Detailed description and illustration of larva, pupa and imago of Holorusia mikado (Westwood, 1876) (Diptera: Tipulidae) from Japan

Abstract Background Holorusia Loew, 1863 (Diptera: Tipulidae) is a relatively large crane fly genus with a wide distribution in the Afrotropic, Australasian–Oceanian, Eastern Palearctic, Oriental and Nearctic Regions. Although the genus is well known to include the largest crane fly species, the immature stages are, thus far, only described for the larva and pupa of the North American Holorusia hesperea Arnaud & Byers, 1990. New information In this study, we describe for the first time the egg, larva and pupae of the Japanese Holorusia mikado (Westwood, 1876). Larvae were collected from semi-aquatic habitats, from slow flowing areas of streams and small waterfalls where leaf litter accumulates; the larvae are detritivores and feed on wet, decomposing leaves. The larvae were reared to adults in the laboratory. Morphological characters of immature stages discussed with comparison with the North American H. hespera. Male and female genitalia are illustrated and described in detail for the first time.


Introduction
Holorusia Loew, 1863 is a large genus of Tipulidae, with 115 species described thus far. The majority of Holorusia species are endemic to the Oriental Region (75 species), while the remaining species occur in the Australasian-Oceanian (20 species), Afrotropical (11 species), Eastern Palearctic (15 species) and Nearctic (one species) Regions (Oosterbroek 2020). Two Holorusia species have been reported so far in Japan: Holorusia mikado (Westwood, 1876) from the Honshu, Kyushu and Shikoku Islands and Holorusia esakii (Takahashi, 1960) from Amami Island (Takahashi 1960, Nakamura 2014, Oosterbroek 2020. The genus includes the largest known crane fly species, with one specimen of "Holorusia mikado", holding The Guinness World Record (2018) as "the world largest specimen of crane fly belonging to the species Holorusia mikado" (https://www.guinness worldrecords.com/). Exact systematic position of the genus Holorusia is not clarified so far. Early studies suggested a close relationship between Prionocera Loew and Holorusia; however, it was questioned by Vane-Wright (1967). Phylogenetic analysis of combined molecular markers (28S rDNA and CAD) and morphological characters (adults, larvae and pupae), revealed a close relationship between the North-American Holorusia hespera andTipula (Nippotipula) abdominalis (Say) (Petersen et al. 2010).
The genus was revised by Vane-Wright (1967) and the following characteristics of the genus established: antenna 12-14 segmented, subserrate or filiform, with short verticils; nasus present or absent; R and R curved towards each other; axillary area of wing well developed, calypter bare; femora with terminal ctenidium (comb of black spike-like setae); male genitalia relatively simple; tergite 9 fleshy, usually deeply emarginate, with long and dense hairs; lobe of gonostylus (outer gonostylus) large, fleshy with fine, short hairs; clasper of gonostylus (inner gonostylus) also simple, dorsally curved, translucent rod, usually with divided tip; cercus strongly sclerotised, narrowed outwardly, with rounded tip; hypogynal valve moderate in length; genital fork Y-shaped with three spermathecae.

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We collected the undescribed larvae of Holorusia mikado from the Honshu and Shikoku Islands and reared them in our laboratory. Here, we describe and illustrate the last instar larva, the pupae of both sexes, habitus and terminalia of the imagos and the unfertilised egg. The morphology of the larva and pupa was compared with those of the Nearctic H. hespera.

Materials and methods
Larvae were collected by hand from a drainage ditch at the side of the road, stream banks, around small waterfalls and small springs in Japan (Fig. 1). Larvae were reared in a 20 cm diameter, round box filled with substrate collected from the larval habitats. When larvae had consumed the decaying leaves, additional leaves were added and the substrate was kept wet.
Male and female terminalia and larva head capsules were photographed in glycerol, after maceration with 10-15% potassium hydroxide (KOH) at room temperature. Pupal exuviae were cleaned in soapy water to remove dirt before photographing. A few larvae were killed by boiling water and preserved in 90% ethanol. Photos were taken using a Zeiss Stemi 508 stereomicroscope, equipped with a Canon Kiss M digital camera. Stack photos were combined using Zerene Stacker software. Illustrations were made in Adobe Photoshop CC 2019. Drawings were created by Takeyuki Nakamura.
The terminology used for larval and pupal morphological features follows Brindle (1960) and the head capsule terminology follows Neugart et al. (2009). General terminology of the Habitats of Holorusia mikado (Westwood, 1876 adult follows Cumming and Wood (2017). In the case of the wing venation, we followed the traditional venation system. The gonostylus terminology follows Ribeiro (2006).
Thorax. Yellowish-brown, with brownish and grey markings. Presutural area of scutum with three grey, longitudinal stripes; median stripe with a dark brown line in the middle; stripes surrounded with brown as in Fig. 2B. V-shaped transverse suture with triangular, dark patch with whitish peaks; surrounded by yellowish-brown area (Fig.  2B). Postsutural area of scutum with two grey stripes. Scutellum grey, with whitish corners. Mediotergite greyish-brown, with longitudinal dark brown line in the middle; laterally yellowish-brown as in Fig. 2B. Lateral part of thorax whitish-yellow to yellowish-brown in living specimens and yellowish-brown in preserved specimens; a dark brown line from the cervical sclerite to base of wing as in Fig. 2A. Base of halter yellowish; steam and knob dark brown ( Fig. 2A and B).
Wing. Tinged with brown. Base of wing dark brown; Sc and R black proximal to crossvein h, remaining veins light brown to brown. Stigma inconspicuous. Calypter bare and well developed. Venation as in Fig. 3.

