The genus Gymnetron from China with description of pre-imaginal stages of G. miyoshii, G. auliense and G. vittipenne (Coleoptera,Curculionidae)

Abstract There are four species of Gymnetron in China recorded to date including Gymnetron miyoshii Miyoshi, 1922, Gymnetron villosipenne Roelofs, 1875, Gymnetron auliense Reitter, 1907 and Gymnetron vittipenne Marseul, 1876, of which the last two are new country records. The pre-imaginal stages including eggs, mature larvae and pupae of Gymnetron miyoshii, Gymnetron auliense and Gymnetron vittipenne are described and illustrated. In addition, their diagnostic characters (larvae and pupae) are discussed and differentiated, and notes on some of their biological parameters are provided. Potential ecological impacts between Gymnetron weevils and their host Veronica spp. also are provided.


Introduction
Gymnetron Schoenherr, 1825 belongs to the tribe Mecinini Gistel, 1848 in the subfamily Curculioninae Latreille, 1802 (Alonso-Zarazaga et al. 1999, Caldara 2001. These weevils are small, distinguished from other Mecinini by the following features taken together: prosternum without median sulcus; elytral margin covering a large portion of the pygidium; elytral striae 3 and 8 joined at apex (Caldara 2008). This genus is widely distributed in the Palaearctic and Afrotropical regions (Alonso-Zarazaga et al. 1999, Caldara 2001, 2003, 2008; distribution in China of G. miyoshii Miyoshi, 1922 andG. villosipenne Roelofs, 1875 is recorded by Caldara (2008). The Palaearctic species of Gymnetron live on Veronica (Caldara 2008), currently included in the Plantaginaceae (Stevens, 2012), while those in the Afrotropical region (Caldara 2003) appear to live on various genera of Scrophulariaceae belonging to the tribes Hemimerideae and Selagineae, Buddleja of the Buddlejeae and Anastrabe of the Stilbaceae, both families very closely related to Plantaginaceae (Stevens, 2012). The immatures of some species of Gymnetron have been studied previously, but without detailed descriptions (van Emden 1938, Scherf 1964, Anderson 1973, May 1993.
The aim of the present study is to describe for the first time all developmental stages of three species of Gymnetron living in China in order to provide further characters for the identification of these taxa.

Materials and methods
Six last instar larvae and ten pupae of Gymnetron miyoshii, five last instar larvae and one pupa of G. villosipenne, and ten last instar larvae and ten pupae of G. vittipenne were examined. Descriptions were made and photographs of pupae were taken with a Canon-5D camera mounted on a Nikon SMZ 1500 microscope. Images of adults were photographed with a CCD Qimagine MicroPublisher 5.0 RTV mounted on a Zeiss SteREO Discovery. V12 microscope; Microscopic slides were studied with a Leica DM 2500 microscope and photos were taken with a Nikon CoolPix 5400. Drawings were made from the original photographs by using the software Adobe Illustrator CS6; photos in the field were taken with Canon G15 and 5D Mark II cameras.
Nomenclature of the larval chaetotaxy mainly follows van Emden (1938), May (1993May ( , 1994, Marvaldi (1999) and Wang et al. (2013), and that of the pupa mainly follows Gosik (2010). The dissecting method used follows May (1979May ( , 1994. Indistinct structures were pigmented with "Chlorazol Black E" for further examination. In pupae, msns and mtns are used as abbreviations of mesonotal setae and metanotal setae, respectively. As msns and mtns are different among the three weevils species examined, these can be added as special diagnostic characters in Gymnetron; in order to differentiate from alar setae and apical setae of the pupa, as and asp are used, respectively. In the descriptions, setae of the thorax and abdomen are described for one side only. After description, all larvae and pupae were mounted using nail polish, a mixture of butyl acetate, ethyl acetate, multipolymer of adipic acid, neopentyl glycol, trimellitic acid and acetyl tributyl citrate. All slides remain together with the adult specimens in the museum of the Institute of Zoology, Chinese Academy of Sciences. Miyoshi, 1922 Gymnetron miyoshii Miyoshi, 1922: 253 Gymnetron villosulum var. orientale Voss, 1955 Description. Adult (Figures 1-2): sides of pronotum in part, mesothoracic epimera, metasternum and urosternite one covered with broad scales; elytral vestiture forming indistinct spots; rostrum in lateral view slightly curved, in female nearly of same width from base to apex (Caldara 2008).
