Descriptions of immature stages of Octodonta nipae (Maulik) (Coleoptera, Chrysomelidae, Cassidinae, Cryptonychini)

Abstract Octodonta nipae (Maulik, 1921), a hispid that damages several species of palm trees, was introduced accidently into China in 2001. The egg, larva, prepupa and pupa of O. nipae are illustrated and described in detail and compared with another invasive species, Brontispa longissima (Gestro, 1885); the scanning electron micrographs of the head capsule, antenna, maxilla, labium and lateral scoli are provided, as well as photos of body of all larval instars and pupa. It is the second description of immature stages in the genus Octodonta Chapuis.


Introduction
The genus Octodonta Chapuis, 1875 belongs to the tribe Cryptonychini (Chrysomelidae: Cassidinae), which contents eight species distributed in Southeast Asia and one species in Pupa New Guinea. Octodonta nipae (Maulik, 1921) is an invasive species which was introduced accidently into Hainan, China in 2001 (Sun et al. 2003), and spread to Fujian in 2007 (Hou and Weng 2010, Tang et al. 2014a, Tang and Hou 2017. O. nipae mainly attacks palm tree (Vassiliou et al. 2011, Staines 2017, such as Nypa fruticans Wurmb, Areca catechu Linn., Metroxylon sagu Rottb., Washingtonia filifera (Lindl.) H. Wendl. and Phoenix canariensis Chabaud, etc. (Maulik 1937, Sun et al. 2003, Li et al. 2014, Meng et al. 2016. In most cases the beetle feeds on young leaves that do not open or on those that open widely (Steiner 2001. This causes the young stems to shrink, curl, or die (Zhang 2003). Effective pest control is hard to achieve, because the pest lives within the leaves of the palm and is therefore hard to reach with contact insecticides (Hou and Weng 2010, Xu et al. 2011, Xi et al. 2013, Feng and Hou 2015. Many larvae and pupae in the subfamily Cassidinae have been described in detail (Maulik 1938, Gressitt 1960a, 1960b, Ford and Cavey 1985, Borowiec and Świętojańska 2003, Świętojańska et al. 2005, 2006, Świętojańska and Borowiec 2007, Świętojańska and Kovac 2007, Świętojańska and Medeiros 2007, Świętojańska and Windsor 2008, Liao et al. 2018, in the genus Octodonta, larvae of O. korthalsiae, O. subparallela, O. maffinensis were keyed and described by Gressitt (1960a); and preimaginal stages of O. depressa were described by Zaitsev (2006). As the only Octodonta species in China, the immature stage of O. nipae is still not described in detail, and we found O. nipae resembles another invasive species, Brontispa longissima (Gestro), especially in the immature stages. Although the morphological and molecular characteristics between B. longissima (Gestro) and O. nipae were compared (Chen et al. 2015 immature stages of these species have not yet been compared. Here we describe the egg, larva and pupa of O. nipae, and pupa of B. longissima, in order to provide diagnostic characters for the identification of this invasive species which will allow us to differentiate it from B. longissima and other species of Octodonta.  (eggs, larvae, prepupae, and pupae) and 31 prepupae of B. longissima were examined.

Adults of
The descriptions and illustrations of O. nipae egg, larva, prepupa and pupa are based upon laboratory reared individuals. Seta of head description follow Borowiec and Świętojańska (2003), forms of description follow Świętojańska and Kovac (2007) and Świętojańska et al. (2015). Photographs of the specimens were made using a Leica MC170 HD digital camera attached to a Leica M165C microscope. Images were produced using the software Zerene Stacker (Zerene Systems LLC, USA). Drawings were traced from images captured with the camera, then edited with the software Photoshop CS2. For the SEM scanning, the specimens were cleared in xylene for 5 hours, then washed in distilled water. Before dehydration through a graded ethanol series, they were cleaned in an ultrasonic cleaner for one minute, then put in a critical point-drier and held using double-sided adhesive tape and coated with gold in a sputter coater. Examinations were done with a JEOL JSM-6380 LV SEM and HITACHI SU3500.
Measurements are given in millimeters as mean ± SD. Data were analyzed with the SPSS Statistics Version 13.0 for Windows (Table 1).

