Ultrastructure of the Tarsus in Oides decempunctatus (Billberg) (Coleoptera: Chrysomelidae) Author(s): Zheng Liu and Ai-Ping Liang Source: Journal of the Kansas Entomological Society, 86(2):122-132. 2013. Published By: Kansas Entomological Society DOI: http://dx.doi.org/10.2317/JKES120825.1 URL: http://www.bioone.org/doi/full/10.2317/JKES120825.1

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. JOURNAL OF THE KANSAS ENTOMOLOGICAL SOCIETY 86(2), 2013, pp. 122–132 Ultrastructure of the Tarsus in Oides decempunctatus (Billberg) (Coleoptera: Chrysomelidae)

1,2 1,3 ZHENG LIU AND AI-PING LIANG

ABSTRACT: The ultrastructure of the tarsus of adult Oides decempunctatus (Billberg) (Coleoptera: Chrysomelidae) was examined using scanning electron microscopy. Structurally the tarsus consists of 5 tarsomeres and a pair of bidentate ungues on the pretarsus. The ventral surfaces of the 1st, 2nd and 3rd tarsomere are covered with dense adhesive setae. Each seta consists of two parts: a setal shaft and a modified apex. Three types of setae, viz. tapered setae, spatulate setae and discoidal setae, are identified based on the shape of the setal tip. The tapered setae are located at the edge of the 1st tarsomeres and on the whole 2nd tarsomeres of the male forelegs and mesolegs and on the whole 1st and 2nd tarsomere of the female legs and male metalegs. They are about 40–60 mm long and 4 mm broad at base, taper from base to apex with a curved, acute and hook-like apex and have a density of approximately 1 seta/100 mm2.The spatulate setae are located at the edge of the 3rd tarsomeres of the male forelegs and mesolegs and on the whole 3rd tarsomeres of the female legs and male metalegs. They are 55–85 mm long and 2.5–4.5 mm broad at base, have a spatulate terminal plate with 7–15 digits on margins, and have a density of approximately 1.5 setae/100 mm2. The discoidal setae are located in the centre of the 1st and 3rd tarsomeres of the male forelegs and mesolegs and are not present on female legs. They are about 45–80 mm long and 2.5–4.5 mm broad at base, have a discoidal terminal plate (7– 9 mm in diameter) and have a density of one seta/100 mm2. These setae are presumed to function as adherence during climbing or mating activities of the . The gland apertures and secretion solidification are also present on the ventral surfaces of the tarsus. KEY WORDS: Ultrastructure, , tarsomere, seta, attachment

Many , such as ants, flies and beetles, are capable of climbing upside down on smooth surfaces. Some of them are even capable of carrying loads equivalent to more than 100 times their own body weight, and can attach or detach freely while running in a high speed (Vogel and Steen, 2010). The mechanism behind the adhesive function of extremities is still a mystery. Legs are the most important organs for insect attachment to substrates. The attachment or detachment is performed by the adhesive structures located on the tarsi and pretarsi of the legs. There are mainly two different ways for insects to attach to substrates: smooth pads and hairy pads. The smooth pads are characterized by the presence of a very soft, bare and flexible cuticle and are found in the ants (Orivel et al., 2001; Federle et al., 2002), honeybees (Federle et al., 2001), cockroaches (van Casteren and Codd 2008) and grasshoppers (Goodwyn et al., 2006). According to their positions on the legs, the smooth pads can be further divided into the smooth arolium (Formicidae, Cicadidae), smooth pulvilli (Coreidae, Pentatomidae) and smooth euplantulae (Blattaria, Orthopteran). The hairy pads are characterized by a dense cover with adhesive setae, and according to their positions, they can also be divided into the hairy soles of tarsomeres (Coleoptera), ‘‘hairy’’ pulvilli (Diptera except for tipulids) and empodium (Bibionidae, Tabanidae), fossula spongiosa (Reduviidae) (Stork, 1980; Gorb, 1998; Beutel and Gorb, 2001; Betz, 2003; Bullock and Federle, 2011b).