Head (Figures 10-11): free, circular in outline, broader than long, broadest at middle; cranial suture undivided, wide, half length of head; frontal suture distinct, not extending to mandibular membrane; endocarinal line short, no more than half as long as frons; frons with three pairs of fs, fs1 and fs3 reduced to basal sensilla, fs5 longest, laterally positioned on epistoma close to antenna, fs4 located near epistoma, half as long as fs5, fs2 located in the middle of frons, half as long as fs4; dorsal epicranium with four pairs of des, des4 reduced to a basal sensillum, des3 longest, located on frontal line, des5 lightly shorter than des3, des2 approximately one quarter as long as des3, des1 slightly shorter than des2; epicranium with two pairs of les, les1 long, les2 short, about half as long as les1; posterior epicranium with three pairs of pes, pes1 minute, pes2 equally as long as pes3; ventral epicranium with one pair of ves, minute; postoccipital condyles indistinct, hypopharyngeal bracon distinct; tentorial bridge narrow, with two small but moderately acute anterior projections and two large, obtuse-angled posterior projections; clypeus transverse, fused to labrum, bearing two pairs of cls, cls1 nearly same length as cls2, located in one line, parallel to clypeus, sensilla absent; antenna (Figure 12) one segment, sensory appendage nearly twice as long as wide, circular in cross-section, contiguous with frontal suture, with one conical and three minute sensilla; ocellus present, not projecting, located below stripe at side, externally close to antenna.
Mouthparts : mandibles ( Figure 13) symmetric, incisor section with two apical teeth and rounded flange posterior to dorsal tooth, molar section with two mds, mds1 nearly same length as mds2, sensilla distinct; labrum ( Figure 14) transverse, fused to clypeus, nearly completely sclerotized, with three pairs of lrms, lrms2 slightly shorter than lrms1, both centrally localized, lrms3 same as lrms1, close to distal margin of labrum; epipharynx ( Figure 15) with all epipharyngeal setae stout and apically rounded, with two pairs of als, three pairs of ams, epipharyngeal sensilla, mes and labral rods (tormae) absent. Labium ( Figure 16) membranous excepting the premental sclerite, labial palpus with one segment, slightly longer than wide, apex of palpus flattened with dense short irregular spiculate setae, and one sensillum; premental sclerite (Pmsc) distinctly posteriorly and laterally dilated, U-shaped, with one pair of sensilla and one pair of long prms. Ligula with two pairs of tiny ligs, ligs1 as long as ligs2. Postlabium (plb) partly sclerotized, with two pairs of plbs at membranous area, plbs1 long, plbs2 short, one quarter long as plbs1; membranous area sparsely and finely asperate. Maxillae with maxillary palpus (mxp) (Figures 16-17) two-segmented, basal segment with one tiny mxps, accessory appendage absent; distal segment sclerotized, apex flattened with dense short irregular spiculate setae, one sensillum; mala with five dorsal robust dms, dms1-5 gradually shorter, with four shorter, more acute vms. Stipes bearing one stps, two pfs and two sensilla, stps strong and long, submedian on venter of base. Pfs1 a little shorter, located near mala, one third as long as pfs2, pfs2 submedian on venter of base, cardo completely divided from stipes. Thorax ( Figure 9): pronotal shield partly pigmented and sclerotized on pale smooth plate. Pronotum with two setae on sclerotized area, dorsopleurum with four dpls; spiracle ( Figure 18) intersegmental between pro-and mesothorax, bicameral, airtube subequal to diameter of circular peritreme; ventropleurum with two vpls; pedal area ( Figure 19) distinct, pedal lobe present, one-segmented, slightly convex, with four setae; mediosternum with two msts. Mesonotum with two folds (prodorsum and postdorsum), prodorsum with two prs, postdorsum with two pds, two setae transversally aligned; alar area with four as, two long, two short; dorsopleurum with one dpls, ventropleurum with one vpls; setae of pedal area and mediosternum same as prothoracic. Metanotum same as mesonotum.
