Description of immature stages and biological notes of Cassidisparelicta Medvedev, 1957, a newly recorded species from China (Coleoptera, Chrysomelidae, Cassidinae, Hispini)

Abstract The first instar and mature larva and pupa of Cassidisparelicta Medvedev, 1957, a newly recorded species from China, are described and figured. The chaetotaxy of the head, mouthparts, legs, and dorsal and ventral surfaces of the body is described. This is the first detailed description of immatures in the genus Cassidispa. Diagnostic characters of this species are compared with other described immatures of some Hispini genera. Biological notes on C.relicta, such as host plants, feeding patterns of adults, structure of larval mines and life history, are also presented.


Introduction
Cassidispa Gestro, 1899 is a leaf beetle genus belonging to the tribe Hispini Gyllenhal, 1813 (Chrysomelidae: Cassidinae), with eight species occurring in China, Russia, Angola, Democratic Republic of Congo and Zimbabwe (Staines 2015). Four species are previously recorded from China: C. bipuncticollis Chen, 1941, C. femoralis Chen & Yu, 1976, C. maderi Uhmann, 1938and C. mirabilis Gestro, 1899(Chen et al. 1986T'an 1993;Hua 2002;Staines 2015). Adults of the genus Cassidispa can be distinguished from all other genera of Hispini by the anterior margin of the pronotum without spines, by the pronotum and elytra with broadly expanded margins, and by the antennae having nine antennomeres (Chen et al. 1986). The main diagnostic characters between Cassidispa and the similar genus Platypria Guérin-Méneville, 1840 are the lateral margins of pronotum expanded from base to anterior angle with irregular translucent patches, the continuous expanded margins of the elytra, the elytra with very low and obtuse tubercles, never with spines, and the lateral margins of elytra with numerous small spines not with long ones (Chen et al. 1986).
There is little published biological information on Cassidispa species. Hua (2002) listed the host of C. bipuncticollis as Betula spp. (Betulaceae) from China. Recently, we discovered that the larvae of C. relicta mine in the leaves of Betula platyphylla Suk. (Betulaceae) and Ulmus pumila Linn. (Ulmaceae) in Inner Mongolia, China. The species reached outbreak levels in 2016-2017 and became a potential pest of the dominant trees in the area. As Dr. Lukáš Sekerka pointed out, the species is Cassidispa relicta Medvedev, 1957 not C. mirabilis. Both species are superficially similar by predominantly black coloration but they are distinct: C. relicta has generally strongly shiny dorsum (but not as shiny in C. mirabilis); C. relicta has shorter and thicker antennae, explanate margin of pronotum largely black (but yellow in C. mirabilis); C. relicta has anterior spots on explanate margin of elytra almost reaching to humeri (but widely separated from humeri in C. mirabilis); elytra of C. relicta is very distinctly constricted in 3/4 length (but weakly in C. mirabilis) (Lukáš Sekerka, Personal Communications). C. relicta is previously reported in Russia (Medvedev 1957;Staines 2015). Therefore, it is a newly recorded species from China and China hosts all five Asian Cassidispa species now.
In this publication, we describe the larvae and pupa of C. relicta and provide the first detailed report on immature morphology and biological information for the genus Cassidispa.

Materials and methods
All immatures and adults were observed and collected at Shanggaotai Forest Farm (Zhuozi County, Inner Mongolia) from March 2016 to October 2017. Immatures and adults of C. relicta were collected on wild plants and some of them were preserved in anhydrous ethanol. Some adults were pinned in the laboratory (Figs 1-3) and determined using the keys in Chen et al. (1986). Four first-instar larvae, three mature larvae and three pupae were examined morphologically. For microscopic study, the heads of the larvae were separated from the rest of the body, boiled in 10% NaOH solution and cleaned in water before dissecting the mouthparts.