1 Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R. China 2 Graduate University of Chinese Academy of Sciences, Beijing 10049, P.R. China 3 Corresponding author, E-mail: [email protected] Accepted 20 March 2013; Revised 27 March 2013 E 2013 Kansas Entomological Society VOLUME 86, ISSUE 2 123

Most Coleopteran species have hairy adhesive soles of tarsomeres, which are the main contact areas between the legs and the substrates (Stork, 1980). Members of the family Chrysomelidae are phytophagous and show advanced climbing skills on their host plants. The tarsal segments (tarsomeres) of chrysomelid species are usually wide and flattened which enables the beetles to increase the contact areas and adhesive forces with their host plants. Previous work on the tarsal ultrastructure in Chrysomelidae is scarce. Stork (1980) observed the tarsal adhesive setae of 24 species in Chrysomeloidea with SEM and found five types of setae, viz. the simple setae, spatulate setae bearing 1 or more digits, spatulate setae with no digits, bifid setae and disco-setae. Oides decempunctatus (Billberg, 1808) (Coleoptera: Chrysomelidae: Galerucine) is a very common and widespread leaf species in Asia and is present in most provinces of China, Korea and Japan. It is oval-shaped, yellow or orange with five black spots on each elytron and is often misidentified as a ladybug (Figs. 1A, 3A). O. decempunctatus is oligophagous with both the adults and larvae feeding on grape (Vitis spp.) leaves, causing dotted or erose holes. Occasionally the entire diachyma is consumed, leaving only the leaf veins (Yu et al., 1996, pp. 117–118). Like many other Chrysomelidae species, O. decempunctatus is good at climbing on smooth surfaces (Liu and Liang, unpubl. data). This paper presents descriptions of the tarsal ultrastructure of both male and female O. decempunctatus for the first time. Information on the external morphology, measurements, density and distribution of the adhesive setae are provided.

Materials and Methods Insects One male and one female adult specimen of O. decempunctatus were examined with the scanning electron microscope. The female was collected in the Beijing Botanical Garden, Beijing, China, in July 2011, and the male was collected in Guangdong Province of China, in July 1960. The specimens were deposited at the Insect Collection of the Institute of Zoology, Chinese Academy of Sciences.

Terminology Morphological terminology mostly follows that of Stork (1980), Beutel and Gorb (2001) and Betz (2003).

SEM The pretarsi and tarsi of forelegs, mesolegs and metalegs were removed from the body, cleaned by 2% phosphate buffered saline, stepwise dehydrated in ethanol (70%,80%,90%,100% 3 3), critical-point dried, coated with gold, and then viewed with a Quanta200 scanning electron microscope (FEI Co. Ltd., Oregon, USA).

Results Gross Morphology of Tarsus The 1st tarsomere (Tar I) is elongate and triangular in ventral view and its width is different in males and females: 1st tarsomeres of male forelegs and mesolegs (Fig. 3B, C) are somewhat wider than those of females (Fig. 1B, C). The 2nd 124 JOURNAL OF THE KANSAS ENTOMOLOGICAL SOCIETY

Fig. 1. Tarsi of the female Oides decempunctatus. A. Female adult, dorsal view. B. Tarsus and pretarsus, ventral view. C. 1st tarsomere, ventral view. D. 2nd tarsomere, ventral view. E. 3rd tarsomere, ventral view. F. 5th tarsomere and ungues, ventral view. Abbreviations: un, ungue (claw); tar I, the 1st tarsomere; tar II, the 2nd tarsomere; tar III, the 3rd tarsomere; tar V, the 5th tarsomere. VOLUME 86, ISSUE 2 125 tarsomere (Tar II) is short and trapeziform (Figs. 1B, D; 3B, D). The 3rd tarsomere (Tar III) is enlarged, bilobed and heart-shaped (Figs. 1B, E; 3B, E). The ventral surfaces of the 1st, 2nd and 3rd trasomeres are covered with dense setae. The 4th tarsomere (Tar IV) is extremely reduced and is hidden inside the 3rd tarsomere. The 5th tarsomere (Tar V) is elongate and slender and is scattered with setae on the ventral, lateral and dorsal surfaces (Fig. 1F).