Abdomen ( Figure 9): with spiracles on segments I-VII, size similar, all anterolateral and unicameral, each with single annulated air-tube, pointing posteriad, subequal to diameter of circular peritreme. Abdominal segments I-VII with tergites with two folds, prodorsum with two prs, prs1 longer than prs2, postdorsum with soft protuberance posteriorly, with one pds, all setae shorter than thoracic setae; spiracular area with two ss, ss1 short, one quarter as long as ss2; dorsopleurum with one dpls, ventropleurum with one vpls, laterosternum with one lsts, mediosternum with three msts, except msts3 in front of msts1, other five setae short and transversally aligned. Abdominal segment VIII with tergite with two folds, prodorsum with one prs, postdorsum with one pds; spiracular area with two ss, ss1 short, one quarter as long as ss2; dorsopleurum with one dpls, ventropleurum with one vpls, laterosternum with one lsts, mediosternum with one msts, except msts2 in front of msts1, other four setae short and transversally aligned. Abdominal segment IX with tergite with two folds, prodorsum with one prs, postdorsum with one pds; pleurum with one pls, sternum with three sts. Abdominal segment X with one tiny seta, anus transverse cleft.  Pupa : Measurements (mm): length: 2.65-3.00 (n = 4), width: 1.00-1.60 (n = 4), height: 1.25-1.50 (n = 4). General appearance: Theca yellow, grayish at apex of antennae, rostrum, legs, wings, elytra, anus and apex and base of ventrites. Setae greatly reduced in number. Ventrites III-X visible in ventral view, tergum I-VIII visible in dorsal view. Head: yellow-gray with one yellow stripe along middle, with one pair of pas, situated at middle margin of eyes; eyes large, one third of length of head, not projecting; rostrum long, twice as long as wide, mesorostrum visibly dilated, mandibular theca weakly projecting, setae absent; antennae applied against prosternum and apically extending to propleurum, subparallel to profemur. Thorax: prothorax bearing one median, lightly pigmented tubercle, apically shallowly bifurcate, with a spiracle between pronotum and mesonotum, but lacking air-tube; pronotum with one as and one sls in ventral view, two pls in dorsal view, as, sls and pls subequal, strong and long, pls1 and pls 2 positioned in one transversal row; mesonotum with two msns on scutellum; metanotum bearing two mtns near hind margins, half shorter in length than pronotal setae. Legs: pro-, meso-and metafemora apically bearing two slightly outcurved fes, fes 1 as long as fes 2, apex with grey circular pigmented area. Abdomen: segments I-VII with tergite bearing one seta, with transversely oval impression, submedian small transverse macula and lateral maculae, segment VII lacking impression. Spiracles present on segments I-VII, spiracular area with one ss, dorsopleurum with one dpls, ventropleurum with one vpls, laterosternum with one lsts, mediosternum with two msts; segment VIII with tergite bearing one fleshy, pigmented, apically narrowing rounded process, with two seta on tergite, sternum with two setae; segment IX with sclerotized, bifurcate, elongate and slightly curved outward pseudocerci, subterminally positioned at ventral abdominal segment IX, invisible in dorsal view; segment X with anus transverse cleft, subterminal, invisible in dorsal view. Biological notes. Veronica anagallis-aquatica L. has been collected with galls on 14-VI-2014 which have been reared in the laboratory. Fifteen pupae were found on 21-VI-2014.

Description. Adult
Egg: unknown.  (Figure 25): pronotum with four pns, dorsopleurum with four dpls. Spiracle bicameral, air-tube equal or shorter than diameter of circular peritreme. Abdomen: with seven spiracles, size similar, all anterolateral and unicameral, each with single annulated air-tube, shorter to diameter of circular peritreme.
Pupa: Measurements (mm): length: 2.80 (n = 1); width: 1.00 (n = 1). Mesonotum with three pairs of msns on scutellum; Pro-, meso-and metafemora of legs apically bearing one slightly outcurved fes, apex with grey circular pigmented area. Remarks. This species has been recorded from Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan. This species is a new record for China.

Gymnetron vittipenne Marseul, 1876
Gymnetron vittipenne Marseul, 1876: 383. Gymnetron apicale Faust 1885: 187. Gymnetron vittipenne var. suturiferum Reitter 1907: 32. (Figures 5-6): Sides of pronotum covered with dense, imbricated, broad scales; uncus of metatibiae strongly enlarged at apex in male; first tarsal segment on venter covered with very dense and long setae in male; ductus of spermatheca sclerotized at base near insertion with spermatheca. Elytra parallel-sided, with reddish and black integument covered with moderately dense, recumbent to erect, seta-like scales arranged in three very irregular rows on each interstria; rostrum in lateral view slightly curved, angulate along dorsal margin at antennal insertion and weakly narrowed at apical third in male, strongly curved, cylindrical and of same length from base to apex in female (Caldara 2008).
