Morphology of immature stages of Helophorus (Gephelophorus) auriculatus (Coleoptera, Helophoridae) Author Minoshima, Yûsuke N. Author Watanabe, Reiya text Acta Entomologica Musei Nationalis Pragae 2020 2020-05-15 60 1 319 332 journal article 10.37520/aemnp.2020.018 1804-6487 3879911 9D78C2BB-8042-4883-9578-A370E8152D9A Helophorus ( Gephelophorus ) auriculatus Sharp, 1884 Description of egg case. Egg-cases ( Figs 1A, B ) oviposited on mud near the edge of water. Egg-case bag-shaped, leaflike mast of greatly variable length; 4.49 ± 1.15 (1.77–7.99, n=32) mm [mean ± SD (minimum–maximum, sample size)]. The mean number of hatched larvae per egg-case was 8.67 ± 1.97 (3–13, n=32) (see also WAŦANAƂൾ et al. (2020)). Mast white in colour; bag covered with mud and sand grains. When hatching, larvae make a hole to escape at the base of the mast. Total length of egg-case 6.98 ± 1.41 (4.39–10.63, n=32) mm. Description of larva. First instar. General morphology similar to third instar; antenna and maxilla proportionally shorter and stouter than third instar. Ventral cuticular projections on nasale absent ( Fig. 1I ). Small to minute thoracic and abdominal sclerites present on third instar often indistinct, undetectable or absent. Basal segment of urogomphus bearing two long setae apically. Primary chaetotaxy of head capsule . Frontale (Figs 2A, 3). Central part with three pairs of sensilla (FR1–3) slightly divergent posteriad; FR1 and FR3 minute seta, FR2 pore-like; FR1 close to frontal line, FR2 behind FR3. Pore-like sensillum FR4 and long seta FR6 situated mesally to antennal socket, FR6 posterolateral to FR4. Minute seta FR5 close to frontal line, posterior to FR6. FR7 minute seta, on inner margin of antennal socket. Rather short seta FR8 behind nasale, short seta FR9 posterolateral to FR8. Pore-like sensilla FR11, FR13, FR14 on epistome, forming oblique row; FR11 anterior to FR13 and FR14, FR13 between FR11 and FR14. Short seta FR10 situated laterally to FR13. Rather long seta FR12 anterior to FR10. FR15 absent. Nasale with group of six setae (gFR1); median two short, stout; lateral two minute. Epistomal lobe with five lanceolate to stout setae and two pore-like sensilla (gFR2); respective setae longer from inside to outside. Fig. 1. Immature stages of Helophorus auriculatus Sharp, 1884 . A, B – egg-case; C – H – whole larva; C, D – first instar, dorsal (C) and ventral (D) view; E – second instar, dorsal view; F–H – third instar, dorsal (F), lateral (G), ventral (H) view; I–K – ventral structure of nasale tooth (I – first instar, from ventral side; J – second instar, from dorsal side; K – third instar, from ventral side). Parietale (Fig. 2). Dorsal surface with a group of five sensilla (PA1–5) forming slightly irregular oblique row in posterolateral part; PA3 pore-like, remaining four short setae. Minute sensillum-like structure between PA4 and PA5 often absent. Pore-like sensillum PA6 and short seta PA7 close to posterior end of frontal line; PA6 behind PA7. Rather long seta PA12 anteromesal to PA5. Seta PA8 extremely long, behind antennal socket. Minute seta PA9, pore-like sensillum PA10 and rather long seta PA11 behind lateral part of antennal socket; PA9 close to outer margin of antennal socket; PA10 between PA8 and PA9; PA11 behind PA10. PA16 rather long seta situated laterally between lateral-most stemma. PA13–15, PA17, PA18 placed laterally on midlength of parietale; PA13 and PA18 very long setae, PA14 rather long seta, PA15 and PA17 pore-like. PA13–15 on dorsolateral surface; PA13 close and dorsal to PA15; PA14 behind PA15; PA17 and PA18 on lateroventral surface, PA18 behind PA17. PA19–21 and PA23 on anterior corner of head capsule; PA19 and PA23 pore-like, PA20 extremely long seta, PA21 rather short seta. PA19 and PA21 on lateral face; PA19 dorsal to PA20; PA21 and PA23 on ventral face, PA21 behind PA23. Pore-like sensilla PA24 and PA25 closely located, behind ventral mandibular articulation; PA24 anterior to PA25. PA22 absent. Rather short seta PA26 situated ventrally on anterior third, posterior to PA25. Pore-like sensillum PA27 and