Legs.
Light brown to brown. Tips of femur and tibia each with a narrow black ring. Tip of femur with comb of dark, strong setae ( Fig. 4A and B). Tip of tibia with less prominent comb of setae. Tibial spurs shorter than width of tibia; tip of spur curved (Fig.  4C); formula: 1, 2, 2. Last tarsomere slightly curved ventrally in males (Fig. 4E), almost straight in females (Fig. 4D). Base of male last tarsomere with hairy lobe (Fig. 4E and G). Female tarsal claw simple (Fig. 4D); male tarsal claw with small sharp tooth at its base and a blunt tooth at ¼ of length of claw ( Fig. 4E and G).  Wing of Holorusia mikado (Westwood, 1876). Scale bar: 1 cm.
Detailed description and illustration of larva, pupa and imago of Holorusia ...

Egg.
A female laid, deformed, unfertilised eggs (not copulated with male) 5 days after it emerged from pupa. Shape of fertilised eggs may differ than that which is described below.  Eggs shiny black, 1.1-1.2 mm long, surface without granulation; micropylar opening (micropyle) at subapical protrusion or in small pit (Fig. 6E).
Lateral sclerotised plates of labrum close situated, distance less than 1/6 of plate width ( Fig. 12A and B). Each plate with two parts anteriorly. Outer-lateral part with 10-15 spine-like setae and with two small pores, one on ventral, one on dorsal side. Innerlateral part membranous covered with hairs; one finger-like black sensory seta on the inner corner; two longer, hyaline, flattened setae laterally to black seta; additional twothree poorly visible papillae amongst hairs. Membranous part between sclerotised plates of labrum, directed ventrally, medially with a notch, covered with stronger hairs (Fig. 12A) and with two papillae, visible only in apical view. Membranous articulation point of antenna with pale, short, dense hairs. Antenna cylindrical, 3-3.5 times longer than wide; slightly curved inwards; sensory pit close to base of antenna (Fig. 12A, B).
Anterior part of clypeus weakly sclerotised. Eight setae around base of antenna; three equal in size, short setae, along outer side of frontal suture; five setae along inner side of frontal suture as: one long, pale seta near base of antenna, two small and one longer setae along frontal suture and one short seta near inner-laterally (Fig. 12B).
Mandible massive, rectangle in lateral view, with two sensory setae at base on lateral side (Fig. 13B). Two blunt teeth on inner side; lacinia mobilis almost as wide as mandible, covered with short blunt setae at base and on middle; tip with longer setae. Additional membranous lobe present below the lacinia mobilis; partly connected to Figure 11.

Figure 12.
Anterior parts of head capsule of the last instar larva of Holorusia mikado (Westwood, 1876). A. labrum, frontoclypeus and antennae after maxillae and mandibles removed, dorsal view; B. anterior part of head capsule, dorsal view; C. anterior part of head capsule, ventral view. Scale bar: 0.5 mm. Maxilla well developed; cardo triangular, slightly curved, with two pale, long setae near to distal end. Long seta with short base at membranous part of maxilla near the joinpoint of maxilla and hypostomal plate (Fig. 12C). Remaining part of maxilla formed by three sclerites. Small triangular sclerite anteriorly to cardo. Inner and outer sclerites with several membranous areas and lobes. Inner sclerite with an apical lobe, covered with long, dorsally curved seate; an inner lateral lobe, covered with long inner-dorsally curved setae; a membranous area on dorsal side near base, with apically directed setae. Outer sclerite with membranous palpifer, with tuft of hairs at the apex, four sensory pits around palp as: two on ventral side next to sclerotised area, one outerlaterally to palp, one inner-laterally to palp; palp short, with a sensory pit at base, tip with membranous sensory ring-like structure; membranous lobe at tip of maxilla densely covered with strong dorsally curved setae, two short, unequal and finger-like papillae barely visible (Figs 12, 13).
Prementum with five blunt teeth; labial area with two papillae on middle, ventral side covered by group of dense hairs originating from posterior premental margin. Hypopharynx membranous, more or less bilobed and covered by short, dense hairs. Hypophayngeal suspensoria (lateral arms of hypopharynx) sclerotised and curved apically (Fig. 14).