The photos of adults were taken using a Cannon EOS 7D camera with macro lenses; the dissection of heads and mouthparts was made with a Motic SMZ-140 and Olympus SZX2-ILLT stereomicroscope; figures and examination were performed using an Optika B-292 microscope and Cannon EOS 70D camera. Our descriptions of immature stages follow Świętojańska et al. (2006). The terminology of the chaetotaxy of the head follows Borowiec and Świętojańska (2003). All studied material (first-instar larvae, mature larvae, and pupae) and adults were deposited at the Leafminer Group, School of Life and Environmental Sciences, Gannan Normal University (Ganzhou, China). Medvedev, 1957 Larva . Length of mature larva 6.1-6.2 mm without head, width 1.6-1.7 mm across pronotum. Length of first instar larva 1.4-1.8 mm without head, width of body 0.7-0.8 mm across pronotum.

Cassidispa relicta
Body distinctly flattened dorso-ventrally. Pronotum of first instar larvae slightly wider than abdominal segments; mature larvae widest across abdominal segments IV-V (Figs 6-7, 16-17). Body color of alcohol-preserved larvae yellowish-white with two irregular brown patches on pronotum (paler and without dark markings in first instar larvae), black anterior margin of abdominal segment IX, dark brown spiracles, yellowish-brown triangular patch on prosternum, brown head and legs. Abdomen of live larvae dark brown or black .
Body with eight pairs of lateral scoli on abdominal segments (Figs 6-7, 16-17). Lateral scoli short and round, approximately of same length; scoli of segments VI-VII with two small simple lateral branches (first instar larvae without lateral branches as in Figs 4-5). All lateral scoli with two setae apically and one seta ventrally.
Granulation of body distinct in all examined specimens including both first instar and mature larvae. Each tergite and sternite (except for sternite VIII) with minute setae on anterior margin; tergites and sternites covered with short pointed setae. Tergites of meso-and metathorax, abdominal segments I-VI and sternites I-VII of  abdomen with transverse grooves (Figs 4-7). Sternites of meso-and metathorax with two short transverse grooves medially, very similar in shape to other tergites and sternites. Posterior margin of each transverse groove and anterior margin of pronotum with distinct asperites.
Pronotum with four setae on each lateral margin, five setae on each lateral side, and three setae close to posterior margin (Figs 4,6). Meso-and metanotum with six minute setae on anterior margin: two pairs at middle and one pair laterally; row of ten setae running across segment; group of six setae laterally. Meso-and metanotum with one seta on slightly visible protuberance laterally. Abdominal tergites I-VII with four setae on anterior margin; two rows of setae running across segment: anterior with two setae, posterior with four setae; three setae close to each spiracle (seta closest to spiracle minute). Abdominal tergite VIII with four minute setae on anterior margin; three rows of setae running across segment: anterior with four setae, median with two setae, and posterior with four setae (between spiracles). Posterior margin of abdominal segment VIII with ten setae: two pairs close to spiracles laterally, three pairs between spiracles medially.
Prosternum with one seta in each anterior angle; one seta laterally at base of leg; and two setae close to posterior margin (Figs 5, 7). Meso-and metasternum with four setae on anterior margin; two rows of setae running across segment medially, both with two setae; and two setae laterally at base of leg. Abdominal sternites I-VII with pair of minute setae on anterior margin medially; with row of six setae running across segment medially; and three setae laterally. Abdominal sternite VIII with row of eight setae across segment anteriorly and two setae posteriorly; with two setae close to each spiracle; four setae along anterior base of anus.
Nine pairs of distinct spiracles (Figs 4, 6, 16): one on thorax and eight on abdomen. Thoracic spiracles distinctly more elevated than abdominal spiracles, diameter of spiracles of abdominal segments I-VII approximately equal, but spiracles of abdominal segment VIII distinctly larger, flattened, slightly elevated.
Five stemmata laterally: four dorsal-laterally, one ventrally (Figs 8-9). Antenna with three antennomeres, set in membranous ring ( Figure 14). First antennomere stout, approximately as wide as long, with three campaniform sensilla; second antennomere stout, slightly longer than wide, with two campaniform sensilla, one promi- nent sensory appendix apically, and one stout seta close to third antennomere; third antennomere very short, with long, pointed seta and three peg-like sensilla.
Labrum approximately three times wider than long, anterior margin slightly emarginate (Figs 10-11), dorsal surface with: three setae and one campaniform sensillum laterally; one pair of campaniform sensilla medially; and four stout setae laterally close to anterior margin. Mid-and anterior areas of ventral surface with numerous stout spines; lateral and posterior areas with tiny spines; two irregular groups of few small sensilla medially.