It differs from G. miyoshii by: General appearance ( Figure 26): size greater. Head: Size greater, endocarinal line long, more than half as long as frons; hypopharingeal bracon distinct; clypeus transverse, bearing two pairs of cls, cls1 nearly same length as cls2, located  Figure 29) transverse, partly sclerotic, anterior margin nearly straight, posterior margin weakly extended medially into clypeal zone, with three pairs of lrms, lrms2 a bit shorter than lrms1, both centrally localized, lrms3 same length as lrms1, close to distal margin of labrum, with one mds, subequal to lrms2; epipharynx ( Figure 30) with two pairs of als, three pairs of ams, one pair of mes, sensilla absent. All epipharyngeal setae stout, short and apically rounded; labium (Figure 31) membranous except sclerotized area. Labial palpus with one segment, longer than wide distinctly, with one pair of sensilla, apically flattened with dense crenulate setae. Premental sclerite (Pmsc) distinctly posteriorly and laterally dilated, U-shaped, with one pair of sensilla and one pair of long prms. Ligulate area with two pairs of tiny lgs, lgs1 same lengtgh as lgs2, with one pair of sensilla. Postlabium partly sclerotized, with two pairs of plbs at membranous area, plbs1 long, plbs2 short, one quarter as long as plbs1.; Maxillae with maxillary palpus (mxp) (Figures 31-32) two segmented, basal segment distinctly wider than long, with one pair of sensilla and one pair of short mxps, accessory appendage absent. Apical segment longer than wide, with one pair of sensilla, apically flattened with dense short irregular speculate setae. Mala with five robust dms, dms1-5 gradually shorter than the former one and four thin vms. Stipes bearing one sts, three palpiferal pfs and two sensilla, sts strong and long, basally medioventral, pfs1 short, located near mala, one third as long as pfs2, pfs2 basally medioventral, same length pfs3, pfs3 lateroventral. Cardo completely divided from stipes. Thorax (Figure 26): Pronotum with six pns. Spiracle (Figure 33) bicameral, air-tube distinctly longer than diameter of circular peritreme, pointing basad. Pedal area (Figure 34) distinct, with five setae; Mesonotum with two folds, prodorsum with one prs. Postdorsum with two pds, one dls transversally aligned. Pedal area same as prothoracic; Metanotum same as mesonotum. Abdomen ( Figure 26): with seven spiracles on segments I-VII, size similar, all anterolateral and unicameral, each with single annulated air-tube, distinctly longer than diameter of circular peritreme, pointing basad. Abd I-VII: tergites with two folds, prodorsum with one tiny prs, postdorsum with soft protuberance posteriorly, with two pds, pds1 short, half as long as pds2. All setae shorter than thoracic setae; Abd VIII: pds1 short, half as long as pds2.
It differs from G. miyoshii by: General appearance: size greater. Head: head yellow-gray with indistinct yellow stripes in middle, two pairs of pas, pas1 situated in middle of frons, pas2 situated at middle margin of eyes. Thorax: prothorax bearing a lightly pigmented tubercle, apically deeply bifurcate. Pronotum with three as, two sls, one ds and two pls. As and sls1, pls subequal, strong and long, pls1 and pls2 in one transverse row, sls2 and ds short; Mesonotum with three msns on scutellum. Abdomen: segment I-VII with tergite bearing two setae. Spiracular area with two ss; Legs: pro-, meso-and metafemora apically bearing one pair of slightly outcurved fes, apex with grey circular pigmented area. Remarks. This species has been recorded from Armenia, Bulgaria, Croatia, Greece, Iran, Iraq, Kazakhstan, Lebanon, Palestine, Slovakia, Syria, Tajikistan, Turkey, Turkmenistan and Uzbekistan. This species is a new record for China.