long, stout seta PA28 located ventrally on posterior two-fifths; pore-like sensillum PA29 posterior to PA28. Pore-like sensillum PA30 situated lateroventrally on posterior fifths. Fig. 3. First instar larva of Helophorus auriculatus Sharp, 1884 . Anterior margin of head capsule, dorsal view. Head appendages . Antenna ( Figs 4A, B ). Antennomere 1 with five pore-like sensilla (AN1–5). AN1 and AN2 situated dorsally on posterior fifth (AN1) and anterior third (AN2). AN3 situated laterally on anterior margin of sclerite. AN4 subapically on inner face of sclerite. AN5 ventrally on intersegmental membrane between antennomeres 1 and 2. Antennomere 2: AN6 situated dorsally on anterior third of sclerite. Setae AN8 and AN9 dorsolaterally on intersegmental membrane between antennomeres 2 and 3; AN8 rather short seta, lateral to minute seta AN9; AN10 and AN11 on inner face of membrane; AN10 short seta, dorsal to rather short seta AN11. AN7 undetectable (see description of third instar). Two sensoria present on lateral face of intersegmental membrane; SE1 conical, moderate in size, SE2 posterior to SE1, conical, much smaller than SE1. Antennomere 3 with group of apical sensilla (gAN) in apical membranous area. Mandibles ( Figs 4 C–E). Three sensilla (MN1–3) forming oblique row at midlength of mandible; MN1 long seta on lateral face, MN2 and MN3 pore-like; MN3 close to incisors area, MN2 between MN1 and MN3. Pore-like sensillum MN4 and minute seta MN5 on dorsolateral face; MN4 anterior to MN2; MN5 anterior to MN4. Pore-like sensillum MN6 present on incisors area, very indistinct. Three sensillum-like structures present on subapical part of dorsal face; three sensillum-like structures ventrally on distal inner tooth. Maxilla ( Figs 4 F–H). Cardo with rather short seta ventrally. Inner face of stipes with five stout, short setae ( MX 7–11). Pore-like sensillum MX 3 on ventral part of inner face. Four sensilla ( MX 2, MX 4–6) situated on lateroventral part. Pore-like sensillum MX 2 and extremely long seta MX 5 on posterior part, pore-like sensillum MX 4 and long seta MX 6 on subapical part. Palpomere 1: MX 16 on inner face of palpomere 1. Three sensilla ( MX 12–14) on subapical part of sclerite; MX 12 pore-like on lateral face; MX 13 very long seta on lateroventral face; MX 14 rather long seta ventrally on lateral part. Pore-like sensilla MX 15 and MX 17 on membrane behind inner appendage, MX 17 dorsally, MX 15 ventrally. Inner appendage with a group of apical sensilla (gAPP). Palpomere 2 with two pores ( MX 18, MX 19) and one minute seta ( MX 27). MX 18 situated ventrally on apical margin of sclerite; MX 27 ventrally at base; MX 19 on inner face of intersegmental membrane between palpomeres 2 and 3. Palpomere 3 with four sensilla ( MX 20–23) on subapical part. MX 20 pore-like, on lateral part; MX 21 very short seta on inner face; MX 22 pore-like, behind MX 21; MX 23 very short seta, on dorsal face. Palpomere 4 with three sensilla ( MX 24–26) and group of apical sensilla (gMX). MX 24 very short seta, situated subbasally on inner face; MX 25 digitiform dorsally on lateral face; MX 26 pore-like ventrally on subapical part of lateral face. Labium (Figs 2B, 4I–K). Submentum with one pair of long setae (LA1) laterally. Pair of minute setae (possible LA2) on membrane between submentum and mentum (Fig. 2B). Ventral surface of mentum with two pairs of sensilla (LA3, LA4); LA3 long seta, LA4 pore-like; LA3 lateral to LA4. Ventral surface of prementum with three pairs of sensilla (LA5–7); LA5 very short seta basally on mesal part; long seta LA6 and pore-like sensillum LA7 closely situated on lateral part. Pore-like sensillum LA8 dorsally on posterior membranous part of prementum. Four sensilla (LA9–12) dorsally on inner part of membrane between prementum and palpomere (anterior membranous area of prementum). LA10 short seta lateral to remaining three sensilla, possible LA9 and LA12 small, digitiform sensilla, anterior one may be LA12; LA11 pore-like. Ventral surface of palpomere 1 with very short seta LA13 at base. Pore-like sensillum LA14 dorsally on intersegmental