Description of immature stages of Laccobius kunashiricus , with a key to genera of the Laccobiini based on larval characters (Coleoptera: Hydrophilidae)

. Immature stages of Laccobius ( Laccobius ) kunashiricus Shatrovskiy, 1984 are described based on specimens reared from adults collected in the ﬁ eld. The egg-case, all larval instars and pupa are described in detail, including primary and secondary chaetotaxy of the larval head. The biology of the species was observed and is brie ﬂ y discussed. We also summarize the knowledge of immature stages of Laccobius Erichson, 1837 and provide a key to identify larvae of the known Laccobiini genera.


Introduction
The aquatic hydrophilid genus Laccobius Erichson, 1837 of the tribe Laccobiini comprises more than 240 species worldwide. The genus represents one of the largest genera in the family Hydrophilidae (SHORT & FIKÁČEK 2011). Laccobius species live in various aquatic habitats such as standing and running waters as well as hygropetric habitats. In contrast to the species richness of the genus, its immature stages are poorly known. Larvae were described for 19 species only; most of the descriptions are moreover rather short and incomplete. Larval chaetotaxy was only briefl y mentioned by PERKINS (1972) andFIKÁČEK et al. (2008) and has never been described in detail (Table 1).
In this study, we obtained immature stages of L. (L.) kunashiricus by rearing adults collected in the fi eld. We herein describe egg-case, all larval instars, and pupa of the species, and provide also the fi rst complete description of the larval chaetotaxy of the genus Laccobius.

Material and methods
Rearing. Adults were kept in a plastic aquarium (15 cm in length, 19 cm in width, 6 cm in height) fi lled with 1-2 cm dechlorinated water and aquatic plants from the collecting site and commercially available (Fig. 1). ARCHANGELSKY (1997) suggested the use of fi sh food in fl akes as a food source for adults. In line with that, we mainly used fi sh food, Hikari Tropical Algae Wafers (Kyorin Co., Ltd., Hyôgo, Japan), which contains plants and algae, as the food source for adults. This tablet-type fi sh food was preferred by adults of Laccobius (and also by Enochrus adults in other rearings). As it did not easily crumble into small pieces, the water did not become dirty soon after introducing the food. Egg cases, once found in the aquarium, were transferred on the tissue paper to small plastic cases (35 mm in diameter, 13 mm in height).
The hatched larvae were reared in the small case. Laccobius larvae do not require large amounts of water as other hydrophilids do; wet tissue paper was suitable for their rearing (Fig.  2B). Although they have cannibalistic behaviour, this was not a major problem as they were provided with plenty of food. Therefore, we reared them as a group. We used commercially available frozen chironomid larvae (Diptera) (Sanmi Co., Ltd., Aso Tropical Fish Co., Ltd., and Kyorin Co., Ltd., Japan) to feed them. An adequate amount of food was placed in the cases twice a day. For pupation, a small quantity of commercial soil (peat moss) was placed on the corner of the cases in which the larvae were reared (Figs 2C,D).
Morphological studies. Specimens were fi xed in 80% ethanol or hot water in the laboratory. Larvae were preserved in screw-cap vials with 80% ethanol. Some larvae were mounted on HS-slides (SHIRAYAMA et al. 1993) (Kanto Rika Co., Ltd., Japan) with Euparal (Waldeck GmbH & Co. KG, Germany). The examined specimens are deposited in the authors' collections.
Our protocol generally follows that of MINOSHIMA &HAYASHI (2011) and. Specimens were soaked in 10% KOH and stained with acid fuchsine when necessary. Observations were carried out using Olympus SZX12 and Leica MZ16 binocular microscopes, and Zeiss Axiophot and Olympus BX40 compound microscopes. Illustrations were prepared with the aid of a drawing tube attached to the BX40 and the MZ16. Line drawings were prepared using the software Paint Tool SAI (Systemax Inc., Japan) and Adobe Photoshop CC (Adobe Systems Inc., USA). Photographs were taken using digital cameras attached to microscopes or directly using digital cameras; the photos were subsequently adapted in Adobe Photoshop Lightroom and Photoshop CC in needed case. Composite images were created using the Image Stacking Software CombineZP (HADLEY 2010).