Biological information
Host plants of Veronica in which larvae of the three species live are widely distributed in China (Zhong 1979). Gymnetron auliense and G. vittipenne were both collected on Veronica oxycarpa Boiss., G. miyoshii was collected on Veronica anagallis-aquatica L.; all host plants live on the banks or in clear slowly flowing streams (Figures 38-40). The adults of G. miyoshii feed on stems near the axils of their host (Figure 41). Females make holes on petals and calyces with mouthparts on the apex of the rostrum (Figure 42), and lay eggs in ovaries of developing flower buds or flowers (Figure 43). One oviposition hole can be found on the surface of one ovary and one larva develops in an ovary. The oval eggs are mostly surrounded by ovules and are evident on the ovary wall (Figure 44-45). As Howden (1995) reported, after oviposition, females seal the hole with fecal material (Figure 46-47). Larvae feed in the ovaries, stimulating ovaries to develop into galls ( Figure 48). Larvae (Figure 49a-b) are active, and if disturbed, their abdomen sways front and back quickly. Mature larvae will not pupate until the ovary Table 1. Diagnostic features of the mature larvae of Gymnetron from China (Characters of G. miyoshi identical in G. auliense/G. vittipenne are not repeated, but indicated by "-").  wall remains as a thin membrane. Pupae (Figure 50) are also active and their abdomen can sway front and back quickly like the larvae. Ovaries attacked by weevils will not produce seeds, but the damage seems not to seriously harm the plant's whole reproductive rate. After observation in the field, we found G. miyoshii mostly live in the ovaries in the middle of the inflorescence, while flowers at top and bottom still produce seeds. Gymnetron auliense, like G. miyoshii, also lays eggs in ovaries of host plants ( Figure Lee et al. (1998) described the larva of G. miyoshii, but after further study, distinct major differences are observed in the head (ocelli, setae and clypeus), mouthparts (labrum, labium and maxillary palpus), setae of thorax and abdomen, and number of spiracles, with comparison in Table 3. The differences maybe due to a misidentification made by Lee et al. (1998). The character of labrum is important in dividing different group in Curculionidae. Usually labrum with one pair of sensilla is the primitive state, but with 0, 1, 3 sensillae is advanced ( van Emden 1938). In our observation, there are 2 sensillae in G. vittipenne, but none in G. miyoshii or G. auliense, which shows the genus of Gymnetron may be not monophyletic. The labrum fused with the clypeus and without the epipharygeal rod is an important character of Gymnetron; the maxillary palpi of these 3 species are 2-segmented, but the basal segment is incomplete in G. miyoshii and G. auliense. We did not observe accessory appendages in any of the 3 species. Caldara (2013) used characters of the host plants to build the phylogenetic tree in Mecinus   Germar, 1821. The immature stages have a close relationship with host plants. Thus, it will be a great help to add characters of immature stages in the phylogenetic studies.

Discussion
In addition, setae on the alar area are found to be variable. There are usually four setae of different lengths on the alar area on each side of each larva. Five setae can be found on the alar area of the metathorax of G. miyoshii. Thus, the setae of the alar area are not useful diagnostic characters. There is only one middle seta on the labrum of G. vittipenne pupae, it is special, and we name it mds. The features of the larva of the genus Gymnetron are as follows: (1) Frontal suture not extending to mandibular membrane; (2) Antennae contiguous with frontal suture; (3) Postoccipital condyles indistinct; (4) meso-, metathorax and abdomen with two tergal folds; (5) Alar area without sclerotized or pigmented areas; (6) Spiracles bicameral; (7) Head brown with pale stripes at side and ecl of head; (8) Accessory sensory appendage of antenna short; (9) Anus, transverse cleft; (10) Living in galls of seeds or stems of Scrophulariaceae or Plantaginaceae.
Parasites of Gymnetron are few (May 1993, Gumovsky 2007, and during this study, only one parasite was found in the larvae of Gymnetron. Low parasitism may due to the following two reasons. First, galls can be a mechanical barrier for escape from natural enemies. Second, iridoid glycosides in host plants can help Gymnetron to protect it from the natural enemies. Iridoid glycosides are unpalatable and denature proteins and DNA (Bowers et al. 1986, Kim et al. 2000. Though there were none of these chemical compounds in adults (Baden et al. 2012), Gymnetron still can use them indirectly. Since larvae and pupae live in galls, the ovary walls with iridoid glycosides can also be a protection against invertebrate and vertebrate predators. Chinese have collected Veronica anagallis-aquatica L. with Gymnetron galls as a Chinese traditional medicine for many years, which can treat some painful and inflammatory human diseases (Zhong 1979). The main active substance in this Chinese traditional medicine is iridoid glycosides (Dong et al. 2011, Guan et al. 2011. Gymnetron feeds on host plants, causing the plants to produce more iridoid glycosides. Baden et al. (2012) only reported there are no iridoid glycosides in adults of Mecinus Germar, 1821 and Rhinusa Stephens, 1829. To confirm whether these chemical substances exist in larvae, pupae and adults of Gymnetron, further studies are needed. While collecting these species from the field, three kinds of host plants only were found, living only in flowing water with little pollution or human disturbance. In spring, there are many host plants in the habitats, but only those with Gymnetron living on them can survive as they begin to flower and seed. We collected plants for rearing weevils in the laboratory, and observed the same phenomenon. Plants with galls of Gymnetron lasted long after seven days, but those without galls began to wilt on the second day and died on the 7th day. The host plants were reared five times under the laboratory conditions. Based on this study, we formulate the hypothesis that Gymnetron feed on Veronica causing them to produce more protective chemicals, which can help the plants to resist environmental stress. Species of Veronica with Gymnetron galls living near water cannot live in unclean polluted water for long, so we can use these two organisms as environmental indicators. So, during the co-evolution of insects and plants, there are relationships not only of plant-herbivores-predator, but herbivores and plants can also help each other to live harmoniously.