-1.05 mm (n = 15), slightly depressed dorsoventrally, bearing a pair of antennae and a group of mandibulate mouthparts directed anteriorly. The posterior part of the head is held in place by the cervical membrane at the articulatory fold. Most parts of the head are dark brown or black, except for the oval less pigmented parts on lateral sides. The exposed anterior parts of the head are strongly sclerotized, with the internalised posterior parts (externolateraliae and internolateralia) are less sclerotized than the exposed parts. The externolateraliae have numerous growth lines near the posterior margins. A pair of sword-shaped incisions extend nearly the posterior 2/5 of the head. Anteriorly each incision is continuous with a deep groove that extends nearly 2/3 of the head. The paired incisions separate lateral genal regions (externolateraliae) from the dorsal fragment (internolateralia) of the head (Cook 1949;Oosterbroek and Theowald 1991). Externolateraliae is widely separated on the ventral surface by a huge incision. Both genal regions are strongly sclerotized at the edges. An inconspicuous larval eye spot is located just behind the base of each mandible.
The frontoclypeus is fused with the internolateralia. The clypeal region of the frontoclypeus is generally pale, less sclerotized, and has a pair of basal darkened areas. Three setae and two or three pores are present on each side: an anterolateral seta is the longest and close to the base of antenna; the other two are located on the lateral darkened areas with the anterior one longer and the posterior one shorter (Fig. 4A, B).
The antenna has only one segment, brownish and cylindrical, and slightly curved inward. Its length is nearly two and a half times as long as the basal width. A large coneshaped and six peg-like sensilla are located on the membranous sensory area at the tip of the antenna (Fig. 4A, B). The basal half of the antenna bears a dorso-mesal pit-like sensillum. The trapeziform labrum is separated from the clypeus region by a clypeolabral suture (Fig. 4A, B). The labrum consists of a membranous part in the middle and a pair of sclerotized lobes on each side. Each sclerotized lobe has two groups of microtrichia along the lateral edge and three long setae on the anterior part. The anterior margin of the labrum The anterior part of the epipharynx is densely covered with posteriorly directed flat hairs ( Fig. 5A-C). The lateral part of the epipharynx is reinforced by a sclerotized and slightly curved tormae, and is covered by dense hairs (Fig. 5A). A pair of rounded cushiony lobes are present on the central part of the epipharynx. Each cushiony lobe is partly covered with a group of hairs (Fig. 5B, D). Two pairs of teeth are arranged in a curved row just behind the two cushiony lobes. The mesal tooth has a pair of peg-like sensilla on the tip, and the lateral tooth has a bifid tip (Fig. 5B,  D).
The mandibles are strongly sclerotized and slightly curved inwards. Each mandible is only one-segmented and operates in the horizontal plane (Fig. 6). Four teeth are present on each mandible, including the largest one pointed anteriorly, a subapical one on the dorsal edge, and two additional (a sharp and a blunt) ones on ventral edge (Fig. 6A, B, D, E). Each mandible bears a triangular lacinia mobilis on the mesal surface. It is sclerotized, fan-shaped, and bordered by a fringe of hairs (Fig. 6C, F). On the mandible, two long setae are present on the posterolateral area, and a sensory pit is posterodorsally (Fig. 6A, B).
Each maxilla consists of a cardo, a stipes, a maxillary palp, a galea and a lacinia (Fig. 7A-D). The cardo is wedgeshaped, brown to black. A pair of setae are present on the distal end of the cardo, almost equal in length (Fig. 7A, C). The galea and lacinia are fused with the stipes. The stipes region has three sclerites. The inner sclerite extends from the ventral surface of the maxilla around the inner margin onto the dorsal surface, while the outer sclerite extends around the outer margin (Fig. 7A, B). The median sclerite is somewhat triangular, extends from the inner sclerite to the distal end of the cardo (Fig. 7A). The one-segmented maxillary palp is situated on the membranous palpifer. The tip of the maxillary palp bears a membranous sensory area, and several small peg-like sensilla are inserted apically (Fig. 7D). The galea region has a cone-shaped sensillum dorsally, and a thin seta ventrally. The lacinia region has a strong seta and a tuft of long paddle-shaped appendages. Almost the whole dorsal surface of the fused regions of the stipes, the galea and the lacinia is covered with groups of dense hairs (Fig. 7D).