Mandibles heavily sclerotized, with two prominent teeth ( Figure 12): anterior distinct and conical, posterior blunt; followed by some tiny teeth. Two long setae very close to each other and one campaniform sensillum.
Maxillae and labium connate ( Figure 15). Each stipes (st) with three campaniform sensilla anteriorly. Palpifer (pp) with one small seta apically and one campaniform sensillum laterally. Maxillary palp (mp) with two palpomeres: first palpomere stout, second palpomere with group of small peg-like sensilla at apex. Mala (mal) with six long pointed setae and one short seta apically, and two setae subapically. Hypopharynx (hyp) covered with numerous small spines. Labial palp (lp) with one palpomere, with group of small peg-like sensilla at apex. Prementum (pre) with two campaniform sensilla anteriorly. Postmentum (post) with two pairs of short setae at base and two campaniform sensilla medially.
Legs oblong, consisting of three segments: coxa, femur, and tibiotarsus ( Figure 13). Tibiotarsus armed apically with claw. Coxa with four setae along base on internal surface, and one seta dorsally and two setae on external surface. Femur with five setae and four campaniform sensilla on basal half, and six long setae on apical half. Tibiotarsus with nine long pointed setae and two campaniform sensilla: two setae at middle laterally, six setae around claw apically, one seta and two sensilla above claw. Base of claw with distinct pulvilli.
Body flattened dorso-ventrally, elongate-oval. Color of live as well as alcohol-preserved pupa brownish yellow, mesothorax with two dark markings at base of wings, apex of abdomen dark brown (Figs 18-19).
Body, especially abdomen, distinctly granulate (Figs 18-21). Abdominal tergites I-III with two short transverse grooves medially. Abdominal tergites IV-VII each with long transverse groove. Tergites of abdominal segments I-VII with several small tubercles around grooves. Tubercles of tergites VI-VII arranged as row close to posterior margin. Sternites of abdominal segments IV-VII each with two transverse ridges close to posterior margin.
Head visible in dorsal view (Figs 18, 20). Prothorax trapezoidal in shape. Thorax without lateral scoli. First abdominal segment without lateral scoli. Segments II-V with single simple scolus laterally and small low tubercle anteriorly armed with pointed  seta. Abdominal segment VI with two lateral scoli. Segments VII-VIII each with three lateral scoli. Each scolus apically armed with one seta. Posterior lateral scolus of segments VI-VIII with one lateral branch directed posteriorly without setae. Segment VIII additionally with two broad, flattened processes on posterior margin; each with six setae apically.
Head with three pairs of setae laterally and four setae anteriorly ( Figure 20). Pronotum with group of nine setae laterally. Mesonotum with three pairs of setae medially. Metanotum with oblique row of four setae on each side medially. Abdominal tergites I-VII with two rows of setae running across segment: anterior with two setae, posterior with four setae; two setae close to each spiracle. Abdominal tergite VIII with three rows of setae running across segment: anterior with four setae, median with four setae, and posterior with two setae medially; one seta very close to each spiracle.
In ventral view (Figure 21), head with group of 14 setae: two setae anteriorly, four setae medially, six setae posteriorly at base of labrum, and two setae at base of mandibles. Tarsus of leg apically armed with one pointed seta. Visible abdominal sternites IV-VII with four setae on anterior margin, row of six setae medially, two setae laterally, and one seta at base of lateral scoli ventrally. Abdominal sternite VIII with rows of six setae anteriorly and six setae along anterior base of anus. Abdominal segments each with one pair of spiracles ( Figure 21). Spiracles of segment I smaller than others, spiracles of segments II-IV and VI-VII similar in shape but approximately twice as large as spiracles of segment I. Spiracles of segment VIII not elevated, oblong. Spiracles of segment V prominent, elongated into cylindrical appendage (respiratory horns), directed posteriorly. Biological notes. The biology of the genus Cassidispa is poorly known. There is only one species having confirmed host plant record. Hua (2002) reported C. bipuncticollis to be associated with Betula (Betulaceae), but it is just a list even without some evidences. Our rearing of C. relicta on Betula and some other trees indicates that there might be more larval host associations for the genus. Additionally, the immature stages of the genus Cassidispa were completely unknown and this paper was the first study about it.