Results and discussion
Genus Laccobius Erichson, 1837 Diagnosis of larva. Surface of head capsule smooth; frontal lines parallel, coronal line absent (Fig. 3A); nasale with median projection bearing a few teeth (Figs 4A, 9A); each epistomal lobe strongly projecting anteriorly, left lobe bearing a group of stout setae anteriorly, right lobe without setae (Figs 4A, 9A); mandibles strongly asymmetrical, apical inner tooth of left mandible with a group of ca. four to six seta-like projections (Figs 5C-D, 10C-D); submentum completely fused to head capsule (not even remnants of submental line are present); labium small, more or less reduced ( The larva of Laccobius is most similar to that of Oocyclus Sharp, 1882, which has similar head morphology. On the basis of the description of the fi rst instar larva of Oocyclus sp. by ARCHANGELSKY (1997), they are distinguishable by the following characters: sensillum SE1 is narrow and long in Laccobius, whereas short and stout in Oocyclus; dorsal cuticular spines on mentum are absent in Laccobius, whereas present in Oocyclus; frontal lines are parallel in Laccobius, whereas lyriform in Oocyclus; MX8 and MX9 slender setae in Laccobius, whereas stout setae bearing subapical tooth in Oocyclus; and mesonotal sclerites of Laccobius proportionally smaller than those of Oocyclus. Description of egg-case. Egg-case white in colour, constructed on substrate in water or at water's surface; spherical with narrow, long fi liform mast of variable length, often extremely long ( Fig. 1). (Fig. 2A) rather thick with weak lateral projections, widest between abdominal segments 2-4. Colour pale brownish white with sclerotised areas darker, tubercles on abdomen dark brown.

Description of larva. General morphology. Third instar. Body
Head. Head capsule (Figs 7A, 8-9) subquadrate; cervical sclerites small. Frontal line almost straight, parallel, almost invisible in third instar; coronal line absent; gular sulcus visible in basal part only. Surface of head capsule smooth. Six stemmata on each anterolateral corner of head capsule. Clypeolabrum (Fig. 9A) asymmetrical. Nasale with median projection bearing three teeth. Lateral lobes of epistome present; left lobe very strongly projecting anteriorly; right lobe strongly projecting anteriorly with membranous area laterally; both lobes projecting further than nasale, left lobe projecting further than right lobe. Left epistomal lobe bearing a group of stout, strongly bent ventrally, seta-like cuticular projections consisting of two rows (dorsal and ventral row) on inner margin, mesally to setae of gFR1. Ventral anterior margin of head capsule slightly asymmetrical. Dorsal and ventral mandibular articulation of left side projecting anteriorly further than right one (Fig. 9).
Antenna (Figs 10A-B) 3-segmented, short, rather slender. Antennomere 1 straight, shorter than antennomere 2; antennomere 2 longest and slightly narrower than antennomere 1; antennomere 3 shortest and narrowest. Mandibles (Figs 10C-D) strongly asymmetrical; left mandible shorter than right one. Right mandible with two inner teeth closely aggregated on median part. Left mandible with one inner tooth; inner tooth bearing a small subbasal tooth anteriorly and a comb composed by fi ve to six spine-like projections posteriorly. Subbasal part of inner face of mandible with several short cuticular spines and minute cuticular teeth. Longitudinal mandibular groove present dorsally on midline of left mandibular base to incisor area. Maxilla (Figs 10E-F) 6-segmented, longer than antenna. Cardo moderate in size, subtriangular. Stipes the longest and widest segment, longer than palpomeres 1-4 combined; inner face with cuticular spines dorsally along inner face. Maxillary palpus 4-segmented; palpomere 1 widest, longer than palpomere 2 and shorter than palpomeres 3 and 4, bearing about two minute cuticular spines on dorsal surface of intersegmental membrane between palpomeres 1 and 2; palpomere 2 the shortest, wider than palpomere 3; palpomere 3 about as long as and wider than palpomere 4; palpomere 4 narrowest; dorsal surface of palpomere 1 incompletely sclerotised; inner process sclerotised. Labium (Figs 10G-H) developed, but small. Submentum fused to head capsule, large, subpentagonal, wider than mentum; submental sulcus hardly visible. Mentum transverse, narrowly cylindrically sclerotised, slightly wider than prementum; dorsal surface bare. Prementum subquadrate, slightly transverse; anterior membranous area with a few short cuticular spines dorsally on lateral face. Ligula reduced as small protuberance. Labial palpus long; palpomere 1 slightly wider than palpomere 2 and distinctly shorter than palpomere 2; intersegmental membrane between palpomeres 1 and 2 with a few short cuticular spines dorsally on median to inner part; palpomere 2 almost straight or slightly curved inwards in apical half.