Egg of O. nipae (Figs 7-9)
Length 1.4 ± 0.31 mm, width 0.5 ± 0.21 mm. The female adults usually lay eggs in pairs, rarely in triplets or as a single egg. The eggs are surrounded by a sticky secretion and covered with debris and excrement. The newly laid eggs are generally ivory-white, covered with a milky-white soft secretion. After several hours, the eggs turn brown and the secretion becomes sclerotized (Fig. 7). Egg are elongate-oval. The egg chorion is covered with raised networks of ridges (Fig. 8), and the two adjoining polygonal cells share the same ridge (Figs 8-9). The anterior pole of the chorion has smaller cells and taller ridges than the lateral chorion (Fig. 8).
Abdomen with eight pairs of lateral scoli on segments I to VIII, and a single pair of supra-anal processes. All lateral scoli not branched, slender and tapering, bearing six club-like setae. Distal segment with supra-anal processes caliper-like hooked, slightly sclerotized, curving slightly inward, each hook with three to four upward directed tiny teeth from base to apex, each teeth with a club-like seta apically.
Spiracles distinctly elevated, round; abdominal spiracles much smaller than mesothoracic spiracle, spiracle of abdominal segment VIII located in inner flank of carina ( Fig. 1, arrows indicated).  (Maulik). 7 eggs 8 egg 9 surface of egg 10 head of first larva, front view 11 head of second instar, front view 12 head of third instar, front view 13 head of fourth instar, front view 14 head of prepupa, front view 15 head of prepupa, dorsal view.
Head well sclerotized, slightly narrower than prothorax, partially retracted into prothorax (Fig. 1). Epicranial stem absent; median endocarina complete and wide ex-tending between two distinct frontal arms, from close to the center of the posterior margin up to the position of the antenna; fronto-clypeal suture present, clypeo-labral suture present. Clypeus distinct, much wider than long. Antenna very short, threesegmented set in membranous ring, anterolaterally directed; segment I and II ring-like, one large sensorial appendage inserted ventrally on segment II, below segment III; segment III parallel-sided and as long as the sensorial appendage (Fig. 10). Five stemmata positioned behind the antenna, four of them in a row, another one positioned further back, pigmented spot placed below row of four distinctly marked stemmata (Fig. 10). Labrum sclerotized, wider than long, six setae positioned dorsally, anterior part with thick stout curve spines.

Second to fourth instar of O. nipae (Figs 2-4, 11-13)
Body length 4.5-7.7 mm, width 1.9-3.2 mm (Table 1). Abdomen wider than head and thorax. Head capsule much wider than long, anterior margin convex and evenly rounded laterally, finely granular. Supra-anal processes strongly carinate and sclerotized, curving slightly inward, each dorsal carina with four to six upward directed large teeth from base to apex, lateral carina bearing with two to four large teeth; two setae positioned on tiny tubercle near inner margin of processes (Fig. 3, arrow indicated).
Body flattened dorso-ventrally, elongate-oval, widest across abdominal segment V. Abdomen with eight pairs of lateral scoli and a pair of short supra-anal processes (Fig. 5). Lateral scoli slender and tapering, finely denticulate (Figs 5, 33) and bearing six long club-like setae (Fig. 25). Supra-anal processes caliper-like hooked, strongly carinate and sclerotized, each dorsal carina with five to six upward directed large teeth from base to apex, lateral carina bearing three to four large laterally directed tubercles with setae at apex, two setae positioned on tiny tubercle near inner margin of processes (Fig. 34, arrow indicated).
Setae of head club-like, blunt apically or pointed, club-like setae more or less of the same length with scoli setae, but some setae of head very short (Figs 22,23,27,28). Setae of tergites short and pointed . Setae of legs club-like or long pointed (Fig. 43).
Dorsal side of prothorax with five pairs of tiny pointed setae (Figs 35,44), four pairs arranged in row near posterior margin, one pair near middle of tergite; three  (Figs 30, 31). Spiracle of last segment abdomen round, larger than other abdominal spiracle, located in inner flank of dorsal carina (Figs 32, 34).
Head (Figs 14,15,17,44) well sclerotized, shallowly retracted into prothorax, distinctly wider than long, but narrower than prothorax, lateral margin strongly rounded; dorsal surface finely granular, median endocarina well developed, widening and deepening from posterior margin to clypeal posterior margin, frontal arms extending from close to the center of the posterior margin up to the position of the antenna.
Stemma (Figs 14, 17). Position of stemmata similar with younger instars. All the stemmata concolorous with surrounding area, pale brown (Fig. 14). Head with numerous setae, distribution of setae as shown in Figure 40. Antenna (Figs 16, 24, 41). Antenna very short, three-segmented, anterolaterally directed set in membranous ring; segment I ring-like, with one tiny seta placed laterally (Figs 16, 41); segment II slightly wider than long, with one large conical sensory appendix apically, three setae laterally (Fig. 24) and one small peg-like sensillum placed between sensory appendage and antennal segment III; segment III as long as segment II, apical portion with six peg-like sensilla (Figs 16, 41).
Mandible heavily sclerotized (Figs 18, 42), triangular, short and compact, with three apical teeth; inner side of mandible sharp, dorsal side of mandible convex, dorsolateral side with one long and one short pointed seta (Figs 19,42).
Leg three-segmented (Figs 29, 43); coxa much wider than long, with setae arranged in three rows: first with two setae, second with three setae, third with two setae and one club-like seta; femur 1.6 times longer than wide, with seven long setae, two club-like setae, and one short seta close to the base, as show in figure 43; tibiotarsus stout, apically with one heavily sclerotized and curved single claw, armed apically six long setae around claw, and one club-like seta at middle. (Figs 6, 45-47) Length 8.6 ± 0.51 mm, width 3.6 ± 0.34 mm. Body long oval, exarate, flattened dorsoventrally; body straight from apex of head to abdominal segment III, bent ventrally at abdominal segment IV (Figs 46, 47). Color light brown, eyes dimly black (Fig. 47).