Thorax and abdomen.
Living specimen greenish-brown, specimen stored in ethanol blackish-brown. Dorsum covered with micro and macro setae, not forming clear patches. Micro setae less dense on ventrum. Pleural area with dense setae, especially conspicuous in specimens stored in ethanol, due to shrinkage of the pleural area. Chaetotaxy of abdominal segment II-VII as in Fig. 16B-D. Setae D6 and V1 bifid. D5 and V2 with patches of dense setae. L3 closer to L2 than to L1. Hypostomium of last instar larva of Holorusia mikado (Westwood, 1876). A, B. Hypostomium plate, ventral view; C. Hypostomium plate, apical view after maxilla and mandibles removed. Scale bar 0.5 mm. Anal division and spiracular disc. Spiracular lobes subequal in length; ventral lobe slightly longer. Lobes capable of closing and hiding spiracles. Spiracles large, more than 1/3 as wide as spiracular disc. Margin of lobe fringed with long pale setae, base of setae black. Dorsal and ventral lobes with dark line along inner margin of lobe; lateral lobe with dark line along ventral margin. Lateral and ventral lobes with short black line, 1/3 of length of lobe. Dorsal lobe with a less noticeable line along the outer margin. Tip of ventral lobe with infuscated area. Sclerotised area ventrally to spiracle narrow line, length half of width of spiracle. Shade of patterns on lobes variable amongst specimens, delicated line on dorsal lobe sometimes difficult to recognise. (Fig. 17A and B.) Sensory setae are very short, very difficult to distinguish them. It is not clear if the short setae are bifid or two separate setae arising from each alveolus.
Dorsal lobe with alveolus at tip of lobe (Fig. 17A). Lateral lobe with alveolus at tip of lobe, dorsally to short mid-line; a sensatory pit at tip of ventral line (Fig. 17E). Ventral lobe with a black spine-like papilla subapically; lobe pale around the base of papilla; additional three alveoli as: one alveolus apical to black papilla, one-one alveolus lateraly to black papilla (Fig. 17C). Two additional alveoli between base of dorsal and lateral lobes, distance between alveoli around 1/3-1/4 of width of spiracle (Fig. 17A).
Anus surrounded by seven yellowish-brown, fleshy, long anal papillae. Three papillae on lateral side, one unpaired papilla at anterior margin of anus (Fig. 18). Three lateral papillae differing in length, anterior papilla longest. In living specimens, papillae about 1.5 times longer than those in Fig. 18A. Lateral papillae bent dorsally, unpaired directed anteriorly (Fig. 18).
General colouration dirty brown. Pupal skin covered with fine particles of substrate (Fig.  19A).
Thorax. Respiratory horns 1.75 times longer than width of thorax in lateral view (Fig.  19A). Horns equal in length, ringed, apical end flattened laterally and slightly widened; with large, longitudinal opening ( Fig. 19B and C). Horn of pupae in wet environments straight; horn of specimens kept in dry condition curved ventrally. Wing sheath reaches posterior end of second abdominal segment, wing venation distinct (Fig. 19A). Leg sheaths extend just beyond posterior end of third abdominal segment. Fore leg sheath shortest, reaching posterior end of fourth tarsomere of mid-leg; hind leg longest. Last tarsomere extended in both sexes, but more prominent in males (Fig. 19E). Tarsal claw sheath prominent in males (Fig. 19E), less in females.