The larvae of C. relicta were found mainly mining in the leaves of Betula platyphylla (Figs 22-25, 27-34)  The life cycle of C. relicta is univoltine based on our field observations throughout 2016-2017. C. relicta overwinters as a mature larva until the temperature rises and the soil thaws in the spring. The mature larvae break dormancy and pupate in the fallen leaves in early April (Figs 33-34). The pupal stage lasts about one month. Adults emerge in late May (the freshly emerged adults are mostly white, with antenna and legs brown, and disc of pronotum black) and feed on the mesophyllic tissue of the upper surface of host leaves (Figs 22-24). The feeding pattern of the adults is usually irregular and may densely cover the leaves (Figs 22-25, 28-32). In early June, the adults start to copulate (Figs 23-24), and fertilized females lay an egg within a shallow hole which was chewed on the lower leaf surface, and then covered with feces (Figs 25-26). Adults have no apparent feeding preferences for young or old leaves, but females generally do not oviposit on new leaves. Females oviposit eggs scatteredly in a one-by-one way (Figs 24-26, 28). The newly deposited eggs are usually milk-white and translucent and its covered feces turn brown over time 28). Sometimes, females will lay some eggs on the tree trunk, perhaps when they could not find any suitable leaves ( Figure 27). The egg stage lasts about 20 days. A freshly hatched larva bores into the mesophyllic tissue and forms a large irregular mine with a resting place (dark color) surrounding the oviposition point ( Figure 29). The larvae deposit their feces in their own mine (Figs 29-32). Each younger larva has its own absolute mine at the early stage which will combine with other mines on the same leaf over time (Figs 29-32). One leaf usually has three larvae or, in an outbreak, up to seven larvae. If one leaf does not likely provide enough food for these larvae to complete their development, they leave their original mine, migrate to a new leaf, and construct a new mine (Figs 31-32). The final larval mine is a large irregular blotch type, almost without any mesophyllic tissue left (Figs 30-32). The mined leaves gradually become yellow and may dry-up or abscise early. In early October the mature larvae leave their mine to enter a fresh uneaten area of the same leaf to construct a pupal mine (Figs 33-34). The mature larvae of C. relicta does not directly go into pupation like other leaf-mining hispines (Świętojańska and Kovac 2007;Lee et al. 2009;Liao et al. 2014;Liao et al. 2018) but into a long dormant period for overwintering. However, it is a shortcoming we did not perform the larval instar observations.
The larva of C. relicta is very similar to that of some Dactylispa species and can be distinguished by the lateral branches of abdominal scoli on segments VI-VII (very small and with a rounded apex), although this character also presented on D. haturanga but distinctly slender and with a pointed apex (Zaitsev 2012). Additionally, the abdominal processes are distinctly round in shape (16)(17), with a pair of spiracles whose opening directed backward (in genus Dactylispa the spiracles usually formed very pointed or triangular respiratory horns). The pupa of C. relicta is most similar to that of Di. testacea. These two species each have a pair of lateral scoli on abdominal segments I-V and have 2-3 pairs of lateral scoli on segments VI-VIII, the abdominal apex has two flattened processes; and the spiracles of the fifth abdominal segment look very similar. The differences are that the body shape of C. relicta is broadly ovate and the anterior margin of the head is slightly straight, but the head of Di. testacea is distinctly emarginate.
The larvae, especially younger larvae, usually returned to the resting place ( Figure 29). Such an interesting and specific behavior was also found in some species of the genus Dactylispa, such as D. subquadrata (Baly, 1874) and D. higoniae (Lewis, 1896) (Yano 1965), but not in D. angulosa (Solsky, 1871) (Yano 1965) and D. approximata Gressitt, 1939(Dai et al. 2012. The leaf areas with obviously deeper color smeared with larval feces which may help the larvae to avoid predators and parasitoids (Hering 1951;Yano 1965 Kimoto et al. 1997 (larva); Liao et al. 2014