Pupa of O. nipae
Head with three stout processes in dorsal view, one central process with apex truncated and bearing two lateral setae (Fig. 45); two lateral processes positioned laterally, with apex acute and bent ventrally, one lateral setae inserted near apex (Fig. 45). In ventral view, eyes, antennae, labrum, mandibles, and maxilla discernible (Fig. 47).
Abdominal segment I to VIII visible in dorsal view, each segment with one pair of spiracles, and a large number of hooked spines and setae (Figs 6, 45). Tergum of first segment with six small spines (Fig. 45). Tergum II to VII marked with eight pairs of hooked spines, five pairs located between two spiracles, each spine bearing one seta (Fig. 45); one pair of spines near spiracle which bears one seta (Fig. 45); two pairs of spines positioned on the lateral margin of tergum, one directed dorsally and one directed ventrally, each of them bearing two setae (Fig. 45). Tergum VIII with supra-anal processes long hooked (Fig. 45), spiracles of tergum VIII in same locality as in larva. Sternum IV to VIII visible, sternum IV to VII bearing 12 hooked spines; sternum VIII without spine, anus positioned centrally, transverse and oval shape, last abdominal segment slim and soft (Figs 45-47).

Prepupa of B. longissima (Figs 49-60)
Body elongate, flattened dorso-ventrally. Abdomen with eight pairs of lateral scoli and a pair of short supra-anal processes (Fig. 59). Lateral scoli short and tapering, finely denticulate and bearing four long club-like setae (Fig. 58). Supra-anal processes caliper-like hooked, strongly carinate and sclerotized, each dorsal carina with five to seven upward directed large teeth from base to apex, lateral carina bearing three to four large laterally directed tubercles with setae at apex (Fig. 59).
Most setae of head hair-like or club-like, blunt apically or pointed, but some setae of head very short (Fig. 57). Setae of tergites short or long, pointed, blunt or club-like (Fig. 52). Setae of legs club-like or long pointed, two setae positioned on tiny tubercle at inner margin of processes (Fig. 59) Dorsal side of prothorax with four pairs of pointed setae arranged in row near posterior margin, one pair near middle of tergite; four pairs of blunt apical setae near anterior margin; six pairs of club-like setae along lateral margin (Fig. 50). Dorsal side of mesothorax with four pairs of pointed setae arranged in row posterolaterally; three pairs of pointed setae arranged in row near middle of tergite; two pairs of tiny pointed setae near anterior margin; one pair of blunt apical setae positioned anterolaterally; three long club-like setae along lateral margin, two of them on the bulge and one behind the bulge (Fig. 50). Dorsal side of metathorax with seven pairs of tiny pointed setae (Fig. 50), four pairs arranged in row posterolaterally; three pairs arranged in row along middle of tergite; one pair of blunt apical setae positioned near anterolaterally; three pairs club-like setae and one pointed seta along lateral margin (Fig. 50).
Nine pairs of spiracles: one pair on the mesothorax and eight on abdomen. Mesothoracic spiracle tubular and distinct (Fig. 50) laterally positioned between pro-and mesothorax; abdominal spiracles small. Spiracle of last segment abdomen round, larger than other abdominal spiracle, located in inner flank of dorsal carina (Fig. 59).
Head (Figs 49, 52) distinctly wider than long, but narrower than prothorax; dorsal surface finely granular, median endocarina well developed, widening and deepening from posterior margin to clypeal posterior margin, frontal arms extending from close to the center of the posterior margin up to the position of the antenna (Fig. 51). Head with numerous setae (Figs 49, 51), distribution of setae as shown in Figure 49. Labrum bent down (Figs 54,55), connected with the anterior clypeal margin, almost as wide as clypeus. Anterior margin with tufted setae (Fig. 53), outer surface of labrum with six long pointed setae (Fig. 55).
Mandible heavily sclerotized (Figs 53, 56), short and compact, with three apical teeth; inner side of mandible sharp, dorsal side of mandible convex, dorsolateral side with one long and one short pointed setae (Fig. 56).
Leg three-segmented (Fig. 51); coxa wider than long, with setae arranged in three rows: first with two pointed setae, second with two pointed setae and one club-like seta, third with one pointed seta and one club-like seta; femur longer than wide, with 11 long pointed setae, one club-like setae, as show in figure 51; tibiotarsus stout, apically with one heavily sclerotized and curved single claw, armed apically nine long setae around claw.