Larval habitat and biology
Holorusia mikado larvae were collected from aquatic and semi-aquatic habitats. The larvae were found along the banks of mountain streams, waterfalls and in drainage ditches at the side of the road, where decaying litter accumulated and the water flow was relatively slow (Fig. 1). Larvae of Pedicia (Pedicia) spp. and Tipula (Platytipula) sp. were collected together with H. mikado in the same microhabitat (Oguni, Honshu Is.). The larvae of Holorusia are detritivores, feed on wet, decomposing leaves. They prefer thinner, softer leaves like maples (Acer spp.) over harder leaves. The habitat niche of the larvae is very similar to those of the Tipula (Acutipula) maxima Poda, 1761 speciesgroup in the Western Palearctic Region. Both groups prefer banks of smaller streams where litter accumulates. The general appearance of larvae of the these two groups are also similar, as in the elongated anal papillae and the general shape of the spiracular field, with relatively long setae on the margin of the spiracular lobes. Both features are characteristics of semi-, to almost freely-aquatic tipulid larvae (Brindle 1957, Keresztes et al. 2018).

Life cycle and activity
Four larvae of Holorusia mikado pupated almost in the same period, with about two and a half days between the earliest and latest pupations. Two males and two females emerged 7-8 days after the pupations, in the morning between 5 and 9 am. (Fig. 23A). The imagos were resting on vertical surfaces during the day and outspread their wings horizontally (Fig. 23B). They were active (flew around) in the insect cage (BugDorm) from the afternoon to midnight. Copulation was not observed. One female was kept separately in a different cage, it laid 70-90 unfertilised eggs to the sieved wall of the cage within three hours. The eggs were deformed and did not stick to any surfaces (Fig. 6E). The female died soon after laying the eggs.
Flying period of imagos: Early May to late August.

Larva
The last instar larvae of H. mikado and H. hespera differ, particularly in the number of anal papillae and the pattern of the spiracular field. H. hespera has three-three lateral anal papillae, while H. mikado has an additional unpaired lobe between the lateral papillae, which is directed anteriorly (Fig. 18B and C). The odd number of anal papillae of H. mikado is quite a unique character within Tipuloidea as crane flies larvae usually have an even number of anal papillae. This is the first known Tipulidae species having odd-numbered anal papillae. The delicate black line on the inner surface of the dorsal lobes are barely indicated in H. hespera, while it is dark and clearly noticeable in H. mikado. Furthermore, the median black line on the ventral lobe is short in H. mikado and does not reach the base of the lobe (Fig. 17), while it is long in H. hespera and almost reaches the base of the lobe (see Alexander 1920 fig. 496 andYoung 2004 fig. 2B). The hypopharynx is six-toothed in H. hespera (see Alexander 1920 fig 494) and five-toothed in H. mikado ( Fig. 12D and E).

Pupa
The pupae of H. hespera and H. mikado differ in the length of their respiratory horns, which are 1.75 times longer than the width of the thorax in H. mikado and about 1.25 times longer in H. hespera. The mid-leg sheath is slightly longer than the hind-leg sheath in H. hespera and clearly shorter than the hind-leg sheath in H. mikado (Fig.  19E). Pleurites have one basal and three posterior spines in the case of H. mikado, but H. hespera has one basal and two posterior spines. The anal spine of the male genital sheath is slender in H. hespera (see Alexander 1920 fig. 497), but short and stout in H. mikado ( Fig. 21B and Fig. 22A).

Notes
The identification of "the largest specimen of crane-fly" as "Holorusia mikado", registered by the Guinness World Records from China (Sichuan) is very questionable, based on the photos that are published on the internet. The specimen has a whitish scutellum and mediotergite, which is greyish-brown on H. mikado specimens. The Chinese specimen obviously belongs to another Holorusia species; therefore, we Holorusia mikado (Westwood, 1876). A. Female emerging from pupa; B. Freshly emerged male.
removed this species from the Chinese checklist. Although the Catalogue of the Craneflies of the World also lists H. mikado from the Island of Taiwan (Oosterbroek 2020), we have not found any evidence that this species was reported from the Island. The species is not listed in the Catalogue of the Diptera of the Oriental Region (Alexander and Alexander 1973) and most probably, the record from Taiwan was accidentally listed in the Catalogue of Palaearctic Diptera (Oosterbroek and Theowald 1992) from Taiwan (Pjotr Oosterbroek, pers. comm.). The only publication that mentions the species and related to Taiwan (as Formosa) is Edwards (1921). The title of this publication was "New and little-known Tipulidae, chiefly from Formosa"; however, the localities of H. mikado, listed in the article, are all from the territory of present-day Japan. Despite long-term studies (second and third authors), we have never found H. mikado on the Ryukyu Islands, the Oriental Island chain lying between Kyushu and Taiwan. For these reasons, we also remove the record of H. mikado from Taiwan in this manuscript and consider that H. mikado occurs only on the Palaearctic Japanese Islands of Honshu, Shikoku and Kyushu, but not on Hokkaido.