Thorax. Thoracic membrane covered with fi ne cuticular pubescence. Prothorax wider than head capsule. Proscutum formed by one large plate subdivided by fi ne sagittal line, anterior part rather weakly sclerotised; whole sclerite bearing fi ne cuticular pubescence. Prosternal sclerite (Fig. 7B) divided into two closely aggregated plates thus can also be considered a subdivided single plate, bearing setae along anterior margin. Mesonotum with three sclerites on each side (Figs 2A, 7C); anterior two small, inner one larger than lateral one; posterior one large, subtriangular, bearing fi ne cuticular spines on lateral to posterior margin and setae of variable length. Two small tubercles present dorsally on each side; mesal one sclerotised, behind each posterior mesonotal sclerite; lateral one membranous, on laterodorsal surface, behind mesonotal spiracle. Metanotum with one pair of subtriangular to oval metanotal sclerites. Three tubercles on each side; mesal one sclerotised, situated behind each sclerite; remaining two membranous, one posterolaterally to sclerite, one on laterodorsal face. Legs (Fig. 11B) moderate in length, slightly visible in dorsal view, 5-segmented; all three pairs similar in shape.
Abdomen ( Fig. 2A) 10-segmented, tapering posteriad, covered with fi ne cuticular pubescence; segments 1 to 7 similar in shape and size. Segment 1 with one pair of small dorsal sclerites bearing one short seta, medially on anterior part; four partially sclerotised tubercles behind dorsal sclerites; two membranous tubercles situated on each lateral face, one laterally to sclerotised tubercles, one on posterior part. Segments 2 to 7 similar to segment 1, dorsal sclerites on segment 2-7 similar or slightly smaller than those on fi rst segment. Spiracular atrium (Fig. 7D) small. Segment 8 with large oval dorsal plate covered with fi ne cuticular projections and setae of variable length; dorsal surface of posterior edge of segment 8 densely covered with fi ne pubescence; procercus incompletely sclerotised, with one long and two short setae. Segment 9 trilobed, partially sclerotised; each lateral lobe without distinct acrocercus; urogomphi short, one-segmented; prostyli absent. Second instar. Closely similar to third instar larva; sclerites on meso-and metathorax and abdominal segments more weakly sclerotised than in third instar.
Head. Frontal lines clearly visible; frontal line nearly straight, connecting lateral margin of antennal socket and posterior margin of head capsule; posterior end of frontal line unclear, possibly, frontal lines curved mesally at the base of head capsule.
Antenna and maxilla (Fig. 6) proportionally stouter than in third instar. Thorax and abdomen. Arrangements of cuticular projections and pubescence on thorax and abdomen similar to third instar but projections and pubescence fi ner than in third instar.
First instar. Similar to second instar larva; sclerites on meso-and metathorax and abdominal segments more weakly sclerotised than in second instar.
Head. Antenna (Figs 5A-B) proportionally stouter than that of second and third instar larvae. Mandible (Figs 5C-D). Distal inner tooth of left mandible with four to fi ve seta-like projections basally on posterior margin. Maxilla (Figs 5E-F). Intersegmental membrane between palpomeres 2 and 3 bearing a few small cuticular spines dorsally. Labium (Figs 5G-H) without small cuticular spines on anterior membranous area of prementum and intersegmental membrane between palpomeres 1 and 2.  Thorax and abdomen. Arrangements of cuticular projections and pubescence on thorax and abdomen similar to third instar but projections and pubescence fi ner than in second instar.
Chaetotaxy of head. Primary chaetotaxy. Frontale (Figs 3A, 4A). Median part with three pairs of sensilla (FR1-3); long seta FR1 situated close to frontal line, minute seta FR3 on mesal part, pore-like sensillum FR2 between FR1 and FR3, posteriorly to line connecting FR1 and FR3. Three setae FR5-7 and pore-like sensillum FR4 situated behind antennal socket, close to each other; FR5 and FR7 rather short, FR6 long; FR7 on inner margin of antennal socket; FR4 and FR6 very close to each other, slightly posteromesally to FR7; FR4 mesally to FR6; FR5 situated posteriorly to FR6. Long seta FR8 situated on mesal part of clypeolabrum, posteriorly to nasale; pore-like sensillum FR15 on median part of nasale; pore-like sensillum FR14 anteriorly to antennal socket. Three setae (FR9-10 and FR12) and two pore-like sensilla (FR11 and FR13) situated mesally on epistome; FR9 long, FR10 short, FR12 very short. FR9-FR11 and FR13 forming a slightly irregular oblique row, FR10 situated posteriorly, FR11 anteriorly, FR9 and FR13 between FR10 and FR11, FR9 posteriorly to FR13; FR12 on inner margin of epistomal lobe, distance between FR12 and FR11 on left side longer than on right. Nasale with group of six short setae, and with (at least) two short ventral setae (gFR1); left one and right two narrow, remaining ones stout. Left epistomal lobe with group of setae (gFR2); lateral two short, remaining ones stout, with small apical tooth; within stout setae, lateral fi ve are true setae but remaining ones could possibly not be true setae (i.e., seta-like cuticular spines); therefore left epistomal lobe may bear two short setae and fi ve stout setae; gFR2 on right lobe absent.