Discussion
The first-instar larva of Octodonta nipae with the combined length of head and thorax make up more than one-third of body length, while in the other larval instars head and thorax account for no more than one-third of body length. The mesothoracic spiracles are invisible in dorsal view in the first and second instar larvae, but the third and fourth instar larvae and prepupa have remarkably long tubular mesothoracic spiracles, which are dorsally visible. The first-instar larva with the supra-anal processes slightly sclerotized and only bearing one to three small teeth; older larvae have a strongly carinate and sclerotized supra-anal processes and more teeth; the last abdominal segment of the pupa is very soft and narrower than in the prepupa (Fig. 1), wrapped closely with crimping exuvium. The third and fourth instar larvae and prepupa have two tubercles which are positioned at the inner margin of the last abdominal segment (Figs 3-5); the first and second instar larvae lack tubercles and only have two tiny setae, which are almost invisible (Figs 1, 2).
The egg and larva of Brontispa longissima were described in detail by Maulik (1938), in his description we found several diagnostic characters distinguishing the immatures of O. nipae and B. longissima. Eggs of both species are elongate and ellipsoidal; similar in size and sculpture. The first instar larva of B. longissima has all lateral scoli bearing two setae while in O. nipae lateral scoli have six club-like setae; the last abdominal segment of B. longissima is caliper-like hooked and has a series of five or six hairs along dorsal margin, but in O. nipae the dorsal margin has three to four club-like setae.
According to our observation, the mature larva of O. nipae resembles the larva of B. longissima, but there are many differences: 1) The body length of O. nipae (7.32 ± 0.06 mm) shorter than B. longissima (8.99 ± 0.38 mm).
2) The setae distribution of head is different as shown in pictures, row of Fa of B.
longissima is only with one seta Fa1, but row of Fa of O. nipae with two setae; row of Fb of B. longissima is with five setae, but row of Fb of O. nipae with four setae. 3) Scoli of O. nipae are conical and slender, bearing six club-like setae; but B. longissima has the scoli which bearing four long club-like setae, and are shorter than the scoli of O. nipae; 4) The setae distribution of thorax is different (Figs 44, 50). 5) The setae distribution of leg is different (Figs 43, 51). 6) The supra-anal processes all caliper-like hooked, O. nipae with two small pointed setae positioned near inner margin of processes (Fig. 34, arrows indicated), but these two setae of B. longissima are closer to the inner margin (Fig. 59, arrows indicated). We also found some different from the description of Maulik (1938), in his description the mandibles of B. longissima are with two teeth, but we found the mandible of B. longissima with three teeth (Fig. 56) as O. nipae; Maulik said "B. longissima with the spiracle of mesothorax is not visible dorsally", but according to our observation the spiracles of mesothorax are visible dorsally (Fig. 50). Gressitt (1960a)  The larva of O. depressa was described and figured by Zaitsev (2006), the mandible is triangular, with one tooth and a sharp inner edge, outer side has one seta; antennae are three-segmented, segment II has one small triangular sensillum apically and segment III long and slender; scoli have seven setae; last abdominal segment with each dorsal carina have five sharp teeth from base to apex, each lateral carina only have one laterally directed tooth near apex. The mandible of O. nipae larva has three teeth at inner side, outer side has two long setae; antennae are three-segmented, but segment II has a large conical sensory appendix apically and one small peg-like sensillum, segment III is conical and apically bearing with six peg-like sensilla; scoli have six setae; last abdominal segment with each dorsal carina have five to six teeth, and each lateral carina have three to four teeth laterally directed.