The prementum is almost entirely covered by the hypostomium. It bears five round blunt teeth at the anterior edge. The middle three teeth are similar in size, and the most lateral teeth are shorter and smaller. The prementum is strongly sclerotized, except for the basal round membranous area, which is covered with dense bristles on the ventral surface, and a pair of labial palps (Fig. 8A, D).
The base of hypopharynx is fused with the prementum (prelabio-hypopharyngeal-complex) ventrally. The hypopharynx is membranous and spoon-shaped, supported below by a pair of U-shaped hypopharyngeal suspensoria (Fig. 8A, B, E). The disc of the hypopharynx is broadly covered with squamaceous hairs, while the remainder is covered with dense pubescence (Fig. 8B, E).
The toothed hypostomium (Fig. 8C, F) is a separate platelike sclerite. It is strongly sclerotized and fused with the ventral margins of the genae. The anterior margin is equipped with seven sharp teeth. The apical tooth is the most prominent and candle-shaped; the lateral three pairs of teeth are much smaller than the apical tooth. The dorsal base of the hypostomium has a group of fine hairs. The posterior part of the hypostomium is divided along the midline by a membranous area. A pair of tiny knee-shaped sclerites are present on the posterior edge (Fig. 3B).

Thorax and abdomen
The thorax and abdomen segments are generally pale yellow, covered with brown microscopic hairs. The second and third thoracic segments and the first abdominal segment are wider than each is long. The first thoracic segment and the 2nd-8th abdominal segments are longer than each is wide ( Fig. 2A-C). The dorsal microscopic hairs on the third thoracic segment, and the first and the eighth abdominal segments are longer than those on the other segments with all placed singly (Figs. 2A, 9E-F, H-I). The lateral surface has several round glabrous patches (Fig. 9G, J). The macrosetae are black basally and brown apically. They are arranged in a roughly similar way on the first to seventh abdominal segments, except for the lateral seta L4 (Fig. 9C, D). Ventral setae V4, V5 and V3 are long, three more times as long as V2. Seta V1 is very short, only half the length as V2. Setae V4 and V5 are very close to each other, and both far from V1-3 (Fig. 9A). Dorsal setae D1-3 are long; D4 and D6 are short; D5 is vestigial or absent. Setae D1-4 are basically at the same horizontal level, and D6 is vertically ahead of D4. Setae D2 and D3 are very close to each other, and they are fairly far from D1 (Fig. 9B). Lateral setae L1 and L4 are long; L2 and L3 are very short. On the first abdominal segment, seta L4 is fairly far from the group of setae L1-3, but it is close to the other three setae on the 2-7th abdominal segments (Fig. 9C, D).
A pair of prominent spiracles are present on the posterior surface of the terminal abdominal segment, together with the surrounding area, called the spiracular disc, with three pairs of spiracular lobes produced from the periphery of the spiracular disc. Dorsal and lateral spiracular lobes are both nearly conical; each lobe is fleshy and glabrous. The paired small dorsal lobes are approximately as long as wide. They are so close to each other that the distance between them is much less than the width of each lobe. A brown elongate sclerotized pattern is present just below the base of each dorsal lobe. The lateral lobe is closer to the dorsal lobe than to the ventral lobe, nearly 1.5 times as long as the dorsal lobe. A tiny spot or stripe is present at the base of the lateral lobe. The ventral lobe is short, broad triangular, about half as long as wide. The paired ventral lobes are directed outward. A transverse trapeziform sclerotized area is present on the base of the upper surface of each ventral lobe. The spiracles are black, round and strongly sclerotized. The distance between the two spiracles is approximately equal to the spiracular diameter. A small black round pit is located in the central area between the two spiracles ( Fig. 10A-D).