Morphology and Types of Tarsal Setae Each seta on the ventral surfaces of the 1st, 2nd and 3rd tarsomere consists of two parts: a setal shaft (sh) and a modified apex that is either tapered or is a spatulate or discoidal terminal plate (tp) (Figs. 2, 4). These three types of setae are separately situated in specific parts of the tarsus. The three female leg pairs and male metalegs only have two types of setae (Figs. 1, 2), but all three types of setae are present on male forelegs and mesolegs (Figs. 3, 4). The distribution of all three types of setae on male forelegs and mesolegs is indicated with three different colors with the tapered, spatulate and discoidal setae highlighted in blue, green and orange, respectively. These setae are presumed to function as adherence during the climbing or mating activities of the beetles. Tapered setae. These setae are located on the whole ventral surfaces of the 1st and 2nd tarsomeres of the three female leg pairs and the male metalegs (Fig. 1C, D) and are present only at the edge of the 1st tarsomeres and on the whole 2nd tarsomeres of the male forelegs and mesolegs (Fig. 3B–D). The seta is elongate, slender, about 40– 60 mm in length and 4 mm in width at base. The setal shaft is straight at base, tapers upwards with curved, acute and hook-like apex (Figs. 2A, B, 3F) and centrally hollow (Fig. 6). There is approximately 1 tapered seta in each 100 mm2 on average. Spatulate setae. These setae are located on the whole ventral surfaces of the 3rd tarsomeres of three female leg pairs and the male metalegs (Fig. 1E) and at the edge of the 3rd tarsomeres of the male forelegs and mesolegs (Fig. 3B, E). The setal shaft is straight, slightly bent, 55–85 mm in length and 2.5–4.5 mm in width. Each seta has a spatulate terminal plate and the widest part of the terminal plate is 6–8 mm in width. The ventral surface of the terminal plate is smooth but its dorsal surface has 7–15 digits (Figs. 2C, F, 4A, B). The spatulate setae are in a high density and there are approximately 1.5 setae in every 100 mm2 on average. Discoidal setae. These setae were only found in the centre of the 1st and 3rd tarsomeres of the male forelegs and mesolegs and are missing from female legs. The setal shaft is straight, about 45–80 mm in length and 2.5–4.5 mm in width. Each seta has a discoidal terminal plate. The terminal plate is approximately 7–9 mmin diameter and its marginal area is slightly higher than the centre (Fig. 4C, F). The terminal plates of the discoidal setae on the 1st tarsomeres usually bear 2 or 1 tips at the edge, while those on the 3rd tarsomeres usually have only one or no tips. The discoidal setae are in a lower density and there is only 1 discoidal seta on every 100 mm2 on the 3rd tarsomeres.

Pretarsus The pretarsus of O. decempunctatus has paired horn-shaped claws (Fig. 1F), which connect to the distal end of the 5th tarsomere. These claws are spread and bidentate. They are approximately 200 mm in length and 310 mm in width. The accessory claws are about 150 mm in length. 126 JOURNAL OF THE KANSAS ENTOMOLOGICAL SOCIETY

Fig. 2. Setae on the tarsi of the female Oides decempunctatus. A. Tapered setae on the 1st tarsomere; B. Tapered setae on the 2nd tarsomere. C–F. Spatulate setae on the 3rd tarsomere. Abbreviations: sh, setal shaft; tp, terminal plate; white arrow indicates the gland apertures. VOLUME 86, ISSUE 2 127

Fig. 3. Tarsi of the forelegs and mesolegs of male Oides decempunctatus. A. Male adult, dorsal view. B. Tarsus and pretarsus, ventral view. The tapered, spatulate and discoidal setae separately present in the blue, green and orange area. C. 1st tarsomere, ventral view. D. 2nd tarsomere, ventral view. E. 3rd tarsomere, ventral view. F. Tapered setae at the edge of the 1st tarsomere. Abbreviations: un, ungue (claw); tar I, the 1st tarsomere; tar II, the 2nd tarsomere; tar III, the 3rd tarsomere; tar V, the 5th tarsomere. 128 JOURNAL OF THE KANSAS ENTOMOLOGICAL SOCIETY

Fig. 4. Setae on the forelegs and mesolegs of male Oides decempunctatus. A, B. Spatulate setae at the edge of the 3rd tarsomere. C, D. Discoidal setae in the centre of the 1st tarsomere. E, F. Discoidal setae in the centre of the 3rd tarsomere. Abbreviations: sh, setal shaft; tp, terminal plate; white arrow indicates the gland apertures. VOLUME 86, ISSUE 2 129

Fig. 5. Secretion solidification on the 3rd tarsomeres of male (A) and female (B) of Oides decempunctatus. Abbreviations: sh, setal shaft; tp, terminal plate.

Secretion Many gland apertures of secretion were seen on the ventral surface of tarsus (white arrows in Figs. 2A, 4C) and the secretion solidification was found around the setae. Substantial secretion was found on the 3rd tarsomeres (Fig. 5A, B).