Labium (Figs 4B,. Submentum with two pairs of setae (LA1-2); LA1 very long, LA2 very short, both situated on anterolateral portion; LA1 very close and posteriorly to LA2. Ventral surface of mentum with one pair of rather long setae (LA3) and pore-like sensilla (LA4); LA4 situated behind LA3, LA3 close to distal margin, LA4 close to proximal margin. Prementum with three pairs of sensilla (LA8-9 and LA10) on dorsal surface and with two pairs of sensilla (LA5-6) on ventral surface. Pore-like sensillum LA8 situated medially, very long seta LA10 on anterior membranous area, anteriorly to LA8; LA9 close to LA8; LA11-12 absent. Minute seta LA5 situated ventrally at base of outer face; very long seta LA6 on median part, on borderline between sclerite and membrane of prementum. One minute seta (LA13) situated lateroventrally on basal margin of palpomere 1; pore-like sensillum LA14 on dorsal surface of intersegmental membrane between palpomeres 1 and 2. LA15 absent. Apical membranous area of palpomere 2 with several setae of variable length and shape (gLA).
Second instar. Primary sensilla on second instar similar to those of fi rst instar, and secondary chaetotaxy on second instar similar to that of third instar. Frontale (e.g., Figs 8A, 9A). Median two setae of gFR1 proportionally longer and stouter than in fi rst instar, rounded apically. Parietale with three secondary setae and one pore-like secondary sensillum (e.g., Fig. 8); two short setae close to lateral edge of antennal socket and PA9, one situate between PA8 and PA9, one on anterolaterally to PA9; one pore-like sensillum very close to PA20-21; one short seta close to PA16.  Antenna (Figs 6A-B). Three minute sensilla-like structures present at base of antennomere 2, two dorsal and one ventral; the presence or absence of these is variable. SE1 stout, slightly shorter than antennomere 3. Mandible (e.g., Figs 10C-D) with four secondary sensilla on each mandible; one minute sensillum situated dorsally on lateral part, close to MN4; one minute to short seta posteriorly to MN1; two rather short setae on lateral face of mandibular base. Maxilla (Figs 6C-D). Stipes with two secondary setae; one rather short seta situated on about posterior third of outer face of sclerite, one long seta close to MX5-6.
Third instar. Similar to second instar. Antennal sensorium SE1 stout, about two-third as long as antennomere 3 (Figs 10A-B). Head. Defl exed ventrally, covered by pronotum in dorsal view. Frontoclypeal sulcus partly visible. Antennae completely covered by head and pronotum in dorsal view; mouthparts visible in ventral view; maxillary palpi long. Styli on head: Two pairs of rather short supraorbital styli close to inner margin of each eye.
Thorax. Pro-, meso-and metathoracic legs visible in ventral view; metathoracic legs partially covered by wingpads, only tibiae to tarsi visible; meso-and metathoracic legs strongly bent ventrally. Apex of tarsi ending with one minute projection. One pair of small projections mesal to metanotal styli. Styli on thorax: Pronotum with 11 pairs of rather short styli (Figs 12D,E). Five pairs of styli on anterior margin of pronotum, three pairs situated laterally, two pairs mesally; mesal two pairs slightly longer than others. Two pairs of styli on median part, one posterior to anterior three pairs, one on mesal part. Four pairs of styli on posterior margin. Meso-and metanotum with one pair of styli on median part.
Abdomen. Abdomen with nine segments, attenuate towards apex. Posterior corner of segment 8 with triangular projection (Figs 12B, F). Segment 9 with well-developed, non-articulated urogomphi; surface of urogomphi fi nely denticulated (Fig. 12F). Styli on abdomen: Abdominal styli rather short, almost equal in size and shape excluding segment 8. Abdominal segment 1 with two pairs of rather short styli, one on median part, one lateral. Segments 2 to 7 with a transverse row of three pairs of styli; one mesal, two on lateral face close to spiracle. Segment 8 with one pair of styli without apical hair on posterior margin, hook-shaped, fi nely denticulate (Fig. 12F).
Biology. Both adults and larvae of L. kunashiricus live in standing water. Two larvae likely belonging to this species were found at the edge of a pond in Hokkaido Prefecture (Y. N. Minoshima, personal observation). Larvae are sluggish, and do not swallow atmospheric air into the alimentary canal (see MINOSHIMA and HAYASHI (2015) for a more general discussion about this behaviour). We placed a living larva into water and confi rmed that it sank in the water as it was not buoyed by air bubbles in the alimentary canal. WILSON (1923) mentioned that the larva of L. (Laccobius) agilis (Randall, 1838) sometimes hangs from the water surface supported by its spiracular atrium.