membrane between palpomeres 1 and 2. Palpomere 2 with two subapical sensilla; LA15 pore-like on dorsolateral face; one additional sensillum digitiform, on lateroventral face. Several short sensilla of variable shape (gLA) on apical membranous area. Second instar. Very similar to third instar; antenna proportionally slightly stouter than in third instar ( Fig. 5A ). Small to minute thoracic and abdominal sclerites present on third instar often indistinct, undetectable or absent. Chaetotaxy of head capsule similar to third instar larva; minute secondary seta, tentatively marked as AN7 present as in third instar (see Fig. 7A ). Fig. 4. First instar larva of Helophorus auriculatus Sharp, 1884 . Head appendages. A, B – antenna, dorsal (A) and ventral (B) view; C–E – mandibles (C – right mandible, dorsal view, D – right mandible, ventral surface (drawn from dorsal), E – left mandible, dorsal view); F–H – maxilla (F – ventral view, gMX omitted; G, H – maxilla, slightly modified by pressure of cover glass for examination, dorsal (G) and ventral (H) face, both drawn from dorsal view). I–K – labium, dorsal (I), dorsolateral (J), ventral (K) view. Fig. 5. Larvae of Helophorus auriculatus Sharp, 1884 . A – head, second instar larva, dorsal (left) and ventral (right) view; B – head, third instar larva, dorsal (left) and ventral (right) view; C – posterior leg, first instar, anterior view; D – posterior leg, third instar, anterior view; E – abdominal apex, third instar, dorsal view. Third instar. General morphology . Body ( Figs 1 E–G) slender; sclerites brownish dorsally, less pigmented ventrally; membranous parts whitish. Head . Head capsule ( Fig. 5B ) subpentagonal, almost parallel-sided. Cervical sclerites absent. Frontal lines complete, lyriform; coronal line absent ( Figs 5B , 6A). Surface of head capsule smooth. Six stemmata on each anterolateral portion of head capsule. Posterior tentorial pits present on anteromedian part of head capsule; gular sulcus visible. Clypeolabrum symmetrical (Fig. 6C). Nasale with single large median projection with smooth lateral margins; ventral cuticular projections on nasale present, well developed ( Fig. 1K ). Epistomal lobe present, projected further than nasale, rounded anteriorly; apical part of epistomal lobe membranous. Antenna ( Fig. 7A ) 3-segmented, slender. Surface of antenna smooth.Antennomere 1 as long as antennomeres 2 and 3 combined. Antennomere 2 widened apically as wide as antennomere 1. Antennomere 3 shortest and narrowest. Approximate ratios of length of antennomeres 1: 2: 3 (including intersegmental membrane) as follows: 1: 0.7: 0.3 (n=1). Two antennal sensoria present (see description of chaetotaxy in detail). Mandibles ( Figs 7B, C ) symmetrical; basal half distinctly wider than apical half; incisor area roughly serrated. Mandible with two inner teeth; basal one smaller than distal one; small aggregated teeth behind basal inner tooth. Groups of short, spine-like projections present dorsally on base of apical inner tooth (retinacular tuft) and on subbasal part of inner face (mandibular penicillus). Small projections present on inner part of ventral surface ( Fig. 7C ). Maxilla ( Fig. 7D ) slender, 6-segmented, longer than antenna. Cardo large, subtriangular. Stipes the longest and widest, shorter than palpomeres 1–4 combined. Stipes not cylindrically sclerotized; dorsal surface of stipes mostly membranous (or very weakly sclerotized); posterior end of dorsal stipes undetectable. Dorsal surface with moderately densely arranged small teeth on membrane; inner face with patch of small teeth on sclerite. Maxillary palpus moderately long, 4-segmented; all segments cylindrically sclerotized. Palpomere 1 longest and widest; dorsal surface membranous in posterior half. Inner process long, sclerotized, as long as palpomere 2. Intersegmental membrane between palpomeres 1 and 2 bearing a few cuticular teeth dorsally. Palpomere 2 similar to palpomere 3 in length, but wider than palpomere 2. Palpomere 4 the shortest and narrowest. Fig. 7. Third instar larva of Helophorus auriculatus Sharp, 1884 .A – antenna, dorsal (left) and ventral (right) surface, both drawn from dorsal view, gAN omitted; B – mandible, dorsal view; C – ventral surface of basal part of mandible; D – maxilla, dorsal (left) and ventral (right) surface, both drawn from dorsal view; E – labium, dorsal view (left) and labium and its adjacent structure, ventral view (right), gAN omitted. Labium ( Fig. 7E ) developed. Submentum fused with head capsule, transverse, subpentagonal. Mentum trapezoid. Dorsal surface of mentum membranous (or very weakly sclerotized); posterior end of dorsal surface undetectable; rather densely arranged cuticular teeth present on dorsal surface; lateral and ventral surface sclerotized. Prementum subpentagonal; posterior margin membranous dorsally; prementum sclerotized between labial palpi, connecting dorsal and ventral surfaces. Small cuticular teeth present laterodorsally on anterior membranous area of prementum. Ligula absent. Labial palpus moderately long, slender, palpomere 1 shorter and slightly wider than palpomere 2. Small cuticular tooth present laterodorsally on intersegmental membrane between palpomeres. Thorax . Prothorax wider than head capsule, widened posteriorly. Proscutum formed by a large plate subdivided mesally by fine sagittal line. Ventral surface of prothorax with a pair of large prosternal sclerites ( Fig. 8 ). One small subtriangular sclerite present mesally behind prosternal sclerite, between coxal cavities; a pair of rather small sclerites behind prosternal sclerite, connecting sclerite of outer margin of procoxal cavity; one small sclerite present posterolaterally to procoxa. Mesonotum with a large plate subdivided mesally by fine sagittal line and two pairs of very narrow, transverse sclerites anteromesally, anterior to large plate. Lateral face of mesothorax with two sclerites; anterior one situated on anterolateral portion, forming a ring around the mesothoracic spiracle; posterior one oval. Ventral surface of mesothorax with sclerites of variable size; two oval sclerites present mesally; anterior one smaller than posterior one; small oval sclerite present laterally to anterior mesal sclerite; associated sclerite of mesocoxa similar to prothoracic one but smaller; one oval sclerite posterolateral to mesocoxa. Metathorax similar to mesothorax with the following two exceptions: narrow mesal sclerites absent on dorsal surface; spiracle absent, thus the lateral sclerite is oval, not forming ring. Legs ( Fig. 5D ) well developed, visible from dorsal view; meso- and metacoxae situated on lateral part of ventral surface, widely separated than procoxae. Ventral face of femur more or less membranous, borderline between membrane and sclerite unclear. Abdomen . Abdomen with 9 well-developed segments ( Figs 1 F–H), segments not subdivided by transverse folds. Segment 1 with several sclerites: dorsal surface with large sclerite present mesally, subdivided by sagittal line; a pair of moderately large sclerites lateral to large sclerite; a pair of small sclerites present anterolaterally. Lateral surface with two sclerites; anterior one smaller than posterior one. Ventral face with 7 sclerites; lateral two pairs oval, median three transverse. Segments 2–7 similar to segment 1, but a minute sclerite present anteromesally to spiracle; anterior two mesal transverse sclerites of ventral face reduced to one anterior very narrow sclerite and small sclerites behind the narrow sclerite. Segment 8 similar to segment 7 but spiracle situated dorsally on posterolateral part; minute sclerite on dorsolateral face absent. Abdominal apex not forming a spiracular atrium ( Fig. 5E ); segment 9 with large, oval plate dorsally, lateral face with one small sclerite; ventral face with small sclerites; a pair of long three-segmented urogomphi present, bearing several long setae. Basal segment of urogomphus bearing three long setae apically. Segment 10 on ventral face of segment 9. Chaetotaxy . Chaetotaxy of head capsule (Fig. 6) and head appendages ( Fig. 7 ) very similar to first instar except that minute secondary seta, tentatively marked as AN7 present