Discussion Presence of Expanded Tarsi and Bidentate Ungues Like many other arboreal Chrysomelidae species, O. decempunctatus has flattened and expanded tarsi covered with extremely dense hairs. In males, the first tarsal segments of forelegs and mesolegs are obviously wider than those of females. O. decempunctatus has a pair of bidentate ungues (claws) and the sharp ungues can stab into the plant tissues or hook the rough surfaces. The ungues are much useful on the rough surfaces, while the hairs are of huge significance for adhesion on the smooth surfaces (Bullock and Federle, 2011a). The tarsal and pretarsal structures of O. decempunctatus are likely well-adapted to the arboreal dwelling environment in which the leaves are smooth and need more adhesive forces for attachment. In order to climb on foliage, their tarsi and claws (the contact organs) have been experiencing a long co-evolution process with the leaf surfaces of their host plants. The host plants have developed tomenta or waxes on their leaves preventing beetles from walking on them (Gorb et al., 2002). On the other hand, the beetles have developed expanded tarsi and bidentate ungues to increase their adhesive forces.

Diversity of Setae Morphology O. decempunctatus has three different types of setae on the tarsus. The morphology, distribution and adhesive force of these setae are diverse. The tapered setae with their acute and hook-like apices and the spatulate setae are for climbing (Stork, 1980). The spatulate setae have some digits on the dorsal surface of their terminal plates, which differ in number (0, 1 or more) in different beetle species (Stork, 1980). According to Stork (1980), these digits may prevent the setae to stick 130 JOURNAL OF THE KANSAS ENTOMOLOGICAL SOCIETY

Fig. 6. Tapered setae of Oides decempunctatus, showing centrally hollow setal shafts. together. Compared with the tapered setae, the spatulate setae are more numerous in density and larger in size, so they should have much more contact area on the substrates and supply more adhesive forces for climbing onto leaf surfaces of their host plants or other smooth surfaces. The discoidal setae are specific for males and may play an important role in the mating behavior. During the mating process, the male beetles need to grasp the females’ elytra tightly and the elytra are particularly smooth. The discoidal setae are found in males of many beetle species and differ in number, shape, diameter and distribution (Stork, 1980; Pelletier and Smilowitz, 1987; Bullock and Federle, 2011b). Stork (1980) studied 4 Galerucine species (Chrysomelidae) and found that the greatest number of the discoidal setae in Sermylassa halensis (L., 1767) was found on the 2nd and 3rd tarsomeres of the fore and middle tarsi. However, in Lochmaea suturalis (Thomson, 1866) and L. caprea (L., 1758), the greatest number of the discoidal setae was found on the 1st tarsomeres of the hind tarsi, and they are absent in Galerucella tenella (L., 1761) (Stork, 1980).

Secretion The gland apertures and secretion solidification around the setae on ventral surface of tarsus are very common in beetle species and they are important in the adhesive process. The contact of the insect adhesive organs and the substrates is mediated by an adhesive liquid film (Federle et al., 2002). The liquid film provides adhesive forces by means of the surface tension and viscosity. Without the liquid film, the adhesive forces will decrease quickly (Langer et al., 2004). There are some canals through the cuticle to release the secretion. However, the mechanism of the wet adhesion is little understood. In O. decempunctatus, the secretion appears to be released onto the surface of the tarsus through the apertures and the centrally hollow setae (Figs. 2A, 4C, 6). Histological sections are, however, required to verify this hypothesis. VOLUME 86, ISSUE 2 131

Many factors, such as the physicochemical property of the secretion, the length and flexibility of the setal materials, the shape of the setal terminal plates, and the width of the tarsus all contribute to the climbing ability of the beetles (Beutel and Gorb, 2001). Setae are made of soft, pliable and flexible materials and are able to adapt to the surface profile of the substrate. Adhesive setae need to be both long enough and sufficiently fine to accommodate the surface roughness (Bullock and Federle, 2011a). The flexibility of these setae allows the minimization of contact distances and maximization of contact areas in order to increase the adhesive force (Beutel and Gorb, 2001). On a single discoidal terminal plate, the central part supplies adhesion approximately twice as strong as the edge part does (Langer et al., 2004).