The anal field is a nearly inverted trapezoid in shape. Two pairs of short, round, fleshy and glabrous anal papillae are produced surrounding the anus. The lateral papillae are relatively longer than the ventral papillae. The papillae expand laterally to make the anal field slightly broader than the spiracular disc ( Fig. 10A-C).

Discussion
Based on Tipula (Pterelachisus) cinereocincta mesacantha and the other eleven described species, the last instar larvae of Pterelachisus may be roughly separated into three groups: The most distinctive features to separate the different groups are the sclerotized areas on the spiracular lobes. In the species of the luridorostris group, the sclerotized areas on dorsal and lateral spiracular lobes are very indistinct or completely disappeared. But the two members can differ from each other by the sclerotized areas on ventral lobes, which are extremely reduced to small spots at the base of the latter is somewhat like that in T. (P.) cinereocincta mesacantha. In the species of the pabulina group, the sclerotized area on each dorsal lobe is small, just located below the base of the lobe, while that on each ventral lobe is also small and never extends to the border of the lobe. In the species of the varipennis group, the sclerotized area on the dorsal lobe is longer, extending from the base of the lobe to the basal 1/3 or even further; the sclerotized area on the ventral Nevertheless, it is not easy to find other evidence to support this categorization of the larval groups of Pterelashisus. The information on the arrangement and length of the abdominal macrosetae is insufficiently known for all described species, though it has shown some variation in the subgenus. In Tipula (Pterelachisus) cinereocincta mesacantha and T. (P.) trivittata, setae V1 and V2 are both extremely short and close to V3, while in most other described species, V2 is equal to V3 in length. In T. (P.) trivittata, seta V2 is very short and significantly shorter than seta V1, while in T. (P.) cinereocincta mesacantha, V2 is slightly longer than V1.
Little research on the head capsule of Pterelashisus has been made previously (Podeniene 2003). We note the variation in the internolateralia, the incision between externolateraliae and internolateralia, the lateral darkened areas on the clypeal region of frontoclypeus, and the toothed hypostomium among the last instar larvae of T. (P.) pseudovariipennis, T. (P.) submarmorata, T. (P.) varipennis, T. (P.) truncorum and T. (P.) cinereocincta mesacantha. These might provide more evidence for discriminating the subgenus and its species after an in-depth study on the morphology of larval head capsule but that is beyond the scope of this paper.
In the present research, we used SEM technology to study the larval head of T. (P.) cinereocincta mesacantha. By contrast with the semi-aquatic species Tipula (Yamatotipula) montium, previously studied by Neugart et al. (2009)  ) montium has a group of long hairs, while the T. (P.) cinereocincta mesacantha does not. Perhaps these differences may be due to feeding in an aquatic environment (montium) versus that in a terrestrial environment (c. mesacantha).
The epipharynx of a crane fly larva has often been neglected in previous work, but has been observed with two pairs of teeth arranged in a transverse row on the disc (Chiswell 1955;Neugart et al. 2009;Oosterbroek and Theowald 1991). In T. (P.) cinereocincta mesacantha, two sensilla inserted on the mesal tooth of the epipharynx were observed in our study by SEM, which has not been recorded in previous works on crane fly larvae. In addition, a pair of fleshy cushiony lobes, anterior to the teeth on the epipharynx, is also recorded by SEM and is a new feature for tipulid larvae. It might be interesting to explore in future studies if there would be additional variations of these larval structures between different species of crane flies and what functions might they have.
In conclusion, the microscopic structures of the last instar larvae of Tipula (Pterelachisus) cinereocincta mesacantha have been investigated with the combination of the LM and SEM in this work. The morphological study of the larvae will provide new insight into the taxonomy and phylogeny of Tipuloidea, and also to the adaption and differentiation of the species in different habitats.