ventrally close to sensorium SE2 ( Fig. 7A ; see also Remarks). Description of pupa. Colour . Transparent milky white with reddish eyes (Figs 9D–F). Body (Fig. 9) elongate with styli bearing apical seta. Head deflexed ventrally; head and appendages covered by pronotum in dorsal view. Head appendages partly visible in ventral view; antenna short; mandibles wide and blunt at apex; maxillary palpus slender. Surface of head capsule bearing three pairs of styli close to inner margin of each eye. Thorax . Pronotum transverse, evenly and weakly convex, bearing eight pairs of styli. Two pairs mesally; one pair on anterior margin and one on posterior margin. Six pairs on lateral margins; three pairs on anterior corner; one pair at midlength; two pairs on posterior corner. Meso- and metanotum with one pair of styli on each median portion. Legs slender, partly visible in dorsal view. Forewing elongate oval, with numerous longitudinal striae; hindwing similar to forewing in size. Fig. 8. Helophorus auriculatus Sharp, 1884 , sclerites on ventral prothoracic segment of third instar larva. Abdomen with nine segments, attenuated towards apex, but anterior third to anterior half almost subparallel-sided. Abdominal segments bearing styli on dorsal and lateral faces. Segment 1 transverse, with one pair of styli on disc, but it is possible that a lateral pair is missing. Segments 2 to 7 transverse, with three pairs of styli; one close to midline, two lateral. Segment 8 with two pairs of styli; one on median part, one on lateral part. Segment 9 with well developed, non -articulated urogomphi sharply pointed at apex. Segments 1 to 7 bearing a pair of spiracles on each side. Table 1. Comparison of primary chaetotaxic characters of Gephelophorus larvae and those of Helophorus and Lihelophorus based on H. ( H. ) liguricus Angus, 1970, H. ( L. ) lamicola Zaitzev, 1908 and H. ( L. ) yangae Angus et al., 2016 described by ANǤUඌ et al. (2016). Based on first instar larvae for all taxa compared.

Gephelophorus	Helophorus	Lihelophorus
FR8	close to each other	close to each other	close to each other / widely separated
FR6	long	minute	long
PA7	short	minute	long/short
PA8	long and stout	long and stout	long and fine
PA12	rather short	rather short	rather short / long
PA13	long and stout	long and fine	long and fine
MX5	much longer than MX6	much longer than MX6	much longer than or same length as MX6
MX7–11	short	short	short / long
MX13	long	rather short	short

Biology. We reared the larvae of H. auriculatus using only Tubifex worms or chironomid larvae for their diet. This stands in contrast to the reports of Chinese population of H. auriculatus as pests of winter wheat (ANǤUඌ & JıA 2015). Based on our rearings, we may confirm that the larvae of Japanese population of this species are carnivorous. For detailed biological information, see WAŦANAƂൾ et al. (2020). Remarks. In helophorid larval antennae, there are two sensoria and two setae (FıĸǍćൾĸ et al. 2008) on the outer face of the intersegmental membrane between antennomeres 2 and 3. Arrangement of the antennal sensilla of H. auriculatus is nearly identical to that of H. ( H. ) liguricus , H. ( Lihelophorus ) yangae and H. ( L. ) lamicola (FıĸǍćൾĸ et al. 2008, ANǤUඌ et al. 2016). However, one secondary seta was present in the area of the second and third instar larvae ( Figs 7A, B ). Although this seta is not a primary sensillum, we have tentatively marked it as AN7 (in parentheses; Fig. 7B ) based on its position. The dorsal surfaces of the stipes and mentum of H. auriculatus were ‘soft’ as described by ANǤUඌ (1992). We illustrated and described these structures as membranous to distinguish them from sclerites ( Figs 4 F–H, I–K, 7D, E) even though it is possible that are weakly sclerotized. This type of modification (‘soft’ dorsal surface) is unusual within the Hydrophiloidea . For example, membranous or weakly sclerotized dorsal surface of the mentum has been reported in the hydrophilid genus Laccobius Erichson, 1837 and the family Epimetopidae (ARർIJANǤൾĿඌĸY 1997; FıĸǍćൾĸ et al. 2011, 2018) which have a reduced mentum.