Acknowledgements We are grateful to Siqin Ge (Institute of Zoology, Chinese Academy of Sciences) for providing the specimens used in this study. We are also grateful to Chunli Li (Institute of Microbiology, Chinese Academy of Sciences) for providing technical assistance with the scanning electron microscopy. This work was supported by the following sources: the National Basic Research Program of China (973 Program) (grant no 2011CB302102), Main Direction Program of Knowledge Innovation of Chinese Academy of Sciences (grant no KSCX2-EW-G-4), the National Natural Science Foundation of China (grant nos 30970400, 31172128), and the National Science Fund for Fostering Talents in Basic Research (Special subjects in taxonomy, NSFC- J1210002), all awarded to APL.

Literature Cited Betz, O. 2003. Structure of the tarsi in some Stenus species (Coleoptera, Staphylinidae): external morphology, ultrastructure, and tarsal secretion. Journal of Morphology 255(1):24–43. Beutel, R. G., and S. N. Gorb. 2001. Ultrastructure of attachment specializations of hexapods (Arthropoda): evolutionary patterns inferred from a revised ordinal phylogeny. Journal of Zoological Systematics and Evolutionary Research 39:177–207. Bullock, J. M. R., and W. Federle. 2011a. The effect of surface roughness on claw and adhesive hair performance in the dock beetle Gastrophysa viridula. Insect Science 18:298–304. Bullock, J. M. R., and W. Federle. 2011b. Beetle adhesive hairs differ in stiffness and stickiness: in vivo adhesion measurements on individual setae. Naturwissenschaften 98(5):381–387. Federle, W., E. L. Brainerd, T. A. McMahon, and B. Ho¨lldobler. 2001. Biomechanics of the movable pretarsal adhesive organ in ants and bees. Proceedings of the National Academy of Sciences, USA 98(11):6215–6220. Federle, W., M. Riehle, A. S. G. Curtis, and R. J. Full. 2002. An integrative study of insect adhesion: mechanics and wet adhesion of pretarsal pads in ants. Integrative and Comparative Biology 42:1100–1106. Goodwyn, P. P., A. Peressadko, H. Schwarz, V. Kastner, and S. Gorb. 2006. Material structure, stiffness, and adhesion: why attachment pads of the grasshopper (Tettigonia viridissima) adhere more strongly than those of the locust (Locusta migratoria) (Insecta: Orthoptera). Journal of Comparative Physiology A 192(11):1233–1243. Gorb, S. N. 1998. The design of the fly adhesive pad: distal tenent setae are adapted to the delivery of an adhesive secretion. Proceedings of the Royal Society of London Series B 265(1398):747–752. Gorb, S. N., R. G. Beutel, E. V. Gorb, Y. Jiao, V. Kastner, S. Niederegger, V. L. Popov, M. Scherge, U. Schwarz, and W. Vo¨tsch. 2002. Structural design and biomechanics of friction-based releasable attachment devices in insects. Integrative and Comparative Biology 42(6):1127–1139. Langer, M. G., J. P. Ruppersberg, and S. Gorb. 2004. Adhesion forces measured at the level of a terminal plate of the fly’s seta. Proceedings of the Royal Society of London Series B 271(1554):2209–2215. 132 JOURNAL OF THE KANSAS ENTOMOLOGICAL SOCIETY

Orivel, J., M. C. Malherbe, and A. Dejean. 2001. Relationships between pretarsus morphology and arboreal life in ponerine ants of the genus Pachycondyla (Formicidae: Ponerinae). Annals of the Entomological Society of America 94(3):449–456. Pelletier, Y., and Z. Smilowitz. 1987. Specialized tarsal hairs on adult male Colorado potato beetles, Leptinotarsa decemlineata (Say), hamper its locomotion on smooth surfaces. Canadian Entomologist 119(12):1139–1142. Stork, N. E. 1980. A scanning electron microscope study of tarsal adhesive setae in the Coleoptera. Zoological Journal of the Linnean Society 68(3):173–306. van Casterena, A., and J. R. Codd. 2010. Foot morphology and substrate adhesion in the madagascan hissing Cockroach, Gromphadorhina portentosa. Journal of Insect Science 10(40):1–12. Vogel, M. J., and P. H. Steen. 2010. Capillarity-based switchable adhesion. Proceedings of the National Academy of Sciences, USA 107(8):3377–3381. Yu, P. Y., S. Y. Wang, and X. K. Yang. 1996. Economic Insect Fauna of China. Fasc. 54. Coleoptera: Chrysomeloidea (II). Science Press, Beijing. 324 pp.